Patent Application: US-4535202-A

Abstract:
prevention and treatment of digestive tract infections in humans and animals by orally administering a single terpene , a terpene mixture or a liposome - terpene composition before or after the onset of a gastro - intestinal infection . such infections may include traveler &# 39 ; s diarrhea , ulcers , anthrax and other bacterial and parasitical infections .

Description:
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 been 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 . iyer , et al ( 1999 ) teach us an oral hygiene antimicrobial product with a combination of 2 or 3 terpenes that showed a synergistic effect . neither of them suggested the use of a terpene , terpene mixture or liposome - terpene ( s ) combination for the prevention or treatment of gastro - intestinal infections i . e . traveler &# 39 ; s diarrhea . several u . s . patents ( u . s . pat . nos . 5 , 547 , 677 , 5 , 549 , 901 , 5 , 618 , 840 , 5 , 629 , 021 , 5 , 662 , 957 , 5 , 700 , 679 , 5 , 730 , 989 ) teach us that certain types of oil - in - water emulsions have antimicrobial , adjuvant and delivery properties . 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 . 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 %. when preparing the emulsion 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 %, once the emulsion is formed this is combined with a carrier in order to be use as a humectant , cream or other suitable carrier for topical applications . the 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 modifications can give us different products with different antimicrobial specificity . by encapsulating terpenes within these emulsions 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 . it will be apparent for those skilled in the art that the aforementioned objects and other advantages may be further achieved by the practice of the present invention . 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 . 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 . 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 including polysorbate - 80 , polysorbate - 20 , polysorbate - 40 , polysorbate - 60 , polyglyceryl esters , polyglyceryl monooleate , 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 %. to form the emulsion a ratio oil to water varying from 10 - 15 parts lipid ( oil phase ) to 35 - 40 parts terpenes ( aqueous phase ) was mixed . 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 . 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 1 without the addition of terpenes in the formulation . 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 . b - 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 - b - 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 . the procedure used was as follows : 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 growth after an absorbance equal to 0 . 9 was reached . cells were harvested , washed with phosphate buffer and resuspended to an absorbance equal to 0 . 5 . one tenth of a ml of the bacteria culture was added to 0 . 9 ml of buffer , warmed to 30 ° c . and then 80 ul of terpenes ( 85 % terpenes and 15 % polysorbate - 80 ), 80 ul water ( background ) or 40 ul chloroform plus 40 ul 1 % sds in water ( positive control ) were added . after the addition of the lysing agents the tubes were mixed for 10 seconds and 0 . 2 ml of onpg ( 4 mg / ml water ) was added , then incubated for 5 minutes . the enzyme activity was stopped with 0 . 5 ml of 1 m sodium carbonate . after being centrifuged for 3 minutes at 1 , 500 × g , supernatant was transferred to cuvettes and read at 420 nm . the relative degree of lysis caused by terpenes was calculated as follows : this shows that dosages can be manipulated to either lyse the cell outright , or in the case of lower dosages , stop bacterial growth without lysis of the cell membrane . the advantage of this controllable result is the ability to prevent lysis and the resultant release of endotoxins where contraindicated . this example demonstrate the effectiveness of the terpene mixture against escherichia coli , salmonella typhimurium , pasteurella mirabilis , staphylococcus aureus , candida albicans and aspergillius fumigatus . each organism , except a . fumigatus , was grown overnight at 35 - 37 ° c . in tryptose broth . a . fumigatus was grown for 48 hours . each organism was adjusted to approximately 10 5 organisms / ml with sterile saline . for the broth dilution test , terpene mixture was 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 . each dilution was added to sterile tubes in 5 ml amounts . three replicates of each series of dilutions were used for each test organism . one half ml of the test organism was added to each series and incubated at 35 - 37 ° c . for 18 - 24 hours . after incubation the tubes were observed for growth and plated onto blood agar . the tubes were incubated an additional 24 hours and observed again . the a . fumigatus test series was incubated for 72 hours . the minimum inhibitory concentration for each test organism was determined as the highest dilution that completely inhibits the organism . this example demonstrates the effectiveness of the terpene mixture at several concentrations against escherichia coli and cultured over time . terpene dilutions ( 1 : 500 , 1 : 1000 , 1 : 2000 , 1 : 4000 , 1 : 8000 , and 1 : 16 , 000 ) were prepared in bhi broth and in saline . these were prepared in 25 ml amounts . e . coli was grown overnight in bhi broth and diluted to a macfarland 0 . 5 concentration in saline . this solution was diluted 1 : 100 to be used to inoculate ( 0 . 5 ml ) each terpene dilution tube . the series that contained the terpene dilution in bhi was tested at 30 min , 90 min , 150 min and 450 min . each tube was mixed and serially diluted in saline . one half milliliters of each dilution was spread plated onto macconkey ( mac ) agar plates . also , 3 drops of the undiluted and the 1 : 100 dilution was added into respective tubes of bhi broth . the tubes and plates were incubated overnight at 35 ° c . the series that contained the terpene &# 39 ; s dilution in saline were tested at 60 min , 120 min , 180 min and 480 min . each tube was mixed and serially diluted in saline . one half milliliters of each dilution was spread plated onto macconkey ( mac ) agar plates . also , 3 drops of the undiluted and the 1 : 100 dilution were added into respective tubes of bhi broth . the tubes and plates were incubated overnight at 35 ° c . [ 0040 ] table 4 subculture from the tubes containing various dilutions of terpenes in saline time dilution 1 : 500 1 : 1000 1 : 2000 1 : 4000 1 : 8000 control 60 min undiluted ng + + + + + 1 : 100 ng ng ng + + + 120 min undiluted ng ng ng + + + 1 : 100 ng ng ng ng + + 180 min undiluted ng ng ng + + + 1 : 100 ng ng ng ng + + 480 min undiluted ng ng ng ng + + 1 : 100 ng ng ng ng ng + [ 0041 ] table 5 the quantitative results of the activity of various terpene dilutions against e . coli ( cfu ) media time 1 : 500 1 : 1000 1 : 2000 1 : 4000 1 : 8000 control broth 30 min 0 0 660 3600 3600 4600 90 min 0 0 12 4600 5400 7600 150 min 0 0 10 8000 12 , 000 14 , 000 450 min 0 0 15 , 000 28 × 10 3 23 × 10 7 16 × 10 8 saline 60 min 0 4 140 4000 2000 1300 120 min 0 0 0 90 3800 2600 180 min 0 0 0 2 2000 5000 480 min 0 0 0 0 104 8000 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 . 1 ) stock solutions of each terpene with polysorbate - 80 were prepared ( 1 . 8 ml terpene plus 0 . 2 ml polysorbate - 80 . 2 ) stock solutions were diluted in brucella broth 10 % ( v / v ) fetal calf serum to a final concentration of stock at 1 : 10 , 1 : 50 , 1 : 100 , 1 : 500 , 1 : 1000 , 1 : 5000 and 1 : 10000 . controls consisted of 10 % ( v / v ) polysorbate - 80 in brucella broth , brucella broth alone and bacteria in brucella broth . 3 ) a total of 1 . 0 × 10 6 bacteria ( 30 ul ) was added to 970 ul terpene dilutions ( final volume of 1 . 0 ml ) in loosely capped tubes and incubated for 24 hours at 37 ° c . with continuous mixing . 4 ) duplicate samples ( 0 . 1 ml ) from each test dilution was titrated onto blood agar plates and incubated for 48 hours at 37 ° c . on 10 % co 2 environment . bacterial colony forming units ( cfu ) were determined visual ( counting ) inspection . recovered bacteria were confirmed to be h . pylori by catalase and urease enzyme activities . the objective of this example was to determine an 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 following by the addition of individual terpenes or as indicated on table 2 ; 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 umol . 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 . it will be apparent for those skilled in the art that a number of modifications and variations may be made without departing for the scope of the present invention as set forth in the appending claims . 1 . bae e a , m j han , n j kim and d h kim , 1998 . anti - helicobacter pylori activity of herbal medicines . biol , pharm . bull 21 ( 9 ) 990 - 992 . 2 . bard , m , m r albert , n gupta , c j guuynn and w stillwell , 1988 . geraniol interferes with membrane functions in strains of candida and saccharomyces . lipids 23 ( 6 ): 534 - 538 . 3 . barranx a , m barsacq , g dufau and j p lauilhe , 1998 . disinfectant or antiseptic composition comprising at least one terpene alcohol and at least one bactericidal acidic surfactant , and use of such a mixture . u . s . pat . no . 5 , 763 , 468 . 4 . boyanova l and g neshev , 1999 . inhibitory effect of rose oil products on helicobacter pylori growth in vivo : preliminary report . j . med . microbiol . 48 : 705 - 706 . 5 . chaumont j p and d leger , 1992 . campaign against allergic moulds in dwellings . inhibitor properties of essential oil geranium “ bourbon ”, citronellol , geraniol and citral . ann pharm fr 50 ( 3 ): 156 - 166 . 6 . crowell , p l and m n gould , 1994 . chemoprevention and therapy of cancer by d - limonene . crit rev oncog 5 ( 1 ): 1 - 22 . 7 . crowell , p l , s ayoubi and y d burke , 1996 . antitumorigenic effects of limonene and perillyl alcohol against pancreatic and breast cancer . adv exp med biol 401 : 131 - 136 . 8 . dupont h . l ., c . d . ericsson , j . j . mathewson , m . w . dupont , z . d . jiang , a . mosavi and f . j . de la cabaña , 1998 . rifaximin : a nonabsorved antimicrobial in the therapy of traveler &# 39 ; 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