Hartmud Neurath

Pegylated granulocyte colony-stimulating factor conveys long-term neuroprotection and improves functional outcome in a model of Parkinson’s disease

Recent proof-of-principle data showed that the haematopoietic growth factor granulocyte colony-stimulating factor (filgrastim) mediates neuroprotection in rodent models of Parkinsons disease. In preparation for future clinical trials, we performed a preclinical characterization of a pegylated derivative of granulocyte colony-stimulating factor (pegfilgrastim) in the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinsons disease. We determined serum and cerebrospinal fluid drug levels after subcutaneous injection. A single injection of pegfilgrastim was shown to achieve stable levels of granulocyte colony-stimulating factor in both serum and cerebrospinal fluid with substantially higher levels compared to repetitive filgrastim injections. Leucocyte blood counts were only transiently increased after repeated injections. We demonstrated substantial dose-dependent long-term neuroprotection by pegfilgrastim in both young and aged mice, using bodyweight-adjusted doses that are applicable in clinical settings. Importantly, we found evidence for the functionally relevant preservation of nigrostriatal projections by pegfilgrastim in our model of Parkinsons disease, which resulted in improved motor performance. The more stable levels of pegylated neuroprotective proteins in serum and cerebrospinal fluid may represent a general advantage in the treatment of chronic neurodegenerative diseases and the resulting longer injection intervals are likely to improve patient compliance. In summary, we found that pegylation of a neuroprotective growth factor improved its pharmacokinetic profile over its non-modified counterpart in an in vivo model of Parkinsons disease. As the clinical safety profile of pegfilgrastim is already established, these data suggest that evaluation of pegfilgrastim in further Parkinsons disease models and ultimately clinical feasibility studies are warranted.

NICOTINE METABOLISM IN ISOLATED PERFUSED LUNG AND LIVER OF PHENOBARBITAL- AND BENZOFLAVONE-TREATED RATS

The kinetics of nicotine elimination was investigated in isolated perfused lung and liver of phenobarbital (PB)- and 5,6-benzoflavone (BF)-pretreated rats. The estimated kinetic parameters demonstrated a high nicotine elimination rate in rat lung approaching the capacity of liver when both organs were in an uninduced state. The concentration-time profiles of cotinine as the main metabolite were almost identical for isolated lung and liver. In both organs the cotinine plasma concentrations reached a plateau level after 60 min of perfusion. Pretreatment of rats with 5,6-benzoflavone did not affect the rate of nicotine elimination and cotinine formation either in the lung or in the liver. Phenobarbital treatment, however, induced nicotine clearance in lung approximately 2-fold. This effect is quantitatively lower than the PB-related 8-fold induction of hepatic nicotine elimination observed in a previous study. The present results also indicate that the turnover of cotinine is markedly enhanced after PB induction. The elimination half-lives and clearance values for cotinine as the substrate were approximately 10-fold increased in rat liver after PB pretreatment. Thus, an important contribution of extrahepatic tissues to nicotine metabolism in rats has to be assumed. Moreover, since cotinine elimination is significantly increased after PB induction it is questionable whether cotinine plasma concentrations can further be used as suitable parameter for nicotine consumption.

Sensitive determination of piritramide in human plasma by gas chromatography

A selective and sensitive method for the determination of piritramide in human plasma is described. A 1-ml aliquot of plasma was extracted with 10 ml of hexane-isoamyl alcohol (99.5:0.5, v/v) (extraction efficiency 86%) after addition of 50 microliters of 2 M ammonia and 20 microliters of aqueous strychnine solution (100 ng per 10 microliters) as internal standard. Gas chromatography was performed with J&W DB-1, 30 m x 0.53 mm I.D. separation column, film thickness 1.5 microns, using an nitrogen-phosphorus-sensitive detector. The assay was linear in the concentration range 3.75-2250 ng/ml (r = 0.999), with a lower limit of detection of 1-2 ng/ml. The precision was determined using spiked plasma samples (10 and 50 ng/ml), with coefficients of variation of 3.5 and 3.1% (intra-day; n = 5) and 4.6 and 4.1% (inter-day; n = 4). In the range 3.75-150 ng/ml, the accuracy of the assay was 3.36%. The method was used for the determination of piritramide plasma concentrations in patients receiving intra- or post-operative analgesia.

High-performance liquid chromatographic method for simultaneous determination of [1-methyl-14C]caffeine and its eight major metabolites in rat urine.

A selective and sensitive reversed-phase liquid chromatographic method was developed for the simultaneous analysis of [1-Me-14C]caffeine and its eight major radiolabelled metabolites in rat urine. The separation of the complex mixture of caffeine metabolites was achieved by gradient elution with a dual solvent system using an endcapped C18 reversed-phase column, which in contrast to commonly used C18 reversed-phase columns also allows the separation of the two isomers of 6-amino-5-(N-formylmethylamino)-1,3-dimethyluracil (1,3,7-DAU), a caffeine metabolite of quantitative importance predominantly occurring in rat. As caffeine is metabolised primarily by members of the cytochrome P450 1A (CYP1A) subfamiliy, determination of the pattern of caffeine metabolites in rat urine enables analysis of activities of this important enzyme subfamily in vivo. Since CYP1A is suggested to be involved in the detoxification of bilirubin, the assay may be applied to search for untoxic inducers of CYP1A which might be of pharmacological interest in the treatment of hyperbilirubinaemia.

Acetonitrile serum concentrations and cyanide blood levels in a case of suicidal oral acetonitrile ingestion

Acute acetonitrile toxicity is mainly dependent on the release of cyanide via hepatic metabolism. Although evaluated in animals, few data are available concerning the toxicokinetic parameters of acetonitrile and acetonitrile-liberated cyanide in human. This paper reports a case of suicidal oral acetonitrile ingestion of about 5 mL without severe symptoms of intoxication in a previously healthy adult male with a body weight of 60 kg. Acetonitrile serum concentrations as well as cyanide blood levels were determined over the whole hospitalization. The elimination half-lives calculated from these data were 32 h for acetonitrile and 15 h for cyanide. After sodium thiosulfate bolus application, the cyanide blood level rapidly decreased to 10% of the initial value, indicating that sodium thiosulfate sufficiently detoxifies acetonitrile-liberated cyanide. Since cyanide levels again increased to maximal values about 4.5 h after sodium thiosulfate application, continued thiosulfate therapy is required as predicted by the long elimination half-lives of acetonitrile and acetonitrile-liberated cyanide. Determination of cyanide and acetonitrile concentrations is recommended for the estimation of optimal individual sodium thiosulfate dosage.

Blood-brain barrier opening with alkylglycerols: Biodistribution of 1-O-pentylglycerol after intravenous and intracarotid administration in rats

Short-chain alkylglycerols have been described to increase the penetration of drugs and macromolecules across the blood–brain barrier (BBB) into the central nervous system (CNS) and were considered to be of potential value in the pharmaceutical treatment of CNS disorders. Due to the lack of information on the pharmacological behavior of these compounds in vivo, pharmacokinetics and biodistribution of [14C]- and [3H]-labeled 1-O-pentylglycerol (49 mg/kg, 100 mM) was investigated in normal male Wistar rats after intravenous as well as intracarotid administration. There was a rapid and predominant renal elimination of 1-O-pentylglycerol and more than 70% of administered dose was found in the urine within 270 min. Analysis of the pharmacokinetic parameters after a single i.v. bolus injection of 1-O-pentylglycerol resulted in a peak blood concentration of 0.58±0.06 μmol/ml, an initial half life of 23±7 min and a terminal half life of 18.8±4.1 h. No accumulation of 1-O-pentylglycerol was observed in the brain or other organs while highest concentrations were found in liver and thymus. This was confirmed by autoradiographic studies. Five minutes after intracarotid administration, high radioactivity was found in the ipsilateral brain, whereas after 30 min radioactivity in the brain has dramatically decreased. Autoradiographic images gave evidence of biliary excretion in addition to the renal elimination. There were no signs of cleavage of the O-alkyl bond in vivo as demonstrated by HPLC analysis. In conclusion, 1-O-pentylglycerol is characterized by pharmacological properties appearing very favorable for in vivo use as a permeabilizing drug for increased drug delivery to the brain.

Acute side effects after consumption of the new synthetic cannabinoids AB-CHMINACA and MDMB-CHMICA

Abstract Introduction: In 2014, the “European Monitoring Centre for Drugs and Drug Addiction” (EMCDDA) reported on 30 novel synthetic cannabinoids (SCs). Among these were indole- and indazole-based valine derivatives with a cyclohexylmethyl side chain (e.g., AB-CHMINACA and MDMB-CHMICA), which represent a new class of SCs. Methods: A prospective observational study of patients treated in emergency departments (EDs) after the intake of SCs was conducted. Clinical and laboratory data were combined and reported to a poison control centre. Serum and/or urine samples of ED patients were analyzed using LC–MS/MS. Results: Forty four patients (39 male, five female, 12–48 years) were included. AB-CHMINACA (MDMB-CHMICA) was identified in 20 (19) serum samples, and in 21 (25) urine samples, respectively. In 19 of the cases, more than one SC was present. Other psychoactive substances (mainly amfetamines) were identified in seven cases, but in five out of these in urine samples only. Based on the Poison Severity Score, severity of poisoning was minor (4), moderate (31) or severe (9). Most frequently reported neuropsychiatric symptoms were CNS-depression (n = 21, 61%), disorientation (n = 20, 45%), generalized seizures (n = 12, 27%), combativeness (n = 8, 18%) and extreme agitation (n = 7, 16%). Duration of symptoms lasting 24 hours or longer occurred in 15 cases (34%). Discussion: The prevalence of certain neuropsychiatric symptoms was higher in our study than in former reports after the intake of SCs of the aminoalkylindole-type (first generation) SCs. In addition, severe poisoning and duration of symptoms were also higher. Conclusions: In this study, the valine derivative AB-CHMINACA and the tert-leucine derivative MDMB-CHMICA (“third generation of SCs”) seem to be associated with more severe clinical toxicity than was previously reported in patients exposed to earlier generation SCs such as JWH-018. However, this observation needs to be confirmed with a larger cohort of patients with analytically confirmed abuse of third generation SCs. The rapid turnover of SCs on the drug market together with the occurrence of SCs such as AB-CHMINACA and MDMB-CHMICA is alarming, especially because of the unexpectedly high frequency of neuropsychiatric symptoms.

The effects of kratom on restraint–stress-induced analgesia and its mechanisms of action

ETHNOPHARMACOLOGICAL RELEVANCE Mitragyna speciosa and its extracts are called kratom (dried leaves, extract). They contain several alkaloids with an affinity for different opioid receptors. They are used in traditional medicine for the treatment of different diseases, as a substitute by opiate addicts, and to mitigate opioid withdrawal symptoms. Apart from their medical properties, they are used to enhance physical endurance and as a means of overcoming stress. PURPOSE The aim of this study was to determine the mechanisms underlying the effects of kratom on restraint-stress-induced analgesia which occurs during or following exposure to a stressful or fearful stimulus. METHODS To gain further insights into the action of kratom on stress, we conducted experiments using restraint stress as a test system and stress-induced analgesia as a test parameter. Using transgenic mu opioid-receptor (MOR) deficient mice, we studied the involvement of this receptor type. We used nor-binaltorphimine (BNT), an antagonist at kappa opioid receptors (KOR), to study functions of this type of receptor. Membrane potential assay was also employed to measure the intrinsic activity of kratom in comparison to U50,488, a highly selective kappa agonist. RESULTS Treatment with kratom diminished stress-induced analgesia in wildtype and MOR knockout animals. Pretreatment of MOR deficient mice with BNT resulted in similar effects. In comparison to U50,488, kratom exhibited negligible intrinsic activity at KOR alone. CONCLUSIONS The results suggest that the use of kratom as a pharmacological tool to mitigate withdrawal symptoms is related to its action on KOR.

Extrahepatic Compared with Hepatic Metabolism of Nicotine in the Rat

Primary oxidation of nicotine exhibits similar rates in isolated rat lung and liver. Secondary metabolism, however, is restricted to the liver as demonstrated by the elimination parameters of 14C-cotinine in isolated lung and liver. Phenobarbital (PB) induction markedly enhances the elimination of cotinine only in liver and not in lung.