| Pancreatic cancer is one of the most malignant and fatal cancers in China and around the world,where the ratio of mortality to morbidity is close to 1.0 and the 5-year survival rate is extremely low.Pancreatic cancer is often insidious and diagnosed at advanced stages because most patients lack early symptoms.Drug therapy is an important option for patients with pancreatic cancer because most of them have lost the opportunity to receive surgical treatment.Currently,FOLFIRINOX,a combination of oxaliplatin,irinotecan,fluorouracil and calcium leucovorin,is a primary chemotherapy option for advanced pancreatic cancer patients.Studies have shown that the median survival time of patients with pancreatic cancer receiving gemcitabine monotherapy is only 6.8 months,while FOLFIRINOX chemotherapy regimen helps to increase the median survival time of patients with pancreatic cancer to 11.1 months,which is still accompanied by severe chemotherapy side effects.The drug treatment for pancreatic cancer patients is yet to be optimistic.Developing novel anti-tumor drugs to improve the outcome of pancreatic cancer patients and reduce drug side effects is currently a hot spot and direction in the field of pancreatic cancer research.Nano-drug is a hotspot in the development of new drugs for pancreatic cancer.Through enhanced permeability and retention(EPR)induced by the increased vascular endothelial cell gap and lack of drainage in tumor tissue,nano-drug can target tumor tissue and be enriched there after entering the systemic circulation.Following cascades of Circulation,Accumulation,Penetration,Internalization,and Release(CAPIR),nano-drug can therefore kill cancer cells.Irinotecan liposome(ONIVYDE injection),the first irinotecan nanomedicine to be approved for the treatment of pancreatic cancer in 2015,exerts slightly improved anti-tumor responses and shows reduced toxicity compared to free irinotecan,even though ONIVYDE delivers irinotecan more efficiently to tumors.The primary reason for this is that irinotecan(CPT-11)is a prodrug of 7-ethyl-10-hydroxycamptothecin(SN-38)and thus must be hydrolyzed,via carboxylase-mediated catalysis to its active form SN-38,in order to exert its antitumor activity.However,humans,and particularly tumor cells,lack this enzyme,resulting in limited SN-38 generation and efficacy.It is promising to increase the anti-tumor efficacy of SN-38-related nano-drugs by constructing nano-drugs directly carrying active SN-38 to reduce the dependence on carboxylesterase catalysis.SN-38 can’t be used directly in clinical practice because of its strong hydrophobicity and tendency to crystallize.Conventional nanocarriers(eg,liposomes,polymer micelles)constructed for SN-38 delivery are considered new clinical entities and require extensive clinical trials prior to being approved for clinical use.Constructing excipient-free nanomedicine is of great significance for clinical translation.CPT-11,a [1,4’-bipiperidine]-1’-carboxylate of SN-38 and thus has a strong affinity for SN-38 via π-π stacking,has been widely used in clinical practice.Therefore,self-assembling nanodispersion constructed by CPT-11 and SN-38 improves the loading efficacy of active SN-38 and excludes any other non-clinical components.Besides,it also matches the concept of “drug-adjuvant homology” and has a good clinical translation prospect.Part Ⅰ Fabrication and characterization of 7-ethyl-10-hydroxycamptothecin-based self-assembling and excipient-free nanodispersion.Aims: CPT-11 has a strong affinity for SN-38 via π-π stacking.We aim to fabricate and characterize SN-38 and CPT-11 based nanodispersion.Methods: SN-38(25 mg)and CPT-11(25 mg)of equal mass are mixed and heated in 200 μL dimethyl sulfoxide(DMSO),and transferred into 4.8 m L 5% glucose solution while stirring(5 m L in total).The mixture was continuously stirred for 30 minutes,followed by 30 minutes of ultrasound(35%w 2s/2s),and finally 10 mg/m L self-assembling and excipient-free S1C1 nanodispersion is made(S1C1 represents the mass ratio of SN-38 to CPT-11 1: 1).The size of S1C1 nanoparticles was measured by a Dynamic Light Scattering(DLS).The nanoparticle structure was observed through a transmission electron microscope(TEM)and an atomic force microscope(AFM).4-(2-Hydroxyethyl)-1-piperazine ethane sulfonic acid(HEPES)buffers with different p H values were obtained by titration with hydrochloric acid and sodium hydroxide,under which nanoparticle stability is observed.Results: The size of S1C1 measured by a Dynamic Light Scattering(DLS)was 79.81 ± 7.03 nm.Microscopic observation under transmission electron microscope(TEM)and atomic force microscope(AFM)showed that S1C1 was homogeneous and around 80 nm in diameter.The size of S1C1 particles was stable at p H = 6 or 7.When the acidity increased to p H = 5,the particle size gradually increased and reached about 600 nm in 48 hours.When the alkalinity increased to p H = 8,the particle size increased rapidly,reaching 2000 nm in 2 hours and 5000 nm in 12 hours.Conclusion: S1C1 nanoparticle obtained from equal SN-38 and CPT-11 via mixing,dissolving,stirring,ultrasonic process is homogeneous and stable in neutrally acidic environment.Part Ⅱ 7-Ethyl-10-Hydroxycamptothecin-based self-assembling excipient-free nanodispersion exerts killing effect on pancreatic cancer cellsAims: In previous studies,we confirmed that S1C1 nanodispersion is homogeneous and stable in neutrally acidic environment.Loaded with more active SN-38,S1C1 is potentially more cytotoxic.We aim to explore whether S1C1 has killing effect on pancreatic cancer cells in vitro and in vivo.Methods:Determine cytotoxicity on pancreatic cancer cells(As PC-1,Bx PC-3,L3.6pl,MIA Pa Ca-2,PANC-1)using 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide(MTT)method.Moreover,the Half-maximal inhibitory concentration(IC50)is calculated by algorithm fitting.Apoptotic effect from S1C1 is analyzed by flow cytometry(FCM)in pancreatic cancer cells(As PC-1,Bx PC-3,L3.6pl,MIA Pa Ca-2,PANC-1).Subcutaneous pancreatic cancer model in nude mice is established to evaluate and compare the therapeutic effects of S1C1 and CPT-11.During the treatment,S1C1 and CPT-11 are both injected every 3 days(q3d)at equivalent concentration of 15 mg/kg SN-38.The first injection starts on the 9th day after tumour cell inoculation,followed by 4 more injections,ending on 21 st day.To construct L3.6pl-luc cells used for in situ injection into the pancreas tail of nude mice,L3.6pl pancreatic cells are infected with Lenti-virus encoding luciferase gene.Orthotopic pancreatic cancer model is later established to evaluate and compare the therapeutic effects of S1C1 and CPT-11 through in-vivo imaging and immunohistochemistry(IHC).Results: The cytotoxicity of S1C1 nanodispersion on pancreatic cancer cell lines(As PC-1,Bx PC-3,L3.6pl,MIA Pa Ca-2,PANC-1)all significantly exceeded CPT-11.According to the converted active SN-38 and IC50 values,S1C1 was 47,110,404,150,4 times more cytotoxic than CPT-11 in As PC-1,Bx PC-3,L3.6pl,MIA Pa Ca-2,and PANC-1 respectively.Flow cytometry showed that S1C1 caused more cell death than CPT-11 at equal concentration of SN-38(p <0.001).Intraperitoneal(i.p.)injection of S1C1 and CPT-11 both could significantly inhibit tumour growth subcutaneously,where tumour volumes were reduced by 90.42% and 73.00% compared with the control group,respectively.Besides,S1C1 caused lighter weight loss(p<0.05)and diarrhea(not statistically significant)than CPT-11.In addition,i.p.injection of the same dose of S1C1 produced better tumour suppressive effects than intravenous(i.v.)injection.Similarly,S1C1 i.p.and CPT-11 i.p.both could significantly inhibit tumour growth in orthotopic pancreatic cancer model.Besides,S1C1 caused lighter weight loss(p<0.05)and diarrhea(not statistically significant)than CPT-11.IHC results showed that smaller residential tumour was observed in S1C1-treated mice than that in CPT-11-treated mice.Lastly,we found the gemcitabine monotherapy hardly showed any therapeutic effect in orthotopic pancreatic cancer model.Conclusion: We confirmed that self-assembling excipient-free nanodispersion S1C1 exerts killing effect on pancreatic cancer cells both in vitro and in vivo.Part Ⅲ 7-Ethyl-10-Hydroxycamptothecin-based self-assembling excipient-free nanodispersion has promising prospects for clinical translationAims: In previous studies,we confirmed that S1C1 exerts better killing effect on pancreatic cancer cells both in vitro and in vivo with lighter side effects than CPT-11.However,CPT-11’s anti-tumour efficacy in murine models does not translate into patients because human lack carboxylesterase which is a key enzyme responsible for CPT-11 hydrolysis and SN-38 generation.Primary animal models cannot precisely predict the therapeutic effect that novel drugs may have in patients.Different carboxylesterase level in mice and human is the reason for CPT-11 efficacy disagreement.This study aims to explore whether S1C1 can overcome the limiting factors of CPT-11 and have a better clinical prospect when animal models are further modified to simulate the internal environment of patients.Methods: Bis-p-nitrophenyl phosphate(BNPP),a carboxylesterase-specific inhibitor,is co-used to achieve carboxylesterase inhibition and mimic low carboxylesterase abundancy in human,based on which we evaluate and compare the therapeutic effects of S1C1 and CPT-11 in pancreatic cancer subcutaneously and orthotopically.BNPP is injected 30 minutes prior to the injection of S1C1 or CPT-11.Injected dose and scheme of S1C1 and CPT-11 are in consistence with Part II.In vivo imaging is performed every 6 days,starting on the 9th day after tumor cells inoculation and ending on 27 th day.To monitor the overall survival of orthotopically inoculated mice,we keep raising mice in cages after q3 d x 5 treatment of S1C1 or CPT-11 is finished.Considering that the majority of clinical pancreatic cancer patients are in advanced stages,we established late-staged murine orthotopic pancreatic cancer model according to the median survival of control mice bearing orthotopic pancreatic cancer.S1C1 is used as monotherapy due to the tumour burden to evaluate the therapeutic effects in late-staged models.What’s more,pancreatic cancer tissue specimens from patients are transplanted subcutaneously in nude mice to construct pancreatic cancer patient-derived xenografts(PDX).Pancreatic cancer tissue is grown subcutaneously in nude mice and passaged down for several generations to form stable PDX models,where therapeutic effects of S1C1 and CPT-11 are evaluated and compared.Injected dose and scheme of S1C1 and CPT-11 are also in consistence with Part Ⅱ.Results: After carboxylesterase inhibition in mice bearing subcutaneous pancreatic cancer,intraperitoneal(i.p.)injection of S1C1 and CPT-11 reduced tumour volume by 90.73% and 59.00% compared with the control group,respectively.In addition,BNPP alone did not produce a significant tumour shrinking effect.In orthotopic pancreatic cancer models,xenografts receiving combined injection of BNPP and S1C1 were significantly smaller than those receiving BNPP and CPT-11 as seen through in-vivo imaging.On 27 th day,tumours in CPT-11 group showed significantly higher tumour activity than those in S1C1 group.In the long run,the median survival of nude mice treated with BNPP and S1C1 was 86 days,while the median survival of those treated with BNPP and CPT-11 was 61 days compared to the median survival of control group being 43 days.Murine model with late-staged pancreatic cancer were established 40 days after orthotopic inoculation and given standard q3 d x 5 S1C1 monotherapy.Four of the six mice were found to have reduced tumour size after S1C1 treatment through in-vivo imaging quantification.Tissue collected from pancreatic cancer patients eventually survived subcutaneously in nude mice and were passaged down for several generations to stabilize xenografts.For the 4th generation PDXs,intraperitoneal injection of S1C1 and CPT-11 groups showed 84.10% and 46.74% smaller than the control group,respectively.Similarly,S1C1 had lighter side effects than CPT-11.Conclusion: We confirmed that the therapeutic effect of S1C1 nanodispersion on pancreatic cancer is more independent of carboxylesterase than CPT-11 and not attenuated upon carboxylesterase inhibition to mimic pancreatic cancer patient internal environment.S1C1 nanodispersion also exerts therapeutic effect in pancreatic cancer PDXs,implying a good prospect of clinical translation. |
PDF Full Text Request