Assessment of murine bladder permeability with fluorescein: validation with cyclophosphamide and protamine.

OBJECTIVES: Bladder hyperpermeability should result in elevated blood levels of intravesically administered agents. Reabsorption from a hyperpermeable bladder should result in prolonged urinary excretion of an agent after parenteral administration. To test these hypotheses, urinary clearance and plasma levels of sodium fluorescein (NaF) were measured in mice before and during cyclophosphamide (CYP) and protamine-induced hemorrhagic cystitis. METHODS: To measure the plasma uptake of NaF from the bladder, 10 mg/mL NaF was instilled, either by catheter or retrograde urethral infusion, 15 minutes before retro-orbital or ventricular sampling. The plasma levels were measured 24 hours and 14 days after exposure to CYP 300 mg/kg or 15 minutes after instillation of protamine 10 mg/mL. Hourly urine concentrations were measured immediately after intraperitoneal administration of 10 mg/kg NaF. Pretreatment samples were compared with those obtained 24 hours after intraperitoneal administration of 300 mg/kg CYP. RESULTS: Urinary NaF excretion was delayed in CYP-exposed mice. A bi-exponential model provided an appropriate fit of the data, both before and after CYP administration. The plasma levels of NaF were significantly elevated at 24 hours and 14 days after CYP exposure when sampled by ventricular nick or retro-orbitally. The median concentration of fluorescein in the protamine-treated mice was significantly higher than in the control mice. CONCLUSIONS: Fluorescein can be used to measure alterations in bladder permeability after bladder mucosal injury in mice. Urinary excretion of NaF is a bi-exponential process that is delayed after bladder mucosal injury, presumably because of increased mucosal permeability and resorption from the urine into the bloodstream.

Stability of methylecgonidine and ecgonidine in sheep plasma in vitro.

BACKGROUND: Crack smokers are exposed to a pyrolysis product, methylecgonidine (MEG), which can be used as an analytical marker for crack smoking. Ecgonidine (EC), a hydrolytic product of MEG, has been identified in urine of crack smokers. MEG undergoes conversion to EC, complicating analysis and perhaps explaining a lack of forensic blood specimens containing MEG. METHODS: We developed gas chromatography-mass spectrometry (GC-MS) assays for MEG and EC. Plasma was collected from sheep blood containing 0, 0.06, or 0.24 mol/L (0%, 0.25%, or 1%) NaF. MEG was added to these plasmas, and they were incubated at -80, 1, 21, or 37 degrees C to determine whether there were temporal, temperature, or storage effects on MEG stability over 48 h. RESULTS: Decreased temperature and increased NaF concentrations limited MEG degradation and EC formation. MEG stored in plasma at -80 degrees C was stable up to 1 month, even in the absence of NaF. CONCLUSIONS: MEG is stable in sheep plasma collected in commercially available, evacuated blood-collection tubes containing NaF and stored at -80 degrees C. In vitro formation of EC can be minimized with appropriate sample handling, and its in vivo formation may provide a better marker of crack smoking than its parent pyrolysis product.

Generation of stable test atmospheres of cocaine base and its pyrolyzate, methylecgonidine, and demonstration of their biological activity.

Generating controlled test atmospheres of known chemical identity and airborne concentration upon demand is a significant technical obstacle that limits the scope and repeatability of studies of inhaled substances. We addressed this problem as applied to the generation of atmospheres that result from heating crack cocaine, which include both cocaine and its pyrolyzate methylecgonidine (MEG). A condensation aerosol generator was used to generate atmospheres comprised of monodisperse particles of cocaine, MEG, or mixtures of both that are of submicron size suitable for deposition in the alveolar region of primates. Compressed air seeded with nanometer-size sodium chloride particles was passed through a constant depth of molten cocaine or MEG in a bead bed, reheated, and condensed to an aerosol within an annulus of cold air. To achieve control of a mixture of both compounds, MEG was condensed onto cocaine particles in a separate coating step. On-line analytical instruments provided verification of airborne concentration, estimates of particle size, and dispersion as well as chemical identity. Specific airway conductance (SGaw), heart rate, and rectal and skin temperatures were measured in squirrel monkeys breathing atmospheres containing condensation aerosols of cocaine or MEG free base. SGaw was reduced after inhalation of either base, and both induced temperature and cardiovascular changes, demonstrating that the aerosols so generated had biological activity.

Methylecgonidine coats the crack particle.

Crack is a form of cocaine base self-administered by smoking. When heated, it volatilizes and may partially pyrolyze to methylecgonidine (MEG). Upon cooling, a condensation aerosol forms. Heating cocaine base in model crack pipes produced particles of about 1 micron in diameter, regardless of the amount heated; however, MEG concentration increased from < or = 2% at 10 mg per heating to as much as 5% at 30 mg per heating. Methylecgonidine was < or = 1% of the recovered material when cocaine was vaporized off a heated wire coil, but the particles were larger (2-5 microns), and the distribution disperse. The vapor pressure of MEG was higher [log P(mm Hg) = 9.994 - 3530/T] than cocaine base, consistent with MEG coating the droplet during condensation, and with evaporation during aging or dilution. Disappearance of MEG from a chamber filled with crack smoke was a two-component process, one proceeding at the rate of cocaine particle removal, and the other at the desorption rate from other surfaces. Particle diameter influences the deposition site in the respiratory tract; thus, the likely different patterns of deposition in the respiratory tract of humans and animals of crack aerosols produced by different techniques warrant consideration, as they may influence our understanding of immediate and delayed sequelae of the inhalation of cocaine and its pyrolysis product, MEG.

Pulmonary effects of the cocaine pyrolysis product, methylecgonidine, in guinea pigs.

The pulmonary effects of the cocaine pyrolysis product, methylecgonidine (MEG; anhydroecgonine methyl ester), were assessed in guinea pigs. Specific airway conductance (SGaw), which decreases during bronchoconstriction, was measured in guinea pigs exposed to atmospheres containing a condensation aerosol of MEG free base (13 +/- 1 mg/liter of air), nebulized MEG fumarate (3 and 12% in phosphate buffered saline) or nebulized acetylcholine chloride (0.2 and 0.4% in phosphate buffered saline). A decrease in SGaw to 24.0 +/- 4.2% (mean +/- 2 S.E.M.) of baseline levels was observed in guinea pigs breathing MEG free base. A decrease to 28.4 +/- 4.5% of baseline was observed following administration of 0.4% acetylcholine. No change in SGaw was measured in guinea pigs exposed to 3% MEG fumarate but SGaw was reduced to 69.3 +/- 5.3% of baseline after exposure to 12% MEG fumarate. MEG free base poses an alkaline challenge to the lung, 3% MEG fumarate is neutral (pH approximately 7.4) and 12% MEG fumarate is acidic (pH approximately 4.3); thus, MEG free-base and 12% MEG fumarate might provoke a reflex bronchoconstriction due to direct pulmonary irritant effects. These results suggest that MEG free base produced during crack pyrolysis may play a role in bronchoconstriction observed in crack smokers.