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2005...pharmakokinetics BAY 59-7939

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					Xenobiotica, September 2005; 35(9): 891–910




Pharmacokinetics of BAY 59-7939 – an oral,
direct Factor Xa inhibitor – in rats and dogs


C. WEINZ1, U. BUETEHORN2, H.-P. DAEHLER2,
C. KOHLSDORFER2, U. PLEISS1, S. SANDMANN2,
K.-H. SCHLEMMER2, T. SCHWARZ2, & W. STEINKE2
1
 Drug Metabolism and Isotope Chemistry and 2Preclinical Pharmacokinetics,
Bayer HealthCare AG, Wuppertal, Germany

(Received 14 March 2005)



Abstract
The pharmacokinetics of BAY 59-7939 – a novel, oral, direct Factor Xa inhibitor – were investigated in
rats and dogs in support of preclinical safety studies and clinical development. BAY 59-7939
was rapidly absorbed after oral dosing, with an absolute bioavailability of 57–66% in rats, and
60–86% in dogs. Plasma pharmacokinetics of BAY 59-7939 were linear across the investigated
dose range (1–10 mg kgÀ1 in rats, 0.3–3 mg kgÀ1 in dogs). Plasma clearance was low: 0.4 l kgÀ1 hÀ1
in rats and 0.3 l kgÀ1 hÀ1 in dogs; volume of distribution (Vss) was moderate: 0.3 l kgÀ1 in rats,
and 0.4 l kgÀ1 in dogs. The elimination half-life after oral administration was short in both species
(0.9–2.3 h). Whole-body autoradiography showed moderate tissue affinity. No retention or small
volume enrichments of BAY 59-7939-related radioactivity were observed. The plasma-protein
binding of BAY 59-7939 was high, species dependent and fully reversible. BAY 59-7939 was rapidly
excreted in rats and dogs, and was not irreversibly retained. A dual mode of excretion (biliary/faecal
and renal) was observed. In summary, BAY 59-7939 had a favourable, predictable pharmacokinetic
profile, with high oral bioavailability and a dual route of excretion.

Keywords: Preclinical, pharmacokinetics, Factor Xa inhibitor, absorption, distribution, excretion, protein
binding, whole-body autoradiography




Introduction
BAY 59-7939 (5-chloro-N-({(5S)-2-oxo-3-[4-(3-oxo-4-morpholinyl)phenyl]-1,3-oxazoli-
din-5-yl}methyl)-2-thiophene-carboxamide) (Figure 1) is a novel, oral, direct
Factor Xa (FXa) inhibitor in clinical development (phase IIb) for the prevention and
treatment of thromboembolic diseases. Approximately one molecule of FXa results in the

Correspondence: C. Weinz, Drug Metabolism and Isotope Chemistry, Bayer HealthCare AG, Building 466, Aprather Weg 18a,
D-42096 Wuppertal, Germany. Tel: 49-202-36-8974. Fax: 49-202-36-4622. E-mail: corinna.weinz@bayerhealthcare.com
ISSN 0049-8254 print/ISSN 1366-5928 online # 2005 Taylor & Francis
DOI: 10.1080/00498250500250493
892     C. Weinz et al.

                          O                     O


                 O        N                 N       O
                                                        H
                                                        N                  Cl
                                                                     S

                                                            O

         Figure 1. Chemical structure of [14C]BAY 59-7939. *Position of the radiolabel.



generation of up to 1000 thrombin molecules (Mann et al. 2003). Therefore, FXa has been
identified as a potential target for intervention in the coagulation cascade. It is integral to
both the intrinsic and extrinsic pathways of the coagulation cascade, and is the point at
which the two pathways converge.
  In vitro, BAY 59-7939 has been shown to selectively and directly inhibit FXa, block
thrombin generation and prolong clotting times (Perzborn et al. 2005). Furthermore,
results from in vivo studies have shown reduced thrombus formation with BAY 59-7939
administered prophylactically in rat and rabbit thrombosis models (Perzborn et al. 2005).
In the current paper, the pharmacokinetics of BAY 59-7939 in vivo in Wistar rats and
beagle dogs are described. In addition, plasma-protein binding was determined in vitro
for several species.


Materials and methods
Materials
[14C]BAY 59-7939. [14C]BAY 59-7939 (Figure 1) was synthesized at Bayer
HealthCare AG (Wuppertal, Germany). If necessary, [14C]BAY 59-7939 was diluted
with unlabelled BAY 59-7939 (assay for use ranging from 98.2 to 99.4%) for use in these
studies. [14C]BAY 59-7939 had a specific activity ranging from 2.66 to 2.69 MBq mgÀ1, and
radiochemical purity ranging from 99.1 to 99.9%.


Plasma samples for protein binding studies. Plasma samples for in vitro protein binding
studies were provided by Bayer HealthCare AG. Pooled plasma from at least three healthy
subjects (male/female), and pooled animal plasma from at least three individual Wistar
(Hsd Cpb:WU) strain rats (male/female) from Harlan Winkelmann (Borchen, Germany);
beagle dogs (female) from Harlan Winkelmann; mini-LEWE pigs (male) from TU Dresden
(Dresden, Germany); rhesus monkeys (female) from Farm of Shunde (China); CHBB
rabbits (female) from Boehringer (Biberach, Germany); and CD-1 mice (male) from
Charles River-Iffa Credo (L’Arbresle Cedex, France) were obtained from heparinized
blood samples by centrifugation. Dog plasma was used immediately after preparation; other
plasma samples were stored below À15 C until use. Human plasma proteins, including
1-acid glycoprotein (AGP, G 9885), were obtained from Sigma (Deisenhofen, Germany),
and serum albumin (HSA, Pentex, 82-323-4) was obtained from Bayer Diagnostica
(Munich, Germany). All other chemicals were of analytical grade and were obtained
from commercial sources.
                                                  Pharmacokinetics of BAY 59-7939        893

Animals
Rats. Male and female Hsd Cpb:WU (Wistar) strain rats, weighing 160–280 g and aged
approximately 8 weeks, were used for the studies on absorption, plasma pharmacokinetics,
distribution and excretion. All animals were purchased from Harlan Winkelmann.
In addition, one male rat of the LE/MOL (Long Evans, pigmented) strain, aged
approximately 12 weeks and weighing 249 g, was purchased from Møllegaard (Skensved,
Denmark), for the distribution study to assess the binding of BAY 59-7939-related
radioactivity to pigmented, melanin-containing tissues. Rats were allowed water ad libitum
and pellet food (NAFAG Nr. 9439, Eberle NAFAG AG; Gossau, Switzerland) restricted to
20 g per rat per day. Food was withdrawn approximately 16 h before drug administration.

Dogs. Female beagle dogs weighing 9–15 kg and aged 2–5 years were purchased
from Harlan Winkelmann or Marshall Farms USA (North Rose, NY, USA). Dogs were
allowed water ad libitum and 300–500 g food per animal per day (Ssniff Hd-Haltung
                                                                       ¨
and Ovator Hundemenu-vital [1 þ 1 v/v]; purchased from Ssniff Spezialdiaten GmbH,
Soest, Germany; and Muskator Werke, Barnewitz/Moll GmbH, Dusseldorf, Germany).
Dogs were fasted for approximately 18 h before drug administration.

Experimental conditions. Rats were acclimatized for approximately 7 days, and the dogs
for at least 4 weeks, to conditions of 20–24 C, 45–65% humidity and a 12-h light/dark cycle
(light from 06.00 to 18.00 h). Rats were housed in MacrolonÕ cages (type IV) or in
metabolism cages, which allow separate sampling of urine, bile and faeces. Dogs were
kept in metabolism cages for the [14C]BAY 59-7939 studies, which allow separate sampling
of urine and faeces, and in standard cages for other studies.
   For the basic pharmacokinetic experiments, each group consisted of either three
animals (plasma pharmacokinetics) or five animals (balance studies). For the whole-body
autoradiography study, only one rat was used per time point. All procedures using
animals were conducted in accordance with the German Animal Protection Act
(Deutsches Tierschutzgesetz).


Dosage form and administration
Rats. For oral and intravenous (i.v.) administration to fasted rats (n ¼ 3 per time point
per route), unlabelled BAY 59-7939 and [14C]BAY 59-7939 were administered in a mixture
of polyethyleneglycol (PEG) 400 and distilled water (60:40). Solutions of BAY 59-7939
up to a concentration of 2 mg mlÀ1 could be prepared with this formulation and were stable
for at least 24 h. The standard administration volume was 5 ml kgÀ1; therefore this
formulation could be used in all rat experiments with the exception of the intraduodenal
(i.d.) study. The target dose of 3 mg kgÀ1 BAY 59-7939 had to be administered in the
i.d. study in a smaller volume of only 1 ml kgÀ1 for technical reasons. Therefore, an
alternative formulation consisting of ethanol, PEG 400 and distilled water (20:60:20) was
developed. Oral doses were administered to rats via gavage, and i.v. doses were administered
as a bolus into a lateral tail vein. Intraduodenal administration in rats was performed
after cannulation of the common bile duct (via a catheter inserted into the distal end of the
common bile duct) under pentobarbital anaesthesia (Duhm et al. 1972).

Dogs. For oral and i.v. administration to fasted dogs (n ¼ 3 per time point per route),
unlabelled BAY 59-7939 and [14C]BAY 59-7939 were administered in a mixture
894       C. Weinz et al.

                    Table I. BAY 59-7939 doses and routes of administration in rats and dogs.

Species      Strain                 Type of study                 Route       Dosage (mg kgÀ1)     Group size
               14
BAY 59-7939/[ C]BAY 59-7939: Plasma pharmacokinetic studies
Rat      Wistar, m  Absorption, bioavailability,       p.o.                     1, 3, 3a, 10     n ¼ 3/time point
Rat      Wistar, m  PK parameters of radioactivity     i.v.                       1, 3, 3a       n ¼ 3/time point
Dog      Beagle, f  and parent compound, elimination, p.o.                       0.3, 1, 3       n ¼ 3/group
Dog      Beagle, f  dose dependency                    i.v.                        0.3, 1        n ¼ 3/group
[14C]BAY 59-7939: Tissue distribution, excretion and balance studies
Rat      Wistar, m    Balance, excretion                     p.o./i.v./i.d.          3           n ¼ 5/group
Rat     Wistar, m, f Distribution (WBA)                      p.o./i.v.              3, 1         n ¼ 1/time point
Rat     LE/MOL, m Distribution (WBA)                         p.o.                    3           n ¼ 1/time point
Dog       Beagle, f   Balance, excretion                     p.o./i.v.               1           n ¼ 3/group

m, Male; f, female; WBA, whole-body autoradiography; p.o., oral; i.v., intravenous; i.d., intraduodenal.
a 14
 [ C]BAY 59-7939 was administered in a separate study to determine the PK of radioactivity and of the parent
compound.


of ethanol, PEG 400 and distilled water (20:60:20), which was stable for at least 24 h.
Solutions with a concentration of up to 3 mg mlÀ1 BAY 59-7939 could be prepared
with this formulation. The standard administration volumes were 0.5 ml in the i.v.
infusion studies and 1 ml kgÀ1 in the oral studies. Oral doses were administered to
dogs via gavage, and i.v. doses by a 15-min infusion after cannulation of a superficial
extremity vein.
   BAY 59-7939 doses and administration routes in rats and dogs are summarized
in Table I.


Specimen collection and preparation
Rat blood samples were collected in heparinized vials following exsanguination (cutting
a carotid artery under deep isoflurane anaesthesia). Secreted bile was collected from rats
at set time intervals using a catheter inserted into the proximal end of the bile duct.
Dog blood samples were collected in heparinized syringes from a punctured superficial
extremity vein or a jugular vein. Plasma was obtained from collected blood samples
by centrifugation.
   All faeces and urine (rat and dog) were collected separately at preset intervals.
Faeces were homogenized in the minimum volume of tap water required to obtain
uniform radioactivity distribution in the samples. In the dog balance studies, the cages
were washed daily after completion of the experiment, to collect excreted radioactive
material that had adhered to the surface of the cage (cage wash). Expired 14CO2 was
adsorbed by ethanolamine and methoxyethanol (1:1). For quantification of radioactivity
in rats, anaesthetized animals (isoflurane inhalation) were exsanguinated and organs
and tissues were removed at autopsy. Selected organs and tissues were wet-weighed
after autopsy. The remaining carcass was also wet-weighed and homogenized mechanically.
The carcass (homogenized), organs and tissues (wet) were weighed again after freeze-drying
and were finally all homogenized mechanically.

Measurement of radioactivity
The concentrations of radioactivity in biological samples (rats and dogs) were determined
by liquid scintillation counting (LSC) using a Beckman LS 6500 Multipurpose
                                                  Pharmacokinetics of BAY 59-7939        895

Counter. Liquid samples, such as plasma, bile and urine, were topped-up with an
LS-cocktail (Ultima GoldTM, Perkin Elmer, Uberlingen, Germany). No further sample
preparation was necessary. The solid samples, such as tissues, whole organs or aliquots,
and faeces, were homogenized, lyophilized, homogenized again, and combusted using
the Canberra Packard System 387 (Oxidizer TRI-CARB 307 and Robot System 80).
The 14C-containing material was oxidized to 14CO2. The 14CO2 was collected in a
column with the adsorbent Carbo-SorbR E (Perkin Elmer), trapped in an LS vial and finally
topped-up with scintillation cocktail (MonophaseR E, Perkin Elmer).


Absorption/bioavailability
The fraction of [14C]BAY 59-7939 absorbed in rats was calculated in three ways: (1) based
on the radioactivity balance in bile-duct cannulated rats after i.d. and i.v. administration;
(2) based on the urinary excretion data after oral and i.v. administration to intact rats;
and (3) based on the ratios of AUCnorm,p.o./AUCnorm,i.v. for the total radioactivity in
plasma (AUCnorm is the AUC divided by dose [mg] kgÀ1 body weight). The fraction of
[14C]BAY 59-7939 absorbed in dogs was derived in two ways: (1) based on the ratio
of AUCnorm,p.o./AUCnorm,i.v for the total radioactivity in plasma; and (2) based on the
urinary excretion data after oral and i.v. administration.
   The experimentally derived absolute bioavailabilities, which were determined from
the ratios of the dose-normalized AUCs after oral and i.v. dosing in rats and dogs,
were then compared with the bioavailability values calculated from the extent of absorption
and the extraction ratio.


Determination of unchanged BAY 59-7939
Plasma concentrations of unchanged BAY 59-7939 were determined by liquid chromatog-
raphy coupled to a tandem mass spectrometer (LC-MS/MS): A modular 2300 HTLC
system, consisting of a binary pump, an isocratic pump and a dual sixport switching valve
module (Cohesive Technologies, Inc., Franklin, MA, USA) was connected to a triple-
stage quadrupole mass spectrometer API 365 (Applied Biosystems, MDS Sciex, Concord,
ON, Canada). The mass spectrometer was equipped with a Turbo IonsprayÕ source. Plasma
samples were precipitated by addition of acetonitrile and the internal standard BAY 60-4758
(5-chloro-N-({3-[3,5-dimethyl-4-(3-oxomorpholin-4-yl)phenyl]-2-oxo-1,3-oxazolidin-5-yl}
methyl)thiophene-2-carboxamide, Bayer HealthCare AG). The lower limit of quantification
was 5 mg lÀ1. Accuracy (deviation from nominal concentrations) ranged from À4.6 to 15.0%,
and precision (relative standard deviation) was less than 10.0%. The method was fully
validated according to the FDA guidelines (2000).


Tissue distribution of [14C]BAY 59-7939 (whole-body autoradiography)
Following the administration of [14C]BAY 59-7939, rats were killed by CO2 inhalation,
deep frozen and embedded in carboxy-methylcellulose at –70 C (Curtis et al. 1981). Sagittal
sections (50 mm) were cut at À25 C using a cryomicrotome (PMV 450, Stockholm, Sweden;
CM 3600, Leica, Microsystems, Nussloch, Germany) and lyophilized. Selected sections
were exposed to Imaging Plates (BAS-SR 2025) (radioluminography) in cassettes (Fuji
Photo Film Ltd, Tokyo, Japan). After exposure, the Imaging Plates were scanned by laser
(BAS 5000, Fuji Photo Film). The autoradiograms were processed and
896     C. Weinz et al.

qualitatively evaluated using the AIDA software (version 2.2). Qualitative evaluation was
performed by visual ranking related to standard radiation sources (14C-spiked blood
standards).


In vitro plasma-protein binding
In vitro plasma-protein binding was evaluated by an equilibrium dialysis method
(Scholtan and Schmid 1963). [14C]BAY 59-7939 was added to each aliquot of mouse,
rat, rabbit, pig, dog, monkey and human plasma to make target concentrations of 0.1, 1.0,
3.0, 10, 30 and 100 mg lÀ1; in addition, a target concentration of 400 mg lÀ1 was prepared,
but only for human plasma. After incubation for 15 min at 37 C, 0.8 ml spiked plasma were
dialysed with an equal volume of phosphate-buffered isotonic solution (PBS, pH 7.4) for 1 h
at 37 C in an equilibrium dialyser (DianormÕ Dialyzer, Dianorm GmbH, Munich,
Germany) equipped with 0.8 ml Teflon half cells separated by a cellulose membrane
(Diachema 10.14 cellulose membrane, molecular weight cut-off of 5000 kDa; Dianorm).
The radioactivity of [14C]BAY 59-7939 in the buffer and the plasma was determined
by LSC. The fraction of unbound BAY 59-7939 (fu, %) was calculated as follows:

                                    fu ¼ Cu =C Â 100,

where Cu is the concentration of unbound BAY 59-7939 and C is the total BAY 59-7939
concentration.
   For the determination of BAY 59-7939 binding to human plasma subfractions, the
isolated subfractions were dissolved in 0.02 mM PBS buffer pH 7.4 (D8537, Sigma).
   To investigate the reversibility of protein binding, [14C]BAY 59-7939 was added to
rat, dog and human plasma and incubated for 15 min. The amount of BAY 59-7939
recovered was determined from the supernatant obtained after precipitation of the protein
using acetonitrile and subsequent washing of the precipitate with 80% ethanol. To investi-
gate the partitioning between blood cells and plasma, whole blood was incubated with
[14C]BAY 59-7939 on a laboratory shaker. The haematocrit (Hk) and the radioactivity
concentration in the blood (CB, mg-eq. mlÀ1) were determined directly after incubation.
Plasma was obtained by centrifugation, and the radioactivity concentration of
BAY 59-7939 in plasma (CP, mg-eq. mlÀ1) was determined; the plasma to blood concentra-
tion ratio (CP/CB) was also calculated. The concentration of BAY 59-7939 in blood
cells (mainly erythrocytes) (CE) was calculated using the following equation:

                             CE ¼ ½CB À CP  ð1 À Hkފ=Hk:

The erythrocyte/plasma partition coefficient (PE/P) was calculated using the equation:

                                      PE=P ¼ CE =CP :



Data evaluation
The pharmacokinetic evaluation of the concentration versus time data was performed
with the aid of non-compartmental analysis using KINCALC software, 2001
(version 2.50.02) (Yamaoka et al. 1978). The area under the concentration–time curve
                                                                 Pharmacokinetics of BAY 59-7939               897

was calculated using the mixed logarithmic–linear trapezoidal rule (Charter 1989).
The pharmacokinetic symbols used are in accordance with the American College of
Clinical Pharmacy (ACCP) nomenclature recommendations (Allen et al. 1982).
Pharmacokinetic parameters and concentrations are given as geometric means ( g ) and     x
                                                         
geometric standard deviations (sg; 1-s interval xg /sg to xg Á sg), whereas the dose (percentage)
and protein binding data are given as arithmetic means with coefficient of variation.


Results
Plasma concentrations and pharmacokinetic parameters of BAY 59-7939
The pharmacokinetic parameters for plasma levels of unchanged BAY 59-7939 after i.v.
and oral administration to Wistar rats and beagle dogs are listed in Tables II and III.
  The mean plasma concentrations following oral dosing in rats and dogs over time
are shown in Figure 2.



Table II. Pharmacokinetic parameters of unchanged BAY 59-7939 [mean (SD)] after single i.v. administration.

                                        Rat (mg kgÀ1)a                                     Dog (mg kgÀ1)

                                  1                        3                        0.3                    1
            À1
AUC (mg h l )                    2.27                     8.51                   1.00 (1.35)           3.18 (1.39)
AUCnorm (kg h lÀ1)               2.27                     2.84                   3.34 (1.35)           3.18 (1.39)
CL (l hÀ1 kgÀ1)                  0.44                     0.35                   0.30 (1.35)           0.31 (1.39)
Vss (l kgÀ1)                     0.32                     0.28                   0.40 (1.33)           0.40 (1.31)
t1/2 (h)                         0.83                     0.94                   0.95 (1.27)           0.97 (1.08)
Intervalb (h)                    1–4                      4–8                      0.25–6                  3–8
a
  PK parameters were calculated from geometric mean concentration versus time data, concentrations were
determined in the plasma of three animals per time point (terminal blood sampling).
b
  Used for regression to determine apparent half-life.



Table III. Pharmacokinetic parameters of unchanged BAY 59-7939 [mean (SD)] after single oral administration.

                                Rat (mg kgÀ1)a                                       Dog (mg kgÀ1)

                          1             3          10                 0.3                      1           3

AUC (mg h lÀ1)          1.49          5.04        14.7            0.61 (2.04)          2.72 (1.24)     6.03 (1.39)
AUCnorm (kg h lÀ1)      1.49          1.68        1.47            2.02 (2.04)          2.72 (1.24)     2.01 (1.39)
Cmax (mg lÀ1)           0.93          3.11        6.01            0.25 (1.35)          1.28 (1.22)     2.72 (1.23)
Cmax,norm (kg lÀ1)      0.93          1.04        0.60            0.85 (1.35)          1.28 (1.22)     0.91 (1.23)
tmax (h)                 0.5           0.5         0.5            0.45 (1.74)          0.57 (1.26)     0.50 (2.00)
t1/2 (h)                2.29          1.41        1.19            0.88 (1.12)          0.92 (1.42)     0.92 (1.09)
Intervalb (h)            4–8           4–8         2–8                2–8                1.5–10           0.5–8
f (%)                   58.2c         65.6c       57.4c           60.4d (1.70)         85.5e (1.39)       63.2e

f, Bioavailability of unchanged compound, calculated with the i.v. AUCnorm value.
a
  PK parameters were calculated from geometric mean concentration versus time data, concentrations were
determined in the plasma of three animals per time point (terminal blood sampling).
b
  Used for regression to determine apparent half-life.
c
  Calculated with the mean AUCnorm of 2.56 kg h lÀ1 after 1 and 3 mg kgÀ1 i.v.
d
  Related to 0.3 mg kgÀ1 i.v. infusion.
e
  Related to 1.0 mg kgÀ1 i.v. infusion.
898     C. Weinz et al.

        (a) 10                                                BAY 59-7939 1 mg/kg
                                                              BAY 59-7939 3 mg/kg
                                                              BAY 59-7939 10 mg/kg
         Concentration (mg/l)


                                  1




                                 0.1




                                0.01
                                       0   2   4          6        8           10
                                               Time (hours)

        (b) 10                                                BAY 59-7939 0.3 mg/kg
                                                              BAY 59-7939 1.0 mg/kg
                                                              BAY 59-7939 3.0 mg/kg


                                  1
         Concentration (mg/l)




                                 0.1




                                0.01




                         0.001
                                       0   2   4          6        8           10
                                               Time (hours)

Figure 2. Comparison of plasma concentration–time curves for BAY 59-7939 after a single oral
administration to (a) male Wistar rats and (b) female beagle dogs at different doses. Data are
geometric means Æ SD from three animals.



Rats
In rats, the pharmacokinetics of unchanged BAY 59-7939 were linear between
1 and 3 mg kgÀ1 after i.v. administration, with an AUC of 2.27 mg h lÀ1 (AUCnorm
2.27 kg h lÀ1) and 8.51 mg h lÀ1 (AUCnorm 2.84 kg h lÀ1), respectively (Table II). Plasma
concentrations of unchanged BAY 59-7939 decreased rapidly to approximately 1% of Cmax
                                                    Pharmacokinetics of BAY 59-7939          899

within 4 h after administration. The half-life determined in the time interval up to 8 h
after administration was approximately 0.9 h. Mean volume of distribution at steady state
was moderate at about 0.3 l kgÀ1; mean plasma clearance was approximately 0.4 l hÀ1 kgÀ1.
The in vitro plasma to blood concentration ratio was 1.53 in rats, and, taking this
into account, whole-blood clearance was low (0.6 l hÀ1 kgÀ1). After oral administration to
rats, maximum BAY 59-7939 plasma concentrations of 0.93, 3.11 and 6.01 mg lÀ1
(Cmax,norm 0.93, 1.04, 0.60 kg lÀ1, respectively) were reached 0.5 h after 1, 3 and 10 mg kgÀ1
BAY 59-7939, respectively; increasing dose proportionally from the low (1 mg kgÀ1) to
the medium (3 mg kgÀ1) doses, and less than dose proportionally from the medium to the
high doses (10 mg kgÀ1) (Table III). The AUC was 1.49, 5.04 and 14.7 mg h lÀ1 (AUCnorm
1.49, 1.68 and 1.47 kg h lÀ1) after administration of 1, 3 and 10 mg kgÀ1 BAY 59-7939,
respectively, and increased dose proportionally. In the interval up to 8 h, the plasma
concentrations of BAY 59-7939 decreased with a half-life of approximately 1–2 h.


Dogs
In dogs, the pharmacokinetics of unchanged BAY 59-7939 were linear for the 0.3
and 1 mg kgÀ1 doses after i.v. administration, with AUC of 1.00 mg h lÀ1 (AUCnorm
3.34 kg h lÀ1) and 3.18 mg h lÀ1 (AUCnorm 3.18 kg h lÀ1), respectively (Table II). Plasma
concentrations decreased within 4 h after administration to concentrations of about 5%
of the Cmax. A half-life of approximately 1 h was determined in the interval up to 8 h after
administration. Mean volume of distribution at steady state was moderate: approximately
0.4 l kgÀ1; mean plasma clearance was approximately 0.3 l hÀ1 kgÀ1. The in vitro plasma
to blood concentration ratio was 1.14 in dogs, and, taking this into account, the whole-blood
clearance was low (0.34 l hÀ1 kgÀ1).
   Following oral administration to dogs, maximum plasma concentrations of 0.25, 1.28
and 2.72 mg lÀ1 (Cmax,norm 0.85, 1.28 and 0.91 kg lÀ1) were reached approximately 0.5 h
after 0.3, 1 and 3 mg kgÀ1 BAY 59-7939, respectively, increasing dose proportionally
from the low (0.3 mg kgÀ1) to the medium dose (1 mg kgÀ1) and to the high dose
(3 mg kgÀ1). The AUC was 0.61, 2.72 and 6.03 mg h lÀ1 (AUCnorm 2.02, 2.72 and
2.01 kg h lÀ1) after administration of 0.3, 1 and 3 mg kgÀ1 BAY 59-7939, respectively,
and increased dose proportionally.
   Up to 10 h, the plasma concentrations of BAY 59-7939 decreased with a half-life of
approximately 0.9 h, and were similar to those observed after i.v. administration in the
same time interval (Table II).


Comparison of pharmacokinetic parameters of total radioactivity and
unchanged BAY 59-7939
The pharmacokinetic parameters for total radioactivity and unchanged BAY 59-7939
after oral and i.v. administration are listed in Tables IV and V for rats and dogs, respectively.
The pharmacokinetic parameters for total radioactivity and unchanged BAY 59-7939 in
rats were obtained from separate studies after oral and i.v. administration of 3 mg kgÀ1
[14C]BAY 59-7939. Thus, there are slight discrepancies between the pharmacokinetic
data presented in Table IV (obtained following administration of [14C]BAY 59-7939) and
those in Tables II and III (obtained following administration of unlabelled BAY 59-7939).
The plasma concentrations of total radioactivity and unchanged BAY 59-7939 after
oral administration to rats and dogs are shown in Figure 3.
900       C. Weinz et al.

Table IV. Comparison of pharmacokinetic parameters of total radioactivity and unchanged BAY 59-7939 (mean)
in plasma after single intravenous and oral administration of [14C]BAY 59-7939 to rats.

                                            3 (mg kgÀ1) i.v.                               3 (mg kgÀ1) p.o.

                              Total radioactivity        BAY 59-7939         Total radioactivity          BAY 59-7939
             À1 a
AUC (mg h l )                        8.78                       6.26                4.20                      2.75
AUCnorm (kg h lÀ1)                   2.82                       2.09                1.35                      0.92
Cmax (mg lÀ1)a                       n.c.                       n.c.                2.04                      1.85
Cmax,norm (kg lÀ1)                   n.c.                       n.c.                0.66                      0.62
tmax (h)                             n.c.                       n.c.                0.25                      0.25
t1/2 (h)                             18.8                       1.11                42.1                      1.25
Intervalb (h)                        8–48                       4–8                24–72                      4–8
AUC0–8 (mg h lÀ1)a                   8.26                       6.25                3.65                      2.74
AUC0–8,norm (kg h lÀ1)               2.66                       2.08                1.17                      0.91
t1/2 apparent (h)                    1.81                       1.11                1.46                      1.25
Intervalb(h)                         4–8                        4–8                 4–8                       4–8
f (%)                                100                        100                 47.9                      44.0

f, Maximum extent absorbed in case of radioactivity, bioavailability in case of unchanged compound,
calculated with the i.v. AUCnorm value; n.c., not calculated.
a
  In case of total radioactivity the units of AUC and Cmax are mg-eq. h lÀ1 and mg-eq. lÀ1, respectively, referring to
the unchanged BAY 59-7939.
b
  Used for regression to determine t1/2.



Table V. Comparison of pharmacokinetic parameters of total radioactivity and unchanged compound [mean (SD)]
in plasma after single i.v. and oral administration of [14C]BAY 59-7939 to dogs.

                                           1 (mg kgÀ1)                                      1 (mg kgÀ1)
                                   i.v. infusion (T ¼ 0.25 h)                                   p.o.

                           Total radioactivity          BAY 59-7939         Total radioactivity           BAY 59-7939
             À1 a
AUC (mg h l )                  5.55 (1.13)               3.18 (1.39)            5.10 (1.10)                2.72 (1.24)
AUCnorm (kg h lÀ1)             5.55 (1.13)               3.18 (1.39)            5.10 (1.10)                2.72 (1.24)
Cmax (mg lÀ1)a                 2.80 (1.07)               2.36 (1.34)            1.67 (1.27)                1.28 (1.22)
Cmax,norm (kg lÀ1)             2.80 (1.07)               2.36 (1.34)            1.67 (1.27)                1.28 (1.22)
tmax (h)                       0.25 (1.00)               0.25 (1.00)            0.57 (1.26)                0.57 (1.26)
t1/2 (h)                       111 (1.56)                0.97 (1.08)            128 (1.14)                 0.92 (1.42)
Intervalb (h)                    48–168                      3–8                  48–168                     1.5–10
t1/2 apparent (h)              6.63 (1.15)                  n.c.                6.21 (1.13)                   n.c.
Intervalb (h)                     8–24                        –                    8–24                         –
t1/2 apparent (h)              1.39 (1.19)               0.97 (1.08)            1.14 (1.08)                0.78 (1.22)
Intervalb (h)                      3–8                       3–8                    3–6                        3–6
f (%)                              100                         100              91.9 (1.20)                85.5 (1.39)

f, Maximum extent absorbed in case of radioactivity, bioavailability in case of unchanged compound,
calculated with the i.v. AUCnorm value; n.c., not calculated.
a
  For total radioactivity, the units of AUC and Cmax are mg-eq. h lÀ1 and mg-eq. lÀ1, respectively, referring to the
unchanged BAY 59-7939.
b
  Used for regression to determine t1/2.


Rats. In male rats, a maximum radioactivity concentration of 2.04 mg-eq. lÀ1
(Cmax, norm: 0.66 kg lÀ1) was measured 15 min after oral dosing of [14C]BAY 59-7939
3 mg kgÀ1 (first sample collection). By comparing the pharmacokinetic parameters for
unchanged BAY 59-7939 with the total radioactivity (unchanged BAY 59-7939 and
                                                                              Pharmacokinetics of BAY 59-7939        901

        (a)                                    10

                                                                                             Total radioactivity
        Equivalent concentration (mg eq/l)                                                   Unchanged BAY 59-7939

                                                1
             or concentration (mg/l)




                                               0.1




                                              0.01




                                             0.001
                                                     0   1       2        3        4          5          6     7
                                                                         Time (hours)

        (b)                                    10

                                                                                             Total radioactivity
        Equivalent concentration (mg eq/l)




                                                                                             Unchanged BAY 59-7939
                                                1
             or concentration (mg/L)




                                               0.1




                                              0.01




                                             0.001
                                                     0       4       8        12        16          20        24
                                                                         Time (hours)

Figure 3. Comparison of plasma concentration–time profiles of total radioactivity and unchanged
BAY 59-7939 after a single oral administration of (a) 3 mg kgÀ1 [14C]BAY 59-7939 to male
Wistar rats and (b) 1 mg kgÀ1 [14C]BAY 59-7939 to female beagle dogs. Data are geometric
means Æ SD from three animals.


radiolabelled metabolites), it was shown that 91% (Cmax 1.85 mg lÀ1; Cmax,norm 0.62 kg lÀ1)
of the total radioactivity was due to unchanged BAY 59-7939, and the remainder was
associated with radioactive metabolites.
  The AUC of total radioactivity was 4.20 mg-eq. h lÀ1 (AUCnorm 1.35 kg h lÀ1) after
oral administration, and 8.78 mg-eq. h lÀ1 (AUCnorm 2.82 kg h lÀ1) after i.v. administration
of 3 mg kgÀ1 [14C]BAY 59-7939. The corresponding AUCs of unchanged BAY 59-7939
902     C. Weinz et al.

were 2.75 mg h lÀ1 (AUCnorm 0.92 kg h lÀ1) after oral administration, and 6.26 mg h lÀ1
(AUCnorm 2.09 kg h lÀ1) after i.v. administration. Thus, the AUC0–8 of parent compound
covered 76% (i.v.) and 75% (oral) of the respective AUC0–8 of radioactivity. Unchanged
BAY 59-7939 and total radioactivity were rapidly eliminated from the plasma of rats,
independently of the route of administration. The apparent elimination half-life of
radioactivity and unchanged BAY 59-7939 was 1.8 and 1.1 h (i.v.), and 1.5 and 1.3 h
(oral), respectively, between 4 and 8 h after administration of a 3 mg kgÀ1 dose.
Concentrations of unchanged BAY 59-7939 were below the lower limit of quantification
24 h after administration. The elimination of radioactivity in plasma was observed until
72 h after oral administration, and was described by a terminal elimination half-life
of 42.1 h at 3 mg kgÀ1. The terminal elimination occurred at plasma concentration
levels between 0.00616 and 0.00280 mg-eq. lÀ1, which is less than 0.3% in relation to the
maximum concentration of radioactivity (CEQmax).

Dogs. In female dogs, the maximum radioactivity concentration (1.67 mg-eq. lÀ1)
was measured 0.57 h after oral [14C]BAY 59-7939 dosing (1 mg kgÀ1). By
comparing the pharmacokinetic parameters for unchanged BAY 59-7939 with total
radioactivity, it was shown that 77% (1.28 mg lÀ1) of the maximum radioactivity
concentration could be accounted for by unchanged BAY 59-7939. The AUCnorm of
unchanged BAY 59-7939 (3.18 kg h lÀ1) covered 57% of the AUCnorm of total
radioactivity (5.55 kg h lÀ1) after i.v. infusion (1 mg kgÀ1). After oral administration,
the AUCnorm of unchanged BAY 59-7939 (2.72 kg h lÀ1) was 53% of the AUCnorm of
total radioactivity (5.10 kg h lÀ1).
   Total radioactivity and unchanged BAY 59-7939 were continuously eliminated
from plasma. The apparent elimination half-life of radioactivity and unchanged
BAY 59-7939 was 1.4 and 0.97 h (i.v.) and 1.1 and 0.78 h (oral) in the intervals 3–8 h
(i.v.) and 3–6 h (oral) after dosing, respectively. The terminal elimination half-life of
radioactivity was 111 h after i.v. infusion and 128 h after oral administration. The terminal
elimination of radioactivity took place at a concentration level of less than 0.1% in relation
to the maximum concentration of radioactivity (CEQmax), and was determined in the time
interval up to 168 h after administration.


In vitro plasma protein binding
The extent of binding of BAY 59-7939 to plasma proteins was high, and species-
specific differences in the degree of binding were observed. For concentrations of
approximately 0.1–3 mg lÀ1 [14C]BAY 59-7939, the fraction of BAY 59-7939 not bound
to plasma proteins (fu) was: 1.3% in rat, 6.5% in mouse, 23.4% in rabbit, 10.4% in dog,
7.1% in pig (investigated concentration range: 0.1–1.4 mg lÀ1), 18.3% in monkey and
5.1% in human (Table VI). Using isolated human plasma proteins, it was found that the
drug was mainly bound to albumin (fu ¼ 19.6%) and, to a lesser extent, to 1-acid
glycoprotein (fu ¼ 67.7%). A concentration-dependent increase in the fu of BAY 59-7939
was observed in all investigated species at concentrations above 10 mg lÀ1. In rats, dogs
and humans, the binding was fully reversible, as shown by 100% recovery of the
radioactivity from the spiked plasma samples after protein precipitation and washing
of the pellet.
  In the blood of rats, dogs and humans [14C]BAY 59-7939 had a low-to-moderate
distribution in blood cells. The erythrocyte/plasma partition-coefficients ranged from
                                                          Pharmacokinetics of BAY 59-7939         903

              Table VI. In vitro plasma-protein binding of BAY 59-7939 in different species.

           Species                            Concentration (mg-eq. lÀ1)                 fu (%)

           Human (m, f)                                0.10–3.09                         5.07
           Wistar rat (m, f)                           0.10–2.97                          1.27
           Beagle dog (f)                              0.10–2.83                         10.40
           Rabbit (f)                                  0.11–3.22                         23.40
           CD-1 mouse (m)                              0.11–3.03                         6.45
           Rhesus monkey (f)                           0.10–3.02                         18.30
           Pig (m)                                     0.10–1.41                          7.13
           HSA                                            1.15                            19.6
           AGP                                            1.13                            67.7

           fu, Unbound fraction; m: male; f: female.



0.08 to 0.24 in rats, from 0.62 to 0.87 in dogs, and from 0.23 to 0.51 in humans.
The corresponding CP/CB ratios were 1.53 (rat), 1.14 (dog) and 1.40 (human).


Tissue distribution of [14C]BAY 59-7939
The distribution of total radioactivity was investigated by means of whole-body
autoradiography after single oral or i.v. doses of [14C]BAY 59-7939 to Wistar rats,
and to one male pigmented rat (Table I). The distribution patterns were similar after
both routes of administration and in both sexes. Radioactivity showed moderate tissue
affinity and was mainly heterogeneously distributed to organs and tissues (Figure 4a).
No penetration across the blood–brain barrier was detected. In general, there were no
relevant differences between the radioactivity distribution in the male albino and the
male pigmented rat 24 h after oral administration. There was no clear evidence for
radioactivity having a specific affinity for melanin-containing tissues in the pigmented
rats. However, compared with the albino rat, slight enrichments (low to moderate
radioactivity concentrations) were seen only in some melanin-bearing tissues of the
pigmented rat, such as the eye wall and Harderian gland.
   A qualitative assessment of the distribution patterns 2–8 h after oral administration
of [14C]BAY 59-7939 showed that the highest levels of radioactivity were detected in
the contents of the gastrointestinal system, the bile duct and the urinary bladder
(Figure 4a). A few organs contained moderate concentrations of radioactivity, such as
the liver, kidneys, skin, intestinal mucosa and coagulation gland. Most of the organs and
tissues – including the heart, lungs, skeletal muscles, testes, salivary glands, lymphatic
tissue and adrenals – had low levels of radioactivity, with concentrations roughly equivalent
to blood levels. Radioactivity was undetectable in some organs, e.g. the brain, spinal cord
and eye lens.
   Twenty-four hours after oral administration of [14C]BAY 59-7939, the highest levels
of radioactivity were detected in the intestinal contents, and moderate concentrations
were detected in the liver and bladder contents (Figure 4b). In the pigmented rat,
moderate concentrations were also detected in the eye wall and the ciliary body.
   After 7 days, elimination of radioactivity was almost complete: moderate-to-low
residual concentrations could only be detected in the liver, kidneys, skin, hair follicles
and gastrointestinal contents. Radioactivity levels in all other organs were below the
autoradiographic detection limit. There was no evidence of accumulation or irreversible
binding of [14C]BAY 59-7939 in organs and tissues of rats.
904     C. Weinz et al.




Figure 4. Whole-body autoradiograms showing the distribution of radioactivity (a) 2,
(b) 24 and (c) 168 h after oral administration of 3 mg kgÀ1 [14C]BAY 59-7939 in male Wistar
rats. (top) Eye–kidney plane; (middle) adrenal gland plane; and (bottom) median or paramedian
plane.




Excretion of radioactivity
Cumulative excretion of radioactivity in the urine and faeces after single administration
of [14C]BAY 59-7939 to rats and dogs is shown in Table VII (oral and i.v.) and Figure 5
(oral only).
                                                                  Pharmacokinetics of BAY 59-7939                  905




                                               Figure 4. Continued.


Table VII. Radioactivity balance after single administration of [14C]BAY 59-7939 to male Wistar (3 mg kgÀ1) and
female Beagle dogs (1 mg kgÀ1) [arithmetic mean (coefficient of variation)].

                Expired        Urine           Bile         Faeces          Residuesa         GIT          Recovery
Route/time      air (%)         (%)            (%)           (%)              (%)             (%)            (%)

Rat
i.v./7 days      n.a.       28.1   (5.68)      n.a.       65.5   (5.09)    0.22   (9.45)   0.02 (12.3)    93.9   (3.22)
p.o./7 days   0.02 (13.6)   24.7   (4.91)      n.a.       66.9   (3.80)    0.15   (26.8)   0.03 (40.3)    91.8   (3.45)
i.d.b/1 day      n.a.       17.1   (27.0)   34.7 (10.2)   46.4   (6.95)    0.42   (13.9)   0.18 (81.8)    98.8   (2.64)
i.v.b/1 day      n.a.       30.3   (22.9)   48.4 (14.8)   12.9   (24.5)    0.76   (38.2)   0.34 (158)     92.7   (4.31)
                             Urine (%)                    Faeces (%)      Cage wash (%)                  Recovery (%)

Dog
i.v./7 days                 50.7 (18.3)                   40.4 (32.5)      1.83 (42.7)                    93.0 (3.55)
p.o./7 days                 52.2 (32.9)                   42.8 (37.4)      2.03 (45.2)                    97.0 (3.94)

GIT, gastrointestinal tract; n.a., not applicable.
a
  Animal excluding GIT.
b
  Bile-duct-cannulated rats.


Rats. In rats, the excretion of radioactivity 7 days after i.v. and oral administration
of 3 mg kgÀ1 [14C]BAY 59-7939 was 28.1% and 24.7% of the dose, respectively, in the
urine, and 65.5% and 66.9%, respectively, in the faeces (Table VII). The radioactive
residues were low – approximately 0.2% of the dose – excluding the gastrointestinal tract, on
day 7. Only 0.02% of the dose was excreted into expiratory air as 14CO2 after oral dosing
of [14C]BAY 59-7939.
   Bile-duct-cannulated rats excreted almost all the radioactivity within 24 h after i.v. and
i.d. administration. After i.d. administration, 17.1% of dose was excreted in the urine,
34.7% in the bile and 46.4% was recovered in the faeces. After i.v. administration of
[14C]BAY 59-7939 to bile-duct-cannulated rats, 30.3% of the administered radioactivity
was found in the urine, 48.4% in the bile and 12.9% in the faeces. Therefore, the
906      C. Weinz et al.

faecal excretion of radioactivity after i.d. administration reflected incomplete absorption of
[14C]BAY 59-7939 in addition to extrabiliary excretion of radioactivity. Overall, recovery of
radioactivity was high: 91.8–98.8% in the various rat studies after 1–7 days.

Dogs. In dogs, the excretion of radioactivity 7 days after i.v. and oral administration
of the 1 mg kgÀ1 dose was 50.7 and 52.2%, respectively, in the urine, and 40.4 and 42.8%,


        (a) 100


                               80
         Percentage of dose




                               60

                                                                                       Urine
                                                                                       Faeces
                               40
                                                                                       Sum


                               20



                                0
                                    0   24   48   72     96       120   144    168
                                                  Time (hours)
        (b) 100


                               80                                                     Urine
                                                                                      Faeces
          Percentage of dose




                                                                                      Sum
                               60



                               40



                               20



                                0
                                    0   24   48   72     96       120    144    168
                                                   Time (hours)

Figure 5. Comparison of cumulative excretion of radioactivity (percentage of administered dose)
in urine and faeces after a single oral administration of [14C]BAY 59-7939 to (a) male Wistar rats at
a 3 mg kgÀ1 dose and (b) female beagle dogs at a 1 mg kgÀ1 dose. Data are arithmetic means from
five rats and three dogs, respectively.
                                                   Pharmacokinetics of BAY 59-7939        907

respectively, in the faeces (Figure 5). The recovery of radioactivity was high: 93% after
i.v. administration and 97% after oral dosing for 7 days.


Discussion
The studies with unlabelled and 14C-labelled BAY 59-7939 summarized in this paper
focus on the absorption, plasma pharmacokinetics, distribution and excretion of this drug
in rats and dogs. The results of the in vitro and in vivo metabolism studies of BAY 59-7939
(Weinz et al. 2004a, b) will be reported separately, but are briefly summarized here. The
biotransformation pathways of BAY 59-7939 in humans were similar to those in rats
and dogs (the species used in the preclinical safety studies), and were similar to those
observed in the in vitro metabolism studies with liver microsomes and hepatocytes from
different species: no major active circulating metabolites, which might contribute to the
pharmacological activity of the compound, were detected in plasma; and BAY 59-7939
was detected mostly as unchanged drug in animal and human plasma. Two major metabolic
pathways of BAY 59-7939 were identified: the oxidative degradation of the morpholinone
ring (via CYP3A4) to the ring-open metabolite M1 (the main metabolite in the excreta,
excreted via renal and faecal/biliary routes); and the hydrolysis of the amide bond to the
corresponding acid, with subsequent conjugation with glycine to form M4 (excreted
renally). In addition, excretion of unchanged drug via the kidneys contributed significantly
to the clearance of BAY 59-7939 in animals and humans. The radioactive dose,
administered in the balance studies, was almost completely recovered in the excreta,
and assigned to unchanged BAY 59-7939 and known metabolites of BAY 59-7939 in rats,
dogs and humans.
   [14C]BAY 59-7939 was rapidly absorbed in rats and dogs: after oral administration,
the maximum plasma concentrations of [14C]BAY 59-7939-associated radioactivity
and unchanged BAY 59-7939 were measured at 0.25–0.5 h in rats (Tables III and IV)
and at 0.45–0.57 h in dogs (Tables III and V). Similarly, rapid absorption was observed
in healthy human subjects receiving BAY 59-7939, with Cmax observed 2 h after single
doses (1.25–80 mg), and 2.5–4 h after multiple dosing (up to 30 mg twice daily)
(Kubitza et al. 2003, 2005).
   In rats, the extent of intestinal absorption of [14C]BAY 59-7939-associated radioactivity
(unchanged BAY 59-7939 and radioactive metabolites) was 62–90% (calculated based on
the radioactivity balance in bile-duct-cannulated rats after i.d. and i.v. administration, as
well as the urinary excretion data after oral and i.v. administration of 3 mg kgÀ1
[14C]BAY 59-7939 to intact rats). The bioavailability was moderate (57–66%). These
experimentally obtained values for the absolute bioavailability in rats can be explained by
the extent of absorption of radioactivity and the estimated extent of first-pass extraction.
Assuming the whole-blood clearance of 0.6 l hÀ1 kgÀ1 is exclusively the result of hepatic bio-
transformation, and that the hepatic blood flow in rats is 4.2 l hÀ1 kgÀ1 (Boxenbaum 1980,
Lin 1998), the extent of first-pass extraction was 14% according to the following equation:
               Extraction ratio ¼ whole-blood clearance=hepatic blood flow:
The following equation was used to calculate bioavailability:
               Bioavailability ¼ extent of absorption  ð1 À extraction ratioÞ:
Taking into account the extent of absorption and the calculated extent of first-
pass extraction, bioavailability in rats is expected to be between 53 and 77%. This agrees
908     C. Weinz et al.

well with the bioavailability determined for BAY 59-7939 in the different rat studies
(57–66%).
   The extent of absorption of radioactivity derived from the AUC ratios for total radio-
activity in plasma after i.v. and oral administration of [14C]BAY 59-7939 was 47.9%
of the dose in rats and, thus, was somewhat lower than the values obtained in the balance
studies. These data, calculated based on the AUC ratios, obviously underestimate
the real extent of intestinal absorption. This discrepancy may indicate biliary excretion of
radioactivity after first-pass extraction.
   In dogs, absorption of radioactivity was determined in the 14C-labelling study and
was found to be almost complete at 92% after oral administration of [14C]BAY 59-7939
(derived from the AUC ratios for total radioactivity in plasma after i.v. and oral
administration). The urinary excretion data also indicate complete absorption of
[14C]BAY 59-7939 in dogs. Absolute bioavailability (derived from the ratios of the
dose-normalized AUCs after oral and i.v. dosing) was high (60–86%). These experimentally
obtained values for the bioavailability can again be explained by the extent of absorption
of radioactivity and the estimated extent of first-pass extraction. The whole-blood
clearance was 0.34 l hÀ1 kgÀ1, and the hepatic blood flow is anticipated to be 2.1 l hÀ1 kgÀ1
(Boxenbaum 1980; Lin 1998). The extent of first-pass extraction was less than 16%.
Based on the extent of absorption and the first-pass extraction, a bioavailability of 77%
would be expected for BAY 59-7939 in dogs. As already shown in the rat study, this
theoretical value agrees strongly with the range of experimentally determined bioavailability
data (60–86%).
   Predictable and dose-proportional pharmacokinetics with low inter-individual variability
were observed in rats and dogs after oral and i.v. administration of BAY 59-7939.
In both species, the radioactivity in plasma was mainly due to unchanged drug, as indicated
by the AUCs: unchanged BAY 59-7939 accounted for 75 and 76% of the AUC(0–8) of
total radioactivity after oral and i.v. administration, respectively, in rats, and 53 and 57%
of the AUC of total radioactivity after oral and i.v. administration in dogs.
   Unchanged BAY 59-7939 was rapidly eliminated from the plasma of rats and dogs.
The short elimination half-life in both species suggests that the effects of BAY 59-7939
are rapidly reversible. The apparent elimination half-life of the radioactivity from the
plasma of both species was similarly short, whereas the terminal elimination of the radio-
activity from the plasma of rats and dogs could be observed until 72 and 168 h after oral
administration. However, this terminal elimination of radioactivity took place at a very
low concentration level in comparison to the maximum concentration of radioactivity.
Major active circulating metabolites that may prolong the pharmacological effects of
BAY 59-7939 were not detected (Weinz et al. 2004b).
   Predictable and dose-proportional pharmacokinetics for BAY 59-7939 have also
been observed in healthy human subjects after multiple dosing (Kubitza et al. 2003).
Again, in the human balance study with [14C]BAY 59-7939, the unchanged drug was
identified as the main compound in human plasma, and no major circulating metabolites
were detected.
   Plasma-protein binding is an important determinant in the evaluation of the pharma-
cokinetics of drugs. It is believed that only the unbound fraction (fu) of the drug produces
its pharmacological effects, and that the fu is related to toxicity and determines the overall
distribution pattern (Rowland and Tozer 1995). A change in protein binding, such as
may be associated with the administration of concomitant medications, may have
an impact on the pharmacological effect, toxicity, and the distribution of a drug.
BAY 59-7939 was highly bound to plasma proteins in vitro. Significant species-specific
                                                 Pharmacokinetics of BAY 59-7939        909

differences in plasma-protein binding were observed, with the lowest fu in rats (1.3%), and
the highest fraction in rabbits (23.4%). The fu was 5.1% in human plasma. A concentration-
dependent increase in fu was observed in all species at concentrations above 10 mg lÀ1,
possibly due to saturation of low-capacity, high-affinity binding sites. In the context of
clinically effective BAY 59-7939 plasma concentrations, the dependence of the fu on the
concentration of BAY 59-7939 is not relevant, as it occurs only at concentrations above
the effective BAY 59-7939 concentrations in humans ( 0.5 mg lÀ1). The major binding
component in human plasma is albumin; other plasma protein subfractions, e.g. 1-acid
glycoprotein, contribute only marginally. The discrepancy between the free fraction of
BAY 59-7939 in human plasma (5.1%) and the fu in human serum albumin (19.6%)
might be explained by the presence of another component in human plasma (e.g. free
fatty acids), whose binding might lead to conformational changes in the albumin molecular
structure, thereby increasing the protein binding of BAY 59-7939. Similar effects were
reported for warfarin (Vorum and Honore 1996; Petitpas et al. 2001), where concentra-
tion-dependent conformational changes of the albumin molecule structure were induced
by the binding of fatty acids to albumin. The protein binding in rat, dog and human
plasma was fully reversible. In human blood, BAY 59-7939 is mainly found in the
plasma; the erythrocyte/plasma partition-coefficient in the in vitro studies was low to
moderate, and in general was consistent with moderate tissue affinity.
   Generally, BAY 59-7939 showed only a moderate tissue affinity and no evidence
of irreversible binding in the organs and tissues of rats. The highest levels of
[14C]BAY 59-7939-associated radioactivity were found in the gastrointestinal contents,
the bile ducts and in the urinary bladder, which is consistent with the rapid faecal/biliary
and renal excretion of BAY 59-7939. These results were confirmed by findings in
healthy human subjects, in which no relevant accumulation of BAY 59-7939 beyond
steady-state was observed after multiple dosing (Kubitza et al. 2003).
   In the current study, no appreciable penetration of [14C]BAY 59-7939 across the
blood–brain barrier was detected. There was no clear evidence for a specific affinity of
[14C]BAY 59-7939 to melanin-containing tissues in pigmented rats. However, in contrast
to albino rats, low-to-moderate radioactivity concentrations were detectable only in some
pigmented tissues of the pigmented rat; therefore, a low binding affinity for melanin
cannot be excluded.
   [14C]BAY 59-7939-associated radioactivity was rapidly excreted in rats and dogs.
Biliary and urinary pathways contributed to the excretion: within 24 h after a single oral
administration of [14C]BAY 59-7939 to rats, 85.1% of the dose was excreted in the urine
(22.6%) and the faeces (62.5%). After i.v. administration in bile-duct-cannulated rats,
approximately 13% of the radioactive dose was recovered in the faeces, indicating
that [14C]BAY 59-7939-associated radioactivity undergoes extrabiliary excretion to a certain
extent. Seven days after administration of [14C]BAY 59-7939, the radioactive residues
in the rats accounted for only 0.2% of the dose. This supports the findings of the tissue
distribution study, and confirms that BAY 59-7939 does not demonstrate irreversible
retention in the body. In dogs, both excretion pathways are of similar importance,
with 52 and 43% of the dose excreted in urine and faeces, respectively, until the 7th day
after administration. In total, 97% of the dose was recovered in the excreta until the
7th day after administration. The dual excretion routes for BAY 59-7939 observed in rats
and dogs have also been observed in humans, with 66 and 28% of the dose excreted
in the urine and faeces, respectively (Weinz et al. 2004b).
   In summary, the present study demonstrates favourable, predictable, dose-
proportional pharmacokinetics for BAY 59-7939 in rats and dogs, with evidence of
910       C. Weinz et al.

high bioavailability, a short half-life, rapid peaking of plasma concentrations and com-
plete elimination. These pharmacokinetic data are supported further by data from
human studies, in which BAY 59-7939 was well tolerated and demonstrated rapid
absorption, dose-proportional pharmacokinetics and no evidence of accumulation
across a wide range of single (1.25–80 mg) and multiple doses (up to 30 mg twice
daily) (Kubitza et al. 2003, 2005).



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