The application of Phase 0 and microtracer approaches in early clinical development: past, present, and future.

Phase 0 microdosing studies were introduced to the drug development community approximately 20 years ago. A microdose is defined as less than 1/100th of the dose calculated based on animal data to yield a pharmacological effect in humans, with a maximum of 100 µg, or 30 nmoles for protein products. In our experience, Phase 0 microdose studies have not been fully embraced by the pharmaceutical industry. This notion is based on the number of Phase 0 studies that we have been involved in. Thus, we conducted at least 17 Phase 0 microdose studies in the Zero's (on average, two per year), but in the years beyond this, it was only 15 studies (1.4 per year); in these latter years, we did conduct a total of 23 studies which employed an intravenous (i.v.) microdose for absolute bioavailability (ABA) assessments (two per year on average), which are the most used and potentially informative type of clinical study using a microdose, albeit they are formally not microdose studies. In the current review, we summarize the past use of and experience with Phase 0 microdose designs in early clinical development, including intravenous 14C microdose ABA studies, and assess what is needed to increase the adoption of useful applications of Phase 0/microdose studies in the near future.

An evaluation of human ADME and mass balance studies using regular or low doses of radiocarbon.

There has been increased interest in conducting human absorption, distribution, metabolism, and excretion (ADME) studies with low doses (up to 0.1 MBq) as opposed to regular doses (1.85-3.7 MBq) of radiocarbon (14 C). This is due to the fact that low-dose human ADME studies may be conducted without dosimetry calculations and will lead to lower human radiation exposure. Here, we sought to compare the outcomes of low-dose versus regular-dose human ADME studies in healthy volunteers. Forty oral human ADME studies conducted at PRA were surveyed, among which 12 were low-dose studies. The fraction of drug material absorbed was 67% ± 7% in the regular-dose studies (data for 13 studies) versus 39% ± 16% in the low-dose studies (data for 5 studies). The average total recovery of 14 C in excreta was 93% ± 5% for regular-dose studies, and 21 of 28 such studies showed recoveries more than 90%. For low-dose studies, average total recovery was 89% ± 9%, and 6 of 12 studies showed recoveries more than 90%. Metabolite profiling was successful in all cases reported (13 regular-dose studies and 5 low-dose studies). There was no obvious relationship between the total recoveries of 14 C in excreta and the proportion of 14 C excreted in feces, or between the total recoveries and the plasma elimination half-lives for parent or total 14 C, neither in the low-dose nor the regular-dose studies. A significant correlation was found between the fraction absorbed and the recovery in feces in the low-dose but not in the regular-dose studies, and no correlation was found between the fractions absorbed and the total recoveries in both types of studies. Low-dose studies were more often conducted on drugs that had a plasma elimination half-life of parent drug more than 100 hours (5 of 12 studies) than regular-dose studies (1 of 26 studies). We conclude that both low-dose as well as regular-dose human ADME studies provide adequate data to support decision making for further drug development.

Controlled release recombinant human interferon-α2b for treating patients with chronic hepatitis C genotype 1: a phase 2a clinical trial.

Better convenience and tolerability and sustained therapeutic concentrations might improve interferon (IFN) treatment for chronic hepatitis C virus (HCV) infection. In an open-label, randomized study, controlled release free (chemically unmodified) recombinant human IFN-α(2b) in poly(ether-ester) microspheres (CR-rhIFN-α(2b)), was injected at doses of 160, 320, 480 or 640 µg every 2 weeks for 12 weeks with concomitant weight-based oral ribavirin in 32 treatment-naïve patients with chronic HCV genotype 1. Treatment was well tolerated, with 31 patients (97%) successfully completing the study. Full doses of CR-rhIFN-α(2b) were administered on 96% of scheduled occasions. Flu-like symptoms were generally mild and brief. Injection site reactions developed in 13 patients (41%), and neutropenia occurred in six of eight patients receiving 640 µg. In the 320, 480 and 640 µg groups, 62-75% of patients achieved a ≥2 log(10) HCV RNA reduction by 4 weeks and 88-100% by 12 weeks. For those groups, the pooled median time to ≥2 log(10) reduction was 11 days (95% confidence interval, 7-35 days). In those groups, viral reduction below the limit of detection was accomplished in 25% of patients by 4 weeks and in 62% by 12 weeks. The 160-µg dose was less potent. After CR-rhIFN-α(2b) injection, stable plateau levels of serum IFN-α(2b) were generally reached within 72 h. Treatment-emergent neutralizing antibodies to IFN-α(2b) were observed in one patient. No antibodies to host plant proteins were detected. CR-rhIFN-α(2b) with ribavirin cotherapy was well tolerated and displayed potent early antiviral activity in patients with chronic HCV genotype 1.

Application of a semipermeable surface column for the determination of amoxicillin in human blood serum.

A high-performance liquid chromatography column-switching system for the automated determination of amoxicillin in human serum was developed as a more efficient alternative for the already existing systems with off-line sample pretreatment. The column-switching system consists of a semipermeable surface (SPS) column and an analytical reversed-phase (RP) C18 column. After centrifuging, pure serum samples were injected into the column-switching system. Clean-up, with regard to removal of proteins, was performed on the SPS column. The fraction containing amoxicillin was concentrated on the analytical RP-C18 column. Finally, chromatography and detection were performed with the RP-C18 column using UV detection at 234 nm. The total analysis time was 15 min. The method has proven to be reliable and to be more time- and resource-efficient compared to previously used methods with off-line sample clean-up. It is now used in bioavailability studies for the development of new amoxicillin formulations.

Transdermal absorption of topical anti-acne agents in man; review of clinical pharmacokinetic data.

BACKGROUND: Apart from oral drug treatment, drug therapy in acne vulgaris comprises topical treatment with agents with a primarily keratolytic action (e.g. tretinoin and benzoylperoxide), and with antibiotics (clindamycin, erythromycin, and erythromycin-zinc complex). The acne grade in the particular patient usually determines the selection of the preferred route of administration, viz. topical or oral, or a combination of both, and topical treatment is usually preferred in mild to moderate acne. The fact that a topically applied compound may also become systemically available to a quantifiable extent, is not generally considered. AIM: The present paper reviews the clinical data on transdermal uptake of anti-acne agents in man, also with respect to their relevance for daily clinical practice. OUTCOME: The majority of published data on transdermal penetration of topical anti-acne agents focuses on the retinoid tretinoin, and on the antimicrobial agent clindamycin. This interest emerges from the fact that these agents have been associated with embryotoxicity/teratogenicity, and pseudomembranous colitis, respectively. For both compounds the extent of systemic availability after topical application is low, viz. 5-7% and 8%, respectively, at its highest. The height and variability in endogenous retinoid levels is very likely to outweigh any contribution of exogenously applied tretinoin, but a full consensus on the safe use of topical tretinoin in pregnancy is still lacking. With respect to clindamycin, the suggested association between its topical use and the occurrence of pseudomembranous colitis appears not to be of clinical relevance. In order to reduce systemic exposure to clindamycin as much as possible, topical application of clindamycin phosphate is to be preferred over clindamycin hydrochloride salt. Regarding other topical anti-acne agents, it has been suggested that topical zinc-erythromycin is to be preferred over erythromycin, both from clinical efficacy and safety viewpoints. With respect to the currently used compounds like benzoylperoxide, azelaic acid, and adapalene, available clinical pharmacokinetic data are scarce, and significant safety concerns did not emerge as yet. CONCLUSION: The limited transdermal uptake of topical anti-acne agents underpins their safe use in daily clinical practice. With respect to topical retinoids, formal consensus is lacking regarding their use in pregnancy.

Transdermal absorption of clindamycin and tretinoin from topically applied anti-acne formulations in man.

The percutaneous absorption of clindamycin was studied in healthy male volunteers, comparing two investigative clindamycin (% w/v)/tretinoin (0.025% w/v) gels, containing clindamycin phosphate ester and clindamycin HCl, respectively, relative to a clindamycin phosphate lotion (1% clindamycin; Dalacin T). Formulations were applied daily for 5 days on the face, according to a balanced complete block design. Redness of the skin was scored visually, and blood and urine were collected. Clindamycin plasma levels did not exceed the limit of quantification (5 ng mL(-1)) with the clindamycin phosphate formulations, but one volunteer who received the clindamycin HCl/tretinoin gel showed plasma levels of up to 13 ng mL(-1). Clindamycin urinary excretion for 12 h after application of the clindamycin phosphate/tretinoin gel was comparable to the values of the reference lotion, whereas the clindamycin HCl/tretinoin gel gave significantly higher values. Erythema appeared to be associated with increased urinary excretion. The formulations were tolerated well. In a separate clinical pilot study in acne patients, the transdermal uptake of tretinoin and clindamycin from the clindamycin phosphate/tretinoin gel was monitored. Plasma samples were collected after 4 and 12 weeks of daily treatment. None of the study plasma samples contained measurable tretinoin levels. Clindamycin levels were not quantifiable in the majority (87%) of samples, the highest plasma level was 11 ng mL(-1). The chemical form of clindamycin proved to modulate skin irritation and percutaneous uptake of clindamycin from a gel formulation in healthy subjects. There was no indications for a notable transdermal uptake of tretinoin during daily application of the gel in patients, nor for an enhancing effect of tretinoin on clindamycin uptake.

Disposition of the selective alpha1A-adrenoceptor antagonist tamsulosin in humans: comparison with data from interspecies scaling.

Tamsulosin-HCl is an alpha1A-adrenoceptor antagonist that is mainly eliminated by metabolism in animals and humans and is highly bound to alpha1-acid glycoprotein in blood plasma. The disposition of the compound (0.4 mg as modified-release granules in a capsule) was determined in male volunteers, using intravenous (iv) infusion of tamsulosin-HCl (0.125 mg over 4 h) as reference treatment for the assessment of absolute oral bioavailability. Disposition parameters of iv tamsulosin in humans was compared with data predicted from animal data by interspecies scaling techniques. Levels after iv dosing in humans showed a biexponential decline, with mean half-lives (+/-SD) of 1.2 +/- 0.6 and 6.8 +/- 3.5 h, respectively. The mean systemic clearance (+/-SD) was low (viz., 48 +/- 24 mL/min). The mean volume of distribution (+/-SD) was rather small (21 +/- 6 L), and was estimated at 16 +/- 4 L in the steady state. The mean absolute oral bioavailability (+/-SD) was approximated at 100 +/- 19%. Systemic clearance in humans was poorly predictable from a logarithmic clearance versus body weight relation of rat, rabbit, and dog data. The prediction improved dramatically (accuracy 213%) when scaling was done with systemic clearance values of unbound drug, and it improved further (accuracy 59%) with the product of unbound clearance and maximum life-span potential. Also, the prediction of volume of distribution improved dramatically (accuracy 81%) after correction for differences in extent of protein binding between species. The terminal disposition half-life of 7.0 h, as predicted after integrating maximum life-span potential and protein binding in scaling of clearance, was very close to the value of 6.8 h established experimentally in humans. The present results with tamsulosin underline the importance of correction for extent of protein binding in allometric scaling of clearance and distribution volume.

Examination of metabolic pathways and identification of human liver cytochrome P450 isozymes responsible for the metabolism of barnidipine, a calcium channel blocker.

1. In a human liver microsomal system, barnidipine was converted into three primary metabolites, an N-debenzylated product (M-1), a hydrolyzed product of the benzyl-pyrrolidine ester (M-3) and an oxidized product of the dihydropyridine ring (M-8). 2. Involvement of CYP3A in the three primary metabolic pathways was revealed by the following studies: (a) inhibition of CYP3A, (b) a correlation study using 10 individual human liver microsomes and (c) cDNA-expression studies. The secondary metabolites, M-2 and M-4 (pyridine forms of M-1 and M-3), were most likely generated from M-8 but were unlikely from M-1 or M-3. Involvement of CYP3A in the secondary pathways of metabolism is also suggested. 3. The possibility of interactions between barnidipine and coadministered drugs was examined in vitro. The formation rate of the primary metabolites was little affected by warfarin, theophylline, phenytoin, diclofenac and amitriptyline at concentrations of 200 microM, but was inhibited by glibenclamide, simvastatin and cyclosporin A. IC50 for the latter drugs was estimated to be > 200, 200 and 20 microM respectively, which was roughly > 200, 6000 and 50 times higher than their respective therapeutic plasma levels, suggesting that interactions with cyclosporin A, a CYP3A inhibitor, are of possible clinical relevance.

In-vitro metabolism of YM17E, an inhibitor of acyl coenzyme A:cholesterol acyltransferase, by liver microsomes in man.

Because YM17E (1,3-bis[[1-cycloheptyl-3-(p-dimethylaminophenyl) ureido]methyl]benzene dihydrochloride) inhibits acyl coenzyme A:cholesterol acyltransferase (ACAT) it has potential application in the treatment of hypercholesterolaemia. In man and animals YM17E is extensively metabolized, via N-demethylation, to five active metabolites (M1, M2-a, M2-b, M3 and M4). The main objectives of this study were to examine inhibition of YM17E metabolism by the products and identify the cytochrome P450 isoforms in liver microsomes which catalyse in-vitro YM17E metabolism in man. In microsomes in man N-demethylation of YM17E to M1 occurred enzymatically; for up to 45 s the rate was linearly proportional to the microsomal protein concentration. This reaction was inhibited by metabolites M2-a, M2-b, M3 and M4. Further, N-demethylation of [14C]-YM17E was also inhibited by its product, M1. These results showed that primary metabolism of YM17E was inhibited by its products, and supported the finding that the non-linear increase in plasma concentration of the parent drug and metabolites observed in an in-vivo study was due to inhibition by these products. Metabolic activity in microsomes from ten individual human livers demonstrated that YM17E N-demethylase activity correlated closely with testosterone 6 beta-hydroxylase activity. When cytochrome P450 isozyme-specific substrates and chemical inhibitors were used to inhibit YM17E N-demethylase activity, CYP3A-specific substrate and inhibitors such as nifedipine, ketoconazole and triacetyloleandomycin strongly inhibited this activity, whereas CYP1A-specific substrate or inhibitor, ethoxyresorufin and alpha-naphthoflavone, inhibited weakly. Other CYP inhibitors, in contrast, had few or no effects. An inhibition study using anti-rat CYP1A1, CYP2B1, CYP2C11, CYP2E1 and CYP3A2 antibodies demonstrated that only anti-rat CYP3A2 antibody inhibited YM17E metabolism, to 40% of control level, with no other antibodies showing an inhibitory effect. Of seven cDNA-expressed P450 isoforms in man (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP2E1 and CYP3A4), CYP3A4, CYP2D6 and CYP1A2 isozyme exhibited substantial catalytic activity of N-demethylation of YM17E. These results indicate the predominant role of CYP3A4 in liver metabolism of YM17E in man.

Multiple dose bioequivalence study with josamycin propionate, a drug with highly variable kinetics, in healthy volunteers.

Josamycin is a macrolide antibiotic with considerable intra- and interindividual variability in kinetics. In the present study bioequivalence of an intact and dispersed josamycin Solutab tablet, containing 1,000 mg of josamycin in the form of josamycin propionate ester, was tested versus a Josacine 1,000 mg reference sachet. The design of this bioequivalence study was adapted to the drug's pharmacokinetic variability, comprising testing in steady-state, testing the reference in replicate, and maintaining a widened bioequivalence margin. The study was performed in a group of 24 male and 12 female healthy subjects, according to a 3-treatment 4-period crossover design. Blood sampling for establishing josamycin propionate and josamycin base serum level profiles were collected during the 12 h dosing interval on day 4. Steady-state serum levels were reached on day 4. With the reference sachet mean peak levels of 1.02 micrograms/ml and 0.36 microgram/ml were observed for parent drug and metabolite, respectively, reached at peak times of 1.5 h and 1.8 h. Comparable profiles were observed with the intact and dispersed Solutab tablets, both tending towards higher serum levels than the sachet. In terms of josamycin propionate levels as well as josamycin base levels, the intact and dispersed Solutab tablet was bioequivalent with the referent sachet within the preset 0.70-1.43 margins. Variability in josamycin kinetics proved to be substantial, maximum differences in peak levels and AUC values being about 10-fold between individuals, and 3-fold within individuals. Retrospectively, the multiple dosing regimen appeared not to result in a clear reduction of intrasubject variability.

Evaluation of the effect of zinc acetate on the stratum corneum penetration kinetics of erythromycin in healthy male volunteers.

Erythromycin with or without additional zinc acetate is used topically in the treatment of acne vulgaris. A potential effect of zinc on the stratum corneum penetration of erythromycin was investigated in human volunteers. Skin surface washings and tape strippings from the skin of the back were collected after drug applications in 12 subjects for quantification of erythromycin levels. Zinc acetate increased the amount remaining on the back skin at 6 h after application from 40 +/- 19 to 56 +/- 15% of the dose and, vice versa, reduced the amount in stratum corneum strips from 22 +/- 7 to 18 +/- 7%, both with statistical significance. The effect varied with body region. Zinc acetate thus provided to prolong the residence time of erythromycin on the skin.

Assay of erythromycin in tape strips of human stratum corneum and some preliminary results in man.

The antibiotic erythromycin is used topically in the treatment of acne vulgaris. In order to evaluate the in vivo cutaneous absorption behavior of erythromycin in man, an analytical procedure for erythromycin in stratum corneum tape strips was developed. Erythromycin was extracted from tape strips using solid-phase extraction, followed by alkaline diethyl ether extraction. Reconstituted extracts were analyzed by RP-HPLC with electrochemical detection. This procedure proved to be adequate in a pilot experiment in man, topical erythromycin appearing to be distributed over the stratum corneum, its upper layers displaying an inward decreasing gradient.

Pharmacokinetics of rectal drug administration, Part II. Clinical applications of peripherally acting drugs, and conclusions.

Part I of this article, which appeared in the previous issue of the Journal, covered general considerations, the physiology of the rectum, spreading of drugs into the colon, rectal absorption, partial avoidance of first-pass elimination, rate-controlled rectal delivery of drugs, irritation of the rectal mucosa and clinical applications of rectal administration, and discussed centrally acting drugs. In Part II, this discussion is extended to drugs which act peripherally and to methods of enhancing rectal drug absorption. The overall summary appeared in Part I.

Topical effects of absorption enhancing agents on the rectal mucosa of rats in vivo.

In the present study, attempts have been made to assess the effects of cefoxitin formulations with various absorption promoters on mucosal integrity after rectal delivery in rats. Observations were made at 2 and 24 h following drug administration. On macroscopic and histologic evaluation, all drug formulations affected mucosal structure in terms of hyperemia, edema, loss of goblet cell vacuoles, detachment of enterocytes, and increase of the number of inflammatory cells; these effects were not reversible in 24 h. The effects of formulations with MGK (a mixture of glyceryl-1-monooctanoate, glyceryl-1,2-dioctanoate, glyceryl-1,3-dioctanoate, glyceryl trioctanoate, glycerol, and octanoic acid), monoglycerides, 3-amino-1-hydroxypropylidene-1,1-diphosphonate, and 4% (w/v) sodium tauro-24,25-dihydrofusidate (STDHF) tended to exceed those observed with sodium salicylate, medium-chain fatty acids, Azone, and lower STDHF concentrations. The clinically used suppository bases Witepsol H15 and PEG 1540/6000 and indomethacin suppositories also affected mucosal structure. Although the interanimal variability in scores was very substantial, results indicate that rectal absorption enhancement is associated with modification of paracellular transport after detachment of enterocytes. However, the extent of drug absorption enhancement appeared not to be directly related to the extent of mucosal damage.

Improvement of drug absorption through enhancers.

The application of enhancers for improvement of drug absorption via the nasal, transdermal and rectal route are outlined. The importance of the relation between the time course of the enhancing effect and the desired plasma - concentration - time profile of the drug is stressed as well as the need to pay more attention to the safety aspects of absorption enhancers.

Absorption enhancement of rectally infused insulin by sodium tauro-24,25-dihydrofusidate (STDHF) in rats.

The bile salt derivative sodium tauro-24,25-dihydrofusidate (STDHF) has been reported to promote nasal absorption of insulin. In the present study the effect of STDHF on rectal insulin absorption was investigated in rats. At concentrations of 1 and 4% (w/v) it enhanced insulin bioavailability from 0.2 +/- 0.2 (control) to 4.2 +/- 3.2 and 6.7 +/- 2.1%, respectively, as assessed by radioimmunoassay. Insulin preparations with STDHF reduced blood glucose concentrations considerably in a concentration-dependent way. Coadministration of STDHF with Na2EDTA (0.25%, w/v) tended to increase further insulin bioavailability and hypoglycemic response. Varying the site of rectal administration did not influence these parameters.

Rectal absorption enhancement of cefoxitin and desglycinamide arginine vasopressin by sodium tauro-24,25-dihydrofusidate in conscious rats.

The effects of sodium tauro-24,25-dihydrofusidate (STDHF), an enhancer of nasal insulin absorption, on the rectal absorption of cefoxitin and desglycinamide arginine vasopressin (DGAVP) were evaluated in the rat. Cefoxitin and DGAVP proved to be poorly absorbed rectally without STDHF, but their bioavailability was considerably increased by STDHF in concentrations of 0.15 to 8% w/v. Both rectal infusion and rectal bolus delivery resulted in complete cefoxitin absorption at 4% w/v of STDHF. Delivery rate appeared to be an important factor in the effect of 4% w/v of STDHF on DGAVP bioavailability; on infusion a mean DGAVP bioavailability (+/- S.D.) of 47 +/- 12% was obtained, whereas after bolus delivery it amounted to 27 +/- 6%. For both compounds the effect of STDHF was significant at 0.5% w/v. It is concluded that STDHF is capable of actively enhancing the rectal absorption of poorly absorbed drugs, including small peptides.

3-Amino-1-hydroxypropylidene-1,1-diphosphonate (APD): a novel enhancer of rectal cefoxitin absorption in rats.

The promoting action of the calcium chelating compound EDTA on intestinal drug absorption is supposed to be based on Ca2+ depletion, inducing widening of tight junctions. The aim of the present study was to evaluate the effects of the calcium-binding agent 3-amino-1-hydroxypropylidene-1,1-diphosphonate disodium salt (APD) on rectal cefoxitin absorption in rats. The extent of rectal cefoxitin absorption was enhanced by 0.5 to 6% w/v of APD, on rectal infusion as well as on bolus delivery, the latter regimen tending to result in lower bioavailabilities. A maximal cefoxitin bioavailability of 85 +/- 10% was achieved by infusion with 4% w/v of APD, compared with 14 +/- 12% without APD.