Jaymin C. Shah

Hydrolysis in pharmaceutical formulations.

This literature review presents hydrolysis of active pharmaceutical ingredients as well as the effects on dosage form stability due to hydrolysis of excipients. Mechanisms and measurement methods are discussed and recommendations for formulation stabilization are listed.

Glyceryl Monooleate Cubic Phase Gel as Chemical Stability Enhancer of Cefazolin and Cefuroxime

The primary objective of this study was to determine the ability of the glyceryl monooleate (GMO) cubic phase gel to protect drugs from chemical instability reactions such as hydrolysis and oxidation. Stability was assessed on cefazolin incorporated in cubic phase gel and in solution at two different concentrations (200 and 50 micrograms/g), at 22, 37, and 50 degrees C. Degradation profiles, plotting percent cefazolin remaining on a logarithmic scale versus time, were constructed and the degradation rate constants calculated from the slopes. At both concentrations, degradation of cefazolin was found to be slower in the cubic phase gel than in solution at 22 and 37 degrees C, but not at 50 degrees C. The degradation rate constants were 3- to 18-fold lower in the gel than in solution at low concentration of cefazolin. At 22 and 37 degrees C, the kinetics of degradation at high concentration of cefazolin was not first-order but showed a lag phase followed by an exponential loss of cefazolin, typical of oxidation. The potential oxidation of the thioether moiety of cefazolin was confirmed by its 18-fold higher stability in the presence of ethylenediaminetetraacetic acid (EDTA) and nitrogen in solution. Cefuroxime, a cephalosporin which degrades solely via beta-lactam hydrolysis, degraded twice as fast in solution as it did in the gel. The enhanced stability of cefazolin and cefuroxime in the GMO cubic phase gel shows its potential as a chemical stability enhancer and this is the first report to demonstrate oxidation, in addition to beta-lactam hydrolysis, as a mechanism for degradation of cefazolin.

Local Delivery of Vancomycin for the Prophylaxis of Prosthetic Device-Related Infections

No HeadingPurpose.To evaluate the in vivo efficacy and pharmacokinetics of vancomycin delivered from glycerylmonostearate (GMS) implants in a prosthetic-device based biofilm infection model.Methods.A biofilm infection model was developed in male Sprague-Dawley rats by implanting a vascular graft on the dorsal side of each rat and infecting it with 1.5 × 108 cfu/ml Staphylococcus epidermidis. The rats were divided into 3 groups of 6 rats each: 1) the control group that received no antibiotics, 2) the IM group that received multiple IM injections of vancomycin at a dose of 25 mg/kg every 6 h for a total of 12 doses, and 3) the implant group that received GMS implants designed to deliver vancomycin at a total dose of 300 mg/kg for a period of 4 days. The pharmacokinetics of vancomycin was determined from IM and implant groups by analyzing for vancomycin in blood using HPLC. In vivo efficacy was studied by evaluation of the wound site and the prosthetic device upon excision, for evidence of infection in the form of purulent discharge at the wound site and yellowish discoloration of the prosthetic device and inflammation as sign of biofilm formation. Microbiological evaluation on the wound site and the prosthetic device was performed by culturing the swabs at the wound site and the prosthetic device in sterile tryptic soy broth for 36–48 h at 37°C.Results.Vancomycin was successfully delivered in a sustained manner for 100 h from GMS implants and the resulting plasma profile showed that the concentrations, after an initial burst, plateaued at about of 4.77 ± 1.43 μg/ml with less fluctuations than the IM group in which the plasma concentrations fluctuated between 2.73 ± 0.94 μg/ml and 19.26 ± 3.67 μg/ml. Upon excision of the wound site, all the animals in the control group developed infection in the form of purulent discharge and yellowish discoloration of the prosthetic device. However, none of the rats in the implant group showed evidence of infection clearly demonstrating the efficacy of the local delivery system in preventing infection. Systemically delivered vancomycin by IM injections failed to prevent infection in four out of six rats. Microbiological evaluation of the wound site and prosthetic device resulted in isolation of biofilm-producing organisms such as Staphylococcus epidermidis, Enterococcus faecalis, and Staphylococcus aureus. These organisms were isolated in greater number of animals in the control group compared to the IM and implant groups.Conclusions.The GMS implants as a delivery system for vancomycin were successful in preventing infection in all the animals compared to the IM and control groups demonstrating the efficacy of a local delivery system in a prosthetic device related biofilm infection model.

Influence of Dosage Form on the Intravitreal Pharmacokinetics of Diclofenac

PURPOSE To prepare a suspension form of diclofenac and compare the influence of the injected form (suspension versus solution) on the intravitreal pharmacokinetics of diclofenac in Dutch belted pigmented rabbits. METHODS Diclofenac acid was prepared and characterized in a suspension formulation. Rabbit eyes were injected with either diclofenac sodium solution (0.3 mg) or diclofenac acid suspension (10 mg) prepared in 0.1 mL balanced salt solution. Rabbits were killed at regular time intervals, the eyes enucleated, and drug content quantified in the vitreous humor and retina-choroid tissue by high-performance liquid chromatography. Pharmacokinetic models were developed for both the dosage forms, and simulations were performed for different doses. RESULTS Diclofenac acid with an approximate 5-mum particle size exhibited 3.5-fold lower solubility in vitreous humor, when compared with its sodium salt. The estimated settling velocity of the suspension in the vitreous humor was 3 cm/h. After diclofenac sodium salt solution injection, drug levels declined rapidly with no drug levels detectable after 24 hours in the vitreous humor and 4 hours in the RC. Throughout the assessed time course, drug levels were higher in the vitreous. However, sustained, high drug levels were observed in both the vitreous humor and the retina-choroid even on day 21 after diclofenac acid suspension injection, with retina-choroid drug levels being higher beginning at 0.25 hour. The elimination half-life of diclofenac suspension was 24 and 18 days in vitreous and retina-choroid, respectively, compared to 2.9 and 0.9 hours observed with diclofenac sodium. The pharmacokinetic models developed indicated a slow-release distribution or depot compartment for the diclofenac acid suspension in the posterior segment. Simulations indicated the inability of a 10-mg dose of diclofenac sodium solution to sustain drug levels in the vitreous beyond 11 days. CONCLUSIONS By choosing a less soluble form of a drug such as diclofenac acid, vitreous elimination half-life can be prolonged up to 24 days, potentially resulting in therapeutic levels in the posterior segment tissues for a few months. Higher detectable drug levels in the retina-choroid suggest rapid settling and persistent retention of suspension in retina-choroid tissue.

In vitro and in vivo characterization of amorphous, nanocrystalline, and crystalline ziprasidone formulations

Ziprasidone, commercially available as Geodon capsules, is an atypical antipsychotic used in the treatment of schizophrenia and bipolar disorder. It is a BCS Class II drug that shows up to a 2-fold increase in absorption in the presence of food. Because compliance is a major issue in this patient population, we developed and characterized solubilized formulations of ziprasidone in an effort to improve absorption in the fasted state, thereby resulting in a reduced food effect. Three formulations utilizing solubilization technologies were studied: (1) an amorphous inclusion complex of ziprasidone mesylate and a cyclodextrin, (2) a nanosuspension of crystalline ziprasidone free base, and (3) jet-milled ziprasidone HCl coated crystals made by spray drying (CCSD) the drug with hypromellose acetate succinate. The formulations were characterized by in vitro methods appropriate to each particular solubilization technology. These studies confirmed that ziprasidone mesylate - cyclodextrin was an amorphous inclusion complex with enhanced dissolution rates. The ziprasidone free base crystalline nanosuspension showed a mean particle size of 274 nm and a monomodal particle size distribution. In a membrane permeation test, the CCSD showed a 1.5-fold higher initial flux compared to crystalline ziprasidone HCl. The three formulations were administered to fasted beagle dogs and their pharmacokinetics compared to Geodon capsules administered in the fed state. The amorphous complex and the nanosuspension showed increased absorption in the fasted state, indicating that solubilized formulations of ziprasidone have the potential to reduce the food effect in humans.

Effect of Cyclodextrin Derivation and Amorphous State of Complex on Accelerated Degradation of Ziprasidone

Inclusion complexes of ziprasidone with several β-cyclodextrins [β-CDs; sulfobutylether-β-cyclodextrins (SBEβCD), hydroxypropyl-β-cyclodextrins (HPβCD), methyl-β-cyclodextrins (MβCD), and carboxyethyl-β-cyclodextrins (CEβCD)] were prepared and solution stability was evaluated at elevated temperature. Solid-state stability was assessed by subjecting various CD complexes of ziprasidone, spray-dried dispersion (SDD), partially crystalline ziprasidone-SBEβCD salts, and the physical mixture of ziprasidone-SBEβCD to γ-irradiation. Degradant I was formed by oxidation of ziprasidone, which upon aldol condensation with ziprasidone formed degradant II in both solution and solid states. In the solution state, CD complexes with electron-donating side chains, such as SBEβCD and CEβCD, produced the highest oxidative degradation followed by HPβCD with 6, 3, and 4 degrees of substitution. In the solid state, crystalline drug substance and physical mixture of crystalline drug-SBEβCD showed very little to no degradation. In contrast, amorphous βCD, MβCD, CEβCD, and SBEβCD complexes as well as the amorphous SDD exhibited greatest extent of oxidative degradation. Results suggest that electron-donating side chains of the derivatized CD interact with transition state of the oxidation reaction and catalyze drug degradation in solution, However, higher mobility in the amorphous state of CD-drug complexes promoted chemical instability of ziprasidone under accelerated conditions irrespective of the chemical nature of the side chain on CD.

Effect of particle size of parenteral suspensions on in vitro muscle damage

Objective: Suspension particle size plays a key role in the release and stability of drugs for oral and parenteral formulations. However, the role of particle size in suspension formulations on tissue damage (myotoxicity) following intramuscular (IM) injection has not been systematically investigated. Materials and methods: Myotoxicity was assessed by the release of cumulative creatine kinase (CCK) from the isolated extensor digitorium longus (EDL) and soleus (SOL) rat muscles for selected suspensions of phenytoin, bupivicane and diazepam. Particle size effects on myotoxicity, independent of any specific drug, were also investigated using characterized non-dissolving polystyrene beads. Results: Myotoxicity was quantitated by the cumulative release of creatine kinase (CCK) from these isolated muscles over 90 or 120 min. The relationship between particle size and myotoxicity was dependent upon the drug in these suspensions. Diazepam and phenytoin suspensions were found to be less myotoxic than bupivicaine. Using unmodified and carboxy modified polystyrene beads, an optimal particle size for reduced myotoxicity following IM injection ranges from approx. 500 nm to 1 µM. Conclusions: The relationship between myotoxicity of IM suspensions and particle size is dependent upon the particular drug and suspension particle size.

A New Vision for the Eye: Unmet Ocular Drug Delivery Needs

ABBREVIATIONS AMD Age related macular degeneration BCRP ATP-binding cassette transporter, sub-family G (WHITE), member 2 CMC Chemistry, manufacturing and controls CMV Cytomegalovirus CNV Choroidal neovascularization DME Diabetic macular edema DR Diabetic retinopathy GCV Ganciclovir IOP Intraocular pressure IVT Intravitreal MEMS Micro-electro-mechanical systems MRP Multidrug resistance-associated protein transporters OAnT Organic anion transporters OCaT Organic cation transporters OCT Optical coherence tomography PDGF Platelet-derived growth factor Pgp p-glycoprotein PLGA Poly-lactic-co-glycolic acid RPE Retinal pigment epithelium TA Triamcinolone acetonide suspension VEGF Vascular endothelial growth factor INTRODUCTION

Abstract 4402: Lazaroid formulations for brain delivery in treatment of glioblastoma multiforme

Lazaroid U-74389G (LAZ) has potential radio-protective and anti-proliferative activities in treatment of glioblastoma, but it exhibits poor brain penetration and high hepatic clearance. To increase the brain exposure of LAZ, we formulated LAZ in co-solvent system and as nanostructured lipid carriers (NLC). The rationale behind this approach is that LAZ formulations due to their high drug payload for administration at higher doses and prolonged circulation results in greater systemic exposure compared to unformulated LAZ solution. The purpose of this study was to comparatively evaluate the brain exposures of unformulated LAZ solution in citrate buffer (pH 3), LAZ co-solvent formulation in mixture of propylene glycol and citrate buffer (pH 3), and LAZ NLC, prepared using ultra-sonication and optimized using Central Composite Design (CCD). Statistical analysis was carried out using Student9s t-test. Male Sprague Dawley rats were randomized into 3 groups and were administered unformulated LAZ solution at 5 mg/kg dose and LAZ co-solvent and LAZ NLC at 15 mg/kg dose, intravenously through jugular vein cannula. The brain tissues were homogenized in normal saline and LAZ in the brain tissue was quantified using Absciex Qtrap 5500 LC/MS/MS with linear range of 1 (LLOQ) - 250 ng/mL. The concentrations of unformulated LAZ solution and the LAZ co-solvent solution were 2 mg/mL and 8.5 mg/mL, respectively. The optimized LAZ NLC had particle size of 150 ± 2.86 nm, zeta potential of (-6.79) ± 0.39 mV, encapsulation efficiency of 81.34 ± 6.06%. The LAZ NLC preparation is monodisperse with polydispersity index of 0.158 ± 0.01 (N = 3). The amount of LAZ in brain tissues after 4 hours is significantly higher in formulation groups (LAZ co-solvent 37.48 ± 7.06 ng/gm and LAZ NLC 27.49 ng/gm) than the unformulated groups (LAZ solution 6.84 ± 1.87 ng/gm). Upon dose normalization there was a significant increase in amount of LAZ in brain tissues (N = 3 each) after 4 hours in LAZ co-solvent group (2.72 ± 0.53 ng/gm/mg) in comparison to unformulated LAZ solution group (1.37 ± 0.37 ng/gm/mg), at p Developing LAZ formulations will offer potential merits of enabling administrations at higher doses, facilitating LAZ delivery to the brain at therapeutic levels and decreasing its systemic clearance. LAZ formulations are capable of incorporating LAZ at higher drug payload than unformulated solution. Upon dose normalization, the amount of LAZ in brain tissue at 4 hours is significantly higher (1.5-2 times) in LAZ formulations in comparison to LAZ unformulated group. This study helps to build a strong foundational background for use of LAZ as an alternative agent in treatment of glioblastoma utilizing its dual properties of lipid peroxidation inhibition and anti-proliferation. Citation Format: Prajakta Gadgil, Diana S-L. Chow, Pamela New, Jaymin Shah. Lazaroid formulations for brain delivery in treatment of glioblastoma multiforme. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4402. doi:10.1158/1538-7445.AM2015-4402

Delineating the role of cooperativity in the design of potent PROTACs for BTK

Significance Proteolysis targeting chimera (PROTAC)-based protein degradation is an emerging field that holds significant promise for targeting the “undruggable” proteome: the vast majority of the proteins that do not exhibit enzymatic activity and are thereby not amenable to classical inhibition. Despite significant progress, a thorough mechanistic characterization of biochemical determinants that underpin efficient PROTAC activity is lacking. Here we address one such question: Is positive cooperativity necessary for potent protein degradation? Through a collection of independent techniques, we show that within a Bruton’s tyrosine kinase/cereblon PROTAC system, potent knockdown correlates with alleviation of steric clashes in the absence of thermodynamic cooperativity. This result broadens the scope of PROTAC applications and affects fundamental design criteria across the field. Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules that simultaneously bind to a target protein and an E3 ligase, thereby leading to ubiquitination and subsequent degradation of the target. They present an exciting opportunity to modulate proteins in a manner independent of enzymatic or signaling activity. As such, they have recently emerged as an attractive mechanism to explore previously “undruggable” targets. Despite this interest, fundamental questions remain regarding the parameters most critical for achieving potency and selectivity. Here we employ a series of biochemical and cellular techniques to investigate requirements for efficient knockdown of Bruton’s tyrosine kinase (BTK), a nonreceptor tyrosine kinase essential for B cell maturation. Members of an 11-compound PROTAC library were investigated for their ability to form binary and ternary complexes with BTK and cereblon (CRBN, an E3 ligase component). Results were extended to measure effects on BTK–CRBN cooperative interactions as well as in vitro and in vivo BTK degradation. Our data show that alleviation of steric clashes between BTK and CRBN by modulating PROTAC linker length within this chemical series allows potent BTK degradation in the absence of thermodynamic cooperativity.