Manipulating the extracellular matrix: an animal model of the bladder pain syndrome.

Bladder pain syndrome (BPS) is associated with breakdown of the protective uroepithelial barrier of the urinary bladder allowing urinary constituents access to bladder sensory neurons. Although there are several animal models of cystitis, none specifically relates to BPS. Here, we aimed to create such a model using enzymatic digestion of the barrier proteoglycans (PGs) in the rat. Twenty female Wistar rats were anaesthetized and transurethrally catheterized. Ten animals were treated with 0.25IU of intravesical chondroitinase ABC and heparanase III to digest chondroitin sulphate and heparin sulphate PGs, respectively. Ten animals received saline. Following PG deglycosylation, bladders showed irregular loss of the apical uroplakin and a significant increase in neutrophils, not evident in the control group. Spinal cord sections were also collected for c-fos analysis. A large and significant increase in fos immunoreactivity in the L6/S1 segments in the treatment vs control bladders was observed. Cystometry was performed on 5 treatment and 5 control animals. Analysis revealed a significant increase in micturition reflex excitability postdeglycosylation. On a further group of 10 animals, von Frey mechanical withdrawal thresholds were tested on abdominal skin before and after PG digestions. There was a significant decrease in abdominal mechanical withdrawal threshold postdeglycosylation compared with controls. The results of this animal study suggest that many of the clinical features of BPS are seen after PG digestion from the bladder lumen. This model can be used to further understand mechanisms of pain in patients with BPS and to test new therapeutic strategies.

Pharmacokinetics and pharmacodynamics of ketoprofen in calves applying PK/PD modelling.

The pharmacokinetics (PK) and pharmacodynamics (PD) of ketoprofen (KTP) were studied in calves following intravenous administration of the drug racemate at a dose rate of 3 mg/kg. To evaluate the anti-inflammatory properties of KTP, a model of acute inflammation, consisting of surgically implanted subcutaneous tissue cages stimulated by intracaveal injection of carrageenan, was used. No differences were observed between disposition curves of KTP enantiomers in plasma, exudate or transudate. This indicates that in calves KTP pharmacokinetics is not enantioselective. S(+)- and R(-)- KTP each had a short elimination half-life (t1/2 beta) of 0.42 +/- 0.08 h and 0.42 +/- 0.09 h, respectively. The volume of distribution (Vd) was low, values of 0.20 +/- 0.06 L/kg and 0.22 +/- 0.06 L/kg being obtained for R(-) and S(+)KTP, respectively. Body clearance (ClB) was high, correlating with the short elimination half-life, 0.33 +/- 0.03 L/kg/h [R(-)KTP] and 0.32 +/- 0.04 L/kg/h [S(+)-KTP]. KTP pharmacodynamics was evaluated by determining the effects on serum thromboxane (TxB2), exudate prostaglandin (PGE2), leukotriene (LTB4) and beta-glucuronidase (beta-glu) and bradykinin (BK)-induced oedematous swelling. Effect-concentration inter-relationships were analysed by PK/PD modelling. KTP did not affect exudate LTB4, but inhibition of the other variables was statistically significant. The mean EC50 values for inhibition of serum TxB2, exudate PGE2 and beta-glu and BK-induced swelling were 0.118, 0.086, 0.06 and 0.00029 microgram/mL, respectively. These data indicate that KTP exerted an inhibitory action, not only as expected, on eicosanoid (TxB2 and PGE2) synthesis but also on exudate beta-glu and BK-induced oedema. The EC50 values for these actions indicate that they are likely to contribute to the overall anti-inflammatory effects of KTP in calves. However, claims that KTP inhibits 5-lipoxygenase and thereby blocks the production of inflammatory mediators such as LTB4 were not substantiated. PK/PD modelling has proved to be a useful tool for analysing the in vivo pharmacodynamics of KTP and for providing new approaches to elucidating its mechanism(s) of action.

Tumor-selective prodrug activation by fusion protein-mediated catalysis.

A two component system, consisting of a fusion protein and an appropriate prodrug, suited to perform selective tumor therapy in vivo is presented. The fusion protein, due to its humanized carcinoembryonic antigen-specific variable region, specifically binds to carcinoembryonic antigen-expressing tumors and has an enzymatic activity comparable to that of human beta-glucuronidase. The prodrug is a nontoxic glucuronide-spacer derivative of doxorubicin decomposing to doxorubicin by enzymatic deglucuronidation. In vivo studies in nude mice bearing human carcinoembryonic antigen-expressing tumor xenografts revealed that 7 days after injection of 20 mg/kg fusion protein a high specificity ratio (> 100:1) was obtained between tumor and plasma or tumor and normal tissues. Injection of 250 mg/kg of prodrug at day 7 resulted in tumor therapeutic effects superior to those of conventional chemotherapy without any detectable toxicity. These superior therapeutic effects which were observed using established human tumor xenografts can be explained by the approximately 4-12-fold higher doxorubicin concentrations found in tumors of mice treated with fusion protein and prodrug than in those treated with the maximal tolerable dose of drug alone. The nondetectable toxicity in the animals treated with fusion protein and prodrug is probably caused by up to 5-fold lower drug concentrations in normal tissues compared to the animals treated with doxorubicin. Thus, a more tumor-selective therapy, resulting in stronger therapeutic effects and reduced toxicity seems to be possible by the appropriate use of the humanized nontoxic fusion protein and the nontoxic prodrug.

Biological monitoring of bidi rollers with respect to genotoxic hazards of occupational tobacco exposure.

Smokeless tobacco habits are associated with a high incidence of oropharyngeal cancer in India. Hence, the biological effects of occupational exposure to smokeless tobacco used for making bidis (the Indian version of cigarettes) were studied in 2 groups of bidi rollers designated BR-K and BR-S and in control subjects with no tobacco habits. Specific tobacco exposure and the electrophilic burden were determined by estimating urinary cotinine and thioethers respectively. Urine mutagenicity was tested with the Ames assay using Salmonella typhimurium strains TA98 and TA100. While cotinine was not detected in control samples, the mean cotinine levels (mmole/mole creatinine) in the BR-K and BR-S groups were 0.79 +/- 0.30 and 0.09 +/- 0.03 respectively. Urinary thioether excretion (mmole/mole creatinine) was significantly elevated in the BR-S group 4.59 +/- 0.52; p less than 0.001) but it was lower in the BR-K group (0.54 +/- 0.08; p less than 0.001) compared to the control (1.83 +/- 0.34). Furthermore, beta-glucuronidase-treated samples from both groups of bidi rollers exhibited increased mutagenicity to TA98 compared to the control group; in addition, BR-S samples exhibited direct mutagenicity to TA98. The results show that occupational tobacco exposure modulates the glutathione conjugation pathway and increases the mutagenic burden of bidi rollers.

Mutagenicity of beta-glucuronidase in the Ames test.

The enzyme preparation beta-glucuronidase/arylsulphatase from Helix pomatia (Boehringer) caused base-pair substitutions in Salmonella typhimurium TA100 and TA1535 strains within the dose range of 0.50-50 microliter per plate. No effect was observed in the TA98 strain. The presence of S9 mix did not substantially affect the mutagenic potential of beta-G. The number of induced revertants decreased continually from experiment to experiment carried out in the course of 12 weeks.

Procedure for mutagenizing spores of Saccharomyces cerevisiae.

A procedure for inducing mutants of a homothallic strain of Saccharomyces cerevisiae is described. The essential parts of the procedure are long incubation in Glusulase, which preferentially kills vegetative cells instead of spores, and treatment in 9% ethyl methanesulfonate, which also preferentially kills vegetative cells instead of spores. Consequently, the viable population is virtually 100% spores.