Characterization of photoreceptor cell types in the little brown bat Myotis lucifugus (Vespertilionidae).

We report the expression of three visual opsins in the retina of the little brown bat (Myotis lucifugus, Vespertilionidae). Gene sequences for a rod-specific opsin and two cone-specific opsins were cloned from cDNA derived from bat eyes. Comparative sequence analyses indicate that the two cone opsins correspond to an ultraviolet short-wavelength opsin (SWS1) and a long-wavelength opsin (LWS). Immunocytochemistry using antisera to visual opsins revealed that the little brown bat retina contains two types of cone photoreceptors within a rod-dominated background. However, unlike other mammalian photoreceptors, M. lucifugus cones and rods are morphologically indistinguishable by light microscopy. Both photoreceptor types have a thin, elongated outer segment. Using microspectrophotometry we classified the absorption spectrum for the ubiquitous rods. Similar to other mammals, bat rhodopsin has an absorption peak near 500 nm. Although we were unable to confirm a spectral range, cellular and molecular analyses indicate that M. lucifugus expresses two types of cone visual pigments located within the photoreceptor layer. This study provides important insights into the visual capacity of a nocturnal microchiropteran species.

Simultaneous determination of tacrine and 1-hydroxy-, 2-hydroxy-, and 4-hydroxytacrine in human plasma by high-performance liquid chromatography with fluorescence detection.

An analytical method was developed for the simultaneous reversed-phase (cyano) high-performance liquid chromatographic determination with fluorescence detection of tacrine and 1-hydroxy-, 2-hydroxy-, and 4-hydroxytacrine in human plasma. Alkalinized human plasma samples were extracted with a mixture of chloroform/l-propanol (90:10, v/v). Extraction recoveries generally ranged from 68% to 83% for all four compounds and did not appear to be concentration dependent over the range examined. Calibration curves were constructed over the following ranges: 0.5-30.0 ng/mL for tacrine (THA) and 4-hydroxytacrine (4-OH-THA), 1.0-30.0 ng/mL for 2-hydroxytacrine (2-OH-THA), and 0.925-46.2 ng/mL for 1-hydroxytacrine (1-OH-THA). The intra- and interassay precision (RSD) for the quality control specimens analyzed during validation were < or = 11.8% and < or = 9.28%, respectively. The intra- and interassay accuracy (RE) for the quality control specimens analyzed during validation ranged from 14.1% to -7.5% and 12.1% to -3.33%, respectively, for all four compounds. The limit of quantitation was estimated as the level of the lowest concentration in the calibration curve for each compound based on acceptable interassay precision and accuracy statistics generated at this concentration. The sensitivity of the method was adequate for the determination of THA, 1-OH-THA, 2-OH-THA, and 4-OH-THA following oral administration of Cognex (40 mg single dose) to normal volunteers.

Receptor-mediated methylprednisolone pharmacodynamics in rats: steroid-induced receptor down-regulation.

Several approaches to receptor down-regulation were examined to extend previous receptor/gene-mediated pharmacokinetic/dynamic models of corticosteroids. Down-regulation of the glucocorticoid receptor was considered as an instantaneous event or as a gradual steroid-receptor-mediated process. Concentrations of plasma methylprednisolone, free hepatic cytosolic receptors, and the activity of hepatic tyrosine aminotransferase (TAT) enzyme were measured for 16 hr following administration of 0, 10, and 50 mg/kg methylprednisolone sodium succinate to 93 adrenalectomized rats. Receptor down-regulation was best described by a fractional decrement in the rate of return of free cytosolic glucocorticoid receptor. Predicted values for free receptor, bound receptor, nuclear bound receptor, and transfer compartments were in accord with the expected rank order values based on the high and low steroid doses. Model parameter estimates were independent of dose and described the rapid depletion of free cytosolic receptor, late-phase return of cytosolic receptor to a new baseline level that was 20-40% lower than control, and the TAT induction/dissipation pattern following steroid dosing. The microscopic association and dissociation constants describing the steroid-receptor interaction were 0.23 L/nmole per hr (k(on)) and 4.74 hr-1 (k(off)) for methylprednisolone compared to previously obtained values of 0.20 L/nmole per hr and 15.7 hr-1 for the related steroid prednisolone. The time course of TAT induction was similar to that observed previously for prednisolone. Efficiency of TAT induction was more closely related to steroid receptor occupancy than plasma methylprednisolone concentrations due to receptor saturability and receptor recycling.

Bioavailability and nonlinear disposition of methylprednisolone and methylprednisone in the rat.

Bioavailability of low (10 mg/kg) and high (50 mg/kg) doses of methylprednisolone was determined after oral administration of the free alcohol of methylprednisolone and iv administration of methylprednisolone sodium succinate. Plasma concentrations of methylprednisolone and methylprednisone (reversible metabolite) were measured by HPLC. Methylprednisolone systemic availability (F) was 49-57% after iv administration and approximately 35% after oral administration. Solubilization of steroids with PEG:ethanol had no effect on their disposition. Apparent systemic clearance (CL) of methylprednisolone was 21 mL/min (low dose), approximately twice the liver blood flow. Dose-dependent changes in steady-state volume of distribution (Vdss) and central volume of distribution (Vdc), volumes, and apparent CL were observed. The methylprednisolone-to-methylprednisone AUC ratio decreased with dose due to saturation of methylprednisone formation clearance (CL12), but this is a minor metabolic pathway. The mean residence time (MRT) increased threefold with dose. Graphical estimates of the Michaelis-Menten capacity (Vmax) and affinity (Km) constants were in reasonable agreement with CL values for the low-dose experimental data. Low systemic availability of iv methylprednisolone sodium succinate was in part due to sequential first-pass hepatic metabolism of the methylprednisolone formed. Methylprednisolone disposition is complex in the rat due to extensive first-pass effects, nonlinear elimination, nonlinear distribution, and reversible metabolism.

Effect of ketoconazole on methylprednisolone pharmacokinetics and receptor/gene-mediated pharmacodynamics.

The disposition of methylprednisolone (MPL) and its metabolite, methylprednisone, and the receptor/gene-mediated pharmacodynamics of methylprednisolone were examined in control and ketoconazole-treated rats. Oral doses of ketoconazole (50 mg/kg/day) for 3 days increased plasma MPL clearance by 50% (NS) with no change in volumes of distribution. The mean residence time decreased from 0.60 +/- 0.15 (control) to 0.43 +/- 0.10 hr with ketoconazole (P less than .05) after 5 mg/kg of MPL (free alcohol). The methylprednisone to MPL area under the curve ratio decreased from 0.19 +/- 0.04 in control to 0.14 +/- 0.03 in ketoconazole-treated rats (P less than .05) due to altered interconversion between these steroids. An improved pharmacokinetic/dynamic receptor/gene-mediated model characterized the steroid receptor binding and induction of tyrosine aminotransferase activity after i.v. MPL sodium succinate (10 mg/kg). In contrast to previous in vitro studies, ketoconazole at maximally tolerated doses failed to antagonize the steroid receptor-mediated activity of MPL. Although ketoconazole at high concentrations competitively inhibited the in vitro binding of steroid to hepatic receptors, no in vivo inhibition was detected after large p.o. ketoconazole doses. Efficiency of tyrosine aminotransferase induction was slightly enhanced in ketoconazole animals. Pharmacokinetic/dynamic factors accounting for the lack of antiglucocorticoid activity primarily include the low ketoconazole receptor binding affinity.

Effects of thyroid dysfunction on prednisolone and prednisone interconversion and disposition in the rat.

The effects of thyroid dysfunction on the reversible metabolism and disposition of prednisolone and prednisone were examined in male Wistar rats. Hyperthyroid rats were produced by daily ip injections of 20 micrograms of I-triiodothyronine/100 g body weight for 6 days. Hypothyroid rats were obtained by providing 0.05% 6-propyl-2-thiouracil in drinking water ad libitum for 21 days. Rats were given 10 mg/kg of prednisolone or prednisone iv, blood samples were collected, and the steroids in plasma were assayed by HPLC. A recently developed pharmacokinetic model encompassing interconversion kinetics was applied. Unexpectedly, the hyperthyroid state increased the AUC (by 163%) of prednisolone (unlike in man) via reduced (66%) prednisolone elimination clearance (CL10), but also caused enhanced (101%) prednisone elimination clearance (CL20). The hypothyroid state increased the AUC (by 117%) of prednisolone via a 56% reduction in CL10 and a reduction in CL20. Prednisone formation of prednisolone (CL21) was about 13-fold greater than the reverse process (CL12), and both interconversion clearances were increased 20-40% by thyroid dysfunction. Experimental thyroid disorders thus alter prednisolone/prednisone pharmacokinetics in rats primarily by selective and sometimes unusual changes in elimination clearance rather than affecting the relative rates of steroid interconversion. Reversible metabolism is much less important in rats compared to man.

Analysis of methylprednisolone, methylprednisone and corticosterone for assessment of methylprednisolone disposition in the rat.

A sensitive, specific and precise high-performance liquid chromatographic assay for the simultaneous determination of methylprednisolone, methylprednisone and corticosterone using betamethasone as the internal standard is reported. Rat serum (0.5 ml) is extracted with methylene chloride, washed with sodium hydroxide, then water and the extract is injected onto a microparticulate silica gel column with ultraviolet detection at 254 nm. Calculated limits of quantitation are less than 10 ng/ml and the intra-day coefficient of variation is less than 5% for each steroid. This assay has been applied to preliminary studies of methylprednisolone disposition in the rat. The plasma concentration-time profile for each steroid was determined following intravenous administration of methylprednisolone (10 mg/kg). Peak serum methylprednisone concentrations of ca. 250 ng/ml occurred within 5 min of methylprednisolone administration and the average area under the curve ratio (methylprednisolone/methylprednisone) was 9.3. These findings demonstrate that methylprednisone is a metabolite of methylprednisolone in the rat and suggest that the metabolic back-conversion of methylprednisone to methylprednisolone may be less than in other species.

Liquid chromatographic analysis of di(2-ethylhexyl) phthalate: application to pharmacokinetic studies in the mongrel dog.

A high-performance liquid chromatographic (HPLC) assay was developed for the determination of di(2-ethylhexyl) phthalate (DEHP) in serum or plasma. Plasma DEHP concentrations that were measured by HPLC in specimens obtained from hemodialysis patients were in good agreement with corresponding concentrations that were measured by gas chromatography with selected ion monitoring (GC-SIM) (r2 = 0.996). Plasma DEHP concentrations were measured after intravenous DEHP administration (1.2-4.4 mg DEHP/kg body weight) to determine the effect of bilateral ureteral ligation on DEHP elimination in the mongrel dog. DEHP plasma clearance (approximately 6.3 ml/min/kg), steady-state distribution volume (approximately 0.21/kg), and terminal half-life (approximately 50 min) were unchanged in two dogs following bilateral ureteral ligation. DEHP terminal half-life and steady-state distribution volume were substantially smaller (25- to 70-fold) than reported previously in the rat or dog.

Pharmacokinetics and toxicity of high-dose human alpha 1-acid glycoprotein infusion in the rat.

The pharmacokinetics of high-dose human alpha 1-acid glycoprotein (AAG) was studied in rats to determine the feasibility of using AAG to alter the tissue distribution of basic drugs. alpha 1-Acid glycoprotein (2.2 g/kg) was administered iv to six male Holtzman rats over a period of 30 min, and serum AAG concentrations were measured by a specific radial immunodiffusion assay. The AAG concentrations were computer fit to a biexponential equation to generate pharmacokinetic constants for an open two-compartment model. The peak serum AAG concentration was 1830 +/- 180 mg/dL at the end of infusion; greater than 20 times the normal value for rats. The central volume of distribution and steady state volume of distribution were 0.09 +/- .02 and 0.15 +/- 0.02 L/kg, respectively. Total body clearance of AAG was 0.065 +/- 0.005 L/kg/h, and the terminal elimination half-life was 19.3 +/- 1.5 h. The AAG administration was tolerated without adverse effect and did not alter systolic blood pressure, the electrocardiogram, creatinine clearance, weight gain, or survival. The results of the histologic examination of various tissues by light microscopy at 30 d post AAG treatment were normal. These data demonstrate that high doses of human AAG can be safely administered to rats and that they produce supraphysiologic serum AAG concentrations.

Protein binding of disopyramide--displacement by mono-N-dealkyldisopyramide and variation with source of alpha-1-acid glycoprotein.

The binding of disopyramide to human serum proteins and human alpha-1-acid glycoprotein (AAG) was determined over a wide drug concentration range. Addition of 3.7 X 10(-6) mol litre-1 mono-N-dealkyldisopyramide caused a 20-100% increase in disopyramide free fraction. The disopyramide free fraction in AAG solutions prepared from various commercially available sources of alpha-1-acid glycoprotein varied up to 2.5 fold at corresponding disopyramide concentrations. Pronounced differences in the calculated binding constants (affinity and capacity) were observed among the commercially available AAG preparations. These findings suggest that binding studies should be performed in appropriately harvested human serum or plasma to avoid possible artifacts associated with the use of commercial preparations of alpha-1-acid glycoprotein for binding studies.

Evaluation of a mouse monoclonal antibody for the determination of serum theophylline by fluorescence polarization immunoassay.

Theophylline concentrations in spiked human serum and serum specimens obtained from patients with normal and impaired renal function were measured by fluorescence polarization immunoassay (FPIA) using a mouse monoclonal antitheophylline antibody. The interday coefficients of variation of the assay after 14 days were 4.2, 3.3, and 2.4% at serum theophylline concentrations in pooled human serum of 7.1, 12.2 and 26.9 mg/L. Theophylline concentrations determined by FPIA and high performance liquid chromatography (HPLC) were used to generate the following linear regression equations relating the corresponding theophylline concentrations measured by each method in serum specimens from 50 patients with normal renal function (FPIA = 1.00 HPLC + 1.01, r = 0.98) and 50 patients with end-stage renal disease (FPIA = 1.04 HPLC + 0.08, r = 0.99). In contrast to previous studies performed with a polyclonal antitheophylline antibody, the precision, accuracy, and specificity of the FPIA were adequate for analysis of theophylline in serum specimens obtained from patients with end-stage renal disease.

Protein binding of disopyramide and elevated alpha-1-acid glycoprotein concentrations in serum obtained from dialysis patients and renal transplant recipients.

A rapid ultrafiltration technique was used to measure the free (unbound) fraction of disopyramide in serum obtained from 14 normal volunteers, 6 chronic hemodialysis patients, and 10 renal transplant recipients. The disopyramide-free fraction varied more than tenfold at a corresponding total (free plus bound) serum disopyramide concentration of 3 micrograms/ml and was related to the concentration of an acute-phase protein, alpha-1-acid glycoprotein (AAG), in patient serum. Moreover, disopyramide-free fraction values were nearly twofold lower than normal in serum specimens obtained from those renal patients and transplant recipients with corresponding AAG serum concentrations greater than 100 mg/100 ml. AAG concentrations varied tenfold in patient serum and were on average nearly three times higher than AAG concentrations in normal volunteer serum. These findings suggested that the free fraction of disopyramide and possibly other drugs which bind extensively to AAG may be lower, and the interpatient variability in drug binding may be much more pronounced in serum obtained from hemodialysis patients and transplant recipients than previously recognized.

Disopyramide pharmacokinetics and bioavailability following the simultaneous administration of disopyramide and 14C-disopyramide.

The pharmacokinetics and bioavailability of total (bound plus unbound) and unbound disopyramide were compared following the simultaneous administration of an oral dose of disopyramide and an intravenous dose of 14C-disopyramide in five normal volunteers and in 11 patients with congestive heart failure. The binding of disopyramide varied between 60 and 92% in patients and between 81 and 88% in normal subjects at postequilibrium drug concentrations of 10(-7) M. The binding of disopyramide to serum protein was concentration-dependent in all study subjects at serum concentrations achieved following drug administration. The association constant for the first binding site in serum from normal subjects and patients averaged 8.7 X 10(5) M-1 and 4.4 X 10(5) M-1, respectively (p less than 0.05). The unbound clearance of disopyramide averaged 277 ml/min and 209 ml/min in normal subjects and in patients (p less than 0.05). When normalized for body weight, the unbound clearance between patients and normal subjects was not significantly different. The elimination half-life of unbound concentrations in normal subjects and in patients averaged 4.9 and 6.1 h, respectively (p less than 0.05). The clearance and elimination half-life of total disopyramide was the same in both groups. Although the bioavailability of disopyramide averaged 0.85 in both groups, it was more variable in patients owing to the variability in the fraction of the dose absorbed. The unbound renal clearance and volume of distribution at steady state of disopyramide was related to cardiac index. The ratio of elimination half-lives of total and unbound disopyramide was related to the extent of serum protein binding.

Analytical specificity of commercially available methods for serum phenytoin determination.

Phenytoin concentrations in serum obtained from uremic patients and patients with normal renal function were measured by fluorescence polarization immunoassay (FPI), rate nephelometric inhibition immunoassay ( RNII ), and homogeneous enzyme immunoassay (EIA) and compared to corresponding values determined by high performance liquid chromatography (HPLC). In serum specimens from patients with normal renal function, phenytoin concentrations measured by FPI, RNII , and EIA averaged within 12% of the corresponding concentrations determined by HPLC. Phenytoin concentrations in uremic serum were 20% (FPI), 60% (EIA), and 83% ( RNII ) higher than corresponding values determined by HPLC. Accumulation of phenytoin metabolites in patients with end-stage renal disease was associated with artifactually elevated measured serum concentrations of phenytoin. FPI was the most specific of the three commercial methods evaluated.

Influence of concentration-dependent protein binding on serum concentrations and urinary excretion of disopyramide and its metabolite following oral administration.

Concentrations of disopyramide (D) and mono-N-dealkyldisopyramide (MND) were measured in serum and urine following administration of 100, 150, 200, and 300 mg D to four volunteers. The free fraction of D in serum was drug concentration-dependent in all four subjects following the administration of the four doses. Areas under the free (unbound) D serum concentration-time curves (AUC free 0-9) were linearly related to dose following 100-200 mg D. AUC free 0-9 values were 30-60 per cent higher than expected in two subjects following 300 mg D due to an apparent dose-dependent decrease in D renal tubular secretion. Renal clearance of free D averaged 10.9 1 h-1. The renal clearance of MND averaged 10.3 1 h-1. The urinary excretion of MND was proportional to disopyramide dose.

Conversion from intravenous to oral dosing using sustained-release theophylline tablets.

Theophylline total body clearance was determined in asthmatic patients, from steady-state serum theophylline concentrations, measured during a constant-rate, intravenous aminophylline infusion. Theo-Dur dosing rates were calculated from the product of theophylline total body clearance and the desired steady-state theophylline concentration. After stopping the aminophylline infusion, oral doses of Theo-Dur were given every 12 hours. Blood samples were obtained 1, 3, 6,9, and 12 hours after the first oral dose and 12 hours after the second oral dose. Patients were evaluated for compliance and theophylline toxicity after receiving Theo-Dur for one week. Total body clearances and theophylline dosing rates varied more than threefold. The difference between the intravenous steady-state serum concentration and the average oral serum concentration was 1.2 micrograms/ml in patients with similar oral and intravenous dosing rates. The difference between peak and trough serum theophylline concentrations after the first oral dose averaged 7.8 micrograms/ml. Eight of nine patients evaluated after leaving the hospital were compliant, as determined from a tablet count during a clinic visit. Serum theophylline concentrations, determined 12 hours after the last Theo-Dur dose, averaged 10.4 micrograms/ml for the compliant patients.

Liquid chromatographic determination of caffeine in biologic fluids.

A high-performance liquid chromatographic procedure was developed for the determination of caffeine in various biologic fluids and coffee. A reversed-phase column and UV detection at 254 nm were used to obtain a sensitivity of 0.1 microgram/ml caffeine in serum and saliva using a sample volume of 0.1 ml. Caffeine metabolites and commonly ingested xanthines do not interfere with the assay. The within-day coefficients of variation were 9.8 and 9.9% at plasma caffeine concentrations of 2 and 10 micrograms/ml, respectively. The day-to-day coefficients of variation were 6.8 and 6.6% at plasma caffeine concentrations of 2 and 10 micrograms/ml, respectively. Serum and saliva caffeine concentrations were determined following a single oral dose of coffee and an intravenous infusion of caffeine in one subject. Computer estimates of caffeine pharmacokinetic parameters in one subject are in excellent agreement with previously published values.

The influence of blood collection technique on serum and plasma protein binding of disopyramide.

Serum and plasma disopyramide (D) protein binding was compared after blood was collected from four normal subjects in various Vacutainer tubes. The fraction of disopyramide bound to proteins in control serum and plasma was drug concentration dependent and correlated well with the capacity factor (N) associated with a high affinity protein binding site. D free fraction increased 60% at a postequilibrium concentration of 2 micrograms/ml in plasma following exposure of blood to green-top Vacutainer stoppers due to a 60% reduction in the affinity constant associated with the high affinity protein binding site. Heparin and EDTA had no effect on the plasma protein binding of D. These results suggest a competitive inhibition of disopyramide binding to alpha 1-acid-glycoprotein following contact of blood with rubber Vacutainer stoppers.