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1.
Clin Pharmacol Drug Dev ; 8(1): 87-94, 2019 01.
Article in English | MEDLINE | ID: mdl-29762909

ABSTRACT

Single-and multiple-dose pharmacokinetics and safety were investigated in this phase 1 study of dichlorphenamide, a carbonic anhydrase inhibitor approved in the United States for treatment of primary periodic paralysis. Dichlorphenamide was administered to 6 cohorts (n = 6 each) of healthy adults. Cohorts A through E received single doses of 25-400 mg followed by 50-800 mg/day in divided doses for 10 total doses. Cohort F (safety analysis only) received up to 28 titrated doses from 100-800 mg/day. Plasma for pharmacokinetics sampling was obtained predose and up to 48 hours postdose. Twenty-five of 36 enrolled subjects completed. Median time to maximum plasma concentration ranged from 1.5-3 hours, and mean half-life from 32-68 hours. Mean area under the concentration-time curve from time 0 to tau (length of the dosing interval estimated using the trapezoidal method) and maximum observed plasma concentration increased dose-proportionally after multiple doses. The incidence and severity of adverse events (AEs) were dose-related, with at least one mild AE reported among 17%, 17%, and 67% of patients in cohorts A, B, and C, respectively; and at least one mild-to-moderate AE among 100% of subjects in cohorts D, E, and F. One serious AE of rash was reported in cohort F. Eleven subjects discontinued; 10 due to AEs at 400 or 800 mg/day (cohorts E and F), including 100% of cohort F. Hypokalemia contributed to 5 of 6 discontinuations in cohort F (all 800 mg/day).


Subject(s)
Carbonic Anhydrase Inhibitors/administration & dosage , Dichlorphenamide/administration & dosage , Adult , Carbonic Anhydrase Inhibitors/adverse effects , Carbonic Anhydrase Inhibitors/blood , Carbonic Anhydrase Inhibitors/pharmacokinetics , Dichlorphenamide/adverse effects , Dichlorphenamide/blood , Dichlorphenamide/pharmacokinetics , Drug Administration Schedule , Female , Healthy Volunteers , Humans , Male , Middle Aged , Single-Blind Method
2.
J Enzyme Inhib Med Chem ; 33(1): 671-679, 2018 Dec.
Article in English | MEDLINE | ID: mdl-29536775

ABSTRACT

Rheumatoid arthritis (RA) is a chronic inflammatory disease caused by a faulty autoimmune response. Recently, it was reported that some human carbonic anhydrases (CAs) isoforms are overexpressed in inflamed synovium of RA patients. New CA inhibitors (CAIs) incorporating CA-binding moiety and the cyclooxygenase inhibitor tail (nonsteroidal anti-inflammatory drug [NSAID] type) were studied. The aim of this work is the evaluation of the chemical stability of NSAID - CAI hybrids towards spontaneous or enzymatic hydrolysis by LC-MS/MS. The analytes are isomer pairs of 6- or 7-hydroxycoumarin, their different fragment ions abundances allowed the development of a mathematical tool (LEDA) to distinguish them. LEDA reliability at ng mL-1 level was checked (>90%), being proved the effectiveness in the correct assignment of the isomer present in the sample. The hybrids resulted stable in all tested matrices allowing us to conclude that these compounds reach the target tissues unmodified, opening perspectives for their development in the treatment of inflammation.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/blood , Carbonic Anhydrase Inhibitors/blood , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Carbonic Anhydrase Inhibitors/chemistry , Carbonic Anhydrase Inhibitors/pharmacology , Carbonic Anhydrases/chemistry , Carbonic Anhydrases/metabolism , Chromatography, Liquid , Humans , Hydrolysis , Stereoisomerism , Structure-Activity Relationship , Tandem Mass Spectrometry
3.
Physiol Rep ; 5(1)2017 Jan.
Article in English | MEDLINE | ID: mdl-28087816

ABSTRACT

The knowledge on real-time neurophysiological effects of acetazolamide is still far behind the wide clinical use of this drug. Acetazolamide - a carbonic anhydrase inhibitor - has been shown to affect the neuromuscular transmission, implying a pH-mediated influence on the central synaptic transmission. To start filling such a gap, we chose a central substrate: hippocampal-prefrontal cortical projections; and a synaptic phenomenon: paired-pulse facilitation (a form of synaptic plasticity) to probe this drug's effects on interareal brain communication in chronically implanted rats. We observed that systemic acetazolamide potentiates the hippocampal-prefrontal paired-pulse facilitation. In addition to this field electrophysiology data, we found that acetazolamide exerts a net inhibitory effect on prefrontal cortical single-unit firing. We propose that systemic acetazolamide reduces the basal neuronal activity of the prefrontal cortex, whereas increasing the afferent drive it receives from the hippocampus. In addition to being relevant to the clinical and side effects of acetazolamide, these results suggest that exogenous pH regulation can have diverse impacts on afferent signaling across the neocortex.


Subject(s)
Acetazolamide/pharmacology , Carbonic Anhydrase Inhibitors/pharmacology , Neurons, Afferent/drug effects , Prefrontal Cortex/drug effects , Acetazolamide/adverse effects , Acetazolamide/blood , Animals , Carbonic Anhydrase Inhibitors/adverse effects , Carbonic Anhydrase Inhibitors/blood , Carbonic Anhydrases/physiology , Electric Stimulation , Hippocampus/drug effects , Hippocampus/enzymology , Hippocampus/physiology , Male , Neuronal Plasticity/drug effects , Prefrontal Cortex/physiology , Rats , Rats, Wistar , Synaptic Transmission/drug effects , Synaptic Transmission/physiology
4.
J Pharm Biomed Anal ; 119: 84-90, 2016 Feb 05.
Article in English | MEDLINE | ID: mdl-26669612

ABSTRACT

In the current study, a rapid and sensitive LC-QTOF-MS/MS method for the determination of brinzolamide in dried blood spots (DBS) was developed and validated. This novel sample collection, storage and transfer technique was suitable for analyzing a drug with high distribution into red blood cells and negligible plasma levels. The method included an isocratic mobile phase consisting of methanol and 10mM ammonium formate (90:10, v/v) and detection in positive electrospray mode (ESI+). The flow rate was adjusted to 0.350mL/min yielding retention times of 1.7min for both brinzolamide and internal standard (IS) rabeprazole on a Cyano analytical column, respectively. The validation of the proposed method over the concentration range 0.500-20.0µg/mL was performed in compliance with EMEA and FDA guidelines, assessing all major performance characteristics. Inter- and intra- assay precisions were less than 14%, while inter- and intra- assay accuracies varied from 92.2 to 111%. No matrix effect was observed and the mean brinzolamide extraction recovery was 93.5%. The method was successfully applied to real DBS samples from patients in steady state condition, receiving brinzolamide ophthalmic suspension 1% (w/v) for several months. Initial concentrations were corrected due to hematocrit effect, using image processing algorithm written in Matlab.


Subject(s)
Carbonic Anhydrase Inhibitors/blood , Chromatography, High Pressure Liquid/methods , Dried Blood Spot Testing/methods , Sulfonamides/blood , Tandem Mass Spectrometry/methods , Thiazines/blood , Administration, Ophthalmic , Aged , Carbonic Anhydrase Inhibitors/administration & dosage , Carbonic Anhydrase Inhibitors/therapeutic use , Chromatography, High Pressure Liquid/instrumentation , Dried Blood Spot Testing/instrumentation , Erythrocytes/chemistry , Humans , Male , Ophthalmic Solutions , Reference Standards , Reproducibility of Results , Sensitivity and Specificity , Sulfonamides/administration & dosage , Sulfonamides/therapeutic use , Tandem Mass Spectrometry/instrumentation , Thiazines/administration & dosage , Thiazines/therapeutic use
5.
J Med Chem ; 56(23): 9623-34, 2013 Dec 12.
Article in English | MEDLINE | ID: mdl-24200125

ABSTRACT

The selective inhibition of cancer-associated human carbonic anhydrase (CA) enzymes, specifically CA IX and XII, has been validated as a mechanistically novel approach toward personalized cancer management. Herein we report the design and synthesis of a panel of 24 novel glycoconjugate primary sulfonamides that bind to the extracellular catalytic domain of CA IX and XII. These compounds were synthesized from variably acylated glycopyranosyl azides and either 3- or 4-ethynyl benzene sulfonamide using Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC). The CA enzyme inhibition profile for all compounds was determined, while in vitro metabolic stability, plasma stability, and plasma protein binding for a representative set of compounds was measured. Our findings demonstrate the influence of the differing acyl groups on these key biopharmaceutical properties, confirming that acyl group protected carbohydrate-based sulfonamides have potential as prodrugs for selectively targeting the extracellular cancer-associated CA enzymes.


Subject(s)
Antigens, Neoplasm/drug effects , Carbonic Anhydrase Inhibitors/chemical synthesis , Carbonic Anhydrases/drug effects , Prodrugs/chemical synthesis , Sulfonamides/chemical synthesis , Antigens, Neoplasm/metabolism , Caco-2 Cells , Carbonic Anhydrase IX , Carbonic Anhydrase Inhibitors/blood , Carbonic Anhydrase Inhibitors/chemistry , Carbonic Anhydrases/metabolism , Catalytic Domain/drug effects , Cell Membrane Permeability , Enzyme Stability , Glycoconjugates/chemical synthesis , Humans , Neoplasms/enzymology , Prodrugs/chemistry , Structure-Activity Relationship
6.
J Pharm Pharm Sci ; 16(1): 89-98, 2013.
Article in English | MEDLINE | ID: mdl-23683608

ABSTRACT

PURPOSE: To characterize the pharmacokinetics (PK) and pharmacodynamics (PD) of acetazolamide (ACTZ) in peritoneal dialysis patients, ACTZ 500 mg was administered intravenously to 7 healthy subjects (HV) and 8 peritoneal dialysis patients (CAPD). METHODS: Population PK/PD modeling was performed with ACTZ serum (total and unbound), urine and dialysate concentrations, intra-ocular pressure (IOP) and covariates. A multi-compartment PK model (accounting for non-linear protein binding) and an inhibitory Emax (maximal change in IOP) PD model were selected. RESULTS: As expected, renal clearance (which almost equals total body clearance) was severely decreased in CAPD (1.2 vs 80.3 L/h) and the elimination half-life of total ACTZ was prolonged (20.6 vs 3.4 hours). The protein binding was significantly altered with a mean free fraction 4.2% in HV and 8.6% in CAPD. Moreover protein binding of ACTZ was concentration dependent in both HV and CAPD. Despite a higher free fraction of ACTZ, the Emax was lower in CAPD: 4.4±1.4 vs 7.4±2.8 mmHg. CONCLUSION: Both PK and PD are significantly altered in dialysis patients.


Subject(s)
Acetazolamide/pharmacology , Carbonic Anhydrase Inhibitors/pharmacology , Models, Biological , Peritoneal Dialysis , Acetazolamide/blood , Acetazolamide/urine , Adult , Blood Proteins/metabolism , Carbonic Anhydrase Inhibitors/blood , Carbonic Anhydrase Inhibitors/urine , Female , Humans , Kidney Function Tests , Male , Middle Aged , Protein Binding , Young Adult
7.
Acta Pharm ; 56(2): 115-42, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16613721

ABSTRACT

This article describes reverse phase high-performance liquid chromatography (RPHPLC) methods for determination of diuretics in different human body fluids (whole blood, plasma, serum or urine). Sample preparation procedures, including solid-phase extraction, liquid-liquid extraction, dilution, precipitation as well as automated RPHPLC procedures, are discussed in order to present the advantages and disadvantages of each type of sample preparation. Also, values of analytical recovery of each procedure used for sample preparation are summarized. The most important RPHPLC parameters (detection mode, stationary phase, mobile phase, sensitivity, etc.) are also summarized and discussed.


Subject(s)
Chromatography, High Pressure Liquid/methods , Diuretics/analysis , Carbonic Anhydrase Inhibitors/analysis , Carbonic Anhydrase Inhibitors/blood , Carbonic Anhydrase Inhibitors/urine , Diuretics/blood , Diuretics/urine , Humans , Reproducibility of Results , Sodium Channel Blockers/analysis , Sodium Channel Blockers/blood , Sodium Channel Blockers/urine , Sodium Potassium Chloride Symporter Inhibitors/analysis , Sodium Potassium Chloride Symporter Inhibitors/blood , Sodium Potassium Chloride Symporter Inhibitors/urine , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet
8.
J Comp Physiol B ; 172(4): 287-95, 2002 May.
Article in English | MEDLINE | ID: mdl-12037591

ABSTRACT

This main purpose of this study was to examine the subcellular distribution and isozyme characteristics of branchial carbonic anhydrase (CA) in Chaenocephalus aceratus, an Antarctic icefish that lacks erythrocytes. The Antarctic fish, Notothenia coriiceps, which possesses erythrocytes, was also studied for comparative purposes. The gills of both species were found to have measurable activity of CA. N. coriiceps also had normal levels of blood CA activity. In contrast, the icefish, C. aceratus, lacked blood CA activity, but was found to possess an endogenous plasma CA inhibitor. The large majority of branchial CA in the gills of these species was located in the cytoplasmic fraction whereas less than 3% was associated with the membrane fraction. In both species, CA from the cytoplasmic gill fraction and membrane fraction differed markedly in terms of their sensitivity to the plasma CA inhibitor from C. aceratus. In addition, treatment with the cleaving enzyme phosphatidylinositol-specific phospholipase C indicated that CA from the branchial membrane fraction of both species is anchored to the membrane via a phosphatidylinositol-glycan linkage. Taken together, these results provide evidence for a CA IV-like isozyme in the gills of Antarctic fish. At present, the functional significance of this membrane-bound CA is unknown, but the relative amount of this isozyme appeared to be greater in the gills of C aceratus, the species that lacked erythrocytes.


Subject(s)
Carbonic Anhydrase Inhibitors/blood , Carbonic Anhydrases/metabolism , Fishes/metabolism , Gills/enzymology , Animals , Antarctic Regions , Carbonic Anhydrases/analysis , Cytoplasm/enzymology , Enzyme Activation/drug effects , Erythrocytes/enzymology , Microsomes/enzymology , Phosphatidylinositol Diacylglycerol-Lyase , Phosphoinositide Phospholipase C , Type C Phospholipases/pharmacology
10.
J Exp Biol ; 203(Pt 19): 3003-9, 2000 Oct.
Article in English | MEDLINE | ID: mdl-10976036

ABSTRACT

The blood serum of the European flounder Platichthys flesus strongly inhibits soluble erythrocytic carbonic anhydrase from the same species. The inhibition is of the uncompetitive type. Hence, the mechanism of the carbonic anhydrase inhibition is different from that of all other known carbonic anhydrase inhibitors. The serum showed no inhibitory effect on carbonic anhydrase from human and bovine red blood cells. By applying the (18)O exchange reaction, it could be demonstrated that the presence of the carbonic anhydrase inhibitor in the extracellular fluid has no effect on carbonic anhydrase in intact red blood cells. Thus, this carbonic anhydrase inhibitor seems to act only within the plasma space of the circulatory system. However, the carbonic anhydrase inhibitor does appear to reduce the bicarbonate permeability of flounder red cells to approximately one-quarter of normal levels as measured by the (18)O exchange reaction. The 28 kDa carbonic anhydrase inhibitor was isolated from the serum by gel filtration. The isolated inhibitor was detected in acrylamide gels as a single band representing a 7 kDa protein. The denaturing conditions used in electrophoresis presumably led to a dissociation of the native protein into subunits.


Subject(s)
Carbonic Anhydrase Inhibitors/blood , Flounder/blood , Animals , Carbonic Anhydrase Inhibitors/chemistry , Carbonic Anhydrase Inhibitors/isolation & purification , Cattle , Erythrocytes/enzymology , Humans , In Vitro Techniques , Molecular Weight , Species Specificity
11.
Am J Vet Res ; 61(8): 965-8, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10951991

ABSTRACT

OBJECTIVE: To determine the pharmacokinetics of acetazolamide administered IV and orally to horses. ANIMALS: 6 clinically normal adult horses. PROCEDURE: Horses received 2 doses of acetazolamide (4 mg/kg of body weight, IV; 8 mg/kg, PO), and blood samples were collected at regular intervals before and after administration. Samples were assayed for acetazolamide concentration by high-performance liquid chromatography, and concentration-time data were analyzed. RESULTS: After IV administration of acetazolamide, data analysis revealed a median mean residence time of 1.71 +/- 0.90 hours and median total body clearance of 263 +/- 38 ml/kg/h. Median steady-state volume of distribution was 433 +/- 218 ml/kg. After oral administration, mean peak plasma concentration was 1.90 +/- 1.09 microg/ml. Mean time to peak plasma concentration was 1.61 +/- 1.24 hours. Median oral bioavailability was 25 +/- 6%. CONCLUSIONS AND CLINICAL RELEVANCE: Oral pharmacokinetic disposition of acetazolamide in horses was characterized by rapid absorption, low bioavailability, and slower elimination than observed initially after IV administration. Pharmacokinetic data generated by this study should facilitate estimation of appropriate dosages for acetazolamide use in horses with hyperkalemic periodic paralysis.


Subject(s)
Acetazolamide/pharmacokinetics , Carbonic Anhydrase Inhibitors/pharmacokinetics , Horses/physiology , Acetazolamide/administration & dosage , Acetazolamide/blood , Administration, Oral , Animals , Area Under Curve , Biological Availability , Carbonic Anhydrase Inhibitors/administration & dosage , Carbonic Anhydrase Inhibitors/blood , Chromatography, High Pressure Liquid/methods , Chromatography, High Pressure Liquid/veterinary , Cross-Over Studies , Female , Half-Life , Injections, Intravenous/veterinary , Least-Squares Analysis , Male
12.
J Pharmacokinet Biopharm ; 27(1): 45-66, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10533697

ABSTRACT

The rate and extent of binding of methazolamide to human erythrocytes was studied in vitro. All experiments were carried out at physiological temperature (37 C) and pH (7.4). Methazolamide (MTZ) buffer concentrations were analyzed by HPLC. Distributional equilibrium between buffer and washed red blood cells was achieved after 1 hr. Results of equilibrium studies were consistent with two classes of binding sites for MTZ within the erythrocyte: a low affinity, high capacity site (CA-I) and a high affinity, low capacity site (CA-II). A two-binding site model was fitted to experimental data generating estimates for binding parameters Ka1 (0.0017 +/- 0.00022 microM-1) nM1 (636 +/- 5.23 microM), Ka2(0.46 +/- 0.0083 microM-1), and nM2(80.9 +/- 0.389 microM). Based upon these findings, kinetic studies were performed in order to characterize the rate of drug distribution. The rate of erythrocyte uptake of MTZ was mathematically modeled using a series of differential equations describing drug diffusion across the red blood cell membrane and subsequent complexation with intracellular binding sites. The model assumed that penetration of MTZ into the red blood cells was passive but drug binding to the carbonic anhydrase isozymes was not instantaneous. Using a novel curve fitting technique, parameter estimates of RBC membrane permeability (0.0102 +/- 0.000618 cm/min), and binding rate constants k-1(0.254 +/- 0.0213 min-1), k1 (0.0022 +/- 0.00020 ml/microgram-min), k-2(1.59 +/- 0.0358 min-1), and k2(3.1 +/- 0.035 ml/microgram-min) were obtained. The model characterized the observed biphasic decline of MTZ buffer concentrations over time and may help explain the prolonged residence of MTZ in vivo.


Subject(s)
Carbonic Anhydrase Inhibitors/blood , Erythrocytes/metabolism , Methazolamide/blood , Models, Biological , Models, Chemical , Binding Sites , Biological Transport , Carbonic Anhydrase Inhibitors/metabolism , Carbonic Anhydrase Inhibitors/pharmacokinetics , Humans , Kinetics , Mathematical Computing , Methazolamide/metabolism , Methazolamide/pharmacokinetics
13.
Int J Clin Pharmacol Ther ; 37(6): 286-93, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10395120

ABSTRACT

OBJECTIVES: In this paper we investigated in humans and in animals the in vitro and in vivo effect of omeprazole upon purified and erythrocyte carbonic anhydrase (CA) I and II isozymes, as well as on gastric mucosa CA IV. METHOD: In vitro, we observed the effect of omeprazole at concentrations between 10(-8)-10(-4) M on purified CA I and CA II, and also on isolated gastric mucosa CA IV, renal and pulmonary CA IV activity, using the dose-response relationship. In vivo, we studied the effect of omeprazole (Losec) on gastric CA I, II and IV, as well as on erythrocyte CA I and CA II, in humans and in animals. RESULTS: In vitro omeprazole inhibits pH-dependent purified CA I and CA II and gastric mucosa CA IV according to dose-response relationship. In vivo, the i.v. administration of omeprazole in rabbits and in humans shows a decrease of erythrocyte CA I and CA II activity as well as of gastric mucosa CA I, II and IV. CONCLUSIONS: Omeprazole in its active form (sulfenamide) selectively inhibits gastric mucosa CA IV and does not modify the activity of the same isozyme from the kidney and lung proving that the enzyme has an organ specificity. Our results lead to the conclusion that omeprazole possesses a dual mechanism of action: both H+K+ATPase and CA inhibition--enzymes that could be in a functional coupling. This dual mechanism of action might explain the higher effectiveness of treatment using substituted benzimidazole inhibitors compared to other therapies.


Subject(s)
Carbonic Anhydrase Inhibitors/pharmacology , Carbonic Anhydrases/drug effects , Erythrocytes/drug effects , Gastric Mucosa/drug effects , Omeprazole/pharmacology , Adult , Animals , Carbonic Anhydrase Inhibitors/blood , Carbonic Anhydrases/metabolism , Cell Line/drug effects , Dose-Response Relationship, Drug , Erythrocytes/enzymology , Gastric Mucosa/enzymology , Humans , Hydrogen-Ion Concentration , Isoenzymes/drug effects , Isoenzymes/metabolism , Male , Omeprazole/blood , Rabbits , Swine
14.
J Pharm Biomed Anal ; 17(8): 1415-21, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9800660

ABSTRACT

A simple and sensitive high-performance liquid chromatographic method for the analysis of acetazolamide (AZ) in rat blood (plasma/serum, whole blood and serum ultrafiltrate), brain tissue and cerebrospinal fluid (CSF) was described. Quantitative extraction of AZ with ethyl acetate from both buffered plasma and brain tissue homogenate (pH 8.0) was achieved. Each extract was evaporated to dryness and the residue was chromatographed on a reversed-phase column. CSF was directly analysed without extraction step. The limits of detection were 0.05 microgram ml-1 for plasma, 0.02 microgram g-1 for brain tissue and 0.004 microgram ml-1 for CSF. Calibration curves were linear over the working ranges of 0.1-100 micrograms ml-1 for plasma, 0.05-50 micrograms g-1 for brain tissue and 0.025-50 micrograms ml-1 for CSF. The reproducibility of AZ assay in the rat biologic media indicated very low relative standard deviations (RSDs). The recoveries of AZ added to plasma and brain tissue were more than 96% with an RSD of less than 5%. The present method was applied to studies of plasma concentration profiles of the drug after administration and its distribution into central nervous system.


Subject(s)
Acetazolamide/analysis , Brain Chemistry , Carbonic Anhydrase Inhibitors/analysis , Chromatography, High Pressure Liquid/methods , Acetazolamide/blood , Acetazolamide/cerebrospinal fluid , Acetazolamide/pharmacokinetics , Animals , Carbonic Anhydrase Inhibitors/blood , Carbonic Anhydrase Inhibitors/cerebrospinal fluid , Carbonic Anhydrase Inhibitors/pharmacokinetics , Injections, Intravenous , Male , Rats , Rats, Wistar , Tissue Distribution
16.
Biopharm Drug Dispos ; 19(6): 373-80, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9737818

ABSTRACT

The pharmacokinetic disposition of methazolamide (MTZ) was studied in five healthy volunteers following administration of a single oral dose. Drug concentrations in blood, plasma, and urine were measured by HPLC. Over the range of plasma concentrations observed in vivo, MTZ free fraction (measured by ultrafiltration) was 0.28. Being a carbonic anhydrase inhibitor, MTZ would be expected to distribute into, and be sequestered by, red blood cells. For this reason, MTZ disposition was characterized utilizing blood concentrations as the reference. Using a two-compartment model, a series of differential equations were simultaneously fitted to blood concentrations and urinary excretion data generating estimates for k10 (0.035 +/- 0.019 h(-1)), k12 (0.200 +/- 0.036 h(-1)), k21 (0.077 +/- 0.046 h(-1)), k(a) (0.304 +/- 0.064 h(-1)), Vc (1.1 +/- 0.18 L) and f(r) (fraction excreted renally, 0.61 +/- 0.14). Total blood clearance was 0.037 +/- 0.020 L h(-1). The model estimate of elimination half-life (126 +/- 61 h) was consistent with drug binding to a high affinity carbonic anhydrase isozyme in the erythocyte. Estimates of MTZ renal clearance and renal excretion ratio were 0.021 +/- 0.010 L h(-1) and 0.16 +/- 0.06, respectively. Overall, the prolonged elimination of MTZ from the blood is the result of extensive erythrocyte distribution and tubular reabsorption by the kidney.


Subject(s)
Carbonic Anhydrase Inhibitors/blood , Carbonic Anhydrase Inhibitors/urine , Erythrocytes/metabolism , Methazolamide/blood , Methazolamide/urine , Administration, Oral , Adult , Area Under Curve , Carbonic Anhydrase Inhibitors/administration & dosage , Cohort Studies , Female , Half-Life , Humans , Male , Methazolamide/administration & dosage , Models, Biological , Prospective Studies
17.
J Pharm Biomed Anal ; 15(7): 1001-8, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9160268

ABSTRACT

A sensitive and specific method for the determination of dorzolamide (I) and its de-ethylated metabolite (II) in human plasma has been developed utilizing high pressure liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection. The analytes and internal standard (III) were isolated from the deproteinized pH 8.0 buffered plasma, using a liquid-liquid extraction with a mixture of ethyl acetate, toluene, and isopropanol. The analytes were then back extracted into 0.085% phosphoric acid (200 microliters) and after washing the acidic extract with hexane, the organic layer was discarded and a fraction (50 microliters) of the acid extract was injected into the LC/MS/MS system. The MS/MS detection was performed on a PE Sciex API III tandem mass spectrometer using a heated nebulizer interface. Multiple reaction monitoring of the parent-->product ion combinations of m/z 325-->199, 297-->199, and 397-->306 were used to quantify I, II, and III, respectively. The assay was validated in the concentration ranges of 0.5-100 and 2.5-100 ng ml-1 of plasma for I and II, respectively. The precision of the assays, expressed as coefficients of variation (C.V.%), were less than 10% over the entire concentration range, with adequate assay specificity and accuracy. The LC/MS/MS method provided a 10-fold increase in the sensitivity of I over the previously reported HPLC/UV method [1].


Subject(s)
Antihypertensive Agents/blood , Carbonic Anhydrase Inhibitors/blood , Sulfonamides/blood , Thiophenes/blood , Antihypertensive Agents/metabolism , Carbonic Anhydrase Inhibitors/metabolism , Chromatography, High Pressure Liquid , Chromatography, Liquid , Humans , Mass Spectrometry , Reproducibility of Results , Sensitivity and Specificity , Spectrophotometry, Ultraviolet , Sulfonamides/metabolism , Thiophenes/metabolism
18.
Biochemistry ; 36(14): 4327-36, 1997 Apr 08.
Article in English | MEDLINE | ID: mdl-9100029

ABSTRACT

The plasma from many vertebrates contains a component that specifically binds and inhibits carbonic anhydrase II with nanomolar affinity. Amino-terminal sequencing of pICA, the previously identified 79-kDa carbonic anhydrase inhibitor isolated from porcine plasma [Roush, E. D., & Fierke, C. A. (1992) Biochemistry 31, 12536-12542], and sequencing of four proteolytic fragments of pICA revealed that each of the partial sequences has 40-80% sequence identity with members of the transferrin protein family. We describe here the isolation of a full-length cDNA clone of pICA from a lambda gt11 porcine liver cDNA library. Heterologous expression of this cDNA clone in a Pichia pastoris expression system led to the secretion into the medium of 5 mg/L of a 79-kDa protein that specifically reacts with anti-pICA antibodies and binds tightly to a carbonic anhydrase-Sepharose affinity column. Pairwise sequential alignment of pICA with various transferrins reveals an amino acid identity as high as 64% and predicts that 16 transferrin disulfide bonds are conserved. However, despite these structural similarities, the properties of pICA are distinct from the properties of transferrin. pICA exhibits a significantly decreased affinity for iron that can be attributed to the loss of one of the eight amino acids that coordinate iron in the transferrins as well as both of the arginine residues responsible for anion binding. In addition, the antigenic determinants of pICA and the transferrins are not identical. These data imply that pICA, along with saxiphilin, is a member of a diverse superfamily of transferrin-like proteins with functions other than iron binding.


Subject(s)
Blood Proteins/chemistry , Carbonic Anhydrase Inhibitors/chemistry , Carbonic Anhydrases/metabolism , Amino Acid Sequence , Animals , Base Sequence , Blood Proteins/analysis , Blood Proteins/pharmacology , Blotting, Western , Carbonic Anhydrase Inhibitors/blood , Carbonic Anhydrase Inhibitors/pharmacology , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Gene Expression , Immunoblotting , Iron-Binding Proteins , Metals/analysis , Molecular Sequence Data , Peptide Fragments/chemistry , Pichia/genetics , Recombinant Proteins/chemistry , Sequence Alignment , Sequence Analysis , Swine , Transferrin/chemistry , Transferrin/genetics , Transferrin-Binding Proteins
19.
J Ocul Pharmacol Ther ; 13(1): 23-30, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9029437

ABSTRACT

Dorzolamide is a powerful inhibitor of carbonic anhydrase (CA) II that penetrates the sclera and cornea to reach the ciliary process and lowers formation of HCO3 and aqueous humor. The usual dose applied to the eye in treatment of glaucoma is 1 drop (30 microL of 2% solution) every 8 hr to each eye, or a total daily dose of 4 mg. On this regime, the red cells accumulated drug over a period of 8 days, reaching a value of 20-25 microM, which corresponds to the concentration of CA II in human red cells. This drug concentration persisted throughout the 18 months of application. The plasma concentration was 0.034 microM, or 1/700 that of the red cells. This plasma concentration corresponds to that calculated from the dilution of administered drug into body water. The data are well fitted into the equilibrium expression for KI of dorzolamide against CA II at 37 degrees C, as 8 x 10(-9) M. The red cells also contain a small amount (5 microM) of the N-des-ethyl metabolite, probably reflecting its modest binding to CA I. In the initial 8-day drug period, virtually none appeared in the urine since CA II sites were being filled. At steady state, renal excretion was 1.3 mg/day and the renal clearance 90 ml/min. These excretion numbers include the small (20%) amount of the des-ethyl metabolite of dorzolamide. The relation of these data to lowering of intraocular pressure is clear. By the systemic route, an inhibitor such as acetazolamide is effective when free drug concentration in plasma is 2.5 microM. In the case of topical drugs, as shown here, the plasma concentration is some 100 x lower, but the concentration in ciliary process is 2-10 microM, comparable to that following systemic drugs (1). In conclusion, the concentration in plasma (reflecting free drug) of dorzolamide is about 1/200 of that needed for systemic effects as seen following acetazolamide or methazolamide. Thus, there is a clear pharmacological basis for the lack of any physiological effects of ocular dorzolamide, except on the eye itself.


Subject(s)
Carbonic Anhydrase Inhibitors/pharmacokinetics , Sulfonamides/pharmacokinetics , Thiophenes/pharmacokinetics , Administration, Topical , Aged , Carbonic Anhydrase Inhibitors/blood , Carbonic Anhydrase Inhibitors/urine , Erythrocytes/metabolism , Female , Glaucoma, Open-Angle/blood , Glaucoma, Open-Angle/urine , Humans , Male , Middle Aged , Ocular Hypertension/blood , Ocular Hypertension/urine , Sulfonamides/blood , Sulfonamides/urine , Thiophenes/blood , Thiophenes/urine
20.
Drug Metab Dispos ; 24(6): 659-63, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8781782

ABSTRACT

The disposition of dorzolamide, a carbonic anhydrase-II (CA-II) inhibitor, was examined in rats after oral and iv administration of 0.05, 0.5, 5, and 25 mg/kg. The area under the blood concentration-time curve (AUC) increased in an approximately dose-proportional fashion up to 0.5 mg/kg. However, at the higher dorzolamide doses there was an unusual 52% decrease in AUC with increasing dose from 0.5 to 25 mg/kg. This was due to a combination of concentration-dependent red blood cell (RBC)/plasma distribution and a competitive drug-metabolite displacement interaction that increased the time-averaged blood clearance by more than 100-fold over the dose range examined. Extensive and saturable binding to the CA-II that is present in RBCs is characteristic of this compound class. However, saturation of binding to the blood enzyme was not sufficient to explain the observed decrease in the AUC at the higher dose levels. A further increase in blood clearance was attributed to the displacement of dorzolamide from the CA-II binding sites by its active N-desethyl metabolite. The proposed mechanism was evaluated in studies that examined the effect of the metabolite on the distribution of dorzolamide between RBCs and plasma. Ex vivo and in vitro assessments of the equilibrium RBC/ plasma concentration ratio indicated that metabolite displacement of dorzolamide from enzyme binding sites in RBC occurs at pharmacologically relevant concentrations.


Subject(s)
Carbonic Anhydrase Inhibitors/pharmacokinetics , Erythrocytes/metabolism , Sulfonamides/pharmacokinetics , Thiophenes/pharmacokinetics , Administration, Oral , Analysis of Variance , Animals , Carbonic Anhydrase Inhibitors/blood , Injections, Intravenous , Male , Rats , Rats, Sprague-Dawley , Sulfonamides/blood , Thiophenes/blood
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