Mefloquine is a highly lipotropic drug which readily crosses the blood-brain barrier 2930 and whose efflux from neuronal tissues is mediated by the efflux pump P-glycoprotein (P-gp) 2931. In rat models a neuronal accumulation of enantiomeric mefloquine supports a stereoselective active neuronal efflux, and in in vivo mouse models, the neuronal concentration of quinine is raised in the presence of mefloquine 32. Mefloquine is, therefore, both a substrate and an inhibitor of P-gp 3133.

Under conditions of long-term chemoprophylaxis, mefloquine levels in plasma have been reported between 1.5 and 3.3 μM 34, but relatively few post-mortem studies have defined concentrations in brain tissue under conditions of chemoprophylaxis. One post-mortem case-series identified mefloquine in brain tissue at concentrations between 8.7 and 14 mg/kg 35, corresponding to significantly higher molar concentrations of between 21 and 34 μM. Another post-mortem study demonstrated ratios of brain white matter to serum mefloquine of 22.9:1 and 55.5:1 for (-) and (+) enantiomeric mefloquine, respectively 30. This same study found grey matter concentrations were approximately 69% of that found in white matter 30. Based on these data it is reasonable to conclude that mefloquine typically accumulates in brain tissue at concentrations approximately 10–30 times higher than found in serum 21.

The MDR1 gene codes for P-gp. Research postulates that polymorphisms in the MDR1 gene that result in a reduced expression of P-gp will result in lower mefloquine efflux from the brain and lead to higher brain tissue concentrations 33 than in individuals lacking these polymorphisms. Studies have demonstrated that the neuropsychiatric effects of mefloquine, as identified by earlier cohort studies 36, are more common among those with MDR1 polymorphisms 33. It is therefore plausible that common MDR1 polymorphisms, by significantly raising neuronal levels of mefloquine, might mediate the propensity towards dose-dependent adverse events.

Mefloquine exhibits a potent dose-dependent ability to interfere with the functioning of gap junctions, which are inter-neuronal channels created by distinct oligomeric proteins called connexins 37. A variety of connexin (Cx) proteins have been identified and characterized, identified by their molecular weight 37. Of these, Cx36 and Cx43 are widely distributed in neuronal tissue and show sensitivity to mefloquine.

Gap junctions formed by Cx36 predominate in neurons 38 in rat models, and play a critical role in the direct transport of ions between adjacent neurons, a process known as neuronal coupling 39. Cx36 is found at high concentrations within the inhibitory GABAergic interneurons of the cortical striatum 39. In mouse and rat models, Cx36 is also found throughout the ventral tegmental area 40, the neocortex 26, thalamic reticular nucleus 26, inferior olive 3841, and possibly hippocampus 26.

Lack of functioning Cx36 has been implicated in epileptogenesis 42, although the underlying mechanism is not clear. In mice lacking Cx36, deficits are found in synchronous firing within cortical, thalamic, and brainstem circuits 26. Gene-transfer research demonstrates inactivation of Cx36 in mature rat models results in measurable degradation in fine temporal coordination of muscle firing 41. Mefloquine blockade of Cx36 also suppresses essential tremor when induced in mouse models 28. At high concentrations, mefloquine also decreases the amplitude of Cx36-mediated cortical spreading depression in rat models in vitro 27.

Significant physiological effects due to mefloquine are noted at concentrations consistent with chemoprophylaxis. At concentrations of 25 μM, mefloquine inhibition of Cx36 in in vivo mouse models is associated with a decrease in GABAergic spontaneous synaptic currents to motor neurons 39. In in vitro studies, mefloquine at concentrations as low as 10 μM significantly reduced cortical coupling, and at 25 μM mefloquine caused increases in spontaneous chemical synaptic activity 26. In in invo studies in rats, mefloquine at concentrations of 25 μM antagonized the stimulatory action of modafinil, likely through decreased gap junction-mediated electrical coupling 43.

The predominant connexin expressed in neuronal glial tissue is Cx43, which is thought to permit the transfer of small molecular weight molecules critical to maintenance of cellular homeostasis 37. Functioning gap junctions in the astrocyte network serves to dilute substances cleared from the extracellular environment, including free calcium 37. At levels of 30 μM, mefloquine strongly inhibits Cx43 26.

Mefloquine in vitro also strongly interferes with neuronal calcium homeostasis, likely through inhibitory action on the endoplasmic reticulum (ER) calcium ATPase, leading to the intracellular release of the ER calcium stores 21. These effects have been consistently noted in rat neurons in vitro at concentrations of between 10–30 μM 2122. In a dog model, mefloquine inhibits inositol-1,4,5-phosphate (IP3)-mediated calcium release from neuronal microsome stores, although at slightly higher molar concentrations 24, suggesting a role for mefloquine in IP3-receptor (IP3-R)-mediated signal transduction processes.

In rat model experiments, mefloquine produces permanent and dose-dependent brain stem lesions in the nucleus gracilis, nuclear cuneatus, and solitary tract 20. These lesions were accompanied by dose-dependent impairments in motor performance consistent with loss of vestibular or nucleus gracilis-dependent proprioceptive function. Inhibition of cytoplasmic calcium homeostasis might trigger a shutdown of protein synthesis 23, resulting in an induction of proapoptotic mediators and contributing to the drug's neurotoxic effects 21. Such lesions were moderately demonstrated at doses of 187 mg/kg, corresponding with rat plasma concentrations of 2.1 μg/mL 20 or approximately 5.6 μM plasma concentration.

Unverricht-Lundborg Disease (ULD) is an inheritable, autosomal recessive, degenerative form of epilepsy characterized as progressive and myoclonic, type 1 (EPM1) 44. ULD is generally diagnosed prior to age 18, and in half of cases the first presentation is tonic-clonic seizure 44. EPM1 homozygosity is characterized by the early onset of progressive ataxia, incoordination, intentional tremor and dysarthria.

Genetic studies have revealed that EPM1 is associated with loss of function in the cystatin B gene (CSTB) in over 90% of cases 45 due to a dodecamer repeat expansion in the CSTB promoter 45 that results in significant downregulation of mRNA expression. CSTB protects against the proteolytic activity of cathepsin cystein proteases, particularly capthepsin B 45, and the main function of cytosolic CSTB in normal physiology is thought to be protection from lysosomal protease leakage 46. However, in CSTB knock-out mice, ataxia, myoclonic seizures, rapid cerebellar degeneration and apoptosis are noted 47 and are associated with significant increases in transcription of cathepsin S and markers of glial activation 48, themselves consistent with reactive changes suggestive of widespread cortical and subcortical grey matter damage 49. CSTB knock-out mice also demonstrate increased susceptibility in vivo to severe seizure, including generalized tonic-clonic seizures, and in vitro exhibit neuronal hyperexcitability characterized by decreased seizure threshold 50.

Additionally, mouse studies demonstrate that EPM1 heterozygosity results in lower cystatin B expression than wild-type mice and is associated also with near-universal mild neurogeneration and neuronal atrophy 51. In some cases heterozygosity also might be associated with mild signs and symptoms of ULD, including ataxia, motor incoordination and myoclonus 51.

These results suggests that CSTB deficiency creates impairments in the normal spectrum of neuronal physiologic safeguards, which predisposes EPM1 heterozygotes to neuronal hyperexcitability and hastens neuronal cell death with seizure 50.

Despite the absence in the literature of such events being reported, and based solely on biologic plausibility, the previous findings support the novel hypothesis that EPM1 heterozygosity might substantially increase the risk of seizure among subjects exposed to mefloquine, particularly in the presence of an MDR1 polymorphism or other condition, including the co-administration of a P-gp inhibitory drug that raises brain concentrations of mefloquine.

Among people with EPM1 mutation and exposed to mefloquine at an as-yet undetermined threshold grey-matter concentration, inherited predisposition to and reductions in physiologic protection from apoptosis are postulated to be exacerbated by a mefloquine-induced dysfunction among a spectrum of ordinarily complementary neuronal physiologic safeguards that become altered in mefloquine's presence. These altered physiologic safeguards include a loss of normal neuronal calcium homeostasis via mefloquine-induced release of ER calcium stores, altered glial cell function via mefloquine-induced Cx43-mediated gap junction dysfunction; and decreased GABAergic cortical inhibition and coupling and increased motor-neuron synaptic activity via mefloquine-induced Cx36-mediated gap junction dysfunction.

Although the precise role of altered astrocyte gap function in mediating apoptosis is not yet clear 52, it might be that in the presence of mefloquine-induced Cx43 dysfunction, increased neuronal cellular calcium resulting from inhibition of ER calcium ATPase is mediated less effectively by the astrocyte network, which then more readily induces neuronal proapoptotic mechanisms themselves promoted by mefloquine.

Simultaneously, it might be that mefloquine-induced Cx36 dysfunction results in decreased inhibitory GABAergic input and increased spontaneous synaptic activity. Supporting this postulate are results from in vivo mouse models, which demonstrate that high-dose mefloquine induces seizure, and that this effect is blocked by potent GABA agonists 53.

Given the simultaneous loss of multiple neuroprotective safeguards associated with mefloquine administration, it is, therefore, plausible that the first manifestation of seizure in EPM1 heterozygotes might occur with mefloquine prophylaxis.