WO1990011072A1 - Drug for treatment of infectious diseases caused by retroviruses as well as for the treatment of tumors - Google Patents

Abstract

The disclosed drug is comprised of D-aspartic acid β monohydroxamate (DAH).

Description

Medicine for the treatment of infectious diseases caused by retroviruses as well as for the treatment of tumors It has been known since the work of KIDD in 1953 that L-asparagmase is an anti-tumor agent. This activity being insufficient, we tried to strengthen it by combining L-asparagmase with other products. In particular, it was proposed in patent application No. 86.08874 of June 19, 1986 to combine with L-asparagmase D-aspartic acid β hydroxamate (DAH).

 However, this association did not bring the expected results, so the research on this association was interrupted and the patent application 86.08874 was abandoned.

 The applicant's work revealed that the DAH used alone had both antiretroviral activity and antitumor activity.

 The present invention relates to the application, as a medicament of a chemical compound: D-aspartic acid β-monohydroxamate (DAH).

HOOC - CH - CH ₂ - CONHOH

I NH- ₂

D-aspartic acid β-monohydroxamate is typically formed by the reaction of an activated acyl group of the D isomer of aspartic acid or one of its derivatives, with the hydroxylamine according to the reaction:

HOOC-CH - CH ₂ - COX + NH ₂ OH

 I

NH ₂

HOOC-CH - CH ₂ - CONHOH + HX

I NH ₂

COX being an amide, ester or anhydride residue. We will expose below, firstly, its antiviral activity, and then, its anti-tumor activity.

 This medicament may be in any suitable form, for example in the form of a lyophilized powder of the active substance to be dissolved extemporaneously in a physiological solution for injection or in a form suitable for oral administration.

 Antiretroviral activity of D-aspartic acid β- monohydroxamate

 Although progress has been made in the fight against viral infections, there is not yet a range of antiviral agents as broad, safe and effective as, for example, antibiotics.

 By their mode of action, antiviral agents can be broadly classified into three categories, the borders between them being often arbitrary: chemical agents, stimulators of the immune system or activators of the immune response, and inducers of the antiviral cellular state.

Chemical agents: This group is the largest and the fastest growing. Since viruses are forced intracellular parasites, the effectiveness of these agents is determined by their ability to differentiate viral metabolism from cellular metabolism, or in other words, between the metabolism of the infected cell and that of the uninfected cell. This amounts to saying that the essential characteristic of this group of agents is their specificity of action. This at the same time determines the strength and weakness of these agents, because if they are often very effective, because they can specifically inhibit a viral enzyme, their spectrum of action is generally very limited and they often end by selecting resistant viral variants, in the same way as antibiotics do with bacteria (De Clercq E., Biochem. J., 20b, 1-1J, 1982; Becker Y. and Hadar J., Prog. Med. Virol., 26, 1-44, 1980). Activators of the immune response: Apart from the different types of vaccines, this group consists of agents capable of activating non-specific defenses (macrophages, natural "killer" cells) or, more rarely, specific defenses (lymphocytes "B" or "T") of the organism. For the most part, these agents are still in the experimental phase. Unlike chemical agents, the essential characteristics of their mode of action are their very broad spectrum of action and the fact that they cannot select resistant variants. However, they are currently limited in number and ineffective, although progress has recently been made in this direction (Koff WC, Showalter SD, Hampar B. and Fidler IJ, Science, 228, 495-496, 1985).

 The inducers of the antiviral cellular state:

This group includes interferon (IFN), IFN inducers and IFN surrogates. It is a still very small group of substances which are characterized by the fact that they awaken in cells a state of resistance to viral infection. These agents combine the power of action at the cellular level with chemical agents and the very broad spectrum of action of activators of the immune system. Because they act on all cells, whether they have been infected or not, a determining factor in their effectiveness is their low toxic power towards the cells.

 The agent under consideration probably belongs, although its precise mode of action has not yet been elucidated, to the first group of substances with a spectrum of action limited to retro-viruses or to viruses whose replication is apparent, such as hepadna viridae.

Retroviruses, like any other virus, cannot reproduce without diverting the biosynthetic apparatus of the cell to their advantage. The peculiarity lies in their ability to reverse the usual flow of genetic information. In retroviruses, the genetic material is made up of ARK, that an enzyme, reverse transcriptase, used to synthesize DNA; this viral DNA can integrate into the genome of the host cell.

 Retroviruses and their carcinogenic power are not a scientific novelty. At the beginning of the century, several researchers had identified, in animals, transmissible agents capable of causing leukemias and solid tumors (Rous P., J. Am. Med. Assoc, 56, 198, 1911). In the following decades, retroviruses were found in many animal species. Prolonged efforts by researchers led to the isolation, in 1980, of the first human retrovirus: the lymphotropic virus of human T lymphocytes type I (HTLV-I) (Poiesz BJ et al, Proc. Natl Acad. Sci. USA, 77, 7415, 1980). HTLV-I causes a rare and extremely malignant cancer, adult T-cell leukemia, which is endemic in parts of Japan, Africa and the Caribbean.

 The first cases of AIDS were diagnosed in 1981 (Gottreb, M. S. et al, Weekly Record, 30, 250, 1981). The agent responsible: the human immunodeficiency virus (HIV), a retrovirus, was discovered in 1983 (Barré Sinoussi F. et al, Science, 220, 868, 1983). This naturally leads to taking stock of the agents active against the retrovirus. Faced with the problems of developing a vaccine in the near future, research into drugs that can inhibit the replication of retroviruses is necessary.

 The applicant's work has made it possible to discover that DAH causes a significant inhibition of the multiplication of viruses belonging to the retrovirus family. This has been demonstrated IN VITRO with the AIDS virus (HIV) and IN VIVO with the FRIEND virus (FLV - Friend Leukemia Virus) which is a murine type C retrovirus.

 Effect of DAH on HIV replication.

The work carried out has made it possible to discover that D-aspartic acid and hydroxamate completely inhibits the viral multiplication of HIV. This inhibition is dose-dependent. This activity has been demonstrated in two models of HIV replication, a) lymphocytes normal infected with the purified virus, and b) coculture of lymphocytes from AIDS patients and normal lymphocytes.

Reverse transcriptase activity was measured for 22 days in the cell culture supernatant as a criterion for viral proliferation. The treatment with DAH lasted 72 hours from OJ to D3 of the manipulation. The figures

1, 2 and 3 show the effect on the production of viral reverse transcriptase, of the dose applied during the first three days, during the treatment of control lymphocytes infected with OJ, either by coculture with patient cells, or with purified virus. It has been found that lymphocytes have proliferated during treatment with the drug, that this drug is not toxic to lymphocytes not infected with the HIV virus. Figures 4 and 5 show the effect of treatment during the first three days, at the doses indicated, on healthy donor lymphocytes. On the other hand, DAH has been shown to be more toxic towards lymphocytes infected with the AIDS virus. Figures 6 and 7 show the effect of treatment during the first three days, at the doses indicated, on healthy donor lymphocytes infected with purified virus.

 Effect of DAH on virus-induced disease

Friend

 The FLV virus is a type C murine retrovirus which produces rapid effects on erythroposis, the erythroid precursors being the target cells of this virus. The Friend complex includes a transforming virus defective for replication, called SFFV (Spleen focus forming virus) because it is capable of inducing macroscopic foci of transformed cells in the spleen of infected adult mice, and a helper virus called the F-

MuLV (Friend Murine Leukemia Virus) which is used for replication. There are two variants of this virus: FV-A which induces anemia and FV-P on which the work has been carried out and which produces polycytemia.

EL-V-induced erythro-leukemia shows two stages: an early stage characterized by the rapid growth in the number of relatively erythropolitic precursors mature with a limited proliferation capacity, and a late stage, characterized by the emergence of undifferentiated precursors with an intense proliferation capacity. In vivo, the manifestations of this disease are splenomegaly accompanied by hepatomegaly, erythroid hyperplasia in the spleen and bone marrow, polycytemia and macroscopic foci in the spleen. The last stages are characterized by the presence of immortal and transplantable erythroid cells.

 During the work carried out, the virus was maintained by successive intraperitoneal passages in the DBA / 2 mouse. This interview was done from the animal's spleen every 21 days. The activity of the drug was evaluated by comparing the survival times of the animals to which the virus is injected, with or without drugs. Treatment started three days after viral infection because it is considered that it takes this period of time for there to be virus sequences in the erythroid precursor genome of the infected mouse. After varying the number of daily injections and the duration of treatment for a given dose, it was established that 3 injections of lg / kg / dose of DAH during the first 10 days and 2 injections of lg / kg / dose of DAH for the next 20 days produces a T / C of 239%. The results are reported in the following table:

 (1) the animals are treated 3 times a day from D 1-10 and 2 times a day from D 11-30.

 (2) T / C = ratio of the survival time of the treated animals (T) and the survival time of the control animals

(VS). Antitumor activity of D-aspartic acid β monohydroxamate

 The applicant's work has made it possible to discover that the D isomer of aspartic acid, when administered in specific dosages, as defined below, has an inhibitory action on tumors.

 In the aforementioned French patent application No. 86.08874, it is reported that D-aspartic acid β hydroxamate has shown no activity in vivo, after treatment with DAH at the rate of 600 mg / kg per day for 5 days, out of three murine tumor models: leukemia 1210, RBL5 lymphoma and melanoma B16, the mice having been inoculated with the abovementioned tumor cells. It has now been observed, on the one hand, that, in the same types of tests carried out in vitro, the inhibition of the growth of tumor cells (CI 50%) takes place for doses of DAH starting between 0.8 and 1 mM per liter of cell medium, and, on the other hand, that, on the murine tumor model of leukemia 1210, reputed to be difficult to cure, an administration of 1 g / kg of body weight, at a rate of 4 times a day for three days already leads to a very significant prolongation of the survival of the treated animals.

 Under these same dosage conditions, L-aspartic acid β hydroxamate is very toxic. Thus, the increase in the doses of the aforementioned derivative D compared to the derivative L reveals an activity of the derivative D, without presenting the toxicity which the derivative L presents at the same dosages (the LD 50% for DAH is 10g / kg then that the LD 50% for LAH is 2.5 g / kg).

 The doses used for the treatment of mice varied from 0.5 g / kg to 1.5 g / kg for a minimum period of 3 days which can extend up to 10 days.

 However, it turns out that it is possible to use higher doses, up to 5 g / kg and that if the dose is low, a longer treatment period is required.

Comparative in vivo study of DAH and LAH The antitumor activity of DAH and LAH was studied using the L1210 leukemia model. L1210 leukemia cells are maintained by intraperitoneal passages in the male B6D2F1 / J mouse. The number of cells used for the therapeutic tests is 105 cells per mouse in 0.5 ml of PBS. The observation period is 30 days.

 The results are reported in the following table:

 (1) The animals are treated 4 times a day.

 (2) T / C = ratio of the survival time of the treated animals (T) and the survival time of the control animals (C).

 (3) at 30 days.

 (4) Weight change (in grams) from D1 - D5.

It is noted that DAH shows significant antitumor activity on this model for administration at a dose greater than or equal to 1 g / kg, 3 or 4 times a day. On the other hand, for LAH, the same protocol is not applicable because of the toxicity, and even using a lighter protocol, no anti-tumor activity was observed, but still a very significant toxicity (death of animals treated with LAH before the control group).

The anti-tumor activity of DAH has also been studied in two other mouse models: L5178Y lymphoma and P388 leukemia. The murine iymphoma cells (L5178Y) are maintained by successive intraperitoneal passages in the DBA / 2 mouse and injected, for the tests, at a dose of 104 cells per mouse.

 The P388 leukemia cells are maintained by intraperitoneal passages in the DBA / 2 mouse and injected, for the tests, at a dose of 105 cells per mouse in the B6D2F1 mouse.

 The results are reported in the following table:

Claims

claims  1. Medicament characterized by the fact that it consists of D-aspartic acid β monohydroxamate (DAH).  2. Medicament according to claim 1, characterized in that it is intended for the treatment of infectious diseases due to retroviruses and for the treatment of tumors.  3. Drug composition according to claims 1 and 2, characterized in that it contains, as active ingredient, D-aspartic acid β monohydroxamate (DAH).  4. Drug composition according to claim 3, characterized in that it is in the form of a lyophilized powder of the active substance to be dissolved extemporaneously in a physiological solution for injection.  5. A drug composition according to claim 4, characterized in that it is in a form suitable for oral administration.