WO1993020806A1

WO1993020806A1 - Use of nitric oxide/nucleophile complexes for the treatment of cancer

Info

Publication number: WO1993020806A1
Application number: PCT/US1993/002922
Authority: WO
Inventors: Chris M. Maragos; Ji Ming Wang; Larry K. Keefer; Joost J. Oppenheim
Original assignee: The United States Of America, Represented By The Secretary, Department Of Health And Human Services
Priority date: 1992-04-13
Filing date: 1993-03-29
Publication date: 1993-10-28
Also published as: AU3969793A

Abstract

A method of inducing cell cytostasis or cytotoxicity by exposing cells to a compound capable of releasing nitric oxide in an aqueous solution, particularly a nitric oxide/nucleophile complex or a derivative thereof, and a pharmaceutical composition containing a therapeutically effective amount of such a compound with a pharmaceutically acceptable carrier.

Description

OF NITRIC OXIDE/NDCLEOPHILE COMPLEXES FOR THE TREATMENT OF CANCER

TECHNICAL FIELD OF THE INVENTION The present invention concerns a method of, and composition for, controlling the proliferation of cells. In particular, the present invention is directed to the use of compounds which release nitric oxide in aqueous solutions, particularly nitric oxide/nucleophile complexes and their derivatives, to induce cytostasis and cytotoxicity so as to attenuate cell proliferation.

BACKGROUND OF THE INVENTION The control of cell proliferation has great usefulness in the treatment of various disorders. Of particular interest in that respect is the attenuation of cell growth or proliferation in the treatment of tumor or cancerous cells, parasites, fungi, bacteria, and other proliferating organisms. Nitric oxide (NO) has recently been implicated in a variety of bioregulatory processes, including normal physiological control of blood pressure, macrophage- induced cytostasis and cytotoxicity, and neurotransmission (Moncada et al., "Nitric Oxide from L- Arginine: A Bioregulatory System," Excerpta Medica. International Congress Series 897 (Elsevier Science Publishers B.V. : Amsterdam, 1990); Marietta et al. , "Unraveling the Biological Significance of Nitric Oxide," Biofactors. 2 , 219-225 (1990); Ignarro, "Nitric Oxide. A Novel Signal Transduction Mechanism for Transcellular Communication," Hypertension (Dallas) . 16. 477-483 (1990)). A number of compounds have been developed which are capable of delivering nitric oxide, including compounds which release nitric oxide upon being metabolized and compounds which release nitric oxide in aqueous solution. Those compounds which release nitric oxide upon being metabolized include the widely used nitrovasodilators glyceryl trinitrate and sodium nitroprusside (Ignarro et al., J. Pharmacol. Exp. Ther.. 218. 739-749 (1981) ; Ignarro, Annu> Rev. Pharmacol. Toxicol.. 30. 535-560 (1990); Kruszyna et al., Toxicol. Appl. Pharmacol.. 91, 429-438 (1987); Wilcox et al., Chem. Res. Toxicol.. 2, 71-76 (1990)). Another compound, S-nitroso-N-acetylpenicillamine, has been reported to release nitric oxide in solution and as being effective at inhibiting DNA synthesis (Garg et al., Biochem. and Biophys. Res. Corom.. 171. 474-479 (1990)) .

Nitric oxide/nucleophile complexes which release nitric oxide in aqueous solution are disclosed in U.S. Patents 4,954,526 and 5,039,705, as well as in pending U.S. patent applications 07/423,279 (filed October 18, 1989), 07/585,793 (filed September 20, 1990), 07/743,892 (filed August 12, 1991), 07/764,906 (filed September 24, 1991), 07/764,908 (filed September 24, 1991), and 07/858,885 (filed March 27, 1992), as being useful cardiovascular agents (see also Maragos et al., J. Med. Chem.. 34. 3242-3247 (1991)).

The cytostatic effect of nitric oxide solutions on tumor cells in vitro has been demonstrated. In particular, it has been shown that solutions of nitric oxide inhibit DNA synthesis and mitochondrial respiration of tumor cells in vitro (Hibbs et al., Biochem. and Biophvs. Res. Comm.. 157. 87-94 (1988) ; Stuehr et al. , J. EXP. Med.. 169. 1543-1555 (1989)). Nitric oxide in its pure form, however, is a highly reactive gas having limited solubility in aqueous media (WHO Task Group on Environmental Health Criteria for Oxides of Nitrogen, Oxides of Nitrogen. Environmental Health Criteria 4 (World Health Organization: Geneva, 1977)). Nitric oxide, therefore, is difficult to introduce reliably into most biological systems without premature decomposition.

The difficulty in administering nitric oxide can be overcome in some cases by administering nitric oxide pharmacologically in prodrug form. The compounds glyceryl trinitrate and sodium nitroprusside are relatively stable but release nitric oxide only on redox activation (Ignarro et al., J. Pharmacol. EXP. Ther.. 218, 739-749 (1981) ; Ignarro, Annu. Rev. Pharmacol. Toxicol. _r 30. 535-560 (1990); Kruszyna et al., Toxicol. Appl. Pharmacol.. 91, 429-438 (1987); Wilcox et al., Chem. Res. Toxicol.. 2, 71-76 (1990)). While this feature may be an advantage in some applications, it can also be a significant liability, as in the development of tolerance to glyceryl trinitrate via the exhaustion of the relevant enzyme/cofactor system (Ignarro et al., Annu. Rev. Pharmacol. Toxicol.. 25. 171-191 (1985) ; Kuhn et al., J. Cardiovasc. Pharmacol.. 14(Suppl. 11), S47- S54 (1989)) and toxicity from metabolically produced cyanide during prolonged administration of nitroprusside (Smith et al., "A Potpourri of Biologically Reactive Intermediates" in Biological Reactive Intermediates IV. Molecular and Cellular Effects and Their Impact on Human Health (Wit er et al., eds.), Advances in Experimental Medicine and Biology Volume 283 (Plenum Press: New York, 1991) , pp. 365-369) .

There remains a need, therefore, for a method of inhibiting the proliferation of tumor cells, parasites, fungi, bacteria, and other proliferating cells or organisms which can be reliably effected i vivo and which preferably does not require undesirable activation mechanisms. It is an object of the present invention to provide such a method. It is another object of the present invention to provide a pharmaceutical composition useful in carrying out that method.

These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION The present invention encompasses a method of controlling cell proliferation by inducing cytostasis and/or cytotoxicity among the cells. Specifically, the present invention involves exposing cells to a compound capable of releasing nitric oxide in an aqueous solution, particularly a nitric oxide/nucleophile complex or derivative thereof.

The present invention also encompasses a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound capable of releasing nitric oxide in an aqueous solution, particularly a nitric oxide/nucleophile complex or derivative thereof. The pharmaceutical composition will generally contain an amount of the nitric oxide releasing compound sufficient to induce cytostasis or cytotoxicity among cells exposed to the pharmaceutical composition and has particular utility in the treatment of tumor cells, as well as in antiparasitic, antifungal, and antibacterial treatments.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph depicting the attenuation of in vitro tumor cell proliferation as reflected by the percent relative ³H-thymidine incorporation by a melanoma subline incubated with various concentrations of various nitric oxide/nucleophile complexes.

Figure 2 is a graph depicting the attenuation of in vitro tumor cell proliferation as reflected by the percent relative ³H-thymidine incorporation by a melanoma subline incubated with various concentrations of the nitric oxide complex of 3-(n-propylamino)propylamine with and without the presence of the nitric oxide scavenger oxymyoglobin. DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is predicated on the discovery that cell proliferation, particularly tumor cells proliferation, can be attenuated or inhibited by exposing cells to a compound that is capable of releasing nitric oxide in an aqueous solution, specifically a nitric oxide/nucleophile complex or a derivative thereof. The present invention concerns a method of controlling cell proliferation by exposing cells to a compound capable of releasing nitric oxide in an aqueous solution, as well as a pharmaceutical composition which includes a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound capable of releasing nitric oxide in an aqueous solution, particularly a nitric oxide/nucleophile complex or a derivative thereof.

The compounds that are capable of releasing nitric oxide in an aqueous solution in the context of the present invention preferably do so spontaneously upon contacting an aqueous environment, e.g., do not require activation through a redox reaction or electron transfer such as required for glyceryl trinitrate and sodium nitroprusside. Some of the nitric oxide/nucleophile complexes useful in the context of the present invention do require activation by particular means, but only as necessary to free the nitric oxide releasing X[N(0)NO]^* group in the vicinity of the particular cells of interest. As an example, covalent attachment of a protecting group to the anionic [N(0)NOJ" function provides a means of postpoining nitric oxide release until the molecule reaches an organ capable of metabolically removing the protecting group. By choosing a protecting group that is selectively cleaved by tumor-specific enzymes, for example, the action of the nitric oxide/nucleophile complex can be targeted to maximize the desired effect. While the nitric oxide releasing compound in the context of the present invention is capable of releasing nitric oxide in an aqueous solution, such a compound preferably releases nitric oxide under physiological conditions.

The compound capable of releasing nitric oxide in an aqueous solution is preferably a nitric oxide/nucleophile adduct, e.g., a complex of nitric oxide and a nucleophile, most preferably a nitric oxide/nucleophile complex which contains the anionic moiety X[N(0)NO]^", where X is any suitable nucleophile residue. The nucleophile residue is preferably that of a primary amine (e.g., X = (CH₃)₂CHNH, as in (CH₃)₂CHNH[N(0)NO]Na) , a secondary amine (e.g., X = (CH₃CH₂)₂N, as in (CH₃CH₂)₂N[N(0)NO]Na) , a polyamine (e.g., X = sper ine, as in the zwitterion H₂N(CH₂)₃NH₂ ⁺(CH₂)₄N[N(0)NO]-(CH₂)₃NH₂, or X 3-(n- propylamino) propylamine, as in the zwitterion CH₃CH₂CH₂N[N(0)NO]-CH₂CH₂CH₂NH₃ ⁺) , or oxide (i.e., X = 0", as in NaO[N(0)NO]Na) , or a derivative thereof. Such nitric oxide/nucleophile complexes are stable solids and are capable of delivering nitric oxide in a biologically usable form at a predictable rate. The nucleophile is preferably not an entity such as sulfite (e.g., X = S0₃ ^", as in NH₄0₃S[N(0)NO]NH) even though the complex is a stable compound, since it is capable of releasing nitric oxide in an aqueous environment only under harsh, nonphysiological conditions.

Other suitable nitric oxide/nucleophile complexes include those having the following formulas:

wherein J is an organic or inorganic moiety, preferably a moiety which is not linked to the nitrogen of the remainder of the complex through a carbon atom, M^+x is a pharmaceutically acceptable cation, where x is the valence of the cation, a is 1 or 2, and b and c are the smallest integers that result in a neutral compound, preferably such that the compound is not a salt of alanosine or dopastin;

R₁-NH⁺- (CH₂) _s-N- [ (CH₂) _yN] _d- [ (CH₂) _Z-N] _b-R₃

I I I I (ID R₂ N₂0₂- R_j R, wherein b and d are the same or different and may be zero or one, R, , R₂, R₃, R,, and R₅ are the same or different and may be hydrogen, C₃__g cycloalkyl, C_W2 straight or branched chain alkyl, benzyl, benzoyl, phthaloyl, acetyl, trifluoroacetyl, p-toluyl, t- butoxycarbonyl, or 2,2,2-trichloro-t-butoxycarbonyl, and x, y, and z are the same or different and are integers from 2 to 12, with the proviso that Rj and R₃ are preferably not both methyl when R₂ is hydrogen, x is 2, and b and d are zero;

wherein B is or -N -N₂0₂",

Re and R₇ are the same or different and may be hydrogen, C^g cycloalkyl, C_ω2 straight or branched chain alkyl, benzyl, benzoyl, phthaloyl, acetyl, trifluoroacetyl, p- toluyl, t-butoxycarbonyl, or 2,2,2-trichloro-t- butoxycarbonyl, f is an integer from 0 to 12, with the proviso that when B is the substituted piperazine moiety

then f is an integer from 2 to 12;

Oj—(CH₂)_g CH₂)_f- H₂ ⁺-R_β (IV)

R>

wherein R₈ is hydrogen, C^ cycloalkyl, C_j.ι₂ straight or branched chain alkyl, benzyl, benzoyl, phthaloyl, acetyl, trifluoroacetyl, p-toluyl, t-butoxycarbonyl, or 2,2,2-tri-chloro-t-butoxycarbonyl, , is hydrogen or a ^cι~^ci₂ straight or branched chain alkyl, and g is 2 to 6;

wherein R_x and

group consisting of a straight chain or branched chain

C_t - C_I2 alkyl group and a benzyl group, with the proviso that no branch occur on the alpha carbon atom, or else _t and ₂ together with the nitrogen atom they are bonded to form a heterocyclic group, M^+t is a pharmaceutically acceptable cation, and x is the valence of the cation;

K[ '(L) _y(R^xR²N-N₂0₂) _g] (VI)

wherein M is a pharmaceutically acceptable metal, or where x is at least two, a mixture of two different pharmaceutically acceptable metals, L is a ligand different from (R^lR²N-N₂0₂) and is bound to at least one metal, R¹ and R² are each organic moieties and may be the same or different (with the proviso that where M is copper, x is one, L is methanol, and y is one, that at least one of R¹ or R² is not ethyl) , x is an integer of from 1 to 10, x' is the formal oxidation state of the metal M, and is an integer of from 1 to 6, y is an integer of from 1 to 18, and where y is at least 2, the ligands L may be the same or different, z is an integer of from_.1 to 20, and K is a pharmaceutically acceptable counterion to render the compound neutral to the extent necessary; and

[R-N(H)N(NO)0-]_yX (VII) wherein R is C^g lower alkyl, phenyl, benzyl, or C₃_₈ cycoloalkyl, any of which R groups may be substituted by one to three substituents, which are the same or different, selected from the group consisting of halo, hydroxy, C_w alkoxy, -NH₂, -C(0)NH₂, -CH(O) , -C(0)OH, and -N0₂, X is a pharmaceutically acceptable cation, a pharmaceutically acceptable metal center, or a pharmaceutically acceptable organic group selected from the group consisting of Cι.g lower alkyl, -C(0)CH₃, and -C(0)NH₂, and y is one to three, consistent with the valence of X.

In addition to the above-recited compounds, pharmaceutically acceptable salts, zwitterions, and derivatives thereof are also useful in the context of the present invention.

The synthesis of nitric oxide/nucleophile complexes generally involves reacting nitric oxide with suitable nucleophiles and has been described in Drago, "Reactions of Nitrogen(II) Oxide," in Free Radicals in Inorganic Chemistry. Advances in Chemistry Series, Number 36 (American Chemical Society: Washington, DC, 1962) , pp. 143-149; Hansen et al., "N-Nitrosation of Secondary Amines by Nitric Oxide via the 'Drago Complex,¹" in N- Nitroso Compounds: Occurrence and Biological Effects (Bartsch et al., eds.), IARC Scientific Publications No. 41 (Int'l Agency for Research on Cancer: Lyon, 1982), pp. 21-29; and Maragos et al., J. Med. Chem.. 34. 3242- 3247 (1991).

The rate at which the nitric oxide/nucleophile complex releases nitric oxide is dependent on at least the pH of the aqueous solution, the temperature, and the specific nature of the nucleophile. In general, the more alkaline the medium and the lower the temperature, the slower the release of nitric oxide. The nature of the nucleophile influences the rate of nitric oxide release over a considerable range (as demonstrated in the Examples herein) . The effect on cell proliferation of the compound capable of releasing nitric oxide, therefore, can be controlled by appropriate selection of the nitric oxide releasing compound. The effect of a compound capable of releasing nitric oxide in an aqueous solution on cells is reversible in the sense that the addition of a compound which is capable of removing or scavenging nitric oxide from an aqueous solution by complexing or reacting with nitric oxide can counteract the inhibitory effect of the compound which releases the nitric oxide. The effect on cell proliferation of the compound capable of releasing nitric oxide, therefore, can be further controlled by use of such a nitric oxide scavenger compound in an appropriate quantity.

While the method of the present invention can be practiced in vitro, it has particular usefulness in in vivo applications. The present inventive method, therefore, includes the administration to an animal, particularly a human, of a therapeutically effective amount of a compound capable of releasing nitric oxide in an aqueous solution, particularly a nitric oxide/nucleophile complex or derivative thereof. The use of such a compound in treating animals, particularly humans, circumvents the disadvantages of the use of pure nitric oxide, aqueous solutions of nitric oxide, and compounds which release nitric oxide but require undesirable activation mechanisms. As regards the in vivo use of the present inventive method, a nitric oxide releasing compound in the context of the present invention can be administered in any suitable manner, preferably with pharmaceutically acceptable carriers. The attenuation of tumor cell proliferation in an animal has utility in, for example, the in vivo treatment of cancerous cells.

One skilled in the art will appreciate that suitable methods of administering a nitric oxide releasing compound in the context of the present invention to an animal are available, and, although more than one route can be used to administer a particular compound, a particular route can provide a more immediate and more effective reaction than another route. Pharmaceutically acceptable carriers are also well-known to those who are skilled in the art. The choice of carrier will be determined in part by the particular compound, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention.

Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water or saline, (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules, (c) suspensions in an appropriate liquid, and (d) suitable emulsions. Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, such carriers as are known in the art. The nitric oxide releasing compounds in the context of the present invention, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

The dose administered to an animal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the animal over a reasonable time frame. The dose will be determined by the strength of the particular compound employed and the condition of the animal, as well as the body weight of the animal to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular compound.

In the treatment of some individuals with the pharmaceutical composition of the present invention, it may be desirable to utilize a "mega-dosing" regimen. In such a treatment, a large dose of the pharmaceutical composition is administered to an individual, time is allowed for the active compound, i.e., the nitric oxide releasing compound, to act, and then a suitable reagent, e.g., a nitric oxide scavenger, is administered to the individual to render the active compound ineffective. The desirable extent of the inhibition of cell proliferation rate will depend on the particular condition or disease being treated, as well as the stability of the patient and possible side-effects. In proper doses and with suitable administration of certain compounds, the present invention provides for a wide range of the inhibition of the normal cell proliferation rate, e.g., from little inhibition to essentially full inhibition. The cytostatic activity of a nitric oxide/nucleophile complex is generally related to the rate and extent of nitric oxide release. It is believed that those compounds which release nitric oxide slowly, such as spermine-bis(nitric oxide) adduct monohydrate and 3(n-propylamino)propylamine bis(nitric oxide) adduct, are more potent inhibitors of DNA synthesis than compounds which release nitric oxide more quickly, such as diethylamine-bis(nitric oxide) adduct sodium salt, isopropylamine-bis(nitric oxide) adduct sodium salt, and sodium trioxodinitrate(II) monohydrate (also known as "Angeli's salt"). It is also believed that a sustained exposure to moderate amounts of nitric oxide has a longer lasting effect than a brief exposure to a greater concentration of nitric oxide. Moreover, the number of molecules of nitric oxide released by a single nitric oxide/nucleophile complex, which will generally vary up to two per [N(0)N0] moiety, apparently affects the potency of the nitric oxide/nucleophile complex, with compounds which release more nitric oxide per molecule having a greater cytostatic effect.

The cytostatic effect of a nitric oxide/nucleophile complex is also dependent on factors in addition to the rate and extent of nitric oxide release. Such factors include the mechanism by which the compound degrades, the degree of uptake by the exposed cells, and the affinity for cellular constituents. For example, spermine binds DNA which may play a role in the nitric oxide/spermine complex having a high cytostatic effect on tumor cells.

While the present inventive method and pharmaceutical composition are primarily directed to the control of tumor cell proliferation, i.e., cytostasis or the inhibition of cell division, the present inventive method and pharmaceutical composition may also have usefulness in controlling tumor cell growth, i.e., by providing a cytotoxic effect to adversely affect cell viability, and in attenuating the proliferation and growth of non-tumor cells, e.g., as antiparasitic, antifungal, and antibacterial agents, as well as in laboratory research. Those compounds which have a cytostatic effect but not a cytotoxic effect can be used with cytotoxic agents in combination therapy. The present invention also has usefulness in prophylatic treatments.

Some of the nitric oxide releasing compounds, in sufficiently high concentration, will have a cytotoxic effect, in addition to a cyctostatic effect, on cells. For example, under at least the conditions set forth in the Exaples below, spermine-bis(nitric oxide) adduct monohydrate has a pronounced cytotoxic effect at a concentration of 500 μM, while 3-(n- propylamino)propylamine bis(nitric oxide) adduct, diethyla ine-bis(nitric oxide) adduct sodium salt, isopropylamine-bis(nitric oxide) adduct sodium salt, sodium trioxodinitrate(II) monohydrate, and N- nitrosohydroxylamine-N-sulfonate do not significantly affect cell viability at concentrations up to 500 μM. The cytotoxic effect of a particular nitric oxide/nucleophile complex is generally related to the nature of the nucleophile and other chemical entities which may be released from the complex, e.g., spermine from the spermine-nitric oxide complex. The following examples further illustrate the present invention but, of course, should not be construed as in any way limiting its scope.

Example 1

The effect on tumor cell proliferation by various nitric oxide/nucleophile complexes was evaluated by examining the effectiveness of such complexes in inhibiting DNA synthesis in A375 human melanoma cells as measured by incorporation of tritiated thymidine. Specifically, A375-C6 human melanoma cells were exposed to 0, 25, 50, 100, 200, 300, and 500 μM of various nitric oxide/nucleophile complexes.

A375-C6 human melanoma cells were harvested with trypsin, washed twice with 10 mM phosphate buffered saline (PBS) , and resuspended in RMPI 1640 medium (Advanced Biotechnologies, Inc., Columbia, Maryland) containing 5% fetal bovine serum (Hyclone) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells were diluted to 5.3 x 10⁴/ml and 0.95 ml aliquots were distributed into microfuge tubes. Fifty microliters of each nitric oxide/nucleophile complex in 100 mM PBS, or PBS control, were added and mixed vigorously. The cell suspensions were distributed onto flat bottom tissue culture clusters (Costar) in the volume of 0.2 ml/well. Tritiated thymidine stock, i.e., ³H- thymidine (³H-TdR) (NEN, Boston, Massachusetts) , with a specific activity of 15.0 Ci/mmol, was diluted in medium and added at a level of 1 μCi/well (25 μl/well) . Following an 8 hour incubation, at 37^βC and 5% Co₂, the cell contents were collected onto filter discs (Model 290 PHD Cell Harvester, Cambridge Technology, Inc., Watertown, Massachusetts) . The radioactivity of the filters was measured (Beckman LS-900) after the addition of 4 ml scintillation cocktail (Aquassure, , NEN) . DNA synthesis was expressed as % relative incorporation of labeled thymidine, i.e., (cpm sample/cpm PBS control) x 100%.

The percent relative ³H-TdR incorporations versus the concentration of the nitric oxide/nucleophile complexes were plotted as shown in Figure 1, and curves were plotted for the data points for equations fitted to each data set with the aid of a commercial computer program (TableCurve, Jandel Scientific, Corte Madera, California) . The data were fit to a dose-response equation of the form:

% Relative Incorporation = a + b/(l + (Dose/c)^d) . This equation is equivalent to the sigmoid E^ model with baseline correction as described in Holford et al. , Clin. Pharmacokinetics. 6_, 429-453 (1981) . Each data point in Figure 1 represents the mean of the results for three separate experiments, with 3-6 wells being used for each experiment (i.e., n = 9-18 wells per data point) . The bars represent 95% confidence intervals based on an evaluation of the standard deviation of the experimental results for each data point.

The following nitric oxide/nucleophile complexes were tested, and the concentration of each of the complexes required to inhibit ³H-TdR incorporation by 50% (IC_JO) was estimated from the equations fit to each data set used to prepare the curves of Figure 1.

Compound

H₂N(CH₂)₃NH₂ ⁺(CH₂)₄N[N(0)NO]-(CH₂)₃NH₂ CH₃CH₂CH₂N [ N ( O ) NO ] -CH₂CH₂CH₂NH₃ ⁺

(CH₃CH₂)₂N[N(0)NO]Na NaO[N(0)NO]Na

(CH₃)₂CHNH[N(0)NO]Na NH₄(0₃S) [N(0)NO]NH₄

IC₅₀ value represents mean of experimental results depicted in Figure 1 as well as from three additional experiments.

Thymidine incorporation was inhibited by 43% at 500 μM (% relative inhibition = 57%) .

The term "NR" indicated no response and means that the compound did not inhibit DNA synthesis at 500 μM.

Controls, all tested concurrently with the nitric oxide/nucleophile complexes at a level of 500 μM, gave the following responses, where n represents the number of wells per data point:

Compound n percent relative thymidine incorporation (mean ± std. dev.) isopropylamine sodium nitrite diethylamine

N-nitrosodiethylamine

3-(n-propylamino)propylamine spermine

For comparison purposes, pure nitric oxide, added as 53 μl of a saturated solution to 950 μl cells (final concentration of 100 μM) resulted in a decrease in thymidine incorporation which was intermediate between the decrease in thymidine incorporation observed for Na₂N₂0₃ and (C^i₅)₂N[N(0)NO]Na. The mean and standard deviation of percent relative incorporation for the pure nitric oxide were 72 ± 15% relative incorporation.

The results of this experiment demonstrate the inhibitory effect of nitric oxide/nucleophile complexes which release nitric oxide in aqueous solution on the proliferation of tumor cells.

Example 2 The effect on tumor cell proliferation by exposure to a nitric oxide/nucleophile complex in the presence of a compound which removes nitric oxide from an aqueous solution was evaluated by examining the effectiveness of the nitric oxide complex of 3-(n- propylamino)propylamine in inhibiting DNA synthesis in A375 human melanoma cells, by itself and in the presence of oxymyoglobin (Mb0₂) , as measured by incorporation of tritiated thymidine as set out in Example 1. A375-C6 human melanoma cells were exposed to 0, 20, 60, 100, 140, 170, and 200 μM of the nitric oxide complex of 3-(n-propylamino)propylamine in accordance with the procedure set out in Example 1, except that 6.3 x 10⁴ cells in 0.90 ml were incubated with 0.05 ml, of 2.5 mM myoglobin (125 μM final concentration after dilution) or PBS control and 0.05 ml of the nitric oxide/nucleophile complex. Those cells exposed to oxymyoglobin were exposed to the oxy yoglobin immediately prior to the addition of the nitric oxide/nucleophile complex. The percent relative ³H-TdR incorporations versus the concentration of the nitric oxide/nucleophile complex were plotted as shown in Figure 2, and curves were plotted for the data points for equations fitted to each data set utilizing the procedure set out in Example 1.

Each data point in Figure 2 represents the mean of the results for three separate experiments, with 3-6 wells being used for each experiment (i.e., n = 9-18 wells per data point) . The bars represent 95% confidence intervals based on an evaluation of the standard deviation of the experimental results for each data point.

The concentration of the nitric oxide/nucleophile complex required to inhibit ³H-TdR incorporation by 50% (IC_JO) was estimated from the equations fit to each data set used to prepare the curves of Figure 2.

* The term "NR" indicated no response and means that the nitric oxide/nucleophile complex did not inhibit DNA synthesis at 200 μM.

The results of this experiment demonstrate that a compound which removes nitric oxide from an aqueous solution, such as oxymyoglobin which forms a nitrosyl- myoglobin (Mb-NO) complex, provides a protective effect against the inhibitory effect on tumor cells of a compound which releases nitric oxide in an aqueous solution.

All of the references, including literature references, patents, and patent applications, cited herein are hereby incorporated in their entireties by reference.

While this invention has been described with an emphasis upon a preferred embodiment, it will be obvious to those of ordinary skill in the art that variations in the preferred method and composition may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of controlling tumor cell proliferation, which method comprises exposing tumor cells to a compound capable of releasing nitric oxide in an aqueous solution.

2. The method of claim 1, wherein said compound is capable of releasing nitric oxide under physiological conditions.

3. The method of claim 2, wherein said compound is a nitric oxide/nucleophile complex or a derivative thereof.

4. The method of claim 3, wherein said compound is a nitric oxide/nucleophile complex.

5. The method of claim 4, wherein said compound contains the anionic moiety X[N(0)N0]", where X is a nucleophile residue.

6. The method of claim 5, wherein X is selected from the group consisting of spermine, 3-(n- propylamino)propylamine, diethylamine, isopropylamine, and oxide.

7. The method of claim 3, wherein said compound is (CH₃)₂CHNH[N(0)NO]Na.

8. The method of claim 3, wherein said compound is CH₃CH₂CH₂N[N(0)NO]"CH₂CH₂CH₂NH₃ ⁺.

9. The method of claim 3, wherein said compound is (CH₃CH₂)₂N[N(0)NO]Na.

10. The method of claim 3, wherein said compound is H₂N(CH₂)₃NH₂ ⁺(CH₂)₄N[N(0)N0]~(CH₂)₃NH₂.

11. The method of claim 3, wherein said compound is NaO[N(0)NO]Na.

12. The method of claim 3, wherein said compound is of the formula I

wherein J is an organic or inorganic moiety, preferably a moiety which is not linked to the nitrogen of the remainder of the complex through a carbon atom, M^+x is a pharmaceutically acceptable cation, where x is the valence of the cation, a is 1 or 2, and b and c are the smallest integers that result in a neutral compound, or pharmaceutically acceptable salts or derivatives thereof.

13. The method of claim 3, wherein said compound is of the formula II

(CH₂)_yN]_d-[ (CH₂)_Z-N]_b-R₃

(II)

N₂0₂ ^"

wherein b and d are the same or different and may be zero or one, R_1# R₂, R₃, R₄, and R₅ are the same or different and may be hydrogen, C₃_₈ cycloalkyl, C_1-1 straight or branched chain alkyl, benzyl, benzoyl, phthaloyl, acetyl, trifluoroacetyl, p-toluyl, t- butoxycarbonyl, or 2,2,2-trichloro-t-butoxycarbonyl, and x, y, and z are the same or different and are integers from 2 to 12, or pharmaceutically acceptable salts or derivatives thereof.

14. The method of claim 3, wherein said compound is of the formula III

wherein B is

R<i and R₇ are the same or different and may be hydrogen, ^c ₃-₈ cycloalkyl, Cj_ι₂ straight or branched chain alkyl, benzyl, benzoyl, phthaloyl, acetyl, trifluoroacetyl, p- toluyl, t-butoxycarbonyl, or 2,2,2-trichloro-t- butoxycarbony1, f is an integer from 0 to 12, with the proviso that when B is the substituted piperazine moiety

then f is an integer from 2 to 12, or pharmaceutically acceptable salts or derivatives thereof.

15. The method of claim 3, wherein said compound is of the formula IV

_S-NH₂ ⁺-R₈ (IV)

wherein R_g is hydrogen, Cj-g cycloalkyl, C ₂ straight or branched chain alkyl, benzyl, benzoyl, phthaloyl, acetyl, trifluoroacetyl, p-toluyl, t-butoxycarbonyl, or 2,2,2-tri-chloro-t-butoxycarbonyl, R, is hydrogen or a ^cι~^ci₂ straight or branched chain alkyl, and g is 2 to 6, or pharmaceutically acceptable salts or derivatives thereof.

16. The method of claim 3, wherein said compound is of the formula

wherein R_t and R₂ are independently selected from the group consisting of a straight chain or branched chain C_x - C₁₂ alkyl group and a benzyl group, with the proviso that no branch occur on the alpha carbon atom, or else R_t and R₂ together with the nitrogen atom they are bonded to form a heterocyclic group, M^+x is a pharmaceutically acceptable cation, and x is the valence of the cation, or pharmaceutically acceptable salts or derivatives thereof.

17. The method of claim 3, wherein said compound is of the formula VI K[ (M) ^ (L) _y(R-R²N-N₂0₂) _z] (VI)

wherein M is a pharmaceutically acceptable metal, or where x is at least two, a mixture of two different pharmaceutically acceptable metals, L is a ligand different from (R^N-N^) and is bound to at least one metal, R¹ and R² are each organic moieties and may be the same or different (with the proviso that where M is copper, x is one, L is methanol, and y is one, that at least one of R¹ or R² is not ethyl) , x is an integer of from 1 to 10, x' is the formal oxidation state of the metal M, and is an integer of from 1 to 6, y is an integer of from 1 to 18, and where y is at least 2, the ligands L may be the same or different, z is an integer of from 1 to 20, and K is a pharmaceutically acceptable counterion to render the compound neutral to the extent necessary, or pharmaceutically acceptable salts or derivatives thereof.

18. The method of claim 3, wherein said compound is of the formula VII

[R-N(H)N(N0)0-]_yX (VII) wherein R is C₂._g lower alkyl, phenyl, benzyl, or C^g cycoloalkyl, any of which R groups may be substituted by one to three substituents, which are the same or different, selected from the group consisting of halo, hydroxy, Cι.g alkoxy, -NH₂, -C(0)NH₂, -CH(O) , -C(0)OH, and -N0₂, X is a pharmaceutically acceptable cation, a pharmaceutically acceptable metal center, or a pharmaceutically acceptable organic group selected from the group consisting of Cj._g lower alkyl, -C(0)CH₃, and -C(0)NH₂, and y is one to three, consistent with the valence of X, or pharmaceutically acceptable salts or derivatives thereof.

19. The method of claim 3, wherein said tumor cells are exposed to said nitric oxide releasing compound in an amount sufficient to inhibit the proliferation of said tumor cells.

20. The method of claim 3, wherein said compound is administered in a therapeutically effective amount with a pharmaceutically acceptable carrier to an animal.

21. The method of claim 20, wherein said animal is a human.

22. The method of claim 2, which method further comprises exposing said tumor cells to a compound capable of removing nitric oxide from an aqueous solution by complexing or reacting with nitric oxide so as to counteract the inhibitory effect of said compound capable of releasing nitric oxide in an aqueous solution.

23. A method of inducing cytostasis or cytotoxicity, which method comprises exposing cells to a compound capable of releasing nitric oxide in an aqueous solution in an amount sufficient to inhibit the proliferation of said cells.

24. The method of claim 23, wherein said compound induces cytostasis.

25. The method of claim 24, wherein said compound is capable of releasing nitric oxide under physiological conditions.

26. The method of claim 25, wherein said compound is a nitric oxide/nucleophile complex or a derivative thereof.

27. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound capable of releasing nitric oxide in an aqueous solution in a therapeutically effective amount sufficient to inhibit the proliferation of cells.

28. The pharmaceutical composition of claim 27, wherein said compound is capable of releasing nitric oxide under physiological conditions.

29. The pharmaceutical composition of claim 28, wherein said compound is a nitric oxide/nucleophile complex or a derivative thereof.

30. The pharmaceutical composition of claim 29, wherein said compound is a nitric oxide/nucleophile complex.

31. The pharmaceutical composition of claim 30, wherein said compound contains the anionic moiety X[N(0)NO]", where X is a nucleophile residue.

32. The pharmaceutical composition of claim 31, wherein X is selected from the group consisting of spermine, 3-(n-propylamino)propylamine, diethylamine, isopropylamine, and oxide.

33. The method of claim 12, wherein said compound is not a salt of alanosine or dopastin.

34. The method of claim 13, wherein R_x and R₃ are not both methyl when R₂ is hydrogen, x is 2, and b and d are zero.