The pharmacokinetics and dosing of oral 4-methylumbelliferone for inhibition of hyaluronan synthesis in mice.

Recently, there has been considerable interest in using 4-methylumbelliferone (4-MU) to inhibit hyaluronan (HA) synthesis in mouse models of cancer, autoimmunity and a variety of other inflammatory disorders where HA has been implicated in disease pathogenesis. In order to facilitate future studies in this area, we have examined the dosing, treatment route, treatment duration and metabolism of 4-MU in both C57BL/6 and BALB/c mice. Mice fed chow containing 5% 4-MU, a dose calculated to deliver 250 mg/mouse/day, initially lose substantial weight but typically resume normal weight gain after 1 week. It also takes up to a week to see a reduction in serum HA in these animals, indicating that at least a 1-week loading period on the drug is required for most protocols. At steady state, more than 90% of the drug is present in plasma as the glucuronidated metabolite 4-methylumbelliferyl glucuronide (4-MUG), with the sulphated metabolite, 4-methylumbelliferyl sulphate (4-MUS) comprising most of the remainder. Chow containing 5% but not 0·65% 4-MU was effective at preventing disease in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis, as well as in the DORmO mouse model of autoimmune diabetes. While oral 4-MU was effective at preventing EAE, daily intraperitoneal injections of 4-MU were not. Factors potentially affecting 4-MU uptake and plasma concentrations in mice include its taste, short half-life and low bioavailability. These studies provide a practical resource for implementing oral 4-MU treatment protocols in mice.

Chitotriosidase activity as additional biomarker in the diagnosis of lysosomal storage diseases.

To date, several genetic variants that lead to a deficiency of chitotriosidase activity have been described. The duplication of 24 bp (dup24bp) in exon 10 of the CHIT1 gene, which causes a complete loss of enzymatic activity of the gene product, is the most common among the European population. The aim of the study was to evaluate the possibility of using chitotriosidase activity as an additional biomarker in diagnosis of lysosomal storage diseases (LSDs) in Ukraine, to determine this parameter in blood plasma of the patients with various lysosomal diseases and to assess the effect of the presence of dup24bp in the CHIT1 gene on this parameter. It has been shown that chitotriosidase activity in blood plasma is a convenient additional biochemical marker in the diagnosis of some LSDs, namely Gaucher disease, Niemann-Pick disease A, B, C and GM1-gangliosidosis. Reference ranges of the normal chitotriosidase activity were determined in blood plasma of Ukrainian population and found to be 8.0-53.1 nmol 4-methylumbelliferone/h·ml of plasma. The total allele frequency of the dup24bp in the CHIT1 gene in Ukrainian population was determined, which amounted to 0.26 (323/1244) that is higher than in European population. It was indicated that moleculargenetic screening of dup24bp in the CHIT1 gene is a necessary stage in a protocol for the laboratory diagnosis of Gaucher disease, Niemann-Pick disease A, B, C as well as GM1-gangliosidosis to avoid incorrect diagnosis.

Systematic screening of human ABCC3 polymorphisms and their effects on MRP3 expression and function.

The present study was undertaken to identify genetic polymorphisms of multidrug resistance-associated protein 3 (MRP3, gene name ABCC3), an ATP-binding cassette transporter that mediates the transport of substrates across the basolateral membrane into the blood, and to investigate their effects on ABCC3 expression and MRP3 function. We identified genetic polymorphisms of ABCC3 and evaluated the effects by (1) a luciferase reporter gene assay, (2) measuring mRNA levels, and (3) a human pharmacogenomics study with 4-methylumbelliferone glucuronide (4-MUG). Overall, 61 genetic variants were identified in three ethnic populations; of these variants 17 were novel (7 were non-synonymous: 61Arg>Cys, 132Gln>Stop, 221Trp>Stop, 270His>Gln, 548Leu>Gln, 600Lys>Arg, and 1324Arg>His). However, these mutations occurred at very low frequencies (max. 4.7%). The observed allele frequencies showed considerable inter-ethnic differences. The reporter gene assay indicated a significant reduction of transcriptional activity with the -1767G>A allele compared to the wild-type allele; however, a decreased expression of ABCC3 mRNA was not detected in human liver samples. A human pharmacokinetic study showed that the ABCC3 genotype in the promoter region was not associated with changes in the pharmacokinetics of 4-MUG, a substrate of MRP3. This is the first study to assess the effects of ABCC3 polymorphisms on human pharmacokinetics; however, further investigations are needed to complete the picture.

Hemoglobin precipitation greatly improves 4-methylumbelliferone-based diagnostic assays for lysosomal storage diseases in dried blood spots.

Derivatives of 4-methylumbelliferone (4MU) are favorite substrates for the measurement of lysosomal enzyme activities in a wide variety of cell and tissue specimens. Hydrolysis of these artificial substrates at acidic pH leads to the formation of 4-methylumbelliferone, which is highly fluorescent at a pH above 10. When used for the assay of enzyme activities in dried blood spots the light emission signal can be very low due to the small sample size so that the patient and control ranges are not widely separated. We have investigated the hypothesis that quenching of the fluorescence by hemoglobin leads to appreciable loss of signal and we show that the precipitation of hemoglobin with trichloroacetic acid prior to the measurement of 4-methylumbelliferone increases the height of the output signal up to eight fold. The modified method provides a clear separation of patients' and controls' ranges for ten different lysosomal enzyme assays in dried blood spots, and approaches the conventional leukocyte assays in outcome quality.

A hyaluronan synthase suppressor, 4-methylumbelliferone, inhibits liver metastasis of melanoma cells.

4-Methylumbelliferone (MU) inhibits the cell surface hyaluronan (HA) formation, and that such inhibition results in suppression of adhesion and locomotion of cultured melanoma cells. Here, we examine the effect of MU on melanoma cell metastasis in vivo. MU-treated melanoma cells showed both decreased cell surface HA formation and suppression of liver metastasis after injection into the mice. Oral administration of MU to mice decreased tissue HA content. These HA knock-down mice displayed suppressed liver metastasis. Thus, both cell surface HA of melanoma cells and recipient liver HA can promote liver metastasis, indicating that MU has potential as an anti-metastatic agent.

Extrahepatic metabolism of 4-methylumbelliferone and lidocaine in the anhepatic rabbit.

Extrahepatic drug metabolism was studied in an anhepatic rabbit model. Plasma concentrations of 4-methylumbelliferone (4-MU) and its major metabolites, 4-methylumbelliferyl-O-glucuronide and 4-methyumbelliferyl sulfate, along with lidocaine and its major metabolites, monoethylglycinexylidide and 3-hydroxylidocaine, were measured in sham rabbits (n = 4) and anhepatic rabbits (n = 4) following bolus intravenous administration of each drug. Along with concentration profiles of the drugs and metabolites, pharmacokinetic analyses of 4-MU metabolism and lidocaine metabolism were used to assess the extrahepatic metabolism of these classical substrates. Total body clearance of 4-MU in the anhepatic rabbits was about 50% that of the sham animals. Extensive extrahepatic glucuronidation of 4-MU was revealed by comparing the AUC ratios of 4-methylumbelliferyl-O-glucuronide and 4-MU in anhepatic and sham rabbit groups. Sulfation of 4-MU was reduced significantly in the anhepatic group, although some extrahepatic sulfation was observed. Total body clearance of lidocaine was reduced 3-fold in anhepatic animals. 3-Hydroxylidocaine was only detected in plasma samples from sham animals. These results emphasize the importance of extrahepatic sites in drug metabolism, especially glucuronidation of phenolic compounds such as 4-MU.

High-performance liquid chromatographic method for the direct determination of 4-methylumbelliferone and its glucuronide and sulfate conjugates. Application to studies in the single-pass in situ perfused rat intestine-liver preparation.

A direct high-performance liquid chromatographic (HPLC) assay was developed for the separation and determination of 4-methylumbelliferone (4MU) and its glucuronide (MUG) and sulfate (MUS) conjugates in the cell-free perfusate ("plasma") from in situ perfused rat intestine-liver preparation. In addition, a procedure was developed to extract and determine 4MU in the whole blood perfusate. Perfusate plasma containing an internal standard (umbelliferone) was precipitated with methanol (1:4, v/v), and injected into a reversed-phase HPLC system with gradient elution. 4MU and the same internal standard were also extracted directly from the whole blood perfusate with ethyl acetate and injected into a reversed-phase HPLC system with isocratic elution. Inter- and intra-day precision studies (n = 5 for each) for both the plasma and whole blood procedures demonstrated relative standard deviation of less than 10% at all concentrations studied. The compounds were stable in either the plasma or blood extracts at room temperature for up to 72 h. The procedures were successfully used to analyze perfusate samples obtained from the single-pass in situ perfusion of rat intestine-liver system with either trace (0.95 nM) or 32.3 microM concentrations of 4MU. The intestine was responsible for the formation of most of the MUG formed by the intestine-liver preparation during steady-state perfusion with either input concentration of 4MU.

Characteristics of leukocyte and plasma methylumbelliferyl-alpha-L-iduronide iduronidase.

Some characteristics of the human leukocyte and plasma alpha-L-iduronidase are described. The enzyme from both sources is sufficiently stable and linear in time to allow accurate determinations. The leukocyte and plasma enzyme have a low acid pH optimum at 3.5 and 4, respectively, which is in agreement with the lysosomal origin of the enzyme in the cell. Both enzymes are inhibited by phenyl-alpha-L-iduronide, heparin, and heparitin sulfate although other mucopolysaccharides also inhibit the leukocyte enzyme. When kept frozen at -20 degrees C, the leukocyte as well as the enzyme in acidified plasma are very stable. We studied the plasma enzyme in more detail. If the plasma is acidified, iduronidase is very stable between 0 and 37 degrees C. CuCl2 and Na2SO4 were very potent inhibitors at concentrations of 10 and 100 mM, respectively. The determination of iduronidase in leukocyte homogenates of patients suspected of Hurler disease together with plasma activities is useful for confirming or corroborating the diagnosis of genetic iduronidase deficiency. Further investigation is needed to determine if the plasma enzyme test would be useful in the biochemical diagnosis of Scheie disease and the Hurler-Scheie compound, two diseases which are also caused by a deficiency of alpha-L-iduronidase.

[The liver and aging. The effect of age on liver function and drug metabolism]. / Leber und Alter. Einfluss des Lebensalters auf Leberfunktion und Arzneimittelmetabolismus.

Theophylline (oxidative phase I metabolism) is biotransformed by the liver at an increasingly delayed rate with advancing age. Hymecromone (phase II metabolism) does not display any dependence of glucuronidisation on age. The indocyanine green half-life and the galactose elimination capacity show a highly significant dependence on age. Drugs with flow-limited elimination or mainly oxidative phase I biotransformation should be administered with reduced dosage level while monitoring the serum concentration levels. Drugs that are transformed via phase II reactions usually do not require dosage adjustment.

Pharmacokinetic study of 4-methylumbelliferone in mice.

The pharmacokinetic behavior of 4-methylumbelliferone (4-MU), a compound known to be excreted as conjugated metabolites, was studied in mice and the results were compared with those reported in rats. Plasma half-life of the terminal phase (t1/2,beta) of mice after an intravenous administration of 4-MU was approximately one-twenty fifth of that of rats. Mice showed a dose dependency in both hepatic and extrahepatic clearances, though dose dependent hepatic clearance is reported to be the main route of elimination in rats. In the same dose range (10-25 mg/kg), the hepatic intrinsic clearance per unit body weight of mice was approximately 5 times larger than that of rats. The blood-to-plasma concentration ratio and the unbound volume of distribution of tissues of mice were smaller than those of rats. It was concluded that the shorter t1/2,beta of mice than that of rats may be due to the larger hepatic clearance, the presence of extrahepatic clearance and the smaller volume of distribution.

Kinetics of hepatic transport of 4-methylumbelliferone in rats. Analysis by multiple indicator dilution method.

Hepatic elimination of 4-methylumbelliferone (4-MU), which has been used as a model compound for conjugative metabolism, was studied by means of a multiple indicator dilution (MID) method in the isolated perfused rat liver. Using this method, three intrinsic hepatic clearances, CLint,inf, CLint,eff, and CLint,seq, which represent the influx, efflux, and sequestration processes, respectively, were obtained. When the dose was increased from a low dose (50 micrograms/rat liver) to a high dose (3000 micrograms/rat liver), the hepatic availability of 4-MU increased from 0.11 to 0.73. With increasing dose, the CLint,eff value increased approximately two times, while the CLint,seq value decreased to approximately one-third. The remarkable dose dependence of hepatic availability was due to nonlinearity in both CLint,eff and CLint,seq values. However, the CLint,inf value was almost independent of dose. The dose-dependent change in CLint,seq might be explained by the saturation of conjugative metabolism of 4-MU, while the increase in the CLint,eff value with increasing dose might be partly explained by the nonlinear tissue binding of 4-MU, since the tissue unbound fraction determined by an ultrafiltration method using liver homogenate increased approximately 1.5 times at higher concentration of 4-MU compared to that at lower concentrations. In addition, based on a comparison of the individual intrinsic clearances, i.e., CLint,inf, CLint,eff, and CLint,seq, the major determining process of the apparent hepatic intrinsic clearance of 4-MU is thought to be the sequestration process at the high dose. However, at the low dose, the membrane transport process (influx and efflux processes) as well as the sequestration process also determine the apparent hepatic intrinsic clearance.

Pharmacokinetic study of 4-methylumbelliferone in rats: influence of dose on its first-pass hepatic elimination.

The dose-dependent first-pass hepatic metabolism and pharmacokinetics of 4-methylumbelliferone (4-MU) were studied at four dose levels (17.6 micrograms-5.29 mg) in rats. 4-MU was given intravenously and intraportally to determine the availability of 4-MU. The availability increased from 0.18 to 1.31 when the dose was increased from 17.6 micrograms to 5.29 mg/rat. The total body plasma clearance of 4-MU was accounted for mostly by the hepatic conjugative metabolism. The contribution of renal clearance to total plasma clearance was 11-35%, depending on the dose. The marked dose-dependency of availability may thus be explained by the saturable conjugative metabolism of 4-MU.

Carrier detection in cystic fibrosis.

We evaluated four methods purported to distinguish between individuals homozygous or heterozygous for cystic fibrosis (CF) and normal controls: (1) detection of a protein in the serum by isoelectric focusing at pH 8.5, (2) detection of a lectinlike factor in the serum by hemagglutination, (3) isolation of CF-lectin from the serum by affinity chromatography, and (4) measurement of MUGB-reactive proteases in the plasma. The results were disappointing. The detection of CF protein by isoelectric focusing was unreliable; it could be identified in only 46% of heterozygotes and 66% of homozygotes, with a false positive rate of 17%. Detection of a lectinlike factor by hemagglutination was also found to be unreliable and irreproducible. The lectin isolated by affinity chromatography was not specific for the CF gene. No significant differences were found in the MUGB titers of the three populations tested. However, low titers (MU less than 200 nmol/ml) were found in 33% of homozygotes and heterozygotes and in 17% of normal controls. We conclude that none of these methods is suitable for carrier detection in cystic fibrosis.

Identification of the major 4-methylumbelliferyl p-guanidinobenzoate-hydrolyzing plasma protein in cystic fibrosis: implication for intrauterine and heterozygote detection.

Measurement of 4-methylumbelliferyl p-guanidinobenzoate (MUGB)-hydrolyzing activity in the plasma of normal controls, cystic fibrosis (CF) heterozygotes, and CF homozygotes did not support previously reported (35) differences in MUGB-hydrolyzing activity. We identified human plasma albumin as the major source of MUGB-hydrolyzing activity by comparison of our plasma results to those obtained with physiologic concentrations of commercial albumin samples. Substantiating evidence was obtained from gel filtration experiments and correlation of albumin levels in CF plasma with MUGB titers. We found essentially no proteolytic activity towards dinitrophenylprotamine sulfate associated with commercial albumin samples. It appears that the reaction between human albumin and MUGB represents a weak esterase activity, perhaps involving the acylation of a specific site(s) on the protein. Hypoalbuminemia has been documented (8) in some CF patients. Low albumin concentrations, indicated by MUGB titers less than 190 nmole methylumbelliferone/ml plasma, were found in 42% of CF homozygotes, 6% of heterozygotes, and 4% of controls. Gel filtration studies of a normal amniotic fluid supernatant indicated that albumin was the major MUGB-hydrolyzing substance in this fluid. We conclude that MUGB abnormalities are not associated with the basic gene defect in CF and cannot be used as the basis of a test for intrauterine or heterozygote detection.

Systematic study of the hydrolysis of 4-methylumbelliferylguanidinobenzoate in plasma from patients with cystic fibrosis and controls.

In contrast to the original reports by others, the hydrolysis of 4-methylumbelliferylguanidinobenzoate ((MUGB), and active-site titrant of serine proteases, was found not to be significantly different in plasma samples from patients with cystic fibrosis (CF), obligate heterozygotes, and normal controls. Since the steady-state turnover of substrate is the dominant term of MUGB hydrolysis, the assay in its present form does not meet the requirements of an active-site titration. The reproducibility and reliability of the assay are hampered by (1) high blank values due to non-specific hydrolysis, (2) the absence of a defined endpoint of the incubation, (3) the non-linearity of the assay with respect to plasma concentration, (4) the temperature-dependent evolution of esterase activity during incubation, and (5) most importantly the loss of activity during storage of plasma samples. Cleavage of MUGB catalysed by the imidazole ring of histidine accounts for a significant portion of the activity in plasma.

Protease deficiency in plasma of patients with cystic fibrosis. Reduced reaction of 4-methylumbelliferylguanidinobenzoate with plasma of patients with cystic fibrosis.

Protease activity in plasma is assayed using 4-methylumbelliferylguanidinobenzoate. The assay is modified by carrying out the reaction in the presence and absence of benzamidine, a competitive inhibitor of trypsin-like proteases. The parameters of the assay are described in detail. Using this assay, our earlier demonstration of a deficiency of protease activity in plasma of patients with cystic fibrosis is confirmed. The activity, corrected for the nonspecific hydrolysis of 4-methylumbelliferylguanidinobenzoate by benzamidine, is expressed as nanomoles of 4-methylumbelliferone released per milliliter plasma. Under standard conditions, the activity in plasma activated with chloroform-ellagic acid was 127.2 +/- 23.1 in 7 controls, 70.4 +/- 11.7 in 11 obligate heterozygotes, and 48.7 +/- 16.6 in 12 patients with cystic fibrosis. Identical results were obtained when unactivated plasma was used. These data demonstrate that the judicious use of specific inhibitors such as benzamidine might be useful in assaying low levels of protease activity in crude systems.