Vinpocetine improved neuropsychiatric and epileptic outcomes in a patient with a GABRA1 loss‐of‐function variant

Abstract Vinpocetine is a synthetic derivative of the alkaloid vincamine and has been used as a dietary supplement for decades. Following a positive report of the use of vinpocetine in a patient with a loss‐of‐function GABRB3 variant, we here describe another patient with a loss‐of‐function GABRA1 variant (p.(Arg112Gln)) who benefited from vinpocetine treatment. This patient was diagnosed with autism spectrum disorder, psychiatric complications, and therapy‐resistant focal epilepsy. Upon add‐on treatment with 40 mg vinpocetine daily for 16 months, the patient experienced an overall improved quality of life as well as seizure freedom. Our findings corroborate that vinpocetine can attenuate epilepsy‐associated behavioral issues in patients with loss‐of‐function GABAA receptor gene variants.


Introduction
Variants in genes encoding subunits that form the major a1b3c2 GABA A receptor, GABRA1, GABRB3, and GABRG2, have been identified to cause different types of epilepsy ranging from mild genetic generalized epilepsy to severe developmental and epileptic encephalopathies. 1 Recently, Billakota et al. 2 reported that vinpocetine (Ethyl (3a,16a)-Eburnamenine-14-Carboxylate), a synthetic derivative of the alkaloid vincamine from the periwinkle plant, Vinca minor, improved clinical outcome of a 29-year-old woman with Lennox-Gastaut syndrome caused by a loss-of-function (LOF) variant in GABRB3. Upon add-on treatment with 20 mg vinpocetine three times daily over a period of 9 months, this patient displayed improved language abilities, behavior, and reduced frequency of spike-wave discharge on electroencephalogram (EEG).
In our clinic, we have been treating a 17-year-old male with de novo LOF variant in GABRA1, who has a history of intractable focal epilepsy and social behavioral disorders. Given the previous success in treating the GABRB3 variant patient and the fact that vinpocetine is generally considered safe for human consumption, we trialed addon treatment with vinpocetine in our patient. A remarkable positive outcome was observed, including reduced seizure frequency and better cognition and behavior.

Clinical Description
The patient is a 17-year-old male born at term by healthy non-consanguineous parents. Early development was unremarkable; however, mild language delay was noticed at the age of 3 years. He suffered from childhood-onset fluency disorder that required support from a speech therapist. At the age of 11 months, he presented with his first febrile seizure (FS). In the following 22 months, he experienced five additional bilateral tonic-clonic seizures, provoked by fever (38-38.5°C). Since the third year of life, the patient started to suffer from afebrile focal seizures characterized by, (1) impaired awareness, occasionally evolving to bilateral TC seizure, or (2) impaired awareness and myoclonic jerks in the neck, shoulder, and arm, typically on the left side.
MRI was normal. EEG revealed occipito-central 2-4 Hz spikes/polyspikes and slow wave complexes accentuated by sleep with anterior spread. Lamotrigine was initiated and he became seizure-free. At the age of 6 years and 6 months, lamotrigine was replaced with valproic acid (VPA) because of possible adverse effect (vocal tics). In the seventh year of life, attention deficit hyperactivity disorder (ADHD) was diagnosed, and methylphenidate was introduced. He was seizure-free until the age of 10 years and 11 months, where he started having monthly generalized tonic-clonic (GTC) seizures and daily FS characterized by: (i) hiccups possibly related to myoclonic jerks of the diaphragm, or (ii) eyes flickering and amaurosis, or (iii) visual aura followed by impaired awareness. VPA increase was associated with severe aggressive behavior. Thus, several drugs (eslicarbazepine, lacosamide, zonisamide) were tried without improvement on seizure frequency; in addition, he started to suffer from anxiety and depression. EEG was unchanged, besides the appearance of photosensitivity. At the age of 13 years, he was diagnosed with obsessive compulsive disorder (OCD), autism spectrum disorder, and mild intellectual disability (IQ score at 53). At 15 years old, implantation of vagus nerve stimulator (VNS) in combination with brivaracetam and VPA lead to a reduction of GTC seizures from monthly to only two GTC seizures a year (Table 1). FS persisted weekly, and anxiety and depressive symptoms did not improve. At this age, whole exome sequencing revealed a de novo missense variant in GABRA1 c.335G>A p.(Arg112Gln), which is a recurrent GABRA1 variant that has previously been reported in 12 individuals with neurodevelopmental disorders and epilepsy. 1,3-9

Treatment with Vinpocetine
Thirteen months after VNS implantation, the introduction of a magistral preparation of vinpocetine 20 mg three times daily was associated with a further reduction of FS, and the patient has currently been seizure-free for 16 months (Table 1). Repeated EEG showed a gradual decrease of posteriorly predominant epileptic abnormalities. In addition, the patient experienced a remarkable improvement in OCD, anxiety, and depression as well as better scores on neuropsychological tests (Table 1). After 9 months, the vinpocetine dose was reduced to 20 mg two times daily due to recurrent headaches, which then ceased. Periodic assessments of ECG, blood pressure, and blood tests did not show any abnormalities. No adverse drug reactions were reported 16 months after vinpocetine was introduced.

Functional Analysis
The Arg112 residue is located in the beta-strand three of the a1 subunit ( Fig. 1A-C). The position of this specific amino acid is not particularly conserved among human GABA A R subunits including the six known alpha subunits (Fig. 1C). While the Arg112 residue does not directly interact with GABA binding, it is speculated to aid in stabilizing the activated state upon GABA binding. 10 Electrophysiological experiments were performed to investigate the functional effects of the p.(Arg112Gln) (a1 R112Q ) variant. Patients with de novo GABRA1 variants are heterozygous for the variant. As pentameric GABA A receptors contain two a1 subunits, patients have mixed receptor populations where each receptor contains either zero (wild type), one (heterozygous), or two (homozygous) variant a1 R112Q subunits. As previously shown, receptors with two variant subunits can be expected to be the more severely affected, while receptors with a single variant subunit display less impairment. 11 As single variant receptors constitute the majority (50%), they are the most important to investigate functionally. Examination of receptors heterozygous for the a1 R112Q subunit was accomplished using concatenated receptor constructs as previously described (Fig. 1D). 12 Electrophysiological analysis revealed that receptors containing the a1 R112Q subunit were approximately 2.5fold less sensitive to GABA but displayed no change in maximal GABA-evoked current amplitudes when compared with wild type receptors (Fig. 1E,F). This significant change in GABA sensitivity demonstrates that receptors with a single variant a1 R112Q subunit display LOF traits, which is in overall agreement with previous studies of receptors with two variant a1 R112Q subunits. 7 Billakota et al. 2 reported that vinpocetine positively modulates GABA-evoked currents, hence this was next investigated. Co-applications of increasing concentrations of vinpocetine with a fixed concentration of GABA caused concentration-dependent increases in GABA-evoked current amplitudes at wild type as well as a1 R112Q containing receptors (Fig. 1G). A maximal fitted efficacy of~400% with accompanying potency of~6 lM was observed in both cases. Finally, no effects were observed upon applications of vinpocetine alone (up to concentrations of 10 lM). These data suggest that vinpocetine can be considered a positive allosteric modulator of a1b3c2 receptors and that the Arg112Gln variant does not influence the actions of vinpocetine.

Discussion
The efficacy of vinpocetine on epileptic seizures has previously been investigated. Meador et al. 13 treated eight patients with focal epilepsy with 20 mg vinpocetine three times daily without observing significant seizure reduction or any significant side effects. In contrast, Dutov et al. 14 and Garza-Morales et al. 15 reported a considerable decrease in seizure frequency with add-on vinpocetine treatment in patients with focal epilepsy. While these are obviously contrasting data, it is important to note that the underlying cause for the patient's epilepsy was not disclosed in these studies and it can therefore not be excluded that vinpocetine is beneficial in specific patient populations.
In the recent study by Billakota et al., the cause of the patient's epilepsy was known to be a LOF variant in the GABRB3 gene. 2 This patient was diagnosed with Lennox-Gastaut syndrome and add-on treatment with vinpocetine resulted in a sustained dose-dependent reduction in spike-wave discharge frequency on EEG as well as an improvement in global impression. Our patient likewise has a LOF variant in a GABA A receptor gene and became seizure free and experienced remarkable improvement in cognition and adaptive skills by adjuvant treatment of 40-60 mg daily vinpocetine. We acknowledge that VNS effects can increase over time; however, the time correlation between the introduction of vinpocetine and the seizure reduction as well as cognitive improvements suggest that these changes were likely related. Vinpocetine has been shown to have extensive pharmacological actions. It has primarily been thought to inhibit calcium/calmodulin-dependent cGMP-PDE 16 and blocking voltage-gated calcium as well as sodium channels. 17 Recently, it was reported that vinpocetine may also potentiate GABA A receptors, and our data supports these findings. 2 Vinpocetine displayed substantial positive modulation of a1b3c2 receptors on par with observations for hypnotics and benzodiazepines. 18 With an observed functional potency of~6 lM, vinpocetine is more potent at GABA A receptors than it is on most other reported targets (IC 50 values of 10-50 lM). 19 Meador et al. 13 observed blood levels of vinpocetine of~15 ng/ml in humans following peroral doses of 20 mg three times daily. This corresponds to a concentration of~40 nM, which appears low in comparison with the observed functional potencies of vinpocetine. However, this observation reflects a single time point and does not reveal the concentrations of vinpocetine available for receptor binding in the human brain. Furthermore, it cannot be excluded that some of the actions of vinpocetine are due to its main metabolite apovincaminic acid which was found in 20-fold higher concentrations. 13 Overall, we believe that it is reasonable to speculate that the potentiation of GABAergic activity constitutes an important component for the effects observed in patients with the GABRB3 and GABRA1 LOF variants. However, if enhancing the GABAergic system is responsible for the effects of vinpocetine, then caution is required when administering vinpocetine to other patients with GABR variants as these can also result in gain-of-function variants. Absalom et al. 12 recently reported adverse reactions to the GABA-potentiating drug vigabatrin in patients with GABRB3 GOF variants and a similar scenario could occur with vinpocetine as add-on treatment in these and other patients that have a GABA A GOF variant.
In conclusion, our findings suggest that vinpocetine may be effective in treating seizure and cognitive behavioral disturbances in patients with LOF GABA A R variants and most importantly improve the patients' quality of life. Further studies in larger cohorts including younger patients and with a longer follow-up are needed to confirm these results and to determine the best dosing practice, long-term effects, and safety profile of vinpocetine.