Bioequivalence Between Generic and Branded Lamotrigine in People With Epilepsy: The EQUIGEN Randomized Clinical Trial.

Importance: Switching between generic antiepileptic drugs is a highly debated issue that affects both clinical care and overall health care costs. Objective: To evaluate the single-dose pharmacokinetic bioequivalence of 3 (1 branded and 2 generic drugs) on-market, immediate-release lamotrigine drug products. Design, Setting, and Participants: The Equivalence Among Antiepileptic Drug Generic and Brand Products in People With Epilepsy (EQUIGEN) single-dose study is a crossover, prospective, sequence-randomized, replicate pharmacokinetic study conducted at 5 US academic epilepsy centers. Fifty adults (≥18 years) with epilepsy who were taking concomitant antiepileptic drugs and not currently receiving lamotrigine were enrolled between July 18, 2013, and January 19, 2015. Every participant was randomly assigned to 1 of 3 equivalent sequences, each comprising 6 study periods, during which they had blood draws before and after medication administration. Forty-nine participants were included in intention-to-treat analyses. Interventions: Participants received a single 25-mg dose of immediate-release lamotrigine at the start of each period, with the branded and the 2 most disparate generic products each studied twice. Lamotrigine was selected as the antiepileptic drug of interest because of its wide use, publications indicating problems with generic switches, and complaints to the US Food and Drug Administration regarding generic products. Both participants and study personnel were blinded to the specific generic products selected. Main Outcomes and Measures: The primary outcome was bioequivalence between products. Maximum plasma concentration (Cmax) and area under the concentration-time curve (AUC) were compared, and average bioequivalence (ABE) was established if the 90% CIs of the ratios of the 2 products were within equivalence limits (80%-125%). Results: Of the 50 randomized participants, 49 (98%) received all 3 lamotrigine products and completed at least 3 pharmacokinetic assessments and 46 (92%) completed all 6 pharmacokinetic assessments. Among the 49 participants, 28 (57%) were men and 21 (43%) were women, 42 (86%) self-identified as white, and 46 (16) years was the mean (SD) age. The 3 drug products were considered bioequivalent because the 90% CIs were within equivalence limits (lowest and highest CI limits for Cmax, 92.6% and 110.4%; for AUC0-96, 96.9% and 101.9%). Replicate testing demonstrated no significant differences in within-subject variability across the 3 products (likelihood ratios, χ22 for log-transformed variables: AUC0-96, 2.58; Cmax, 0.64; and AUC0-∞, 4.05; P ≥ .13) and that the 3 products were also bioequivalent according to scaled ABE and individual bioequivalence criteria with no subject × formulation interaction (Cmax, 0.00; AUC0-96, 0.54; and AUC0-∞, 0.36; P ≥ .76). Conclusions and Relevance: This study provides evidence that the disparate lamotrigine products studied are bioequivalent when tested in people with epilepsy taking concomitant antiepileptic drugs. Trial Registration: clinicaltrials.gov Identifier: NCT01733394.

HOXA11 mutation in amegakaryocytic thrombocytopenia with radio-ulnar synostosis syndrome inhibits megakaryocytic differentiation in vitro.

Homeobox genes encode for regulatory proteins central to hematopoietic differentiation and proliferation. Previously, we identified an inherited syndrome of congenital amegakaryocytic thrombocytopenia and radio-ulnar synostosis that is associated with a point mutation in the third helix of HOXA11 homeodomain (HOXA11-DeltaH3). Here, we demonstrate that this mutation results in a significantly truncated protein with impaired DNA-binding efficiency. Electrophoretic mobility shift assays (EMSA) confirm that wild-type HOXA11 (HOXA11-WT) interacts in vitro with the DNA-binding consensus sequence for HOXA11, and that this interaction is most efficient when the TALE transcription factor, Meis1b, is also present. However, the binding between HOXA11-DeltaH3 and DNA is abrogated even in the presence of Meis1b, suggesting the point mutant causes a disruption in the DNA-binding capacity. We investigated whether the point mutation also affected the physical protein-protein interaction between HoxA11 and Meis1b. Using GST pulldown assays, we find Meis1b interactions with both HOXA11-WT and HOXA11-DeltaH3 in the presence of DNA. DNAse treatment decreased these interactions, suggesting that the interaction is a protein-protein association, and DNA may serve to stabilize this interaction. Stable expression of FLAG-HOXA11-WT or -DeltaH3 in K562 cells significantly impacts megakaryocytic differentiation. Staurosporine (STSP) induced K562 cells to differentiate into a megakaryocytic phenotype. Treatment leads to an increase in surface expression of the megakaryocytic/platelet-specific antigen, CD61, and causes morphological changes consistent with megakaryocytic differentiation. CD61 surface expression on STSP treated HOXA11-WT and -DeltaH3 expressing cells was significantly reduced as compared to untransfected K562 cells. Interestingly, we found only a slight difference in CD61 expression between wild-type and mutant HOXA11 K562. These data suggest that HoxA11 inhibition of differentiation may involve nonhomeodomain sequences. Finally, our laboratory has detected a small amount of HoxA11 mRNA in cells isolated from unfractionated human cord blood and murine ES cell culture cocultured on OP9 for 6 days in the absence of leukemia inhibitory factor (LIF). This finding suggests HoxA11 may be endogenously expressed in very early hematopoietic precursor cells. Taken together, these data begin to give us insight into the molecular mechanisms by which HoxA11 may be involved in regulating megakaryocytic differentiation.