Topically applied novel TRPV1 receptor antagonist, ACD440 Gel, reduces evoked pain in healthy volunteers, a randomized, double-blind, placebo-controlled, crossover study.

BACKGROUND: The TRPV1 receptor is a key molecule in pain generation. Previous development of oral TRPV1-antagonists was halted due to systemic heat insensitivity and body temperature alterations. The present Phase 1b study investigated the efficacy, safety and plasma exposure of a topically administered TRPV1-antagonist (ACD440 Gel) in healthy subjects. METHODS: The study comprised two parts. In part 1, 24 healthy subjects were included in this randomized double-blind, placebo-controlled, crossover trial. ACD440 Gel or Placebo was applied once daily and wiped off after 1 h, for 5 consecutive days. Assessments were done in normal skin, skin optimized for penetration (by stripping and occlusive gel application) and UVB-irradiated skin. Pain induced by thermo-nociceptive CO2 laser impulses generated laser-evoked potentials (LEPs), with readouts of peak-to-peak (PtP) amplitude in vertex-EEG and pain assessments by VAS (0-100). Endpoints include effects at 1 hour post-dose, AUC(Days 1-5) and AUC(0-24, Day 4). In UVB-irradiated skin, also pain on pinprick and skin redness were assessed. Part 2 explored the plasma pharmacokinetics of ACD440. RESULTS: ACD440 Gel reduced LEP PtP amplitude and VAS pain, p < 0.001, in all skin conditions, versus placebo. In UVB-irradiated skin, pinprick pain was also reduced, p = 0.047. Effects were significant after 1 h, maintaining for at least 9 h. There were no adverse events or drug-induced erythema. Plasma exposures of ACD440 were too low to establish an elimination half-life of ACD400. CONCLUSIONS: Topical ACD440 Gel demonstrated a significant analgesic effect on LEP, VAS score and pinprick pain, with low systemic exposures, supporting further clinical development. SIGNIFICANCE: This study demonstrates that the topical administration of a TRPV1-antagonist, ACD440 Gel, has potential as a new treatment for painful conditions affecting the skin, such as chronic peripheral neuropathic pain, without any local or systemic side effects.

Safety, Tolerability, Pharmacokinetics and Quantitative Electroencephalography Assessment of ACD856, a Novel Positive Allosteric Modulator of Trk-Receptors Following Multiple Doses in Healthy Subjects.

BACKGROUND: ACD856 is a positive allosteric modulator of tropomyosin receptor kinase (Trk) receptors which has shown to have pro-cognitive and anti-depressant-like effects in various animal models. It is currently in clinical development for the treatment of Alzheimer's disease and other disorders where cognition is impaired and is also considered for indications such as depression or other neuropsychiatric diseases. ACD856 has a novel mechanism of action modulating the activity of the Trk-receptors, resulting in increased stimulation of the neurotrophin signaling pathways. Previous studies applying single intravenous and oral doses of ACD856 indicate that ACD856 is safe and well-tolerated by healthy volunteer subjects, and that it has suitable safety and pharmacokinetic properties for further clinical development. OBJECTIVES: To investigate the safety and tolerability of 7 days of treatment with multiple ascending oral doses of ACD856 in healthy subjects, and to characterize its pharmacokinetic (PK) properties. In addition, pharmacodynamic effects of ACD856 using quantitative electroencephalography (qEEG) as an indicator for central target engagement were assessed. DESIGN: This was a prospective, phase I, double-blind, parallel-group, placebo-controlled, randomized study of the safety, tolerability, PK and pharmacodynamics of multiple ascending oral doses of ACD856 in healthy subjects. ACD856 or placebo were administered in 3 ascending dose cohorts of 8 subjects. Within each cohort, subjects were randomized to receive either ACD856 (n=6) or placebo (n=2). SETTING: The study was conducted at a First-in-Human unit in Sweden. PARTICIPANTS: Twenty-four healthy male and female subjects. INTERVENTION: The study medication was administered as an oral solution, with ACD856 or the same contents without the active ingredient (placebo). The dose levels ranged from 10 mg to 90 mg. ACD856 was administered once daily for 7 days, targeting steady state. MEASUREMENTS: Safety and tolerability assessments included adverse events, laboratory, vital signs, 12-lead electrocardiogram (ECG), physical examination, assessment of stool frequency and questionnaires to assess symptoms of anxiety, depression, as well as suicidal ideation and behavior. In addition, cardiodynamic ECGs were extracted to evaluate cardiac safety. PK parameters were calculated based on measured concentrations of ACD856 in plasma, urine, and cerebrospinal fluid (CSF) samples. Metabolite profiling, characterization and analysis was performed based on and urine samples. qEEG was recorded for patients in the two highest dose cohorts (30 and 90 mg/day) as a pharmacodynamic assessment to explore central target engagement. RESULTS: Treatment with ACD856 was well tolerated with no serious adverse events. No treatment emergent or dose related trends were observed for any of the safety assessments. ACD856 was rapidly absorbed and reached maximum plasma exposure at 30 to 45 minutes after administration. Steady state was reached before Day 6, with an elimination half-life at steady state of approximately 20 hours. At steady state, ACD856 exhibited accumulation ratios for Cmax and AUC of approximately 1.6 and 1.9 respectively. The exposure, Cmax and AUC0-24, increased proportionally with the dose. There was no unchanged ACD856 detected in urine. The metabolic pattern in urine and plasma was similar, and in alignment with the metabolites observed in preclinical toxicology studies. The level of ACD856 measured in CSF at steady state increased with dose, indicating Central Nervous System (CNS) exposure at relevant levels for pharmacodynamic effects. ACD856 demonstrated significant dose-dependent treatment-associated changes on qEEG parameters. Specifically, increase of the relative theta power and decrease of the fast alpha and beta power was observed, leading to an acceleration of the delta+theta centroid and an increase in the theta/beta ratio. CONCLUSIONS: ACD856 was well tolerated at the tested dose levels (10-90 mg/daily for 7 days) in healthy subjects. The compound has a robust pharmacokinetic profile, with rapid absorption and dose-dependent exposure. ACD856 was shown to pass the blood-brain-barrier, reach relevant exposure in the CNS and to induce dose-dependent treatment-related changes on qEEG parameters, indicating central target engagement.

Clinical testing of three novel transient receptor potential cation channel subfamily V member 1 antagonists in a pharmacodynamic intradermal capsaicin model.

BACKGROUND: The transient receptor potential cation channel subfamily V 1 (TRPV1) is involved in nociception and has thus been of interest for drug developers, as a target for novel analgesics. However, several oral TRPV1 antagonists have failed in development, and novel approaches to target TRPV1 with innovative chemistry are needed. METHOD: This work describes an intradermal microdosing approach in humans for pharmacodynamic deductions and pharmacological profiling of compounds. First, a human capsaicin model was developed, to generate pharmacodynamic translational data (Study Part A, n = 24). Then, three small molecule TRPV1 antagonists (AZ11760788, AZ12048189 and AZ12099548) were investigated in healthy volunteers (Study Part B, n = 36), applying the established model. Pain and flare were assessed by Visual Analogue Score and laser Doppler, respectively. RESULTS: The developed model proved useful for pharmacologic deductions; all compounds caused a dose-dependent inhibition of capsaicin-induced pain and flare responses, with a rank order potency of AZ11760788 > AZ12048189 â‰« AZ12099548. In addition, the dose-response data showed that the minimal antagonist concentrations needed to inhibit TRPV1 was ≥6-7 times the equilibrium dissociation constant for each compound. CONCLUSION: With careful design of a pharmacodynamic translational human pain model, it was possible to rank order TRPV1 efficacy among three investigational TRPV1 antagonists, and to estimate human efficacious concentrations. SIGNIFICANCE: This fast and cost-effective translational approach allows for generation of human target engagement information early in drug development. This could be of value for other development programmes where pharmacological targets are expressed in peripheral sensory nerves.

Preclinical characterization of three transient receptor potential vanilloid receptor 1 antagonists for early use in human intradermal microdose analgesic studies.

BACKGROUND: The transient receptor potential vanilloid receptor 1 (TRPV1) is a nonselective cation channel involved in the mediation of peripheral pain to the central nervous system. As such, the TRPV1 is an accessible molecular target that lends itself well to the understanding of nociceptive signalling. This study encompasses preclinical investigations of three molecules with the prospect to establish them as suitable analgesic model compounds in human intradermal pain relief studies. METHODS: The inhibitory effectiveness was evaluated by means of in vitro assays, TRPV1 expressing Chinese hamster ovary cells (CHO-K1) and rat dorsal root ganglion cultures in fluorescent imaging plate reader and whole cell patch clamp systems, as well as in vivo by capsaicin-evoked pain-related behavioural response studies in rat. Secondary pharmacology, pharmacokinetics and preclinical safety were also assessed. RESULTS: In vitro, all three compounds were effective at inhibiting capsaicin-activated TRPV1. The concentration producing 50% inhibition (IC50 ) determined was in the range of 3-32 nmol/L and 10-501 nmol/L using CHO-K1 and dorsal root ganglion cultures, respectively. In vivo, all compounds showed dose-dependent reduction in capsaicin-evoked pain-related behavioural responses in rat. None of the three compounds displayed any significant activity on any of the secondary targets tested. The compounds were also shown to be safe from a toxicological, drug metabolism and pharmacokinetic perspective, for usage in microgram doses in the human skin. CONCLUSION: The investigated model compounds displayed ideal compound characteristics as pharmacological and translational tools to address efficacy on the human native TRPV1 target in human skin in situ. SIGNIFICANCE: This work details the pharmaceutical work-up of three TRPV1-active investigational compounds, to obtain regulatory approval, for subsequent use in humans. This fast and cost-effective preclinical development path may impact research beyond the pain management area, as it allows human target engagement information gathering early in drug development.

The impact of the site of blood sampling on pharmacokinetic parameters following sublingual dosing to dogs.

INTRODUCTION: Drugs are most commonly administered orally, but some potential drug candidates are not suited for oral administration due to poor absorption, high first pass metabolism or gastrointestinal side effects. The interest for transmucosal dosing for systemic drug delivery is increasing, e.g. buccal, sublingual and nasal routes. The evaluation of the systemic plasma concentration and the derivation of the pharmacokinetic parameters of candidate compounds in preclinical studies are essential for drug development. The effect of site of blood sampling on the measured drug concentration, in both animals and humans, is to some extent known but it is not always taken into consideration in the design of pharmacological and toxicological studies. METHODS: Blood samples were collected both from leg and jugular veins from beagle dogs following a single sublingual dosing of Compound A in order to determine the impact of different sites of blood sampling on plasma pharmacokinetics. Plasma was prepared by centrifugation and plasma concentrations of Compound A were determined by protein precipitation and liquid chromatography followed by mass spectrometric detection. The pharmacokinetic parameters were calculated by non-compartment methods. RESULTS: Sampling from the jugular vein resulted in higher and more variable exposure during the absorption phase compared to sampling from a leg vein. The plasma exposure in the jugular vein, in terms of C(max), was 4-fold compared to that in the leg vein and an approximately 2-fold bioavailability was observed. DISCUSSION: The aim of this investigation was to determine the impact of the different sites of blood sampling on assessing systemic plasma exposure and pharmacokinetic parameters for Compound A following sublingual dosing to dogs. The results demonstrate the significant impact that the site of blood sampling has on PK parameters, and raise concerns of using the jugular vein as a site of sampling after sublingual and other transmucosal routes of dosing in the head region.

The metabolic syndrome: prevalence and association to leisure-time and work-related physical activity in 60-year-old men and women.

BACKGROUND AND AIM: This study examined the prevalence of the metabolic syndrome and its association to lifestyle factors in 60-year-old men and women, with special emphasis on physical activity (PA). METHODS AND RESULTS: Every third 60-year-old man and woman in the Stockholm County, Sweden, was invited to a survey of cardiovascular risk factors. Seventy-seven percent of the sample, 4228 individuals, agreed to participate (2036 men and 2192 women). Participants underwent physical examination and laboratory tests, and completed a questionnaire. After excluding 364 subjects suffering from cardiovascular disease and/or cancer, the prevalence of the metabolic syndrome was 24% and 19% in men and women, respectively. The adjusted odds ratio for having the metabolic syndrome in the high leisure-time PA group was 0.33 (95% confidence interval: 0.22-0.51) using the low leisure-time PA group as reference. However, no such inverse association was noted for work-related PA. CONCLUSIONS: This cross-sectional survey of 60-year-old men and women demonstrates a high prevalence of the metabolic syndrome. The robust inverse dose-response relationship between leisure-time PA and the metabolic syndrome emphasises the role of PA in the prevention and treatment of the metabolic syndrome.

A trial with N-carbamylglutamate may not detect all patients with NAGS deficiency and neonatal onset.

N-acetylglutamate synthase (NAGS) deficiency is a rare urea cycle disorder. An effective treatment, N-carbamoyl-L-glutamic acid (NCGA), is now available, increasing the importance of identifying and treating these patients early. We describe a case with genetically verified NAGS deficiency and neonatal onset of severe hyperammonaemia. The ammonia levels increased above 1400 micromol/L. The patient did not respond to NCGA treatment during the first 15 h, indicating that a delayed response or no response cannot be used as a safe indicator for excluding NAGS deficiency in the acute situation. Hence, conventional treatment should not be delayed by a diagnostic procedure, such as a loading test. Furthermore, at 3 years of age this patient has normal psychomotor development, underlining the possibility of a favourable outcome despite markedly elevated ammonia levels, coma, and seizures in the neonatal period. Including NCGA early in the treatment of patients with hyperammonaemia may be of clinical importance. In order to detect patients with NAGS deficiency and neonatal onset and to optimize care, it is important to use the available treatment strategies to reduce plasma ammonia concentrations without delay. We propose the use of combined symptomatic treatment, i.e. glucose infusion, sodium benzoate, arginine or citrulline, and when indicated haemodialysis, as well as NCGA treatment in all neonates presenting with severe hyperammonaemia. The treatment should be continued until laboratory investigations are complete or indicate another disorder.

Increased lipolysis in LCHAD deficiency.

An increasing number of fatty acid oxidation defects are being detected owing to diagnostic improvements and a greater awareness among clinicians. The metabolic block leads to energy disruption, fatty infiltration, and toxic effects on organ functions exerted by beta-oxidation metabolites. This investigation was undertaken to assess the influence of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency on lipolysis and energy turnover. We addressed the question whether the lipolysis and glucose production rates would be altered in the fasting state in a child with this disease. Lipolysis, glucose production and resting energy expenditure (REE) were studied in a 17-month-old girl with LCHAD deficiency and her healthy twin sister. Lipolysis and glucose production were determined after a 4-6 h fast by constant-rate infusion of [1,1,2,3,3-(2)H(5)]glycerol and [6,6-(2)H(2)]glucose and analysis by gas chromatography-mass spectrometry. REE was estimated by indirect calorimetry. The affected girl showed 50% higher lipolysis than did her sister, whereas the glucose production rates were similar. Plasma levels of dicarboxylic acids of 6-12 carbon atoms chain length, 3-hydroxy fatty acids of 6-18 carbon atoms chain length, total free fatty acids, and acylcarnitines were increased in the patient, as was REE. Since glucose production rates and plasma glucose levels were similar in the two girls, the increased lipolysis observed in the patient probably represents a compensatory mechanism for energy generation. This is achieved at the price of an augmented risk for fatty acid infiltration and toxic effects of beta-oxidation intermediates. This highlights the importance of avoiding fasting in these patients.

Insulin sensitivity and lipolysis in adolescent girls with poorly controlled type 1 diabetes: effect of anticholinergic treatment.

OBJECTIVES: Increased GH secretion could be one factor behind the impaired glycaemic control often seen in adolescent girls with type 1 diabetes. Because GH induces insulin resistance, treatment with anticholinergic agents, such as pirenzepine (PZP), has been used to reduce GH secretion. However, in a previous study of adolescent girls with type 1 diabetes, we observed an improvement in glycaemic control during 12 weeks of PZP therapy despite unchanged excretion of GH in urine. Considering the complex mechanisms behind urinary GH excretion, the effects of PZP on pituitary GH secretion or secretory pattern cannot be excluded. Thus, to assess the effect of anticholinergic treatment on metabolic control in adolescent girls with diabetes, we have investigated GH secretion, insulin sensitivity and lipolysis before and during treatment with PZP. PATIENTS: Eleven adolescent girls with type 1 diabetes and poor metabolic control were investigated before and after treatment with PZP, 100 mg orally, twice a day for 3 weeks. DESIGN: Serum samples for analysis of haemoglobin A1c and IGF-I were obtained in addition to serum profiles of GH, insulin and IGFBP-1 before and after 3 weeks of PZP treatment. Effects on insulin sensitivity and lipolysis were also assessed. MEASUREMENTS: IGFBP-1 was measured every hour, whereas serum GH and insulin were measured every 20 min for 24 h. Insulin sensitivity was analysed with the hyperinsulinaemic euglycaemic clamp technique. The rate of lipolysis was assessed under basal conditions following a constant rate infusion of [1,1,2,3,3-2H5]-glycerol. In five girls, lipolysis was also estimated during the hyperinsulinaemic euglycaemic clamp. RESULTS: There was a significant reduction in haemoglobin A1c levels (9.9 +/- 0.2%vs. 9.1 +/- 0.2; P < 0.0001) during 3 weeks of PZP treatment. In additional, the glucose requirement during the euglycaemic hyperinsulinaemic clamp increased by more than 30% (72.5 +/- 4.9 vs. 96.8 +/- 8.5 mg/m2/min; P = 0.003). However, we could not demonstrate any significant changes in GH secretion (area under the curve, basal levels or peak amplitude) or in the GH secretory pattern (peak height, peak length or interpeak interval). Concordantly, the IGF-I levels were statistically unchanged, as were IGFBP-1 concentrations. The rate of lipolysis did not change under basal conditions (3.40 +/- 0.53 vs. 3.04 +/- 0.54 micro mol/kg/min, n = 11, P = 0.54) or during the hyperinsulinaemic euglycaemic clamp (1.58 +/- 0.21 vs. 2.08 +/- 0.26 micro mol/kg/min; n = 5, P = 0.32). CONCLUSIONS: Our observations of an increased glucose requirement during the clamp as well as a decrease in haemoglobin A1c demonstrate improved insulin sensitivity in the adolescent girls with diabetes following pirenzepine therapy. The mechanism behind the improvement is not clear, as neither secretion nor the secretory pattern of GH changed significantly. The persistently high levels of GH might explain the unaltered rate of lipolysis despite the improved insulin sensitivity. The observed improvement in glycaemic control in adolescent girls with type 1 diabetes following pirenzepine therapy is promising, although more studies on this topic are needed.

Anticholinergic treatment improves glycaemic control in adolescent girls with insulin-dependent diabetes mellitus.

UNLABELLED: Metabolic control often deteriorates during puberty in girls with insulin-dependent diabetes. It is well accepted that there is an abnormality in the growth hormone (GH)-insulin-like growth factor-I (lGF-I) axis in these girls, resulting in reduced IGF-I levels and elevated GH. As GH antagonizes insulin, attempts have previously been made to reduce excess GH secretion through anticholinergic treatment. However, most of these studies have been performed on adult patients. The aim of the present study was to evaluate the effects of 12 wk of oral anticholinergic treatment with Pirenzepine, 100 mg twice daily, in 16 adolescent girls with diabetes. Serum samples of IGF-I, glycated haemoglobin and fasting IGF-binding protein 1 were analysed at initiation and after 3, 8 and 12 wk of Pirenzepine therapy. Nocturnal urinary GH excretion was also examined. Glycated haemoglobin declined significantly after 3 wk of Pirenzepine therapy (9.8 +/- 0.18 vs 9.2 +/- 0.17; p < 0.001) and was still improved at the end of the study. Unexpectedly, nocturnal urinary GH excretion did not change. Serum IGF-I continuously increased during the study, while IGF-binding protein 1 levels were not significantly altered. CONCLUSION: Anticholinergic treatment with Pirenzepine improves glycaemic control in adolescent girls with diabetes. Although nocturnal urinary GH excretion was unchanged there may still be changes in pituitary GH secretion to explain the improvement. Effects of Pirenzepine on gastrointestinal motility can represent other possible mechanisms behind the improved metabolic control.

Metabolism of sameridine to monocarboxylated products by hepatocytes isolated from the male rat.

The metabolism of sameridine (LPB) (an amide-type local anesthetic-analgesic agent with a hexyl side chain) to carboxylic acid derivatives by isolated male rat hepatocytes was studied using gradient reversed-phase HPLC and mass spectrometry. Incubation of sameridine with hepatocytes resulted in the formation of numerous different metabolites. Two carboxylic acids, i.e., the C(6) and C(4) carboxylated derivatives of sameridine (LPB-6'-oic acid and LPB-4'-oic acid), were found to be produced from the intermediate omega-hydroxy metabolite (6'-hydroxy-LPB). Shortening of the alkyl chain in LPB-6'-oic acid by two carbon atoms resulted in LPB-4'-oic acid. However, incubation of rat hepatocytes with 5'-hydroxy-LPB [the (omega-1)-hydroxy derivative of sameridine] did not give rise to any carboxylated derivative. Addition of SKF525A inhibited the metabolism of sameridine by rat hepatocytes, indicating that the initial step is catalyzed by cytochrome P450. Furthermore, the metabolism of sameridine to LPB-4'-oic acid was enhanced in hepatocytes isolated from rats treated with clofibrate, an up-regulator of peroxisomal fatty acid beta-oxidation and of microsomal cytochrome P450 4A. L-Carnitine (which increases the rate of mitochondrial fatty acid beta-oxidation) had no effect on the level of LPB-4'-oic acid produced by isolated rat hepatocytes. The metabolism of 6'-hydroxy-LPB to LPB-6'-oic acid was inhibited almost completely by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase. Considered together, our findings suggest that cytochrome P450 4A, cytosolic dehydrogenases, and the enzymes involved in peroxisomal fatty acid beta-oxidation catalyze the metabolism of sameridine to LPB-4'-oic acid.

Profound changes in the GH-IGF-I system in adolescent girls with IDDM: can IGFBP1 be used to reflect overall glucose regulation?

Disturbances in the relations between insulin, growth hormone (GH) and insulin-like growth factor I (IGF-I) may be a major cause behind deteriorated metabolic control in adolescent girls with type I diabetes. These patients have increased GH secretion and low IGF-I concentrations. The aim of this study was to identify possible endocrine mechanisms behind good and poor glycaemic control in such girls, focusing on the insulin-GH-IGF-I axis. Ten girls with well-controlled insulin-dependent diabetes mellitus (IDDM), hemoglobin A1c (HbA1c) 6.5+/-0.4% (normal range 3.9-5.2%) and nine healthy controls were investigated and compared with 11 girls with poor glucose regulation, HbA1c 10.9+/-0.4%, and their corresponding controls. Serum profiles of glucose, insulin, GH and IGF-binding protein 1 (IGFBP1) were analysed in addition to IGF-I and HbA1c. Two interesting observations were made. GH concentrations were equally elevated in the two diabetic groups regardless of metabolic control (mean 24 h GH - girls with poorly controlled diabetes 10.0+/-1.0 mU/L vs 9.8+/-1.7 - girls with well-controlled diabetes; p=ns). Likewise, the IGF-I concentrations were reduced to the same extent (233+/-19 vs 242+/-23 microg/L; p=0.75). Secondly, despite similar insulin concentrations (mean 24 h insulin - girls with poorly controlled diabetes 22.9+/-2.6 and girls with well-controlled diabetes 27.3+/-2.9 mU/L, respectively; p=0.26), there was a marked difference in IGFBP1 concentrations between the two groups with IDDM (mean IGFBP1 - girls with poorly controlled diabetes 70.5+/-9.1 microg/L vs girls with well-controlled diabetes 28.6+/-3.3; p<0.001). Despite equally elevated GH concentrations that may induce insulin resistance, the markedly lower concentrations of IGFBP1 in the well-controlled group indicate a higher hepatic insulin sensitivity in these girls compared with those with a poor control. Furthermore, in spite of similar total IGF-I concentrations, the lower IGFBP1 concentrations may result in higher IGF-I bioactivity in the well-controlled group. This may be reflected in better growth of the well-controlled group whose height of 168.7+/-0.9 vs 163.6+/-1.2 cm was significantly different (p<0.004). IGFBP1 may be a marker of overall insulinization in adolescents with type 1 diabetes, independent of the absolute insulin dose used for therapy.

Liquid chromatographic bioanalytical determination of ropivacaine, bupivacaine and major metabolites.

Bioanalytical methods for the determination of ropivacaine, bupivacaine and their major metabolites in urine and blood plasma are presented. Ropivacaine is a new local anaesthetic drug mainly used for surgery and for postoperative pain relief. The samples are hydrolysed and cleaned using solid-phase extraction and analysed using ion-pair reversed-phase liquid chromatography with gradient elution. The analytes are detected using UV at 210 nm. The methods are highly selective and the limits of quantification were 1 microM in urine and 0.1 microM in plasma, respectively. The between-day variance was generally below 3% (RSD).

Is growth hormone hypersecretion in diabetic adolescent girls also a daytime problem?

OBJECTIVES: Glycaemic control often deteriorates during puberty in girls with insulin dependent diabetes mellitus (IDDM). This may be due in part to the normal psychosocial changes associated with adolescence. Puberty is, however, also characterized by rapid somatic development, orchestrated by hormonal changes. Some of these hormones play a major role in glucose homeostasis. We have examined the insulin-GH-IGF-I axis in 11 adolescent girls with poorly controlled insulin dependent diabetes and compared the data with those of 10 non-diabetic girls matched for age, pubertal stage and body mass index (BMI). METHODS: Serum profiles of glucose, insulin, GH and IGF binding protein 1 (IGFBP1) were analysed in addition to IGF-I in serum and nocturnal urinary excretion of GH. MEASUREMENTS: Serum glucose, insulin and IGFBP1 were measured every hour for 24 h, whereas GH in serum was measured every 30 minutes during the same period. Nocturnal urinary GH was analysed as a mean of three consecutive nights. RESULTS: The insulin profiles of the IDDM patients were flat with low post-prandial peaks, corresponding to only one-third of the peaks of the non-diabetic girls. The integrated insulin levels, both during 24-h sampling and during daytime, were significantly lower in the diabetic group. There were no differences during night-time. The diabetic patients had elevated mean baseline levels of serum GH (IDDM 2.8 +/- 0.5 mU/l, controls 0.7 +/- 0.2; P < 0.001), a higher 24-h mean serum GH level (9.8 +/- 1.7 mU/l vs. 4.4 +/- 0.7; P < 0.001), significantly more peaks and a urinary GH excretion twice as high as in the non-diabetic group. An interesting observation was the finding of marked differences in daytime GH concentrations between the groups, both regarding overall integrated levels (GH AUC 103 +/- 15.8 and 35.9 +/- 7.1 mU/l x 12 h, respectively; P < 0.005) as well as baseline levels (3.8 +/- 0.6 mU/l vs. 0.7 +/- 0.2; P < 0.001). In contrast, during night-time only the mean basal levels of GH differed. The level of IGF-I was reduced in the diabetic group compared with the healthy controls (IDDM 233 +/- 19 micrograms/l vs. controls 327 +/- 21; P < 0.005). In addition, the IDDM patients had significantly increased concentrations of IGFBP 1, but kept a normal diurnal rhythm with a pronounced night peak. CONCLUSION: Hypoinsulinaemia in adolescent IDDM patients, particularly in the portal hepatic circulation, results in decreased IGF-I and increased IGFBP 1 production in the liver. High levels of IGFBP 1 may, in turn, reduce the bioactivity of IGF-I even further. Low levels of IGF-I will lead to increased GH secretion. Earlier studies on the relationship between GH and diabetic control have focused on elevated GH levels during the night. In this study we have observed markedly elevated levels of GH also during daytime in adolescent IDDM patients. This indicates increased insulin resistance and insulin demand also during the day in diabetic subjects. The increased insulin resistance may result in hyperglycaemia leading to additional insulin resistance. A vicious circle may thus be induced, accelerating metabolic impairment in poorly controlled adolescent IDDM girls.

Comparative local anesthetic efficacy and pharmacokinetics of epidurally administered ropivacaine and bupivacaine in the sheep.

BACKGROUND AND OBJECTIVES: Ropivacaine is the S(-) propyl homolog of bupivacaine and mepivacaine. Studies in humans have confirmed the results of studies in laboratory animals that ropivacaine is a long-acting local anesthetic with an anesthetic profile similar to bupivacaine. Acute, intravenous systemic toxicity studies have been conducted in sheep and dogs. Local anesthetic efficacy has been studied after epidural administration in the dog. This study was initiated to determine the local anesthetic efficacy and pharmacokinetics of ropivacaine and bupivacaine after epidural administration in an experimental sheep model and to evaluate the sheep model as a model of experimental epidural anesthesia. METHODS: Twelve adult nonpregnant ewes were prepared with chronically implanted lumbar epidural catheters and arterial lines. Each sheep received injections of 5.0 mL ropivacaine and bupivacaine (0.5% and 0.75%) in a blinded, random, cross-over fashion. Onset and duration of sensory and motor blockade were evaluated. Arterial blood samples were drawn for serum drug concentration determinations and pharmacokinetic analysis. RESULTS: Onset and duration of motor blockade were similar for comparable concentrations of both drugs. Both concentrations of ropivacaine and bupivacaine 0.5% demonstrated differential neural blockade. The peak serum concentrations generally occurred within 8 minutes after administration. The terminal elimination half-life in serum was about 3.5-4.0 hours and 6 hours for ropivacaine and bupivacaine, respectively. No signs of systemic toxicity were observed. Results of sensory and motor blockade were consistent with previous studies in laboratory animals and humans. CONCLUSIONS: Ropivacaine produces sensory and motor blockade which is similar to that produced by equal concentrations of bupivacaine after epidural administration in the sheep. Peak serum concentrations produced no signs of systemic toxicity. The results of this study are consistent with previously published data from studies in laboratory animals and humans. The sheep model of experimental epidural anesthesia appears to be a clinically relevant method to evaluate experimental local anesthetics.

Toxicity of bupivacaine and ropivacaine in relation to free plasma concentrations in pregnant rats: a comparative study.

The relationship between free drug concentration and toxicity of bupivacaine and ropivacaine, a new local anaesthetic agent, was studied in a pregnant rat model. The compounds were given subcutaneously to rats in late pregnancy. Dose levels (bupivacaine 5.5 to 24 mg/kg and ropivacaine 5.3 to 26 mg/kg) were selected based upon the proposed human dosage and the known pharmacological activity of the compounds. Chewing, spasm, dyspnoea, drowsiness, salivation and convulsions were observed in a dose-dependent manner in the animals given 14 to 24 mg/kg of bupivacaine, while only a few animals receiving 26 mg/kg of ropivacaine showed less severe symptoms. Deaths from clonic convulsions were occasionally seen in animals receiving 14 mg/kg or more of bupivacaine. No animals receiving ropivacaine died. No effects on litter size offspring loss or weight of the offspring at birth were observed with one exception. After 24 mg/kg of bupivacaine an increased postnatal loss of the offsprings were noticed, most likely due to impaired maternal care. Protein binding, at expected Cmax, were significantly lower for ropivacaine (around 49%) compared with bupivacaine (around 69%) at dose levels. The results suggest an increased safety margin before onset of toxic side effects after treatment with ropivacaine, compared to bupivacaine, in pregnant rase.

A GC-MS study of three major acidic metabolites of delta 1-tetrahydrocannabinol.

The major urinary metabolite of delta 1-tetrahydrocannabinol (delta 1-THC) (1), delta 1-THC-7-oic acid (2), has been extensively studied for several purposes, including testing in the workplace for drug abuse. Immunoassays in combination with more specific methods such as gas chromatography-mass spectrometry (GC-MS), are commonly used for verification of positive results in the screening. Two additional and recently synthesized acidic metabolites of 1, 4",5"-bisnor-delta 1-THC-7,3"-dioic acid (3) and 4"-hydroxy-delta 1-THC-7-oic acid (4), were studied to widen the scientific basis in the analysis. Five different derivatives were examined using GC-MS. In addition, a new deuterated internal standard for 2, [2H10]-2, was evaluated. According to our results, suitable derivatives of 2, 3, and 4, according to chromatographic properties, are the methyl ester/silyl ether (procedure a), the methyl ester/trifluoroacetate (procedure b), or the silyl ester/silyl ether (procedure c). The estimated recoveries of [2H5]-3 and [2H6]-4 using liquid-liquid extraction were 24% and 50%, respectively. The properties of [2H10]-2 as internal standard were equivalent to those of [2H9]-2 and, under the conditions used, did not appear to give rise to a significantly higher chromatographic resolution from that of 2. However, [2H10]-2 produces ions at different mass numbers, which makes it useful as a complement to the existing deuterated internal standards of 2.

Metabolism and excretion of ropivacaine in humans.

The pharmacokinetics, biotransformation, and urinary excretion of ropivacaine (Naropin), a new local anesthetic agent, have been studied in six healthy male volunteers after a 15-min iv infusion of 152 mumol (50 mg) of [14C]ropivacaine, with a specific radioactivity of 22.5 kBq/mumol (8.8 kBq/mg). Blood, urine, and feces were collected for up to 96 hr after administration. The plasma and urine samples were analyzed for unchanged ropivacaine and for four of its metabolites, i.e. 3-OH-2',6'-pipecoloxylidide (3-OH-PPX), 4-OH-ropivacaine, 3-OH-ropivacaine, and the N-dealkylated metabolite PPX, using GC and HPLC methods. The presence of 2,6-xylidine in plasma was also analyzed. The metabolites were quantified after acidic hydrolysis. The radioactivity could be followed in plasma for up to 14 hr after administration, with ropivacaine being the predominant compound in the early samples. The concentrations of the aforementioned metabolites in plasma were below or just above the lower limit of quantification, and no 2,6-xylidine was detected. The maximum plasma concentration of ropivacaine was 5.9 +/- 2.6 microM (1.6 +/- 0.7 mg/liter), with an elimination half-life of 2.0 +/- 0.3 hr and a total plasma clearance of 397 +/- 127 ml/min. The maximum plasma concentration value for the total radioactivity was 5.5 +/- 2.4 microM (1.5 +/- 0.7 mg/liter) and the elimination half-life was 5.4 +/- 2.9 hr. [14C]Ropivacaine and its metabolites were mainly excreted in the urine, with a total recovery of 86 +/- 3% in the urine and 9 +/- 1% in the feces after 96 hr. Most of the radioactivity (about 68%) was excreted within 12 hr. Ropivacaine was extensively metabolized, and only 1 +/- 0.6% of the dose was excreted unchanged in the urine. The major metabolite identified in the urine was conjugated 3-OH-ropivacaine, which was excreted to an extent of 37 +/- 3% of the dose. The urinary excretion of 4-OH-ropivacaine was < 1%, whereas the N-dealkylated metabolites PPX and 3-OH-PPX accounted for 2 and 3% of the dose, respectively. An additional hydroxylated metabolite, 2-OH-methyl-ropivacaine, was tentatively identified in the urine of some volunteers, accounting for about 4-15% of the dose.

A urinary metabolite of delta 1-tetrahydrocannabinol. The first synthesis of 4",5"-bisnor-delta 1-tetrahydrocannabinol-7,3"-dioic acid, and a deuterium labelled analogue.

The first synthesis of unlabelled and [2H5]-labelled 4",5"-bisnor-delta 1-THC-7,3"-dioic acid, the major dicarboxylated urinary metabolite of delta 1-THC in man, is presented (preliminary results of this work have been presented in part at the Melbourne Symposium on Cannabis, Australia, September 1987, Ref. 1). The synthesis of methyl 3-(3,5-dihydroxyphenyl)-[3,3-2H2]-propanoate (8) is described in a nine step sequence from 3,5-dimethoxybenzoic acid in an overall yield of 24%. Compound 8 is condensed with a terpene synthon 9 under acidic conditions, acetylated and hydrolyzed with red HgO and HgCl2 to afford the 1-formyl-4",5",7-trisnor-delta 1-THC-3"-oic acid derivative (11). Compound 11 is oxidized using NaClO2 in 2-methyl-2-butene and hydrolyzed to give (+/-)-4",5"-bisnor-delta 1-THC-7,3"-dioic acid (12). The same approach has been used to prepare both the labelled and unlabelled metabolite.