C1-Esterase inhibitor: an anti-inflammatory agent and its potential use in the treatment of diseases other than hereditary angioedema

C1-esterase inhibitor (C1-Inh) therapy was introduced in clinical medicine about 25 years ago as a replacement therapy for patients with hereditary angioedema caused by a deficiency of C1-Inh. There is now accumulating evidence, obtained from studies in animals and observations in patients, that administration of C1-Inh may have a beneficial effect as well in other clinical conditions such as sepsis, cytokine-induced vascular leak syndrome, acute myocardial infarction, or other diseases. Activation of the complement system, the contact activation system, and the coagulation system has been observed in these diseases. A typical feature of the contact and complement system is that on activation they give rise to vasoactive peptides such as bradykinin or the anaphylatoxins, which in part explains the proinflammatory effects of either system. C1-Inh, belonging to the superfamily of serine proteinase inhibitors (serpins), is a major inhibitor of the classical complement pathway, the contact activation system, and the intrinsic pathway of coagulation, respectively. It is, therefore, endowed with anti-inflammatory properties. However, inactivation of C1-Inh occurs locally in inflamed tissues by proteolytic enzymes (e.g., elastase) released from activated neutrophils or bacteria thereby leading to increased local activation of the various host defense systems. Here we will give an overview on the biochemistry and biology of C1-Inh. We will discuss studies addressing therapeutic administration of C1-Inh in experimental and clinical conditions. Finally, we will provide an explanation for the therapeutic benefit of C1-Inh in so many different diseases.     

In vivo effects of cellulose sulphate on plasma kininogen, complement and inflammation

1. The in vivo action of cellulose sulphate was studied in an attempt to clarify the role of complement and kinin formation in inflammation.2. Inflammatory oedema was produced in the rat paw by heat (45.5 degrees C), and on the ear by xylene. The oedema was assessed by comparing the ratio of fresh wet weight to dry weight of corresponding injured and non-injured parts.3. Following cellulose sulphate (6.5 mg/kg i.v.), plasma kininogen concentrations were promptly reduced by 90% or more. The reduction in complement titres was statistically significant and ranged from 17 to 65%. No toxic effects were observed. The oedema caused by heat or xylene was not reduced in these rats.4. Cellulose sulphate (80 mg/kg i.p.) given over 3 days depleted plasma kininogen by about 90%, but reduced complement titres only slightly. These rats gained less weight and their condition was poor. Blood clotting was impaired and widespread haemorrhages were found. Heat and xylene produced significantly less oedema than in control rats. This diminished response is attributed to toxic side effects of cellulose sulphate, rather than depleted plasma kininogen and reduced plasma complement.5. The results suggest that the inflammatory reactions to thermal and chemical injury can fully develop when plasma kininogen and complement are lowered.     

Effects of dextran sulphate (DXS) on lymphocyte localization in complement-deficient mice: evidence that the fifth component of complement is not implicated in the DXS activity

The effects of subcutaneously or intraperitoneally administered dextran sulphate (DXS) (50 mg/Kg) on the subsequent 1 h localization of intravenously injected radiolabelled lymph node cells was investigated in complement deficient mice which lack C5. DXS proved to be equally as potent in depressing cell localization in deficient as compared to normal mice. These findings indicate that the terminal complement components are not essential for DXS activity.     

Inhibition of complement by covalent attachment of rosmarinic acid to activated C3b.

Rosmarinic acid has been reported to inhibit complement activation in vivo as well as in vitro. Previous studies suggested that the inhibitory effect was due to inhibition of C3/C5 convertases, but inhibition of C3b attachment would yield the same results. Recent work in our laboratory demonstrated that compounds with polyhydroxylated phenyl rings are highly reactive with the thioester bond in nascent C3b. These compounds block complement activation by preventing attachment of C3b to the activating surface. Because rosmarinic acid contains two 3,4-dihydroxyphenyl groups, the current study was undertaken to re-examine the mechanism of inhibition by analyzing the effect of rosmarinic acid on C3b attachment. In assays using purified complement proteins, rosmarinic acid inhibited covalent attachment of C3b to cells with an 1C50 = 34 microM. Inhibition of C5 convertase activity required 1500 microM rosmarinic acid, and no significant inhibition of the C3 convertase enzyme, which produces C3b from C3, was observed at 10,000 microM. In hemolytic assays using human serum, rosmarinic acid was shown to inhibit activation of both the classical (IC50 = 180 microM) and the alternative (IC50 = 160 microM) pathways of complement. Rosmarinic acid concentrations up to 10,000 microM did not cause direct inactivation of C3. Radioiodination of rosmarinic acid was used to demonstrate covalent activation-dependent incorporation of rosmarinic acid specifically into the thioester-containing alpha'-chain of nascent C3b. These findings indicate that inhibition of complement activation by rosmarinic acid is due to the reaction of rosmarinic acid with the activated thioester of metastable C3b, resulting in covalent attachment of the inhibitor to the protein.     

Pharmacology of calcium-induced long-term potentiation in rat hippocampal slices

A transient increase (10 min) in extracellular calcium concentration (4 mM) causes a long-lasting (greater than 2 hr) enhancement of population spike responses evoked by radiatum fibers to CA1 pyramidal neurons in rat hippocampal slices. This phenomenon is similar to tetanic long-term potentiation (LTP), and is also related to memory processes. The influence of various drugs was investigated on calcium-induced LTP. The NMDA antagonist 2 amino-5-phosphonopentanoic acid (AP5; 100 microM) was able to prevent the calcium-induced LTP, while atropine sulphate (10 microM), propranolol hydrochloride (10 microM) and verapamil hydrochloride (100 microM) were ineffective. The results suggest an involvement of the NMDA receptor in the development of calcium-induced LTP.     

Effect of oral iron supplementation on oxidative stress and colonic inflammation in rats with induced colitis

BACKGROUND: Iron supplementation may increase disease activity in ulcerative colitis, possibly through the production of reactive oxygen species from the Fenton reaction. AIM: To assess the effects of two doses of oral iron on intestinal inflammation and oxidative stress in experimental colitis. METHODS: Colitis was induced in rats by giving 5% dextran sulphate sodium in drinking water for 7 days. First, using a 2 x 2 factorial design, rats with or without dextran sulphate sodium received the regular diet or a diet containing iron 3%/kg diet. Second, rats with dextran sulphate sodium-induced colitis were supplemented with iron 0.3%/kg diet and compared with rats on dextran sulphate sodium and regular diet. The body weight change, histological scores, colon length, rectal bleeding, plasma and colonic lipid peroxides, colonic glutathione peroxidase and plasma vitamin E and C were measured. Faecal analysis for haem and total, free and ethylenediaminetetra-acetic acid-chelatable iron was also performed. RESULTS: Iron 3% and iron 0.3% increased the activity of dextran sulphate sodium-induced colitis, as demonstrated by higher histological scores, heavier rectal bleeding and further shortening of the colon. This was associated with increased lipid peroxidation and decreased antioxidant vitamins. Faecal iron available to the Fenton reaction was increased in a dose-dependent manner. CONCLUSIONS: Iron supplementation taken orally enhanced the activity of dextran sulphate sodium-induced colitis and is associated with an increase in oxidative stress.     

E3040 sulphate,a novel thromboxane synthase inhibitor,blocks the Cl- secretion induced by platelet-activating factor in isolated rat colon

1. E3040 (6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl)benzothiazole), is a novel dual inhibitor of 5-lipoxygenase (5-LOX) and thromboxane synthase (Tx synthase). Here, we examined the effects of E3040 sulphate, a sulphate conjugate of E3040, on these enzyme activities in cell-free systems and on the thromboxane A2 (TxA2)-mediated Cl- secretion induced by platelet-activating factor (PAF) in isolated rat colons. 2. E3040 sulphate inhibited Tx synthase activity in a concentration-dependent manner (IC50=0.013 microM), whereas it induced little effects on 5-LOX and cyclo-oxygenase activities (IC50>100 microM) with the cell-free enzyme assay. 3. With isolated rat colonic mucosa, E3040 sulphate in a concentration-dependent manner (IC50=1.8 microM) inhibited the Cl- secretion induced by 10 microM PAF. On the other hand, E3040 sulphate (30 microM) induced no effect on the prostaglandin E2 (0.5 microM)- and leukotriene D4 (1 microM)-induced Cl- secretion in the colon. 4. PAF (10 microM) increased a release of TxB2, a stable metabolite of TxA2, from the colonic mucosa. This increase was significantly inhibited by subsequent addition of E3040 sulphate (30 microM). 5. Probenecid (100 microM), an inhibitor of organic anion transporter, abolished the inhibitory effect of E3040 sulphate on the PAF-induced Cl- secretion. Another inhibitor, sulphobromophthalein (30 microM) partially but significantly attenuated the effect of E3040 sulphate. p-aminohippuric acid (1 mM) had no effect. 6. These findings suggest that E3040 sulphate is a novel Tx synthase inhibitor, and that E3040 sulphate taken up into the colonic cells by organic anion transporters inhibits the PAF-induced Cl- secretion by blocking a release of TxA2.     

Effects of ethanol ingestion on the metabolism of a hepatotoxic dose of paracetamol in mice

After administration to mice of a hepatotoxic dose of paracetamol (400 mg/kg body wt, p.o.) peak plasma concentrations of the drug and its glucuronide were approximately 900 microM around one hour. Corresponding levels of the sulphate, mercapturate and cysteine conjugates were approximately 100, 35 and 20 microM, respectively. Urinary excretion accounted for 55% of the administered drug 31 h after dosing. Of this total, 64.7% was paracetamol glucuronide, 17.9% paracetamol cysteine, 10.4% paracetamol sulphate, 0.5% paracetamol mercapturate and 6.5% unchanged drug. One hour after acute ethanol administration (3 g/kg, p.o., concomitantly with paracetamol) plasma levels of the glucuronide, cysteine and mercapturate conjugates were decreased by approximately 50%. There were reductions in the urinary excretion of the glucuronide (-13%) and cysteine conjugates (-24%), but increases in the amounts of mercapturate (+52%), sulphate +11%) and unchanged drug (+81%). Chronic ethanol ingestion (15 g/kg per d for 28 d) caused a transient initial increase in plasma paracetamol cysteine (+32%) and mercapturate (+41%) concentrations, but the only substantial change in urinary excretion was a 29% increase in the amount of paracetamol glucuronide. After chronic ethanol consumption, acute ethanol administration had a transient inhibitory effect on paracetamol mono-oxygenation, but glucuronidation was unaffected (as judged by plasma concentrations). Only paracetamol mercapturate excretion was substantially affected (+64%).     

Four motor effects of capsaicin on guinea-pig distal colon.

The motor effects of capsaicin on the guinea-pig distal colon have been investigated in vivo and in vitro. Capsaicin (0.1-10 micrograms kg-1 i.v.) produced a transient relaxation which was reduced by pretreatment with capsaicin itself, atropine, hexamethonium, phentolamine or guanethidine and almost abolished by tetrodotoxin (TTX). Topically applied capsaicin produced a transient inhibition of tone and spontaneous activity prevented by topically applied TTX. In isolated preparations of distal colon, capsaicin produced a transient, TTX- and atropine-sensitive contraction which was followed by a depression of the contractile activity. The depressant effect was unaffected by atropine plus guanethidine but was greatly reduced by TTX, indicating activation of intramural non-adrenergic, non-cholinergic (NANC) mechanisms. The depressant effect on the first exposure to capsaicin (1 microM) was greater than that produced by a second, third or fourth exposure. In preparations excised from capsaicin-pretreated animals, capsaicin (1 microM) only produced an inhibitory effect on spontaneous contractions. Desensitization did not occur to this inhibitory effect. In preparations pre-exposed to capsaicin (1 microM, 1 h before), capsaicin (1-30 microM) produced a concentration-related inhibition of spontaneous contractions (IC50 = 19 microM) and of the high K+-induced tonic contraction (IC50 = 23 microM). A similar effect on spontaneous motility was produced by capsaicin in colonic segments excised from capsaicin-pretreated guinea-pigs (IC50 = 16 microM) or guinea-pigs treated with TTX (IC50 = 20 microM). It is concluded that, in vivo, capsaicin activates inhibitory reflexes, presumably due to stimulation of primary afferent fibres. This effect involves, at least in part, activation of sympathetic nerves to this organ. The contractile effect of capsaicin on the isolated colon involves activation of intramural cholinergic neurones, whereas the TTX-sensitive component of the inhibitory effect involves either release of an inhibitory transmitter through an axon reflex arrangement or activation of NANC neurones. In addition, at high concentrations capsaicin produces a direct depression of smooth muscle contraction.     

Omeprazole weakly inhibits CYP1A2 activity in man

BACKGROUND AND OBJECTIVES: Omeprazole is an inducer of human cytochrome P450 1A (CYP1A) enzymes, but shows inhibitory effects on CYP2C19 and CYP3A4. In this study, a potential inhibitory effect of omeprazole on caffeine metabolism, a validated CYP1A2 marker, was examined. METHODS: A randomized, balanced crossover single-dose study was conducted in 16 healthy volunteers comprising 12 extensive (EM) and 4 poor metabolizers (PM) for CYP2C19. All volunteers received a 40 mg omeprazole dose or placebo 0.5 h prior to caffeine 3 mg/kg body weight. Six EMs were re-tested with 80 mg of omeprazole. In vitro, effects of omeprazole on caffeine N3-demethylation were determined in human liver microsomes. RESULTS: In vivo, non-parametric point estimates (90% confidence intervals) for the ratios of caffeine pharmacokinetics with/without co-administration of the 40 mg omeprazole dose were: AUC 1.08 (1.04 - 1.13), MRT 1.09 (0.99 - 1.19), and plasma ratio of paraxanthine/caffeine 6 h post-dose 0.91 (0.80 - 1.00). Inhibition of caffeine N3-demethylation by omeprazole was slightly more pronounced in PM than in EM of CYP2C19. Estimates for the 80 mg omeprazole dose were: AUC 1.12 (1.05 -1.18), MRT 1.18 (1.07 - 1.30), and paraxanthine/caffeine ratio 0.83 (0.74 -0.94). In vitro, omeprazole was mainly a competitive CYP1A2 inhibitor with K(i) values of around 150 microM. CONCLUSIONS: Omeprazole exerts a concentration-dependent inhibition of CYP1A2 activity in man. However, even after single oral doses up to 80 mg, this effect is weak and without clinical relevance.     

Some pharmacodynamic properties of carrageenin in the rat

1. Carrageenin oedema is suppressed by pre-treating the rats with cellulose sulphate, a kininogen depleting agent. This inhibition is closely related to the dose of cellulose sulphate and to the time course of kininogen depletion.2. Oedema induced by egg white or by dextran, in which the mediators are histamine and 5-hydroxytryptamine, is quite unaffected by cellulose sulphate treatment.3. Carrageenin injected intravenously lowers the arterial blood pressure of rats. This hypotensive effect is unaffected by histamine antagonists and is abolished by protease inhibitors and thus seems to be due to kinin release from plasma substrates.4. Like cellulose sulphate, carrageenin enhances the esterolytic activity of the blood from treated rats when incubated with benzoyl-arginine ethyl ester.5. The ability of carrageenin to activate the kinin-forming system could account for both its inflammatory and hypotensive effects.     

Triggering endogenous immunosuppressive mechanisms by combined targeting of Dipeptidyl peptidase IV (DPIV/CD26) and Aminopeptidase N (APN/ CD13)--a novel approach for the treatment of inflammatory bowel disease

The ectopeptidases Dipeptidylpeptidase IV and Alanyl-Aminopeptidase N, strongly expressed by both, activated and regulatory T cells were shown to co-operate in T cell regulation. Based on the findings that DPIV and APN inhibitors induce the TGF-beta1 and IL-10 production and a suppression of T helper cell proliferation in parallel, and that particularly APN inhibitors amplify the suppressing activity of regulatory T cells, both peptidases represent a promising target complex for treatment of diseases associated with an imbalanced T cell response, such as inflammatory bowel diseases (IBD). The aim of the present study was to analyze the therapeutic potential of DPIV and APN inhibitors in vivo in a mouse model of colitis. Balb/c mice received 3% (w/v) dextran sulphate sodium with the drinking water for 7 days. After onset of colitis symptoms, inhibitor treatment started at day 3. Disease activity index (DAI) was assessed daily, supplemented by histological and immunological analysis. While the DPIV inhibitor Lys-[Z(NO])(2)]-pyrrolidide or the APN-inhibitor Actinonin alone had marked but no significant therapeutic effects, the simultaneous administration of both inhibitors reduced colitis activity in comparison to placebo treated mice, significantly (DAI 4.8 vs. 7.7, p<0.005). A newly developed compound IP12.C6 with inhibitory capacity toward both enzymes significantly attenuated the clinical manifestation of colitis (DAI 3.2 vs. 7.6, p<0.0001). TGF-beta mRNA was found to be up-regulated in colon tissue of inhibitor-treated animals. In summary our results strongly suggest that combined DPIV and APN inhibition by synthetic inhibitors represents a novel and efficient approach for the pharmacological therapy of IBD by triggering endogenous immunosuppressive mechanisms.     

Anti-HIV activity of dextran sulphate as determined under different experimental conditions

Dextran sulphate is a potent and selective inhibitor of human immunodeficiency virus type 1 (HIV-1). Its anti-HIV-1 activity has been investigated under varying experimental conditions. MT-4 cells were infected with HIV-1 at different multiplicities of infection (MOI), and treated with either dextran sulphate, 3'-azido-2',3'-dideoxythymidine (AZT), or anti-HIV-1 serum obtained from an ARC patient. Dextran sulphate suppressed HIV-1 replication (as monitored by viral antigen expression) when the MOI was 0.01 or 0.1. It was ineffective at an MOI of 1.0. The anti-HIV-1 serum was only partially effective at an MOI of 0.01 and ineffective at an MOI of 0.1 or 1.0. AZT proved effective at all three MOIs. Co-cultures of uninfected and HIV-1-infected MT-4 cells were protected against destruction by dextran sulphate at a concentration of 10 and 100 micrograms/ml. To fully suppress viral antigen expression a concentration of 100 micrograms/ml was needed. When used at this concentration, a 1-h contact of dextran sulphate with the cells during the virus adsorption period sufficed to suppress HIV-1 antigen expression. In this sense, dextran sulphate behaved like the anti-HIV-1 serum. Dextran sulphate also behaved like OKT-4A in that they both inhibited HIV-1 attachment to the MT-4 cells, whereas OKT-4 failed to do so. However, dextran sulphate did not affect the binding of OKT-4A to the cells. The present results support the concept that dextran sulphate owes its anti-HIV-1 activity mainly to inhibition of virus binding to its target cells. The anti-HIV-1 activity of dextran sulphate is highly dependent on its sulphate content.     

In vitro and in vivo inhibitory effect of stiripentol on clobazam metabolism.

A metabolic interaction between stiripentol (STP), an anticonvulsant agent that inhibits the activity of several cytochromes P450 (P450s), and clobazam (CLB), a 1,5-benzodiazepine, used in association with STP in severe myoclonic epilepsy in infancy was observed in vivo. This interaction was characterized in vitro using cDNA-expressed CYP3A4 and CYP2C19 (main P450 involved in CLB metabolism) to calculate K(i) and IC(50) of stiripentol in comparison with ketoconazole (CYP3A4 inhibitor) and omeprazole (CYP2C19 inhibitor). STP inhibited N-demethylation of CLB to N-desmethylclobazam (NCLB) mediated by CYP3A4 (noncompetitively) and CYP2C19 (competitively) with K(i) = 1.59 +/- 0.07 and 0.516 +/- 0.065 microM and IC(50) = 1.58 microM [95% confidence interval (CI95%) = 1.20-2.08] and 3.29 microM (CI95% = 1.87-5.79), respectively. STP inhibited also more strongly the 4'-hydroxylation of NCLB to 4'-hydroxy-N-desmethylclobazam by CYP2C19 [competitive interaction with K(i) = 0.139 +/- 0.025 microM and IC(50) = 0.276 microM (CI95% = 0.206-0.371)]. The inhibitory effect of STP on CLB demethylation by CYP3A4 was much weaker than that of ketoconazole [IC(50) = 0.023 microM (CI95% = 0.016-0.033)], whereas its effect on NCLB hydroxylation by CYP2C19 was much higher than that of omeprazole [IC(50) = 2.99 microM (CI95% = 2.11-4.24)]. The major in vitro inhibitory effect of STP on CLB metabolism and mostly on NCLB biotransformation is consistent with the changes in vivo in CLB and NCLB plasma concentrations in children treated by the association CLB/STP.     

Enhancement of gastrointestinal absorption of ovalbumin caused by spermine induces an increase in plasma histamine levels in mice sensitized to ovalbumin

The aim of this study was to determine whether a spermine (SPM)-induced increase in gastrointestinal absorption of an allergen leads to an anaphylactic response in sensitized mice. First, we examined the enhancing effect of SPM on the gastrointestinal absorption of ovalbumin (OVA) in an in situ jejunum loop study in rats and an in vivo oral absorption study in mice. Second, we investigated whether enhancement of gastrointestinal absorption of OVA caused by SPM induces an anaphylactic response in mice sensitized to OVA. In the in situ jejunum loop study in rats, a significant amount of immune-reactive OVA was detected in the plasma after co-administration of OVA and SPM. Oral co-administration of OVA and SPM to mice in vivo also increased plasma OVA concentrations in an SPM dose-dependent manner. Furthermore, in sensitized mice, a significant increase in plasma histamine levels occurred along with the increase in plasma OVA levels after co-administration of OVA with SPM. This finding suggests that an SPM-induced increase in gastrointestinal absorption of OVA leads to an anaphylactic response. These results indicate that excess oral ingestion of SPM may have widespread health effects, including the induction of food allergies, via modulation of the function of the gastrointestinal epithelial barrier.     

Trimethoprim and sulfamethoxazole are selective inhibitors of CYP2C8 and CYP2C9, respectively.

To evaluate the inhibitory effects of trimethoprim and sulfamethoxazole on cytochrome P450 (P450) isoforms, selective marker reactions for CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 were examined in human liver microsomes and recombinant CYP2C8 and CYP2C9. The in vivo drug interactions of trimethoprim and sulfamethoxazole were predicted in vitro using [I]/([I] + K(i)) values. With concentrations ranging from 5 to 100 microM, trimethoprim exhibited a selective inhibitory effect on CYP2C8-mediated paclitaxel 6alpha-hydroxylation in human liver microsomes and recombinant CYP2C8, with apparent IC(50) (K(i)) values of 54 microM (32 microM) and 75 microM, respectively. With concentrations ranging from 50 to 500 microM, sulfamethoxazole was a selective inhibitor of CYP2C9-mediated tolbutamide hydroxylation in human liver microsomes and recombinant CYP2C9, with apparent IC(50) (K(i)) values of 544 microM (271 microM) and 456 microM, respectively. With concentrations higher than 100 microM trimethoprim and 500 microM sulfamethoxazole, both drugs lost their selectivity for the P450 isoforms. Based on estimated total hepatic concentrations (or free plasma concentrations) of the drugs and the scaling model, one would expect in vivo in humans 80% (26%) and 13% (24%) inhibition of the metabolic clearance of CYP2C8 and CYP2C9 substrates by trimethoprim and sulfamethoxazole, respectively. In conclusion, trimethoprim and sulfamethoxazole can be used as selective inhibitors of CYP2C8 and CYP2C9 in in vitro studies. In humans, trimethoprim and sulfamethoxazole may inhibit the activities of CYP2C8 and CYP2C9, respectively.     

Inhibitory effects of H1-antihistamines on CYP2D6- and CYP2C9-mediated drug metabolic reactions in human liver microsomes

OBJECTIVE: To screen the inhibitory effects of H1-antihistamines on hepatic bufuralol 1'-hydroxylation and on tolbutamide 4-methylhydroxylation in human liver microsomes. METHODS: Bufuralol 1'-hydroxylation and tolbutamide 4-methylhydroxylation were used as index reactions for CYP2D6 and CYP2C9, respectively. The metabolites of both reactions were measured using high-performance liquid chromatography and were used as indicators of whether CYP2D6 or CYP2C9 activities were inhibited or unaffected by the agents. RESULTS: All five H1-antihistamines studied showed a concentration-dependent inhibition of CYP2D6-mediated bufuralol 1'-hydroxylation with 50% inhibitory concentration (IC50) values of 32-109 microM. Cyclizine and promethazine showed inhibitory effects on tolbutamide 4-methylhydroxylation with IC20 values of 85 microM and 88 microM, respectively. Tripelennamine, chlorpheniramine, and diphenhydramine showed no inhibitory effects on CYP2C9. CONCLUSION: All five H1-antihistamines studied inhibited CYP2D6 markedly, but only cyclizine and promethazine inhibited CYP2C9 at concentrations above that usually seen in plasma. Promethazine and chlorpheniramine inhibited CYP2D6 at concentrations that are very close to their therapeutic plasma concentrations. Further studies in humans, especially in poor metabolizers of CYP2D6, will be required to confirm these findings.     

Effect of probenecid on the formation and elimination kinetics of the sulphate and glucuronide conjugates of diflunisal

The effect of probenecid on the pharmacokinetics of diflunisal and its glucuronide and sulphate conjugates was studied in 8 healthy volunteers. Diflunisal 250 mg b. d. was administered p. o. for 15 days and its steady state pharmacokinetics was evaluated on Day 16 after the last dose (control phase). Probenecid 500 mg b. d. was co-administered throughout the entire study period in the treatment phase of the study.The steady state plasma concentration of diflunisal was significantly higher during the probenecid treatment phase as compared to the control phase (104.0 vs. 63.1 g·ml^–1). This was the result of a significant decrease in the plasma clearance of diflunisal from 5.8 (control) to 3.4 ml·min^–1 (probenecid co-administration). The metabolite formation clearances of both glucuronides were significantly decreased by probenecid, -45 % and -54 % for the phenolic and acyl glucuronide, respectively. The metabolite formation clearance of the sulphate conjugate was not affected by probenecid co-administration.Steady state plasma concentrations of the sulphate and glucuronide conjugates of diflunisal were 2.5- to 3.1-fold higher during probenecid co-administration, due to a significant reduction in the renal clearance of the three diflunisal conjugates. Probenecid also reduced the plasma protein binding of diflunisal, but only to a minor extent; the unbound plasma fraction of diflunisal at steady state averaged between 5 and 30 % higher during probenecid co-administration.     

Structure and function of eritadenine and its 3-deaza analogues: potent inhibitors of S-adenosylhomocysteine hydrolase and hypocholesterolemic agents

d-Eritadenine (DEA) is a potent inhibitor of S-adenosyl-l-homocysteine hydrolase (SAHH) and has hypocholesterolemic activity. We have hypothesized that 3-deaza-DEA (C3-DEA) and its analogues retain high level of SAHH inhibitory activity and have resistance to deamination and glycosidic bond hydrolysis in vivo. Such C3-DEA analogues would have much higher hypocholesterolemic activity. C3-DEA, and its methyl ester (C3-OMeDEA) and its methyl amido (C3-NMeDEA) were synthesized to examine their SAHH inhibitory and hypocholesterolemic activities. A crystal structure of SAHH containing C3-DEA was determined and confirmed that DEA and C3-DEA bound to the same site of SAHH with the same binding mode. The SAHH inhibitory activities of C3-DEA (K(I)=1.5 microM) and C3-OMeDEA (K(I)=1.5 microM) are significantly lower than that of DEA (K(I)=30 nM), while rats fed by C3-DEA and C3-OMeDEA decrease the total plasma cholesterol and phospholipids by 36-40% and 23%, respectively, which is similar to the level of reductions (42% and 27%) by DEA. C3-NMeDEA lost most of the SAHH inhibitory activity (K(I)=30 microM) and dietary C3-NMeDEA does not decrease cholesterol and phospholipid in plasma but decreases the triacylglycerol level by 16%. DEA and C3-DEA analogues are neither substrates nor inhibitors of adenosine deaminase.     

Caffeine induces a profound and persistent tachycardia in response to MDMA ("Ecstasy") administration

Caffeine promotes hyperthermia and lethality when co-administered with the recreational drug 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy") to rats. In the present study, co-administration of caffeine (10 mg/kg, s.c.) with MDMA (10 mg/kg, s.c.) induced a profound tachycardic response compared to rats treated with either drug alone. However, neither caffeine (30 microM) nor MDMA (1-30 microM), alone or in combination, affected the electrocardiogram of the isolated heart suggesting that central and sympathomimetic actions, rather than direct actions of these drugs on the heart, are responsible for the tachycardia observed in vivo. This is a serious drug interaction, which could have important health consequences for recreational drug users.