Maged W. Helmy

Spray-dried casein-based micelles as a vehicle for solubilization and controlled delivery of flutamide: Formulation, characterization, and in vivo pharmacokinetics

Novel casein (CAS)-based micelles loaded with the poorly soluble anti-cancer drug, flutamide (FLT), were successfully developed in a powdered form via spray-drying technique. Genipin (GNP) was used to crosslink CAS micelles as demonstrated by color variation of the micelles. Drug solubilization was enhanced by incorporation within the hydrophobic micellar core which was confirmed by solubility study and UV spectra. Spherical core-shell micelles were obtained with a particle size below 100 nm and zeta potential around -30 mV. At low drug loading, FLT was totally incorporated within micellar core as revealed by thermal analysis. However, at higher loading, excess non-incorporated drug at micelle surface caused a significant reduction in the surface charge density. Turbidity measurements demonstrated the high physical stability of micelles for 2 weeks dependent on GNP-crosslinking degree. In a dry powdered form, the micelles were stable for 6 months with no significant changes in drug content or particle size. A sustained drug release from CAS micelles up to 5 days was observed. After i.v. administration into rats, CAS micelles exhibited a prolonged plasma circulation of FLT compared to drug solution. Furthermore, a more prolonged drug systemic circulation was observed for GNP-crosslinked micelles. Overall, this study reports the application of spray-dried natural protein-based micelles for i.v. delivery of hydrophobic anti-cancer drugs such as FLT.

Novel ionically crosslinked casein nanoparticles for flutamide delivery: formulation, characterization, and in vivo pharmacokinetics.

A novel particulate delivery matrix based on ionically crosslinked casein (CAS) nanoparticles was developed for controlled release of the poorly soluble anticancer drug flutamide (FLT). Nanoparticles were fabricated via oil-in-water emulsification then stabilized by ionic crosslinking of the positively charged CAS molecules below their isoelectric point, with the polyanionic crosslinker sodium tripolyphosphate. With the optimal preparation conditions, the drug loading and incorporation efficiency achieved were 8.73% and 64.55%, respectively. The nanoparticles exhibited a spherical shape with a size below 100 nm and a positive zeta potential (+7.54 to +17.3 mV). FLT was molecularly dispersed inside the nanoparticle protein matrix, as revealed by thermal analysis. The biodegradability of CAS nanoparticles in trypsin solution could be easily modulated by varying the sodium tripolyphosphate crosslinking density. A sustained release of FLT from CAS nanoparticles for up to 4 days was observed, depending on the crosslinking density. After intravenous administration of FLT-CAS nanoparticles into rats, CAS nanoparticles exhibited a longer circulation time and a markedly delayed blood clearance of FLT, with the half-life of FLT extended from 0.88 hours to 14.64 hours, compared with drug cosolvent. The results offer a promising method for tailoring biodegradable, drug-loaded CAS nanoparticles as controlled, long-circulating drug delivery systems of hydrophobic anticancer drugs in aqueous vehicles.

Ionically-crosslinked milk protein nanoparticles as flutamide carriers for effective anticancer activity in prostate cancer-bearing rats

In this study, casein (CAS) nanoparticles were used to encapsulate the hydrophobic anticancer drug, flutamide (FLT), aiming at controlling its release, enhancing its anti-tumor activity, and reducing its hepatotoxicity. The nanoparticles were prepared by emulsification of CAS, at pH below its isoelectric point, and stabilized via ionic-crosslinking with sodium tripolyphosphate (TPP). The nanoparticles were spherical and positively charged with a size below 100 nm and exhibited a sustained drug release up to 4 days. After intravenous administration into prostate cancer-bearing rats for 28 days, FLT-loaded CAS nanoparticles showed a higher anti-tumor efficacy as revealed by a significantly higher % reduction in PSA serum level (75%) compared to free FLT (55%). Moreover, the nanoparticles demonstrated a marked reduction in the relative weights of both prostate tumor and seminal vesicle (43% and 32%) compared to free FLT (12% and 18%), respectively. A significantly higher anti-proliferative, anti-angiogenic, and apoptotic effects was demonstrated by the nanoparticles compared to drug solution as evidenced by their ability to decrease the expression of the proliferative marker (Ki-67) and reduce the level of tumor angiogenic markers (VEGF and IGF-1) as well as their ability to activate caspase-3 with subsequent induction of apoptosis in prostate cancer cells. Conclusively, these novel ionically-crosslinked milk protein nanovehicles offer a promising carrier to allow controlled intravenous delivery of hydrophobic anticancer drugs.

Celecoxib offsets the negative renal influences of cyclosporine via modulation of the TGF-β1/IL-2/COX-2/endothelin ETB receptor cascade

Endothelin (ET) signaling provokes nephrotoxicity induced by the immunosuppressant drug cyclosporine A (CSA). We tested the hypotheses that (i): celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, counterbalances renal derangements caused by CSA in rats and (ii) the COX-2/endothelin ET(B) receptor signaling mediates the CSA-celecoxib interaction. Ten-day treatment with CSA (20 mg/kg/day) significantly increased biochemical indices of renal function (serum urea, creatinine), inflammation (interleukin-2, IL-2) and fibrosis (transforming growth factor-β₁, TGF-β₁). Histologically, CSA caused renal tubular atrophy along with interstitial fibrosis. These detrimental renal effects of CSA were largely reduced in rats treated concurrently with celecoxib (10 mg/kg/day). We also report that cortical glomerular and medullary tubular protein expressions of COX-2 and ET(B) receptors were reduced by CSA and restored to near-control values in rats treated simultaneously with celecoxib. The importance of ET(B) receptors in renal control and in the CSA-celecoxib interaction was further verified by the findings (i) most of the adverse biochemical, inflammatory, and histopathological profiles of CSA were replicated in rats treated with the endothelin ETB receptor antagonist BQ788 (0.1 mg/kg/day, 10 days), and (ii) the BQ788 effects, like those of CSA, were alleviated in rats treated concurrently with celecoxib. Together, the data suggest that the facilitation of the interplay between the TGF-β1/IL-2/COX-2 pathway and the endothelin ET(B) receptors constitutes the cellular mechanism by which celecoxib ameliorates the nephrotoxic manifestations of CSA in rats.

Superiority of aromatase inhibitor and cyclooxygenase-2 inhibitor combined delivery: Hyaluronate-targeted versus PEGylated protamine nanocapsules for breast cancer therapy

Despite several reports have revealed the beneficial effect of co-administration of COX-2 inhibitors with aromatase inhibitors in managing postmenopausal breast cancer; no nanocarriers for such combined delivery have been developed till now. Therefore, protamine nanocapsules (PMN-NCs) have been developed to co-deliver letrozole (LTZ) that inhibits aromatase-mediated estrogen biosynthesis and celecoxib (CXB) that synergistically inhibits aromatase expression. Inspired by the CD44-mediated tumor targeting ability of hyaluronate (HA), we developed HA-coated PMN-NCs (HA-NCs) via electrostatic layer-by-layer assembly. Moreover, multi-compartmental PEGylated phospholipid-CXB complex bilayer enveloping PMN-NCs (PEG-NCs) were designed for conferring biphasic CXB release from the phospholipid corona and oily core as well as enabling passive-targeting. The NCs demonstrated excellent stability, prolonged circulation and could be scaled up with the aid of spray-drying technology. Hemolysis, serum stability and cytotoxicity studies confirmed the superiority of combined LTZ-CXB nano-delivery. Mechanistically, the NCs especially HA-NCs and PEG-NCs demonstrated precious anti-tumor effects in vivo revealed as reduction in the tumor volume and aromatase level, increased apoptosis, as well as inhibition of VEGF, NF-κB and TNF-α augmented by histopathological and immunohistochemical studies. Overall, our approach provided for the first time a potential strategy for targeted LTZ-CXB combined therapy of hormone-dependent breast cancer via singular nanocapsule delivery system.

Shell-crosslinked zein nanocapsules for oral codelivery of exemestane and resveratrol in breast cancer therapy

AIM Oral administration of exemestane (EXM) and resveratrol (RES) for breast cancer therapy has been limited by their poor solubility and low permeability. METHODS In this study, these issues were tackled using zein nanocapsules (ZNCs) for oral EXM/RES codelivery combining drug solubilization within oily core and resistance to digestion via hydrophobic protein shell. Furthermore, higher oral stability and sustained release could be enabled by glutaraldehyde crosslinking of zein shell. RESULTS & CONCLUSION EXM/RES-ZNCs showed enhanced cytotoxicity against MCF-7 and 4T1 breast cancer cells compared with free drug combination with higher selectivity to cancer cells rather than normal fibroblasts. In vivo, crosslinked EXM/RES-ZNCs markedly reduced the percentage increase of Ehrlich ascites mammary tumor volume in mice by 2.4-fold compared with free drug combination.

Endothelin ETA receptor/lipid peroxides/COX-2/TGF-β1 signalling underlies aggravated nephrotoxicity caused by cyclosporine plus indomethacin in rats.

Cyclosporine (CSA) and non‐steroidal anti‐inflammatory drugs (NSAIDs) are co‐prescribed for some arthritic conditions. We tested the hypothesis that this combined regimen elicits exaggerated nephrotoxicity in rats via the up‐regulation of endothelin (ET) receptor signalling.

Celecoxib, but not indomethacin, ameliorates the hypertensive and perivascular fibrotic actions of cyclosporine in rats: Role of endothelin signaling

The immunosuppressant drug cyclosporine (CSA) is used with nonsteroidal antiinflammatory drugs (NSAIDs) in arthritic conditions. In this study, we investigated whether NSAIDs modify the deleterious hypertensive action of CSA and the role of endothelin (ET) receptors in this interaction. Pharmacologic, protein expression, and histopathologic studies were performed in rats to investigate the roles of endothelin receptors (ETA/ETB) in the hemodynamic interaction between CSA and two NSAIDs, indomethacin and celecoxib. Tail-cuff plethysmography measurements showed that CSA (20 mg kg(-1) day(-1), 10 days) increased systolic blood pressure (SBP) and heart rate (HR). CSA hypertension was associated with renal perivascular fibrosis and divergent changes in immunohistochemical signals of renal arteriolar ETA (increases) and ETB (decreases) receptors. While these effects of CSA were preserved in rats treated concomitantly with indomethacin (5 mg kg(-1) day(-1)), celecoxib (10 mg kg(-1) day(-1)) abolished the pressor, tachycardic, and fibrotic effects of CSA and normalized the altered renal ETA/ETB receptor expressions. Selective blockade of ETA receptors by atrasentan (5 mg kg(-1) day(-1)) abolished the pressor response elicited by CSA or CSA plus indomethacin. Alternatively, BQ788 (ETB receptor blocker, 0.1 mg kg(-1) day(-1)) caused celecoxib-sensitive elevations in SBP and potentiated the pressor response evoked by CSA. Together, the improved renovascular fibrotic and endothelin receptor profile (ETA downregulation and ETB upregulation) mediate, at least partly, the protective effect of celecoxib against the hypertensive effect of CSA. Clinically, the use of celecoxib along with CSA in the management of arthritic conditions might provide hypertension-free regimen.

Phytosomal bilayer-enveloped casein micelles for codelivery of monascus yellow pigments and resveratrol to breast cancer

AIM Multireservoir nanocarriers were fabricated for delivering antineoplastic drug cocktail from herbal and fungal origin. Monascus yellow pigments (MYPs), monascin and ankaflavin, were isolated from red-mold rice, and incorporated within casein micelles (CAS MCs) along with the herbal drug, resveratrol (RSV). Both drugs (MYPs and RSV) were simultaneously incorporated into the hydrophobic core of CAS MCs. Alternatively, MYPs-loaded CAS MCs were enveloped within RSV-phytosomal bilayer elaborating multireservoir nanocarriers. RESULTS Cytotoxicity studies confirmed the superiority of multireservoir nanocarriers against MCF-7 breast cancer cells. The in vivo antitumor efficacy was revealed by reduction of the tumor volume and growth biomarkers. CONCLUSION Multireservoir CAS nanocarriers for codelivery of both MYPs and RSV may be promising alternative to traditional breast cancer therapy.

Additive Renoprotection by Pioglitazone and Fenofibrate against Inflammatory, Oxidative and Apoptotic Manifestations of Cisplatin Nephrotoxicity: Modulation by PPARs

Nephrotoxicity is a major side effect for the antineoplastic drug cisplatin. Here, we employed pharmacological, biochemical, and molecular studies to investigate the role of peroxisome proliferator-activated receptors (PPARs) in cisplatin nephrotoxicity. Rats were treated with a single i.p. dose of cisplatin (5 mg/kg) alone or combined with pioglitazone (PPARγ agonist), fenofibrate (PPARα agonist), pioglitazone plus fenofibrate, or thalidomide (Tumor necrosis factor-α inhibitor; TNF-α). Cisplatin nephrotoxicity was evidenced by rises in renal indices of functional (blood urea nitrogen, BUN, and creatinine), inflammatory (TNF-α, interleukin 6, IL-6), oxidative (increased malondialdehyde, MDA, and decreased superoxide dismutase, SOD and nitric oxide metabolites, NOx), apoptotic (caspase 3), and histological (glomerular atrophy, acute tubular necrosis and vacuolation) profiles. Cisplatin effects were partly abolished upon concurrent exposure to pioglitazone, fenofibrate, or thalidomide; more renoprotection was observed in rats treated with pioglitazaone plus fenofibrate. Immunostaining showed that renal expressions of PPARα and PPARγ were reduced by cisplatin and restored to vehicle-treated values after simultaneous treatment with pioglitazone or fenofibrate. Fenofibrate or pioglitazone renoprotection remained unaltered after concurrent blockade of PPARα (GW6471) and PPARγ (GW9662), respectively. To complement the rat studies, we also report that in human embryonic kidney cells (HEK293 cells), increases caused by cisplatin in inflammatory, apoptotic, and oxidative biomarkers were (i) partly improved after exposure to pioglitazone, fenofibrate, or thalidomide, and (ii) completely disappeared in cells treated with a combination of all three drugs. These data establish that the combined use of pioglitazone and fenofibrate additively improved manifestations of cisplatin nephrotoxicity through perhaps GW6471/GW9662-insensitive mechanisms.