Dentin primer based on a highly functionalized gelatin-methacryloyl hydrogel.

Gelatin-methacryloyl hydrogels (GelMA) have demonstrated their utility as scaffolds in a variety of tissue engineering applications. OBJECTIVES: In this study, a highly functionalized GelMA hydrogel was synthesized and assessed for degree of functionalization. As the proposed GelMA hydrogel was coupled to a visible-light photoinitiator, we hypothesized it might serve as base to formulate a model dentin primer for application in restorative dentistry. METHODS: GelMA was mixed with photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), photopolymerized for 0-40 s using a dental light-curing device and tested for extrudability, degree of photo-crosslinking (DPxlink), water sorption/solubility/swelling (WS/SL/SW) and apparent modulus of elasticity (AE). Model dentin primer was prepared by mixing GelMA+LAP with a primer of a commercial three-step etch-and-rinse adhesive. After application of GelMA-based primer to acid-etched dentin, samples were bonded with correspondent adhesive agent, photopolymerized and had their immediate bond strength compared to control samples primed and bonded with the same commercial material. RESULTS: Extrudability of hydrogel was confirmed using a microsyringe to write the acronym "CDMI". DPxlink of GelMA+LAP changed significantly as a function of photopolymerization time (20 s < 30 s ≤ 40 s). WS, SL and SW were significantly reduced in hydrogels polymerized for 30 and 40 s. AE of hydrogels varied significantly as a function of photopolymerization time (20 s < 30 s ≤ 40 s; 20 s ‡ 40 s). Bond strength of dentin primed with GelMA-based primer was lower (∼29.3 MPa) but not significantly of that of control (∼34.6 MPa). CONCLUSIONS: Optimization of a GelMA-based dentin primers can lead to the development of promising biomimetic adhesives for dentin rehabilitation.

Selective antagonism of CRF1 receptor by a substituted pyrimidine.

The corticotrophin-releasing factor (CRF) and its type 1 receptor (CRF1R) regulate the hypothalamic-pituitary-adrenal axis, as well as other systems, thus playing a crucial role in the maintenance of homeostasis. Non-peptide CRF1R-selective antagonists exert therapeutic effects on experimental animals with abnormal regulation of their homeostatic mechanisms. However, none of them is as yet in clinical use. In an effort to develop novel small non-peptide CRF1R-selective antagonists, we have synthesized a series of substituted pyrimidines described in a previous study. These small molecules bind to CRF1R, with analog 3 having the highest affinity. Characteristic structural features of analog 3 are a N,N-bis(methoxyethyl)amino group at position 6 and a methyl in the alkythiol group at position 5. Based on the binding profile of analog 3, we selected it in the present study for further pharmacological characterization. The results of this study suggest that analog 3 is a potent CRF1R-selective antagonist, blocking the ability of sauvagine, a CRF-related peptide, to stimulate cAMP accumulation in HEK 293 cells via activation of CRF1R, but not via CRF2R. Moreover, analog 3 blocked sauvagine to stimulate the proliferation of macrophages, further supporting its antagonistic properties. We have also constructed molecular models of CRF1R to examine the interactions of this receptor with analog 3 and antalarmin, a prototype CRF1R-selective non-peptide antagonist, which lacks the characteristic structural features of analog 3. Our data facilitate the design of novel non-peptide CRF1R antagonists for clinical use.

Pathogen-mimicking vaccine delivery system designed with a bioactive polymer (inulin acetate) for robust humoral and cellular immune responses.

New and improved vaccines are needed against challenging diseases such as malaria, tuberculosis, Ebola, influenza, AIDS, and cancer. The majority of existing vaccine adjuvants lack the ability to significantly stimulate the cellular immune response, which is required to prevent the aforementioned diseases. This study designed a novel particulate based pathogen-mimicking vaccine delivery system (PMVDS) to target antigen-presenting-cells (APCs) such as dendritic cells. The uniqueness of PMVDS is that the polymer used to prepare the delivery system, Inulin Acetate (InAc), activates the innate immune system. InAc was synthesized from the plant polysaccharide, inulin. PMVDS provided improved and persistent antigen delivery to APCs as an efficient vaccine delivery system, and simultaneously, activated Toll-Like Receptor-4 (TLR-4) on APCs to release chemokine's/cytokines as an immune-adjuvant. Through this dual mechanism, PMVDS robustly stimulated both the humoral (>32 times of IgG1 levels vs alum) and the cell-mediated immune responses against the encapsulated antigen (ovalbumin) in mice. More importantly, PMVDS stimulated both cytotoxic T cells and natural killer cells of cell-mediated immunity to provide tumor (B16-ova-Melanoma) protection in around 40% of vaccinated mice and significantly delayed tumor progression in rest of the mice. PMVDS is a unique bio-active vaccine delivery technology with broader applications for vaccines against cancer and several intracellular pathogens, where both humoral and cellular immune responses are desired.

Synthesis of 2-imino and 2-hydrazono thiazolo[4,5-d]pyrimidines as corticotropin releasing factor (CRF) antagonists.

Corticotropin-releasing factor (CRF) is an important neuropeptide hormone which controls the body's overall response to stress. It plays a crucial role in regulating the behavioral, cardiovascular, immune and gastrointestinal systems. Over-activation of the CRF system has been implicated in many disorders including anxiety, depression, drug addiction, hypertension, Irritable Bowel Syndrome (IBS), peptic ulcers, inflammation and others. Thus, binding of CRF to its receptors is an attractive target to develop new medications which aim at treating ailments associated with chronic stress. Numerous small-molecule non-peptide CRF receptor antagonists were developed and many are in various stages in clinical trials. Many showed great promise in treatment of anxiety, depression, peptic ulcers, inflammation, IBS and drug addiction. In our recent previous work, the development of two series of pyrimidine and fused pyrimidine CRF antagonists were described. In continuation of our efforts in this direction, in the current manuscript, the synthesis of a third series of CRF receptor antagonists is described. The binding affinities of select compounds for the type 1 receptor of CRF (CRF1R) were determined and compared to a standard CRF antagonist drug antalarmin. A lead compound was identified and further evaluated by measuring its effect on the inhibition of the agonist-stimulated accumulation of second messengers.

Structure and Function of Small Non-Peptide CRF Antagonists and their Potential Clinical Use.

Corticotropin-releasing factor (CRF) can be considered a very important hormone or a chemical mediator. It works closely with other systems to regulate the manner through which the body may respond to stress. Thus it affects many biological processes associated with stress. Dysfunction of this system has also been correlated with various diseases such as major depression, anxiety, drug addiction and eating disorders. Rationally, this means that interfering with binding of CRF to its intended receptors can be an attractive target for drug design aiming at developing new medications for many ailments that are associated with stress such as depression, anxiety and stress-induced relapse in drug addiction. Hundreds of accounts of small molecule antagonists have appeared in the literature and the preclinical and clinical pharmacology have been reported for many of these agents. Several classes of small molecule CRF receptor antagonists which belong to the non-peptide class have been developed with many being widely used for research purposes. Currently several major pharmaceutical companies are pursuing development of small non-peptide CRF1 receptor antagonists. In this review article we explain the importance of development of non-peptide CRF antagonists and their clinical relevance with emphasis on those members that showed great promise or those that were advanced to clinical trials.

Thiazolo[4,5-d]pyrimidines as a privileged scaffold in drug discovery.

Thiazolo[4,5-d]pyrimidines are fused heterocyclic ring-systems that can be viewed at the first glance as purine isosteres. They are the 7 thia-analogs of purines via the replacement of the nitrogen at position 7 of the purine ring by a sulfur atom. Because of the structural resemblance to adenine and guanine and their related derivatives as adenosine, guanosine, cAMP, cGMP and similar biomolecules, many thiazolo[4,5-d]pyrimidines scaffold were developed and utilized by medicinal chemists to design novel therapeutics. Many were found to have a broad range of pharmacological activities. The outstanding development of thiazolo[4,5-d]pyrimidines within a short time span shows its magnitude of usefulness for medicinal chemistry research. Despite their importance from pharmacological and synthetic point of views, hardly there is a comprehensive review of thiazolo[4,5-d]pyrimidines applications in medicinal research to date. Thus, this review article describes the structures and medicinal significance of all classes of thiazolo[4,5-d]pyrimidines reported in literature to date. It describe the development of thiazolo[4,5-d]pyrimidines as immune-modulators, Corticotropin Releasing Factor (CRF) receptor antagonists, anti-Parkinson's, antiviral, anticancer, antibacterial, antifungal, analgesic, anti-inflammatory agents including COX inhibitors, chemokines antagonists and Fractlkine receptor antagonists.

Discovery of inulin acetate as a novel immune-active polymer and vaccine adjuvant: synthesis, material characterization, and biological evaluation as a toll-like receptor-4 agonist.

Vaccine adjuvants are an essential part of modern vaccine design, especially against intracellular pathogens such as M. tuberculosis, malarial parasite, HIV, influenza virus and Ebola. The present work offers a unique approach to designing novel vaccine adjuvants by identifying polymers that mimic "pathogen associated molecular patterns" (PAMPS) and engineering an immune-active particulate vaccine delivery system that uses the polymer. By using this strategy, we report the discovery of the first plant polymer based toll-like receptor-4 (TLR-4) agonist, inulin acetate (InAc). InAc was synthesised from the plant polysaccharide inulin. Inulin acetate as a polymer and particles prepared using InAc were characterised using various physicochemical techniques. The TLR-4 agonistic activity of InAc was established in multiple immune, microglial, dendritic, peripheral blood mononuclear (human and swine) and genetically modified epithelial cells (HEK293) that exclusively express TLR-4 on their surface. InAc activated all the above-mentioned cells to release proliferative cytokines; however, InAc failed to activate when the were cells either pre-incubated with a TLR-4 specific antagonist or isolated from mice deficient in adapter proteins involved in TLR signalling (Mal/MyD88). Antigen encapsulated microparticles prepared with TLR-4 agonist InAc mimicked pathogens to offer improved antigen delivery to dendritic cells compared to soluble antigen (47 times) or antigen encapsulated poly(lactic-co-glycolic acid) (PLGA) particles (1.57 times). In conclusion, InAc represents a novel polymer-based modern vaccine adjuvant targeting specific signalling pathways of the innate immune system, which could be formulated into a platform vaccine delivery system against cancer and viral diseases.

Synthesis of new thiazolo[4,5-d]pyrimidines as Corticotropin releasing factor modulators.

Corticotropin-releasing factor (CRF) is a neurohormone that plays a crucial role in integrating the body's overall response to stress. It appears necessary and sufficient for the organism to mount functional, physiological and endocrine responses to stressors. CRF is released in response to various triggers such as chronic stress. The role of CRF and its involvement in these neurological disorders suggest that new drugs that can target the CRF function or bind to its receptors may represent a new development of neuropsychiatric medicines to treat various stress-related disorders including depression, anxiety and addictive disorders. Based on pharmacophore of the CRF1 receptor antagonists, a new series of thiazolo[4,5-d] pyrimidines were synthesized as Corticotropin-releasing factor (CRF) receptor modulators and the prepared compounds carry groups shown to produce optimum binding affinity to CRF receptors. Twenty two compounds were evaluated for their CRF1 receptor binding affinity in HEK 293 cell lines and two compounds 5o and 5s showed approximately 25% binding affinity to CRF1 receptors. Selected compounds (5c and 5f) were also evaluated for their effect on expression of genes associated with depression and anxiety disorders such as CRF1, CREB1, MAO-A, SERT, NPY, DatSLC6a3, and DBH and significant upregulation of CRF1 mRNA has been observed with compound 5c.

Synthesis of substituted pyrimidines as corticotropin releasing factor (CRF) receptor ligands.

Corticotropin releasing factor (CRF) is a neuropeptide hormone produced from the hypothalamus that controls the secretion of corticotropin (ACTH) from the anterior pituitary gland that, in turn, prompts the adrenal glands to secrete glucocorticoids. This involvement in the hypothalamic-pituitary-adrenal axis (HPA) in response to stress and also playing a key role in behavioral, cardiovascular, immune and gastrointestinal systems made CRF binding to its receptors an important target in drug discovery aiming to develop lead compounds with the potential to treat various stress-related disorders including depression, anxiety and addictive disorders. Several non-peptide CRF1 receptor antagonists were developed by pharmaceutical companies and are currently in clinical trials with the aim of improving the health consequences of chronic stress and for use in the clinical management of anxiety and stress. Many showed promising results not only in treatment of anxiety and depression but also in treatment of CRF-induced hypertension, as well as in treatment of arthritis, irritable bowel syndrome and peptic ulcers. In this manuscript, we describe the synthesis of substituted pyrimidines with close structural similarities to reported lead compounds with promising CRF1 receptor affinities and carrying groups known to be associated with optimum affinity to CRF1 receptors. The affinity of the newly prepared compounds in comparison to antalarmin, a potent CRF1 receptor antagonist in clinical trials as a standard, is also described. Four compounds from the new series showed promising CRF1 receptor affinity.

Sarcophine-diol, a skin cancer chemopreventive agent, inhibits proliferation and stimulates apoptosis in mouse melanoma B16F10 cell line.

Sarcodiol (SD) is a semi-synthetic derivative of sarcophine, a marine natural product. In our previous work, we reported the significant chemopreventive effects of SD against non-melanoma skin cancer both in vitro and in vivo mouse models. In this investigation, we extended this work to study the effect of sarcodiol on melanoma development, the more deadly form of skin cancer, using the mouse melanoma B16F10 cell line. In this study we report that SD inhibits the de novo DNA synthesis and enhances fragmentation of DNA. We also evaluated the antitumor effect of SD on melanoma cell viability using several biomarkers for cell proliferation and apoptosis. SD inhibits the expression levels of signal transducers and activators of transcription protein (STAT-3) and cyclin D1, an activator of cyclin-dependent kinase 4 (Cdk4). SD treatment also enhances cellular level of tumor suppressor protein 53 (p53) and stimulates cleavage of the nuclear poly (ADP-ribose) polymerase (cleaved-PARP). SD also enhances cellular levels of cleaved Caspase-3, -8, -9 and stimulates enzymatic activities of Caspase-3, -8 and -9. These results, in addition to inhibition of cell viability, suggest that SD inhibits melanoma cell proliferation by arresting the cell-division cycle in a Go quiescent phase and activates programmed cell death (apoptosis) via extrinsic and intrinsic pathways. Finally, these studies demonstrate that SD shows a very promising chemopreventive effect in melanoma B16F10 tumor cells.