WO2002036133A1

WO2002036133A1 - New use

Info

Publication number: WO2002036133A1
Application number: PCT/SE2001/002363
Authority: WO
Inventors: Peter Wiklund; Eddie Weitzberg; Jon Lundberg
Original assignee: Peter Wiklund; Eddie Weitzberg; Jon Lundberg
Priority date: 2000-11-03
Filing date: 2001-10-29
Publication date: 2002-05-10
Also published as: AU2002211170A1; SE0004026D0

Abstract

The present invention relates to use of a nitrite donor in the preparation of a medicament for the treatment, profylaxis or management of a disorder involving neoplastic cells and also a method for treating a human or non-human animal with a disorder involving neoplastic cells, comprising the step of administering a composition comprising a nitrite donor.

Description

New use

Background to the invention

Nitric oxide (NO) is an important biological mediator and cell signalling molecule in the vascular, nervous and immune system and is known to have bacteriostatic, antiviral and anti-tumour properties. NO, in addition to its major importance in blood flow regulation

(Furchgott & Zawadski, 1980, Nature, 288, p 373-376 and Moncada et al., 1991, Pharmacol. Rev., 43, p 109-142) is known to take part in host-defence reactions (Nathan & Hibbs, 1991, Curr. Op. Immunol., 3, p 65-70) and is involved in nerve transmission (Gillespie et al., 1989, Br. J. Pharmacol., 98, p 1080-1082). In the cardiovascular system, large amounts of NO are produced from the endothelium in response to endothelium-dependent vasoactive substances (e.g. acetylcholine, substance P) or to shear stress (Moncada et al., 1991, supra) . It has also been suggested that the prophylactic effects of urinary acidification against lower urinary tract infection may be medicated via induction of non-enzymatic NO formation from acidified nitrite (Lundberg et al, 1997, Urology, p 189-191). Nitrites have long been used as NO-donors, which are known to act vasodilating and sodium nitrite was used as a NO-donor in studies of EDRF/NO where it was shown that acidified nitrite (~pH2) evoked larger vasorelaxation than neutral nitrite (Furchgott et al., 1987, Fed. Proc, 46, p 385) which is believed to be due to the production of the vasoactive molecule NO. Nitrite has also been regarded as carcinogenic by the evolution of N-Nitroso compounds, (NOC)² , formed upon nitrite exposure (Bartsch, H et al " Inhibitors of endogenous nitrosation:

Mechanisms and implications in human cancer prevention". Mutat. Res., 202, 307-324). Surprisingly, e have now showed that a nitrite donor may be used against cancers as set forth below.

Increasing evidence has shown that NO is involved in many aspects of tumour biology and tumour cell differentiation (Maeda & Akaike, 1998, Biochemistry (Mosc), 63, p 854-*).

NO has a dual action in the growth of urinary bladder cancer cells (Morcos et al, 1999, Urology, 53, p 1252-*). Low concentrations of NO produced by calcium-dependent NOS activity have a role in the growth of T24 and MBT-2 bladder cancer cell lines whereas enzymatic formation of NO by the INOS isoform has been suggested to exert cytotoxic effects on T24 and MBT-2 bladder cancer cells lines (Morcos et al., 1999, supra). Exogenous administration of NO donors in high concentrations inhibits the growth of bladder cell lines

(Jansson et al., 1998, Br. J. Cancer, 78, p 588-*). However there are no reports on the effect of non-enzymatic NO formation from urinary nitrite on the growth of bladder cancer cells.

In all of the above described research in the instances in which non-enzymatic NO is formed, this has been achieved in an acidified environment. Weitzberg Lundberg (1998, Nitric Oxide: Biology and Chemistry, 2(1), p 1-7) provide a review of the synthesis of NO non- enzymatically. In vivo NO results from the ingestion of nitrates or nitrites in dietary sources.

Nitrate is partly reduced to nitrite by nitrate reductase expressed in bacteria in the oral cavity.

Nitrite is also excreted in saliva. Nitrite is present throughout the body in fluids and tissues in concentrations in the μM range. In the acidic stomach nitrite is reduced to NO. NO has been observed in various parts of the body through acidification of nitrite, namely in urine which had been acidified (optionally with the addition of nitrite), in the stomach, in the oral cavity, in the heart after ischemia-reperfusion injury and on the slightly acidic surface of the skin.

In all cases acidification (even in the presence of reducing agents) was required for the conversion of nitrite to NO. For example, Weitzberg & Lundberg (1998, supra) note in connection with NO production in ischemia or hypoxia that a threshold of pH 6 is required, above which NO production is detected.

Further, through WO 93/20806 treatment of neoplastic cells with a NO-releasing agent is disclosed. Regarding US 6,103,275 there is described treatment of wounds wherein a nitrite salt and a reducing agent (e.g. ascorbic acid) and an acid with pKa between 1 and 4 is combined on the skin. Cancer is also mentioned as a possible desease to be treated as well. In Jay L. Zweier et al (Biochemica et Biophysica Acta (BBA) energetics, volume 1411, No. 2-3, May 1999, page 250-262, there is discussed that nitrite is a NO-source and that the NO-release is higher the lower pH-value. In US 6,110,453, US 5,691,423 and WO 96/15797, respectively, there are further disclosed nitric oxide releasing compositions comprising a N₂0₂ ^"-functional group associated with a polymer.

However there are no reports on the effect of nitrite or a nitrite donor on the growth of cancer cells, whereby there is a delivery of nitrite specifically at an acidic pH-range.. A problem, among others, when treating cancer disorders is to get a targeted action of the medicament that is administrated, i.e. enable bringing the curative activity specific to a target with neoplastic cells. However, it has now surprisingly been found that a nitrite donor may be used for targeted treatment, profylaxis or management of disorders involving neoplastic cells whereby nitrite is converted non-enzymatically to NO and reactive nitrogen intermediates (RNIs) at the locus for neoplastic cells. This conversion is preferably performed in an acidic environment.

Tumours are known to generally exhibit a mildly acidic environment, thus enabling a targeted action of the nitrite donor. By the appropriate administration of a nitrite donor, NO and other reactive nitrogen intermediates (RNTs) may be allowed to be produced non-enzymatically at that site with neoplastic cells. In view of the cytotoxic and cytostatic properties of NO and the reactive nitrogen intermediates (RNIs), anti-tuniour effects may be achieved.

As mentioned above, nitrite is converted non-enzymatically to NO and other reactive nitrogen intermediates (RNIs) at the locus for neoplastic cells, which in turn generally has a lowered pH. The process, which may preferably take place in an acidic environment, may be summarized as follows:

NO₂ ^' + H⁺ ^ HNO₂ 2HNO₂ - zr^ N₂O₃ + H₂O

N₂O₃ =-=^ NO +NO₂

Summary of the invention The present invention solves the above problems by providing use of a nitrite donor in the preparation of a medicament for the treatment, profylaxis or management of a disorder involving neoplastic cells. Further alternatively stated the present invention provides a method for treating a human or non-human animal with a disorder involving neoplastic cells, comprising the step of administering a composition comprising a nitrite donor. Detailed description of invention

The expression "nitrite donor " is meant to include in the present description a compound or complexes which when present in the recipient (subject to the mode of administration) may release nitrite ions into said recipient (at any location within said recipient, but preferably at or near the site of the disorder. The compound may also be a nitrite such as sodium nitrite.

The expression "treatment" is meant to include in the present description reducing, alleviating or eliminating one or more symptoms of the condition which is being treated, relative to the symptoms prior to treatment. The expression "disorder involving neoplastic cells " is meant to include in the present description a disorder which is typified by the presence of a tumour. Appropriately, tumours which may be treated include malignant and pre-malignant or benign tumours and include carcinomas, sarcomas, glioma, melanoma and Hodgkins disease, including cancers of the stomach, bladder, kidney, pancreas, brain, head and neck, breast, gut,prostate, lung and ovary and lymphomas, preferably bladder cancer. Preferably the tumour is solid. Appropriate treatment of tumours according to the invention results in decreased or maintained tumour size (e.g. by halting proliferation, causing differentiation, enhancing or inducing anti-tumour immune responses or causing some cell death). The disorder is normally occuring in an acidic environment. Neoplastic cells involved in the above indications generally are surrounded by an acidic, often mildly acidic, environment. This acidic condition is mainly due to a low blood circulation within the area for neoplastic cells. The pH in the area comprising neoplastic cells thus is below 7 (pH from 0.01 to 7.0), preferably the pH is from 4 to 7.

The expression "reducing agents" is meant to include in the present description an agent which may be included in compositions of the invention as an aid. Appropriate reducing agents include ascorbyl pahnitate, vitamin C, vitamin E, manganese, selenium, beta-carothene, pro- anthocyanidins, polyphenols, urat, co-enzyme Q-10, thioles and bioflavonoids. The reducing agent may preferably give a reducing environment, most preferred a strong reducing environment, when administrated together with a nitrite donor.

The expression "pharmaceutically acceptable" is meant to include in the present description ingredients that are compatible with other ingredients of the compositions as well as physiologically acceptable to the recipient. Compositions for use according to the present invention comprise a nitrite donor together with a pharmaceutically acceptable diluent, carrier or excipient.

The expressions " reactive nitrogen intermediates" and "RNIs" are meant to include in the present description peroxynitrite, and nitrous acid (HN0₂), nitrosothiols, nitrosonium (NO⁺) and other nitrogen oxides than NO. The RNIs of which some may evolve from nitrous acid (HN0₂), may need an additional compound for their formation, such as peroxynitrite which needs the presence of a superoxide.

According to one preferred embodiment of the present invention the nitrite donor is releasing nitrite only at an acidic pH. According to one preferred embodiment of the present invention at least one reducing agent is incorporated into the medicament. Preferably the reducing agent is ascorbic acid which also is pharmaceutically suitable. This compound has several advantages, for example it is freely soluble in water and is therefore readily distributed in body fluids, excess amounts are excreted in urine, it is non-toxic and its physiological effect and behaviour are well documented. However, in a preferred embodiment of the invention, the compositions contain only one nitrite donor or a combination of nitrite donors.

As will be appreciated, when using nitrite donor compounds the toxicity of these compounds must be taken into account. Administration of too much nitrite donor may render into methaemoglobinaemia. Non-human animals which may be treated preferably include mammals, particularly livestock and domestic animals such as dogs, cats, rabbits, guinea pigs, hamsters, mice, rats, horses, goats, sheep, pigs and cows.

Depending on the mode of administration, various forms of the compositions may be used. Thus, pharmaceutical compositions may be formulated in conventional manner using readily available ingredients. The active ingredients (i.e. the nitrite donor), may be incorporated, optionally together with other active substances, with one or more conventional carriers, diluents and/or excipients, to produce conventional galenic preparations such as tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatine capsules, suppositories, sterile injectable solutions, sterile packaged powders, and the like. Compositions may additionally as set out above comprise molecules which aid the conversion of nitrite to NO and RNIs, such as reducing agents. In addition, compositions may contain other molecules which assist or augment the action of the NO and the RNIs, e.g. in methods of treating tumours other anti-tumour agents may be used. Examples of suitable carriers, excipients, and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatine, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, water, water/ethanol, water/glycol, water/polyethylene glycol, propylene glycol, methyl cellulose, methylhydroxybenzoates, propyl hydroxybenzoates, talc, magnesium stearate, mineral oil or fatty substances such as hard fat or suitable mixtures thereof. The compositions may additionally include lubricating agents, wetting agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavouring agents, and the like. The compositions of the intention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art. Compositions may be in an appropriate dosage form, for example as an emulsion or in liposomes, niosomes, microspheres, nanoparticles or the like. If the target disorder is not present in the stomach, the composition comprising a nitrite donor according to the present invention and the composition is taken orally, said composition is enterically coated for passage through the stomach; enteric coatings as such are well known in the art. Examples of enteric coating polymers are cellulose acetate phtalate, hydroxy propyl methyl cellulose phtalate, polyvinyl acetate phtalate carboxy methyl ethyl cellulose, co-polymerized methacrylic acid and methacrylic acid/methacrylic acid methyl esters such as compounds known under the trade name Eudragit (Rohm Pharma).

If required, the compositions may also contain targeting moieties attached to the active ingredient, e.g. a ligand which binds specifically and selectively to an endogenous receptor to allow targeting to a particular cell type or location, such as targeting to certain specific tumour cells. Thus an even more targeted action may be accomplished.

Administration may be performed by local or systemic application as appropriate. Administration of compositions for use in the invention may take place by, any of the conventional routes, e.g. by inhalation, orally, rectally or parenterally, such as by intramuscular, subcutaneous, intraperitoneal or intravenous injection, although this depends on the condition to be treated. Local administration may be performed, e.g. at a tumour site e.g. by use of a catheter or syringe. Treatment by topical application of a composition, e.g. an ointment, to the skin is also possible for appropriate conditions, e.g. actinic keratosis. optionally administration may be performed at intervals, e.g. 2 or more applications, e.g. 2-4 applications at hourly, daily, weekly or monthly intervals, e.g. several times a day, or every 3-5 days, or at fortnightly, monthly or quarterly intervals.

5 The active ingredient in compositions used in the intention may comprise from about

0.01 % to about 99% by weight of the formulation, preferably from about 0.1 to about 50%, for example 10%. The compositions are preferably formulated in a unit dosage form, e.g. with each dosage containing from about 0.01 mg to about 1 g of the active ingredient, e.g. 0.05 mg to

0.5 g, for a human, e.g. 1 - 100 mg. The precise dosage of the active compound to be

L 0 administered and the length of the course of treatment will, of course, depend on a number of factors including for example, the age and weight of the patient, the specific condition requiring treatment and its severity, and the route of administration. Generally however, an effective dose may lie in the range of from about 0.1 μg/kg to about 10 mg/kg, preferably 0.01 to 1 mg/kg, e.g. from about 1 mg to 1 g of nitrite donor per day, depending on the animal to be treated and

L5 the dosage form, taken as a single dose. Thus for example, an appropriate daily dose for an adult may be from 1 mg to 1 g per day, e.g. 2.5 to 500 mg of the nitrite donor per day.

We will now describe the present invention by using Figures and one Example but they are only for purposes of illustration and shall not in any way limit the scope of the appended set of claims.

2 0

Figures

Figure 1 shows gaseous NO formation from nitrite in acidic urine. Low concentrations of NO were formed from urine at all pH values tested. Addition of nitrite resulted in release of NO in large concentrations in acidic urine (p <0.05). NO formation, after ascorbate addition in

25 the presence of nitrite, was further increased (p <0.05);

Figure 2 shows the effect of urinary nitrite on the growth of T24 cells. Incubation of cells at lowering urine pH (4.5-6.5) resulted in inhibition of [³H] thymidine incorporation Nitrite addition resulted in significant reduction of [³H] thymidine incorporation (p <0.05). The addition of ascorbate had an augmentary effect on the nitrite ,induced reduction of [³H ]

30 thymidine incorporation (p <0.05);

Figure 3 shows the effect of urinary nitrite on the growth of MBT-2 cells. Incubation of cells at lowering urine pH (4.5-6.5) resulted in inhibition of [³H ] thymidine incorporation

Nitrite addition resulted in significant reduction of [³H ] thymidine incorporation (p <0.05). The addition of ascorbate had an augmentary effect on the nitrite, induced reduction of [³H ] thymidine incorporation (p <0.05); and Figure 4 shows the effect of ascorbate addition to acidic urine on the growth of T24 cells.

EXAMPLE 1: INHIBITION OF BLADDER CANCER CELLS IN NITRITE SUPPLEMENTED. ACIDIFIED URINE

INTRODUCTION

Increasing evidence has shown that NO is involved in many aspects of tumour biology and tumour cell differentiation (as mentioned hereinbefore). However, there are no reports on . the effect of non-enzymatic NO and RNI formation from urinary nitrite on the growth of bladder cancer cells, fn this study, we investigated effects of nitrite in acidified urine on the growth of bladder cancer cells in vitro.

MATERIALS AND METHODS

Measurement of NO (gaseous phase) formation from nitrite in urine.

NO formation from nitrite in acidic urine was measured by a NO chemiluminescence analyzer as previously described (Lundberg et al., 1997, supra). In brief, 10 ml urine with or without nitrite and/or ascorbate was incubated in 60 ml closed plastic syringe with a 50 ml headspace at 37°C for 30 minutes. The head space air was withdrawn and immediately injected onto a NO chemiluminescence analyzer. In this measurement NO acts as an indication of the acidic conversion of nitrite, thus NO is an indirect indicator of RNIs. Urinary nitrite and nitrate measurement by capillary electrophoresis.

Fresh midstream urine samples were collected from volunteers. Nitrite and nitrate were measured by capillary electrophoresis as previously described (Leone et al., 1994, Biochem. Biophys. Res. Commun., 200, p 951-957). All plastics and tubes were rinsed with de-ionized water (18.2 MΩ) three times. Ultrafree-MC centrifuge filters (5000 nominal molecular weight limit) were rinsed with de-ionized water and centrifuged at 5000 g for 30 minutes.

Samples were diluted in de-ionized water and centrifuged in pre-washed filters at 5000 g for 30 minutes. After centrifugation, samples were kept at O°C and analysed immediately.

A Hewlett Packard (HP 3D CE) system with UN-detection at 214 nm was used for detection of nitrite and nitrate. The background electrolyte consisted of 25 mM Νa₂SO₄ and 5% CIA-Pak ™ OFM Anion (osmotic flow modifier) dissolved in deionized water. Samples were injected by a negative voltage of -6 kN for 20 seconds onto extended light path capillaries (104 cm effective length and 75 μm ID). Analysis was performed at an applied negative voltage of 300 N/cm. Data were analysed by a Hewlett Packard (HP3D) data system as peak areas.

Cell Culture. T24 human bladder cancer cells and MBT-2 mouse bladder cancer cells were cultured in RPMI-1640 cell culture medium supplemented with 10% fetal calf serum, 10 mM Ν-2-hydroxyethylpiperazine-Ν'-2-ethane sulphonic acid (HEPES), 2 mM L-glutamine and benzylpenicillin/streptomycin (100 IU/ml and 100 mg/ml respectively). Cells were grown in 5% C0₂ at 37°C and medium was replaced every second day. At confluence cells were detached by trypsinization. T24 cells were from the American type culture collection (ATCC ' number: HTB-4) and were mycoplasma free. To study the effect of urinary nitrite on the growth of T24 and MBT-2 cell lines, cells were plated in 48 well culture plates at a density of 10,000 cells/ml and allowed to grow for 24 hours. Then, the medium was discarded and T24 cells were incubated twice at 37°C for 15 minutes with two hours interval in between each incubation in 500 μl of urine. Fresh midstream urine was collected from volunteers, pH was adjusted by HC1 and NaOH (10 M) over the range of 4.5-7 and sterilized by filtration. Basal urinary nitrite concentrations were 0.5+ 0.02 μM as measured by capillary electrophoresis. Sodium nitrite (NaNO₂ ^") was added to urine in a concentration of 50 μM where the ionic nitrite (NO,^") was at a concentration of 75 μM. Ascorbate was added in a concentration of 10 mM/ml identical to urinary excretion of ascorbate following oral intake of 1-2 g/day ascorbic acid (Brandt et al., 1997, Am. J. Clin. Pathol., 68, 592). After each incubation with urine, cells were washed with phosphate buffer solution

(PBS), cell culture medium was added and cells were re-incubated.

I^s HJ thymidine incorporation. [³H] thymidine (1 μCi. per well) was added to the growing cells 2 hours before harvesting^ At harvest, the medium was discarded, cells were washed with PBS and the supernatant was discarded. One milliliter of ice-cold 10% trichloroacetic acid (TCA) was added to each well and incubated for 15 minutes. Cells were washed with PBS and lyzed with 0.1 M NaOH. The suspension was dissolved in scintillation liquid and counted in a beta-counter.

Statistical analysis. Differences between NO concentrations released from urine with or without nitrite and/or ascorbate at different pH were analysed by ANOVA for repeated ^■ measures. The effect of nitrite and/or ascorbate on the growth of T24 and MBT-2 cells were statistically examined by ANONA for repeated measures followed by the Tuckey post-hoc test.

RESULTS

Gaseous NO formation from nitrite in urine. NO release from normal urine or acidified urine was low (Figure 1). Nitrite addition (50 μM) resulted in massive increase of NO formation at acidic urine pH (p <0.05) (Figure 1). Addition of ascorbate in the presence of nitrite resulted in further increase of NO formation (p <0.05) (Figure 1). However, addition of ascorbate to urine without nitrite had no significant effect on NO formation at any of the pH values tested (Figure 1).

Effect of nitrite on the growth ofT24 and MBT-2 bladder cancer cell lines. A pH-dependent decrease in [³H]thymidine incorporation in T24 and MBT-2 cells was seen when cells were incubated with urine at lowering pH (Figures 2 and 3). At acidic pH, there was a further reduction of [³H] thymidine incorporation when cells were incubated with sodium nitrite (50 μM) (p <0.05). Addition of ascorbate (10 mM) to cells treated with nitrite (50 μM) resulted in a further reduction of [³H]thymidine incorporation compared to cells incubated with nitrite only (p <0.05). Sodium nitrite had no inhibitory effect on T24 or

MBT-2 cell grown at pH 7.0 (data not shown). Ascorbate alone had no effect on the growth of T24 or MBT-2 cells (Figure 4).

DISCUSSION We demonstrated growth inhibition of T24 and MBT-2 bladder cancer cells in the presence of nitrite in acidic urine. Further reduction of the cell growth was seen when ascorbate was added to nitrite containing urine, whereas ascorbate alone had no inhibitory effect on T24 or MBT-2 cell growth. In line with this inhibitory effect of nitrite, NO formed from nitrite was greatly enhanced in acidic urine (Figure 1). Figure 2 and 3 show that even at pH 6.5, [³H] thymidine incorporation was reduced to <40% of control levels illustrating that anti-proliferative effects occur under acidified conditions which appears to coincide with NO production. Furthermore, addition of ascorbate resulted in further increases in NO formation from nitrite in acidic urine.

NO release from acidified nitrite may explain the growth inhibitory effect of acidified nitrite on T24 and MBT-2 cells. However, acidification of nitrite results in the formation of NO and other nitrogen oxides, peroxynitrite, sodium nitrite and nitrous acid (HN0₂) (RNIs)

(Kaplan et al, 1996, Infect, hnmun., 64, p69-*). Peroxynitrite is rather cytotoxic and has been shown to induce DNA damage and subsequent activation of apoptosis and carcinogenesis

(Szabo & Ohshima, 1997, Nitric Oxide, 1, p 373-*). Sodium nitrite and nitrous acid have been suggested to have bacteriostatic effects (Kaplan et al., 1996, supra; Kono et al., 1994, Arch. Biochem. Biophys., 311, p 153-*). There are no reports on the effect of such compounds on

_ tumour cells. Our results show that sodium nitrite in the concentration used had essentially no effect on T24 or MBT-2 cell growth in unacidifϊed urine (pH 7). Thus, the growth inhibitory effect of acidified nitrite on T24 cells may be contributed to the formation of NO as well as other compounds, RNIs, which includes peroxynitrite, nitrous acid, nitrosothiols, nitrosonium (NO⁺) and other nitrogen oxides.

It should be understood that modifications can be made to the embodiments disclosed herein. Therefore the above description should not be construed as limiting, but merely as exemplification of preferred embodiments. Those skilled in the art will envision other modifications within the scope of the claims appended hereto.

Claims

1. Use of a nitrite donor in the preparation of a medicament for the treatment, profylaxis or management of a disorder involving neoplastic cells, wherein the nitrite donor is releasing nitrite only at an acidic pH.

2. Use according to claim 1, wherein at least one reducing agent is incorporated into the medicament.

3. Use according to claim 2, wherein the reducing agent is ascorbic acid.

4. Use according to claim 1, wherein the disorder is occuring in an acidic environment.

5. A method for treating a human or non-human animal with a disorder involving neoplastic cells, comprising the step of administering a composition comprising a nitrite donor wherein the nitrite donor is releasing nitrite only at an acidic pH.