Formulation and Characterization of Raloxifene Nanostructured Lipid Carriers for Permeability and Uptake Enhancement Applications.

Raloxifene (RLX), a biopharmaceutical classification system (BCS) class II drug, is a selective estrogen receptor modulator (SERM) having an estrogenic effect on the bone and an antiestrogenic effect on the endometrium and breast. Low solubility, high permeability, high metabolism, and low bioavailability are the characteristics of raloxifene. Although 60% is absorbed orally, raloxifene shows extremely poor bioavailability (2%) owing to its low solubility and extensive (>90%) intestinal/hepatic first-pass metabolism. Hence, it becomes important to increase the solubility of raloxifene to enhance its bioavailability. In this study, raloxifene nanostructured lipid carriers (RNLCs) were prepared using the melt dispersion ultrasonication method. The prepared RNLCs were characterized, and the in vitro studies were carried out in the human epithelial breast cancer cell line (MCF-7). The RNLCs had a size of 114.8 ± 0.98 nm and a zeta potential of +9.21 ± 0.58 mV. Transmission electron microscopy (TEM) images showed particle size ranging from 65 to 120 nm. With an entrapment efficiency of 75.04% ± 2.75%, the RNLCs showed sustained release over 7 days compared with the raloxifene drug solution. The prepared RNLCs were successfully taken up by the MCF-7 cells in a time-dependent manner, and the RNLCs showed increased cell cytotoxicity compared with the raloxifene drug. Using the parallel artificial membrane permeability assay (PAMPA), the permeability rate for raloxifene solution was calculated to be 8 × 10-6 cm/s, and for the RNLCs, it was calculated to be 17.8 × 10-6 cm/s. Hence, from the permeability rate calculated, we could conclude that raloxifene, when formulated as nanostructured lipid carriers, showed increased permeability. Overall, the prepared RNLCs were found to be superior to the raloxifene drug as such.

First Total Synthesis and Pharmacological Potential of a Plant Based Hexacyclopeptide.

A new bioactive proline-rich cyclohexapeptide - diandrine C (6), previously isolated from whole plant of Drymaria diandra (Caryophyllaceae), was synthesized through coupling reactions of tetrapeptide unit Boc-Gly--Pro--Tyr--Trp-OH with dipeptide unit -Pro-Gly-OMe using N,N-diisopropylcarbodiimide (DIPC) as the coupling agent, followed by cyclization of linear hexapeptide unit under alkaline condition. Structure of cyclohexapeptide was confirmed by means of chemical, and spectroscopic analyses and also was screened for its antimicrobial and anthelmintic properties. Bioevaluation results indicated that the newly synthesized hexacyclopeptide exhibited potent antimicrobial activity against Gram-negative bacteria Pseudomonas aeruginosa, Klebsiella pneumoniae and pathogenic Candida albicans at 6 µg/mL. Moderate to good level of antihelmintic activity against three earthworm species Megascoplex konkanensis, Pontoscotex corethruses and Eudrilus eugeniae was also observed at concentration of 2 mg/mL.

Aminoglycoside Allergic Reactions.

Aminoglycosides are antimicrobial agents that are primarily used for infections caused by Gram-negative pathogens. The purpose of this article is to review the allergic reactions reported in the published literature to aminoglycoside antibiotics. A thorough PubMed search was conducted and excluded non-allergic adverse reactions to aminoglycosides. Allergic reactions to aminoglycosides occur infrequently, but can include cutaneous reactions as well as systemic reactions, including anaphylaxis. Of the evaluated aminoglycosides, gentamicin had the most reported allergic reactions, including the most reports of anaphylaxis, followed by tobramycin, and then amikacin. Most reports of allergic reactions occurred in patients who had a prior exposure to some dosage form of an aminoglycoside. Cross-reactivity among aminoglycosides is common and occurs due to the similarities in their chemical structures. Desensitization protocols to tobramycin have been described in the literature.

Macrolide Allergic Reactions.

Macrolides are antimicrobial agents that can be used to treat a variety of infections. Allergic reactions to macrolides occur infrequently but can include minor to severe cutaneous reactions as well as systemic life-threatening reactions such as anaphylaxis. Most reports of allergic reactions occurred in patients without prior exposure to a macrolide. Cross-reactivity among macrolides may occur due to the similarities in their chemical structures; however, some published literature indicates that some patients can tolerate a different macrolide. Most published reports detailed an allergic reaction to erythromycin. Desensitization protocols to clarithromycin and azithromycin have been described in the literature. The purpose of this article is to summarize macrolide-associated allergic reactions reported in published literature. An extensive literature search was conducted to identify publications linking macrolides to hypersensitivity reactions.

GADD45α-targeted suicide gene therapy driven by synthetic CArG promoter E9NS sensitizes NSCLC cells to cisplatin, resveratrol, and radiation regardless of p53 status.

Background: GADD45α is a tumor suppressor protein often upregulated by environmental stresses and DNA-damage agents to cause growth arrest, apoptosis, tumor growth inhibition, and anti-angiogenesis. A novel suicide gene therapy vector pE9NS.G45α was engineered by cloning GADD45α opening reading frame downstream to the synthetic CArG promoter E9NS, which contains nine repeats of CArG element with modified core A/T sequence and functions as a molecular switch to drive the expression of GADD45α. The current study aims to determine the efficacy of this suicide gene therapy vector in combination with cisplatin, resveratrol, and radiation in NSCLC cell lines with various p53 statuses. Methods: Three NSCLC cell lines, H1299 (deleted p53), A549 (wild-type p53), and H23 (mutated p53), were examined in the present investigation to represent NSCLC with different p53 functions. MTT assay was conducted to select suitable doses of cisplatin, resveratrol, and radiation for gene therapy, and dual luciferase assay was performed to validate the activation of promoter E9NS. The efficacy of gene therapy combinations was evaluated by the amount of GADD45α expression, cell survival, and apoptosis. Results: All the combinations successfully activated promoter E9NS to elevate intracellular GADD45α protein levels and subsequently enhanced cell viability reduction and apoptosis induction regardless of p53 status. Conclusion: Our study demonstrates that GADD45α-targeted suicide gene therapy controlled by synthetic promoter E9NS sensitizes NSCLC cells to cisplatin, resveratrol, and radiation and is effective against NSCLC at least in vitro.

Role of CYP2B6 pharmacogenomics in bupropion-mediated smoking cessation.

WHAT IS KNOWN AND OBJECTIVE: Pharmacogenomics holds promise in smoking cessation because of its potential to shed light on the complexity of drug metabolism and improve treatments using therapeutic agents. The cytochrome P450 2B6 gene (CYP2B6) encodes CYP2B6 enzyme that has been found to mediate the hydroxylation of bupropion, a smoking cessation aid. CYP2B6 exhibits a range of polymorphic variants that alter the pharmacokinetics and pharmacodynamics of bupropion. Genetic variations in CYP2B6 may influence the risk of adverse effects or efficacy of treatment with bupropion. The objective of this review was to investigate the influence of pharmacogenomics on smoking cessation therapy. METHODS: A thorough literature search was conducted on PubMed, SCOPUS and EMBASE using keywords related to bupropion, smoking cessation, pharmacogenomics and CYP2B6. Research and review articles, case reports and clinical and preclinical studies pertinent to the research topic were identified, evaluated and summarized. Cited articles within the above-mentioned sources also provided pertinent information. RESULTS: There is strong literature evidence to prove that CYP2B6 polymorphisms affect pharmacokinetic and pharmacodynamic properties of bupropion, thus affecting the therapeutic outcome of smoking cessation therapy. WHAT IS NEW AND CONCLUSIONS: Complete understanding of pharmacogenetic variation of bupropion pharmacokinetics and pharmacodynamics will be beneficial for designing safer and more personalized smoking cessation therapy with improved outcomes.

Aflibercept Nanoformulation Inhibits VEGF Expression in Ocular In Vitro Model: A Preliminary Report.

Age-related macular degeneration (AMD) is one of the leading causes of blindness in the United States, affecting approximately 11 million patients. AMD is caused primarily by an upregulation of vascular endothelial growth factor (VEGF). In recent years, aflibercept injections have been used to combat VEGF. However, this treatment requires frequent intravitreal injections, leading to low patient compliance and several adverse side effects including scarring, increased intraocular pressure, and retinal detachment. Polymeric nanoparticles have demonstrated the ability to deliver a sustained release of drug, thereby reducing the necessary injection frequency. Aflibercept (AFL) was encapsulated in poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) via double emulsion diffusion. Scanning electron microscopy showed the NPs were spherical and dynamic light scattering demonstrated that they were uniformly distributed (PDI < 1). The encapsulation efficiency and drug loading were 75.76% and 7.76% respectively. In vitro release studies showed a sustained release of drug; 75% of drug was released by the NPs in seven days compared to the full payload released in 24 h by the AFL solution. Future ocular in vivo studies are needed to confirm the biological effects of the NPs. Preliminary studies of the proposed aflibercept NPs demonstrated high encapsulation efficiency, a sustained drug release profile, and ideal physical characteristics for AMD treatment. This drug delivery system is an excellent candidate for further characterization using an ocular neovascularization in vivo model.

Salts of Therapeutic Agents: Chemical, Physicochemical, and Biological Considerations.

The physicochemical and biological properties of active pharmaceutical ingredients (APIs) are greatly affected by their salt forms. The choice of a particular salt formulation is based on numerous factors such as API chemistry, intended dosage form, pharmacokinetics, and pharmacodynamics. The appropriate salt can improve the overall therapeutic and pharmaceutical effects of an API. However, the incorrect salt form can have the opposite effect, and can be quite detrimental for overall drug development. This review summarizes several criteria for choosing the appropriate salt forms, along with the effects of salt forms on the pharmaceutical properties of APIs. In addition to a comprehensive review of the selection criteria, this review also gives a brief historic perspective of the salt selection processes.

Current and evolving approaches for improving the oral permeability of BCS Class III or analogous molecules.

The Biopharmaceutics Classification System (BCS) classifies pharmaceutical compounds based on their aqueous solubility and intestinal permeability. The BCS Class III compounds are hydrophilic molecules (high aqueous solubility) with low permeability across the biological membranes. While these compounds are pharmacologically effective, poor absorption due to low permeability becomes the rate-limiting step in achieving adequate bioavailability. Several approaches have been explored and utilized for improving the permeability profiles of these compounds. The approaches include traditional methods such as prodrugs, permeation enhancers, ion-pairing, etc., as well as relatively modern approaches such as nanoencapsulation and nanosizing. The most recent approaches include a combination/hybridization of one or more traditional approaches to improve drug permeability. While some of these approaches have been extremely successful, i.e. drug products utilizing the approach have progressed through the USFDA approval for marketing; others require further investigation to be applicable. This article discusses the commonly studied approaches for improving the permeability of BCS Class III compounds.

Monoamine Oxidase Inhibitory Activity: Methyl- versus Chlorochalcone Derivatives.

Numerous studies have shown that chalcones are promising scaffolds for the development of new monoamine oxidase-B (MAO-B) inhibitors. As a continuation of our ongoing research into the development of reversible human MAO-B (hMAO-B) inhibitors, two series of twenty chalcones containing electron-donating and electron-withdrawing substituents were synthesized. All compounds were found to be competitive, selective, and reversible inhibitors of hMAO-B except (2E)-1-(4-methylphenyl)-3-(4-nitrophenyl)prop-2-en-1-one (P7) and (2E)-1-(4-chlorophenyl)-3-(4-nitrophenyl)prop-2-en-1-one (P17), which were found to be selective inhibitors of hMAO-A. The most potent hMAO-B inhibitor, (2E)-1-(4-chlorophenyl)-3-(4-ethylphenyl)prop-2-en-1-one (P16), showed a Ki value of 0.11±0.01 µm. Molecular docking simulations were carried out to identify the hypothetical binding mode for the most potent compounds in the active sites of hMAO-A and B. The ability of the compounds to cross the blood-brain barrier was assessed by parallel artificial membrane permeability assay (PAMPA). Additionally, the most potent hMAO-B inhibitor P16 showed no toxicity in cultured hepatic cells at concentrations of 5 and 25 µm.

Role of Cytochrome P450 2B6 Pharmacogenomics in Determining Efavirenz-Mediated Central Nervous System Toxicity, Treatment Outcomes, and Dosage Adjustments in Patients with Human Immunodeficiency Virus Infection.

For treatment-naive patients with human immunodeficiency virus infection, efavirenz (EFV), together with tenofovir and emtricitabine, was once widely prescribed given its efficacy and ease of administration in a combination pill. However, the high rate of central nervous system (CNS) toxicities from EFV prompted the U.S. Department of Health and Human Services to move the EFV-based regimen from the recommended to the alternative category. For patients who do meet the criteria for newer recommended antiretroviral treatments, EFV is a viable option and often the mainstay of treatment outside the United States because newer antiretroviral treatments are more expensive. CNS toxicity occurring with the recommended standard dose of EFV remains a challenge and may in part be attributable to polymorphisms in cytochrome P450 (CYP) 2B6, the enzyme involved in the major metabolic pathway for converting EFV to inactive metabolites. Functionally deficient alleles of CYP2B6 such as CYP2B6*6, *18, and *22 may be responsible for significantly higher therapeutic concentrations of EFV at a standard dose of 600 mg/day. We conducted a thorough review of the reported studies to elucidate the relationship between polymorphisms in CYP2B6 with adverse events and treatment response, including virologic suppression, immunologic response, resistance, and discontinuation of treatment. Compelling evidence exists to support the case for CYP2B6 genotype-guided EFV therapy while acknowledging the need for prospective controlled clinical trials to evaluate its clinical utility.

Withaferin A Regulates LRRK2 Levels by Interfering with the Hsp90- Cdc37 Chaperone Complex.

Leucine-Rich Repeat Kinase 2 (LRRK2) is a large, multi-domain protein that has been found to be mutated in patients with familial and sporadic Parkinson's disease, Alzheimer's disease and Crohn's disease. While the functions of LRRK2 are still largely unclear and mutations in LRRK2 are associated with adverse gain-of-function activities such as increased kinase activity, increased levels of LRRK2 alone are associated with toxicity in neurons. Consequently, exploring mechanisms to decrease levels of LRRK2 using pharmacological inhibitors would be highly advantageous. Previous work has shown that the chaperone heat shock protein 90 (Hsp90) and its co-chaperone Cdc37 interact with and stabilize LRRK2. In the current study, we explore the regulation of LRRK2 by withaferin A (WA), a potent inhibitor of the interaction between Hsp90 and Cdc37. We report that treatment of the microglial cell line N9 with WA causes a decrease in cellular levels of LRRK2 in a dose- and time-dependent manner. We also find that treatment with WA disrupts the interaction between Hsp90, its co-chaperone Cdc37 and LRRK2, which leads to the destabilization and decreased levels of LRRK2. Additionally, treatment with celastrol, which is also an inhibitor of the Hsp90-Cdc37 complex, decreased LRRK2 levels. Interestingly, treatment with WA in the presence of celastrol enhanced the clearance of LRRK2. Overall, our data suggest that LRRK2 levels can be regulated by targeting the Hsp90-Cdc37 complex, which may have implications in the search for therapeutic strategies for Alzheimer's disease, Parkinson's disease and other LRRK2 proteinopathies.

Increasing oral absorption of polar neuraminidase inhibitors: a prodrug transporter approach applied to oseltamivir analogue.

Poor oral absorption is one of the limiting factors in utilizing the full potential of polar antiviral agents. The neuraminidase target site requires a polar chemical structure for high affinity binding, thus limiting oral efficacy of many high affinity ligands. The aim of this study was to overcome this poor oral absorption barrier, utilizing prodrug to target the apical brush border peptide transporter 1 (PEPT1). Guanidine oseltamivir carboxylate (GOCarb) is a highly active polar antiviral agent with insufficient oral bioavailability (4%) to be an effective therapeutic agent. In this report we utilize a carrier-mediated targeted prodrug approach to improve the oral absorption of GOCarb. Acyloxy(alkyl) ester based amino acid linked prodrugs were synthesized and evaluated as potential substrates of mucosal transporters, e.g., PEPT1. Prodrugs were also evaluated for their chemical and enzymatic stability. PEPT1 transport studies included [(3)H]Gly-Sar uptake inhibition in Caco-2 cells and cellular uptake experiments using HeLa cells overexpressing PEPT1. The intestinal membrane permeabilities of the selected prodrugs and the parent drug were then evaluated for epithelial cell transport across Caco-2 monolayers, and in the in situ rat intestinal jejunal perfusion model. Prodrugs exhibited a pH dependent stability with higher stability at acidic pHs. Significant inhibition of uptake (IC(50) <1 mM) was observed for l-valyl and l-isoleucyl amino acid prodrugs in competition experiments with [(3)H]Gly-Sar, indicating a 3-6 times higher affinity for PEPT1 compared to valacyclovir, a well-known PEPT1 substrate and >30-fold increase in affinity compared to GOCarb. The l-valyl prodrug exhibited significant enhancement of uptake in PEPT1/HeLa cells and compared favorably with the well-absorbed valacyclovir. Transepithelial permeability across Caco-2 monolayers showed that these amino acid prodrugs have a 2-5-fold increase in permeability as compared to the parent drug and showed that the l-valyl prodrug (P(app) = 1.7 × 10(-6) cm/s) has the potential to be rapidly transported across the epithelial cell apical membrane. Significantly, only the parent drug (GOCarb) appeared in the basolateral compartment, indicating complete activation (hydrolysis) during transport. Intestinal rat jejunal permeability studies showed that l-valyl and l-isoleucyl prodrugs are highly permeable compared to the orally well absorbed metoprolol, while the parent drug had essentially zero permeability in the jejunum, consistent with its known poor low absorption. Prodrugs were rapidly converted to parent in cell homogenates, suggesting their ability to be activated endogenously in the epithelial cell, consistent with the transport studies. Additionally, l-valyl prodrug was found to be a substrate for valacyclovirase (K(m) = 2.37 mM), suggesting a potential cell activation mechanism. Finally we determined the oral bioavailability of our most promising candidate, GOC-l-Val, in mice to be 23% under fed conditions and 48% under fasted conditions. In conclusion, GOC-l-Val prodrug was found to be a very promising antiviral agent for oral delivery. These findings indicate that the carrier-mediated prodrug approach is an excellent strategy for improving oral absorption of polar neuraminidase inhibitors. These promising results demonstrate that the oral peptide transporter-mediated prodrug strategy has enormous promise for improving the oral mucosal cell membrane permeability of polar, poorly absorbed antiviral agents and treating influenza via the oral route of administration.