D. K. Walker

Design of drugs involving the concepts and theories of drug metabolism and pharmacokinetics.

Drug metabolism input to the discovery process had historically been on an empirical case-by-case basis, since, detailed descriptors of the effect on pharmacokinetics of a change in structure or physicochemical property were not available. Considerable advances have been made in recent years, such that basic rules can be applied to predict the behavior of a compound in man based on physicochemistry and structure. This is particularly true in the areas of absorption, distribution, and clearance. In particular, knowledge of the reactions catalyzed by the enzymes of drug metabolism, including the cytochrome P450 super family, can be used in the design of new chemical entities, together with the usual pharmacological-derived SAR. The combination of both pharmacokinetics and pharmacodynamics at the discovery stage leads to drugs with optimum performance characteristics. Such drugs are easier to develop, representing a huge saving in resources. Moreover, the marketed compound is much more likely to find high clinical utilization. This review uses dofetilide, fluconazole, and amlodipine to highlight the multifaceted consequences of changing chemical structure, in terms of drug disposition, and reinforces these principles with examples from the literature.

PEG and PEG conjugates toxicity: towards an understanding of the toxicity of PEG and its relevance to PEGylated biologicals

PEG is used to improve pharmacokinetic properties of biologicals. Concern has been expressed about the toxicological effect and/or fate of the PEG. This paper reviews the available toxicity, metabolism and clearance data of PEG and PEGylated products in order to place such concerns in to appropriate context. The available data demonstrates that PEG itself only shows toxicity at high, parenteral doses and the usual target organ is the kidney as this is the route of excretion for unchanged PEG. A large therapeutic window (approximately 600-fold) exists between the maximum PEG burden from a current biological agent and the doses of PEG associated with human toxicity. Pathological changes which results in no functional deficit, PEG containing vacuoles in cells, have been observed with PEGylated biologicals. There is evidence that these PEG vesicle can resolve with time. In conclusion the doses used clinically for current and many future PEGylated biologicals are low and will result in exposures to PEG significantly lower than that required to elicit PEG toxicity. In all cases the routine regulatory toxicology studies would identify relevant pathology should it occur.

Assessment of the absorption, metabolism and absolute bioavailability of maraviroc in healthy male subjects

AIMS Two studies were conducted to: (i) quantify the amount of drug-related radioactivity in blood, plasma, urine and faeces following a (14)C-labelled dose of maraviroc; and (ii) investigate the pharmacokinetics, safety and tolerability of intravenous (i.v.) maraviroc and determine the absolute bioavailability of oral maraviroc. Metabolite profiling was also conducted. Data from both of these studies were used to construct a mass-balance model for maraviroc. METHODS Study 1 was an open-label study in three healthy male subjects. All subjects received a single 300-mg oral solution dose of (14)C-labelled maraviroc. Study 2 included two cohorts of subjects. Cohort 1 involved a double-blind (third party open), four-way crossover study where eight subjects received escalating i.v. doses of maraviroc (3, 10 and 30 mg) with placebo insertion. Cohort 2 involved an open, two-way crossover study where 12 subjects received 30 mg maraviroc by i.v. infusion and 100 mg maraviroc orally in random order. In study 1, blood samples and all urine and faeces were collected up to at least 120 h postdose. In study 2, blood samples were taken at intervals up to 48 h postdose. Urine was also collected up to 24 h postdose in cohort 1 only. RESULTS After oral administration in study 1, maraviroc was rapidly absorbed with a plasma T(max) reached by 2 h postdose for all three subjects. The maximum concentrations of radioactivity also occurred within 2 h for all subjects. There was a higher amount of radioactivity in plasma than in blood (blood/plasma ratio approximately 0.6 for AUC(t) and C(max)). Unchanged maraviroc was the major circulating component in plasma, accounting for approximately 42% of the circulating radioactivity. Following a 300-mg (14)C-labelled maraviroc dose, means of 76.4% and 19.6% of radioactivity were recovered in the faeces and urine, respectively. The mean total recovery of dosed radioactivity was 96%, with the majority of radioactivity being recovered within 96 h postdose. Profiling of the urine and faeces showed similar and extensive metabolism in all subjects. Unchanged maraviroc was the major excreted component (33%). The major metabolic pathways were determined and involved oxidation and N-dealkylation. Intravenous doses of maraviroc (3-30 mg) were well tolerated in study 2, and drug exposure was approximately proportional to dose within the studied range. Approximately 23% of total clearance (44 l h(-1)) was accounted for by renal clearance (10.2 l h(-1)). Mean volume of distribution at steady state was 194 l. Absolute bioavailability of a 100-mg oral tablet dose, by comparison with a 30-mg i.v. dose, was calculated to be 23.1%. CONCLUSIONS Maraviroc is rapidly absorbed and extensively metabolized, although unchanged maraviroc is the major circulating component in plasma and is the major excreted component after oral dosing. The pharmacokinetics of maraviroc after i.v. administration is approximately proportional over the dose range studied. Renal clearance contributes 23% of total clearance. The absolute bioavailability of 100 mg oral maraviroc is 23%.

Pharmacokinetics and metabolism of dofetilide in mouse, rat, dog and man

1. Pharmacokinetics of dofetilide were studied in man, dog, rat and mouse after single i.v. and oral doses of dofetilide or 14C-dofetilide. 2. Dofetilide was absorbed completely in all species. Low metabolic clearance in man resulted in complete bioavailability following oral administration. Higher metabolic clearance in rodents, and to a lesser extent dogs, resulted in decreased bioavailability because of first-pass metabolism. 3. Following i.v. administration, the volume of distribution showed only moderate variation in all species (2.8-6.3 l/kg). High plasma clearance in rodents resulted in short half-life values (mouse 0.32, male rat 0.5 and female rat 1.2 h), whilst lower clearance in dog and man gave longer terminal elimination half-lives (4.6 and 7.6 h respectively). 4. After single i.v. doses of 14C-dofetilide, unchanged drug was the major component excreted in urine of all species with several metabolites also present. 5. Metabolites identified in urine from all species were formed by N-oxidation or N-dealkylation of the tertiary nitrogen atom of dofetilide. 6. After oral and i.v. administration of 14C-dofetilide to man, parent compound was the only detectable component present in plasma and represented 75% of plasma radioactivity. No single metabolite accounted for greater than 5% of plasma radioactivity.

Preclinical assessment of the distribution of maraviroc to potential human immunodeficiency virus (HIV) sanctuary sites in the central nervous system (CNS) and gut-associated lymphoid tissue (GALT)

1. Growing knowledge of the pathogenesis of human immunodeficiency virus (HIV)-1 infection has led to the identification of potential virus sanctuary sites within the central nervous system and gut-associated lymphoid tissue. 2. Maraviroc is a novel CCR5 antagonist for the treatment of HIV-1 infection. Disposition studies have been performed within the preclinical testing of maraviroc to determine its distribution to these anatomical sites. 3. Maraviroc, which is a substrate of the efflux transporter P-glycoprotein, shows limited distribution to the central nervous system as evidenced by cerebrospinal fluid concentrations that were 10% of the free plasma concentration following intravenous infusion to rats. Tissue distribution studies also indicated limited distribution of radioactivity into brain tissue of rats. 4. Radioactivity in gut-associated lymphoid tissue lymph nodes exceeded the concentrations in blood and concentrations in the contents of thoracic ducts of the lymphatic system were similar to blood levels following intravenous administration to rats.

Pharmacokinetic/pharmacodynamic assessment of the effects of E4031, cisapride, terfenadine and terodiline on monophasic action potential duration in dog

1. Torsades de pointes (TDP) is a potentially fatal ventricular tachycardia associated with increases in QT interval and monophasic action potential duration (MAPD). TDP is a side-effect that has led to withdrawal of several drugs from the market (e.g. terfenadine and terodiline). 2. The potential of compounds to cause TDP was evaluated by monitoring their effects on MAPD in dog. Four compounds known to increase QT interval and cause TDP were investigated: terfenadine, terodiline, cisapride and E4031. On the basis that only free drug in the systemic circulation will elicit a pharmacological response target, free concentrations in plasma were selected to mimic the free drug exposures in man. Infusion regimens were designed that rapidly achieved and maintained target-free concentrations of these drugs in plasma and data on the relationship between free concentration and changes in MAPD were obtained for these compounds. 3. These data indicate that the free ED50 in plasma for terfenadine (1.9 nM), terodiline (76 nM), cisapride (11 nM) and E4031 (1.9 nM) closely correlate with the free concentration in man causing QT effects. For compounds that have shown TDP in the clinic (terfenadine, terodiline, cisapride) there is little differentiation between the dog ED50 and the efficacious free plasma concentrations in man (<10-fold) reflecting their limited safety margins. These data underline the need to maximize the therapeutic ratio with respect to TDP in potential development candidates and the importance of using free drug concentrations in pharmacokinetic/pharmacodynamic studies.

Potential role for P-glycoprotein in the non-proportional pharmacokinetics of UK-343,664 in man.

1. UK-343,664 is a potent and specific PDE5 inhibitor. Following single oral doses to human volunteers, it exhibited non-proportional pharmacokinetics over the dose range 30-800mg. Over this 27-fold dose range, Cmax and AUCt increased 247- and 287-fold respectively. The half-life (4-6 h) was similar at all doses. No systemic exposure was quantifiable at doses <10 mg. 2. UK-343,664 is a lipophilic molecule (log D7.4 = 3.1) and as such is expected to be cleared mainly by metabolism. Based on studies with expressed human P450 enzymes it was concluded that the metabolism of UK-343,664 was predominantly mediated by CYP3A4. With a moderate Km

The use of 96-well Scintiplates to facilitate definitive metabolism studies for drug candidates

Semi-quantitative analysis of the drug-related components in biological samples collected during definitive metabolism studies using radiolabelled drug candidates is commonly achieved by HPLC profiling, using either on-line radiochemical detection or off-line liquid scintillation counting (LSC) following collection of the HPLC eluent into vials. However, although the use of LSC with vials has high sensitivity, the approach is time-consuming, laborious and destructive, whilst on-line detection methods are inappropriate for samples with low-levels of radioactivity (commonly the case with plasma samples). The use of 96-well microtitre plates (Scintiplates) for fraction collection during HPLC profiling provides a sensitive, effective and efficient alternative method for the semi-quantitative analysis of radiolabelled components in biological samples. Furthermore, the approach is non-destructive, such that subsequent identification of the isolated components can be achieved. Although the Scintiplate methodology is not appropriate for the analysis of excreta samples, where quenching of the radiochemical signal by endogenous components was observed, the approach was demonstrated to be valid for the relative quantification of [14C]-labelled material in plasma samples for all species investigated. In addition, good sensitivity was observed, with a counting efficiency of 79% for [14C], such that a drug-related component accounting for 10-15 dpm is quantifiable. The utility of the methodology for profiling circulating metabolites was demonstrated by the analysis of a rat plasma sample following oral administration of [14C]-UK-349,862. The Scintiplate approach and subsequent mass spectrometric analysis resulted in the relative quantitation and specific characterisation of circulating metabolites accounting for 93% of the total plasma radioactivity.

Measurement of the class III antidysrhythmic drug, UK-68,798, in plasma by radioimmunoassay.

A sensitive radioimmunoassay (RIA) for the specific determination of 1-(4-methanesulphonamidophenoxy)-2-[N-(4-methanesulphonamido -phenethyl)-N- methylamino]ethane (UK-68,798), a novel class III antidysrhythmic agent, in human plasma is described. Specific antisera were raised in sheep using desmesyl-UK-68,798-succinate-ovalbumin conjugate as the antigenic hapten carrier protein. The antisera produced exhibited high specificity for UK-68,798 compared with known metabolites from animals, other antidysrhythmic agents and co-administered drugs. Good correlation was found in a comparison of the RIA method with a high-performance liquid chromatography (HPLC) method (r = 0.997) and a 10-fold lower limit of determination was observed for the RIA method compared with the HPLC method (0.05 and 0.5 ng ml-1, respectively). The RIA method was applied to the analysis of UK-68,798 in plasma obtained from human volunteers receiving the compound.

Automated 96-well solid phase extraction for the determination of doramectin in cattle plasma

Automated standard and sample preparation have been coupled with 96-well solid phase extraction (SPE) technology to produce a cost effective, high throughput system for the analysis of drugs in biological media. The system was originally designed using the Packard Multiprobe 104DT robotic sample processor (RSP) to improve throughput for the assay of doramectin in cattle plasma, and the assay has since been validated (0.5-100 ng ml[-1]) using the Tecan Genesis RSP 150/8. The robotic processor conducts all liquid handling procedures involved in sample extraction. These comprise preparation of calibration standards in plasma, dispensing and diluting of plasma samples and addition of internal standard. In addition, the robot primes the 96-well SPE block, applies calibration standards and samples, draws the mixtures through the 96-well SPE block, and finally washes the block ready for manual elution. The doramectin assay involves high-performance liquid chromatography (HPLC) with fluorescence detection, and requires the sample extracts to be derivatised prior to analysis. The derivatisation procedure is performed manually in situ in the polypropylene deep 96-well block into which the samples have been eluted from the SPE-block. The derivatised samples are taken directly from the deep well block and injected into the HPLC for analysis. This type of batch processing keeps sample transfer to a minimum. Automated sample preparation, in combination with the use of 96-well technology, has reduced both cost and effort required in the analysis of doramectin in cattle plasma samples, and has resulted in improved sample throughput.