HU197212B - Process for producing pharmaceuticals with antiviral and antitumour effect - Google Patents

Abstract

The invention relates to antiviral and antitumor compositions for use as medicaments. According to the invention, the novel compositions contain {elimination; Improvement; Alleviation or alleviation of pathological conditions caused by viruses or tumors such as infirmity; Pain; infections}

Description

The present invention relates to a method of combating viral and cancerous diseases, that is, to eliminating viruses and tumors, and to eliminating, ameliorating, ameliorating and ameliorating the symptoms, conditions, such as falls, infections, malaise, of these diseases, for the preparation of novel pharmaceutical compositions. In addition to the xanthate compound having antiviral and antitumor activity, they contain an adjuvant containing an ionic, lipophilic and hydrophilic moiety which enhances or enhances the antiviral and antitumor activity of the former.

No. 3,146,772. German Patent Publication No. 2,091,244; United Kingdom Nos. 1,174,978 and 1,175,047; Canadian and U.S. Patent No. 4,602,037. U.S. Patents (issued July 22, 1986) disclose xanthate compounds of interesting pharmacological activity, particularly antiviral and antitumor activity.

It has now been found that certain adjuvant agents which have no antiviral or antitumor activity per se are xanthate compounds having antiviral or antitumor activity, particularly those disclosed in U.S. Patent No. 3,146,772. German Patent Publication No. 2 September 1982, U.S. Patent No. 2,091,244. United Kingdom (published July 28, 1982 and published February 6, 1985), U.S. Patent Nos. 1,174,978 and 1,175,047. Canadian (both September 25, 1984) and the compounds described in the above-mentioned U.S. Patents.

More particularly, the present invention relates to pharmaceutical compositions having antiviral and antitumor activity which ameliorate the condition of viral and antitumor diseases, as well as a known xanthate antiviral and antitumor compound, in particular a compound of formula I, wherein

R ¹ is tricyclodecyl, mantyl, bicycloheptyl, cyclododecyl, 4-isobornylcyclohexyl

R ² is an alkali metal atom and contains an ionic adjuvant containing at least one lipophilic and hydrophilic moiety.

These two components are of paramount importance to the invention and accordingly the pharmaceutical compositions of the invention contain or consist essentially of these two components *.

You are the ionic adjuvant. a potentiator compound comprising both lipophilic and hydrophilic groups, preferably a compound wherein the lipophilic group is a straight or branched C8-C16 aliphatic group and the hydrophilic group is a carboxylic, sulfate, sulfonate phosphonic acid, phosphate group; preferably an aliphatic monocarboxylic acid having 8 to 16 carbon atoms or an alkali metal salt thereof, alkylene sulfate, sulfonate or phosphate, phosphonate or an alkali metal salt thereof having from 10 to 14 carbon atoms. It is preferable to use stearic acids such as deoxycholic acid, preferably in the form of an alkali metal salt such as sodium or potassium. Especially preferred are naturally occurring fatty acids or fatty alcohol sulphates having from 8 to 16, especially from 8 to 14, particularly preferably from 10 to 12 carbon atoms. In this series, C 11 compounds appear to be optimal. In general, the adjuvant compound, which is preferably ca. 8- kb It may have 16, preferably 8 to 14 carbon atoms, and is preferably used in the form of an anionic compound, preferably in the form of a pharmaceutically acceptable salt such as an alkali metal salt such as a sodium or potassium salt.

More particularly, the present invention relates to a process for the preparation of a pharmaceutical composition having an antiviral and antitumor activity comprising a known xanthate antiviral and antitumor compound and an adjuvant compound having an ionic nature, comprising both a lipophilic and a hydrophilic moiety. The adjuvant compound further extends the action of xanthate with antiviral and antitumor activity to a broader pH range, such as physiological pH range. The present invention further describes the use of these pharmaceutical compositions for viral and antitumor therapy, and a method for combating a viral or cancerous disease comprising the step of simultaneously increasing the amount of a xanthate compound having a viral or antitumor effect to a specified amount in a viral or antitumor disease. an ionic-type adjuvant compound is added. The adjuvant compound contains both a hydrophilic and a lipophilic group. The combined amounts are effective for these antiviral and antitumor targets and may be administered either individually or in the form of a unitary formulation containing the same amounts. Other aspects of the invention will be apparent to those skilled in the art from the following.

Preferably the following compounds are used as xanthate: sodium or potassium 1-adamantyl xanthate (D424);

sodium or potassium-8 (9) -tricyclo [5-2.1.1]. ^2-6] -decyl xanthate (D609);

sodium or potassium 2-endo or exo-bicyclo [2.2-l ^L4] heptyl xanthate (D611); sodium or potassium cyclododecyl xanthate (D435);

sodium or potassium -4-isobornylcyclohexyl xanthate (D622) and the adjuvant compound is an aliphatic monocarboxylic acid having from 8 to 16 carbon atoms, preferably from 10 to 14 carbon atoms, or its sodium or potassium salt,

C 10 -C 14 alkyl sulfate, phosphate, phosphonate, or their sodium or potassium salts, or alkali metal deoxycholate; a preferred adjuvant is the sodium or potassium salt of decanoic acid, undecanoic acid, dodecenoic acid, deoxycholic acid, dodecylsulfate or dodecylphosphonic acid.

In the compositions of the invention, the molar ratio of xanthate to adjuvant compound is from 1: 6 to 1: 0.25, preferably from 1: 3 to 1: 0.5, more preferably from 1: 1 to 1: 2.

The compositions of the invention may be formulated and used for topical, oral or parenteral administration, in particular as an ointment, gel or spray, or as a tablet, capsule or suppository, or as an infusion or injection.

The pharmaceutical compositions of the present invention are used as antiviral and antitumor agents for combating viral or cancerous diseases by administering a quantity of xanthate and a lipophilic and hydrophilic substance to a chemotherapeutic agent that is susceptible to viral or cancerous disease. an adjuvant composition having both groups, suitable for the above viral and antitumor purposes; or a viral or cancerous disease susceptible to such a composition is treated with a composition comprising an xanthate compound having an antiviral and antitumor activity and an anionic adjuvant effective to alleviate said condition.

According to the invention, it is preferred that the hydrophilic group present in the adjuvant compound, such as the carboxyl, sulfate, sulfonate or phosphate group, is preferably a long aliphatic chain.

which represents the lipophilic group, is present at one end, forming a polar molecule having a hydrophilic "head and lipophilic" body. Of these types of carboxylic acids, decane, undecanoic and dodecanoic acids are particularly preferred. Thus, as an adjuvant compound, it is preferable to use compounds having an anionic hydrophilic group 45, preferably in the form of their pharmaceutically acceptable salts, in particular their alkali metals such as sodium or potassium; although many cationic-type compounds have been reported in the prior art

Thus, quaternary ammonium salts are disclosed and may also be advantageously used in some cases, provided that they satisfy the above criteria, such as those containing a lipophilic and hydrophilic moiety, and are discussed below.

The compositions of the present invention are prepared with conventional pharmaceutically acceptable excipients, carriers and diluents. 60

The dosage to be employed will essentially depend on the form of application and the purpose and subject matter of the application, such as whether the agent is to be used therapeutically or prophylactically. The single dose magnitude barrier and route of administration are suitably determined individually in the circumstances. In general, a therapeutically effective amount of a pharmaceutical composition of the invention for an injectable composition will be in the range of ca. 0.005 to approx. 10 mg / kg body weight, preferably approx. 0.01 to 0.1 mg / kg body weight.

In addition to the above ingredients, the oral or parenteral pharmaceutical formulations of the present invention will typically comprise a buffer, the pH of which may be about. 7 to 8, preferably ca. It also contains sodium chloride, mannitol, or sorbitol at 7.4 to adjust the isotonic value. These compositions may also be in the form of a lyophilized or solid preparation. Obviously, if the purpose is to kill a virus outside a living animal, the preparation can be used without therapeutic or pharmacological considerations.

Preparations for topical use. in aqueous form, for example by dissolving the combination of the invention in an aqueous buffer solution and then adding a polymeric binder such as polyvinylpyrrolidone to the resulting solution.

Oily formulations for topical use may be formulated, for example, by suspending the combination of the present invention in an oil followed by a swelling agent such as aluminum stearate and / or a plant surfactant or surfactant such as a fatty acid monoester of a polyhydric alcohol such as glycerol. monostearate or sorbitol monooleate. The latter agent is called the so-called. HLB (hydrophilic-lipophilic equilibrium) value up to about 10

For example, a fatty ointment composition may be prepared by suspending the two active compounds of the present invention in an ointment base and then on. up to about 10 HLB.

An emulsion ointment composition can be prepared in a similar manner, for example, by mixing an aqueous solution of the active ingredient of the invention in a soft ointment base and then adding a surfactant of up to about 10 HLB. All such topical formulations may also contain a preservative. Concentrations of the two active ingredients are generally about 100 mg per 100 mg of total formulation. It may be between 0.05 and 5 mg, preferably between 0.25 and 1 mg, and in the extreme up to 10 mg.

The xanthate of formula (I) are known and conventional manner, such as ^Rl OMe alkoxides of the formula - wherein R ¹ is as defined above and Me is an alkali metal atom of carbon disulphide or equivalent to those alcoholate alcohol carbon disulphide a-37

Table 1 is prepared in the presence of such a strong alkaline base. Such methods of preparation are described in U.S. Patent No. 3,146,772. German Patent Application Publication No. 4,684,500 and the patents cited above. In this sense, explicit reference is made to published documents.

Another component of the composition of the present invention, the ionic adjuvant compound having both a lipophilic and a hydrophilic moiety, is illustrated by the following compounds: commercially available linear or branched, saturated or unsaturated, e.g. Monocarboxylic acids, sulfates, sulfonates, phosphates containing from 8 to 16 aliphatic groups. Preferably, these compounds can be converted into the corresponding salt using methods known in the art using a base to form a pharmaceutically acceptable salt. 20

Some of the results of the pharmacological tests are described below.

1. Cell toxicity studies 25

Test compounds: Potassium 8 (9) -tricyclo [5.2.1.0 ² - ⁶ ] decyl xanthate (D609), abbreviated as .DEXA, which was combined with the carboxylic acids of various chain lengths shown in Table 1.

Cell cultures used: common hamster embryo fibroblast (HEF); hamster embryo fibroblast (HEF-BPV) transformed with bovine papilloma virus; 35

Medium: Eagle's basic medium supplemented with Earle's salts (BME) supplemented with 10% fetal calf serum (FBS), 1% penicillin + streptomycin; pH 7.4. 40

Method: The above cell cultures were incubated in vitro on the respective medium. To each sample was added 20 µl of the above DEXA and gradually increasing amounts of the fatty acids shown in Table 1. After 3 days, the cultures were examined microscopically and the minimum toxic concentration was determined. The results are summarized in Table 1. 5 °

Test HEF Culture HEF-BPV Culture Combination Minimum DEXA + Toxic Concentration of 20 mg / ml DEXA + Adjuvant + Adjuvant Adjuvant (µg / ml) (Mg / ml)

+ undecanoic acid 100 10 DEXA + + dodecanoic acid 40 10 DEXA + + myristic acid 30 10

Table 1 shows that the combination of the active ingredient DEXA and the fatty acids shown exhibits a significantly more toxic effect on the transformed than on the untransformed cell cultures. This is especially true for combinations with decanoic acid and undecanoic acid, where the toxicity of the combination to transformed cell cultures is ten times that of conventional cell cultures.

The cellular toxicity of various combinations of the invention was also determined in another model, mouse embryonic fibroblast (EEF) and transformed mouse embryo fibroblast cell cultures (EEF-K1). These cell cultures were incubated in medium containing BME, 5% calf plasma, penicillin and streptomycin at pH 7.4. The assay was performed as described above, but microscopic evaluation was performed after 24 hours. The results obtained are summarized in Table 2.

Table 2

Combination tested

DEXA ++ adjuvant

EEF EEF-K1

Minimum Toxic Concentration of Mg / ml DEXA + Adjuvant Adjuvant Adjuvant (Mg / ml) (Mg / ml)

Table 1 ___ 55

HEF Culture Tested HEF-BPV Culture Combination Minimum DEXA + Toxic Concentration of 20 mg / ml DEXA + Adjuvant + Adjuvant Adjuvant (Mg / ml) (Mg / ml)

DEXA ++ decanoic acid 100 10

DEXA +

DEXA + K-

decanoate 80 10th DEXA + decanoic acid 40 DEXA + + undecanoic acid 80 10 DEXA + + K-undecanoo 80 10 DEXA + + dodecanoic acid 60 20 DEXA + + Tridecanoic acid 60 10 DEXA + + tetradecanoic acid 80 10-20

The data in Table 2 show that the combinations tested show significantly more toxic effects on the transformed cell cultures than on the conventional cultures.

Selectivity of the toxic effects of the combination of DEXA undecanoic activity against transformed cell cultures further demonstrates uridine tritiated experiment based on ⁽³ H-uridine) recording, which was performed as follows.

Common mouse embryo fibroblasts (EEF) and bovine papilloma virus transformed mouse embryo fibroblasts (EEF-SZFV) were plated on Linbro plates (2x10 ⁵ cells / well) and in 20, 40, 60 and 80 pg / ml) of the combination containing undecanoic acid. The medium used was Ecl. 1µl supplemented with Earle's salt, 5% calf plasma at pH 7.5, 5% carbon dioxide. Two days later, ³ H-uridine with 1 pCi / ml activity was added, but no new medium containing the above active ingredient was added. Two hours later, the amount of ³ H-uridine, which can be precipitated with acid, was assayed in each culture. Before carrying out the total count of the low molecular weight components - washed - unincorporated ³ H-uridine is. Counting was then done with dry culture. The average radioactivity measured in two cultures is

1.

Figure 1 illustrates that the combination of the invention selectively kills or inactivates the transformed cell cultures, as a result of the observation that radioactivity, i.e. ³ H-uridine, was found only in normal cell cultures, whereas in the transformed cultures it was not observed.

the table

% Pfu tested for virus-free inhibitor-free inhibitor

DEXA (10 pg / ml) + + Decanoic acid pg / ml 8.7 pg / ml 0 pg / ml 0.3 Decanoic acid pg / ml 18.9 pg / ml 24 pg / ml 35

The table shows that the combination of DEXA and decanoic acid has a significantly stronger inhibitory effect than the ingredients of the combination alone.

The combination of DEXA and dodecyl sulfate sodium salt (SDS) similarly strongly inhibits the reproduction of HSV1.

Sejtek: Rita P 0/37;

Medium: BME, 5% FBS, 1% penicillin + + streptomycin; pH 7.4; 5%

CO2 atmosphere;

Inhibitors: 10 pg / ml DEXA + 10, 20, 40, 80 pg / ml dodecyl sulfate (SDS);

Rita cells were infected with MOI (0.05 pfu / cell) and the inhibitor was applied 2 hours post-infection (2 cultures each). Virus propagation was harvested 24 hours after infection.

Control value: 7.7x10 ⁶ pfu / ml.

The results are shown in Table 3 b.

2. Examination of antiviral activity

The inhibitory effect of the active ingredient DEXA, the combination of DEXA and decanoic acid, and only the decanoic acid, at different concentrations, in different herpes simplex virus

1 in vitro cultures (HSV1 culture). The pH of the medium is 7.4. At 2 days post-incubation, virus yield was determined and expressed as% of the appropriate culture without inhibitor. plaque-forming unit (pfu) was calculated. The results are summarized in Table 3 (a).

the table

Table b)

Active Ingredients The combination titres pfu / ml inhibition factor 10 pg / ml DEXA + + 10 pg / ml SDS 2.5x10 ² 3x10 ⁴ 10 µg / ml DEXA + + 20 pg / ml SDS 25 3x10 ⁵ 10 pg / ml DEXA + + 40 pg / ml SDS 25 · 3x10 ⁵ 10 pg / ml DEXA + + 80 pg / ml SDS 2.5 3x10 "

The growth of uninfected Rita cells was virtually inhibited by the same combination of agents.

% Pfu tested for inhibitor-free virus culture

DEXA (20 pg / ml) 22.6

Similar results were obtained with a combination of cyclododecyl xanthate (D435) and undecanoic acid. This combination is also examined.

-511 ^í for needles, for example, human embryonic lung confluent cells infected with HSV-l was observed very pronounced inhibition. We achieved the same effect by using D424, D611 or D622 instead of D609.

It has been found that the active ingredient combination described above inhibits RNA viruses (single-stranded viruses) such as the viruses causing bladder mucositis.

The following is an experiment in which the healing effect of the combination of the invention and the adjuvant in a guinea pig HSV-induced injury was investigated.

The substances listed in Table 4 were applied twice daily to the porcine HSV-infected skin surface in the form of a petroleum jelly-based ointment. The results are summarized in Table 4.

Table 4

Test substance number of injuries at the start of treatment (96 hours after infection) after) 36 hours after treatment MEDICAL fruit LASIK % petroleum jelly 10% 8 8 decanoic acid 10 10 0 9 9 petroleum jelly 5% 6 4 DEXA and 23 14 10% Dean's 16 5 42 acid 11 8 8 6

It is clear from the data in the above table that the best healing results were achieved with the combination according to the invention.

The antiviral activity of the combination was also tested in six guinea pigs infected with Wal-HSV-1. Twice daily treatment was started 18 hours after infection with a petroleum jelly containing 5% DEXA and 5% decanoic acid. After two days of treatment (i.e., 72 hours after infection, the animals were sacrificed and the infected skin was removed. The skin was frozen in liquid nitrogen and finely ground using a Micro Dismembrator (tm) (two samples per animal) and collected fold amount (by weight / volume) of tissue culture medium Short centrifugation stone essentially -. Eppendorf - barman peak-centrifuge for 1 min - of the supernatant virus titer was determined by the test virus is not detectable by the vakkontroil value per tissue lxlO ⁵ pfu / gram.. value.

Hatás The effect of the carbon chain length of monocarboxylic acids on antiviral activity and cytotoxicity in combinations of various monocarboxylic acids with D609 is shown in Figure 3. HSV-1-infected () and uninfected (a) Rita cells were treated at pH 7.4 with combinations containing 10 pg / ml D6O9 and 40 pg / ml monocarboxylic acid for each monocarboxylic acid. plaque technique was defined separately in the two cultures. From the margin of error, standard deviation was inferred. In double uninfected treated and untreated Rita cell cultures, cell density was determined by hematocytometry after staining with trypan blue.

3. Examination of the anti-tumor effect

Lymphatic leukemic mice were treated. 6 week old DBA-2 mice were infected intravenously lxlO ⁵ NCI Egg types of leukemia cells. 18 hours later, three groups of 10 animals were formed each. One group that received no treatment represented the control group. The second group was treated only with DEXA (4x15 mg / kg, then 6x11 mg / kg). The third group was performed with the combination according to the invention, DEXA and undecanoic acid (DEXA was added as described in the second group and an additional 4x7.5 mg / kg, followed by 6x5.5 mg / kg undecanoic acid). All treatments were given hourly by intravenous injection. The following day (2 days after tumor cell infection), blood was collected from each animal and lymphocyte concentration was determined.

The results are summarized in Table 5.

Table 5

lymphocytes x 10 ~ ⁶ / ral untreated DEXA DEXA + + undecanoic acid 13.8 17.9 8.6 11.1 15.0 11.7 15.8 24.1 12.0 17.3 13.0 9.8 12.0 18.6 12.8 15.7 15.6 18.4 23.0 10.7 11.6 18.3 14.6 12.4 20.2 13.7 8.4 10.8

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Table 5

lymphocyte count x 10 ⁶ / ml untreated DEXA DEXA + + undecanoic acid average value x 10 ⁶ / ml 16.3 15.9 11.7 significant P <0.01 Table 5 shows that the find- combination reduces lymphocyte counts. significantly

In rats with autochthonous skin, regression was observed after systemic treatment with the combination of DEXA and undecanoic acid according to the invention.

Skin cancer was induced in 7-week-old female NMRI mice in a manner known per se. A single treatment with 25.6 mg of DMBA in 0.1 ml of acetone, followed by 7 days of treatment with 6.16 mg of TPA in 0.1 ml of acetone twice daily for 23 weeks, was performed. Three weeks after the end of the promoter treatment, chemotherapeutic treatment was initiated by intravenous injection at a dose of 10 mg / kg, at a concentration of 0.9% sodium chloride at pH 7.4, at a concentration of 1 mg / ml. The pH was adjusted with 0.15 m sodium hydroxide solution. 40 cancerous animals were selected and 4 groups were formed as desired. Eleven animals were not treated with ten DEXA, ten undecanoic acid, and nine with a combination of DEXA and undecanoic acid (1 mg / ml and 10 mg / kg for each compound).

In Figure 2, the time axis represents the time of injection at the small Δ. Three-dimensional size of each tumor was determined by caliper: thus, 35 of the untreated animals, 40 of the animals treated with undecanoic acid, 37 of the animals treated with DEXA and 35 of the animals treated with the combination according to the invention. The initial size of the tumors ranged from 2 to 400 mm ³ . All single tumors were observed throughout the study. Growth and regression were expressed as a percentage of baseline size. The mean of each group is shown in Figure 2. Significance of regression of tumors treated with the combination of the present invention was determined by Student's T test. Two days after treatment, the treated group the tumors kezeletlenekétől significantly different, p <10 'to ^10, and even after five days of treatment, P <10'''.

Subcutaneous treatment of autochthonous skin tumors was performed as follows.

A test animal (belonging to the above species) was chemically developed a skin tumor as described above, and the animals were divided into four groups as desired. Treatment was given directly to the tumor by subcutaneous injection: 0.5 ml every two hours for two days. The solution for injection contained 5 mg / ml DEXA and 5 mg / ml undecanoic acid. Six days after the start of treatment, photographic and visual evaluation was performed. As a result of the treatment, all tumors were significantly regressed or even completely disappeared, and in all cases, the initial tumor size was reduced by at least 50%.

The regression effect on systemic treatment of a combination of DEXA and undecanoic acid according to the invention is shown below for mouse autochthonous cancers.

NMRI mice developed a skin tumor as described above. Two days after the last TPA treatment, chemotherapy treatment was started. The treatment agent was administered intravenously three times daily. Each injection contained 10 mg / kg DEXA and 10 mg / kg undecanoic acid (each compound was dissolved in 0.9% sodium chloride at a concentration of 1 mg / ml). The pH of the solution was adjusted to pH 7.4 with 0.15 M sodium hydroxide solution. The animals were photographed at the start and end of treatment and the number of tumors in each animal was determined. Four weeks after treatment, the presence of tumors was re-examined: no evidence of recurrence was observed at sites where the tumors had disappeared. The results are summarized in Table 6.

Table 6

The test animal the number of tumors the agent used for treatment treatment before treatment after 1 3 3 3 5 3 4 14 4 5 11 8 6 5 3 7 5 0 DEXA + 8 15 10 undecanoic 9 13 0 acid 10 14 4 arc. 11 6 2 altogether 91 37 = 59% regression

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Table 7

Antiviral activity of D609 / undecanoic acid on RNA viruses at pH 7.4

Table 6

The number of tumors examined is the number of tumors the agent used for treatment animal treatment 1 before tezelés after 1 11 10 2 4 3 3 8 7 4 4 2 6 4 0 control 7 7 7 8 5 5 9 10 8 10 4 1 11 7 5 altogether 64 48 = 25%

regression

The data in Table 6 show that the combination of the present invention comprising DEXA and undecanoic acid has a regression of 59% for skin tumors and only 25% for untreated animals.

The test results for the antiviral and antitumor activity of the compositions of the present invention are described below.

(A) Antiviral activity of D609 / undecanoic acid

Xanthan compounds are known to be active against various DNA and RNA viruses at acidic pH [Sauer, G., Amtmann, E., Melber, K., Knapp, A., Müller, K., Hmmm, K., and Scherm, A .: DNA RNA virus species are inhibited by xanthates, a source of antiviral compounds with unique properties. Proc. Natl. Acad. Sci. USA 81: 3263-3267 (1984)]. However, in the present case, it has been possible to extend the unique broad-spectrum antiviral activity of these compounds to higher and physiological (7,4) pH values by administering certain ionic adjuvant compounds containing both lipophilic and hydrophilic groups simultaneously or in combination, combinations of tricyclodecan-9-yl xanthate (D609) with sodium deoxycholate, sodium dodecyl sulfate, and certain fatty acids such as adjuvants which have no antiviral activity in themselves, various DNA and RNA viruses such as Cystic herpes and B4 - replication is inhibited in vitro at pH 7.4.

Virus D609 unde- alkanoic viral yield species [Pg / ml] [Pg / ml] [Pfu / ml] VSV ^b 0 0 (4.3il) xl0 « 5 0 (05.06 ± 0.05) xl0® 0 40 (4.3 ± 0.3) x10 ⁶ 5 40 (1.3 ± 0.2) * xl0 Coxsalkie B4 0 0 (1:15 ± 0.2) xl0 " 10 40 (07.07 ± 0.25) * xl0

^b Values double experiments, the virus yield was also determined from the data of double tests.

VSV offspring were harvested at 10 hours and Coxsalkie B4 24 hours after infection.

For saturated fatty acids of different chain lengths, there was some benefit in terms of carbon number: the efficiency of undecanoic acid containing 11 carbon atoms was three orders of magnitude higher than that of the shorter (C8) or longer (C18) monocarboxylic acids. Based on the dose-effect kinetics, it was determined that the dose which inhibits the replication of herpes viruses by a factor of 1000 has no effect on the mitotic activity of non-infected growing control cultures. The 1: 1 combination of D609 undecanoic acid proved to be most preferred as shown in Figure 4 (black circles).

Similar results were obtained for HTLV III virus, which could be inhibited in tissue culture by the combination of 10 pg D609 and 10 pg undecanoic acid / ml after 3 and 5 day application periods (Figure 5). Combination treatment does not affect the mitotic activity of mitogen-activated peripheral human blood lymphocytes, nor does it affect concentrations at which B- and T-cell lymphomas die ([Chapter 1 (B) and Figure 6]). makes the D609 / undecanoic acid combination potentially useful for the chemotherapeutic treatment of AIDS disease.

The inhibitory effect of D609 and undecanoic acid at different concentrations on the herpes virus is shown in Figure 4. Concentrations of both components were expressed as abscissae-817. D609 and undecanoic acid were mixed in a ratio of 1: 1, ol: 2, 1: 3, 1: 1 (undecanoic acid).

Combinations with an inhibitory factor greater than 10 ³ also had cellular toxic effects. However, at lower concentrations, no untoward cell toxic effects were observed on uninfected control cells.

The antiviral activity of D609 / undecanoic acid 1: 1 combination (denoted by · in the figure) was found to be most favorable for any compound or other combination. Thus, a 1: 1 combination of the components is recommended for use.

Figure 5 illustrates the following. HTLV III-specific nucleic acid (as a replication intermediate, superhelical DNA) was isolated and visualized by gel electrophoresis and cloning with authentic, radioactively labeled HTLV III DNA. After 3 and 5 days of treatment (lines (a) and (c)), the HTLV III DNA characterization line was completely broken off, but HTLV III specific DNA was seen in untreated infected (K37 T-lymphoma) cultures (lines b and d). (both super-helical Form I and relaxed circular γ) After cleavage with single-site cleavage endonuclease, I and ΙΙ were converted to linear III (f and h lines) The results demonstrate that the above treatment can completely inhibit the replication of HTLV III.

(B) Antitumor activity of D609 / andecanoic acid

Combinations of xanthate antiviral compounds with monocarboxylic acids of a certain chain length, preferably of 11 or 12 carbon atoms, have a strong antitumor activity in vitro.

Tricyclodecan-9-yl xanthpgenate (D609) or cyclododecyl xanthogenate (D435), when administered in combination with undecanoic acid or dodecanoic acid, can cause cell death (usually with low serum requirements). When the combination was administered at the same concentration to healthy cells from which the transformed cells were generated, no or only a mild effect was observed.

The above is illustrated in Table 8, where the effect of the D609 / undecanoic acid combination on healthy and transformed cells is compared. The selective 'killing' of tumor cells (and those transformed chemically or virally) is clearly due to the difference in the 'survival rate - a factor ΙΟ' of ⁶ , ie detectable in the system tested. While healthy cells remained virtually unchanged after treatment, their transformed derivatives (which exhibit low serum requirements) died after the same treatment.

Meth-A tumor (fibrosarcoma) was successfully prevented by intraperitoneal treatment with D609 / undecanoic acid (Figure 6). In addition, therapeutic efficacy was demonstrated even at advanced stage (5 days post-infection) (Figure 7). When treated with D609 alone without undecanoic acid, essentially no antitumor activity could be demonstrated.

Figure 8 illustrates the selective killing of various B and T cell lymphomas (same treatment with peripheral human blood lymphocytes (pBL)).

Table 8 shows the effect of xanthate / monocarboxylic acid treatment on the growth characteristics of different cell types.

Cells were plated at a density of 3.8x10 ⁴ / cm ^{2 in} plastic containers or Petri dishes and after 4 hours a new tissue culture medium [Eagle's basic medium containing 2.2 g of sodium bicarbonate, 1% penicillin and streptomycin (BME) ) and supplemented with 5% fetal bovine serum (FBS) with or without the combination of 10 pg / ml of D609 and 40 pg / ml of undecanoic acid. The resulting culture was incubated in a 5% carbon dioxide atmosphere (pH 7.4 ± 0.05) at 37 ° C (two experiments with both the control and the treated culture. After 24 hours, the tissue culture medium was 10%). We recharged with BME supplemented with FBS.

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Table 8

Notation denomination Cell type Species Kétszere- contract! time (Sun)* Colony formation at low serum concentrations ** survival gold RITA kidney monkey 1.9 - 0.73? 1.20 5 CV-1 kidney monkey 1:21 - C-6 SV40 transformed monkey 1.65 + <4x10-655 MEF embryo fibroblast mouse 1:14 - 0.60-5 SV3T3 SV40 transformed mouse 0:53 + <2xl0- "§5 MEF-K1 BPV-1 was transformed mouse 0:58 + <3x10-6 55 MCA-3F keratinocyte mouse 1.5 - 0.25 ^§ 0.81 ⁵ MCA-3D keratinocyte mouse 1:45 - HEL-30 chemically transformed keratinocytes mouse 0.78 + < ^2x10-5§§ A PDV chemically transformed keratinocytes mouse 0.65 + <3x10- ^{6 55} HRD fibroblast hamster 1.73 - S 0.67 ^s HEF-BPV BPV-1 was transformed hamster 0:48 + <2xl0-6§5 Hela cervix carcinoma human 1.0 + <3x10-6 55 HEL fibroblast human 2:35 - 0.54 5 HTB72 malignant melanoma human 2.9 + 0.087 ⁵ MRC-5 SV-80 fibroblast SV-40 transformed fibroblast human human 2.9 + 0.80 5 <4x10-6 55. Wi-38 fibroblast human 2.85 - $ 0.73

Table 8 legend 24 hours after removal of the D609 / undecanoic acid combination, the cells were trypsinized and the number of germ cells was determined by trypan blue staining with a Neubauer hematite. Survival rates were expressed as the ratio of cell numbers obtained in untreated and treated cultures.

After 10 days of incubation at 37 ° C, the cultures were fixed for 2 minutes in 2% formaldehyde and stained with 0.5% crystal violet for 1 minute. Cells that were inoculated into Petri dishes prior to treatment showed the highest survival rates. In no case were we able to detect surviving cells.

4x10 ⁵ cells were introduced into 6 cm Petri dishes and incubated with BME, 5% FBS, 5% carbon dioxide. After 1 and 2 days, cell counts in two Petri dishes were determined for each cell lineage. The doubling time was determined from the observed growth curves by extrapolation.

** 6 cm Petri dishes of each cell type were arranged at 10 ³ cells, and incubated for two weeks with 1%, 2%, 5% or 10% FBS-containing medium. Colonies were visualized by staining with crystal violet after fixation in 1% formaldehyde.

Figure 6 shows treatment of Meth-A tumor immediately after transplantation. Male, 8-10 weeks old with a body weight of 20 g Balb-c mice were inoculated ip administration lxlO ⁶ tumor cells. At 60 hours after transplantation, the animals were treated. Treatment was performed once daily for 5 days with isotonic glucose solution containing 50 mg / kg D609, mg / kg potassium undecanoate and 25 U-insulin / kg. The substances were administered ip in 1 ml / 20 g body weight solution. Ten animals were treated with a solution of insulin in isotonic glucose alone. Eight animals in the D609 / undecanoic acid treatment group survived more than 90 days.

Figure 7 illustrates treatment of Meth-A tumor with D609 / / undecanoic acid for a prolonged period after transplantation. 10-12 weeks old, weighing 20 g

-1021 female Balb-c mice with 1x10® Meth-A tumor cells i.p. treatment. Five days after transplantation, ten animals received 50 mg / kg D609, 50 mg / kg potassium undecanoate twice daily. and treated with isotonic glucose solution containing 25 IU insulin / kg. The substances were dissolved in 1 ml / 20 g body weight solution. Ten animals were not treated as a control group. Two animals from the treated group (empty circles) survived for more than 65 days.

In the above studies, insulin was administered to provide the animal with one of the growth factors necessary to increase glucose production and enhance cell growth, proliferation, and proper functioning.

Figure 8 shows the susceptibility of healthy blood lymphocytes and T and B cell lymphomas to the D609 / undecanoic acid combination. Peripheral blood lymphocytes (pBL) were harvested by centrifugation on a limphoprep gradient and incubated in RPMI 1640 medium supplemented with 10% bovine fetal calf serum and 5 pg / ml plant agglutinin (PHA). After 1 day, the cells were separated by centrifugation and so-called. microtiter plates (2x10 ⁵ cells / well) and medium containing no PHA but interleukin-2 (IL-2) was added. D609 and increasing concentrations of undecanoic acid were added as shown. In parallel, two T-cell lymphomas (Jurkat and K37) and two B-cell lymphomas (Raji and P3HR1) were treated. Day after incubation, the number of live cells in each well was determined by the resin displacement method (using trypan blue). In Jurkat, Raji and K37 cell cultures treated with pg D609 and 40 pg / ml undecanoic acid, no live cells could be detected. However, human peripheral blood lymphocytes, although treated in the same way, retained their mitotic dividing capacity.

The antiviral activity of the D609 / undecanoic acid and D609 / dodecyltrimethylammonium bromide combinations was compared at pH 7.4. The results are summarized below.

Compound antivirals hatás® Cellular toxicity ^b undecanoic acid (Cll) 5.8x10 ³ 1.6 dodecyl trimethyl ammonium bromide (TNCB) 15 1.6

In two experiments, Rita cells (MOI = 0.01 pfu / cell) infected with HSV-1 for 23 hours after the adsorption period were treated with 10 pg / ml of D609 and either 40 pg / ml of Cn or 20 µg / ml of TNCB in BME, 5% FBS, 1% penicillin cs in stroptomycin medium, pH 7.4. Virus yield was determined by plaque assay and compared with that of untreated culture. The antiviral activity was expressed as the factor of viral titer inhibition (the mean of the two cultures).

^b > Uninfected Rita cells were treated as described in (a) and cell counts were determined using a Neubauer counting chamber. The ratio of untreated and treated cultures (mean of the two experiments) is given in the table.

The results show that the cationic adjuvant (TNCB) has a much less specific antiviral activity compared to undecanic acid (Cn).

Although the xanthate compounds of the present invention having antiviral and antitumor activity, particularly D609, when used alone, their antiviral activity at pH 6.8 appears to be higher than the slightly higher pH of 7.25-7, 8 values; however, their combination with an ionic adjuvant of the present invention results in viral growth at a physiological ca. Effectively inhibits at pH 7.4. In further experiments, accordingly, Rita cell cultures were treated with D609 for 1 day following infection with HSV-1 and simultaneously with one of the adjuvant compounds described in Table 9. (D609: adjuvant ratio was selected as the basis for preliminary experiments. 1: 4). The combination of D609 with any of the first three compounds of Table S resulted in the most striking HSV-1 growth inhibition results when treated at pH 7.4. The increased antiviral effect cannot be attributed to the increased cellular toxicity, since 62.5-83% of the uninfected cells subjected to the same treatment were able to divide as shown in the table. The last three adjuvants in the table showed minimal antiviral activity compared to the first three compounds.

-1 123

Table 9

The antiviral activity of D609 in combination with various adjuvant compounds at pH 7.4

Name of Compound Structure Inhibition Factor Antiviral Activity Cell Toxicity ^b

undecanoic CHsfCHzbCOOH 5.8x10 ³ 1.6 Sodium deoxycholate C23H39O2CO0Na l.OxlO ³ 1.2 Nétrium dodecyl sulphate CH3 (CH) nOSO3Na 7.2x10 ³ 1.6 Dodecyl phosphate CH3 (CH) nOPO3Hz 10.4 n.d. Dodecyl acid CH3 (CH) nPO3Hz 2.8 1.4 Dodecyl tr trimethyl ammonium m- -bromides (TNCB) CH3 (CH2) LLN (CH 3) 3 + Br- 15 1.6

In two replicate experiments, Rita cell cultures infected with HSV-1 were treated with 10 pg / D609 compound and 40 pg / ml detergent for 23 hours after the adsorption period (1 hour). Virus growth was determined by duplicate plaque assays and compared with the yield of untreated culture. The antiviral effect was expressed by the factor inhibition of viral yield.

Uninfected Rita cells were treated as described above in two experiments, and cell counts were determined using a Neubauer chamber. The ratio of the values obtained for untreated and treated cultures (mean of the two experiments) is given.

Further evidence is HTLV-III virus

Inhibition of DEXA / undecanoic acid combination 30

K37 (human T-cell lymphoma) cells were used for HTLV-ΙΠ virus reproduction.

K37 cells infected with HTLV-III were treated with 10 pg DEXA 35 and 10 pg undecanoic acid / ml for 3 or 5 days post-infection. Under these conditions, K37 cells continued to grow, as determined from pre- and post-treatment cell counts (after vital staining). Furthermore, the same amount of cellular m RNA (.Northern Biot ') was found in the treated and untreated cells.

Simultaneously, DNA was isolated from the above treated and untreated K37-45

cell cultures, and hybridized with ³² P-labeled cloned authentic HTLV III DNA, and the results were determined by autoradiography. Like all retroviruses, the HTLV ΙΠ-virus replication intermediate is superhelical DNA. Both this (Form I) and the extended (relaxed) Form II in both untreated cultures.

After 3 days and 5 days, the autoradiogram shows black spots (Figures 1.5b 55 and d). After cutting with a single-cut restriction enzyme, which converts annular DNA to unit-length linear DNA, 3 and 5 days later, the characteristic signals were again obtained (Figures 5 f and h 60). However, after 3 or 5 days in the treated cultures, neither super-helical DNA (Figs. 5a and c) nor linear DNA (Figs. 5e and f) were detected. Thus, the results indicate that DEXA / undecanoic acid combination treatment completely inhibits HTLV III virus replication.

The inhibitory effect described above occurs in K37 cells at a concentration that does not cause toxic damage to uninfected K37 cells, and only at higher concentrations does selective damage occur not only in K37-T lymphoma cells but also in other human T- and B- lymphomas as shown in Figure 8. However, the DEXA / undecanoic acid combination does not damage myocyte-stimulated human peripheral lymphocytes (pBL) at concentrations that would selectively kill tumor cells.

The experiment was carried out as follows. Peripheral blood lymphocytes were isolated by lymphoprep gradient centrifugation. Cells were incubated with RPMI 1640 medium containing 10% fetal calf serum and 5 pg / ml plant agglutinin (PHA). After 1 day, the cells were spheronized by centrifugation and plated on microtiter plates (2x10 ⁵ cells / well) in new medium containing no PHA but containing 10% interleukin-2. Then DEXA and increasing concentrations of undecanoic acid were added as shown in Figure 8. In parallel, T-cell lymphomas (Jurkat and K37) and B-lymphomas (Raji and P3HR1) were treated in the same manner. After 4 days of incubation, the number of live cells was determined by a synaptic technique (trypan blue). No live cells were found in Jurkat, Raji and K37 cultures treated with 5 pg DEXA and 40 pg / ml undecanoic acid. In contrast, treated 13

Even under these conditions, -1225 human peripheral blood lymphocytes were capable of mitotic division.

The specific inhibition of HTLV III replication and the selective cytotoxicity of T- and B-human lymphoma 5 tumor cells make it possible to use the DEXA / undecanoic acid combination as an effective agent in AIDS chemotherapy.

In summary, the present invention relates to the preparation of novel pharmaceutical compositions having a viral and antitumor activity, containing a known xanthate antiviral and an ionic-type adjuvant having both lipophilic and hydrophilic activity. . Many of the features and benefits of these formulations, their use, are described in detail above. 20

The invention is not limited to the precise details of the operations, nor to the exact formulations, processes, methods, or details of the invention presented and described, as the obvious changes and equivalents will be apparent to those skilled in the art, .

Description of the figures

Figure 1 shows the uptake of radioactive uridine into transformed and untransformed (healthy) mouse embryo fibroblast cells treated with combinations of 20 μg / ml of active ingredient (D609) and varying amounts of adjuvant between 10 and 100 μg / ml.

Figure 2: Changes in induced skin tumors following intravenous treatments at concentrations comparable to those of the present invention and the components of the compositions.

Figure 3 illustrates the effect of the carbon chain length of monocarboxylic acids on the antiviral and cellular toxicity of 45 combinations with D609 in both uninfected and HSV-1 infected Rita cells. 50

Figure 4: Combinations of D609 in varying concentrations and different ratios of undecanoic acid in herpes. rus inhibits growth. 55

Figure 5: Gel electrophoresis showing complete inhibition of HTLV IH virus replication.

Figure 6: Combination of D609 with undecanoic acid shows treatment of Meth-A tumor gQ immediately after transplantation.

Figure 7 shows treatment with D609 / undecanoic acid for a prolonged period after transplantation of Meth-A tumor. g5 14

Figure 8: Sensitivity of healthy blood lymphocytes and T-cell and B-cell lymphomas to the combination of D609 / undecanoic acid.

Claims (7)

PATENT CLAIMS A process for the preparation of a medicament having antiviral and antitumor activity and for the treatment of AIDS, characterized in that the xanthate derivative having the antiviral activity of the formula I, wherein ^R1 triciklodecil-, adamantyl, bicycloheptyl, isobornyl, cyclohexyl, cyclododecyl, R ² represents an alkali metal, an adjuvant and a monocarboxylic acid or alkali metal salt of 8-16 carbon atoms, 10-14 carbon atoms, sulfate, phosphate, phosphonate or alkali metal salt or an alkali metal deoxycholate, 1: 6-1 weight ratio of 00:25 taking conventional pharmaceutical carrier, mixed with diluents and / or other excipients and optionally mixed with a cell growth and glucose boosting agent to restore the normal functioning of the body. The process according to claim 1, wherein the xanthate compound is sodium or potassium adamantyl xanthate, sodium or potassium-8 (9) -tricyclo [5.2.1.0. ² - ⁶ '] -decyl xanthate, sodium or potassium 2-endo- or exo-bicyl; [2.2.1 ^b4 '] heptyl xanthate, sodium or potassium cyclododecyl xanthate, sodium or potassium; -4-isobornyl-cyclohexyl xinate, and as an adjuvant a C 8 -C 16 aliphatic monocarboxylic acid or its sodium or potassium salt, C 10 -C 14 alkyl sulfate, phosphate or phosphonate, or the sodium or potassium salt thereof deoxycholate is used. 3. The process according to claim 1 or 2, wherein the adjuvant compound is sodium or potassium salt of decanoic acid, undecanoic acid, dodecanoic acid, deoxycholic acid, dodecyl sulfate or dodecyl phosphonic acid. 4. The process according to any one of claims 1 to 4, characterized in that the xanthate compound is preferably used in a molar ratio of 1: 3 to 1: 0.5, more preferably 1: 1 to 1: 2, relative to the adjuvant compound. 5. Process for the preparation of a pharmaceutical composition for topical, oral or parenteral administration according to any one of claims 1 to 13, in particular in the form of an ointment, gel or spray, tablets, capsules, suppository preparations or solutions for infusion or injection, characterized in that and / or other excipients. 6. A process according to any one of claims 1 to 3, characterized in that a growth promoter is added to the composition. 7. The method of claim 6, wherein the growth promoter is insulin.