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Background: Today knowledge about pharmacokinetics of anticancer drugs in human malignant tumors is poor. Data from in vivo studies are limited and difficult to obtain due to ethical aspects. An ex vivo isolated perfused and ventilated human lung model however allows pharmacological studies of human bronchial carcinoma inside their host organ, the lung, under physiological conditions without compromising the patient., Methods: Following surgery for bronchial carcinoma human lung preparations were reperfused and ventilated extracorporally for 2-3 hours. During the reperfusion anticancer drugs are added to the perfusion solution and their uptake into tumor, normal lung tissue, and lymph nodes is studied., Results: An initial study showed that lung reperfusion under physiological circumstances over a period of 2-3 hours did not interfere with histo-pathological diagnostics and staging; an important precondition for potential adjuvant treatment. Pharmacokinetics of cyclophosphamid, adriamycin, and a water soluble adriamycin prodrug (HMR 1826) were measured. Final tissue concentrations of adriamycin and cyclophosphamid in peripheral lung parenchyma turned out to be 10 times higher compared to tumor tissue. However, following perfusion with adriamycin prodrug final tissue concentrations of adriamycin were in the same range in lung and tumor., Conclusions: The ex-vivo isolated human lung perfusion model (IHLP) has proven to be an ideal scientific model for pharmacological investigations of human tumors as an intermediate step between cell culture and in-vivo situation without any disadvantage for the patient. The tumor-to-host interaction is completely saved in this model. However, first pass reactions of drugs in other organs must not play a role for the substances studied with the IHLP. The role and future applications of the isolated perfused human lung model for other indications is discussed.

Purpose: The alkylating cytostatic prodrug cyclophosphamide is bioactivated by the human cytochrome P450 enzyme system. Since these enzymes are not only expressed in human liver, but also in extrahepatic tissue, local bioactivation of this drug may play an important role in its antineoplastic effects, e.g., chemotherapy of lung tumors. This would require uptake of significant amounts of cyclophosphamide into tumor tissue, which has not yet been demonstrated., Methods: We used a recently developed, ex vivo isolated, ventilated and perfused human lung model to study cyclophosphamide uptake into bronchial carcinoma and healthy lung tissue. Following a standard lobectomy, lung samples containing the tumor were perfused with buffer containing 2 mM cyclophosphamide for 2 h. Cyclophosphamide concentrations in perfusate and healthy peripheral tissue were measured during the perfusion and in tumors at the end of perfusion., Results: In all tissue samples, cyclophosphamide uptake was relatively poor, indicated by a tissue to perfusate ratio of 0.021. Moreover, in tumor samples, cyclophosphamide concentrations were significantly lower (P < 0.05) than in healthy lung tissue and showed pronounced interindividual variability. Median concentrations were 36.8 microg/g (26.9 44.2 microg/g) in healthy tissue and 5.1 microg/g (0.0-26.8 microg/g) in tumor samples. Tumor cyclophosphamide concentrations varied between 0 and 75% of those reached in healthy tissue., Conclusions: Our results indicate that CP tumor concentrations are modulated by factors different from dose and that expression of bioactivating enzymes in human lung or transfection of genes encoding these enzymes into tumor cells does not necessarily lead to local bioactivation of cyclophosphamide.

Purpose: The alkylating agent cyclophosphamide (CP) is a prodrug that is metabolized to both cytotoxic and inactive compounds. We have previously shown that following dose escalation from conventional-dose (CD) to high-dose (HD) levels; the fraction of the dose cleared by bioactivation is significantly decreased (66% versus 48.5%) in favor of inactivating elimination pathways when the HD is given as a single 1-h infusion. Based on the concept of bioactivating enzyme saturation with increasing doses, we investigated the influence of fractionated application of HD-CP on dose-dependent changes in metabolism., Patients and Methods: Plasma concentrations of CP (measured by high-performance liquid chromatography, HPLC) and urinary concentrations of CP and its major metabolites (quantified by [31P]-nuclear magnetic resonance spectroscopy; [31P]-NMR spectroscopy), were determined in four patients with high-risk primary breast cancer who received adjuvant chemotherapy including both CD-CP (500 mg/ m2 infused over 1 h) and split HD-CP (50 mg/kg infused over 1 h on each of 2 consecutive days (d): d1 and d2., Results: (Data are given as mean values for CD and d1/d2 of HD, respectively). Systemic clearance (CL) of CP was similar during CD and d1 of HD, but significantly increased on d2 of HD (CL: 83 and 78/115 ml/min; P < 0.01 for d1 versus d2). The latter was translated into an increase in formation CL of both active (+ 16.4 ml/min) and inactive metabolites (+ 17.6 ml/ min) and reflects autoinduction of metabolism. As compared with CD-CP, no statistically significant decrease was observed in the relative contribution of bioactivation CL to overall CL during both days of HD (63% versus 57%/53%). Recovery of intact CP in 24-h urine corresponded to 24%, 29%, 22% of the dose (P < 0.05 for d1 versus d2 of HD)., Conclusions: Following dose escalation of CP, dividing the high dose over 2 days instead of one single infusion may favorably impact the metabolism of CP in terms of bioactivation. In addition, on day 2 of a split regimen, renal elimination of CP is decreased, which implies that more drug is available for metabolism.

Trofosfamide and its congeners ifosfamide and cyclophosphamide are cell-cycle-nonspecific alkylating agents that undergo bioactivation catalyzed by liver cytochrome P450 (CYP) enzymes. Two NADPH-dependent metabolic routes for the anticancer drug trofosfamide, i.e., 4-hydroxylation and N-dechloroethylation, were studied in human liver microsomes and in seven recombinant human CYP isoforms (i.e., CYP1A1, 1A2, 2A6, 2B6, 2D6, 2E1, and 3A4-OR) to identify the CYP enzymes involved. Recombinant human CYP3A4 and CYP2B6 exhibited catalytic activity with respect to both pathways of trofosfamide. Enzyme kinetic analyses revealed the dominant role of human CYP3A4 in 4-hydroxylation and N-dechloroethylation of trofosfamide. This was confirmed by the observation that only the CYP3A4 contents of five samples of human liver microsomes correlated with both pathways of trofosfamide. Furthermore, ketoconazole, a selective inhibitor of CYP3A4, substantially inhibited microsomal trofosfamide 4-hydroxylation and N-dechloroethylation (50% inhibitory concentration < 1 microM for both reactions). The present study indicates that human liver microsomal CYP3A4 preferentially catalyzes the two NADPH- dependent metabolic routes of trofosfamide, which emphasizes the necessity for awareness of potential interactions with any coadministered drugs that are CYP3A4 substrates.

Purpose: The alkylating anticancer agent cyclophosphamide (CP) is a prodrug that undergoes a complex metabolism in humans producing both active and inactive metabolites. In parallel, unchanged CP is excreted via the kidneys. The aim of this study was to investigate the influence of dose escalation on CP pharmacokinetics and relative contribution of activating and inactivating elimination pathways., Patients and Methods: Pharmacokinetics of CP were assessed in 12 patients with high-risk primary breast cancer who received an adjuvant chemotherapy regimen that included four courses of conventional-dose CP (500 mg/m2 over 1 hour every 3 weeks) followed by one final course of high-dose CP (100 mg/kg over 1 hour). Plasma concentrations of CP were analyzed by high-performance liquid chromatography (HPLC), 24-hour urinary concentrations of CP, and its inactive metabolites (carboxyphosphamide, dechloroethylcyclophosphamide [dechlorethylCP], ketocyclophosphamide [ketoCP]) were determined by 31-phosphorus-nuclear magnetic resonance (31P-NMR)-spectroscopy., Results: There was no difference in dose-corrected area under the concentration-time curve (AUC) (216 v 223 [mumol.h/[mL.g]), elimination half-life (4.8 v 4.8 hours), systemic clearance (79 v 77 mL/min) and volume of distribution (0.49 v 0.45 L/kg) of CP between conventional- and high-dose therapy, respectively. However, during high-dose chemotherapy, we observed a significant increase in the renal clearance of CP (15 v 23 mL/min; P < .01) and in the formation clearance of carboxyphosphamide (7 v 12 mL/min; P < .05) and dechloroethylCP (3.2 v 4.2 mL/min; P < .05), whereas metabolic clearance to ketoCP remained unchanged (1.3 v 1.2 mL/min). Consequently, metabolic clearance to the remaining (reactive) metabolites decreased from 52 to 38 mL/min (P < .001). The relative contribution of the different elimination pathways to overall clearance of CP demonstrated wide interindividual variability., Conclusion: Overall pharmacokinetics of CP are apparently not affected during eightfold dose escalation. However, there is a shift in the relative contribution of different clearances to systemic CP clearance in favor of inactivating elimination pathways, thereby indicating saturation of bioactivating enzymes during dose escalation. Besides individual enzyme capacity, hydration and concomitant medication with dexamethasone modulated CP disposition.

A high-performance liquid chromatographic method for the quantification of acrolein following incubation of cyclophosphamide (CP) with human liver microsomes was developed. Based on the formation of the fluorescent derivative 7-hydroxyquinoline by condensation of acrolein with 3-aminophenol quantitation was performed without prior extraction or other sample cleanup procedures. The method showed sufficient sensitivity with a limit of detection of 5 ng/ml and a limit of quantification of 10 ng/ml. The suitability of the method is shown for enzyme kinetic studies.

Objective: Cyclophosphamide (CP) is an antineoplastic prodrug which requires bioactivation (4-hydroxylation) by the cytochrome P450 (CYP) enzymes in human liver. In parallel, P450-mediated side-chain oxidation (N-dealkylation) leads to the formation of the non-alkylating dechloroethylcyclophosphamide (DCI-CP) and chloroacetaldehyde, the latter being a potential neurotoxic agent. The enzyme responsible for side-chain oxidation has not been identified yet. We therefore used an in vitro approach to characterize the enzyme involved in N-dealkylation of CP., Methods: CP was incubated with the microsomal fraction of human liver in the presence of specific inhibitors for some P450 enzymes and in the presence of stable expressed P450 enzymes. Dechloroethylcyclophosphamide was analysed using gas chromatography and nitrogen-phosphorus detection., Results: Formation of DCl-CP increased linearly with substrate concentration over the entire concentration range (20 mumol.l-1 to 36 mmol.l-1). Saturation of the enzyme was not observed. Incubation with stable expressed P450 enzymes and inhibition experiments indicated that CYP3A4 was the major enzyme involved in side-chain oxidation of CP., Conclusion: Our in vitro data indicate that side-chain oxidation of CP occurs in dose-dependent fashion in men with no saturation of this pathway even following dose escalation. Thus enhanced neurotoxicity following CP administration may result in the setting of high-dose chemotherapy. Moreover, we conclude that CP has the potential to interact with other CYP 3A4 substrates.

A method for the sensitive determination of dechloroethylcylclophosphamide (3-DCl) in microsomal incubation mixtures was developed. 3-DCl, a side-chain oxidation product of cyclophosphamide (CP), was isolated by extraction with acetic acid ethyl ester following solid-phase extraction on C8 cartridges. Quantification of the metabolite was performed by direct capillary gas chromatography with a nitrogen-phosphorus detector without prior derivatization. The method showed good sensitivity and reproducibility with a detection limit of 1 ng/ml and a limit of quantification of 5 ng/ml. The suitability of the method is shown for the quantification of 3-DCl following incubation of CP with human liver microsomes.