TGF-β receptor type I (TGF-βRI) kinase (IC50 = 56 nM)

In SK-Sora, HepG2, and Hep3B cell lines, galunisertib (LY2157299) (0.1, 1, 10, and 100 μM) somewhat potentiates Bay 43-9006 in a dose-dependent manner; however, this effect is not observed in JHH6, SK-HEP1, or HuH7 cell lines[2].

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Galunisertib (LY2157299) is a selective ATP-mimetic inhibitor of TGF-β receptor (TβR)-I activation currently under clinical investigation in hepatocellular carcinoma (HCC) patients. Our study explored the effects of galunisertib in vitro in HCC cell lines and ex vivo on patient samples. Galunisertib was evaluated in HepG2, Hep3B, Huh7, JHH6 and SK-HEP1 cells as well as in SK-HEP1-derived cells tolerant to sorafenib (SK-Sora) and sunitinib (SK-Suni). Exogenous stimulation of all HCC cell lines with TGF-β yielded downstream activation of p-Smad2 and p-Smad3 that was potently inhibited with galunisertib treatment at micromolar concentrations. Despite limited antiproliferative effects, galunisertib yielded potent anti-invasive properties. Tumor slices from 13 patients with HCC surgically resected were exposed ex vivo to 1 µM and 10 µM galunisertib, 5 µM sorafenib or a combination of both drugs for 48 hours. Galunisertib but not sorafenib decreased p-Smad2/3 downstream TGF-β signaling. Immunohistochemistry analysis of galunisertib and sorafenib-exposed samples showed a significant decrease of the proliferative marker Ki67 and increase of the apoptotic marker caspase-3. In combination, galunisertib potentiated the effect of sorafenib efficiently by inhibiting proliferation and increasing apoptosis.

Subcutaneous implants of human xenografts, Calu6 (non-small cell lung cancer) and MX1 (breast cancer), are made in nude mice. Galunisertib (LY2157299) causes a 70% reduction in pSmad for both kinds of cell lines when taken orally at a dose of 75 mg/kg. The recovery of pSmad to 80% of baseline occurred about 6 hours post-administration [3].

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Human xenografts Calu6 (non-small cell lung cancer) and MX1 (breast cancer) were implanted subcutaneously in nude mice and LY2157299, a new type I receptor TGF-beta kinase antagonist, was administered orally. Plasma levels of LY2157299, percentage of phosphorylated Smad2,3 (pSmad) in tumour, and tumour size were used to establish a semi-mechanistic pharmacokinetic/pharmacodynamic model. An indirect response model was used to relate plasma concentrations with pSmad. The model predicts complete inhibition of pSmad and rapid turnover rates [t(1/2) (min)=18.6 (Calu6) and 32.0 (MX1)]. Tumour growth inhibition was linked to pSmad using two signal transduction compartments characterised by a mean signal propagation time with estimated values of 6.17 and 28.7 days for Calu6 and MX1, respectively. The model provides a tool to generate experimental hypothesis to gain insights into the mechanisms of signal transduction associated to the TGF-beta membrane receptor type I.[3]

Recently, kinase inhibitors have shown great potential against fibrotic diseases and, specifically, the transforming growth factor-β receptor (TGF-βR) was found as a new and promising target for scleroderma therapy. In the current study, we propose that the large pool of existing kinase inhibitors could be exploited for inhibiting the TGF-βR to suppress scleroderma. In this respect, we developed a modeling protocol to systematically profile the inhibitory activities of 169 commercially available kinase inhibitors against the TGF-βR, from which five promising candidates were selected and tested using a standard kinase assay protocol. Consequently, two molecular entities, namely the PKB inhibitor MK-2206 and the mTOR C1/C2 inhibitor AZD8055, showed high potency when bound to the TGF-βR, with IC50 values of 97 and 86 nM, respectively, which are close to those of the recently developed TGF-βR selective inhibitors SB525334 and galunisertib/LY2157299 (IC50 = 14.3 and 56 nM, respectively). We also performed atomistic molecular dynamics simulations and post-molecular mechanics/Poisson–Boltzmann surface area analyses to dissect the structural basis and energetic properties of intermolecular interactions between the TGF-βR kinase domain and these potent compounds, highlighting intensive nonbonded networks across the tightly packed interface of non-cognate TGF-βR-inhibitor complexes[1].

Cell cytotoxicity assay[2]
Cell survival was determined using the MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide). The conversion of yellow water-soluble tetrazolium MTT into purple insoluble formazan is catalyzed by mitochondrial dehydrogenases and used to estimate the number of viable cells. In brief, cells were seeded in 96-well tissue culture plates at a density of 2 × 103 cells/well. After drug exposure, cells were incubated with 0.4 mg/mL MTT for 4 hours at 37°C. After incubation, the supernatant was discarded, insoluble formazan precipitates were dissolved in 0.1 mL of DMSO, and the absorbance was measured at 560 nm by use of a microplate reader. Wells with untreated cells or with drug-containing medium without cells were used as positive and negative controls respectively. For proliferation assay, MTT assay was done daily to determine the number of viable cells in untreated control and galunisertib-treated group.

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Ex Vivo tissue profiling (TIPCAN®)[2]
The effects of galunisertib were tested on freshly resected tumors from HCC patients which can be cultured alive in specific conditions of culture medium and atmosphere, depending on available tumor resection from the surgical department. After pathological evaluation by the hospital pathologist, the tumor samples were extemporaneously sliced using Tissue Slicer® instrument into 300μm-thick slices and cultured “alive” at 37°C into the William’s E medium, complemented with in-house proprietary dedicated components including foetal calf serum, glucose, gentamicin and HEPES, under normoxic conditions. The samples were prepared using tissue-slicer technology and treated for 24 to 72 hours with 1 and 10 μM galunisertib or 5 μM sorafenib. After 24 to 72 hours treatment, the explanted HCC was paraffin embedded and assessed for expression of selected markers. The tests comprised assessment of cancer cell proliferation (MIB1/Ki67), death (active caspase- 3), and several changes in cell signalling (phospho-kinases). Tissue quality was assessed by a pathologist. If tissue integrity was not maintained over time (>20% necrosis induction), tissues were discarded.

PK/PD experiments[3]
Calu6[3]
LY2157299 was given orally as a single dose (data from eight independent studies were combined) or in a multiple dosing design (one study). The value of the dose levels given in a single dose manner was 10 (n = 3), 30 (n = 8), 50 (n = 26), 75 (n = 69), 100 (n = 3), 150 (n = 21) and 300 (n = 3) mg/kg. Animals were sacrificed at the following times: 0.5, 1, 1.5, 2, 4, 8 and 16 h after administration, then the tumour was removed and blood was recovered. In the multiple dosing study, LY2157299 was administered twice a day (bid) at the dose of 75 mg/kg every 12 h for 20 consecutive days to 31 mice. Animals were sacrificed at 2 h after the last administration at days 10, 15, 20 and 25, and the tumour was removed for pSmad determination and the blood was recovered for determination of drug levels in plasma. [3]

MX1[3]
Twelve mice involved in a single study were treated with a single 75 mg/kg dose of LY2157299. Animals were sacrificed at 0.5, 1, 2, 4 and 16 h after drug administration, tumours were removed and the blood was collected. [3]

Determination of LY2157299 in plasma[3]
Venous blood samples (1 ml) was drawn into sodium-heparinised tubes for measurement of LY2157299. Plasma samples were analysed using a validated method involving protein precipitation with turbo ion spray LC/MS/MS detection. The validated range of measurement in plasma was 5–1000 ng/ml (a 50-fold dilution was validated to demonstrate the ability of the assay to analyse samples at higher concentrations). The value of the limit of quantification of the assay was 1.14 ng/ml. The accuracy of the assay was <15% and the intra and interassay coefficients of variation were less than 10%.[3]

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Tumour growth experiments[3]
Calu6[3]
Data from two studies are presented. The first available data came from a study where 20 mice were treated bid with either saline (control group; n = 10) or 75 mg/kg of LY2157299 (treated group; n = 10) for 20 consecutive days. Tumour size was measured every 4–6 days for one month after the first drug administration and afterwards the animals were sacrificed. The data from this study were used to develop the tumour growth model (index dataset). Later, data from a second study also became available and were used for model validation purposes (test dataset). Seventy-six mice were treated bid with either saline (control group; n = 36) or 75 mg/kg of LY2157299 for 10 (n = 10) 15 (n = 10) or 20 (n = 20) consecutive days. Tumour size was measured once a week for one month.

MX1[3]
Data were obtained from a single study where mice were treated three times a day with either saline (control group, n = 10) or 75 mg/kg of LY2157299 (treated group; n = 10) for 20 consecutive days. Tumour size was measured every 3–4 days for one month after the first drug administration and afterwards the animals were killed.

Dissolved in DMSO and diluted in saline; 75 mg/kg/day; oral gavage

Nude mice implanted subcutaneously with Calu6 or MX1 cells

Pharmacokinetic parameters were determined for patients administered galunisertib during the first 14 days of the 28-day intermittent treatment cycle (2 weeks on/2 weeks off schedule). The PK profile of galunisertib was characterized by rapid absorption, with median t max ranging from 0.5 to 2 h following oral dosing with 80 or 150 mg BID (Fig. 2). At steady state, on Day 14 in Cycle 1, the mean t 1/2 was 8.90 h and the mean CLss/F and Vz,ss/F during the terminal phase were 30.2 L/h and 388 L, respectively, for 150 mg BID (Table 3). Although the number of patients in the 2 cohorts was small and imbalanced (Cohort 1, n = 3; Cohort 2, n = 9), high interpatient variability for galunisertib exposure [AUC(0−48) coefficient of variation (CV) %] was observed (Cohort 1 CV % = 35 %; Cohort 2 CV % = 88 %). [5]

Galunisertib administered to 12 Japanese patients with advanced solid tumors was well tolerated and had a favorable safety profile; no DLTs or cardiovascular toxicities were reported. Dose escalation was successfully performed within the 2 dosing cohorts (80 and 150 mg BID) and galunisertib exposure data confirmed that exposure could be maintained within the predefined therapeutic window for the majority of patients during treatment with galunisertib. All patients completed at least one cycle of galunisertib treatment before discontinuing due to disease progression; no patients had a clinical response to treatment, however, two patients had stable disease. [5]

The favorable tolerability and safety profile of 80 and 150 mg BID doses of galunisertib in Japanese patients was confirmed based on the TEAE profile reported during the study. Overall, there were no CTCAE Grade ≥3 study drug-related toxicities reported. Possible drug-related TEAEs included two patients with increased BNP levels, two patients with leukopenia, and two patients with rash. The two patients with increased BNP (Grade 1) did not experience any cardiotoxicities, and no febrile neutropenia was reported for any patient. Possible study drug-related leukopenia (n = 3 events) was also reported in the FHD study in patients who received a combination of galunisertib and lomustine; however, causality could not be specifically attributed to either drug. Therefore, it is unclear if the reported leukopenia was related to galunisertib treatment.

[1]. Targeting the TGF-β receptor with kinase inhibitors for scleroderma therapy. Arch Pharm (Weinheim). 2014 Sep;347(9):609-15.

[2]. Effects of TGF-beta signalling inhibition with galunisertib (LY2157299) in hepatocellular carcinoma models and in ex vivo whole tumor tissue samples from patients. Oncotarget. 2015 Aug 28;6(25):21614-27.

[3]. Semi-mechanistic modelling of the tumour growth inhibitory effects of LY2157299, a new type I receptor TGF-beta kinase antagonist, in mice. Eur J Cancer. 2008 Jan;44(1):142-50.

[4]. Clinical development of galunisertib (LY2157299 monohydrate), a small molecule inhibitor of transforming growth factor-beta signaling pathway. Drug Des Devel Ther. 2015 Aug 10;9:4479-99.

[5]. Phase 1 study of galunisertib, a TGF-beta receptor I kinase inhibitor, in Japanese patients with advanced solid tumors. Cancer Chemother Pharmacol . 2015 Dec;76(6):1143-52.

LY-2157299 is a pyrrolopyrazole that is 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole which is substituted at positions 2 and 3 by 6-methylpyridin-2-yl and 6-(aminocarbonyl)quinolin-4-yl groups, respectively. A Transforming growth factor-betaRI (TGF-betaRI) kinase inhibitor, it blocks TGF-beta-mediated tumor growth in glioblastoma. It has a role as a TGFbeta receptor antagonist and an antineoplastic agent. It is a member of quinolines, a pyrrolopyrazole, a member of methylpyridines, an aromatic amide and a monocarboxylic acid amide.
Galunisertib has been used in trials studying the basic science and treatment of Glioma, Neoplasms, Solid Tumor, GLIOBLASTOMA, and Prostate Cancer, among others.
Galunisertib is an orally available, small molecule antagonist of the tyrosine kinase transforming growth factor-beta (TGF-b) receptor type 1 (TGFBR1), with potential antineoplastic activity. Upon administration, galunisertib specifically targets and binds to the kinase domain of TGFBR1, thereby preventing the activation of TGF-b-mediated signaling pathways. This may inhibit the proliferation of TGF-b-overexpressing tumor cells. Dysregulation of the TGF-b signaling pathway is seen in a number of cancers and is associated with increased cancer cell proliferation, migration, invasion and tumor progression.

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Galunisertib (LY2157299) is a selective ATP-mimetic inhibitor of TGF-β receptor (TβR)-I activation currently under clinical investigation in hepatocellular carcinoma (HCC) patients. Our study explored the effects of galunisertib in vitro in HCC cell lines and ex vivo on patient samples. Galunisertib was evaluated in HepG2, Hep3B, Huh7, JHH6 and SK-HEP1 cells as well as in SK-HEP1-derived cells tolerant to sorafenib (SK-Sora) and sunitinib (SK-Suni). Exogenous stimulation of all HCC cell lines with TGF-β yielded downstream activation of p-Smad2 and p-Smad3 that was potently inhibited with galunisertib treatment at micromolar concentrations. Despite limited antiproliferative effects, galunisertib yielded potent anti-invasive properties. Tumor slices from 13 patients with HCC surgically resected were exposed ex vivo to 1 µM and 10 µM galunisertib, 5 µM sorafenib or a combination of both drugs for 48 hours. Galunisertib but not sorafenib decreased p-Smad2/3 downstream TGF-β signaling. Immunohistochemistry analysis of galunisertib and sorafenib-exposed samples showed a significant decrease of the proliferative marker Ki67 and increase of the apoptotic marker caspase-3. In combination, galunisertib potentiated the effect of sorafenib efficiently by inhibiting proliferation and increasing apoptosis. Our data suggest that galunisertib may be active in patients with HCC and could potentiate the effects of sorafenib.[2]

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Human xenografts Calu6 (non-small cell lung cancer) and MX1 (breast cancer) were implanted subcutaneously in nude mice and LY2157299, a new type I receptor TGF-beta kinase antagonist, was administered orally. Plasma levels of LY2157299, percentage of phosphorylated Smad2,3 (pSmad) in tumour, and tumour size were used to establish a semi-mechanistic pharmacokinetic/pharmacodynamic model. An indirect response model was used to relate plasma concentrations with pSmad. The model predicts complete inhibition of pSmad and rapid turnover rates [t(1/2) (min)=18.6 (Calu6) and 32.0 (MX1)]. Tumour growth inhibition was linked to pSmad using two signal transduction compartments characterised by a mean signal propagation time with estimated values of 6.17 and 28.7 days for Calu6 and MX1, respectively. The model provides a tool to generate experimental hypothesis to gain insights into the mechanisms of signal transduction associated to the TGF-beta membrane receptor type I.[3]

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Transforming growth factor-beta (TGF-β) signaling regulates a wide range of biological processes. TGF-β plays an important role in tumorigenesis and contributes to the hallmarks of cancer, including tumor proliferation, invasion and metastasis, inflammation, angiogenesis, and escape of immune surveillance. There are several pharmacological approaches to block TGF-β signaling, such as monoclonal antibodies, vaccines, antisense oligonucleotides, and small molecule inhibitors. Galunisertib (LY2157299 monohydrate) is an oral small molecule inhibitor of the TGF-β receptor I kinase that specifically downregulates the phosphorylation of SMAD2, abrogating activation of the canonical pathway. Furthermore, galunisertib has antitumor activity in tumor-bearing animal models such as breast, colon, lung cancers, and hepatocellular carcinoma. Continuous long-term exposure to galunisertib caused cardiac toxicities in animals requiring adoption of a pharmacokinetic/pharmacodynamic-based dosing strategy to allow further development. The use of such a pharmacokinetic/pharmacodynamic model defined a therapeutic window with an appropriate safety profile that enabled the clinical investigation of galunisertib. These efforts resulted in an intermittent dosing regimen (14 days on/14 days off, on a 28-day cycle) of galunisertib for all ongoing trials. Galunisertib is being investigated either as monotherapy or in combination with standard antitumor regimens (including nivolumab) in patients with cancer with high unmet medical needs such as glioblastoma, pancreatic cancer, and hepatocellular carcinoma. The present review summarizes the past and current experiences with different pharmacological treatments that enabled galunisertib to be investigated in patients.[4]

C22H19N5O

369.42

369.158

C, 71.53; H, 5.18; N, 18.96; O, 4.33

700874-72-2

700874-72-2;924898-09-9 (hydrate);

10090485

White to yellow solid powder

1.4±0.1 g/cm3

619.0±55.0 °C at 760 mmHg

328.2±31.5 °C

0.0±1.8 mmHg at 25°C

1.751

1.73

1

4

3

28

585

0

IVRXNBXKWIJUQB-UHFFFAOYSA-N

InChI=1S/C22H19N5O/c1-13-4-2-5-18(25-13)21-20(19-6-3-11-27(19)26-21)15-9-10-24-17-8-7-14(22(23)28)12-16(15)17/h2,4-5,7-10,12H,3,6,11H2,1H3,(H2,23,28)

4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline-6-carboxamide

LY2157299; LY2157299; 4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline-6-carboxamide; UNII-3OKH1W5LZE; ly2157299(galunisertib); LY 2157299

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 5.75 mg/mL (15.56 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.63 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.63 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 4: ≥ 2.08 mg/mL (5.63 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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Solubility in Formulation 5: 2% DMSO+30% PEG 300+ddH2O:5 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)