The effect of intradermal microdosing of a transient receptor potential cation channel subfamily V member 1 antagonist on heat evoked pain and thermal thresholds in normal and ultraviolet-C exposed skin in healthy volunteers.

BACKGROUND: Three TRPV1 (Transient Receptor Potential Vanilloid Receptor 1) antagonists were developed for testing in situ in human skin (Sjögren et al., 2016; Sjögren et al., 2018; Sjögren et al., 2018). The first human study using these compounds and capsaicin, was performed to determine the required local antagonist concentrations needed for target engagement (Proof of Mechanism, PoM) (Sjögren et al., 2018). In this paper, the aim was to address a TRPV1 antagonist's ability to inhibit a more complex pain signal and to define translational endpoints that could be used in further drug development, when progressing orally bioavailable TRPV1 antagonists as novel analgesic medications. METHOD: This was a single centre, placebo-controlled, clinical proof of principle (PoP) study in 25 healthy volunteers. The subjects were exposed to UV irradiation, causing a local tissue inflammation. Three different doses of AZ12048189 were administered to assess pain perception through quantitative sensory testing (QST) and erythema using Laser Doppler scanning. RESULTS: AZ12048189 increased the warmth detection threshold (WDT) and the heat pain threshold (HPT) and decreased the intensity of supra threshold heat pain (STHP). AZ12048189 did not, however, have any significant effects as assessed using mechanical stimulation or Laser Doppler. CONCLUSIONS: This study validated translational tools to confirm target engagement for TRPV1 antagonists; WDT, HPT and STHP have utility in this respect, after oral administration of a TRPV1 antagonist. This study also proved that TRPV1 antagonists can inhibit a more complex, non-capsaicin dependent thermally induced pain signal.

Comparing sensibility for touch, cold, and warmth in different skin areas.

The primary objective of this pilot study was to assess if the magnitude estimation of suprathreshold brushing, warmth (40 °C), and cold (25 °C) stimuli of the skin over the dorsum of the hand and the dorsum of the foot are comparable to the perceived intensity for the same stimuli applied to the skin over any of the following areas: forehead, m. trapezius, m. deltoideus, thoracic back, and lumbar back, respectively. Thirty-two subjects aged 18-64 years were included. Participants were examined by two physicians on two different occasions, 1-58 days apart. Participants rated the magnitude of the perceived sensation of each stimulus on an 11-point numerical rating scale (NRS) 0-10, where 0 was anchored to "no sensation at all for touch/cold/warmth" and 10 anchored to "the most intense imaginable non-painful sensation of touch/cold/warmth". The criterion for sensory equivalence for one modality was arbitrarily considered satisfactory if two regions had the same numerical rating ±1 point in at least 85% of the individuals. Based on the pre-study criteria for sensory equivalence applied in this study only one area was found to be equivalent to the foot skin for the percept of brushing, that is, the skin over the deltoid muscle and one area for the hand, that is, the skin over the forehead. We failed to find any area with equivalent sensitivity to the hand or the foot for the cold or warm stimuli.

Ganciclovir concentrations in the cerebral extracellular space after valganciclovir treatment; a case study.

Nearly all glioblastomas (GBMs), brain tumours with very poor prognosis, are infected with human cytomegalovirus (CMV). The anti-CMV drug valganciclovir (VGCV) has shown promise as a treatment option for patients with GBM, but its penetration into the central nervous system (CNS) is unknown. Here we describe a patient with GMB receiving VGCV in whom an intracerebral microdialysis catheter was implanted and ganciclovir (GCV) concentrations in brain extracellular fluid (BECF) and serum were monitored. GCV was rapidly absorbed. Cmax values (at 3 h) in serum and BECF were 19.6 and 10.2 µmol/L, T½ values were 3.2 and 4.5 h, and plasma and BECF AUC0-∞ values were 90.7 and 75.9 µmol h/L, respectively. Thus, VGCV treatment results in significant intracerebral levels of GCV that may be sufficient for therapeutic effects. Further studies of this drug in patients with GBM are warranted.

Towards Development of a Dermal Pain Model: In Vitro Activation of Rat and Human Transient Receptor Potential Ankyrin Repeat 1 and Safe Dermal Injection of o-Chlorobenzylidene Malononitrile to Rat.

During clinical development of analgesics, it is important to have access to pharmacologically specific human pain models. o-Chlorobenzylidene malononitrile (CS) is a selective and potent agonist of the transient receptor potential ankyrin repeat 1 (TRPA1), which is a transducer molecule in nociceptors sensing reactive chemical species. While CS has been subject to extensive toxicological investigations in animals and human beings, its effects on intradermal or subcutaneous injection have not previously been reported. We have investigated the potential of CS to be used as an agonist on TRPA1 in human experimental pain studies. A calcium influx assay was used to confirm the capacity of CS to activate TRPA1 with >100,000 times the selectivity over the transient receptor potential vanilloid receptor 1. CS dose-dependently (EC50 0.9 µM) released calcitonin gene-related peptide in rat dorsal root ganglion cultures, supporting involvement in pain signalling. In a local tolerance study, injection of a single intradermal dose of 20 mM CS to rats resulted in superficial, circular crusts at the injection sites after approximately 4 days. The histopathology evaluation revealed a mild, acute inflammatory reaction in the epidermis and dermis at the intradermal CS injection site 1 day after administration. After 14 days, the epidermal epithelium was fully restored. The symptoms were not considered to be adverse, and it is suggested that doses up to 20 µL of 20 mM CS can be safely administered to human beings. In conclusion, our data support development of a CS human dermal pain model.

High-Dose Ribavirin Enhances Early Virological Response in Hepatitis C Genotype 1-Infected Patients.

BACKGROUND: The aim of the study was to investigate whether patients with a previous nonresponse to standard of care treatment with ribavirin dosed according to body weight would respond to a high individualized dose of concentration-monitored ribavirin. METHODS: Previous nonresponders to standard of care treatment with peginterferon (peg-IFN) and ribavirin were included. Ribavirin was dosed aiming at a plasma concentration of >15 µmol/L. The initial ribavirin dose was calculated from a formula based on renal function and body weight. Erythropoietin treatment was started 2 weeks before antiviral therapy. RESULTS: Twenty patients (16 men and 4 women) with a mean age of 52 years were included. Sixty percent had advanced fibrosis. Eighty percent of patients achieved an early viral response, and 60% were negative for hepatitis C virus ribonucleic acid (HCV RNA) at treatment week 24. High-dose ribavirin resulted in a significantly increased HCV RNA drop at week 12 (mean: 3.13 versus 2.05 IU/mL; P < 0.001). Nine patients were negative for HCV RNA at the end of treatment, and 1 achieved sustained viral response. The final steady-state daily dose of ribavirin varied from 1400 to 4400 mg. Hemoglobin levels decreased during treatment, mean Hb 163, 134, and 110 g/L at week 0, 4, and 12, respectively. Two patients received blood transfusions. No other severe adverse events were recorded. CONCLUSIONS: An individualized high ribavirin dose resulted in a more pronounced early viral HCV RNA decline than a standard-dose ribavirin scheme. This regime is safe provided that close monitoring of anemia is undertaken and that treatment with erythropoietin is given.

Assessment of interaction potential of AZD2066 using in vitro metabolism tools, physiologically based pharmacokinetic modelling and in vivo cocktail data.

PURPOSE: Static and dynamic (PBPK) prediction models were applied to estimate the drug-drug interaction (DDI) risk of AZD2066. The predictions were compared to the results of an in vivo cocktail study. Various in vivo measures for tolbutamide as a probe agent for cytochrome P450 2C9 (CYP2C9) were also compared. METHODS: In vitro inhibition data for AZD2066 were obtained using human liver microsomes and CYP-specific probe substrates. DDI prediction was performed using PBPK modelling with the SimCYP simulator™ or static model. The cocktail study was an open label, baseline, controlled interaction study with 15 healthy volunteers receiving multiple doses of AD2066 for 12 days. A cocktail of single doses of 100 mg caffeine (CYP1A2 probe), 500 mg tolbutamide (CYP2C9 probe), 20 mg omeprazole (CYP2C19 probe) and 7.5 mg midazolam (CYP3A probe) was simultaneously applied at baseline and during the administration of AZD2066. Bupropion as a CYP2B6 probe (150 mg) and 100 mg metoprolol (CYP2D6 probe) were administered on separate days. The pharmacokinetic parameters for the probe drugs and their metabolites in plasma and urinary recovery were determined. RESULTS: In vitro AZD2066 inhibited CYP1A2, CYP2B6, CYP2C9, CYP2C19 and CYP2D6. The static model predicted in vivo interaction with predicted AUC ratio values of >1.1 for all CYP (except CYP3A4). The PBPK simulations predicted no risk for clinical relevant interactions. The cocktail study showed no interaction for the CYP2B6 and CYP2C19 enzymes, a possible weak inhibition of CYP1A2, CYP2C9 and CYP3A4 activities and a slight inhibition (29 %) of CYP2D6 activity. The tolbutamide phenotyping metrics indicated that there were significant correlations between CLform and AUCTOL, CL, Aemet and LnTOL24h. The MRAe in urine showed no correlation to CLform. CONCLUSIONS: DDI prediction using the static approach based on total concentration indicated that AZD20066 has a potential risk for inhibition. However, no DDI risk could be predicted when a more in vivo-like dynamic prediction method with the PBPK with SimCYP™ software based on early human PK data was used and more parameters (i.e. free fraction in plasma, no DDI risk) were taken into account. The clinical cocktail study showed no or low risks for clinical relevant DDI interactions. Our findings are in line with the hypothesis that the dynamic prediction method predicts DDI in vivo in humans better than the static model based on total plasma concentrations.

A positron emission tomography study in healthy volunteers to estimate mGluR5 receptor occupancy of AZD2066 - estimating occupancy in the absence of a reference region.

AZD2066 is a new chemical entity pharmacologically characterized as a selective, negative allosteric modulator of the metabotropic glutamate receptor subtype 5 (mGluR5). Antagonism of mGluR5 has been implicated in relation to various diseases such as anxiety, depression, and pain disorders. To support translation from preclinical results and previous experiences with this target in man, a positron emission tomography study was performed to estimate the relationship between AZD2066 plasma concentrations and receptor occupancy in the human brain, using the mGluR5 radioligand [(11)C]-ABP688. The study involved PET scans on 4 occasions in 6 healthy volunteers. The radioligand was given as a tracer dose alone and following oral treatment with different doses of AZD2066. The analysis was based on the total volume of distribution derived from each PET-assessment. A non-linear mixed effects model was developed where ten delineated brain regions of interest from all PET scans were included in one simultaneous fit. For comparison the analysis was also performed according to a method described previously by Lassen et al. (1995). The results of the analysis showed that the total volume of distribution decreased with increasing drug concentrations in all regions with an estimated Kipl of 1170 nM. Variability between individuals and occasions in non-displaceable volume of distribution could explain most of the variability in the total volume of distribution. The Lassen approach provided a similar estimate for Kipl, but the variability was exaggerated and difficult to interpret.

Effects of food or sleep deprivation during civilian survival training on clinical chemistry variables.

OBJECTIVE: To describe clinical chemistry and weight changes after short-term food or sleep deprivation or multiple deprivations during civilian survival training. METHODS: Data from one baseline-controlled two-period crossover study designed to compare sleep deprivation for up to 50 hours with food deprivation for up to 66 hours (n = 12) and data from regular multiple-deprivations survival training comparing participants (n = 33) with nondeprived instructors (n = 10). RESULTS: Food deprivation was associated with decreased body weight, blood glucose, serum triglycerides, sodium, chloride, and urine pH, and there were increases in blood and urine ketones and serum free fatty acids. Sleep deprivation was associated with a minor decrease in hemoglobin and erythrocyte particle count and volume fraction and an increase in leukocytes. CONCLUSIONS: The clinical chemistry and body weight changes associated with food deprivation were qualitatively similar to those observed in fasting obese patients but developed quicker in the survival training setting. Sleep deprivation had few effects on the clinical chemistry profile except for hematological variables. Physicians evaluating clinical chemistry data from patients subjected to short-term food or sleep deprivation should take the physiological state into account in their assessment.

Circulating human hepcidin-25 concentrations display a diurnal rhythm, increase with prolonged fasting, and are reduced by growth hormone administration.

BACKGROUND: Hepcidin-25 reduces iron absorption by binding to the intestinal iron transporter ferroportin and causing its degradation. Currently, little is known about the basal regulation of circulating hepcidin-25. In addition, although erythropoietin administration has been reported to decrease the circulating hepcidin concentration, information is limited regarding how other stimulators of erythropoiesis, such as growth hormone (GH), might alter hepcidin-25 concentrations. METHODS: We used a sensitive and specific hepcidin-25 dual-monoclonal antibody sandwich immunoassay to measure hepcidin-25 in healthy human volunteers at various time points throughout the day and during 3 days of fasting and subsequent refeeding. We also measured hepcidin-25 concentrations in healthy volunteers after GH administration. RESULTS: In healthy individuals, hepcidin-25 concentrations displayed a diurnal variation, with concentrations being lowest in the early morning and steadily increasing throughout the day before declining during the evening hours, a pattern that was not influenced by food intake. Prolonged fasting produced statistically significant increases in hepcidin-25 concentrations. Refeeding reversed this process, and GH administration markedly decreased hepcidin-25 concentrations. CONCLUSIONS: Our results indicate that in humans, hepcidin-25 exhibits diurnal changes that can be altered by prolonged fasting, which increases hepcidin-25 concentrations approximately 3-fold after 3 days of fasting, possibly owing to a suppression of erythropoiesis that may occur during the fasting state to preserve tissue iron concentrations. In contrast, GH administration decreased hepcidin-25 concentrations by approximately 65%, presumably by stimulating erythropoiesis. These results indicate that circulating hepcidin-25 concentrations display much more dynamic and rapid variation than might have been anticipated previously.

Effects of sleep or food deprivation during civilian survival training on cognition, blood glucose and 3-OH-butyrate.

OBJECTIVES: The study was designed to compare effects of food deprivation (FD) and sleep deprivation (SD) on cognition during survival training. METHODS: In a cross-over design (n=12), the effects of FD (up to 66 hours followed by 500 kcal intake over 24 hours) and SD (up to 50 hours) on cognitive variables, blood glucose, and 3-OH-butyrate were studied. RESULTS: Food deprivation and SD impaired attention-dependent tasks. The FD impairment of simple reaction time was independent of blood glucose levels, which were normalized by a 500 kcal intake over 24 hours while the reaction time was not. Sleep deprivation and FD impaired maze-solving performance on all variables except rule breaks, which were significantly occurring after 50 hours of SD. Delayed word recall was impaired by SD for 50 hours. On the Balloon Analogue Risk Task, SD was associated with reduced risk-taking. In a gambling task, both SD for 50 hours and FD for 66 hours were associated with a tendency to make early choices when presented with consecutive choices, but the risk-taking was not affected. CONCLUSIONS: Sleep deprivation has multiple cognitive effects, including attention, memory, visual-spatial ability, and risk-taking. Food deprivation had no affect on risk-taking, while the other tasks were affected in a way similar to SD but were less pronounced. The FD effects on cognition did not appear to depend on blood sugar levels. The need to sleep should be prioritized in survival situations to avoid cognitive impairment.

Long term perturbation of endocrine parameters and cholesterol metabolism after discontinued abuse of anabolic androgenic steroids.

AIMS: To study the long-term impact of anabolic androgenic steroid (AAS) abuse on the cholesterol profile, and the potential to suppress endocrine activity in men working out at gym facilities. To study the relation between urinary biomarkers for testosterone and nandrolone abuse and the UGT2B17 genotype and time profile. EXPERIMENTAL DESIGN: Subjects (N = 56) were recruited through Anti-Doping Hot-Line. Serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), plasma levels of low density lipoprotein (LDL), high density lipoprotein (HDL) and urinary steroid profile were regularly measured for a period of up to one year after cessation of intramuscular AAS abuse. RESULTS AND DISCUSSION: A sustained suppression of LH, and FSH was observed for several months. The nandrolone urinary biomarker 19-NA was detectable several months after the last nandrolone intake and was correlated to the levels of LH and FSH. Testosterone abuse on the other hand was detectable only for a few weeks, and some of the testosterone abusers did not test positive due to a genetic deletion polymorphism of the UGT2B17. Significantly increased levels of HDL and decreased levels of LDL were observed for 6-months after cessation of AAS abuse. CONCLUSION: Some individuals had a sustained suppression of LH and FSH for a period of 1 year whereas the cholesterol profile was normalized within 6 month. The long term consequences of these findings remain to be established.

Circulating proprotein convertase subtilisin kexin type 9 has a diurnal rhythm synchronous with cholesterol synthesis and is reduced by fasting in humans.

OBJECTIVE: To gain insight into the function of proprotein convertase subtilisin kexin type 9 (PCSK9) in humans by establishing whether circulating levels are influenced by diurnal, dietary, and hormonal changes. METHODS AND RESULTS: We monitored circulating PCSK9 in a set of dynamic human experiments and could show that serum PCSK9 levels display a diurnal rhythm that closely parallels that of cholesterol synthesis, measured as serum lathosterol. In contrast to these marked diurnal changes in cholesterol metabolism, serum low-density lipoprotein (LDL) cholesterol levels remained stable during the diurnal cycle. Depletion of liver cholesterol by treatment with the bile acid-binding resin, cholestyramine, abolished the diurnal rhythms of both PCSK9 and lathosterol. Fasting (>18 hours) strongly reduced circulating PCSK9 and lathosterol levels, whereas serum LDL levels remained unchanged. Growth hormone, known to be increased during fasting in humans, reduced circulating PCSK9 in parallel to LDL cholesterol levels. CONCLUSIONS: Throughout the day, and in response to fasting and cholesterol depletion, circulating PCSK9 displays marked variation, presumably related to oscillations in hepatic cholesterol that modify its activity in parallel with cholesterol synthesis. In addition to this sterol-mediated regulation, additional effects on LDL receptors may be mediated by hormones directly influencing PCSK9.

A novel mutation in the biliverdin reductase-A gene combined with liver cirrhosis results in hyperbiliverdinaemia (green jaundice).

BACKGROUND: Hyperbiliverdinaemia is a poorly defined clinical sign that has been infrequently reported in cases of liver cirrhosis or liver carcinoma, usually indicating a poor long-term prognosis. AIMS: To clarify the pathogenesis of hyperbiliverdinaemia in an extended case report. METHODS: A 64-year-old man with alcoholic cirrhosis was admitted to hospital with severe bleeding from oesophageal varices. Ultrasonography showed ascites, but no dilatation of the biliary tree. The skin, sclerae, plasma, urine and ascites of the patient showed a greenish appearance. Bilirubin levels were normal, and there were no signs of haemolysis. Biliverdin was analysed in plasma and urine with liquid chromatography coupled to mass spectrometry. The seven exonic regions of the biliverdin reductase-A (BVR-A) gene was amplified by polymerase chain reaction and sequenced. RESULTS: Biliverdin was present in plasma and urine. In nucleotide 52 of exon I of the DNA isolated from the hyperbiliverdinaemic patient, we discovered a novel heterozygous C-->T nonsense mutation converting an arginine (CGA) in position 18 into a stop codon (TGA) (R18Stop) predicted to truncate the protein N-terminally to the active site Tyr97. Two children of the proband were heterozygous for the identical mutation in the BVR-A gene, but had no clinical signs of liver disease and had normal levels of biliverdin. The BVR-A gene mutation was not found in 200 healthy volunteers or nine patients with end-stage liver cirrhosis. CONCLUSION: Hyperbiliverdinaemia (green jaundice) with green plasma and urine may be caused by a genetic defect in the BVR-A gene in conjunction with decompensated liver cirrhosis.

Reexamining transaminase elevation in Phase I clinical trials: the importance of baseline and change from baseline.

PURPOSE: The aims of the present analysis were to determine prevalence of transaminase elevation in placebo-treated healthy volunteers in our historical phase I clinical trials and to assess which factors were associated with it. METHODS: We performed a retrospective analysis of alanine transaminase (ALT) levels in 481 placebo-treated healthy volunteers from 20 phase I trials by examining ALT elevation rates using the upper limit of normal values (ULN) as the cutoff as well as changes from baseline in actual ALT values. RESULTS: The ULN for ALT ranged from 32 to 72 IU/L across the studies. Although the overall ALT elevation rate (4.4%) from pooled datasets was low, the elevation rates were higher in more recent studies than in earlier ones. While elevation rates at baseline and during placebo treatment did not differ significantly, ALT maximal levels during placebo treatment were significantly higher than baseline levels. Moreover, baseline ALT levels were found to be more important in predicting ALT elevation during placebo treatment than demographic and study design factors. CONCLUSION: Baseline level and changes from baseline in transaminase are important variables to examine in addition to elevation above ULN for more reliably interpreting liver signals in Phase I clinical trials.

Transfer of buprenorphine into breast milk and calculation of infant drug dose.

Little is known about the safety of buprenorphine (BUP) in breastfeeding. The aim of this work was to investigate the transfer of buprenorphine and its main active metabolite, norbuprenorphine (n-BUP), into human milk and to determine the drug dose and effects in exposed infants. Seven lactating women, who were maintained on BUP treatment because of previous opiate addiction, were studied in an open observational study. All mothers had a strong wish to breastfeed their newborn infants. Buprenorphine samples for analysis were collected from the urine of 6 infants together with breast milk, blood, and urine from their mothers during a 24-hour period in the week after birth. One mother-infant pair was studied at 9 months of age. Buprenorphine and n-BUP were analyzed by a liquid chromatography/mass spectrometry method suitable for handling different matrices. Buprenorphine and n-BUP were found in low levels in the infants' urine. Breastfed infants were exposed to a calculated BUP dose per kg bodyweight less than 1%, with an average milk/plasma area under the curve of 1.7 (range, 1.1-2.8) for BUP and 0.7 (range, 0.4-1.2) for n-BUP. These data support the use of BUP during breastfeeding. However, the authors recommend that infants be monitored closely.

Biological accelerator mass spectrometry at Uppsala University.

A new research programme for the biological applications of accelerator mass spectrometry has been initiated at Uppsala University and the first results are presented. A (14)C-labelled pharmaceutical substance has been dissolved in human blood, plasma and urine and diluted over 3 orders of magnitude. The measured drug concentrations were found to be in good agreement with the predicted values. Furthermore, the effect of the sample preparation background contribution has been studied as the sample amount was varied down to sub-microl sizes.

AAPS-FDA workshop white paper: microdialysis principles, application and regulatory perspectives.

Many decisions in drug development and medical practice are based on measuring blood concentrations of endogenous and exogenous molecules. Yet most biochemical and pharmacological events take place in the tissues. Also, most drugs with few notable exceptions exert their effects not within the bloodstream, but in defined target tissues into which drugs have to distribute from the central compartment. Assessing tissue drug chemistry has, thus, for long been viewed as a more rational way to provide clinically meaningful data rather than gaining information from blood samples. More specifically, it is often the extracellular (interstitial) tissue space that is most closely related to the site of action (biophase) of the drug. Currently microdialysis (microD) is the only tool available that explicitly provides data on the extracellular space. Although microD as a preclinical and clinical tool has been available for two decades, there is still uncertainty about the use of microD in drug research and development, both from a methodological and a regulatory point of view. In an attempt to reduce this uncertainty and to provide an overview of the principles and applications of microD in preclinical and clinical settings, an AAPS-FDA workshop took place in November 2005 in Nashville, TN, USA. Stakeholders from academia, industry and regulatory agencies presented their views on microD as a tool in drug research and development.