AU783029B2 - Anhydrovinblastine for the treatment of cervical and lung cancer - Google Patents

Description

P/00/011 28/5/91 Regulation 3.2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Name of Applicant: Actual Inventors Address for service is: University of British Columbia Bruce SCHMIDT, James KUTNEY and Lawrence MAYER WRAY ASSOCIATES 239 Adelaide Terrace Perth, WA 6000 Attorney code: WR Invention Title: "Anhydrovinblastine for the Treatment of Cervical and Lung Cancer" This application is a Divisional Application by virtue of Section 39 of Australian Patent Application 63885/98.

The following statement is a full description of this invention, including the best method of performing it known to me:- "ANHYDROVINBLASTINE FOR THE TREATMENT OF CERVICAL AND LUNG CANCER" TECHNICAL FIELD The present invention is related generally to the use ofantineoplastic vinca alkaloids as antitumour agents. More particularly, the present invention is related to providing use for a derivative of vinblastine, anhydrovinblastine (hereinafter AHVB), as an antineoplastic agent with improved therapeutic properties, demonstrating a significantly higher maximum tolerated dose and less toxicity than its parent and related compounds.

BACKGROUND OF INVENTION Due to a high degree of unpredictability, classic techniques of drug development are inventive.

Mostly through aprocess of elimination, a large number of natural products and synthetic chemical compounds are screened for desired effects, using aseries of increasingly complex systems, beginningwith simple in vitro cell-level assays, progressingto animals and finally human clinical trials. But, due to essential characteristics such as adsorption, distribution and metabolism, the initial in vitro tests that can not take these features into account could eliminate a powerful drug that does not perform well in such simple systems. The drug could be metabolized to different compounds in animal models than in humans, which may also demonstrate different adsorption or distribution patterns. Or finally, compounds can look very promising all the way through clinical trials, butthen demonstrate unpleasant side effects or a high degree of tolerance when used by the human population at large. It is never obvious which compound will continue to look promising as each stage of tests and development are initiated.

Control of tumorous growthhas been achieved to acertain degree using oncolytic vinca alkaloids as antitumour agents alone or in combination with other antineoplastic drugs in cancer chemotherapy for more than 20 years. Approximately 30 alkaloids with a wide range of pharmacological activities have been extracted from the Vincarosea( Catharanthus roseus), commonly known as the periwinkle plant Ofthese, only vinleurosine, vinrosidine, vinblastine and vincristine possess significant anti-tumour activity. In particular, vinblastine andvincristine have been usedwidely as single agents and in combination with outer antineoplastic drugs in cancer chemotherapy. In addition to the naturally occurring alkaloids, some vinca alkaloid analogues have been synthesized by functional transformation or by semisynthetic processes (R.J.

Cersosimo, et al., Pharmacotherapy 3:359-274, 1983; P. Mangency, et al., Org. Chem.

44:3765-3768, 1979; R. Maral, et al., Cancer Lett. 22:49-54, 1984).

Chemically, these vinca alkaloids have adimeric asymmetric structure composed of 2 nuclei linked by a carbon-carbon bond; adihydroindole nucleus (vindoline), which is the major alkaloid contained in the periwinkle, andthe indole nucleus catharanthine (Figure The structural difference between vincristine and vinblastine exists atthe R1 position while vinblastine and vindesine differ with regard to the R2 and R3 substituents.

The mode of action of the antineoplastic vinca alkaloids has yet to be completely understood.

However, it has been established that the antitumour activity is directly related to the highbinding affinity ofthese compounds for tubulin, the basic protein subunit ofmicrotubules (RA. Bender and B. Chabner, In: Chabner (ed) Pharmacol. Princ. of CancerTreat., Saunders, Phil, PA, p.

256,1982; W.A. Creasey, In: Hahn(ed) Antibiotica, Vol. 2, Springer, Berlin, p. 4 1 4,1979).

The consensus is that these agents arrest cell mitosis at metaphase by preventing tubulin polymerizationto foimmicrotubules andby inducing depolymerization (RJ. Owellen and CA.

Hartke, Cancer Res., 36:1499-1504, 1976; R.H. Himes and R.N. Kersey, Cancer Res., 36:3798-3806,1976; R.S. Camplejohn, CellTissue Kinet. 13:327-332,1980). As such, the vincaalkaloids arecllcycle-specific anti-mitotic agents, orspindle poisons. Thebinding affinity of the vinca alkaloids to tubulin correlates poorly with the relative ability ofvincristine, vinblastine and vindesine to inhibit cell growth S. Camplejohn, supra; P.J. Ferguson and C.E. Cass, Cancer Res., 45:5480-5488,1985). The major difference in anti-tumour activity between these drugs appears, therefore, to relate to theirretentionin tumourtissue(P. Ferguson, supra;

J.K.

Horton et al., Biochem. Pharmacol. 37:3995-4000,1988). In asimilarvein, the differenttoxicity profiles ofthe vinca alkaloids seems related to tissue uptake and retention properties rather than to inherent tubulinbinding affinity. For example, studies have demonstratedthatvincristine is more potentthan vinblastine or vindesine in blocking fast axoplasmic transport in nerve cells (S.

Ochs and R. Worth, Proc. Am. Assoc. Cancer Res., 16:70, 1975; S.Y. Chan et al., J.

Neurobiol. 11:251-264,1980). In addition, it is taken up into nerves 4 times faster than the other drugs (Z.Iqbal andS. Ochs, J.Neurobiol., 11:251-264, 1980) andexhibits an extended terminal elimination phase of plasma clearance, suggesting a more prolonged exposure to vincristine thanto the othervinca alkaloids Nelson etal., CancerTreat. Rev., 7:17-24, 1980).

The in vitro and in vivo differences observedbetween the vinca alkaloids are striking given the subtle chemical alterations displayedby the various agents relative to their large, complex molecular structure. For example, vincristine is very effective in treating humanrhabdosarcomas transplanted in nude mice whereas vinblastine is not active in this system Bruchovskyetal., Cancer Res. 25:1232-1238, 1965). This difference is obtained simply as a result of the substitution of an aldehyde group for amethyl group at the R1 position. Further, this chemical substitution leads to a shift in the toxicology profile such that peripheral neuropathy (in the absence ofhematological toxicity) is dose limiting in humans forvincristne whereas anemiaand leucopenia are typically dose limiting for vinblastine Brads, Proc. Int. Vincaalkaloid Symposium, 95-123, 1980; S.S. Legha, Med. Toxicol., 1:421-427, 1986). A particularly interesting therapeutic profile has been observed for anew semisynthetic vinca alkaloidnamed Navelbine (vinorelbine, 5'-noranhydroblastine). This compound is less potentthan vinblastine andvincristine against murine P388 and L1210 leukemia but is active against cells derived from human lung cancerwhereas the othervinca alkaloids are inactive Cros, etal., Seminars in Oncology, 16:15-20, 1989). As well, clinical trials onNavelbine support its utility in treating non-small cell lung cancer Depierre et al., Am. J. Clin. Oncol., 14:155- 119, 1991; A.

Yokoyama etal., Am. Soc. Clin. Oncol., 11:957, 1992). The toxicity profile of this agent appears similarto that ofvinblastine, where hematological toxicities and notneurologcal side effects are dose limiting.

Vincristine has proved particularly useful as anintravenously administered oncolytic agent in combination with other oncolytic agents forthe treatment ofvarious cancers including centralnervous-system leukemia, Hodgkin's disease, lymphosarcoma, reticulum-cell sarcoma, rhabdomyosarcoma, neuroblastoma, and Wilma tumor. It is for intravenous use only andthe intratecal administration is uniformly fatal. Following single weekly doses, the most common adverse reaction is hair loss; the most troublesome are neuromuscularin origin. When single weekly doses of the drug are employed, the adverse reactions of leukopenia, neuritic pain, constipation, and difficulty in walking can occur. Other adverse reactions that have been reported are abdominal cramps, ataxia, foot drop, weight loss, optic atrophy with blindness, transient cortical blindness, fever, cranial nerve manifestations, parehesia andnumbness of the digits, polyuria, dysuria, oral ulceration, headache, vomiting, diarrhea, andintestinal necrosis and/or perforation.

Navelbine T (vinorelbine tartrate) is a novelvinca alkaloid in which the catheranthine unit is the site ofstructuralmodification. Its anti-tumour activity is also thought to be due primarily to its ability to interfere with microtubule activity thereby inhibiting mitosis atmetaphase through its interactionwith tubulin. It is indicated in the treatment of advancednon-small cell lung cancer as asingle agent or in combination, administeredby intravenous route only. Its side effects include phlebitiaor extravasion injury as itis amoderate vasicant. Studies on adverse reactions based on use ofNavelbine™ as a single agent indicate Granculocytopenia as the major dose-limiting toxicity, although itwas generally reversible andnot cumulative over time. Mildto moderate peripheral neutopathy manifested by pareathesia and hypesthesia are the most frequentlyreportedneurologic toxicities, occurringin 10% ofpatients. Mild tomoderatenausea occurs inroughly one-third of patients treated with Navelbine M with a slightly lesser fraction experiencing constipation, vomiting, diarrhea, anorexia, and stomatitis.

Compounds exhibiting lessened toxic effects with equal or greater chemotherapeutic activity remain to be achieved. Thus, aneedremains for a drug providing improved anti-tumour efficacy for the treatment of cancer.

It is, therefore, an objectof the pres ent Invention to provide a method of tea~ng cancerwhich comprises ,n*ste-ring toa human vatient suffering frm cance-r and in nee~dof-,eattent n amrount of -ARHVB, effective to arrest Or signifiatyslow the prog-ess of the disease.

It is anothaerobje-t ofthe pre sentinvention to provide amnethod of using AIVB as an antiurour agenitcomrising therapeutic amount oft.-he chemcal substance ofthe ore-sn etinvention to airest tumnorous growth.

The above and various other obj ects and advantages of the present invention are achi eved by adininistration of a dervtv fiba~e H Other objects and ~advant ages will become evident from the following, detailed description of the p-rese-nt invention.

SUIVLkRY OF RIYENTION The present invention is particularly directed to the use of a derivative of vinblastine, 3 anhydroviriblastne (AHYB), which differs from viablastne in that itpossesses a double bond at the 3 position of the caranthine, nucleus rather than the hydroxyl group that is present in the parent structure, as an antineoplastic agent in the therapeuti-c tr.atrnent of cancer.

One embodiment of the present invention involves the use of 3 ',4'-anihydrovinblasbne, or varants thereof, as an antineoplastic agent in the treatmtent of cancer.

Another emodimrent of the present invention involves thaeuse of ,4r' hydrovinblasnflne as an antineoplastic agent in the treatment of cancer, wherein the concentration of anhydrovinblastine is at significantly higher maxomum conce-ntation than theralpeutically acceptable concentrations for vincristine or Navelbinem for use in the treatment of cancer.

Yet another embodiment of the present invention involves the use of anhydrovinblastine as an antineoplastic agent in the treatment of cervical cancer.

Yet a further embodiment of the present invention involves the use of anhydrovinblastine as an antineoplastic agent in the treatment of lung cancer.

The invention further provides for the use of 3',4'-anhydrovinblastine or a pharmaceutically acceptable salt thereof in the treatment of cancer in a mammal, or the manufacture of a medicament for the treatment of cancer, wherein the cancer is other than leukemia or lymphoma, or the cancer is leukemia or lymphoma. Preferably the higher maximum concentration of 3',4'-anhytdrovinblastine is approximately 10 times higher than the therapeutically acceptable concentrations for vincristine for use in the treatment of cancer in a mammal.

The invention also provides a therapeutic composition comprising a unit dosage amount of 3',4'-anhydrovinblastine or its pharmaceutically acceptable salt that is approximately times greater than the therapeutically acceptable unit dosage amount for vincristine, and one or more pharmaceutically acceptable, physiologically inert or active diluents or adjuvants.

TABLES AND FIGURES Table 1 shows relative cytotoxicity of vincristine, AHVB and Navelbine TM on tumor cell l. .lines.

Table 2 depicts estimates of subacutely toxic dosages of vincristine sulfate, NavelbineM, and AHVB when administered to healthy male Nb rats as a single, intraperitoneal injection.

Table 3 depicts C-4 solid tumor delay in growth data.

Figure 1 depicts the chemical structure of some vinca alkaloids.

6/2 Figure 2 depicts comparison of effects of administering a single intraperitoneal injection, at a subacutely toxic dose, of vincristine, Navelbine M and AHVB to Nb rats bearing single welldeveloped, subcutaneous Nb2-U17 tumor transplants on average tumor weight and average weight of the rats as a function of time.

Figure 3 depicts comparison of the effects of administering a single intraperitoneal injection, at a half subacutely toxic dose of vincristine, NavelbineTM and AHVB to Nb rats bearing single well-developed, subcutaneous Nb2-U17 tumor transplants on average tumor weight and average weight of the rat as a function of time.

Figure 4 depicts changes in mean animal weight of BDFl mice bearing intraperitoneal P388 tumours following i.v. administration of saline, vincristine, NavelbineM and AHVB.

oo ::oo• Figure 5 depicts an example cytotoxicity curve used to estimate the ICo of various vica alkaloids.

Figure 6 depicts P388 anti-turnour activity of selected formulations of inca alkaloids.

Figure 7 depicts a dose response curve obtained for AHVB when used to treat BDF mice bearing P388 tumours.

Figure 8 depicts cytotoxicity curves usedto estimate the IC5o ofAHVB on the celllines SKOV3 and C-4.

Figure 9 depicts mean tumour weight in grams over time (30 days period) following administration at days 1,5, and9, ofNavelbin e M,bisulphate AHVB, ditartrate AHVB, and control.

DETAILED DESCRIPTION OF TIHE

INVENTION

There are many possible derivatives orvariations ofvinblastine possible. However, there is no certainty, even to those skilled in the area of anti-cancer drug development, that any such derivatives willbe as efficacious or even more efficacious than the parent compound- This takes much testing and experimentation.

The term"variants" for purposes of 3' 4,'anhydrovinblastine means any chemical structure that is a derivative of 3',4'-anhydrovinblastine achieved through conservative substitution of side groups, yet still exhibits the same or similar antineoplastic properties as 3 ',4'-anhydrovinblastine.

Characterization of AHVB Anti-tumour Activity In Vitro Cytotoxicity experiments on AHVB were performed as direct comparisons with vincrisne and Navelbine M in order to assess its inherent antineoplastic profile against a variety of tumour cell types relative to other relevant vincaalkaloids. The cytotoxicity ofAHVB was investigated in vitro against a panel of tumour cell lines of varying lineage in order to determine the specificity of its antitumour activity with respect to cell type. The our lines studied were P38 ymphocytic leukemia (amurine lymphocytic leukemia), Noble (Nb) ratU 17 lymphoma, MCF7 human breast carcinoma, H460 human non-small cell lung carcinoma, K562 human erythrokeukeria andLS 180 human colon carcinomabased on establishedNCI in vitronew anti-cancer drug cytotoxicity screening protocols.

Standarddose response cytotoxicity assays Mosmass, J. Immunol. Meth., 65:55-64,1983) were utilizedto determine the IC50 (drug concentration required to induce 50% inhibition of tumour cell growth) forvincristine, Navelbine andAHVB. The result arepresentedinTable 1. The indicatedcell lines were obtained from either the ATCC orNCI tumourrepository and were culturedin tissue culture mediaby standard techniques well known to those skilled in the art, prior to dilution to a defined cell concentration required for the studies in 96 well plates.

A wide range of drug concentrations were exposedto tumour cells growing at log phase in 96well microtitreplates. Cellconcentrations depended on the cell line aswell as the length oftime to be cultured. Typically, P3 88 cells were plated at a concentration of30,000, 2,000 and 750 cells per well for studies lasting 1, 3 and7 days, respectively. MCF7 cells were plated at a concentration of7,000 and 1,500 cells perwell for studies lasting 3 and7 days, respectively.

H460 cells were plated at a concentration of 2,500 and 1,000 cells perwell for studies lasting 3 and7 days, respectively. K562 cells were plated at a concentration of 1,500 and 10,000 cells per well for studies lasting 1 and 3 days, respectively. LS180 cells were plated at a concentration of 5,000 and 20,00 cells perwell for studies lasting 3 and7 days, respectively.

After plating all cell lines were incubated (C02 incubator at 37oC, 5% CO2) for 24 hours prior to addition of the cytotoxic agent (See Table 1).

RELATIVE CYTOTOXICITY

OF

VINCRISTINE, AHVB AND

NAVELBINE

M

ON TUMOR CELL

LINES

DRUG ICo (nM) CELL TYPE EXPOSURE DRUG

IC

50 (nM) LINES TIME V CRIST E NAVELBNE

AHVB

(days) P38____8 m i 1 11.0 3.6 20.0 ±10.0 140.0-53.0 leukemia 3 1.0 0.3 0.7 0.3 15.0 8.7 leukemia 20.0 7 2.0 2.5 20.0

N.DN.D.N.D.

MCF7 human N.D. N.D>2500 breast 3 >2500 >2500 >2500 7 2.6 1.6 2.6 1.6 31.3 12.4 z 6 ha Ntilc.

H460 human N.D. N.D.

N.

lung 3 3.5 0.3 10.0 3 3.5 7 2.5 >0.5 >50.0>50.0 50.) >50.0 K562 eyth o 3 1.50.42.5 2.2 18.8 8.8 leukemia 7 N.D

N.D.

LS 180 human 1 N.D. ND. N>50.0D.

colon 3 50.0 >50.0 >50.0 7 1.50.5 17.5 Table 1 Subsequently the plates were incubated for the indicated time period. At specified times, cells were washed and subsequently exposed to the dye inclusion marker

MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium), which accumulatedinto viable cells.

MTT was added to the cells at a final concentration of50 g per well. After a4 hour incubation, the cells were washed free ofmedia and unreacted MT, prior to addition ofDMSO whichwas required to s olubilize the insoluble formazan precipitatethat formedin viable cells. After the s ample was mixedthrough repeatedpipetting, the colouredproductwas measured using aplate reader operating at 570 nm. The absorbance values obtained for cells cultured inthe absence of drug was assumed to represent 100% viability. Experiments were repeated to substantiate any differences noted between AHVB and other vinca alkaloids.

Characterization of AHVB Antitumour Activity In Vivo Evaluation of in vitro cell cytotoxicity was followedby studies regarding the antineoplastic activities ofAHVB in three in vivo rodentmodels. Thus, anti-tumour activity ofAHVB was determinedusing arat solid tumour model (U17 lymphoma)themurine P388 tumourodel(R Noble, et al., Cancer Res., 37:1455-1460, 1977; P.W. Gout et al., Biochem Cell Biol., 64:659-666, 1986), and a H460 SC Tumour mouse model.

The U17 celllinewas originally derived from a transplantable malignant lymphomathat arose spontaneously in maleNoble rats (British Columbia Cancer Research Centre Joint Animal Breeding Facility with parents obtained fromtheNationalInstitutes ofHealth, Bethesda,

MD).

The cell line is prolactin dependent and can readily be cultured in vitro. U17 derived solid tumours are generatedby subcutaneous injection (viathe trocar method) of asmall (2mm 2) piece of tumour tissue obtained from male Noble rat Tumour tissue used for the implants arose two weeks after injection of 5 x 10 U17 cells (from culture) subcutaneously in the nape oftheneck.

For assessing the anti-tumou activity ofAHVB, tumour bearing animals (2-4 g tumours) were given asingletreatment of drug and tumour size was measuredas afunct of e following treatment. The anti-tumour activity was assessed at a series of different doses in order to determine the maximum therapeutic dose ofAHVB. Comparative studies between vincristine, vinblastine andAHVB were performed. For these studies anti-tumour activity was determined at the maximum therapeutic dose of each drug.

Antitumour studies on mice focussed, in one case, on the P388 leukemia model. This is a standardNCI model for evaluation ofnew anti-cancer agents andit has been demonstrated to be sensitive to treatmentwith vinca alkaloids. This is an ascitic tumour model thatwas generated by intraperitonealinoculation of I x 106 P388 cells (derived from culture, with an original cell line obtained from the NCI tumour repository) in BDF1 mice (Charles Rivers). One day after tumour cell inoculation, micewere treatedwith a single intravenous injection of drug. Animal weight was monitored daily and tumour progressionwas measured as an increase in animal weight andthrough estimation of survival time. Therapywas describedby adecrease intumour progression and an increase in survival time relative to an untreated control group. Initial studies established the maximum therapeuticdose for AHVB. Subsequently comparative studies with vincristine andNavelb were initiated where animals were treatedwith each drug at the maximum therapeutic dose.

The Canadian Council on Animal Care Guidelines were strictly adhered to and all animal protocols employedwere approvedby the Animal Care Committees of UBC and the B CCA.

Animals were evaluated twice daily for any signs of stress (tumour or drug related) and if an animal appeared to be suffering (excessive weight loss or gain, lethargy, scruffy coat, etc.) than the animal was terminated.

Identification of Maximum Tolerated Dose of AHVB Range-finding acute (14 day), single dose toxicity studies wereperformedinhealthy male Nb rats in order to determine the maximum tolerated dose ofvincristine silfate, Navelbine n t and AHVB when administered as a single, intraperitoneal injection in these rodents (see Table 2).

12 Dru Dose (mg/kg) Mortality (surviving rats/in ected rats) I mSaline H4.3 n/a 3/3 Vincristine sulfate 0.7 3/3 Navelbine 1 (Vinorelbine tartrate) 5.0 0/3 Anhydrovinblastife 10.0 0/3 7

T

4.4 0/1 3/3 Table 2: Estimation of subacutely toxic dosages ofvincristine sulfate, Navelbine TM, and AHVB when administered to healthy male Nb rats as a single, intraperitoneal injection.

To this end, healthy non-tumour bearing male Nb rats (weight range 333-399 grams) were divided in groups of 3 animals. Each group was used to test one drug at one dosage. In a group, each animal received one intraperitoneal injection at aparticular dose, as indicatedin 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 ranged from 0. 1 1.O ml.

Salinewas used as a control. The highest dose of each drug which allowed survival of all animals in a group (3 out of3) was taken as the subacutely toxic dosage forthe drug, i.e. 0.7 mg/kg for vincristine, 2.0 mg/kg for NavelbineTM and 3.0 mg/kg for AIVB.

The health of the animals was assessedby daily weightmeasurements in addition to behavioural indications ofstress. Animals continuedto be monitoredthroughoutthe complete 14 day study period. Animals were euthanized in the event of signs of severe stress orweight loss in excess of20%. All animals were necrops ied at the end of the study period or atthe time of premature euthanasia. Once weight loss in excess of 20 or premature animal death was noted at a dose 13 level, the dosewas decreaseduntil theweight loss nadirwas less than 20% andno premature animal deaths were observed.

Studies in the Rat U17 Lymphoma Model Cultures ofthe non-metastatic, pre-TNb2 lymphoma line originally developed at The University ofBritish Columbiaand designated Nb2-U17 (Anticancer Research 14:2485-2492,1994), and are available fromthe British Columbia Cancer Research Centre. Cells from exponentially growing Nb2-U 17 suspension cultures were injected subcutaneously into methoxyfluraneanesthetized, mature male Nb rats (5 rats; 3 10- 380 grams; 5 x 106 cells/ratin 1 ml of culture medium) at the nape of the neck using a 1.5" 20-gauge needle. At about 3 weeks, when the tumours reached a size of4 -7 cm (length width), the animals were sacrificed and the tumours used for transplantation as described below.

A tumour from aratwas excised, minced and the tumour tissue was put into trocars 13 gauge). The tissue samples were implanted subcutaneously in the nape of the neck of methoxyflurane-anesthetizedmaleNb rats (248 -404 grams; 1 trocarperrat). This procedure was repeated 5 times to get atotal of 60 tumour-bearing rats to be used for efficacy studies of the 3 drugs.

When the tumours were well established (1.5 2weeks later), three separate groups of20 rats, as closely matched as possible in terms ofboth tumourweight and ratweight, were selected for administration of the three test articles one group for each test article).

Vincristine was administered to rats weighing 281 -384 grams, bearing tumours weighing 6.3 16.3 grams. Navelbine^ M was administered to rats weighing 274 -389 grams bearing tumours weighing 9.1 -23.3 grams. AHVB was administeredto rats weighing 303 -400 grams, bearing tumours weighing 7.9 25.9 grams. Tumourweights were estimatedusing the hemi-ellipsoid model (weight in grams length x depth x 7/6 in cm).

The oncolytic effects of each of the three drugs were assessed at a subacutely toxic dose, determined for each drug in preliminary studies using non-tumour-bearing, mature male Nb rats, i.e. 3.0, 2.0 and 0.7 mg/kg forAHVB,Navelbine M andvincrisfine, respectively as illustated in Figure 2. In addition, each drugwas assessed at 50% and25% of its subacutely toxic dose.

Five turnour-bearing rats were us edto evaluate the effect at each dose level. The drugs were administered intraperitoneally as a single bolus in a volume of0.19 -0.31 ml, as indicated bythe weight of the animals. To this end, drugpreparations were diluted to appropriate concentrations using sparged saline adjusted with acetic acid to pH 4.2. For each drug, a group of 5 control rats received an intraperitoneal injection of the equivalent amount of saline (pH The tumour-bearing rats were organized in the following groups: Group Drug/Saline Dose(mg/kg) 1 saline 2 AHVB 3 AHVB 4 AHVB 0.75 4 saline 6 Navelbine- 7 Navelbiner" 8 NavelbineTM 9 saline vincristine 0.7 11 vincristine 0.35 12 vincristine 0.175 Following administrtion ofthe test articles, the animals; weight and tumour size (using calipers) were determined daily until the tumour reached an estimatedweight of 35 grams, or started to ulcerate, atwhich times the animals were sacrificed (by carbon dioxide inhalation) andsubj ected to necropsy. Animals were also monitored atleast daily for signs of stress forthe full length of the study. Animals manifesting severe symptoms of stress (rapidweight loss, panting, hunched posture, scruffy coat) were also sacrificed and a necropsy performed.

Anhydrovinblastine Sulfate (3',4'-dehydrovinblastine) was obtained fromthe British Columbia Cancer Agency (BCCA), Investigational Drug Section. Vincristine Sulfate (Sulfate of 22-oxovincaleukoblastine) was obtained from David Bull Laboratories Ltd., Australia.

NavelbineTM (vinorelbine tartrate; 3 ,4'-didehydro- 4 deoxy-C'-novincaleukoblastine-di-L-tartrate) was purchased from Burroughs WellcomeInc., Canada; 0.9% Sodium Chloride Injection USP, pH 4.2 was purchased from Baxter.

The methodology involving animals was approvedby the BCCA's Institutional Animal Care Committee (IACC) atUBC prior to conducting the studies (Animal Care CertificateNo. A94- 1602). During the study the care, housing and use of animals was performed in accordance with the Canadian Council on Animal Care Guidelines.

The results of the efficacy studies are given in Figures 2 3. Figures 2 3 present averages of data from 5 or fewer animals.

The effect of administering asingle intraperitoneal, subacutely toxic dose ofAHVB, Navelbine andvincristine on the size of single, well-establishedNb2-U1 7 lymphoma transplants (average weight 10 13 grams) and the weight ofthe animals, as a function of time are demonstrated in Figure 2. Whereas the tumours in the control animals continued to increase in size to an average weight of about 40 grams in 6 days, the tumours in the drug-treated animals in each case regressed to essential non-palpability within 5 days of drug administration. After day recurrence of tumours inNavelbineM- andAHVB-treated animals occurredto aboutthe same extent In contrast, recurrence of tumours was not observed in vincristine-treated animals (not evenonday29). Figure 2 also shows thatthe animals lostweight following drug administration.

However, most of the weight was regained after about 17 days. As controls for each drug, Nb2-U 1 7 tumourtransplant-bearing rats injectedwith saline were used. For each of the six groups five animals were used Vincristine sulfate (0.7 mg/kg) was administered in avolume of 0.20 0.23 ml torats weighing 281 331 grams bearing tumours weighing 7.6 14.2 grams.

Navelbine" (2.0 mg/kg) was administered in a volume of 0.24 0.31 ml to rats weighing 297 389 grams bearing tumours weighing 11.5 13.7 grams. AHVB (3.0 mg/kg) was administered in a volume of 0.20- 0.24 ml to rats weighing 314 374 grams bearing tumours weighing 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 bearing tumours weighing 9.4 14.6 grams.

Navelbine T M controls: saline was administered in a volume of 0.25 0.29 ml to rats weighing 310 -365 grams bearing tumours weighing 9.5 18.2 grams. AHVB controls: saline was administeredin avolume of 0.19 0.25 ml torats weighing 303 400 grams bearing tumours weighing 7 9 16.6 grams. The efficacies of each drugwere determined separately at three different dosages versus a control.

Figure 3 shows the anti-tumour effects of the three drugs at 50% of their individual maximum tolerateddoses. The datashowthatNavelbine was less potentthan AHVB which in tuwas less potent than vincristine.

Nb2-U17 turourtransplant-bearing rats injectedwith salinewere used as controls. For each of the six groups five animals were used. Vincristine sulfate (0.35 mg/kg)was administered in avolume of 0.23 0.27 ml to rats weighing 327 384 grams bearing tumours weighing 6.4 -13.4 grams. Navelbine T (1.0 mg/kg) was administeredin avolume of0.2 4 -0.28 mltorats weighing 296 351 grams bearing tumors weighing9.1 14.1 grams. AHVB (1.5mg/kg) was administeredin avolume of 0.20 -0.23 mlto rats weighing 308 359 grams bearing tumors weighing 9.7 19.5 grams. Vincristine sulfate controls: salinewas administered in avolume of 0.21 0.26 ml to rats weighing 294 370 grams bearing tumours weighing 9.4 14.6 grams Navelbine M controls: saline was administered in avolume of 0.25 0.29 m to rats weighing 310 365 grams bearing tumours weighing 9.5 18.2 grams. AHVB controls: salinewas administered in avolume of 0.19 -0.25 mltorats weighing 303 400 grams bearing tumors weighing 7.9 16.6 grams. The efficacies of each drug were determined separately at three 17 different dosages versus acontrol. In Figure 3, results of the three drugs at equivalent, i.e. half subacutely toxic, dosages are compared- The controls in Figure 3 are the same as in Figure 2.

Studies in the Murine P388 Model A cytotoxicity curvewas generatedto estimate the IC 5 o ofvincristine, Navelbine andAHVB in the murine P388 cell line (see Figure In this study, P388 cells derived from an ascitic tumour growninBDFl were first separated from red cells employing Ficoll-Paque. Isolated white cells were washed twice then placed in serunm containing tissue culture media(1 x 10 cells perml of RPMI 1640 supplementedwith L-glutamine, penicillin, streptomycin and 10% fetal bovine serum) and cultured for 2 hours. All non adherent cells were collected and that cell population was defined as P388 cells and used for cytotoxicity assays 24 hours later.

Cytotoxicity assays were performed as describedin the the section entitled Characterization of AHVB Anti-tumour Activity In Vitro. The drug concentations used are indicated on the X-axis.

V'-cristine is representedby the filled circles, Navelbinel by the filled triangles andAHVB by the filled squares.

The in vivo anti-tumour activity ofAHVB was comparedto that ofvincristine, Navelbine M in the BDF1-murine P388 model inthe procedure asfollows. P388 cells were derived from the ascities of previously injected female BDF1 mice (19 21 grams) P388 cells, from theNCItumour repositorywere inoculated directly into mice. The cells arrive fromNCI frozen in 1 ml aliquots. These samples were thawed rapidly at 37 0 C and subsequently injected (within 1 hour) intraperitoneally into two mice, 0.5 mlpermouse. One week (7 days) after inoculation, the tumour cells were harvestedby removingperitoneal fluid using a sterile syringe with a22 gauge needle. The cells, pooled from two animals, were counted using aheamocytometer, diluted (RPMI media) to aconcentration of2 x 106 cels/ml ml was thenre-inj ected into each oftwo BDF1 mice. Remaining cells were washed andplaced into a DMSO containing media and frozen (in freezerpacks that cool at a defined rate). This process was repeatedweekly over a2-week period. Cells used for anti-tumour studies were collected fromthe third passage to the 20th passage. After the20thpassage the cellswereno longerus ed for experimental studies. Newly established cells were derived from the frozen cells prepared as described above.

Groups (five mice per group) of female BDF1 mice (Charles Rivers, Canada) were injected (intraperitoneal) with 106 P388 cells (as described above). One day after tumor cell inoculation, the mice were given abolus intravenous injection of indicated drug via the lateral tail vein.

Control groups were injected with saline. Free drug samples were prepared on the day of injection such that the final concentrations were sufficient to deliverthe indicated drug dose in a volume of200 All dilutions were made using 0.9% Sodium Chloride Injection USP. The mice were briefly (less than 30 sec.) restrained during intravenous injections. Dilation of the vein was achievedby holding the animals under aheat lamp for a period ofbetween five and ten minutes. Following administration of the test articles, animals were weighed daily for fourteen days andmonitoredforsigns ofstress twice daily for the first 14 days (once daily on weekends) and once daily forthe remainder of thestudy. Severely distressedanimals wereterminatedby COz asphyxiation andthe time of death was recordedto occur on the following day. Although complete dose titrations were completed for each drug, the data shown in Figure 6 is that obtained after administration of the free drugs attheirmaximum tolerated dose. This was 3,40 and 40 mg/kg for vincristine, Navelbin e M and AHVB, respectively.

Figure 4 presents the results of a study demonstrating vinca alkaloid induced weight loss following asingle intravenous injection ofthe indicated drug atthe maximumtolerated dose (see Figure These data were obtained as part of the study detailed in Figure 6. After treating mice (bearing the P388 tumour) with asingle dose of the indicated drug, animals were examined twice daily forthe first 14 days (once daily on weekends). Mean body weightwas determined daily over this time period and the results are shown in Figure 4. Weight gain in the control is an indication oftumourprogression. Results indicate thatAHVB, administeredat 40 mg/kg, is the least toxic of the three drugs evaluated.

The dose response curve obtained for AHVB when used to treat BDF1 mice bearing P388 tumours is presentedin Figure 7. The studies were conducted as described for Figure 6. The maximum tolerated dose ofAHVB (40 mg/kg) as specified in these studies reflects a very acute (within 1 hour) toxic reaction that limits further dose excalatin for i.v. administration ofAHVB.

This contrasts the more prolonged toxicity observed for Navelbine M at its maximum tolerated dose and suggests that an ability to circumvent the acute toxicity of AHVB could lead to significant increases in its maximum tolerated dose.

Based on observation of the in vitro drug screen studies, it is surprising that AHVB would performwell as an antineoplastic agent foruse in cancertherapy. The in vitro tests indicate that AHVB is consistently 10 to 15 foldless active on permolar basis (Table 1 andFigure 5) than vincristine andNavelbine Theseresults suggestthatAHVB wouldnotperformwell as an anti-tumour agent However, in an efficacy study, also employing the P388 cell line (see Figure the anti-tumour activity of AHVB at the maximum tolerated dose (40 mg/kg, single i.v.

injection) is significantly better than that observed for vincristine (administered at the maximum tolerated dose of the free drug of 3 mg/kg). Improved anti-tumour activity, in this case, is measuredby the number oflong term survivors (>60 days). It is important to stress that, for this example, AHVB is approximately 10 times less toxic (on a weight basis) than vincristine.

Therefore, 10 times more drug can be given and it is at this dose that improvements were observed in the long term survival of animals with P388 tumours. When compared to Navelbine

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the in vivo results are even more surprising as the maximumtolerated dose of the two drugs in animals bearing P388 tumours are about the same (40 mg/k).

Figure 8 shows the cytotoxicity ofAVHB on SKOV3 cells and C-4 cellswith a3 day incubation.

The ICs forthe SKOV3 andC-4 cells were 4.0 1 .M and 0.02.M respectively. Both celllines were obtained fromthe ATCC and grown using standard growth techniques andmedium as described above. The ICss were determined through standard cytoxicity assays described above, with each well containing approximately 104 cells.

Studies in the H460 SC Tumour Mouse Model Cultures ofH460 Human Lung cells are available from the British Columbia Cancer Research Center. Cells were injected subcutaneously twice into mature male Rag-2 mice (24 mice, I x 106 cells/mouse) using a 26-gauge needle. The H460 cells were suspended in a Hank's Balanced Salt Solutionwithout calcium Tumours were allowed to form in the mice for 11 days.

When the tumours were well established, four separate groups of mice, were selected for administration of the three test articles one group for each test article ofAHVB bisulphate, AHVB ditartrate, and Navelbine") and one control.

AHVB bisulphate and ditartrate, andNavelbine M were solubilizedusing 5% dextrose saturated withArgon. Both of these articles were at a concentration of20 mg/ml. Any dose dilutions were made with 5% dextrose.

The articles were administered intravenously on the days 1, 5 and 9, as were controls of dextrose. Body weights andtumourmeasurements with calipers weretaken every day forthe first 10 days and then every other day for the remainder of the study.

Following administraion ofthe test articles, the animals; weight andtumour size (using calipers) were determined daily forthe first 10 days and then every other day for the remainder of the study. Ifthe tumour size reached 1 graminweight orthe tumourstartedtoulcerate,the animals were sacrificed (by carbon dioxide inhalation) andsubj ectedto necropsy. Animals were also monitored at least daily for signs of stress for the full length ofthe study. Animals manifesting severe symptoms of stress (rapidweight loss, panting, hunchedposture, scruffy coat) were also sacrificed and a necropsy performed.

Anhydrovinblastine Sulfate 3 ',4'-dehydrovinblastine) was obtained fromthe British Columbia Cancer Agency (BCCA), Investigational Drug Section. Navelbine (vinorelbine tartrate; 3',4'-didehydro- 4 deoxy-C'-norvincaleukoblastine-di-L-tartrate) was purchased from Glaxo/Burroughs Wellcome Inc., Canada- The methodology involving animals was approvedby the BCCA's Institutional Animal Care Committee (IACC) at UBC prior to conducting the studies (Animal Care CertificateNo. A94- 1602). During the study the care, housing anduse of animals was performedin accordance with the Canadian Council on Animal Care Guidelines.

Theresults oftheefficacy studies are givenin Figure9 andpresentaveragesofdatafrom6 or fewer animals. Each mouse in a given article group had two subcutaneous tumours on its back.

Each tumourwas measured in length andwidth and the volume of each tumour was calculated by (L X W) 2 The two tumourvolumes were then averaged The volume averages of all the mice/group were averaged to yield a mean for the single date point appears on the graph in Figure 9. The calculation was performed each day the tumours were measured. The standard deviation of the mean andthe standard error of the mean were calculated with the error bars appearing in the graph in Figure 9.

Studies in the C-4 (Cervical) Solid Tumour Model Cultures of C-4 HumanCervical Carcinomacells are availablefromthe British ColumbiCancer Research Centre. Cells were injected subcutaneously twice into mature male Rag-2 mice (24 mice, 1 x 106 cells/mouse) using a26-gaugeneedle. The C-4 cells were suspended in a Hank's Balanced Salt Solution without calcium. Tumours were allowed to form in the mice for 31 days.

When the turnours were well established, four separate groups of mice, were selected for administration ofthe three test articles one group for each test article of AHVB bisulphate, AHVB ditartrate, and Navelbine

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and one control.

AHVB bisulphate and ditatrate, andNavelbi n e wer e solubilizedusing 5% dextose saturated with Argon. These articles were administeredat doses of20 mg/Kg I.V. Any dose dilutions were made with 5% dextrose.

The articles were administeredintravenously onhe days 1,5 and 9, as were controls of dextose. Body weights andtumourrmeasurements with calipers were taken regularly over the period of the study of 69 days.

Following administration of the test articles, the animals, weight andtumour size (using calipers) were determinedregulary over the period of the study. Ifthe tumour size reached I gram in weight or the tuour started to ulcerate, the animals were sacrificed (by carbon dioxide inhalation) and subjectedto necropsy. Animals were also monitored at least daily for signs of stress for the full leng th of the study. Animals manifesting severe symptoms of stress (rapid weight loss, panting, hunched posture, scruffy coat) were also sacrificed and a necropsy performed.

Anhydrovinblastine Sulfate 3 ',4'-dehydrovinblastine) was obtained fromthe British Coumbi Cancer Agency (BCCA), Investigational Drug Section. NavelbineM (vinorelbinetatate; 3',4'-didehydro-4'- deoxy-C'-nor in c aleukoblastine-di-L-t artr ate) was purchased from Glaxo/Burroughs Wellcome Inc.; Canada.

The methodology involving animals was approvedby the B CCA's Institutional Animal Care Committee(IACC) at UBC priorto conducting the studies (Animal Care Certificate No. A94- 1602). During the study the care, housing anduseof animal was performed in accordance with the Canadian Council on Animal Care Guidelines.

Theresults ofthe efficacy studies are givenin Table 3 andpresentaverages odataom 6 or fewer animals. Each mouse in a given article group had two subcutaneous turnours on its back.

Eachtumour was measured in length andwidth andthe volume of each tumourwas calculated by (X W 2 /2.Thetwotumor vlume wee ten averaged. The volume averages of all the MIce/groUp were averaged to yield a mean for each single date point.Thcaulaofws Performed each day the tumnours were measured' Navelbinemtum~our reachedtheir observable .'growth th-reshold' at day 41 and continued to gow teailywhrea th AIVBditatate reached the threshold on day 5 5. The tumnour groestedilyth erea b tuhe AHV 0 B eflegligible tumour growth through day 6 9. Navelbine

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had an 84% delay in growth in the tumour, AH diraowthh a f extee dela of206%, and AHVB bisulphate exhibited a marked delay in tumour grwt of i gr at tha ond9% Tumour growth didnot reach the observable growth threshold over 7 0 days.Tidaasfon in Table 3 below.

Table 3: Solid Tumour Delay in Growth Data basis of in io Cyotol (Stuines.8 Theprese th in ets proesntedmhere otat 4HVB~O cnaiingl disclosed ithe cam nc mbn to ih n rm r p r vm e tsi h acepabe i an tu or physioloclly ave, diluents or adjuvaits. The compounds of the invention can be freeze dried and, if desired, combined with other pharmaceutically acceptable excipients to prepare formulations for administration. These compositions may be presented in any form appropriate for the administration route envisaged. The parenteral and the intravenous route are the preferential routes for administration.

3',4'-anhydrovinblastine may be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulatins containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrastemal injection or infusion techniques formulation comprising 4-anhydrovinblastine In addition, there is provided apharmaceutical formulaion comprising 4 a dro bstin and a pharmaceutically acceptable carrier. 3',4'-nhdroviblastie may be present in association with one or more non-toxic pharmaceuticallY acceptable carriers and/or diluents and/or adjuvants and if desired other active ingredients. The pharmaceutical compositions containing 3 ',4'-anhydrovinblastine may be in aform suitable fororal use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersiblepowders or granules emulsion hard or soft capsules, or syrups or elixirs.

Compositions intended of oral use may be prepared according to any known to the art forthe manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting ofsweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceuticallY elegant and palatable preparations. Tablets containthe active ingredientin admixture withnon-toxicpharmaceuticallY Sacceptable excipients which are suitable forthemanufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodiumiphosphate: granulating and disintegrating agents for example, corn starch, or alginic acid: binding agents, for example starch, gelatin or acacia, andlubricating agents, for example magesiumstearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorptin in the gastrointestinal tract For example, atine delay mate

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ial and thereby provide asustained action over aloner pe eample,a time deaymatal such as glyceryl monosterate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredientis mixedwith an inert solid diluent, for example, calcium carbonate, calcium phosphate orkaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methyl cellulose, hydroropylmetyellulose, sodium alginate, polyvinylpyrrolidone, gurtragacanth andgum acacia: dispersing orwetting agents maybe a 1 polyvinylpyrroidone, gum alkylene naturally-occrinphophatide, for example, lecithin, condensation product oan a lene oxide with fatty acids, for example polyoxyethyene stearate, or condensation products o ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensaionpructs of ethylene oidewithartial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitolmonooleate, or condensation products of ethylene oxide with partial esters derivedfrom fatty acids andhexitol anhydrides, for example polyethylene sorbitan monooleat e aqueous suspensions may also contain one ormore prese vesfor example ethyl, or n-propyl p-hydroxybenzoate, one or more colouring agents, one ormore flavouring agents or one or more sweetening agents, such as sucrose or saccharin.

Oily suspensionsmaybe formulatedby suspending the active ingredients in avegetable oil, for example arachis oil, olive oil, sesame oil or coconoil, or ai oil such as qidparaffin The oily suspension may contain a thickening agent, for examplebeeswa r cety alcohol. sweetening agents such as those set forth above, andflavoring agents may be added to provide palatable oral preparations These composition may bepresvedb the addition of an anti-oxidant such as ascorbic acid.

Diprible powder5and granules suitable for preparatinoanquosupesobth dition ofaervide the acti ve ingredient in admixture with a dispersing or wetng agent, aupdingo agentnonoroe preservatives Suitable dispersing r wtigaet n supedig getsae exempifiby thoe alreadymentioned above Additioal excipents for exape 5 eeeilg laouig andnt colurng agents may also be present.

Pharmaceutical composition of the invention may als bei h orm ofah oil, i un era mulfons The oils phas e may be a vegetable oil, for example Olive oi or arachis il, or an au allyo example liquidparfin~ or mixtures of these. Sutal emlsfyn aensma b atrally 0 c c u r in g g u i s fo r e x a m p le g u m a c a c ia o r g u rn tra g a c a n th n a u a l c u r n for xml syba, eihn and esters or partial esters derived fromfatty acids and hexitol, anhydride, for example sorbitan monoleate, and condensationroutofheaipilst with ethylene oxide, for example polyoxYethylene sorbitanmonoleate. The emulsions may also contain sweetenling and flavourinig agrentssyrups and elixirs may be formulatedwith sweetenling agents for examplel glyertiv prplne gycol sorito or ucroe. uch forulations may also contain a demulce, apresatvan g~lyolhi an criolOrurosng agS. The pharmaceutical cormposition may be inthe form of astenle njctbl aqeus or oleagirous suspension. this suspension may be formulation ccordin'to ~fl~fl1-usig tos sutale isprsng ~wtfllg gets ndsuspending agents which have been mentioned above. The sterile inj ectable prepart o mysoent steril netale a solution ano~toic arentally acceptable diuetoslvnfreapesasuin or suspension in ao- vnstamyb mlydaewtr in 1,3 butanediol. AmTong the acceptable vehicles andsolven hadtin brie mploedl are tr Ringer's solution and isotonic sodium chlori de solution. In adtisronste ld fixed l r coventionaly employed as a solvent Or sus For dtirone fany anids fixeda oil my be~ploydincudi syntheti mono- or diglycerides I diin at cd uha oleic acid find use in the Preparation of injectables.

also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drugwit a suitable nonirritating excipientwhich is solid at ordinary temperatures but liquid at the rectal suitable non-irritatidg. Suchmaterials are cocoa temperature andwill therefore melt in the rectum to release the drug. Suchm butter and polyethylene glycols.

3', 4 '-anhydrovinblastine may be administered parenterally in sterile medium. The drug, depending on the vehicle and concentration used, caneitherbe suspended or dissolvedin the vehicle. Advantageously, adjuvants such as local anaesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

of this invention, te dose be administered, whether asingle dose, multiple For the compounds ofthis invention, the dose o .onsider usbeing used. Factors to ClDo dos e, or a daily dose, will vary withthe particular compounbeingus ed. F actors to consider when deciding upon dose reimeninclude potency of the compound route of administration, size of the recipient and the nature of the patient's condition.

The dosagetobe adiistered notubjectto defined limits, but in will usually be an effective Taepdosaeotobecadministered isnot subjectto a amount Itwill usually be the equivalent, on amolarbasis of the pharmacologicall active free form produced from a dosage formulation upon the metabolic release of the active free drug to achieve its desired pharmacological and physiological effects.

An oncologist skilled in the art of cancer treatent will be ableto ascertain, without undue expeimentatons, appropriate protocols for effective administration of the compounds ofthis present invention by referring to the earlier studies of vinblastie and its derivatives.

AHVB a derivative ofthe Vinca Akaloid Vinblastine has wn s against a panel ofhuman cancer celllines, andsignificant activity againstthehumanH460 non small cell lung carcinoma tmurxenaphin SCID/Rag-2 ice.n vitro cytotoxicity assays utismall cell lung cyttarcinomassaywith adrug exposure time of 72 hours have shown that utilizing the MTT cytotoxicity assay with a drug exposure tune B is an ve cytotoxic with IC values ranging from 20-24 aM againstthe H460 AH-VB is an active cytotoi X r1 wi s human non-small cell lung carcinoma C-4 human cervical carcinom, K562 human leukemia, and the A43 I human eaidermoidcell lines. AHVB was aporoximately 10-fold less active than Navelbine" when tested in vitro against the same cell lines. Sur-risingly, however when.

AHVB was tested in vivo in solid tumour efficacy experiments it was found to be more potent -than NavelbineT. Male SCID/Rag-2 mice were inoculatedsc. with H460 cells and after 12 days oftumour rowth AHVB and Navelbine^ were deliveredi.v. at doses of I 0mg/kg and mg/kg on days 1, 5,9. In this model, AHVB caused greater tour grow inhibition and was less toxic than Navelbine". These results suggest that AHVB may have desirable pharmacological properties for therapeutic applications.

Itis to be understood that the examples described above are not meant to limit the scope of the present invenion. Itis exoected that numerous variantswillbeobvioustothenersonskilledin the artto which the present invention pertains, without any departuie from the spirit ofthe present invention. Tne appended claims, properly constued, form the only limitation upon the scope of the present invenution.

Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such'as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Claims (14)

1. The use of 3',4'-anhydrovinblastine or a pharmaceutically acceptable salt thereof, in the treatment of cancer in a mammal, wherein the cancer is other than leukemia or lymphoma.

2. The use of 3',4'-anhydrovinblastine or a pharmaceutically acceptable salt thereof, in the treatment of lymphoma in a mammal.

3. The use of 3',4'-anhydrovinblastine or a pharmaceutically acceptable salt thereof, in the treatment of leukemia in a mammal.

4. The use of 3',4'-anhydrovinblastine or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of cancer in a mammal, wherein the cancer is other than leukemia or lymphoma.

The use of 3',4'-anhydrovinblastine or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of lymphoma in a mammal.

6. The use of 3',4'-anhydrovinblastine or a pharmaceutically acceptable salt thereof, -for the manufacture of a medicament for the treatment of leukemia in a mammal.

7. The use according to any one of claims 1-6, wherein the higher maximum concentration of -anhydrovinblastine is approximately ten times higher than the therapeutically acceptable concentrations for vincristine for use in the treatment of cancer in a mammal.

8. The use according to any one of claims 1-7, wherein said salt is anhydrovinblastine sulfate, 3',4'-anhydrovinblastine bisulfate or anhydrovinblastine ditartrate.

9. The use according to claim 1 or 4, wherein said cancer is a carcinoma.

The use according to claim 1 or 4, wherein said cancer is a sarcoma.

11. The use according to claim 1 or 4, wherein said cancer is selected from the group of breast cancer, cervical cancer, lung cancer, and colon cancer.

12. A therapeutic composition comprising a unit dosage amount of anhydrovinblastine or its pharmaceutically acceptable salt that is approximately times greater than the therapeutically acceptable unit dosage amount for vincristine, and one or more pharmaceutically acceptable, physiologically inert or active diluents or adjuvants.

13. The therapeutic composition of claim 12, wherein said salt is 3',4'-anhydrovinblastine •sulfate, 3',4'-anhydrovinblastine bisulfate or 3',4'-anhydrovinblastine ditartrate.

14. A use according to claim 1 substantially as herein described with reference to the examples. A therapeutic composition according to claim 12 substantially as herein described with reference to the examples. DATED this TWENTY-FIFTH day of JULY 2005. S.University of British Columbia Applicant Wray Associates Perth, Western Australia Patent Attorneys for the Applicant.