Irreversible Protein Binding Research Articles

L-Arginine sulfate reduces irreversible protein binding in immobilized metal affinity chromatography

Immobilized metal affinity chromatography (IMAC) is a powerful technique for capture and purification of relevant biopharmaceuticals in complex biological matrices. However, protein recovery can be drastically compromised due to surface induced spreading and unfolding of the analyte, leading to fouling of the stationary phase. Here, we report on the kinetics of irreversible adsorption of a protease on an IMAC resin in a time span ranging from minutes to several hours. This trend correlated with the thermal data measured by nano differential scanning calorimetry, and showed a time-dependent change in protein unfolding temperature. Our results highlight that ‘soft’ proteins show a strong time dependent increase in irreversible adsorption. Furthermore, commonly used co-solvents for preservation of the native protein conformation are tested for their ability to reduce fouling. Thermal data suggests that the amino acid l-arginine is beneficial in preventing unfolding, which was confirmed in batch adsorption experiments. The choice of counter-ions has to be considered when using this amino acid. These results show that l-arginine sulfate decelerates the irreversible adsorption kinetics of proteins on the IMAC stationary phase to a greater extent than l-arginine chloride.

Immune dysregulation increases the incidence of delayed-type drug hypersensitivity reactions.

Delayed-type, T cell-mediated, drug hypersensitivity reactions are a serious unwanted manifestation of drug exposure that develops in a small percentage of the human population. Drugs and drug metabolites are known to interact directly and indirectly (through irreversible protein binding and processing to the derived adducts) with HLA proteins that present the drug-peptide complex to T cells. Multiple forms of drug hypersensitivity are strongly linked to expression of a single HLA allele, and there is increasing evidence that drugs and peptides interact selectively with the protein encoded by the HLA allele. Despite this, many individuals expressing HLA risk alleles do not develop hypersensitivity when exposed to culprit drugs suggesting a nonlinear, multifactorial relationship in which HLA risk alleles are one factor. This has prompted a search for additional susceptibility factors. Herein, we argue that immune regulatory pathways are one key determinant of susceptibility. As expression and activity of these pathways are influenced by disease, environmental and patient factors, it is currently impossible to predict whether drug exposure will result in a health benefit, hypersensitivity or both. Thus, a concerted effort is required to investigate how immune dysregulation influences susceptibility towards drug hypersensitivity.

Electrochemistry/liquid chromatography/mass spectrometry to demonstrate irreversible binding of the skin allergen p‐phenylenediamine to proteins

para-Phenylenediamine (PPD) is a potent and well-known allergen, which is commonly used in hair or fur dyes and can cause severe allergic contact dermatitis. In this work, the skin-sensitizing potential of PPD with respect to the conjugation of proteins was evaluated using an approach without animal testing. Electrochemistry (EC) coupled offline to liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS) was employed to convert the pre-hapten PPD into its reactive hapten analogs. A previous study had already shown that this purely instrumental method is suitable for accelerating and simulating the various oxidation processes, which PPD may undergo, and that the emerging products are prone to react with soft thiol groups of small nucleophiles like glutathione and cysteine. This investigation was extended by successfully demonstrating adduct formation between EC-generated PPD oxidation products and the three proteins β-lactoglobulin A (β-LGA), human serum albumin and human hemoglobin. A tryptic digest of modified β-LGA provided evidence for irreversible protein binding of monomeric PPD, a PPD dimer and the PPD trimer known as Bandrowski's Base. It was shown that the main oxidation product p-phenylene quinone diimine, and the reactive oligomerized species, primarily attack the free thiol function of proteins rather than other nucleophilic amino acid residues. The pre-hapten PPD was efficiently activated upon EC oxidation and the resulting species were further reacted with different proteins leading to diverse hapten-protein complexes. Thereby, problems related to the complex matrix present in conventional in vitro or in vivo methods could effectively be avoided.

Niveau de preuve du suivi thérapeutique pharmacologique du cisplatine

Cisplatin is an anticancer agent widely used in clinical practice. Cisplatin undergoes irreversible protein binding in plasma and presents a major nephrotoxicity. Some studies determined unbound and bound platin concentrations using flameless atomic absorption spectrophotometry or chromatography. These studies showed a relationship between cisplatin exposition, notably its maximal concentration, and nephrotoxicity. However, the relationship between cisplatin exposition and its efficacy is not yet established. The population approach using a nonlinear mixed effects model showed a low variability of drug exposition parameters. In some cases, Bayesian adaptative dosing were proposed. However, some particular populations will have to be taken into account such as children, elderly patients, acute or chronic renal failure. The impact of cisplatin pharmacogenetics on its therapeutic monitoring need to be studied. Therapeutic cisplatin monitoring is today possibly useful measuring maximal concentration and using described ajusted-modelling. Some prospective multicentric validations are required.

Adsorption kinetics of hemoglobin onto silicate adsorbents

A comparative study was performed of the adsorption kinetics of hemoglobin onto four silicate adsorbents that differed in parameters of porous structure: industrial silochrome, and three silicates obtained by template synthesis. The existence of a reversible stage of the process preceding irreversible protein binding was shown by kinetic analysis. Rate constants for the reversible stage of the process and the effective constants of first order rate were calculated. It was concluded that the adsorption rate is the highest on silochrome and the lowest on silicate of MCM-41 type, the pore size of which is comparable to the diameter of a hemoglobin molecule. The constant of the desorption rate for the first reversible stage of the process was shown in the case of mesoporous silicates to be lower by a factor of 3.5–4 than in the case of silochrome.

Metabolic and Chemical Origins of Cross-Reactive Immunological Reactions to Arylamine Benzenesulfonamides: T-Cell Responses to Hydroxylamine and Nitroso Derivatives

Exposure to sulfamethoxazole (SMX) is associated with T-cell-mediated hypersensitivity reactions in human patients. T-cells can be stimulated by the putative metabolite nitroso SMX, which binds irreversibly to protein. The hydroxylamine and nitroso derivatives of three arylamine benzenesulfonamides, namely, sulfamethozaxole, sulfadiazine, and sulfapyridine, were synthesized, and their T-cell stimulatory capacity in the mouse was explored. Nitroso derivatives were synthesized by a three-step procedure involving the formation of nitro and hydroxylamine sulfonamide intermediates. For immune activation, female Balb-c strain mice were administered nitroso sulfonamides four times weekly for 2 weeks. After 14 days, isolated splenocytes were incubated with the parent compounds, hydroxylamine metabolites, and nitroso derivatives to measure antigen-specific proliferation. To explore the requirement of irreversible protein binding for spleen cell activation, splenocytes were incubated with nitroso derivatives in the presence or absence of glutathione. Splenocytes from nitroso sulfonamide-sensitized mice proliferated and secreted interleukin (IL)-2, IL-4, IL-5, and granulocyte monocyte colony-stimulating factor following stimulation with nitroso derivatives but not the parent compounds. Splenocytes from sensitized mice were also stimulated to proliferate with hydroxylamine and nitroso derivatives of the structurally related sulfonamides. The addition of glutathione inhibited the nitroso-specific T-cell response. Hydroxylamine metabolites were unstable in aqueous solution: Spontaneous transformation yielded appreciable amounts of nitroso and azoxy compounds as well as the parent compounds within 0.1 h. T-cell cross-reactivity with nitroso sulfonamides provides a mechanistic explanation as to why structurally related arylamine benzenesulfonamides are contraindicated in hypersensitive patients.

Novel Bis-platinum Complexes Endowed with an Improved Pharmacological Profile

Multinuclear platinum complexes are characterized by a peculiar DNA binding mode and higher cytotoxic potency than the mononuclear complexes, and efficacy against a wide range of preclinical tumor models. To reduce the high irreversible plasma protein binding and improve the chemical and metabolic drug stability, novel bis-platinum complexes were designed starting from the parent compound CT-3610. The novel second-generation bis-platinum complexes utilize alkylcarboxylate as leaving groups to improve their pharmacokinetic and pharmacodynamic profiles, thus overcoming the limitations of the previously developed multinuclear compounds. The selected compounds [CT-47518 and CT-47463, respectively (bis-capronate) platinum and (bis-butyrate) platinum], have similar in vitro degradation kinetics in human and murine plasma and, above all, an increased stability when compared to CT-3610, particularly in human plasma. In addition, both compounds exhibited a marked cytotoxic potency as compared with cisplatin and oxaliplatin. Interestingly, they were capable of overcoming resistance mediated by DNA mismatch repair defects in different cellular models. The complexes showed marked antitumor efficacy in Pt-refractory tumor xenografts, with remarkable activity in terms of tumor growth inhibition and tumor growth delay. The improved stability profile in human plasma compared to early bis- and triplatinum complexes together with the marked activity in cellular systems as well as in in vivo models, make CT-47518 and CT-47463 attractive candidates for further development.

A Mechanistic Investigation into the Irreversible Protein Binding and Antigenicity of p-Phenylenediamine

Exposure to the skin sensitizer p-phenylenediamine (PPD) is associated with allergic contact dermatitis; however, the ability of PPD to modify protein has not been fully investigated. The aims of this study were to characterize the reactions of PPD and the structurally related chemical 2,5-dimethyl-1,4-benzoquinonediamine with model nucleophiles, a synthetic peptide (DS3) containing each of the naturally occurring amino acids and His-tagged glutathione-S-transferase pi (GSTP), and to explore the effect of dimethyl substitution on PPD-specific T-cell responses using lymphocytes from allergic patients. The reductive soft nucleophiles N-acetyl cysteine and glutathione prevented PPD self-conjugation reactions and Bandrowski's base formation, but no adducts were detected. N-Acetyl lysine, a hard nucleophile, did not alter the rate of PPD degradation or form PPD adducts. With PPD and 2,5-dimethyl-1,4-benzoquinonediamine, only cysteine was targeted in the DS3 peptide. PPD and 2,5-dimethyl-1,4-benzoquinonediamine were also found to selectively modify the reactive Cys 47 residue of GSTP, which has a pK(a) of 3.5-4.2 and therefore exists in a largely protonated form. Glutathione formed mixed disulfides with the DS3 peptide, reducing levels of PPD binding. Lymphocytes from PPD allergic patients proliferated in the presence of PPD but not with 2,5-dimethyl-1,4-benzoquinonediamine. These results reveal that PPD and 2,5-dimethyl-1,4-benzoquinonediamine bind selectively to specific cysteine residues in peptides and proteins. Lymphocytes from PPD allergic patients were capable of discriminating between the different haptenic structures, suggesting that the hapten, but not the peptide moiety associated with MHC, is an important determinant for T-cell recognition.

COMPARATIVE PROTEIN BINDING, STABILITY AND DEGRADATION OF SATRAPLATIN, JM118 AND CISPLATIN IN HUMAN PLASMA IN VITRO

1. Satraplatin is an investigational orally administered platinum-based antitumour drug. The present study compared the plasma protein binding, stability and degradation of satraplatin with that of its active metabolite JM118 and cisplatin. 2. The platinum complexes were incubated in human plasma for up to 2 h at 37 degrees C and quantified in plasma fractions by inductively coupled plasma-mass spectrometry on- or off-line to high-performance liquid chromatography. 3. All three platinum drugs became irreversibly bound to plasma proteins and showed negligible reversible protein binding. They were also unstable in plasma and generated one or more platinum-containing degradation products during their incubation. However, the three platinum complexes differed in the kinetics of their instability and protein binding, as well as in the number of degradation products formed during their incubation. 4. In conclusion, the plasma protein binding, instability and degradation of satraplatin and its active metabolite JM118 are qualitatively similar to that of cisplatin and other clinically approved platinum-based drugs. Quantitative differences in their irreversible protein binding and degradation were related to their respective physiochemical properties and bioactivation mechanisms.

Comparative CYP-dependent binding of the adrenocortical toxicants 3-methylsulfonyl–DDE and o,p′-DDD in Y-1 adrenal cells

The environmental pollutant 3-MeSO(2)-DDE [2-(3-methylsulfonyl-4-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethene] is an adrenocortical toxicant in mice, specifically in the glucocorticoid-producing zona fasciculata, due to a cytochrome P450 11B1 (CYP11B1)-catalysed bioactivation and formation of covalently bound protein adducts. o,p'-DDD [2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethane] is toxic and inhibits steroidogenesis in the human adrenal cortex after bioactivation by unidentified CYPs, but does not exert any toxic effects on the mouse adrenal. As a step towards determining in vitro/in vivo relationships for the CYP-catalysed binding and toxicity of 3-MeSO(2)-DDE and o,p'-DDD, we have investigated the irreversible protein binding of these two toxicants in the murine adrenocortical cell line Y-1. The irreversible binding of 3-MeSO(2)-DDE previously demonstrated in vivo was successfully reproduced and could be inhibited by the CYP-inhibitors etomidate, ketoconazole and metyrapone. Surprisingly, o,p'-DDD reached similar levels of binding as 3-MeSO(2)-DDE. The binding of o,p'-DDD was sensitive to etomidate and ketoconazole, but not to metyrapone. Moreover, GSH depletion increased the binding of 3-MeSO(2)-DDE, but not of o,p'-DDD, indicating an important role of GSH conjugation in the detoxification of the 3-MeSO(2)-DDE-derived reactive metabolite. In addition, the specificity of CYP11B1 in activating 3-MeSO(2)-DDE was investigated using structurally analogous compounds. None of the analogues produced histopathological lesions in the mouse adrenal in vivo following a single i.p. injection of 100 mg/kg body weight, but two of the compounds were able to decrease the irreversible binding of 3-MeSO(2)-DDE to Y-1 cells. These results indicate that the bioactivation of 3-MeSO(2)-DDE by CYP11B1 is highly structure-dependent. In conclusion, both 3-MeSO(2)-DDE and o,p'-DDD bind irreversibly to Y-1 cells despite differences in binding and adrenotoxicity in mice in vivo. This reveals a notable in vitro/in vivo discrepancy, the contributing factors of which remain unexplained. We consider the Y-1 cell line as appropriate for studies of the cellular mechanisms behind the adrenocortical toxicity of these substances.

FORMATION AND PROTEIN BINDING OF THE ACYL GLUCURONIDE OF A LEUKOTRIENE B4ANTAGONIST (SB-209247): RELATION TO SPECIES DIFFERENCES IN HEPATOTOXICITY

SB-209247 [(E)-3-[6-[[(2,6-dichlorophenyl)-thio]methyl]-3-(2-phenylethoxy)-2-pyridinyl]-2-propenoic acid], an anti-inflammatory leukotriene B4 receptor antagonist, was associated in beagle dogs but not male rats with an inflammatory hepatopathy. It also produced a concentration-dependent (10-1000 microM) but equal leakage of enzymes from dog and rat precision-cut liver slices. The hepatic metabolism of SB-209247 was investigated with reference to the formation of reactive acyl glucuronides. [14C]SB-209247 (100 micromol/kg) administered i.v. to anesthetized male rats was eliminated by biliary excretion of the acyl glucuronides of the drug and its sulfoxide. After 5 h, 1.03 +/- 0.14% (mean +/- S.E.M., n = 4) of the dose was bound irreversibly to liver tissue. The sulfoxide glucuronide underwent pH-dependent rearrangement in bile more rapidly than did the SB-209247 conjugate. [14C]SB-209247 was metabolized by sulfoxidation and glucuronidation in rat and dog hepatocytes, and approximately 1 to 2% of [14C]SB-209247 (100 microM) became irreversibly bound to cellular material. [14C]SB-209247 sulfoxide and glucuronide were the only metabolites produced by dog, rat, and human liver microsomes in the presence of NADPH and UDP-glucuronic acid (UDPGA), respectively. V(max) values for [14C]SB-209247 glucuronidation by dog, rat, and human microsomes were 2.6 +/- 0.1, 1.2 +/- 0.1, and 0.4 +/- 0.0 nmol/min/mg protein, respectively. Hepatic microsomes from all three species catalyzed UDPGA-dependent but not NADPH-dependent irreversible binding of [14C]SB-209247 (100-250 microM) to microsomal protein. Although a reactive acyl glucuronide was formed by microsomes from every species, the binding did not differ between species. Therefore, neither the acute cellular injury nor glucuronidation-driven irreversible protein binding in vitro is predictive of the drug-induced hepatopathy.

Effects of a cisplatin-chondroitin sulfate A complex in reducing the nephrotoxicity of cisplatin.

To assess the effects of a macromolecular prodrug in reducing the nephrotoxicity of cisplatin (CDDP), chondroitin sulfate A (CSA) with a mean molecular weight of 23,000 Da was used to form a complex with CDDP, and the pharmacokinetics and toxicology of the resulting complex were examined in rats in comparison with those of CDDP. The total plasma platinum levels and urinary accumulation were determined up to 3 h following a bolus injection of 2 mg/kg. The results of the pharmacokinetic analysis showed that the complex suppressed the rapid distribution of CDDP, decreased the renal clearance and resulted in over fivefold higher AUC values within 3 h in comparison with CDDP treatment. In addition, the plasma levels of the drug following administration of the complex decreased greatly with time throughout the experimental period (3-24 h), whereas a slow elimination was observed following CDDP administration, which was due to the irreversible protein binding of CDDP. The tissue-to-plasma partition ratio at 10 min also indicated that the CDDP-CSA complex controlled the perfusion of CDDP to tissues, especially to the kidney. The accumulation in various tissues was evaluated at 3 h and 24 h following the injection of 5 mg/kg. Marked differences in renal accumulation were found within 3 h. Significant reductions in accumulation in the kidney, lung, muscle and whole blood were found within 24 h of administration of the complex. The renal toxicity of the CDDP-CSA complex was evaluated by measuring blood urea nitrogen (BUN), serum creatinine (Cr) and the ratio of terminal kidney weight to body weight at doses of 2 mg/kg and 5 mg/kg. The complex displayed a much lower nephrotoxicity at 5 mg/kg in comparison to CDDP, and similar results were obtained at 2 mg/kg. This suggests that the complex changed the toxicodynamics of CDDP. Moreover, the anticancer activity of the CDDP-CSA complex, tested against SW 4800 human colon cancer cells and HeLa human cervix cancer cells in vitro, showed no decrease as compared with that of free CDDP. We conclude that the CDDP-CSA complex had the same activity as the parent drug but showed reduced nephrotoxicity at high doses of CDDP through an improvement in the pharmacokinetics of CDDP, which resulted from both the minimization of entry into normal tissues and renal clearance. In addition, it is also possible that different intracellular interactions in renal cells play a role in protection against the nephrotoxicity of high doses of CDDP.

The effect of cytochromes P4501A induction and inhibition on the disposition of the cognition activator tacrine in rat hepatic preparations.

1. The disposition of tacrine 1,2,3,4-tetrahydro-9-acridinamine monohydrochloride monohydrate (THA, Cognex), was studied using livers obtained from control, phenobarbital (PB), isosafrole (ISO), and 3-methycholanthrene (3-MC) treated rats. 2. Pretreatment of rats with PB, ISO, and 3-MC reduced AUC(10-120 min) of THA in liver perfusates by 28, 32, and 86% respectively. 3. Elimination of [14C]-THA-derived radioactivity into bile was 7.6 +/- 1.2%, 11.7 +/- 2.9%, 14.8 +/- 2.0%, and 46.3 +/- 9.7% (mean +/- SD) of the infusion dose for control PB, ISO, and 3-MC pretreated isolated perfused rat livers, respectively. 4. In perfusion experiments using 3-MC pretreated livers, a marked increase in irreversible protein binding of 3-, 7-, and 8-fold was observed to microsomal, cytosolic and total liver proteins, respectively, compared to control. Only a slight effect was observed on protein binding in perfusion experiments using PB and ISO pretreated animals. 5. Co-incubations of [14C]-THA with the metabolic inhibitors enoxacin, ethimizol, and furafylline in hepatocyte preparations obtained from 3-MC pretreated rats markedly inhibited THA-derived irreversible protein binding. Furafylline, a specific inhibitor of cytochrome P4501A2, had the greatest inhibitory effect (approximately 70%). 6. These results are consistent with a major role of cytochrome P4501A in the metabolism and irreversible protein binding of THA in rat liver and demonstrate the utility of isolated liver perfusion and hepatocyte models for examining the effect of metabolic modulators.

Adrenocorticolytic DDT-metabolites: studies in mink, Mustela vison and otter, Lutra lutra

: The irreversible binding and toxicity of the DDT metabolites p,p'-DDD and o,p-DDD in the adrenal cortex of female mink Mustela vison were studied. Histological examination of adrenals from mink given a single i.p. injection of p,p'-DDD or o,p'-DDD (125 mg per kg body weight) showed vacuolation, necrosis and focal bleedings in the zonae fasciculata and reticularis. Autoradiograms of solvent-extracted tissue sections of minks given a single i.v. injection of p,p'-[(14)C]DDD (0.7 mg per kg body weight) revealed a high level of irreversibly bound radio-activity in the adrenal cortex. Microautoradiography showed that the irreversibly bound radio-activity was confined to the zonae fasciculata and reticularis. Incubation of p,p'-[(14)C]DDD and o,p'-[(14)C]DDD with mink or otter, Lutra lutra, adrenal homogenate (300 x g supernatant) resulted in an irreversible binding of radioactivity to protein from both species. The irreversible protein binding of the DDD isomers in mink and otter was decreased by addition of the cytochrome P450 inhibitors metyrapone and carbon monoxide, indicating a cytochrome P450 dependent metabolic activation. In contrast, 3-methylsulfonyl-[(14)C]DDE, a potent adrenocortical toxicant in mice, does not appear to be metabolized to a reactive metabolite in the adrenal cortex of mink or otter. In conclusion, both p,p'-DDD and o,p'-DDD are toxic to the mink adrenal zona fasciculata and reticularis following activation in situ to reactive, tissue-binding metabolites. The results suggest that p,p'-DDD and o,p'-DDD are adrenocortical toxicants also in otter. The involvement of environmental pollutants in the generation of the adrenocortical hyperplasia observed among Baltic seals is discussed.

In vitro and in vivo irreversible plasma protein binding of beclobric acid enantiomers.

Acyl glucuronides are known to be labile conjugates, which undergo hydrolysis and bind irreversibly to proteins. The lipid-regulating agent (+/-)-beclobrate is immediately converted to the free acid after oral administration. Further metabolism leads to formation of the corresponding diastereomeric acyl glucuronides. Beclobric acid glucuronides were quantified by indirect measurement with an HPLC method based on chiral fluorescent derivatization of the carboxylic acid and subsequent normal-phase chromatography. The renal clearance of unchanged drug is low, with almost all drug excreted into urine as glucuronic acid conjugates. Beclobric acid glucuronide is also detectable in plasma. In vitro degradation studies with beclobric acid glucuronide (at a concentration of 5 microM in 150 mM phosphate buffer pH 7.4) exhibited a minor tendency for acyl migration and hydrolysis, i.e., a higher stability than has been observed for the acyl glucuronides of most other drugs. The in vitro degradation half-lives of the two beclobric acid beta-1-O-acyl glucuronides were 22.7 and 25.7 h. After incubation with pooled plasma and human serum albumin in buffer pH 7.4 irreversible binding was measured in vitro. No significant difference between the two enantiomers was detected with respect to the magnitude of in vitro irreversible binding. In 3 healthy male volunteers the extent of irreversible binding of both beclobric acid enantiomers to plasma proteins was investigated after single and multiple oral doses of racemic beclobrate (100 mg once daily). Irreversible binding of both enantiomers was observed in all volunteers. The adduct densities for (-)- and (+)-beclobric acid after single 100 mg beclobrate doses were 0.147 x 10(-4) and 0.177 x 10(-4) mol/mol protein. Multiple dosing increased irreversible binding 3- to 4-fold.

Pharmacokinetics and irreversible binding of tolmetin and its glucuronic acid esters in the elderly.

In a previous study, the disposition and irreversible plasma protein binding of tolmetin have been described in young volunteers. Significant levels of tolmetin glucuronide and its isomer(s) were found in plasma, and irreversible binding was shown to occur in all subjects. In the present study, the pharmacokinetics and irreversible binding in 5 elderly volunteers are investigated after a single dose (400 mg) of tolmetin. No difference of physiological importance has been found, unless weight correction is employed, when compared with the young subjects. Very strong correlations were present between the level of binding and exposure to the glucuronide or its isomer. An elderly patient currently treated with tolmetin (400 mg b.i.d.) also participated in the study. The results show an accumulation of the irreversible binding to levels 4-17 times higher than after a single dose. Nevertheless, neither toxic nor allergic reactions have been observed in this patient.

Ethanol‐Induced Enhancement of Cocaine Bioactivation and Irreversible Protein Binding: Evidence Against a Role of Cytochrome P‐450IIE1

Chronic ethanol consumption potentiates cocaine-induced liver injury in rodents. Since cocaine has to be bioactivated by a cytochrome P-450-dependent N-oxidative pathway to exert its hepatotoxic effects, we studied the role of the ethanol-inducible P-450IIE1 for cocaine metabolism. Male Sprague-Dawley rats were pretreated with either a liquid diet containing ethanol (30% of calories) for 4 weeks or injected with pyrazole (200 mg/kg/day, ip, for 3 days). Both agents induced microsomal p-nitrophenol hydroxylation which is a probe for the catalytic activity of P-450IIE1. However, only ethanol, but not pyrazole, increased both microsomal cocaine N-demethylase activity (by 47%) and the extent of irreversible binding of [3H]-cocaine to microsomal proteins (by 100%), which was taken as a quantitative endpoint for the formation of a reactive metabolite. Cocaine N-demethylation and irreversible protein binding of cocaine were not inhibited by P-450IIE1 isozyme-selective substrates, nor was the rate of cocaine metabolism and binding decreased by functionally active polyclonal anti-rat P-450IIE1 antibodies. Furthermore, pyrazole pretreatment sensitized cultured hepatocytes to the glutathione-dependent cytotoxic effects of nontoxic concentrations of cocaine. These results indicate that (a) cocaine is not a major substrate for the ethanol-inducible P-450IIE1, (b) the enhancing effects of ethanol on cocaine bioactivation may be due to induction of other P-450 isoforms, and (c) induction of P-450IIE1 may potentiate cocaine-induced hepatocellular toxicity in vitro independently of cocaine metabolism, e.g., by P-450IIE1-dependent oxidative stress.

Adrenocortical toxicity of 3-methylsulfonyl-DDE in mice: II. Mitochondrial changes following ecologically relevant doses

Transmission electron microscopy was used to characterize early ultrastructural lesions in the adrenal zona fasciculata of female C57BL mice given a single ip injection of the adrenocorticolytic DDT-metabolite 3-methylsulfonyl-DDE (MeSO 2-DDE). Following 3 mg/kg, mitochondrial changes were observed 6 hr after dosing. At 12 and 24 hr the mitochondrial changes were conspicuous, with disorganization and disappearance of central cristae. At doses of 6, 12, and 25 mg/kg body wt initial (6 hr) mitochondrial vacuolization was observed, followed by disappearance of mitochondria (6–12 mg/kg) or cellular necrosis (25 mg/kg). The metabolic activation and binding of MeSO 2-[ 14C]DDE in adrenal homogenates were determined in vitro. The irreversible binding of MeSO 2-[ 14C]DDE to the mitochondria-containing adrenal S-9 pellet fraction was 50 times higher than that to the postmitochondrial S-12 supernatant fraction. The apparent K m was 2.1 μ m and the apparent V max was 104 pmol/mg protein/30 min for the binding of MeSO 2-[ 14C]DDE to S-0.3 supernatants. The irreversible protein binding was inhibited by metyrapone ( K i = 1 μ m) and 11-deoxycorticosterone ( K i = 3 μ m). In conclusion, the adrenal metabolic activation of MeSO 2-DDE is suggested to be mediated by a mitochondrial cytochrome P450 form, presumably P450 (11β). A primary mitochondrial lesion develops and subsequently leads to degeneration and necrosis of the zona fasciculata.

Disposition and immunogenicity of penicillin in the rabbit.

The immunogenicity, disposition and irreversible protein binding of benzylpenicillin (BP) were studied in male New Zealand White rabbits. There was an increase in IgG anti-benzylpenicilloyl (BPO) antibody activity, as detected by enzyme-linked immunosorbent assay (ELISA) following daily intramuscular administration (for 4 consecutive days) of BP (2.7 and 1.6 mmol/kg) freshly dissolved in 0.15 M NaCl. Antibody activity reached a maximum approximately 14 days after the last injection. There was a smaller immune response when a dose of 270 mumol/kg was administered. The specificity of the IgG antibody response for the BPO determinant was confirmed by inhibition of binding by BPO-aminocaproate. [3H]BP, administered intravenously to rabbits at a dose of 2.7 mmol/kg was rapidly cleared from plasma, and unchanged BP was not detected at 1 h. After 3 h, irreversible binding accounted for less than 0.004% of the dose bound per milliliter of plasma, and this represented all the radioactivity present in plasma at this time. Covalent binding of BP to plasma proteins, in vitro, after 3 h was of the same magnitude for rabbit, rat and human plasma. Therefore, BP can induce a specific antibody response in the rabbit in contrast to the lack of immunogenicity observed previously in the rat.

Disposition and irreversible plasma protein binding of tolmetin in humans.

The pharmacokinetics and irreversible plasma protein binding of tolmetin were studied in six healthy subjects after the administration of a single, 400 mg dose of tolmetin. With HPLC analysis, tolmetin, tolmetin glucuronide, and the isomers of tolmetin glucuronide, which result from intramolecular acyl migration in vivo, were detected in the plasma up to 4 hours after administration, whereas these conjugates were present in the urine up to 24 hours. Irreversible binding of tolmetin to plasma proteins occurred in all subjects. Irreversible binding exhibited a better correlation with exposure to tolmetin glucuronide (r = 0.5618) and the isomers of tolmetin glucuronide (r = 0.8200) than with exposure to tolmetin (-0.3635). This is consistent with the hypothesis that covalent binding occurs via the acyl glucuronide.