CA2079803A1 - Use of trinitrobenzenes or carminic acid in the treatment of cancer or viral diseases - Google Patents

Abstract

Compounds or radicals based on trinitrobenzene, carminic acid and derivatives thereof are useful as anti-cancer and anti-viral agents. Such compounds and formulations based thereon may be administered to a human or animal patient orally or parenterally in a substantially pure aqueous solution/suspension ranging at dilutions between about 10-3-10-15 molar concentrations. In preferred embodiments, one or more of said compounds and an anthraquinone (such as carminic acid) are used. Carminic acid alone exhibits significant anti-viral effects.

Description

WO 91/lStOO
,~ PCT/GB91 /OOSl 7 ,f ~ :-r .
Z~ '~3 ~
USE OF TRINITROBENZENES OR CARMINIC ACID
IN THE TREATMENT OF CANCER OR VIRAL DISEASES

The present invention relates generally to compounds and therapeutic formulat~on~ ba6ed on trinitrobenzene and~or carminic acid (or their derivatives~ which ln certain low concentrations exhibit anti-cancer or anti-vlral activity.

Traditional cancer treatments generslly involve surgery, radiotherapy, chemotherapy, or some combination ~hereof.
w~ e these treatments are often effecti~e in lengthening a pBtient' 8 life or sometime6 eradicating the cancer, they ha~e well-known serious side effectq. More recently, alternative attempt6 to treat cert in cancera have been developed. One such technique, ~immunotherapy,~ is designed to strengthen the innate ability of the patlent's immune sy~tem to flght cancer.

While ~iagnostlc and treatment techniques have improved significantly over the last decades, there has been very little impro~ement in the overall survi~al rate in patients, especially those with solid tumours treated by these orthodox methods. And, although alternative techniques ~uch as "immunotherapy" show promise, the treatments de~elopsd thus far can only help a limited number of patients, 6till exhibit toxic side effects and~or are complex and e~pensive to per~orm.

53U~3~;TITUTE 5~EE~

:~ W O 91/~5200 PC~r/G891/00517 ` 2 In the area of anti-viral medicine, many anti-viral drugs are known, but therapy msy involve hi~h doses or be of limited effect. In HIv treatment, AZ~ is the only significantly effective drug foun~ to date, but treatment re6ults are variable and the drug i~ expen6ive.

The present invention approache6 these problems through the low dose use of readily avallable trinitrobenzene compounds ~nd~or carminic acid (either singularly or in combination). lt has now been found, most surprisingly, that such compound3 are efficaciou~ a~ anti-cancer or ant~-viral agents when administered at low concentrationq, reg~rdless of patient bodyweight. Toxicity and expense problems a~sociated with the prior art thus do not apply.

Thus, in one aspect the invention proYideq for use in the therapy or prophylasis of neopl~sm or viral lnfectlon in human snd non-human animals, a formulation comprising a compound dis~ol~ed or di6persed in an aqueous medium at a concentration of 10-3 to 1~-15 moles per litre and having the general formula:
~1~
>~ O~L

~J2.

f 3 Z~

wherein X is ~elected from OH, NH2 halogen, a 6ulfo group, a carboxyl group, OCH3, or a ~ubstituted or un6ubstitute~
hydrazyl group of the formuls:

Z - N - N -Y A
wherein A i5 hydrogen or an unpaired electron of the nitrogen atom, Y is hydrogen or an organic group and Z i~ an organic group, or Y and Z together with the ad~acent nitroqen atom form a nitrogen-contalning heterocycle; provided that when X
is a 6ubstituted or un~ubstituted hydrazyl group as aforesaid one of the NO2 groups may be replaced by a sulfo group. The ~ulfo group is prefer~bly a sulphonate salt group, optionally, sodium or potassium sulphonate (SO~Na or SO3R) and the carboxyl group is ~referably a carbo~ylato salt group, optionally, sodium or potas~ium carboxylate.

In another aspect the in~ention provide~ for use in the therapy or prophyla~is of neoplasm or viral infection in human and non-human anlmals, a formulation comprising:

(a) one or more compounds of the g~neral formula:
X - P
wherein P is a nitrophenyl, and X i~ ~elected from O~, NH2 halogen, a sulfo group, a carbo~yl ~roup, OCH3 or a substituted or unsubstituted hydrazyl group of the formula:

WO 91/15200 PCI'/GB91/00517 2J`7~

Z - N - N -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 nitrog~n atom ~orm a nitrogen-containing heterocycle;
provided that when X is a substituted or unsub~tituted hydrazyl group QS afore~aid one of the NO2 groups may be replaced by a sulfo group; and ~b) a quinone, such a~ carminic acid or its derivative3.

A further aspect of the invention i8 a pharmaceutical or veterinary formulation wherein a compound as defined above i5 di~olved or dispersed in an aqueous medium at a concentration of from about 10-3 to 10-15 moles per litre.

Signiftcantly, carmlnic acid has been found to axhibit anti-viral effects when used alon~ at low molar concentration~
accord~ng to the invention. Thus, an important aspect of the invention ia the use of carminic acid and its derivetives in the preparation of a medicament for the prophylaxis or therapy 5 2~?7~ 3 of viral disease such as AID5. Such derivatives have the following general formula:

R~o~l ~ o~
where ~ is COO~ (i.e., carminic acid) or 60me other or~anic or inorganic functional group such as NH2, SO3LX, H or Na], snd the C-glycoside is any sugar. The anthraquinone may optionally be a benzo~uinone (single ring) or napthaquinone (double ring).

Without being tied to an exact mechani~m, it i3 believed thst the compounds referred to above function by initiating and propagating a free radical mechanlsm, thereby producing active chemical species that ~electively attack abnormal cell structures. In Ç~SQL ~a~rch 36 (1978), 1745-1750, Bachur et al describe possible free radical mech~nisms in connection with the known biological actionc of quinone-containing anti-cancer drugs. It is postulated that these drugs may generate oxygen-dependent free radicals ~uch as superoxide or hydro2yl radicals. In the present invention, it is likely thst the abo~e mentioned compound~ serve as cataly6ts for a redo~-recycling mechanism which continuou~ly generates free radicals such as superoxides. The free radicals, or their by-p~oducts, selectively attack cancer cell~ or viruses.

2~ J~ 6 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 carmlnic acid or a derivative thereof), or an admixture of one or more trinitrobenzene derivatives and an anthraquinone glycoside. Carminlc acid has been used in the laboratory for staining nucleic acids and, interestingly, its effect on DNA ls inhibited by, in5~r ~li~, free radical scavengers (Lown et al., B1ooraanic ~hQm.
(1979), l7-29). Again, such a formulation contains the actiYe ingred~ents dissolved or su pended in an aqueou~ medlum at a concentration in the range from 10-3 to lO-1s moles per litre, and may be admin~stered orally or parenterally. Carminic acid is especially useful for anti-viral treatment.

For 3 more complete understsnding of the present invention and the advantage6 thereof, reference ~hould be made to the following detailed description taken in connection with the accompanying figures in which:

FIGURE l is a plot of the a~erage survival ti~e in days of NMRI mice transplanted with MAC16 colon carclnoma cells after treatment wlth various therapeutically-~ffective concentrations of picryl chloride according to the teachings of the pre~ent invention; and ~ ~ 2~

F~GURE 2 is a plot of the dose response of a GM892 cell line to diphenyl picrylhydrazyl ~DPPZ) and diphenyl picrylhydrazine (DPPH) according to the teachings of the invention.

Turning now to the various specific aspectQ of the invention, a compound cspable of initiating and propagating a free radical mechanism ~ay be dissolv~ad or disper6ed in an aqueous medium at a therapeutically-effective concentratiol~ in the range of from about 10-3 - 10-15 moles per litre. Such compounds catalytically trigger free radical chain reactions, thereby producing active chemical ~pecie6 that selectively attsck abnormsl cells. One such set of compound~ include derivati~es of nitrobenzene o~ the following general formula:

X ~~ ~JOL

wherein X is selected ~rom ~H, N~2 halogen, a sulo group, a csrboxyl group, OCH3 or a ~ubstituted or unsubstituted hydrazyl group. When X i5 the hydroxyl radicsl, the compound is picric acid. ~hen X is chloride, picryl chloride is formed, and so forth. The Sulro group is preferably a ~ulphonate salt group, optionally, sodium or potassium ~ulphsnate (SO3Na or SO3K) and the carboxyl group is pr~ferably a carboxylate salt group, optionslly, sodium or 2~ ~ ~8`~ 8 ~ ~
potasslum carboxylate. The halogen is Cl, Br or F.
Preferably, the hydrazyl group or derivative ls a radical of the followinq general formula:

Y A
wherein A is hydrogen or the unpaired electron of one nitrogen atom, Y i~ hydrogen or an organic group and Z i6 an organic group, or Y znd Z together with the adja~ent nitrogen atom form a nitrogen-containing heterocycle (e.g., a carbazyl group); provlded that when X is a sub6tltuted or unsubstituted hydrazyl group as afore~aid one of the NO2 groups may be replaced by a sulfo group. For example, where A i9 hy~rogen and Y and Z are phenyl groups, the overall compound become~
diphenyl picrylhydrazine (DPP~). Where A i5 an unpaired electron and Y and Z are phenyl groups, th~ diphenyl picrylhydrazyl (DPPZ) radical ia formed. Carbazyl picrylamine (CPZ) i~ formed when A i6 hydrogen and Y and Z are paired phenyl groups, thus forming a nitrogen-containing heterocycle. One particularly deairable water-soluble derivative has the following Rtructural formula for the hydrazyl-derived group:

Y A

WO91/152~ PCT/GB91/~517 9 2~
wherein Y and Z are phenyl groups, and potassium i6 optionally hydrogen or sodium.

Another particularly ~uitable water soluble derivative is diphenyl dinitro~ulfonate phenylhydrazyl (DDSH), which includes a sulfonats potas6ium salt group SO3R (substituted for the 5-NO2 group) and i9 preferably syntheslzed by the interactlon of diphenylhydrazine with the pota6sium salt o~
2-chloro-3, 5-dinitro-benzene ~ulfonic acid in dilute alcohol or dilute dioxane, with subsequent o~idation of the resultin~
hydrazin~ by lead dioxide. Additional detall6 concerninq the synthesis of the DDS~ radical are set forth in In~estigation In The Field Of Th~ Chemistry Of F~99L~ 18 Of The Hy~r~ u~ SeLiQ~ ~ VITI, by M.A. Ikrina et al, Zhurnal Obschei ~himii, Volume 32, No. 12 at 3952-3957, Decsmber, 1962, incorporated here by reference.

It has been shown that trinitrobenzene deriv~tives as above, e.g., dip.henyl picrylhydrazine, are effective when admini~tered to a host in a substantially pure aqueous solution/.~u~pension ranging at dilutions between about 10-3 -l0-15 molar concentration (i.e., ~therapeutically-effective concentrations~). FIGURE l is a pl~t of the average survival tlme ln days of NMRI mice transplanted with MAC16 colon carcinoma cells after treatment by subcutaneous lnjection with various therapeutically-effective concentrations of picryl W091/152~ PCTtGB9l/~517 2~

chloride according to the teachings of the pre~ent lnvention.
Aq seen in FIGURE 1, the untreated control anlmals lived an average of les~ than 10 days, whereas animal~ treated with the variou6 ~pecified concentration~ of picryl chloride had significant survival rates. The best results were obtained at 10-1Z ~olar 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 dsy 60.

FIGURE 2 i~ a plot of the dose response of 3 GM892 cell line to diphenyl picrylhydrszyl (DPPZ) and diphenyl picrylhydrazine (DPPH). The figure represent6 an average of 3-6 different experiments, with a Coulter counter u~ed to determine the cell count. As seen in F~GURE 2, both agents provide signlficant in-YiSLQ anti-tumor effects on this cell line when admini6tered in a ~ubstantially pure aqueous solution/3uspension ranging at dilution6 between about 10-3 -10-l5 molar concentration.

Sig~ificant therapeutic resultq are obtaine~ u61ng pharmaceutical or Yeterinary compositions ba~ed on the above compounds, singularly or in combination, di~salYed or di6per6ed in an squeous medium in the concentrations pre~iously described. For example, carminic acid alone (or a deriYatl~e thareof) is especially us~ful as sn anti-vl~al WO91/15200 2~ t2~'n ~ PCT/GB91/00517 agent. Carminic acld has a C-glyco~ide (C6H~1O5) side-linked to a polyhydroxyanthraquinone 88 e~ldenced by the following formula:

0~
c I~
tn combinatlon form, one preferred compo~itlon i8 an admisture of one or more trinitrobenzene derl~atives and an anthraquinone having a glycosidic moiety, optlonally carmlnic ac~d. For e~ample, one such composition i5 an admi~ture of picryl chloride (or picryl 6ulfonate) ana car~inic aci~.
Although not meant to be limitin~, the preferred ratio of picryl chlorlde to carminic acid i9 preferably between l:l and 1:2 but with the concentration of the acti~e ingredients being in a therapeutically-effecti~e conc~antration of between about 10-3 - lO-15 molar concentratlon. A
therapeutically-effective amount of the ph~rmaceutically composition in ~olutton or suspension i~ betw~en 2.0-5.0 mls, and this amount i8 3pparently substantt~lly tndependent of the bo~yweight of the ho6t 3nimal.

I~ desired, more than one trinltrobenzene can be advanta~eously incorporated into the admixture. For e~ample, when plcric acid and DPPH are uBed, these trinitroben2enes msy act synergi6tically in gen0rating free radicals and a fre-2~ 2 radical chain reaction mechanism. The qulnone, if used, canalso be a -Qource of OH free radicals. In one embodiment, predetermined amounts of picric acid, DPPH and carminic acid are mi~ed in a substantially pure aqueous solution/suspenslon ranging at dilutions gi~ing between about 10-3 - 10-15 molar concentration. The DPPH has the highest dilution, followQa by the carminic acid and then the picric acid. ~hile not meant to be limiting, a pharmaceutical or veterinary composition may be formed by firRt dissolving the hydrazine derivative in double-distilled, deionized water in a clean gla6s container under ~terile condition~. Therea~ter, the carminic acid is added and mixed into the solution. The picric acid is then added and the solutlon throughly mixed. Serial dilution can then be used to obtain the desired molar concantration.
Alternatively, the three constituents are mi~ed together prior to dissolution in the carrier.

According to other features of the invention, the efficacy of a free radical chain reactlon mechanlEm may be enhanced through administration of iron or any other transitional metal, especially copper. Although not descrlbed in detail, it i~ also envisioned that the anti-tu~or agent~
described abo~e can be admini~tered to the host Qubcutaneously, intravenously or using an acceptable carrier or e~cipient. Also, while double-distilled, deionized water i~ the preferred solutlon/suspension liquid, other dilutants, .

WO91/152~ 2~ ~ 9~ PCT/GB91/00517 f ` 1 3 such as a dimethylsulfoxide/water solution, arachis oil, olive oil, vegetable oil or corn oil, may be useful as well. Yet another useful catalyst for the free radical mechanism i9 a (low concentration) polyunsaturated fatty acid, which i6 a long chain free carboxylic acid typically found in a lipld.

It should be spprecisted that whlle 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 al~o suitable catalysts for the free radical mechanism. Such compounds are included within the above general formula.

In the above general formula, when A is hydrogen and Y is a phenyl group, 2 may be, for e~ample, 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 appendi~ Table III. Another ~et of "phenyl" ~erivatives is derived from the compounds ~hown in appendis Table IV and a set of "carbazyl~ deri~atives is defined by the formulae set forth in appendix Table V.
;~ Althou~h not meant to be limiting, the preferred hydrazine derivatives are diphenyl picrylhydrazine (DPPH) and d~rivatives thereo~ ~uch a~ diphenyl picrylhydrazyl (DPPZ), 2~ 3~ ~ 1 4 pnenyl picrylhydrazine ~PPH), carbazyl picrylamine (C~Z) and 2-sulfophenyl, 2-sulfophenyl, picrylhydrazine.

Moreover, while the emphasis in the above discu~6ion has been on novel anti-cancer and anti-viral therapies, it should be appreciated that the active chelnical ~pecies generated by the trinitrobenzene and carminic acid derivatives accordinq to the invention may al60 evidence ~ignificant anti-fungal and anti-bacterial effect3. Use of such low concentration therapies is also being investigated in connection with treatment of genetic and autoimmune di orders.

In one pilot study of the invention, a sixty four year old fem~le with advanced cervical cancer wa~ treated with diphenyl dinitrosulfonate phenylhydrazyl (DDSH). On ~ystematic ex~mination, the patient pre~ented wlth lower abdominal pain, anaemia, and vaginal bleedin~. On gynecological examination, a ldrge mass was located protrubing from the birth canal and extendlng to the lateral pelvic wall. The patient w3s dlagnosed with stage 3 cervical cancer (squamou6 cell carcinoma), a finding later confirmed during exploratory surgery in which extenslve metastatic masses throughout the abdomen were al-qo discovered. After diagnosed as terminal, the patient was treated with a ~ingle 2.0 ml.
subcutaneous injection of DDSH di~solved in double-distilled, deionized water at 10-9 molar concentration. No other WO91/15200 2~ PCT/GB91/00517 f" ' : 1 5 tre~tm~nt was undertaken. Upon recent clinical examinatlon, the patient was found to be ree from the cancer, with no evldence remaining of the m~tastatic masses.

In another pilot study a sixteen year old male, diagnosed with metastatic pheochromocytoma involving the liver and jawbone, was al~o treated with DDSH by subcutaneous injection at 10-9 molar concentration. When fir~t examined, the patient had extremely high blood pressure, 180/l60, an abnormal heart rate, and extensive psin ln the jaw area. A first subcutaneous injection wa~ given in April, 1990, with a follow-up injection provided in November, 1990. Upon recent investigation, the patient~s blood pressure is normal, the extensive jawbone paln has subsided, and general health i~
consldered good.

~ a8ed on ongoing pilot studie~, carminic acid has evidenced slgnificant anti-~iral effects when dissolved in an aqueou~ medium at low concentrations. In one pilot study, a thirty seven year old male was diagnosed as HIV positive by the 6tandard ELISA test. When fir~t esamined ln November, 199~, the patient had oral thrush, very severe herpes zoost~r of the lsft facial nerve with invol~ement of the left orbital region, hard bilateral cervical lymph nodes, and an enlarged liver and ~pleen. Thereafter, ~he patient was treated with carminic acid, dissolved in double-distilled, ~eionized water 2 ~ 6 at 10-6 molar concentration, via su~cutaneous injection~. For five ~ays, the patient received a Ringle 2.0 ml. ln~ection per day. Ater five days, a similar five day course (one injection per day for five days) wa~ repeated. After the ourth cour~e (20 in~ections), the patient wa~ in good general condition, W8~ no longer anaemlc, and the oral thru5h, cer~ical lymph nodes and herpes zooster infectton had cleared. The ~pleen and liv~r were normal and the patient had gainea weight. Signlfic~ntly, the patient's white blood count (~3C) had 1ncreased rom 2,000 cells/mm3 to l2,qO0 cell~/mm3, wtth a correspondlng increase in hemoglobin (Hb) rom ll.3 to l9.7 grams per deciliter.

TheYe findlngs are significAnt and in~icate that carminic scid, tn therapeutically-effective concentrations a~
described, appe3rs to stimulate the immune system. It it~
believed that other quinones ha~ing ~ide-chained sugars (and derivatives thereof) msy al~o e~hiblt anti-viral activity when admlnistered ac w rding to the teachings herein. Thus, an important a~pect of the in~ention i8 the use of carminic acl~
and it~ deri~atives in the preparation of ~ medicament for the prophyla2ls or therapy of viral disease such as AIDS. Such ~Qrivstive~ have the followlng ~ene~al formula:
G~
R 7~~

~

WO91/15200 ~ PCT/GB91/~517 where R is COO~ (carminic acld) or other organic or lnorganic functional group 3uch as NH2, SO3[~, ~ or Ns], and the C-glycoside is any .qugar. The anthraquinone may optionally be a benzoquinone (single ring) or napthaquinone (double ring).

While the inv~ntion has been described in part by reference to various preferred embodiments, those skilled in the art will appreciate that various modificationfi, substitution6, omi6sions and changes may be made without departing therefrom.

Claims (15)

1. For use in the therapy or prophylaxis of neoplasm or viral infection in human and non-human animals, a formulation comprising a compound dissolved or dispersed in an aqueous medium at a concentration in the range from 13-3 to 10-15 moles per litre and having the general formula:

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:

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.

2. For use in the therapy or prophylaxis of neoplasm or viral infection in human and non-human animals, a formulation comprising:

(a) one or more compounds of the general formula:
X - P
wherein P is trinitrophenyl and X is selected from OH, NH2 halogen, a sulfo group, a carboxyl group, OCH3 or a substituted or unsubstituted hydrazyl group of the formula:

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; and (b) a quinone, optionally an anthraquinone having a glycosidic moiety, preferably, carminic acid.

3. A formulation as claimed in Claim 1 or Claim 2 wherein the sulfo group is a sulphonate salt group, optionally, sodium or potassium sulphonate.

4. A formulation as claimed in Claim 1 or Claim 2 wherein the carboxyl group is a carboxylate salt group, optionally, sodium or potassium carboxylate.

5. A formulation as claimed in Claim 1 or Claim 2 wherein halogen in Cl, or or F.

6. A formulation as defined in any one of Claims 1 through 5 including picric acid, picryl chloride, picryl sulfonate or diphenyl picrylhydrazine or any two or more thereof.

7. A formulation as defined in any one of Claims 1 through 6 in unit dosage form, optionally each unit comprising between 2.0-5.0 mls. of the solution or dispersion.

8. A pharmaceutical or veterinary formulation wherein a radical or compound as defined in Claim 1 is dissolved or dispersed in an aqueous medium at a concentration in the range from 10-3 to 10-15 moles per litre.

9. A formulation as claimed in Claim 8 and in unit dosage form, optionally each unit comprising from 2.0-5.0 ml of the solution or dispersion.

10. A formulation as claimed in Claim 8 or Claim 9 also comprising an anthraquinone glycoside, optionally carminic acid.

11. The use of a radical or compound having a formula as defined in Claim 1, optionally together with an enthraquinone glycoside, e.g. carminic acid, in the preparation of a medicament for the prophylaxis or therapy of cancer or viral disease.

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

13. For use in the therapy of viral infection in human animals, a formulation comprising an anthraquinone glycoside, optionally carminic acid, dissolved in an aqueous medium at a concentration in the range from 10-3 to 10-15 moles per litre.

14. The use of an anthraquinone glycoside, optionally carminic acid, in the preparation of a medicament for the prophylaxis or treatment of a viral infection.

15. The use of Claim 14 wherein the medicament is for the prophylaxis or treatment of an HIV infection.