Preclinical development of an automated injection device for intradermal delivery of a cell-based therapy.

Current methods for intradermal delivery of therapeutic products in clinical use include manual injection via the Mantoux technique and the use of injection devices, primarily developed for the delivery of vaccines and small molecules. A novel automated injection device is presented specifically designed for accurate delivery of multiple doses of product through a number of adjustable injection parameters, including injection depth, dose volume and needle insertion speed. The device was originally conceived for the delivery of a cell-based therapy to patients with skin wounds caused by epidermolysis bullosa. A series of preclinical studies was conducted (i) to evaluate the performance of the pre-production model (PreCTCDV01) and optimise the final design, (ii) to confirm that a cell therapy product can be effectively delivered through the injection system and (iii) to test whether the device can be safely and effectively operated by potential end-users. Results from these studies confirmed that the device is able to consistently deliver repeated doses of a liquid to the intradermal layer in an ex vivo skin model. In addition, the device can support delivery of a cell therapy product through a customised microbore tubing without compromising cell viability. Finally, the device was shown to be safe and easy to use as evidenced by usability testing. The clinical device has since been granted European market access and plans for clinical use are currently underway. The device is expected to find use in the emerging area of cell therapies and a broad spectrum of traditional parenteral drug delivery applications.

Automated large-scale culture and medium-throughput chemical screen for modulators of proliferation and viability of human induced pluripotent stem cell-derived neuroepithelial-like stem cells.

The aim of this study was to demonstrate proof-of-concept feasibility for the use of human neural stem cells (NSCs) for high-throughput screening (HTS) applications. For this study, an adherent human induced pluripotent stem (iPS) cell-derived long-term, self-renewing, neuroepithelial-like stem (lt-NES) cell line was selected as a representative NSC. Here, we describe the automated large-scale serum-free culture ("scale-up") of human lt-NES cells on the CompacT SelecT cell culture robotic platform, followed by their subsequent automated "scale-out" into a microwell plate format. We also report a medium-throughput screen of 1000 compounds to identify modulators of neural stem cell proliferation and/or survival. The screen was performed on two independent occasions using a cell viability assay with end-point reading resulting in the identification of 24 potential hit compounds, 5 of which were found to increase the proliferation and/or survival of human lt-NES on both occasions. Follow-up studies confirmed a dose-dependent effect of one of the hit compounds, which was a Cdk-2 modulator. This approach could be further developed as part of a strategy to screen compounds to either improve the procedures for the in vitro expansion of neural stem cells or to potentially modulate endogenous neural stem cell behavior in the diseased nervous system.

Identification of an Orally Bioavailable, Potent, and Selective Inhibitor of GlyT1.

Amalgamation of the structure-activity relationship of two series of GlyT1 inhibitors developed at Merck led to the discovery of a clinical candidate, compound 16 (DCCCyB), which demonstrated excellent in vivo occupancy of GlyT1 transporters in rhesus monkey as determined by displacement of a PET tracer ligand.

Optimisation of a series of potent, selective and orally bioavailable GlyT1 inhibitors.

A series of heterocyclic sulfonamides have been developed which are potent and selective inhibitors of hGlyT1. SAR studies to optimise the in vitro and in vivo properties are described. Optimisation of the central scaffold resulted in cyclohexane sulfones 28 and 29, which have good PK properties and show promise for further development.

Sarcosine based indandione hGlyT1 inhibitors.

A series of sarcosine based indandione hGlyT1 inhibitors has been developed. Optimization of substitution around the indandione and sarcosine moieties has led to highly potent inhibitors at hGlyT1, which show selectivity over a number of other receptors.

A new pyridazine series of GABAA alpha5 ligands.

Screening of the Merck compound collection identified 6 as an unusually simple, low molecular weight hit with moderate affinity for GABAA receptors. The structural novelty of 6, compared to our advanced series of GABAA alpha5 inverse agonists, made it an attractive molecule for further exploration. This paper will describe the evolution of 6 into a new series of ligands with nanomolar affinity and functional selectivity for GABAA alpha5 receptor subtypes.

Multiple structural features of steroids mediate subtype-selective effects on human alpha4beta3delta GABAA receptors.

Neurosteroids have been shown to mediate some of their physiological effects via a modulatory site on type A inhibitory gamma-aminobutyric acid (GABAA) receptors. In particular, recent evidence has implicated selective potentiation of the delta subunit of GABAA receptors as an important mediator of in vitro and in vivo neurosteroid activity. However, this has been demonstrated for only a very small number of steroids, so both the generality of this finding, and the structural features of steroids which mediate functional delta-selectivity, are unclear. We have used a potentiometric assay based on fluorescence resonance energy transfer to measure GABA-activated responses in L(tk-) cells stably transfected with human GABAA receptor alpha4beta3delta and alpha4beta3gamma2 receptor subtypes. A set of 28 steroids were evaluated on these subtypes to characterise their functional potency and efficacy in modulating GABA responses. For most compounds there was a clear separation of their efficacy profiles between the receptor subtypes, with a substantially larger maximal response at the alpha4beta3delta receptor. 5beta-Pregnan-3beta-ol-20-one, 5beta-pregnane-3alpha,20beta-diol and 5beta-pregnane-3alpha,17alpha-diol-11,20-dione showed particularly high efficacy for alpha4beta3delta. No compounds were identified that simply inhibited responses at delta-containing receptors. However, 5beta-pregnane-3alpha,17alpha,20beta-triol, prednisolone 21-acetate, 4-pregnene-17alpha,20alpha-diol-3-one-20-acetate, 4-pregnen-20alpha-ol-3-one, and 5beta-pregnane-3alpha,17alpha,21-triol-20-one inhibited, though did not abolish, GABA responses at the alpha4beta3gamma2 subtype, while evoking modest-amplitude potentiation of alpha4beta3delta responses. Molecular modelling on this compound series using principal components analysis indicates that several structural features of steroids underlie their relative functional selectivity for potentiation of delta-containing GABAA receptors.

Compounds exhibiting selective efficacy for different beta subunits of human recombinant gamma-aminobutyric acid A receptors.

Inhibitory GABA(A) receptor modulators are widely used therapeutic agents for a variety of central nervous system disorders. Ltk(-) cells stably expressing human recombinant GABA(A) subunits (alpha1beta1-3gamma2s) were seeded into 96-well plates, loaded with chlorocoumarin-2-dimyristoyl phosphatidylethanolamine and bis(1,3-diethyl-2-thiobarbiturate)trimethineoxonol, and rapid fluorescence resonance energy transfer technique (FRET) measurements were made of GABA-evoked depolarizations in low-Cl(-) buffer using a voltage/ion probe reader. The influence of different betasubunits on the ability of agents to modulate and directly activate the ion channel was examined. GABA evoked concentration-dependent decreases in FRET, increasing fluorescence emission ratio (460/580 nm) at alpha1beta1gamma2, alpha1beta2gamma2, and alpha1beta3gamma2 receptors with similar maximal amplitude (P > 0.05, n = 17) and EC(50) values of 2.4 +/- 0.2, 2.5 +/- 0.2, and 1.3 +/- 0.1 microM, respectively. Piperidine-4-sulfonic acid and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol were less potent, with EC(50) values of 8.7 +/- 0.9, 9.2 +/- 0.5, and 11.7 +/- 1.2, and 43.7 +/- 6.4, 24.8 +/- 1.6, and 26.1 +/- 2.4 microM, respectively. Potency and maximal efficacy of propofol, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, pentobarbital, and steroids, 5alpha-pregnan-3alpha-ol-20-one and 5beta-pregnan-3alpha-ol-20-one, were unaffected by the beta isoform present in the receptor complex. However, several compounds displayed beta2/3 subunit selectivity, notably loreclezole, R(-)-etomidate, and a group of anti-inflammatory agents including mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, niflumic acid, and diflunisal. The anti-inflammatories exhibited varying levels of efficacy at beta2/3 subunits, with micromolar potency, while having antagonist or weak inverse agonist profiles at alpha1beta1gamma2. Diflunisal was the most efficacious compound, eliciting greater potentiation than loreclezole (90 +/- 14% and 109 +/- 14% at beta3 and beta2, respectively, compared with 62 +/- 6% and 56 +/- 3%), whereas niflumic acid exhibited the lowest efficacy. An additional agent, olsalazine, weakly potentiated responses at all three receptors without any selectivity. This study identifies and characterizes a variety of allosteric modulators for which betasubunits are an important determinant of efficacy and potency.

3,4-Dihydronaphthalen-1(2H)-ones: novel ligands for the benzodiazepine site of alpha5-containing GABAA receptors.

A series of substituted 3,4-dihydronaphthalen-1(2H)-ones with high binding affinity for the benzodiazepine site of GABAA receptors containing the alpha5-subunit has been identified. These compounds have consistently higher binding affinity for the GABAA alpha5 receptor subtype over the other benzodiazepine-sensitive GABAA receptor subtypes (alpha1, alpha2 and alpha3). Compounds with a range of efficacies for the benzodiazepine site of alpha5-containing GABAA receptors were identified, including the alpha5 inverse agonist 3,3-dimethyl-8-methylthio-5-(pyridin-2-yl)-3,4-dihydronaphthalen-1(2H)-one 22 and the alpha5 agonist 8-ethylthio-3-methyl-5-(1-oxidopyridin-2-yl)-3,4-dihydronaphthalen-1(2H)-one 19.