HPLC-UV and GC-MS Methods for Determination of Chlorambucil and Valproic Acid in Plasma for Further Exploring a New Combined Therapy of Chronic Lymphocytic Leukemia.

High performance liquid chromatography with ultra-violet detection (HPLC-UV) and gas chromatography-mass spectrometry (GC-MS) methods were developed and validated for the determination of chlorambucil (CLB) and valproic acid (VPA) in plasma, as a part of experiments on their anticancer activity in chronic lymphocytic leukemia (CLL). CLB was extracted from 250 µL of plasma with methanol, using simple protein precipitation and filtration. Chromatography was carried out on a LiChrospher 100 RP-18 end-capped column using a mobile phase consisting of acetonitrile, water and formic acid, and detection at 258 nm. The lowest limit of detection LLOQ was found to be 0.075 µg/mL, showing sufficient sensitivity in relation to therapeutic concentrations of CLB in plasma. The accuracy was from 94.13% to 101.12%, while the intra- and inter-batch precision was ≤9.46%. For quantitation of VPA, a sensitive GC-MS method was developed involving simple pre-column esterification with methanol and extraction with hexane. Chromatography was achieved on an HP-5MSUI column and monitored by MS with an electron impact ionization and selective ion monitoring mode. Using 250 µL of plasma, the LLOQ was found to be 0.075 µg/mL. The accuracy was from 94.96% to 109.12%, while the intra- and inter-batch precision was ≤6.69%. Thus, both methods fulfilled the requirements of FDA guidelines for the determination of drugs in biological materials.

Spray-Dried Silica Xerogel Nanoparticles as a Promising Gastroretentive Carrier System for the Management of Chemotherapy-Induced Nausea and Vomiting.

PURPOSE: The current work aimed to develop spray-dried silica xerogel nanoparticles (SXNs) as a gastroretentive carrier for the dual delivery of chlorambucil (CHL) and granisetron hydrochloride (GR). As a low-density system, it was proposed to float over gastric fluids; allowing for the retention of CHL in the acidic medium where it is more stable while ensuring the solubility of GR. METHODS: Silica xerogels were developed by sol-gel process, using Tetraethyl orthosilicate (TEOS) water and acetic acid, followed by spray drying. SXNs were evaluated for particle size, zeta potential, entrapment efficiency (EE%), CHL and GR release after 1 hr (P1h) and after 8 hrs (P8h). The best achieved system (SXN4) was evaluated for morphology, pore diameter, total porosity, bulk density, wetting time, floating characteristics. Furthermore, the pharmacokinetics of the loaded drugs were evaluated in rats; relative to an aqueous CHL suspension containing GR. RESULTS: SXN4 system had the highest desirability (0.69); showing spherical nanoparticles (181.63 nm), negative zeta potential (-5.18 mV), promising EE% of 59.39% and 73.94% (for CHL and GR, respectively) and sustained CHL and GR release profiles characterized by low P1h (22.75% and 30.74%) and high P8h (60.36% and 99.33%), respectively. It had a mean pore diameter of 8.622 nm, a total porosity of 62.27%, a bulk density of 0.605 g/mL, a wetting time of 292 sec, zero lag time and a floating duration of at least 8 h. CONCLUSION: The prolongation in the mean residence time (MRT(0-∞)) and the promotion of the relative oral bioavailabilities of both drugs could unravel the potential of this system for the management of chemotherapy-induced nausea and vomiting.

In vitro effect of chlorambucil on human glioma cell lines (SF767 and U87-MG), and human microvascular endothelial cell (HMVEC) and endothelial progenitor cells (ECFCs), in the context of plasma chlorambucil concentrations in tumor-bearing dogs.

The objective of this study was to investigate a possible mechanism of action of metronomic chlorambucil on glioma by studying the in vitro cytotoxicity and anti-angiogenic effects on glioma and endothelial cells, respectively. The in vitro LD50 and IC50 of chlorambucil were determined using human SF767 and U87-MG glioma cell lines, human microvascular endothelial cells (HMVECs) and human endothelial colony forming cells (ECFCs). Results were analyzed in the context of chlorambucil concentrations measured in the plasma of tumor-bearing dogs receiving 4 mg m-2 metronomic chlorambucil. The LD50 and IC50 of chlorambucil were 270 µM and 114 µM for SF767, and 390 µM and 96 µM for U87-MG, respectively. The IC50 of chlorambucil was 0.53 µM and 145 µM for the HMVECs and ECFCs, respectively. In pharmacokinetic studies, the mean plasma Cmax of chlorambucil was 0.06 µM. Results suggest that metronomic chlorambucil in dogs does not achieve plasma concentrations high enough to cause direct cytotoxic or growth inhibitory effects on either glioma or endothelial cells.

Validated LC-MS/MS method for the simultaneous determination of chlorambucil and its prodrug in mouse plasma and brain, and application to pharmacokinetics.

A simple, sensitive and rapid LC-MS/MS method was developed and validated for the simultaneous determination of chlorambucil (CHL) and the prodrug of chlorambucil (CHLS) in mouse plasma and brain tissue. Detection was performed on a Diamonsil ODS chromatography column using gradient elution with a mobile phase of 0.2% aqueous formic acid and acetonitrile. Mass spectrometry was carried out in multiple reaction monitoring mode using a positive electrospray ionization interface. Good linearity was found for CHLS and CHL in plasma and brain tissue in different linear ranges (r>0.9996). Intra-day and inter-day precision was within 9.11% and accuracy was not more than 11.07%. The validated method was successfully applied to the pharmacokinetic study of CHLS and CHL in mice after intravenous administration.

Characterization and in vivo evaluation of novel lipid-chlorambucil nanospheres prepared using a mixture of emulsifiers for parenteral administration.

PURPOSE: The purpose of the study was to develop and evaluate different lipid-based formulations for parenteral administration, as potential novel carrier systems for lipophilic drugs, and to turn an unstable drug such as chlorambucil into a useful one. METHODS: A two-stage, high-pressure homogenizer was used to yield a very fine monodispersed lipid nanosphere. The strategy of combining egg yolk phospholipid and nonionic emulsifier (Lutrol F 68 and Tween 80) as an emulsifier mixture was adopted to increase safety and tolerance. The final lipid nanospheres, in a lipophilic mixture consisting of three components, monostearin, medium-chain triglycerides and soya oil, were evaluated for physicochemical properties, such as particle size, surface morphology, drug-entrapment efficiency, drug-loading capacity, lyophilization and in vivo drug-release behavior. RESULTS: A monodispersed lipid nanosphere with a mean particle size ranging from 90 to 150 nm was achieved. The optimized injectable cryoprotectants for lipid nanosphere were sucrose (7.5%) and mannitol (7.5%), which can stabilize the particle size (LD50) at approximately 129 nm after reconstitution. The results show that the formulation can effectively administer anticancer drugs and thus improve patient quality of life. CONCLUSIONS: The novel lipid nanosphere complex developed is a useful anticancer drug delivery vehicle for parenteral administration. The formulation strategy has the potential for the development of further methods of drug delivery for a wide variety of anticancer drugs.

Pharmacokinetics and pharmacodynamics of chlorambucil delivered in long-circulating nanoemulsion.

Chlorambucil was incorporated into a nanoemulsion modified with poly(ethylene glycol) to improve its pharmacokinetics and tissue distribution, and thus enhance its therapeutic efficacy. A long-circulating nanoemulsion (LNE) was prepared using soybean oil, egg lecithin, cholesterol and PEG(2000)DSPE. The LNE had an oil droplet size <200 nm with a surface charge of -32.2 to -35.6 mV. Approximately, 97% of the chlorambucil was encapsulated in the LNE. Intravenous (i.v.) administration of the chlorambucil LNE to C57 B/6 mice showed improved pharmacokinetic parameters with 1.4-fold higher area under the plasma concentration-time curve (AUC) and 1.3-fold longer half-life compared to a non-PEG-modified nanoemulsion, and 2.7-fold higher AUC and 7.6-fold longer half-life compared to chlorambucil solution. Tissue distribution studies after i.v. administration with LNE showed a considerable decrease in drug uptake in the reticulo-endothelial system containing organs compared to non-PEG-modified nanoemulsion. Additionally, the chlorambucil delivered in LNE significantly enhanced therapeutic efficacy in the subcutaneous colon-38 adenocarcinoma tumor mouse model with no apparent increase in toxicity. This study suggests that LNE could produce remarkably improved pharmacokinetic profile and therapeutic efficacy of chlorambucil compared to non-PEG-modified nanoemulsion and solution.

Rapid determination of the anti-cancer drug chlorambucil (Leukeran) and its phenyl acetic acid mustard metabolite in human serum and plasma by automated solid-phase extraction and liquid chromatography-tandem mass spectrometry.

A bioanalytical method for the determination of the anticancer drug chlorambucil (Leukeran) and its phenyl acetic acid mustard metabolite in human serum and plasma is described. Automated solid-phase extraction of the analytes is carried out with C18 sorbent packed in a 96 well format microtitre plate using a robotic sample processor. The extracts are analysed by isocratic reversed-phase liquid chromatography using pneumatically and thermally assisted electrospray ionisation (TurboIonspray) with selected reaction monitoring. The method is specific and sensitive, with a range of 4-800 ng/ml in human serum and plasma for both parent drug and metabolite (sample volume 200 microl). The method is accurate and precise with intra-assay and inter-assay precision (C.V.) of <15% and bias <15% for both analytes. The automated extraction procedure is significantly faster than manual sample pre-treatment methods, a batch of 96 samples is extracted in 50 min allowing for faster sample turnaround. The method has been used to provide pharmacokinetic support to biocomparability studies of Leukeran following single doses of oral tablet formulations.

Kinetics of chlorambucil in vitro: effects of fluid matrix, human gastric juice, plasma proteins and red cells.

The mechanisms involved in the bioavailability of chlorambucil or 4-[p-(bis[2-hydroxyethyl]amino)phenyl]-butyric acid are poorly understood. The effects of different matrices on the disintegration of chlorambucil were investigated by HPLC, 1H NMR, 31P NMR, and mass spectrometry. Cellular incorporation and protein binding of the drug in vitro was assessed with [3H]-chlorambucil. Decomposition of chlorambucil and its major metabolite, phenylacetic acid mustard, to mono- and dihydroxy derivatives, was significantly faster in water than in PBS, (phosphate-buffered saline, pH 7.4). The hydrolysis of chlorambucil was as fast in plasma ultrafiltrate as in PBS; plasma proteins, preferentially albumin, prevented this disintegration. In phosphate-buffered media, two additional stabile hydrolysis products were found which were characterised as the mono- and bis-phosphates of 4-[p-(bis[2-hydroxyethyl]amino)phenyl]butyric acid, results of the reaction of nucleophilic buffer species with the aziridinium ion intermediates. Chlorambucil bound covalently to plasma proteins and was incorporated into red cells. These interactions are likely to have a significant role in vivo, reducing the bioavailability of the drug. High H+ concentration associated with high chloride concentration in human gastric juice had a stabilizing effect on chlorambucil. Incorporation of [3H]-chlorambucil into red cells was inhibited in a concentration-dependent fashion by whole human plasma as well as by albumin. We conclude that the chemico-biological interactions demonstrated in the present investigation provide explanations for the remarkable pharmacokinetic differences observed intra- and inter-individually in the clinical use of chlorambucil. The present information is important, when clinical or in vitro evaluation of efficacy and bioavailability of chlorambucil is considered.

A pharmacokinetic study of prednimustine as compared with prednisolone plus chlorambucil in cancer patients.

The pharmacokinetic characteristics of prednisolone and of chlorambucil and its beta-oxidized metabolite, phenylacetic mustard (PAM) were studied in plasma after the oral administration of 200 mg prednimustine (Sterecyt) and a regimen consisting of 20 mg prednisolone plus 20 mg chlorambucil, respectively. A total of 12 cancer patients completed this trial. The drugs were given in a cross-over study as single doses, and serial plasma samples were collected for 32 h. Chlorambucil and PAM were assayed by a gas chromatographic/mass spectrometry method and prednisolone, by radioimmunoassay. The median relative availability of the prednisolone and chlorambucil moiety in prednimustine was 19% and 16%, respectively. Prednisolone, as well as chlorambucil and PAM, appeared later and at a significantly lower concentration in plasma after treatment with prednimustine as compared with the mixture of chlorambucil and prednisolone. We also found that the elimination phase of chlorambucil and PAM in plasma is prolonged after the administration of prednimustine as compared with chlorambucil per se. In contrast, the elimination of the prednisolone moiety of prednimustine and that following the administration of a plain prednisolone tablet did not seem to differ. The modified plasma profile of the alkylating components following prednimustine administration may be important for the clinical efficacy of prednimustine.

Studies on the pharmacokinetics of chlorambucil and prednimustine in patients using a new high-performance liquid chromatographic assay.

Following the oral administration of either chlorambucil/prednisolone or prednimustine to patients, the plasma levels of free chlorambucil and phenylacetic acid mustard, the beta-oxidation product of chlorambucil, were measured using a new high-performance liquid chromatographic (HPLC) assay. This assay permitted the simultaneous detection of the analyzed compounds with a lower limit of detection of 30 ng/ml. The pharmacokinetics of chlorambucil and phenylacetic acid mustard were found to be entirely different when prednimustine was administered as opposed to its components chlorambucil and prednisolone together. After the ingestion of the conjugate, the plasma concentration-time curves of chlorambucil and phenylacetic acid mustard showed a "delayed" pattern compared with those obtained after the administration of the components. The mean area under the concentration-time curves (AUCs) of prednimustine-derived chlorambucil and phenylacetic acid mustard were 25% and 40%, respectively, of the areas obtained after a stoichiometrically equivalent dose of chlorambucil. Free plasma prednimustine could not be detected at any time. This different pharmacokinetic behavior might offer an explanation for the superior therapeutic effects of prednimustine demonstrated by clinical studies.

Inter- and intraindividual differences in oral chlorambucil pharmacokinetics.

Inter- and intraindividual variation in the pharmacokinetics of oral chlorambucil was investigated in patients with chronic lymphocytic leukaemia. Five patients were given in randomized order 15, 40, 60 and 70 mg chlorambucil p.o. and plasma was analyzed for chlorambucil and its cytotoxic metabolite phenyl acetic acid mustard. The area under the plasma concentration-time curve of chlorambucil varied two- to fourfold between patients at each dose level. The AUC of the metabolite was higher and showed twofold interindividual variation. The intraindividual dose-corrected AUCcs also varied twofold. No dose dependency of the dose corrected AUC was seen either for chlorambucil or the metabolite. Elimination of both compounds form plasma was rapid, with half-lives of 1.01 and 1.94, respectively.

[A new anti-cancer agent, bestrabucil (KM 2210): I. Concentration in the serum and tissue in urogenital tumors].

Serum and tissue concentrations of bestrabucil (KM 2210), a combined agent of 17-estradiol and Chlorambucil, were examined in patients with urogenital cancers including those of the kidney, bladder, prostate and testis. We administered orally 100mg (50 mg X 2/day of bestrabucil for 3 days), and determined its plasma levels and metabolites. A maximum drug concentration, i.e., 9.25 ng/ml, was noted 3 hours after administration; a constant plasma level of 5 ng/ml was maintained and the concentration of free chlorambucil was low. After single or consecutive oral administration of bestrabucil (100-300 mg), tumor specimens contained significantly large amounts of bestrabucil in comparison with adjacent normal tissue. Selective accumulation of the active component in tumor tissue suggests the clinical usefulness of bestrabucil.

Can food affect the bioavailability of chlorambucil in patients with haematological malignancies?

Pharmacokinetic studies in ten patients with haematological disorders were undertaken on the first and second days of one course of chemotherapy. Patients received chlorambucil under fasting and non-fasting conditions. Plasma concentrations of chlorambucil were determined by a reversed-phase high-performance liquid chromatography assay. Statistical analysis by the Wilcoxon signed rank test for non-parametric data indicated that food caused a significant reduction in peak plasma levels (P less than 0.01), elimination rate constants (P less than 0.01) and area under the plasma chlorambucil/time curve (P = 0.01). Food was also found to prolong the time taken to attain peak plasma levels (P less than 0.01). Regression analysis of renal function with elimination rate constants showed that chlorambucil elimination was independent of renal function (n = 8; r = -0.007; P = 0.72). In view of these results we suggest that chlorambucil is given on an empty stomach.

Drug level monitoring: cytostatics.

The present review on the quantification of cytostatic drugs has mainly been focussed on chromatographic techniques. Special attention has been paid to the precautions that have to be taken into account to ensure the selectivity and accuracy of the various methods. The various cytostatics that have been dealt with are: alkylating agents, antimetabolites, vinca alkaloids, antibiotics, cis-diamminedichloroplatinum, podophyllotoxine derivatives, and nitrosoureas.

[Bestrabacil: a possible target-oriented anticancer agent].

Bestrabucil, the benzoate of an estradiol-chlorambucil conjugate, was initially developed as a target-oriented anticancer agent for breast cancers with positive estrogen receptors, by conjugating a tumoricidal agent, chlorambucil, to a vehicle, estradiol. Further studies, however, revealed that regardless of the presence of estrogen receptors, bestrabucil selectively accumulates in malignant tumor cells. The unique feature of bestrabucil, selective affinity to tumor cells, was demonstrated in in vitro and in vivo studies. Significantly larger amounts of 3H-bestrabucil accumulated in malignant cells (3T3-SV40 transformed) than in normal cells (3T3). In vitro inhibition effects of bestrabucil on cell growth was observed only in malignant cells. Selective accumulation of bestrabucil in malignant tumor tissues was also demonstrated in in vivo experiments. After a single oral administration of 100 mg/kg of bestrabucil to female Wistar rats bearing Walker 256 carcinoma, bestrabucil accumulated significantly occurred in tumor tissues with little or no accumulation in normal tissues and blood. Anti-tumor effects and toxicities of bestrabucil and chlorambucil, were compared using Walker 256 carcinoma. Bestrabucil exerted its antitumor effects with little change in leucocyte counts in the peripheral blood, whereas chlorambucil showed significant side effects. Finally, selective accumulation of bestrabucil in malignant tumor tissues, was demonstrated clinically. Tumor specimens obtained during the operation of patients with various types of cancer, 24 hours after oral administration of 100 mg of bestrabucil, contained significantly larger amounts of bestrabucil compared with adjacent normal tissues. Clinical trials of bestrabucil are being carried out at present. Bestrabucil seems to be a promising target-oriented anticancer agent and deserves further investigation.