GB1561226A - Pharmaceutical compositions containing 4-deacetoxy-vinblastine - Google Patents

Description

(54) PHARMACEUTICAL COMPOSITIONS CONTAINING 4-DEACETOXY-VINBLASTINE (71) We, RICHTER GEDEON VEGYESZETI GYAR RT. a body corporate organised under the laws of Hungary of 21 Gyomroi u., Budapest X., Hungary, do hereby declare the invention for which we pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following statement: - This invention concerns a pharmaceutically active diindole alkaloid and acid addition salts thereof. Furthermore, the invention relates to a process for preparing pharmaceutical compositions containing such compounds as active principles. More specifically, the invention relates to a process for isolating 4-deacetoxy-vinblastine, a minor alkaloid of Vinca rosea L., or its acid addition salts, as well as pharmaceutical compositions thereof.

It is known that the minor alkaloids of Vinca rosea L. with diindole skeleton possess valuable cytostatic effects (U.S. patent specifications nos. 3,097,137 and 3,205,220; Hungarian patent specifications Nos. 153,200, 154,715 and 160,967). Of these minor alkaloids vincristine, the one regarded as the most valuable, makes up some hundredths of one per cent of the total alkaloid content, whereas the less effective vinblastine and leurosine are present in an amount of about 1 to 2 per cent calculated on the total alkaloid content.

In the recent past the demand for the latter two alkaloids has increased considerably, which can be attributed to the recognition that vinblastine can be converted into the more valuable vincristine by a semisynthetic process (Hungarian patent specification No.

165,599), and that the N-formyl derivative of leurosine (Hungarian patent specification No.

165,986 also inhibits the growth of tumors resistant towards the cytostatic agents known before.

The more recent investigations in this field have been directed to the preparation of further effective semisynthetic alkaloid derivatives, such as the carboxamide and deacetyl derivatives, such as the carboxamide and deacetyl derivatives (published German patent applications Nos. 1,795,763 and 2,415,890), as well as to the isolation of further minor alkaloids, such as vincadioline, leurocolombine (U.S. patent specifications Nos. 3,887,565 and 3,890,325) and 4-deacetoxy-vinblastine (N. Neuss, A.J. Barnes and L.L. Huckstep: Experimentia 31/1, 18-19 /1975/).

The isolation of the further minor alkaloids was rendered possible by improvements in chromatographic techniques, more particularly by the use of high-pressure chromatography. Using such equipment even compounds with rather similar physical constants and chemical structures can be separated successfully from each other. Nowadays high-pressure chromatography can be applied successfully for laboratory scale operations, but its utilization is, however, extreinely complicated and expensive in large-scale manufacture.

N. Neuss et al. [Experimentia 31/1, 18-19 (1975)] succeeded in isolating 4-deaxetoxyvinblastine, an accompanying alkaloid of vinblastine not known before, by subjecting vinblastine to two chromatographic separations and two recrystallizations. The U.V. and I.R. spectra of vinblastine and 4-deacetoxy-vinblastine are very closely related, and the difference between their retention factors amounts to only 0.03. In the cited publication the authors described the separation technique of the two alkaloids and the difficulties which had arisen in connection with the structural determination. They did not give, however, any infomation on the pharmacological or other effects of the new alkaloid.

This invention is based on the recognition that 4-deacetoxy-vinblastine has surprising pharmacological advantages over vinblastine and other diindole alkaloids having antitumor activity; thus the invention provides a new pharmaceutical composition useful in the therapy of malignant tumors, which comprises an effective amount of 4-deacetoxyvinblastine as active material or a pharmeutically acceptable salt thereof and a pharmaceutically acceptable carrier. Preferably the composition is substantially free of vinblastine. Owing to the difficulty in separating 4-deacetoxy-vinblastine from related alkaloids, known pharmaceutical compositions of the latter may have contained minor amounts of 4-deacetoxy-vinblastine. It will be appreciated however that such compositions did not contain effective amounts of 4-deacetoxy-vinblastine and are thus clearly distinguished from the compositions of the invention.

The invention provides in another aspect a new process for the isolation of 4-deacetoxy-vinblastine and acid addition salts thereof and formulation thereof into pharmacutical compositions.

In the process of the invention an enriched mixture of the sulfate or other acid addition salts of minor diindole alkaloids can be used as starting substance. This enriched mixture may be isolated from the plant (Vinca rosea L.) according to the method described in Hungarian patent No. 160,967.

According to a newly elaborated and preferred method (defined more generally hereinafter), the enriched alkaloid salt mixture was processed as follows: An alkanolic solution of the alkaloid salt mixture was treated with an organic base to liberate the alkaloid bases. The leurosine base, which separatec from the reaction mixture, was filtered off, and the filtrate was evaporated under reduced pressure. The residue was dissolved in an organic solvent, upon which the salt of the organic base applied to liberate the alkaloid bases separated. The separated by-product was filtered off and the filtrate was extracted with an aqueous phosphate buffer solution (pH = 3.7 to 4.3). The aqueous-acidic extract was acidified to pH 3.7 to 4.1 with phosphoric acid, and then the acidic solution was extracted with a chlorinated hydrocarbon. The major portion of vinblastine was isolated from the chlorinated hydrocarbon phase, and the aqueous-acidic phase was subjected to further treatment.

It has been found that the above treatment causes the alkaloids accompanying vinblastine to be enriched in the aqueous-acidic phase.

Furthermore, it has been found that, besides the already vincristine, N-demethylvinblastine and minor amounts of residual vinblastine, a fourth alkaloid, the aforesaid 4-deacetoxy-vinblastine, can also be detected in the mixture of the accompanying alkaloids.

Upon examining the separation possibilities of the four alkaloids it has been found that when the alkaloid mixture if formylated in order to convert 4-demethyl-vinblastine, a substance difficult to separate from vinblastine, into vincristine, the resulting alkaloid mixture, now containing only three components, can be separated easily into the individual components by subjecting it to simple extraction procedures and chromatography performed on a conventional column chromatograph.

Upon examining the pharmacological properties of 4-deacetoxy-vinblastine sulfate it has been found that the tumor inhibiting effects of this compound are roughly identical to those of vinblastine. The deacetoxy compound is, however, far less toxic than vinblastine.

Neurtoxicity accompanied with paralytic symptoms, a characteristic adverse side-effect of the structurally related compounds (such as vincristine and vinblastine), did not appear at all during the administration of the deacetoxy compound in our tests.

The pharmacological tests were performed on transplantable tumors of rats and mice.

The effects on L 1210/VS (vinca-sensitive) mouse lymphoid leukemia and L 1210/Ref (vinca-refractory) mouse lymphoid leukemia were tested on BDF1 mice, the effects on Ehrlich as cites carcinomatous tumors and S 37 ascites sarcomatous tumors were examined on Swiss mice, whereas the effects on Novikoff hepatoma were examined on Wistar rats.

The 4-deacetoxy-vinblastine sulfate was dissolved in physiological saline and the solution was administered intraperitoneally into the animals to be tested. In the tests the average prolongation of survival period and the tumor-inhibiting effect were examined in relation to the untreated controls. The results are listed in Table 1. In the Table the symbol T/c represents the ratio of treated animals to the controls, and the term "limit-days" means the day ending the period of four times the average survival period observed in the control group. The number of animals surviving and those tumor-free on the limit day was noted.

Table 1 Daily dosage Number of Average survival period On the limit-day of 4-deacet- Frequency of treat- (days) T/c x 100 (day 36 to Tumor oxy-vinblas- tratment ments treated control (day 40) tine sulfate lives tumormg/kg i.p. -free L 1210/VS 0.9 once a day 7 11.5 8.8 128 0/6 0/6 1.8 " 7 11.3 8.8 128 0/6 0/6 2.0 " 16 24.5 10.3 238 0/6 0/6 4.0 " 16 27.7 10.3 269 0/6 0/6 4.0 once in every 11 27.5 10.3 267 0/6 0/6 two days 4.0 " 8 17.7 8.3 213 0/6 0/6 5.0 " 8 20.8 8.3 251 0/6 0/6 6.0 " 8 24.2 8.3 291 0/6 0/6 L 1210/ref 2.0 once a day 7 9.5 8.0 119 0/6 0/6 4.0 " 7 11.5 8.0 144 0/6 0/6 4.0 once in every 7 11.7 8.0 146 0/6 0/6 two days Ehrlich ascites carcinoma 3.0 once a day 9 30 17.4 174 10/10 6/10 S 37 ascites 1.8 once a day 8 68.5 182. 376 6/8 6/8 sarcoma 2.7 " 8 67.9 18.2 373 7/10 7/10 Novikoff 0.5 once a day 8 10.3 8.2 126 0/6 0/6 hepatome 1.0 " 8 27.7 8.2 338 1/6 1/6 1.5 " 8 24.7 7.8 317 2/6 2/6 NK/Ly ascites 2.0 once a day 8 48.1 14.4 334 0/10 0/10 lymphoma 4.0 " 8 55.0 14.4 382 6/10 0/10 6.0 " 8 47.2 14.4 328 0/10 0/10 It appears from the table that L210/Ref leukemia, resistant towards the known vinca alkaloids, is not sensitive towards 4-deacetoxy-vinblastine, either. On the other hand, a significant dosage-dependent inhibiting effect can be observed for all the other transplanted tumors, especially for Novikoff hepatoma. When administering 4-deacetoxy-vinblastine sulfate to rats or mice in daily dosages of 1 to 10 mg/kg, preferably 1 to 6 mg/kg, the survival period increases by about 50 to 250 per cent.

In human therapy the intravenous or infusion methods of administration are recommended. The compound can be administered e.g. in daily dosages of 0.1 to 0.5 mg/kg.

The toxicity data of 4-deacetoxy-vinblastine, compared to those of other known vinca alkaloids, are listed in Table 2.

Table 2 Compound LD50 mg/kg 4-deacetoxy-vinblastine (i.p., mice) > 23 Vinblastine (i .p., mice) 7.5 Vincristine (i.p., mice) 4.2 N-Formyl-leurosine (i.p., mice) 29.0 Thus in broad terms the present invention relates to a process for preparing a pharmaceutical composition which comprises separating 4-deacetoxy-vinblastine from a mixture thereof with N-dimethyl-vinblastine, vincristine and a minor amount of vinblastine by a process wherein said mixture is formylated, the pH of the formylated product, in solution, is adjusted to 7.5 to 10, the solution is extracted with a water-immiscible organic solvent, and the alkaloids in said extract are subjected to chromatography to separate the desired 4-deacetoxy-vinblastine, and admixing said 4-deacetoxy-vinblastine or a pharmaceutically acceptable acid addition salt thereof with a pharmaceutically acceptable carrier. Preferably said mixture of 4-deacetoxy-vinblastine with N-demethyl-vinblastine, vincristine and vinblastine has been prepared by basifying a solution of an enriched mixture of acid addition salts of minor diindole alkaloids, separating the precipitated leurosine from the resulting mixture of alkaloid bases, evaporating the filtrate, taking the residue of said mixture into solution in an organic solvent, extracting said solution with an aqueous buffer at pH 3.7 to 4.3 to obtain an aqueous extract, extracting the major part of the vinblastine from said aqueous extract with an organic solvent, adjusting the pH of the residual aqueous extract to 7.5 to 10, and extracting said residual aqueous extract with an organic solvent to obtain a solution of said mixture of 4-deacetoxy-vinblastine with N-demethyl-vinblastine vincristine and vinblastine.

Preferred ways of performing the various process steps described above will now be discussed. In the first step of the process the enriched mixture of the acid addition salts of minor diindole alkaloids, preferably the mixture of the respective sulfates, is dissolved in an organic solvent. As solvent preferably acetone or a C1 aliphatic alcohol, particularly methanol can be applied. The dissolution is performed at a temperature of 0 C to 50"C, preferably at room temperature.

The solution is treated with a base, such as monoethylamine, diethylamine or pyridine, to liberate the minor diindole alkaloid bases. The leurosine base separates from the solution at 0 to 25"C. The solution can be cooled to 0 C in order to achieve a complete separation. The separated leurosine is filtered off, washed with the same solvent used in the dissolution step, and dried. The resulting crude leurosine can be purified, if desired, by known measures, such as by recrystallization. If desired, the leurosine base can be converted into its acid addition salts, preferably into the sulfate.

The mother liquor obtained when separating the leurosine base from the mixture is evaporated under reduced pressure. The residue is dissolved in an organic solvent, preferably in a mononuclear aromatic hydrocarbon solvent, such as benzene, toluene or xylene. Benzene proved to be a particularly preferred solvent. Upon this step the salt (e.g. the sulfate) of the organic base (e.g. monomethylamine, diethylamine or pyridine), used to liberate the diindole alkaloid bases, is left as a residue and can be removed. After the removal of the solids the solution in the organic solvent is extracted with an aqueous phosphate or other buffer solution of pH 3.7 to 4.3, preferably of pH 4 + 0.1. The aqueous-acidic extracts are combined, and the pH of the solution is adjusted with an acid, preferably with phosphoric acid, to 3.5 to 4.1, preferably to 4.0 + 0.1. The resulting aqueous-acidic solution is extracted with a chlorinated hydrocarbon, preferably with methylene chloride, and the phases are separated. The methylene chloride extracts are combined, dried over sodium sulfate, filtered, and the filtrate is evaporated to dryness under reduced pressure. This operation yields vinblastine as a residue, which can be converted optionally into its acid addition salts, preferably into the sulfate.

Thereafter the pH of the aqueous-acidic phase is adjusted to 7.5 to 10, preferably to 8.5 to 9, by adding a base, preferably aqueous ammonia, to the mixture.

The alkaline solution is extracted with a water-immiscible organic solvent, preferably with a chlorinated hydrocarbon, such as methylene chloride or chloroform. The extracts are combined, evaporated, and the obtained residue which contains vincristine, N-demethylvinblastine, a minor amount of vinblastine and 4-deacetoxy-vinblastine is formylated. As formylating agent preferably a mixture of formic acid and acetic anhydride is used. Upon this reaction N-demethyl-vinblastine converts quantitatively into vincristine. When the reaction is over the mixture is diluted with a 5 to 15 fold, preferably 10 fold amount of water, and the pH of the diluted solution is adjusted to 7.5 to 10, preferably to 8.5 to 9.

The alkaline solution is extracted with a water-immiscible organic solvent, preferably with a mononuclear aromatic hydrocarbon or a chlorinated hydrocarbon, such as benzene, toluene, xylene, methylene chloride or chloroform. Benzene proved to be a particularly preferred extracting agent. The extracts are combined and evaporated under reduced pressure. The residue which contains vincristine, vinblastine and 4-deacetoxy-vinblastine is dissolved in an organic solvent or solvent mixture, preferably in a mixture of a mononuclear aromatic hydrocarbon and a chlorinated hydrocarbon, particularly in a mixture of benzene and chloroform, and the solution is subjected to column chromatography. As adsorbent alumina, particularly partially deactivated alumina, may be used. The elution is performed preferably with an organic solvent mixture, particularly with mixtures of benzene and chloroform containing the two components in varying amounts. The chromatographic fractions are examined preferably by thin layer chromatography, and the fractions containing the same alkaloid components are combined. The fractions containing vincristine and vinblastine, respectively, are processed according to known techniques, whereas the fractions containing 4-deacetoxy-vinblastine are processed by evaporating to dryness the combined solutions. If desired, the resulting crude 4-deacetoxy-vinblastine can be purified by recrystallization or can be converted into its acid addition salts.

4-Deacetoxy-vinblastine and/or its acid addition salts can be administered orally and/or parenterally either in single daily dosage units or for taking several times a day in subdivided dosage units. The parenteral route, primarily the intravenous route proved to be a particularly preferred manner of administration.

In order to prepare a parenterally administerable composition, which should of course be sterile, the active agent can be dissolved in distilled water or physiological saline and/or optionally in a organic solvent, such as in a lower aliphatic alcohol or glycol ether (e.g. ethyleneglycol monoethyl ether). The injectable solutions may contain optionally dissolution aids, such as polyoxyethylene sorbitance derivatives (e.g. Tween 20, Tween 60, Tween 80, Tween is a registered Trade Mark) and other auxiliary agents, such as preservatives (e.g. benzyl alcohol or p-oxy-benzoic acid esters, antioxidants (e.g. ascorbic acid, sodium pyrosulfite or tocopherol), complexing agents (e.g. ethylenediamine tetraacetates) and buffers. The pharmaceutical compositions may also contain local anaesthetic agents, such as lidocaine. The solid compositions may contain various carriers, e.g. lactose.

The process of the invention is illustrated in detail by the aid of the following non-limiting Examples.

The physical constants of 4-deacetoxy-vinblastine have been determined as follows: The optical rotation has. been measured on an Opton (registered Trade Mark) polarimeter; whereas the PMR spectra were recorded on a Varian (registered Trade Mark) EM 360 spectrophotometer using deuterochloroform as solvent and tetramethylsilane as internal standard.

Example 1 Crude 4-deacetoxy-vinblastine 50 g. of a mixture of crude diindole alkaloid sulfates are dissolved in 300 ml. of methanol at room temperature, and 13 ml. of diethylamine are added to the solution with stirring.

The solution is maintained at 0 C for one hour, whereupon the crude leurosine base separates in crystalline form. The crystals are filtered off, washed with a small amount of methanol, and dried in vacuo.

Yield: 6.4 g. of crude leurosine base, which contains 90 to 95% of the leurosine present originally in the starting diindole alkaloid sulfate mixture.

The crude leurosine base is purified by recrystallization. If desired, this compound is converted into its sulfate.

The filtrate obtained when separating the crude leurosine base is evaporated to dryness under reduced pressure, and the residue is dissolved in 2500 ml. of benzene. During this operation diethylamine sulfate separates in crystalline form. The crystals are filtered off, washed with a small amount of benzene, and stored. The diethylamine base can be removed from this salt.

The benzene filtrate is extracted with 3 x 2500 ml. of an aqueous phosphate buffer solution (pH = 4.0 + 0.1). The phosphate buffer solution is prepared by dissolving 10% by weight of dry sodium dihydrophosphate in water, and adjusting the pH of the solution with 0.1 N-phosphoric acid to the above value. The phases formed in the extraction step are allowed to separate, and the aqueous solutions are combined. The pH of the aqueous solution is adjusted to 4.0 + 0.1 by adding a further amount of 0.1 N phosphoric acid, and then the solution is extracted with 4 x 2500 ml. of methylene chloride. The methylene chloride phases are combined, dried over sodium sulfate, and evaporated to dryness under reduced pressure.

Yield: 25 g.of vinblastine, which can be used directly, i.e. without any further purification, in the semisynthetic prepartion of vincristine.

The obtained 25 g. of vinblastine are dissolved in 80 ml. of methanol, and the solution is acidified to pH = 5 with 0. 3% by volume ethanolic sulfuric acid. Upon standing, vinblastine sulfate separates in crystalline form.

The pH of the aqueous-acidic phase is adjusted to 8.5 to 9.0 with concentrated aqueous ammonia, and the alkaline solution is extracted with 4 x 800 ml. of chloroform. The chloroform extracts are combined, dried over sodium sulfate, and filtered. The filtrate is evaporated under reduced pressure, and the residue (8 g. of an amorphous alkaloid mixture) is dissolved in a mixture of 45 ml. of formic acid and 11 ml. of acetic anhydride.

The solution is allowed to stand at room temperature for 5 to 15 minutes, and then it is diluted with a tenfold amount of distilled water cooled previously to 50C. The pH of the diluted solution is adjusted to 8.5 to 9.0 with concentrated aqueous ammonia, and the alkaline solution is extracted with 4 x 800 ml. of benzene. The benzene extracts are combined, evaporated under reduced pressure, and the residue (6.9 g. of an amorphous alkaloid mixture) is dissolved in 42 ml. of 1:1 mixture of benzene and chloroform. The alkaloids are separated from each other by column chromatography. A suspension of 345 g. of alumina (activity grade: IV-V) in a 1:1 mixture of benzene and chloroform is filled into a column with a diameter of 4 cm., and the solution to be treated is poured onto the column.

The alkaloids adsorbed on the column are eluted as follows: elution is started with a 9:1 mixture of benzene and chloroform, and the effluent is collected into fractions of 100 ml. each. The fractions are subjected to thin layer chromatography [adsorbent: alumina on aluminum foil (Merck; registered Trade Mark) developing agent: a mixture of 100 ml; of benzene, 50 ml. of chloroform and 7.5 ml. of diethylamine]. Fractions Nos. 3 to 10, which contain 4-deacetoxy-vinblastine, are combined and evaporated under reduced pressure. 1.7 g. of crude 4-deacetoxy-vinblastine are obtained.

Fractions Nos. 10 to 36 are eluted with a 4:1 mixture of benzene and chloroform, and the individual fractions are examined by thin layer chromatography as described above.

Fractions Nos. 11 to 19 are combined and evaporated under reduced pressure. 0.6 g. of' an alkaloid mixture are obtained, which contains vinblastine and 4-deacetoxy-vinblastine in a ratio of 1:1. This mixture is subjected to a second chromatographic treatment. The residue is dissolved in a 1:1 mixture of benzene and chloroform, this solution is applied onto a column filled with alumina (activity grade: IV-V), and then the elution is performed as described above. Yield: 0.18 g. of vinblastine and 0.20 g. of 4-deacetoxy-vinblastine.

If desired, this latter chromatographic step can be performed on the combined residues obtained from 10 parallel runs.

Fractions Nos. 20 to 24 are combined and evaporated under reduced pressure. 0.9 g. of vinblastine are obtained.

Fractions Nos. 25 to 36 contain 0.3 g. of deacetoxy-vinblastine.

Fractions Nos. 36 to 45 are eluted with a mixture of benzene and chloroform containing the two components in equal amounts. The effluents are combined and evaporated under reduced pressure. 1.7 g. of vincristine are obtained.

Example 2 Purification of 4-deacetoxy-vinblastine 1.7 g. of crude 4-deacetoxy-vinblastine, obtained as described in Example 1, are dissolved in 25 ml. of a 3:2 mixture of methanol and water. The solution is cooled to 50C and allowed to stand for 16 hours. The separated crystals are filtered off, washed with 3 to 4 ml. of a 3:2 mixture of methanol and water cooled to 5"C, and dried. 1.3 g. of 4-deacetoxy-vinblastine are obtained; m.p.: 190-193"C, [a] 20 = + 107 (c = 1%, in chloroform).

Analysis: calculated for C44H56N407: C: 70.19%, H: 7.50%, N: 7.44%, 0: 14.87%, Found: C: 69.85%, H: 7.52%, N: 7.23%, 0: 14.97%, Mass spectrum The mass spectrum of 4-deacetoxy-vinblastine was examined in comparison with that of vinblastine. For the deacetoxy compound the molecular ion peak appears at m/e = 752, whereas for vinblastine it appears at m/e = 810. The difference amount to 58 mass units is equal to the mass of the acetoxy group.

Both diindole alkaloids consist of a velbamine and a vindoline moieties. Since the mass numbers characteristic of the velbamine fragment and the base peak of the velbamine moiety (m/e = 355 and 154) also apear in the mass spectrum of 4-deacetoxy-vinblastine, it follows that the acetoxy group in equestion is missing from the vindoline fragment. Based on the splitting paths (m/e = 122, 135, 224) it is probable that the deacetoxy compound lacks the acetoxy group coupled to position 4 of vinblastine.

PMR-spectrum When comparing the PMR spectrum of 4-deacetoxy-vinblastine with that of vinblastine it appears that the peak observable in the spectrum of vinblastine at = 2.09 ppm, which is characteristic of the 4-acetoxy group, cannot be observed in the spectrum of the deacetoxy compound.

The characteristic peaks of the PMR spectrum of 4-deacetoxy-vinblastine are as follows: = 2.73 ppm (N-methyl protons) a = 3.60 ppm (carbomethoxy protons) = 3.81 ppm (aromatic -OCH3 protons) = 3.85 ppm (carbomethoxy protons) Example 3 4-Deacetoxy-vinblastine sulfate 1.25% ethanolic sulfuric acid is added to 1.1 g. of crude 4-deacetoxy-vinblastine (prepared as described in Example 1) until the pH of the mixture reaches the value of 4.5.

The mixture is allowed to stand at 50C for 2 hours. The separated crystals are filtered off and washed with 2 to 3 ml. of 5"C ethanol. 0.95 g. of crude 4-deacetoxy-vinblastine sulfate are obtained. If desired, this compound can be recrystallized as follows: 0.95 g. of crude 4-deacetoxy-vinblastine sulfate are dissolved in 25 ml. of methanol, and 125 ml. of acetone are added to the solution. The solution is allowed to stand at 5"C for 2 hours, and the separated crystals are filtered off. The crystals are washed with 2 to 3 ml. of cold acetone, and dried. 0.88 g. of purified 4-deacetoxy-vinblastine sulfate are obtained; [a] 20 = + 29.00 (c = 1%, in methanol).

Example 4 Pharmaceutical composition containing 4-deacetoxy-vinblastine sulfate An injectable solution containing 5 ml. per ampoule of physiological saline, 50 mg. per ampoule of benzyl alcohol, 50 mg. per ampoule of lastose and 1, 5 or 7 mg. per ampoule of freeze-dried 4-deacetoxy-vinblastine sulfate is prepared. The solvent is sterilized by filtration prior to filling it into the ampoules.

Claims (33)

WHAT WE CLAIM IS:-

1. A process for preparing a pharmaceutical composition which comprises separating 4-deacetoxy-vinblastine from a mixture thereof with N-demethyl-vinblastine, vincristine and a minor amount of vinblastine by a process wherein said mixture is formylated, the pH of the formylated product, in solution, is adjusted to 7.5 to 10, the solution is extracted with a water-immiscible organic solvent, and the alkaloids in said extract are subjected to chromatography to separate the desired 4-deacetoxy-vinblastine, and admixing said 4-deacetoxy-vinblastine or a pharmaceutically acceptable acid addition salt thereof with a pharmaceutically acceptable carrier.

2. A process according to claim 1 wherein said formylation is performed with a mixture of formic acid and acetic anhydride whereafter the reaction mixture is diluted with water.

3. A process according to claim 1 or 2 wherein the pH of said formylated product, in solution, is adjusted to 8.5 to 9.

4. A process according to any of the preceding claims wherein said water-immiscible organic solvent is a mononuclear aromatic hydrocarbon or a chlorinated hydrocarbon.

5. A process according to claim 4 wherein said organic solvent comprises toluene, xylene, methylene chloride or chloroform.

6. A process according to claim 4 wherein said organic solvent is benzene.

7. A process according to any of the preceding claims wherein said chromatography is performed on alumina.

8. A process according to claim 7 wherein partially deactivated alumina is employed.

9. A process according to claim 7 or 8 wherein the desired 4-deacetoxy-vinblastine is eluted with a mixture of benzene and chloroform.

10. A process according to any of the preceding claims wherein said mixture of 4-deacetoxy-vinblastine with N-demethyl-vinblastine, vincristine and vinblastine has been prepared by basifying a solution of enriched mixture of acid addition salts of minor diindole alkaloids, separating the precipitated urosine from the resulting mixture of alkaloid bases, evaporating the filtrate, taking the residue of said mixture into solution in an organic solvent, extracting said solution with an aqueous buffer at pH 3.7 to 4.3 to obtain an aqueous extract, extracting the major part of the vinblastine from said aqueous extract with an organic solvent, adjusting the pH of the residual aqueous extract to 7.5 to 10, and extracting said residual aqueous extract with an organic solvent to obtain a solution of said mixture of 4-deacetoxy-vinblastine with N-demethyl-vinblastine, vincristine and vinblastine.

11. A process according to claim 10 wherein sulfate salts of said minor diindole alkaloids are employed.

12. A process according to claim 10 or 11 wherein said enriched mixture of acid addition salts is in solution in acetone or a C1--5 alcohol.

13. A process according to claim 12 wherein said C1--5 alcohol is methanol and wherein the leurosine is allowed to separate at 0 to 250C.

14. A process according to any of claims 10- 13 wherein the residue of the said mixture, after evaporation of solvent, is taken into solution in a mononuclear aromatic hydrocarbon as organic solvent.

15. A process according to claim 14 wherein said aromatic hydrocarbon is benzene.

16. A process according to any of claims 10 - 15 wherein said aqueous buffer at pH 3.7 to 4.3 is a phosphate buffer.

17. A process according to claim 16 wherein the pH of the aqueous extract in said phosphate buffer is adjusted to 4 + 0.1 before extracting said vinblastine therefrom.

18. A process according to any of claims 10 - 17 wherein said major part of the vinblastine is extracted with a chlorinated hydrocarbon.

19. A process according to claim 18 wherein said chlorinated hydrocarbon is methylene chloride.

20. A process according to any of claims 10- 19 wherein the pH of said residual aqueous extract is adjusted to 8.5 to 9.

21. A process according to any of claims 10- 20 wherein the pH of said residual aqueous extract is adjusted with aqueous ammonia.

22. A process according to any of claims 10- 21 wherein said residual aqueous extract is extracted with a chlorinated hydrocarbon.

23. A process according to claim 22 wherein said chlorinated hydrocarbon is methylene chloride or chloroform.

24. A process for preparing a pharmaceutical composition comprising 4-deacetoxyvinblastine or acid addition salts thereof, wheren an enriched mixture of acid addition salts of minor diindole alkaloids is treated with a base to liberate the minor diindole alkaloid bases, the precipitated leurosine is separated from the mixture of alkaloid bases, the resulting mother liquor is evaporated, the residue is dissolved in an organic solvent, the resulting solution is extracted with an aqueous phosphate buffer solution of pH 3.7 to 4.3, the separated aqueous-acidic extract is extracted with a chlorinated hydrocarbon, and the chlorinated hydrocarbon phase, which contains the major amount of vinblastine, is separated from the aqueous-acidic phase, which contains the other minor diindole alkaloid, the pH of the aqueous-acidic phase obtained in the above step is adjusted with a base to 7.5 to 10, the aqueous-alkaline solution is extracted with a chlorinated hydrocarbon, the chlorinated hydrocarbon phase, which contains N-demethyl-vinblastine, vincristine, minor amounts of vinblastine and 4-deacetoxy-vinblastine, is evaporated, the residue is formylated, the pH of the mixture obtained after formylation is adjusted with a base to 7.5 to 10, the alkaline solution is extracted with mononuclear aromatic hydrocarbon or with a chlorinated hydrocarbon, the extract is evaporated, the residue, which contains 4deacetoxy-vinblastine, vinblastine and vincristine, is subjected to chromatography on partially deactivated alumina, the separated 4-deacetoxy-vinblastine is optionally converted into an acid addition salt thereof, and said 4-deacetoxy-vinblastine or salt thereof is admixed with a pharmaceutically acceptable carrier.

25. A process according to claim 1 or 10, wherein said 4-deacetoxy-vinblastine is separated and said 4-deacetoxyl-vinblastine or salt thereof is admixed with a pharmaceutically acceptable carrier, substantially as hereinbefore described with reference to Example 1.

26. A process according to claim 1 or 10, wherein 4-deacetoxy-vinblastine is separated substantially as hereinbefore described with reference to Examples 1 and 2.

27. Compositions of 4-deacetoxy-vinblastine when obtained by the process of any of the preceding claims.

28. A pharmaceutical composition comprising an effective amount of 4-deacetoxy vinblastine or a pharmaceutically acceptable acid addition salt thereof and a pharmaceuticall acceptable carrier.

29. A pharmaceutical composition comprising an effective amount of 4-deacetoxyvinblastine or a pharmaceutically acceptable acid addition salt thereof and a pharmaceutically acceptable carrier, said composition being substantially free of vinblastine.

30. A composition according to claim 28 or 29 in unit dosage form.

31. A composition according to claim 28, 29 or 30 in a sterile form intended for parenteral administration by injection or by infusion.

32. A composition according to claims 30 and 31 wherein each unit does contains 1 to 7 mg of 4-deacetoxy-vinblastine or a pharmaceutically acceptable acid addition salt thereof.

33. A composition according to claim 28 or 29, substantially as described herein.