Patent Application: US-98049397-A

Abstract:
a method and composition for treatment of hepatitis c virus , administered as two component treatment , involving 1 ) an orally administered squalene in combination with 2 ) the inhalation of squalene medication . each medication component must be administered concurrently .

Description:
a medicinal chemistry study was conducted in which the prediction of anti - hcv compounds is done using appropriate chemical information data management and analysis systems computer - assisted techniques which are widely used to store information in databases in a form of that so - called molecular spread sheets . this is used for structure - activity relationships ( sar ) studies . a study aiming at the correlations between intrinsic , physical and chemical or biological molecular properties , structure - property correlation ( spc ) ( woldawer et al . in 1993 and iute in 1990 ). the study is conducted using software available in the market . these are also called quantitative structure - property relationships ( qspr .) ( hansch in 1981 ). when biological properties or activities are involved , quantitative structure activity relationship ( qsar ) forms a subset of this . many antiviral compounds for hiv have been developed so far , where the protease sites were known for a long time . the similarity between hiv and hcv is due to that hiv - protease is an essential enzyme for the replication of hiv and is widely regarded as one of the most promising targets for the design of new drugs for the treatment of aids . as a result many hundred of inhibitor classes have already been identified and crystal structures have been determined for at least 160 complexes of inhibitors with the enzyme ( hansch in 1984 ). the hcv molecular site is a protease enzyme also ( love et al . and kim et al . in 1996 ). although they are different proteases , however , using this similarity has benefitted the search for a drug for hcv . if we have a drug candidate , it is table 1______________________________________hiv - protease inhibitor antivirals . antiviral molecular formula manufacturer______________________________________1 . acyclovir c . sub . 8 h . sub . 11 n . sub . 5 o . sub . 3 merck2 . bms - 187071 c . sub . 32 h . sub . 49 n . sub . 3 o . sub . 6 bristol myers squib3 . bms - 182193 c . sub . 30 h . sub . 45 n . sub . 3 o . sub . 6 bristol myers squib4 . bms - 186318 c . sub . 36 h . sub . 54 n . sub . 4 o . sub . 9 bristol myers squib5 . calanolide a c . sub . 22 h . sub . 26 o . sub . 56 . indinavir c . sub . 36 h . sub . 47 n . sub . 5 o . sub . 4 merck7 . kni - 272 c . sub . 33 h . sub . 41 n . sub . 5 o . sub . 6 s . sub . 2 nikko kyodo co . 8 - kni - 227 c . sub . 35 h . sub . 45 n . sub . 5 o . sub . 6 s . sub . 2 nikko kyodo co . 9 - ritonavir c . sub . 37 h . sub . 48 n . sub . 6 o . sub . 5 s . sub . 2 abbott10 - samquinavir c . sub . 38 h . sub . 50 n . sub . 6 o . sub . 5 hoffman laroche11 - xm - 323 c . sub . 35 h . sub . 38 n . sub . 2 o . sub . 5 dupont merck pharmaceutical12 - zidovudine ( azt ) c . sub . 10 h . sub . 13 n . sub . 5 o . sub . 4 burrough welcome______________________________________ advantageous to start the molecular modeling with a preliminary structure - property correlation ( spc ) studies . therefore , we have conducted in - depth spc . study prior to molecular modeling study which helped us finger pointing the compounds which have antiviral activity against hcv . in the spc study we used protease inhibitor anti - hiv drugs for correlation with some chosen squalene isomers as anti - hcv of drug molecules . the anti - hiv drugs used are : ______________________________________ 1 ! acyclovir , 2 ! bms - 187071 , 3 ! bms - 182193 , 4 ! bms - 186318 , 5 ! calanolide a , 6 ! indinavir , 7 ! kni - 272 , 8 ! kni - 227 , 9 ! ritonavir , 10 ! samquinavir , 11 ! xm - 323 , 12 ! zidovudine ( azt ). ______________________________________ where bms refers to bristol myers squib co . and associated numbers are code numbers made by the company . kni refers to kyodo nikko co . and xm - 323 is an antiviral made by dupont - merck pharmaceutical . table 2______________________________________the computer printout of the press test . compounds with highest predicted values : ______________________________________sq11 ( 8 . 411494 ) cis - cis squalenesq21 ( 8 . 394141 ) trans - cis squalenesq22 ( 8 . 392908 ) trans - trans squalenesq12 ( 8 . 375626 ) cis - trans squalenesq31 ( 7 . 488726 ) 2 - aza - 2 , 3 - dihydrosqualene , antifungalbm + 51 . 0836 ( 7 . 187380 ) antiviral ; inhibits hiv - 1 reverse transcriptase made by bohringer mannheim . l - 702019 ( 6 . 773015 ) antiviral ; inhibitor of hiv - 1 reverse transcriptase , made by merck . sq32 ( 6 . 632001 ) 2 - aza - 2 , 3 - dihydrosqualene hcl , antifungals - 2720 ( 6 . 263846 ) antiviral inhibits hiv - 1 reverse transcriptase and replication , made by hoescht . thiazolobenzimidazole ( 5 . 999923 ) antiviral ; inhibits hiv - 1 , also ( tbz ) known as nsc 625487 . sulfoxamine ( 5 . 619261 ) antiviral ; inhibits hiv - 1 , also known as nsc 287474 . r86183 ( 5 . 445646 ) antiviral ; hiv - 1 , replication inhibitor ; synergistic with 2 &# 39 ;, 3 &# 39 ;- dideoxynucleoside analogs , made by janssen . cl - tibo ( r82913 ) ( 5 . 444047 ) antiviral ; inhibits hiv - 1 reverse transcriptase , made by janssen . npps ( 5 . 304927 ) antiviral ; inhibits hiv - 1 reverse transcriptase , c . sub . 12 h . sub . 9 nso . sub . 4 . ______________________________________ our isomers of squalene , which are sq11 , sq12 , sq21 , and sq22 refers to cis - cis , cis - trans , trans - cis and trans - trans squalene respectively , which will be refered to as sqxx . as we know that log p is the predominant descriptor in many structure - property correlation studies which is a function of the penetration of the drug molecule through the cell membrane . the log p values of our sqxx series of the squalene isomers are significantly higher than that of hiv - protease inhibitors used in the study . other similar spr studies are conducted using other variables as percent of hydrophilic surface , molecular volume , surface area , solubility parameter density , vapor pressure , molar refraction { mr = mw * index of refraction - 1 !/ density squared * ( index of refraction + 2 )! for liquids }, and water solubility . these intrinsic and physico - chemical properties are functions of biological properties of drug molecules . all these correlation studies were done using multiple linear regression analysis ( mlr ) showing similar results of the superiority of squalene over the other known antiviral compounds . we do not know if sqxx series is itself the hcv - protease inhibitor or one of its metabolites or by - products , which could be further investigated , however it is a good start as lead compounds . a printout of the contribution to press ( predictive residual sum of squares ) is listed in table 2 ., which shows that squalene is on the top of the list due to its high log p value . a number of reviews have documented the history , strategy and success of quantitative drug design , i . e . design using spc / qsar methods ( van de waterbeemd , stanon et al ., waldawer et al ., tute , hansch and fujita ). the theoretical expectation of the possibility that squalene is an antiviral compound has been confirmed by the in vitro assay where its results testifies that squalene has antiviral activity using the ifn as a reference . therefore , the present invention introduces the squalene as antiviral compound specifically for hepatitis c virus administered orally as 500 mg twice / day and 2 - 3 drops ( 10 - 15 mg .) through the nasal membrane three times per day for a period of two to six months depending on the viral load in the patient which could be tested periodically by the polymerase chain reaction { pcr ( hcv ) rna } quantitative hepatitis c test . therefore , it is the principal object of the invention to introduce the squalene as an antiviral compound for hepatitis c virus . it is another object of the invention to include other compounds which are structurally related to squalene i . e ., any compounds having aliphatic conjugated π bonding , as they might have antiviral activity against hepatitis c virus . another object of the invention is the advantage of using squalene , where it is available as squalene soft gel capsules sold in any natural food store for $ 15 / 100 capsules , 500 mg . each , safe vegetable extract and squalene from shark liver extract . it is a further object of the invention to provide a method of treatment of hcv by providing a procedure for administration of the squalene . still another object of the invention is to provide improved elements and arrangements thereof in a method and composition for treating hepatitis c for the purposes described which is inexpensive , dependable , not toxic and fully effective in accomplishing its intended purposes . the patient in this case is in the medical profession , was infected by a contaminated syringe on may 21 , 1984 . he has been diagnosed then as hbv case and received γ - globulin , and the follow - up showed negative hbv infection . on a late stage , early 1990 , the patient start complaining from abdominal pain , liver enlargement , tendency to sleep for a long periods of time , gastric and intestinal discomfort and general weakness in his body . his periodic blood chemistry showed that his alanine aminotransferase ( alt ) normal level is 0 - 45 u / l ! was 137 on january of 1990 , 150 on november of 1993 and 139 on january of 1994 . at that time he went for explicit diagnostic tests which concluded that the patient had cirrhosis with chronic hepatitis c , portal hypertension with varices and duodenal ulcer . it was prescribed for the patient omeprizole and indoral for treatment of ulcer and portal hypertension respectively and an advice for seeking liver transplant was given . the time was against him , where it results in progressively worsening liver inflamation . the herbal treatment was the only alternative for him , then he found by the analysis of the herbal extract that it contains toxic substances and concluded that squalene was the ingredient that causes the reduction in the viral load . therefore he switched to squalene treatment . table 3 . shows the progress of the alt and the decrease in the virus load as indicated by pcr - rna quantitative test . where the alt is an enzyme located in the liver cells . it leaks out and make their way into the general circulation when liver cells are injured . it is thought to be a specific indicator of liver inflamation . as noted in table 3 , hcv - pcr - rna quantitative test taken one month after starting the treatment showed that the hepatitis c viral load was more than 30 , 000 copies per ml serum , which is strongly positive . four month later , the viral load went down to 20 , 000 copies per ml serum . four month later , it showed negative hcv . the viral load quantitative test showed undetected hcv since april of 96 until now . furthermore , the alt continued to normalize . another fact is the alpha fetoprotein was found to be down to 17 , lower than the results of 61 on january , 1995 . although the liver cirrhosis remains , complete recovery from ulcers or varices was found , and most importantly , no hepatitis c virus remained . in 1997 the patient has been diagnosed as having an end stage liver disease secondary to post necrotic cirrhosis and has been listed for liver transplant . after receiving table 3______________________________________date alt pcr - rna______________________________________analysis before start using the squalene treatment . 1 / 23 / 90 137 no pcr - rna hcv quantitative analysis11 / 11 / 93 150 no pcr - rna hcv quantitative analysis1 / 1 / 94 139 no pcr - rna hcv quantitative analysisstarting the treatmeny with squalene on june 1 . sup . st . 1995 . 6 / 29 / 95 67 no pcr - rna hcv quantitative analysis7 / 14 / 95 47 no pcr - rna hcv quantitative analysis7 / 28 / 95 55 no pcr - rna hcv quantitative analysis8 / 11 / 95 42 no pcr - rna hcv quantitative analysis8 / 18 / 95 -- 30 , 120 copies / ml serum9 / 7 / 95 55 no pcr - rna hcv quantitative anaiysis10 / 12 / 95 51 no pcr - rna hcv quantitative analysis11 / 22 / 95 50 no pcr - rna hcv quantitative analysis12 / 15 / 95 -- 19 , 940 copies / ml serum4 / 15 / 96 53 & lt ; 2 , 000 copies / ml serum6 / 17 / 96 49 & lt ; 2 , 000 copies / ml serum the patient continued using6 / 10 / 97 40 & lt ; 2 , 000 copies / ml serum the squalene . ______________________________________ a liver transplant , the patient continued on testing for hcv - pcr - rna , with negative results . usually most of liver transplant recipients who have history of hcv infection , and use ifn as medication , continue to have hcv in their blood stream after receiving a new liver . this is because the hcv hides inside the leucocytes which work as reservoir for the virus . by using the squalene as a medication , the hcv is completely abolished in the blood serum and in the leucocytes as well . this is an advantage of the squalene treatment over the interferon . it is to be understood that the present invention is not limited to the embodiment and example described above , but encompasses any and all embodiments within the scope of the claims . 1 . purcell , r . h . hepatitis c virus ; historical perspective and current concepts , fems microbial rev . 14 , 181 - 192 , ( 1994 ). 2 . van der poel , c . l . hepatitis c virus . epidemiology , transmission and prevention . in hepatitis c virus . current studies in hematology and blood transfusion , h . w . reesink ed . 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