Optimization, characterization and in vivo evaluation of mupirocin nanocrystals for topical administration.

Treatment of infectious skin conditions resulting from wounds and burns with topical antibiotics is challenging, particularly those caused by methicillin-resistant Staphylococcus aureus bacteria (MRSA). This is due to the formation of bacterial biofilms characterized by antimicrobial resistance. Mupirocin (MP), a widely used topical antibiotic, is active against gram-positive bacteria including MRSA. However, MP suffers from sub-optimal therapeutic efficacy due to its poor water-solubility and the significant rise in MP-resistant S. aureus. In this study, the physico-chemical characteristics of MP were modified through nanocrystallization to improve its therapeutic efficacy for the treatment of skin infections. Mupirocin-nanocrystals (MP-NC) were prepared using a nanoprecipitation technique and optimized using a D-optimal response surface design. The optimization of MP-NC produced ultra-small monodisperse spherical particles with a mean diameter of 70 nm and a polydispersity index of 0.2. The design resulted in two optimal MP-NC formulations that were evaluated by performing series of in vitro, ex vivo, microbiological, and in vivo studies. In-vitro results showed a 10-fold increase in the saturation solubility and a 9-fold increase in the dissolution rate of MP-NC. Ex vivo permeation studies, using pig ears skin, showed a 2-fold increase in the dermal deposition of MP-NC with the highest drug deposition occurring at 500-µm skin depth. Moreover, the optimal MP-NC formulations were lyophilized and incorporated into a 2% w/w cream. Microbiological studies revealed a 16-fold decrease in the minimum inhibitory concentration and the minimum bactericidal concentration of MP-NC. In vivo studies, using a rat excision burn wound model, demonstrated rapid and complete healing of infected burn wounds in rats treated with MP-NC cream in comparison to marketed Avoban ointment. Our results suggest that nanocrystallization of MP may provide an avenue through which higher levels of a topically applied MP can be permeated into the skin to reach relevant infectious areas and exert potential local antibacterial effects.

In Situ-Forming Microparticles for Controlled Release of Rivastigmine: In Vitro Optimization and In Vivo Evaluation.

In this work, sucrose acetate isobutyrate (SAIB) and polylactic co-glycolic acid (PLGA) were used alone or in combination as a matrix-former (MF) to prepare long-acting injectable rivastigmine (RV) in situ-forming microparticles (ISM). RV-ISM were prepared by the emulsification of an internal phase, containing the drug and the matrix former(s), into an external oily phase containing a stabilizer. The statistical design, Central Composite Design (CCD), was adopted as a quality by design (QbD) approach to optimize the formulation of RV-ISM systems. The fabricated RV-ISM systems was designed to minimize the initial burst drug release and maximize the sustainment of RV release from the ISM and ease of injection. The influence of critical formulation variables such as the matrix-former to drug (MF/D) ratio and SAIB to PLGA (S/P) ratio in the internal phase with respect to critical quality attributes (CQAs), such as the percentage drug release within the first day (Q1), the time required for 50% drug release (T50%) and the rate of injection, were studied using the CCD. The optimal RV-ISM system with the highest desirability value (0.74) was predicted to have an MF/D ratio of 11.7:1 (w/w) and an S/P ratio of 1.64:1 (w/w). The optimal RV-ISM system was assessed for its release profile, injectability, rheological properties, morphology, effect on cell viability, tolerance to γ-sterilization and in vivo performance in male albino rabbits. In vitro release studies revealed that the optimal RV-ISM system released 100% of its drug content throughout a release period of 30 days with only 15.5% drug release within the first day (Q1) and T50% of 13.09 days. Moreover, the optimal system showed a high injection rate of 1.012 mL/min, pseudoplastic flow, uniform spherical globules with homogenous particle size, minimal cytotoxicity and high tolerability to γ-sterilization. In vivo pharmacokinetic (PK) studies revealed that the rate of absorption of RV from the optimal RV-ISM system was controlled compared to a drug solution following either intramuscular (IM) or subcutaneous (SC) injection. Furthermore, the optimal RV-ISM was found to follow flip-flop PK with poor correlation between in vitro release and in vivo findings. These findings suggest that the optimal RV-ISM is a promising tool to achieve a sustained release therapy for RV; however, further investigation is still required to optimize the in vivo performance of RV-ISM.

Nanocrystals of Fusidic Acid for Dual Enhancement of Dermal Delivery and Antibacterial Activity: In Vitro, Ex Vivo and In Vivo Evaluation.

With the alarming rise in incidence of antibiotic-resistant bacteria and the scarcity of newly developed antibiotics, it is imperative that we design more effective formulations for already marketed antimicrobial agents. Fusidic acid (FA), one of the most widely used antibiotics in the topical treatment of several skin and eye infections, suffers from poor water-solubility, sub-optimal therapeutic efficacy, and a significant rise in FA-resistant Staphylococcus aureus (FRSA). In this work, the physico-chemical characteristics of FA were modified by nanocrystallization and lyophilization to improve its therapeutic efficacy through the dermal route. FA-nanocrystals (NC) were prepared using a modified nanoprecipitation technique and the influence of several formulation/process variables on the prepared FA-NC characteristics were optimized using full factorial statistical design. The optimized FA-NC formulation was evaluated before and after lyophilization by several in-vitro, ex-vivo, and microbiological tests. Furthermore, the lyophilized FA-NC formulation was incorporated into a cream product and its topical antibacterial efficacy was assessed in vivo using a rat excision wound infection model. Surface morphology of optimized FA-NC showed spherical particles with a mean particle size of 115 nm, span value of 1.6 and zeta potential of -11.6 mV. Differential scanning calorimetry and powder X-ray diffractometry confirmed the crystallinity of FA following nanocrystallization and lyophilization. In-vitro results showed a 10-fold increase in the saturation solubility of FA-NC while ex-vivo skin permeation studies showed a 2-fold increase in FA dermal deposition from FA-NC compared to coarse FA. Microbiological studies revealed a 4-fofd decrease in the MIC against S. aureus and S. epidermidis from FA-NC cream compared to commercial Fucidin cream. In-vivo results showed that FA-NC cream improved FA distribution and enhanced bacterial exposure in the infected wound, resulting in increased therapeutic efficacy when compared to coarse FA marketed as Fucidin cream.

High-fat diet induced alteration in lipid enzymes and inflammation in cardiac and brain tissues: Assessment of the effects of Atorvastatin-loaded nanoparticles.

Treatment with Lipitor is associated with several adverse impacts. Here we investigated the effects of low Lipitor nanoparticles (atorvastatin calcium nanopartilcle [AC-NP]), with size less than 100 , on enzymes of lipid metabolism and inflammation in cardiac, hepatic, and brain tissues of hypercholestremic adult male rats. Adult male rats were divided into five experimental groups. In group 1, the intact control (normal pellet diet), animals were fed a normal control diet; the other four groups were fed a high-fat diet (HFD) for 6 weeks. After 6 weeks, groups from 2 to 5 were assigned as a positive control (HFD), HFD + Lipitor, HFD + AC-NP-R1, or HFD + AC-NP-R2. Different treatments were administrated orally for two regimen periods (R1 daily and R2 once every 3 days). The treatment was conducted for two consecutive weeks. The HFD group faced a significant elevation in 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA), associated with a significant reduction in low-density lipoprotein receptor (LDL-R) along with cholesterol 7 α-hydroxylase enzyme in hepatic tissues, compared with the control group. Also, the HFD group induced hepatic, cardiac, and brain inflammation, evidenced by increased hepatic oxidative stress markers and cardiac homocysteine, together with elevated proinflammatory cytokines interleukin-1ß (IL-1ß) and IL-6 levels in brain tissue, compared with the control group. Different AC-NP treatments significantly augmented both mRNA LDL-R and mRNA 7α-hydroxylase expression in hepatic tissues, associated with significant depletion in mRNA HMG-CoA expression, compared with HFD + Lipitor. The inflammation symptoms were ameliorated by the AC-NP treatments, compared to HFD + Lipitor. Lipitor encapsulation in NP formulation results in increased efficiency and reduced dose-related adverse effects known to be associated with the Lipitor chronic administration.

Superhydrophobic Substrates for Ultrahigh Encapsulation of Hydrophilic Drug into Controlled-Release Polyelectrolyte Complex Beads: Statistical Optimization and In Vivo Evaluation.

: In this work, ultrahigh drug-loaded chitosan (Ch)/K-carrageenan (Kc) polyelectrolyte complex (PEC) beads were formed in situ by cross-linking in a glutaraldehyde-saturated atmosphere and were prepared on superhydrophobic substrates fabricated by spraying glass surfaces with ready-made spray for domestic use (NeverWet®). Verapamil hydrochloride (VP), a highly hydrophilic drug with a short biological half-life, was incorporated into a series of Ch-based and/or Ch/Kc-PEC-based beads to control its release profile in vivo. The formulation of VP-loaded beads was optimized using stepwise statistical designs based on a prespecified criterion. Several characteristics of the prepared beads, such as entrapment efficiency (EE%), in vitro drug release, swelling ratio, size and surface microstructure as well as molecular interactions between the drug and formulation ingredients, were investigated. In vivo pharmacokinetic (PK) studies were carried out using the rabbit model to study the ability of the optimized VP-loaded beads to control the absorption rate of VP. Results revealed that the prepared superhydrophobic substrates were able to fabricate VP-loaded beads with extremely high EE exceeding 90% w/w compared to only 27.80% when using conventional ionotropic gelation technique. PK results showed that the rate of VP absorption was well controlled following oral administration of the optimized beads to six rabbits compared to a marketed VP immediate release (IR) tablet, as evidenced by a 2.2-fold increase in mean residence time (MRT) and 5.24-fold extension in half value duration (HVD) over the marketed product without any observed reduction in the relative oral bioavailability.

Thermogelling Platform for Baicalin Delivery for Versatile Biomedical Applications.

Baicalin (BG) is a natural glycoside with several promising therapeutic and preventive applications. However, its pharmaceutical potential is compromised by its poor water solubility, complex oral absorption kinetics, and low bioavailability. In this work, BG was incorporated in a series of chitosan (Ch)/glycerophosphate (GP)-based thermosensitive hydrogel formulations to improve its water solubility and control its release profile. Molecular interactions between BG and GP were investigated using Fourier transform infrared spectroscopy (FT-IR), and the ability of GP to enhance the water solubility of BG was studied in different release media. Drug-loaded Ch/GP hydrogels were prepared and characterized for their gelation time, swelling ratio, and rheological properties in addition to surface and internal microstructure. Polyethylene glycol (PEG) 6000 and hydroxypropyl methyl cellulose (HPMC) were incorporated in the formulations at different ratios to study their effect on modulating the sol-gel behavior and the in vitro drug release. In vivo pharmacokinetic (PK) studies were carried out using a rabbit model to study the ability of drug-loaded Ch/GP thermosensitive hydrogels to control the absorption rate and improve the bioavailability of BG. Results showed that the solubility of BG was enhanced in the presence of GP, while the incorporation of PEG and/or HPMC had an impact on gelation time, rheological behavior, and rate of drug release in vitro. PK results obtained following buccal application of drug-loaded Ch/GP thermosensitive hydrogels to rabbits showed that the rate of BG absorption was controlled and the in vivo bioavailability was increased by 330% relative to BG aqueous oral suspension. The proposed Ch/GP thermosensitive hydrogel is an easily modifiable delivery platform that is not only capable of improving the solubility and bioavailability of BG following buccal administration but also can be suited for various local and injectable therapeutic applications.

Efficacy and Safety Profiles of Oral Atorvastatin-Loaded Nanoparticles: Effect of Size Modulation on Biodistribution.

Atorvastatin calcium (AC)-loaded nanoparticles (NPs) of mean particle diameter <100 nm and narrow distribution were prepared and characterized. Their in vivo PK as well as PD measures following oral administration in different dosage regimens in hyperlipidemic rats were evaluated. The results revealed that the oral bioavailability of two selected AC-NPs formulations was 235% and 169% relative to Lipitor. However, the treatment regimens were not superior in reducing serum total cholesterol (TC), low-density lipoproteins (LDL), and triglycerides (TG) levels compared to Lipitor. Moreover, the AC-NPs treatments were associated with significant adverse effects observed biochemically and histologically. These results were contradictory with those obtained from a previous study in which similarly formulated AC-NPs of mean particle diameter >200 nm were found to be more safe and effective in reducing TC, LDL, and TG levels when administered to hyperlipiemic rats at reduced dosing frequency compared to daily dose of Lipitor despite their lower oral bioavailability. The discrepant correlation between pharmacokinetics (PK) and pharmacodynamics (PD) results was suggested to pertain to the different biodistribution profiles of AC-NPs depending on their sizes. Hereby, we provide a simple approach of particle size modulation to enhance the efficacy and safety of atorvastatin.

PD-PK evaluation of freeze-dried atorvastatin calcium-loaded poly-ε-caprolactone nanoparticles.

In this work lyophilized poly-ε-caprolactone nanoparticles (NPs) loaded with atorvastatin calcium (AC) were developed in an attempt to improve the in-vivo performance of AC following oral administration. The individual and combined effects of several formulation variables were previously investigated using step-wise full factorial designs in order to produce optimized AC-NPs with predetermined characteristics including particle size, drug loading capacity, drug release profile and physical stability. Four optimized formulations were further subjected in this work to lyophilization to promote their long-term physical stability and were fully characterized. The pharmacodynamics (PD)/pharmacokinetics (PK) properties of two optimized freeze-dried AC-NPs formulations showing acceptable long-term stability were determined and compared to a marketed AC immediate release tablet (Lipitor(®)) in albino rats. PD results revealed that the two tested formulations were equally effective in reducing low density lipoproteins (LDL) and triglycerides (TG) levels when given in reduced doses compared to Lipitor(®) and showed no adverse effects. PK results, on the other hand, revealed that the two freeze-dried AC-NPs formulations were of significantly lower bioavailability compared to Lipitor(®). Taken together the PD and PK results demonstrate that the improved efficacy obtained at reduced doses from the freeze-dried AC-NPs could be due to increased concentration of AC in the liver rather than in the plasma.

Effect of lyophilized grapefruit juice on P-glycoprotein-mediated drug transport in-vitro and in-vivo.

The administration of grapefruit juice (GFJ) has been postulated to inhibit the activity of P-glycoprotein (P-gp) transport system and thus can enhance the uptake of substrate drugs. However, for various reasons, the results obtained have been always swaying between confirmation and refutation. This study aims at re-evaluating the effect of lyophilized freshly-prepared grapefruit juice (LGFJ) prepared from the whole peeled fruit on P-gp activity using the model drug doxorubicin (DOX) in-vitro and timolol maleate (TM) in-vivo. Human uterine sarcoma MES-SA/DX5v cells, grown under nanomolar concentration of DOX and highly expressing P-gp, were used as model cells for in-vitro studies whereas white New Zealand male rabbits were used for in-vivo studies. Results showed that the accumulation of DOX in MES-SA/DX5v cells was increased by 18.3 ± 2.0% in presence of LGFJ compared to control experiments. Results from in-vivo absorption studies showed that the relative oral bioavailability of TM ingested with LGFJ was significantly higher by 70% and 43% compared to the oral bioavailability of TM ingested with saline and a commercial GFJ, respectively. This study as such confirms the inhibitory effects of LGFJ on P-gp efflux proteins and highlights the superiority of using lyophilized freshly prepared juices over the commercially available juices in research studies. Also, the results call for further studies to assess the possibility of co-administrating LGFJ with anti-cancer agents to modulate multidrug resistance in their cellular environment or incorporating LGFJ in solid dosage forms to improve oral bioavailability of drugs.

Enhanced gene transfection using calcium phosphate co-precipitates and low-intensity pulsed ultrasound.

The capability to controllably disrupt the cell membrane by ultrasound (US), thus facilitating entry of exogenous species, has now reached a state of some maturity. However, a compelling question asks whether there is a residual role for US in enhancing transfection: that is, once the genetic material has been delivered to the cytosol, can US assist in its transport into the nucleus? The present experiment was designed with a view to addressing this question. As such, our experimental setup discriminates between: (i) the precursor cell membrane permealization step, and (ii) any subsequent intracellular trafficking into the nucleus. In this study, calcium phosphate co-precipitates (CaP) were used to internalize plasmid DNA encoding for luciferase (pDNA-Luc) (>90%) in HeLa cells. After 2h incubation with the CaP-pDNA-Luc, cells were washed and insonated for varying durations. The results showed that US can indeed enhance the intracellular trafficking of previously internalized genes when longer insonation periods are implemented, culminating with an increased probability for successful nuclear localization, as inferred from an enhanced luciferase expression. Moreover, the results suggest that the intracellular role of US might be mediated through a pathway that appears not to be limited to destabilizing the endosomal vesicles. The study thus provides new information regarding the intracellular effects of US, and in effect represents a new modality combining US and CaP carriers for improved efficiency in gene delivery.

Comparison of in vitro and in vivo performance of a colonic delivery system.

The aim of this work is to compare in vitro to in vivo performance of a colonic drug delivery system, made of small pectin-ethylcellulose coated drug beads. The delivery system was evaluated in vitro by conducting drug release studies in different dissolution media to mimic transit times and pH conditions in the stomach, small intestine and colon and in vivo by using gamma-scintigraphic studies in dogs and absorption studies in human volunteers under fed and fasted conditions. In vitro release studies indicated that drug release rate depended on the ratio of the pectin to ethylcellulose in the coat and the thickness of the coat. In vivo release studies obtained by deconvolution of biostudy data did not correlate with in vitro results obtained from most coat formulations. Beads showing ideal release profiles in vitro showed very poor performance in vivo and only those beads showing colonic premature drug release in vitro might be able to deliver the drug to the colon. Scintigraphic studies of a selected formulation showed that the labeled beads had an estimated gastric emptying time of 3 h, an estimated small intestine transit time of 2 h and an estimated colonic transit time of 36 h. Average in vivo lag times of the selected formulation from absorption studies in humans were found to be 6.1 h and 4.8 h under fed and fasted conditions, respectively. The C(max) was also observed at 6.8 h and 5.5 h on average, under fed and fasted conditions, respectively, which might indicate that release of drug from the beads, resulted from degradation of pectin in the coat by enzymatic action in the colon rather than by simple diffusion. Deconvolution of biostudy data showed that drug absorption continued on average for at least 12 h under both fed and fasted conditions.

Bioavailability of riboflavin from a gastric retention formulation.

A gastric retention formulation (GRF) made of naturally occurring carbohydrate polymers and containing riboflavin was tested in vitro for swelling and dissolution characteristics as well as in fasting dogs for gastric retention. The bioavailability of riboflavin, a drug with a limited absorption site in the upper small intestine, from the GRF was studied in fasted healthy humans and compared to an immediate release formulation. It was found that when the GRF is dried and immersed in gastric juice it swells rapidly and releases its drug content in a zero-order fashion for a period of 24 h. In vivo studies in dogs showed that a rectangular shaped GRF stayed in the stomach of fasted dogs for more than 9 h, then disintegrated and reached the colon in 24 h. Endoscopic studies in dogs showed that the GRF hydrates and swells back to about 75% of its original size in 30 min. These in vivo results correlated with in vitro results. Pharmacokinetic parameters determined from urinary excretion data from six human subjects under fasting conditions showed that bioavailability depended on the size of the GRF. The biostudy indicated that bioavailability of riboflavin from a large size GRF was more than triple that measured after administration of an immediate release formulation. Deconvolved input functions from biostudy data suggest that the large size GRF stayed in the stomach for about 15 h.