FR2645020A1 - MEDICINE FOR THE TREATMENT OF INFECTIOUS DISEASES DUE TO RETROVIRUS AND FOR THE TREATMENT OF TUMORS - Google Patents

Abstract

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

Description

Drug for the treatment of infectious diseases due to retroviruses as well as for the treatment of tumors
It has been known since the work of KIDD in 1953 that L-asparaglose is an antitumour agent. This activity proving insufficient. we tried to strengthen it by associating L-asparaginase with other products. In particular, it has been proposed in patent application No. 86.e8874 of 19 November 1986 to associate * the
L-asperaginase D-aspartic acid ss hydroxamate (DAH).

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

 The Applicant's work has found that DHA used alone has both antiretroviral and antitumor activity.

The present invention relates to the application as a medicament of a chemical compound: Ss-monohydroxamate acid daspartic acid (DAH).

The D-aspartic acid ss-monchydroxamate is typically formed by the reaction of an active acyl group of the isomer D of aspartic acid or a derivative thereof with hydroxylamine following the reaction:

COX being an amide, ester or anhydride residue.

 Hereafter, first, its antiviral activity, and then its antitumor activity.

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

Antiretroviral activity of D-aspartic acid 8monohydroxamate
Although progress has been made in the fight against viral infections, a range of antiviral agents as extensive, safe and effective as antibiotics are not yet available.

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

Chemical Agents: This group is the largest and the fastest growing group. Since viruses are obligate intracellular parasites, the effectiveness of these agents is determined by their ability to differentiate viral metabolism from cellular metabolism, or otherwise, between the metabolism of the infected cell and that of the cell. uninfected cell. Ge is to say that the essential characteristic of this group of agents is their specificity of action. This, at the same time, determines the strength and the weakness of these agents, because 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 up selecting resistant viral variants, in the same way that antibiotics do with bacteria (De Clercq E.,
Biochem. J., 26, 1-13, 1982; Becker Y. and Hadar J.,
Prog. Med.Virol., 26, 1-44, 198>.

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 body 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 can not select resistant variants.

But for the moment they are limited in number and inefficient, although progress has recently been made in this direction (Koff WC, Showalter SD,
Hampar B. and Fidler IJ, Science, 228, 495-496, 1965v.

Inductors of the antiviral cellular state: This group includes interferon (IFN), IFN inducers, and IFN substitutes. It is a still very small group of substances that are characterized by the fact that they awaken in the cells a state of resistance to viral infection. These agents combine the potency of action at the cellular level of the chemical agents and the very broad spectrum of activators of the immune system. Because they wilt on all cells, whether infected or not, a determining factor in their effectiveness is their low toxicity to 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 retroviruses or to viruses whose replication is related to it. , such as hepadna viridae.

 Retroviruses, like any other virus, can not reproduce without detracting from the biosynthetic device of the cell. The peculiarity lies in their ability to reverse the usual flow of genetic information. In retroviruses, the genetic material is RNA, which an enzyme, reverse transcriptase, uses to synthesize DNA; this viral DNA can integrate into the genome of the host cell.

Retroviruses and their carcinogenic potential are not new to science. By the beginning of the century, several researchers had identified transmissible agents in animals that could cause leukemias and solid tumors (Rous P.,
J. Am. Med. Assoc., 56, 198, 1911). In the following decades, retroviruses have been found in many animal species. The prolonged efforts of researchers led to the isolation, in 198e, 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). The
HTLV-I causes a rare and extremely malignant cancer, T-cell leukemia in adults, 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. 25Z, 1981). The causative agent: the human immunodeficiency virus (HIV), a retrovirus, was discovered in 1983 (Barré Sinoussi F. et al., Science, 22X, 868, 1983). This naturally leads to an update on the active agents against the retrovirus.

In view of the problems of developing a vaccine in the short term, research into drugs that can inhibit retroviral replication is needed.

The work of the applicant has revealed that DHA causes a significant inhibition of the multiplication of viruses belonging to the family of retroviruses. This has been demonstrated IN VITRO with the AIDS virus (HIV) and IN VIVO with the FRIEND virus CFLV
Friend Leukemia Virus) which is a type C murine retrovirus.

Effect of DHA on HIV replication.

Work has shown that D-aspartic acid ss hydroxamate completely inhibits HIV viral multiplication. This inhibition is dose-dependent. This activity was highlighted on two replication models of the
HIV, a> normal lymphocytes infected with the purified virus, and b) coculture of lymphocytes of patients with
AIDS and normal lymphocytes. The reverse transcriptase activity was measured for 22 days in the cell culture supernatant as a viral proliferation criterion. Treatment with DAH lasted 72 hours from the manipulation. Figures t, 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 at OJ, either by coculture with cells of the patient, 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. FIGS. 4 and 5 show the effect of a treatment during the first three days, at the doses indicated, on healthy donor lymphocytes. On the other hand, DAM was more toxic towards infected lymphocytes. by the AIDS virus. Figures 6 and 7 show the effect of treatment during the first three days, at the indicated doses, on healthy donor lymphocytes, infected with purified virus.

Effect of DAH on the patient induced by the
Friend
FLV is a type C murine retrovirus that produces rapid effects on erythropoiesis, with erythrod precursors being the target cells of this virus. The Friend complex includes a defective transforming virus for replication, called the
SFFV <Spleen focus forming virus) because it is able to induce macroscopic foci of transformed cells in the spleen of the infected adult mouse, and a helper virus called F-MuLV <Friend Murine
Leukemia Virus) that is used for replication. There are two variants of. this virus: the FV-A which induces anemia and the FV-P on which the work was carried out and which produces a polycytemia. FL-V-induced erythroid leukemia has two early-stage stages characterized by the rapid growth in the number of relatively mature erythropoietic precursors with limited proliferation capacity. and a late stage, characterized by the emergence of undifferentiated precursors with an intense proliferative capacity. In vivo, the manifestations of this disease are spiénégalie 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 immortal and transplantable erythroid cells.

During the work done, the virus was maintained by successive intraperitoneal passages in the DBA / 2 mouse. Get maintenance was done from the animal's spleen every 21 days. The activity of the drug a. was evaluated by comparing the survival times of animals injected with the virus, with or without drugs. The treatment started three days after the viral infection because it is considered that this period of time is necessary for there to be sequences of the virus in the genome of the erythroid precursors of the infected mouse. After having varied the number of daily injections and duration of treatment for a given dose, it has been established that 3 injections of 1 g / kg / dose of DAH during the first 1 days and 2 injections of 1 g / kg / dose of DAH during the following 2 days, produce a T / C of 239%. The results are reported in the following table
Substance Dose (g / kg Days of T / C x 100 Number
active weight treatment (%) of animals
corporal) (2) survivors
DAH lx 3 / d J 1-3 239.86 (i>
1 x 2 / d
(1) the animals are treated 3 times a day of J 1-10 and twice daily of J 11-3.

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

Antitumor activity of D-spartique acid fi monohydroxamate
The work of the Applicant has found that the isomer D of aspartic acid, when administered in specific assays, as defined below, has an inhibitory action on tumors.

 In the aforementioned French patent application No. 86.e8874, it is reported that D-aspartic acid 8 hydroxamate showed no activity in vivo, after treatment with DAH at a rate of 600 mg / kg per day for 5 days. on three murine 12 leukemia tumor models, RBL5 lymphoma and B16 melanoma, the mice having been inoculated with the aforementioned tumor cells.It has now been observed, on the one hand, that in the same types of tests conducted in vitro Inhibition of tumor growth <5% IC> occurs at doses of DAH starting between 0.8 and 1 mM per liter of cell medium, and on the other hand, that in the murine tumor model leukemia 1210, considered difficult to cure, an administration of 1 g / kg of body weight, 4 times a day for three days, already leads to a very significant prolongation of the survival of the treated animals.

 Under these same dosing conditions, Laspartic acid ss hydroxamate is very toxic. Thus, the increase in the doses of the above-mentioned derivative D relative to the derivative L reveals an activity of the derivative D, without presenting the toxicity that the derivative L exhibits at the same doses. The LD 50% for the DAH is 10 g / kg. that the LD 50% for LAH is 2.5 g / kg>.

 The doses used for the treatment of mice ranged from 1.5g / kg to 1.5g / kg for a minimum period of 3 days 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, it takes a longer treatment period.

In vivo comparative study of DAH and LAH
The antitumor activity of DAH and LAH was studied on the L1210 leukemia model. The L121 leukemia cells are maintained by intraperitoneal passages in the B6D2F1 / J mule mouse. The number of cells used for the therapeutic trials is 10 cells per mouse in 8.5 ml of PBS. The observation period is 30 days.

The results are reported in the following table
Substance Dose (g / kg Days of T / 0x100 Nomore (3) Evolution
active weight treatment (%) of animal weight
corporal) (2> survivors (4)
DAH 1x3 / d 1-10 347 3/5 +1.300
DAH 1.5x4 / d (1) 1,2,3,4 347 3,5 + 1,000
DAH 1,5x4 / d 1,2,3 276 0/5 +1,200
DAH 1x4 / d 1,2,3 171 0/5 +1,100
DAH 0.5x4 / d 1,2,3 113 0/5 +1,400 LAH 1,5x4 / d 1 1 e, 5
LAH 1x4 / day 1,2 <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 J1 - J5.

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

The antitumor activity of DAH has also been studied in two other murine models: lymphoma
L5178Y and P388 leukemia.

 Murine lymphoma cells (L5178Y) were maintained by successive intraperitoneal passages in DBA / 2 mice and injected for the assays at a dose of 10 cells per mouse.

P388 leukemia cells are maintained by intraperitoneal passages in mice
DBA / 2 and injected, for the tests, at the dose of 10 cells per mouse on the mouse * 6D2F1.

The results are reported in the following table:
Lymphoma L5178Y
Substance Dose (g / kg) Days of T / Cx100 Number
active weight treatment (%) of animals
corporal) survivors #
DAH 1 x 4 / day 1,2,3,4,5 576 5/5
DAH 1 x 3 / day 1,2,3,4,5 576 5/5 # g 128 days.

Leukemia P388
Substance Dose <g / kg Days of T / Cx100 Number
active weight treatment (%) of animals
corporal) survivors @
DAH 1.5 x 4 / d 1,2,3,4,5 190 0/5
DAH 1.5 x 41d 1,2,3 155 # at 30 days.

Claims (5)

Revendicatlons  1. Drug characterized by the fact that it consists of D-aspartic acid ss monohydroxamate (DAM).  2. Medicament according to claim 1, characterized in that tir is intended for the treatment of infectious diseases due to retroviruses and the treatment of tumors.  3. Drug composition according to claims 1 and 2, characterized in that it contains as active ingredient D-aspartic acid ss monohydroxamate (DAH).  4. The medicinal 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. Drug composition according to claim 4, characterized in that it is in a form suitable for oral administration.