JPH02502730A - Use of colchicine and related compounds for retrovirus suppression - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Col for retrovirus suppression Use of hitine and related compounds The present invention relates to retroviruses of the tentivirus subgroup. The present invention relates to a method of treating a human infected with a virus. Retroviruses are other viruses It is a group of RN viruses instead of the NA genome that is characteristic of viruses. lentivirus is characterized by the many years it takes for the onset of infection in humans. It is a subgroup of retroviruses. Viruses persist in numbers for a long time after the initial invasion. It remains dormant even for years and can suddenly become malignant when the right environmental conditions are met. There is.

The covert nature of these lentiviruses is known and recognized.

These include, for example, diseases known as acquired immunodeficiency syndromes (AIDs). Involved human immunodeficiency viruses HIV-1 and HIV-2 and T-cell lymphoma HTLV human T cell cytophilic lymphocytes (phototropic) virus include.

The replication cycle of the virus, i.e. the growth of the virus or in the human system. Spread of infection is through cell receptors on T cells (T4 cells in the case of the HIV virus) attachment, cell wall penetration, removal of retrovirus cell wall, reverse transcription on viral RNA DNA transcription by the action of enzymes and by known and understood mechanisms, The process ends with the generation of new RNA-containing particles (performed through several other steps). be exposed.

suppressing and preventing replication by inhibition at one of its several steps would be possible.

The currently appropriate method for treating AIDS is AZT (3'-Azito 3'-Deodorant). This is due to treatment with xythymidine).

It is the nucleoside triphosphate of reverse transcriptase of HIV or other retroviruses. It is a nucleoside analog that is a competitive inhibitor of ’, 5’ Phosphosulfonate is added to the growing DNAg by However, its own 3' position is blocked by an azide group, making it difficult for further addition to the DNA chain. addition is not possible. This results in chain termination and replication failure.

In human treatment, the high doses required for oral administration result in up to 20% of patients Significant hesotoxilogy with severe leukopenia cal) is highly toxic. Other side effects include rash puritis, Includes CNS toxicity with nausea, headache and sometimes localized cancer persistence.

Another drug used to treat AIDS is Suramin. This drug is a well-known anti-trypanosomal drug. It's an investment It is a model drug that has been shown to be effective against HIV in vitro. Ivy. Enzyme template-brimer binding site and appearance in vitro Because of the above interaction, retrovirus reverse transcriptase activity in vitro Potential inhibitor. Side effects in humans include fever, rash, pyuria (P)+ur ia), proteinuria, neutropenia, baratesias ( Parathesius) and adrenal insufficiency.

Strategies including the use of drugs such as AZT and Shuramin are into the cell at the stage of reverse transcription to A, that is, DNA synthesis on the RNA template. The aim is to inhibit HIV replication in the human body. However, these compounds also It also interferes with normal NA-dependent DNA synthesis. This is especially true for long-term use This results in serious side effects on tissues such as the bone marrow over a period of time.

Clearly, improved methods of treating HIV and other retroviral infections are needed. considered essential.

Colchicine is (S)-N-(5,6,7,9-tetrahydro-9-oxabenzo Hebutaten-7-yl)acetamide.

It is used as a therapeutic agent in humans, especially as a gout suppressant and as a familial medicinal agent. It has long been known for the treatment of Tarrenian fibre. It can also be digested orally It can also be safely administered extravascularly. Its activity in the mammalian body is well known and well-understood. It is understood. Colchiceine is the 10- The theory is that it is a methylated homolog. It has also been used for the same purpose. Colchicine is Due to its known efficacy, it is a more suitable therapeutic agent. It relates to the use in the present invention. is also a preferred agent. Because it is easily available at a reasonable cost, This is because it is extremely effective for newly discovered uses.

There are several other analogs and homologues of colchicine known to have the same activity. Exists. these are, are also useful in the practice of the present invention.

! -Skin Colchicine and its homologs and analogs were administered to humans, and lentivirus It has been discovered that infections caused by a subgroup of retroviruses can be treated. It was seen. The therapeutic agent is conveniently administered orally, but intravenously or intramuscularly. Parenteral administration, including intra-injection, may also be employed. The therapeutic agent may also be a suppository or It may be administered in a variety of sustained release compositions, including transdermal compositions.

As shown above, once a human system is infected, retrovirus replication requires many steps. There is a tip. Theoretically, it is possible to inhibit replication at any one of the steps. Any of these can produce organic results if you interfere with them enough.

actin-like microfilaments, intermt41ate filaments Viral uptake and morphogenesis of the cytoskeletal system consisting of microtubules and microtubules. phogenesis) has not been widely studied. , microtubules are responsible for receptor mobility and protein Camping (Edelman, G. M. (1976) Science 1 92 = 218-226) and intercellular transport and exocytosis (Butc her aru! Goldman (1974), Gordon and Webb (1976)). limited number of , microtubule depolymerizing agents including colchicine for the morphogenesis of enveloped viruses. The effect of was investigated in cell culture. The results were mixed. love dowi rus vesicular stomatitis (Gentr. y, N., and Bussereau, F. (1980) J. Vir. al, 34277-281), but on the contrary, it is not effective against alphavirus. Semliki Forest virus was inhibited (R4chardson, C, D, an d Vance, D, E.

(1978) J, Biol, Chell, 253: 4584 45 89) Chronic infection with Haloney murine leukemia virus In certain cell lines, treatment of cultures with low levels of colchicine inhibits virus production. (Satake and Libtiy (1) 982) J. Gen. Viral, 58: 339-349).

Knowledge about the effects of drugs on the skeletal structure of cells, including the effects of drugs on viral replication and Not providing a basis for predicting the ability to usefully interact with the ability to transmit infectious disease This is clear from the above.

The present invention for inhibiting the replication of retroviruses of the Lentivirus subclass. T4 lymphoblastoid cell lines (particularly) (permanently added to IV-1) to confirm efficacy of therapeutic agents. known to be continuously infected. Known and readily available H9 cellular No known infection (cited by Poporic etat (1984)) Used with cell lines HOT and CEM.

All cell lines were grown every 4 days by removing the supernatant medium and adding fresh growth medium, i.e. 0% fetal bovine serum, 10,000 units/1d penicillin and 50ugss/ d was replaced and maintained with RPM 11640 containing streptomycin.

RPMI is a standard growth medium.

Li1ly Co, Indian as a preparation for intravenous administration at a concentration of 0.5 ■/d Colchicine, a drug obtained from A-Polis Indiana, is properly diluted. of 0.1, 1.0 and 108 m, which is equivalent to the plasma levels that can be reached in humans. level was added to the medium. The cells were grown in maintenance media (maintenance media). m) pre-treated with colchicine for 60 minutes before dyeing without further addition of drugs. Alternatively, add colchicine to the maintenance medium and then change the medium every 4 days. At that time, it was included. Cultures are usually observed for 20 days and several In the experiment, observations were made until 24 days after infection. Standard Tribane Blue Exclusion Test ( All viability was determined by trypan blue exclusion test) was measured.

Viral replication is dependent on specific structural components associated with retroviral peptide replication. was evaluated by measuring the concentration of that released into the culture supernatant. These are reverse transcription enzymes and P24 antigen. P24 antigen is Viral reverse transcriptase and P24 are collected by precipitation or centrifugation for measurement. .

Reverse transcriptase was described by O' Shaughnessy et al. By law, Yoshida et al. al (1982) P NAs USA 79: 2031), Measured.

By this method, polyA-dependent reverse transcriptase activity can be determined in cells using the following reaction mixture. was measured in virus particle preparations derived from media lacking .

50ttl of 50mM) Squirrel HC1! , pH 7,8; 56m dithiothlate 100mM MC1, 10mM HgC1z 10. crm (H) DTTP (160λ/mM), template containing 7-7-2p 0. 1% TritonX-100, 2ttgs polyA, 0.4μgs dT12 -18 and appropriate amount of virus preparation (as RT source) 0 reaction mixture at 37 °C for 1 h. The ('H)dTMP incorporated into the polymer was incubated at 0°C. precipitated with 10% trichloroacetic acid, collected on a glass fiber filter, Measured using a liquid scintillation counter.

Abbott antigen kits are readily available commercially. Operation is standard EL I SA method, in which surface-immobilized antibodies are incubated with a test composition. , and after appropriate washing, incubate again with another antibody labeled with an enzyme conjugate. It is intended to be

The enzyme is then dissociated under conditions that make the reaction products detectable.

In one test, colchicine at the above dilution was added to Hlv-1 infected cells. InH9 was added to a growing culture. In the first test 4 days after adding colchicine There was a 67% decrease in reverse transcriptase production compared to control values, and by day 8, The value in the treated cell line is that of the control that remained throughout the culture period. It was less than 10%.

For P24, the concentration decreased to 50% of the control by the 122nd day, and by the 24th day It continued to drop until the eyes. On the 24th day, the measurements showed a slight increase; It is not sensitive enough to detect small changes in virus particle concentration. This may be due to a different measurement method.

In another test, uninfected cell lines HυT and CEM were tested first. pre-treated with colchicine at the concentration described for 1 hour, then treated with phosphate-buffered saline for 3 hours. Washed twice. They were then used as a source of fresh staining with the addition of supernatant from H9-infected cells. Resuspend in nutrient medium. After incubation for 1 hour, the colchicine-containing medium was reduced to 0. 01 or 0.1 μm. These concentrations were maintained throughout the test period. The upper level was tested every 4 days. Cell line pretreatment with colchicine and its Infection of the line with HTV-1 leads to decreased P24 antigen production and This resulted in a continued decline until day 24. Reverse transcriptase levels are also It decreased like that.

The amount of silver that will be effective in treating people suffering from any of the previously mentioned infections is determined by the physician. (It will vary depending on many factors, which are known and recognized.) et al., which may include, for example, the patient's age, weight, and disease status. Amounts of approximately oral and extraintestinal dosage units can be prepared by standard methods; Also contains selected active compounds as the only or major active ingredient in the composition A wide variety of known inert excipients may be used to formulate compositions useful in the practice of this invention. It can be used for manufacturing.

These include, for example, dextrose, starch, talc, various types of clay (c lay), mineral oil, cotton seed or sesame oil as well as therapeutic agents. , or may be suspended with the aid of known surfactants. water or various miscible or Including immiscible aqueous compositions.

For buccal and sublingual administration, the active ingredient may be combined with lactose or other suitable carbohydrates. It can be formulated in tablet form with a water-soluble binder.

For rectal administration, such as cocoa butter, petrolatum, or other natural lubricants. agent or polyethylene glycol 1000 or polyethylene glycol Herbal medicines containing active ingredients dispersed in synthetic emollients such as Recall 4000 or inserts may be used.

The active agents of the present invention can be used in sustained release dosage forms. It is convenient to administer via ace dosage forms. This is always Avoiding the need to look at the clock or intervene in normal daily activities. suitable for such preparations. Several compositions are known and may also be used.

For oral administration, the therapeutic agents of choice include Brazilian wax, palm wax, and cellulose. esters and ethers, fats, oils, keratin, gluten or various natural or Tablets that disintegrate over time or are coated with known materials of varying thickness such as synthetic esters. may be placed in pellets, such as stearic acid or castor oil cores. Tablets containing the selected drug in a slowly dissolving core are useful. the drug itself and coated with substances that dissolve at different rates, such as dehydrogenated castor oil or fatty acids. Mixed release granule tablets consisting of a mixture of drugs in separate particles may also be used. . Others include active substances and ion exchange resins, such as sulfuric acid-type cation exchange resins. It can also be combined with

Several transdermal formulations may be used in the practice of this invention. they are built It is a discrete dosage form with a system that applies to the skin. Once absorbed, the therapeutic agent is released through the skin at a controlled rate and circulated throughout the body. So The system typically includes an outer coated barrier, a drug reservoir that may have a release rate controlling membrane. Kurabe, a part or parts of the system at the system/skin JIB interface Adhesive contacts applied to parts of and protection removed before passing through the system Consists of layers.

The drug reservoir is usually polyvinylpyrrolidone or polyvinylpyrrolidone from which the drug is slowly released. or some type of polymer matrix, such as silicone polymer. Po Microporous membranes such as lipropylene film can serve as membranes to control release rates. I can get it.

Procedural amendment (salmon) March 6th, 1990

Claims (4)

[Claims] 1. Treating human patients infected with retroviruses of the lentivirus subgroup A method of treating a human with a therapeutically effective amount of colchicine or colchicine. A method comprising treating with a compound having similar activity. 2. 2. The method of claim 1, wherein the therapeutically effective compound is colchicine. 3. 3. The method of claim 2, wherein the therapeutically effective amount is about 1 to 2 mgs daily. 4. Claim 1, wherein the retrovirus is HIV-1, HIV-2 or HTLV. , method 2 or 3.