Elevated peripheral levels of receptor-interacting protein kinase 1 (RIPK1) and IL-8 as biomarkers of human amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating fatal neurodegenerative disease with no cure. Receptor-interacting protein kinase 1 (RIPK1) has been proposed to mediate pathogenesis of ALS. Primidone has been identified as an old drug that can also inhibit RIPK1 kinase. We conducted a drug-repurposing biomarker study of primidone as a RIPK1 inhibitor using SOD1G93A mice and ALS patients. SOD1G93A mice treated with primidone showed significant delay of symptomatic onset and improved motor performance. One-hundred-sixty-two ALS participants dosed daily with primidone (62.5 mg) completed 24-week follow-up. A significant reduction was showed in serum levels of RIPK1 and IL-8, which were significantly higher in ALS patients than that of healthy controls (P < 0.0001). Serum RIPK1 levels were correlated positively with the severity of bulbar symptoms (P < 0.05). Our study suggests that serum levels of RIPK1 and IL-8 in peripheral can be used as clinical biomarkers for the activation of RIPK1 in central nervous system in human ALS patients. Repurposing primidone may provide a promising therapeutic strategy for ALS. The effect of primidone for the treatment of other inflammatory diseases may also be considered, since the activation of RIPK1 has been implicated in mediating a variety of inflammatory diseases including COVID-19-associated cytokine release syndrome (CRS). (ChiCTR2200060149).

Beneficial effects of primidone in Niemann-Pick disease type C (NPC)-model cells and mice: Reduction of unesterified cholesterol levels in cells and extension of lifespan in mice.

Niemann-Pick disease type C (NPC) is caused by a loss of function of either NPC1 or NPC2 protein, resulting in the accumulation of unesterified, free-cholesterol (free-C) in cells/tissues and thus leading to cell/tissue damage. In the brain of patients/animals with NPC, as a consequence of the accumulation of free-C in late endosomes/lysosomes (LE/LY) in cells, multiple lipids including complex sphingolipids are accumulated, and almost all patients/animals ultimately develop progressive/fatal neurodegeneration. Several reagents that are considered to act in the brain show beneficial effects on NPC-model animals. In the present study, we investigated the effects of antiepileptic drugs, such as primidone and valproic acid, on the accumulation of free-C in NPC1-null CHO cells and NPC1* fibroblasts, human fibroblasts established from a patient with NPC1 mutation. Like valproic acid, treatment with primidone reduced free-C levels in LE/LY in NPC1-null/mutant cells. Down-regulation of cholesterol ester levels in NPC1-null cells and up-regulation of HMG-CoA reductase and low-density lipoprotein receptor mRNA levels in NPC1* cells were partially recovered by primidone treatment. Thus, primidone was suggested to enhance free-C trafficking from LE/LY to endoplasmic reticulum in NPC1-null/mutant cells. In NPC1-null mice, oral application of primidone (100 mg/kg/day) extended lifespan by approximately 5 days, although the first days showing ataxia, a typical symptom of neuromotor dysfunction, were not affected. Our findings suggest the potential of primidone for the treatment of NPC.

TRPM3 channel activation inhibits contraction of the isolated human ureter via CGRP released from sensory nerves.

AIMS: Sensory nerve activation modulates ureteral contractility by releasing neuropeptides including CGRP and neurokinin A (NKA). TRPM3 is a recently discovered thermosensitive channel expressed in nociceptive sensory neurons, and plays a key role in heat nociception and chronic pain. The aim of this study is to examine the role of TRPM3 activation in human ureter motility. MAIN METHOD: Human proximal ureters were obtained from fourteen patients undergoing nephrectomy. Spontaneous or NKA-evoked contractions of longitudinal ureter strips were recorded in an organ bath. Ureteral TRPM3 expression was examined by immunofluorescence. KEY FINDINGS: Spontaneous contractions were observed in 60% of examined strips. TRPM3 activation using pregnenolone sulphate (PS) or CIM0216 (specific TRPM3 agonists) dose-dependently reduced the frequency of spontaneous and NKA-evoked contractions, with IC50s of 241.7 µM and 4.4 µM, respectively. The inhibitory actions of TRPM3 agonists were mimicked by CGRP (10 to 100 nM) or a cAMP analogue (8-Br-cAMP; 1 mM). The inhibitory actions of TRPM3 agonists (300 µM PS or 30 µM CIM0216) were blocked by pretreatment with primidone (TRPM3 antagonist; 30 µM), tetrodotoxin (sodium channel blocker; 1 µM), olcegepant (CGRP receptor antagonist; 10 µM), or H89 (non-specific PKA inhibitor; 30 µM). TRPM3 was co-expressed with CGRP in nerves in the sub-urothelial and intermuscular regions of the ureter. SIGNIFICANCE: TRPM3 channels expressed on sensory terminals of the human ureter involve in inhibitory sensory neurotransmission and modulate ureter motility via the CGRP-cAMP-PKA signal pathway. Targeting TRPM3 may be a pharmacological strategy for promoting the ureter stone passage.

Primidone blocks RIPK1-driven cell death and inflammation.

The receptor-interacting serine/threonine protein kinase 1 (RIPK1) is a key mediator of regulated cell death and inflammation. Recent studies suggest that RIPK1 inhibition would fundamentally improve the therapy of RIPK1-dependent organ damage in stroke, myocardial infarction, kidney failure, and systemic inflammatory response syndrome. Additionally, it could ameliorate or prevent multi-organ failure induced by cytokine release in the context of hyperinflammation, as seen in COVID-19 patients. Therefore, we searched for a RIPK1 inhibitor and present the aromatic antiepileptic and FDA-approved drug primidone (Liskantin®) as a potent inhibitor of RIPK1 activation in vitro and in a murine model of TNFα-induced shock, which mimics the hyperinflammatory state of cytokine release syndrome. Furthermore, we detected for the first time RIPK1 activation in the respiratory tract epithelium of hospitalized patients who tested positive for SARS-CoV-2 infection. Our data provide a strong rationale for evaluating the drug primidone in conditions of hyperinflammation in humans.

Primidone inhibits TRPM3 and attenuates thermal nociception in vivo.

The melastatin-related transient receptor potential (TRP) channel TRPM3 is a nonselective cation channel expressed in nociceptive neurons and activated by heat. Because TRPM3-deficient mice show inflammatory thermal hyperalgesia, pharmacological inhibition of TRPM3 may exert antinociceptive properties. Fluorometric Ca influx assays and a compound library containing approved or clinically tested drugs were used to identify TRPM3 inhibitors. Biophysical properties of channel inhibition were assessed using electrophysiological methods. The nonsteroidal anti-inflammatory drug diclofenac, the tetracyclic antidepressant maprotiline, and the anticonvulsant primidone were identified as highly efficient TRPM3 blockers with half-maximal inhibition at 0.6 to 6 µM and marked specificity for TRPM3. Most prominently, primidone was biologically active to suppress TRPM3 activation by pregnenolone sulfate (PregS) and heat at concentrations markedly lower than plasma concentrations commonly used in antiepileptic therapy. Primidone blocked PregS-induced Cai influx through TRPM3 by allosteric modulation and reversibly inhibited atypical inwardly rectifying TRPM3 currents induced by coapplication of PregS and clotrimazole. In vivo, analgesic effects of low doses of primidone were demonstrated in mice, applying PregS- and heat-induced pain models, including inflammatory hyperalgesia. Thus, applying the approved drug at concentrations that are lower than those needed to induce anticonvulsive effects offers a shortcut for studying physiological and pathophysiological roles of TRPM3 in vivo.

Antiepileptic drugs: Impacts on human serum paraoxonase-1.

Serum paraoxonase (PON1) is a key enzyme related to high-density lipoprotein (HDL)-cholesterol particle. It can prevent the oxidation of low-density lipoprotein (LDL) and HDL. The present article focuses on the in vitro inhibition role of some antiepileptic drugs (AEDs) such as valproic acid, gabapentin, primidone, phenytoin, and levetiracetam on human paraoxonase (hPON1). Therefore, PON1 was purified from human serum with a specific activity of 3976.36 EU/mg and 13.96% yield by using simple chromatographic methods. The AEDs were tested at various concentrations, which showed reduced in vitro hPON1 activity. IC50 values for gabapentin, valproic acid, primidone, phenytoin, and levetiracetam were found to be 0.35, 0.67, 0.87, 6.3, and 53.3 mM, respectively. Ki constants were 0.261 ± 0.027, 0.338 ± 0.313, 0.410 ± 0.184, 10.3 ± 0.001, and 43.01 ± 0.003 mM, respectively. Gabapentin exhibited effective inhibitory activity as compared with the other drugs. The inhibition mechanisms of all compounds were noncompetitive.

Zebrafish larvae exposed to ginkgotoxin exhibit seizure-like behavior that is relieved by pyridoxal-5'-phosphate, GABA and anti-epileptic drugs.

The etiology of epilepsy is a very complicated, multifactorial process that is not completely understood. Therefore, the availability of epilepsy animal models induced by different mechanisms is crucial in advancing our knowledge and developing new therapeutic regimens for this disorder. Considering the advantages of zebrafish, we have developed a seizure model in zebrafish larvae using ginkgotoxin, a neurotoxin naturally occurring in Ginkgo biloba and hypothesized to inhibit the formation of the neurotransmitter γ-aminobutyric acid (GABA). We found that a 2-hour exposure to ginkgotoxin induced a seizure-like behavior in zebrafish larvae. This seizure-like swimming pattern was alleviated by the addition of either pyridoxal-5'-phosphate (PLP) or GABA and responded quickly to the anti-convulsing activity of gabapentin and phenytoin, two commonly prescribed anti-epileptic drugs (AEDs). Unexpectedly, the ginkgotoxin-induced PLP depletion in our experimental setting did not affect the homeostasis of folate-mediated one-carbon metabolism, another metabolic pathway playing a crucial role in neural function that also relies on the availability of PLP. This ginkgotoxin-induced seizure behavior was also relieved by primidone, which had been tested on a pentylenetetrazole-induced zebrafish seizure model but failed to rescue the seizure phenotype, highlighting the potential use and complementarity of this ginkgotoxin-induced seizure model for AED development. Structural and morphological characterization showed that a 2-hour ginkgotoxin exposure did not cause appreciable changes in larval morphology and tissues development. In conclusion, our data suggests that this ginkgotoxin-induced seizure in zebrafish larvae could serve as an in vivo model for epileptic seizure research and potential AED screening.

[Anticonvulsants as bioantioxidants under stress conditions].

The action of heterocyclic amides series (ethosuximide, phenytoin, primidone) on lipid peroxidation and membrane bound monoamine oxidases A and B under stress condition has been studied. The intraperitoneal injection of the drugs resulted in enhancement of SOD, decrease of brain malondialdehyde content and mitochondrial activity of monoamine oxidases A and B.

The antiepileptic primidone impairs male rat sexual behavior.

Many antiepileptic drugs (AEDs) produce sexual impairments. Of commonly prescribed AEDs, primidone produces the greatest impairments. Here we examined the effects of primidone on male rat sexual behavior. Sexually-experienced male rats received administration of either vehicle or primidone. After baseline measures were obtained, the effects of daily primidone treatment on home cage sexual performance were assessed three times over the course of 14 days. Motor activity and sucrose preference were also assessed during this time period. Results indicate that primidone impaired copulation but not sexual motivation. Specifically, animals receiving primidone displayed fewer ejaculations, required more time to achieve an intromission, and displayed fewer intromissions per attempted mount as evidenced by a lower intromission ratio. However, animals treated with primidone also chose a goal box containing a sexually-receptive female in an x-maze as often as animals receiving vehicle. The lower intromission ratio suggests an inability to achieve intromissions perhaps as a result of impaired erectile function. Primidone did not affect motor activity or sucrose consumption, an additional measure of natural reward. Together, these data indicate that primidone impairs male sexual activity and suggest that these impairments result primarily from changes in erectile function and not changes to mechanisms mediating motivation.

Lacosamide, the new anticonvulsant, effectively reduces harmaline-induced tremors in rats.

The present study aimed at evaluating the efficacy of lacosamide (0.3 to 30 mg/kg), a new anticonvulsant drug, in a model of essential tremor in comparison to the reference compounds propranolol and primidone. We observed a high tremorlytic effect of lacosamide reducing the intensity of tremors following harmaline administration in a dose-dependent manner. The highest dose also modified the latency and intensity of tremor at onset. The effect of lacosamide was equal or even superior to propranolol and primidone indicating that lacosamide may be a new antitremorgenic drug that merits further clinical investigation.

Genetic essential tremor in gamma-aminobutyric acidA receptor alpha1 subunit knockout mice.

Essential tremor is the most common movement disorder and has an unknown etiology. Here we report that gamma-aminobutyric acidA (GABA(A)) receptor alpha1-/- mice exhibit postural and kinetic tremor and motor incoordination that is characteristic of essential tremor disease. We tested mice with essential-like tremor using current drug therapies that alleviate symptoms in essential tremor patients (primidone, propranolol, and gabapentin) and several candidates hypothesized to reduce tremor, including ethanol; the noncompetitive N-methyl-D-aspartate receptor antagonist MK-801; the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA); the GABA(A) receptor modulators diazepam, allopregnanolone, and Ro15-4513; and the L-type Ca2+ channel antagonist nitrendipine. Primidone, propranolol, and gabapentin reduced the amplitude (power) of the pathologic tremor. Nonsedative doses of ethanol eliminated tremor in mice. Diazepam, allopregnanolone, Ro15-4513, and nitrendipine had no effect or enhanced tremor, whereas MK-801 and CCPA reduced tremor. To understand the etiology of tremor in these mice, we studied the electrophysiological properties of cerebellar Purkinje cells. Cerebellar Purkinje cells in GABA(A) receptor alpha1-/- mice exhibited a profound loss of all responses to synaptic or exogenous GABA, but no differences in abundance, gross morphology, or spontaneous synaptic activity were observed. This genetic animal model elucidates a mechanism of GABAergic dysfunction in the major motor pathway and potential targets for pharmacotherapy of essential tremor.

Benefits and risks of pharmacological treatments for essential tremor.

Essential tremor can cause significant functional disability in some patients. The arms are the most common body part affected and cause the most functional disability. The treatment of essential tremor includes medications, surgical options and other forms of therapy. Presently there is no cure for essential tremor nor are there any medications that can slow the progression of tremor. Treatment for essential tremor is recommended if the tremor causes functional disability. If the tremor is disabling only during periods of stress and anxiety, propranolol and benzodiazepines can be used during those periods when the tremor causes functional disability. The currently available medications can improve tremor in approximately 50% of the patients. If the tremor is disabling, treatment should be initiated with either primidone or propranolol. If either primidone or propranolol do not provide adequate control of the tremor, then the medications can be used in combination. If patients experience adverse effects with propranolol, occasionally other beta-adrenoceptor antagonists (such as atenolol or metoprolol) can be used. If primidone and propranolol do not provide adequate control of tremor, occasionally the use of benzodiazepines (such as clonazepam) can provide benefit. Other medications that may be helpful include gabapentin or topiramate. If a patient has disabling head or voice tremor, botulinum toxin injections into the muscles may provide relief from the tremor. Botulinum toxin in the hand muscles for hand tremor can result in bothersome hand weakness and is not widely used. There are other medications that have been tried in essential tremor and have questionable efficacy. These drugs include carbonic anhydrase inhibitors (e.g. methazolamide), phenobarbital, calcium channel antagonists (e.g. nimodipine), isoniazid, clonidine, clozapine and mirtazapine. If the patient still has disabling tremor after medication trials, surgical options are usually considered. Surgical options include thalamotomy and deep brain stimulation of the thalamus. These surgical options provide adequate tremor control in approximately 90% of the patients. Surgical morbidity and mortality for these procedures is low. Deep brain stimulation and thalamotomy have been shown to have comparable efficacy but fewer complications have been reported with deep brain stimulation. In patients undergoing bilateral procedures deep brain stimulation of the thalamus is the procedure of choice to avoid adverse effects seen with bilateral ablative procedures. The use of medication and/or surgery can provide adequate tremor control in the majority of the patients.

Homocysteine concentrations and methionine loading in patients on antiepileptic drugs.

OBJECTIVES: A few reports have shown elevated fasting total plasma homocysteine (tHcy) in patients taking antiepileptic drugs (AEDs). In this study we determined the influence of AEDs on plasma tHcy levels prior to and following methionine loading. MATERIAL AND METHODS: Thirty-four patients on different AEDs and 34 matched controls were recruited. Blood samples were drawn prior to and 6 h post-methionine loading (6h-PML). RESULTS: The patients on AEDs inducing the cytochrome P450 (carbamazepine, phenytoin, phenobarbital, primidone), had higher fasting and 6h-PML plasma tHcy concentrations than the controls (P = 0.01 and P<0.001). Patients on AED inhibiting the cytochrome P450 (valproate [VPA]), had lower 6h-PML p-tHcy concentrations than controls (P = 0.01). CONCLUSIONS: Our data indicate that not only fasting but also 6h-PML tHcy levels should be determined in order to identify hyperhomocysteinemia among patients on AEDs. Inducer AEDs seem to have an opposite effect than the inhibitor VPA on plasma tHcy, erythrocyte folate and serum folate levels.

Accelerated recovery and disposition from rocuronium in an end-stage renal failure patient on chronic anticonvulsant therapy with sodium valproate and primidone.

An end-stage renal failure patient, receiving chronic treatment with the anticonvulsants, sodium valproate and primidone, showed accelerated recovery with enhanced elimination (T1/2(z) = 52 min) and clearance (Cl = 14.4 ml min-1 kg-1) of rocuronium. The pharmacokinetic and pharmacodynamic effects of rocuronium in this patient are compared with those published for healthy and renal failure patients. Increased hepatic binding of rocuronium rather than metabolism is suggested as the possible cause of this effect.

Clinically significant pharmacokinetic drug interactions with carbamazepine. An update.

Carbamazepine is one of the most commonly prescribed antiepileptic drugs and is also used in the treatment of trigeminal neuralgia and psychiatric disorders, particularly bipolar depression. Because of its widespread and long term use, carbamazepine is frequently prescribed in combination with other drugs, leading to the possibility of drug interactions. The most important interactions affecting carbamazepine pharmacokinetics are those resulting in induction or inhibition of its metabolism. Phenytoin, phenobarbital (phenobarbitone) and primidone accelerate the elimination of carbamazepine, probably by stimulating cytochrome P450 (CYP) 3A4, and reduce plasma carbamazepine concentrations to a clinically important extent. Inhibition of carbamazepine metabolism and elevation of plasma carbamazepine to potentially toxic concentrations can be caused by stiripentol, remacemide, acetazolamide, macrolide antibiotics, isoniazid, metronidazole, certain antidepressants, verapamil, diltiazem, cimetidine, danazol and (dextropropoxyphene) propoxyphene. In other cases, toxic symptoms may result from elevated plasma concentrations of the active metabolite carbamazepine-10,11-epoxide, due to the inhibition of epoxide hydrolase by valproic acid (sodium valproate), valpromide, valnoctamide and progabide. Carbamazepine is a potent inducer of CYP3A4 and other oxidative enzyme system in the liver, and it may also increase glucuronyltransferase activity. This results in the acceleration of the metabolism of concurrently prescribed anticonvulsants, particularly valproic acid, clonazepam, ethosuximide, lamotrigine, topiramate, tiagabine and remacemide. The metabolism of many other drugs such as tricyclic antidepressants, antipsychotics, steroid oral contraceptives, glucocorticoids, oral anticoagulants, cyclosporin, theophylline, chemotherapeutic agents and cardiovascular drugs can also be induced, leading to a number of clinically relevant drug interactions. Interactions with carbamazepine can usually be predicted on the basis of the pharmacological properties of the combined drug, particularly with respect to its therapeutic index, site of metabolism and ability to affect specific drug metabolising isoenzymes. Avoidance of unnecessary polypharmacy, selection of alternative agents with lower interaction potential, and careful dosage adjustments based on serum drug concentration monitoring and clinical observation represent the mainstays for the minimisation of risks associated with these interactions.

Seizure control after chronic pharmacotherapy in epileptic disorders beginning after 40 years of age.

Few studies have been published on the pharmacologic response to treatment of patients whose seizures begin after 40 years of age. The purpose of this study was to assess the impact of chronic pharmacotherapy on the seizure control of such patients. We retrospectively studied the seizure frequency recorded during a 12-month period in a group of 94 outpatients whose seizure disorders began after 40 years of age (median age of seizure onset 56.5 years) and who had been treated with anticonvulsant medication for a median period of 6 years (range 18 months to 12 years). We assessed the relationship between the patients' seizure frequency during the last 12 months of treatment using (a) the present and previously prescribed pharmacologic regimens, (b) anticonvulsant blood levels of present regimen, (c) etiology and duration of seizure disorder, (d) age at onset of seizures, and (e) presence of electrographic (EEG) and neuroradiologic abnormalities. We only identified side effects occurring at blood levels within or below the drug's therapeutic range. Seventy-eight patients (83%) were seizure free during the last 12 months of treatment, 11 (11%) had rare seizures, and five (6%) had more than four seizures per year. Seizure frequency was not affected by duration and etiology of seizure disorder, age at onset of seizures, seizure type, neuroradiologic or electroencephalographic abnormalities, and present or previously prescribed pharmacologic regimens. Persistent side effects were reported in seven of 76 (9%) monotherapy regimens and in two of 12 (17%) polytherapy regimens. Our data suggest that seizures beginning after the age of 40 have a favorable prognosis after chronic pharmacotherapy.

Na(+)-dependent biotin transport into brush-border membrane vesicles from human kidney cortex.

Renal reabsorption of biotin was investigated in human kidney by means of the isolated brush-border membrane vesicle technique. Biotin uptake into the vesicles was sodium-dependent producing a typical overshoot when incubated under sodium-gradient conditions (external concentration greater than internal). This effect was not observed in the presence of gradients of KCl, LiCl or choline-chloride, nor in the absence of any salt. Using the K+/valinomycin voltage-clamp method biotin uptake remained uninfluenced, i.e. was electroneutral, whereas glucose uptake (which is known to be electrogenic in kidney of other species) was greatly increased. When biotin transport was investigated as a function of external sodium concentration a stoichiometic coupling factor of 1 for the Na(+)-biotin- cotransport was determined. Increasing the biotin concentration in the incubation medium up to 200 mumol/l led to saturation with the kinetic parameters of 31 mumol/l for the apparent Michaelis constant and 82 nmol g protein-1 30 s-1 for the maximal transport rate. Uptake was not saturable in the concentration range of 0.001-1 mumol/l. Inhibition of the biotin uptake (25 mumol/l) was observed in the presence of 250 mumol/l dethiobiotin, bisnorbiotin, thioctic acid, and probenecid, whereas biocytin, propionic acid, lactic acid, succinic acid, citric acid, ascorbic acid, primidone and carbamazepine had no effect. We conclude that renal biotin reabsorption in human kidney is specifically sodium-dependent, saturable and electroneutral. It therefore fulfills the requirements for a secondary active carrier-mediated transport system. The results suggest that biocytin is not an inhibitor of renal biotin reabsorption.

Anticonvulsant effects of phenobarbital and primidone during ontogenesis in rats.

The influence of phenobarbital (PHB, 10, 20, 40 or 80 mg/kg i.p.) and primidone (PRI, 40 or 80 mg/kg i.p.) on metrazol-induced motor seizures was studied in rats 7, 12, 15, 18, 25 and 90 days old. PHB blocked both types of seizures induced by metrazol-minimal (mMS) as well as generalized tonic-clonic (MMS)--in all age groups where they appeared under control conditions. The effect against major seizures was always better expressed than against mMS. Pretreatment with PHB led to the appearance of mMS in 7- and 12-day-old rat pups, where control animals did not exhibit this type of seizure. Combined administration of the 2 high doses of PHB and metrazol resulted in the appearance of behavioral automatisms in young rats. PRI abolished MMS in adult rats only, no changes were seen in 25-day-old animals and specific suppression of the tonic phase of MMS was observed in younger rats. mMS were influenced only in 7- and 12-day-old rats, where an increase in their incidence was recorded. Pretreatment with PRI never induced automatisms. The different actions of PHB and PRI speak in favor of an anticonvulsant action of PRI itself.