WO2011086424A1

WO2011086424A1 - Pharmaceutical use of multicyclic compounds mixtures as concomitant anti-cancer, anti-inflammatory and anti-pain agents

Info

Publication number: WO2011086424A1
Application number: PCT/IB2010/050199
Authority: WO
Inventors: Luiz Francisco Pianowski; João Batista CALIXTO; Claudio Paulino Chaves
Original assignee: Amazonia Fitomedicamentos Ltda
Priority date: 2010-01-15
Filing date: 2010-01-15
Publication date: 2011-07-21

Abstract

This invention refers to a pharmaceutical use of a mixture of multicyclic compounds, namely ingenol and lanosta-8,24-dien-3-ol compounds, as concomitant anti-cancer, anti-inflammatory and anti-pain agents.

Description

PHARMACEUTICAL USE OF MULTICYCLIC COMPOUNDS MIXTURES AS CONCOMITANT ANTI-CANCER, ANTI-INFLAMMATORY AND ANTI-PAIN AGENTS.

This invention generally refers to a pharmaceutical use of a mixture of multicyclic compounds, namely ingenol and lanosta-8,24-dien- 3-ol compounds , as concomitant anti-cancer, anti-inflammatory and anti- pain agents.

BACKGROUND

Certain ingenol related compounds are known as anti-cancer agents, for instance as mentioned in patent US7,378,445, ingenol-3- angelate, 20-deoxy-ingenol-3-angelate and 20-O-acetyl-ingenol-3- angelate.

Certain lanosta-8,24-dien-3-ol compounds, such as euphol, are known as anti-inflammation agents, such as reported in J. Pharm. Pharmacol. 2000 Jan;52(l):119-24.

There is no known disclosure in the art of compounds known to be concomitantly effective against cancer, inflammation and pain, as in the mixtures of the present invention, namely mixtures of ingenol and lanosta-8,24-dien-3-ol compounds.

The meaning of concomitant anti-cancer, anti-inflammatory and anti-pain effects relates to any amelioration obtained against cancer conditions, along with decrease in inflammation and pain, compared to what would be observed without the administration of the mixture of the invention.

DESCRIPTION OF THE FIGURES

Figures 1 and 2 respectively present the concentration/activity curves for the assayed substance and for doxorubicin, a chemotherapeutical product used as a positive control for cells in different concentrations (250 to 0.25 -g/ml), relating the percentage of cell growth to the concentration of the utilized product.

Figures 3A, 3B and 3C: 3A is a graph of time vs. frequency of response. 3B and 3C are representations of the areas under the curves, allowing a percentage comparison among tested compounds, after acute treatment from 0 to 48h (3B) and prolonged (3rd to 24th day) after injection of CFA.

Figures 4A and 4B: 4A being a graph of time vs. frequency of response, and 4B the expression of the area under the curve of 4A for each tested compound, allowing a percentage comparison among them. DESCRIPTION OF THE INVENTION

The present invention in general concerns the pharmaceutical use of mixtures of ingenol and lanosta-8,24-dien-3-ol compounds as concomitant anti-cancer, anti-inflammatory and anti-pain agents.

Without excluding any other, adequate ingenol compounds are one or more of 3-(2,4,6-dodecatrienoyl)-ingenol, 3-(2,4,6,8- tetradecatetranoyl)-ingenol, pharmaceutically acceptable salts thereof, isomers, polymorphs, solvates or hydrates, prodrugs or metabolites thereof.

Ingenol compounds can be obtained for instance from Euphorbiaceae plants, or by chemical synthesis, the path being irrelevant to the invention. Adequate Ingenol compounds may, for instance, be provided as a fraction of a polar solvent extract of an Euphorbiaceae plant latex according to international patent publication WO 2007000618.

Without excluding any other, adequate lanosta-8,24-dien-3- ols are one or more of euphol (RN 514-47-6), tirucallol (RN 514-46-5) and lanosterol (RN 79-63-0), pharmaceutically acceptable salts thereof, isomers, polymorphs, solvates or hydrates, prodrugs or metabolites thereof. Lanosta-8,24-dien-3-ols can be obtained for instance from Euphorbiaceae plants, or by chemical synthesis, the path being irrelevant to the invention.

Metabolites of euphol are one or more of the ones shown in table I.

TABLE I - euphol metabolites

444

Adequate mixtures of ingenols and lanosta-8,24-dien-3-ols range from 1: 100 to 100: 1, particularly 1:50 to 50:1, more particularly 1: 10 to 10:1, and even more particularly 1:4 to 4: 1.

Therefore, in a first aspect, the invention concerns the use of mixtures of ingenol and lanosta-8,24-dien-3-ol compounds for the production of pharmaceutical compositions useful as concomitant anticancer, anti- inflammation and anti-pain agents.

According to the invention, mixtures of ingenol and lanosta-8,24- dien-3-ol compounds of the invention, as well as compositions comprising them can be administered to the subject in need of treatment in any adequate way, enteral or parenteral, including oral, topical, transdermal, subcutaneous, intraperitonial, intravenous, by infiltration, by inhalation, transdermal, transmucosal, intramuscular, intrapulmonary, vaginal, rectal, intraocular, and sublingual. Particularly adequate ways of administration in the present invention are topically and systemically (infiltration, oral, inhalation by spray, transdermal). The ingenol compounds of the invention can be comprised in slow or controlled release compositions. Known adjuvants and excipients can be utilized in ingenol compounds containing compositions - a reference for pharmaceutical dosage forms useful for the compositions related to the invention can be found in the publication Remington's Pharmaceutical Sciences, Mack Publishing, 1965- 1990.

The mixtures and compositions of the invention can be administered to patients as solids, liquids or semi-liquids, tablets, capsules, pills, powder, granules, suspensions, emulsions, dispersions and any other useful known pharmaceutically acceptable form. The compositions might contain further active agents, for instance antibiotics, depending on the desired effect. For oral administration as tablets or capsules (both soft and hard capsules), the ingenol compounds can be combined with pharmaceutically acceptable inert vehicles, such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium phosphate, mannitol, sorbitol, and similar; for oral administration in the liquid form, the ingenol compounds can be combined with ethanol, glycerol, water, and similar. When desired or necessary, agglomerating agents, lubricant agents, disintegrating agents, color and fragrance can be added to the mixture. Common agglomerating agents are glucose, beta-lactose, corn sweeteners, natural or synthetic gums such as gum arabica, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, wax and similar. Lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride. Disintegrants include starch, methyl cellulose, agar, bentonite, xanthan gum, and similar.

The compositions concerned in the invention can also be administrated as liposomes or coupled with soluble polymers as vehicles.

Liquid dosage forms for oral administration may comprise colorants and edulcorants to increase acceptance by patients. Acceptable vehicles for water dosage forms are, water, an appropriate oil, a saline solution, aqueous dextrose, other sugar solutions and glycols as propylene glycol or polyethylene glycols, phosphate buffer.

In another aspect, the invention concerns a method of treating cancer, particularly when inflammation and pain conditions are present, comprising the administration to a patient a pharmacology effective amount of a mixture of ingenol and lanosta-8,24-dien-3-ol compounds in a pharmacologically acceptable carrier. A example of adequate amount of a mixture of the invention comprising one or more ingenols and one or more lanosta-8,24-dien-3-ols, is 0.001 to 2000 mg per kg of patient weight of such mixture, administered to such a patient one or more times a day.

In another aspect, the invention concerns a method of treating cancer, particularly when inflammation and pain conditions are present, comprising the administration to a patient a pharmacology effective amount of a mixture of ingenol and lanosta-8,24-dien-3-ol compounds in a pharmacologically acceptable carrier.

EXAMPLES

Though the following examples are concrete embodiments of the invention, they do not in any way impose limitations to it, other than what is expressed in the claims presented further on.

PREPARATION OF A SAMPLE OF TH E MIXTURE OF TH E I NVENTION

The following is a description of an extract made from the plant Euphorbia tirucalli, utilized in examples below as an example of mixture of the invention: to fresh latex diluted 1: 1 in water, hexane is added, to promote coagulation. The insolubilized material is washed several times with water and filtered. The obtained material, referred to as AM-10, is submitted to a mixture of butanol and hexane, the more polar substances remaining in the butanol. Butanol is then separated from the hexane, filtrated and finally withdrawn by evaporation.

About 70% of the weight of the extract obtained as described above comprises about 70 % euphol, about 10% tirucallol, about 10% of 3-(2,4,6- dodecatrienoyl)-ingenol and about 10% of 3-(2,4,6,8- tetradecatetranoyl)- ingenol.

EFFECT AGAINST CANCER

EXAM PLE 1

I N VITRO MODEL TO EVALUATE TH E ANTI PROLI FERATION ACTIVITY OF H UMAN TUMOR CELL LIN ES BY USI NG TH E SULFORHODAMI NE B ASSAY.

To perform this test, cancer cell lines U251, UACC-62, MCF-7, NCI- ADR, 786-0, NCI-H460, PC-3, OVCAR -03, HT-29, K562 and VERO were selected, cultivated in RPMI with 5% SFB (RPM I refers to RPM I 1640 - Roswell Park Memorial Institute cultivation medium, as per J. Surg. Oncol. 1969; 1 (2); 153-66; SBF stands for inactivated bovine fetal serum); the mentioned cell lines were supplied by the National Cancer Institute NCI, United States of America (Table 1):

Table 1. Cell panel to evaluate antiproliferation activity.

Cell line expressing resistance phenotype to multiple drugs. Cells were kept in 25 cm² flasks with 5 ml of RPMI/SFB at 37 under 5% C0₂ and 100% humidity atmosphere, replicated whenever the formed carpet reached about 80% confluence.

ASSAY TO DETERMINE THE ANTI PROLIFERATION ACTIVITY OF ASSAYED SUBSTANCES.

100 μΙ of cells in RPMI/SFB/gentamicin were inoculated under their corresponding inoculation densities (pre-established through growth curves) in 96-well plates. After 24 hours incubation at 37 °C in 5% C0₂ and 100% humidity atmosphere, the assay substances (0.25 to 250 μg/ml) in 100 μg/ml volume were added. At that moment, a control plate was fixed to determine the absorbency at the moment of addition of the assay substance (value T₀ is represented in the graphs on figs. 1 and 2 by the full line on point zero). After 48 hours incubation, the other plates were fixed to determine the protein content.

SAMPLE DILUTION

To produce stock solutions, samples were diluted in sodium dimethylsulfoxide (DMSO) in concentration of 100 mg/ml. For addition to the experimental plates, those solutions were diluted 400 times in RPMI/SFB/gentamicin.

COLORIMETRIC ASSAY WITH SULFORHODAMINE-B (SRB) This assay was run according to Skehan et al, "New Colorimetric Cytotoxicity Assay for Anticancer-Drug Screening", in J. Natl. Cancer Inst. 82: 1107- 1112 (1990).

After 48 hours of incubation, cells were fixed with 50 μΙ of 50% trichloroacetic acid (TCA) at 4°C. To complete cell fixation, plates were incubated for one hour at 4°C. After being fixed with trichloroacetic acid, plates were submitted to four washes with distilled water to remove TCA residues, cultivation medium, bovine fetal serum and secondary metabolites, and subsequently kept at room temperature until fully dried.

Plates were then colored by adding 50 μΙ of sulforhodamine (SRB) at 0.4% (weight/volume) dissolved in 1% acetic acid and incubated for 30 minutes at 4 °C. They were then washed for four consecutive times with 1% acetic acid. The residue of the washing solution was removed and the plates dried again, at room temperature. The coloring agent linked to cell proteins was solubilized with tris(hydroxymethyl) aminomethane buffer (Trizma base ^®, supplied by Sigma Aldrich Fine Chemicals, U. S. A.), with 10 μΜ concentration and pH 10.5 for five minutes in ultrasound. Spectrophotometric reading of absorbency was achieved with 560 nm in an ELISA reader.

RESULT ANALYSIS The average absorbencies discounted from their respective blanks were calculated and the growth inhibition (Gl) of each assayed samples was determined with the help of the formula below. Results obtained were analyzed, considering that:

- if T > C, cell growth was stimulated;

- if T < T₀ but < C, there was cytostatic activity (growth inhibition) and the used formula is 100 X [(T - T₀)/(C - T₀)];

- if T < T₀, there was cytocidal activity (cell death) and the used formula is 100 X [(T- T₀)/(C - T₀)]; wherein T is the average absorbency of the treated cell, C is the cell control and T₀ is the control of cells on the day of addition.

Finally, it was also possible to subtract the obtained result from 100%, thus obtaining the growth inhibition (Gl) percentage.

The graphs on Figures 1 and 2 respectively present the concentration/activity curves for the assayed substance and for doxorubicin, a chemotherapeutical product used as a positive control for cells in different concentrations (250 to 0.25 μg/ml), relating the percentage of cell growth to the concentration of the utilized product.

Samples are considered as active when they present growth inhibition of more than 50% (represented in the graph by the line on point 50) in a concentration-dependent form and preferably presenting cell selectivity (different activity between the cell lines or specific activity for one of the cell lines).

EFFECT AGAINST INFLAMMATION AND PAIN TESTED ANIMALS

Male 20-30g mice were kept in filtered-air ventilated cages, with controlled temperature (22±2°C) and humidity (50-60%), 12h light/12h dark cycles, with free access to water and food. The animals remained in the lab for an adaptation period of at least 1 hour before the pharmacological tests, held between 8:00 and 17:00 h.

INFLAMMATION AND NOCICEPTION

Tested animals' nociceptive mechanical threshold was evaluated as the response frequency of paw withdrawal after ten applications of a Von Frey filament (VH F, Stoelting, Chicago, USA). The animals were individually placed in 9x7x11 cm transparent acrylic compartments over an elevated wire mesh platform, to allow access to the paw plantar surfaces. The Von Frey filament was applied to the right hind paw, watching the criteria of (1) application perpendicular to the plantar surface with enough pressure to bend the filament, so as to ensure total pressure; (2) the animals were evaluated when all four paws were accommodated on the wire mesh; (3) a paw withdrawal response was considered when the animal removed the paw entirely from the wire mesh; (4) each animal was stimulated 10 consecutive times, with 1 second duration each stimulation; (5) each paw withdrawal was considered as 10% of a response, with 10 withdrawals corresponding to 100% response.

EXAMPLE 2 - PERSISTENT I NFLAM MATORY NOCICEPTION I NDUCED BY CFA (COMPLETE FREUN D'S ADJ UVANT) - emphasis on pain.

The animals were intraplantarly injected lmg CFA, a dose that produces hypernociception and increase in the size of the injected paw (Neuropharmacology, 41:1006-1012, 2001; Anesth Analg., 101: 1763-1769, 2005).

Animals were treated orally with 100 mg/kg AM10 or 70 mg/kg gabapentin (positive control), 1 hour before the CFA injection. The mechanical hypernociception was measured through stimulation with a 0,6 g Von Frey filament in time intervals of 1, 2, 4, 6, 8, 24 and 48 hours after the CFA injection, and up to the re-establishment of the nociceptive response. After that - 3rd day - a chronic treatment was started to evaluate the prolonged treatment with euphol. For that, animals received daily 100 mg/kg AM10, orally, for 5 days, and hypernociception was evaluated once a day, 4 hours after the first administration. Mechanical hypernociception was evaluated until the return of the painful response. After that, the once-a-day treatment started again, for 5 days, to evaluate the development of tolerance for the compound, and hypernociception was evaluated up to the return of the nociceptive response.

The results are shown in figures 3A, 3B and 3C. 3A is a graph of time vs. frequency of response. 3B and 3C are representations of the areas under the curves, allowing a percentage comparison among tested compounds, after acute treatment from 0 to 48h (3B) and prolonged (3rd to 24th day) after injection of CFA.

As can be seen in figures 3A, 3B and 3C, the acute treatment with AM 10 reduced significantly the mechanical hypernociception induced by CFA, close to the effect obtained with gabapentin.

When AM-10 was administered once a day for 5 days, one observes the inhibition of the nociceptive response caused by CFA during the 6 following days of treatment. The mechanical hypernociception was reduced again with the start of the prolonged treatment, as seen with the aid of curves from the 3rd to the 24th day after the injection of CFA. Similar inhibition was observed compared with prolonged treatment with gabapentin.

EXAMPLE 3 - Inflammatory nociception induced by carrageenan - emphasis on inflammation

For the inducement of inflammatory pain, each animals received a 20 μΙ intraplantar carrageenan injection (300 μg/paw) in the right hind paw. Animals treated with a 0.9% (20 μΙ/paw) PBS (phosphates buffered saline) solution were used as control. That carrageenan dosage produces edema, nociception and significant size increase in the injected paw.

The animals were treated orally with AM10 (100 g/l<g) one hour before the carrageenan injection. Animals treated with subcutaneous injections of 0.5 mg/kg dexamethasone 4 hours before the carrageenan injection were used as positive control. Hypernociception was evaluated hourly during 8 hours with a 0.6g Von Frey filament, as well as after 24 and 48 hours after carrageenan injection.

The results are shown in figures 4A and 4B, 4A being a graph of time vs. frequency of response, and 4B the expression of the area under the curve of 4A for each tested compound, allowing a percentage comparison among them.

As can be seen in those figures, the treatment with AM10 significantly reduced the inflammatory mechanical hypernociception induced by carrageenan, particularly during the acute treatment. AM10 lead to a reduction of nociceptive response similar to the control group treated with dexamethasone.

Examples 1, 2 and 3 show that, according to the invention, mixtures of ingenol and lanosta-8,24-dien-3-ol compounds are concomitantly anti-cancer, anti-inflammatory and anti-pain agents.

It is well understood that, with the help of the teachings and examples presented herein, a person skilled in the art is able to reproduce the invention in equivalent ways, using the same functions to obtain similar results, without departing from the scope of the invention defined in the attached claims.

Claims

Pharmaceutical use of mixtures of multicyclic compounds as concomitant anti-cancer, anti-inflammatory and anti-pain agents, wherein such multicyclic compounds are ingenol and lanosta-8,24- dien-3-ol compounds.

Pharmaceutical use according to claim 1 characterized in that said ingenol compounds are one or more of 3-(2,4,6- dodecatrienoyl)-ingenol, 3-(2,4,6,8- tetradecatetranoyl)-ingenol, pharmaceutically acceptable salts thereof, isomers, polymorphs, solvates or hydrates, prodrugs or metabolites thereof.

Pharmaceutical use according to claim 1 characterized in that said lanosta-8,24-dien-3-ols are or more of euphol, tirucallol, lanosterol, pharmaceutically acceptable salts thereof, isomers, polymorphs, solvates or hydrates, prodrugs or metabolites thereof.

Pharmaceutical use according to claim 1 characterized in that said mixtures of one or more ingenols and one or more lanosta- 8,24-dien-3-ols range from 1:100 to 100:1, particularly 1:50 to 50:1, more particularly 1:10 to 10:1, and even more particularly 1:4 to 4:1.

Pharmaceutical use according to claim 1 characterized in that said mixture comprises about 70% euphol, 10% tirucallol, 10% 3- (2,4,6-dodecatrienoyl)-ingenol and 10% 3-(2,4,6,8- tetradecatetranoyl)-ingenol, in weight.

6. USE of mixtures of multicyclic compounds comprising one or more ingenol compounds and one or more lanosta-8,24-dien-3-ol compounds for the production of pharmaceutical compositions useful in the concomitant treatment of cancer, inflammation and pain.

7. USE according to claim 6, wherein said mixtures are described in one of claims 2 to 5.

8. A METHOD OF TREATING CANCER, particularly when inflammation and pain conditions are present, comprising the administration to a patient a pharmacology effective amount of a mixture of ingenol and lanosta-8,24-dien-3-ol compounds in a pharmacologically acceptable carrier.

9. A METHOD according to claim 8, wherein the said mixture is as described in one of claims 2 to 5.

10. A METHOD according to claim 8, wherein the amount of said mixture is 0.001 to 2000 mg per kg of patient weight, administered to such a patient one or more times a day.