Methods and compositions for healing ulcers

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METHODS AND COMPOSITIONS FOR HEALING ULCERS

TECHNICAL FIELD 5

The present invention relates to the healing and/or prevention of ulcers in general, and more specifically, to the use of glycyl-L-histidyl-L-lysine, L-lysyl-L-histidylglycine, glycyl-L-histidyl-L-lysine:copper(II), L-lysyl- 1Q L-histidyl-glycine:copper(II), and derivatives thereof within a method for healing and/or prevention of ulcers in warm-blooded animals.

BACKGROUND OF THE INVENTION

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The treatment of stomach ulcers remains a major health problem despite the development of numerous anti-ulcer medications. Traditionally, digestive ulcers have been treated through neutralization of excess stomach acid or through diet and behavioral or emo- 20 tional modification. Well-known stomach acid neutralizers include sodium bicarbonate, magnesium hydroxide, calcium carbonate, aluminum hydroxide, aluminum phosphate, magnesium trisilicate, and tribasic calcium phosphate. Certain polyamine methylene resins have 25 also been tried. Attempts have also been made to inhibit the flow of gastric acid, although these attempts are characterized by rather serious side effects. More specifically, while a compound referred to as cimetidine has been effective in stopping the secretion of stomach 30 acid by blocking histamine sites, it has been found to have certain undesirable characteristics, including impairment of kidney function and mental confusion.

While certain low molecular weight compositions, such as salicylate-copper of diisopropylsalicylate-cop- 35 per, have been reported to inhibit the production of stomach ulcers, these complexes tend to easily dissociate in the stomach into free copper and salicylate, which limits their practical use. In addition, these small copper complexes tend to be poorly soluble under aqueous conditions and must be administered with tissue-irritating solubilizing agents. Another such agent, the penicillamine-copper complex, often produces skin rashes and personality changes ("penicillamine psychosis"). 45

More recent medical treatments involve the use of H2 receptor blockers such as cimetidine, or the use of growth factors such as Epiderman Growth Factor (EGF, Urogastrone) or peptide fragments of EGF. However, these treatments suffer from a number of 50 disadvantages, including instability, difficulty in synthesis and administration, and high production costs.

Therefore, there is a need in the art for an improved composition for healing and/or preventing the formation of ulcers. The present invention provides such 55 composition, and further provides other related advantages.

DISCLOSURE OF THE INVENTION

Briefly stated, the present invention discloses a van- 60 ety of pharmaceutical compositions suitable for use within the methods hereinafter described: (a) a method for reducing the formation of stomach ulcers in warmblooded animals; (b) a method for reducing the secretion of stomach acid in warm-blooded animals; (c) a 65 method for increasing the secretion of cytoprotective mucous in the stomach of warm-blooded animals; and (d) a method for healing established gastric (stomach)

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or duodenal (intestinal) ulcers in warm-blooded animals.

The compositions described herein include glycyl-Lhistidyl-L-lysine (GHL), L-lysyl-L-histidyl-glycine (LHG), glycyl-L-histidyl-L-lysine:copper(II) (GHLCu), L-lysyl-L-histidyl-glycine:copper(II) (LHG-Cu), and various derivatives of GHL-Cu and LHG-Cu.

The derivatives of GHL-Cu have the general formula:

o
II

[glycyl-L-histidyl-lysine-C—R]:copper(II)

wherein R is selected from the group consisting of alkyl moieties containing from 1 to 18 carbon atoms, aryl moieties containing from 6 to 12 carbon atoms, alkoxy moieties containing from 1 to 18 carbon atoms, and aryloxy moieties containing from 6 to 12 carbon atoms, or where R is L-prolyl-L-valyl-L-phenylalanyl-Lvaline or L-valyl-L-phenylalanyl-L-valine.

In addition to the derivatives described above, other chemical modifications may be made to alter the biological activity of GHL and GHL-Cu derivatives. For instance, glycine may be replaced by a variety of other small amino acids, including alanine, serine, and valine. Further, the copper(II) binding affinity of the molecule may be increased by addition of an N-terminal amino acid, such as glycine, to convert glycyl-L-histidyl-Llysine to glycyl-L-glycyl-L-histidyl-L-lysine. In addition, glycine could be added to a derivative as described above to create the corresponding tetrapeptide. The binding affinity for copper(II) of the imadazole group in the histidyl residue may be modified by substitution of 3-methylhistidine for histidine or by extending the lysyl side chains by adding additional carbon atoms to the chain.

The derivatives of LHG-Cu have the general formula:

o
II

[L-lysyl-L-histidyl-glycine-C—R]:copper(II)

wherein R is selected from the group consisting of alkyl moieties containing from 1 to 18 carbon atoms, aryl moieties containing from 6 to 12 carbon atoms, alkoxy moieties containing from 1 to 18 carbon atoms, and aryloxy moieties containing from 6 to 12 carbon atoms, or where R is L-prolyl-L-valyl-L-phenylalanyl-Lvaline or L-valyl-L-phenylalanyl-L-valine.

In addition to the derivatives described above, other chemical modifications may be made to alter the biological activity of LHG and LHG-Cu derivatives. For instance, lysine may be replaced by a variety of other small amino acids, including alanine, serine, and valine. Further, the copper(II) binding affinity of the molecule may be increased by addition of an N-terminal amino acid, such as glycine, to convert L-lysyl-L-histidyl-glycine to glycyl-L-lysyl-L-histidyl-glycine. In addition, glycine could be added to a derivative as described above to create the corresponding tetrapeptide. The binding affinity for copper(II) of the imadazole group in the histidyl residue may be modified by substitution of 3-methylhistidine for histidine or by extending the lysyl side chains by adding additional carbon atoms to the chain.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a photograph of rat stomachs, illustrating 10 the ulcerations in control animals as compared to treated animals. The circled black dots in the stomach wall are stomach ulcers.

FIG. 2 depicts the increase in stomach pH (indicating a stomach acid secretion) with an increasing dosage of 15 GHL-Cu.

FIG. 3 illustrates the decrease in visible ulcer formation with an increasing dosage of GHL-Cu.

FIG. 4 depicts the healing of duodenal ulceration observed following drinking water treatment.

FIG. 5 is a photograph of rat stomachs, illustrating the inhibition of stomach ulcer formation by pretreatment with GHL-Cu.

FIG. 6 illustrates the healing of ethanol-induced gas- ^ trie ulcers.

BEST MODE FOR CARRYING OUT THE
INVENTION

As described herein, GHL, LHG, GHL-Cu, LHG- 3Q Cu and various derivatives thereof may be used in methods for (a) reducing the secretion of stomach acid in warm-blooded animals, (b) increasing the secretion of cytoprotective mucous in the stomach of warmblooded animals, (c) reducing the formation of stomach 35 ulcers in warm-blooded animals, and (d) healing established stomach or intestinal ulcers in warm-blooded animals. Derivatives of the present invention are described in detail in U.S. Pat. No. 4,665,054 and U.S. pat. application Ser. No. 312,295, now U.S. Pat. No. 40 4,877,770, which are hereby incorporated by reference. The derivatives of the present invention may be prepared by esterification, by the removal of a water molecule, or by the addition of a group (either an alcohol, such as octanol, methanol, benzyl alcohol, or NH3) to 45 the carboxylic acid terminus of GHL or LHG, resulting in the formation of a more lipophilic derivative.

GHL, LHG, GHL-Cu, LHG-Cu and several analogs have been tested in animal models of both gastric and duodenal ulcers. GHL-Cu, LHG-Cu and their analogs 50 have been found to prevent gastric ulcer formation caused both by excessive acid secretion and by 95% ethanol ingestion. In the excessive acid (Shay) model system, increasing concentrations of GHL-Cu, LHGCu or analogs thereof decreased stomach acid secretion 55 and resulted in fewer visible lesions when compared with saline controls. Pretreatment with GHL-Cu, LHG-Cu and analogs thereof was found to prevent gastric irritation and ulceration caused by ingestion of 95% ethanol. Administration of GHL-Cu, LHG-Cu 60 and its analogs in the drinking water also accelerate the healing of ulcers caused by the ethanol.

GHL-Cu, LHG-Cu, and analogs thereof also accelerate the healing of established duodenal ulcers. In this model system, ulcers are induced by cysteamine injec- 65 tion. Briefly, rats with surgically confirmed ulcers were treated with GHL-Cu or LHG-Cu in the drinking water. At the end of the study, the treated rats exhibited

GHL—OH+R—H—GHL—R+H20.

In practice, the reaction is most readily carried out by adding the R group to the amino acid lysine prior to the combination of lysine with the other two amino acids to GHL. After the formation and isolation of GHL-R, the copper (II) is chelated to the molecule to form the bioactive complex.

The overall reaction to form the more lipophilic derivatives of GHL-Cu may be characterized:

(1) lysine-OH+R-H->lysine-R+H20

(2) lysine-R -(-blocked L-histidine->blocked L-histidineL-lysine-R

(3) blocked L-histidine-L-lysine-R—>-partially blocked L-histidine-L-lysine-R

(4) partially blocked L-histidine-L-lysine-R+blockedglycine->-blocked glycyl-L-histidine-L-lysine-R

(5) . blocked gycyl-L-Wstidine-L-lysine-R—►glycyl-Lhistidine-L-lysine-R

(6) glycyl-L-histidine-L-lysine-R+copper(II)—>glycylL-histidine-L-lysine-R:copper(II).

The overall reaction to form the more lipophilic derivatives of LHG-Cu is the same as outlined above for GHL-Cu, except glycine-OH is the initial reaction component instead of lysine-OH, and blocked lysine is used in place of blocked glycine in step 4.

The results disclosed herein suggest that GHL, LHG, GHL-Cu, LHG-Cu, and derivatives thereof will exert healing actions on a variety of gastrointestinal diseases, such as colonic healing after anastomosis, lesions occurring subsequent to intestinal and bowel ischemia, necrotizing enterocolitis, and wounds of the mouth, throat, and esophagus. The general healing properties of GHLCu are described in U.S. Pat. Nos. 4,810,693 and 4,760,051, herein incorporated by reference.

Within a preferred embodiment, GHL or LHG, or a derivative of GHL-Cu or LHG-Cu are present in a 1:1 to 2:1 ratio. Within the present invention, it is generally preferred to administer the compositions described herein orally and in a capsule form. Methods for encapsulating compositions (such as in a coating of hard gelatin) for oral administration are well known in the art (Baker, Richard, Controlled Release of Biologically Active Agents, John Wiley and Sons, 1986). It is also generally preferred to administer the compositions in dosages from about 0.1 to 100 mg/kg of host body weight, although the dosage may be influenced by the condition of the patient. Further, it may be preferable to initially begin using a treatment of GHL-Cu or LHG-Cu, and then continue with treatment using the free peptide (GHL or LHG) with or without a small amount of copper(II).

To summarize the examples that follow, Example 1 illustrates the synthesis of glycyl-L-histidyl-L-lysine benzyl estencopper(II). Example 2 demonstrates the synthesis of glycyl-L-histidyl-L-lysine n-octyl ester:copper(II). Example 3 illustrates (A) the synthesis of glycyl-L-histidyl-L-lysine n-stearyl estencopper(II), and (B) its synthesis by an alternative procedure. Based upon either procedure, one skilled in the art could sub. 5

stitute n-palmityl alcohol (16 carbons) for the n-stearyl alcohol (18 carbons) to yield glycyl-L-histidyl-L-lysine n-stearyl ester:copper(II). Example 4 illustrates (A) the synthesis of glycyl-L-histidyl-L-lysyl-L-prolyl-L-valylL-phenylalanyl-L-valine:copper(II) and glycyl and gly- 5 cyl-L-histidyl-L-lysyl-L-valyl-L-phenylalanyl-Lvaline:copper(II) by solid-phase synthesis, and (B) the preparation of glycyl-L-histidyl-L-lysyl-L-valyl-Lphenylalanyl-L-valine by solution synthesis. Example 7 demonstrates the inhibition of stomach acid accumula- io tion, the stimulation of cytoprotective mucous secretion, and a reduction in the formation of stomach ulcers in warmblooded animals. Example 8 demonstrates the healing of established stomach ulcers with GHL-Cu, LHG-Cu and derivatives thereof. Example 9 illustrates i5 the healing of duodenal ulcers following oral administration with compounds of the present invention. Example 10 demonstrates the healing and prevention of ethanol-induced ulcers.

The following examples are offered by way of illus- 2q tration and not by way of limitation.

EXAMPLES

Preparation of GHL.LHG, GHL-Cu and LHG-Cu for

Use in Animals 25

GHL and LHG were purified by dissolving, in glass, distilled water (50 mg/ml), then centrifuging at 20,000 Xg for 1 hour at 3° C. This removes poorly water-soluble material remaining from the synthetic procedure. The supernatant is lyophilized, then passed through a 30 Sephadex G-10 column at 3° C. in a solvent of 0.5% acetic acid. The main peak that elutes behind the solvent front (monitored by absorption at 254 nanometers) is lyophilized to dryness. GHL-Cu and LHG-Cu were prepared by combining purified GHL or LHG with 35 equimolar amounts of cupric acetate and sodium hydroxide, then precipitated by use of ethanol addition and low temperature by published methods (Perkins et al., Inorg. Chim. Acta 67:93-99, 1984).

Sources of chemicals 40

Chemicals and peptide intermediates utilized in the following examples may be purchased from the following suppliers: Sigma Chemical Co. (St. Louis, Mo.); Peninsula Laboratories (San Carlos, Calif.); Aldridge 45 Chemical Co. (Milwaukee, Wis.); Vega Biochemicals (Tucson, Ariz.); Pierce Chemical Co. (Rockford, 111.); Research Biochemicals (Cleveland, Ohio); Van Waters and Rogers (South San Francisco, Calif.); Bachem, Inc. (Torrance, Calif.). 50

EXAMPLE 1

Synthesis of glycyl-L-histidyl-L-lysine benzyl
estencopper(II)

Ne-benzyloxycarbonyl-L-lysine benzyl ester was dis- 55 solved in 1:1 hexane-ethyl acetate and coupled to N°-tbutyloxycarbonyl-N'^-benzyloxycarbonyl-L-histidine using dicyclohexylcarbodiimide as a coupling agent. Sodium bicarbonate (10%) was added and the product extracted into the organic layer. The product, Na-t- 60 butyloxycarbonyl-N'm-benzyloxycarbonyl-L-histidylNc-benzyloxycarbonyl-L-lysine benzyl ester, was crystallized from solution. The N-terminal group of the blocked dipeptide was removed by stirring in 50% trifluoroacetic acid in dichloromethane for 30 minutes, 65 then vacuum evaporated. The product, N'm-benzyloxycarbonyl-L-histidyl-Ne-benzoylcarbonyl-L-lysine benzyl ester, was coupled to lysine with dicyclohexylcar

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bodiimide as a coupling agent. Blocking groups were removed by catalytic hydrogenation using 10% palladium on carbon in glacial acetic acid. After lyophilization, the product, glycyl-L-histidyl-L-lysine benzyl ester, was dissolved in water and purified by ion-exchange chromatography on Dowex 50 X-4 cationexchange resin and elution with 0.1M ammonium hydroxide, the eluate being immediately neutralized with acetic acid. A further passage through an anionexchange column BioRex 63 at neutral pH removed breakdown products with free carboxylic acid groups.

The glycyl-L-histidyl-L-lysine benzyl ester was dissolved in water with equimolar copper acetate added. The pH was raised to neutrality with sodium hydroxide. The solution was centrifuged at 20,000Xg for 1 hour at 3° C. to remove poorly water-soluble material. The supernatant was lyophilized to obtain glycyl-L-histidylL-lysine benzyl ester:copper(II).

EXAMPLE 2

Synthesis of glycyl-L-histidyl-L-lysine n-octyl
estencopper(II)

A mixture of Ne-benzyloxycarbonyl-L-lysine, noctanol, benzene, and p-toluenesulfonic acid monohydrate was refluxed overnight using a DeanStark trap to remove water. After cooling, dry ethyl ether was added. The solution was then allowed to precipitate at 0° C. overnight. A portion of the precipitated solid was added to 50 ml potassium carbonate solution and 50 ml dichloromethane. After extraction, the layers were separated and the organic phase washed with water and brine, then dried with anyhdrous magnesium sulfate. Filtration, evaporation and purification by flash column chromatography gave n-octyl Ne-benzyloxycarbonylL-lysinate. The product was dissolved in tetrahydrofuran and mixed with Na-t-butyloxycarbonyl-L-N'm-benzyloxycarbonyl-L-histidine, isobutyl chloroformate and N-methylmorpholine. After evaporation, water and ethyl acetate were added. The product was extracted into the organic phase, which was dried with anhydrous magnesium sulfate. Filtration, evaporation and purification by flash column chromatography gave n-octyl N°-t-butyloxycarbonyl-N'm-benzyloxycarbonyl-L-histidyl-Ne-benzyloxycarbonyl-L-lysinate.

The product was dissolved in 50% trifluoroacetic acid in dichloromethane for 30 minutes, then evaporated, forming n-octyl N'm-benzyloxycarbonyl-L-histidyl-Ne-benzyloxycarbonyl-L-lysinate. This was dissolved in tetrahydrofuran, and isobutyl chloroformate, N-methylmorpholine and benzyloxycarbonylglycine were added to form n-octyl benzyloxycarbonylglycineN'm-benzyloxycarbonyl-L-histidyl-Nc-benzyloxycarbonyl-L-lysinate. This was dissolved in glacial acetic acid and hydrogenated overnight.

The resultant n-octyl ester of glycyl-L-histidyl-Llysine was converted to the copper complex by the addition of an equimolar quantity of copper diacetate. The pH was raised to neutrality with sodium hydroxide. The solution was centrifuged at 20,000Xg for 1 hour at 3° C. to remove poorly water-soluble material. The supernatant was lyophilized to obtain glycyl-L-histidylL-lysine n-octyl ester:copper(H).

EXAMPLE 3

A. Synthesis of glycyl-L-histidyl-L-lysine n-stearyl ester:copper(II)