SIBP-03, a novel anti-HER3 antibody, exerts antitumor effects and synergizes with EGFR- and HER2-targeted drugs.

HER3 (human epidermal growth factor receptor 3) acts through heterodimerization with EGFR (epidermal growth factor receptor) or HER2 to play an essential role in activating phosphoinositide 3-kinase (PI3K) and AKT signaling-a crucial pathway that promotes tumor cell survival. HER3 is a promising target for cancer therapy, and several HER3-directed antibodies have already entered into clinical trials. In this study we characterized a novel anti-HER3 monoclonal antibody, SIBP-03. SIBP-03 (0.01-10 µg/mL) specifically and concentration-dependently blocked both neuregulin (NRG)-dependent and -independent HER3 activation, attenuated HER3-mediated downstream signaling and inhibited cell proliferation. This antitumor activity was dependent, at least in part, on SIBP-03-induced, cell-mediated cytotoxicity and cellular phagocytosis. Importantly, SIBP-03 enhanced the antitumor activity of EGFR- or HER2-targeted drugs (cetuximab or trastuzumab) in vitro and in vivo. The mechanisms underlying this synergy involve increased inhibition of HER3-mediated downstream signaling. Collectively, these results demonstrated that SIBP-03, which is currently undergoing a Phase I clinical trial in China, may offer a new treatment option for patients with cancers harboring activated HER3, particularly as part of a combinational therapeutic strategy.

Targeting ST8SIA6-AS1 counteracts KRASG12C inhibitor resistance through abolishing the reciprocal activation of PLK1/c-Myc signaling.

BACKGROUND: KRASG12C inhibitors (KRASG12Ci) AMG510 and MRTX849 have shown promising efficacy in clinical trials and been approved for the treatment of KRASG12C-mutant cancers. However, the emergence of therapy-related drug resistance limits their long-term potential. This study aimed to identify the critical mediators and develop overcoming strategies. METHODS: By using RNA sequencing, RT-qPCR and immunoblotting, we identified and validated the upregulation of c-Myc activity and the amplification of the long noncoding RNA ST8SIA6-AS1 in KRASG12Ci-resistant cells. The regulatory axis ST8SIA6-AS1/Polo-like kinase 1 (PLK1)/c-Myc was investigated by bioinformatics, RNA fluorescence in situ hybridization, RNA immunoprecipitation, RNA pull-down and chromatin immunoprecipitation. Gain/loss-of-function assays, cell viability assay, xenograft models, and IHC staining were conducted to evaluate the anti-cancer effects of co-inhibition of ST8SIA6-AS1/PLK1 pathway and KRAS both in vitro and in vivo. RESULTS: KRASG12Ci sustainably decreased c-Myc levels in responsive cell lines but not in cell lines with intrinsic or acquired resistance to KRASG12Ci. PLK1 activation contributed to this ERK-independent c-Myc stability, which in turn directly induced PLK1 transcription, forming a positive feedback loop and conferring resistance to KRASG12Ci. ST8SIA6-AS1 was found significantly upregulated in resistant cells and facilitated the proliferation of KRASG12C-mutant cancers. ST8SIA6-AS1 bound to Aurora kinase A (Aurora A)/PLK1 and promoted Aurora A-mediated PLK1 phosphorylation. Concurrent targeting of KRAS and ST8SIA6-AS1/PLK1 signaling suppressed both ERK-dependent and -independent c-Myc expression, synergistically led to cell death and tumor regression and overcame KRASG12Ci resistance. CONCLUSIONS: Our study deciphers that the axis of ST8SIA6-AS1/PLK1/c-Myc confers both intrinsic and acquired resistance to KRASG12Ci and represents a promising therapeutic target for combination strategies with KRASG12Ci in the treatment of KRASG12C-mutant cancers.

Discovery, structural optimization, and anti-tumor bioactivity evaluations of betulinic acid derivatives as a new type of RORγ antagonists.

Betulinic acid (BA) is a natural pentacyclic triterpenoid that has a wide range of biological and pharmacological effects. Here, computational methods such as pharmacophore screening and reverse docking were used to predict the potential target for BA. Retinoic acid receptor-related orphan receptor gamma (RORγ) was confirmed as its target by several molecular assays as well as crystal complex structure determination. RORγ has been the focus of metabolic regulation, but its potential role in cancer treatment has only recently come to the fore. In this study, rationale optimization of BA was performed and several new derivatives were generated. Among them, the compound 22 showed stronger binding affinity with RORγ (KD = 180 nM), good anti-proliferative activity against cancer cell lines, and potent anti-tumor efficacy with a TGI value of 71.6% (at a dose of 15 mg/kg) in the HPAF-II pancreatic cancer xenograft model. Further RNA-seq analysis and cellular validation experiments supported that RORγ antagonism was closely related to the antitumor activity of BA and 22, resulting in suppression of the RAS/MAPK and AKT/mTORC1 pathway and inducing caspase-dependent apoptosis in pancreatic cancer cells. RORγ was highly expressed in cancer cells and tissues and positively correlated with the poor prognosis of cancer patients. These results suggest that BA derivatives are potential RORγ antagonists worthy of further exploration.

Design and Synthesis of Triazole-Containing HDAC Inhibitors That Induce Antitumor Effects and Immune Response.

Histone deacetylase (HDAC) is an epigenetic antitumor drug target, but most existing HDAC inhibitors show limited antitumor activity and their use is often accompanied by serious adverse effects. To overcome these problems, we designed and synthesized a series of triazole-containing compounds as novel HDAC inhibitors. Among them, compound 19h exhibited potent and selective inhibition of HDAC1, with good antiproliferative activity in vitro and an excellent pharmacokinetic profile. Compound 19h significantly inhibited the growth of human tumor xenografts in nude mice and murine tumor growth in immune-competent mice bearing MC38 colon cancer. In the MC38 model, 19h increased the ratio of splenic CD4+ T effector cells and promoted complete tumor regression in 5/6 animals when combined with the mPD-1 antibody. These results suggested that selective class I HDAC inhibitors exert direct tumor growth inhibition and indirect immune cell-mediated antitumor effects and are synergistic with immune checkpoint inhibitors.

143D, a novel selective KRASG12C inhibitor exhibits potent antitumor activity in preclinical models.

The KRASG12C mutant has emerged as an important therapeutic target in recent years. Covalent inhibitors have shown promising antitumor activity against KRASG12C-mutant cancers in the clinic. In this study, a structure-based and focused chemical library analysis was performed, which led to the identification of 143D as a novel, highly potent and selective KRASG12C inhibitor. The antitumor efficacy of 143D in vitro and in vivo was comparable with that of AMG510 and of MRTX849, two well-characterized KRASG12C inhibitors. At low nanomolar concentrations, 143D showed biochemical and cellular potency for inhibiting the effects of the KRASG12C mutation. 143D selectively inhibited cell proliferation and induced G1-phase cell cycle arrest and apoptosis by downregulating KRASG12C-dependent signal transduction. Compared with MRTX849, 143D exhibited a longer half-life and higher maximum concentration (Cmax) and area under the curve (AUC) values in mouse models, as determined by tissue distribution assays. Additionally, 143D crossed the blood‒brain barrier. Treatment with 143D led to the sustained inhibition of KRAS signaling and tumor regression in KRASG12C-mutant tumors. Moreover, 143D combined with EGFR/MEK/ERK signaling inhibitors showed enhanced antitumor activity both in vitro and in vivo. Taken together, our findings indicate that 143D may be a promising drug candidate with favorable pharmaceutical properties for the treatment of cancers harboring the KRASG12C mutation.

BCL-2 inhibitor synergizes with PI3Kδ inhibitor and overcomes FLT3 inhibitor resistance in acute myeloid leukaemia.

Inhibitors targeting the antiapoptotic molecule BCL-2 have therapeutic potential for the treatment of acute myeloid leukaemia (AML); however, BCL-2 inhibitors such as venetoclax exhibit limited monotherapy efficacy in relapsed or refractory human AML. PI3Kδ/AKT signalling has been shown to be constitutively active in AML patients. Here, we demonstrate that the combination of BCL-2 and PI3Kδ inhibitors exerts synergistic antitumour effects both in vitro and in vivo in AML. Cotreatment with venetoclax and the specific PI3Kδ inhibitor idelalisib significantly enhanced antiproliferative effects and induced caspase-dependent apoptosis in a panel of AML cell lines. The synergistic effects were mechanistically based on the inactivation of AKT/4E-BP-1 signalling and the reduction of MCL-1 expression, which diminished the binding of Bim to MCL-1. Notably, compared with the parental FLT3-ITD-positive MV-4-11, the acquired FLT3 inhibitor quizartinib-resistant xenograft model carrying the F691L mutation, exhibited a markedly higher sensitivity to venetoclax. Furthermore, venetoclax combined with idelalisib led to tumour regression in all animals in this quizartinib-resistant AML model. Thus, these data indicate that combined inhibition of BCL-2 and PI3Kδ may be a promising strategy in AML, especially for patients with FLT3-ITD and/or FLT3-TKD mutations.

Dual-acting antitumor agents targeting the A2A adenosine receptor and histone deacetylases: Design and synthesis of 4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-amine derivatives.

Based on its inhibition by antagonists, the A2A adenosine receptor (A2AAR) has attracted attention as an anti-tumor drug target; however, in preclinical models and clinical trials, A2AAR antagonists have so far shown only limited efficacy as standalone therapies. The design of dual-acting compounds, targeting the A2AAR and histone deacetylases (HDACs), is used here as an approach to the discovery of novel and more potent antitumor agents. Based on the core structures of the A2AAR antagonists V-2006 and CPI-444, novel 4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-amine derivatives were designed as such dual-acting compounds. The binding affinities for A2AAR of all the new compounds were tested, and their HDAC inhibitory activity was evaluated. Compounds with balanced A2AAR antagonism and HDAC inhibition were tested for their in vitro anti-proliferative activity and pharmacokinetic properties. One of the compounds, 14c (4-(2-(6-Amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-N-(2-amino-phenyl)benzamide) showed an overall favorable pharmacokinetic profile; in the mouse MC38 xenograft model, it showed potent anti-tumor effects with inhibition rates of 44% (90 mg/kg, po, bid) and 85% (60 mg/kg, ip, bid), respectively.

Structure-Based Design of Dual-Acting Compounds Targeting Adenosine A2A Receptor and Histone Deacetylase as Novel Tumor Immunotherapeutic Agents.

Adenosine is an immunosuppressive factor in the tumor microenvironment mainly through activation of the A2A adenosine receptor (A2AR), which is a mechanism hijacked by tumors to escape immune surveillance. Small-molecule A2AR antagonists are being evaluated in clinical trials as immunotherapeutic agents, but their efficacy is limited as standalone therapies. To enhance the antitumor effects of A2AR antagonists, dual-acting compounds incorporating A2AR antagonism and histone deacetylase (HDAC) inhibitory actions were designed and synthesized, based on co-crystal structures of A2AR. Compound 24e (IHCH-3064) exhibited potent binding to A2AR (Ki = 2.2 nM) and selective inhibition of HDAC1 (IC50 = 80.2 nM), with good antiproliferative activity against tumor cell lines in vitro. Intraperitoneal administration of 24e (60 mg/kg, bid) inhibited mouse MC38 tumor growth with a tumor growth inhibition rate of 95.3%. These results showed that dual-acting compounds targeting A2AR and HDAC are potentially immunotherapeutic agents that are worth further exploring.

In vivo flow cytometry reveals a circadian rhythm of circulating tumor cells.

Circulating tumor cells (CTCs) is an established biomarker of cancer metastasis. The circulation dynamics of CTCs are important for understanding the mechanisms underlying tumor cell dissemination. Although studies have revealed that the circadian rhythm may disrupt the growth of tumors, it is generally unclear whether the circadian rhythm controls the release of CTCs. In clinical examinations, the current in vitro methods for detecting CTCs in blood samples are based on a fundamental assumption that CTC counts in the peripheral blood do not change significantly over time, which is being challenged by recent studies. Since it is not practical to draw blood from patients repeatedly, a feasible strategy to investigate the circadian rhythm of CTCs is to monitor them by in vivo detection methods. Fluorescence in vivo flow cytometry (IVFC) is a powerful optical technique that is able to detect fluorescent circulating cells directly in living animals in a noninvasive manner over a long period of time. In this study, we applied fluorescence IVFC to monitor CTCs noninvasively in an orthotopic mouse model of human prostate cancer. We observed that CTCs exhibited stochastic bursts over cancer progression. The probability of the bursting activity was higher at early stages than at late stages. We longitudinally monitored CTCs over a 24-h period, and our results revealed striking daily oscillations in CTC counts that peaked at the onset of the night (active phase for rodents), suggesting that the release of CTCs might be regulated by the circadian rhythm.

Design, Synthesis and Pharmacological Evaluation of Novel Hsp90N-terminal Inhibitors Without Induction of Heat Shock Response.

Heat shock protein 90 (Hsp90) is a potential oncogenic target. However, Hsp90 inhibitors in clinical trial induce heat shock response, resulting in drug resistance and inefficiency. In this study, we designed and synthesized a series of novel triazine derivatives (A1-26, B1-13, C1-23) as Hsp90 inhibitors. Compound A14 directly bound to Hsp90 in a different manner from traditional Hsp90 inhibitors, and degraded client proteins, but did not induce the concomitant activation of Hsp72. Importantly, A14 exhibited the most potent anti-proliferation ability by inducing autophagy, with the IC50 values of 0.1 µM and 0.4 µM in A549 and SK-BR-3 cell lines, respectively. The in  vivo study demonstrated that A14 could induce autophagy and degrade Hsp90 client proteins in tumor tissues, and exhibit anti-tumor activity in A549 lung cancer xenografts. Therefore, the compound A14 with potent antitumor activity and unique pharmacological characteristics is a novel Hsp90 inhibitor for developing anticancer agent without heat shock response.

Dysoxylactam A: A Macrocyclolipopeptide Reverses P-Glycoprotein-Mediated Multidrug Resistance in Cancer Cells.

A 17-membered macrocyclolipopeptide, named dysoxylactam A (1) comprising an unprecedented branched C19 fatty acid and an l-valine, was isolated from the plants of Dysoxylum hongkongense. The challenging relative configuration of 1 was established by means of residual dipolar coupling-based NMR analysis. The absolute configuration of 1 was determined by single-crystal X-ray diffraction on its p-bromobenzoate derivative (2). Compound 1 dramatically reversed multidrug resistance in cancer cells with the fold-reversals ranging from 28.4 to 1039.7 at the noncytotoxic concentration of 10 µM. The mode-of-action study of 1 revealed that it inhibited the function of P-glycoprotein (P-gp), a key mediator in multidrug resistance.

Preclinical characterization of SHR6390, a novel CDK 4/6 inhibitor, in vitro and in human tumor xenograft models.

Inhibition of the cyclin-dependent kinase (CDK) 4/6-retinoblastoma (RB) pathway is an effective therapeutic strategy against cancer. Here, we performed a preclinical investigation of the antitumor activity of SHR6390, a novel CDK4/6 inhibitor. SHR6390 exhibited potent antiproliferative activity against a wide range of human RB-positive tumor cells in vitro, and exclusively induced G1 arrest as well as cellular senescence, with a concomitant reduction in the levels of Ser780-phosphorylated RB protein. Compared with the well-known CDK4/6 inhibitor palbociclib, orally administered SHR6390 led to equivalent or improved tumor efficacy against a panel of carcinoma xenografts, and produced marked tumor regression in some models, in association with sustained target inhibition in tumor tissues. Furthermore, SHR6390 overcame resistance to endocrine therapy and HER2-targeting antibody in ER-positive and HER2-positive breast cancer, respectively. Moreover, SHR6390 combined with endocrine therapy exerted remarkable synergistic antitumor activity in ER-positive breast cancer. Taken together, our findings indicate that SHR6390 is a novel CDK4/6 inhibitor with favorable pharmaceutical properties for use as an anticancer agent.

Pharmacologic characterization of fluzoparib, a novel poly(ADP-ribose) polymerase inhibitor undergoing clinical trials.

Poly(ADP-ribose) polymerase (PARP) enzymes play an important role in repairing DNA damage and maintaining genomic stability. Olaparib, the first-in-class PARP inhibitor, has shown remarkable clinical benefits in the treatment of BRCA-mutated ovarian or breast cancer. However, the undesirable hematological toxicity and pharmacokinetic properties of olaparib limit its clinical application. Here, we report the first preclinical characterization of fluzoparib (code name: SHR-3162), a novel, potent, and orally available inhibitor of PARP. Fluzoparib potently inhibited PARP1 enzyme activity and induced DNA double-strand breaks, G2 /M arrest, and apoptosis in homologous recombination repair (HR)-deficient cells. Fluzoparib preferentially inhibited the proliferation of HR-deficient cells and sensitized both HR-deficient and HR-proficient cells to cytotoxic drugs. Notably, fluzoparib showed good pharmacokinetic properties, favorable toxicity profile, and superior antitumor activity in HR-deficient xenografts models. Furthermore, fluzoparib in combination with apatinib or with apatinib plus paclitaxel elicited significantly improved antitumor responses without extra toxicity. Based on these findings, studies to evaluate the efficacy and safety of fluzoparib (phase II) and those two combinations (phase I) have been initiated. Taken together, our results implicate fluzoparib as a novel attractive PARP inhibitor.

Aberrant modulation of ribosomal protein S6 phosphorylation confers acquired resistance to MAPK pathway inhibitors in BRAF-mutant melanoma.

BRAF and MEK inhibitors have shown remarkable clinical efficacy in BRAF-mutant melanoma; however, most patients develop resistance, which limits the clinical benefit of these agents. In this study, we found that the human melanoma cell clones, A375-DR and A375-TR, with acquired resistance to BRAF inhibitor dabrafenib and MEK inhibitor trametinib, were cross resistant to other MAPK pathway inhibitors. In these resistant cells, phosphorylation of ribosomal protein S6 (rpS6) but not phosphorylation of ERK or p90 ribosomal S6 kinase (RSK) were unable to be inhibited by MAPK pathway inhibitors. Notably, knockdown of rpS6 in these cells effectively downregulated G1 phase-related proteins, including RB, cyclin D1, and CDK6, induced cell cycle arrest, and inhibited proliferation, suggesting that aberrant modulation of rpS6 phosphorylation contributed to the acquired resistance. Interestingly, RSK inhibitor had little effect on rpS6 phosphorylation and cell proliferation in resistant cells, whereas P70S6K inhibitor showed stronger inhibitory effects on rpS6 phosphorylation and cell proliferation in resistant cells than in parental cells. Thus regulation of rpS6 phosphorylation, which is predominantly mediated by BRAF/MEK/ERK/RSK signaling in parental cells, was switched to mTOR/P70S6K signaling in resistant cells. Furthermore, mTOR inhibitors alone overcame acquired resistance and rescued the sensitivity of the resistant cells when combined with BRAF/MEK inhibitors. Taken together, our findings indicate that RSK-independent phosphorylation of rpS6 confers resistance to MAPK pathway inhibitors in BRAF-mutant melanoma, and that mTOR inhibitor-based regimens may provide alternative strategies to overcome this acquired resistance.

Pharmacologic characterization of CT-711, a novel dual inhibitor of ALK and c-Met.

Anaplastic lymphoma kinase (ALK) is a validated molecular target for patients harboring ALK rearrangement, which triggers the development of ALK inhibitors. However, the activation of mesenchymal-epithelial transition factor (c-Met) has emerged as a common cause of acquired resistance induced by selective ALK inhibitors. Herein, we report the first preclinical characterization of CT-711, a novel dual inhibitor of ALK and c-Met. CT-711 demonstrates potent inhibitory activity against ALK kinase activity. Moreover, CT-711 profoundly inhibits ALK signal transduction and thereby induces G1 phase arrest and apoptosis, and results in remarkable anti-proliferative activity against ALK-driven cancer cells. Furthermore, CT-711 effectively inhibits c-Met kinase activity and potently overcomes the resistance mediated by c-Met activation. When orally administered to nude mice bearing xenografts, CT-711 exhibits favorable pharmacokinetic properties and robust antitumor activity. It is noteworthy that CT-711 is superior to crizotinib, the first-in-class ALK inhibitor, in the treatment of ALK-driven cancers in various models. The results of the current study provide a solid foundation for the clinical investigation of CT-711 in patients with tumors harboring ALK rearrangement.

Pharmacological characterization of hetrombopag, a novel orally active human thrombopoietin receptor agonist.

Nonpeptide thrombopoietin receptor (TPOR/MPL) agonists, such as eltrombopag, have been used to treat thrombocytopenia of various aetiologies. Here, we investigated the pharmacological properties of hetrombopag, a new orally active small-molecule TPOR agonist, in preclinical models. Hetrombopag specifically stimulated proliferation and/or differentiation of human TPOR-expressing cells, including 32D-MPL and human hematopoietic stem cells, with low nanomolar EC50 values through stimulation of STAT, PI3K and ERK signalling pathways. Notably, hetrombopag effectively up-regulated G1 -phase-related proteins, including p-RB, Cyclin D1 and CDK4/6, normalized progression of the cell cycle, and prevented apoptosis by modulating BCL-XL/BAK expression in 32D-MPL cells. Moreover, hetrombopag and TPO acted additively in stimulating TPOR-dependent signalling, promoting cell viability, and preventing apoptosis. Orally administered hetrombopag specifically promoted the viability and growth of 32D-MPL cells in hollow fibres implanted into nude mice with much higher potency than that of the well-known TPOR agonist, eltrombopag, in association with activation of TPOR-dependent signal transduction in vivo. Taken together, our findings indicate that, given its favourable pharmacological characteristics, hetrombopag may represent a new, orally active, small-molecule TPOR agonist for patients with thrombocytopenia.

DCZ3112, a novel Hsp90 inhibitor, exerts potent antitumor activity against HER2-positive breast cancer through disruption of Hsp90-Cdc37 interaction.

Hsp90 regulates the stability of oncoproteins important in tumor development and progression, and represents a potential therapeutic target. However, all Hsp90 inhibitors currently in clinical trials target Hsp90 ATPase activity and exhibit low selectivity and high toxicity. In this study, we discovered a new Hsp90 inhibitor, DCZ3112, with a novel mechanism of action. DCZ3112 directly bound to the N-terminal domain of Hsp90 and inhibited Hsp90-Cdc37 interaction without inhibiting ATPase activity. DCZ3112 inhibited the proliferation predominantly in HER2-positive breast cancer cells, including those resistant to the classical Hsp90 inhibitor geldanamycin, which mainly targets ATPase. DCZ3112 produced synergistic in vitro activity in inhibiting cell proliferation, inducing G1-phase arrest and apoptosis, and reducing AKT and ERK phosphorylation. Consistent with this, DCZ3112 alone inhibited the growth of HER2-positive BT-474 xenografts, and exhibited enhanced antitumor activity when combined with the anti-HER2 antibody trastuzumab. Importantly, DCZ3112 also significantly inhibited the growth of trastuzumab-resistant BT-474 cells, and combined treatment retained synergistic antitumor activity. Thus, our findings show that disrupting Hsp90-Cdc37 interaction may represent a promising strategy against HER2-positive breast cancer, especially those with acquired resistance to trastuzumab.

Monitoring circulating prostate cancer cells by in vivo flow cytometry assesses androgen deprivation therapy on metastasis.

It remains controversial whether surgical castration prolongs survival rate and improves therapy prospects in patients suffering from prostate cancer. We used PC3 cell line to establish prostate tumor models. In vivo flow cytometry and ultrasonic imaging were used to monitor the process of prostate cancer growth, development and metastasis. We found out that the number of circulating tumor cells (CTCs) in orthotopic tumor model was higher than that in subcutaneous tumor model. The CTC number in orthotopic tumor model was due to burst growth, while CTC number in subcutaneous tumor model showed a gradual increase with tumor size. After androgen deprivation therapy (ADT) through testicular extraction, we constructed GFP-PC3 subcutaneous tumor models and orthotopic tumor models. We found dramatically decreased CTC number, relieved symptoms caused by the tumor, and significantly prolonged survival time after testicular extraction in orthotopically transplanted prostate tumor model, while the carcinogenesis process and metastases were little influenced by ADT in subcutaneous tumor model. ADT treatment can restrict tumor growth, decrease the CTC number significantly and inhibit distant invasion through inhibition of tumor proliferation and tumor angiogenesis in orthotopical prostate tumor model. © 2018 International Society for Advancement of Cytometry.

Pharmacokinetics and disposition of anlotinib, an oral tyrosine kinase inhibitor, in experimental animal species.

Anlotinib is a new oral tyrosine kinase inhibitor; this study was designed to characterize its pharmacokinetics and disposition. Anlotinib was evaluated in rats, tumor-bearing mice, and dogs and also assessed in vitro to characterize its pharmacokinetics and disposition and drug interaction potential. Samples were analyzed by liquid chromatography/mass spectrometry. Anlotinib, having good membrane permeability, was rapidly absorbed with oral bioavailability of 28%-58% in rats and 41%-77% in dogs. Terminal half-life of anlotinib in dogs (22.8±11.0 h) was longer than that in rats (5.1±1.6 h). This difference appeared to be mainly associated with an interspecies difference in total plasma clearance (rats, 5.35±1.31 L·h-1·kg-1; dogs, 0.40±0.06 L·h-1/kg-1). Cytochrome P450-mediated metabolism was probably the major elimination pathway. Human CYP3A had the greatest metabolic capability with other human P450s playing minor roles. Anlotinib exhibited large apparent volumes of distribution in rats (27.6±3.1 L/kg) and dogs (6.6±2.5 L/kg) and was highly bound in rat (97%), dog (96%), and human plasma (93%). In human plasma, anlotinib was predominantly bound to albumin and lipoproteins, rather than to α1-acid glycoprotein or γ-globulins. Concentrations of anlotinib in various tissue homogenates of rat and in those of tumor-bearing mouse were significantly higher than the associated plasma concentrations. Anlotinib exhibited limited in vitro potency to inhibit many human P450s, UDP-glucuronosyltransferases, and transporters, except for CYP3A4 and CYP2C9 (in vitro half maximum inhibitory concentrations, <1 µmol/L). Based on early reported human pharmacokinetics, drug interaction indices were 0.16 for CYP3A4 and 0.02 for CYP2C9, suggesting that anlotinib had a low propensity to precipitate drug interactions on these enzymes. Anlotinib exhibits many pharmacokinetic characteristics similar to other tyrosine kinase inhibitors, except for terminal half-life, interactions with drug metabolizing enzymes and transporters, and plasma protein binding.