Showing papers in "Pharmaceutical Research in 1993"

The preparation and characterization of poly(lactide-co-glycolide) microparticles. II: The entrapment of a model protein using a (water-in-oil)-in-water emulsion solvent evaporation technique

Abstract: Poly(lactide-co-glycolide) (PLG) microparticles with entrapped antigens have recently been investigated as controlled-release vaccines. This paper describes the preparation of PLG microparticles with an entrapped model antigen, ovalbumin (OVA), using a (water-in-oil)-in-water emulsion solvent evaporation technique. In a series of experiments, the effects of process parameters on particle size and OVA entrapment were investigated. It was found that smooth, spherical microparticles 1–2 µm in diameter containing up to 10% (w/w) OVA could be produced using a small volume of external aqueous phase containing a high concentration of emulsion stabilizer and a 1:5 antigen:polymer ratio. PAGE analysis, isoelectric focusing, and Western blotting of OVA released from the microparticles in vitro confirmed that the molecular weight and antigenicity of the protein remained largely unaltered by the entrapment procedure.

Upper Gastrointestinal pH in Seventy-Nine Healthy, Elderly, North American Men and Women

Abstract: Gastric and duodenal pH levels were measured in 79 healthy, elderly men and women (mean ± SD = 71 ± 5 years) under both fasted and fed conditions using the Heidelberg capsule technique The pH was recorded for 1 hr in the fasted state, a standard liquid and solid meal of 1000 cal was given over 30 min, then the pH was measured for 4 hr postprandially Results are given as medians and interquartile ranges: fasted gastric pH, 13 (11–16); gastric pH during the meal, 49 (39–55); fasted duodenal pH, 65 (62–67); and duodenal pH during the meal, 65 (64–67) Although fasted gastric pH, fasted duodenal pH, and duodenal pH during the meal differ statistically from those observed in young subjects, the differences are not expected to be clinically significant in terms of drug absorption for the majority of elderly subjects Following a meal, gastric pH decreased from a peak pH of 62 (58–67) to pH 20 within 4 hr in most subjects This rate of return was considerably slower than in young, healthy subjects Nine subjects (11%) had a median fasted gastric pH >50, and in five of these subjects the median pH remained >50 postprandially In this group, drugs and dosage forms which require an acidic environment for dissolution or release may be poorly assimilated

Selective paracellular permeability in two models of intestinal absorption: cultured monolayers of human intestinal epithelial cells and rat intestinal segments.

Abstract: New data on the permeabilities of hydrophilic markers in two commonly used in vitro models, i.e., excised intestinal segments from the rat and monolayers of Caco-2 cells, are presented. The results are compared to human in vivo data. Two groups of hydrophilic marker molecules were tested: (1) monodisperse polyethylene glycols of molecular weights ranging from 194 to 502 g/mol and (2) a heterogeneous group of molecules consisting of urea, creatinine, erythritol, and mannitol (60–182 g/mol). The permeabilities of the marker molecules showed a nonlinear dependence on the molecular weight and decreased in the order rat ileum > rat colon > Caco-2 cells. Surprisingly, the polyethylene glycols permeated more easily than the other marker molecules, indicating that characteristics other than molecular weight, e.g., the flexibility of the structure, may also be important for permeation through the membrane. Comparisons with the published permeability profiles of polyethylene glycols in human intestinal segments in vivo (i.e., calculated permeability coefficients as a function of molecular weight) indicate that the human intestine is more permeable than the in vitro models. However, the permeability profiles of the corresponding segments in the human intestine and the in vitro models were comparable. Thus, good correlations were established between permeabilities of the human ileum and rat ileum and between those of human colon, rat colon, and the Caco-2 cells. We conclude that the paracellular absorption in humans can be studied mechanistically in these in vitro models.

A saturable transport mechanism in the intestinal absorption of gabapentin is the underlying cause of the lack of proportionality between increasing dose and drug levels in plasma

Abstract: Gabapentin (l-(aminomethyl)cyclohexaneacetic acid) is a neuroprotective agent with antiepileptic properties. The structure is small (molecular weight less than 200), is zwitterionic, and resembles an amino acid with the exception that it does not contain a chiral carbon and the amino group is not alpha to the carboxylate functionality. Gabapentin is not metabolized by humans, and thus, the amount of gabapentin excreted by the renal route represents the fraction of dose absorbed. Clinical trials have reported dose-dependent bioavailabilities ranging from 73.8 ± 18.3 to 35.7 ± 18.3% when the dose was increased from 100 to 1600 mg. The permeability of gabapentin in the rat intestinal perfusion system was consistent with carrier-mediated absorption, i.e., a 75 to 80% decrease in permeability when the drug concentration was increased from 0.01 to 50 mM (0.46 ± 0.05 to 0.12 ± 0.04). Excellent agreement was obtained between the actual clinical values and the predicted values from in situ results for the fraction of dose absorbed calculated using the theoretically derived correlation, Fabs = 1 - exp(−2Peff) by Ami-don et al. (Pharm. Res. 5:651–654, 1988). The permeability values obtained for gabapentin correspond to 67.4 and 30.2% of the dose absorbed at the low and high concentrations, respectively. In the everted rat intestinal ring system, gabapentin shared an inhibition profile similar to that of L-phenylalanine. Characteristics of gabapentin uptake included cross-inhibition with L-Phe, sensitivity to inhibition by L-Leu, stereoselectivity as evidenced by incomplete inhibition by D-Phe, and lack of effect by Gly. Our findings support absorption of gabapentin by a saturable pathway, system L, shared by the large hydrophobic amino acids, L-Phe and L-Leu. The saturable absorption pathway makes a major contribution to the lack of proportionality in plasma levels of drug with increasing dose ob-served in the clinic.

In Vitro Evaluation of Calcium Pectinate: A Potential Colon-Specific Drug Delivery Carrier

Abstract: Calcium pectinate (CaP)—the insoluble salt of pectin—can potentially be used as a colon-specific drug delivery system. The use of CaP as a carrier was based on the assumption that, like pectin, it can be decomposed by specific pectinolytic enzymes in the colon but that it retains its integrity in the physiological environment of the small bowel. The biodegradation of the carrier was characterized by monitoring the percent cumulative release of the insoluble drug indomethacin, incorporated into pectin or CaP matrices. Compressed tablets of pectin and indomethacin were analyzed for degradation in the presence of Pectinex 3XL, a typical pectinolytic enzyme mixture, and in the presence of the human colonic bacterium Bacteroides ovatus. The degradation of CaP-indomethacin tablets was assessed in the presence of Pectinex 3XL and in rat cecal contents. The release of indomethacin was significantly increased (end-time percentage cumulative release vs control) in the presence of Pectinex 3XL (89 ± 20 vs 16 ± 2 for CaP tablets), Bacteroides ovatus (12 and 22 vs 5.2 for pectin tablets), and rat cecal contents (61 ± 16 vs 4.9 ± 1.1 for CaP tablets). The weight loss of tablet mass was significantly higher (end-time dry weight vs control) in the presence of Pectinex 3XL (0 vs 75 ± 6% of initial weight for CaP tablets). These findings indicate the potential of CaP, compressed into tablets with insoluble drug, to serve as a specific drug delivery system to the colon.

Estimating the fraction dose absorbed from suspensions of poorly soluble compounds in humans: a mathematical model.

Abstract: A microscopic mass balance approach has been developed to predict the fraction dose absorbed of suspensions of poorly soluble compounds. The mathematical model includes four fundamental dimensionless parameters to estimate the fraction dose absorbed: initial saturation (Is), absorption number (An), dose number (Do), and dissolution number (Dn). The fraction dose absorbed (F) increases with increasing Is, An, and Dn and with decreasing Do. At higher Dn and lower Do, the fraction dose absorbed reaches the maximal F, which depends only on An. The dissolution number limit on F can appear at both lower Do and lower Dn. Likewise, at higher Do and Dn, the fraction dose absorbed reaches a Do limit. Initial saturation makes a significant difference in F at lower Do and Dn. It is shown that the extent of drug absorption is expected to be highly variable when Dn and Do are approximately one. Furthermore, by calculating these dimensionless groups for a given compound, a formulation scientist can estimate not only the extent of drug absorption but also the effect, if any, of particle size reduction on the extent of drug absorption.

Determinants of Release Rate of Tetanus Vaccine from Polyester Microspheres

Abstract: Controlled-release formulations based on poly(lactic) (PLA) and poly(lactic/glycolic) acid (PLGA) microspheres containing tetanus vaccine were designed. The polymers forming the microspheres were L-PLA of different molecular weights and DL-PLGA, 50:50. These microspheres were prepared by two solvent elimination procedures, both using a double emulsion, and were characterized for size, morphology, and toxoid release kinetics. The influence of formulation variables such as polymer type, vaccine composition, and vaccine/polymer ratio was also investigated. Both techniques yielded microspheres with similar size, morphology, and release properties. Microsphere size was dependent on the type of polymer and the presence of the surfactant L-α-phosphatidylcholine, which led to a reduction in microsphere size. On the other hand, the release kinetics of encapsulated protein were affected by the polymer properties (ratio lactic/glycolic acid and molecular weight) as well as by the vaccine composition, vaccine loading, and microsphere size. Moreover, for some formulations, a decrease in microsphere size occurred simultaneously, with an increase in porosity leading to an augmentation of release rate. The changes in the PLA molecular weight during in vitro release studies indicated that release profiles of tetanus toxoid from these microspheres were only marginally influenced by polymer degradation. A significant fraction of protein (between 15 and 35%) was initially released by diffusion through water-filled channels. In contrast, the decrease in the PLGA molecular weight over the first 10 days of incubation suggested that erosion of the polymer matrix substantially affects protein release from these microspheres. Among all formulations developed, two differing in microsphere size, polymer hydrophobicity, and release profile were selected for in vivo administration to mice. Administration of both formulations resulted in tetanus neutralizing antibody levels that were higher than those obtained after administration of the fluid toxoid.

Prolongation of the circulation time of doxorubicin encapsulated in liposomes containing a polyethylene glycol-derivatized phospholipid: pharmacokinetic studies in rodents and dogs.

Abstract: The pharmacokinetics of doxorubicin (DOX) encapsulated in liposomes containing polyethylene glycol-derivatized distearoylphosphatidylethanolamine (PEG/DSPE) were investigated in rodents and dogs. The plasma levels of DOX obtained with PEG/DSPE-containing liposomes were consistently higher than those without PEG/DSPE or when PEG/DSPE was replaced with hydrogenated phosphatidylinositol (HPI). Despite the inclusion of PEG/DSPE in liposomes, there was a significant drop in the plasma levels of DOX when the main phospholipid component, hydrogenated phosphatidylcholine, was replaced with lipids of lower phase transition temperature (dipalmitoylphosphatidylcholine, egg phosphatidylcholine), indicating that phase transition temperature affects the pharmacokinetics of liposome-encapsulated DOX. In beagle dogs, clearance was significantly slower for DOX encapsulated in PEG/DSPE-containing liposomes than in HPI-containing liposomes, with distribution half-lives of 29 and 13 hr, respectively. In both instances, almost 100% of the drug measured in plasma was liposome-associated. The apparent volume of distribution was only slightly above the estimated plasma volume of the dogs, indicating that drug leakage from circulating liposomes is insignificant and that the distribution of liposomal drug is limited mostly to the intravascular compartment in healthy animals.

Sodium caprate elicits dilatations in human intestinal tight junctions and enhances drug absorption by the paracellular route.

Abstract: The effects of the absorption enhancer sodium caprate on human intestinal epithelial cells were investigated using Caco-2 cell monolayers. The effects on epithelial integrity and drug transport are dependent on time and concentration and are decreased by Ca2+, most likely through the formation of Ca22+ soaps. Morphological data indicate that exposure to sodium caprate results in cytoskeletal changes and in structural alterations of the tight junctions in the form of dilatations, while the effects on the apical cell membranes are limited. We conclude that sodium caprate increases the absorption of drugs mainly by the paracellular route.

A floating controlled-release drug delivery system: in vitro-in vivo evaluation.

Abstract: A novel floating controlled-release drug delivery system was formulated in an effort increase the gastric retention time of the dosage form and to control drug release. The buoyancy was attributed to air and oil entrapped in the agar gel network. A floating controlled-release 300-mg theophylline tablet having a density of 0.67 was prepared and compared in vitro and in vivo to Theo-dur. The in vitro release rate of the floating tablet was slower. In vivo scintigraphic studies for a floating and a heavy nonfloating tablet, under fasting and nonfasting conditions, showed that the presence of food significantly increased the gastric retention time for both tablets, and tablet density did not appear to make a difference in the gastric retention time. However, the positions of the floating and nonfloating tablets in the stomach were very different. Bioavailability studies in human volunteers under both fasting and nonfasting conditions showed results comparable to those with Theo-dur. The floating controlled-release theophylline tablet maintained constant theophylline levels of about 2 mg/mL for 24 hr, which may be attributable to the release from the agar gel matrix and the buoyancy of the tablet in the stomach.

In Vitro Extended-Release Properties of Drug-Loaded Poly(DL-Lactic Acid) Nanoparticles Produced by a Salting-Out Procedure

Abstract: Savoxepine-loaded poly(DL-lactic acid) (PLA) nanoparticles were prepared using an emulsion technique involving a salting-out process which avoids surfactants and chlorinated solvents. After their formation, the nanoparticles were purified by cross-flow microfiltration and subsequently freeze-dried. The drug loading and the drug entrapment efficacy were improved by using savoxepine base rather than the methanesulfonate salt and by modifying the pH of the aqueous phase. A drug entrapment efficacy as high as 95% was obtained with a 9% drug loading. The overall yield of the procedure can rise up to 93%. In vitro release studies have demonstrated that by varying the mean size of the nanoparticles and their drug loading, the release of the drug from the nanoparticles can be modulated to last from several hours to more than 30 days, thus allowing the preparation of an injectable extended-release dosage form.

Drug Absorption Limited by P-Glycoprotein-Mediated Secretory Drug Transport in Human Intestinal Epithelial Caco-2 Cell Layers

Abstract: The hypothesis was tested that the operation of an ATP-dependent export pump localized at the apical (brush border) surface of the intestinal epithelium may limit substrate absorption kinetics. Human intestinal Caco-2 cell-layers display saturable secretion of vinblastine from basal to apical surfaces (Km, 18.99 ± 5.55 µM; Vmax, 1285.9 ± 281.2 pmol cm−2 hr−1) that is inhibited by verapamil, consistent with the expression of the ATP-dependent P-glycoprotein drug efflux pump at the apical brush border membrane. Inhibition of P-glycoprotein by a variety of modulators (verapamil, 1,9-dideoxyforskolin, nifedipine, and taxotere) is associated with an increased vinblastine absorptive permeability. Vinblastine absorption displayed a nonlinear dependence upon luminal (apical) vinblastine concentration, and vinblastine absorption increased markedly at concentrations where vinblastine secretory flux was saturated (>20 µM). Upon inhibition of P-glycoprotein by verapamil and 1,9-dideoxyforskolin, vinblastine absorption increased and was linearly dependent on vinblastine concentration. The limitation of P-glycoprotein substrate absorption by active ATP-dependent export via P-glycoprotein is discussed, together with the possibility that other classes of substrate may be substrates for different ATP-dependent export pumps.

Zero-order drug release from hydrocolloid matrices.

Abstract: Matrices are manufactured by direct compression of a powder mixture of a polymer, e.g., methylhydroxypropyl cellulose (MHPC) or polyvinylalcohol (PVAI), and a drug. The following factors that can influence the drug release mode were investigated at constant surface: (i) polymer solution viscosity, glass transition temperature, and swelling; (ii) drug concentration in the matrix and solubility; and (iii) conditions of release experiment (hydrodynamics). In the case of zero-order release profiles (hydrocolloids with low viscosities), only the dissolution of the polymer appears to control the drug release rate. Factors accelerating polymer dissolution resulted in higher release rates. Comparison of swollen and dry hydrocolloid matrices shows that the duration and kinetics of drug release were not controlled by the swelling front moving into the dry polymer, and water penetration and relaxation were not rate controlling. Therefore, the glass transition temperature had no effect on drug release from these hydrocolloids. The higher the hydrodynamic stress exerted on the eroding hydrocolloid, the faster the resulting drug release as a result of accelerated polymer dissolution. With hydrocolloids of very high viscosity the polymer dissolution is slow, and drug release from the swollen gel appears to be controlled by diffusion according to kinetics of the Higuchi type.

Site-specific drug delivery to pilosebaceous structures using polymeric microspheres

Abstract: In order to improve the therapeutic index of adapalene, a new drug under development for the treatment of acne, site-specific delivery to the hair follicles using 50:50 poly(DL-lactic-co-glycolic acid) microspheres as particulate carriers was investigated in vitro and in vivo. The percutaneous penetration pathway of the microspheres was shown to be dependent on their mean diameter. Thus, after topical application onto hairless rat or human skin, adapalene-loaded microspheres (5-µm diameter) were specifically targeted to the follicular ducts and did not penetrate via the stratum corneum. The in vitro release of adapalene from the microspheres into artificial sebum at 37°C was controlled and faster than the in vivo sebum excretion in humans. Aiming to reduce either the applied dose of drug or the frequency of administration, different formulations of adapalene-loaded microspheres were evaluated in vivo in the rhino mouse model. A dose-related comedolytic activity of topical formulations of adapalene-loaded microspheres was observed in this model. Furthermore, by applying a site-specific drug delivery system (0.1% adapalene) every other day or by administering a 10-fold less concentrated targeted formulation (0.01%) every day, a pharmacological activity equivalent to a daily application of an aqueous gel containing drug crystals (0.1% adapalene) was observed. Since an aqueous gel containing 10% adapalene-loaded microspheres was not irritating in a rabbit skin irritancy test, this formulation was applied onto forearms of human volunteers. Site-specific drug delivery was further evidenced by follicular biopsy. These results support the view that follicular drug targeting using 5-µm polymeric microspheres may represent a promising therapeutic approach for the treatment of pathologies associated with pilosebaceous units.

A new method for estimating dermal absorption from chemical exposure. 1. General approach.

Abstract: To evaluate systemic chemical exposure from dermal absorption, one must know the mass of chemical absorbed including the portion that has entered the skin but not yet entered the body's interior system. Algebraic equations are presented for estimating dermal absorption including the effects of exposure time and chemical nature of the compound, in particular lipophilicity and molecular weight. The proposed equations account for larger absorption rates during the initial exposure period as well as the hydrophilic barrier which the viable epidermis presents to lipophilic chemicals. These algebraic expressions are shown to represent adequately the exact solution of the unsteady-state diffusion equations for a two-membrane composite. Finally, procedures are proposed for estimating a priori the required physicochemical data when experimental values are not available. Specifically, the Potts and Guy permeability correlation is split into parts separately representing stratum corneum partitioning and diffusivity.

Comparison of the Permeability Characteristics of a Human Colonic Epithelial (Caco-2) Cell Line to Colon of Rabbit, Monkey, and Dog Intestine and Human Drug Absorption

Abstract: The in vitro permeabilities of Caco-2 monolayers and permeabilities in tissue sections from colon of monkey, rabbit, and dog were compared using a series of compounds. The selected compounds differed in their physicochemical properties, such as octanol/water partition coefficient, water solubility, and molecular weight. Their structure included steroids, carboxylic acids, xanthins, alcohols, and polyethylene glycols. A linear permeability relationship was established between Caco-2 and colon tissue from both rabbit and monkey. The results suggest that Caco-2 is twice as permeable as rabbit and five times as permeable as monkey colon. However, no clear relationship could be established between Caco-2 monolayers and dog colon permeability. A relationship between permeability in Caco-2 monolayers and human absorption was found. The results suggest that within certain limits, permeability of Caco-2 monolayers may be used as a predictive tool to estimate human drug absorption.

Peptide Stability in Drug Development. II. Effect of Single Amino Acid Substitution and Glycosylation on Peptide Reactivity in Human Serum

Abstract: The determination of peptide stability in human serum (HS) or plasma constitutes a powerful screening assay for eliminating unstable peptides from further development. Herein we report on the stability in HS of several major histocompatibility complex (MHC)-binding peptides. Some of these peptides are in development for the novel treatment of selected autoimmune disorders such as rheumatoid arthritis and insulin-dependent diabetes. For most of the l-amino acid peptides studied, the predominant degradation mechanism is exopeptidase-catalyzed cleavage. Peptides that were protected by d-amino acids at both termini were found to be more stable than predicted, based on additivity of single substitutions. In addition, N-acetylglucosamine glycopeptides were significantly stabilized, even when the glycosylation site was several amino acids from the predominant site(s) of cleavage. This indicates that long-range stabilization is possible, and likely due to altered peptide conformation. Finally, the effect of single amino acid substitutions on peptide stability in HS was determined using a model set of poly-Ala peptides which were protected from exopeptidase cleavage, allowing the study of endopeptidase cleavage pathways.

Controlled Delivery Systems for Proteins Using Polyanhydride Microspheres

Abstract: A method to provide near-constant sustained release of high molecular weight, water-soluble proteins from polyanhydride microspheres is described The polyanhydrides used were poly (fatty acid dimer) (PFAD), poly(sebacic acid) (PSA), and their copolymers [P(FAD-SA)] P(FAD-SA) microspheres containing proteins of different molecular sizes—lysozyme, trypsin, heparinase, ovalbumin, albumin, and immunoglobulin—were prepared by a solvent evaporation method using a double emulsion The microspheres containing proteins were spherical, with diameters of 50–125 µm, and encapsulated more than 80% of the protein, irrespective of the protein used Enzymatic activity studies showed that encapsulation of enzymes inside polyanhydride microspheres can protect them from activity loss When not placed inside polyanhydride microspheres, trypsin lost 80% of its activity in solution at 37°C at pH 74 in 12 hr, whereas inside the polyanhydride microspheres the activity loss was less than 10% under these conditions About 47% of the enzymatic activity of heparinase encapsulated in the microspheres was lost at 37°C in 24 hr, while in solution it lost over 90% of its activity The protein-loaded microspheres displayed near-zero-order erosion kinetics over 5 days as judged by the release of sebacic acid (SA) from the microspheres The microspheres degraded to form SA and FAD monomers All proteins were released at a near-constant rate without any large initial burst, irrespective of polymer molecular weight and protein loading The period of protein release was longer than that of SA and continued protein release was observed even after the microsphere matrix had completely degraded Differential scanning calorimetric studies demonstrated an interaction between protein and the FAD monomers produced with microsphere degradation It is likely that the protein interaction with FAD monomers permits formation of water-insoluble protein aggregates which slowly dissolve and diffuse out of the matrix, leading to delayed protein release For trypsin-loaded microspheres, trypsin lost 40% of its activity during microsphere preparation Activity studies demonstrated that the sonication process was primarily responsible for activity loss A reduction in the period of ultrasound exposure decreased the loss of protein activity to around 20%

Biodistribution of micelle-forming polymer-drug conjugates

Abstract: Polymeric micelles have potential utility as drug carriers. To this end, polymeric micelles based on AB block copolymers of polyethylene oxide (PEG) and poly(aspartic acid) [p(Asp)] with covalently bound Adriamycin (ADR) were prepared. The micelle forming polymer–drug conjugates [PEO-p(Asp(ADR)] were radiolabeled and their biodistribution was investigated after intravenous injection in mice. Long circulation times in blood for some compositions of PEO-p[Asp(ADR)] conjugates were evident, which are usually atypical of colloidal drug carriers. This was attributed to the low interaction of the PEO corona region of the micelles with biocomponents (e.g., proteins, cells). Low uptake of the PEO-p(Asp(ADR)] conjugates in the liver and spleen was determined. The biodistribution of the PEO-p[Asp(ADR)] conjugates was apparently dependent on micelle stability; stable micelles could maintain circulation in blood, while unstable micelles readily formed free polymer chains which rapidly underwent renal excretion. Long circulation times in blood of PEO-p(Asp(ADR)] conjugates are thought to be prerequisite for enhanced uptake at target sites (e.g., tumors).

Introduction to backpropagation neural network computation.

Abstract: Neurocomputing is computer modeling based, in part, upon simulation of the structure and function of the brain. Neural networks excel in pattern recognition, that is, the ability to recognize a set of previously learned data. Although their use is rapidly growing in engineering, they are new to the pharmaceutical community. This article introduces neurocomputing using the backpropagation network (BPN).

Microdialysis Calibration Using Retrodialysis and Zero-Net Flux: Application to a Study of the Distribution of Zidovudine to Rabbit Cerebrospinal Fluid and Thalamus

Abstract: A retrodialysis (RD) method for the real-time calibration of on-line microdialysis (MD) procedures was investigated in vitro and in vivo. Calibration by retrodialysis was simultaneously validated through the use of a zero-net flux (ZNF) method, which assumes directional independence of diffusion of the solute. In RD, a calibrator with dialysance (PeA; effective permeability–surface area product) similar to that of the compound of interest is introduced into the perfusate. If the calibrator is suitable, its loss from the perfusate during RD is identical to the recovery of the solute of interest determined simultaneously by normal MD. Two antiviral nucleosides (AZT and AZdU) which differ structurally by only a methylene group were utilized as solute and calibrator, respectively. Both nucleosides exhibited similar recovery and loss at flow rates of 0.5 to 5 (µL/min in vitro, indicating a similar PeA product in this flow domain. Furthermore, both compounds showed similar loss into the lateral ventricle or thalamus of rabbits (n = 4) during RD at a flow rate of 1 µL/min for 6 hr. The relative loss decreased rapidly within the first hour, reaching a relatively stable value after 2 hr. The significant reduction in the loss of AZdU and AZT in vivo compared with that in vitro likely results from a lower diffusion coefficient in tissue. The distribution of AZT between plasma and cerebrospinal fluid (CSF) in the ventricle and extracellular fluid (ECF) in thalamus was determined at steady state using calibration by RD and ZNF simultaneously. The relative loss of AZdU during continuous RD was not significantly different from the recovery of AZT determined by ZNF in the same animal. Since RD may allow for continuous monitoring of microdialysis recovery in real time, it may offer an advantage over the ZNF method of system calibration. The steady-state C csf/C p and C ecf/C p ratios for AZT in this study were 0.26 ± 0.08 and 0.18 ± 0.08. That these ratios are much less than unity suggests that carrier-mediated transport of AZT exists in the brain-to-plasma direction.

Decreased protein-stabilizing effects of cryoprotectants due to crystallization.

Abstract: The stabilizing effects of various additives against inactivation of an enzyme (β-galactosidase from Aspergillus oryzae) during freeze-drying were studied, with a focus on their crystallinity. The crystalline morphology of mannitol and inositol in freeze-dried cakes depended on the solute concentrations before freezing and the freeze-drying method used. The additives in their amorphous state showed concentration-dependent stabilization of the enzyme, whereas additive crystallization during freeze-drying decreased their effects. Heat treatment before freeze-drying also caused crystallization and diminished the stabilizing effects. Noncovalent soluble aggregates were observed in the inactivated enzyme solution. These results show the importance of maintaining the amorphous state of additives used as stabilizing agents during freeze-drying.

Design of New Formulations for Topical Ocular Administration: Polymeric Nanocapsules Containing Metipranolol

Abstract: To investigate the potential of polymeric nanocapsules for ocular delivery of beta-blockers, several formulations of polyisobutylcyanoacrylate and polyepsiloncaprolactone nanocapsules containing metipranolol base were developed. These formulations differed in the polymer forming the coating and in the type and volume of the oil encapsulated. Analysis of particle-size distribution, electrophoretic mobility, and loading efficiency of the nanocapsules revealed that the type of oil is the most important factor influencing these properties. From the in vitro release studies, we concluded that drug diffusion through a dialysis membrane is delayed as a consequence of the encapsulation process. However, the release profiles were not influenced by the polymeric coating, suggesting that drug release from these systems is governed mainly by the partition of the drug between the oily core and the aqueous release medium. Nevertheless, despite the inability of the polymer coat to control the release of the drug, its contribution to the stabilization of the emulsion was noted. Finally, the suitability of these formulations for ophthalmic administration was investigated. Although the pharmacologic response was not affected by the encapsulated metipranolol compared with the commercial eye drops, a drastic reduction of the drug's systemic side effects was observed.

Pulmonary Delivery of Free and Liposomal Insulin

Abstract: The effects of oligomerization and liposomal entrapment on pulmonary insulin absorption were investigated in rats using an intratracheal instillation method. The results indicated that both dimeric and hexameric insulins can be rapidly absorbed into the systemic circulation, producing a significant hypoglycemic response. Intratracheal instillation of insulin in two different oligomerized states has not resulted in any significant difference in the duration of hypoglycemic effect. However, the initial hypoglycemic response (first 10 min) obtained from intratracheal administration of 25 IU/kg hexameric insulin appears to be slower than that from the 25 IU/kg dimeric insulin, thereby suggesting that hexameric insulin may have a lower permeability coefficient across alveolar epithelium than the dimeric insulin. Intratracheal administration of insulin liposomes (dipalmitoylphosphatidyl choline:cholesterol, 7:2) led to facilitated pulmonary uptake of insulin and enhanced the hypoglycemic effect. Nevertheless, similar insulin uptake and pharmacodynamic response were obtained from both the physical mixture of insulin and blank liposomes and liposomally entrapped insulin.

Convective solvent flow across the skin during iontophoresis

Abstract: Enhanced flux of neutral solutes during transdermal iontophoresis is attributed largely to electroosmotic volume flow. In this study, the iontophoretic fluxes of tritiated water (3H2O) and 14C-labeled mannitol through hairless mouse skin (HMS) were determined. The following questions were addressed: (i) What is the variability of water flux during iontophoresis? (ii) To what extent is the iontophoretic flux of a neutral solute correlated with water flux? (iii) Does the intrinsic permeability of the skin to neutral solutes change following iontophoresis? (iv) What is the effect of low pH on electroosmotic volume flow? and (v) Does the skin remain permselective after removal of the stratum corneum? Transport of both water and mannitol reached steady-state levels during 10 hr of constant-current iontophoresis (0.36 mA/cm2). Anodal fluxes exceeded cathodal values. Cathodal mannitol flux was retarded, relative to passive transport, by net volume flow in the opposite direction, such that transport of this molecule increased significantly after the termination of current passage. Anodal equivalent volume flows for water and mannitol, respectively, were 2.7 (±1.3) and 1.23 (±0.59) µL/hr cm2, indicating that only ~50% of the water flux participated in the electroosmosis of mannitol. The passive permeability of water and mannitol after 10 hr of iontophoresis was, respectively, 6 and 30 times greater than the pretreatment values. At pH 7, the cationic permselectivity of HMS was marginal [the Na+ transport number ( $$t_{Na} + $$ ) was determined to be 0.46] and less than that reported for human skin. Lowering the pH values of the solutions on either side of the skin to slightly less than 4 reversed the direction of net volume flow; cathodal flux was greater than anodal flux. When the donor solution was at pH 3.8 and the receptor was pH 7.4, the flux profile was complicated and net volume flow was not obvious. Finally, it was found that electroosmosis from anode to cathode was retained even following removal of the stratum corneum by tape-stripping.

Chemical Pathways of Peptide Degradation. IV. Pathways, Kinetics, and Mechanism of Degradation of an Aspartyl Residue in a Model Hexapeptide

Abstract: In this study the hexapeptide Val-Tyr-Pro-Asp-Gly-Ala (Asp-hexapeptide) was used as a model to investigate the kinetics of aspartate degradation in aqueous solution. The apparent rate of degradation of the Asp-hexapeptide was determined as a function of pH, buffer concentration, and temperature. At very acidic pH levels (0.3, 1.1, 1.5, 2.0, and 3.0), the apparent rate of degradation followed pseudo-first-order kinetics. In this pH region, the Asp-hexapeptide predominantly underwent specific acid-catalyzed hydrolysis of the Asp-Gly amide bond (Asp-X hydrolysis) to form a tetrapeptide (Val-Tyr-Pro-Asp) and a dipeptide (Gly-Ala). In addition, parallel formation of a cyclic imide intermediate could be observed, although no isoAsp-hexapeptide was detected. At pH 4.0 and 5.0, the Asp-hexapeptide simultaneously isomerized via the cyclic imide to form the iso-Asp-hexapeptide and underwent Asp-X hydrolysis to produce the cleavage products. The pH-rate profiles (pH 0.3–5.0) for the Asp-X hydrolysis and the formation of cyclic imide revealed that the degree of ionization of the carboxylic acid side chain of Asp residue significantly altered the rate of reaction, with the ionized form being more reactive than the unionized form. Little or no buffer catalysis was observed for either pathway. Solvent isotope experiments were used to probe the mechanism of the Asp-X hydrolysis reaction. At pH values above 6.0, the apparent rate of degradation of the Asp-hexapeptide followed pseudo-first-order reversible kinetics, with the isoAsp-hexapeptide being the only observed product (isomerization). Above pH 8.0, the isomerization kinetics were found to be independent of pH and buffer concentration. The kinetics of degradation of Asp-hexapeptide (Val-Tyr-Pro-Asp-Gly-Ala) and Asn-hexapeptide (Val-Tyr-Pro-Asn-Gly-Ala) were compared to determine the relative instability of the Asp and Asn residues and to understand the mechanism of formation of cyclic imide at near neutral to basic pH.

Biotechnology and bone graft substitutes.

Abstract: Trauma, disease, developmental deformities, and tumor resection frequently cause bone defects that seriously challenge the skills of orthopedic and maxillofacial surgeons. Currently, repairing osseous deficiencies involves various medical surgical techniques, including autogenous grafts, allografts, internal and external fixation devices, electrical stimulation, and alloplastic implants. The existing technology, though effective in many cases, still is beset with numerous difficulties and disadvantages. A critical need for improved treatment methods exists today. Biotechnology now provides access to new bone repair concepts via administration of protein growth and morphogenic factors. Implantable device and drug delivery system technologies also have advanced. The converging biopharmaceutical, device, and delivery technologies represent an opportunity to improve the quality of health care for individuals with orthopedic and maxillofacial deficiencies. This report reviews current concepts in fracture healing and bone repair and examines existing treatment modalities. It also addresses novel protein drugs that stimulate osseous regeneration and delivery systems for these drugs.

Reverse iontophoresis : development of a noninvasive approach for glucose monitoring

Abstract: Solvent flow generated during iontophoresis can be used to convect neutral molecules through the skin, thereby greatly enhancing their flux. This concept was exploited to realize noninvasive glucose measurement by its iontophoretic extraction from the subcutaneous tissue. The hypothesis was tested in vitro using hairless mouse skin. The dermal surface was bathed with a glucose solution; chambers on the epidermal surface housed the current delivery electrodes. Iontophoresis (at 0.36 mA/cm2) was performed for 2 hr, at the end of which the solutions in contact with the electrodes were analyzed. The amount extracted was proportional to the glucose solution concentration bathing the dermis. Higher radioactivity levels were found at the anode than at the cathode, possibly because of glucose metabolism during its outward transport across the skin. Glucose biotransformation results in negatively charged metabolites which migrate to the anode. Two sensitive glucose sensors were developed; one was selective for glucose, the other for glucose and related compounds. Both sensors indicated the presence of glucose at the cathode but an abnormally high value was also recorded at the anode. This signal, however, was not due to glucose but rather to electroactive ascorbate withdrawn from the skin. Finally, a system has been developed with which glucose can be extracted noninvasively from the subcutaneous tissue and unambiguously measured. Whether iontophoretic glucose sampling in vivo will be equally successful remains to be answered.

The interconversion kinetics, equilibrium, and solubilities of the lactone and hydroxyacid forms of the HMG-CoA reductase inhibitor, CI-981

Abstract: The pH dependence of the interconversion kinetics, equilibrium, and solubilities of the lactone and hydroxyacid forms of the HMG-CoA reductase inhibitor, CI-981 ([R-(R*,R*)]-2-(4-fluorophenyl)- beta,delta-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenylamino)carbonyl ]- 1H-pyrrole-1-hepatonic acid), are important considerations when choosing and developing one of the forms of these compounds. Over a pH range of 2.1 to 6.0 and at 30 degrees C, the apparent solubility of the sodium salt of CI-981 (i.e., the hydroxyacid form) increases about 60-fold, from 20.4 micrograms/mL to 1.23 mg/mL, and the profile yields a pKa for the terminal carboxyl group of 4.46. In contrast, over a pH range of 2.3 to 7.7 and also at 30 degrees C, the apparent solubility of the lactone form of CI-981 varies little, and the mean solubility is 1.34 (+/- 0.53) micrograms/mL. The kinetics of interconversion and the equilibrium between the hydroxyacid and the lactone forms have been studied as a function of pH, buffer concentration, and temperature at a fixed ionic strength (0.5 M) using a stability-indicating HPLC assay. The acid-catalyzed reaction is reversible, whereas the base-catalyzed reaction can be treated as an irreversible reaction. More specifically, at pH 6, the equilibrium reaction is no longer detectable and greatly favors the hydroxyacid form.(ABSTRACT TRUNCATED AT 250 WORDS)