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DRUG METABOLISM AND DISPOSITION
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
DMD 28:503–513, 2000 /1746/
Vol. 28, No. 5
Printed in U.S.A.
METABOLISM, DISPOSITION, EXCRETION, AND PHARMACOKINETICS OF
LEVORMELOXIFENE, A SELECTIVE ESTROGEN RECEPTOR MODULATOR, IN THE RAT
RICHARD J. MOUNTFIELD, BENEDICTE KIEHR,
AND
BRIAN A. JOHN
Departments of Drug Metabolism (R.J.M.) and Pharmacokinetics (B.K.), Novo Nordisk A/S, Novo Nordisk Park, Maaloev, Denmark; and
Department of Drug Metabolism, HLS Ltd, Huntingdon, Cambridgeshire, England (B.A.J.)
(Received July 22, 1999; accepted January 10, 2000)
This paper is available online at http://www.dmd.org
ABSTRACT:
elimination was long (24 h) and a doubling in dose resulted in an
approximate doubling in exposure. The majority of the drug was
excreted as norlevormeloxifene; the 7-desmethyl metabolite of
levormeloxifene, via the formation of phase II metabolites (glucuronides) and excretion into the bile. Unchanged drug was also
excreted, mainly from 0 to 24 h, and accounted for about 6 to 12%
of the dose. Together these two components accounted for approximately 50% of the radioactivity excreted. Additional metabolites isolated and identified by liquid chromatography-tandem
mass spectrometry, and accounting for 1 to 5% of the excreted
radioactivity in rat feces during the first 24 h, included two monohydroxylevormeloxifene species, a pyrrolidinone ring-opened metabolite of levormeloxifene, and desmethylnorlevormeloxifene.
Estrogen replacement therapy (ERT)1 has been shown to be effective in both preventing postmenopausal osteoporosis and in reducing
the risk of cardiovascular disease (Witt and Lousberg, 1997). However, without the concomitant administration of progesterone supplements, ERT has been associated with an increased stimulation of the
endometrium, causing hyperplasia and risk of cancer. Thus, there is an
interest in developing oral drugs that possess the beneficial effects of
ERT, such as osteoporosis prevention, but do not have any detrimental
effect on the uterus.
One such candidate, levormeloxifene ((⫺)-3,4-trans-7-methoxy-2,2dimethyl-3-phenyl-4-{4-[2-(pyrrolidin-1-yl)ethoxy]phenyl}chromane,
hydrogenfumarate), is a selective estrogen receptor modulator, with low
intrinsic estrogenicity that has been shown to prevent osteopenia in the
ovariectomized rat model of human osteoporosis (Bain et al., 1997), and
to prevent aortic cholesterol accumulation in the ovariectomized rabbit
model (Holm et al., 1997). In addition, levormeloxifene has an apparently
unique estrogenic effect on the uterus of ovariectomized animals whereby
uterine weight is increased with no evidence of epithelial proliferation or
glandular stimulation (Bain et al., 1997; Korsgaard et al., 1997).
Levormeloxifene was selected as a development candidate for the
prevention and treatment of postmenopausal osteoporosis and it has
been postulated that it could provide an alternative to current ERTs
because no epithelial or glandular proliferation in the uterus or associated tissue has been observed in animal species or postmenopausal
human volunteers treated with levormeloxifene. The drug, whose
structure is shown in Fig. 1, is the l-enantiomer of ormeloxifene, and
the following preclinical studies were performed to characterize the
1
Abbreviations used are: ERT, estrogen replacement therapy; LC-MS-MS,
liquid chromatography-tandem mass spectrometry; WBA, whole body autoradiography; AUC, area under the plasma concentration versus time curve; APCI,
atmospheric pressure chemical ionization.
Send reprint requests to: Dr. R.J. Mountfield, F. Hoffman-La Roche Ltd,
Pharmaceuticals Division, Granzacher Str. 124, CH 4002 Basel, Switzerland.
E-mail: [email protected]
503
FIG. 1. The structure of levormeloxifene.
ⴱ, indicates position of radiolabel.
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 11, 2017
The tissue distribution, pharmacokinetics, metabolism, and excretion of the selective estrogen receptor modulator levormeloxifene
have been investigated after oral administration of [14C]levormeloxifene to male and female Sprague-Dawley rats. The
quantitative distribution of radiolabeled levormeloxifene and/or
metabolites was confirmed by whole body autoradiography.
Levormeloxifene was absorbed from the gastrointestinal tract and
was widely distributed into tissues, with peak radioactive concentrations generally being observed 4 h after administration in the
intestine, liver, lung, kidney, spleen, pancreas, adrenals, and ovary
(females). Fecal elimination was the major excretion route of radioactivity. In a separate pharmacokinetic study, plasma Cmax was
generally observed 6 h after dose administration and the half-life of
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MOUNTFIELD ET AL.
disposition and excretion of this new selective estrogen receptor
modulator, because there were very little preclinical metabolic data
available for the new antiestrogens that are currently in clinical trials
(Lindstrom et al., 1984; Tanaka et al., 1994; O’Donnell et al., 1998).
However, the development of this compound has recently been
stopped due to a number of adverse events being reported during
phase III clinical trials, but new indications are currently being pursued as preclinical testing is near completion. It is anticipated that data
generated within drug metabolism may contribute to the overall
evaluation of new indications.
Materials and Methods
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Chemicals. 14C-Radiolabeled levormeloxifene (3,4-trans-7-methoxy-2,2dimethyl-3-phenyl-4{4-[2-pyrrolidin-1-yl)ethoxy]phenyl}chromane hydrogenfumarate) (Fig. 1) was synthesized at Amersham (Amersham, UK) and purified in the Department of Isotope Chemistry, Novo Nordisk A/S. The
radiochemical purity was ⬎99%, as determined by HPLC analysis, and the
specific activity was 53 mCi/mmol. Nonradiolabeled levormeloxifene and
chromatographic reference compounds were synthesised by Dr. S. Treppendal
(Department of Chemistry, Novo Nordisk A/S, Maaloev, Denmark). Reference
compounds included (⫹, ⫺)-3, 4-trans-2, 2-dimethyl-3-phenyl-4-[4-{2-(pyrrolidin-1-yl)ethoxy}phenyl]-7-hydroxychromane hydrochloride; (⫹, ⫺)-3, 4-trans 3phenyl-4-[4-{2-(pyrrolidin-1-yl)ethoxy}phenyl]-7-hydroxychromane hydrochloride; (⫹, ⫺)-3, 4-trans-2, 2-dimethyl-3-phenyl-4-(4-hydroxyphenyl)-7-hydroxychromane; and 3, 4-trans-2, 2-dimethyl-3-phenyl-4-(4-hydroxyphenyl)-7-methoxychromane, with respective codes NNC 46 – 0002, NNC 46 – 0003, NNC
46 – 0004, and NNC 46 – 0005.
Animals and Dosing. Sprague-Dawley rats weighing 200 to 230 g were
obtained from Charles River UK Ltd. (Margate, UK) or from Moellegards
Breeding Laboratories (Lille Skensved, Denmark). Rats were housed in groups
of six or fewer in stainless steel cages or singly in glass metabolism cages
(Jencons Ltd., Leighton Buzzard, UK) in air conditioned rooms maintained at
19 –23°C, 40 to 60% relative humidity, and a 12-h light/dark cycle. Rats were
acclimatized under these conditions for at least 2 days before dosing.
[14C]Levormeloxifene was prepared by dissolution in 0.1 M H2SO4, [10%
(v/v)] Tween 80, and injection grade water, and the pH of the solution was
adjusted to 4 with sodium hydroxide. For excretion balance studies, three male
and three female Sprague-Dawley rats received single oral doses of [14C]levormeloxifene (0.7 mg/kg or 50 mg/kg b.wt.), with each animal being
administered approximately 25 ␮Ci of radioactivity. The dose levels were
selected based on previous pharmacokinetic and toxicokinetic studies. After
dose administration, animals were housed in metabolism cages to allow the
collection of urine and feces separately. At the end of 168 h the rats were
sacrificed by cervical dislocation and the carcasses retained after removal of a
limited number of organs/tissues.
For a disposition study, 15 male and 15 female Sprague-Dawley rats
received a single oral dose of [14C]levormeloxifene (0.7 mg/kg or 50 mg/kg
b.wt.), with each animal being administered approximately 25 ␮Ci of radioactivity, and at 2, 4, 24, 48, and 72 h, three male and three female rats had
blood collected into heparinized tubes, plasma was prepared by centrifugation,
and animals were sacrificed by cervical dislocation. A selected number of
tissues were removed for additional analysis, including adrenal glands, brain,
eye, heart, kidney, lacrimal gland, large intestine contents, large intestine wall,
liver, lung, ovary (female), pancreas, pituitary, prostate (male), salivary gland,
small intestine contents, small intestine wall, spleen, stomach contents, stomach wall, testis (male), thymus, thyroid, urinary bladder, and uterus (female).
The final group of animals were used in the excretion balance experiment
described above and were sacrificed at 168 h after dose administration. For
whole body autoradiography (WBA) experiments, five male, five female, and
five pregnant Sprague-Dawley rats received a single oral dose of [14C]levormeloxifene (1.4 mg/kg b.wt., approximately 50 ␮Ci per animal). At 2, 4,
24, 48, and 72 h animals were sacrificed and prepared for WBA.
For biliary excretion experiments, four male and four female SpragueDawley rats were surgically cannulated, resulting in animals with permanent
bile fistulas as described by Remie et al. (1991) and Kuipers et al. (1985a,b).
After recovery from surgery, collection of bile for up to 24 h post dose
administration was performed. Dose levels and formulation were as described
for excretion-balance experiments.
For pharmacokinetic experiments, levormeloxifene was dissolved in ethanol
[1% (v/v)] and diluted with purified water. Final concentrations of dose
material were 0.5 and 1 mg/ml for p.o. dosing. Female rats (n ⫽ 12 for each
of two groups) received levormeloxifene at a dose level of 0.5 or 1.0 mg/kg
b.wt., three times weekly for a period of 5 weeks, corresponding to the
minimum dose levels where bone efficacy was observed.
Blood Sampling and Drug Analysis for Pharmacokinetic Experiments.
Blood samples were collected before, 4, 6, 24, and 48 h after the sixth
administration (day 13), and before, 4, 6, 24, 48, 54, 72, and 96 h after the final
administration (day 34). In general, three blood samples, one on days 13 to 15
and two on days 34 to 38 (1000 ␮l each) were collected per animal by removal
of blood from the opthalmic venous plexus into heparinized Eppendorf tubes.
Blood samples were centrifuged and the supernatant (plasma) aspirated and
stored frozen (⫺18°C). Plasma (400 ␮l) was applied to mixed-mode columns
(SPEC C8/SCX, 30 mg, 3 ml (Ansys, Irvine, CA) conditioned with 0.5 ml of
methanol followed by 0.5 ml of phosphate buffer (0.1 M, pH 2.0). The
cartridges were then rinsed with 0.5 ml of 1 M acetic acid, 0.5 ml of
acetonitrile, and 0.5 ml of buffer in that order. The analytes were finally eluted
from the cartridges by 1 ml of methanol-triethylamine (98:2, v/v). The eluate
was evaporated to dryness in a TurboVap LV evaporator and redissolved in
100 ␮l of acetonitrile/water (40:60), and 75 ␮l was applied to the HPLC
system.
The chromatographic system consisted of a Waters LC Module I System
(Waters, Milford, MA), a Micro-Lab Universal-Thermostat Column Heater
(Micro-Lab, Hoejbjerg, Denmark) and a Jasco 821-FP flourescence detector
(Jasco, Tokyo, Japan) operated at 279 and 305 nm excitation and emission
wavelengths, respectively. The chromatograph was interfaced to an Expert
Ease V 3.1 data system (Waters, Milford, MA) installed on a Digital Equipment Vax computer. The stationary phase consisted of a LichroSpher 100
RP-C18, 5 ␮m endcapped, 250 ⫻ 4 mm analytical column and a LichroSpher
100 RP-18, 5 ␮m endcapped, 4 ⫻ 4 mm precolumn, both supplied by Merck,
Darmstadt, Germany. The mobile phase consisted of acetonitrile-ammonium
formate (0.1 M, pH 3.3). The initial composition of the mobile phase was
40:60 of acetonitrile/ammonium formate. Within 10 min, the concentration of
the organic solvent (acetonitrile) increased to 60% (linear increase). This
concentration was maintained for 5 min, after which the concentration of
acetonitrile declined to the initial value of 40% (linear decline in 5 min). For
the purpose of equilibration this concentration (40%) was kept constant for 5
min. The flow rate was 1 ml/min, column temperature was 60°C, and run time
was approximately 25 min. The assay was validated with respect to linearity,
precision, and accuracy (intra- and total assay), lower and upper limit of
quantitation, recovery, and interference from endogenous substances. For both
levormeloxifene and its major metabolite, 7-desmethyllevormeloxifene, the
assay was found to be linear in the range 2.5 to 500 ng/ml. The lower limit of
quantitation was set to 2.5 ng/ml (both analytes) as the intra-assay precision
was 7.3 and 9.1% (c.v.) and intra-assay accuracy was 83 and 108% for
levormeloxifene and 7-desmethyllevormeloxifene, respectively. The total (intra- ⫹ interassay) precision at 2.5 ng/ml was below 7.3% (c.v.), and the total
accuracy ranging between 92 and 99% for the parent compound and its major
metabolite. At 500 ng/ml (upper limit of quantitation) the c.v. was below 4.8%
and the accuracy was between 98 and 99% for levormeloxifene and 7-desmethyllevormeloxifene, respectively. The recovery of levormeloxifene and
7-desmethyllevormeloxifene from plasma was 86 and 78%, respectively. No
interference from endogenous substances were present at the retention time of
levormeloxifene and 7-desmethyllevormeloxifene.
Data Handling for Pharmacokinetic Experiments. Mean concentration/
time data were calculated and analyzed by noncompartmental methods using
the software Topfit (version 2.0; Heinzel et al., 1993). The apparent maximal
concentration (Cmax) and the corresponding time (tmax) were determined visually from the concentration-time profile. Calculation of terminal half-life (t1/2)
was based on data obtained during days 34 to 38. The terminal half-life was
calculated by means of log-linear regression using at least six data points. The
total area under the plasma concentration versus time curve (AUC) was
determined by the linear trapezoidal rule from time zero to last sampling point
equal to or above the lower limit of quantitation, AUCt, added as the residual
area as estimated by log-linear extrapolation to infinity.
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METABOLISM AND DISPOSITION OF LEVORMELOXIFENE IN RATS
TABLE 1
The maximal concentration of radioactivity (microgram equivalents per gram) in selected tissues of the rat after administration of
(mean ⫾ S.D.)
Dose
Tissue
Stomach wall
Small intestine wall
Large intestine wall
Liver
Kidney
Lung
Plasma
a
0.7 mg/kg
14
[C]levormeloxifene
50 mg/kg
Male
Female
Male
Female
1.275 ⫾ 0.349
(2)a
3.917 ⫾ 1.455
(2)
0.594 ⫾ 0.169
(4)
2.741 ⫾ 0.158
(4)
0.724 ⫾ 0.039
(4)
2.748 ⫾ 0.397
(4)
0.051 ⫾ 0.001
(4)
1.132 ⫾ 0.342
(4)
4.428 ⫾ 0.232
(2)
0.843 ⫾ 0.152
(24)
4.004 ⫾ 0.342
(4)
0.928 ⫾ 0.058
(4)
3.503 ⫾ 0.215
(4)
0.047 ⫾ 0.215
(4)
112.1 ⫾ 44.8
(2)
210.1 ⫾ 43.4
(2)
81.34 ⫾ 11.37
(24)
115.7 ⫾ 5.5
(4)
46.97 ⫾ 2.57 g
(4)
163.2 ⫾ 20.8
(4)
2.57 ⫾ 0.15
(4)
370.9 ⫾ 371.2
(2)
236.9 ⫾ 24.7
(2)
68.95 ⫾ 22.22
(24)
189.6 ⫾ 5.01
(4)
64.53 ⫾ 6.36
(4)
207.1 ⫾ 44.52
(4)
2.27 ⫾ 0.24
(4)
numbers in parenthesis refer to time (h) of maximal radioactivity.
Data shown for 4-h time point. SI, small intestine; LI, large intestine.
Radioanalysis. Radioactivity in liquid samples (urine, plasma, bile, metabolism cage washes, and expired air trap solutions) was quantified by mixing
aliquots with scintillation system MI-31 or Pico Aqua (Packard Instruments
Ltd., Pangbourne, UK and Downers Grove, IL, respectively) and conventional
liquid scintillation counting. Rat whole body digestion was carried out at 50°C
in a solution containing NaOH, water, methanol, and Triton X-405 and
samples (1 g) were mixed with scintillation system MI-31.
Feces were homogenized to a paste in distilled water and samples (0.2– 0.3
g) were burned in oxygen with an Automatic Sample Oxidizer (model 307;
Packard Instruments Ltd., Pangbourne, UK). The products of combustion were
absorbed in Optisorb I (Fisons plc, Loughborough, UK) and mixed with
Optisorb S scintillator (Fisons plc). Large tissues were homogenized, with the
addition of a known weight of water, using an Ultra-Turrax laboratory homogenizer [Semat Technical (UK) Ltd., St Albans, UK]. Other tissues were
finely scissor-minced or in the case of bone, ground with a pestle and mortar.
Radioactivity was measured by liquid scintillation counting using a Wallac
1409 or 1410 (Pharmacia-Wallac Oy, Turku, Finland) or a Packard 200CATriCarb (Packard Instruments, Downers Grove, IL) automatic liquid scintillation
analyzer with appropriate quench correction.
WBA. Sprague-Dawley rats received single oral doses of [14C]levormeloxifene (1.4 mg/kg b.wt.) and WBA was performed at 2, 4, 24, 48, and 72 h after
dose administration, essentially as described by Ullberg and Larrson (1981).
Sections were prepared using a 9400 Cryostat Microtome (Bright Instruments
Co., Huntingdon, UK). Sagittal sections (30 ␮m) were cut at six levels through
the carcass, between the levels of the kidneys (males) or ovaries (females) and
the spinal cord. Sections were mounted on Cellux tape (Aston Clinton, St.
Albans, UK) and freeze-dried in a Lyolab B freeze-drier (Life Sciences
Laboratories Ltd., Luton, UK) before placing them in contact with Kodak
DEF5 film (Kodak Ltd., Hemel Hempstead, UK) and ␤max X-ray film
(Amersham International, Amersham, UK). The film was exposed for 41 days
FIG. 3. Proportions of dose present in combined selected tissues of male (m) and
female (f) rats after oral administration of [14C]levormeloxifene (0.7 or 50
mg/kg) to rats.
Selected tissues (including GI tract contents) as described in Materials and
Methods. Data shown as mean ⫾ S.D. f, f (0.7 mg/kg); Œ, m (0.7 mg/kg); , f (50
mg/kg); ⽧, m (50 mg/kg).
at ⫺20°C before its development. Autoradiographs were evaluated by visual
inspection.
Analysis of Metabolites. Tentative identification of metabolites in fecal
extracts, urine, plasma, bile, and selected tissue extracts for the low and high
doses was achieved by HPLC cochromatography with authentic standards. The
elution times for levormeloxifene, NNC 46 – 0002, NNC 46 – 0003, NNC
46 – 0004, and NNC 46 – 0005 were typically 36, 22, 29, 32, and 55 min,
respectively, although it was apparent on some of the HPLC runs that there was
a shift in retention time, possibly due to the matrix in the injected samples.
Additional identification was achieved by mass spectroscopy. Certain bile,
urine, and plasma samples were deconjugated by mixing in equal proportions
with ␤-glucuronidase (Type H1, 2000 U/ml) provided by Sigma, and incubating overnight at 37°C in acetate buffer (pH 5). Feces and tissue extracts were
made by homogenization of samples in diethylether, centrifugation to obtain
supernatants, evaporation of solvent, and resuspension in the HPLC mobile
phase. Plasma samples (native or enzyme-treated) were applied to IsoluteConfirm HCX mixed-mode solid-phase extraction columns (3 ml/300 mg size;
Jones Chromatography, Hengoed, UK), which were preconditioned with methanol and phosphate buffer (0.1 M; pH 2.0). After sample loading, the column
was rinsed with acetic acid (1 M) and phosphate buffer (0.1 M; pH 2.0) and the
analytes were eluted with methanol/triethylamine (98:2, v/v).
HPLC conditions. Samples were analyzed using a LiChrospher 100 C18
column (particle size 5 ␮m, 250 ⫻ 4 mm id; Merck, Darmstadt, Germany) with
a ␮BondaPak C18 Guard-Pak precolumn (Millipore, Waters, MA). A Thermo
Separation Products HPLC system was used (Thermo Separation Products,
Stone, UK) consisting of a pump, interface, and a UV 2000 variable wavelength UV detector and a Ramona-5 or ␤-RAM on-line radioactivity detector
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FIG. 2. Mean concentrations (⫾S.D.) of radioactivity (tissue/plasma ratio) in
selected tissues after oral administration of [14C]levormeloxifene (0.7 mg/kg or
50 mg/kg b.wt.) to male (m) and female (f) rats.
506
MOUNTFIELD ET AL.
Results
FIG. 4. WBA of a female rat 4 h after oral administration of [14C]levormeloxifene.
ad, adrenal; b, brain; bf, brown fat; bl, blood; bm, bone marrow; cac, caecum
contents; elg, exorbital lacrimal gland; fa, fat; Hd, Harderian gland; ilg, intraorbital
lacrimal gland; k, kidney; l, liver; lic, large intestine contents; lu, lung; mb,
meibomian gland; my, myocardium; mu, muscle; ov, ovary; p, pancreas; pb, pineal
body; pg, preputial gland; pit, pituitary; s.c., spinal cord; sg, salivary gland; sic,
small intestine contents; sk, skin; sp, spleen; stc, stomach contents; th, thymus; ty,
thyroid; ut, uterus.
(supplied by LabLogic Systems Ltd., Sheffield, UK) fitted with a solid scintillator flow cell. For the biliary excretion experiments a Hitachi-Merck HPLC
system (supplied by Kebo Struers, Copenhagen, Denmark) consisting of a
pump, interface, UV detector, and on-line radioactivity detector (Canberra
Flo-One, Canberra, Copenhagen, Denmark) was used. The absorption (for
detection of reference standards) was measured at 279 nm. A mobile phase
gradient was used: from 0 to 55 min, a 3:7 ratio of 0.1 M ammonium formate,
pH 3.3/acetonitrile; from 55.1 to 57.0 min, a 1:1 ratio was used; and from 57.1
to 59.0 min, a 7:3 ratio was used. A flow rate of 1.0 ml/min was used except
from 27.6 to 55.1 min, when the flow rate was increased to 1.3 ml/min. The
above conditions were modified slightly during the isolation of metabolites for
mass spectrometry analysis. Individual radioactive peaks were collected manually, solvent was removed under nitrogen, and residues were redissolved in
acetonitrile/water (1:1, v/v) before mass spectrometry. The radiochemical
purity of each metabolite was assessed by both reinjection on HPLC and
thin-layer chromatography analysis (not shown).
Mass spectrometry. Selected metabolites were analyzed by atmospheric
pressure chemical ionization (APCI) mass spectrometry in both positive and
negative ionization modes, where appropriate, using a TSQ7000 (Finnigan
MAT, San Jose, CA) or an API 300 triple quadrapole liquid chromatographytandem mass spectrometry (LC-MS-MS) mass spectrometer (Perkin-Elmer
Sciex Instruments, Beaconsfield, UK) equipped with an Ionspray interface
Quantitative Tissue Distribution. Radiolabeled levormeloxifene
was distributed throughout body tissues after oral administration at
dose levels of 0.7 and 50 mg/kg b.wt., and the rate of absorption and
the general distribution of radioactivity was similar at both dose
levels. The concentrations of radioactivity in the tissues increased in
a dose-proportional manner. Not surprisingly, the greatest amounts of
radioactivity were found in the gastrointestinal tract contents and in
those organs responsible for absorption and elimination soon after
administration (Table 1).
However, high concentrations of radioactivity were also found in
the lungs. Maximal radioactivity concentrations were generally found
4 h postdose, but concentrations peaked in the testes and large
intestine wall (50-mg/kg dose) of male rats at 24 h and in the fat (s.c.),
lacrimal gland, large intestine wall, mammary gland, skin, and thymus
of female rats at 24 h postdose. The proportion of the dose distributed
into the tissues of female rats was greater than in male rats at all time
points, at both dose levels (Fig. 2).
Excluding radioactivity in the gastrointestinal tract contents, radioactivity in the tissues of male rats reached a maximum level of 29.2
and 21.4% of the administered dose at 4 h and thereafter declined to
2.9 and 1.6% at 72 h postdose for the 0.7 mg/kg and 50 mg/kg b.wt.
dose, respectively. Figure 3 shows the proportion of the dose in
selected tissues, including the gastrointestinal tract contents, described
in Materials and Methods. A higher proportion of radioactivity was
retained in the tissues of female rats with a maximum level of 38.0 and
30.8% at 4 h, declining to 10.5 and 6.51% at 72 h postdose for the 0.7
mg/kg and 50 mg/kg b.wt. dose, respectively.
Peak radioactivity concentrations were generally 1.2 to 1.7 times
greater in female rat tissues than in the corresponding male tissues
with an even greater difference (2.2-fold) for fat observed. Mean peak
concentrations in the whole blood, plasma, skin, small intestine wall,
and stomach wall were similar between sexes. The concentration
difference of radiolabeled levormeloxifene between male and female
rats generally increased with time, and at 72 h the concentrations were
4- to 6-fold greater in the corresponding tissues of females than males.
Even higher differences of 8-, 9-, and 23-fold were found for the
spleen, pituitary gland, and salivary gland, respectively.
Qualitative Distribution. The general distribution of radioactivity
from WBA agreed with the quantitative distribution data. The only
tissues found to contain notable concentrations of radioactivity that
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(Perkin-Elmer Sciex Instruments, Thornhill, Canada). Ionization conditions
were optimized by varying the octapole offset (range ⫺2.9 to ⫺3.3 V in the
positive ion mode, ⫹ 3.0 V in the negative ion mode), capillary (⫹20 to ⫹91.8
in the positive ion mode, ⫺20 to ⫺96.8 V in the negative ion mode), and tube
lens voltages (⫹72 to ⫹181.5 V in positive ion mode, ⫺72 to –187.1 V in
negative ion mode). A portion of the sample (ca. 50 ␮l) was injected into the
APCI interface at 1 ml/min, in acetonitrile/water (1:1, v/v). Mass spectra of the
compounds of interest were recorded over an appropriate mass range for 2 min
at a scan rate of 1 s/scan. An APCI mass spectrum of the compound of interest
was obtained by averaging several scans across the region of the mass chromatogram where a response was observed, with appropriate background subtraction. This mass spectrum was examined to identify a candidate molecular
ion, [M⫹H]⫹ or [M-H]⫺, for the compound of interest, and any other structurally significant fragment ions resulting from in-source collisionally induced
dissociation. For metabolite identification experiments from bile, the mass
spectrometer scanned in the range m/z 100 to 700 with a dwell time of 0.5 ms
and a step size of 0.1 amu. The electrospray and orifice voltages were set to
5000 and 30 V, respectively. In the tandem mass spectrometry mode (product
ion scan) the mass spectrometer scanned in the range m/z 50 to 500 (or higher,
dependent on the mass of the precursor ion) with a dwell time of 0.5 ms and
a step size of 0.1 amu. The electrospray and orifice voltages were set to 5000
and 30 V, respectively, and the fragmentation energy was ⫹35 V.
METABOLISM AND DISPOSITION OF LEVORMELOXIFENE IN RATS
507
bm, bone marrow; cb, cerebellum; ce, cerebrum; ch, choroid plexus; cq, copora quadrigemina; ol, olfactory lobe; pit, pituitary.
were not sampled in the quantitative analysis were the pineal body,
brown fat, and the preputial, meibomain, and Harderian glands.
At 2 h after administration of [14C]levormeloxifene (1.4 mg/kg
b.wt.), the greatest radioactivity concentrations were found in the
contents of the upper gastrointestinal tract, liver, lung, spleen, pancreas, adrenal gland, pineal body, and in the renal cortex. Slightly
lower concentrations were found in the lower intestinal tract wall,
lacrimal glands, salivary glands, thyroid, pituitary, and brown fat.
Moderately high concentrations were found in the bone marrow,
preputial gland, Harderian gland, myocardium, and prostate. Maximal
concentrations in most tissues were noted at 4 h postdose, in agreement with the quantitative results. Concentrations of radioactivity in
the ovaries of female rats were relatively high at all time points
(comparable with the levels seen in the spleen and kidneys) (Fig. 4).
Concentrations in the uterus were also moderately high but declined
more rapidly than from the ovaries.
After 24 h, concentrations of radioactivity had markedly decreased
in many tissues. However, as with the quantitative analysis, concen-
trations of radioactivity in tissues of female rats were markedly higher
than concentrations in corresponding tissues of male rats, indicating a
slower rate of elimination in the female rat. This was substantiated in
a separate toxicokinetic experiment (not shown) where the minimum
trough concentrations of levormeloxifene were significantly higher in
female rats than in males, at all sampling periods, after daily dosing
for 28 days.
After 72 h radioactivity in the gastrointestinal tract was mainly
localized to the lower part of the tract. Notably high concentrations
were still observed in the Harderian gland and in the meibomian
gland.
Photographic enlargements of the brain of female rats showed that
radioactivity was distributed relatively uniformly throughout the cerebrum, cerebellum, and olfactory lobe (Fig. 5). Higher concentrations
were observed in the pineal body and in the pituitary gland, and in the
choroid plexus shortly after administration.
14
C-Excretion. After oral administration of radiolabeled levormeloxifene (0.7- or 50-mg/kg dose), radioactivity was excreted pre-
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 11, 2017
FIG. 5. Enlargement of female rat brain 2 h after oral administration of [14C]levormeloxifene.
508
MOUNTFIELD ET AL.
TABLE 2
TABLE 3
Excretion of radioactivity after oral administration of [14C]levormeloxifene to
rats (mean ⫾ S.D. values)
Excretion of radioactivity into bile after oral administration of
[14C]levormeloxifene to rats (mean values)
Dose
0.7 mg/kg
Time (h)
Male n ⫽ 3
Feces
0–24
24–48
48–72
72–96
96–120
120–144
144–168
Total feces
51.1 ⫾ 13.1
33.1 ⫾ 13.6
9.4 ⫾ 1.2
3.1 ⫾ 0.4
1.8 ⫾ 0.2
0.9 ⫾ 0.1
0.5 ⫾ 0.2
99.95 ⫾ 2.3
50 mg/kg
Female n ⫽ 3
Male n ⫽ 3
Dose
Female n ⫽ 3
23.5 ⫾ 4.1
29.3 ⫾ 2.9
18.2 ⫾ 0.8
10.2 ⫾ 1.4
5.9 ⫾ 0.7
4.0 ⫾ 0.4
2.4 ⫾ 0.5
93.6 ⫾ 1.5
46.0 ⫾ 3.3
34.2 ⫾ 3.8
12.5 ⫾ 0.8
3.5 ⫾ 0.6
0.9 ⫾ 0.3
0.5 ⫾ 0.1
0.2 ⫾ 0.1
97.7 ⫾ 0.5
21.9 ⫾ 9.4
26.0 ⫾ 1.1
19.1 ⫾ 5.4
12.5 ⫾ 0.9
8.2 ⫾ 1.8
4.4 ⫾ 1.3
2.5 ⫾ 1.1
94.5 ⫾ 2.3
0.7 mg/kg
Time
h
50 mg/kg
Male
Female n ⫽ 2
Male n ⫽ 2
Female n ⫽ 2
4.3
4.3
5.1
5.1
5.4
5.4
3.2
3.2
0.2
0.2
0.2
0.2
0.5
0.5
0.3
0.3
6.0
11.2
9.1
6.5
32.8
2.8
5.0
4.4
3.6
15.8
7.5
8.7
7.1
5.6
28.9
3.7
5.5
5.8
4.8
19.8
Carcass
60.2
78.0
61.1
73.7
Total recovery
97.5
99.0
95.8
96.8
Urine
0–6
6–24
24–48
48–72
72–96
96–120
120–144
144–168
Total urine
0.04 ⫾ 0.03
0.3 ⫾ 0.1
0.2 ⫾ 0.03
0.04 ⫾ 0.1
0.02 ⫾ 0.01
0.01 ⫾ ⬍0.01
0.01 ⫾ ⬍0.01
0.01 ⫾ ⬍0.01
0.6 ⫾ 0.1
0.1 ⫾ 0.1
0.3 ⫾ 0.02
0.3 ⫾ 0.1
0.2 ⫾ 0.1
0.2 ⫾ 0.1
0.1 ⫾ 0.1
0.01 ⫾ 0.02
0.04 ⫾ 0.01
1.3 ⫾ 0.2
0.1 ⫾ 0.04
0.4 ⫾ 0.04
0.3 ⫾ 0.03
0.1 ⫾ 0.01
0.04 ⫾ 0.01
0.01 ⫾ 0.01
0.01 ⫾ ⬍0.01
0.01 ⫾ 0.01
0.8 ⫾ 0.04
0.1 ⫾ 0.03
0.4 ⫾ 0.04
0.3 ⫾ 0.1
0.2 ⫾ 0.03
0.2 ⫾ 0.02
0.1 ⫾ 0.02
0.1 ⫾ 0.02
0.03 ⫾ 0.01
1.3 ⫾ 0.1
Cage wash
0.02 ⫾ 0.01
0.04 ⫾ 0.02
0.05 ⫾ 0.04
0.06 ⫾ 0.04
1.5 ⫾ 0.3
5.8 ⫾ 0.9
0.7 ⫾ 0.03
2.7 ⫾ 1.2
Dose
Cmax
tmax
98.6 ⫾ 1.2
mg/kg
ng/ml
h
ng 䡠 h/
ml
ng 䡠 h/ml
26a
6a
799a
971a
22b
4b
710b
918b
64a
6a
1580a
1968a
74b
6b
1631b
2171b
Carcass
Total recovery
102 ⫾ 2.3
100.8 ⫾ 2.1
99.3 ⫾ 0.5
dominantly in the feces (Table 2). Mean fecal excretion accounted for
68.5 to 64.1% of the administered dose during the first 48 h, and 96.8
to 96.1% of the dose at the end of 168 h for the low (0.7 mg/kg)- and
high (50 mg/kg)-dose groups, respectively, with male and female data
combined at each dose level. Approximately 1.0% of the administered
dose was excreted in the urine in the low- and high-dose groups. In a
comparison of male and female rats, at both dose levels radioactivity
was excreted more rapidly by males than by females (Table 2).
Interestingly, a lower proportion of the dose was excreted in the urine
of males than females, 0.6 to 0.8% compared with 1.3 to 1.3% (low
and high doses, respectively) over 168 h, respectively, and the amount
retained by the carcasses was also lower in males at both dose levels.
After oral administration of [ 14 C]levormeloxifene to bilecannulated rats, 16 to 20% of the radioactivity was excreted into the
bile from female rats within 24 h, at the low and high doses, respectively (Table 3). In male animals, 33 to 29% of the dose was excreted
into bile during the first 24 h (low and high doses, respectively). Less
than 5.0% (mean, male and female animals, both dose levels) and
0.5% (mean, male and female animals, both dose levels) of the total
radioactivity was found in, respectively, feces or urine (Table 3). The
majority of the radioactivity was retained in the body up to 24 h, with
over 60% of the recovered radioactivity being detected in the animal
carcasses.
Pharmacokinetics. After drug administration, Cmax was generally
observed 6 h after dosing. Pharmacokinetic parameter estimates are
presented in Table 4. The half-life of elimination was long (24 h) and
a doubling in dose resulted in an approximate doubling in exposure.
Metabolite Profiling and Identification. A number of different
radioactive metabolites were tentatively identified by HPLC analysis,
with subsequent structural confirmation by LC-MS-MS.
Bile. Proportions of radioactive components were generally similar
at both the low- and high-dose groups in male and female animals
(0 –24 h post dose). One major metabolite (M1), in addition to two
minor metabolites (M2 and M4) were isolated from bile. Typical
HPLC chromatograms of bile from male (a) and female (b) rats, 0 to
TABLE 4
Pharmacokinetic parameter estimates for levormeloxifene in rats after oral
administration (mean values)
AUC(0–48h)
AUCc
0.5
1.0
a
b
c
␭z
t1/2
h⫺1
h
0.0299b
23.2b
0.0288b
24.0b
estimates based on data obtained during days 13 to 15.
estimates based on data obtained during days 34 to 38.
AUC ⫽ AUC (0 –⬁).
6 h after oral administration of 0.7 mg/kg b.wt. [14C]levormeloxifene,
are shown in Fig. 6. Mass fragmentation patterns for four metabolites
are shown in Table 5 and the proportions of identified metabolites in
the bile samples analyzed are shown in Table 6. The remaining
radioactivity was excreted into feces during the experimental time
period. Bile isolated from both male and female rats (both dose levels)
consisted mainly of 7-desmethyllevormeloxifene glucuronide (M1) at
all time points, with the additional quantitatively minor metabolites
being identified as the glucuronides of hydroxylevormeloxifene (M2)
and levormeloxifene (M4). Glucuronidase treatment of bile resulted in
the identification of the aglucans for the isolated metabolites (M3
shown for reference purposes).
The highest concentration of M1 was detected during the first 6 h
post dose administration in males and accounted for 57.4 to 56.2% of
the sample radioactivity (low- and high-dosing groups, respectively),
whereas in females higher concentrations of M1 were seen during 6to 12-h post dose administration (62.5 to 61.3% of the sample radioactivity in the low- and high-dose groups, respectively). Minor sex
differences were observed for the proportion of hydroxylevormeloxifene glucuronide (M2).
Feces. Proportions of radioactive components and metabolite profiles were generally similar at both the low- and high-dose groups in
male and female animals. At least 11 metabolites were detected in
feces, based on fraction collection data and on-line radioactivity
monitoring; however some of these metabolites remained unidentified
during the study (Fig. 7, representative on-line HPLC chromatograms
of feces from male (a) and female (b) rats, 0 to 24 h after oral
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 11, 2017
Feces
0–24
Total feces
Urine
0–24
Total urine
Bile
0–6
6–12
12–18
18–24
Total bile
METABOLISM AND DISPOSITION OF LEVORMELOXIFENE IN RATS
14
administration of 0.7 mg/kg b.wt. [ C]levormeloxifene). There was
some shift in retention time during HPLC analysis but this was
attributed to a matrix effect and was compensated for by cochromatography with the authentic reference standards. Subsequent HPLC
runs (minus reference standards) were then used for isolation of
metabolites for LC-MS-MS analysis. Feces consisted mainly of 7-desmethyllevormeloxifene (M6, norlevormeloxifene) at all time points,
but unchanged drug was the second most prevalent component (Fig.
7 and Table 6). Mass fragmentation patterns for six of the isolated
metabolites are shown in Table 5. The proportions of identified
metabolites (M5–M9, and levormeloxifene) in feces are shown in
Table 6.
Unchanged drug in feces accounted for 7.3 to 9.9% of the total dose
in male rats from 0 to 24 h, decreasing to 0.5 to 1.8% in the 24- to 48-h
sample in the low- and high-dose groups, respectively. In female rats,
unchanged drug accounted for 7.8 to 3.9% of the total dose in the 0-
Radioactivity in each collected fraction was determined by off-line liquid scintillation counting. Letter indicates metabolite assignment. Minor metabolites not
shown in figure.
to 24-h fecal radioactivity but only 1.7 to 1.1% of the 24- to 48-h
samples, in the low- and high-dose groups, respectively. Total fecal
excretion of unchanged drug from 0 to 48 h in male rats and from 0
to 72 h in female rats amounted to 7.8 to 11.7% (low- and high-dose
group, respectively) and 10.4 to 6.1% (low- and high-dose group,
respectively) of the dose, respectively.
The major metabolite in feces, 7-desmethyllevormeloxifene (M6),
accounted for 32.1 to 27.6% of the total dose in male rats in the lowand high-dose groups, respectively, from 0 to 48 h. In female rats,
7-desmethyllevormeloxifene isolated in feces accounted for 28.3 to
25.6% of the administered dose (0 –72 h) in the low- and high-dose
groups, respectively. A number of less quantitatively significant metabolites (M5, M7-M9) were also isolated and identified (Tables 5 and
6; Fig. 7).
Urine. After both 0.7 and 50 mg/kg b.wt. doses of 14C-levormeloxifene, radioactivity excreted in urine, accounting for approximately
1% of the administered dose, was largely associated with chromatographically polar metabolites. There were marginal sex differences in
the polar metabolites, although there were no differences between
dosing groups. For example, polar components made up 91% of the
total urinary radioactivity excreted from 0 to 24 h (Fig. 8A) in male
rats after dosing at 0.7 mg/kg b.wt. In urine excreted by female rats
24- to 48-h postdose (0.7 mg/kg b.wt. dose), a slightly lower proportion of urinary radioactivity was associated with these polar components (about 73–75%) (Fig. 8B).
After incubation with ␤-glucuronidase, proportions of polar components decreased with a concomitant increase in the less polar
components, most notably 7-desmethyllevormeloxifene (M6) and monohydroxylevormeloxifene (M5 and M7). Proportions of unchanged
drug also increased after enzyme treatment from only 1.4 and 0.4% of
sample radioactivity before treatment, in urine from males and females, respectively, to 19.7 and 8.3%, respectively, at the low dose
level.
These data indicate that radioactivity excreted in urine is predominantly associated with glucuronic acid conjugates, including conjugates of 7-desmethyllevormeloxifene (the major fecal metabolite),
parent drug, and monohydroxylevormeloxifene.
Plasma. Unchanged drug was the major radioactive component
present in the systemic circulation, at both dose levels, 4 and 24 h after
dose administration. At 4 h, unchanged drug in the low- and high-dose
groups, respectively, accounted for 71.9 to 37.7 and 61.5 to 49.4% of
plasma extract radioactivity in male and female rats, respectively.
Figure 9 shows HPLC profiles of plasma from male (a) and female (b)
rats 4 h after oral administration of 0.7 mg/kg b.wt. [14C]levormeloxifene. There was again some shift in retention time during analysis of
the different samples; however, this was compensated for by inclusion
of reference standards. Other metabolites present in the systemic
circulation included monohydroxylevormeloxifene (M5), and the mi-
TABLE 5
Mass spectra profiles of levormeloxifene metabolites isolated from rat bile and feces
Metabolite
M1
M2
M3
M4
M5
M6
M7
M8
M9
Levormeloxifene
a
sample treated with ␤-glucuronidase.
Metabolite Source
m/z
bile
bile
bile, treateda
bile
feces extract
feces extract
feces extract
feces extract
feces extract
feces extract
620
650
458
634
474
444
474
490
430
458
Product Ions
444,
474,
267,
458,
283,
241,
283,
472,
239,
267,
294,
269,
176,
326,
271,
192,
255,
326,
211,
189,
151,
181,
143,
252,
192,
119,
215,
267,
159,
175,
98
98
119, 98, 91, 71
151, 112
181, 143, 119, 98, 71
98, 91, 71
165, 135, 107, 98, 71
229, 223, 165, 130, 112, 91, 87, 84, 69, 44,
118, 98, 91, 71
165, 119, 98, 91, 71,
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 11, 2017
FIG. 6. HPLC of bile from male (A) and female (B) rats, 0 to 6 h after oral
administration of 0.7 mg/kg b.wt. [14C]levormeloxifene.
509
510
MOUNTFIELD ET AL.
TABLE 6
Proportions of metabolites in individual samples (0 –24 h)
Results expressed as percent dose.
Bile
0.7 mg/kg
Feces
50 mg/kg
Metabolite
M1
M2
M3a
M4
M5
M6
M7
M8
M9
M10
M
14.31
2.49
F
8.82
0.76
M
13.36
2.70
F
10.75
1.53
5.90
ND
ND
ND
ND
ND
3.39
ND
ND
ND
ND
ND
5.08
ND
ND
ND
ND
ND
Levormeloxifene
ND
ND
ND
0.7 mg/kg
50 mg/kg
4.88
ND
ND
ND
ND
ND
M
ND
ND
ND
ND
4.96
18.66
5.42
0.92
1.94
F
ND
ND
ND
ND
0.02
9.06
1.06
0.09
1.39
M
ND
ND
ND
ND
3.03
15.67
4.27
1.38
0.87
F
ND
ND
ND
ND
ND
9.43
1.88
0.44
0.48
ND
7.26
7.76
9.93
3.92
FIG. 8. HPLC, with on-line radioactivity detection, of urine from male (A) and
female (B) rats, 0 to 24 and 24 to 48 h, respectively, after oral administration of
0.7 mg/kg b.wt. [14C]levormeloxifene.
Letters indicate metabolite assignment.
FIG. 7. HPLC, with on-line radioactivity detection, of feces from male (A) and
female (B) rats, 0 to 24 h after oral administration of 0.7 mg/kg b.wt.
[14C]levormeloxifene.
Letters indicate metabolite assignment. Minor metabolites not shown in figure.
nor metabolites (not shown in Fig. 9), 7-desmethyllevormeloxifene
(M6), desmethylnorlevormeloxifene (M9), and 7-desmethyllevormeloxifene glucuronide (M1, which increased over time). Additionally, an unknown metabolite was also evident at both levels at all time
points. After 24 h the proportion of radioactivity associated with
unchanged drug decreased, and proportions of metabolites correspondingly increased. Interestingly, greater concentrations of the parent drug remained in the circulation of female rats at increasing time
reflecting a possible lower rate of metabolism.
Tissues. Up to 72 h after administration of [14C]levormeloxifene,
radioactivity was predominantly associated with the parent compound
in nongastrointestinal tract tissues, in both sexes, at both dose levels.
Figure 10 shows representative HPLC chromatograms of liver extracts
from female rats 24 (a) and 72 h (b) after oral administration of 0.7 mg/kg
b.wt. [14C]levormeloxifene. For example, [14C]levormeloxifene accounted for 85.8 to 93.9% of the total radioactivity in the liver of male
and female rats after dosing at 0.7 mg/kg b.wt., declining to 76.2 to
88.4%, respectively, at 24 h. At later time points (24-h), there was an
increase in the proportion of quantitatively minor metabolites, primarily
7-desmethyllevormeloxifene (M6) and monohydroxylevomeloxifene
(M5 and M7). M6 was quantified by off-line radioactivity monitoring of
collected fractions, because with the on-line radioactivity profile the
response for M6 was only slightly above background. The metabolite
profiles in lung and kidney were very similar to those in liver with
unchanged drug accounting for the majority of the total radioactivity in
the 2- to 4-h lung and 4-h kidney extracts.
Discussion
Paramount to the development of a suitable selective estrogen
receptor modulator for the treatment of osteoporosis is an understanding of the distribution, metabolism, and excretion of the compound
after oral administration to preclinical species, because the pharmacological profile of compounds that bind to estrogen receptors may be
altered due to the formation of metabolites with higher estrogenic
activity than the parent compound (Dodge et al., 1997).
The results from the current study indicate that within 2 h after oral
dose administration, radioactivity was higher in all tissues than in the
blood, indicating rapid distribution of levormeloxifene and/or metab-
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 11, 2017
ND, below limit of detection.
a
Metabolite M3 was isolated from bile samples after treatment with ␤-glucuronidase and was
the aglucan of M4.
METABOLISM AND DISPOSITION OF LEVORMELOXIFENE IN RATS
511
FIG. 10. HPLC, with on-line radioactivity detection, of liver extracts from female
rats 24 (A) and 72 (B) h after oral administration of 0.7 mg/kg b.wt.
[14C]levormeloxifene.
Letters indicate metabolite assignment. Minor metabolites not shown in figure.
Letters indicate metabolite assignment. Minor metabolites not shown in figure.
olites. However, based on HPLC analysis of tissue extracts (liver,
kidney, and lung), much of this radioactivity, up to 94% of the sample
radioactivity in some tissue samples, was associated with parent
compound (Fig. 10), as indeed was the radioactivity associated with
the systemic circulation (Fig. 9). Peak radioactivity concentrations
were generally achieved 4 h postdose in both male and female animals
at dose levels of 0.7 and 50 mg/kg b.wt. Interestingly, peak radioactivity concentrations were generally circa 1.2 to 1.7 times greater in
female rat tissue than in corresponding tissues from male animals (not
shown), and this was also reflected in a slower elimination of radioactivity from female animals. The reason for these differences are
unclear, although this may be related primarily to fundamental differences in the metabolism between sexes (Mugford and Kedderis,
1998). Alternatively differences in elimination rates may be related to
the compound class, that of a selective estrogen receptor modulator
and differences in tissue binding and discrete receptor interactions
(Dodge et al., 1997) with respect to the distribution and concentration
of estrogen receptors (Kuiper et al., 1997). However, after single oral
doses any differences between the rate of elimination of drug is
probably more likely attributable to sex differences in metabolism.
Radioactivity was slowly excreted into feces, presumably after
conjugation of metabolites and excretion into bile (Tables 2, 3, and 6)
with only approximately 1% of the administered dose being excreted
via the renal route. The major metabolite isolated from feces was
characterized by HPLC and LC-MS-MS as 7-desmethyllevormeloxifene (norlevormeloxifene), indicating a typical cytochrome P450
demethylation reaction on the methoxy group of levormeloxifene, and
accounted for about 34 to 43% of fecal extract radioactivity and for
about 25 to 33% of the dose. Unchanged drug was also excreted,
mainly from 0 to 24 h, and accounted for about 6 to 12% of the dose.
Together these two components accounted for approximately 50% of
the radioactivity excreted in those feces samples analyzed. Additional
metabolites isolated and identified by LC-MS-MS, and accounting for
up to 5% of the excreted radioactivity in rat feces during the first 24 h
included two separate monohydroxylevormeloxifene (hydroxylated
on different benzene rings), and desmethylnorlevormeloxifene. The
formation of this metabolite is highly unusual because the proposed
structure would arise from C-demethylation and O-demethylation of
levormeloxifene. C-demethylation is an unexpected metabolic reaction and in the absence of definitive evidence, the proposed structure
should be regarded with caution. Additionally, a pyrrolidinone ringopened metabolite of levormeloxifene was also isolated and identified. A proposed route of metabolism is shown in Fig. 11.
The pharmacokinetics of levormeloxifene were also determined in
female rats, and drug measurements were performed on animals dosed
with 1.0 and 0.5 mg/kg b.wt. levormeloxifene, reflecting the anticipated therapeutic dose range. Results indicated that Cmax was generally observed 6 h after dosing, and the AUC values increased fairly
proportionally to the dose. The half-life of elimination was long, being
approximately 1 day. The plasma levels of the major metabolite
7-desmethyllevormeloxifene were in all pharmacokinetic samples below the lower limit of quantitation, confirming tissue distribution
experiments, indicating that parent compound was the major circulating species and the major species in tissues even though the major
metabolite excreted was 7-desmethyllevormeloxifene.
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 11, 2017
FIG. 9. HPLC, with on-line radioactivity detection, of plasma from male (A) and
female (B) rats 4 h after oral administration of 0.7 mg/kg b.wt.
[14C]levormeloxifene.
512
MOUNTFIELD ET AL.
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 11, 2017
FIG. 11. Proposed biotransformation pathway.
Metabolite M3 not shown because this was identified as the aglucan of M4.
In conclusion, it would appear that levormeloxifene is an orally
active compound and it can be predicted that the main site of metabolism is in the liver, with the major excretion pathway of parent
compound and metabolites being via the fecal route in rodents. There
appeared to be some minor gender differences in the distribution,
metabolism, and excretion of radioactivity. However, there were no
apparent changes in metabolism between dose levels. Similar studies
in monkeys and human volunteers have indicted a similar excretion
pathway and the formation of a number of comparable metabolites to
those found in the rat species (Mountfield et al., 1999). What has not
been established during these preclinical studies is the effect of the
long half-life of this compound (219 h in volunteers; B.K., personal
communication) on the overall consequences of repeated administra-
tion to patients. Interestingly, a similar compound being developed for
osteoporosis, idoxifene, also has a long half-life, and clinical development was recently stopped due to adverse events in the clinic
(SCRIP, 1999, 2431, p21).
Acknowledgments. Bile duct cannulation procedures were performed by T. Pederson. Mass spectra were obtained by O. Wassmann
and Dr. D. Watson.
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