Whole blood or plasma: what is the ideal matrix for pharmacokinetic-driven drug candidate selection?

In the present era of drug development, quantification of drug concentrations following pharmacokinetic studies has preferentially been performed using plasma as a matrix rather than whole blood. However, it is critical to realize the difference between measuring drug concentrations in blood versus plasma and the consequences thereof. Pharmacokinetics using plasma data may be misleading if concentrations differ between plasma and red blood cells (RBCs) because of differential binding in blood. In this review, factors modulating the partitioning of drugs into RBCs are discussed and the importance of determining RBC uptake of drugs for drug candidate selection is explored. In summary, the choice of matrix (plasma vs whole blood) is an important consideration to be factored in during drug discovery.

Design of cationic nanostructured heterolipid matrices for ocular delivery of methazolamide.

Solid lipid nanoparticles (SLNs) formulated from one type of lipid (homolipid) suffer from low drug encapsulation and drug bursting due to crystallization of the lipid into the more ordered ß modification, which leads to decreased drug entrapment and faster drug release. This study assessed the feasibility of using nanostructured lipid matrices (NLMs) for ocular delivery of methazolamide-(MZA) adopting heterolipids composed of novel mixtures of Compritol (®) and cetostearyl alcohol (CSA), and stabilized by Tween 80(®). The systems were prepared using the modified high shear homogenization followed by ultrasonication method, which avoids the use of organic solvents. A 3(2) full factorial design was constructed to study the influence of two independent variables, namely the ratio of CSA:Compritol and the concentration of Tween 80, each in three levels. The dependent variables were the entrapment efficiency percentages (EE%), mean particle size (PS), polydispersity index (PDI), and zeta potential (ZP). In vivo intraocular pressure (IOP) lowering activity for the selected formulae was compared to that of MZA solution. The results showed that increasing the ratio of CSA to Compritol increased the EE% and PS, while increasing the concentration of Tween 80, decreased PS with no significant effect on EE%. The ZP values of all formulae were positive, and greater than 30 mV. The best formula, composed of 4% CSA, 2% Compritol, 0.15% stearylamine, and 2% Tween 80, with EE% of 25.62%, PS of 207.1 nm, PDI of 0.243, and ZP of 41.50 mV, showed in vitro sustained release properties for 8 hours and lowered the intraocular pressure by 8.3 mmHg within 3 hours, with this drop in pressure lasting for 12 hours.

Determination of memantine in rat plasma by HPLC-fluorescence method and its application to study of the pharmacokinetic interaction between memantine and methazolamide.

A sensitive high-performance liquid chromatographic method with fluorescence detection was developed to determine memantine (MT) in rat plasma. The method consists of pre-column labeling of MT with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) and a clean-up step with solid-phase extraction. A good separation of DIB-MT was achieved within 12 min on an octadecylsilica (ODS) column (150 × 4.6 mm i.d.; 5 µm) with a mobile phase of acetonitrile-water (70:30, v/v). The calibration curve prepared with fluoxetine as an internal standard showed good linearity in the range of 10-400 ng/mL (r = .999). The limits of detection and quantitation at signal-to-noise ratios of 3 and 10 were 2.0 and 6.6 ng/mL, respectively. The method was shown to be reliable with precisions of <5% for intra-day and <9% for inter-day as relative standard deviation. The fluorescence property and reaction yield of authentic DIB-MT were also examined. The proposed method was successfully applied to study the pharmacokinetic interaction between MT and methazolamide.

The metabolism of methazolamide - identification of metabolites in guinea pig urine.

The in vivo metabolism of methazolamide, a carbonic anhydrase inhibitor, was studied using guinea pigs as the animals. (14)C-Labeled methazolamide was synthesized. Eighty percent of intraperitoneally injected radioactivity was recovered from urine and feces within 24 hours. HPLC analysis on a C(18) column detected 2 radioactive metabolites (Peaks A and B). The Peaks A and B were isolated from the urine of the animals dosed with non-radioactive methazolamide.They were purified on the C(18) column. Their chemical structure was revealed by UV-absorbance spect a and LC/MS, and confirmed by comparing it with that of chemically synthesized compound. They were a glucuronide, (2-acetylimino-3-methyl-Δ(4)-1,3,4-thiadiazol-5-yl)-1-thio-ß-D-glucopyranosiduronic acid, and a sulfonic acid, N-[3-methyl-5-sulfo-1,3,4-thiadiazol-2(3H)-ylidene]acetamide.

A potential new therapeutic system for glaucoma: solid lipid nanoparticles containing methazolamide.

Methazolamide (MTA) is an antiglaucoma drug; however, there are many side effects of its systemic administration with insufficient ocular therapeutic concentrations. The aim of this study was to formulate MTA-loaded solid lipid nanoparticles (SLNs) and evaluate the potential of SLNs as a new therapeutic system for glaucoma. SLNs were prepared by a modified emulsion-solvent evaporation method and their physicochemical characteristics were evaluated. The pharmacodynamics was investigated by determining the percentage decrease in intraocular pressure. The ocular irritation was studied by Draize test. Despite a burst release of SLNs, the pharmacodynamic experiment indicated that MTA-SLNs had higher therapeutic efficacy, later occurrence of maximum action, and more prolonged effect than drug solution and commercial product. Formulation of MTA-SLNs would be a potential delivery carrier for ocular delivery, with the advantages of a more intensive treatment for glaucoma, lower in doses and better patient compliance compared to the conventional eye drops.

Preparation and evaluation of in situ gelling ophthalmic drug delivery system for methazolamide.

PURPOSE: In this study, a thermosensitive in situ gelling vehicle was prepared to increase the precorneal resident time and the bioavailability of methazolamide (MTA). METHOD: Poloxamer analogs were used as the gelling agents, and the in situ gel was obtained by using a cold method. The gelation temperature, rheological properties, in vitro release as well as in vivo evaluation (the elimination of MTA in aqueous humor and intraocular-lowering effect) of the optimized formulations were investigated. RESULTS: The optimum concentrations of poloxamer analogs for the in situ gel-forming delivery system were 21% (w/w) poloxamer 407 and 10% (w/w) poloxamer P188. This formulation was able to flow freely under nonphysiological conditions and underwent sol-gel transition in the cul-de-sac upon placement into the eye. In vitro release studies demonstrated a diffusion-controlled release of MTA from the poloxamer solutions over a period of 10 hours. In vivo evaluation indicated that the poloxamer solutions had a better ability to retain drug than MTA eyedrops did. CONCLUSION: These results suggested that in situ gelling ophthalmic drug delivery system may hold some promise in ocular MTA delivery.

Cyclodextrin solubilization of carbonic anhydrase inhibitor drugs: formulation of dorzolamide eye drop microparticle suspension.

Topically applied carbonic anhydrase inhibitors (CAIs) are commonly used to treat glaucoma. However, their short duration of action requiring multiple daily dosing can hamper patient compliance. The aim of this study was to develop novel aqueous CAI eye drop formulation containing self-assembled drug/cyclodextrin (D/CD) microparticles that enhance and prolong drug delivery to the eye. Phase-solubility of each drug tested (i.e. methazolamide, brinzolamide and dorzolamide HCl) was determined in either pure water or an aqueous eye drop medium. The pH was adjusted to maximize the fraction of unionized drug. Dorzolamide had the highest affinity for γ-cyclodextrin (γCD) and, thus, was selected for further investigation. Hydroxypropyl methylcellulose (HPMC) was the most effective polymer tested for stabilization of the dorzolamide/γCD complexes and gave the highest mucoadhesion at 0.5% w/v concentration. Thus, the dorzolamide eye drop vehicle containing γCD (18% w/v) and HPMC (0.5% w/v) was developed. The physicochemical properties of this formulation complied with the specifications of the eye drop suspension monograph of the European Pharmacopoeia. The in vivo testing of the formulation showed that the drug was delivered to the aqueous humor in rabbits for at least 24h with the maximum drug concentration at 4h. Furthermore, this formulation delivered the drug to the posterior segment of the eye after topical administration. These results indicate that this CAI eye drop formulation has the potential of being developed into a once-a-day product.

Methazolamide calcium phosphate nanoparticles in an ocular delivery system.

A new system for the local delivery of methazolamide to the eye has been developed based on calcium phosphate (CaP) nanoparticles. The methazolamide loaded CaP nanoparticles were prepared through the formation of an inorganic core of CaP and further adsorption of the methazolamide. The maximum loading of methazolamide studied using UV-vis spectrophotometry was about 0.2% (w/w). The drug-loaded particles had a negative surface charge at about -30 mV while their mean particle diameter was estimated to be 256.4 nm. In vitro release studies demonstrated diffusion-controlled release of methazolamide from the CaP nanoparticles over a period of 4 h. In vivo studies indicated that the intraocular pressure (IOP)-lowering effect of the inorganic nanoparticle eye drops lasted for 18 h, which was significantly better than the effect of 1% brinzolamide eye drops (6 h). Physical stability studies indicated that the preparation was stable for 6 months at 40 degrees C. These findings suggested that methazolamide bound to CaP nanoparticles might be useful in the local treatment of glaucoma.

In vitro characterization of the erythrocyte distribution of methazolamide: a model of erythrocyte transport and binding kinetics.

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.

The ocular distribution of methazolamide after corneal and scleral administration: the effect of ionization state.

The accession of methazolamide in ionized and unionized form to cornea, sclera, aqueous humor and ciliary process was studied 10 minutes following separate application to either sclera or cornea of a 1 mM solution. Cornea and ciliary process concentrations were 27.3 and 14.5 microM for unionized application to cornea and 10.1 and 7.1 microM for ionized application. Bulk aqueous humor concentrations were much lower, 3.1 and 1.1 microM, and cannot account for drug found in ciliary process on this time scale. Scleral application of drug, by contrast, gave undetectable ciliary process concentrations. These results are presented as a model for drug disposition following single drop topical sulfonamide therapy. The scleral pathway for drug delivery to ciliary process was further tested by application to sclera of 300 microL of either a 1 mM ionized or unionized solution for 30 minutes or as a 2% (85.5 mM) ionized solution for 30 minutes. In these series, red cell carbonic anhydrase was presaturated at -24 hours with drug to remove a possible route for loss of drug from sclera, that of the systemic circulation and the high concentration of carbonic anhydrase in red cells. After either ionized or unionized application, approximately 1 microM was detected in ciliary process, but all drug was attributable to the blood content of the tissue and the drug bound to red cell carbonic anhydrase. After 2% dosing, 4 microM was detected in ciliary process after allowance for drug in red cells. This concentration is below that necessary for inhibition of ciliary process carbonic anhydrase, suggesting that especially with regard to topical sulfonamide therapy, the corneal route of drug delivery to ciliary process predominates greatly over the scleral route.

Determination of methazolamide concentrations in human biological fluids using high performance liquid chromatography.

Methazolamide is a carbonic anhydrase inhibitor used to treat glaucoma. In vivo, methazolamide readily distributes into red blood cells. Therefore, both blood and plasma concentration data are needed in order to characterize the pharmacokinetics of methazolamide. In the present study, an analytical method using high performance liquid chromatography was validated for determination of methazolamide concentrations in several biological fluids. Through slight modification of a previously reported method for acetazolamide, another carbonic anhydrase inhibitor, methazolamide was readily quantitated in whole blood, plasma and urine. Sample preparation involved liquid-liquid extraction with ethyl acetate followed by a washing step using phosphate buffer (pH 8.0). After back extraction into glycine buffer (pH 10.0), samples were then washed with ether and injected onto the chromatograph. Chromatography was performed using a C-18, 5 microns reverse-phase column with UV detection at a wavelength of 285 nm. Mobile phase consisted of 0.05 M sodium acetate (pH 4.0) and acetonitrile (20%). The assay was validated over two standard concentration ranges from 1 to 100 micrograms ml-1, concentrations reflective of those expected in vivo, Calibration curves were linear for all biological fluids and coefficients of variation for interday and intraday reproducibility studies were less than 8% (range 3.1-7.9%). The method was used to measure methazolamide concentrations in blood, plasma and urine following oral administration to five human subjects.

Penetration into the anterior chamber via the conjunctival/scleral pathway.

The importance of the conjunctival/scleral pathway as a route of entry into the ciliary body, and in particular uptake and deposition by vessels, was investigated. A constant concentration of methazolamide analogs as well as 6-carboxyfluorescein (6-CB) and rhodamine B (RB) was maintained on either the cornea or the conjunctiva/sclera tissue, the latter excluding the cornea. The solutions were applied with the use of a cylindrical well affixed to the cornea of an anesthetized white rabbit. After two hours, concentrations of drug or dye were measured in cornea, aqueous humor or iris/ciliary body for both routes of entry. Confocal microscopy methods were used to determine reflected fluorescence images for 6-CB and RB. Carbonic anhydrase inhibition, partitioning, solubility and intraocular pressure (IOP) measurements were also determined. Permeability calculations were estimated for drug diffusing against aqueous flow within the posterior chamber. The conjunctival/scleral route of entry produced higher iris/ciliary body concentrations for all compounds except for the lipophilic RB. Confocal microscopy results suggested that drug is gaining entry into the ciliary body through vessel uptake in the sclera. Following entry of drug into the conjunctival/scleral tissue, a significant portion enters scleral vessels and deposits within the ciliary body. Calculations are given that indicate that once drug penetrates the cornea it is highly unlikely drug diffuses through the pupil against aqueous flow to enter the posterior chamber and reach the ciliary body.