Pemetrexed pharmacokinetics and pharmacodynamics in a phase I/II study of doublet chemotherapy with vinorelbine: implications for further optimisation of pemetrexed schedules.

The purpose of this study was to investigate the utility of plasma pharmacokinetic and pharmacodynamic measures including plasma deoxynucleosides, homocysteine and methylmalonic acid concentrations in understanding the time course and extent of the inhibition of thymidylate synthase (TS) by pemetrexed in the context of a phase I/II combination study with vinorelbine. Eighteen patients received supplementation with folic acid and Vitamin B(12) 1 week before beginning treatment with pemetrexed and vinorelbine administered in a dose-escalating manner on a 21-day cycle. Heparinised blood samples were collected from consenting patients in the first cycle for pharmacokinetic analyses and in the first two cycles for determination of plasma thymidine, deoxyuridine, homocysteine and methylmalonic acid concentrations. These values were correlated with response and toxicity. Plasma deoxyuridine was used as a measure of TS inhibition, and concentrations of deoxyuridine were significantly elevated relative to baseline on days 1 (P<0.01), 2 (P<0.001) and 3 (P<0.05) after treatment at all pemetrexed dose levels (400-700 mg m(-2)). The magnitude of deoxyuridine elevation correlated with pemetrexed area under the plasma concentration-time curve (AUC) (r(2)=0.23, P<0.05). However, deoxyuridine concentrations returned to baseline between 8 and 15 days after treatment with pemetrexed, suggesting that inhibition of TS was not durable. Pemetrexed AUC correlated with the percentage decline (relative to baseline) in both platelets (r(2)=0.58, P<0.001) and leucocytes (r(2)=0.26, P<0.05) at day 8. Baseline homocysteine was also significantly correlated with these measures of haematological toxicity (r(2)=0.37, P<0.01 and r(2)=0.39, P<0.01, respectively). In addition, there was a significant reduction of plasma homocysteine on days 8 (P<0.005) and 15 (P<0.05) in cycle 1 compared to baseline values. The results suggest that the TS inhibitory effects of pemetrexed are short-lived and make the case for a more frequent schedule of administration such as every 2 weeks. The lack of protracted TS inhibition may be due to concomitant vitamin administration, and this may be the mechanism by which vitamins prevent life-threatening toxicity from pemetrexed. Baseline homocysteine concentration remains a predictive marker for haematological toxicity even following folate supplementation.

Imatinib disposition and ABCB1 (MDR1, P-glycoprotein) genotype.

The aim of this study was to explore the impact of individual variation in drug elimination on imatinib disposition. Twenty-two patients with gastrointestinal stromal tumor or chronic myeloid leukemia initially received imatinib 600 mg daily with dosage subsequently toxicity adjusted. Pharmacokinetic parameters on day 1 and at steady-state were compared with elimination phenotype and single-nucleotide polymorphisms of CYP3A5 and ABCB1. A fivefold variation in estimated imatinib clearance (CL/F) was present on day 1 and mean CL/F had fallen by 26% at steady state. This reduction in imatinib CL/F was associated with ABCB1 genotype, being least apparent in thymidine homozygotes at the 1236T>C, 2677G>T/A and 3435C>T loci. Toxicity-related dose reduction also tended to be less common in these individuals. ABCB1 genotype was associated with steady-state CL/F due to an apparent genotype-specific influence of imatinib on elimination. Further evaluation of ABCB1 genotype and imatinib dosage is warranted.

Wash-in methodology and modeling to determine hepatocellular D-glucose transport in the perfused rat liver.

Wash-in experiments, which may be useful for the study of the disposition of substrates in the liver, have not been well described. To investigate physiological models for wash-in curves, we performed experiments on the perfused livers of male Wistar rats anesthetized with pentobarbital. Test perfusate contained (14)C-sucrose as the extracellular marker and (3)H-glucose. Liver perfusions were performed with background glucose concentrations of 5.7, 10.7, 51.2, or 108.5 mM. Outflow time-activity curves were analyzed with the use of four models. The V(max) and K(m) for the influx of glucose were 1.1 +/- 0.03 micromol/s/g and 41 +/- 3 mM with the Crone-Renkin early extraction model; 1.4 +/- 0.04 micromol/s/g and 36 +/- 3 mM with dispersion model analysis; 1.8 +/- 0.1 micromol/s/g and 25 +/- 4 mM with the Goresky distributed model to fit differentiated wash-in curves; and 2.7 +/- 0.6 micromol/s/g and 28 +/- 21 mM with compartmental analysis. There was reasonable agreement between the four models, and they yielded results similar to those reported for glucose uptake in other preparations.

Carbon monoxide disposition and permeability-surface area product in the foetal circulation of the perfused term human placenta.

In order to estimate the placental barrier to gas transfer, a novel carbon monoxide (CO) wash-in method was used to estimate the permeability-surface area (PS) product for the transfer of gas across the foetal circulation in the perfused human term placenta. The PS product for CO was 0.0096+/-0.006 ml/s/g or 0.012+/-0.007 ml/s/g using compartmental or Crone-Renkin analysis, respectively. Using this result and a published estimate of the placental capillary surface area, the permeability coefficient to CO across the foetal circulation was found to be approximately 4 x 10(-5)cm/s. This result is compatible with the hypothesis that the foetal circulation of the human placenta imposes a potentially significant barrier to gas transfer.

Hepatic cytochrome P450 3A drug metabolism is reduced in cancer patients who have an acute-phase response.

Inflammatory disease states (infection, arthritis) are associated with reduced drug oxidation by the cytochrome P450 3A system. Many chemotherapy agents are metabolised through this pathway, and disease may therefore influence inter-individual differences in drug pharmacokinetics. The purpose of this study was to assess cytochrome P450 3A function in patients with advanced cancer, and its relation to the acute-phase response. We evaluated hepatic cytochrome P450 3A function in 40 patients with advanced cancer using the erythromycin breath test. Both the traditional C(20min) measure and the recently proposed 1/T(MAX) values were estimated. The marker of acute-phase response, C-reactive protein and the pro-inflammatory cytokines IL-6, IL-1beta, TNFalpha and IL-8 were measured in serum or plasma at baseline. Cancer patients with an acute phase response (C-reactive protein >10 mg x l(-1), n=26) had reduced metabolism as measured with the erythromycin breath test 1/T(MAX) (Kruskal-Wallis Anova, P=0.0062) as compared to controls (C-reactive protein < or =10 mg x l(-1), n=14) Indeed, metabolism was significantly associated with C-reactive protein over the whole concentration range of this acute-phase marker (r=-0.64, Spearman Rank Correlation, P<0.00001). C-reactive protein serum levels were significantly correlated with those of IL-6 (Spearman coefficient=0.58, P<0.0003). The reduction in cytochrome P450 3A function with acute-phase reaction was independent of the tumour type and C-reactive protein elevation was associated with poor performance status. This indicates that the sub-group of cancer patients with significant acute-phase response have compromised drug metabolism, which may have implications for the safety of chemotherapy in this population.

Toxicity of irinotecan (CPT-11) and hepato-renal dysfunction.

Various clinical and laboratory parameters have been investigated for their ability to predict toxicity arising from the use of the anticancer drug, irinotecan (CPT-11). In particular, patients deficient in the conjugation of SN-38, a metabolite of CPT-11, are known to be at greater risk. We describe one case of a patient with metastatic colorectal cancer treated with a single dose of CPT-11 at 125 mg/m(2). Although this patient lacked any known predictive factors for toxicity, he experienced severe side-effects several days later. We hypothesized that the toxicity in this patient was due to compromised SN-38 conjugation. Plasma samples were analyzed by reversed-phase high-performance liquid chromatography assay for CPT-11 and its metabolites at 96, 144, 168, 192 and 288 h post-administration. We observed that the concentrations of both the parent drug and its metabolites were markedly raised (11- to 60-fold expected). Additionally the estimated terminal half-lives were 1.5-7 times those expected (29.5, 101, 39.6 and 41.8 h for CPT-11, APC, SN-38G and SN-38, respectively). We conclude that the toxicity in this patient was not caused by deficient SN-38 conjugation, but by decreased drug excretion through both hepatic and renal routes.

High-performance liquid chromatography-electrospray ionization mass spectrometry method for the measurement of moclobemide and two metabolites in plasma.

A rapid and sensitive liquid chromatography-electrospray ionisation mass spectrometry (HPLC-ESI-MS) assay has been developed for the measurement of moclobemide and metabolites, Ro12-5637 and Ro12-8095, in human plasma. Sample preparation (0.5 ml plasma) involves solid-phase extraction using C18 cartridges. A Nova-Pak phenyl column (Waters, 4 microm, 150x2 mm I.D.) was employed for analyte separation with a mixture of 0.2 M ammonium formate buffer, pH 3.57 and acetonitrile as the mobile phase. The within- and between-day precisions of the assay were <18% and the limit of quantification for all analytes was 0.01 microg/ml. The total run-time was 6 min. The method described was used to measure moclobemide, Ro12-5637 and Ro12-8095 in human plasma following an oral 300 mg dose.

The erythromycin breath test for the prediction of drug clearance.

The erythromycin breath test (EBT) is a putative probe of cytochrome P450 (CYP) 3A4 activity in vivo. Therefore, the EBT might prove useful for the individualisation of doses of drugs that have a low therapeutic window (for example the immunosuppressants or cytotoxics) and are metabolised by CYP3A4. However, there is a lack of consensus as to how the EBT should be used to predict total body clearance (CL), and the results so far have been largely disappointing. We argue that the required assumption that individuals produce 5 mmol of CO2/min per m2 at rest is one of the problems with the existing EBT, as the literature suggests significant variability and possible gender differences in this parameter. An examination of the EBT with a simple compartment model suggests that alternative parameters could be more useful in the prediction of CL. In particular, there is theoretical support for the use of the time-point at which breath radioactivity is maximal (tmax) as a correlate for CL. This is in agreement with our recent study of the pharmacokinetics of erythromycin in patients with cancer.

Carbon monoxide wash-in method to determine gas transfer in vascular beds: application to rat hindlimb.

The vascular barrier to gas transfer is an important physiological parameter; however, no readily applicable technique exists to quantitate the process. A simple technique to measure the permeability-surface area (PS) product for gas transfer in vascular beds is proposed using wash in of carbon monoxide (CO) and Crone-Renkin analysis. Wash-in experiments were performed on the perfused hindlimbs of male Wistar rats (n = 15) by using CO as a surrogate marker for oxygen and technetium-99m-labeled albumin as the vascular marker. The use of CO and erythrocyte-free perfusate and the collection of outflow samples into tubes preloaded with erythrocytes obviated the need for an anaerobic collection device or consideration of Hb binding in the analysis. The PS product for CO was determined from the early extraction as 0.013 +/- 0.006 ml. s(-1). g(-1). Compartmental analysis revealed that the fractional recovery of CO was 0.45 +/- 0.14 and the volume of distribution was 2.31 +/- 0.76 ml/g. This technique detected a small measurable barrier to the transfer of CO across the hindlimb vasculature and is potentially applicable to other vascular beds in health and disease.

Highly sensitive analysis of the antifolate pemetrexed sodium, a new cancer agent, in human plasma and urine by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatography method was developed and validated for the quantitation of pemetrexed (LY231514, ALIMTA) in human urine and plasma. Plasma samples were spiked with the internal standard lometrexol and extracted using Certify II columns. Pemetrexed was assayed in diluted urine by an external calibration method. A C8 column was used for the separation of analytes with a mobile phase composed of sodium formate buffer and acetonitrile. Between- and within-day precision and accuracy were acceptable down to the limit of quantitation of 5 ng/ml in plasma. This method was used successfully for an investigation of the disposition of pemetrexed in patients receiving 500 mg/m2 as a 10-min infusion.

Optimizing the erythromycin breath test for use in cancer patients.

The erythromycin breath test (EBT) is a putative in vivo probe for drug metabolism by cytochrome P450 3A4 (CYP3A4). Because many anticancer drugs are metabolized by this system, we sought to further develop the EBT as a tool for predicting the clearance, in cancer patients, of drugs metabolized by CYP3A4. Sixteen adult patients with incurable cancer were studied. The EBT was performed on day 1 and breath sampled after the i.v. injection of 4 microCi of 14C-erythromycin. The breath 14CO2 flux (CERt) was estimated at 11 time points over 2 h. On day 2, the EBT was repeated midway through a 10-min infusion of 100 mg of erythromycin lactobionate, and the plasma pharmacokinetics of erythromycin were determined. The infusion of 100 mg of erythromycin did not modify the EBT results significantly. The values of the conventional EBT parameter CER20 min obtained on day 1 were comparable for most subjects (0.03-0.06% dose/min), with the exception of an individual receiving the known CYP3A4 inducers dexamethasone and phenytoin who returned a value of 0.14% dose/min. There was no significant correlation between any of the conventional EBT parameters and erythromycin clearance. However, two parameters reflecting early emergence of breath radioactivity (1/TMAX and CER3 min/CERMAX) correlated significantly with erythromycin clearance (P = 0.005 and 0.006, respectively). Novel parameters derived from the EBT are significantly correlated with the clearance of erythromycin even in the presence of confounding factors, such as metastatic liver disease, altered protein binding, and comedication. These parameters may enable dose optimization of cytotoxics metabolized by CYP3A4.

Frequency of cytochrome P450 3A4 variant genotype in transplant population and lack of association with cyclosporin clearance.

OBJECTIVE: Cytochrome P450 3A4 (CYP3A4) plays a vital role in the oxidative metabolism of many xenobiotics. Some recent reports have provided circumstantial evidence in support of an association between a genetic polymorphism (A-->G) in the 5'-flanking region (-290) of CYP3A4 and altered enzyme activity. We sought to determine whether genotyping patients for CYP3A4-G could assist with the dose optimisation of drugs metabolised by this system. METHODS: Normal subjects and renal-transplant patients receiving cyclosporin for immune modulation were genotyped for the CYP3A4-G variant. A surrogate for cyclosporin clearance was estimated from the ratio of the cyclosporin dose, normalised for body weight and the corresponding trough concentration. The association between genotype and clearance was examined in patients who received twice-daily doses of cyclosporin and who were not on concurrent medication known to modify CYP3A4 function. RESULTS: The allelic frequencies of the CYP3A4-G variant were estimated to be 2.6% and 3% in transplant patients and normal subjects, respectively. The median cyclosporin pseudo-clearance of transplant patients with wild-type CYP3A4 was 0.90 l/h/kg (range: 0.35-3.8 l/h/kg; n = 86), whereas the corresponding value for the five patients heterozygotic for the CYP3A4-G variant was 0.71 l/h/kg (range 0.35-0.91 l/h/kg). The distribution of the pseudo-clearance according to genotype was not found to be significant according to a Fisher's exact test (P = 0.15). CONCLUSION: Genotyping for the CYP3A4-G polymorphism is unlikely to assist cyclosporin dose selection in transplant patients.

Clinical pharmacokinetics of the irinotecan metabolite 4-piperidinopiperidine and its possible clinical importance.

PURPOSE: To investigate the clinical relevance of 4-piperidinopiperidine (4PP) in the activity of irinotecan (CPT-11), a high-performance liquid chromatography-turboionspray-tandem mass spectrometry assay for plasma 4PP was developed. METHODS: Plasma samples were prepared for analysis following C18 solid-phase extraction. Chromatography was performed on a Waters Nova-Pak Phenyl column. Selected reaction monitoring with the mass transitions m/z 169.2 --> 84.2 and 139.2 --> 98.1 was used for the detection of 4PP and the internal standard (IS), 1-piperidineproprionitrile, respectively. RESULTS: The assay was linear from 14.8 to 591.0 nM with absolute recoveries of 4PP (59.1 nM) and IS (143.7 nM) of 85.7% (n = 10) and 86.7% (n = 10), respectively. The accuracy and imprecision of the method (total) was > or = 96.8% and < or = 8.5% over the concentration range studied, respectively. 4PP was detectable in plasma following the administration of 125, 350, 500 mg/m2 and 600 mg/m2 CPT-11 to patients, with AUC(4PP) correlated with the dose (r2 = 0.66). Plasma concentrations of 4PP declined slowly with a long terminal half-life (33.4 +/- 17.1 h). CONCLUSIONS: Overall, the concentrations of 4PP in plasma were in the sub-micromolar range (< 206.9 nM) and substantially lower than those capable of inducing apoptosis of cancer cells.

Clinical and preclinical pharmacokinetics of raltitrexed.

Raltitrexed is a specific, folate-based inhibitor of thymidylate synthase with activity in advanced colorectal cancer comparable with that of fluorouracil (5-fluorouracil) plus folinic acid. Its activity is enhanced by rapid cellular entry and polyglutamation, with the polyglutamated derivatives having approximately 100-fold greater inhibitory potency than the parent compound. A number of phase I/pharmacokinetic studies have been performed, including schedules involving a 15-minute infusion every 3 weeks, weekly x 6 every 8 weeks, and every 2 weeks. The maximum tolerated dose (MTD) for the 3-weekly schedule was 3.5 to 4.5 mg/m2 in adults and 6 mg/m2 in a paediatric population. The MTDs for the other schedules have not yet been reported. The disposition of raltitrexed in patients is best described by a 3-compartment model with a terminal half-life (t1/2gamma) of 260 hours, the latter being subject to significant interpatient variability. A similar protracted t1/2gamma has been detected in all of the animal species studied. Together with evidence from the mass-balance studies performed, this delayed elimination suggests considerable sequestration of raltitrexed in tissues, predominantly as polyglutamate forms. Nevertheless, there has been no pharmacokinetic evidence of drug accumulation in plasma following repeated administration. On the basis of animal experiments, the oral bioavailability and penetration of raltitrexed into cerebrospinal fluid are both likely to be limited in the clinical setting. Raltitrexed is over 90% bound to plasma protein over the concentration range of 20 to 100 micromol/L. Apart from poly-glutamation, raltitrexed does not appear to be metabolised to a significant extent, and most of the excreted drug (approximately 20% of the administered dose) is recovered unchanged in the urine within the first 24 hours post-administration. The average clearance of raltitrexed is 2.4 L/h (40 ml/min), and this value is significantly reduced in patients with compromised renal function (glomerular filtration rate of 25 to 65 ml/min). These patients are more likely to experience severe antiproliferative toxicity with raltitrexed. A careful evaluation of renal function, particularly in the elderly, is warranted. It has not been possible to establish strong correlations between the plasma pharmacokinetics of raltitrexed and toxicity, and the cellular pharmacokinetics of raltitrexed may be more predictive. Studies in mice have demonstrated that delayed administration of folinic acid can assist in the recovery of animals from antiproliferative toxicity, possibly by promoting the release of polyglutamated drug from tissues. This approach should be evaluated as a rescue regimen in patients with severe proliferative toxicity.

Metabolism of CPT-11. Impact on activity.

Irinotecan (CPT-11) is a semi-synthetic camptothecin with a broad spectrum of clinical activity. It is a prodrug that is cleaved by esterases to the potent topoisomerase I poison, SN-38. In humans, this activation is relatively inefficient, but this may result in a more protracted formation of SN-38 lactone. Some intratumoral activation may also occur, but the significance of this process is uncertain. CPT-11 is metabolized by cytochrome P450 3A to yield a number of comparatively inactive compounds. SN-38 is glucurono-conjugated in the liver, and this metabolite, although inactive, may participate in the enterohepatic cycling of SN-38 after hydrolysis in the intestinal lumen. Overall, the production of SN-38 from CPT-11 is the result of the complex interplay of several metabolic pathways and the source of considerable interpatient variability.

Hepatic disposition of neurotoxins and pesticides.

The hepatic disposition of pesticides and neurotoxins may influence susceptibility to Parkinson's disease. Therefore we examined the behaviour of paraquat, dichlorodiphenyltrichloroethane (DDT), malathion and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in perfused rat liver using the multiple indicator-dilution technique. The values for the recovery of paraquat, DDT, malathion and MPTP were 1.05+/-0.12, 0.32+/-0.01, 0.11+/-0.02 and 0.02+/-0.01, respectively. The volumes of distribution were 0.28+/-0.13, 0.69+/-0.12, 3.30+/-0.58 and 5.10+/-6.00 ml/g, respectively. The permeability-surface area products suggest that transport of DDT and MPTP across cell membranes is by simple diffusion. However, there may be a specific influx mechanism for malathion and a specific efflux mechanism for paraquat. There is considerable variability in the hepatic disposition of putative neurotoxins such as MPTP and pesticides. Factors that influence the hepatic disposition of neurotoxins may alter susceptibility to neurotoxic diseases however the effects will be diverse.

The mechanism for the inhibition of acetylcholinesterases by irinotecan (CPT-11).

Irinotecan (CPT-11) is an anticancer drug that occasionally produces acute cholinergic side effects. Preliminary findings suggest that these are mediated through the inhibition of acetylcholinesterase (AChE). In this study, the inhibition of various AChEs by CPT-11 was studied. The lactone form of CPT-11 resulted in apparent noncompetitive inhibition of electric eel and both human recombinant and erythrocyte AChE with K(i) values of 0.065, 0.19, and 0.29 microM, respectively. The carboxylate form of CPT-11 was approximately 10 times less potent. Apparent noncompetitive inhibition of AChE may arise through several mechanisms, and those relevant to CPT-11 were identified from key experimental findings. First, the inhibition by CPT-11 was found to be instantly reversible in dilution studies. Second, incubation of the enzyme with CPT-11 before the introduction of neostigmine protected the enzyme from inactivation. Third, regeneration of the active enzyme after preincubation with neostigmine was totally suppressed by the addition of 2 microM CPT-11, indicating that CPT-11 is a potent inhibitor of decarbamoylation and, by inference, deacylation. Additional experiments with tacrine revealed functional differences between these two inhibitors. Also, preliminary molecular modeling of the interaction between AChE and CPT-11 indicated that the latter does not bind at the same site as tacrine. Displacement studies with the peripheral site-specific ligand, propidium, confirmed that CPT-11 binds at this site. The rapid reversibility of the inhibition of AChE by CPT-11 and the lower activity of the carboxylate form are likely reasons for the transient nature of the cholinergic toxicity observed clinically.

Carbon monoxide disposition in the perfused rat liver.

A simple method for determining carbon monoxide (CO) disposition in the rat liver perfused with erythrocyte-free buffer was developed. Wash-in experiments were performed with buffer containing tracer quantities of [14C]sucrose and 3H2O and equilibrated with CO. Outflow samples were collected into tubes containing human erythrocytes, which avidly bind CO. Outflow curves were analyzed using compartmental models. Fractional recovery of CO was 1.07 +/- 0. 17, and the apparent volume of distribution was 1.37 +/- 0.30 ml/g of liver (n = 8). A flow-limited model fitted the data most effectively, although estimates of the permeability-to-surface area product were attempted using a barrier-limited model. This technique will facilitate investigation of the effects of disease on gaseous substrate disposition in perfused organs.