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Objectives: The purpose of this study was to evaluate the steady-state pharmacokinetics of amprenavir and ritonavir in HIV-infected patients with different degrees of hepatic impairment., Methods: HIV-positive patients receiving fosamprenavir/ritonavir (700/100 mg twice daily) were included. Patients were classified into three groups: (i) chronic hepatitis; (ii) liver cirrhosis; (iii) normal liver function. Serial blood samples for steady-state amprenavir and ritonavir pharmacokinetics (>14 days on treatment) were collected in the fasting state before the morning dose (C(trough)) and then 1, 2, 3, 4, 6, 8, 10 and 12 h after drug intake. Amprenavir and ritonavir plasma concentrations were determined by HPLC., Results: Twenty-one HIV-infected patients were included. Seven had chronic hepatitis, eight had liver cirrhosis and six patients were in the control group. Amprenavir AUC(0-12), AUC(0-infinity), C(max) and C(ss) were increased by 50% to 60% in the cirrhotic group when compared with controls, whereas CL/F was decreased by 40%. Patients with chronic hepatitis showed a significant increase in AUC(0-12), C(max) and C(ss) values when compared with controls. Ritonavir pharmacokinetics was different only in cirrhotic patients when compared with controls. Liver function parameters at weeks 4, 12 and 24 were not different from baseline in any of the groups. Overall, a significant correlation between amprenavir AUC(0-12) and total bilirubin values on the day of pharmacokinetic analysis was found (r = 0.64, P = 0.003)., Conclusions: On the basis of these data and also of data available in the literature, it seems reasonable to adapt the dose of fosamprenavir and/or ritonavir exclusively in the presence of adverse events, possibly related to protease inhibitors (i.e. liver toxicity), in subjects with high drug plasma levels. Therapeutic drug monitoring is advised in the management of these patients.

Acute liver toxicity is a frequent adverse event that occurs during antiretroviral therapy and was observed in 6-30% of the patients on treatment, especially in presence of HCV coinfection (Cooper et al., 2002, Maida et al., 2006, Sulkowski et al., 2000). A correlation between HCV-associated liver-fibrosis severity and the risk of HAART associated hepatoxicity has been demonstrated (Aranzabal et al., 2005, Sulkowski et al., 2004). This high liver toxicity rate might be due to increased drug exposure in patients with liver disease (Veronese et al., 2000). It has been reported that patients with chronic hepatitis C show significantly reduced CPY3A4 and CYP2D6 activity in comparison with healthy volunteers (Becquemont et al., 2002). The aim of this study was to evaluate the liver function tests in HCV-co-infected patients treated with fos-amprenavir and ritonavir.

Objectives: The aim of this study was to evaluate the correlation between liver function markers (necrosis and cholestasis) and plasma lopinavir levels in a cohort of HIV-infected patients treated with lopinavir and ritonavir., Patients and Methods: The blood samples for determining steady-state C(trough) lopinavir levels and analysing liver function were drawn from fasting patients. Steady-state C(trough) lopinavir levels, liver function and immuno-virological markers were assessed on the same day. Plasma lopinavir and ritonavir levels were determined by means of high-performance liquid chromatography., Results: One hundred and forty-nine patients were included in the analysis [57 were HCV co-infected (34%) and 10 were HBV co-infected (6.7%)]; they had been treated with lopinavir/ritonavir for a median of 232 days (range 132-282). All patients received lopinavir/ritonavir [400/100 mg twice daily or 533/133 mg twice daily if amprenavir or a non-nucleoside reverse transcriptase inhibitor (NNRTI) was part of therapy] and concomitant therapy with NRTI(s). Median (interquartile) lopinavir trough levels were 6391 ng/mL (4121-8726), 5662 (3585-8893) and 6819 ng/mL (5324-8726) in the patients with HIV alone and those with HIV/HCV (or HBV) co-infection, respectively (P = not significant). Univariate analysis showed a significant association between the cholestasis markers and C(trough) lopinavir level. Multivariate analysis selected only gamma glutamyltranspeptidase (GGT) (OR = 1.010, 95% CI: 1.002-1.021) as being independently associated with plasma lopinavir levels of >6425 ng/mL; alkaline phosphatase (OR = 1.004, 95% CI: 1.000-1.010; P = 0.08) and total bilirubin (OR = 3.118, 95% CI: 0.980-11.715; P = 0.07) were not associated., Conclusions: Elevated lopinavir concentrations are associated with raised GGT.

A reversed-phase high-performance liquid chromatography (HPLC) method with fluorimetric detection, which allows the simultaneous determination of plasma concentrations of four selective serotonin reuptake inhibitors (SSRIs) is presented. Fluvoxamine, paroxetine, sertraline, and fluoxetine were extracted from plasma with ethyl acetate and then derivatized with dansyl chloride. The analytes were separated using Hypersyl ODS C18 (5 microm) 250 x 4.6 mm column (ThermoQuest, Runcorn, UK). For continuous gradient separation, the mobile phase consists of two eluents, acetonitrile and potassium phosphate buffer (10 mmol/L, pH 7.2) at total flow rate of 1.5 mL/min. Detection was carried out at lambda exc = 366 nm and lambda em = 490 nm. The authors found recoveries of 90% to 95% for fluvoxamine, 94% to 100% for paroxetine, 88% to 95% for sertraline, 93% to 100% for fluoxetine, and 97% to 100% for internal standard (nortriptyline). Imprecision of the method ranged from 2.5% to 8.9%. The assay was linear from 10 to 1500 ng/mL for sertraline, and from 5 to 1500 ng/mL for the other drugs. The authors conclude that this method is suitable for monitoring antidepressant therapy. In addition, the authors report the effects of adding paroxetine to fluvoxamine on plasma levels in a group of patients in combined drug therapy.

Human PF4 is a heparin-binding chemokine known to be capable of inhibiting endothelial cell proliferation and angiogenesis. To explore the biological mechanisms responsible for this action, we investigated the effect of PF4 on epidermal growth factor (EGF)-stimulated human umbilical vein endothelial cells (HUVEC), a model system in which stimulation is essentially independent of interaction with cell-surface glycosaminoglycans. Based on previous findings that PF4 blocks endothelial cell cycle entry and progression into S phase, we studied the molecular mechanism(s) of PF4 interference with cell cycle machinery. PF4 treatment of EGF-stimulated HUVEC caused a decrease in cyclin E-cyclin-dependent kinase 2 (cdk2) activity with resulting attenuation of retinoblastoma protein phosphorylation. PF4-dependent downregulation of cyclin E-cdk2 activity was associated with increased binding of the cyclin-dependent kinase inhibitor, p21(Cip1/WAF1), to the cyclin E-cdk2 complex. Analysis of total cellular p21(Cip1/WAF1) showed that in the presence of PF4, p21(Cip1/WAF1) levels were sustained at time points when p21(Cip1/WAF1) was no longer detectable in cells stimulated by EGF in the absence of PF4. These findings indicate that PF4 inhibition of HUVEC proliferation in response to EGF is associated with impaired downregulation of p21(Cip1/WAF1) and provide the first evidence for interference with cell cycle mechanisms by a chemokine.

Although thrombocytopenia associated with the use of histamine H2 receptor (H2R) antagonists has been described, a drug-dependent, platelet-reactive antibody has not previously been identified in such cases. We studied serum from a patient who developed acute, severe thrombocytopenia after exposure to the H2 receptor antagonist, ranitidine, and identified an antibody that reacted with normal platelets in the presence of this drug at pharmacologic concentrations. In flow cytometric and immunoprecipitation studies, the antibody was shown to be specific for the glycoprotein Ib/IX complex (GPIb/IX). From the pattern of monoclonal antibody (MoAb) inhibition and the reactions of antibody with Chinese hamster ovary (CHO) cells transfected with GPIX and GPIbbeta, we found that the patient's antibody is specific for an epitope on GPIX close to, or identical with a site recognized by the MoAb SZ1 that is a common target for antibodies induced by quinine and quinidine, drugs structurally unrelated to ranitidine. These findings provide evidence that immune thrombocytopenia can be caused by sensitivity to an H2 R antagonist and suggest that the SZ1 binding site on GPIX may be a common target for drug-induced antibodies. Further studies of the epitope for which SZ1 is specific may provide clues to the mechanism(s) by which drugs promote tight binding of antibody to a membrane glycoprotein and cause platelet destruction in patients with drug sensitivity.

1. In sensitized guinea-pigs, the effects of gamma-aminobutyric acid (GABA) and GABAmimetic drugs have been investigated on tracheal segments contracted by cumulative application of an allergen (ovoalbumin, OA) and on serosal mast cells. The same drugs have also been tested on activation of alveolar macrophages isolated from unsensitized guinea-pigs. 2. Superfusion with GABA (1-1000 microM) reduced the contraction intensity of tracheal strips. The effect of GABA (100 microM) was not affected by the carrier blockers, nipecotic acid and beta-alanine (300 microM each). It was mimicked by the GABAB agonist (-)-baclofen (100 microM) but not 3-aminopropanephosphinic acid (100 microM, 3-APA). The GABAA agonist, isoguvacine (100 microM) did not exert any effect. GABA (10 microM)-induced inhibition of tracheal contractions was reduced by the GABAB antagonist, 2-hydroxysaclofen (100 microM, 2-HS), but not by the GABAA antagonist, bicuculline (30 microM). 3. The reduction in contraction intensity induced by GABA (100 microM) was prevented by a 40 min preincubation of tracheal strips with capsaicin (10 microM), but not tetrodotoxin (TTX, 0.3 microM). The effect of GABA (1000 microM) was absent after preincubation with indomethacin (2.8 microM) but unmodified when nordihydroguaiaretic acid (NDGA, 3.3 microM) was used. Finally, removal of the epithelium prevented the GABA effect. 4. Anaphylactic histamine release from serosal mast cells isolated from sensitized animals was not affected either by GABA (10-1000 microM) or the selective receptor agonists (-)-baclofen (0.1-1000 microM) and isoguvacine (10-1000 microM). The release of platelet-activating factor (PAF) from alveolar macrophages stimulated by formyl-Met-Leu-Phe (FMLP; 1 microM) was modified neither by GABA (100 microM)nor by (-)-baclofen (100microM).5. In conclusion, these data show that GABA can inhibit allergic phenomena in the guinea-pig airways through activation of GABAB receptors. An involvement of neuropeptidergic sensory structures is suggested but a role for epithelial cells and arachidonate metabolites is not definitely proved.

Compounds derived from glucocorticoids, 21-aminosteroids, were reported to inhibit in vitro lipid peroxidation in CNS tissue. In order to evaluate the possible scavenging and/or iron chelating activities in vivo of the 21-aminosteroid U74500A (pregna-1,4,9(11)-triene-3,20-dione, 21-(4-(5,6-bisdiethylamino)-2-pyridinyl)-1-piperazinyl)-16-methyl- , HCl (16 alpha)), the drug was administered for seven days to rats. These rats had been induced by iron-saccharate complex injection a slow process of lipid peroxidation into their right brain hemicortex. The drug was injected also to intact rats (normal rats). Seven days after the operation the extent of iron-induced lipid peroxidation in both the hemicortices and the effect of the drug, were assessed by the evaluation of lipid-soluble fluorescence and of conjugated diene formation. The assessment was performed both in vehicle (control) and in U74500A-treated rats. In the iron-injected rat groups the drug induced a significant dose-related reduction of fluorescence values. Formation of conjugated dienes showed a significant decrease when U74500A (48 mg/kg every 48 hr) was administered to cortico-cerebrally iron-injected animals. The lipid peroxidation of cortices in normal rats was evaluated as thiobarbituric acid reactant substances in both the drug-treated and the control animals. In normal rats, U74500A (48 mg/kg every 48 hr) caused a significant decrease of TBARS values, as compared to those observed in the control group. The iron content in the iron-injected hemicortices, which was evaluated by the ferrozine method, was not modified by drug treatment. U74500A appears to have in vivo antioxidant properties and not to affect the iron content in the neural tissue. An interaction of this drug with the metal, however, cannot be excluded.

Salmeterol (1 nM-100 microM) showed an inhibitory action on anaphylactic histamine release from mast cells, isolated from pleural and peritoneal cavities of actively sensitized guinea-pigs and stimulated by incubation with allergen. The effect is concentration-dependent and is reduced by the beta-adrenoceptor antagonist propranolol (1 microM). This study supports the hypothesis of an anti-inflammatory property of salmeterol, which concerns cells involved in the early phases of asthma.

D-Penicillamine, a trifunctional amino acid known for its ability to form metal complexes and for being a radical scavenger, has been investigated "in vitro" and "in vivo" in the rat brain cortex. At 50 microM the drug facilitated lipid hydroperoxides and TBARS formation in brain cortex homogenates, while at higher concentrations a clear inhibition of the lipid peroxidative process was observed. The activity of the D-penicillamine (25 and 50 mg/Kg i.p.) was evaluated "in vivo" after a 7-day treatment in rats in whose brain cortex a slow process of lipid peroxidation was induced by iron-saccharate injection. Lipid hydroperoxides, lipid soluble fluorescent compounds and the iron content of both iron-injected and contralateral hemicortices showed a significant decrease in comparison to rats untreated with D-penicillamine. The higher dose also induced in normal rats a significant decrease in basal TBARS and iron content of the brain cortex. In the iron-injected cortex the observed Fe2+/Fe3+ ratio was significantly different from that of normal rats. On the contrary ratios obtained form D-penicillamine treated animals were higher in comparison to both normal and iron-injected animals. These results suggest that D-penicillamine, acting as a reducing agent, inhibits the iron redox system and, as a chelating agent, can remove metal from action sites where lipid peroxidation may occur.

The spontaneous motility of longitudinal muscle of human jejunum was recorded and the effect of gamma-aminobutyric acid-ergic (GABAergic) drugs was tested. GABA and (-)-baclofen (10(-6)-10(-4) M) dose dependently reduced the amplitude and frequency of the spontaneous contractions; muscimol and 3-aminopropanesulfonic acid (3 x 10(-5) M) were ineffective. The effect of 3 x 10(-5) M GABA was reduced by 3 x 10(-3) M 5-aminovaleric acid but not by 3 x 10(-5) M picrotoxin. The dose-response curve for GABA was shifted to the right by 3 x 10(-3) M 3-aminopropanesulfonic acid. Tetrodotoxin 3 x 10(-7) M prevented the GABAergic action, whereas various receptor antagonists tested did not affect it. GABAergic drugs did not influence the spontaneous motility of either circular or longitudinal muscles of human colon. It is suggested that GABAB receptor activation induces the inhibition of human jejunum longitudinal muscle motility by a neurogenic mechanism. The possible involvement of postganglionic cholinergic neurons is to be evaluated by other techniques.

In view of the emerging role of metals and particularly iron in the pathogenesis of several ischemic or degenerative CNS diseases, via a lipid peroxidative process, a model of slow iron-induced peroxidative damage in the rat brain cortex has been carried out. Iron-carbohydrate complexes were injected in the right brain cortex, and biochemical assays were performed on ipsilateral and contralateral samples two hours or seven days after injection. Iron-saccharate caused a significant increase in the ipsilateral cortex in TBARS, conjugated dienes and fluorescent substances seven days after injection, whereas no biochemical alteration was observed two hours after treatment. In order to prevent or to limit lipid peroxidation, some drugs known for chelating and/or scavenging activity were administered to iron-injected rats. DL-alpha-tocopherol, methylprednisolone, D-penicillamine significantly decreased the value of fluorescent products formed by iron-saccharate, whereas desferrioxamine was not effective.

Further evidence is reported on the influence exerted by capsaicin on the anaphylactic reaction evoked in actively sensitized guinea-pigs. In Herxheimer microshock induced by ovalbumin aerosol, pretreatment of animals with 100 micrograms/kg i.p. capsaicin prolonged the preconvulsion time when the drug was administered 3 h before antigen challenge. In contrast, the same dose of capsaicin injected 30 min before aerosol caused a shortening of latency of the respiratory symptomatology. The influence of the drug is no longer evident after 24 h. In "in vitro" experiments desensitization to capsaicin of tracheal preparations caused a reduction of histamine and SRS-A released during antigen challenge, in comparison to controls. Moreover, anaphylactic histamine release was increased in preparations perfused with 10(-8) M substance P. In conclusion, our findings confirm that neuropeptides may be involved in the pathogenesis of asthma by affecting release of mediators.

1) Responsiveness of guinea-pig ileal longitudinal smooth muscle-myenteric plexus preparations to drugs activating GABA-B receptors was studied in morphine-tolerant animals. For this purpose morphine pellets (75 mg each) were implanted subcutaneously in guinea-pigs and experiments were performed three days later in electrically-stimulated ileal strips. 2) Activation of GABA-B receptors with GABA (10(-6) -10(-3) M) or (-)-baclofen (10(-6)-10(-3) M) caused a dose-related inhibition of twitch response that was about 80% lower in preparations from morphine-tolerant animals than in controls. This was found both in preparations maintained in the presence of morphine (10(-6) M) and in morphine-free Krebs. The effect was evident also in ileal preparations from morphine-tolerant animals in which a withdrawal syndrome was induced by the administration of naloxone before sacrifice. 3) The phenomenon was specific since the dose-response curve of the adenosine-inhibitory effect was comparable in preparations from tolerant animals and controls. 4) The hyporesponsiveness to GABA-B receptor activation began 12 h after pellet implantation and was maximal on the third day. 5) It is concluded that during tolerance to and withdrawal from morphine there is a hyporesponsiveness of GABA-B receptors in "in vitro" guinea-pig ileal longitudinal muscle-myenteric plexus preparations.

The influence of capsaicin on anaphylactic reactions in the guinea-pig was studied both in vivo and in vitro. In guinea-pigs actively sensitized with ovalbumin, Herxheimer microshock was elicited by antigen aerosol and the preconvulsion time recorded. The preconvulsion time was reduced by about 30% in animals pretreated with capsaicin (1 mg/kg) injected i.p. 30 min before antigen aerosol, whereas it remained unchanged when the drug was administered two days before aerosol treatment. Capsaicin shows a partial protective effect when the provocative aerosol was administered 3 h after the last of three doses of capsaicin (100 micrograms/kg, i.p.), which had been injected for three consecutive days. Ileum longitudinal muscle strips were used for in vitro anaphylaxis studies. These were isolated from guinea-pigs actively sensitized with ovalbumin and histamine release evoked by antigen was measured. Preparations perfused with capsaicin (10(-6)-10(-4) M) and desensitized to the drug, showed a lower anaphylactic release of histamine. This effect was dose-dependent, with the histamine release reduced by 35% at higher concentrations (10(-5)-10(-4) M) of capsaicin. The mechanism of the influence of capsaicin on anaphylactic reactions is discussed briefly.