LV10574B

LV10574B - The therapeutic use of low concentrations of trinitrobenzene, carminic acid and their derivatives as anti-cancer and anti-viral agents

Info

Publication number: LV10574B
Application number: LVP-92-149A
Authority: LV
Inventors: Ayuko Washington Odur
Original assignee: Radopath Ltd
Priority date: 1990-04-03
Filing date: 1992-10-05
Publication date: 1995-08-20
Also published as: EP0524212A1; LV10574A; HUT62785A; CA2079803A1; AU7560491A; NO923824L; BR9106310A; MC2246A1; FI924475A; HU9203148D0; JPH06501449A; WO1991015200A2; FI924475A0; AU662883B2; WO1991015200A3; NO923824D0

Description

LV 10574

THE THERAPEUTIC USE OF LOW CONCENTHATIONS OF TRINITROBENZENE, CARMINIC ACID AND THE IR DERIVATIVES AS ANTI-CANCER AND ANTI-VIRAL AĢENTS

The present invention relates generally to compounds and therapeutic formulations based on trinitrobenzene and/or carminic aeid (or their derivatives) which in certain low concentrations exhibit anti-cancer or anti-viral activity.

Traditional cancer treatments generally involve surgery, radiotherapy, chemotherapy, or some combination thereof. While these treatments are often effective in lengthening a patient‘8 life or sometimes eradicating the cancer, they have well-known serious side effects. More . recently, alternative attempts to treat certain cancers have been developed. One such technique, "immunotherapy,” is designed to strengthen the innate ability of the patient's immune system to fight cancer.

While diagnostic and treatment techniques have improved significantly over the last decades, there has been very little improvement in the overall survival rāte in patients, especially those with solid tumours treated by these orthodox methods. And, although alternative techniques such as "immunotherapy" show promise, the treatments developed thus far can only help a limited number of patients, stili exhibit toxic side effects and/or are complex and expensive to perform. 1

In the area of anti-viral medicine, many anti-viral drugs are known, but therapy may involve high doses or be of limited effect. In Hiv treatment, AZT is the only significantly effective drug found to datē, but treatment results are variable and the drug is expensive.

The present invention approaches these problems through the low dose use of readily available trinitrobenzene compounds and/or carminic ecid (either singularly or in combination) . It has now been found, most surprisingly, that such compounds are efficacious as anti-cancer or anti-viral aģents when administered at low concentrations, regardless of patient bodyweight. Toxicity and expense problems associated with the prior art thus do not apply.

Thus, in one aspect the invention provides for use in the therapy or prophylaxis of neoplasm or virai infection in human and non-human animals, a formulation comprising a compound dissolved or di6persed in an aqueous medium at a concentration of 10"3 to 10“15 moles per litre and having the general formula:

2 LV 10574 wherein X is selected from OH, NH2> halogen, a sulfo group, a carboxyl group, OCH3# or a substituted or unsubstituted hydrazyl group of the formula: 2 - N - N -

I I

Y A wherein A is hydrogen or an unpaired electron of the nitrogen atom, Y is hydrogen or an organic group and Z is an organic group, or Y and Z together with the adjacent nitrogen atom form a nitrogen-containing heterocycle; provided that when X is a substituted or unsubstituted hydrazyl group as aforesaid one of the NO2 groups may be replaced by a sulfo group. The sulfo group is preferably a sulphonate salt group, optionally, sodium or potassium sulphonate (S03Na or S03K) and the carboxyl group is preferably a carboxylate salt group, optionally, sodium or potassium carboxylate.

In another aspect the invention provides for use" in the therapy or prophylaxis of neoplasm or virai infection in human and non-human animals, a formulation comprising: (a) one or more compounds of the general formula:

X - P wherein P is a nitrophenyl, and X is selected from OH, NHZi halogen, a sulfo group, a carboxyl group, OCHg^ or a substituted or unsubstituted hydrazyl group of the formula: 3 Ζ - Ν - Ν -

I I Υ Α wherein Α is hydrogen or βη unpaired electron of the nitrogen atom, Y is hydrogen or an organic group and z is an organic group, or Y and Z together with the adjacent nitrogen atom form a nitrogen-containing heterocycle; provided that when X is a substituted or unsubstituted hydrazyl group as aforesaid one of the N02 groups may be replaced by a sulfo group; and (b) a quinone, such as carminic acid or its derivatives. A further aspect of the invention is a pharmaceutical or veterinary formulation wherein a compound as defined above is dissolved or dispersed in an aqueous medium at a concentration of from about 10"3 to 10"15 moles per litre.

Significantly, carminic acid has been found to exhibit anti-viral effects when used alone at low molar concentrations according to the invention. Thus, an important aspect of the invention is the use of carminic acid and its derivetiveB in the preparation of a medicament for the prophylaxis or therapy 4 LV 10574 of virai disease such as AIDS. Such derivatives have the following general formula:

where R is COOH (i.e., carminic acid) or aome other organic or inorganic functional group such as NH2, S03[K, H or Na], and the C-glycoside is any sugar. The anthraquinone may optionally be a benzoquinone (single ring) or napthaquinone (double ring).

Without being tied to an exact mechanism, it is believed that the compounds referred to above function by initiating and propagating a free radical mechanism, thereby producing active Chemical epecies that selectively attack abnormal celi structures. In Cancer Research 36 (197.8), 1745-1750, Bachur et al describe possible free radical mechanisms in connection with the known biological actions of quinone-containing anti-cancer drugs. It is 'postulated that these drugs may generate oxygen-dependent free radicals such as superoxide or hydroxyl radicals. In the present invention, it is likely that the above mentioned compounds serve as catalysts for a redox-recycling mechanism which continuously generates free radicals such as superoxides. The free radicals, or their by-products, selectively attack cancer celis or viruses. 5

Preferred formulations according to the invention· comprise a trinitrobenzene derivative (such as picryl chloride or diphenyl picrylhydrazine (DPPH)), an anthraquinone having a glycosidic moiety (such as carminic acid or a derivative thereof), or an adraixture of one or more trinitrobenzene derivatives and an anthraguinone glycoside. Carminic acid has been used in the laboratory for staining nucleic acids and, interestingly, its effect on DNA is inhibited by, inter alia. free radical scavengers (Lowņ et al., Biooroanic Chem. 8 (1979), 17-24). Again, such a formulation contains the active ingredients dissolved or euspended in an agueous medium at a concentration in the range from 10~3 to 10"15 moles per litre, and may be administered orally or parenterally. Carminic acid is especially useful for anti-viral treatment.

For a more complete understanding of the present invention and the advantages thereof, reference should be made to the following detailed description taken in connection with the accompanying figurēs in which: FIGURĒ 1 is a plot of the average survival time in days of NMRI mice transplanted with MAC16 colon carcinoma celis after treatment with various therapeutically-effective concentrations of picryl chloride according to the teachings of the present invention; and 6 LV 10574 FIGURĒ 2 is a plot of the dose response of a GM892 celi' line to diphenyl picrylhydrazyl (DPPZ) and diphenyl picrylhydrazine (DPPH) according to the teachings of the invention.

Turning now to the various specific aspects of the invention, a compound capable of initiating and propagating a free radical mechanisra may be dissolved or dispersed in an aqueous medium at a therapeutically-effective concentration in the range of frorn about 10'3 - 10"15 moles per litre. Such compounds catalytically trigger free radical chain reactions, thereby producing active Chemical species that 6electively attack abnormal celis. One such set of compounds include derivatives of nitrobenzene of the following general formula; AIOj.

wherein X is selected from OH, ,NH2/ halogen, a sulfo group, a carboxyl group, 0CH3i or a substituted or unsubstituted hydrazyl group. when X is the hydroxyl radical, the compound is picnc acid. When X is chloride, picryl chloride is formed, and so forth. The sulfo group is preferably a sulphonate Balt group, optionally, sodium or potassium sulphonate (S03Na or S03K) and the carboxyl group is preferably a carboxylate salt group, optionally, sodium or 7 potassium carboxylate. The haiogen is Cl, Br oc P. Preferably, the hydrazyl group or derivative is a radical of the following generai formula: Z - N - N -

II

Y · A wherein A is hydrogen or the unpaired electron of one nitrogen atom, Y is hydrogen or an organic group and Z is an organic group, or Y znd Z together with the adjacent nitrogen atom form a nitrogen-containing heterocycle (e.g., a carbazyl group); provided that when X is a substituted or unsubstituted hydrazyl group as aforesaid one of the NO2 groups may be replaced by a sulfo group. For example, where A is hydrogen and Y and Z are phenyl groups, the overall compound becomes diphenyl picrylhydrazine (DPPH). Where A is an unpaired electron and Y and Z are phenyl groups, the diphenyl picrylhydrazyl (DPPZ) radical is formed. Carbazyl picrylamine (CPZ) is formed when A is hydrogen and Y and Z are paired phenyl groups, thus forming a nitrogen-containing heterocycle. One particularly desirable water-soluble derivative has the £ollowing structural formula for the hydrazyl-derived group: LV 10574 wherein Y and Z are phenyl groups, and potassium is optionally >·. hydrogen or sodium.

Another particularly suitable water soluble derivative is diphenyl dinitrosulfonate phenylhydrazyl (DDSH)/ which includes a sulfonate potassium salt group S03K (substituted for the 5-NO2 group) and is preferably synthesized by the interaction of diphenylhydrazine with the potassium salt of 2-chloro-3, 5-dinitro-benzene sulfonic acid in dilute alcohol or dilute dioxane, with subseguent oxidation of the resulting hydrazine by lead dioxide. Additional details concerning the synthesis of the DDSH radical are set forth in Investioation

In_Thfi_Field Of The Chemistrv Qf_Free RaflicalS—Qf_Ih£

Hy.dragi,Ilg_Series No. VIII, by M.A. Ikrina et al, Zhurnal

Obschei Khimii/ Volume 32, No. 12 at 3952-3957, December, 1962, incorporated here by reference.

It has been shown that trinitrobenzene derivatives ae above, e.g., diphenyl picrylhydrazine, ere effective when administered to a host in a substantially pure agueous solution/suspension ranging at dilutions between about 10“3 -10"15 molar concentration (i.e., *'therapeutically-ef f ective concentrations") . FIGURĒ 1 is a plot of the average survival time in dsys of NMRI mice transplanted with MAC16 colon carcinoma celis after treatment by subcutaneous injection with various therapeutically-effective concentrations of picryl 9 chloride according to the teachinge of the present invention.. Aa seen in FIGURĒ 1, the untreated control animals lived an average of less than 10 days, whereas animals treated with the various specified concentrations of picryl chloride had significant survival rātes. The best results were obtained at 10"1Z molar concentration when the animals were injected subcutaneously for five (5) days (one injection per day). At this concentration, most of the treated animals were tumor-free on day 60. FIGURĒ 2 is a plot of the dose response of a GM892 celi line to diphenyl picrylhydrazyl (DPPZ) and diphenyl picrylhydrazine (DPPH). The figurē rep'resents an average of 3-6 different experiments, with a Coulter counter used to determine the celi count. As seen in FIGURĒ 2, both aģents provide significant in-vitro anti-tumor effects on this celi line when administered in a substantially pure agueous solution/suspension ranging at dilutions between about 10“3 -10”15 molar concentration.

Significant therapeutic results are obtained using pharmaceutical or veterinary compositions based on the above compounds, singularly or in combination, dissolved or di6persed in an aqueous medium in the concentrations previou3ly described. For example, carminic acid alone (or a derivative thereof) is especially useful as an anti-viral 10 LV 10574 aģent. Carminic acid has a C-glycoside (CfiHn05) side-linked to a polyhydroxyanthraquinone es evidenced by the following formula:

In combination form, one preferred composition is an admixture of one or more trinitrobenzene derivatives and an anthraquinone having a glycosidic moiety, optionally carminic acid. For example, one such composition is an admixture of picryl chloride (or picryl sulfonete) and carminic acid. Although not meant to be limiting, the preferred ratio of picryl chloride to carminic acid is preferably between 1:1 and 1:2 but with the concentration of the active ingrediants being in a therepeutically-effective concentration of between about 10“3 - 10"15 molar concentration. A therapeutically-effective amount of the pharmaceutically composition in solution or suspension is between 2.0-5.0 mis, and this amount is apparently substantially independent of the bodyweight of the host animal.

If desired, more than one trinitrobenzene can be advantageously incorporated into the admixture. For esample, when picric acid and DPPH are used, these trinitrobenzenes may act synergistically in generating free radicals and a free 11 radical chain reaction mechanism. The quinone, if used, can elso be a source of OH free radicals. In one embodiment, predetermined araounts of picric acid/ DPPH and carminic acid are mixed in a substantially pure aqueous solution/suspension ranging at dilutions giving between about ΙΟ”3 - 10"15 molar concentration. The DPPH has the highest dilution, followed by the carminic acid and then the picric acid. While not meant to be limiting, a pharmaceutical or veterinary compoeition may be formed by first dissolving the hydrazine derivative in double-distilled, deionized water in a clean glass Container under sterils conditions. Thereafter, the carminic acid is added and mixed into the solution. The picric acid is then added and the solution throughly mixed.' Serial dilution can then be used to obtain the desired molar concentration. Alternatively, the three constituents are mixed together prior to dissolution in the carrier.

According to other features of the invention, the efficacy of a free radical chain reaction mechanism may be enhanced through administration of iron or any other transitional mētai/ especially copper. Although not described in detail, it is also envisioned that the anti-tumor aģents described above can be administered to the host subcutaneously/ intravenously or using an acceptable carrier or excipient. Also, while double-distilled, deionized water is the preferred solution/suspension liguid, other dilutante, 12 LV 10574 such as a dimethylsulfoxide/water solution, arachis oil/ olive oil, vegetable oil or corn oil, may be useful as well. Yet another useful cattflyst for the free radical mechanism is a (low concentration) polyunsaturated fatty acid, which ie a long Chain free carboxylic acid typically found in a lipid.

It should be appreciated that while the preferred trinitrobenzene derivatives useful in accordance with the present invention are picric acid, picryl chloride, picryl sulfonate, diphenyl picrylhydrazine and - diphenyl picrylhydrazyl, other trinitrobenzene compounds are also suitable catalysts for the free radical mechanism, Such compounds are included within the above general formula. (DPPZ),

In the above general formula, when A is hydrogen and Y is a phenyl group, Z may be, for example, any aromatic group such as shown in appendix Table I or an aliphatic group as shown in appendix Table II. Alternatively, Y and Z may be any of the aromatic compounds shown in appendix Table III. Another set of "phenyl" derivatives is derived from the compounds shown in appendix Table IV and a set of "carbazyl” derivatives is defined by the formulae set forth in eppendix Table V. Although not meant to be limiting, the preferred hydrazine derivatives are diphenyl picrylhydrazine (DPPH) and derivatives thereof such as diphenyl picrylhydrazyl 13 phenyl picrylhydrazine (PPH), carbazyl piCrylamine (CP2) and 2-sulfophenyl/ 2-sul£ophenyl, picrylhydrazine.

Moreover, while the emphasis in the above discussion has been on novel anti-cancer and anti-viral therapies, it should be appreciated that the active Chemical species generated by the trinitrobenzene and carminic acid derivatives according to the invention may also evidence significant anti-fungal and anti-bacterial effects. Use of such low concentration therapies is also being inveetigated in connection with treatment of genetic and autoimmune disorders.

In one pilot study of the invention, a sixty four year old female with advanced cervical cancer was treated with diphenyl dinitrosulfonate phenylhydrazyl (DDSH). On systematic examination, the patient presented with lower abdominal pain, anaemia, and vaginai bleeding. On gynecological examination, a large mass was located protrubmg from the birth canal and extending to the lateral pelvic wall. The patient was diagnosed with stage 3 cervical cancer (squamous celi carcinoma), a finding later confirmed during exploratory surgery in which extensive metastatic masses throughout the abdotnen were also discovered. After diagnosed as termiņai, the patient «as treated with a single 2.0 ml. subcutaneous injection of DDSH dissolved in double-distilled, deionized water at 10“9 molar concentration. No other 14 LV 10574 treatment was under’taken. Upon recent clinicai ezamination, the patient was found to be free from the cancer, with no evidence remaining of the metastatic raasses.

In another pilot study a sixteen year old mele, diagnosed with metastatic pheochromocytoma involving the liver and jawbone, was also treated with DDSH by subcutaneous injection at 10-9 molar concentration. when first examined, the patient had extremely high blood pressure, 180/160, an abnormal heart rāte, and extensive pain in the jaw area. A first subcutaneous injection was given in April, 1990, with a follow-up injection provided in November, 1990. Upon recent investigation, the patient’s blood pressure is normai, the extensive jawbone pain has subsided, and general health is considered good.

Based on ongoing pilot studies, carminic acid has evidenced significant anti-viral effects when dissolved in an aqueous medium at low concentrations. In one pilot study, a thirty seven year old male was diagnosed as Hiv positive by the Standard ELISA tēst. When first examined in November, 1990, the patient had oral thrush, very severe herpes zooster of the left facial nerve with involvement of the left orbital region, hard bilateral cervical lymph ņodes, and an enlarged liver and spleen. Thereafter, the patient was treated with carminic acid, dissolved in double-distilled, deionized water 15 at 10“6 molar concentration, via subcutaneous injections. For l· * five days, the patient received a single 2.0 ml. injection per day. After five . days, a similar five day course (one injection per day for five days) was repeated. After the fourth course (20 injections)/ the patient was in good general condition, was no longer anaemic, and the oral thrush, cervical lymph nodēs and herpes zooster infection had cleared. The spleen and liver were normai and the patient had gained weight. Significantly, the patient’s white blood count (WBC) had increased from 2,000 cells/mm3 to 12,400 cells/mm3, with a corresponding increase in hemoglobin (Hb) from 11.8 to 14.7 grams per deciliter.

These findings are significant and indicate that carmihic acid, in therapeutically-effective concentrations as described, appears to Btimulate the immune system. It is believed that other quinones having side-chained sugars (and derivatives thereof) may also exhibit anti-viral activity when administered according to the teachings herein. Thus, an important aspect of the invention is the use of carminic acid and its derivatives in the preparation of a medicament for the prophylaxis or therapy of virai disease such as AIDS. Such derivatives have the follovring general formula:

16 LV 10574 where R is COOH (carminic acid) or other organic or inorganic functional group such as NH2, SOjtK, H or Ne], and the C-glycoside is any sugar. The anthraguinone may optionally be a benzoquinone (single ring) or napthaquinone (double ring).

While the invention has been described in part by reference to variou6 preferred erobodiments, thoBe skilled in the art will appreciate that various modif ications, substitutions, omissions and changes may be made without departing therefrom. 17

Claims

EN 10574 PATENT FORMULA POINTS 1. Finished form for use in the treatment or prophylaxis of malignant neoplasms or viral infections consisting of a compound dissolved or dispersed in an aqueous medium at a concentration of 3 to 15 oli in 10 to 10 moles per liter with the general formula: no3

wherein X is selected from OH, NHg, halogen, sulpho, carboxy, OCH and substituted or unsubstituted hydrazilyl, of formula:, Z-N- N - II YA wherein A is hydrogen or an unseparated electron of nitrogen, Y is hydrogen or an organic group, and Z is an organic group, or Y and Z together with the adjacent nitrogen atom form a nitrogen-containing heteroalkyl; provided that when X is a substituted or unsubstituted hydrazil group, one of the NOg groups may be replaced by a sulpho group.

2. Form for use in the treatment or prophylaxis of human or animal malignant neoplasms or viral infections consisting of: (a) one or more compounds of the general formula: X-P -2- wherein P is trinitrophenyl and X is selected from OH, NOg, , halogen, sulcoxy, carboxyl, OCH 2 and substituted or unsubstituted hydrazyl group of the formula: Z - N - N - i IYA wherein A is hydrogen or an unbranched electron of nitrogen, Y is hydrogen or an organic group, and Z is an organic group, or Yun Z, together with the adjacent nitrogen atom, forms a nitrogen-containing heteroalkyl; provided that when X is a substituted or unsubstituted hydrazyl group, one of the NOg groups may be replaced by a sulpho group; and (b) quinone, if desired, glycosylated anthraquinone, preferably carmine acid.

The finished form according to claim 1 or 2, wherein the sulcogroup is a sulfonate salt group, if desired, sodium or potassium sulfonate.

The finished form according to claim 1 or 2, wherein the carboxyl group is a carboxylate salt group, if desired, sodium or potassium carboxylate.

A finished form according to claim 1 or 2, wherein the halogen is 01, Br or P.

A finished form as defined in any one of Claims 1 to 5 of the patent formula, including picric acid, picryl chloride, picryl sulfonate or diphenylchrylhydrazine, or any two or more thereof.

7. The finished form as defined in any one of claims 1 to 6 in the form of dosage units; if desired, each unit may contain from 2.0 to 5.0 ml of solution or dispersion.

A pharmaceutical or veterinary finished form wherein the radical or compound as defined in claim 1 of formula LV 10574 is dissolved or dispersed in an aqueous medium in the range of 10 to " 3 to 10 ", 15 moles / liter. i ·

A finished form according to claim 8 in the form of dosage units; , 1a, each unit may contain from 2.0 to 5.0 ml of solution or dispersion.

10. The finished form according to claim 8 or 9, further comprising anthraquinone glycoside, if desired, carminic acid.

Use of a formula as defined in claim 1 for a radical or combination with a patent, if desired, together with anthraquinone glycoside such as carmine acid, cancer or virus for prophylaxis or therapy for the preparation of therapeutic agents.

The use of claim 11, wherein said 3 radical or compound is in an aqueous medium at a concentration of from 10 to 15 moles per liter.

13. Finished form containing anthraquinone glycoside, if desired, carminic acid, dissolved in water in a cono-centered interlayer — 3—15 to 10 to 10 moles per liter, for use in the treatment of oilvec virus infection.

Use of antraquinone glycoside, if desired, carminic acid for the preparation of medicaments for the prevention or treatment of viral infection.

The use of claim 14, wherein the medicament is for the prophylaxis or treatment of a human immunodeficiency virus infection.