Direct toxicity of nonsteroidal antiinflammatory drugs for renal medullary cells

Antipyretic analgesics, taken in large doses over a prolonged period, cause a specific form of kidney disease, characterized by papillary necrosis and interstitial scarring. Epidemiological evidence incriminated mixtures of drugs including aspirin (ASA), phenacetin, and caffeine. The mechanism of toxicity is unclear. We tested the effects of ASA, acetaminophen (APAF, the active metabolite of phenacetin), caffeine, and other related drugs individually and in combination on mouse inner medullary collecting duct cells (mIMCD3). The number of rapidly proliferating cells was reduced by ≈50% by 0.5 mM ASA, salicylic acid, or APAF. The drugs had less effect on confluent cells, which proliferate slowly. Thus, the slow in vivo turnover of IMCD cells could explain why clinical toxicity requires very high doses of these drugs over a very long period. Caffeine greatly potentiated the effect of acetaminophen, pointing to a potential danger of the mixture. Cyclooxygenase (COX) inhibitors, indomethacin and NS-398, did not reduce cell number except at concentrations greatly in excess of those that inhibit COX. Therefore, COX inhibition alone is not toxic. APAF arrests most cells in late G1 and S and produces a mixed form of cell death with both oncosis (swollen cells and nuclei) and apoptosis. APAF is known to inhibit the synthesis of DNA and cause chromosomal aberrations due to inhibition of ribonucleotide reductase. Such effects of APAF might account for renal medullary cell death in vivo and development of uroepithelial tumors from surviving cells that have chromosomal aberrations.

Cellular mechanisms of neuropathic pain, morphine tolerance, and their interactions

Compelling evidence has accumulated over the last several years from our laboratory, as well as others, indicating that central hyperactive states resulting from neuronal plastic changes within the spinal cord play a critical role in hyperalgesia associated with nerve injury and inflammation. In our laboratory, chronic constriction injury of the common sciatic nerve, a rat model of neuropathic pain, has been shown to result in activation of central nervous system excitatory amino acid receptors and subsequent intracellular cascades including protein kinase C translocation and activation, nitric oxide production, and nitric oxide-activated poly(ADP ribose) synthetase activation. Similar cellular mechanisms also have been implicated in the development of tolerance to the analgesic effects of morphine. A recently observed phenomenon, the development of “dark neurons,” is associated with both chronic constriction injury and morphine tolerance. A site of action involved in both hyperalgesia and morphine tolerance is in the superficial laminae of the spinal cord dorsal horn. These observations suggest that hyperalgesia and morphine tolerance may be interrelated at the level of the superficial laminae of the dorsal horn by common neural substrates that interact at the level of excitatory amino acid receptor activation and subsequent intracellular events. The demonstration of interrelationships between neural mechanisms underlying hyperalgesia and morphine tolerance may lead to a better understanding of the neurobiology of these two phenomena in particular and pain in general. This knowledge may also provide a scientific basis for improved pain management with opiate analgesics.

Agonist-selective endocytosis of mu opioid receptor by neurons in vivo.

Opiate alkaloids are potent analgesics that exert multiple pharmacological effects in the nervous system by activating G protein-coupled receptors. Receptor internalization upon stimulation may be important for desensitization and resensitization, which affect cellular responsiveness to ligands. Here, we investigated the agonist-induced internalization of the mu opioid receptor (MOR) in vivo by using the guinea pig ileum as a model system and immunohistochemistry with an affinity-purified antibody to the C terminus of rat MOR. Antibody specificity was confirmed by the positive staining of human embryonic kidney 293 cells transfected with epitope-tagged MOR cDNA, by the lack of staining of cells transfected with the delta or kappa receptor cDNA, and by the abolition of staining when the MOR antibody was preadsorbed with the MOR peptide fragment. Abundant MOR immunoreactivity (MOR-IR) was localized to the cell body, dendrites, and axonal processes of myenteric neurons. Immunostaining was primarily confined to the plasma membrane of cell bodies and processes. Within 15 min of an intraperitoneal injection of the opiate agonist etorphine, intense MOR-IR was present in vesicle-like structures, which were identified as endosomes by confocal microscopy. At 30 min, MOR-IR was throughout the cytoplasm and in perinuclear vesicles. MOR-IR was still internalized at 120 min. Agonist-induced endocytosis was completely inhibited by the opiate antagonist naloxone. Interestingly, morphine, a high-affinity MOR agonist, did not cause detectable internalization, but it partially inhibited the etorphine-induced MOR endocytosis. These results demonstrate the occurrence of agonist-selective MOR endocytosis in neurons naturally expressing this receptor in vivo and suggest the existence of different mechanisms regulating cellular responsiveness to ligands.

Impacto no sistema imunológico da utilização prolongada de morfina para tratamento da dor

Introdução. Apesar das evidências dos efeitos imunomodulatórios da morfina, não há na literatura estudos que tenham comparado a interação entre citocinas, imunidade celular (linfócitos T, B e NK) e a administração prolongada de morfina administrada pelas vias oral ou intratecal em doentes com dor crônica neuropática não relacionada ao câncer. Foram avaliados de forma transversal e comparativa 50 doentes com diagnóstico de dor lombar crônica e com presença de radiculopatia (dor neuropática) previamente operados para tratar hérnia discal lombar (Síndrome Dolorosa Pós- Laminectomia), sendo 18 doentes tratados prolongadamente com infusão de morfina pela via intratecal com uso de sistema implantável no compartimento subaracnóideo (grupo intratecal); 17 doentes tratados prolongadamente com morfina pela via oral (n=17) e 15 doentes tratados com fármacos mas sem opióides (grupo sem opioide). Foram analisadas as concentração das citocinas IL-2, IL-4, IL-8, TNFalfa, IFNy, IL-5, GM-CSF, IL-6, IL-10 e IL-1beta no plasma e no líquido cefalorraquidiano; imunofenotipagem de linfócitos T, B e células NK e avaliados os Índice de Escalonamento de Opióide (em percentagem de opióide utilizada e em mg), dose cumulativa de morfina (mg), duração do tratamento em meses, dose final de morfina utilizada (em mg), e equivalente de morfina por via oral (em mg). Resultados. Não houve diferença estatisticamente significativa entre o número de linfócitos T, B e NK nos doentes com morfina administrada pelas vias IT, VO e os não usuários de morfina. Houve correlação positiva entre as concentrações de linfócitos T CD4 e o Índice de Escalonamento de Opióide (em % e mg) nos doentes tratados com morfina por via intratecal. Houve correlação negativa entre as concentrações de células NK (CD56+) e o Índice de Escalonamento de Opióide (em % e mg) nos doentes tratados com morfina por via intratecal. Houve correlação positiva entre o número de células NK (CD56+) e a dose cumulativa de morfina (em mg) administrada pelas vias intratecal e oral. Houve correlação positiva entre as concentrações de linfócitos T CD8 e a duração do tratamento em meses nos doentes tratados com morfina pela via oral. As concentrações de IL-8 e IL-1beta foram maiores no LCR do que no plasma em todos os doentes da amostra analisada. As concentrações de IFNy no LCR foram maiores nos doentes que utilizavam morfina pela via oral e nos não usuários de morfina do que nos que a utilizavam pela via intratecal. As concentrações de plasmáticas de IL-5 foram maiores nos doentes utilizavam morfina pela via oral ou intratecal do que nos que não a utilizavam. A concentração de IL-5 no LCR correlacionou-se negativamente com a magnitude da dor de acordo com a EVA nos doentes tratados com morfina pelas via oral ou intratecal. Nos doentes tratados com morfina pelas via oral ou intratecal, a concentração de IL-2 no LCR correlacionou-se positivamente com a magnitude da dor de acordo com a EVA e negativamente com o Índice de Escalonamento de Opióide (em % e mg) e a dose cumulativa de morfina (em mg). As concentrações plasmáticas de GMCSF foram maiores nos doentes utilizavam morfina pela via oral ou intratecal do que nos não a utilizavam. A concentração de TNFalfa no LCR nos doentes tratados com morfina pela via intratecal correlacionou-se negativamente com o Índice de Escalonamento de Opióide (em % e mg), a dose cumulativa de morfina (em mg) e dose equivalente por via oral (em mg) de morfina. A concentração plasmática das citocinas IL-6 e IL-10 correlacionou-se negativamente com a duração do tratamento (em meses) nos doentes tratados com morfina administrada pela via oral. O Índice de Escalonamento de Opióide (em mg e %) correlacionou-se negativamente com as concentrações no LCR de IL-2 e TNFalfa nos doentes tratados com morfina administrada pela via intratecal. O Índice de Escalonamento de Opióide (em mg e %) correlacionou-se negativamente com as concentrações no LCR de IL-2 e IL-5 nos doentes tratados com morfina administrada pela via oral. Houve correlação negativa entre a intensidade da dor de acordo com a EVA e as concentrações de IL-5 e IL-2 no LCR nos doentes tratados com morfina administrada pelas vias oral e intratecal. Houve correlação negativa entre a intensidade da dor de acordo com a EVA e as concentrações plasmáticas de IL-4 nos doentes tratados com morfina administrada pela via intratecal. Houve correlação negativa entre a intensidade da dor de acordo com a EVA e as concentrações plasmáticas de IL-1beta nos doentes tratados com morfina administrada pela via intratecal. Conclusões: Os resultados sugerem associações entre citocinas e imunidade celular (células T , B e NK) e o tratamento prolongado com morfina administrada pela via oral ou intratecal. Estes resultados podem contribuir para a compreensão da imunomodulação da morfina administrada por diferentes vias em doentes com dor neuropática crônica não oncológica . São necessários mais estudos sobre os efeitos da morfina sobre o sistema imunológico

Inequality in analgesic prescription in Spain: a gender development issue

Objectives: It is well known that sex differences in analgesic prescription are not merely the logical result of greater prevalence of pain in women, since this therapeutic variability is related to factors such as educational level or social class. This study aims to analyse the relationship between analgesic prescription and gender development in different regions of Spain. Methods: Cross-sectional study of sex-differences in analgesic prescription according to the gender development of the regions studied. Analgesic prescription, pain and demographic variables were obtained from the Spanish Health Interview Survey in 2006. Gender development was measured with the Gender Development Index (GDI). A logistic regression analysis was conducted to compare analgesic prescription by sex in regions with a GDI above or below the Spanish average. Results: Once adjusted by pain, age and social class, women were more likely to be prescribed analgesics than men, odds ratio (OR) = 1.74 (1.59-1.91), as residents in regions with a lower GDI compared with those in region with a higher GDI: ORWomen = 1.26 (1.12-1.42), ORMen = 1.30 (1.13-1.50). Women experiencing pain in regions with a lower GDI were more likely than men to be treated by a general practitioner rather than by a specialist, OR = 1.32 (1.04-1.67), irrespective of age and social class. Conclusions: Gender bias may be one of the pathways by which inequalities in analgesic treatment adversely affect women's health. Moreover, research into the adequacy of analgesic treatment and the possible medicalisation of women should consider contextual factors, such as gender development.

Ototoxicidade

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Crioterapia como tratamento de entorses de tornozelos de atletas: revisão sistemática

A crioterapia é prática comum na medicina esportiva, pela praticidade, facilidade de acesso e baixo custo, possuindo vantajoso uso na Saúde Pública. No entanto, os efeitos analgésicos do gelo tem pouca base objetiva e sistematizada em termos de técnicas, duração e frequência. O objetivo deste estudo foi sintetizar através da revisão sistemática (RS) as evidências relativas à efetividade da crioterapia para o tratamento das entorses de tornozelo de atletas. A RS é um método de pesquisa observacional e retrospectivo, pelo qual se tratam artigos, preferencialmente Ensaios Clínicos Aleatórios – ECA, como sujeitos da investigação, com rigorosos critérios de inclusão e exclusão e, quando possível, realiza-se uma macro estatística dos resultados – metaanálise. No presente RS, foram consultados cinco bancos de dados - Medline, Embase, Cochrane, Lilacs e PEDro para buscar ECA sobre crioterapia com os desfechos dor, edema, rigidez e função. Resultados: 289 estudos foram identificados inicialmente, dos quais nove com tratamentos isolados ou associados à crioterapia, porém apenas um preencheu aos critérios de inclusão, cujo N era 121 atletas, dos quais 64 receberam a crioterapia (funcional) e 57 no grupo controle (imobilização). Maior probabilidade para o evento dor foi observada no grupo controle, após 3 e 12 meses. A RS revelou uma lacuna em ECA dentro do tema, mas não encontrou efeito adverso na prática da crioterapia, sendo um princípio analgésico importante, sobretudo em lesões de tecidos moles.

Sleepiness, sleep-disordered breathing, and accident risk factors in commercial vehicle drivers

Sleep-disordered breathing and excessive sleepiness may be more common in commercial vehicle drivers than in the general population. The relative importance of factors causing excessive sleepiness and accidents in this population remains unclear. We measured the prevalence of excessive sleepiness and sleep-disordered breathing and assessed accident risk factors in 2,342 respondents to a questionnaire distributed to a random sample of 3,268 Australian commercial vehicle drivers and another 161 drivers among 244 invited to undergo polysomnography. More than half (59.6%) of drivers had sleep-disordered breathing and 15.8% had obstructive sleep apnea syndrome. Twenty-four percent of drivers had excessive sleepiness. Increasing sleepiness was related to an increased accident risk. The sleepiest 5% of drivers on the Epworth Sleepiness Scale and Functional Outcomes of Sleep Questionnaire had an in-creased risk of an accident (odds ratio [OR] 1.91, p = 0.02 and OR 2.23, p < 0.01, respectively) and multiple accidents (OR 2.67, p < 0.01 and OR 2.39, p = 0.01), adjusted for established risk factors. There was an increased accident risk with narcotic analgesic use (OR 2.40, p < 0.01) and antihistamine use (OR 3.44, p = 0.04). Chronic excessive sleepiness and sleep-disordered breathing are common in Australian commercial vehicle drivers. Accident risk was related to increasing chronic sleepiness and antihistamine and narcotic analgesic use.

Paracetamol [acetaminophen]-induced gastrotoxicity: Revealed by induced hyperacidity in combination with acute or chronic inflammation

Paracetamol is regarded as a relatively safe drug in the gastro-duodenal region of humans but recent epidemiological investigations have suggested that at high doses there may be an increased risk of ulcers and bleeding. To investigate the possibility that inflammatory conditions and gastric acidity may play a role in potentiating development of gastric mucosal injury from paracetamol in rats (as noted previously with various non-steroidal anti-inflammatory drugs) we studied the gastric irritant effects of paracetamol and some phenolic and non-phenolic analgesics and antipyretics in rats with adjuvant or collagen II induced arthritis or zymosan-induced paw inflammation and given 1.0 ml hydrochloric acid (HCl) 0.1 M and/or an i. p. injection of the cholinomimetic, acetyl-β-methyl choline chloride 5.0 mg/kg. Gastric lesions were determined 2 h after oral administration of 100 or 250 mg/kg paracetamol or at therapeutically effective doses of the phenolic or non-phenolic analgesics/antipyretics. The results showed that gastric mucosal injury occurred with all these agents when given to animals that received all treatments so indicating there is an adverse synergy of these three factors, namely: (i) intrinsic disease; (ii) hyperacidity; and (iii) vagal stimulation for rapidly promoting gastric damage, both in the fundic as well as the antral mucosa, for producing gastric damage by paracetamol, as well as the other agents. Removing one of these three predisposing factors effectively blunts/abolishes expression of this paracetamol-induced gastrotoxity in rats. These three factors, without paracetamol, did not cause significant acute gastropathy.

Regional differences in the in vitro penetration of hydrocortisone through equine skin

Little is known about the transdermal penetration of hydrocortisone in the horse and, although commercial formulations containing hydrocortisone are registered for topical use in the horse, there have been no studies investigating the movement of this glucocorticoid through different regions of equine skin. Skin was harvested from the thorax, groin and leg (dorsal metacarpal) regions of five Thoroughbred geldings and frozen (-20 degrees C) until required. Defrosted skin was placed in Franz-type diffusion cells and the amount of radiolabelled (H-3) hydrocortisone, in a saturated solution of unlabelled hydrocortisone in 50% ethanol (w/w), which penetrated through and remained within skin samples was measured over 24 h. Significantly higher (P < 0.001) maximum flux (J(max); mol/cm(2)/h) was measured when hydrocortisone was applied to skin from the leg, compared to thorax and groin, although significantly less hydrocortisone (P < 0.001) was retained within skin from the leg at 24 h. Topical application of hydrocortisone in a vehicle containing ethanol would penetrate faster through leg skin from the lower leg when compared with the thorax or groin, which depending on cutaneous blood flow, may result in higher systemic drug concentrations or greater efficiency in treating local inflamed tissue.

WOMAC 20, 50, 70 response levels in patients treated with Rofecoxib for osteoarthritis

Aim of study: The goal of this post-hoc analysis was to examine the difference between treatment groups when varying the target response level from at least a 20% improvement from baseline, to at least 50% and 70% improvements in Phase III studies of rofecoxib in patients with osteoarthritis. Methods: The analysis focused on results from two 6-week, placebo-controlled, ibuprofen-comparator, Phase III osteoarthritis studies. These studies employed a flare design requiring a minimum level of symptoms at entry following discontinuation of prior analgesics. Two definitions of ‘‘patient improved’’ from baseline were used: (1) WOMAC-P: a reduction in the WOMAC pain score and (2) WOMAC-PFS: a reduction in the WOMAC pain score and either a reduction in the WOMAC stiffness or function score. The improvement target was increased from 20% to 50% to 70%, relative to baseline, to investigate how the increase affects the ability to detect the differences between treatment groups. Analyses were conducted on the average and last of all measurements collected during a 6-week treatment period. Results: In the ibuprofen-comparator studies, 1545 patients were randomized to placebo, rofecoxib 12.5 mg once daily, rofecoxib 25 mg once daily, and ibuprofen 800 mg three times daily in a 1:3:3:3 ratio. The percentages of patients who met the improvement targets decrease as the target increases from 20% to 50% to 70%. There were meaningful differences between the active treatment and placebo that were inversely related to the improvement target. For example, there was a 31 (P ! 0.001), 21 (P ! 0.001), and 12 (P ! 0.001) percentage-point difference between rofecoxib 25 mg and placebo for the 20%, 50%, and 70% targets for WOMAC-P. For WOMAC-PFS, the differences between rofecoxib 25 mg and placebo were 33 (P ! 0.001), 18 (P ! 0.001), and 9 (P ! 0.01) percentage points for the 20%, 50%, and 70% improvement targets. Conclusions: Meaningful differences between active treatments and placebo were detected at all three response levels associated with the WOMAC-P and WOMAC-PFS endpoints. The differences between groups were more dramatic at the 20% and 50% response levels. The WOMAC (20,50,70)-P and WOMAC (20,50,70)-PFS endpoints further confirm, at an individual patient level, the clinical benefit of rofecoxib in the treatment of osteoarthritis that was previously reported as a difference in means.

Prevalence of patient with low intensity pain symptom severity states during treatment with Rofecoxib and Ibuprofen for osteoarthritis

Aim of study: To examine the prevalence of low intensity symptom severity states in patients taking placebo, rofecoxib 12.5 mg once daily, rofecoxib 25 mg once daily, or ibuprofen 800 mg three times daily using a post-hoc definition of low pain intensity states (BLISS Index) based on the WOMAC Index. Methods: Two 6-week, double-blind, parallel-group, placebocontrolled, ibuprofen-comparator studies were conducted to measure the efficacy of rofecoxib in patients with knee or hip osteoarthritis. These studies employed a flare design requiring a minimum level of symptoms at entry following discontinuation of prior analgesics. The WOMAC Pain subscale (100 mm visual analog scale) was used as the pain measure. In separate analyses, WOMAC pain subscale scores from each patient were compared to five thresholds of pain:%5 mm, %10 mm, %15 mm, %20 mm, and %25 mm. The percent of patients with BLISS states (1) on average over 6 weeks, (2) at any time during the study, and (3) at week 6 was computed for each treatment group and threshold. The treatment group percentages were compared using Fisher’s exact test. Results: During the study, patients received placebo (N Z 143), rofecoxib 12.5 mg (N Z 461), rofecoxib 25 mg (N Z 459), or ibuprofen (N Z 465). For each pain threshold and treatment group, the percent of patients with BLISS states at any time (e.g., 50% for rofecoxib 25 mg) exceeded the percentage at week 6 (e.g., 40% for rofecoxib 25 mg) which, in turn, exceeded the percentage with BLISS states on average (e.g., 32% for rofecoxib 25 mg). The percentages of patients in the active treatment groups with BLISS states on average were significantly different than observed in the placebo group at the%15 mm threshold (8–11% points vs placebo, P ! 0.01), %20 mm level (10–15% points, P ! 0.01), and %25 mm level (14–17% points, P ! 0.001). Significant differences between the active treatments and placebo were also observed at the %10 mm threshold (8–9% points, P ! 0.05) for measurements at week 6 and at the%10 (12–14% points, P !0.001) and%5 mm thresholds (5–7% points, P ! 0.05) for patients with BLISS states at any time. Conclusion: These measures of BLISS states differentiate all three active treatment groups from placebo and further confirm, at an individual patient level, the clinical benefit of rofecoxib in the treatment of osteoarthritis. Furthermore, they provide information on the prevalence of patients achieving low (%15 mm, %20 mm, %25 mm), and very low (%5 mm, %10 mm) pain severity states.

Opioids and NMDA receptors: Neuro-excitation and abnormal pain behaviours

In the clinical setting, chronic administration of high doses of systemic morphine may result in neuro-excitatory behaviours such as myoclonus and allodynia in some patients. Additionally, high doses of m-opioid agonists such as morphine administered chronically by the intrathecal route in both rats and humans, as well as DAMGO in rats, have been reported to produce neuro-excitatory behaviours. However, more recently, it has begun to be appreciated that even at normal analgesic doses, opioids such as morphine are capable not only of activating pain inhibitory systems (analgesia/antinociception), but they also activate pain facilitatory systems such that post-opioid allodynia/hyperalgesia may be evident after cessation of opioid treatment. Whilst it is well documented that opioid receptors mediate the inhibitory effects of opioid analgesics, the excitatory and pro-nociceptive effects of opioids appear to involve indirect activation of N-methyl-D-aspartate (NMDA) receptors, such that the extent of pain relief produced may be the net effect of these two opposing actions. Apart from the NMDA-nitric oxide (NO) pro-nociceptive signaling cascade, considerable evidence also implicates dynorphin A as well as the endogenous anti-opioid peptides cholecystokinin (CCK), neuropeptide FF (NPFF) and orphanin FQ/nociceptin, in mediating opioid-induced neuro-excitation and abnormal pain behaviours. Apart from the neuro-excitatory effects that may be produced by the parent opioid, systemic administration of some opioid analgesics such as morphine and hydromorphone in rats and humans results in their rapid conversion to 3-glucuronide metabolites that also contribute significantly to the neuro-excitatory and abnormal pain behaviours produced