Endocannabinoid analysis in GlucoEXACT plasma: Method validation and sample handling recommendations.

Endocannabinoids (ECs), such as anandamide and 2-arachidonyl glycerol (2-AG), contribute to the pathology of inflammatory, malignant, cardiovascular, metabolic and mental diseases. The reliability of quantitative analyses in biological fluids of ECs and endocannabinoid-like (EC-like) substances depends on pre-analytical conditions such as temperature and "time-to-centrifugation". Standardization of these parameters is critical for valid quantification and implementation in clinical research. In this study, we compared concentrations obtained with GlucoEXACT blood collection tubes versus K3EDTA tubes and employed the optimized procedure to assess ECs profiles in patients with inflammatory skin disease and healthy controls. A UHPLC-MS/MS method was validated for human plasma from GlucoEXACT blood collection tubes according to EMA and FDA guidelines, and pre-analytical conditions were systematically modified to assess analyte stability and optimize the procedures. The results showed significantly lower concentrations of ECs and EC-like substance concentrations with GlucoEXACT tubes compared with K3EDTA tubes, and GlucoEXACT extended the time window of stable concentrations. The strongest method-disagreement occurred for 1/2-AG suggesting that GlucoEXACT delayed ex vivo isomer rearrangement. Hence, GlucoExact tubes were superior in terms of stability and reliability. However, although absolute concentrations obtained with GlucoExact and K3EDTA differed, linear regression studies showed high agreement (except for 1/2-AG), and both methods showed similar EC profiles and similar disease-dependent pro-inflammatory patterns in dermatology patients. Hence, despite the obstacles in EC analyses, implementation of optimized pre-analytical blood collection and sample processing procedures provide reliable insight into peripheral ECs.

Current evidence of cannabinoid-based analgesia obtained in preclinical and human experimental settings.

Cannabinoids have a long record of recreational and medical use and become increasingly approved for pain therapy. This development is based on preclinical and human experimental research summarized in this review. Cannabinoid CB1 receptors are widely expressed throughout the nociceptive system. Their activation by endogenous or exogenous cannabinoids modulates the release of neurotransmitters. This is reflected in antinociceptive effects of cannabinoids in preclinical models of inflammatory, cancer and neuropathic pain, and by nociceptive hypersensitivity of cannabinoid receptor-deficient mice. Cannabis-based medications available for humans mainly comprise Δ9 -tetrahydrocannabinol (THC), cannabidiol (CBD) and nabilone. During the last 10 years, six controlled studies assessing analgesic effects of cannabinoid-based drugs in human experimental settings were reported. An effect on nociceptive processing could be translated to the human setting in functional magnetic resonance imaging studies that pointed at a reduced connectivity within the pain matrix of the brain. However, cannabinoid-based drugs heterogeneously influenced the perception of experimentally induced pain including a reduction in only the affective but not the sensory perception of pain, only moderate analgesic effects, or occasional hyperalgesic effects. This extends to the clinical setting. While controlled studies showed a lack of robust analgesic effects, cannabis was nearly always associated with analgesia in open-label or retrospective reports, possibly indicating an effect on well-being or mood, rather than on sensory pain. Thus, while preclinical evidence supports cannabinoid-based analgesics, human evidence presently provides only reluctant support for a broad clinical use of cannabinoid-based medications in pain therapy. SIGNIFICANCE: Cannabinoids consistently produced antinociceptive effects in preclinical models, whereas they heterogeneously influenced the perception of experimentally induced pain in humans and did not provide robust clinical analgesia, which jeopardizes the translation of preclinical research on cannabinoid-mediated antinociception into the human setting.

Dysregulation of lysophosphatidic acids in multiple sclerosis and autoimmune encephalomyelitis.

Bioactive lipids contribute to the pathophysiology of multiple sclerosis. Here, we show that lysophosphatidic acids (LPAs) are dysregulated in multiple sclerosis (MS) and are functionally relevant in this disease. LPAs and autotaxin, the major enzyme producing extracellular LPAs, were analyzed in serum and cerebrospinal fluid in a cross-sectional population of MS patients and were compared with respective data from mice in the experimental autoimmune encephalomyelitis (EAE) model, spontaneous EAE in TCR1640 mice, and EAE in Lpar2 -/- mice. Serum LPAs were reduced in MS and EAE whereas spinal cord LPAs in TCR1640 mice increased during the 'symptom-free' intervals, i.e. on resolution of inflammation during recovery hence possibly pointing to positive effects of brain LPAs during remyelination as suggested in previous studies. Peripheral LPAs mildly re-raised during relapses but further dropped in refractory relapses. The peripheral loss led to a redistribution of immune cells from the spleen to the spinal cord, suggesting defects of lymphocyte homing. In support, LPAR2 positive T-cells were reduced in EAE and the disease was intensified in Lpar2 deficient mice. Further, treatment with an LPAR2 agonist reduced clinical signs of relapsing-remitting EAE suggesting that the LPAR2 agonist partially compensated the endogenous loss of LPAs and implicating LPA signaling as a novel treatment approach. Graphical summary of lysophosphatidic signaling in multiple sclerosis.

Pro-inflammatory obesity in aged cannabinoid-2 receptor-deficient mice.

BACKGROUND AND OBJECTIVES: Cannabinoid-1 receptor signaling increases the rewarding effects of food intake and promotes the growth of adipocytes, whereas cannabinoid-2 receptor (CB2) possibly opposes these pro-obesity effects by silencing the activated immune cells that are key drivers of the metabolic syndrome. Pro- and anti-orexigenic cannabimimetic signaling may become unbalanced with age because of alterations of the immune and endocannabinoid system. METHODS: To specifically address the role of CB2 for age-associated obesity, we analyzed metabolic, cardiovascular, immune and neuronal functions in 1.2-1.8-year-old CB2(-/-) and control mice, fed with a standard diet and assessed effects of the CB2 agonist, HU308, during high-fat diet (HFD) in 12-16-week-old mice. RESULTS: The CB2(-/-) mice were obese with hypertrophy of visceral fat, immune cell polarization toward pro-inflammatory subpopulations in fat and liver and hypertension, as well as increased mortality despite normal blood glucose. They also developed stronger paw inflammation and a premature loss of transient receptor potential responsiveness in primary sensory neurons, a phenomenon typical for small fiber disease. The CB2 agonist HU308 prevented HFD-evoked hypertension, reduced HFD-evoked polarization of adipose tissue macrophages toward the M1-like pro-inflammatory type and reduced HFD-evoked nociceptive hypersensitivity, but had no effect on weight gain. CONCLUSIONS: CB2 agonists may fortify CB2-mediated anti-obesity signaling without the risk of anti-CB1-mediated depression that caused the failure of rimonabant.

Multiple rodent models and behavioral measures reveal unexpected responses to FTY720 and DMF in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a widely-used rodent model for multiple sclerosis (MS), but a single model can hardly capture all features of MS. We investigated whether behavioral parameters in addition to clinical motor function scores could be used to assess treatment efficacy during score-free intervals in the relapsing-remitting EAE model in SJL/J mice. We studied the effects of the clinical reference compounds FTY720 (fingolimod, 0.5mg/kg/day) and dimethyl fumarate (DMF, 20-30 mg/kg/day) on clinical scores in several rodent EAE models in order to generate efficacy profiles. SJL/J mice with relapsing-remitting EAE were studied using behavioral tests, including rotarod, gait analysis, locomotor activity and grip strength. SJL/J mice were also examined according to Crawley's sociability and preference for social novelty test. Prophylactic treatment with FTY720 prevented clinical scores in three of the four EAE rodent models: Dark Agouti (DA) and Lewis rats and C57BL/6J mice. Neither prophylactic nor late-therapeutic treatment with FTY720 reduced clinical scores or reversed deficits in the rotarod test in SJL/J mice, but we observed effects on motor functions and sociability in the absence of clinical scores. Prophylactic treatment with FTY720 improved the gait of SJL/J mice whereas late-therapeutic treatment improved manifestations of reduced social (re)cognition or preference for social novelty. DMF was tested in three EAE models and did not improve clinical scores at the dose used. These data indicate that improvements in behavioral deficits can occur in absence of clinical scores, which indicate subtle drug effects and may have translational value for human MS.

Congenital taurine deficiency in mice is associated with reduced sensitivity to nociceptive chemical stimulation.

The amino acid taurine is required for development and functioning of the central and peripheral nervous system where it exerts osmoregulatory, neuromodulatory and anti-apoptotic actions. It is subject to cellular import by the taurine transporter slc6a6. Absence of the transporter and consequently, absence of taurine leads to several neurologic deficits and sensory losses. In a slc6a6 knock-out mouse model, consequences of congenital taurine deficiency were assessed in nociceptive sensory processes. The formalin assay, hot plate assay, and summated generator potentials in response to local nociceptive stimulation with gaseous CO2 were applied. Reduced responsiveness of slc6a6(-/-) mice to nociceptive stimulation was observed in particular to chemical nociceptive stimuli. Scl6a6 knock-out mice spent significantly less time licking the formalin injected paw and displayed smaller amplitudes of the nociceptive nasal mucosa potentials than wild-type mice (p=0.002 and 0.01 respectively). In contrast, withdrawal latencies on a hot plate did not significantly differ, suggesting that intracellular taurine deficits lead in particular to a hyposensitivity of nociceptive sensory neurons sensitive to noxious chemical stimulation. As hereditary absence of taurine affects biological processes of anatomical structure development, the altered nociceptive responses likely reflect consequences of compromised peripheral nervous system development.

Contribution of genetic variants to pain susceptibility in Parkinson disease.

BACKGROUND: Pain is a one of the most disturbing non-motor symptoms of Parkinson disease (PD). The susceptibility to pain varies substantially among patients with PD. The aim of this study was to assess a potential association of genetic variants to PD-related pain. METHODS: We analysed 20 candidate SNPs from 12 genes previously reported to be associated with various pain phenotypes in a homogeneous group of 229 Israeli Jewish PD patients, with and without pain (n = 165 and 64, respectively). RESULTS: The statistical analysis accounted for the potential influence of demographic and clinical factors. The non-synonymous rs6746030 single nucleotide polymorphism (SNP) of the SCN9A gene, which alters the coding sequence of the sodium channel Nav1.7 (arginine to tryptophan), was nominally associated with PD-related pain susceptibility (p = 0.037), as well as with central and musculoskeletal pain subtypes independently. The synonymous rs324419 SNP of the FAAH gene which encodes fatty acid amide hydrolase, a cannabinoid metabolizing enzyme, was associated with PD-related pain (p = 0.006) and specifically with the musculoskeletal subtype. The FAAH haplotype of rs324419 and rs2295633 SNPs, which was previously associated with the variability in pain response in humans, was also associated with PD-related pain (p = 0.012) and specifically with PD-related musculoskeletal pain. CONCLUSIONS: Variants within in the SCN9A and FAAH genes were associated with the risk of pain in PD patients. These findings may contribute to our understanding of pain mechanisms of PD and to direct future therapies.

Genetically polymorphic OCT1: another piece in the puzzle of the variable pharmacokinetics and pharmacodynamics of the opioidergic drug tramadol.

We investigated whether tramadol or its active metabolite, O-desmethyltramadol, are substrates of the organic cation transporter OCT1 and whether polymorphisms in OCT1 affect tramadol and O-desmethyltramadol pharmacokinetics. Tramadol showed high permeability through parallel artificial membrane permeability assays (PAMPAs). Tramadol uptake in HEK293 cells did not change after OCT1 overexpression, and the concentrations of tramadol in the plasma of healthy volunteers were independent of their OCT1 genotypes. In contrast, O-desmethyltramadol showed low membrane permeability, and OCT1 overexpression increased O-desmethyltramadol uptake 2.4-fold. This increase in uptake was reversed by OCT1 inhibitors and absent when loss-of-function OCT1 variants were overexpressed. Volunteers carrying loss-of-function OCT1 polymorphisms had significantly higher plasma concentrations of O-desmethyltramadol (P = 0.002, n = 41) and significantly prolonged miosis, a surrogate marker of opioidergic effects (P = 0.005, n = 24). In conclusion, polymorphisms in OCT1 influence the pharmacokinetics of O-desmethyltramadol, presumably by affecting its uptake into liver cells, and thus may modulate the efficacy of tramadol treatment.

Accumulation of phosphorylated I kappaB alpha and activated IKK in nodes of Ranvier.

AIMS: Nuclear factor-kappaB (NF-kappaB) is an ubiquitously expressed transcription factor that modulates inducible gene transcription crucial for the regulation of immunity, inflammatory processes, and cell survival. In the mammalian nervous system, constitutive NF-kappaB activation is considered to promote neuronal cell survival by preventing apoptosis. Increasing evidence suggests a critical role for NF-kappaB activation in acute and chronic neurodegenerative diseases. Recently, a striking enrichment of phosphorylated I kappaB alpha (pI kappaB alpha) and activated I KappaB Kinase (IKK), two key components of the NF-kappaB activation pathway, was demonstrated in the axon initial segment (AIS) of neurons. As the AIS shares fundamental features with nodes of Ranvier (NR), we examined whether pI kappaB alpha and activated IKK are also enriched in NR. METHODS: Double-immunofluorescence labelling was performed with vibratome sections of the rodent central and peripheral nervous system. Sections were analysed using confocal laser scanning microscopy and preembedding electron microscopy. RESULTS: Here we report a remarkable accumulation of pI kappaB alpha and activated IKK in NR in the central and peripheral nervous system. Immunolabelling for both proteins extended from NR into the adjacent paranode. pI kappaB alpha predominantly accumulated within the cytoplasm and was associated with fasciculated microtubules. This association was confirmed by electron microscopy. By comparison, activated IKK preferentially clustered beneath the cytoplasmic membrane. CONCLUSION: In conclusion, the coincident accumulation of pI kappaB alpha and activated IKK in AIS and NR suggests that these specific axonal compartments contribute to neuronal NF-kappaB activation.

The partial 5-hydroxytryptamine1A receptor agonist buspirone does not antagonize morphine-induced respiratory depression in humans.

Based on experiments in rats, serotonin receptor 5-hydroxytryptamine (5-HT)(1A) agonists have been proposed as a potential therapeutic strategy for the selective treatment of opioid-induced respiratory depression. We investigated the clinical applicability of this principle in healthy volunteers. Twelve subjects received 0.43 mg/kg morphine (30 mg for 70 kg body weight) administered intravenously (i.v.) over approximately 2 h. At the start of the morphine infusion, they received in a randomized, double-blind cross-over design 60 mg p.o. buspirone or placebo. Respiratory depression (hypercapnic challenge) and pain (electrical stimuli: 5 Hz sinus 0-20 mA; chemical stimuli: 200 ms gaseous CO(2) pulses applied to the nasal mucosa) were assessed at baseline, at the end of the morphine infusion, and a third time after antagonizing the opioid effects by i.v. administration of 2 mg naloxone. The linear relationship between the minute ventilation and the CO(2) concentration in the inspired air of 1.07+/-0.27 l/mm Hg CO(2) at baseline conditions became shallower (0.45+/-0.23 l/mm Hg CO(2)) after morphine administration (P<0.001), indicating respiratory depression, which was significantly reversed by naloxone (0.95+/-0.43 l/mm Hg CO(2); P=0.001). Co-administration of buspirone had no effect on morphine-induced respiratory depression (slope 0.45+/-0.23 l/mm Hg CO(2) under morphine plus placebo versus 0.38+/-0.25 l/mm Hg CO(2) under morphine plus buspirone; P=0.7). Significant morphine-induced analgesia was observed in both pain models and was reversed by naloxone but unaffected by buspirone. Buspirone significantly increased the nausea induced by morphine (P=0.011). Oral co-administration of a high dose of the clinically available 5-HT(1A) agonist buspirone cannot be advised as a remedy for opioid-induced respiratory depression. This is indicated by its lack of anti-respiratory depressive effects and by the buspirone-associated increase of morphine-induced nausea.

Cyclooxygenase 2 expression in the spared nerve injury model of neuropathic pain.

Cyclooxygenase-2 (COX-2) after induction peripherally, and within the CNS, plays an important role in producing inflammatory pain. However, its role in neuropathic pain models is controversial. Recently a robust and persistent model of partial nerve injury pain, the spared nerve injury (SNI) model, has been developed. The aim of the present study was to examine the regulation of COX-2 in the rat SNI model and to evaluate the effectiveness of the selective COX-2 inhibitor rofecoxib in preventing neuropathic allodynia and hyperalgesia. RNase protection assays revealed only a very small and transient increase in COX-2 mRNA in the dorsal horn of the spinal cord in the SNI model with a maximum change at 24 h. Immunohistochemical analysis showed a small increase in COX-2 protein in the deep layers of the dorsal horn 10 h following SNI surgery. Rofecoxib (100 microM) did not affect spontaneous excitatory postsynaptic currents or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propanoic acid (AMPA) and N-methyl-d-aspartate (NMDA) responses in lamina II neurons from spinal cords of animals with SNI indicating no detectable action on transmitter release or postsynaptic activity. Furthermore, rofecoxib treatment (1 and 3.2 mg/kg for 5 and 3 days respectively starting on the day of surgery) failed to modify the development of allodynia and hyperalgesia in the SNI model. However, rofecoxib significantly reduced inflammatory hypersensitivity evoked by injection of complete Freund's adjuvant into one hindpaw, indicating that the doses used were pharmacologically active. The pain hypersensitivity produced by the SNI model is not COX-2-dependent.

Modulation of spinal nociceptive processing through the glutamate transporter GLT-1.

GLT-1 is the predominant glutamate transporter in most brain regions and therefore plays a major role in terminating synaptic transmission and protecting neurons from glutamate neurotoxicity. In the present study we assessed (i) the regulation of GLT-1 expression in the spinal cord after peripheral nociceptive stimulation and (ii) the nociceptive behavior of rats following inhibition or transient knockdown of spinal GLT-1. Formalin injection into one hindpaw caused a rapid transient upregulation of GLT-1 protein expression in the spinal cord which did not occur when rats were pretreated with morphine (10 mg/kg, i.p.) suggesting that the nociceptive input specifically caused the increase of GLT-1 transcription. Inhibition of GLT-1 by the transportable inhibitor trans-pyrrolidine-2,4-dicarboxylic acid resulted in a significant reduction of nociceptive behavior in the rat formalin assay. Similar results were obtained with a transient reduction of GLT-1 protein expression by antisense oligonucleotides. These data suggest that inhibition of GLT-1 activity or expression reduces excitatory synaptic efficacy and thereby nociception. Mechanisms that might explain this phenomenon may include activation of inhibitory metabotropic glutamate receptors, postsynaptic desensitization or disturbance of glutamate recycling.

Effects of selective COX-1 and -2 inhibition on formalin-evoked nociceptive behaviour and prostaglandin E(2) release in the spinal cord.

Nociception evoked prostaglandin (PG) release in the spinal cord considerably contributes to the induction of hyperalgesia and allodynia. To evaluate the relative contribution of cyclooxygenase-1 (COX-1) and COX-2 in this process we assessed the effects of the selective COX-1 inhibitor SC560 and the selective COX-2 inhibitor celecoxib on formalin-evoked nociceptive behaviour and spinal PGE(2) release. SC560 (10 and 20 mg/kg) significantly reduced the nociceptive response and completely abolished the formalin-evoked PGE(2) raise. In contrast, celecoxib (10 and 20 mg/kg) was ineffective in both regards, i.e. the flinching behaviour was largely unaltered and the formalin-induced PGE(2) raise as assessed using microdialysis was only slightly, not significantly reduced. This suggests that the formalin-evoked rapid PG release was primarily caused by COX-1 and was independent of COX-2. Mean free spinal cord concentrations of celecoxib during the formalin assay were 32.0 +/- 4.5 nM, thus considerably higher than the reported IC50 for COX-2 (3-7 nM). Therefore, the lack of efficacy of celecoxib is most likely not to be a result of poor tissue distribution. COX-2 mRNA and protein expression in the spinal cord were not affected by microdialysis alone but the mRNA rapidly increased following formalin injection and reached a maximum at 2 h. COX-2 protein was unaltered up to 4 h after formalin injection. The time course of COX-2 up-regulation suggests that the formalin-induced nociceptive response precedes COX-2 protein de novo synthesis and may therefore be unresponsive to COX-2 inhibition. Considering the results obtained with the formalin model it may be hypothesized that the efficacy of celecoxib in early injury evoked pain may be less than that of unselective NSAIDs.

Determination of celecoxib in human plasma and rat microdialysis samples by liquid chromatography tandem mass spectrometry.

Methods for the determination of celecoxib in human plasma and rat microdialysis samples using liquid chromatography tandem mass spectrometry are described. Celecoxib and an internal standard were extracted from plasma by solid-phase extraction with C18 cartridges. Thereafter compounds were separated on a short narrow bore RP C18 column (30 x 2 mm). Microdialysis samples did not require extraction and were injected directly using a narrow bore RP C18 column (70 x 2 mm). The detection was by a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray interface. The compounds were detected in the negative ion mode using the mass transitions m/z 380-->316 and m/z 366-->302 for celecoxib and internal standard, respectively. The assay was validated for human plasma over a concentration range of 0.25-250 ng/ml using 0.2 ml of sample. The assay for microdialysis samples (50 microl) was validated over a concentration range of 0.5-20 ng/ml. The method was utilised to determine pharmacokinetics of celecoxib in human plasma and in rat spinal cord perfusate.

Simultaneous fitting of R- and S-ibuprofen plasma concentrations after oral administration of the racemate.

AIMS: To assess the pharmacokinetic equivalence of two different formulations of ibuprofen lysinate with special focus on the expected effects. METHODS: Sixteen healthy volunteers received cross-over ibuprofen lysinate as either one tablet of 400 mg ('test') or two tablets of 200 mg ('reference'). Ibuprofen plasma concentrations were followed up for 10 h. Bioequivalence was assessed by standard noncompartmental methods. Ibuprofen plasma concentrations were fitted with a model that took bioinversion of R- to S-ibuprofen into account. RESULTS: Peak plasma concentrations of R- and S-ibuprofen were 18.1 and 20 microg ml(-1) (test), and 18.2 and 20 microg ml(-1) (reference). Areas under the plasma concentration vs. time curves were 39.7 and 67.5 microg ml(-1) h (test), and 41.1 and 68.2 microg ml(-1) h (reference). Clearance of R-ibuprofen was 5.2 (test) and 5 l h(-1) (reference). A specific plasma concentration was reached with the test formulation about 5 min later than with the reference. Parameters from compartmental modelling were (given for R-and then for S-ibuprofen): body clearance: 4.9 and 4.64 l h(-1), central volume of distribution: 2.8 and 4.1 l, intercompartment clearance: 5.1 and 5.45 l h(-1), peripheral volume of distribution: 4.1 and 5.2 l. The absorption rate constant was 1.52 h(-1), and the test but not the reference formulation had a lag time of 0.1 h. Simulations showed similarity between formulations of the expected effects except for a calculated delay of 6 min with the test formulation. CONCLUSIONS: Ibuprofen formulations were bioequivalent. The pharmacokinetic model may serve as a basis for future pharmacokinetic/pharmacodynamic calculations after administration of racemic ibuprofen.

Cyclooxygenase-independent actions of cyclooxygenase inhibitors.

Several studies have demonstrated unequivocally that certain nonsteroidal anti-inflammatory drugs (NSAIDs) such as sodium salicylate, sulindac, ibuprofen, and flurbiprofen cause anti-inflammatory and antiproliferative effects independent of cyclooxygenase activity and prostaglandin synthesis inhibition. These effects are mediated through inhibition of certain transcription factors such as NF-kappaB and AP-1. The respective NSAIDs might interfere directly with the transcription factors, but their effects are probably mediated predominantly through alterations of the activity of cellular kinases such as IKKbeta, Erk, p38 MAPK, or Cdks. These effects apparently are not shared by all NSAIDs, since indomethacin failed to inhibit NF-kappaB and AP-1 activation as well as Erk and Cdk activity. In contrast, indomethacin was able to activate PPARgamma, which was not affected by sodium salicylate or aspirin. The differences in cyclooxygenase-independent mechanisms may have consequences for the specific use of these drugs in individual patients because additional effects may either enhance the efficacy or reduce the toxicity of the respective compounds.

COX-2 independent induction of cell cycle arrest and apoptosis in colon cancer cells by the selective COX-2 inhibitor celecoxib.

The regular use of various nonsteroidal anti-inflammatory drugs (NSAIDs) was shown to decrease the incidence of colorectal cancer. This effect is thought to be caused predominantly by inhibition of cyclooxygenase-2 (COX-2) and, subsequently, prostaglandin synthesis. However, recent studies have suggested that COX-independent pathways may contribute considerably to these antiproliferative effects. To evaluate the involvement of COX-dependent and COX-independent mechanisms further, we assessed the effects of celecoxib (selective COX-2 inhibitor) and SC560 (selective COX-1 inhibitor) on cell survival, cell cycle distribution, and apoptosis in three colon cancer cell lines, which differ in their expression of COX-2. Both drugs induced a G0/G1 phase block and reduced cell survival independent of whether or not the cells expressed COX-2. Celecoxib was more potent than SC560. The G0/G1 block caused by celecoxib could be attributed to a decreased expression of cyclin A, cyclin B1, and cyclin-dependent kinase-1 and an increased expression of the cell cycle inhibitory proteins p21Waf1 and p27Kip1. In addition, celecoxib, but not SC560, induced apoptosis, which was also independent of the COX-2 expression of the cells. In vivo, celecoxib as well as SC560 reduced the proliferation of HCT-15 (COX-2 deficient) colon cancer xenografts in nude mice, but both substances had no significant effect on HT-29 tumors, which express COX-2 constitutively. Thus, our in vitro and in vivo data indicate that the antitumor effects of celecoxib probably are mediated through COX-2 independent mechanisms and are not restricted to COX-2 over-expressing tumors.

Comparison of tissue concentrations after intramuscular and topical administration of ketoprofen.

PURPOSE: To assess whether topical ketoprofen, which has been reported to provide analgesic effects in clinical studies, reaches predictable tissue concentrations high enough to account for the reported analgesia. Intramuscular ketoprofen was used as positive control. METHODS: Muscle and subcutaneous tissue concentrations were assessed by microdialysis. Plasma and tissue concentrations after intramuscular injection were described using a three-compartment population pharmacokinetic model. The prediction performance of the model was assessed by superimposing tissue concentrations of 12 subjects that did not participate in the present study. RESULTS: Most dialysate concentrations after topical dosing of ketoprofen (100 mg) were below the quantification limit of 0.47 ng/ml. Plasma concentrations increased slowly and reached an apparent plateau of 7-40 ng/ml at 10-12h. No decline was observed up to 16 h. Tissue concentrations after intramuscular injection (100 mg) were about 10 times higher than those after topical dosing. Tissue concentrations measured in the majority of the 12 subjects that did not participate in the present study were found within the range of two-thirds of the predicted concentrations. CONCLUSION: Predictable and cyclooxygenase-inhibiting concentrations of ketoprofen were achieved in subcutaneous and muscle tissue after intramuscular but not after topical dosing. Thus, the tissue concentrations of ketoprofen after topical administration can hardly explain the reported clinical efficacy of topical ketoprofen.

Kinetics and dose-response of photosensitivity in cream psoralen plus ultraviolet A photochemotherapy: comparative in vivo studies after topical application of three standard preparations.

BACKGROUND: Topical photochemotherapy with bath psoralen plus ultraviolet (UV) A irradiation (PUVA) has been developed to reduce possible side-effects of oral PUVA therapy. Although the efficacy of bath PUVA therapy appears to be similar to oral PUVA therapy, provision of bathing facilities has obvious economic, logistic and sanitary implications. Cream PUVA therapy has recently been developed as a variation of topical PUVA. OBJECTIVES: To understand the photobiological effects and to increase the safety and effectiveness of this novel topical PUVA therapy, we assessed the kinetics and dose-response of phototoxicity of 8-methoxypsoralen (8-MOP) cream in order to develop a treatment schedule for this treatment option. METHODS: Ninety-eight patients (63 men and 35 women) undergoing cream PUVA therapy were studied. The phototoxic properties of topically applied 8-MOP in three different water-in-oil creams as vehicles were assessed. In a dose-response study, four concentrations of 8-MOP cream (0.0006-0.005%) were used for determination of the minimal phototoxic dose (MPD). The kinetics of photosensitization were tested by determination of MPDs after different application times of 8-MOP cream (10, 20, 30 and 60 min). The persistence of phototoxicity was assessed by UVA exposure at defined time intervals after application of 8-MOP cream (0, 30, 60 and 120 min). RESULTS: The concentration required to produce sufficient but not undue photosensitization of the skin was 0.001% 8-MOP. The duration of application leading to the lowest MPD was 30 min. Greatest photosensitization was achieved when UVA irradiation was performed between 0 and 30 min after 8-MOP removal. These findings showed no significant difference between the three vehicles used. CONCLUSIONS: Based on our data we recommend application of 0.001% 8-MOP in a water-in-oil cream for 30 min. Irradiation with UVA should be performed within 30 min after removal of 8-MOP cream, as there is a rapid decrease in photosensitivity thereafter.