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The interactions of the vinca alkaloid drugs catharanthine and vindoline with tubulin have been investigated and compared with those of vinblastine and vincristine. Both drugs were found to be less effective in bringing about the inhibition of tubulin self-assembly into microtubules than vincristine and vinblastine, the drug to protein molar ratio required being 3 orders of magnitude greater. An analytical ultracentrifuge study has shown that catharanthine can induce the self-association of tubulin into linear indefinite polymers with an efficacy that is 75% that of vinblastine or vincristine, the intrinsic dimerization constant for the liganded protein being K2 congruent to 1 x 10(5) M-1. The effect of vindoline was marginally detectable. Binding studies of catharanthine using the gel batch and fluorescence perturbation techniques showed a polymerization-linked binding of one catharanthine molecule per tubulin alpha-beta dimer with a binding constant of (2.8 +/- 0.4) x 10(3) M-1. For vindoline, binding to tubulin was marginally detectable by fluorescence spectroscopy, although addition of vindoline to tubulin did generate a difference spectrum. It was concluded that the binding of vinblastine/vincristine to tubulin and its consequences are determined by the interaction of the indole part of catharanthine with tubulin, the role of vindoline being that of an anchor.

The pharmacokinetic behavior of navelbine was investigated in 19 patients presenting with advanced cancers (mainly women with breast cancer). Navelbine was given orally at seven dose levels of up to 200 mg/week. For a given dose, patients received four successive weekly treatments. Five subjects also received two different doses. After drug administration, plasma was collected for 48 or 72 h and monitored for navelbine concentration by radioimmunoassay. Absorption of navelbine was very rapid after oral administration: maximal drug concentrations were reached within the first 1 or 2 h (Tmax, 0.9-1.75 h; cmax, 70.9-832.6 ng/ml), with absorption constants ranging from 0.85 to 2.42 l/h. A comparison of dose-normalised plasma concentration profiles revealed significant time dependence in six evaluable patients (P less than 0.001). Only four subjects who received low doses (less than or equal to 100 mg/week) exhibited time-independent kinetics. All of the five patients who were treated at different doses displayed apparent dose dependence (P less than 0.001). No individual profile was characterised by both time- and dose-independent pharmacokinetics. In all, 18 patients presented biphasic plasma concentration-decay patterns, and only 1 subject exhibited monophasic decay kinetics. The navelbine pharmacokinetic parameters obtained following oral administration were similar to those observed after i.v. bolus injection and were characterised by high oral clearance (0.43-1.45 1 h-1 kg-1), a large apparent volume of distribution (27.4-45.9 1/kg), and a long terminal half-life (24.2-56.5 h). Large intra- and inter-individual variations in pharmacokinetic parameters were observed. Moreover, after a high dose of 200 mg, an enterohepatic cycle and/or a delay in navelbine's absorption at a distal intestinal site as evidenced by a marked plasma level rebound was observed.

The possibility of reducing toxicity of Vinblastine by fixing it in polybutyl-2-cyanoacrylate nanoparticles was studied. A significant reduction of mortality of mice was recorded after a single intraperitoneal (i. p.) injection of nanoparticle-associated Vinblastine. A wide range of doses of Vinblastine (1.04-6.25 mg/kg) were used. There was a considerable decrease of leucopenia caused by Vinblastine-loaded polybutyl-2-cyanoacrylate nanoparticles as compared to that induced by free Vinblastine.

Radiation nephritis is the principle late toxicity seen after total body irradiation in barrier-maintained rats when hematologic toxicity is prevented by bone marrow transplantation. Renal dysfunction is observed for single doses as low as 7.5 Gy. Hepatic blood flow, as measured by indocyanine green clearance, is decreased after 8.5-9.5 Gy single-dose total body irradiation. Serum albumin levels are decreased after 9.5 Gy single-dose total body irradiation. Hypoalbuminemia is a symptom of hepatic damage, but can also be caused by renal damage or edema. No decrease in total serum protein is observed, indicating that proteinuria resulting from renal damage is not the cause of hypoalbuminemia. No edema and some dehydration are observed. These data indicate that hepatic damage as well as renal damage may be occurring after total body irradiation plus bone marrow transplantation. Animals given total body irradiation plus bone marrow transplantation show decreased tolerance to a wide variety of immunosuppressive and cytotoxic drugs, even when these drugs are given months after total body irradiation. Altered drug clearance after total body irradiation plus bone marrow transplantation is observed for cis-platinum, vincristine, and adriamycin. The increase in cis-platinum toxicity after total body irradiation plus bone marrow transplantation is caused by decreased renal drug clearance. The decrease in vincristine tolerance and the alterations in adriamycin and vincristine pharmacokinetics are caused by altered drug distribution after total body irradiation plus bone marrow transplantation. These results indicate that bone marrow transplant survivors may show altered clearance of, and decreased tolerance to, a wide variety of drugs that are used after bone marrow transplantation.

Mouse C127 cells, transfected with the chicken calmodulin (CaM) gene and overexpressing CaM protein, were used to evaluate the effect of elevated levels of CaM on the sensitivity of these cells to various anticancer drugs. Clones C2 and C3 overexpress CaM mRNA by 40- and 80-fold, respectively, and CaM protein 3- and 8-fold, respectively. These cell lines were tested for their sensitivity to vincristine, vinblastine, bleomycin, and Adriamycin by comparing the 50% inhibitory concentration in a 72-h growth inhibition assay. The 50% inhibitory concentration values for vincristine with C2 and C3 cells were 6.27 +/- 0.56 nM and 6.60 +/- 0.96 nM, respectively. These values were significantly lower than 13.9 +/- 0.79 nM for the parental C127 cells and 14.0 +/- 1.55 nM for clone 6.8 (the control cell line for transfection without the chicken CaM gene) at P less than or equal to 0.005. The proliferation of C2 and C3 cells was inhibited at lower concentrations of vinblastine as well. The 50% inhibitory concentration values for the C2 and C3 cell lines were approximately one-half those required for C127 or clone 6.8 cells. However, no significant difference in the sensitivity to the DNA-binding drugs, bleomycin and Adriamycin, was observed between the different cell lines. The uptake of [3H]vinblastine was evaluated and found to be increased 1.6- and 2.8-fold in C2 and C3 cells, respectively, as compared with that value obtained for C127 cells. Moreover, the efflux of [3H]vinblastine from vinblastine-loaded cells was also observed to be decreased in the C2 and C3 cell lines. These data suggest that the increase in CaM expression in the C2 and C3 cell lines might be related to the higher sensitivity of these cells to Vinca alkaloids. This increased sensitivity appears to be due to the increase in intracellular concentration of the Vinca alkaloids as a result of an increase in drug uptake and a decrease in efflux. Moreover, the increased sensitivity of clones C2 and C3 to Vinca alkaloids appears to be specific to this class of drugs in that no collateral effects were observed for the DNA-damaging drugs, Adriamycin and bleomycin.

Vinblastine and Vincristine pharmacokinetics, including tissue distribution, metabolism and biliary excretion, were investigated, using both "in vitro" and "in vivo" models, after i.v. injections in rats. Plasma kinetic curves were best fitted to a two-compartment open model. The average terminal half-lives of VLB and VCR were 14.3 h and 7.5 h, respectively. The systemic clearance and apparent distribution volume for VLB, respectively 1.49 l/h/kg and 11.46 l/kg, were significantly greater than those of VCR, 0.12 l/h/kg and 0.41 l/kg. VCR was found to be widely distributed in tissues after i.v. injections in rats. The highest drug accumulation site was the intestine (122.0 ng/g wet tissue at 24 h). Liver and kidneys also retained high proportions of drug (respectively, 47.0 ng/g and 44.4 ng/g at 24 h). Biliary excretion was more rapid for VCR (42.7% of total radioactivity excreted over 24 h) than VLB (28.2% of total dose over 24 h). For both molecules, the percentage of radioactivity excreted in bile over 30-48 h ranged between 40-50% of total dose. At high doses, either biliary excretion rate or cumulated excretion was reduced. High performance liquid chromatography analysis of bile samples revealed four biotransformation products for VLB and three for VCR. When incubated in freshly isolated rat hepatocytes, VLB penetrated more rapidly and intensely into the cells (more than 90% of total dose taken up over 20 min) than VCR (only about 40% accumulated), probably through a passive diffusion mechanism followed by tight cellular binding. "In vitro" metabolism patterns were similar to those found "in vivo", except for the most polar metabolites observed "in vitro". Two anti-Vinca monoclonal antibodies with different specificities were used to test VCR metabolite immunoreactivities. The results suggested that some structural modifications occurred in the catharantine moiety of the molecule but that the dimeric structure seemed to be well conserved after biotransformation.

A high-performance liquid chromatographic procedure, including sample pretreatment, is presented for the analysis of the experimental antitumour drug vintriptol in plasma. The sample pretreatment involved liquid-liquid extraction of the buffered (pH 3) sample with chloroform. Vinblastine was used as internal standard. Separation was achieved on a Hypersil ODS (5 microns) column with a mobile phase of acetonitrile-phosphate buffer. Electrochemical detection (at +0.70 V) was used, giving a detection limit of 2 micrograms/l. The applicability of the assay was demonstrated in a pharmacokinetic study with eight cancer patients who received 45 or 50 mg/m2 vintriptol in a phase I study. A three-compartment model was used to fit the plasma concentration-time curves. Pharmacokinetic parameters are presented.

The interaction of etoposide (VP-16), Vinca alkaloids, and verapamil with the P-glycoprotein (P-gp) was studied in human breast (MCF-7) and Chinese hamster lung (DC3F) cell lines and the corresponding multidrug-resistant MCF-7/ADR and DC3F/ADX tumor cell lines, selected for resistance to Adriamycin and actinomycin D, respectively, and overexpressing P-gp. Verapamil (10 microM) markedly reversed resistance to vincristine (11-fold in DC3F/ADX and 125-fold in MCF-7/ADR; 1-hr exposure), but it had a very modest effect on resistance to VP-16 (3- to 4-fold; 1-hr exposure). Resistant cells accumulated 2- to 4-fold less VP-16 and vincristine than the parental cell lines. Verapamil (10 microM) significantly increased accumulation and retention of vincristine, but not of VP-16, in resistant cell lines. Photoaffinity labeling of resistant cell lines with radioactive analogs of verapamil [N(p-azido-3-125I-salicyl)-N'-beta-aminoethylverapamil (NASVP)] and vinblastine[N-(p-azido-3-125I-salicyl)-N'-beta-aminoethylvindesine (NASV)] showed distinctly labeled P-gp bands in both resistant cell lines, compared with wild-type cells. Excess nonradioactive vinblastine or verapamil effectively competed with the P-gp photolabeling by either NASVP or NASV, with IC50 levels of 0.6 and 10 microM, respectively. In contrast, nonradioactive VP-16 was 100- to 500-fold less potent than vinblastine in competing with P-gp photolabeling, suggesting that VP-16 has significantly lower affinity for P-gp than Vinca alkaloids have. Taken together, our data indicate that P-gp glycoprotein by itself may not be important in the transport/efflux of VP-16 and, thus, in the mechanism of resistance to VP-16 in these cells.

Treatment of drug-resistant human KB carcinoma cells (KB-V1) with 0.2 microM phorbol 12-myristate 13-acetate (PMA) resulted in increases of 4-fold in both membrane-associated protein kinase C activity and phosphorylation of P-glycoprotein. The response was essentially complete after 30 min and was relatively stable, since both of these parameters remained elevated above basal levels in cells exposed to PMA for 24 hours. In contrast, long-term PMA treatment of drug-sensitive KB-3 cells caused complete depletion of protein kinase C. The rate of accumulation of [3H]vinblastine in KB-V1 cells was 0.8 +/- 0.1 pmol/mg/30 min in the absence, and 1.9 +/- 0.2 pmol/mg/30 min in the presence, of 20 microM verapamil. Preincubation of cells with PMA resulted in a time-dependent decrease, up to 60% after 24 hours, in both of these values. These results suggest that protein kinase C mediated phosphorylation stimulates the drug transport activity of P-glycoprotein.

The plasma pharmacokinetics of the monoclonal antibody-vinca conjugate KS 1/4-desacetylvinblastine hydrazide (DAVLB-hyd; [3H]LY203725) have been evaluated in rats (17 mg/kg) and monkeys (15 mg/kg) after i.v. dosing. Plasma concentrations of radioactivity 1 hr after dosing were higher in monkeys than in rats. The biphasic elimination of radioactivity in rats was characterized by half-lives (T1/2) of 10 and 143 hr, whereas the elimination of radioactivity in monkeys was characterized by T1/2 values of 11 and 66 hr. Plasma total antibody and radioactivity concentrations were similar within 6 (rat) and 24 (monkey) hr after dosing; however, total antibody concentrations were greater than radioactivity concentrations thereafter, indicating the presence of free antibody in the plasma. Plasma elimination T1/2 values and areas under the curve of total antibody in rats and monkeys were greater than those of radioactivity. The presence of free antibody implies the presence of free DAVLB-hyd; however, plasma concentrations of free DAVLB-hyd were at least 3 orders of magnitude less than those of radioactivity in both species. The T1/2 of free DAVLB-hyd in plasma of LY203725 dosed monkeys was 16 hr. Hydrolysis of the conjugate to yield free DAVLB-hyd was observed upon incubation of conjugate with rat plasma in vitro. Administration of DAVLB-hyd to rats resulted in a rapid initial decrease in plasma DAVLB-hyd followed by a slower (T1/2 = 1.4 hr) elimination rate. Fecal excretion was the predominant mode of elimination of radioactivity in both rats and monkeys dosed with [3H]LY203725.

Vinblastine sulfate (VLB) suspended within a collagen matrix (CM) as a diffusion limiting drug delivery vehicle was examined in vitro, as well as in mouse subcutaneous and brain tumor models. Against RIF-1 and KHT subcutaneous tumors, there was enhancement of antitumor activity with intratumoral (i.t.) delivery of VLB when it was combined with CM and/or epinephrine (epi) provided as a vasoactive agent to limit diffusion of VLB away from the injection site. Furthermore, in pharmacokinetic studies an 3-fold enhancement of tumor exposure to drug (AUC) with the CM-formulation was observed relative to the administration of free VLB i.t. Craniotactic injection of VLB into mouse brain in doses from 0.2 to 2 mg/kg revealed that the CM association markedly reduced the acute toxicity of VLB in normal mouse brain. Furthermore, mice with stereotactically implanted KHT brain tumors treated with 0.2 mg/kg VLB in CM had less tumor present in the brain histologically compared to the free VLB and untreated control groups.

One major feature of multidrug resistance is the reduced cellular level of drugs maintained by MDR cells. Although there is now strong evidence that drugs are actively pumped out of MDR cells, transport experiments have indicated decreased initial rates of influx at the earliest times at which measurements could be made. We have used a pulsed quench-flow apparatus to study transport characteristics of colchicine resistant MDR cells on a very fast time scale. A rapid association of daunomycin with drug sensitive cells occurred within 0.11 sec. This association is virtually absent in MDR cells. In efflux experiments performed on the same rapid time scale, greater than 50% of daunomycin efflux occurred within 0.1 sec. No substantial efflux from B1, drug sensitive cells was observed. On the other hand, vinblastine accumulation by both cell types was similar for approx. 10 seconds. Thus, kinetically, not all drugs are handled in a similar fashion by MDR cells. The pulsed quench-flow apparatus was useful in making fast time measurements of drug influx and efflux and in demonstrating the differences between drug recognition patterns by MDR cells.

Unidirectional blood-brain barrier transfer of the lipophilic anticancer agents vincristine and vinblastine was studied in anesthetized rats, using an isolated, in situ brain perfusion technique. Drug binding to plasma constituents was also measured. Despite the high lipophilicity of these agents (the log octanol/physiological saline partition coefficient equalled 2.14 and 1.68, respectively), the cerebrovascular permeability-surface area product, PA, of vincristine in plasma was only 0.49 x 10(-4) ml s-1 g-1 for parietal cerebral cortex, whereas that of vinblastine was too low for determination. These values are similar to those of water-soluble, poorly diffusible nonelectrolytes. The PAs were significantly higher in the absence of plasma protein, being 1.24 x 10(-4) and 5.36 x 10(-4) ml s-1 g-1, respectively. Even these values, determined by brain perfusion of protein-free buffer, were lower than would be expected from the lipophilicity of the agents. The results suggest that additional factors, such as steric hindrance and molecular charge distribution, related to the chemical and geometric structure and the large size of vincristine and vinblastine (molecular weight, 825 and 814 daltons, respectively) restrict their passage across the blood-brain barrier. As a consequence of their paradoxically low permeability at the blood-brain barrier and restrictive binding to plasma and blood constituents, doses of both agents that cause significant inhibition of extracerebral Walker 256 carcinosarcoma tumor implants in rat have no effect on tumor located in the brain.

The pharmacokinetics of a murine monoclonal antibody vinca conjugate (KS1/4-DAVLB) was investigated in 13 patients with adenocarcinomas who received single intravenous doses ranging from 40 to 250 mg/m2 and in three patients who were administered 1.5 mg/kg every 48 to 72 hours for up to 15 doses. Five patients in the single-dose study also received 100 microCi of [3H]-KS1/4-DAVLB. Overall mean values for the pharmacokinetic variables were as follows: elimination half-life, 31.5 hours; distribution volume, 4.43 L; and clearance, 0.09 L/hr. KS1/4-DAVLB demonstrated linear elimination kinetics in both the single- and multiple-dose studies. Significant concentrations of KS1/4-DAVLB were noted in a pleural effusion. Ten percent of the radioactive dose was recovered in the urine and 20% in the feces over a 5-day period. Small molecular weight vinca species were detected in the feces but not in the serum.

Navelbine (NVB) (5'-noranhydrovinblastine) is a new semi-synthetic vincaalkaloid (VA) exhibiting a high affinity for tubulin and considerable anticancer activity in patients. A better hematologic tolerance and a weak neurotoxicity have been reported for this drug as compared to other VA. Moreover, NVB presents a relatively high bioavailability and a good digestive tolerance, thus offering original perspectives for the treatment of ambulatory cancer patients. A clinical pharmacokinetic study of NVB was carried out on 12 patients after oral administration of the drug. The pharmacokinetic parameters were similar to those of intravenous administration and also showed a high interindividual variability. Studies on the in vitro metabolism of NVB using hepatic microsomal fractions from 22 different donors demonstrated the formation of 3 metabolites. The biotransformation rate quantitatively varies from one human liver specimen to another, a fact which could be, in part, at the origin of the interindividual variability of the therapeutic response.

A limited sampling model was developed for vinblastine to estimate the total area under the concentration time curve (AUC) using only two timepoints. Detailed pharmacokinetic analysis (16 timepoints) was performed in 30 patients treated with a small bolus dose (3 mg/m2) of vinblastine. A model for the total AUC was developed by multiple linear regression, using the first 15 patients as the training data set: AUC = 38.0 C10 + 73.8 C36 - 12.9, where C10 and C36 represent the serum vinblastine concentration at 10 hours and 36 hours, respectively (r = 0.99, P less than 0.0001). The model was validated on the other 15 patients, the test data set (r = 0.94, P less than 0.0001), with a mean predictive error of 13%. Limited sampling models may facilitate large-scale pharmacodynamic studies of new anticancer drugs, in order to relate the estimated AUC to toxicity and/or response without the need for detailed pharmacokinetic analysis.

Multidrug-resistant clones of a drug-sensitive human malignant melanoma cell line were isolated by single-step selection in culture medium containing either vincristine (4.5 ng/ml or 7.5 ng/ml), vinblastine (3 ng/ml), or colchicine (8 ng/ml). This protocol yielded primary colonies showing relatively low (4- to 24-fold) levels of drug resistance. These clones exhibit the classical multidrug resistance (MDR) phenotype, being cross-resistant to Vinca alkaloids, anthracyclines, colchicine, and actinomycin D. The appearance of an MDR phenotype in these cells was linked to a decreased accumulation and increased efflux of the drug [3H]vinblastine when compared to the drug-sensitive melanoma cell line. This increased drug efflux was dependent on the presence of cellular ATP and could be reduced by treatment of the cells with rotenone and deoxyglucose. A partial human mdr complementary DNA clone was used to monitor the degree of amplification and the level of transcription of this gene in the cloned lines. All 5 MDR sublines expressed increased levels of the specific 4.5-kilobase mdr mRNA, but did not show mdr gene amplification. Our results indicate that relatively low levels of drug resistance, similar to those observed clinically and in experimental xenografts, can be achieved by single-step drug selection and result from increased expression of at least one member of the mdr gene family.

Drugs that disorganize or overstabilize cytoplasmic microtubules (colchicine, vinblastine, griseofulvin, or taxol) can at certain concentrations totally block proliferation of SV40 and polyoma virus transformants with only a minimal effect on the proliferation of the parental rat 3T3 cells. This difference in sensitivity is not due to a more active drug uptake by transformed cells. Examination of cytoplasmic microtubules in actively proliferating normal or transformed cells reveals two categories in each case: cells with microtubules and cells without distinct microtubules. The proportion of cells without distinct microtubules did not differ much between normal and transformed cells. However, transformed cells with a clear microtubule network appear to have fewer microtubules than normal cells. This may contribute to the higher sensitivity of transformed cells. These results render even more rational the use of antimicrotubule drugs in cancer chemotherapy.

To examine the effect of vinblastine on the movement of calcium and macromolecules through the enamel organ in the secretion zone and through the odontoblast layer, 45CaCl2 and [125I]-insulin were used as radioautographic tracers. Vinblastine did not alter the localization of either labelled Ca or insulin in the enamel organ and underlying enamel, but eliminated both labels in the pulp, odontoblasts and dentine. It is concluded that vinblastine has no effect on the passage of Ca and macromolecules in the enamel organ and secretory ameloblast layers, whereas its effect on the pulp and odontoblasts prevents passage of these tracers into the predentine and dentine.

Eight lines of human breast tumors implanted in nude mice were treated with various antitumor agents at two different doses, maximum tolerated doses (MTD) and rational doses (RD) that were pharmacokinetically equivalent to the clinical doses; the response rates to both doses were compared. With MTD, the response rates to mitomycin C and vinblastine were 100%, and those to other agents including cyclophosphamide, nimustine (a water-soluble nitrosourea), vincristine, Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH), 5-fluorouracil (5-FU), and methotrexate were 30%-50%, indicating high responsiveness to the former two agents. In contrast, when the RD were used, the response rates to the majority of these agents were 25%-40%, and those to vincristine and nimustine were 13% and 0%, respectively. These results agree with the reported clinical results compared with those with MTD, suggesting the importance of the use of clinically equivalent doses in the evaluation of antitumor efficacy in a human tumor/nude mouse system.

To reproduce clinical effects of various antitumor agents in the human tumor/nude mouse model, we investigated the responsiveness of 11 lines of human gastric tumor xenografts to doses of the agents pharmacokinetically equivalent to the respective clinical doses, which we designated the "rational dose" (RD). We found that the response rates to mitomycin C, 3-[(4-amino-2-methyl-5-pyrimidinyl]methyl-1-[2-chloroethyl]-1- nitrosourea (ACNU), adriamycin, 5-fluorouracil were 18%, and that to vinblastine was 30%; on the other hand, those to vincristine, methotrexate, and cyclophosphamide were poor. In contrast, in our previous study using the maximum tolerated doses, response rates to mitomycin C, ACNU, and vinblastine were as high as 64-82%, and those to adriamycin and 5-fluorouracil were 18%. When these results were compared with the clinical response rates of gastric tumors, as a whole, the results with RD's exhibited much better coincidence with the clinical data in terms of relative therapeutic potency, indicating the validity of the use of clinically equivalent doses instead of maximum tolerated doses in the human tumor model.

The purpose of present study was to analyze the factors involved in the tissue distribution of vinblastine (VBL). The specific binding of VBL to mouse tissue cytosol determined by a charcoal method correlated well with the tissue concentration of tubulin, a target protein for the pharmacological activity of Vinca alkaloids. The calculated tissue-to-plasma partition coefficients (Kp) in various tissues, based on the VBL binding to 100000 x g cytosols showed a good correlation with the corresponding in vivo Kp values of rats reported in the literature, however, the calculated Kp values were greatly underestimated. The total binding (including specific and non-specific bindings) to cytosols from the liver and kidney, determined with the ultrafiltration method, were approximately 5 times higher than those determined with the charcoal method for both tissues. However, the total bindings to cytosol cannot explain the high Kp values in vivo. Considering the intracellular distribution of VBL, it was found that cytosol is not the main binding component for VBL since approximately one-half of the VBL in the liver homogenate was associated with the nuclear fraction. The Kp value in the liver, calculated by considering the intracellular distribution, became close to the in vivo Kp value. It was concluded that the tissue distribution of VBL cannot be accounted for only by the binding to tubulin, and that the bindings to other intracellular components should also be included.

The reversed-phase high-performance liquid chromatography with electrochemical detection was used to quantify plasma and urine levels of vinblastine, vincristine, vindesine and a metabolite of vinblastine, desacetylvinblastine. Sample clean-up consisted of solid-phase extraction with a Bond Elut CN column. The extracts were separated on a Hypersil ODS column. The mobile phase consisted of a mixture of methanol and 10 mM phosphate buffer (pH 7.0). The limit of sensitivity using electrochemical detection was 100 pg on-column for all compounds with a signal-to-noise ratio of 3. Quantification of the compounds in human plasma and urine was possible down to 1 ng/ml (ca. 1 pmol). Pharmacokinetic results show that the sensitivity of the method is adequate for drug monitoring in clinical research.

Resistance to etoposide, which was expressed following exposure of a human tumour cell line (HN-I) to fractionated X-irradiation (II fractions to a total dose of 50Gy), was found to be exhibited after delivery of only 5 fractions (total dose of 22.5Gy). In addition, 2 new etoposide-resistant sublines of these HN-I cells have been developed by continuous exposure in vitro to sublethal drug concentrations. No significant differences in growth characteristics were shown between all these resistant sublines and the parental line. The drug-treated line, HN-I/VP-2, expressed cross resistance to vincristine, adriamycin and daunomycin, and marginal cross resistance to vinblastine and cisplatin. The X-irradiation-treated subline (HN-I/DXR-II) also proved cross-resistant to vincristine and marginally cross-resistant to vinblastine, but showed unaltered responses to adriamycin and daunomycin, and expressed marginal collateral sensitivity to cisplatin. Comparisons of drug-uptake characteristics showed that only the HN-I/VP2 cells and not the HN-I/DXR-II cells had reduced uptake of vincristine, vinblastine and daunomycin. However, etoposide uptake was not altered in either resistant subline. Further investigations have shown that the approximately 4-fold level of resistance to etoposide in these HN-I/VP-2 and HN-I/DXR-II cells was associated with a reduction in etoposide-induced DNA single-strand breakage. However, repair of these lesions, after drug removal, was rapid and similar in the parental and drug-resistant sublines, with 50% having resealed within 20-26 min. Resistance to etoposide was also associated with significantly elevated (p less than 0.01) glutathione peroxidase activity in both sublines, whilst glutathione S-transferase activity was marginally elevated (117%) only in the HN-I/DXR-II cells. There were no significant alterations in total glutathione levels. These results suggest that not only do patterns of response to anti-tumour drugs differ depending upon the agent employed to "induce" resistance, but that multiple mechanisms appear to be associated with these altered responses.

Eleven patients with advanced cancer received navelbine (15 mg/m2) as a single i.v. bolus injection. At least 1 week later, the patients were given a 2-fold increased dose of navelbine (30 mg/m2) and, for seven of them, the 30-mg/m2 dose was repeated after a delay longer than a week. After each administration, plasma and urine were collected for 72 h and monitored for navelbine concentration by radioimmunoassay. The comparison of dose-normalized plasma level profiles showed significant time dependence (P less than 0.05) in four of the seven assessable patients. Some patients also exhibited significant (P less than 0.05) nonlinear (dose dependent) kinetic profiles. Only 3 of the 10 appreciable patients were characterized by both time independent and linear profiles. However, the plasma concentration decay curves presented a triphasic shape similar to that obtained with other antitumor Vinca alkaloids and the data were consistent with a three-compartment pharmacokinetic model. The dose and/or time dependence evidenced for most of the patients did not result in marked changes in pharmacokinetic parameters among courses. The pharmacokinetics of navelbine were characterized by a high plasma clearance (0.27 to 1.49 liter.h-1.kg-1), a large distribution volume (8.2 to 48.2 liter.kg-1), and a long terminal half-life (22.1 to 67.8 h). Urine excretion was low (less than 7.9%). Thus, navelbine pharmacokinetics resembles that of other antitumor Vinca alkaloids.

Vinca alkaloids (VA) represent a family of closely related molecules, which includes vincristine (VCR), vinblastine (VLB), vindesine (VDS) and navelbine (NVB), a new synthetic VA presently in phase II clinical trial. Development of sensitive and specific analytical tools (polyclonal and monoclonal antibodies) enabled us to investigate the kinetic behavior of VDS and NVB after IV bolus or long term administration. Following IV bolus injection, the mean pharmacokinetic parameters are: total plasma clearance: 0.53 l/h-1kg-1, 0.72 l/h-1kg-1 and apparent elimination half-life: 23.2 h, 39.5 h for VDS and NVB, respectively. Chronic treatment reveals time- and dose-dependence relationships and detailed observations of individual kinetics demonstrate an important interindividual variability for both drugs. Renal excretion of VDS and NVB is low (from 5 to 12% of the total dose), suggesting the important role of the liver in their rapid elimination.

The dimeric Vinca alkaloid vinblastine (VLB) undergoes metabolic transformation to three products in a reaction catalyzed by the human serum copper oxidase ceruloplasmin. The enzyme reaction requires chlorpromazine as a shuttle oxidant, and the course of the oxidation reaction appears to be subject to the nature of the shuttle oxidant used. Preparative-scale incubations have resulted in the isolation of three products, which were characterized by chemical and spectral analyses. The metabolites were identified as the ring fission product catharinine, obtained by oxidation of the Iboga ring system; an enamine/ether derivative obtained by oxidation of the Aspidosperma portion of VLB; and a metabolite embodying the same structural changes in both parts of the vinblastine dimeric structure. Catharinine is identical with the product of VLB oxidation obtained by peroxidase oxidation. The other two products are new metabolites and are derivatives of VLB. All of the metabolites are less active than VLB when tested in vitro vs the human T-cell leukemic cell line (CRFF-CEM).

The effect of interferon (IF) on the activity of multipurpose hepatic oxidases (MHO) in animals in the process of metastatic tumour development as well as interaction of IF and vinblastine (VBL) in the cell culture have been studied. IF activates MHO under conditions of their inhibition at the beginning of the tumour development and inhibits them to the normal level under activation in the postmetastatic period. Contrary to the data obtained in vivo IF does not decrease the toxicity of VBL for the tumour cells in vitro. The data obtained evidence for the modulating effect of IF on the MHO system under the tumour development.

The clinical pharmacokinetics of vinblastine administered by continuous 5-day infusion (3 mg/m2/day) was studied in 12 patients with primary testicular cancer. Serum vinblastine concentrations were determined by radioimmunoassay on serum collected over a 10-day period. Steady-state vinblastine concentrations were achieved within 60 to 108 hours (median, 72 hours). Vinblastine pharmacokinetics were analyzed and correlated to hematologic and nonhematologic toxicity. Hematologic toxicity was severe (granulocytopenia of less than 500/microL) in all patients; however, no correlation of vinblastine pharmacokinetics to duration of granulocytopenia or nadir was noted. Nonhematologic toxicity, however, showed a direct correlation to steady-state vinblastine concentrations. Two distinct groups of patients were identified by a toxicity score evaluating nonhematologic toxicity: as low (group A) or high (group B). The toxicity score was calculated for each patient based on accumulated toxicity during the course of treatment. The mean toxicity score for all patients was 7.11 and for groups (A and B) it was 4.0 and 9.6, respectively (P = .02). Steady-state vinblastine concentration for each patient was compared with toxicity where the mean steady-state vinblastine concentration was 7.3 ng/mL for all patients, and 5.8 ng/mL and 8.5 ng/mL for groups A and B, respectively (P = .01). These steady-state vinblastine concentrations correlated directly with the mean toxicity scores revealing that patients with high steady-state vinblastine concentrations demonstrated more nonhematologic toxicity. Application of these data to pharmacokinetically directed studies are warranted to investigate this relationship and designate dosages of vinblastine to avoid excessive toxicity.

The conjugate, KS1/4-DAVLB, of the murine monoclonal antibody KS1/4 with the vinca alkaloid 4-desacetylvinblastine (DAVLB) was administered intravenously to rats and monkeys. Terminal plasma half-life (t1/2) values were measured as radioequivalents and as functionally immunoreactive antibody conjugate after dosing with KS1/4-[3H]DAVLB. The t1/2 values, determined radiometrically, were 145 hr and 62 hr in male rats after 10 and 100 mg/kg doses and 92 hr and 90 hr in male and female monkeys after a 40 mg/kg dose. Comparable results were obtained when the functionally immunoreactive conjugate concentrations were determined by an enzyme-linked immunosorbent assay technique. The ratio of 35S:3H in the plasma after dosing rats with 100 mg/kg [35S]KS 1/4-[3H]DAVLB remained reasonably constant during 336 hr. Less than 1% of the total vinca alkaloid equivalents present in the plasma at any time could be extracted as free vinca species; the major vinca alkaloid metabolities present at early time points were hemisuccinate derivatives of DAVLB, whereas, at later times, DAVLB and its N-oxide were equally as concentrated. The major pathway of elimination was fecal with about one-half of the administered radioactivity cleared in 150-250 hr. After dosing with [35S]KS 1/4-[3H]DAVLB, the ratio of 35S:3H radioactivity in the bile was substantially less than that in the plasma. Evaluation of radioactivity eluted from the bile by size-exclusion HPLC showed that almost all of the tritium was associated with material of lower molecular weight than that of KS 1/4-DAVLB.(ABSTRACT TRUNCATED AT 250 WORDS)

A study was designed to investigate the fate of navelbine (NVB) and its excretion routes in two cancer patients treated with tritiated NVB (30 mg/m2) by i.v. bolus injection. Plasma and red blood cell concentrations and urine and stool elimination were monitored over long periods of time. NVB plasma and urine concentrations were measured by both radioimmunoassay (RIA) and direct radioactive (RA) determination. Samples were also analyzed by high-performance liquid chromatography to evaluate the importance of NVB metabolism. Whereas the major excretion route for NVB was the stool (from 34% to 58.4% of the total dose given over 21 days), urinary excretion was low (about 21% within the same time period), corresponding mainly to that of unchanged drug. Thus, a good correlation was found between RIA and RA determinations in urine. In contrast, plasma area under the curve (AUC) values obtained after RA and RIA analysis differed markedly (AUC RIA/AUC RA = 0.23-0.31), demonstrating that a significant proportion of the plasma-circulating drug was biotransformed, mainly during the last elimination phase. This could have important pharmacological and toxicologic implications in clinical practice.

Vinorelbine (Navelbine) is a new semisynthetic vinca alkaloid which chemically differs from vinblastine by substitutions on the catharantine moiety of the molecule. It has shown promising experimental antitumor activity against experimental murine tumors as well as continuous cell lines of human neoplastic origin and human tumor xenografts in nude mice. Acute subacute and chronic toxicity extensively studied in rodents, dogs and primate has shown that hematotoxicity was almost the sole side-effect; neurotoxicity appears very limited. Almost exclusive affinity of NVB for mitotic tubulin and tubulin associated protein accounts for this pattern of toxicity. Phase I and II studies have been conducted in humans. Dose limiting side-effect appears to be neutropenia: the drug is slightly emetogenic, induces little alopecia, almost no neurotoxicity, and no other toxicity. Although preliminary, results of phase II studies already suggest significant activity of NVB in non small lung cancer (33% response rate in 78 evaluable patients), advanced breast cancer (53% response rate in 33 pts without significant chemotherapy for the target progression) and Hodgkin's disease (90% response rate after 4 weekly courses in 31 pts). Thus extensive pharmacological studies and ongoing clinical studies confirm that chemical modifications of the catharantine moiety of vinca alcaloid can lead to active agents with broader spectrum of activity and easily manageable side effects.