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WO2009143475A9 - Methods and compositions related to aglycosidic esculentoside a - Google Patents

Methods and compositions related to aglycosidic esculentoside a Download PDF

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Publication number
WO2009143475A9
WO2009143475A9 PCT/US2009/045056 US2009045056W WO2009143475A9 WO 2009143475 A9 WO2009143475 A9 WO 2009143475A9 US 2009045056 W US2009045056 W US 2009045056W WO 2009143475 A9 WO2009143475 A9 WO 2009143475A9
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Prior art keywords
esa
radiation
och
cancer
subject
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PCT/US2009/045056
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French (fr)
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WO2009143475A1 (en
Inventor
Paul Okunieff
Zhang Lurong
Yang Shanmin
Zhang Mei
Chaomei Liu
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University Of Rochester
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Publication of WO2009143475A1 publication Critical patent/WO2009143475A1/en
Publication of WO2009143475A9 publication Critical patent/WO2009143475A9/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/194—Carboxylic acids, e.g. valproic acid having two or more carboxyl groups, e.g. succinic, maleic or phthalic acid

Definitions

  • This invention relates generally to methods and compositions related to new selective COX-2 inhibitors.
  • Cyclooxygenase is an enzyme that catalyzes the rate-limiting step in the conversion of arachidonic acid to prostaglandins.
  • COX-I is constitutively expressed at low levels in many cell types. Specifically, COX-I is known to be essential for maintaining the integrity of the gastrointestinal epithelium. COX-2 expression is stimulated by growth factors, cytokines, and endotoxins. The cyclooxygenase 2 isoform is not expressed in most tissues (e.g., liver) under physiological conditions but is highly upregulated under certain conditions. For example, COX-2 is upregulated in inflammatory processes and cancer, for example. Up-regulation of COX-2 is responsible for the increased formation of prostaglandins associated with inflammation. What is needed in the art are novel compositions and methods for inhibiting COX-2.
  • this invention in one aspect, relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound having Formula I: wherein, R 1 and R 2 are, independent of one another, H, OH, alkyl (e.g., CH 3 , C 2 H 5 , C 3 H 7 , CH(CH 3 ) 2 ,
  • alkoxide e.g., OCH 3 , OC 2 H 5 , OC 3 H 7 , OCH(CH 3 ) 2 , OC 4 H 9 , OCH(CH 3 )C 2 H 5 , OCH 2 CH(CH 3 ) 2 , OC(CH 3 ) 3 ,
  • aryl e.g., C 6 H 5 , C 6 H 4 OCH 3 , C 6 H 4 OH, C 6 H 4 ONH 2 , CH 2 C 6 H 5
  • hydroxyl alkyl e.g., -CH 2 OH, -C 2 H 4 OH
  • OSO 2 CH 3 OSO 2 C 6 H 5 CH 3
  • OC 5 -cyclic ether e.g., THF
  • OC 6 -cyclic ether e.g., THP
  • O-linear ether e.g., MOM, MEM
  • O-silyl ether e.g., TBDMS, TMS, TES
  • ester e.g., CH 3 C(O)O (acetate), CH 3 CH 2 C(O)O (propionate)
  • acyl e.g. , CH 3 C(O), CH 3 CH 2 (O), acetal, OLi, ONa, OK, or OCa
  • aryl
  • Each R 3 is, independent of one another, H, OH, alkoxide (e.g., OCH 3 , OC 2 H 5 , OC 3 H 7 , OCH(CH 3 ) 2 , OC 4 H 9 , OCH(CH 3 )C 2 H 5 , OCH 2 CH(CH 3 ) 2 , OC(CH 3 ) 3 , OC 5 Hn), hydroxyl alkyl (e.g., -CH 2 OH, C 2 H 4 OH), C(O)H, CO 2 H, CO 2 Na, CO 2 K, CO 2 Li, CO 2 Ca, CO 2 R 8 , Or -CH 2 OR 8 ;
  • Each R 4 is, independent of one another, H, alkyl (e.g.
  • R 5 and R 6 are, independently of one another, H, CH 3 , OH, CO 2 H, or CO 2 R 8 ;
  • Each R 7 is, independent of one another, H or together are an oxo group;
  • R 8 is an alkyl (e.g. , CH 3 , C 2 H 5 , C 3 H 7 , CH(CH 3 ) 2 , C 4 H 9 , CH(CH 3 )C 2 H 5 , CH 2 CH(CH 3 ) 2 ,
  • R 2 does not comprise a saccharide.
  • the compound comprises Formula II:
  • Also disclosed is a method of reducing inflammation in a subject comprising administering to the subject an effective amount of any one of the compositions disclosed above.
  • a method of inhibiting a cytokine in a subject comprising administering to the subject an effective amount of any one of the compositions disclosed above.
  • the cytokine can be selected from the group consisting of ILl, IL6, TNF ⁇ , TNF ⁇ ,
  • VEGF vascular endothelial growth factor
  • MCPl vascular endothelial growth factor
  • a method of reducing radiation damage in a subject comprising administering to the subject an effective amount of any one of the compositions disclosed above.
  • the radiation damage can be caused by radiation therapy.
  • the radiation therapy can also be used to treat cancer.
  • the radiation damage can be caused by nuclear radiation.
  • the radiation can be caused by a weapon.
  • a method of treating neoplasias in a subject comprising administering to the subject an effective amount of any one of the compositions disclosed above.
  • the neoplasia can be selected from lung cancer, breast cancer, gastrointestinal cancer, bladder cancer, head and neck cancer and cervical cancer.
  • a method of treating a subject with arthritis the method comprising administering to the subject an effective amount of any one of the compounds disclosed above, hi one example, the arthritis can be rheumatoid arthritis.
  • Figure 1 shows the structure of EsA, h-EsA, and Celebrex®.
  • Figures 2 and 3A show Celebrex® effectively inhibited the early phase of IR demonists (up to day 19 with the onset on day 14), but gradually it loss its effect after day
  • Figure 3B shows that EsA dramatically reduced the IR-induced soft tissue fibrosis evidenced by the much less shortening of IR leg as compared to the vehicle control and Celebrex® group.
  • Figure 4A shows alopecia induced by COX-2 inhibitors.
  • Figure 4B shows that alopecia recovers faster in the EsA group compared to the Celebrex® group.
  • Figure 5 A shows the effect of EsA on collagen induced arthritis (CIA).
  • Figure 5B shows the effect of EsA on swollen paws.
  • Figure 5C shows the effect of EsA on the climbing (grab force) of a joint.
  • Figures 6A-C show that at the intermediate phase (6 weeks after onset of symptom), the grab force of paw was measured and the difference among the control and treatment groups can be well distinguished. While the normal mice could hold on the bar for 12 minutes, the CIA mice could only hold for a few seconds. This significant impairment in CIA paw function could be reversed by both EsA and h-ESA to a greater degree than was observed for Celebrex®.
  • Figure 7 shows h-EsA inhibits CIA relapsing.
  • Figure 8 shows h-EsA reduces joint deformation.
  • Figure 9 shows pathological analysis of CIA.
  • Figure 10 shows EsA inhibits anticollagen II production.
  • Figure 11 shows IR-induced increase of ILl ⁇ , TNF ⁇ , MCP-I and VEGF in macrophages were reduced by EsA in a dose-dependent manner.
  • Figure 12 shows IR-induced increase of IL-I ⁇ and TNF ⁇ in macrophages in vitro were reduced by EsA in a dose-dependent manner.
  • Figure 13 shows EsA reduced IL- l ⁇ and TNF ⁇ in IR skin.
  • Figure 14 shows that EsA reduces VEGF in irradiated lung.
  • FIG. 26 shows that h-EsA does not significantly reduce IL-6 in lung 3 weeks post-radiation.
  • Figure 16 shows the effect of EsA on production of IL-l ⁇ . After treatment without or with 0.1 or 1 ug/ml EsA for 18 hr, the Raw 264.7 macrophage cells or A431 epidermoid cells were irradiated at different IR doses. The protein level of IL-l ⁇ was measured by ELISA. At doses of 2-4Gy, IL-l ⁇ was greatly induced (A), but could be reduced by EsA at a concentration of 0.1 ug/ml (B and C). 28.
  • Figure 17 shows EsA-reduced IR-pneumonitis and fibrosis.
  • Figure 18 shows the effect of Dexamethasone, Celebrex® and h-EsA after 8 weeks of treatment on the breath rate of ionized lung (18 Gy) in C57BL/6 mouse measured at 4 months.
  • Figure 19 shows the effect of h-EsA, GA(8 weeks) on the compliance of ionized (18 Gy) lung in C57BL/6 mice at 6.7 months.
  • Figure 20 shows h-EsA and GA in HPLC.
  • Figure 21 shows plasma and liver PK of h-EsA and EsA.
  • Figure 22 shows that the CBCT can obtain a complete 3D 650x650x428 scan (i.e. 428 slices) in ⁇ 10 seconds with isotropic resolution of 270 ⁇ m (central slice shown), encompassing a volume approximately 15 cm 3 .
  • Figure 23 shows the CBCT imaging data was analyzed using custom MATLAB software. The lungs were segmented automatically (Fig 23A) and a 3D lung region was obtained for each mouse (Fig 23B). In order to reduce the effect of cardiac motion on the value of mean lung density, the boundary of the lung was excluded from the analysis.
  • Figure 24 shows CBCT scans in mice. For each mouse, a histogram of the voxel intensity (pulmonary tissue density) was created (Fig 24A). The lower density unit (HU) is, the health the lung is. The data from two independent study (separated in 2 years) showed that: 1) normal range of CBCT was relatively consistent (mean value -454 to -473); 2) 7- 12 months post lung IR ( 15- 18 Gy), the HU increased (mean value -348 to
  • FIG. 25 shows the experimental procedure for the effect of h-EsA on radiation-induced pneumonitis and lung fibrosis. The specific results are shown in Figures 26-39.
  • Figure 26 shows the effects of plasma on IL-I alpha.
  • Figure 27 shows the effect of h-EsA on lung PF-4. 39.
  • Figure 28 shows the effect of h-EsA on lung TNF-alpha.
  • Figure 29 shows the effect of h-EsA on lung VACAM-I.
  • Figure 30 shows the effect of h-EsA on p-selectin (day 17 after lung IR).
  • Figure 31 shows the effect of h-EsA on lung TNF-alpha.
  • Figure 32 shows the effect of h-EsA and h-GA on ionized lung.
  • Figure 33 shows the effect of h-EsA on lung p-selectin 48 hours post lung IR.
  • Figure 34 shows the effect of h-EsA on secretion of p-selectin in ionized whole lung.
  • Figure 35 shows the effect of h-EsA on the breath rate of ionized lung (8 weeks).
  • Figure 36 shows the effect of h-EsA on the breath rate of ionized whole lung.
  • Figure 37 shows the effect of h-EsA on the compliance of ionized lung at 7 months.
  • Figure 38 shows the effect of h-EsA on the compliance of ionized lung at 6.7 months.
  • Figure 39 shows the effect of h-EsA (8 weeks) on the compliance of ionized lung at 6.7 months.
  • Figure 40 shows the experimental procedure for showing the effect of h-EsA on radiation-induced dermatitis and soft tissue fibrosis. The results are seen in Figures 41-43. 52.
  • Figure 41 shows the effect of h-EsA on the scoring of the right hind leg ionized with 30 Gy.
  • Figure 42 shows the effect of h-EsA on the skin score of ionized right leg measured at 2 months.
  • Figure 43 shows the effect of h-EsA on the skin score of right hind leg measured at 7 months.
  • Figure 44 shows the experimental procedure for showing the effect of h-EsA on collagen-induced arthritis (CIA). The results are shown in figures 45-58.
  • Figure 45 shows the effect of h-EsA on the secretion of IL-I alpha at 72 hours.
  • Figure 46 shows the effect of h-EsA on swollen hind paws at 5 months.
  • Figure 47 shows the effect of h-EsA on swollen hind paws in relapsing CIA model at 5 weeks.
  • Figure 48 shows the effect of h-EsA on swollen hind paws in a CIA model measured at 48 hours.
  • Figure 49 shows the effect of h-EsA on extension ability on hind ankle in relapsing CIA at 5 months.
  • Figure 50 shows the effect of h-EsA on relapsing CIA model at 5 months.
  • Figure 51 shows the effect of h-EsA on climbing of hind paws at 6 weeks.
  • Figure 52 shows the effect of h-EsA on the extension ability of hind ankle in relapsing CIA model.
  • Figure 53 shows the effect of h-EsA on swollen paws in relapsing CIA.
  • Figure 54 shows the effect of EsA and h-EsA on swollen paws in CIA at three weeks.
  • Figure 55 shows measuring the extent angle of an ankle.
  • Figure 56 shows that when CIA mouse were treated with vehicle alone, there was a lot of infiltrated inflammatory cells and erosion of cartilage. In contrast, when CIA mice were treated with h-ES A , there were much less infiltrated inflammatory cells and cartilage erosion.
  • Figure 57 shows CIA model in DBA/1 J mouse treated with CelebrexTM. It was shown that most of the cartilage was normal, but there was a foci of erosions in bone and cartilage.
  • Figure 58 shows CIA model in DBA/1 J mouse treated with h-ESA. It was shown that most of the cartilage was normal, but there was a foci of erosions in bone and cartilage
  • Figure 59 shows h-EsA reduces inflammatory cells infiltrated in the irradiated lung in the pneumonitis phase. At day 2.5 and 17 after lung radiation, the lung lavage was collected from each mouse in all groups. The cellular portion was assessed for the numbers of infiltrated macrophage and neutrophils. The h-EsA significantly inhibited the number of infiltrated macrophage on day 2.5. Shown is the effect of agents on lavage macrophage at 2.5 days post- 15 Gy lung IR. 71.
  • Figure 60 shows the effect of agents on lavage macrophage at 17 days post-
  • Figure 61 shows h-EsA reduces inflammatory cells infiltrated in the irradiated lung in the pneumonitis phase. The effects of agents on lavage neutrophils 17 days post- lung IR is shown, and it can be seen that on day 17, the infiltrated neutrophils were reduced (PO.05).
  • Figure 62 shows that h-EsA reduced radiation-induced lung pathological alterations in acute phase.
  • the pathological analysis on day 2.5 and 17 post-IR demonstrated that h-EsA reduced the infiltrated inflammatory cells in lung.
  • FIG. 63 shows that h-EsA reduces radiation-induced lung fibrosis.
  • the lung of mice treated with either vehicle alone or h-EsA were assessed for the content of collagen with two assays. Collagen trichrome blue staining showed that the IR-induced collagen deposit was reduced by h-EsA (less collagen blue staining as compared to vehicle control).
  • Figure 64 also shows that h-EsA reduces radiation-induced lung fibrosis.
  • Hydroxyproline a key component of collagen, was measured with chemical reaction (HCl digestion of protein and then assaying the small collagen component). The results showed that hydroxyproline in the lung was reduced in the h-EsA group compared with IR + vehicle control (P ⁇ 0.05). Taken together, it was shown that h-EsA inhibited radiation induced both pneumonitis and lung fibrosis.
  • Figure 65 shows the effect of h-EsA on radiation-induced dermonitis and soft tissue contraction.
  • Right hind legs of mice (8 mice/group) were subjected to 30 Gy radiation and then the vehicle alone or h-EsA was given orally at 10 mg/kg every other day for one month .
  • the skin score was measured at 2 months after treatment (skin score), and the results show that h-EsA treated mice had significantly less radiation-induced dermonitis.
  • Figure 66 shows the effect of h-EsA on leg Conscture 2 months after irradiated hind leg of C57BL/6 mice. It is clearly seen that the h-EsA group had significantly less contracture of the hind leg.
  • Figure 67 shows the results of an acute toxicity test. The acute toxicity of EsA and h-EsA on C57 and ICR mice is shown. The therapeutic does of h-EsA is 10-15 mg/kg, and the therapeutic window can be > 50.
  • Ranges may be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. 82.
  • the terms “higher,” “increases,” “elevates,” or “elevation” refers to levels above control levels.
  • the terms “low,” “lower,” “reduces,” or “reduction” refers to levels below control levels. For example, control levels can be normal in vivo levels prior to, or in the absence of, inflammation or the addition of an agent which causes inflammation.
  • Inflammation or "inflammatory” is defined as the reaction of living tissues to injury, infection, or irritation. Anything that stimulates an inflammatory response is said to be inflammatory.
  • Inflammatory disease is defined as any disease state associated with inflammation.
  • the inflammation can be associated with an inflammatory disease.
  • inflammatory disease include, but are not limited to, asthma, systemic lupus erythematosus, rheumatoid arthritis, reactive arthritis, spondyarthritis, systemic vasculitis, insulin dependent diabetes mellitus, multiple sclerosis, experimental allergic encephalomyelitis, Sjogren's syndrome, graft versus host disease, inflammatory bowel disease (including Crohn's disease and ulcerative colitis) and scleroderma, myasthenia gravis, Guillain-Barre disease, primary biliary cirrhosis, hepatitis, hemolytic anemia, uveitis, Grave's disease, pernicious anemia, thrombocytopenia, Hashimoto's thyroiditis, oophoritis, orchitis, adrenal gland diseases, anti-phospholipid syndrome, Wegener's granulomatos
  • infectious process is defined as the process by which one organism is invaded by any type of foreign material or another organism. The results of an infection can include growth of the foreign organism, the production of toxins, and damage to the host organism.
  • Cancer therapy is defined as any treatment or therapy useful in preventing, treating, or ameliorating the symptoms associated with cancer. Cancer therapy can include, but is not limited to, apoptosis induction, radiation therapy, and chemotherapy.
  • Transplant is defined as the transplantation of an organ or body part from one organism to another.
  • Transplant rejection is defined as an immune response triggered by the presence of foreign blood or tissue in the body of a subject. In one example of transplant rejection, antibodies are formed against foreign antigens on the transplanted material.
  • inhibition means to reduce at least one activity as compared to a control (e.g., activity in the absence of such inhibition). It is understood that inhibition or suppression can mean a slight reduction in activity to the complete ablation of all activity. Inhibition or suppression also includes prevention.
  • An "inhibitor” or “suppressor” can be anything that reduces the targeted activity, or has the potential to reduce the targeted activity in the preventative sense. For example, inhibition of COX-2 by a composition such as h-EsA or a derivative thereof can be determined by assaying the amount of COX-2 activity present in a cell.
  • the composition can be administered to the cell before it is exposed to circumstances that would cause an elevation in COX-2 activity, and the levels of COX-2 activity can be measured before and after the exposure.
  • the composition can be said to inhibit COX-2 activity if the amount of COX-2 activity is reduced in the presence of the composition as compared to the amount of COX-2 activity in the absence of the composition.
  • a “subject” is meant an individual.
  • the "subject” can include domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.) and birds.
  • livestock e.g., cattle, horses, pigs, sheep, goats, etc.
  • laboratory animals e.g., mouse, rabbit, rat, guinea pig, etc.
  • the subject is a mammal such as a primate, and, more preferably, a human.
  • aglycosidic EsA is meant an Esculentoside A (EsA) molecule that is aglycosidic.
  • EsA Esculentoside A
  • An example of such a molecule is h-EsA, which is described in greater detail below.
  • the aglycosidic EsA molecule and deriviatives thereof are useful in reducing radiation damage, cytokine inhibition by radiation damage, brain edema, pain, and inflammation, for example. They can also be used to treat neoplasias, as disclosed in US Patent Publication Nos. 20040053935, 20040053934, 20040053900, 20030225150, 20030162829, 20020169195, and 20020086894.
  • Saponins are a large family of naturally occurring glycoconjugate compounds with considerable structural diversity. Saponins are glycosidic natural plant products, composed of a ring structure (the aglycone) to which is attached one or more sugar chains. The saponins are grouped together based on several common properties. In particular, saponins are surfactants which display hemolytic activity and form complexes with cholesterol. Although saponins share these properties, they are structurally diverse.
  • the aglycone can be a steroid, triterpenoid or a steroidal alkaloid and the number of sugars attached to the glycosidic bonds vary greatly, or can be entirely absent, as is the case with h-EsA and the derivatives thereof discussed herein.
  • Saponins have been used in pharmaceutical compositions for a variety of purposes.
  • U.S. Pat. No. 5,118,671 describes the use of aescin, a saponin obtained from Aesculus hippocastanum seeds, in pharmaceutical and cosmetic compositions as an anti-inflammatory.
  • U.S. Pat. No. 5,147,859 discusses the use of Glyccyrrhiza glabra saponin/phospholipid complexes as anti-inflammatory and anti-ulcer agents
  • U.S. Pat. No. 5,166,139 describes the use of complexes of saponins and aglycons, obtained from Centella asiatica and Terminalia sp., with phospholipids in pharmaceutical compositions.
  • International Publication No. WO 91/04052, published 4 Apr. 1991 discusses the use of solid Quillaja saponaria saponin/GnRH vaccine compositions for immunocastration and immunospaying.
  • the saponin family includes Esculentosides A, B, C, D and E, and are isolated from Phytolacca esculent.
  • Esculentoside A Esculentoside A (EsA, 3-0- [ ⁇ -D-glucopyranosyl- (Hensley et al. (1999); Felemovicius et al. (1995))- ⁇ -D-xylo-pyranosyl] phytolaccagenin, Figure 1) is a highly purified saponin from Phytolacca esculent.
  • EsA has a molecular weight of 826 Daltons (Yi et al. Chinese Herb Medicine, 15 (2): 7-11 (1984)).
  • EsA Because of its hydrophilic radices such as hydroxide and carboxyl, EsA has a high water-solubility. Derivatives and analogs of EsA are also contemplated and are discussed below. Specifically disclosed herein is h-EsA, an aglycoside analog of Esculentoside A. Esculentoside A does not have a cross reaction with sulfonamide antibiotics, unlike many non-steroidal anti-inflammatory drugs (NSAIDS). h-EsA has anti-inflammatory effects with mechanisms differing from currently used anti-inflammatory drugs.
  • NSAIDS non-steroidal anti-inflammatory drugs
  • Inflammation is a complex stereotypical reaction of the body expressing the response to damage of its cells and vascularized tissues.
  • the discovery of the detailed processes of inflammation has revealed a close relationship between inflammation and the immune response.
  • the main features of the inflammatory response are vasodilation, i.e. widening of the blood vessels to increase the blood flow to the infected area; increased vascular permeability, which allows diffusible components to enter the site; cellular infiltration by chemotaxis, or the directed movement of inflammatory cells through the walls of blood vessels into the site of injury; changes in biosynthetic, metabolic, and catabolic profiles of many organs; and activation of cells of the immune system as well as of complex enzymatic systems of blood plasma.
  • Acute inflammation can be divided into several phases. The earliest, gross event of an inflammatory response is temporary vasoconstriction, i.e. narrowing of blood vessels caused by contraction of smooth muscle in the vessel walls, which can be seen as blanching (whitening) of the skin. This is followed by several phases that occur over minutes, hours and days later. The first is the acute vascular response, which follows within seconds of the tissue injury and lasts for several minutes. This results from vasodilation and increased capillary permeability due to alterations in the vascular endothelium, which leads to increased blood flow (hyperemia) that causes redness (erythema) and the entry of fluid into the tissues (edema).
  • Activated cells can also be identified at the site of inflammation. “Activated cells” are defined as cells that participate in the inflammatory response.
  • T-cells and B-cells examples include, but are not limited to, T-cells and B-cells , macrophages, NK cells, mast cells, eosinophils, neutrophils, Kupffer cells, antigen presenting cells, as well as vascular endothelial cells.
  • Macrophages release cytokines (e.g., tumor necrosis factor, interleukin-1), which heighten the intensity of inflammation by stimulating inflammatory endothelial responses; these endothelial changes help recruit large numbers of T cells to the inflammatory site.
  • cytokines e.g., tumor necrosis factor, interleukin-1
  • Damaged tissues release pro-inflammatory mediators (e.g., Hageman factor (factor XII) that trigger several biochemical cascades.
  • the clotting cascade induces fibrin and several related fibrinopeptides, which promote local vascular permeability and attract neutrophils and macrophages.
  • the kinin cascade principally produces bradykinin, which promotes vasodilation, smooth muscle contraction, and increased vascular permeability.
  • h-EsA shows a strong inhibition of inflammation.
  • mice studies during the acute phase (2 weeks), swelling of the paw was reduced in EsA/h-EsA treated mice as compared to the untreated CIA control.
  • the mean volume in normal mice was about 10.5 mm3 while CIA increased it to 20 mm3, whereas EsA and h-EsA reduced it to 16 mm3 (Example 1, Figure 3).
  • EsA/h-EsA had a similar effects compared to Celebrex (Fig 3).
  • h-EsA is equally effective as Celebrex at early stage of CIA and can maintain its effectiveness for a longer period of time; 2) the triterpenoid portion of EsA is the functional element of EsA; and 3) h-EsA can be a better choice than EsA (depending on the route of administration and other factors) due to elimination of the surfactant sugar group. It has been shown that h-EsA can effectively reduce the effects of CIA during peak inflammation (second boost) as a mitigation/treatment agent. It is also believed that it can also act to reduce or prevent its occurrence of CIA from time of onset (first immunization period). 103.
  • cytokine can be selected from the group consisting of angiogenic, growth, fibrogenic, and inflammatory cytokines. Examples of such cytokines include, but are not limited to, ILl, IL6, TNF ⁇ , TGF ⁇ , VEGF, and MCPl or any combination thereof.
  • the aglycodisic EsA can be administered in a variety of ways, as disclosed below.
  • Inhibiting a cytokine refers to blocking or reducing at least one cytokine mediated event.
  • EsA inhibited the production of prostaglandin E 2 (PGE 2 ), platelet-activating factor (PAF), and nitric oxide (NO).
  • PGE 2 is known to play a major role in acute inflammation.
  • Nitric Oxide has a key role in perpetual inflammation (Fang et al. Yao Xue Xue Bao. 26(10):721-4 (1991)).
  • Inflammation can be associated with a number of different diseases and disorders. Examples of inflammation include, but are not limited to, inflammation associated with hepatitis, inflammation associated with the lungs, and inflammation associated with an infectious process. Inflammation can also be associated with liver toxicity, which can be associated in turn with cancer therapy, such as apoptosis induction or chemotherapy, or a combination of the two, for example.
  • cancer therapy such as apoptosis induction or chemotherapy, or a combination of the two, for example.
  • the infectious process can be associated with a viral infection.
  • viral infections include, but are not limited to, Herpes simplex virus type-1, Herpes simplex virus type-2, Cytomegalovirus, Epstein-Barr virus, Varicella-zoster virus, Human herpesvirus 6, Human herpesvirus 7,
  • Human herpesvirus 8 Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus, Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St.
  • the infectious process can also be associated with a bacterial infection.
  • bacterial infections include, but are not limited to, M. tuberculosis, M. bovis, M. bovis strain BCG, BCG substrains, M. avium, M. intracellular, M. africanum, M. kansasii, M. marinum, M. ulcerans, M. avium subspecies paratuberculosis, Nocardia asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Salmonella typhi, other Salmonella species.
  • Staphylococcus aureus Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, Bacillus anthracis, Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Clostridium tetani, other Clostridium species, Yersinia enterolitica, and other Yersinia species.
  • the infectious process can also be associated with a parasitic infection.
  • parasitic infections include, but are not limited to, Toxoplasma gondii, Plasmodium species such as Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and other Plasmodium species, Trypanosoma brucei, Trypanosoma cruzi,
  • Leishmania species such as Leishmania major
  • Schistosoma such as Schistosoma mansoni and other Shistosoma species
  • Entamoeba histolytica a histolytica
  • the infectious process can also be associated with a fungal infection.
  • fungal infections include, but are not limited to, Candida albicans, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneomocystis carnii, Penicillium marneffi, and Alternaria alternata.
  • transplant rejection is associated with transplant rejection in a transplant recipient.
  • antibodies are formed against foreign antigens on the transplanted material.
  • the transplantation can be, for example, organ transplantation, such as liver, kidney, skin, eyes, heart, or any other transplantable organ of the body or part thereof.
  • Transplantation immunology refers to an extensive sequence of events that occurs after an allograft or a xenograft is removed from a donor and then transplanted into a recipient. Tissue is damaged at both the graft and the transplantation sites. An inflammatory reaction follows immediately, as does activation of biochemical cascades. A series of specific and nonspecific cellular responses ensues as antigens are recognized. Antigen-independent causes of tissue damage (i.e., ischemia, hypothermia, reperfusion injury) are the result of mechanical trauma as well as disruption of the blood supply as the graft is harvested. In contrast, antigen-dependent causes of tissue damage involve immune-mediated damage.
  • tissue damage i.e., ischemia, hypothermia, reperfusion injury
  • graft antigens are recognized by T cells; the resulting cytokine release eventually leads to tissue distortion, vascular insufficiency, and cell destruction. Histologically, leukocytes are present, dominated by equivalent numbers of macrophages and T cells within the interstitium. These processes can occur within 24 hours of transplantation and occur over a period of days to weeks.
  • pathologic tissue remodeling results from peritransplant and posttransplant trauma. Cytokines and tissue growth factor induce smooth muscle cells to proliferate, to migrate, and to produce new matrix material. Interstitial fibroblasts are also induced to produce collagen.
  • Transplant rejection may occur within 1-10 minutes of transplantation, or within 10 minutes to 1 hour of transplantation, or within 1 hour to 10 hours of transplantation, or within 10 hours to 24 hours of transplantation, within 24 hours to 48 hours of transplantation, within 48 hours to 1 month of transplantation, within 1 month to 1 year of transplantation, within 1 year to 5 years of transplantation, or even longer after transplantation.
  • IR IR- induced local damage of normal tissue
  • radiation therapy does cause IR- induced local damage of normal tissue (radiation toxicity), leading to a temporary or persistent impairment of irradiated tissues, which lowers the life quality of cancer patients.
  • Some severe side effects can even result in the discontinuation of the life-saving radiation therapy (Johansen et al. Radiother Oncol. 40: 101-9 (1996), Niemierko et al. Int J Radiat Oncol Biol Phys. 25: 135-45, 1993., Wiess et al.
  • the radiation damage can be caused by radiation therapy, such as that used to treat cancer.
  • the radiation damage can also be caused by nuclear radiation, or by a weapon, such as a terrorist agent.
  • the compositions herein can be admininstered prior to, after, or during exposure to radiation.
  • Radiation toxicity can be divided into two main stages: early toxicity and late toxicity (MacKay et al. Radiother Oncol. 46:215-6 (1998), Rubin et al. Radiother Oncol. 35: 9-10, (199?), Dubray et al. Cancer Radiother.
  • ER kills cell through the production of free radicals.
  • the IR toxicity is a result of counteraction of host defense system that responds to IR physical insult.
  • the cells are damaged by free radicals, and undergo either repair or apoptosis/death, which initiates the cascade of signal transduction pathways (such as Nuclear factor- ⁇ B (NFK ⁇ ), etc.).
  • signal transduction pathways such as Nuclear factor- ⁇ B (NFK ⁇ ), etc.
  • IR up-regulates the expression of inflammatory mediators (such as cytokines, lymphokines and chemokines) and immunomodulatory molecules (MHC, co- stimulatory molecules, adhesion molecules, death receptors, heat shock proteins) in irradiated tumor, stromal, and vascular endothelial cells (Friedman et al.).
  • inflammatory mediators such as cytokines, lymphokines and chemokines
  • MHC immunomodulatory molecules
  • MHC co- stimulatory molecules
  • adhesion molecules adhesion molecules
  • death receptors heat shock proteins
  • heat shock proteins heat shock proteins
  • ILl ⁇ is a key cytokine in the IR inflammation process. As one of the effects, ILl ⁇ enhances the expression of COX-2, and together they markedly induce inflammatory angiogenesis (Kuwano et al. FASEB J.
  • IL-l ⁇ -induced activation of the COX-2 gene is modulated by NF-k ⁇ (Kirtikara et al. (2000), Crofford et al. Arthritis Rheum. 40,226-236 (1997)).
  • the COX-2 selective inhibitors can block ILl ⁇ induced angiogenesis but only partially block VEGF-induced angiogenesis.
  • the ILl ⁇ induced angiogenesis is much less in the COX-2 knockout mice than wild-type mice (Kuwano et al. (2004)).
  • Cyclooxygenase is the rate-limiting step in the conversion of arachidonic acid to prostaglandins.
  • COX-I is constitutively expressed at low levels in many cell types. Specifically, COX-I is known to be essential for maintaining the integrity of the gastrointestinal epithelium. COX-2 expression is stimulated by growth factors, cytokines, and endotoxins.
  • the cyclooxygenase 2 isoform (COX-2) is not expressed in most tissues (e.g., liver) under physiological conditions but is highly upregulated in inflammatory processes and cancer, for example. Up-regulation of COX- 2 is responsible for the increased formation of prostaglandins associated with inflammation.
  • compositions disclosed herein can also be used to treat cancer.
  • a neoplasm, or tumor is an abnormal, unregulated, and disorganized proliferation of cell growth.
  • a neoplasm is malignant, or cancerous, if it has properties of destructive growth, invasiveness and metastasis.
  • Invasiveness refers to the local spread of a neoplasm by infiltration or destruction of surrounding tissue, typically breaking through the basal laminas that define the boundaries of the tissues, thereby often entering the body's circulatory system.
  • Metastasis typically refers to the dissemination of tumor cells by lymphotics or blood vessels. Metastasis also refers to the migration of tumor cells by direct extension through serous cavities, or subarachnoid or other spaces.
  • Prostaglandins are arachidonate metabolites produced in virtually all mammalian tissues and possess diverse biologic capabilities, including vasoconstriction, vasodilation, stimulation or inhibition of platelet aggregation, and immunomodulation, primarily immunosupression (Moskowitz and Coughlins, Stroke 1981; 12: 882-86; Leung and Mihich. Nature 1980; 597-600; Brunda et al., J. Immunol. 1980; 124: 2682-7).
  • Cyclooxygenase-2 (COX-2) is induced by diverse inflammatory stimuli (Isakson et al., Adv. Pros. Throm. Leuk Res. 1995, 23, 49-54).
  • Prostaglandin-mediated effects at both the microenvironmental and cellular levels have been implicated in the modulation of such response. Inhibition of prostaglandin synthesis also induces an accumulation of cells in the G phase of the cell cycle, which are generally considered to be the most sensitive to ionizing radiation. With the inhibition of prostaglandin synthesis, prostaglandin-induced immunosuppressive activity was diminished and antitumor immunologic responses were able to potentiate tumor response to radiation. Finally, prostaglandins are vasoactive agents and are thus can regulate tumor blood flow and perfusion.
  • COX-2 inhibitors have been described for the treatment of cancer (WO98/16227) and for the treatment of tumors (EP 927,555).
  • Celecoxib® a specific inhibitor of COX-2, exerted a potent inhibition of fibroblast growth factor-induced corneal angiogenesis in rats.
  • COX-2 specific inhibitors prevent angiogenesis in experimental animals, but their efficacy in enhancing in vivo tumor response to radiation has not been established.
  • COX-2 is overexpressed in adenocarcinoma (Tsuji et al. (1998), Sano et al. Cancer Res.
  • COX-2 inhibitors can act as chemopreventive agents.
  • ILl ⁇ -stimulated COX-2 expression can be found in almost all types of cells, including monocytes/macrophages (Caivano et al. J. Immunol. 164: 3018-3025 (2000), vascular endothelial cells (Kirtikara et al. (2000)), stromal cells (Bamba et al. Int. J. Cancer 83: 470-475 (1999)), epithelial cells and nonepithelial cells, showing that this interaction is critical for all types of tissue damage/ inflammation processes. The blocking of these paired molecules has therefore not been restricted to a specific tissue.
  • neoplasia disorders selected from the group consisting of acral lentiginous melanoma, actinic keratoses, adenocarcinoma, adenoid cycstic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, astrocytic tumors, bartholin gland carcinoma, basal cell carcinoma, bronchial gland carcinomas, capillary, carcinoids, carcinoma, carcinosarcoma, cavernous, cholangiocarcinoma, chondrosarcoma, choriod plexus papilloma/carcinoma, clear cell carcinoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal, epitheloid, Ewing
  • Intracellular edema is defined by cellular swelling, usually of astrocytes, and classically is seen following cerebral ischema caused by cardiac arrest or head injury.
  • the blood brain barrier is intact.
  • Extracellular edema is a consequence of vascular injury with disruption of the blood brain barrier. Causes include trauma, tumor, and abscess. Ultimately, these changes can lead to herniation. Brain edema can also be radiation induced.
  • Disclosed are methods of inhibiting angiogenesis in a subject comprising administering to the subject an aglycosidic EsA.
  • An increase in the expression of COX-2 has been correlated with a poor clinical outcome in patients with colorectal and other cancers. It has been shown that the COX-2 expressed in the epithelial cell compartment regulates angiogenesis in the stromal tissues of the mammary gland and that it is critical during mammary cancer progression (Chang et al. PNAS, DOI: 10.1073/pnas.2535911100, December 15, 2003.).
  • the effect of inhibition of prostanoid synthesis on COX-2 transgenic mice was determined, using a strain that develops spontaneous mammary tumors.
  • indomethacin strongly decreased microvessel density and inhibited tumor progression. Indomethacin also inhibited upregulation of angiogenic regulatory genes in COX-2 transgenic mammary tissue.
  • prostaglandin E2 stimulated the expression of angiogenic regulatory genes in mammary tumor cells isolated from COX-2 transgenic mice and treated with celecoxib, a COX-2-specific inhibitor, and reduced tumor growth and microvessel density.
  • Aglycosidic EsAs have molecular similarity to steroid hormones. This molecule can block enzymes related to steroid metabolism and interconversion. An example of such an enzyme is the aromatase enzyme, which converts androgens to estrogens. Agents that block this enzyme are the preferred treatment for many women with post-menopausal breast cancer. As disclosed herein, aglycosidic EsA and its derivatives can have antiinflammatory effects, which can be related to steroid effects and include cytokine-modifying effects. Aglycosidic EsA and its derivatives can also have a therapeutic effect on the endometritis and breast adenoma (two types of estrogen related chronic diseases). C. COMPOSITIONS
  • Disclosed herein and useful in the methods described are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that, while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular molecule, such as EsA, is disclosed and discussed and a number of modifications that can be made to a number of places within the molecule can be made, specifically contemplated is each and every combination and permutation of the molecule unless specifically indicated to the contrary.
  • the term "substituted" is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds.
  • Illustrative substituents include, for example, those described below.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms, such as nitrogen can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms.
  • substitution or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g. , a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • alkyl as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, w-propyl, isopropyl, /t-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like.
  • the alkyl group can also be substituted or unsubstituted.
  • the alkyl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxamate, hydroxy, ketone, nitro, silyl, sulfo- oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below. 137.
  • alkyl is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group.
  • halogenated alkyl specifically refers to an alkyl group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine, or iodine.
  • alkylalcohol specifically refers to an alkyl group that is substituted with one or more hydroxyl groups, as described below.
  • alkylthiol specifically refers to an alkyl group that is substituted with one or more thiol groups, as described below.
  • alkylalkoxy specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below.
  • alkylamino specifically refers to an alkyl group that is substituted with one or more amino groups, as described below, and the like. 138. This practice is also used for other groups described herein.
  • cycloalkyl refers to both unsubstituted and substituted cycloalkyl moieties
  • the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an "alkylcycloalkyl.”
  • a substituted alkoxy can be specifically referred to as, e.g., a "halogenated alkoxy”
  • a particular substituted alkenyl can be, e.g., an "alkenylalcohol”
  • a particular substituted alkynyl can be, e.g., an "alkynylsilyl
  • a particular substituted aryl can be, e.g., a "nitroaryl”
  • a particular substituted cycloalkyl can be, e.g., a "cycloalkylether”
  • a particular substituted heterocycloalkyl can be, in
  • alkoxy as used herein is an alkyl group bound through a single, terminal ether linkage; that is, an “alkoxy” group may be defined as -OA where A is alkyl as defined above.
  • alkenyl as used herein is a hydrocarbon group of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond.
  • the alkenyl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxamate, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.
  • groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxamate, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfox
  • alkynyl as used herein is a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon triple bond.
  • the alkynyl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxamate, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.
  • aryl as used herein is a group that contains any carbon-based aromatic group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, phenoxybenzene, and the like.
  • aryl also includes "heteroaryl,” which is defined as a group that contains an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus.
  • non-heteroaryl which is also included in the term “aryl,” defines a group that contains an aromatic group that does not contain a heteroatom.
  • the aryl group can be substituted or unsubstituted.
  • the aryl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxamate, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.
  • the term "biaryl” is a specific type of aryl group and is included in the definition of aryl. Biaryl refers to two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.
  • cycloalkyl as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.
  • heterocycloalkyl is a cycloalkyl group as defined above where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus.
  • the cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted.
  • the cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxamate, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.
  • Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like.
  • heterocycloalkenyl is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkenyl,” where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus.
  • the cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted.
  • the cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxamate, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.
  • cyclic group is used herein to refer to either aryl groups, non-aryl groups (i.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups), or both. Cyclic groups have one or more ring systems that can be substituted or unsubstituted. A cyclic group can contain one or more aryl groups, one or more non-aryl groups, or one or more aryl groups and one or more non-aryl groups.
  • aldehyde as used herein is represented by the formula -C(O)H.
  • amine or “amino” as used herein are represented by the formula NAA 1 A 2 , where A, A 1 , and A 2 can be, independently, hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
  • esters as used herein is represented by the formula -OC(O)A or - C(O)OA, where A can be an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above. 150.
  • ether as used herein is represented by the formula AOA 1 , where A and A 1 can be, independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
  • ketone as used herein is represented by the formula AC(O)A 1 , where A and A 1 can be, independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
  • halide refers to the halogens fluorine, chlorine, bromine, and iodine.
  • hydroxamate as used herein is represented by the formula -
  • hydroxyl as used herein is represented by the formula -OH.
  • nitro as used herein is represented by the formula -NO 2 .
  • sil as used herein is represented by the formula -SiAA 1 A 2 , where A, A 1 , and A 2 can be, independently, hydrogen, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
  • sulfo-oxo as used herein is represented by the formulas -S(O)A, -S(O) 2 A, -OS(O) 2 A, or -OS(O) 2 OA, where A can be hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
  • sulfonyl is used herein to refer to the sulfo-oxo group represented by the formula -S(O) 2 A, where A can be hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
  • sulfonylamino or "sulfonamide” as used herein is represented by the formula -S(O) 2 NH-.
  • sulfone as used herein is represented by the formula AS(O) 2 A 1 , where A and A 1 can be, independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above. 161.
  • sulfoxide as used herein is represented by the formula AS(O)A 1 , where A and A 1 can be, independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above. 162.
  • thiol as used herein is represented by the formula -SH.
  • R 1 is a straight chain alkyl group
  • one of the hydrogen atoms of the alkyl group can optionally be substituted with a hydroxyl group, an alkoxy group, an alkyl group, a halide, and the like.
  • a first group can be incorporated within second group or, alternatively, the first group can be pendant ⁇ i.e., attached) to the second group.
  • an alkyl group comprising an amino group the amino group can be incorporated within the backbone of the alkyl group.
  • the amino group can be attached to the backbone of the alkyl group. The nature of the group(s) that is(are) selected will determine if the first group is embedded or attached to the second group.
  • pharmaceutically acceptable salts and esters of compounds represented by Formula I are also described herein.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual along with the selected compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • Pharmaceutically acceptable salts can be prepared, for example, by treating the compound with an appropriate amount of a pharmaceutically acceptable base.
  • Representative pharmaceutically acceptable bases include ammonium hydroxide, sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide, ferrous hydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide, ferric hydroxide, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2- diethylaminoethanol, lysine, arginine, histidine, and the like. See for example, S. M. Berge, et at, "Pharmaceutical Salts," J Pharm.
  • the reaction is conducted in water, alone or in combination with an inert, water-miscible organic solvent, at a temperature of from about 0°C to about 100°C, such as at room temperature.
  • the molar ratio of compounds represented by Formula I to be used is chosen to provide the ratio desired for any particular salts.
  • the compound can be treated with approximately one equivalent of a pharmaceutically acceptable base to yield a neutral salt.
  • esters include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, phenyl, pyridinyl, benzyl, and the like.
  • esters can be prepared by, for example, by treating the compound with an appropriate amount of carboxylic acid, ester, acid chloride, acid anhydride, or mixed anhydride agent that will provide the corresponding pharmaceutically acceptable ester.
  • Typical agents that can be used to prepare pharmaceutically acceptable esters include, for example, acetic acid, acetic anhydride, acetyl chloride, benzylhalide, benzaldehyde, benzoylchloride, methyl ethylanhydride, methyl phenylanhydride, methyl iodide, and the like.
  • Suitable compounds are aglycone saponins (e.g., sapogenins).
  • suitable aglycone saponins are the acid saponins (i.e., triterpenoid saponins). These compounds can be obtained by removing the glycosidic moieties at R 2 in commercial or naturally isolable saponins.
  • saponins disclosed in U.S. Patents 6,528,058, 6,645,495, 6,753,414, 6,524,584, 6,231,859, 5,688,772, 5,597,807, and 5,057,540 (which are incorporated by reference herein in their entirety) can be treated with acid or base to remove the glycoside moiety.
  • aglycoside saponins e.g., the triterpenoid saponins
  • plant sources e.g., Phytolacca esculenta, Panax notoginsing, Akebia trifoliate
  • extraction methods see e.g., U.S. Patents 4,879,376, 5,977,081, WO/2006/116656
  • suitable compounds can have the following formula.
  • R 1 and R 2 are, independent of one another, H, OH, alkyl (e.g., CH 3 , C 2 H 5 , C 3 H 7 , CH(CH 3 ) 2 , C 4 H 9 , CH(CH 3 )C 2 H 5 , CH 2 CH(CH 3 ) 2 , C(CH 3 ) 3 , C 5 Hi 0, alkoxide (e.g. , OCH 3 ,
  • acyl e.g., CH 3 C(O), CH 3 CH 2 (O), acetal, OLi, ONa, OK, or OCa;
  • Each R 3 is, independent of one another, H, OH, alkoxide (e.g., OCH 3 , OC 2 H 5 , OC 3 H 7 , OCH(CH 3 ) 2 , OC 4 H 9 , OCH(CH 3 )C 2 H 5 , OCH 2 CH(CH 3 ) 2 , OC(CH 3 ) 3 , OC 5 H 11 ), hydroxyl alkyl (e.g., -CH 2 OH, C 2 H 4 OH), C(O)H, CO 2 H, CO 2 Na, CO 2 K, CO 2 Li, CO 2 Ca, CO 2 R 8 , or -CH 2 OR 8 ;
  • alkoxide e.g., OCH 3 , OC 2 H 5 , OC 3 H 7 , OCH(CH 3 ) 2 , OC 4 H 9 , OCH(CH 3 )C 2 H 5 , OCH 2 CH(CH 3 ) 2 , OC(CH 3 ) 3 ,
  • Each R 4 is, independent of one another, H, alkyl (e.g., CH 3 , C 2 H 5 , C 3 H 7 , CH(CH 3 ) 2 , C 4 H 9 , CH(CH 3 )C 2 H 5 , CH 2 CH(CH 3 ) 2 , C(CH 3 ) 3 , C 5 H 11 ), or aryl (e.g., C 6 H 5 , C 6 H 4 OCH 3 , C 6 H 4 OH, C 6 H 4 ONH 2 , CH 2 C 6 H 5 );
  • R 5 and R 6 are, independently of one another, H, CH 3 , OH, CO 2 H, or CO 2 R 8 ;
  • Each R 7 is, independent of one another, H or together are an oxo group;
  • R 8 is an alkyl (e.g., CH 3 , C 2 H 5 , C 3 H 7 , CH(CH 3 ) 2 , C 4 H 9 , CH(CH 3 )C 2 H 5 , CH 2 CH(CH 3 ) 2 ,
  • R 2 does not comprise a saccharide.
  • glycyrrhetinic acid glycyrrhizinic acid, ⁇ -amyrin, astragaloside IV, astragaloside II, astragaloside I and acetylastragaloside I, saikosaponin-d, maesabalide III, arvensoside C, bidentatoside II, chikusetsusaponin V methyl ester, capilliposide G, capilliposide H, aglycosidyl esculentoside A, B, C, or D.
  • a suitable compound can have the following formula.
  • compositions comprising a compound represented by Formula I.
  • compositions prepared by or with compounds represented by Formula I can be used as monomers in peptide synthesis.
  • peptides comprising at least one compound represented by Formula I. 169.
  • Compounds represented by Formula I can be optically active or racemic.
  • the stereochemistry at the tertiary carbon shown in Formula I can vary and will depend upon the spatial relationship between the substituents on that carbon. In one aspect, the stereochemistry at the tertiary carbon shown in Formula I is S. In another aspect, the stereochemistry at the tertiary carbon shown in Formula I is R. 170. Using techniques known in the art, it is possible to vary the stereochemistry at the tertiary carbon shown in Formula I.
  • the compound represented by Formula I is the substantially pure S enantiomer.
  • the compound represented by Formula I is the substantially pure R enantiomer.
  • other carbon stereocenters can exist in compounds represented by Formula I.
  • the S and R isomers of such additional stereocenters are contemplated herein.
  • Formula I includes enantiomers, diastereomers, and meso forms of the compounds represented thereby. 171.
  • Compounds represented by Formula I can be readily synthesized using techniques generally known to those of skill in the art.
  • the starting materials and reagents used in preparing these compounds are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, NJ.), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St.
  • compositions of the invention can be administered in vivo in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • compositions comprising aglycosidic EsAs or a derivative thereof and a pharmaceutical carrier. Pharmaceutical carriers are known to those skilled in the art.
  • compositions can be administered in a number of ways, as described below. Other compounds will be administered according to standard procedures used by those skilled in the art. 175.
  • Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. 176.
  • the pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.
  • Administration may be topically (including opthamalically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.
  • the disclosed compositions can be administered intravenously, intraperitoneally, intramuscularly, intraarticularly, intrathecally, subcutaneously, intracavity, or transdermally.
  • the pharmaceutical compositions can also be admininstered in the form of an intraoperative wash.
  • compositions disclosed herein can also be administered through topical intranasal administration or administration by inhalant.
  • topical intranasal administration means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization. The latter may be effective when a large number of animals are to be treated simultaneously.
  • Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation.
  • compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition.
  • Parenteral administration of the composition is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.
  • Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
  • Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, propionic acid, glyco
  • the dosage ranges for the administration of the compositions are those large enough to produce the desired effect of the methods disclosed herein.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. While individual needs vary, determination of optimal ranges of effective amounts of the vector is within the skill of the art.
  • Typical dosages comprise about 0.01 to about 100 mg/kg-body wt.
  • the preferred dosages comprise about 0.1 to about 100 mg/kg-body wt.
  • the most preferred dosages comprise about 1 to about 100 mg/kg-body wt.
  • h- EsA is administered in the amount of 2-40 mg/kg.
  • h-EsA is administered in the amount of 5-30 mg/kg.
  • h-EsA is administered in the amount of 5-20 mg/kg.
  • An appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
  • Dosages can be given every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, or 72 hours, or any amount in between. It can also be given weekly, biweekly, monthly, or yearly, depending on the condition being treated and the individual needs of the subject receiving treatment. Dosages can also be administered in the form of a bolus. Dosages can also be administered preventatively in an effective amount that can be determined by one of ordinary skill in the art.
  • the materials may be in solution or suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, Br. J. Cancer, 58:700-703, (1988); Senter, Bioconjugate Chem., 4:3-9, (1993); Battelli, Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog.
  • Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of cells in vivo.
  • stealth and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of cells in vivo.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes, Cancer Research, 49:6214- 6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)).
  • receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin- coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes.
  • the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor- level regulation.
  • receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).
  • Liposomes are vesicles comprised of one or more concentrically ordered lipid bilayers which encapsulate an aqueous phase. They are normally not leaky, but can become leaky if a hole or pore occurs in the membrane, if the membrane is dissolved or degrades, or if the membrane temperature is increased to the phase transition temperature.
  • Current methods of drug delivery via liposomes require that the liposome carrier ultimately become permeable and release the encapsulated drug at the target site. This can be accomplished, for example, in a passive manner wherein the liposome bilayer degrades over time through the action of various agents in the body. Every liposome composition will have a characteristic half-life in the circulation or at other sites in the body and, thus, by controlling the half-life of the liposome composition, the rate at which the bilayer degrades can be somewhat regulated.
  • liposome membranes can be constructed so that they become destabilized when the environment becomes acidic near the liposome membrane (see, e.g., Proc. Natl. Acad. Sci. USA 84:7851 (1987); Biochemistry 28:908 (1989), which is hereby incorporated by reference in its entirety).
  • liposomes When liposomes are endocytosed by a target cell, for example, they can be routed to acidic endosomes which will destabilize the liposome and result in drug release.
  • the liposome membrane can be chemically modified such that an enzyme is placed as a coating on the membrane which slowly destabilizes the liposome. Since control of drug release depends on the concentration of enzyme initially placed in the membrane, there is no real effective way to modulate or alter drug release to achieve "on demand” drug delivery. The same problem exists for pH-sensitive liposomes in that as soon as the liposome vesicle comes into contact with a target cell, it will be engulfed and a drop in pH will lead to drug release.
  • This liposome delivery system can also be made to target B cells by incorporating into the liposome structure a ligand having an affinity for B cell- specific receptors.
  • compositions including the liposomes in a pharmaceutically acceptable carrier are also contemplated.
  • Transdermal delivery devices have been employed for delivery of low molecular weight compositions by using lipid-based compositions (i.e., in the form of a patch) in combination with sonophoresis.
  • transdermal delivery can be further enhanced by the application of an electric field, for example, by ionophoresis or electroporation.
  • an electric field for example, by ionophoresis or electroporation.
  • Using low frequency ultrasound which induces cavitation of the lipid layers of the stratum corneum higher transdermal fluxes, rapid control of transdermal fluxes, and drug delivery at lower ultrasound intensities can be achieved.
  • Still further enhancement can be obtained using a combination of chemical enhancers and/or magnetic field along with the electric field and ultrasound.
  • Implantable or injectable protein depot compositions can also be employed, providing long-term delivery of, e.g., an aglycosidic EsA or derivatives thereof.
  • an injectable depot gel composition which includes a biocompatible polymer, a solvent that dissolves the polymer and forms a viscous gel, and an emulsifying agent in the form of a dispersed droplet phase in the viscous gel.
  • a gel composition can provide a relatively continuous rate of dispersion of the agent to be delivered, thereby avoiding an initial burst of the agent to be delivered. 193.
  • kits that can be used in practicing the methods disclosed herein.
  • a kit can comprise an aglycosidic EsA or a derivative thereof.
  • the kit can further comprise instructions.
  • the kits can include any reagent or combination of reagent discussed herein or that would be understood to be required or beneficial in the practice of the disclosed methods.
  • Example 1 h-EsA, a Novel Anti-inflammation Agent
  • COX-2 inhibitors have dominated the market for nonsteroidal anti-inflammatory agents (NSAIDs).
  • NSAIDs nonsteroidal anti-inflammatory agents
  • Vioxx® a major COX-2 inhibitor, was removed from the marketplace due to cardiac toxicity.
  • Celebrex® the other major COX-2 inhibitor, now has almost no competition and therefore can remain inhibitively expensive.
  • Celebrex® has a lower but still measurable rate of thromboembolic complications. There is therefore a powerful need for a new NSAID.
  • h-EsA an aglycoside analog of Esculentoside A (EsA, a saponin isolated from Phytolacca esculenta)
  • EsA an aglycoside analog of Esculentoside A
  • EsA dramatically reduced the IR-induced soft tissue fibrosis evidenced by the much less shortening of IR leg as compared to the vehicle control and Celebrex® group (Fig 3B); 4)
  • the side effect of EsA and Celebrex® was different. For example, it was observed that 45 days after treated C57B1/6 mice (that had a radiation-induced demonitis) with either EsA (10 mg/kg qd) or Celebrex® (30 mg/kg, qd) for one month, both groups had developed alopecia (Fig 4A), strongly suggesting that in vivo, EsA has a similar biological activity as Celebrex. However, the area of hair loss in front head was bigger in Celebrex® group than that in the EsA group (Fig 4A).
  • the first is a measurement of joints swollen at acute phase.
  • Novel plethysmometer for mouse Water replacement has been the working principle for measuring the volume of swollen paws for decades.
  • the commercially available plethysmometer is only useful for rat given the 10 ⁇ l precision of the devices. Since the mouse is 1/10 to 1/15 the weight of a rat, a plethysmometer with a precision as low as 1 ⁇ l in volume was needed.
  • the front chamber of the device has a limited open space that allows for only the entry and emersion of a mouse paw;
  • the front chamber connects to a very fine tube which is calibrated so that the volume of fluid displaced from the chamber is read by the distance that the displaced fluid is forced into the tube (1 mm change in distance in the tube is equivalent to 1 ⁇ L), and
  • the volume of displaced fluid can easily be measured through the clear tubing using a ruler with millimeter gradations. Using this device, the extents of paw swelling among the different treatments was observed. 200.
  • the measurement of grab force (dropping time) at the intermediate stage was the second device used.
  • the grab force of the mouse hind paws is assessed by the time an animal is able to hold to a metal net standing at an 85 degree angle.
  • the average time until the mouse drops (dropping time) is 10-12 minutes.
  • the paw grab force for a mouse experiencing the symptoms of CIA is dramatically reduced.
  • the result is presented as a shortening of dropping time.
  • This new apparatus allows us to objectively to determine the function of the joints in the two back paws (note: the two front paws were taped to prevent their use in holding to the metal net).
  • the third device measures a deformed ankle at late stage.
  • the progression of CIA disease in the mouse is characterized by an increasing stiffness of the ankle joint that limits the extension of the joint to values below the normal extension range of 135 degrees.
  • the extension of ankle joint is gauged against a circular rule that measures the extension angle of the joint.
  • mice were immunized with 0.1 ml/mice of an emulsion of 100 ⁇ g collagen II (in 50 ul) in Freund's complete adjuvant (in 50 ul) through tail s.c. injection. Five weeks (35 days) later, to accelerate the CIA process, the mice were boosted with 20 ⁇ g collagen II (in 50 ul) with Freund's incomplete adjuvant.
  • mice were randomly divided into eight groups (8 mice per group) with five groups treated with the same dose of EsA (10 mg/kg) through five different administration routes, namely, p.o., i.p., i.v. (using a liposomal form of EsA), s.c. and i.m., one group treated p.o. with vehicle alone and one group an untreated control.
  • the positive control group was treated with Celebrex® at an oral dose of 30 mg/kg.
  • the results of the plethysmometer measurements of paw swelling at 6 weeks (post-boost) showed that the p.o. route was the best at reducing swelling compared with the other four routes and the Celebrex® control group.
  • Acute toxicities of EsA and its derivative h-EsA Once the administration path (p.o.) was determined, an acute toxicity test was conducted to see the dose limitation. Mice (6 groups) were fed with different doses of EsA once and the number of lethal events was recorded daily for 2 weeks. The LD 50 was about 55 mg/kg, which seems too low for an anti-CI agent (unless effective dose can be lowered to 1 mg/kg). It was speculated that this unexpectedly low LD 50 was due to the sugar function on EsA that confers a saponin (surfactant) property to the compound. This surfactant- like property can cause disruption of cell membranes as was evidenced by the occurrence of hemolysis in the toxicity study.
  • saponin saponin
  • h-EsA When h-EsA was administered p.o. to mice, there was no lethality observed up to a dose of 190 mg/kg. In fact, some mice were able to tolerate a doses up to 220 mg/kg. Based on the toxicity data, we assigned an LD 50 of 195 mg/kg for h-Es A. With this LD 50 , a therapeutic index for h-EsA of at least 20 is now achievable with a higher index expected since the therapeutic dose is expected to be ⁇ 5 mg/kg using an optimized dose schedule. Given the large therapeutic index for h-EsA in comparison to EsA, h-EsA was used in treatment studies whereas EsA was be used as a positive control.
  • mice Male, 7 weeks old immunized for 4 weeks were randomly divided into groups (10/group): 1) vehicle alone; 2) Celebrex® (14 mg/kg, 1.8 mM), as a positive control; 3) EsA (15 mg/kg, 0.9 mM) as parental agent control; and 4) h-EsA (15 mg/kg, 1.4 mM; or 7.5 mg/kg, 0.7 mM).
  • the negative controls were the normal mice with same age and sex without immunization.
  • h-EsA As a CIA mitigation agent, the treatment was started three days after the second collagen II boost was given to trigger CIA symptoms. The symptoms normally appeared 7 days after boost. The schedule for treatment was q.o.d. (every other day) for 3 weeks. One week after boost, paw swelling was measured weekly. The dropping time (grab force) was measured at week 4. Three months later, the deformed ankle angle was measured in relapsing CIA mice. The results were very promising. In the acute phase (2 weeks), swelling of the paw was reduced in EsA/h-EsA treated mice as compared to the untreated CIA control.
  • mice The mean volume in normal mice was about 10.5 mm 3 while CIA increased it to 20 mm 3 , whereas EsA and h-EsA reduced it to 16 mm 3 (Fig 3). EsA/h-EsA had a similar effects compared to Celebrex® (Fig 3). Although not statistically significant, CIA score was only slightly reduced in the EsA treated group compared to the Celebrex® group whereas there was a 17% reduction in
  • CIA score for the h-EsA group when measured at week 3 after treatment (Fig 4). It was noted that the CIA paw scoring was not reflective of the differences between the groups due to the subjective nature of the observers' judgment.
  • Fig 9 The results (Fig 9) showed that the joints of EsA, h-EsA, and Celebrex® treated mice had less severity in bone/cartilage erosion/destruction, synovium hyperplasia, and infiltration of mono- and polymorphonuclear cells as compared to CIA mice treated with vehicle alone. In addition, collagen deposition was significantly reduced in mice treated with EsA as measured by anti-collagen II immunoassay (Fig 10).
  • h-EsA is equally effective as Celebrex® at early stage of CIA and can maintain its effectiveness for a longer period of time; 2) the triterpenoid portion of EsA is the functional element of EsA; and 3) h-EsA may be safer than EsA due to elimination of the surfactant sugar group. It has been shown that h-EsA can effectively reduce the effects of CIA during peak inflammation (second boost) as a mitigation/treatment agent. It is also believed that it can also act to reduce or prevent its occurrence of CIA from time of onset (first immunization period). 209.
  • TNF ⁇ (Fig 12) and other cytokines was greatly reduced by EsA in a dose- dependent manner; and 2) at the tissue level samples collected from mouse study), IR- induced increases in IL-6, TNF ⁇ , and other EvIs, such as MCP-I and IL-6 in skin were reduced by EsA to a greater extent than Celebrex® (Fig 13).
  • h-EsA (10 mg/kg po) reduced the levels of vascular endothelial growth factor (VEGF), part of the platlet- derived growth factor (PDGF) pathway, to control levels 1 week post irradiation (12.5
  • IL-I can better account for EsA-related IR protection than COX-2 inhibition.
  • IL- l ⁇ induced by 2 or 4 Gy IR in A431 human epidermoid carcinoma cells was also completely inhibited by 0.1 ug/ml EsA (P ⁇ .01, Fig 16B and 16C).
  • other proinflammatory cytokines were also studied in a panel of normal cell lines including macrophages, epithelial cells, and fibroblasts from human and mouse origins.
  • EsA potently inhibited the production and release of IL-I ⁇ , IL- l ⁇ , IL-6, TNF ⁇ , TGF ⁇ , and (cytokines involved in the IL-I network).
  • IL-I IL-I ⁇ , IL- l ⁇ , IL-6, TNF ⁇ , TGF ⁇ , and (cytokines involved in the IL-I network).
  • cytokines involved in the IL-I network The effect on IL-I suppression is not cell specific and appears to be universal in human and murine cell lines.
  • IL-I and COX-2 were elevated following IR and are a component of early IR dermatitis, pneumonitis, and brain edema; 2) EsA alleviated radiation toxicity of the lung, skin, and brain in mice; 3) EsA inhibits COX-2 activity specifically; and 4) EsA reduces IR-related IL-I, IL-6, TNF ⁇ ,
  • mice were irradiated with a 18 Gy dose to the lungs and treated p.o.
  • mice were anesthetized and the trachea was surgically exposed and incised. A 16-gauge, 1 cm stainless steel tube was inserted into the trachea and then secured with surgical silk. The animal was then connected to a Harvard rodent ventilator. For the studies a respiratory rate of 150 breaths per minute was used, and a tidal volume calculated based on the weight of the mouse.
  • the formula used for this calculation is 0.01 ml per gram body weight.
  • the animal was then placed in a plethysmograph, and pressure- volume measurements were taken.
  • the results of lung compliance testing in the treated mice are shown in Fig 19.
  • the data show that early inhibition of pneumonitis by EsA can prevent progression to late stage fibrosis in irradiated lung and should have some utility in treating both IR pneumonitis and pulmonary fibrosis.
  • the data also suggest that h-EsA out-performs Celebrex® in controlling radiation effects on lung tissues.
  • the current approach to quantifying h-EsA in plasma and tissues is a single extraction method using liquid- liquid extraction with analysis of the extracts by HPLC analysis.
  • Recoveries of h-EsA for human plasma spiked at 1, 5 or 10 ⁇ g/mL h-EsA and GA (5 ⁇ g/mL) using solvent extraction techniques with n-butanol (BuOH) as the solvent followed by solid phase extraction was found to range from 82.1-101% for h-EsA and 75-80% for GA.
  • the recoveries of h-EsA from spiked human plasma were found to decrease with h-EsA concentration.
  • Figure 20 shows a typical chromatogram obtained for a plasma extract sample at both 205 nm (upper panel) and 252 nm (lower panel) wavelengths.
  • the plasma is spiked with h-EsA and GA at a concentration of 5 ⁇ g/mL.
  • h-EsA is shown to elute at 18.8 min, whereas GA elutes at 30.3 min, as shown in the chromatogram obtained at 252 nm.
  • These chromatograms demonstrate the ability to resolve h-EsA and GA from plasma components. Similar resolution is obtained for liver extracts using a modified HPLC gradient method as described above.
  • the limits of detection (LOD), as calculated from the calibration-design-dependent approach, for h-EsA in plasma and liver extracts are defined as 5 and 10 times of the background in the chromatograms, respectively.
  • LOD and LOQ for h-EsA in spiked plasma are 0.040 ⁇ g/mL and 0.08 ⁇ g/mL, respectively, using solvent extraction and HPLC analytical methods to measure h-EsA.
  • the peak area for this concentration is
  • the plasma and liver samples were both solvent extracted using n-butanol and SPE purified and then analyzed by HPLC, as described in section Cl .4.3.
  • the mass of EsA or h-EsA in the re-suspended plasma and liver extracts was calculated using GA as the internal standard loaded at a mass of 6.25 ⁇ g in plasma samples and 9.8 to 14.3 ⁇ g in liver homogenates, depending on the mass of the sample.
  • the mass of EsA or h-EsA is then normalized to the volume (for plasma) or tissue mass (for liver) extracted and expressed as ⁇ g/mL plasma or ng/mg liver tissue.
  • h-EsA the simultaneous measurement of h-EsA in plasma obtained in the EsA studies shows h-EsA to be at low levels in plasma, with a C max below 1.8 ⁇ g/mL between 1 to 6 hours. This result indicates that at most approximately 8-10% of EsA undergoes hydrolysis of the sugar portion of the compound in the gut and liver. Therefore, the therapeutic and toxicological effects observed for EsA are likely due to the parent compound and not the hydrolysis product, h-EsA.
  • Example 2 Effect of h-EsA on radiation-induced lung fibrosis:
  • CBCT Cone Beam Computed Tomography
  • the CBCT can obtain a complete 3D 650x650x428 scan (i.e. 428 slices) in ⁇ 10 seconds with isotropic resolution of 270 ⁇ m (central slice shown in Fig 22), encompassing a volume approximately 15 cm 3 .
  • the scanner achieves a CT density sensitivity of ⁇ 5 HU, which is capable of discerning density changes in the mouse lung due to radiation pneumonitis and fibrosis.
  • the imaging data was analyzed using custom MATLAB software.
  • the lungs were segmented automatically (Fig 23 A) and a 3D lung region was obtained for each mouse (Fig 23B).
  • Fig 23A a histogram of the voxel intensity (pulmonary tissue density) was created (Fig 24A).
  • the lower density unit (HU) is, the health the lung is.
  • Boquillon M Boquillon JP
  • Bralet J Photochemically induced, graded cerebral infarction in the mouse by laser irradiation evolution of brain edema. J Pharmacol Toxicol Methods. 1992;27(l):l-6.
  • Interleukin-lbeta induces cyclooxygenase-2 and prostaglandin E(2) synthesis in human neuroblastoma cells: involvement of p38 mitogen-activated protein kinase and nuclear factor-kappaB. J. Neurochem. 75,2020-2028
  • Manetti F, Maccari L, Corelli F, Botta M 3D QSAR models of interactions between beta- tubulin and microtubule stabilizing antimitotic agents (MSAA): a survey on taxanes and epothilones. Curr Top Med Chem. 2004;4(2):203-17.
  • TGF-bl is an important contributing factor in the development of radiation induced fibrosis, Int. J. Radiat. Oncol. Biol. Phys. 46:, 2000. Olson JJ, Beck DW, Warner DS, Coester H.: The role of new vessels and macrophages in the development and resolution of edema following a cortical freeze lesion in the mouse. J Neuropathol Exp Neurol. 1987;46 (6):682-94

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Abstract

Disclosed are compositions related to water soluble selective COX-2 inhibitors and methods of using the inhibitors (including h-EsA and derivatives thereof.)

Description

METHODS AND COMPOSITIONS RELATED TO AGLYCOSIDIC
ESCULENTOSIDE A I. CROSS REFERENCE TO RELATED APPLICATIONS 1. This application claims the benefit of priority of U.S. Provisional Application
No. 61/055,755, filed May 23, 2008, which application is incorporated herein by this reference in its entirety.
II. BACKGROUND OF THE INVENTION
A. FIELD OF THE INVENTION
2. This invention relates generally to methods and compositions related to new selective COX-2 inhibitors.
B. BACKGROUND ART 3. Cyclooxygenase is an enzyme that catalyzes the rate-limiting step in the conversion of arachidonic acid to prostaglandins. There are two known types of cyclooxygenase, COX-I and COX-2. COX-I is constitutively expressed at low levels in many cell types. Specifically, COX-I is known to be essential for maintaining the integrity of the gastrointestinal epithelium. COX-2 expression is stimulated by growth factors, cytokines, and endotoxins. The cyclooxygenase 2 isoform is not expressed in most tissues (e.g., liver) under physiological conditions but is highly upregulated under certain conditions. For example, COX-2 is upregulated in inflammatory processes and cancer, for example. Up-regulation of COX-2 is responsible for the increased formation of prostaglandins associated with inflammation. What is needed in the art are novel compositions and methods for inhibiting COX-2.
III. SUMMARY OF THE INVENTION
4. In accordance with the purpose(s) of this invention, as embodied and broadly described herein, this invention, in one aspect, relates to a pharmaceutical composition comprising a compound having Formula I:
Figure imgf000004_0001
wherein, R1 and R2 are, independent of one another, H, OH, alkyl (e.g., CH3, C2H5, C3H7, CH(CH3)2,
C4H9, CH(CH3)C2H5, CH2CH(CH3)2, C(CH3)3, C5H11), alkoxide (e.g., OCH3, OC2H5, OC3H7, OCH(CH3)2, OC4H9, OCH(CH3)C2H5, OCH2CH(CH3)2, OC(CH3)3,
OC5Hn), aryl (e.g., C6H5, C6H4OCH3, C6H4OH, C6H4ONH2, CH2C6H5), hydroxyl alkyl (e.g., -CH2OH, -C2H4OH), OSO2CH3, OSO2C6H5CH3, OC5-cyclic ether (e.g., THF), OC6-cyclic ether (e.g., THP), O-linear ether (e.g., MOM, MEM), O-silyl ether (e.g., TBDMS, TMS, TES), ester (e.g., CH3C(O)O (acetate), CH3CH2C(O)O (propionate)), acyl (e.g. , CH3C(O), CH3CH2(O), acetal, OLi, ONa, OK, or OCa;
Each R3 is, independent of one another, H, OH, alkoxide (e.g., OCH3, OC2H5, OC3H7, OCH(CH3)2, OC4H9, OCH(CH3)C2H5, OCH2CH(CH3)2, OC(CH3)3, OC5Hn), hydroxyl alkyl (e.g., -CH2OH, C2H4OH), C(O)H, CO2H, CO2Na, CO2K, CO2Li, CO2Ca, CO2R8, Or -CH2OR8; Each R4 is, independent of one another, H, alkyl (e.g. , CH3, C2H5, C3H7, CH(CH3)2, C4H9, CH(CH3)C2H5, CH2CH(CH3)2, C(CH3)3, C5H11), or aryl (e.g., C6H5, C6H4OCH3, C6H4OH, C6H4ONH2, CH2C6H5);
R5 and R6 are, independently of one another, H, CH3, OH, CO2H, or CO2R8; Each R7 is, independent of one another, H or together are an oxo group; R8 is an alkyl (e.g. , CH3, C2H5, C3H7, CH(CH3)2, C4H9, CH(CH3)C2H5, CH2CH(CH3)2,
C(CH3)3, C5H11), aryl (e.g., C6H5, C6H4OCH3, C6H4OH, C6H4ONH2, CH2C6H5), Li, Na, K, or Ca; and The line between atoms a and b, shown as ===:, is a single or double bond; and a pharmaceutically acceptable carrier.
4. In one example, R2 does not comprise a saccharide. In another embodiment, the compound comprises Formula II:
Figure imgf000005_0001
5. Also disclosed is a method of reducing inflammation in a subject comprising administering to the subject an effective amount of any one of the compositions disclosed above.
6. Further disclosed is a method of inhibiting a cytokine in a subject comprising administering to the subject an effective amount of any one of the compositions disclosed above. The cytokine can be selected from the group consisting of ILl, IL6, TNFα, TNFβ,
VEGF, and MCPl or any combination thereof.
7. Disclosed herein is a method of reducing radiation damage in a subject comprising administering to the subject an effective amount of any one of the compositions disclosed above. In one example, the radiation damage can be caused by radiation therapy. The radiation therapy can also be used to treat cancer.The radiation damage can be caused by nuclear radiation. The radiation can be caused by a weapon. 8. Further disclosed is a method of treating neoplasias in a subject, the method comprising administering to the subject an effective amount of any one of the compositions disclosed above. The neoplasia can be selected from lung cancer, breast cancer, gastrointestinal cancer, bladder cancer, head and neck cancer and cervical cancer. 9. Also disclosed is a method of treating a subject with arthritis, the method comprising administering to the subject an effective amount of any one of the compounds disclosed above, hi one example, the arthritis can be rheumatoid arthritis.
III. BRIEF DESCRIPTION OF THE DRAWINGS 10. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the principles of the invention.
11. Figure 1 shows the structure of EsA, h-EsA, and Celebrex®.
12. Figures 2 and 3A show Celebrex® effectively inhibited the early phase of IR demonists (up to day 19 with the onset on day 14), but gradually it loss its effect after day
23 (late phase of IR demonists). In contrast, EsA reduced the severity of IR demonists both in early and late phases.
13. Figure 3B shows that EsA dramatically reduced the IR-induced soft tissue fibrosis evidenced by the much less shortening of IR leg as compared to the vehicle control and Celebrex® group.
14. Figure 4A shows alopecia induced by COX-2 inhibitors.
15. Figure 4B shows that alopecia recovers faster in the EsA group compared to the Celebrex® group.
16. Figure 5 A shows the effect of EsA on collagen induced arthritis (CIA). Figure 5B shows the effect of EsA on swollen paws. Figure 5C shows the effect of EsA on the climbing (grab force) of a joint.
17. Figures 6A-C show that at the intermediate phase (6 weeks after onset of symptom), the grab force of paw was measured and the difference among the control and treatment groups can be well distinguished. While the normal mice could hold on the bar for 12 minutes, the CIA mice could only hold for a few seconds. This significant impairment in CIA paw function could be reversed by both EsA and h-ESA to a greater degree than was observed for Celebrex®.
18. Figure 7 shows h-EsA inhibits CIA relapsing.
19. Figure 8 shows h-EsA reduces joint deformation. 20. Figure 9 shows pathological analysis of CIA.
21. Figure 10 shows EsA inhibits anticollagen II production.
22. Figure 11 shows IR-induced increase of ILl α, TNFα, MCP-I and VEGF in macrophages were reduced by EsA in a dose-dependent manner.
23. Figure 12 shows IR-induced increase of IL-I α and TNFα in macrophages in vitro were reduced by EsA in a dose-dependent manner.
24. Figure 13 shows EsA reduced IL- lα and TNFα in IR skin.
25. Figure 14 shows that EsA reduces VEGF in irradiated lung.
26. Figure 15 shows that h-EsA does not significantly reduce IL-6 in lung 3 weeks post-radiation. 27. Figure 16 shows the effect of EsA on production of IL-lβ. After treatment without or with 0.1 or 1 ug/ml EsA for 18 hr, the Raw 264.7 macrophage cells or A431 epidermoid cells were irradiated at different IR doses. The protein level of IL-lβ was measured by ELISA. At doses of 2-4Gy, IL-lβ was greatly induced (A), but could be reduced by EsA at a concentration of 0.1 ug/ml (B and C). 28. Figure 17 shows EsA-reduced IR-pneumonitis and fibrosis.
29. Figure 18 shows the effect of Dexamethasone, Celebrex® and h-EsA after 8 weeks of treatment on the breath rate of ionized lung (18 Gy) in C57BL/6 mouse measured at 4 months.
30. Figure 19 shows the effect of h-EsA, GA(8 weeks) on the compliance of ionized (18 Gy) lung in C57BL/6 mice at 6.7 months.
31. Figure 20 shows h-EsA and GA in HPLC.
32. Figure 21 shows plasma and liver PK of h-EsA and EsA.
33. Figure 22 shows that the CBCT can obtain a complete 3D 650x650x428 scan (i.e. 428 slices) in ~10 seconds with isotropic resolution of 270 μm (central slice shown), encompassing a volume approximately 15 cm3. 34. Figure 23 shows the CBCT imaging data was analyzed using custom MATLAB software. The lungs were segmented automatically (Fig 23A) and a 3D lung region was obtained for each mouse (Fig 23B). In order to reduce the effect of cardiac motion on the value of mean lung density, the boundary of the lung was excluded from the analysis.
35. Figure 24 shows CBCT scans in mice. For each mouse, a histogram of the voxel intensity (pulmonary tissue density) was created (Fig 24A). The lower density unit (HU) is, the health the lung is. The data from two independent study (separated in 2 years) showed that: 1) normal range of CBCT was relatively consistent (mean value -454 to -473); 2) 7- 12 months post lung IR ( 15- 18 Gy), the HU increased (mean value -348 to
-364); and 3) after mitigation agent treatment, the fibrosis was partially blocked as the density of lung was reduced towards normal range (mean value -447 to —458). Both hEsA and Celebrex® that were used for 4-6 months could block the lung fibrosis formed in 7-12 months (Fig 24B). 36. Figure 25 shows the experimental procedure for the effect of h-EsA on radiation-induced pneumonitis and lung fibrosis. The specific results are shown in Figures 26-39.
37. Figure 26 shows the effects of plasma on IL-I alpha.
38. Figure 27 shows the effect of h-EsA on lung PF-4. 39. Figure 28 shows the effect of h-EsA on lung TNF-alpha.
40. Figure 29 shows the effect of h-EsA on lung VACAM-I.
41. Figure 30 shows the effect of h-EsA on p-selectin (day 17 after lung IR).
42. Figure 31 shows the effect of h-EsA on lung TNF-alpha.
43. Figure 32 shows the effect of h-EsA and h-GA on ionized lung. 44. Figure 33 shows the effect of h-EsA on lung p-selectin 48 hours post lung IR.
45. Figure 34 shows the effect of h-EsA on secretion of p-selectin in ionized whole lung.
46. Figure 35 shows the effect of h-EsA on the breath rate of ionized lung (8 weeks). 47. Figure 36 shows the effect of h-EsA on the breath rate of ionized whole lung. 48. Figure 37 shows the effect of h-EsA on the compliance of ionized lung at 7 months.
49. Figure 38 shows the effect of h-EsA on the compliance of ionized lung at 6.7 months. 50. Figure 39 shows the effect of h-EsA (8 weeks) on the compliance of ionized lung at 6.7 months.
51. Figure 40 shows the experimental procedure for showing the effect of h-EsA on radiation-induced dermatitis and soft tissue fibrosis. The results are seen in Figures 41-43. 52. Figure 41 shows the effect of h-EsA on the scoring of the right hind leg ionized with 30 Gy.
53. Figure 42 shows the effect of h-EsA on the skin score of ionized right leg measured at 2 months.
54. Figure 43 shows the effect of h-EsA on the skin score of right hind leg measured at 7 months.
55. Figure 44 shows the experimental procedure for showing the effect of h-EsA on collagen-induced arthritis (CIA). The results are shown in figures 45-58.
56. Figure 45 shows the effect of h-EsA on the secretion of IL-I alpha at 72 hours. 57. Figure 46 shows the effect of h-EsA on swollen hind paws at 5 months.
58. Figure 47 shows the effect of h-EsA on swollen hind paws in relapsing CIA model at 5 weeks.
59. Figure 48 shows the effect of h-EsA on swollen hind paws in a CIA model measured at 48 hours. 60. Figure 49 shows the effect of h-EsA on extension ability on hind ankle in relapsing CIA at 5 months.
61. Figure 50 shows the effect of h-EsA on relapsing CIA model at 5 months.
62. Figure 51 shows the effect of h-EsA on climbing of hind paws at 6 weeks.
63. Figure 52 shows the effect of h-EsA on the extension ability of hind ankle in relapsing CIA model.
64. Figure 53 shows the effect of h-EsA on swollen paws in relapsing CIA. 65. Figure 54 shows the effect of EsA and h-EsA on swollen paws in CIA at three weeks.
66. Figure 55 shows measuring the extent angle of an ankle.
67. Figure 56 shows that when CIA mouse were treated with vehicle alone, there was a lot of infiltrated inflammatory cells and erosion of cartilage. In contrast, when CIA mice were treated with h-ES A , there were much less infiltrated inflammatory cells and cartilage erosion.
68. Figure 57 shows CIA model in DBA/1 J mouse treated with Celebrex™. It was shown that most of the cartilage was normal, but there was a foci of erosions in bone and cartilage.
69. Figure 58 shows CIA model in DBA/1 J mouse treated with h-ESA. It was shown that most of the cartilage was normal, but there was a foci of erosions in bone and cartilage
70. Figure 59 shows h-EsA reduces inflammatory cells infiltrated in the irradiated lung in the pneumonitis phase. At day 2.5 and 17 after lung radiation, the lung lavage was collected from each mouse in all groups. The cellular portion was assessed for the numbers of infiltrated macrophage and neutrophils. The h-EsA significantly inhibited the number of infiltrated macrophage on day 2.5. Shown is the effect of agents on lavage macrophage at 2.5 days post- 15 Gy lung IR. 71. Figure 60 shows the effect of agents on lavage macrophage at 17 days post-
15 Gy lung IR. As described above in Figure 59, it can be seen that h-EsA significantly inhibited the number of infiltrated macrophage on days 2.5 and 17.
72. Figure 61 shows h-EsA reduces inflammatory cells infiltrated in the irradiated lung in the pneumonitis phase. The effects of agents on lavage neutrophils 17 days post- lung IR is shown, and it can be seen that on day 17, the infiltrated neutrophils were reduced (PO.05).
73. Figure 62 shows that h-EsA reduced radiation-induced lung pathological alterations in acute phase. The pathological analysis on day 2.5 and 17 post-IR demonstrated that h-EsA reduced the infiltrated inflammatory cells in lung. The effect of h-EsA on pathohistological changes at 2.5 days and 17 days after 15 Gy lung-IR
(C57BL/6 mouse model) is shown. 74. Figure 63 shows that h-EsA reduces radiation-induced lung fibrosis. To determine the effect of h-EsA on lung fibrosis, the lung of mice treated with either vehicle alone or h-EsA were assessed for the content of collagen with two assays. Collagen trichrome blue staining showed that the IR-induced collagen deposit was reduced by h-EsA (less collagen blue staining as compared to vehicle control).
75. Figure 64 also shows that h-EsA reduces radiation-induced lung fibrosis. Hydroxyproline, a key component of collagen, was measured with chemical reaction (HCl digestion of protein and then assaying the small collagen component). The results showed that hydroxyproline in the lung was reduced in the h-EsA group compared with IR + vehicle control (P<0.05). Taken together, it was shown that h-EsA inhibited radiation induced both pneumonitis and lung fibrosis.
76. Figure 65 shows the effect of h-EsA on radiation-induced dermonitis and soft tissue contraction. Right hind legs of mice (8 mice/group) were subjected to 30 Gy radiation and then the vehicle alone or h-EsA was given orally at 10 mg/kg every other day for one month .The skin score was measured at 2 months after treatment (skin score), and the results show that h-EsA treated mice had significantly less radiation-induced dermonitis.
77. Figure 66 shows the effect of h-EsA on leg contratcture 2 months after irradiated hind leg of C57BL/6 mice. It is clearly seen that the h-EsA group had significantly less contracture of the hind leg.
78. Figure 67 shows the results of an acute toxicity test. The acute toxicity of EsA and h-EsA on C57 and ICR mice is shown. The therapeutic does of h-EsA is 10-15 mg/kg, and the therapeutic window can be > 50.
IV. DETAILED DESCRIPTION OF THE INVENTION
79. The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the Examples included therein and to the Figures and their previous and following description.
A. DEFINITIONS 80. As used in the specification and the appended claims, the singular forms "a,"
"an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a small molecule" includes mixtures of one or more small molecules, and the like.
81. Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. 82. The terms "higher," "increases," "elevates," or "elevation" refers to levels above control levels. The terms "low," "lower," "reduces," or "reduction" refers to levels below control levels. For example, control levels can be normal in vivo levels prior to, or in the absence of, inflammation or the addition of an agent which causes inflammation.
83. "Inflammation" or "inflammatory" is defined as the reaction of living tissues to injury, infection, or irritation. Anything that stimulates an inflammatory response is said to be inflammatory.
84. "Inflammatory disease" is defined as any disease state associated with inflammation. The inflammation can be associated with an inflammatory disease. Examples of inflammatory disease include, but are not limited to, asthma, systemic lupus erythematosus, rheumatoid arthritis, reactive arthritis, spondyarthritis, systemic vasculitis, insulin dependent diabetes mellitus, multiple sclerosis, experimental allergic encephalomyelitis, Sjogren's syndrome, graft versus host disease, inflammatory bowel disease (including Crohn's disease and ulcerative colitis) and scleroderma, myasthenia gravis, Guillain-Barre disease, primary biliary cirrhosis, hepatitis, hemolytic anemia, uveitis, Grave's disease, pernicious anemia, thrombocytopenia, Hashimoto's thyroiditis, oophoritis, orchitis, adrenal gland diseases, anti-phospholipid syndrome, Wegener's granulomatosis, Behcet's disease, polymyositis, dermatomyositis, multiple sclerosis, vitiligo, ankylosing spondylitis, Pemphigus vulgaris, psoriasis, and dermatitis herpetiformis.
85. "Infectious process" is defined as the process by which one organism is invaded by any type of foreign material or another organism. The results of an infection can include growth of the foreign organism, the production of toxins, and damage to the host organism.
86. "Cancer therapy" is defined as any treatment or therapy useful in preventing, treating, or ameliorating the symptoms associated with cancer. Cancer therapy can include, but is not limited to, apoptosis induction, radiation therapy, and chemotherapy.
87. "Transplant" is defined as the transplantation of an organ or body part from one organism to another.
88. "Transplant rejection" is defined as an immune response triggered by the presence of foreign blood or tissue in the body of a subject. In one example of transplant rejection, antibodies are formed against foreign antigens on the transplanted material.
89. Herein, "inhibition" or "suppression" means to reduce at least one activity as compared to a control (e.g., activity in the absence of such inhibition). It is understood that inhibition or suppression can mean a slight reduction in activity to the complete ablation of all activity. Inhibition or suppression also includes prevention. An "inhibitor" or "suppressor" can be anything that reduces the targeted activity, or has the potential to reduce the targeted activity in the preventative sense. For example, inhibition of COX-2 by a composition such as h-EsA or a derivative thereof can be determined by assaying the amount of COX-2 activity present in a cell. The composition can be administered to the cell before it is exposed to circumstances that would cause an elevation in COX-2 activity, and the levels of COX-2 activity can be measured before and after the exposure. In this example, if the amount of COX-2 activity is reduced in the presence of the composition as compared to the amount of COX-2 activity in the absence of the composition, the composition can be said to inhibit COX-2 activity.
90. As used throughout, by a "subject" is meant an individual. Thus, the "subject" can include domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.) and birds. Preferably, the subject is a mammal such as a primate, and, more preferably, a human.
91. By "aglycosidic EsA" is meant an Esculentoside A (EsA) molecule that is aglycosidic. An example of such a molecule is h-EsA, which is described in greater detail below. The aglycosidic EsA molecule and deriviatives thereof are useful in reducing radiation damage, cytokine inhibition by radiation damage, brain edema, pain, and inflammation, for example. They can also be used to treat neoplasias, as disclosed in US Patent Publication Nos. 20040053935, 20040053934, 20040053900, 20030225150, 20030162829, 20020169195, and 20020086894. A more detailed disclosure of how aglycosidic EsA can be used to treat various diseases and disorders, and ameliorate various conditions, follows.
B. AGLYCOSIDIC ESA MOLECULES AND METHODS OF USING
92. The compositions disclosed herein offer additional advantages over known COX-2 inhibitors. Saponins are a large family of naturally occurring glycoconjugate compounds with considerable structural diversity. Saponins are glycosidic natural plant products, composed of a ring structure (the aglycone) to which is attached one or more sugar chains. The saponins are grouped together based on several common properties. In particular, saponins are surfactants which display hemolytic activity and form complexes with cholesterol. Although saponins share these properties, they are structurally diverse. In particular, the aglycone can be a steroid, triterpenoid or a steroidal alkaloid and the number of sugars attached to the glycosidic bonds vary greatly, or can be entirely absent, as is the case with h-EsA and the derivatives thereof discussed herein.
93. Saponins have been used in pharmaceutical compositions for a variety of purposes. For example, U.S. Pat. No. 5,118,671, describes the use of aescin, a saponin obtained from Aesculus hippocastanum seeds, in pharmaceutical and cosmetic compositions as an anti-inflammatory. Similarly, U.S. Pat. No. 5,147,859, discusses the use of Glyccyrrhiza glabra saponin/phospholipid complexes as anti-inflammatory and anti-ulcer agents and U.S. Pat. No. 5,166,139, describes the use of complexes of saponins and aglycons, obtained from Centella asiatica and Terminalia sp., with phospholipids in pharmaceutical compositions. International Publication No. WO 91/04052, published 4 Apr. 1991, discusses the use of solid Quillaja saponaria saponin/GnRH vaccine compositions for immunocastration and immunospaying.
94. The saponin family includes Esculentosides A, B, C, D and E, and are isolated from Phytolacca esculent. Esculentoside A (EsA, 3-0- [β-D-glucopyranosyl- (Hensley et al. (1999); Felemovicius et al. (1995))-β-D-xylo-pyranosyl] phytolaccagenin, Figure 1) is a highly purified saponin from Phytolacca esculent. EsA has a molecular weight of 826 Daltons (Yi et al. Chinese Herb Medicine, 15 (2): 7-11 (1984)). Because of its hydrophilic radices such as hydroxide and carboxyl, EsA has a high water-solubility. Derivatives and analogs of EsA are also contemplated and are discussed below. Specifically disclosed herein is h-EsA, an aglycoside analog of Esculentoside A. Esculentoside A does not have a cross reaction with sulfonamide antibiotics, unlike many non-steroidal anti-inflammatory drugs (NSAIDS). h-EsA has anti-inflammatory effects with mechanisms differing from currently used anti-inflammatory drugs.
95. Inflammation is a complex stereotypical reaction of the body expressing the response to damage of its cells and vascularized tissues. The discovery of the detailed processes of inflammation has revealed a close relationship between inflammation and the immune response. The main features of the inflammatory response are vasodilation, i.e. widening of the blood vessels to increase the blood flow to the infected area; increased vascular permeability, which allows diffusible components to enter the site; cellular infiltration by chemotaxis, or the directed movement of inflammatory cells through the walls of blood vessels into the site of injury; changes in biosynthetic, metabolic, and catabolic profiles of many organs; and activation of cells of the immune system as well as of complex enzymatic systems of blood plasma.
96. There are two forms of inflammation, acute and chronic. Acute inflammation can be divided into several phases. The earliest, gross event of an inflammatory response is temporary vasoconstriction, i.e. narrowing of blood vessels caused by contraction of smooth muscle in the vessel walls, which can be seen as blanching (whitening) of the skin. This is followed by several phases that occur over minutes, hours and days later. The first is the acute vascular response, which follows within seconds of the tissue injury and lasts for several minutes. This results from vasodilation and increased capillary permeability due to alterations in the vascular endothelium, which leads to increased blood flow (hyperemia) that causes redness (erythema) and the entry of fluid into the tissues (edema).
97. Examples of chronic inflammatory diseases include tuberculosis, chronic cholecystitis, bronchiectasis, rheumatoid arthritis, Hashimoto's thyroiditis, inflammatory bowel disease (ulcerative colitis and Crohn's disease), silicosis and other pneumoconiosis, and implanted foreign body in a wound. 98. Activated cells can also be identified at the site of inflammation. "Activated cells" are defined as cells that participate in the inflammatory response. Examples of such cells include, but are not limited to, T-cells and B-cells , macrophages, NK cells, mast cells, eosinophils, neutrophils, Kupffer cells, antigen presenting cells, as well as vascular endothelial cells.
99. Macrophages release cytokines (e.g., tumor necrosis factor, interleukin-1), which heighten the intensity of inflammation by stimulating inflammatory endothelial responses; these endothelial changes help recruit large numbers of T cells to the inflammatory site.
100. Damaged tissues release pro-inflammatory mediators (e.g., Hageman factor (factor XII) that trigger several biochemical cascades. The clotting cascade induces fibrin and several related fibrinopeptides, which promote local vascular permeability and attract neutrophils and macrophages. The kinin cascade principally produces bradykinin, which promotes vasodilation, smooth muscle contraction, and increased vascular permeability.
101. Dislcosed herein are methods of treating inflammation in a subject by administering to the subject an effective amount of an aglycosidic EsA or a derivative thereof.. In various kinds of animal inflammatory models, h-EsA shows a strong inhibition of inflammation. In mice studies, during the acute phase (2 weeks), swelling of the paw was reduced in EsA/h-EsA treated mice as compared to the untreated CIA control. The mean volume in normal mice was about 10.5 mm3 while CIA increased it to 20 mm3, whereas EsA and h-EsA reduced it to 16 mm3 (Example 1, Figure 3). EsA/h-EsA had a similar effects compared to Celebrex (Fig 3).
102. Taken together, in the mouse CIA (Collagen Induced Arthritis) model, it was demonstrated: 1) h-EsA is equally effective as Celebrex at early stage of CIA and can maintain its effectiveness for a longer period of time; 2) the triterpenoid portion of EsA is the functional element of EsA; and 3) h-EsA can be a better choice than EsA (depending on the route of administration and other factors) due to elimination of the surfactant sugar group. It has been shown that h-EsA can effectively reduce the effects of CIA during peak inflammation (second boost) as a mitigation/treatment agent. It is also believed that it can also act to reduce or prevent its occurrence of CIA from time of onset (first immunization period). 103. Disclosed are methods of inhibiting a cytokine in a subject comprising administering to the subject an aglycosidic EsA or a derivative thereof. The cytokine can be selected from the group consisting of angiogenic, growth, fibrogenic, and inflammatory cytokines. Examples of such cytokines include, but are not limited to, ILl, IL6, TNFα, TGFβ, VEGF, and MCPl or any combination thereof. The aglycodisic EsA can be administered in a variety of ways, as disclosed below.
104. "Inhibiting a cytokine" refers to blocking or reducing at least one cytokine mediated event.
105. Also disclosed are methods of inhibiting PGE2 in a subject comprising administering to the subject an aglcyosidic EsA or a derivative thereof. Also disclosed are methods of inhibiting nitric oxide (NO) in a subject comprising administering to the subject an aglycosidic EsA or a derivative thereof. EsA inhibited the production of prostaglandin E2 (PGE2), platelet-activating factor (PAF), and nitric oxide (NO). PGE2 is known to play a major role in acute inflammation. Nitric Oxide has a key role in perpetual inflammation (Fang et al. Yao Xue Xue Bao. 26(10):721-4 (1991)).
106. Inflammation can be associated with a number of different diseases and disorders. Examples of inflammation include, but are not limited to, inflammation associated with hepatitis, inflammation associated with the lungs, and inflammation associated with an infectious process. Inflammation can also be associated with liver toxicity, which can be associated in turn with cancer therapy, such as apoptosis induction or chemotherapy, or a combination of the two, for example.
107. When the inflammation is associated with an infectious process, the infectious process can be associated with a viral infection. Examples of viral infections include, but are not limited to, Herpes simplex virus type-1, Herpes simplex virus type-2, Cytomegalovirus, Epstein-Barr virus, Varicella-zoster virus, Human herpesvirus 6, Human herpesvirus 7,
Human herpesvirus 8, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus, Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency cirus, Human T-cell Leukemia virus type-1, Hantavirus, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, and Human Immunodeficiency virus type-2.
108. The infectious process can also be associated with a bacterial infection. Examples of bacterial infections include, but are not limited to, M. tuberculosis, M. bovis, M. bovis strain BCG, BCG substrains, M. avium, M. intracellular, M. africanum, M. kansasii, M. marinum, M. ulcerans, M. avium subspecies paratuberculosis, Nocardia asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Salmonella typhi, other Salmonella species. Shigella species, Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida, other Pasteurella species, Actinobacϊϊlus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus, other Brucella species, Cowdria ruminantium, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, other Rickettsial species, Ehrlichia species,
Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, Bacillus anthracis, Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Clostridium tetani, other Clostridium species, Yersinia enterolitica, and other Yersinia species.
109. The infectious process can also be associated with a parasitic infection. Examples of parasitic infections include, but are not limited to, Toxoplasma gondii, Plasmodium species such as Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and other Plasmodium species, Trypanosoma brucei, Trypanosoma cruzi,
Leishmania species such as Leishmania major, Schistosoma such as Schistosoma mansoni and other Shistosoma species, and Entamoeba histolytica.
110. The infectious process can also be associated with a fungal infection. Examples of fungal infections include, but are not limited to, Candida albicans, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneomocystis carnii, Penicillium marneffi, and Alternaria alternata.
111. Also disclosed are methods of reducing transplant rejection in a recipient by administering to the recipient an aglycosidic EsA or a derivative thereof. Inflammation is associated with transplant rejection in a transplant recipient. In one example of transplant rejection, antibodies are formed against foreign antigens on the transplanted material. The transplantation can be, for example, organ transplantation, such as liver, kidney, skin, eyes, heart, or any other transplantable organ of the body or part thereof.
112. Transplantation immunology refers to an extensive sequence of events that occurs after an allograft or a xenograft is removed from a donor and then transplanted into a recipient. Tissue is damaged at both the graft and the transplantation sites. An inflammatory reaction follows immediately, as does activation of biochemical cascades. A series of specific and nonspecific cellular responses ensues as antigens are recognized. Antigen-independent causes of tissue damage (i.e., ischemia, hypothermia, reperfusion injury) are the result of mechanical trauma as well as disruption of the blood supply as the graft is harvested. In contrast, antigen-dependent causes of tissue damage involve immune-mediated damage.
113. Rejection is the consequence of the recipient's alloimmune response to the nonself antigens expressed by donor tissues. In hyperacute rejection, transplant subjects are serologically presensitized to alloantigens (i.e., graft antigens are recognized as nonself). Histologically, numerous polymorphonuclear leukocytes (PMNs) exist within the graft vasculature and are associated with widespread microthrombin formation and platelet accumulation. Little or no leukocyte infiltration occurs. Hyperacute rejection manifests within minutes to hours of graft implantation. Hyperacute rejection has become relatively rare since the introduction of routine pretransplantation screening of graft recipients for antidonor antibodies.
114. In acute rejection, graft antigens are recognized by T cells; the resulting cytokine release eventually leads to tissue distortion, vascular insufficiency, and cell destruction. Histologically, leukocytes are present, dominated by equivalent numbers of macrophages and T cells within the interstitium. These processes can occur within 24 hours of transplantation and occur over a period of days to weeks. 115. In chronic rejection, pathologic tissue remodeling results from peritransplant and posttransplant trauma. Cytokines and tissue growth factor induce smooth muscle cells to proliferate, to migrate, and to produce new matrix material. Interstitial fibroblasts are also induced to produce collagen. Histologically, progressive neointimal formation occurs within large and medium arteries and, to a lesser extent, within veins of the graft. Leukocyte infiltration usually is mild or even absent. AU these result in reduced blood flow, with subsequent regional tissue ischemia, fibrosis, and cell death. (Prescilla et al. http://www.emedicine.com, Immunology of Transplant Rejection, updated June 20, 2003).
116. Transplant rejection may occur within 1-10 minutes of transplantation, or within 10 minutes to 1 hour of transplantation, or within 1 hour to 10 hours of transplantation, or within 10 hours to 24 hours of transplantation, within 24 hours to 48 hours of transplantation, within 48 hours to 1 month of transplantation, within 1 month to 1 year of transplantation, within 1 year to 5 years of transplantation, or even longer after transplantation.
117. Disclosed herein are methods of treating radiation damage. Ionizing radiation (IR) remains a main stream therapy for cancer, since it controls both primary and metastatic cancer without significant systemic damage. However, radiation therapy does cause IR- induced local damage of normal tissue (radiation toxicity), leading to a temporary or persistent impairment of irradiated tissues, which lowers the life quality of cancer patients. Some severe side effects can even result in the discontinuation of the life-saving radiation therapy (Johansen et al. Radiother Oncol. 40: 101-9 (1996), Niemierko et al. Int J Radiat Oncol Biol Phys. 25: 135-45, 1993., Wiess et al. Toxicology 15;189(l-2):l-20 (2003 JuI) Protection against ionizing radiation by antioxidant nutrients and phytochemicals. Toxicology. 15: 189(1-2): 1-20 (2003 JuI), Goiten et al. Cancer 55: 2234-9 (1985)). Radiation damage can also occur by exposure to nuclear radiation, or exposure to a weapon that causes radiation.
118. Disclosed herein are methods of reducing radiation damage in a subject comprising administering to the subject an effective amount of an aglycosidic EsA. As disclosed above, the radiation damage can be caused by radiation therapy, such as that used to treat cancer. The radiation damage can also be caused by nuclear radiation, or by a weapon, such as a terrorist agent. The compositions herein can be admininstered prior to, after, or during exposure to radiation. 119. Radiation toxicity can be divided into two main stages: early toxicity and late toxicity (MacKay et al. Radiother Oncol. 46:215-6 (1998), Rubin et al. Radiother Oncol. 35: 9-10, (199?), Dubray et al. Cancer Radiother. 1: 744-52 (1997), Vozenin-Brotons et al. Radiat Res. 152: 332-7 (1999), Lefaix et al. Br J Radiol Suppl. 19: 109-13 (1986), Lefaix et al. Soc Biol FiI. 191: 777-95 (1997), Verola et al. Br J Radiol Suppl. 19: 104-8 (1986)). For example, in the irradiated soft tissue, there is early radiation dermatitis (ERD) that occurs within one month after IR, and late radiation fibrosis (LRF) which develops two months later. In the irradiated lung, there is pnuemonitis (early) and lung fibrosis (late) (Chen et al. IntJRadiat Oncol Biol Phys. 49: 641-8 (2001), Chen et al. Seminars in Radiation Oncology 12: 26-33 (2002), Marks et al. Int J Radiat Biol. 76: 469-75 (2000)). In the irradiated brain, there is brain edema (early) and brain degeneration (late). The pathophysiological mechanisms underlying these phenomena have been studied, but remain unclear.
120. In general, ER kills cell through the production of free radicals. The IR toxicity is a result of counteraction of host defense system that responds to IR physical insult. Upon IR, the cells are damaged by free radicals, and undergo either repair or apoptosis/death, which initiates the cascade of signal transduction pathways (such as Nuclear factor-κB (NFKβ), etc.). As a result, IR up-regulates the expression of inflammatory mediators (such as cytokines, lymphokines and chemokines) and immunomodulatory molecules (MHC, co- stimulatory molecules, adhesion molecules, death receptors, heat shock proteins) in irradiated tumor, stromal, and vascular endothelial cells (Friedman et al.). Among them, for example, ILl, IL6, MCP-I, COX-2 and TGFq play critical roles in IR toxicity (Chen et al. (2001) Hallahan et al. Important Adv Oncol.:7l-S0 (1993)). The accumulated cytokines and chemokines attract the immune cells (such as macrophages, dendritic cells, T cells and B cells) to the irradiated spot to engulf the apoptotic and necrotic cellular debris. After internalizing the debris, some of the mutated normal tissue "self antigens can be presented by dendritic cells to T cells (McBride et al. Radiat Res.162(1): 1-19 (2004 JuI)). The interaction of sensitized T cells with the existing IR-induced mutated "wrong proteins" or "wrong genes" (which can pass to daughter cells) in irradiated normal tissues triggers a new wave of mass production of cytokines, which occurs a few months after IR, which may be the driving force for the late toxicity (chronic inflammation). This process is evidenced by the several waves of mass production of secretory molecules (cytokines and inflammatory mediators) at the stages of early and late toxicity.
121. A network exists between IR-induced molecules, such as interaction among NO, NF-Kβ, cytokines and COX. An interaction loop and feed-back control exists among these molecules. Upon IR, the NO and the signaling of DNA breakage directly activate NF- kβ, which induces ILlβ. The ILlβ binds to its receptors, which again triggers NFkβ and P38 pathways to enhance its production, a positive feed-back to amplify the inflammation signaling. ILlβ is a key cytokine in the IR inflammation process. As one of the effects, ILlβ enhances the expression of COX-2, and together they markedly induce inflammatory angiogenesis (Kuwano et al. FASEB J. 18(2):300-10 (2004)), a critical process in IR inflammation (toxicity). As a control, IL-lβ-induced activation of the COX-2 gene is modulated by NF-kβ (Kirtikara et al. (2000), Crofford et al. Arthritis Rheum. 40,226-236 (1997)). The COX-2 selective inhibitors can block ILlβ induced angiogenesis but only partially block VEGF-induced angiogenesis. Similarly, the ILlβ induced angiogenesis is much less in the COX-2 knockout mice than wild-type mice (Kuwano et al. (2004)).
Overexpression of COX-2 also is accompanied by up-regulation of nitric oxide synthases (Tsuji et al. Nippon Rinsho. 56: 2247-2252 (1998)), which can intensify local damage.
122. Cyclooxygenase is the rate-limiting step in the conversion of arachidonic acid to prostaglandins. There are two known genes of cyclooxygenase, COX-I and COX-2. COX-I is constitutively expressed at low levels in many cell types. Specifically, COX-I is known to be essential for maintaining the integrity of the gastrointestinal epithelium. COX-2 expression is stimulated by growth factors, cytokines, and endotoxins. The cyclooxygenase 2 isoform (COX-2) is not expressed in most tissues (e.g., liver) under physiological conditions but is highly upregulated in inflammatory processes and cancer, for example. Up-regulation of COX- 2 is responsible for the increased formation of prostaglandins associated with inflammation.
123. The compositions disclosed herein can also be used to treat cancer. A neoplasm, or tumor, is an abnormal, unregulated, and disorganized proliferation of cell growth. A neoplasm is malignant, or cancerous, if it has properties of destructive growth, invasiveness and metastasis. Invasiveness refers to the local spread of a neoplasm by infiltration or destruction of surrounding tissue, typically breaking through the basal laminas that define the boundaries of the tissues, thereby often entering the body's circulatory system. Metastasis typically refers to the dissemination of tumor cells by lymphotics or blood vessels. Metastasis also refers to the migration of tumor cells by direct extension through serous cavities, or subarachnoid or other spaces. Through the process of metastasis, tumor cell migration to other areas of the body establishes neoplasms in areas away from the site of initial appearance. 124. Prostaglandins are arachidonate metabolites produced in virtually all mammalian tissues and possess diverse biologic capabilities, including vasoconstriction, vasodilation, stimulation or inhibition of platelet aggregation, and immunomodulation, primarily immunosupression (Moskowitz and Coughlins, Stroke 1981; 12: 882-86; Leung and Mihich. Nature 1980; 597-600; Brunda et al., J. Immunol. 1980; 124: 2682-7). They are implicated in the promotion of development and growth of malignant tumors (Honn et al., Prostaglandins 1981;21:833-64; Furuta et al., Cancer Res. 1989, 48, 3002-7; Taketo; J. Natl. Cancer Inst. 1998, 90, 160920). They are also involved in the response of tumor and normal tissues to cytotoxic agents such as ionizing radiation (Milas and Hanson, Eur. J. Cancer 1995, 31 A, 1580-5). Prostaglandin production is mediated by two cyclooxygenase enzymes: COX-I and COX-2. Cyclooxygenase- 1 (COX-I) is constitutively expressed and is ubiquitous.
Cyclooxygenase-2 (COX-2) is induced by diverse inflammatory stimuli (Isakson et al., Adv. Pros. Throm. Leuk Res. 1995, 23, 49-54).
125. Prostaglandin-mediated effects at both the microenvironmental and cellular levels have been implicated in the modulation of such response. Inhibition of prostaglandin synthesis also induces an accumulation of cells in the G phase of the cell cycle, which are generally considered to be the most sensitive to ionizing radiation. With the inhibition of prostaglandin synthesis, prostaglandin-induced immunosuppressive activity was diminished and antitumor immunologic responses were able to potentiate tumor response to radiation. Finally, prostaglandins are vasoactive agents and are thus can regulate tumor blood flow and perfusion.
126. COX-2 inhibitors have been described for the treatment of cancer (WO98/16227) and for the treatment of tumors (EP 927,555). Celecoxib®, a specific inhibitor of COX-2, exerted a potent inhibition of fibroblast growth factor-induced corneal angiogenesis in rats. (Masferrer et al., Proc. Am. Assoc. Cancer Research 1999, 40, 396). COX-2 specific inhibitors prevent angiogenesis in experimental animals, but their efficacy in enhancing in vivo tumor response to radiation has not been established. COX-2 is overexpressed in adenocarcinoma (Tsuji et al. (1998), Sano et al. Cancer Res. 55: 3785-3789 (1995), Murata et al. Am. J. Gastroenterol. 94: 451-455 (1999)). The enhanced COX-2-induced synthesis of prostaglandins stimulates cancer cell proliferation (Sheng et al. J. Biol. Chem. 276: 18075- 18081 (2001), Achiwa et al. Clin. Cancer Res. 5: 1001-1005 (1999), promotes angiogenesis (Ben-Av et al. FEBS Lett. 372: 83-87 (1995), Tsuji et al. J. Exp. Clin. Cancer Res. 20: 117- 129 (2001)), inhibits apoptosis (Sheng et al. Cancer Res. 58:362-366 (1998)) and increases metastatic potential (Kakiuchi et al. (2002), Xue et al. World J. Gastroenterol. 9: 250-253 (2003)).
127. The inhibition of COX-2 has dual benefits: protecting the normal tissues and inhibiting the cancer cells. In addition, COX-2 inhibitors can act as chemopreventive agents. ILlβ-stimulated COX-2 expression can be found in almost all types of cells, including monocytes/macrophages (Caivano et al. J. Immunol. 164: 3018-3025 (2000), vascular endothelial cells (Kirtikara et al. (2000)), stromal cells (Bamba et al. Int. J. Cancer 83: 470-475 (1999)), epithelial cells and nonepithelial cells, showing that this interaction is critical for all types of tissue damage/ inflammation processes. The blocking of these paired molecules has therefore not been restricted to a specific tissue.
128. The methods and combinations of the present invention may be used for the treatment of neoplasia disorders selected from the group consisting of acral lentiginous melanoma, actinic keratoses, adenocarcinoma, adenoid cycstic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, astrocytic tumors, bartholin gland carcinoma, basal cell carcinoma, bronchial gland carcinomas, capillary, carcinoids, carcinoma, carcinosarcoma, cavernous, cholangiocarcinoma, chondrosarcoma, choriod plexus papilloma/carcinoma, clear cell carcinoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal, epitheloid, Ewing's sarcoma, fibrolamellar, focal nodular hyperplasia, gastrinoma, germ cell tumors, glioblastoma, glucagonoma, hemangiblastomas, hemangioendothelioma, hemangiomas, hepatic adenoma, hepatic adenomatosis, hepatocellular carcinoma, insulinoma, intaepithelial neoplasia, interepithelial squamous cell neoplasia, invasive squamous cell carcinoma, large cell carcinoma, leiomyosarcoma, lentigo maligna melanomas, malignant melanoma, malignant mesothelial tumors, medulloblastoma, medulloepithelioma, melanoma, meningeal, mesothelial, metastatic carcinoma, mucoepidermoid carcinoma, neuroblastoma, neuroepithelial adenocarcinoma nodular melanoma, oat cell carcinoma, oligodendroglial, osteosarcoma, pancreatic polypeptide, papillary serous adenocarcinoma, pineal cell, pituitary tumors, plasmacytoma, pseudosarcoma, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, small cell carcinoma, soft tissue carcinomas, somatostatin-secreting tumor, squamous carcinoma, squamous cell carcinoma, submesothelial, superficial spreading melanoma, undifferentiatied carcinoma, uveal melanoma, verrucous carcinoma, vipoma, well differentiated carcinoma, and Wilm's tumor.
129. Disclosed herein are methods of inhibiting COX-2 in a subject comprising administering to the subject an aglycosidic EsA or a derivative thereof. Also disclosed herein are methods of treating pain in a subject by administering to the subject an effective amount of an aglycosidic EsA or a derivative thereof. Pain is often associated with inflammation and the presence of COX-2. Aglycosidic EsAs and derivatives thereof can be used as analgesics for pain management. 130. Also contemplated are methods of inhibiting or preventing brain edema in a subject comprising administering to the subject an effective amount of an aglycosidic EsA. Cerebral edema occurs due to an increase in brain water content. It can be either intracellular or extracellular. Intracellular edema is defined by cellular swelling, usually of astrocytes, and classically is seen following cerebral ischema caused by cardiac arrest or head injury. The blood brain barrier is intact. Extracellular edema is a consequence of vascular injury with disruption of the blood brain barrier. Causes include trauma, tumor, and abscess. Ultimately, these changes can lead to herniation. Brain edema can also be radiation induced.
131. Disclosed are methods of inhibiting angiogenesis in a subject comprising administering to the subject an aglycosidic EsA. An increase in the expression of COX-2 has been correlated with a poor clinical outcome in patients with colorectal and other cancers. It has been shown that the COX-2 expressed in the epithelial cell compartment regulates angiogenesis in the stromal tissues of the mammary gland and that it is critical during mammary cancer progression (Chang et al. PNAS, DOI: 10.1073/pnas.2535911100, December 15, 2003.). 132. The effect of inhibition of prostanoid synthesis on COX-2 transgenic mice was determined, using a strain that develops spontaneous mammary tumors. It was observed that indomethacin strongly decreased microvessel density and inhibited tumor progression. Indomethacin also inhibited upregulation of angiogenic regulatory genes in COX-2 transgenic mammary tissue. In addition, it was shown that prostaglandin E2 stimulated the expression of angiogenic regulatory genes in mammary tumor cells isolated from COX-2 transgenic mice and treated with celecoxib, a COX-2-specific inhibitor, and reduced tumor growth and microvessel density.
133. Aglycosidic EsAs have molecular similarity to steroid hormones. This molecule can block enzymes related to steroid metabolism and interconversion. An example of such an enzyme is the aromatase enzyme, which converts androgens to estrogens. Agents that block this enzyme are the preferred treatment for many women with post-menopausal breast cancer. As disclosed herein, aglycosidic EsA and its derivatives can have antiinflammatory effects, which can be related to steroid effects and include cytokine-modifying effects. Aglycosidic EsA and its derivatives can also have a therapeutic effect on the endometritis and breast adenoma (two types of estrogen related chronic diseases). C. COMPOSITIONS
134. Disclosed herein and useful in the methods described are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that, while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular molecule, such as EsA, is disclosed and discussed and a number of modifications that can be made to a number of places within the molecule can be made, specifically contemplated is each and every combination and permutation of the molecule unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.
135. As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms "substitution" or "substituted with" include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g. , a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
136. The term "alkyl" as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, w-propyl, isopropyl, /t-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group can also be substituted or unsubstituted. The alkyl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxamate, hydroxy, ketone, nitro, silyl, sulfo- oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below. 137. Throughout the specification "alkyl" is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group. For example, the term "halogenated alkyl" specifically refers to an alkyl group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine, or iodine. The term "alkylalcohol" specifically refers to an alkyl group that is substituted with one or more hydroxyl groups, as described below. The term "alkylthiol" specifically refers to an alkyl group that is substituted with one or more thiol groups, as described below. The term "alkylalkoxy" specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below. The term "alkylamino" specifically refers to an alkyl group that is substituted with one or more amino groups, as described below, and the like. 138. This practice is also used for other groups described herein. That is, while a term such as "cycloalkyl" refers to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an "alkylcycloalkyl." Similarly, a substituted alkoxy can be specifically referred to as, e.g., a "halogenated alkoxy," a particular substituted alkenyl can be, e.g., an "alkenylalcohol," a particular substituted alkynyl can be, e.g., an "alkynylsilyl," a particular substituted aryl can be, e.g., a "nitroaryl," a particular substituted cycloalkyl can be, e.g., a "cycloalkylether," a particular substituted heterocycloalkyl can be, e.g., a "heterocycloalkylnitro," a particular substituted cycloalkenyl can be, e.g., a "alkylcycloalkenyl," a particular substituted heterocycloalkenyl can be, e.g., a "heterocycloalkenylthiol," and the like.
139. The term "alkoxy" as used herein is an alkyl group bound through a single, terminal ether linkage; that is, an "alkoxy" group may be defined as -OA where A is alkyl as defined above.
140. The term "alkenyl" as used herein is a hydrocarbon group of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond. Asymmetric structures such as (AB)C=C(CD) are intended to include both the E and Z isomers. This may be presumed in structural formulae herein wherein an asymmetric alkene is present, or it may be explicitly indicated by the bond symbol C=C. The alkenyl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxamate, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.
141. The term "alkynyl" as used herein is a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon triple bond. The alkynyl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxamate, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.
142. The term "aryl" as used herein is a group that contains any carbon-based aromatic group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, phenoxybenzene, and the like. The term "aryl" also includes "heteroaryl," which is defined as a group that contains an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term "non-heteroaryl," which is also included in the term "aryl," defines a group that contains an aromatic group that does not contain a heteroatom. The aryl group can be substituted or unsubstituted. The aryl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxamate, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein. The term "biaryl" is a specific type of aryl group and is included in the definition of aryl. Biaryl refers to two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.
143. The term "cycloalkyl" as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The term "heterocycloalkyl" is a cycloalkyl group as defined above where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxamate, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.
144. The term "cycloalkenyl" as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and contains at least one double bound, e.g., C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like. The term "heterocycloalkenyl" is a type of cycloalkenyl group as defined above, and is included within the meaning of the term "cycloalkenyl," where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxamate, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.
145. The term "cyclic group" is used herein to refer to either aryl groups, non-aryl groups (i.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups), or both. Cyclic groups have one or more ring systems that can be substituted or unsubstituted. A cyclic group can contain one or more aryl groups, one or more non-aryl groups, or one or more aryl groups and one or more non-aryl groups.
146. The term "aldehyde" as used herein is represented by the formula -C(O)H.
147. The terms "amine" or "amino" as used herein are represented by the formula NAA1A2, where A, A1, and A2 can be, independently, hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
148. The term "carboxylic acid" as used herein is represented by the formula -C(O)OH.
149. The term "ester" as used herein is represented by the formula -OC(O)A or - C(O)OA, where A can be an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above. 150. The term "ether" as used herein is represented by the formula AOA1, where A and A1 can be, independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
151. The term "ketone" as used herein is represented by the formula AC(O)A1, where A and A1 can be, independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
152. The term "halide" as used herein refers to the halogens fluorine, chlorine, bromine, and iodine. 153. The term "hydroxamate" as used herein is represented by the formula -
C(O)NHOH.
154. The term "hydroxyl" as used herein is represented by the formula -OH.
155. The term "nitro" as used herein is represented by the formula -NO2.
156. The term "silyl" as used herein is represented by the formula -SiAA1A2, where A, A1, and A2 can be, independently, hydrogen, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
157. The term "sulfo-oxo" as used herein is represented by the formulas -S(O)A, -S(O)2A, -OS(O)2A, or -OS(O)2OA, where A can be hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
158. The term "sulfonyl" is used herein to refer to the sulfo-oxo group represented by the formula -S(O)2A, where A can be hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
159. The term "sulfonylamino" or "sulfonamide" as used herein is represented by the formula -S(O)2NH-.
160. The term "sulfone" as used herein is represented by the formula AS(O)2A1, where A and A1 can be, independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above. 161. The term "sulfoxide" as used herein is represented by the formula AS(O)A1, where A and A1 can be, independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above. 162. The term "thiol" as used herein is represented by the formula -SH.
163. "Cy," "R1,'' "R2," and "L" as used herein can, independently, possess one or more of the groups listed above. For example, if R1 is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group can optionally be substituted with a hydroxyl group, an alkoxy group, an alkyl group, a halide, and the like. Depending upon the groups that are selected, a first group can be incorporated within second group or, alternatively, the first group can be pendant {i.e., attached) to the second group. For example, with the phrase "an alkyl group comprising an amino group," the amino group can be incorporated within the backbone of the alkyl group. Alternatively, the amino group can be attached to the backbone of the alkyl group. The nature of the group(s) that is(are) selected will determine if the first group is embedded or attached to the second group.
164. Also described herein are the pharmaceutically acceptable salts and esters of compounds represented by Formula I (described below). By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual along with the selected compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. Pharmaceutically acceptable salts can be prepared, for example, by treating the compound with an appropriate amount of a pharmaceutically acceptable base. Representative pharmaceutically acceptable bases include ammonium hydroxide, sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide, ferrous hydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide, ferric hydroxide, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2- diethylaminoethanol, lysine, arginine, histidine, and the like. See for example, S. M. Berge, et at, "Pharmaceutical Salts," J Pharm. Sci., 66:1-19, 1977, which is incorporated herein by reference for its teaching of pharmaceutically acceptable salts. In one aspect, the reaction is conducted in water, alone or in combination with an inert, water-miscible organic solvent, at a temperature of from about 0°C to about 100°C, such as at room temperature. The molar ratio of compounds represented by Formula I to be used is chosen to provide the ratio desired for any particular salts. For preparing, for example, the ammonium salt of a compound represented by Formula I, the compound can be treated with approximately one equivalent of a pharmaceutically acceptable base to yield a neutral salt. Pharmaceutically acceptable esters include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, phenyl, pyridinyl, benzyl, and the like. Pharmaceutically acceptable esters can be prepared by, for example, by treating the compound with an appropriate amount of carboxylic acid, ester, acid chloride, acid anhydride, or mixed anhydride agent that will provide the corresponding pharmaceutically acceptable ester. Typical agents that can be used to prepare pharmaceutically acceptable esters include, for example, acetic acid, acetic anhydride, acetyl chloride, benzylhalide, benzaldehyde, benzoylchloride, methyl ethylanhydride, methyl phenylanhydride, methyl iodide, and the like.
Suitable compounds are aglycone saponins (e.g., sapogenins). In specific examples, suitable aglycone saponins are the acid saponins (i.e., triterpenoid saponins). These compounds can be obtained by removing the glycosidic moieties at R2 in commercial or naturally isolable saponins. For example, saponins disclosed in U.S. Patents 6,528,058, 6,645,495, 6,753,414, 6,524,584, 6,231,859, 5,688,772, 5,597,807, and 5,057,540, (which are incorporated by reference herein in their entirety) can be treated with acid or base to remove the glycoside moiety. Alternatively, aglycoside saponins (e.g., the triterpenoid saponins) can be isolated directly from a variety of plant sources (e.g., Phytolacca esculenta, Panax notoginsing, Akebia trifoliate) by known extraction methods (see e.g., U.S. Patents 4,879,376, 5,977,081, WO/2006/116656
In many examples, suitable compounds can have the following formula.
Figure imgf000034_0001
where
R1 and R2 are, independent of one another, H, OH, alkyl (e.g., CH3, C2H5, C3H7, CH(CH3)2, C4H9, CH(CH3)C2H5, CH2CH(CH3)2, C(CH3)3, C5Hi 0, alkoxide (e.g. , OCH3,
OC2H5, OC3H7, OCH(CH3)2, OC4H9, OCH(CH3)C2H5, OCH2CH(CH3)2, OC(CH3)3, OC5H11), aryl (e.g., C6H5, C6H4OCH3, C6H4OH, C6H4ONH2, CH2C6H5), hydroxyl alkyl (e.g., -CH2OH, -C2H4OH), OSO2CH3, OSO2C6H5CH3, OC5-cyclic ether (e.g., THF), OC6-cyclic ether (e.g., THP), O-linear ether (e.g., MOM, MEM), O-silyl ether (e.g. , TBDMS, TMS, TES), ester (e.g. , CH3C(O)O (acetate), CH3CH2C(O)O
(propionate)), acyl (e.g., CH3C(O), CH3CH2(O), acetal, OLi, ONa, OK, or OCa;
Each R3 is, independent of one another, H, OH, alkoxide (e.g., OCH3, OC2H5, OC3H7, OCH(CH3)2, OC4H9, OCH(CH3)C2H5, OCH2CH(CH3)2, OC(CH3)3, OC5H11), hydroxyl alkyl (e.g., -CH2OH, C2H4OH), C(O)H, CO2H, CO2Na, CO2K, CO2Li, CO2Ca, CO2R8, or -CH2OR8;
Each R4 is, independent of one another, H, alkyl (e.g., CH3, C2H5, C3H7, CH(CH3)2, C4H9, CH(CH3)C2H5, CH2CH(CH3)2, C(CH3)3, C5H11), or aryl (e.g., C6H5, C6H4OCH3, C6H4OH, C6H4ONH2, CH2C6H5);
R5 and R6 are, independently of one another, H, CH3, OH, CO2H, or CO2R8; Each R7 is, independent of one another, H or together are an oxo group;
R8 is an alkyl (e.g., CH3, C2H5, C3H7, CH(CH3)2, C4H9, CH(CH3)C2H5, CH2CH(CH3)2,
C(CH3)3, C5Hn), aryl (e.g., C6H5, C6H4OCH3, C6H4OH, C6H4ONH2, CH2C6H5), Li, Na, K, or Ca; and the between atoms a and b, shown as ZZ^L, is a single or double bond.
In certain examples, R2 does not comprise a saccharide.
Some specific compounds contemplated herein are glycyrrhetinic acid, glycyrrhizinic acid, β-amyrin, astragaloside IV, astragaloside II, astragaloside I and acetylastragaloside I, saikosaponin-d, maesabalide III, arvensoside C, bidentatoside II, chikusetsusaponin V methyl ester, capilliposide G, capilliposide H, aglycosidyl esculentoside A, B, C, or D. In another specific example, a suitable compound can have the following formula.
Figure imgf000035_0001
165. Examples of compounds, adjuvants, and derivatives of saponins can be found, for example, in US Patents 6,528,058, 6,645,495, 6,753,414, 6,524,584, 6,231,859, 5,688,772, 5,597,807, and 5,057,540. Each is herein specifically incorporated by reference for their teaching in regard to saponins and derivatives thereof which are useful with the methods disclosed herein.
166. Reference will now be made in detail to specific aspects of the disclosed materials, compounds, compositions, components, and methods, examples of which are illustrated in the following description and examples. 167. Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer and diastereomer, and a mixture of isomers, such as a racemic mixture. 168. In one aspect, described herein are compositions comprising a compound represented by Formula I. In another aspect, described herein are compositions prepared by or with compounds represented by Formula I. For example, compounds represented by Formula I can be used as monomers in peptide synthesis. The use of amino acid monomers to synthesize peptides is well known in the art. Techniques for generating peptides from various amino acids, like those represented by Formula I, can involve solution based chemistry or solid phase chemistry, and can be performed on automated peptide synthesizers. Reviews of peptide syntheses that can be used to prepare peptides from the compounds disclosed herein can be found in Angew. Chem. Int. Ed. Engl., 24:799, 1985; Ace. Chem. Res., 22:47, 1989; Angew. Chem. Int. Ed. Engl, 30:1437, 1991; Pure & Appl. Chem., 59:331, 1987; Synthesis, 453, 1972; Angew. Chem. Int. Ed. Engl. 24:719, 1985, which are incorporated herein by reference for their teachings of peptide synthetic techniques. Disclosed herein are peptides comprising at least one compound represented by Formula I. 169. Compounds represented by Formula I can be optically active or racemic. The stereochemistry at the tertiary carbon shown in Formula I can vary and will depend upon the spatial relationship between the substituents on that carbon. In one aspect, the stereochemistry at the tertiary carbon shown in Formula I is S. In another aspect, the stereochemistry at the tertiary carbon shown in Formula I is R. 170. Using techniques known in the art, it is possible to vary the stereochemistry at the tertiary carbon shown in Formula I. While such enantioselective and enantiospecific techniques typically provide the one isomer, the presence of a minor amount of the other isomer can sometimes occur. As such, in one aspect, the compound represented by Formula I is the substantially pure S enantiomer. Alternatively, the compound represented by Formula I is the substantially pure R enantiomer. Also, depending on the particular R1 and/or L group, other carbon stereocenters can exist in compounds represented by Formula I. The S and R isomers of such additional stereocenters are contemplated herein. Accordingly, Formula I includes enantiomers, diastereomers, and meso forms of the compounds represented thereby. 171. Compounds represented by Formula I can be readily synthesized using techniques generally known to those of skill in the art. The starting materials and reagents used in preparing these compounds are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, NJ.), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser' s Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989). 172. While the synthetic routes discussed above can be performed as solution-phase multiple parallel syntheses, which involves the synthesis of compounds in individual reaction vessels, other methods can be performed. For example, combinatorial based syntheses or solid phase syntheses can be used and will depend on the particular compounds to be synthesized, the availability of reagents, or preference.
D. ADMINISTRATION/PHARMACEUTICAL CARRIERS
173. The compositions of the invention can be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. 174. Disclosed are compositions comprising aglycosidic EsAs or a derivative thereof and a pharmaceutical carrier. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered in a number of ways, as described below. Other compounds will be administered according to standard procedures used by those skilled in the art. 175. Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. 176. The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including opthamalically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed compositions can be administered intravenously, intraperitoneally, intramuscularly, intraarticularly, intrathecally, subcutaneously, intracavity, or transdermally. The pharmaceutical compositions can also be admininstered in the form of an intraoperative wash.
177. The compositions disclosed herein can also be administered through topical intranasal administration or administration by inhalant. As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization. The latter may be effective when a large number of animals are to be treated simultaneously. Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition.
178. Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.
179. Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
180. Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
181. Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
182. Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
183. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect of the methods disclosed herein. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. While individual needs vary, determination of optimal ranges of effective amounts of the vector is within the skill of the art. Typical dosages comprise about 0.01 to about 100 mg/kg-body wt. The preferred dosages comprise about 0.1 to about 100 mg/kg-body wt. The most preferred dosages comprise about 1 to about 100 mg/kg-body wt.
184. For example, .01, .I, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 75, or 100 mg/kg or any amount in between of an aglycosidic EsA or a derivative thereof can be administered to a subject for treatment of inflammation, pain, brain edema, angiogenesis, and COX-2 inhibition, for example, hi one embodiment, h- EsA is administered in the amount of 2-40 mg/kg. hi another embodiment, h-EsA is administered in the amount of 5-30 mg/kg. hi another embodiment, h-EsA is administered in the amount of 5-20 mg/kg. An appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
185. Dosages can be given every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, or 72 hours, or any amount in between. It can also be given weekly, biweekly, monthly, or yearly, depending on the condition being treated and the individual needs of the subject receiving treatment. Dosages can also be administered in the form of a bolus. Dosages can also be administered preventatively in an effective amount that can be determined by one of ordinary skill in the art.
186. The materials may be in solution or suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, Br. J. Cancer, 58:700-703, (1988); Senter, Bioconjugate Chem., 4:3-9, (1993); Battelli, Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes, Cancer Research, 49:6214- 6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). hi general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin- coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor- level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).
187. Liposomes are vesicles comprised of one or more concentrically ordered lipid bilayers which encapsulate an aqueous phase. They are normally not leaky, but can become leaky if a hole or pore occurs in the membrane, if the membrane is dissolved or degrades, or if the membrane temperature is increased to the phase transition temperature. Current methods of drug delivery via liposomes require that the liposome carrier ultimately become permeable and release the encapsulated drug at the target site. This can be accomplished, for example, in a passive manner wherein the liposome bilayer degrades over time through the action of various agents in the body. Every liposome composition will have a characteristic half-life in the circulation or at other sites in the body and, thus, by controlling the half-life of the liposome composition, the rate at which the bilayer degrades can be somewhat regulated.
188. hi contrast to passive drug release, active drug release involves using an agent to induce a permeability change in the liposome vesicle. Liposome membranes can be constructed so that they become destabilized when the environment becomes acidic near the liposome membrane (see, e.g., Proc. Natl. Acad. Sci. USA 84:7851 (1987); Biochemistry 28:908 (1989), which is hereby incorporated by reference in its entirety). When liposomes are endocytosed by a target cell, for example, they can be routed to acidic endosomes which will destabilize the liposome and result in drug release.
189. Alternatively, the liposome membrane can be chemically modified such that an enzyme is placed as a coating on the membrane which slowly destabilizes the liposome. Since control of drug release depends on the concentration of enzyme initially placed in the membrane, there is no real effective way to modulate or alter drug release to achieve "on demand" drug delivery. The same problem exists for pH-sensitive liposomes in that as soon as the liposome vesicle comes into contact with a target cell, it will be engulfed and a drop in pH will lead to drug release. This liposome delivery system can also be made to target B cells by incorporating into the liposome structure a ligand having an affinity for B cell- specific receptors.
190. Compositions including the liposomes in a pharmaceutically acceptable carrier are also contemplated.
191. Transdermal delivery devices have been employed for delivery of low molecular weight compositions by using lipid-based compositions (i.e., in the form of a patch) in combination with sonophoresis. However, as reported in U.S. Patent No. 6,041,253 to Ellinwood, Jr. et al., which is hereby incorporated by reference in its entirety, transdermal delivery can be further enhanced by the application of an electric field, for example, by ionophoresis or electroporation. Using low frequency ultrasound which induces cavitation of the lipid layers of the stratum corneum, higher transdermal fluxes, rapid control of transdermal fluxes, and drug delivery at lower ultrasound intensities can be achieved. Still further enhancement can be obtained using a combination of chemical enhancers and/or magnetic field along with the electric field and ultrasound.
192. Implantable or injectable protein depot compositions can also be employed, providing long-term delivery of, e.g., an aglycosidic EsA or derivatives thereof. For example, U.S. Patent No. 6,331,311 to Brodbeck, which is hereby incorporated by reference in its entirety, reports an injectable depot gel composition which includes a biocompatible polymer, a solvent that dissolves the polymer and forms a viscous gel, and an emulsifying agent in the form of a dispersed droplet phase in the viscous gel. Upon injection, such a gel composition can provide a relatively continuous rate of dispersion of the agent to be delivered, thereby avoiding an initial burst of the agent to be delivered. 193. Disclosed herein are kits that can be used in practicing the methods disclosed herein. For example, a kit can comprise an aglycosidic EsA or a derivative thereof. The kit can further comprise instructions. The kits can include any reagent or combination of reagent discussed herein or that would be understood to be required or beneficial in the practice of the disclosed methods.
194. Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.
195. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.
E. EXAMPLES
Example 1 : h-EsA, a Novel Anti-inflammation Agent
196. Since their discovery, COX-2 inhibitors have dominated the market for nonsteroidal anti-inflammatory agents (NSAIDs). In 2004, Vioxx®, a major COX-2 inhibitor, was removed from the marketplace due to cardiac toxicity. Celebrex®, the other major COX-2 inhibitor, now has almost no competition and therefore can remain inhibitively expensive. Celebrex® has a lower but still measurable rate of thromboembolic complications. There is therefore a powerful need for a new NSAID. It has recently been discovered that h-EsA, an aglycoside analog of Esculentoside A (EsA, a saponin isolated from Phytolacca esculenta), has multifactorial anti-inflammatory activity with an improved toxicity profile when compared to analogs of h-EsA. 197. Experiments with a lung inflammation model have been carried out, and the optimal condition for HPLC analysis for PK study has been performed. These data are summarized below. Using the COX inhibitors screen kit (cat# 560131 Cayman Inc. Ann Arbor, MI), it was found that the inhibitory effect of EsA on COX-2 activity was consistently obtained and this has been subsequently confirmed. When compared with current first line drug for anti-COX2, Celebrex®, the several differences are found to be significant: 1) Structurally, EsA differs from Celebrex® (Fig 1 in right); 2) EsA preferentially acts on COX2 with a IC50 that differs from Celebrex® (Table 1), which can lead to a difference in their side effects; 3) Experimentally, Celebrex® effectively inhibited the early phase of IR demonists (up to day 19 with the onset on day 14), but gradually it loss its effect after day 23 (late phase of IR demonists). In contrast, EsA reduced the severity of IR demonists both in early and late phases (Fig 2 and Fig 3A). In addition, EsA dramatically reduced the IR-induced soft tissue fibrosis evidenced by the much less shortening of IR leg as compared to the vehicle control and Celebrex® group (Fig 3B); 4) The side effect of EsA and Celebrex® was different. For example, it was observed that 45 days after treated C57B1/6 mice (that had a radiation-induced demonitis) with either EsA (10 mg/kg qd) or Celebrex® (30 mg/kg, qd) for one month, both groups had developed alopecia (Fig 4A), strongly suggesting that in vivo, EsA has a similar biological activity as Celebrex. However, the area of hair loss in front head was bigger in Celebrex® group than that in the EsA group (Fig 4A). In addition, after 2 weeks, the hair in mice treated with EsA re-grew back quickly while the mice in Celebrex® group were still in alopecia for another 5 weeks (Fig 4B), showing that the side effect of EsA can be much more reversible than that of Celebrex®.
Table 1. Comparison of IC50 between EsA and other two cox-2 inhibitors
Figure imgf000044_0001
198. The high potency of Celebrex® on inhibition of COX2 might break the balance of COX2 and COXl at much great extent and cause greater side effect than EsA. Overall, the in vitro and in vivo data strongly support that EsA is structurally and biologically different from Celebrex®. The unique characteristic of EsA in the treatment of IR-induced soft tissue damage are: 1) it reduced the early demonitis with an equivalent effect as Celebrex®; 2) it prevents the progression of acute IR demonitis to late fibrosis; 3) at therapeutic dose that exerts a similar effect as Celebrex®, EsA has a less severe and more reversible of side effect.
199. While working on CIA model, it was found that that most of the methods used to determine the severity of CIA are either not objective or not sufficiently quantitative, such as the joint score that assesses the degree of swelling, redness and deformation of a join based on a semi-quantitative 1-4 scale that is determined by eye. hi order to adequately determine the agent effects, a set of assays that can precisely represent the severity of disease in a non-objective and quantitative fashion was needed. For this, three devices were developed to measure the severity of CIA at different stages.
The first is a measurement of joints swollen at acute phase. Novel plethysmometer for mouse Water replacement has been the working principle for measuring the volume of swollen paws for decades. However, the commercially available plethysmometer is only useful for rat given the 10 μl precision of the devices. Since the mouse is 1/10 to 1/15 the weight of a rat, a plethysmometer with a precision as low as 1 μl in volume was needed.
Yet, a device with this level of precision was not commercially available. Therefore, a low volume plethysmomete was developed with the following properties: A) the front chamber of the device has a limited open space that allows for only the entry and emersion of a mouse paw; B) the front chamber connects to a very fine tube which is calibrated so that the volume of fluid displaced from the chamber is read by the distance that the displaced fluid is forced into the tube (1 mm change in distance in the tube is equivalent to 1 μL), and C) the volume of displaced fluid can easily be measured through the clear tubing using a ruler with millimeter gradations. Using this device, the extents of paw swelling among the different treatments was observed. 200. The measurement of grab force (dropping time) at the intermediate stage was the second device used. In this measurement technique the grab force of the mouse hind paws is assessed by the time an animal is able to hold to a metal net standing at an 85 degree angle. For control animals, the average time until the mouse drops (dropping time) is 10-12 minutes. However, due to the pain and stiffness of the joints, the paw grab force for a mouse experiencing the symptoms of CIA is dramatically reduced. The result is presented as a shortening of dropping time. This new apparatus allows us to objectively to determine the function of the joints in the two back paws (note: the two front paws were taped to prevent their use in holding to the metal net).
201. The third device measures a deformed ankle at late stage. The progression of CIA disease in the mouse is characterized by an increasing stiffness of the ankle joint that limits the extension of the joint to values below the normal extension range of 135 degrees. The extension of ankle joint is gauged against a circular rule that measures the extension angle of the joint. These methods that have been developed provide an ability to sensitively and objectively measure the severity of CIA in the CIA mouse models and more accurately determine how different treatments affect CIA at different stages of the disease. However, to compare the results of the CIA studies with the results presented in the literature, the traditional paw scoring system was used, in addition to the three newly developed methods described above. The scoring was performed by three scientists in a double blind fashion. The results were expressed by a standard scoring system: 0 = no evidence of erythema and swelling, 1 = mild redness and swelling of joint and ankle, 2 =defmite swelling, 3 = severe swelling of entire limb, 4 = limb burned out and deformed or ankylosed. The severity score is calculated for the 4 limbs and the maximum is 16 points. With high confidence on reliable and precise techniques for measurement of severity of CIA, the following experiments were sequentially carried out to determine: 1) the administration route for EsA; 2) its acute toxicity; and 3) its effect on the treatment of CIA.
202. The efficacy of five different administration routes (p.o., i.p., i.v., s.c, and i.m.) was determined for EsA. DBA/J1 mice were immunized with 0.1 ml/mice of an emulsion of 100 μg collagen II (in 50 ul) in Freund's complete adjuvant (in 50 ul) through tail s.c. injection. Five weeks (35 days) later, to accelerate the CIA process, the mice were boosted with 20 μg collagen II (in 50 ul) with Freund's incomplete adjuvant. One week after the initial immunization, the mice were randomly divided into eight groups (8 mice per group) with five groups treated with the same dose of EsA (10 mg/kg) through five different administration routes, namely, p.o., i.p., i.v. (using a liposomal form of EsA), s.c. and i.m., one group treated p.o. with vehicle alone and one group an untreated control. The positive control group was treated with Celebrex® at an oral dose of 30 mg/kg. The results of the plethysmometer measurements of paw swelling at 6 weeks (post-boost) showed that the p.o. route was the best at reducing swelling compared with the other four routes and the Celebrex® control group. Consistent with these results are the data obtained from the grab force measurements for the hind paw (presented as dropping time in log scale) demonstrating that the p.o. and i.p. routes (between 0.61-0.71 min) were better than the s.c. and i.m. routes at maintaining hind paws function, whereas the i.v. route provided the least protection of hind paw function (0.05 min, Fig 5). Since EsA given by both p.o. and i.p. pass through the portal vein to the liver, it appears that certain modifications of EsA occur in the liver.
203. Acute toxicities of EsA and its derivative h-EsA: Once the administration path (p.o.) was determined, an acute toxicity test was conducted to see the dose limitation. Mice (6 groups) were fed with different doses of EsA once and the number of lethal events was recorded daily for 2 weeks. The LD50 was about 55 mg/kg, which seems too low for an anti-CI agent (unless effective dose can be lowered to 1 mg/kg). It was speculated that this unexpectedly low LD50 was due to the sugar function on EsA that confers a saponin (surfactant) property to the compound. This surfactant- like property can cause disruption of cell membranes as was evidenced by the occurrence of hemolysis in the toxicity study. However, administration of a liposomal form of EsA was found to be effective in protecting against hemolysis. Given that the most effective administration routes were through p.o. and i.p. and the expectation that glycosylases in the gastrointestinal tract and/or the liver metabolism can cleave the sugar function from EsA, it appears that the triterpenoid structure of EsA can be the active portion of the compound. To test this, the sugar function was hydro lyzed from EsA using 10% HCl to obtain the triterpenoid portion of the compound, otherwise known as h-EsA (see Fig 1). The h-EsA was obtained as a precipitate after neutralization of the acid hydrozylate with an overall yield of 81.1%. When h-EsA was administered p.o. to mice, there was no lethality observed up to a dose of 190 mg/kg. In fact, some mice were able to tolerate a doses up to 220 mg/kg. Based on the toxicity data, we assigned an LD50 of 195 mg/kg for h-Es A. With this LD50, a therapeutic index for h-EsA of at least 20 is now achievable with a higher index expected since the therapeutic dose is expected to be <5 mg/kg using an optimized dose schedule. Given the large therapeutic index for h-EsA in comparison to EsA, h-EsA was used in treatment studies whereas EsA was be used as a positive control.
204. The CIA in DAB/1 mice was induced by bovine collagen II. The mice (male, 7 weeks old) immunized for 4 weeks were randomly divided into groups (10/group): 1) vehicle alone; 2) Celebrex® (14 mg/kg, 1.8 mM), as a positive control; 3) EsA (15 mg/kg, 0.9 mM) as parental agent control; and 4) h-EsA (15 mg/kg, 1.4 mM; or 7.5 mg/kg, 0.7 mM). The negative controls were the normal mice with same age and sex without immunization.
205. For testing of h-EsA as a CIA mitigation agent, the treatment was started three days after the second collagen II boost was given to trigger CIA symptoms. The symptoms normally appeared 7 days after boost. The schedule for treatment was q.o.d. (every other day) for 3 weeks. One week after boost, paw swelling was measured weekly. The dropping time (grab force) was measured at week 4. Three months later, the deformed ankle angle was measured in relapsing CIA mice. The results were very promising. In the acute phase (2 weeks), swelling of the paw was reduced in EsA/h-EsA treated mice as compared to the untreated CIA control. The mean volume in normal mice was about 10.5 mm3 while CIA increased it to 20 mm3, whereas EsA and h-EsA reduced it to 16 mm3 (Fig 3). EsA/h-EsA had a similar effects compared to Celebrex® (Fig 3). Although not statistically significant, CIA score was only slightly reduced in the EsA treated group compared to the Celebrex® group whereas there was a 17% reduction in
CIA score for the h-EsA group when measured at week 3 after treatment (Fig 4). It was noted that the CIA paw scoring was not reflective of the differences between the groups due to the subjective nature of the observers' judgment.
206. At the intermediate phase (6 weeks after onset of symptom), the grab force of paw was measured. The difference among the control and treatment groups could be well distinguished (Fig 6). While the normal mice could hold on the bar for 12 minutes, the CIA mice could only hold for a few seconds. This significant impairment in CIA paw function could be reversed by both EsA and h-ESA to a greater degree than was observed for Celebrex® (Fig 6). 207. Therapeutic effect of EsA and h-EsA on relapsing CIA: Considering CIA is a recurrent chronic disease, the clinical situation was mimicked by giving CIA mice a third boost (50 μg/collagen II/mice) with LPS as the adjuvant (2.5 mg/kg) 3 months after all the symptoms caused by the second boost had completely disappeared. The relapsing CIA was treated with EsA, h-EsA and Celebrex®. The results showed that h-EsA could reduce the degree of relapsing CIA, as evidenced by reduced paw swelling (Fig 7) and joint deformity (Fig 8). These data also suggest that repeated use of EsA or h-EsA do not result in any tolerance or insensitivity in the host. Finally, a histopathological analysis was performed on ankle tissues in the CIA models. Mice were sacrificed and ankles were fixed in 4% formalin for 24 hr. After decalcification for 2 weeks in a 10% EDTA solution the ankles were embedded in paraffin. The paws were sectioned and stained with H & E. Evaluation of disease was made according to the following scale: 1, synovial hyperplasia; 2, start of pannus development; 3, erosions of bone and cartilage; 4, severe inflammation and erosions. The results (Fig 9) showed that the joints of EsA, h-EsA, and Celebrex® treated mice had less severity in bone/cartilage erosion/destruction, synovium hyperplasia, and infiltration of mono- and polymorphonuclear cells as compared to CIA mice treated with vehicle alone. In addition, collagen deposition was significantly reduced in mice treated with EsA as measured by anti-collagen II immunoassay (Fig 10).
208. Taken together, in the mouse CIA model, it was demonstrated: 1) h-EsA is equally effective as Celebrex® at early stage of CIA and can maintain its effectiveness for a longer period of time; 2) the triterpenoid portion of EsA is the functional element of EsA; and 3) h-EsA may be safer than EsA due to elimination of the surfactant sugar group. It has been shown that h-EsA can effectively reduce the effects of CIA during peak inflammation (second boost) as a mitigation/treatment agent. It is also believed that it can also act to reduce or prevent its occurrence of CIA from time of onset (first immunization period). 209. The results of this set study are consistent with that of IR-soft tissue damage model, in which Celebrex® works well at the beginning and then gradually loses its protective effect, while the effect of EsA can be observed from acute phase to late fibrosis. Again, this indicates that EsA has a relatively long-lasting effect than Celebrex®. 210. Effect of EsA on inflammatory molecules (IMs): Since EsA/h-EsA had a prolonged effect on three CI models (CIA, IR-induced soft tissue and lung acute and chronic inflammation), it was believed that it can act through inhibition of inflammatory molecules (IM). To test this hypothesis, the Ms at both cell and tissue levels were assessed. The data showed that: 1) at the cellular level, the production of ILl α, TNFa, MCP-I, VEGF (Fig 11) and other cytokines was greatly reduced by EsA in a dose- dependent manner; and 2) at the tissue level (samples collected from mouse study), radiation induced increased IL lα, MCP-I, TNFa, IL6 and VEGF in the soft tissue were reduced at a greater extent than Celebrex® (Fig 12). Since EVIs play an important role in CI progression, the inhibition of IMs by EsA confers the therapeutic effects.
211. Since EsA and h-EsA had a prolonged effect on CI in three CI models (CIA, IR-induced cutaneous tissue, and acute and chronic lung inflammation), it was speculated that these two compounds may mediate their effects on CI through the inhibition of inflammatory molecules (IM). To test this hypothesis, the IMs were assessed at both cell and tissue levels. The data showed that: 1) at the cellular level, the production of IL-I α, and 212. TNFα (Fig 12) and other cytokines was greatly reduced by EsA in a dose- dependent manner; and 2) at the tissue level samples collected from mouse study), IR- induced increases in IL-6, TNFα, and other EvIs, such as MCP-I and IL-6 in skin were reduced by EsA to a greater extent than Celebrex® (Fig 13). In lung, h-EsA (10 mg/kg po) reduced the levels of vascular endothelial growth factor (VEGF), part of the platlet- derived growth factor (PDGF) pathway, to control levels 1 week post irradiation (12.5
Gy) (Fig 14) but was unable to reduce levels of IL-I α below levels of irradiation only mice 3 weeks post-irradiation (18 Gy) when administered at a dose of 15 mg/kg po (Fig 15). Therefore, the results show that the inhibition of one or more IM pathways by EsA and h-EsA is one mechanism through which the compounds are able to reduce the effects of CI in tissues.
213. From the work on EsA and radiation dermatitis, the protective effect of EsA appears to result from reduction of IL-I by the cells within the exposed tissues, for example epidermis or vascular epithelial cells, rather than by cytokines produced by macrophages. To test this hypothesis, it was first determined that IR powerfully induced IL-I in vitro in kerotinocytes but had no effect on Raw macrophage. It was then demonstrated that if these macrophages were first activated to produce IL-I using LPS with radiation, IL-I was decreased in all radiation doses and at all EsA doses tested (Fig 16A and l6B).
214. Therefore, the inhibition of IL-I can better account for EsA-related IR protection than COX-2 inhibition. In another set of experiments, IL- lβ induced by 2 or 4 Gy IR in A431 human epidermoid carcinoma cells was also completely inhibited by 0.1 ug/ml EsA (P< .01, Fig 16B and 16C). Finally, using a sensitive ELISA, other proinflammatory cytokines were also studied in a panel of normal cell lines including macrophages, epithelial cells, and fibroblasts from human and mouse origins. EsA potently inhibited the production and release of IL-I α, IL- lβ, IL-6, TNFα, TGFβ, and (cytokines involved in the IL-I network). Thus the effect on IL-I suppression is not cell specific and appears to be universal in human and murine cell lines.
215. Taken together, it has been demonstrated that: 1) IL-I and COX-2 were elevated following IR and are a component of early IR dermatitis, pneumonitis, and brain edema; 2) EsA alleviated radiation toxicity of the lung, skin, and brain in mice; 3) EsA inhibits COX-2 activity specifically; and 4) EsA reduces IR-related IL-I, IL-6, TNFα,
VEGF, and TGFβ production.
216. From the results of these studies it has been demonstrated: 1) that EsA and h- EsA are effective in reducing inflammation in our CIA model; 2) EsA and h-EsA act through inhibition of COX-2 activity; however, compared to Celebrex®, EsA and h-EsA have an advantage of not only reducing the symptoms related to CIA but also successfully blocking the progression of CIA, resulting in better treatment efficacy; 3) the oral path was the best route of administration compared with four other routes (i.v., s.c, Lm., i.p.) for treating CIA; 4) the therapeutic window was fairly large for h-EsA (> 20) and the mice treated for up to 2 months had no obvious side effect. 217. Work has also been conducted that examined the more global effects of EsA and h-EsA on the protection of other soft tissues from the effects of chronic inflammation. Of special interest was determining the ameliorating effects of EsA and h- EsA on radiation-induced chronic inflammation and the impact that this can have on reducing late radiation toxicities in lung and cutaneous soft tissues. The results of these studies show that both EsA and h-EsA have an ability to control both inflammation and fibrosis related to radiation exposures of these tissues. In fact, for some end-points such as radiation pneumonitis, pulmonary fibrosis, and lung function, the use of EsA or h-EsA were able to either maintain or return these end-points to levels that were either near or statistically equivalent to unirradiated controls.
218. Therapeutic effects of EsA and h-EsA on lung inflammation induced by IR: fibrosis-prone C57BL/6 mice were irradiated with 12.5 (experiment 1) or 18 Gy
(experiment 2) on whole chest and treated with EsA (10 mg/kg) or h-EsA (15mg/kg) via p.o. (q.o.d.) for 1 or 2 months. Histological analysis of lung tissue was conducted at 4, 6 and 42 weeks post-irradiation. The results showed that the signs of pneumonitis (evidenced by increased infiltration of inflammatory cells and exudates) and fibrosis (evidenced by increased fibroblast and thickened alveolar walls) were significantly reduced in mice administered EsA in comparison to mice receiving IR only (Fig 17). hi another set of experiments, mice were irradiated with a 18 Gy dose to the lungs and treated p.o. with either with Dexamethasone (10 mg/kg), Celebrex® (30 mg/kg) or h-EsA (15 mg/kg) 3 times a week for 4 or 8 weeks. Breathing rate and lung compliance were used to assess lung function as a function of treatment regime. Fig 18 shows the effect of treatment on breathing rate where only h-EsA reduces rate to normal control levels (p<0.05). To determine lung compliance and resistance, mice were anesthetized and the trachea was surgically exposed and incised. A 16-gauge, 1 cm stainless steel tube was inserted into the trachea and then secured with surgical silk. The animal was then connected to a Harvard rodent ventilator. For the studies a respiratory rate of 150 breaths per minute was used, and a tidal volume calculated based on the weight of the mouse.
219. The formula used for this calculation is 0.01 ml per gram body weight. The animal was then placed in a plethysmograph, and pressure- volume measurements were taken. The results of lung compliance testing in the treated mice are shown in Fig 19. The data show that early inhibition of pneumonitis by EsA can prevent progression to late stage fibrosis in irradiated lung and should have some utility in treating both IR pneumonitis and pulmonary fibrosis. The data also suggest that h-EsA out-performs Celebrex® in controlling radiation effects on lung tissues.
220. HPLC analysis for h-EsA level in biological samples: To develop EsA and h- EsA into anti-CI agents, the pharmacokinetics of these compounds must be known, which in turn guides the design of an optimal drag schedule with a goal to maximize the beneficial effect and reduced the toxic side effects of the two anti-CI agents.
221. Optimization of sample extraction and HPLC analytical methods: The current approach to quantifying h-EsA in plasma and tissues is a single extraction method using liquid- liquid extraction with analysis of the extracts by HPLC analysis. Recoveries of h-EsA for human plasma spiked at 1, 5 or 10 μg/mL h-EsA and GA (5 μg/mL) using solvent extraction techniques with n-butanol (BuOH) as the solvent followed by solid phase extraction was found to range from 82.1-101% for h-EsA and 75-80% for GA. The recoveries of h-EsA from spiked human plasma were found to decrease with h-EsA concentration. Mean (+/- 1 S.D.) percent recovery for h-EsA decreased with concentration with values of 101 (8.0), 92.9 (9.3) and 82.1 (8.1) calculated for samples containing 1, 5 and 10 μg/mL, respectively. The percent relative standard deviation (RSD) in the inter-day recovery of h-EsA from spiked plasma (5 μg/mL) using BuOH extraction alone on five separate days was calculated to be 8.89. 222. Using sample extraction and HPLC analytical methods, good resolving power was achieved allowing for the identification and quantification of h-EsA and GA in plasma and liver extracts tested to date. Figure 20 shows a typical chromatogram obtained for a plasma extract sample at both 205 nm (upper panel) and 252 nm (lower panel) wavelengths. In this example, the plasma is spiked with h-EsA and GA at a concentration of 5 μg/mL. In the chromatogram taken at 205 nm, h-EsA is shown to elute at 18.8 min, whereas GA elutes at 30.3 min, as shown in the chromatogram obtained at 252 nm. These chromatograms demonstrate the ability to resolve h-EsA and GA from plasma components. Similar resolution is obtained for liver extracts using a modified HPLC gradient method as described above. The limits of detection (LOD), as calculated from the calibration-design-dependent approach, for h-EsA in plasma and liver extracts are defined as 5 and 10 times of the background in the chromatograms, respectively. Using this definition, the LOD and LOQ for h-EsA in spiked plasma are 0.040 μg/mL and 0.08 μg/mL, respectively, using solvent extraction and HPLC analytical methods to measure h-EsA. Based a preliminary CMAX of 2.48 μg/mL for h-EsA in plasma at 2 h for a single 15 mg/kg (p.o.) dose in BALB/c mice, the peak area for this concentration is
31 times greater than the LOQ for the current assay. Obviously, the sugar portion of EsA plays a role in its toxicity. Because EsA is water-soluble while h-EsA is hydrophobic, it was speculated that their difference in toxicity can be due to the rate of absorption after taken orally. A pilot PK study was conducted with a single po dose of 15 mg/kg of EsA or h-EsA to mice. The plasma and liver as well as 12 different organs (lung, heart, kidney, spleen, thymus, muscle, brain, skin, bone marrow, abdominal fat, pancreas, and testis) were collected at different time points (0.5, 1, 2, 3, 6, 24 hr). The plasma and liver samples were both solvent extracted using n-butanol and SPE purified and then analyzed by HPLC, as described in section Cl .4.3. The mass of EsA or h-EsA in the re-suspended plasma and liver extracts was calculated using GA as the internal standard loaded at a mass of 6.25 μg in plasma samples and 9.8 to 14.3 μg in liver homogenates, depending on the mass of the sample. The mass of EsA or h-EsA is then normalized to the volume (for plasma) or tissue mass (for liver) extracted and expressed as μg/mL plasma or ng/mg liver tissue.
223. The results of the analysis of h-EsA and EsA in plasma (right panel) and liver (left panel) as a function of time are shown in Fig. 21. The mass of h-EsA recovered from the plasma was found to be highly variable. Based on QC studies of spiked human plasma, the extraction and HPLC analytical methods were performing within specifications, that is, the mass of h-EsA and EsA calculated in the QC samples was within +/- 5% of the target mass (5 μg/200 μL of plasma). Therefore, the variability appears to be due to the differences in the absorption of these compounds from the gut between animals. This can be verified with additional replicates and time points using a refined extraction method that uses a secondary solid phase extraction step to minimize the contribution of background from plasma and liver in quantification of h-EsA.
224. Based on a fit of the h-EsA data by eye, a Cmax of approximately 2.5 μg/mL was found to occur at 3.5 hours in plasma. An AUCt of 39 μg»h/mL was calculated by the linear trapezoid rule as applied to the fitted curve. For EsA, a fit of the data by eye, gave a Cmax of approximately 20 μg/mL at 6.5 hours in plasma. An AUCt of 92 μg#h/mL was calculated by the linear trapezoid rule as applied to the fitted curve. In the liver, a crude fit of the h-EsA concentration data by eye gave a Cmax of approximately 12 ng/mg tissue with a tmax of approximately 0.5 hours. Additionally, the simultaneous measurement of h-EsA in plasma obtained in the EsA studies shows h-EsA to be at low levels in plasma, with a Cmax below 1.8 μg/mL between 1 to 6 hours. This result indicates that at most approximately 8-10% of EsA undergoes hydrolysis of the sugar portion of the compound in the gut and liver. Therefore, the therapeutic and toxicological effects observed for EsA are likely due to the parent compound and not the hydrolysis product, h-EsA.
225. Summary of EsA/h-EsA in IR-induced CI in tissues: The data obtained from in vitro and in vivo IR studies strongly suggest that EsA and h-EsA are new anti-CI agents for controlling IR-induced intermediate and late toxicities in soft tissues. In murine lung and skin models, EsA was shown to reduce both radiation pneumonitis and pulmonary fibrosis in lung, and early and late dermatitis and late stage fibrosis in skin..
Therefore, the data obtained support further development of EsA as an agent to control early and late IR-induced tissue damage. Given that the triterpenoid portion of EsA appears to be the active functional group for mediating inflammation effects in soft tissues, h-EsA can have similar biological effects to those observed for EsA. 1. Example 2: Effect of h-EsA on radiation-induced lung fibrosis:
CBCT results
226. 1) Cone Beam Computed Tomography (CBCT) is the best for monitoring lung structure alterations: As indicated above, it has been found that CBCT is noninvasive, low risk, and can be performed many times in the same treated mouse. Due to its high quality (resolution) lung images, it can "see" fibrosis clearly, which allows for the observance of treatment effect from time to time.
227. The pilot studies were done three times. At each time, differences were seen between normal and IR, as well as vehicle and agent treatments. As shown in Fig 22, the CBCT can obtain a complete 3D 650x650x428 scan (i.e. 428 slices) in ~10 seconds with isotropic resolution of 270 μm (central slice shown in Fig 22), encompassing a volume approximately 15 cm3. The scanner achieves a CT density sensitivity of ~5 HU, which is capable of discerning density changes in the mouse lung due to radiation pneumonitis and fibrosis.
228. The imaging data was analyzed using custom MATLAB software. The lungs were segmented automatically (Fig 23 A) and a 3D lung region was obtained for each mouse (Fig 23B). In order to reduce the effect of cardiac motion on the value of mean lung density, the boundary of the lung was excluded from the analysis. For each mouse, a histogram of the voxel intensity (pulmonary tissue density) was created (Fig 24A). The lower density unit (HU) is, the health the lung is. The data from two independent study (separated in 2 years, Table 2) showed that: 1) normal range of CBCT was relatively consistent (mean value -454 to -473); 2) 7-12 months post lung IR (15-18 Gy), the HU increased (mean value -348 to -364); and 3) after mitigation agent treatment, the fibrosis was partially blocked as the density of lung was reduced towards normal range (mean value -447 to -458). It seems that both hEsA and Celebrex that used for 4-6 months could block the lung fibrosis formed in 7-12 months (Table 2 and Fig 24B).
Figure imgf000056_0001
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Claims

What is claimed is:Claims
1. A pharmaceutical composition comprising a compound having Formula I:
Figure imgf000069_0001
wherein,
R1 and R2 are, independent of one another, H, OH, alkyl (e.g., CH3, C2H5, C3H7, CH(CH3)2, C4H9, CH(CH3)C2H5, CH2CH(CH3)2, C(CH3)3, C5Hn), alkoxide (e.g., OCH3, OC2H5, OC3H7, OCH(CH3)2, OC4H9, OCH(CH3)C2H5, OCH2CH(CH3)2, OC(CH3)3, OC5H11), aryl (e.g., C6H5, C6H4OCH3, C6H4OH, C6H4ONH2, CH2C6H5), hydroxyl alkyl (e.g., - CH2OH, -C2H4OH), OSO2CH3, OSO2C6H5CH3, OC5-cyclic ether (e.g., THF), OC6- cyclic ether (e.g., THP), O-linear ether (e.g., MOM, MEM), O-silyl ether (e.g., TBDMS, TMS, TES), ester (e.g., CH3C(O)O (acetate), CH3CH2C(O)O (propionate)), acyl (e.g., CH3C(O), CH3CH2(O), acetal, OLi, ONa, OK, or OCa;
Each R3 is, independent of one another, H, OH, alkoxide (e.g., OCH3, OC2H5, OC3H7,
OCH(CH3)2, OC4H9, OCH(CH3)C2H5, OCH2CH(CH3)2, OC(CH3)3, OC5H11), hydroxyl alkyl (e.g., -CH2OH, C2H4OH), C(O)H, CO2H, CO2Na, CO2K, CO2Li, CO2Ca, CO2R8, Or -CH2OR8;
Each R4 is, independent of one another, H, alkyl (e.g., CH3, C2H5, C3H7, CH(CH3)2, C4H9, CH(CH3)C2H5, CH2CH(CH3),, C(CH3)3, C5H11), or aryl (e.g., C6H5, C6H4OCH3, C6H4OH, C6H4ONH2, CH2C6H5);
R5 and R6 are, independently of one another, H, CH3, OH, CO2H, or CO2R8;
Each R7 is, independent of one another, H or together are an oxo group;
R8 is an alkyl (e.g., CH3, C2H5, C3H7, CH(CH3)2, C4H9, CH(CH3)C2H5, CH2CH(CH3)2, C(CH3)3, C5H11), aryl (e.g., C6H5, C6H4OCH3, C6H4OH, C6H4ONH2, CH2C6H5), Li, Na, K, or Ca; and
The line between atoms a and b, shown as ^111, is a single or double bond; and a pharmaceutically acceptable carrier.
2. The composition of claim 1, wherein R2 does not comprise a saccharide.
3. The composition of claim 1, wherein the compound comprises Formula II:
Figure imgf000070_0001
4. A method of reducing inflammation in a subject comprising administering to the subject an effective amount of the composition of claim 1.
5. A method of inhibiting a cytokine in a subject comprising administering to the subject an effective amount of the composition of claim 1.
6. The method of claim 5, wherein the cytokine is selected from the group consisting of ILl, IL6, TNFα, TNFβ, VEGF, and MCPl or any combination thereof.
7. A method of reducing radiation damage in a subject comprising administering to the subject an effective amount of the composition of claim 1.
8. The method of claim 7, wherein the radiation damage is caused by radiation therapy.
9. The method of claim 8, wherein the radiation therapy is used to treat cancer.
10. The method of claim 7, wherein the radiation damage is caused by nuclear radiation.
11. The method of claim 7, wherein the radiation is caused by a weapon.
12. A method of treating neoplasias in a subject, the method comprising administering to the subject an effective amount of the composition of claim 1.
13. The method of claim 12, wherein the neoplasia is selected from lung cancer, breast cancer, gastrointestinal cancer, bladder cancer, head and neck cancer and cervical cancer.
14. A method of treating a subject with arthritis, the method comprising administering to the subject an effective amount of the composition of claim 1.
15. The method of claim 14, wherein the arthritis is rheumatoid arthritis.
PCT/US2009/045056 2008-05-23 2009-05-22 Methods and compositions related to aglycosidic esculentoside a WO2009143475A1 (en)

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