Use of 5-aminosalicylates as antimicrobial agents

BACKGROUND OF THE INVENTION
 Throughout this application various publications are referenced within
 parentheses. The disclosures of these publications in their entireties are
 hereby incorporated by reference in this application in order to more
 fully describe the state of the art to which this invention pertains.
 1. The Field of the Invention
 This invention relates to the medical arts. In particular, it relates to a
 method of inhibiting bacterial growth in the gastrointestinal tract of a
 human or non-human vertebrate by using an antimicrobial agent.
 2. Discussion of the Related Art
 Antimicrobial or antibiotic agents are used in the treatment of bacterial
 infections, especially of the gastrointestinal tract. Gastrointestinal
 infections affect millions of people world-wide, especially children, and
 pose an increasing health hazard in hospital settings.
 Of course, bacteria inhabit healthy intestines to the benefit of their
 human and animal hosts. Anaerobic bacteria, including the Bacteroides
 fragilis group and Clostridium species are common members of the
 intestinal microflora of healthy individuals, and non-toxigenic strains
 can be transmitted without causing disease symptoms. (B. A. Cunha,
 Nosocomial diarrhea, Crit. Care Clin. 14(2):329-38 [1998]; H. Kato et al.,
 Application of typing by pulsed-yield gel electrophoresis to the study of
 Clostridium difficile in a neonatal intensive care unit, J. Clin.
 Microbiol. 32(9):2067-70 [1997]; V. O. Rotimi and B. I. Duerden,
 Bacteroides species in the normal neonatal faecal flora, J. Hyg. [Lond.]
 87(2):299-304 [1981]; B. I. Duerden, The isolation and identification of
 Bacteroides spp. From the normal human faecalflora, J. Med. Microbiol.
 13(1):69-78 [1980]; S. S. Long and R. M. Swenson, Development of anaerobic
 flora in healthy newborn infants, J. Pediatr. 91(2):298-301 [1977]). Other
 pathogenic bacterial strains have an adverse effect on their hosts.
 For example, enterotoxigenic strains of Bacteroides fragilis are associated
 with diarrhea in humans. (S. K. Niyogi et al., Association of
 enterotoxigenic Bacteroides fragilis with childhood diarrhoea, Indian J.
 Med. Res. 105:167-69 [1997]; R. B. Sack et al., Enteroloxigenic
 Bacteroides fragilis: epidemiologic studies of its role as a human
 diarrhoeal pathogen, J. Diarrhoeal Dis. Res. 10(1):4-9 [1992]). And
 patients with Crohn's disease were reported to have higher numbers of B.
 fragilis group bacteria in their intestines than healthy controls. (J. G.
 Ruseler-van Embden and H.C. Both-patoir, Anaerobic gram-negative faecal
 flora in patients with Crohn's disease and healthy subjects, Antonie van
 Leeuwenhoek 49(2):125-32 [1983]).
 More prominent agents of gastrointestinal disease than Bacteroides, are the
 Clostridium species, especially C. difficile and C. perfringens.
 Clostridium species are gram-positive, spore-forming anaerobes; some
 strains that colonize the human intestines can, under certain
 circumstances, release potent protein exotoxins that induce inflammation
 of the intestinal mucosa. (M. L. Job and N. F. Jacobs, Jr., Drug-induced
 Clostridium difficile-associated disease, Drug Saf. 17(1):37-46 [1997]).
 For example, antibiotics and other chemotherapeutic agents can induce the
 expression of Toxins A and B from Clostridium difficile. (B. A. Cunha
 [1998]). Agents known to have a high potential to induce C.
 difficile-associated disease are aminopenicillins, cephalosporins and
 clindamycin. (M. L. Job and N. F. Jacobs, Jr., Drug-induced Clostridium
 difficile-associated disease, Drug. Saf. 17(1):37-46 [1997]; Y. Hutin et
 al., Prevalence of and risk factors for Clostridium difficile colonization
 at admission to an infectious diseases ward, Clin. Infect. Dis.
 24(5):920-24 [1997]; C. D. Settle and M. H. Wilcox [1996]).
 In developed countries, the great majority of cases of C. difficile
 infection are hospital-acquired, and the number of nosocomial clostridial
 infections is reported to be rising. (C. D. Settle and M. H. Wilcox,
 Review article: antibiotic-induced Clostridium difficile infection,
 Aliment. Pharmacol. Ther. 10(6):835-41[1996]; J. S. Brazier, The
 epidemiology and typing of Clostridium difficile, J. Antimicrob.
 Chemother. 41 Suppl. C:47-57 [1998]; S. Tabaqchali and M. Wilks,
 Epidemiological aspects of infections caused by Bacteroides fragilis and
 Clostridium difficile, Eur. J. Clin. Microbiol. Infect. Dis. 11(11):
 1049-57 [1992]; C. R. Clabots et al., Acquisition of Clostridium difficile
 by hospitalized patients: evidence for colonized new admissions as a
 source of infection, J. Infect. Dis. 166(3):561-67 [1992]).
 A nosocomial pleural infection with C. difficile, following surgical
 insertion of a chest drain has also been reported (A. J. Simpson et al.,
 Nosocomial empyema caused by Clostridium difficile, J. Clin. Pathol.
 49(2):172-73 [1996]), but intestinal infections are the greatest problem.
 Nosocomial diarrhea due to gastrointestinal infection with C. difficile has
 become a major health care problem, causing 20-30% of all nosocomial
 diarrheas and affecting up to 8% of hospitalized patients. (L. R. Peterson
 and P. J. Kelly, The role of the clinical microbiology laboratory in the
 management of Clostridia difficile-associated diarrhea, Infect. Dis. Clin.
 North Am. 7(2):277-93 [1993]). Clostridium difficile is considered to be
 the premier cause of diarrhea among hospitalized patients. (M. Delmee et
 al., Treatment of Clostridium difficile colitis. Summary of a round table
 held in Brussels on Mar. 3, 1994, Acta Clin. Belg. 50(2):114-116 [1995]).
 An infection of C. difficile can add an average of three weeks to a
 patient's hospital stay. (C. D. Settle and M. H. Wilcox [1996]). Symptoms
 may include, diarrhea, self-limited colitis, toxic megacolon or
 potentially lethal fulminant pseudomembranous colitis. Intestinal
 infection with C. difficile has also been linked to reactive arthritis.
 (I. H. Kocar et al., Clostridium infection in patients with reactive
 arthritis of undetermined etiology, Scand. J. Rheumatol. 27(5):357-62
 [1998]; R. K. Cleary, Clostridium difficile-associated diarrhea and
 colitis: clinical manifestations, diagnosis, and treatment, Dis. Colon.
 Rectum 41(11):1435-49 [1998]). Bacteraemia and subsequent sepsis is
 another possible complication of intestinal infection by C. difficile. (P.
 Naaber et al., Bacterial translocation, intestinal microflora and
 morphological changes of intestinal mucosa in experimental models of
 Clostridium difficile infection, J. Med. Microbiol. 47(7):591-98 [1998];
 R. J. Feldman et al., Bacteremia due to Clostridium difficile: case report
 and review of extraintestinal C. Difficile infections, Clin. Infect. Dis.
 20(6):1560-62 [1995]). In at least one nosocomial outbreak, 17 patients
 died from C. difficile infection. (C. D. Settle and M. H. Wilcox [1996]).
 Clostridium difficile intestinal infections in children, unassociated with
 antibiotic use or hospital stays, can cause chronic diarrhea and failure
 to grow. (T. E. Liston, Clostridium difficile toxin associated with
 chronic diarrhea and failure to gain weight, Clin. Pediatr. (Phila.)
 22(6):458-60 [1983]).
 In developing countries, C. difficile is also thought to be a causal agent
 of wide-spread acute cliarrheal disease. (S. K. Niyogi et al., Prevalence
 of Clostridium difficile in hospitalized patients with acute diarrhoea in
 Calcutta, J. Diarrhoeal Dis. Res. 9(1):16-19 [1991]; S. Q. Akhtar,
 Isolation of Clostridium difficile from diarrhoea patients in Bangladesh,
 J. Trop. Med. Hyg. 90(4):189-92 [1987]).
 Enterotoxigenic strains of C. perfringens are linked with a significant
 number of cases of antibiotic-associated diarrhea, especially among
 elderly hospitalized patients, children, and infants. (A. Wada et al.,
 Nosocomial diarrhoea in the elderly due to enterotoxigenic Clostridium
 perfringens, Microbiol. Immunol. 40(10):767-71 [1996]; M. M. Brett et al.,
 Detection of Clostridium perfringens and its enterotoxin in cases of
 sporadic diarrhoea, J. Clin. Pathol. 45(7):609-11 [1992]; S. C. Samuel et
 al., An investigation into Clostridium perfringens enterotoxin-associated
 diarrhoea, J. Hosp. Infect. 18(3):219-30 [1991]; S. P. Boriello et al.,
 Epidemiology of diarrhoea caused by enterotoxigenic Clostridium
 perfringens, J. Med. Microbiol. 20(3):363-72 [1985]; R. Willliams et al.,
 Diarrhoea due to entertoxigenic Clostridium perfringens: clinical features
 and management of a cluster of 10 cases, Age Aging 14(5):296-302 [1985]).
 Clostridium perfringens has been implicated as a possible contributor to
 sudden infant death syndrome (SIDS) in susceptible infants. (R. R. Meer et
 al., Human disease associated with Clostridium perfringens enterotoxin,
 Rev. Environ. Contam. Toxicol. 150:75-94 [1997]).
 Clostridium perfringens is well known as a causative agent of
 non-gastrointestinal gangrene, a special problem for many elderly and
 diabetic patients with poor blood circulation. But also in more extreme
 cases of gastrointestinal infection, C. perfringens can cause enteritis
 necroticans, a gangrene of the bowel resulting in necrosis, sepsis, and
 hemolysis, in humans and domesticated animals. (L. E. Clarke et al.,
 Enteritis necroticans with midgut necrosis caused by Clostridium
 perfringens, Arch. Surg. 129(5):557-60 [1994]; D. Bueschel et al.,
 Enterotoxigenic Clostridium perfringens type A necrotic enteritis in a
 foal, J. Am. Vet. Med. Assoc. 213(9):1305-07 [1998]; E. G. Pearson et al.,
 Hemorrhagic enteritis caused by Clostridium perfringens type C in afoal,
 J. Am. Vet. Med. 188(11):1309-10 [1986]; F. Al-Sheikhy and R. B. Truscott,
 The interaction of Clostridium perfringens and its toxins in the
 production of necrotic enteritis of chickens, Avian Dis. 21(2):256-63
 [1977]).
 Although rare in developed countries, clostridial enteritis necroticans in
 humans is more common in some developing countries. (D. A. Watson et al.,
 Pig-bel but no pig: enteritis necroticans acquired in Australia, Med. J.
 Aust. 155(1):47-50 [1991]). In New Guinea, enteritis necroticans, known
 locally as pigbel, has been a major cause of illness and death among
 children. (G. W. Lawrence et al., Impact of active immunisation against
 enteritis necroticans in Papua New Guinea, Lancet 336(8724): 1165-67
 [1990]). Clostridium perfringens type C, the etiologic agent of enteritis
 necroticans, was also isolated from Bangladeshis with bloody or watery
 diarrheal illness. (F. P. van Loon et al., Clostridium perfringens type C
 in bloody and watery diarrhea in Bangladesh, Trop. Geogr. Med.
 42(2):123-27 [1990]).
 Entertoxigenic strains of C. perfringens have also been linked to
 nosocomial and non-nosocomial outbreaks of food poisoning, due to
 heat-resistant spores and a rapid growth rate in warm food. (A. M. Pollack
 and P. M. Whitty, Outbreak of Clostridium perfringens food poisoning, J.
 Hosp. Infect. 17(3):179-86 [1991]; M. Van Damme-Jongsten et al., Synthetic
 DNA probes for detection of enterotoxigenic Clostridium perfringens
 strains isolated from outbreaks of food poisoning, J. Clin. Microbiol.
 28(1):131-33 [1990]).
 Spores of Clostridium botulinum germinating in warm food can cause another
 form of food poisoning called botulism. Growing particularly in non-acidic
 foods lacking nitrites, and protected from oxygen, the vegetative cells of
 C. botulinum release an exotoxin that when consumed with the food is
 activated by trypsin in the stomach, and is absorbed intact by the blood
 stream. The exotoxin binds to nerve cells, preventing the release of the
 neurotransmitter acetylcholine. Resulting symptoms of botulism include
 blurred vision, difficulty in swallowing and speaking, and increasing
 muscular weakness, and usually nausea and vomiting. Death often results
 from paralysis of the muscles required for breathing. (R. Y. Stanier et
 al., The Microbial World, 5 th ed., Prentice Hall, Englewood Cliffs, N.J.
 pp.626-27 [1986]). Clostridium botulinum sometimes colonizes the
 intestines of infants and can cause infantile botulism, which can lead to
 respiratory paralysis and sudden infant death.
 Botulism is a problem for the food packaging industry. Spores of C.
 botulinum may not be killed if canning is done at too low a temperature.
 High temperature autoclave treatment may be unsuitable for some foods
 Mayonnaise and other non-acidic foods are particularly prone to foster the
 growth of C. botulinum. Now with increasing health concerns about the use
 of nitrite as a food preservative, alternative antimicrobial agents are
 needed against the growth of C. botulinum and other food poisoning
 bacterial pathogens.
 Antimicrobial agents with selective toxicity for a specific spectrum or
 range of pathogenic microorganisms are well known in the art. One class of
 antimicrobial agents is the antibiotics, which are compounds, synthesized
 and excreted by various microorganisms, that are selectively toxic to
 other microorganisms, specifically bacteria. In addition, some antibiotics
 can be artificially modified to produce antimicrobial agents that are more
 effective and/or more able to overcome antibiotic resistance.
 The first line antibiotic treatment for diseases associated with
 gastrointestinal infections of Clostridium has been a 10-day course of
 metronidazole or vancomycin, which may be administered orally,
 intravenously, or rectally. (R. K. Cleary [1998]; C. P. Kelly and J. T.
 LaMont, Clostridium difficile infection, Annu. Rev. Med. 49:375-90 [1998];
 C. M. Reinke and C. R. Messick, Update on Clostridium difficile-induced
 colitis, Part 2, Am. J. Hosp. Pharm. 51(15):1892-1901 [1994]).
 Neither of these antibiotics has been completely satisfactory. While
 metronidazole (flagyl) is effective against obligate anaerobes (e.g., P.
 Muir et al., Breath hydrogen excretion by healthy cats after oral
 administration of oxytetracycline and metronidazole, Vet. Rec. 138:635-39
 [1996]; B. Lembcke et al., Different actions of neomycin and metronidazole
 on breath hydrogen (H) exhalation, Z. Gastroenterol. 18(3):155-60 [1980]),
 it yields an unpleasant after-taste to many patients, even when delivered
 intravenously. Other common side effects of metronidazole are neuropathy
 and gastrointestinal distress. Also, metronidazole is a known reproductive
 toxicant affecting mammalian sperm cells. (R. E. Linder et al., Endpoints
 of spermatotoxicity in the rat after short duration exposures to fourteen
 reproductive toxicants, Reprod. Toxicol. 6(6):491-505 [1992]).
 On the other hand, a course of vancomycin is prohibitively expensive (10-50
 times more expensive than metronidazole), and there are concerns about the
 rapid development of vancomycin-resistance among pathogenic Clostridium,
 Enterococcus, Pediococcus, Citrobacter, Klebsiella, Enterobacter, and
 Staphylococcus species, because the plasmid-borne vancomycin resistance
 gene (VanR) is readily transmissible. (ASHP therapeutic position statement
 on the preferential use of metronidazole for the treatment of Clostridium
 difficile-associated disease, Am. J. Health Syst. Pharm. 55(13):1407-1
 [1998]; S. H. Cohen et al., Isolation of a toxin B-deficient mutant strain
 of Clostridium difficile in a case of recurrent C. difficile-associated
 diarrhea, Clin. Infect. Dis. 26(2):1250 [1998]; C. Edlund et al., Effect
 of vancomycin on intestinal flora of patients who previously received
 antimicrobial therapy, Clin. Infect. Dis. 25(3):729-32 [1997]; C. A.
 O'Donovan et al., Enteric eradication of vancomycin-resistant Enterococcus
 faecium with oral bacitracin, Diagn. Microbiol. Infect. Dis. 18(2):105-09
 [1994]; E. Yamaguchi et al., Colonization pattern of vancomvcin-resistant
 Enterococcus faecium, Am. J. Infect. Control 22(4):202-06 [1994]; C. P.
 Kelly and J. T. LaMont [1998]).
 In addition, relapses of clostridial infections occur in about 5-42% of
 those treated with metronidazole or vancomycin and are believed to be
 caused by persistent endogenous clostridial spores, which are antibiotic
 resistant. (S. H. Cohen et al. [1998]; R. Fekety et al., Recurrent
 Clostridium difficile diarrhea: characteristics of and risk factors for
 patients enrolled in a prospective, randomized, double-blinded trial,
 Clin. Infect. Dis. 24(3):324-33 [1997]; S. Johnson et al., Treatment of
 asymptomatic Clostridium difficile carriers fecal excretors with
 vancomycin and metronidazole. A randomized, placebo-controlled trial, Ann.
 Intern. Med. 117(4):297-302; M. J. Zimmerman et al., Review article:
 treatment of Clostridium difficile infection, Aliment. Pharmacol. Ther.
 11(6):1003-12 [1997]).
 Teicoplanin is another antibiotic found to be effective against gram
 positive anaerobes such as Propionibacterium acnes, Clostridium
 perfringens, C. difficile, and other Clostridium spp., Peptococcus spp.,
 Peptostreptococcus spp. (H. Hassan et al., In vitro activity of
 teicoplanin, vancomycin, A16686, clindamycin, erythromycin and fusidic
 acid against anaerobic bacteria, Singapore Med. J. 31(1):56-58 [1990]; The
 Swedish CDAD Study Group, Treatment of Clostridium difficile associated
 diarrhea and colitis with an oral preparation of teicoplanin; a dose
 finding study, Scand. J. Infect. Dis. 26(3):309-16 [1994]; C. Wenisch et
 al., Comparison of vancomycin, teicoplanin, metronidazole, and fusidic
 acid for the treatment of Clostridium difficile-associated diarrhea, Clin.
 Infect. Dis. 22(5):813-18 [1996]).
 However, teicoplanin is not widely available. The peptide antibiotic
 bacitracin is also reported to be effective in treating C.
 difficile-induced diarrhea, but it is not widely available in an enteric
 formulation. (M. N. Dudley et al., Oral bacitracin vs. vancomycin
 therapyfor Clostridium difficile-induced diarrhea, A randomized
 double-blind trial, Arch. Intern. Med. 146(6):1101-04 [1986]).
 Other adjunct treatments are reportedly effective for refractory C.
 difficile-related disease, including whole-bowel irrigation and enteric
 administration of the non-pathogenic yeast Saccharomyces boulardii. (C. A.
 Liacouras and D. F. A. Piccoli, Whole-bowel irrigation as an adjunct to
 the treatment of chronic, relapsing Clostridium difficile colitis, J.
 Clin. Gastroenterol. 22(3):186-89 [1996]; C. M. Surawicz, Clostridium
 difficile disease: diagnosis and treatment, Gastroenterologist 6(1):60-65
 [1998]). But such treatments are uncomfortable or distasteful for many
 patients and are less suitable than easily administered antibiotics as a
 first line treatment regimen.
 Accordingly, there remains a definite need for a modestly priced
 antimicrobial agent for treating gastrointestinal infections, without the
 commonly unpleasant side effects and bacteria resistance associated with
 metronidazole and vancomycin.
 Pharmaceutical preparations of 4-(p)-aminosalicylic acid (i.e., 4-ASA or
 para-aminosalicylic acid) or 4-(p)-aminosalicylate sodium salt (e.g.,
 Nemasol-Sodium.RTM. or Tubasal.RTM.) have been used systemically in cases
 of tuberculosis as antimicrobial chemotherapeutic agents against
 Mycobacterium tuberculosis.
 On the other hand, the 5-aminosalicylates are known as anti-inflammatory
 chemotherapeutic agents and have not been used as antimicrobial agents.
 These compounds include 5-aminosalicylic acid (i.e., 5-ASA, mesalamine, or
 mesalazine) and conjugated derivatives thereof, known for their
 anti-inflammatory properties. These anti-inflammatory agents are
 commercially available in various pharmaceutical preparations such as
 Asacol.RTM., Rowasa.RTM., Claversal.RTM., Pentasa.RTM., Salofalk.RTM.,
 Dipentum.RTM., Azulfidine.RTM. (SAZ) and others.
 5-Aminosalicylates have been used widely to reduce mucosal inflammation in
 inflammatory bowel disease, ulcerative colitis and Crohn's disease. (S. B.
 Hanauer and FB. Baert, Medical Therapy of Inflammatory Bowel Disease,
 Inflamm. Bowel Dis. 78(6):1413-25 [1994]; C. J. Mulder et al., Drug
 therapy. dose-response relationship of oral mesalazine in inflammatory
 bowel disease, Mediators Inflamm. 7(3):135-36 [1998]; W. Kruis et al.,
 Olsalazine versus mesalazine in the treatment of mild to moderate
 ulcerative colitis, Aliment. Pharmacol.12(8):707-15 [1998]; J. N. Healey,
 Gastrointestinal transit and release of mesalazine tablets in patients
 with inflammatory bowel disease, Scand. J. Gastroenterol. 172:47-51
 [1990]).
 The mechanism underlying the anti-inflammatory properties of the
 5-aminosalicylates is unknown, but it may result from their ability to
 inhibit oxidation at the surface of endothelial membranes, perhaps through
 radical scavenging, and to prevent lipid peroxidation. (D. C. Pearson et
 al., The anti-oxidant properties of5-aminosalicylic acid, Free Radic.
 Biol. Med. 21(3):367-73 [1996]). 5-Aminosalicylates may be able to act
 synergistically with endogenous antioxidants such as alpha-tocopherol to
 prevent the oxidative damage implicated in the pathogenesis of
 inflammatory bowel diseases. (E. Goncalves et al., Antioxidant activity of
 5-aminosalicylic acid against peroxidation of phosphotidylcholine
 liposomes in the presence of alpha-tocopherol: a synergistic effect?, Free
 Radic. Res. 29(1):53-66 [1998]).
 There are several reports that 5-aminosalicylic acid also inhibits
 fimbriae-mediated cellular adhesion by enteroaggregative Escherischia coil
 strains, associated with both acute and persistent diarrhea in infants and
 children. (G. Kang et al., Salicylate inhibits fimbriae mediated Hep-2
 cell adherence ofand haemagglutination by enteroaggregative Escherischia
 coli, FEMS Microbiol. Lett. 166(2):257-65 [1998]; D. Law and H. Chart,
 Enteroaggregative Escherischia coil, J. Appl. Microbiol. 84(5):685-97
 [1998]; Y. Germani et al., Prevalence of enteropathogenic,
 enteroaggregative Escherischia coli among isolates from children with
 diarrhea in New Caledonia, J. Infect. Dis. 174(5):124-26 [1996]; S.
 Knutton et al., Ability of enteroaggregative Escherischia coil strains to
 adhere in vitro to human intestinal mucosa, Infect. Immun. 60(5):2083-91
 [1992]).
 Antimicrobial growth inhibitory properties of the 5-aminosalicylates and
 other features and advantages of the present invention will be described
 herein.
 SUMMARY OF THE INVENTION
 The method of the present invention employs the antimicrobial properties of
 5-aminosalicylates in a method of inhibiting bacterial growth in a human
 or non-human vertebrate. By administering to a patient a pharmaceutical
 composition comprising a 5-aminosalicylate, the growth of bacterial
 species can be arrested, including the growth of anaerobic pathogens of
 the genus Clostridium, for example, C. perfringens, C. difficile, C.
 botulinum and C. tetani. The present method can be used to treat the
 gastrointestinal tract or any other non-gastrointestinal body site or
 tissue to inhibit bacterial growth.
 The present method also has veterinary applications and can be used to
 treat a wide variety of non-human vertebrates, including wild, domestic
 and farm animals.
 The present invention is also related to an antimicrobial method for
 inhibiting the growth of a bacterial species in a foodstuff and to
 foodstuffs containing a 5-aminosalicylate compound, useful for preventing
 food poisoning or botulism. The present invention is also related to food
 containers and food-handling implements with bacteriostatic properties,
 intended for holding a foodstuff, and to cleansers, polishes, paints,
 sprays, soaps, and detergents for inhibiting the growth of a bacterial
 species on surfaces.
 The 5-aminosalicylates are generally well-tolerated and competitively
 priced compounds that can fill a definite need for an antimicrobial agent,
 which is selectively effective against gastrointestinal and
 non-gastrointestinal clostridial diseases.
 These and other advantages and features of the present invention will be
 described more fully in a detailed description of the preferred
 embodiments which follows.
 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
 The present invention is directed to the use of 5-aminosalicylates as
 antimicrobial agents in a method of inhibiting bacterial growth in a human
 or non-human vertebrate, in the gastrointestinal tract or any other body
 site or tissue. The method involves administering to a human or non-human
 vertebrate subject a pharmaceutically acceptable composition of the
 present invention.
 The pharmaceutically acceptable compositions in accordance with the present
 invention are formulated for acceptable delivery to a human or non-human
 vertebrate gastrointestinal tract or other body site or tissue. Topical
 delivery to gastrointestinal mucosa and/or systemic delivery thereto are
 intended. Also included are pharmaceutically acceptable formulations for
 systemic or topical delivery to a non-gastrointestinal body site or tissue
 subject to bacterial growth, and particularly subject to clostridial
 growth.
 The present invention is also related to pharmaceutically acceptable
 compositions containing a 5-aminosalicylate compound.
 Five-aminosalicylate compounds include 5-aminosalicylic acid or any
 compound containing a 5-aminosalicylate moiety, or any conjugated
 derivative thereof, that is effective in inhibiting the growth of a
 bacterial species in a human or non-human vertebrate gastrointestinal
 tract or any other body site, limb, organ, such as heart, lung or kidney,
 or any other tissue of a vertebrate body.
 A most preferred 5-aminosalicylate compound is 5-aminosalicylic acid, also
 known as mesalamine or mesalazine, and commercially available, for
 example, in pharmaceutical preparations known as Asacol.RTM. (Procter and
 Gamble), Pentasa .RTM.(Ferring A/S Vanlose), Claversal.RTM., Mesasal.RTM.,
 Rowasa.RTM., and Salofalk.RTM. (Dr. Falk GmbH). (R. N. Brogden and E. M.
 Sorkin, Mesalazine. A review of its pharmacodynamic and pharmacokinetic
 properties, and therapeutic potential in chronic inflammatory bowel
 disease, Drugs 38(4):500-23 [1989]).
 Also preferred are 5-aminosalicylate salts, including the sodium or
 potassium salts of conjugated 5-aminosalicylates.
 Other preferred 5-aminosalicylate compounds, for purposes of the present
 invention, are compounds containing a 5-aminosalicylic acid moiety, or a
 5-aminosalicylate moiety, conjugated to another 5-aminosalicylic acid
 moiety or 5-aminosalicylate moiety, or conjugated to a pharmaceutically
 acceptable carrier molecule.
 Preferred conjugated 5-aminosalicylate compounds include, but are not
 limited to, olsalazine or Poly-ASA. Olsalazine is known variously as
 disodium-diazosalicylate, azodisalicylate, or Dipentum.RTM. (Pharmacia).
 Olsalazine comprises two 5-aminosalicylate moieties linked through a
 diazobond. Poly-ASA comprises a water-soluble polymer that contains
 5-aminosalicylate moieties linked through diazobonds to an inert
 sulfanilamide ethylene polymer. (F. Martin, Oral 5-aminosalicylic acid
 preparations in treatment of inflammatory bowel disease: an update,
 Digestive Dis. Sci. 32(12 Suppl.):57S-63S [1987]; H. Allgayer,
 Sulfasalazineand 5-ASA compounds, GastrointestinalPharmacol. 21(3):643-658
 [1992]).
 Balsalazide (balsalazine) is another preferred conjugated 5-aminosalicylate
 compound. (J. R. Green et al., Balsalazide is more effective and better
 tolerated than mesalamine in the treatment of acute ulcerative colitis.
 The Abacus Investigator Group, Gastroenterol. 114(1):15-22 [1998a]; J. R.
 Green et al., Maintenance and remission of ulcerative colitis: a
 comparison between balsalazide 3 g and mesalamine 1.2 g daily over 12
 months. Abacus Investigator Group, Aliment. Pharmacol. Ther.
 12(12):1207-16 [1998]b).
 Another preferred conjugated 5-aminosalicylate compound is sulphasalazine,
 also known as sulfasalazine, salazosulfapyridine, or Azulfidine.RTM., in
 which a 5-aminosalicylic acid moiety is linked through a diazobond to
 sulfapyridine, the carrier molecule. (H. P. Osterwald, Pharmaceutic
 development: mesalazine, Scand. J. Gastroenterol Suppl. 172:43-46 [1990]).
 The skilled practitioner will be aware that patients to whom
 sulphasalazine is administered should be monitored especially carefully
 for symptoms of drug intolerance or hypersensitivity reactions that can
 occur in some patients. These symptoms are related to the sulfapyridine
 moiety and may include headache, nausea, vomiting, fever, rash,
 epidermolysis, hemolytic anemia, pancreatitis, pulmonary fibrosis,
 agranulocytosis, or liver toxicity; the use of sulphasalazine should be
 discontinued immediately when these symptoms occur. (S. B. Hanauer and G.
 Stathopoulos, Risk-benefit assessment of drugs used in the treatment of
 inflammatory bowel disease, Drug Saf. 6(3):192-219 [1992]; H. Allgayer
 [1992]). Impairment of male fertility is another possible side effect of
 sulphasalazine related to sulfapyridine. V. A. Botoman and G. F. Bonner,
 Management of inflammatory bowel disease, Am. Fam. Physician 57(l):57-68
 [1998]).
 Other preferred conjugated 5-aminosalicylate compounds are ipsalazine
 (ipsalazide) and salicylazobenzoic acid; these are also well tolerated
 molecules comprising a 5-aminosalicylic acid moiety or 5-aminosalicylate
 moiety linked by a diazobond to another pharmaceutically acceptable
 chemical unit. (M. C. Rijk et al., Disposition of mesalazine-delivering
 drugs in patients with inflammatory bowel disease, with and without
 diarrhoea, Scand. J. Gastroenterol. 27(10):863-68 [1992]; S. N. Rasmussen,
 Bioavailability of controlled release mesalazine [5-ASA]preparations, J.
 Gastroenterol. 30(Suppl. 8):112-114 [1995]).
 Other preferred conjugated 5-aminosalicylate compounds for gastrointestinal
 use are bile acids conjugated with a 5-aminosalicylic acid moiety or
 5-aminosalicylate moiety. For example, in ursodeoxycholic
 acid-5-aminosalicylic acid (UDCA-5-ASA), ursodeoxycholic acid is linked
 through an amide bond to 5-aminosalicylic acid. The conjugated
 5-aminosalicylate compound is synthesized by adding a basic solution of
 5-aminosalicylic acid into the mixed anhydride of ursodeoxycholic acid and
 ethyl chloroformate. (A. K. Batta et al., Synthesis and intestinal
 metabolism of ursodeoxycholic acid conjugate with an antiinflammatory
 agent, 5-aminosalicylic acid, J. Lipid Res. 39(8);1641-46 [1998]).
 Five-aminosalicylate bile acid conjugates are not substantially absorbed
 in the duodenum but can be partially hydrolyzed in the colon by intestinal
 bacteria, such as Clostridium perfringens, to ursodeoxycholic acid and
 5-aminosalicylic acid. Id.
 As the skilled artisan is aware, bacterial azoreductases can reduce diazo
 bonds linking 5-aminosalicylic acid or 5-aminosalicylate moieties to
 another chemical moiety, thus releasing 5-aminosalicylic acid or
 5-aminosalicylate. Similarly, certain hydrolases, e.g., cholylglycine
 hydrolase, can deconjugate 5-aminosalicylic acid moieties or
 5-aminosalicylate moieties from other carriers, such as bile acids. Other
 particular varieties of 5-aminosalicylate conjugate can be subject to
 deconjugation by other enzyme systems. Whether or not a 5-aminosalicylic
 acid or 5-aminosalicylate moiety is actually deconjugated from a carrier
 molecule or from another 5-aminosalicylate moiety, in a human or non-human
 vertebrate gastrointestinal tract or other body site, does not limit the
 embodiments of a 5-aminosalicylate compound contemplated by the present
 invention. The present invention is not committed to any particular
 mechanism by which a particular 5-aminosalicylate compound operates to
 inhibit bacterial growth.
 The 5-aminosalicylate compound is administered by any suitable method.
 Representative methods include giving, providing, feeding or
 force-feeding, dispensing, inserting, prescribing, furnishing, treating
 with, taking, swallowing, eating or applying a pharmaceutical composition
 of the present invention.
 As well as a 5-aminosalicylate compound, the pharmaceutical compositions of
 the present invention can optionally contain pharmaceutically acceptable
 solvent(s), adjuvant(s) or non-medicinal carrier(s), binder(s),
 thickener(s), or filler substance(s) that are known to the skilled
 artisan. Common fillers include, but are not limited to, sucrose or
 lactose, or polymeric substances like starch. Also contemplated are
 additional medicinal or nutritive additives in combination with a
 5-aminosalicylate compound, as may be desired to suit the more particular
 needs of the practitioner.
 A dose effective to inhibit the growth of a bacterial species is such dose
 as sufficient to prevent cellular proliferation of a bacterial species, by
 either killing bacterial cells or by preventing or slowing cellular growth
 or reproduction of a bacterial species, compared to the rate of growth or
 reproduction in the absence of a 5-aminosalicylate compound. By way of
 example, 12.5 mg or more of 5-aminosalicylic acid have been found
 sufficient to inhibit the growth of Clostridium perfringens on 10-mL blood
 agar plates.
 The effective dose for each human or non-human vertebrate subject will
 depend upon the size and physiologic reactions of the subject to whom or
 to which the pharmaceutical preparations of the present invention are
 administered. And these reactions and the antimicrobial activity of the
 administered dose can be monitored by the prescribing physician or
 veterinarian. The pharmaceutical compositions of the present invention can
 be formulated and manufactured at more than one concentration, such that
 modular incremental amounts of 5-aminosalicylate compound are easily
 administered.
 For example, currently available preparations of 5-aminosalicylate
 compounds can be obtained in variable dosage units, including, for
 example: 250 mg (e.g., Salofalk.RTM., Pentasa.RTM., Azulfadine.RTM.,
 Dipentum.RTM., or olsalazine); 400 mg (e.g., Asacol.RTM.); 0.5, 1.5 g or
 3.0 g (e.g., Pentasa.RTM., Rowasa.RTM., Azulfadine.RTM., balsalazide).
 Suppositories containing 5-aminosalicylic acid in dosage units ranging
 from 0.2 to 1.0 grams are also available; enema solutions containing 1 to
 4 g of 5-aminosalicylic acid are also known. (M. A. Peppercorn, Advances
 in drug therapy for inflammatory bowel disease, Ann. Intern. Med.
 112:50-60 [1990]).
 The foregoing are merely illustrative of the possible dosage units that can
 be employed in the pharmaceutical compositions of the present invention,
 and smaller or larger dosage units than these are also contemplated.
 Larger dosage units are especially useful for large non-human vertebrates,
 such as, but not limited to, bovine animals, horses, pachyderms, or large
 marine mammals; smaller dosage units are especially useful for pediatric
 application and for small vertebrates, such as, but not limited to, mice
 or chickens.
 A minimum effective dose is as little as between 6.25 and 12.5 mg of a
 5-aminosalicylate compound per day. Effective doses of a 5-aminosalicylate
 compound at 12.5 to 150 mg per day and 150 to 250 mg per day are
 sufficient for smaller human adults, children, and smaller non-human
 vertebrates, such as rodents, canines, chickens and turkeys. A higher
 effective dose for an adult human is 250-6400 mg of a 5-aminosalicylate
 per day. Pediatric doses are typically 10-20% of effective doses for adult
 humans. Higher effective doses, from 6,450 mg/day, up to 1,000 mg/kg body
 mass per day can be used for large non-human vertebrates, for example, for
 sheep and larger animals such as cattle, horses, and elephants.
 Antimicrobial activity by 5-aminosalicylate compounds against a specific
 bacterial species of interest is determined by routine means well known to
 the skilled practitioner. For example, a "lawn" of a bacterial species can
 be plated on an appropriate solid medium, and zones of growth inhibition
 around sterile cellulose disks impregnated with a 5-aminosalicylate
 compound of interest can be measured. Similarly, inhibition assays in
 liquid media are also routinely accomplished.
 Inhibition of bacterial growth in a gastrointestinal tract is measured by
 conventional means well known to the skilled artisan. Many fermentative
 bacterial species found in the gastrointestinal tract produce detectable
 quantities of hydrogen or methane gas in the presence of certain sugars,
 which gases enter the blood stream of the host and are exhaled. This is
 the basis for intestinal bacterial growth detection means, such as, but
 not limited to, the lactulose, glucose, or lactose breath hydrogen tests.
 (E.g., P. Kerlin and L. Wong, Breath hydrogen testing in bacterial
 overgrowth of the small intestine, Gastroenterol. 95(4):982-88 [1988]; A.
 Strocchi et al., Detection of malabsorption of low doses of carbohydrate:
 accuracy of various breath H.sub.2 criteria, Gastroenterol.
 105(5):1404-1410 [1993]).
 Alternatively, bacterial growth in a gastrointestinal tract is measured by
 detection of .sup.13 CO.sub.2 or .sup.14 CO.sub.2 breath emissions after
 administering an isotope-labeled sugar that is metabolizable by
 gastrointestinal bacteria but non-digestible by the host, such as, but not
 limited to, xylose or lactulose in humans. (E.g., G. R. Swart and J. W.
 van den Berg, .sup.13 C breath test in gastrointestinal practice, Scand.
 J. Gastroenterol. [Suppl.] 225:13-18 [1998]; C. E. King and P. P. Toskes,
 Breath tests in the diagnosis of small intestinal bacterial overgrowth,
 Crit. Rev. Lab. Sci. 21(3):269-81 [1984]; C. S. Chang et al., Increased
 accuracy of the carbon-14 D-xylose breath test in detecting
 small-intestinal bacterial overgrowth by correction with the gastric
 emptying rate, Eur. J. Nucl. Med. 22(10):1118-22 [1995]; A. Schneider et
 al., Value of the .sup.14 C-D-xylose breath test in patients with
 intestinal bacterial overgrowth, Digestion 32(2):86-91 [1985]).
 Direct gastrointestinal sampling or biopsy from any body site or tissue can
 also be used to measure the inhibition of bacterial growth in a
 gastrointestinal tract or other body site or tissue. As the skilled
 artisan is aware, direct sampling at time intervals provides information
 about the growth inhibition of specific bacterial species of interest, to
 which breath testing is not well-suited. Samples are diluted and bacterial
 numbers can be assessed by conventional microbiological means such as, but
 not limited to colony plating or Most Probable Number (MPN) techniques, or
 direct counting of bacterial cells. For direct bacterial cell counts,
 cells can optionally be labeled with specific markers, and counts can be
 accomplished manually or by devices such as fluorescence activated cell
 sorting (FACS).
 Alternatively, evidence of inhibition of bacterial growth can be inferred
 by the practitioner treating a bacterial infection or intestinal bacterial
 overgrowth in a human or nonhuman vertebrate subject with observation of
 an improvement in various infection-or overgrowth-related symptoms in
 response to the administration of an antimicrobial composition of the
 present invention.
 Among the bacterial species inhibited in accordance with the present
 inventive method are obligate anaerobes such as, but not limited to,
 Clostridium species. It is a particular advantage of the present invention
 that 5-aminosalicylic acid is an antimicrobial agent that does not affect
 many beneficial or commensal gastrointestinal bacteria but selectively
 inhibits potentially pathogenic clostridial species, such as, but not
 limited to, C. perfringens, C. difficile, C. tetani and C. botulinum.
 The method of the present invention is also useful for veterinary purposes.
 An antimicrobial composition comprising a 5-aminosalicylate compound can
 be administered to a non-human vertebrate including, but not limited to, a
 wild, domestic or farm animal. The present method is useful for treating a
 mammal such as a non-human primate, mouse, rat, rabbit, gerbil, hamster,
 canine, feline, ovine, bovine, swine, pachyderm, equine, or marine mammal.
 Also, the method is useful to inhibit the growth of bacteria in a bird
 (avian) or poultry, such as a duck, chicken, goose, turkey, ostrich, emu,
 dove, pigeon, quail, pheasant, peafowl, or guinea fowl.
 In one embodiment, the pharmaceutically acceptable composition is
 administered by a non-systemic delivery route to the site of bacterial
 infection or overgrowth that is not primarily by way of the blood stream
 of a human or non-human vertebrate.
 Some non-systemic delivery routes and pharmaceutically acceptable
 non-systemic delivery systems that could be employed by one of skill in
 the art in practicing the methods and compositions of the present
 invention are now exemplified. The following are presented merely to
 illustrate and in no way to limit the possible delivery means
 contemplated.
 For gastrointestinal bacterial infection or bacterial overgrowth, suitable
 non-systemic delivery routes include, but are not limited to, an ingestive
 delivery route or a colonic delivery route. A most preferred delivery
 route is an ingestive delivery route, whereby the antimicrobial agent
 enters the gastrointestinal or digestive tract by way of voluntary or
 forced ingestion through the mouth. The organs of a gastrointestinal tract
 include the esophagus, stomach, large intestine, small intestine, or
 rectum. The skilled artisan will be aware that in a non-human vertebrate
 the digestive tract may include a rumen, crop, gullet, cecum, or other
 specialized organ as pertains to a particular vertebrate species.
 Another non-systemic delivery route is useful for non-gastrointestinal
 bacterial infections, particularly infections of the skin or externally
 accessible wounds; this delivery route is topical application to the
 affected area of an antimicrobial cream, gel, or ointment.
 Another preferred embodiment of the pharmaceutical compositions of the
 present invention, employing a non-systemic delivery route, is a
 suppository or foam for delivery of a composition comprising a
 5-aminosalicylate compound via anus or rectum to the colon. Once
 delivered, a 5-aminosalicylate compound of the present invention will act
 topically at the intestinal mucosa. Such suppository or foam delivery
 systems are known in the art; a commercially available example is
 Rowasa.RTM. mesalamine suppositories for anti-inflammatory purposes.
 Together with a 5-aminosalicylate compound the pharmaceutical preparation
 of the present invention, such as a suppository, can employ a variety of
 conventional thickeners, such as alginate, xanthan gum, or petrolatum.
 Also contemplated are suppositories or foams comprising hydrophilic
 polymers, such as hydroxypropyl cellulose, hydroxypropyl methylcellulose,
 hydroxyethylcellulose, dextran, pectin, polyvinyl pyrrolidone, starch,
 gelatin, or any of a number of other polymers known to be useful for this
 purpose.
 Another preferred embodiment of the compositions of the present invention
 is a gel for non-systemic delivery of a composition comprising a
 5-aminosalicylate compound via the colon, similar to gels commonly used
 for the delivery of other chemotherapeutic agents. Hydrogel matrices are
 known for this purpose. (Feijen, Biodegradable hydrogel matrices for the
 controlled release of pharmacologically active agents, U.S. Pat. No.
 4,925,677). Such biodegradable gel matrices may be formed, for example, by
 cross-linking a proteinaceous component and a polysaccharide or
 mucopolysaccharide component, then loading with a 5-aminosalicylate
 compound, for deliverability over an extended period.
 Another preferred embodiment of the present pharmaceutical composition
 formulated for a colonic non-systemic delivery system is a composition
 comprising a 5-aminosalicylate compound in an osmotically suitable enema
 solution. Commercially available preparations include Rowasa.RTM.
 mesalamine enemas for anti-inflammatory purposes. (See, R. N. Brogden and
 E. M. Sorkin, Mesalazine. A review of its pharmacodynamic and
 pharmacokinetic properties, and therapeutic potential in chronic
 inflammatory bowel disease, Drugs 38(4):500-24 [1989]).
 A most preferred embodiment of the pharmaceutical composition of the
 present invention is formulated for a non-systemic ingestive delivery
 system, such as, but not limited to a tablet, capsule, or caplet.
 Preferably, the non-systemic ingestive delivery system comprises an enteric
 coating to prevent esophageal or gastric release of 5-aminosalicylate
 compound. Such enteric-coated pharmaceuticals disintegrate after leaving
 the stomach, resulting in drug dispersion in the small intestine or colon
 where 5-aminosalicylates will act topically at the intestinal mucosa. As
 the skilled artisan will be aware, enteric-coated drug delivery systems
 are typically pH-sensitive, polymer-coated tablets, capsules, or caplets.
 A polymer coating can be selected that will direct release of a
 5-aminosalicylate compound to a particular region of the gut. Such
 polymers include, but are not limited acrylic polymers such as Eudragit-L
 or Eudragit-S, and cellulosic polymers, such as ethylcellulose.
 Commercially available examples of enteric-coated 5-aminosalicylates are
 mesalamine preparations known as Asacol.RTM. and Claversal.RTM., used as
 anti-inflammatory agents. The acrylic polymer coating of Claversal.RTM.,
 starts to dissolve at pH 6.0 after passing through the far more acidic
 milieu of the stomach, and as a result, mesalamine is reliably released in
 the distal small intestine (ileum) and colon of a human patient. (H. P.
 Osterwald, Pharmaceutic development: mesalazine, Scand. J. Gastroenterol.
 Suppl. 172:43-46 [1990]; D. Claussen, Asacol [mesalamine], Gastroenterol.
 Nurs. 15(l):33-34 [1992]). The acrylic polymer coating (i.e., Eudragit-S)
 of Asacol.RTM. is degraded at above pH 7.0. Thus, it is carried to the
 right side of the human colon where intraluminal pH is elevated above 7.0.
 (M. J. Dew et al., An oral preparation to release drug in the human colon,
 Br. J. Clin. Pharmacol. 14:405-08 [1982]). Salofalk.RTM. is another
 example; it is coated first with a semipermeable layer of ethylcellulose
 and second with an acrylic polymer (i.e., Eudragit-L), which is degraded
 at above pH 5.6 in the distal small intestine and colon to release
 mesalamine there. (F. Martin [1987]).
 Pentasa.RTM. contains 5-aminosalicylic acid microgranules coated with a
 semipermeable membrane of amphionic ethylcellulose, which can dissolve at
 either acidic or basic pH to release the microgranules. (F. Martin
 [1987]).
 In addition, such tablets, capsules, or caplets can be formulated in a
 multi-layer configuration for slow release over an extended period. Again,
 Pentasa.RTM. is an example of a commercially available ingestible slow
 release form of a 5-aminosalicylate compound. (See P. Fockens et al.,
 Comparison ofthe efficacy and safety of 1.5 compared with 3.0 g oral slow
 release mesalazine [Pentasa] in the maintenance treatment of ulcerative
 colitis. Dutch Pentasa Study Group, Eur. J. Gastroenterol. Hepatol.
 7(11):1025-30 [1995]).
 Another preferred ingestive delivery system comprises a lavage system,
 whereby a patient will ingest a large volume of an osmotically balanced
 flushing solution, containing 5-aminosalicylic acid, or a conventional
 flushing solution in conjunction with another ingestible form of a
 5-aminosalicylate antimicrobial agent. Such a lavage system can virtually
 eliminate bacterial populations from the intestine. This may be especially
 desirable in refractory cases of bacterial overgrowth or in preparing a
 patient for abdominal surgery.
 Another preferred ingestive delivery system is especially, but not
 exclusively, useful for veterinary applications. In this embodiment a
 pharmaceutical preparation of the present invention is formulated and
 prepared to be ingested by an animal along with its food, as part of a
 pharmaceutically acceptable feed mixture. A pharmaceutically acceptable
 food additive for humans is also contemplated.
 For some applications, a preferred embodiment of the antimicrobial method
 of the present invention involves a systemic delivery route, i.e., a route
 whereby delivery of a 5-aminosalicylate compound to the site of infection
 or bacterial growth is primarily via the blood stream. This embodiment can
 be used to inhibit bacterial growth in any body site or tissue, including
 the gastrointestinal tract. A systemic delivery route is also
 particularly, but not exclusively useful for gastrointestinally infected
 patients who are unable to effectively ingest a non-systemic formulation
 of the composition of the present invention due to persistent nausea,
 difficulty in swallowing, or other ingestion-preventing conditions, or
 who, due to resection or other condition of the bowel cannot accept a
 colonic delivery system.
 Alternatively, a systemic delivery route can be employed to deliver a
 5-aminosalicylate compound to body sites or tissues other than those of
 the gastrointestinal tract, including, but not limited to, skin, heart,
 lung, blood, kidney, liver, brain, arms, legs, digits, sexual organs,
 trunk, head, neck, or tail. Applications can include but are not limited
 to treating or preventing clostridial infections at any body site or
 tissue of a vertebrate. Such clostridial infections include, but are not
 limited to, gangrene or tetanus, caused, respectively, by C. perfringens
 and C. tetani, when these species grow in wounds and damaged tissues with
 low oxygen tension.
 Entry of a 5-aminosalicylate compound into the blood stream of a human or
 non-human vertebrate patient can occur by any route, system, device,
 method or mechanism. For the purposes of the present invention, a systemic
 delivery route can also include delivery through the skin, mucosa or
 epithelium of the mouth including the sublingual epithelium, the rectum,
 or the vaginal epithelium.
 Systemic delivery systems that are contemplated by the present invention
 include, but are not limited to, ingestion, injection, or intravenous
 drip, most conventionally. Other useful systemic delivery systems are
 known and include, but are not limited to, implant; adhesive transdermal
 patches; topical creams, gels or ointments for transdermal delivery;
 transmucosal delivery matrices or suppositories or gels. It is
 contemplated that the compositions of the present invention are formulated
 to deliver an effective dose of a 5-aminosalicylate compound by these or
 any other pharmaceutically acceptable systemic delivery system.
 A preferred embodiment of the compositions of the present invention
 employing a systemic delivery route is a topical cream, ointment or gel to
 be applied to the skin. In this embodiment, a composition of the present
 invention comprises a 5-aminosalicylate compound in a pharmaceutically
 acceptable delivery system comprising a permeation or penetration
 enhancer, such as polyethylene glycol monolaurate, dimethyl sulfoxide,
 N-vinyl-2-pyrrolidone, N-(2-hydroxyethyl)-pyrrolidone, or
 3-hydroxy-N-methyl-2-pyrrolidone. A variety of conventional thickeners
 often used in creams, ointments and gels, such as, but not limited to,
 alginate, xanthan gum, or petrolatum, may also be employed in this
 embodiment of a composition of the present invention.
 Another preferred embodiment of the compositions of the present invention
 is a formulation for systemic transmucosal delivery of a 5-aminosalicylate
 compound. A variety of pharmaceutically acceptable systems for
 transmucosal delivery of therapeutic agents are known in the art and are
 compatible with the practice of the present invention. (Heiber et al.,
 Transmucosal delivery of macromolecular drugs, U.S. Pat. Nos. 5,346,701
 and 5,516,523; Longenecker et al., Transmembrane formulations for drug
 administration, U.S. Pat. No. 4,994,439). Transmucosal delivery devices
 may be in free form, such as a cream, gel, or ointment, or may comprise a
 determinate form such as a tablet, patch, or troche. For example, delivery
 of a 5-aminosalicylate compound may be via a transmucosal delivery system
 comprising a laminated composite of, for example, an adhesive layer, a
 backing layer, a permeable membrane defining a reservoir containing a
 5-aminosalicylate compound a peel seal disc underlying the membrane, one
 or more heat seals, and a removable release liner. (Ebert et al.,
 Transdermal delivery system with adhesive overlay and peel seal disc, U.S.
 Pat. No. 5,662,925; Chang et al., Device for administering an active agent
 to the skin or mucosa, U.S. Pat. Nos. 4,849,224 and 4,983,395).
 Alternatively, a tablet or patch for delivery through the oral mucosa can
 comprise an inner layer containing the therapeutic agent of choice, a
 permeation enhancer, such as a bile salt or fusidate, and a hydrophilic
 polymer, such as hydroxypropyl cellulose, hydroxypropyl methylcellulose,
 hydroxyethylcellulose, dextran, pectin, polyvinyl pyrrolidone, starch,
 gelatin, or any of a number of other polymers known to be useful for this
 purpose. This inner layer can have one surface adapted to contact and
 adhere to the moist mucosal tissue of the oral cavity and may have an
 opposing surface adhering to an overlying non-adhesive inert layer.
 Optionally, such a transmucosal delivery system can be in the form of a
 bilayer tablet, in which the inner layer also contains additional binding
 agents, flavoring agents, or fillers. Some useful systems employ a
 non-ionic detergent along with a permeation enhancer. These examples are
 merely illustrative of available transmucosal delivery technology and are
 not limiting of the present invention.
 Another preferred embodiment of the compositions of the present invention
 is a gel for systemic delivery of a 5-aminosalicylate via the rectal or
 vaginal mucosa, similar to gels commonly used for the delivery of various
 other therapeutic agents. Hydrogel matrices are known for this purpose.
 (Feijen, Biodegradable hydrogel matricesfor the controlled release of
 pharmnacologically active agents, U.S. Pat. No. 4,925,677). Such
 biodegradable gel matrices can be formed, for example, by cross-linking a
 proteinaceous component and a polysaccharide or mucopolysaccharide
 component, then loading with a 5-aminosalicylate compound to be delivered.
 Another preferred embodiment of the compositions of the present invention
 is the systemic delivery of a 5-aminosalicylate compound via a
 biodegradable matrix implanted within the body or under the skin of a
 human or non-human vertebrate. The implant matrix may be a hydrogel
 similar to those described above. Alternatively, it may be formed from a
 poly-alpha-amino acid component. (Sidman, Biodegradable, implantable drug
 delivery device, and process for preparing and using same, U.S. Pat. No.
 4,351,337).
 A preferred embodiment of the composition of the present invention
 employing a systemic delivery route is a transdermal delivery system of a
 kind known in the art for delivering various drugs. Transdermal delivery
 systems can be of any number of varieties known in the art. For example,
 delivery of a 5-aminosalicylate compound can be via a transdermal delivery
 system comprising a laminated composite of an adhesive layer, a backing
 layer, a permeable membrane defining a reservoir containing a
 5-aminosalicylate compound, a peel seal disc underlying the membrane, one
 or more heat seals, and a removable release liner. (Ebert et al.,
 Transdermal delivery system with adhesive overlay and peel seal disc, U.S.
 Pat. No. 5,662,925; Chang et al., Device for administering an active agent
 to the skin or mucosa, U.S. Pat. Nos. 4,849,224 and 4,983,395).
 Alternatively, a transdermal delivery device can be a matrix type
 transdermal patch. (Chien et al., Transdermal estrogen/progestin dosage
 unit, system and process, U.S. Pat. Nos. 4,906,169 and 5,023,084; Cleary
 et al., Diffusion matrix for transdermal drug administration and
 transdermal drug delivery devices including same, U.S. Pat. No. 4,911,916;
 Teillaud et al., EVA-based transdermal matrix system for the
 administration of an estrogen and/or a progestogen, U.S. Pat. No.
 5.605,702; Venkateshwaran et al., Transdermal drug delivery matrix for
 coadministering estradiol and another steroid, U.S. Pat. No. 5,783,208;
 Ebert et al., Methods for providing testosterone and optionally estrogen
 replacement therapy to women, U.S. Pat. No. 5,460,820). The matrix of the
 patch can comprise a basal support layer, such as an acrylic or
 ethylene/vinyl acetate copolymer or a polyurethane foam or cellulosic
 material, and an adhesive, such as, but not limited to, polysiloxane. In
 the compositions of the present invention, the polymer matrix also
 contains a 5-aminoslaicylate compound, as described above, and optionally,
 a penetration-enhancing vehicle or carrier, such as N-vinyl-2-pyrrolidone,
 N-(2-hydroxyethyl)-pyrrolidone, or 3-hydroxy-N-methyl-2-pyrrolidone. The
 adhesive patch may be pressure-sensitive, to release the 5-aminosalicylate
 compound across the skin of the patient when the patch matrix has been
 applied to the skin. The patch may optionally comprise an inert backing
 layer in addition to a matrix layer, or can comprise multiple dosage units
 or layers.
 The present invention is also related to a method of inhibiting the growth
 of a bacterial species on a foodstuff. A foodstuff for purposes of the
 present invention is any food or beverage that can be ingested. This
 method is intended for bacteriostatic food packaging or handling and
 relies on the antimicrobial properties of 5-aminosalicylates. The present
 method involves treating a food-contacting surface of a material that is
 pharmaceutically acceptable for food packaging or food handling purposes,
 with a 5-aminosalicylate compound in an amount effective to inhibit the
 growth of a bacterial species on a food contacting the surface.
 Acceptable materials for food packaging or handling include, but are not
 limited to, paper, wood, cardboard, or other cellulosic polymers,
 including transparent and non-transparent cellulosic polymers; plastic
 polymers; waxes; glass or silica; pottery, earthenware, or other ceramic;
 or metallic materials.
 A food-contacting surface of a suitable polymeric material is treated with
 a 5-aminosalicylate compound by enmeshing, implanting, or impregnating the
 compound within the polymeric material, by means known to the artisan
 skilled in food packaging and handling materials.
 Alternatively, a suitable polymeric material is treated with a
 5-aminosalicylate compound by covalent linkage, such as by conjugation of
 the 5-aminosalicylate compound to the polymeric material through, for
 example, diazo bonds or amide bonds.
 Alternatively, a polymeric or non-polymeric material is treated with a
 5-aminosalicylate compound by coating the material with a coating
 formulation suitable for a desired coating means, such as, but not limited
 to, dipping, spraying or brushing onto a surface intended for food
 contact. A suitable coating formulation contains, in addition to a
 5-aminosalicylate compound, an appropriate carrier(s), which carriers are
 known in the art. For example, the skilled practitioner can employ as a
 carrier a non-toxic polymeric resin, additionally containing an effective
 amount of a 5-aminosalicylate compound, which resin can be used to coat
 the food-contacting surface of the material, hardening in place upon it.
 An effective amount of a 5-aminosalicylate compound for treating the
 food-contacting surface of the material is between 0.1 and 10 mg per
 cm.sup.2 of the surface. Most preferably, the amount of 5-aminosalicylate
 compound is 0.4 to 2 mg per cm.sup.2 of the surface. This is sufficient
 concentration to inhibit the growth of a food-poisoning or
 botulism-causing bacterial species as demonstrated in the detailed
 examples described herein. While the amount of the 5-aminosalicylate
 compound on the food-contacting surface of the material is relatively low,
 it is preferable not to use sulphasalazine as the 5-aminosalicylate
 compound, due to the possibility of sulfapyridine-related reactions in a
 minority of food consumers, as described above. Also, it is preferable not
 to use a 5-aminosalicylate-conjugated bile acid, as this may adversely
 affect the stability or taste of the food contacting the surface.
 The present invention is also related to food containers and food-handling
 implements for holding a foodstuff, which includes containing, packaging,
 covering, storing, displaying, processing, cutting, chopping, impaling,
 kneading, manipulating or otherwise handling the foodstuff, such that a
 surface of the food container or implement comes in contact with the
 foodstuff,
 The present food containers and food-handling implements comprise a
 material suitable for contact with food and have a food-contacting surface
 treated with a 5-aminosalicylate compound, as described above, in an
 amount effective to inhibit the growth of a bacterial species. The
 containers and implements are in any suitable disposable (i.e.,
 single-use) or non-disposable (i.e., multi-use) configuration capable of
 holding a foodstuff. These configurations include, but are not limited to,
 foils, shear wraps, sheets, papers, waxed papers, bags, cartons, trays,
 plates, bowls, covered and uncovered storage vessels, serving dishes,
 cups, cans, jars, bottles, or any other suitable container configuration
 for a particular foodstuff. Additional configurations especially useful
 for food handling purposes include, but are not limited to, gloves or
 mitts; utensils such as forks, spoons, knives, slicers, processors,
 juicers, grinders, chippers, hooks, presses, screws, openers, cutters,
 peelers, tongs, ladles, scoops, cups, chutes or spatulas; and cutting
 boards, kneading boards, mixing bowls, drying or cooling racks, or
 shelves.
 The present method, containers, and implements are especially useful in
 inhibiting the growth of bacterial species that can release pathogenic
 exotoxins, such as, but not limited to, Clostridium perfringens, or
 Clostridium botulinum, the exotoxins of which cause botulism. They are
 especially, but not exclusively, useful in commercial and institutional
 food preparation contexts where food is handled and packaged in bulk, such
 as, but not limited to, food processing factories, canning plants,
 slaughterhouses, restaurants, cafeterias, salad bars, grocery outlets, and
 hospitals. The present method, containers and implements are useful for
 any kinds of foodstuff. They are particularly useful in situations where
 food is processed, packaged, stored, or displayed at a temperature, at or
 above room temperature, but insufficiently hot to kill bacterial cells and
 spores, for example, when food is kept under a warming light. But the
 present method, containers and implements are also useful in the home
 kitchen.
 The present invention is also related to antimicrobial cleansers, polishes,
 paints, sprays, soaps, or detergents formulated for application to
 surfaces to inhibit the growth of a bacterial species thereon. These
 surfaces include, but are not limited to surfaces, such as, countertops,
 desks, chairs, laboratory benches, tables, floors, bed stands, tools or
 equipment, doorknobs, and windows. The present cleansers, polishes,
 paints, sprays, soaps, or detergents contain a 5-aminosalicylate compound
 that provides a bacteriostatic property to them. They can optionally
 contain suitable solvent(s), carrier(s), thickeners, pigments, fragrances,
 deodorizers, emulsifiers, surfactants, wetting agents, waxes, or oils. A
 preferred embodiment is a formulation for external use as a
 pharmaceutically acceptable skin cleanser, particularly for the surfaces
 of human hands.
 In the present cleansers, polishes, paints, sprays, soaps, and detergents,
 the concentration of the 5-aminosalicylate compound is between 0.625 and
 200 mg per mL. Most preferably the concentration of 5-aminosalicylate
 compound is 1.25 to 50 mg per mL.
 The present cleansers, polishes, paints, sprays, soaps, and detergents are
 useful in homes and institutions, particularly but not exclusively in
 hospital settings for the prevention of nosocomial infections.
 The present invention is also related to an antimicrobial method for
 inhibiting the growth of a bacterial species in a foodstuff and to
 foodstuffs containing a 5-aminosalicylate compound. Bacterial growth in
 foodstuff, if uninhibited, can result in the release of bacterial
 exotoxins that can cause illness or death in a human or non-human
 vertebrate that consumes the foodstuff. The present method and foodstuffs
 are particularly useful in preventing clostridial food poisoning by, for
 example, Clostridium perfringens, or by Clostridium botulinum, the
 exotoxins of which cause botulism.
 The present method employs a 5-aminosalicylate compound added to the
 foodstuff. Any foodstuff can be treated using the present method, but
 foods for which the present method is especially useful include non-acidic
 foods, such as mayonnaise or other egg products, potato products, and
 other vegetable or meat products. Five-aminosalicylates are autoclavable
 and thus may be used effectively in canned foods.
 The 5-aminosalicylate compound for adding to the foodstuff can be part of
 any comestible formulation that can also include a suitable medium or
 carrier for convenient mixing or dissolving into a particular foodstuff.
 The medium or carrier is preferably one that will not interfere with the
 familiar flavor of the food of interest, such as are known by the artisan
 skilled in food processing techniques.
 An effective amount of a 5-aminosalicylate compound to be added to a
 foodstuff is less than that required for administration to a vertebrate
 subject. The present foodstuffs contain a concentration of a
 5-aminosalicylate compound between 0.625 and 10 mg per gram of the
 foodstuff. Most preferably the concentration of 5-aminosalicylate compound
 is 1.25 to 5 mg per gram of foodstuff. This is sufficient concentration to
 inhibit the growth of a food-poisoning or botulism-causing bacterial
 species as demonstrated in the detailed examples described herein.
 While the concentration of the 5-aminosalicylate compound in the foodstuff
 is relatively low, it is again, preferable not to use sulphasalazine as
 the 5-aminosalicylate compound, due to the possibility of
 sulfapyridine-related reactions in a minority of food consumers, as
 described above. Also, it is preferable not to use a
 5-aminosalicylate-conjugated bile acid, as this may adversely affect the
 stability or taste of the foodstuff before consumption.
 The present antimicrobial method for inhibiting bacterial growth in a
 foodstuff and foodstuffs employing the antimicrobial properties of
 5-aminosalicylates are useful alternative means of preventing food
 poisoning or botulism, instead of nitrite preservatives or gamma
 irradiation of food, which are disfavored by a sizeable number of
 consumers.
 The methods and pharmaceutical compositions of the present invention
 provide a much needed addition to the antimicrobial armamentarium against
 bacterial overgrowths and infections, especially clostridial infections
 that affect great numbers of humans and animals worldwide.
 Five-aminosalicylate compounds are modestly priced relative to other
 antibiotics and are conveniently administered.
 The administration of 5-aminosalicylates is well tolerated by the vast
 majority of patients with only rare side effects, which, of course, the
 practitioner should monitor in each individual patient. For example,
 5-aminosalicylic acid is known to pose no substantial teratogenic risk in
 humans and can be administered safely to pregnant women. (O. Diav-Citrin
 et al., The safety of mesalamine in human pregnancy: a prospective
 controlled cohort study, Gastroenterol. 114(1):23-28 [1998]; P. Marteau et
 al., Foetal outcome in women with inflammatory bowel disease treated
 during pregnancy with oral mesalazine microgranules, Aliment. Pharmacol.
 Ther. 12(11):1101-08 [1998]; C. M. Bell and F. M. Habal, Safety of topical
 5-aminosalicylic acid in pregnancy, Am. J. Gastroenterol. 92(12):2201-02
 [1997]). The use of sulphasalazine requires closer monitoring, as
 mentioned above.

The foregoing applications for the methods and compositions of the present
 invention are illustrative and by no means exhaustive. The invention will
 now be described in greater detail by reference to the following
 non-limiting examples.
 EXAMPLES
 The antimicrobial properties of a 5-aminosalicylate compound, mesalamine,
 were tested using five organisms, commonly, but not exclusively, found in
 gastrointestinal tracts.
 Example 1
 Bacterial Cultures
 Bacteroides fragilis (Gram negative anaerobe), Clostridium perfringens
 (Gram positive obligate anaerobe), Escherichia coli (Gram negative
 facultative anaerobe), a Lactobacillus isolate (Gram positive aerotolerant
 anaerobe), and Enterococcus faecalis (Gram negative facultative anaerobe)
 were examined, because each produces a detectable gaseous fermentation
 product under certain anaerobic growth conditions. All cultures were
 maintained on standard blood agar plates ([g/L distilled H.sub.2 O]:
 pancreatic digest of casein, 15.0; papale digest of soybean meal, 5.0;
 NaCl, 5.0; agar, 20.0; yeast extract, 5.0; hemin, 0.003; vitamin K1, 0.01;
 L-cystine, 0.4; defibrinated sheeps' blood, 5.0%) at 37.degree. C. in an
 anaerobic CO.sub.2 chamber (Bacteroides, Lactobacillus and Clostridium) or
 aerobically (Escherichia and Enterococcus). Inocula for plating
 experiments were taken aseptically by probe from distinct colonies on
 culture plates that were no more than 72 hours (anaerobic) or 24 hours
 (aerobic) old.
 Example 2
 Plating and Colony Counting
 Mesalamine was obtained commercially in the form of Asacol (Procter and
 Gamble). The entenic coating on 400 mg Asacol tablets was removed with
 acetone. After the coating was dissolved, intact tablets were air dried
 and sterilized by autoclaving at 121.degree. C., 15 psi, for 15 minutes.
 The sterile tablets were aseptically dissolved in sterile distilled water.
 One milliliter-aliquots of serial dilutions containing 0, 3.125, 6.25,
 12.5, 25, 50, and 100 mg/mL of mesalamine in sterile distilled water were
 placed onto anaerobic and aerobic blood 10-mL agar plates. To facilitate
 impregnation into the agar, the plates were allowed to dry for about 6
 hours.
 Aliquots of 0.5 McFarland units of cell suspensions of each bacterial
 species were separately diluted 1:106 in sterile saline (0.15 M NaCl), and
 1 mL of each diluted cell suspension was plated on 4 replicate plates at
 each concentration of mesalamine. All plates were incubated at 37.degree.
 C. Plates inoculated with E. coli or Enterococcus faecalis were incubated
 aerobically for 24 hours, and plates inoculated with C. perfringens, B.
 fragilis, or Lactobacillus were allowed to incubate anaerobically in
 GasPak chambers for 48 hours. Colony counts were assessed from each of the
 four plates of each concentration of mesalamine for each species. Paired
 t-test was used to compare groups of readings. Four replicate control
 plates at each concentration of mesalamine were incubated as described
 above after plating with 1 mL of sterile saline. (All controls were
 negative for bacterial colonies.)
 Example 3
 Results
 Resulting colony counts for E. coli and a C. perfringens isolate are
 tabulated in Table 1. In the concentration range between 12.5 and 100
 mg/mL, mesalamine inhibited the growth of the C. perfringens isolate but
 had no effect on E. coli colony counts. Similar growth inhibition by
 mesalamine was detected for Clostridium perfringens ATCC 13124 and
 Clostridium difficile ATCC 9689. (Data not shown.) But mesalamine did not
 affect the colony numbers of Lactobacillus, Enterococcus, or Bacteroides.
 TABLE 1
 Growth inhibition of a Clostridium perfringens isolate by
 mesalamine.
 Results: (mean colony count .+-. SE)
 Mesalamine
 (mg/mL) 0 3.125 6.25 12.5 25
 50 100
 E. coli 28.5 .+-. 2.3 29.3 .+-. 1.9 29.0 .+-. 2.2 24.0 .+-. 4.8
 28.3 .+-. 4.6 33.3 .+-. 2.6 27.8 .+-. 2.5
 C. perfringens 29.5 .+-. 1.3 38.7 .+-. 33.2 24.8 .+-. 13.9 1.0 .+-. 0.8*
 2.0 .+-. 4.0* 1.8 .+-. 2.4* 1.3 .+-. 2.5*
 *p, 0.0001
 These results demonstrate that 5-aminosalicylates exert a selective
 antimicrobial action, for example on the growth of Clostridium. This
 selectivity of action is one of the advantages of the present invention, a
 feature which is useful in fighting the serious problem of clostridial
 infections and botulism food poisoning.