Mark R. Bowles

Conversion of irinotecan (CPT-11) to its active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), by human liver carboxylesterase

We have investigated the conversion of the novel anti-topoisomerase I agent CPT-11 (irinotecan; 7-ethyl-10[4-(1-piperidino)-1-piperidno]carbonyloxycamptothecin ) to its active metabolite, SN-38 (7-ethyl-10-hydroxycamptothecin), by human liver carboxylesterase (HLC). Production of SN-38 was relatively inefficient and was enzyme deacylation rate-limited with a steady-state phase occurring after 15-20 min of incubation. This later phase followed Michaelis-Menten kinetics with an apparent Km of 52.9 +/- 5.9 microM and a specific activity of 200 +/- 10 mumol/sec/mol. However, the total enzyme concentration estimated from the intercept concentrations of SN-38 was much lower than that estimated directly from the titration of active sites with paraoxon (0.65 vs. 2.0 microM, respectively). Because deacylation rate-limiting kinetics result in the accumulation of inactive acyl-enzyme complex, we postulated that incubation of CPT-11 with HLC would result in an inhibition of the HLC-catalysed hydrolysis of p-nitrophenylacetate (p-NPA), an excellent substrate for this enzyme. Indeed, this was found to be the case although complete inhibition could not be attained. Analysis of possible kinetic schemes revealed that the most likely explanation for the disparity in estimated enzyme concentrations and the incomplete inhibition of p-NPA hydrolysis is that CPT-11 also interacts at a modulator site on the enzyme, which profoundly reduces substrate hydrolysis. Furthermore, loperamide, a drug often used for the treatment of CPT-11-associated diarrhea, was found to inhibit both CPT-11 and p-NPA HLC-catalysed hydrolysis, most likely by a similar interaction. These observations have direct implications for the clinical use of CPT-11.

Evaluation of equilibrium constants for antigen-antibody interactions by solid-phase immunoassay: the binding of paraquat to its elicited mouse monoclonal antibody

A procedure is devised for simple graphical evaluation of the association constant for antibody-antigen interactions from data obtained by conventional solid-phase immunoassay for antigen. Application of the method to situations involving a univalent antigen is illustrated by means of ELISA data for the interactions of paraquat with its elicited murine monoclonal antibody, and the Fab fragment derived therefrom. Although the completely general situation with both antibody and antigen multivalent is not amenable to study by the present procedure, the quantitative expression is readily modified to accommodate antigen multivalency provided that the univalent Fab fragment may be substituted for the multivalent antibody (IgG or IgM) as partitioning species in the solid-phase immunoassay.

Large scale production and purification of paraquat and desipramine monoclonal antibodies and their Fab fragments

We describe the rapid, large scale purification of Fab fragments from mouse monoclonal antibodies. Antibodies against two clinically important and often fatal toxins, paraquat and desipramine, were isolated from mouse ascites fluid by preparative high performance hydroxylapatite (HPHT) or ion exchange (DEAE) high performance liquid chromatography. A competitive inhibition ELISA was used to determine the cross-reactivity of the antibody with analogs of the antigens. Papain digests of the IgGs were subjected to further HPHT followed by Sephadex G-100 chromatography to yield homogeneous Fab fragment preparations. The high purity of these preparations, demonstrated by SDS polyacrylamide gel electrophoresis, has only been achieved previously by affinity chromatography. Intrinsic association constants for the intact IgG and the Fab fragment--antigen interactions, determined by competitive inhibition ELISA, were similar. This indicates that antigen-binding activity was conserved during the production and purification of the Fab fragments.

Human Papillomavirus Type 6b Virus-Like Particles Are Able To Activate the Ras-MAP Kinase Pathway and Induce Cell Proliferation

ABSTRACT The initial step in viral infection is the attachment of the virus to the host cell via an interaction with its receptor. We have previously shown that a receptor for human papillomavirus is the α6 integrin. The α6 integrin is involved in the attachment of epithelial cells with the basement membrane, but recent evidence suggests that ligation of many integrins results in intracellular signaling events that influence cell proliferation. Here we present evidence that exposure of A431 human epithelial cells to human papillomavirus type 6b L1 virus-like particles (VLPs) results in a dose-dependent increase in cell proliferation, as measured by bromodeoxyuridine incorporation. This proliferation is lost if VLPs are first denatured or incubated with a monoclonal antibody against L1 protein. The MEK1 inhibitor PB98059 inhibits the VLP-mediated increase in cell proliferation, suggesting involvement of the Ras-MAP kinase pathway. Indeed, VLP binding results in rapid phosphorylation of the β4 integrin upon tyrosine residues and subsequent recruitment of the adapter protein Shc to β4. Within 30 min, the activation of Ras, Raf, and Erk2 was observed. Finally, the upregulation of c-myc mRNA was observed at 60 min. These data indicate that human papillomavirus type 6b is able to signal cells via the Ras-MAP kinase pathway to induce cell proliferation. We hypothesize that such a mechanism would allow papillomaviruses to infect hosts more successfully by increasing the potential pool of cells they are able to infect via the initiation of proliferation in resting keratinocyte stem and suprabasal cells.

Cloning and analysis of a cDNA encoding a human liver carboxylesterase

A human liver carboxylesterase (CE)-encoding cDNA has been cloned using synthetic oligodeoxyribonucleotides (oligos) based on the known amino acid (aa) sequences of rabbit and rat liver CEs. The oligos hybridize specifically to DNA encoding liver CEs. The longest cDNA obtained from screening several cDNA libraries encodes about 80% of the protein and translates into an aa sequence which has a high degree of similarity with the sequences of liver CEs from other species. On hybridization to mRNA isolated from human liver, the cDNA gave a single band of about 2.0 kb consistent with its encoding a protein of less than 68 kDa. DNA obtained from a number of human livers and probed with the CE cDNA gave identical hybridization patterns. These patterns were moderately complex by comparison with published data.

Quantitation of Paraquat in Biological Samples by Radioimmunoassay Using a Monoclonal Antibody

We have developed a sensitive and specific radioimmunoassay for the quantitative determination of paraquat in plasma, urine, and bronchoalveolar lavage fluid using a monoclonal antibody. The synthesis of the iodinated paraquat tracer is novel and less complicated than a previously reported method. Regardless of the type of biological sample analyzed, the sensitivity of the assay was 0.46 ng/ml in a 200-microliters aliquot. The results correlated well with those of another assay performed in a separate laboratory. Paraquat concentrations were determined in plasma and in urine of a dog over several days after the intravenous administration of 7.48 mg paraquat cation/kg and in bronchoalveolar lavage fluid obtained 34 hr after the 2-hr infusion was discontinued. Concentrations of paraquat measured ranged from 14.1 to 0.03 micrograms/ml in plasma and 2034 to 0.36 micrograms/ml in urine. Concentrations of paraquat in plasma obtained at various times after the ingestion of paraquat by three patients ranged from 51.0 to 0.010 micrograms/ml.

Purification and characterization of two human liver carboxylesterases

1. Two carboxylesterases (EC 3.1.1.1) purified from human livers were distinguished by pI (isoelectric point), nondenaturing polyacrylamide gel electrophoresis, molecular weight, catalytic activity, N-terminus and immunological cross-reactivity. 2. The low pI carboxylesterase has not been reported previously. 3. Numerous bands seen when each enzyme was focused on analytical IEF gels could not be separated. 4. When sections of the band pattern was refocused, the original complete band pattern was generated. 5. Both the mid and low pI carboxylesterases had catalytic activity for xenobiotics as well as medium and long chain fatty acid esters.

Prevention of paraquat toxicity in suspensions of alveolar type II cells by paraquat-specific antibodies

1 The herbicide, paraquat, is accumulated by the energy-dependent polyamine uptake pathway of alveolar type II cells. There it undergoes redox cycling that results in an amplified production of toxic reactive oxygen species and depletion of NADPH and other reducing equivalents. These processes account for the lung being the major target organ for paraquat toxicity. 2 We postulated that paraquat-specific antibodies would inhibit the uptake of the herbicide by type II cells and prevent its toxicity. Accordingly, we examined the effects of paraquat-specific monoclonal antibodies and Fab fragments on the uptake, efflux and cytotoxicity of 50 μM paraquat in suspensions of alveolar type II cells isolated from the rat. 3 The uptake of paraquat was linear over 40 min. Over this time, the uptake rate was inhibited significantly (% inhibition, 73-89) by IgG (25 or 50 μM) or Fab fragments (50 or 100 μM). 4 The apparent efflux rate of paraquat, studied over 16 h, was increased significantly from 0.12 h-1 for the control cells in medium to 0.17 h-1 by paraquat-specific Fab fragments but was unaffected by the specific IgG. 5 Cytotoxicity was determined by measuring the release of 51Cr from the cells. The cytotoxicity of 50 μM paraquat was decreased significantly (percent decrease, 56-80%) in the presence of specific antibodies. 6 These studies in vitro suggest some potential for immunotherapy in selected cases of paraquat poisoning.

Survival After Unexpected High Serum Methotrexate Concentrations in a Patient with Osteogenic Sarcoma

SummaryAn 18-year-old female patient receiving adjuvant chemotherapy for osteogenic sarcoma developed a pruritic erythematous rash during infusion of the eighth dose of methotrexate (8 g/m2) in the series. In other respects, the infusion proceeded normally but the 24-hour serum concentration of methotrexate was unexpectedly and extremely high, 574 /μmol/L. Dosing error was excluded, as was the hypothesis that the high concentrations were due to the presence of methotrexate-specific antibodies. Acute oliguria and renal failure were the primary manifestations of the drug-induced toxicity and the high concentrations can be attributed to decreased renal elimination of the drug over the first 24 hours. Treatment consisted of folinic acid rescue, forced diuresis, sequential charcoal haemoperfusion and haemodialysis, and repeated oral doses of activated charcoal. After examination of the contribution of the extracorporeal procedures and the charcoal to the elimination of the drug, the relative lack of morbidity was attributed primarily to the folinic acid rescue and the intensive supportive care.

Competition between paraquat and putrescine for uptake by suspensions of rat alveolar type II cells

Paraquat and the structurally similar polyamines, such as putrescine and spermidine, are accumulated actively and selectively by the alveolar type II cells via the polyamine uptake system. We report the uptake kinetics of paraquat and putrescine and their mutual inhibition in freshly isolated rat type II cell suspensions. The uptake of paraquat by type II cells exhibited saturation kinetics and could be inhibited in a concentration-dependent manner by putrescine. By applying enzyme kinetic analysis to our experimental data it was demonstrated that the uptake of paraquat or putrescine is inhibited in a partially competitive manner by the respective inhibitor. Thus, we postulate that the polyamine uptake pathway in type II cells for paraquat and putrescine has two separate sites, one for each substrate, and that binding of one leads to a conformational change in the other.