Carbamazepine, Sodium Valproate and Levetiracetam Modulate Wnt Inhibitors in Indian Women with Epilepsy

Introduction

Wnt signaling plays an important role in development and maintenance of many organs and tissues, including bone. Wnt/β-catenin or canonical pathway appears to be particularly important for bone biology (1). Wnt signaling is tightly regulated by members of several families of secreted antagonists including sclerostin and dickkopf-1 (DKK-1) (2). Previous reports have identified vitamin-D inactivation as a major cause of antiepileptic drugs (AEDs) induced metabolic bone diseases (3). However, it is required to explore newer mechanisms that may aid in understanding the pathophysiology of AED induced bony consequences better. The objective of our study was primarily to perform sub-group analysis on our previously published report on the modulation of Wnt inhibitors and receptor activator of nuclear factor kappaB ligand (RANKL) following AED monotherapy. It is well-known that DKK-1 and sclerostin, secreted by osteocytes, block the effect of Wnt on osteoblasts thereby reducing bone formation. Sclerostin has been reported as a strong and independent risk factor for osteoporosis related fractures among postmenopausal women (4). On the other hand, RANK/RANKL pathway plays a critical role in bone remodeling and higher RANKL levels corresponds to increased bone resorption. Several studies provide evidence of association of these markers with bony consequences (5-7). This study aims to look for evidence of any association between Wnt inhibitors and three AEDs.

Method

The study includes sub-group analysis of data of a cross-sectional analytic study conducted at Neuroscience Centre, AIIMS, New Delhi and has been published recently (8). Parveen et al included 32 adult women with epilepsy treated with sodium valproate (SVP), carbamazepine (CBZ) or levetiracetam (LTM) as monotherapy for at least a year. All women gave their consent in accordance with declaration of Helsinki. Serum was employed for the analysis of Wnt inhibitors (Sclerostin and DKK-1) and RANKL using human specific ELISA kit from Sincere Biotech (Beijing, People's Republic of China) and 25OHD procured from Siemens (Mumbai, India).

Data contained both continuous and categorical variables. Therefore, mean±SD or median with interquartile range (IQR) were used for continuous data whereas frequency with proportions were used for categorical data for their presentation. Normality of the continuous variables was tested by the Kolmogorov- Smirnov and Shapiro-Wilk tests. Analysis of variance (ANOVA) test was used between treatment groups and post-hoc analysis was done by using Tukey's honestly significant difference test. ANOVA test and post-hoc Dunnett t-test was used for comparison between individual drug and control. For this comparison, RANKL was converted to log form as data was not normally distributed. The Spearman rank correlation was used to assess the correlation between the variables. The two-sided p-value less than 0.05 was considered as statistical significant. The statistical software IBM SPSS Version 22.0 (Chicago, USA) was used for entire analysis.

Results

Discussion

There is increasing evidence that AED therapy is associated with alteration in bone metabolism and mineralization suggesting bone loss. However, there is no generalized mechanism responsible for such consequences. In our previous study, alteration in Wnt inhibitors was reported following AED therapy in Wistar rats (9) and in Indian women with epilepsy (8). The present study analyzed the effect of individual drugs viz SVP, CBZ and LTM on modulation of Wnt inhibitors in persons with epilepsy. Similar to previous study by Gifre et al (10) evaluating the effect of glucocorticoids on modulation of Wnt inhibitors, the present study also reported differential effects with the type of Wnt inhibitor evaluated. While there were higher sclerostin levels with all three AEDs, DKK-1 levels were lowered with CBZ and LTM. Highest sclerostin levels were reported with LTM while lowest DKK-1 levels with CBZ. However, in our study, the observed lower DKK- 1 levels following AED therapy in persons with epilepsy was not significant with SVP in individual AED analysis. DKK-1 plays a critical role during skeletal development but it is not highly expressed in adult bone unless activated by an insult (11). Sclerostin expression is limited primarily to the skeleton whereas expression of DKK-1 is more widespread (12).

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Fig. 1:

Positive correlation between Sclerostin and RANKL in treatment with CBZ. Correlation is significant at the 0.01 level (2-tailed).

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The higher serum RANKL observed in our study following SVP is in agreement with pre-clinical study demonstrating increased RANKL levels in rats treated with valproate (13). RANKL activates and/or induces various transcription factors including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), c-Fos and nuclear factor of activated T- cells c1 (NFATc1), which act as positive modulators of osteoclast differentiation (14). RANKL increases NFATc1 protein levels by post-translational modification. RANKL stimulates NFATc1 acetylation via histone acetyltransferases (HATs). RANKL-mediated NFATc1 acetylation is increased by the histone deacetylase (HDAC) inhibitors (15). Various AEDs such as valproic acid, CBZ, oxcarbazepine, lamotrigine and levetiracetam are known to exert histone deacetylase inhibitory (HDACi) properties (16). Hence, increased RANKL levels by SVP and LTM could also be related to its HDAC inhibiting property (17). Though, CBZ didn't exert significant rise in serum RANKL levels but showed an increasing trend. The role of Wnt inhibitors and RANKL in bony consequences following AED therapy is proven in our previous report where sclerostin was positively correlated with RANKL, while no such effects were observed with DKK-1 (8). Now the individual analysis of drugs showed that the positive correlation observed between sclerostin and RANKL is due to CBZ. 25OHD was lowered with all three AEDs compared to control, however, the serum levels were not in the deficient range. Interestingly, our study showed that decrease in 25OHD was highest in SVP group as compared to CBZ and LTM treatment group. However, our study had limitation as the baseline data for 25OHD was not available and we compared the results with healthy controls.

To conclude, AEDs may compromise bone health through alterations in Wnt pathway via enhanced sclerostin levels which may or may not be related to RANKL depending upon the type of AED investigated.