Protein engineering to increase the potential of a therapeutic antibody Fab for long-acting delivery to the eye

To date, ocular antibody therapies for the treatment of retinal diseases rely on injection of the drug into the vitreous chamber of the eye. Given the burden for patients undergoing this procedure, less frequent dosing through the use of long-acting delivery (LAD) technologies is highly desirable. These technologies usually require a highly concentrated formulation and the antibody must be stable against extended exposure to physiological conditions. Here we have increased the potential of a therapeutic antibody antigen-binding fragment (Fab) for LAD by using protein engineering to enhance the chemical and physical stability of the molecule. Structure-guided amino acid substitutions in a negatively charged complementarity determining region (CDR-L1) of an anti-factor D (AFD) Fab resulted in increased chemical stability and solubility. A variant of AFD (AFD.v8), which combines light chain substitutions (VL-D28S:D30E:D31S) with a substitution (VH-D61E) to stabilize a heavy chain isomerization site, retained complement factor D binding and inhibition potency and has properties suitable for LAD. This variant was amenable to high protein concentration (>250 mg/mL), low ionic strength formulation suitable for intravitreal injection. AFD.v8 had acceptable pharmacokinetic (PK) properties upon intravitreal injection in rabbits, and improved stability under both formulation and physiological conditions. Simulations of expected human PK behavior indicated greater exposure with a 25-mg dose enabled by the increased solubility of AFD.v8.     

Reformulation of a new vancomycin analog: An example of the importance of buffer species and strength

The purpose of this research was to use our previously validated dynamic injection apparatus as a rapid method for screening pH-adjusted formulations of a new vancomycin analog, Van-An, for their potential to precipitate upon dilution. In 1 vial, Van-An was reconstituted according to the manufacturer’s instructions. In a separate vial, the Van-An formulation’s existing phosphate buffer species was supplemented with acetate buffer, which has a pKa in the desired range: between the pH values of the formulation (pH 3.9) and blood (pH 7.4). The formulations were injected using the dynamic injection apparatus into a flowing stream of isotonic Sorensen’s phosphate buffer at rates of 0.25, 0.5, 1, and 2 mL/min. The peaks obtained with the spectrophotometer were reproducible for each injection rate/formulation combination. For the phosphate-buffered formulation, the least amount of precipitation was obtained at the 0.25 mL/min injection rate. Acetate buffer was able to substantially reduce such precipitation, even at the highest injection rate. The opacity peaks for the formulation with the acetate addition were significantly smaller (P<.05) than those obtained for the unaltered formulation at all 4 injection rates. The results suggest that acetate is a better buffer species than phosphate for the pH range defined. Furthermore, we present evidence to support a generally applicable approach to screening new formulations of drug products that may be clinically useful for reducing the incidence of phlebitis in humans. Published: January 13, 2006     

Comparison of Suprachoroidal Drug Delivery with Subconjunctival and Intravitreal Routes Using Noninvasive Fluorophotometry

Purpose

To determine whether exposure of sodium fluorescein (NaF) to the choroid-retina region in the posterior segment of the eye is greater with suprachoroidal injection when compared to intravitreal and transscleral routes.

Methods

Suprachoroidal injection, a new approach for drug delivery to the posterior segment of the eye was validated using a 34 G needle and Indian ink injections in Sprague Dawley rats, followed by histology. Delivery of NaF was compared in Sprague Dawley rats after suprachoroidal, posterior subconjunctival, or intravitreal injections. NaF levels were monitored noninvasively up to 6 hours using Fluorotron Master™, an ocular fluorophotometer Pharmacokinetic parameters were estimated using WinNonlin.

Results

Histological analysis indicated localization of India ink to the suprachoroidal space below sclera, following injection. NaF delivery to choroid-retina was in the order: suprachoroidal > intravitreal >posterior subconjunctival injection. Peak NaF concentration (C_max) in choroid-retina was 36-fold (p = 0.001) and 25-fold (p = 0.001) higher after suprachoroidal (2744±1111 ng/ml) injection when compared to posterior subconjunctival (76±6 ng/ml) and intravitreal (108±39 ng/ml) injections, respectively. NaF exposure (AUC_0–360min) to choroid-retina after suprachoroidal injection was 6-fold (p = 0.001) and 2-fold (p = 0.03) higher than posterior subconjunctival and intravitreal injections, respectively. Choroid-retina T_max was observed immediately after dosing with suprachoroidal injections and at 10 and 27.5 minutes, respectively, with subconjunctival and intravitreal injections.

Conclusions

Suprachoroidal injections are feasible in a rat model. Suprachoroidal injections resulted in the highest bioavailability, that is, the extent and rate of delivery of NaF to choroid-retina, when compared to intravitreal and posterior subconjunctival injections. Ocular fluorophotometry is useful for noninvasive monitoring of NaF in rats following administration by various routes including suprachoroidal route.     

Preparation and evaluation of niosome gel containing acyclovir for enhanced dermal deposition

Niosomes suggest a versatile vesicle delivery system with possible transport of drugs via topical route for skin delivery. The aim of the present research was to optimize niosome gel formulation of acyclovir and to evaluate in both in vitro and in vivo rabbit model. Niosome formulations were formulated by coacervation phase separation technique with different ratios of nonionic surfactants, phospholipids and cholesterol using 3² factorial design. Altering the surfactant concentration has influenced the drug entrapment, but not vesicle size. At high surfactant combinations, the acyclovir release from niosomes was strongly influenced by cholesterol:lecithin ratio. Ex vivo drug permeation data indicate substantial difference in flux values and was influenced by the niosome composition. Ex vivo studies using formulation (B₈) for drug deposition indicate greater amount of niosome being diffused into the skin layers and formed a depot, compared to commercial acyclovir cream (control). Two distinct dermatopharmacokinetic profiles were observed, in vivo, for niosome gel formulation (B₈) and control, which were analog to the profiles observed with ex vivo deposition studies. In vivo plasma drug level suggests low systemic exposure of acyclovir (C_max: 9.44?±?2.27?ng/mL and 14.54?±?3.11?ng/mL for niosome formulation and control, respectively). Comparison of kinetic data of acyclovir in the stratum corneum and plasma signifies that the niosome formulation forms a depot in the epidermis or dermis region. This study concludes that the niosome gel formulation (B₈) could be a viable vesicular system for an impressive transdermal delivery of acyclovir by topical application.     

Determination of PF-04928473 in human plasma using liquid chromatography with tandem mass spectrometry

A simple, rapid and sensitive liquid chromatography/tandem mass spectrometric (LC/MS/MS) analytical method was developed for quantification of Hsp90 inhibitor PF-04928473 in human plasma, following administration of its prodrug, PF-04929113. Sample processing involved protein precipitation by addition of 0.4 mL of methanol containing internal standard (PF-04972487) to 50 μL volume of plasma sample. Chromatographic separation of PF-04928473 and PF-04972487 was achieved on a Phenomenex^® Luna C18(2) (2.0mm × 50 mm, 5 μm) column using a gradient elution method with mobile phase solvents: methanol containing 0.1% formic acid and 0.1% formic acid at a flow rate of 0.25 mL/min. Detection was performed in electrospray positive ionization mode, monitoring the ion transitions from m/z 465.1 → 350.1 (PF-04928473) and m/z 447.0 → 329.1 (PF-04972487). The retention times for PF-04928473 and PF-04972487 were 1.86 and 2.85 min, respectively. Calibration curves were generated in the range of 2–2000 ng/mL. The accuracy and precision ranged from 94.1 to 99.0% and 86.7 to 97.6%, respectively, which were calculated using quality control samples of three different concentrations analyzed in quintuplicate on four different days.     

Remediation of undesirable secondary interactions encountered in hydrophilic interaction chromatography during development of a quantitative LC–MS/MS assay for a dipeptidyl peptidase IV (DPP-IV) inhibitor in monkey serum

PF-00734200 (3,3-Difluoropyrrolidin-1-yl)-((2S,4S)-4-(4-(pyrimidin-2-yl) piperazin-1-yl)pyrrolidin-2-yl)methanone) is an inhibitor of dipeptidyl peptidase IV (DPP-IV) for the treatment of diabetic complications and other disorders. A sensitive and selective LC–MS/MS assay capable of quantifying PF-00734200 in monkey serum was required to support regulated safety studies. Due to the polar nature of this compound and for ease of sample processing, hydrophilic interaction chromatography (HILIC) was identified as an ideal assay technique. During the initial phase of method development significant peak tailing was observed. The effects of polar organic modifier percentage, buffer concentration, column particle size, and flow rate were assessed to determine the final optimal conditions. PF-00734200 demonstrated a strong dependence on buffer concentration with respect to height equivalent to a theoretical plate (HETP), capacity factor (k′), and tailing factor (T). Improvements in chromatography were observed with increasing buffer concentration due to reduction of electrostatic secondary interactions with ionized silanols. A plot of log k′ versus percentage organic modifier at an elevated buffer concentration, produced a linear fit with a correlation coefficient of 0.996, indicating that the primary chromatographic retention mechanism was partitioning. A LC–MS/MS assay was successfully developed and validated for GLP bioanalysis of PF-00734200 in monkey serum utilizing the optimized HILIC conditions. Additionally, carryover was effectively minimized through fortification of ethylene glycol to the sample extract.     

Tissue pharmacokinetics and pharmacodynamics of AmBisome® (L-AmBis) in uninfected and infected animals and their effects on dosing regimens

Abstract

By selecting a unique combination of lipids and amphotericin B, the liposome composition for AmBisome® (L-AmBis) has been optimized resulting in a formulation that is minimally toxic, targets to fungal cell walls, and distributes into and remains for days to weeks in various host tissues at drug levels above the MIC for many fungi. Procedures have been standardized to ensure that large scale production of the drug retains the drug’s low toxicity profile, favorable pharmacokinetics and antifungal efficacy. Tissue accumulation and clearance with single or multiple intravenous administration is similar in uninfected and infected animal species, with tissue accumulation being dose-dependent and the liver and spleen retaining the most drug. The efficacy in animals appears to be correlated with drug tissue levels although the amount needed in a given organ varies depending upon the type of infection. The long-term tissue retention of bioactive L-AmBis in different organs suggests that for some indications, prophylactic and intermittent drug dosing would be efficacious reducing the cost and possible toxic side-effects. In addition, preliminary preclinical studies using non-intravenous routes of delivery, such as aerosolized L-AmBis, catheter lock therapy, and intravitreal administration, suggest that alternative routes could possibly provide additional therapeutic applications for this antifungal drug.     

In vivo characterization of a novel GnRH (gonadotropin-releasing hormone) antagonist, LXT-101, in normal male rats

LXT-101 is a newly developed GnRH (gonadotropin-releasing hormone) analogue. In this study, the in vivo pharmacological profile in intact male rats and binding characters of LXT-101 were illustrated, and regulation of mRNA of hormone receptors related to the pituitary-gonadal axis during and after administration was observed to reveal its molecular mechanism of potent effect and reversibility. After single subcutaneous injections, LXT-101 produced a dose- and time-dependent suppression of serum testosterone level. Multiple administrations and osmotic pump implantation revealed that the time of onset and dose needed to maintain the effect of chemical castration decreased as the frequency of injection increased and gave direct proof that depot formulation could significantly improve the duration of antagonist delivery and pharmacological activities compared to the injectable formulation. And LXT-101 showed excellent character of regulating the pituitary-gonadal axis quickly and reversibly. Competitive binding assay showed that LXT-101 could specifically bind a pituitary GnRH receptor with high affinity. These results indicated that LXT-101 is fit for sustained-release formulation and it might possibly be developed as an ideal candidate for treating sex hormone-sensitive tumors and other disorders.     

Prolonged Corticotrophic Action of a Synthetic Substituted α1-18 ACTH

The adrenocorticotrophic effects of a synthetic corticotrophin analogue, α^1-18 ACTH with D-serine¹, lysine¹⁷, and lysine amide¹⁸ substitutions has been studied. It is effective after both intramuscular and subcutaneous administration and compared with tetracosactrin depot (Synacthen depot, Cortrosyn depot) it has a similarly prolonged time course of action—about 24 hours after 0·5 mg and 30 hours after 1 mg. Unlike tetracosactrin depot, however, it is well absorbed when given intranasally and does not produce painful reactions at the site of injection. Its prolonged time course of action does not depend on a formulation designed to delay its release from the injection site but most probably on a decreased rate of degradation in the circulation.     

Induction of local inflammation by recombinant human platelet factor 4 in the mouse

Platelet factor 4 (PF-4) has been shown to be chemotactic for neutrophils and monocytes in vitro. To assess whether these observations have in vivo relevance, we tested the ability of recombinant human PF-4 (rPF-4) to induce acute and chronic dermal inflammation in the mouse. When injected as a single dose intradermally, rPF-4 induced an acute inflammatory response that peaked at 6 to 12 hr and which resolved by 36 hr. Injection of an equivalent amount of cytochrome c, buffer alone, or an amino-terminal PF-4 peptide failed to elicit a significant inflammatory response; however, the carboxy-terminal PF-4 peptide retained proinflammatory properties. The inflammatory infiltrate induced by a single injection of either rPF-4 or the 41 amino acid carboxy-terminal peptide was composed of neutrophils and smaller numbers of mononuclear cells. Repeated injection of rPF-4 resulted in nearly equal numbers of neutrophils and mononuclear cells. Moreover, marked dermal fibrosis developed after only 5 days of daily injection of rPF-4. Although relatively high concentrations of rPF-4 were required to elicit an inflammatory response, these concentrations may be locally attainable during platelet aggregation. Our findings thus support the hypothesis that PF-4 may contribute to the development of inflammatory responses at sites of platelet aggregation.     

Measurement of the rate of aqueous humor flow

Techniques which estimate the rate of aqueous flow generally require the use of tracer substances. Determination of the distribution of the tracer in the relevant body compartments permits calculation of the rate of flow within the limits of accuracy of the method used. The underlying theory, as well as the advantages and limitations of methods employing systemic, topical, intracameral, and intravitreal administration of tracer substances are reviewed. Since these methods all assume that the rate of aqueous secretion is constant, yet the presence of a diurnal rhythm of flow has been demonstrated in both rabbits and humans, a compartmental model of a circadian system based upon the vitreous depot technique is presented. This model estimates the degree to which a continuously changing rate of aqueous flow limits the ability to determine aqueous flow rate accurately by this particular method and illustrates this limitation, which is common to all tracer methods.     

A new extra long acting depot preparation of the LHRH analogue Zoladex®. First endocrinological and pharmacokinetic data in patients with advanced prostate cancer

A new depot formulation of the LHRH analogue Zoladex® (goserelin acetate) has been developed which releases the drug over a period of at least 3 months as judged by measurement of drug content in depots at intervals after insertion in male rats and by the suppression of oestrogen secretion and oestrus in female rats. This formulation is based on the lactide/glycolide polymer system used for the standard 1-month Zoladex® depot, but the dose has been increased to 10.8 mg and the characteristics have been modified to enable a longer release of drug to be achieved.

Thirty-eight patients with histologically proven, locally advanced (stage T3 or T4) and/or metastatic prostate cancer were treated with this new longer acting LHRH analogue depot formulation containing 10.8 mg Zoladex®. After initial increase of serum testosterone in the first week of therapy, castration levels were reached in all patients after 4 weeks and this was maintained for more than 14 weeks. At the time of depot exhaustion, when escape from castration levels of androgen occurred, all patients received a single injection of a standard 1-month depot containing 3.6 mg Zoladex® which restored castration levels of androgen thus showing that the pituitary gland was again suppressed. The tolerance and acceptability of the longer-acting depot is high and comparable to the 1-month depot. Taking into account social and psychological factors, patients with advanced prostate carcinoma will soon be able to be treated with a longer acting LHRH depot formulation every 3 months an alternative of the 1-month depot now widely used clinically.     

The Influence of Modified Pluronic F127 Copolymers with Higher Phase Transition Temperature on Arsenic Trioxide-Releasing Properties and Toxicity in a Subcutaneous Model of Rats

Pluronic F127 (PF-127) shows thermoreversible property, which is of the utmost interest in optimizing drug formulation and delivery. However, its hitherto unresolved drawback of a low phase transition temperature (T (tr)) has limited its application in injectable drug delivery systems. We have recently synthesized a new type of PF-127 copolymers with higher T (tr) using a simple oxidative method. Here, we have investigated the drug-releasing feature of oxidized PF-127 and oxidized PF-127-containing silver nanoparticles (SNPs), carrying arsenic trioxide (ATO), in a subcutaneous model of rats. Injectable hydrogels prepared with oxidized PF-127s were less viscous and easier to inject, at the same concentration, than their precursor. Addition of SNPs further elevated T (tr), resulting in even lower viscosity of the injectable hydrogel prepared from SNP-containing oxidized PF-127. The oxidized PF-127 copolymers did not differ significantly in ATO-releasing ability, compared with parental PF-127, but the addition of SNPs altered the ATO-releasing feature of oxidized PF-127 to some extent. ATO-carrying oxidized PF-127s had similar toxicity, but the addition of SNPs enhanced the hepatotoxicity of ATO, as evidenced by elevated serum levels of alanine aminotransferase and aspartate aminotransferase and histological alterations, compared to parental PF-127. The results presented herein warrant further investigation of the modified PF-127 copolymers to deliver ATO or other drugs in the form of injectable hydrogels.     

Modeling the influence of cyclodextrins on oral absorption of low‐solubility drugs: I. Model development

The ability to quantitatively predict the influence of a solubilization technology on oral absorption would be highly beneficial in rational selection of drug delivery technology and formulation design. Cyclodextrins (CDs) are cyclic oligosaccharides which form inclusion complexes with a large variety of compounds including drugs. There are many studies in the literature showing that complexation between CD and drug enhances oral bioavailability and some demonstrating failure of CD in bioavailability enhancement, but relatively little guidance regarding when CD can be used to enhance bioavailability. A model was developed based upon mass transport expressions for drug dissolution and absorption and a pseudo‐equilibrium assumption for the complexation reaction with CD. The model considers neutral compound delivered as a physical mixture with CD in both immediate release (IR) and controlled release (CR) formulations. Simulation results demonstrated that cyclodextrins can enhance, have no effect, or hurt drug absorption when delivered as a physical mixture with drug. The predicted influence depends on interacting parameter values, including solubility, drug absorption constant, binding constant, CD:drug molar ratio, dose, and assumed volume of the intestinal lumen. In general, the predicted positive influence of dosing as a physical mixture with CD was minimal, alluding to the significance of dosing as a preformed complex. The model developed enabled examination of which physical and chemical properties result in oral absorption enhancement for neutral drug administered as a physical mixture with CD, demonstrating the utility of modeling the influence of a drug delivery agent (e.g., CD) on absorption for rational dosage form design. Biotechnol. Bioeng. 2010; 105: 409–420. © 2009 Wiley Periodicals, Inc.     

Intravitreal Docosahexaenoic Acid in a Rabbit Model: Preclinical Safety Assessment

Purpose

The purpose of the present study was to evaluate the retinal toxicity of a single dose of intravitreal docosahexaenoic acid (DHA) in rabbit eyes over a short-term period.

Methods

Sixteen New Zealand albino rabbits were selected for this pre-clinical study. Six concentrations of DHA (Brudy Laboratories, Barcelona, Spain) were prepared: 10 mg/50 µl, 5 mg/50 µl, 2’5 mg/50 µl, 50 µg/50 µl, 25 µg/50 µl, and 5 µg/50 µl. Each concentration was injected intravitreally in the right eye of two rabbits. As a control, the vehicle solution was injected in one eye of four animals. Retinal safety was studied by slit-lamp examination, and electroretinography. All the rabbits were euthanized one week after the intravitreal injection of DHA and the eyeballs were processed to morphologic and morphometric histological examination by light microscopy. At the same time aqueous and vitreous humor samples were taken to quantify the concentration of omega-3 acids by gas chromatography. Statistical analysis was performed by SPSS 21.0.

Results

Slit-lamp examination revealed an important inflammatory reaction on the anterior chamber of the rabbits injected with the higher concentrations of DHA (10 mg/50 µl, 5 mg/50 µl, 2''5 mg/50 µ) Lower concentrations showed no inflammation. Electroretinography and histological studies showed no significant difference between control and DHA-injected groups except for the group injected with 50 µg/50 µl.

Conclusions

Our results indicate that administration of intravitreal DHA is safe in the albino rabbit model up to the maximum tolerated dose of 25 µg/50 µl. Further studies should be performed in order to evaluate the effect of intravitreal injection of DHA as a treatment, alone or in combination, of different retinal diseases.     

Tolerability of High-Volume Subcutaneous Injections of a Viscous Placebo Buffer: A Randomized,Crossover Study in Healthy Subjects

Monoclonal antibody biotherapeutics are often administered by subcutaneous (SC) injection. Due to dose requirements and formulation limitations, SC injections >1 mL are often required. We used a viscous placebo buffer (5 cP), characteristic of a high-concentration antibody formulation, to investigate the effect of dose volume and injection rate on the tolerability of higher-volume SC injections. In this randomized, crossover, single-center study, 48 healthy adults received one 1.2-mL bolus injection over 5 s and three 3.5-mL injections over 1, 4, and 10 min in different abdominal quadrants, with each injection separated by approximately 2 h. The primary objective was to compare pain scores associated with the injections, immediately after administration and 1 h later, using a 100-mm visual analog scale (VAS). Secondary objectives included assessment of adverse events, including injection site reactions and swelling. Mean age was 38.4 (11.6) years and 20 subjects (42%) were female. Lowest mean VAS score was for the 10-min (6.83 mm) and highest for the 1-min injection (19.13 mm). One hour after administration, mean VAS scores were <3.5 mm for all injections. Swelling was similar among the three 3.5-mL injections. After needle removal, leakage occurred following 14 (29%) 1.2-mL injections, eight (17%) 4-min injections, five (10%) 1-min injections, and four (8%) 10-min injections. Fifteen subjects (31%) experienced an adverse event, none of which was serious, fatal, or led to study discontinuation. All injection durations were well tolerated, suggesting a single large-volume SC injection of a biotherapeutic agent could be used instead of multiple injections.KEY WORDS: placebo buffer, subcutaneous injections, tolerability, viscous     

Transscleral Sustained Vasohibin-1 Delivery by a Novel Device Suppressed Experimentally-Induced Choroidal Neovascularization

We established a sustained vasohibin-1 (a 42-kDa protein), delivery device by a novel method using photopolymerization of a mixture of polyethylene glycol dimethacrylate, triethylene glycol dimethacrylate, and collagen microparticles. We evaluated its effects in a model of rat laser-induced choroidal neovascularization (CNV) using a transscleral approach. We used variable concentrations of vasohibin-1 in the devices, and used an enzyme-linked immunosorbent assay and Western blotting to measure the released vasohibin-1 (0.31 nM/day when using the 10 μM vasohibin-1 delivery device [10VDD]). The released vasohibin-1 showed suppression activity comparable to native effects when evaluated using endothelial tube formation. We also used pelletized vasohibin-1 and fluorescein isothiocyanate-labeled 40 kDa dextran as controls. Strong fluorescein staining was observed on the sclera when the device was used for drug delivery, whereas pellet use produced strong staining in the conjunctiva and surrounding tissue, but not on the sclera. Vasohibin-1 was found in the sclera, choroid, retinal pigment epithelium (RPE), and neural retina after device implantation. Stronger immunoreactivity at the RPE and ganglion cell layers was observed than in other retinal regions. Significantly lower fluorescein angiography (FA) scores and smaller CNV areas in the flat mounts of RPE-choroid-sclera were observed for the 10VDD, VDD (1 μM vasohibin-1 delivery device), and vasohibin-1 intravitreal direct injection (0.24 μM) groups when compared to the pellet, non-vasohibin-1 delivery device, and intravitreal vehicle injection groups. Choroidal neovascularization can be treated with transscleral sustained protein delivery using our novel device. We offer a safer sustained protein release for treatment of retinal disease using the transscleral approach.     

Pharmaceutical development and manufacturing of a parenteral formulation of a novel antitumor agent, VNP40101M

The objective of this study was to develop and manufacture a stable parenteral formulation for Phase I clinical trials of VNP40101M (1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(2-methylamino)carbonyl] hydrazine), a novel antitumor agent. The solubility and stability of the drug was determined. Solubility studies suggested that VNP40101M exhibited poor aqueous solubility but showed appreciable solubility in nonaqueous solvents. The aqueous solubility of the drug could not be increased by adjusting the pH. At a pH above 7, basecatalyzed decomposition of VNP40101M occurred. The low octanol-water partition coefficient of 0.75 suggested poor solubility in lipophilic solvents. Based on these preformation observations, a parenteral formulation containing 10 mg/mL of VNP40101M was prepared in a solvent system consisting of 30% ethyl alcohol and 70% polyethylene glycol-300 (PEG-300). To minimize base-catalyzed hydrolytic degradation. citric acid at 0.6% concentration was included to acidify the formulation. Rubber closures, filter membranes, and liquid transfer tubing were selected on the basis of compatibility studies and absence of loss of drug the of adsorption of these components. The formulation was subjected to accelerated stability studies and dilution studies with large volume parenteral (LVP) solutions, normal saline, and 5% dextrose injection (D5W). The results of the dilution study indicated that the formulation could be diluted in these solutions up to 2 mg/mL for 8 hours without drug precipitation and degradation. Accelerated stability studies suggested that the product should be kept at 2°C to 8°C for long-term storage. The developed formulation was successfully scaled up and manufactured for use in clinical trials. Published: August 26, 2001.     

Quantification of triamcinolone acetonide in ocular tissues after intravitreal injection to rabbit using liquid chromatography-tandem mass spectrometry

A sensitive and selective liquid chromatographic-tandem mass spectrometric method was developed for quantification of triamcinolone acetonide (TA) in rabbit ocular tissues. After a simple liquid–liquid extraction using tert-butyl methyl ether, TA and internal standard methylprednisolone were separated on a C₁₈ column with a mobile phase of acetonitrile:water:formic acid (60:40:0.1, v/v/v) and quantified by the use of selected reaction monitoring mode with a total run time of 4 min. The method was validated in tissue homogenates with a daily working range of 1–1000 ng/mL with correlation coefficient of more than 0.99 and a sensitivity of 1 ng/mL as lower limit of quantification, respectively. The mean intra-day and inter-day precisions were less than 10% and accuracy values were higher than 90%. This method was fully validated for the accuracy, precision and stability studies. The method proved to be accurate and specific, and was applied to an in vivo biodistribution study of TA after intravitreal injection to rabbits. Values of mean residence time in vitreous humor, crystalline lens and aqueous humor were 27.7, 35.8 and 20.0 days, respectively.     

Design and development of multivesicular liposomal depot delivery system for controlled systemic delivery of acyclovir sodium

The aim of the present study was to design a depot delivery system of acyclovir sodium using multivesicular liposomes (MVLs) to overcome the limitations of conventional therapies and to investigate its in vivo effectiveness for sustained delivery. MVLs of acyclovir were prepared by the reverse phase evaporation method. The loading efficiency of the MVLs (45%–82%) was found to be 3 to 6 times higher than conventional multilamellar vesicles (MLVs). The in vitro release of acyclovir from MVL formulations was found to be in a sustained manner and only 70% of drug was released in 96 hours, whereas conventional MLVs released 80% of drug in 16 hours. Following intradermal administration to Wistar rats, the MVL formulations showed effective plasma concentration for 48 hours compared with MLVs and free drug solution (12–16 hours). C_max values of MVL formulations were significantly less (8.6–11.4 μg/mL) than MLV and free drug solution (12.5 μg/mL). The AUC_0–48 of the MVL formulations was 1.5- and 3-fold higher compared with conventional liposomes and free drug solution, respectively. Overall, formulations containing phosphatidyl glycerol as negatively charged lipid showed better results. The MVL delivery system as an intradermal depot offers the advantage of a very high loading and controlled release of acyclovir for an extended period of time. The increase in AUC and decrease in C_max reflects that the MVL formulations could reduce the toxic complications and limitations of conventional IV and oral therapies. Published: September 20, 2005