Pulmonary delivery and tissue distribution of aerosolized antisense 2'-O-Methyl RNA containing nanoplexes in the isolated perfused and ventilated rat lung.

Pulmonary delivery of drugs, particularly in the treatment of lung cancer, is an attractive strategy for future targeted therapy. In this context, inhalation of nanoplexes might offer a new mode for drug delivery in gene therapy. However, limited data are currently available demonstrating pulmonary delivery, cellular uptake as well as local tolerability in lung tissue. The aim of this study was to elucidate the pulmonary delivery, tissue distribution and local tolerability of aerosolized chitosan-coated poly(lactide-co-glycolide) based nanoplexes containing antisense 2'-O-Methyl RNA (OMR). Therefore, an aerosol of OMR-nanoplexes or OMR alone was administered intra-tracheally using the model of the isolated perfused and ventilated rat lung. Localization of OMR in rat lung tissue was examined by immunohistochemistry. Administration of the OMR-nanoplex formulation resulted in significantly higher cellular OMR uptake of the respiratory epithelium in contrast to the administration of OMR alone, indicating that drug administration via aerosolized nanoplexes is able to target lung tissue. No prominent changes in lung physiology parameters were observed following inhalation, suggesting good local tolerability of OMR-nanoplex formulation.

Treatment of lung cancer via telomerase inhibition: self-assembled nanoplexes versus polymeric nanoparticles as vectors for 2'-O-Methyl-RNA.

Antisense oligonucleotide, 2'-O-Methyl-RNA (OMR), is known as potent telomerase inhibitor for the treatment of lung cancer but limited by poor intracellular uptake. Chitosan-coated polymeric nanoparticles were compared to chitosan solution as non-viral vectors for OMR. The study investigated the role of chitosan properties and concentration in improving the efficiency of the nanocarriers in terms of loading, viability, cellular uptake, and telomerase inhibition in human lung cancer cell lines. Certain concentration of chitosan on nanoparticle surface is necessary to significantly increase the cellular uptake. However, excessive chitosan negatively affected the transfection efficiency. Self-assembled nanoplexes with chitosan polymer are preferentially adsorbed to the cell membrane rather than being internalized. Thus, polymeric nanoparticles proved to be superior to cationic polymers as carrier for antisense oligonucleotides. Charge cannot be considered the principle factor behind improved transfection. Uptake studies carried out on air-interface cell cultures to mimic in vivo conditions supported the results on normal cultures showing enhanced uptake of nanoplexes over naked oligonucleotides. OMR nanoplexes reduced telomerase activity by ∼50% in A549 cells concluding the potential of the system as a safe, non-invasive, and efficient treatment for lung carcinoma. These data are prerequisites for the ongoing studies on lung perfusion model and in vivo experiments.

Activity levels of tamoxifen metabolites at the estrogen receptor and the impact of genetic polymorphisms of phase I and II enzymes on their concentration levels in plasma.

The therapeutic effect of tamoxifen depends on active metabolites, e.g., cytochrome P450 2D6 (CYP2D6) mediated formation of endoxifen. To test for additional relationships, 236 breast cancer patients were genotyped for CYP2D6, CYP2C9, CYP2B6, CYP2C19, CYP3A5, UGT1A4, UGT2B7, and UGT2B15; also, plasma concentrations of tamoxifen and 22 of its metabolites, including the (E)-, (Z)-, 3-, and 4'-hydroxymetabolites as well as their glucuronides, were quantified using liquid chromatography-tandem mass spectrometry (MS). The activity levels of the metabolites were measured using an estrogen response element reporter assay; the strongest estrogen receptor inhibition was found for (Z)-endoxifen and (Z)-4-hydroxytamoxifen (inhibitory concentration 50 (IC50) 3 and 7 nmol/l, respectively). CYP2D6 genotypes explained 39 and 9% of the variability of steady-state concentrations of (Z)-endoxifen and (Z)-4-hydroxytamoxifen, respectively. Among the poor metabolizers, 93% had (Z)-endoxifen levels below IC90 values, underscoring the role of CYP2D6 deficiency in compromised tamoxifen bioactivation. For other enzymes tested, carriers of reduced-function CYP2C9 (*2, *3) alleles had lower plasma concentrations of active metabolites (P < 0.004), pointing to the role of additional pathways.

The influence of chitosan content in cationic chitosan/PLGA nanoparticles on the delivery efficiency of antisense 2'-O-methyl-RNA directed against telomerase in lung cancer cells.

Tailorable cationic chitosan/PLGA nanoparticles (CPNP) were used for the delivery of an antisense 2'-O-methyl-RNA (2OMR) directed against RNA template of human telomerase. Here, we describe the influence of the chitosan content on binding efficiency, complex stability, uptake in different human lung cell types and finally demonstrate the efficacy of this nanoplex system. CPNPs were prepared by the emulsion-solvent evaporation method using different amounts of chitosan and purified by preparative size exclusion chromatography. The characterization by photon correlation spectroscopy and zeta potential measurements showed a small increase in size and an increase of zeta potential with increasing amounts of chitosan. Binding efficiency and complex stability with 2OMR was high in water and correlated well with the chitosan content of particles but was weak in physiologically relevant media (PBS and RPMI cell culture medium). However, flow cytometry analysis showed that the uptake of 2OMR into A549 lung cancer cells was considerably higher in combination with nanoparticles and dependent on the amount of chitosan when compared to 2OMR alone. Confocal laser scanning microscopy revealed that the uptake into A549 cells is mediated via complexes of 2OMR and chitosan/PLGA nanoparticles despite the weak binding in cell culture medium. The nanoparticles were well tolerated and efficient in inhibiting telomerase activity.

Decomposition of the telomere-targeting agent BRACO19 in physiological media results in products with decreased inhibitory potential.

The stability of the acridine-based telomere-targeting agent BRACO19, a G-quadruplex stabilizing substance, was tested at different pH, temperature and in different dissolution media. Analysis was performed by HPLC. Decomposition products were examined by LC/MS and NMR. The TRAP assay was used to determine the inhibitory potential of the decomposition products on telomerase activity. The results show that the stability of BRACO19 strongly depends on pH and temperature. Decomposition was fastest at physiological pH and temperature while the type of dissolution medium had no major influence on stability. The most probable mechanism for this decomposition seems to be a hydrolysis of the amide bonds in position 3 and 6 of the acridine ring and/or a deamination of the phenyl ring. The decomposition products showed a reduced inhibitory potential compared to the parent compound BRACO19. The results demonstrate that the preparation of dosage forms and their storage conditions will have an important influence on the stability--and hence biological efficacy--of BRACO19 and related substances.

Biopharmaceutical characterization of the telomerase inhibitor BRACO19.

PURPOSE: To characterize the telomerase inhibitor and G-quadruplex stabilizing substance 9-[4-(N,N-dimethylamino)phenylamino]-3,6-bis (3-pyrrolodino-propionamido) acridine x 3HCl (BRACO19) in terms of biopharmaceutical properties such as solubility, protein binding, interaction with membrane lipids, cytotoxicity, and permeability across pulmonary epithelial cells. METHODS: Protein binding and interaction with membrane lipids were investigated by two high-performance liquid chromatography methods with immobilized human serum albumin and immobilized phosphatidylcholine, respectively. Cytotoxicity (methyl-thiazolyl-tetrazolium assay) and transport studies were performed with the bronchial cell lines 16HBE14o- and Calu-3, primary human alveolar epithelial cells, and the intestinal cell line Caco-2. Transport experiments were also done in the presence of cyclosporin A (10 microM) and tetraethylammonium chloride (5 mM) and at low temperature (4 degrees C). RESULTS: BRACO19 has good solubility of at least 2 mg/mL in water and in physiological buffers of pH 7.4 and below. Protein binding to human serum albumin was 38%. No interaction with membrane lipids could be found. Cytotoxicity in 16HBE14o-, Calu-3, and human alveolar epithelial cells was in the range of IC50 = 3.5 to 13.5 microM. Caco-2 cells were not affected at concentrations up to 50 microM. No transport of BRACO19 was detected across either cell monolayer in absorptive direction. In secretory direction, permeability was very low, with P (app) values in the range of 0.25 x 10(-7) to 0.98 x 10(-7) cm/s for all epithelial cell cultures tested. The transport was not influenced by cyclosporin A or tetraethylammonium chloride or at 4 degrees C, indicating that no efflux/influx systems or active transport are involved. CONCLUSIONS: From these results, we conclude that the very poor permeability of BRACO19 is its main biopharmaceutical limitation. Further applications will require a suitable formulation to warrant adequate delivery across cellular barriers.

Combined cytotoxic action of Viscum album agglutinin-1 and anticancer agents against human A549 lung cancer cells.

BACKGROUND: Viscum album agglutinin-1 (VAA-1) is assumed to be the biologically most active ingredient of misteltoe extracts that are often used as adjuvant cancer therapy. To develop new approaches for lung cancer treatment, we evaluated the antineoplastic activity of VAA-1 alone and in combination with other chemotherapeutic drugs, including doxorubicin, cisplatin and taxol in the human lung carcinoma cell line A549. MATERIALS AND METHODS: Cytotoxicity was determined by 5-bromo-2'-deoxyuridine (BrdU) ELISA-assays and drug interaction assessed by the isobologram method. Analysis of cell cycle distribution was obtained using flow cytometry. RESULTS: For all drug combinations tested the outcome was additive with the combination of VAA-1 and cycloheximide showing strong synergistic effects. Moreover, VAA-1 induced G1-phase accumulation mechanisms without causing apoptosis. CONCLUSION: Our findings suggest that the simultaneous administration of VAA-1 with all anticancer agents tested is advantageous since cytotoxic effects are enhanced. These data may provide new clinicalperspectives in future mistletoe therapy.

Sensitive and rapid quantification of busulfan in small plasma volumes by liquid chromatography-electrospray mass spectrometry.

BACKGROUND: High-dose busulfan is widely used in conditioning regimens before hematopoietic stem cell transplantation in both adults and children. Large interindividual variability in pharmacokinetics after oral administration has been reported; therefore, therapeutic drug monitoring of busulfan may decrease the incidence of drug-related toxicity (for example, hepatic venoocclusive disease) and may also improve therapeutic efficacy. METHODS: Busulfan concentrations were quantified using 200 microL of plasma and liquid-liquid extraction with diethyl ether after the addition of [2H8]busulfan as the internal standard. Separation and detection of busulfan and [2H8]busulfan were achieved with a LUNA C8 column (5 microm; 150 x 2 mm i.d.) at 30 degrees C, a HP 1100 liquid chromatography system, and a HP 1100 single-quadrupole mass spectrometer. Busulfan and [2H8]busulfan were detected as ammonium adducts in selected-ion monitoring mode at m/z 264.2 and 272.2, respectively. RESULTS: The calibration curve was linear at 5-2000 microg/L busulfan. Intra- and interassay imprecision (CV) and bias were both <11%. The limits of detection and quantification were 2 and 5 microg/L, respectively. Extraction recovery of busulfan was >87%. Analysis of pharmacokinetics in four patients receiving high-dose busulfan indicated that minimum busulfan concentrations before the next dose were 405-603 microg/L, with no interference observed. CONCLUSIONS: The new rapid and sensitive liquid chromatographic-mass spectrometric assay is an appropriate method for quantification of busulfan in human plasma, making therapeutic drug monitoring of busulfan faster and easier in clinical practice.

Microsomal epoxide hydrolase expression as a predictor of tamoxifen response in primary breast cancer: a retrospective exploratory study with long-term follow-up.

PURPOSE: It has been suggested that estrogen receptor-independent high-affinity binding sites for antiestrogens could limit their local bioavailability and response. Microsomal epoxide hydrolase (mEH) was recently shown to be a component of the antiestrogen binding site complex. We investigated whether mEH expression in primary breast tumors is related to disease outcome and to the efficacy of tamoxifen treatment. PATIENTS AND METHODS: Expression of mEH was semiquantitatively assessed by immunohistochemistry in sections prepared from archival paraffin blocks of primary breast cancers from 179 patients with a mean follow-up time of 81 months. RESULTS: Expression of mEH was correlated with poor disease outcome in all patients (P: < .01; n = 179) and in patients receiving tamoxifen (P: < .01; n = 78), but not in patients not treated with tamoxifen. Moreover, mEH was an independent prognostic factor by Cox regression analysis. CONCLUSION: The results of this first exploratory study suggest that mEH expression in primary breast cancer could be of predictive value for response to tamoxifen treatment and/or may be a novel independent prognostic factor for survival. The results are in agreement with the model that mEH participates in an estrogen receptor-independent tamoxifen- binding complex.

Distribution of microsomal epoxide hydrolase in humans: an immunohistochemical study in normal tissues, and benign and malignant tumours.

Microsomal epoxide hydrolase is a biotransformation enzyme which is involved in the hydrolysis of various epoxides and epoxide intermediates. In the present study, its distribution was investigated in both normal human tissues and human tumours of different histogenetic origin using immunohistochemical techniques. In normal tissue, epithelial cells were more often and more intensely immunostained than mesenchymal cells. The main epithelial cell types expressing microsomal epoxide hydrolase were hepatocytes, acinus cells of the pancreas, and cells of salivary and adrenal glands. Immunostained cells of mesenchymal origin included monocytes, fibrocytes, fibroblasts, vessel endothelium, muscle cells, and cells of the reproductive system. Three patterns of expression were observed in tumour tissues: (1) moderate or strong in hepatocellular carcinomas, tumours of the adrenal gland, and theca-fibromas of the ovary; (2) inhomogeneous staining pattern of variable intensity in breast cancer, lung cancer, colorectal carcinomas, carcinoid tumours, and some tumours of mesenchymal origin; and (3) no expression in malignant melanomas, malignant lymphomas, and renal carcinomas. These data indicate that microsomal epoxide hydrolase expression is not restricted to tissue of any particular histogenetic origin. Nonetheless, immunohistochemical identification of microsomal epoxide hydrolase may be helpful in some well-defined histological settings, for example, confirmation of hepatocellular carcinoma.

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. One strategy to overcome these adverse effects is application of nontoxic glucuronide prodrugs from which the active moiety is released by beta-glucuronidase within or near the tumor. The use of glucuronide prodrugs in pancreatic cancer requires increased expression of the enzyme in the diseased tissue, a problem that has not been addressed so far. We therefore investigated function and expression of beta-glucuronidase in tissue samples from human healthy pancreas (n=7) and pancreatic adenocarcinoma (n=8), respectively. Comparing the ability of tissue homogenates to cleave the standard substrate 4-methylumbelliferyl-beta-D-glucuronide, we found a significantly increased specific beta-glucuronidase activity (P<0.05) in pancreatic cancer (median: 133; 75% percentile: 286; 25% percentile: 111 nmol/mg per h) as compared to healthy pancreas (median: 74; 75% percentile: 113; 25% percentile: 71 nmol/mg per h). Enzyme kinetic experiments with the model prodrug N-[4-beta-glucuronyl-3-nitrobenzyloxycarbonyl] doxorubicin (HMR 1826) demonstrated bioactivation of HMR 1826 by pancreatic beta-glucuronidase. Enzymatic activity was found to be closely related to enzyme contents (r=0.87) as assessed by Western blot analysis. Our data indicate that increased beta-glucuronidase activity in pancreatic cancer seems to be due to an elevated steady-state level of the protein. This may be the basis for new therapeutic strategies in treatment of pancreatic carcinoma by using glucuronide prodrugs of anticancer agents.

[Pharmacokinetics of cyclophosphamide, adriamycin and adriamycin prodrug (HMR 1928) using an ex vivo isolated perfused human lung model (IHLP)]. / Pharmakokinetik von Cyclophosphamid, Adriamycin und Adramycin-Prodrug (HMR 1826) im ex-vivo-isolierten perfundierten humanen Lungenresektionsmodell (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. METHODS: Following surgery for bronchial carcinoma human lung preparations were reperfused and ventilated extracorporally for 2-3 hours. During the reperfusion anticancer drugs are added to the perfusion solution and their uptake into tumor, normal lung tissue, and lymph nodes is studied. RESULTS: An initial study showed that lung reperfusion under physiological circumstances over a period of 2-3 hours did not interfere with histo-pathological diagnostics and staging; an important precondition for potential adjuvant treatment. Pharmacokinetics of cyclophosphamid, adriamycin, and a water soluble adriamycin prodrug (HMR 1826) were measured. Final tissue concentrations of adriamycin and cyclophosphamid in peripheral lung parenchyma turned out to be 10 times higher compared to tumor tissue. However, following perfusion with adriamycin prodrug final tissue concentrations of adriamycin were in the same range in lung and tumor. CONCLUSIONS: The ex-vivo isolated human lung perfusion model (IHLP) has proven to be an ideal scientific model for pharmacological investigations of human tumors as an intermediate step between cell culture and in-vivo situation without any disadvantage for the patient. The tumor-to-host interaction is completely saved in this model. However, first pass reactions of drugs in other organs must not play a role for the substances studied with the IHLP. The role and future applications of the isolated perfused human lung model for other indications is discussed.

Ligands for Viscum album agglutinin and galectin-1 in human lung cancer: is there any prognostic relevance?

Viscum album agglutinin (VAA) is an extract component of mistletoe. It belongs to the plant lectin family and exerts various biological effects such as cytotoxic properties for tumor cells in culture. VAA as well as galectin-1, an endogenous lectin, possess galactose-specific surface-binding sites. We therefore investigated 159 cases of lung cancer for their capacity to bind VAA and galectin-1 and for Lewis antigen reactivity. Three different methods were used for detection of VAA: a two-step method with biotinylated VAA; an immune complex three-step method, and a four-step method. The most sensitive results were obtained with the four-step method utilising VAA, a goat-anti-VAA antibody and a biotinylated rabbit-anti-goat antibody. Intensity and distribution of staining were assessed using an immunoreactive score index (0-12). Approximately 70% of all tumors exhibited moderate to strong binding capacity for VAA. Adenocarcinomas and bronchiolo-alveolar carcinomas were more frequently labeled than squamous carcinomas. No relationship between expression of binding sites for VAA and galectin-1 as well as of Lewis antigens was found. Moreover, there was no correlation between VAA-binding capacity and survival, whereas expression of galectin-1-binding sites was of prognostic significance. Patients showing expression of galectin-1-binding sites revealed a better prognosis than those lacking binding sites or showing a weak reactivity (P = 0.0257 log rank test of Kaplan-Meier statistics).

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. 2. Developed, therefore, is a quantitative, image-analysis-based immunohistochemical technique for the analysis of GSTM1 protein in paraffin-embedded tissue samples. It was applied to the determination of the GSTM1 phenotype using liver biopsies taken from 70 patients. 3. Of the 70 cases (depending on the cut-off point), 51-54% were deficient in GSTM1. A single 27 kD band characteristic for GSTM1 was found in seven of 16 cases analysed by Western blotting using the same GSTM1 antibody as in the immunohistochemical analysis. There was a good correlation (r = 0.87) between the staining intensity of the GSTM1 band and the staining intensity evaluated by immunohistochemistry. 4. It is concluded that this quantitative immunohistochemical method permits accurate determination of the GSTM1 phenotype and is well suited for retrospective analysis of GSTM1 expression in specific tissues in situ.

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. Compounds were separated by isocratic high-performance liquid chromatography on a LiChrospher 100 RP18 column and a mobile phase consisting of citric acid buffer-acetonitrile-methanol-tetrahydrofuran within 30 min and quantified with fluorescence detection. The method showed good recoveries for all compounds (86-99%) and a linear calibration range of 20 ng/g-80 microg/g for doxorubicin and 1-600 microg/g for HMR 1826.

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. One potential method to overcome the cardiac toxicity of Dox is to apply a nontoxic, glucuronide prodrug (HMR 1826) from which Dox is released by the action of beta-glucuronidase, an enzyme present at high levels in many tumors. Using a recently developed, isolated, perfused human lung model, we compared the uptake of Dox into normal lung and lung tumors after a 2.5-h lung perfusion with doxorubicin (n = 8) and with the novel doxorubicin glucuronide prodrug (n = 8). Dox showed a poor uptake into lung tumors as compared with normal lung [mean Dox concentration at the end of perfusion, 1.78 +/- 3.11 (median, 0.66) microg/g versus 22.03 +/- 10.4 (median, 18.5) microg/g; P < 0.001]. However, after perfusion with HMR 1826, the level of Dox in tumor tissue was about 7-fold higher than after perfusion with Dox itself [14.04 +/- 12.9 (median, 12.9) microg/g versus 1.78 +/- 3.11 (median, 0.66) microg/g, P < 0.05, n = 8]. In vitro experiments showed a significantly higher beta-glucuronidase expression and activity in the tumors. The extent of in vitro cleavage of HMR 1826 by homogenized lung tissue was closely related to the content of beta-glucuronidase (r = 0.9834, P < 0.0001). When D-saccharolactone, a specific inhibitor of beta-glucuronidase, was added to the perfusate containing HMR 1826, no accumulation of Dox in lung tissue was seen. These data indicate that the high Dox levels achieved in the tumors with HMR 1826 resulted from cleavage of the prodrug by beta-glucuronidase at the tumor site. Thus, the problem of poor Dox uptake into lung tumors could be circumvented by applying the doxorubicin glucuronide prodrug. Several lines of evidence based on both ex vivo and in vitro results indicate that the approach described using a glucuronide prodrug may be useful in facilitating more selective delivery of chemotherapy to tumors in humans.

Interindividual variability in expression and activity of human beta-glucuronidase in liver and kidney: consequences for drug metabolism.

Glucuronidation of drugs represents a major pathway of human drug metabolism. Numerous studies show that the glucuronides formed can accumulate during chronic therapy and/or have direct pharmacological activity. In both cases, cleavage of the glucuronide by human beta-glucuronidase (beta-Gluc) would release the parent compound, thereby modifying drug disposition. Variability in expression of beta-Gluc could therefore be a confounding factor for interindividual variability in drug disposition both in the setting of accumulating glucuronides or for the use of glucuronides as prodrugs, such as the nontoxic glucuronide-spacer derivative of doxorubicin (Dox-S-G). We therefore investigated expression and function of beta-Gluc in human liver (n = 30) and human kidney (n = 18). Cleavage of the model compound 4-methylumbelliferyl-beta-D-glucuronide (MUG) revealed a wide range of activities in liver (0.32-1.85 mumol/mg/h, mean value 0.87 +/- 0.34 mumol/mg/h) and kidney (0.07-1.00 mumol/mg/h, mean 0.39 +/- 0.21 mumol/mg/h), which followed a log normal distribution. Variable enzyme activity was closely correlated to enzyme expression as assessed by Western blotting (r = 0.80, P < .001 and r = 0.71, P < .05 for liver and kidney, respectively). Glycyrrhizin (Ki = 470 and 570 microM), estradiol 3-glucuronide (Ki = 0.9 and 1.2 mM) and paracetamol glucuronide (Ki = 1.6 and 2 mM) were found to inhibit beta-Gluc activity competitively in liver and kidney, respectively. Enzyme kinetics were investigated in detail for MUG and Dox-S-G. Whereas MUG followed monophasic Michaelis-Menten kinetics in liver (K(m) = 1.32 +/- 0.25 mM, Vmax = 1201 +/- 462 nmol/mg/h, n = 3) and kidney (K(m) = 1.04 +/- 0.05 mM, Vmax = 521 +/- 267 nmol/mg/h, n = 3), cleavage of Dox-S-G was best described by the Hill equation, which indicated a cooperative substrate binding pattern of Dox-S-G. In summary, beta-Gluc function shows wide interindividual variability in human liver and kidney that is due to different steady-state levels of the enzyme. Moreover, enzyme kinetics are substrate-dependent, with Dox-S-G showing a cooperative binding. These data indicate the possibility of wide interindividual variability in beta-Gluc-mediated cleavage of drug glucuronides in the human.