A sensitive LC-MS/MS assay to quantitate free payload Aur0101 from ADC PYX-201 in rat and monkey plasma.

Aur0101 is a cytotoxic and small-molecule microtubule depolymerizing agent, and is the payload conjugated to antibody-drug conjugate PYX-201. Developing and validating a sensitive bioanalytical method to quantitate Aur0101 was novel and crucial in preclinical PYX-201 studies. Reference standard Aur0101 and its stable isotope labelled internal standard Aur0101-d were used in this LC-MS/MS method.

Quantitation of total antibody (tAb) from antibody drug conjugate (ADC) PYX-201 in rat and monkey plasma using an enzyme-linked immunosorbent assay (ELISA) and its application in preclinical studies.

PYX-201 is an investigative ADC oncology drug composed of a monoclonal human immunoglobulin G (IgG) antibody targeting the extra domain B splice variant of fibronectin (EDB + FN) conjugated to an auristatin payload through a cleavable linker. Effective measurement of PYX-201 tAb is the key to ADC drug PYX-201 preclinical pharmacokinetics (PK) assessment. PYX-201 monoclonal antibody (mAb) was used as the reference standard, goat anti-human IgG polyclonal antibody (pAb) or rabbit anti-human Kappa light chain mAb was employed as the capture antibody, and mouse mAb or goat pAb anti-human IgG the crystallizable fragment (Fc) (horseradish peroxidase (HRP)) was utilized as the detection antibody in this ELISA.

Quantification of antibody-drug conjugate PYX-201 in rat and monkey plasma via ELISA and its application in preclinical studies.

PYX-201 is a novel antibody-drug conjugate targeting the extra domain B splice variant of fibronectin in the tumor microenvironment. Accurate quantification of PYX-201 is critical for PYX-201 pharmacokinetics profiling in preclinical studies. ELISA was performed using reference standard PYX-201, mouse monoclonal anti-monomethyl auristatin E antibody, mouse IgG1, mouse monoclonal anti-human IgG horseradish peroxidase and donkey anti-human IgG horseradish peroxidase.

Nanomedicines for back of the eye drug delivery, gene delivery, and imaging.

Treatment and management of diseases of the posterior segment of the eye such as diabetic retinopathy, retinoblastoma, retinitis pigmentosa, and choroidal neovascularization is a challenging task due to the anatomy and physiology of ocular barriers. For instance, traditional routes of drug delivery for therapeutic treatment are hindered by poor intraocular penetration and/or rapid ocular elimination. One possible approach to improve ocular therapy is to employ nanotechnology.

In vitro transport and partitioning of AL-4940, active metabolite of angiostatic agent anecortave acetate, in ocular tissues of the posterior segment.

Purpose: The purpose of this study was to evaluate partitioning into and transport across posterior segment tissues (sclera, retinal pigment epithelium (RPE)-choroid) of AL-4940, the active metabolite of angiostatic cortisene anecortave acetate (AL-3789).

Methods: Transport of [(14)C]-AL-4940 was measured through RPE-choroid-sclera (RCS) and sclera, excised from Dutch Belted pigmented rabbits' eyes, in the directions of scleral to vitreal (S-->V) and vitreal to scleral (V-->S) for 3 h at 37 degrees C using Ussing chambers. Tissue integrity was monitored by transepithelial electrical resistance (TEER), potential difference (PD), and biochemical assay (LDH).

Delivery of celecoxib for treating diseases of the eye: influence of pigment and diabetes.

Importance Of The Field: Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are two major causes of blindness. In these disorders, growth factors such as vascular endothelial growth factor (VEGF) are upregulated, leading to either enhanced vascular permeability or proliferation of endothelium. While corticosteroid therapies available at present suffer from side effects including cataracts and elevated intraocular pressure, anti-VEGF antibody therapies require frequent intravitreal injections, a procedure that can potentially lead to retinal detachment or endophthalmitis.

Effect of diabetes on transscleral delivery of celecoxib.

Purpose: To investigate the effects of diabetes on transscleral retinal delivery of celecoxib in albino and pigmented rats.

Methods: Albino (Sprague Dawley-SD) and pigmented (Brown Norway-BN) rats were made diabetic by a single intraperitoneal injection of streptozotocin (60 mg/kg) following 24 h of fasting and diabetes was confirmed (blood glucose>250 mg/dL). Two months after diabetes induction, the integrity of blood-retinal-barrier in control versus diabetic rats from both strains was compared by using FITC-dextran leakage assay.

Effect of circulation on the disposition and ocular tissue distribution of 20 nm nanoparticles after periocular administration.

Purpose: Our previous studies indicated that while 20 nm particles are rapidly cleared from the periocular space of the rat following posterior subconjunctival injection, 200 nm particles persisted for at least two months. To understand faster clearance of 20 nm particles, the purpose of this study was to determine transscleral permeability and in vivo disposition in the presence and absence of circulation. Further, it was the purpose of this study to simulate sustained retinal drug delivery after periocular administration of rapidly cleared and slowly cleared nanoparticles.

Effect of eye pigmentation on transscleral drug delivery.

Purpose: To determine the influence of eye pigmentation on transscleral retinal delivery of celecoxib.

Methods: Melanin content in ocular tissues of both the strains was determined by sodium hydroxide solubilization

Method: The affinity of celecoxib to synthetic and natural melanin was estimated by co-incubating celecoxib and melanin in isotonic phosphate-buffered saline. The binding affinity (k) and the maximum binding (r(max)) for celecoxib to both natural and synthetic melanin were estimated.

Modeling of corneal and retinal pharmacokinetics after periocular drug administration.

Purpose: To develop pharmacokinetics models to describe the disposition of small lipophilic molecules in the cornea and retina after periocular (subconjunctival or posterior subconjunctival) administration.

Methods: Compartmental pharmacokinetics analysis was performed on the corneal and retinal data obtained after periocular administration of 3 mg of celecoxib (a selective COX-2 inhibitor) to Brown Norway (BN) rats. Berkeley Madonna, a differential and difference equation-based modeling software, was used for the pharmacokinetics modeling.

Celecoxib inhibits proliferation of retinal pigment epithelial and choroid-retinal endothelial cells by a cyclooxygenase-2-independent mechanism.

Age-related macular degeneration (ARMD) is a leading cause of blindness. The major reason for severe vision loss in ARMD is choroidal neovascularization due to an elevation in the expression of angiogenic factors such as vascular endothelial growth factor (VEGF). Drugs with anti-VEGF and antiproliferative activities can be beneficial for the treatment of this disorder.

Single periocular injection of celecoxib-PLGA microparticles inhibits diabetes-induced elevations in retinal PGE2, VEGF, and vascular leakage.

Purpose: To determine whether celecoxib inhibits VEGF secretion from ARPE-19 cells and to investigate further the safety and effectiveness of periocular celecoxib-poly (lactide-co-glycolide; PLGA) microparticles in inhibiting elevations in retinal PGE(2), VEGF, and blood-tissue barrier leakage at the end of 60 days in a streptozotocin diabetic rat model.

Methods: VEGF mRNA and protein expression in ARPE-19 cells was evaluated in the presence of 0 to 10 microM celecoxib, and cytotoxicity of celecoxib on ARPE-19 and RF6A cells was evaluated over a 0- to 100-microM concentration range. Celecoxib-PLGA microparticles were prepared by a modified solvent evaporation technique, sterilized by 25 kGy of gamma-irradiation, and characterized for size, zeta potential, drug loading, and in vitro release.

Size-dependent disposition of nanoparticles and microparticles following subconjunctival administration.

The purpose of this study was to determine the retention and ocular distribution of subconjunctivally administered nanoparticles and microparticles. Fluorescent polystyrene particles (carboxylate modified, negatively charged) of various sizes (20 nm, 200 nm and 2 microm; Fluospheres, dose 400 microg) were administered to male Sprague-Dawley rats by subconjunctival injection under anaesthesia. The disposition of the particles in the periocular and ocular tissues was studied for up to 60 days by quantifying the particle amounts using liquid extraction followed by spectrofluorimetric analysis.

Inhibition of cyclooxygenase-2, but not cyclooxygenase-1, reduces prostaglandin E2 secretion from diabetic rat retinas.

Up-regulation of cyclooxygenase-2 occurs in retinal cells during the early onset of diabetic retinopathy. Under these conditions, prostaglandin production is elevated, which in turn leads to an increased expression of vascular endothelial growth factor (VEGF)--a growth factor implicated in vascular leakage and neovascularization. In this ex vivo study, we tested whether cyclooxygenase-1 or cyclooxygenase-2 is responsible for diabetes-induced secretion of prostaglandin E2 from isolated rat retinas.