JPH0940580A - Anti-hiv agent ifn-omega - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a medicine compound capable of effectively suppressing replication of HIV and treating a HIV infectious disease, containing omega interferon (IFN-ω). SOLUTION: This agent contains an effective amount of IFN-ω. A dose and a dose ratio are preferably 1×10<6> to 10×10<6> unit three times daily or a week. IFN-ω, typically, can be lyophilized and made into a pharmaceutical preparation filled in a vial. In this case, a human serum albumin is preferably added as a stabilizer. IFN-ω is parenterally administered and further another active component having anti-retrovirus activity such as a nucleic acid derivative and a non-nucleic acid reverse transcriptase inhibitor may be orally administered.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD In mammals, the type I interferon gene is an alpha-interferon (I
FN-α), beta-interferon (IFN-β)
And omega-interferon (IFN-ω) form a superfamily that includes a family of three genes. In humans, there are only monofunctional genes encoding IFN-β and IFN-ω, whereas the IFN-α family contains 13 functional genes. All of the interferons mentioned above have antiviral and antiproliferative effects, but IFN-β and IFN-α
Antiviral activity in ivo is limited. further,
Interestingly, no significant qualitative difference was reported between IFN-α and IFN-ω when interferon activity was measured by inhibition of the replication of vesicular stomatitis virus or encephalomyocardial virus. ing. (Adol
f et al. J. Biol. Chem. 265, 9290-9295; 1990). Interferon-mediated suppression of HIV expression is thought to be due to the interaction of a complex between the protein encoded by the interferon-induced cellular gene and the viral determinant. In acute in vitro infections, the main antiviral effect of IFN is the proviral D
In chronically infected cells, IFN appears to suppress viral aggregation, whereas it occurs in the pre-formation stage of NA synthesis. In vivo, elevated serum levels of IFN-α appear to contribute to the inhibition of HIV replication in the early stages, but in the later stages of infection, the acquisition rate of IFN-α resistance contributes to the progression of AIDS. It will increase greatly with it. Recent evidence suggests that HIV-specific Tat and Rev proteins may play a role in evading the antiviral effects of IFN. (Shirazi et al. J. Interferon
Res. 14, 259-263; 1994). This result indicates that Tat protein is an enzyme that plays an important role in the antiviral action of IFN, dsRNA-dependent protein kinase (PK).
It may be related to the finding that it is possible to suppress Tar-mediated induction of R). (Hovanessian,
J. Interferon Res. 9, 644-647; 1989).

Surprisingly, IFN-ω is an active suppressor of HIV replication, and both the laboratory and primary isolates of HIV-1 have been shown to have different levels of I than other isolates when used at the same dose.
More effectively than IFN-type interferon
Suppressed by −ω. Presumably, IFN-ω is capable of suppressing the replication of IFN-α2 resistant isolates by inducing more sustained expression of various IFN-stimulating genes. The gene encoding IFN-ω has already been isolated and sequenced, E. coli (see E. coli, EP-B-0,170,204), yeast (EP-A-0,264,
790), insect cells (Voss et al. Eur. J. Biochem. 217,
913-919; 1993) and Chinese hamster ovary cells (Adolf et al. Biochem. Biophys. Acta 1089, 167-17).
4; 1991). They are described in glycosylated and non-glycosylated form. Accordingly, the present invention provides a novel method of treating HIV-1 infections, comprising administering an effective amount of glycosylated and unglycosylated IFN-ω and containing an effective amount of glycosylated and unglycosylated IFN-ω. To a corresponding pharmaceutical composition manufacturing method.

[0003]

BEST MODE FOR CARRYING OUT THE INVENTION The activity of IFN-ω to suppress HIV-1 replication was tested as follows. 2 × 10 ⁶ peripheral blood mononuclear cells (PBMCs) were treated with 8 of virus challenge.
5 to 500 units of IFN-ω per ml before time
Processed in. Duplicate culture
Together with the T cell trophic HIV-1 isolate NL4-3 and / or its macrophage variant AD81.
Inoculated at a reverse transcriptase (RT) activity of ³ cpm, 12
Maintained in the continuous presence of IFN-ω for days. HIV-1 replication was monitored in culture supernatants by RT assay at various times post infection. The average of duplicate RT activity measurements in the presence and absence of IFN-ω was plotted against time. IFN-ω was also found to suppress the replication of T cell trophic HIV-1 laboratory isolate NL4-3 and its macrophage trophic variant AD81 at all concentrations (FIGS. 1A and 1B). . Suppression was dose-dependent, but the decrease in supernatant RT activity was not linearly proportional to the concentration of IFN.
Cell viability was greater than 90% throughout the infection. I
Comparison with FN-ω and IFN-α2 was performed as follows. Three IFN-α2-sensitive strains (Fig. 2A, Fig. 2B,
FIG. 2C) and three IFN-α2-insensitive strains (FIG. 3).
A, FIG. 3B, FIG. 3C) of the HIV-1 primary isolate IF
Tests for sensitivity to N-α2 and IFN-ω were performed at the same dose (100 U / ml). IFN-ω or IFN-
Mean RT activity of duplicate cultures in the presence or absence of α2 was plotted against time and the percent inhibition (% I) was calculated from the difference in the areas under the curves obtained. The IFN-α2-sensitive primary isolate was IFN-ω.
Inhibition of viral replication at 100 U / ml is more sensitive to IFN- than to IFN-α2.
It has been found that ω is larger. Similar results were obtained when using the glycosylated form of IFN-ω against isolates NL4-3 and AD81. Furthermore, IFN-ω
Suppresses the replication of 3 isolates resistant to IFN-α2 at the same dose (less than 20% suppression)
It was possible. These findings are summarized in Table 1 below.

[0004]

[Table 1] Table 1 Suppression rate Phenotype HIV-1 isolate IFN-α2 IFN-ω IFN-α2 IFN-ω NL4-3 67 86 S S AD81 78 93 S S PI8659 70 85 S S PI8377 48 72 S S PI8637 75 89 S S PI7633 9 43 R S PI7700 17 39 R S PI7602 20 51 R S S = IFN sensitivity (> 20% inhibition) R = IFN resistance (<20% inhibition)

The suppression mechanism was investigated as follows. H
In order to further investigate the mechanism of suppression of IV-1 replication, IF in the protein synthesis of unicycle ring virus
The effect of N-ω was examined and compared to that observed in IFN-α2. PBMCs were pretreated with IFN and treated with 1.5 × 10 ³ cpm / ml of HIV-in the presence or absence of 100 U / ml of IFN-ω or IFN-α2.
1, challenged with NL4-3. HIV-1 protein levels were measured 96 hours post infection. Western blot analysis detected high levels of p55 and p24 in the absence of IFN, and also traces of p160. In the presence of IFN-α2, the levels of these viral proteins are significantly lower, but IFN-ω
Was barely detectable in the presence of the same dose (Fig. 4).

Next, the expression of the IFN-inducible gene will be described. The antiviral activity of IFN is mediated by a number of cellular genes triggered by the binding of IFN to its receptor. Therefore, HI by IFN-ω and IFN-α2
It was examined whether the difference in the inhibition rate of V-1 replication corresponds to the difference in the expression of IFN-induced cellular genes. PB
MCs were treated with 100 U / ml of IFN-ω or IFN-α2, total cellular RNA was separated 2, 4 and 8 hours after the treatment, and the induction kinetics of IFN-stimulating gene was analyzed by Northern blotting. (Fig. 5). 2 ', 5'-oligoadenylate synthase (2', 5'-OAS) mRNA
Was detectable in cells treated with IFN-ω and IFN-α2 for up to 2 hours. However, the transcription level of 2 ', 5'-OAS reached a maximum at 4 hours after treatment with IFN-α2 and decreased by 8 hours. On the other hand, in the cells treated with IFN-ω, it became constant after the peak.
Similarly, transcription levels from the IRF-1 and 9-27 genes were constant during induction in cells treated with IFN-ω, whereas IFN-α2 treated cells decreased by 8 hours. But the most obvious qualitative difference is the ISG
It was found with the induction of the gene encoding -15. Expression of this gene follows the same pattern as the gene encoding 2 ', 5'-OAS in IFN-α2-treated cells, but IFN-ω shows high levels of ISG-15mR.
NA was induced and these levels were constant during induction. Thus, upon viral challenge 8 hours after contact with IFN, 2 ', 5'-oligoadenylate synthase, IRF-1, 9-27 and ISG-15 mRNA.
Is more than IFN-α2 treated cells.
Higher levels were found in cells treated with N-ω.

The following interferons, cells, virus stocks and general methods were used. IFN-ω and I
The antiviral activity of FN-α2 was measured by MTT (Cheung et al. J.
Immunol. 146, 121-127; 1991) and tested in a cytopathic assay (Hansen et al. J. Immunol. Method).
s 119, 203-210; 1989). EBTr (NBL-
4) Bovine tracheal cells were used as indicator cells. In this assay, activity was normalized using IFN-α2 as an indicator. Peripheral blood mononuclear cells (PBMCs) were collected from standard hemapheresed blood donors according to the manufacturer's instructions, and Ficoll (Organon Teknica Cap
pel, Durham, NC) and 5 mg / ml phytohemagglutinin (DIFCO Laboratories, Detroit, MI)
Stimulation for 48 hours with 15% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 50 mg / ml gentamicin and 10 units / ml interleukin 2
(GIBCO BRL) in RPMI-1640 medium (GIBCO BRL)
BRL, Grand Island, NY). CEMx174
Cells were maintained in RPMI-1640 medium, 2 mM L-glutamine and 50 mg / ml gentamicin.

HIV-1NL4-3 (Adachi et al. J. Vio
rl. 59, 284-291 (1986)) and the macrophage trophic variant AD81 (Freed et al. Nature 369,
107-108; 1994) in Jurkat cells and the respective infected clone molecules (pNL4-3 and pAD81).
Was prepared by the electroporation method. HIV primary isolate is HI
Derived from a V seropositive donor. The virus stock was then added to PHA-stimulated uninfected standard donor PB.
Supernatants were harvested unconcentrated from short-term co-cultures of PBMCs infected with MCs (Kunzi et al. J. Inf. Diseas.
es 171, 822-828; 1995). Quantitation of all stocks was determined by reverse transcriptase (RT) assay, cpm / ml
It is represented by. To determine susceptibility to IFN, cells were treated 8 hours before viral challenge with 100 units / ml recombinant IFN-α2 or recombinant IFN.
-Maintained within ω. 2 × in the presence or absence of IFN
10 ⁶ cells were challenged with a virus inoculum of RT activity of 1.5 × 10 ³ cpm / ml. The infection is duplicated,
Culture supernatants were assayed for RT activity 4, 8 and 12 days after inoculation.

The reverse transcriptase (RT) assay was performed as follows. Reverse transcriptase activity with virions was measured essentially as described above (Shirazi et al. J. Vior
l. 66, 1321-1328; 1992). Briefly, 10
μl of the culture supernatant was added to 50 mM TRIS (pH 7.
8), 7.5 mM KCl, 5 mM MgCl ₂ , 2 mM dithiothreitol (DTT), 0.05% Nonidet
t) P-40 (Sigma, St. Louis, MO) and 0.5 μCi [α ³² P]
In TTP (3000 Ci / mmol) (ICN, Irvine, CA), po
ly (A) and oligo (dT) (1.57 μg / ml) (Pharmacia, Piscata
wy, NJ) and mixed with 50 μl of RT reaction buffer containing template primer. The reaction solution was incubated at 37 ° C. for 2 hours. The reaction product was loaded onto DE81 paper using a dot-blot apparatus (Whatman International Ltd., Maidstone, Englan.
Blotted in d). Test paper with 20 x SSC (0.3 M Na
Cl, 0.63 M sodium citrate) and washed twice, 95
Washed once with% ethanol. After drying, the radioactivity was measured with a scintillation counter. To confirm the accuracy and reproducibility of the assay, a virus stock with a known titer (NL4-3) was used as a positive control. Mean values of RT activity in the presence or absence of IFN are plotted against time, inhibition rate (% I)
Is expressed as the difference in area under the resulting curves. (Kunzi et al. J. Inf. Diseases 171, 822-828;
1995).

Western blot analysis was performed as follows. Cells are washed with phosphate buffered saline and 50 mM T
ris, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Nonidet P-40, 0.
5% sodium deoxycholate and 2 mM phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO)
Was dissolved. 100 mg of cellular protein was added to SDS-
Polyacrylamide (Bio-Rad Laboratories, Richmon
d, CA) Separation by electrophoresis and transfer by electroblotting onto nitrocellulose filters (Millipore, Bedford, MA). The filters were then incubated with sera from HIV seropositive donors and the HIV-specific antigen-antibody complex labeled with ¹²⁵ I protein A (New England).
Nuclear, Boston, MA). New anti-HIV
In its -1 action, IFN-ω is effective alone or in combination with other compounds having antiretroviral activity in treating infections by HIV-1. Other compounds having antiretroviral activity include nucleic acid derivatives (eg, zidovudine,
1000-1200mg / day), DDI (200-750mg / day), DD
C), a non-nucleic acid reverse transcriptase inhibitor (for example, nevirapine (100-1000 mg / day)), or a protease inhibitor (for example, Ro34-8959 (200-2000 mg / day)).

Therefore, another object of the present invention is IFN-ω.
Of the active ingredient and the active ingredient active against HIV, which may be administered separately. IFN-ω may be administered parenterally to a subject in need of treatment. The dose and rate is about 1 × 10 ⁶ -10 × 10 ⁶ units daily or three times a week,
At this time, an effective amount of the active ingredient for HIV may be orally administered. A typical pharmaceutical formulation of IFN-ω is
Lyophilized and placed in vials. The dosage form includes
It contains human serum albumin as a stabilizer. The composition of the reconstituted solution is shown as an example. IFN-ω 16.5 mcg (≡ 6.6 × 10 ⁶ IU) Human albumin 5.00 mg / ml Potassium chloride 0.20 mg / ml Potassium dihydrogen phosphate 0.20 mg / ml Sodium chloride 8.00 mg / ml Disodium hydrogen phosphate dodecahydrate 2.89 mg / ml Water for injection 1.00 ml

The IFN-ω of the invention can be formulated according to known methods of making useful pharmaceutical compositions, wherein the polypeptide is combined with a mixture with a pharmaceutically acceptable carrier vehicle. For suitable vehicles and morphologies, see EW Martin, Remington's Pharmaceut.
ical Science and reference examples are clearly described. IFN-ω is used to prepare a pharmaceutically acceptable composition suitable for effective administration to a recipient (patient),
It is mixed with a suitable amount of vehicle. The preferred method of administration is parenteral administration.

[Brief description of drawings]

FIG. 1. IFN-ω-mediated suppression of HIV-1 replication. PHA-stimulated PBMCs were untreated or treated with 5, 50 or 500 U / mL IFN-ω for 8 hours. 1 × 10 ⁶ cells / mL of duplicate culture was added to the RT
It was challenged with HIV-1 NL4-3 having an activity of 1.5 × 10 ³ cpm / mL. Culture supernatants were assayed for RT activity every 4 days, cells were split 1: 2 each time and cultured in fresh medium in the presence or absence of IFN-ω.
Mean RT activity is expressed in cpm / mL and plotted against time. A: T cell trophic HIV-1 NL4-3 laboratory isolate. B: Macrophage trophic mutant of NL4-3, AD81.

FIG. 2: Comparison of IFN-ω and IFN-α2 mediated suppression against HIV-1 primary isolates. PHA-stimulated PBMCs are untreated or 100 U / mL I
It was treated with FN-ω for 8 hours. The HIV-1 primary isolate was infected, and the RT activity of the supernatant was assayed and plotted in the same manner as in FIG. A: IFN-α2 sensitive primary isolate 8659. B: IFN-α2 sensitive primary isolate 8377. C: IFN-α2 sensitive primary isolate 8637.

FIG. 3. Replication of IFN-α2-resistant HIV-1 primary isolates is suppressed by IFN-ω. PBMCs were stimulated as described in Figure 1 and challenged with IFN-α2-resistant HIV-1 primary isolates. Viral replication was assayed and recorded as in FIG. A: IFN-α2 resistant primary isolate 7533. B: IFN-α2 resistant primary isolate 7700. C: IFN-α2 resistant primary isolate 7602.

FIG. 4: IFN-for levels of viral proteins
The effect of ω. 5 × 10 ⁶ PHA-stimulated PBMCs were untreated (lane a) or 100 before challenge with HIV-1 NL4-3 with RT activity (+) of 1.5 × 10 ³ cpm / mL. U / mL IFN-α2 (A) (lane b) or IFN-ω (W) (lane c) was treated for 8 hours. Proteins were isolated 96 hours post infection and HIV specific protein expression was detected by Western blot analysis with sera from HIV seropositive donors.

FIG. 5: Northern blot analysis of the induction kinetics of IFN-stimulated genes in cells treated with IFN-α2 or IFN-ω. HIV with RT activity (+) of 1.5 × 10 ³ cpm / mL
Prior to challenge with 1NL4-3, 1 × 10 ⁷ uninfected PHA-stimulated PBMCs were treated with 100 U / mL IF.
It was treated with N-α2 (A42) or IFN-ω (W).
Total cellular RNA is separated at given intervals after IFN treatment,
5 μg of RNA was added to 2 ′, 5′-OAS, IRF-1,9-
27, ISG-15 and actin-specific probes were used for Northern hybridization (Nothern hybridizatio
n) was analyzed.

 ─────────────────────────────────────────────────── ———————————————————————————————— Inventor Paula Em Pisa Row, Maryland, USA 21210 Baltimore Huntley Square 5503

Claims (9)

[Claims] 1. HIV containing an effective amount of IFN-ω
A pharmaceutical composition for treating an infection. 2. The pharmaceutical composition according to claim 1, which suppresses HIV replication by administering an effective amount of IFN-ω. 3. The pharmaceutical composition according to claim 1, which suppresses HIV replication by sustained expression of an IFN-inducible gene. 4. The pharmaceutical composition according to claim 1, which further comprises administering an effective amount of a component more active against HIV. 5. A method of treating HIV infection, which comprises administering an effective amount of IFN-ω. 6. An effective amount of IFN-ω is administered and HIV is administered.
6. The method for treating HIV infection according to claim 5, which comprises suppressing the replication of the. 7. The method according to claim 5, wherein HIV replication is suppressed by sustained expression of an IFN-inducible gene. 8. Use of an effective amount of IFN-ω for the manufacture of the pharmaceutical composition according to any of claims 1-4. 9. The method for producing a pharmaceutical composition according to claim 1, wherein an effective amount of IFN-ω is contained in a suitable carrier.