Bruce A. Mico

Selective inactivation of cytochrome P-450 isozymes by suicide substrates

Abstract The autocatalytic destruction of cytochrome P-450 by the following six substrates has been investigated in vivo and in vitro with microsomal and purified, reconstituted rat liver enzymes: 2-isopropyl-4-pentenamide (AIA), 1-ethinylcyclopentanol, 17α-propadienyl-19-nortestosterone, fluroxene, 5,6-dichloro-1,2,3-benzothiadiazole (DCBT), and 1-aminobenzotriazole (ABT). Administration of the first three substrates to rats pretreated with either phenobarbital (Pb) or 3-methylcholanthrene (3-MC), or their incubation with hepatic microsomes from such rats, produced a larger decrease in cytochrome P-450 levels in the membranes from Pb- than 3-MC-treated rats. Comparable losses, however, were observed in microsomes from rats pretreated with both Pb and 3-MC when the last three agents were used. Similar experiments were carried out using the major cytochrome P-450 isozymes purified from liver microsomes of Pb- or 3-MC-treated rats. The Pb isozyme was inactivated during catalytic turnover of all six substrates while only three substrates (DCBT, ABT, and fluroxene) were found to inactivate the 3-MC isozyme. Oxygen consumption studies with purified enzymes have shown that AIA is not a measurable substrate for the 3-MC isozyme, a fact which explains its failure to inactivate this isozyme. Similar studies with the Pb isozyme establish that one enzyme molecule is inactivated for approximately every 230–320 AIA molecules processed by the enzyme.

Destruction of cytochrome P-450 by allylisopropylacetamide is a suicidal process.

Abstract The destruction of cytochrome P -450 by allylisopropylacetamide (2-isopropyl-4-pentenamide) in microsomes from phenobarbital-pretreated rats has been shown to require oxygen, to be inhibited by NADP through inhibition of cytochrome P -450 reductase, and to be slightly stimulated by NADH. Glutathione (1 m m ) does not inhibit destruction, but methyl 4,5-epoxy-2-isopropylpentanoate (5 m m ), an analog of the epoxide of allylisopropylacetamide, does. The inactivation of cytochrome P -450 is both time dependent and saturable, although no more than approximately 40% of the microsomal enzyme appears to be normally destructible. However, mechanical perturbation of the microsomal suspension by rehomogenization initiates renewed destruction. Kinetic analysis shows that the destructive process is pseudo-first-order, with an apparent inactivation rate constant of 1.4 × 10 −3 s −1 and an apparent K m of 1.14 m m . Approximately 230 molecules of substrate are turned over for each destructive event. These results, in conjunction with previously reported data, clearly and unambiguously establish that inactivation of cytochrome P -450 by allylisopropylacetamide is a suicidal process.

Suicidal inactivation of cytochrome P-450. Formation of a heme-substrate covalent adduct

Abstract The green pigment accumulated in the livers of phenobarbital pretreated rats after administration of 2-( 14 C)-2-isopropyl-4-pentenamide (allylisopropylacetamide, AIA) is radiolabeled. The single primary green prophyrin component isolated by HPLC (λ max (CHCl 3 ) 417, 512, 545, 594, 652 nm) is cleanly converted to a zinc complex (λ max (CHCl 3 ) 431, 547, 591, 634, 669 nm). The radiolabel quantitatively shifts with the chromophore on TLC and HPLC upon formation of the zinc complex. Correlation of chromophore absorbance with radiolabel content suggests the formation of a 1:1 porphyrin-AIA adduct. Cytochrome P-450 is therefore destroyed by self-catalyzed addition of AIA to its heme prosthetic group.

Destruction of cytochrome P-450 by 2-isopropyl-4-pentenamide and methyl 2-isopropyl-4-pentenoate: mass spectrometric characterization of prosthetic heme adducts and nonparticipation of epoxide metabolites.

Abstract Incubation of hepatic microsomes from phenobarbital-treated rats with methyl 2-isopropyl-4-pentenoate results in rapid destruction of the microsomal cytochrome P-450. The destruction does not occur in the absence of NADPH or with methyl 2-isopropylpentanoate. Administration of methyl 2-isopropyl-4-pentenoate to phenobarbital-pretreated rats leads to hepatic accumulation of a “green” pigment which, after methylation and purification, yields an abnormal porphyrin chromatographically and spectroscopically indistinguishable from that similarly obtained with 2-isopropyl-4-pentenamide (allylisopropylacetamide). Field desorption mass spectrometry showed that both abnormal porphyrins exhibited molecular ions at m e 730. The mass spectrum of the zinc and copper complexes confirmed this value. Esterification in deuterated methanol of the amide-derived porphyrin showed that only two methyl esters were formed. Finally, methyl 4,5-epoxy-2-isopropylpentanoate and the known metabolites of 2-isopropyl-4-pentenamide were shown not to destroy cytochrome P-450. These results clearly establish that the carbonyl groups of the two destructive substrates are intimately involved in formation of the isolated porphyrin adducts, and exclude participation of the corresponding epoxide metabolites in the destruction of cytochrome P-450.

Dose-dependent bioavailability of prazosin in beagle dogs.

Summary Prior to the introduction of an intravenous dosage form for use in humans, prazosin pharmacokinetic studies emphasizing clearance, hepatic extraction, and bioavailability were carried out in dogs. Two such canine studies reported significantly different values for the oral bioavailability of prazosin. This study investigated the differences in prazosin oral availability in beagle dogs. Three male animals were administered an intravenous (1 mg/kg) and three different oral doses (15, 5, and 1 mg) with a 7-day washout between study days. The mean predicted bioavailability, based on hepatic clearance and an estimate for liver blood flow, was 74%. The mean absolute bioavailabilities, determined for each dose in each animal by comparing dose-corrected areas under the plasma concentration–time curve, were 82, 27, and 23%. Although good agreement was evident in bioavailability between the 15-mg oral dose and what was predicted, calculated availabilities for the 5-mg and 1-mg oral doses were approximately one-third the predicted value. The results obtained from this study, together with data from the two previous studies, indicate that the bioavailability of prazosin in dogs is dose-dependent. Possible mechanisms for this observation are also presented.

INACTIVATION OF CYTOCHROME P-450 BY SUICIDE SUBSTRATES. PARTIAL STRUCTURE OF THE RESULTING PROSTHETIC-HEME ADDUCTS

The following substrates alkylate the prosthetic-heme of cytochrome P-450 during catalytic interaction with the enzyme: 2-isopropyl-4-pentenamide, methyl 2-isopropyl-4-pentenoate, norethisterone, 1-ethynylcyclohexanol, 1-ethynylcyclopentanol, and 3-phenoxy-1-propyne. Mass spectrometric studies suggest that an oxygen atom is incorporated into each substrate prior to or during the alkylation process.