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Antimicrobial Agents and Chemotherapy, May 2005, p. 1813-1822, Vol. 49, No. 5
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.5.1813-1822.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Clinical Pharmacokinetics of Alamifovir and Its Metabolites

Clark Chan,1* Eyas Abu-Raddad,2 Georg Golor,3 Hikari Watanabe,4 Akira Sasaki,4 Kwee Poo Yeo,1 Danny Soon,1 Vikram P. Sinha,2 Shawn D. Flanagan,2,{dagger} Minxia M. He,2 and Stephen D. Wise1

Lilly-NUS Centre for Clinical Pharmacology, National University of Singapore, Singapore, Republic of Singapore,1 Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana,2 PAREXEL Institute of Clinical Pharmacology, Berlin, Germany,3 Mitsubishi Pharma Corporation, Tokyo, Japan4

Received 24 August 2004/ Returned for modification 15 November 2004/ Accepted 27 January 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Alamifovir, a purine nucleotide analogue prodrug, and its hydrolyzed derivatives have shown preclincal efficacy activity against wild-type and lamivudine-resistant hepatitis B virus. Two studies were conducted to examine the single- and multiple-dose alamifovir pharmacokinetics after oral administration in healthy males. In study 1, subjects were given single doses (0.2 to 80 mg), with a subset receiving 20 mg in a fed state. Study 2 subjects were dosed with 2.5 to 15 mg twice daily for 15 days. Plasma samples were collected over 72 h in study 1 and over 24 h on days 1 and 15 in study 2. Concentrations of alamifovir and its major metabolites were determined using liquid chromatography/tandem mass spectrometry methods. The data were analyzed using a noncompartmental technique. Although alamifovir was rapidly absorbed, there was limited systemic exposure due to its rapid hydrolysis and formation of at least three metabolites, suggesting that alamifovir acts as a prodrug. The major metabolites detected were 602074 and 602076, with 602075 detectable only in higher-dose groups. Maximum 602074 plasma concentration was achieved at approximately 0.5 h (Tmax) and declined with a 1- to 2-h terminal half-life (t1/2). Maximum concentrations of 602076 (Cmax) averaged 10% of the 602074 Cmax and reached Tmax in 2.5 h with a 4-h t1/2. Food appeared to decrease the extent of absorption of the compound. Multiple dosing resulted in minimal accumulation, and the concentrations following multiple doses could be predicted using the single-dose data. Alamifovir was well tolerated and the pharmacokinetics were characterized in these studies.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Despite safe and effective vaccines, over 350 million people worldwide are chronic carriers of the hepatitis B virus (10). The greatest prevalences of hepatitis B (HBV) infection are in Asia, the Middle East, and Africa (2). The chronic carrier state of the virus can lead to potentially fatal complications such as end-stage liver disease, cirrhosis, and hepatocellular carcinoma (16). Successful therapy of patients with hepatitis B requires long-term suppression of viral replication. Lamivudine has become the standard oral therapy for chronic carriers of the virus. Lamivudine is a nucleoside analogue and has been shown to be effective in suppressing hepatitis B viral replication by inhibiting reverse transcriptase (3, 19, 21). However, prolonged lamivudine monotherapy results in the emergence of lamivudine-resistant mutants in over 50% of patients after 2 years and 70% after 4 years (1, 6, 11, 12, 13, 18). Therapy with alpha interferon is only beneficial for a well-defined group and has also shown high long-term remission rates and low overall response rates (8, 20). New antiviral treatments, especially those effective against lamivudine-resistant strains, need to be investigated.

Alamifovir {MCC-478; 2-amino-9-[2-(phosphonomethoxy)ethyl]-6-(4-methoxyphenylthio)purine bis(2,2,2-trifluoroethyl)ester} has been shown to be active against wild-type HBV as well as lamivudine-resistant mutants in vitro. The effective concentration of alamifovir required to reduce replication by 50% (EC50) for the wild-type was 0.027 µM, which is approximately 20 times less than lamivudine, and the EC50 for the lamivudine-resistant mutants was 2.6 to 3.3 µM (14). The mechanism of action of alamifovir appears to be via the inhibition of the protein priming reaction and the packaging reaction, thereby decreasing HBV replication (T. Shaw, D. Colledge, V. Sozzi, S. Locarnini, Abstr. 54th Annu. Meet. Am. Assoc. Study Liver Dis., abstr. 716A, 2003). This appears to be a novel mechanism of action compared to the conventional nucleoside/nucleotide reverse transcriptase inhibitors.

To date, alamifovir has been studied in rats, mice, cynomolgus monkeys, ducks (S. Yuasa, and N. Kamiya, Abstr. 52nd Annu. Meet. Am. Assoc. Study Liver Dis., abstr. 587, 2001), and woodchucks (S. Yuasa, N. Kamiya, K. Yamabe, and Y. Yamaguchi, Abstr. 52nd Annu. Meet. Am. Assoc. Study Liver Dis., abstr. 591, 2001). In rat and monkey studies, orally administered [14C]alamifovir was rapidly absorbed with peak plasma concentrations of radioactivity at approximately 0.3 h and 0.7 h, respectively. The oral bioavailability of alamifovir, as assessed by radioactivity of 14C-labeled compound in rats and cynomolgus monkeys, was approximately 10% and 35%, respectively (Eli Lilly and Co., Indianapolis, IN, unpublished data). Preliminary data in animals also indicates that the bioavailability of alamifovir may decrease when it is given with food. In contrast, the oral bioavailability of the broad-spectrum antiviral adefovir was approximately 4% in cynomolgus monkeys (5, 17) and less than 12% in humans (4). Therefore, in comparison to adefovir, alamifovir has shown greater oral bioavailability.

In animal species, alamifovir is quickly and extensively hydrolyzed to monoester 602074 (GCD-187) and then oxidized to form the O-desmethyl product 602075 (GCD-231) (Fig. 1) (9). The major circulating metabolite in the rat is 602075, and the major metabolite in monkeys and woodchucks is 602074. Metabolite 602074 was further hydrolyzed to form the free acid 602076 (GCD-189). In the woodchuck model, the exposure to 602076 metabolite was approximately 20% of 602074, with 602075 detectable only in small amounts (Eli Lilly and Co., Indianapolis, IN, unpublished data). The metabolites have also been shown to have anti-HBV activity (9). In the animal studies, since alamifovir is quickly and extensively hydrolyzed, with limited amounts detectable in the systemic circulation, it is likely that the metabolites significantly contribute to in vivo efficacy.



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  		FIG. 1. Proposed metabolic pathway of alamifovir.

 
Orally administered [14C]alamifovir in rats and monkeys is predominantly excreted via the fecal route (due to biliary excretion and unabsorbed dose), which accounted for 86.7% ± 4.2% (mean ± standard deviation) of the total radiocarbon administered in the monkey and 95.8% ± 1.7% in the rat. The metabolites 602074 and 602076 are both extensively protein bound. Both these metabolites were greater than 90% protein bound in humans and monkeys (Eli Lilly and Co., Indianapolis, IN, unpublished data). Therefore, only a relatively small amount of the absorbed drug would be expected to be filtered through the glomerulus.

A preclinical safety profile of alamifovir was established in rats, monkeys, and woodchucks after single- and repeat-dose toxicology studies for up to 9 months' duration. There was no evidence of mitochondrial toxicity in vitro or of specific organ toxicity in the animal models. Although alamifovir exhibited renal toxicity in rats with regard to increases in serum creatinine and elevations in urinary enzyme lactate dehydrogenase and beta-N-acetylglucosamine, these findings were observed at a dose significantly higher than the toxic doses of adefovir dipivoxil (Eli Lilly and Co., Indianapolis, IN, unpublished data). Plasma concentrations over the spectrum of effects in animals have identified a safe and appropriate target range of exposure for clinical studies.

To date, the clinical pharmacokinetic properties of alamifovir have not been described in detail. To be able to study alamifovir in a patient population, pharmacokinetic properties, dose proportionality, and the food effect on drug exposure need to be assessed. The aim of the studies presented is to examine the tolerability and clinical pharmacokinetics of alamifovir after oral administration of single and multiple doses in healthy subjects.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects. Eighty (study 1) and 32 (study 2) healthy adult males, as determined by a medical history and physical examination and a 12-lead electrocardiogram, were enrolled after giving written informed consent to participate in the studies. Subjects were free to withdraw from the study at any time. Subject demographics in both studies are summarized in Table 1. Study 1 was approved by the Ethics Committee at the Medical Board (Ärztekammer) of Berlin, Germany, and study 2 was approved by the Research and Ethics Committee of the National University Hospital, Singapore. Both studies were performed in accordance with the Declaration of Helsinki for biomedical research in humans, International Conference on Harmonisation/Good Clinical Practice standards, and the respective local laws and regulations.


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  		TABLE 1. Subject demographics

 
The sample size determination for these studies followed empirical and safety considerations; therefore, no formal statistical sample size calculations were necessary.

Study design. Study 1 was a double-blind, randomized, placebo-controlled, single-dose escalating study. The subjects were allocated to 11 treatment groups (0.2-, 0.6-, 2-, and 6-mg alamifovir capsule formulation and 5-, 10-, 20-, 40-, 60-, and 80-mg tablet formulation) with a total of eight subjects in each group. Subjects were randomly assigned, at a ratio of 3 to 1, to receive either a single oral dose of alamifovir (n = 6) or a matching placebo (n = 2). No food intake was allowed from 12 h before until 4 h after dosing. The doses were swallowed entirely with 200 ml of room temperature water. Blood, urine, and fecal samples were collected during the subsequent 72 h. Dose-escalation steps were separated by a minimum of 7 days and moved forward only if the preceding lower doses were well tolerated.

Following a 4-week washout, the eight subjects from the 20-mg treatment who had received the study drug in a fasted state were enrolled into a 20-mg fed treatment group. The 20-mg fed group received a standardized high-fat breakfast (62% fat, 24% carbohydrate, and 14% protein) approximately 30 min prior to dosing. The same subjects who received alamifovir in the fasted state received alamifovir in the fed state, and the subjects that received the placebo control continued to receive the placebo. All other study procedures and conditions for the 20-mg fed group were kept consistent with the previous treatments.

Study 2 was a single-blind, randomized, placebo-controlled, multiple-dose escalation study of alamifovir. The subjects were allocated to one of four treatment groups (2.5, 5, 10, and 15 mg of alamifovir in a tablet formulation) with a total of eight subjects in each group. Each treatment period was 15 days in duration. On day 1 of the treatment, the subjects in group 1 were randomly assigned, at a ratio of 3 to 1, to receive either 2.5-mg oral doses of alamifovir (n = 6) or placebo (n = 2) for the duration of the study. After the initial dosing day, 14 subsequent days of twice-daily (BID) oral doses of alamifovir or placebo administration commenced. As with the first dosing day, on the last day of dosing (day 15), the subjects received only the morning dose. Serial venous blood samples were also collected at predose and for the subsequent 24 h from dosing on days 1 and 15. After a review of the safety data, the dose was escalated for the next group.

Safety. For both study 1 and study 2, subjects were monitored with regard to their clinical and laboratory safety throughout the study. This included a routine physical examination, assessment of vital signs, 12-lead electrocardiogram, and clinical laboratory tests. Further routine follow-up medical assessments were conducted at approximately 2 weeks after the final dose.

Sampling. For study 1, serial venous blood samples (15 ml) for the determination of alamifovir, 602074, 602075, and 602076 concentrations in plasma were collected into glass tubes containing sodium heparin via an indwelling cannula. Blood samples were collected just prior to dose and at 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 24, 36, 48, and 72 h postdose during each treatment period. Samples were immediately centrifuged for 10 min at 2,000 x g and at 4°C. Plasma was divided into two aliquots of 3.5 ml, with acetic acid (70 µl) added to one of the aliquots to stabilize the parent compound. Each 3.5 ml was further separated into two tubes, with one containing 2 ml for the assay; the remaining portion of the sample was stored as a backup. All samples, including the backup aliquots, were stored at –20°C. The 2-ml plasma samples with and without acetic acid were used for the assay of alamifovir and its metabolites, respectively. Urine samples were collected from predose until 96 h postdose. Fecal samples were collected only from the 10-mg treatment group from 1 day prior to dosing until 5 days after dosing.

For study 2, on the first dosing day (day 1) and the last dosing day (day 15) when subjects received only the morning dose of alamifovir, serial venous blood samples (6 ml) were collected just prior to dosing and at 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 16, and 24 h. Samples were immediately centrifuged for 10 min at 3,000 x g and at 4°C. For every 1 ml of plasma obtained, 50 µl of hydrochloric acid was added to each sample before storage at –80°C.

Drug assay. For study 1, the determination of alamifovir, 602074, 602075, and 602076 concentrations in plasma and urine samples were performed using a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method (Mitsubishi Kagaku Bio-Clinical Laboratories, Inc., Tokyo, Japan). A structural analogue of alamifovir was used as the internal standard (GCD-199). The internal standard, 10 µl of acetonitrile, and 500 µl of water were added to plasma and urine samples before they were placed on an OASIS HLB extraction plate (Waters Corporation, Milford, Mass.). The plate was eluted with 200 µl of acetonitrile-water (1:1), and the eluate was filtered under 5,000 x g centrifugation for 2 min. The supernatant was injected on a Shiseido CAPCELL PAK C18 (2.0 by 150 mm; particle size, 5 µm) column and eluted with an acetonitrile-water (1:1) mobile phase at a flow rate of 0.2 ml/min. Alamifovir and its internal standard were detected in the positive-ion mode, and the metabolites were detected in the negative-ion mode. The calibration curves were validated and were linear over the concentration range (lower limit of quantification to upper limit of quantification) of 0.348 to 34.757 nmol/liter for the parent, 1.013 to 202.675 nmol/liter for 602074, 4.172 to 417.211 nmol/liter for 602075, and 4.862 to 486.172 nmol/liter for 602076. The intraday and interday variability of the assays was 10.6% or less. The determination of alamifovir in feces was performed by LC/MS/MS (Mitsubishi-Tokyo Pharmaceuticals, Inc., Tokyo, Japan).

For study 2, alamifovir, 602074, 602075, and 602076 concentrations in plasma were assayed by a validated Turbo IonSpray LC/MS/MS detection method (Advion BioSciences, Inc., Ithaca, N.Y.). Alamifovir and its internal standard structural analogue (602079) and 602074, 602075, 602076, and their internal standard analogue (602078) were extracted from human plasma samples by precipitating plasma proteins. The supernatants were evaporated to dryness and then reconstituted in methanol-water (20:80). The analytes were placed on a Zorbax Eclipse XDB-C8 (2.1 by 50 mm; 5 µm) column and eluted with both a 20:80 methanol:water and 80:20 methanol:water mobile phase at a flow rate of 0.3 ml/min. Concentrations of alamifovir and three metabolites were determined using a Turbo IonSpray LC/MS/MS. Alamifovir and its internal standard 602079 were detected in the positive-ion mode, and 602074, 602075, 602076, and their internal standard 602078 were detected in negative-ion mode. The calibration curves were validated and were linear over the concentration range (lower limit of quantification to upper limit of quantification) of 0.869 to 130.338 nmol/liter for alamifovir, 1.013 to 152.006 nmol/liter for 602074, 1.043 to 156.454 nmol/liter for 602075, and 1.215 to 182.315 nmol/liter for 602076. The intraday and interday variability of the precision of the assay was 12.5% or less for 602076 and 7.2% or less for alamifovir, 602074, and 602075.

In general, alamifovir and its metabolites were stable in the plasma and urine samples for at least 20 h at room temperature and at least 6 months when placed in the freezer.

Pharmacokinetic analysis. Using WinNonlin Professional version 3.1 (Pharsight Corporation, Mountain View, Calif.), noncompartmental pharmacokinetic parameters were derived from each subject's data using the actual sampling times. Maximum concentration (Cmax) of drug in plasma after a single dose and at steady state (Cmax,ss) and the time taken to reach Cmax and Cmax,ss (Tmax and Tmax,ss, respectively) were recorded from the observed data. The elimination rate constant (kel) was derived from a linear regression of the terminal log-linear disposition phase of the concentration-time curve for each subject. The elimination half-life (t1/2) was calculated as ln(2)/kel. The area under the plasma concentration-time curve from time of dosing to the last measurable concentration (AUC0-t) for the single dose and the area under the concentration-time curve at steady state on day 15 (only morning dose administered) during the 24-h postdose period (AUC{tau},ss, where {tau} = 24 h) were calculated using the linear/log trapezoidal rule, where the linear trapezoidal rule and the log trapezoidal rule were applied up to and after the Tmax, respectively. The area under the concentration-time curve from time of dosing to infinity (AUC0-{infty}) was calculated as AUC0-t + C(t)/kel, where extrapolation is based on the predicted concentration at the last quantifiable time point [C(t)]. The predicted value was based on the linear regression performed to estimate the terminal elimination rate constant and was dependent on three or more data points. The accumulation ratio for the steady-state data was calculated as AUC{tau},ss/AUC{tau},Day 1, where AUC{tau},Day 1 is the area under the concentration-time curve from dosing to 24 h postdose on day 1. For the purpose of the pharmacokinetic analysis, the data were converted to molar units using the molecular weights of alamifovir, 602074, 602075, and 602076 as 575.425, 493.4011, 479.3743, and 411.3772, respectively.

The amount of each analyte excreted (Ae) into the urine was defined as the sum of the amount recovered within 96 h of dosing, with the assumption that no relevant drug amounts were excreted beyond this collection period. The renal clearance was obtained by calculating the Ae/AUC0-{infty} ratio.

Statistical analysis. The pharmacokinetic parameters Cmax and AUC0-{infty} were log transformed prior to statistical analysis. Food effect analysis was performed using analysis of variance (22). The inter- and intrasubject coefficients of variation (CV%) were obtained from the variance components of a mixed effects model that included subject as a random factor. For dose proportionality assessment of the single-dose data, a power model (7) was used with a log-transformed pharmacokinetic parameter as the dependent variable, log-transformed dose level as a continuous covariate, and study 1 and study 2 as categorical covariates. Where appropriate, the 90% or 95% confidence intervals (CI) were determined.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pharmacokinetics. (i) Study 1, single-dose study. The subject demographics for study 1 are presented in Table 1. The parent compound, alamifovir, was rapidly absorbed and metabolized to form at least three metabolites. Concentrations of the parent compound were detected only in the higher-dose groups (5 to 80 mg) within 1 h of dosing. Due to the high proportion of samples that were below the limit of quantification (approximately 94%), it was only possible to determine a limited number of pharmacokinetic parameters for the parent compound (Table 2). There were no detectable concentrations of alamifovir in the urine or the feces.


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  		TABLE 2. Alamifovir and metabolite pharmacokinetic parameter values following single oral doses in study 1

 
The major metabolite observed following single oral doses of alamifovir was the 602074 metabolite. Plasma concentrations of 602074 were quantifiable in all dose groups, with concentrations two orders of magnitude higher than those of the parent compound (Table 2). The metabolite was rapidly formed, reaching a peak plasma concentration usually in 0.5 to 1 h in the fasted state. Following the peak, 602074 declined monoexponentially, with an apparent terminal half-life of approximately 1 h in the lower-dose groups (0.2 to 20 mg). In the higher-dose groups (40 to 80 mg), plasma concentrations of 602074 were quantifiable even at later time points, and the terminal half-life appeared prolonged to nearly 2 h (Fig. 2). The greatest variability in the 602074 mean values occurred at the first two sampling time points, and the overall median (range) CV% for the mean points at all dose levels was 54.2 (16.4 to 171). The metabolite 602074 was detectable in the urine in all dose groups, the amount excreted in the urine increased with dose, and the renal clearance appeared independent of dose.



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  		FIG. 2. Mean plasma concentrations of 602074 and 602076 metabolites following single oral doses of alamifovir in study 1.

 
The metabolite 602075 could not be quantified in subjects receiving the 0.2- to 6-mg oral dose or the 20-mg dose in the fed state. In the other dose groups, 602075 reached a peak plasma concentration approximately 0.5 to 1 h after dosing. In all treatment groups, the 602075 concentrations were below the lower limit of detection of the assay by 1.5 h. Urinary excretion of 602075 was only observed in the 10-, 60-, and 80-mg dose groups. Similar to the results for the 602074 metabolite, renal clearance was less than the standard glomerular filtration rate (15). The amount of 602075 excreted in the urine and other derived pharmacokinetic parameters are presented in Table 2.

Quantifiable concentrations of 602076 were observed in all subjects given an oral dose of alamifovir greater than 2 mg (Table 2). The maximum 602076 plasma concentration was approximately 18-fold higher than that of the parent compound in the two highest dose groups but averaged only 10% of the 602074 metabolite. The maximum plasma concentration of 602076 was reached at approximately 2 to 4 h postdose, which is generally later than the time taken to reach the maximum alamifovir, 602074, and 602075 plasma concentrations (Fig. 2). The overall median (range) CV% for the mean 602076 points at all dose levels was 51.7 (14.2 to 117). The geometric mean terminal half-life ranged from 2 to 5.4 h and did not show evidence of dose dependency. Measurement of 602076 urinary excretion was not conducted in this study.

(ii) Study 1, formulation comparison and food effect. As apparent in Table 2, the exposure of the metabolites following the administration of the tablet formulation appeared slightly higher than that of the capsule formulation.

A comparison of fed and fasted conditions at the 20-mg dose level was used to assess the food effect on plasma exposure of alamifovir metabolites. The estimated ratio of fed/fasted for 602074 AUC0-{infty} was 0.48 (90% CI, 0.30 to 0.79) and 0.38 (90% CI, 0.19 to 0.74) for the Cmax. This indicates that food decreases the overall exposure of 602074 by approximately 50%. Food causes an even greater decrease in the 602076 exposure with the fed/fasted ratios for the 602076 AUC0-t and Cmax of 0.18 (90% CI, 0.10 to 0.31) and 0.24 (90% CI, 0.16 to 0.36), respectively. There is no evidence of a significant difference in Tmax for both metabolites.

(iii) Study 2, single-dose study. The subject demographics for study 2 are presented in Table 1. Alamifovir was rapidly absorbed with a median Tmax of 0.5 h postdose. Consistent with the results of study 1, the parent compound was only quantifiable in a limited number of samples within 1 h of dosing and only at the highest 15-mg dose group (Table 3).


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  		TABLE 3. Alamifovir and metabolite pharmacokinetic parameter values following single and multiple doses in study 2

 
The major metabolite observed was 602074, which was quantifiable even at the lower-dose levels. The time to maximum plasma concentrations across all treatments in this study was approximately 0.5 h, and the terminal half-life of 602074 was approximately 1 to 1.5 h. Plasma concentrations of 602074 were quantifiable up to approximately 10 h after dosing, and the terminal half-life appeared to increase with increasing doses. The mean concentration-time profiles at each dose group on day 1 are shown in Fig. 3, with the derived pharmacokinetic parameters summarized in Table 3.



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  		FIG. 3. Mean plasma concentrations of 602074 and 602076 metabolites following single and multiple oral doses of alamifovir in study 2.

 
Only a limited number of samples were quantifiable for 602075 in the 5-mg and 10-mg dose groups. Noncompartmental pharmacokinetic parameters for this metabolite could be derived only for the 15-mg dose group (Table 3). The metabolite was formed rapidly with a median Tmax of 0.75 h and a terminal half-life of approximately 1.3 h.

The median Tmax of 602076 across all treatments was approximately 2.5 h, which is greater than the values for the 602074 and 602075 metabolites. The geometric mean terminal half-life of 602076 was 4 to 5 h, which was consistent across the dose levels. The derived 602076 pharmacokinetic parameters are presented in Table 3, and the mean concentration-time plots at each treatment group are illustrated in Fig. 3.

(iv) Study 2, multiple-dose study. The pharmacokinetic properties of the parent compound were not altered following 15 days of twice-daily alamifovir dosing. Similarly, there were no changes in the 602074 Cmax, Tmax, and the area under the curve following multiple doses (Table 3). There was no accumulation of the 602074 metabolite following multiple dosing due to the short half-life of 602074 relative to the dosing interval.

Similarly, there were no apparent differences in the 602076 Cmax, Tmax, and area under the curve following multiple doses of alamifovir compared to single-dose administration. Of all the analytes measured, 602076 had the longest terminal half-life of 4 to 5 h. However, the 602076 terminal half-life was still short relative to the dosing period, and therefore accumulation of this metabolite was similarly minimal.

The mean concentration-time profiles of 602074 and 602076 for each dose group on day 1 and on the last dosing day (day 15) are shown in Fig. 3. The overall median CV% (range) for the mean 602074 and 602076 points at all dose levels were 45.0 (16.9 to 102) and 38.0 (5.64 to 82.9), respectively.

Assessments of inter- and intrasubject variability were based on the pooling of the single- and multiple-dose data in study 2. The intersubject CV% for the AUC0-{infty} and Cmax of 602074 was less than or equal to 31%, and the intrasubject CV% was 32% or less. For the 602076 AUC0-{infty} and Cmax, the intersubject CV% was less than or equal to 25%, and the intrasubject CV% was 37% or less.

(v) Dose proportionality. With the exclusion of the 20-mg (fed) treatment in study 1, single-dose data from study 1 and study 2 were combined for the dose proportionality assessment of Cmax and AUC0-{infty} for the 602074 and 602076 metabolites (Fig. 4). For the 602074 metabolite, the estimation of the slope parameter over the 0.2- to 80-mg dose range was 1.11 (95% CI, 1.06 to 1.16) for Cmax and 1.15 (95% CI, 1.07 to 1.23) for AUC0-{infty}. Since the lower bound of the 95% CI was greater than 1, this indicates that over the 0.2- to 80-mg dose range with a doubling of dose there would be a slightly greater than doubling of the Cmax and AUC0-{infty}. When considering a smaller and more therapeutically relevant 2.5- to 20-mg dose range, as used in a 4-week patient study (S. Lowe, J. McGill, V. Sinha, D. Soon, C. H. Teng, S. D. Wise, and K. Sathirakul, Abstr. Digestive Disease Week, abstr. 1369, 2002), the slope estimations of Cmax and AUC0-{infty} were 1.05 (95% CI, 0.87 to 1.24) and 1.11 (95% CI, 0.87 to 1.35), respectively. Since the 95% CI contains 1, there would be no evidence of deviation from a slope of unity over the 2.5- to 20-mg dose range.



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  		FIG. 4. Dose proportionality assessment of Cmax and AUC0-{infty} for the 602074 and 602076 metabolites.

 
For the 602076 metabolite, the estimate of the slope parameter over the 0.2- to 80-mg dose range was 0.88 (95% CI, 0.75 to 1.01) for Cmax, which included unity. However for AUC0-{infty} the estimate was 0.76 (95% CI, 0.59 to 0.94), where the upper bound of the 95% CI was less than 1, indicating that over the 0.2- to 80-mg dose range, with a doubling of dose the AUC0-{infty} would be slightly less than doubled. As with the 602074 metabolite, when an estimation of the slope parameter is made over a narrower dose range (2.5 to 20 mg), both the slope parameter estimation of Cmax (1.09 [95% CI, 0.84 to 1.34]) and the AUC0-{infty} (0.89 [95% CI, 0.58 to 1.19]) contains unity, although the 95% CI became wider.

Safety. From the 80 subjects enrolled in study 1, a total of 17 adverse events were reported. Common adverse events were headaches, rhinitis, and dizziness, and the number and incidence of these events were not related to dose (Table 4). There were no serious adverse events or withdrawals from the study due to adverse events. Overall, there were no clinically significant abnormalities in clinical laboratory evaluation parameters, vital signs, and 12-lead electrocardiograms.


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  		TABLE 4. Common adverse events reported after single doses of alamifovir in study 1

 
From the 32 subjects in study 2, there were a total of 162 adverse events reported, of which 82 were possibly related to the study drug. Of these, the most commonly reported events were upper abdominal pain, diarrhea, and nausea, but these were not related to increases in the dose (Table 5). There were no serious adverse events or withdrawals from the study due to adverse events. There appeared to be a drug-related change in serum alanine transaminase (ALT) elevation in one subject in the 10-mg BID group and two subjects in the 15-mg BID treatment group. The maximum extent of ALT elevations attributable to alamifovir was approximately three times the upper limit of normal (70 U/liter) but in all cases the ALT levels returned to a normal range in the subsequent weeks. There were no other clinically significant abnormalities in clinical laboratory evaluation parameters, vital signs, and 12-lead electrocardiograms throughout the study.


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  		TABLE 5. Common adverse events reported after single and multiple doses of alamifovir in study 2

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Alamifovir is a potential agent for the treatment against wild-type HBV as well as lamivudine-resistant mutants. The aim of these studies was to examine the clinical pharmacokinetics of alamifovir after administration of single and multiple oral doses in healthy subjects. The overall discussion of the results summarizes both study 1 and study 2 data together, though it is not the intention to make direct comparisons between these studies, as two different assay methods were used. The parent compound was rapidly absorbed following oral administration, with no unchanged drug detected in the feces. Peak plasma concentrations were generally reached within a half hour of dosing, although this could have been earlier given the limitation of the sampling schedule used. The overall low exposure of the parent compound can be accounted for by its rapid enzymatic and/or chemical hydrolysis and further oxidation to form at least three metabolites. Since there is very little systemic exposure to alamifovir following administration, anti-HBV activity of the compound in vivo would be assumed to be mainly due to the hydrolyzed derivatives of alamifovir, and alamifovir itself acts primarily as a prodrug.

The plasma metabolite profiles of alamifovir in humans are comparable to the preclinical woodchuck model rather than the rat model, in that the 602075 metabolite was detected only in limited amounts, 602074 contributed to the greatest metabolic fraction, and 602074 and 602076 were the major metabolites.

The 602074 metabolite was quantifiable at all dose levels. The maximum concentration for 602074 was rapidly reached within 0.5 to 1 h after dosing in the fasted state and was generally a half hour later than the peak plasma concentration of the parent compound. Following the peak, 602074 declined monoexponentially, with a terminal half-life of approximately 1 h in the lower-dose groups (0.2 to 20 mg) and was prolonged to nearly 2 h in the higher-dose groups. The apparent slight prolongation of the half-life is likely due to the presence of a second phase of decline that could not be observed at lower doses due to assay limitations. The metabolite 602074 was excreted unchanged in the urine at a dose-independent clearance rate. This rate of renal clearance is less than the standard glomerular filtration rate for a healthy adult (15) and would, therefore, indicate that net renal reabsorption of the 602074 metabolite occurs.

The 602075 metabolite was only quantifiable in treatments of alamifovir equal to or greater than 10 mg. Similar to 602074, the 602075 metabolite was rapidly formed, reaching a peak concentration approximately 1 h after dosing of the parent. This would indicate that hydrolysis of the parent to the 602074 and the oxidation of 602074 to 602075 is very rapid.

Metabolite 602076 was the second most abundant metabolite observed in humans, averaging 10% of the amount of 602074. Peak concentrations of 602076 occurred approximately 2.5 h after dosing, which is a pronounced delay compared to the other metabolites. This is consistent with the proposed metabolic pathway, where the formation of the 602076 metabolite is limited by the rate of formation of the 602074 metabolite (Fig. 1). Although a direct conversion of alamifovir to 602076 could be proposed, the fact that 602076 is the acid of the monoester 602074 suggests that the formation of 602076 from alamifovir occurs indirectly through 602074. Following the peak, 602076 declines monoexponentially, with a half-life of approximately 2.5 to 5.5 h, which was the longest of all the analytes.

A comparison of fed (high-fat meal) and fasted conditions at the 20-mg dose level was used to assess the food effect on alamifovir pharmacokinetics. Given the limited quantifiable concentrations and high variability for the 20-mg fed treatment (geometric CV, 304%), it was not possible to determine the food effect on the exposure of the parent compound. For the 602074 metabolite, food appears to decrease the overall exposure by 50% and causes an even greater decrease in 602076 exposure by over 75%. The observed food effect may be compounded by the potential nonlinearity of 602074 and 602076 exposure. From this preliminary assessment based indirectly on the metabolite data, the presence of food in the stomach appears to decrease the bioavailability of alamifovir.

The 602074 and 602076 metabolites were assessed for dose proportionality using the power model (7). With the exclusion of the 20-mg (fed) treatment, single-dose data from study 1 and study 2 were combined for the dose proportionality assessment. Although both assays were validated, interpretation of the pooled data should be limited by the fact that two different assay methods were used. This analysis indicates that over more than two orders of magnitude of dose (0.2 to 80 mg) there is a slightly greater than linear increase in exposure of 602074. Over the same dose range, the exposure of the 602076 metabolite indicated a less than linear increase in exposure with increases in dose. However, with a narrower therapeutic dose range (2.5 to 20 mg), as used in a 4-week trial in HBV patients (Lowe et al., Abstr. Digestive Disease Week 2002), the exposure of both major metabolites did not appear to deviate from unity. However, the 95% CI was larger with the narrower dose range, reflecting the small number of subjects. An adequately powered study, using a single alamifovir formulation, is required to formally assess proportionality of the derivatives.

Multiple twice-daily administration of alamifovir did not result in accumulation of the 602074 metabolite and resulted in only minor accumulation of the 602076 metabolite. This is probably due to the short half-life of the major metabolites compared to the dosing interval and suggests time-independent pharmacokinetic properties. Given the reported half-life of the major metabolites, steady state would be expected to be attained after the second day of dosing with little to no accumulation. Similar to adefovir pharmacokinetics, the relatively short half-life of the study drug in plasma may not necessarily reflect the duration of action of the drug, which is dependent on the active metabolites present within the cell (4). Therefore, intracellular and effect site kinetics, as well as the dynamics of the pharmacological response, need to be considered when determining the optimal dosing regimen.

In summary, alamifovir acting as a prodrug is rapidly absorbed and metabolized to form 602074, 602075, and 602076 in humans. These hydrolyzed derivatives are likely to contribute significantly to the in vivo anti-HBV efficacy. Multiple dosing resulted in minimal accumulation, and the concentrations following multiple doses could be predicted using the single-dose data. Alamifovir was well tolerated by the healthy subjects in both of the studies.


   ACKNOWLEDGMENTS
 
The work in study 1 and study 2 was financially supported by Mitsubishi-Tokyo Pharmaceuticals, Inc., and Eli Lilly and Company, respectively.

We thank all those involved at PAREXEL GmbH for leading the way with the first study and also the staff at Lilly-NUS Centre for Clinical Pharmacology Pte Ltd for their extraordinary effort with the recruitment and conduct of study 2. Also special mention goes to K. Sathirakul who played a role in the planning of study 2.


   FOOTNOTES
 
* Corresponding author. Mailing address: Lilly-NUS Centre for Clinical Pharmacology, Level 6 Clinical Research Centre (MD11), National University of Singapore, 10 Medical Dr., Singapore 117597, Republic of Singapore. Phone: 65 6413 9802. Fax: 65 6779 0587. E-mail: clark_chan{at}lilly.com. Back

{dagger} Present address: Ligand Pharmaceuticals, San Diego, CA 92121. Back


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Antimicrobial Agents and Chemotherapy, May 2005, p. 1813-1822, Vol. 49, No. 5
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.5.1813-1822.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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