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).

Dose-dependent apoptotic and necrotic myocyte death induced by the beta2-adrenergic receptor agonist, clenbuterol.

We have investigated the dose- and time-dependency of myocyte apoptosis and necrosis induced by the beta2-adrenergic receptor agonist, clenbuterol, with the aim of determining whether myocyte apoptosis and necrosis are two separate processes or a continuum of events. Male Wistar rats were administered subcutaneous injections of clenbuterol, and immunohistochemistry was used to detect myocyte-specific apoptosis and necrosis. Myocyte apoptosis peaked 4 h after, and necrosis 12 h after, clenbuterol administration. In the soleus, peak apoptosis (5.8 +/- 2.0%; P < 0.05) was induced by 10 mug and peak necrosis (7.4 +/- 1.7%; P < 0.05) by 5 mg x kg(-1) clenbuterol. Twelve hours after clenbuterol administration, 73% of damaged myocytes labeled as necrotic, 27% as apoptotic and necrotic, and 0% as purely apoptotic. Administrations of clenbuterol (10 microg x kg(-1)) at 48-h intervals induced cumulative myocyte death over 8 days. These data show that the phenotype of myocyte death is dependent on the magnitude of the insult and the time at which it is investigated. Only very low doses induced apoptosis alone; in most cases apoptotic myocytes lysed and became necrotic and the magnitude of necrosis was greater than that of apoptosis. Thus, it is important to investigate both apoptotic and necrotic myocyte death, contrary to the current trend of only investigating apoptotic cell death.

Effects of encapsulation of primidone on its oxidative metabolism in rats.

The aim of this study was to evaluate the influence of primidone (PRM) nanoencapsulation on its metabolism. Suspensions of PRM powder and PRM-loaded poly-epsilon-caprolactone nanocapsules were administered orally in the same way to rats. Primidone-loaded poly-epsilon-caprolactone nanocapsules were prepared according to the interfacial deposition technique. Free PRM suspensions were obtained by addition of PRM powder to a suspension of 0.212% carboxymethylcellulose CMC 12H in water. The dose was 20 mg/kg, n = 6, for each experiment. Urinary and faecal levels of PRM and of its three major metabolites, phenylethylmalonamide (PEMA), phenobarbital (PB), and p-hydroxyphenobarbital (p-HO-PB), were determined. Concentrations were evaluated by high-performance liquid chromatography (HPLC) according to a validated analytical method. After PRM nanocapsule administration, non-metabolised PRM urinary levels were increased compared to those observed after administration of a suspension of primidone powder (43.7+/-8.8% after PRM-loaded nanocapsule and 37.7+/-8.1% after free PRM administration). For phenylethylmalonamide, no difference was observed in urinary excretion in the two cases. For two of the oxidised metabolites, PB and p-HO-PB, excretion was delayed and shortened. The amount of these oxidised metabolites was lowered from 0.95% after free PRM administration to 0.25% after PRM-loaded nanocapsule administration. No difference was noted in non-metabolised primidone excretion in faeces. These results suggest that primidone-loaded nanocapsules could be used as a vehicle for oral primidone administration in order to minimise the phenobarbital metabolic pathway.

Simultaneous determination of primidone and its three major metabolites in rat urine by high-performance liquid chromatography using solid-phase extraction.

A new high-performance liquid chromatographic method for simultaneous determination of primidone (PRM) and of its three major metabolites, phenobarbital (PB), p-hydroxyphenobarbital (p-HO-PB) and phenylethylmalonamide (PEMA), in rat urine, was developed. After acid hydrolysis, these compounds were extracted from urine by means of a Bond Elut Certify LRC column with good clean-up. The extracts were chromatographed on a C18 reversed-phase column using isocratic elution at 40 degrees C, with UV detection at 227 nm. The limit of detection was 0.5 mg/ml for the four compounds. Good linearity (r2>0.99) was observed within the calibration ranges studied: 37.4-299.3 microg/ml for PRM, 26.4-211.2 microg/ml for PB, 12.5-100.2 microg/ml for p-HO-PB and 12.1-97.0 microg/ml for PEMA. Repeatability was in the range 3.1-6.8%. This method constitutes a useful tool for studies on the influence of various parameters on primidone metabolism.

Phenobarbital, drug metabolism, and human cancer.

To investigate the possible influence of anticonvulsant treatment on cancer risk, a nested case-control study of 104 lung cancers, 18 bladder cancers, and 322 cancer-free controls was conducted. The background for the study was previous observations among 8004 epileptics in Denmark with a significantly high risk for lung cancer and a significantly low risk for bladder cancer. Cigarette smoking appears to explain the lung cancer excess but not the low risk for bladder cancer, another tobacco-related disease. Information was abstracted on 94 and 95% of the cases and controls, respectively. Lung cancer was not associated with any anticonvulsant drug, but bladder cancer was inversely related to use of phenobarbital (PB). The apparent protective effect of PB was further evaluated in a study of rats given 4-aminobiphenyl (ABP), a bladder carcinogen. The levels of 4-aminobiphenyl adducts in hemoglobin and in bladder and liver DNA were significantly lower in rats given PB prior to 4-aminobiphenyl, compared to controls. These studies suggest that PB may induce drug-metabolizing enzymes of the liver that deactivate bladder carcinogens found in cigarette smoke and provide clues to the role of activation and detoxification of carcinogens in humans.

Urinary metabolites of phenobarbitone, primidone, and their N-methyl and N-ethyl derivatives in humans.

1. Phenobarbitone (1) and three of its N-alkyl derivatives, and primidone (10) and four of its N-alkyl derivatives, were orally administered separately to two human volunteers. Total urine was collected for approximately 2 weeks following each dose, and the drugs and their metabolites were assayed by g.l.c.-mass spectrometry. 2. Recoveries in the phenobarbitone series increased from approximately 40% to approximately 50% as alkylation of (1) increased. There was a linear relationship between the extent of p-hydroxylation and the lipophilicity (log P) of the substrates. The increased total recovery was largely attributable to increased p-hydroxylation. 3. Urinary recoveries in the primidone series decreased from approximately 80% for (10) to approximately 30% for its diakyl derivatives, despite a slight increase in p-hydroxylation with increasing alkylation (and increasing lipophilicity). The decreased recovery was mainly the result of decreased urinary excretion of the drug.

The disposition of primidone in elderly patients.

1. The pharmacokinetics and metabolism of primidone at steady-state were studied in 10 elderly patients aged 70-81 years and eight control subjects aged 18-26 years. 2. Primidone half-lives and clearance values (mean +/- s.d.) were similar in the elderly and in the young (12.1 +/- 4.6 vs 14.7 +/- 3.5 h and 34.8 +/- 9.0 vs 33.2 +/- 7.2 ml h-1 kg-1 respectively. 3. The serum concentrations of the metabolites phenylethylmalonamide (PEMA) and phenobarbitone relative to those of parent drug were higher in the elderly than in the young, the difference being significant (P less than 0.01) in the case of PEMA. 4. The renal clearances of primidone, phenobarbitone and PEMA were moderately decreased in the elderly but this reduction was statistically significant only for PEMA. Elderly patients excreted a reduced proportion of unchanged primidone and an increased proportion of PEMA in urine. 5. Ageing is associated with a greater accumulation of PEMA, which is unlikely to have a major clinical significance.

Metabolic studies with phenobarbitone, primidone and their N-alkyl derivatives: quantification of substrates and metabolites using chemical ionization gas chromatography-mass spectrometry.

Metabolic studies with phenobarbitone, primidone and some of their N-alkyl derivatives required the concurrent assay of any mixture of these substrates (twelve compounds) and their major metabolites (an additional twenty-two compounds) in urine. The method described in the present report met this requirement by incorporating two complementary derivatization techniques into a gas chromatographic-mass spectrometric (GC-MS) assay procedure. Following hydrolysis of conjugates with beta-glucuronidase, urine samples were extracted with ethyl acetate (3 X 5 ml). The combined extracts were dried over sodium sulphate, divided into two equal portions, and the solvent was removed. One residue was derivatized by propylation using 1-iodopropane with base catalysis. The other residue was silylated using methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide. The derivatives in each case were analysed by GC-MS, using temperature-programmed packed-column GC and chemical ionization MS. Mass spectra were acquired over an appropriate mass range, and peak areas for the compounds of interest were determined from specific mass chromatograms. Satisfactory precision, accuracy, specificity and sensitivity were obtained for all analytes. All compounds produced satisfactory derivatives by at least one procedure; twelve compounds could be analysed by both techniques. The method illustrates the utility of chemical ionization GC-MS for the simultaneous quantitative analysis of multiple related analytes in complex biological samples.

Primidone and essential tremor.

To clarify whether primidone itself, and not only its metabolite phenobarbitone, suppresses essential tremor, the effect of a high single dose of primidone was tested. Of 11 patients, 8 showed a reduction of their tremor by 54%-69% for up to 28 h. The serum concentration of primidone was as expected, whereas those of the metabolites phenyl-ethyl-malonamide and phenobarbitone were very low. The tremor suppression can thus be considered to be an effect of primidone. Three of the 11 patients did not show a reduction of tremor.

Effect of primidone in somatic and germ cells of mice.

Primidone, an anti-convulsant drug, was tested in mice for mutagenicity in somatic cells by the micronucleus test and in germ cells by the sperm-head abnormality assay. Mice were treated orally with the drug at doses of 4.37, 8.75 and 13.11 mg/mouse. The results indicate that the drug is capable of inducing mutations both in somatic and germ cells of mice.

Nursing interventions for anticonvulsant drug interactions.

The management of epilepsy or seizure disorder commonly includes drug therapy. Optimal treatment may involve single-drug regimens, but multiple agents are often required. Although any pharmacological agent can be involved in a drug interaction, anticonvulsants are often implicated. As more drugs are prescribed for the patient, the potential for drug interactions increases. This article reviews the background of anticonvulsant drug interactions, the most common interactions encountered, and nursing interventions useful in monitoring potential interactions. Suggestions for nursing research are given.

Poor correlation between single-dose data and steady-state kinetics for phenobarbitone, primidone, carbamazepine and sodium valproate in children during monotherapy. Possible reasons for the lack of correlation.

An investigation was performed to determine the relationship between the serum drug concentration/dose ratio at 24 hours following a first dose and that at steady-state for phenobarbitone, primidone (as phenobarbitone and as primidone), carbamazepine and sodium valproate, in order to assess the utility of this method in clinical practice. The drugs were given as monotherapy to 63 children for the treatment of epilepsy or febrile convulsions. The correlation between concentration/dose ratios, instead of between serum concentrations, was investigated with the aim of allowing the use of variable doses. The correlation coefficients were: r = 0.30 for phenobarbitone; r = 0.05 for phenobarbitone derived from primidone; r = 0.38 for primidone; r = 0.19 for carbamazepine; and r = 0.52 for sodium valproate. None of these correlation coefficients differed statistically from 0. These low correlation coefficients contrast with the acceptable results found by other authors for other drugs, indicating that several factors may have a greater influence on this correlation than earlier investigations suggest. The poor correlation obtained emphasises the need for clinical verification of mathematical models based on theoretical considerations which do not always apply in practice.

Phenylethylmalonamide serum levels in patients treated with primidone and the effects of other antiepileptic drugs.

Data are presented for the serum levels of 2-ethyl-2-phenylmalonamide (PEMA) in patients receiving anticonvulsant medication. Statistical analysis of these data indicates that the serum level of PEMA, which is a metabolite of primidone, is affected not only by the dose of primidone but also by the serum levels of other prescribed anticonvulsant drugs. In particular, phenobarbitone is shown to be a major perturbation upon the PEMA serum level.

Carbamazepine drug interactions.

Carbamazepine (CBZ) is commonly prescribed as an anticonvulsant or for the pain of trigeminal neuralgia. The potential for clinically important drug interactions exists because CBZ may induce the hepatic metabolism of other drugs or, conversely, other drugs may induce or inhibit the metabolism of CBZ. Studies and case reports demonstrate that CBZ may accelerate the metabolism of phenytoin, phenobarbital (PB), primidone, valproic acid, and warfarin. Likewise, phenytoin, PB, and primidone may increase the hepatic metabolism of CBZ. Inhibition of the metabolism of CBZ has been caused by triacetyloleandomycin, erythromycin, propoxyphene, isoniazid, and cimetidine. Future investigations will document the clinical significance of the CBZ interactions as well as reveal new interactions.

Effects of pregnancy on antiepileptic drug utilization.

Pregnancy is associated with characteristic changes in the disposition of antiepileptic drugs; recent findings on this aspect of drug utilization are presented. In one study involving 48 pregnancies, the mean level-dose ratio of phenytoin decreased by 34%. In another study of 111 patients, phenytoin clearance increased gradually over the first 32 weeks of pregnancy and reached twice the preconception value. In two studies with phenobarbital, levels tended to decrease, although this effect was less pronounced than for phenytoin. Similarly for primidone, pregnancy had little effect on steady-state levels; however, levels of phenobarbital formed from primidone exhibited large decreases during pregnancy followed by increases after delivery. This effect was quite consistent. Carbamazepine clearance tended to increase to a relatively small extent. Limited data indicate that valproate levels decrease by 30 to 40% during pregnancy. The mechanisms responsible for these effects have not been elucidated and possibly include decreased bioavailability or compliance, increased metabolic clearance, or decreased plasma protein binding. Since the patient at risk of an increase in seizure frequency cannot be identified prior to conception, therapeutic monitoring is imperative during and after pregnancy.