WO1995025513A1

WO1995025513A1 - Zinc compound and cyclodextrine for treatment of gastric disorders

Info

Publication number: WO1995025513A1
Application number: PCT/AU1995/000146
Authority: WO
Inventors: Kim Drummond Rainsford; Richard H. Hunt; V. Joyce Goldie
Original assignee: Bellara Medical Products Limited
Priority date: 1994-03-18
Filing date: 1995-03-17
Publication date: 1995-09-28
Also published as: AUPM456894A0

Abstract

A pharmaceutical composition and a method for the treatment of gastric disorders and Helicobacter pylori in the stomach or duodenum comprising a zinc compound and a cyclodextrin or cyclodextrin derivative for instance selected from α-cyclodextrin, β-cyclodextrin or η-cyclodextrin. The pharmaceutical composition may also include a pharmaceutically acceptable acidic compound such as a carboxylic acid.

Description

Zinc compound and cyclodextrin for treatment of gastric disorders.

TECHNICAL FIELD

This invention relates to compositions and methods for the treatment of gastric disorders particularly peptic ulcers.

BACKGROUND ART

Peptic ulcers are ulcers of the stomach or duodenum, and it has been recently discovered that a major contributor to their cause, in the majority of instances, is the presence and action of Helicobacter pylori, a Gram- negative, spiral-shaped microaerophilic bacteria. A great deal of interest has been directed to this primate-specific pathogen, and it has been suggested to be a causative agent of chronic active gastritis (reviewed by Blaser MJ, J Infect Dis 1990; 161:626-633), and gastric or duodenal ulcers (reviewed by Graham DY, J Gastroenterol Hepatol 1991; 6:105-113). More recently, Helicobacter pylori infections have also been identified as a significant risk factor for gastric cancer (The Eurogast Study Group, Lancet 1993; 341:1359-1362).

Helicobacter pylori infection is almost always associated with inflammation. However, peptic ulcer disease and gastric carcinoma occur in only a subset of individuals infected chronically with Helicobacter pylori.

Prior to the discovery that Helicobacter pylori is associated with peptic ulcer disease, it was believed that healing of peptic ulcers, particularly gastric ulcers, could be achieved solely through use of H2-receptor blocking agents such as cimetidine and ranitidine which serve to reduce secretion of gastric acid to levels which allow healing of the ulcer. Although healing is achieved with a high level of success in the first instance, relapses occur at a very high rate, with continued recurrence despite further treatment with the H2-blockers. A more recent development has been the use of gastric acid (proton) pump inhibitors which inhibit the secretion of acid through the membranes of the parietal cells. Omeprazole is the first of the proton pump inhibitors to have been commercialized, and it is singularly effective in suppressing gastric acid secretion. Similarly to the H2-blockers, the proton pump inhibitors allow healing of ulcers, but also with relapses after some months. The cause of the relapses in ulceration after treatment with established therapies, eg H2-blockers or proton pump inhibitors has been attributed to the presence of Helicobacter pylori in the gastrointestinal tract of these patients. The Helicobacter pylori infection is widespread among patients with peptic ulcers: over 80% of patients with gastric ulcers and with duodenal ulcers have the infection. To dispel the theory that the infection may be caused by the ulcers, studies have shown that relapses are reduced to extremely low levels if Helicobacter pylori is eliminated as part of the treatment. Treatment of Helicobacter pylori is commonly achieved by triple therapies such as a H2-blocker or a proton pump inhibitor in combination with metronidazole and one antibiotic, or with two antibiotics (either clarithromycin or amoxycillin). This treatment has replaced the prior standard of bismuth, tetracycline and metronidazole. Although they have shown to be effective, the triple therapies treatments are unsatisfactory as they use antibiotics which are prone to development of resistance, they produce side effects which are sufficiently severe to require withdrawal of the therapy, they are very expensive and they are inconvenient as they require taking a number of doses for each of the three medications at different times of the day.

In view of the overwhelming evidence that Helicobacter pylori eradication results in cure of peptic ulcer disease, and the strong risk factor of gastric cancer in patients with Helicobacter pylori, there is a need for a convenient, safe and relatively cheap therapy for eradicating the Helicobacter pylori disease. Although the following description refers to treatment of peptic ulcer disease, the eradication of Helicobacter pylori is also a requirement for gastric cancer prevention in patients at risk to this disease: the described invention, therefore, applies to all situations where eradication of Helicobacter pylori is required.

There are two problems which need to be solved in patients who have peptic ulcer disease in association with Helicobacter pylori;

(i) provision of an environment in which peptic ulcers will heal at an acceptable rate, and

(ii) eradication of Helicobacter pylori from the gastrointestinal tract.

The invention described herein encompasses the discovery of a pharmaceutical delivery system which delivers a treatment to the gastrointestinal tract in a form and at a rate which is optimal for successfully solving both these problems.

Zinc is an essential trace element, which in humans and animals serves as both a nutrient and a drug. Zinc has two very important basic properties which allow it to be so important physiologically and biochemically. Firstly, zinc is virtually non-toxic compared with iron, copper, mercury, and other metals. Secondly, its physical and chemical properties, including its generally stable association with macromolecules and its coordination flexibility, make it highly adaptable to meeting the needs of proteins and enzymes that carry out diverse biological functions.

Zinc has been found to have antibacterial activity. All living systems need zinc for growth, with bacteria generally requiring 10"^M to lO^M. At higher concentrations zinc inhibits cellular function. In bacteria, concentrations of zinc between lO^'-^M and 10^~3_v_ reportedly decrease sugar transport, amino acid uptake, and electron transfer. Inhibition of all of these is probably mediated, at least in part, by the binding of zinc to sulfhydryl or histidine residues and the subsequent inhibition of enzyme function. Thus, microbial metabolism is inadequate without trace amounts of zinc but in the presence of concentrations of zinc that exceed the normal physiologic range these concentrations of zinc are inhibitory or toxic to cellular activities and growth of bacteria.

Zinc has been available pharmaceutically for many years as a variety of salts: eg chloride, sulphate, acetate, gluconate, orotate, and more recently zinc acexamate and zinc L-carnosine. These salts can be toxic to mucosal cells if administered in a form which allows formation of a concentrated solution of zinc ions, and this can occur in the presence of gastric acid when administered orally. It has been found that these salts are ineffective in treating bacteria such as Helicobacter pylori in acidic media.

Zinc sulphate has been reported to enhance the healing of gastric ulcers in patients with gastric ulcer (Frommer DJ, Med J Aust 1975; 2:793-796). Zinc salts and complexes with amino acids have been shown to have anti-ulcer activity in a variety of experimentally-induced ulcer models in laboratory animals (Cho CH and Pfeiffer CJ. In: Pfeiffer CJ (Ed) Drugs and Peptic Ulcer Vol 1. Therapeutic Agents for Peptic Ulcer Disease. CRC Press. Boca Raton, 1982, pp 147-153). Zinc acexamate has also been used successfully in the treatment of gastric and duodenal ulcers in man. Apart from the consistently positive results with zinc acexamate, zinc salts have not been found to give reliable and consistent efficacy in the treatment of gastric or duodenal ulcers. One problem that occurs at the doses used (greater than 40 mg zinc) is that oral zinc can produce a metallic taste, nausea and epigastric pain or mucosal irritation, and can cause mucosal erosions and overt copper deficiency anaemia. In addition, the problem of eradicating Helicobacter pylori in peptic ulcer disease has not been overcome with the zinc compounds tested.

A new form of zinc has been recently introduced; zinc monoglycerolate which is produced by mixing zinc oxide or zinc hydroxide together with glycerol at temperatures in excess of 200°C (Taylor R M and Brock A J, AU Patent 554151, 1980; AU Patent 591706, 1986). Zinc monoglycerolate is a white lubricious powder, the latter property being imparted by its polymeric two-dimensional structure. The compound is highly insoluble in water, but it is slowly soluble in a variety of biological fluids which impart controlled release properties to the zinc (Fairlie D, Whitehouse MW and Taylor RM, Agents Actions 1992; 36:152-158). The formula for zinc monoglycerolate is (C3H6θ3Zn)_n, and its structure is:

In a series of studies using rats, zinc monoglycerolate was found to be an effective agent in preventing ulcer development in a wide range of model systems and it may be more effective than zinc salts because of the controlled slow-release of zinc from the complex (Rainsford and Whitehouse, J Pharm Pharmacol 1992; 44:476-482). Zinc monoglycerolate has been shown in in vitro studies to have a strong bacteriostatic action against Staphylococcus aurens and Escherichia coli and good bacteriostatic action against Pseudomonas aeruginosa and the yeast Candida albicans. In addition, zinc monoglycerolate has been shown to have very effective antifungal activity against Trichophyton rubrum and a Mixed Mildew which is a mixture of Aspergillus niger, Aspergillus flavas, Syncephalastrutn racmosum, Alternaria radicina and Paecilomyces varioti.

DISCLOSURE OF THE INVENTION

In vitro studies were performed to test the antibacterial activity of zinc monoglycerolate against Helicobacter pylori. It was found that zinc monoglycerolate had no antibacterial activity against Helicobacter pylori when used as a suspension of the powder, or when it was dissolved in citric acid solution. On the other hand, it has been surprisingly found that addition of β-cyclodextrin to a solution of zinc monoglycerolate in citric acid caused the zinc monoglycerolate to be remarkably effective in inhibiting the growth of Helicobacter pylori.

It has since been established that the β-cyclodextrin is surprisingly effective in enhancing the solubilizing of the zinc monoglycerolate, thus providing a delivery system for the zinc monoglycerolate, so that zinc ions can be released at a rate which is sufficient to provide concentrations of zinc ions which are bacteriostatic against Helicobacter pylori. It has also been discovered that formulating zinc monoglycerolate with cyclodextrins provides a much more effective formulation for healing peptic ulcers in rats than is provided by zinc monoglycerolate powder alone.

Zinc monoglycerolate therefore has the two properties required in the treatment of peptic ulcer disease which is associated with Helicobacter pylori: (i) provision of an environment in which peptic ulcers will heal at an acceptable rate, and (ii) eradication of Helicobacter pylori from the gastrointestinal tract.

It has also been surprisingly found that zinc sulphate, when formulated together with β-cyclodextrin, is effective in inhibiting a wide range of strains of Helicobacter pylori, whereas zinc sulphate is ineffective when β- cyclodextrin is excluded. In one form therefore the invention is said to reside in a pharmaceutical composition for the treatment of gastric disorders comprising a zinc compound and a cyclodextrin or cyclodextrin derivative.

The pharmaceutical composition may further include a pharmaceutically acceptable acidic compound and preferably a carboxylic acid.

The zinc compound may be selected from the group comprising zinc acetate, zinc acexamate, zinc bacitracin, zinc caprylate, zinc carbonate, zinc L- carnosine, zinc chloride, zinc citrate, zinc lactate, zinc monoglycerolate, zinc oleate, zinc orotate, zinc oxalate, zinc oxide, zinc permanganate, zinc p- phenolsulphate, zinc phosphate, zinc propionate, zinc stearate, zinc sulphate, zinc tartrate, and zinc valerate.

The cyclodextrin may be selected from α-cyclodextrin, β-cyclodextrin or γ- cyclodextrin or derivatives thereof.

The zinc compound may be present in an amount of from 0.15 mmole to 4 mmole. The molar ratio of cyclodextrin to zinc compound may be from 10:1 to 1:100. The molar ratio of carboxylic acid to zinc compound may be from 5:1 to 1:5.

In an alternative form the invention may be said to reside in a pharmaceutical composition for the eradication of Helicobacter pylori in the stomach or duodenum of human patients comprising a zinc compound and a cyclodextrin or cyclodextrin derivative.

In an alternative form the invention may be said to reside in a method of treatment of gastric disorders including the step of oral administration of a pharmaceutical composition comprising a zinc compound and a cyclodextrin or cyclodextrin derivitive.

In an alternative form the invention may be said to reside in a method of for the eradication of Helicobacter pylori in the stomach or duodenum of human patients including the step of oral administration of a pharmaceutical composition comprising a zinc compound and a cyclodextrin or cyclodextrin derivitive. This generally describes the invention but to assist with understanding of the invention reference will now be made to the best method known of performing the invention and examples of the invention.

BEST MODE OF CARRYING OUT THE INVENTION

Studies of the inhibitory effects on Helicobacter pylori growth, of zinc monoglycerolate or zinc sulphate with β-cyclodextrin as outlined in the included examples, have shown that the antibacterial effect of both zinc compounds is enhanced by the presence of the cyclodextrin. The effects of zinc monoglycerolate or zinc sulphate could be due to Zn*-*+ ions, the association with the cyclodextrin providing a system which enables penetration or uptake of Zn2+ ions into the bacterial cell where the zinc ion exerts its inhibitory effects. Irrespective of the exact mechanism of action, it has been found that a lipophilic and water insoluble zinc compound, zinc monoglycerolate, and a hydrophilic and water soluble zinc compound, zinc sulphate, are equally effective in inhibiting the growth of Helicobacter pylori when formulated with a cyclodextrin and optionally a carboxylic acid, as shown with β-cyclodextrin and optionally citric acid.

The invention therefore includes all the practical zinc compounds for oral administration, and these include: zinc acetate, zinc acexamate, zinc bacitracin, zinc caprylate, zinc carbonate, zinc L-carnosine, zinc chloride, zinc citrate, zinc lactate, zinc monoglycerolate, zinc oleate, zinc orotate, zinc oxalate, zinc oxide, zinc permanganate, zinc p-phenolsulphate, zinc phosphate, zinc propionate, zinc stearate, zinc sulphate, zinc tartrate, and zinc valerate. These examples do not restrict or limit the range of possible zinc compounds that could be used in the invention, for example there are a range of possibilities for forming useful compounds of zinc with higher polyols than the glycerol used in making zinc monoglycerolate.

Cyclodextrins are enzymatically modified starches made up of glucopyranose units. Three different basic cyclodextrins are known: the α- cyclodextrin consists of six glucopyranose units, the β-cyclodextrin of seven units, and the γ-cyclodextrin of eight units. All of the cyclodextrins are crystalline and nonhygroscopic, and they feature a cylinder-shaped, macro- ring structure with a large internal axial cavity. The outer surface of a cyclodextrin molecule is hydrophilic, whereas the internal cavity is apolar. When the cavity inside a cyclodextrin is filled by a molecule of another substance, the result is an inclusion complex, an entity consisting normally of two molecules in which the host molecule contains the guest molecule, totally or in part, using only physical forces. No covalent bonding is involved. Cyclodextrins can contain a variety of guest molecules of the size of one or two benzene rings to form crystalline inclusion complexes. Larger guest molecules, with side chains of comparable size, can also be contained.

In aqueous solution, the slightly apolar cyclodextrin cavities are occupied by water molecules. Water molecules are polar, and polar-apolar interaction makes them energetically unfavourable as guest molecules. For this reason, the high-enthalpy water molecules can be replaced by appropriate guest molecules which are less polar than water. Although the host:guest ratio is usually 1:1, one or two (or sometimes even three) cyclodextrin molecules can contain one or more guest molecules.

Inclusion complexes can be isolated as stable crystalline substances. When these complexes are dissolved, an equilibrium is established between dissociated and associated species.

Cyclodextrin molecules are relatively large with an outer surface that is strongly hydrophilic. The molecule brings the hydrophobic guest molecules into solution, keeps them in the dissolved state, and releases them as they are removed from solution in accordance with a specific dissociation equilibrium constant. When a cyclodextrin is administered orally, only insignificant amounts are absorbed intact through the intestinal tract. Most of the cyclodextrin is metabolised by the microflora in the colon. The primary metabolites (acyclic maltodextrins, maltose, and glucose) are then further metabolised and absorbed in the same way as starch and are finally excreted as carbon dioxide and water.

Natural cyclodextrins have various drawbacks, one of which is their cavity size and another is poor water solubility. α-Cyclodextrin, with six glucopyranose units, has the smallest cavity (internal diameter almost 5 A), which is mostly too small for a pharmaceutical molecule. β-Cyclodextrin, with seven glucopyranose units, is more convenient (internal diameter almost 6 A). γ-Cyclodextrin, with eight glucopyranose units and an internal diameter of almost 8 A, should be the best one, but it is not in fact intensively produced and it is too expensive to use on an industrial scale. β-cyclodextrin is the most promising drug-complexing agent due to its cavity size and the fact that it is also the cheapest to manufacture, β-cyclodextrin, however, has a surprisingly low solubility in water compared with the other cyclodextrins. At room temperature β-cyclodextrin has a water solubility of 1.85 g/100 mL, compared with α-cyclodextrin at 14.5 g/100 mL and γ- cyclodextrin at 23.3 g/100 mL. The β-cyclodextrin also has the property that it is more inclined to crystallize than the other cyclodextrins.

The above problems with β-cyclodextrin mean that it is preferable to modify it to improve its solubility and limit its strong tendency to crystallize, if it is to be a practical proposition for pharmaceutical delivery systems.

Fortunately, any replacement of hydroxyl groups by an alkyl or aryl ether or amino or ester group results in a dramatic increase in solubility, even if the replacement group is hydrophobic. There have been two approaches; random replacement of hydroxyl groups which is in some circles regarded as being more technically feasible; and preparation of a well-defined homogeneous derivative. A large number of derivatives of cyclodextrins have been prepared, but only the non-toxic and relatively simple derivatives are possibilities for pharmaceutical applications.

Derivatives which have an increased water solubility have been obtained by alkylation of the hydroxyl groups (methyl-, hydroxypropyl-, hydroxyethyl-, and carboxymethyl-cyclodextrins), by substitution of primary hydroxyl groups by saccharides (glycosyl- and maltosyl-cyclodextrins), by esterifying one or more free hydroxyl groups (eg succinylated-β-cyclodextrin), by polymerization of cyclodextrins, and by substituting primary or secondary hydroxyl groups by amine groups (eg for primary hydroxyl group substitution, 6-^-amino-6*^-deoxy-β-cyclodextrin) .

The cyclodextrin derivatives are able to provide a wider range of possibilities for changes in solubility, bioavailability, and stability than are possible with their parent cyclodextrins. Most of the cyclodextrin derivatives are highly water soluble and can therefore be expected to provide a greater increase in the water solubility of the guest molecule than can the natural cyclodextrins.

It is not known, however, whether zinc monoglycerolate forms inclusion complexes with cyclodextrins, or whether the presence of the cyclodextrin causes an additional activity which, in the presence of zinc ions, combines to provide an enhanced antibacterial activity which inhibits the Helicobacter pylori.

The function of the citric acid is to solubilize the zinc compound, essentially by hydrolysis to zinc ions. Thus the requirement for an acidic component depends on the type of zinc compound being considered and the gastric environment of a particular patient. If the zinc compound is a soluble zinc salt there will be no need for an accompanying acid to aid in solubilizing the zinc compound.

Suitable organic acids include saturated or unsaturated aliphatic mono-, di- and tricarboxylic acids and hydroxycarboxylic acids, such as acetic acid, propionic acid, succinic acid, adipic acid, sorbic acid, citric acid, malic acid, lactic acid, glutaric acid, tartaric acid, and fumaric acid; and also aminoacids, such as glycine, glutamic acid and aspartic acid; and furthermore optionally substituted aromatic carboxylic acids, such as benzoic acid, gallic acid, bile acids and resin acid; and also polysaccharides such as alginic acid; and where appropriate the acid alkali salts of any of these organic acids. Also suitable are boric acid, salts that can be hydrolyzed through an acidic reaction such as aluminium sulphate, acidic phosphates such as sodium dihydrogen phosphate and potassium dihydrogen phosphate, and acidic sulfates such as sodium hydrogen sulfate. Also possible are nicotinic acid, ascorbic acid and salicylic acid and salicylates, but these compounds are unlikely to be used in practice for this purpose as they have therapeutic activity of their own.

Preferred acids include the food acids: citric, tartaric, malic, fumaric, adipic and succinic. Acid anhydrides can be derived from certain of these acids. When mixed with water they are hydrolyzed to the corresponding acid, for example succinic anhydride and citric anhydride.

As the Helicobacter pylori bacterium resides in the gastrointestinal tract, a treatment using the compounds of the present invention will preferably be oral, and any of the usual pharmaceutical media, aids and excipients may be used. For liquid oral preparations, one may utilize water, glycols, oils, alcohols or the like, along with colouring agents, preservatives, flavours or the like, to prepare solutions, elixirs, syrups, suspensions, etc. For solid oral preparations, one may utilize conventional granulating agents, lubricants, binders, disintegrating agents, fillers, glidants or the like, to prepare such solid dosage forms as capsules, tablets, sachets, granules or powders. A pharmaceutical composition according to the present invention may have a range of proportions of constituents as set out below:

Zinc dosage: 10 mg to 250 mg elemental zinc equivalent, or 0.15 mmole to 4 mmole; preferably 25 mg to 100 mg elemental zinc equivalent, or 0.4 to 1.5 mmole.

BCD:ZMG from 10:1 to 1:100, preferably 2:1 to 1:20 on a molar basis

Carboxylic acid:ZMG from 5:1 to 1:5, preferably 2:1 to 1:2 on a molar basis

BCD is β-cyclodextrin and ZMG is zinc monoglycerolate

It should be noted that the carboxylic acid is only required if the zinc compound is insoluble or poorly soluble in aqueous media. Example 1

A solid phase agar plate qualitative technique was used to determine whether various formulations of zinc compounds were effective in inhibiting the growth of a number of strains of Helicobacter pylori.

Plates were first poured with an agar layer containing the zinc compounds at the required concentration and pH. The inoculum containing the Helicobacter pylori at a density of approximately 10'--' organisms /mL was then delivered onto the agar layer using a multipipettor. The plates were then incubated under microaerobic conditions at 37°C for 5 days when they were assessed. Details of the preparation of the solutions are as follows.

Buffered Peptone Water (BPW)

Proteose Peptone No. 3 Difco 10 g NaCl 5 g

Na₂HPθ4.12H₂0 3.5 g

KH₂P0₄ 1.5 g

Distilled water 1 L

These components were dissolved and the pH adjusted with NaOH to pH 5.3 or 7.4.

Zinc Stock Solutions

155 mg of zinc monoglycerolate (ZMG, CsHgOsZn, MW=155.4), or 161 mg ZnSO 7H2θ (MW= 287.6), was dissolved in 10 mL citric acid (0.1M, pH 3.3) and filter sterilized using a Gelman 0.22 μm disposable filter. A citric acid control was used with no addition of zinc compound, and this control was diluted and poured onto the plates in an equivalent manner to the zinc-containing solutions.

1 in 10 dilutions were made with BPW to achieve concentrations of

1.55 mg/mL for zinc monoglycerolate and 0.903 mg/mL for ZnSθ4-

For those solutions requiring β-cyclodextrin (BCD), 100 mg was added to each 100 mL of solution, giving 1.0 mg/mL of BCD. The weight ratio of ZMG to BCD was therefore 1.55:1, and this was constant through all dilutions. Similarly for ZnSθ4 the weight ratio was

0.903:1 for ZnSO^BCD. The molecular weight for β-cyclodextrin is 1135 daltons, and the molar ratios of ZMG and ZnSθ4 to BCD were therefore 11.35:1 and 6.36:1, respectively.

Speed of preparation and subsequent handling of the solutions was necessary and strictly adhered to as zinc monoglycerolate hydrolyzes in aqueous media. The total procedure was usually completed within 30 minutes. Buffered Peptone Medium for Plates CBPM)

Oxoid purified agar was added to BPW (1 g agar to 100 mL BPW) and the solution was sterilized by autoclaving at 121°C for 20 minutes. The medium was allowed to cool to 50°C and the following were added if required in the experiment: horse serum: 5 mL horse serum made up to 100 mL with BPW triphenyltetrazolium chloride (TTC): 5 mg of 1% aqueous solution /L medium

Dilutions for Pouring onto Plates:

The stock solutions were diluted twice with BPW, and finally 2 mL of each diluted solution was mixed with 18 mL of BPM to give the required final concentration of metal compound for pouring onto the plates.

The final pH was checked and found to be 4.9-5.4 for a starting pH of 5.3 for the BPW, or 6.9-7.2 for the staring pH of 7.4 for the BPW.

Plates of media only, of media plus horse serum, and of media plus

BCD were run as controls throughout the experiments against all the strains of Helicobacter pylori. These controls showed no inhibition to growth of the bacteria.

Inoculum

Twenty six strains of Helicobacter pylori were used: 5 reference strains (from LCDC Ottawa, Canada), and 21 clinical strains. Suspensions of each strain of Helicobacter pylori were made in BPW to a density comparable to McFarland 0.5 standard to approximately 1 x lO*-*¹ organisms /mL. The inoculum was then surface inoculated on the plates using a multipipettor. The organisms had been grown for 48 hours on chocolate agar prior to making the suspensions.

Incubation Conditions The plates were incubated under microaerobic conditions at 37°C.

They were examined after 3 days, and finally assessed after 5 days incubation. Results

The results of these experiments are summarized in Tables 1-3. In the assessments the following designations were given:

Assessment: + confluent growth

± uncountable colonies

- no growth

NT: not tested at this concentration

Where possible, ED50 values for the results given in Tables 1-3 were calculated by probit analysis from the results given in Tables 1-3 using the probit method of Finney (D J Finney 1971, "Probit Analysis", 3rd Edition, Cambridge University Press). These values are given in Table 4, together with limit values where these estimates could not be made. This approach has been used to quantitate the effectiveness of inhibition, recognizing at the same time that each strain of Helicobacter pylori will have a different sensitivity. The estimates of ED50 are therefore not applicable elsewhere; they are used here simply for qualitative comparisons between the different combinations of zinc salts, with or without BCD. The same 26 strains of Helicobacter pylori were used in each experiment.

Referring to Table 4, it will be seen that in all experiments, the addition of BCD greatly enhanced the inhibitory effect of each zinc agent against growth of Helicobacter pylori. Both with and without the addition of BCD the results were similar when comparing ZMG and ZnSθ4- It was also observed that the inhibitory effect was greater at the higher pH for both zinc compounds.

It was concluded from these experiments that β-cyclodextrin is effective in enhancing the inhibitory effect of zinc compounds against growth of Helicobacter pylori. Table 1:

Zinc monoglycerolate

Concentration of ZMG (mmole/L)

Other Total pH 0.50 0.32 0.25 0.193 0.161 0.097 components No. of strains

Horse serum 26 5.0 + 18 21 22 24 24 25

TTC + 0 0 1 0 0 0

- 8 5 3 2 2 1

BCD 26 5.0 + 0 0 0 0 0

TTC + 0 0 0 0 0 NT

- 26 26 26 26 26

Horse serum 26 7.2 + 1 25 13 17 16

TTC ± 10 0 3 2 2 NT

- 15 1 10 7 8

BCD 26 7.2 + 0 0 0 0 0

TTC + 0 0 0 0 0 NT

- 26 26 26 26 26

Table 2:

Zinc monoglycerolate, including lower concentrations

Concentration of ZMG (mmole/L)

Other Total PH 0.50 0.32 0.193 0.129 0.064 0.032 0.016 components No. of strains

Horse serum 26 5.1 + 26 26 26 26 26 26 26

TTC + 0 0 0 0 0 0 0

- 0 0 0 0 0 0 0

BCD 26 5.1 + 0 0 1 3 0 0 0

TTC ± 0 0 0 0 1 0 0

- 26 26 25 23 25 26 26

Horse serum 26 7.2 + 4 5 12 15 26 26 26

TTC + 7 7 5 6 0 0 0

- 15 14 9 5 0 0 0

BCD 26 7.2 + 0 0 0 0 0 0 0

TTC ± 0 0 0 0 0 0 0

- 26 26 26 26 26 26 26 Table 3: Zinc sulphate

Concentration of ZJ1SO4 (mmole/L)

Other Total pH 0.281 0.181 0.111 0.070 0.035 0.017 0.009 components No. of strains

Horse serum 26 5.1 + 26 26 26 26 26 26 26

TTC ± 0 0 0 0 0 0 0

- 0 0 0 0 0 0 0

BCD 26 5.1 + 0 1 1 1 1 1 5

TTC ± 0 3 2 1 0 3 10

- 26 22 23 24 25 22 11

Horse serum 26 7.2 + 5 16 25 25 25 25 25

TTC ± 3 9 0 1 1 1 0

- 18 1 1 0 0 0 1

BCD 26 7.2 + 0 0 2 0 0 0 0

TTC ± 0 0 1 0 0 0 0

- 26 26 23 26 26 26 26

Table 4:

Estimated values for ED50 for experiments from Tables 1-3

Example 2

To obviate matrix /diffusion effects which might occur with solid agar media as used in Example 1, experiments were performed using a liquid phase system in Microtiter plates. Buffered peptone water (BPW) pH 5.1 and pH 7.2 were prepared as described in Example 1 and they were used without addition of agar. Horse serum (5% of final concentration) or β-cyclodextrin (1 mg/mL of final concentration) were added to the BPW. Triphenyltetrazolium chloride (TTC) was added to most of the individual experiments, with omission from some to demonstrate that TTC had no influence on the effects of the zinc compounds.

Five clinical isolates and one reference standard isolate of Helicobacter pylori were used as test organisms.

The results are presented in Table 5.

Table 5

Liquid phase inhibition of Helicobacter pylori by ZMG, with and without

BCD

The results in Table 5 show that ZMG with CD produced inhibition of growth of Helicobacter pylori in all strains at pH 7.2 and in 5/6 strains at pH 5.1.

ZMG alone in serum had no effect at pH 5.1; with inhibition being shown in 3 of the strains at pH 7.2, and partial inhibition in a further 1 strain at pH 7.2.

The results again demonstrated the beneficial effect of β-cyclodextrin on ZMG in inhibiting the growth of Helicobacter pylori.

Claims

1/ A pharmaceutical composition for the treatment of gastric disorders comprising a zinc compound and a cyclodextrin or cyclodextrin derivative.

2/ A pharmaceutical composition as defined in Claim 1 further including a pharmaceutically acceptable acidic compound.

3/ A pharmaceutical composition as defined in Claim 1 wherein the zinc compound is selected from the group comprising zinc acetate, zinc acexamate, zinc bacitracin, zinc caprylate, zinc carbonate, zinc L-carnosine, zinc chloride, zinc citrate, zinc lactate, zinc monoglycerolate, zinc oleate, zinc orotate, zinc oxalate, zinc oxide, zinc permanganate, zinc /?-phenolsulphate, zinc phosphate, zinc propionate, zinc stearate, zinc sulphate, zinc tartrate, and zinc valerate.

4/ A pharmaceutical composition as defined in Claim 1 wherein the cyclodextrin is selected from α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin or derivatives thereof.

5/ A pharmaceutical composition as defined in Claim 2 wherein the acidic compound is a carboxylic acid.

6/ A pharmaceutical composition as defined in Claim 1 wherein the zinc compound is present in an amount of from 0.15 mmole to 4 mmole.

7/ A pharmaceutical composition as defined in Claim 1 wherein the molar ratio of cyclodextrin to zinc compound is from 10:1 to 1:100.

8/ A pharmaceutical composition as defined in Claim 5 wherein the molar ratio of carboxylic acid to zinc compound is from 5:1 to 1:5.

9/ A pharmaceutical composition for the eradication of Helicobacter pylori in the stomach or duodenum of human patients comprising a zinc compound and a cyclodextrin or cyclodextrin derivative.

10/ A pharmaceutical composition as defined in Claim 9 further including a pharmaceutically acceptable acidic compound. 11/ A pharmaceutical composition as defined in Claim 9 wherein the zinc compound is selected from the group comprising zinc acetate, zinc acexamate, zinc bacitracin, zinc caprylate, zinc carbonate, zinc L-carnosine, zinc chloride, zinc citrate, zinc lactate, zinc monoglycerolate, zinc oleate, zinc orotate, zinc oxalate, zinc oxide, zinc permanganate, zinc p-phenolsulphate, zinc phosphate, zinc propionate, zinc stearate, zinc sulphate, zinc tartrate, and zinc valerate.

12/ A pharmaceutical composition as defined in Claim 9 wherein the cyclodextrin is selected from α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin or derivatives thereof.

13/ A pharmaceutical composition as defined in Claim 10 wherein the acidic compound is a carboxylic acid.

14/ A pharmaceutical composition as defined in Claim 9 wherein the zinc compound is present in an amount of from 0.15 mmole to 4 mmole.

15/ A pharmaceutical composition as defined in Claim 9 wherein the molar ratio of cyclodextrin to zinc compound is from 10:1 to 1:100.

16/ A pharmaceutical composition as defined in Claim 13 wherein the molar ratio of carboxylic acid to zinc compound is from 5:1 to 1:5.

17/ A method of treatment of gastric disorders including the step of oral administration of a pharmaceutical composition comprising a zinc compound and a cyclodextrin or cyclodextrin derivitive.

18/ A method as defined in Claim 17 wherein the cyclodextrin is selected from α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin or derivatives thereof.

19/ A method as defined in Claim 17 wherein the pharmaceutical composition further includes a pharmaceutically acceptable acidic compound.

20/ A method as defined in Claim 19 wherein the acidic compound is a carboxylic acid. 21/ A method as defined in Claim 17 wherein the zinc compound is selected from the group comprising zinc acetate, zinc acexamate, zinc bacitracin, zinc caprylate, zinc carbonate, zinc L-carnosine, zinc chloride, zinc citrate, zinc lactate, zinc monoglycerolate, zinc oleate, zinc orotate, zinc oxalate, zinc oxide, zinc permanganate, zinc p-phenolsulphate, zinc phosphate, zinc propionate, zinc stearate, zinc sulphate, zinc tartrate, and zinc valerate.

22/ A method as defined in Claim 17 wherein the zinc compound is administered in a dosage of from 0.15 mmole to 4 mmole.

23/ A method as defined in Claim 17 wherein the molar ratio of cyclodextrin to zinc compound administered is from 10:1 to 1:100.

24/ A method as defined in Claim 20 wherein the molar ratio of carboxylic acid to zinc compound administered is from 5:1 to 1:5.

25/ A method of for the eradication of Helicobacter pylori in the stomach or duodenum of human patients including the step of oral administration of a pharmaceutical composition comprising a zinc compound and a cyclodextrin or cyclodextrin derivitive.

26/ A method as defined in Claim 25 wherein the cyclodextrin is selected from α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin or derivatives thereof.

27/ A method as defined in Claim 25 wherein the pharmaceutical composition further includes a pharmaceutically acceptable acidic compound.

28/ A method as defined in Claim 27 wherein the acidic compound is a carboxylic acid.

29/ A method as defined in Claim 25 wherein the zinc compound is selected from the group comprising zinc acetate, zinc acexamate, zinc bacitracin, zinc caprylate, zinc carbonate, zinc L-carnosine, zinc chloride, zinc citrate, zinc lactate, zinc monoglycerolate, zinc oleate, zinc orotate, zinc oxalate, zinc oxide, zinc permanganate, zinc p-phenolsulphate, zinc phosphate, zinc propionate, zinc stearate, zinc sulphate, zinc tartrate, and zinc valerate. 30/ A method as defined in Claim 25 wherein the zinc compound is administered in a dosage of from 0.15 mmole to 4 mmole.

31/ A method as defined in Claim 25 wherein the molar ratio of cyclodextrin to zinc compound administered is from 10:1 to 1:100.

32/ A method as defined in Claim 28 wherein the molar ratio of carboxylic acid to zinc compound administered is from 5:1 to 1:5.