WO2002056879A1 - Utilisation de terpenes dans le traitement des infections du tube digestif - Google Patents

Utilisation de terpenes dans le traitement des infections du tube digestif Download PDF

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WO2002056879A1
WO2002056879A1 PCT/GB2002/000015 GB0200015W WO02056879A1 WO 2002056879 A1 WO2002056879 A1 WO 2002056879A1 GB 0200015 W GB0200015 W GB 0200015W WO 02056879 A1 WO02056879 A1 WO 02056879A1
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terpene
liposome
mixture
combination
terpenes
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PCT/GB2002/000015
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English (en)
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Michael Wyllie
David Ernest Young
Julio Pimentel
Lanny Franklin
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Medpharma Plc
Ximed Group Plc
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Priority claimed from GB0100059A external-priority patent/GB0100059D0/en
Priority claimed from GB0108246A external-priority patent/GB0108246D0/en
Application filed by Medpharma Plc, Ximed Group Plc filed Critical Medpharma Plc
Priority to AU2002217316A priority Critical patent/AU2002217316A1/en
Publication of WO2002056879A1 publication Critical patent/WO2002056879A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/075—Ethers or acetals
    • A61K31/085—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/11—Aldehydes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes

Definitions

  • the present invention relates to the treatment of microbial infections, especially the prevention and treatment of digestive tract infections in humans and animals, by orally administering a single terpene, a terpene mixture or a liposome-terpene (s) composition before or after the onset of the infection.
  • Digestive tract infections are mainly caused by pathogenic and opportunistic microorganisms and toxins produced by them. These illnesses are present in all types of animals and humans .
  • Anthrax is considered a potential agent for use in biological warfare.
  • Anthrax is an acute infectious disease caused by the spore-forming bacteria Bacillus anthracis .
  • Anthrax is primarily a disease of domesticated and wild animals, particularly herbivorous animals . Humans become infected with anthrax by handling products from infected animals or by inhaling anthrax spores from contaminated animal products. Anthrax can also be spread by eating undercooked meat from infected animals.
  • Anthrax infection can occur in three forms: cutaneous, inhalation, and gastrointestinal.
  • the most common form is the cutaneous anthrax infection, which occurs when bacteria enter a cut or abrasion on the skin. This infection begins as a raised itchy bump that develops into a vesicle and then a painless ulcer, usually 1-3 cm in diameter, with a characteristic black necrotic area in the center. About 20% of untreated cases of cutaneous anthrax result in death. Deaths may be prevented with prompt antimicrobial treatment.
  • the inhalation form has early symptom similar to a common cold which progressively results in severe breathing problems . This type of anthrax is usually fatal.
  • the intestinal form is characterized by an acute inflammation of the intestinal tract. The initial signs are nausea, loss of appetite, vomiting, and fever followed by abdominal pain, vomiting of blood and severe diarrhea. Intestinal anthrax results in death in 25% to 60% of cases. Anthrax is treated with antimicrobials and can be prevented with vaccination. The Department of Defense in the USA has a mandatory anthrax vaccination of all active military personnel.
  • Traveler's diarrhea defined as the passage of more than 3 unformed stools in a 24-hour period, is a self-limiting illness lasting 3 - 5 days.
  • the illness may be presented either as (1) acute watery diarrhea (2) diarrhea with blood (dysentery) or (3) chronic diarrhea, often with clinical nutrient malabsorption.
  • Several factors contribute to the development of diarrhea in travelers, including personal (age, socioeconomic status, body weight, preexisting gastrointestinal illnesses) , behavioral (mode of travel, standard of accommodation, eating in public places, dietary errors) and travel related (destination, duration of stay, country of origin, season) .
  • Approximately 85% of lithe diarrheas among international travelers are produced by bacterial enteropathogens .
  • pathogens are usually acquired through ingestion of fecally contaminated food or water. Sometimes dirty hands or insects are the vectors of fecal contamination. Cooked food is safe to consume as long as the temperature at the interior of the food reaches 160°F or more. An undercooked hamburger is risky food, because ground meat can become contaminated at the processing plant and during preparation.
  • the common pathogens that produce traveler's diarrhea include Clostridium difficile, Yersenia enteroli tica, Shxgella sp . , Campylobacter sp . , Salmonella sp . , ETEC (enterotoxigenic) and EAEC (enteroaggregative) Escherichia coli . Traveler's diarrhea produced by Shxgella sp. or Salmonella sp . tend to cause a more severe and longer lasting disease than that caused by the most common cause, enterotoxigenic E. coli (ETEC) . Campylobacter jejuni is a relativelly common cause of traveler's diarrhea especially in the winter. Viruses such as rotavirus, cytomegalovirus and Norwalk agent are less common causes.
  • ETEC Enterotoxigenic
  • Verocytotoxic E. coli (VTEC) strains produce toxins that destroy the gut ucosa and can cause kidney damage; E. coli 0157 H:7 is the most publicised example of this type.
  • E. coli Enteropathogenic E. coli
  • EIEC Enteroinvasive E. coli
  • EIEC and VTEC strains can cause very serious disease ( aemorrhagic colitis and renal failure) and require hospitalisation. Milder cases are usually treated by fluid and electrolyte replacement and rest.
  • antibiotics limits the course of diarrhea to a little over a day compared with an average of over 3 - 5 days when diarrhea remains untreated.
  • the widespread resistance of the traditional antimicrobial agent, Trimethoprim plus sulfamethoxazole (TMP/SMX) , and fluoroquinolones are the main reasons of concern about the continuous use of antimicrobials for the treatment of traveler's diarrhea (Dupont et al, 1998).
  • TMP/SMX Trimethoprim plus sulfamethoxazole
  • fluoroquinolones are the main reasons of concern about the continuous use of antimicrobials for the treatment of traveler's diarrhea (Dupont et al, 1998).
  • the extensive use of antibiotics can also lead to overgrowth syndromes, Candida vagini tis can occur, the overgrowth of Clostridium difficile due to less competitive environment in the gastrointestinal tract can also result in diarrhea.
  • H. pylori a gram-negative, microaerophilic spiral bacteria is the major cause of gastro-duodenal disease, including chronic gastritis, gastric and duodenal ulcers and gastric neoplasia. Greater than 50% of North American adults over 50 years of age are infected with H. pylori .
  • H. pylori is characterized by very high urease activity that may be associated with virulence, in the absence of urea H. pylori is sensitive to acidic pH. Urease activity may be an important colonization and survival factor by generating ammonia in the immediate bacterial microenvironment .
  • H. pylori has been classified as a type 1 carcinogen by the World Health Organization because of the danger of persistent infection with the bacterium causing gastric cancer.
  • H. pylori infection is of extreme importance in the causation of peptic ulcer disease. By initiating a gastritis or dyspeptic symptoms, it can predispose to subsequent episode of either gastric lymphoma or stomach cancer.
  • H. pylori The eradication of H. pylori has been obtained with combination therapy, triple therapy using bismuth plus two antibiotics (metronidazole and either amoxicillin or tetracycline has been effective) .
  • problems due to development of antimicrobial resistant and side effects may explain why the use of antibiotics has not become a preferred treatment for gastritis and peptic ulcers due to H. pylori .
  • Antibacterial treatment of H. pylori is difficult because of the habitat occupied by the organism below the layer of the mucus adherent to the gastric mucosa. Access of antibacterial agents to this site is limited from the lumen of the stomach and also from the gastric blood supply.
  • medium chain fatty acids and medium chain triglycerides have been shown to inhibit the growth of H. pylori in vitro.
  • the mechanism by which they exert antibacterial effect is thought to involve: 1) damage to the bacterial outer membrane leading the increase membrane fluidity and permeability, 2) Incorporation of these fatty acids, making the bacterial membrane unstable, 3) Production of peroxides due to oxidation of fatty acids.
  • H. pylori The mode of transmission of H. pylori in humans is still poorly understood. There are reports of detection of this microorganism in the oral cavity and in the feces . If H. pylori is harvested in the oral cavity or bowel, these might represent important reservoir for the reinfection and transmission with consequences from treatment.
  • One vector for the transmission of H. pylori are flies, they can carry viable H. pylori in their external surf ces and alimentary tracts .
  • Another animal of economic importance is swine.
  • the incidence of diarrhea in neonates and weaned piglets is very high.
  • E. coli and Salmonella are the main microorganisms involved in diarrhea in swine.
  • One of the preferred methods is segregated early weaning (SEW) .
  • SEW segregated early weaning
  • the basis of early weaning is that the earlier piglets are weaned from the sow the less are the chances of crossover diseases between sow and piglets. This method requires the use of antibiotics.
  • antibiotics In both cases, calf and piglet scours, the preferred method of treatment is antibiotics.
  • the European Community has banned the use of 5 antibiotics and in the Unites States the FDA is banning the use of fluoroquinolone in animals due to the development of Campylobacter resistant to this antibiotic. Bacteria resistance has encouraged the development of antibiotic- alternative products.
  • Terpenes are widespread in nature, mainly in plants as constituents of essential oils. Their building block is the hydrocarbon isoprene (C 5 H 8 ) n . Terpenes have been found to be effective and nontoxic dietary antitumor agents which act through a variety of mechanisms of action (Crowell and Gould, 1994 and Crowell et al, 1996). Terpenes, i.e. geraniol, tocotrienol, perillyl alcohol, b-ionone and d-limonene, suppress hepatic HMG-COA reductase activity, a rate limiting step in cholesterol synthesis, and modestly lower cholesterol levels in animals (Elson arid Yu, 1994) .
  • C 5 H 8 hydrocarbon isoprene
  • D-limonene and geraniol reduced mammary tumors (Elegbede et al, 1984 and 1986 and Karlson et al, 1996) and suppressed the growth of transplanted tumors (Yu et al, 1995) .
  • Terpenes have also been found to inhibit the in-vitro growth of bacteria and fungi (Chaumont and Leger, 1992, Moleyar and Narasimham, 1992 and Pattnaik, et al, 1997) and some internal and external parasites (Hooser, et al, 1986) .
  • Geraniol was found to inhibit growth of Candida albicans and Saccharomyces cerevisiae strains by enhancing the rate of potassium leakage and disrupting membrane fluidity (Bard, et al, 1988) .
  • B-ionone has antifungal activity which was determined by inhibition of spore germination, and growth inhibition in agar (Mikhlin et al, 1983 and Salt et al, 1986) .
  • Teprenone (geranylgeranylacetone) has an antibacterial effect on H. pylori (Ishii, 1993) . Solutions of 11 different terpenes were effective in inhibiting the growth of pathogenic bacteria in in-vitro tests; levels ranging between 100 ppm and 1000 ppm were effective.
  • terpenes were diluted in water with 1% polysorbate 20 (Kim et al, 1995) .
  • Diterpenes, i.e. trichorabdal A from R. Trichocarpa has shown a very strong antibacterial effect against H. pylori (Kadota, et al, 1997) .
  • Rosanol a commercial product with 1% rose oil has been shown to inhibit the growth of several bacteria (Pseudomona, Staphylococus , E. coli and Hpylori) .
  • Geraniol is the active component (75%) of rose oil.
  • Some extracts from herbal medicines have been shown to have an inhibitory effect on H. pylori , the most effective being decursinol angelate, decursin, magnolol, berberine, cinnamic acid, decursinol and gallic acid (Bae, et al 1998) .
  • Extracts from cashew apple, anacardic acid and (E) -2-hexenal have shown bactericidal effect against H. pylori .
  • Digestive tract infections not only are an uncomfortable illness for humans but also are of economic importance for the animal industry. In some cases the illness can cause death in children, elderly and immune-compromised people.
  • the preferred treatment of the disease is antibiotics.
  • the extensive use of antibiotics in humans and the animal industry has created the development of antibiotic-resistant bacteria.
  • the increased antibiotic resistance has beer the main reason to seek new antimicrobial alternatives .
  • the European Community has banned the use of 5 antibiotics in animals and in the Unites States the FDA is banning the use of fluoroquinolone in animals due to the development of Campylobacter resistant to this antibiotic.
  • Terpenes which are GRAS (Generally Recognized As Safe) have been found to inhibit the growth of cancerous cells, decrease tumor size, decrease cholesterol levels and have a biocidal effect on microorganisms in vitro. Onawunmi (1989) showed that growth media with more than 0.01 % citral reduced the concentration of E. coli and at 0.08% there was a bactericidal effect.
  • Barranx, et al (1998) teach us a terpene formulation, based on pine oil, used as a disinfectant or antiseptic cleaner.
  • Koga, et al (1998) teach that a terpene found in rice has antifungal activity.
  • the present invention provides a composition for preventing or treating gastro-intestinal infections, wherein said composition comprises a terpene or a mixture of terpenes .
  • said composition comprises a terpene or a mixture of terpenes .
  • certain mixture of terpenes are synergistically effective, relative to the effects of the component terpenes administered separately.
  • terpenes having biocidal activity which in combination with two or more other terpenes synergistically increase the biocidal effectiveness are of especial interest .
  • One composition of interest comprises a mixture of carvone and geraniol, optionally together with other terpenes.
  • the content of carvone and geraniol may each be from 10 to 90% (by weight) , but is preferably 10 to 60% by weight.
  • Other terpenes which may be present include citral, b- ionone, eugenol, terpeniol, carvacrol, anethole or the like. These optional additional terpenes may be present at 5 to 50% by weight, for example 10 to 40% by weight.
  • the terpenes may be presented in the form of liposomes.
  • Liposomes are microscopic structures consisting of concentric lipid bilayers enclosing an aqueous space. Liposomes are classically prepared from phospholipids which occur naturally in animal cell membranes, but several synthetic formulations are now commonly used. The lipid composition of the liposome can be varied to give liposomes different physical characteristics i.e. size and stability.
  • Liposomes can be prepared by the reverse-phase evaporation or dehydration-rehydration vesicle methods using a mixture of dipalmitoyl phosphatidyl choline, cholesterol, dipalmitoyl phosphatidyl glycerol, dipalmitoyl phosphatidyl ethanolamine and other synthetic fatty acids and emulsifiers.
  • liposomes When making liposomes first multilamellar vesicles are formed spontaneously when amphipathic lipids are hydrated in an aqueous medium. Unilamellar vesicles are often produced from multilamellar vesicles by the application of ultrasonic waves.
  • Multilamellar vesicles can be prepared by the procedure known as dehydration-rehydration. Briefly, egg phosphatidylcholine and cholesterol are mixed in chloroform, dried in a rotary evaporator, dilute with water and sonificated to form unilamellar vesicles. The solution is freeze dried and rehydrated with the terpene solution in order to embed the terpene inside the liposome. Another method to produce liposomes is by mixing together lipids, an emulsifier and the terpenes. The emulsion is obtained by using a Polytron homogenizer with special flat rotor that creates an emulsion.
  • the lipids could consist of soybean oil, any commercial or pharmaceutical oil; the emulsifier consist of egg yolk lecithin, plant sterols or synthetic including polysorbate-80, polysorbate-20, polysorbate-40, polysorbate-60, polyglyceryl esters, polyglyceryl monooleate, decaglyceryl monocaprylate, propylene glycol dicaprilate and triglycerol monostearate.
  • the lipid concentration in the oil phase is 75-95% and the emulsifier concentration from 5-25%.
  • a ratio oil to water could vary from 10-15 parts lipid to 35-40 parts terpenes diluted in water at a concentration of 0.5% to 50%.
  • emulsion concentration use for topical application varies from 0.0055 through 1.0% of the final product.
  • Several modifications to the emulsion can be achieved by simply varying the concentration and type of terpenes used. This modification can give us different products with different antimicrobial specificity.
  • the antimicrobial effect will be increased: (1) the liposome will disrupt the bacterial membrane and (2) the terpenes will be more effective in disrupting cytoplasmatic enzymes.
  • the terpene, terpene mixture or liposome-terpene (s) combination consists of a blend of generally recognized as safe (GRAS) terpenes with a GRAS surfactant.
  • the ratio of terpenes is from 1-99% and the surfactant ratio from 1-99% of the mixture.
  • the terpenes, comprised of natural or synthetic terpenes, are citral, b-ionone, geraniol, eugenol, carvone, terpeniol, carvacrol, anethole or other terpenes with similar properties.
  • the surfactant is preferably polysorbate-80 or other suitable GRAS surfactants.
  • lipids used are all food-grade or pharmaceutical-grade.
  • a set amount of lipids, an emulsifier and the terpenes was used to prepare an emulsion.
  • the emulsion was obtained by using a Polytron homogenizer with special flat rotor that created an emulsion.
  • the lipids consisted of soybean oil, any commercial or pharmaceutical oil; the emulsifier consist of egg yolk lecithin, plant sterols or synthetic emulsifiers including polysorbate-80, polysorbate-20, polysorbate-40, polysorbate-60, polyglyceryl esters, polyglyceryl onooleate, decaglyceryl monocaprylate, propylene glycol dicaprilate and triglycerol monostearate.
  • a solution containing 75-95% lipids (oil) and 5-25% emulsifier consisted of the oil phase.
  • the aqueous phase consisted of the terpene diluted in water at a rate of 0.5% to 50%.
  • lipid phase lipid phase
  • terpenes aqueous phase
  • Any standard method for the preparation of liposomes can be followed with the knowledge that the lipids used are all food-grade or pharmaceutical-grade.
  • the suspension containing a lipid, an emulsifier and the terpenes is emulsified with a Posytron homogenizer until a complete milky solution is obtained.
  • This step consists of the preparation of the terpene (s) -liposome combination by mixing 99% of liposome and 1% of terpene mixture.
  • terpene (s) -liposome combination by mixing 99% of liposome and 1% of terpene mixture.
  • Several combinations of this formulation can be obtained by varying the amount of terpene and liposome from 1% to 99%.
  • the liposomes are prepared as in Example 2 without the addition of terpenes in the formulation.
  • EXAMPLE 4 In-vitro effectiveness of terpenes against E. coli This example demonstrates the effect of terpenes on the cell membrane fragility of E. coli , which is considered indicative of other pathogenic bacteria such as Salmonella and Listeria . Lysis of the cell membrane was monitored by the determination of galactosidase activity.
  • ⁇ -galactosidase is a well-characterized cytosolic enzyme in bacteria. This enzyme is inducible in the presence of isopropyl-1-thiogalactosidase (IPTG) and assayed colorimetricaly with substrate o-nitro-phenyl- ⁇ -D-galactoside (ONPG) .
  • ONPG is cleaved to release o-nitrophenol with peak absorbance at 420 nm. Since intact E. coli is impermeable to both ONPG and the enzyme, the cells have to be lysed prior to enzymatic assay. Therefore the ability of terpenes to lyse E. coli can be measured with this enzymatic assay and compared to known lysing agents .
  • E. coli strains AW574 or AW405 were cultured overnight in 10 ml tryptone broth with 1 nM IPTG at 35°C. Cells were allowed to grow until an absorbance equal to 0.9 was reached. Cells were harvested, washed with phosphate buffer and resuspended to an absorbance equal to 0.5. 0.1 ml of the bacteria culture was added to 0.9 ml of buffer, warmed to 30°C and then 80 ⁇ l of terpenes (85% terpenes and 15% polysorbate-80) , 80 ⁇ l water (background) or 40 ⁇ l chloroform plus 40 ⁇ l 1% SDS in water (positive control) were added.
  • the tubes were mixed for 10 seconds and 0.2 ml of ONPG (4 mg/ml water) was added, then incubated for 5 minute .
  • the enzyme activity was stopped with 0.5 ml of 1 M sodium carbonate. After being centrifuged for 3 minutes at 1,500 x g, supernatant was transferred to cuvettes and read at 420 nm.
  • the relative degree of lysis caused by terpenes was calculated as follows:
  • This example demonstrate the effectiveness of terpenes against Escherichia coli , Salmonella typhimurium, Pasteurella mirabilis, Staphylococcus aureus, Candida albicans and Aspergillius fumigates.
  • Salmonella typhimurium Salmonella typhimurium
  • Pasteurella mirabilis Staphylococcus aureus
  • Candida albicans Aspergillius fumigates.
  • Each organism, except A. fumigatus was grown overnight at 35-37°C in tryptose broth.
  • a . fumigates was grown for 48 hours. Each organism was adjusted to approximately 10 5 organisms/ml with sterile saline.
  • terpenes were diluted in sterile tryptose broth to give the following dilutions: 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16,000, 1:32,000, 1:64,000 and 1:128,000.
  • EXAMPLE 6 In in-vitro effectiveness of terpenes against Escheri ⁇ hia coli over time.
  • This example shows the bactericidal effect of selected terpenes on the viability of H. pylori .
  • Five terpenes (anethole, carvone, citral, geraniol and b-ionone) were used for this study. Terpenes were mixed to a ratio of 90% terpene plus 10% polysorbate-80.
  • the H. pylori used was strain #26695 of porcine origin, this bacteria is a motile, cag A, vac A cytotoxin-positive gram negative bacteria which colonizes gnotobiotic piglets and indefinitely persists within the gastric microenvironment as a superficial infection of the gastric mucosa and mucus layer.
  • EXAMPLE 8 In vitro effectiveness of single or 19 combination of terpenes against E. coli . 20 21 The objective of this example was to determine an 22 optimum terpene mixture which could have a greater biocidal effect.
  • E. coli strain AW574 was grown in tryptone broth to an exponential growth phase (O.D. between 0.4 and 1.0 at 590 nm) . One tenth of this growth was inoculated to 10 ml of tryptone broth followed by the addition of individual terpenes as indicated in Example 5; then incubated for -24 hours at 35-37°C and the O.D. determined in each tube.
  • the concentration of terpenes was 1 or 2 ⁇ Mol . Each treatment was repeated in triplicate. The results are expressed as percentage bacterial growth as compared to the control treatment. It is observed that the combination of terpenes give better biocidal effect than single terpenes, with geraniol and carvone better than b-ionone .
  • EXAMPLE 9 In vitro effectiveness of a combination of terpenes against different E. Coli strains
  • Bacteria were sub-cultured from original American Type Culture Collection (ATCC) freeze-dried material. They included E. coli strains 8739, 25922 and 700728 (Serotype group 0: 157 H:7), which are BioSafety class 1 organisms and E. coli 12795 (Serotype group 0: 26) which is a BioSafety class 2 organism. All the bacteria were cultured on Tryptone Soya Agar (TSA) , supplied by Oxoid Ltd, Hampshire and Mueller Hinton Agar (MHA) , supplied by Merck Ltd. The incubation temperature was 35 °C.
  • TSA Tryptone Soya Agar
  • MHA Mueller Hinton Agar
  • E. coli cultures were prepared in nutrient broth and allowed to grow until exponential growth phase was achieved (16 hours at 35 °C) . 1 ml of this culture was transferred to each of a series of pre-sterilised Duran bottles containing 100 ml nutrient broth, 0.5 % w/v Polysorbate 80 and this gave an initial inoculum of approximately 10 8 microbial cells per ml of broth.
  • the Duran bottles were agitated on a vortex shaker to produce good mixing and the Optical Density (OD) at 590 nm read on a calibrated Unicam UV 300 spectrophotometer controlled by Vision 32 software. The OD of a sample of a placebo broth was also recorded. The bottles were then placed in an incubator at 35°C. The bottles were removed at 30 minute intervals and placed on a vortex shaker at level three for 30 seconds. The bottles were then returned to the incubator. The OD was recorded at hourly intervals, for up to 24 hours.
  • OD Optical Density
  • the broths were autoclaved on programme 4 of an AVX240 autoclave (132 °C for 30 minutes) to sterilise them.
  • the terpenoids tested in this series of exemplary experiments included I-carvone, citral and geraniol in varying proportions.
  • One exemplary formulation, constituting the test formulation, is given in Table 8, below
  • the 16 hour old E. coli culture used as the inoculum had an OD at 590 nm of 0.697 units.
  • Table 9 summarises the results of the 50 and 100 ⁇ l/100 ml broth test. These results indicated good activity against E. coli 8739. Table 11 indicates that the test formulation showed good activity when challenged with other strains of E. coli including two pathogenic strains 700728, 12795.
  • test formulation* had the lowest OD readings, therefore indicating greater inhibition of cell proliferation.
  • 50 ⁇ l/100 ml broth of the test formulation appeared to have both slowed cell proliferation and reduced the final number of cells present in the broth. Where no test formulation was present, growth was rapid for all strains tested, especially in the first 4 hours after inoculation.
  • test formulation is only one of a range of terpene formulations investigated so far and it is clearly very active. Clear inhibition of E. coli growth has been observed in broth tests conducted at 50 ⁇ l and 100 ⁇ l/100 ml broth, both against anti microbial assay strains and against pathogenic strains.
  • Formulations have been developed now which show very great activity against potentially lethal strain 0157: H7 of E. coli , both at very high innocula which are not sustainable in life and at levels which, though likely to be fatal, are found.
  • Three 100 ml bottles were each filled with McConkey broth to which was added one of either 20 ⁇ g/ml oxacillin, or 10 ⁇ g/ml of amoxicillin, or 1 ⁇ g/ml of the exemplary test terpene formulation. Each bottle was then inoculated with 10 4 E. coli 0157 :H: 7 and incubated for 24 hours at 35°C.
  • the McConkey broth containing the oxacillin had lost its magenta colour and become yellowish and turbid, indicating that the antibiotic had been overwhelmed by the E. coli .
  • the McConkey broth in the bottle containing the amoxicillin had only slightly reduced magenta colour, indicating that the antibiotic had contained the ⁇ . coli , whereas the McConkey broth in the bottle containing the terpene sample had an undiminished magenta colour.

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Abstract

L'invention concerne la prévention et le traitement des infections du tube digestif chez les humains et les animaux par administration orale d'un seul terpène, d'un mélange de terpènes ou d'une composition de liposome-terpène(s) avant ou après le début d'une infection gastro-intestinale. Ces infections peuvent comprendre la diarrhée du voyageur, les ulcères, l'infection à l'anthrax et d'autres infections bactériennes et parasitaires.
PCT/GB2002/000015 2001-01-03 2002-01-03 Utilisation de terpenes dans le traitement des infections du tube digestif WO2002056879A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002217316A AU2002217316A1 (en) 2001-01-03 2002-01-03 Use of terpenes for the treatment of digestive tract infections

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
GB0100059.5 2001-01-03
GB0100059A GB0100059D0 (en) 2001-01-03 2001-01-03 Anti-infectives for human and animal use
GB0108246.0 2001-04-02
GB0108246A GB0108246D0 (en) 2001-04-02 2001-04-02 Anti-bacterial preparations
US32681801P 2001-10-04 2001-10-04
US60/326,818 2001-10-04

Publications (1)

Publication Number Publication Date
WO2002056879A1 true WO2002056879A1 (fr) 2002-07-25

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PCT/GB2002/000015 WO2002056879A1 (fr) 2001-01-03 2002-01-03 Utilisation de terpenes dans le traitement des infections du tube digestif

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AU (1) AU2002217316A1 (fr)
WO (1) WO2002056879A1 (fr)

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US7070815B2 (en) 2002-11-27 2006-07-04 Loders Croklaan Usa Llc Anti diarrhoea compositions
WO2006120565A2 (fr) * 2005-05-13 2006-11-16 Advanced Scientific Developements Combinaison pharmaceutique comprenant un agent antifongique et un actif choisi parmi le carveol, l'eugenol, le thymol, le borneol, le carvacrol, et les i0n0nes alpha- et beta-.
EP1765318A2 (fr) * 2004-06-30 2007-03-28 Nutrition Sciences N.V./S.A. Acides gras a chaine moyenne pouvant etre utilises comme agents antimicrobiens
WO2009016026A1 (fr) * 2007-07-27 2009-02-05 Basf Se Aliment pour animaux contenant du tétrahydrogéraniol
EP2368547A1 (fr) * 2010-03-26 2011-09-28 Cesa Alliance S.A. Compositions antivirales comprenantes la géraniol et la carvone
JP2012077033A (ja) * 2010-10-01 2012-04-19 Kinki Univ ヘリコバクターピロリの運動能阻害剤
CN103458883A (zh) * 2011-03-28 2013-12-18 切萨联盟有限公司 用于体内治疗用途的病毒抑制剂组合物
AU2008302667B2 (en) * 2007-06-15 2014-07-10 Board Of Regents, The University Of Texas System Methods and compositions to inhibit edema factor and adenylyl cyclase
US9439416B2 (en) 2005-11-30 2016-09-13 Eden Research Plc Compositions and methods comprising terpenes or terpene mixtures selected from thymol, eugenol, geraniol, citral, and l-carvone
US9655360B2 (en) 2004-01-23 2017-05-23 Eden Research Plc Nematicidal compositions and methods of using them
US9668500B2 (en) 2012-04-24 2017-06-06 Purina Animal Nutrition Llc Feeding methods and systems for young livestock animals using sensory compounds
US10383329B2 (en) 2012-11-21 2019-08-20 Eden Research Plc Preservatives
US10638750B2 (en) 2004-05-20 2020-05-05 Eden Research Plc Compositions containing a hollow glucan particle or a cell wall particle encapsulating a terpene component, methods of making and using them
KR20200049883A (ko) * 2012-06-06 2020-05-08 상하이 지아오통 유니버시티 장내 미생물 개체군 개선을 위한 방법 및 조성물
US10667512B2 (en) 2005-11-30 2020-06-02 Eden Research Plc Terpene-containing compositions and methods of making and using them
US10940172B2 (en) 2017-01-03 2021-03-09 Purina Animal Nutrition Llc Methods of feeding animals phytogenic products
US11213051B2 (en) 2014-07-02 2022-01-04 Purina Animal Nutrition Llc Milk replacer products containing halides and sources of hydrogen peroxide and methods of feeding same

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US7070815B2 (en) 2002-11-27 2006-07-04 Loders Croklaan Usa Llc Anti diarrhoea compositions
US10004229B2 (en) 2004-01-23 2018-06-26 Eden Research Plc Nematicidal compositions and methods of using them
US9655360B2 (en) 2004-01-23 2017-05-23 Eden Research Plc Nematicidal compositions and methods of using them
US10729130B2 (en) 2004-01-23 2020-08-04 Eden Research Plc Nematicidal compositions and methods of using them
US10638750B2 (en) 2004-05-20 2020-05-05 Eden Research Plc Compositions containing a hollow glucan particle or a cell wall particle encapsulating a terpene component, methods of making and using them
EP1765318A2 (fr) * 2004-06-30 2007-03-28 Nutrition Sciences N.V./S.A. Acides gras a chaine moyenne pouvant etre utilises comme agents antimicrobiens
US8962683B2 (en) 2004-06-30 2015-02-24 Nutrition Sciences Nv Medium chain fatty acids applicable as anti-microbial agents
WO2006120565A2 (fr) * 2005-05-13 2006-11-16 Advanced Scientific Developements Combinaison pharmaceutique comprenant un agent antifongique et un actif choisi parmi le carveol, l'eugenol, le thymol, le borneol, le carvacrol, et les i0n0nes alpha- et beta-.
WO2006120565A3 (fr) * 2005-05-13 2007-08-23 Advenced Scient Developements Combinaison pharmaceutique comprenant un agent antifongique et un actif choisi parmi le carveol, l'eugenol, le thymol, le borneol, le carvacrol, et les i0n0nes alpha- et beta-.
EA014068B1 (ru) * 2005-05-13 2010-08-30 Адванст Сайентифик Дивелопментс Фармацевтическая композиция, содержащая противогрибковый агент и активный ингредиент, выбранный из карвеола, тимола, эвгенола и карвакрола
US10667512B2 (en) 2005-11-30 2020-06-02 Eden Research Plc Terpene-containing compositions and methods of making and using them
US10258033B2 (en) 2005-11-30 2019-04-16 Eden Research Plc Compositions and methods comprising terpenes or terpene mixtures selected from thymol, eugenol, geraniol, citral and L-carvone
US9439416B2 (en) 2005-11-30 2016-09-13 Eden Research Plc Compositions and methods comprising terpenes or terpene mixtures selected from thymol, eugenol, geraniol, citral, and l-carvone
AU2008302667B2 (en) * 2007-06-15 2014-07-10 Board Of Regents, The University Of Texas System Methods and compositions to inhibit edema factor and adenylyl cyclase
EP2025244A1 (fr) * 2007-07-27 2009-02-18 Basf Se Nourriture pour animaux comportant du tétrahydrogéraniol
WO2009016026A1 (fr) * 2007-07-27 2009-02-05 Basf Se Aliment pour animaux contenant du tétrahydrogéraniol
US8980945B2 (en) * 2010-03-26 2015-03-17 Cessa Alliance Viral inhibitor compositions for in vivo therapeutic use comprising a combination of (−)-carvone, geraniol and a further essential oil component
US20130225676A1 (en) * 2010-03-26 2013-08-29 Christine Coppens Viral inhibitor compositions for in vivo therapeutic use comprising a combination of (-) -carvone, geraniol and a further essential oil component
AU2011231511B2 (en) * 2010-03-26 2014-03-20 Cesa Alliance S.A. Viral inhibitor compositions for in vivo therapeutic use comprising a combination of (-) -carvone, geraniol and a further essential oil component
EP2368547A1 (fr) * 2010-03-26 2011-09-28 Cesa Alliance S.A. Compositions antivirales comprenantes la géraniol et la carvone
CN102946876B (zh) * 2010-03-26 2016-04-06 切萨联盟有限公司 用于体内治疗应用的包含(-)-香芹酮、香叶醇和进一步的精油成分的组合病毒性抑制剂组合物
WO2011117424A1 (fr) * 2010-03-26 2011-09-29 Cesa Alliance S.A. Compositions inhibitrices virales pour une utilisation thérapeutique in vivo comprenant une combinaison de (-)-carvone, de géraniol et d'un autre composant d'huile essentielle
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CN102946876A (zh) * 2010-03-26 2013-02-27 切萨联盟有限公司 用于体内治疗应用的包含(-)-香芹酮、香叶醇和进一步的精油成分的组合病毒性抑制剂组合物
US8883859B1 (en) * 2010-03-26 2014-11-11 Cesa Alliance S.A. Viral inhibitor compositions for in vivo therapeutic use comprising a combination of (-) -carvone, geraniol and a further essential oil component
JP2012077033A (ja) * 2010-10-01 2012-04-19 Kinki Univ ヘリコバクターピロリの運動能阻害剤
KR101818286B1 (ko) 2011-03-28 2018-01-12 세사 알리안스 에스.에이. 생체내 치료 용도를 위한 바이러스 억제제 조성물
CN103458883A (zh) * 2011-03-28 2013-12-18 切萨联盟有限公司 用于体内治疗用途的病毒抑制剂组合物
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