Colin Pouton

Adenovirus: a blueprint for non-viral gene delivery

Publisher: Elsevier BV

Date: 10-2010

DOI: 10.1016/J.COPBIO.2010.06.011

Abstract: Although adenoviral vectors may not find a direct clinical role in gene therapy, an understanding of the mechanisms of DNA delivery that adenoviruses use is of vital importance to the design of next-generation non-viral gene delivery systems. Adenoviruses overcome a series of biological barriers, including endosomal escape, intracellular trafficking, capsid dissociation, and nuclear import of DNA, to deliver their genome to the host cell nucleus. The understanding of these processes at the molecular level is progressing and is set to inform the design of synthetic gene delivery systems.

Synthesis and evaluation of new N6-substituted adenosine-5′-N-methylcarboxamides as A3 adenosine receptor agonists

Publisher: Elsevier BV

Date: 05-2010

DOI: 10.1016/J.BMC.2010.03.047

Abstract: A number of N(6)-substituted adenosine-5'-N-methylcarboxamides were synthesised and their pharmacology, in terms of their receptor affinity, selectivity and cardioprotective effects, were explored. The first series of compounds, 4a-4f and 5a-5f, showed modest receptor affinity for the A(3)AR with K(i) values in the low to mid muM range. However, the incorporation of a 4-(2-aminoethyl)-2,6-di-tert-butylphenol group in the N(6)-position (in compounds 4g and 5g) significantly improved the affinity with K(i) values of 30 and 9 nM, respectively. Improvements in affinity, as well as selectivity were seen when a functionalized linker was introduced. The N(6)-phenyl series, compounds 7a-7d, demonstrated low to mid nanomolar receptor affinities (K(i)=2.3-45.0 nM), with 7b displaying 109-fold selectivity for the A(3)AR (vs A(1)). The N(6)-benzyl series 9a-9c also proved to be potent and selective A(3)AR agonists and the longer chain length linker 13 was tolerated at the A(3)AR without abrogation of affinity or selectivity. Cardioprotection was demonstrated by a simulated ischaemia cell culture assay, whereby 7b, 7c, 9a, 9b and 9c all showed cardioprotective effects at 100 nM comparable or better than the benchmark A(3)AR agonist IB-MECA, but which were indistinguishable by statistical analysis. For ex le, compound 9c reduced cell death by 68.0+/-3.6%.

Synthesis and Pharmacological Evaluation of Noscapine-Inspired 5-Substituted Tetrahydroisoquinolines as Cytotoxic Agents.

Publisher: American Chemical Society (ACS)

Date: 29-08-2018

DOI: 10.1021/ACS.JMEDCHEM.8B00986

Abstract: A series of 5-substituted tetrahydroisoquinolines was synthesized via a 10-step linear synthesis to assess whether replacement of noscapine's southern isobenzofuranone with other moieties resulted in retained cytotoxic activity. One such molecule, 18g, bearing a para-methoxybenzyl functionality with N-ethylcarbamoyl substitution, produced cell-cycle arrest at the G2/M phase with an EC

Digestion of phospholipids after secretion of bile into the duodenum changes the phase behavior of bile components

Publisher: American Chemical Society (ACS)

Date: 16-07-2014

DOI: 10.1021/MP500193G

Abstract: Bile components play a significant role in the absorption of dietary fat, by solubilizing the products of fat digestion. The absorption of poorly water-soluble drugs from the gastrointestinal tract is often enhanced by interaction with the pathways of fat digestion and absorption. These processes can enhance drug absorption. Thus, the phase behavior of bile components and digested lipids is of great interest to pharmaceutical scientists who seek to optimize drug solubilization in the gut lumen. This can be achieved by dosing drugs after food or preferably by formulating the drug in a lipid-based delivery system. Phase diagrams of bile salts, lecithin, and water have been available for many years, but here we investigate the association structures that occur in dilute aqueous solution, in concentrations that are present in the gut lumen. More importantly, we have compared these structures with those that would be expected to be present in the intestine soon after secretion of bile. Phosphatidylcholines are rapidly hydrolyzed by pancreatic enzymes to yield equimolar mixtures of their monoacyl equivalents and fatty acids. We constructed phase diagrams that model the association structures formed by the products of digestion of biliary phospholipids. The micelle-vesicle phase boundary was clearly identifiable by dynamic light scattering and nephelometry. These data indicate that a significantly higher molar ratio of lipid to bile salt is required to cause a transition to lamellar phase (i.e., liposomes in dilute solution). Mixed micelles of digested bile have a higher capacity for solubilization of lipids and fat digestion products and can be expected to have a different capacity to solubilize lipophilic drugs. We suggest that mixtures of lysolecithin, fatty acid, and bile salts are a better model of molecular associations in the gut lumen, and such mixtures could be used to better understand the interaction of drugs with the fat digestion and absorption pathway.

Targeted delivery to the nucleus

Publisher: Elsevier BV

Date: 10-08-2007

DOI: 10.1016/J.ADDR.2007.06.010

Abstract: Macromolecules and supramolecular complexes are frequently required to enter and exit the nucleus during normal cell function, but access is restricted and exchange to and from the nucleus is tightly controlled. We describe the mechanisms which regulate nuclear import of endogenous molecules and indicate how viruses exploit these mechanisms during their life cycle. Opportunities exist to make use of natural pathways for delivery of therapeutic entities, in particular to develop safe and effective methods for gene therapy, although past attempts to design non-viral nuclear delivery systems have met with limited success. To increase the likelihood of success scientists will need an appreciation of the mechanisms by which viruses deliver their genomes to the nucleus, and will need a commitment to control the architecture of non-viral delivery systems at the molecular level. Effective delivery systems will require several attributes to facilitate endosomal escape, microtubular transport and uptake through the nuclear pore complex. The published literature provides a strong foundation for design of nuclear targeting systems. The challenge faced by delivery scientists is to assemble a system which is as effective as, for ex le, the adenovirus but which lacks its immunogenicity. This article reviews the relevant literature and indicates key areas for future research.

Aqueous phase behavior of the PEO-containing non-ionic surfactant C₁₂E₆: A molecular dynamics simulation study

Publisher: Elsevier BV

Date: 04-2021

DOI: 10.1016/J.JCIS.2020.12.032

Toward the establishment of standardized in vitro tests for lipid-based formulations, Part 1: Method parameterization and comparison of in vitro digestion profiles across a range of representative formulations

Publisher: Elsevier BV

Date: 09-2012

DOI: 10.1002/JPS.23205

Pharmaceutical and biological properties of poly(amino acid)/DNA polyplexes

Publisher: Informa UK Limited

Date: 1999

DOI: 10.3109/10611869909085498

Abstract: Physicochemical properties of polyplexes formed between pRSVlacZ and poly(amino acid)s were investigated as a paradigm of more complex, synthetic virus-like, DNA delivery systems, that are of interest to many gene delivery laboratories. We observed the interaction between polymer and DNA using ethidium exclusion, and determined the size distributions and the zeta potentials of polyplexes. We correlated these properties with their fundamental interactions with cultured B16 murine melanoma cells, and the resulting efficiency of transfection. A variety of poly(amino acid)s each condensed DNA to produce particles with mean hydrodynamic diameters of approximately 100 nm (a typical span of a population was 80-120nm). Poly(amino acid) polyplexes were unstable in electrolyte solutions such as cell culture media. The apparent particle size increased in electrolyte, depending on the charge ratio, to diameters up to 700 nm. This was thought to be due to aggregation, since neutral particles were most sensitive. When the charge ratio (+/-) exceeded unity polyplexes had positive zeta potentials (which peaked at approximately +30 mV), bound non-specifically to cells, were internalised and in the presence of an endosomolytic agent were able to transfect cells. Though all cationic poly(amino acid)s investigated formed polyplexes with similar physical properties, their biological properties were significantly different. Polyplexes prepared with poly-L-ornithine were the most effective transfection agents, but poly(lys-co-ala, 1: 1) systems appeared to be inactive. This may reflect the differences in uncoupling of DNA and polymer, which is expected to be necessary for passage through the nuclear pore. Uncoupling of polycation and DNA was investigated by exposing the complexes to dextran sulphate. Release of DNA was detected by increased fluorescence at 600 nm in the presence of ethidium. Release of DNA was incomplete from polyplexes formed with high molecular weight polylysine. This may explain the lower levels of transfection observed with high molecular weight polylysine. The significance of these observations for design of advanced non-viral gene delivery systems is discussed.

The impact of size and charge on the pulmonary pharmacokinetics and immunological response of the lungs to PLGA nanoparticles after intratracheal administration to rats

Publisher: Elsevier BV

Date: 11-2020

DOI: 10.1016/J.NANO.2020.102291

Colloidal characteristics and formulation of pure protein particulate vaccines

Publisher: Oxford University Press (OUP)

Date: 29-05-2012

DOI: 10.1111/J.2042-7158.2012.01513.X

Abstract: We recently reported that dense gas processing of the protein ovalbumin (OVA) resulted in the formation of particles that were insoluble in water and which retained their immunogenicity in vivo. In the present study, the colloidal properties of these pure protein particles were investigated to in part inform rational formulation approaches. The colloidal properties of the particles, in terms of size, zeta potential and pH-dependent surface and solution properties, were examined. In phosphate-buffered saline (pH 7.4), flocculation of the particles was observed, which was prevented when particles were suspended in acetate buffer at pH lower than 4. The resulting particle size was 300 nm with low polydispersity and zeta potential of 22.9 ± 3.1 mV (mean ± SEM, n = 3) at pH 3. Dense gas OVA particles were also prevented from flocculation using steric stabilisation with Pluronic F127. In this form the particles were stable in Krebs–Henseleit solution for 48 h at room temperature. These findings indicate that insoluble pure protein particles produced by dense gas processing have desirable characteristics as particulate vaccines, including consistency of particle size under controlled conditions and high colloid stability.

Genetic reporter cell lines

Publisher: Wiley

Date: 03-12-2015

DOI: 10.1002/9781118308295.CH25

A targeted NKX2.1 hesc reporter line enables identification of human basal forebrain derivatives

Publisher: Oxford University Press (OUP)

Date: 03-2011

DOI: 10.1002/STEM.587

Abstract: We have used homologous recombination in human embryonic stem cells (hESCs) to insert sequences encoding green fluorescent protein (GFP) into the NKX2.1 locus, a gene required for normal development of the basal forebrain. Generation of NKX2.1-GFP+ cells was dependent on the concentration, timing, and duration of retinoic acid treatment during differentiation. NKX2.1-GFP+ progenitors expressed genes characteristic of the basal forebrain, including SHH, DLX1, LHX6, and OLIG2. Time course analysis revealed that NKX2.1-GFP+ cells could upregulate FOXG1 expression, implying the existence of a novel pathway for the generation of telencephalic neural derivatives. Further maturation of NKX2.1-GFP+ cells gave rise to γ-aminobutyric acid-, tyrosine hydroxylase-, and somatostatin-expressing neurons as well as to platelet-derived growth factor receptor α-positive oligodendrocyte precursors. These studies highlight the ersity of cell types that can be generated from human NKX2.1+ progenitors and demonstrate the utility of NKX2.1GFP/w hESCs for investigating human forebrain development and neuronal differentiation.

From influenza to COVID-19: Lipid nanoparticle mRNA vaccines at the frontiers of infectious diseases

Publisher: Elsevier BV

Date: 09-2021

DOI: 10.1016/J.ACTBIO.2021.06.023

Transformation of Biopharmaceutical Classification System Class I and III Drugs Into Ionic Liquids and Lipophilic Salts for Enhanced Developability Using Lipid Formulations

Publisher: Elsevier BV

Date: 2018

DOI: 10.1016/J.XPHS.2017.05.019

Abstract: Higher lipid solubility of lipophilic salt forms creates new product development opportunities for high-dose liquid-filled capsules. The purpose of this study is to determine if lipophilic salts of Biopharmaceutical Classification System (BCS) Class I amlodipine and BCS Class III fexofenadine, ranitidine, and metformin were better lipid formulation candidates than existing commercial salts. Lipophilic salts were prepared from lipophilic anions and commercial HCl or besylate salt forms, as verified by

Location of Solvated Probe Molecules Within Nonionic Surfactant Micelles Using Molecular Dynamics

Publisher: Elsevier BV

Date: 2019

DOI: 10.1016/J.XPHS.2018.10.055

Abstract: An iconic textbook that pharmaceutical scientists encounter in undergraduate courses is "Martin's Physical Pharmacy and Pharmaceutical Sciences." Within the chapter on Colloids, a figure indicates the location of solubilization of molecules within spherical, nonionic surfactant micelles. The surfactant consists of polyethylene glycol (PEG) hydrophilic headgroups and alkane chains for the hydrophobic tail. The figure shows benzene and toluene within the alkane core, salicylic acid (2-hydroxybenzoic acid) at the interface between the core and PEG chains, and then para-hydroxybenzoic acid (4-hydroxybenzoic acid) located between the PEG chains. Molecular dynamics simulations of octaethylene glycol monododecyl ether micelles were performed with a series of probe molecules, including those within the Martin's figure, to determine their solubilization location. Relative placement of molecules within the micelle was correct however, some specifics were different. In particular, benzene and toluene are excluded from the core, and 4-hydroxybenzoic acid prefers to maintain contact with the core. A series of molecules containing 6 carbon atoms were also studied to determine the effects of cyclization (moves out of core), polar functionalization (anchored to interface), and aromatization (excluded from central core). Molecular dynamics was found to be a useful tool for gaining insight into interactions important in solubilization of molecules.

Transcriptional signature in microglia associated with Aβ plaque phagocytosis

Publisher: Springer Science and Business Media LLC

Date: 21-05-2021

DOI: 10.1038/S41467-021-23111-1

Abstract: The role of microglia cells in Alzheimer’s disease (AD) is well recognized, however their molecular and functional ersity remain unclear. Here, we isolated amyloid plaque-containing (using labelling with methoxy-XO4, XO4 + ) and non-containing (XO4 − ) microglia from an AD mouse model. Transcriptomics analysis identified different transcriptional trajectories in ageing and AD mice. XO4 + microglial transcriptomes demonstrated dysregulated expression of genes associated with late onset AD. We further showed that the transcriptional program associated with XO4 + microglia from mice is present in a subset of human microglia isolated from brains of in iduals with AD. XO4 − microglia displayed transcriptional signatures associated with accelerated ageing and contained more intracellular post-synaptic material than XO4 + microglia, despite reduced active synaptosome phagocytosis. We identified HIF1α as potentially regulating synaptosome phagocytosis in vitro using primary human microglia, and BV2 mouse microglial cells. Together, these findings provide insight into molecular mechanisms underpinning the functional ersity of microglia in AD.

In vitro maturation of dopaminergic neurons derived from mouse embryonic stem cells: Implications for transplantation

Publisher: Public Library of Science (PLoS)

Date: 22-02-2012

DOI: 10.1371/JOURNAL.PONE.0031999

Enhanced Extravasation, Stability and in Vivo Cardiac Gene Silencing via in Situ siRNA–Albumin Conjugation

Publisher: American Chemical Society (ACS)

Date: 19-12-2011

DOI: 10.1021/MP2002522

Abstract: A potential barrier to progression of siRNA therapeutics to the clinic is the ability of these agents to cross the vascular endothelium to reach target cells. This study aimed to bypass the endothelial barrier by harnessing the extravasation capability of the serum protein albumin to allow siRNA to reach cardiomyocytes. A strategy for conjugating siRNA to albumin in vivo was developed that involved activating 3'-amine, 2'-O-methyl, phosphorothioate modified siRNA with succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) to yield maleimide-functionalized siRNA ("activated siRNA") this thiol-reactive species can then irreversibly link to the single surface-exposed cysteine residue of endogenous albumin following intravenous administration. An IGF-I-receptor (IGF-IR) siRNA sequence which was effective in vitro was used to test the ability of the siRNA-albumin conjugate to bypass the endothelial barrier in Balb/C mice and produce silencing. In situ conjugation of maleimide-functionalized siRNA to albumin in mouse serum occurred within minutes of addition, and the resulting conjugate was found to be nuclease stable by SDS-PAGE analysis. In Sprague-Dawley rats, activated siRNA showed a significantly enhanced elimination half-life (75.9 ± 18.2 min) compared to unactivated siRNA (5.1 ± 0.2 min). Intravenous injection of this activated siRNA (1 mg/kg daily for four days) significantly reduced left ventricle IGF-IR mRNA to 64.1 ± 4.1% of that in vehicle-treated animals (mean ± SEM), while the control siRNA (unconjugated) had no effect (n = 4, P > 0.05). Imaging of microvessels from mice treated with fluorescein-labeled activated siRNA showed clear evidence of extravasation and cellular uptake in capillary endothelial cells, cardiomyocytes and vascular smooth muscle cells for mice treated with the activated siRNA but not mice treated with the unactivated siRNA. siRNA-albumin constructs are therefore capable of extravasation to the myocardium resulting in silencing in this otherwise silencing-resistant organ.

No title found

Publisher: Royal Society of Chemistry (RSC)

Date: 2022

DOI: 10.1039/D2BM00168C

Abstract: Ionizable cationic lipids play a critical role in developing new gene therapies for various biomedical applications, including COVID-19 vaccines. However, it remains unclear whether the formulation of lipid nanoparticles (LNPs) using DLin-MC3-DMA, an optimized ionizable lipid clinically used for small interfering RNA (siRNA) therapy, also facilitates high liver-selective transfection of other gene therapies such as plasmid DNA (pDNA). Here we report the first investigation into pDNA transfection efficiency in different mouse organs after intramuscular and intravenous administration of lipid nanoparticles (LNPs) where DLin-MC3-DMA, DLin-KC2-DMA or DODAP are used as the ionizable cationic lipid component of the LNP. We discovered that these three benchmark lipids previously developed for siRNA delivery followed an unexpected characteristic rank order in gene expression efficiency when utilized for pDNA. In particular, DLin-KC2-DMA facilitated higher

Synthesis and Pharmacological Evaluation of Dual Acting Antioxidant A_2A Adenosine Receptor Agonists

Publisher: American Chemical Society (ACS)

Date: 29-03-2012

DOI: 10.1021/JM300206U

Abstract: A series of adenosine-5'-N-alkylcarboxamides and N(6)-(2,2-diphenylethyl)adenosine-5'-N-alkylcarboxamides bearing antioxidant moieties in the 2-position were synthesized from the versatile intermediate, O(6)-(benzotriazol-1-yl)-2-fluoro-2',3'-O-isopropylideneinosine-5'-N-alkylcarboxamide (1). These compounds were evaluated as A(2A) adenosine receptor (A(2A)R) agonists in a cAMP accumulation assay, and a number of potent and selective agonists were identified. Three of these compounds were evaluated further in an ischemic injury cell survival assay and a reactive oxygen species (ROS) production assay whereby 15b and 15c were shown to reduce ROS activity and cell death due to ischemia.

Isolation of LMX1a Ventral Midbrain Progenitors Improves the Safety and Predictability of Human Pluripotent Stem Cell-Derived Neural Transplants in Parkinsonian Disease

Publisher: Society for Neuroscience

Date: 22-10-2019

DOI: 10.1523/JNEUROSCI.1160-19.2019

Abstract: Human pluripotent stem cells (hPSCs) are a promising resource for the replacement of degenerated ventral midbrain dopaminergic (vmDA) neurons in Parkinson's disease. Despite recent advances in protocols for the in vitro generation of vmDA neurons, the asynchronous and heterogeneous nature of the differentiations results in transplants of surprisingly low vmDA neuron purity. As the field advances toward the clinic, it will be optimal, if not essential, to remove poorly specified and potentially proliferative cells from donor preparations to ensure safety and predictable efficacy. Here, we use two novel hPSC knock-in reporter lines expressing GFP under the LMX1A and PITX3 promoters, to selectively isolate vm progenitors and DA precursors, respectively. For each cell line, unsorted, GFP + , and GFP − cells were transplanted into male or female Parkinsonian rodents. Only rats receiving unsorted cells, LMX1A-eGFP + , or PITX3-eGFP − cell grafts showed improved motor function over 6 months. Postmortem analysis revealed small grafts from PITX3-eGFP + cells, suggesting that these DA precursors were not compatible with cell survival and integration. In contrast, LMX1A-eGFP + grafts were highly enriched for vmDA neurons, and importantly excluded expansive proliferative populations and serotonergic neurons. These LMX1A-eGFP + progenitor grafts accelerated behavioral recovery and innervated developmentally appropriate forebrain targets, whereas LMX1A-eGFP − cell grafts failed to restore motor deficits, supported by increased fiber growth into nondopaminergic target nuclei. This is the first study to use an hPSC-derived reporter line to purify vm progenitors, resulting in improved safety, predictability of the graft composition, and enhanced motor function. SIGNIFICANCE STATEMENT Clinical trials have shown functional integration of transplanted fetal-derived dopamine progenitors in Parkinson's disease. Human pluripotent stem cell (hPSC)-derived midbrain progenitors are now being tested as an alternative cell source however, despite current differentiation protocols generating % correctly specified cells for implantation, resultant grafts contain a small fraction of dopamine neurons. Cell-sorting approaches, to select for correctly patterned cells before implantation, are being explored yet have been suboptimal to date. This study provides the first evidence of using 2 hPSC reporter lines (LMX1A-GFP and PITX3-GFP) to isolate correctly specified cells for transplantation. We show LMX1A-GFP + , but not PITX3-GFP + , cell grafts are more predictable, with smaller grafts, enriched in dopamine neurons, showing appropriate integration and accelerated functional recovery in Parkinsonian rats.

Self-emulsifying drug delivery systems: formulation and biopharmaceutic evaluation of an investigational lipophilic compound.

Publisher: Springer Science and Business Media LLC

Date: 1992

DOI: 10.1023/A:1018987928936

Abstract: Self-emulsifying drug delivery systems (SEDDSs) represent a possible alternative to traditional oral formulations of lipophilic compounds. In the present study, a lipophilic compound, WIN 54954, was formulated in a medium chain triglyceride oil/nonionic surfactant mixture which exhibited self-emulsification under conditions of gentle agitation in an aqueous medium. The efficiency of emulsification was studied using a laser diffraction sizer to determine particle size distributions of the resultant emulsions. An optimized formulation which consisted of 25% (w/w) surfactant, 40% (w/w) oil, and 35% (w/w) WIN 54954 emulsified rapidly with gentle agitation in 0.1 N HCl (37 degrees C), producing dispersions with mean droplet diameters of less than 3 microns. The self-emulsifying preparation was compared to a polyethylene glycol 600 (PEG 600) solution formulation by administering each as prefilled soft gelatin capsules to fasted beagle dogs in a parallel crossover study. Pharmacokinetic parameters were determined and the absolute bioavailability of the drug was calculated by comparison to an i.v. injection. The SEDDS improved the reproducibility of the plasma profile in terms of the maximum plasma concentration (Cmax) and the time to reach the maximum concentration (tmax). There was no significant difference in the absolute bioavailability of WIN 54954 from either the SEDDS or the PEG formulations.

High Molecular Weight DNA Enrichment with Peptide Nucleic Acid Probes.

Publisher: Springer New York

Date: 2017

DOI: 10.1007/978-1-4939-6750-6_4

Abstract: Here, we describe how peptide nucleic acid (PNA) probes can be used to enrich genomic DNA fractions to facilitate downstream analysis, such as the haplotype phasing of the isolated genomic pieces. This method enriches for polymorphic regions of fragmented chromosomes by physically separating the desired sequence and flanking regions. The PNA probes used for enrichment are novel synthetic nucleic acids with highly specific targeting and hybridization properties. Using a enrichment technique, we capture high molecular weight genomic DNA using nothing more than a simple modification to standard genomic DNA extraction from blood.

Midbrain and forebrain patterning delivers immunocytochemically and functionally similar populations of neuropeptide Y containing GABAergic neurons

Publisher: Elsevier BV

Date: 02-2011

DOI: 10.1016/J.NEUINT.2011.02.016

Abstract: Neurons differentiated in vitro from embryonic stem cells (ESCs) have the potential to serve both as models of disease states and in drug discovery programs. In this study, we use sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF-8) to enrich for forebrain and midbrain phenotypes from mouse ESCs. We then investigate, using Ca(2+) imaging and [(3)H]-GABA release studies, whether the GABAergic neurons produced exhibit distinct functional phenotypes. At day 24 of differentiation, reverse transcriptase-PCR showed the presence of both forebrain (Bf-1, Hesx1, Pgc-1α, Six3) and midbrain (GATA2, GATA3) selective mRNA markers in developing forebrain-enriched cultures. All markers were present in midbrain cultures except for Bf-1 and Pgc-1α. Irrespective of culture conditions all GABA immunoreactive neurons were also immunoreactive to neuropeptide Y (NPY) antibodies. Forebrain and midbrain GABAergic neurons responded to ATP (1 mM), L-glutamate (30 μM), noradrenaline (30 μM), acetylcholine (30 μM) and dopamine (30 μM), with similar elevations of intracellular Ca(2+)([Ca(2+)](i)). The presence of GABA(A) and GABA(B) antagonists, bicuculline (30 μM) and CGP55845 (1 μM), increased the elevation of [Ca(2+)](i) in response to dopamine (30 μM) in midbrain, but not forebrain GABAergic neurons. All agonists, except dopamine, elicited similar [(3)H]-GABA release from forebrain and midbrain cultures. Dopamine (30 μM) did not stimulate significant [(3)H]-GABA release in midbrain cultures, although it was effective in forebrain cultures. This study shows that differentiating neurons toward a midbrain fate restricts the expression of forebrain markers. Forebrain differentiation results in the expression of forebrain and midbrain markers. All GABA(+) neurons contain NPY, and show similar agonist-induced elevations of [Ca(2+)](i) and [(3)H]-GABA release. This study indicates that the pharmacological phenotype of these particular neurons may be independent of the addition of the patterning factors that direct neurons toward forebrain and midbrain fates.

Dynein light chain association sequences can facilitate nuclear protein import

Publisher: American Society for Cell Biology (ASCB)

Date: 08-2007

DOI: 10.1091/MBC.E07-01-0030

Abstract: Nuclear localization sequence (NLS)-dependent nuclear protein import is not conventionally held to require interaction with microtubules (MTs) or components of the MT motor, dynein. Here we report for the first time the role of sequences conferring association with dynein light chains (DLCs) in NLS-dependent nuclear accumulation of the rabies virus P-protein. We find that P-protein nuclear accumulation is significantly enhanced by its dynein light chain association sequence (DLC-AS), dependent on MT integrity and association with DLCs, and that P-protein-DLC complexes can associate with MT cytoskeletal structures. We also find that P-protein DLC-AS, as well as analogous sequences from other proteins, acts as an independent module that can confer enhancement of nuclear accumulation to proteins carrying the P-protein NLS, as well as several heterologous NLSs. Photobleaching experiments in live cells demonstrate that the MT-dependent enhancement of NLS-mediated nuclear accumulation by the P-protein DLC-AS involves an increased rate of nuclear import. This is the first report of DLC-AS enhancement of NLS function, identifying a novel mechanism regulating nuclear transport with relevance to viral and cellular protein biology. Importantly, this data indicates that DLC-ASs represent versatile modules to enhance nuclear delivery with potential therapeutic application.

Interaction of viruses with host cell molecular motors

Publisher: Elsevier BV

Date: 10-2010

DOI: 10.1016/J.COPBIO.2010.06.009

Abstract: Viral particles are generally too large to diffuse freely within the crowded environment of the host cell cytoplasm. They depend on mammalian cell transport systems, in particular the microtubular molecular motor dynein, to deliver their nucleic acids to the vicinity of the nucleus. An understanding of how viruses interact with dynein, and its many accessory proteins, may reveal targets for drug discovery and will unlock the toolbox required to improve the performance of synthetic gene delivery systems.

Human pluripotent stem cell derived midbrain PITX3eGFP/w neurons: a versatile tool for pharmacological screening and neurodegenerative modeling

Publisher: Frontiers Media SA

Date: 31-03-2015

DOI: 10.3389/FNCEL.2015.00104

Comparing mouse and human pluripotent stem cell derived cardiac cells: Both systems have advantages for pharmacological and toxicological screening

Publisher: Elsevier BV

Date: 07-2015

DOI: 10.1016/J.VASCN.2015.04.009

Abstract: Pluripotent stem cells offer an unparalleled opportunity to investigate cardiac physiology, pharmacology, toxicology and pathophysiology. In this paper we describe the use of both mouse (Nkx2-5(eGFP/w)) and human (NKX2-5(eGFP/w)) pluripotent stem cell reporter lines, differentiated toward cardiac lineage, for live single cell high acquisition rate calcium imaging. We also assess the potential of NKX2-5(eGFP/w) cardiac lineage cells for use toxicological screening as well as establish their sensitivity to a shift between low and high oxygen environments. Differentiated mouse Nkx2-5(eGFP/w) cells demonstrated a wide range of spontaneous oscillation rates that could be reduced by ryanodine (10μM), thapsigargin (1μM) and ZD7288 (10μM). In contrast human NKX2-5(eGFP/w) cell activity was only reduced by thapsigargin (1μM). Human cell survival was sensitive to the addition of trastuzumab and doxorubicin, while the switch from a low to a high oxygen environment affected oscillation frequency. We suggest that the human NKX2-5(eGFP/w) cells are less suitable for studies of compounds affecting cardiac pacemaker activity than mouse Nkx2-5(eGFP/w) cells, but are very suitable for cardiac toxicity studies.

Structure and dynamics of glyceride lipid formulations, with propylene glycol and water

Publisher: No publisher found

Date: 2009

DOI: 10.1021/MP8001667 CCC

Modulation of nucleocytoplasmic trafficking by retention in cytoplasm or nucleus

Publisher: Wiley

Date: 15-08-2009

DOI: 10.1002/JCB.22218

Abstract: Nuclear protein transport processes have largely been studied using in vitro semi-intact cell systems where high concentrations of nuclear localizing substrates are used, and cytoplasmic components such as the microtubule (MT) network, are either absent or damaged. Here we use the fluorescence recovery after photobleaching (FRAP) technique to analyze the nucleocytoplasmic flux of distinct fluorescently tagged proteins over time in living cultured cells. FRAP was performed in different parts of the cell to analyze the kinetics of nucleocytoplasmic trafficking and intranuclear/cytoplasmic mobility of the tumor suppressor Rb protein and a SV40 large tumor antigen (T-ag) derivative containing the nuclear localization sequence (NLS), both fused to green fluorescent protein (GFP). The results indicate that proteins carrying the T-ag NLS are highly mobile in the nucleus and cytoplasm. Rb, in contrast, is largely immobile in both cellular compartments, with similar nuclear import and export kinetics. Rb nuclear export was CRM-1-mediated, with its reduced mobility in the cytoplasm in part due to association with MTs. Overall our results show that nuclear and cytoplasm retention modulates the rates of nuclear protein import and export in intact cells.

Polymeric Precipitation Inhibitors Promote Fenofibrate Supersaturation and Enhance Drug Absorption from a Type IV Lipid-Based Formulation

Publisher: American Chemical Society (ACS)

Date: 16-04-2018

DOI: 10.1021/ACS.MOLPHARMACEUT.8B00206

Abstract: The ability of lipid-based formulations (LBFs) to increase the solubilization, and prolong the supersaturation, of poorly water-soluble drugs (PWSDs) in the gastrointestinal (GI) fluids has generated significant interest in the past decade. One mechanism to enhance the utility of LBFs is to prolong supersaturation via the addition of polymers that inhibit drug precipitation (polymeric precipitation inhibitors or PPIs) to the formulation. In this work, we have evaluated the performance of a range of PPIs and have identified PPIs that are sufficiently soluble in LBF to allow the construction of single phase formulations. An in vitro model was first employed to assess drug (fenofibrate) solubilization and supersaturation on LBF dispersion and digestion. An in vitro-in situ model was subsequently employed to simultaneously evaluate the impact of PPI enhanced drug supersaturation on drug absorption in rats. The stabilizing effect of the polymers was polymer specific and most pronounced at higher drug loads. Polymers that were soluble in LBF allowed simple processing as single phase formulations, while formulations containing more hydrophilic polymers required polymer suspension in the formulation. The lipid-soluble polymers Eudragit (EU) RL100 and poly(propylene glycol) bis(2-aminopropyl ether) (PPGAE) and the water-soluble polymer hydroxypropylmethyl cellulose (HPMC) E4M were identified as the most effective PPIs in delaying fenofibrate precipitation in vitro. An in vitro model of lipid digestion was subsequently coupled directly to an in situ single pass intestinal perfusion assay to evaluate the influence of PPIs on fenofibrate absorption from LBFs in vivo. This coupled model allowed for real-time evaluation of the impact of supersaturation stabilization on absorptive drug flux and provided better discrimination between the different PPIs and formulations. In the presence of the in situ absorption sink, increased fenofibrate supersaturation resulted in increased drug exposure, and a good correlation was found between the degree of in vitro supersaturation and in vivo drug exposure. An improved in vitro-in vivo correlation was apparent when comparing the same formulation under different supersaturation conditions. These observations directly exemplify the potential utility of PPIs in promoting drug absorption from LBF, via stabilization of supersaturation, and further confirm that relatively brief periods of supersaturation may be sufficient to promote drug absorption, at least for highly permeable drugs such as fenofibrate.

Delivery and Expression of mRNA in the Secondary Lymphoid Organs Drive Immune Responses to Lipid Nanoparticle-mRNA Vaccines after Intramuscular Injection

Publisher: American Chemical Society (ACS)

Date: 26-07-2023

DOI: 10.1021/ACS.MOLPHARMACEUT.2C01024

Transient Supersaturation Supports Drug Absorption from Lipid-Based Formulations for Short Periods of Time, but Ongoing Solubilization Is Required for Longer Absorption Periods

Publisher: American Chemical Society (ACS)

Date: 13-01-2017

DOI: 10.1021/ACS.MOLPHARMACEUT.6B00792

Abstract: The current studies sought to explore the impact of drug supersaturation and precipitation during the dispersion and digestion of lipid-based formulations (LBFs), on in vivo absorption using a coupled in vitro digestion-in vivo perfusion absorption model. Fenofibrate absorption was evaluated from a number of LBFs with different solubilization and supersaturation capacities, and conditions at the absorptive membrane manipulated by changing perfusion conditions, intestine segment lengths, and by the conduct of experiments in the presence or absence of suspended recipitated drug. LBF dispersion and digestion resulted in varying periods of supersaturation across the different formulations. Even fleeting (5-10 min) periods of supersaturation were able to drive flux across a perfused 10 cm intestinal segment for up to 60 min, although over longer infusion periods (60-80 min) flux dropped in the absence of ongoing drug solubilization and supersaturation. In contrast, the presence or absence of precipitated/suspended drug, had little impact on drug flux. When perfused intestinal segment lengths were extended, the role of initial supersaturation was attenuated and ongoing solubilization conditions became the primary driver of absorptive flux. The data suggest that for highly permeable drugs such as fenofibrate, a short period of supersaturation at the absorptive membrane may be sufficient to drive absorptive drug flux in spite of significant drug precipitation on formulation dispersion or digestion in vitro. In contrast, where longer periods of absorption are required, for ex le, at higher doses, the requirement for ongoing solubilization and supersaturation becomes more apparent.

SIRPA, VCAM1 and CD34 identify discrete lineages during early human cardiovascular development

Publisher: Elsevier BV

Date: 07-2014

DOI: 10.1016/J.SCR.2014.04.016

Abstract: The study of human cardiogenesis would benefit from a detailed cell lineage fate map akin to that established for the haematopoietic lineages. Here we sought to define cell lineage relationships based on the expression of NKX2-5 and the cell surface markers VCAM1, SIRPA and CD34 during human cardiovascular development. Expression of NKX2-5(GFP) was used to identify cardiac progenitors and cardiomyocytes generated during the differentiation of NKX2-5(GFP/w) human embryonic stem cells (hESCs). Cardiovascular cell lineages sub-fractionated on the basis of SIRPA, VCAM1 and CD34 expression were assayed for differentiation potential and gene expression. The NKX2-5(pos)CD34(pos) population gave rise to endothelial cells that rapidly lost NKX2-5 expression in culture. Conversely, NKX2-5 expression was maintained in myocardial committed cells, which progressed from being NKX2-5(pos)SIRPA(pos) to NKX2-5(pos)SIRPA(pos)VCAM1(pos). Up-regulation of VCAM1 was accompanied by the expression of myofilament markers and reduced clonal capacity, implying a restriction of cell fate potential. Combinatorial expression of NKX2-5, SIRPA, VCAM1 and CD34 can be used to define discrete stages of cardiovascular cell lineage differentiation. These markers identify specific stages of cardiomyocyte and endothelial lineage commitment and, thus provide a scaffold for establishing a fate map of early human cardiogenesis.

Interaction with biliary and pancreatic fluids drives supersaturation and drug absorption from lipid-based formulations of low (saquinavir) and high (fenofibrate) permeability poorly soluble drugs

Publisher: Elsevier BV

Date: 03-2021

DOI: 10.1016/J.JCONREL.2021.01.007

Design of lipid-based formulations for oral administration of poorly water-soluble drugs: precipitation of drug after dispersion of formulations in aqueous solution

Publisher: Elsevier BV

Date: 10-2009

DOI: 10.1002/JPS.21659

Endothelin-1 and angiotensin II modulate rate and contraction amplitude in a subpopulation of mouse embryonic stem cell-derived cardiomyocyte-containing bodies

Publisher: Elsevier BV

Date: 2011

DOI: 10.1016/J.SCR.2010.09.001

Abstract: Embryonic stem cell-derived cardiomyocytes (ESC-CMs) have applications in understanding cardiac disease pathophysiology, pharmacology, and toxicology. Comprehensive characterization of their basic physiological and pharmacological properties is critical in determining the suitability of ESC-CMs as models of cardiac activity. In this study we use video microscopy and quantitative PCR to investigate the responses of mouse ESC-CMs to adrenoceptor, muscarinic, angiotensin II (Ang II), and endothelin-1 (ET-1) receptor activation. Isoprenaline (10 nM-10 μM) increased beating rate and contraction litude in all beating bodies (BBs), whereas carbachol (up to 1 μM) and the I(f) channel blocker ZD-7288 (10 μM) decreased contraction frequency. ET-1 (0.01-100 nM) reduced contraction litude in all BBs and increased contraction frequency in 50% of BBs these effects were blocked by the ET(A) receptor antagonist BQ123 (250 nM). Ang II (0.01 nM-1 μM) increased both contraction litude (all BBs) and frequency (in 50% of BBs), effects blocked, respectively, by losartan (100 nM) and PD123,319 (200 nM). These results indicate the presence of functional ET(A) and both AT₁ and AT₂ receptors in murine ESC-CMs, but their expression and or activity appears to be evident only in a limited set of BBs.

Quantifying the Endosomal Escape of pH-Responsive Nanoparticles Using the Split Luciferase Endosomal Escape Quantification Assay

Publisher: American Chemical Society (ACS)

Date: 29-12-2021

DOI: 10.1021/ACSAMI.1C18359

Abstract: All nanoparticles have the potential to revolutionize the delivery of therapeutic cargo such as peptides, proteins, and RNA. However, effective cytosolic delivery of cargo from nanoparticles represents a significant challenge in the design of more efficient drug delivery vehicles. Recently, research has centered on designing nanoparticles with the capacity to escape endosomes by responding to biological stimuli such as changes in pH, which occur when nanoparticles are internalized into the endo-/lysosomal pathway. Current endosomal escape assays rely on indirect measurements and yield little quantitative information, which hinders the design of more efficient drug delivery vehicles. Therefore, we adapted the highly sensitive split luciferase endosomal escape quantification (SLEEQ) assay to better understand nanoparticle-induced endosomal escape. We applied SLEEQ to evaluate the endosomal escape behavior of two pH-responsive nanoparticles: the first with a poly(2-diisopropylamino ethyl methacrylate) (PDPAEMA) core and the second with 1:1 ratio of poly(2-diethylamino ethyl methacrylate) (PDEAEMA) and PDPAEMA. SLEEQ directly measured the cytosolic delivery and showed that engineering the nanoparticle disassembly pH could improve the endosomal escape efficiency by fivefold. SLEEQ is a versatile assay that can be used for a wide range of nanomaterials and will improve the development of drug delivery vehicles in the future.

Increasing the proportional content of surfactant (Cremophor EL) relative to lipid in self-emulsifying lipid-based formulations of danazol reduces oral bioavailability in beagle dogs

Publisher: Springer Science and Business Media LLC

Date: 15-02-2007

DOI: 10.1007/S11095-006-9194-Z

Abstract: To investigate the impact of a change in the proportions of lipid, surfactant and co-solvent on the solubilisation capacity of self-emulsifying formulations of danazol during in vitro dispersion and digestion studies and correlation with in vivo bioavailability in beagle dogs. Formulations from within the phase diagram of the pseudo-ternary system composed of soybean oil:maisine 35-1 (1:1 w/w), Cremophor EL and ethanol were assessed in vitro on dispersion and digestion. The relative bioavailability of danazol after administration of a series of these formulations was also determined. All formulations formed microemulsions in the presence of water and no drug precipitation was observed on dispersion. In contrast, drug solubilisation was markedly affected by lipase-mediated digestion and a reduction in lipid (and increase in surfactant) content resulted in increased drug precipitation. Consistent with these data, the bioavailability of danazol decreased significantly when the lipid content in the formulations was reduced. A rank-order correlation was observed between the patterns of solubilisation obtained during in vitro digestion and the in vivo performance of self-emulsifying formulations of danazol. In general a decrease in the lipid content and an increase in the proportions of surfactant and co-solvent resulted in reduced danazol bioavailability.

Specification of murine ground state pluripotent stem cells to regional neuronal populations

Publisher: Springer Science and Business Media LLC

Date: 22-11-2017

DOI: 10.1038/S41598-017-16248-X

Abstract: Pluripotent stem cells (PSCs) are a valuable tool for interrogating development, disease modelling, drug discovery and transplantation. Despite the burgeoned capability to fate restrict human PSCs to specific neural lineages, comparative protocols for mouse PSCs have not similarly advanced. Mouse protocols fail to recapitulate neural development, consequently yielding highly heterogeneous populations, yet mouse PSCs remain a valuable scientific tool as differentiation is rapid, cost effective and an extensive repertoire of transgenic lines provides an invaluable resource for understanding biology. Here we developed protocols for neural fate restriction of mouse PSCs, using knowledge of embryonic development and recent progress with human equivalents. These methodologies rely upon naïve ground-state PSCs temporarily transitioning through LIF-responsive stage prior to neural induction and rapid exposure to regional morphogens. Neural subtypes generated included those of the dorsal forebrain, ventral forebrain, ventral midbrain and hindbrain. This rapid specification, without feeder layers or embryoid-body formation, resulted in high proportions of correctly specified progenitors and neurons with robust reproducibility. These generated neural progenitors/neurons will provide a valuable resource to further understand development, as well disorders affecting specific neuronal subpopulations.

In vitro differentiation of pluripotent stem cells towards either forebrain GABAergic or midbrain dopaminergic neurons

Publisher: Wiley

Date: 03-12-2015

DOI: 10.1002/9781118308295.CH10

In vitro assessment of drug-free and fenofibrate-containing lipid formulations using dispersion and digestion testing gives detailed insights into the likely fate of formulations in the intestine

Publisher: Elsevier BV

Date: 07-2013

DOI: 10.1016/J.EJPS.2013.04.036

Abstract: The solubilizing properties of lipid-based formulations (LBFs) can change dramatically following dispersion and digestion of the formulation components. This study investigated the performance of self-emulsifying LBFs consisting of four different long-chain (LC)/medium-chain (MC) lipid blends formulated with the lipophilic drug fenofibrate and either a water-insoluble surfactant polysorbate 85 (Tween 85) or its more hydrophilic relative, polysorbate 80 (Tween 80). These components allowed closely related Type II and IIIA LBFs of fenofibrate to be evaluated during in vitro dispersion and in vitro digestion testing. Initial assessment of the solvent capacity of drug-free LBFs during dispersion and digestion revealed that the solubility of fenofibrate was more dependent on the surfactant type rather than lipid composition. Type II LBFs in the dispersed state were generally better at solubilizing fenofibrate than equivalent Type IIIA LBFs, regardless of lipid composition. However, even when high drug loadings were used, supersaturation/drug precipitation after dispersion of Type II or Type IIIA LBFs was only moderate. In contrast, digestion of both Type II and IIIA LBFs led to much higher levels of drug supersaturation, and this resulted in drug precipitation. After digestion the ability of each LBF to maintain drug in a solubilized state was highly dependent on lipid composition as well as the choice of surfactant. Notably, MC lipids exhibited very good solubilizing properties in the dispersed state, but resulted in a higher degree of supersaturation on digestion, leading to higher susceptibility to drug precipitation. This study showed that replacing LC lipids with MC lipids in Type II and IIIA LBF, in the proportions used here has little effect on fenofibrate solubilization during dispersion, but is likely to promote supersaturation on digestion. Without careful consideration of drug loading and choice of surfactant in Type II/IIIA MC lipid formulations, there is a high risk of precipitation of drug in the intestine.

Inhibition of β-catenin dependent WNT signalling upregulates the transcriptional repressor NR0B1 and downregulates markers of an A9 phenotype in human embryonic stem cell-derived dopaminergic neurons:

Publisher: Public Library of Science (PLoS)

Date: 23-12-2021

DOI: 10.1371/JOURNAL.PONE.0261730

Abstract: In this study we investigate how β-catenin-dependent WNT signalling impacts midbrain dopaminergic neuron (mDA) specification. mDA cultures at day 65 of differentiation responded to 25 days of the tankyrase inhibitor XAV969 (XAV, 100nM) with reduced expression of markers of an A9 mDA phenotype ( KCNJ6 , ALDH1A1 and TH ) but increased expression of the transcriptional repressors NR0B1 and NR0B2 . Overexpression of NR0B1 and or NR0B2 promoted a loss of A9 dopaminergic neuron phenotype markers ( KCNJ6 , ALDH1A1 and TH ). Overexpression of NR0B1 , but not NR0B2 promoted a reduction in expression of the β-catenin-dependent WNT signalling pathway activator RSPO2 . Analysis of Parkinson’s disease (PD) transcriptomic databases shows a profound PD-associated elevation of NR0B1 as well as reduced transcript for RSPO2 . We conclude that reduced β-catenin-dependent WNT signalling impacts dopaminergic neuron identity, in vitro , through increased expression of the transcriptional repressor, NR0B1 . We also speculate that dopaminergic neuron regulatory mechanisms may be perturbed in PD and that this may have an impact upon both existing nigral neurons and also neural progenitors transplanted as PD therapy.

Self-Crosslinking Lipopeptide/DNA/PEGylated Particles: A New Platform for DNA Vaccination Designed for Assembly in Aqueous Solution

Publisher: Elsevier BV

Date: 09-2018

DOI: 10.1016/J.OMTN.2018.05.025

Pharmaceutical applications of embryonic stem cells

Publisher: Elsevier BV

Date: 12-12-2005

DOI: 10.1016/J.ADDR.2005.08.003

Abstract: Embryonic stem cells (ESCs) will become a source of all adult differentiated cells once reliable protocols for directed differentiation have been established. This resource will revolutionise laboratory cell biology and will provide much improved cell culture models for discovery and development of drugs, and fundamental studies of the genetic basis of disease. These are early days, and there are few ex les of practical outcomes in the pharmaceutical world, though in recent years some drug-like molecules, which direct differentiation, have been discovered. At this stage the focus is on understanding the signalling systems and transcription factors that drive differentiation, on characterisation and isolation of precursor cells, and on establishing methods to improve the homogeneity of differentiated cells derived from ESCs. There is particular interest in establishing methods for deriving fully differentiated human cells from human ESCs (HESCs), but there are challenges to be met before HESC technology can be taken up in a widespread manner. Protocols for expansion of HESC cultures are labour-intensive at present. There is an urgent need for discovery of factors that will allow serum-free (and feeder cell-free) expansion of HESCs, and which can be applied to the majority of HESC lines. Our expectation is that adequate protocols will emerge in the coming years opening the way for development of many cell culture tools in the pharmaceutical industry. Here we review the current status and future prospects for this exciting field and encourage pharmaceutical scientists to play a role in directing its development.

Synthesis and Biological Evaluation of Adenosines with Heterobicyclic and Polycyclic N⁶‐Substituents as Adenosine A₁ Receptor Agonists

Publisher: Wiley

Date: 08-06-2012

DOI: 10.1002/CMDC.201200208

Abstract: A concise synthesis of a series of N(6)-substituted adenosines with bicyclo[3.2.1]octan-6-yl and polycyclic N(6)-substituents has been developed. The adenosine A(1) receptor (A(1)R) affinity and potency of these compounds was initially assessed using competitive binding assays and cyclic adenosine monophosphate (cAMP) accumulation assays in DDT(1) MF-2 cells. The potency and receptor subtype selectivity of selected ex les was further evaluated by measuring their effects on cAMP accumulation at all human adenosine receptor subtypes expressed in CHO cells. The results of these assays indicated that all of the synthesised N(6)-substituted adenosines are full agonists at A(1) R and activate this receptor selectively over the other adenosine receptor subtypes. The two standout compounds in terms of potency were N(6)-(3-thiabicyclo[3.2.1]octan-6-yl)adenosine and N(6)-(cubanylmethyl)adenosine with EC(50) values at human A(1)R of 2.3 nM and 1.1 nM, respectively. The cubanylmethyl derivative in particular proved to be highly receptor subtype selective. These two compounds were further evaluated in a simulated ischaemia model in cultured cardiomyoblasts, where they were found to impart protective effects under hypoxic conditions that resulted in a significant reduction in cell death.

Chronic stress in mice remodels lymph vasculature to promote tumour cell dissemination

Publisher: Springer Science and Business Media LLC

Date: 03-2016

DOI: 10.1038/NCOMMS10634

Abstract: Chronic stress induces signalling from the sympathetic nervous system (SNS) and drives cancer progression, although the pathways of tumour cell dissemination are unclear. Here we show that chronic stress restructures lymphatic networks within and around tumours to provide pathways for tumour cell escape. We show that VEGFC derived from tumour cells is required for stress to induce lymphatic remodelling and that this depends on COX2 inflammatory signalling from macrophages. Pharmacological inhibition of SNS signalling blocks the effect of chronic stress on lymphatic remodelling in vivo and reduces lymphatic metastasis in preclinical cancer models and in patients with breast cancer. These findings reveal unanticipated communication between stress-induced neural signalling and inflammation, which regulates tumour lymphatic architecture and lymphogenous tumour cell dissemination. These findings suggest that limiting the effects of SNS signalling to prevent tumour cell dissemination through lymphatic routes may provide a strategy to improve cancer outcomes.

Resolving subcellular pH with a quantitative fluorescent lifetime biosensor

Publisher: Cold Spring Harbor Laboratory

Date: 13-04-2022

DOI: 10.1101/2022.04.13.488146

Abstract: Changes in sub-cellular pH play a key role in metabolism, cell growth, membrane transport, and can also be exploited to control cargo release from therapeutic delivery systems. Most methods to measure pH rely on intensity changes of pH sensitive fluorophores, however these measurements are h ered by high uncertainty in the inferred pH and the need for multiple fluorophores. To address this, we have developed a method to accurately quantify sub-cellular pH in in idual vesicles using fluorescent lifetime imaging microscopy (pHLIM). pHLIM exploits the linear pH dependant lifetime of the fluorescent protein mApple and uses deep learning models to automatically identify and measure the pH of subcellular compartments. We have engineered mApple fusion proteins to measure the pH of the cytosol, endosomes, lysosomes and demonstrated the utility of pHLIM by measuring pH changes induced by drugs (bafilomycin A1) and polyethylenimine (a common transfection reagent). pHLIM is a simple and quantitative method to measure sub-cellular pH that has the potential to help with the design of the next generation of controlled drug release systems and to understand drug action and disease progression.

Evaluation of the impact of surfactant digestion on the bioavailability of danazol after oral administration of lipidic self-emulsifying formulations to dogs

Publisher: Elsevier BV

Date: 02-2008

DOI: 10.1002/JPS.21246

Acute or delayed systemic administration of human amnion epithelial cells improves outcomes in experimental stroke

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 03-2018

DOI: 10.1161/STROKEAHA.117.019136

Abstract: Human amnion epithelial cells (hAECs) are nonimmunogenic, nontumorigenic, anti-inflammatory cells normally discarded with placental tissue. We reasoned that their profile of biological features, wide availability, and the lack of ethical barriers to their use could make these cells useful as a therapy in ischemic stroke. We tested the efficacy of acute (1.5 hours) or delayed (1–3 days) poststroke intravenous injection of hAECs in 4 established animal models of cerebral ischemia. Animals included young (7–14 weeks) and aged mice (20–22 months) of both sexes, as well as adult marmosets of either sex. We found that hAECs administered 1.5 hours after stroke in mice migrated to the ischemic brain via a CXC chemokine receptor type 4-dependent mechanism and reduced brain inflammation, infarct development, and functional deficits. Furthermore, if hAECs administration was delayed until 1 or 3 days poststroke, long-term functional recovery was still augmented in young and aged mice of both sexes. We also showed proof-of-principle evidence in marmosets that acute intravenous injection of hAECs prevented infarct development from day 1 to day 10 after stroke. Systemic poststroke administration of hAECs elicits marked neuroprotection and facilitates mechanisms of repair and recovery.

8-(omega-aminoalkyl)theophyllines and their use in preparing fluorescently labeled derivatives for applications in immunoassay

Publisher: American Chemical Society (ACS)

Date: 07-1997

DOI: 10.1021/BC9700648

Glyceride lipid formulations: Molecular dynamics modeling of phase behavior during dispersion and molecular interactions between drugs and excipients

Publisher: Springer Science and Business Media LLC

Date: 03-10-2013

DOI: 10.1007/S11095-013-1206-1

Abstract: Little is known about the microstructure of lipid-based formulations, or how their structure changes as they disperse in the lumen of the gastrointestinal tract. We used molecular dynamics (MD) simulation to study such formulations at the molecular level as they interact with water during dispersion. We studied a simple lipid formulation, by itself and in the presence of drugs. The formulation contained mono- and di-lauroyl glycerides at 0-75% (w)/w water. Acyclovir, danazol, hydrocortisone, ketoprofen or progesterone, were included to investigate their dynamic behavior and localization during dispersion. Micro-structuring of the formulation was evident at all water concentrations. As the water content increased, the microstructure evolved from a continuous phase containing isolated water molecules, to a reverse micellar solution and finally to a system containing lamellar lipids with large pools of free water. Drugs partitioned into the aqueous and lipid domains principally under the influence of hydrogen bonding and hydrophobic interactions. Drugs located preferentially to the interfaces between water and lipid where they are able to make both hydrophobic and hydrophilic interactions. Molecular dynamics simulations offer an unprecedented view of the structure of lipid-based formulations and has considerable potential as an in silico tool for formulators.

Directed expression of gata2, mash1, and foxa2 synergize to induce the serotonergic neuron phenotype during in vitro differentiation of embryonic stem cells

Publisher: Oxford University Press (OUP)

Date: 23-05-2011

DOI: 10.1002/STEM.640

Abstract: Investigation of serotonergic neuronal activity and its relationship to disease has been limited by a lack of physiologically relevant in vitro cell models. Serotonergic neurons derived from embryonic stem cells (ESCs) could provide a platform for such studies and provide models for use in drug discovery. Here, we report enhancement of serotonergic differentiation using a genetic approach. Expression of Gata2 increased the yield of serotonergic neurons. Enhancement was only achieved when Gata2 was expressed under the control of the tissue-specific promoter of the transcription factor Nkx6.1. High levels of Gata2 expression in ESCs compromised pluripotency and induced non-neuronal differentiation. Combined directed expression of Gata2, proneural gene Mash1, and forkhead transcription factor Foxa2 further enhanced serotonergic neural differentiation, resulting in a 10-fold increase in serotonin content. These neurons were also capable of depolarization (KCl, 30 mM)-induced elevations of intracellular Ca2+. The presence of sonic hedgehog during differentiation produced a further modest increase in numbers (1.5-fold). Transgene expression did not influence the number of tyrosine hydroxylase positive neurons in the cultures after 20 days, implying that Gata2, Mash1, and Foxa2 modulate in vitro differentiation at a time beyond the decision-point for dopaminergic or nondopaminergic commitment. This study demonstrates that the directed expression of specific transcription factors enhances serotonergic neuron differentiation in vitro and highlights the importance of transgene expression at the right stage of ESC differentiation to effect the generation of a desired neural subtype.

Targeting Melanocortin Receptors Using S_NAr-Type Macrocyclization: A Doubly Orthogonal Route to Cyclic Peptide Conjugates

Publisher: American Chemical Society (ACS)

Date: 21-02-2023

DOI: 10.1021/ACS.JMEDCHEM.2C01587

Formulation of poorly water-soluble drugs for oral administration: physicochemical and physiological issues and the lipid formulation classification system

Publisher: Elsevier BV

Date: 11-2006

DOI: 10.1016/J.EJPS.2006.04.016

Abstract: Poorly water-soluble drug candidates often emerge from contemporary drug discovery programs, and present formulators with considerable technical challenges. The absorption of such compounds when presented in the crystalline state to the gastrointestinal tract is typically dissolution rate-limited, and the drugs are typically BCS class II or class IV compounds. Class IV compounds, which have low membrane permeability as well as poor aqueous solubility, are often poor candidates for development, unless the dose is expected to be low. The rate and extent of absorption of class II compounds is highly dependent on the performance of the formulated product. These drugs can be successfully formulated for oral administration, but care needs to be taken with formulation design to ensure consistent bioavailability. Essentially the options available involve either reduction of particle size (of crystalline drug) or formulation of the drug in solution, as an amorphous system or lipid formulation. The performance of amorphous or lipid formulations is dependent on their interaction with the contents of the gastrointestinal tract, therefore, a formulation exercise should involve the use of techniques which can predict the influence of gut physiology. A major consideration is the fate of metastable supersaturated solutions of drug, which are formed typically after dispersion of the formulation and its exposure to gastrointestinal digestion. A better understanding of the factors which affect drug crystallization is required, and the introduction of standardised predictive in vitro tests would be valuable. Although many bioavailability studies have been performed with poorly water-soluble drugs, thus far this research field has lacked a systematic approach. The use of a lipid formulation classification system combined with appropriate in vitro tests will help to establish a database for in vitro-in vivo correlation studies.

Enhancing the Oral Absorption of Kinase Inhibitors Using Lipophilic Salts and Lipid-Based Formulations

Publisher: American Chemical Society (ACS)

Date: 30-10-2018

DOI: 10.1021/ACS.MOLPHARMACEUT.8B00858

Abstract: The absolute bioavailability of many small molecule kinase inhibitors (smKIs) is low. The reasons for low bioavailability are multifaceted and include constraints due to first pass metabolism and poor absorption. For smKIs where absorption limits oral bioavailability, low aqueous solubility and high lipophilicity, often in combination with high-dose requirements have been implicated in low and variable absorption, food-effects, and absorption-related drug-drug interactions. The current study has evaluated whether preparation of smKIs as lipophilic salts/ionic liquids in combination with coadministration with lipid-based formulations is able to enhance absorption for ex les of this compound class. Lipophilic (docusate) salt forms of erlotinib, gefitinib, ceritinib, and cabozantinib (as ex le smKIs demonstrating low aqueous solubility and high lipophilicity) were prepared and isolated as workable powder solids. In each case, the lipophilic salt exhibited high and significantly enhanced solubility in lipidic excipients (>100 mg/g) when compared to the free base or commercial salt form. Isolation as the lipophilic salt facilitated smKI loading in model lipid-based formulations at high concentration, increased in vitro solubilization at gastric and intestinal pH and in some cases increased oral absorption (∼2-fold for cabozantinib formulations in rats). Application of a lipophilic salt approach can therefore facilitate the use of lipid-based formulations for ex les of the smKI compound class where low solubility limits absorption and is a risk factor for increased variability due to food-effects.

Dual acting antioxidant A1 adenosine receptor agonists

Publisher: Elsevier BV

Date: 10-2007

DOI: 10.1016/J.BMCL.2007.07.035

Abstract: Herein we report the synthesis and biological evaluation of some potent and selective A(1) adenosine receptor agonists, which incorporate a functionalised linker attached to an antioxidant moiety. N(6)-(2,2,5,5-Tetramethylpyrrolidin-1-yloxyl-3-ylmethyl)adenosine (VCP28, 2e) proved to be an agonist with high affinity (K(i)=50nM) and good selectivity (A(3)/A(1) > or = 400) for the A(1) adenosine receptor. N(6)-[4-[2-[1,1,3,3-Tetramethylisoindolin-2-yloxyl-5-amido]ethyl]phenyl]adenosine (VCP102, 5a) has higher binding affinity (K(i)=7 nM), but lower selectivity (A(3)/A(1)= approximately 3). All compounds bind weakly (K(i)>1 microM) to A(2A) and A(2B) receptors. The combination of A(1) agonist activity and antioxidant activity has the potential to produce cardioprotective effects.

Characterising the developmental profile of human embryonic stem cell-derived medium spiny neuron progenitors and assessing mature neuron function using a CRISPR-generated human DARPP-32WT/eGFP-AMP reporter line

Publisher: Elsevier BV

Date: 06-2017

DOI: 10.1016/J.NEUINT.2017.01.003

Abstract: In the developing ventral telencephalon, cells of the lateral ganglionic eminence (LGE) give rise to all medium spiny neurons (MSNs). This development occurs in response to a highly orchestrated series of morphogenetic stimuli that pattern the resultant neurons as they develop. Striatal MSNs are characterised by expression of dopamine receptors, dopamine-and cyclic AMP-regulated phosphoprotein (DARPP32) and the neurotransmitter GABA. In this study, we demonstrate that fine tuning Wnt and hedgehog (SHH) signaling early in human embryonic stem cell differentiation can induce a subpallial progenitor molecular profile. Stimulation of TGFβ signaling pathway by activin-A further supports patterning of progenitors to striatal precursors which adopt an LGE-specific gene signature. Moreover, we report that these MSNs also express markers associated with mature neuron function (cannabinoid, adenosine and dopamine receptors). To facilitate live-cell identification we generated a human embryonic stem cell line using CRISPR-mediated gene editing at the DARPP32 locus (DARPP32

The mRNA Vaccine Technology Era and the Future Control of Parasitic Infections

Publisher: American Society for Microbiology

Date: 23-03-2023

DOI: 10.1128/CMR.00241-21

Abstract: Despite intensive long-term efforts, with very few exceptions, the development of effective vaccines against parasitic infections has presented considerable challenges, given the complexity of parasite life cycles, the interplay between parasites and their hosts, and their capacity to escape the host immune system and to regulate host immune responses. For many parasitic diseases, conventional vaccine platforms have generally proven ill suited, considering the complex manufacturing processes involved and the costs they incur, the inability to posttranslationally modify cloned target antigens, and the absence of long-lasting protective immunity induced by these antigens.

Computational Models of the Gastrointestinal Environment. 2. Phase Behavior and Drug Solubilization Capacity of a Type I Lipid-Based Drug Formulation after Digestion

Publisher: American Chemical Society (ACS)

Date: 15-02-2017

DOI: 10.1021/ACS.MOLPHARMACEUT.6B00887

Abstract: Lipid-based drug formulations can greatly enhance the bioavailability of poorly water-soluble drugs. Following the oral administration of formulations containing tri- or diglycerides, the digestive processes occurring within the gastrointestinal (GI) tract hydrolyze the glycerides to mixtures of free fatty acids and monoglycerides that are, in turn, solubilized by bile. The behavior of drugs within the resulting colloidal mixtures is currently not well characterized. This work presents matched in vitro experimental and molecular dynamics (MD) theoretical models of the GI microenvironment containing a digested triglyceride-based (Type I) drug formulation. Both the experimental and theoretical models consist of molecular species representing bile (glycodeoxycholic acid), digested triglyceride (1:2 glyceryl-1-monooleate and oleic acid), and water. We have characterized the phase behavior of the physical system using nephelometry, dynamic light scattering, and polarizing light microscopy and compared these measurements to phase behavior observed in multiple MD simulations. Using this model microenvironment, we have investigated the dissolution of the poorly water-soluble drug danazol experimentally using LC-MS and theoretically by MD simulation. The results show how the formulation lipids alter the environment of the GI tract and improve the solubility of danazol. The MD simulations successfully reproduce the experimental results showing the utility of MD in modeling the fate of drugs after digestion of lipid-based formulations within the intestinal lumen.

Embryonic stem cells as a source of models for drug discovery

Publisher: Springer Science and Business Media LLC

Date: 08-2007

DOI: 10.1038/NRD2194

Abstract: Embryonic stem cells (ESCs) will become a source of models for a wide range of adult differentiated cells, providing that reliable protocols for directed differentiation can be established. Stem-cell technology has the potential to revolutionize drug discovery, making models available for primary screens, secondary pharmacology, safety pharmacology, metabolic profiling and toxicity evaluation. Models of differentiated cells that are derived from mouse ESCs are already in use in drug discovery, and are beginning to find uses in high-throughput screens. Before analogous human models can be obtained in adequate numbers, reliable methods for the expansion of human ESC cultures will be needed. For applications in drug discovery, involving either species, protocols for directed differentiation will need to be robust and affordable. Here, we explore current challenges and future opportunities in relation to the use of stem-cell technology in drug discovery, and address the use of both mouse and human models.

Computational Models of the Gastrointestinal Environment. 1. The Effect of Digestion on the Phase Behavior of Intestinal Fluids

Publisher: American Chemical Society (ACS)

Date: 15-02-2017

DOI: 10.1021/ACS.MOLPHARMACEUT.6B00888

Abstract: Improved models of the gastrointestinal environment have great potential to assist the complex process of drug formulation. Molecular dynamics (MD) is a powerful method for investigating phase behavior at a molecular level. In this study we use multiple MD simulations to calculate phase diagrams for bile before and after digestion. In these computational models, undigested bile is represented by mixtures of palmitoyl-oleoylphosphatidylcholine (POPC), sodium glycodeoxycholate (GDX), and water. Digested bile is modeled using a 1:1 mixture of oleic acid and palmitoylphosphatidylcholine (lysophosphatidylcholine, LPC), GDX, and water. The computational phase diagrams of undigested and digested bile are compared, and we describe the typical intermolecular interactions that occur between phospholipids and bile salts. The diffusion coefficients measured from MD simulation are compared to experimental diffusion data measured by DOSY-NMR, where we observe good qualitative agreement. In an additional set of simulations, the effect of different ionization states of oleic acid on micelle formation is investigated.

Toward the establishment of standardized in vitro tests for lipid-based formulations, part 6

Publisher: Springer Science and Business Media LLC

Date: 02-10-2014

DOI: 10.1208/S12248-014-9672-X

Preparation and in vitro evaluation of novel lipopeptide transfection agents for efficient gene delivery

Publisher: American Chemical Society (ACS)

Date: 12-03-2008

DOI: 10.1021/BC700463Q

Abstract: Gene therapy by delivery of nonviral expression vectors is highly desirable, due to their safety, stability, and suitability for production as bulk pharmaceuticals. However, low transfection efficiency remains a limiting factor in application on nonviral gene delivery. Despite recent advances in the field, there are still major obstacles to overcome. In an attempt to construct more efficient nonviral gene delivery vectors, we have designed a series of novel lipopeptide transfection agents, consisting of an alkyl chain, one cysteine, 1 to 4 histidine and 1 to 3 lysine residues. The lipopeptides were designed to facilitate dimerization (by way of the cysteine residues), DNA binding at neutral pH (making use of charged lysine residues), and endosomal escape (by way of weakly basic histidine residues). DNA/lipopeptide complexes were evaluated for their biophysical properties and transfection efficiencies. The number and identity of amino acids incorporated in the lipopeptide construct affected their DNA/lipopeptide complex forming capacity. As the number of lysine residues in the lipopeptide increased, the DNA complexes formed became more stable, had higher zeta potential (particle surface charge), and produced smaller mean particle sizes (typically 110 nm at a charge ratio of 5.0 and 240 nm at a charge ratio of 1.0). The effect of inclusion of histidines in the lipopeptide moiety had the opposite effect on complex formation to lysine, but was necessary for high transfection efficiency. In vitro transfection studies in COS-7 cells revealed that the efficiency of gene delivery of the luciferase encoding plasmid, pCMV-Luc, mediated by all the lipopeptides, was much higher than poly(L-lysine) (PLL), which has no endosomal escape system, and in two cases was slightly higher than that of branched polyethylenimine (PEI). Lipopeptides with at least two lysine residues and at least one histidine residue produced spontaneous transfection complexes with plasmid DNA, indicating that endosomal escape was achieved by incorporation of histidine residues. These low molecular weight peptides can be readily synthesized and purified and offer new insights into the mechanism of action of transfection complexes.

Toward the establishment of standardized in vitro tests for lipid-based formulations, part 4: Proposing a new lipid formulation performance classification system

Publisher: Elsevier BV

Date: 08-2014

DOI: 10.1002/JPS.24067

'Stealth' lipid-based formulations: Poly(ethylene glycol)-mediated digestion inhibition improves oral bioavailability of a model poorly water soluble drug

Publisher: Elsevier BV

Date: 10-2014

DOI: 10.1016/J.JCONREL.2014.07.037

Abstract: For over 20years, stealth drug delivery has been synonymous with nanoparticulate formulations and intravenous dosing. The putative determinants of stealth in these applications are the molecular weight and packing density of a hydrophilic polymer (commonly poly(ethylene glycol) (PEG)) that forms a steric barrier at the surface of the nanoparticle. The current study examined the potential translation of the concepts learned from stealth technology after intravenous administration to oral drug delivery and specifically, to enhance drug exposure after administration of oral lipid-based formulations (LBFs) containing medium-chain triglycerides (MCT). MCT LBFs are rapidly digested in the gastrointestinal tract, typically resulting in losses in solubilisation capacity, supersaturation and drug precipitation. Here, non-ionic surfactants containing stealth PEG headgroups were incorporated into MCT LBFs in an attempt to attenuate digestion, reduce precipitation risk and enhance drug exposure. Stealth capabilities were assessed by measuring the degree of digestion inhibition that resulted from steric hindrance of enzyme access to the oil-water interface. Drug-loaded LBFs were assessed for maintenance of solubilising capacity during in vitro digestion and evaluated in vivo in rats. The data suggest that the structural determinants of stealth LBFs mirror those of parenteral formulations, i.e., the key factors are the molecular weight of the PEG in the surfactant headgroup and the packing density of the PEG chains at the interface. Interestingly, the data also show that the presence of labile ester bonds within a PEGylated surfactant also impact on the stealth properties of LBFs, with digestible surfactants requiring a PEG Mw of ~1800g/mol and non-digestible ether-based surfactants ~800g/mol to shield the lipidic cargo. In vitro evaluation of drug solubilisation during digestion showed stealth LBFs maintained drug solubilisation at or above 80% of drug load and reduced supersaturation in comparison to digestible counterparts. This trend was also reflected in vivo, where the relative bioavailability of drug after administration in two stealth LBFs increased to 120% and 182% in comparison to analogous digestible (non-stealth) formulations. The results of the current study indicate that self-assembled "stealth" LBFs have potential as a novel means of improving LBF performance.

Human leukocyte antigen haplotype phasing by allele-specific enrichment with peptide nucleic acid probes.

Publisher: Wiley

Date: 19-01-2014

DOI: 10.1002/MGG3.65

A stably engineered, suicidal strain of listeria monocytogenes delivers protein and/or DNA to fully differentiated intestinal epithelial monolayers

Publisher: American Chemical Society (ACS)

Date: 26-05-2009

DOI: 10.1021/MP800153U

Abstract: We have generated a recombinant stable, suicidal Listeria monocytogenes strain (rsDelta2) capable of delivering antigens as protein or DNA into non iding intestinal epithelial cells. The rsDelta2 strain was generated by inserting a cell wall hydrolysin gene, "ply118" together with its associated holin gene from a Listeria-specific phage, into the attenuated L. monocytonegenes genome of strain Delta2. The hol118 ly118 gene was placed under the control of the Listeria promoter PactA, inducing bacteria to undergo autolysis in eukaryotic cells. The rsDelta2 strain had normal growth rate in rich bacterial growth medium, but its replication in eukaryotic cells was limited, and its autolysis was used to deliver its contents to the cytoplasm of eukaryotic cells. The delivery potential of rsDelta2 was explored using engineered shuttle vectors designed to facilitate expression of a transgene, either in rsDelta2 (driven by Phly) or in the mammalian cell (driven by P(CMV)), or both (using our engineered dual Listeria and mammalian expression vector, pDuLX). The luciferase reporter was used to demonstrate that pDuLX vector allowed delivery of both protein and DNA to iding Caco-2 human epithelial cells. As expected, non iding fully differentiated Caco-2 monolayers were resistant to transfection with Lipofectamine, which can be explained by lack of access to the cell nucleus. We demonstrated that when Caco-2 monolayers were treated with rsDelta2, the bacteria were able to deliver a significant quantity of luciferase protein. By implication the bacteria were also able to deliver DNA, but expression driven by the eukaryotic promoter in host Caco-2 cells was not observed. When the rsDelta2 strain was taken up by Caco-2 cells, there was little or no bacterial growth, whereas the control Delta2 strain was viable and grew by approximately three log cycles within the Caco-2 cells. A small mass of protein or DNA was delivered by the Delta2 strain perhaps because some bacteria died, but despite the level of growth the mass of protein delivered to iding Caco-2 cells by the Delta2 strain was considerably less than that delivered by the rsDelta2 strain. We concluded that the Listeria delivery system has prospects for oral vaccination using antigens synthesized by the bacterium itself.

Structure and dynamics of glyceride lipid formulations, with propylene glycol and water

Publisher: American Chemical Society (ACS)

Date: 23-03-2009

DOI: 10.1021/MP8001667

Abstract: We report on the aggregation and dynamic behavior of excipients in type I and surfactant-free lipid formulations containing water-soluble cosolvents. Specifically we have investigated the internal structure of mixed glyceride formulations, with and without propylene glycol, in the anhydrous state and during dilution into water. We performed molecular dynamics (MD) simulations using GROMACS 3.1.4 (www.gromacs.org) to investigate the aggregation structures and phase changes which would occur on dispersion and dilution of the product in the gastrointestinal tract. MD experiments on mixed glyceride lipid formulations, revealed that they form microstructural features even in the presence of trace amounts of water, typical of what would be anticipated in capsule formulations in practice. These formulations are typically thought of as homogeneous mixtures, a view which has prevailed to some extent because the chemical ersity (chain length and degree of saturation) in excipients derived from vegetable oils prevents analysis of their microstructure by spectroscopic techniques. Our MD simulations suggest that a considerable depth of structure exists in the formulations, and that drugs partition into the various domains under the influence of intermolecular interactions, often dominated in the presence of water by hydrogen bonding. The lipid formulations consist of distinct regions of hydrophobic and hydrophilic character, essentially exhibiting reverse micellar character under low-water-content conditions. MD modeling has great potential as a predictive tool, in particular to identify drugs which may be prone to precipitation on dilution or dispersion of the lipid formulation.

Viral Delivery of GDNF Promotes Functional Integration of Human Stem Cell Grafts in Parkinson's Disease.

Publisher: Elsevier BV

Date: 04-2020

DOI: 10.1016/J.STEM.2020.01.010

Tissue-specific Calibration of Real-time PCR Facilitates Absolute Quantification of Plasmid DNA in Biodistribution Studies

Publisher: Elsevier BV

Date: 2016

DOI: 10.1038/MTNA.2016.79

Investigations into the binding affinities of different human 5-HT 4 receptor splice variants

Publisher: S. Karger AG

Date: 2010

DOI: 10.1159/000280418

Abstract: This study examined whether the drug-receptor-binding sites of 5 selected human 5-HT sub /sub receptor splice variants [h5-HT sub (a) /sub , h5-HT sub (b) /sub , h5-HT sub (c) /sub , h5-HT sub (d) /sub and h5-HT sub (g) /sub ] display preferential affinities towards agonists. The agonists selected on the basis of chemical ersity and clinical relevance were: 5-HT sub /sub benzamides, renzapride, zacopride and prucalopride the benzimidazolones, DAU 6236 and BIMU 1 the aromatic ketone, RS67333, and the indole carbazimidamide tegaserod. The rank order of affinities ranging across the splice variants was: tegaserod (pK sub i /sub : 7.38–7.91) ≧ Y-36912 (pK sub i /sub : 7.03–7.85) = BIMU 1 (pK sub i /sub : 6.92–7.78) ≧ DAU 6236 (pK sub i /sub : 6.79–7.99) ≧ 5-HT (pK sub i /sub : 5.82–7.29) ≧ 5-MeOT (pK sub i /sub : 5.64–6.83) ≧ renzapride (pK sub i /sub : 4.85–5.56). We obtained affinity values for the 5-HT sub (b) /sub , (d) and (g) variants for RS67333 (pK sub i /sub : 7:48–8.29), prucalopride (pK sub i /sub : 6.86–7.37) and zacopride (pK sub i /sub : 5.88–7.0). These results indicate that the ligands interact with the same conserved site in each splice variant. Some splice variants have a higher affinity for certain agonists and the direction of selectivity followed a common trend of lowest affinity at the (d) variant. However, this trend was not evident in functional experiments. Our findings suggest that it may be possible to design splice variant selective ligands, which may be of relevance for experimental drugs but may be difficult to develop clinically.

Solubilisation behaviour of poorly water-soluble drugs during digestion of solid SMEDDS

Publisher: Elsevier BV

Date: 09-2018

DOI: 10.1016/J.EJPB.2018.07.006

Abstract: Lipid based-formulations can enhance the bioavailability of poorly water-soluble lipophilic drugs through enhanced solubilisation of drugs in the gastrointestinal (GI) tract during digestion. This study investigates the solubilisation behaviour of poorly water-soluble drugs upon digestion of solid self-microemulsifying drug delivery system (S-SMEDDS). The S-SMEDDS were prepared using two different core lipids, Gelucire® 44/14 (GEL) or glyceryl monooleate (GMO), and were loaded with two model drugs, fenofibrate (FEN) and cinnarizine (CINN). S-SMEDDS formulations were characterized using wide-angle X-ray scattering (WAXS) and Raman spectroscopy, and their structural behaviour and drug solubilisation behaviour were monitored using drug-related diffraction peaks during digestion under fasted and fed simulated intestinal conditions using time-resolved small and wide-angle X-ray scattering (SAXS/WAXS). The concentrations of FEN and CINN released into the aqueous phase (AP) during digestion were quantified using high-performance liquid chromatography (HPLC). Both model drugs, FEN and CINN, had greater solubility in the GMO-based S-SMEDDS formulations and were partially solubilised into lipid matrix and uniformly distributed in solid formulations. The extent of digestion was greater for the GEL-based systems (92-94%) than GMO-based systems (65-75%) as was the rate of digestion. GEL-based S-SMEDDS formulations formed a lamellar phase during digestion in the fasted state and formed mixed micelles in the fed state. In contrast, the GMO-based system formed the mixed micelles in both intestinal conditions. The time-resolved SAXS profiles revealed solubilisation of crystalline drugs into the lipolysis products. Synchrotron SAXS results were in correlation with the HPLC measurements, confirming the ability of the SAXS technique to monitor drug behaviour and showing that the digestion of S-SMEDDS can enhance drug solubilisation.

A new in vitro lipid digestion – in vivo absorption model to evaluate the mechanisms of drug absorption from lipid-based formulations

Publisher: Springer Science and Business Media LLC

Date: 24-12-2015

DOI: 10.1007/S11095-015-1843-7

Abstract: In vitro lipid digestion models are commonly used to screen lipid-based formulations (LBF), but in vitro-in vivo correlations are in some cases unsuccessful. Here we enhance the scope of the lipid digestion test by incorporating an absorption 'sink' into the experimental model. An in vitro model of lipid digestion was coupled directly to a single pass in situ intestinal perfusion experiment in an anaesthetised rat. The model allowed simultaneous real-time analysis of the digestion and absorption of LBFs of fenofibrate and was employed to evaluate the influence of formulation digestion, supersaturation and precipitation on drug absorption. Formulations containing higher quantities of co-solvent and surfactant resulted in higher supersaturation and more rapid drug precipitation in vitro when compared to those containing higher quantities of lipid. In contrast, when the same formulations were examined using the coupled in vitro lipid digestion - in vivo absorption model, drug flux into the mesenteric vein was similar regardless of in vitro formulation performance. For some drugs, simple in vitro lipid digestion models may underestimate the potential for absorption from LBFs. Consistent with recent in vivo studies, drug absorption for rapidly absorbed drugs such as fenofibrate may occur even when drug precipitation is apparent during in vitro digestion.

Lipid-Based Formulations and Drug Supersaturation: Harnessing the Unique Benefits of the Lipid Digestion/Absorption Pathway

Publisher: Springer Science and Business Media LLC

Date: 04-07-2013

DOI: 10.1007/S11095-013-1126-0

Abstract: Drugs with low aqueous solubility commonly show low and erratic absorption after oral administration. Myriad approaches have therefore been developed to promote drug solubilization in the gastrointestinal (GI) fluids. Here, we offer insight into the unique manner by which lipid-based formulations (LBFs) may enhance the absorption of poorly water-soluble drugs via co-stimulation of solubilization and supersaturation. Supersaturation provides an opportunity to generate drug concentrations in the GI tract that are in excess of the equilibrium crystalline solubility and therefore higher than that achievable with traditional formulations. Incorporation of LBF into lipid digestion and absorption pathways provides multiple drivers of supersaturation generation and the potential to enhance thermodynamic activity and absorption. These drivers include 1) formulation dispersion, 2) lipid digestion, 3) interaction with bile and 4) lipid absorption. However, high supersaturation ratios may also stimulate drug precipitation and reduce exposure where re-dissolution limits absorption. The most effective formulations are likely to be those that generate moderate supersaturation and do so close to the site of absorption. LBFs are particularly well suited to these criteria since solubilization protects against high supersaturation ratios, and supersaturation initiation typically occurs in the small intestine, at the absorptive membrane.

Cooperative Cardioprotection Through Adenosine A1 and A2A Receptor Agonism in Ischemia-Reperfused Isolated Mouse Heart

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 10-2010

DOI: 10.1097/FJC.0B013E3181F03D05

A rare SMAD9 mutation identifies the BMP signalling pathway as a potential osteoanabolic target

Publisher: Cold Spring Harbor Laboratory

Date: 25-02-2019

DOI: 10.1101/560565

Abstract: To identify targets for novel anabolic medicines for osteoporosis, we recruited a large cohort with unexplained high bone mass (HBM). Exome sequencing identified a rare (minor allele frequency 0.0014) missense mutation in SMAD9 (c.65T C, p.Leu22Pro) segregating with HBM in an autosomal dominant family. The same mutation was identified in another two unrelated in iduals with HBM. In-silico protein modelling predicts the mutation severely disrupts the MH1 DNA-binding domain of SMAD9. Affected in iduals have bone mineral density [BMD] Z-Scores +3 to +5, with increased volumetric cortical and trabecular BMD, increased cortical thickness, and low/normal bone turnover. Fractures and nerve compressions are not seen. Both genome-wide, and gene-based association testing of heel estimated-BMD in ,924 UK-Biobank British subjects showed strong associations with SMAD9 (P GWAS =6×10 −16 P GENE =8×10 −17 ). Smad9 is highly expressed in murine osteocytes and zebrafish bone tissue. Our findings support SMAD9 as a novel HBM gene, and a potential novel osteoanabolic target.

Compartmentalized microfluidic chambers enable long-term maintenance and communication between human pluripotent stem cell-derived forebrain and midbrain neurons

Publisher: Royal Society of Chemistry (RSC)

Date: 2021

DOI: 10.1039/D1LC00505G

Abstract: Open coculture microfluidic chambers embedding Matrigel for 3D culture of hPSCderived forebrain and midbrain neurons over 5 months with functional neural activity.

Ultrafast star-shaped acoustic micromixer for high throughput nanoparticle synthesis

Publisher: Royal Society of Chemistry (RSC)

Date: 2020

DOI: 10.1039/C9LC01174A

Abstract: Ultrafast acoustic micromixers can effectively homogenize fluids in 4 milliseconds, at 8 ml min −1 flowrates providing a 50-fold improvement in throughput.

If Human Brain Organoids Are the Answer to Understanding Dementia, What Are the Questions?

Publisher: SAGE Publications

Date: 13-04-2020

DOI: 10.1177/1073858420912404

Abstract: Because our beliefs regarding our in iduality, autonomy, and personhood are intimately bound up with our brains, there is a public fascination with cerebral organoids, the “mini-brain,” the “brain in a dish”. At the same time, the ethical issues around organoids are only now being explored. What are the prospects of using human cerebral organoids to better understand, treat, or prevent dementia? Will human organoids represent an improvement on the current, less-than-satisfactory, animal models? When considering these questions, two major issues arise. One is the general challenge associated with using any stem cell–generated preparation for in vitro modelling (challenges lified when using organoids compared with simpler cell culture systems). The other relates to complexities associated with defining and understanding what we mean by the term “dementia.” We discuss 10 puzzles, issues, and stumbling blocks to watch for in the quest to model “dementia in a dish.”

The synthesis and biological evaluation of multifunctionalised derivatives of noscapine as cytotoxic agents

Publisher: Wiley

Date: 11-12-2014

DOI: 10.1002/CMDC.201300395

Abstract: Noscapine, a phthalideisoquinoline alkaloid derived from Papaver somniferum, is a well-known antitussive drug that has a relatively safe in vitro toxicity profile. Noscapine is also known to possess weak anticancer efficacy, and since its discovery, efforts have been made to design derivatives with improved potency. Herein, the synthesis of a series of noscapine analogues, which have been modified in the 6', 9', 1 and 7-positions, is described. In a previous study, replacement of the naturally occurring N-methyl group in the 6'-position with an N-ethylaminocarbonyl was shown to promote cell-cycle arrest and cytotoxicity against three cancer cell lines. Here, this modification has been combined with other structural changes that have previously been shown to improve anticancer activity, namely halo substitution in the 9'-position, regioselective O-demethylation to reveal a free phenol in the 7-position, and reduction of the lactone to the corresponding cyclic ether in the 1-position. The incorporation of new aryl substituents in the 9'-position was also investigated. The study identified interesting new compounds able to induce G2/M cell-cycle arrest and that possess cytotoxic activity against the human prostate carcinoma cell line PC3, the human breast adenocarcinoma cell line MCF-7, and the human pancreatic epithelioid carcinoma cell line PANC-1. In particular, the ethyl urea cyclic ether noscapinoids and a compound containing a 6'-ethylaminocarbonyl along with 9'-chloro, 7-hydroxy and lactone moieties exhibited the most promising biological activities, with EC50 values in the low micromolar range against all three cancer cell lines, and these derivatives warrant further investigation.

Pluripotent stem cell-derived dopaminergic neurons as models of neurodegeneration

Publisher: Future Medicine Ltd

Date: 11-2013

DOI: 10.2217/FNL.13.50

Abstract: Researchers utilize a number of models of Parkinson’s disease ranging in complexity from immortalized cell lines to nonhuman primates. These models are used to investigate everything from the mechanisms underlying neurodegeneration, to drugs that may improve patient outcomes. Each model system has advantages and disadvantages, depending on their application. In this review, the authors assess the potential value of embryonic stem and induced-pluripotent stem cells as additions to the crowded Parkinson’s disease in vitro model landscape.

An in vitro digestion test that reflects rat intestinal conditions to probe the importance of formulation digestion vs first pass metabolism in danazol bioavailability from lipid based formulations

Publisher: American Chemical Society (ACS)

Date: 29-09-2014

DOI: 10.1021/MP500197B

Abstract: The impact of gastrointestinal (GI) processing and first pass metabolism on danazol oral bioavailability (BA) was evaluated after administration of self-emulsifying drug delivery systems (SEDDS) in the rat. Danazol absolute BA was determined following oral and intraduodenal (ID) administration of LFCS class IIIA medium chain (MC) formulations at high (SEDDSH-III) and low (SEDDSL-III) drug loading and a lipid free LFCS class IV formulation (SEDDS-IV). Experiments were conducted in the presence and absence of ABT (1-aminobenzotriazole) to evaluate the effect of first pass metabolism. A series of modified in vitro lipolysis tests were developed to better understand the in vivo processing of SEDDS in the rat. Danazol BA was low ( 45% after oral administration of SEDDS-III or SEDDS-IV. In contrast, previous studies in dogs suggest that danazol BA is less dependent on first pass metabolism and more sensitive to changes in formulation processing. In vitro digestion models based on likely rat GI conditions suggest less drug precipitation on formulation digestion when compared to equivalent dog models, consistent with the increases in in vivo exposure (fraction absorbed) seen here in ABT-pretreated rats.

Purification and characterization of adenovirus core protein VII: a histone-like protein that is critical for adenovirus core formation

Publisher: Microbiology Society

Date: 07-2017

DOI: 10.1099/JGV.0.000817

Abstract: Adenovirus protein VII is a highly cationic core protein that forms a nucleosome-like structure in the adenovirus core by condensing DNA in combination with protein V and mu. It has been proposed that protein VII could condense DNA in a manner analogous to mammalian histones. Due to the lack of an expression and purification protocol, the interactions between protein VII and DNA are poorly understood. In this study we describe methods for the purification of biologically active recombinant protein VII using an E. coli expression system. We expressed a cleavable fusion of protein VII with thioredoxin and established methods for purification of this fusion protein in denatured form. We describe an efficient method for resolving the cleavage products to obtain pure protein VII using hydroxyapatite column chromatography. Mass spectroscopy data confirmed its mass and purity to be 19.4 kDa and >98 %, respectively. Purified recombinant protein VII spontaneously condensed dsDNA to form particles, as shown by dye exclusion assay, electrophoretic mobility shift assay and nuclease protection assay. Additionally, an in vitro bioluminescence assay revealed that protein VII can be used to enhance the transfection of mammalian cells with lipofectamine/DNA complexes. The availability of recombinant protein VII will facilitate future studies of the structure of the adenovirus core. Improved understanding of the structure and function of protein VII will be valuable in elucidating the mechanism of adenoviral DNA condensation, defining the morphology of the adenovirus core and establishing the mechanism by which adenoviral DNA enters the nucleus.

PI3K Activation in Neural Stem Cells Drives Tumorigenesis which can be Ameliorated by Targeting the cAMP Response Element Binding (CREB) Protein.

Publisher: Oxford University Press (OUP)

Date: 30-04-2018

DOI: 10.1093/NEUONC/NOY068

A Novel Highly Selective Adenosine A1 Receptor Agonist VCP28 Reduces Ischemia Injury in a Cardiac Cell Line and Ischemia–Reperfusion Injury in Isolated Rat Hearts at Concentrations That Do Not Affect

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 09-2010

DOI: 10.1097/FJC.0B013E3181EB8563

Efficiently Specified Ventral Midbrain Dopamine Neurons from Human Pluripotent Stem Cells Under Xeno-Free Conditions Restore Motor Deficits in Parkinsonian Rodents

Publisher: Oxford University Press (OUP)

Date: 14-10-2016

DOI: 10.5966/SCTM.2016-0073

Abstract: Recent studies have shown evidence for the functional integration of human pluripotent stem cell (hPSC)-derived ventral midbrain dopamine (vmDA) neurons in animal models of Parkinson's disease. Although these cells present a sustainable alternative to fetal mesencephalic grafts, a number of hurdles require attention prior to clinical translation. These include the persistent use of xenogeneic reagents and challenges associated with scalability and storage of differentiated cells. In this study, we describe the first fully defined feeder- and xenogeneic-free protocol for the generation of vmDA neurons from hPSCs and utilize two novel reporter knock-in lines (LMX1A-eGFP and PITX3-eGFP) for in-depth in vitro and in vivo tracking. Across multiple embryonic and induced hPSC lines, this “next generation” protocol consistently increases both the yield and proportion of vmDA neural progenitors (OTX2/FOXA2/LMX1A) and neurons (FOXA2/TH/PITX3) that display classical vmDA metabolic and electrophysiological properties. We identify the mechanism underlying these improvements and demonstrate clinical applicability with the first report of scalability and cryopreservation of bona fide vmDA progenitors at a time amenable to transplantation. Finally, transplantation of xeno-free vmDA progenitors from LMX1A- and PITX3-eGFP reporter lines into Parkinsonian rodents demonstrates improved engraftment outcomes and restoration of motor deficits. These findings provide important and necessary advancements for the translation of hPSC-derived neurons into the clinic.

A microtubule-facilitated nuclear import pathway for cancer regulatory proteins

Publisher: Wiley

Date: 16-05-2007

DOI: 10.1111/J.1600-0854.2007.00564.X

Abstract: Nuclear protein import is dependent on specific targeting signals within cargo proteins recognized by importins (IMPs) that mediate translocation through the nuclear pore. Recent evidence, however, implicates a role for the microtubule (MT) network in facilitating nuclear import of the cancer regulatory proteins parathyroid hormone-related protein (PTHrP) and p53 tumor suppressor. Here we assess the extent to which MT and actin integrity may be generally required for nuclear protein import for the first time. We examine 10 nuclear-localizing proteins with erse IMP-dependent nuclear import pathways, our results indicating that the cytoskeleton does not have a general mechanistic role in nuclear localization sequence-dependent nuclear protein import. Of the proteins examined, only the p110(Rb) tumor suppressor protein Rb, together with p53 and PTHrP, was found to require MT integrity for optimal nuclear import. Fluorescence recovery after photobleaching experiments indicated that the MT-dependent nuclear transport pathway increases both the rate and extent of Rb nuclear import but does not affect Rb nuclear export. Dynamitin overexpression experiments implicate the MT motor dynein in the import process. The results indicate that, additional to IMP/diffusion-dependent processes, certain cancer regulatory proteins utilize an MT-enhanced pathway for accelerated nuclear import that is presumably required for their nuclear functions.

Solubilisation partial characterisation of the alpha-MSH receptor on primary rat Schwann cells

Publisher: Wiley

Date: 20-12-1993

DOI: 10.1016/0014-5793(93)81619-B

Abstract: The ACTH/MSH melanocortin core peptide sequence possesses neurotrophic properties in peripheral nerve. During functional neuroanatomical recovery after damage to peripheral nerves, Schwann cells play a significant role in facilitating regeneration. Here we employ a modified super-potent alpha-MSH analogue to solubilise alpha-MSH receptor proteins from cultured primary rat Schwann cells. [125I-Tyr2,Nle4,D-Phe7,ATB-Lys11]-alpha-MSH photoaffinity labelled proteins from Schwann cells were analyzed by SDS-PAGE followed by autoradiography. The results indicate that the alpha-MSH receptor proteins labelled have a molecular weight of 42-45 kDa. These data are the first to demonstrate solubilisation and characterisation of alpha-MSH receptors from non-melanoma cells.

TGFβ3, dibutyryl cAMP and a notch inhibitor modulate phenotype late in stem cell-derived dopaminergic neuron maturation.

Publisher: Frontiers Media SA

Date: 02-2023

DOI: 10.3389/FCELL.2023.1111705

Abstract: The generation of midbrain dopaminergic neurons (mDAs) from pluripotent stem cells (hPSC) holds much promise for both disease modelling studies and as a cell therapy for Parkinson’s disease (PD). Generally, dopaminergic neuron differentiation paradigms rely on inhibition of smad signalling for neural induction followed by hedgehog signalling and an elevation of β-catenin to drive dopaminergic differentiation. Post-patterning, differentiating dopaminergic neuron cultures are permitted time for maturation after which the success of these differentiation paradigms is usually defined by expression of tyrosine hydroxylase (TH), the rate limiting enzyme in the synthesis of dopamine. However, during maturation, culture media is often supplemented with additives to promote neuron survival and or promote cell differentiation. These additives include dibutyryl cyclic adenosine monophosphate (dbcAMP), transforming growth factor β3 (TGFβ3) and or the γ-secretase inhibitor (DAPT). While these factors are routinely added to cultures, their impact upon pluripotent stem cell-derived mDA phenotype is largely unclear. In this study, we differentiate pluripotent stem cells toward a dopaminergic phenotype and investigate how the omission of dbcAMP, TGFβ3 or DAPT, late in maturation, affects the regulation of multiple dopaminergic neuron phenotype markers. We now show that the removal of dbcAMP or TGFβ3 significantly and distinctly impacts multiple markers of the mDA phenotype ( FOXA2, EN1, EN2, FOXA2, SOX6 ), while commonly increasing both MSX2 and NEUROD1 and reducing expression of both tyrosine hydroxylase and WNT5A . Removing DAPT significantly impacted MSX2, OTX2, EN1, and KCNJ6. In the absence of any stressful stimuli, we suggest that these culture additives should be viewed as mDA phenotype-modifying, rather than neuroprotective. We also suggest that their addition to cultures is likely to confound the interpretation of both transplantation and disease modelling studies.

Mouse and human microglial phenotypes in Alzheimer’s disease are controlled by amyloid plaque phagocytosis through Hif1α

Publisher: Cold Spring Harbor Laboratory

Date: 17-05-2019

DOI: 10.1101/639054

Abstract: The important role of microglia, the brain’s resident immune cells, in Alzheimer’s disease (AD) is now well recognized, however their molecular and functional ersity and underlying mechanisms still remain controversial. To transcriptionally and functionally characterize the ersity of microglia in AD and aging, we isolated the amyloid plaque-containing (XO4 + ) and non-containing (XO4 − ) microglia from an AD mouse model. Transcriptomics analysis unveiled independent transcriptional trajectories in ageing and AD. XO4 + microglial transcriptomes linked plaque phagocytosis to altered expression of bona fide late onset AD genetic risk factors. We further revealed that the XO4 + transcriptional program is present in a subset of human microglia from AD patients and is a direct and reversible consequence of Aβ plaque phagocytosis. Conversely, XO4 − microglia in AD displayed an accelerated ageing signature and contained more intracellular post synaptic material than plaque-containing microglia, despite reduced active synaptosome phagocytosis. Mechanistically, we predicted HIF1α as a core regulator of the XO4 − /XO4 + axis, and further validated the mechanism in vitro using human stem cell-derived microglia like cells and primary human microglia. Together these findings unveiled the molecular mechanism underpinning the functional ersity of microglia in AD, providing opportunities to develop treatments targeted at subset specific manipulation of the microglial niche.

Lipophilic Salts and Lipid-Based Formulations: Enhancing the Oral Delivery of Octreotide

Publisher: Springer Science and Business Media LLC

Date: 06-2021

DOI: 10.1007/S11095-021-03063-3

Abstract: Successful oral peptide delivery faces two major hurdles: low enzymatic stability in the gastro-intestinal lumen and poor intestinal membrane permeability. While lipid-based formulations (LBF) have the potential to overcome these barriers, effective formulation of peptides remains challenging. Lipophilic salt (LS) technology can increase the apparent lipophilicity of peptides, making them more suitable for LBF. As a model therapeutic peptide, octreotide (OCT) was converted to the docusate LS (OCT.DoS LS formulation improved the solubility and loading of OCT in LBF excipients and OCT.DoS The combination of LS and LBF enhanced the in vivo oral absorption of OCT primarily via the protective effect of LBF sheltering the peptide from gastrointestinal degradation.

NKX2-5eGFP/w hESCs for isolation of human cardiac progenitors and cardiomyocytes

Publisher: Springer Science and Business Media LLC

Date: 23-10-2011

DOI: 10.1038/NMETH.1740

Abstract: NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs) NKX2-5(eGFP/w) hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP(+) cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages.

In vitro digestion testing of lipid-based delivery systems: Calcium ions combine with fatty acids liberated from triglyceride rich lipid solutions to form soaps and reduce the solubilization capacity of colloidal digestion products

Publisher: Elsevier BV

Date: 2012

DOI: 10.1016/J.IJPHARM.2012.11.024

Abstract: In vitro digestion testing is of practical importance to predict the fate of drugs administered in lipid-based delivery systems. Calcium ions are often added to digestion media to increase the extent of digestion of long-chain triglycerides (LCTs), but the effects they have on phase behaviour of the products of digestion, and consequent drug solubilization, are not well understood. This study investigates the effect of calcium and bile salt concentrations on the rate and extent of in vitro digestion of soybean oil, as well as the solubilizing capacity of the digestion products for two poorly water-soluble drugs, fenofibrate and danazol. In the presence of higher concentrations of calcium ions, the solubilization capacities of the digests were reduced for both drugs. This effect is attributed to the formation of insoluble calcium soaps, visible as precipitates during the digestions. This reduces the availability of liberated fatty acids to form mixed micelles and vesicles, thereby reducing drug solubilization. The use of high calcium concentrations does indeed force in vitro digestion of LCTs but may overestimate the extent of drug precipitation that occurs within the intestinal lumen.

Human longevity is associated with regular sleep patterns, maintenance of slow wave sleep, and favorable lipid profile

Publisher: Frontiers Media SA

Date: 24-06-2014

DOI: 10.3389/FNAGI.2014.00134

Colloidal aspects of dispersion and digestion of self-dispersing lipid-based formulations for poorly water-soluble drugs

Publisher: Elsevier BV

Date: 03-2019

DOI: 10.1016/J.ADDR.2019.01.008

Abstract: Self-dispersing lipid-based formulations, particularly self-microemulsifying drug delivery systems (SMEDDS) have gained an increased interest in recent times as a means to enhance the oral bioavailability of poorly water-soluble lipophilic drugs. Upon dilution, SMEDDS self-emulsify in an aqueous fluid and usually form a kinetically stable oil-in-water emulsion or in some rare cases a true thermodynamically stable microemulsion. The digestion of the formulation leads to the production of hiphilic digestion products that interact with endogenous hiphilic components and form self-assembled colloidal phases in the aqueous environment of the intestine. The formed colloidal phases play a pivotal role in maintaining the lipophilic drug in the solubilised state during gastrointestinal transit prior to absorption. Thus, this review describes the structural characterisation techniques employed for SMEDDS and the recent literature studies that elucidated the colloidal aspects during dispersion and digestion of SMEDDS and solid SMEDDS. Possible future studies are proposed to gain better understanding on the colloidal aspects of SMEDDS and solid SMEDDS.

Resolving subcellular pH with a quantitative fluorescent lifetime biosensor

Publisher: Springer Science and Business Media LLC

Date: 12-10-2022

DOI: 10.1038/S41467-022-33348-Z

Abstract: Changes in sub-cellular pH play a key role in metabolism, membrane transport, and triggering cargo release from therapeutic delivery systems. Most methods to measure pH rely on intensity changes of pH sensitive fluorophores, however, these measurements are h ered by high uncertainty in the inferred pH and the need for multiple fluorophores. To address this, here we combine pH dependant fluorescent lifetime imaging microscopy (pHLIM) with deep learning to accurately quantify sub-cellular pH in in idual vesicles. We engineer the pH sensitive protein mApple to localise in the cytosol, endosomes, and lysosomes, and demonstrate that pHLIM can rapidly detect pH changes induced by drugs such as bafilomycin A1 and chloroquine. We also demonstrate that polyethylenimine (a common transfection reagent) does not exhibit a proton sponge effect and had no measurable impact on the pH of endocytic vesicles. pHLIM is a simple and quantitative method that will help to understand drug action and disease progression.

Unravelling cytosolic delivery of cell penetrating peptides with a quantitative endosomal escape assay

Publisher: Springer Science and Business Media LLC

Date: 17-06-2021

DOI: 10.1038/S41467-021-23997-X

Abstract: Cytosolic transport is an essential requirement but a major obstacle to efficient delivery of therapeutic peptides, proteins and nucleic acids. Current understanding of cytosolic delivery mechanisms remains limited due to a significant number of conflicting reports, which are compounded by low sensitivity and indirect assays. To resolve this, we develop a highly sensitive Split Luciferase Endosomal Escape Quantification (SLEEQ) assay to probe mechanisms of cytosolic delivery. We apply SLEEQ to evaluate the cytosolic delivery of a range of widely studied cell-penetrating peptides (CPPs) fused to a model protein. We demonstrate that positively charged CPPs enhance cytosolic delivery as a result of increased non-specific cell membrane association, rather than increased endosomal escape efficiency. These findings transform our current understanding of how CPPs increase cytosolic delivery. SLEEQ is a powerful tool that addresses fundamental questions in intracellular drug delivery and will significantly improve the way materials are engineered to increase therapeutic delivery to the cytosol.

Cyclosporin Structure and Permeability: From A to Z and Beyond

Publisher: American Chemical Society (ACS)

Date: 03-09-2021

DOI: 10.1021/ACS.JMEDCHEM.1C00580

Abstract: Cyclosporins are natural or synthetic undecapeptides with a wide range of actual and potential pharmaceutical applications. Several members of the cyclosporin compound family have remarkably high passive membrane permeabilities that are not well-described by simple structural metrics. Here we review experimental studies of cyclosporin structure and permeability, including cyclosporin-metal complexes. We also discuss models for the conformation-dependent permeability of cyclosporins and similar compounds. Finally, we identify current knowledge gaps in the literature and provide recommendations regarding future avenues of exploration.

Cardioprotection Induced by Adenosine A1 Receptor Agonists in a Cardiac Cell Ischemia Model Involves Cooperative Activation of Adenosine A2A and A2B Receptors by Endogenous Adenosine

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 05-2009

DOI: 10.1097/FJC.0B013E3181A443E2

Assessing the cellular toxicity of peptide inhibitors of intracellular protein-protein interactions by microinjection

Publisher: Elsevier BV

Date: 2021

DOI: 10.1016/J.BMC.2020.115906

Toward the establishment of standardized in vitro tests for lipid-based formulations. 2. The effect of bile salt concentration and drug loading on the performance of type I, II, IIIA, IIIB, and IV formulations during in vitro digestion

Publisher: American Chemical Society (ACS)

Date: 22-10-2012

DOI: 10.1021/MP300331Z

Abstract: The LFCS Consortium was established to develop standardized in vitro tests for lipid-based formulations (LBFs) and to examine the utility of these tests to probe the fundamental mechanisms that underlie LBF performance. In this publication, the impact of bile salt (sodium taurodeoxycholate, NaTDC) concentration and drug loading on the ability of a range of representative LBFs to generate and sustain drug solubilization and supersaturation during in vitro digestion testing has been explored and a common driver of the potential for drug precipitation identified. Danazol was used as a model poorly water-soluble drug throughout. In general, increasing NaTDC concentrations increased the digestion of the most lipophilic LBFs and promoted lipid (and drug) trafficking from poorly dispersed oil phases to the aqueous colloidal phase (AP(DIGEST)). High NaTDC concentrations showed some capacity to reduce drug precipitation, although, at NaTDC concentrations ≥3 mM, NaTDC effects on either digestion or drug solubilization were modest. In contrast, increasing drug load had a marked impact on drug solubilization. For LBFs containing long-chain lipids, drug precipitation was limited even at drug loads approaching saturation in the formulation and concentrations of solubilized drug in AP(DIGEST) increased with increased drug load. For LBFs containing medium-chain lipids, however, significant precipitation was evident, especially at higher drug loads. Across all formulations a remarkably consistent trend emerged such that the likelihood of precipitation was almost entirely dependent on the maximum supersaturation ratio (SR(M)) attained on initiation of digestion. SR(M) defines the supersaturation "pressure" in the system and is calculated from the maximum attainable concentration in the AP(DIGEST) (assuming zero precipitation), ided by the solubility of the drug in the colloidal phases formed post digestion. For LBFs where phase separation of oil phases did not occur, a threshold value for SR(M) was evident, regardless of formulation composition and drug solubilization reduced markedly above SR(M) > 2.5. The threshold SR(M) may prove to be an effective tool in discriminating between LBFs based on performance.

Strategies to Address Low Drug Solubility in Discovery and Development

Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)

Date: 2013

DOI: 10.1124/PR.112.005660

Abstract: Drugs with low water solubility are predisposed to low and variable oral bioavailability and, therefore, to variability in clinical response. Despite significant efforts to "design in" acceptable developability properties (including aqueous solubility) during lead optimization, approximately 40% of currently marketed compounds and most current drug development candidates remain poorly water-soluble. The fact that so many drug candidates of this type are advanced into development and clinical assessment is testament to an increasingly sophisticated understanding of the approaches that can be taken to promote apparent solubility in the gastrointestinal tract and to support drug exposure after oral administration. Here we provide a detailed commentary on the major challenges to the progression of a poorly water-soluble lead or development candidate and review the approaches and strategies that can be taken to facilitate compound progression. In particular, we address the fundamental principles that underpin the use of strategies, including pH adjustment and salt-form selection, polymorphs, cocrystals, cosolvents, surfactants, cyclodextrins, particle size reduction, amorphous solid dispersions, and lipid-based formulations. In each case, the theoretical basis for utility is described along with a detailed review of recent advances in the field. The article provides an integrated and contemporary discussion of current approaches to solubility and dissolution enhancement but has been deliberately structured as a series of stand-alone sections to allow also directed access to a specific technology (e.g., solid dispersions, lipid-based formulations, or salt forms) where required.

Local inflammation alters the lung disposition of a drug loaded pegylated liposome after pulmonary dosing to rats

Publisher: Elsevier BV

Date: 08-2019

DOI: 10.1016/J.JCONREL.2019.05.043

Abstract: The development of inhalable 'nanomedicines' based on biocompatible lipids and polymers is attracting increasing interest worldwide. Our understanding of how pulmonary inflammation impacts on lung distribution and clearance kinetics however, is limited. Similarly, there is limited information on how the inhaled delivery of biocompatible nanomaterials affects existing respiratory disease. We have addressed these knowledge gaps by describing and comparing the pulmonary pharmacokinetic behaviour of a

Unravelling cytosolic delivery of endosomal escape peptides with a quantitative endosomal escape assay (SLEEQ)

Publisher: Cold Spring Harbor Laboratory

Date: 20-08-2020

DOI: 10.1101/2020.08.20.258350

Abstract: Endosomal escape is an essential requirement but a major obstacle to efficient delivery of therapeutic peptides, proteins and nucleic acids. Current understanding of endosomal escape mechanisms remains limited due to significant number of conflicting reports, which are compounded by low sensitivity and indirect assays. To resolve this, we developed a highly sensitive Split Luciferase Endosomal Escape Quantification (SLEEQ) assay to probe mechanisms of cytosolic delivery. We applied SLEEQ to evaluate the endosomal escape of a range of widely studied putative endosomal escape peptides (EEPs). We demonstrated that positively-charged EEPs enhanced cytosolic delivery as a result of increased non-specific cell membrane association, rather than increased endosomal escape efficiency. These findings transform our current understanding of how EEPs increase cytosolic delivery. SLEEQ is a powerful tool that addresses fundamental questions in intracellular drug delivery and will significantly improve the way materials are engineered to increase therapeutic delivery to the cytosol.

Tetraspanins in viral infections: a fundamental role in viral biology?

Publisher: American Society for Microbiology

Date: 09-2005

DOI: 10.1128/JVI.79.17.10839-10851.2005

Extended periods of neural induction and propagation of embryonic stem cell-derived neural progenitors with EGF and FGF2 enhances Lmx1a expression and neurogenic potential

Publisher: Elsevier BV

Date: 09-2011

DOI: 10.1016/J.NEUINT.2011.04.002

Abstract: Neural stem (NS) cells are multipotent cells defined by their capacity to proliferate and differentiate into all neuronal and glial phenotypes. NS cells can be obtained from specific regions of the adult brain, or generated from embryonic stem cells (ESCs). NS cells differentiate into neural progenitor (NP) cells and subsequently neural precursors, as transient steps towards terminal differentiation into specific mature neuronal or glial phenotypes. When cultured in EGF and FGF2, ESC-derived NS cells have been reported to be stable and multipotent. Conditions that enable differentiation of NS cells through the committed progenitor and precursor stages to specific neuronal subtypes have not been fully established. In this study we investigated, using Lmx1a reporter ESCs, whether the length of neural induction (NI) dictated the phenotypic potential of cultures of ESC-derived NS cells or NP cells. Following 4, 7 or 10 day periods of NI, ESCs in monolayer culture were harvested and cultured as neurospheres, prior to replating as monolayer cultures for several passages in EGF and FGF2. The NS/NP cultures were then directed towards mature neuronal fates over 16-17 days. 4 and 7-day NS cell cultures could not be differentiated towards dopaminergic, serotonergic or cholinergic fates as determined by the absence of tyrosine hydroxylase, 5-HT or choline acetyltransferase (ChAT) immunolabelling. In contrast NS/NP cultures derived after 10 days of NI were able to generate tyrosine hydroxylase and 5-HT positive neurons (24±6 and 13±1% of the βIII-tubulin positive population, respectively, n=3). Our data suggest that extended periods of neural induction enhanced the potential of mouse ESC-derived NS/NP cells to generate specific subtypes of neurons. NS/NP cells derived after shorter periods of NI appeared to be lineage-restricted in relation to the neuronal subtypes observed after removal of EGF.

Identification of a melanocortin receptor expressed by murine brain microvascular endothelial cells in culture

Publisher: Elsevier BV

Date: 07-1995

DOI: 10.1006/MVRE.1995.1035

Abstract: Microvascular endothelial cells were isolated from the brains of C57 mice and cultured in selective growth media. The isolation and culture techniques employed in this study minimised the contamination by nonendothelial cells such as astrocytes, pericytes, and smooth muscle cells. Microvascular endothelial cells examined using phase contrast light microscopy grew as small colonies of spindle-shaped cells which merged together to form typical contact-inhibited monolayers. The endothelial origin of these cells was determined using several established characterisation techniques. Preliminary receptor binding studies at 4 degrees using [125I-Tyr2, Nle4, D-Phe7]alpha-melanocyte-stimulating hormone ([125I-Tyr2, Nle4, D-Phe7]alpha-MSH) suggested the possibility that melanocortin receptors were present on the surface of brain microvascular endothelial cells. Subsequent binding isotherms confirmed that a small population of high-affinity melanocortin receptors was expressed. The existence of a specific binding site for alpha-MSH was confirmed by photoaffinity labeling with the 4-(1-azi-2,2,2,-trifluoroethyl)benzoic acid (ATB) derivative, [125I-Tyr2, Nle4, D-Phe7, (ATB)-Lys11] alpha-MSH. SDS-PAGE analysis identified the presence of a specific band with a molecular mass of approximately 45 kDa, which was consistent with previous data on melanoma melanocortin receptors, and represented a ligand-receptor complex. This study suggests that a receptor for alpha-MSH is expressed on the extracellular surface of murine brain microvascular endothelial cells however, the physiological role of this receptor is as yet unknown.

Advances in targeted delivery

Publisher: Elsevier BV

Date: 12-2001

DOI: 10.1016/S0169-409X(01)00235-6

Stem Cell Technology

Publisher: Springer New York

Date: 2013

DOI: 10.1007/978-1-4614-6486-0_25

Broad immunity to SARS-CoV-2 variants of concern mediated by a SARS-CoV-2 receptor-binding domain protein vaccine

Publisher: Elsevier BV

Date: 06-2022

DOI: 10.1016/J.EBIOM.2023.104574

Computational and Experimental Models of Type III Lipid-Based Formulations of Loratadine Containing Complex Nonionic Surfactants

Publisher: American Chemical Society (ACS)

Date: 22-11-2021

DOI: 10.1021/ACS.MOLPHARMACEUT.1C00547

50 years of oral lipid-based formulations: Provenance, progress and future perspectives

Publisher: Elsevier BV

Date: 06-2016

DOI: 10.1016/J.ADDR.2016.04.007

Abstract: Lipid based formulations (LBF) provide well proven opportunities to enhance the oral absorption of drugs and drug candidates that sit close to, or beyond, the boundaries of Lipinski's 'rule-of-five' chemical space. Advantages in permeability, efflux and presystemic metabolism are evident however, the primary benefit is in increases in dissolution and apparent intestinal solubility for lipophilic, poorly water soluble drugs. This review firstly details the inherent advantages of LBF, their general properties and classification, and provides a brief retrospective assessment of the development of LBF over the past fifty years. More detailed analysis of the ability of LBF to promote intestinal solubilisation, supersaturation and absorption is then provided alongside review of the methods employed to assess formulation performance. Critical review of the ability of simple dispersion and more complex in vitro digestion methods to predict formulation performance subsequently reveals marked differences in the correlative ability of in vitro tests, depending on the properties of the drug involved. Notably, for highly permeable low melting drugs e.g. fenofibrate, LBF appear to provide significant benefit in all cases, and sustained ongoing solubilisation may not be required. In other cases, and particularly for higher melting point drugs such as danazol, where re-dissolution of crystalline precipitate drug is likely to be slow, correlations with ongoing solubilisation and supersaturation are more evident. In spite of their potential benefits, one limitation to broader use of LBF is low drug solubility in the excipients employed to generate formulations. Techniques to increase drug lipophilicity and lipid solubility are therefore explored, and in particular those methods that provide for temporary enhancement including lipophilic ionic liquid and prodrug technologies. The transient nature of these lipophilicity increases enhances lipid solubility and LBF viability, but precludes enduring effects on receptor promiscuity and off target toxicity. Finally, recent efforts to generate solid LBF are briefly described as a means to circumvent the need to encapsulate in soft or hard gelatin capsules, although the latter remain popular with consumers and a proven means of LBF delivery.

Molecular Dynamics Simulation Studies of Bile, Bile Salts, Lipid-Based Drug Formulations, and mRNA–Lipid Nanoparticles: A Review

Publisher: American Chemical Society (ACS)

Date: 17-05-2023

DOI: 10.1021/ACS.MOLPHARMACEUT.3C00049

Toward the establishment of standardized in vitro tests for lipid-based formulations. 5. Lipolysis of representative formulations by gastric lipase

Publisher: Springer Science and Business Media LLC

Date: 07-10-2015

DOI: 10.1007/S11095-014-1532-Y

Abstract: Lipid-based formulations (LBF) are substrates for digestive lipases and digestion can significantly alter their properties and potential to support drug absorption. LBFs have been widely examined for their behaviour in the presence of pancreatic enzymes. Here, the impact of gastric lipase on the digestion of representative formulations from the Lipid Formulation Classification System has been investigated. The pHstat technique was used to measure the lipolysis by recombinant dog gastric lipase (rDGL) of eight LBFs containing either medium (MC) or long (LC) chain triglycerides and a range of surfactants, at various pH values [1.5 to 7] representative of gastric and small intestine contents under both fasting and fed conditions. All LBFs were hydrolyzed by rDGL. The highest specific activities were measured at pH 4 with the type II and IIIA MC formulations that contained Tween®85 or Cremophor EL respectively. The maximum activity on LC formulations was recorded at pH 5 for the type IIIA-LC formulation. Direct measurement of LBF lipolysis using the pHstat, however, was limited by poor LC fatty acid ionization at low pH. Since gastric lipase initiates lipid digestion in the stomach, remains active in the intestine and acts on all representative LBFs, its implementation in future standardized in vitro assays may be beneficial. At this stage, however, routine use remains technically challenging.

DNA-Dependent Protein Kinase Is a Context Dependent Regulator of Lmx1a and Midbrain Specification

Publisher: Public Library of Science (PLoS)

Date: 23-10-2013

DOI: 10.1371/JOURNAL.PONE.0078759

Overcoming biological barriers to in vivo efficacy of antisense oligonucleotides

Publisher: Cambridge University Press (CUP)

Date: 03-2009

DOI: 10.1017/S1462399409001021

Abstract: Antisense oligonucleotides as a therapeutic platform have been slow to progress since the approval of the first antisense drug in 1998. Recently, there have been several ex les of convincing antisense interventions in animal models and promising clinical trial data. This review considers the factors determining the success of antisense oligonucleotides as therapeutic agents. In order to produce target knockdown after systemic delivery, antisense oligonucleotides must avoid nuclease degradation, reticuloendothelial-system uptake and rapid renal excretion, and extravasate to the target cell type outside the vasculature. They then must enter the target cell, and escape the endosome–lysosome pathway so as to be free to interact with the target mRNA. We consider the significance of these limiting factors based on the literature and our own experience using systemic administration of antisense oligonucleotides.

A CX3CR1 Reporter hESC Line Facilitates Integrative Analysis of In-Vitro-Derived Microglia and Improved Microglia Identity upon Neuron-Glia Co-culture

Publisher: Elsevier BV

Date: 06-2020

DOI: 10.1016/J.STEMCR.2020.04.007

Computational Models of the Intestinal Environment. 3. The Impact of Cholesterol Content and pH on Mixed Micelle Colloids

Publisher: American Chemical Society (ACS)

Date: 05-10-2017

DOI: 10.1021/ACS.MOLPHARMACEUT.7B00446

Abstract: In this study, we use molecular dynamics (MD) and experimental techniques (nephelometry and dynamic light scattering) to investigate the influence of cholesterol content and pH on the colloidal structures that form in the gastrointestinal (GI) tract upon lipid digestion. We demonstrate that the ionization state of the molecular species is a primary driver for the self-assembly of aggregates formed by model bile and therefore should be considered when performing in silico modeling of colloidal drug delivery systems. Additionally, the incorporation of physiological concentrations of cholesterol within the model systems does not affect size, number, shape, or dynamics of the aggregates to a significant degree. The MD data shows a reduction in aggregate size with increasing pH, a preference for glycodeoxycholate (GDX) to occupy the aggregate surface, and that the mixed micellar aggregates are oblate spheroids (disc-like). The results obtained assist in understanding the process by which pH and cholesterol influence self-assembly of mixed micelles within the GI tract. The MD approach provides a platform for investigation of interactions of drugs and formulation excipients with the endogenous contents of the GI tract.

VIRAL DELIVERY OF GDNF PROMOTES FUNCTIONAL INTEGRATION OF HUMAN STEM CELL GRAFTS IN PARKINSON’S DISEASE

Publisher: Cold Spring Harbor Laboratory

Date: 10-12-2019

DOI: 10.1101/870725

Abstract: The derivation of neurotransmitter and region-specific neuronal populations from human pluripotent stem cells (PSC) provides impetus for advancing cell therapies into the clinic. At the forefront is our ability to generate ventral midbrain (VM) dopaminergic (DA) progenitors, suitable for transplantation in Parkinson’s disease (PD). Pre-clinical studies, however, have highlighted the low proportion of DA neurons within these grafts and their inferior plasticity by comparison to human fetal donor transplants. Here we sought to examine whether modification of the host environment, through viral delivery of a developmentally critical molecule, glial cell line-derived neurotrophic factor (GDNF), could improve graft survival, integration and function in Parkinsonian rodents. Utilising LMX1A- and PITX3-GFP hPSC reporter lines, we tracked the response of DA progenitors implanted into either a GDNF-rich environment, or in a second group, after a 3-week delay in onset of exposure. We found that early exposure of the graft to GDNF promoted survival of DA and non-DA cells, leading to enhanced motor recovery in PD rats. Delayed overexpression of intrastriatal GDNF also promoted motor recovery in transplanted rats, through alternate selective mechanisms including enhanced A9/A10 specification, increased DA graft plasticity, greater activation of striatal neurons and elevated DA metabolism. Lastly, transcriptional profiling of the grafts highlighted novel genes underpinning these changes. Collectively these results demonstrate the potential of targeted neurotrophic gene therapy strategies to improve human PSC graft outcomes.

Enhancing intestinal drug solubilisation using lipid-based delivery systems

Publisher: Elsevier BV

Date: 03-2008

DOI: 10.1016/J.ADDR.2007.10.014

Abstract: Lipid-based delivery systems are finding increasing application in the oral delivery of poorly water-soluble, lipophilic drugs. Whilst lipidic dose forms may improve oral bioavailability via several mechanisms, enhancement of gastrointestinal solubilisation remains argueably the most important method of absorption enhancement. This review firstly describes the mechanistic rationale which underpins the use of lipid-based delivery systems to enhance drug solubilisation and briefly reviews the available literature describing increases in oral bioavailability after the administration of lipid solution, suspension and self-emulsifying formulations. The use of in vitro methods including dispersion tests and more complex models of in vitro lipolysis as indicators of potential in vivo performance are subsequently described, with particular focus on recent data which suggests that the digestion of surfactants present in lipid-based formulations may impact on formulation performance. Finally, a series of seven guiding principles for formulation design of lipid-based delivery systems are suggested based on an analysis of recent data generated in our laboratories and elsewhere.

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

Publisher: MyJove Corporation

Date: 23-08-2014

DOI: 10.3791/51764

Heterogeneous population of dopaminergic neurons derived from mouse embryonic stem cells: Preliminary phenotyping based on receptor expression and function

Publisher: Wiley

Date: 04-2007

DOI: 10.1111/J.1460-9568.2007.05489.X

Abstract: The possibility exists that directed differentiation of mouse embryonic stem (mES) cells is capable of yielding enriched populations of dopaminergic neurons, but at present there is little understanding of the pharmacological properties of these cells or whether such cells represent a pharmacologically, phenotypically similar population. In this study we used a simple culture protocol to generate dopaminergic neurons and offer a preliminary pharmacological investigation of these cells using Ca2+ imaging and [3H]-dopamine release studies. In fluo-4 AM loaded cells, 13-17 days postplating, and after the addition of tetrodotoxin some of the population of mouse embryonic stem cell-derived neurons responded to adenosine triphosphate (ATP), noradrenaline (NA), acetylcholine (ACh) and L-glutamate (L-glut) with elevations of Ca2+ influx. Within the microtubule-associated protein and tyrosine hydroxylase (TH)-positive cell population adenosine triphosphate, noradrenaline, acetylcholine and L-glutamate elicited positive elevations of Ca2+ in 74, 66, 58 and 67% of the population cells could be further sub ided into three major pharmacologically distinct populations based on the combinations of agonist they responded to. Acetylcholine (30 microM) and noradrenaline (30 microM) were the only agonists to elicit significant tritium overflow from [3H]-dopamine loaded cells. The acetylcholine effect was blocked by atropine (1 microM) and tetrodotoxin (1 microM) and elevated by haloperidol (100 nM). The noradrenaline effects were reduced by cocaine (10 microM), but not by tetrodotoxin (100 nM). These data indicate that the dopaminergic neurons derived from mouse embryonic stem cells represent a heterogeneous population possessing combinations of purinergic, adrenergic, cholinergic and glutamatergic receptors located on the cell soma.

Formulation of lipid-based delivery systems for oral administration: materials, methods and strategies

Publisher: Elsevier BV

Date: 03-2008

DOI: 10.1016/J.ADDR.2007.10.010

Abstract: Oral lipid-based drug delivery systems may include a broad range of oils, surfactants, and cosolvents. This ersity makes comparison of lipid-based formulations difficult. Although the relationship between formulation and drug absorption is understood at a conceptual level, performance in vivo cannot be predicted with confidence at present. The Lipid Formulation Classification System (LFCS) identifies the factors which are likely to affect performance in vivo. There is now a need to establish performance criteria which will facilitate in vitro-in vivo correlation studies. In this review we discuss the properties of excipients, and identify criteria for selection of excipients for lipid-based formulations. Excipients are discussed in the context of the LFCS, our existing knowledge of the fate of these materials during dispersion and digestion, and the likely consequences of their use in formulations. We outline the formulation strategies that can be used for each type of lipid formulation, and suggest a framework for the in vitro testing of each type. Finally we address the choice of lipid formulations in relation to the physicochemical properties of the drug.

Commercially Supplied Amine-Modified siRNAs May Require Ultrafiltration prior to Conjugation with Amine-Reactive Compounds

Publisher: Hindawi Limited

Date: 2011

DOI: 10.4061/2011/154609

Abstract: Conjugation of siRNA to macromolecules such as serum albumin has multiple potential benefits, including enhanced extravasation via albumin-mediated transcytosis across endothelial cells and reduced renal clearance. In attempting to conjugate siRNA to albumin, we used commercially sourced amine-modified siRNA and reacted it with the heterobifunctional linker succinimidyl 4-[ N -maleimidomethyl]cyclohexane-1-carboxylate (SMCC) to introduce a maleimide group suitable for conjugation to the thiol group of the surface-exposed cysteine residue (Cys 34) within albumin. We found the conjugation of the SMCC-treated siRNA to bovine serum albumin (BSA) to be very inefficient and investigated the cause of the low yield of conjugate. Ultrafiltration with phosphate-buffered saline prior to activation with SMCC dramatically increased the yield of siRNA-albumin conjugate (~15-fold). Communication with the commercial supplier revealed that ammonium acetate buffer was used in a desalting step as part of the siRNA purification process prior to supply, likely resulting in ammonium counterions to the siRNA polyanion, which would interfere with conjugation by consuming the SMCC. After ultrafiltration, a greatly reduced amount of SMCC could be used to affect conjugation, without significant reduction in yield. These data indicate that amine-modified siRNA sourced commercially may require ultrafiltration or dialysis prior to use in conjugation reactions.

Evaluation of the structural determinants of polymeric precipitation inhibitors using solvent shift methods and principle component analysis

Publisher: American Chemical Society (ACS)

Date: 02-07-2013

DOI: 10.1021/MP300576U

Abstract: The presence of polymers within solid dose forms, such as solid dispersions, or liquid or semisolid formulations, such as lipid-based formulations, can promote the maintenance of drug supersaturation after dissolution or dispersion/digestion of the vehicle in the gastrointestinal tract. Transiently stable supersaturation delays precipitation, increases thermodynamic activity, and may enhance bioavailability and reduce variability in exposure. In the current study a erse range of 42 different classes of polymers, with a total of 78 polymers across all classes, grades, and molecular weights were examined, to varying degrees, as potential polymeric precipitation inhibitors (PPIs) using a solvent shift method to initiate supersaturation. To provide a deeper understanding of the molecular determinants of polymer utility the data were also analyzed, along with a range of physicochemical descriptors of the polymers employed, using principle component analysis (PCA). Polymers were selectively tested for their ability to stabilize supersaturation for nine poorly water-soluble model drugs, representing a range of nonelectrolytes, weak acids, and weak bases. In general, the cellulose-based polymers (and in particular hydroxypropylmethyl cellulose, HPMC, and its derivatives) provided robust precipitation inhibition across most of the drugs tested. Subsequent PCA indicate that there is consistent PPI behavior of a given polymer for a given drug type, with clear clustering of the performance of polymers with each of the nonelectrolytes, weak bases, and weak acids. However, there are some exceptions to this, with some specific drug type-polymer interactions also occurring. Polymers containing primary amine functional groups should be avoided as they are prone to enhancing precipitation rates. An inverse relationship was also documented for the number of amide, carboxylic acid, and hydroxyl functional groups therefore for general good PPI performance the number of these contained within the polymer should be minimized. Molecular weight is a poor predictor of performance, having only a minor influence, and in some cases a higher molecular weight enhances the precipitation process. The importance of ionic interactions to the ability of a PPI to stabilize the supersaturated state was demonstrated by the advantage of choosing a polymer with an opposite charge with respect to the drug. Additionally, when the polymer charge is the same as the supersaturated drug, precipitation is likely to be enhanced. A PCA model based on polymer molecular properties is presented, which has a central oval region where the polymer will general perform well across all three drug types. If the polymer is located outside of this region, then they either show compound-specific inhibition or enhance precipitation. Incomplete separation of the PPI performance based on the molecular properties on the polymers indicates that there are some further molecular properties that might improve the correlation.

Synthesis and biological evaluation of N-substituted noscapine analogues

Publisher: Wiley

Date: 10-10-2012

DOI: 10.1002/CMDC.201200365

Abstract: Noscapine is a phthalideisoquinoline alkaloid isolated from the opium poppy Papaver somniferum. It has long been used as an antitussive agent, but has more recently been found to possess microtubule-modulating properties and anticancer activity. Herein we report the synthesis and pharmacological evaluation of a series of 6'-substituted noscapine derivatives. To underpin this structure-activity study, an efficient synthesis of N-nornoscapine and its subsequent reduction to the cyclic ether derivative of N-nornoscapine was developed. Reaction of the latter with a range of alkyl halides, acid chlorides, isocyanates, thioisocyanates, and chloroformate reagents resulted in the formation of the corresponding N-alkyl, N-acyl, N-carbamoyl, N-thiocarbamoyl, and N-carbamate derivatives, respectively. The ability of these compounds to inhibit cell proliferation was assessed in cell-cycle cytotoxicity assays using prostate cancer (PC3), breast cancer (MCF-7), and colon cancer (Caco-2) cell lines. Compounds that showed activity in the cell-cycle assay were further evaluated in cell viability assays using PC3 and MCF-7 cells.

A Nonionic Polyethylene Oxide (PEO) Surfactant Model: Experimental and Molecular Dynamics Studies of Kolliphor EL

Publisher: Elsevier BV

Date: 2019

DOI: 10.1016/J.XPHS.2018.11.028

Abstract: Polyethoxylated, nonionic surfactants are important constituents of many drug formulations, including lipid-based formulations. In an effort to better understand the behavior of formulation excipients at the molecular level, we have developed molecular dynamics (MD) models for the widely used surfactant Kolliphor EL (KOL), a triricinoleate ester of ethoxylated glycerol. In this work, we have developed models based on a single, representative molecular component modeled with 2 force field variations based on the GROMOS 53A6

Adenovirus Terminal Protein Contains a Bipartite Nuclear Localisation Signal Essential for Its Import into the Nucleus

Publisher: MDPI AG

Date: 24-03-2021

DOI: 10.3390/IJMS22073310

Abstract: Adenoviruses contain dsDNA covalently linked to a terminal protein (TP) at the 5′end. TP plays a pivotal role in replication and long-lasting infectivity. TP has been reported to contain a nuclear localisation signal (NLS) that facilitates its import into the nucleus. We studied the potential NLS motifs within TP using molecular and cellular biology techniques to identify the motifs needed for optimum nuclear import. We used confocal imaging microscopy to monitor the localisation and nuclear association of GFP fusion proteins. We identified two nuclear localisation signals, PV(R)6VP and MRRRR, that are essential for fully efficient TP nuclear entry in transfected cells. To study TP–host interactions further, we expressed TP in Escherichia coli (E. coli). Nuclear uptake of purified protein was determined in digitonin-permeabilised cells. The data confirmed that nuclear uptake of TP requires active transport using energy and shuttling factors. This mechanism of nuclear transport was confirmed when expressed TP was microinjected into living cells. Finally, we uncovered the nature of TP binding to host nuclear shuttling proteins, revealing selective binding to Imp β, and a complex of Imp α/β but not Imp α alone. TP translocation to the nucleus could be inhibited using selective inhibitors of importins. Our results show that the bipartite NLS is required for fully efficient TP entry into the nucleus and suggest that this translocation can be carried out by binding to Imp β or Imp α/β. This work forms the biochemical foundation for future work determining the involvement of TP in nuclear delivery of adenovirus DNA.

Inclusion of Digestible Surfactants in Solid SMEDDS Formulation Removes Lag Time and Influences the Formation of Structured Particles During Digestion

Publisher: Springer Science and Business Media LLC

Date: 23-01-2017

DOI: 10.1208/S12248-016-0036-6

Abstract: Solid self-microemulsifying drug delivery systems (SMEDDS) have received considerable attention in recent times attempting to overcome the drawbacks of liquid SMEDDS. Earlier literature reports on solid SMEDDS have focussed on formulation development however, the digestibility and propensity for self-assembly of the digested components with endogenous bile salts and phospholipids are unknown. Therefore, as a starting point, previously reported solid SMEDDS containing Gelucire® 44/14 (GEL) and the non-digestible surfactants, Vitamin E TPGS (TPGS) and Lutrol® F 127 (F 127), were prepared, and their dispersion and digestion behaviours were studied using an in vitro lipolysis model, coupled with small-angle X-ray scattering (SAXS) to determine the formed colloidal structures during digestion in real time. GEL alone was digested (89%) and formed a lamellar phase (Lα). When surfactants were added at a 40:60% w/w lipid to surfactants ratio, digestion was inhibited with a significant lag time being evident. However, increasing the fraction of GEL to 50% w/w enabled digestion with reduced lag time. The substitution of the non-digestible surfactants with digestible surfactants, sucrose esters S-1670 (S-1670) and Span® 60 (S-60), eliminated the digestion lag time, and the formation of colloidal structures was more similar to that of GEL alone.

Using molecular dynamics to study liquid phase behavior: simulations of the ternary sodium laurate/sodium oleate/water system

Publisher: American Chemical Society (ACS)

Date: 26-08-2011

DOI: 10.1021/LA2022903

Abstract: The prediction of surfactant phase behavior has applications in a wide range of areas. An accurate modeling of liquid phase behavior can aid our understanding of colloidal process or be used to design phases that respond in a defined way to their environment. In this work, we use molecular dynamics to model the phase behavior of the ternary sodium laurate/sodium oleate/water system and compare the simulation results to experimental data. Simulations were performed with the GROMOS 53A6 united-atom force field and cover the entire ternary phase diagram, producing micellar, hexagonal, and lamellar phases. The aggregate simulation time for the 33 simulations performed during this study is 4.4 μs. We find that the simulations were able to model the experimentally observed liquid phase behavior accurately, showing that the carboxylate and lipid parameters of the 53A6 force field give very good quality results for the in silico prediction of liquid system phase behavior.

Non-linear increases in danazol exposure with dose in older vs. younger beagle dogs: The potential role of differences in bile salt concentration, thermodynamic activity, and formulation digestion

Publisher: Springer Science and Business Media LLC

Date: 30-01-2014

DOI: 10.1007/S11095-013-1260-8

Abstract: To explore the possibility that age-related changes in physiology may result in differences in drug bioavailability after oral administration of lipid based formulations of danazol. Danazol absorption from lipid formulations with increasing drug load was examined in younger (9 months) and older (8 years) beagles. Age related changes to hepatic function were assessed via changes to systemic clearance and serum bile acid concentrations. Changes to lipolytic enzyme activity and intestinal bile salt concentration were evaluated using in vitro lipolysis. Drug exposure increased linearly with dose in younger animals. In older animals, bioavailability increased with increasing dose to a tipping point, beyond which bioavailability reduced (consistent with initiation of precipitation). No differences in hepatic function were apparent across cohorts. Changes to enzyme concentrations in lipolysis studies had little impact on drug precipitation/solubilisation. In contrast, higher bile salt concentrations better supported supersaturation at higher drug loads. Differences in animal cohort can have a significant impact on drug absorption from lipid based formulation. For danazol, bioavailability was enhanced under some circumstances in older animals. In vitro experiments suggest that this was unlikely to reflect changes to metabolism or lipolysis, but might be explained by increases in luminal bile salt hospholipid concentrations in older animals.

Mechanism of microtubule-facilitated fast track nuclear import

Publisher: Elsevier BV

Date: 04-2011

DOI: 10.1074/JBC.M110.210302

A suicidal strain of Listeria monocytogenes is effective as a DNA vaccine delivery system for oral administration

Publisher: Elsevier BV

Date: 09-2017

DOI: 10.1016/J.VACCINE.2017.08.014

Abstract: In this study we determined the in vivo activity of model ovalbumin vaccines delivered by direct intramuscular delivery of plasmid DNA or oral delivery using a recombinant suicidal Listeria monocytogenes strain (rsΔ2). In a previous report we described how rsΔ2 is capable of delivering luciferase, as protein or DNA, in vitro, into non- iding intestinal epithelial cells (Kuo et al., 2009). This is achieved by engineering a dual expression shuttle vector, pDuLX-Luc, that replicates in E. coli and rsΔ2 and drives gene expression from the Listeria promoter (Phly) as well as the eukaryotic cytomegalovirus promoter (CMV), thereby delivering both protein and plasmid DNA to the cell cytoplasm. For the current in vivo study rsΔ2 containing pDuLX-OVA was used to deliver both ovalbumin protein and the mammalian expression plasmid by the oral route. Controls were used to investigate the activity of this system versus positive and negative controls, as well as quantifying activity against direct intramuscular injection of expression plasmids. Oral administration of rsΔ2(pDuLX-OVA) produced significant titres of antibody and was effective at inducing targeted T-cell lysis (approximately 30% lysis relative to an experimental positive control, intravenous OVA-coated splenocytes+lipopolysaccharide). Intramuscular injection of plasmids pDuLX-OVA or p3L-OVA (which lacks the prokaryotic promoter) also produced significant CTL-mediated cell lysis. The delivery of the negative control rsΔ2 (pDuLX-Luc) confirmed that the observed activity was induced specifically by the ovalbumin vaccination. The data suggest that the oral activity of rsΔ2(pDuLX-OVA) is explained by delivery of OVA protein, expressed in rsΔ2 from the prokaryotic promoter present in pDuLX-OVA, but transfection of mammalian cells in vivo may also play a role. Antibody titres were also produced by oral delivery (in rsΔ2) of the p3L-OVA plasmid in which does not include a prokaryotic promoter.

PI3K activation in neural stem cells drives tumorigenesis which can be suppressed by targeting CREB

Publisher: Cold Spring Harbor Laboratory

Date: 30-05-2017

DOI: 10.1101/143388

Abstract: Hyperactivation of the PI3K signaling is common in human cancers, including gliomas, but the precise role of the pathway in glioma biology remains to be determined. Some limited understanding of PI3K signaling in brain cancer come from studies on neural stem rogenitor cells (NSPCs) where signals transmitted via the PI3K pathway cooperate with other intracellular pathways and downstream transcription factors to regulate NSPC proliferation. To investigate the role for the PI3K pathway in glioma initiation and development, we generated a mouse model targeting the inducible expression of a Pik3ca H1047A oncogenic mutation and simultaneous deletion of the PI3K negative regulator, Pten, in NSPCs. We show that the expression of a Pik3ca H1047A was sufficient to initiate tumorigenesis but that simultaneous loss of Pten, was required for the development of invasive, high-grade glioma. Mutant NSPCs exhibited enhanced neurosphere forming capacity which correlated with increased Wnt signaling. We also show that loss of CREB in Pik3ca-PTEN tumors led to a longer symptom-free survival in mice. Taken together, our findings present a novel mouse model for high-grade glioma with which we demonstrate that the PI3K pathway is important for initiation of tumorigenesis and that disruption of downstream CREB signaling attenuates tumor expansion.

Electrical and neurotransmitter activity of mature neurons derived from mouse embryonic stem cells by Sox-1 lineage selection and directed differentiation

Publisher: Wiley

Date: 12-2004

DOI: 10.1111/J.1460-9568.2004.03782.X

Abstract: Sx1TV2/16C is a mouse embryonic stem (ES) cell line in which one copy of the Sox1 gene, an early neuroectodermal marker, has been targeted with a neomycin (G418) selection cassette. A combination of directed differentiation with retinoic acid and G418 selection results in an enriched neural stem cell population that can be further differentiated into neurons. After 6-7 days post-plating (D6-7PP) most neurons readily fired tetrodotoxin (TTX)-sensitive action potentials due to the expression of TTX-sensitive Na(+) and tetraethylammonium (TEA)-sensitive K(+) channels. Neurons reached their maximal cell capacitance after D6-7PP however, ion channel expression continued until at least D21PP. The percentage of cells receiving spontaneous synaptic currents (s.s.c.) increased with days in culture until 100% of cells received a synaptic input by D20PP. Spontaneous synaptic currents were reduced in litude and frequency by TTX, or upon exposure to a Ca(2+)-free, 2.5 mm Mg(2+) saline. S.s.c. of rapid decay time constants were preferentially blocked by the nonNMDA glutamatergic receptor antagonists CNQX or NBQX. Ca(2+) levels within ES cell-derived neurons increased in response to glutamate receptor agonists l-glutamate, AMPA, N-methyl-d-aspartate (NMDA) and kainic acid and to acetylcholine, ATP and dopamine. ES cell-derived neurons also generated cationic and Cl(-)-selective currents in response to NMDA and glycine or GABA, respectively. It was concluded that ES-derived neurons fire action potentials, receive excitatory and inhibitory synaptic input and respond to various neurotransmitters in a manner akin to primary central neurons.

Unlocking the full potential of lipid-based formulations using lipophilic salt/ionic liquid forms

Publisher: Elsevier BV

Date: 03-2019

DOI: 10.1016/J.ADDR.2019.05.008

Abstract: Lipid-based formulations (LBF) are widely used by industry and accepted by the regulatory authorities for oral drug delivery in the pharmaceutical and consumer healthcare market. Innovation in the LBF field is however needed in order to meet the demands of modern drugs, their more challenging problem statements and growing needs for achieving optimal pharmacokinetics (i.e., no food-effects, low variability) on approval. This review describes a new lipophilic salt / ionic liquid approach in combination with LBF, and how this salt strategy can be used to better tailor the properties of a drug to LBFs. The potential advantages of lipophilic salts are discussed in the context of dose escalation studies during toxicological evaluation, reducing the pill burden, increasing drug absorption of new drugs and in life-cycle management. Commentary on lipophilic salt synthesis, scale-up, LBF design and the regulatory aspects are also provided. These topics are discussed in the broad context of bringing the widely recognized advantages of LBFs to a broader spectrum of drugs.

Haplotyping the human leukocyte antigen system from single chromosomes

Publisher: Springer Science and Business Media LLC

Date: 27-07-2016

DOI: 10.1038/SREP30381

Abstract: We describe a method for determining the parental HLA haplotypes of a single in idual without recourse to conventional segregation genetics. Blood s les were cultured to identify and sort chromosome 6 by bivariate flow cytometry. Single chromosome 6 lification products were confirmed with a single nucleotide polymorphism (SNP) array and verified by deep sequencing to enable assignment of both alleles at the HLA loci, defining the two haplotypes. This study exemplifies a rapid and efficient method of haplotyping that can be applied to any chromosome pair, or indeed all chromosome pairs, using a single sorting operation. The method represents a cost-effective approach to complete phasing of SNPs, which will facilitate a deeper understanding of the links between SNPs, gene regulation and protein function.

Improvement in the Predicted Partitioning of Alcohol and Polyethylene Oxide Groups Between Water and Octanol (logP) in Molecular Dynamics Simulations

Publisher: Elsevier BV

Date: 2019

DOI: 10.1016/J.XPHS.2018.11.016

Abstract: Molecular dynamics simulations can be applied to explore the complex liquid phase behavior of lipid-based formulations and the gastrointestinal tract lumen. In order for the results from these simulations to be of value, the manner in which molecules interact with both aqueous and oil phases present needs to be as correct as possible. An existing molecular dynamics force field, GROMOS 53a6, was demonstrated to poorly reproduce the partitioning of straight-chain alcohol and short-chain polyethylene glycol (PEG) molecules between octanol and water phase (logP), with the molecules too hydrophobic. Force field parameters for Lennard-Jones interactions between CH2 and CH3 with water oxygen were adjusted to reproduce the experimental octanol logP, with all other Lennard-Jones and force field parameters left untouched. This parameter set, called 53a6

The potential of oily formulations for drug delivery to the gastro-intestinal tract

Publisher: Elsevier BV

Date: 04-1997

DOI: 10.1016/S0169-409X(96)00486-3

7^th Australasian gene therapy society meeting

Publisher: Wiley

Date: 07-2011

DOI: 10.1002/JGM.1582

Polymeric Nanotubes as Drug Delivery Vectors─Comparison of Covalently and Supramolecularly Assembled Constructs

Publisher: American Chemical Society (ACS)

Date: 12-06-2036

DOI: 10.1021/ACS.BIOMAC.2C00063

Generic construction of single component particles that elicit humoural and cellular immune responses without the need for adjuvants

Publisher: Elsevier BV

Date: 12-2008

DOI: 10.1016/J.VACCINE.2008.09.087

Abstract: Insoluble, pure protein particles could be advantageous as single-entity vaccines or as carriers for small peptide epitopes. Dense gas anti-solvent precipitation was employed to produce pure protein particles which were found to be insoluble in water. As particulate and multimerized antigens are more immunogenic and hence more advantageous for vaccination, particles were produced via this method using ovalbumin as a model antigen. The particles produced had a mean diameter of approximately 300nm, and remained as discrete particles at low pH. At neutral pH or in the presence of electrolyte, the particles exhibited predictable flocculation behaviour to produce aggregates 1-5microm in diameter. Immunisation of mice with these flocculates elicited specific ovalbumin antibody production, T-cell proliferation and a cytotoxic T-cell response, all in the absence of adjuvant. Thus, dense gas processing could be used as a generic method to produce pure protein particulate vaccines.

Comparison of 5-HT4 and 5-HT7 receptor expression and function in the circular muscle of the human colon

Publisher: Elsevier BV

Date: 03-2007

DOI: 10.1016/J.LFS.2006.12.025

Abstract: Serotonin receptors are potential targets for treating functional bowel disorders. This study investigated the functional roles and expression of the 5-HT4 and the 5-HT7 receptor, which coexist in human colon circular smooth muscle. 5-HT3 receptor expression was also investigated. Part of the relaxant response to 5-HT was due to activation of 5-HT4 receptors as the apparent pKB value of the selective 5-HT4 antagonist, GR 113808, was 9.36. 5-HT4 mRNA levels were low in five tissues and undetectable in four others, but all responded to 5-HT with an EC50 value of 102.54+/-19.32 nM. The contribution of 5-HT7 receptors to the response was not readily demonstrated using the selective 5-HT7 antagonist, SB-269970, as its apparent pKB value of 7.19 (5-HT4 block with 1 microM GR 113808) was lower than the value obtained using the 5-HT7 guinea pig ileum assay (8.62). Nevertheless, the 5-HT7 receptor was expressed more consistently than the 5-HT4, but at similar levels. The 5-HT(3Ashort) and 5-HT(3B) subunits were co-expressed at similar levels, but the 5-HT(3Along) subunit was detected in only five of the nine s les tested. The findings show that 5-HT4-induced relaxation occurs at low to undetectable levels of tissue mRNA, as measured by qPCR. Although 5-HT7 receptor mRNA is detected at low, but consistent levels, the functional activity of this receptor is not readily identified given the currently available drugs.

Molecular dynamics simulations of spontaneous bile salt aggregation

Publisher: Elsevier BV

Date: 06-2006

DOI: 10.1016/J.COLSURFA.2006.02.009

Elastomeric nanocomposites as cell delivery vehicles and cardiac support devices

Publisher: Royal Society of Chemistry (RSC)

Date: 2010

DOI: 10.1039/C0SM00213E

Toward the establishment of standardized in vitro tests for lipid-based formulations, part 3: understanding supersaturation versus precipitation potential during the in vitro digestion of type I, II, IIIA, IIIB and IV lipid-based formulations

Publisher: Springer Science and Business Media LLC

Date: 10-05-2013

DOI: 10.1007/S11095-013-1038-Z

Abstract: Recent studies have shown that digestion of lipid-based formulations (LBFs) can stimulate both supersaturation and precipitation. The current study has evaluated the drug, formulation and dose-dependence of the supersaturation - precipitation balance for a range of LBFs. Type I, II, IIIA/B LBFs containing medium-chain (MC) or long-chain (LC) lipids, and lipid-free Type IV LBF incorporating different doses of fenofibrate or tolfenamic acid were digested in vitro in a simulated intestinal medium. The degree of supersaturation was assessed through comparison of drug concentrations in aqueous digestion phases (APDIGEST) during LBF digestion and the equilibrium drug solubility in the same phases. Increasing fenofibrate or tolfenamic acid drug loads (i.e., dose) had negligible effects on LC LBF performance during digestion, but promoted drug crystallization (confirmed by XRPD) from MC and Type IV LBF. Drug crystallization was only evident in instances when the calculated maximum supersaturation ratio (SR(M)) was >3. This threshold SR(M) value was remarkably consistent across all LBF and was also consistent with previous studies with danazol. The maximum supersaturation ratio (SR(M)) provides an indication of the supersaturation 'pressure' exerted by formulation digestion and is strongly predictive of the likelihood of drug precipitation in vitro. This may also prove effective in discriminating the in vivo performance of LBFs.

Using polymeric precipitation inhibitors to improve the absorption of poorly water-soluble drugs: A mechanistic basis for utility

Publisher: Informa UK Limited

Date: 26-10-2010

DOI: 10.3109/1061186X.2010.525652

A PITX3-EGFP Reporter Line Reveals Connectivity of Dopamine and Non-dopamine Neuronal Subtypes in Grafts Generated from Human Embryonic Stem Cells

Publisher: Elsevier BV

Date: 09-2017

DOI: 10.1016/J.STEMCR.2017.08.002

Choice of nonionic surfactant used to formulate type IIIA self-emulsifying drug delivery systems and the physicochemical properties of the drug have a pronounced influence on the degree of drug supersaturation that develops during in vitro digestion

Publisher: Elsevier BV

Date: 04-2014

DOI: 10.1002/JPS.23856

Monash University

Location: Australia

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Standardising Protocols For The Differentiation And Integration Of Human Pluripotent Stem Cell-derived Neural Transplants In Parkinson's Disease

Start Date: 2016

End Date: 2019

Funder: National Health and Medical Research Council

The Nanoparticle Network

Start Date: 2003

End Date: 2003

Funder: Australian Research Council

Discovery Projects - Grant ID: DP140103743

Start Date: 2014

End Date: 12-2016

Amount: $393,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP120100600

Start Date: 01-2012

End Date: 12-2014

Amount: $330,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0884059

Start Date: 01-2009

End Date: 02-2014

Amount: $350,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0775530

Start Date: 03-2007

End Date: 12-2011

Amount: $93,354.00

Funder: Australian Research Council

Special Research Initiatives - Grant ID: SR0354658

Start Date: 01-2004

End Date: 12-2003

Amount: $10,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP180103661

Start Date: 2018

End Date: 12-2020

Amount: $501,024.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0455327

Start Date: 09-2004

End Date: 06-2007

Amount: $398,733.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP160100705

Start Date: 11-2016

End Date: 12-2019

Amount: $515,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0455474

Start Date: 02-2005

End Date: 08-2008

Amount: $257,803.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE200100163

Start Date: 06-2020

End Date: 05-2022

Amount: $700,000.00

Funder: Australian Research Council

Industrial Transformation Training Centres - Grant ID: IC170100016

Start Date: 05-2018

End Date: 12-2024

Amount: $3,123,492.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE110100125

Start Date: 10-2011

End Date: 10-2012

Amount: $500,000.00

Funder: Australian Research Council

Special Research Initiatives - Grant ID: SR1101002

Start Date: 07-2011

End Date: 12-2019

Amount: $21,000,000.00

Funder: Australian Research Council