A regulatory viewpoint on transporter-based drug interactions.

1. Pharmacokinetic drug interactions can lead to serious adverse events and the evaluation of a new molecular entity's (NME) drug-drug interaction potential is an integral part of drug development and regulatory review before its market approval. Clinically relevant interactions mediated by transporters are of increasing interest in clinical development and research in this emerging area and it has been revealed that drug transporters can play an important role in modulating drug absorption, distribution, metabolism and elimination. 2. Acting alone or in concert with drug-metabolizing enzymes transporters can affect the pharmacokinetics and/or pharmacodynamics of a drug. The newly released drug interaction guidance by the US Food and Drug Administration (USFDA) includes new information addressing drug transporter interactions with a primary focus on P-glycoprotein (P-gp, ABCB1). 3. This paper provides a regulatory viewpoint on transporters and their potential role in drug-drug interactions. It first outlines information that might be needed during drug development and ultimately included in new drug application (NDA) submissions to address potential transporter-mediated drug interactions. Next, it explains criteria that may warrant conduct of in vivo P-gp-mediated drug interaction studies based on in vitro assessment. In addition, it includes a review case that describes the evaluation of data suggesting a P-gp-based induction interaction.

Application of a simple competitive protein-binding assay technique to the pharmacokinetics of N-(phosphonacetyl)-L-aspartate in humans.

A competitive protein-binding assay for N-(phosphonacetyl)-L-aspartate (PALA) using aspartate transcarbamylase as the receptor protein and [14C]PALA as the radioactive ligand is described here and has been applied to study the pharmacokinetics of PALA in humans. A protein-free ultrafiltrate of plasma, prepared by centrifugation of 1-ml samples through Amicon Centriflo membrane cones, was used in the assay, which had a maximun sensitivity of 0.7 microM PALA in plasma. At this level, the coefficient of variation was less than 10%. Comparison of the competitive binding assay to a gas chromatographic-mass spectrometric technique shows that the two methods yield equivalent results in the concentration range of 1 microM to 1 mM. However, the competitive binding assay possesses practical advantages because of its simplicity and the ease with which multiple samples may be assayed. PALA disappearance from plasma was studied in seven patients and was found to be consistent with a two-compartment open model. The t1/2 alpha (elimination half-life for initial phase) and t1/2 beta (elimination half-life for terminal phase) were 0.93 +/- 0.73 (S.D.) hr and 4.82 +/- 1.48 hr, respectively. The cumulative urinary excretion of PALA int two patients was 70 and 90% of the administered dose 16 hr after the infusion was completed.

Pharmacokinetics of intraventricular and intravenous N,N',N''-triethylenethiophosphoramide (thiotepa) in rhesus monkeys and humans.

The cerebrospinal fluid (CSF) and plasma pharmacokinetics of N,N',N"-triethylenethiophosphoramide (thiotepa), an alkylating agent used for treatment of carcinomatous meningitis, were determined in rhesus monkeys in order to assess the relative advantage of intraventricular versus systemic administration of the drug. Following an i.v. thiotepa dose of 0.9 mg/kg (11 mg/sq m), peak plasma levels of parent drug reached approximately 1 microgram/ml. Thiotepa was rapidly equilibrated with lumbar and ventricular CSF. Systemic, lumbar, and ventricular exposure to the drug, measured as area under the curve (AUC), were similar in all cases. After a 1-mg intraventricular dose of thiotepa, peak ventricular levels were greater than 100 micrograms/ml. However, peak levels in the lumbar CSF at 1 h after intraventricular administration were less than 10 micrograms/ml. The AUC for ventricular CSF was nearly 100-fold greater for the intraventricular route than for the i.v. route; however, the AUC for lumbar CSF following intraventricular delivery was only 5% of the AUC for ventricular CSF. N,N',N''-Triethylenephosphoramide, an active metabolite of thiotepa observed in all fluids, appeared to have a much slower total body clearance than thiotepa. Comparison of the data obtained from monkey experiments with data from a patient with meningeal disease supports the use of the monkey as a model for intraventricular pharmacokinetics. The data presented indicate that there is no relative advantage to intraventricular administration of thiotepa at the doses currently used in clinical trials.

2-Chloroethyl (methylsulfonyl)methanesulfonate (NSC-338947), a more selective DNA alkylating agent than the chloroethylnitrosoureas.

The novel chloroethylating agent 2-chloroethyl (methylsulfonyl)methanesulfonate (CIEtSoSo) has been shown to act like the chloroethylnitrosoureas (CIEtNu's) in its DNA damaging and cytotoxic effects in human cell lines and has similar activity to the CIEtNu's in the National Cancer Institute antitumor screening tests. Its simpler chemistry, however, suggests that it may alkylate DNA more selectively than do the CIEtNu's. The DNA base adducts produced in calf thymus DNA by CIEtSoSo have been compared to a representative, non-carbamoylating CIEtNu, 1-(2-chloroethyl)-3-(cis-2-OH)cyclohexyl-1-nitrosourea, using high-pressure liquid chromatography. Two major modified base peaks were observed from the nitrosourea treated sample which have been subsequently identified by high-pressure liquid chromatography comparison of synthesized standards and by electron impact gas chromatography/mass spectrometric analysis to be 7-hydroxyethylguanine and 7-chloroethylguanine. In contrast only 7-chloroethylguanine was obtained from the CIEtSoSo treated DNA at equimolar doses. Thus CIEtSoSo was found to be more specific in its reaction with DNA in that it produced less variety of products than the nitrosourea, with no apparent generation of hydroxyethyl products, which are major side reactions of the CIEtNu's.

Phase I trial of N-(phosphonacetyl)-L-aspartate.

A Phase I clinical trial of N-(phosphonacetyl)-L-aspartate, an antimetabolite which inhibits a key enzyme in the de novo pathway of pyrimidine biosynthesis, was conducted. N-(Phosphonacetyl)-L-aspartate was given as an i.v. 15-min infusion once daily for five days; cycles of treatment were repeated every three weeks. Thirty-four patients received treatment. Dose-limiting toxicity was observed at 1500 to 2000 mg/sq m/day and was manifested by skin rash, diarrhea, and stomatitis. Rash and diarrhea usually began during the first week of treatment and persisted up to Day 17 of a cycle of therapy. No consistent hematopoietic, hepatic, or renal toxicity was observed. One partial response in a patient with colon carcinoma was seen and continues at more than eight months. Stable disease was observed in three patients with colon carcinoma, two patients with hypernephroma, one patient with pancreatic carcinoma, and one patient with melanoma. The predictability and reversibility of toxicity and the suggestion of antitumor activity in humans are observations which support the further evaluation of N-(phosphonacetyl)-L-aspartate in Phase II studies.

4-Demethylpenclomedine, an antitumor-active, potentially nonneurotoxic metabolite of penclomedine.

Penclomedine [3,5-dichloro-4,6-dimethoxy-2-(trichloromethyl)pyridine], an antitumor agent, is currently in Phase I clinical trials and is believed to be a prodrug. In these studies, cerebellar effects have been dose limiting. Previous studies identified 4-demethylpenclomedine (4-DM-PEN) as the major plasma metabolite in rodents and humans. 4-DM-PEN was demonstrated to be an antitumor-active metabolite of penclomedine in vivo when evaluated against the penclomedine-sensitive MX-1 human breast tumor xenograft implanted either s.c. or intracerebrally and is believed to be on the metabolic activation pathway of penclomedine. Because earlier studies revealed an absence of neurotoxic cerebellar effects for 4-DM-PEN in contrast to penclomedine in a rat model, this metabolite may be a candidate for an alternative to penclomedine in the clinic for treatment of breast cancer or brain tumors, if the cerebellar effects of penclomedine preclude its further clinical development. Because neither penclomedine nor 4-DM-PEN were very active in vitro, the metabolism of penclomedine was also investigated using rat liver microsomes in an attempt to identify the ultimate active form of the drug. Metabolites and putative metabolites were prepared by chemical synthesis for antitumor evaluation in vitro and in vivo. A reductive metabolite, alpha,alpha-didechloro-PEN, was observed to be much more cytotoxic than penclomedine or 4-DM-PEN in vitro, but evaluation of this and the other metabolites and putative metabolites in vivo against the MX-1 tumor failed to identify any active metabolite among the structures evaluated other than 4-DM-PEN. The limited activity of 4-DM-PEN in vitro indicates that it, like penclomedine, is also a prodrug, demonstrating a need for additional studies on the metabolic activation of penclomedine to identify the ultimate active form of the drug.

Murine and human in vivo penclomedine metabolism.

Penclomedine is a multichlorinated alpha-picoline derivative which has shown prominent activity in murine breast cancer models and is currently undergoing clinical development. Previous in vitro research has identified several penclomedine metabolites. In this study, human and murine in vivo penclomedine metabolism was examined. Upon i.v. administration to mice, no penclomedine was detectable in plasma at time points as early as 1 h postinfusion. The principle metabolite was demethyl-penclomedine [3, 5-dichloro-2-methoxy-4-hydroxy-6-(trichloromethyl)pyridine]. Both penclomedine and demethyl-penclomedine could be recovered from tissues. Greater than 60% of the penclomedine dose remaining in the body at 22 h was indelibly bound to tissue and plasma proteins. Urinary metabolites of penclomedine consisted mainly of penclomic acid and additional polar metabolites. The results obtained after p. o. administration were nearly identical to i.v. administration with respect to the extent, level, and type of metabolites found in the plasma, tissues, and urine and with respect to the extent of protein binding. In human subjects administered penclomedine daily for 5 consecutive days, demethyl-penclomedine could be detected in plasma and accumulated with successive doses of penclomedine, reaching peak plasma concentrations of up to 10 times that of penclomedine itself and plasma exposures of nearly 400 times that of the parent drug. It appears that patients eliminate penclomedine largely through metabolism and that this drug may be amenable to p.o. administration.

Tissue and tumor distribution of C-penclomedine in rats.

Penclomedine, a lipophilic alpha-picoline derivative, is undergoing clinical development presently because of its pronounced antitumor activity against intracerebral (i.c.) tumor xenografts. Penclomedine may be metabolized in vivo to a more potent compound. Although it may be useful in the treatment of brain tumors, the drug has caused significant neurotoxicity in early clinical trials. The possibility that antitumor activity and neurotoxicity may be mediated by different mechanisms prompted a study assessing the differential distribution of penclomedine and penclomedine metabolites to brain and i.c.-implanted tumors in rats. In the present study, quantitative autoradiographic analysis demonstrated a homogenous distribution of 14C-penclomedine in all organs within 1 h of administration. Levels of 14C-penclomedine in both i.c. and s.c. tumors were three times higher than in normal brain tissue. High-performance liquid chromatography combined with gas chromatography and mass spectrophotometry demonstrated that two metabolites, O-demethyl penclomedine and penclomic acid, were responsible for most of the plasma radioactivity. Penclomic acid was also the most common urinary metabolite of penclomedine. In liver samples, although a large number of metabolite peaks were detected, no parent compound could be identified. However, in tumors and all other tissues, penclomedine was the main compound detected. The finding of penclomedine in normal brain tissue indicates not only that this drug may be useful in tumors with normal blood-brain barrier function, but also that it may be directly neurotoxic.

Cyclophosphamide and 4-Hydroxycyclophosphamide/aldophosphamide kinetics in patients receiving high-dose cyclophosphamide chemotherapy.

Using a recently developed gas chromatography and mass spectrometry method to determine whole-blood cyclophosphamide (CP) and 4-hydroxycyclophosphamide/aldophosphamide (4-HO-CP/AP) concentrations, we investigated their pharmacokinetics in women receiving CP therapy. Patients (n = 18) received one or two courses of CP: (a) a 90-min i.v. infusion (4 g/m2) followed by a 96-h i.v. infusion (6 g/m2) in combination with high-dose thiotepa; or (b) a 96-h i.v. infusion (6 g/m2) in combination with high-dose thiotepa. Whole-blood exposures to CP [area under the whole blood concentration versus time curve (AUCCP)] and 4-HO-CP/AP (AUC4HOCP) between courses 1 and 2 were compared after normalization to dose (g/m2). A nonproportional increase was observed for the AUCCP between the first course [1112 micrometer. h/g/m2 +/- 14% coefficient of variation (CV)] and the second course (1579 micrometer . h/g/m2 +/- 28% CV) (P < 0.001). In contrast, the AUC4HOCP (27 micrometer . h/g/m2 +/- 25% CV) determined for the first course was 29% higher than the AUC4HOCP (21 micrometer . h/g/m2 +/- 26% CV) for the second course (P < 0.01). The interpatient whole-blood exposures to both CP and 4-HO-CP/AP were remarkably consistent in this patient population with percent CVs ranging from 14 to 28%. Because thiotepa (800 mg/m2) was administered simultaneously with CP during the second course of treatment, possible inhibition of CP metabolism by thiotepa was investigated using human liver microsomes in vitro. IC50 values determined for inhibition of CP metabolism in three individual liver donors ranged from 1.0 to 40 micrometer. However, the clinical relevance of this observation has not been established.

Antiarrhythmic potency of N-acetylprocainamide.

Compared to procainamide in an animal arrhythmic model, the antiarrhythmic potency of the N-acetylated metabolite of procainamide (NAPA) was 92% with respect to dose and 70% with respect to plasma level. The antiarrhythmic effects of combinations of the drugs were additive. Measurements of procainamide and NAPA plasma levels needed to suppress ventricular extrasystoles suggested that both compounds are nearly equipotent in patients as well. The average plasma level required for arrhythmia control in these patients was equivalent to 5.1 mcg/ml procainamide. Since patients on long-term procainamide therapy have plasma concentrations of NAPA that are usually comparable to, and occasionally greater than, their procainamide levels, dose regiments based on procainamide levels alone need revision to include consideration of the levels of this metabolite.

Procainamide and N-acetylprocainamide kinetics investigated simultaneously with stable isotope methodology.

The pharmacokinetics of procainamide (PA) and N-acetylprocainamide (NAPA) were compared in 3 normal subjects after simultaneous intraveous injection of PA and NAPA-13C. The distribution kinetics of both compounds were modeled with a 3-compartment mamillary system, and it was found that their steady-state distribution volumes were not significantly different, averaging 1.41 L/kg for PA and 1.46 L/kg for NAPA. However, the intercompartmental clearances of NAPA were slower than those of PA. In these normal subjects, the average elimination t1/2 and total elimination clearance for PA were 2.5 hr and 589.8 ml/min, and for NAPA were 6.2 hr and 233.7 ml/min. Mean renal clearances of PA (346.7 ml/min) and of NAPA (199.5 ml/min) exceeded the usual rate of glomerular filtration, which suggests that both compounds are eliminated in part by renal tubular secretion. All subjects were phenotypic rapid acetylators of isoniazid and converted approximately one fourth of the administered PA dose to NAPA-12C. The fate of 15.4% of the administered PA and 14.5% of the administered NAPA-13C was not determined.

Absolute bioavailability in man of N-acetylprocainamide determined by a novel stable isotope method.

Absorption of a single oral dose of N-acetylprocainamide (NAPA) was studied in 3 normal subjects. Approximately 85% of the oral dose was absorbed and peak plasma NAPA concentrations were reached in 45 to 90 min. In 2 subjects, NAPA was absorbed at a fast initial rate, then more slowly, prolonging the apparent elimination phase half-life. Absolute bioavailability was determined by a new stable isotope method that entailed intravenous injection of NAPA 13C at the same time that an unlabeled NAPA capsule was given orally. Plasma levels and urine excretion of both compounds were determined by mass fragmentography. Bioavailability was assessed by deconvoluting the plasma level vs time curves resulting from intravenous and oral drug administration, and also by comparing the relative percentage of NAPA and NAPA-13C excreted unchanged in the 24 hr after simultaneous administration.

Antiarrhythmic efficacy of N-acetylprocainamide in patients with premature ventricular contractions.

Oral administration of a 1.5-gm dose of N-acetylprocainamide (NAPA) to 9 patients with premature ventricular contractions (PVCs) confirmed previous indirect evidence that this metabolite of procainamide has antiarrhythmic efficacy and potency comparable to those of procainamide. Although the mechanism by which NAPA acts as an antiarrhythmic drug is not known, it was found that the 6 patients with coupled PVCs responded to NAPA therapy and that the 3 patients without coupled PVCs failed to respond. Coupling interval prolongation also occurred during NAPA therapy in 4 of the 6 responding patients. These observations suggest that NAPA may terminate coupled PVCs by slowing and then interrupting conduction of re-entrant impulses, as has been proposed for procainamide. NAPA plasma concentrations of 7.4-17.2 mug/ml were well tolerated by the patients and produced an average fall of 3 mm Hg in mean arterial pressure and a 7.6% mean increase in corrected QT interval.

Clinical pharmacology of 5-iodo-2'-deoxyuridine and 5-iodouracil and endogenous pyrimidine modulation.

We describe the clinical pharmacology and metabolism of 5-iodo-2'-deoxyuridine (IdUrd) during and after a 12-hour infusion. The kinetics of IdUrd were linear between 250 and 1200 mg/m2. The plasma IdUrd concentration reached steady state in less than 1 hour. Total body clearance of IdUrd was 750 ml/min/m2 and the disappearance t1/2 at the end of the infusion was less than 5 minutes. The primary metabolite, 5-iodouracil (IUra), did not reach steady state during the infusion. At the end of the 1200 mg/m2 infusion, the maximum plasma IUra concentration was 100 mumol/L, or about 10 times the simultaneous IdUrd plasma concentration. During the infusion there was at least a fifty- to 100-fold increase in uracil and thymine plasma concentrations. After the infusion, IUra disappearance from plasma was nonlinear, with an apparent Michaelis constant of 30 mumol/L. Plasma uracil and thymine levels slowly decreased after the IdUrd infusion until IUra fell to less than 30 mumol/L. There was subsequently a parallel and more rapid decrease in the plasma concentrations of uracil and thymine. Uridine, 2'-deoxyuridine, and thymidine plasma levels did not change significantly as a result of IdUrd therapy. These changes in endogenous pyrimidine pools are consistent with competitive inhibition of dihydrouracil dehydrogenase by IUra. An in vitro human bone marrow assay was used to determine the relative toxicity of IdUrd and IUra. Although exposure to IUra was tenfold higher than that to IdUrd, IdUrd was at least 100 times more cytotoxic to marrow cells.

Pharmacological activity, metabolism, and pharmacokinetics of glycinexylidide.

Glycinexylidide (GX) is a metabolite of lidocaine that is frequently present in mug/ml concentrations in the plasma of patients treated with lidocaine infusions for 24 hr or more. Plasma levels of GX have 26% the antiarrhythmic activity of lidocaine in an animal model, and GX adversely affects the mental performance of normal subjects at plasma concentrations comparable to those found in patients. The total volume of GX distribution in man is similar to that of lidocaine but the plasma clearance is less, so that the 10-hr elimination phase half-life of GX is much longer than the 1 1/2 hr half-life reported in normal subjects for lidocaine. About half of an administered dose of GX is excreted unchanged in urine, roughly 15% appears in urine as conjugates of xylidine and p-OH xylidine, and the fate of the rest is unknown.

Pharmacokinetics and metabolism of lidocaine in patients with renal failure.

The kinetics of distribution and elimination of lidocaine and two of its metabolites, monoethylglycinexylidide (MEGX) and glycinexylidide (GX), were studied in 4 uremic patients on chronic hemodialysis. Each patient received a loading dose of 75 mg of lidocaine, followed by a 30 mug/kh/min lidocaine infusion. No toxic side effects from lidocaine were seen during the study. Average values for lidocaine steady-state plasma levels (2.3 mug/ml) clearance (12.3 ml/min/kg), terminal half-life (148 min), and total volume of distribution (1.9 L/kg) were found, and are similar to those values reported for normal subjects MFGX and after lidocaine infusion averaged 1/5-2/3 of the corresponding lidocaine level, as in nonuremic subjects, and plateaued by 6-8 hr. GX levels did not reach plateau by 12 hr and remained relatively unchanged after infusion. It is concluded that lidocaine infusion in uremic patients is safe, with no abnormal cumulation of lidocaine or MEGX. GX levels, however, may increase progressively, even after 12 hr.

Dose-ranging trial of N-acetylprocainamide in patients with premature ventricular contractions.

Ten patients with chronic premature ventricular contractions (PVCs) received short-term oral therapy with N-acetylprocainamide (NAPA) to determine its antiarrhythmic efficacy and side effects under the conditions of a placebo-controlled, dose-ranging trial. NAPA was effective in suppressing PVCs in 8 patients but caused a paradoxical increase in PVC frequency in one. Results were equivocal in the remaining patient because PVCs did not recur when NAPA therapy was withdrawn. Mean NAPA plasma levels as high as 41.1 microng/ml did not have untoward hypotensive or myocardial depressant effects, as judged by electrocardiographic and systolic time intervals. There was, in fact, a consistent reduction in PEP/LVET ratio, indicating that NAPA increases the force of myocardial contraction. The mean NAPA elimination half-life of 10.9 hr was longer than the 6.2 hr half-life reported for normal subjects, but its prolongation was predictably correlated with reductions in creatinine clearance. Gastrointestinal side effects experienced by 3 patients and insomnia noted by 2 patients are similar to known adverse reactions to procainamide.

Initial pharmacology and toxicology of intravenous desmethylmisonidazole.

Since January 1981, 52 patients have entered the Radiation Therapy Oncology Group Phase I trial with intravenous (i.v.) desmethylmisonidazole (DMM). DMM is less lipophilic than misonidazole (MISO) and theoretically will be less neurotoxic due to lower penetration into neural tissue and more rapid elimination. The drug is administered intravenously to achieve the maximum drug concentration in tumor for a given dose. The protocol slowly escalates the total dose of drug administered. At this time the planned dose on the three week schedule is 1g/m2 five times per week to a total of 15g/m2, and on the seven week schedule is 1.25g/m2 twice weekly to a total dose of 17.5g/m2. The preliminary plasma pharmacokinetic data demonstrates high peak plasma levels within five minutes of the end of the drug infusion. Compared to MISO the percent of DMM excreted in the urine is increased, 63% vs 10%, and the elimination half-life is decreased: DMM, i.v. 5.3h; MISO, i.v. 9.3h; MISO, oral 10 to 13h. Neurotoxicity has been observed in approximately 30% of patients given a cumulative dose of greater than 11g/m2. This is in comparison to a 50% incidence in the RTOG Phase I study with oral MISO at doses of 12g/m2. There is not sufficient data to evaluate the relationship between neurotoxicity and drug exposure. Further patient accrual on this study is required to better define the properties of DMM.

Bioavailability of penclomedine and systemic exposure to 4-O-demethylpenclomedine in patients receiving oral and intravenous penclomedine.

PURPOSE: Oral administration of penclomedine was investigated based on preclinical studies indicating that an oral schedule of penclomedine treatment may prevent the neurotoxicity observed in phase I studies of an intravenous (i.v.) formulation, possibly by reducing maximum plasma concentrations (Cmax) of the neurotoxic parent species. METHODS: Penclomedine was administered i.v. (200 mg/m2) and orally (250 mg/m2) in alternate sequences to patients with solid tumor malignancies. Plasma concentrations of parent drug and the principal metabolite, 4-O-demethylpenclomedine, were determined by a reversed-phase HPLC assay. RESULTS: Penclomedine was detectable in the plasma of all patients within 1 h of oral penclomedine treatment and Cmax was reached within 1 to 4 h. Consistent with the hypothesis that an oral schedule of administration may circumvent neurotoxicity, a paired data analysis demonstrated a significant reduction in Cmax values following oral administration (P=0.017). However the magnitude of this reduction was highly variable. Similarly an extensive range in the relative exposure to both parent drug and metabolite were observed. The bioavailability of penclomedine ranged from 28% to 98% (median 73%). CONCLUSIONS: Oral penclomedine does produce systemic exposure, but substantial interpatient variability in absorption and systemic exposure is present which may limit the clinical role of the oral route of administration.

Analysis of tissue progesterone residues by gas chromatography - mass spectral and radioimmunological detection methods.

Methods for the extraction, isolation and analysis of tissue concentrations of progesterone suitable for studying residue levels from livestock treated with this steroid for the control and synchronization of estrus are presented. The system employs biphasic partitioning for the extraction and silica gel chromatography for the isolation and demonstrates 80 to 90% recovery of 14C-labeled progesterone added as an internal standard. Residue analysis of fat, kidney, liver and muscle tissue samples from ovariectomized non-treated and progesterone treated ewes are compared employing a competitive inhibition radioimmunoassay system which appears to be less specific for progesterone than the gas chromatography-mass spectrometry method employing selective ion monitoring detection.