Effects of mycophenolic acid alone and in combination with its metabolite mycophenolic acid glucuronide on rat embryos in vitro.
Mycophenolic acid (MPA) is an immunosuppressive agent that acts as a selective, non-reversible inhibitor of the enzyme inosine-5'-monophosphate dehydrogenase (IMPDH). Malformations have been described in children after maternal exposure to mycophenolate. However, the causal link is unclear in most cases because women had been treated with a combination of drugs and birth defects may have other causes. Therefore, it is important to study the action of this drug and its main metabolite on embryonic tissue. We studied the teratogenic potential of MPA and its major metabolite, the mycophenolic acid glucuronide (MPAG) in the rat whole-embryo culture. A total of 147 day 9.5 embryos were cultivated for 48 h in the standard medium containing 85 % serum. We tested MPA at concentrations of 0.1; 0.25; 0.5; 0.75 mg/l (0.31; 0.78; 1.56; 2.34 μM) and MPA glucuronide at concentrations of 3; 10; 30; 100 mg/l (6.04; 20.14; 60.43; 201.43 μM). Both substances are highly protein bound, and MPA glucuronide might displace MPA from protein binding. Therefore, we examined whether the effects of MPA can be enhanced when studied in combination with the glucuronide. Furthermore, the focus was on additional endpoints to the standard evaluation of cultivated embryos, such as development of cranial nerves [trigeminal nerve (V), facial nerve (VII), glossopharyngeal nerve (IX), vagus nerve (X)] after staining with an antibody against 2H3 neurofilament. Ultrastructural changes were evaluated by electron microscopy. At a concentration of 0.75 mg MPA/l medium, all embryos showed dysmorphic changes. Embryos exposed to 0.25 mg MPA/l medium showed impaired development of nerves, and at 0.1 mg/l, no effects were detectable. Concentration-dependent ultrastructural changes, such as signs of apoptosis, were found by electron microscopy. The examination of the metabolite in this assay showed that at a concentration of 100 mg MPAG/l, the embryos exhibited distinct malformations. This is probably caused by MPA, which was detectable at 0.6 % in the material used for our experiments. The combination of the parent compound (0.03; 0.1; 0.25 mg/l) with its metabolite MPAG (3 mg/l) did not cause enhanced toxicity under our experimental conditions. IMPDH, the target enzyme of MPA, could be detected in rat embryos on day 9.5 of embryonic development as well as at the end of the culture period 48 h later. In summary, MPA impairs embryonic development at low, therapeutically relevant concentrations, but the glucuronide does not exhibit such a potential. Activity of MPA is not enhanced by MPAG.
Institute for Clinical Pharmacology and Toxicology, Charité, Universitätsmedizin, Luisenstraße 7, 10117 Berlin, Germany.
Flavia
F
Schmidt
Kathrin
K
Eckardt
Mehdi
M
Shakibaei
Petra
P
Glander
Ralf
R
Stahlmann
D000014:Abnormalities, Drug-Induced / Q000209:etiology*; D000818:Animals; D003391:Cranial Nerves / Q000002:abnormalities / Q000187:drug effects / Q000648:ultrastructure; D004305:Dose-Response Relationship, Drug; D004359:Drug Therapy, Combination; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian / Q000187:drug effects* / Q000201:enzymology; D047108:Embryonic Development / Q000187:drug effects* / Q000502:physiology; D020719:Glucuronides / Q000378:metabolism / Q000633:toxicity*; D007168:IMP Dehydrogenase / Q000378:metabolism; D007166:Immunosuppressive Agents / Q000378:metabolism / Q000633:toxicity*; D009173:Mycophenolic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000633:toxicity; D051381:Rats; D013723:Teratogens / Q000633:toxicity*; D018675:Toxicity Tests
D020719:Glucuronides; D007166:Immunosuppressive Agents; D013723:Teratogens; C113145:mycophenolic acid glucuronide; D007168:IMP Dehydrogenase; D009173:Mycophenolic Acid