CYX-5, a G-protein biassed MOP receptor agonist, DOP receptor antagonist and KOP receptor agonist, evokes constipation but not respiratory depression relative to morphine in rats.

BACKGROUND: Strong opioid analgesics such as morphine alleviate moderate to severe acute nociceptive pain (e.g. post-surgical or post-trauma pain) as well as chronic cancer pain. However, they evoke many adverse effects and so there is an unmet need for opioid analgesics with improved tolerability. Recently, a prominent hypothesis has been that opioid-related adverse effects are mediated by ß-arrestin2 recruitment at the µ-opioid (MOP) receptor and this stimulated research on discovery of G-protein biassed opioid analgesics. In other efforts, opioids with MOP agonist and δ-opioid (DOP) receptor antagonist profiles are promising for reducing side effects c.f. morphine. Herein, we report on the in vivo pharmacology of a novel opioid peptide (CYX-5) that is a G-protein biassed MOP receptor agonist, DOP receptor antagonist and kappa opioid (KOP) receptor agonist. METHODS: Male Sprague-Dawley received intracerebroventricular bolus doses of CYX-5 (3, 10, 20 nmol), morphine (100 nmol) or vehicle, and antinociception (tail flick) was assessed relative to constipation (charcoal meal and castor oil-induced diarrhoea tests) and respiratory depression (whole body plethysmography). RESULTS: CYX-5 evoked naloxone-sensitive, moderate antinociception, at the highest dose tested. Although CYX-5 did not inhibit gastrointestinal motility, it reduced stool output markedly in the castor oil-induced diarrhoea test. In contrast to morphine that evoked respiratory depression, CYX-5 increased tidal volume, thereby stimulating respiration. CONCLUSION: Despite its lack of recruitment of ß-arrestin2 at MOP, DOP and KOP receptors, CYX-5 evoked constipation, implicating a mechanism other than ß-arrestin2 recruitment at MOP, DOP and KOP receptors, mediating constipation evoked by CYX-5 and potentially other opioid ligands.

Cough as an adverse effect on inhalation pharmaceutical products.

Cough is an adverse effect that may hinder the delivery of drugs into the lungs. Chemical or mechanical stimulants activate the transient receptor potential in some airway afferent nerves (C-fibres or A-fibres) to trigger cough. Types of inhaler device and drug, dose, excipients and formulation characteristics, including pH, tonicity, aerosol output and particle size may trigger cough by stimulating the cough receptors. Release of inflammatory mediators may increase the sensitivity of the cough receptors to stimulants. The cough-provoking effect of aerosols is enhanced by bronchoconstriction in diseased airways and reduces drug deposition in the target pulmonary regions. In this article, we review the factors by which inhalation products may cause cough.

Intracerebroventricular administration of CYX-6, a potent µ-opioid receptor agonist, a δ- and κ-opioid receptor antagonist and a biased ligand at µ, δ & κ-opioid receptors, evokes antinociception with minimal constipation and respiratory depression in rats in contrast to morphine.

Mu opioid receptor (MOPr) agonists are thought to produce analgesia via modulation of G-protein-coupled intracellular signalling pathways whereas the ß-arrestin2 pathway is proposed to mediate opioid-related adverse effects. Here, we report the antinociception, constipation and respiratory depressant profile of CYX-6, a potent MOPr agonist that is also a delta and a kappa opioid receptor (DOPr/KOPr) antagonist and that lacks ß-arrestin2 recruitment at each of the MOPr, DOPr and the KOPr. In anaesthetised male Sprague Dawley rats, an intracerebroventricular (i.c.v.) guide cannula was stereotaxically implanted. After 5-7 days post-surgical recovery, rats received a single i.c.v. bolus dose of CYX-6 (3-30 nmol), morphine (100 nmol) or vehicle. Antinociception was assessed using the warm water tail flick test (52.5 ± 0.5 °C). Constipation was assessed using the charcoal meal gut motility test and the castor oil-induced diarrhoea test. Respiratory depression was measured by whole-body plethysmography in awake, freely moving animals, upon exposure to a hypercapnic gas mixture (8% CO2, 21% O2 and 71% N2). The intrinsic pharmacology of CYX-6 given by the i.c.v. route in rats showed that it produced dose-dependent antinociception. It also produced respiratory stimulation rather than depression and it had a minimal effect on intestinal motility in contrast to the positive control, morphine. CYX-6 is an endomorphin-2 analogue that dissociates antinociception from constipation and respiratory depression in rats. Our findings provide useful insight to inform the discovery and development of novel opioid analgesics with a superior tolerability profile compared with morphine.

Study Protocol for a Pilot, Open-Label, Prospective, and Observational Study to Evaluate the Pharmacokinetics of Drugs Administered to Patients during Extracorporeal Circulation; Potential of In Vivo Cytochrome P450 Phenotyping to Optimise Pharmacotherapy.

Pharmacokinetic alterations of medications administered during surgeries involving cardiopulmonary bypass (CPB) and extracorporeal membrane oxygenation (ECMO) have been reported. The impact of CPB on the cytochrome P450 (CYP) enzymes' activity is the key factor. The metabolic rates of caffeine, dextromethorphan, midazolam, omeprazole, and Losartan to the CYP-specific metabolites are validated measures of in vivo CYP 1A2, 2D6, 3A4, 2C19, and 2C9 activities, respectively. The study aim is to assess the activities of major CYPs in patients on extracorporeal circulation (EC). This is a pilot, prospective, open-label, observational study in patients undergoing surgery using EC and patients undergoing laparoscopic cholecystectomy as a control group. CYP activities will be measured on the day, and 1-2 days pre-surgery/3-4 days post-surgery (cardiac surgery and Laparoscopic cholecystectomy) and 1-2 days after starting ECMO, 1-2 weeks after starting ECMO, and 1-2 days after discontinuation from ECMO. Aforementioned CYP substrates will be administered to the patient and blood samples will be collected at 0, 1, 2, 4, and 6 h post-dose. Major CYP enzymes' activities will be compared in each participant on the day, and before/after surgery. The CYP activities will be compared in three study groups to investigate the impact of CYPs on EC.

Sorafenib N-Oxide Is an Inhibitor of Human Hepatic CYP3A4.

The multi-kinase inhibitor sorafenib (SOR) is clinically important in the treatment of hepatocellular and renal cancers and undergoes CYP3A4-dependent oxidation in liver to the pharmacologically active N-oxide metabolite (SNO). There have been reports that kinase inhibitors such as SOR may precipitate pharmacokinetic interactions with coadministered drugs that compete for CYP3A4-mediated biotransformation, but these occur non-uniformly in patients. Clinical evidence also indicates that SNO accumulates in serum of some patients during prolonged SOR therapy. In this study undertaken in hepatic microsomes from individual donors, we assessed the possibility that SNO might contribute to pharmacokinetic interactions mediated by SOR. Enzyme kinetics of CYP3A4-mediated midazolam 1'-hydroxylation in individual human hepatic microsomes were analyzed by non-linear regression and appropriate replots. Thus, SNO and SOR were linear-mixed inhibitors of microsomal CYP3A4 activity (Kis 15 ± 4 and 33 ± 14 µM, respectively). To assess these findings, further molecular docking studies of SOR and SNO with the 1TQN crystal structure of CYP3A4 were undertaken. SNO elicited a larger number of interactions with key amino acid residues located in substrate recognition sequences of the enzyme. In the optimal docking pose, the N-oxide moiety of SNO was also found to interact directly with the heme moiety of CYP3A4. These findings suggest that SNO could contribute to pharmacokinetic interactions involving SOR, perhaps in individuals who produce high circulating concentrations of the metabolite.

Cyclooctatetraene: A Bioactive Cubane Paradigm Complement.

Cubane was recently validated as a phenyl ring (bio)isostere, but highly strained caged carbocyclic systems lack π character, which is often critical for mediating key biological interactions. This electronic property restriction associated with cubane has been addressed herein with cyclooctatetraene (COT), using known pharmaceutical and agrochemical compounds as templates. COT either outperformed or matched cubane in multiple cases suggesting that versatile complementarity exists between the two systems for enhanced bioactive molecule discovery.

An improved LC-MS/MS method for simultaneous evaluation of CYP2C9, CYP2C19, CYP2D6 and CYP3A4 activity.

AIM: To develop an LC-MS/MS assay to quantitate well-tolerated substrates; midazolam (CYP3A), omeprazole (CYP2C19), dextromethorphan (CYP2D6), losartan (CYP2C9) and their respective metabolites' concentrations in plasma samples. PATIENTS & METHODS: A solid-phase extraction method was optimized to extract analytes of interest simultaneously from human plasma samples. The assay analyzed plasma samples collected from patients who received equal or lower than therapeutic doses of CYP substrates. RESULTS: This assay was validated based on the European Medicines Agency guideline for bioanalytical method validation and was sensitive, linear, accurate and precise with acceptable recovery and matrix effects. CONCLUSION: Small sample volume and dose of cytochrome P450 substrates, short-run time, using stable isotope internal standards and being cost effective are the major advantages of the assay.

Progress in understanding mechanisms of opioid-induced gastrointestinal adverse effects and respiratory depression.

Opioids evoke analgesia through activation of opioid receptors (predominantly the µ opioid receptor) in the central nervous system. Opioid receptors are abundant in multiple regions of the central nervous system and the peripheral nervous system including enteric neurons. Opioid-related adverse effects such as constipation, nausea, and vomiting pose challenges for compliance and continuation of the therapy for chronic pain management. In the post-operative setting opioid-induced depression of respiration can be fatal. These critical limitations warrant a better understanding of their underpinning cellular and molecular mechanisms to inform the design of novel opioid analgesic molecules that are devoid of these unwanted side-effects. Research efforts on opioid receptor signalling in the past decade suggest that differential signalling pathways and downstream molecules preferentially mediate distinct pharmacological effects. Additionally, interaction among opioid receptors and, between opioid receptor and non-opioid receptors to form signalling complexes shows that opioid-induced receptor signalling is potentially more complicated than previously thought. This complexity provides an opportunity to identify and probe relationships between selective signalling pathway specificity and in vivo production of opioid-related adverse effects. In this review, we focus on current knowledge of the mechanisms thought to transduce opioid-induced gastrointestinal adverse effects (constipation, nausea, vomiting) and respiratory depression.

Differential effects of hepatic cirrhosis on the intrinsic clearances of sorafenib and imatinib by CYPs in human liver.

The tyrosine kinase inhibitors sorafenib and imatinib are important in the treatment of a range of cancers but adverse effects in some patients necessitate dosage modifications. CYP3A4 has a major role in the oxidation of sorafenib to its N-oxide and N-hydroxymethyl metabolites and also acts in concert with CYP2C8 to mediate imatinib N-demethylation. CYP3A4 expression and function are impaired in patients with advanced liver disease, whereas the functions of CYP2C enzymes are relatively preserved. We evaluated the biotransformation of sorafenib and imatinib in well-characterized microsomal fractions from 17 control subjects and 19 individuals with hepatic cirrhosis of varying severity. The principal findings were that liver disease impaired the microsomal oxidation of sorafenib to its major metabolites to 40-44% of control (P<0.01), whereas the N-demethylation of imatinib was relatively unimpaired. The impairments in sorafenib biotransformation were correlated with decreased serum albumin concentrations and increased serum bilirubin concentrations in patients with liver disease, but not with the overall grade of liver disease according to the Child-Pugh system. In contrast, there was no relationship between imatinib N-demethylation and clinicopathologic factors in liver disease patients. These findings were accounted for in terms of the differential roles of CYPs 3A4 and 2C8 in the intrinsic clearance of the drugs. CYP3A4 has the major role in the intrinsic clearance of sorafenib but plays a secondary role to CYP2C8 in the intrinsic clearance of imatinib. In agreement with these findings CYP2C protein expression and CYP2C8-mediated paclitaxel 6α-hydroxylation were unimpaired in cirrhotic livers. This information could be adapted in individualized approaches such as in vivo CYP3A4 phenotyping to optimize sorafenib safety and efficacy in cancer patients with liver dysfunction.

Effect of cardiopulmonary bypass on cytochrome P450 enzyme activity: implications for pharmacotherapy.

For patients undergoing cardiopulmonary bypass (CPB) during cardiac surgery, there are well-documented changes in the pharmacokinetics (PK) of commonly administered drugs. Although multiple factors potentially underpin these changes, there has been scant research attention on the impact of CPB to alter the activities of cytochrome P450 (CYP) isoenzymes. PK changes during cardiac surgery with CPB have the potential to adversely affect the safety and efficacy of pharmacotherapy and increase the risk of drug-drug interactions. Clinically significant changes in drug PK during CPB are likely to be prominent for drugs where CYP metabolism is a major clearance (CL) mechanism. However, clinical data from patients undergoing CPB surgery in support of this hypothesis are lacking, leaving a significant knowledge gap. In this review, we address the effects of CPB on the release of pro-inflammatory cytokines, in surgeries with and without CPB, both pre and post initiation of surgery. We reviewed literature to explore the relationship between the release of pro-inflammatory cytokines, and the expression and activities of CYP enzymes. Through this approach, we provide new insight on the effects of CPB on the PK of drugs administered to patients in the clinical setting. Future research to address this knowledge gap will have considerable impact to assist clinicians with optimizing pharmacotherapy in this patient population.

An improved liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantification of dexmedetomidine concentrations in samples of human plasma.

Dexmedetomidine (DMET) is a sedative, analgesic and anxiolytic with minimum adverse respiratory effects. An LC-MS/MS bioanalytical method has been developed and validated to accurately measure DMET concentrations in samples of human plasma. The method overcomes difficulties in the extraction and quantification of DMET due to the fact that it binds strongly to glass and plastic tubes, as well as solid phase extraction (SPE) cartridges. Human plasma (50 µL) was mixed with the internal standard (IS) (DMET-d4) solution (100 µL) and 0.1% formic acid (50 µL) and extracted using Oasis HLB 1 CC (30 mg) solid phase extraction (SPE) cartridges (Waters®). The glass tubes were coated with bovine serum albumin (BSA) 0.5% (20 µL) before eluting DMET and the IS. After evaporation under nitrogen at room temperature, the analytes were reconstituted in 20% acetonitrile in 0.1% formic acid in water and transferred to silanized glass vials. An electrospray ionisation (ESI) mass spectrometry method in positive mode was created and the precursor/product transitions (m/z) were 201.1 → 95.0 (DMET) and 204.9 → 99.0 (IS). The method was robust and fully validated based on the 2012 EMEA guideline for bioanalytical method validation in the concentration range of 0.5-20 ng/mL. Using this assay, we showed that DMET binds strongly to Extracorporeal Membrane Oxygenation (ECMO) circuits, consistent with expectations for small lipophilic compounds.

High-throughput assay for quantification of the plasma concentrations of thiopental using automated solid phase extraction (SPE) directly coupled to LC-MS/MS instrumentation.

Most previous assays for thiopental are time-consuming due to laborious sample extraction steps prior to analysis using gas chromatography or high pressure liquid chromatography. Here, we describe the first high-throughput liquid chromatography - tandem mass spectrometry (LC-MS/MS) method for quantification of thiopental concentrations in samples of human plasma. Robotic on-line solid phase extraction (SPE) was used to elute the analytes of interest from samples of human plasma (50µL) loaded onto C18 SPE cartridges to which were added aliquots (50µL) of internal standard solution (thiopental-d5 100ng/mL) and 0.5% formic acid in water (100µL). Cartridges were washed using 10% methanol in ammonium acetate buffer (50mM, pH 7) before elution with mobile phase comprising 0.1% formic acid in water and acetonitrile with a flow rate of 0.55mL/min using a 7.2min run time. The analytes were separated on a C18 XTerra® analytical column. Mass spectrometry detection was performed using a QTrap 5500 mass spectrometer (AB Sciex) with negative ionisation. The multiple reaction monitoring (MRM) transitions for thiopental and the internal standard were 241→58, and 246→58, respectively. The calibration curve was linear over a range of 6-600ng/mL. Thiopental was stable in human plasma samples for at least 36h in the autosampler, as well as after three cycles of freeze and thaw, and after 3h storage at room temperature. The absolute recovery and matrix effect were 102% and 6.9%, respectively, and the within-run and between-run precision and accuracy were ≤15%. Our method is fully-validated and satisfies the requirements of the 2012 European Medicines Agency (EMEA) guideline for Bioanalytical Method Validation.

The effect of 1 mg folic acid supplementation on clinical outcomes in female migraine with aura patients.

BACKGROUND: Migraine is a common neurovascular condition that may be linked to hyperhomocysteinemia. We have previously provided evidence that reduction of homocysteine with a vitamin supplementation can reduce the occurrence of migraine in women. The current study examined the occurrence of migraine in response to vitamin supplementation with a lower dose of folic acid. METHODS: This was a 6 month randomised, double blinded placebo controlled trial of daily vitamin supplementation containing 1 mg of folic acid, 25 mg of Vitamin B6 and Vitamin B12, on reduction of homocysteine and the occurrence of migraine in 300 female patients diagnosed with migraine with aura. RESULTS: Vitamin supplementation with 1 mg of folic acid, did not significantly decrease homocysteine levels (P = 0.2). The treatment group did not show a significant decrease in the percentage of participants with high migraine disability, severity or frequency at the end of the 6 month intervention (P > 0.1). CONCLUSION: 1 mg of folic acid in combination with vitamin B6 and B12 is less effective in reducing migraine associated symptoms compared to the previously tested dosage of 2 mg folic acid in combination with 25 mg of vitamin B6 and 400 µg of vitamin B12.

In Vitro Metabolic Stability and in Vivo Biodistribution of 3-Methyl-4-furoxancarbaldehyde Using PET Imaging in Rats.

Painful diabetic neuropathy (PDN) is a type of peripheral neuropathic pain that is currently difficult to treat using clinically available analgesics. Recent work suggests a progressive depletion of nitric oxide (NO) in nerve cells may be responsible for the pathobiology of PDN. The nitric oxide donor, 3-methyl-4-furoxancarbaldehyde (PRG150), has been shown to produce dose-dependent analgesia in a rat model of PDN. To gain insight into the mechanism of analgesia, methods to radiolabel PRG150 were developed to assess the in vivo biodistribution in rats. The furoxan ring was labeled with (13)N to follow any nitric oxide release and the 3-methyl substituent was labeled with (11)C to track the metabolite using PET imaging. The in vitro metabolic stability of PRG150 was assessed in rat liver microsomes and compared to in vivo metabolism of the synthesized radiotracers. PET images revealed a higher uptake of (13)N over (11)C radioactivity in the spinal cord. The differences in radioactive uptake could indicate that a NO release in the spinal cord and other components of the somatosensory nervous system may be responsible for the analgesic effects of PRG150 seen in the rat model of PDN.

Evaluation of a High-Throughput Peptide Reactivity Format Assay for Assessment of the Skin Sensitization Potential of Chemicals.

The direct peptide reactivity assay (DPRA) is a validated method for in vitro assessment of the skin sensitization potential of chemicals. In the present work, we describe a peptide reactivity assay using 96-well plate format and systematically identified the optimal assay conditions for accurate and reproducible classification of chemicals with known sensitizing capacity. The aim of the research is to ensure that the analytical component of the peptide reactivity assay is robust, accurate, and reproducible in accordance with criteria that are used for the validation of bioanalytical methods. Analytical performance was evaluated using quality control samples (QCs; heptapeptides at low, medium, and high concentrations) and incubation of control chemicals (chemicals with known sensitization capacity, weak, moderate, strong, extreme, and non-sensitizers) with each of three synthetic heptapeptides, viz Cor1-C420 (Ac-NKKCDLF), cysteine- (Ac-RFAACAA), and lysine- (Ac-RFAAKAA) containing heptapeptides. The optimal incubation temperature for all three heptapeptides was 25°C. Apparent heptapeptide depletion was affected by vial material composition. Incubation of test chemicals with Cor1-C420, showed that peptide depletion was unchanged in polypropylene vials over 3-days storage in an autosampler but this was not the case for borosilicate glass vials. For cysteine-containing heptapeptide, the concentration was not stable by day 3 post-incubation in borosilicate glass vials. Although the lysine-containing heptapeptide concentration was unchanged in both polypropylene and borosilicate glass vials, the apparent extent of lysine-containing heptapeptide depletion by ethyl acrylate, differed between polypropylene (24.7%) and glass (47.3%) vials. Additionally, the peptide-chemical complexes for Cor1-C420-cinnamaldehyde and cysteine-containing heptapeptide-2, 4-dinitrochlorobenzene were partially reversible during 3-days of autosampler storage. These observations further highlight the difficulty in adapting in vitro methods to high-throughput format for screening the skin sensitization potential of large numbers of chemicals whilst ensuring that the data produced are both accurate and reproducible.

Validating Eaton's Hypothesis: Cubane as a Benzene Bioisostere.

Pharmaceutical and agrochemical discovery programs are under considerable pressure to meet increasing global demand and thus require constant innovation. Classical hydrocarbon scaffolds have long assisted in bringing new molecules to the market place, but an obvious omission is that of the Platonic solid cubane. Eaton, however, suggested that this molecule has the potential to act as a benzene bioisostere. Herein, we report the validation of Eaton's hypothesis with cubane derivatives of five molecules that are used clinically or as agrochemicals. Two cubane analogues showed increased bioactivity compared to their benzene counterparts whereas two further analogues displayed equal bioactivity, and the fifth one demonstrated only partial efficacy. Ramifications from this study are best realized by reflecting on the number of bioactive molecules that contain a benzene ring. Substitution with the cubane scaffold where possible could revitalize these systems, and thus expedite much needed lead candidate identification.

A novel fully validated LC-MS/MS method for quantification of pyridoxal-5'-phosphate concentrations in samples of human whole blood.

Quantification of pyridoxal-5'-phosphate (PLP) in biological samples is challenging due to the presence of endogenous PLP in matrices used for preparation of calibrators and quality control samples (QCs). Hence, we have developed an LC-MS/MS method for accurate and precise measurement of the concentrations of PLP in samples (20µL) of human whole blood that addresses this issue by using a surrogate matrix and minimizing the matrix effect. We used a surrogate matrix comprising 2% bovine serum albumin (BSA) in phosphate buffer saline (PBS) for making calibrators, QCs and the concentrations were adjusted to include the endogenous PLP concentrations in the surrogate matrix according to the method of standard addition. PLP was separated from the other components of the sample matrix using protein precipitation with trichloroacetic acid 10% w/v. After centrifugation, supernatant were injected directly into the LC-MS/MS system. Calibration curves were linear and recovery was >92%. QCs were accurate, precise, stable for four freeze-thaw cycles, and following storage at room temperature for 17h or at -80°C for 3 months. There was no significant matrix effect using 9 different individual human blood samples. Our novel LC-MS/MS method has satisfied all of the criteria specified in the 2012 EMEA guideline on bioanalytical method validation.

In vitro methods for hazard assessment of industrial chemicals - opportunities and challenges.

Allergic contact dermatitis (ACD) is a delayed-type hypersensitivity immune reaction mediated by T-lymphocytes as a result of repeated exposure of an allergen primarily on skin. ACD accounts for up to 95% of occupational skin diseases, with epoxy resins implicated as one of the most common causes of ACD. Efficient high-throughput in vitro screening for accurate identification of compounds and materials that may pose hazardous risks in the workplace is crucial. At present, the murine local lymph node assay is the 'method of choice' for predicting the sensitizing potency of contact allergens. As the 3Rs principles of reduction, refinement, and replacement in animal testing has gained political and economic momentum, several in vitro screening methods have been developed for identifying potential contact allergens. To date, these latter methods have been utilized primarily to assess the skin sensitizing potential of the chemical components of cosmetic products with scant research attention as to the applicability of these methods to industrial chemicals, particularly epoxy resins. Herein we review the currently utilized in vitro methods and identify the knowledge gaps with regard to assessing the generalizability of in vitro screening methods for assessing the skin sensitizing potential of industrial chemicals.