Multiseed liposomal drug delivery system using micelle gradient as driving force to improve amphiphilic drug retention and its anti-tumor efficacy.

To improve drug retention in carriers for amphiphilic asulacrine (ASL), a novel active loading method using micelle gradient was developed to fabricate the ASL-loaded multiseed liposomes (ASL-ML). The empty ML were prepared by hydrating a thin film with empty micelles. Then the micelles in liposomal compartment acting as 'micelle pool' drove the drug to be loaded after the outer micelles were removed. Some reasoning studies including critical micelle concentration (CMC) determination, influencing factors tests on entrapment efficiency (EE), structure visualization, and drug release were carried out to explore the mechanism of active loading, ASL location, and the structure of ASL-ML. Comparisons were made between pre-loading and active loading method. Finally, the extended drug retention capacity of ML was evaluated through pharmacokinetic, drug tissue irritancy, and in vivo anti-tumor activity studies. Comprehensive results from fluorescent and transmission electron microscope (TEM) observation, encapsulation efficiency (EE) comparison, and release studies demonstrated the formation of ML-shell structure for ASL-ML without inter-carrier fusion. The location of drug mainly in inner micelles as well as the superiority of post-loading to the pre-loading method , in which drug in micelles shifted onto the bilayer membrane was an additional positive of this delivery system. It was observed that the drug amphiphilicity and interaction of micelles with drug were the two prerequisites for this active loading method. The extended retention capacity of ML has been verified through the prolonged half-life, reduced paw-lick responses in rats, and enhanced tumor inhibition in model mice. In conclusion, ASL-ML prepared by active loading method can effectively load drug into micelles with expected structure and improve drug retention.

Optimization of Weight Ratio for DSPE-PEG/TPGS Hybrid Micelles to Improve Drug Retention and Tumor Penetration.

PURPOSE: To enhance therapeutic efficacy and prevent phlebitis caused by Asulacrine (ASL) precipitation post intravenous injection, ASL-loaded hybrid micelles with size below 40 nm were developed to improve drug retention and tumor penetration. METHODS: ASL-micelles were prepared using different weight ratios of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethyleneglycol-2000 (DSPE-PEG2000) and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) polymers. Stability of micelles was optimized in terms of critical micelle concentration (CMC) and drug release properties. The encapsulation efficiency (EE) and drug loading were determined using an established dialysis-mathematic fitting method. Multicellular spheroids (MCTS) penetration and cytotoxicity were investigated on MCF-7 cell line. Pharmacokinetics of ASL-micelles was evaluated in rats with ASL-solution as control. RESULTS: The ASL-micelles prepared with DSPE-PEG2000 and TPGS (1:1, w/w) exhibited small size (~18.5 nm), higher EE (~94.12%), better sustained in vitro drug release with lower CMC which may be ascribed to the interaction between drug and carriers. Compared to free ASL, ASL-micelles showed better MCTS penetration capacity and more potent cytotoxicity. Pharmacokinetic studies demonstrated that the half-life and AUC values of ASL-micelles were approximately 1.37-fold and 3.49-fold greater than that of free ASL. CONCLUSIONS: The optimized DSPE-PEG2000/TPGS micelles could serve as a promising vehicle to improve drug retention and penetration in tumor.

Optimization of the formation of embedded multicellular spheroids of MCF-7 cells: How to reliably produce a biomimetic 3D model.

To obtain a multicellular MCF-7 spheroid model to mimic the three-dimensional (3D) of tumors, the microwell liquid overlay (A) and hanging-drop/agar (B) methods were first compared for their technical parameters. Then a method for embedding spheroids within collagen was optimized. For method A, centrifugation assisted cells form irregular aggregates but not spheroids. For method B, an extended sedimentation period of over 24 h for cell suspensions and increased viscosity of the culture medium using methylcellulose were necessary to harvest a dense and regular cell spheroid. When the number was less than 5000 cells/drop, embedded spheroids showed no tight cores and higher viability than the unembedded. However, above 5000 cells/drop, cellular viability of embedded spheroids was not significantly different from unembedded spheroids and cells invading through the collagen were in a sun-burst pattern with tight cores. Propidium Iodide staining indicated that spheroids had necrotic cores. The doxorubicin cytotoxicity demonstrated that spheroids were less susceptible to DOX than their monolayer cells. A reliable and reproducible method for embedding spheroids using the hanging-drop/agarose method within collagen is described herein. The cell culture model can be used to guide experimental manipulation of 3D cell cultures and to evaluate anticancer drug efficacy.

Improving drug retention in liposomes by aging with the aid of glucose.

This paper describes a novel method to improve drug retention in liposomes for the poorly water-soluble (lipophilic) model drug asulacrine (ASL). ASL was loaded in the aqueous phase of liposomes and the effects of aging conditions and drug loading levels on drug retention were investigated using an in vitro bio-relevant drug release test established in this study. The status of intra-liposomal drug was investigated using differential scanning calorimetry (DSC) and cryo-transmission electron microscopy (cryo-TEM). Pharmacokinetics and venous tolerance of the formulations were simultaneously studied in rabbits following one-hour intravenous infusion via the ear vein. The presence of glucose during aging was found to be crucial to accelerate drug precipitation and to stabilize the liposomal membrane with high drug loading (8.9% over 4.5% w/w) as a prerequisite. Although no drug crystals were detected, DSC showed a lower phase-transition peak in the glucose-assisted aged ASL-liposomes, indicating interaction of phospholipids with the sugar. Cryo-TEM revealed more 'coffee bean' like drug precipitate in the ASL-liposomes aged in the glucose solution. In rabbits, these liposomes gave rise to a 1.9 times longer half-life than the fresh liposomes, with no venous irritation observed. Inducing and stabilizing drug precipitation in the liposome cores by aging in the presence of sugar provided an easy approach to improve drug retention in liposomes. The study also highlighted the importance of bio-relevance of in vitro release methods to predict in vivo drug release.

Barriers to positive policy change that aims to increase access to medicines through reclassification: the case of oseltamivir in New Zealand.

OBJECTIVES: This study aims to identify and explore emergent barriers to consumers accessing oseltamivir without prescription following policy change introduced in New Zealand to increase access via community pharmacies. METHODS: Semi-structured interviews were conducted with 26 community pharmacists immediately following the first season of oseltamivir availability without prescription in October 2007. Interviews were transcribed verbatim and coded using a framework approach to identify themes. KEY FINDINGS: Non-prescription sales of oseltamivir were slow during this period. Participants acknowledged that they may have missed opportunities to recommend oseltamivir and attributed this to a range of reasons. Pharmacy-related barriers identified included limited pharmacist confidence, concerns about efficacy and safety of the product, location of the product in the pharmacy, affordability and the role of support staff. Many pharmacists adopted a 'risk-benefit analysis' that balanced symptom severity with perceived value for money. Consumer barriers included cost, limited awareness of availability and limited ability to correctly self-diagnose and manage influenza. CONCLUSIONS: Complexity in the factors that influenced pharmacist motivation to supply oseltamivir without prescription highlighted the potential for positive policy change to be hindered by multiple barriers. Greater understanding of such barriers is important for effective transition of medicines from prescription to non-prescription availability to achieve increased consumer access through reclassification. Concerns that pharmacists are influenced by commercial priorities when medicines are newly reclassified were not substantiated in this study.

Post-insertion of poloxamer 188 strengthened liposomal membrane and reduced drug irritancy and in vivo precipitation, superior to PEGylation.

The ultimate aim of this study was to develop asulacrine (ASL)-loaded long-circulating liposomes to prevent phlebitis during intravenous (i.v.) infusion for chemotherapy. Poly(ethylene)glycol (PEG) and poloxamer 188-modified liposomes (ASL-PEGL and ASL-P188L) were developed, and ASL was loaded using a remote loading method facilitated with a low concentration of sulfobutyl ether-ß-cyclodextrin as a drug solubilizer. The liposomes were characterized in terms of morphology, size, release properties and stability. Pharmacokinetics and venous tissue tolerance of the formulations were simultaneously studied in rabbits following one-hour i.v. infusion via the ear vein. The irritancy was assessed using a rat paw-lift/lick model after subplantar injections. High drug loading 9.0% w/w was achieved with no drug leakage found from ASL-PEGL or ASL-P188L suspended in a 5% glucose solution at 30days. However, a rapid release (leakage) from ASL-PEGL was observed when PBS was used as release medium, partially related to the use of cyclodextrin in drug loading. Post-insertion of poloxamer 188 to the liposomes appeared to be able to restore the drug retention possibly by increasing the packing density of phospholipids in the membrane. In rabbits (n=5), ASL-P188L had a prolonged half-life with no drug precipitation or inflammation in the rabbit ear vein in contrast to ASL solution. Following subplantar (footpad) injections in rats ASL solution induced paw-lick/lift responses in all rats whereas ASL-P188L caused no response (n=8). PEGylation showed less benefit possibly due to the drug 'leakage'. In conclusion, drug precipitation in the vein and the drug mild irritancy may both contribute to the occurrence of phlebitis caused by the ASL solution, and could both be prevented by encapsulation of the drug in liposomes. Poloxamer 188 appeared to be able to 'seal' the liposomal membrane and enhance drug retention. The study also highlighted the importance of bio-relevant in vitro release study in formulation screening.

Strategies to maximize liposomal drug loading for a poorly water-soluble anticancer drug.

PURPOSE: To develop a liposomal system with high drug loading (DL) for intravenous (i.v.) delivery of a poorly water-soluble basic drug, asulacrine (ASL). METHODS: A thin-film hydration and extrusion method was used to fabricate the PEGylated liposomal membranes followed by a freeze and thaw process. A novel active drug loading method was developed using ammonium sulphate gradient as an influx driving force of ASL solubilized with sulfobutyl ether-ß-cyclodextrin (SBE-ß-CD). DL was maximized by optimizing liposomal preparation and loading conditions. Pharmacokinetics was evaluated following i.v. infusion in rabbits. RESULTS: Freeze-thaw resulted in unilamellar liposome formation (180 nm) free of micelles. Higher DL was obtained when dialysis was used to remove the untrapped ammonium sulphate compared to ultracentrifuge. The pH and SBE-ß-CD level in the loading solution played key roles in enhancing DL. High DL ASL-liposomes (8.9%w/w, drug-to-lipid mole ratio 26%) were obtained with some drug "bundles" in the liposomal cores and were stable in a 5% glucose solution for >80 days with minimal leakage (<2%). Surprisingly, following administration of ASL-liposomes prepared with or without SBE-ß-CD, the half-lives were similar to the drug solution despite an increased area under the curve, indicating drug leakage from the carriers. CONCLUSIONS: High liposomal DL was achieved with multiple strategies for a poorly-water soluble weak base. However, the liposomal permeability needed to be tailored to improve drug retention.

Anticoagulation management by community pharmacists in New Zealand: an evaluation of a collaborative model in primary care.

OBJECTIVES: Despite the introduction of new oral anticoagulants, vitamin K antagonists remain the mainstay of the prevention and treatment of thromboembolism. The advent of affordable point-of-care testing presents an opportunity for community pharmacists to provide anticoagulation management services, better utilizing their training, reducing the workload on medical practices and improving accessibility and convenience for patients. This study aimed to determine the effectiveness of anticoagulation management by community pharmacists. METHODS: All patients enrolled in a pilot programme for a community pharmacy anticoagulation management service using point-of-care international normalized ratio testing and computer-assisted dose adjustment were included in a follow-up study, including before-after comparison. Outcomes included time in therapeutic range (TTR), time above and below range, number and proportion of results outside efficacy and safety thresholds, and a comparison of care led by pharmacists and care led by a primary-care general practitioner (GP). KEY FINDINGS: A total of 693 patients were enrolled, predominantly males over 65 years of age with atrial fibrillation. The mean TTR was 78.6% (95% CI 49.3% to 100%). A subgroup analysis (n = 221) showed an increase in mean TTR from 61.8% under GP-led care to 78.5% under pharmacist-led care (P < 0.001), reflecting a reduction in the time above and, in particular, below the range. The mean TTR by pharmacy ranged from 71.4% to 84.1%. The median number of tests per month was not statistically different between GP- and pharmacist-led care. CONCLUSIONS: Community-pharmacist-led anticoagulation care utilizing point-of-care testing and computerized decision support is safe and effective, resulting in significant improvements in TTR. Our results support wider adoption of this model of collaborative care.

Physicochemical characterization of asulacrine towards the development of an anticancer liposomal formulation via active drug loading: stability, solubility, lipophilicity and ionization.

To facilitate the development of a liposomal formulation for cancer therapy, the physicochemical properties of asulacrine (ASL), an anticancer drug candidate, were characterized. Nano-liposomes were prepared by thin-film hydration in conjugation with active drug loading using ammonium sulphate and post-insertion with Poloxamer 188. A stability-indicating HPLC assay with diode array detection was developed for the determination of ASL concentrations. The U-shaped pH-solubility profile in aqueous solutions, with a lowest solubility at pH 7.4 (0.843 µg/mL), indicated that ASL is an ampholyte, and dilution or neutralization of acidic drug solutions used in clinical trials with physiological fluids may cause drug precipitation. The basic pKa value measured by absorbance spectroscopy was 6.72. The logD value at pH 3.8 was 1.15 which increased to 3.24 as pH increased to 7.4. ASL was found to be the most stable in acidic conditions and degraded most rapidly in alkaline conditions. An extra-liposomal pH of 5.6 during drug loading was found to be optimal to achieve the highest drug loading (DL) of 4.76% and entrapment efficiency (EE) of 99.9%. At this pH, >90% of ASL was ionized conferring high drug solubility (1mg/mL) and acted as a reservoir of unionized ASL to be transported into liposomal cores. As a suspension the optimized liposomes showed great physicochemical stability for five months at 4°C. In summary, the obtained physicochemical parameters provided insightful information useful to maximise DL into the liposomes, and explain a tendency of drug precipitation of pH-solubilized formulations following intravenous infusion.

In-vitro prediction of bioavailability following extravascular injection of poorly soluble drugs: an insight into clinical failure and the role of delivery systems.

OBJECTIVES: To investigate the feasibility of using an in-vitro model to simulate the incidence of post-injection drug precipitation (PDP), and to identify the roles of drug properties and delivery systems in its occurrence. METHODS: A literature review on incomplete absorption following extravascular injection (subcutaneous and intramuscular) was conducted. Six model drugs in nine different formulations were studied for an in-vitro/in-vivo correlation. A rapid in-vitro dilution method using a 96-well plate was used for predicting PDP by dilution with a physiological buffer. New formulations based on hydroxypropyl-ß-cyclodextrin (CD), with and without co-solvents or pH control, were developed and tested on the in-vitro model. KEY FINDINGS: The occurrence of precipitation detected from the in-vitro dilution model appeared to be correlated with clinical reports and animal studies. The formulation components played an important role in determining the potential for drug precipitation on dilution or pH neutralization. CD was found to reduce the tendency for precipitation. The addition of co-solvents may reduce the effect of CD, depending on the solvent used. CONCLUSIONS: The in-vitro model can be used as a cost-effective screening tool in injectable formulation development for safe and effective delivery of poorly soluble drugs. PDP can be circumvented with a well-designed formulation.

Preparation and characterization of a lovastatin-loaded protein-free nanostructured lipid carrier resembling high-density lipoprotein and evaluation of its targeting to foam cells.

This study was designed to investigate whether a non-protein nanostructured lipid carrier (NLC) resembling high-density lipoprotein (HDL) could deliver a hydrophobic anti-atherogenic drug, lovastatin, to foam cells. Lovastatin-loaded NLC (LT-NLC) was prepared by a nanoprecipitation/solvent diffusion method. The LT-NLC-apoprotein (LT-NLC-apo) was prepared by incubating LT-NLC with native HDL. The physicochemical parameters of LT-NLC were characterized in terms of particle size, zeta potential, morphology, entrapment efficiency, and crystallization behavior. Targeting behavior and mechanism were demonstrated by the incubation of LT-NLC-apo with a RAW 264.7 macrophage-derived foam cell model in the presence or absence of very-low-density lipoprotein (VLDL) and lipase. The results showed that LT-NLC was solid spherical or oval in shape with an average diameter of 13.8 ± 2.2 nm, zeta potential of -29.3 ± 0.2 mV and entrapment efficiency of 96.2 ± 1.3%. Phagocytosis studies showed that uptake of LT-NLC-apo by macrophages was significantly lower than LT-NLC (p < 0.01), suggesting that LT-NLC-apo could possibly escape recognition from macrophages in vivo. The uptake was increased twofold when LT-NLC-apo was incubated with transfected foam cells containing VLDL and lipase. These results indicated that non-protein NLC resembling HDL could be a useful tool to deliver lipophilic anti-atherogenic drugs to foam cells, and that uptake could be enhanced by the VLDL receptor pathway.

Synthesis and pharmacokinetics of strontium fructose 1,6-diphosphate (Sr-FDP) as a potential anti-osteoporosis agent in intact and ovariectomized rats.

A novel strontium compound has been synthesized by the reaction of fructose-1,6-diphosphate with strontium (Sr-FDP). The compound was characterized and confirmed with elemental analyses and spectroscopic (IR, NMR) methods. The pharmacokinetic profiles of Sr-FDP were investigated in Sprague-Dawley rats following oral administration at a dose of 110, 220, and 440mg/kg respectively. Pharmacokinetic differences were also compared in intact rats and ovariectomized rats with and without estrogen supplement. Strontium concentrations in plasma, urine, tissue and feces were determined by graphite furnace atomic absorption spectroscopy (GFAAS). The results showed that Sr-FDP was absorbed rapidly with T(max)<1h in all the groups with AUC(0-∞) proportional to the oral dose. The pharmacokinetic profiles were characterized by long half-life, a large apparent volume of distribution. The highest Sr concentration was observed in the bone at 6h, and the level of Sr decreased close to the baseline in heart, liver, spleen, lung, intestine, brain and kidney after 12h. The cumulative amounts of Sr over 96h were found to be ~3% in urine, but ~70% in feces suggesting that the parent drug was mainly excreted from the intestine. The C(max) and AUC(0-∞) of Sr-FDP in ovariectomized rats were significantly decreased compared to those in intact rats, and this trend was ameliorated by using 17-beta-estradiol (E(2)) treatment in the ovariectomized rats.

Assessing the responsiveness of the Asthma Quality of Life Questionnaire with pharmaceutical care.

OBJECTIVE: To assess the responsiveness of the Asthma Quality of Life Questionnaire (AQLQ) in the context of pharmaceutical care delivery New Zealand community pharmacy setting. SETTING: Community pharmacy practices in three locations in the Otago and Southland region of New Zealand. METHOD: About 62 patients with asthma (17-80 years of age) were recruited in five community pharmacies in the Otago and the Southland region of New Zealand. Patients were randomly assigned to two groups (Group 1 and Group 2). The AQLQ and a study-specific outcomes questionnaire were administered to both groups at baseline (T1), then again (with an additional global Self-Assessment of change question) 3 months later (at T2) after providing the service to Group 1. Responsiveness of the AQLQ was assessed by measuring the ability of the AQLQ to detect within-subject change in patients who subjectively indicated change, and to distinguish between two groups of patients: those who indicated change and those who indicated no change. Additionally, the correlation between the change of the AQLQ scores at T2 and the patients' self-assessment of change at T2 was estimated and used as means for assessing the AQLQ responsiveness. MAIN OUTCOME MEASURE: Asthma-specific quality of life as measured by the AQLQ and change in quality of life as perceived by participants. RESULTS: The results supported the responsiveness of three out of the four domains of the Asthma Quality of Life Questionnaire. The fourth domain, Environmental stimuli, showed weaker responsiveness, and the reasons of this were discussed. CONCLUSION: This study provided data supporting the responsiveness of the AQLQ when used in the context of pharmaceutical care. However, while the AQLQ's Activity Limitation, Symptoms and Emotional domains reflected adequate sensitivity to change in QoL over time, its Environmental domain was less sensitive. Researchers conducting longitudinal studies utilising the AQLQ in pharmaceutical care interventions should bear this in mind, and should consider the possible reasons for this apparent lack of responsiveness, and its implications.

Health-related quality of life measurement in pharmaceutical care. Targeting an outcome that matters.

The shift in emphasis of healthcare from dealing only with disease and death to also managing illness, meant that healthcare providers started to realise the importance of assessing the quality of the patient's life as a new therapeutic outcome. This is equally true in the evolving concept of pharmaceutical care, the ultimate target of which is improving the patient's quality of life (QoL) through a cooperative alliance between the pharmacist and the patient. This article discusses the place of QoL assessment in today's healthcare environment, with special emphasis on its use in the practice of pharmaceutical care.

Stereoselective urinary excretion of formoterol and its glucuronide conjugate in human.

AIMS: Formoterol is an inhaled beta2-adrenoceptor agonist used as a racemic mixture of the active (R; R)- and inactive (S; S)-enantiomers (rac-formoterol). Glucuronidation is an important route of metabolism in humans which occurs faster for (S; S)-formoterol in human liver microsomes. The aim of this study was to investigate the stereoselectivity of urinary excretion of formoterol and its glucuronide conjugate after oral dosing with rac-formoterol. METHODS: Seven nonsmoking volunteers (six males, one female) were included in the study. After an overnight fast, a single 60 micro g oral dose of rac-formoterol fumarate dihydrate was ingested. Urine samples were collected at 1 h intervals for the first 4 h, and at 6, 8, 12 and 24 h after dosing. Formoterol enantiomers were analysed by chiral h.p.l.c. assay and formoterol glucuronides were determined as formoterol enantiomers after enzymatic cleavage with beta-glucuronidase. RESULTS: The female subject displayed a different pattern of metabolism and statistical analysis was therefore limited to data for the six males. The median (range) of the total urinary excretion of formoterol was 37.8% (20.9-51.2%) of the dose. The medians (ranges) of the amounts of (R; R)- and (S; S)-formoterol and of (R; R)- and (S; S)-formoterol glucuronide excreted were 2.1 (1.0-2.9), 3.5 (2.6-3.8), 21.0 (13.1-31.0) and 10.3 (4.2-14.6)%, respectively, of the dose. Unchanged (S; S)-formoterol excretion was significantly greater than that of unchanged (R; R)-formoterol and (R; R)-formoterol glucuronide excretion was significantly greater than that of (S; S)-formoterol glucuronide. The total RR-formoterol (unchanged drug plus glucuronide) excreted was significantly greater than the total (S; S)-formoterol. CONCLUSIONS: Our study demonstrates that the urinary excretion of formoterol in male humans after oral administration of rac-formoterol is stereoselective with preferential excretion of the active (R; R)-formoterol as unchanged drug and glucuronide. The different pattern of metabolism in the female subject provides impetus for further studies of the effect of gender on the stereoselective metabolism and pharmacokinetics of formoterol.

Community pharmacists' perspectives on pharmaceutical care implementation in New Zealand.

OBJECTIVE: There were three objectives to this study: to establish New Zealand community pharmacists' level of understanding of the pharmaceutical care process, to determine their attitudes to the concept of pharmaceutical care; and to determine the barriers to commencing pharmaceutical care practice. Comparisons were made between proprietors (pharmacy owners) and employees, males and females, and younger and older pharmacists. METHOD: The research tool was a questionnaire instrument, encompassing a total of 67 questions designed to determine community pharmacists' understanding, attitudes and appreciation of the opportunities and barriers inherent in the pharmaceutical care process. A total of 490 pharmacists representing 286 proprietors and 204 employees randomly selected from the Pharmaceutical Society register were sent a questionnaire. RESULTS: The total responses numbered 377, which was a 76.9% overall response rate. Over 60% of the pharmacists surveyed had a correct understanding of pharmaceutical care. Approximately the same percentage felt the future of pharmacy would depend on the provision of services other than dispensing. Insufficient time, as a barrier to implementation, was identified by 87% of respondents, and an absence of a reimbursement system by a further 82%. Lack of: therapeutic knowledge; clinical problem solving skills; finance; appropriate space, patient demand; access to patient medical records, and data on the value of PC were identified as major barriers by over 50% of all respondents. There were significant differences in response to a number of issues recorded by males and females, proprietors and employees, and pharmacists above and below the mean sample age of 45 years. CONCLUSION: This study found that the community pharmacy environment in New Zealand had a high level of understanding of the pharmaceutical care process, but identified some significant barriers to implementation.