scholarly journals THU0080 PRECLINICAL CHARACTERIZATION OF TLL018, A NOVEL, HIGHLY POTENT AND SELECTIVE JAK1/TYK2 INHIBITOR FOR TREATING AUTOIMMUNE DISEASES

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 252.1-252
Author(s):  
X. Liu ◽  
F. Tan ◽  
C. Liang
Keyword(s):  
Bone Resorption ◽  
Autoimmune Diseases ◽  
Whole Blood ◽  
Significant Inhibition ◽  
Clinical Development ◽  
Therapeutic Window ◽  
Collagen Induced Arthritis

Background:Janus kinases (JAKs) are important regulators of intracellular responses triggered by many key proinflammatory cytokines and are clinically validated therapeutic targets for treating various autoimmune diseases. However, current approved JAK inhibitors failed to achieve maximal clinical benefit in part due to their unfavorable selectivity for individual JAKs such as JAK2 and/or JAK3, leading to dose-limiting toxicities or severe toxicities (e.g., thrombosis, anemia, immune suppression). Selective inhibition of JAK1 and/or TYK2 may minimize or avoid some of the toxicities and potentially offer a better therapeutic window for treating autoimmune diseases. No highly selective JAK1/TYK2 inhibitor has been reported to date.Objectives:Discovery of a highly selective JAK1/TYK2 inhibitor that maximally avoids JAK2 and JAK3 inhibition. We described preclinical characterization of a novel, highly potent and selective JAK1/TYK2 inhibitor TLL018 and its potential utility in treating autoimmune diseases such as rheumatoid arthritis (RA).Methods:Using predicting SAR, TLL018 was designed to achieve exquisite selectivity for both JAK1 and TYK2 while sparing JAK2, JAK3 and other human kinases. Its enzyme and cell activities, kinase selectivity, andin vivoefficacy were assessed in a battery of relevant enzyme, cell and whole blood assays, andin vivoarthritis animal models. Additional preclinical DMPK and toxicology studies were conducted to support its clinical development.Results:TLL018 is a highly potent and selective, orally bioavailable JAK1/TYK2 inhibitor against JAK1 (IC50= 4 nM) and TYK2 (IC50= 5 nM) as measured inin vitrokinase assays with ATP concentrations at individual Km. Its potency against JAK2 or JAK3 is greater than 1 µM. Profiling against a panel of over 350 human kinase showed that TLL018 is exclusively selective for JAK1 and TYK2, with ≥ 90-fold selectivity against all other kinases tested. TLL018 exhibited potent cellular activity for JAK1-mediated IL-6 signaling (IC50= 0.6 µM) with greater than 100-fold selectivity against JAK2-mediated cytokine (e.g., TPO) signaling in human whole blood-based assays.Oral administration of TLL018 demonstrated dose-dependent efficacy in commonly studied rat adjuvant-induced arthritis (rAIA) model and mouse collagen-induced arthritis (mCIA) model. Significant inhibition of inflammation, bone resorption, splenomegaly and body weight change was observed in adjuvant-induced disease in rats. In addition, significant inhibition of inflammation, cartilage destruction, bone resorption and histological signs was demonstrated in collagen-induced arthritis in mice. Noticeably, TLL018 exhibited significant anti-inflammation activity at doses that only blocked JAK1 and TYK2 and exerted little inhibition of JAK2 and JAK3.In support of clinical development of TLL018, preclinical ADME and PK studies and IND-enabling toxicology and safety pharmacology studies were completed, confirming that TLL018 possesses excellent ADME and PK properties, and exhibits a clean on-target safety profile.Conclusion:TLL018 is a highly potent and selective JAK1/TYK2 inhibitor that demonstrated excellent efficacy and tolerability in relevant mouse and rat arthritis models. The collective data of its preclinical pharmacology, PK and toxicology showed a favorable pharmaceutical profile, further supporting its development for treating autoimmune diseases including RA. Clinical evaluation of TLL018 is ongoing.Disclosure of Interests:Xiangdong Liu Shareholder of: I own shares of TLL Pharmaceutical LLC, Employee of: I am employed by TLL Pharmaceutical LLC, Fenlai Tan Shareholder of: I own shares of TLL Pharmaceutical LLC, Employee of: I am employed by TLL Pharmaceutical LLC, Chris Liang Shareholder of: I own shares of TLL Pharmaceutical LLC, Employee of: I am employed by TLL Pharmaceutical LLC

scholarly journals AB0095 PRECLINICAL CHARACTERIZATION OF CJ-15314, A HIGHLY SELECTIVE JAK1 INHIBITOR, FOR THE TREATMENT OF AUTOIMMUNE DISEASES

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1347.2-1347
Author(s):  
S. Y. Ki ◽  
H. Shin ◽  
Y. Lee ◽  
H. R. Bak ◽  
H. Yu ◽  
...  
Keyword(s):  
Autoimmune Disease ◽  
Autoimmune Diseases ◽  
Whole Blood ◽  
Inflammatory Diseases ◽  
Selective Inhibition ◽  
Janus Kinase ◽  
Dependent Manner ◽  
Human Whole Blood

Background:Janus kinases (JAK1, JAK2, JAK3, and TYK2) play critical roles in mediating various cytokine signaling, and has been developed as a target for autoimmune diseases such as RA. Tofacitinib, oral Pan-JAK inhibitor, demonstrated efficacy in RA patients, but its widespread use is limited by safety issues. Baricitinib, JAK1/2 inhibitor, is also known to interfere with the hematopoiesis system, such as anemia and thrombocytopenia associated with suppression of JAK2 signals. Therefore, it is necessary to develop a new potent compound that selectively inhibits JAK1 over JAK2, 3Objectives:To identify the pharmacological characteristic based on efficacy of CJ-15314 as potent and selective JAK1 inhibitor for treatment of autoimmune disease.Methods:In vitro, cell-based, kinase panel, Kd value and human whole blood assay were performed to determine the inhibition potency and selectivity for JAK subfamily kinases. In vivo therapeutic potential was evaluated by RA model including rat Adjuvant-Induced Arthritis (AIA) and collagen-induced arthritic (CIA). To confirm the possibility of further expansion into the autoimmune disease, BioMAP® Diversity PLUS® Panel was performed by discoverX.Results:In vitro assay, CJ-15314 inhibited JAK kinase family in a concentration-dependent manner with IC50 values of 3.8 nM against JAK1, Selectivity for JAK1 over JAK2, 3 was approximately 18, 83 fold greater for CJ-15314. In 1mM ATP condition, CJ-15314 has been confirmed to have the highest JAK1 selectivity over competing drugs, under 1 mM ATP condition that reflects the physiological environment in the body. Similarly, Kd values has also confirmed the selectivity of JAK1, which is 10 fold higher than JAK2, 3. Accordingly, in human whole blood assays, CJ-15314 is 11 fold more potent against IL-6 induced pSTAT1 inhibition through JAK1 (IC50 value: 70 nM) than GM-CSF-induced pSTAT5 inhibition (JAK2) whereas baricitinib and filgotinib exhibited only 2 fold and 7 fold respectively.In vivo efficacy model, CJ-15314 inhibited disease severity scores in a dose dependent manner. In the rat AIA model, CJ-15314 at 30 mg/kg dose showed 95.3% decrease in arthritis activity score, 51.2% in figotinib at 30 mg/kg, 97.7% showed baricitinib at 10 mg/kg. CJ-15314 showed superior anti-arthritic efficacy than filgotinib. CJ-15314 also minimally affected anemia-related parameters but not bricitinib end of the 2-week treatment. In the rat CIA model, like 10 mg/kg of bricitinib, 30 mg/kg of CJ-15314 also has a similar effect, with a significant reduction in histopathological scores.In biomap diversity panel, CJ-15314 inhibited the expression of genes such as MCP-1, VCAM-1, IP-10, IL-8, IL-1, sTNF-α and HLA-DR confirming the possibility of expansion into other diseases beyond arthritis.Conclusion:CJ-15314 is a highly selective JAK1 inhibitor, demonstrates robust efficacy in RA animal model and is good candidate for further development for inflammatory diseases.* CJ-15314 is currently conducting a phase I trial in south Korea.References:[1]Clark JD et al. Discovery and development of Janus kinase (JAK) inhibitors for inflammatory diseases. J Med Chem. 2014; 57(12):5023-38.[2]Burmester GR et al. Emerging cell and cytokine targets in rheumatoid arthritis. Nat Rev Rheumatol. 2014; 10(2):77-88[3]Jean-Baptiste Telliez et al. Discovery of a JAK3-selective inhibitor: functional differentiation of JAK3-selective inhibition over pan-JAK or JAK1-selective inhibition. ACS Chem. Biol., 2016; 11 (12):3442-3451Disclosure of Interests:so young Ki Employee of: CJ healthcare, hyunwoo shin Employee of: CJ healthcare, yelim lee Employee of: CJ healthcare, Hyoung rok Bak Employee of: CJ healthcare, hana yu Employee of: CJ healthcare, Seung Chan Kim Employee of: CJ healthcare, juhyun lee Employee of: CJ healthcare, donghyun kim Employee of: CJ healthcare, Dong-hyun Ko Employee of: CJ Healthcare, dongkyu kim Employee of: CJ healthcare

scholarly journals Characterization of bone resorption in novel in vitro and in vivo models of oral squamous cell carcinoma

Oral Oncology ◽  
2012 ◽  
Vol 48 (6) ◽  
pp. 491-499 ◽  
Author(s):  
Chelsea K. Martin ◽  
Wessel P. Dirksen ◽  
Sherry T. Shu ◽  
Jillian L. Werbeck ◽  
Nanda K. Thudi ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Bone Resorption ◽  
Oral Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
In Vivo Models

scholarly journals Antibody to Receptor Activator of NF-κB Ligand Ameliorates T Cell-Mediated Periodontal Bone Resorption

2010 ◽  
Vol 79 (2) ◽  
pp. 911-917 ◽  
Author(s):  
Xiaoping Lin ◽  
Xiaozhe Han ◽  
Toshihisa Kawai ◽  
Martin A. Taubman
Keyword(s):  
T Cell ◽  
Bone Resorption ◽  
Alveolar Bone ◽  
Immune Cell ◽  
Significant Inhibition ◽  
Dependent Manner ◽  
Igg Antibody ◽  
Receptor Activator

ABSTRACTActivated T and B lymphocytes in periodontal disease lesions express receptor activator of NF-κB ligand (RANKL), which induces osteoclastic bone resorption. The objective of this study was to evaluate the effects of anti-RANKL antibody on periodontal bone resorptionin vitroandin vivo. Aggregatibacter actinomycetemcomitansouter membrane protein 29 (Omp29) andA. actinomycetemcomitanslipopolysaccharide (LPS) were injected into 3 palatal gingival sites, and Omp29-specific T clone cells were transferred into the tail veins of rats. Rabbit anti-RANKL IgG antibody or F(ab′)2antibody fragments thereof were injected into the palatal sites in each rat (days −1, 1, and 3). Anti-RANKL IgG antibody significantly inhibited soluble RANKL (sRANKL)-induced osteoclastogenesisin vitro, in a dose-dependent manner, but also gave rise to a rat antibody response to rabbit IgGin vivo, with no significant inhibition of periodontal bone resorption detected. Lower doses (1.5 and 0.15 μg/3 sites) of F(ab′)2antibody were not immunogenic in the context of the experimental model. Periodontal bone resorption was inhibited significantly by injection of the anti-RANKL F(ab′)2antibody into gingivae. The sRANKL concentrations for the antibody-treated groups were decreased significantly compared to those for the untreated group. Osteoclasts on the alveolar bone surface were also diminished significantly after antibody injection. Gingival sRANKL concentration and bone loss showed a significant correlation with one another in animals receiving anti-RANKL F(ab′)2antibody. These results suggest that antibody to RANKL can inhibitA. actinomycetemcomitans-specific T cell-induced periodontal bone resorption by blockade and reduction of tissue sRANKL, providing an immunological approach to ameliorate immune cell-mediated periodontal bone resorption.

scholarly journals Preventive CCL2/CCR2 Axis Blockade Suppresses Osteoclast Activity in a Mouse Model of Rheumatoid Arthritis by Reducing Homing of CCR2hi Osteoclast Progenitors to the Affected Bone

2021 ◽  
Vol 12 ◽  
Author(s):  
Darja Flegar ◽  
Maša Filipović ◽  
Alan Šućur ◽  
Antonio Markotić ◽  
Nina Lukač ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Clinical Score ◽  
Differentiation Potential ◽  
Ccr2 Expression ◽  
Molecule Inhibitor ◽  
Intravascular Staining

Detailed characterization of medullary and extramedullary reservoirs of osteoclast progenitors (OCPs) is required to understand the pathophysiology of increased periarticular and systemic bone resorption in arthritis. In this study, we focused on identifying the OCP population specifically induced by arthritis and the role of circulatory OCPs in inflammatory bone loss. In addition, we determined the relevant chemokine axis responsible for their migration, and targeted the attraction signal to reduce bone resorption in murine collagen-induced arthritis (CIA). OCPs were expanded in periarticular as well as circulatory compartment of arthritic mice, particularly the CCR2hi subset. This subset demonstrated enhanced osteoclastogenic activity in arthritis, whereas its migratory potential was susceptible to CCR2 blockade in vitro. Intravascular compartment of the periarticular area contained increased frequency of OCPs with the ability to home to the arthritic bone, as demonstrated in vivo by intravascular staining and adoptive transfer of splenic LysMcre/Ai9 tdTomato-expressing cells. Simultaneously, CCL2 levels were increased locally and systemically in arthritic mice. Mouse cohorts were treated with the small-molecule inhibitor (SMI) of CCR2 alone or in combination with methotrexate (MTX). Preventive CCR2/CCL2 axis blockade in vivo reduced bone resorption and OCP frequency, whereas combining with MTX treatment also decreased disease clinical score, number of active osteoclasts, and OCP differentiation potential. In conclusion, our study characterized the functional properties of two distinct OCP subsets in CIA, based on their CCR2 expression levels, implying that the CCR2hi circulatory-like subset is specifically induced by arthritis. Signaling through the CCL2/CCR2 axis contributes to OCP homing in the inflamed joints and to their increased osteoclastogenic potential. Therefore, addition of CCL2/CCR2 blockade early in the course of arthritis is a promising approach to reduce bone pathology.

The Development of Homologous (Canine/Anti-Canine) Antibodies in Dogs with Haemophilia A (Factor VIII Deficiency): A Ten-Year Longitudinal Study

1993 ◽  
Vol 69 (01) ◽  
pp. 021-024 ◽  
Author(s):  
Shawn Tinlin ◽  
Sandra Webster ◽  
Alan R Giles
Keyword(s):  
Factor Viii ◽  
Canine Model ◽  
Significant Inhibition ◽  
Human Condition ◽  
Haemophilia A ◽  
Variable Expression ◽  
The Family ◽  
Over Time

SummaryThe development of inhibitors to factor VIII in patients with haemophilia A remains as a serious complication of replacement therapy. An apparently analogous condition has been described in a canine model of haemophilia A (Giles et al., Blood 1984; 63:451). These animals and their relatives have now been followed for 10 years. The observation that the propensity for inhibitor development was not related to the ancestral factor VIII gene has been confirmed by the demonstration of vertical transmission through three generations of the segment of the family related to a normal (non-carrier) female that was introduced for breeding purposes. Haemophilic animals unrelated to this animal have not developed functionally significant factor VIII inhibitors despite intensive factor VIII replacement. Two animals have shown occasional laboratory evidence of factor VIII inhibition but this has not been translated into clinical significant inhibition in vivo as assessed by clinical response and F.VIII recovery and survival characteristics. Substantial heterogeneity of inhibitor expression both in vitro and in vivo has been observed between animals and in individual animals over time. Spontaneous loss of inhibitors has been observed without any therapies designed to induce tolerance, etc., being instituted. There is also phenotypic evidence of polyclonality of the immune response with variable expression over time in a given animal. These observations may have relevance to the human condition both in determining the pathogenetic factors involved in this condition and in highlighting the heterogeneity of its expression which suggests the need for caution in the interpretation of the outcome of interventions designed to modulate inhibitor activity.

Dipyridamole Inhibits Platelet Aggregation in Whole Blood

1983 ◽  
Vol 50 (04) ◽  
pp. 852-856 ◽  
Author(s):  
P Gresele ◽  
C Zoja ◽  
H Deckmyn ◽  
J Arnout ◽  
J Vermylen ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Significant Negative Correlation ◽  
Significant Inhibition ◽  
Oral Drug ◽  
Human Blood Samples ◽  
Induced Aggregation

SummaryDipyridamole possesses antithrombotic properties in the animal and in man but it does not inhibit platelet aggregation in plasma. We evaluated the effect of dipyridamole ex vivo and in vitro on platelet aggregation induced by collagen and adenosine- 5’-diphosphate (ADP) in human whole blood with an impedance aggregometer. Two hundred mg dipyridamole induced a significant inhibition of both ADP- and collagen-induced aggregation in human blood samples taken 2 hr after oral drug intake. Administration of the drug for four days, 400 mg/day, further increased the antiplatelet effect. A significant negative correlation was found between collagen-induced platelet aggregation in whole blood and dipyridamole levels in plasma (p <0.001). A statistically significant inhibition of both collagen (p <0.0025) and ADP-induced (p <0.005) platelet aggregation was also obtained by incubating whole blood in vitro for 2 min at 37° C with dipyridamole (3.9 μM). No such effects were seen in platelet-rich plasma, even after enrichment with leukocytes. Low-dose adenosine enhanced in vitro inhibition in whole blood.Our results demonstrate that dipyridamole impedes platelet aggregation in whole blood by an interaction with red blood cells, probably involving adenosine.

Репрограммированые in vitro на М3 фенотип макрофаги останавливают рост солидной карциномы in vivo

2018 ◽  
pp. 41-46
Author(s):  
А.А. Раецкая ◽  
С.В. Калиш ◽  
С.В. Лямина ◽  
Е.В. Малышева ◽  
О.П. Буданова ◽  
...  
Keyword(s):  
Solid Tumor ◽  
Antitumor Effect ◽  
Tumor Development ◽  
Significant Inhibition ◽  
The Body ◽  
Ehrlich Carcinoma ◽  
Solid Carcinoma ◽  
Ec Cells

Цель исследования. Доказательство гипотезы, что репрограммированные in vitro на М3 фенотип макрофаги при введении в организм будут существенно ограничивать развитие солидной карциномы in vivo . Методика. Рост солидной опухоли инициировали у мышей in vivo путем подкожной инъекции клеток карциномы Эрлиха (КЭ). Инъекцию макрофагов с нативным М0 фенотипом и с репрограммированным M3 фенотипом проводили в область формирования солидной КЭ. Репрограммирование проводили с помощью низких доз сыворотки, блокаторов факторов транскрипции STAT3/6 и SMAD3 и липополисахарида. Использовали две схемы введения макрофагов: раннее и позднее. При раннем введении макрофаги вводили на 1-е, 5-е, 10-е и 15-е сут. после инъекции клеток КЭ путем обкалывания макрофагами с четырех сторон область развития опухоли. При позднем введении, макрофаги вводили на 10-е, 15-е, 20-е и 25-е сут. Через 15 и 30 сут. после введения клеток КЭ солидную опухоль иссекали и измеряли ее объем. Эффект введения макрофагов оценивали качественно по визуальной и пальпаторной характеристикам солидной опухоли и количественно по изменению ее объема по сравнению с группой без введения макрофагов (контроль). Результаты. Установлено, что M3 макрофаги при раннем введении от начала развития опухоли оказывают выраженный антиопухолевый эффект in vivo , который был существенно более выражен, чем при позднем введении макрофагов. Заключение. Установлено, что введение репрограммированных макрофагов M3 ограничивает развитие солидной карциномы в экспериментах in vivo . Противоопухолевый эффект более выражен при раннем введении М3 макрофагов. Обнаруженные в работе факты делают перспективным разработку клинической версии биотехнологии ограничения роста опухоли, путем предварительного программирования антиопухолевого врожденного иммунного ответа «в пробирке». Aim. To verify a hypothesis that macrophages reprogrammed in vitro to the M3 phenotype and injected into the body substantially restrict the development of solid carcinoma in vivo . Methods. Growth of a solid tumor was initiated in mice in vivo with a subcutaneous injection of Ehrlich carcinoma (EC) cells. Macrophages with a native M0 phenotype or reprogrammed towards the M3 phenotype were injected into the region of developing solid EC. Reprogramming was performed using low doses of serum, STAT3/6 and SMAD3 transcription factor blockers, and lipopolysaccharide. Two schemes of macrophage administration were used: early and late. With the early administration, macrophages were injected on days 1, 5, 10, and 15 following the injection of EC cells at four sides of the tumor development area. With the late administration, macrophages were injected on days 10, 15, 20, and 25. At 15 and 30 days after the EC cell injection, the solid tumor was excised and its volume was measured. The effect of macrophage administration was assessed both qualitatively by visual and palpation characteristics of solid tumor and quantitatively by changes in the tumor volume compared with the group without the macrophage treatment. Results. M3 macrophages administered early after the onset of tumor development exerted a pronounced antitumor effect in vivo , which was significantly greater than the antitumor effect of the late administration of M3 macrophages. Conclusion. The observed significant inhibition of in vivo growth of solid carcinoma by M3 macrophages makes promising the development of a clinical version of the biotechnology for restriction of tumor growth by in vitro pre-programming of the antitumor, innate immune response.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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