Preventing agent for human immunodeficiency virus infection comprising menfegol.

BACKGROUND OF THE INVENTION

1) Field of the Invention:

0001This invention relates to a preventing agent for human immunodeficiency virus (HIV) infection suitable for external application, which comprises menfegol as an active ingredient. More specifically, this invention is concerned with a preventing agent for HIV infection which contains menfegol as an active ingredient in a form of a jelly preparation, foaming tablets, an ointment, vaginal suppositories or the like. This invention also embraces therein condoms applied with said jelly preparation.

2) Description of the Related Art:

0002Acquired immunodeficiency syndrome (AIDS) is a disease caused by cytopathogenicity and decrease of helper T cells (Cluster of Differentiation type 4 antigen positive cells) due to infection by HIV. AIDS patients will go on to develop opportunistic infections such as pneumocytis carinii pneumonia, and malignant tumors like Kaposi's sarcoma, etc., resulting in their deaths eventually. Although HIV infection may take place by breast-feeding, the administration of a blood preparation, etc., it occurs above all by heterosexual or homosexual intercourse. Thus, the increase of AIDS patients has become a serious social problem in recent years. A wide variety of research has been conducted for the prevention and treatment of AIDS, but no AIDS drug whose effectiveness has been confirmed has yet been developed. For the prevention of AIDS, it is hence important to avoid HIV infection. Use of condoms is recommended for this purpose. condoms are however accompanied by a potential danger of breakage or slip-off and are not considered to be perfect for the prevention of HIV infection.

SUMMARY OF THE INVENTION

0003An object of this invention is to provide a preventing agent for HIV infection suitable for external application.

0004Another object of this invention is to provide a condom with such an HIV infection preventing agent.

0005It has now been found that menfegol has strong anti-HIV activity,and is useful for preventing AIDS.

0006Menfegol, i.e. p-menthanylphenyl polyoxy-ethylene (8.8) ether having an average molecular weight of 620.03, is a nonionic surfactant. Menfegol has been used widely as a spermatocide, but other medical uses are yet unknown.

0007In one aspect of this invention, there is thus provided a preventing agent for HIV infection comprising menfegol as an active ingredient. The HIV infection preventive may preferably be in the form of a jelly preparation, foaming tablets, ointment or vaginal suppositories.

0008In another aspect of this invention, there is also provided a condom applied with said jelly preparation.

0009According to this invention, menfegol can be formulated into a preparation form suitable for usual external application, for example, jelly preprations, foaming tablets, ointment, vaginal suppositories, etc. Application of such a preparation into the vagina before coitus makes it possible to prevent HIV infection. In the case of a jelly preparation, it can be used along with a condom to prevent HIV infection doubly.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

0010Menfegol can be prepared, for example, by using turpentine oil as a starting material. Turpentine oil is converted into p-menthanylphenol via p-menthadiene and p-menthene. p-Menthanylphenol is then reacted with ethylene oxide in the presence of an alkali catalyst to obtain menfegol. [see K. Furuse: Perfumerie Cosmetique Savons, <u style="single">10</u>, 342 (1967)].

0011Although menfegol is considered to be effective when used in an amount of 2 mg per time in view of its anti-HIV activities and the sperm volume (normally, about 10 mℓ) per ejaculation, it is preferable to use it in an amount of 10-100 mg per each time to ensure its sufficient effect.

0012Anti-HIV effects of menfegol and menfegol-containing preparations according to this invention will be demonstrated in the following examples.

Example 1:

Anti-HIV Test

0013In this test, there was used, as an HIV strain, lymphadenopathy associated virus (LAV) which was allowed to grow in CEM cells, a cell strain of the CD4+T cell line (Cluster of Differentiation type 4 antigen positive cell line). The LAV was furnished by Pasteur Institute, France.

0014The following cells and virus culture operations were all carried out at 37°C in a CO₂ incubator.

0015CEM cells (1 x 10⁷ cells/mℓ) infected with LAV (antigen expression rate: 80-100%) were cultured overnight in RPMI-1640 medium containing 10 mM of N-2-hydroxyethyl­piperazine-N-2-ethanesulfonic acid and 10% of fetal calf serum (hereinafter called the "medium A"). The virus preparation in culture medium was divided into 6 portions. Five portions were added with menfegol to give final concentrations of 2.5 mM, 1.0 mM, 0.25 mM, 0.0625 mM and 0.0156 mM, respectively. The remaining one portion was added with same volume of the medium A to provide a control. After incubating those mixtures of virus and menfegol at room temperature for 5 minites to allow menfegol act on the LAV, those were applied on Sephadex G-200 (trade name; Product of Pharmacia AB) which was swallen by distilled water and equilibrated with medium A. By the spin column technique (2,000 rpm, 30 seconds), menfegol was removed from the respective mixtures. Removal of menfegol was carried for eliminating any direct cytotoxicity of menfegol against T-lymphocytes.

0016On the other hand, 100 µℓ of MT-4 cells (a T lymphocyte strain of the Cluster of Differentiation type 4 antigen positive cell line) suspended at 5 x 10⁵ cells/mℓ in the medium A were added to the respective wells of a plastic plate (8 rows x 12 columns) for tissue culture. Then, the wells were added respectively with 100 µℓ portions of tenfold stepwise (10⁰-10⁻⁹ diluted solutions of the menfegol-removed virus sample. After 10-days incubation, the virus titer (TCID₅₀) in each sample was determined by the triple blank test, using the indirect fluorescent antibody technique.

0017The results are shown in the following table. <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="col1" align="center"><u style="single">Amount of menfegol added, mM</u></entry><entry namest="col2" nameend="col2" align="center"><u style="single">Virus Titer, (TCID₅₀/mℓ)</u></entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">0 (Control)</entry><entry namest="col2" nameend="col2" align="right">10^5.13</entry></row><row><entry namest="col1" nameend="col1" align="left">2.5</entry><entry namest="col2" nameend="col2" align="right">&lt;10^0.3</entry></row><row><entry namest="col1" nameend="col1" align="left">1.0</entry><entry namest="col2" nameend="col2" align="right">&lt;10^0.3</entry></row><row><entry namest="col1" nameend="col1" align="left">0.25</entry><entry namest="col2" nameend="col2" align="right">&lt;10^0.3</entry></row><row><entry namest="col1" nameend="col1" align="left">0.0625</entry><entry namest="col2" nameend="col2" align="right">&lt;10^3.13</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">0.0156</entry><entry namest="col2" nameend="col2" align="right">&lt;10^4.47</entry></row></tbody></tgroup></table></tables>

0018From the foregoing results, menfegol has been found to complete inactivate the HIV at a concentration of at least 0.25 mM (0.016% w/v).

Example 2:

Foaming Tablets

0019<tables id="tabl0002" num="0002"><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="col1" align="center"><u style="single">Ingredient</u></entry><entry namest="col2" nameend="col2" align="center"><u style="single">Weight per tablet, mg</u></entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">Menfegol</entry><entry namest="col2" nameend="col2" align="char" char=".">60.0</entry></row><row><entry namest="col1" nameend="col1" align="left">Dioctyl sodium sulfosuccinate</entry><entry namest="col2" nameend="col2" align="char" char=".">5.0</entry></row><row><entry namest="col1" nameend="col1" align="left">Dried egg albumen</entry><entry namest="col2" nameend="col2" align="char" char=".">12.5</entry></row><row><entry namest="col1" nameend="col1" align="left">Light silicic anhydride</entry><entry namest="col2" nameend="col2" align="char" char=".">105.0</entry></row><row><entry namest="col1" nameend="col1" align="left">Crystalline cellulose</entry><entry namest="col2" nameend="col2" align="char" char=".">30.0</entry></row><row><entry namest="col1" nameend="col1" align="left">Potassium hydrogentartrate</entry><entry namest="col2" nameend="col2" align="char" char=".">326.0</entry></row><row><entry namest="col1" nameend="col1" align="left">Sodium bicarbonate</entry><entry namest="col2" nameend="col2" align="char" char=".">160.0</entry></row><row><entry namest="col1" nameend="col1" align="left">Polyvinylpyrrolidone (K-30)</entry><entry namest="col2" nameend="col2" align="char" char=".">17.0</entry></row><row><entry namest="col1" nameend="col1" align="left">Corn starch</entry><entry namest="col2" nameend="col2" align="char" char="."><u style="single">84.5</u></entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">TOTAL</entry><entry namest="col2" nameend="col2" align="char" char=".">800.0</entry></row></tbody></tgroup></table></tables>

Preparation

0020Light silicic anhydride (2/3 portion), potassium hydrogentartrate and corn starch were mixed, to which menfegol, a solution of dioctyl sodium sulfosuccinate in a mixture of water and ethanol (1 : 1 V/V) and an aqueous solution of polyvinylpyrrolidone (K-30) were added successively. The resulting mixture was mixed thoroughly, granulated, and then dried to obtain menfegol-containing granules.

0021Light silicic anhydride (1/3 portion), sodium bicarbonate and crystalline cellulose were mixed thoroughly, to which the menfegol-containing granules prepared above were added, followed by the addition of dried egg albumen. The resulting mixture was mixed uniformly. The thus-obtained mixture was compressed into tablets having a weight of 800 mg. The thus-obtained tablets contain 60.0 mg of menfegol.

Example 3:

Jelly Preparation

0022<tables id="tabl0003" num="0003"><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="col1" align="center"><u style="single">Ingredient</u></entry><entry namest="col2" nameend="col2" align="center"><u style="single">Weight, mg</u></entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">Menfegol</entry><entry namest="col2" nameend="col2" align="char" char=".">20.00</entry></row><row><entry namest="col1" nameend="col1" align="left">Polyethylene glycol #400</entry><entry namest="col2" nameend="col2" align="char" char=".">519.40</entry></row><row><entry namest="col1" nameend="col1" align="left">Polyethylene glycol #4000</entry><entry namest="col2" nameend="col2" align="char" char=".">60.00</entry></row><row><entry namest="col1" nameend="col1" align="left">Methylparaben</entry><entry namest="col2" nameend="col2" align="char" char=".">0.45</entry></row><row><entry namest="col1" nameend="col1" align="left">Propylparaben</entry><entry namest="col2" nameend="col2" align="char" char="."><u style="single">0.15</u></entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">TOTAL</entry><entry namest="col2" nameend="col2" align="char" char=".">600.00</entry></row></tbody></tgroup></table></tables>

Preparation

0023Polyethylene glycol #400 and polyethylene glycol #4000 were molten at 60 - 65°C. To the melt, menfegol, methylparaben and propylparaben were added successively under stirring, at same temperature. The resulting mixture was stirred intimately and then allowed to cool gradually to room temperature, thereby obtaining a jelly preparation.

Example 4:

Condom with jelly preparation

0024Jelly preparation (600 mg), which had been formulated in accordance with Example 3, was heated to about 80°C to enhance its flowability. In this state, the jelly preparation was added dropwise to an inner tip wall portion (semen catch) of a main body of a condom and was then allowed to cool to room temperature gradually. A condom applied with the jelly preparation was thus obtained.