MXPA99008108A - Anhydrovinblastine for the treatment of cervical and lung cancer - Google Patents

Abstract

The present invention is particularly directed to the use of a derivative of vinblastine, 3',4'-anhydrovinblastine (AHVB), which differs from vinblastine in that it possesses a double bond at the 3',4'position of the caranthine nucleus rather than the hydroxyl group that is present in the parent structure, as an anti-neoplastic agent in the therapeutic treatment of cancer.

Description

ANTHDROVINBI.ASTINA FOR THE TREATMENT OF CERVICAL AND LUNG CANCER TECHNICAL FIELD The present invention generally refers to alkaloids an t i neop 1 per cent of vincapervinca as an an t i t umo a 1 is agents. More particularly, the present invention relates to providing the use of a vinblastine derivative, anhydr ov inb 1 asyna (hereinafter AHVB), as an antineoplastic agent with improved therapeutic properties, which demonstrates a maximum dose tolerated much higher and less toxicity than its compounds of origin or with which it is related.

BACKGROUND OF THE INVENTION Due to a higher degree of difficulty in predicting, the classical techniques of drug development are inventive.

Mainly through a process of elimination, in search of the desired effects, a large number of natural products and synthetic chemical compounds are examined, using a series of increasingly complex systems, which begin with simple in vi tro level tests. cellular, 5 which are taken to animals and finally to clinical trials in humans. However, due to essential characteristics such as adsorption, distribution and metabolism, the initial in vi tro tests that can take into account these aspects can eliminate a powerful drug that does not perform well in such systems. The drug can be metabolized into different compounds in animal models and in humans, -15 which can also demonstrate different patterns of adsorption or distribution, or finally, the compounds can be very expensive at all times until they reach clinical judgments, but then they can demonstrate unpleasant side effects or a high degree of tolerance when used on a large scale in the human population. It is never obvious which compound will continue to be promising as each stage of tests and development. For more than 20 years, it has been possible to control tumor growth to a certain degree by using vinca vinca oncolytic alkaloids alone or in it as a agent in combination with other drugs. "t 5 an t i neop 1 ás t i eos Approximately 30 alkaloids have been extracted with a wide range of pharmacological activities from Vincapervinca rosea (Ca t t thus roseus), commonly known as maiden grass. these, only vinleurosine, vinrosidine, vinblastine and vincristine have an important antitumor activity. In particular, vinblastine and vincristine have been widely used as simple agents or in combination with other non-specific drugs in cancer chemotherapy. In addition to the naturally occurring alkaloids, some vincapervi alkaloid analogs have been synthesized by functional transformation or by means of semi-synthetic processes (RJ Cersosimo, and 3- others, Pharmaco t herapy 3: 359-274, 1983; P. Mangency, et al., Org. Chem. 44: 3765-3768 , 1979: R. Maral, et al., Cancer Lett.22: 49- -t 25 54, 1984). % j Chemically, these vincapervinca alkaloids have an asymmetric dimorphic structure composed of 2 nuclei joined by means of a carbon-carbon bond; a dihydroindole nucleus (vindoline); which is the main alkaloid contained in the herb 5 maiden, and the indole nucleus of catarantin (Figure 1). The structural difference between vincristine and vinblastine exists in the Rl position while vinblastine and vindesine differ with respect to the substituents R2 and R3. The mode of action of the alkaloids an t i neop 1 to t i eos of vinca pervinca still needs to be fully understood. However, it has been established that its activity antitumor is directly related to the higher binding affinity of these compounds with tubulin, the basic subunit *. Microtubule protein (R.A. Bender and B. Chabnerm In: Chabner (de) Pharmacol. • 4 20 of Cancer Treat. , Saunders, Phil, PA, p. 256, 1982; W.A. Creasy, In: Hahn (de) Antibiotica, Vol. 2, Springer, Berlin, p. 414, 1979). The consensus is that these agents stop cell mitosis in metaphase avoiding the polymerization of tubulin to form microtubules T inducing depolymerization (R.J. Owellen and C.A.

- JL Hartke, Cancer Res., 36: 1499-1504, 1976; RH. Himes and R N. Kersey, Cancer Res,, 36.37 8.3806, 197; R.S. Camplejohn, Cell Tissue Kmet, 13: 327-332, 1980 =. Thus, the vincapervinc alkaloids are specific cell cycle agents, or spindle poisons. The binding affinity of the vincapervinca alkaloids with tubulin correlates sparingly with the relative capacity of vincpstine, vinblastine and vindesine to inhibit cell growth (R.S. Camplejohn, supra; P.J. Ferguson and C.E. Cass, Cancer Res., 45: 5480-5488, 1985). Therefore, the The main difference in antitumor activity between these drugs seems to be related to their retention in the tumor tissue (P. Ferguson, supra; J. Horton et al., Biochem. Pharmacol. 37: 3995-4000, 1988), In a similar vein, the different toxicity profiles of periwinkle alkaloids appear to be related to the JT tissue absorption and retention properties rather than binding affinity inherent in tubulin. For example, studies have shown that vincristine is * more powerful than vinblastine or vindesine - $ -, in the rapid blockade of axoplasmic transport in nerve cells (S. Ochs and R. Worth, Proc.A. Assoc.Cancer Res., 16:70, 1975; SY, Chan et al., J. Neurobiol. 5 11: 251-264, 1980). In addition, the nerves are taken 4 times faster than the other drugs (Z. Iqbal and S. Ochs, J. Neurobiol., 11: 251-264: 1980) and show an extended phase of terminal elimination of plasma suppression, suggesting a longer exposure to vincristine than to the other periwinkle alkaloids (RL Nelson et al., Cancer Trear, Res., 7: 17-24, 1980), 15 The differences in vi In vi tro observed among the vincapervinca alkaloids surprisingly give the subtle chemical alterations exhibited by the various agents relating to their large and complex molecular structure. For example, vincristine is very effective in the treatment of human combats transplanted to nude mice, whereas vinblastine is not active in this system (N. Bruchovsky et al., Cancer Res. • 1 * 25: 1232-123 1965) This difference is obtained simply as a result of the substitution of an aldehyde group with a methyl group in the Rl position. Furthermore, this chemical substitution leads to a change in the toxicological profile so that Peripheral neuropathy (in the absence of acute toxicity) limits the dose of vincristine in humans while anemia and leukopenia usually limit the dose of vinblastine (WP Brads, Proc. Int. Vin caalkal oid Symposium , 95-123, 1980; SS Legha, Med. Toxicol., 1: 421-427, 1986). A therapeutic profile of particular interest has been observed for a new semi-synthetic alkaloid of periwinkle named navelbine (vinorelbine, 5'-noranhydroblastine). This compound is less potent than vinblastine and vincristine against murine P388 and L1210 leukemia but is active against cells derived from human lung cancer while the other vincapervinca alkaloids are inactive (S, Cros, and others, Seminars in Oncology, 16: 15-20, 1989). While the clinical judgments on navelbine base their utility on the treatment of non-small cell lung cancer (A. Depierre et al., Am. J. Clin. Oncol., 14: 155-119, 1991; A. Yokoyama et al. , Am.

Soc. Clin. Oncol. , 11: 957, 1992). The toxicity profile of this agent seems similar to that of vinblastine, when the dose does not limit the toxicity of the blood and the side effects and not the side effects. Vincristine has proven to be particularly useful as an oncolytic agent administered intravenously in combination with other oncolytic agents for the treatment of several cancers including leukemines of the central nervous system., Hodgkin's disease, 1 i n f aar coma, reticular cell sarcoma, rabdomi dare coma, neurob l as t orna and Wilma tumor. It is exclusively for intravenous use and intrathecal administration is uniformly fatal. Following simple weekly doses, the most common adverse reaction is hair loss; and the most problematic are those of neuromuscular origin. When simple weekly doses are used, adverse reactions of leukopenia, neuritic pain, constipation and difficulty walking may occur. Other adverse reactions that have been reported are abdominal cramps, ataxia, gout, weight loss, optic atrophy with blindness, transient cortical blindness, fever. manifestations of the cranial nerve, parks and digital numbness, polyuria, dysuria, oral ulceration, headache, vomiting, diarrhea and necrosis and / or intestinal perforation. Navelbine (vinorelbine tartrate) is an ervinca vinca alkaloid in which the catherantin unit is the site of structural modification. It is also thought that its antitumor activity is mainly due to its ability to infer with microtubule activity, thus inhibiting mitosis in the metaphase through its interaction with tubulin. It is indicated in the treatment of advanced non-small cell lung cancer as a single agent or in combination, only administered intravenously. Its side effects include felbitis or extravasation injury as a vasicante. Studies on adverse reactions based on the use of Navelbine as a single agent indicate that it was the main dose-limiting toxicity, although it was generally reversible and did not accumulate over time. . The neurological toxicities reported most frequently are mild to moderate neuropathy manifested by pareatasia and hypesthesia, which occur in 10% of patients. The mild to moderate nausea occurs scarcely in a third of patients treated with Navelbine with a slightly smaller fraction is experienced constipation, vomiting, diarrhea, anorexia and stomatitis. Lack of achieving compounds that show decreased toxic effects with equal or greater chemotherapeutic activity. In this way, the need to provide improved, anti-tumor efficacy for the treatment of cancer continues. Therefore, an object of the present invention is to provide a cancer treatment method comprising administration to human patients suffering from cancer and in need of treatment, an amount of AHVB, effective in stopping or significantly diminishing "the evolution Another aspect of the present invention is to provide a method for using AHVB as an antitumor agent, comprising the therapeutic amount of the chemical substance of the present invention to arrest tumor growth. Advantages of the present invention are achieved by the administration of a vinblastine derivative, AHVB Other objects and advantages will be apparent from the detailed description of the present invention.

BRIEF DESCRIPTION OF THE INVENTION The present invention is directed in particular to the use of a vinblastine derivative, 3 ', 4'-anhydrovinblastine (AHVB), which differs from vinblastine because it possesses a double bond at the 3', 4 'position of the carantine nucleus instead of the hydroxyl group that is present in the original structure, as an agent more likely to be used in the therapeutic treatment of cancer. One embodiment of the present invention involves the use of 3 ', 4' -anh i drovinb la t i na, or variants thereof, as an inelastic agent in the treatment of cancer. Another embodiment of the present invention involves the 3 ', 4' -anh i dr ovi nb 1 as a na agent as an agent for the treatment of cancer, when the concentration of 3 ', 4' -anh i dr or vi nb 1 as ti is found in a maximum concentration greater than the age-speci fi cally acceptable concentrations of vinblastine or navelbine for use in the treatment of cancer. Still another embodiment of the present invention involves the use of 3 ', 4' -a nh i r or v i n b 1 as t a na agent as an an agent in the treatment of lung cancer.

PICTURES AND FIGURES Table 1 shows the relative toxicity of vincristine, AHVB and navelbine in tumor cell lines. Table 2 shows calculations of sub-acute toxic dosages of vincristine sulfate, navelbine and AHVB when administered to healthy male Nb rats as a simple aperitoneal int injection. Table 3 represents the delay of the solid tumor C - 4, in information on growth.

Figure 1 depicts the chemical structure of some periwinkle alkaloids. Figure 2 compares the effects of administering a simple intraperitoneal injection in a subacute toxic dose of vincristm, navelbine and AHVB to Nb rats that have subcutaneous tumor transplants. Well-developed simple Nb2-U17 with the average tumor weight and average rat weight as a function of time. Figure 3 shows a comparison of the effects of administering a simple intraperitoneal injection, in a highly toxic dose of vincristine, navelbine and AHVB to Nb rats that have well developed subcutaneous simple subcutaneous tumor transplants Nb2- U17 with the average tumor weight and average weight of the rat as a function of time. Figure 4 shows the changes in the average animal weight of BDF1 mice having P388 tumors i n t r ap er i t o ne 1 is that they have i.v. of saline solution, vincristine, navelbine and AHVB.

Figure 5 represents an example of a curve of c 11 or t oxiity used to calculate IC50 of several vinca vinca alkaloids. Figure 6 shows the P388 antitumor activity of selected formulations of vinca vinca alkaloids. Figure 7 depicts a dose response curve obtained with AHVB when used to treat BDF1 mice having P388 tumors. Figure 8 represents the cytotoxicity curves used to calculate the IC50 of AHVB in SKOV3 and C-4 cell lines. Figure 9 shows the average weight of a tumor in grams over time (period of 30 days) following administration on days 1, 5 and 9 of navelbine, AHVB bisulfate, AHVB ditartrate and control. Figure 10 represents the average weight of a tumor in grams over time (period of 69 days) followed by administration on days 1, 5 and 9 of navelbine, AHVB bisulfate, AHVB ditartrate and control.

DETAILED DESCRIPTION OF THE INVENTION There are many possible derivatives or possible variations of vinblastine. However, there is no certainty, even for experts in the area of drug development, that any one of these derivatives is as effective or even more effective than the original compound. Experiments The term I variants! for the purposes of 3 ', 4' -anh i drovinb 1 as ti na means any chemical structure that is a derivative of 3 ', 4' -anh i drov i nb 1 as ti na achieved through of substitution of collateral groups, which still shows the same properties as anneopic 1 or more similar than 3 ', 4'-anhydrovinblastine.

CHARACTERIZATION OF ANTITUMORAL ACTIVITY OF AHVB GN VI TE O The cytotoxicity experiments in AHVB were carried out as direct comparisons with vincristine and navelbine in order to achieve their inherently anti-tumor profile against a variety of tumor cell types in relation to the other relevant vincapermine alkaloids. . The toxicity of AHVB was investigated in vi tro against a panel of antigen-like cell lines of varied lineage in order to determine the specificity of its antitumor activity with respect to type d? cell. The studied tumor lines were lymphocytic leukemia P388 (a murine lymphocytic leukemia), noble rat lymphoma (Nb) U17, human carcinoma of breast MCF7, human carcinoma of non-small cell lung H460, er itr oqueu cen ia humana K562 and colon human carcinoma LS180 based on established protocols in vi tro examination of cytotoxicity of new anticancer drug. Acitot oxy-dose response assays are used regularly (R. Mosmass, J. Immunol.Meth,, 65: 55-64, 1983) to determine the IC50 (drug concentration required to induce 50% inhibition in growth). of tumor cells) for vincristine, navelbine and AHVB. The results are shown in table 1. The indicated cell lines were obtained from ATCC or NCI tumor deposit and were cultured in tissue culture medium by means of normal techniques well known to those skilled in the art., before dilution at a defined cell concentration required for studies in 96-well plates. A wide variety of drug to tumor growth concentrations were exposed in a long phase in 96-well microtiter plates. The cellular concentrations depended on the cell line as well as on the duration of the culture. Commonly, P388 cells were placed in a concentration of 30,000, 2,000 and 750 cells per well for studies that lasted 1, 3 and 7 days, respectively. The MCF7 cells were placed in a concentration of 7,000 and 1,500 cells per well for studies that lasted 3 and 7 days, respectively. The H460 cells were placed in concentrations of 2,500 and 1,000 cells per well for studies that lasted 3 and 7 days, respectively. K562 cells were placed in concentrations of 1,500 and 10,000 cells per well for studies that lasted 1 and 3 days, respectively. The LS180 cells were placed in a concentration of 5,000 and 20,000 cells per well for studies that lasted 3 and 7 days, respectively. After placing on the plates all the cell lines were incubated (C02 incubator at 37 ° C, 5% C02) for 24 hours before adding the cytotoxic agent (See Table 1).

CITOTOXTCTDAD RET.ATTVA D VINCRTSTINA. AHVR Y_ NAVELBINA IN TUMOR CELL LINES Subsequently the plates were incubated during the indicated period. At specific times, the cells were washed and then exposed to an inclusion dye labeling MTT (tetrazolium 3- (4,5-d ime i 11 iazol-2-i1) -2,5-dif in i 1 o), which accumulated in viable cells. MTT was added to the cells at a final concentration of 50 μ per well. After a 4 hour incubation, the medium and the unreacted MTT were removed from the cells before adding DMSO which was needed to solubilize the insoluble formazan precipitate that formed in the viable cells. After the mixture was mixed from one test tube to another, the colored product was measured using a plate reader operating at 570 nm. The absorbance values obtained by the cells cultured in the absence of the drug were assumed to represent 100% viability. The experiments for substances were repeated whatever differences would be noted between AHVB and other periwinkle alkaloids.

CHARACTERIZATION OF ANTITUMQRAL ACTIVITY AHVB TN VI VQ The cellular evaluation was continued in vitro by means of studies that take into account the AHTB activities in three different models of rodents in vi. In this way, the antitumor activity of AHVB was determined using a rat solid tumor model (U17 lymphoma); the murine P388 tumor model (R. Noble, et al., Cancer Res,, 37: 1455-1460, 1977; P.W. Gout et al.

Biochem. Cell Biol., 64:65-666, 1986), and a mouse H460 SC tumor model. The U17 cell line was originally derived from a. malignant lymphoma t ransp 1 an t ab 1 e that arose spontaneously in male Noble rats (British Columbia Cancer Research Center Joint Animal Breeding Facility with pacifiers obtained from National Institutes of Health, Bethesda, MD). The cell line depends on prolactin and can be cultured easily. The U17 derived solid tumors are generated by subcutaneous injection (by the trocar method) of a small piece (2mm2) of tumor tissue obtained from male Noble rat. The tumor tissue used for implants arises two weeks after subcutaneously injecting U17 5xl06 cells (from the culture) into the neck. To achieve the anti-tumor activity of AHVB, animals that have tumors (2-4gm tumors) were given a single drug treatment and the tumor size was measured as a function of time following the treatment. The antitumor activity was achieved in a series of different doses in order to determine the maximum therapeutic dose of AHVB. Comparative studies between vincristine, vinblastine and AHVB were carried out. For these studies, the antitumor activity was determined at the maximum dose of each drug. The original studies in mice focus, in one case, on the P388 leukemia model. This is a regular NCI model for the evaluation of new agents at the same time and has been shown to be sensitive to vinca pervin alkaloid treatment. This is an ascitic tumor model that was generated by intraperitoneal inoculation of P388 lxl0s cells (derived from the culture, with an original cell line obtained from the NCI tumor deposit) in BDF1 mice (Charles Rivers). One day after the inoculation of the tumor cell, the mice were treated with a single intravenous injection of the drug. The weight of the animal was monitored daily and the evolution of the tumor was measured as an increase in the weight of the animal and through the calculation of survival time. Therapy is described as a decrease in tumor evolution and an increase in the time of survival in c a ation with an untreated control group. Initial studies established the maximum therapeutic sosis for AHVB. Subsequently, studies began comparisons with vincristine and Navelbine in which the animals were treated with each drug at the maximum therapeutic dose. The Canadian Council on Standards for Animal Care adhered strictly to all animal protocols used and these were approved by the Animal Care Committees of UBC and the BCCA. Animals were evaluated twice a day for any signs of stress (related to the tumor or the drug) and if an animal appeared to be suffering (loss or excessive weight gain)., lethargy, mistreated hair, etc.) then sacrificed to the an ima 1.

IDENTIFICATION OF THE MAXIMUM TOLERANT DOSE OF AHVB Acute scale (14 days) and single dose toxicity studies were performed on male Nb rats in order to determine the maximum tolerated dose of vincristine sulfate, Navelbine and AHVB when administered as a single intraperitoneal injection. in these rodents (see table 2).

Table 2: Subacute toxic dosage calculation of vincristine sulfate, Navelbine and AHVB when administered to healthy male Nb rats as a single intraperitoneal injection. For this purpose, healthy male Nb rats without tumor (weight scale 333-399 grams) were divided into groups of 3 animals. House group was used to test a drug in a dose. In each group, each animal received an intrapeptic injection ne a 1 at a particular dose, as indicated in Table 2. The volumes within which the drugs were administered depended on the concentration of the drug solution (in saline) and the weight of the animals, and varied from 0.1-1.0 mi. The saline was used as a control. The highest dose of each drug that allowed the survival of all the animals in one group (3 of 3) was taken as the subacute toxic dosage for the drug, ie 0.7 mg / kg for vincristine, 2.0 mg / kg for Navelbine and 3.0 mg / kg for AHVB. The health of the animals was achieved by daily weight measurement as well as an indication of the behavior due to stress. The animals continued to be monitored throughout the entire study period of 14 days. The animals were sacrificed in the case of signs of severe stress or excessive weight loss of 20%. All animals were necropsied at the end of the study period or at the time of premature euthansia. Once excessive weight loss of 20% or premature death of the animal was noted at a dose level, the dose was decreased until the nadir of the weight loss was less than 20% and no premature animal deaths were observed.

STUDIES IN THE MODEL OF LYMPHOMA Ul.7 IN RATS Cultures of the non-metastatic pre-Nb2 lymphoma line developed at the University of British Columbia and designated Nb2-U17 (Anticancer Research 14: 2485-2492, 1994), are available from the British Columbia Cancer Research Center. Cells from Nb2-U17 suspension cultures growing expo nen cia lly were injected their cutaneously into mature male Nb rats treated with methoxyfuran (5 rats, 310-380 grams, 5 x 10 6 cells / rat in 1). my culture medium) in the neck using a 1.51 gauge 20 needle, at approximately 3 o'clock, when the tumors reached a size of 4-7 cm (length + thickness), the animals and animals were sacrificed. Tumors were used for transplants as described below. The tumor of a rat was excised, shredded and the tumor tissue was placed in trocars (21, caliber 13). Tissue samples were implanted subcutaneously in the neck of male Nb rats anesthetized with me t oxi f 1 ur anus (284-404 grams, 1 trocar per rat). This procedure was repeated 5 times to reach a total of 60 rats with tumor for use in studies on the efficacy of the 3 drugs. When the tumors were well established (1.5-2 weeks later), three groups of 20 rats were selected, as coincident as possible in terms of tumor weight and rat weight, for the administration of three test items (i.e. , a group for each test item). Vincristine was administered to rats weighing 281-384 grams, with tumors weighing 6.3-16.3 grams. Navelbine was administered to rats weighing 274-389 grams with tumors weighing 9.1-23.3 grams. AHVB was administered to rats weighing 303-400 grams, with tumors weighing 7.9-25.9 grams. The weight of the tumors was calculated using the model h em i e 1 i p s i da 1 (weight in grams = length x depth x p? / 6 in cm). The oncolytic effects of each of the three drugs were estimated in a toxic dose subcutaneously, determined for each drug in preliminary studies using mature male Nb rats without tumor, ie 3.0, 2.0 and 0.7 mg / kg for AHVB, Naveline and vincristine, respectively as illustrated in Figure 2. In addition, each drug was fixed at 50 % and 25% of its dose subcutaneously toxic. ST used five rats with tumor to evaluate the effect of each dose level. The drugs were administered intraperitoneally as an individual pill in a volume of 0.19-0.31 ml, as indicated by the weight of the animals. For this purpose, the drug preparations were diluted to appropriate concentrations using spiked saline solution adjusted with acetic acid to a pH of 4.2. For each drug, a carbon dioxide) and underwent necropsy. The animals were also monitored at least daily to see their signs of tension during the entire duration of the study. Animals that manifested severe symptoms (accelerated weight loss, panting, stooped posture, battered fur) were also sacrificed and necropsied. Anhydrovinblastine sulfate (3 ', 4' -desh i dr ovi nb 1 as t i na) was obtained from the drug research section of the British Columbia Cancer Agency (BCCA) (Cancer Agency of British Columbia). Vincristine sulfate (22-oxovi sulfate, 1 eu-co-t-na) was obtained from David Bull Laboratories Ltd., Australia. The NavelbinaMR (vinorelbine tartrate); 3 ', 4'-d i desh i dr o-4' -deoxy-C'-norvincaleucoblastin-di-L-tartrate) was purchased from Borroughs Wellcome Inc., Canada; 0.9% USP sodium chloride injection, pH 4.2 was purchased from Baxter. The BCCA Institutional Committee for Animal Care (IACC) at the UBC approved the methodology that involves animals before conducting the studies (Animal Care Certificate No. A94-1602). During the study, lodging and use of the animals were carried out in accordance with the Canadian Council on Standards for Animate Care 1. The results of the efficacy studies are given in figures 2-3. Figures 2-3 present data averages of 5 animals or less. Figure 2 demonstrates the effect of administering a subcutaneously toxic single intraperitoneal dose of AHVB, Navelbine®, and vincristine on the size of well-established unique transplants of Nb2-U17 lymphoma (average weight 10.13 grams) and the weight of the animals. , as a function of time. While the tumors in the control animals continued to increase in size to an average weight of approximately 40 grams in 6 days, the tumors in the animals treated with drugs in each case had a regression to essential non-palpability within 5 days of the administration. of the drug After day 10, recurrence of tumors occurred in animals treated with Navelbina "* and AHVB in approximately the same degree. In contrast, no recurrence of tumors was observed in the animals treated with vincristine (not even on day 29). Figure 2 also shows that the animals lost weight after the administration of the drug, however, most of the weight was recovered after about 17 days.As controls for each drug, Nb2-U17 rats were used with tumor transplant injected with saline solution For each of the six groups, five animals were used: vincristine sulfate (0.7 mg / kg) in a volume of 0.20-0.23 ml was administered to rats weighing 281-331 grams with tumors weighing 7.6-14.2 grams Navelbina ™ (2.0 mg / kg) in a volume of 0.24-0.31 ml was administered to rats weighing 297-389 grams with tumors weighing 11.5-13.7 grams, AHVB (3.0 mg / kg) was administered in a volume of 0.20 -0.24 mi to rats who weighed 314-374 grams with tumors that weighed 8.2-14.2 grams. Vincristine sulfate controls: saline was administered in a volume of 0.21-0.26 ml to rats weighing 294-370 grams with tumors weighing 9.4-14.6 grams. Navelbine Controls ": saline was administered in a volume of 0.25-0.29 ml to rats weighing 310-365 grams with tumors weighing 9.5-18.2 grams AHVB controls: saline was administered in a volume of 0.19-0.25 ml In rats weighing 303-400 grams with tumors weighing 7.9-16.6 grams, the efficacies of each drug were determined separately in three different dosages against a cont ol. three drugs in 50% of their maximum tolerated individual doses.The data show that Navelbina0 was less potent than AHVB which in turn was less potent than vincristine.Nb2-U17 rats with tumor transplantation injected with saline were used As controls, five animals were used for each of the six groups: vincristine sulfate (0.35 mg / kg) in a volume of 0.23-0.27 ml was administered to rats weighing 327-384 grams with tumors weighing 6.4- 13.4 gram Navelbina ™ (1.0 mg / kg) in a volume of 0.24-0.28 was administered to rats weighing 296-351 grams with tumors weighing 9.1-14.1 grams. AHVB (1.5 mg / kg) in a volume of 0.20-0.23 ml was administered to rats weighing 308-359 grams with tumors weighing 9.7-19.5 grams. Vincristine sulfate controls: saline was administered in a volume of 0.21-0.26 ml to rats weighing 294-370 grams with tumors weighing 9.4-14.6 grams. Navelbine Controls ": saline was administered in a volume of 0.25-0.29 ml to rats weighing 310-365 grams with tumors weighing 9.5-18.2 grams AHVB controls: saline was administered in a volume of 0, 19-0.25 ml to rats weighing 303-400 grams with tumors weighing 7.9-16.6 grams. The efficiencies of each drug were determined separately in three different tests against one control. In Figure 3, the results of the three drugs are compared in equivalents, that is to say, half of subacute toxic dosages. The controls in figure 3 are the same as in figure 2.

STUDIES IN THE P38S MODEL OF MURINQ A cytotoxicity curve was generated to calculate the ICS0 of vincristine, Navelbine ™ and AHVB in the murine P388 cell line (see figure 5). In this study, P388 cells derived from ascitic tumor growth in BDF1 were first separated from red blood cells using Ficoll-Paque. The isolated white blood cells were washed twice, then placed in serum containing tissue culture medium (1 x 10 5 cells per ml of RPMI supplemented with L-glutamine, penicillin, etomicine and 10% fetal bovine serum) and they cultivated for two hours. All non-adrenoreceptive cells were harvested and the cell population was defined as P388 cells and 24 hours later it was used for cytotoxic assays. The assays were performed as ST described in the section titled Characterization of the Antitumor Activity of AHVB In Vi tro. The drug concentrations used are indicated on the X axis. The vincristine is represented by filled circles, the NavelbinaMR by means of filled triangles and the AHVB by means of filled squares. The antitumor activity in vi ve of AHVB was compared with that of vincristine, Navelbina0 in the P388 model of murine BDF1 as follows in the procedure. The P388 cells were derived from ascites of P388 cells of previously injected female BDF1 mice (19-21 grams), from the reservoir. "NCI tumor cells were inoculated directly into the mice.The cells arrived from NCI frozen in aliquots of 1 ml. These samples were rapidly thawed at 37 ° C and subsequently injected (within 1 hour) in vitro into 1 in 2 mice, 0.5 ml per mouse, 1 week (7 days) after inoculation. , the tumor cells were collected by removing the peritoneal fluid using a sterile syringe with a 22 gauge needle. The cells, combined from two animals, were counted using a citrus heme, diluted (RPMI medium) to a concentration of 2xl06 cells / ml and then re-injected 0.5 ml into each of the two BDF1 mice.The remaining cells were washed or placed in a medium containing DMSO and frozen (in freezer packs that are cooled at a certain rate). a) This process was repeated every week for a period of 2 weeks. The cells used for the an i t umor a 1 studies were collected from the third passage to the twentieth passage. After the twentieth passage the cells were not used again for experimental studies. Established cells were derived from frozen cells prepared as described below. Groups (five mice per group) of female BDF1 mice (Charles Rivers, Canada) ST injected (intraperitoneally) with P388 cells 106 (as described below). One day after the inoculation of the tumor cell, the mice were given a large intravenous injection of the indicated drug by means of the lateral vein of the tail. The control groups were injected with a saline solution. Free drug samples were prepared on the day of injection so that the final concentrations were sufficient to deliver the indicated drug dose in a volume of 200 μl. All dilutions were made using USP injection of 0.9% sodium chloride, ST mice were briefly confined (less than 30 seconds) during intravenous injections. Dilatation of the vein was achieved by keeping the animals under a heat lamp for a period of between five and ten minutes. In the next administration of the test items, the animals were weighed daily for fourteen days and tension signs were monitored twice a day for the first 14 days (once a day on weekends) and once a day for the rest of the study, severely stressed animals were eliminated by asphyxia with C02 and the moment was recorded the next dayAlthough the titrations of complete doses were completed for each drug, the data shown in Figure 6 is obtained after the administration of the free drugs at their maximum tolerated dose. This was 3, 40 and 40 mg / kg for vincristine. Ship 1 b i aMR and AHVB, respectively, Figure 4 presents the results of a study demonstrating that the vinca pervin alkaloids induce weight loss following a single intravenous injection of the indicated drug at the maximum tolerated dose (see figure 6). These data were obtained as part of the detailed study in figure 6, After treating the mice (with the P388 tumor) with a single dose of the indicated drug, the animals were examined twice a day (once a day for the purposes of week), the average daily body weight was determined during this period and the results are shown in figure 4. The weight gain in the control is an indication of the evolution of the tumor. The results indicate that AHVB, administered at 40 mg / kg, is the least toxic of the three drugs evaluated. Figure 7 shows the dose response curve obtained for AHVB when used to treat BDF1 mice with P388 tumors. The studies were conducted as described for Figure 6. The maximum tolerated dose of AHVB (40 mg / kg) as specified in these studies reflects a very acute toxic reaction (within 1 hour) which limits the subsequent increase in the dose for administration i.v. of AHVB. This contrasts with the longer toxicity observed in Navelbina1"1 at its maximum tolerated dose and suggests that an ability to avoid the acute toxicity of AHVB could lead to significant increases in its maximum tolerated dose, based on observation of the studies examined in the literature. drug in vi tro, it is surprising that AHVB worked well as a neoplastic agent for use in cancer therapy.In vi tro tests indicate that AHVB TS isistently 10 to 15 times less active on a molar basis (Table 1 and Figure 5). ) than vincristine and Navelbine.RTM These results suggest that AHVB would not perform well as an antitumor agent, however, in an efficacy study, which also employs the P388 cell line (see Figure 6), the antitumor activity of AHVB in the maximum tolerated dose (40 mg / kg, single iv injection) is much better than that observed for vincristine (administered at the maximum tolerated dose of the free drug d 3 mg / kg) The improved antitumor activity, in this case, is measured by the number of long-term survivors (>;60 days) . It is important to note that, for this example, AHVB is approximately 10 times less toxic (at a basis weight) than vincristine. Therefore, 10 times more drug can be given and it is in this dose that the improvements in the long-term survival of animals with P388 tumors were observed.

When compared to Navelbina ™, the results in vi ve are even more surprising since the maximum tolerated doses of the two drugs in animals that have P388 tumors are more or less the same (40 mg / kg). Figure 8 shows the cytotoxicity of AHVB in SK0V3 cells and C-4 cells with a three-day incubation. The IC50 for the SK0V3 and C-4 cells was 4.0 μM and 0.02 μM respectively. Both cell lines were obtained from the ATCC and grown using regular growth and media techniques as described below. The IC50s were determined through normal cytotoxicity assays described below, with each well containing approximately 104 cells.

STUDIES IN THE MODEL OF MOUSE WITH TUMOR H460 The crops d? Human lung H460 cells are available from the British Columbia Cancer Research Center. ST injected cells subcutaneously twice in mice Mature male Rag-2 (24 mice, lxlO4 cells / mouse) using a 26 gauge needle. H460 ST cells suspended in saline d? Hank balanced without calcium. Tumors were formed in the mice for 11 days. When the tumors were well established, four separate groups of mice were selected for the administration of the three test articles (ie one group for each item tested for AHBB bisulfate, AHVB ditartrate and Navelbine) and one control. Bisulphate and the ditartrate of AHVB and Navelbina® were placed under the use of 5% dextrose saturated with argon.These two articles were in a concentration of 20 mg / ml, Any dilution of the dose was made with d T t 5% rose The articles were administered intravenously on days 1, 5 and 9, as dextrose controls at 5% Body weights and tumor measurements were taken daily with calibrators during the first 10 days and After every third day during the remainder of the study, continuing the administration of the items to be tested, the animals were determined daily, the weight and size of the tumor (using calibrators) during the first 10 days and then each t During the rest of the study. If the tumor size reached 1 gram of weight or the tumor began to ulcerate, the animals were sacrificed (by inhalation of carbon dioxide) and subjected to a necropsy. The animals were also watched at least once a day to see signs d? tension throughout the duration of the study. Animals that manifested severe symptoms (accelerated weight loss, panting, stooped posture, battered fur) were also sacrificed and necropsied. The sulphate of anhydride or vi nb 1 as t i na (3 ', 4'-dehydrovinblastine) d was obtained. section d? Research on drugs from the British Columbia Cancer Agency (BCCA) (Cancer Agency of British Columbia). The Navelbine ™ (vinorelbine tartrate; 3 ', 4' -d i desh i dr o-4 '-deoxy-C'-norvincaleucoblastin-di-L-tar treatment) was purchased from Borroughs Wellcome Inc., Canada. The BCCA Institutional Committee for Animal Care (IACC) at the UBC approved the methodology that involves animals before conducting the studies (Certificate for Animal Care No. A94-1602). During the study, feeding and use of the animals were carried out in accordance with the Canadian Council on Standards for Animal Care. The results of the studies d? efficacy are given in Figure 9 and present data averages of 6 animals or less. Each mouse in a given article group had two subcutaneous tumors on its back. Each tumor was measured in length and thickness or volume of each tumor was calculated by means of (LXW) / 2. Then the two tumor volumes were averaged. The volume averages of all mice / ST group averaged to result in a mean for the date point on the graph in Figure 9. The calculation was made every day that tumors were mediated. The normal deviation of the average and the normal error were calculated with the error bars in the graph in Figure 9.

STUDIES IN THE MODEL OF SOLID TUMOR Q ^ A (CERVICAL) Crops d? C-4 human cervical carcinoma cells are available from the British Columbia Cancer Research Center. ST subcutaneously injected cells twice in mature male Rag-2 mice (24 mice, lxlO6 cells / mouse) using a 26-gauge needle. C-4 cells were suspended in a Hank's balanced salt solution without calcium. ST tumors were allowed to form in the mice for 31 days. When the tumors were well established, four separate groups of mice were selected for the administration of the three items tested (i.e., one group for each item tested for AHVB bisulfate, AHVB ditartrate and Navelbine) and one control group. The bisulfate and Ditartrate of AHVB and Navelbina® were placed on a 5% dextrose solution saturated with argon.These articles were added in a dose of 20 mg / kg IV Any dilution of the dose was made with dext rose at 5% The articles were administered intravenously on days 1, 5 and 9, as dextrose controls at 5% Body weights and tumor measurements were taken regularly with calibrators during the study period of 69 days.Continuing the administration of the articles to be tested, the animals were regularly determined the weight and size of the tumor (using calibrated is) during the period of the study.If the tumor size reached 1 gram of weight or the tumor began to ulcerate, ST sacrificed the animals (by means of inhalation d? carbon dioxide) and underwent a necropsy. The animals were also monitored at least once a day to see signs of stress throughout the duration of the study. Animals that manifested severe symptoms were also sacrificed (accelerated weight loss, panting, stooped posture, battered fur) and also a necropsy. Anhydrovinblastine sulfate (3 ', 4' -desh i dr ovi nbl as t i na) was obtained from the drug research section of the British Columbia Cancer Agency (BCCA) (Cancer Agency of British Columbia). Navelbine "(vinorelbine tartrate; 3 ', 4'-didehydro-4' - deoxy-C'-norvincaleucoblastine-di-L-tartrate) was purchased from Borroughs Wellcome Inc., Canada The BCCA Institutional Committee for Care Animal (IACC) in the UBC approved the methodology that involves animals before conducting the studies (Certificate for Animal Care No. A94-1602), During the study, the care, accommodation and use of the animals were made according to The Canadian Council on Standards for Animal Care The results of the efficacy studies are given in Table 3 and present data averages of 6 animals or less.Each mouse in a given article group had two subcutaneous tumors. On the back, each tumor was measured in length and thickness and the volume of each tumor was calculated by means of (LXW) 2/2, then the two tumor volumes were averaged.The volume averages of all the mice / group were averaged to result in an average for the each date point. The ST calculation performed every day that the tumors were measured. The tumors treated with Navelbine "" reached their 'growth threshold' observable on day 14 and continued with constant growth while the distartrate of AHVB reached its threshold? The tumor treated with AHBB bisulfate showed negligible tumor growth until day 69. Navelbine "8 had a delay of 84% in growth in the tumor, AHVB ditartrate had an extended delay of 106%, and bisulfate of AHVB showed a marked delay in tumor growth of more than 209% .The growth of the tumor did not reach the observable growth threshold during 70 days.This data is found in Table 3 below.

TABLE 3: DELAY OF SOLID TUMOR IN GROWTH DATA Taken together, the results presented here show that AHVB has important and unique pharmacological properties that lead to significant improvements in the antitumor efficacy in vi v relative to other vincapervinca alkaloids such as vincristine and Navelbina ™. These results are unique and new because the activity of AHVB was predicted to be much lower based on cytotoxic studies in vi ro. The present invention also provides pharmaceutical compositions containing compounds as described in the claims in combination with one or more pharmaceutically acceptable diluents or auxiliaries., inert or physiologically active. The compounds of the invention can be freeze-dried and, if so desired, be combined with other pharmaceutically acceptable excipients to prepare the formulations for administration.

These compositions may be presented in any form appropriate for the contemplated route of administration. The parental and intravenous routes are the preferred routes of administration. 3 ', 4' -anh i dr ovi nb 1 as tina can be administered orally, topically, parentally, by inhalation or aspersion or rectally in unit formulations. of dosage containing pharmaceutically acceptable agents, auxiliaries and non-toxic vehicles. The term "parental" as used herein includes subcutaneous, intravenous, int amuscular, i n t r a s t er na 1 or infusion techniques. In addition, a pharmaceutical formulation comprising 3 ', 4'-anh i dr or vi nb 1 as t i na and a pharmaceutically acceptable carrier is provided. The 3 ', 4'-anhydrovinblastine may be present together with one or more non-toxic pharmaceutically acceptable carriers and / or diluents and / or auxiliaries and if desired other active ingredients. Pharmaceutical compositions containing 3 ', 4'-anhydrovinblastine may be in a form suitable for oral use, for example, as tablets, troches. dragees, aqueous or oily suspensions, dispersible powders or prules, hard or soft emulsion capsules, or syrups or elixirs. Compositions intended for oral use can be prepared according to any known technique for the manufacture of pharmaceutical compositions and said compositions can contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and conservative agents in order to provide pharmaceutically elegant and tastable preparations. The tablets contain the active ingredient mixed with pharmaceutically acceptable non-toxic excipients which are suitable for the manufacture d? tablets For example, these excipients may be inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; agents d? granulation or disintegration, for example, corn starch or alginic acid; binding agents, for example, starch, gelatin or acacia and lubricating agents, for example, magnesium stearate, stearic acid or talc, The tablets may be uncoated or they may be covered by known techniques to delay disintegration and absorption in the trointestinal tract and thus provide sustained action over a longer period. For example, can you use a material d? delay such as glyceryl monostearate or diesterate d? glyceryl. Formulations for oral use may also be presented as hard gelatine capsules in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatine capsules in which the active ingredient is mixed with water or an oily medium, for example, peanut oil, liquid paraffin or olive oil, The aqueous suspensions contain active materials mixed with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, 1 or 1 osose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and acacia gum; The dispersing or emulsifying agents may be naturally occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example, potassium stearate or oxygenation, or condensation products. of ethylene oxide with long-chain aliphatic alcohols, for example hep t a-de cae ti 1 eneoxi ce t ano 1, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as mono-oleate 1 i oxi eti 1 ensor bito 1, or products d? condensation of ethylene oxide with partial esters derived d? fatty acids and hexitol, for example, monooleate of pol i e t i 1 ensor b i t an. The aqueous suspensions may also contain one or more preservatives is, for example, ethyl or n-propyl-phi or oxy oxybenzoate, one or more coloring agents, one or more flavoring agents or one or more sweetening agents, such as sucrose or saccharin. ST oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, oyster oil or coconut oil, or in a mineral oil such as liquid paraffin. Oily suspensions may contain a thickener, for example beeswax, hard paraffin or alcol cetyl, Sweetening agents such as those set forth above, and flavoring agents may be added to provide tastase oral preparations. These compositions can be preserved by adding an antioxidant such as ascorbic acid. Dispersible powders and granules suitable for preparation of an aqueous suspension by adding water provide the active ingredient mixed with a dispersing agent or emulsifier, suspending agent and one or more preservatives. The dispersing or emulsifying agents are and the suspending agents ST exemplify through the aforementioned. Additional excipients may also be present, for example sweetening, flavoring and coloring agents. The syrups and elixirs can be formulated with sweetening agents, for example, glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain an emollient, a preservative and sweetening and flavoring agents. The pharmaceutical compositions may also be in the form of a sterile aqueous or oleaginous injectable suspension. This suspension can also be formulated in accordance with the known art using those agents d sper sador es em em 1 s i f i cad or es and suitable suspending agents which have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or injection in a non-toxic diluent or solvent in an acceptable form, for example as a solution in 1,3-butanediol. Among the vehicles and acceptable solvents that ST can use are water, Ringer's solution and isotonic solution d? sodium chloride. In addition, fixed sterile oils are conventionally used as a solvent or suspension medium. For this purpose any fixed oil can be employed including mono-or synthetic diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables. It can also be administered 3 ', 4'-anh i dr ovi nb 1 as t i na in the form of sweetening and flavoring agents. The pharmaceutical compositions may also be in the form of a sterile aqueous or oleaginous injectable suspension. This suspension can also be formulated d? In accordance with the known art using those TS or emulsifier agents and suitable suspending agents which have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or injection in a non-toxic diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, fixed sterile oils are conventionally used as a solvent or suspension medium. For this purpose any fixed oil can be employed including mono-or synthetic diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables. It can also be administered 3 ', 4'-anh i dr ovi nb 1 as t i na in the form of suppositories for rectal administration. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and, therefore, it will melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols. It can also be administered 3 ', 4' -anh i d r ov i n b 1 as t i na parentally in a sterile environment. The drug, depending on the vehicle and the concentration used, can be suspended or dissolved in the vehicle. Conveniently, auxiliaries such as anesthetic agents, preservatives and pH regulators ST can dissolve in the v? H? C u 1. For the compounds of this invention, the dose to be administered, either a single dose, multiple dose or a daily dose, will vary with the particular compound being used. Factors that should be considered when deciding a dose regimen include potency of the compound, route of administration, size of the container and the nature of the patient's condition.

The dosage to be administered is not subject to defined limits, but will usually be an effective amount. Commonly it will be the equivalent, on a molar basis of the free pharmacologically active form produced from a formulation of. dosage with metabolic release of the free active drug to achieve the desired pharmacological and physiological effects. An oncologist skilled in the art of cancer treatment will be able to ascertain, without undue experimentation, the appropriate protocols for the effective administration of the compounds of the present invention with reference to previous studies d? vinblastine and its derivatives. AHVB, a derivative of vincastin vinca pervinca alkaloid has shown an important cytotoxic potential against a panel of human cancer cell lines, and a significant activity against xenograft of lung carcinoma tumor of non-small H460 cells in SCID / Rag-2 mice. In vitro cytotoxicity assays using the MTT cytotoxicity assay with a drug exposure time of 72 hours have shown that AHVB is an active cytotoxic drug with ICS0 values ranging from 20-24 nM against carcinoma against lung carcinoma of non-small human H460 cells, human cervical carcinoma C-4m human K562 leukemia, and human epidermoid A431 cell lines. AHVB was approximately 10 times less active than Navelbine "" when tested against the same cell lines. But D? surprisingly, when AHVB was tested in vitro in ST solid tumor efficacy experiments found that it is more potent than NavelbinaMR. Male SCID / Rag-2 mice were inoculated. with H460 cells and after 12 days of tumor growth, iv was delivered. AHVB and Navelbina "" in doses? 10 mg / kg and 20 mg / kg on days 1.5 and 9. In this model, AHVB caused greater inhibition of tumor growth and was less toxic than NavelbinaMR. These results suggest that AHVB may have pharmacologically convenient properties for therapeutic applications. It should be understood that the examples described above are not intended to limit the scope of the present invention. It is expected that numerous variants which are part of the present invention will be obvious to the person skilled in the art, without departing from the spirit of the present invention. The appended claims, properly analyzed, form the only limitation within the scope of the present invention.

Claims (5)

f? £ (? D > '^ ^ >? ^

1 . - The use of 3 ', 4'-anhydrovinblastine, or variants thereof, as an agent in the treatment of cancer.

2. - The use of 3 ', 4'-anhydrovinblastine as an agent for the treatment of cancer, in which the concentration of 3', 4 '-anh i dr ovi nb 1 asti na is at an ignificantly higher concentration than therapeutically acceptable concentrations for vinblastine or Navelbine0 for use in the treatment of cancer

3. A therapeutic composition comprising (the compound of the claim 1) 3 ', 4' -anh i dr ovi nb 1 as t i na and one or more physiologically inert or pharmaceutically acceptable diluents or auxiliaries.

4. A therapeutic composition comprising (1) compound of the claim 2) 3 ', 4'-anhydrovinblastine and one or more pharmaceutically acceptable physiologically inert or active diluents or auxiliaries.

5. - The use of the composition of claims 3 or 4 for the treatment of cancer. 6.- The use d? the composition of claims 3 or 4 for the treatment of cervical cancer. 7. The use of the composition of claims 3 or 4 for the treatment of lung cancer. 8, - The use of 3 ', 4'-anhydrovinblastine for the manufacture of a medicament for the treatment of cancer. 9. The use of the composition of claims 3 or 4 for the manufacture of a medicament for the treatment of cancer.