Cephalosporin-induced alteration in hepatic glutathione redox state. A potential mechanism for inhibition of hepatic reduction of vitamin K1,2,3-epoxide in the rat.

Hypoprothrombinemia is a serious adverse effect of antimicrobial therapy that occurs after administration of some second- and third-generation cephalosporins which contain the methyltetrazole-thiol (MTT) group. Previous studies have shown that in vitro MTT directly inhibits microsomal gamma-carboxylation of a synthetic pentapeptide. Since MTT is a thiocarbamide, a type of compound that can increase oxidation of glutathione, the present studies were carried out to determine whether alterations in hepatic glutathione redox state might interfere with vitamin K metabolism. Dose-related increases in biliary efflux and hepatic concentration of oxidized glutathione (GSSG) occurred after intravenous administration of MTT or MTT-containing antibiotics to rats. This finding suggested that these compounds could alter the hepatic glutathione redox state in vivo. Microsomal reduction of vitamin K epoxide occurred in the presence of 100 microM dithiothreitol (DTT), but was inhibited by preincubation with GSSG at concentrations as low as 10 microM. At higher concentrations of DTT (1.0 mM) inhibition by GSSG persisted, but higher concentrations were required, suggesting that the thiol/disulfide ratio, rather than the absolute concentration of GSSG was important. By contrast, GSSG did not effect microsomal gamma-carboxylation of a pentapeptide, using either vitamin K1 or its hydroquinone as a cofactor. These findings suggest a novel mechanism for the hypoprothrombinemia occurring after administration of MTT-containing antibiotics.

Systemic and intestinal pharmacokinetics of methotrexate in patients with inflammatory bowel disease.

BACKGROUND: The pharmacokinetics of low-dose subcutaneous methotrexate have not been determined throughout the standard weekly dosing interval. It is not known whether methotrexate concentrations in the gastrointestinal tract are sufficient for pharmacologic activity in inflammatory bowel disease. METHODS: Ten patients with inflammatory bowel disease participated in the study. After the patients started taking 15 or 25 mg subcutaneous methotrexate once a week, erythrocyte methotrexate concentration was measured every 2 weeks. The absorption, rectal distribution, metabolism, and elimination of methotrexate were measured. The effect of methotrexate on proliferation of an intestinal epithelial cell line was determined. RESULTS: After weekly subcutaneous administration of methotrexate was begun, trough erythrocyte concentration rose to reach a plateau after 6 to 8 weeks, ranging from 150 to 300 nmol/L. More than 90% of subcutaneously administered methotrexate was rapidly excreted in the urine. The methotrexate plasma time course after subcutaneous administration fit a 2-compartment first-order model with biphasic elimination and trough concentration of about 1 nmol/L. Trough and peak methotrexate concentrations (mean value +/- SD) were 64 +/- 33 and 206 +/- 64 fmol/mg in the rectal mucosa and 4 +/- 3 and 51 +/- 26 nmol/L in the rectal lumen. These methotrexate concentrations were in the range found to be pharmacologically active against Caco-2 cell growth, that is, a 50% inhibitory concentration from 10 to 46 nmol/L. CONCLUSION: Subcutaneous methotrexate was well absorbed and distributed to the site of the lesions in patients with inflammatory bowel disease. Methotrexate was concentrated intracellularly in blood and in the rectum. The methotrexate concentration in the rectal mucosa remained within a pharmacologically active range throughout the dosing interval. The findings represent a pharmacologic explanation for the sustained efficacy of weekly methotrexate therapy.

Diadenosine 5',5"-P1,P5-pentaphosphate harbors the properties of a signaling molecule in the heart.

Dinucleotide polyphosphates (ApnA) have emerged as signaling molecules in rapidly dividing cells. The presence and role of Ap5A in the heart remain unknown. Here, we report that the myocardium contains abundant amounts of diadenosine 5',5"-P1,P5-pentaphosphate (Ap5A), a member of the ApnA family. Ischemia induced 10-fold decrease in the myocardial concentration of Ap5A. A target of Ap5A action was identified to be the cardiac ATP-sensitive K+ (K(ATP)) channel, a metabolism-sensitive ion conductance activated in ischemia. At levels found in hearts prior to ischemia, Ap5A maintained a low probability of K(ATP) channel opening, but at levels found in hearts following ischemia, Ap5A allowed a high probability of K(ATP) channel opening. Taken together, the present data suggest that Ap5A harbors the properties of a signaling molecule involved in the cardiac response to metabolic stress.

Prospective randomized double-blind comparison of moxalactam and tobramycin in treatment of urinary tract infections.

In a prospective, randomized, double-blind trial, a regimen of 250 mg of moxalactam every 12 hours was compared with 1.0 mg/kg of tobramycin every eight hours in the treatment of urinary tract infections. One hundred and eleven patients were entered into the study; results in 63 (18 men and 45 women) were evaluable for both efficacy and toxicity. Thirty evaluable patients received moxalactam, and 33 received tobramycin. The mean duration of therapy in each group was seven days. There were six treatment failures in the moxalactam group and 10 failures in the tobramycin group (p greater than 0.4). Nephrotoxicity, defined as an increase in serum creatinine levels to 0.5 mg/dl or more, did not occur in either group. Thirteen patients in the moxalactam group and one in the tobramycin group had enterococci isolated from a urine culture specimen during or after therapy. It is concluded that use of the moxalactam regimen is as effective and safe as use of the tobramycin regimen in the treatment of urinary tract infections. The clinical significance of the enterococcal isolates associated with moxalactam therapy is yet to be determined.

Clinical outcome and pharmacokinetics after addition of low-dose cyclosporine to methotrexate: a case study of five patients with treatment-resistant inflammatory bowel disease.

INTRODUCTION: This study reports the clinical outcome, toxicity, and methotrexate pharmacokinetics after the addition of low-dose cyclosporine to methotrexate in patients with ulcerative colitis or Crohn's disease. METHODS: Three patients with steroid-refractory ulcerative colitis and two patients with steroid refractory Crohn's disease who failed monotherapy with subcutaneous methotrexate 25 mg/week for 16 weeks were treated with the combination of methotrexate and low-dose oral cyclosporine (3 mg/kg/day) for an additional 16 weeks. Clinical response was measured with the Inflammatory Bowel Disease Questionnaire (IBDQ) score. Concentrations of erythrocyte methotrexate, plasma methotrexate, and plasma 7-hydroxymethotrexate were also determined. RESULTS: Both patients with Crohn's disease withdrew from the study for toxicity (headaches, seizure). The three patients with ulcerative colitis experienced clinical improvement with a mean increase in the IBDQ score from 164 to 190 points, p = 0.01. The mean serum creatinine in the three patients who completed the study increased from 0.9 mg/dL at baseline to 1.2 mg/dL at week 16. p = 0.04. One patient developed hypertension. There was no significant change from baseline in the concentrations of erythrocyte methotrexate, plasma methotrexate, and plasma 7-hydroxymethotrexate. CONCLUSIONS: Combination therapy with methotrexate and low-dose oral cyclosporine did not alter methotrexate pharmacokinetics and resulted in high rates of cyclosporine-associated toxicity.

Plasma and rectal adenosine in inflammatory bowel disease: effect of methotrexate.

In animal models, the antiinflammatory mechanism of action of methotrexate has been attributed to elevation of the extracellular concentration of the endogenous autocoid, adenosine. Our goal was to determine if methotrexate elevates adenosine concentrations in plasma and at the site of disease in patients with inflammatory bowel disease. In 10 patients with Crohn's disease or ulcerative colitis, rectal adenosine and plasma adenosine concentrations were measured before and immediately after a subcutaneous injection of methotrexate, 15 or 25 mg. The mean predose rectal adenosine concentration of 2.4 mumol/l was not significantly different from the postdose concentration of 2.1 mumol/l, p = 0.17, (paired two-tailed t test). Rectal adenosine concentration tended to correlate with rectal endoscopic disease activity, r = 0.59, p = 0.067 (Spearman rank order correlation). After methotrexate injection, neither the mean daily plasma adenosine concentration, nor the plasma adenosine at any individual time point, were significantly different from preinjection values. In patients with inflammatory bowel disease, an injection of methotrexate in the clinically effective dose range does not raise rectal or plasma adenosine concentrations. A role for adenosine as the mediator of the antiinflammatory action of methotrexate is not supported.

Ability of 1-methyltetrazole-5-thiol with microsomal activation to inhibit aldehyde dehydrogenase.

Antibiotics that contain the 1-methyltetrazole-5-thiol (MTT) leaving group are associated with an adverse effect when alcohol is ingested after their administration. Therefore, the ability of MTT to inhibit an enzyme in alcohol metabolism, aldehyde dehydrogenase (ALDH), was examined. In the absence of microsomes, MTT did not inhibit ALDH obtained from either yeast or rat liver. In the presence of rat hepatic microsomes, MTT was able to inhibit the enzyme from both sources. The characteristics of the inhibition were studied, using the yeast enzyme, and found to be dependent upon the length of incubation with the hepatic microsomes and upon the concentration of MTT. Inhibition required the presence of NADH and was not detected if the microsomes were heat treated. Dilution did not reverse the inhibition. Intact antibiotics which contain the MTT moiety did not cause an inhibition of yeast ALDH unless the antibiotics were first treated with potassium hydroxide and then incubated with microsomes. Inhibition of ALDH activity measured in the mitochondrial plus microsomal fractions of rat liver also required NADH and was prevented by glutathione and heat treatment of the microsomes. These results indicate that microsomal activation of MTT is necessary for inhibition of aldehyde dehydrogenase. The behavior of MTT described here may explain the adverse effect observed if alcohol is ingested following administration of MTT-containing antibiotics.

Role of disulfiram in the in vitro inhibition of rat liver mitochondrial aldehyde dehydrogenase.

The alcohol aversion therapy drug disulfiram has been shown to inhibit hepatic aldehyde dehydrogenase (ALDH), one of the key enzymes involved in ethanol metabolism. It is believed by some that disulfiram could be one of the active inhibitors in vivo. However, the actual interaction between disulfiram and ALDH remains ambiguous. We report here that when disulfiram inhibited recombinant rat liver mitochondrial ALDH (rlmALDH) in vitro, no significant molecular mass increase was detected during the first 30 min as determined by on-line HPLC-electrospray ionization mass spectrometry (LC-MS). This indicated that the inhibition in vitro was not caused directly by covalent adduct formation on the enzyme. We subsequently subjected both control and disulfiram-inhibited rlmALDH to Glu-C proteolytic digestion. LC-MS analysis of the Glu-C digestion of disulfiram-inhibited enzyme revealed that one peptide of M(r) = 4821, which contained the putative active site of the enzyme, exhibited a mass decrease of 2 amu as compared with the same peptide found in the Glu-C digestion of the control (M(r) = 4823). We believe that the loss of 2 amu indicated that inhibition of rlmALDH in vitro was due to formation of an intramolecular disulfide bond between two of the three adjacent cysteines in the active site, possibly via a very rapid and unstable mixed disulfide interchange reaction. Further confirmation of the intramolecular disulfide bond formation came from the fact that by adding dithiothreitol (DTT) we were able to recover partial enzyme activity. In addition, the peptide of M(r) = 4821 observed in the Glu-C digestion of the disulfiram-treated ALDH reverted to M(r) = 4823 after treatment with DTT, which indicated that the disulfide bond was reduced. We, thereby, conclude that disulfiram inhibited rlmALDH by forming an intramolecular disulfide, possibly via a fast intermolecular disulfiram interchange reaction.

Inhibition of aldehyde dehydrogenase by disulfiram and its metabolite methyl diethylthiocarbamoyl-sulfoxide.

Disulfiram (DSF) is presently the only available drug used in the aversion therapy of recovering alcoholics. It acts by inhibiting aldehyde dehydrogenase (ALDH), leading to high blood levels of acetaldehyde. The in vitro inhibition of ALDH by DSF and its metabolites was systematically studied by combined enzyme inhibition assay with direct molecular weight determination of the same sample using electrospray ionization-mass spectrometry (ESI-MS). Enzyme activity was measured after incubating yeast ALDH (yALDH) with excess concentrations of DSF, methyl diethyldithiocarbamate (MeDDC) and methyl diethylthiocarbamoyl-sulfoxide (MeDTC-SO) and then subjected to analysis by ESI-MS. Addition of DSF resulted in complete enzyme inhibition; however, ESI-MS analysis demonstrated no discernible shift in molecular weight, indicating that no intermolecular adduct was formed with the protein. Treatment of yALDH with MeDTC-SO also completely abolished yALDH activity with a concomitant increase of + approximately 100 Da in the molecular mass of the enzyme. This indicated formation of a covalent carbamoyl protein adduct. Furthermore, the effects of dithiothreitol (DTT) were examined on samples of inhibited protein in vitro. At pH 7.5, DTT completely reversed inhibition after DSF treatment. yALDH inhibited by MeDTC-SO could not be recovered by DTT at pH 7.5, but at pH 9 the enzymic activity was fully restored and a mass loss of approximately 100 Da was noted. This observations are consistent with mechanisms where inhibition of yALDH by DSF in vitro involves oxidation of the active site, whereas MeDTC-SO forms a covalent adduct with the protein in vitro resulting in cessation of enzyme activity.

Determination of in vivo adducts of disulfiram with mitochondrial aldehyde dehydrogenase.

Extensive use for disulfiram (DSF) has been found in the aversion therapy treatment of recovering alcoholics. Although it is known to irreversibly inhibit hepatic aldehyde dehydrogenase (ALDH), the specific mechanism of in vivo inhibition of the enzyme by the drug has not been determined yet. We have demonstrated in this report a novel, but simple and rapid method for structurally characterizing in vivo derived protein-drug adducts by linking on-line sample processing to HPLC-electrospray ionization mass spectrometry (HPLC-MS) and HPLC-tandem mass spectrometry (HPLC-MS/MS). Employing this approach, rats were administered DSF, and their liver mitochondria were isolated and solubilized. Both native and in vivo DSF-treated mitochondrial ALDH (mALDH) were purified in one step with an affinity cartridge. The in vivo DSF-treated mALDH showed 77% inhibition in enzyme activity as compared with that of the control. Subsequently, the control and DSF-inhibited mALDH were both subjected to HPLC-MS analyses. We were able to detect two adducts on DSF-inhibited mALDH, as indicated by the mass increases of approximately 71 and approximately 100 Da. To unequivocally determine the site and structure of these adducts, on-line pepsin digestion-HPLC-MS and HPLC-MS/MS were performed. We observed two new peptides at MH(+) = 973.7 and MH(+) = 1001.8 in the pepsin digestion of DSF-inhibited enzyme. These two peptides were subsequently subjected to HPLC-MS/MS for sequence determination. Both peptides possessed the sequence FNQGQC(301)C(302)C(303), derived from the enzyme active site region, and were modified at Cys(302) by N-ethylcarbamoyl (+71 Da) and N-diethylcarbamoyl (+99 Da) adducts. These findings indicated that N-dealkylation may be an important step in DSF metabolism, and that the inhibition of ALDH occurred by carbamoylation caused by one of the DSF metabolites, most likely S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO). Finally, there was no evidence of the presence of an intramolecule disulfide bridge modification on the peptide FNQGQCCC.

S-methyl N,N-diethylthiocarbamate sulfone, a potential metabolite of disulfiram and potent inhibitor of low Km mitochondrial aldehyde dehydrogenase.

Disulfiram inhibits hepatic aldehyde dehydrogenase (ALDH) causing an accumulation of acetaldehyde after ethanol ingestion. It is thought that disulfiram is too short-lived in vivo to directly inhibit ALDH, but instead is biotransformed to reactive metabolites that inhibit the enzyme. S-Methyl N,N-diethylthiocarbamate (MeDTC) sulfoxide has been identified in the blood of animals given disulfiram and is a potent inhibitor of ALDH (Hart and Faiman, Biochem Pharmacol 46: 2285-2290, 1993). MeDTC sulfone is a logical metabolite of MeDTC sulfoxide. Therefore, we investigated the effects of MeDTC sulfone on the activity of rat hepatic low Km mitochondrial ALDH, the major enzyme in the metabolism of acetaldehyde. MeDTC sulfone inhibited the low Km mitochondrial ALDH in vitro with an IC50 of 0.42 +/- 0.04 microM (mean +/- SD, N = 5) compared with disulfiram, which had an IC50 of 7.5 +/- 1.2 microM under the same conditions. The inhibition of ALDH by MeDTC sulfone was time dependent. The decline in ALDH activity followed pseudo first-order kinetics with an apparent half-life of 2.1 min at 0.6 microM MeDTC sulfone. Inhibition of ALDH by MeDTC sulfone was apparently irreversible; dilution of the inhibited enzyme did not restore lost activity. The substrate (acetaldehyde, 80 microM) and cofactor (NAD, 0.5 mM) together completely protected ALDH from inhibition by MeDTC sulfone; substrate alone partially protected the enzyme. Addition of either thiol-containing compound glutathione (GSH) or dithiothreitol (DTT) to MeDTC sulfone before incubation with the enzyme increased the IC50 of MeDTC sulfone by 7- to 14-fold. Neither GSH nor DTT could restore lost ALDH activity after exposure of the enzyme to MeDTC sulfone. Results of these studies indicate that MeDTC sulfone, a potential metabolite of disulfiram, is a potent, irreversible inhibitor of low Km mitochondrial ALDH.

Photolysis of sulfiram: a mechanism for its disulfiram-like reaction.

Sulfiram, a drug applied topically to treat scabies, produces effects similar to those of disulfiram after subsequent ingestion of ethanol. Disulfiram, used in aversion therapy in the treatment of alcoholism, inhibits hepatic aldehyde dehydrogenase (ALDH) causing an accumulation of acetaldehyde after ethanol ingestion. The increased tissue levels of acetaldehyde cause a spectrum of undesirable side-effects including flushing, nausea, vomiting, and tachycardia, which are referred to as the disulfiram reaction. Previous studies have shown that in vitro sulfiram is a very weak inhibitor of ALDH, but solutions of sulfiram markedly increase in potency with time. In the present study, fresh solutions of sulfiram were exposed to fluorescent room light under ambient conditions and analyzed at timed intervals by HPLC. At least eight products, including disulfiram, were formed in the light-exposed sulfiram solutions, but not in solutions kept in the dark. Structural characterization of two of the photolysis products was obtained by on-line microbore HPLC-mass spectrometry (mu LC-MS) and on-line microbore HPLC-tandem mass spectrometry (mu LC-MS/MS) using continuous flow-liquid secondary ion mass spectrometry (CF-LSIMS) as the primary ionization method. Sulfiram was converted to disulfiram at an initial rate of 0.7%/hr, and the formation of disulfiram correlated with the increase in ALDH inhibition in vitro. The results of this investigation show that while sulfiram is a weak inhibitor of ALDH in vitro, it is readily photoconverted to disulfiram, a very potent inhibitor of ALDH, which may explain the adverse reaction to ethanol after sulfiram therapy.

Inhibition of recombinant human mitochondrial aldehyde dehydrogenase by two intermediate metabolites of disulfiram.

Disulfiram is used in aversion therapy for alcoholism. S-Methyl-N,N-diethylthiocarbamate (MeDTC) sulfoxide, a potent inhibitor of the target enzyme mitochondrial aldehyde dehydrogenase (ALDH2), is thought to be the principal active metabolite of disulfiram in vivo. We examined the effects on recombinant human ALDH2 of two intermediate metabolites of disulfiram, S-methyl-N,N-diethyldithiocarbamate (MeDDC) sulfoxide and MeDDC sulfine. MeDDC sulfoxide was a potent inhibitor of ALDH2 with an IC50 of 2.2 +/- 0.5 microM (mean +/- SD, N = 4) after preincubation with enzyme for 30 min. MeDDC sulfine was a relatively weak inhibitor of ALDH2 under the same conditions with an IC50 value of 62 +/- 14 microM. The inhibition of ALDH2 by both compounds was irreversible and did not require the cofactor NAD. The latter finding demonstrates that inactivation of ALDH2 is independent of the dehydrogenase activity of the enzyme. GSH blocked almost completely the inhibition by 20 microM of MeDDC sulfoxide and greatly diminished the inhibition by 200 microM of MeDDC sulfine. Inactivation by MeDDC sulfoxide was time dependent. MeDTC sulfoxide was a more potent inhibitor of recombinant human ALDH2 (IC50 = 1.4 +/- 0.3 microM after preincubation for 15 min) than either of the intermediate metabolites, and its inhibition was unaffected by GSH. Our results suggest that these newer intermediate metabolites of disulfiram, especially the more potent MeDTC sulfoxide, have the potential to inhibit the target enzyme ALDH2 in patients receiving disulfiram. However, until the significance of the interactions of the inhibitors with GSH is more fully understood, the contribution of MeDDC sulfine and MeDDC sulfoxide to the pharmacological effects of disulfiram in vivo is uncertain.

Inhibition of human mitochondrial aldehyde dehydrogenase by the disulfiram metabolite S-methyl-N,N-diethylthiocarbamoyl sulfoxide: structural characterization of the enzyme adduct by HPLC-tandem mass spectrometry.

S-Methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO) is a known metabolite of the aversion therapy drug disulfiram (DSF). MeDTC-SO is also a potent inhibitor of human mitochondrial aldehyde dehydrogenase (hmALDH) with an IC50 of 1.5 microM. Inhibition of the enzyme by MeDTC-SO resulted in the addition of approximately 100 Da to the molecular mass of the intact protein, as determined by on-line HPLC-electrospray ionization MS (LC-MS). Dialysis of the inhibited protein did not reverse the inhibition, and the molecular mass of 54,533 Da (+/- 0.01%) remained unchanged, indicating that a covalent modification of the protein had occurred. Proteolytic digestion of hmALDH under basic conditions using trypsin at pH 7.8 revealed that the adduct was base labile. However, treating the adducted protein with endopeptidase-Glu-C at pH 3.7 produced a peptide adduct at MH+ = 4924, tentatively attributable to a carbamoylated peptide. This peptide contains three adjacent cysteines, one of which has been implicated as a key amino acid in the highly conserved active site region of ALDH. A pepsin digestion of hmALDH carried out at pH 3.7 and subsequent LC-MS analysis revealed an ion at MH2(2+) = 501.5, corresponding to the carbamoylated peptide FNQGQC1C2C3. This peptide contains the same adjacent active site cysteines. This latter peptide was subjected to LC-MS/MS, which enabled us to determine that the site of carbamoylation was at Cys2. The MS/MS product ion data also confirmed the presence of a carbamoyl group as the adduct species.

Effect of enzyme inhibitors on protein quaternary structure determined by on-line size exclusion chromatography-microelectrospray ionization mass spectrometry.

Aldehyde dehydrogenases (ALDH) are a family of enzymes primarily involved in the oxidation of various aldehydes. Most ALDH enzymes derived from mammalian sources have been shown to exist as homotetramers, consisting of four identical subunits of approximately 54 kDa. The presence of the homotetramer appears to be necessary for enzyme activity. In this study, recombinant rat liver mitochondrial ALDH (rmALDH) was inhibited in vitro with four different inhibitors, namely, disulfiram (MW, 296.5), prunetin (MW, 284.3), benomyl (MW, 290.3), and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) (MW, 351.8). Subsequently, inhibited rmALDH was analyzed by a novel approach of on-line size exclusion chromatography-microelectrospray ionization-mass spectrometry (SEC-muESI-MS) to examine the noncovalent quaternary structural stability of the inhibited enzyme. Analysis of native rmALDH by SEC-muESI-MS revealed predominantly the homotetramer (Mr = approximately 217,457 Da, +/- 0.01%) with some in-source, skimmer-induced dissociation to afford monomer (Mr = approximately 54,360 Da, +/- 0.01%). Both disulfiram and prunetin inhibited rmALDH by >70% and >90%, respectively, but did not disrupt the quaternary structure of rmALDH. Furthermore, there was no detectable change within experimental error (+/- 0.01%) of the disulfiram or the prunetin homotetramers (Mr = approximately 217,448 Da and Mr = approximately 217,446 Da). This may possibly indicate that inhibition occurred via formation of intramolecular disulfide bond at the enzyme active site, or weak affinity noncovalent binding. In contrast, benomyl-inhibited rmALDH homotetramer (>90% inhibition) exhibited a Mr = approximately 217,650 Da (+/- 0.01%) corresponding to two butylcarbamoyl adducts on two of the four enzyme subunits. The skimmer-induced monomer afforded a mixture of unmodified rmALDH (Mr = approximately 54,365 Da, +/- 0.01%) and butylcarbamoylated enzyme (Mr = approximately 54,459 Da, +/- 0.01%). Finally, TPCK (>90% inhibition) modified all four subunits of rmALDH to give Mr = approximately 218,646 Da (+/- 0.01%). In all four cases while significant enzyme inhibition occurred, no destabilization of the quaternary complex was detected.

Nutritional sources of vitamin K.

OBJECTIVE: To review the function of vitamin K in clotting and methods of its analysis, to present results of previous studies on the role of dietary vitamin K in humans and animals, and to reanalyze these data in light of current methods. DESIGN: A review of assumptions stated in the literature is presented, including the incorrect theory that a diet-induced deficiency of vitamin K is nonexistent and the unsubstantiated hypothesis that antibiotics can cause vitamin K deficiency by destroying intestinal bacteria. CONCLUSION: The insistent belief that intestinal bacteria are an important source of vitamin K has led to erroneous conclusions about the sources of vitamin K for human nutrition. In the future, the importance of various sources of vitamin K, their pathways of absorption, and their susceptibility to administration of antibiotics should be evaluated without recourse to current assumptions.

Drug-grapefruit juice interactions.

Grapefruit juice, a beverage consumed in large quantities by the general population, is an inhibitor of the intestinal cytochrome P-450 3A4 system, which is responsible for the first-pass metabolism of many medications. Through the inhibition of this enzyme system, grapefruit juice interacts with a variety of medications, leading to elevation of their serum concentrations. Most notable are its effects on cyclosporine, some 1,4-dihydropyridine calcium antagonists, and some 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors. In the case of some drugs, these increased drug concentrations have been associated with an increased frequency of dose-dependent adverse effects. The P-glycoprotein pump, located in the brush border of the intestinal wall, also transports many cytochrome P-450 3A4 substrates, and this transporter also may be affected by grapefruit juice. This review discusses the proposed mechanisms of action and the medications involved in drug-grapefruit juice interactions and addresses the clinical implications of these interactions.

Methylphenidate: its pharmacology and uses.

Methylphenidate is a commonly used medication in the United States. This central nervous system stimulant has a mechanism of action distinct from that of amphetamine. The Food and Drug Administration has approved methylphenidate for the treatment of attention-deficit/hyperactivity disorder and narcolepsy. Treatment with methylphenidate has been advocated in patients with traumatic brain injury and stroke, cancer patients, and those with human immunodeficiency virus infection. Placebo-controlled trials have documented its efficacy as an adjunctive agent in the treatment of depression and pain. This article reviews the current understanding of the mechanism of action and efficacy of methylphenidate in various clinical conditions.

Transcutaneous drug delivery: a practical review.

OBJECTIVE: To describe the advantages, disadvantages, practical considerations, and future developments of transcutaneous drug delivery. MATERIALS AND METHODS: The physiochemical properties of the drug preparation that are factors in the effectiveness of transcutaneous transport are drug stability or volatility, use of a solvent carrier or vehicle, use of a penetration enhancer, and type of delivery device. Because a drug should remain on the skin without evaporating or becoming otherwise inactive, it is suspended in a vehicle--any gel, lotion, or paste used to apply the drug to the skin. Penetration enhancers include several compounds that are mixed into vehicles to alter the molecular environment of the epidermis and facilitate absorption. The delivery system itself occasionally proves to be the ultimate determinant of transdermal drug flow. RESULTS: The advantages of transcutaneous drug delivery are avoidance of the gastrointestinal tract and hepatic first-pass biotransformation and metabolism, control of absorption, availability of multiple skin sites to avoid local irritation and toxicity, and improved patient compliance. The disadvantages include the potential for localized irritant and allergic cutaneous reactions, systemic toxicity, and difficulties associated with the time necessary for a drug to diffuse through the skin. CONCLUSION: Transdermal drug regimens are safe and effective. They provide clinicians the opportunity to offer more therapeutic options to their patients to optimize their care.

Theophylline: recent advances in the understanding of its mode of action and uses in clinical practice.

Theophylline, a drug that has been used for several decades, has several different actions at a cellular level, including inhibition of phosphodiesterase isoenzymes, antagonism of adenosine, enhancement of catecholamine secretion, and modulation of calcium fluxes. Recently, theophylline was found to have several immunomodulatory and anti-inflammatory properties, and thus interest in its use in patients with asthma has been renewed. The use of theophylline in the treatment of asthma and chronic obstructive pulmonary disease has diminished with the advent of new medications, but theophylline remains beneficial, especially in the patient with difficult refractory symptoms. In the future, theophylline may be used as treatment for bradyarrhythmias after cardiac transplantation, prophylactic medication to reduce the severity of nephropathy associated with intravenous administration of contrast material, therapy for breathing problems during sleep, and treatment for leukemias.