Ong Yung Sheng, Benjamin EVALUATION OF ADVANCED PACLITAXEL DRUG DELIVERY IMPLANTS FOR CONTROLLED RELEASE POST-SURGICAL TREATMENT AGAINST GLIOBLASTOMA MULTIFORME IN THE BRAIN ONG YUNG SHENG, BENJAMIN MSc, DIC, BEng (Hons) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING DEPARTMENT OF CHEMICAL & BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE Page Ong Yung Sheng, Benjamin CONTENT I Acknowledgements II Abstract 1.0 Introduction 1.1 2.0 Background & Literature Review 2.1 2.2 2.3 3.0 Motivation, Objectives & Organization Development of Controlled Release Implants for Chemotherapy to the Brain Paclitaxel Local Implants for Paclitaxel Delivery Evaluation of Paclitaxel Foams for local implants 11 3.1 Materials and Methods 3.1.1 Paclitaxel Foam Formulations 3.1.2 In vitro release of Paclitaxel in PBS 3.1.3 Cell Culture Maintenances 3.1.4 Cell Growth, Viability and Apoptotic activity Studies 3.1.5 Animal Care 3.1.6 In vivo release of Paclitaxel 3.1.7 Intracranial Survivability Analysis 3.1.8 In vivo intracranial bio-distribution Studies 11 11 12 12 12 13 14 14 15 3.2 Results and Discussion 3.2.1 In vivo Release 3.2.2 In vitro Cell Proliferation & Apoptotic Activity 3.2.3 Intracranial Survivability Studies 3.2.4 In vivo Bio-distribution 16 16 20 23 23 3.3 Conclusions 25 4.0 Evaluation of EHDA Microparticles 4.1 Materials and Methods 4.2 In Vivo Release 4.3 Tumor Volume Response Study 26 26 27 30 5.0 Evaluation of Spray-Dried 0.8% Paclitaxel Loaded Discs 32 6.0 7.0 8.0 Conclusions and Recommendations List of Figures Reference 35 37 39 Appendix A: Raw Data from Experiments 42 Page Ong Yung Sheng, Benjamin ACKNOWLEDGEMENTS The author would like to thank his group members in the drug delivery lab in particularly, Ms Laiyeng Lee and Mr Jingwei Xie for providing the formulations and other technical support Thanks go to Mr Sudhir Hulikal Ranganath, Ms Dawn Ng, Ms Meijia Ng and Mr Junjie Huang for laboratory assistance Also thanks to Ms Fan Lu and A/Prof Lee How Sung, from Dept of Pharmacology, NUS, for carrying out the LCMSMS analysis To A/Prof Gavin Dawe, Ms Alice Ee and Ms Han Siew Peng, Dept of Pharmacology, NUS, for technical training and advise in intracranial surgery, to Ms Kho Jia Yen, NUMI histology Lab, NUS, for consultation on tissue staining and preparations, and A/Prof Ong Wei Yi, Dept of Anatomy, NUS, for his valuable inputs and time on experiment design and concept Final thanks go to Prof Nick Sahinidis, UIUC, and A/Prof Wang Chi-Hwa, NUS as thesis advisors Page Ong Yung Sheng, Benjamin ABSTRACT In this thesis, evaluation of three different Paclitaxel controlled release biodegradable implants for post-surgical implantation was carried out The Poly-(DL-lactic-co-glycolic acid) (PLGA) based implants were fabricated in the form of Pressure Quenched Foams, Electro-Hydrodynamic Atomized microparticles and Spray-Dried Discs Two formulations of foams with different functions were evaluated The formulations were (F1) 5% Paclitaxel loaded PLGA 85:15 foams as the slower but prolonged releasing implant and (F2) 10% Paclitaxel PLGA 50:50 foam for faster drug release Experiments carried out were in vitro cell cultures to compare controlled release from foams vs acute Paclitaxel exposure over 24 hours in terms of cell proliferation response and apoptotic activity We were able to show through the biodistribution in brain tissue experiment that Paclitaxel levels were sustained at ~ mm from the site of implantation over a period of 28 days Electro-hydrodynamic microparticles were showed to agree with in vitro release within an in vivo environment releasing Paclitaxel for up to 28 days after implantation In an tumor response study, the results suggests enhanced tumor suppression by prolonging time taken to reach max tumor volume of 3,000mm3 by days over the commercial Taxol® product The in vivo release of Sprayed Discs was carried out and the results show some correlation to the published in Wang et al (2003) [18] The results suggest that in an in vivo environment, sustained release can be achieve for up to 42 days with a peak release into systemic circulation observable at 21 days after implantation Page Ong Yung Sheng, Benjamin CHAPTER 1: INTRODUCTION One of the main challenges of modern pharmacology has been the delivery of the therapeutic agent to the site of action (where the agent is need) and to reach concentration levels high enough to achieve the desired treatment response Often in many cases, duration of exposure at these levels over prolong periods of time are essential to prevent a relapse back into the diseased state and to provide a sustainable environment for patient recovery Moreover, control of drug levels below toxicity limits are crucial to prevent/reduce side effects to an acceptable level Many of these requirements and challenges are not unlike those encountered in the field of chemical engineering The encapsulation drugs in a biodegradable matrix from which the drug can diffuse out from is analogues to chemical reactants diffusing into a catalyst pore By changing constituent block concentrations in the polymer matrix, control of the rate of polymer degradation can be achieved thereby changing rate of release of the drug Our study focuses on developing controlled releases implants in the form of discs for Paclitaxel to be surgically inserted to remove remaining tumor cells after a debulking surgery (to remove the main tumor bulk) in the brain The discs are inserted into the cavity (where the resected tumor was removed from) and the wound is closed Over time, the discs will release Paclitaxel into the peripheral tissue up to the durations of more than a month It is hoped that by applying this strategy, tumor cells around the cavity would be eliminated and tumor remission avoided Page Ong Yung Sheng, Benjamin 1.1 MOTIVATION, OBJECTIVES & ORGANIZATION The long-term vision beyond the scope of this project would be to develop accurate computational models that would aid in the analysis and design of controlled release implants Results from here can be extrapolated to consider synergetic treatments e.g changes in transport of the drug under environment of periodic irradiation therapy which is know to induce interfering physiological changes Irradiation is known to increase blood brain barrier (BBB) permeability which affects drug transport from the implant through drug loss from interstial tissue across BBB into cerebral spinal fluid (CSF) circulation besides drug diluting effects due to CSF coming into the interstial space Modeling such dynamics can help medical practitioners and scientist explain causes for or lack of treatment efficacy of strategies undertaken To begin on this vision, the goal of this MEng project was to carry out preliminary in vivo experiments to evaluate the treatment with novel Paclitaxel release foam developed by Ms Lai Yeng, a fellow research group member, based on a high pressure quench and rapid solidification of drug-polymer melt This thesis presents a step-by-step approach in the analysis of the use of foams for controlled release of Paclitaxel as implants within the brain for the post-surgical treatment of glioblastoma multiforme through combining cell culture and in vivo experiments to evaluate the efficacy of this treatment within the body Key issues for evaluation of the foams carried out in this thesis involve (i) In vitro drug release in PBS to examine at the degradation rate of the polymer and hence the drug release profile This section was undertaken by Ms Lee Lai Yeng but is presented in this thesis for completeness (ii) In vivo release subcutaneously in mice to obtain release profiles within the body This experiment reconfirms the release profile in a physiologically lipophilic environment This Page Ong Yung Sheng, Benjamin was thought to be significant since degradation rate of PLGA is likely to change according to proportions of hydrophilic and lipophilic blocks Moreover, this step evaluates the safety of the implants against bulk release of the drug resulting in systemic toxicity Weights of the animals were regularly to check this point (iii) Level of toxicity response of tumor cells cultures to sustained release from the foams through cell growth and relative caspase activity levels Design of controls compared the recovery of the (a) cells to acute exposure (over 24 hrs) with commercial taxol and (b) the experimental foams Experimental design was on the basis of two times the Area Under the Curve (AUC) levels An AUC level is the area under the curve of a plot of axis between Concentration of the drug vs the duration of exposure to the drug and is used here to provide consistency in the design of the control groups with commercial Taxol® This study attempts to show the value of sustained release on a cellular level (iv) Intracranial biodistribution of the drug in the brain over time This study presents the ability of the foams to maintain therapeutic levels of paclitaxel at distances away from the implant over a period of one month This is important as it illustrates sustain release and penetration distance of the drug from the site of implantation It also serves as raw data for computational model validation (v) Intracranial Survivability of tumor-laden rats treated with Paclitaxel laded and placebo (blank PLGA polymer without Paclitaxel) PLGA implants Prolong survivability of the experimental groups over the placebo groups indicate enhanced treatment by the foams Besides the foams implants, evaluation of two other implant formulations was undertaken Namely, 16.8% Paclitaxel loaded EHDA (ElectroHydroDynamic Atomization) microparticles where we analysed the in vivo release profile as well as tumor volume response and 0.8% Paclitaxel loaded Spray-dried compressed discs in an in vivo release study Page Ong Yung Sheng, Benjamin CHAPTER 2: BACKGROUND & LITERATURE REVIEW This section provides a summary of research in the development of controlled release implants to the brain Section 2.1 provides an outline of the development and challenges to effective treatment, Section 2.2 covers the background of Paclitaxel, which is the chemotherapeutic agent to be delivered and Section 2.3 will give a review of the research to date specifically of controlled release implants for Paclitaxel 2.1 DEVELOPMENT OF CONTROLLED RELEASE IMPLANTS FOR CHEMOTHERAPY TO THE BRAIN Over the last three decades there has been a rise in brain cancers like glioblastoma multiforme (GBM), oligodendroglioma, anaplastic astrocytoma, medulloblastoma, and mixed glioma has been on the rise Of these, GBM is the most frequent accounting for 16,797 cases out of 38,453 cases per year of malignant brain tumors between 1973 and 2001 in America alone [1] The conventional clinical treatment for glioma is by surgical debulking of the accessible tumor from the patient’s brain The amount of tumor removed is often limited by proximity to critical regions for brain function and this presents a risk of tumor re-growth from residual tumor The approach for limiting cancer remission is carried out by conventional systemic post-surgical chemotherapy and radiotherapy courses Unfortunately, these have resulted in limited clinical effectiveness due to restricted transport of chemotherapy agent across the BBB (blood brain barrier) and significant PgP (P-glycoprotein) mediated efflux barrier effects [2] To overcome barriers to effective drug transport, biodegradable controlled-release polymers implants could be surgically located at the site of tumor removal during the debulking surgery Commercial implants Page Ong Yung Sheng, Benjamin ® like the Gliadel Wafer delivering BCNU (Carmustine) has enjoyed limited successes in improving patient survival rates Clinical trials with Gliadel® Wafer vs placebo wafers have been shown to prolong survival in people with newly diagnosed high-grade malignant gliomas (in addition to surgery and radiation) from a median survival of 11.6 months to 13.9 months With recurrent glioblastoma multiforme in addition to surgery, median survival increased to 6.4 months from 4.6 months [3, 4] Since only one third of GBM patients are responsive to BCNU [5] with other Gliadel wafer associated complications like cerebal edema [6], several groups have been working on controlled release for other drugs such as doxorubicin [1] and paclitaxel 2.2 Paclitaxel Paclitaxel (see Figure A for chemical structure), a chemotherapeutic drug originating from the pacific yew Taxus brevifolia, and other members of the Taxaceae family [7] is commonly used as a chemotherapeutic agent of for ovarian and breast cancer Paclitaxel functions through promotion of the assembly and stabilization of microtubules inhibiting cellular division It also prevents de-polymerization of the assembled microtubules and thereby halts mitosis or cell division and binds to Bcl-2 [8, 9] which normally blocks the process of apoptosis, allowing apoptosis to proceed Unfortunately, Paclitaxel is highly hydrophobic and exhibits a fast plasma clearance when administered by infusion [10] Absorption across the BBB was also poor due to pglycoprotein (p-gp) efflux effects [11, 12, 13] However, studies have over showed that prolong exposure to Paclitaxel for more than 24 hrs can provide significant clinical efficacy [14] Figure A: Chemical Structure of Paclitaxel Page Ong Yung Sheng, Benjamin 2.3 Local Implants for Paclitaxel Delivery Several studies have been carried out using different materials to achieve controlled release of Paclitaxel from surgical implants Von Eckardstein et al used a nitrosoureas liquid crystalline cubic phase encapsulating carboplatin and paclitaxel and reported reduction in tumor sizes in F98 rat brains Brain tissue concentration of Paclitaxel showed little or no drug in the vicinity of mm beyond days Clinical observations of the same formulations have suggested feasible and safe usage if < 15 mg paclitaxel was used [15, 16] Li et al used implants based on polyphsophoester p(DAPG-EOP) polymer at 10% drug loading into Polilactofate microspheres which were combined with PEG-100 Brain tissue concentrations after 30 days showed drug concentrations above LD90 (Drug concentration need to kill 90% of tumor cells) of a depth between to mm and enhanced survivability [17] Wang et al reported the in vitro release profiles of discs released from Poly (DL-lactic-co-glycolic) acid 50:50 (MW 45,000- 75,000) fabricated by spray-drying followed by ton compression, a delay of 15 days before drug release was observed [18] Elkharraz et al fabricated injectable Poly (DL-lactic-co-glycolic) acid 50:50 based microparticles from oil-in-water extraction/evaporation method and glycerol tripalmitate-based implants with 29 and 60% w/w and showed that release of 73 to 87 % of the encapsulate drug within days in the presence of N,N-Diethylnicotinamide (DENA), a hydrotropic agent for paclitaxel, significantly increase the release of paclitaxel increased due to elevated hydrolysis rate of PLGA polymers and the paclitaxel solubility [19, 20] However, how DENA would be used in drug delivery seemed to be in question since DENA affects the central nervous system expressed in seizures and behavioral changes [21] Ho et al., was able to show that a constant zero-order in vitro release of 0.92+/-0.03 pg/day Paclitaxel over days was achievable using Chitosan-egg phosphatidylcholine (chitosan-ePC) Page ... of drug- polymer melt This thesis presents a step-by-step approach in the analysis of the use of foams for controlled release of Paclitaxel as implants within the brain for the post- surgical treatment. .. developing controlled releases implants in the form of discs for Paclitaxel to be surgically inserted to remove remaining tumor cells after a debulking surgery (to remove the main tumor bulk) in the. .. sustained release on a cellular level (iv) Intracranial biodistribution of the drug in the brain over time This study presents the ability of the foams to maintain therapeutic levels of paclitaxel