KR20010020318A - Pharmaceutical compositions of arglabin and arglabin derivatives - Google Patents

Abstract

The present invention discloses a pharmaceutical composition comprising agglombin and agglombin derivatives. The composition may be in unit dosage form and is useful for the treatment of tumors in the human body. The present invention discloses compositions and kits containing a first chemotherapeutic agent and a second chemotherapeutic agent comprising arglabin or a derivative thereof. The compositions and kits are useful for treating tumors in human patients. The present invention also provides various derivatives of agglabin effective for inhibiting tumor growth in mammals.

Description

Pharmaceutical compositions of arglabin and arglabin derivatives}

Cancer is the leading cause of death in the United States and affects people worldwide. Surgical procedures, radiation therapy and chemotherapy are the most widely used treatments. Chemotherapeutic agents create conditions that inhibit the growth and proliferation of cells. DNA synthesis can be inhibited by blocking purine biosynthesis, pyrimidine biosynthesis, conversion of ribonucleotides to deoxyribonucleotides, metabolism inhibition, insertion or crosslinking. For example, RNA synthesis can be inhibited by antimetabolic agents. For example, protein synthesis can be inhibited by agents that deaminize asparagine. In addition, drugs that inhibit the function of microtubules can also be used as chemotherapeutic agents.

In general, chemotherapeutic agents affect both tumor cells and rapidly growing cells of normal tissues, such as bone marrow, hair vesicles and epithelium of the gastrointestinal tract. Side effects associated with chemotherapeutic agents include loss of appetite, nausea, vomiting, diarrhea, inhibition of bone marrow function, and hair loss. There is a need for development of chemotherapy agents that are effective as anticancer agents and have minimal toxicity.

Figure 1 shows a synthesis example of the arglobin derivatives 2 to 9.

Figure 2 shows a synthesis example of the arglabin derivatives 10 to 13.

FIG. 3 shows a synthesis example of the arglobin derivatives 14a-14d, 15a-15d, and 16,

4 depicts the structures of compounds 17-21.

Figure 5 shows the effect of increasing the concentration of dimethylaminoarglobin hydrochloride on the viability of transformed cells.

FIG. 6 shows the effect on the proliferation of transformed cells with increasing dimethylaminoarglobin hydrochloride concentration.

Figure 7 shows the effect of increasing the concentration of dimethylaminoarglobin hydrochloride on the viability of normal cells.

8 is a spectral index plot of naphthol cleavage product. FIG. 8A shows the case where no drug is present and FIG. 8B shows the case where a drug is present.

Detailed description of the preferred embodiment

The present invention provides novel compounds that can inhibit tumor growth in the human body. These compounds can be synthesized using the parent compound, arglabin (Fig. 1), which is separated from Artemisia glabella. Various arglabin derivatives can be prepared using a series of chemical theories. For example, epoxy argazine can be prepared by epoxidation of an olefin double bond triple-substituted with peracetic acid. Dichlorohydrin can be prepared by treating the epoxy arglobin with ether-acetic hydrochloric acid solution. Dibromoarglobin can be prepared by reacting arglobin with Br ₂ and carbon tetrachloride. Arglobin chlorohydrin can be prepared by reacting arglobin with methanol solution of hydrochloric acid. By epoxidation of argvin chlorohydrin with peracetic acid and chloroform, an epoxy argvin chlorohydrin separable by chromatograph can be prepared. Arglobin diols, their isomers and dienes can be prepared by hydrolysis of arglobin. Epiarglabin, a 1, 10 epimer of arglabin, can be prepared by treating arglabin diol with POCl ₃ . Benzylaminoarglobin and benzylaminoepoxyaglavine can be prepared by treating arglobin and epoxy argvin with benzeneamine. Dimethylaminoarglobin and dimethylaminoepoxyarglobin can be prepared by treating argazine and epoxy argazine with dimethylamine. Morpholine-aminoarglobin and morpholine-aminoepoxyaglavine can be prepared by treatment of argolin and epoxyarglobin with morpholine. Pharmaceutically acceptable salts of these compounds can be prepared by standard methods and can be used as anticancer agents. For example, dimethylaminoarglobin hydrochloride and dimethylaminoepoxyaglabin hydrochloride can be prepared by hydrochlorination. Dihydroarglobin can be prepared by treating arglabin with ethanol and H ₂ / Ni. Various arglabin derivatives as described above are shown in FIGS. 1-4.

The present invention also relates to a method for inhibiting tumor growth in a patient diagnosed with a cancer comprising administering to the patient arglabin or a derivative thereof. This method is commonly used for the treatment of cancers such as breast cancer, colon cancer, rectal cancer, gastrointestinal cancer, pancreatic cancer, lung cancer, liver cancer, ovarian cancer, esophageal cancer, leukemia and lymphoma, but certain types of cancer such as lung cancer, liver cancer or ovarian cancer In this case, such therapy is particularly preferred. The compounds may be topically, orally, intravenously, intraperitoneally, intraperitoneally, intratracheally, subcutaneously, intramuscularly, intranasally, inhaled or by suppository, depending on the type of cancer and the various conditions of the patient. May be administered. For example, intraperitoneal administration can be applied to patients with ascites. Intrapleural administration may be applied to speakers with lung cancer. Administration with suppositories can be applied to patients with rectal cancer. Arglabin or a derivative thereof may be administered about 40 mg or about 480 mg daily, preferably about 175 mg to about 315 mg, more preferably about 240 mg to 280 mg. Typical dosages are about 0.5 mg / kg to about 7 mg / kg. In the worst case, up to 20 mg / kg of agglabin or its derivatives may be administered. Once administered, these compounds act as anticancer agents to inhibit tumor growth or to degrade tumors.

Even without binding by special biochemical mechanisms, these compounds can interfere with farnesylation of proteins such as ras protein to stop or inhibit the growth of cancer cells. The ras gene is a protooncogen that plays an important role in various forms of human tumors, including colorectal cancer, exocrine pancreatic cancer and spinal leukemia (Barbacid, 1987, Ann. Rev. Biochem. 56: 779). About 20-30% of all tumors in the human body are due to the activation of the ras proto-oncogene. The ras gene forms a multi-gene family that transforms cells through the action of a 21 kDa protein named ras p21 (also referred to herein as "ras"). ras acts as a G-regulatory protein that hydrolyzes GTP to GDP. If ras is inactive, it is combined with GDP. When the proliferative factor receptor is activated, ras changes GDP to GTP and changes itself. When ras is bound to GTP, wild-type ras connects activated proliferation factor receptor signals to downstream mitogen agonists. Ras' inherent GTP degradation activity returns to inactivation coupled with GDP. In tumor cells, the ras gene is mutated, resulting in loss of regulatory function, resulting in transmission of proliferative factor stimulus signals and activation of oncogenes.

For both normal function and oncogenic function, ras must be placed in the plasma membrane in the process depending on proper post-translational processing of ras (Hancock, 1989, cell 57: 1167). In the first stage of the post-translational process of ras, farnesyl groups react with farnesyl pyrophosphate to attach to cysteine residues at position 186 of the protein. In the second step, the three carboxy-terminal amino acids of the protein are cleaved by the action of specific proteases. In the third step, the carboxylic acid ends are alkylated with methyl groups and converted to methyl esters.

Post-translational modifications of ras are controlled by amino acid sequence motifs often referred to as "CAAX boxes". In this sequence motif, C represents cysteine, A represents aliphatic amino acids, and X represents other amino acids such as methionine, serine or glutamine. This motif, depending on the specific sequence of the CAAX box, acts as a signal sequence for the farnesyl-protein transferase or geranilegeranyl-protein transferase catalyzing alkylation of the cysteine residues of the CAAX sequence. Farnesylation of ras is necessary for the proteolytic process, palmitoylation and strong binding of ras proteins to cell membranes.

In the absence of farnesylation, the oncogene form of ras cannot transform the cells oncogeneally. Farnesyl-protein transferase inhibitors have been shown to inhibit the growth of ras-transformed cells in soft agar. Thus, inhibitors of farnesyl-protein transferase and inhibitors of ras activity are generally considered to be useful as anticancer agents for many forms of cancer (Gibbs et al., 1984, proc. Natl. Acad. Sci. USA 81: 5704-). 5708; Jung et al., 1994, Mol. Cell. Biol. 14: 3707-3718; Predergast et al., 1994, Mol. Cell. Biol. 14: 4193-4202; Vogt et al., 1995, J. Biol Chem. 270: 660-664; and Maron et al., 1995, J. Biol. Chem. 270: 22263-22270).

As described below, arglabin and its derivatives inhibit protein farnesylation.

In another embodiment, there is provided a pharmaceutical composition comprising from about 40 mg to about 480 mg, preferably from about 175 mg to about 315 mg, more preferably from about 240 mg to about 280 mg of agglomazine or a derivative thereof in unit dosage form. . Generally, the dosage of such pharmaceutical compositions is about 0.5 mg / kg to about 7 mg / kg. In the worst case, about 20 mg / kg may be administered. Pharmaceutically acceptable lyophilized salts, such as lyophilized dimethylaminoarglobin and dimethylaminoarglobin hydrochloride, are useful as pharmaceutical compositions. In pharmaceutically acceptable compositions, the optimal concentration of arglabin and its derivatives will vary, including a number of preferred dosages of the compound to be administered, chemical properties of the compound used, formulation through excipients, and route of administration. Depends on the argument The optimal dosage of the pharmaceutical composition to be administered also depends on various factors such as the metastasis form and extent of the tumor, the general health of the particular patient and the relative biological efficacy of the compound administered. Such compositions may be useful for the treatment of tumors, particularly for lung cancer, liver cancer and ovarian cancer, and other tumors such as breast cancer, rectal cancer, colon cancer, gastric cancer, pancreatic cancer or esophageal cancer may be preferred with the composition. In addition, hematopoietic tumors such as leukemias and lymphomas may also be preferred for treatment with the compositions.

The compounds of the present invention can be prepared into pharmaceutical compositions by mixing with pharmaceutically acceptable non-toxic excipients or carriers. Such compounds and compositions are suitable for parenteral administration, in particular aqueous solutions, aqueous suspensions mixed in physiological buffers; For oral administration, in particular tablets or capsules; Or for intranasal administration, in particular powders, nasal drops, aerosols. Compositions for other routes of administration can be prepared as desired using standard methods.

The compounds of the present invention may be conveniently administered in unit dosage form and may be administered to any of the methods well known in the art of pharmacy, such as, for example, the method described in Remington's Pharmacy (Mack Pub. Co., Easton, PA, 1980). Can be prepared by Formulations for parenteral administration may include sterile water or sterile saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalene, and the like as conventional excipients. In particular, biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers or polyoxyethylene-polyoxypropylene copolymers are examples of excipients that control the release of the compounds of the invention in vivo. Other suitable parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation administration can be, for example, aqueous solutions comprising polyoxyethylene-9-lauryl ether, glycocholate and deoxcholate, or they can be oily solutions for administration to nasal dropping agents. If desired, the compounds may be formulated in a gel that can be applied in the nasal cavity. In addition, formulations for parenteral administration may include glycocholate for buccal administration.

The present invention further relates to a packaging material and a product which contains agglomerates or derivatives thereof contained in the packaging material. Arglabin or its derivatives are therapeutically effective in inhibiting tumor growth in the human body. The packaging material includes a label or package insert that indicates whether or not agglomerates or derivatives thereof can be used to inhibit tumor growth. Dimethylaminoarglobin and pharmaceutically acceptable salts thereof are particularly useful arglabin derivatives in the product of production.

In another embodiment, the present invention is directed to compositions and kits comprising a first chemotherapeutic agent and a second chemotherapeutic agent comprising arglabin or a derivative thereof. The second chemotherapeutic agent is not arglabin or a derivative thereof. Such compositions are useful for inhibiting tumor growth in the human body. Dimethylaminoarglobin or a pharmaceutically acceptable salt thereof is a particularly useful arglabin derivative. Alkylating agents, antimetabolic agents, vinca alkaloids, antibiotics or platinum coordination complexes can be used in the compositions of the present invention in various groups. For example, alkylating agents such as nitrogen mustard cyclophosphamide and sarcolysine can be used, and other alkylating agents such as methylnitrosourea are also suitable for use. Anti-metabolites such as vinca alkaloids such as vinblastine or vincristine, as well as folic acid-like methotrexate, or pyrimidine analogs such as fluorouracil or 5-fluorouracil, can be used. Antibiotics, such as rubidomycin, as well as platinum coordination complexes such as cisplatin may be suitable chemotherapeutic agents. Combination chemotherapeutic agents can be combined with agglombin or derivatives thereof. For example, vincristine and cyclophosphamide, or vincristine and vinblastine, may be combined with agglombin or derivatives thereof.

In addition, in a human patient, the present invention provides a tumor by administering to a patient a predetermined amount of a composition comprising a first chemotherapeutic agent and a second chemotherapeutic agent comprising agglomazine or a derivative thereof effective for inhibiting tumor growth in a human body. It relates to a method of inhibiting the growth of. The second chemotherapeutic agent is not arglabin or its derivatives. Such compositions show improved anticancer effects and also inhibit tumor metastasis. In particular, such compositions are useful for treating tumors with drug resistance. Such agents may be administered separately or in combination. Toxicity can be reduced by administering agglomin or derivatives thereof several hours before administration of the chemotherapeutic agent. The composition can be administered by any route.

In addition, in the treatment with a chemotherapeutic agent, the present invention provides a method for inhibiting the immune decay effect of a chemotherapeutic agent by administering to a patient an amount of arglabin or a derivative thereof effective to enhance the patient's immune system. . For example, the immune system can be enhanced by increasing the total number of leukocytes, T-lymphocytes, B-lymphocytes or immunoglobin.

Hereinafter, the present invention will be described in more detail with reference to examples which do not limit the scope of the invention described in the claims.

Summary of the Invention

The present inventors have found that agglombin and various derivatives of agglombin can function as chemotherapeutic agents with fewer side effects than the common side effects of the use of other chemotherapeutic agents.

One aspect of the present invention is to provide a pharmaceutical composition in unit dosage form suitable for the treatment of a human tumor. The composition essentially comprises about 40 mg to about 480 mg of arglabin or a derivative thereof. For example, the unit dosage of the composition may include about 175 mg to about 315 mg or about 240 mg to 280 mg of arglabin or a derivative thereof. Arglabin or its derivatives can be used in the manufacture of a medicament for the treatment of tumors. The composition can be used in the manufacture of a medicament for the treatment of tumors. The composition is effective for the treatment of various tumors including breast cancer, colon cancer, rectal cancer, gastrointestinal cancer, pancreatic cancer, lung cancer, liver cancer, ovarian cancer, esophageal cancer, leukemia and lymphoma. In particular, the composition is useful for the treatment of lung cancer, liver cancer and ovarian cancer.

Dimethylaminoarglobin or a pharmaceutically acceptable salt thereof is a particularly useful arglabin derivative that can be used in the pharmaceutical compositions of the present invention. Dimethylaminoarglobin or pharmaceutically acceptable salts thereof can be lyophilized.

The present invention also provides a composition comprising a first chemotherapeutic agent and a second chemotherapeutic agent comprising arglabin or a derivative thereof. The second chemotherapeutic agent is not arglabin or its derivatives. The composition is effective in inhibiting tumor growth in the human body. Among the agglombin derivatives are methylaminoarglabin or pharmaceutically acceptable salts thereof. For example, the second chemotherapeutic agent is an alkylating agent such as cyclophosphamide, sarcolysine or methylnitrosouurea, an anti-metabolizer such as methotrexate or fluorouracil, a vinca alkaloid such as vinblastine or vincristine, ruby It may be an antibiotic such as domycin or a platinum coordination complex such as cisplatin. Two or more chemotherapeutic agents may be included in the composition together in the arglabin or derivatives thereof. The composition can be used in the manufacture of a medicament for treating a tumor.

The present invention also provides compounds that inhibit tumor growth in mammals. These compounds are selected from the group consisting of compounds represented by the following formula (1), (2), (3), (3), (5) and (6).

Wherein RR ₁ is NHCH ₂ Ph or N (CH ₂ CH ₂ ) ₂ O, RR ₂ is NHCH ₂ Ph, N (CH ₂ CH ₂ ) ₂ O, N (CH ₃ ) ₂ or pharmaceutically acceptable Possible salts thereof, X, are OH or Cl. Such agglombin derivatives include dimethylaminoepoxyaglavine, dibromoarglobine chlorohydrin, 11,13-dihydroarglobine, benzylaminoarglabin, morpholine-aminoarglobin, benzylaminoepoxyar Glassine, morpholine-aminoepoxyarglobin, epoxyarglobinchlorohydrin or pharmaceutically acceptable salts thereof. The compounds of Formulas 1, 2, 3, 4, 5 or 6 may be used in the manufacture of a medicament that inhibits tumor growth in mammals.

Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those disclosed herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of inconsistency with the original meaning, a separate definition is made herein. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other aspects and advantages of the invention will be apparent from the following detailed description and claims.

Example 1 Isolation of Arglabin

Artemisia glabella kar. Et kir., A plant of the genus Artemisia, is a perennial plant that is widespread on dry hills in Kazakhstan. The ground portion of A. glabella, which includes leaves, buds, buds and stems, contains cisquiterpene lactones containing arglabin throughout the growth phase of the plant (Table 1).

Growth stage Plant organs Dry plant (g) Argrabin Extracted (g) Arglabin in dry plants (%) Rosette leaf 1900 6.4 0.34 Buttering Leaf stem 100017001000 6.11.286.0 0.610.080.60

Various solvents were used to extract cisquiterpene lactones from the dry plant material (Table 2). It was found that the highest yield was obtained when chloroform was extracted three times at 45-50 ° C. during the flowering stage of plants.

menstruum Ejected substance (g) Separated lactone (g) Lactone% in dried plants water 1.5 Arglabin argolide dihydrogargolide 0.02trace trace Petrol-diethyl ether 1: 1 3.2 Arglabin argolide dihydrogargolide 0.002 trace race benzene 3.4 Arglabin Trace Diethyl ether 4.8 Arglabin argolide dihydrogargolide 0.110.007 Trace chloroform 6.7 Arglabin argolide dihydrogargolide 0.1500.00750.0006 ethanol 5.1 Arglavin Argolide 0.080.01

Extraction was performed using a counter-flow continuous extractor, a loading device and an extraction device consisting of three containers isolated from the environment. The solvent vessel has a filter, a distiller with an evaporator and a condenser and a buffer capacity. The desiccant container is composed of a dryer, a cyclone, a cooler, a ventilator and a heater. The coolant vessel includes a saltpan with a vent. The extraction device also has a deodorization device with a ventilator, a waste tank and an extract collector.

About 7.7 kg of dry matter of Artemisia glabella was placed in the extraction apparatus and continuously mixed with the solvent as the material moved through the extractor column. The solvent moved away from the dry plant material and gradually the solvent was saturated with the extract material. The saturated solvent was transferred and filtered first to remove plant particulates and then evaporated. The filtered plant particles were reextracted by recycling to the extractor. The vapor produced by evaporation was sent to the condenser. Pure solvent was recovered from the condenser and recycled to the extraction unit. External air was blown from the fan pumped from the salt pan to cool the condenser surface of the condenser with pre-cooled water. By cooling by evaporation of air in the salt pan, the water can be cooled to a temperature significantly below ambient temperature.

The extract purified from the solvent is in tar form. About 7% (539 g) of plant material was recovered during this process.

The tar was dissolved by adding twice the 60 ° C. ethanol (about 1.08 L) with continuous stirring to purify the tar. Distilled water heated to about 70 ° C. was added so that the ratio of alcohol to water was 2: 1. The tar-alcohol-water solution was stirred thoroughly for 30 minutes and then left at room temperature for about 24 hours or until a precipitate formed. The water alcohol solution was filtered through a ceramic filter under vacuum. The procedure was repeated for precipitates remaining after filtration.

The filtrate was rotary evaporated and the alcohol was vacuum distilled in the form of an azeotrope with water containing 68-70% alcohol. After distillation of the alcohol, an aqueous solution containing about 286 g of purified tar was obtained.

Purified tar was separated into individual components through KCK silica gel column under pressure using benzene as eluent. Benzene fractions were collected and analyzed for arglobin using thin layer chromatography (TLC) (silofol, benzene-ethanol, 9: 1). The arglobin-containing fractions were distilled off to remove benzene. Arglabin at this stage is yellow. 33 g of arglobin was obtained in a yield of about 11.7%.

Arglabin was recrystallized by dissolution in hexane and heating to a product to hexane ratio of 1:10 (w / v). The filtrate was obtained by filtration under reduced pressure with a solution containing arglobin. Arglabin crystals were separated from the filtrate at room temperature. At this stage, about 21 g of arglabin was recovered. Arglobin is 1 (R), 10 (S) -epoxy-5 (S), 6 (S), 7 (S) -guaia-3 (4), 11 (13) -diene-6,12 It has an old structure. The conformational structure of arglabin was determined through X-ray analysis.

The linkage of the pentene and heptane rings and the heptane and γ-lactone rings into two crystallographically independent agglomerate molecules is a transoid. The twist angles of O ₃ C ₁ C ₅ H ₅ are -142 (1) and -136 (2) °, and the twist angles of H ₆ C ₆ C ₇ H ₇ are -167 (2) and -159 (3), respectively. °. The pentene ring forms a 1α-envelope (ΔC ¹ _S = 2.9 and 1.5 °) locus and the heptane ring is a 7α, 1, 10β-chain (ΔC ⁷ _S = 2.7 and 4.7 °). The methyl group by C-10 atom has the α-orientation of the equator bond. The formation of the γ-lactone ring is between the 7α-envelope and the 6β, 7α-semichair, but close to the latter (ΔC ¹² ₂ = 2.0 and 6.1 °).

NMR spectra of arglabin were recorded on a Varian HA-100D instrument in CDCl. Chemical shifts are given in δ-scale from the TMC signal that is acknowledged for zero. There are two 3-proton singlets at 1.34 ppm (methyl of epoxide) and 1.94 ppm (methyl of double bond). Single-proton doublets were recorded at 2.95 ppm (protons of carbon) with J = 10 Hz. Single-proton triplets were detected centrally at 3.97 ppm (lactone protons) with J = 10 Hz. Two single-proton doublets were obtained at 5.42 ppm at J = 3 Hz and 6.1 ppm (exomethylene of the lactone cycle) at J = 3 Hz, and the single-proton signal at 5.56 (vinyl proton). The structure of arglobin (FIG. 1) was confirmed based on the NMR spectrum of the isolated compound and the NMR spectrum of cisquiterpene lactone arboresciene and rudartin associated with the compound.

Summary of Arglabin Properties: Colorless, Melting Point of about 100-102 ° C. (hexanes). [a] ²⁰ _D = + 45.6 ° (C 0.3, CHCl ₃ ); IR band (KBr) 1760, 1660, 1150, 1125 cm ⁻¹ ; ¹ H-NMR (400 MHz, CDCl ₃ ) δ1.34 (3H, s, H-14), 1.94 (3H, s, H-15), 2.95 (1H, d, J 10 Hz, H-5), 3.97 (1H, t, J 10 Hz, H-6), 5.56 (1H, br s, H-3), 5.42 (1H, d, J 3 Hz, H-13a), 6.10 (1H, d, J 3 Hz, H- 13b).

Example 2: Arglabin Derivatives

For ease of understanding, the names of various compounds are numbered to easily identify identity with the compounds shown in the figures, as shown below.

Arglabin derivatives were prepared using reagents that affect the epoxide or olefin group of arglabin. Epoxidation of the triple-substituted olefinic double bonds of arglabin 1 with peracetic acid (FIG. 1) showed high yield and 95% stereoselectivity, with 3β, 4β-epoxyarglobin 2 (1 (10), 3 (4) -diepoxy-guai-11 (13) -ene-6,22-old). Epoxy argvin 2 was recovered in about 65% yield using silica gel column chromatography with ethyl ether. IR and NMR spectra were used to confirm the structure of epoxyglybin 2.

Summary of Properties of Epoxy Arglobin 2:

Melting point ca. 149-151 ° C. (Et ₂ O—CH ₂ Cl ₂ ). [a] ²² _D = + 94.0 ° (c 1.7, CHCl ₃ ); IR band (KBr) 1760, 1670 cm ⁻¹ ; ¹ H-NMR (400 MHz, py-d ₅ ) δ 1.30 (3H, s, H-14), 1.68 (3H, s, H-15), 3.31 (1H, s, H-3), 4.11 ( 1H, t, J 10 Hz, H-6), 5.43 (1H, d, J 3 Hz, H-13a), 6.16 (1H, d, J 3 Hz, H-13b).

Dichlorohydrin 3 and 4 were prepared by treatment of epoxyarglobin 2 with ether-acetone hydrochloric acid solution (FIG. 1). Two epoxy groups were opened in 60% yield and 95% regioselectivity was observed. Dichlorohydrin 3 and 4 were diluted with water, washed with NaHCO ₃ and purified by silica gel column chromatography using Et ₂ O-iPr ₂ O in a 1: 1 ratio. The structure was confirmed by IR and NMR spectral data.

Summary of the properties of dichlorohydrin 3 and 4:

3 (60%) melting point 190-191 ° C .; [α] ²⁰ _D -90.1 ° (c 0.34, acetone); IR band (KBr) 3465, 1750, 1670 cm ⁻¹ ; ¹ H-NMR (400 MHz, py-d ₅ ) δ1.61 (3 Hz, s, H-14), 1.62 (3H, s H-15), 4.42 (3H, dd, J10, 7 Hz, H-3) 4.48 (1H, t, J 10 Hz, H-6), 5.52 (1H, d, J 3 Hz, H-13a), 6.04 (1H, d, J 3 Hz, H-13b).

4 (5%) melting point 176-178 ° C. (CH ₂ Cl ₂ —Et ₂ O); [α] ²⁴ _D + 23.25 ° (c 0.43, CHCl ₃ ); IR band (KBr) 3680, 1770, 1670 cm ⁻¹ ; ¹ H-NMR (400 MHz, py-d ₅ ) δ1.41 (3H, s, H-14), 1.57 (3H, s, H-15), 3.39 (1H, d, J 10 Hz, H-5) , 3.42 (1H, s, 10-OH), 4.05 (1H, d, J 5.5 Hz, H-3), 4.40 (1H, s, 4-OH), 4.55 (1H, t, J 10 Hz, H-6 ), 5.54 (1H, d, J 3 Hz, H-13a), 6.21 (1H, d, J 3 Hz, H-13b).

Other derivatives were prepared by the formation of mobile bromohydrin to triple-substituted double bonds by interaction of bromination with N-bromosuccinimide in aqueous acetone and partial bromination of exomethylene groups. 3,4-Dibromoarglabin 5 was prepared by treating arglobin 1 with bromine and carbon tetrachloride at 0 ° C.

Treatment of arglobin 1 with methanol hydrochloric acid solution afforded a chromatographically separated mixture in which the chlorohydrin 6/7 was mixed at a ratio of about 6: 1. At the same time a partial attachment of the hydrochloric acid factor to the exomethylene double bond was observed by Michael type reaction. The dominant regioisomer 6 was epoxidized with peracetic acid in chromoform to produce a mixture of separable epoxide argazine chlorohydrin isomers 8/9 mixed in a 1: 1 ratio. The previously unknown structure of chlorohydrin 6-9 was determined based on elemental and spectral analysis with reference to the X-ray results for epoxide 8.

About 15 ml of acetonitrile and about 550 mg of arglobin 1 with HBF ₄ were refluxed to prepare diol 10, and their isomers 11 and 12, in low yield as main products (FIG. 2). The reaction was neutralized, diluted with water, extracted with chloroform and purified by column chromatography (10, petrol-ethyl ether 2: 1, 11, petrol-ethyl ether 1: 1, 12, petrol-ethyl ether 1: 3). .

Summary of the properties of diol 10, isomer 11 and diene 12:

10, melting point 184-185 ° C. (ethyl ether); [α] ²¹ _D + 72.3 ° (c 0.3, CHCl ₃ ); IR band (KBr) 3440, 1770, 1680 cm ⁻¹ ; ¹ H-NMR (400 MHz, py-d ₅ ) δ 1.30 (3H, s, H-14), 1.92 (3H, br s, H-15), 4.18 (1H, dd, J 10, 1 Hz, H -6), 5.43 (3H, broad s, H-3), 5.38 (1H, d, J 3.5 Hz, H-13a), 6.14 (1H, d, J 3.5 Hz, H-13b).

11, melting point 149-151 ° C. (CH ₂ Cl ₂ -Et ₂ O); [α] ²¹ _D + 108.6 ° (c 0.3, CHCl ₃ ); IR band (KBr) 3460, 1770, 1670 cm ⁻¹ ; ¹ H-NMR (400 MHz, py-d ₅ ) δ1.35 (3 Hz, s, H-14), 1.92 (3 H, br s, H-15), 3.10 (1 H, d, J 10 Hz, H-5 ), 4.39 (1H, t, J 10 Hz, H-6), 5.48 (1H, br s, H-3), 5.44 (1H, d, J 3.5 Hz, H-13a), 6.14 (1H, d, J 3.5 Hz, H-13b).

12, melting point 220-222 ° C. (EtOH); [α] ²² _D + 80.6 ° (c 0.57, CHCl ₃ ); IR band (KBr) 3350, 1770, 1680, 1550 cm ⁻¹ ; ¹ H-NMR (400 MHz, py-d ₅ ) δ 1.47 (3H, s, H-14), 1.92 (3H, br s, H-15), 4.32 (1H, t, J 10.5 Hz, H- 6), 5.24 (1H, br s, H-2), 5.50 (1H, br s, H-3), 5.41 (1H, d, J 3.5 Hz, H-13a), 6.15 (1H, d, J 3.5 Hz, H-13b).

Epiarglabin 13, a 1,10-epimer of arglabin, was synthesized by adding about 0.1 ml of POCl ₃ to a cooled pyridine solution (about 0 ° C.) of diol 10 (FIG. 2). After stirring for 24 hours at about -5 ℃, the reaction was terminated by extraction with ethyl ether. After washing with 5% hydrochloric acid and water, the residue was crystallized from petroleum ethyl ether to give 40 mg of 1,10-epiaglibin 13.

Summary of the characteristics of 1,10-epiarglavin 13:

Melting point 193-194 ° C. (EtOH); [α] ²⁰ _D + 78.4 ° (c 0.43, CHCl ₃ ); IR band (KBr) 1760, 1665, 1650, 1150 cm ⁻¹ ; ¹ H-NMR (400 MHz, py-d ₅ ) δ 1.30 (3H, s, H-14), 1.90 (3H, br s, H-15), 2.66 (1H, m, H-5), 4.18 (1H, dd, J 14.5 12.5 Hz, H-6), 5.43 (1H, m, H-3), 5.38 (1H, m, H-3), 6.14 (1H, d, J 3.5 Hz, H-13b ).

The reaction of benzeneamine, dimethylamine and morpholine with argvin 1 and epoxy argvin 2 in an alcohol medium results in a chemiselective reaction of the Michael reactions to the activated double bonds of these molecules to the corresponding derivatives 14a-d and 15a-d was prepared 56-85% (FIG. 3). The α array of aminomethyl residues was confirmed by spectrum.

Synthesis of Dimethylaminoarglobin 14b: Arglabin 1 was mixed with 0.21 L of alcohol and heated to 40 ° C. to completely dissolve the arglabin. After filtration, a 33% solution of dimethylamine (0.023L) was added dropwise with stirring. The mixture was left at room temperature for 24 hours. The reaction was monitored by TLC in cilofol plates. After completion of the amination reaction the mixture was heated to 52 ° C. and vacuum distilled with alcohol. About 0.63 L of chloroform was added to the residual solution and stirred for 30 minutes. The mixture was poured into a separating funnel and the chloroform collected at the bottom of the funnel. Chloroform extraction on the aqueous layer was repeated two more times. The collected chloroform was dried using magnesium sulfate. The chloroform-magnesium sulfate mixture was stirred for 30 minutes and then filtered under reduced pressure to remove chloroform. About 22 g of dimethylaminoaglabin 14b was prepared.

First, dimethylaminoarglobin 14b was purified to 5 volumes of chloroform (w / v) and then mixed with about 3 volumes of KCK silica gel (w / w). After evaporating the solvent, the dried material was separated by KCK silica gel column chromatography prepared using adduct to adsorbent ratio 1:22. The column was eluted with a mixture of petroleum diether and sulfur ether (1: 1, 1: 2). About 14-17 ml of fractions were collected and monitored by TLC. From this fraction dimethylaminoarglobin 14b was recrystallized from chloroform and ether (1: 1).

Summary of the properties of dimethylaminoarglobin 14b:

Melting point 94.5-95.5 ° C .; [α] ²¹ _D + 47 ° (c 1.7, CHCl ₃ ); Atomic analysis 70.41% C, 8.7% H, 4.28% N (C ₁₂ H ₂₅ O ₃ N); IR band (≥ CHCl max) 3050-3000 (Shoulder), 2940, 2860, 2835, 2780, 2410, 1770 (carbonyl lactone), 1650 (double bond), 1550-1530 (wide band), 1470, 1450, 1385 , 1335, 1180, 1150, 1140, 1125 cm ^-1 (epoxy group); MS (m / z,% strength) M + HCl 291 (5.07, HCl), 247 (0.5), 188 (1.2), 155 (2.19), 105 (1.6), 97 (3.2), 77 (3.5), 70 ( 6.2), 67 (2.9), 58 (100); NMR (200 MHz, CDCl ₃ , δ scale; multiplex, PPM KCCB) 1.90 (3H), 2.27 (6H), 4.00 (1H,) = 9.5), broadened singlet 5.53 (1H), dm 2.66 (2H, d , J4 = J2 = 5.5).

Dimethylaminoarglobin hydrochloride 14d was prepared by dissolving dimethylaminoarglobin 14b in 0.22 L of alcohol and heating to 40 ° C. After filtration under reduced pressure, 0.2 kg of sodium chloride was added and concentrated sulfuric acid was added dropwise to prepare hydrochloric acid gas. The reaction was monitored by TLC. When the reaction was complete, the mixture was heated to 52 ° C. and ethanol was vacuum distilled. About 0.9 L of ethyl acetate was added to the remaining tar with vigorous stirring. A precipitate of about 21 g of dimethylaminoaglabin hydrochloride 14d was obtained.

About 0.1 L of chloroform was added to dissolve 14d of dimethylaminoaglabin hydrochloride and then distilled to remove chloroform. While stirring vigorously, the residual tar and 0.83 L of ethyl acetate were mixed. The mixture was cooled to precipitate the product completely. The resulting precipitate was filtered under reduced pressure to remove all solvents. The final product was vacuum dried to anhydrous and then dissolved in pyrogen-free distilled water in a ratio of 2 g of dry matter to 100 ml of water. About 20 g of 14d of dimethylaminoarglobin hydrochloride were obtained (95% of the amount expected in this step).

Summary of the Properties of Dimethylaminoarglobin Hydrochloride 14d:

Melting point 203-204 ° C. (ethanol-ether); [α] ²¹ _D + 61.53 ° (c 0.52, CHCl ₃ ); IR 33050-3000 (wide band), 2980, 2970 (strong wide band, NH); 2890, 2970, 2360-2300 (wide band), 1775 (carbonyl lactone), 1650 (weak band), 1480, 1450, 1385, 1345, 1185, 1140-1120, 1100, 1065, 1040, 1010 cm ⁻¹ ; MS (m / z,% strength) 291 (3.01, M + HCl), 115 (2.19), 105 (1.5), 97 (3.2), 91 (4.0), 77 (3.5), 70 (16.2), 67 (2.9 ), 58 (100); NMR (200 MHz, CDCl ₃ , δ scale; multiplex, PPM KCCB) c. 1.30 (3 H), c. 1.87 (3 H), c. 2.87 (6H), dm 4.17 (1H, J1 =, J2 = 10 Hz), broadened singlet 5.55 (1H).

11,13 dihydroarglobin 16 was prepared by treatment of agglomerate 1 with ethanol and H / Ni.

Example 3: Lyophilization

An aqueous solution of dimethylaminoarglabin hydrochloride was filtered through a cotton-gauze plug or eight layers of gauze, and a sterile Millipore filter in a sterile glass jar. The solution was vacuum pumped from the jar into the measuring burette and filled into 2 ml vials or ampoules. Filled vials or ampoules were kept at −40 ° C. on a sterilized shelf for 24 hours before drying in a KC-30 lyophilizer or LS-45 lyophilizer. After this softening period the drying process was started. The temperature was kept at -40 ° C for 2 hours and then gradually increased to about 50 ° C (error of about ± 5 ° C). The transition occurred at 50 ° C. over 12-13 hours of drying time. The final temperature did not exceed 60 ° C. Total drying time was 24 hours. After this procedure, the vial containing the dried compound was immediately capped and sealed. The ampoule was sealed. Each vial or ampoule contains about 44 g of preparation.

Sterile unfiltered vials or ampoules were sterilized in an autoclave at 120 ° C. and 1.2 atm for 20 minutes.

Alternatively, the prepared aqueous dimethylaminoaglabin hydrochloride solution was filtered through a cotton-gauze plug or an eight-layer gauze. About 200 ml of the solution was poured into a 500 ml bottle, covered with a cotton-gauze plug and wrapped in oil paper. The filled bottles were sterilized for 20 minutes at 120 ° C. and 1.2 atm in an autoclave. The sterile solution was cooled to room temperature. Using a sterile preparation method, a sterilized 10 ml vial was filled with 2 ml of solution. The vial was then lyophilized as described above. After lyophilization, each vial contains 0.04 g of the compound.

17 g of the compound were prepared, yield 88.2% for this step and yield 0.22% for dry natural products. The lyophilized material is pale yellow and has a bitter taste. The authenticity of the product was confirmed by measuring the melting point and recording the IR spectrum, mass spectrum and NMR spectrum. The quality of the preparation was controlled by diluting 1 mg of preparation with 0.2 ml of water. Adding a drop of saturated vanillin solution in concentrated sulfuric acid turned the mixture purple, which confirmed the presence of terpenes. Lyophilized material can be stored for three years.

Example 4 Isolation of Other Cisquiterpene Lactones

The structures of compounds 17-21 are shown in FIG. 4. Glabelline 17 was also isolated from Artemisia glabella. The yield of the compound from the dried raw material was about 0.016%. The structure of glabella was determined through IR spectrum, NMR spectrum, C13 NMR spectrum, mass spectrum and chemical transfer.

Summary of Characteristics of Glabellin: Melting Point 130-131 ° C. (Petrol-Diether); [α] ²⁰ _D + 90.9 ° (SO, 17, chloroform).

3-keto-edudeme-1 (2), 4 (5), 11 (13) -triene-6,12-oxide (1) 18 were prepared by selective dehydrogenation of α-sathonin, It can be prepared from more than 20 species of the genus plants. The structure was determined by IR spectrum, UV spectrum and NMR spectrum.

Summary of characteristics of 18: Melting point 145-147 ° C. (methanol); [α] ¹⁸ _D -10.4 ° (1, 12, chloroform).

Anobin 19 was extracted from Achilles nobilis L. The 2α, 3α-epoxy-4α, 10α-deoxy-5,7 (H), 6β (H) -gui-11 (13) -ene-6, 12-oxide structures of anovin 19 have an IR spectrum, It was determined by NMR spectrum, mass spectrum and chemical transfer.

Epoxy estapiatone 20 was prepared by isomerization of useful perphen lactones such as estapiatin via isomerization of trifluoride boron with etherate followed by epoxidation with re-chlorobenzoyl acid. 3-keto-10α (14) -epoxy-1, 5,7α (H) 4, 6β (H) -guwai-11 (13) -ene-6, 12-old structure of IR spectrum, NMR spectrum, mass Determined by spectra.

Gaigranin 21 was prepared by extracting the ground portion of Gailllardio grandiflora with chloroform and then separating it by silica gel column chromatography. The structure of Gaigranin 21 was determined by IR spectrum, UV spectrum, NMR spectrum and Cesy spectrum.

Example 5 In Vitro Activity of Arglabin and Derivatives—Cell Viability

Primary cultures of transformed cells and normal cells were incubated with various concentrations of dimethylaminoarglobin hydrochloride to determine the effect on cell viability.

Mouse mast cell tumors (P-185), bone marrow tumor cells (Z-P3xAg8.653 and Pai) and leukemia human cell lines were used. Primary cultures of normal mouse hepatocytes were isolated from the liver of mice using a collagenase. Splenocytes were isolated using a free homogenizer. Bone marrow cells can be obtained by washing the bone marrow. See, eg, Shears, S.B. and Kirk, C.J. (1984), Biochem. J. 219: 375-382.

Cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum, 100 mM L-glutamine and 50 μg / ml of gentamycin at 37 ° C. under carbon dioxide 5%. Cells were seeded in 24-well plates at a density of 50,000 cells per well, grown for approximately 2 days until near confluency, and then transferred to 96-well plates at the same density. Transformed cell lines were incubated for 18 hours with dimethylaminoarglobin hydrochloride at a concentration of 1.5 μg / ml to 100 μg / ml. Cell viability was measured using trypan blue exclusion test. As shown in FIG. 5, the viability was reduced by half at the concentration of 6 μg / ml for X-653 and K-562 cells and 12 μg / ml for P-815 cells. About 25% of X-653 and K-562 cells survived at a concentration of 12 μg / ml, while P-815 cells survived the same proportion of cells at a concentration of about 25 μg / ml.

At high concentrations of dimethylaminoaglabin hydrochloride, all of the transformed cells had further reduced viability.

Proliferation of the transformed cells was measured by incubating for 18 hours with ³ H-labeled thymidine in the medium. After a certain period of time, the number of ³ H-thymidine bound cells was counted and measured for proliferation. The rate of DNA synthesis from thymidine binding can be measured quantitatively, which is generally directly proportional to the rate of cell division. 6 shows that proliferation of X-653 and P-815 cells is effectively blocked at concentrations of 6 μg / ml and 12 μg / ml, respectively.

Primary cultures of normal cells were incubated for 18 hours with dimethylaminoaglabin hydrochloride at a concentration of 10 μg / ml to 2560 μg / ml. Viability was measured by trypan blue exclusion test. FIG. 7 shows that the viability of normal cells decreases as the concentration of dimethylaminoaglabin hydrochloride increases, and that at too high concentrations, normal cells may die as compared to transformed cells. At the concentration of 320 μg / ml, the number of viable splenocytes was reduced by 50% compared to the control sample. At 640 μg / ml and 1280 μg / ml concentrations, 40% and 10% splenocytes were still alive. About 50% and 25% of the hepatocytes were still alive at this same concentration. Myeloid cells were more sensitive to dimethylaminoaglabin hydrochloride. Only 50% of myeloid cells were still alive at the concentration of 160 μg / ml. Increasing the concentration to 320 μg / ml reduced viable cells to about 25%.

Protein prenylation

Mouse myeloma Pai cells were cultured in the presence of 60 μM of dimethylaminoarglabin hydrochloride. Cells were collected by centrifugation at 600 xg for 10 minutes and then washed twice with PBS. Control cells were cultured in the absence of drug. Cells were lysed in hydrolysis buffer (Tris 50 mM, pH 7.4, EDTA 25 mM, Tween 0.05%, NaCl 0.01M) on ice for 30 minutes. The lysates were prepared by uniformly mixing at 4 ° C. for 5 minutes and precipitated by centrifugation at 12,000 × g for 10 minutes. Supernatants were collected. The protein was precipitated with trichloroacetic acid and then washed successively with ethanol and ethyl ether. The binding between isoprenoids and proteins was selectively cleaved with naphthol as disclosed in Epstein, WW et al., (1991) Proc. Natl. Acad. Sci. USA 88: 9668-9670. In general, 5 mg of a mixture of potassium naphthoxide and naphthol 4: 1 was added to about 10 mg of precipitated protein. After 50 μl of dimethylformamide was added, the tube was filled with argon gas, covered with a cap, and heated to 100 ° C. for 8 to 15 hours. The reaction product was extracted with hexane and analyzed by HPLC (Waters System) using a 0.4 x 15 cm reverse phase Nova-Pac C ₁₈ column. The column was eluted with a 20% mixture of water in acetonitrile at a flow rate of 1.0 ml / min. The product of selective cleavage by naphthol was detected at 360 nm (FIG. 8) with full-scale deflection in 0.01 A units. In the case of the control sample (FIG. 8A), the farnesistaine derivative was eluted at 4.5 minutes, and the geranylgeraniylcysteine derivative was eluted at 6 minutes. The molar ratio of geranylgeranyl to farnesestein was 6.

The effect of dimethylaminoarglobin hydrochloride on the prenylation of cells is shown in Figure 8B. When 60 μm of dimethylaminoaglabin hydrochloride was used, the peaks corresponding to the farnesysteine derivatives were not shown in the chromatograph, while the geranylgeranyl peaks appeared as in the control sample. This indicates that dimethyl-iminoarglobin hydrochloride has little effect on the geranylgeranilation of the protein but prevents farnesylation.

Example 6 In Vivo Activity of Arglabin and Its Derivatives

All of the compounds in this group have low toxicity and are resistant at doses above the therapeutic dose. Using a conventional toxicity measurement method, LD ₅₀ was injected by injecting a solution of 2% of dimethylaminoarglobin hydrochloride in dimethyl sulfoxide (DMSO) into mice (20-22 g of worms) and rats (120-130 g of body weight). Measured. LD ₅₀ for mice was 190-220 mg / kg and rats were 280-310 mg / kg. The dissection of the animals revealed hypertension of the internal organs and mesenteric and gastrointestinal vasodilation.

Administration of drug-resistant doses to rats and rabbits did not interfere with liver, kidney, cardiovascular, circulatory or peripheral nervous system function. Blood pressure was also maintained. In addition, no fever, allergic reactions, teratogenicity or fetal toxicity were observed in the animals.

The maximum tolerated dose (MTD) of arglobin or its derivatives was determined by intraperitoneal administration to rats, guinea pigs or mice on a daily basis over 5 to 20 days and intravenously to rabbits. In general, it was determined that the MTD of all compounds tested was about 20 mg / kg to 50 mg / kg. Reversible changes in glycolysis and tissue respiration were observed in serum and liver tissues. Changes in hormonal equilibrium and increased protein levels in urine were intraperitoneally administered daily with prolonged periods of 2% aqueous solution of dimethylaminoarglobin hydrochloride. Appeared later.

Inhibition of Tumor Growth in Rats

Human tumors were transplanted into mice and rats (sarcoma M-1; Fleece's sarcoma; Walker's carcinosarcoma; Geren's carcinoma; Sarcoma 45; Sarcoma 180; Sarcoma 37; Bonsai liver cancer PC-1; Erich's solid adenocarcinoma Breast cancer (PMK); Lymphocytic leukemia P-388; Pseudo-lymphocytic leukemia L-1210; Various lymphomas of fleece that are resistant to rubidomycin, prosperidin and leucoefdine; Sarcolysin, 5-fluorouracil, pro Various sarcomas resistant to speedin and rubidomycin 45). In case of Mice, treatment was started 24 hours after transplantation, and in rats, it was started at the time when observation of tumor nodules was measured. Test animals were grouped from 10-15 for control specimens. Tumor growth inhibition rate was measured after the type of treatment to evaluate the anticancer activity of the compounds. The results were analyzed statistically using t-test. Histologically, inhibition of tumors involves atrophy and necrosis of tumor cells, disturbed blood supply of tumor tissues, and replacement of connective tissues.

Tables 3 and 4 summarize the tumor growth inhibition efficiencies of agglombin and various derivatives for both drug-resistant and drug-resistant tumors. In addition, for comparison, Table 3 includes the tumor growth inhibition rate of colchicine known to have anticancer activity. Introduction of halides such as bromine and chlorine has been shown to enhance anticancer activity. Epoxidation of C3-C4 double bonds of arglabin has also been shown to enhance anticancer activity. Dimethylaminoarglobin and dimethylaminoarglobin hydride are effective against various tumors. Dimethylaminoarglobin hydrochloride has the advantage of being water soluble.

Combination therapy

Animal experiments have devised the most ideal treatment regimen by the combination of dimethylaminoarglobin hydrochloride with other anticancer agents.

Combination treatment with dimethylaminoarglabin hydrochloride, cisplatin and methotrexate completely eliminated tumors in which 60% of the rats were resistant to prosperidin and rubidomycin. This combination treatment also overcomes the cross resistance of sarcoma 45 to methotrexate, the cross resistance of sarcoma 45 to 5-fluorouracil, and the cross resistance of fleece lymphosarcoma to rubidomycin. There was no animal that died for this treatment.

After administration of sarcolysine, 60% completely disappeared from rats by the secondary sensitivity of Leucofin resistant fleece lymphosarcoma. Combination of dimethylaminoarglobin and sarcolysine inhibited DNA synthesis in half of the MTD (synthesis inhibition index 94.1-97.1%). The concentration of blood cells did not decrease for this combination treatment.

Combination treatment of dimethylaminoarglobin hydrochloride with methylnitrosourea followed the administration of two drugs at 2, 4, or 24 hour intervals. Administration of dimethylaminoarglobin hydrochloride 2 hours prior to methylnitrosourea administration proved most appropriate. Cross resistance of sarcoma-45 to prospidine, cross-resistance of sarcoma 45 to 5-fluorouracil, cross-resistance of fleece lymphosarcoma to rubymycin and cross-resistance of fleece lymphosarcoma to prospindine Overcoming treatment with aminoarglobin hydrochloride and methylnitrosourea was overcome. About 60% of tumors disappeared in rats without drug side effects. Administration of methylnitrosourea prior to dimethylaminoarglobin hydrochloride reduces anticancer effects and increases toxicity.

Histologically, little nuclear enriched transformed cells with no apparent structure were observed after the combination treatment of dimethylaminoarglobin hydrochloride and methylnitrosourea compared to the control population. Toxicity has been shown to be reduced when dimethylaminoarglobin hydrochloride is administered 2 hours prior to methylnitrosourea administration.

The same results were obtained for the breeding of guerene and the carcinosarcoma of walker when dimethylaminoarlabin hydrochloride was administered 2 hours before the mixture of vincristine and vinblastine.

In animals with drug-resistant and non-drug-resistant tumors, dimethylaminoarglobin hydrochloride significantly prolonged survival. Combination treatment of dimethylaminoarglobin hydrochloride with other anticancer agents further prolonged survival. For example, the combination treatment of dimethylaminoarglobin hydrochloride and vincristine prolonged survival by 114% for animals with methylnitrosourea resistant tumors. In the case of the combination treatment of dimethylaminoarglobin hydrochloride and cisplatin, the survival time was extended by 117% for animals with methotrexate resistance L1210 at half the MTD. Triple combination of dimethylaminoarglobin hydrochloride and vincristine or dimethylaminoarglobin hydrochloride and cyclophosphamide in combination with dimethylaminoarglobin hydrochloride, vincristine and cyclophosphamide In case of treatment, it showed excellent therapeutic effect. Triple combination treatment extended the survival period by 209%. The quadruple combination treatment of dimethylaminoarglobin hydrochloride, vincristine, cyclophosphamide and cisplatin was less effective than the triple combination treatment. This is because the toxicity of anticancer drugs is increased.

Treatment with dimethylaminoarglobin hydrochloride alone and in combination with other drugs was modeled after drug-resistant metastatic fleece lymphosarcoma. In inguinal lymph nodes, metastasis was the most sensitive among the early and drug resistant lymph nodes. It did not occur within 10% of the test cases. Treatment with dimethylaminoarglobin hydrochloride alone extended 128% survival compared to the control sample. Combination treatment of dimethylaminoarglobin with vincristine inhibited tumor lysis and double tumor growth for 30% of rats. Survival was prolonged 174% without incidence of new metastases in inguinal lymph nodes.

Combination treatment of dimethylaminoarglobin hydrochloride with methotrexate prolonged survival by 300% for animals with propidine resistant fleece lymphosarcoma. This combination treatment reduced the frequency of metastasis by eight times.

In order to elucidate possible mechanisms of dimethylaminoarglobin high chloride on early tumors, drug resistant tumors and their metastases, sarcoma 45 and dimethylaminoarglobin and sarcolysine were treated alone and in combination to investigate DNA synthesis inhibition. . For non-drug resistant sarcoma 45, favorable results were observed in the treatment of sarcolysine alone and in the combination of sarcolysine and dimethylaminoarglobin. For drug resistant sarcoma 45, treatment with dimethylaminoarglobin hydrochloride alone was very effective (DMA inhibition index 94%). In addition, DNA inhibition increased after 24 hours with daily administration over subsequent 5 and 10 days. This has a cumulative anti-cancer effect than repeated administration of dimethylaminoarglobin hydrochloride than one dose of MTD.

Immunity of rats to early tumors and propidine resistant tumors with metastasis was studied after treatment with dimethylaminoarglobin hydrochloride alone and in combination with other cell growth inhibitors. Improvements in immunosuppression have been found after treatment with sarcolysine and cisplatin and after treatment of animal tumors with dimethylaminoaglabin hydrochloride. In the case of the combination treatment of dimethylaminoaglabin hydrochloride and sarcolysine or cisplatin, the immunological index was increased, especially when dimethylaminoaglabin hydrochloride was administered 2 hours before cell growth inhibitor administration. These results indicate that dimethylaminoaglabin attenuates the immunosuppressive effects of cell growth inhibitors and normalizes the immune balance in the body. These data show that, when treated alone and in combination with dimethylaminoaglabin hydrochloride, cytotoxicity is reduced and effects on drug resistant tumors are enhanced.

Immune system changes

The effect of dimethylaminoarglobin hydrochloride on intact mice and immunosuppressed mice was measured. Cyclophosphamide was administered at 200 mg / kg to inhibit the mice's immunity. Injection of cyclophosphamide resulted in severe leukopenia associated with lymphopenia. Cell-mediated immunity was less suppressed while humoral immunity in animals was significantly suppressed. A 2% dimethylaminoaglabin hydrochloride solution was injected 50 mg / kg and 100 mg / kg intraperitoneally into white mongrerel mice. Hemagglutination tests and prolonged form hypersensitivity were measured before and after drug administration. A single dose of 50 mg / kg of dimethylaminoaglabin hydrochloride did not alter the hemagglutination titer or delayed-type hypersensitivity response. In the case of dose of 100 mg / kg, hemagglutination titer was slightly reduced.

Repeated intraperitoneal administration of 10-50 mg / kg of dimethylaminoaglabin hydrochloride over 5-10 days measured the effect of sustained administration. Low doses, such as 10 and 20 mg / kg, for 5 and 10 days increased hemagglutination titers in both 5 and 10 days. High doses such as 30 mg / kg for 10 days decreased the hemagglutination index. For the delayed-type hypersensitivity reaction, 5 days of administration was ineffective, but 10 days of administration increased.

Injecting 10 mg / kg into the intact mice increased the absolute number of lymphocytes and increased the total number of white blood cells. The relative number of neutrophils decreased slightly. There was no overall increase in white blood cell counts when the dose was increased to 20 mg / kg. The function of neutrophils was measured using nitroblue netrazolium assay. When 10 mg / kg was administered, the number of neutrophils was reduced while the activity of neutrophils was not changed. At high doses, 20 mg / kg, the number of nitroblue tetrazolium-positive neutrophils was reduced, indicating a decrease in neutrophil function.

Significant changes in T-lymphocytes, B-lymphocytes, and natural killer cells occurred when intraperitoneal administration of 10 and 20 mg / kg of dimethylaminoaglabin daily intraperitoneally for 10 days. At 10 mg / kg, B-lymphocytes were stable while natural killer cells and T-lymphocytes were increased, resulting in an increase in the total number of leukocytes. The increase in T-lymphocytes was the result of an increase in the number of T-helper subgroups. The number of T-suppressor subgroups did not change. At high doses (20 mg / kg), the number of B-lymphocytes decreased and the number of T-lymphocytes decreased slightly, but the total white blood cell count did not change. Natural killer cells increased. Nitroblue tetrazolium analysis also decreased.

The effect of dimethylaminoarglobin hydrochloride on the whole immune system was dependent on the dose administered. At low doses (10 mg / kg) of dimethylaminoarglobin hydrochloride, T-lymphocytes, B-lymphocytes, and natural killer cell numbers were increased. The increase in T-lymphocytes is due to an increase in the number of subgroup T-helpers in the T-lymphocytes. At high doses (20 mg / kg) of dimethylaminoarglobin hydrochloride, the number of T-lymphocytes and B-lymphocytes decreased, but the number of other cells such as natural killer cells increased.

Injecting 10 mg / kg of dimethylaminoaglabin hydrochloride into immunosuppressed mice for 10 days reduced leukopenia and lymphopenia in the mice. The total white blood cell count of mice that were immunosuppressed by treatment for 10 days was not different from the values obtained from the intact mice. The increase in the number of T-lymphocytes is attributable to an increase in the number of subgroup T-helpers as well as the subgroup T-suppressors. The number of B-lymphocytes did not fully restore to normal values.

Other immunological data were obtained from the "August" line of rats having and weighing 140-160 g fleece lymphosarcoma and no rats. For the four indices: spontaneous rosette formation of erythrocytes, nitroblue tetrazolium analysis, delayed-type hypersensitivity and hemagglutination before, during (5 and 10) and after 5 days of treatment with dimethylaminoarglabin hydrochloride Tested. Approximately 50 mg / kg of a 2% aqueous solution of lyophilized dimethylaminoaglabin hydrochloride was intraperitoneally injected daily for 10 days. The results are summarized in Table 4. Infusion of lyophilized dimethylaminoaglabin hydrochloride in intact rats promoted delayed type hypersensitivity, but all other measured indices decreased. In rats with fleece lymphosarcoma, the immune system was promoted because all measured indices increased. An advantage of lyophilized dimethylaminoaglabin hydrochloride is that it mitigates the immunosuppression of known cell growth inhibitors such as 5-fluorouracil and sarcolysine.

Rats without Fleece lymphosarcoma Rats with Fleece Lympharcoma Indices After treatment After treatment Spontaneous-erythrocyte rosette formation (E-ROK) 64-84% reduction 94.8-162.5% increase Nitroblue Tetrazolium (NBT) 67-91% reduction 79.2-175.4% increase Delayed type hypersensitivity 153-207% reduction 89.9-132.2% increase Red blood cell aggregation (RHG) 43-53% reduction 82.2-142.6% increase

Pharmacokinetics

Thirty of the rat females and males bred were randomized to obtain animal kinetic experimental data for dimethylaminoarglobin hydrochloride. Rats weighed 200-220 g. For analysis of all pharmacokinetic data, gas chromatograph and FARM modeling program were used. The maximum concentration in serum was 30 μg / ml after 1 hour of intravenous administration of 2 mg / kg of dimethylaminoaglabin hydrochloride. Dimethylaminoarglobin hydrochloride quickly spread through the organs from blood to peripheral tissues. The apparent distribution was large, indicating that dimethylaminoaglabin hydrochloride can cross cell membranes and tissue membranes. After 1 hour of administration, the maximum concentration of the drug appeared in the lungs and spleen. Maximum concentrations in lung and spleen were 149.4 and 159 μg / g, respectively. The concentrations in liver and skeletal muscle within 3 hours of administration were 228.6 and 176.4 μg / g, respectively. The drug accumulated in the liver and lasted longer than other tissues. Dimethylaminoarglobin hydrochloride can cross the blood-brain barrier. The concentration in brain tissue was 23.9 μg / g after 1 hour and stabilized at 15.6 μg / g after 24 hours.

Dimethylaminoarglobin hydrochloride was excreted very slowly, with a biological half life of about 46.8 hours in rats and an average retention time of 67 hours. Kidney excretion was slow. The concentration in the kidneys peaked after 3 hours. The maximum concentration in the kidneys was maintained for 24 hours and was 56.6 μg / g. Total clearance was 0.05 ml / min at low drug migration into the blood from peripheral tissues.

Clinical Data of Dimethylaminoarglobin Hydrochloride

The first clinical trial of dimethylaminoaglabin hydrochloride was performed in 51 patients with late stage tumor (III-IV). In the first clinical trial, 20.7% of the patients had lung cancer, 17% liver cancer, 17% gastric cancer, 9.4% rectal cancer, 5.7% ovarian cancer, 5.7% esophageal cancer patients, and the remaining salivary gland cancer, lymphoma, breast cancer or colon Was a cancer patient. 67.9% of the patients were male and 32.1% were female. In general, patients were injected intravenously with an aqueous solution of dimethylaminoaglabin hydrochloride. Patients with ascites were administered intraperitoneally. Patients with pleurisy were administered intrapleurally. Before proceeding with the full-scale experiment, a small dose was administered to the patient to monitor whether there was an allergic reaction. Initially, the dose was administered at 80 mg once daily and gradually increased the maximum concentration. After 30-35 days the dose was increased to 480 mg per day. At this high dose, patients developed nausea and vomiting. The usual daily dose was measured to be about 240-280 mg. Typically over the course of treatment, the total dose was 5-6 g of dimethylaminoaglabin hydrochloride, but a high dose of about 20 g was administered. Immediately after administration of the compound, patients felt a bitter taste that quickly disappeared. Additional treatments have been performed in some patients. The data of the first clinical trial is summarized in Table 6. The patient's condition was indicated as 1 to 3 before treatment, with 1 representing only lying patients, 2 representing significantly limited activity, and 3 representing fully active patients. Treatment results ranged from 0 to 3, where 0 did not improve, 1 improved or improved within a month, 2 significantly improved (25-50% reduction in tumor size), and 3 very improved. Case (tumor size reduced by 50-100%).

Overall, 30% of patients improved significantly. After treatment with dimethylaminoaglabin hydrochloride, 55.6% of liver cancer patients improved significantly. The treatment of dimethylaminoarglobin hydrochloride was particularly good for patients with lung and ovarian cancer, with about 64% of lung cancer patients and about 66% of ovarian cancer patients improving significantly. Dimethylaminoarglobin hydrochloride has little toxicity and does not inhibit blood formation. No adverse reactions were recorded in the gastrointestinal tract or hair follicles during the experiment.

In patients with primary hepatocellular malignancies, liver size decreased by more than 50% in two patients and about 50% in other patients after treatment with lyophilized dimethylaminoarlabin hydrochloride. Patients showed improved mental state and appetite. The pain in the lower right side disappeared.

The immunological status of the patients was assessed using standard methods of rosette-forming and phagocytosis. These indices were measured before, during and after treatment. Analysis of the mean immunological values for this patient population was positive for the treatment. At 3-5 days of treatment, the number of T-lymphocytes decreased from 57% to 40.1%, the number of T-helper lymphocytes decreased from 50% to 37.3%, and neutrophil adhesion increased from 42% to 28.5%. . Undifferentiated lymphocytes increased from 21.5% to 42.2%. The general change in the ratio of T-helper lymphocytes to T-suppressor lymphocytes is due to an increase in T-suppressor lymphocytes. The number of B-lymphocytes and phagocytic activity remained stable. The total number of leukocytes increased to 9.4 x 10 ⁹ / L, and the total number of lymphocytes also increased. All forms of immunoglobin increased.

At 20 days of treatment, all indices returned to normal. Some patients returned to normal at 14 days. Patients analyzed after 30 days of treatment showed a significant increase in the number of T-lymphocytes and adhesion of neutrophils.

The first clinical trial was discontinued because it took three to six months to produce dimethylaminoarglobin. In the second clinical trial, dimethylaminoarglobin hydrochloride was administered to 72 patients with stage 4 cancer (61.1% male, 38.9% female) with cancer at various sites. 25% of the patients were gastric cancer, 16.7% liver cancer, 18.1% lung cancer, 40.2% esophageal cancer, breast cancer, ovarian cancer, pancreatic cancer, brain tumor or lymphoma. Patients in worse condition had metastases to the liver (25%), peritonitis lymph nodes (25%), ascites (22.2%) and exudative pleurisy (11.1%). Some of these patients have already been treated with dimethylaminoaglabin hydrochloride in the first clinical trial. The results of the second clinical trial are summarized in Table 7.

reaction % Of patients A complete improvement - Partial improvement with 50% or more reduction in tumor or metastases 61.1 No tumor progression or stabilization 31.9 Tumor progression 7.0

The use of dimethylaminoarglobin hydrochloride as an anti-tumor cell inhibitor in solid tumors has many advantages. These drugs have no side effects and do not inhibit blood formation. It normalizes the function of the immune system and does not cause allergic reactions. As a cytostatic agent, dimethylaminoarglobin hydrochloride is particularly effective in primary liver cancer and other solid tumors with multiple growth meningitis as complications. 61.1% of patients showed partial improvement of tumor, 31.9% of patients with stabilized tumor progression, and only 7.6% of patients with relapse. 88.9% (64 out of 72) responded to treatment and 11.1% (8 out of 72) did not respond.

The following is a summary of the experimental records of patients who received only dimethylaminoarglobin hydrochloride as a chemotherapeutic agent.

Patient M, age 55 and case number 305, was admitted to the skin of the chest and abdomen with multiple nodules, ulcers in the breast area and sclerosis of the right breast. During my previous hospital stay, I had a radio mastectomy for breast cancer at the Sakhalinsk Cancer Center. After surgery, she received six courses of combined chemotherapy with cyclophosphamide and methotrexate.

A symptomatic treatment was recommended because of the recurrence of the cancer. The abdominal and thoracic skin had multiple metastatic nodules ranging in size from 0.5 to 1 cm prior to treatment. The left chest surface had an ulcer approximately 10 x 12 cm in area. The right breast deformed due to invasive metastasis. Edema was a bit severe.

The blood test before treatment showed the following results: Hb-29, ESR-6 mm / hy, L-3.3, Er-3.8ml, juv.ne-4, seg. ne-78, mon-1. This patient received 5 courses of dimethylaminoarglobin hydrochloride at a total dose of 6.0-7.3 mg. The blood test after chemotherapy showed the following results: Hb-122, ESR-20 mm / h, L-10.9, Er-3.8ml, eos-1, stab ne-3, seg. ne-64, lym-34, mon-2.

During treatment, the ulcers were epithelialized, metastatic nodules disappeared, and invasiveness of the right breast was reduced by 50%. Slightly severe edema disappeared.

A four month old patient with case number N2225 was observed to recover completely from liver cancer. This young patient was admitted to Karaganda Cancer Treatment Center Surgery in an extremely severe condition and was diagnosed with fetal liver cancer. Jaundice was present on the outer skin of the skin. The patient had difficulty breathing. The heart sound was clear and regular. Ps-150 per minute. The tongue was moist and clear. The abdomen was large. The liver is large, protruding from the lower edge of the upper flaring of the ilium, on a smooth surface.

Ultrasonography of the liver (UST) revealed that the liver was large and occupied throughout the abdominal cavity. The structure was asymmetrical because of the lesions of asymmetric structures with hydrophilic rims of 5-6 cm or less, meaning liver cancer.

Pretreatment blood tests showed the following results: Hb-84, ESR-4 mm / h, L-10.9, Er-3.3ml, eos-1, juv, ne-55, stab ne-45, seg. ne-14, lym-30, mon-5.

Paracentesis was performed on the liver while controlled by UST. The exposed nuclei of tumor cells were observed against the background of liver cells with degenerative changes. The patient was diagnosed with fetal liver cancer.

The course of treatment of dimethylaminoaglabin hydrochloride began with an intravenous dose of 120 mg daily. The total dose was 2040 mg during the course of treatment. During the treatment, it improved significantly. The abdomen became symmetrical and smaller in size, with reduced liver size.

According to the UST results, the liver protruded 4 cm below the rib cage along the central clavicle line, and not only its structure but also its contour was asymmetric due to the lesion of the asymmetric structure with a hydrophilic rim and the lesion with a maximum echo diameter of 2.0-2.5 cm. . In conclusion, it was a tumor with lesion change.

Post-treatment blood tests showed the following results: Hb-177, ESR-4 mm / h, er-4.0ml, z-9.8, eos-3, seg. ne-28, lym-53, mon-6. The child was discharged from a satisfactory state. Two weeks after the treatment, the patient showed good resistance.

By early 1997, the child was satisfied. Her mother did not report any signs of relapse. As a result of palpation, the liver was soft and protruded 2 cm below the edge of the rib arch. The child was thought to be cured.

Patient A, age 27, case number 543, was diagnosed with a brain tumor. In neurosurgery, the patient's condition was poor and the possibility of surgery was excluded. The patient was very weak and had a bradykinesia in the form of a severe aikineticorigid syndrom. Exopthalm appeared on both sides. After the first course of dimethylaminoarglobin hydrochloride treatment its condition stabilized and the headache disappeared. The state after the second process was stable. Headaches were gone and meat was restored. In this case it was experimentally confirmed that the compound crosses the blood-brain barrier.

The efficacy of the treatment of dimethylaminoarglobin hydrochloride monochemotherapeutic agent was evaluated according to the 1997 Karnofsky scale (Table 8). No side effects of dimethylaminoaglabin hydrochloride treatment have been reported. The average index of peripheral blood is shown in Table 9.

Karnovsky scale Evaluation of effectiveness,% Description of the effect Number of patients Absolute number % 90 Maintain normal activities, minimize symptoms of illness 30 41.7 80 Difficulty in normal activities, slight symptoms of illness. 10 13.9 70 Can work on their own but cannot work 24 33.3 60 Sometimes you need help but can work on your own. 5 6.9 30 Severely weak. 3 4.2

Experiment. Indices Before treatment 10 ¹² / 1-10 ^1/9 During treatment After treatment Red blood cells 2.3-3.0 2.4-3.0 3.0 leukocyte 4.5-5.5 3.5-4.0 4.0 Lymphocytes 8-15 28-35 15-20 Platelets 2.0 (thsd) 2.3 2.8

The index of the immune system was measured using rosette formation and phagocytosis methods. The subjects were examined in 57 patients who received dimethylaminoaglabin hydrochloride. The 11 indexes of cellular immunity and humoral immunity were measured for each patient to assess immune status. The following indices were measured in 0.05 ml of peripheral blood: absolute and relative amounts of T- and B-lymphocytes, the amount of undifferentiated "zero" cells, neutrophil adhesion and phagocytic activity, hemograms, serum immunoglobin concentrations. . The indices were measured before treatment, on days 2, 5 and 14 of treatment and after treatment. The measurement results before and after the treatment are summarized in Table 10.

On days 2-5 of treatment, the percentage of T- and T-helper lymphocytes decreased significantly. The concentration of undifferentiated "zero" cells increased. This population of undifferentiated cells consisted of mature and immature B-lymphocytes, mature and immature T-lymphocytes, and neutral killer cells. No significant change was recorded in the hemogram.

Beginning on Days 6-10, almost all the indexes returned to their initial values. At 2 weeks of treatment, the ratio of T-lymphocytes and T-helper populations was increased, but the number of B-lymphocytes was decreased. At this time, there was no change in serum immunoglobin concentration.

After treatment, there was a statistically significant increase in the percentage of T-lymphocyte content. The absolute number of T-lymphocytes was increased as well as an increase in the number of T-helper lymphocytes and an improvement in phagocytosis of neutrophils. The number of B-lymphocytes increased and the amount of immunoglobin A, M and G also increased. The number of undifferentiated cells decreased.

The total number of lymphocytes in peripheral blood was elevated. When these parameters are examined after dimethylaminoarglobin hydrochloride treatment, both quantitative and qualitative changes in tumors can occur in blood cells. Quantitative changes, such as a decrease in the number of neutrophils and an increase in the number of lymphocytes, were observed, but no change in the qualitative (morphological) composition of blood cells was observed. This means reducing the lymphotoxic effect by the tumor. Immunological indices correlate with clinical trial results in most cases.

Immunity Index of Patients Before and After Arglabin Treatment Indices Before treatment (range) After Treatment (Range) T-lymphocytes,% 48.00 ± 1.74 (28-72) 52.00 ± 1.42 (36-72) T-lymphocytes, absolute 0.72 ± 0.08 (0.13-1.67) 1.14 ± 0.17 (0.18-2.99) B-lymphocytes,% 19.12 ± 1.02 (8-12) 18.50 ± 10.0 (4-32) B-lymphocytes, absolute 0.29 ± 0.05 (0.09-0.73) 0.45 ± 0.07 (0.05-1.16) T-helper cells,% 39.12 ± 1.25 (24-52) 45.30 ± 1.26 (28-60) T-suppressor,% 10.48 ± 1.60 (00-60) 10.50 ± 0.95 (00-24) Undifferentiated lymphocytes,% 32.64 ± 1.74 (8-52) 26.00 ± 2.21 (4-60) D-phagocytes,% 41.36 ± 0.95 (28-52) 48.1 ± 0.93 (26-85) D-phagocytic, absolute number 1.69 ± 0.26 (0.64-7.45) 2.32 ± 0.19 (0.66-5.69) Adherent cells, N / Ph 39.68 ± 1.74 (24-68) 42.40 ± 1.72 (24-66) Immunoglobin A, g / l 1.48 ± 0.02 (1.06-2.0) 1.97 ± 0.08 (0.90-2.84) G 14.33 ± 0.43 (11.0-22.0) 17.91 ± 0.41 (11.6-22.0) M 1.36 ± 0.03 (1.18-1.84) 1.53 ± 0.03 (1.20-1.84) White blood cells, 10 ^9/1 5.97 ± 0.60 (3.40-18.6) 9.14 ± 0.61 (3.40-18.9) Neutrophils,% 4.72 ± 0.91 (0.0-23.0) 5.45 ± 0.79 (0-20) Segmentation nuclei 61.68 ± 45.0 (23-85) 63.05 ± 1.79 (38-88) Eosinophil 2.72 ± 0.71 (0-18) 3.35 ± 1.30 (0-33) Monocytes 5.28 ± 2.48 (0-12) 4.45 ± 0.44 (1-12) Lymphocytes 25.52 ± 2.02 (6-57) 23.15 ± 1.7 (4.0-47)

Mean values of immunological indices were evaluated by regression analysis. The functional state of the immune system was assessed using integration indices such as mean activity (correlation) expressed in relative units. Increased exponential-activity of the immune system during treatment means that the immune system responds to treatment.

The correlation between these data shows that the total number of true binding parameters increased during treatment (the number of bonds with r> 0.7 increased and the number of negative bonds decreased).

The number of correlations between immune elements, ie lymphocytes and neutrophil elements, has increased.

Therefore, statistical data analyzed by various statistical analytical methods indicate that dimethylaminoarglabin hydrochloride is effective as an agent to stimulate some immune factors and improve the functional state of the immune system. This represents the immunostimulatory effect of these agents.

Another embodiment

The present invention has been described with their detailed description, but the foregoing description is intended to illustrate the invention, not to limit the scope of the invention, and the scope of the invention is defined by the appended claims. It is obvious. Other aspects, advantages and modifications of the invention are within the scope of the following claims.

Claims (29)

A pharmaceutical composition in unit dosage form suitable for treating a tumor of the human body, characterized in that it comprises from about 40 mg to about 480 mg of arglabin or a derivative thereof. The pharmaceutical composition of claim 1, wherein the tumor is selected from breast cancer, colon cancer, rectal cancer, gastric cancer, pancreatic cancer, lung cancer, liver cancer, ovarian cancer, leukemia, lymphoma and esophageal cancer. The pharmaceutical composition of claim 2, wherein the tumor is selected from lung cancer, liver cancer and ovarian cancer. 2. A pharmaceutical composition according to claim 1, wherein said derivative is dimethylaminoarglobin or a pharmaceutically acceptable salt thereof. 5. The pharmaceutical composition according to claim 4, wherein the dimethylaminoarglobin or a pharmaceutically acceptable salt thereof is lyophilized. 2. The pharmaceutical composition of claim 1, wherein the unit dosage form comprises about 175 mg to 315 mg of arglabin or a derivative thereof. 7. A pharmaceutical composition according to claim 6, wherein said unit dosage form comprises about 240 mg to 280 mg of agglombin or a derivative thereof. Use of the pharmaceutical composition of claim 1 in the manufacture of a medicament for the treatment of tumors. Use of arglabin or its derivatives in the manufacture of a medicament for the treatment of tumors. An effective composition for inhibiting tumor growth in a human body, the composition comprising a first chemotherapeutic agent and a second chemotherapeutic agent comprising arglabin or a derivative thereof. 11. The composition of claim 10, wherein said second chemotherapeutic agent is selected from the group consisting of alkylating agents, antimetabolic agents, vinca alkaloids, antibiotics and platinum coordination complexes. 12. The composition of claim 11, wherein said alkylating agent is cyclophosphamide, sarcolysine or methylnitrosouurea. 12. The composition of claim 11, wherein said antimetabolic agent is methotrexate or fluorouracil. 12. The composition of claim 11, wherein the vinca alkaloid is vinblastine or vincristine. 15. The composition of claim 14, wherein said chemotherapeutic agent further comprises cyclophosphamide. 12. The composition of claim 11, wherein the antibiotic is rubidomycin. 12. The composition of claim 11, wherein said platinum coordination complex is cisplatin. 11. A composition according to claim 10, wherein said derivative is dimethylaminoarglobin or a pharmaceutically acceptable salt thereof. Use of the composition of claim 10 in the manufacture of a medicament for the treatment of tumors. A compound which inhibits tumor growth in a mammal and is selected from the group consisting of the following Chemical Formulas 1, 2, 3, 4, 5 and 6. <Formula 1> <Formula 2> <Formula 3> <Formula 4> <Formula 5> <Formula 6> Wherein RR ₁ is NHCH ₂ Ph or N (CH ₂ CH ₂ ) ₂ O, RR ₂ is NHCH ₂ Ph, N (CH ₂ CH ₂ ) ₂ O, N (CH ₃ ) ₂ or pharmaceutically Acceptable salts thereof; X is OH or Cl. 21. A compound according to claim 20, wherein said compound is dimethylaminoepoxyagrabine or a pharmaceutically acceptable salt thereof. 21. A compound according to claim 20, wherein said compound is dibromoarglabin. 21. A compound according to claim 20, wherein said compound is arglabin chlorohydrin. 21. A compound according to claim 20, wherein said compound is 11,13-dihydroarglobin. 21. A compound according to claim 20, wherein said compound is benzylaminoaglabin. 21. A compound according to claim 20, wherein said compound is morpholine-aminoarglobin. 21. A compound according to claim 20, wherein said compound is benzylaminoepoxyagrabine. 21. A compound according to claim 20, wherein said compound is morpholine-aminoepoxyagrabine. 21. A compound according to claim 20, wherein said compound is epoxy argvin chlorohydrin.