Anti-tumor Efficacy And Safety Of New Small Molecule Anti-angiogenic Drugs In Non-small Cell Lung Cancer

Globally,lung cancer is a threat to human health which is associated with the highest morbidity and mortality among all cancers.It is reported by national cancer center that there were 787,000 new cases of lung cancer in China and 631,000 deaths in 2015.According to the type of tissue,lung cancer is usually divided into non-small cell lung cancer(NSCLC)and small cell lung cancer.NSCLC accounts for 80–85%of lung cancers.Most lung cancer patients already have advanced or metastatic disease when diagnosed,missing opportunity to perform surgery.The main drug treatments of lung cancer include chemotherapy,molecular targeted therapy,immunotherapy and anti-angiogenic therapy.For a long time,platinum-based chemotherapy has been the standard treatment for patients with advanced NSCLC.In recent years,with deep understanding of tumor molecular typing and immune escape mechanisms,targeted therapy and immunotherapy have become important strategy for advanced NSCLC.In the meanwhile,with the advancement of the concept of anti-tumor angiogenesis,anti-angiogenic therapy has become an indispensable treatment for advanced NSCLC.Angiogenesis plays an important role in tumor development and progression.The VEGF/VEGFR pathway is an important pro-angiogenic signal,which can promote endothelial cell proliferation,induce endothelial cell migration and tubule formation,increase the permeability of blood vesselsthe and promote the proliferation,migration,survival of cancer.Blockade of EGFR and VEGFR pathway is a key method in inhibiting tumor growth.Angiogenesis inhibitors include monoclonal antibodies and small molecule tyrosine kinase inhibitors which are mainly against VEGF or VEGFR.In the treatment of lung cancer,angiogenesis inhibitors of monoclonal antibodies have developed rapidly.Bevacizumab was approved as early as 2006 while the small molecule anti-angiogenic drugs nintedanib and anlotinib were approved in 2014 and 2018,respectively.Anlotinib is the only small molecule anti-angiogenic drug and the only monotherapy of anti-angiogenic drug approved for the treatment of lung cancer in China.However,there are still many unresolved questions in the phase III clinical ALTER0303study that prompts anlotinib to be approved.Whether patients over 75 years old and patients with Eastern Cooperative Oncology Group performance status>1 can use anlotinib?Do different clinicopathological characteristics affect the efficacy of anlotinib?What is the efficacy and safety of anlotinib combined with other anti-tumor drugs in the treatment of lung cancer?In recent years,more and more anti-angiogenic drugs are developed.However,most anti-angiogenic drugs have limited anti-tumor activity.Combining other anti-tumor drugs has become the main method of anti-angiogenic drugs for the treatment of lung cancer.Anti-angiogenic drugs combined with chemotherapy and targeted therapy for lung cancer patients have achieved remarkable results.As the efficacy of anti-angiogenic drugs continues to be recognized,their adverse events have also received increasing attention.Whether it is a monoclonal antibody or a small molecule tyrosine kinase inhibitor,the long t_1/2 poses a challenge to the management of side effects and limits the clinical application of anti-angiogenic drugs.Most of the previous small molecule anti-angiogenic drugs have a long t_1/2and tissue accumulation.Cinical study demonstrates that anlotinib has a long t_1/2(>95 h),and the treatment stratedy in advanced NSCLC is 12 mg once daily with 2 weeks on and 1 week off.Sunitinib also has a long t_1/2(>40 hours)and the approved dose of sunitinib in treatment of metastatic renal cell carcinoma is 50 mg once daily with 4 weeks on and 2 weeks off.CM082 is a novel small molecular anti-angiogenic drugs,developed on the same chemical framework with sunitinib,has a short t_1/2 in human(<6.5 h),and can be administered daily.CM082 has less adverse effects and provides more possibilities for combination therapy.At present,the basic research on the anti-vascular and anti-tumor activity of CM082 has not been reported,and the efficacy of CM082 in the treatment of advanced NSCLC remains to be further studied.This study firstly evaluated the anti-vascular activity of CM082 and explored the anti-tumor effect of CM082 on NSCLC.At the same time,due to the lack of clinical data of efficacy and safety of CM082 in patients with advanced NSCLC and few data of anlotinib in patients with advanced NSCLC in real-world setting,we futher investigated the efficacy and safety of anlotinib under different treatment modes in the treatment of patients with advanced NSCLC.Part?The anti-tumor activity of CM082 in NSCLC:preclinical studyBackground and PurposeThe macromolecular monoclonal antibody bevacizumab or ramucirumab combined with the epidermal growth factor receptor-tyrosine kinase inhibitor(EGFR-TKI)as first-line treatment improves the median progression free survival(m PFS)of patients with EGFR mutant advanced NSCLC Compared with EGFR-TKI,and this treatment strategy has been recommended by multiple guidelines.The European Society of Medical Oncology held in September 2020 firstly reported that small molecule anti-angiogenic drug apatinib combined with gefitinib also prolonged the m PFS of patients with EGFR-mutant NSCLC patients compared with gefitinib alone.However,while the combination therapy brings survival benefits,it also increases the occurrence of adverse effects,which has become one of the important reasons for limiting clinical use of anti-angiogenic drugs.CM082 is a small molecule anti-angiogenic drug with less adverse effects,and its efficacy in the treatment of advanced NSCLC is still unknown.Our research aimed to evaluate the anti-vascular activity of CM082 and to explore the effect of CM082 combined with gefitinib in EGFR mutant NSCLC via preclinical experiments.MethodsThe inhibitory effects of CM082 and sunitinib on the proliferation,migration,and tubule formation of human umbilical vein endothelial cells(HUVEC)with high expression of VEGFR2 were compared to evaluate the anti-vascular activity of CM082in vitro.Then the effects of CM082 in combination with gefitinib on cell proliferation,clone formation,apoptosis,cell cycle and cell growth in three dimensional of HCC827(19Del)and NCI-H3255(L858R)cells were compared to gefitinib.Finally,in the NCI-H3255 xenograft model,the effects of CM082 combined with gefitinib on the tumor growth,cell proliferation,apoptosis and angiogenesis and signal moleculars were compared to gefitinib.Results1.CM082 inhibited the growth of VEGF-stimulated HUVECs with an IC₅₀ of0.031±0.005?mol/L,while the IC₅₀ of sunitinib was 0.012±0.003?mol/L.Notably,the IC₅₀ values of CM082 and sunitinib for inhibiting FBS-stimulated HUVEC growth were29.9±3.9?mol/L and 4.4±0.5?mol/L,respectively.CM082 and sunitinib had similar inhibitory activity on VEGF-induced activation of VEGFR and downstream signaling molecules in HUVEC cells.CM082 inhibited cell migiration and tubule formation of HUVECs in a concentration-dependent manner.At the concentration of 0.01?mol/L and0.1?mol/L,the the inhibition rate of CM082 on cell migiration were 64.5%and 88.3%respectively.At the concentration of 0.1?mol/L and 1?mol/L,the the inhibition rate of CM082 on tubule formation were 33.1%and 65.1%respectively.2.CM082,administered at 10?mol/L or 20?mol/L,showed a synergistic inhibitory effect in combination with gefitinib on the growth of HCC827 and NCI-H3255 cells.The combined index of the two drugs is less than one.The inhibitory effect of the co-administration on NCI-H3255 colony formation was stronger than that of monotherapy(P=0.0079 vs gefitinib,P=0.0015 vs CM082).Surprisingly,co-administration completely inhibited colony formation by HCC827 cells(P=0.0002 vs gefitinib,P=0.0011 vs CM082).CM082 combined with gefitinib was superior to monotherapy in promoting the apoptosis of HCC827 cells(P=0.018 vs gefitinib,P=0.005 vs CM082)and NCI-H3255 cells(P=0.003 vs gefitinib,P=0.0007 vs CM082).CM082 arrested the HCC827/NCI-H3255 cell cycle in the S phase,gefitinib blocked the HCC827/NCI-H3255 cell cycle in the G₀/G₁ phase,and when combined,it blocked the cells in the G₀/G₁ phase and S phase.CM082 combined with gefitinib was superior to monotherapy in inhibiting the cell growth of HCC827 cells(P=0.012 vs gefitinib,P=0.005 vs CM082)and NCI-H3255 cells(P=0.009 vs gefitinib,P=0.004 vs CM082)in three dimensional cell culture.3.CM082,administered at 80 mg/kg and 160 mg/kg twice daily,had a similar inhibitory effect on tumor growth compared to that in the control group.The TGI was51.4%(80 mg/kg)and 53.6%(160 mg/kg),which was lower than that of sunitinib(66.8%),but there was no statistically significant difference(the P values were 0.22 and0.29,respectively).The TGI by CM082(80 mg/kg twice daily)combined with gefitinib was 107.7%and was significantly higher than that by monotherapy with gefitinib(93.6%)(P<0.0001)and CM082(51.4%)(P<0.0001).At the same time,the administration of CM082 combined with gefitinib had no difference on the weight of the mice compared to monotherapy.CM082 combined with gefitinib inhibited tumor proliferation(P=0.0005 vs gefitinib,P=0.0001 vs CM082),promoted tumor apoptosis(P=0.01 vs gefitinib,P=0.008 vs CM082),and inhibited angiogenesis(P=0.0001 vs gefitinib,P=0.0025 vs CM082)compared to monotherapy.At the same time,both CM082 and gefitinib could inhibit the expression of VEGF-A,and combination therapy exhibited a synergistic inhibitory effect on VEGF-A compared to monotherapy(P=0.0002 vs gefitinib,P=0.0054 vs CM082).CM082 and gefitinib both inhibited the phosphorylation of ERK1/2,AKT,and STAT3 in NCI-H3255 tumor xenograft model.However,CM082significantly inhibited the phosphorylation of ERK1/2 and AKT,and gefitinib obviously inhibited the phosphorylation of ERK1/2 and STAT3.More importantly,co-administration was superior to monotherapy in inhibiting the phosphorylation of STAT3(P=0.0015 vs gefitinib,P=0.0002 vs CM082).Conclusions1.CM082 exhibited similar anti-vascular activity and anti-tumor effects as sunitinib.CM082 inhibited VEGF-indued HUVEC cells proliferation and FBS-stimualted tubule formation and cell migration.2.Whether in HCC827 or NCI-H3255 cells,CM082 enhanced the antitumor effect of gefitinib by inhibiting proliferation,promoting apoptosis and cell cycle arrest.3.CM082 enhanced the antitumor effect of gefitinib on NCI-H3255 xenograft tumors by inhibiting proliferation,promoting apoptosis,and inhibiting angiogenesis.Part?The efficacy and safety of anlotinib in advanced NSCLC:a real-world studyBackground and PurposeAnlotinib is the only small-molecule anti-angiogenic drug approved for the treatment of lung cancer in China.The ALTER0303 trial that prompts the approval of anlotinib still has many unresolved problems,which cannot meet the drug needs of clinical patients.In the real world,more than 90%of cancer patients cannot participate in clinical trials.The process of patients receiving treatment is not as strict and standardized as clinical trials.The patient population and treatment methods are not completely consistent with clinical trials.This single-center,retrospective,observational study was conducted to illustreate the efficacy and safety of anlotinib on advanced NSCLC and analyse the relationship between baseline characteristic and efficacy of anlotinib.MethodsMedical records of patients with advanced NSCLC who were treated with anlotinib in the Department of Pulmonary Oncology of the Fifth Medical Center of the People's Liberation Army from June 2018 to May 2020 were collected.The objective response rate(ORR),disease control rate(DCR),the median progression free survival(m PFS),median overall survival(m OS)and treatment related adverse events of patients were caculated.Survival curves were derived using the Kaplan–Meier method.Univariate analysis was performed by log-rank testing.Cox regression analysis was used to evaluate the significance of factors obtained from the univariate analysis.ResultsA total of 174 patients received anlotinib,including 121 patients adminisrated anlotinib in third-line and further treatment and 20 patients who continued to use EGFR-TKI with anlotinib beyond gradual progression of first-line EGFR-TKI.The rest were not analyzed due to the small number of cases.1.The efficacy and safety of anlotinib in advanced NSCLC:among 109 patients who could be evaluated for efficacy,the ORR was 13.8%and the DCR was 82.6%.The median PFS was 4.5 months(95%confidence interval[CI]:4.1-4.9),and the median OS was 9.5months(95%CI:8.7-10.3).Treatment-related grade 3 adverse events included hypertension(8.3%),hand-foot syndrome(2.5%),proteinuria(1.7%),etc.No serious and fatal adverse events occurred.8 patients(6.6%)discontinued anlotinib due to side effects,and 10 patients(8.3%)reduced the dose due to side effects.Patients with ECOG PS=2or liver metastases have shorter PFS and OS.2.The efficacy and safety of continuing EGFR-TKI with anlotinib beyond gradual progression in patients with advanced NSCLC:among 19 patients who could be evaluated for efficacy,the ORR was 21.1%and the DCR was 100%.The median PFS was 6.3months(95%CI:5.5-7.1),and the median OS was 20.2 months(95%CI:13.1-27.3).There were no relationship between baseline characteristic and median PFS.The grade3/4 treatment-related adverse events were hypertension(15%),Proteinuria(5%),hand-foot syndrome(5%)and fatigue(5%).No severe or life-threatening adverse events were reported.3 patients(15%)discontinued the drugs and another 3 patients reduced the dosage of anlotinib due to side effects.Conclusions1.Anlotinib was well tolerated and effective in patients with advanced NSCLC under real-world conditions,which was similar to the results of ALTER0303 trial.Elderly patients older than 75 years also benefited from the use of anlotinib,while patients with liver metastasis and ECOG PS=2 had limited benefit from anlotinib treatment.2.The clinical efficacy of continuing EGFR-TKI with anlotinib in advanced NSCLC patients who had gradual progression in first-line EGFR-TKI treatment was similar to contimuing EGFR-TKI plus chemotherapy and superior to continuing EGFR-TKI alone.Future prospective studies in this regard will be beneficial in validating our results.Anlotinib combined with EGFR-TKI was well-tolerated in patients with advanced NSCLC,which is similar to previous studies.