In vivo cyclopamine exposure has been commonly used to inhibit Hh signaling in murine models of development and disease. However, administration routes widely vary and a comparison of these approaches has not been published. Here, incorporating data from three administration routes, we characterized a two-compartment PK model yielding route- and dose-specific toxicity and PK profiles.
We found that administration by ip injection and oral gavage was limited by toxicity and a relatively short elimination half-life. Administration by oral gavage was particularly suboptimal given associated toxicity at higher concentrations and limited bioavailability (0.33 relative to ip administration). The toxicity associated with ip injection manifested as severe dystonia within 2 h of administration while toxicity associated with po and OsP administration manifested as lethargy at least 6 h after administration. The data, presented in suggests that toxicity of was not a direct function Cmax or area under curve (AUC) values.
Visual inspection of the oral and ip bolus plots in may suggest that the terminal elimination slope should be shallower, reflecting a longer elimination half-life. These late, sustained concentrations may in fact be outliers, as most serum cyclopamine concentrations 24 h after the bolus were below the LLOQ.
As opposed to bolus dosing, OsP infusion yielded sustained concentrations without high, transient peak concentrations. However, detailed analysis yielded somewhat unexpected results. OsP infusion of 20 and 160 mg/kg/day yielded Css levels and AUC values proportional to dose, while values from 10 mg/kg/day infusion was less than proportional () due to increased clearance. Further, there appears to be a gradual decline in the measured concentrations of cyclopamine over time, particularly notable in the 101-h infusion (). It is not known whether this is a real effect, and if so if it is due to a decrease in pump efflux, degradation or adsorption of drug within the pump, or perhaps enzyme induction. Enzyme induction does not appear likely as there is no evidence of decreasing concentrations during tile daily × five ip injections. Similarly, with an elimination half-life of 4 h, there was no significant accumulation of drug in serum with daily ip injections.
Overall, however, OsP infusion yielded mostly stable concentrations and generally circumvented the toxicity of ip and po administration likely caused by large initial concentration spikes. Moreover, periods of sustained concentrations may be better suited for certain studies as opposed to intermittent concentration spikes produced by bolus administration ().
Here, we set out to establish an administration regimen for cyclopamine-induced teratogenicity in the mouse. We found that in vivo infusion of 160 mg/kg/day yielded amniotic concentrations of ~1.5μM (), while the in vitro LOEL for reduction of FNP expansion was 2.0μM (). Achieved in vivo concentrations approximating the in vitro LOEL may explain the incomplete inter- and intralitter penetrance of teratogenicity. Inter- and intralitter variations in embryonic staging may also contribute to the incomplete penetrance. In affected litters, overall body size of embryos with gross defects was slightly smaller than unaffected littermate embryos. It is unclear whether this reflects increased sensitivity of earlier staged embryos within a litter at time of exposure or if it is a subsequent effect of cyclopamine exposure in sensitive embryos.
While the Veratrum
alkaloids, cyclopamine (11-deoxyjervine) and jervine, have been considered “universal” teratogens, the mouse appears to be somewhat resistant. Comparing the interspecies teratogenic activity of cyclopamine and jervine, Keeler (1975)
found that while golden hamster fetuses were extremely sensitive, rats were less sensitive and Swiss Webster mice were apparently resistant to both related compounds.
The cyclopamine-induced facial defects described here mimic those reported by Omnell et al. (1990)
who found that of 20 litters of C57BL/6J dams exposed to 70 mg/kg jervine via po at E8.5, 53 of 126 living fetuses were abnormal, predominately displaying cleft lip/palate often accompanied by open eyelid and limb and digit defects. We attempted to precisely replicate these experimental conditions but did not find defects in a total of 22 fetuses from four surviving litters (Lipinski, unpublished data). However, this sample size may have been insufficient given the low frequency of cyclopamine-induced defects produced by the cyclopamine administration regimen described here.
Omnell also reported an approximate LD50
concentration for jervine of 120 mg/kg, and Keeler (1975)
found that teratogenic doses of cyclopamine caused varying lethality in hamsters. These data, along with our finding that while cyclopamine infusion at 160 mg/kg/day caused teratogenicity, infusion at 240 mg/kg/day caused dam toxicity, suggests that the concentration required for Veratrum
alkaloid-induced terato-genicity approaches dam-toxic concentrations.
The mouse serves as a valuable tool to study craniofacial development because of the high fidelity between embryonic development of the face in mouse and man (Diewert and Wang, 1992
). The methodology for teratogen-induced CL/P described here provides an additional model, which along with genetic mouse models (reviewed in Juriloff and Harris, 2008
) provide tools to study the morphological processes underlying CL/P. However, the low frequency of affected embryos by the presented administration methodology mitigates its practical utility. The mouse strain utilized in this study appears particularly resistant as spontaneous CL/P is not seen in C57BL/6J colonies. Conversely, some mouse strains demonstrate reliable spontaneous frequencies of CL/P, such as A strain (2-24%) and CL/Fr (36%) (Juriloff and Harris, 2008
; Millicovsky et al., 1982
; Trasler and Marchado, 1979
). In fact, using embryo transfer techniques, Martin et al. (1995)
found that the frequency and severity of CL/P was significantly reduced in CL/Fr blastocysts implanted into C57BL/6J dams compared to donor strain dams. Given these observations, exploration of relative strain sensitivity to cyclopamine-induced CL/P may yield a more tractable model.
CL/P arises from failed fusion of the median nasal prominence with the maxillary prominences. Several factors may contribute, including failed growth of the FNP effectively preventing or delaying contact of the prominences, causing failed or deficient fusion (Juliloff and Hmis, 2008
). Our in vitro
whole-mouse embryo assays demonstrated that exposure of 2μM cyclopamine caused a subtle but significant decrease in the mediolateral expansion of the FNP, providing a likely mechanism for the CL/P defects presented by embryos exposed in vivo
expression in the neuroectoderm is required for induction of Hh signaling in the adjacent face and for expansion of the FNP in chick (Marcucio et al., 2005
). Hh signaling blockade following establishment of Shh
in the forebrain but prior to its induction in the face results in facial defects without detectable effects on the forebrain (Cordero et al., 2004
). Similarly, the findings here demonstrate that chemical inhibition temporally targeting Hh signaling during FNP expansion induces isolated facial clefting in the mouse that phenotypically mimic human anomalies.
While HPE is a rare clinical occurrence (~1/15,000 live births), non-syndromic CL/P is much more common (~1/700 live births). The etiological bases for CL/P in humans appear complex cmd multifactorial, likely involving genetic and environmental factors (reviewed in Murray, 2002
). The finding here that tsansient inhibition of Hh signaling induces CL/P in mice is significant given recent findings that numerous structurally diverse small molecules inhibit Hh signaling with varying potencies (Chen et al., 2002
; Frank Kamenetsky et al., 2002
, Lipinski et al., 2007
; Williams et al., 2003
). Taken together, these findings argue tllat further efforts to identify and characterize Hh signaling inhibitors of human exposure may provide important insights into the underlying etiology of cleft lip/palate, one of the most common and morbid human birth defects.