Endocannabinoid System of the Blood-Brain Barrier: Current Understandings and Therapeutic Potentials.

The endocannabinoid system (ECS) has been found at the blood-brain barrier (BBB), as Cannabinoid receptors were characterized in human brain microvascular endothelial cells and astrocytes. In several in vitro and in vivo studies, cannabinoids decreased BBB permeability and enhanced membrane integrity, which may be achieved through endothelial tight junctions and other mechanisms. These permeability regulation effects of cannabinoids suggested that the ECS may protect the brain by enhancing barrier integrity. Related questions about cannabinoid-drug interaction and drug distribution across the BBB are also raised. Specifically, can cannabinoids significantly reduce drug bioavailability to the brain? More in-depth and systematic investigations are needed to characterize and quantify these effects of cannabinoids on brain microvasculature physiopathology. Therefore, this review summarizes literatures from different disciplines to promote more research on assessing the therapeutic benefits and risks of using cannabinoids to protect BBB from dysfunctions or breakdown, and to avoid consequent brain damages due to inflammation, neurodegenerations, hemorrhage, ischemia, or other causes.

Quality Assessment of Expired Naloxone Products from First-Responders' Supplies.

Objective: Naloxone is an opioid receptor antagonist that reverses life-threatening effects of opioid overdose. Since the 1970s, naloxone products have been developed as injectable solutions, and more recently as nasal sprays. Naloxone products have saved many lives in emergency settings. These products are routinely carried by public safety first-responders including fire fighters (FF), law enforcement officers (LEO), and emergency medical services (EMS). Now, they are also distributed through community access programs to the public. While public safety medications are monitored, those publically distributed are not, so expired products can be possibly found on-hand in an emergency. This study analyzed the quality and stability of expired Naloxone HCl Solutions for Injection, to assess their remaining efficacies and potential risks. Methods: The samples were collected from EMS or law enforcement training supplies and expired returns, with expiration dates ranging from 1990 to 2018. Using standardized techniques, the remaining naloxone was quantified, and the main degradation products, nornaloxone (also known as noroxymorphone) and other possible species, were monitored and quantified systematically. Results: Most tested samples were found containing more than 90% of labeled naloxone, including those stored for nearly 30 years. The naloxone degradation was slow, but generally correlated with storage time length. There was no significant amount of degradation products detected across all samples. Nornaloxone was detected from some older samples, but all less than 1%. Therefore, although it is an opioid agonist, the risk caused by nornaloxone should be low. Conclusion: This quality assessment demonstrates that expired naloxone products may still meet USP standards, even after many years. Further pharmaceutical, clinical, and regulatory investigation should be conducted to confirm our findings, especially for new naloxone products with different formulations and routes of administration. Extending the shelf-life of naloxone products may have important financial and public health consequences in addressing future drug shortages and meeting the needs for this critical drug.

Current Practices in Global/International Advanced Pharmacy Practice Experiences: Home/Host Country or Site/Institution Considerations.

International outreach by schools and colleges of pharmacy is increasing. In this paper, we provide current practice guidelines to establish and maintain successful global/international advanced pharmacy practice experiences (G/I APPEs) with specific recommendations for home/host country and host site/institution. The paper is based on a literature review (2000-2014) in databases and Internet searches with specific keywords or terms. Educational documents such as syllabi and memoranda of understanding (MoUs) from pharmacy programs were also examined. In addition, a preliminary draft was developed and the findings and recommendations were reviewed in a 90-minute roundtable discussion at the 2014 American Association of Colleges of Pharmacy Annual Meeting. Recommendations for the host country include travel considerations (eg, passport, visa, air travel), safety, housing, transportation, travel alerts and warnings, health issues, and financial considerations. For the home country, considerations for establishment of G/I APPE site (eg, vetting process, MoU, site expectations) are described. The paper is a resource for development of new G/I APPEs and provides guidance for continuous quality improvement of partnerships focusing on G/I pharmacy education.

Do formulation differences between the reference listed drug and generic piperacillin-tazobactam impact reconstitution?

Pharmaceutical differences between the reference listed drug (RLD) and generic formulations of piperacillin-tazobactam may impact the reconstitution process for intravenous administration. This study evaluated the RLD against three generic formulations and measured their reconstitution times using a standardized process. The mean (standard deviation [SD]) reconstitution time for one generic formulation was 5.57 (1.49) min, which was 35% to 42% longer (P < 0.002) than that for the RLD and two other formulations. Observable microscopic differences in powder particle morphology may explain these findings.

Protein transduction domain-containing microemulsions as cutaneous delivery systems for an anticancer agent.

In this study, we developed cationic microemulsions containing a protein transduction domain (penetratin) for optimizing paclitaxel localization within the skin. Microemulsions were prepared by mixing a surfactant blend (BRIJ:ethanol:propylene glycol 2:1:1, w/w/w) with monocaprylin (oil phase) at 1.3:1 ratio, and adding water at 30% (ME-30), 43% (ME-43), and 50% (ME-50). Electrical conductivity and viscosity measurements indicated that ME-30 is most likely a bicontinuous system, whereas ME-43 and ME-50 are water continuous. Their irritation potential, studied in bioengineered skin equivalents, decreased as aqueous content increased. Because ME-50 was not stable in the presence of paclitaxel (0.5%), ME-43 was selected for penetratin incorporation (0.4%). The microemulsion containing penetratin (ME-P) displayed zeta potential of +5.2 mV, and promoted a 1.8-fold increase in paclitaxel cutaneous (but not transdermal) delivery compared with the plain ME-43, whereas the enhancement promoted by another cationic microemulsion containing phytosphingosine was 1.3-fold. Compared with myvacet oil, ME-P promoted a larger increase on transepidermal water loss (twofold) than the plain or the phytosphingosine-containing microemulsions (1.5-fold), suggesting that penetratin addition increases the barrier-disrupting and penetration-enhancing effects of microemulsions. The ratio Δcutaneous/Δtransdermal delivery promoted by ME-P was the highest among the formulations, suggesting its potential for drug localization within cutaneous tumor lesions.

Influence of internal structure and composition of liquid crystalline phases on topical delivery of paclitaxel.

This study aimed to evaluate whether and how the internal structure and composition of liquid crystalline systems can be tailored to maximize paclitaxel cutaneous delivery. Liquid crystalline phases of water, Brij-97, and medium-chain mono/diglycerides (MCG) were characterized by polarized light microscopy. Lamellar phases containing 20% (w/w) water and MCG at 10% (LP-10) or 20% (LP-20), and a hexagonal phase (HP) with 45% water and 10% MCG were selected; paclitaxel was incorporated at 0.5% (w/w). Compared with drug solution in myvacet oil, LP-20 provided the highest paclitaxel cutaneous delivery (threefold), and LP-10 the highest transdermal delivery (fourfold). Using a fluorescent drug derivative [at 0.5%, (w/w)], we observed that penetration occurred through intact stratum corneum. To evaluate whether penetration results relate to drug release differences, paclitaxel self-diffusion coefficient (D) and in vitro release were studied. D was the highest in LP-20, but release from LP-20 and LP-10 was similar. The low D in HP was associated with the lowest drug release. As an index of efficacy, we assessed the cytotoxicity of paclitaxel-loaded LP-20 against fibroblasts. Cell viability was 1.3-2 times smaller with LP-20 than with drug solution. Our results demonstrate that LP-20 provides optimization of paclitaxel cutaneous delivery and efficient cytotoxicity.

Topical delivery of lycopene using microemulsions: enhanced skin penetration and tissue antioxidant activity.

Topical delivery of lycopene is a convenient way to supplement cutaneous levels of antioxidants. In this study, lycopene was incorporated (0.05%, w/w) in two microemulsions containing BRIJ-propylene glycol (2:1, w/w, surfactant blend) but different oil phases: mono/diglycerides of capric and caprylic acids (MG) or triglycerides of the same fatty acids (TG). Microemulsions containing MG and TG were isotropic, fluid, and clear, with internal phase diameters of 27 and 52 nm, respectively. Both MG- or TG-containing microemulsions markedly increased lycopene penetration in the stratum corneum (6- and 3.6-fold, respectively) and in viable layers of porcine ear skin (from undetected to 172.6 +/- 41.1 and 103.1 +/- 7.2 ng/cm(2), respectively) compared to a control solution. To assure that lycopene delivered to the skin was active, the antioxidant activity of skin treated with MG-containing microemulsion was determined by CUPRAC assay, and found to be 10-fold higher than untreated skin. The cytotoxicity of MG-containing microemulsion in cultured fibroblasts was similar to propylene glycol (considered safe) and significantly less than of sodium lauryl sulfate (a moderate-to-severe irritant) at 1-50 microg/mL. These results demonstrate that the MG-containing microemulsion is an efficient and safe system to increase lycopene delivery to the skin and the antioxidant activity in the tissue.

Oral microemulsions of paclitaxel: in situ and pharmacokinetic studies.

The overall goal of this study was to develop cremophor-free oral microemulsions of paclitaxel (PAC) to enhance its permeability and oral absorption. The mechanism of this enhancement, as well as characteristics of the microemulsions relevant to the increase in permeability and absorption of the low solubility, low permeability PAC was investigated. Phase diagrams were used to determine the macroscopic phase behavior of the microemulsions and to compare the efficiency of different surfactant-oil mixtures to incorporate water. The microemulsion region on the phase diagrams utilizing surfactant-myvacet oil combinations was in decreasing order: lecithin: butanol: myvacet oil (LBM, 48.5%)>centromix CPS: 1-butanol: myvacet oil (CPS, 45.15%)>capmul MCM: polysorbate 80: myvacet oil (CPM, 27.6%)>capryol 90: polysorbate 80: myvacet oil (CP-P80, 23.9%)>capmul: myvacet oil (CM, 20%). Oil-in-water (o/w) microemulsions had larger droplet sizes (687-1010 nm) than the water-in-oil (w/o) microemulsions (272-363 nm) when measured using a Zetasizer nano series particle size analyzer. Utilizing nuclear magnetic resonance spectroscopy (NMR), the self-diffusion coefficient (D) of PAC in CM, LBM and CPM containing 10% of deuterium oxide (D(2)O) was 2.24x10(-11), 1.97x10(-11) and 0.51x10(-11) m(2)/s, respectively. These values indicate the faster molecular mobility of PAC in the two w/o microemulsions (CM and LBM) than the o/w microemulsion--CPM. The in situ permeability of PAC through male CD-IGS rat intestine was 3- and 11-fold higher from LBM and CM, respectively, than that from the control clinical formulation, Taxol (CE, cremophor: ethanol) in a single pass perfusion study. PAC permeability was significantly increased in the presence of the pgp/CYP3A4 inhibitor cyclosporine A (CsA). This enhancement may be attributed to the pgp inhibitory effect of the surfactants, oil and/or the membrane perturbation effect of the surfactants. The oral disposition of PAC in CM, LBM and CPM compared to CE was studied in male CD-IGS rats after a single oral dose (20 mg/kg). The area-under-the-curve of PAC in CM was significantly larger than LBM, CPM and CE. Oral microemulsions of PAC were developed that increased both the permeability and AUC of PAC as compared to CE.

A transmembrane helix-bundle from G-protein coupled receptor CB2: biosynthesis, purification, and NMR characterization.

The cannabinoid receptor subtype 2 (CB2) is a member of the G-protein coupled receptor (GPCR) superfamily. As the relationship between structure and function for this receptor remains poorly understood, the present study was undertaken to characterize the structure of a segment including the first and second transmembrane helix (TM1 and TM2) domains of CB2. To accomplish this, a transmembrane double-helix bundle from this region was expressed, purified, and characterized by NMR. Milligrams of this hydrophobic fragment of the receptor were biosynthesized using a fusion protein overexpression strategy and purified by affinity chromatography combined with reverse phase HPLC. Chemical and enzymatic cleavage methods were implemented to remove the fusion tag. The resultant recombinant protein samples were analyzed and confirmed by HPLC, mass spectrometry, and circular dichroism (CD). The CD analyses of HPLC-purified protein in solution and in DPC micelle preparations suggested predominant alpha-helical structures under both conditions. The 13C/15N double-labeled protein CB2(27-101) was further verified and analyzed by NMR spectroscopy. Sequential assignment was accomplished for more than 80% of residues. The 15N HSQC NMR results show a clear chemical shift dispersion of the amide nitrogen-proton correlation indicative of a pure double-labeled polypeptide molecule. The results suggest that this method is capable of generating transmembrane helical bundles from GPCRs in quantity and purity sufficient for NMR and other biophysical studies. Therefore, the biosynthesis of GPCR transmembrane helix bundles represents a satisfactory alternative strategy to obtain and assemble NMR structures from recombinant "building blocks."

Expression, purification, and isotope labeling of cannabinoid CB2 receptor fragment, CB2(180-233).

To develop an approach to obtain milligram quantities of purified isotope-labeled seven transmembrane G-protein coupled cannabinoid (CB) receptor for NMR structural analysis, we chose a truncated CB receptor fragment, CB2(180-233), spanning from the fifth transmembrane domain (TM5) to the associated loop regions of cannabinoid CB2 receptor. This highly hydrophobic membrane protein fragment was pursued for developmental studies of membrane proteins through expression and purification in Escherichia coli. The target peptide was cloned and over-expressed in a preparative scale as a fusion protein with a modified TrpDeltaLE1413 (TrpLE) leader sequence and a nine-histidine tag at its N-terminal. An experimental protocol for enzyme cleavage was developed by using Factor Xa to remove the TrpLE tag from the fusion protein. A purification process was also established using a nickel affinity column and reverse-phase HPLC, and then monitored by SDS-PAGE and MS. This expression level is one of the highest reported for a G-protein coupled receptor and fragments in E. Coli, and provided a sufficient amount of purified protein for further biophysical studies.