Raman based chemometric model development for glycation and glycosylation real time monitoring in a manufacturing scale CHO cell bioreactor process.

The Quality by Design (QbD) approach to the production of therapeutic monoclonal antibodies (mAbs) emphasizes an understanding of the production process ensuring product quality is maintained throughout. Current methods for measuring critical quality attributes (CQAs) such as glycation and glycosylation are time and resource intensive, often, only tested offline once per batch process. Process analytical technology (PAT) tools such as Raman spectroscopy combined with chemometric modeling can provide real time measurements process variables and are aligned with the QbD approach. This study utilizes these tools to build partial least squares (PLS) regression models to provide real time monitoring of glycation and glycosylation profiles. In total, seven cell line specific chemometric PLS models; % mono-glycated, % non-glycated, % G0F-GlcNac, % G0, % G0F, % G1F, and % G2F were considered. PLS models were initially developed using small scale data to verify the capability of Raman to measure these CQAs effectively. Accurate PLS model predictions were observed at small scale (5 L). At manufacturing scale (2000 L) some glycosylation models showed higher error, indicating that scale may be a key consideration in glycosylation profile PLS model development. Model robustness was then considered by supplementing models with a single batch of manufacturing scale data. This data addition had a significant impact on the predictive capability of each model, with an improvement of 77.5% in the case of the G2F. The finalized models show the capability of Raman as a PAT tool to deliver real time monitoring of glycation and glycosylation profiles at manufacturing scale.

6-Substituted quinolines as RORγt inverse agonists.

We identified 6-substituted quinolines as modulators of the retinoic acid receptor-related orphan receptor gamma t (RORγt). The synthesis of this class of RORγt modulators is reported, and optimization of the substituents at the quinoline 6-position that produced compounds with high affinity for the receptor is detailed. This effort identified molecules that act as potent, full inverse agonists in a RORγt-driven cell-based reporter assay. The X-ray crystal structures of two full inverse agonists from this chemical series bound to the RORγt ligand binding domain are disclosed, and we highlight the interaction of a hydrogen-bond acceptor on the 6-position substituent of the inverse agonist with Glu379:NH as a conserved binding contact.

Identification and structure activity relationships of quinoline tertiary alcohol modulators of RORγt.

A high-throughput screen of the ligand binding domain of the nuclear receptor retinoic acid-related orphan receptor gamma t (RORγt) employing a thermal shift assay yielded a quinoline tertiary alcohol hit. Optimization of the 2-, 3- and 4-positions of the quinoline core using structure-activity relationships and structure-based drug design methods led to the discovery of a series of modulators with improved RORγt inhibitory potency and inverse agonism properties.

Perfil citopatológico de mulheres atendidas nas unidades básicas do município de Uruguaiana, RS / Cytopathological profile of women met in the basic units of the municipality of Uruguaiana, RS

O câncer do colo de útero tem sido apontado como o terceiro tipo de câncer mais comum entre as mulheres e sua relação com a infecção por papilomavírus humano (HPV) é bem estabelecida. Objetivo: conhecer o perfil citopatológico de mulheres atendidas nas Unidades Básicas de Saúdedo município de Uruguaiana, RS. Métodos: análise retrospectiva de corte transversal e descritivo dos laudos de exames citopatológicos de pacientes querealizaram a citopatologia ginecológica (Papanicolaou) e respectivos prontuários, emitidos entre os anos de 2003 e 2011. Foram selecionadas as variáveis referentes à idade e a alteração citopatológica e classificadas segundo Bethes da, 2001. Resultados: da totalidade de laudos de exames citopatológicos analisados, 15,5% possuem alguma alteração celular e a que apresentou maior prevalência foi a atipia de células escamosas de significado indeterminado(ASC-US), com 50,28%, seguida das lesões intraepiteliais de baixo grau (LIEBG), com 35,45%. Em ambos os casos as mulheres com idade inferior a 25anos foram as que apresentaram maior prevalência. Cerca de 13% das pacientes apresentaram infecção pelo HPV e a maior prevalência foi observada nafaixa etária inferior a 25 anos (47,84%). Conclusão: a faixa etária de maior prevalência de lesões cervicais está em mulheres com idade inferior a 25 anose muitas das alterações celulares estão associadas à infecção pelo HPV. Salienta-se, portanto, a necessidade de políticas de rastreamento de lesões cervicais em mulheres nesta faixa etária, evitando assim a progressão das lesões que evoluem ao câncer.

Cancer of the cervix has been named as the third most common type of cancer among women and its relation to infection by human papillomavirus (HPV) is well established. Objective: cytopathologic know the profile of women attending the Basic Health Units in the municipality of Uruguaiana, RS. Methods: retrospective analysis of cross-sectional and descriptive the reports of cytopathology from patients who underwent gynecologic cytology (Papanicolaou) and their medical records, issued between the years 2003 and 2011. We selected the variables related to age and change and cytopathological sorted by Bethesda, 2001. Results: of all reports of reports of cytopathology analyzed, 15.5% have a cell phone and some change withthe highest prevalence was atypical squamous cells of undetermined significance (ASC-US), with 50.28%, followed by low-grade intraepithelial lesions(LSIL), with 35.45%. In both cases, women younger than 25 years showed the highest prevalence. About 13% of patients had HPV infection and the highestprevalence was observed in the age group below 25 years (47.84%). Conclusion: the age group with the highest prevalence of cervical lesions in womenis under the age of 25 years and many of the cellular changes associated with HPV infection. It is noteworthy, therefore, the need for political screening ofcervical lesions in women in this age group, thus preventing the progression of lesions that evolve to cancer.

Optimization of a pyrazole hit from FBDD into a novel series of indazoles as ketohexokinase inhibitors.

A series of indazoles have been discovered as KHK inhibitors from a pyrazole hit identified through fragment-based drug discovery (FBDD). The optimization process guided by both X-ray crystallography and solution activity resulted in lead-like compounds with good pharmaceutical properties.

Electron density guided fragment-based drug design--a lead generation example.

We describe here a method using protein crystallography as the sole detection tool for fragment-based lead discovery. The methodology consists of iterative design, synthesis, and X-ray crystallographic screening of three libraries of compounds. Target-specific compound design, by way of active site electron density in the presence of a bound fragment hit and the intentional lack of solution activity bias form the basis of our approach. We provide an example of this alternative fragment-based drug design (FBDD) method, detailing results from a campaign using ketohexokinase to generate a unique lead series with promising drug-like properties.

Identification of diaryl ether-based ligands for estrogen-related receptor α as potential antidiabetic agents.

Estrogen-related receptor α (ERRα) is an orphan nuclear receptor that has been functionally implicated in the regulation of energy homeostasis. Herein is described the development of diaryl ether based thiazolidenediones, which function as selective ligands against this receptor. Series optimization provided several potent analogues that inhibit the recruitment of a coactivator peptide fragment in in vitro biochemical assays (IC(50) < 150 nM) and cellular two-hybrid reporter assays against the ligand binding domain (IC(50) = 1-5 µM). A cocrystal structure of the ligand-binding domain of ERRα with lead compound 29 revealed the presence of a covalent interaction between the protein and ligand, which has been shown to be reversible. In diet-induced murine models of obesity and in an overt diabetic rat model, oral administration of 29 normalized insulin and circulating triglyceride levels, improved insulin sensitivity, and was body weight neutral. This provides the first demonstration of functional activities of an ERRα ligand in metabolic animal models.

Inhibitors of Ketohexokinase: Discovery of Pyrimidinopyrimidines with Specific Substitution that Complements the ATP-Binding Site.

Attenuation of fructose metabolism by the inhibition of ketohexokinase (KHK; fructokinase) should reduce body weight, free fatty acids, and triglycerides, thereby offering a novel approach to treat diabetes and obesity in response to modern diets. We have identified potent, selective inhibitors of human hepatic KHK within a series of pyrimidinopyrimidines (1). For example, 8, 38, and 47 exhibited KHK IC50 values of 12, 7, and 8 nM, respectively, and also showed potent cellular KHK inhibition (IC50 < 500 nM), which relates to their intrinsic potency vs KHK and their ability to penetrate cells. X-ray cocrystal structures of KHK complexes of 3, 8, and 47 revealed the important interactions within the enzyme's adenosine 5'-triphosphate (ATP)-binding pocket.

Electron density guided fragment-based lead discovery of ketohexokinase inhibitors.

A fragment-based drug design paradigm has been successfully applied in the discovery of lead series of ketohexokinase inhibitors. The paradigm consists of three iterations of design, synthesis, and X-ray crystallographic screening to progress low molecular weight fragments to leadlike compounds. Applying electron density of fragments within the protein binding site as defined by X-ray crystallography, one can generate target specific leads without the use of affinity data. Our approach contrasts with most fragment-based drug design methodology where solution activity is a main design guide. Herein we describe the discovery of submicromolar ketohexokinase inhibitors with promising druglike properties.

Potency variation of small-molecule chymase inhibitors across species.

Chymases (EC 3.4.21.39) are mast cell serine proteinases that are variably expressed in different species and, in most cases, display either chymotryptic or elastolytic substrate specificity. Given that chymase inhibitors have emerged as potential therapeutic agents for treating various inflammatory, allergic, and cardiovascular disorders, it is important to understand interspecies differences of the enzymes as well as the behavior of inhibitors with them. We have expressed chymases from humans, macaques, dogs, sheep (MCP2 and MCP3), guinea pigs, and hamsters (HAM1 and HAM2) in baculovirus-infected insect cells. The enzymes were purified and characterized with kinetic constants by using chromogenic substrates. We evaluated in vitro the potency of five nonpeptide inhibitors, originally targeted against human chymase. The inhibitors exhibited remarkable cross-species variation of sensitivity, with the greatest potency observed against human and macaque chymases, with K(i) values ranging from approximately 0.4 to 72nM. Compounds were 10-300-fold less potent, and in some instances ineffective, against chymases from the other species. The X-ray structure of one of the potent phosphinate inhibitors, JNJ-18054478, complexed with human chymase was solved at 1.8A resolution to further understand the binding mode. Subtle variations in the residues in the active site that are already known to influence chymase substrate specificity can also strongly affect the compound potency. The results are discussed in the context of selecting a suitable animal model to study compounds ultimately targeted for human chymase.

Narrowband tunable filter based on velocity-selective optical pumping in an atomic vapor.

We demonstrate a tunable narrowband filter based on optical-pumping-induced circular dichroism in rubidium vapor. The filter achieves a peak transmission of 14.6%, a linewidth of 80 MHz, and an out-of-band extinction of >or=35 dB. The transmission peak can be tuned within the range of the Doppler linewidth of the D1 line of atomic rubidium at 795 nm. While other atomic filters work at frequencies far from absorption, the presented technique provides light resonant with atomic media, useful for atom-photon interaction experiments. The technique could readily be extended to other alkali atoms.

4-Amino-6-arylamino-pyrimidine-5-carbaldehyde hydrazones as potent ErbB-2/EGFR dual kinase inhibitors.

Members of a novel class of 4-amino-6-arylamino-pyrimidine-5-carbaldehyde hydrazones were identified as potent dual ErbB-2/EGFR kinase inhibitors using concept-guided design approach. These compounds inhibited the growth of ErbB-2 over-expressing human tumor cell lines (BT474, N87, and SK-BR-3) in vitro. Compound 15 emerged as a key lead and showed significant ability to inhibit growth factor-induced receptor phosphorylation in SK-BR-3 cells (IC(50)=54 nM) and cellular proliferation in vitro (IC(50)=14, 58, and 58 nM for BT474, N87, and SK-BR-3 respectively). The X-ray co-crystal structure of EGFR with a close analog (17) was determined and validated our design rationale.

Discovery of novel 4-amino-6-arylaminopyrimidine-5-carbaldehyde oximes as dual inhibitors of EGFR and ErbB-2 protein tyrosine kinases.

We herein disclose a novel series of 4-aminopyrimidine-5-carbaldehyde oximes that are potent and selective inhibitors of both EGFR and ErbB-2 tyrosine kinases, with IC(50) values in the nanomolar range. Structure-activity relationship (SAR) studies elucidated a critical role for the 4-amino and C-6 arylamino moieties. The X-ray co-crystal structure of EGFR with 37 was determined and validated our design rationale.

7-fluoroindazoles as potent and selective inhibitors of factor Xa.

We have developed a novel series of potent and selective factor Xa inhibitors that employ a key 7-fluoroindazolyl moiety. The 7-fluoro group on the indazole scaffold replaces the carbonyl group of an amide that is found in previously reported factor Xa inhibitors. The structure of a factor Xa cocrystal containing 7-fluoroindazole 51a showed the 7-fluoro atom hydrogen-bonding with the N-H of Gly216 (2.9 A) in the peptide backbone. Thus, the 7-fluoroindazolyl moiety not only occupied the same space as the carbonyl group of an amide found in prior factor Xa inhibitors but also maintained a hydrogen bond interaction with the protein's beta-sheet domain. The structure-activity relationship for this series was consistent with this finding, as the factor Xa inhibitory potencies were about 60-fold greater (DeltaDelta G approximately 2.4 kcal/mol) for the 7-fluoroindazoles 25a and 25c versus the corresponding indazoles 25b and 25d. Highly convergent synthesis of these factor Xa inhibitors is also described.

Structural basis for elastolytic substrate specificity in rodent alpha-chymases.

Divergence of substrate specificity within the context of a common structural framework represents an important mechanism by which new enzyme activity naturally evolves. We present enzymological and x-ray structural data for hamster chymase-2 (HAM2) that provides a detailed explanation for the unusual hydrolytic specificity of this rodent alpha-chymase. In enzymatic characterization, hamster chymase-1 (HAM1) showed typical chymase proteolytic activity. In contrast, HAM2 exhibited atypical substrate specificity, cleaving on the carboxyl side of the P1 substrate residues Ala and Val, characteristic of elastolytic rather than chymotryptic specificity. The 2.5-A resolution crystal structure of HAM2 complexed to the peptidyl inhibitor MeOSuc-Ala-Ala-Pro-Ala-chloromethylketone revealed a narrow and shallow S1 substrate binding pocket that accommodated only a small hydrophobic residue (e.g. Ala or Val). The different substrate specificities of HAM2 and HAM1 are explained by changes in four S1 substrate site residues (positions 189, 190, 216, and 226). Of these, Asn(189), Val(190), and Val(216) form an easily identifiable triplet in all known rodent alpha-chymases that can be used to predict elastolytic specificity for novel chymase-like sequences. Phylogenetic comparison defines guinea pig and rabbit chymases as the closest orthologs to rodent alpha-chymases.

Structural determination of estrogen-related receptor gamma in the presence of phenol derivative compounds.

We screened the ligand-binding domain of estrogen-related receptor (ERR) gamma in ThermoFluor, in an effort to develop chemical tools and decipher the biology of this orphan nuclear receptor. Several ligands were found to stabilize thermodynamically the protein. Amongst the ligands were bisphenol A (BPA) and 4-chloro-3-methyl phenol (ClCH3Ph). These ligands were further characterized and found to be competitive for 4-hydroxytamoxifen (4OHT) binding, a known reported antagonist ligand for ERRgamma, but functionally they did not enhance or disrupt affinity of the receptor for co-activator peptides. The preservation of the constitutive active conformation of the receptor in the presence of these two ligands was confirmed upon the determination of the co-crystal structures. The structures of BPA and ClCH3Ph were determined to a resolution of 2.1 and 2.3A, respectively, and the antagonist 4OHT was refined to 2.5A resolution. In the presence of BPA and ClCH3Ph the receptor maintained the transcriptional active conformation as reported previously for the apo-protein in the presence of a co-activator peptide fragment. In addition the ERRgamma-BPA structure identifies an interaction between the phenolic-OH and the side chain of N346. The preservation of the constitutive active conformation of the receptor in the presence of the small phenol compounds suggest that the biological activity of the receptor might be regulated by a natural occurring ligand.

Human anterior lens capsule as a biologic substrate for the ex vivo expansion of limbal stem cells in ocular surface reconstruction.

PURPOSE: To study the potential use of human anterior lens capsule as a scaffold for stem cell transplantation in treatment of limbal cell deficiency. METHODS: Limbal biopsies and anterior lens capsules were obtained (same eye) from 30 patients during cataract surgery. Biopsies were suspended in Dulbecco modified Eagle medium under sterile conditions and stored at 4 degrees C. Capsules were treated in distilled water under strict asepsis for 2 hours to eliminate the crystalline epithelium and stored at 4 degrees C. After initial processing, the limbal biopsy was plated epithelial-side down (48 hours) on the capsular specimen in a 35-mm culture dish. Samples were sorted into 4 groups. Group 1 was made up of 10 samples in which limbal biopsies were allowed to grow on corresponding capsules from the same eye (autologous). Group 2 was 10 limbal biopsies that were allowed to grow on capsules of different eye (allogeneic). The remaining specimens were randomized into 2 groups. Group 3 included 10 capsules on which an ex vivo expanded cell line was allowed to grow. Group 4 harbored 10 limbal biopsies that were allowed to grow on polystyrene culture plates. All specimens were incubated for 2 weeks at 37 degrees C and 5% CO2. Cell density, viability, morphology, and adherence of the cell-capsule complex were evaluated at 1, 3, 7, and 14 days. RESULTS: Rate of cell growth and density in groups 1 and 2 were comparable to the control groups. Cell viability was 95% or superior in all groups, and desmosomes developed between growing cells. CONCLUSIONS: Human anterior lens capsule is a potential scaffold for ex vivo expansion of limbal epithelial cells, possibly providing a substrate for ocular surface reconstruction.

Use of autologous platelet-rich plasma in the treatment of dormant corneal ulcers.

PURPOSE: To investigate the potential role of autologous platelet-rich plasma in promoting healing in dormant corneal ulcers. DESIGN: Prospective, consecutive, interventional, noncomparative, nonrandomized, observational study. PARTICIPANTS: Forty eyes of 38 patients with dormant corneal ulcers. METHODS: Autologous platelet-rich plasma was used in a total of 40 eyes with dormant corneal ulcers divided into 2 groups: group I, 26 eyes treated with topical eyedrops of autologous platelet-rich plasma (12 neurotrophic, 9 herpetic, and 5 immunological ulcers), and group II, 14 eyes treated surgically with a solid clot of autologous platelet-rich plasma combined with amniotic membrane transplantation in perforated corneas or with impending perforation. The treatment was used in patients with chronic nonhealing ulcers (mean, 2 years of evolution) that had been unresponsive to conventional topical therapy. Autologous blood from each patient was obtained by venipuncture, and platelet-rich plasma was prepared from each blood sample without additives. MAIN OUTCOME MEASURES: Ulcer size, inflammation, healing, visual acuity, and patient's subjective symptoms. RESULTS: Autologous platelet-rich plasma promoted healing of ulcers. In group I, 13 eyes healed, 11 eyes improved significantly, and 2 eyes showed no change. In group II, 10 eyes healed and 4 eyes improved significantly. Inflammation and subjective symptoms, particularly pain, improved in all patients. Vision remained stable or improved in all cases. CONCLUSION: Autologous platelet-rich plasma promoted healing of dormant corneal ulcers even in eyes threatened by corneal perforation and was accompanied by a reduction in pain and inflammation.

Novel tetrahydroisoquinolines are histamine H3 antagonists and serotonin reuptake inhibitors.

A series of novel 4-aryl-1,2,3,4-tetrahydroisoquinoline-based histamine H(3) ligands that also have serotonin reuptake transporter inhibitor activity is described. The synthesis, in vitro biological data, and select pharmacokinetic data for these novel compounds are discussed.

Discovery, SAR, and X-ray structure of novel biaryl-based dipeptidyl peptidase IV inhibitors.

The discovery, SAR, and X-ray crystal structure of novel biarylaminoacyl-(S)-2-cyano-pyrrolidines and biarylaminoacylthiazolidines as potent inhibitors of dipeptidyl peptidase IV (DPP IV) are reported.