Targeted antimicrobial photochemotherapy

This study explores a new approach for antimicrobial therapy with light activation of targeted poly-L-lysine (pL)-chlorin e6 (ce6) conjugates. The goal was to test the hypothesis that these conjugates between pL and ce6 would efficiently target photodestruction towards gram-positive (Actinomyces viscosus) and gram-negative (Porphyromonas gingivalis) oral species while sparing an oral epithelial cell line (HCPC-1). Conjugates of ce6 with pL (average molecular weight, 2,000) having a positive, neutral, or negative charge were prepared. Illumination with red light (lambdamax = 671 nm) from a diode array produced a dose-dependent loss of CFU from the bacteria, under conditions that did not affect the viability of the epithelial cells. For P. gingivalis, the cationic conjugate produced 99% killing, while the neutral conjugate killed 91% and the anionic conjugate killed 76% after 1 min of incubation and exposure to red light for 10 min. For A. viscosus, the cationic conjugate produced >99.99% killing while HCPC-1 cells remained intact. The importance of the positive charge was shown by the effectiveness of ce6-monoethylenediamine monoamide (a monocationic derivative of ce6) in killing both bacteria. The clinically employed benzoporphyrin derivative under the same conditions killed epithelial cells while leaving P. gingivalis relatively unharmed. A mixture of ce6 with pL did not show phototoxicity comparable with that of the cationic conjugate. These results were explained by the selective uptake of the conjugates by bacteria (20- to 100-fold) compared to that by mammalian cells, while free ce6 showed much less selectivity for bacteria (5- to 20-fold). The data suggest that the cationic pL-ce6 conjugate may have an application for the photodynamic therapy of periodontal disease.     

Mediators of change in physical activity following an intervention in primary care: PACE

Lysozyme is able to lyse Gram-positive bacteria acting as muramidase on the peptidoglycan polymer. Gram-negative bacteria in vitro are not lysed by lysozyme. It was assumed that the peptido-glycan is protected by the outer membrane and thus that Gram-negative bacteria are not affected by lysozyme without the aid of other factors such as EDTA or complement which enable lysozyme to penetrate the outer membrane. Accidentally, Pellegrini et al. [(1992) J. Appl. Bacteriol., 72:180-187] found that lysozyme per se is able to kill some Gram-negative bacteria. On the basis of morphological and immunocytochemical findings obtained from chemically fixed bacteria, it was concluded that lysozyme does not lyse Gram-negative bacteria but affects the cytoplasm of for example, Escherichia coli, leading to its disintegration, whilst the membranes do not break down. In an attempt to clarify the action of lysozyme on E. coli, we employed cryotechniques including ultrarapid freezing, cryomicroscopy and freeze substitution, and immunolabeling. Bacteria that were immediately frozen after exposure to lysozyme remained morphologically intact. Individual bacteria plated on agar after exposure to lysozyme were mostly intact when frozen within a few seconds. However, inner and outer membranes of 80% of the bacteria were disrupted, whereas the cytoplasm of only a few bacteria showed signs of disintegration when bacteria were frozen with a delay of only 5 min of plating onto pure agar or agar containing growth medium. After a period of time of 15 min between plating onto agar and freezing, about 97% of the bacteria showed changes of disintegration of various extent. Immunolabeling showed that lysozyme binds to the outer cell membrane and may penetrate the membrane, reaching the periplasmic space and possibly the inner cell membrane. The ultrastructural findings and the results of antibacterial assays suggest that lysozyme is bactericidal for E. coli but is not able to induce disintegration. Disintegration is accomplished by changes of the environment starting at the cell membranes. The mechanism by which lysozyme penetrates the membrane, the way it acts to be bactericidal, and the way disintegration is initiated remain to be clarified.     

Bile acid conjugation in fetal hepatic organ cultures

A new technique has been developed in which mammalian fetal liver can be maintained in organ culture for prolonged periods with intact structure and function. Near-term rat fetal liver explants were incubated in vitro for periods of up to 3 wk with preservation of normal cellular morphology and intercellular (organ) relationships. [14C]cholate was incorporated into tissue and medium conjugates at a constant rate during 21 days in vitro. During a 24-h incubation with radioactively labeled cholic acid, bile acid conjugates accumulated in tissues to a maximum value by 6 h and maintained this value through 24 h. During the same 24-h incubation with [14C]cholate, conjugates were secreted into the medium at a constant rate. Addition of 8 X 10(-4) M taurine to the medium during a 4-day incubation produced a threefold enhancement in the rate of conjugate formation in tissues and medium. Enhanced conjugation in the presence of additional taurine was due almost entirely to increased taurocholate formation and no significant difference was observed in the amount of glycocholate formed. Exposure of explants to 3.6 X 10(-4) M cycloheximide for prolonged periods resulted in inhibition of conjugate formation, but when this concentration of cycloheximide was maintained for only 24 h a significantly (P less than 0.001) increased rate of conjugate formation was observed. The results indicate that metabolic processes in the organ-culture system are in a state of dynamic equilibrium and that morphologic integrity and specific hepatocytic function are maintained after 21 days in vitro. Preferential taurocholate formation was demonstrated in rat fetal liver, and the data suggest that glycine and taurine interact with separate enzymatic systems in bile acid conjugation. The possible mechanisms that mediate the effect of cycloheximide are discussed.     

Mechanism of carbamoyl phosphate synthetase from Escherichia coli--binding of the ATP molecules used in the reaction and sequestration by the enzyme of the ATP molecule that yields carbamoyl phosphate

A directly energized vacuolar pump for glutathione (GS) conjugates has been described for several plant species. Since glucuronate conjugates also occur in plants, we addressed the question whether plant vacuoles take up the abiotic glucuronate conjugate estradiol 17-(beta-glucuronide) (E217G) via a GS conjugate pump, which in some cases has been reported to accept various organic anions as substrates, or via a distinct glucuronate transporter. Uptake studies into vacuoles from rye and barley were performed with E217G and metolachlor-GS (MOC-GS), a substrate of the GS conjugate ATPase, to compare glucuronate conjugate transport into vacuoles containing endogenous flavone glucuronides with those lacking specific glucuronate conjugates, respectively. Our results indicate that E217G and MOC-GS are taken up into vacuoles of both plants via a directly energized mechanism since transport was (i) strictly ATP-dependent; (ii) inhibited by vanadate but not by bafilomycin A1, azide, verapamil, nor by dissipation of the vacuolar DeltapH or DeltaPsi; (iii) E217G uptake into rye vacuoles was partially driven by other nucleotides in the following order of efficiency: ATP > GTP > UTP congruent with CTP, whereas the non-hydrolyzable ATP analogue 5'-adenylyl-beta,gamma-imidodiphosphate, ADP, or PPi did not energize uptake. E217G transport into rye vacuoles was saturable (Km approximately 0.2 mM). The rye-specific luteolin glucuronides decreased uptake rates of E217G and MOC-GS into rye and barley vacuoles to comparable degrees with the mono- and diglucuronidated derivatives (40-60% inhibition) being more effective than the triglucuronide. Inhibition of E217G uptake by luteolin 7-O-diglucuronide was competitive (Ki = 120 microM). Taurocholate had no effect on E217G transport, and uptake of MOC-GS was not inhibited by E217G. Although GS conjugates and oxidized GS decreased MOC-GS transport, E217G uptake into rye and barley vacuoles was stimulated up to 7-fold in a concentration-dependent manner by these substances, with dinitrobenzene-GS being most effective. The stimulation of the GS conjugates was not due to detergent or redox effects and was specific for the E217G pump. GS conjugate stimulation of glucuronate uptake was unique for plants as E217G uptake into yeast microsomal vesicles was not affected. By comparison with a DeltaYCF1 yeast mutant, defective in vacuolar transport of GS conjugates mediated by YCF1, it was shown that E217G was taken up into yeast vesicles via a YCF1-independent directly energized pump. These results indicate that E217G as a glucuronate conjugate is transported across the vacuolar membranes of plants and yeast by a carrier distinct from the GS conjugate ATPase.     

Comparison of pharmacokinetic parameters of a polypeptide, the Bowman-Birk protease inhibitor (BBI), and its palmitic acid conjugate

PURPOSE: The alteration of the pharmacokinetic parameters of the polypeptide BBI through conjugation with palmitic acid was examined. METHODS: 125I-BBI or 125I-Pal-BBI was administered iv to 6 week old CF-1 mice at a dose of 3 mg/kg. The mice were sacrificed at 5, 10, 20, 60, 120, 240, 360, and 480 min and the total radioactivity was determined for blood and each organ. The blood was analyzed on a Sephadex G-50 size-exclusion column to determine the amount of intact polypeptide present in the blood. From the amount of intact polypeptide at each time point, the pharmacokinetic parameters were determined. RESULTS: By conjugating three palmitic acids to each BBI molecule, the area under the curve (AUC) and mean residence time (MRT) increase by a factor of 10.8 and 2.8, respectively. There was also a difference in the organ distribution between the two treatments; while 125I-BBI was rapidly cleared from the kidneys, 125I-Pal-BBI was predominantly to the liver. Subsequent studies suggested that the binding of the conjugate to non-albumin serum proteins was most likely the cause of the altered pharmacokinetics. CONCLUSIONS: The residence time in the blood and the lipophilicity of BBI were increased upon conjugation with palmitic acid through a reversible disulfide linkage. Pharmacokinetic studies showed an increase in the AUC and a decrease in kidney clearance in palmitic acid conjugates, indicating a potential increase of the therapeutic efficacy of the polypeptide drug.     

Posttranslational modifications of trichomonad tubulins; identification of multiple glutamylation sites

A method of conjugating poly(glutamic acid) poly(phosphorothioate)-cross-linked alkaline phosphatase to maleimide-derivatized immunoglobulin is described. Intramolecular autocatalyzed cross-linking of alkaline phosphatase at 2:1 to 4:1 polymer:enzyme ratios introduced 32-68 thiolates on the surface of the enzyme. Depending on the stoichiometry of polymer to enzyme, the cross-linked alkaline phosphatase retained 75-90% of its native catalytic activity. The cross-linked thiolate-functionalized alkaline phosphatase was conjugated to maleimide-derivatized immunoglobulin. Compared to a control prepared using non-cross-linked alkaline phosphatase, these conjugates were smaller in size and more stable to heat. The enzymatic activity of the cross-linked conjugates after incubation at 45 degrees C and pH 7.5 for 25 days was 35% higher than those of the highest-activity control conjugates. The conjugation process could be controlled by varying the stoichiometries of poly(glutamic acid) poly(phosphorothioate), alkaline phosphatase, and immunoglobulin.     

Antigenic crossreactivity and lipopolysaccharide neutralization properties of bovine immunoglobulin G

We investigated a possible mechanism by which immunization against core and lipid A determinants of lipopolysaccharide reduced clinical cases of mastitis and symptoms commonly associated with heterologous Gram-negative IMI. The IgG fraction of sera from cows immunized with either Escherichia coli J5 bacterin, E. coli J5 lipopolysaccharide conjugate vaccine, or unimmunized controls was purified by precipitation with caprylic acid and ammonium sulfate. The degree of IgG crossreactivity with Gram-negative bacteria that were isolated from clinical quarters was greater than that with Gram-positive isolates of Staphylococcus aureus. The highest magnitude of crossreactivity was against smooth strain E. coli isolates, followed by heterologous species of Enterobacter, Serratia, and Klebsiella isolates. Serum IgG from cows immunized with conjugate was highly crossreactive to E. coli J5, E. coli O111:B4, Serratia marcescens, Klebsiella pneumoniae, and Salmonella typhimurium lipopolysaccharides. The magnitude of antibody crossreactivity with lipopolysaccharides coincided with the ability of IgG to suppress the mitogenic effect of lipopolysaccharides on bovine lymphocytes.     

Specific delivery of captopril to the kidney with the prodrug captopril-lysozyme

Low-molecular-weight proteins (LMWPs) accumulate in the proximal tubular cells of the kidney, which makes these proteins interesting tools for renal drug targeting. We studied this approach using the LMWP lysozyme as a carrier for the angiotensin-converting enzyme inhibitor captopril. Captopril was conjugated to lysozyme via a disulfide bond. The pharmacokinetics of the captopril-lysozyme conjugate was studied in the rat. Only intact conjugate could be detected in the circulation. The total amount of captopril disulfides in the kidney was six times higher after administration of the conjugate than after the administration of an equivalent amount of free captopril. The conjugate was recovered in the urine partially as intact conjugate and partially as low-molecular-weight disulfides. The excretion of conjugate in the urine was not a consequence of the coupling of captopril to lysozyme because an equivalent bolus dose of native lysozyme was similarly excreted into the urine. By determination of the renal angiotensin-converting enzyme activity, we showed that the conjugate was degraded to the pharmacologically active captopril in vivo. We conclude that the coupling of captopril to the LMWP lysozyme results in increased captopril concentrations in the kidney and reduced captopril concentrations in the circulation.     

Inhibition of N-nitrosodimethylamine demethylase in rat and human liver microsomes by isothiocyanates and their glutathione, L-cysteine, and N-acetyl-L-cysteine conjugates

Natural and synthetic isothiocyanates and their conjugates were examined for their inhibitory effects toward rat and human liver microsomal N-dimethylnitrosoamine demethylase (NDMAd) activity using a radiometric NDMAd assay. Substrate concentrations of 30 and 60 microM were used to probe the activity of cytochrome P4502E1 isozyme through the alpha-hydroxylation of NDMA. It was found that alkyl isothiocyanates such as sulforaphane and allyl isothiocyanate displayed very weak inhibition, whereas the arylalkyl isothiocyanates such as benzyl and phenethyl isothiocyanate showed significant inhibition toward rat liver NDMAd activity with IC50 values of 9.0 and 8.3 microM, respectively. More interestingly, glutathione conjugates of benzyl, phenethyl, and 6-phenylhexyl isothiocyanates all inhibited NDMAd at the comparable concentrations. In the phenethyl isothiocyanate conjugates series, there exist marked differences in their inhibitory activity; i.e., its conjugates with L-cysteine (IC50 = 4.3 microM) and with glutathione (IC50 = 4.0 microM) are more potent than its conjugate of N-acetylcysteine (IC50 = 24.0 microM). The same trend was also observed for the human liver microsomal NDMAd activity. The half-lives of these conjugates were determined in the presence of other free thiols from L-cysteine or glutathione using an HPLC system. It was shown that isothiocyanates are released from their conjugates and react with the free thiols present in the solution. The longer half-life of N-acetylcysteine conjugate of phenethyl isothiocyanate as compared to the other conjugates is consistent with its lower inhibitory activity. The inhibition of NDMAd, and therefore cytochrome P4502E1, by isothiocyanate conjugates is most likely due to the action of the free isothiocyanates released from the conjugates. Since cytochrome P4502E1 and other isozymes play important roles in the activation of the tobacco-specific nitrosoamines, these results provide a basis for investigating the potential of isothiocyanate conjugates as chemopreventive agents.     

Synthesis and in vitro efficacy of transferrin conjugates of the anticancer drug chlorambucil

One strategy for improving the selectivity and toxicity profile of antitumor agents is to design drug carrier systems employing soluble macromolecules or carrier proteins. Thus, five maleimide derivatives of chlorambucil were bound to thiolated human serum transferrin which differ in the stability of the chemical link between drug and spacer. The maleimide ester derivatives 1 and 2 were prepared by reacting 2-hydroxyethylmaleimide or 3-maleimidophenol with the carboxyl group of chlorambucil, and the carboxylic hydrazone derivatives 5-7 were obtained through reaction of 2-maleimidoacetaldehyde, 3-maleimidoacetophenone, or 3-maleimidobenzaldehyde with the carboxylic acid hydrazide derivative of chlorambucil. The alkylating activity of transferrin-bound chlorambucil was determined with the aid of 4-(4-nitrobenzyl)pyridine (NBP) demonstrating that on average 3 equivalents were protein-bound. Evaluation of the cytotoxicity of free chlorambucil and the respective transferrin conjugates in the MCF7 mammary carcinoma and MOLT4 leukemia cell line employing a propidium iodide fluorescence assay demonstrated that the conjugates in which chlorambucil was bound to transferrin through non-acid-sensitive linkers, i.e., an ester or benzaldehyde carboxylic hydrazone bond, were not, on the whole, as active as chlorambucil. In contrast, the two conjugates in which chlorambucil was bound to transferrin through acid-sensitive carboxylic hydrazone bonds were as active as or more active than chlorambucil in both cell lines. Especially, the conjugate in which chlorambucil was bound to transferrin through an acetaldehyde carboxylic hydrazone bond exhibited IC50 values which were approximately 3-18-fold lower than those of chlorambucil. Preliminary toxicity studies in mice showed that this conjugate can be administered at higher doses in comparison to unbound chlorambucil. The structure-activity relationships of the transferrin conjugates are discussed with respect to their pH-dependent acid sensitivity, their serum stability, and their cytotoxicity.     

Carrier-mediated active transport of the glucuronide and sulfate of 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040) into rat liver: quantitative comparison of permeability in isolated hepatocytes, perfused liver and liver in vivo

The hepatic uptake of glucuronic acid and sulfate conjugates of 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040), a dual inhibitor of 5-lipoxygenase and thromboxane A2 synthetase, was investigated in rats. The biliary excretion clearance values for the glucuronide and the sulfate, obtained after i.v. administration of E3040, were similar and corresponded to approximately 30% of the hepatic blood flow rate. The influx clearance values of E3040 conjugates in the presence of 3% bovine serum albumin, measured by a multiple indicator dilution method in the perfused liver, were 1.20 ml/min/g liver for the glucuronide and 0.74 ml/min/g liver for the sulfate, which were twice and equal to the normal hepatic plasma flow rate, respectively, which suggests the presence of an efficient transport system(s). The uptake of E3040 conjugates into the isolated hepatocytes is mediated by Na(+)-independent active transport system(s), which is inhibited by dibromosulfophthalein and bile acids. The uptake for the sulfate had high-affinity and high-capacity transport activity (Km = 25 microM; Vmax = 7.8 nmol/min/10(6) cells) compared with that for the glucuronide (Km = 59 microM; Vmax = 2.2 nmol/min/10(6) cells). The uptakes of E3040 conjugates (glucuronide, sulfate) exhibited a mutual competitive inhibition. It is suggested that both conjugates share a multispecific organic anion transporter located on the sinusoidal membrane.     

Trajectory and current situation of biomedical research in Argentina]

A cationic manganese porphyrin-peptide nucleic acid (PNA) conjugate has been prepared and used to cleave a double-stranded DNA target. Cleavage experiments were performed with a 247-base pair restriction DNA fragment containing a 10-base pair homopurine binding target for the PNA. Oxidative activation by this Mn porphyrin-PNA conjugate leads to sequence specific, 3'-staggered cleavage of both DNA strands near the strand displacement junction. Furthermore, the Mn porphyrin-PNA porphyrin conjugates bind over 100-fold better to double-stranded DNA compared to the native PNA.     

Reversal of chloroquine resistance in murine malaria parasites by prostaglandin derivatives

An oligomeric ester of prostaglandin B2 (OC-5186) was found to reverse chloroquine resistance in the murine malarial parasite Plasmodium berghei. When mice were infected with either chloroquine-sensitive or -resistant P. berghei on day 0 (by intraperitoneal injection of 1 x 10(6) parasitized erythrocytes), they died before day 23. When treated with 15 mg/kg/day of chloroquine for the first four days of infection, all mice infected with the sensitive-strain survived, while all those infected with the resistant strain died before day 23. When OC-5186 (3-12 mg/kg/day) was administered in combination with chloroquine for the first four days, 60% of the animals infected with the resistant strain survived. The differences in the survival rate between the group treated with chloroquine only and the group treated with a combination of drugs (chloroquine plus 3-12 mg/kg/day of OC-5186) were significant. There was also a significant inhibition of parasitemia in the group treated with the combination of drugs. The combinations of chloroquine and a monomer ester of prostaglandin B2 (OC-5181) had some antimalarial activity, but the differences between the chloroquine-treated group and the combination treatment group were not significant in terms of both the parasitemia and the survival rate. Another oligomeric ester of prostaglandin E1 (MR-356) as well as unesterified monomer prostaglandins (PGA2 and PGB2) were ineffective by themselves and in combination with chloroquine.     

Synthesis of carboxymethylpullulan-peptide-doxorubicin conjugates and their properties

The amino group of doxorubicin (DXR) was found to be bound to the carboxyl group of carboxymethylpullulan (CMPul) either directly or through tetrapeptide spacers, including Gly-Gly-Phe-Gly, Gly-Phe-Gly-Gly and Gly-Gly-Gly-Gly. These conjugates had DXR contents of 6.1-7.1%, with the degree of substitution of carboxymethyl groups being 0.6 per sugar moiety. These conjugates associate in phosphate-buffered saline (PBS) (pH 7.4), forming micelles with hydrophobic DXR inside and hydrophilic CMPul on the outside. The amounts of DXR released from the conjugates in the presence of rat liver lysosomal enzymes were determined by HPLC. The rate of the drug release differed among the conjugates tested. CMPul-DXR conjugate bound through Gly-Gly-Phe-Gly released 35% of its DXR over 24 h. On the other hand, CMPul-DXR conjugate without spacer released no free DXR. The antitumor effect of each conjugate in rats bearing Walker 256 was studied by monitoring the tumor weights after a single intravenous injection. Compared with DXR, CMPul-DXR conjugates bound through Gly-Gly-Phe-Gly and Gly-Phe-Gly-Gly spacers significantly suppressed the tumor growth, while CMPul-DXR conjugate bound through Gly-Gly-Gly-Gly showed less antitumor effect than DXR. CMPul-DXR conjugate bound through Gly-Gly-Gly-Gly showed less antitumor effect than DXR. CMPul-DXR conjugate without spacer showed no in vivo antitumor effect even at a dose equivalent to as much as 20 mg/kg of DXR.     

Comparative cytotoxicity of 14 novel selenocysteine se-conjugates in rat renal proximal tubular cells

Recently, Se-substituted selenocysteine conjugates were proposed as potential prodrugs to target biologically active selenol compounds to tissues containing high activities of cysteine conjugate beta-lyases, such as the kidneys. However, several selenium compounds are known to be relatively toxic compounds. In the present study, the cytotoxicity of 14 selenocysteine Se-conjugates was determined in freshly isolated rat renal proximal tubular cells (RPTC). The results of this study show that four selenocysteine Se-conjugates with alkyl substituents (methyl, ethyl, n-propyl, and n-butyl) did not cause significant cytotoxicity to RPTC up to concentrations of 500 microM after 90 min of incubation. Also, no effect was observed on mitochondrial functioning as indicated by the unaffected mitochondrial membrane potential (delta psi). Se-(i-Propyl)-selenocysteine, however, appeared to be a cytotoxic compound, causing time- and dose-dependent cytotoxicity, and caused a decrease of delta psi in remaining viable cells. Aminooxyacetic acid (AOAA) provided significant protection against cell death of Se-(i-propyl)-selenocysteine, pointing to involvement of cysteine conjugate beta-lyase. AOAA, however, did not prevent the decrease of delta psi. Differentially substituted Se-(phenyl)-L-selenocysteine and Se-(benzyl)-L-selenocysteine conjugates appeared to be cytotoxic to RPTC at a concentration of 200 microM, as indicated by increased cell death and a decreased delta psi in remaining viable cells. Within the Se-benzyl-series, Se-(4-methoxybenzyl)-L-selenocysteine was the most toxic conjugate, whereas Se-(4-chlorophenyl)-L-selenocysteine was the most toxic conjugate of the Se-phenyl compounds. The selenocysteine Se-conjugates with nonsubstituted phenyl and benzyl substituents were nontoxic at 200 microM, but caused significant cell death at a concentration of 500 microM. Preincubation with AOAA, an inhibitor of cysteine conjugate beta-lyase, provided only partial protection against the cytotoxicity of Se-(phenyl)-L-selenocysteine (500 microM) and Se-(4-methoxybenzyl)-L-selenocysteine (200 microM). AOAA did not protect against cytotoxicity of the other conjugates, suggesting direct effects of these compounds or involvement of alternative routes of bioactivation. This study demonstrates that cytotoxicity of selenocysteine Se-conjugates is strongly dependent on the nature of the Se-bound substituent. The nontoxic Se-(alkyl)-Se-conjugates may be promising candidates for further evaluation for chemopreventive activities.     

Role of cross-linking agents in determining the biochemical and pharmacokinetic properties of Mgr6-clavin immunotoxins

Several immunotoxins (ITs) were synthesized by the attachment of clavin, a recombinant toxic protein derived from Aspergillus clavatus, to the monoclonal antibody Mgr6 that recognizes an epitope of the gp185(HER-2) extracellular domain expressed on breast and ovarian carcinoma cells. Conjugation and purification parameters were analyzed in an effort to optimize the antitumor activity and stability of the ITs in vivo. To modulate the in vitro and in vivo properties of the immunotoxins, different coupling procedures were used and both disulfide and thioether linkages were obtained. Unhindered and hindered disulfide with a methyl group linkage ethyl S-acetyl 3-mercaptopropionthioimidate ester hydrochloride (AMPT) or ethyl S-acetyl 3-mercaptobutyrothioimidate ester hydrochloride (M-AMPT) were obtained by reaction with recombinant clavin, while the monoclonal antibody Mgr6 was derivatized with ethyl 3-[(4-carboxamidophenyl)dithio]propionthioimidate ester hydrochloride (CDPT). To achieve higher hindrance (a disulfide bond with a geminal dimethyl group), Mgr6 was derivatized with the N-hydroxysuccinimidyl 3-methyl-3-(acetylthio)butanoate (SAMBA) and clavin with CDPT. To evaluate the relevance of the disulfide bond in the potency and pharmacokinetic behavior of the ITs, a conjugate consisting of a stable thioether bond was also prepared by derivatizing Mgr6 with the N-hydroxysuccinimidyl ester of iodoacetic acid (SIA) and clavin with AMPT. The immunotoxins were purified and characterized using a single-step chromatographic procedure. Specificity and cytotoxicity were assayed on target and unrelated cell lines. The data indicate that the introduction of a hindered disulfide linkage into ITs has little or no effect on antitumor activity and suggest that disulfide cleavage is essential for activity; indeed, the intracellularly unbreakable thioether linkage produced an inactive IT. Analysis of IT stability in vitro showed that the release of mAb by incubation with glutathione is proportional to the presence of methyl groups and increases exponentially with the increase in steric hindrance. Analysis of the pharmacokinetic behavior of ITs in Balb/c mice given intravenous bolus injections indicated that ITs with higher in vitro stability were eliminated more slowly; i.e., the disulfide bearing a methyl group doubled the beta-phase half-life (from 3.5 to 7.1 h) compared with that of the unhindered, while a geminal dimethyl protection increased the elimination phase to 24 h. The thioether linkage showed its intrinsic stability with a beta-phase half-life of 46 h. The thioether linkage also increased the distribution phase from 17 to 32 min. The in vitro characteristics and in vivo stability of Mgr6-clavin conjugates composed of a methyl and dimethyl steric hindered disulfide suggest clinical usefulness.     

Glucuronidation of N-hydroxy heterocyclic amines by human and rat liver microsomes

The food-borne carcinogenic and mutagenic heterocyclic aromatic amines undergo bioactivation to the corresponding N-hydroxy (OH)-arylamines and the subsequent N-glucuronidation of these metabolites is regarded as an important detoxification reaction. In this study, the rates of glucuronidation for the N-OH derivatives of 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP), 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) by liver microsomal glucuronosyltransferase were compared to that of the proximate human urinary bladder carcinogen, N-OH-aminobiphenyl (N-OH-ABP) and the proximate rat colon carcinogen N-OH-3,2'-dimethyl-4-amino-biphenyl (N-OH-DMABP). Human liver microsomes catalyzed the uridine 5'-diphosphoglucuronic acid (UDPGA)-dependent glucuroidation of N-OH-IQ, N-OH-PhIP, N-OH-Glu-P-1 and N-OH-MeIQx at rates of 59%, 42%, 35% and 27%, respectively, of that measured for N-OH-ABP (11.5 nmol/min/mg). Rat liver microsomes also catalyzed the UDPGA-dependent glucuronidation of N-OH-PhIP, N-OH-Glu-P-1 and N-OH-IQ at rates of 30%, 20% and 10%, respectively of that measured for N-OH-DMABP (11.2 nmol/min/mg); activity towards N-OH-MeIQx was not detected. Two glucuronide(s) of N-OH-PhIP, designated I and II, were separated by HPLC. Conjugate II was found to be chromatographically and spectrally identical with a previously reported major biliary metabolite of PhIP in the rat, while conjugate I was identical with a major urinary metabolite of PhIP in the dog. Hepatic microsomes from rat, dog and human were found to catalyze the formation of both conjugates. The rat preferentially formed conjugate II (I to II ratio of 1:15), while the dog and human formed higher relative amounts of conjugate I (I to II ratio of 2.5:1.0 and 1.3:1.0 respectively). Fast atom bombardment mass spectrometry of conjugates I and II gave the corresponding molecular ions and showed nearly identical primary spectra. However, collision-induced spectra were distinct and were consistent with the identity of conjugates I and II as structural isomers. Moreover, the UV spectrum of conjugate I exhibited a lambda max at 317 nm and was essentially identical to that of N-OH-PhIP, while conjugate II was markedly different with a lambda max of 331 nm. Both conjugates were stable in 0.1 N HCl and were resistant to hydrolysis by rat, dog and human liver microsomal beta-glucuronidases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Development of a streptavidin-anti-carcinoembryonic antigen antibody, radiolabeled biotin pretargeting method for radioimmunotherapy of colorectal cancer. Reagent development

With pretargeting, radioisotope delivery to tumor is decoupled from the long antibody localization process, and this can increase tumor:blood ratios dramatically. Several reagents were prepared for each step of a "two-step" pretargeting method, and their properties were investigated. For pretargeting tumor, streptavidin-monoclonal antibody (StAv-mab) conjugates were prepared by cross-linking sulfo-SMCC-derivatized streptavidin to a free thiol (SH) group on MN-14 [a high-affinity anti-carcinoembryonic antigen (CEA) mab]. Thiolated mabs were generated either by reaction of 2-iminothiolane (2-IT) with mab lysine residues or by reduction of mab disulfide bonds with (2-mercaptoethyl)amine (MEA). Both procedures gave protein-protein conjugates isolated in relatively low yields (20-25%) after preparative size-exclusion (SE) chromatography purification with conservative peak collection. Both StAv-MN-14 conjugates retained their ability to bind to CEA, to an anti-idiotypic antibody to MN-14 (WI2), and to biotin, as demonstrated by SE-HPLC. Two clearing agents, WI2 mab and a biotin-human serum albumin (biotin-HSA) conjugate, were developed to remove excess circulating StAv-MN-14 conjugates in animals. Both clearing proteins were also modified with galactose residues, introduced using an activated thioimidate derivative, to produce clearing agents which would clear rapidly and clear primary mab rapidly. At least 14 galactose residues on WI2 were required to reduce blood levels to 5.9 +/- 0.7% ID/g in 1 h. Faster blood clearance (0.7 +/- 0.2% ID/g) was observed in 1 h using 44 galactose units per WI2. For the delivery of radioisotope to tumor, several biotinylated conjugates consisting of biotin, a linker, and a chelate were prepared. Conjugates showed good in vitro and in vivo stability when D-amino acid peptides were used as linkers, biotin-peptide-DOTA-indium-111 had a slightly longer blood circulation time (0.09 +/- 0.02% ID/g in 1 h) than biotin-peptide-DTPA-indium-111 (0.05 +/- 0.03% ID/g in 1 h) in nude mice. A longer circulation time with the neutral DOTA complex might allow higher tumor uptake.     

Bone marrow and renal injury associated with haloalkene cysteine conjugates in calves

Almost 40 years ago, it was reported that cattle-feed which had been extracted with hot trichloroethylene and then fed to calves produced renal injury and a fatal aplastic anaemia. The toxic factor was subsequently identified as S-(1,2-dichlorovinyl)-L-cysteine (DCVC). These original findings have been confirmed, a single intravenous dose of DCVC at 4 mg/kg, or 0.4 mg/kg intravenously per day administered for 10 days to calves produced aplastic anaemia, and renal injury after a single dose of 4 mg/kg. The toxicity to calves of a number of other haloalkene cysteine conjugates has been examined to ascertain whether, like DCVC, they produce bone marrow and renal injury. Intravenous administration of the N-acetyl cysteine conjugate of DCVC produced renal but not bone marrow injury at a molar equivalent dose to DCVC, indicating that the calf can deacetylate the mercapturic acid and further that sufficient chemical had reached the kidney to be a substrate for the enzyme cysteine conjugate beta-lyase. However, intravenous administration of the alpha-methyl analogue of DCVC, which cannot undergo metabolism via the enzyme cysteine conjugate beta-lyase, was without toxicity at doses about five-fold higher than DCVC. These latter findings provide strong evidence that metabolism of DCVC via the enzyme beta-lyase is necessary for bone marrow and renal injury to occur. The cysteine conjugates of perchloroethylene and hexachloro-1,3-butadiene(HCBD) when given intravenously to calves at molar equivalent doses to DCVC, or above, did not produce either bone marrow or renal injury. In contrast, intravenous administration of the cysteine conjugate of tetrafluoroethylene (TFEC) produced severe renal tubular injury in calves without affecting the bone marrow. In vitro studies with these haloalkene cysteine conjugates showed, like DCVC, that they were good substrates for calf renal cysteine conjugate beta-lyase and toxic to renal cells as judged by their ability to reduce organic anion and cation transport by slices of calf renal cortex and inhibit the renal enzyme glutathione reductase. Calves were also dosed either orally or intravenously with HCBD to assess its toxicity. HCBD at higher molar equivalent doses than DCVC produced mid-zonal necrosis in the liver, renal tubular necrosis but no bone marrow injury in calves. The key findings emerging from these studies are (1) that none of the other cysteine conjugates, at molar equivalent doses to DCVC and above, produce bone marrow injury in calves, (2) TFEC produced only renal injury, suggesting that sufficient of the other conjugates had not reached the kidney for metabolism by beta-lyase to produce cytotoxicity and (3) that HCBD itself is more toxic than its cysteine or mercapturic acid conjugate, suggesting that pharmaco-kinetics and disposition are important factors in determining the toxicity of these conjugates to calves. Further studies are needed to understand the basis for the selective toxicity of DCVC to the bone marrow of calves.