The nonsteroidal anti-inflammatory drug indomethacin induces heterogeneity in lipid membranes: potential implication for its diverse biological action.

BACKGROUND: The nonsteroidal anti-inflammatory drug (NSAID), indomethacin (Indo), has a large number of divergent biological effects, the molecular mechanism(s) for which have yet to be fully elucidated. Interestingly, Indo is highly amphiphilic and associates strongly with lipid membranes, which influence localization, structure and function of membrane-associating proteins and actively regulate cell signaling events. Thus, it is possible that Indo regulates diverse cell functions by altering micro-environments within the membrane. Here we explored the effect of Indo on the nature of the segregated domains in a mixed model membrane composed of dipalmitoyl phosphatidyl-choline (di16:0 PC, or DPPC) and dioleoyl phosphatidyl-choline (di18:1 PC or DOPC) and cholesterol that mimics biomembranes. METHODOLOGY/PRINCIPAL FINDINGS: Using a series of fluorescent probes in a fluorescence resonance energy transfer (FRET) study, we found that Indo induced separation between gel domains and fluid domains in the mixed model membrane, possibly by enhancing the formation of gel-phase domains. This effect originated from the ability of Indo to specifically target the ordered domains in the mixed membrane. These findings were further confirmed by measuring the ability of Indo to affect the fluidity-dependent fluorescence quenching and the level of detergent resistance of membranes. CONCLUSION/SIGNIFICANCE: Because the tested lipids are the main lipid constituents in cell membranes, the observed formation of gel phase domains induced by Indo potentially occurs in biomembranes. This marked Indo-induced change in phase behavior potentially alters membrane protein functions, which contribute to the wide variety of biological activities of Indo and other NSAIDs.

Exposure to nonsteroidal anti-inflammatory drugs and weight loss and hospitalization in non-small cell lung cancer patients

Background. Cancer cachexia is a common syndrome complex in cancer, occurring in nearly 80% of patients with advanced cancer and responsible for at least 20% of all cancer deaths. Cachexia is due to increased resting energy expenditure, increased production of inflammatory mediators, and changes in lipid and protein metabolism. Non-steroidal anti-inflammatory drugs (NSAIDs), by virtue of their anti-inflammatory properties, are possibly protective against cancer-related cachexia. Since cachexia is also associated with increased hospitalizations, this outcome may also show improvement with NSAID exposure. ^ Design. In this retrospective study, computerized records from 700 non-small cell lung cancer patients (NSCLC) were reviewed, and 487 (69.57%) were included in the final analyses. Exclusion criteria were severe chronic obstructive pulmonary disease, significant peripheral edema, class III or IV congestive heart failure, liver failure, other reasons for weight loss, or use of research or anabolic medications. Information on medication history, body weight and hospitalizations was collected from one year pre-diagnosis until three years post-diagnosis. Exposure to NSAIDs was defined if a patient had a history of being treated with NSAIDs for at least 50% of any given year in the observation period. We used t-test and chi-square tests for statistical analyses. ^ Results. Neither the proportion of patients with cachexia (p=0.27) nor the number of hospitalizations (p=0.74) differed among those with a history of NSAID use (n=92) and those without (n=395). ^ Conclusions. In this study, NSAID exposure was not significantly associated with weight loss or hospital admissions in patients with NSCLC. Further studies may be needed to confirm these observations.^

Structure and function study of prostaglandin H synthase

Prostaglandin H synthase (PGHS) is a key enzyme in biosynthesis of prostaglandins, thromboxane, and prostacyclin. It has two activities, cyclooxygenase and peroxidase. "PGHS" means PGHS-1. A current hypothesis considers the cyclooxygenase reaction to be a free radical chain reaction, initiated by interaction of the synthase peroxidase with hydroperoxides leading to the production of a tyrosyl free radical. According to this hypothesis, tyrosyl residue(s) may play a key role in the cyclooxygenase reaction. Tetranitromethane (TNM) can relatively selectively nitrate tyrosines at pH 8.0. The effect of TNM on both cyclooxygenase activity and peroxidase activity has been examined: reaction of the synthase holoenzyme with TNM at pH 8.0 led to inactivation of both activities, with the cyclooxygenase activity being lost rapidly and completely, while the peroxidase activity was lost more slowly. Indomethacin, a non-steroidal anti-inflammatory agent, can protect the synthase from the inactivation of TNM. Amino acid analyses indicated that a loss of tyrosine and formation of nitrotyrosine residues occurred during reaction with TNM, and that TNM-reacted holoenzyme with $<$10% residual cyclooxygenase activity had about 2.0 nitrotyrosine/subunit.^ PGH synthase is known to be an endoplasmic reticulum membrane-associated protein. Antibodies directed at particular PGHS peptide segments and indirect immunofluorescence have been used to characterize the membrane topology of crucial portions of PGHS. PGHS was expressed in COS-1 cells transfected with the appropriate cDNA. Stably-transfected human endothelial cells were also used for the topology study. The cells were treated with streptolysin-O, which selectively permeabilizes the plasma membrane, or with saponin to achieve general membrane disruption, before incubation with the antipeptide antibodies. Bound antipeptide antibody was stained by FITC-labelled secondary antibody and visualized by fluorescence microscopy. With the antipeptide antibodies against residues 51-66, 156-170 or 377-390, there was a significant reticular and perinuclear pattern of staining in cells permeabilized with saponin but not in cells permeabilized with SLO alone. Antibodies directed against the endogenous C-terminal peptide or against residues 271-284 produced staining in cells permeabilized with saponin, and also in a lower, but significant fraction of cells permeabilized with SLO. Similar results were obtained when COS-1 cells expressing recombinant PGHS with a viral reporter peptide inserted at the C-terminus were stained with antibody against the reporter epitope.^ The PGHS C-terminal sequence is similar to that of the consensus KDEL ER retention signal. The potential function of the PGHS C-terminus segment in ER retention was examined by mutating this segment and analyzing the subcellular distribution of the mutants expressed in COS-1 cells. None of the mutants had an altered subcellular distribution, although some had greatly diminished the enzyme activities. (Abstract shortened by UMI.) ^

Self medication behaviors of illegal immigrants living along the U.S.-Mexico border

Mexican immigrants make up the largest subgroup of Hispanics living in the United States. The largest percentage of illegal immigrants comes from México. As such they are a subpopulation with limited access to health care and social services; their health seeking behaviors including self-medication behaviors that, aside from the intake of antibiotics, have not been studied in depth. The analysis of the data presented sought to document the medication behaviors of illegal immigrants living in El Paso County along the U.S.-México border. Of the 80 participants, 31 were taking medication on a regular basis. Of these, 28 claimed that at least one of the medications had been prescribed by a physician, 13 people had bought at least one of their medications in México, nine participants claimed that they had not paid for at least one of the medications they were taking, ten participants reported that they had skipped the doses of at least one of their medications due to monetary constraints. Participants were also asked if they had purchased medication in México during the year prior to the study, 68 of the 80 (85%) participants had bought 295 pharmaceutical products across the border themselves or through a third party. The most frequently purchased medications were antibiotics (17%), followed by syrups, pomades, creams, eardrops, and cold medicine as a group (15%), followed by analgesics (13%) and other non steroidal anti-inflammatory drugs (12%) and oral hypoglycemic agents (6%). ^

Effect of indomethacin on bile acid-phospholipid interactions: implication for small intestinal injury induced by nonsteroidal anti-inflammatory drugs.

The injurious effect of nonsteroidal anti-inflammatory drugs (NSAIDs) in the small intestine was not appreciated until the widespread use of capsule endoscopy. Animal studies found that NSAID-induced small intestinal injury depends on the ability of these drugs to be secreted into the bile. Because the individual toxicity of amphiphilic bile acids and NSAIDs directly correlates with their interactions with phospholipid membranes, we propose that the presence of both NSAIDs and bile acids alters their individual physicochemical properties and enhances the disruptive effect on cell membranes and overall cytotoxicity. We utilized in vitro gastric AGS and intestinal IEC-6 cells and found that combinations of bile acid, deoxycholic acid (DC), taurodeoxycholic acid, glycodeoxycholic acid, and the NSAID indomethacin (Indo) significantly increased cell plasma membrane permeability and became more cytotoxic than these agents alone. We confirmed this finding by measuring liposome permeability and intramembrane packing in synthetic model membranes exposed to DC, Indo, or combinations of both agents. By measuring physicochemical parameters, such as fluorescence resonance energy transfer and membrane surface charge, we found that Indo associated with phosphatidylcholine and promoted the molecular aggregation of DC and potential formation of larger and isolated bile acid complexes within either biomembranes or bile acid-lipid mixed micelles, which leads to membrane disruption. In this study, we demonstrated increased cytotoxicity of combinations of bile acid and NSAID and provided a molecular mechanism for the observed toxicity. This mechanism potentially contributes to the NSAID-induced injury in the small bowel.

Novel Use of Dual Anti-Inflammatory Therapy to Overcome Drug Resistance and Improve Functional Recovery Following Spinal Cord Injury

Over 1.2 million Americans are currently living with a traumatic spinal cord injury (SCI). Despite the need for effective therapies, there are currently no proven effective treatments that can improve recovery of function in SCI patients. Many therapeutic compounds have shown promise in preclinical models of SCI, but all of these have fallen short in clinical trials. P-glycoprotein (Pgp) is an active transporter expressed on capillary endothelial cell membranes at the blood-spinal cord barrier (BSCB). Pgp limits passive diffusion of blood-borne drugs into the CNS, by actively extruding drugs from the endothelial cell membrane. Pgp can become pathologically up-regulated, thus greatly impeding therapeutic drug delivery (‘multidrug resistance’). Importantly, many drugs that have been evaluated for the treatment of SCI are Pgp substrates. We hypothesized that Pgp-mediated drug resistance diminishes the delivery and efficacy of neuroprotective drugs following SCI. We observed a progressive, spatial spread of Pgp overexpression within the injured spinal cord. To assess Pgp function, we examined spinal cord uptake of systemically-delivered riluzole, a drug that is currently being evaluated in clinical trials as an SCI intervention. Blood-to-spinal cord riluzole penetration was reduced following SCI in wild-type but not Pgp-null rats, highlighting a critical role for Pgp in mediating spinal cord drug resistance after injury. Others have shown that pro-inflammatory signaling drives Pgp up-regulation in cancer and epilepsy. We have detected inflammation in both acutely- and chronically-injured spinal cord tissue. We therefore evaluated the ability of the dual COX-/5-LOX inhibitor licofelone to attenuate Pgp-mediated drug resistance following SCI. Licofelone treatment both reduced spinal cord Pgp levels and enhanced spinal cord riluzole bioavailability following SCI. Thus, we propose that licofelone may offer a new combinatorial treatment strategy to enhance spinal cord drug delivery following SCI. Additionally, we assessed the ability of licofelone, riluzole, or both to enhance recovery of locomotor function following SCI. We found that licofelone treatment conferred a significant improvement in hindlimb function that was sustained through the end of the study. In contrast, riluzole did not improve functional outcome. We therefore conclude that licofelone holds promise as a potential neuroprotective intervention for SCI.

Drug metabolism in human brain: high levels of cytochrome P4503A43 in brain and metabolism of anti-anxiety drug alprazolam to its active metabolite.

Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism. Metabolism in brain specific nuclei may play a role in pharmacological modulation of drugs acting on the CNS and help explain some of the diverse response to these drugs seen in patient population. P450 enzymes are also present in brain where drug metabolism can take place and modify therapeutic action of drugs at the site of action. We have earlier demonstrated an intrinsic difference in the biotransformation of alprazolam (ALP) in brain and liver, relatively more alpha-hydroxy alprazolam (alpha-OHALP) is formed in brain as compared to liver. In the present study we show that recombinant CYP3A43 metabolizes ALP to both alpha-OHALP and 4-hydroxy alprazolam (4-OHALP) while CYP3A4 metabolizes ALP predominantly to its inactive metabolite, 4-OHALP. The expression of CYP3A43 mRNA in human brain samples correlates with formation of relatively higher levels of alpha-OH ALP indicating that individuals who express higher levels of CYP3A43 in the brain would generate larger amounts of alpha-OHALP. Further, the expression of CYP3A43 was relatively higher in brain as compared to liver across different ethnic populations. Since CYP3A enzymes play a prominent role in the metabolism of drugs, the higher expression of CYP3A43 would generate metabolite profile of drugs differentially in human brain and thus impact the pharmacodynamics of psychoactive drugs at the site of action.

Hypertensive nephropathy treatment by heart-protecting musk pill: a study of anti-inflammatory therapy for target organ damage of hypertension.

This study was designed to investigate the protective effect of the heart-protecting musk pill (HMP) on inflammatory injury of kidney from spontaneously hypertensive rat (SHR). Male SHRs aged 4 weeks were divided into SHR model group, HMP low-dosage group (13.5 mg/kg), and HMP high-dosage group (40 mg/kg). Age-matched Wistar-Kyoto rats were used as normal control. All rats were killed at 12 weeks of age. Tail-cuff method and enzyme-linked immunosorbent assay were used to determine rat systolic blood pressure and angiotensin II (Ang II) contents, respectively. Renal inflammatory damage was evaluated by the following parameters: protein expressions of inflammatory cytokines, carbonyl protein contents, nitrite concentration, infiltration of monocytes/macrophages in interstitium and glomeruli, kidney pathological changes, and excretion rate of urinary protein. HMP did not prevent the development of hypertension in SHR. However, this Chinese medicinal compound decreased renal Ang II content. Consistent with the change of renal Ang II, all the parameters of renal inflammatory injury were significantly decreased by HMP. This study indicates that HMP is a potent suppressor of renal inflammatory damage in SHR, which may serve as a basis for the advanced preventive and therapeutic investigation of HMP in hypertensive nephropathy.

Drug metabolism in human brain: high levels of cytochrome P4503A43 in brain and metabolism of anti-anxiety drug alprazolam to its active metabolite.

Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism. Metabolism in brain specific nuclei may play a role in pharmacological modulation of drugs acting on the CNS and help explain some of the diverse response to these drugs seen in patient population. P450 enzymes are also present in brain where drug metabolism can take place and modify therapeutic action of drugs at the site of action. We have earlier demonstrated an intrinsic difference in the biotransformation of alprazolam (ALP) in brain and liver, relatively more alpha-hydroxy alprazolam (alpha-OHALP) is formed in brain as compared to liver. In the present study we show that recombinant CYP3A43 metabolizes ALP to both alpha-OHALP and 4-hydroxy alprazolam (4-OHALP) while CYP3A4 metabolizes ALP predominantly to its inactive metabolite, 4-OHALP. The expression of CYP3A43 mRNA in human brain samples correlates with formation of relatively higher levels of alpha-OH ALP indicating that individuals who express higher levels of CYP3A43 in the brain would generate larger amounts of alpha-OHALP. Further, the expression of CYP3A43 was relatively higher in brain as compared to liver across different ethnic populations. Since CYP3A enzymes play a prominent role in the metabolism of drugs, the higher expression of CYP3A43 would generate metabolite profile of drugs differentially in human brain and thus impact the pharmacodynamics of psychoactive drugs at the site of action.

The relationship of mutations in alpha- and beta-tubulin to microtubule assembly and anti-mitotic drug resistance

The mechanisms responsible for anti-cancer drug (including Taxol) treatment failure have not been identified. In cell culture model systems, many β-tubulin, but very few α-tubulin, mutations have been associated with resistance to Taxol. To test what, if any, mutations in α-tubulin can cause resistance, we transfected a randomly mutagenized α-tubulin cDNA into Chinese hamster ovary (CHO) cells and isolated drug resistant cell lines. A total of 12 mutations were identified in this way and all of them were confirmed to confer Taxol resistance. Furthermore, all cells expressing mutant α-tubulin had less microtubule polymer. Some cells also had abnormal nuclei and enlarged cell bodies. The data indicate that α-tubulin mutations confer Taxol resistance by disrupting microtubule assembly, a mechanism consistent with a large number of previously described β-tubulin mutations. ^ Because α- and β-tubulin are almost identical in their three dimensional structure, we hypothesized that mutations discovered in one subunit, when introduced into the other, would produce similar effects on microtubule assembly and drug resistance. 9 α- and 2 β-tubulin mutations were tested. The results were complex. Some mutations produced similar changes in microtubule assembly and drug resistance irrespective of the subunit in which they were introduced, but others produced opposite effects. Still one mutation produced resistance when present in one subunit, yet had no effect when present on the other; and one mutation that produced Taxol resistance when present in α-tubulin, resulted in assembly-defective tubulin when it was present in β-tubulin. The results suggest that in most cases, the same amino acid modification in α- and β-tubulin affects the microtubule structure and assembly in a similar way. ^ Finally, we tested whether three β-tubulin mutations found in patient tumors could confer resistance to Taxol by recreating the mutations in a β-tubulin cDNA and transfecting it into CHO cells. We found that all three mutations conferred Taxol resistance, but to different extents. Again, microtubule assembly in the transfectants was disrupted, suggesting that mutations in β-tubulin are a potential problem in cancer therapeutics. ^

Trends in anti-tuberculosis drug resistance from 2003--2007 at Pham Ngoc Thach tuberculosis and lung disease hospital, Ho Chi Minh City, Vietnam

Vietnam is one of the countries with the highest prevalence and incidence of tuberculosis (TB) in the world (1). Although Vietnam has had many successes in TB control, it still faces the challenge of drug resistant and multidrug-resistant tuberculosis (MDR-TB). MDR-TB appears to be relatively stable, but data on MDR-TB continues to be scarce and routine testing of all isolates for drug susceptibility is not performed under Vietnam's National Tuberculosis Program (6). Pham Ngoc Thach Hospital (PNT), the leading tuberculosis and lung disease hospital in Ho Chi Minh City, serves as a reference hospital and laboratory for both Ho Chi Minh City and the Southern Vietnam region. This study is an unmatched, nested case-control study consisting of a secondary analysis of a previously created dataset composed of drug susceptibility and basic demographic data from a cohort of patients diagnosed with tuberculosis at PNT from 2003 through 2007 in order to calculate the prevalence of resistance among acid-fast bacilli smear-positive patients. The susceptibility records for the years 2003-2004 were not representative of the entire population, but over the years 2005-2007 the investigator found a decrease in resistance to all primary TB drugs on which records were available, as well as MDR-TB. Overall, females showed a higher proportion of resistance to TB drugs than males, and females had a greater likelihood of presenting with MDR-TB than males (OR=1.77). Persons 35-54 had greater likelihood of having MDR-TB than younger and older age groups. Among the population with HIV data, HIV-positivity was associated with greater likelihood of MDR-TB (OR=1.70, 95% CI=0.97-3.11). This study shows that rates of TB drug resistance are high, but declining, in one of Vietnam's largest TB hospitals, and that females and HIV-positive individuals are possible high-risk groups in this population.^

Ketamine-induced hepatoprotection: the role of heme oxygenase-1.

Lipopolysaccharide (LPS) causes hepatic injury that is mediated, in part, by upregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Ketamine has been shown to prevent these effects. Because upregulation of heme oxygenase-1 (HO-1) has hepatoprotective effects, as does carbon monoxide (CO), an end product of the HO-1 catalytic reaction, we examined the effects of HO-1 inhibition on ketamine-induced hepatoprotection and assessed whether CO could attenuate LPS-induced hepatic injury. One group of rats received ketamine (70 mg/kg ip) or saline concurrently with either the HO-1 inhibitor tin protoporphyrin IX (50 micromol/kg ip) or saline. Another group of rats received inhalational CO (250 ppm over 1 h) or room air. All rats were given LPS (20 mg/kg ip) or saline 1 h later and euthanized 5 h after LPS or saline. Liver was collected for iNOS, COX-2, and HO-1 (Western blot), NF-kappaB and PPAR-gamma analysis (EMSA), and iNOS and COX-2 mRNA analysis (RT-PCR). Serum was collected to measure alanine aminotransferase as an index of hepatocellular injury. HO-1 inhibition attenuated ketamine-induced hepatoprotection and downregulation of iNOS and COX-2 protein. CO prevented LPS-induced hepatic injury and upregulation of iNOS and COX-2 proteins. Although CO abolished the ability of LPS to diminish PPAR-gamma activity, it enhanced NF-kappaB activity. These data suggest that the hepatoprotective effects of ketamine are mediated primarily by HO-1 and its end product CO.