 Chemical Structure.png)
Galunisertib (formerly known as LY2157299; LY-2157299) is a selective, ATP-mimetic, and orally bioavailable small molecule inhibitor of the TGFβ receptor I (TβRI) with potential anticancer activity. It inhibits TGFβ with an IC50 of 56 nM in cell-free assays. Galunisertib inhibited HCC cell migration on Laminin-5, Fibronectin, Vitronectin, Fibrinogen and Collagen-I and de novo phosphorylation of pSMAD2. Galunisertib inhibited HCC migration and cell growth independently of the expression levels of TGF-βRII. Galunisertib has antitumor activity in tumor-bearing animal models such as breast, colon, lung cancers,and hepatocellular carcinoma. Galunisertib was evaluated by Lilly Pharmaceuticals in multiple clinical trials either as a single agent or in combination with various agents such as gemcitabine, paclitaxel, sorafenib, or durvalumab. Galunisertib was in a phase II clinical trial for treatment of hepatocellular carcinoma but was discontinued in January 2020.
Physicochemical Properties
| Molecular Formula | C22H19N5O |
| Molecular Weight | 369.42 |
| Exact Mass | 369.158 |
| Elemental Analysis | C, 71.53; H, 5.18; N, 18.96; O, 4.33 |
| CAS # | 700874-72-2 |
| Related CAS # | 700874-72-2;924898-09-9 (hydrate); |
| PubChem CID | 10090485 |
| Appearance | White to yellow solid powder |
| Density | 1.4±0.1 g/cm3 |
| Boiling Point | 619.0±55.0 °C at 760 mmHg |
| Flash Point | 328.2±31.5 °C |
| Vapour Pressure | 0.0±1.8 mmHg at 25°C |
| Index of Refraction | 1.751 |
| LogP | 1.73 |
| Hydrogen Bond Donor Count | 1 |
| Hydrogen Bond Acceptor Count | 4 |
| Rotatable Bond Count | 3 |
| Heavy Atom Count | 28 |
| Complexity | 585 |
| Defined Atom Stereocenter Count | 0 |
| InChi Key | IVRXNBXKWIJUQB-UHFFFAOYSA-N |
| InChi Code | 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) |
| Chemical Name | 4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline-6-carboxamide |
| Synonyms | 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 |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
TGF-β receptor type I (TGF-βRI) kinase (IC50 = 56 nM) Galunisertib (LY2157299) specifically targets transforming growth factor-beta (TGF-β) receptor type I (ALK5) (ALK5 IC50 = 56 nM) [3] Galunisertib (LY2157299) shows weak or no inhibition of other ALK receptors (ALK1, ALK2, ALK3, ALK4: IC50 > 1 μM) and unrelated kinases (PKA, PKC, ERK1/2: IC50 > 10 μM) [3][4] |
| ln Vitro |
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]. 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. In human hepatocellular carcinoma (HCC) cell lines (HepG2, Huh7, PLC/PRF/5), Galunisertib (LY2157299) (10 μM) inhibits TGF-β1-induced Smad2 phosphorylation by 80-85% after 24 hours. It downregulates TGF-β target genes (CTGF, PAI-1, Snail) at mRNA level by 60-70% and inhibits cell proliferation by 45-60% after 72 hours (MTT assay) [2] - In human dermal fibroblasts isolated from scleroderma patients, Galunisertib (LY2157299) (5 μM) reduces TGF-β1-induced collagen type I synthesis by 65% and α-SMA expression by 70% at protein level, suppressing myofibroblast differentiation [1] - In ex vivo whole tumor tissue samples from HCC patients, Galunisertib (LY2157299) (20 μM) inhibits Smad2 phosphorylation by 75% and reduces the expression of proliferation marker Ki-67 by 50% after 48 hours of culture [2] - In normal human hepatocytes (NHHs), Galunisertib (LY2157299) shows low toxicity at concentrations up to 50 μM (cell viability > 85% vs. control) [2][5] |
| ln Vivo |
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]. 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] In nude mice bearing subcutaneous Huh7 HCC xenografts, oral administration of Galunisertib (LY2157299) (100 mg/kg/day for 21 days) significantly inhibits tumor growth. Tumor volume was reduced by 62% compared to vehicle-treated mice, and tumor weight decreased by 58%. Tumor tissues show downregulated p-Smad2 (70% reduction) and Ki-67 (55% reduction) [2][3] - In a mouse model of liver fibrosis induced by CCl₄, oral Galunisertib (LY2157299) (50 mg/kg/day for 8 weeks) reduces hepatic collagen deposition by 55% and α-SMA-positive myofibroblasts by 60%, improving liver function (ALT and AST levels reduced by 40-45%) [1] - In rats bearing orthotopic HCC xenografts, intraperitoneal administration of Galunisertib (LY2157299) (75 mg/kg/day for 14 days) inhibits tumor invasiveness, with metastatic nodules in the liver reduced by 65% [2] |
| Enzyme Assay |
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]. ALK5 kinase activity assay: Purified recombinant human ALK5 was incubated with Smad2-derived substrate peptide and Galunisertib (LY2157299) (0.1 nM-1 μM) in assay buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.1 mM ATP) at 30°C for 60 minutes. Phosphorylated substrate was detected by radiolabeled ATP counting, and IC50 values were calculated from dose-response curves [3] - Kinase selectivity assay: Galunisertib (LY2157299) (10 μM) was screened against a panel of 50+ kinases (including ALK1-4, PKA, PKC, ERK1/2, EGFR) using respective substrate peptides and assay buffers. Kinase activity was quantified by colorimetric assay, with no significant off-target inhibition (>50% activity reduction) observed [3][4] |
| Cell Assay |
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. 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. HCC cell proliferation and TGF-β signaling assay: HepG2, Huh7, and PLC/PRF/5 cells were seeded in 6-well plates at 2×10⁵ cells/well and treated with Galunisertib (LY2157299) (1-20 μM) for 1 hour, then stimulated with TGF-β1 (5 ng/mL) for 24-72 hours. Western blot detected p-Smad2 and total Smad2; qPCR analyzed CTGF/PAI-1/Snail mRNA levels; MTT assay measured cell viability [2] - Scleroderma fibroblast fibrosis assay: Human dermal fibroblasts from scleroderma patients were seeded in 6-well plates at 1×10⁵ cells/well and activated with TGF-β1 (10 ng/mL) for 24 hours. Galunisertib (LY2157299) (1-10 μM) was added, and cells were cultured for 48 hours. Collagen type I synthesis was measured by ELISA; α-SMA expression was detected by Western blot [1] - Ex vivo patient tumor tissue assay: Fresh HCC tumor tissues from patients were cut into 2-mm slices and cultured in medium containing Galunisertib (LY2157299) (20 μM) for 48 hours. Tissue sections were used for p-Smad2 immunostaining and Ki-67 immunohistochemistry [2] |
| Animal Protocol |
Dissolved in DMSO and diluted in saline; 75 mg/kg/day; oral gavage Nude mice implanted subcutaneously with Calu6 or MX1 cells 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] 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. Subcutaneous Huh7 xenograft model: 6-8 weeks old nude mice were subcutaneously inoculated with Huh7 cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were randomly divided into vehicle and Galunisertib (LY2157299) groups. The drug was suspended in 0.5% carboxymethylcellulose sodium and administered orally at 100 mg/kg/day for 21 days. Vehicle group received carboxymethylcellulose sodium. Tumor volume was measured every 3 days; tumors were excised for Western blot (p-Smad2) and Ki-67 immunostaining [2][3] - Mouse CCl₄-induced liver fibrosis model: C57BL/6 mice were injected intraperitoneally with CCl₄ (1 mL/kg, 1:1 v/v in olive oil) twice weekly for 8 weeks. Concurrently, Galunisertib (LY2157299) was suspended in 0.5% carboxymethylcellulose sodium and administered orally at 50 mg/kg/day for 8 weeks. Vehicle group received carboxymethylcellulose sodium. Liver tissues were collected for Masson’s trichrome staining (collagen content), α-SMA immunostaining, and ALT/AST measurement [1] - Rat orthotopic HCC model: Male Wistar rats were implanted with Huh7 cells into the liver parenchyma. One week post-implantation, Galunisertib (LY2157299) was dissolved in saline and administered intraperitoneally at 75 mg/kg/day for 14 days. Vehicle group received saline. Livers were harvested to count metastatic nodules and analyze tumor invasiveness [2] |
| ADME/Pharmacokinetics |
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] Absorption: Oral administration of Galunisertib (LY2157299) in mice and rats shows moderate oral bioavailability (35-45% in mice; 40-50% in rats) [3][4] - Distribution: The drug distributes widely in tissues, with tumor tissue concentration 2.5-3.0 fold higher than plasma concentration in HCC xenograft mice [3] - Metabolism: Galunisertib (LY2157299) is primarily metabolized in the liver via cytochrome P450 enzymes, with no major active metabolites identified [4][5] - Excretion: Approximately 60% of the administered dose is excreted in feces and 30% in urine within 72 hours [4] - Half-life: The plasma elimination half-life (t₁/₂) is 4-6 hours in mice and 5-7 hours in rats [3][4] |
| Toxicity/Toxicokinetics |
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. In vitro, Galunisertib (LY2157299) shows low toxicity to normal human cells (NHHs IC50 > 50 μM; human dermal fibroblasts IC50 > 60 μM) [2][5] - In in vivo studies, oral or intraperitoneal administration of Galunisertib (LY2157299) at tested doses (50-100 mg/kg/day) causes no significant body weight loss (<5% vs. baseline) or overt lethality in mice and rats [1][2][3] - No significant changes in liver function (ALT, AST) or renal function (creatinine, BUN) were observed in Galunisertib (LY2157299)-treated animals compared to vehicle controls [1][3] - Clinical toxicity (Phase 1 study in Japanese patients): The most common adverse events (AEs) are fatigue (35%), nausea (28%), diarrhea (25%), and vomiting (20%), all of grade 1-2. The maximum tolerated dose (MTD) is 300 mg/day orally, with dose-limiting toxicity (DLT) being grade 3 elevated AST/ALT in 1 patient [5] - Plasma protein binding rate: Galunisertib (LY2157299) has a high plasma protein binding rate of 92-95% in humans, mice, and rats (in vitro plasma binding assay) [4][5] |
| References |
[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. |
| Additional Infomation |
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. 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] 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] 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] Galunisertib (LY2157299) is a potent, selective small-molecule inhibitor of TGF-β receptor type I (ALK5), targeting the TGF-β signaling pathway [3][4] - Its mechanism of action involves competitive binding to the ATP-binding pocket of ALK5, inhibiting its kinase activity and blocking downstream Smad2/3 phosphorylation and TGF-β-mediated transcriptional activation of pro-fibrotic, pro-tumorigenic, and pro-invasive genes [1][2][3][4] - Galunisertib (LY2157299) exhibits in vitro anti-proliferative, anti-fibrotic, and anti-invasive activities, as well as in vivo antitumor effects in HCC models and anti-fibrotic effects in liver fibrosis models [1][2][3] - It has undergone clinical development for the treatment of advanced solid tumors, including HCC, pancreatic cancer, and scleroderma (a TGF-β-driven fibrotic disease) [4][5] - The drug’s favorable pharmacokinetic profile (moderate oral bioavailability, wide tissue distribution, acceptable half-life) and manageable toxicity support its clinical application potential [4][5] |
Solubility Data
| Solubility (In Vitro) |
|
|||
| Solubility (In Vivo) |
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. 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. 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. 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. Solubility in Formulation 5: 2% DMSO+30% PEG 300+ddH2O:5 mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7069 mL | 13.5347 mL | 27.0695 mL | |
| 5 mM | 0.5414 mL | 2.7069 mL | 5.4139 mL | |
| 10 mM | 0.2707 mL | 1.3535 mL | 2.7069 mL |