WO1995000170A1

WO1995000170A1 - Inhibition of human immunodeficiency virus reproduction by deoxyribonuclease i

Info

Publication number: WO1995000170A1
Application number: PCT/US1994/007115
Authority: WO
Inventors: Rudolf I. Salganik
Original assignee: Rufeld, Inc.
Priority date: 1993-06-24
Filing date: 1994-06-23
Publication date: 1995-01-05
Also published as: AU7212694A

Abstract

The present invention is related to the development of means for inhibiting human immunodeficiency virus (HIV) reproduction in HIV-infected cells. Application of pancreatic deoxyribonuclease I (DNase I) results in a sharp decrease of the HIV-specific total antigens, p24 antigen and HIV reverse transcriptase activity in HIV-infected MT-4 and in an increase of the cell viability. The optimal dose of DNase I for inhibition of HIV in the MT-4 cells is 150-1500 Kunitz units per ml of the cell medium. Although HIV is an RNA-containing virus, pancreatic ribonuclease A (RNase A), contrary to expectation, has no effect on the HIV reproduction and even interferes with the DNase I HIV-inhibiting effect.

Description

INHIBITION OF HUMAN IMMUNODEFICIENCY VIRUS REPRODUCTION BY DEOXYRIBONUCLEASE I

BACKGROUND OF THE INVENTION

1. FIELD OF THE INVENTION:

This invention relates to agents and methods capable of inhibiting reproduction of the human immunodeficiency virus (HIV).

Numerous synthetic and natural compounds were assayed for their anti-HIV activity in a search of means for treatment of the acquired immune deficiency syndrome (AIDS) caused by HIV. The level of anti-HIV activity was established for azidothymidine (AZT) and its analogs, aurintricarboxyhc acid, ribovirin, pyridinone. The most effective anti-HIV agents belong to the nucleoside derivatives such as AZT, ddl and other dideoxynucleosides which terminate the nucleic acids synthesis catalyzed by the viral reverse transcriptase (RT). That is the background of apphcation of AZT for treatment of AIDS. However, AZT's effectiveness in inWbiting HIV reproduction in chronically infected macrophages/monocytes is poor. Under AZT apphcation, HIV mutants resistant to this compound of AZT arise in a short time. This is the reason AZT has only a transitory therapeutic effect. In addition, AZT and its derivatives have harmful side effects. For this reason, large-scale research to develop other anti-HIV agents continues.

2. ART BACKGROUND:

It was shown previously by R.I. Salganik et al. that pancreatic deoxyribonuclease I (DNase I) efficiently inhibits reproduction of DNA- containing viruses (DNA viruses) such as vaccinia, adenovirus, and herpes, by selectively suppressing the viral DNA synthesis. See, e.g., Trukharev and Salganik, Virology 33:552-555 (1967); Salganik et al., in Inhibitory virusnoy aktivnosti (Inhibitors of the viral activity) Riga, USSR, Sanatne, 1972, pp. 147-152 (in Russian), Salganik, Current Trends in Life Sciences, 12:115-123 (1984). DNase I is devoid of cytopathogenic, antimitotic or mutagenic effects when added to nutritive media of cell cultures or administered parenterally in high doses to animals. See e.g., Lapik et al. Pharmakologiya i Toxikologiya (Pharmacology and Toxicology), 2:210-214 (1970) (in Russian). Additionally, no embryotoxic, teratogenic or other toxic effects were observed with these enzymes in animals. Clinical trials have demonstrated high therapeutic efficiency of DNase I in treatment of herpes zoster, herpes simplex, herpes keratltis, infectious mononucleosis, adenoviral conjuctivitis and nasopharingitis caused by DNA viruses. See e.g., Colain et al., Ann. Oculist., 203:371-378, (1970); Gutorov et al., Zhurn. Nevropathologii i Psildiiatirii (J. Neurology and Psychiatry)., 11:75-78 (1976) (in Russian); Krivosheev et al., Vestnik Dermatologii i venerologii (Bulletin of Dermatology and Venereal Diseases) No. 1:29-35 (1987) (in Russian).

Numerous experiments performed by Salganik et al. and others have also shown that pancreatic ribonuclease A (RNase A), which splits RNA molecules, inhibits reproduction of a number of RNA- containing viruses (RNA viruses), such as influenza virus, tick-borne encephalitis virus, foot-and-mouth diseases virus. See, e.g., LeClerk, Nature 177:578-579 (1956); Salganik et al., Doklady Academii Nauk SSSR (Proc. USSR Acad. Sci.) 180:1473-1475 (1968); Salganik, Current Trends in Life Sciences 12, 115-123 (1989). It was demonstrated that RNase A is efficient in the treatment of diseases caused by RNA viruses, such as tick-borne encephalitis, meningitis caused by enteroviruses and parotitis virus, respiratory viral diseases caused by ECHO viruses, parainfluenza virus and enteroviruses. See, e.g. Lobzin and Sichko, Vrachebnoe Delo (Physician's Affairs) No. 10:38-41 (1969) (in Russian); Glukhov et al., Arch. Neurol., 33:598-603 (1976); Chernobrova, Pediatriya (Pediatry) Nl:38-42 (1978) (in Russian). The apphcation of RNase A was also shown to be safe.

It was important to find out whether nucleases (DNase I or RNase A) are able to inhibit reproduction of HIV. The structure of the HIV genome and the HIV reproduction cycle are quite distinct from those of other viruses. HIV is a an example of a rerrovirus. As with other retroviruses, it possesses genomic RNA which serves as a template for reverse transcription. The newly synthesized reversely transcribed DNA is integrated into the cellular genomic DNA. Thereafter, it serves as a template for generation of viral genomic RNA which, when replicated, provides synthesis of new viral RNA genomes and when translated ensures synthesis of new HIV proteins both forming together a multitude of new viral particles.

It was not obvious that DNase I and/or RNase A would be effective in inhibiting the reproduction of HIV. As described above, DNase I is effective against DNA viruses, while HTV is an example of an RNA virus. Moreover, other compounds which inhibit reproduction of DNA viruses, such as 5-iododeoxyuridine, arabinosyl cytosine and acyclovir, do not have significant effect on RNA viruses and do not affect the reproduction of HIV.

Thus, HIV does not respond to the drugs which inhibit reproduction of DNA viruses. Moreover, because it had previously been demonstrated that (1) DNase I is highly effective in inhibiting DNA viruses but not RNA viruses, and (2) RNase A is highly effective in inhibiting the reproduction of RNA viruses but not DNA viruses, one would have expected that RNase A would have inhibited reproduction of HIV but DNase I would not. Therefore, it is quite surprising that in fact the reverse is true: DNase I is very efficient as an anti-HIV agent, whereas RNase A is not.

The precise molecular mechanism by which DNase I interrupts the HTV life cycle is the subject of current investigation. Because of the unique nature of the retrovirus reproductive patterns, it may be that DNase I interrupts the reverse transcription stage of HIV reproduction by digestion of the DNA copy of the viral genomic RNA when the DNA copy becomes, for reasons as yet unknown, exposed to the DNase I action. At the same time, it is also unknown why HIV RNA is much less exposed to the action of RNase A.

Because of this remarkable finding, we feel obligated to proceed immediately to more advanced clinical phases using DNase I as a promising anti-HIV therapy, either alone or in combination with other established anti-HIV therapies. SUMMARY OF THE INVENTION

Methods for the suppression of HIV reproduction are provided. The methods consist in administration to the cell medium of an effective amount of pancreatic DNase I with a suitable divalent cation activator before, simultaneously with or after HIV infection of the cell.

The effective amount preferably provides a local concentration of DNase I between approximately 30 and 1500 Kunitz units of activity per ml of cell medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE I is a graph illustrating the Anti-HIV Activity of DNase I On HIV-infected MT-4 CeUs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods of inhibiting HIV reproduction in human or animal cells. The anti-HIV agent provided is pancreatic DNase I, preferably with a divalent cation activator. The method consists of administering DNase I into the cell medium before, simultaneously with or after infection of the cells with HIV. In one embodiment, the anti-HIV agent is DNase I isolated from an animal's pancreas. It has a specific activity 3000 Kunitz units per mg, lyophilized preparation, research grade (Serva) or a preparation with a much lower specific activity, produced by St. Petersburg's Drug Factory for medical apphcation, for local and parenteral administration. It is administered into the cell medium into a delivery system to achieve a final concentration between 30 and 1500 Kunitz units per ml.

In another embodiment, the present invention is directed to methods for treating patients with AIDS caused by HTV.

As used herein, the term "systemic", with regard to concentration, means the concentration of the antiviral agents per ml of cell medium or body fluids.

As used herein, "cation activator" refers to divalent cations which are required for DNase I activity as a cofactor or to augment the level of DNase I activity. Suitable divalents for use in the present invention include, but are not limited to, metal cations such as magnesium and manganese. Cation activators supplied as their corresponding salt are also included within this definition.

Although the method of this invention is described with respect to pancreatic DNase I, practice of the principles of this invention is contemplated with antiviral agents which are equivalent in their property to hydrolyze DNA. DNase I is extracted preferably from cattle or pig pancreases by methods well known in the art. Such methods include extraction of the enzymatic proteins from the minced pancreatic glands by a weak solution of sulfuric acid, step-wise precipitation of the enzymatic protein by ammonium sulfate, purification of DNase I by chromatography and lyophilization of the enzyme. Alternatively, the nuclease compound of the present invention can be produced by DNA recombinant methods providing high level expression of a cloned gene coding for DNase I in a prokaryotic or eukaryotic expression system. The following examples are intended to illustrate but not limit the invention.

Example I

This example shows the inhibitory effect of DNase I on reproduction of HIV in the human MT-4 cells judging by the activity of HIV-specific reverse transcriptase.

The MT-4 ceUs were cultured in 96-well microplates in RPMI medium supplemented by 10% fetal bovine serum, 300 mg/ml L-glutamine, 100 mg/ml glutamycine MT-4 cells (5 x 10⁵ cells/ml) were infected with HIV (multiplicity of infection of 0.2-0.5). Incubation was carried out at 37° C in a 5% CO2 atmosphere. The compounds under study, pancreatic

DNase I (Serva) and AZT, were diluted in the culture medium to their final concentrations. Their inhibitory effect on the HIV reproduction was evaluated after 96 hours of cultivation. Pancreatic DNase I in amounts of 0.5, 1, 5, 10, 50, 100, 500 or 1000 micrograms per ml was added to the medium at a time with the HIV administration. In some experiments, the enzyme was added I hour before or 1 hour after the HIV administration.

To study the reverse transcriptase (RT) activity, 15 ul of each supernatant from the culture medium was added to the 96-well microtitere plates that contained 40 ml of virus dilution buffer (50 mM Tris-HCl, ph 7.8, 150 mM KCl, 5mM EGTA, 0.3 mM glutatione, 0.1% Triton x- 100 and 25 mg/ml each of poly (A) and oligo (dTi6). Microplates were incubated for 10 minutes in the cold, thereafter 10 ul of H] dTTP (10-20 uCi) were added and the mixture was incubated for one hour at 37 ^° C. After incubation the content of each well was transferred to nitrocellulose filters. The filters were air dried, washed six times with 0.5mM phosphate buffer, water and 96 ^° ethanol and their radioactivity was determined by scintillation counter.

As shown in Table 1 and Fig. 1, addition of 10 and 50 micrograms of DNase I per ml of the ceU medium simultaneously with HIV infection decreases the activity of RT by about 70%. Administration of 500 or 1000 mg of DNase I per ml of the cell medium decreases the RT activity by more than 80%. When DNase I was added 1 hour before or 1 hour after the HIV administration, its inhibitory effect was lower than under simultaneous administration of the HIV and DNase I. Administration of AZT in a dose of 0.1 mg per ml of the cell medium results in a decrease of RT activity by 72%. Example π

The data presented demonstrate the effect of DNase I on reproduction of HIV monitored by the expression of viral antigens in the HIV-infected ceU culture. The conditions of the experiments were similar to the described in Example 1.

The amount of HIV-antigens was determined by direct ELISA assay. Tween 80 was added to the HTV-infected cell samples to inactivate the virus. Further inactivation was achieved by incubation at 4 ^* C f or 24 hours. A series of twofold dilutions of the HrV-containing material was prepared. Anti-HIV-1, and anti-p24 antibodies conjugated to horse radish peroxidase were used for the ELISA assay provided by reaction with O-phenylendiamine.

When MT-4 cells were infected with HIV, the content of viral- specific p24 antigen was 2278 ng/ml. A significant decrease of the p24 amount was found under the effect of 5 and 10 micrograms of DNase I. When 100, 500 or 1000 micrograms/ml of DNase I was added to the culture medium, the p24 antigen expression was almost completely suppressed; it made up 260-430 ng/ml. The effect of AZT was close to these results (Table 1). Similar data were obtained when anti-HIV- 1-lgG was used to estimate the amount of HIV proteins in the cell medium (Table 1). Example HI

This example shows the inhibitory effect of DNase I treatment on reproduction of HIV in the human MT-4 cells judging by HTV- induced cytopathic effect.

The MT-4 cells were cultured and infected by HIV as described in Example 1. Stock solution of DNase I was diluted in the culture medium to the final concentrations. DNase I in amounts given in Example 1 was added at a time with administration of HIV. The inhibitory effect of DNase I on the HIV reproduction was evaluated after 96 hours of cultivation. The number of viable cells was counted. The viable cells were distinguished by dyeing with 0.4% solution of trypan blue.

The percentage of viable cells in uninfected cell culture after three days was 83%. In the HTV-infected cell culture the corresponding percentage had decreased to 59%. When 100, 500 or 1000 micrograms of DNase I was added, the percentage of viable cells almost approached the control level. Adding 5, 10 or 100 micrograms of DNase I was less efficient, although it also afforded some protection to the cells from the cytopathogenic effect of HTV infection (Table 1 and Fig. 1). When AZT was added to the cell medium in the amount of 0.1 μg/ml, the ceU viability increased from 59% to only 74%.

It is important that DNase I even in high concentration does not adversely affect the cell viability. All anti-HIV effects of DNase I are summarized in Fig. 1. Table 1

Inhibitory Effect Of DNase 1 On The Expression Of HTV-Specific

Eac igure presents t e average o ive experiments ± s.e. n.d. = (none detected)

Example IV The DNase I index of selectivity (IS) was estimated. The index is an important characteristic of the compound as a promising therapeutic agent. We define IS herein as a ratio between the concentration of DNase I resulting in a 50% decrease of cell viability (it describes the rate of DNase I toxicity) and the concentration of the enzyme leading to 50% inhibition of HTV reproduction. The concentration of DNase I resulting in a 50% decrease of the cell viability (CD 50) is 5000

Kunitz units per ml of the cell medium. The concentration of DNase I leading to a 50% rate of inhibition of HTV reproduction is 1.8 micrograms/ml when estimated by RT activity or 7.0 μg/ml when estimated by a decrease of 50% of p24. Accordingly, the IS measured by RT is 2778, while the IS measured by p24 is 7140. In either case the very high IS demonstrates the safety of the applied doses of DNase I.

Example V This example demonstrates the failure of pancreatic RNase A to affect the reproduction of HTV. RNase A was obtained from Sigma (specific activity 72 Kunitz units/mg). It is well known that HIV is an RNA virus. The viral RNA programs synthesis of all viral proteins which provide reverse transcription, regulation of the viral nucleic acids synthesis viral coat formation. HTV RNA functions during the whole period of HTV reproduction and therefore may be continuously exposed to the effects of RNase A. HIV DNA is a short-hving intermediary product which, when integrated into the cellular genome, becomes protected from the DNase I action. A number of data partially presented above (see, Background of Invention) demonstrates that, for RNA viruses other than HTV, RNase A effectively inhibits their viral RNA synthesis and reproduction of the viruses, whereas DNase I does not. Therefore, one would have expected that RNase A might be an efficient anti-HIV agent. However, the data presented in Table 2 demonstrate that RNase A practically does not suppress the expression of the HTV antigens and, compared to DNase I and AZT, has a rather weak protective effect on the viability of HIV-infected cells. Under the effect of RNase A, even a concentration as high as 1,000 μg/ml, the HTV reverse transcriptase activity diminishes only shghtly. When moderate and low concentrations of RNase A are applied, the HTV reverse transcriptase activity actually increases shghtly. (N.B.: this increase in the HIV reverse transcriptase activity does not reflect a stimulatory effect of RNase A on HIV reproduction, since there is no increase in the amounts of total HTV antigens and p24 antigen.)

Therefore, unexpectedly, RNase A has a very weak effect on HTV reproduction, if any effect at all, while DNase I is a potent inhibitor of HTV multiplication.

Table 2

Inhibitory Effect Of RNase A On The Expression Of HTV- Specific

n.d. = (none detected) Example VI

This example demonstrates the combined action of DNase I and RNase A on HTV reproduction. Two nucleases were apphed at a time in equal amounts. Table 3 shows that in the presence of RNase A the inhibitory effect of DNase I on the expression of HTV antigens is weakened. The RNase A interferes also with the positive effect of DNase I on the viabihty of the HTV-infected cells.

The mechanism of the interference of two nucleases is not clear. However, the data support the conclusion that, contrary to expectations, DNase I but not RNase A has weU-manifested anti-HTV effects. Indeed, RNase A even decreases the anti-HTV activity of DNase I.

Table 3

Combined Effect Of DNase I and RNase A On The Expression Of HTV- Specific

Each figure presents the average of five experiments ± s.e. n.d. = (none detected)

Example vπ This example demonstrates the combined action of DNase I and AZT on HTV reproduction. DNase I and AZT were apphed both alone and in combination at the concentrations shown in Table 4. Table 4 shows that AZT alone inhibits HTV reproduction (as measured by p24 antigen content) by 87.8%, while DNase I alone inhibits HTV reproduction by 80%. The combination of AZT and DNase I inhibits HTV reproduction by 90.5%. Therefore, DNase I does not appear to interfere with the anti- HTV effectiveness of AZT, and in fact appears to enhance it.

TABLE 4

PUBLICATIONS

1. Trukhachev, A. & Salganik, R., Virology 33:552-555 (1967.)

2. Salganik, R. et al., in "Inhibitory virusnoy aktivnosti (Inhibitors of the viral activity), Riga, Sinatne, 1972, pp. 147-152.

3. Salganik, R., Current Trends in Life Sciences, 12: 115-123 (1984).

4. Lapik, A. et al., Pharmakol. Toksikol. (Pharmacol. Toxicol.) 2: 210-214 (1970).

5. Colain, A. et al., Ann. Oculist, Paris., 203:371-378.

6. Gutorov, A. et al., Zhurn. Nevropat. Psikhiatz. (J. Neuropath. Psychiatr.), 11:75-78 (1976).

7. Krivosheev, B. et al., Vestnik Dermat. Venerol. (Bull. Dermat. Venerol.) Nl:29-35 (1987).

8. Le Clerk, J., Nature 177:578-579 (1956).

9. Salganik, R. Dokl. Akad. Nauk SSSR (Proc. USSR Acad. Sci.) 129:212-214 (1959).

10. Salganik, R. ibid., 180:1473-1475 (1968).

11. Lobzin, V., and Sichko, Zh., Vrachebnoe Delo (Physician's Affairs) No. 10:38-41 (1969).

12. Glukhov, B. et al., Arch. Neurol, 33:598-603 (1976).

13. Chernobrova, , Pediatriya (Pediatry) No. 1:38-42 (1978).

Claims

CLAIMS What is claimed is:

1. A method for treating HIV infections with a DNA hydrolyzing compound to inhibit HTV reproduction.

2. The method of claim 1, wherein the DNA hydrolyzing compound is administered parenterally.

3. The method as in claim 1 or 2, in which the DNA hydrolyzing compound is Pancreatic Deoxyribonuclease I.

4. The method of claim 3, wherein the Pancreatic Deoxyribonuclease I is present in the concentration of 10 to 500 micrograms per milliliter.

5. The method of claim 3, wherein a second anti-viral compound is added to boost the inhibition of HTV reproduction.

6. The method of claim 4, wherein the second anti-viral compound is AZT.

7. The method as in claim 1, 2, 4, 5, or 6, in which the infected ceUs are human cells.

8. The method of claim 7, wherein the human cells are MT-4 cells.