Introduction

Top Abstract Introduction Materials and Methods Design of Studies in... Design of Studies in... Sample Analysis Pharmacokinetic Assessments. Results Discussion References

Thrombin plays a central role in the processes of hemostasis and thrombus formation. Among its numerous functions are the catalysis of the transformation of fibrinogen to fibrin monomers producing the fibrin network that reinforces the fragile, platelet-rich plug and the stimulation of platelet aggregation. A number of agents, including warfarin and low-molecular-weight heparins, are available for the prevention and treatment of thromboembolic complications. Indeed, warfarin and other vitamin K antagonists are the only oral anticoagulants available today. However, patients receiving these agents require frequent monitoring of prothrombin time because of the narrow therapeutic window and slow on- and offset of action of these agents (Hirsh et al., 2001), characteristics that make it difficult to predict their anticoagulant effect. Warfarin is also subject to large variability in its pharmacokinetic (PK¹) properties due to drug and food interactions (Hirsh et al., 2001). Despite monitoring, these agents are associated with a high incidence of bleeding and drug-related deaths (Anonymous, 1993). Low-molecular-weight heparins can be administered without routine coagulation monitoring (Hull and Pineo, 1995), but the fact that they can only be administered parenterally limits their use. Currently available anticoagulants therefore have a number of drawbacks, and there is an ongoing quest for a new anticoagulant that is effective, safe, and can be administered orally without routine coagulation monitoring.

Melagatran is a novel, direct thrombin inhibitor that binds rapidly, reversibly, and competitively to the active site of thrombin with a K_i of 0.002 µM (Gustafsson et al., 1998). It has shown promise as an antithrombotic drug, both in animal models of experimental thrombosis (Elg et al., 1997, 1999; Eriksson et al., 1997; Mattsson et al., 1997; Gustafsson et al., 1998; Metha et al., 1998) and in clinical trials in orthopedic surgery patients (Heit et al., 2001; Eriksson et al., 2002a,b,c) and patients with deep venous thrombosis (Eriksson et al., 1999a, 2003). Because of low membrane permeability, melagatran is not suited to oral administration, however, its favorable pharmacodynamic properties were the impetus for the development of ximelagatran, a novel, direct thrombin inhibitor that can be administered orally, whereupon it is rapidly transformed to melagatran, its active form (Gustafsson el al., 2001). Melagatran may be formed from ximelagatran via two different pathways, as shown in Fig. 1. The primary compounds formed during the bioconversion of ximelagatran to melagatran are OH-melagatran (formed by hydrolysis of the ethyl ester) and ethyl-melagatran (formed by reduction of the hydroxyamidine), which are both subsequently converted to melagatran. The thrombin-inhibiting activity of ximelagatran and OH-melagatran is about 1% of that of melagatran, whereas ethyl-melagatran has about the same inhibitory potency as melagatran (Gustafsson et al., 2001).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   The proposed metabolic pathway of ximelagatran showing the formation of melagatran via two intermediate compounds, ethyl-melagatran, and OH-melagatran, by reduction of the OH-group and hydrolysis of the ethyl ester. The 14C- and tritium (T)-labeled positions are indicated in the figure.

The aim of the studies described here was to examine the metabolism and excretion patterns and the PK properties of ximelagatran and melagatran following the administration of single oral and i.v. doses to animals and humans. The main focus was on evaluating the bioavailability of melagatran, the active thrombin inhibitor, following oral administration of ximelagatran.

	Materials and Methods

Top Abstract Introduction Materials and Methods Design of Studies in... Design of Studies in... Sample Analysis Pharmacokinetic Assessments. Results Discussion References

Ximelagatran and melagatran were synthesized by AstraZeneca (Mölndal, Sweden), using methods analogous to those described previously (Antonsson et al., 1994, 1997). Ethyl-melagatran and OH-melagatran, used as reference compounds for metabolite identification in biological samples, and radiolabeled compounds were prepared at the Department of Medicinal Chemistry, AstraZeneca. The chemical structures of these compounds are shown in Fig. 1. The radiolabeled compounds (specific radioactivity and radiochemical purity given in parentheses) used were [¹⁴C]ximelagatran (2.0 kBq/nmol, >97%), [³H]ximelagatran (55 kBq/nmol, >97%), [³H]melagatran (521 kBq/nmol, 97%), OH-melagatran-³H (55 kBq/nmol, >95%), and ethyl-melagatran-³H (900 kBq/nmol, >98%). In essentially all studies, aqueous solutions (pH adjusted to 3-5) of melagatran (chemical purity 99.3%) and ximelagatran (chemical purity >97%) were used for dosing to animals and humans. In the food interaction study for melagatran in human subjects, an immediate-release tablet of melagatran was administered. All other chemicals used were of analytical grade.

	Design of Studies in Animals

Top Abstract Introduction Materials and Methods Design of Studies in... Design of Studies in... Sample Analysis Pharmacokinetic Assessments. Results Discussion References

Sprague-Dawley rats and beagle dogs were used in studies to examine the mass balance and PK properties of melagatran and ximelagatran after i.v. and oral (by gastric gavage) administration. All animal studies described were approved by the regional ethics committee.

Mass Balance and Pharmacokinetics in Rats. The rats were starved overnight until after drug administration but retained free access to drinking water at all times. In mass balance studies, the rats were placed in all-glass metabolic cages that allowed separate collection of urine and faeces. PK studies were performed in separate groups of rats. Two days before dosing, these rats were anesthetized, and a catheter was placed in the carotid artery for collection of blood samples. For i.v. dosing, a catheter was also inserted in the right jugular vein. The catheters were exteriorized at the nape of the neck and sealed. After surgery, the rats were housed individually and had free access to water and food.

Mass balance studies. Single, 2 µmol/kg (3.5 MBq/kg) i.v. doses of [³H]melagatran were administered (via a catheter placed in the right vena jugularis) to one group of four rats, and single, 30 µmol/kg (2 MBq/kg), oral doses of [³H]melagatran were administered by gavage to a separate group of four rats. The rats used were 12 to 14 weeks old and weighed 339 to 355 g. Urine and feces were collected during the 19-h period before dosing and at 24-h intervals for 7 days after dosing. Urine samples collected during the 0- to 24-h time period and feces collected during the 0- to 24- and 24- to 48-h time periods postdosing were used in the analysis of metabolic patterns of melagatran.

PK studies. Unlabeled melagatran was administered as an i.v. bolus dose (2 µmol/kg) or orally (30 µmol/kg) to separate groups of four male or four female rats. A higher oral dose of 100 µmol/kg was also given to another group of four male rats. The rats used were 12- to 14-weeks old. At the time of dosing, the females weighed from 209 to 240 g and the males from 338 to 365 g. Blood samples (0.2 ml) were collected from all rats 60 min or less before and at regular intervals until 240 min after i.v. dosing and 480 min after oral dosing.

Mass Balance and Pharmacokinetics in Dogs. Male and female dogs that were 2- to 6-years old and weighing 10 to 17 kg were used in the experiments. Two groups of four dogs (two males and two females) were administered single doses of [³H]melagatran either as an i.v. infusion over 1 min in a front leg vein (2 µmol/kg, 2MBq/kg), or orally by gavage (30 µmol/kg, 6 MBq/kg). Just before administration of both the oral and i.v. doses of melagatran, the dogs were placed in metabolic cages. Blood samples (5 ml) were collected from a front leg vein before and then at frequent intervals until 300 min after i.v. dosing and 360 min after oral dosing. Additional blood samples were then collected every 24 h for 5 days. Urine was collected in the cage during the 0- to 12- and 12- to 24-h time periods postdosing and thereafter at 24-h intervals for 5 days. Feces were collected at 24-h intervals for 5 days postdosing.

	Design of Studies in Humans

Top Abstract Introduction Materials and Methods Design of Studies in... Design of Studies in... Sample Analysis Pharmacokinetic Assessments. Results Discussion References

Subjects. Young healthy white male subjects, from whom informed consent was obtained prior to enrollment, were included in the studies that were performed in compliance with current good clinical practice guidelines and the Declaration of Helsinki. The subjects included in three melagatran studies were 20- to 40-years old and weighed 66 to 86 kg. These studies were performed in Sweden and approved by the Medical Products Agency of Sweden and the Göteborg University Ethics Committee. For the ximelagatran study, the subjects were 31- to 50-years old and weighed 76 to 86 kg. This study was performed in Scotland and approved by the local independent ethics committee.

Melagatran Studies.

Mass balance and pharmacokinetics Fixed doses of [³H]melagatran were administered to 12 healthy male volunteers in an open-label, crossover study. Each subject was to receive two single doses of [³H]melagatran, one as an iv infusion and the second as an oral solution, with at least 2 weeks between doses. The iv dose consisted of 5.3 µmol (2.3 mg) melagatran containing 5.2 MBq of [³H]melagatran in a volume of 15 ml infused over 10 min. The oral dose consisted of 256 µmol (110 mg) melagatran containing 7.4 MBq [³H]melagatran in a volume of 40 ml followed by 60 ml of water to rinse the vial. On the evening before each investigational day, subjects were instructed to have dinner no later than 7:00 PM and to abstain from food and drink from 10:00 PM until a standardized meal (lunch) was served 3 h after drug administration. Other standardized meals were served 6 h (snack), 10 h (dinner) and 13 h (snack) after drug administration. Venous blood samples were drawn by repeated venipuncture (Venoject; Terumo Europe N.V., Leuven, Belgium) from a forearm (the forearm contralateral to that of the infusion for subjects receiving i.v. melagatran) before and then frequently during the 24-h period after administration of both the i.v. and the oral doses of melagatran. Urine was collected predose, during the 0- to 4-, 4- to 12-, and 12- to 24-h time periods postdosing and thereafter at 24-h intervals for 5 days postdosing. Feces were collected at 24-h intervals over the 5-day period postdosing. The amounts of urine and feces collected were determined by weighing.

Dose linearity. This was examined in two studies as follows: 1) an open-label, dose-escalation study in 26 healthy male volunteers, in which melagatran was administered as gradually increasing single i.v. doses (administered as a constant infusion over 10 min) from 1.7 to 82 µg/kg (0.004-0.19 µmol/kg) to groups of two or four subjects per dosing level; 2) an open-label, dose-escalation study in 23 healthy, male volunteers, in which melagatran was administered as gradually increasing single oral doses (in solution) from 0.02 to 3.28 µg/kg (0.05-7.6 µmol/kg) to groups of two or four subjects per dosing level. Each subject received a maximum of two doses of melagatran separated by at least 1 week. On the evening before each investigational day, subjects were instructed to have dinner no later than 7:00 PM and to abstain from food and drink from 10:00 PM until a standardized meal was served postdrug administration. In the i.v.-dosing study, standardized meals were served 1 h (breakfast) and 4 h (lunch) after the start of the i.v. infusion, whereas in the oral dosing study, they were served 3 h (lunch), 6 h (snack), 10 h (dinner), and 13 h (snack) after drug administration. In the i.v.-dosing study, blood samples were collected immediately before and then frequently until 4 h after dosing for doses up to 50 nmol/kg, and additionally at 6 and 8 h after dosing for doses of 50 nmol/kg and above. In the oral dosing study, blood samples were drawn before and at frequent intervals until 24 h after dosing.

Effect of food. Six healthy human volunteers received melagatran as a 50-mg tablet on three investigational days separated by washout periods of at least 1 week, in an open-label, three-way, randomized crossover study. Administration of melagatran occurred under fasting conditions, together with breakfast, or 2 h after breakfast, in randomized order.

Ximelagatran study. The mass balance and pharmacokinetics of ximelagatran was examined in an open-label, sequential, nonrandomized study, in which each subject received a single, 50 mg (105 µmol), oral dose of [¹⁴C]ximelagatran (2.52 MBq), at a concentration of 1.25 mg/ml. This was followed 20 days later by a single, 10 mg (21 µmol), i.v. dose of unlabeled ximelagatran infused over 10 min with the aid of an infusion pump. Subjects were required to remain at the study site for 7 to 9 days following drug administration in the first study session and for 24 h following drug administration in the second study session. Subjects were required to fast from 11:00 PM on the evenings before drug administration until a standardized lunch was served 4 h postdosing. Subjects also abstained from food for 2 h before each laboratory investigation.

Plasma Protein Binding and BloodPlasma Partitioning.

The plasma protein binding and the partitioning between blood and plasma were determined in vitro in freshly collected blood and plasma. For ximelagatran and the two intermediates, ethyl-melagatran and OH-melagatran, blood from four dogs and four healthy human subjects (two male and two female animals/subjects) were used in the experiments. For melagatran, pooled blood from three male dogs, ten male rats, and individually in four healthy human subjects (two male and two female) was used. The blood samples from the different species were treated separately, and all experiments were carried out in triplicate. Blood and plasma were incubated separately with [³H]ximelagatran at concentrations of 0.1, 1, and 10 µM, and with ³H-labeled ethyl-melagatran and OH-melagatran at concentrations of 0.05, 0.5, and 5 µM. [³H]melagatran was incubated at the following concentrations: 0.05, 0.5, and 5 µM in humans; 0.05, 5, and 100 µM in dogs; and 0.05, 5, and 30 µM in rats. Plasma protein binding, determined using ultrafiltration, was given as the ratio of concentrations in ultrafiltrate and plasma. No adsorption to the membrane or the ultrafiltration device was observed for any of the compounds. The whole blood to plasma concentration ratio was calculated as the concentration of radioactivity in whole blood divided by the concentration of radioactivity in the corresponding plasma samples.

	Sample Analysis

Top Abstract Introduction Materials and Methods Design of Studies in... Design of Studies in... Sample Analysis Pharmacokinetic Assessments. Results Discussion References

Handling and Stability of Biological Samples. All urine and feces samples were directly frozen and stored at 20°C until analysis. Blood samples were collected in heparinized test tubes, kept on ice until plasma was separated by centrifugation (within 1 h of collection), and then kept frozen at 20°C until analysis. The stability of ximelagatran in freshly collected dog and human blood was evaluated by incubation for 1 h at 37°C. In these incubation experiments, more than 90% of the initial ximelagatran concentrations were remaining, supporting that the stability of ximelagatran was satisfactory when the blood samples were handled as described above.

Concentration of Total Radioactivity. In melagatran studies, the concentration of total radioactivity in the biological samples was determined by liquid scintillation counting after mixing the samples with liquid scintillation fluid. Fecal samples were homogenized in approximately twice their volume in tap water. For ximelagatran studies, all samples except whole blood and bile were analyzed as described for melagatran studies. Whole blood, feces homogenate, and bile were combusted using a Packard Tri-Carb Automatic Sample Oxidizer (PerkinElmer Life Sciences, Boston, MA). For mass balance studies, the amounts of radioactivity in urine and feces were expressed as a percentage of the administered dose.

Metabolic Patterns in Urine, Bile, and Feces. For both melagatran and ximelagatran studies, a reversed-phase gradient LC system with on-line radioactivity detection was used to determine the metabolic pattern in urine and feces. Urine samples were injected directly onto the LC column. Bile samples were diluted with mobile phase A and centrifuged at 15,000g for 10 min. Feces homogenates (100-mg aliquot from samples containing >3% of the dose) were mixed with 0.5 ml of phosphoric acid (1 M), shaken for 5 min, and centrifuged at 10,000g. The resultant supernatant was injected onto the LC column. Complete recovery of radioactivity for the extraction from fecal samples was demonstrated by liquid scintillation counting.

Plasma and Urine Concentrations. For the melagatran PK studies, the plasma concentration of melagatran was determined by reversed-phase liquid chromatography and positive electrospray ionization mass spectrometry. For quantification, an analog to melagatran was used as internal standard. Melagatran was isolated from plasma by solid-phase extraction on octylsilica. LC separation of the extracts was performed on an octadecylsilica column using a mobile phase consisting of 40% acetonitrile and 0.08% formic acid in 0.0013 mol/l ammonium acetate solution. Molecular ions (M + H)⁺ of melagatran and the internal standard were measured by selected ion monitoring at m/z 430.2 and 444.2, respectively.

	Pharmacokinetic Assessments.

Top Abstract Introduction Materials and Methods Design of Studies in... Design of Studies in... Sample Analysis Pharmacokinetic Assessments. Results Discussion References

A bi-exponential model was fit to the data obtained after i.v. dosing in the human melagatran study whereas noncompartmental analysis was used for all other data. Weighted least-squares nonlinear regression using PCNONLIN (version 4.2; Statistical Consultants Inc., Lexington, KY) was used to fit the bi-exponential model to the data for i.v. melagatran in human subjects. The weights used were (Y_pred)² or (Y_pred)¹, where Y_pred is the model-predicted plasma concentration. Basic PK parameters were derived from the bi-exponential model in a standard manner. The C_max was the highest plasma concentration observed, and t_max was the time at which C_max occurred. The area under the plasma concentrationtime curve (AUC) was calculated using the log-linear trapezoidal rule from time 0 to t_last, the last sampling time with a measurable plasma concentration, and extrapolated to infinity by addition of the quantity C_last/k, where C_last was the predicted plasma concentration at t_last, and k was the elimination rate constant. C_last and k were estimated by linear least-squares regression of log plasma concentrationtime data in the terminal phase of the decline. The t_1/2 was calculated as ln2/k. The area under the first moment curve (the curve of the product of concentration and time versus time; AUMC) was calculated using the linear trapezoidal rule and extrapolated to infinity by adding the quantity (C_last · t_last/k) + (C_last/k²). Plasma clearance (CL) and volume of distribution at steady state (V_ss) were also calculated following i.v. dosing; CL as dose_i.v./AUC_i.v. and V_ss as CL × MRT, where MRT is the mean residence time calculated as AUMC_i.v./AUC_i.v..

Oral bioavailability was calculated as 100 · (AUC_oral/AUC_i.v.)/(dose_i.v./dose_oral), where the oral and i.v. suffixes denote the quantities for oral and i.v. dosing, respectively. After both oral and i.v. administration of ximelagatran, the bioavailability of melagatran (F_Mel) was calculated as (AUC × CL_Mel)/dose, where CL_Mel is the mean value of melagatran clearance. Renal clearance (CL_R) of melagatran was estimated as A_e/AUC, where A_e was the amount of melagatran excreted in urine.

In the food interaction study in dogs, differences in the parameters C_max, AUC, and t_1/2 between the two treatments (administration with and without food) were tested by the Student's paired t test. Log-transformed values were used for AUC and C_max. In the human study, analysis of variance analysis was used to test for between-treatment differences in C_max and AUC using log-transformed values. Differences were considered significant at p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Design of Studies in... Design of Studies in... Sample Analysis Pharmacokinetic Assessments. Results Discussion References

Mass Balance and Metabolic Profile of Melagatran. The mean cumulative recovery of total radioactivity excreted in urine and feces, following i.v. and oral administration of [³H]melagatran to rats, dogs, and human subjects, is shown in Table 1. In rats and dogs, the major portion of radioactivity excreted in feces and urine following both oral and i.v. administration of melagatran was identified as unchanged melagatran. However, a few minor peaks in the chromatogram eluted from the reversed-phase LC system with a shorter retention time than melagatran, suggesting that metabolites with a more polar nature than melagatran were formed. The concentration of these metabolites was low in relation to melagatran, except in the urine of dogs, where they were present in somewhat larger amounts. The structure of these polar metabolites remains to be identified.

Mass Balance and Metabolic Profile of Ximelagatran. The mean cumulative recovery of total radioactivity in urine and feces, following oral and i.v. administration of [¹⁴C]ximelagatran is shown in Table 2. The excretion of radioactivity was rapid and essentially complete for both animals and human subjects.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Cumulative amounts of ximelagatran, melagatran, ethyl-melagatran, and OH-melagatran excreted in urine in the 0- to 24-h period following oral and i.v. administration of ximelagatran to rats, dogs, and humans (as determined using radiochemical detection methods in rats and dogs and LC-MS in humans). Results are expressed as mean percentages of administered dose.

Pharmacokinetics of Melagatran. The mean plasma concentrationtime profiles in rats, dogs, and human subjects, after oral and i.v. administration of melagatran, are shown in Fig. 3. The PK parameters of melagatran are presented in Tables 4 and 5. For rats, the apparent t_1/2 of melagatran after oral administration (mean values of 1.1-1.6 h) was longer than after i.v. administration indicating absorption-rate limited elimination. Renal clearance of melagatran was estimated to 8.6 ml/min/kg for male rats and 119 ± 10 ml/min for human subjects.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Mean plasma concentrations (log scale) of total radioactivity (B and C only) and of unchanged melagatran (A, B, and C) versus time (h) since oral and i.v. administration of melagatran to rats (A), dogs (B), and humans (C).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   AUC and Cmax versus i.v. dose of melagatran and dose-normalized AUC and Cmax versus oral dose of melagatran in humans.

Pharmacokinetics of Ximelagatran. The mean plasma concentrationtime profiles for those compounds with measurable plasma concentrations in rats, dogs, and human subjects are shown in Fig. 5. Melagatran was formed rapidly and was the predominant compound in plasma after both oral and i.v. administration. Consequently, the PK analysis was focused on melagatran. The PK parameters of melagatran following oral and i.v. administration of ximelagatran are presented in Tables 6 and 7. CL_R of melagatran after i.v. administration of ximelagatran was 23.1 and 4.37 ml/min per kg body weight for rats and dogs, respectively, and 120 ml/min for humans.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Mean plasma concentrations (log scale) of ximelagatran, melagatran, ethyl-melagatran, and OH-melagatran versus time (h) since oral and i.v. administration of ximelagatran to rats, dogs, and humans.

Plasma Protein Binding and BloodPlasma Partitioning. The degree of plasma protein binding and the partitioning between blood and plasma was low and concentration-independent for all species and compounds. The mean values for the plasma protein binding of melagatran were 10.6 and 10.0% in dogs and rats, respectively, with low variability. Of the human subjects, one male and one female had essentially no plasma protein binding of melagatran, whereas the other two had values of 13.3 and 15.3%. For ximelagatran, the mean percentage binding was 79% in humans and 76% in dogs. For ethyl-melagatran, the corresponding estimates were 44 and 63%, and for OH-melagatran they were 10 and 12% in humans and dogs, respectively. The mean blood-to-plasma concentration ratios of melagatran were 0.55 in dogs and 0.62 in rats. The individual mean values in humans ranged from 0.56 to 0.62. The mean ratios of ximelagatran, ethyl-melagatran, and OH-melagatran ranged from 0.54 to 0.67 in dogs and humans.

	Discussion

Top Abstract Introduction Materials and Methods Design of Studies in... Design of Studies in... Sample Analysis Pharmacokinetic Assessments. Results Discussion References

The PK properties of the oral direct thrombin inhibitor, ximelagatran, and in particular the formation and bioavailability of its active form, melagatran, were examined in animals and healthy male human subjects. Ximelagatran was rapidly absorbed and metabolized following oral administration to rats, dogs, and humans. Melagatran was the dominant compound in the plasma of all three species. It was demonstrated that the bioconversion of ximelagatran to its active form, melagatran, occurs by ester hydrolysis and reduction via two intermediates, ethyl-melagatran and OH-melagatran (Fig. 1). Ethyl-melagatran is formed by reduction of the hydroxyamidine and OH-melagatran by ester hydrolysis. In vivo reduction of an amidoxime derivative, pentamidoxime, to its corresponding amidine has been demonstrated previously (Clement, 1998). The PK properties of melagatran, following oral and i.v. administration, were also consistent between the animal species and humans. Melagatran has a relatively low plasma clearance, negligible plasma protein binding, a small volume of distribution, and a short elimination half-life. Renal excretion of unchanged melagatran was the main route of elimination. In humans, linear PK of melagatran were observed after i.v. dosing over the entire dose range studied with low interindividual variability. The calculated renal clearance of melagatran in rats and humans was similar to the glomerular filtration rate (Davies and Morris, 1993). The polar nature of melagatran, which prevents passive reabsorption from the proximal and distal tubules, and the fact that plasma protein binding is negligible, suggests that glomerular filtration is the mechanism of elimination in the kidneys. After i.v. administration of ximelagatran to rats, the renal clearance of melagatran was higher than that determined after administration of melagatran and higher than the glomerular filtration rate. This may be due to metabolism of ximelagatran and the formation of melagatran in the kidneys. In dogs and humans, CL_R of melagatran was after i.v. administration of ximelagatran approximately equal to the glomerular filtration rate.

The low and dose-dependent oral bioavailability of melagatran in rats and humans is consistent with its polar properties and poor membrane permeability determined in the Caco-2 cell line (Gustafsson et al., 2001). The markedly higher bioavailability observed in dogs compared with rats and humans is probably the result of greater absorption across the gut wall in this species. This is consistent with a recent report reviewing the absorption data for a large number of compounds, which concluded that the fraction absorbed was markedly higher in dogs than in humans, whereas the absorption data obtained in rats were in much better agreement with data in humans (Chiou et el, 2000). The interindividual variability in melagatran exposure following its oral administration to humans was also large. The combination of low interindividual variability after i.v. administration and the strong correlation between C_max and AUC after oral administration suggest that the variability in plasma levels after oral administration is mainly due to variability in the extent of absorption. In the presence of food, the bioavailability of melagatran was significantly reduced in both dogs and humans.

Ximelagatran, for which the membrane permeability is 80 times higher than for melagatran (Gustafsson et al., 2001), was rapidly absorbed and metabolized following oral administration. In rats, the bioconversion of ximelagatran was so rapid that no detectable concentrations of ximelagatran were found in plasma after either i.v. or oral administration. Ethyl-melagatran was also undetectable in rat plasma. However, by using sodium dodecyl sulfate as an esterase inhibitor in the blood collection test tube (Holm et al., 1985), it was shown that both male and female rats had measurable concentrations of ximelagatran and ethyl-melagatran. This finding indicates that ester hydrolysis of ximelagatran occurred to some extent ex vivo in blood after collection of the sample. The levels of ximelagatran and ethyl-melagatran were much lower than that of melagatran, and the clearance of ximelagatran exceeded cardiac output (data on file), suggesting a high esterase activity in blood and tissues of rats. Also, OH-melagatran was present in rat plasma with high peak concentrations after both i.v. and oral administration but declined rapidly. In dogs and humans, the plasma concentrations of the intermediates were low compared with that of melagatran. The plasma concentration of OH-melagatran appeared to be higher than that of ethyl-melagatran, suggesting that the ester hydrolysis occurred more readily than the reduction.

In dogs and rats, there was a dose-dependent increase in the bioavailability of melagatran after both oral and i.v. administration of ximelagatran. In a previous study in which healthy male human subjects received escalating single oral doses of ximelagatran ranging from 5 to 98 mg, the bioavailability of melagatran was dose-independent and estimated to be about 20% (Eriksson et al., 1999b). This is consistent with the value of 19% observed at the dose of 50 mg (equivalent to approximately 0.3 µmol/kg) given to the male human subjects in the present study. Consequently, the dose-dependent increase in F_Mel observed in the animal experiments is likely due to the higher doses (40 µmol/kg) that were given to the rats and dogs. Possible reasons for the observed dose-dependent increase in F_Mel are an increase in the fraction of ximelagatran absorbed, increased metabolism of ximelagatran to melagatran, or decreased elimination of melagatran at higher doses. Dose proportional PK for i.v. melagatran was shown in humans and has also been demonstrated in rats and dogs for a wide range of doses (data on file), which supports that the elimination of melagatran is independent of dose.

The bioavailability of melagatran and the amount of melagatran recovered in urine was higher after i.v. than after oral administration of ximelagatran, which suggests incomplete absorption. The relative bioavailability of melagatran after oral versus i.v. administration of ximelagatran, obtained as the ratio of the AUC estimates of melagatran and corrected for difference in dose, was 40 to 70% at the 10 µmol/kg dose given to rats and dogs. In humans, the relative bioavailability of melagatran was 43%. Similar estimates were obtained for the relative bioavailability of the intermediates, ethyl-melagatran (34%) and OH-melagatran (37%). Assuming that the metabolism of ximelagatran is the same after oral and i.v. administration, this means that 40 to 70% of the oral dose of ximelagatran is absorbed. As F_Ximel after oral administration to humans was only 21.9%, this suggests that first-pass metabolism of ximelagatran during absorption reduces its oral bioavailability. If the absorption of ximelagatran is incomplete, there is a possibility that it may be increased at the higher doses that were given to animals.

The estimate of the fraction of oral ximelagatran absorbed is uncertain because presystemic metabolism and sequential biliary excretion of ximelagatran or its products appeared to occur. It is therefore likely that the fraction absorbed is even higher than 40 to 70%. The principal route of excretion of the orally administered dose of ximelagatran was fecal in rats, dogs, and humans. High recovery of radioactivity in the feces was also observed after i.v. administration to rats and dogs, suggesting biliary excretion of ximelagatran and/or formed metabolites. In fact, this was demonstrated by the substantial fraction of the dose excreted in bile collected after oral administration of ximelagatran to rats and one dog. The predominant compound in the bile of rats was melagatran whereas about equal amounts of melagatran and ethyl-melgatran were found in bile from the dog. The dose-dependent increase in F_Mel observed in the animal experiments could therefore be caused by saturation of the biliary excretion of melagatran and ethyl-melagatran.

In addition to being the major circulating compound in the plasma of all three species following oral and i.v. administration of ximelagatran, melagatran was also the major product found in urine and feces collected after oral and i.v. administration. Appreciable quantities of ethyl-melagatran were also recovered in fecal samples, whereas neither OH-melagatran nor ximelagatran were detected. Polar metabolites were also found that in general were present in larger amounts in feces than in urine. This may be due to instability of ximelagatran in the gastrointestinal tract. Anaerobic incubation of ximelagatran in human feces homogenate showed rapid disappearance of ximelagatran and formation of melagatran, ethyl-melagatran, and unknown polar metabolites. In the human subjects, the concentration of total radioactivity in plasma was higher than the sum of the plasma concentrations of ximelagatran, the intermediates, and melagatran. This supports the premise that unknown metabolites were present in plasma, but it is also possible that this is an artifact, as the two assay methods may give slightly different results.

The plasma protein binding of ximelagatran, ethyl-melagatran, OH-melagatran, and melagatran was relatively low in both human and dog plasma, although that of ximelagatran and ethyl-melagatran was higher than that of OH-melagatran, the binding of which was similar to that of melagatran. The low blood-to-plasma concentration ratios for ximelagatran, ethyl-melgatran, OH-melagatran, and melagatran suggest a low affinity for, and a low penetration into, red blood cells.

In conclusion, ximelagatran was rapidly absorbed and converted to melagatran, the predominant compound in plasma and the active form of ximelagatran, following oral administration. The bioavailability of melagatran in humans was about 20%, presumably because of incomplete oral absorption of ximelagatran, but also first-pass metabolism of ximelagatran with subsequent biliary excretion of the formed metabolites.