SA93130529B1 - Anti-hepatitis B virus treatment with purine 3,2-dioxynucleosides - Google Patents

Abstract

Abstract: This invention shows a method for the treatment of hepadnavirus infection in animals. Animals infected with duck hepatitis B virus can be treated with 2',3'-dideoxy nucleoside adenine, guanine, hepoxanthine, or 6,2-diaminopurine. 2,6-diaminopurine, or various analogues of substituted purines. 2',3'-doxypurine nucleosides inhibited multiplication of duck hepatitis B virus in cultured liver cells by more than 99% at a dose of 1 μg/ml. Also, the extremely strong efficacy of 2,6-diaminopurine-2',3'-dideoxynucleoside was demonstrated in the complete disinfection of sera of pekin ducks. from duck hepatitis B vims. And that the selective effect of 2',3'-dideoxypurinenucleosides-'3,'2'-dideoxypurinenucleosides on hepadnavirus replication depends on discovering the unexpected effect of hepadnavirus with 2',3'-dideoxypurine nucleosides. '3,'2dideoxypurinenucleoside. These compounds provide a new antiviral therapy for the treatment of acute or chronic hepadnavirus infections.

Description

2 Y

Hepatitis B virus Anti-hepatitis B virus 2,3-Dideoxypurine nucleosides AY JY with purine compounds Full description BACKGROUND DOXY ALITY This invention relates to a method for the use of purine compounds Or its pharmaceutically acceptable derivatives 2",3"-dideoxypurine nucleoside nucleosides as an antiviral therapy in the treatment of acute or chronic infection with hepatitis B virus in animals. global scale. The virus that causes this disease is transmitted through blood, blood products, and contamination of syringe needles by abusers from the Lady’s mothers, and through sexual contact and transmission “IV drug abusers.” Or carriers of it to infants, more prevalent in Southeast Asia, Africa and parts of South America. In those areas - 0 from mothers to children at an early age to convert a high rate of Lady virus transmission leads to hepatitis B virus. the chance of chronic carriers becoming chronic carriers 56 deaths of males infected with hepatitis B virus; as infants; It's about

Primary hepatocellular or cirrhosis die from cirrhosis of the liver as a result of chronic infection with hepatitis B virus. Females who develop this carcinoma ve from chronic Sian Tiga disease at birth have about a 110% chance of dying from hepatitis B virus over Jala million YA with hepatitis B virus. It is estimated that there is a worldwide range. Since the reproduction of Gawd The field of antiviral chemotherapy is considered a recent 0 locating fan viruses mainly includes the host cell. It was hard © 4 Z Z

v Specialized viral therapy for specialized antiviral chemotherapy. No attempts were made to treat pregnant women

Adenine infection with chronic hepatitis B virus has met with little success. Adenine arabinoside has been used to treat chronic pregnant women. Three controlled studies of interferon and arabinoside interferon in the treatment of chronic infection (placebo) were conducted with a placebo control (DNA gin J) to compare the effect of adenine M on hepatitis B virus. In two of these three studies, the response rate to adenine arabinoside was significantly better in patients treated with it than in patients in the control group (the Gastrointestinal Study) and Bassendine who received a placebo (Bassendine).

Yokosuta for six 1941 and Yokosuta 0171-1011 01 p. A+ vol. “Gastroeutorology” (VAC p. 301-746 of the year (AY “Diseases of the stomach and intestines”); ple Aad vol. Controlled No significant benefit observed in patients treated with adenine arabinoside 157-1590 ya (AT Gastroenterology “; vol. ale” and co-administration (Hoofnagle) (Hoofnagle) prednisone It has been reported that giving patients an introductory dose regimen of prednisone (VAS) for a year (Perrillo, RP.) gradually decreasing before treatment with adenine arabinoside is beneficial (RP Briley).

YAT=YA+ Gaia AA vol. “Gastroeutorology and intestines sazall diseases ale” and those with it However, in most open and controlled studies no benefit was shown from treatment (VAAC for year 0s plus fas only Of the treated patients, even these results were not convincing. Most trials of using adenine arabinoside to treat chronic neurotoxicity with hepatitis B virus due to the low success rate and moderate toxicity of this antiviral agent. However, with very limited success and interferon toxicity is significant. Thus, there is a lack of effective anti-hepatitis B virus treatment. as antiviral agents.” Indeed, 707-dioxycytidine > vo

¢ 0 is now actively being studied as an antiviral agent for retroviruses including HIV and other 767-doxynucleosides have been shown; But not 767-2,3-dideoxy thymidine. They are potent inhibitors of human immunodeficiency virus (HIV) (Mitsuya et al. (Mitsuya et al. PNAS Journal > AY Vol. p. YY + 1-7695 of ¢Y3A0, Mitsuya & Broder Journal (PNAS vol. AY pg. 1519-1511 of 1985). Burroughs Wellcome disclosed in its European patent application with serial number 8077657-01 reported that dioxynucleosides may be useful in the treatment of infections with hepatic DNA viruses such as hepatitis B © virus, however, they did not provide any experimental evidence to support this hypothesis nor did they mention our discovery It is not expected that purine 77 - 2,3-dideoxynucleosides are much more effective than pyrimidine 7-dideoxynucleosides, although it has been known for WY compounds. Doxynucleosides have antiviral activity, the work that was revealed (die) focused on a compound of this type being effective against retroviruses, especially human immunodeficiency virus (SL) (HIV) compounds were 767 - dioxynucleosides The most effective against human immunodeficiency virus (HIV) are 707-1-dideoxycytidine (ddC) | 231--dideoxyadenosine 3 2 "(ddA) and pyrimidine L "-pyrimidine 2 3 -dideoxynucleoside 0 and purine SY CY purine 2,3 -dideoxynucleosides, respectively. It is clearly impossible to predict the specific effect of hepatitis DNA viruses on 767 SE deoxynucleosides based on the known retroviral effects of these compounds. It is not surprising that retroviruses are RNA viruses, while hepatic DNA viruses are reductive DNA viruses of the Yo family, which are clearly different and multiply by different AL replicates. ¥i4

GENERAL DESCRIPTION OF THE INVENTION According to the objective FAY (op) a method of medical treatment of an animal infected with hepatic DNA virus includes giving this animal a composition containing Yad amount of a biologically active compound 767-2,3-dideoxynucleoside mixed with a biologically acceptable carrier ; Provided that this m is the aforementioned compound 707 dioxynucleoside represented by the formula: Y NF T << l l l R, 001120 11 11 i 11 il 83 where (x) is either an atom a hydrogen, an alkyl group (Ry), an amine group (NI), or a halogen; or NHR; or NR), or OH or OR, or SH or SR; and is (not) either a hydrogen atom or an alkyl group (Ri) or an amine group ( NH) or halogen 00 | or NHR, or NR): or OR, sl OH or SH or SR; (Ry) is a reduced alkyl group of 1 to 8 carbon atoms. It is either a hydrogen atom or an acceptable ester (Lia shan) Wikia) or a salt of said ester that gives Ll bioacceptable. Brief Explanation of Drawings Preferred embodiments of the invention are graphically illustrated as follows: Figure 1: Shows response curves to doses of selected purines and pyrimidines Sr Y deoxynucleosides. 7

1b Figure Y: dot-blot chromatography hybridization of DNA from control virus (ve) and drug-treated duck hepatitis B virus (DHBV)-infected liver cells. a figure? : It is an analysis of Southern M51 [Ua] Southern intracellular viral DNA ° that was separated on an agarose gel. ANS (agarose gel). Figure; It is a Southern analysis of an extracellular virion DNA stain that was separated on an agarose gel 71,9. Figure E: dot-blotanalysis of sera from animals infected with duck hepatitis B virus (01187). Detailed Description This detailed description of preferred embodiments of the invention demonstrates the discovery that compounds of Formula (V) are useful antiviral agents in the treatment of hepatitis D virus infections. Eg it is easier to prove the efficacy of these compounds in animals, especially because the duck hepatitis B virus behaves in ducks as tan silas as the hepatitis B virus in humans. Therefore, these compounds were tested for their unexpected efficacy in ducks infected with duck hepatitis B virus (01287). These compounds are estimated to be effective in treating animals infected with hepatic DNA virus; As humans and non-humans. Non-human animals include ducks, marmots, woodchucks, beachy ground squirrels, and kangaroos. In previous studies of antiviral agents, it did not differentiate between the activities of purine CNY Y. purine 2,3 -dideoxynucleosides and pyrimidine EY JY pyrimidine 2,3 -dideoxynucleosides. Difference in Us in current strain of duck hepatitis B virus. Wag purine 2,3 -dideoxynucleoside It is assumed that compounds of formula (0) can bind vo-genome-linked protein that initiates the formation of the DNA strand. negative. And since pg v ' (Will et al) has to form the positive strand (Will and his cohort) template, the negative strand acts as a template, so the prevention (VAAY Volume 1)<p. 111-4504 for the year (jof Virology Journal of Virology At this early stage of DNA formation, the formation of the negative and positive strands of DNA blockage lad will differ from the reproduction of other DNA viruses. Gla The reproduction of the hepatitis DNA virus differs in a similar way 5 to what is seen in reverse transcription viruses, and a clear difference lies in Reverse transcriptase DNA virus ASS mechanism (Summers and Mason) Summers and Mason discovered hepatic DHBV retrovirus vol. 749; page first in a duck hepatitis virus model American B; page PNAS magazine (Mason et al) and Mason et al. “VAY for the year 15-67, and then it was found that it is similar in the hepatitis B virus (VAY for the year 4001-9892 0 pp. 87/-17; year 09 alas cvirology ¢ (Blum et al) (Blum et al) 1-07 p. and Miller and those with him “VAS .77-7 4 p. OY Virology; vol. dae and Miller and from 1985 of the year After the hepatitis DNA virus penetrates the target cells (liver cells) it removes its envelope and in the nucleus it transforms The DNA virus is partially double-stranded into the virus nucleus, and the DNA enters the nucleus in a circular, covalently locked, double-stranded form. The negative strand plays the role of a template to form from the strand (kb) which is larger than ;,? thousand bases) pregepomic pre-genetic RNA thousand base RNA). It forms pre-genetic RNA by means of the negative cellular RNA polymerase (TY) enzyme; DNA Gall, and pre-genetic RNA acts as a template for the formation of the strand cellular RNA polymerase (activity of the viral DNA polymerase transcription enzyme, which is performed by the enzyme Viral DNA polymerase © and initiates negative strand formation; covalently coupled protein (reverse transcriptase) pp. 005-801 of 71 cells; vol. Robinson and Gerlich co-authored AVY=110 £0 volume “lug pill ale” and Molnar-Kemper et al. magazine 80 pages

H During formation, RNA is degraded before the genome with an efficiency similar to that of the enzyme ribonuclease (1 54) of the viral DNA polymerase enzyme. When the negative strand is formed, the (ribonuclease H) vo

Jr A to the 082-negative-strand region and performs a small DRI of pre-genome RNA from the L region Journal of Virology; Volume (Will et al) in the role of the precursor to the formation of the positive strand (Will and those with him) and the positive strand may not be formed before it is enveloped (VIAY) page 111-904 of the year 71 and the export of viral particles. nucleo eapsids with protein nuclear covers for the virus

° Hence, the possible explanation for the intensity of the unexpected effect of hepatitis DNA viruses on purine 7 - purine 2,3 -dideoxynucleoside compared to pyrimidine compounds SEY Y pyrimidine 2,3 -dideoxynucleosides is that purine 27 Purine 2,3 -dideoxynucleosides bind to the genome-binding protein that initiates negative strand formation. In hepatitis B 18 virus, hepatitis

0 HUMANS Terminal 5 in the negative strand cascade is (GAAAAAGT.) 5-4 (Will et al.; Journal of Virology; Vol. YAAY ud 911-464 dad ia TY 5d GTAATTCTT... The nucleotide at the 5th end that binds to the protein is a purine nucleotide, and the current results, Ne, in hepatocyte cultures indicate that 01197, compared to other hepatic DNA viruses, is more effective than purine 767 - a deoxychloride purine. Z Ly 2,3 -dideoxynucleoside is at least 100 to 0001 times more pyrimidine 2,3' dideoxynucleosides This may be the unique step of association with the DNA strand initiation protein Negative Als is a possible mechanism by which (0° Als) explains this unexpected finding. However, there may be other alllls in this regard. In any case, the results of the tests clearly prove the efficacy of compounds of formula ((). This suggests that The unexpected effect of hepatic DNA on purine 7607-2,3-dideoxynucleosides without causing any damage to host cells at concentrations much higher (100 to 1000-fold) suggests that these antiviral agents ve uniquely affect virus replication. It allows the imposition of a protein that blocks the initiation of DNA synthesis

LE 9 viral purine SY JY deoxynucleosides purine 2.31 -dideoxynucleosides applied. It is widely used to treat infections with other viruses such as polioviruses and adenoviruses, which are also known to have Gig pa starts to form their DNA. -

The useful compounds in this invention are represented by the following formula (1): Q Y NZ << No | Gel N R, 0Clia 0 | i 11 i 11 3 11 and in In this formula, (X) is a hydrogen atom (H), an alkyl group (Ry), an amine (NEL), a halogen, (.2138), or NR: or OH or SH or OR, or (Y)5 SR is H or B group or NH; or a halogen; or NHR; or NR): or OH or OR, or SH or (Rt) SR: is a reduced alkyl group with 1 to 4 carbons; (Re) is either a hydrogen atom (H) or a :0 bioavailable ester or a salt of this ester that gives a bioavailable tale. 4 2

1 The following table shows examples of the compounds of formula (1) shown above: Z 1 Table (Y) What the symbol represents X Number of the compound What the symbol represents

OHH 1

NH; H7

NH, etc.) C,H, CH=R)R y

OH NH, 3 etc.) C,H; CH;=R)OR NH, o

NH, NH, 1

H NH, no :

SH NH, A (&C,H;,CH;=R)NHRNH; 45 McCartt etc.) CH=R)N(R), NH, 0" (I,Br,CLF) Halogen | y NH, 11 (J C;Hs,CH;=R) SR NH, yy z NH (IBr.CLF) Halogen Vv z NH;© LG CH CH=R)NER ve oo

NH, etc.) C,H; CH=R)N(R), yo

NH, OH5

NH, (2) petkin ,CH;=R)OR 1 ] NH; Sh Ya

NH, etc.) CH, CH;=R) SR 14 hypoxanthine guanine and guanine adenine The preferred formula 0 compounds are the adenine of formula 1 honey 2,6-diamino purine or 6-7 derivatives Diaminopurine hepoxanthine °

JE 1 i.e. in which (X) is hydrogen (H) and (NO) (X) 5 NH; amino NH, and (7) hydroxyl (OH) (KX) hydrogen formation and (7a) hydroxyl formation; (X) is eNH, (Y) NH, the order. Many of the above compounds listed in Table 1 perform the function of pro-drugs that are metabolized in organisms to the most potent SE 07 deoxynucleosides such as SE TY de° dideoxy guanosine 3 2. It may be (Re) a hydrogen atom to form a hydroxyl or an acyl group or its esters or z-esters which are terminated esters and esters-salts or salts by themselves provided that they are all biologically acceptable. This ester may be straight or branched chain. It is preferred that the aster be short, the length of the chain being from 1 to; carbon atoms. One or more of these 0 compounds may be administered to provide an effective blood stream concentration (JH) of 50 µg/mL (micrograms per milliliter); The preferred concentration range for a dose is 1.1 to 10 µg/mL. These compositions are given by the usual methods Jie tablets and oral liquids or (suppository) suppository or injection liquids or aerosol inhalation spray etc. The absence of an effective antiviral agent for the treatment of viral hepatitis B has led to the use of duck liver cell cultures to grow DHBV and to test the effectiveness of chemotherapeutic agents against the virus. Then, duck liver cell cultures are used to grow Z77 and test the effectiveness of chemotherapy agents against the virus. Although the methods are short-handed in explaining DHBY treatment, it should be recognized that this treatment method is effective against viruses reproducing with a similar replication mechanism. Hence, the purpose of these following examples is to clarify various aspects of the invention by way of representation and not to limit the objective of the invention, as the objective of the invention is clearly defined in the elements of protection. Dose-response curves for Purine 767 - purine 2,3 -dideoxynucleosides and pyrimidine GS Y pyrimidine 2,3 -dideoxynucleosides were prepared using hepatocytes infected with vO virus. Duck liver DHBV Hepatocyte cultures infected with virus 011877 were prepared in the manner that

os "7 is described in Example 1. Dioxycycloside homologues were added to the media of the hepatocyte cultures on the second day and preserved in the cultures with the media changed every two days. On the sixteenth day, DNA was extracted from the cells and a drop chromatography was performed. The virus using a DHBV probe tagged with radioactive phosphorus (PP) and according to the radioactivity of each point, it was quantified by cutting the point and counting its radioactivity.The following is an explanation of the abbreviations used in Figure 1 to distinguish the dose response curves: ddA = 7 -dideoxyadenosine 2,3 6 - 7 -dideoxyguanosine -dideoxyguanosine 2',3 dar > 727 -dideoxyinosine 2,3 ddDAPR = 17 -diaminopurine 2 3 -dideoxyriboside vy -2,6-dideoxyriboside 7 > 207 ga-dideoxythymidine "2,3 SET = ddC -dideoxycytidine 2.3 From the results shown in Figure 1, the nucleoside analog (Silas) concentrations required to inhibit viral replication by 0 78 were calculated as follows: Z 0 1.077 μg/ml for EYE Aminopurine 07 - Doxyriboside; Z and 00 µg/ml for 707-doxyguanosine and 17 µg/ml for 77-doxyadenosine; 1.05 µg/ml for 77-doxyenosine; and 0 µg/mL for 77 SUE deoxycytidine. 727-dioxythymidine did not inhibit DHBV replication: Vo | Example 01 Newborn ducklings were exposed to DHBV virus infection, and after Y = 0 days after infection, blood samples were taken from the ducklings and Bing scanned for DHBV DNA using point 0 z hybridization with a specialized DNA probe (Masson (Mason and those with him are lecturers of the work of the Academy of Natural Sciences, via

7 3 ¢ (Proc, Natl.

Acad.

Sci) USA; VA volume p. 50601-79917 for the year 1 7) Livers were removed from ducklings that gave a positive result on dot-blot and were used to produce cultures of primary liver cells infected with virus 01187 as described above (Tattleman) Tuttleman et al. Aas Virology; OA vol. pp. 5-17° (0) After culture growth 2 days, antiviral agents were added to culture media. Media were changed every 2 days, cells were removed at selected times, and total DNA was extracted. .

Stain (put in the form of spots) Lal on nitrocellulose paper and probe

Gay (2P) radioactive phosphorus-tagged DNA probe for the following method: ball was extracted from DHBV-infected liver cells by sputtering on a 0-nitrocellulosic filter and the radioactive phosphorus-tagged probe described above was used by Penick translated-DHBV DNA (pDH-010=DHBV) and Gall were extracted from +1 cm diameter culture dishes at varying intervals after cultivation. In the control group infected with virus (VC) without treatment, cells were harvested on days Y, 7, 48, 10, 41, 81 and Cal Gg Ye two copies of samples on days 16, 18 and 70. In cultures In the drug-treated groups, ve cells were harvested on days A, 14, and Ye. In Figure 7, the gradient of days is shown from left to right. The drugs were added to the cultures on the second day after cultivation; co Bing throughout the medium changes every two days. The used concentrations (micrograms / ml) were shown in the figure. For the drugs 27-dioxythymidine (AT) and 77-deoxycytidine AE (400)

and adenine arabinoside (ara A), 77-dioxyadenosine (ddA), and 77-dioxyguanosine (400). All intracellular DNA was extracted from the cells using the standard 0-phenol extraction method, and the cells were lysed in a 7-cm diameter Petri dish (about ½ cell axe) in a lysis buffer. Contains SDS Ly, 1001 mM of Tris-HCI pH eA, 01 mM EDTA © 5, EGTA 5 104 mM NaCl digested as a lysate ve cells with 00+ mg/mL prunase-8 (available from Sigma) at PY °C for

JVE

2 hours, then the protein was extracted by extraction with an equal volume of saturated phenol with a mM molecular Yo solution (ul «Tris-HCI pH 0,¥ (pH=7.5) 04, + mM 70.1 5 EDTA -hydroxy-quinoline # Sanam0n:8-070:0:70 A concentrated ammonium acetate solution [molecular pH (molecular (Y,0)] was added to the aqueous layer to produce a 78 molecular ammonium acetate solution and 0 nucleic acids were precipitated by adding two volumes of JY ethanol er and the DNA disk was washed with ethanol and dried. The DNA was dissolved in a solution containing VY,0-mL-methylcellulose (Tris-HCI, pH 05, 10-mL-methylene EDTA, 1077 glycerol, 70.00 bromophenol blue, and stains a portion of one twelfth ) fe 3a 11 from the DNA sample on nitrocellulose for analysis by dot chromatography. Both ddA 106; ddC and ddT were purchased from Pharmacia 0 and JECT can be prepared as aminopurine SEY D oxy-riboside [or 7-diamino-4-;7-dioxy-7-8-glycero-pentofhoranosyl)-purine (ddDAPR) [2,6-diamino-9-2,3-dideoxy-B-D- glycero-pentofuranosoyl-purine by a Bias method for efficient conversion of adenosine to its ddAdo derivative (Robins et al. Tetrahedron Lett. Hansske and Robins, Tetrahedron Lett., 26, 4295-4298, 1985 1 Ao) crystalline ddDAPR has a melting point of 144 to 190 °C; and a k/charge mass spectrum

MS m/z 250.1180 [M+(C1oH14Ne0)=250.1178] This lineage demonstrated an unexpectedly high susceptibility to DHBV virus ©727-dioxyadenosine (ddA) and 77-dioxyguanosine (416). ). And as shown in the figure? 727 dioxythymidine (ddT) did not inhibit DHBV DNA formation even at a concentration of 10 μg/mL. 767-Dioxycytidine inhibits viral DNA production by approximately 707% at 01 µg/mL but has little inhibition at 1 µg/mL. Adenine arabinose at a concentration of 10 μg/ml inhibits the formation of DHBV-DNA vo by about 757. Each of the 5 ddG ddA inhibits the formation of DHBV-DNA by more than 4"

SL Yeo µg/mL. These results are consistent in the results of triple experiments (repeating 1 on 794 at three times concentration). See Table 7 below: Table? Comparison of the effectiveness of Purine 767-doxynucleosides and that of pyrimidine 707-doxynucleosides | Nucleosides in the inhibition of hepatic DNA virus replication. For hybridization of droplets on the 20th day after cultivation. (densitometry scans) based on densitometry scans Comparative Compound Residual Ratio / Control Media without Drug Zero iy μg/ml ) 1 +) 7 1 µg/mL (0) 1) ddC

Lov (ddC 01 μg/ml) above 01 (ara A 7959 μg/ml) above V+ ddA (734 μg/ml) above 1 (ddA 794 μg/ml) above 01 (6 734 μg/mL) above 1 (6 (*) μg/mL) 1(-dioxyriboside FY-diiminopurine 7) (*) 741 μg /ml) 1( 7 Do the hybridization spots of the two compounds not appear in the figure?)*( Example?: De AEYEY. The effectiveness of southern Purine homologues was confirmed by Southernon chromatography analysis as shown in the figure. It has DHBV virus DNA is specialized against dia-oxynucleoside 1. This information was collected in the following way: Intracellular viral DNA was collected on an agarose gel, then DNA Lal was separated by Southern chromatography analysis in (DHBV) in Jap and extracted. DNA from hepatocytes infected with hepatitis 6 virus, as shown in Lane, a 1 cm diameter plate and one-fifth of the total amount of DNA was placed on each strip on the day. 1) The DNA of the viral control group was extracted on the second day (the lane). YO Figure 1

Jy A (the path;). Aggregates Yeo (trail “*) were harvested on the VE day and on the eighth day Y (trail) after cultivation in dishes. DNA samples were from 01 and VE treated with the drug on days 0 (0 (trail). VE microg/mL (day 01) ddA drug-treated hepatocytes as follows: ( trail 10 and day V ( trail VE microg/mL ) day 1 ( 1 ) ddA Trajectory Ye and Day 1 (Trail 01) Yo (Trail 3) and Day VE µg/mL (Day 01) ddG (A © DNA pled com Zag) VY (V path yo and day) 11 (V path µg/ml) day covalently and covalently closed circular (RC) Relaxed Circular in shape? (SS) single-stranded (CCC) closed circular bacteria agile a” aga Hin d 111 7 Kilobases (thousands of bases) markers (bacteriophage) DNA acid laal 0 and the relaxed circular covalent circular (RC) DHBV DNA forms of ddG or ddA were increased and DHBV inhibited LDA-infected liver by incubation in single-stranded (SS) and CCC ) in triplicate experiments, ddA was inhibited and 006 was more effective than .01137 µg/mL DNA formulation. 1 At a concentration of 011817 DNA, the formation of all forms of Tan occurred in a strong form, ddA to day 16 of day ddA. Hepatocytes treated with the drug ERC DNA had a slight increase in |.

DHBV DNA in all forms of Lalli 006, on the other hand, treatment with the drug 0.0 induced a more effective antiviral factor ddG on the second day. On the basis of these studies, we see that 0 ad and 106 are considered antiviral agents for ddA, however, both DHBV in the treatment of ddA from: or other cycloside analogues such as AT adenine and ddC are much more effective. From D J JY and in similar experiments in which the two drugs adenine arabinoside and arabinoside 7 were used

SEY - dioxyriboside JY $23 -dideoxy inosine (ddI) oxyinosine These two drugs, 2,6-diamino purine 2,3 -dideoxyriboside (ddDAPR) turned out to be purine 2, 3-dideoxynucleosides +1-dioxynucleosides 1-purine compounds (inhibition above 746 at DHBV against Ta a also two active antiviral agents stripped ddDAPR µg/ml) see table 7. It is well known that the sums of Amin Yo Yaa

7 VY (Balzarini and co. adenosine deaminase by means of the enzyme adenosine deaminase) (Balzarini » yald 15/87 p. 771-754 of the year 046 Biochem. Same Biochem, Biophs. Res. Commun.145, 269-276, 1987 ((Balzarini et al, Biochem, Biophs. Res. Commun. 145, 277-283, 1987) YAY=YVY p. Prodrug acts as a ddDAPR primer and thus it is likely that similarly 0.000 for an example drug? The figure shows the ability of 67-dioxynucleoside analogues to inhibit extracellular production. Southern DNA chromatography analysis separated an extracellular virion on 17 days after Ye to day 11. Culture media were collected from day 7.1.9 agarose gel with pronase enzyme and the DNA pelleted virions were cultured in dishes. Granular virions were digested in a control group sample (1 all) 4 of the whole sample on each track. As shown in figure (f) trail ddA µg/ml of 1 virus and (track 7) a sample of hepatocytes treated with de hepatocytes and (track) ddG µg/ml of 1 die treated hepatocytes 1 μg/ml from 407 and (Trail 0) 1 die treated hepatocytes sample 16 Fig.; on DNA in a loose circular shape. ARC The symbol .006 ge or 6 denotes extracellular virus production significantly reduced at ddA and 040 failed to inhibit extracellular virus production at ddT µg/mL; z either 1/µg/mL. 1 When DHBV LS deoxynucleosides A = CY, Ye purine compounds are inhibited by DHBV purine compounds when used at low concentrations. The mechanism of selective inhibition of the reproduction of SCA-dioxynucleosides is not known, compared to pyrimidine compounds. 707-Di-707 ABU Behind this ABU advance Leg has mentioned (BY) on Tian closides. However, then one explanation is the discovery. Another possible mechanism is that pyrimidine compounds 77-Di-Di Oxynucleosides Doxynucleosides in Sr 1 efficiently; like phosphorylated purines, they do not phosphorylate Yo

Yaa pr YA for purine compounds 77 - Dioxy Tan However, even assuming good phosphorylation (dad) liver cells compete Doxycycloside triphosphate to compete with SEY nucleosides; It contains purine and doxy-adenosine triphosphate (dGTP) to inhibit viral DNA polymerase. However, the inhibition efficiency (dATP) observed in these experiments suggests a unique mechanism for the action of purine-707 dinucleosides. Also, purine analogues (HBV) deoxynucleosides should selectively inhibit the replication of hepatitis B virus type JUTY. On the basis of a very similar replication mechanism, hepatitis virus type Uap is expected to be expressed by these antiviral agents. Silas Til (HBV) B (HBV) This therapeutic method has been shown to be effective against viruses that replicate in a similar way as 011837 such as hepatitis B virus type in humans. 0 deoxynucleoside form 1 as AE 707 Analogues may be given a bioacceptable ester or salt of this ester or bioacceptable salt. This compounds may be given

Goh Invention of usual methods of treatment including oral, anal, parenteral (intravenous, intramuscular, or subcutaneous) or inhalation aerosol inhalation | 0 It is known that purine 767 dioxynucleosides are subject to cleavage at acidic pH values such as those normally found in the stomach. Therefore, the cleavage must be able to exceed the acidic pH in the oral formulation. ' 0 Stomach. This may be achieved by using a capsule from which the drug is released after a certain time, or by adjusting the gastric pH before oral administration of Purine 767-Doxynucleosides. 0 for aqueous or non-aqueous injections. Since these compounds dissolve in ALE, they are prepared in the form of sterile solutions. Water, it is most likely that the optimal composition for intravenous injection is an equal aqueous solution

pe 19 | Osmosis, isotonic, pH neutral, contains “buffer La” ais appropriate, le prevents the proliferation of bacteria acceptable. Z It is desired to achieve drug levels in the blood plasma of 1 to 10 μg/mL. Some cases may require higher levels but it is expected that administration levels will not exceed 0 1 μg/mL in the bloodstream. And on the basis of published studies of other 707-dioxynucleosides; Lower levels in the range of 1 to 10 µg/mL can easily be reached by administering 1-9 mg/kg (milligrams of drug per kg of body weight) orally, provided variable pH limits are observed. Tability mentioned above. The basis for this 0 is the known experience of other nucleoside analogues such as YaJY-deoxythymidine (AZT)-azido-3-deoxythymidine 3 which gives peak plasma levels of 1 μg/mL after 1 infusion. mg/kg of the drug intravenously or administering 7 mg/kg of the drug by al halls Effective plasma levels for ASS inhibition of human immunodeficiency virus (HIV) are achieved by 040 immediately following drug administration Oral. vo The toxicity of purine 767-dioxynucleosides was tested in duck livers and monkey kidney cells (Vero). It was 100 µg/mL. As it is known, many lymphocyte cell lines have been examined previously to see the extent of their vitality and their functioning in the presence of SEY deoxynucleosides. These cell lines did not show a decrease in viability and function. These cell lines did not show viable Lali and only showed moderate TS of immune function even when exposed to doses of 100 μg/mL of 707-deoxynucleosides (Mitsuya et al. PNAS vol. (AY) p.186 Lid 2001-7095 (Mitsuya et al, PNAS, 82,7096-7 100, 1985).

Y. Example; Additional tests were conducted to verify efficacy in vivo. Four Pekingese ducks that proved to be chronically infected with duck hepatitis virus type B (011817) were treated with Doxyriboside and injected these animals with AE 767 purine sind JETT with the drug AE 767 mg/kg of the drug two injections per day. intramuscularly for two weeks. This 10 m amount of treatment caused a rapid purification of the sera of these animals from the virus, as shown in Figure 0 + and row “A” in Figure 0 shows the results of examining the sera of 0 animals before treatment in the form of impregnated spots. It is clear that the animals The five suffer from chronic infection with duck hepatitis virus diay. The sera of the four animals > bled dai' showed complete clearing of the virus in one week. The untreated duck (No. The sera of the four treated ducks were completely cleared of the virus. The duck (No. ©) remained strongly positive after two weeks (row C control animal (© column yo) and we verified .ddG Our results indicated that it metabolized rapidly to metabolized a3 ddDAPR (a3 ddDAPR). Our results indicated that ddG was converted to ddDAPR by using whole blood from a duck and measuring its conversion rate in a matter of minutes. ddG has been metabolized. This test indicates ddDAPR over 0 in test tubes Tas to be effective in ddDAPR These tests provide great reinforcement of the fact that the drug is both a prodrug and is likely to be a prodrug in vivo and in vivo. Biology in vitro Y. 006. Although detailed embodiments of the invention have been described in detail above, those experienced: The invention Tae with this technique will realize that it is possible to make various modifications to it without deviating from or within its scope specified in the enclosed claims. ..

CY

Claims (1)

2 Yh Elements of protection Hepatic Lal A method for medically treating an animal that needs such a method and is infected with a virus hepadnavirus tv is biologically active in conjugation with 2,3-dideoxynucleoside SEY JY from 3 2,3-doxynucleoside SEY SY and represents the Lila compound (acceptable carrier 0) mentioned by the formula: dideoxynucleoside 1 7 N p | | v oAR,CCH; 0 H H H H H H in which both 7 and 7 are: A Y X OH H 1 NH, H Y ; OH NH, 1 (C.Hs «CH; = R) OR NH, 1 NH, NH, © H NH, 1 SH NH, for (C;Hs «CH; = R) NHR NH, A (I Br «CI F) Halogen NH, 4 (C;Hs «CH; = R) SR NH, Ye oF YY 0 be 17 or 1h acyl group containing 1 to ; carbon atoms forming an ester : .. mentioned to provide a bioacceptable salt. ester is a bioacceptable salt or ester salt of the aforementioned ester 1 hepatic hapadnavirus Ball where virus (1) of the protection requirement Wy method - “7 1 | .hepatitis B virus is hepatitis B virus Y HOS Ry of claim (1) where Gp method -* 0 1 ;- method according to claim (1) where Ry is of the bioacceptable ester OCH; Ys is NH, be X where oF) for protection claim Wy method — 1 0 (OH, 7 be NH, be X where oF) method according to claim 1 -1 OH and 7 be H be X for claim (?) where Ty method 7 -1 0 CNH; Ys be NH, be X Cua (7) method according to claim -8 + 1 01 4- method Ty for claim (1); where the said effective amount in the animal's bloodstream is L is less than 10 µg/mL.-0 1 method ad, of protection claim 4) where the effective amount reported in the bloodstream of animal Y is in the range 1.1 to 1 0 µg/mL Ml. So YY Claimant method (©) where the said effective amount in the animal's bloodstream = (1 µg/mL.) 01 to 1.1 is in the range of Claimant method (1) Where the stated effective amount in the animal's bloodstream is -17 0 µg/mL. 01 to 1.1 is in the range from Y the stated effective amount in the animal's bloodstream Cua (V) for protection claim Way Method VY 1 µg/mL .01 to 1.1 is in the range of the stated effective amount in the animal's bloodstream (A) of protection claim Gig Method 1 -14 µg /ml. 01 to 11 is in the range from where the said animal is human. (VY) For protection claim Gag method —v0 > 1 Hepadnavirus is Cua ( hepatic DNA virus VY) of the protection claim Gay method 5-1 hepatitis B virus for the aforementioned hepatitis B virus hepadnavirus where the hepatic DNA virus (VY) of the protection claim la method VY Co hepatitis B virus It is the aforementioned hepatitis B virus, the Gall hepatitis B virus, and the Cua virus (VY) is required for protection. hepadnavirus The hepatic DNA virus Cua (14) is a method according to the protection requirement 4-1. hepatitis 3 virus is a hepatitis B virus. J Y ¢ wherein said composition is in the form of tablets to be administered (V1) by way of protection — Y 001 — by mouth, liquid, suppository, injectable solution, or in the form of aerosol Admin: A. Y where said composition is on Tablet form to be administered (VY) method according to Claim 71-1. aerosol for injection or in the form of oral Ju spray, liquid, suppository or Y solution where the said composition is in the form of tablets to be given (VA) For protection claim Gig method YY 1 — oral, liquid, suppository, injectable solution, or in the form of a topical spray. YY 001. JE aerosol spray, oral liquid, suppository, injectable solution, or on 7 : Where both * and 7 are as 1 (1) ;?*- Method 885) of Claim 1 Y X OH H 1 oo OH NH, Y OCH; NH, or OCH; NH, ¢ H NH, °NH, NH, 1 NHCH; NH, v NHC,H; NH, A F NH, 4 Cl NH, Yo : Br NH, 1) I NH, VY oF Yo Z NH; and 7 be NH, be X where (VE) for protection claim Gay method —Yo 1 z | (OH and 7 be NHy be X of the claim (?) where Gi, method 71-1 OCH; and 7 be NH, be X Cum (V6) method according to the claim - 77 0 OCH; and 7 be NH be th (V4) for protection request thy method -78 0 nhch; Ys is NH, X Cua (YE) is a method according to claim 4 - 1 Cl and 7 is NH, where 7 is (YE) method according to claim =F 01 AN z