AU2011100052A4 - Method and preparartion for binding acetaldehyde in the stomach - Google Patents

Description

I AUSTRALIA FB RICE & CO Patent and Trade Mark Attorneys Patents Act 1990 BIOHIT OYJ COMPLETE SPECIFICATION INNOVATION PATENT Invention Title: Method and preparation for binding acetaldehyde in the stomach The following statement is a full description of this invention including the best method of performing it known to us:- 1IA Method and preparation for binding acetaldehyde in the stomach The object of the invention is obtaining a pharmaceutical preparation according to the claims for locally binding the acetaldehyde in the stomach. Another object of the invention 5 is a method for decreasing the risk of developing cancer of the stomach and the large intestine. It has been found that acetaldehyde causes cancer in animals (Feron et al, (1982) Eur J Cancer Clin Oncol 18:13-31). It has also been found that acetaldehyde is a local 10 carcinogen, when occurring in human saliva and the alimentary tract. This is supported by the fact that Asian heavy consumers of alcohol, who have a familial low-activity modification of aldehyde dehydrogenase-2 (ALDH2) enzyme, have both an increased risk of developing cancer of the mouth, the pharynx, and the alimentary tract, and an increased acetaldehyde content of the saliva after consuming alcohol (Vakevainen et al, (2000) 15 Alcohol Clin Exp Res 24:873-877). In the organism, acetaldehyde is formed from alcohol as a consequence of hepatic metabolism and, according to recent research, locally in the alimentary tract through a microbial alcohol dehydrogenase (Salaspuro et al, (1996) Ann Med 28:195-200). 20 After moderate consumption of ethanol, for example, high acetaldehyde contents of a microbial origin have been found in human saliva; in other words, acetaldehyde builds up in saliva as an intermediate product of microbial metabolism (Homann et al, Carcinogenesis (1997) 18:1739-1743). 25 Acetaldehyde is also formed in the mouth, the pharynx, and the upper airways as a consequence of smoking and exposure to air contamination. It has been proven that chronic smoking increases the acetaldehyde production of saliva originated in microbes. 30 The acetaldehyde formed in the saliva is then conducted to the stomach together with the saliva. Acetaldehyde also builds up in the stomach as a consequence of microbial metabolism in a situation, where there is no acid in the stomach or the acid has been removed by 2 medication (Va.kevainen et al, (2000) Alimentary Pharmacology Therapeutics, in press). It has also been shown that acetaldehyde builds up in the large intestine, because its bacteria that represent the normal flora are capable of converting ethanol into acetaldehyde (Jokelainen et al, (1996) Gut 39:100-104). 5 The effective substances used in the present invention are known from before. However, the pharmaceutical compositions according to the present invention containing these substances are not known, and the alleged effect of the prior art publications is systemic, and based on the reaction of the effective substances to the acetaldehyde inside the blood 10 and/or the cells of the organism. On the basis of these prior art publications it is doubtful whether the preparations in question are even capable of decreasing the acetaldehyde content of blood originating in alcohol. The known preparations also have such a composition that they would not be able to bind, on a long-term basis, the acetaldehyde, which is locally generated in the organism and which locally occurs in high contents (see 15 publications US 5 202 354, US 4 496 548, US 4 528 295, US 5 922 346). The acetaldehyde, which forms in the organism during the consumption of alcohol and afterwards, also causes physiological symptoms called a hangover. Previously, an effort has been made to decrease the symptoms caused by acetaldehyde by taking preparations 20 containing ascorbic acid, thiamine, cysteine or cysteic acid, and flavonoids or flavonoid complexes in a form of tablets swallowed orally in connection with, before or after consuming alcohol. It is believed that the method in question functions because, when swallowed, the effective substances are transported into the blood circulation. The tablets used in the method contained small amounts of effective substances only, and therefore 25 had no effect on the acetaldehyde in the stomach (Matsuoka, US Pat No 5,202,354 and Moldowan et al, US Pat No 4,496,548). It has been suggested that preparations containing amino acids, such as L-cysteine, methionine, taurine or arginine, ascorbic acid and vitamins A and E, which are sucked or 30 chewed in the mouth be used to decrease the effect of the harmful free radical compounds that are formed in connection with using tobacco products or being exposed to them. It is believed that amino acids affect various tissues after being absorbed (Hersch, US Pat No 5,922,346, Hersch, International Patent Application No PCT/US98/12617). This is believed to be caused by the amino acids cysteine, glutamine and glycine combining to 3 form glutathione in the liver cells. The formed glutathione will then protect the cells against oxidative damage caused by free radicals. Therefore, the effect is systemic. Further, there is no binding of acetaldehyde by these preparations, and the effect very short-term. Hence, we are not talking about a local long-term effect. 5 So far, neither a method nor a preparation has been presented, which would locally decrease the acetaldehyde content of the stomach. The methods and preparations according to prior art contain acetaldehyde-binding substances in small amounts only, or their impact is systemic or very short-term, whereby the content of acetaldehyde either does not change 10 or quickly regains its previous level after the effect of the substances has ended. The object of the invention is to provide a method and a preparation for decreasing or removing the acetaldehyde content of the stomach, and consequently that of the large intestine, the latter caused at least partly by acetaldehyde not bound in the stomach having 15 been transported to the large intestine. The use, the composition, and the method according to the invention are very useful in locally binding the increased levels of acetaldehyde that occur in connection with consuming alcoholic drinks or foodstuff, or in connection with smoking. In principle, the acetaldehyde can originate from any source, such as a foodstuff containing acetaldehyde; the acetaldehyde can have been formed from the ethanol 20 contained by the foodstuff or it can have been formed from an endogenous ethanol occurring in the organism. The purpose of the invention is to decrease the risk of contracting cancers of the stomach, and consequently of the large intestine, which are caused by the acetaldehyde in the said areas. 25 The invention is based on the surprising observation that the harmful amount of acetaldehyde locally occurring in the stomach can be bound locally, quickly and in large concentrations into a chemically safe form by using the preparations according to the present invention. As the substances that bind it are released in contents high enough throughout the entire period of effect of the acetaldehyde, the local acetaldehyde content 30 remains low. In this way, the local risk of contracting cancer caused by acetaldehyde decreases.

4 According to the invention, cysteine in the form of L- or D-cysteine, preferably L-cysteine, is used to prepare a pharmaceutical preparation containing a composition, which is used to locally bind the acetaldehyde in the stomach. 5 According to the invention, the pharmaceutical composition comprises cysteine, as bound to two or more pharmaceutically acceptable additive substances. The substances contained by the composition, such as the carriers, binders and other additives, are selected so that the cysteine is released within a long period of time. 10 According to the method of the invention, for decreasing the carcinogenous effect of acetaldehyde, the acetaldehyde contained by the stomach is locally bound into a safe form by using said pharmaceutical composition that releases the cysteine. The invention provides considerable advantages. The pharmaceutical compositions 15 comprising the acetaldehyde-binding cysteine can be used to decrease the risk of developing cancer of the stomach. In particular, the compositions according to the invention can be used for large-scale consumers of alcohol and especially for those, who have a familial low-activity modification of the aldehyde dehydrogenase-2 (ALDH2) enzyme, or those who have no acid in the stomach or the acid has been removed by 20 medication. The use of the compositions according to the invention is also of benefit to those who consume moderate amounts of alcohol or who consume foodstuffs that contain small contents of alcohol or acetaldehyde. Furthermore, the use of the compositions according to the invention also benefits smokers. 25 In the following, the present invention is examined more closely with the aid of a detailed description, examples and the appended drawings. Figure 1 is a graph showing the results of a dissolution test carried out using L-cysteine. 30 Figure 2 is a graph showing the concentration of acetaldehyde in the gastric juice after administration of L-cysteine or placebo. Figure 3 is a graph showing the mean concentration of L-cysteine in the gastric juice after administration of ethanol and either L-cysteine or placebo.

5 "The acetaldehyde-binding substance" refers to the active cysteine compound, which can be L- or D-cysteine, preferably L-cysteine. 5 "The binding of acetaldehyde" refers to a chemical reaction between the acetaldehyde and the cysteine, wherein the acetaldehyde jointly with the cysteine forms a larger molecule, while water is formed. This larger molecule is 2-methyl-L-thiazolidine-4-carboxylic acid, which is safe for the organism. 10 "The long-term binding of acetaldehyde" means keeping the acetaldehyde content for at least 30 minutes, preferably over 60 minutes, and most preferably over 120 minutes below a limit that is considered harmful, or preferably on a lower level than in a case where no pharmaceutical composition is used. 15 "A harmful/carcinogenic content of acetaldehyde" in the human mouth, oesophagus, stomach, and large intestine is 20-800 pmol/l of saliva or the contents of the intestine. Keeping the acetaldehyde content essentially lower than without the use of the pharmaceutical composition means keeping the acetaldehyde content at a level that is at 20 least 20%, preferably over 40%, and most preferably over 60% lower than when not using the pharmaceutical composition. Such a harmful or carcinogenic content of acetaldehyde in the human stomach can be obtained in connection with consuming alcoholic drinks, particularly strong alcoholic 25 drinks, or foodstuffs containing alcohol, as a consequence of smoking or when consuming other preparations containing acetaldehyde. "Alcoholic drinks" are ethanol-containing drinks, the ethanol content varying within 0.7% by volume and 84% by volume." 30 "Alcoholic foodstuffs" refer to foodstuffs containing at least 0.7% of ethanol. Such foodstuffs can be, for example, fermented juices or preserves, or foodstuffs preserved with small amounts of alcohol, pastries, jellies, and mousse seasoned with liqueur or corresponding preparations containing alcohol.

6 The use of the preparations according to the invention can be of benefit even, when light alcoholic drinks or foodstuffs are consumed, which contain small amounts of alcohol. Some foodstuffs can also already contain acetaldehyde. Acetaldehyde-containing 5 foodstuffs, which have ethanol that is generated in connection with fermentation, such as beer, cider, wine, home-brewed beer, and other alcoholic drinks, as well as many juices. As for alcoholic drinks, sherry contains an especially large amount of acetaldehyde. Therefore, the compositions according to the invention are preferably consumed or 10 administered in connection with consuming alcohol, most suitably in the form of alcoholic drinks or alcohol-containing foodstuff, or in connection with smoking. "In connection with consuming alcohol" herein refers to the period of time that begins from starting to enjoy alcohol and ends, when there is no more alcohol in the blood. 15 "In connection with smoking" herein refers to the period of time that begins from starting to smoke and ends, when smoking is stopped. "A long-acting preparation that has a local effect on the stomach" refers to capsules having 20 a multiparticular content, and which, when wetted under the influence of the gastric juices form a gel that floats in the contents of the stomach, as a consequence of which their residence time in the stomach is prolonged and thus enables a prolonged release in and a local effect of the drug on the stomach. 25 A special property required of the pharmaceutical composition that has a local effect on the stomach is that it remains in the stomach for as long as possible. Technically, this is solved by making a preparation that floats in the contents of the stomach. These preparations are provided by using polymer capsules, such as capsules made from hydroxypropyl methyl cellulose, that form a gel under the influence of gastric acid. 30 A single dose of the pharmaceutical composition having a local effect on the stomach comprises 50-500 mg of acetaldehyde-binding substance; preferably the amount of acetaldehyde-binding substance is 50-300 mg, and most preferably 100-200 mg.

7 When needed, the dosage is renewed at 4 to 10-hour intervals, preferably at 6 to 8-hour intervals. The amount of compound released in the conditions of the stomach is preferably 40-80 mg 5 in an hour. The preparation according to the invention, which releases its contents in the stomach, contains at least one - preferably two - polymers, in the form of additives, such as carriers, fillers or binders, which have the task of keeping the drug as long as possible, for two 10 hours minimum, in the stomach so that it forms a gel that floats in the contents of the stomach. Another task of the polymers is to prolong the release of the effective substance. The preparation contains an encapsulated composition comprising a mixture of powder or granules, and that forms a gel in the stomach. In addition to the cysteine, the composition 15 comprises said polymers and optional further additives. The amount of polymers in the composition is 10-50%, preferably 15-40%, and most preferably 20-30%. 20 The cysteine in the composition according to the invention is preferably mixed with the fillers needed and, after that, granulated by using enteric polymers as binders. The filler is preferably in the form of calcium hydrogen phosphate (CaHPO 4 ), which has the advantages of not swelling in the stomach content and of being suitable for direct 25 compression. The amount of filler in the granules is preferably 30-70%, most suitably 40 60%. The binder used can be any known enteric polymer, preferably a methacrylate derivative, more preferably a derivative known by the trade names Eudragit, and most suitably 30 Eudragit RS-PO. The amount of enteric polymer in the composition is preferably 2-5%, most preferably 3-4%. The composition can also contain other conventional additives, such as titanium dioxide, preferably in minor amounts of 2 - 5 % of the granules.

8 The composition of the preparation comprising granules can be as follows, for example: L-Cysteine 100 mg 5 contained in an HPMC capsule, and mixed and granulated with Calcium hydrogen phosphate 30 - 50 mg Eudragit RS-PO 40 -60 mg titanium dioxide 5 - 10 mg 10 The content of acetaldehyde formed in saliva as a consequence of consuming alcoholic drinks, smoking or for some other reason can be decreased so that, for example, in connection with consuming alcoholic drinks or smoking, a preparation (i.e. a unit of the composition according to the invention) is swallowed, whereby it releases the cysteine in the stomach at a suitable rate, whereby the acetaldehyde content locally increased in the 15 stomach as a consequence of consuming alcoholic drinks or foodstuff, or as a consequence of smoking, is decreased by more than 20%, preferably over 40%, most preferably over 60%, typically 60-80% compared with consuming a placebo. Simultaneously, the acetaldehyde content formed from consumed or endogenous ethanol in 20 the large intestine can be decreased by over 20%, preferably over 40%, most preferably 60 80% compared with consuming a placebo. In the following, the invention is examined with the aid of examples. 25 Example 1 - preparation of the capsules The capsules were prepared by mixing 500 g of L-cysteine (Gonmisol S.A., Spain), 500 g of Eudragit RS-PO, forming a matrix structure (Evonik R6hm GmbH, Germany), and 1 kg of calcium hydrogen phosphate (Emcompress@ Anhydrous; Mendell a Penwest Company, 30 Lakeville, MN) in a Turbula Powder Blender (Glen Mills Inc., Clifton, NJ) for 10 minutes. The mixture was wet-granulated using ethanol. The obtained wet granules were sieved using a 2-mm sieve, and thereafter allowed to dry at room temperature in a fume hood for 9 24 hours. The dried granules were sieved using a 1.68 mm and 1.18 mm sieves, and the obtained middle fraction was collected for capsulation. Simultaneously, a placebo formulation, where the L-cysteine was replaced by the same 5 amount of CaHPO 4 , was prepared following the exact same procedure. The obtained matrix granules were weighed into HPMC capsules so that the desired amount of cysteine per capsule was obtained. The L-cysteine concentration of the granules was determined using a capillary method (400 mg of granules contained 98 mg of L 10 cysteine). The amount of L-cysteine per capsule was left at 50 mg in order to ease the selection of a suitable dosage (for a dosage of 100 mg or 200 mg of L-cysteine, 2 or 4 capsules were administered at essentially the same time to the subject). Similar capsules containing also titanium dioxide were prepared, and this excipient was 15 found not to have an effect on the desired function of the capsule. The capsules prepared above are ingested to decrease the risk of cancer locally caused by acetaldehyde in occasions, which are favourable for an increase in the acetaldehyde content of the stomach, such as in connection with consuming alcoholic drinks. The dosage 20 is given at 4 to 6-hour intervals as long as there is alcohol in the blood. Example 2 - dissolution test for the capsules Dissolution tests were carried out on the capsules of Example 1 according to the USP I 25 method (USP 24) (The United States Pharmacopeia 2001). A standard curve was prepared between 0.01 and 0.6 mg/ml (y = 2.196 + 0.0016, r 2 = 0.9999). The medium used was 500 ml of pH 1.2 HCI buffer. The rotation rate of the baskets was 100 rpm, and the temperature of the medium was +37*C (+ 0.5). Samples were taken at 5-minute intervals for the first half hour and thereafter at 10-minute intervals for the remaining 2 hours. L-cysteine was 30 detected in flow-through cells (10 mm) at a wavelength of 213 nm. The results were calculated by using dissolution software. The system was equipped with a bath and a pump (Sotax AT7 UV Dissolution System, Stax, Allschwil, Switzerland) and a spectrophotometer (PerkinElmer, Lambda 25, PerkinElmer, Inc., Waltham, MA), the software used for the test and for calculating the results was WinSotax (Sotax).

10 This dissolution test showed that the formulation released L-cysteine at a controlled rate, yet fast enough to have time to react with acetaldehyde before leaving the stomach. These results are shown in Fig. 1. When not granulated, the L-cysteine was dissolved rapidly 5 (100 % in 5 minutes). Example 3 - Acetaldehyde-binding Study procedure: 10 Seven volunteers (2 men, 5 women) with achlorhydric atrophic gastritis participated in the study. Their mean age ± SD was 57 ±7 years and mean body weight 75 ± 22 kg. All volunteers were non-smokers and normal social drinkers, with an average consumption of 50 g or less of ethanol per week. 15 A randomized double-blinded placebo-controlled study design was used, and each participant served as his/her own control. The 2 study days were separated by at least a 3 day interval. The volunteers were told to refrain from alcohol intake for 24 hours and food intake for 12 hours prior to the study. 20 A nasogastric tube (Duodenal tube Levin, CH10, Unomedical, Denmark) was inserted into the subjects to a depth of 55 cm at the beginning of each study day. The tube was lubricated with Xylocain gel (AstraZeneca, S6dertalje, Sweden) containing no ethanol. During the tube placement, the volunteers were given 100 ml of water to facilitate 25 swallowing of the tube. The subjects were given four capsules, containing either cysteine (50 mg in each capsule) or placebo, as prepared according to Example 1, orally double blindly with 200 ml of water. Immediately thereafter, ethanol (0.3 g/kg body weight) diluted in water to 15 vol%, 30 was infused via the nasogastric tube into the stomach of the volunteers. Samples of gastric juice (5 ml) were aspirated through the tube at 5-minute intervals up to 60 minutes after the ethanol infusion or until the stomach had emptied, as indicated by I1 unsuccessful aspiration. The samples were analyzed for pH and acetaldehyde, ethanol and cysteine concentrations. Analysis: 5 To measure the acetaldehyde concentration, 450 gl of gastric juice was immediately transferred into a headspace vial containing 50 pl of 6 mol/l perchloric acid. Perchloric acid does not hydrolyze the cysteine-acetaldehyde bond. 10 For the ethanol analysis, the gastric juice was diluted 10-fold in purified water, and 500 pl of diluted gastric juice was transferred into a headspace vial. Two parallel samples were used for the measurements, and the mean value was calculated. The levels of acetaldehyde and ethanol were analyzed by headspace gas chromatography, 15 as previously described (Vakevainen et al., 2002, Scand J Gastroenterol, 37:648-655). L-cysteine concentration of the gastric juice samples were determined by using an HPLC method. A standard curve was prepared between concentrations of 0.0625 and 2.0 mg/ml (y = 851.06x + 8.52, r 2 = 0.9704). Two parallel samples were again prepared. 60 pl of 20 gastric juice was measured into a test tube, and 30 p of pH 7.4 phosphate-buffered saline solution and 30 g1 of 20 vol-% tri-n-butyl phosphine in dimethylformamide were added. The samples were incubated for 30 minutes at +4*C, after which 90 pl of cold 10% trichloroacetic acid containing 1 mM Na 2 EDTA was added, and the samples were vortexed for 2 minutes and then centrifuged for 10 minutes at 4500 rpm. 50 pl of supernatant was 25 pipette into a test tube containing 125 pl of pH 9.5 borate buffer with 4mM Na 2 EDTA, 10 p of 1.55 M sodium hydroxide, and 50 pl of 2 mg/ml 4-fluoro-7-sulfobenzofurazan, ammonium salt (SBD-F) solution in borate buffer. The samples were incubated for 60 minutes at +60*C so that a yellow derivate was formed. Thereafter, 150 pl of the solution was pipette into HPLC inserts, and used for the measurements. The isocratic mobile phase 30 was pH 7.0 phosphate buffer and methanol (95:5). The flow rate was 1 ml/min and the retention time was 6 minutes. The L-cysteine concentration was determined using a fluorescence detector (excitation 385 nm, emission 515 nm).

12 Results: Fig. 2 shows the effect of the L-cysteine administration (or the placebo administration) on the acetaldehyde levels. In all measurements, the average acetaldehyde concentration of 5 the gastric juice was 2.6 times higher with placebo than with cysteine. No significant differences existed in ethanol concentrations between cysteine and placebo treatments. The average ethanol concentration in the gastric juice was 5.0 vol-% in the first sample, declining to 0.9 vol-% in the 40-minute sample. A positive correlation emerged between the acetaldehyde concentration and the ethanol concentration. 10 L-cysteine was detected in the gastric juice of all volunteers after the administration of study formulations containing L-cysteine. The mean cysteine concentrations are represented in Fig. 3. After administration of placebo formulations, no L-cysteine was detected. No significant correlation was found between the cysteine concentration and the 15 acetaldehyde concentration.

Claims (5)

1. A locally acting sustained release pharmaceutical preparation comprising a granulated composition encapsulated in a capsule made of hydroxyl propyl methyl cellulose (HPMC), 5 the granulated composition containing, as its active ingredient L- or D-cysteine, further containing calcium hydrogen phosphate as a filler, Eudragit RS-PO as a binder, and optionally containing further fillers, binders or other additives.

2. The pharmaceutical preparation according to Claim 1, wherein the cysteine is L cysteine, and is present in an amount of 100 - 200 mg per administered dose. 10

3. The pharmaceutical preparation according to Claim 1, further containing titanium dioxide as a further additive.

4. A method for decreasing the risk of contracting cancer of the stomach, and consequently the large intestine, caused by the presence of acetaldehyde in these areas, wherein the preparation according to Claim 1 is administered to a subject in connection 15 with the subject smoking or in connection with the subject consuming alcoholic drinks or consuming alcohol-containing foodstuffs.

5. The method according to claim 4, wherein the preparations are administered at an interval of 4 - 10 hours, for as long as there is ethanol in the blood, or in connection with smoking. 20