Paul Richard Gellert

Polylactide-poly(ethylene Glycol) Micellar-like Particles as Potential Drug Carriers: Production, Colloidal Properties and Biological Performance

The micellar-like particle systems produced from poly-D,L-lactide-poly(ethylene glycol) (PLA-PEG) copolymers have been assessed using a range of physicochemical characterisation methods, followed by in vivo studies of their biodistribution after intravenous administration to the rat. The size of the PEG chain was kept constant at 5 or 2 kDa, while the PLA size increased within a series from 2 to 25 kDa. The results obtained reveal, that in an aqueous medium the copolymers assembled into micellar-like structures, with the PLA segments forming the core and the PEG segments the surrounding corona. The size of the PLA segments dominated the process of assembly of the molecules and the characteristics of the resultant micellar-like particles. The PLA-PEG micellar particles were found to be less dynamic than those obtained from conventional surfactants. Particles formed from the lower molecular weight PLA polymers allowed a level of chain mobility while the cores of the micellar particles formed from higher molecular weight PLA appeared to be solid-like in nature. The size of the micellar particles was dependent on the copolymer molecular weight and the z-average diameter increased from 25 to 76 nm as the molecular weight of the PLA moiety increased. This provides an ability to control the particle size by adjusting the molecular weight of the PLA moiety. Following intravenous administration to the rat model, micellar-like particles smaller than approximately 70 nm accumulated in the liver, despite the fact that the PEG corona provided an effective steric stabilization effect. Micellar-like particles with a diameter of more than approximately 70 nm exhibited prolonged systemic circulation and reduced liver uptake, although the steric stabilisation of these particles was shown to be less effective. These findings agree with recent observations from other research groups; that indicate a possibility that very small particulates can pass through the sinusoidal fenestrations in the liver and gain access to the parenchymal cells of the liver.

Enhanced oral absorption of hydrophobic and hydrophilic drugs using quaternary ammonium palmitoyl glycol chitosan nanoparticles.

As 95% of all prescriptions are for orally administered drugs, the issue of oral absorption is central to the development of pharmaceuticals. Oral absorption is limited by a high molecular weight (>500 Da), a high log P value (>2.0) and low gastrointestinal permeability. We have designed a triple action nanomedicine from a chitosan amphiphile: quaternary ammonium palmitoyl glycol chitosan (GCPQ), which significantly enhances the oral absorption of hydrophobic drugs (e.g., griseofulvin and cyclosporin A) and, to a lesser extent, the absorption of hydrophilic drugs (e.g., ranitidine). The griseofulvin and cyclosporin A C(max) was increased 6- and 5-fold respectively with this new nanomedicine. Hydrophobic drug absorption is facilitated by the nanomedicine: (a) increasing the dissolution rate of hydrophobic molecules, (b) adhering to and penetrating the mucus layer and thus enabling intimate contact between the drug and the gastrointestinal epithelium absorptive cells, and (c) enhancing the transcellular transport of hydrophobic compounds. Although the C(max) of ranitidine was enhanced by 80% with the nanomedicine, there was no appreciable opening of tight junctions by the polymer particles.

Multiplexing Spheroid Volume, Resazurin and Acid Phosphatase Viability Assays for High-Throughput Screening of Tumour Spheroids and Stem Cell Neurospheres

Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money.

In silico modelling of drug-polymer interactions for pharmaceutical formulations

Selecting polymers for drug encapsulation in pharmaceutical formulations is usually made after extensive trial and error experiments. To speed up excipient choice procedures, we have explored coarse-grained computer simulations (dissipative particle dynamics (DPD) and coarse-grained molecular dynamics using the MARTINI force field) of polymer–drug interactions to study the encapsulation of prednisolone (log p = 1.6), paracetamol (log p = 0.3) and isoniazid (log p = −1.1) in poly(l-lactic acid) (PLA) controlled release microspheres, as well as the encapsulation of propofol (log p = 4.1) in bioavailability enhancing quaternary ammonium palmitoyl glycol chitosan (GCPQ) micelles. Simulations have been compared with experimental data. DPD simulations, in good correlation with experimental data, correctly revealed that hydrophobic drugs (prednisolone and paracetamol) could be encapsulated within PLA microspheres and predicted the experimentally observed paracetamol encapsulation levels (5–8% of the initial drug level) in 50 mg ml−1 PLA microspheres, but only when initial paracetamol levels exceeded 5 mg ml−1. However, the mesoscale technique was unable to model the hydrophilic drug (isoniazid) encapsulation (4–9% of the initial drug level) which was observed in experiments. Molecular dynamics simulations using the MARTINI force field indicated that the self-assembly of GCPQ is rapid, with propofol residing at the interface between micellar hydrophobic and hydrophilic groups, and that there is a heterogeneous distribution of propofol within the GCPQ micelle population. GCPQ–propofol experiments also revealed a population of relatively empty and drug-filled GCPQ particles.

Using Smartphones and Health Apps to Change and Manage Health Behaviors: A Population-Based Survey

Background Chronic conditions are an increasing challenge for individuals and the health care system. Smartphones and health apps are potentially promising tools to change health-related behaviors and manage chronic conditions. Objective The aim of this study was to explore (1) the extent of smartphone and health app use, (2) sociodemographic, medical, and behavioral correlates of smartphone and health app use, and (3) associations of the use of apps and app characteristics with actual health behaviors. Methods A population-based survey (N=4144) among Germans, aged 35 years and older, was conducted. Sociodemographics, presence of chronic conditions, health behaviors, quality of life, and health literacy, as well as the use of the Internet, smartphone, and health apps were assessed by questionnaire at home visit. Binary logistic regression models were applied. Results It was found that 61.25% (2538/4144) of participants used a smartphone. Compared with nonusers, smartphone users were younger, did more research on the Internet, were more likely to work full-time and more likely to have a university degree, engaged more in physical activity, and less in low fat diet, and had a higher health-related quality of life and health literacy. Among smartphone users, 20.53% (521/2538) used health apps. App users were younger, less likely to be native German speakers, did more research on the Internet, were more likely to report chronic conditions, engaged more in physical activity, and low fat diet, and were more health literate compared with nonusers who had a smartphone. Health apps focused on smoking cessation (232/521, 44.5%), healthy diet (201/521, 38.6%), and weight loss (121/521, 23.2%). The most common app characteristics were planning (264/521, 50.7%), reminding (188/521, 36.1%), prompting motivation (179/521 34.4%), and the provision of information (175/521, 33.6%). Significant associations were found between planning and the health behavior physical activity, between feedback or monitoring and physical activity, and between feedback or monitoring and adherence to doctor’s advice. Conclusions Although there were many smartphone and health app users, a substantial proportion of the population was not engaged. Findings suggest age-related, socioeconomic-related, literacy-related, and health-related disparities in the use of mobile technologies. Health app use may reflect a user’s motivation to change or maintain health behaviors. App developers and researchers should take account of the needs of older people, people with low health literacy, and chronic conditions.

ToF-SIMS analysis of chemical heterogenities in inkjet micro-array printed drug/polymer formulations.

Three different formulations comprising two drugs, felodipine and hydrochlorothiazide (HCT) and two polymers, poly(vinyl pyrolidone) (PVP) and poly(lactic-co-glycolic acid) (PLGA) were inkjet printed as micro-dot arrays and analysed on an individual micro-spot basis by time-of-flight secondary ion mass spectrometry (ToF-SIMS). For the HCT/PLGA formulation, the spots showed heterogeneity of the drug and other chemical constituents. To further investigate these heterogeneities, multivariate curve resolution was applied to the ToF-SIMS hyperspectral image datasets. This approach successfully identified distinct chemical components elucidating the HCT, PLGA, substrate material, and contaminants based on sulphur, phosphorous and sodium chloride. Spots printed using either of the drugs with PVP exhibited full substrate coverage and a uniform distribution of the active ingredient along with all other constituents within the printed spot area. This represents the preferred situation in terms of stability and controlling the release of a drug from a polymer matrix.

Structural basis of Lewis(b) antigen binding by the Helicobacter pylori adhesin BabA.

X-ray structure of BabA bound to Lewisb reveals molecular interactions used by H. pylori to colonize the gastric mucosa. Helicobacter pylori is a leading cause of peptic ulceration and gastric cancer worldwide. To achieve colonization of the stomach, this Gram-negative bacterium adheres to Lewisb (Leb) antigens in the gastric mucosa using its outer membrane protein BabA. Structural information for BabA has been elusive, and thus, its molecular mechanism for recognizing Leb antigens remains unknown. We present the crystal structure of the extracellular domain of BabA, from H. pylori strain J99, in the absence and presence of Leb at 2.0- and 2.1-Å resolutions, respectively. BabA is a predominantly α-helical molecule with a markedly kinked tertiary structure containing a single, shallow Leb binding site at its tip within a β-strand motif. No conformational change occurs in BabA upon binding of Leb, which is characterized by low affinity under acidic [KD (dissociation constant) of ~227 μM] and neutral (KD of ~252 μM) conditions. Binding is mediated by a network of hydrogen bonds between Leb Fuc1, GlcNAc3, Fuc4, and Gal5 residues and a total of eight BabA amino acids (C189, G191, N194, N206, D233, S234, S244, and T246) through both carbonyl backbone and side-chain interactions. The structural model was validated through the generation of two BabA variants containing N206A and combined D233A/S244A substitutions, which result in a reduction and complete loss of binding affinity to Leb, respectively. Knowledge of the molecular basis of Leb recognition by BabA provides a platform for the development of therapeutics targeted at inhibiting H. pylori adherence to the gastric mucosa.

3D inkjet printing of tablets exploiting bespoke complex geometries for controlled and tuneable drug release

Abstract A hot melt 3D inkjet printing method with the potential to manufacture formulations in complex and adaptable geometries for the controlled loading and release of medicines is presented. This first use of a precisely controlled solvent free inkjet printing to produce drug loaded solid dosage forms is demonstrated using a naturally derived FDA approved material (beeswax) as the drug carrier and fenofibrate as the drug. Tablets with bespoke geometries (honeycomb architecture) were fabricated. The honeycomb architecture was modified by control of the honeycomb cell size, and hence surface area to enable control of drug release profiles without the need to alter the formulation. Analysis of the formed tablets showed the drug to be evenly distributed within the beeswax at the bulk scale with evidence of some localization at the micron scale. An analytical model utilizing a Fickian description of diffusion was developed to allow the prediction of drug release. A comparison of experimental and predicted drug release data revealed that in addition to surface area, other factors such as the cell diameter in the case of the honeycomb geometry and material wettability must be considered in practical dosage form design. This information when combined with the range of achievable geometries could allow the bespoke production of optimized personalised medicines for a variety of delivery vehicles in addition to tablets, such as medical devices for example. Graphical abstract Figure. No Caption available.

Potent cyclic peptide inhibitors of VLA‐4 (α4β1 integrin)‐mediated cell adhesion. Discovery of compounds like cyclo(MePhe‐Leu‐Asp‐Val‐d‐Arg‐d‐Arg) (ZD7349) compatible with depot formulation

Additional structure–activity relationship studies on potent cyclic peptide inhibitors of very late antigen‐4 (VLA‐4) are reported. The new N‐ to C‐terminal cyclic hexa‐, hepta‐ and octapeptide inhibitors like cyclo(MeIle/MePhe‐Leu‐Asp‐Val‐X) (X=2–4 amino acids containing hydrophobic and/or basic side chains) were synthesized using solid phase peptide synthesis methods. The peptides were evaluated in in vitro cell adhesion assays and in in vivo inflammation models. Many of the peptides like cyclo(MePhe‐Leu‐Asp‐Val‐d‐Arg‐d‐Arg) (ZD7349) (17), cyclo(MeIle‐Leu‐Asp‐Val‐d‐Arg‐d‐Arg‐d‐Phe) (20), cyclo(MeIle‐Leu‐Asp‐Val‐d‐Arg‐d‐Arg‐MePhe) (21) and cyclo(MePhe‐Leu‐Asp‐Val‐d‐Arg‐d‐Arg‐d‐Ala‐d‐Ala) (23) were potent inhibitors of VLA‐4‐mediated cell adhesion and inhibited ovalbumin‐induced delayed type hypersensitivity (DTH) response in mice. The more potent compounds were highly selective and did not affect U937 cell adhesion to fibronectin (VLA‐5), phorbolmyristate acetate or PMA‐differentiated U937 cell adhesion to intercellular cell adhesion molecule‐1 (ICAM‐1)‐expressing Chinese hamster ovary cells (LFA‐1) and adenosine diphosphate (ADP)‐induced platelet aggregation (GPIIb/IIIa). In contrast to the inhibitors like Ac‐cyclo(d‐Lys‐d‐Ile‐Leu‐Asp‐Val) and cyclo(CH2CO‐Ile‐Leu‐Asp‐Val‐Pip‐CH2CO‐Ile‐Leu‐Asp‐Val‐Pip) described earlier, the new compounds were much more compatible with the depot formulations based on poly(dl‐lactide‐co‐glycolide) polymers. The hexapeptide cyclo(MePhe‐Leu‐Asp‐Val‐d‐Arg‐d‐Arg) (ZD7349) (17) inhibited MOLT‐4 cell adhesion to fibronectin and vascular cell adhesion molecule‐1 (VCAM‐1) with IC50 values of 260 and 330 nm, respectively, and did not show any significant effect against other integrins (IC50>300 μm). ZD7349 inhibited ovalbumin‐induced DTH response in mice when administered continuously using a mini‐pump (ED50 0.01 mg/kg/day) or when given as an s.c. or i.v. bolus injection at a dose of 1–10 mg/kg. ZD7349 was also active in type II collagen‐induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE) tests at a dose of 3–10 mg/kg. The peptide was released from some formulations over a period of 10–20 days. ZD7349 is currently undergoing pre‐clinical investigation. Copyright

Potent Cyclic Monomeric and Dimeric Peptide Inhibitors of VLA-4 (?4?1 Integrin)-Mediated Cell Adhesion Based on the Ile-Leu-Asp-Val Tetrapeptide

Potent monomeric and dimeric cyclic peptide very late antigen‐4 (VLA‐4) inhibitors have been designed based on a tetrapeptide (Ile‐Leu‐Asp‐Val) sequence present in a 25‐amino acid peptide (CS‐1) reported in the literature. The peptides, synthesized by the SPPS techniques, were evaluated in the in vitro cell adhesion assays and in the in vivo inflammation models. The N‐ to C‐terminal cyclic peptides such as cyclo(Ile‐Leu‐Asp‐Val‐NH‐(CH2)2‐S‐(CH2)2‐CO) (28) and cyclo(MeIle‐Leu‐Asp‐Val‐D‐Ala‐D‐Ala) (31), monomeric and dimeric peptides containing piperazine (Pip) or homopiperazine (hPip) residues as linking groups, e.g. cyclo(MeIle‐Leu‐Asp‐Val‐Pip‐CH2CO‐NH‐(CH2)2‐S‐CH2‐CO) (49) and cyclo(MeIle‐Leu‐Asp‐Val‐hPip‐CH2CO‐MeIle‐Leu‐Asp‐Val‐hPip‐CH2CO) (58) and cyclic peptides containing an amide bond between the side chain amino group of an amino acid such as Lys and the C‐terminal Val carboxyl group, e.g. Ac‐cyclo(D‐Lys‐D‐Ile‐Leu‐Asp‐Val) (62) and β‐Ala‐cyclo(D‐Lys‐D‐Leu‐Leu‐Asp‐Val) (68) were more potent than CS‐1 in inhibiting the adhesion of the VLA‐4‐expressing MOLT‐4 cells to fibronectin. The more potent compounds were highly selective and did not affect U937 cell adhesion to fibronectin (VLA‐5), PMA‐differentiated U937 cell adhesion to intercellular cell adhesion molecule‐1‐expressing Chinese hamster ovary cells (LFA‐1) and ADP‐induced platelet aggregation (GPIIb/IIIa). A number of the more potent compounds inhibited ovalbumin‐induced delayed type hypersensitivity in mice and some were 100–300 times more potent (ED50=0.003–0.009 mg/kg/day, s.c.) than CS‐1. Two peptides, Ac‐cyclo(D‐Lys‐D‐Ile‐Leu‐Asp‐Val) (62) and cyclo(CH2CO‐Ile‐Leu‐Asp‐Val‐Pip‐CH2CO‐Ile‐Leu‐Asp‐Val‐Pip) (55), were formulated in poly(DL‐lactide‐co‐glycolide) depots and the release profile was investigated in vitro over a 30‐day period. Copyright