Milan Slavik

Pyridoxine therapy for palmar-plantar erythrodysesthesia associated with continuous 5-fluorouracil infusion

SummaryThe limiting toxicity of low dose continuous infusion 5-fluorouracil (200–300 mg/m2/day) is often palmarplantar erythrodysesthesia (PPE). PPE developed in 16/25 patients (exact 95% confidence interval of 42% –82%) with metastatic colon cancer enrolled in a phase II trial. In this trial, 5-FU was given continuously at a dose of 200 mg/m2/day until toxicity or progressive disease forced discontinuation.The first signs of the syndrome developed at a median of 2 months following infusion initiation and, unless treatment was interrupted, became progressively worse. The incidence of moderate to severe PPE was 71% in the 14 previously untreated patients (exact 95% confidence intervals of 42–92%). Seventy-eight percent of the responders in the no prior treatment group developed PPE. The incidence of moderate to severe PPE was only 27% in the 11 previously treated patients (exact 95% confidence intervals of 6–61%). The higher incidence of PPE in the previously untreated patients probably resulted from a longer total infusion time (median = 7.3 months) than the previously treated (median = 4.5 months). The longer infusion time in turn was a result of the higher response rates (64 vs 18%) in the previously untreated versus treated groups.Five previously untreated patients who developed PPE received 50 or 150 mg of pyridoxine/day when moderate PPE changes were noted. Reversal of PPE without interruption of the 5-FU was seen in 4/5 patients. Four of these patients who received pyridoxine had responded to 5-FU treatment. No adverse affect of pyridoxine on clinical response was noted.The five previously untreated patients who received pyridoxine for PPE continued 5-FU for a median of 6 months after development of the syndrome. The six previously untreated patients who did not receive pyridoxine when they developed PPE were able to continue 5-FU for a median of only 2.5 months after development of the syndrome. A similar number of clinical responders to 5-FU were present in both groups.Considering the high incidence of PPE and response in previously untreated colon cancer patients who receive protracted continuous 5-FU, prophylactic pyridoxine in conjunction with this treatment modality might be useful.

Spirogermanium: A new investigational drug of novel structure and lack of bone marrow toxicity

SummarySpirogermanium (NSC 192965) is a new metallic investigational anticancer drug of novel heterocyclic structure. Although its mode of action has not been fully elucidated, it appears that spirogermanium is not a phase or cell cycle specific drug and inhibits DNA, RNA and protein synthesis, the protein synthesis being the most susceptible to this agent. Spirogermanium has shown cytotoxic activity in vitro against several human tumor cell lines at concentrations (1 μg/ml) that were also found toxic to the cultured rat neurons. Although spirogermanium has no effect on normal bone marrow colony forming cells in mice, dogs, or man, it has revealed cytotoxic activity in vitro against human myeloid leukemia cell line K 562 at clinically achievable concentrations. These in vitro findings, indicating selective cytotoxic activity against leukemic cells suggest this drug as a candidate for clinical studies in acute and chronic leukemias.Spirogermanium has revealed activity in vivo against intraperitoneally implanted Walker 256 sarcoma, 13762 mammary adenocarcinoma, and 11095 prostatic carcinoma in rats, but no antitumor activity in vivo was found in the murine tumors used in the past by the NCI screen (L 1210 and P 388 leukemia, B 16 melanoma, Lewis lung carcinoma).Spirogermanium is remarkable for its lack of bone marrow toxicity confirmed in preclinical toxicology and clinical studies; moderate, predictable, and reversible CNS toxicity is dose-limiting. Activity in malignant lymphoma, ovarian cancer, breast cancer, large bowel cancer, and prostatic cancer was reported in the clinical studies. The drug is currently under clinical investigation against the wide spectrum of solid tumors and malignant lymphomas.The dose of 80–120 mg/m2, given by 60′ infusion three times a week, is currently used and tolerated in Phase II clinical studies.The recently introduced five days continuous infusion schedule has been also under clinical investigation and the doses of 250–300 mg/m2/day are recommended for Phase II studies.Of interest are results reported in this paper of spirogermanium in vitro preferential activity against the resistant strains of Plasmodium falciparum at clinically achievable concentrations suggesting this drug as a possible new antimalarial agent of novel structure.

High sensitivity ELISA determination of taxol in various human biological fluids

Taxol (paclitaxel)--the natural product isolated from Pacific yew (Taxus brevifolia)--is a novel agent with high activity in the treatment of patients with several malignant tumors including those resistant to other cytotoxic drugs. The therapeutic index of this promising anticancer drug could be further increased by the exploration of its pharmacokinetic pharmacodynamic relationship in cancer patients. Since taxol is highly protein bound, a very specific and highly sensitive analytical method is required in order to determine free, protein unbound and biologically active taxol species in human physiological fluids: plasma; plasma ultrafiltrate; and salivary fluids. In order to accomplish this, a new indirect competitive enzyme-linked immunosorbent assay (ELISA), for quantitating such a low bioactive taxol concentration level, has been developed in our laboratories. This method uses taxol competitive inhibition of mouse anti-taxol antibodies binding to the solid phase coated antigen 7-succinyltaxol-bovine serum albumin. This indicates recognition of the active taxol in the solution phase, where a diluted horseradish peroxidase labeled goat anti-mouse enzyme conjugate is used. While employing this technique, after systematic optimization of the experimental conditions, we are able to detect the anticipated taxol in plasma ultrafiltrate and salivary fluids at the concentration level of subpicogram per milliliter. The working range of the assay is approximately five orders in magnitude, i.e. from pg ml(-1) to 100 ng ml(-1). The clinical part of this study verified the working range of the ELISA method using samples of physiological fluids from a cancer patient treated with 3 h intravenous (i.v.) infusion of this drug. Our results of taxol determination in plasma, plasma ultrafiltrate and saliva demonstrate the applicability of the newly developed ELISA method for further pharmacokinetic studies of free, biologically active taxol species in cancer patients.

Phase I trial and pharmacokinetic study of intravenous and oral α-Difluoromethylornithine

Eflornithine-HCl (α-difluoromethylornithine or DFMO), an irreversible inhibitor of ornithine decarboxylase, blocks polyamine synthesis and has demonstrated antitumor activity in cell culture and animal tumor models. This phase I study was designed to determine and compare toxicity and the maximally tolerated dose of a 4-day course of DFMO given to patients in oral, continuous intravenous infusion or pulse intravenous infusion forms. Twenty-four patients were entered into this study: 8 received intravenous pulse drug, 10 intravenous continuous infusion of drug, and 6 oral DFMO. The most frequent toxicity was nausea and vomiting which occurred in 9 courses of oral drug. Only two patients receiving intravenous DFMO had nausea and vomiting. Clinically significant thrombocytopenia and audiometric abnormalities were not encountered in contrast to previous experience with 28-day courses of oral DFMO. The maximally tolerated dose of a four-day course of oral DFMO was 3.75 gm/M2 every 6 hours. The maximally tolerated dose of intravenous pulse and continuous infusion DFMO was not attained. Pharmacokinetic studies demonstrated that the intravenous schedules achieved higher plasma levels of DFMO than those previously obtained with chronic oral dosing.

Degradation of dacarbazine in aqueous solution

The effects of initial concentration (0.05-5.0 mg ml-1, 2.5 x 10(-4)-0.025 M) (pH 1-13), buffer concentration (0.01-0.075 M), light, antioxidants and co-solvents on the degradation of dacarbazine in aqueous solution were investigated at 37 degrees C. Liquid chromatography was used to monitor the degradation of dacarbazine as well as the appearance of degradation products. The kinetics of hydrolysis of dacarbazine in the dark were pseudo first-order and independent of the initial concentration of the drug. The degradation of dacarbazine was accelerated by light and at low concentration proceeded by pseudo zero-order kinetics. The pH-rate profiles showed that both the photolytic and the hydrolytic reactions were dependent on the ionization state of the molecule. The main degradation product of both hydrolysis and photolysis was detected by liquid chromatography and confirmed by mass spectrometry to be 2-azahypoxanthine.

Plasma analysis of α-difluoromethylornithine using pre-column derivatization with naphthalene-2,3-dicarboxaldehyde/CN and multidimensional chromatography

A procedure for the plasma analysis of alpha-difluoromethylornithine (DFMO) has been developed that utilizes pre-column derivatization with naphthalene-2,3-dicarboxaldehyde/cyanide (NDA/CN) in pH 9.2 borate buffer. Selective derivatization of delta-amine of DFMO followed by quenching of the reaction results in the formation of a cyanobenz [f] isoindole (CBI) derivative that is stable for 24 h. Plasma was prepared for derivatization by a single step procedure which resulted in an ultrafiltrate compatible with derivatization and analysis. The DFMO derivative (CBI-DFMO) was separated from plasma interferences by multidimensional chromatography with an analysis time of 28 min. The response for DFMO in plasma was linear over the range of 2.1 x 10(-8) 2.1 x 10(-6) M after derivatization. This procedure encompasses a useful linear range and offers the advantages of minimal sample preparation and production of a stable fluorophor.

Stereoselective pharmacokinetics and metabolism of the enantiomers of cyclophosphamide : preliminary results in humans and rabbits

[R(+),S(-)]-Cyclophosphamide [(R,S)-CP] is an anticancer drug, containing a chiral phosphorous atom, which is prepared and used clinically as the racemic mixture. A new high-performance liquid chromatographic assay suitable for pharmacokinetic studies of CP enantiomers in plasma has been reported recently by this laboratory (Reid et al., Anal Chem 61: 441-446, 1989). Briefly, the assay involves ethyl acetate extraction of CP enantiomers from plasma followed by derivatization to diastereomers in a two-step process utilizing chloral and (+)-naproxen acid chloride. Chromatographic analysis was performed on a reversed phase (ODS) column with detection at 232 nm. In the present study, preliminary results on the applicability of this assay to pharmacokinetic studies are presented. Several rabbits were used to compare the influence of i.p., i.v., and oral routes of administration on the stereoselective disposition of (R,S)-CP. Following i.p. administration, S-CP was cleared faster than R-CP. Following oral administration, only R-CP was detectable in plasma, while i.v. administration resulted in minor or no stereoselective disposition. These results indicated that there was a marked stereoselective metabolism of the S-CP enantiomer, with the i.p. and oral routes producing the greatest differences due to first-pass metabolism. Incubation of rabbit-liver microsomes with (R,S)-CP demonstrated that the monooxygenase system can exhibit marked stereoselectivity in its metabolism of CP. The ratio of R-CP to S-CP in the incubation medium increased during the incubation period from 1:1 initially to 4.5:1 after 60 min. The results from the experiments with rabbits indicate that the first-pass metabolism of this drug is highly stereoselective; in contrast, cancer patients who had received (R,S)-CP as an i.v. infusion showed no stereoselectivity in the elimination of the enantiomers. Pharmacokinetic studies with cancer patients, receiving (R,S)-CP as an oral dose, are in progress in order to determine if stereoselective first-pass metabolism of this drug also occurs in humans.

Analysis of 5-flourouracil in plasma by precolumn derivatization with 4-bromomethyl-7-methoxycoumarin, followed by multi-dimensional high-performance liquid chromatography

Abstract An assay for 5-fluorouracil (5- FU ) has been developed that utilizes a double extraction with ethyl acetate, followed by precolumn derivatization with 4-bromomethyl-7-methoxycoumarin. The reaction mixture was quenched with 5% acetic acid, extracted with hexane, and analyzed by multi-dimensional high-performance liquid chromatography. D erivatized 5- FU was injected into a cyanopropyl column and a heart cut containing the analyte was then switched to an octadecyl column and quantitated by fluorescence detection. The assay had a limit of detection of 0.5 ng 5- FU /ml plasma and was linear to 20 μ/ml. It was shown to be free of interferences from the other anticancer agents commonly used in combination with 5- FU . This assay should have the sensitivity needed to measure the low levels that occur after low-dose, continuous infusion of 5- FU .

High-performance liquid chromatography of 5-fluorouracil after derivatization with 4-bromomethyl-7-methoxycoumarin. Characterization of the derivative and the use of column switching for the improvement of resolution and the enhancement of sensitivity

The derivatization of 5-fluorouracil with 4-bromomethyl-7-methoxycoumarin has been reported previously; however, the structure of the derivative was not confirmed. The synthesis and purification of the 5-FU derivative is described along with the spectroscopic (MS and NMR) determination that it is labelled at both heterocyclic nitrogens as expected. A column switching HPLC system is also presented which consists of primary separation on a cyanopropyl column followed by a final separation on an ODS column with fluorescence detection. This system removes all interferences from the derivatization system and has a limit of detection for the pure derivative of less than 50 fmol (injection volume = 100 microliters).

Stability of intravenous admixtures of 5-fluorouracil and spirogermanium, a novel combination of cytotoxic agents

Abstract When used in combination, spirogemianium (SG) and 5-fluorouracil (5-FU) have to be administered at separate intravenous-infu- sion sites. To ease the administration of this potentially useful combination of antineoplastic agents, the stability of SG and 5-FU admixtures was studied. Direct mixing of SG and 5-FU formulations resulted in the formation of a precipitate. This precipitate was the free base form of SG, which has a low solubility at the pH of the i.v. admixtures. The appropriate dilution in D5W needed to prevent the precipitation was calculated from the pH-solubility curves of SG and 5-FU and the stability of 250 mg SG and 1000 mg 5-FU in 250 ml infusion containers was then investigated. There was no loss of SG or 5-FU in glass bottles after storage for 7 days at 4° C followed by 2 days at room temperature. However, in Polyvinylchloride (PVC) and polyolefin infusion bags at pH > 8.0, the SG concentration decreased by 68 and 28%, respectively, after 7 days at room temperature. This decrease was independent of the 5-FU concentration and was believed to be due to sorption or adsorption of SG by the plastic. Loss of SG was also found when the admixtures were passed through PVC or polyolefin tubing at 20 ml h −1 . By lowering the pH of the admixture to a value of less than 6.0, the loss of SG to PVC containers or tubing was prevented. Simultaneous administration of SG and 5-FU is possible if adequate dilution is maintained to prevent precipitation and the resulting pH of the admixture is kept below 6.0.