FORWARDS-1: an adaptive, single-blind, placebo-controlled ascending dose study of acute baclofen on safety parameters in opioid dependence during methadone-maintenance treatment-a pharmacokinetic-pharmacodynamic study.

BACKGROUND: Treatment of opiate addiction with opiate substitution treatment (e.g. methadone) is beneficial. However, some individuals desire or would benefit from abstinence but there are limited options to attenuate problems with opiate withdrawal. Preclinical and preliminary clinical evidence suggests that the GABA-B agonist, baclofen, has the desired properties to facilitate opiate detoxification and prevent relapse. This study aims to understand whether there are any safety issues in administering baclofen to opioid-dependent individuals receiving methadone. METHODS: Opiate-dependent individuals (DSM-5 severe opioid use disorder) maintained on methadone will be recruited from addiction services in northwest London (NHS and third sector providers). Participants will be medically healthy with no severe chronic obstructive pulmonary disease or type 2 respiratory failure, no current dependence on other substances (excluding nicotine), no current severe DSM-5 psychiatric disorders, and no contraindications for baclofen or 4800 IU vitamin D (placebo). Eligible participants will be randomised in a 3:1 ratio to receive baclofen or placebo in an adaptive, single-blind, ascending dose design. A Bayesian dose-escalation model will inform the baclofen dose (10, 30, 60, or 90 mg) based on the incidence of 'dose-limiting toxicity' (DLT) events and participant-specific methadone dose. A range of respiratory, cardiovascular, and sedative measures including the National Early Warning Score (NEWS2) and Glasgow Coma Scale will determine DLT. On the experimental day, participants will consume their usual daily dose of methadone followed by an acute dose of baclofen or placebo (vitamin D3) ~ 1 h later. Measures including oxygen saturation, transcutaneous CO2, respiratory rate, QTc interval, subjective effects (sedation, drug liking, craving), plasma levels (baclofen, methadone), and adverse events will be obtained using validated questionnaires and examinations periodically for 5 h after dosing. DISCUSSION: Study outcomes will determine what dose of baclofen is safe to prescribe to those receiving methadone, to inform a subsequent proof-of-concept trial of the efficacy baclofen to facilitate opiate detoxification. To proceed, the minimum acceptable dose is 30 mg of baclofen in patients receiving ≤ 60 mg/day methadone based on the clinical experience of baclofen's use in alcoholism and guidelines for the management of opiate dependence. TRIAL REGISTRATION: Clinicaltrials.gov NCT05161351. Registered on 16 December 2021.

Drying of open animal joints in vivo subsequently causes cartilage degeneration.

OBJECTIVES: During open orthopaedic surgery, joints may be exposed to air, potentially leading to cartilage drying and chondrocyte death, however, the long-term effects of joint drying in vivo are poorly understood. We used an animal model to investigate the subsequent effects of joint drying on cartilage and chondrocytes. METHODS: The patellar groove of anaesthetised rats was exposed (sham-operated), or exposed and then subjected to laminar airflow (0.25m/s; 60 minutes) before wounds were sutured and animals recovered. Animals were monitored for up to eight weeks and then sacrificed. Cartilage and chondrocyte properties were studied by histology and confocal microscopy, respectively. RESULTS: Joint drying caused extensive chondrocyte death within the superficial regions of cartilage. Histology of dried cartilage demonstrated a loss of surface integrity at four weeks, fibrillations at eight weeks, and an increased modified Mankin score (p < 0.001). Cartilage thickness increased (p < 0.001), whereas chondrocyte density decreased at four weeks (p < 0.001), but then increased towards sham-operated levels (p < 0.01) at eight weeks. By week eight, chondrocyte pairing/clustering and cell volume increased (p < 0.05; p < 0.001, respectively). CONCLUSIONS: These in vivo results demonstrated for the first time that as a result of laminar airflow, cartilage degeneration occurred which has characteristics similar to those seen in early osteoarthritis. Maintenance of adequate cartilage hydration during open orthopaedic surgery is therefore of paramount importance.Cite this article: Dr A. Hall. Drying of open animal joints in vivo subsequently causes cartilage degeneration. Bone Joint Res 2016;5:137-144. DOI: 10.1302/2046-3758.54.2000594.

Self-assembly of gold supraparticles with crystallographically aligned and strongly coupled nanoparticle building blocks for SERS and photothermal therapy.

A new method is introduced for self-assembling citrate-capped gold nanoparticles into supraparticles with crystallographically aligned building blocks. It consists in confining gld nanoparticles inside a cellulose acetate membrane. The constituent nanoparticles are in close contact in the superstructure, and therefore generate hot spots leading to intense Surface-Enhanced Raman Scattering (SERS) signals. They also generate more plasmonic heat than the nanoparticle building blocks. The supraparticles are internalized by cells and show low cytotoxicity, but can kill cancer cells when irradiated with a laser. This, along with the improved plasmonic properties arising from their assembly, makes the gold supraparticles promising materials for applications in bioimaging and nanomedicine.

Airflow accelerates bovine and human articular cartilage drying and chondrocyte death.

OBJECTIVE: Exposure of articular cartilage to static air results in changes to the extracellular matrix (ECM) and stimulates chondrocyte death, which may cause joint degeneration. However during open orthopaedic surgery, cartilage is often exposed to laminar airflow, which may exacerbate these damaging effects. We compared drying in static and moving air in terms of cartilage appearance, hydration and chondrocyte viability, and tested the ability of saline-saturated gauze to limit the detrimental effects of air exposure. DESIGN: Articular cartilage from bovine metatarsophalangeal joints (N = 50) and human femoral heads (N = 6) was exposed for 90 min to (1) static air (2) airflow (up to 0.34 m/s), or (3) airflow (0.18 m/s), covered with gauze. Following air exposure, cartilage was also rehydrated (0.9% saline; 120 min) to determine the reversibility of drying effects. The influence of airflow was assessed by studying macroscopic appearance, and quantifying superficial zone (SZ) chondrocyte viability and cartilage hydration. RESULTS: Airflow caused advanced changes to cartilage appearance, accelerated chondrocyte death, and increased dehydration compared to static air. These effects were prevented if cartilage was covered by saline-saturated gauze. Cartilage rehydration reversed macroscopic changes associated with drying but the chondrocyte death was not altered. Chondrocytes at the cut edge of cartilage were more sensitive to drying compared to cells distant from the edge. CONCLUSIONS: Airflow significantly increased articular cartilage dehydration and chondrocyte death compared to static air. As laminar airflow is routinely utilised in operating theatres, it is essential that articular cartilage is kept wet via irrigation or by covering with saline-saturated gauze to prevent chondrocyte death.

Fat transforms ascorbic acid from inhibiting to promoting acid-catalysed N-nitrosation.

BACKGROUND: The major potential site of acid nitrosation is the proximal stomach, an anatomical site prone to a rising incidence of metaplasia and adenocarcinoma. Nitrite, a pre-carcinogen present in saliva, can be converted to nitrosating species and N-nitroso compounds by acidification at low gastric pH in the presence of thiocyanate. AIMS: To assess the effect of lipid and ascorbic acid on the nitrosative chemistry under conditions simulating the human proximal stomach. METHODS: The nitrosative chemistry was modelled in vitro by measuring the nitrosation of four secondary amines under conditions simulating the proximal stomach. The N-nitrosamines formed were measured by gas chromatography-ion-trap tandem mass spectrometry, while nitric oxide and oxygen levels were measured amperometrically. RESULTS: In absence of lipid, nitrosative stress was inhibited by ascorbic acid through conversion of nitrosating species to nitric oxide. Addition of ascorbic acid reduced the amount of N-nitrosodimethylamine formed by fivefold, N-nitrosomorpholine by >1000-fold, and totally prevented the formation of N-nitrosodiethylamine and N-nitrosopiperidine. In contrast, when 10% lipid was present, ascorbic acid increased the amount of N-nitrosodimethylamine, N-nitrosodiethylamine and N-nitrosopiperidine formed by approximately 8-, 60- and 140-fold, respectively, compared with absence of ascorbic acid. CONCLUSION: The presence of lipid converts ascorbic acid from inhibiting to promoting acid nitrosation. This may be explained by nitric oxide, formed by ascorbic acid in the aqueous phase, being able to regenerate nitrosating species by reacting with oxygen in the lipid phase.

Validation of techniques to detect illicit heroin use in patients prescribed pharmaceutical heroin for the management of opioid dependence.

BACKGROUND: The clinical implementation and evaluation of heroin substitution programmes have been confounded by the lack of objective and validated biomarkers for illicit heroin use in patients prescribed pharmaceutical heroin. This study examined the capacity to detect illicit heroin use by gas chromatography-mass spectrometry (GC-MS) analysis of urine samples for the presence of opium impurities common to illicit, but not pharmaceutical heroin. AIMS: To characterize the diagnostic properties of the metabolites of noscapine and papaverine in comparison to morphine as a gold-standard marker of illicit heroin use; and to examine the relationships between the self-reported time since most recent heroin use and the detection of these opioids in urine. DESIGN: A cross-sectional study of 52 opioid-dependent patients in treatment (not prescribed heroin), who self-reported illicit heroin use within the preceding 2 weeks. Self-report data regarding recent drug use and a urine sample were collected. GC-MS analyses of urines were conducted and reported by laboratory staff blinded to self-report data. FINDINGS: The metabolites of papaverine (hydroxypapaverine and dihydroxypapeverine) were found to have high sensitivity, specificity and negative predictive values as markers for illicit heroin use compared to the 'gold-standard' morphine. Other opioids, including 6-mono-acetylmorphine (6-MAM), codeine and noscapine metabolites (e.g. meconine) were less adequate in detecting heroin use. CONCLUSIONS: GC-MS detection of papaverine metabolites in urine appears to be suitable method of identifying illicit heroin use for clinical and research purposes.

Is there a cloud in the silver lining for imatinib?

Imatinib mesylate (Gleevec) or Glivec), a small molecule tyrosine kinase inhibitor for the treatment of chronic myeloid leukaemia, has been said to herald the dawn of a new era of rationally designed, molecularly targeted oncotherapy. Lurking on the same new horizon, however, is the age-old spectre of drug resistance. This review sets the intoxicating clinical perspective against the more sobering laboratory evidence of such divergent mechanisms of imatinib resistance as gene amplification and stem cell quiescence. Polychemotherapy has already been considered to combat resistance, but a more innovative, as yet unformulated, approach may be advocated.

The impact of sporadic campylobacter and salmonella infection on health and health related behaviour: a case control study.

The aim of the work was to explore the impact on general and psychological health of those with a proven bacterial gastrointestinal infection and to compare this with controls from whom no bacterial pathogen was identified. A case control study was conducted using an interviewer-administered questionnaire. Thirty-nine cases from whose faeces salmonella or campylobacter had been cultured were compared with matched controls. Reported gastrointestinal symptoms, general health and self-reported hygiene practices were compared. At the time of acute illness the General Household Questionnaire suggested similar levels of morbidity, though by follow up the controls were substantially more likely to be distressed. Cases were more likely to have changed their food preparation practices, to avoid certain eating places and to have been given advice about food preparation. In this small study a positive diagnosis of salmonella or campylobacter seems to have had a reassuring effect when compared with those for whom no diagnosis was made.

Analysis of urine for drugs of abuse using mixed-mode solid-phase extraction and gas chromatography-mass spectrometry.

A method for the simultaneous analysis of urine for the major drugs of abuse is described. The analytical procedure uses solid-phase extraction (SPE), gas chromatography-mass spectrometry (GC-MS) and a semi-automated identification process. It allows simultaneous extraction, derivatization and analysis of acidic, neutral and basic drugs from urine. Urine samples were subjected to enzymatic hydrolysis followed by SPE using Bakerbond narc-2 columns. The eluant was selectively derivatized with N-methyl-bis-trifluoroacetamide (MBTFA) and N-methyl-N-trimethylsilyltrifluoroacetamide + 1% trimethylchlorosilane. Analysis was performed using a GC-MS system operating in full scan mode. A simple macro programme was written to enhance the mass spectra identification capabilities of the MS software by producing extracted ion chromatograms (EIC) for the drugs of interest. Once a suspect compound was indicated by EIC, the mass spectrum of the compound was searched manually against reference libraries for positive identification and the retention time checked against that of the standard. This procedure has increased both the amount and the reliability of information given to clinicians without increasing the cost per sample. The system has been in routine operation for 24 months, processing up to 40 urine samples per day, with a usual turn-around time of 48 h.

Treatment of superficial ulcerative squamous cell carcinoma in three horses with topical 5-fluorouracil.

Three horses with superficial ulcerative squamous cell carcinoma were successfully treated with topical 5-fluorouracil. Two of them remain in remission after a single course of treatment and the third case is controlled by the application of the cream for seven days every six weeks. No systemic side-effects were observed but there was a localised inflammatory reaction in the skin in each case.

The maintenance of genetic polymorphism in small island populations: large mammals in the Hebrides.

Conventionally, small populations living on islands are expected to lose genetic variation by drift. Fluctuations in population size, combined with polygynous mating systems, are expected to contribute to the process by increasing sampling effects on genetic variation. However, in individually monitored populations of Red deer on Rum and Soay sheep on St. Kilda, which experience fluctuations in population size, two processes have been identified which mitigate loss of genetic variation. First, in a number of examples, population reductions are associated with selection. Selection may be in favour of heterozygotes, or, as we have documented in several cases, it may fluctuate in direction temporally. Second, in Soay sheep, in which mortality over population crashes is male-biased, ostensibly leading to low effective numbers of males, molecular studies show that there are systematic changes in the reproductive success of young males, and in variance in male success, that broaden genetic representation compared with expectation.

Orbital fat prolapse in the dog.

Five dogs presented with varying degrees of subconjunctival swelling and enophthalmos. Biopsy revealed the subconjunctival tissue to be fat. Surgical excision in two cases resulted in resolution of the clinical signs and demonstrated that the fat came from the retrobulbar region.

Enhancement of retinal acetylcholine release by DAMGO: possibly a direct opioid receptor-mediated excitatory effect.

1. An eye-cup preparation in anaesthetized rabbits was used to examine opioid modulation of acetylcholine (ACh) release from cholinergic neurones in the retina. 2. The mu-opioid receptor agonist, [D-Ala2, MePhe4, Gly-ol5]-enkephalin (DAMGO), when applied locally to the retina at concentrations between 1-30 microM significantly increased the light-evoked release of ACh. The effect of DAMGO was completely blocked by the selective mu-receptor antagonist CTOP but the kappa-receptor antagonist nor-binaltorphimine (norBNI) did not affect the action of DAMGO on ACh release indicating that the opioid produced its effect by activation of mu-receptors (the rabbit retina has negligible delta-receptors). 3. Blockade with bicuculline and strychnine of GABAergic and glycinergic inputs to the cholinergic neurones did not affect the action of DAMGO on ACh release. Also DAMGO did not reduce the potassium-evoked release of either GABA or glycine from rat isolated retinas. 4. Exposure of the rabbit retina to a combination of an A1-adenosine receptor antagonist, 8-cyclopentyl-1,3 dipropylxanthine (DPCPX), and adenosine deaminase did not affect the enhancing action of DAMGO on the light-evoked release of ACh. 5. When the retina in the rabbit eye-cup was exposed to kainate, the release of ACh was increased by approximately three times the resting release. In the presence of DAMGO the kainate-evoked release of ACh was enhanced by 44%. 6. These experiments show that activation of mu-opioid receptors by DAMGO increases the release of ACh elicited by physiological stimulation (flickering light). Since we could find no evidence thatDAMGO reduces inhibitory inputs to the cholinergic neurones, it seems that the enhancing action ofDAMGO on the light-evoked release of ACh involves a direct excitatory effect rather than disinhibition.This conclusion is supported by the enhancing action of DAMGO on the kainate-evoked release of ACh because kainate is thought to act directly on the cholinergic neurones.

Receptor binding in Japanese quail selected for long or short tonic immobility.

Japanese quail, selectively bred for long (LTI) and short (STI) tonic immobility (TI) responses, are thought to represent high and low fear groups, respectively. To study the neurochemical mechanisms underlying the behavioral distinctions, binding parameters were determined at the benzodiazepine, 5-HT1A, 5-HT3, alpha 2, and opioid receptor sites in the forebrains of the two lines. No differences were found in 5-HT1A, 5-HT3, alpha 2, mu- or kappa-opioid receptor binding between the lines. The KD for the binding of [3H]-flunitrazepam at the benzodiazepine receptor was significantly greater in the LTI than in the STI birds, indicating lower affinity for benzodiazepine ligands. The lines did not differ in benzodiazepine receptor number. Using [3H]-naltrindole, the LTI line was found to have fewer delta-opioid receptors than the STI line; the birds did not vary with respect to the affinity of these receptors. Thus, the selective breeding of the two lines has resulted in differences in benzodiazepine and delta-opioid binding, and these could produce differences in activity levels, fear, and pain responses, all of which could contribute to the tonic immobility response.

Characterization and amino acid sequence of Neisseria gonorrhoeae dihydrofolate reductase.

Dihydrofolate reductase has been purified from a trimethoprim-resistant strain of Neisseria gonorrhoeae. The enzyme showed a single component on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 18,000) and on isoelectric focusing in 5 M urea (pI = 6.8). Although gel electrophoresis under nondenaturing conditions resolved the preparation into two enzymatically active proteins (called form 1 and form 2), they were not genetically determined isozymes. Both had a similar dihydrofolate Km (2 microM), NADPH Km (10 microM), and trimethoprim Ki (20 nM), and form 2 (the slower migrating species) was shown to be generated from form 1 by the electrophoresis conditions. The complete covalent structure of the enzyme has also been determined. It is a single polypeptide composed of 162 residues and containing 4 cysteines. The gonococcal dihydrofolate reductase shares a 35% homology with the chicken liver enzyme and a 40% homology with the Escherichia coli enzyme. Most of these identities are residues that have been implicated in the binding of NADPH and methotrexate to the E. coli and Lactobacillus casei reductases.

Species-specific irreversible inhibition of Neisseria gonorrhoeae dihydrofolate reductase by a substituted 2,4-diamino-5-benzylpyrimidine.

Neisseria gonorrhoeae dihydrofolate reductase undergoes a time-dependent, irreversible inactivation by 2,4-diamino-5-[3,5-dimethoxy-4-(p-bromoacetamidophenoxy)benzyl] pyrimidine. The kinetics of inactivation are consistent with the reversible formation of an enzyme-inhibitor complex followed by covalent binding to the enzyme. The reversible component is competitive with dihydrofolate and has an inhibitor binding constant of 10 nM. Irreversible inactivation proceeds as a pseudo first-order process with a minimum inactivation half-time of 20 min and a Ki of 28 nM. Using radiolabeled inhibitor, it was shown that approximately 1 mol of ligand was covalently bound to the enzyme/mol of methotrexate binding site when the enzyme was completely inhibited. Radiolabeled inhibitor remained associated with the enzyme following denaturation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cyanogen bromide cleavage of the 14C-labeled enzyme-inhibitor complex yielded only one radioactive polypeptide, and sequence determinations showed that His-25 was modified by covalent attachment of the inhibitor. When dihydrofolate reductases from Lactobacillus casei, Streptococcus faecium, Escherichia coli, SR-1 rodent lymphoma, and chicken liver were tested with the affinity label, only the L. casei enzyme showed a time-dependent increase in inhibition. These data, along with comparisons of known amino acid sequences and x-ray crystal structures, were used to make predictions concerning the three-dimensional conformation of the gonococcal enzyme.

Intravesicular pH and iron uptake by immature erythroid cells.

The intravesicular pH of intact rabbit reticulocytes was measured by two methods; one based on the intracellular:extracellular distribution of DMO (5, 5, dimethyl + oxazolidin-2,4-dione), methylamine, and chloroquine and the other by quantitative fluorescence microscopy of cell-bound transferrin. The latter method was also applied to nucleated erythroid cells from the fetal rat liver. A pH value of approximately 5.4 was obtained with both methods and in both types of cells. Treatment of the cells with lysosomotrophic agents, metabolic inhibitors, and ionophores elevated the intravesicular pH and inhibited iron uptake from transferrin. When varying concentrations of NH4Cl were used, a close correlation was observed between the inhibition of iron uptake and elevation of the intravesicular pH. At pH 5.4 iron release from rabbit iron-bicarbonate transferrin in vitro was much more rapid than from iron-oxalate transferrin. The bicarbonate complex donates its iron to rabbit reticulocytes approximately twice as quickly as the oxalate complex. It is concluded that the acidic conditions within the vesicles provide the mechanism for iron release from the transferrin molecule after its endocytosis and that the low vesicular pH is dependent on cellular metabolism.

Immunity to canine adenovirus respiratory disease: effect of vaccination with an inactivated vaccine.

Nine puppies without maternal antibody to canine adenovirus (CAV) were divided into two groups. The first consisted of six puppies, each of which was given two doses of a commercial inactivated CAV-1 vaccine, 14 days apart. Eight days after administration of the second dose of vaccine, all six puppies, together with the second group, consisting of three unvaccinated controls, were challenged with an aerosol of virulent CAV-2. One dog from each group was killed on the third, fifth and 10th days after challenge and the three additional vaccinates killed at intervening times. All of the dogs developed respiratory signs, mainly coughing and tachypnoea, but the vaccinated dogs made a more rapid recovery. The lungs of both groups were consolidated, the areas affected being more extensive in the controls, and histological examination revealed the main lesion to be a severe necrotising bronchiolitis. Virus was isolated from the respiratory tissues and from throat swabs collected from both groups of dogs. The presence of neutralising antibody in the serum was not, of itself, sufficient to control viral replication and oblate the disease.

Inhibition of lipid peroxidation by the iron-binding protein lactoferrin.

Lactoferrin containing physiological amounts of iron is an inhibitor of lipid peroxidation induced by iron(III) salts and ascorbic acid. It might therefore help to protect neutrophils, inflammatory foci and secretions from metal-ion-dependent oxidative damage.