WO2011139237A1

WO2011139237A1 - Composition for the treatment of helicobacter pylori (h. pylori) infectious

Info

Publication number: WO2011139237A1
Application number: PCT/SG2011/000178
Authority: WO
Inventors: Brian Damian Pethica; Anthony Morris Roberton; Charmian Jocelyn O'connor
Original assignee: Synergy Pharmaceuticals Pte Ltd
Priority date: 2010-05-05
Filing date: 2011-05-05
Publication date: 2011-11-10
Also published as: GB201007446D0; TW201200154A

Abstract

A pharmaceutical composition comprising a surfactant and a fatty acid, wherein the molar ratio of surfactant to fatty acid is in the range of 0.2:1 to 3:1 for the treatment of a H. pylori infection is provided. The use of the composition, a pharmaceutical dosage form and a kit is also provided.

Description

COMPOSITION FOR THE TREATMENT OF HELICOBACTER PYLORI (H. PYLORI) INFECTIOUS

Technical Field

The invention is directed to the treatment of gastric 5 and/or duodenal Helicobacter pylori infections. In particular, the invention is directed to a pharmaceutical composition for use in such a treatment.

Background

10 H. pylori is a Gram-negative bacterium that can inhabit various areas of the stomach and duodenum. It causes a chronic low-level inflammation of the stomach lining and is strongly linked to the development of duodenal and gastric ulcers and stomach cancer. Over 80%

15 of individuals- infected with the bacterium are asymptomatic.

More than 50% of the world's population harbour H. pylori in their upper gastrointestinal tract. Infection is more prevalent in -developing countries, and incidence 20 is decreasing in .western countries. The route of transmission . is unknown, although it is known that individuals typically become infected in childhood.

Ή. pylori -has been shown subsequently to be the cause of more than 90% of duodenal ulcers, the majority of 25 gastric ulcers, and is an important causal factor of gastric carcinoma. There is also evidence to suggest that H. pylori colonisation in the stomach could be involved in the aetiology of a variety _o_f _ _non^ga^..trointestinal conditions.

30 Eradication of H. pylori from the stomach often relies on a combination therapy, usually a proton pump- inhibitor (for acid suppression) and two or more types of antibiotic. Increasing antibiotic resistance has made treating the infection significantly more difficult and side effects are common. In addition to this, there are issues of patient compliance with the treatment regime. The standard triple therapy requires use of the drugs by the patient for one to two weeks.

The antibiotics used in conventional triple therapy have to be absorbed in the intestine and then secreted from stomach tissue after blood circulation in order to reach the site of H. pylori colonisation. The stomach mucus layer prevents them from reaching the H. pylori colonisation site from the direction of the stomach lumen.

More recently, topical gastric therapies for the treatment of gastric . H. pylori ^■ infections have been developed that use both a proton pump inhibitor in a systemically available dosage form and a proton pump inhibitor in a gastrically . available form. In particular, such therapies have also included a fatty acid and/or monoglyceride . The microbicidal efficacy of fatty acids is known to be greater with decreasing pH, i.e. increasing acidity [Ref. Sun- CQ et al. Antibacterial actions of fatty acids and monoglycerides against Helicobacter pylori. FEMS Immunology and Medical Microbiology 36(2003), 9-17].

The use of fatty acids in such formulations presents a number of challenges. Achieving an, adequate microbicidal concentration requires a small volume of stomach contents, making concentrated dosage forms preferable, such as a solid dosage form. However, fatty acids are generally insoluble in^~ aqueous environments, and tend to crystallise out in acidic environments. Gastric emptying, times are shorter following an overnight fast or with liquid stomach contents, so that highly efficient dissolution of the formulations is required to achieve a microbicidal effect while the fatty acid still remains in the stomach. For example, half of the amount of inert liquids such as water will empty from the stomach in a period of 8-18 minutes, an.d emptying of nutrient liquids exhibits an initial rapid phase lasting 5-30 minutes [ref: pp205-214 Textbook of Gastroenterology, ed Tadataka Yamada. Lippincott Williams and Wilkins 2003] . Thus, the solubility of fatty acid formulations in an aqueous medium in the stomach or prior to oral administration is a critical part of any fatty acid H. pylori treatment. Accordingly, known H. pylori topical treatments comprising lauric acid are unsuitable for therapeutic use as the lauric acid does not dissolve quickly enough in the stomach prior to gastric emptying. Thus, the lauric acid is removed from the stomach before any treatment of H. pylori can be provided. ,^■

A range of surfactants are commonly used as excipients to aid solubilisation of pharmaceuticals, such as the non- ionic surfactants described herein. As an example, the recognised range for the poloxyethylene sorbitan fatty acid esters (commonly referred to as Tweens) used as a solubilising agent for poorly soluble active constituents in lipophilic- bases is from 1—10% [Ref Handbook of Pharmaceutical Excipients 5^th Edition. Eds Rowe R.C, Sheskey P.J and Owen S.C, Pharmaceutical Press] .

Accordingly, there is a need to provide an improved composition for the treatment of H. pylori infections that overcomes, or at least ameliorates, one or more of the disadvantages described above.

There is a need to provide an improved ^■ composition which in which a fatty acid component is soluble in an aqueous medium, including at a mildly acidic pH.

Summary

According to a first aspect, there is provided a pharmaceutical composition comprising a surfactant and a fatty acid, wherein the molar ratio of surfactant to fatty acid is in the range of 0.2:1 to 3:1. Advantageously, the molar ratio of the surfactant to • fatty acid is such that - the fatty acid is rapidly soluble in an aqueous medium at a pH of about 3 to about 7.

Advantageously, as the composition does not rely on .antibiotics to elicit a therapeutic effect, drug resistant H. pylori are obviated.

Advantageously, because the surfactant is present in an amount that permits the fatty acid to be soluble in an aqueous medium at a mildly acidic pH, the composition in accordance with the disclosure obviates the requirement to administer proton pump inhibitor, H2 antagonist or oral antacid to raise the pH of the stomach.

According to a second aspect, there is provided use of a composition according to the first aspect, in the preparation or manufacture of a medicament . for the treatment of a H. pylori infection.

According to a third aspect, there is provided a pharmaceutical dosage form- comprising a composition according to the first aspect. ^■

According to a fourth aspect, there is provided a kit comprising the pharmaceutical composition according, to the first aspect and directions for use.

^■According to a fifth aspect, there is provided a kit comprising the pharmaceutical dosage form according to the third aspect and directions for use- According to a sixth aspect, there is provided a method for treating a disease or disorder comprising administering a therapeutically effective amount of the composition according to the first aspect to a patient in need thereof.

According to a seventh aspect, there is provided a composition according to the first aspect for use as a medicament. According to an eighth aspect, there is provided a pharmaceutical composition comprising a surfactant and a fatty acid, wherein the molar ratio of surfactant to fatty acid is in the range of 0.2:1 to 3:1 for the treatment of a H. pylori infection.

Def ini-tions

The following are some definitions that may be helpful in understanding the description of the present invention. These are intended as general definitions and should in no way limit the scope of the present invention to those terms alone, but are put forth for a better understanding of the following description.

Unless the context requires otherwise or it is specifically stated to the contrary, integers, steps, or elements of the invention recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers, but not the exclusion of any other step or element or integer or group of elements or integers. Thus, in the context of this specification, the term "comprising" means "including principally, but not necessarily solely",..

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to · be understood that the invention includes- all such variations and modifications.

The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps -or features.

In the context of this invention the term "administering" and variations of that term including "administer" and "administration", includes contacting, applying, delivering or providing a compound or composition of the invention to an organism, or a surface by any appropriate means.

In the context of this specification, the term "patient" includes humans and individuals of any species of social, economic or research importance including but not limited to members of the type ovine, bovine, equine, porcine, feline, canine, primates (including human and non-human primates), rodents, murine, caprine, leporine, and avian.

In the context of this specification, the term ^■ "treatment", -refers to any and all uses which remedy a disease state or symptoms, prevent the establishment of. disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever.

In the context of this specification the term "therapeutically effective amount", include meaning a sufficient but non-toxic amount of a compound or composition of the invention to provide the desired^' therapeutic or diagnostic effect_._ The., .exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered, the mode of administration, and so forth. Thus, it is not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective ^"amount" may be determined by one of. ordinary skill in the art using only routine experimentation.

The language "pharmaceutically acceptable carrier" is intended to include solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional medium or agent is incompatible with the compound, use thereof in the therapeutic compositions and methods of treatment and prophylaxis is contemplated. Supplementary active compounds may also be incorporated into the compositions according to the present invention.

"Unit dose form" or "dosage form" or "dosage unit form" as used herein refers to physically discrete units suited as unitary dosages for the individual to be treated; each unit containing a predetermined quantity of ^■ compound^ s ) is calculated to produce^' the desired therapeutic effect in association with the required pharmaceutical carrier. The compound (s) may be formulated for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in an acceptable dosage unit. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

The^■ term "soluble" as used herein., relates to any physical form that permits the killing efficacy of a fatty acid against H. pylori. This may include, for_. instance, an aqueous solution, dispersion, emulsion, micro-emulsion, micellar solution, liposomal solution, and the like. "Soluble" does not include fatty acid in a crystalline form or where it forms obvious lumps or flakes visible to the eye. In ^'one embodiment, the ..term "soluble" does not include fatty acid in a separated lipid phase such that it ^■ is not readily accessible to the aqueous ^' phase^' (for example, lauric acid dissolved in corn oil droplets) . ^'

The term "rapidly" relates to a time frame of from 1 to 30 minutes of the formulation reaching an aqueous environment, and preferably within 5 minutes.

The word "substantially" does not exclude "completely" e.g. a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from the definition of the invention.

Unless specified otherwise, the terms "comprising" and "comprise", and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, unrecited elements.

As used herein, the term "about", in the context of concentrations · of components -of- the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.

With respect to pH, the term "about" is intended to encompass a difference of ±0.5 pH units from the stated ^■ value and preferably ±0.3 pH units from the stated value.

The - term mole (mol) means -^~the~mean^—mol^'ecul^'ar-weight in grams, and millimole (mmol) the molecular weight in milligrams. The measurement units used in the text have their usual technical meanings, with rounding of numbers- usually to the second digit.

Throughout^" this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible . limitation on the scope of the disclosed ranges. Accordingly, the description of. a range should be. considered- to have specifically disclosed all the possible ^' sub-ranges as well as individual numerical values within that range. For example, description of a range such -as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range,- for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Certain embodiments may also be described broadly and generically herein. Each of the narrower species and- subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso or .negative^■ limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Disclosure of Optional Embodiments

Exemplary, non-limiting embodiments of a .pharmaceutical composition will now be disclosed. The composition comprises a surfactant and a fatty acid, wherein the molar ratio of surfactant to fatty acid is in the range of 0.2 : 1 to 3 : 1.

In one embodiment, the fatty acid may be selected from one or more of the following saturated fatty acids: butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid or any straight chain saturated fatty acid with chain length from 4 carbons to 16 carbons. In another embodiment the fatty acid may be selected from one or more of the following unsaturated fatty acids: myristoleic acid, palmitoleic acid, oleic acid, linoleic acid or linolenic acid. In one embodiment, the unsaturated fatty acids are all cis.

In one embodiment, the fatty acids are linolenic acid, palmitoleic acid or lauric acid. In another embodiment, the fatty acid is lauric acid.

In another embodiment, a combination of two or more of the fatty acids listed above may be provided in the composition.

In an alternative -embodiment a monoglyceride may be used instead of a fatty acid. In one embodiment, the monoglycerides may be selected from the group consisting of 1-monocaprylin, 1-monocaprin, 1-monolaurin, 1-monomyristin, 1-monopalmitin and 1-monostearin.

The surfactant may be a non-ionic or ionic surfactant. The surfactant may be selected from a Brij surfactant, a Tween surfactant, an IPEGAL surfactant, a MERPOL surfactant., a Triton surfactant, a bile salt, lecithin or sorbitan monopalmitate .

In one embodiment, the surfactant may be selected from Tween 20 or Tween 80. In one embodiment, the surfactant is Tween 20 (polysorbate 20).

Without being bound by theory, it is believed that a fatty acid is not easily soluble at an acidic pH such as that found in the stomach. It is essential that the fatty acid is provided in solution to allow contact between the fatty acid and H. pylori. Previously,_^ai_s_ing__ of the pH of the stomach to aid solubilisation of the fatty acid was commonly achieved by the administration of a systemically available proton pump inhibitor, H2 antagonist or oral antacid .

The inventors have found that a ratio of Tween 20: lauric acid in a molar ratio independently selected from the group consisting of about 0.2:1 to 3:1; about 0.3:1 to 2:1; about 0.4:1 to 1:1 and about 0.5:1 to 1:1 permits lauric acid to be soluble in an aqueous medium, including at a pH of between about 3 and about 7,. at physiological temperatures. In particular, the surfactant provides the rapid solubility of lauric acid in the aqueous medium of the stomach.

In another embodiment, the ratio of Tween 20: lauric acid is 0.7:1. At this molar ratio of 0.7:1, Tween 20 makes up greater than 80% w/w of the composition. At a molar ratio of 0.2:1, Tween 20 makes up greater than 54% w/w of the composition, which is far above the recognized range . for solubilising poorly soluble active constituents in lipophilic bases. The accepted upper limit '[for reference see Background] of 10% w/w Tween 20 would be a ^■ molar ratio of less than 0.02:1 with lauric acid.

The composition may be used in combination with other known treatments or antimicrobial ^■ agents, including antibiotics and the like. Suitable agents are listed, for example, in The Merck Index: An Encyclopedia of Chemicals , Drugs, and Biologicals, 14th Edition, 2006, the entire contents of which are incorporated herein by reference. Combinations of active agents, including compositions of the invention, may be synergistic.

The composition may further comprise a mucolytic preparation. In one embodiment, the mucolytic preparation may be selected from an enzyme such as a proteinase (e.g. Pronase) . Alternatively, in another _ embodiment , the mucolytic preparation may be a sulfhydryl compound selected from an N-alkyl-cysteine, penicillamine or N-(2- mercaptopropionyl ) -glycine . In one embodiment, the sulfhydryl compound is N-alkyl-cysteine. In another embodiment, the sulfhydryl compound is N-acetyl-cysteine . Mucolytic agents such as Pronase may be administered in a dosage range from about 18,000 - 36,000 tyrosine units for a single administration as disclosed in Kimura et^'al., Am.- J. Gastroenterol. 90: 60-63, 1995. N-acetyl- - cysteine may be administered in a dosage range of from about 0.1 to 10 g for a single administration. Preferably, the N-acetyl-cysteine is administered orally at a dose of up to about 9-10 grams for an adult. In another embodiment, the N-acetyl-cysteine is administered0 orally at · a dose of about 1-2 grams.

In one embodiment, the composition may further comprise at least one proton pump inhibitor. The proton pump inhibitor may be selected from one or more of omeprazole, lansoprazole, pantoprazole, esomeprazole,5 timoprazole, rabeprazole or picoprazole.

In another embodiment, the composition may further comprise an antibiotic. The antibiotic may be selected from one or more of metronidazole, tetracycline, ^■ - clarithromycin-, amoxicillin, erythromycin.

0 In a further embodiment, the composition may further comprise a H₂-receptor antagonist. The H₂-receptor antagonist may be selected from one or more of cimetidine, ranitidine, famotidine or nizatidine.

There is provided use of a composition as defined5 above, in the preparation or manufacture of a medicament for the treatment of a disease or disorder. In one embodiment, the disease or disorder is a H. pylori infection.

In one embodiment, the mucolytic preparation is to be0 administered prior to said fatty acid and said surfactant.

The timeframe in which the mucolytic preparation is to be administered prior to said fatty acid and said surfactant may be independently selected from the group . consisting of about 1 to about 90 minutes, about !^■ to about 80 minutes; about, 1 to about 70 minutes; about 1 to about 60 minutes; about 1 to about 50 minutes; about 1 to about 40 minutes; about 1 to about 30 minutes; about 1 to about.20 minutes; about 1. to about 10 minutes and about 1 to about 5 minutes.

In one embodiment, the mucolytic preparation is to be administered about 10 minutes before said fatty acid and said surfactant.

In another embodiment, the mucolytic preparation is to be administered simultaneously with said fatty acid and said surfactant. As used herein, the administration of the mucolytic agent and fatty acid and surfactant in less than about 45 minutes apart is considered simultaneous administration.

There is provided a pharmaceutical dosage form comprising a pharmaceutical composition ^' as defined above.

The dosage form may be prepared, for example, in unit dose forms, such as tablets, capsules, sachets, dragees .

They may be prepared in a conventional manner, for example by means ^" of conventional mixing, granulating, confectioning^', or dissolving processes.

Liquid formulations for oral administration may be prepared in the form of solutions, syrups or suspensions.

Liquid formulations .typically comprise the pharmaceutical composition with an excipient such as sugar or sugar alcohols, and a carrier such as ethanol, water, glycerol, propylene glycol, polyethylene glycol.

If desired, such liquid formula.tiojis _. may. also contain colouring . .agents, flavouring agents, saccharine, thickening agents (e.g. carboxy methyl cellulose), suspending agents (e.g. sorbitol syrup, methylcellulose , ■ hydrogenated edible fats), emulsifying agents (e.g. lecithin, acacia), and/or .preservatives (e.g. methyl p- hydroxy benzoates, propyl p-hydroxy benzoates, sorbic acid). Liquid formulations for administration may also be prepared in the form of a dry powder to be reconstituted with water or another suitable vehicle prior to use. _r

In addition to the active- ^' ingredients, the pharmaceutical composition may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques, for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton Pa.).

The composition may be administered orally, for example, with an inert diluent or an assimilable edible carrier. In one embodiment, the composition is administered orally after a period of fasting or before the ingestion of food.

The composition and other ingredients may also be enclosed i a hard or soft shell- gelatin capsule or compressed into tablets. For oral therapeutic administration, the composition may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

In one embodiment said dosage form is a capsule. In one embodiment, the capsule is a hard gelatine capsule such as a Licaps® capsule.

Tablets, troches, pills, capsules and the like can also contain the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and- a sweetening agent such as sucrose, lactose or saccharin or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.

When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials can be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules can be coated with shellac, sugar or both. A syrup or elixir can contain the composition, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange .flavour.

In one embodiment, the pharmaceutical composition may further include a suitable buffer. Suitable buffer agent agents are well known to those skilled in the art and include, but are not limited to, phosphates, citrates, carbonates and mixtures thereof.

Single or multiple- administrations of the pharmaceutical composition may- - be- -carried out. One skilled in the art would be able, by routine experimentation, to determine effective, non-toxic dosage levels of the composition and an administration pattern which would be suitable for treating a H. pylori infection .

Further, it will be apparent to one of ordinary skill in the ^'art that the optimal course^' of treatment, such as the number of doses of the composition given per day for a defined number of days, ^' can be _^ascertained using conventional course of treatment determination tests.

Generally, an effective dosage of fatty acid per day may be selected from the range consisting of about 0.025- 2.5 mmol; about 0.1-2.0 mmol; about 0.2-1.75 mmol; about 0.3-1.5 mmol; about 0.4-1.5 mmol about 0.5-1.5 mmol; about 0.6-1.5 mmol and about 0.7-1.5 mmol. In one embodiment, the effective -dose of fatty acid is 0.75-1.5 mmol.

Generally, an effective dosage of surfactant in moles to rapidly solubilise the fatty acid ^' is in the range of about ^' 0.2-3.0 times the molar amount of fatty acid administered.

In one embodiment an effective dosage is a mixture of 0.75 mmol (150mg) lauric acid and 0.53 mmol (645mg) of Tween 20, with one or two doses given once daily being the administered dose .

In another embodiment, the effective dosage may be repeated one or more times in any 24 hour period of treatment .

In one embodiment, the dosage form according to the invention is one or more capsules containing an effective dose of the composition. In one embodiment, the effective dose in each capsule is about 0.53 mmol of surfactant and 0.75 mmol of fatty acid. In another embodiment, the effective^' dose is a capsule containing a mixture of about 0.53 mmol (645 mg) of Tween 20 and about 0.75 mmol. (150 mg) lauric acid.

There is provided a kit comprising the pharmaceutical composition as- defined above and directions for use.

There is provided a kit comprising the pharmaceutical dosage form as defined above and directions for use.

There is provided a method for treating a disease or disorder comprising administering a therapeutically effective amount of the composition accordjjr^_to_t e first aspect to a patient in need thereof. In one embodiment the disease or disorder is a H. pylori infection.

There is also provided a composition as defined above for use as a medicament.

There is also provided a pharmaceutical composition comprising a surfactant and a fatty acid, wherein the molar ratio of surfactant to fatty acid is in the range of .0.2:1 to 3:1^' for the treatment- of a H. pylori infection._.

Brief Description of Drawings

The accompanying drawings illustrate a disclosed embodiment and serve to explain the principles of the disclosed embodiment.. It is to be understood, however, that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the invention.

Fig. 1 shows the dissolution of Tween 20/lauric acid at different timepoints up to 20 minutes (methodology: see Example 1) , with dissolution shown by the buffering effect of the lauric acid against an amount of caustic soda added such .that the pH is 7 ^· if all the lauric acid is dissolved.

Fig. .2 shows a timeline of survival of - H. pylori cells (in units of log cfu/mL) from which reduction in viability due to the Tween 20/lauric acid pharmaceutical formulation can be calculated..

Examples

Non-limiting examples of the invention, including the- best mode, and a comparative example will be further described in greater detail by reference to specific Examples, which . should not be construed as in any way limiting, the scope of the invention.

Example 1: Speed of dissolution of Tween 20/lauric acid mixtures under physiological-like conditions and a range of molar ratios of Tween 20 (TW) to lauric acid (I±A)

The mixtures were prepared as follows. Lauric acid 80 mg (400 micromol) and selected amounts of Tween 20 were premixed in a capped 100 mL bottle in the absence of water, heated to 50°C for 10 minutes with mixing, then allowed to cool to room temperature (18°C) for more than 15 hours. The molar ratios of Tween:LA selected were (1)0:1; (H) 0.1:1; (G) 0.2:1; (F) , 0.3:1; (E) 0.5:1; (D) 0.7:1; (C) 1.0:1; (A) 2.0:1 and bottle (B) contained 400 micromol HC1 instead of TW/LA to test the speed of equilibration of the electrode. As a negative control bottle (J) contained no LA or TW.

A total volume of 480 mL was prepared, containing

19.2 mL of 100 mM NaOH and water. This was preheated to 37.4°C. At zero time, 40 mL of this solution was added to the TW/LA mixture in each bottle of TW/LA mixture, and maintained in a rotary shaking waterbath (100 rpm, 37.4°C) . The pH was read after 30 sec of equilibration of the pH electrode, at 1 min, 4 min, 7 min, 10 min^' and 20 min after the NaOH addition. The amount of NaOH was targeted to give pH 7.0 when the LA was completely dissolved, based -on earlier slower-- titrations. In solution, the lauric. acid concentration would be 10 millimolar (400 micromol in 40 mL) , which is expected to have a microbicidal effect on H. pylori based on prior art. The pH results are shown in Table 1 as follows: Table.1

The pH results are graphically shown in Fig. 1, with molar ratio ^' of Tween 20 to lauric acid plotted against pH at the specified timepoints.

The data demonstrate that there, is a cutoff in. rapid solubility of the T /LA mixture, with good solubility within 4.-7 minutes when the Tween: LA molar ratio is above 0.2, and poor solubilisation even at 20 min when the ratio is lower than this (such that there is not neutralisation of the NaOH by LA reaching solution) .

In parallel, the appearance of .the solution was observed at the same timepoints that pH was measured. When the Tween 20: LA molar^' ratio was 0.5:1 or above, by 10 minutes the solution was either clear or with mild to moderate haziness. At the 0.2 : 1 molar ratio there were a few suspended flakes in clear solution. Below this ratio there were a few small floating flakes in clear, solution but with solid residual lauric acid still stuck to the bottom of the bottle at 20 minutes.

Example 2 : Maintenance of lauric acid in solution during acidification, varying according to Tween 20/lauric acidmolar ratios .

Methods: Sodium laurate 400 micromol (88.9 mg) was mixed with a range of Tween 20 amounts to give molar ratios of Tween 20 to laurate of 1:1; 0.7:1; 0.5:1; 0.3:1; 0.2:1; 0.1:1 and 0:1 in 100 mL Schott bottles with screw top lids. For example, the amount of Tween 20 added for the 1 :^" 1^{" "}ratio was 4^"91.2 mg (400 micromol). Each was heated to 50°C for 10-12 min and mixed as far as possible. They were then cooled to 18°C for at least 15 hours.

Prewarmed water was added to each mixture, and the pH followed for 7 min. At 8 min 0.4 mL of 100 mM^' HC1 was added (40 micromol) and the pH recorded after 1 min. The solution was gently , rotated in a water bath _: for a^' further 2 min, when another 0.4 mL of 100 mM HC1 was added and the pH recorded after a further 1 minute etc. The bottle was rotated at 100 rpm in a rotary water bath (held at 37.4°C) between the^■ acid addition and the pH reading, until the next addition of hydrochloric acid.

Results

The Table 2 shows the pH values during^' titrations, with the appearance of the solution as indicated by the different fonts (see table footnote).

Table 2. pH values during titrations of selected Tween

20: sodium laurate molar ratios .

Clear solution (normal font)

Hazy & birefringent crystals (Underlined italic)

Thick precipi-tate forming- (Bold)-

The soapy mixture of Tween 20/sodium laurate dissolves fairly -easily in water at 37.4°C at all the molar ratios studied. For the 0:1 molar ratio the pH at baseline was 7.63, with somewhat higher pH up to 8.26 in the presence of Tween 20 suggesting that dissolution is aided by the Tween.. 20. As_. acid .is added to the_..solution there is conversion of the laurate_. to iauric acid . When the Tween 20: sodium . laurate molar ratio is above 0.3:1 in this experiment, the solution remains clear^' down to pH 3 for at least 20 minutes. With a ratio of 0.3:1, the LA remains in solution until pH 5.9, with haze and birefringent crystals at pH 6.35. At a ratio of 0.2:1 precipitate starts to form at mildly acidic pH of 6.67 and below, with haze and birefringent crystals at close to neutral pH at lower ratios.

Conclusion

A ratio of Tween 20 to lauric acid of 0.3:1 or above is necessary to keep the lauric acid in solution at mildly acidic pH, with a ratio of 0.5:1 if the pH is down to 3.

Example 3 : Timeline Killing of H. pylori Experiment

Materials and Methods .

Killing Solution:

Solution 1: 25 mL of water was^' added to 0.025 mL 0.1M HC1 (H⁺ = 10^"4, or pH. 4). 5 mL of Aurum Injection N- acetylcysteine and 10 mL Lipton Ice Tea (peach) and 10 mL water was then prepared. 250 mg NaHCC>3 was then addedto the N-acetylcysteine/Lipton Ice Tea solution, followed by addition of the HC1, and the solution was heated at 37 °C for .10 mins . 2.941 grams Zegerid® (20 mg Omeprazole and excipients) was then added to the solution together with 1:0...mL of., water . - - --- ---

On the previous day 150 mg iauric acid and 645 mg Tween 20 were premixed and heated to 50°C and left at room temperature overnight to form a glassy solid.

On the day of the experiment solution 1 was added to the Tween 20/lauric acid and heated to 37°C for 5 min with gentle occasional swirling. The 'lauric- acid and Tween 20 could be observed to be dissolving.

0.5 grams of citric acid.lt^O was mixed with 15 mL water and added to the solution. The pH was measured as 6.73. Another 2 mL of 0.5M citric acid solution was added to give a final pH of 6.54 (measured) .

The Lauric acid concentration was 150 mg/75 mL, which is equivalent to 10 mM concentration.

The Tween 20 concentration will be 645 mg/75 mL, which is equivalent to 7 mM concentration or 0.86 % (w/v) .

0.06 mL of the "Killing Solution" prepared above was added to 1.065 mL Isosensitest broth (pH 6.5 containing 5% (v/v) horse serum, previously pre-heated for 10 min at 65°C to denature bactericidal contents), and equilibrated under microaerophilic conditions (5% oxygen, 2% hydrogen, 10% carbon dioxide and 83% nitrogen) .

H. pylori inoculum (0.075 mL) was added to 1.125 mL of Isosensitest broth/serum and 1/20 "diluted Killing Solution". The final concentrations ^· of lauric acid and Tween 20 in the incubation will be 0.5 mM and 0.35 mM or 0.043 % (w/v) respectively. This mixture was incubated at 37°C under microaerophilic conditions for selected times.

At the end of the preselected time, a 20 μL aliquot was removed and suitably diluted. Duplicate 20 ]i aliquots were then plated on Columbia agar containing 5% horse serum. Plates were incubated under microaerophilic conditions at 37°C for 3 days, . and colonies counted.

Control Solution:^{' '}

25 mL of water was added to 0.025 mL 0.1M HC1 (H⁺ = 10^" ⁴,- or pH ) . 10 mL Lipton Ice Tea and 15 mL water was then added. 250 mg NaHC0₃ was further added to the

solution. The solution was then heated for 10 minutes at 37 °C. The pH was then adjusted to 6.5 with 0.5 M citric acid (final pH 6.5-2 measured) . and the total volume made up to 75 mL. ^' ._.. . ^'

0.06 mL of the "Control Solution" was then ^'added to 1.065 mL. Isosensitest broth (pH 6.5 containing 5% (v/v) horse serum, previously pre-heated for 10 min at 65°C to denature bactericidal contents). 0.075 mL of H. pylori inoculum was added to 1.125 mL of Isosensitest broth/serum and 1/20 "diluted Control Solution". The final

concentrations of lauric acid and Tween 20 in the

incubation were 0 mM and 0 mM, respectively. The

bacterial cells were then incubated microaerophilically at 37 °C for preselected times. At the end of the preselected time, a 20 pL aliquot was suitably diluted, and plated on Columbia agar containing 5% horse serum.

■

RESULTS :

Inoculum.

Calculation from 10^"5 dilution. Undiluted suspension will have 2.68 x 10⁸ cfu/mL.

Calculation from 10^"6 dilution. Undiluted suspension will have 4.50 x lO⁸ cfu/mL.

If 0.075 mL of inoculum is suspended in 1.2 mL incubation, concentration of cells will be 2.24 x 10⁷ cfu/mL, or log ^■ 7.35.

Control incubation, 0 min sample (~1 min)

Calculation from 10^~5 dilution. Undiluted suspension will 'have 6.17- x 10⁷ cfu/mL.

Calculation from 10^~4 dilution. Undiluted suspension will have 3.35 x 10⁷ cfu/mL.

Average 4.67 x 10⁷ cfu/mL, or log 7.67.

Control incubation, 40 min sample Calculation from 10^~4. dilution . Undiluted suspension will have 2.23 x 10⁷ cfu/mL. ·

Calculation from 10^~3 dilution. Undiluted suspension will have 3. ^"02 x 10⁷ cfu/mL.

Average 2.62 x 10⁷ cfu/mL, or log 7.42.

Experimental incubation, 0 min sample (more likely 1 min) Calculation from 10^~4 dilution. Undiluted suspension will have 7.00 x 10⁶ cfu/mL.

Calculation from 10^"3 dilution. Undiluted suspension will have 5.35 x 10⁶ cfu/mL.

Average 6.18 x 10⁶ cfu/mL, or log 6.79. Experimental incubation, 10 min sample

20 L aliquots of all dilutions (undiluted, 10^"1, 10^-2, 10^" ³, 10^~4) gave 0 colonies.

Average 0 cfu/mL.

Assuming <0.5 cfu in undiluted 20 μΐ aliquot, concentration of live cells in incubation is <25 cfu/mL or <log 1.4

Experimental incubation, 20 min sample

All dilutions (undiluted, 10^_1, 10^"2, 10^~3) gave 0 colonies. Average 0 cfu/mL.

Assuming <0.5 cfu in undiluted 20 μΐι aliquot, concentration of live cells in incubation is <25 cfu/mL or <log 1.4

Experimental incubation, 40 min sample

All dilutions (undiluted,^' 10^"1, 10^"2, 10^~3) gave 0 colonies. Average 0 cfu/mL.

Assuming <0.5 cfu in undiluted 20 aliquot, concentration of live cells in incubation is <25 cfu/mL or <log 1.4 Conclusion :

The control, incubations showed that H. pylori was stable and viable over 40 min in the presence of Isosensitest broth pH 6.5, attenuated serum, ice tea, citric acid, under microaerophilic conditions in the absence .of the N-acetylcysteine, omeprazole, lauric acid, Tween 20, and (see Fig. 2) .

The experimental incubations showed that <log 1.4 cfu/mL (out of the initial log 7.35 cfu/mL) survived for 10 min or longer in the presence of the N-acetylcysteine, omeprazole, lauric acid, Tween 20, and■ in the presence of IsoSensitest broth pH 6.5, attenuated serum, Ice Tea, citric acid, under microaerophilic conditions (see Fig. 2) . Thus, within 10 minutes all H. pylori bacteria were killed.

The zero time experimental incubation suggested that approximately 1 log cfu/mL had been killed within approximately 1 minute (see Fig. 2).

The 1:20 dilution of "Killing Solution" is obviously extremely potent against H. pylori.

Accordingly, it can be seen that the composition in accordance with the present invention demonstrates superior anti-Helicobacter ■ activity and that the composition of the invention is effective for treating H. pylori infections.

Applications

The composition disclosed herein provides an effective treatment for H. pylori infections.

Advantageously, the composition of the disclosure provides a more convenient treatment to . the presently recommended triple therapy. The composition may also encourage stricter adherence to a treatment regimen for H. pylori infections.

Advantageously, as the composition does not rely on antibiotics to elicit a therapeutic effect, drug resistant H. pylori are obviated and antibiotic side effects are avoided.

Advantageously, because the molar ratio of the fatty acid and surfactant permits the fatty acid to be rapidly solubilised at mildly acidic pH (pH values between pH 3 and pH 7) the composition in accordance with the disclosure obviates the requirement to administer proton pump inhibitor, H2 antagonist or oral antacid to raise the pH -of the stomach to^' a pH at which lauric acid becomes soluble in the aqueous phase.

It will^■ be apparent that various other modifications and adaptations of the invention will be apparent, to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it ^' is ^■ intended that all such modifications and adaptations come within the scope of the appended claims.

Claims

1. A pharmaceutical composition comprising a surfactant and a fatty acid, wherein the molar ratio of surfactant to fatty acid is in the range of 0.2:1 to 3:1. -

2. The composition according to claim 1, wherein the molar ratio of surfactant to fatty acid is in the range of from 0.5:1 to 1:1.

3. The composition according to claim 1 or claim 2, wherein the molar ratio of surfactant to fatty acid is 0.7:1.

4. The composition according to any one of the preceding claims, wherein the fatty acid is selected from one or more of the following saturated fatty . acids: butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid or other straight chain saturated fatty acid with chain length from 4 carbons to 16 carbons.

5. The composition according to claim 4, wherein the fatty acid is selected from one or more of the following unsaturated fatty acids: myristoleic acid, palmitoleic acid, oleic acid, linoleic acid or linolenic acid.

6. The composition according to claim 5, wherein the unsaturated fatty acids are all cis.

7. The composition according to any one of claims 4 to 6, wherein the fatty acid is selected from one or more of linolenic acid, palmitoleic acid or lauric acid.

8. The composition according to claim 7, wherein the fatty acid is lauric acid.

9. The composition according to any one of claims 1 to 8, wherein the surfactant is a non-ionic surfactant or a bile salt.

The composition according to claim 9, wherein the non-ionic surfactant is selected from a Brij surfactant, a Tween surfactant, an IPEGAL surfactant, a ^' MERPOL surfactant, a Triton surfactant, or sorbitan monopalmitate.

The composition according to claim 10, wherein the surfactant is a Tween -surfactant .

The composition according to claim 11, wherein the Tween surfactant is selected from Tween 20 or Tween 80.

The composition according to claim 12, wherein the Tween surfactant is Tween 20.

The composition according to any one of claims 1 to 13, wherein the composition further comprises a mucolytic preparation.

The composition according to claim 14, wherein the mucolytic preparation is selected from an enzyme or a sulfhydryl compound.

The composition according to claim 15, wherein the sulfhydryl . compound is selected from an N-alkyl- cysteine, penicillamine or N- ( 2-mercaptopropionyl ) - glycine.

The composition according to claim 15, wherein the enzyme is a proteinase.

The composition according to claim 15 or 16, wherein the sulfhydryl compound is N-acetyl-cysteine .

The composition according to any one^■ of the preceding claims, wherein the composition further comprises at least one proton pump inhibitor selected from . omeprazole, lansoprazole, pantoprazole, esomeprazole , timoprazole, rabeprazoleor picoprazole.

20. The composition according to any one of the preceding claims, wherein the composition further comprises an antibiotic.

21. The composition according to claim 20, wherein the antibiotic is selected from metronidazole, tetracycline, clarithromycin, erythromycin or amoxicillin.

22. The composition according to any one. of the preceding claims wherein the composition further comprises a H₂-receptor antagonist selected from cimetidine, ranitidine, famotidine or nizatidine.

23. Use of. a composition according to any one of claims 1 to 22, in the preparation or manufacture of a medicament for the treatment of a disease or disorder.

^'24. The use - according to claim 23, wherein the disease or disorder is a H. pylori infection.

25. A pharmaceutical dosage form comprising a composition according to any one of claims 1 to 22.

26. The pharmaceutical dosage form according to claim 24, wherein said dosage form is a capsule.

27. A kit comprising the pharmaceutical composition according to any one of claims 1 to 22 and directions for use.

28. A kit - comprising the pharmaceutical dosage form according^' to claim 25 or 26 and ^' directions for use.

29. A composition according to any one of claims 1 to 22 for use as a medicament.

30. A pharmaceutical composition comprising a surfactant and a fatty acid, wherein the molar . ratio of surfactant to fatty acid is in the range of 0.2:1 to 3:1 for the treatment of a H. pylori infection.