Physiologically active substances, a process for preparation and pharmaceutical compositions thereof.

The present invention relates to physiologically active substances extracted from infected tissues, a process for the preparation and pharmaceutical compositions thereof.

As a result of investigations for physiologically active substances extracted from infected tissues and produced by inoculation with a poxvirus to animal tissues, organs or cultured cells, the novel substances extracted from infected tissues have been found having excellent pharmacological actions such as analgesic, sedative and anti-inflammatory actions, especially on diseased animals, for example, animals under stresses.

Oral drugs are easy to be taken for patients and preferable to other preparations such as injections. But many drugs do not exhibit their effects at oral administrations. It has been elucidated that the physiologically active substances of the present invention show evident therapeutic effects at both oral and parenteral administrations.

An object of the present invention is to provide novel physiologically active substances extracted from infected tissues. Another object of the invention is to provide a process for the preparation of the substances. A further object of the invention is to provide pharmaceutical compositions containing the physiologically active substances as an active ingredient.

The physiologically active substances of the present invention can be prepared as follows: <ul id="ul0001" list-style="none"><li>1) Infected tissues are homogenized with an extraction medium, and tissue fragments are removed.</li><li>2) Extracted solution thus obtained is subjected to treatment to remove proteins.</li><li>3) An adsorbent is added to the deproteinized solution, and then the material adsorbed onto the adsorbent is eluted.</li><li>4) The eluted material in aqueous solution is added to ethanol, and the precipitate is collected.</li><li>5) The resulting product is further fractionated on molecular weight basis to give the substances of the present invention.</li></ul>

The term "infected tissues" as used in this specification is defined as meaning: animal tissues, organs or cultured cells inoculated or infected with a poxvirus.

A poxvirus, for example, orthopoxvirus such as vaccinia virus, cowpox virus, variola virus, infectious ectromelia virus or monkeypox virus, parapoxvirus such as orf virus, paravaccinia virus or bovine papular stomatitis virus, capricopoxvirus such as sheeppox virus, goatpox virus or lumpy skin disease virus, avipoxvirus such as fowlpox virus or hare fibroma virus, leporipoxvirus such as rabbit myxoma virus or rabbit fibroma virus, swinepoxvirus, Yaba monkey tumor virus or Tarapox virus, can be used.

To obtain the infected tissues, various kinds of animals or birds can be utilized, for example, rabbit, sheep, goat, pig, cow, horse, monkey, hamster, guinea pig, rat, mouse or hen can be employed. The animal or bird can be selected according to a specie of poxvirus and other conditions. Also any kind of cultured cell, in which the selected poxvirus can multiply, is available, for example, cultured cell or tumor cell of kidney, skin, lung, testis, liver, muscle, adrenal, thyroid gland, brain, nerve cell or blood cell of rabbit, sheep, goat, pig, cow, horse, monkey, hamster, guinea pig, rat, mouse, hen or their embryo, cultured cell derived from human such as Hela cell, or decidua of the hatching egg can be employed.

The infected tissues are collected under aseptic conditions and ground to as small a size as possible. An extraction medium is added to the ground material which is then homogenized. As an extraction medium, distilled water, physiological saline, weakly acidic or basic buffer etc. may be used, and if desired, a stabilizer such as glycerin, a disinfectant or preservative such as phenol, or an inorganic salt such as sodium chloride, potassium chloride or magnesium chloride can be added to the medium. At that time, the extraction can be facilitated by a procedure to disintegrate cell tissues, such as freeze-thaw extraction, sonication or treatment with a detergent or an enzyme dissolving cell membrane.

The resulting emulsion is filtered or centrifuged to remove tissue fragments. The filtrate or supernatant is deproteinized which can be carried out according to a known method, for example, heating, sonication, treatment with a protein-denaturant such as an acid, a base, urea, guanidine, an organic solvent or a detergent, iso-electric point precipitation or salting-out technique. Subsequently, the denatured proteins thereby precipitated are removed by filtration using a filter paper such as cellulose or nitrocellulose, a glass filter, sellaite, Seitz's filter etc., ultrafiltration, gel filtration, ion-­exchange chromatography or centrifugation.

The resulting extract containing the active substances is acidified, preferably to pH 3.5 - 5.5, by addition of an acid such as hydrochloric acid, sulfuric acid or hydrobromic acid, and then subjected to adsorption to an adsorbent such as active carbon, kaolin or an ion-­exchange resin. The adsorbent can be added to the extracted solution and it is stirred, or the extracted solution can be passed through a column of the adsorbent.

To elute the material containing the active substances of the present invention, a basic solution is added to the absorbent, preferably adjusting the suspension to pH 9 - 12, and then the mixture is incubated or stirred at room temperature or at a suitable temperature above room temperature by heating. The elution is achieved by removing the absorbent according to a known method such as filtration or centrifugation.

The eluate thus obtained is concentrated, or concentrated to dryness and dissolved in water. The aqueous solution of eluted material is added to ethanol to be from 60% to 95%, preferably from 70% to 90% of ethanol concentration. The precipitated material is collected by a conventional way such as filtration or centrifugation.

After the collected precipitate is dissolved in water, fractionation based on molecular weight such as ultrafiltration or gel filtration is carried out to give the active substances of the present invention.

The following examples, which are illustrative only and not intended to limit the scope of the invention, describe the preparation of the physiologically active substances of the present invention.

Example 1

Vaccinia virus was inoculated to the skin of healthy adult rabbit. The inflamed skin was cut off under aseptic conditions and well ground. Aqueous phenol solution was added to this ground material and subjected to homogenization, and the emulsion was filtered by centrifugation. The resulting filtrate was adjusted to pH 4.8 - 5.5, and then heated in a stream of 100°C steam. After removing proteins thereby precipitated by filtration, the filtrate was adjusted to about pH 9.0 by addition of sodium hydroxide, heated at 100°C and filtered. The filtrate was adjusted to pH 3.5 - 5.0 and 7.5% - 15% active carbon was added thereto. After stirring for 3 - 5 hrs, the suspension was filtered. Water was added to the resulting active carbon and the suspension was adjusted to pH 10.5 - 11.5 by addition of sodium hydroxide. The extraction procedure was carried out by stirring for 1.5 - 3.0 hrs at 45 - 60°C. The suspension was filtered to remove the active carbon. The filtrate was adjusted to near neutral pH by addition of hydrochloric acid and concentrated to dryness under reduced pressure. The yield of the product was 4 - 6 g from 1 kg of infected tissues.

Example 2

The product obtained in Example 1 was dissolved in distilled water. The aqueous solution was added to about 2.5 to 9 times volume of ethanol and the precipitate was collected by filtration. The collected precipitate was dissolved in distilled water and any insoluble matter was removed by filtration. Subsequently, the solution was filtered through a ultrafiltration membrane eliminating substances molecular weight of which is more than 10,000. To the remaining solution concentrated to small amount, distilled water was added and it was filtrated again. The procedure was repeated several times. As the resulting filtrate, the physiologically active substances of the present invention were obtained.

The substances of the invention thus obtained was concentrated to dryness under reduced pressure. As a result of weighing the dried substances, the yield of the substances of the present invention is 1.0 - 1.5 g when 1 kg of infected skin-tissues of mature rabbit are employed.

The physiologically active substances obtained in the above examples is pale yellowish brown and hygroscopic powder with a slightly peculiar smell; having a molecular weight of less than 10,000; having a UV adsorption maximum at 265 - 275 nm; comprising amino acids such as tyrosine, phenylalanine and alanine, organic acids such as urocanic acid, lactic acid and oxalic acid, bases of nucleic acid such as adenine, thymine, guanine and cytosine, sugar, uracil, xanthine, phosphorus; being negative on various methods of protein analysis; being stable for digestive enzymes such as pepsin, trypsin, chymotrypsin, amylase and lipase; and having no antigenicity.

The following descriptions serve to illustrative pharmaceutical studies of the substances of the present invention.

I. Toxicity test

The physiologically active substances of the present invention were administered to groups each consisting of 10 SD-JCL strain female rats, ddy strain female mice and Japanese white rabbits orally. LD₅₀ of the substances of this invention were more than 10,000 mg/kg independently of species of animals and differences between the sexes. It was found that the substances of the present invention had very low toxicity.

As a result of subacute toxicity tests, no abnormality was observed at any organs. The reproduction tests showed no effect on pregnant animal, fetus, newborn and reproductivity of offspring (F₁).

II. Pharmacological study

(1) Analgesic effect

Analgesic effect of the substances of the present invention was studied by analgesic test methods such as Acetic acid method and Randall-Selitto method.

Acetic acid method: after intraperitoneal injection of acetic acid in physiological saline to mice, the frequency of writhing was counted.

Randall-Selitto method: pressure was applied to the tail of mice and the analgesic coefficients are determined by the minimum pressure by which animals started to escape.

ED₅₀ value of the substances of the present invention in SART-stressed animal [Folia Pharmacol. Jap.; <u style="single">71</u>, 195-­210 (1975)] was from one third to two fifth of the value in experiments using normal animals. The substances of the present invention showed more remarkable effect shown in analgesic tests using SART-stressed animals than in experiments using normal animals. Furthermore, SART-­stressed animals showed significant lowering of the pain thresholds compared with the normal animals, i.e. they are falling into hyperalgesic state. In SART-stressed animals the substances of the present invention evidently restored the pain threshold to normal level.

(2) Sedative effect

Significant supression of spontaneous motor activity and exploratory movement was observed in mice administered with the substances of the invention in comparison to a control group.

(3) Clinical study

Comparative clinical studies were carried out between drugs on the market and the pharmaceutical composition containing the substances of the present invention as an active ingredient. Therapeutic effects of the pharmaceutical composition of the present invention in the treatment of low back pain were compared with Ketoprofen. 16 mg daily of the active ingredient as the substances of the present invention was orally administered for 2 weeks to patient suffering from low back pain. In the case of Ketoprofen, 150 mg daily was administered in the same manner. Improvement rate of the group of the pharmaceutical composition of the present invention was equal to that of the Ketoprofen group, but safety of this pharmaceutical composition was significantly superior to that of Ketoprofen.

Also the comparative test with Indomethacin was done in osteoarthritis of the knee. Equal dosage of the physiologically active substances of the invention as mentioned above and 75 mg daily of Indomethacin were orally administered for 4 weeks to patents suffering from osteoarthritis of the knee. Improvement rate of the group of the pharmaceutical composition of the present invention was equal to that of the Ketoprofen group, but with respect to safety the present composition was clearly superior to Indomethacin.

As shown by the above-mentioned results, the physiologically active substances of the present invention have excellent therapeutic effects on dolorous diseases such as neuralgia, osteoarthritis of knee, low back pain, articular rheumatism and pain of gout, especially chronic symptoms of these diseases.

The above pharmacological studies using the animal models for human disease, such as stressed animals, showed that the substances of the present invention have a characteristic effect improving the abnormal hyperalgesic state under the conditions of the lowered pain threshold, and restoring the pain threshold to normal level. Namely the substances of this invention show remarkable effects especially on diseased animals which are not in a normal state.

The physiologically active substances of the present invention have therapeutic effects at even oral administration as well as have low toxicity and great safety compared with drugs on the market such as Ketoprofen and Indomethacin. Therefore, the substances of the invention can be used for long-term continuous administrations and so have the advantage of treatment for a chronic diseases.

The substances of the present invention can be made into pharmaceutical compositions by combination with appropriate medicinal carriers or diluents, and can be formulated into preparations in solid or liquid form such as tablets, capsules, powders, granules, syrups, injections or suppositories in usual ways for oral or parenteral administrations.

In pharmaceutical dosage forms, the substances of the present invention can be used in the form of their pharmaceutically acceptable salts, and also can be used alone or in appropriate association, as well as in combination with other pharmaceutically active components.

In case of oral preparations, the substances can be used alone or combined with appropriate additives to make tablets, capsules, powders, granules or syrups, e.g. with conventional additives such as lactose, mannitol, corn starch or potato starch; with binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators such as corn starch, potato starch or calcium carboxymethylcellulose; with lubricants such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

The substances of the present invention can be formulated into a preparations for injections or suppositories according to the conventional method.

The desirable dose of the substances of the present invention varies with the subject, form of the drug, method and period of administration. However, in order to obtain desirable effects, generally it is recommended to administer orally 1 to 100 mg, preferably 4 to 40 mg daily for an adult.

In case of parenteral administrations such as injections, doses of the substances in the order of one tenth to one third of the above dose are preferable as daily doses.

A prescription of the pharmaceutical compositions is shown below as one of examples which contain the substances of the present invention as an active ingredient. <tables id="tabl0001" num="0001"><table frame="all"><tgroup cols="2" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="78.75mm" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><thead valign="top"><row><entry namest="col1" nameend="col2" align="left">Prescription example 1 (tablet)</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="center">Component</entry><entry namest="col2" nameend="col2" align="center">Content in a tablet (mg)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">substances of this invention</entry><entry namest="col2" nameend="col2" align="right">4</entry></row><row><entry namest="col1" nameend="col1" align="left">lactose</entry><entry namest="col2" nameend="col2" align="right">106</entry></row><row><entry namest="col1" nameend="col1" align="left">crystalline cellulose</entry><entry namest="col2" nameend="col2" align="right">40</entry></row><row><entry namest="col1" nameend="col1" align="left">calcium carboxymethylcellulose</entry><entry namest="col2" nameend="col2" align="right">20</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">magnesium stearate</entry><entry namest="col2" nameend="col2" align="right">10</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="right">Total 180 mg</entry></row></tbody></tgroup></table></tables>