* Celgene Corp., Warren, New Jersey; and Argus Research, Horsham, Pennsylvania 19044 and Milestone Biomedical Associates, Frederick, Maryland 21701, Divisions of Charles River Discovery and Development Services
Received December 17, 2003; accepted June 9, 2004
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ABSTRACT |
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Key Words: thalidomide; developmental; perinatal; postnatal; rabbit.
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INTRODUCTION |
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Thalidomide's teratogenic effects in mice, rats, rabbits, monkeys, and humans have been extensively studied and documented (Brent and Holmes, 1988; Fratta et al., 1965
; Newman, 1985
; Newman et al., 1993
; Schumacher et al., 1968
). Oral developmental no observed adverse effect levels (DNOAEL) were estimated to be less than 31, 25, and 12 mg/kg/day for various strains of mice, rats, and rabbits, respectively (Newman et al., 1993
). No DNOAEL have been determined for monkeys and humans. Apart from the monkey, the rabbit is the most sensitive species in manifesting thalidomide-induced terata (Schumacher et al., 1968
; Staples and Holtkamp, 1968
). In rats, both teratogenic and non-teratogenic results have been obtained, with the most common observation being an increased number of resorptions (Fratta et al., 1965
). The lowest doses and shortest treatment period where characteristic birth defects in human fetuses have been documented were 25 mg/day (0.5 mg/kg, based on a 50 kg human) for 23 days and 50 mg/day (1 mg/kg/day) for only 1 day (Newman et al., 1993
). Current therapeutic doses for ENL are much higher at 100400 mg/day (28 mg/kg/day) and 200400 mg/day (48 mg/kg/day) for inflammatory and oncology indications respectively (Govindarajan, 2002
; LaDuce and Gaspara, 2001
; Singhal et al., 1999
; Teo et al., 2002a
). While these doses are known to be teratogenic in humans when taken during the organogenesis period (days 2055 after fertilization), it is not known what effect they would have on peri- and postnatal reproductive functions. A related concern is whether thalidomide's teratogenic activity is heritable (McBride, 1994
). Limited clinical evidence, however, shows that this is not the case (Smithells, 1998
; Stromland et al., 2002
). There have not been any comprehensive animal studies to assess completely these reproductive and heritability issues.
The present GLP study was performed at the request by the U.S. Food and Drug Administration (FDA) to determine the effects of thalidomide on developmental, peri-, and postnatal function (Segment III). New Zealand White rabbits were used in this study because they are more sensitive to the teratogenic effects of thalidomide than rodents and because it is a species other than man in which thalidomide has produced teratogenic effects. The study was designed with extensive evaluations and conformed to ICH Harmonized Tripartite Guideline stages D (closure of hard palate) through F (lactation and weaning) of the reproductive process (ICH, 1994). Observations were made on gestation, parturition, lactation, and maternal behavior and on the development of the offspring. There was no evaluation of the Caesarean-delivered fetuses (stages C and D), as this was performed in another study (Teo et al., 2004b
). Since thalidomide-induced adverse effects during this period may be delayed and inherited in the offspring, observations continued through to sexual maturity of the F1 generation rabbits and examination of the preterm F2 fetuses.
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MATERIALS AND METHODS |
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Environmental conditions. Temperature and relative humidity in animal rooms were regularly measured and ranged between 64 and 79°F and 30 and 70%, respectively. A 12-h light/12-h dark cycle was used throughout the study.
Thalidomide formulation. Thalidomide is a white powder that is stable at room temperature and insoluble in water. It is a chiral compound and readily undergoes enantiomeric inversion in plasma (Eriksson et al., 1995). The present study will therefore use a racemic mixture of thalidomide. The batch used had a purity of 100% as determined by HPLC (Teo et al., 2001
). The dosing vehicle used was 1% carboxymethylcellulose (sodium salt, medium viscosity; Sigma Chemical Company, St. Louis, MO) dissolved in deionized water. Vehicle and thalidomide dosing formulations were prepared at least weekly and kept refrigerated while not in use. Daily aliquots were allowed to equilibrate to room temperature overnight. Formulations were stirred at room temperature for at least 2 h prior to and during dosing. Dose analyses showed the formulations to be stable at room temperature over 24 h (within ± 10% of target concentration), homogenous (
5% relative standard deviation) and accurate (within ±10%).
Dose selection study. Doses for the present studies were selected based on findings from a dose ranging study. Female rabbits (five/dose) were administered 0 (vehicle control), 30, 150, 300, and 500 mg/kg/day of thalidomide 14 days prior to mating through sacrifice for a total of 55 consecutive days as described previously (Teo et al., 2004b). Briefly, there was minimal maternal toxicity, based on clinical observations, body weight, and feed consumption at the highest dose. Doses of 150, 300, and 500 mg/kg appeared to inhibit ovulation with no viable litters available for evaluation in the 300 and 500 mg/kg groups. Inhibition of ovulation or an effect in litter size is not anticipated since, in the current study design, initial dosing started on day 18 of presumed gestation. Based on reduced weight gain and feed consumption at 300 and 500 mg/kg, doses for females in the present definitive study were set at 0, 30, 150, and 500 mg/kg. The low dose is expected to be the no observed adverse effect level (NOAEL), and the high dose expected to produce minimal toxicity. Plasma levels in the dose selection study indicated that exposure to thalidomide will be significant in the definitive study. The 500 mg/kg top dose was 125 times that of the average therapeutic dose of 4 mg/kg (200 mg for a 50 kg patient; Celgene internal document). Corresponding plasma exposure in the female rabbit as measured by the area under the plasma concentration versus time curve (AUC) was 16 times that in humans (data not shown).
Study Design
Dosing of F0 maternal rabbits. Twenty-five F0 rabbits/dose group were orally gavaged with vehicle, 30, 150, or 500 mg/kg from DG 18 to DP 28, for a total of approximately 42 days. An additional six rabbits/dose group were gavaged at the same time and served as satellite animals for blood and milk collection. F1 pups were not dosed but may have been exposed in utero during maternal gestation or via milk from the lactation period (Fig. 1). Weaning day was defined as the day when does were removed from the litter. The dose volume used was 10 ml/kg.
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F1 generation, pup measurements and observations. Day 1 of lactation (postpartum) was defined as the day of birth. Each litter was evaluated for viability at least twice daily. Pups in each litter were counted daily. Clinical observations were performed once daily during the preweaning period beginning on DP 4. Pup weights were recorded on DPs 4, 7, 14, 21, 28, 35, 42, and 49 for all litters. Male and female pups were also examined for viability and clinical observations, and body weights were taken weekly and at sacrifice for males and at DGs 0, 7, 10, 14, 17, 21, and 24 for females during the postweaning period. Feed consumption was also recorded daily. Male pups were evaluated for sexual maturation starting at 3 months of age. Females were examined for age of vaginal patency from DP 36.
F1 generation, reproductive evaluation. At approximately 6 months of age, 12 males and 12 females in each group were randomly selected for reproductive evaluation. One male and one female were paired within each maternal dose group. On the mating day, female rabbits were intravenously administered 200 USP units/kg of human chorionic gonadotropin to induce ovulation prior to mating. Within a 3-h window of this, each female was placed in the cage of the assigned male and monitored for 1 h until mating was confirmed by observation to have occurred at least once. The day of mating was designated DG 0. If a given pair of rabbits was observed fighting, another male was substituted. When the first pair did not mate during the first observation period, the female was placed with the same male on the following day and monitored until mating was confirmed. When the pair failed to mate within the second observation period on the second day, the female was placed with another male on the third day. When mating is again not confirmed, the female rabbit was sacrificed, Caesarean-sectioned, and examined for uterine contents 29 days after the last day of attempted mating. Males that did not mate were continued on study until scheduled sacrifice.
Gross necropsy, F0 generation rabbits. After completion of the 29-day postpartum period, F0 females assigned to the main study were sacrificed by an intravenous injection of euthanasia solution (Beuthanasia®D Special, Schering-Plough Animal Health), and a gross necropsy of the thoracic, abdominal, and pelvic viscera was performed. The number and distribution of implantation sites was recorded. Rabbits that did not deliver a litter were sacrificed on day 34 of presumed gestation and examined for gross lesions. Uteri were stained with 10% ammonium sulfide to confirm the absence of implantation sites (Salewski, 1964). Does with no surviving pups were sacrificed after the last pup was found dead or presumed cannibalized. A gross necropsy of the viscera was performed. Rabbits that died or were sacrificed because of abortion were examined for the cause of death on the day the observation was made. Pups from two separate litters were discovered out of their cages. The does and litters for these pups were sacrificed because the pups could not be identified with their respective litters. Pregnancy status and uterine contents were recorded. Satellite rabbits were sacrificed after the 29-day postpartum period and discarded.
Gross necropsy, F1 generation pups. F1 pups that died before initial examination of the litter for pup viability were evaluated for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that sank were considered stillborn; pups with lungs that floated were considered liveborn and to have died shortly after birth. Pups with lesions were retained in neutral buffered 10% formalin. Pups found dead or sacrificed because of moribund condition were examined for gross lesions and for the cause of death or moribund condition. All pups observed with limb splay were removed from study. They were euthanized, perfused, and fixed for radiologic and neurohistopathologic evaluation by Consultants in Veterinary Pathology Inc., (Murrysville, PA). In addition, three normal male and female pups from the vehicle group were selected and similarly processed and evaluated. Radiographs were taken of the front and hind limbs. Following examination of these radiographs, one affected pup and three normal male and female pups from the vehicle dose and three normal male and female pups from the 500 mg/kg group were also evaluated. All pups in the main and satellite studies that were not selected for continued evaluation were sacrificed after DP 49 and examined for gross lesions. The heart was examined using a variation of the microdissection technique (Staples, 1974). A single cross section was made between the parietal and frontal bones, and the brain was examined in situ.
Gross necropsy, F1 generation rabbits. F1 rabbits that survived were sacrificed after completion of the mating period, and a gross necropsy of the viscera was performed. Testes and epididymides were excised and paired organ weights were recorded. The epididymides were retained in formalin while the testes were fixed in Bouin's solution from 48 to 96 h and then retained in formalin. All rabbits not selected for the reproductive evaluation at 6 months of age were sacrificed and examined for gross lesions. All surviving female rabbits were sacrificed on DG 29, Caesarean-sectioned, and a gross necropsy of the viscera performed. Uteri of apparently nonpregnant rabbits were stained with ammonium sulfide. The rabbits were examined for the number and distribution of corpora lutea, implantation sites, live and dead fetuses, and early and late resorptions. A live fetus was defined as one that responded to stimuli, while a dead fetus was defined as a term fetus that did not respond to stimuli and was not markedly autolyzed. Dead fetuses demonstrating marked to extreme autolysis were considered to be late resorptions. A conceptus was defined as a late resorption if it was grossly evident that organogenesis had occurred; if this was not the case, the conceptus was defined as an early resorption. Each fetus was weighed and examined externally for gross external alterations and internally for sex. At necropsy, sections of the sciatic, tibial, fibular, and sural nerves were excised from all F1 male and female rabbits selected for continued postweaning evaluation and retained in formalin. Females without a confirmed mating date were sacrificed on an estimated DG 29. Uteri of apparently nonpregnant rabbits were stained with ammonium sulfide. Rabbits that died or were sacrificed because of moribund condition, abortion, or premature delivery were examined for the cause of death or moribundity on the day the observation was made. They were examined for gross lesions. Testes and epididymides were excised, and paired organ weights recorded and processed as described earlier. Pregnancy status and uterine contents were recorded. Aborted fetuses, concepti in utero, and delivered pups were examined to the extent possible using the same methods described for term fetuses. Uteri of apparently nonpregnant rabbits were stained as described earlier. In an ancillary study, 5-month-old rabbits from both sexes were selected from each dose to evaluate learning and memory, using eyeblink classical conditioning (manuscript in preparation).
Radiography and neurohistopathology of F1 pups with splayed limbs. All pups exhibiting splayed limbs were removed from the study, euthanized, and fixed by systemic perfusion. The vehicle and 500 mg/kg pups were evaluated by radiologic and neurohistologic procedures. Full-body radiographs were taken using an InnoVet SelectTM X-ray machine (Summit Industries, Chicago, IL) using both mammography and medium screen films. After radiography, carcasses were further dissected by removing the dorsal arches from each spinal column. The brains were removed from the cranial vaults and eight standardized coronal slices prepared, starting at the frontal pole and ending at the level of the medulla oblongata. After removal of each spinal cord, all of the spinal nerve roots with associated dorsal root ganglia were dissected off of the cord and approximately half submitted for processing. Two cross sections were taken from each of the cervical, thoracic, and lumbar spinal cord regions. One horizontal and one para-sagittal section were also taken from the cervical cord. The brachial plexus and its branches were harvested from one leg (usually the left), as was the sciatic nerve. Representative longitudinal and/or transverse sections were taken from the following skeletal muscles of the front leg: pectoral muscle (transverse and longitudinal) and triceps (transverse). From the hind leg, muscle sections were taken from the adductor magnus (transverse and longitudinal), semi-membranosus/semi-tendinosis (transverse), and gastrocnemius (transverse) muscles. For most of the rabbits, the skeletal muscle sections were also taken from the left leg. The tissue sections just described were processed and embedded in blocks. They were processed for embedding in paraffin, following standardized procedures, using a Shandon CitadelTM tissue processor (Pacific Southwest Lab Equipment, San Diego, CA). After the paraffin blocks had been prepared, they were sectioned on rotary microtomes set at thicknesses of 45 m. All sections were stained with hematoxylin and eosin. All microscopic findings were assigned one of five severity grades (minimal, mild, moderate, marked, or severe). A distribution pattern of focal, multifocal, or diffuse was also used for each microscopic lesion.
Data analysis. All adult and pup/fetal incidence data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution (Snedecor and Cochran, 1967). Body weights, body weight changes, feed consumption data, duration of gestation, litter averages for pup and fetal body weights and percent male pups/fetuses and mortality, cumulative survival, and parameters involving continuous data were analyzed using Bartlett's test of homogeneity of variance and the analysis of variance (ANOVA) when Bartlett's was not statistically significant (p > 0.001) (Snedecor and Cochran, 1967
; Sokal and Rohlf, 1969
). If the ANOVA was significant (p
0.05), the Dunnett's test was used to identify the statistical significance of the individual groups (Dunnett, 1955
). If the ANOVA was not appropriate (that is the Bartlett's was significant, p
0.001), the Kruskal-Wallis test was used when 75% or fewer ties were present (Sokal and Rohlf, 1969
). When more than 75% ties were present, the Fisher's exact test was used (Siegel, 1956
). In cases where the Kruskal-Wallis test was statistically significant (p
0.05), the Dunn's method of multiple comparisons was used to identify the statistical significance of the individual groups (Dunn, 1964
). All other natural delivery data involving discrete and count data obtained at Caesarean-sectioning of the F1 generation does were evaluated using the Kruskal-Wallis test.
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RESULTS |
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Maternal body weight, body weight changes, and feed consumption. Body weight and weight gain were comparable among all groups during the gestation (DGs 1829) and lactation periods. Significant (p 0.05) reductions in absolute and relative feed consumption were observed from DGs 1829 for 150 and 500 mg/kg groups compared to control. Reductions were present during DPs 14 and 47 (p
0.05) for 150 mg/kg and every interval (p
0.05 to p
0.01) for the entire lactation period (DLs 121) for 500 mg/kg.
Natural delivery and litter observations. The numbers of does with stillborn pups significantly (p 0.01) increased to 7 (31.8% of total) and 10 (43.5%) in the 150 and 500 mg/kg groups, respectively, from 1 (4.3%) in control. Does with all pups dying from DPs 14 was also significantly (p
0.05 to p
0.01) increased to 7 (33.3%) and 13 (56.5%) at 150 and 300 mg/kg, respectively, from 0 in control. The mean number of liveborn pups and the percentage of live pups were significantly (p
0.01) decreased at 500 mg/kg (Table 1). The number of pups found dead, presumed cannibalized, or moribund sacrificed was significantly (p
0.01) increased at 150 and 500 mg/kg at certain intervals. All deaths resulted in significantly (p
0.05 to p
0.01) reduced viability index at 150 and 500 mg/kg. The lactation index was not significantly reduced in the same dose groups. The number of surviving pups and the live litter size at weighing from DPs 1 to 49 were reduced to significantly reduced (p
0.05 to p
0.01) at 150 and 500 mg/kg. All pups died between DPs 1 and 29 in 0, 1, 9, and 16 litters at 0, 30, 150, and 500 mg/kg, respectively. Pup weights were increased to significantly (p
0.01) increased (due to the small litter size) from postpartum day 21 and were significantly (p
0.05 to p
0.01) reduced at 500 mg/kg at postpartum days 3542. All other delivery observations were comparable.
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Plasma and milk levels of thalidomide. There were dose-dependent increases in plasma and milk concentrations of thalidomide 3 h postdose over the 4 weeks (Table 2). These increases, however, were not dose proportional. Milk levels of thalidomide were generally greater than plasma, with the magnitude being dose dependent. Milk concentrations were 1.162.11, 1.052.43, and 0.643.6 times that of plasma at 30, 150, and 500 mg/kg, respectively.
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Body weight, body weight changes and feed consumption. Body weight and body weight changes (only for female) were reduced or significantly (p 0.05 to p
0.01) reduced in F1 males and females (precohabitation period only) with F0 maternal does dosed with 500 mg/kg (F1 males) and 150 or 500 mg/kg (F1 females) thalidomide. While there were significant (p
0.05 to p
0.01) decreases or increases in absolute and relative feed consumption for F1 males and females from litters where F0 does were administered 500 mg/kg thalidomide, these changes were considered unrelated to treatment, since there was no dose-response relationship.
Sexual maturation. Sexual maturation for F1 male and female rabbits was unaffected by maternal thalidomide dosing. The average day on which maturation or vaginal patency occurred was not affected by maternal dosing. There were no statistically significant or biologically important differences in learning and memory from F1 generation rabbits (manuscript in preparation).
Mating and fertility. Fertility of F1 male and female offspring was not significantly affected by maternal doses of thalidomide. The pregnancy index may have been reduced from 12/12 (rabbits pregnant/rabbits selected for mating) to 10/13 in the vehicle and 500 mg/kg maternal thalidomide dose, respectively.
Terminal body weights and testes and epididymides measurements. All body weight, testes, and epididymides measurements were comparable among the four groups.
Caesarean-sectioning and litter observations. All litter averages for corpora lutea, implantation, litter sizes, resorptions, percent male fetuses, percent resorbed conceptuses, fetal body weights, and the number of does with resorptions were similar among all four dose groups. Litter size, the average number of live fetuses per litter, and the average female and male fetal body weights were also comparable. No doe had a litter with only resorbed conceptuses and there were no dead fetuses. All placentae appeared normal.
Fetal gross external alterations. There were no fetal gross external alterations related to maternal dosing of thalidomide at up to 500 mg/kg.
Radiographic and neurohistopathologic of F1 pups with splayed limbs. There were no apparent radiological differences between the vehicle and 500 mg/kg maternal dose group F1 pups. Treatment-related microscopic findings included minimal nerve fiber degeneration (brachial plexus and sciatic), mild to moderate skeletal muscle fiber atrophy, and mild muscle fiber degeneration. The minimal neuropathy may be due to thalidomide treatment. Muscle changes were considered secondary to the splay leg rather than the cause of the splay. Muscle fiber degeneration and atrophy were present to a biologically significant degree at 500 mg/kg. These histologic findings were also considered secondary to the splay leg.
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DISCUSSION |
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Thalidomide administered during the post-organogenesis period in rabbits did not produce any teratogenicity in F1 pups, as there are no major morphological changes in organs and tissues during this stage of fetal development. The apparent non-statistically significant increase in abortion in rabbits, particularly at 500 mg/kg, correlated with the significant rise in abortion or resorption seen with Holtzman rats and Green Monkeys orally dosed with 50150 and 1030 mg/kg from days 914 and 2833 of gestation, respectively (Hendrickx and Sawyer, 1978; Schumacher et al., 1968
). The abortions and/or resorptions could be due to thalidomide's anti-angiogenic activity on neovascularization of the fetus through its suppression of pro-angiogenic growth factors such as bFGF and VEGF (D'Amato et al., 1994
). This anti-angiogenic activity could also be occurring in the dose range finding study where a dose-dependent decrease in ovulation was observed at 150 mg/kg and higher. It is possible that a similar mechanism of action is involved in rodents, as resorption is a major finding in pregnant rats treated with thalidomide (Kenyon et al., 1997
; Schumacher et al., 1968
). Abortions could also be due to thalidomide's immunomodulatory activity in shifting the body's balance of T helper (Th) cell subsets. Clones of T helper cells normally exist as two subsets depending on the specific cytokines secreted in response to antigenic stimulation (Mosmann and Coffman, 1989
). Th1 cells participate in cell-mediated immunity for controlling intracellular pathogens, while Th2 cells are involved with antibody-mediated immunity for controlling extracellular parasites. Pregnancy is thought to be predominantly a Th2 event providing for `tolerance' of the fetus (Kidd, 2003
). This is supported by increased plasma levels of the Th2 cytokine IL-10 in normal human pregnancy and decreased number of Th2 cells at the deciduas basalis from aborted fetuses (Holmes et al., 2003
; Michimata et al., 2003
). Thalidomide has been shown to shift the Th1Th2 balance toward a preponderance of Th1 cells (Oliver, 2000
; Verbon et al., 2000
). This imbalance is thought to be a cause of recurrent spontaneous abortions in humans and could play a role in abortions observed in the present study (Michimata et al., 2003
).
The cause of the tan livers in two does at 500 mg/kg that aborted is unknown. No histopathology was performed. Studies in other species did not show similar liver discoloration. Nonpregnant mice and rats dosed at up to 3000 mg/kg/day for 13 weeks and dogs at up to 1000 mg/kg/day for 1 year exhibited hepatic centrilobular hypertrophy and accumulation of bile pigment in liver canaliculi, respectively, both with no liver discoloration (Teo et al., 1999, 2001
). A direct effect of thalidomide on pregnant rabbit livers cannot be discounted. Red substances in the maternal stomach and uterus are probably remnants of cannibalized fetuses and blood, respectively. The incidence of thick endometrium was a spontaneous event, as it was only seen in one fetus. The significant changes in some of the natural delivery parameters and litter observations were thalidomide related (Table 1). These consisted of increased number of does with stillborn pups and all pups dying from DPs 14 at 150 and 500 mg/kg, decreased number of liveborn pups and percentage of live pups at 500 mg/kg, increased number of dead pups at 150 and 500 mg/kg (resulting in reduction of viability index at 150 and 500 mg/kg), and reduced number of surviving pups and live litter size. These findings generally related to effects on survival of the offspring and may have been mediated through the doe or via direct exposure of the conceptuses to thalidomide. While thalidomide has been shown to decrease thyroxine (T4) levels in rats and dogs (Teo et al., 1999
, 2001
), the normal maturation and subsequent lack of effect on the fecundity of the F1 generation in this study demonstrates that thalidomide does not have any significant adverse effects on the production and secretion of reproductive hormones such as chorionic gonadotropin, estrogen, and progesterone in F0 does and F1 pups and rabbits.
There was an apparent increase in the incidence of splay limbs in the 150 and 500 mg/kg dose groups. Splayed limbs are a known spontaneous alteration in young rabbits. Lindsey and Fox (1974) noted that the term `splay leg' is the descriptive name commonly applied to rabbits which lack the ability to adduct one or all legs and come to a standing position. Affected animals assume the typical "splay leg" appearance in ventral recumbency and, at best, are able to move horizontally for only short distances by making weak, clumsy movements of the legs. In more severe cases, all legs may be completely paralyzed. Affected animals appear mentally alert. "Splay leg" is probably an inherited recessive achondroplasia and remains a descriptive clinical term without clearly established pathological meaning(s). Further systematic investigation of the condition is warranted. The animal supplier for this study noted that young animals with this condition are culled prior to shipment and no incidence could be provided (personal communication). We speculate that the splay may be due to treatment-related decrease in litter size resulting in superior nutritional status for rabbits in smaller litters where musculoskeletal development provided inadequate support for F1 pups with quick weight gains. The faster growth in these smaller litters would therefore not be a direct effect of administration of thalidomide to the does or via the milk to the pups. The nerve and muscle fiber degeneration and skeletal muscle atrophy observed in F1 pups were probably a secondary finding attributed to thalidomide but could also be a cause for the observed splay. Thalidomide causes reversible sensory peripheral neuropathy in humans characterized by distal axonopathy. The axonal degeneration without myelination gradually moves proximally toward the nerve cell body (Aronson et al., 1984
). Morphologic changes in rat dorsal root ganglion have been observed in vitro after exposure to thalidomide (Aronson et al., 1984
). No effects, however, were seen in mouse and rat sciatic nerve and spinal cord after oral dosing of up to 3000 mg/kg for 2 years (manuscript in preparation). No effects were also seen in the sciatic and sural nerves, dorsal root ganglion, and spinal cord in dogs dosed with 1000 mg/kg for 1 year (Teo et al., 2001
). There was no evidence showing that the splay limbs and muscle degeneration were related. The muscle fiber degeneration could be caused by thalidomide, as higher concentrations were observed in milk compared to plasma indicating fetal exposure (Table 2).
The lack of effect in species other than rabbits may indicate a species difference in nerve degeneration and peripheral neuropathy, with the rabbit potentially being similar to man. This is supported by initial findings of a species difference in the hydrolytic breakdown of thalidomide (Schumacher et al., 1965a). No subsequent comprehensive interspecies comparisons of metabolism and breakdown products have been performed due to the production of numerous chiral hydrolysis products (Schumacher et al., 1965b
). A recent report, however, showed that urine samples from multiple myeloma patients and mice showed different metabolite and hydrolysis profiles (Lu et al., 2003
). It is possible that a species-specific breakdown product is responsible for the nerve degeneration and neuropathy observed in rabbits and humans. The muscle degeneration and atrophy observed could also be mediated by a breakdown product found only in rabbits and man or may be due to increased exposure of rabbit pups to thalidomide through the milk at a critical or sensitive time of muscle development. These levels of either thalidomide or a breakdown product may be higher than those achieved in plasma in other species. Muscle abnormalities are not adverse events associated with thalidomide use in humans. The only such incidence was in a single infant rhesus monkey after prenatal dosing of 10 mg/kg on days 3335 of gestation (Theisen et al., 1979). The histopathology, however, was different from the current rabbit study, with disruptive condensation and splitting of premuscle masses evident. It is doubtful that the muscle findings in the rabbit and monkey are applicable to humans. It is not known if the thalidomide-induced nerve and muscle abnormalities are affected by the pregnancy, direct exposure, or a unique sensitivity of the F1 pups.
Thalidomide possesses central and peripheral nervous activities in humans. It crosses the bloodbrain barrier as evidenced by its sedative and anti-emetic properties (Govindarajan et al., 2000; Kanbayashi et al., 1999
). It also causes dose-limiting neuropathy in peripheral nerves (Ochonisky et al., 1994
). The present study has presented an opportunity to study the neurobehavioral effect of thalidomide in rabbits. Assessment of neurological function and integrity is usually performed in a battery of behavioral tests in rodents. In an ancillary study, some F1 male and female rabbits from the present study were evaluated for learning and memory using a novel validated eyeblink classical conditioning system. No statistically significant or biologically important differences in learning and memory were evident at all F0 maternal doses of thalidomide (manuscript in preparation).
Plasma sampling revealed that F1 fetuses and pups were exposed to thalidomide in utero and ex utero through placental blood and maternal milk respectively. This showed that thalidomide is transported and secreted into major bodily fluids including semen (Teo et al., 2001b, 2004b). Due to its low aqueous solubility, thalidomide exhibits absorption-rate-limited pharmacokinetics in humans at oral doses at or greater than 200 mg, with the prolonged terminal phase representing absorption (Teo et al., 2004c). This was also observed in the rabbit dose ranging study (data not shown). Single time-point plasma and milk sampling at one day of each week of the preweaning period were only performed here, since extensive sampling and pharmacokinetic analysis were already completed in the dose ranging study (Teo et al., 2004b
). The ranging study in pregnant rabbits used the same doses as the present study and showed similar significant plasma levels at the time of maximum plasma levels (Tmax) (Table 1). Currently, most prescriptions of thalidomide are for multiple myeloma with the Maximum Recommend Human Daily Dose (MRHDD) of 10 mg/kg (600 mg/day). No plasma pharmacokinetics is yet available from these patients to allow for exposure comparison between species. However based on the body-weight-normalized dose, the lowest and highest doses of 30 and 500 mg/kg used in the present study were 3 and 50 times the MRHDD, respectively. In similar comparisons using the average daily dose of 160 mg/day or 2.67 mg/kg (Celgene internal document) for multiple myeloma, the 30 and 500 mg/kg F0 rabbit doses were 11 and 187 times greater than humans. In terms of exposure as assessed by the area under the plasma concentration versus time curve (AUC), rabbits were exposed at 3 and 15 times that of the average human dose, respectively (Table 3). Exposure comparisons between other species are discussed elsewhere (Teo et al., 2004b
).
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom all correspondence should be addressed at Celgene Corp., 7 Powder Horn Drive, Warren, NJ 07059. Fax: (732) 805-3616. E-mail: steo{at}celgene.com.
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REFERENCES |
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Brent, R., and Holmes, L. (1988). Clinical and basic science lessons from the thalidomide tragedy: What have we learned about the cause of limb defects? Teratology 38, 241251.[ISI][Medline]
D'Amato, R., Loughnan, M., Flynn, E., and Folkman, J. (1994). Thalidomide is an inhibitor of angiogenesis. Proc. Natl. Acad. Sci., U.S.A. 91, 40824085.[Abstract]
Davies, F., Raje, N., Hideshima, T., Lentzsch, S., Young, G., Tai, Y., Lin, B., Podar, K., Gupta, D., Chauhan, D., et al. (2001). Thalidomide and immunomodulatory derivatives augment natural killer cell cytotoxicity in multiple myeloma. Blood 98, 210216.
Dunn, O. (1964). Multiple comparisons using rank sums. Technometrics 6, 241252.[ISI]
Dunnett, C. (1955). A multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 50, 10961121.[ISI]
Eriksson, T., Bjorkman, S., Roth, B., Fyge, A., and Hoglund, P. (1995). Stereospecific determination, chiral inversion in vitro and pharmacokinetics in humans of the enantiomers of thalidomide. Chirality 7, 4452.[ISI][Medline]
Fratta, I., Sigg, E., and Maiorana, K. (1965). Teratogenic effects of thalidomide in rabbits, rats, hamsters and mice. Toxicol. App. Pharmacol. 7, 268286.[ISI]
Govindarajan, R., Heaton, K., Broadwater, R., Zeitlin, A., Lang, N., and Hauer-Jensen, M. (2000). Effect of thalidomide on gastrointestinal toxic effects of irinotecan. Lancet 356, 566567.[CrossRef][ISI][Medline]
Govindarajan, R. (2002). Irinotecan/thalidomide in metastatic colorectal cancer. Oncology 16, 2326.[CrossRef]
Hendrickx, A., and Sawyer, R. (1978). Developmental staging and thalidomide teratogenicity in the Green Monkey (Cercopithecus aethiops). Teratology 18, 393404.[ISI][Medline]
Holmes, V., Wallace, J., Gilmore, W., McFaul, P., and Alexander, H. (2003). Plasma levels of the immunomodulatory cytokine interleukin-10 during normal human pregnancy: A longitudinal study. Cytokine 21, 265269.[CrossRef][ISI][Medline]
Hwu, W., Krown, S., Menell, J., Panageas, K., Merrell, J., Lamb, L., Williams, L., Quinn, C., Foster, T., Chapman, P., et al. (2003). Phase II study of temozolomide plus thalidomide for the treatment of metastatic melanoma. J. Clin. Oncol. 21, 33513356.
ICH Harmonized Tripartite Guideline (1994). Detection of toxicity to reproduction for medicinal products. S5A, S5B.
Kanbayashi, T., Shimizu, T., Takahashi, Y., Kitajima, T., Takahashi, K., Saito, Y., and Hishikawa, Y. (1999). Thalidomide increases both REM and stages 34 sleep in human adults: A preliminary study. Sleep 22, 113115.[ISI][Medline]
Kenyon, B., Browne, F., and D'Amato, R. (1997). Effects of thalidomide and related metabolites in a mouse corneal model of neovascularization. Exp. Eye Res. 64, 971978.[CrossRef][ISI][Medline]
Kidd, P. (2003) Th1/Th2 balance: The hypothesis, its limitations and implications for health and disease. Altern. Med. Rev. 8, 223246.[Medline]
LaDuce, J., and Gaspari, A. (2001). Targeting tumor necrosis factor alpha. New drugs used to modulate inflammatory diseases. Dermatol. Clin. 19, 617635.[ISI][Medline]
Lindsey, R., and Fox, R. (1974). Inherited diseases and variations. In The Biology of the Laboratory Rabbit (S. Weisbroth, R. Flatt, and A. Krauss, Eds), pp. 395396. Academic Press, New York.
Lu, J., Palmer, B., Kestell, P., Browett, P., Baguley, B., Muller, G., and Ching, L.-M. (2003). Thalidomide metabolites in mice and patients with multiple myeloma. Clin. Cancer. Res. 9, 16801688.
Marriott, J., Clarke, I., Dredge, K., Muller, G., Stirling, D., and Dalgleish, A. (2002). Thalidomide and its analogues have distinct and opposing effects on TNF- and TNFR2 during co-stimulation of both CD4(+) and CD8(+) T cells. Clin. Exp. Immunol. 130, 7584.[CrossRef][ISI][Medline]
McBride, W. (1994). Thalidomide may be a mutagen. Brit. Med. J. 308, 16351636.
Michimata, T., Sakai, M., Miyazaki, S., Ogasawara, M., Suzumori, K., Aoki, K., Nagata, K., and Saito, S. (2003). Decreases of T-helper 2 and T-cytotoxic 2 cells at implantation sites occurs in unexplained recurrent spontaneous abortion with normal chromosomal content. Human Reprod. 18, 15231528.
Mosmann, T., and Coffman, R. (1989). Th1 and Th2 cells: Different patterns of lymphokine secretion lead to different functional properties. Ann. Rev. Immunol. 7, 145173.[CrossRef][ISI][Medline]
Newman, C. (1985). Teratogen update: clinical aspects of thalidomide embryopathya continuing preoccupation. Teratology 32, 133144.[ISI][Medline]
Newman, L., Johnson, E., and Staples, R. (1993). Assessment of the effectiveness of animal developmental toxicity testing for human safety. Repro. Toxicol. 7, 359390.[CrossRef][ISI][Medline]
NIH (1985). Guide for the Care and Use of Laboratory Animals; DHEW, Publication No. (NIH) 8623.
Ochonisky, S., Vernoust, J., Bastuji-Garin, S., Gheradi, R., and Revuz, J. (1994). Thalidomide neuropathy incidence and clinicoelectrophysiologic findings in 42 patients. Arch. Dermatol. 130, 6669.[Abstract]
Oliver, S. (2000). The Th1/Th2 paradigm in the pathogenesis of scleroderma and its modulation by thalidomide. Curr. Rheumatol. Rep. 2, 486491.[Medline]
Raje, N., and Anderson, K. (2002). Thalidomide and immunomodulatory drugs as cancer therapy. Curr. Opin. Oncol. 14, 635640.[CrossRef][ISI][Medline]
Rajkumar, S., Hayman, S., Gertz, M., Dispenzieri, A., Lacy, M., Greipp, P., Geyer, S., Iturria, N., Fonseca, R., Lust, J., et al. (2002). Combination therapy with thalidomide plus dexamethasone for newly diagnosed myeloma. J. Clin. Oncol. 20, 43194323.
Salewski, E. (1964). Farbemethode zum makroskopischen nachweis von implantation stellen am uterus der ratte. Archiv. Pharmakol. Experiment. Pathol. 247, 367372.
Schumacher, H., Smith, R., and Williams, R. (1965a). The metabolism of thalidomide: The fate of thalidomide and some of its hydrolysis products in various species. Brit. J. Pharmacol. 25, 338351.[Medline]
Schumacher, H., Smith, R., and Williams, R. (1965b). The metabolism of thalidomide: The spontaneous hydrolysis of thalidomide in solution. Brit. J. Pharmacol. 25, 324337.[Medline]
Schumacher, H., Blake, D., Gurian, J., and Gillette, J. (1968). A comparison of the teratogenic activity of thalidomide in rabbits and rats. J. Pharmacol. Exp. Ther. 160, 189199.[ISI][Medline]
Siegel, S. (1956). Nonparametric Statistics for the Behavioral Sciences. The Fisher's Exact Probability Test, McGraw-Hill Company, New York, pp. 96105.
Singhal, S., Mehta, J., Desikan, R., Ayers, D., Roberson, P., Eddlemon, P., Munshi, N., Anaissie, E., Wilson, C., Dhodapkar, M., et al. (1999). Antitumor activity of thalidomide in refractory multiple myeloma. N. Engl. J. Med. 341, 15651571.
Smithells, D. (1998). Does thalidomide cause second generation birth defects? Drug Safety 19, 339341.[ISI][Medline]
Snedecor, G., and Cochran, W. (1967). Statistical Methods, 6th ed., Iowa State University Press, Ames.
Sokal, R., and Rohlf, F. (1969). Kruskal-Wallis Test. Biometry. W. H. Freeman and Company, San Francisco.
Staples, R. (1974). Detection of visceral alterations in mammalian fetuses. Teratology 9, A37A38.
Staples, R., and Holtkamp, D. (1968). Effects of thalidomide treatment on gestation and fetal development. Exp. Mol. Pathol. 2, 81106.
Stromland, K., Philipson, E., and Grondlund, M. (2002). Offspring of male and female parents with thalidomide embryopathy: Birth defects and functional anomalies. Teratology 66, 115121.[CrossRef][ISI][Medline]
Teo, S., Trigg, N., Shaw, M., Morgan, M., and Thomas, S. (1999). Subchronic toxicity of thalidomide in rodents after 13-weeks oral administration. Intl. J. Toxicol. 18, 337352.[CrossRef][ISI]
Teo, S., Evans, M., Brockman, M., Ehrhart, J., Morgan, M., Stirling, D., and Thomas, S. (2001a). Safety profile of thalidomide after 53 weeks oral administration in Beagle dogs. Toxicol. Sci. 59, 160168.
Teo, S., Harden, J., Burke, A., Noormohamed, F., Youle, M., Johnson, M., Peters, B., Stirling, D., and Thomas, S. (2001b). Thalidomide is distributed into human semen after oral dosing. Drug Metab. Dispos. 29, 13551357.
Teo, S., Resztak, K., Scheffler, M., Kook, K., Zeldis, J., Stirling, D., and Thomas, S. (2002a). Thalidomide in the treatment of leprosy. Microbes Infect. 4, 11931202.[CrossRef][ISI][Medline]
Teo, S., Chandula, R., Harden, J., Stirling, D., and Thomas, S. (2002b). Sensitive and rapid method for the determination of thalidomide in human plasma and semen using solid phase extraction and LC-tandem mass spectrometry. J. Chrom. B 767, 145151.[ISI]
Teo, S. (2004a). Immunomodulatory and non-immunomodulatory properties of thalidomide and its analogues: Implications for anticancer therapy. AAPS J. (online), www.aapspharmsci.org (in press).
Teo, S., Denny, K., Stirling, D., Thomas, S., Morseth, S., and Hoberman, A. (2004b). Effects of thalidomide on reproductive function and early embryonic development in male and female New Zealand White rabbits. Birth Defects Res. Develop. Reprod. Toxicol. 71, 116.[CrossRef][ISI]
Teo, S., Colburn, W., Tracewell, G., Kook, K., Stirling, D., Jaworsky, M., Scheffler, M., Thomas, S., and Laskin, O. (2004c). Clinical pharmacokinetics of thalidomide. Clin. Pharmacokin. 43, 311327.[ISI][Medline]
Theisen, C., Bodin, J., Svoboda, J., and Pettinelli, M. (1976). Unusual muscle abnormalities associated with thalidomide treatment in a rhesus monkey: A case report. Teratology 19, 313319.
Verbon, A., Juffermans, N., Speelman, P., van Deventer, S., ten Berge, I., Guchelaar, H., and van der Poll, T. (2000). A single oral dose of thalidomide enhances the capacity of lymphocytes to secrete gamma interferon in healthy humans. Antimicrob. Agents Chemother. 44, 22862290.
Wines, N., Cooper, A., and Wines, M. (2002). Thalidomide in dermatology. Australas. J. Dermatol. 43, 229240.[CrossRef][Medline]
Zeldis, J., Williams, B., Thomas, S., Elsayed, M. (1999). S.T.E.P.S.TM: A comprehensive program for controlling and monitoring access to thalidomide. Clin. Ther. 21, 319330.[CrossRef][ISI][Medline]
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