Pharmacokinetics of Monoclonal Antibody and Antibody Fragments in The Mouse Eye Following Intravitreal Administration.

Mice are rarely used in pharmacokinetic (PK) studies of ocular therapeutics due to the small size of their eyes and challenges in drug administration, tissue collection, and analysis of drug concentrations. Therefore, ocular PK of protein therapeutics in mouse eye following intravitreal (IVT) administration is not known. Here, we have presented the first of its kind investigation, to study the PK of 4 different size non-binding protein therapeutics in mouse plasma, cornea/ICB, vitreous humor, retina, and posterior cup (including choroid) following IVT administration. Administered proteins include trastuzumab (150 kDa) and F(ab)2 (100 kDa), Fab, and scFv (27 kDa) fragments of trastuzumab. An imaging and injection apparatus suitable for performing small (50 nL) IVT injections in mice was developed, and techniques for enucleation of the eye and dissection of ocular tissues were developed. Furthermore, a sensitive enzyme-linked immunosorbent assay (ELISA) for detection of proteins in very small amounts of ocular tissues were developed. It was observed that elimination from the vitreous chamber was the primary driver of PK in the cornea/ICB, retina, posterior cup, and plasma. Trastuzumab displays first-order kinetics in the vitreous humor with a half-life of 18.8 h. F(ab)2, Fab, and ScFv show biphasic PK profiles with distribution phases becoming more rapid as molecular weight decreases, and terminal elimination becoming longer as molecular weight decreases, with terminal half-lives of 16.3, 20.6, and 48.9 h, respectively. The mean residence times of trastuzumab, F(ab)2, Fab, and scFv in the vitreous humor were 26.0, 12.2, 10.7, and 8.16 h, respectively. It was found that the mean residence time in vitreous humor doubles with an increase in molecular weight of ∼69 kDa. Interestingly, the PK of proteins measured in the un-injected eye suggest the presence of a pathway for drug transfer between the eyes, which needs to be further validated. Overall, the findings presented here pave the way for drug discovery and development studies of protein therapeutics for ophthalmic indications in mice.

Heparan sulfate-dependent RAGE oligomerization is indispensable for pathophysiological functions of RAGE.

RAGE, a druggable inflammatory receptor, is known to function as an oligomer but the exact oligomerization mechanism remains poorly understood. Previously we have shown that heparan sulfate (HS) plays an active role in RAGE oligomerization. To understand the physiological significance of HS-induced RAGE oligomerization in vivo, we generated RAGE knock-in mice (AgerAHA/AHA) by introducing point mutations to specifically disrupt HS-RAGE interaction. The RAGE mutant demonstrated normal ligand-binding but impaired capacity of HS-binding and oligomerization. Remarkably, AgerAHA/AHA mice phenocopied Ager-/- mice in two different pathophysiological processes, namely bone remodeling and neutrophil-mediated liver injury, which demonstrates that HS-induced RAGE oligomerization is essential for RAGE signaling. Our findings suggest that it should be possible to block RAGE signaling by inhibiting HS-RAGE interaction. To test this, we generated a monoclonal antibody that targets the HS-binding site of RAGE. This antibody blocks RAGE signaling in vitro and in vivo, recapitulating the phenotype of AgerAHA/AHA mice. By inhibiting HS-RAGE interaction genetically and pharmacologically, our work validated an alternative strategy to antagonize RAGE. Finally, we have performed RNA-seq analysis of neutrophils and lungs and found that while Ager-/- mice had a broad alteration of transcriptome in both tissues compared to wild-type mice, the changes of transcriptome in AgerAHA/AHA mice were much more restricted. This unexpected finding suggests that by preserving the expression of RAGE protein (in a dominant-negative form), AgerAHA/AHA mouse might represent a cleaner genetic model to study physiological roles of RAGE in vivo compared to Ager-/- mice.

Pharmacokinetics of Monoclonal Antibody and Antibody Fragments in the Mouse Eye Following Systemic Administration.

The ocular pharmacokinetics (PK) of antibody-based therapies are infrequently studied in mice due to the technical difficulties in working with the small murine eye. This study is the first of its kind to quantitatively measure the PK of variously sized proteins in the plasma, cornea/ICB, vitreous humor, retina, and posterior cup (including choroid) of the mouse and to evaluate the relationship between molecular weight (MW) and antibody biodistribution coefficient (BC) to the eye. Proteins analyzed include trastuzumab (150 kDa), trastuzumab-vc-MMAE (T-vc-MMAE, 155 kDa), F(ab)2 (100 kDa), Fab (50 kDa), and scFv (27 kDa). As expected, ocular PK mirrored the systemic PK as plasma was the driving force for ocular exposure. For trastuzumab, T-vc-MMAE, F(ab)2, Fab, and scFv, respectively, the BCs in the cornea/ICB were 0.610%, 0.475%, 1.74%, 3.39%, and 13.7%; the BCs in the vitreous humor were 0.0198%, 0.0427%, 0.0934%, 0.234%, and 5.56%; the BCs for the retina were 0.539%, 0.230%, 0.704%, 2.44%, and 20.4%; the BCs for the posterior cup were 0.557%, 0.650%, 1.47%, 4.06%, and 13.9%. The relationship between BC and MW was best characterized by a log-log regression in which BC decreased as MW increased, with every doubling in MW leading to a decrease in BC by a factor of 3.44 × , 6.76 × , 4.74 × , and 3.43 × in cornea/ICB, vitreous humor, retina, and posterior cup, respectively. In analyzing the disposition of protein therapeutics to the eye, these findings enhance our understanding of the potential for ocular toxicity of systemically administered protein therapeutics and may aid in the discovery of systemically administered protein therapeutics for ocular disorders.

Effect of variable domain charge on in vitro and in vivo disposition of monoclonal antibodies.

A growing body of evidence supports the important role of molecular charge on antibody pharmacokinetics (PK), yet a quantitative description of the effect of charge on systemic and tissue disposition of antibodies is still lacking. Consequently, we have systematically engineered complementarity-determining regions (CDRs) of trastuzumab to create a series of variants with an isoelectric point (pI) range of 6.3-8.9 and a variable region (Fv) charge range of -8.9 to +10.9 (at pH 5.5), and have investigated in vitro and in vivo disposition of these molecules. These monoclonal antibodies (mAbs) exhibited incrementally enhanced binding to cell surfaces and cellular uptake with increased positive charge in antigen-negative cells. After single intravenous dosing in mice, a bell-shaped relationship between systemic exposure and Fv charge was observed, with both extended negative and positive charge patches leading to more rapid nonspecific clearance. Whole-body PK experiments revealed that, although overall exposures of most variants in the tissues were very similar, positive charge of mAbs led to significantly enhanced tissue:plasma concentration ratios for most tissues. In well-perfused organs such as liver, spleen, and kidney, the positive charge variants show superior accumulation. In tissues with continuous capillaries such as fat, muscle, skin, and bone, plasma concentrations governed tissue exposures. The in vitro and in vivo disposition data presented here facilitate better understanding of the impact of charge modifications on antibody PK, and suggest that alteration in the charge may help to improve tissue:plasma concentration ratios for mAbs in certain tissues. The data presented here also paves the way for the development of physiologically based pharmacokinetic models of mAbs that incorporate charge variations.

A Two-Pore Physiologically Based Pharmacokinetic Model to Predict Subcutaneously Administered Different-Size Antibody/Antibody Fragments.

Quantitative modeling of the subcutaneous absorption processes of protein therapeutics is challenging. Here we have proposed a "two-pore" PBPK model that is able to simultaneously characterize plasma PK of different-size protein therapeutics in mice. The skin compartment is evolved to mechanistically account for the absorption pathways through lymph and blood capillaries, as well as local degradation at the SC injection site. The model is developed using in-house plasma PK data generated following subcutaneous administration of 6 different-size protein therapeutics (13-150 kDa) in mice. The model was able to capture plasma PK of all molecules following intravenous and subcutaneous administration relatively well. From the observed plasma PK profiles, as well as from the model simulation result, several important PK descriptors were found to be dependent on protein size for FcRn nonbinding molecules. A positive correlation was found between Tmax and protein size. A "U" shape relationship was found between Cmax and protein size. Negative correlations were observed between bioavailability (F) and local degradation rate (kdeg,SC), and F and protein size. Pathway analysis of the model was conducted for the subcutaneous absorption process, and continuous relationships were established between the percentage of absorption through lymphatic and vascular pathways and protein size. This PBPK model could serve as a platform for the development of different-size protein therapeutics and will be scaled up to humans for translational studies in the future.

Evolution of the Systems Pharmacokinetics-Pharmacodynamics Model for Antibody-Drug Conjugates to Characterize Tumor Heterogeneity and In Vivo Bystander Effect.

The objective of this work was to develop a systems pharmacokinetics-pharmacodynamics (PK-PD) model that can characterize in vivo bystander effect of antibody-drug conjugate (ADC) in a heterogeneous tumor. To accomplish this goal, a coculture xenograft tumor with 50% GFP-MCF7 (HER2-low) and 50% N87 (HER2-high) cells was developed. The relative composition of a heterogeneous tumor for each cell type was experimentally determined by immunohistochemistry analysis. Trastuzumab-vc-MMAE (T-vc-MMAE) was used as a tool ADC. Plasma and tumor PK of T-vc-MMAE was analyzed in N87, GFP-MCF7, and coculture tumor-bearing mice. In addition, tumor growth inhibition (TGI) studies were conducted in all three xenografts at different T-vc-MMAE dose levels. To characterize the PK of ADC in coculture tumors, our previously published tumor distribution model was evolved to account for different cell populations. The evolved tumor PK model was able to a priori predict the PK of all ADC analytes in the coculture tumors reasonably well. The tumor PK model was subsequently integrated with a PD model that used intracellular tubulin occupancy to drive ADC efficacy in each cell type. The final systems PK-PD model was able to simultaneously characterize all the TGI data reasonably well, with a common set of parameters for MMAE-induced cytotoxicity. The model was later used to simulate the effect of different dosing regimens and tumor compositions on the bystander effect of ADC. The model simulations suggested that dose-fractionation regimen may further improve overall efficacy and bystander effect of ADCs by prolonging the tubulin occupancy in each cell type. SIGNIFICANCE STATEMENT: A PK-PD analysis is presented to understand bystander effect of Trastuzumab-vc-MMAE ADC in antigen (Ag)-low, Ag-high, and coculture (i.e., Ag-high + Ag-low) xenograft mice. This study also describes a novel single cell-level systems PK-PD model to characterize in vivo bystander effect of ADCs. The proposed model can serve as a platform to mathematically characterize multiple cell populations and their interactions in tumor tissues. Our analysis also suggests that fractionated dosing regimen may help improve the bystander effect of ADCs.

Antibody Coadministration as a Strategy to Overcome Binding-Site Barrier for ADCs: a Quantitative Investigation.

It has been proposed that the binding-site barrier (BSB) for antibody-drug conjugates (ADCs) can be overcome with the help of antibody coadministration. However, broad utility of this strategy remains in question. Consequently, here, we have conducted in vivo experiments and pharmacokinetics-pharmacodynamics (PK-PD) modeling and simulation (M&S) to further evaluate the antibody coadministration hypothesis in a quantitative manner. Two different Trastuzumab-based ADCs, T-DM1 (no bystander effect) and T-vc-MMAE (with a bystander effect), were evaluated in high-HER2 (N87) and low-HER2 (MDA-MB-453) expressing tumors, with or without the coadministration of 1, 3, or 8-fold higher Trastuzumab. The tumor growth inhibition (TGI) data was quantitatively characterized using a semi-mechanistic PK-PD model to determine the nature of drug interaction for each coadministration regimen, by estimating the interaction parameter ψ. It was found that the coadministration strategy improved ADC efficacy under certain conditions and had no impact on ADC efficacy in others. The benefit was more pronounced for N87 tumors with very high antigen expression levels where the effect on treatment was synergistic (a synergistic drug interaction, ψ = 2.86 [2.6-3.12]). The benefit was diminished in tumor with lower antigen expression (MDA-MB-453) and payload with bystander effect. Under these conditions, the coadministration regimens resulted in an additive or even less than additive benefit (ψ ≤ 1). As such, our results suggest that while antibody coadministration may be helpful for ADCs in certain circumstances, one should not broadly apply this strategy to all the scenarios without first identifying the costs and benefits of this approach.

Estrogen receptor activation in response to Azadirachtin A stimulates osteoblast differentiation and bone formation in mice.

The positive effects of the sex hormone in sustaining bone homeostasis are exercised by maintaining the equilibrium between cell activity and apoptosis. In this regard, the importance of estrogen receptors in maintaining the bone is that it is an attractive drug target, if devoid of known side effects. In this study, we show that a natural pure compound Azadirachtin A (Aza A) isolated from Azadirachta indica binds selectively to a site in the estrogen receptor, identifying itself to be a selective tissue modifier. Using computational and medicinal chemistry, we show that Aza A binds potentially and selectively to estrogen receptor-α (ERα) as compared with ERß. This preferential binding of Aza A to ERα with good pharmacokinetic distribution in the body forms metabolites, showing that it is well absorbed. In in vivo estrogen deficiency models for osteoporosis, Aza A at a much lower dose enhances new bone formation at both sites of the trabecular and cortical bone with increased bone strength and presents with no hyperplastic effect in the uterus.

A Cell-Level Systems PK-PD Model to Characterize In Vivo Efficacy of ADCs.

Here, we have presented the development of a systems pharmacokinetics-pharmacodynamics (PK-PD) model for antibody-drug conjugates (ADCs), which uses intracellular target occupancy to drive in-vivo efficacy. The model is built based on PK and efficacy data generated using Trastuzumab-Valine-Citrulline-Monomethyl Auristatin E (T-vc-MMAE) ADC in N87 (high-HER2) and GFP-MCF7 (low-HER2) tumor bearing mice. It was observed that plasma PK of all ADC analytes was similar between the two tumor models; however, total trastuzumab, unconjugated MMAE, and total MMAE exposures were >10-fold, ~1.6-fold, and ~1.8-fold higher in N87 tumors. In addition, a prolonged retention of MMAE was observed within the tumors of both the mouse models, suggesting intracellular binding of MMAE to tubulin. A systems PK model, developed by integrating single-cell PK model with tumor distribution model, was able to capture all in vivo PK data reasonably well. Intracellular occupancy of tubulin predicted by the PK model was used to drive the efficacy of ADC using a novel PK-PD model. It was found that the same set of PD parameters was able to capture MMAE induced killing of GFP-MCF7 and N87 cells in vivo. These observations highlight the benefit of adopting a systems approach for ADC and provide a robust and predictive framework for successful clinical translation of ADCs.

Prevention of articular cartilage degeneration in a rat model of monosodium iodoacetate induced osteoarthritis by oral treatment with Withaferin A.

Withaferin A (WFA), a highly oxygenated withanolide is used for anti-osteoporotic, fracture healing, obesity control as medicine and dietary supplement in Ayurveda and Unani medicine but its potential remains to be investigate for the osteoarthritis studies. In the present study, chondro-protective effects of WFA, under in vitro and in vivo conditions were evaluated. In-vitro pharmacological activity of WFA was tested on rat articular chondrocytes through MTT, DPPH, different staining, FACS and translation studies. In-vivo studies of WFA were evaluated through monosodium iodoacetate (MIA) induced osteoarthritis studies. DPPH assay, alcian blue and toluidine blue staining indicated the chondrogenic potential of WFA. Similarly, WFA enhance chondrogenesis through up-regulation of SOX9 protein. In addition, WFA reduced the ROS generation, mitochondrial depolarization and apoptosis induced by inflammatory cytokines IL-1ß and TNF-α. Furthermore, WFA treatment in MIA treated rats alleviated cartilage erosion and improvement in sub-chondral bone micro-architecture by decrease in Tissue volume (∼32%), and trabecular bone pattern factor (∼28%). Taken together, our study provides convincing evidence for the candidature of WFA (10 mg kg-1 day-1) as a potential agent for the treatment of cartilage degenerative diseases like osteoarthritis.

Heartwood extract from Dalbergia sissoo promotes fracture healing and its application in ovariectomy-induced osteoporotic rats.

OBJECTIVES: This study was undertaken to investigate the effects of a heartwood ethanolic extract (HEE) made from the Dalbergia sissoo on facture healing and in the prevention of pathological bone loss resulting from estrogen deficiency in ovariectomized (Ovx) rats. METHODS: Heartwood ethanolic extract (250, 500 and 1000 mg/kg per day) was administered orally immediately next day after drill-hole injury and continued for 2 weeks. Ovx rats received HEE at same doses for 12 weeks and compared with 17-ß estradiol (E2; 100 µg/kg for 5 days/week subcutaneously) group. Confocal imaging for fracture healing, micro-architecture of long bones, biomechanical strength, formation of mineralized nodule by bone marrow osteoprogenitor cells, bone turnover markers and gene expression were studied. One-way ANOVA was used to test significance. KEY FINDINGS: Heartwood ethanolic extract treatment promoted fracture healing, formation of new bone at the drill-hole site and stimulated osteogenic genes at callus region. HEE administration to the Ovx rats exhibited better micro-architectural parameters at various anatomical positions, better bone biomechanical strength and more osteoprogenitor cells in the bone marrow compared with Ovx + vehicle group. HEE exhibited no uterine estrogenicity. CONCLUSIONS: Oral administration of HEE was found to promote fracture healing and exhibited osteoprotective effect by possibly stimulation of osteoblast function.

Vitamin B6 Tethered Endosomal pH Responsive Lipid Nanoparticles for Triggered Intracellular Release of Doxorubicin.

This study reports the development of Vitamin B6 (VitB6) modified pH sensitive charge reversal nanoparticles for efficient intracellular delivery of Doxorubicin (DOX). Herein, VitB6 was conjugated to stearic acid, and the nanoparticles of the lipid were formulated by solvent injection method (DOX-B6-SA-NP). Because of the pKa (5.6) of VitB6, DOX-B6-SA-NP showed positive charge and enhanced release of DOX at pH 5. Confocal microscopy illustrated that DOX-B6-SA-NP treatment kept higher DOX accumulation inside the cells than conventional pH insensitive lipid nanoparticles (DOX-SA-NP). The cationic charge of nanoparticles subsequently facilitated the endosomal escape and promoted the nuclear accumulation of DOX. Furthermore, in vitro cytotoxicity, apoptosis, cell cycle arrest, and mitochondrial membrane depolarization studies supported the enhanced efficacy of DOX-B6-SA-NP in comparison to free DOX and DOX-SA-NP. Intravenous pharmacokinetics and biodistribution investigations indicated that pH sensitive nanoparticles can significantly prolong the blood circulation time of DOX in biological system and increase the drug accumulation to tumor site. Consequent to this, DOX-B6-SA-NP also exhibited much enhanced therapeutic efficacy and lower toxicity in tumor-bearing rats compared to free DOX. The reduction in toxicity was confirmed by histological and survival analysis. In conclusion, these results suggest that the VitB6 modified charge reversal nanoparticles can be a novel platform for the successful delivery of anticancer drugs.

Vitamin B12 Grafted Layer-by-Layer Liposomes Bearing HBsAg Facilitate Oral Immunization: Effect of Modulated Biomechanical Properties.

Adhesion forces of nanoparticulate materials toward biological membrane are crucial for designing a delivery system for therapeutic molecules and vaccines. The present study aims to investigate the impact of surface roughness of the nanoparticulate system in oral delivery of antigen and its targeting to toward intestinal antigen presenting cells. To evaluate this hypothesis, layer-by-layer coated liposomes (LBL-Lipo) were fabricated using sodium alginate and Vitamin B12 conjugated Chitosan (VitB12-Chi) as anionic and cationic polyelectrolyte, respectively. Change in surface roughness was observed on changes in pH from gastric to intestinal conditions attributed to increase and decrease in charge density on VitB12-Chi. Surface roughness was measured in terms of root-mean-square measured by topographical analysis using atomic force microscopy. LBL-Lipo were further characterized for their size, zeta potential, and release behavior to evaluate the potential for oral vaccine delivery. In vitro cell uptake in macrophage cells (J-744) shows about 2- and 3.1-fold increased uptake of rough LBL-Lipo over smooth LBL-Lipo at 37 °C (endocytosis) and 4 °C (endocytosis inhibition) indicating improved biological interaction. Further in vivo immunization study revealed that prototype formulations were able to produce 4.8- and 3.3-fold higher IgG and IgA levels in serum and feces, respectively, in comparison to smooth LBL-Lipo.

Kaempferol targets Krt-14 and induces cytoskeletal mineralization in osteoblasts: A mechanistic approach.

Kaempferol (KEM) has been observed to stimulate Krt-14 protein which subsequently contributes to matrix maturation and mineralization in rat primary osteoblast cells. Incorporation of Krt-14 siRNA results in reduced mRNA and protein expression after 48h post transfection and remained low for 9days. At day 9 Krt-14 siRNA significantly reduced mineralization without concomitant change in the cell number. ColI and OCN gene expression was reduced in Krt-14 siRNA-treated osteoblast cells. Soluble osteocalcin and collagen levels were markedly decreased in conditioned medium as well as in acid-salt soluble cell-ECM layer treated with Krt-14 siRNA compared to control siRNA treated cells corroborated at the ultrastructral level by AFM. Further, knockdown of Krt-14 and inhibitors against AMPK and mTOR, repressed the activation of mTOR and mineralization attenuated by KEM confirmed the role of Krt-14 in mineralization. These findings strongly suggest that Krt-14 regulates osteoblast mineralization by organizing osteoblast derived ECM.

Vitamin B12 functionalized layer by layer calcium phosphate nanoparticles: A mucoadhesive and pH responsive carrier for improved oral delivery of insulin.

The present study investigates the potential of layer by layer coated calcium phosphate nanoparticles - for oral delivery of insulin where Vitamin B12 grafted chitosan and sodium alginate have been used as cationic and anionic polyelectrolyte respectively. The major emphasis has been given on the role Vitamin B12 conjugated chitosan as cationic polyelectrolyte (VitB12-Chi) in the delivery system. VitB12-Chi conjugate was prepared by carbodiimide reaction. The formulated VirB12-Chi-CPNPs were tested for in vitro and in vivo efficacy studies carried out in Caco-2 monolayers and diabetic rats. VitB12-Chi-CPNPs with particle size <250nm and zeta potential+32.56(±2.34) exhibited pH responsive insulin release at simulated gastric fluid and simulated intestinal fluid. Fluorescence microscopy and flow cytometry studies revealed higher uptake of VitB12-Chi-CPNPs in Caco-2 monolayer in comparison to Chi-CPNPs. Further reduction in TEER supported paracellular transport of insulin because of opening of tight epithelial junctions. In vivo intestinal uptake of FITC tagged Vit-B12-Chi-CPNPs from different intestinal segments supported paracellular and receptor mediated uptake of VitB12-Chi-CPNPs. Plasma insulin and blood glucose levels were measured in diabetic rats and showed about four fold increases in insulin bioavailability and sustained hypoglycemic effects up to 12h of administration with VitB12-Chi-CPNPs in comparison to Chi-CPNPs. Results of the study revealed the potential of layer by layer nanoparticles for oral insulin delivery. The study also specifically highlighted the role of VitB12 as a pH sensitive and targeting ligand which significantly participated in enhancing insulin oral bioavailability. STATEMENT OF SIGNIFICANCE: Oral delivery of insulin is always the most desirable approach for diabetic patients however it's also the most challenging in respect to formulation development due to harsh gastrointestinal conditions. Several groups have been working from decades for oral delivery of insulin. However the beauty of this prototype formulation is that it exhibits the pH responsive behavior in natural condition of gastrointestinal tract. It resists the release of insulin at gastric condition however stimulate the release at intestinal conditions. Apart from pH responsive behavior it utilizes multiple pathways to improve the overall bioavailability of insulin including paracellular transport and receptor mediated endocytosis.

Investigating the role of Pluronic-g-Cationic polyelectrolyte as functional stabilizer for nanocrystals: Impact on Paclitaxel oral bioavailability and tumor growth.

Paclitaxel (PTX) is a potent anticancer drug which suffers limitations of extremely low oral bioavailability due to low solubility, rapid metabolism and efflux by P-gp transporters. The main objective of this study was to overcome the limitation of PTX by designing delivery systems that can enhance the absorption using multiple pathways. A novel Pluronic-grafted chitosan (Pl-g-CH) copolymer was developed and employed as a functional stabilizer for nanocrystals (NCs) and hypothesized that it would improve PTX absorption by several mechanisms and pathways. Pl-g-CH was synthesized and characterized using (1)H NMR and then used as a stabilizer during nanocrystal development. To establish our proof of concept the optimized formulation having a particle size 192.7 ± 9.2 nm and zeta potential (+) 38.8 ± 3.12 mV was studied extensively on in vitro Caco-2 model. It was observed that nanocrystals rendered higher PTX accumulation inside the cell than Taxol™. P-gp inhibitory potential of Pl-g-CH was proved by flow cytometry and fluorescence microscopy where the much enhanced fluorescence intensity of Rhodamine 123 (Rho-123, P-gp substrate) was observed in the presence of Pl-g-CH. In addition, a significant decrease in Trans Epithelial Electrical Resistance (TEER) of Caco-2 cell monolayers was observed with nanocrystals as well as with Taxol™ (in the presence of free Pl-g-CH compared to only Taxol™). This supports the role of the stabilizer in reversible opening of tight junctions between cells which can allow paracellular transport of drug. The in vivo results were in complete corroboration with in vitro results. Nanocrystals resulted in much enhanced absorption with 12.6-fold improvement in relative bioavailability to that of Taxol™. Concomitantly efficacy data in B16 F10 murine melanoma model also showed a significant reduction in tumor growth with nanocrystals compared to Taxol™ and control. Based on the results it can be suggested that nanocrystals with functional stabilizers can be a promising approach for the oral delivery of anticancer drugs which are P-gp substrates STATEMENT OF SIGNIFICANCE: Nanocrystals are currently one of the most explored novel drug delivery systems especially for oral delivery of drugs because of ease in synthesis and high drug loading. But their use is still limited for oral delivery of anticancer drugs which are P-gp substrates. This particular study aims at widening the scope of nanocrystals by using a functional stabilizer which participates in enhancing the oral absorption of anticancer drugs and controlling the tumor growth.

An insight into functionalized calcium based inorganic nanomaterials in biomedicine: Trends and transitions.

Over the recent years the use of biocompatible and biodegradable nanoparticles in biomedicine has become a significant priority. Calcium based ceramic nanoparticles like calcium phosphate (CaP) and calcium carbonate (CaCO3) are therefore considered as attractive carriers as they are naturally present in human body with nanosize range. Their application in tissue engineering and localized controlled delivery of bioactives for bones and teeth is well established now, but recently their use has increased significantly as carrier of bioactives through other routes also. These delivery systems have become most potential alternatives to other commonly used delivery system because of their cost effectiveness, biodegradability, chemical stability, controlled and stimuli responsive behaviour. This review comprehensively covers their characteristic features, method of preparation and applications but the thrust is to focus their recent development, functionalization and use in systemic delivery. On the same platform mineralization of other nanoparticulate delivery system which has widened their application drug delivery will be discussed. The emphasis has been given on their pH dependent properties which make them excellent carriers for tumour targeting and intracellular delivery. Finally this review also attempts to discuss their drawback which limits their clinical utility.

Surface-engineered dendrimeric nanoconjugates for macrophage-targeted delivery of amphotericin B: formulation development and in vitro and in vivo evaluation.

The present study aimed to develop an optimized dendrimeric delivery system for amphotericin B (AmB). Fifth-generation (5.0 G) poly(propylene imine) (PPI) dendrimers were synthesized, conjugated with mannose, and characterized by use of various analytical techniques, including Fourier transform infrared spectroscopy (FTIR), (1)H nuclear magnetic resonance ((1)H-NMR) spectroscopic analysis, and atomic force microscopy (AFM). Mannose-conjugated 5.0 G PPI (MPPI) dendrimers were loaded with AmB and evaluated for drug loading efficiency, in vitro drug release profile, stability, hemolytic toxicity to human erythrocytes, cytotoxicity to and cell uptake by J774A.1 macrophage cells, antiparasitic activity against intracellular Leishmania donovani amastigotes, in vivo pharmacokinetic and biodistribution profiles, drug localization index, toxicity, and antileishmanial activity. AFM showed the nanometric size of the MPPI dendrimers, with a nearly globular architecture. The conjugate showed a good entrapment efficiency for AmB, along with pH-sensitive drug release. Highly significant reductions in toxicity toward human erythrocytes and macrophage cells, without compromising the antiparasitic activity of AmB, were observed. The dendrimeric formulation of AmB showed a significant enhancement of the parasiticidal activity of AmB toward intramacrophagic L. donovani amastigotes. In the in vitro cell uptake studies, the formulation showed selectivity toward macrophages, with significant intracellular uptake. Further pharmacokinetic and organ distribution studies elucidated the controlled delivery behavior of the formulation. The drug localization index was found to increase significantly in macrophage-rich organs. In vivo studies showed a biocompatible behavior of MPPIA, with negligible toxicity even at higher doses, and promising antileishmanial activity. From the results, we concluded that surface-engineered dendrimers may serve as optimized delivery vehicles for AmB with enhanced activity and low or negligible toxicity.

Development of docetaxel nanocapsules for improving in vitro cytotoxicity and cellular uptake in MCF-7 cells.

The aim of this study was to fabricate docetaxel loaded nanocapsules (DTX-NCs) with a high payload using Layer-by-Layer (LbL) technique by successive coating with alternate layers of oppositely charged polyelectrolytes. Developed nanocapsules (NCs) were characterized in terms of morphology, particle size distribution, zeta potential (ζ-potential), entrapment efficiency and in vitro release. The morphological characteristics of the NCs were assessed using transmission electron microscopy (TEM) that revealed coating of polyelectrolytes around the surface of particles. The developed NCs successfully attained a submicron particle size while the ζ-potential of optimized NCs alternated between (+) 34.64 ± 1.5 mV to (-) 33.25 ± 2.1 mV with each coating step. The non-hemolytic potential of the NCs indicated the suitability of the developed formulation for intravenous administration. A comparative study indicated that the cytotoxicity of positively charged NCs (F4) was significant higher (p < 0.05) rather than negative charged NCs (F3), plain drug (DTX) and marketed preparation (Taxotere®) when evaluated in vitro on MCF-7 cells. Furthermore, cell uptake studies evidenced a higher uptake of positive NCs (≥1.2 fold) in comparison to negative NCs. In conclusion, formulated NCs are an ideal vehicle for passive targeting of drugs to tumor cells that may result in improved efficacy and reduced toxicity of encapsulated drug moiety.

Development of stabilized Paclitaxel nanocrystals: In-vitro and in-vivo efficacy studies.

OBJECTIVE: The aim of the study was to develop stable Paclitaxel nanocrystals (PTX/NCs) for enhanced oral delivery of Paclitaxel (PTX) by circumventing its difficult solubilization properties and rapid metabolism. METHODS: Preparation of nanocrystals (NCs) was carried out using high pressure homogenizer (Microfluidizer™) without using any organic solvent. Effect of various process and formulation parameters on development and stability of nanocrystals (NCs) were investigated. Particle characteristics, stability studies, in-vitro cellular studies and oral pharmacokinetics in male Wistar rats were examined. RESULTS: It was found that different stabilizer used had different effect on size reduction and stability. Surfactants (Tween 80) and low molecular weight synthetic polymer sodium poly styrene sulfonate (PSS) found more suitable and efficient compared to high molecular weight polymers glycol chitosan (GC) and sodium alginate (SA). In-vitro cytotoxicity and cell cycle arrest studies on MCF7 and MDA-MB breast cancer cell lines revealed that PTX/NCs retained the activity even after processing at high pressure and also NCs were more potent and efficacious than PTX solution. The oral in-vivo pharmacokinetic studies demonstrated that PTX/NCs exhibit significant increase in AUC0-t, Cmax, MRT and decrease in Tmax, compared to plain PTX crystals. The increase in AUC was almost 9-10 fold compared to plain PTX crystals. CONCLUSION: Altogether study showed that PTX/NC can be a clinically relevant drug delivery system for oral chemotherapy as it can remarkably increases the pharmacological effect by increasing oral bioavailability.