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Sustained Release of a Soft Glucocorticoid Loteprednol Etabonate Using PLA Microspheres for the Prevention of Islet Tran...

Permanent Link: http://ufdc.ufl.edu/UFE0022665/00001

Material Information

Title: Sustained Release of a Soft Glucocorticoid Loteprednol Etabonate Using PLA Microspheres for the Prevention of Islet Transplantation Rejection
Physical Description: 1 online resource (121 p.)
Language: english
Creator: Pinto, Elanor
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: controlled, delivery, drug, glucocorticoid, infusion, islet, loteprednol, microspheres, parenteral, pla, plga, polymer, solvent, sustained
Pharmacy -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Type I Diabetes Mellitus is an autoimmune disease in which the insulin producing beta cells are permanently destroyed leaving the patient dependant on exogenous insulin for the rest of his/her life. Islet transplantation can offer a possible treatment for the most severe forms of the disease. The islet allograft replaces the destroyed beta cells with viable ones. Dr. Buchwald's group, at the University of Miami's Diabetes Research Institute, has developed a novel method of islet transplantation. A suitable, long-term immunosuppressant regimen is needed to prevent the alloimmune rejection of the transplanted islets. We proposed that by incorporating the immunosuppressant loteprednol etabonate (LE) into a biodegradable polymer (PLGA) matrix, we can have controlled release of the drug to the localized islet cells. This project investigated the efficacy of the PLGA microspheres drug delivery system of LE in preventing islet rejection.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Elanor Pinto.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Hochhaus, Guenther.
Local: Co-adviser: Hughes, Jeffrey.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022665:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022665/00001

Material Information

Title: Sustained Release of a Soft Glucocorticoid Loteprednol Etabonate Using PLA Microspheres for the Prevention of Islet Transplantation Rejection
Physical Description: 1 online resource (121 p.)
Language: english
Creator: Pinto, Elanor
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: controlled, delivery, drug, glucocorticoid, infusion, islet, loteprednol, microspheres, parenteral, pla, plga, polymer, solvent, sustained
Pharmacy -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Type I Diabetes Mellitus is an autoimmune disease in which the insulin producing beta cells are permanently destroyed leaving the patient dependant on exogenous insulin for the rest of his/her life. Islet transplantation can offer a possible treatment for the most severe forms of the disease. The islet allograft replaces the destroyed beta cells with viable ones. Dr. Buchwald's group, at the University of Miami's Diabetes Research Institute, has developed a novel method of islet transplantation. A suitable, long-term immunosuppressant regimen is needed to prevent the alloimmune rejection of the transplanted islets. We proposed that by incorporating the immunosuppressant loteprednol etabonate (LE) into a biodegradable polymer (PLGA) matrix, we can have controlled release of the drug to the localized islet cells. This project investigated the efficacy of the PLGA microspheres drug delivery system of LE in preventing islet rejection.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Elanor Pinto.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Hochhaus, Guenther.
Local: Co-adviser: Hughes, Jeffrey.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022665:00001


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1 SUSTAINED RELEASE OF A SOFT GLUCOCORTICOID LOTEPREDNOL ETABONATE USING PLA MICROSPHERES FOR THE PR EVENTION OF ISLET TRANSPLANTATION REJECTION By ELANOR PINTO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Elanor Pinto

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3 To my family: my parents (Henry Stany Pinto an d Bridget Francisca Pinto), my sister (Eloise Pinto), and my brother (Eri c Pinto). Thank you for all your support and encouragement.

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4 ACKNOWLEDGMENTS I would first like to than k my advisor Dr. Gnther Hochhaus for accepting me into his group, for guiding and pushing me to become a better scientist, and for not giving up on me when my project was not going well I learned a lot from him and really appreciate all his help and guidance. I would also lik e to thank my supervisory committee members: Dr. Nicholas Bodor, Dr. Jeffrey Hughes, Dr. Ha rtmut Derendorf, and Dr. Rajiv Si ngh. A special thanks to Dr. Sihong Song and Dr. Jeffrey Hughes for the use of th eir facilities and equipment. I would like to thank Dr. Peter Buchwald and Dr. Antonello Pileggi for conducting the animal studies. I would also like to thank Dr. Emy Wu for all the guida nce she gave me on the animal studies and in graduate school. Thanks to Dr. Anthony Palmieri III for his advice on my project and my career. Special thanks to Dr. Kevin Powers for bei ng a great mentor during my undergraduate and graduate years. Special thanks to Yufei Tang for all the gui dance she gave me on developing the HPLC assays and equipment maintenan ce. A special thanks also to Dr. Katauon Derakhshandeh for all the help and guidance she gave me in my project and for her friendship. Thanks to Dr. Bin Zhang on training and guiding me on the cell culture studie s. Also thanks to Dr. Yuanqing Lu and Don Blair for also helping me with the equipment main tenance. I would like to thank Dr. Vishal Shinde and Dr. Sreedharan Sabarinath for their guidance on my HPLC studies. Special thanks to Gill Brubaker for training and helping me with pa rticle size analysis. Special thanks also to Kerry Siebein for all those hours she spent on SEM analysis with me. Thanks to Jung Hun Jang for the PXRD analysis. I would especially like to thank the Major Analytical Instrumentation Center (MAIC) and Particle Engi neering Research Center (PERC) for the use of their facilities and expertise.

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5 There are so many people at the Department of Pharmaceutics that I would like to thank. Special thanks to the office staff, especially Patr icia Khan and Andrea Tuck er, for all their help. I would also like to thank my labmates (Navin Goyal, Gina Patel, Dr. Kai Wu, Nasha Wang, and Dr. Srikumar Sahasranaman) for always offering a helpful hand when I needed one and for the great company. I would love to thank all my frie nds inside and outside th e department who made my graduate years fun. I would especially lik e to thank Sharonda Amaye-Obu, Dr. Nathalie Toussaint, Dana Mahabee, and Maria Seabra fo r being there through the good and bad times and for their generous friendship. Special thanks to Dr. Nathalie Toussaint, Dr. Wouter Driessen, Dr. Leah Villegas, and Dr. William Millard for SERIS (South Eastern Regional International Symposium). I could not have asked fo r better teammates to work with. Finally, I would love to give a special thanks to my parent s, my sister Eloise, and my brother Eric for all the support they gave me. Words cannot expr ess the gratitude I have for them.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF TABLES ...........................................................................................................................8LIST OF FIGURES .........................................................................................................................9LIST OF ABBREVIATIONS ........................................................................................................ 12ABSTRACT ...................................................................................................................... .............15 CHAP TER 1 INTRODUCTION .................................................................................................................. 17Type 1 Diabetes ......................................................................................................................17Islet Transplantation ...............................................................................................................17History .............................................................................................................................17Current Methods of Islet Transplantation ........................................................................ 18Novel Bioartificial Pancreas ............................................................................................19Immunosuppressant Therapy ..................................................................................................20Problems with Current Immunosuppressive Agents .......................................................20Glucocorticoids as Immunosuppressants ........................................................................21Loteprednol Etabonate .....................................................................................................21Localized Sustained Release Technologies ............................................................................22Outline of This Dissertation ....................................................................................................232 PREPARATION AND CHAR ACTERIZATION OF THE SUSTAINED RELEASE LE-PLGA MICROSPHERES ................................................................................................ 27Introduction .................................................................................................................. ...........27Hypothesis ..............................................................................................................................29Materials and Methods ...........................................................................................................29Chemicals ........................................................................................................................29Solvent Evaporation ........................................................................................................30Encapsulation Efficiency .................................................................................................30Loading Efficiency ..........................................................................................................31Particle Size Analysis ......................................................................................................31Scanning Electron Microscope (SEM) ............................................................................32Powder X-Ray Diffraction (PXRD) ................................................................................ 32In-Vitro Drug Release ...................................................................................................... 32Statistical Analysis .......................................................................................................... 34

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7 Results and Discussion ........................................................................................................ ...34Emulsification Method .................................................................................................... 34Theoretical Drug Loading ............................................................................................... 34Surfactant in Wash Media ............................................................................................... 36Factorial Design Analysis: Inner Diameter of Infusion Tube vs. Infusion Rate of Oil Phase ......................................................................................................................36Factorial Design Analysis: Percent Pol yvinyl Alcohol vs. Inner Diameter of Infusion Tube ...............................................................................................................38Factorial Design Analysis: Percent Lactic Acid vs. Percent Polyvinyl Alcohol ............. 39The Effect of Polydispersity (PR) on the In Vitro Drug Release Profile .........................42Conclusion .................................................................................................................... ..........433 CYTOTOXICITY OF SUSTAINE D RELEASE MICROSPHERES ................................... 82Introduction .................................................................................................................. ...........82Hypothesis ..............................................................................................................................83Materials and Methods ...........................................................................................................83Chemicals ........................................................................................................................83MIN-6 Cell Culture ......................................................................................................... 84MIN-6 Cell Viability Determination ............................................................................... 84Results and Discussion ........................................................................................................ ...85Conclusion .................................................................................................................... ..........874 IN VIVO CHARACTERIZATI ON OF SUSTAI NED RELE ASE MICROSPHERES ......... 91Introduction .................................................................................................................. ...........91Hypothesis ..............................................................................................................................92Materials and Methods ...........................................................................................................93Islet Transplantation ........................................................................................................93Statistical Analysis .......................................................................................................... 94Results and Discussion ........................................................................................................ ...94Conclusion .................................................................................................................... ..........985 SUMMARY ....................................................................................................................... ...105REFERENCES .................................................................................................................... ........108BIOGRAPHICAL SKETCH .......................................................................................................121

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8 LIST OF TABLES Table page 2-1 Experimental design (ID vs. IR) ........................................................................................ 542-2 Microsphere formulation variables and properties (ID vs. IR) .......................................... 542-3 Experimental design (PVA vs. ID) .................................................................................... 602-4 Microsphere formulation variables and properties (PVA vs. ID) ...................................... 602-5 Experimental design (LA vs. PVA) ................................................................................... 662-6 Microsphere formulation variables an d physical parameters (LA vs. PVA) .....................662-7 Particle size properties of LE-PLGA (50:50) microspheres .............................................. 722-8 Particle size properties of LE-PLGA (75:25) microspheres .............................................. 732-9 Particle size propertie s of LE-PLA microspheres .............................................................. 742-10 Particle size propertie s of LE-PLGA microspheres ........................................................... 752-11 Particle size properties of LEPLA microspheres with varying PV and PN and constant PR .........................................................................................................................792-12 Particle size properties of LE-PLA microspheres with same PV but varying PR ...............802-13 Particle size properties of LE-PLA microspheres with same PN and varying PR ..............814-1 Best fit equations of commonl y used dissolution models for the in vitro drug release of the LE-PLA microsphere formulation ......................................................................... 1014-2 Volume and number distribution statistics of LE-PLA microspheres ............................. 104

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9 LIST OF FIGURES Figure page 1-1 Current method of islet transplantation .............................................................................. 251-2 Neovascularized biohybrid implant devi ce to be used in the animal study ....................... 262-1 Different emulsification methods used: (A) sonication and (B) infusion method ............. 442-2 SEM pictures showing the surface mo rphology of LE-PLA microspheres prepared by (A) sonication and (B) infusion method ....................................................................... 452-3 In vitro drug release of LE-PLA microsphere s prepared by sonication (F1) and infusion method (F2) (n=3). Studies were preformed under accelerated conditions and correlated to expected real-time conditions. ............................................................... 462-4 Encapsulation efficiency of LE-PLA mi crospheres prepared with 5, 10, 20, and 30% loteprednol etabonate in formulation (n =3) Significant difference determined by Student t-test (P<0.05). ......................................................................................................472-5 Loading efficiency of LE-PLA micros pheres prepared with 5, 10, 20, and 30% loteprednol etabonate in formulation (n =3) Significant difference determined by Student t-test (P<0.05). ......................................................................................................482-6 Initial burst of LE-PLA microspheres prepared with 5, 10, 20, and 30% loteprednol etabonate in formulation (n=3) Significan t difference determined by Student t-test (P<0.05). ..................................................................................................................... .......492-7 PXRD patterns of LE, physical mixture, and LE-PLA microspheres of varying drug loadings ...................................................................................................................... ........502-8 Initial burst of the LE-PLA microspheres prepared with 0% or 1% SDS in wash media (n=3). Significant difference dete rmined by Student t-test (P<0.05). ..................... 532-9 Encapsulation Efficiency (EE) of PLA mi crospheres prepared with variable inner diameter of infusion tube (ID) and infusion rate (IR) ........................................................ 552-10 Loading Efficiency (LdE) of PLA micr ospheres prepared w ith variable inner diameter of infusion tube (ID) and infusion rate (IR) ........................................................ 562-11 Mean particle diameter based on volume distribution (PV) of PLA microspheres prepared with variable inner diameter of infusion tube (ID) and infusion rate (IR) .......... 572-12 Mean particle diameter based on number distribution (PN) of PLA microspheres prepared with variable inner diameter of infusion tube (ID) and infusion rate (IR) .......... 58

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10 2-13 Mean particle diameter ratio (PR) of PLA microspheres prepar ed with variable inner diameter of infusion tube (ID) and infusion rate (IR) ........................................................ 592-14 Encapsulation efficiency (EE) of PLA mi crospheres prepared with variable % of polyvinyl alcohol (PVA) a nd inner diameter of infusion tube (ID) ................................... 612-15 Loading efficiency (LdE) of PLA microspheres prepared w ith variable % of polyvinyl alcohol (PVA) a nd inner diameter of infusion tube (ID) ................................... 622-16 Mean particle diameter based on volume distribution (PV) of PLA microspheres prepared with variable % of polyvinyl alcohol (PVA) and inner diameter of infusion tube (ID) .............................................................................................................................632-17 Mean particle diameter based on number distribution (PN) of PLA microspheres prepared with variable % of polyvinyl alcohol (PVA) and inner diameter of infusion tube (ID) .............................................................................................................................642-18 Mean particle diameter ratio (PR) of PLA microspheres prepar ed with variable % of polyvinyl alcohol (PVA) a nd inner diameter of infusion tube (ID) ................................... 652-19 Encapsulation efficiency (EE) of PLA mi crospheres prepared with variable % of lactic acid in the PLGA polymer (LA) and % of polyvinyl alcohol (PVA) in the aqueous phase ....................................................................................................................672-20 Loading efficiency (LdE) of PLA microsphe res prepared with variable % of lactic acid in the PLGA polymer (LA) and % of polyvinyl alcohol (PVA) in the aqueous phase 682-21 Mean particle diameter based on volume distribution (PV) of PLA microspheres prepared with variable % of lactic acid in the PLGA polymer (LA) and % of polyvinyl alcohol (PVA) in the aqueous phase ..................................................................692-22 Mean particle diameter based on number distribution (PN) of PLA microspheres prepared with variable % of lactic acid in the PLGA polymer (LA) and % of polyvinyl alcohol (PVA) in the aqueous phase ..................................................................702-23 Mean particle diameter ratio (PR) of PLA microspheres prepar ed with variable % of lactic acid in the PLGA polymer (LA) and % of polyvinyl alcohol (PVA) in the aqueous phase ....................................................................................................................712-24 In vitro drug release profile for LE-PLGA (50:50) microspheres prepared with variable % of polyvi nyl alcohol (PVA) ............................................................................. 722-25 In vitro drug release profile for LE-PLGA (75:25) microspheres prepared with variable % of polyvi nyl alcohol (PVA) ............................................................................. 732-26 In vitro drug release profile for LE-PLA microspheres prepared with variable % of polyvinyl alcohol (PVA) .................................................................................................... 74

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11 2-27 In vitro drug release prof ile for LE-PLGA micros pheres prepared with variable % of polyvinyl alcohol (PVA) .................................................................................................... 752-28 Particle morphology of the LEPLGA 50:50 micros pheres during the in vitro drug release studies at (A) 0, (B) 4, (C) 8, and (D) 12 months .................................................. 762-29 Particle morphology of the LEPLGA 75:25 micros pheres during the in vitro drug release studies at (A) 0, (B) 4, (C) 8, and (D) 12 months .................................................. 772-30 Particle morphology of the LE-PLA microspheres during the in vitro drug release studies at (A) 0, (B) 4, (C) 8, and (D) 12 months .............................................................. 782-31 In vitro drug release profile for LE-PLA microspheres with varying mean volume (PV) and number (PN) diameter and same polydispersity (PR) ........................................... 792-32 In vitro drug release profile for LE-PLA microspheres with the same mean volume diameter (PV) but varying ratio (PR) .................................................................................. 802-33 In vitro drug release profile for LE-PLA microspheres with the same mean number diameter (PN) and varying ratio (PR) .................................................................................. 813-1 Effect of loteprednol et abonate on MIN-6 cell viability after 1 day (a) and 4days (b) of incubation (n=4). (*P<0.05, **P<0.01) ......................................................................... 883-2 Effect of PLA microspheres on MIN-6 cell viability after 1 day (a) and 4 days (b) of incubation (n=4). (**P<0.01; ***P<0.001) .......................................................................893-3 Effect of PLA microspheres on MIN-6 cell viability after 1 and 4 days of incubation (n=4). (***P<0.001) ..........................................................................................................904-1 Islet transplantation using the novel biohybrid device. ..................................................... 994-2 Percent survival of chemically diabet ic rats receiving islet transplantation in conjunction with the following local i mmunosuppressant therapy: saline solution with no drug (control), LE solution infused with Alzet pump (LE Infusion), and LEPLA microspheres inserted into device (LE-PLA Microspheres) ................................... 1004-3 Curve-fitting of the in vitro drug release (Q) of the LE-PL A microsphere formulation 1024-4 Rate of drug released (dQ/dt) from the LE-PLA microsphere formulation ..................... 1034-5 Particle size distributi on of the LE-PLA microsphere formulation based on the (A) volume and (B) number distribution ................................................................................ 104

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12 LIST OF ABBREVIATIONS AE 1-cortienic acid etabonate ANOVA Analysis of variance BAD Proapoptotic Bcl-2-associat ed death promoter protein CA 1-cortienic acid Ca2+ Calcium ion cmc Critical micelle concentration CO2 Carbon dioxide DCM Dicholoromethane DDS Drug delivery system DDW Double distilled water DMEM Dulbeccos modified Eagles medium DMSO Dimethyl sulphoxide DNA Deoxyribonucleic acid dQ/dt Rate of drug released DT Total amount of drug added to the formulation DW Amount of drug found in the wash solution EDTA Ethylenediaminetetraacetic acid EE Encapsulation efficiency FBS Fetal bovine serum G Glucocorticoid GA Glycolic acid GR Glucocorticoid receptor H2O Water HCl Hydrochloric acid

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13 HFIP Hexafluoroisopropanol HPLC High performance liquid chroma tography with UV-VIS (Ultraviolet visible) detection HSP90 Heat-shock protein 90 LA Lactic acid monomer LdE Loading efficiency LE Loteprednol etabonate LE-PLA Poly(D,L-lactic) acid microspheres dope d with loteprednol etabonate LE-PLGA Poly(D,L-lactic-co-glycolic) acid micr ospheres doped with loteprednol etabonate IC50 50% inhibitory concentration ID Inner diameter of infusion tube IEQ Islet equivalents IL Interleukin IR Infusion rate of organic phase into aqueous phase IS Immunosuppressant MTT 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide MW Molecular weight PBS Phosphate buffer saline PLA Poly(D,L-lactic acid) PLGA Poly(D,L-lactic-co-glycolic) acid PN Mean particle diameter ba sed on the number distribution PP-2B Calcineurin PR Polydispersity index; Ratio of PV/PN PTFE Polytetrafluoroethylene PV Mean particle diameter ba sed on the volume distribution

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14 PVA Polyvinyl alcohol PXRD Powder X-ray diffraction Q In vitro drug release amount Q Total amount of drug in the microspheres (mg) Qt Amount of drug released as a function of time (mg) R2 Correlation coefficient SDS Sodium dodecyl sulfate SE Standard error SEM Scanning electron microscope t1/2 Biological half-life US United States xexp Amount of drug quantified for a given amount of the formulation xLE Total amount of drug added in preparing the formulation xm Amount of formulation analyzed xP Total amount of polymer added in preparing the formulation

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15 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SUSTAINED RELEASE OF A SOFT GLUCOCORTICOID LOTEPREDNOL ETABONATE USING PLA MICROSPHERES FOR THE PR EVENTION OF ISLET TRANSPLANTATION REJECTION By Elanor Pinto December 2008 Chair: Gnther Hochhaus CoChair: Jeffrey Hughes Major: Pharmaceutical Sciences Type I Diabetes Mellitus is an autoimmune disease in which the insulin producing cells are permanently destroyed leaving the patient dependant on exogenous insu lin for the rest of his/her life. Islet transplantation can offer a possi ble treatment for the most severe forms of the disease. The islet allograft replaces the destroyed cells with viable ones. Dr. Buchwalds group, at the University of Miamis Diabetes Research Institute, has developed a novel method of islet transplantation. The donor islets are infused in to a subcutaneous, neova scularized biohybrid implant device. The neovascularized device locali zes the islets allowing for ease of implantation and retrieval of donor islet cells. This procedure s till has complications that need to be refined one being a need for a suitable, long-ter m immunosuppressant regimen to prevent the alloimmune rejection of the islets w ith low side effects to the patient. The soft glucocorticoid loteprednol etabonate (LE) was c hosen as the immunosuppressant since it breaks down into inact ive metabolites reducing systemic accumulation of the drug and adverse side effects. LE and the islets are pl aced into the neovascularized device allowing for localized delivery which also minimizes systemic side effects as opposed to other routes of

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16 administration (i.e. oral). It is desired to have a therapeutic drug concentr ation within the device that can last over a range of three months to one year. The overall objective was to develop a sustaine d release formulation of LE suitable for use in the biohybrid transplant device for islet tr ansplantation. By incorporating the drug into a biodegradable polymer matrix, we can have controlled release of the drug to the localized islet cells. Poly(D,L-lactic) acid (PLA) and poly(D,L-lactic-co-glycolic) acid (PLGA) microspheres prepared by solvent evaporation we re used for sustained delivery of LE. The formulation process parameters were optimized using factorial design analysis to produce 5 to 50 micron-sized, monodispersed, smooth microspheres having sustaine d drug release ranging from three months to a few years. The LE-PLA microspheres were analyzed for its in vitro cell toxicity and in vivo efficacy. The drug LE had a threshold concentration of 10 M and IC50 of 20 M on the MIN-6 insuloma cell line. However, the LE-PLA microspheres had no cytotoxicity. The LE-PLA microspheres were tested to find the immunosuppressive activity in chem ically diabetic rats receiving islet transplantation using the novel implant device. Th e survival duration of the rats with the LEPLA microspheres was double that of the control (no drug). These results show a great promise in the sustained release LE-PLA microspheres improving the success of is let transplantation by making the way for a more patient compliant and effective immunosuppressant treatment.

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17 CHAPTER 1 INTRODUCTION Type 1 Diabetes In 2002, diabetes was listed as the sixth lead ing cause of death on United States (U.S.) death certificates [1]. About 5 to 10 % of all diagnosed cases had type 1 diabetesestim ating that about one in every 400 to 600 children and adol escents in the U.S. suffered from the disease [1]. In type 1 diabetes, the bodys immune system destroys the insulin-producing pancreatic beta cells. Risk factors may be autoimmune, genetic [2], viral infections [3, 4], or environmental factors [5]. Patients suffer from a lower quality of life as a result of th e long-term hyperglycemic complicationsdiabetic retinopathy, nephropath y, neuropathy, and vasculopathy. More serious complications can include hypoglycemic episodes wh ich can lead to a coma, seizure, or even death. Daily administration of exogenous insulin is needed to maintain glycemic control. Islet transplantation is reco mmended treatment for those suffering from the more severe forms of type 1 diabetes. Currently, islet transplant ation is given to patients suffering from brittle or unstable diabetes or to thos e undergoing other transplantations (i.e. kidney or liver) requiring immunosuppressive therapy [6]. Patients who have received islet tran splants have improved glycemic control and do not suffer from severe hypoglycemia [7, 8]. In addition, they have improved cardiovascular, renal, neural, and retinal functions [9-11]. Isle t transplantation has been used over the past three decades and could be a very promising treatment or cure for type 1 diabetes. Islet Transplantation History In 1967, Lacy et al. cam e across a collangenase-b ased method for isolating islets [12] and was successful, in 1972, in transplanting the cells in to rodent models. In 1974, the first clinical

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18 allogeneic islet transplantation was performed at the University of Minnesota [13]. However, no patients achieved insulin independence. Over the years, experts all over from Miami, Milan, Edmonton, Minnesota, and Pittsburg attempted to improve the islet purification methods, immunosuppressant protocols, and ultimately the clin ical success of islet tr ansplantation [14-16]. A major success occurred in 2000 when Shapiro et al. developed the Edmonton protocol which was 100% successful in seven recipients achieving insulin independence following islet transplantation using a strong immunosuppressant protocol. [17]. Follow-up studies revealed that 80% of the recipients remained insulin independ ent after one year [18, 19] and 10% after 5 years [20]. The median duration of insu lin independence was 15 months. Wh ile there is still a need for improvement, the Edmonton protocol brought ba ck interest in islet transplantation. Current Methods of Islet Transplantation For a succes sful transplant, about one million islets are needed and isolated from two or three donor pancreases. Ricordi et al. devised a suitable method for the large-scale isolation of human islets [21]. A collagenase so lution is inserted into the main pancreatic duct and the gland is placed in a warm digestion chamber where th e collagenase digests th e exocrine tissue. The digested tissue is centrifuged and the islets (characterized by a lower density) migrate to the upper layer. The purified islets are infused into th e portal vein of the liver using a closed gravity fed bag system [22] as shown in Figure 1-1. Th e liver (as opposed to the pancreas) makes a better residence site for the transplanted isle ts due decreased organ morbidity, damage during surgery, and a greater physiological advantage (since the liver is a major site of insulin function) [23]. While intrahepatic islet infu sion through the portal vein is the recommended route of transplantation, the procedure is still associated with problem s. Bleeding is the most common followed by portal vein thrombosis and elevated pressure [16, 24]. In addition, the lack of

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19 vacularization and oxygen increas e islet loss and impairment [25]. Alternative sites of transplantation could improve is let engraftment and long-term function. Various sites tested include the spleen [26], omental pouch [27], intr amuscular tissue [28], and subcutaneous tissue [29]. Novel Bioartificial Pancreas At the Univ ersity of Miamis Diabetes Research Institute, Pileggi et al. [30] decided to focus on subcutaneous islet transpla ntation since it is less invasive as compared to the other sites. However, subcutaneous tissue still has the pr oblem of vascularization serving as an ill environment for the transplanted islets in providi ng insufficient nutrients from the blood stream and waste removal [31, 32]. Neovascularized biohybrid devices for subcutaneous islet transplantation have been succe ssful in providing a suitable environment for islets [31, 33]. Hence, Pileggi et al. developed their ow n neovascualarized biohybrid implant device. The biohybrid implant device (Figure 1-2) consis ted of a stainless-stee l mesh cylinder (2 x 0.6 cm) with two polytetrafluoroethylene (PTFE) stoppers and a plunger [30]. The device (with the plunger insert) is implanted in to the subcutaneous tissue in the dorsal region of the rat forty days before islet transplantation. This period is sufficient for the connective tissue to surround the device and for vascularization to occur. The plunger is removed prior to transplantation to provide a bed/void for inserti ng the islets. The device is th en plugged with the stopper. Pileggi et al. [30] was able to maintain normal non-fasting blood gl ucose levels (< 200 mg/dL) in chemically diabetic rat recipients wi th the biohybrid device. In addition, the recipients had long-term survival duration (> 5 months) and islet function. Hence the reversal of diabetes was successful with the novel biohybrid device. However, an immunosuppressant is still needed to reduce alloimmune rejecti on of the device and islets.

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20 Immunosuppressant Therapy As with all transplan tations, immunosuppre ssants are necessary to prevent alloimmune rejection of transplants. Initial islet transpla nts utilized the immunosuppressants cyclosporine A, prednisone, azathioprine, and 15-deoxyspergualin [34, 35]. However, these immunosuppressant regimens were not successful considering that fewer than 10% of patients achieved insulin independence [36]. The first successful regime n for islet transplantation came with the Edmonton protocol. The Ed monton protocol consisted of sirolimus (rapamycin or Rapamune), FK506 (tacrolimus or Prograf), and humanized anti-IL-2 receptor mAb antibody (daclizumab or Zenapax) [17]. More recent successful studies have included everolimus, basiliximab, FTY720, and lisofylline [37-39] in immunosuppressant regimens. Problems with Current Immunosuppressive Agents The risk-to-benefit ratio has to be taken into con sideration when selecting effective immunosuppressive agents. Even with the success of the Edmonton protocol, the immunosuppressant regimen contained potent drugs th at are associated with serious side effects [40, 41]. The calcineurin inhibi tor tacrolimus has been asso ciated with neurotoxicity, nephrotoxicity, and impaired islet graft function [42-45]. Sirolimus is known to cause oral ulcers, dyslipidemia, myelotoxicity, and impaired islet graft function [46, 47]. Other commonly used immunosuppressants are al so associated with side effects. Cyclosporine causes nephrotoxicity and impairs islet graft function [45, 48]. Azathioprine can cause nephrotoxicity and pulmonary toxicity [49, 50]. 15-deoxyspergualin can cause anorexia, diarrhea, leucopenia, and sepsis [51]. Daclizumab is associated with significant morbidity and mortality since it can increase the risks of inf ections [52]. While glucocorticoids are also

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21 affiliated with serious side effects, a more localized, low-dose delivery (within the biohybrid implant) with the newer glucocorticoids ma y be therapeutically beneficial [53-57]. Glucocorticoids as Immunosuppressants Glucocorticoids elicit their immunosuppressive effects by att acking the cells that control the bodys natural imm une response. Dexamethasone induces apoptosis in lymphocytes (T cells and B cells) and thymocytes (T cell precursors) [58-60]. Glucocorticoids cause cell shrinkage, chromatin condensation, and DNA cleavage by ca spase-3 activation [61] They induce their immunosuppressive effects through th e expression of cytoplasmic glucocorticoid receptor to repress genes involved in elicit ing an immune response. For ex ample, glucocorticoids inhibit NF-B, antiapoptotic transcription fact or, by increasing production of NF-B inhibitor, IB [6264]. In addition, glucocorticoids down-re gulate proinflammatory mediators TNF, IL-1 and IL-6 that are secreted by monocytes and macr ophages during an immune response [65-68]. The immunosuppressive effects of glucocorticoids make them suitable for use in preventing allograft rejection. Loteprednol Etabonate Loteprednol etabonate (LE) is a soft gluc ocorticoid that breaks down into inactive m etabolites 1-cortienic acid etabonate (AE) and then into 1-cortienic acid (CA) in-vivo [69]. The clearance mechanism of LE reduces accumulati on of the drug and systemic side effects. LE is 1.5, 8, and 15 times more potent than dexame thasone, prednisolone, and hydrocortisone [70, 71]. LE has been used for treatment of ophthalm ic [72-76], pulmonary [ 77, 78], dermatological [79], arthritic [80], and gastro intestinal dis eases [81]. A current immunosuppressant protoc ol looks at reducing the rejec tion of transplanted islets by infusing the islets and the anti-inflammato ry drug LE into a neovascularized biohybrid

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22 implant device [53]. It wa s found that the dose (45 g for 3 months to 180 g for one year) of LE necessary to maintain therapeutic levels (~0.5 g/day) within the devi ce over a three-month to one-year period is fatal to the be ta islet cells. In add ition, due to the soft properties of the drug, the stability of LE (t1/2 ~ 18 hrs at 37oC in dog blood and plasma) [82] is also a problem. This specific project aims to develop a su stained release formulation of LE. Localized Sustained Release Technologies There are various commercially available d rug delivery systems (DDS) that focus on localized sustained delivery. They range from infusion pumps [83] to monolithic devices [84, 85] to biodegradable microspheres [86], etc. This project focuses on using biodegradable microspheres for localized drug delivery due to thei r versatility of different administration routes (oral, intramuscular, subcutaneous, etc), exce llent storage stability, suitability for industrial production, and safe and ease of elimination of the DDS by biodegradation [87, 88]. Biodegradable microspheres have been developed for the following types of polymers: gelatin, albumin, polyorthoesters, polyanhydr ides, and polyesters. Most commonly used are polyesters specifically poly(D,L-lactic) acid (PLA) and poly(D,L-lactic-co-glycolic)acid (PLGA). PLGA Microspheres PLA and PLGA m icrospheres have been used as DDS for years [86]. They are able to encapsulate and provide sustai ned release of both hydrophilic a nd lipophilic drugs [89]. PLA and PLGA microspheres can decrease unwanted side effects while maintaining therapeutic effects since they can be utilized for target ed delivery [90]. The polymers degrade in vivo by hydrolysis of their ester linkages to CO2 and H2O making the polymer biodegr adable and biocompatible [91]. The biodegradation rate of PLGA can vary fr om less than one month to a period of a few years depending on the copolymer composition [92].

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23 Outline of This Dissertation The purpose of this study was to develop a su stained release f ormulation of LE suitable for use in a biohybrid implant device for islet tran splantation. We proposed that by incorporating the drug into a biodegradable polymer matrix, we can have a controlled release of the drug to the localized islet cells. The sustai ned release formulation was de veloped by solvent evaporation using the biodegradable PLGA microspheres as the drug delivery vehicle. In developing the formulation, the process parameters were optimized to produce 5 to 50 micron-sized, monodispersed, smooth microspheres having sustai ned release ranging from three months to a few years. Chapter 2 of this dissertation investigated the effect of process parameters on the development of the LE-PLGA microspheres. The microspheres were characterized for their encapsulation efficiency, drug loading efficiency initial burst of the microspheres in the dissolution media, partic le size and morphology, and in vitro drug release profile. Factorial design analysis was performed to optimize th e process using the infusion method during the emulsification stage. In addition, varying the percentage of emul sifier polyvinyl alcohol (PVA) in the aqueous phase and the monomer compositi on of the copolymer PLGA was investigated to determine a formulation with an optimal drug release profile. Chapter 3 investigated the cytotoxicity of the LE-PLA microspheres on the MIN-6 insuloma beta cells. A dose of 45 g for a 3 month period of LE is necessary to maintain therapeutic levels for a one-time administrati on of the immunosuppressant into the implant. However, exposing the beta cells to such a high dose is toxic. It is hypothesized that by incorporating the drug into the biodegradable pol ymer matrix will reduce the dose exposure to

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24 the islets hence protecting them from the toxi c effects. In addition, the polymer microspheres should not cause any additional cytotoxicity. Chapter 4 investigated the in vivo efficacy of the LE-PLA micr ospheres. The three month release of LE-PLA microspheres with a mean partic le diameter of 5 microns were inserted into the novel biohybrid implant device. The device was implanted into chemically diabetic rats receiving islet transplantation. The mean survival duration of the rats was used as an indicator of the transplant rejection time point. It is hypothe sized that the mean survival duration of the recipients receiving the LE-PLA microspheres will be significantly greater than the control. In addition, the mean survival time of the recipients should last for three months. The final chapter, Chapter 5, will summarize th e findings and address future works for the project.

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25 Figure 1-1. Current method of islet transplantation ( http://www.cumc.columbia.edu/news/in -vivo/Vol1_Iss4_feb25_02/pictures/isletisola tion.jpg )

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26 Figure 1-2. Neovascularized biohybrid implant device to be used in the animal study ( http://www.diabetesresearch.org/Newsr oom /NewsReleases/DRI/NewDeviceforIslet Transplantation.htm )

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27 CHAPTER 2 PREPARATION AND CHARAC TERIZATION OF THE SUST AINED RELEASE LE-PLGA MICROSP HERES Introduction Poly(DL-lactic) acid (PLA) and poly(DL-lactic-co-glycolic) acid (PLGA) are commonly used biodegradable synthetic polym ers for sustained release drug delivery. The polymers have excellent tissue biocompatibilit y, biodegradation property, and safety profile [93-95]. Solvent evaporation is a commonly used method for pr eparing PLGA microspheres. Sustained release PLGA microspheres have been previously made using this method having drug release over a month [94, 96-99] and will be used in this pr oject for developing the LE-PLGA microspheres. The goal of this study was to develop LE-PL GA microspheres having sustained release ranging from three months to one year. The reason for using microspheres, as opposed to nanospheres, is that nanospheres may migrate out through the pores of the biohybrid implant device. In addition, nanospheres exhibit much faster drug release over a shor ter period of time as opposed to microspheres [100, 101]. However, mi crospheres that are very large have a disadvantage of getting stuck in the syringe needle. To avoid th e problems of using nanospheres and very large microspheres, the LE-PLGA microsphe res will be made in the size range of 5 to 50 microns. In developing the LE-PLGA microspheres, various process parameters were varied to determine the effect on formulation properties: encapsulation efficiency, loading efficiency, particle diameter, particle morphology, and in vitro drug release. The process parameters varied are: a) The type of emulsification method used b) The theoretical drug loading c) Use of surfactant in wash solution d) Inner diameter of infusion tube (ID) e) Infusion rate of oil phase into aqueous phase (IR)

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28 f) The percentage of emulsifier polyvinyl alcohol (PVA) in the aqueous phase g) The LA:GA monomer composition of the copolymer (% LA) Various emulsification methods can be empl oyed in solvent evaporation to produce PLGA microspheres. Bath sonication and the infusi on method were chosen in this study. Bath sonication produces low levels of sonic waves that provide enough energy to the system to form microspheres and not nanospheres [102]. The infusion method utilizes a needle based system that infuses droplets of the oil phase into an aque ous phase forming an o/w emulsion [103]. Based on the size of the emulsion droplets, which can be controlled by the stirre r speed, microspheres can be formed. The ideal LE-PLGA microspheres would have a very high drug content or drug loading. One way to control this factor would be to star t off with a high theore tical/initial drug loading. However formulations with high drug loading ha ve a problem a high initial burst [104]. An initial burst is undesirable since it can result in acute toxicity. So in developing the LE-PLGA microspheres, the formulation with the highest drug loading have to be determined without having a high initial burst. Aside from modifying the theoretical drug lo ading, there are other ways of reducing the initial burst. Akbugcara was able to reduce the initial burst by modifying the ratio of various polymers Eudragit L, Eudragit RS, and Eudragit S in the furosemide loaded microspheres [105]. Xu et al. produced hydroxyapatite-coated PL GA microspheres loaded with amoxicillin [106]. The coating produced microspheres with low initial burst. In addition, Xu et al. used an anionic surfactant sodium dodecyl sulfate (SDS) which was also able to produce microspheres with low initial burst. In this project, we investigated whether adding SDS to the wash media will reduce the initial burst.

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29 Due to the promising results with the infusi on method, factorial design analysis was used to obtain a mathematical model for predicting the optimal formulation. We investigated the effect on the LE-PLGA microsphere s physicochemical properties by varying the ID versus IR, ID versus % PVA, and % PVA versus % LA. Chac on et al. reported that using slower infusion rates and larger diameter needle s produced larger microspheres [107]. Chu et al. noticed that increasing the percentage of PVA in the aque ous phase produced smaller microspheres with lower encapsulation efficiency [108]. Increasi ng the lactic monomer composition in the PLGA copolymer produced microspheres that had more sustained release [109, 110]. By varying the four parameters, we should be able to find LE-PLGA microspheres in th e ideal size range and having the ideal release profile. Hypothesis We hypothesized that w e can produce m onodisperse LE-PLGA microspheres having sustained release ranging from three months to one year utilizing solvent evaporation and by varying the above mentioned process parameters. Materials and Methods Chemicals Loteprednol etabonate (LE) was generous ly donated by Dr. Nicholas Bodor. Poly(D,Llactic) acid (PLA) (0.68 dL/g inhere nt viscosity in chloroform @ 30oC), poly(D,L-lactic-coglycolic) acid 75:25 (PLGA 75:25) (0.68 dL/g in herent viscosity in chloroform), and poly(D,Llactic-co-glycolic) acid 50:50 (PLGA 50:50) (0.55 to 0.75 dL/g inherent viscosity in HFIP) were purchased from DURECT Corporation, Pelh am, Alabama. The polyvinyl alcohol (MWavg 30,000 70,000, 87 to 90% hydrolyzed) was purchased from Sigma Chemical Co. (St. Louis, MO). The remaining chemicals were of analytical grade an d were purchased from Fisher Scientific Inc. (Suwanee, GA).

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30 Solvent Evaporation The drug LE and polym er PLA/PLGA were added to the organic solvent dichloromethane (DCM) and sonicated until both were solubilized. The organic solution was added to the aqueous solution containing a certain pe rcentage of the emulsifier polyvinyl alcohol (PVA) and emulsified either by (a) sonication in a bath soni cator for 5 minutes or (b) infusion of the organic phase into the aqueous phase stirred magnetically at room temperature. Figure 2-1 shows the setup of the two emulsification methods. The emul sion was constantly stirred overnight using the Bellco Multistir 9 (Catalog #7760-00303) to allow the DCM to evaporate off and form a suspension. The suspension was centrifuged at 3000 rpm for 15 minutes using the 50 mL polypropylene tubes, Beckman centrifuge (Model # J2-21), and Beckman ro tor (Model # JA-20). The supernatant was collected separately and th e residue resuspended in the wash solution. The collected microspheres were washed three more times. The residue was resuspended in the minimal volume of double distille d water (DDW) and frozen at oC for at least two hours. The samples were then lyophilized using Labconco Freeze Dry System 4.5 (Kansas City, MO) for three days and stored at 4oC in a desiccator until use. Encapsulation Efficiency The wash suspension so lution was collected in a beaker. One mL of the wash solution was dried off by vacuum centrifugation in triplicate. The dried resi due was redissolved in 1 mL mobile phase and 100 L of 10X dilution was injected into a reverse-phase Waters C18 150 x 4.6 mm 5 micron HPLC column. The mobile phase c onsisted of acetonitrile: DDW: glacial acetic acid (60:40:0.4). The flowrate used was 0.8 mL/min and wavelength 254 nm. From a 100 g/mL stock in acetonitrile, calibration samples of 30, 20, 10, 5, 1, 0.5, and 0.1 g/mL were made with the mobile phase. The encapsulation efficiency (EE) was calculated as follows:

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31 where DT is the total (or initial) amount (mg) of drug added to the formulation DW is the amount (mg) of drug found in the wash solution Loading Efficiency A known a mount of the LE-PLGA microspheres wa s dissolved in 2 mL of DCM by bath sonication for 15 minutes. The DCM was removed by vacuum centrifugation. The dried residue was redissolved in 2 ml mobile phase and 100 L of (10X or 100X) dilution was injected into a reverse-phase Waters C18 150 x 4.6 mm 5 micron HPLC column. The mobile phase consisted of acetonitrile: DDW: glacial acetic acid (60:40:0.4). The flowrate used was 0.8 mL/min and wavelength 254 nm. From a 100 g/mL stock in acetonitrile, calibra tion samples of 30, 20, 10, 5, 1, 0.5, and 0.1 g/mL were made with the mobile phase. The loading efficiency (LdE) was calculated as follows: where xexp is the amount (mg) of drug quantifie d by HPLC analysis for a given amount of the formulation xm is the amount (mg) of formulation analyzed xLE is the total amount (mg) of drug added in preparing the formulation xP is the total amount (mg) of polymer added in preparing the formulation Particle Size Analysis A known a mount of the LE-PLGA microspheres was dispersed in de ionized water and sonicated for 60 seconds to break apart the aggregates. The susp ension was then subjected to

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32 analysis using the Coulter LS13320 (Beckman Coulter, Fullerton, CA). The Coulter LS13320 can analyze particles in the size range of 400 nm to 2 mm u tilizing laser diffraction. The refractive index was set at 1.46 (0.01 i ) and run time at 60 seconds. The runs were done in triplicate per sample. Scanning Electron Microscope (SEM) The LE-PLGA m icrospheres were analyzed using a JEOL (Model 6335F) scanning electron microscope (SEM) to obtain the size, shape, and surface morphology. The microspheres were mounted on aluminum SEM stubs with do uble stick carbon tape. A thin layer of carbon, approximately 10 to 15 nm thick, was evaporated onto the surface of the particles prior to SEM analysis. Characterization was perf ormed at 2-5 kEV under vacuum. Powder X-Ray Diffraction (PXRD) A known a mount of LE-PLGA microspheres were stuck onto a microscope slide using double sided tape. The Philips APD X-ray diffractometer was used. The taped samples were exposed to Cu radiation (40 kV, 20 mA) and scanned from 5o to 40o, 2 at a step size of 0.02o and step time of 1 second. In-Vitro Drug Release The in vitro drug release studies were perform ed at 37oC using 200 mL dissolution media in a capped 250 mL erlenmeyer flask and contin uously stirred. The dissolution media consisted of 1X phosphate buffer saline (pH 7.4), 0.025% s odium azide, and 1% sodium dodecyl sulfate (SDS). Sodium azide was used as a preservative SDS was used to enhanc e the stability of the drug and to keep the in vitro drug release assay under sink conditions. For accessing the drug released within the implant device, the sample a nd separate technique was used with a stir rate of 30 rpm. However, considering that the sustai ned release formulations will take 6 months to

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33 one year for the drug to release, an accelerated in vitro release assay was used (i.e. the stir rate used was around 550 rpm) to make the study more f easible and correlated to the assay performed under real time conditions (data not shown). A predetermined amount of the LE-PLGA micros pheres were added to the bulk media. The amount of sample used was adjusted to keep the total released drug concentration in the dissolution media below 15% of the drugs saturation solubility (126 g/mL). At distinct time intervals, 0.5 mL of the bulk media was taken out and analyzed using the HPLC. The samples (0.5 mL) were centrifuged at 13,200 rpm for 10 minutes. The supernatant (100 L) was injected into a reverse-phase Waters C18 150 x 4.6 mm 5 micron HPLC column. The mobile phase consisted of acetonitrile: DDW: glacial acetic acid (60:40:0.4). The flowrate used was 0.8 mL/min and wavelength 254 nm. Previous stability studies (data not shown) of LE showed that the degradation of the drug in aqueous media to AE only and not to CA over time indicating no need for detecting CA. Calibration samples of 20, 10, 5, 1, 0.5, 0.1, and 0.05 g/mL were prepared by diluting a 100 g/mL LE and AE stock (in 100% acet onitrile) in dissolution media. Quality control samples consisted of 10, 1, and 0.1 g/mL (LE and AE). The calibration curve was plotted in Excel using peak heights of the absorbance of the standard solutions. The trendline was used to calculate the sample concentrations. At the end of the study, the solids were coll ected by centrifugation to access the amount of drug remaining. DCM was added to the solids to dissolve the PLA and release the remaining drug. The suspension was vortexed and dried by vacuum centrifugation. The dried residue was reconstituted with 0.5 mL mobile phase and 100 L was injected into the HPLC using the conditions mentioned above. Calibration sta ndards consisted of 30, 20, 10, 5, 1, 0.5, 0.1, and

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34 0.05 g/mL of LE and AE. Quality control standards consisted of 10, 1, and 0.1 g/mL of LE and AE. Statistical Analysis Design-Expert Version 7.1.5 (Stat-Ease, In c., Minneapolis, MN) and Sigm aPlot for Windows Version 10.0 (Systat Software, Inc., Sa n Jose, CA) were used for curve fitting the experimental data. The goodness of fit was de termined by the correlation coefficient (R2). The analysis of variance (ANOVA) was determined using Design-Expert Results and Discussion Emulsification Method The type of em ulsification method used ma de a difference in the morphology of the microspheres produced. Emulsification using soni cation (Figure 2-2A) produced a polydispersed formulation of deformed/deflated microspheres due to the energy generated by sonication and premature precipitation of the emulsion droplets into solid microspheres. Emulsification by infusion of the organic phase into the aqueous ph ase stirred at a set speed (Figure 2-2B) formed monodispersed, smoother microspheres due the slow er precipitation of the emulsion droplets allowing more time for more uniform droplets to form. As shown in Figure 2-3, the more monodispersed microspheres (F2) had a more su stained zero-order rele ase as opposed to the polydispersed microspheres (F1) pr epared by sonication. Formulation F1 had faster release since it contained clusters of nanospheres while F2 did not. Nanospheres have much larger specific surface area than microspheres so drugs can diffuse out of nanospheres at a faster rate [100]. Theoretical Drug Loading The theoretical drug loading of the LE-PLA m icrospheres were analyzed at 5, 10, 20, and 30% LE. Increasing the theoretical drug loadi ng from 5% to 10% had a decrease in the

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35 encapsulation efficiency (Figure 2-4) and loading efficiency (Figure 2-5) However from 10% to 30% drug loading, there was an increase in the encapsulation and loading efficiency. The initial decrease in the encapsulation and loading efficien cy was a result of the p oorer incorporation of the drug into the polymer matrix due to a low affinity of the drug to th e polymer [111]. Further increasing the drug loading provided a more saturated environment in the organic phase which forced more drug to incorporat e into the polymer matrix. Hen ce the results were microspheres with higher encapsulation and loading efficiency. There was a dramatic difference in the initial burst (Figure 2-6) for the four formulations. The 5% LE loaded microspheres had a very low initial burst of 0.2% ( 0.4%) and was significantly different from the 10% 30% LE loaded microspheres which had an initial burst of 78.5% to 94.4%. The PXRD plots gave a clearer explanation for the ini tial bursts observed. The pure drug LE powder (Figure 2-7A) had di stinct sharp peaks at 16, 17, and 19 2 angles. The blank PLA microspheres (Figure 2-7B) did not ha ve any distinct peaks. When looking at the physical mixtures containing 5, 10, 20, and 30% drug and blank PLA microspheres (Figure 2-7C, E, G, and I), the three 2 angle peaks had increased intensity as the % drug in the mixture increased. The 5% LE loaded PLA microspheres (Figure 2-7D) showed no peaks at the three 2 angles. However the 10, 20, and 30% LE loaded PLA microspheres did. The absence of peaks for the 5% LE loaded PLA microspheres indicated that there was very little or no drug on the surface of the microspheres. Since the 10, 20, a nd 30% drug loaded PLA microspheres (Figure 27F, H, and J) contained peaks, this indicates that there was still unencapsulated drug in the form of drug crystals on the surface of the microsphere s [112]. The initial burst observed with the 10, 20, and 30% LE loaded PLA microspheres was due to the immediate dissolution of the unencapsulated drug.

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36 Surfactant in Wash Media Using the surfactant sodiu m dod ecyl sulfate (SDS) in the wash media significantly reduced the initial burst (Figure 2-8) as compared to the formulation washed with double distilled water (DDW). At 1% SDS or 1 g/100 mL, the concentrat ion of SDS in water was above its critical micelle concentration (cmc) whic h is 0.24 g/100 mL [113]. So the wash solution contained SDS micelles which have a highly hydrophobic core. The hydrophobic core attracts and solubilizes the unencapsulated drug by making it easier to remove the drug not imbedded in the PLA microspheres. Micelles are commonly utilized to extract unwanted hydrophobic chemicals from aqueous solutions [114]. So utilizing this tec hnology was an effective and practical mean of producing formulations with a low initial burst. Factorial Design Analysis: Inner Diameter of In fusion Tube vs. Infusion Rate of Oil Phase A factorial design analysis study was perfor m ed to optimize the properties of the LEPLGA microspheres using the infusion emulsifica tion method (Figure 2-1). The inner diameter of the infusion tube (ID) and the infusion rate (I R) of the organic phase into the aqueous phase were varied according to the conditions listed in Table 2-1. The % PVA used was 0.2%. And, the microspheres were made with PLA. Table 2-2 lis ts the formulations encapsulation efficiency (EE), loading efficiency (LdE), mean partic le diameter based on the volume distribution (PV), mean particle diameter base d on the number distribution (PN), and the polydispersity (PR), the ratio of the means of the two distributions, which is calculated as follows: The number distribution is a statis tical representation of the actual mean diameter of the sample. Visually it would depict the pa rticle size distribution observed if the microspheres diameter were individually measured under SEM. The volume distribution is commonly used when

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37 modeling or understanding systems that are concentration dependent (in our case the in vitro drug release studies). The volume distribution is weighed to empha size the larger microspheres. When looking at concentration dependent systems, the larger particles contain a significantly greater amount of drug. However, a considerably sm aller particle contains a minute or negligible amount of drug in comparison. Since the large part icles contain more drug, they tend to dictate the concentration dependent charact eristics of a formulation making PV an important parameter to investigate. The ratio PR represents the polydispersity of a sample with 1 being monodisperse and greater than 2.0 being polydispersed. The contour plot in Figure 2-9 is a graphical representation of the encapsulation efficiency results listed in Table 2-2. It can be observed th at the highest encapsulation efficiency occurred when the ID was 0.020 inches and the IR was ei ther 1.0 or 0.8 mL/min. The similar trend was also observed with the loading efficiency (Figur e 2-10). The contour plot Figure 2-11, showed a more parabolic relationship wh ere the ID of 0.015 and 0.055 inches produced microspheres with a larger mean diameter (based on the volume distribution PV) and where the IR of 0.8 mL/min produced the smaller microspheres. The PN (Figure 2-12) also showed the same trend with the ID significantly increasing as PN increased (P<0.01). No noticeable trend was observed with the IR. With the PR (Figure 2-13), using larger IDs produced more polydispersed microspheres (P<0.05) whereas the IR had no significant effect. As previously reported by Chacon et al., larger microspheres were formed with an increase in ID [107]. However, so did the polydispersity (PR) making the larger ID s not suitable in optimizing the formulation. The IR had no effect on the PV, PN, and PR. However, an IR of 1.0 or 0.8 mL/min gave the highest encapsulation and loadi ng efficiency. In the future studies, an IR of 0.8 mL/min was chosen in optimizing the formulations.

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38 Factorial Design Analysis: Percent Polyvinyl Alcohol vs. Inner Diameter o f Infusion Tube Further studies were performed using factorial design analysis looking at the effect of the percent of polyvinyl alcohol (PVA) and the inner diameter of the infusion tube (ID) on the physicochemical properties of the LE-PLA microspheres. The process parameters were varied according to the conditions listed in Table 2-3. Th e infusion rate (IR) used was 0.8 mL/min. And, the microspheres were made with PLA. Table 2-4 lists the formulations encapsulation efficiency (EE), loading efficiency (LdE), mean partic le diameter based on the volume distribution (PV), mean particle diameter based on the number distribution (PN), and the polydispersity (PR). The encapsulation efficiency (Figure 2-14) de creased as the % PVA decreased. With the loading efficiency (Figure 2-15), the same tr end was observed. The formulations made with 0.2% PVA had a much lower loading efficiency as opposed to those made with the 0.5% and 1.0% PVA. The PV (Figure 2-16) significantly decreas ed as the % PVA decreased (P<0.005) with IDs above 0.020 inches. The ID relationship with PV was a parabolic one (P<0.05) with an ID of 0.030 inches producing the smallest microsphere s and the 0.055 and 0.015 inches producing the larger microspheres. Th e similar trend was observed with PN (Figure 2-17). The PN generally decreased with decreasing % PVA (P<0.05). A parabolic relationship was again observed with varying IDs with 0.030 inches producing the smallest microspheres. The PR (Figure 2-18) was smallest when using higher % of PVA (P<0.05) and lower ID. As reported by Chu et al., increasing the % PVA in the aqueous solution did produce smaller microspheres [108]. PVA reduced the su rface tension between the aqueous and organic phase by forming emulsions where the hydrophobic end of PVA was absorbed in the oil phase and hydrophilic end was absorbed in the aqueous phase. The more PVA added, the more stable and smaller the emulsion droplets [115]. The formation of the smaller emulsion droplets was due

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39 to the PVA increasing the viscosity of the aqueou s solution. The increase in the viscosity of the aqueous solution reduced the frequency of the em ulsion droplets collisions which prevented the coalescence of the emulsion droplets into larger ones. As DCM evaporated, the polymer in the emulsion droplets precipitated into the small microspheres. Factorial Design Analysis: Percent Lact ic Acid vs. Percent Polyviny l Alcohol Further studies were performed using factorial design analysis looking at the effect of the percent of monomer lactic acid (LA) and polyvinyl alcohol (PVA) on the physicochemical properties of the LE-PLGA microspheres. The pro cess parameters were va ried according to the conditions listed in Table 2-5. The infusion ra te (IR) used was 0.8 mL/min. Since the 0.015 inches inner diameter of the infusion tube (ID) produced larger more monodispersed microspheres, this tubing was used for the study. The LE-PLGA microsphe res were made with PLA, PLGA 75:25, and PL GA 50:50. Table 2-6 lists the formul ations encapsulation efficiency (EE), loading efficiency (LdE), mean partic le diameter based on the volume distribution (PV), mean particle diameter based on the number distribution (PN), and the polydispersity (PR). The encapsulation efficiency (Figure 2-19) and loading efficiency (Figure 2-20) initially decreased as the % PVA decreased to 2.0%, incr eased to 1.0%, and decreased to 0.2%. The % PVA had a significant effect on the encapsulation efficiency (P<0.0005) and loading efficiency (P<0.05). Varying the % LA had no significant effect. With the PV (Figure 2-21), smaller microspheres were formed with increasing % P VA (P<0.005). No noticeable effect was observed with varying % LA. With the PN (Figure 2-22), smaller microspheres were again formed with increasing % PVA (P<0.05). However with 0.2% to 0.5% PVA for PLGA 50:50 and PLGA 75:25, the microspheres diameter decrease d. With decreasing % PVA (P<0.05), the PR (Figure 2-23) increased. No significant effect was observed with varying % LA.

PAGE 40

40 Figure 2-24 shows the in vitro drug release profiles of the LE-PLGA 50:50 microspheres. Even though the microsphere diameter (listed in Table 2-7) increased when the % PVA increased from 0.2% to 0.3%, the LE release rate increased and the duration of the release shortened. This indicates that the release profile was not just based on the particle size of the microspheres but also on another phenomena. Enhanced internal porosity (or decreased density) of the LE-PLGA microspheres can have a higher drug release rate as opposed to microspheres that are completely dense [112]. There was a decrease in the LE release rate when the % PVA increased from 0.3% to 2.0%. This indicated that the LE-PLGA microspheres formed at 2.0% PVA were denser than the ones formed at 0.5% and at 0.3% PVA. When the % PVA increased from 2.0% to 5.0%, the microsphere diameter decreased and the LE rele ase rate increased. This resembles expected release rates based on the diameter of the mi crospheres indicating that the density of the microspheres play little or no role on the release profiles. With the PLGA 50:50 microsphere LE release profiles, the smallest microspheres (5% PVA) had a concave downward profile. This was typical of diffusion-controlled release. In other words, the drug diffusion rate was much faster than the polymer degradation rate. The fast diffusion rate was probably due to smaller mi crospheres having a larger specific surface area [100] and increased water uptake resulting in sw elling of the microspheres [101]. The larger microspheres (0.2% to 2.0% PVA) had more of an S-shaped release profile. The reason for the S-shaped release profile was due to two differe nt phases [100]. First, there was an initial diffusion of the drug from the immediate surf ace layer of the microsphere. Since large microspheres have low specific surface area, this diffusion rate was slow. The drug that was not in immediate exposure to the aqueous bulk enviro nment had to travel through the dense polymer matrix to reach the bulk media. Second, the mi crospheres swelled due to water penetration.

PAGE 41

41 During this phase, more drug was exposed to the aqueous bulk environment so the diffusion and deadsorption of the drug from the microspheres occurred very rapidly. Polymer erosion also played a role since it can increase drug diffusivi ty through the enlargement of aqueous pores in the polymer matrix [116]. A similar trend was observed with the LE-PLGA 75:25 microspheres, the in vitro drug release profiles (Figure 2-25) were more lin ear. When the % PVA increased from 0.3% to 0.5%, the microsphere diameter (listed in Table 2-8) increased and the LE release rate decreased. Again there was a decrease in the LE release rate when the % PVA increased from 0.5% to 1.0% possibly due to a change in the microsphere de nsity. When the % PVA increased from 1.0% to 5.0%, the microsphere diameter decreased and the LE release rate increased. The similar trend continued with the LE-PLA microspheres. The in vitro drug release profiles (Figure 2-26) were more linear. Even th ough the microspheres diameter (listed in Table 2-9) increased when the % PVA increased from 0.2% to 0.3%, the LE rel ease rate increased due to a change in the density of the microsphere s. When the % PVA increased from 0.3% to 0.5%, the microsphere diameter decreased and so di d the LE release rate. Finally, when the % PVA increased from 0.5% to 5.0%, the microsphere di ameter decreased and the LE release rate increased. As expected, the LE release rates of the LEPLGA microspheres (Figure 2-27) decreased as the percentage of lactic acid (LA) increase d as a result of an increase in the polymer decomposition and erosion [109, 110]. As shown in Table 2-10, the microspheres chosen had similar mean volume (PV) and number (PN) diameters and polydispersity (PR). SEM pictures were taken of the PLGA 50:50 (Figure 2-28), PL GA 75:25 (Figure 2-29), and PLA (Figure 2-30) microspheres at 0, 4, 8, and 12 months during the in vitro drug release study. For all three types

PAGE 42

42 of microspheres, the surface was initially ve ry smooth. With the LE-PLGA 50:50 microspheres, rippling on the surface was observed at month 4 a nd the microspheres structure collapsed and eroded at month 8 and 12. After the microsphere s collapsed, they seemed to be sticky and agglomerated with other microspheres to form la rger particles which could be seen by the eye (picture not shown). The LE-PLGA 75:25 microsphe res maintained their structure but some bubbling on the surface was seen at 12 months. The LE-PLA microspheres were smooth up to 4 months but afterwards the micros pheres ruptured possibly due to internal pressure buildup within the microspheres over time as a result of osmotic forces [117]. The Effect of Polydispersity (PR) on the In Vitro Drug Release Profile An interesting observation was made in relation to the polydispersity (PR) and the in vitro drug release profiles of the LE-PLA microspheres. Figure 2-31 shows the release of LE from PLA-microspheres having varying PV and PN and constant PR (Table 2-11). When the PV diameter ranged from 39 to 56 m with constant PR, the LE release rate did not vary at all. Figure 2-32 shows the release of LE fr om PLA-microspheres having similar PV and varying PR (Table 2-12). With increasing PR, the LE release rate increased. Figure 2-33 shows the release of LE from PLA-microspheres having similar PN and varying PR (Table 2-13). Increasing the PR, decreased the LE release rate. But this decrease seems to be saturable when the PV is above 24 m. Polydisperse PLGA microspheres usually have two distinct phases [112] as observed in Figure 2-33. The first phase consisted of a faster drug release rate foll owed by a slower rate. During the first phase, the smaller microspheres released the drug more rapidly since smaller microspheres have much larger specific surf ace area for diffusing out the drug [100]. Larger

PAGE 43

43 microspheres released the drug at a much slower rate because of the lower specific surface area and the slow diffusion of the drug through a greate r distance (the radius of the microsphere). Conclusion Em ulsification using the infusion method produced more monodispersed, spherical microspheres with a more sust ained and zero-order release. 5% drug loading produced LE-PLA microspheres w ith the lowest initia l burst due to low or no drug adsorbed onto the surface of the microsphere. Using 1% SDS in the wash solution did a gr eat job of removing the drug adsorbed on the microsphere surface. The resultant micros pheres had a very low initial burst. The optimal IR was 0.8 mL/min. Increas ing the ID produced larger LE-PLA microspheres but also increased the polydispersity. Increasing the % of PVA in the aqueous so lution, produced smaller microspheres with higher polydispersity. The encap sulation and loading efficiency was also increased with increasing % PVA. In looking at the in vitro drug release of the LE-PLGA microspheres, another parameter beside the size (possibly the density/porosity) of the microspheres influenced the drug release. Varying the composition of the lactic and gl ycolic acid monomer in the PLGA copolymer had no significant effect on the encapsulation and loading efficiency, the particle size, and polydispersity of the microspheres. Ho wever, in decreasing the % LA, the drug release rates also was increased. SEM pictures show that the LE-PLGA 50:50 microspheres internal structure collapsed and eroded over time. LE-PLA microspheres ruptured after 4 months of in vitro drug release. Monodisperse LE-PLA microspheres had very little difference on the in vitro LE release rates when varying the size of the micros pheres. However, varying the mean volume diameter, mean number diameter, and the polydispersity made a big difference and should to be taken in consid eration when predicting the in vitro drug release profiles. Formulation M7 was chosen for the in vitro cell toxicity studies (Chapter 3) and the in vivo feasibility study (Chapter 4) since it had a desired durati on of release of 3 months.

PAGE 44

44 Figure 2-1. Different emulsifi cation methods used: (A) sonicat ion and (B) infusion method

PAGE 45

45 Figure 2-2. SEM pictures showing the surf ace morphology of LE-PLA microspheres prepared by (A) sonication and (B) infusion method

PAGE 46

46 Time ( months ) 04812162024 % Drug Release 0 20 40 60 80 100 F1 F2 Figure 2-3. In vitro drug release of LE-PLA microsphere s prepared by sonication (F1) and infusion method (F2) (n=3). Studies were preformed under accelerated conditions and correlated to expected real-time conditions.

PAGE 47

47 Figure 2-4. Encapsulation efficiency of LE-PL A microspheres prepared with 5, 10, 20, and 30% loteprednol etabonate in formulation (n=3) Significant difference determined by Student t-test (P<0.05). Drug Loading (%) 1234 Encapsulation Efficiency (%) 0 20 40 60 80 100 51 02 03 0

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48 Figure 2-5. Loading efficiency of LE-PLA microspheres prepared with 5, 10, 20, and 30% loteprednol etabonate in formulation (n=3) Significant difference determined by Student t-test (P<0.05). Drug Loading (%) 1234 Loading Efficiency (%) 0 20 40 60 80 100 120 51 02 03 0

PAGE 49

49 Figure 2-6. Initial burst of LEPLA microspheres prepared with 5, 10, 20, and 30% loteprednol etabonate in formulation (n=3) Significant difference determined by Student t-test (P<0.05). Drug Loading (%) 1234 Initial Burst (%) 0 20 40 60 80 100 120 51 02 03 0

PAGE 50

50 Figure 2-7. PXRD patterns of LE, physical mixture, and LE-PLA microspheres of varying drug loadings 510152025303540 Intensity 0 200 400 600 800 1000 1200 Loteprednol Etabonate 510152025303540 Intensity 0 200 400 600 800 1000 1200 Blank PLA Microspheres 510152025303540 Intensity 0 200 400 600 800 1000 1200 5% Loteprednol Etabonate + PLA Microsphere Mixture 2 Angle 510152025303540 Intensity 0 200 400 600 800 1000 1200 5% Loteprednol Etabonate Encapsulated PLA MicrospheresA B D C

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51 Figure 2-7 Continued. 510152025303540 Intensity 0 200 400 600 800 1000 1200 10% Loteprednol Etabonate + PLA Microsphere Mixture 510152025303540 Intensity 0 200 400 600 800 1000 1200 10% Loteprednol Etabonate Encapsulated PLA Microspheres 510152025303540 Intensity 0 200 400 600 800 1000 1200 20% Loteprednol Etabonate + PLA Microsphere Mixture 2 Angle 510152025303540 Intensity 0 200 400 600 800 1000 1200 20% Loteprednol Etabonate Encapsulated PLA MicrospheresE F G H

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52 Figure 2-7 Continued. 510152025303540 Intensity 0 200 400 600 800 1000 1200 30% Loteprednol Etabonate + PLA Microsphere Mixture 2 Angle 510152025303540 Intensity 0 200 400 600 800 1000 1200 30% Loteprednol Etabonate Encapsulated PLA MicrospheresI J

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53 Figure 2-8. Initial burst of the LE-PLA microspheres prepared with 0% or 1% SDS in wash media (n=3). Significant difference dete rmined by Student t-test (P<0.05).

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54 Table 2-1. Experimental design (ID vs. IR) Factor Levels 1 2 3 4 5 Inner Diameter of Tube (inches) 0.015 0.020 0.030 0.040 0.055 Infusion Rate (mL/min) 0.5 0.6 0.8 1.0 1.4 Table 2-2. Microsphere formulation va riables and propert ies (ID vs. IR) # ID (inches) IR (mL/min) EE (%) LE (%) PV ( m) PN ( m) PR 1 0.015 0.5 64.7 79.5 35.3 0.7 47.2 2 0.015 0.6 53.8 71.5 36.2 26.2 1.4 3 0.015 0.8 44.8 58.3 44.5 37.7 1.2 4 0.015 1.0 46.9 59.3 37.9 26.4 1.4 5 0.015 1.4 65.7 78.3 47.4 41.4 1.1 6 0.020 0.5 61.0 73.0 39.1 31.3 1.3 7 0.020 0.6 47.6 63.7 48.8 42.5 1.1 8 0.020 0.8 78.1 92.5 32.0 0.8 41.0 9 0.020 1.0 84.8 100.0 48.9 42.5 1.2 10 0.020 1.4 45.1 63.4 35.4 12.1 2.9 11 0.030 0.5 58.2 63.6 23.8 1.1 21.3 12 0.030 0.6 58.3 67.9 34.3 0.9 37.6 13 0.030 0.8 60.3 66.7 22.6 1.2 19.7 14 0.030 1.0 75.5 92.9 47.2 41.3 1.1 15 0.030 1.4 49.5 63.1 30.4 21.5 1.4 16 0.040 0.5 62.8 80.4 29.9 0.8 35.3 17 0.040 0.6 62.8 83.2 41.3 0.8 54.9 18 0.040 0.8 59.6 64.4 22.2 0.9 25.0 19 0.040 1.0 54.2 62.4 31.4 0.8 39.4 20 0.040 1.4 64.6 80.6 37.1 0.9 40.2 21 0.055 0.5 41.7 59.4 36.1 10.8 3.4 22 0.055 0.6 54.1 68.7 39.5 23.4 1.9 23 0.055 0.8 62.4 73.9 30.1 0.9 32.6 24 0.055 1.0 50.1 62.1 36.2 0.8 45.7 25 0.055 1.4 70.4 87.2 36.1 0.8 45.4

PAGE 55

55 29.0, min 74.1 min 03.1 1 06.0 03.0 1 %8.64 (%)2 2 2 R mL mL IR inches inches ID EE Figure 2-9. Encapsulation Efficiency (EE) of PL A microspheres prepared with variable inner diameter of infusion tube (ID) and infusion rate (IR)

PAGE 56

56 18.0, min% 95.10 % 43.417 min% 95.10 % 43.417%14.61(%)2 2 2 2 2 2 RIR mL ID inches IR mL ID inches LdE Figure 2-10. Loading Efficiency (LdE) of PL A microspheres prepared with variable inner diameter of infusion tube (ID) and infusion rate (IR)

PAGE 57

57 49.0, min 86.1 2.15972 min 72.6 87.1270 03.53)(22 2 2 2 2 RIR mL m ID inches m IR mL m ID inches m m mPV Figure 2-11. Mean particle diam eter based on volume distribution (PV) of PLA microspheres prepared with variable inner diameter of infusion tube (ID) and infusion rate (IR)

PAGE 58

58 62.0, min 6.1 4.118528 min 4.98 min 7.2499 min 7.1 9341 min 1.64 min 7.0 1.283 4.3 )( 12 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 RIR mL m ID inches m IRID inches mL m IRID inches mL m IR mL m ID inches m IRID inchesmL m IR mL m ID inches m m mPN Figure 2-12. Mean particle diam eter based on number distribution (PN) of PLA microspheres prepared with variable inner diameter of infusion tube (ID) an d infusion rate (IR)

PAGE 59

59 61.0, min 1.10 491990 min 2.371 min 6.10452 min 2.17 5.38804 min 7.195 min 3.6 6.11434.1223 3 3 33 2 2 2 2 2 2 2 2 22 1 RIR mL ID inches IRID inchesmL IRID inchesmL IR mL ID inches IRID inchesmL IR mL ID inches PR Figure 2-13. Mean particle diameter ratio (PR) of PLA microspheres prepar ed with variable inner diameter of infusion tube (ID) and infusion rate (IR)

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60 Table 2-3. Experimental design (PVA vs. ID) Factor Levels 1 2 3 4 5 Polyvinyl Alcohol (%) 0.2 0.5 1.0 --Inner Diameter of Tube (inches) 0.015 0.020 0.030 0.040 0.055 Table 2-4. Microsphere formulation va riables and properties (PVA vs. ID) # PVA (%) ID (inches) EE (%) LE (%) PV ( m) PN ( m) PR 1 0.2 0.015 44.8 58.3 44.5 37.7 1.2 2 0.2 0.020 78.1 92.5 32.0 0.8 41.0 3 0.2 0.030 60.3 66.7 22.6 1.2 19.7 4 0.2 0.040 59.6 64.4 22.2 0.9 25.0 5 0.2 0.055 62.4 73.9 30.1 0.9 32.6 6 0.5 0.015 65.8 94.9 48.8 44.3 1.1 7 0.5 0.020 63.7 90.9 46.7 41.0 1.1 8 0.5 0.030 76.8 93.2 27.3 0.8 34.1 9 0.5 0.040 88.2 108.6 43.1 33.3 1.3 10 0.5 0.055 78.7 99.4 40.3 26.1 1.5 11 1.0 0.015 75.6 85.7 40.7 27.6 1.5 12 1.0 0.020 95.5 115.2 41.1 26.5 1.6 13 1.0 0.030 73.9 88.0 38.6 26.7 1.4 14 1.0 0.040 87.0 89.0 43.7 35.7 1.2 15 1.0 0.055 76.4 89.5 35.8 0.5 66.2

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61 73.0, % 1.17916 % 8.36 % 2.1396 25.69%57.26(%)22 2 2 RID inches PVA ID inches PVA EE Figure 2-14. Encapsulation efficiency (EE) of PLA microspheres prepared with variable % of polyvinyl alcohol (PVA) and inner diameter of infusion tube (ID)

PAGE 62

62 76.0, % 6.2601 % 16.114 % 64.181 41.163%61.41(%)22 2 2 RID inches PVA ID inches PVA LdE Figure 2-15. Loading efficiency (LdE) of PL A microspheres prepared with variable % of polyvinyl alcohol (PVA) and inner diameter of infusion tube (ID)

PAGE 63

63 89.0 63.931 % 88.60 % 78.0 28.152 % 02.0 % 45.5 38.7 % 04.005.0 )( 12 3 3 2 2 2 2 2 2 2 2 R ID inches m IDPVA inches m IDPVA inches m ID inches m PVA m IDPVA inches m ID inches m PVA m m m PV Figure 2-16. Mean particle diam eter based on volume distribution (PV) of PLA microspheres prepared with variable % of polyvinyl alcohol (PVA) and inner diameter of infusion tube (ID)

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64 82.0 643.4 % 524.1 % 94.1387 553.5 % 50.56 % 83.10434 66.21752 % 27.4159.238)(2 3 3 2 2 2 2 2 2 2 2 R ID inches m E IDPVA inches m E IDPVA inches m ID inches m E PVA m IDPVA inches m ID inches m PVA m m mPN Figure 2-17. Mean particle diam eter based on number distribution (PN) of PLA microspheres prepared with variable % of polyvinyl alcohol (PVA) and inner diameter of infusion tube (ID)

PAGE 65

65 80.0, 10.92505 %55.2880 %97.54 90.11654 %38.0 )(%53.267 32.467 %99.126.5/1233 2 2 1 2 1 2 22 2 2 1 1 1 RID inches IDPVA inches IDPVA inches ID inches PVA IDPVA inches ID inches PVA PR Figure 2-18. Mean particle diameter ratio (PR) of PLA microspheres prepar ed with variable % of polyvinyl alcohol (PVA) and inner diameter of infusion tube (ID)

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66 Table 2-5. Experimental design (LA vs. PVA) Factor Levels 1 2 3 4 5 Lactic Acid composition (%) 50 75 100 --Polyvinyl Alcohol (%) 0.2 0.5 1.0 2.0 5.0 Table 2-6. Microsphere formul ation variables and physical parameters (LA vs. PVA) # LA (%) PVA (%) EE (%) LE (%) PV ( m) PN ( m) PR 1 50 0.2 43.6 73.0 37.7 16.1 2.4 2 50 0.5 61.9 92.9 37.8 20.2 1.9 3 50 1.0 76.2 100.9 40.9 27.4 1.5 4 50 2.0 49.1 84.5 27.9 7.1 3.9 5 50 5.0 69.0 93.1 8.0 2.8 2.8 6 75 0.2 35.7 72.2 32.2 0.8 41.1 7 75 0.5 51.8 81.0 38.2 27.2 1.4 8 75 1.0 69.4 93.7 38.9 22.4 1.8 9 75 2.0 57.0 86.9 31.3 10.4 3.0 10 75 5.0 82.7 97.3 11.5 3.5 3.2 11 100 0.2 44.8 58.3 44.5 37.7 1.2 12 100 0.5 75.0 93.9 49.3 42.6 1.2 13 100 1.0 75.6 85.7 40.7 27.6 1.5 14 100 2.0 58.6 86.9 22.2 0.9 25.0 15 100 5.0 77.5 100.2 17.0 4.6 3.8

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67 95.0, %45.8 %388.5 %3741.0 %37.60 %02.0 %16.103.6177.2%86.116(%)23 2 2 2 22 2 1 21 1 RPVA PVALA E PVALA E PVA LA PVALA PVA LA EE Figure 2-19. Encapsulation efficiency (EE) of PLA microspheres prepared with variable % of lactic acid in the PLGA polymer (LA) and % of polyvinyl alcohol (PVA) in the aqueous phase

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68 83.0,%79.5 %01.0 %484.7 %72.41 %352.2 %26.0 80.6163.0%27.88(%)23 2 2 2 22 2 1 21 1 RPVA PVALA PVALA E PVA LA E PVALA PVA LA LdE Figure 2-20. Loading efficiency (LdE) of PLA mi crospheres prepared with variable % of lactic acid in the PLGA polymer (LA) and % of polyvinyl alcohol (PVA) in the aqueous phase

PAGE 69

69 99.0, % 349.1 % 434.1 % 02.0 % 457.4 % 05.0 % 451.205.0)(23 3 2 3 2 2 2 RPVA m E PVALA m E PVA m PVALA m E PVA m LA m Em mPV Figure 2-21. Mean particle diam eter based on volume distribution (PV) of PLA microspheres prepared with variable % of lactic acid in the PLGA polymer (LA) and % of polyvinyl alcohol (PVA) in the aqueous phase

PAGE 70

70 86.0, % 86.4 % 09.0 % 330.5 % 06.40 % 02.0 % 25.0 % 58.57 % 21.262.65)(23 3 2 3 2 3 2 2 2 2 2 RPVA m PVALA m PVALA m E PVA m LA m PVALA m PVA m LA m m mPN Figure 2-22. Mean particle diam eter based on number distribution (PN) of PLA microspheres prepared with variable % of lactic acid in the PLGA polymer (LA) and % of polyvinyl alcohol (PVA) in the aqueous phase

PAGE 71

71 77.0, %11.0 %373.1 %408.1 %90.0 %444.3 %301.5 %33.1 %04.028.1 123 3 2 3 23 2 2 22 2 1 1 RPVA PVALA E PVALA E PVA LA E PVALA E PVA LA PR Figure 2-23. Mean particle diameter ratio (PR) of PLA microspheres prepar ed with variable % of lactic acid in the PLGA polymer (LA) and % of polyvinyl alcohol (PVA) in the aqueous phase

PAGE 72

72 Figure 2-24. In vitro drug release profile for LE-PLGA (50:50) microspheres prepared with variable % of polyvi nyl alcohol (PVA) Table 2-7. Particle size properties of LE-PLGA (50:50) microspheres Formulation PV (m) PN ( m) PR 0.2% PVA 37.7 ( 1.3) 16.1 ( 2.1) 2.4 ( 0.2) 0.3% PVA 42.4 ( 0.2) 27.5 ( 0.4) 1.5 ( 0.0) 0.5% PVA 37.8 ( 0.3) 20.2 ( 0.9) 1.9 ( 0.1) 2.0% PVA 27.9 ( 2.0) 7.1 ( 0.8) 3.9 ( 0.2) 5.0% PVA 8.0 ( 0.1) 2.8 ( 0.0) 2.8 ( 0.0) Time (month) 024681012 % Drug Release 0 20 40 60 80 100 0.2% PVA 0.3% PVA 0.5% PVA 2.0% PVA 5.0% PVA

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73 Figure 2-25. In vitro drug release profile for LE-PLGA (75:25) microspheres prepared with variable % of polyvi nyl alcohol (PVA) Table 2-8. Particle size properties of LE-PLGA (75:25) microspheres Formulation PV (m) PN ( m) PR 0.3% PVA 27.3 ( 0.4) 12.5 ( 0.4) 2.2 ( 0.0) 0.5% PVA 38.2 ( 0.4) 27.2 ( 0.8) 1.4 ( 0.0) 1.0% PVA 38.9 ( 0.4) 22.4 ( 5.1) 1.8 ( 0.4) 2.0% PVA 31.3 ( 1.4) 10.4 ( 1.2) 3.0 ( 0.2) 5.0% PVA 11.5 ( 0.1) 3.5 ( 0.0) 3.2 ( 0.0) Time (month) 024681012 % Drug Release 0 20 40 60 80 100 0.3% PVA 0.5% PVA 1.0% PVA 2.0% PVA 5.0% PVA

PAGE 74

74 Figure 2-26. In vitro drug release profile for LE-PLA microspheres prepared with variable % of polyvinyl alcohol (PVA) Table 2-9. Particle size propert ies of LE-PLA microspheres Formulation PV (m) PN ( m) PR 0.2% PVA 44.5 ( 0.9) 37.7 ( 3.1) 1.2 ( 0.1) 0.3% PVA 55.9 ( 0.1) 49.8 ( 0.1) 1.1 ( 0.0) 0.5% PVA 49.3 ( 0.2) 42.6 ( 0.3) 1.2 ( 0.0) 1.0% PVA 40.7 ( 0.2) 27.6 ( 0.2) 1.5 ( 0.0) 2.0% PVA 22.2 ( 0.5) 0.9 ( 0.0) 25.0 ( 0.8) 5.0% PVA 17.0 ( 0.8) 4.6 ( 0.1) 3.8 ( 0.2) Time (month) 024681012 % Drug Release 0 20 40 60 80 100 0.2% PVA 0.3% PVA 0.5% PVA 1.0% PVA 2.0% PVA 5.0% PVA

PAGE 75

75 Figure 2-27. In vitro drug release profile for LE-PLGA micr ospheres prepared with variable % of polyvinyl alcohol (PVA) Table 2-10. Particle size prope rties of LE-PLGA microspheres Formulation PV ( m) PN ( m) PR PLGA 50:50 37.8 ( 0.3) 20.2 ( 0.9) 1.9 ( 0.1) PLGA 75:25 38.2 ( 0.4) 27.2 ( 0.8) 1.4 ( 0.0) PLA 38.6 ( 0.2) 26.7 ( 0.6) 1.4 ( 0.0)

PAGE 76

76 Figure 2-28. Particle morphology of th e LE-PLGA 50:50 micr ospheres during the in vitro drug release studies at (A) 0, (B ) 4, (C) 8, and (D) 12 months A B C D

PAGE 77

77 Figure 2-29. Particle morphology of th e LE-PLGA 75:25 micr ospheres during the in vitro drug release studies at (A) 0, (B ) 4, (C) 8, and (D) 12 months A B C D

PAGE 78

78 Figure 2-30. Particle morphology of the LE-PLA microspheres during the in vitro drug release studies at (A) 0, (B) 4, (C) 8, and (D) 12 months A B C D

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79 Figure 2-31. In vitro drug release profile for LE-PLA microspheres with varying mean volume (PV) and number (PN) diameter and same polydispersity (PR) Table 2-11. Particle size properties of LE-PLA microspheres with varying PV and PN and constant PR Formulation PV (m) PN ( m) PR M1 38.6 ( 0.2) 26.7 ( 0.6) 1.4 ( 0.0) M2 43.4 ( 0.0) 40.2 ( 0.0) 1.1 ( 0.0) M3 55.9 ( 0.1) 49.8 ( 0.1) 1.1 ( 0.0) Time (month) 024681012 % Drug Released 0 20 40 60 80 100 M1 M2 M3

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80 Figure 2-32. In vitro drug release profile for LE-PLA micr ospheres with the same mean volume diameter (PV) but varying ratio (PR) Table 2-12. Particle size properties of LE-PLA microspheres with same PV but varying PR Formulation PV (m) PN ( m) PR M4 30.4 ( 0.8) 21.5 ( 2.1) 1.4 ( 0.1) M5 17.0 ( 0.8) 4.6 ( 0.1) 3.8 ( 0.2) M6 24.1 ( 0.4) 0.9 ( 0.0) 28.1 ( 0.4) Time (month) 024681012 % Drug Released 0 20 40 60 80 100 M4 M5 M6

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81 Figure 2-33. In vitro drug release profile for LE-PLA microspheres with the same mean number diameter (PN) and varying ratio (PR) Table 2-13. Particle size properties of LE-PLA microspheres with same PN and varying PR Formulation PV (m) PN ( m) PR M7 5.4 ( 0.4) 1.0 ( 0.0) 5.4 ( 0.4) M8 24.1 ( 0.4) 0.9 ( 0.0) 28.1 ( 0.4) M9 37.4 ( 0.1) 0.8 ( 0.0) 46.3 ( 0.2) Time (month) 024681012 % Drug Released 0 20 40 60 80 100 M7 M8 M9

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82 CHAPTER 3 CYTOTOXICITY OF SUSTAI NED RELEASE MICROSPHERES Introduction Glucocorticoids can induce apoptosis in -cells by the m itochondr ial apoptotic pathway [118]. Glucocorticoids activate the glucocor ticoid receptor by releasing the molecular chaperones heat-shock protein (HSP90) and FK -binding protein from the receptor complex [119]. HSP90 activates PP-2B (calci neurin) which increases the [Ca2+] leading to the dephosphorylation of the proapoptotic Bcl-2-associated death promoter (BAD) protein [120, 121]. The dephosphorylated BAD leads to mitochondr ial pore formation resu lting in the release of cytochrome c into the cytoso l [122]. Cytosolic cytochrome c activates the caspase cascade causing cleavage of various prot eins, degradation of nuclear DNA, and eventually cell death [123, 124]. Glucocorticoids can also slow down cell growth [125]. Dexamethasone was shown to diminish cell prolifer ation by arresting the G1 cell cycle [126] and down-regulating growthpromoting factors [127]. Since preservation of the transplanted cells is a major concern, it is important to know the -cell cytotoxic dose of loteprednol etabonate in the therap eutic range necessary with the LEPLA microspheres (~50 g for 3 month release). It is also important to know if the PLA microspheres will negate the cytot oxic effect of loteprednol eta bonate or enhance it. Previous studies performed on PLA/PLGA microspheres s howed that the microspheres could induce a different degree of cytotoxicity on different t ypes of cells. Gomes et al. investigated the cytotoxicity of blank PLGA (50:50) microsphe res on rat peritoneal exudate cells in the concentration range of 0.1 to 1.0 mg/mL polymer [128]. There was no significant difference in the percent cell viability as compared to th e control containing no polymer. Xie et al. investigated the cytotoxicity of cisplatin loaded PLA/PLGA mi crospheres on C6 glioma cell line

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83 in the concentration range of 0.125 to 2 mg/mL polym er [129]. It was observed that the cisplatin loaded polymer microspheres had a 1.6 to 2 fold higher cytotoxicity as compared to the unencapsulated drug. In addition, longer exposure to cells (3 days as opposed to 1), further decreased the cell viability. Ma nca et al. looked at the rifa mpicin loaded PLGA (75:25) cytotoxicity on the human A549 alveolar cells [130]. Incorporating the drug into the polymer matrix did significantly reduce the cytotoxicity of the drug. However, there was an increase in the cytotoxicity with an increase in the formulation concentration. Unfortunately, no studies have been perfor med on the PLA microsphere toxcity on the cells. This project investigat ed the cytotoxicity of lote prednol etabonate loaded PLA microspheres on the MIN-6 mouse insulinoma ce ll line. Cytotoxicity was determined by a colorimetric method based on the MTT assay. Viab le cells are unable to induce MTT to colored formazan serving as an indirect m easurement of cell viability [131]. Hypothesis We hypothesized that by encapsulating the gl ucocorticoid lotepre dnol etabonate (LE) within th e polymer poly(D,L-lactic) acid (PLA) microspheres, th e cell toxicity to the beta cells will be decreased as compared to the unencapsulated LE formulation at equivalent drug concentrations. Materials and Methods Chemicals The MIN-6 mouse insulinom a cell line wa s obtained from Dr. Sihong Songs lab. The Dulbeccos modified Eagles medium (Cat. # 10-013-CV), Trypsin EDTA (Cat. # 25-052-Cl), phosphate buffer saline solution (1X PBS), fetal bovine serum, penicillin and streptomycin solution (Cat. # 30-002-CI) were purchased from Cellgro (Manassas, VA). Dimethyl sulphoxide

PAGE 84

84 (DMSO), Isopropanol, and Hydrochloric acid (HCl) were purchased from Fisher Scientific Inc. (Suwanee, GA). Tween 80 was purchased from Sigma Chemical Co. (St. Louis, MO). MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazol ium bromide) was purchased from Calbiochem (EMD Chemicals, Inc., Gibbstown, NJ). MIN-6 Cell Culture The MIN-6 cells were cultured in 10 cm plat es in 4.5g/L glucose Dulbeccos modified Eagles medium (DMEM) containing 15% fetal bovine serum (FBS) and 1% penicillin and streptomycin. The cells were incubated at 37oC under conditions of 5% CO2. The medium was changed every 2 to 3 days and subcultured when the plate was confluent (~weekly). MIN-6 Cell Viability Determination The MTT assay was used to estim ate cell viability. The MIN-6 cells (10,000 cells/well) were seeded in a 96-well flat-bottom plate (C orning Inc., Corning, NY) in 4.5-g/L glucose DMEM for 24 hours. The medium was removed and replaced with DMEM containing unencapsulated drug or LE-PLA microspheres at th e following concentrations of LE: 100, 10, 1, 0.1, and 0.01 M. The control used was a blank having 0 M LE in DMEM media. After incubation for 1 and 4 days, the medium was removed and replaced with filtered MTT (100 L, 0.5 mg/mL in DMEM) and incubated for 3 hours at 37oC. The cells were treated with 100 L isopropyl alcohol containing 0.04 mol/L HCl for 30 minutes under dark at room temperature. The absorbance was measured using the Dyne x Technologies microplate spectrophotometer model MRXTM (Chantilly, VA) at a wavelength of 550 nm. The percent cell viability was calculated to be:

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85 The percent cell viability of the samples was presented as mean SE. Statistical significance of the difference in the percent cel l viability between each concentrations of unencapsulated LE or LE-PLA microspheres were te sted using the Student t-test (SigmaPlot for Windows Version 10.0, Systat Soft ware, Inc., San Jose, CA). Results and Discussion The MIN-6 cells were exposed to vary ing concentrations (0.01, 0.1, 1, 10, and 100 M) of LE. Af ter one day, the unencapsulated drug decrease d the cell viability of the MIN-6 cells with a threshold concentration of 10 M LE (Figure 3-1a). After 4 da ys, the threshold concentration was still 10 M LE but the decrease in the cell viabil ity was more pronounced (Figure 3-1b). The half maximal inhibito ry concentration (IC50), which could only be determined at day 4, was 20 M LE. The blank PLA microspheres at 0.1, 1, 10, 100, a nd 1000 mg/mL concentrations were also exposed to the MIN-6 cells for 1 and 4 days. Ther e was no decrease in the cell viability at day 1 (Figure 3-2a) and day 4 (Figure 32b) indicating that there was no -cell cytotoxicity. Interestingly, there was an increase in the ce ll viability with a thresh old concentration of 100 mg/mL at day 1 and 1000 mg/mL at day 4. A possi ble explanation for the increase in cell viability was due to the degradation product of the PLA polymer (lactic acid) feeding the citrate cycle by producing pyruvate through the lactate dehydrogenase reaction [132]. The citrate cycle (also known as the Krebs cycle) is a metabolic pathway that produces energy in the mitochondria of living cells. The MTT assay is based on ce ll proliferation by determining the formazan production from active mitochondria (only found in living cells). The hi gher concentration of lactic acid, from a higher concentration of polymer, would produce more pyruvate. A higher concentration of pyruvate could enhance th e mitochondria activity hence producing more

PAGE 86

86 formazan resulting in an increase in the cell viabi lity. Ignatius et al. noticed an increase in the succinate dehydrogenase activity, a mitochondrial enzyme used in the citrate cycle, when exposing Clone L929 mouse fibroblast cells to th e degradation products of PLGA (70:30) and PLGA (90:10) [132]. This observati on suggests that PLA could in crease mitochondria activity. By incorporating the drug into the PLA micr ospheres, there was no significant decrease in the cell viability even with an increase in th e formulation concentra tion at day 1 and day 4 (Figure 3-3). The insignificant decrease in cell viability could be due to minimal crystalline drug on the surface on the PLA microspheres and hence a low initial burst. Figure 2-7 shows the PXRD analysis of LE-PLA microspheres. There are small crystalline peaks at the 16, 17, 19, and 24 2 angle for the 5% loteprednol etabonate and blank PLA microsphere mixture which correlated to the prominent peaks for the unencapsu lated drug at the same angles. Note that the blank PLA microspheres are amorphous hence there were no crystalline peaks. However, with the LE-PLA microspheres containing 5% loteprednol etabonate, no crystalline peaks were observed indicating that the drug was incorporat ed within the microspheres and not residing on the surface. The initial burst of the tested formulation is 1.7% at 0 hours which correlates to 1.7 M of LE being exposed to the -cells. This concentration is below the 10 M threshold concentration. Murillo et al. also observed a simila r trend when investigati ng the cytotoxicity of PLGA (50:50) microspheres containing Hot Saline antigenic extract on th e macrophage cell line J774.2 [133]. The formulation had 24.4 (.3) % cell viability and 40.3 (.7) % initial burst. The low cell viability was due to the high initial burst of the formulation which meant an exposure of a higher dose of th e toxic drug to the cells.

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87 Conclusion LE was cytotoxic to the MIN-6 insu linoma ce lls at a threshold concentration of 10 M and with an IC50 of 20 M (only observed at day 4). The blank PLA microspheres had no -cell cytotoxicity in the 0.01 to 1 mg/mL concentration range. In fact, the blank microsphere s increased the cell viability of the MIN-6 insulinoma cell line indicating that PLA could enhance the mitochondrial activity of the -cells. Since the LE-PLA microspheres had no cr ystalline drug on the surface of the microspheres and a low initial burst, there wa s no observed cytotoxicity indicating that the LE-PLA microspheres can prevent -cell cytotoxicity induced by the drug.

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88 Figure 3-1. Effect of loteprednol etabonate on MIN-6 cell viability after 1 day (a) and 4days (b) of incubation (n=4). (*P<0.05, **P<0.01) Unencapsulated Loteprednol Etabonate ( M) 123456 Cell Viability (% of Control) 0 20 40 60 80 100 120 C0.010.1110100* Unencapsulated Lote prednol Etabonate ( M) 123456 Cell Viability (% of Control) 0 20 40 60 80 100 120 C0.010.1110100** ** A B

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89 Figure 3-2. Effect of PLA micros pheres on MIN-6 cell viability after 1 day (a) and 4 days (b) of incubation (n=4). (**P<0.01; ***P<0.001) PLA Microspheres( g/ml) 123456 Cell Viability (% of Control) 0 20 40 60 80 100 120 140 C** ***0.11101001000 PLA Microspheres( g/ml) 123456 Cell Viability (% of Control) 0 20 40 60 80 100 120 140 C0.11101001000** A B

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90 Figure 3-3. Effect of PLA micr ospheres on MIN-6 cell viability af ter 1 and 4 days of incubation (n=4). (***P<0.001) LE-PLA Microspheres( g/ml) 123456789 Cell Viability (% of Control) 0 20 40 60 80 100 120 140 C 101001000C101001000*** *** ***Day 1 Day 4

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91 CHAPTER 4 IN VIVO CHARACTERIZATION OF SU STAI NED RELEASE MICROSPHERES Introduction Life-long im munosuppression is required for pa tients receiving islet transplantation. Current regimens are associated with a lot of complications lowering a pa tients quality of life [47, 48, 55, 134-140]. Localized, versus system ic, delivery of immunosuppressants reduce systemic toxic effects while preventing rejecti on of transplanted organs. Wang et al. looked at the intraportal delivery of immunosuppressants in a rat model receiving intrahepatic islet allografts [141]. Intraportal deliv ery of tacrolimus significantly increased the mean survival duration as compared to the control (no immunosuppressant) and intravenously administered tacrolimus. In addition, the intraportal administra tion of tacrolimus, as opposed to intravenous administration, had lower mean systemic levels possibly resulting in lower systemic toxicity. Ruers et al. observed the same results when co mparing cardiac allograft survival times and systemic drug levels in rats using systemic (j ugular vein) and local (car otid artery) routes of administration of budesonide [142]. Localized delivery of immunosuppressants co uld be possible by many drug delivery systems (DDS) ranging from liposomes to drug-eluting stents to hydrogels [143-147]. Bocca et al. analyzed the possibility of applying of three DDS for localized immunosuppression longterm delivery within the neovasc ularized islet biohybrid device : (1) sustained-release implant (similar to RetisertTM); (2) infusion using an Alzet implantable osmotic minipump; and (3) sustained-release microspheres [53]. RetisertTM is a nonbiodegradable im plant (3 x 2 x 5 mm) containing a fluocinolone acetonide microcrystalline cellulose tablet [148]. As the tablet breaks down, the drug is released through an orifice in the implant into the surrounding environment. The drug release profile has an initial releas e rate of 0.6 g/day over the first month followed by

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92 0.3 to 0.4 g/day over 2.5 years. Implantable infusi on pumps have been used for localized delivery of various therapeutics including 6-me rcaptopurine, prostaglandin E (PGE1), heparin, tacrolimus, and 15-deoxyspergualin into transplanted organs [51, 149-152]. The Alzet minipumps are filled with the drug solution and usually implan ted subcutaneously with the catheter inserted into the nearby artery or into the transplant. Release profiles range from 1 day to 6 weeks with delivery rates between 0.11 to 10 L/hr [153]. Sustained-release microspheres have been previously used as a localized DDS of immunosuppressants fo r preventing transplant rejection [144, 154, 155]. In this project, we studied the feasibility of using biodegradable PLA microspheres for localized sustained-release delivery of lotepre dnol etabonate (LE). Using various considerations, a drug release rate of approximately 1 nmol/day or 0.5 g/day was obtained as a first estimate for the local dose needed to provide sufficient immunosuppression by maintaining a therapeutic concentration of 5 500 nM within the biohybrid device [53]. The LE-PLA microspheres DDS was compared to the 2 week Alzet osmotic mi nipump DDS. The animal studies were performed at the Diabetes Research Instit ute at the Miller School of Medi cine, University of Miami in collaboration with Dr. Peter Buchwalds Drug Discovery group and Dr. Antonello Pileggis Translational Cell Processing a nd Transplant Models Core. Hypothesis We hypothesized that th e 3-month release LEPLA microsphere formulation will prevent the rejection of the islet allografts (as indicated by the mean survival duration of the chemically diabetic rats) for around a 3-month period.

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93 Materials and Methods Islet Transplantation Islet transplantation using the neovascularized islet biohybrid device was perform ed at the Diabetes Research Institute us ing the method developed by Pileggi et al. [30]. The project and animal care was conducted under protocols a pproved by the Institutional Animal Care Committee. To summarize the procedure (Fig ure 4-1), the rats were put u nder general anesthesia using isoflurane USP (Baxter, Deerfield, IL) prior to the surgery. The sterilized islet biohybrid device was subcutaneously implanted into the dorsal regi on of the rats. The stainless steel device was 2 cm long with a diameter of 0.6 cm and pore size of 450 m with a polytetrafluoroethylene (PTFE) plug and inserted plunger (which filled the inner void of the device). After forty days, the biohybrid device was neovascularized and preparations for islet tr ansplantation were started. Donor islets were obtained from male Wistar Furth rats (Indianapolis, IN) and purified by enzyme digestion using Liberase (Roche; Indi anapolis, IN) and density gradient separation (Mediatech; Herndon, VA). Isle t grafts were cultured in CMRL-1066 medium (GibcoInvitrogen; Carlsbad, CA) at 37oC in 5% CO2. Female Lewis rats of body weights around 200 g were administered 60 mg/kg intravenously the -cell toxin streptozotocin (Sigma Aldrich, St. Louis, MO) at day one and day two/three prior to transplantation. Only rats having non-fasting glucose levels 350 mg/dL were used. The PTFE plunger was removed from the biohybrid device through a cutaneous incisi on. Islet grafts of 3000 islet equi valents (IEQ) were suspended in PBS and inserted into the inner void of the device using a syringe (Hamilton; Reno, NV) and polyethylene catheter. Another PTFE plug was used to seal the device. The skin was then sutured to close the incision.

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94 Animals were then maintained on a twoto three-week regimen of systemic immunosuppression in addition to the localized delivery. The systemic immunosuppression was then gradually withdrawn leaving only the localized one. For the control, no immunosuppressants were inserted into the bioh ybrid device. For loca lized delivery of LE by infusion, 10 mg/L LE (in 10% ethanol and 90% 1X PBS) was inserted into an Alzet Osmotic Pump Model #1002, 0.25 L/h (Durect Corp., Cupertino, CA). The pump was implanted subcutaneously and catheter inserted into the or ifice of one of the PTFE plugs and replaced every two weeks as needed. For localized delivery of LE-PLA microspheres, 4. 5 mg loading dose of LE-PLA microspheres (sterilized by radiation) were inserted in to the biohybrid device prior to sealing the device. The delivery rate was estimated to be around 3.0 g/day based on a 4% drug content released over appr oximately 60 day period. Statistical Analysis The statistical sign ificance of difference in the percent survival of islet recipients between rats receiving different immunos uppressant treatments was tested using the Student t-test (SigmaPlot for Windows Version 10.0, Systat Softwa re, Inc., San Jose, CA). Curve fitting of the in vitro drug release profile was preformed with SigmaPlot 10.0 and Excel software programs. Results and Discussion The m ean survival duration of the chemically diabetic rats was increased with the use of the sustained release formulation as compared to the control (sal ine) and infused drug (Figure 42). The chemically diabetic rats received two to three weeks weaning of systemic immunosuppression following islet transplantat ion in conjunction with the localized immunosuppressant therapies. The localized im munosuppression was maintained after weaning of the systemic administration. The localized immunosuppressant therapies used was: no drug

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95 (control), LE solution delivered with 2-week release Alzet pump, and LE-PLA microspheres inserted into the implant. The mean survival duration was 9.3 3.6 days (n = 10), 13.6 4.9 days (n = 5), and 20 6.6 days (n = 4) for the control, LE infusion, and LE-PLA microspheres respectively. The survival duration for the contro l was not significantly di fferent from the rats receiving LE infusion but was significantly diffe rent from those receiving LE-PLA microspheres (P < 0.01). There was no significant difference in the survival durations of the rats receiving the LE infusion and LE-PLA microspheres. The LE-PLA microspheres tested had a three month in vitro drug release profile (Figure 43). The in-vitro drug release duration did not match the in vivo mean survival duration of the recipients. Possible reasons for the differences coul d be due to (1) the drug release rate from the formulation being below the therapeutic range, (2 ) the size of the microspheres not being large enough, and (3) the in vitro in vivo drug release time scale not co rrelating to each other. In order to investigate if the drug release rate fell below the therapeutic range, the in vitro drug release profile of the LEPLA microspheres tested was compared to various commonly used dissolution models to find one that had the best fit [156]. Table 4-1 shows the best fit equations and the correlation coefficient (R2) of in vitro release profile to the various dissolution models. The Second-order exponential and Korsmeye r-Peppas dissolution models gave the best fit as shown in Figure 4-3. The Korsmeyer-Peppas model was chosen for future analysis due to the simplicity of the model as compared to th e second order exponential dissolution model. In taking the differential of the Korsmeyer-Pe ppas model, we get the drug release rate: (4-1) where Qt is the amount of drug released as a function of time (mg) Q is the total amount of dr ug in the microspheres (mg) Kk is a constant = 0.696

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96 n is the order of the model = 0.32 t is the time of drug released in months The drug release rate (Equation 4-1) is plotte d as shown in Figure 4-4. Bocca et al. [53] recommended a drug release rate of 0.5 g/day in order to maintain therapeutic concentrations based on in vitro cytotoxicity studies and computational modeling. The release rate was determined under the assumption that no local drug metabolism occurred which may not be true in the case of LE considering that it is a soft drug that hydrolyzes in aqueous solution. LE has a half life of 1.4 days in aqueous solution (data no t shown) meaning a minimal drug release rate of l.6 g/day may be needed to maintain therapeutic concentrations. The baseline 0.5 g/day (Figure 4-4) indicates that th e formulation should last 3 m onths. However, the baseline 1.6 g/day indicates that the formulation should la st 1.1 months or 33 days which is more representative of the in vivo data. Since the device was prevas cularized, it is qu ite possible for the enzymes that promote hydrolysis to enter the device and degrade the drug faster. So the minimal drug release rate is not certain. Figure 4-5a depicts the partic le size distribution of the LEPLA microspheres based on the % volume (meaning the distribution has been nor malized to emphasize the larger particles). Figure 4-5b depicts the particle size distribution based on the % num ber (the distribution has not been normalized and would represent the distri bution observed under SEM). Both have different mean averages implying that the formula tion is polydispersed as indicated by a high meanvolume/meannumber ratio (Table 4-2). The subcutan eous tissue surrounding the device contained arteries, veins, capillaries, and adipose tissue which could carry the smaller microspheres into the blood str eam [157]. With a lower amount of microspheres within the device, the total drug concentrati on would be lower. Also, the drug release rate would be lower

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97 since larger microspheres have slower rates. Future studies will be conducted with larger microspheres. A good correlation with the in vitro dissolution profile and the in vivo plasma concentrations can reduce product development cost and time by predicting beforehand the expected in vivo duration of drug release [157]. The in vitro drug release profile of the LE-PLA microspheres was determined from a dissolution assay performed at 37oC under sink conditions at 30 rpm using a magnetic stirrer. Using low stir rates (as opposed to the standard 100 rpm) is common when determining the drug release of drug loaded microsphe res injected into muscle or subcutaneous tissue. The low stir rates would represent the environment in the subcutaneous tissue which is dense and would have low flux. C hu et al. used a bath shaker at 40 rpm to conduct in vitro dissolution studies for huperzine A loaded PLGA microspheres [158]. Four different formulations were in jected either subcutaneously or intramuscularly. All had an in vitro in vivo correlation (IVIVC) coefficient (R2) of > 0.96 indicating a great correlation. The enzymatic degradation of the polymers also n eed to be considered when developing the in vitro release study [159]. While the dissolution assay was performed under conditions suitable for drug release within a subcutaneous implant, it would advisable to determine the in vitro in vivo correlation. Unfortunately, the rats receiving is let transplantation undergo a lot of stress from the surgery that obtaining blood samples at sufficient time point s for a proper pharmacokinetic study could be detrimental to the rat and hence the study. Othe r alternatives include determining the drug content remaining in the biohybr id implant device which would tell us how much drug was released. Also determining the cumulative am ount of drug and metabol ites (as a function of time) excreted from urine and feces could be an indirect correlati on to the amount of drug

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98 remaining in the device [160]. The drug content in the device is cu rrently being investigated. We may look at the cumulative drug and metabolites amount in the waste in the future. Conclusion The LE-PLA m icrospheres was successful in im peding islet transplant ation rejection as indicated by the mean survival time of 20 6.6 days. The survival time of the microspheres was significantly differe nt from the control (no drug). The LE-PLA microspheres did not maintain ther apeutic concentrations over the expected 3 month duration. Future studies have to be done with la rger, more monodisperse microspheres (> 10 m and meanvolume/meannumber ratio < 1.5). Future studies have to be done to determine the in vitro in vivo drug release correlation by either analyzing the drug remaining in the device at the e nd of the study or the feces and urine for the amount of drug and metabolites expelled from the body.

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99 Figure 4-1. Islet transplantation using the novel biohybrid device.

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100 Days after Immunosuppressant Treatment 051015202530 % Survival 0 20 40 60 80 100 Control (Saline) LE Infusion LE-PLA Microspheres Figure 4-2. Percent survival of chemically diabetic rats receiving islet transplantation in conjunction with the following local i mmunosuppressant therapy: saline solution with no drug (control), LE solution infused with Alzet pump (LE Infusion), and LE-PLA microspheres inserted into device (LE-PLA Microspheres)

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101 Table 4-1. Best fit equations of comm only used dissolution models for the in vitro drug release of the LE-PLA microsphere formulation

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102 Figure 4-3. Curve-fitting of the in vitro drug release (Q) of the LE-PLA microsphere formulation

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103 Figure 4-4. Rate of drug released (dQ/dt ) from the LE-PLA microsphere formulation

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104 Figure 4-5. Particle size dist ribution of the LE-PLA microsphere formulation based on the (A) volume and (B) number distribution Table 4-2. Volume and number distribu tion statistics of LE-PLA microspheres Volume Distribution Number Distribution Mean ( m) 5.059 0.124 Median ( m) 3.412 0.094 Standard Deviation ( m) 6.839 0.139 d10 ( m) 0.729 0.059 d90 ( m) 9.295 0.209 Meanvolume/Meannumber 40.80 A B

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105 CHAPTER 5 SUMMARY Poly(D,L-lactic-co-glycolic) acid (PLGA) micros pheres developed by solvent evaporation was successful as a sustained drug deliver y system for localized delivery of the immunosuppressant loteprednol etabonate (LE) within an implant device. The solvent evaporation process parameters (Chapter 2) we re optimized to produce 5 to 50 micron-sized, monodispersed, smooth microspheres having sustai ned release ranging from three months to a few years. The type of emulsification method us ed made a difference in the morphology and the release profile of the microspheres produced. The infusion rate of the organic phase into the aqueous phase and the inner diam eter of the infusion tube, duri ng the emulsification step, also made a difference in the smoothness, encapsulati on efficiency, drug loading, and polydispersity of the microspheres produced. Emulsificati on using the infusion method produced more monodispersed, spherical microspheres with a more sustained and zero-order release. The drug loading at 5% produced LE-PLA mi crospheres with the lowest init ial burst due to low or no drug adsorbed onto the surface of the microspheres. Using 1% SDS in the wash phase did a great job of removing the drug adsorbed on the microsphe re surface. The resultant microspheres had a very low initial burst. A factorial design analysis study was performe d to more thoroughly determine the optimal formulation. The optimal infusion rate (IR) wa s determined to be 0.8 mL/min. Increasing the inner diameter of the infusion tube (ID) produced larger LE-PLA microspheres but also increased the polydispersity. In creasing the % of polyvinyl al cohol (PVA) in the aqueous solution, produced smaller microspheres with higher polydispersity. Th e encapsulation and loading efficiency also increased with increasing % PVA. In looking at the in vitro drug release of the LE-PLGA microspheres, another parameter be side the size (possibly the density/porosity)

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106 of the microspheres influenced the drug releas e. Varying the composition of the lactic and glycolic acid in the PLGA polymer had no significant effect on the encapsulation and loading efficiency and the particle size and polydispers ity of the microspheres. However, in decreasing the % of the lactic acid monomer, the drug release rates incr eased. SEM pictures show that the LE-PLGA 50:50 microspheres internal structur e collapsed and eroded over time. LE-PLA microspheres ruptured after 4 months of in vitro drug release. Monodisperse LE-PLA microspheres had very little difference on the in vitro LE release rates when varying the size of the microspheres. However, varying the mean volume diameter, mean number diameter, and the polydispersity made a big differe nce and should be taken into c onsideration when predicting the in vitro drug release profiles. Formulation M7 was chosen for the in vitro cell toxicity studies (Chapter 3) and the in vivo feasibility study (Chapter 4) sin ce it had a desired duration of release of 3 months. The LE-PLA microspheres were analyzed for its in vitro cell toxicity using the M TT assay. The drug LE had a threshold concentration of 10 M and IC50 of 20 M on the MIN-6 insuloma cell line. However, the LE-PLA microspheres had no cytotoxic ity. The LE-PLA microspheres were tested to find the immunosuppressive activity in chem ically diabetic rats receiving islet transplantation using the novel implant device. The microspheres were succe ssful in preventing islet transplantation by increasing the mean survival duration to 20 6.6 days. The survival duration of the microspheres was twice as much as the control (no drug). The LE-PLA microspheres did not maintain ther apeutic concentrations over the expected 3 month duration. Future studies have to be done with larger, more monodisperse microspheres (> 7 m and meanvolume/meannumber ratio < 1.5). Future studies have to also be done to determine the in vitro in vivo drug release correlation by ei ther analyzing the drug remaining in the device at

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107 the end of the study or the feces and urine for the amount of drug and metabolites expelled from the body. However, these results show a great promise in the sustained release LE-PLGA microspheres improving the success rate of isle t transplantation by making the way for a more patient compliant and effectiv e immunosuppressant therapy.

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121 BIOGRAPHICAL SKETCH Elanor Pinto was born in Kuwait City, Ku wait on Septem ber 21, 1980. She graduated St. Johns Lutheran High School, Ocala, Florida in 1997. She received her B.S. in Chemical Engineering from University of Florida, Gaines ville, Florida in 2002. After her bachelors, Elanor worked for almost a year as a res earch technician for the Particle Engineering Research Center (PERC), Gainesvi lle, Florida. She entered the pharmaceutical sciences program in 2003 under the supervision of Dr. Gnther Hochhaus. She received her Ph.D. in pharmaceutical scie nces in December 2008. While in undergraduate school, El anor participated in the Pa rticle Engineering Research Center (PERC) Undergraduate Research Assistantship program under th e supervision of Dr. Kerry Johanson. In addition, she participated in the selective Integr ated Product & Process Design (IPPD) course in which she worked on a Kimberly Clark Co. sponsored project. The feasibility project was successful and resulted in a promising processing technology for the company. While in graduate sc hool, Elanor attended a summe r internship at Boehringer Ingelheim in Ridgefield, Connecticut under the supervisi on of Dr. Xiaohui Mei. Elanor was an active member of the American Association of Pharmaceutical Scientists (AAPS). During her first year as a member in 2004, she was the Chair of AAPS University of Floridas Student Chapter. The following year she was the co-chair and co-founder of the graduate student initiated South East Regional Interdisciplinary Symposium (SERIS) which was held in Gainesville, Florida in 2006. In 2006, she became the first student representative of AAPS Modified Release Focus Group Steering Team Also while in graduate school, she was the Pharmaceutical Sciences representative for Un iversity of Floridas Graduate Student Council.