Efficacy and pharmacokinetics of intravitreal non-steroidal anti-inflammatory drugs for intraocular inflammation.

OBJECTIVE: To determine the efficacy and pharmacokinetics of intraocularly delivered non-steroidal anti-inflammatory drugs in an animal model of ocular inflammation. METHODS: Lipopolysaccharide was injected into the vitreous of rabbit eyes to induce inflammation. Treated eyes were injected with 3 mg of ketorolac or 0.3 mg of diclofenac. Twenty-four hours later, total leucocyte concentrations and prostaglandin E2 concentrations were determined. For intraocular pharmacokinetics, 0.1 ml of ketorolac (3 mg) and 0.1 ml of diclofenac (0.3 mg) were injected into rabbit eyes. Reverse-phase high-performance liquid chromatography was used to analyse drug levels within the retina/choroid at 0.25 (15 min), 1, 2, 4, 24, and 48 h after injection. RESULTS: Eyes treated with ketorolac and diclofenac demonstrated reduced aqueous leucocyte concentrations of 62% and 64% respectively, compared with untreated controls (p<0.05). Ketorolac and diclofenac reduced aqueous prostaglandin E2 levels by 85% (p<0.005) and 59% (p<0.005), respectively. Ketorolac and diclofenac achieved a peak vitreous concentration of 234 and 73 microg/ml, respectively. After 48 h, ketorolac was barely detectable (0.06 microg/ml) in the vitreous, and diclofenac was undetectable. The peak concentration of each drug in the retina/choroid was 201 microg/g for ketorolac and 4.1 microg/g for diclofenac. Both drugs were undetectable in the retina/choroid after 48 h. CONCLUSIONS: Both ketorolac and diclofenac have potent anti-inflammatory effects after intraocular injection. Pharmacokinetic analysis demonstrated good penetration into the retina/choroid but rapid clearance by 48 h.

Intravenous transferrin, RGD peptide and dual-targeted nanoparticles enhance anti-VEGF intraceptor gene delivery to laser-induced CNV.

Choroidal neovascularization (CNV) leads to loss of vision in age-related macular degeneration (AMD), the leading cause of blindness in adult population over 50 years old. In this study, we developed intravenously administered, nanoparticulate, targeted nonviral retinal gene delivery systems for the management of CNV. CNV was induced in Brown Norway rats using a 532 nm laser. We engineered transferrin, arginine-glycine-aspartic acid (RGD) peptide or dual-functionalized poly-(lactide-co-glycolide) nanoparticles to target delivery of anti-vascular endothelial growth factor (VEGF) intraceptor plasmid to CNV lesions. Anti-VEGF intraceptor is the only intracellularly acting VEGF inhibitory modality. The results of the study show that nanoparticles allow targeted delivery to the neovascular eye but not the control eye on intravenous administration. Functionalizing the nanoparticle surface with transferrin, a linear RGD peptide or both increased the retinal delivery of nanoparticles and subsequently the intraceptor gene expression in retinal vascular endothelial cells, photoreceptor outer segments and retinal pigment epithelial cells when compared to nonfunctionalized nanoparticles. Most significantly, the CNV areas were significantly smaller in rats treated with functionalized nanoparticles as compared to the ones treated with vehicle or nonfunctionalized nanoparticles. Thus, surface-functionalized nanoparticles allow targeted gene delivery to the neovascular eye on intravenous administration and inhibit the progression of laser-induced CNV in a rodent model.

Budesonide reduces vascular endothelial growth factor secretion and expression in airway (Calu-1) and alveolar (A549) epithelial cells.

Vascular endothelial growth factor (VEGF), a cytokine expressed in the respiratory epithelial cells, induces vascular hyperpermeability and edema, symptoms that are alleviated by budesonide, an anti-asthma corticosteroid. However, modulation of VEGF levels by budesonide in the respiratory epithelium has not been studied. In this study, we investigated the mechanisms of VEGF secretion using brefeldin A and monensin in human airway (Calu-1) and alveolar (A549) epithelial cells, and further determined whether budesonide inhibits VEGF secretion and mRNA expression through a glucocorticoid receptor-mediated mechanism. In both cell types, VEGF secretion was inhibited by brefeldin A and monensin, suggesting vesicular transport of VEGF through endoplasmic reticulum (ER)-golgi pathway. At concentrations devoid of cytotoxicity, budesonide reduced VEGF secretion and VEGF mRNA expression in both cell types and these effects were inhibited by mifepristone (RU 486), a glucocorticoid receptor antagonist, suggesting that budesonide reduces VEGF secretion and expression through its glucocorticoid receptor-mediated action. Also, budesonide-mediated inhibition of VEGF mRNA was time- and protein synthesis-dependent. Thus, budesonide may be of potential value in treating disorders of the respiratory tract that are associated with VEGF elevation.

Expression of multidrug resistance-associated protein (MRP) in human retinal pigment epithelial cells and its interaction with BAPSG, a novel aldose reductase inhibitor.

PURPOSE: The objective of this study was to determine the expression and activity of multidrug resistance-associated protein (MRP) in the retinal pigment epithelial (RPE) cells and to further assess whether BAPSG, a novel anionic aldose reductase inhibitor, interacts with MRP. METHODS: Functional and biochemical evidence for MRP was obtained in a human retinal pigment epithelial (ARPE-19) cell line and primary cultures of human retinal pigment epithelial (HRPE) cells. Fluorescein accumulation and efflux in the presence and absence of MRP inhibitors was used to obtain functional evidence for MRP. Western blots and RT-PCR were used to obtain biochemical evidence for MRP1. The influence of MRP inhibitors on BAPSG accumulation and efflux in ARPE-19 cells was determined to understand its interaction with MRP. RESULTS: MRP inhibitors increased fluorescein accumulation and reduced efflux in RPE cells. Both cell types exhibited a 190-kDa western blot band corresponding to MRP1 protein and a 287 bp RT-PCR band corresponding to MRP1 mRNA. MRP inhibitors reduced BAPSG efflux and increased its accumulation in ARPE-19 cells. CONCLUSIONS: MRP is functionally and biochemically active in human RPE cells. Anionic BAPSG is a likely substrate for MRP.

Preparation of drug delivery systems using supercritical fluid technology.

Small changes in temperature and pressure near the critical region induce dramatic changes in the density and solubility of supercritical fluids, thereby facilitating the use of environmentally benign agents such as CO2 for their solvent and antisolvent properties in processing a wide variety of materials. While supercritical fluid technologies have been in commercial use in the food and chromatography industries for several years, only recently has this technology made inroads in the formulation of drug delivery systems. This review summarizes some of the recent applications of supercritical fluid technology in the preparation of drug delivery systems. Drugs containing polymeric particles, plain drug particles, solute-containing liposomes, and inclusion complexes of drug and carrier have been formulated using this technology. Also, polymer separation using this technology is enabling the selection of a pure fraction of a polymer, thereby allowing a more precise control of drug release from polymeric delivery systems.

Delivery systems for penetration enhancement of peptide and protein drugs: design considerations.

This paper discusses the challenges to be met in designing delivery systems that maximize the absorption of peptide and protein drugs from the gastrointestinal and respiratory tracts. The ideal delivery system for either route of administration is one that will release its contents only at a favorable region of absorption, where the delivery system attaches by virtue of specific interaction with surface determinants unique to that region and where the delivery system travels at a rate independent of the transitory constraints inherent of the route of administration. Such a delivery system, which is as yet unavailable, will benefit not only peptide and protein drugs, but other poorly absorbed drugs.

Interaction of [D-Trp6, Des-Gly10] LHRH ethylamide and hydroxy propyl beta-cyclodextrin (HPbetaCD): thermodynamics of interaction and protection from degradation by alpha-chymotrypsin.

PURPOSE: The purpose of this study is to investigate the mechanisms and thermodynamics of the interaction between hydroxypropyl beta-cyclodextrin (HPdetaCD) and [D-Trp6, des-Gly10] LHRH ethylamide (deslorelin), a peptide drug. METHODS: We used UV and fluorescence spectroscopy to study the interaction of HPbetaCD and deslorelin. Circular dichroism was used to study the conformational changes induced in deslorelin upon interaction with HP beta CD. The thermodynamics of the interaction of deslorelin and HPbetaCD was studied using isothermal titration calorimetry (ITC). We also determined the effect of HPbetaCD on the degradation of deslorelin by alpha-chymotrypsin. RESULTS: UV and fluorescence spectroscopy indicated that HPbetaD induced a change in polarity of the environment surrounding the chromophores of deslorelin. Wavelength selective fluorescence indicated an increase in the fluorescence polarization of deslorelin with an increase in excitation wavelength in the presence of HPbetaCD suggesting that tryptophan is present in a media of reduced mobility. Circular dichroism studies indicated that HPbetaCD stabilizes the conformation of deslorelin. In addition, ITC indicated an exothermic reaction between deslorelin and HPbetaCD with a low enthalpy of binding of approximately -600 cal/mol and a binding affinity of approximately -1.25 x 10(2) M-1. Finally, the rate of degradation of deslorelin by alpha-chymotrypsin was decreased by 33% in the presence of HPbetaCD. CONCLUSIONS: These results indicate that there is an interaction between HPbetaCD and deslorelin, which involves the inclusion of aromatic amino acids of deslorelin into the hydrophobic cavity of the cyclodextrin. This inclusion, providing steric hindrance, may be one of the mechanisms by which HPbetaCD reduces enzymatic hydrolysis of deslorelin.

In-vitro hydrolysis, permeability, and ocular uptake of prodrugs of N-[4-(benzoylamino)phenylsulfonyl]glycine, a novel aldose reductase inhibitor.

To enhance the ocular uptake of N-[4-(benzoylamino)phenylsulfonyl]glycine (BAPSG), two ester (methyl and isopropyl) prodrugs were synthesized and evaluated for their stability in various buffers (pH 1-9), hydrolysis in rabbit ocular tissues (cornea, conjunctiva, iris-ciliary body, lens, aqueous humor, and vitreous humor), transport across cornea and conjunctiva, and in-vivo uptake following topical administration. Over the pH range of 1-9, the rate constants for degradation ranged from 5.67 to 218.9 x 10(-3) h(-1) for the methyl ester and from 3.14 to 4.45 x 10(-3) h(-1) for the isopropyl ester. At all pH conditions, the isopropyl ester was more stable when compared with the methyl ester. A change in buffer concentration at pH 7.4 did not influence the stability of the prodrugs. The prodrugs were rapidly hydrolysed in the tissue homogenates, with the rate constants for hydrolysis ranging from 1.98 to 7.2x 10(-3) min(-1) for the methyl ester and 3.32 to 6.53 x 10(-3) min(-1) for the isopropyl ester. The in-vitro permeability of the methyl ester was less than the parent drug across cornea and conjunctiva. Isopropyl ester levels were not detectable in the receiver chamber even at the end of the 4-h transport study. Following topical administration of BAPSG and the two prodrugs at a dose of 60 microg/eye, the lowest levels were seen in vitreous humor for parent compound and its methyl ester. In general, the tissue uptake of methyl ester was less than BAPSG. Isopropyl ester levels were below detection limits in all the ocular tissues. Lipophilic ester prodrugs of BAPSG showed good aqueous solution stability in tissue homogenates. However, these prodrugs lacking the free carboxylate anion exhibited reduced in-vitro permeability and in-vivo uptake, suggesting the importance of free carboxylate anion in the delivery of BAPSG.

Drug efflux pumps: challenges and opportunities. 14-18 November 1999, New Orleans, LA, USA.

A symposium entitled 'Drug efflux pumps: challenges and opportunities' addressed the detection and functional characterization of drug efflux pumps, their influence on the cytochrome P450 (CYP450) system and their role in blood-brain barrier (BBB) permeability. Drug efflux pumps utilize ATP as the energy source in exporting solutes, and belong to the family of ATP-binding cassette transporters (ABC transporters). P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP) are members of this family of transporters. These transporters are overexpressed in cancer cells leading to decreased drug accumulation and hence drug resistance. Another causative factor for drug resistance is increased elimination mechanisms, metabolism by CYP450 enzymes being the most important. The multidrug resistance gene (mdr) is a key determinant in the expression of CYP3A enzymes, the most predominant subclass of CYP450. New techniques are being developed to identify and characterize drug efflux pumps, the latest being gamma-scintigraphy and hypertonic saline (HTS). Efflux pumps play a major role in restricting the drug transport across the BBB. Currently, structural domains that are responsible for the functional activity of the efflux proteins are being investigated to design better inhibitors for drug efflux pumps.

Rabbit corneal and conjunctival permeability of the novel aldose reductase inhibitors: N-[[4-(benzoylamino)phenyl] sulphonyl]glycines and N-benzoyl-N-phenylglycines.

Corneal and conjunctival permeability has been investigated for novel aldose reductase inhibitors (ARIs) of the N{[4-(benzoylamino)phenyl]sulphonyl}glycine (benzoylaminophenylsulphonylglycine) and N-benzoyl-N-phenylglycine (benzoylphenylglycine) series, compounds developed for prevention of cataract formation in diabetic subjects. Six benzoylaminophenylsulphonylglycines were synthesized with modifications either of the phenyl group or of the glycine structure and three benzoylphenylglycines were synthesized with modification in the phenyl group of the benzoyl moiety. Transport of ARIs in the mucosal to serosal direction was evaluated across rabbit cornea and conjunctiva bathed in glutathione-bicarbonate Ringer's solution maintained at pH 7.4 and 37 degrees C. The permeability coefficients of the novel ARIs across cornea and conjunctiva ranged from 1.87 to 8.95 x 10(-6) cm s(-1) and from 4.6 to 19.15 x 10(-6) cm s(-1), respectively. The ratio of corneal to conjunctival permeability ranged from 0.12 to 0.79. The calculated log partition coefficient (log P) values for the ARIs were in the range 0.84 to 2.78. The log distribution coefficients (log D) were in the range -2.87 to -0.89. There was no apparent relationship between log P or log D and the permeability coefficients of the ARIs for either tissue. Cornea was more resistant to ARI transport than was conjunctiva. Substitution of a phenyl group for hydrogen in the glycine methylene group reduced the permeability coefficient. Permeability coefficients were different for different stereoisomers. Compared with the permeability coefficient of benzoylaminophenylsulphonylglycine, that of 4-fluorobenzoylaminophenylsulphonylglycine was lower in the cornea but similar in the conjunctiva. In both tissues, the permeability coefficient of 2-nitrobenzoylaminophenylsulphonylglycine was less than that of 4-nitrobenzoylaminophenylsulphonylglycine. There was no significant difference between the permeability coefficients of 3-nitro- and 4-nitrobenzoylphenylglycines through either tissue and the permeability coefficients of these compounds were greater than that of the more lipophilic 4-methylbenzoylphenylglycine. The lack of dependence of the permeability coefficients on log P or log D and the different permeabilities of stereoisomers imply the existence of specialized transport processes for the ARIs tested in this study.

Influence of pH and temperature on kinetics of ceftiofur degradation in aqueous solutions.

The objective of this study was to evaluate the stability of ceftiofur (1 mg mL(-1)) in aqueous solutions at various pH (1, 3, 5, 7.4 and 10) and temperature (0, 8, 25, 37 and 60 degrees C) conditions. The ionic strength of all these solutions was maintained at 0.5 M. Ceftiofur solutions at pH 5 and 7.4 and in distilled water (pH = 6.8) were tested at all the above temperatures. All other solutions were tested at 60 degrees C. Over a period of 84 h, the stability was evaluated by quantifying ceftiofur and its degradation product, desfuroylceftiofur, in the incubation solutions. HPLC was used to analyse these compounds. At 60 degrees C, the rate of degradation was significantly higher at pH 7.4 compared with pH 1, 3, 5 and distilled water. At both 60 degrees C and 25 degrees C, degradation in pH 10 buffer was rapid, with no detectable ceftiofur levels present at the end of 10 min incubation. Degradation rate constants of ceftiofur were 0.79+/-0.21, 0.61+/-0.03, 0.44+/-0.05, 1.27+/-0.04 and 0.39+/-0.01 day(-1) at pH 1, 3, 5, 74 and in distilled water, respectively. Formation of desfuroylceftiofur was the highest (65%) at pH 10. The rate of degradation increased in all aqueous solutions with an increase in the incubation temperature. At pH 7.4 the degradation rate constants were 0.06+/-0.01, 0.06+/-0.01, 0.65+/-0.17, and 1.27+/-0.05 day(-1) at 0, 8, 25, 37 and 67 degrees C, respectively. The energy of activation for ceftiofur degradation was 25, 42 and 28 kcal mol(-1) at pH 5, 7.4 and in distilled water, respectively. Desfurylceftiofur formation was the greatest at alkaline pH compared with acidic pH. Ceftiofur degradation accelerated the most at pH 7.4 and was most rapid at pH 10. The results of this study are consistent with rapid clearance of ceftiofur at physiological pH.

Ceftiofur distribution in plasma and joint fluid following regional limb injection in cattle.

The objective of this study was to evaluate the efficacy of regional intravenous (i.v.) injection of ceftiofur in delivery of this drug to joint fluid and plasma in a limb distal to a tourniquet in five, healthy, adult, mixed breed beef cattle. A tourniquet was positioned in the mid-metacarpal region, and 500 mg of ceftiofur was administered through a catheter in the dorsal common digital vein (DCDV). Plasma samples were collected from the catheter at 15, 30 and 45 min postinjection, and from the abaxial proper palmar vein (APPV) at 15 min postinjection. Synovial fluid was collected from the metacarpal phalangeal joint at 45 min postinjection. Ceftiofur concentrations were estimated in plasma and synovial fluid using high-pressure liquid chromatography (HPLC) and a microbiological assay utilizing Pasteurella haemolytica as the test organism. Both assays indicated highest plasma concentrations of ceftiofur at 15 min, with the concentrations declining with time. Concentrations of ceftiofur in plasma obtained from the DCDV were not significantly different from APPV levels, indicating rapid distribution of ceftiofur within the limb. Microbiological assay always demonstrated higher concentrations of ceftiofur compared with HPLC assay, because the former probably also detected the active metabolites of ceftiofur as well as the parent compound. At 45 min, ceftiofur concentrations determined by HPLC were 251+/-97 and 15+/-5 microg/mL in plasma and synovial fluid, respectively. Regional intravenous injection appears to be a feasible technique to produce rapid distribution of ceftiofur within the limb well above therapeutic concentrations.

Disposition of non-viral gene delivery systems and oligonucleotides.

At the 1998 annual meeting of the American Association of Pharmaceutical Scientists (AAPS), held in San Francisco, there was a roundtable discussion meeting entitled 'Disposition of Non-viral Gene Delivery Systems and Oligonucleotides'. Four speakers presented the advances made by their research groups in this area. The non-viral delivery systems utilized lipid, peptide, and polymeric carriers. The disposition of naked plasmid DNA was compared with complexed and condensed DNA. Although the disposition kinetics were more favorable with cationic systems, the expression levels obtained from naked DNA were superior. Interpolyelectrolyte complexes (IPECs) containing pluronic units are efficient for transfection. With the FDA approval of the first antisense oligonucleotide for therapeutic applications, there is growing interest in the development of antisense oligonucleotides. ISIS Pharmaceuticals Inc (Carlsbad, CA, USA) is evaluating various routes of delivery for oligonucleotides, including oral, rectal, inhalation, intravitreal and dermal routes.

Drug delivery applications of supercritical fluid technology.

At the 1998 annual meeting of the American Association of Pharmaceutical Scientists (AAPS) held in San Francisco, a symposium entitled 'Drug Delivery Applications of Supercritical Fluid Technology' was presented. Five speakers presented the advances made by their research groups in this area. Various approaches using supercritical fluids as solvents, antisolvents or mechanical agents were employed in forming particles or delivery systems of various drugs, including antibiotics, steroids and macromolecules. The particles obtained with this technology were small, free-flowing, easily wettable and have minimal surface charges. A coating unit employing supercritical CO2 to fluidize the substrates and to remove solvent has been reported. Nasal delivery of insulin was enhanced when the formulations were processed with supercritical CO2. Macromolecules such as insulin and trypsin dissolved in a carrier liquid could be incorporated in preformed microspheres upon supercritical fluid exposure. Thus supercritical fluid technology appears to be a promising new approach to process drugs and their dosage forms.

Nasal route for direct delivery of solutes to the central nervous system: fact or fiction?

During this century, several investigators reported that certain viruses, metals, drugs, and other solutes could bypass systemic circulation and enter the brain and/or cerebrospinal fluid directly following nasal administration. Although evidence clearly suggests that the olfactory epithelium and its olfactory cells play a major role, little is known about the mechanisms of direct transport of solutes into the brain. An overview of what is known about these mechanisms may aid in further research in this field, including studies of direct drug delivery to the central nervous system. This review, in addition to summarizing the literature to date, clearly describes the intricate association of the anatomical features involved in direct entry of solutes into the brain following nasal administration. To aid in the understanding of the possible routes a solute can take after nasal administration, the anatomy of the olfactory epithelium and surrounding tissues is described, and a detailed scheme delineating the emerging pathways is presented. Techniques used in delineating these pathways and studies supporting a particular pathway are discussed in greater detail. Finally, some factors influencing the direct transport of solutes to the cerebrospinal fluid and brain are summarized.

Inhibition of corneal metabolism of deslorelin by EDTA and ZnCl2.

It was the aim of this study to determine whether deslorelin is degraded by the rabbit corneal tissue and to further delineate the mechanisms. Deslorelin was incubated with intact cornea either alone or in the presence of 0.1 mM ouabain, 0.1% 2,4-dinitrophenol, 0.1 mM phosphoramidon, 0.1 mM N-tosyl-L-phenylalanine chloromethylketone (TPCK), 0.1-2% EDTA, 0.1-1% ZnCl2, 0.1% dithiothreitol (DTT), or 0.1% N-ethylmaleimide (NEM) at 37 degrees C. In addition, deslorelin alone was incubated with cornea at 4 degrees C. Following a 90-min incubation, the supernatants were analyzed using a reversed-phase HPLC. Metabolite peaks observed in controls at 37 degrees C were not detected in the low-temperature study, suggesting inhibition of metabolism at low temperature. Intact drug remaining in the supernatant was not altered by ouabain and dinitrophenol, suggesting that energy-dependent corneal uptake is not likely for deslorelin. Phosphoramidon and TPCK failed to alter deslorelin levels, indicating that phosphoramidon and TPCK-sensitive endopeptidases did not contribute to the observed metabolism. DTT and NEM also failed to affect deslorelin levels. However, 2% EDTA and 1% ZnCl2 significantly elevated the intact deslorelin levels by 44 and 60%, respectively, and the metabolite peaks almost completely disappeared. These observations are consistent with the corneal metabolism of deslorelin by either metallo-peptidases or metal-dependent peptidases.

Metabolism of [des-Gly10, D-Trp6]LHRH ethylamide in the rabbit conjunctiva.

It was the objective of this study to determine whether [des-Gly10, D-Trp6]LHRH ethylamide, an LHRH agonist known as deslorelin, is degraded by the rabbit conjunctiva. Intact conjunctiva was incubated with deslorelin either alone or in the presence of 0.1 mM ouabain, 0.1% 2,4-dinitrophenol, 0.1 mM phosphoramidon, 0.1 mM N-tosyl-L-phenylalanine chloromethylketone (TPCK), 2% EDTA, 1% ZnCl2, 0.1% dithiothreitol (DTT), or 0.1% N-ethylmaleimide (NEM) at 37 degrees C. Furthermore, deslorelin alone was incubated with conjunctiva at 4 degrees C. All incubation solutions were made isotonic, and the pH was adjusted to 5.0. A reversed-phase HPLC was used to analyze supernatants collected at the end of 90 min. Deslorelin metabolism was inhibited at low temperature, as suggested by the disappearance of metabolite peaks at low temperature. Both ouabain and dinitrophenol failed to alter the intact drug remaining in the supernatant, indicating that energy-dependent cellular uptake of deslorelin is unlikely in the conjunctiva. Phosphoramidon- and TPCK- sensitive endopeptidases did not contribute to the observed metabolism, as suggested by the lack of effect of phosphoramidon and TPCK on deslorelin levels. DTT and NEM also failed to affect deslorelin levels. On the other hand, EDTA and ZnCl2 significantly elevated the intact deslorelin levels by 61 and 53%, respectively, and almost completely abolished the metabolite peaks, indicating a possible role for either metaldependent peptidases or metallo-peptidases in the conjunctival metabolism of deslorelin.