Smart design of patient-centric long-acting products: from preclinical to marketed pipeline trends and opportunities.

INTRODUCTION: We see a development in the field of long-acting products to serve patients with chronic diseases by providing benefits in adherence, efficacy, and safety of the treatment. This review investigates features of long-acting products on the market/pipeline to understand which drug substance (DS) and drug product (DP) characteristics likely enable a successful patient-centric, low-dosing frequency product. AREAS COVERED: This review evaluates marketed/pipeline long-acting products with greater than 1 week release of small molecules and peptides by oral and injectable route of administration (RoA), with particular focus on patient centricity, adherence impact, health outcomes, market trends, and the match of DS/DP technologies which lead to market success. EXPERT OPINION: Emerging trends are expected to change the field of long-acting products in the upcoming years by increasing capability in engineered molecules (low solubility, long half-life, high potency, etc.), directly developing DP as long-acting oral/injectable, increasing the proportion of products for local drug delivery, and a direction toward more subcutaneous, self-administered products. Among long-acting injectable products, nanosuspensions show a superiority in dose per administration and dosing interval, overwhelming the field of infectious diseases with the recently marketed products.

Discovery of a novel 2-aminopyrazine-3-carboxamide as a potent and selective inhibitor of Activin Receptor-Like Kinase-2 (ALK2) for the treatment of fibrodysplasia ossificans progressiva.

Inhibition of mutant activin A type-1 receptor ACVR1 (ALK2) signaling by small-molecule drugs is a promising therapeutic approach to treat fibrodysplasia ossificans progressiva (FOP), an ultra-rare disease leading to progressive soft tissue heterotopic ossification with no curative treatment available to date. Here, we describe the synthesis and in vitro characterization of a novel series of 2-aminopyrazine-3-carboxamides that led to the discovery of Compound 23 showing excellent biochemical and cellular potency, selectivity over other BMP and TGFß signaling receptor kinases, and a favorable in vitro ADME profile.

Enhanced tendon healing by a tough hydrogel with an adhesive side and high drug-loading capacity.

Hydrogels that provide mechanical support and sustainably release therapeutics have been used to treat tendon injuries. However, most hydrogels are insufficiently tough, release drugs in bursts, and require cell infiltration or suturing to integrate with surrounding tissue. Here we report that a hydrogel serving as a high-capacity drug depot and combining a dissipative tough matrix on one side and a chitosan adhesive surface on the other side supports tendon gliding and strong adhesion (larger than 1,000 J m-2) to tendon on opposite surfaces of the hydrogel, as we show with porcine and human tendon preparations during cyclic-friction loadings. The hydrogel is biocompatible, strongly adheres to patellar, supraspinatus and Achilles tendons of live rats, boosted healing and reduced scar formation in a rat model of Achilles-tendon rupture, and sustainably released the corticosteroid triamcinolone acetonide in a rat model of patellar tendon injury, reducing inflammation, modulating chemokine secretion, recruiting tendon stem and progenitor cells, and promoting macrophage polarization to the M2 phenotype. Hydrogels with 'Janus' surfaces and sustained-drug-release functionality could be designed for a range of biomedical applications.

Clinically established biodegradable long acting injectables: An industry perspective.

Long acting injectable formulations have been developed to sustain the action of drugs in the body over desired periods of time. These delivery platforms have been utilized for both systemic and local drug delivery applications. This review gives an overview of long acting injectable systems that are currently in clinical use. These products are categorized in three different groups: biodegradable polymeric systems, including microparticles and implants; micro and nanocrystal suspensions and oil-based formulations. Furthermore, the applications of these drug delivery platforms for the management of various chronic diseases are summarized. Finally, this review addresses industrial challenges regarding the development of long acting injectable formulations.

Einstein-Podolsky-Rosen Paradox and Quantum Entanglement at Subnucleonic Scales.

In 1935, Einstein, Podolsky, and Rosen (EPR) formulated an apparent paradox of quantum theory [Phys. Rev. 47, 777 (1935)PHRVAO0031-899X10.1103/PhysRev.47.777]. They considered two quantum systems that were initially allowed to interact and were then later separated. A measurement of a physical observable performed on one system then had to have an immediate effect on the conjugate observable in the other system-even if the systems were causally disconnected. The authors viewed this as a clear indication of the inconsistency of quantum mechanics. In the parton model of the nucleon formulated by Bjorken, Feynman, and Gribov, the partons (quarks and gluons) are viewed by an external hard probe as independent. The standard argument is that, inside the nucleon boosted to an infinite-momentum frame, the parton probed by a virtual photon with virtuality Q is causally disconnected from the rest of the nucleon during the hard interaction. Yet, the parton and the rest of the nucleon have to form a color-singlet state due to color confinement and so have to be in strongly correlated quantum states-we thus encounter the EPR paradox at the subnucleonic scale. In this Letter, we propose a resolution of this paradox based on the quantum entanglement of partons. We devise an experimental test of entanglement and carry it out using data on proton-proton collisions from the Large Hadron Collider. Our results provide a strong direct indication of quantum entanglement at subnucleonic scales.

Multifunctional Carrier Based on Halloysite/Laponite Hybrid Hydrogel for Kartogenin Delivery.

A novel carrier system based on halloysite nanotubes (HNT), for the potential intraarticular delivery of kartogenin (KGN) by means laponite (Lap) hydrogel (HNT/KGN/Lap), is developed. The drug was first loaded into HNT, and the hybrid composite obtained was used as filler for laponite hydrogel. Both the filler and the hydrogel were thoroughly investigated by several techniques and the hydrogel morphology was imaged by transmission electron microscopy. Furthermore, the gelating ability of laponite in the presence of the filler and the rheological properties of the hybrid hydrogel were also investigated. The kinetic release of kartogenin from HNT and HNT/Lap hybrid hydrogel was studied both in physiological conditions and in ex vivo synovial fluid. In the last case, the kinetic results highlighted that HNT carrier can effectively release KGN in a sustained manner for at least 38 days. Finally, a preliminary biological assays showed that the HNT/KGN/Lap hybrid hydrogel did not exhibit any cytotoxic effect.

Pharmacological Characterization of a Novel 5-Hydroxybenzothiazolone-Derived ß 2-Adrenoceptor Agonist with Functional Selectivity for Anabolic Effects on Skeletal Muscle Resulting in a Wider Cardiovascular Safety Window in Preclinical Studies.

The anabolic effects of ß 2-adrenoceptor (ß 2-AR) agonists on skeletal muscle have been demonstrated in various species. However, the clinical use of ß 2-AR agonists for skeletal muscle wasting conditions has been limited by their undesired cardiovascular effects. Here, we describe the preclinical pharmacological profile of a novel 5-hydroxybenzothiazolone (5-HOB) derived ß 2-AR agonist in comparison with formoterol as a representative ß 2-AR agonist that have been well characterized. In vitro, 5-HOB has nanomolar affinity for the human ß 2-AR and selectivity over the ß 1-AR and ß 3-AR. 5-HOB also shows potent agonistic activity at the ß 2-AR in primary skeletal muscle myotubes and induces hypertrophy of skeletal muscle myotubes. Compared with formoterol, 5-HOB demonstrates comparable full-agonist activity on cAMP production in skeletal muscle cells and skeletal muscle tissue-derived membranes. In contrast, a greatly reduced intrinsic activity was determined in cardiomyocytes and cell membranes prepared from the rat heart. In addition, 5-HOB shows weak effects on chronotropy, inotropy, and vascular relaxation compared with formoterol. In vivo, 5-HOB significantly increases hind limb muscle weight in rats with attenuated effects on heart weight and ejection fraction, unlike formoterol. Furthermore, changes in cardiovascular parameters after bolus subcutaneous treatment in rats and rhesus monkeys are significantly lower with 5-HOB compared with formoterol. In conclusion, the pharmacological profile of 5-HOB indicates superior tissue selectivity compared with the conventional ß 2-AR agonist formoterol in preclinical studies and supports the notion that such tissue-selective agonists should be investigated for the safe treatment of muscle-wasting conditions without cardiovascular limiting effects.

Developmentally inspired programming of adult human mesenchymal stromal cells toward stable chondrogenesis.

It is generally accepted that adult human bone marrow-derived mesenchymal stromal cells (hMSCs) are default committed toward osteogenesis. Even when induced to chondrogenesis, hMSCs typically form hypertrophic cartilage that undergoes endochondral ossification. Because embryonic mesenchyme is obviously competent to generate phenotypically stable cartilage, it is questioned whether there is a correspondence between mesenchymal progenitor compartments during development and in adulthood. Here we tested whether forcing specific early events of articular cartilage development can program hMSC fate toward stable chondrogenesis. Inspired by recent findings that spatial restriction of bone morphogenetic protein (BMP) signaling guides embryonic progenitors toward articular cartilage formation, we hypothesized that selective inhibition of BMP drives the phenotypic stability of hMSC-derived chondrocytes. Two BMP type I receptor-biased kinase inhibitors were screened in a microfluidic platform for their time- and dose-dependent effect on hMSC chondrogenesis. The different receptor selectivity profile of tested compounds allowed demonstration that transient blockade of both ALK2 and ALK3 receptors, while permissive to hMSC cartilage formation, is necessary and sufficient to maintain a stable chondrocyte phenotype. Remarkably, even upon compound removal, hMSCs were no longer competent to undergo hypertrophy in vitro and endochondral ossification in vivo, indicating the onset of a constitutive change. Our findings demonstrate that adult hMSCs effectively share properties of embryonic mesenchyme in the formation of transient but also of stable cartilage. This opens potential pharmacological strategies to articular cartilage regeneration and more broadly indicates the relevance of developmentally inspired protocols to control the fate of adult progenitor cell systems.

Influence of environmental information in natural scenes and the effects of motion adaptation on a fly motion-sensitive neuron during simulated flight.

Gaining information about the spatial layout of natural scenes is a challenging task that flies need to solve, especially when moving at high velocities. A group of motion sensitive cells in the lobula plate of flies is supposed to represent information about self-motion as well as the environment. Relevant environmental features might be the nearness of structures, influencing retinal velocity during translational self-motion, and the brightness contrast. We recorded the responses of the H1 cell, an individually identifiable lobula plate tangential cell, during stimulation with image sequences, simulating translational motion through natural sceneries with a variety of differing depth structures. A correlation was found between the average nearness of environmental structures within large parts of the cell's receptive field and its response across a variety of scenes, but no correlation was found between the brightness contrast of the stimuli and the cell response. As a consequence of motion adaptation resulting from repeated translation through the environment, the time-dependent response modulations induced by the spatial structure of the environment were increased relatively to the background activity of the cell. These results support the hypothesis that some lobula plate tangential cells do not only serve as sensors of self-motion, but also as a part of a neural system that processes information about the spatial layout of natural scenes.

3-alkoxy-pyrrolo[1,2-b]pyrazolines as selective androgen receptor modulators with ideal physicochemical properties for transdermal administration.

We describe the synthesis and characterization of 3-alkoxy-pyrrolo[1,2-b]pyrazolines as novel selective androgen receptor (AR) modulators that possess excellent physicochemical properties for transdermal administration. Compound 26 bound to human AR with an IC50 of 0.7 nM with great selectivity over other nuclear hormone receptors and potently activated AR in a C2C12 muscle cell reporter gene assay with an EC50 of 0.5 nM. It showed high aqueous solubility of 1.3 g/L at pH 7.4, and an in silico model as well as a customized parallel artificial membrane permeability assay indicated good skin permeation. Indeed, when measuring skin permeation through excised human skin, an excellent flux of 2 µg/(cm(2)·h) was determined without any permeation enhancers. In a 2 week Hershberger model using castrated rats, the compound showed dose-dependent effects fully restoring skeletal muscle weight at 0.3 mg/kg/day after subcutaneous administration with high selectivity over prostate stimulation.

Texture-defined objects influence responses of blowfly motion-sensitive neurons under natural dynamical conditions.

The responses of visual interneurons of flies involved in the processing of motion information do not only depend on the velocity, but also on other stimulus parameters, such as the contrast and the spatial frequency content of the stimulus pattern. These dependencies have been known for long, but it is still an open question how they affect the neurons' performance in extracting information about the structure of the environment under the specific dynamical conditions of natural flight. Free-flight of blowflies is characterized by sequences of phases of translational movements lasting for just 30-100 ms interspersed with even shorter and extremely rapid saccade-like rotational shifts in flight and gaze direction. Previous studies already analyzed how nearby objects, leading to relative motion on the retina with respect to a more distant background, influenced the response of a class of fly motion sensitive visual interneurons, the horizontal system (HS) cells. In the present study, we focused on objects that differed from their background by discontinuities either in their brightness contrast or in their spatial frequency content. We found strong object-induced effects on the membrane potential even during the short intersaccadic intervals, if the background contrast was small and the object contrast sufficiently high. The object evoked similar response increments provided that it contained higher spatial frequencies than the background, but not under reversed conditions. This asymmetry in the response behavior is partly a consequence of the depolarization level induced by the background. Thus, our results suggest that, under the specific dynamical conditions of natural flight, i.e., on a very short timescale, the responses of HS cells represent object information depending on the polarity of the difference between object and background contrast and spatial frequency content.

Competitive reporter monitored amplification (CMA)--quantification of molecular targets by real time monitoring of competitive reporter hybridization.

BACKGROUND: State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. METHODOLOGY AND PRINCIPAL FINDINGS: The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. CONCLUSIONS AND SIGNIFICANCE: The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2), we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls and targets into a single assay and to perform the assay on simple and robust instrumentation is a prerequisite for the development of novel molecular point of care tests.

Chiral separation of the ß2-sympathomimetic fenoterol by HPLC and capillary zone electrophoresis for pharmacokinetic studies.

The development of methods for the separation of the enantiomers of fenoterol by chiral HPLC and capillary zone electrophoresis (CZE) is described. For the HPLC separation precolumn fluorescence derivatization with naphthyl isocyanate was applied. The resulting urea derivatives were resolved on a cellulose tris(3,5-dimethylphenylcarbamate)-coated silica gel column employing a column switching procedure. Detection was carried out fluorimetrically with a detection limit in the low ng/mL range. The method was adapted to the determination of fenoterol enantiomers in rat heart perfusates using liquid-liquid extraction. As an alternative a CE method was used for the direct separation of fenoterol enantiomers comparing different cyclodextrin derivatives as chiral selectors.

Discriminative stimulus properties of naloxone in Long-Evans rats: assessment with the conditioned taste aversion baseline of drug discrimination learning.

RATIONALE: The characterization of the discriminative stimulus properties of naloxone has focused primarily on its actions at the mu opioid receptor, although naloxone also displays an affinity for delta and kappa receptor subtypes. OBJECTIVES: The present study extends this characterization of the naloxone cue by investigating if relatively specific antagonists for the mu (naltrexone: 0.10-0.56 mg/kg), delta (naltrindole: 1-18 mg/kg), and kappa (MR2266: 1.8-10 mg/kg) opioid receptor subtypes will substitute for naloxone in animals trained to discriminate naloxone from its vehicle. The temporal nature of the naloxone cue was examined by varying pretreatment time points (15, 30, 45, 60 min). Finally, various doses of naltrexone methobromide (1-18 mg/kg) were assessed to determine peripheral mediation of the cue. MATERIALS AND METHODS: Female Long-Evans rats (N = 30) received an injection of naloxone (1 mg/kg; i.p.) 15 min prior to a pairing of saccharin (20-min access) and the emetic LiCl (1.8 mEq; i.p.; n = 16, group NL) or vehicle (n = 14, group NW); on other days, they were injected with saline prior to saccharin alone. Substitution tests with compounds with various receptor affinities and selective CNS and PNS actions were then assessed. RESULTS: Only naloxone and naltrexone produced dose-dependent decreases in saccharin consumption. Naloxone administered at 15 and 30 min before saccharin produced decreases in consumption similar to that displayed on training days. Naltrexone methobromide substituted only at the highest dose tested (18 mg/kg). CONCLUSIONS: Naloxone's stimulus effects appear to be mediated centrally via activity at the mu opioid receptor.

Regulation and traffic of ceramide 1-phosphate produced by ceramide kinase: comparative analysis to glucosylceramide and sphingomyelin.

Ceramide 1-phosphate (C1P) has been characterized as a sphingolipid that participates in cell signaling. Although C1P synthesis is thought to occur via phosphorylation of ceramide by ceramide kinase (CerK), the processes that regulate C1P formation and fate remain largely unknown. In this study we analyzed bone marrow-derived macrophages (BMDM) from CerK-null mice (Cerk(-/-)) and found significant levels of C1P, suggesting that previously unrecognized pathways may also lead to C1P formation. After these experiments we used an overexpression system, BMDM from Cerk(-/-) mice, and short-chain fluorescent ceramides to trace CerK-dependent formation of C1P. Because the ceramide analogs can also be converted to glucosylceramide (GlcCer) and sphingomyelin (SM), they allowed us to directly compare all three metabolites. We found that C1P produced by CerK is turned over rapidly when serum is removed or upon calcium chelation, whereas GlcCer and SM are stable under these conditions. We further demonstrated that ceramide must be transported to the Golgi complex to be phosphorylated by CerK. Inhibition of the ceramide transfer protein slowed down SM formation without decreasing C1P, suggesting an alternate route of ceramide transport. Other experiments indicated that, like GlcCer and SM, C1P traffics along the secretory pathway to reach the plasma membrane. Furthermore, in BMDM C1P was secreted more readily than was GlcCer or SM. Altogether, our results indicate that CerK is essential to C1P formation via phosphorylation of Cer, providing the first insights into mechanisms underlying ceramide access to CerK and C1P trafficking as well as clarifying C1P as a signaling entity.

Synthesis and immobilization of erythro-C14-omega-aminosphingosine-1-phosphate as a potential tool for affinity chromatography.

A sphingosine-1-phosphate (S1P) analogue containing a terminal alkyl chain amino group is synthesized in a few steps via olefin cross-metathesis of an optically resolved intermediate and subsequent phosphorylation. Regioselective acylation of this intermediate at its N terminus in solution is demonstrated as a model reaction and provides a biologically active derivative. Finally, the omega-amino intermediate is immobilized on an affinity matrix. The choice of a UV-active phosphate protecting group allows for quantification of resin loading after cleavage which amounted to 66 %.

An efficient, one-pot synthesis of various ceramide 1-phosphates from sphingosine 1-phosphate.

An efficient, one-pot procedure for the synthesis of ceramide 1-phosphates with varying N-acyl substituents, to serve as tool compounds for analytical and biological investigations, was developed. Sphingosine 1-phosphate was silylated in situ to increase its solubility and to protect the 3-hydroxy functionality and then allowed to react with activated acid derivatives in the presence of diisopropylethylamine. Simultaneous cleavage of the silyl protecting groups and separation from reagents and by-products was achieved by medium pressure chromatography on reversed phase material. Thus, ceramide 1-phosphates with various fatty acid chains and with fluorescent and affinity labels attached to the sphingoid backbone were prepared in good yields.

Synthesis of borondipyrromethene (BODIPY)-labeled sphingosine derivatives by cross-metathesis reaction.

A new efficient and flexible synthesis of fluorescently labeled sphingosine derivatives from commercially available Garner aldehyde (8) is described. For this, appropriate alkenylated borondipyrromethene (BODIPY) dyes were synthesized and used for the first time in a cross-metathesis reaction, the key step of the approach. The labeled sphingosines with appropriate chain length were accepted as substrates by sphingosine kinases (SPHKs), yielding the corresponding phosphorylated products. One of these derivatives (11d) was identified as the first reported selective substrate for SPHK-1.

Serotonin 2A receptor antagonist treatment reduces dopamine D1 receptor-mediated rotational behavior but not L-DOPA-induced abnormal involuntary movements in the unilateral dopamine-depleted rat.

Previous experiments have demonstrated that serotonin (5-HT) 2A receptor antagonists suppress hyperkinetic behaviors associated with dopamine (DA) D1 receptor supersensitivity in rats with 6-hydroxydopamine (6-OHDA) lesions. Since l-DOPA induced dyskinesia (LID) may be mediated by over-sensitive D1-mediated signaling, the present study examined the effects of the selective 5-HT2A antagonist M100907 on LID behaviors in DA-depleted rats. Adult male Sprague-Dawley rats with unilateral 6-OHDA lesions received daily l-DOPA treatments to produce dyskinetic behaviors as measured by abnormal involuntary movements (AIMs) testing. In these animals, M100907 (0.01, 0.1 or 1.0mg/kg, ip) given 30 min before l-DOPA did not alter the appearance or intensity of AIMs behaviors. Because l-DOPA induced AIMs in rats are dependent upon D1 and D2 receptor activation, a second study was performed to determine if M100907 could suppress D1- or D2-mediated rotational behaviors. Contralateral rotations induced by the D1 agonist SKF82958 were significantly reduced by pre-treatment with M100907. However, M100907 was ineffective in reducing rotations induced by the D2 agonist quinpirole. The finding that M100907 suppresses rotations induced by D1, but not D2, agonists may provide a partial explanation for the lack of effect of a selective 5-HT2A antagonist on l-DOPA-induced AIMs behaviors.