Site-specific 99mTc-labeling of antibody using dihydrazinophthalazine (DHZ) conjugation to Fc region of heavy chain.

The development of an antibody labeling method with 99mTc is important for cancer imaging. Most bifunctional chelate methods for 99mTc labeling of antibody incorporate a 99mTc chelator through a linkage to lysine residue. In the present study, a novel site-specific 99mTc labeling method at carbohydrate side chain in the Fc region of 2 antibodies (T101 and rabbit anti-human serum albumin antibody (RPAb)) using dihydrazinophthalazine (DHZ) which has 2 hydrazino groups was developed. The antibodies were oxidized with sodium periodate to produce aldehyde on the Fc region. Then, one hydrazine group of DHZ was conjugated with an aldehyde group of antibody through the formation of a hydrazone. The other hydrazine group was used for labeling with 99mTc. The number of conjugated DHZ was 1.7 per antibody. 99mTc labeling efficiency was 46-85% for T101 and 67-87% for RPAb. Indirect labeling with DHZ conjugated antibodies showed higher stability than direct labeling with reduced antibodies. High immunoreactivities were conserved for both indirectly and directly labeled antibodies. A biodistribution study found high blood activity related to directly labeled T101 at early time point as well as low liver activity due to indirectly labeled T101 at later time point. However, these findings do not affect practical use. No significantly different biodistribution was observed in the other organs. The research concluded that DHZ can be used as a site-specific bifunctional chelating agent for labeling antibody with 99mTc. Moreover, 99mTc labeled antibody via DHZ was found to have excellent chemical and biological properties for nuclear medicine imaging.

Influence of metabolic activation on the induction of micronuclei by antihypertensive drugs in L929 cells.

The influence of metabolic activation on the genotoxic activity of the antihypertensive drugs hydralazine and dihydralazine was investigated. An in vitro micronucleus test for estimating the genotoxic activity of these drugs was used. The results obtained indicated that hydralazine and dihydralazine induce micronuclei formation in L929 cells. When L929 cell cultures were treated with drugs together with liver membrane fraction (S9 fraction) from polychlorinated biphenyl (Aroclor 1254) induced rat liver, the number of micronucleated cells decrease, however, almost to the level found in control cultures. The experiments with modified S9 mix allow the conclusion that the antioxidant enzymes catalase and superoxide dismutase present in S9 liver fraction play a role in the protection of cells from the genotoxic action of hydralazine and dihydralazine.

Mechanism-based inactivation of cytochrome P450s 1A2 and 3A4 by dihydralazine in human liver microsomes.

Dihydralazine is known to induce immunoallergic hepatitis. Since anti-liver microsome (anti-LM) autoantibodies found in the serum of the patients react with P450 1A2, it is suggested that dihydralazine is biotransformed into a reactive metabolite, which covalently binds to cytochrome P450 1A2 and triggers an immunological response as a neoantigen. We investigated inactivation of P450 enzymes, including P450 1A2, during the metabolism of dihydralazine to evaluate the selectivity of P450 1A2 as a catalyst and a target of dihydralazine. Human liver microsomes or microsomes from lymphoblastoid cells expressing P450 enzymes were preincubated with dihydralazine in the presence of NADPH, followed by an assay of several monooxygenase activities. Preincubation of human liver microsomes with dihydralazine in the presence of NADPH resulted in decreases in phenacetin O-deethylase activity (an indicator of P450 1A2 activity) and testosterone 6beta-hydroxylase activity (P450 3A4), but not in diclofenac 4'-hydroxylase activity (P450 2C9), an indication of inactivation of P450s 1A2 and 3A4 during the dihydralazine metabolism. The inactivation of both of the P450s followed pseudo-first-order kinetics and was saturable with increasing dihydralazine concentrations. Similar time-dependent decreases in the activities were obtained in the case for use in microsomes expressing P450 1A2 and P450 3A4 instead of the human liver microsomes. The data presented here demonstrated that dihydralazine was metabolically activated not only by P450 1A2 but also by P450 3A4, and the chemically reactive metabolite bound to and inactivated the enzyme themselves, suggesting that dihydralazine is a mechanism-based inactivator of P450s 1A2 and 3A4. The data support the postulated covalent binding of a reactive metabolite of dihydralazine to P450 1A2 as a step in the formation of anti-LM antibodies in dihydralazine hepatitis, but it is not the unique factor for determining the specificity of the autoantibodies.

Epitope mapping of human CYP1A2 in dihydralazine-induced autoimmune hepatitis.

Dihydralazine-induced hepatitis is characterized by the presence of anti-liver microsomal (anti-LM) autoantibodies in the sera of patients. Cytochrome P450 1A2 (CYP1A2), involved in the metabolism of dihydralazine, was shown to be a target for autoantibodies. In order to investigate further the relationship between drug metabolism and the pathogenesis of this drug-induced autoimmune disease, and since the specificity of anti-LM autoantibodies towards CYP1A2 has been determined, the antigenic site was further localized. By constructing fragments derived from CYP1A2 cDNA and probing the corresponding proteins with several anti-LM sera, we were able to define a region (amino acid 335-471) which was immunoreactive with 100% of sera. An internal deletion in this region led to the loss of recognition by anti-LM autoantibodies, confirming that the epitope was conformational. Epitope mapping studies had previously been performed for CYP2D6, CYP17, CYP21A2, and recently for CYP3A1 and CYP2C9. Those data were compared with results obtained in the present study for CYP1A2.

Interactions of dihydralazine with cytochromes P4501A: a possible explanation for the appearance of anti-cytochrome P4501A2 autoantibodies.

The antihypertensive drug dihydralazine may, on rare occasions, cause immunoallergic hepatitis characterized by anti-cytochrome P450 (P450)1A2 autoantibodies. To understand the first steps leading to this immune reaction, we studied the covalent binding fo dihydralazine metabolites to microsomes from rat and human livers. Upon incubation with NADPH and microsomes, dihydralazine formed metabolites that reacted with heme (as evidenced by destruction of heme, formation of 445-nm light-absorbing complexes, and covalent binding of heme to P450 apoprotein) and covalently bound to microsomal proteins. Formation of these metabolites was shown (by NADPH dependence, induction by beta-naphthoflavone, and immunoinhibition by anti-P4501A antibodies) to be mediated by P4501A. Finally, these metabolites appeared to bind to P4501A2, which produced them. These results support the following scheme for the first steps of this autoimmune reaction: P4501A2 metabolizes dihydralazine into reactive metabolites that then bind to it, forming a neoantigen that triggers an immune response characterized by autoantibodies against P4501A2.

Immunotoxicology and expression of human cytochrome P450 in microorganisms.

Drug-induced hepatitis can be caused by an abnormal immunological response. In the case of tienilic acid- and dihydralazine-induced hepatitis, we postulated a scheme in which a P450 produced a reactive metabolite (step 1); this reactive metabolite bound to the P450 producing it (step 2) leading to a neoantigen triggering the immune response (step 3); the autoantibodies produced during the immune response recognized the P450 producing the reactive metabolite (step 4). The use of microorganisms (yeast or bacteria) expressing cloned human P450 helped in proving some steps of this postulated scheme, particularly steps 1 and 4.

[The quantitative determination of dihydralazine and selected metabolites in man]. / Zur quantitativen Bestimmung von Dihydralazin und ausgewählter Metabolite beim Menschen.

Dihydralazine and its metabolites were estimated in the steady-state in 9 hypertonic patients by gas chromatography. Serum levels of both dihydralazine and metabolites were very low and particularly below the detection limit. In urine and faeces about half of the dose was found mainly as metabolites, 2/3 of this in faeces. Acid labile hydrazones of dihydralazine (about 6% of the dose), primary acetylated and primary oxidized metabolites (both about 20%) were identified as main metabolites. Secondary metabolites (hydrazones, acetylated and oxidized products) were measured, too.

[The quantitative identification of metabolites of dihydralazine in the human]. / Zum qualitativen Nachweis von Metaboliten des Dihydralazins beim Menschen.

The metabolism of dihydralazine sulfate was studied in 11 hypertonic patients treated with that drug chronically. The metabolites were identified in urine with gc, dc, and hplc by comparison with synthesized reference compounds. Following metabolites could be verified: acetylated products, oxidation products, hydrazones and products of decomposition. Products resulting from reaction with nitrites could be not detected.

[Quantitative determination and kinetics of dihydralazine in hypertension patients]. / Quantitative Bestimmung und Kinetik von Dihydralazin bei Hypertoniepatienten.

For the quantitative determination of dihydralazine (1) a derivative with acetylacetone in biological material was formed at pH = 4.9, extracted with n-hexane, and measured gaschromatographically with N-P-FID. Acid labile 1 was hydrolyzed with HCl (1 mol/l) for 24 h. The detection limit was 25 nmol/l plasma. Kinetic studies were performed in 16 patients with essential hypertension under steady-state conditions after the oral application of 50 mg 1. The acetylator phenotype was determined with sulfamethazine. Complete dihydralazine plasma level-time courses were found in only 5 cases. The concentrations were below the detection limit in 4 patients for the whole period. Only single values could be registered in the remaining patients. Maximal plasma levels of the free (58-314 nmol/l) and acid labile 1 (147-367 nmol/l) were reached 20-40 min after the application. The elimination half life was 23-47 min for the free 1, 55-92 min for the acid labile 1. Less than 0.5% of the applied drug were excreted into the 24 h urine in its free form, about 0.4% as acid labile derivatives. No correlation could be found between the acetylator phenotype of the patients and the kinetic behaviour of the drug. Preliminary studies concerning the biliary excretion of 1 after i. m. application in two patients with T-drain showed an accumulation of the free compound with bile/plasma ratios up to 7.4.

The influence of the acetylator phenotype for the clinical use of dihydralazine.

Dihydralazine is a substrate of the human N-acetyltransferase. Therefore the acetylator phenotype could influence the pharmacodynamic response of dihydralazine and/or side effects of this drug. In this study it could be shown that: among patients with dihydralazine incompatibility slow acetylators preponderated; the risk of early side effects was higher in females than in males; and the ratio of fast/slow acetylators was higher in dihydralazine treated patients than in patients treated with other antihypertensives. Dihydralazine should be given very cautiously to female hypertonic patients that are slow acetylators.

[Role of dihydralazine in the treatment of chronic cardiac insufficiency]. / La place de la dihydralazine dans le traitement de l'insuffisance cardiaque chronique.

Dihydralazine, sold under the name of Nepressol, is the derivative of the hydrazinophthalazines used in France for the treatment of severe cardiac failure. The hydrazinophthalazines act in cardiac failure by decreasing systemic arterial resistance and increasing cardiac output without causing tachycardia or increasing myocardial contractility in normal or hypotensive subjects. The hydrazinophthalazines are metabolised by N-acetyl-transferase in the liver. The hepatic concentration of this enzyme is genetically determined. The immediate haemodynamic results observed with 100 to 300 mg/daily of Nepressol are impressive: an 86 p. 100 increase in cardiac output; a 75 p. 100 decrease in systemic valvular resistance; a 25 p. 100 fall in left ventricular filling pressures. These haemodynamic results are maintained at long-term (3 to 48 months). This treatment is associated with an improvement in symptoms and functional capacity. However, its efficacy in improving survival has not been demonstrated. Immunological complications giving rise to a lupic syndrome result only in biological changes and do not influence management.

Dihydralazine therapy and acetylator phenotype.

The acetylator phenotype was studied in 21 hypertensive patients receiving chronic treatment with dihydralazine and other antihypertensive drugs. Plasma dihydralazine concentrations were measured 6 hours after the morning dose of the drug. There were no significant differences between the slow and fast acetylators 1) in the daily dose of dihydralazine needed, 2) in the concentrations of dihydralazine achieved in plasma, 3) in the dose/concentration-relationship of dihydralazine or 4) in the appearance of side effects. Antinuclear antibodies were found in five patients. Four of them were fast acetylators. The lowest dose/concentration relationship was found in a patient with impaired renal function. According to these results the acetylator phenotype seems to be an unimportant factor in therapy with dihydralazine. More investigation will be needed in order to elucidate the metabolism of dihydralazine.