Use of porphyrins and metalloporphyrins in the treatment of diseases caused by Human Immunodeficiency Viruses.

BACKGROUND OF THE INVENTION

0001The present invention relates to the treatment of diseases caused by Human Immunodeficiency Viruses (hereinafter, referred to as HIVs).

0002HIVs have been known as the causative viruses for AIDS (Acquired Immunodeficiency Syndrome). AIDS is a viral disease in which T-cells are attacked and immunological mechanism is directly destroyed. As the result, opportunistic infections such as pneumocystis carinii pneumonia and candidosis, malignant tumors such as Kaposi sarcoma or mental disorders appear in patients, and once onset of AIDS has occurred, patients die within a short period of half or one year unless any effective therapeutic measure is taken. Therefore, it has been dread as a disease with unfavorable prognosis.

0003Accordingly, prevalence of AIDS as dangerous disease is becoming a problem of utmost importance on public health as it causes social unrest and other inconvenient situation such as discrimination against patients. The development of a medicine for treating AIDS is thus a matter not only of great importance but also of exceptional urgency.

0004Examples of medicines presently known to have antiviral effect against HIVs are nucleic acid antiviral agents such as Azidothymidine (AZT) and special plant products such as Glycyrrhizin (from Glycyrrhiza). These medicines, however, have deficiencies in that the nucleic acid antiviral agents have serious toxity and the plant products have not sufficient efficacy. Consequently, there has been a continuous need for anti-HIV agents having sufficient activity and less side effect.

0005The present inventors have searched for substances having anti-HIV activity within known products having higher safety and successfully found out that Protoporphyrin disodium salt, which has been marketed as hepatic and hence has established safety, has a potent anti-HIV activity and that also some of its analogues have similar activity.

SUMMARY OF THE INVENTION

0006In one aspect, the present invention provides a method of treatment for diseases caused by Human Immunodeficiency Viruses (HIVs), which comprises administering, to a human subject in need of such treatment, a therapeutically effective amount of a compound of the formula; <chemistry id="chem0001" num="0001"><img file="EP0337598A2_D0001.tif" /></chemistry> wherein either R₁, R₂, R₃, R₄, R₅, R₆, R₇ and R₈ are the same or different, each hydrogen atom, a lower alkyl group, a lower alkenyl group, a hydroxy(lower)alkyl group, a carboxy group or a carboxy(lower)alkyl group, R₉ is hydrogen atom or a carboxy(lower)alkyl group, with the proviso that at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a hydroxy(lower)alkyl group, a carboxy group or a carboxy(lower)alkyl group, and R₁₀ is hydrogen atom or a lower alkyl group, or R₆ and R₁₀ together form the group -CO-CH₂- and R₁, R₂, R₃, R₄, R₅, R₇, R₈ and R₉ are as defined above, and the symbol of a line and a dotted line means a single bond or a double bond, an N-(lower)alkyl derivative, a metal coordination compound, a physiologically acceptable and hydrolyzable ester or a physiologically acceptable salt thereof.

0007In another aspect, the present invention provides a use of the compound of the formula; <chemistry id="chem0002" num="0002"><img file="EP0337598A2_D0002.tif" /></chemistry> wherein either R₁ R₂, R₃, R₄, R₅, R₆, R₇ and R₈ are the same or different, each hydrogen atom, a lower alkyl group, a lower alkenyl group, a hydroxy(lower)alkyl group, a carboxy group or a carboxy(lower)alkyl group, R₉ is hydrogen atom or a carboxy(lower)alkyl group, with the proviso that at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a hydroxy(lower)alkyl group, a carboxy group or a carboxy(lower)alkyl group, and R₁₀ is hydrogen atom or a lower alkyl group, or R₆ and R₁₀ together form the group -CO-CH₂- and R₁, R₂, R₃, R₄, R₅, R₇, R₈ and R₉ are as defined above, and the symbol of a line and a dotted line means a single bond or a double bond, an N-(lower)alkyl derivative, a metal coordination compound, a physiologically acceptable and hydrolyzable ester or a physiologically acceptable salt thereof for the manufacture of a medicament for treatment of diseases caused by HIVs.

0008In a further aspect, the present invention provides a pharmaceutical composition comprising the compound of the formula; <chemistry id="chem0003" num="0003"><img file="EP0337598A2_D0003.tif" /></chemistry> wherein either R₁, R₂, R₃, R₄, R₅, R₆, R₇ and R₈ are the same or different, each hydrogen atom, a lower alkyl group, a lower alkenyl group, a hydroxy(lower)alkyl group, a carboxy group or a carboxy(lower)alkyl group, R₉ is hydrogen atom or a carboxy(lower)alkyl group, with the proviso that at least one of R₁, R₂, R₃, R₄, R₅, R₇, R₈ and R₉ is a hydroxy(lower)alkyl group, a carboxy group or a carboxy(lower)alkyl group, and R₁₀ is hydrogen atom or a lower alkyl group, or R₆ and R₁₀ together form the group -CO-CH₂- and R₁, R₂, R₃, R₄, R₅, R₇, R₈ and R₉ are as defined above, and the symbol of a line and a dotted line means a single bond or a double bond, an N-(lower)alkyl derivative, a metal coordination compound, a physiologically acceptable and hydrolyzable ester or a physiologically acceptable salt thereof as an active ingredient in association with a pharmaceutically acceptable carrier, diluent or excipient.

DETAILED DESCRIPTION OF THE INVENTION

0009The terms and definitions described herein are illustrated as follows.

0010Porphyrins are well known to lie in tautomeric forms. It is to be understood that all the tautomeric forms are regarded as the same and hence included in the scope of the invention and that the above formula is used only for the sake of convenience.

0011The term "lower" is used to intend a group having up to 6 carbon atoms, unless otherwise provided.

0012The term "lower alkyl group" includes straight or branched chain saturated hydrocarbon groups having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl.

0013The term "lower alkenyl group" includes straight or branched chain unsaturated hydrocarbon groups having 2 to 6 carbon atoms, such as vinyl, allyl, 1-propenyl, butenyl, pentenyl and hexenyl.

0014The term "hydroxy(lower)alkyl group" includes lower alkyl groups as defined above having more than one and preferably up to three hydroxy groups at any possible positions.

0015The term "carboxy(lower)alkyl group" includes lower alkyl groups as defined above having more than one and preferably less than three carboxy groups at any possible positions.

0016Preferred compounds are the compounds of the formula (I), in which either R₁, R₂, R₃, R₄, R₅, R₆, R₇ and R₈ are, the same or different, each hydrogen atom, methyl, ethyl, vinyl, 1-hydroxyethyl, 3-hydroxypropyl, carboxy, caboxymethyl or 2-carboxyethyl, R₉ is hydrogen atom or caboxymethyl, with the proviso that at least one of them is 1-hydroxyethyl, 3-hydroxypropyl, carboxy, caboxymethyl or 2-carboxyethyl, and R₁₀ is hydrogen atom or methyl, or R₆ and R₁₀ together form the group -CO-CH₂- and R₁, R₂, R₃, R₄, R₅, R₇, R₈ and R₉ are as defined above, and the symbol of a line and a dotted line is a single bond or a double bond, an N-methyl derivative or a physiologically acceptable salt thereof.

0017More preferred compounds are the compounds of the formula (I), in which either R₁ is methyl, carboxy, or carboxymethyl, R₂ is hydrogen atom, methyl, ethyl, vinyl, 1-hydroxyethyl, or 2-carboxyethyl, R₃ is methyl, ethyl, carboxymethyl or 2-carboxyethyl, R₄ is hydrogen atom, methyl, ethyl, vinyl, 1-hydroxyethyl, carboxymethyl or 2-carboxyethyl, R₅ is methyl, vinyl, carboxymethyl or 2-carboxyethyl, R₆ is hydrogen atom, methyl, 3-hydroxypropyl, carboxy, carboxymethyl or 2-carboxyethyl R₇ is methyl, 3-hydroxypropyl, carboxymethyl or 2-carboxyethyl, R₈ is methyl, carboxymethyl or 2-carboxyethyl, R₉ is hydrogen atom or carboxymethyl, and R₁₀ is hydrogen atom or methyl, or R₆ and R₁₀ together form the group -CO-CH₂- and R₁, R₂, R₃, R₄, R₅, R₇, R₈ and R₉ are as defined above.

0018The most preferred compounds are the compounds of the formula (I), in which the symbol of a line and a dotted line is a single bond in the case of Chlorophyllin ligand or a double bond in the other cases, and having the groups shown in the following Table. <tables id="tabl0001" num="0001"><img file="EP0337598A2_D0004.tif" /></tables><tables id="tabl0002" num="0002"><img file="EP0337598A2_D0005.tif" /></tables>

0019While preferred porphyrins used in the invention are naturally occurring isomers, a mixture containing such isomer can also be used in the invention.

0020The N-(lower)alkyl derivatives of the porphyrins of the formula (I) include compounds wherein at least one of N-atoms has a lower alkyl group as defined above.

0021The metal coordination compounds of the porphyrins of the formula (I) include those containing, for example, Mg, Fe(II) (e.g. Heme), Fe(III), FeCl (e.g. Hemin), Co, Cu etc. coordinated as a central atom or ion.

0022The above compounds of formula (I) and the coordination compounds thereof include novel and known compounds. The novel compounds can be prepared by a process similar to that for the known one.

0023As the porphyrins of the formula (I) are amphoteric, the physiologically acceptable salts thereof include both of the salts with acids and the salts with bases. The term "physiologically acceptable salts" refers to the salts which do not show significant toxity at the doses of administration and have not physical or chemical property that makes the administration difficult. Within such salts, the salts with acids include those with inorganic (or mineral) acids such as hydrochloric acid, sulfuric acid, phosphoric acid etc., and those with organic acids such as carboxylic acid, e.g. acetic acid, tartaric acid, citric acid, succinic acid, fumaric acid etc. and sulfonic acid, e.g. methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid etc. The salts with bases include those with inorganic base salt such as alkali metal salts, e.g. sodium salt, potassium salt etc., alkali-earth metal salts, e.g. calcium salt, magnesium salt etc., polyvalent metal salts, e.g. aluminum salt, manganese salt etc. and organic base salts such as organic amine salts, e.g. trimethylamine salt, triethylamine salt, cyclohexylamine salt, ethanolamine salt, diethanolamine salt, tromethamine salt, morpholine salt, piperidine salt, procaine salt, caffeine salt etc. Where plurality of carboxy group are present, any number of said groups may be in the salt form. These salts can be prepared by the conventional process, e.g. by neutralization, salt exchange or ion exchange.

0024The term "physiologically acceptable and physiologically hydrolyzable ester" refers to the esters which do not show significant toxicity at the doses of administration and have not physical or chemical property that makes the administration difficult, and further, can produce the corresponding acids and alcohols, which do not show significant toxicity at the amount to be produced, by hydrolysis under physiological conditions, for example under conditions within human or animal bodies. Such esters include lower aklyl esters such as methyl ester, ethyl ester, propyl ester, isopropyl ester, t-butyl ester, lower alkanoyloxy(lower)aklyl esters such as acetoxymethyl ester, pivaloyloxymethyl ester, 1-[(lower)alkoxycarbonyloxy]-­(lower)alkyl ester such as 1-(ethoxycarbonyloxy)ethyl ester, phthalidyl ester, 5-indanyl ester, 2-(3-phthalidylidene)­ ethyl ester, (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl ester etc. Where plurality of carboxy groups are present, any number of said groups may be in the ester form. These ester can be prepared by the conventional process, e.g. by condensation between a carboxylic acid or a reactive derivative at the carboxy group thereof with an alcohol or a reactive derivative at the hydroxy groups thereof, by ester exchange etc. These esters may have an improved property such as solubility, stability, absorbablity etc. as compared with the free acids.

0025The term "HIVS" includes Lymphadenopathy Associated Virus Type 1 (LAV-1) and Type 2 (LAV-2), Human T-Lymphotropic Virus Type III (HTLV-III), AIDS-Associated Retrovirus (ARV) etc., including variant different in property such as infectiosity to cultivated cells.

0026The term "diseases caused by HIVs" includes AIDS, ARC, PGL and LAS, and in an alternative classification, acute symtoms due to HIV infection, generalized lymphadenopathy, persistent pyrexia, diarrhea, reduction of weight, dementia, bulbar disorder, peripheral nervous disorder, pneumocystis carinii pneumonia, toxoplasmosis, candidosis, Herpesvirus infeciton, Kaposi sarcoma, non-Hodgkin lymphoma, cancer etc.

0027The term "treatment" includes all kinds of control of disease including prophylaxis and therapy such as prevention of infection, prevention of pathopoiesis where infection is established, prevention of deterioration of disease (sustention), reduction of symptoms, changing to asymptomatic carrier and complete recovery.

0028For the usages for carrying out the above treatment, the required dose of the porphyrins or salts thereof will, of course, vary depending on age and condition of particular patient, the compound actually used, the mode of administration and treatment desired. In general, however, satisfactory results are obtained in adminstration at a dosage from 10 to 1000 mg, preferably 50 to 500 mg, conveniently administered in 2 to 4 divided doses a day or in sustained release form.

0029For administration, the porphyrins may be mixed with a pharmaceutical carrier such as organic or inorganic solid or liquid excipient e.g. suitable for local, nasal, peroral, rectal, external administration or injection, and administered in the form of the conventional pharmaceutical preparation. Such preparation includes solids (e.g., tablet, granule, powder, capsule, etc.) and liquids (e.g., liquid, emulsion, suspension, etc.), suppository and ointment. The above carriers include starch, lactose, glucose, sucrose, dextrin, cellulose and derivatives thereof, paraffin, fatty acid glyceride, water, alcohol, gum arabic, etc. If necessary, auxiliary, stabilizer, humectant, emulsifier, lubricant, binder, pH regulating agent, isotonicity agent, and other additives in ordinary use may be added.

0030The toxicity of typical porphyrins, metal coordination compound, salts or ester thereof used in the invention is known and extremely low. For example, toxicity of Protoporphyrin disodium salt is shown below. <tables id="tabl0003" num="0003"><table frame="all"><title>Table 2</title><tgroup cols="3" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="center">Route of Administration</entry><entry namest="col2" nameend="col2" align="center">Animal</entry><entry namest="col3" nameend="col3" align="center">LD₅₀ (mg/kg)</entry></row></thead><tbody valign="top"><row rowsep="0"><entry namest="col1" nameend="col1" morerows="3" rowsep="1" align="left">intravenous</entry><entry namest="col2" nameend="col2" align="left">mouse</entry><entry namest="col3" nameend="col3" align="right">347.3</entry></row><row rowsep="0"><entry namest="col2" nameend="col2" align="left">rat</entry><entry namest="col3" nameend="col3" align="right">240.3</entry></row><row rowsep="0"><entry namest="col2" nameend="col2" align="left">rabbit</entry><entry namest="col3" nameend="col3" align="right">220.0</entry></row><row><entry namest="col2" nameend="col2" align="left">cat</entry><entry namest="col3" nameend="col3" align="right">215.5</entry></row><row><entry namest="col1" nameend="col1" align="left">subcutaneous</entry><entry namest="col2" nameend="col2" align="left">mouse</entry><entry namest="col3" nameend="col3" align="right">1,147.0</entry></row><row><entry namest="col1" nameend="col1" align="left">intraperitoneal</entry><entry namest="col2" nameend="col2" align="left">mouse</entry><entry namest="col3" nameend="col3" align="right">1,028.6</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">oral</entry><entry namest="col2" nameend="col2" align="left">mouse</entry><entry namest="col3" nameend="col3" align="right">> 25,000.0</entry></row></tbody></tgroup></table></tables>

0031The following Examples will illustrate the present invention in further detail. In the examples, the term "active ingredient" refers to any one of the porphyrins, metal coordination compound, salt or ester used in the invention.

Example 1

0032<tables id="tabl0004" num="0004"><table frame="all"><tgroup cols="3" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">Active Ingredient</entry><entry namest="col2" nameend="col2" /><entry namest="col3" nameend="col3" align="right">20 mg</entry></row><row><entry namest="col1" nameend="col1" align="left">Microcrystalline Cellulose</entry><entry namest="col2" nameend="col2" /><entry namest="col3" nameend="col3" align="right">25 mg</entry></row><row><entry namest="col1" nameend="col1" align="left">Hydroxypropyl Cellulose</entry><entry namest="col2" nameend="col2" /><entry namest="col3" nameend="col3" align="right">1 mg</entry></row><row><entry namest="col1" nameend="col1" align="left">Carboxymethyl Cellulose Calcium Salt</entry><entry namest="col2" nameend="col2" /><entry namest="col3" nameend="col3" align="right">5 mg</entry></row><row><entry namest="col1" nameend="col1" align="left">Magnecium Stearate</entry><entry namest="col2" nameend="col2" /><entry namest="col3" nameend="col3" align="right">2 mg</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">Lactose</entry><entry namest="col2" nameend="col2" align="left">q.s. to</entry><entry namest="col3" nameend="col3" align="right">150 mg</entry></row></tbody></tgroup></table></tables>

0033The above ingredients are mixed, granulated by wet process, compressed into tablets and sugar-coated according to the conventional method.

Example 2

0034<tables id="tabl0005" num="0005"><table frame="all"><tgroup cols="3" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">Active Ingredient</entry><entry namest="col2" nameend="col2" /><entry namest="col3" nameend="col3" align="right">50 mg</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">Physiological Saline</entry><entry namest="col2" nameend="col2" align="left">q.s. to</entry><entry namest="col3" nameend="col3" align="right">10 ml</entry></row></tbody></tgroup></table></tables>

Test Example 1

(Preparation of test samples)

0035Test samples were prepared by diluting samples with RPMI-1640 medium supplemented with 10% fetal calf serum (FCS).

(Assay of anti-HIV activity)

0036MT-4 cells (MT-4) were challenged with LAV strain of HIV (10²˙⁷ median tissue culture infectious dose (TCID₅₀/ml) at the multiplicity of infection (m.o.i.) of 0.001 TCID₅₀/cell (or 0.01 TCID₅₀/cell or 0.1 TCID₅₀/cell) for adsorption period of 1 hour, incubated in the presence of test compounds until cytopathic effect (CPE) was observed in control (without the test compounds), and assayed for HIV production. All the procedures were carried out in th dark. Assay was carried out as follows. A microtiter plate (96 wells) was innoculated with MT-4 at 2x10⁴/0.1ml/well, then with the virus suspension obtained above at 0.1 ml/well and observed for CPE during 7 days. Amount of HIV production was shown in terms of TCID₅₀/ml.

0037Compounds were judged as antivirally active when production of HIV was reduced as compared with the control.

0038Protoporphyrin disodium salt (PPNa) was used as the test compound, sodium dextran sulfate (DSNa), glycyrrhizin (GR) and AZT as the references.

(Results)

0039The results are shown in the following Table, wherein numerical values indicate TCID₅₀/ml. Tox means results were not obtained due to cell toxicity. <tables id="tabl0006" num="0006"><img file="EP0337598A2_D0006.tif" /></tables><tables id="tabl0007" num="0007"><img file="EP0337598A2_D0007.tif" /></tables>

Test Example 2

0040The procedure of Test Example 1 was repeated except that, as the test compounds, hemin (HM), hematoporphyrin disodium (HPNa), Protoporphyrin dimethyl ester (PPMe), Reduced Protoporphyrin (Table 1, PPOH), Coproporphyrin III tetramethyl ester (CPIIIMe), Coproporphyrin III tetrasodium (CPIIINa), Uroporphyrin I dihydrochloride (UPIHCl), Deuteroporphyrin dihydrochloride (DPHCl), Mesoporphyrin dihydrochloride (MPHCl), Phylloporphyrin (Phl), Pyrroporphyrin (PyP), Phytoporphyrin (Pht), N-methylmesoporphyrin* (MeMP), N-methylprotoporphyrin * (MePP), Chlorophyllin ligand(Chl) and Chlorophyl(Ch) were used in place of PPNa.

0041The results are shown in the following Table, wherein asterisk (*) indicates a mixture of isomers. <tables id="tabl0008" num="0008"><img file="EP0337598A2_D0008.tif" /></tables><tables id="tabl0009" num="0009"><img file="EP0337598A2_D0009.tif" /></tables><tables id="tabl0010" num="0010"><img file="EP0337598A2_D0010.tif" /></tables>

0042From the above results, it was concluded that the porphyrins used in the invention were antivirally active against HIV.

Test example 3

0043In view of the disclosure of EP 196 515 A1 in which a therapeutic protein composition suspected to contain viruses was exposed to a light with a divided wave length in the presence of various photo-sensitizers including porphyrins, some experiments were conducted to see 1) whether or not other photo-sensitizers than porphyrins have anti-HIV activity in the absence of light and 2) whether or not the porphyrins of the present invention having anti-HIV activity also have antiviral activity against viruses other than HIVs, e.g. against Herpes Simplex Virus (HSV).

1) Test for anti-HIV activity with other photo-sensitizers:

0044The procedure of Test Example 1 was repeated except that, as the test compounds, Methylene Blue (MB), Quinoline Yellow (QY) and Toluidine Blue (TB) were used in place of PPNa. The results are shown in the following Table. <tables id="tabl0011" num="0011"><table frame="all"><title>Table 5</title><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 rowsep="1"><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" rowsep="0" align="center">Concentration (10µg/ml)</entry></row><row><entry namest="col1" nameend="col1" align="left"><u style="single">m.o.i. 0.01</u></entry><entry namest="col2" nameend="col2" /></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">MB</entry><entry namest="col2" nameend="col2" align="right">10⁶</entry></row><row><entry namest="col1" nameend="col1" align="left">QY</entry><entry namest="col2" nameend="col2" align="right">10⁶</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">TB</entry><entry namest="col2" nameend="col2" align="right">10⁶</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">Control</entry><entry namest="col2" nameend="col2" align="right">10⁶</entry></row></tbody></tgroup></table></tables>

0045The above results indicate that they have no antiviral activity.

2) Test for anti-HSV activity with the porphyrins:

Cell and Virus:

0046Herpes simplex virus (HSV) type 1, strain WT-51, and BS-C-40, an African green monkey kidney cell line, were used. HSV stock was prepared in BS-C-40 and the infectivity of the virus was measured on BS-C-40 monolayers by plaque method. Eagle's minimal essential medium (MEM) supplemented with 8% newborn calf serum was used as the culture medium and cells were incubated at 37°C in a humidified CO₂-incubator (5% CO₂, 95% air).

Determination of antiviral activity of protoporphyrin:

0047BS-C-40 monolayers were infected with HSV at the m.o.i. of 0.01 at 37°C for 1 hour and the monolayers were washed with MEM, and fresh culture medium was added. After 18 hours of incubation in the presence or absence of protoporpyrin, the virus infectivity in the culture fluid was measured by plaque method.

Plaque assay:

0048Two tenths ml aliquots of serially 10-fold diluted virus were inoculated onto confluent monolayer of cells in 35 mm plastic petridishes. After adsorption of the virus at 37°C for 1 hour, the monolayers were overlaid with 2 ml of MEM with 1% methylcellulose and 2% newborn calf serum. On day 3, the monolayers were stained with neutral red and plaque counts were made.

0049The results indicated that PPNa has no anti-HSV activity.

0050It can be concluded from above experiments that the activity of porphyrins used in the present invention against HIVs is specific.