Chandrasekar Durairaj

Nanosized dendritic polyguanidilyated translocators for enhanced solubility, permeability, and delivery of gatifloxacin.

PURPOSE Dendrimeric polyguanidilyated translocators (DPTs) are nanosized novel dendrimers that efficiently translocate molecules across biological barriers. The purpose of this study was to develop a DPT that could serve as an ophthalmic delivery vehicle for gatifloxacin and to evaluate its in vitro and in vivo delivery after topical application. METHODS The gatifloxacin (GFX) solubility-enhancing property of a six-guanidine group-containing dendrimer (g6 DPT) was investigated as a function of pH and dendrimer concentration. Mechanisms of drug interaction with the dendrimer were investigated by using isothermal titration calorimetry (ITC), Fourier-transformed infrared spectroscopy (FTIR), and nuclear magnetic resonance spectroscopy (NMR). Permeability of the dendrimer was assessed in human corneal epithelial cells (HCECs) and across isolated bovine sclera-choroid-RPE (SCRPE). In vitro efficacy of the dendrimer formulation was evaluated with a time-to-kill assay for methicillin resistant Staphylococcus aureus (MRSA). In vivo delivery of GFX in a dendrimer eye drop formulation was studied in New Zealand White rabbits after a single dose or multiple doses over 3 weeks. Drug levels in various ocular tissues were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS An optimized DPT-GFX formulation (final pH 5.9, no preservative) increased GFX solubility by fourfold. The dendrimer formed isotonically stable, nanosized (346-nm) complexes with GFX via ionic bond, hydrogen bond, and hydrophobic interactions. The dendrimer gained rapid entry into the HCECs (within 5 minutes) and increased the transport of GFX by 40% across the SCRPE in 6 hours. DPT-GFX exhibited a three times faster killing rate for MRSA when compared with GFX alone. In vivo administration of DPT-GFX (1.2% wt/vol) resulted in ∼13-fold, and ∼2-fold higher areas under the curve (AUCs) for tissue concentrations in conjunctiva and cornea, respectively, when compared with GFX (0.3%) after a single dose. Further, a single dose of DPT-GFX sustained aqueous humor and vitreous humor drug levels during the 24-hour study, with a t(1/2) of 9 and 32 hours, respectively. After multiple doses, similar advantages were seen with DPT-GFX. CONCLUSIONS The DPT forms stable complexes with GFX and enhances its solubility, permeability, anti-MRSA activity, and in vivo delivery, potentially allowing a once-daily dose regimen.

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 μg/ml, respectively. After 48 h, ketorolac was barely detectable (0.06 μg/ml) in the vitreous, and diclofenac was undetectable. The peak concentration of each drug in the retina/choroid was 201 μg/g for ketorolac and 4.1 μg/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.

Targeted drug and gene delivery systems for lung cancer therapy

Purpose: To evaluate the efficacy of a novel docetaxel derivative of deslorelin, a luteinizing hormone–releasing hormone (LHRH) agonist, and its combination in vivo with RGD peptide conjugated nanoparticles encapsulating an antiangiogenic, anti–vascular endothelial growth factor (VEGF) intraceptor (Flt23k; RGD-Flt23k-NP) in H1299 lung cancer cells and/or xenografts in athymic nude BALB/c mice. Experimental Design: The in vitro and in vivo efficacy of the deslorelin-docetaxel conjugate was evaluated in H1299 cells and xenografts in athymic nude mice. Coadministration of deslorelin-docetaxel conjugate and RGD-Flt23k-NP was tested in vivo in mice. Tumor inhibition, apoptosis, and VEGF inhibition were estimated in each of the treatment groups. Results: The conjugate enhanced in vitro docetaxel efficacy by 13-fold in H1299 cells compared with docetaxel at 24 hours, and this effect was inhibited following reduction of LHRH receptor expression by an antisense oligonucleotide. Combination of the conjugate with the RGD-Flt23k-NP in vivo resulted in an 82- and 15-fold tumor growth inhibition on day 39 following repeated weekly i.v. injections and a single intratumoral (i.t.) injection, respectively. These effects were significantly greater than individual targeted therapies or docetaxel alone. Similarly, apoptotic indices for the combination therapy were 14% and 10% in the i.v. and i.t. groups, respectively, and higher than the individual therapies. Combination therapy groups exhibited greater VEGF inhibition in both the i.v. and i.t. groups. Conclusions: Docetaxel efficacy was enhanced by LHRH receptor–targeted deslorelin conjugate and further improved by combination with targeted antiangiogenic nanoparticle gene therapy. Combination of novel targeted therapeutic approaches described here provides an attractive alternative to the current treatment options for lung cancer therapy. (Clin Cancer Res 2009;15(23):7299–308)

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.

Luteinizing hormone-releasing hormone receptor–targeted deslorelin-docetaxel conjugate enhances efficacy of docetaxel in prostate cancer therapy

Docetaxel, a chemotherapeutic agent currently used for improving survival of prostate cancer patients, suffers from low therapeutic index. The objective of this study was to prepare a new docetaxel derivative conjugated to deslorelin, a luteinizing hormone-releasing hormone (LHRH) superagonist, and to determine whether it enhances docetaxel potency in vitro and in vivo. Because docetaxel is not amenable for conjugation with peptides, we introduced a -COOH group in docetaxel, forming docetaxel-hemiglutarate, and subsequently conjugated this to serine in deslorelin, forming deslorelin-docetaxel. Fourier-transform IR, 1H-nuclear magnetic resonance, and liquid chromatography-mass spectrometry analyses confirmed deslorelin-docetaxel formation. Antiproliferative efficacy in LNCaP and PC-3 cell lines over 24, 48, and 72 hours exhibited the order deslorelin-docetaxel > docetaxel, whereas deslorelin alone had no effect, with deslorelin-docetaxel potency being 15-fold greater than docetaxel at 72 h. Further, cells pretreated with antisense oligonucleotide against LHRH receptor exhibited decreased deslorelin-docetaxel efficacy, without any change in docetaxel efficacy. Thus, deslorelin-docetaxel efficacy is likely mediated via LHRH receptor. Cell cycle analysis showed that docetaxel treatment led to arrest in G2-M phase, whereas deslorelin-docetaxel treatment allowed greater progression to apoptosis in both cell lines, with deslorelin-docetaxel exerting 5-fold greater apoptosis compared with docetaxel in prostate cancer cell lines. Antitumor efficacy studies in PC-3 prostate xenograft-bearing mice indicated the efficacy order deslorelin-docetaxel > docetaxel ≫ deslorelin > PBS, with deslorelin-docetaxel exerting ∼5.5-fold greater tumor growth inhibition than docetaxel alone. Thus, deslorelin-docetaxel prepared in this study retains pharmacologic effects of both docetaxel and deslorelin while enhancing the antiproliferative, apoptotic, and antitumor efficacy of docetaxel by several folds in prostate cancer therapy. [Mol Cancer Ther 2009;8(6):1655–65]