| Cancer is an evident public health problem worldwide and it is the second leading cause of death in the United States.Cancer has also been the leading cause of death since 2010 in China.The WHO has estimated that 27 million cancer incidences and 17 million cancer deaths will occur by the year 2030.Systemic chemotherapy is the most commonly used methods of cancer treatment.However,intravenously administered anticancer drugs must overcome transport barriers created by the high tumor interstitial fluid pressure before reaching the lesion side.As a result,only a small fraction of the administered drugs could access the tumor site.On the other hand,higher systemic doses can result in undesirable side effects to normal tissues.Local chemotherapy is considered as an alternative of conventional anticancer treatment where anticancer drugs are released directly at the tumor site.The development of polymer-based local drug delivery systems that target therapy specifically at the tumor site can greatly improve antitumor efficacy and minimize systemic side effects.Several implantable sustained-release drug delivery systems have been commercially available and had a great success in clinic.Gliadel(?)is the first locally delivered antitumor implant containing carmustine approved by the FDA to treat recurrent malignant glioma in the USA.Other commercially available devices include Decapeptyl(?),Lupron Depot(?),Zoladex(?),Eligard(?),viadur(?),OncoGel(?)and InGell(?)Delta.Furthermore,Sinofuan(?)(fluorouracial implants)have been widely used in peritoneal interstitial chemotherapy to treat alimentary system cancers in China.Etoposide is the first semi-synthetic topoisomerase ? inhibiting anticancer agent derived from podophyllotoxin and approved for use by the FDA in the USA in 1983.Etoposide was widely used to treat acute myeloid leukaemia,Hodgkin's disease,non-Hodgkin's lymphoma,small cell lung cancer,non-small cell lung cancer,gastric cancer,breast cancer and ovarian cancer.Now there are two commercial products of etoposide on the market:etoposide injections and oral soft capsules.However,both of these formulations have disadvantages.Etoposide has a tendency to precipitate when diluted for intravenous administration because of its low aqueous solubility.Excipients used for etoposide injections such as ethanol,benzyl alcolhol,polysorbate 80 and polyethylene glycol are related to hypersensitivity reactions.Moreover,the oral administration of etoposide capsules exhibits a low bioavailability along with high inter-and intra-patient variability.In addition,etoposide related secondary leukaemia has also been reported when it was used to treat lung cancer,non-Hodgkin lymphoma,neuroblastoma,acute lymphoid leukemia,Wilms tumor,and rhabdomyosarcoma.Additionally,the conventional continuous intravenous infusion over 24-34 hours is inconvenient to practice and causes pain to patients.Recently,many drug delivery systems have been developed with various pharmaceutical and pharmacological approaches to overcome the limitations of etoposide and have shown enhanced antitumor efficacy,including etoposide-loaded micoremulsions,polymer particles,micelles,liposomes,nanoparticles and etoposide-loaded poly(?-caprolactone)implants.In the present study,we fabricated the sustained-release etoposide-loaded implants containing 40%etoposide,50%poly(L-lactid acid)(PLLA)and 10%polyethylene glycol 4000(PEG4000)(w/w)by the direct compression method.The pharmaceutical research of the implants included drug-excipient compatibility,content uniformity,morphology,in vitro and in vivo release profiles,thermal analyses,Fourier Transform Infrared Spectroscopy(FTIR),sterility and stability investigation.To explore the local drug distribution,the implants were implanted into the liver of the miniature pig,the UPLC-MS/MS was used to determine the amount of etposide in the liver tissue surrounding the implants.Then the antitumor activity of intratumoral implantation of the implants was tested in xenograft model of A549 human non-small cell lung cancer.Furthermore,histological study of the tumor was carried out to evaluate the necrosis in the tumor and the local inflammatory response in the implantation site.The drug-excipient compatibility testing was conducted at an early stage of preparation of the sustained-release etoposide-loaded implants.The samples were stored at different stress conditions for 10 days:60? in hot air oven,25?/90%± 5%relative humidity(RH)and exposure to artificial daylight fluorescent lamp(4500Lx±500Lx).Then the drug content was determined using HPLC method.We found that during the storage period,no morphological changes and significant changes in drug content were observed in drug-excipient combinations.The sustained-release etoposide-loaded implants were prepared by direct compression of dry blends containing 40%etoposide,50%PLLA and 10%PEG4000(w/w).The direct compression method is most widely used in drug preparation without using of organic solvents which are often toxic to environment and patients.The implants were molded into cylinder with an average weight of 1.33 ± 0.03 mg and an average length of 1.92 ± 0.14 mm.Moreover,the mean diameter of the implants was 0.9 mm.The SEM images of the implants demonstrated the homogenous drug distribution in the formulation.Content uniformity testing is a pharmaceutical analysis parameter for the quality control of solid dosage.In this work,the content uniformity of the sustained-release etoposide implants was tested accrording to the guidelines of the Chinese Pharmacopoeia.The calculated acceptance value of content uniformity was 6.3 which met the requirements for content uniformity of the Chinese Pharmacopoeia.The in vitro and in vivo drug release were studied and the results showed that the implants exhibited initial burst effect followed by sustained-release profiles both in vitro and in vivo.The burst release may be due to fast dissolution and diffusion of etoposide from the surface of the implants.The drug release rate gradually declined.This can be explained by the fact that both PLLA and etoposide are hydrophobic,so it is not easy for etoposide to be released from the PLLA and diffusion into the medium.After being implanted into the xenograft tumor,the duration of drug release became longer compared with in vitro settings.It is known that the optimal drug release profile is characterized by the ability to release large amount of drug early to reach the therapeutic concentration followed by sustained release to maintain the therapeutic concentration.The drug release profiles of the implants ensured the prolonged exposure of etoposide to cancerous cells which produce a longer period of enzyme inhibition and increase anticancer efficacy.The thermal analyses(DSC and TGA)and FTIR were carried out to investigate the physicochemical properties of the implants and its components.The results indicated that no significant interaction between the etoposide and the excipients were observed in the implants.Gamma radiation from 60Co was used to sterilize the etoposide-loaded implants.Exposure to gamma radiation at dose of 25 KGy caused no changes in the drug content of etoposide.Membrane filtration method was used for detection of microbial contaminants of the radiated implants.During a 14-day incubation period,microbial growth was not observed in any tube containing etoposide-loaded implants.According to the Chinese Pharmacopoeia,influencing factors test was used to investigate the stability of the sustained-release etoposide-loaded implants.The samples were stored at different stress conditions for duration of 10 days:60 ? in hot air oven,25 ?/90%± 5%relative humidity(RH)and exposure to artificial daylight fluorescent lamp(4500Lx±500Lx).We found that no significant changes happened in the appearance,the related substances,drug content and in vitro drug release of the implants,indicating that etoposide was stable in the excipients during the observe period.To explore the local drug distribution of the sustained-release etoposide-loaded implants,we implanted the implants into the liver of the miniature pig.The local drug distribution profiles showed that high etoposide concentrations near the implantation site of the implants.The drug concentrations were 401.6ng/g,296.4 ng/g,68.4 ng/g at the distance of lcm from the implant boundary on day 1,5,10 post implantation,respectively.The results demonstrated that high level of etoposide could be detected within 1 cm from the bo undry of the implantation site.In this study,we investigated the antitumor efficacy of the sustained-release etoposide-loaded implants using a human non-small cell lung cancer xenograft mouse model.We found that intratumoral implantation of the implants could efficiently delay the tumor growth.The tumor growth curve indicated that the etoposide-loaded implants could inhibit tumor growth in a dose-dependent manner.Additionaly,the tumor suppression rate increased when higher doses of the implants were given because a larger amount of etoposide released from the implants,accumulated in the tumor site and resulted in strong antitumor efficacy.It is interesting that escalating the drug content of etoposide-loaded implants elicited significant antitumor effect without additional toxicity.Histological evaluation of tumor tissues confirmed the antitumor activity of the sustained-release etoposide-loaded implants.Higher-dose implants resulted in more severe tumor cell destruction.It is worth noting that no infiltration of inflammatory cells was observed in the tumor tissue.Moreover,we did not find fibrous capsule formation around the implantation site.Inflammation reaction is the host response to the implanted biomaterial and the degree of the response depends on the properties of the implant,such as size,morphology,composition,stability,sterility,contact duration and degradation.An implant with good biocompatibility must not be recognized as foreign by immune system and induce inflammatory reactions.The histological studies revealed that PLLA-based sustained-release etoposide-loaded implants have provided an acceptable histocompatibility after implantation into the tumor.In summary,the sustained-release etoposide-loaded implants provided a continuous drug release of etoposide and enabled high drug concentrations at the target site,while reducing systemic toxicity.We believe that the sustained-release etoposide-loaded implants have the potential to be used as a new targeted chemotherapy method to treat lung cancer in humans.Furthernore,implantaion of the implants after primary surgical resection will play a positive role in preventing the recurrence of the tumor. |
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