Published-Ahead-of-Print September 18, 2008, DOI:10.2164/jandrol.108.004994
Journal of Andrology, Vol. 30, No. 1, January/February 2009
Copyright © American Society of Andrology
DOI: 10.2164/jandrol.108.004994
Adjudin Targeting Rabbit Germ Cell Adhesion as a Male Contraceptive: A Pharmacokinetics Study
GUO-XIN HU*,
LU-FENG HU*,
DAI-ZHENG YANG*,
JUN-WEI LI*,
GUO-RONG CHEN
,
BING-BING CHEN*,
,
DOLORES D. MRUK,
MICHELE BONANOMI
,
BRUNO SILVESTRINI,
C. YAN CHENG AND
REN-SHAN GE*,
From the * Institute of Molecular Toxicology and
Pharmacology, School of Pharmacy, and the
Institute of Molecular Pathology, 1st
Affiliated Hospital, Wenzhou Medical College, Wenzhou, China;
Population Council and Rockefeller University,
New York, NY; and Bioprogress Pharmaceuticals
S.p.A., Rome, Italy.
|
Correspondence to: Ren-Shan Ge or C. Yan Cheng, Population Council, 1230 York
Avenue, New York, NY 10065 (e-mail:
rge{at}popcbr.rockefeller.edu
or
Y-Cheng{at}popcbr.rockefeller.edu). |
| Received for publication January 17, 2008;
accepted for publication September 10, 2008. |
 |
Abstract
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Adjudin (1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide;
formerly called AF-2364) has been shown to inhibit spermatogenesis by
disrupting anchoring junctions at the Sertoligerm cell interface. This, in
turn, leads to germ cell loss from the seminiferous epithelium, and transient
infertility. Adjudin's efficacyin inhibiting spermatogenesis, the recovery of
spermatogenesis after cessation of the drug, and side effects were examined in
adult male Japanese rabbits. The pharmacokinetics profiles of adjudin in
rabbits after oral administration and after intravenous injection were
compared. Rabbits received 25 mg/kg adjudin once weekly for 4 consecutive
weeks either by intravenous injection or by gavage. Vehicle-treated rabbits
were used as controls. At 1, 2, 3, 4, and 8 weeks after treatment, testes were
removed for microscopic examination to assess the status of spermatogenesis.
Four weeks after intravenous cessation of adjudin, the recovery of
spermatogenesis also was monitored. Blood was withdrawn after first
administration to measure plasma concentrations of adjudin by high-performance
liquid chromatography. Four weeks after intravenous treatment, examination of
testis sections showed rapid exfoliation of elongated/elongating spermatids
and the presence of large multinucleated cells; more than 95% of germ cells
were absent from the seminiferous epithelium. Intravenous treatment showed a
more severe disturbance of spermatogenesis compared with gavage treatment,
which was correlated with bioavailability of the drug. The areas under the
curve for intravenous injection and gavage were 20.11 ± 1.90 and 2.23
± 0.45 mg·h·L–1, respectively. These
results illustrate the potential of adjudin as a male contraceptive, and the
efficacy is associated with the bioavailability of the drug.
Key words: Male contraception, testis, Sertoli-germ cell adhesion
During spermatogenesis, preleptotene and leptotene spermatocytes residing
below the blood-testis barrier, which is formed by the inter-Sertoli cell
tight junctions near the basal lamina, will migrate from the basal to the
adluminal compartment of the seminiferous epithelium for further development
(Russell, 1977;
Mruk and Cheng, 2004b). This
process is associated with extensive restructuring of cell-cell actin-based
anchoring junctions. One such anchoring junction is the ectoplasmic
specialization, which is found between Sertoli and germ cells at step 8 and
beyond (Mruk and Cheng, 2004b).
Thus, the ectoplasmic specialization could be a specific target for male
contraception, because its disruption can lead to germ cell loss from the
epithelium. Adjudin
(1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide; formerly known
as AF-2364) is a derivative of lonidamine, which specifically targets the
apical ectoplasmic specialization (Cheng et
al, 2005). Adjudin has been shown to remarkably disrupt anchoring
junctions between Sertoli cells and elongated/elongating, and round spermatids
in adult rats (Cheng et al,
2005). Although it also affects anchoring junctions between
Sertoli cells and spermatocytes but to a lesser extent, adjudin does not seem
to affect the blood-testis barrier and adherens junctions between Sertoli
cells and spermatogonia (Mruk and Cheng,
2004b; Lee and Cheng,
2005; Lee et al,
2005). Extensive studies from rat models showed that
adjudin-induced infertility is not associated with substantial changes in
serum levels of follicle-stimulating hormone, luteinizing hormone, and
testosterone (Cheng et al,
2001). Furthermore, adjudin-induced inhibition of spermatogenesis
is also reversible, and normal spermatogenesis and fertility resume
progressively after cessation of the compound
(Cheng et al, 2001). Although
adjudin-induced disruption of spermatogenesis has been extensively evaluated
in rats, the antifertility and pharmacokinetics profiles have not been
examined in other species. The present study evaluated the effects on
fertility and recovery of spermatogenesis after cessation of treatment. The
pharmacokinetics profiles after intravenous and oral administration of the
adjudin were compared in adult male Japanese rabbits.
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Materials and Methods
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Chemicals and Animals
Adjudin was synthesized as described previously
(Cheng et al, 2001). The
compound was suspended in corn oil and used at a concentration of 25 mg/mL for
gavage treatment. For intravenous injection, the compound was suspended in
ethanol/DMSO (2:1, vol/vol). Carbamazepine, the internal standard, was
purchased from Sigma Co (St Louis, Missouri). All other chemicals and reagents
were high-performance liquid chromatography (HPLC) grade. Male adult Japanese
rabbits (2.3–2.6 kg) were raised from Wenzhou Medical College Laboratory
Animal Center (Wenzhou, China).
Animal Treatment
Adult male rabbits were divided into 3 groups: vehicle control, 25 mg/kg
per week intravenous administration, and 25 mg/kg per week oral
administration. The 25 mg/kg per week dose was selected based on the minimal
effective dose of gavage in rats (Cheng et
al, 2001). All animals were housed at Wenzhou Medical College
Laboratory Animal Research Center. All experimental procedures and protocols
were reviewed and approved by the Animal Care and Use Committee of Wenzhou
Medical College and were in accordance with the Guide for the Care and Use of
Laboratory Animals. Rabbits were individually housed and maintained with water
and standard rabbit feed ad libitum. Control animals were treated with
intravenous injection ethanol/DMSO (2:1, vol/vol) solution. Animals were
treated intravenously or orally with 25 mg/kg per week adjudin (1 injection
each week) for 4 consecutive weeks. Some animals were housed for an additional
4 weeks after cessation of intravenous or oral treatment. Blood samples
(0.5
1 mL) were directly collected into the heparinized tube from the
marginal ear vein at predetermined times before or after the first
administration of adjudin and centrifuged at 800 x g for 10
minutes. Plasma samples were stored at –20°C for further
analysis.
Sample Preparation and HPLC Detection of Adjudin
The plasma concentrations of adjudin were measured using reversed-phase
HPLC and a diode array detector (DAD). Adjudin and the internal standard
(carbamazepine, 160 ng) were extracted twice from 300 µL plasma by mixing
with an organic solvent (n-hexane to acetoacetate, 4:1, vol/vol) and
centrifugation at 2500 x g for 5 minutes. After evaporation of
the solvent under nitrogen gas at 50°C, the residue was reconstituted in
mobile phase A (100 µL), and a 40-µL aliquot was injected.
Chromatographic separations were performed by an Agilent HPLC system (Agilent,
Santa Clara, California) equipped with a quaternary pump and autosampler
coupled to a DAD detector (G1315). Chromatographic separations were achieved
on an XDB-C-18 column (inner diameter, 4.5x150 mm; particle size 5
µm; Agilent). The mobile phases used were: phase A solution with
acetonitrile, water, and trifluoroacetic acid at 5:95:0.1 (vol/vol/vol), and
phase B solution with acetonitrile, water, and trifluoroacetic acid at
95:5:0.1 (vol/vol/vol) operated under isocratic conditions at a flow rate of
1.0 mL/min at 30°C with following A and B ratios: 0–3.5 minutes, A/B
= 66:34; 3.5–5.5 minutes, A/B = 60:40; and 5.5–7.5 minutes, A/B =
40:60. Adjudin and internal standard were detected at 302 nm. Carbamazepine
and adjudin were separated with different retention times
(Figure 1). Calibration
standards of adjudin were prepared by serial dilution to obtain concentrations
0.125, 0.25, 0.5, 1.0, 2.5, 5, 10, and 20 µg/mL
(Figure 2). Stock solutions
were prepared weekly throughout the validation period and on each day of
sample analysis. Calibration curves were prepared by spiking blank plasma.
Calibration curves were linear over the range 0.15 to 20 µL/mL (r
= 0.99) (Figure 2). Adjudin
quality control samples corresponding to low, medium, and high concentrations
on the calibration curve were 0.25, 1, and 5 µL/mL, respectively. The
intra-assay coefficients of variation at 0.25, 1, and 5 mg/L of adjudin were
3.15%, 2.88%, and 1.04% (n = 5), respectively. The interassay coefficients of
variation at the above concentrations were 7.41%, 11.21%, and 3.55% (n = 5 in
all cases), respectively. The method is selective for adjudin, with no
interference from other plasma components. The recoveries of 0.25, 1, and 5
mg/L are listed in Table 1.
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Figure 1. Chromatographic separation of adjudin and carbamazepine (internal control).
Adjudin and carbamazepine peaks were well separated. (A) Chromatograph of
carbamazepine (peak 1, retention time 4.7 minutes) and adjudin (peak 2,
retention time 7.2 minutes) dissolved in mobile phase (x-axis is the retention
time in minutes and y-axis is the value of absorbance at 302 nm). (B)
Chromatograph of blank plasma without adjudin and/or carbamazepine. (C)
Chromatograph of adjudin and carbamazepine in a rabbit plasma sample. mAU
indicates milli-absorbance unit.
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Figure 2. Standard curve of adjudin. This is estimated by the high-performance liquid
chromatography method as described in "Materials and Methods." Ai
indicates absorbance of adjudin; As, absorbance of the internal standard,
carbamazepine.
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Histologic Procedures
The animals were anesthetized by 25% ethyl carbamate (4 mL/kg), and testes
were removed immediately at specified time points after adjudin treatment.
Testes and epididymedes were examined by light microscopy to assess the status
of spermatogenesis. In brief, 6-µm–thick cross sections were prepared
and mounted on glass slides and stained with hematoxyslin and eosin.
Data Processing
Following intravenous or oral administration, the adjudin plasma
concentration-time data were fitted to any compartmental models (Drug and
Statistics, version 2.0, Clinical and Drug Research Center, Anhui, China),
which were then used to calculate different pharmacokinetics parameters with
the noncompartment model. All pharmacokinetics parameters were summarized
using descriptive statistics. The 95% confidence intervals for the differences
between the means of the pharmacokinetics parameters were calculated using the
commercially available computer program Confidence Interval Analysis
(Altman et al, 2000).
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Results
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Pharmacokinetics of Adjudin in Male Rabbits
In the pharmacokinetics study shown in
Figure 1, we developed an HPLC
method to measure the plasma concentrations of adjudin using carbamazepine as
internal control. The method detected plasma adjudin with good precision, and
the detection limit for adjudin was 125 ng/mL in plasma. The standard curve
used to calibrate adjudin and to interpolate unknown samples is shown in
Figure 2.
Table 1 also illustrates the
recovery of adjudin in rabbit plasma samples, confirming the validity of this
HPLC-based method to quantify adjudin in rabbit plasma samples.
Concentrations of adjudin in rabbit plasma peaked at 7.669 mg/L 15 minutes
after a single intravenous injection of 25 mg/kg adjudin. The plasma adjudin
level declined rapidly thereafter, and it became undetectable in rabbits
within 24 hours. The mean plasma concentration-time profile for intravenous
administration was best described by a 2-compartment curve
(Figure 3). Pharmacokinetics
parameters of adjudin based on the curves are shown in
Table 2. The overall
elimination constant (k21) was 2.363 ± 2.062 h–1, and
the terminal half-life (t
β) of adjudin was 2.284 ± 0.599
hours. This proved that adjudin was rapidly distributed to peripheral tissues.
We calculated the area under the curve (AUC), maximum concentration
(Cmax), and time to maximum concentration (tmax) for
rats treated orally with the same doses of adjudin as for intravenous
injection. The AUC results showed that oral administration is about 8.055%
± 1.453% as effective as intravenous injection
(Table 2;
Figure 3A vs 3B). Moreover, the
tβ (5.645 ± 1.512 hours) of adjudin in the systemic
circulation after gavage administration was longer than that of intravenous
administration (2.284 ± 0.599 hours;
Table 2), showing that
orally-administered adjudin had a slower clearance rate.
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Figure 3. Plasma time curve of adjudin after intravenous or oral administration of
single dose of 25 mg/kg adjudin on 6 male Japanese rabbits. (A) Adjudin plasma
profile after intravenous administration. (B) Adjudin plasma profile after
gavage. Adjudin level in plasma was estimated by HPLC as shown in Figures
1 and
2.
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Table 2. Pharmacokinetics parameters after single-dose 25 mg/kg intravenous
or oral administration of adjudin in male rabbit
plasmaa
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Efficacy of Adjudin
The efficacy of adjudin in depleting germ cells from the seminiferous
epithelium of rabbit testes versus normal rabbits
(Figure 4A) was evaluated by
histologic analysis of testis and epididymis sections. About 70% of tubules
were devoid of sperm 2 weeks after intravenous administration of adjudin
(Figure 4B). Four weeks after
intravenous treatment, more than 95% of round and elongate spermatids and more
than 50% of spermatocytes had disappeared
(Figure 4C). Sperm numbers in
the epididymis were significantly reduced after 2 weeks of intravenous
treatment versus control (normal) rabbits
(Figure 5A, B). No sperm except
some cellular debris were found in the epididymis after 4 weeks of intravenous
administration (Figure 5C).
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Figure 4. Testis sections after intravenous or oral administration of adjudin. Adult
rabbits were treated with adjudin (25 mg/kg b.w.) intravenously or orally and
killed 2, 3, 4, or 8 weeks after treatment for histologic analysis by
hematoxylin and eosin staining. Cross section of control (A), 2 (B) or 4 (C)
weeks after IV administration, 4 weeks (D) after oral administration, or 4
weeks after cessation of adjudin by intravenous (E) or oral (F)
administration. Scale bar = 50 µm.
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Figure 5. Epididymis sections after intravenous or oral administration of adjudin.
Adult rabbits were treated with adjudin (25 mg/kg b.w.) intravenously or
orally and killed 2, 3, 4, or 8 weeks after treatment for histologic analysis
by hematoxylin and eosin staining. Cross section of control (A),2 (B) or 4 (C)
weeks after intravenous administration, 4 weeks (D) after oral administration,
or 4 weeks after cessation of adjudin by intravenous (E) or oral (F)
administration. Scale bar = 50 µm.
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The effects of the 4-week oral administration were comparable to those of
the 2-week intravenous administration, as shown in
Figure 4D, and sperm numbers in
the epididymis section (Figure
5D). This suggests that oral administration is far less effective
than intravenous administration.
Systemic Toxicity
Treatment had no effects on body weight
(Table 3) or behavior, and it
did not disturb gastrointestinal function.
Recovery of Spermatogenesis
Four weeks after cessation of intravenous treatment, 50% of tubules showed
signs of spermatogenesis recovery (Figure
4E). Some spermatozoa were also found in the epididymal lumen
(Figure 5E). In comparison, the
recovery was faster in the gavage group, as spermatogenesis was not completely
inhibited after 4 weeks of oral administration.
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Discussion
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The present study demonstrated for the first time that adjudin effectively
depleted elongating/elongated spermatids from the seminiferous epithelium in
rabbit testes. This was followed by the loss of most spermatocytes from the
seminiferous epithelium, as shown in an earlier report
(Chen et al, 2003). The
efficacy of the drug as a male contraceptive, however, is dependent on its
bioavailability. Pharmacokinetic data showed that only about 8% of the drug
was bioavailable after oral administration of the drug, which was consistent
with an earlier report in rats that assessed the tissue disruption of
[3H]-adjudin following its administration by gavage
(Cheng et al, 2005).
The adjudin-induced, stage-dependent germ cell loss possibly was caused by
activation of the mechanism that causes the cleavage of anchoring junctions
between late spermatids and Sertoli cells
(Mruk and Cheng, 2004a;
Cheng et al, 2005). Indeed, the
adjudin-induced disturbance of junction restructuring during spermatogenesis
is associated with a complex process regulated by a multitude of molecules and
MAPK-dependent signal transduction pathways
(Xia and Cheng, 2005;
Xia et al, 2007).
Adjudin-induced changes in the steady-state levels of junction proteins that
are the crucial proteins of the disrupted signaling pathways (eg, the ERK
signaling pathway) appear to be associated with testin, because a disruption
of Sertoli-germ cell anchoring junctions is associated with a significant
surge in testin expression both in vivo and in vitro
(Cheng et al, 2001).
Significant changes in expression of multiple genes were detected by
approximately 8 hours after administration of adjudin in a subsequent study
using gene profiling techniques in adult rats
(Xia et al, 2007). As in rats,
orally administrated adjudin had low bioavailability in rabbits. As reported
herein, in rabbits the orally administrated bioavailability of adjudin was
about 8% of that dose given intravenously. We suggest that adjudin is poorly
absorbed or extensively metabolized in the gastrointestinal tract. The
pharmacokinetics profiles in rabbits were similar to those in rats
(Cheng et al, 2005). When
[3H]-adjudin was administered orally to adult rats, the drug was
found to distribute uniformly among the testis, kidney, liver, brain, heart,
small intestine, prostate, epididymis, seminal vesicles, pancreas, and thyroid
after its absorption via the gastrointestinal tract
(Cheng et al, 2005). These
findings show that adjudin is not specifically taken up by any organ. By 72 to
96 hours after administration, adjudin had been virtually removed via the
urine from all organs examined. Our data in rabbits have confirmed these
earlier findings in rats, because 24 hours after a single intravenous
injection of adjudin at 25 mg/kg body weight (b.w.), the serum adjudin level
was already undetectable. Earlier studies in which adjudin was given by gavage
showed that less than 5% of [3H]-adjudin was taken up by all organs
combined and only 0.035% of administered [3H]-adjudin reached the
testis (Cheng et al, 2005);
these studies corroborate our finding that this compound has a low oral
bioavailability. The mean plasma concentration-time profile for intravenous
administration as reported herein was best described by a 2-compartment curve
and was consistent with an extensive distribution in peripheral organs.
In summary, adjudin induced germ cell loss from the seminiferous epithelium
in rabbits, as previously shown for rats, and it is a potential male
contraceptive when delivered intravenously or orally. The pharmacokinetics
analysis, however, indicates that this drug has low bioavailability when
delivered orally.
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Footnotes
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Supported in part by grants from the National Institutes of Health
(National Institute of Child Health and Human Development U54 HD029990 Project
5 and U01 HD045908) to C.Y.C. and the Wenzhou Medical College Fund to
R.-S.G.
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Abstract
Materials and Methods
