Involvement of AP-2 binding sites in regulation of human beta-glucuronidase.

The lysosomal hydrolase beta-glucuronidase (beta-gluc) can be used for the bioactivation of non-toxic glucuronide prodrugs of anticancer agents. The enzyme is present at high levels in many tumours and hence may lead to an enhanced drug targeting by tumour-selective release of the active anticancer drug. Individual expression and regulation of this enzyme is one factor modulating the bioactivation of glucuronide prodrugs.

Nuclear factor-kappaB mediates up-regulation of cathepsin B by doxorubicin in tumor cells.

Anthracyclines such as doxorubicin remain among the most effective agents for the treatment of solid tumors and hematological malignancies. To overcome dose-limiting side effects like cardiotoxicity, an intensive effort has been undertaken to develop promising doxorubicin prodrugs that are specifically activated at the tumor site. One approach is the application of peptide prodrugs of doxorubicin.

Expression of multidrug resistance proteins in rat and human chronic pancreatitis.

Background And Aims: The expression of the ABC-transporters MDR-1, MRP1, and MRP-2 was investigated in healthy pancreas and in chronic pancreatitis tissue samples in rats and humans to evaluate their possible involvement in a multidrug resistance of the pancreas with consequences for the pharmacologic treatment of pancreatic diseases.

Methods: Human pancreatic tissue samples of healthy tissue and chronic pancreatitis were collected during pancreas surgery. In rats, the time-course of the expression of transporter proteins was studied 14, 28, and 56 days after experimental induction of chronic pancreatitis.

Verapamil regulates activity and mRNA-expression of human beta-glucuronidase in HepG2 cells.

A promising development in tumor therapy is the application of non-toxic prodrugs from which the active cytostatic is released by endogenous enzymes such as beta-glucuronidase (beta-gluc). Regulation of beta-gluc expression is one crucial factor modulating bioactivation of prodrugs. Recent experiments in rats indicate regulation of beta-gluc activity by the calcium channel blocker verapamil.

Busulfan induces activin A expression in vitro and in vivo: a possible link to venous occlusive disease.

Purpose: Hepatic venous occlusive disease is a severe side effect after administration of busulfan before hematopoietic stem cell transplantation. The syndrome is characterized by liver enlargement, fluid retention, jaundice, and weight gain. Endothelial injury has been described as the precipitating factor.

Functional significance of a hereditary adenine insertion variant in the 5'-UTR of the endothelin-1 gene.

Endothelin-1 (ET-1) is known as a potent vasoconstrictor peptide and stimulator of cell proliferation. The human preproendothelin-1 mRNA contains a frequent adenine insertion polymorphism (allele frequency = 0.28) within the 5'-untranslated region (5'-UTR), 138 bp downstream of the transcription start site, which was assumed to be related to hypertension.

Overexpression of glutathione S-transferase A1-1 in ECV 304 cells protects against busulfan mediated G2-arrest and induces tissue factor expression.

1. The antineoplastic drug busulfan is frequently used in preconditioning regimens for bone marrow transplantation. Pharmacokinetics vary tremendously between patients due to extensive metabolism in the liver via conjugation to glutathione catalysed by glutathione S-transferase (GST) A1-1.

The effects of the human MDR1 genotype on the expression of duodenal P-glycoprotein and disposition of the probe drug talinolol.

Background And Objectives: A single-nucleotide polymorphism (SNP) of the human multidrug-resistance gene in wobble position of exon 26 reportedly predicts expression and function of P-glycoprotein in human enterocytes and lymphocytes. Several other allelic variants of MDR1 have been identified, some of which lead to amino acid exchange with as yet unknown functional relevance.

Methods: In healthy white volunteers, we investigated the influence of the hereditary variants C3435T in exon 26 and G2677T/A (Ala893Ser/Thr) in exon 21 and the influence of 7 frequent or putative functional SNPs on duodenal MDR1 messenger ribonucleic acid (n = 32) and immunoreactive P-glycoprotein (n = 37) expression.

Expression and localization of P-glycoprotein in human heart: effects of cardiomyopathy.

ABC-type transport proteins, such as P-glycoprotein (P-gp), modify intracellular concentrations of many substrate compounds. They serve as functional barriers against entry of xenobiotics (e.g.

Effect of levothyroxine administration on intestinal P-glycoprotein expression: consequences for drug disposition.

Objective: Thyroid function alters the pharmacokinetics of many drugs; one example is the cardiac glycoside digoxin. Because digoxin disposition is affected by intestinal expression of P-glycoprotein, we hypothesized that thyroid hormones may regulate P-glycoprotein and influence disposition of P-glycoprotein substrates.

Methods: Duodenal expression of P-glycoprotein measured by reverse transcriptase-polymerase chain reaction of MDR1 messenger ribonucleic acid (mRNA) and by immunohistochemical examination was studied in 8 healthy volunteers (4 men and 4 women; age range, 22-29 years; body weight, 59-89 kg) before and after coadministration with levothyroxine (200 microg orally for 17 days), which resulted in suppression of thyroid-stimulating hormone.

Expression of active human beta-glucuronidase in Sf9 cells infected with recombinant baculovirus.

Antibody directed enzyme prodrug therapy (ADEPT) using glucuronide prodrugs is an experimental approach to reduce systemic toxicity of anti-cancer agents. Bioactivation of such prodrugs is achieved by fusion proteins consisting of targeting moieties (e.g.

Verapamil decreases glucuronidase activity in the gut.

The present investigation addressed the role of verapamil for oral pharmacokinetics of morphine-6-beta-glucuronide (M6G). Male Sprague-Dawley rats received 62.5 mg kg(-1) M6G-dihydrate orally w/wo pre-treatment with 70 mg kg(-1) verapamil.

Dose optimization of a doxorubicin prodrug (HMR 1826) in isolated perfused human lungs: low tumor pH promotes prodrug activation by beta-glucuronidase.

HMR 1826 (N-[4-beta-Glucuronyl-3-nitrobenzyl-oxycarbonyl]doxorubicin) is a nontoxic glucuronide prodrug from which active doxorubicin is released by beta-glucuronidase. Preclinical studies aimed at dose optimization of HMR 1826, based on intratumoral pharmacokinetics, are important to design clinical studies. Using an isolated perfused human lung model, the uptake of doxorubicin into normal tissue and tumors after perfusion with 133 microg/ml (n = 6), 400 microg/ml (n = 10), and 1200 microg/ml (n = 6) HMR 1826 was compared.

Regulation of human beta-glucuronidase by A23187 and thapsigargin in the hepatoma cell line HepG2.

A novel approach to reducing organ toxicity of anticancer agents is the application of nontoxic glucuronide prodrugs from which the active drug is released by human beta-glucuronidase, an enzyme present at high levels in many tumors. In view of high interindividual variability in beta-glucuronidase expression, regulation of this enzyme is an essential factor modulating bioactivation of glucuronide prodrugs. However, data on regulation of human beta-glucuronidase expression are not available.

[Pharmacokinetics of cyclophosphamide, adriamycin and adriamycin prodrug (HMR 1928) using an ex vivo isolated perfused human lung model (IHLP)].

Background: Today knowledge about pharmacokinetics of anticancer drugs in human malignant tumors is poor. Data from in vivo studies are limited and difficult to obtain due to ethical aspects. An ex vivo isolated perfused and ventilated human lung model however allows pharmacological studies of human bronchial carcinoma inside their host organ, the lung, under physiological conditions without compromising the patient.

Expression and function of beta-glucuronidase in pancreatic cancer: potential role in drug targeting.

Improvement of non-surgical strategies is a pivotal task in the treatment of pancreatic cancer. Response to treatment with most anticancer agents has been very poor, probably due to insufficient drug concentration in tumor tissue. Increased response rates during chemotherapy might be achieved by dose escalation; however, this approach is often hampered by severe side effects.

Does pH 6 beta-galactosidase activity indicate cell senescence?

Apparently two forms of beta-galactosidase (beta-GAL) in cells or tissue sections can be detected by enzyme histochemical staining (X-GAL). Using a sensitive and specific HPLC method we have determined the pH dependent activity of beta-GAL in cell lines of lung carcinoma (A549), colon carcinoma (Caco2-TC7), promyelocytic leukemia (HL60), hepatoma (HepG2) and human liver homogenates. The HPLC method has been validated and the influence of pH and substrate concentration was studied.

Quantitative immunohistochemical analysis of the glutathione S-transferase GSTM1: in situ phenotyping in archival material.

1. GSTM1 is present in only approximately 50% of Caucasian individuals and deficiency of GSTM1 is associated with susceptibility to a growing number of diseases, especially cancer. Thus, a method that would allow accurate, retrospective determination of the GSTM1 phenotype in different patient populations would have many applications.

Determination of tetrahydrothiophene formation as a probe of in vitro busulfan metabolism by human glutathione S-transferase A1-1: use of a highly sensitive gas chromatographic-mass spectrometric method.

A method for the sensitive determination of tetrahydrothiophene (THT) in cytosolic incubation mixtures was developed. Busulfan conjugation with glutathione was predominantly catalysed by glutathione S-transferase A1-1 (GST A1-1) and THT was released from the primary metabolite by alkalization. After liquid-liquid extraction using n-pentane separation and quantification of the product was performed by gas chromatography with a mass-selective detector.

In vitro cleavage of paracetamol glucuronide by human liver and kidney beta-glucuronidase: determination of paracetamol by capillary electrophoresis.

A capillary electrophoresis (CE) method was developed using paracetamol glucuronide as a novel probe for human beta-glucuronidase activity. Using UV detection without prior sample clean-up procedures, fast and reliable quantitation of the released paracetamol was possible. The method showed good precision, accuracy and sensitivity with a limit of detection of 0.

Simultaneous high-performance liquid chromatographic determination of a glucuronyl prodrug of doxorubicin, doxorubicin and its metabolites in human lung tissue.

A rapid and sensitive method was developed for the simultaneous determination of the new doxorubicin glucuronide prodrug HMR 1826, the parent drug doxorubicin and its metabolites in human lung tissue samples. Homogenization of frozen tissue samples with the micro-dismembrator was followed by a silver nitrate precipitation step. By removing the exceeding silver ions with sodium chloride further purification steps could be omitted.

Elucidation of the mechanism enabling tumor selective prodrug monotherapy.

Elucidation of the mechanism enabling tumor selective PMT in vivo with appropriate glucuronyl-spacer-doxorubicin prodrugs, such as HMR 1826, is important for the design of clinical studies, as well as for the development of more selective drugs. Enzyme histochemistry, immunohistochemistry, and the terminal deoxytransferase technique were applied using human cryopreserved cancer tissues, normal human, monkey, and mouse tissues, and human tumor xenografts to examine mechanisms underlying the selectivity of successful PMT with HMR 1826. It could unambiguously be shown by enzyme histochemistry that necrotic areas in human cancers are the sites in which lysosomal beta-glucuronidase is liberated extracellularly in high local concentrations.

The role of beta-glucuronidase in drug disposition and drug targeting in humans.

Glucuronides of drugs often accumulate during long term therapy. The hydrolysis of glucuronides can be catalysed by beta-glucuronidase, an enzyme expressed in many tissues and body fluids in humans. The possible contribution of beta-glucuronidase to drug disposition in humans has not been assessed in a systematic manner, but this enzyme may be able to release, locally or systemically, the active or inactive parent compound from drug glucuronides, thereby modifying the disposition and action of these drugs.

Enhanced uptake of doxorubicin into bronchial carcinoma: beta-glucuronidase mediates release of doxorubicin from a glucuronide prodrug (HMR 1826) at the tumor site.

Lack of tumor selectivity is a severe limitation of cancer chemotherapy. Consequently, reducing dose-limiting organ toxicities such as the cardiac toxicity of doxorubicin (Dox) is of major clinical relevance. Approaches that would facilitate a more tumor-selective anticancer therapy by using nontoxic prodrugs that are converted to active anticancer agents at the tumor site have been the subject of intensive research.