Method and composition for the treatment of diarrhea and gastrointestinal spasms

Anti-diarrheal and/or gastrointestinal anti-spasmodic pharmaceutical compositions containing &lsqb;A&rsqb; a polyamine of the formula: R 1 —N 1 (R 2 )—(CH 2 )x-N 2 H—Q—N 3 H—(CH 2 )y-N 4 (R 3 )—R 4 (I) wherein: R 1 , R 2 , R 3 and R 4 may be the same or different and are H, alkyl, cycloalkyl or aralkyl having from 1 to 12 carbon atoms, or a heterocyclic group having from 3 to 10 atoms wherein the hetero atom is said N 1 or N 4 ; Q is a cycloalkyl group having from 3 to 10 carbon atoms; x is an integer from 3 to 6, inclusive; and y is an integer from 3 to 6, inclusive; or (II) a salt thereof with a pharmaceutically acceptable acid; and &lsqb;B&rsqb; a pharmaceutically acceptable carrier therefor as well as methods of treatment utilizing the polyamines are disclosed.

SYNTHESIS OF CHX(3,4,3)-trans N,N′-Bis&lsqb;3-(ethylamino)propyl&rsqb;-trans-1,4-cyclohexanediamine tetrahydrochloride &lsqb;CHX(3,4,3)-trans, 1&rsqb; was assembled via fragment synthesis (Scheme 1). Disulfonamide 3, available in 90% yield from reaction of trans-1,4-diaminocyclohexane (2) with mesitylenesulfonyl chloride (2 equiv) under biphasic conditions, was alkylated with N-(3-bromopropyl)-N-ethylmesitylenesulfonamide (2 equiv, NaH, DMF) to provide protected polyamine 4 in 73% yield. The amino groups of 4 were unmasked with 30% HBr in acetic acid and phenol, furnishing final product 1 in 64% yield. &lsqb;Bergeron et al, J. Med. Chem., Vol. 7, pp3464-3476 (1984)&rsqb; 
 SUMMARY OF THE INVENTION The foregoing and other objects are realized by the present invention, one embodiment of which is an anti-diarrheal, anti-secretory, or gastrointestinal anti-spasmodic pharmaceutical composition comprising an anti-diarrheal or gastrointestinal antispasmodic (hereinafter “GI anti-spasmodic”) effective amount of a compound of the formula set forth below and a pharmaceutically acceptable carrier therefor. An additional embodiment of the present invention comprises a method of treating a human or nonhuman animal in need thereof comprising administering to the animal an anti-diarrheal or GI antispasmodic effective amount of a compound of the formulae below. Suitable methods for the preparation of polyamines for use in the composition and method of the invention are those described in application Ser. No. 07/210,520 filed Jun. 23, 1988, now U.S. Pat. No. 5,091,576, the entire contents and disclosure of which is incorporated herein by reference. The polyamines suitable in the practice of the invention include those having the formula: R 1 —N 1 (R 2 )—(CH 2 )x-N 2 H—Q—N 3 H—(CH 2 )y-N 4 (R 3 )—R 4 (I) wherein: R 1 , R 2 , R 3 and R 4 may be the same or different and are H, alkyl, cycloalkyl or aralkyl having from 1 to 12 carbon atoms, or a heterocyclic group having from 3 to 10 atoms wherein the hetero atom is said N 1 or N 4 ; Q is a cycloalkyl group having from 3 to 10 carbon atoms; x is an integer from 3 to 6, inclusive; and y is an integer from 3 to 6, inclusive; or (II) a salt thereof with a pharmaceutically acceptable acid. 
 DETAILED DESCRIPTION OF THE INVENTION The present invention is predicated on the discovery that polyamines of the above formula act to inhibit the potential for the large and small intestines to contract. While not wishing to be bound by any theory as to the mechanism of action of the polyamines as inhibitors of this action of the intestines, it is hypothesized that the polyamines function via a receptor-dependent regulation mechanism whereby the myoelectric activity of the muscle tissue of the colon and small intestine and the secretion of fluid and electrolytes by these organs are modulated. In addition, some of these above effects may be directly or indirectly mediated through the release of nitric oxide or through the activation of nitric oxide synthase. For each of the utilities mentioned herein, the amount required of active agent, the frequency and mode of its administration will vary with the identity of the agent concerned and with the nature and severity of the condition being treated and is, of course, ultimately at the discretion of the responsible physician or veterinarian. In general, however, a suitable dose of agent will lie in the range of about 0.0001 mg to about 500 mg per kilogram of mammal body weight being treated. Administration by the parenteral route (intravenously, intradermally, intraperitoneally, intramuscularly or subcutaneously) is preferred for a period of time of from one to ten days; although the agent may also be administered orally. For chronic problems, the drug is administered as needed, subcutaneously, intravenously, or orally. While it is possible for the agents to be administered as the raw substances, it is preferable, in view of their potency, to present them as a pharmaceutical formulation. The formulations of the present invention, both for veterinary and human use, comprise the agent together with one or more acceptable carriers therefor and optionally other therapeutic ingredients. The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Desirably, the formulations should not include oxidizing agents and other substances with which the agents are known to be incompatible. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the agent with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the agent with the carrier(s) and then, if necessary, dividing the product into unit dosages thereof. Formulations suitable for parenteral administration conveniently comprise sterile aqueous preparations of the agents which are preferably isotonic with the blood of the recipient. Suitable such carrier solutions include phosphate buffered saline, saline, water, lactated ringers or dextrose (5% in water). Such formulations may be conveniently prepared by admixing the agent with water to produce a solution or suspension which is filled into a sterile container and sealed against bacterial contamination. Preferably, sterile materials are used under aseptic manufacturing conditions to avoid the need for terminal sterilization. The pharmaceutical compositions according to the invention are those which are suitable for enteral, such as oral, administration and for parenteral, such as subcutaneous, administration to warm-blooded animals, especially humans, and which contain the pharmacologically active substance on its own or together with a pharmaceutically acceptable carrier. The dosage of the active substance depends on the species of warm-blooded animal and on the age and individual condition the illness to be treated and also on the mode of administration. The novel pharmaceutical preparations contain from approximately 10% to approximately 95%, and preferably from approximately 20% to approximately 90%, of the active substance. Pharmaceutical compositions according to the invention can, for example, be in unit dose form, such as drages, tablets, capsules, suppositories or ampoules, and contain from approximately 0.05 g to approximately 10.0 g, and preferably from approximately 0.3 g to approximately 1.0 g, of the active ingredient. The pharmaceutical compositions of the present invention are manufactured in a manner known per se, for example, by means of conventional mixing, granulating, confectioning, dissolving or lyophilizing processes. Pharmaceutical compositions for oral use can be obtained by combining the active substance with one or more solid carriers, if desired, granulating a resulting mixture and processing the mixture or granulate, if desired or necessary after the addition of suitable adjuncts, to form tablets or dragee cores. In so doing, they can also be incorporated into plastic carriers which release the active substances or allow them to diffuse in controlled amounts. Suitable carriers are especially fillers such as guars, for example, lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen phosphate, also potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, and/or, if desired, disintegrators such as the above-mentioned starches, also carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate. Adjuncts are especially useful, such as flow-regulating and lubricating agents, for example, silica, talc, stearic acid or salts thereof such as magnesium or calcium stearate, and/or polyethylene glycol. Dragee cores are provided with suitable coatings that are, if desired, resistant to gastric juice, there being used, inter alia, concentrated sugar solutions which optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions in suitable organic solvents or solvent mixtures or, for the manufacture of coatings that are resistant to gastric juice, solutions of suitable cellulose, preparations such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate. Coloring substances or pigments can be added to the tablets or coatings, for example for the purpose of identification or for indicating different doses of active substance. Other orally administrable pharmaceutical compositions are dry-filled capsules made of gelatin and also soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The dry-filled capsules may contain the active ingredient in the form of a granulate, for example, in admixture with fillers such as corn starch, binders and/or glidants such as talc or magnesium stearate and optionally, stabilizers. In soft capsules, the active ingredient is preferably dissolved or suspended in suitable liquids or wax-like substances such as fatty oils, paraffin oil or polyethylene glycols, it being possible also for stabilizers to be added. Other forms of oral administration are, for example, syrups prepared in a customary manner that contain the active ingredient in, for example, suspended form and in a concentration of approximately from 5% to 20%, and preferably approximately 10%, or in a similar concentration that provides a suitable single dose when administered, for example, in measures of 5 or 10 ml. Also suitable are, for example, powdered or liquid concentrates for preparing shakes, for example, in milk. Such concentrates can also be packed in single-dose quantities. Particularly suitable dosage forms for parenteral administration are sterile aqueous solutions of an active ingredient in water-soluble form, for example, a water-soluble salt, or sterile aqueous injection suspensions which contain substances increasing the viscosity, for example, sodium, carboxymethyl cellulose, sorbitol and/or dextran, and optionally stabilizers. In addition, the active ingredient, with or without adjuvants, can also be in lyophilized form and brought into solution prior to parenteral administration by the addition of suitable solvents. Such formulations may optionally contain one or more additional ingredients among which may be mentioned preservatives, such as methyl hydroxybenzoate, chlorocresol, metacresol, phenol and benzalkonium chloride. Such materials are of special value when the formulations are presented in multi-dose containers. Buffers may also be included to provide a suitable pH value for the formulation and suitable materials include sodium phosphate and acetate. Sodium chloride or glycerin may be used to render a formulation isotonic with the blood. If desired, the formulation may be filled into the containers under an inert atmosphere such as nitrogen or may contain an anti-oxidant and are conveniently presented in unit dose or multi-dose form, for example, in a sealed ampoule. It will be appreciated that while the agents described herein form acid addition salts and carboxyl acid salts, the biological activity thereof will reside in the agent itself. These salts may be used in human and in veterinary medicine and presented as pharmaceutical formulations in the manner and in the amounts (calculated as the base) described herein above, and it is then preferable that the acid moiety be pharmacologically and pharmaceutically acceptable to the recipient. Examples of such suitable acids include (a) mineral acids: hydrochloric, hydrobromic, phosphoric, metaphosphoric, and sulfuric acids; (b) organic acids: tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycollic, gluconic, gulonic, succinic and arylsulfonic, for example, p-toluenesulfonic acids. In compounds of the invention, R1 and R2 are preferably methyl, ethyl, propyl, benzyl, etc., it being understood that the term “aralkyl” is intended to embrace any aromatic group, the chemical and physical properties of which do not adversely affect the efficacy and safety of the compound for therapeutic applications. Preferred, however, are the hydrocarbyl aralkyl groups, i.e., comprised only of C and H atoms. Polyamines of the above formula are synthesized according to the methods described in application Ser. No. 07/210,520 filed Jun. 23, 1988, now U.S. Pat. No. 5,091,576, and Ser. No. 07/870,441 filed Oct. 9, 1991, the entire contents and disclosures of both of which are incorporated herein by reference. The invention is illustrated by the following non-limiting examples. 
 EXAMPLE 1 
 Synthesis of CHX(3,4,3)-trans Referring to FIG. 1 , N,N′-Bis&lsqb;3-(ethylamino)propyl&rsqb;-trans-1,4-cyclohexanediamine tetrahydrochloride &lsqb;CHX(3,4,3)-trans, 1&rsqb; was assembled via a fragment synthesis (Scheme 1) Disulfonamide 3, available in 90% yield from reaction of trans- 1,4-diaminocyclohexane (2) with mesitylenesulfonyl chloride (2 equiv) under biphasic conditions, was alkylated with N-(3bromopropyl)-N-ethylmesitylenesulfonamide (2 equiv, NaH, DMF) to provide protected polyamine 4 in 73% yield. The amino groups of 4 were unmasked with 30% HBr in acetic acid and phenol, furnishing the final product 1 in 64% yield. 
 EXAMPLE 2 
 Castor Oil-induced Diarrhea in Rats Male Sprague-Dawley rats (350-400 g, Harlan Sprague-Dawley, Indianapolis, Ind.) were fasted overnight in hanging wire cages and allowed free access to water. A typical experiment involved 20 rats: 5 untreated controls and 5 pretreated with polyamine analogues at each of three doses (typically equivalent on a molar basis to 1, 5, 10, or 25 mg/kg of DEHSPM) as either a sc injection or a po gavage 30 min prior to castor oil. All animals were then challenged with castor oil (purchased from a local drugstore) as a gastric gavage of 5 mL/kg of body weight at t&equals;0 and monitored for the onset and duration of diarrhea at 30-min intervals for a 6-h period during which they received no food or water (Eaker, E. Y.; Bixler, G. B.; Mathias, J. R., J. Pharm Exp. Ther. 1988, 246, 786-789). Onset of diarrhea for the control rats was between 30 and 90 min and lasted for at least 6 h. The animal weight and stool weight were recorded at 2, 4, and 6 h. 
 EXAMPLE 3 
 Irritable Bowel Syndrome in Rats Male Sprague-Dawley rats (200-350 g, Harlan Sprague-Dawley, Indianapolis, Ind.) were housed in hanging wire cages in a temperature- and humidity-controlled room with a 12hour light/dark cycle; the animals were fasted overnight (unless non-fasted rats were utilized in experiments involving PO administration) and allowed free access to water. Animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee. A typical experiment involved 20 rats: 5 untreated controls and 5 pretreated with polyamine analogues at each of three doses as either a SC injection or a PO gavage 30 min prior to commencement of the stress. All animals were then housed in individual polycarbonate cages containing a clear 70×50 mm Pyrex crystallization dish inverted in the center. To begin the stress, water was added to each cage to within 0.5 cm of the top of the Pyrex dish. The fecal output of the animals was recorded at 30-min intervals for a 6-h period, during which they received no food or water. Stool output was expressed as the number of fecal pellets excreted over the 6-h collection period. Percent reduction was calculated by dividing the mean value from the treated animals (T) by the mean value from the control animals (C), subtracting the resulting quotient from 1.0, and multiplying by 100 &lsqb;i.e., (1.0−T/C)×100&rsqb;. 
 EXAMPLE 4 
 Efficacy of CHX(3,4,3)-trans in Rats CHX(3,4,3)-trans (1) was effective in the castor oil-induced diarrhea model. When administered sc at doses ranging from 0.0078 mg/kg to 24.9 mg/kg, there was a significant reduction in weight loss at all dose levels (P<0.005 to P<0.001, Table 1). In addition, the compound also significantly decreased stool output relative to controls at sc doses of 0.0156 mg/kg to 24.9 mg/kg (P<0.001 for all doses, Table 1). CHX(3,4,3)-trans was also remarkably effective at controlling IBS. When administered sc at doses ranging from 0.03 mg/kg to 0.99 mg/kg, there was a significant reduction in fecal output at all dose levels (P<0.001, Table 2). Oral administration of this compound also diminished fecal output at doses&gE;5 mg/kg (P&lE;0.001, Table 2). 1 TABLE 1 Antidiarrheal Activity of Polyamine Analogues Predicated on a (3,4,3) Backbone a compd. structure/ dose 15 per kg weight P- % stool P- % no, abbreviation mg :mol n loss value c reduction d output e value c reduction d 1 0 0 20 11.6 ∀ 3.7 — — 4.9  2.0 — — 0.0078 0.0181 5 8.8 ∀ 1.2 <0.005 24 3.6 ∀ 1.3 >0.05 NS 0.0156 0.036 5 3.1 ∀ 1.2 <0.001 73 0.4 ∀ 0.9 <0.001 92 0.03125 0.0726 5 4.2 ∀ 1.7 <0.001 64 0 ∀ 0 <0.001 100 0.625 0.145 5 3.7 ∀ 0.8 <0.001 68 0 ∀ 0 <0.001 100 0.125 0.29 5 4.1 ∀ 0.7 <0.001 65 0 ∀ 0 <0.001 100 0.25 0.58 4 2.7 ∀ 0.7 <0.001 77 0 ∀ 0 <0.001 100 0.5 1.16 5 2.9 ∀ 0.1 <0.001 75 0 ∀ 0 <0.001 100 0.99 2.3 9 2.7 ∀ 1.3 <0.001 77 0 ∀ 0 <0.001 100 4.97 11.6 5 1.6 ∀ 1.1 <0.001 86 0 ∀ 0 <0.001 100 24.87 57.8 5 0.8 ∀ 0.7 <0.001 93 0 ∀ 0 <0.001 100 a Polyamine analogues were administered sc to rats at the doses shown in the table. Thirty minutes later, the rats were given castor oil, 5 mL/kg, by gavage. Stool output was monitored for 6-h after castor oil administration. b Weight loss is expressed as g of weight lost per 350 g of rat weight over the 6-h experimental period. c A one-tailed t-test assuming unequal variance was performed on the data of the treated vs control (0 mg/kg) animals for each compound. A value of P <0.05 was considered significant. d Percent reduction was calculated by dividing the mean value from the treated animals (T) by the mean value from the control animals (C), subtracting the resulting quotient from 1.0, and multiplying by 100 &lsqb;i.e., (1.0 − T/C) × 100&rsqb;. NS, not significant. 2 TABLE 2 Activity of Polyamine Analogues Against Stress-Induced Irritable Bowel Syndrome Dose, kg −1 Stool P- % Structure/Abbreviation mg &mgr;mol n Output b Value Reduction d 1 0 0.03125 0.0625 0.125 0.25 0.5 0.99 0 0.0726 0.145 0.29 0.58 1.16 2.3 10 5 5 5 5 5 5 5 # 11.3 ± 4.1 5.4 ± 2.1 2.2 ± 1.3 0.6 ± 0.9 0 ± 0 0 ± 0 0 ± 0 — 0.001 <0.001 <0.001 <0.001 <0.001 <0.001 —52.2 80.5 94.7 100 100 100 a Polyamine analogues were administered sc to rats at the doses shown in the table. Thirty minutes later, the rats were subjected to stress, i.e., a cage filled with water to within 1 cm of the height of a small dish. Stool output was monitored for 6 h after commencement of the stress. b A cone-tailed t-test assuming unequal variance was performed on the data of the treated vs control (0 mg/kg) animals for each compound. A value of P < 0.05 was considered significant. c Stool output is expressed as the number of fecal pellets excreted over the 6-h collection period. d Percent reduction was calculated by dividing the mean value from the treated animals (T) by the mean value from the control animals (C), subtracting the resulting quotient from 1.0, and multiplying by 100 &lsqb;i.e., (1.0-T/C) × 100&rsqb;. NS, not significant.