JPH0686383B2 - Hepatitis B virus infection therapeutic agent containing a pyrimidine nucleoside compound as an active ingredient - Google Patents

Description

Detailed Description Background Hepatitis B virus particles (HBV), known as Dane particles, consist of outer lipoprotein coatings (hepatitis B surface antigen, HB _S Ag) and internal core (B). Hepatitis B core antigen, HBcA
G) and a 42 nm composite spherical particle consisting of This core contains circular partially double-stranded DNA and DNA polymerase. In vitro, DNA polymerase fills in the large single-stranded portion of the genome, creating a complete double-stranded portion of the genome. That is, the DNA is completely double-stranded.

However, the properties of Dane particles with respect to DNA polymerases remain uncertain. HBV D by pyrophosphate analog of intercalating agent and arabinofuranosyl nucleotide
It is already known that NA polymerase is selectively inhibited, indicating that it can inhibit the replication of hepatitis B virus particles in patients suffering from chronic hepatitis B. This invention is already known in vivo as an anti-herpesvirus agent, and Lopez et al.
It relates to the inhibition of HBV DNA polymerase by the 5-substituted-1- (2′-deoxy-2′-substituted-β-D-arabinofuranosyl) compound reported in 4,211,773.

A very similar thing to HBV is in Woodchuck, known as Woodchuck Hepatitis B Virus (WHV). Both belong to a new class of the same virus, sometimes described as Hepa-DNA viruses. As already thought, both HBV and WHV DNA polymerases have the same basic properties. WHV DNA
The polymerase activity is therefore initially in
It was studied in parallel with that of HBV in in vitro studies.

Brief Description This invention relates to a composition for treating hepatitis virus infection in vitro. More specifically, the present invention describes woodchuck hepatitis virus infection in woodchuck and hepatitis B virus infection in human as described in US Pat. No. 4,211,773, which is a 5-position substitution with antiviral activity-1- (2 '). -Deoxy-2'-position substitution-β-D
-Arabinofuranosyl) pyrimidine nucleosides are provided for compositions for treatment.

Detailed Description Preparation of Viral Particles HBV particles containing DNA polymerase activity were purified from serum of HBsAg-positive immunosuppressed patients. WHV was obtained from 5 woodchuck (Marmota manax) chronic virus carriers. The woodchuck was originally transferred from Pennsylvania (Dutchland Lad. Pennsylvania, USA), but belonged to a group that had been serologically tested for over a year. (Hantz et al, J. Virol. Metho. 7, 45-55 (19
83)].

The virus was purified by centrifugation through a density gradient of sucrose and CsCl of the same density. (Posted by Hantz et al.).

Reagents Triphosphorus oxide was used for in vitro studies. This is necessary to expect the metabolic processes of the cell.

Unlabeled nucleoside triphosphate was obtained from Sigma (reagent company, St. Louis, USA). Tritiated deoxyribonucleoside triphosphate [ ³ H] dTTP (30 Ci / mmo
l), [ ³ H] dATP (24 Ci / mmol), [ ³ H] dCTP (17 Ci / mmo
l), [ ³ H] dGTP (9.5 Ci / mmol) is Amersham (Amershal
France). ACV is Burroughs Wellcome, Chape
Obtained from l Hill NC. DRVLERCO et al.
(Proc. Natl. Acad. Sci. USA, 76, 2947-2951 (197
9)]. FIAC synthesis is described in US Patent No. 4,211,773 to Lopez et al. All three nucleoside analogues are allaudeen et al [Proc. Natl. Aca
d. Sci. USA, 78, 2698-2702 (1981)], and converted into the corresponding 5'-triphosphates. The purity of triphosphorus oxide is Bruker HX-27
¹ H NMR on a 0 spectrometer and high performance liquid chromatography on an Altex Model 332 gradient liquid chromatograph.

Analysis of DNA polymerase activity DNA polymerase was analyzed by Kaplan et al [J. Virol, 12,995 (1
973)] was slightly modified and analyzed. The measurement is 50m
M Tris-HCl (pH 7.6), 40 mM MgCl ₂ , 60 mM NH ₄ Cl,
Reaction solution containing 100 μM of dATP, dCTP, and dGTP, 0.2 to 0.7 μM of [ ³ H] dTTP (30 Ci / mmol), 10 mM of 2-mercaptoethanol, 0.5% Nonicet P 40, and enzyme.
It was performed in 50 μl. The reaction was started by adding virus particles containing 2-mercaptoethanol and Nonidet P40. The reaction was carried out at 37 ° C for 1 hour. Acid-insoluble radioactive material is filtered through a glass fiber filter (GF / C
Whatman). Wash the filter 5 times with cold 5% trichloroacetic acid containing 10 mM sodium pyrophosphate, then 95%
Washed with ethanol. The radioactivity of the dried filters was measured with a liquid scintillation counter.

Agarose gel electrophoresis of [ ³² P] DNA [ ³² P] DN generated by an endogenous DNA polymerase reaction
A product is Summers et al [Proc.Natl.Acad.Sci.USA 7
2, 4597-4601 (1975)] and analyzed by agarose gel electrophoresis. Standard D containing 1 μM [ ³² P] dCTP (500 Ci / mmol) as radiolabeled nucleotide
The reaction was performed with 15 μl of NA polymerase mixture.

After incubating at 37 ℃ for 2 hours, 10mM Tris, 10
mM EDTA, 0.1 mg / ml proteinase K, 0.1% (wt / vo
The reaction was stopped by adding 20 μl of l) sodium dodecyl sulfate and incubated for another 60 minutes at 37 ° C. 5%
(Wt / vol) 5% sucrose, 1% bromphenol blue
Add μl and heat the whole reaction mixture at 65 ° C for 5 minutes and immediately
C. and then electrophoresed on a 1% agarose horizontal slab gel. After electrophoresis the gel was dried and detected by autoradiography.

Discussion HBV and WHV DNA polymerase reaction products. The specificity of the HBV and WHV particle preparation DNA polymerase was regulated by efficiency by analyzing the labeled DNA produced during the endogenous reaction. The measurement of DNA polymerase was performed by a method using [ ³² P] dCTP as the wobbled nucleotide. The reaction products were analyzed by agarose gel electrophoresis and autoradiography. The results in FIG. 1 show the time-dependent synthesis of HBV and WHV DNA after 15, 30, 120 and 180 minutes of reaction. For HBV the full 3.3kb length
A smaller form (from 1.8 kb) corresponding to an unfilled single-stranded portion of the DNA and viral genome
2.8 kb) was obtained. The product of the WHV DNA polymerase reaction was more heterogeneous and ran a little faster. After reacting for 180 minutes, the long WHV DNA had a length of 3.1 kb.

Relative sensitivities of HBV and WHV DNA polymerases to ACVTP, FIACTP and BVdUTP The relative sensitivities of the two enzymes to CVTP, FIACTP and BVdUTP were compared using excess nucleotides. The concentration of the corresponding radioactive triphosphate was maintained at 2-3 times the Km value, while the remaining nucleotides were sufficiently in excess (50 μM) for each inhibitor. In Fig. 2, FIACTP is HB
The most potent inhibitor of V DNA polymerase,
It shows that ara-CTP is the least effective. BVdUTP
And ara-TTP showed similar inhibition.

We obtained similar results with WHV DNA polymerase. HB
The concentrations of triphosphate required to inhibit V and WHV DNA polymerases by 50% are shown in Table 1. FIACTP
The ID ₅₀ of BVdUTP and ACVTP were very low (0.05 μM) compared with those of 5 and 18 times, respectively.

Kinetics of inhibition ACVTP, FIACTP, and BVdUTP inhibit the viral DNA synthesis catalyzed by HBV and WHV DNA polymerases, although the action mode for various compounds is known to be different by increasing the substrate concentration. It was investigated by measuring the extent of inhibition. Analysis with ACVTP revealed that [ ³ H] dVTP was the rate-limiting substrate, while the other three nucleotides were in excess. The same conditions were used for [ ³ H] -dCTP for inhibition of FIACTP and [ ³ H] -dTTP for inhibition of BVdUTP.
The Lineweaver-Burk plot shows that the inhibition of HBV DNA polymerase by ACVTP, FIACTP and BVdUTP antagonized with dVTP, dCTP and dTTP, respectively (Fig. 3). Similar results were obtained with WHV DNA polymerase. Km of dGTP, dCTP, dTTP for DNA polymerases of both viruses
Values and ACVTP and BVdUTP Ki values in Table 2A (HBV) and Table 2B (WH
V). It was shown that WHV DNA polymerase had a lower affinity for ACVTP, FIACTP and BVdUTP than HBV DNA polymerase. However, the Km-Ki ratio was similar for the two enzymes.

Effect of BVdUTP, FIACTP, and ACVTP on in vitro DNA synthesis To measure the effect of three analogs on viral DNA synthesis, a time course experiment was carried out using HBV DNA polymerase. [ ³ H] -dATP as radiolabeled substrate
Was used. For all four nucleoside triphosphate substrates, the reaction increased linearly up to 60 minutes (Figure 4). If dTTP, dCTP and dGTP are removed from the reaction mixture, DN
Almost no A synthesis occurred. When dTTP was replaced with BVdUTP, the viral DNA synthesis was 4% after the reaction for 3 hours.
It progressed to 6% (Fig. 4A). This value increased to 67% of the optimum activity after 4 hours of reaction (Table 3). However, in the same experiment, when dTTP was replaced with ara-TTP, no DNA synthesis was observed (Fig. 4A).

When doing the same experiment with FIACTP instead of dCTP [ ³ H] dAMP
Uptake of ara-C was obtained instead of dCTP, although only a small amount of uptake was obtained (about 10% of the control after 3 hours of reaction).
Almost no uptake was observed when TP was used (Fig. 4B). Replacing dGTP with ACVTP did not increase the polymerization process. HBV and WHV DNA
The effects of these various analogs on surrogate substrates for polymerases are summarized in Table 3. BVdUTP was the most effective surrogate substrate for both enzymes. DNA synthesized using BVdUTP as a surrogate substrate was analyzed by agarose gel electrophoresis (Fig. 5). BVdUTP only yielded incompletely filled single-stranded regions of HBV or WHV DNA.

The effect of ACVTP on HBV DNA synthesis was also studied in a time course experiment and the results are shown in Figure 6. [ ³ H] dTTP
Was the labeled substrate. Reaction is without dGTP or dGTP
Was started with 5 μM ACVTP instead. Uptake of [ ³ H] dTMP could not be measured under these conditions. reaction
After 120 minutes 100 μM dGTP was added. In the system lacking only dGTP, DNA synthesis increased as expected with the addition of dGTP. However, when first tested with 5 μM ACVTP instead of dGTP, no DNA synthesis was seen even after the addition of 100 μM dGTP. AC in the same proportion as reaction in comparison
Starting with VTP (5 μM) and dGTP (100 μM), considerable DNA synthesis was observed (Fig. 6A). Ara similar experiment
-ATP (Fig. 6B) and ara-CTP (Fig. 6C). In both cases, addition of normal substrates (dATP and dCTP) in the presence of analogs after 120 minutes of reaction increased HBV DNA synthesis. Thus AC instead of the natural triphosphate substrate
Upon addition of VTP, dGTP appears to suppress further DNA elongation without enhancing DNA synthesis.

Observations Nucleoside analogs that exhibit antiviral activity must be converted into their triphosphate derivatives in cells in order to inhibit viral DNA synthesis. It has been previously reported that CVTP, BVdUTP and FIACTP selectively inhibit the herpes simplex virus DNA polymerase, which may partially explain the selective antiviral activity of the nucleoside analogs ACV, BVdUTP and FIAC. In vitro as described above
Evidence in shows that all three compounds inhibit HBV and WHV DNA polymerases, and that inhibition antagonizes the corresponding natural triphosphate substrates. ACVTP,
The respective inhibitory activities of BVdUTP, FIACTP and two other nucleotide analogues, ara-CTP and ara-TTP, were compared. The inhibitory activities of HBV and WHV against DNA polymerase decrease in the following order: FIACTP, BVdUTP, ara-TTP, ACVTP, ara
-CTP.

In vitro inhibition of HBV replication by ACV is associated with chronic B
It has been shown in patients with hepatitis C [Weller et al. Lancet,
1,273 (1982); Weller et al, J. Antimicrob. Chemothe
r.11, 223-231 (1983)]. Whether the in vivo effect of ACV is associated with the in vitro inhibitory effect of ACVTP on HBV DNA polymerase is unconfirmed. This is because the role of endogenous DNA polymerases in the recovery of HBV in infected cells remains unknown. Moreover, a key step in the action of anti-herpesviruses such as AOV, FIAC, and BVdU is in cells infected with herpesviruses encoding thymidine kinase, where these analogs selectively phosphorylate. is there. However, HBV and WHV have not been reported to have viral thymidine kinase activity. Thus this in vitro study tested these compounds in v
It is not directly related to the possibilities of using ivo. Also, because the action patterns of individual compounds are different,
It is unpredictable that in vivo activity may be related to other in vivo activities.

However, since there is no cell culture system for HBV growth,
Studies on the inhibition of the endogenous DNA polymerase reaction of BV have left an efficient search to identify potential anti-HBV agents.

Based on this data, pyrimidine nucleoside compounds effective for FIAC and related anti-herpesviruses (5-position substitution-1
-(2'-deoxy-2 'substituted-β-D-arabinofuranosyl) pyrimidine nucleoside compounds) will be used to treat hepatitis B virus infection in humans and woodchuck hepatitis virus infection in woodchuck . FIACs and related compounds are selected for their blistering activity compared to other drugs tested in vitro. The activity of these compounds was reported in vitro as reported above.
It would be significantly superior to the activity of other groups of compounds treated in vitro.

In Vivo Testing Testing is done on humans and woodchucks. Compounds to be tested step on the following pyrimidine nucleotide compounds; FIAC
(2'-fluoro-2'-deoxy-arabinosyl-5
-Iodocytosine), FIAU (2'-fluoro-2'-deoxy-arabinosyl-5-iodouracil), FMAU
(2'-fluoro-2'-deoxy-arabinosyl-5
-Methyluracil or 2'-fluoro-2'-deoxy-arabinosyl-5-thymine).

These compounds are soluble in water and therefore aqueous or water-based solutions are the solvent of choice for treating patients. The solubilities are as follows.

The solubility of these compounds is increased by adding acid or alkali to the aqueous solution. Thus, the solubility of FIAC can be increased to about 250 mg / ml by adding about 1 molar equivalent of hydrochloric acid, although some hydrochloric acid will act. FIAU can be multiplied many times by adding 1 equivalent of sodium hydroxide to form the sodium salt of FIAU.

The doses for treating humans are as follows:

FIAC up to 400mg / m ² per day, preferably 120mg / day
m ² is used as needed for each treatment for up to 7 days. Approximately half the desired dose is the lowest practical dose. Below this amount the effect of the drug is too small. Thus, about 60 mg / m ² is the lowest practical dose for FIAC.

FIAU is used in the same dose range as FIAC. It will be appreciated that FIAU is a metabolite of FIAC and is expected to be useful in the same dose range.

FMAU up to 32mg / m ² daily, preferably 16mg / m ^{2 as} needed
m ² (to avoid side effects), and most preferably about a day
4 mg / m ^{2 is used} for each treatment for up to 7 days. About half the most desirable dose, or about 2 mg / m ^2, is the minimum practical dose.

Doses above these maximum doses have undesirable side effects.

Similar treatments are performed in woodchucks to inhibit replication of hepatitis B type viruses as follows.
The amount used is in the same range as used in humans 1
Measured in mg / m ² per day.

The description and examples are illustrative and not limiting of the invention, and other embodiments within the spirit and scope of the invention are suggested to those skilled in the art. It will be understood that.

[Brief description of drawings]

FIG. 1 is an agarose gel electrophoresis diagram of [ ³² P] DNA synthesis, and FIG. 2 is a graph showing the inhibitory activity of nucleotide analogues on HBV DNA polymerase. Figure 3 shows HB
It is a graph which shows the inhibition rate of V DNA polymerase. Figure 4 shows BVdUT as a surrogate substrate for HBV DNA polymerase.
It is a graph which shows the test result of P, ara-TTP, FIACTP, and ara-CTP. FIG. 5 is an agarose gel electrophoresis diagram of DNA synthesized by BVdUTP. FIG. 6 is a graph showing the effect of ACVTP on HBV DNA synthesis.

Front Page Continuation (72) Inventor Carlos Lopez, United States, 10021 New York, New York, East Sixty Nine Street 333 (72) Inventor Kristian Jie. Trepo France 69500 Bron, Berri-Uesalis 22 No. 22 (56) References JP-A-55-49335 (JP, A)

Claims (4)

[Claims] 1. A formula Where A is OR ³ , SR ³ , NR ³ R ⁴ or NH acyl and R ³ and R ⁴ are the same or different and are hydrogen, lower alkyl having 1 to 7 carbon atoms, aralkyl or aryl; NH acyl is alkanoyl or aroylamide B is oxygen or sulfur; X is halogen, alkylsulfonyl or arylsulfonyl; Y is halogen, amino, monoalkyl- or monoaralkylamino, dialkylamino, aminomethyl, hydroxymethyl, lower alkyl, aryl, aralkyl,
Vinyl, substituted vinyl, ethynyl or substituted ethynyl; Z is methine or nitrogen; R ¹ and R ² are the same or different and are hydrogen, acyl or aroyl. A therapeutic agent for hepatitis B virus infection, which comprises an effective amount of a compound having: 2. A compound represented by the formula is 2'-fluoro-
The drug according to claim 1, which is 2'-deoxy-arabinosyl-5-iodocytosine. 3. A compound of formula 2'-fluoro-
The drug according to claim 1, which is 2'-deoxy-arabinosyl-5-iodouracil. 4. A compound represented by the formula is 2'-fluoro-
The drug according to claim 1, which is 2'-deoxy-arabinosyl-5-methyluracil.