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Design of a stealth liposome delivery system for a novel glycinamide ribonucleotide formyltransferase inhibitor

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Design of a stealth liposome delivery system for a novel glycinamide ribonucleotide formyltransferase inhibitor

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Content INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. ProQuest Information and Learning 300 North Zeeb Road, Ann Arbor, M l 48106-1346 USA 800-521-0600 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DESIGN OF A STEALTH LIPOSOME DELIVERY SYSTEM FOR A NOVEL GLYCINAMIDE RIBONUCLEOTIDE FORMYLTRANSFERASE INHIBITOR by Wen-Chin Tsai A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (PHARMACEUTICAL SCIENCES) December 2001 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number 3065858 ___ < 8 UMI U M I Microform 3065858 Copyright 2002 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor. M l 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UNIVERSITY OF SOUTHERN CALIFORNIA THE GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES. CALIFORNIA 90007 This dissertation, loritten by under the direction of f t & s Dissertation Committee, and approved by all its members, has been presented to and accepted by The Graduate School, in partial fulfillment of re quirements for the degree of W en-Chin T s a i DOCTOR OF PHILOSOPHY 5SEK M TO N COMM ITTEE Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Wen-Chin Tsai Robert T. Koda ABSTRACT DESIGN OF A STEALTH LIPOSOME DELIVERY SYSTEM FOR A NOVEL GLYCINAMIDE RIBONUCLEOTIDE FORMYLTRANSFERASE INHIBITOR AG2034 is a novel GARFT inhibitor which was synthesized using the three dimensional structure of the GARFT domain of the human trifunctional enzyme as determined by X-ray crystallography. Inhibition of GARFT results in a selective blockade of de novo purine biosynthesis, a process which is critical for tumor cell growth. According to preclinical studies, the gastrointestinal tract has been found to be the primary target organ for toxicity in mice. The rationale for liposome delivery is to alter the pharmacokinetics and biodistribution of AG2034 to improve the therapeutic efficacy and safety. A stealth liposome delivery system was developed and optimized to reduce the toxicity in vivo. The effect of cholesterol on encapsulation efficiency showed a DSPC/CHOL ratio of 1:0.25 formulation had higher percent drug encapsulated. Upon the addition of 5% PEG(2k)-DPPE, encapsulation efficiency was increased from 5% to 15% compared to the formulation without PEG(2k)-DPPE. The impact of the manufacturing method on encapsulation efficiency indicated that the extrusion method had much higher efficiency than the remote loading method in terms of drug encapsulation. The encapsulation efficiency was increased up to 21% as a result of increasing the lipid concentration from 40 mM to 60 mM, with only a slight increase occurring when further increasing the lipid concentration to 80 mM. The effect of AG2034 loading on encapsulation l Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. efficiency showed that there were minimal differences in encapsulation efficiency when increasing the AG2034 loading in formulations. The pharmacokinetic study indicated that in contrast to the rapid blood clearance observed for free AG2034. the liposomal delivery systems extended AG2034 existence in blood. In the case of stealth liposomes, 50% of AG2034 was present in the blood 57 hours after i.v. injection. Administration of free AG2034 was found to be more toxic, resulting in mortality at earlier time points than that found for AG2034 encapsulated in stealth liposomes. The acute toxicity study demonstrated that the stealth liposome delivery system significantly reduces the in vivo toxicity when compared to free AG2034. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DEDICATION To My Parents and Wife For Their Unceasing Support and Love Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS I would sincerely thank my advisor, Dr. Robert T. Koda, for his guidance and support during my study at the School of Pharmacy, USC. My sincere thanks also go to my committee members. Dr. Wei-Chiang Shen for his keen interest in my research project and his helpful suggestions and Dr. Stanley P. Azen for being so kind to serve as the external member on my committee and for his statistical advice. My thanks to Dr. Praveen Tyle, a scientist with great vision and full enthusiasm. I thank him not only for his guidance and advice but also for his encouragement to go to graduate school. I would like to thank Dr. Eric J. Lien and Dr. Vincent H. L. Lee for their precious advice in my early dissertation research. My heartfelt thanks to my boss, Ms. Terry L. Wilke, at Pfizer Global Research and Development, La Jolla Laboratories, not only for her generous support, care and patience during the last five years at my graduate study, but also for her excellent editorial help and review on this dissertation and friendship in developing my professional career. Finally, I would like to thank my wife, Dr. Yali Tsai, for her constant encouragement and scientific discussions in my dissertation throughout the years. t U Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS CONTENTS PAGE DEDICATION ii ACKNOWLEDGEMENTS iii LISTOFTABLES. . . .. ix LIST OF FIGURES ................................................................................................ xi CHAPTER I. INTRODUCTION .................. I LA. Specific Aim s................................................................................................2 LB. Historical Perspective of GARFT Inhibitors..............................................3 I.C. AG2034 .........................................................................................................5 I.D. Acute Toxicity in Mice and D ogs...............................................................6 I.E. Liposomes.....................................................................................................7 1. Background of Liposomes.................................................................... 7 2. Techniques for Preparation of Liposomes....................................... 17 1. Large Liposomes......................................................................... 17 1. Multilamellar Vesicles............................................................ 17 2. Large Uni- or Oligolamellar Vesicles.....................................17 2. Small Unilamellar Liposomes..................................................... 18 3. Formation of Liposomes...................................................................... 18 4. Phase Behavior of Liposomes.............................................................. 19 5. Background of Stealth Liposomes.......................................................20 6. Interactions of Liposomes with C ells................................................. 22 1. Intermembrane Transfer................................................................22 2. Contact Release..............................................................................22 3. Adsorption......................................................................................23 4. Fusion.............................................................................................23 5. Phagocytosis/Endocytosis.............................................................24 7. Rationale for the Development of a Stealth AG2034 Liposome Formulation_____________________________________24 0. DEVELOPMENT AND CHARACTERIZATION OF A STEALTH AG2034 LIPOSOME DELIVERY SYSTEM......................... 26 O.A. A b s tr a c t .........................................................................................27 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 113. Background..................................... 29 II.C. Experimental..............................................................................................30 1. Materials................................................................................................ 30 1. Chemicals.......................................................................................30 2. HPLC Analysis of AG2034 .............................................................. 31 1. HPLC System...............................................................................3 1 2. Mobile Phase................................................................................ 3 1 3. Preparation of 25 mM Ammonium Phosphate, pH = 3 .0 .........31 4. Preparation of 0.1 M Phosphate Buffer. pH = 8 .0 .......................32 5. Preparation of Standards...............................................................32 6. Preparation of Samples................................................................. 32 3. Assay Validation for AG2034 ........................................................... 33 1. Linearity.........................................................................................33 2. Accuracy and Precision.................................................................33 4. UV-Vis Analysis of Phospholipids .....................................................34 1. UV-Vis Spectrophotometric System............................................34 2. Preparation of Ammonium Ferrothiocyanate Solution...............34 3. Preparation of Standards...............................................................34 1. Preparation of DPPC Standards...............................................34 2. Preparation of DSPC Standards...............................................35 3. Preparation of DSPC/PEK(2k)-DPPE with the Molar Ratio of 15.2:1 Standards........................................................ 35 4. Preparation of DSPC/PEG(2k)-DPPE with the Molar Ratio of 7.2:1 Standards.......................................................... 36 4. Preparation of Samples..................................................................36 5. Assay Validation for Phospholipids....................................................37 1. Linearity......................................................................................... 37 2. Accuracy and Precision.................................................................37 6. pH-Solubility Profile............................................................................38 7. Heating Time Effect on Stability of AG2034 during Preparation of Liposomal Formulations..................................................................38 8. Preparation of Liposomes....................................................................39 1. Extrusion M ethod.......................................................................... 39 2. Remote Loading Method...............................................................40 3. Extraction of AG2034 from Liposomal Formulations................41 9. Encapsulation Efficiency......................................................................42 10. Development of A Stealth Liposome Delivery System.....................43 1. Effect of Phospholipids and Cholesterol on Encapsulation Efficiency................ 43 2. Influence of PEG(2k)-DPPE Loading on Encapsulation Efficiency__________________________________ 43 3. Impact of Manufacturing Method of Liposomes on Encapsulation Efficiency_______________________________ 44 V Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4. Effect of Phospholipids Loading on Encapsulation Efficiency „ 44 5. Effect of AG2034 Loading on Encapsulation Efficiency 44 1L Determination of Liposome Size....................................................... 45 12. Determination of the Phase Transition Temperature........................45 13. Leakage Study.....................................................................................46 14. Cell Growth Study...............................................................................46 15. In Vitro Release Study........................................................................47 16. Efficacy Study of Liposome-Associated AG2034 Treated with Human Serum....................................................................................... 48 n.D. Results....................................................................................................... 49 1. HPLC Analysis of AG2034 .............................................................. 49 1. Linearity..........................................................................................50 2. Accuracy and Precision..................................................................50 2. UV-Vis Analysis of DPPC..................................................................52 1. Linearity..........................................................................................52 2. Accuracy and Precision..................................................................52 3. UV-Vis Analysis of DSPC..................................................................54 1. Linearity..........................................................................................54 2. Accuracy and Precision..................................................................55 4. UV-Vis Analysis of DSPC/PEG(2k)-DPPE with the Molar Ratio of 15.2:1 Standards...............................................................................56 1. Linearity..........................................................................................56 2. Accuracy and Precision..................................................................57 5. UV-Vis Analysis of DSPC/PEG(2k)-DPPE with the Molar Ratio of 7.2:1 Standards.................................................................................59 1. Linearity..........................................................................................59 2. Accuracy and Precision..................................................................59 6. pH-Solubility Profile......................................................................... 6 1 7. Extraction of AG2034 from Liposomal Formulations......................62 8. Heating Time Effect on Stability of AG2034 during Preparation of Liposomal Formulations..................................................................63 9. Liposomes............................................................................................64 10. Development of a Stealth Liposome Delivery System ..................... 65 1. Effect of Phospholipids and Cholesterol on Encapsulation Efficiency....................... 65 2. Influence of PEG(2k)-DPPE Loading on Encapsulation Efficiency________________________________ 67 3. Impact of Manufacturing Method of Liposomes on Encapsulation Efficiency................ 68 4. Effect of Phospholipids Loading on Encapsulation Efficiency68 5. Effect of AG2034 Loading on Encapsulation Efficiency........... 69 11. Determination of Liposomes Size......................................... 70 12. Differential Scanning Calorimetric Analysis ........................ 72 vt Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 13. Leakage Study............................................................................ _ ......... 74 14. Cell Growth Study............................................................................... 75 15. In Vitro Release Study.........................................................................76 16. Efficacy Study of Liposome-Associated AG2034 Treated with Human Serum 77 HI. EVALUATION OF PLASMA CLEARANCE AND TOXICITY OF AG2034 STEALTH LIPOSOME DELIVERY SYSTEM.............................79 HI.A. Experimental............................................................................................80 1. Materials............................................................................................... 80 1. Chemicals....................................................................................... 80 2. Anim als................................... 80 3. Equipment...................................................................................... 80 2. Preparation of Formulations................................................................ 81 1. Preparation of 5 mg/mL and 15 mg/mL of AG2034 Solutions . 81 2. Preparation of Liposomal Formulation of AG2034 ................... 81 3. Pharmacokinetics................................................................................. 82 4. HPLC Analysis of AG2034 In V ivo.....................................................83 1. Liquid Chromatography.................................................................83 2. Chromatographic Conditions.........................................................83 3. Preparation of 1% (v/v) Formic A cid............................................83 4. Preparation of 2% (v/v) Ammonium Hydroxide......................... 84 5. Preparation of Standards and Quality Control Samples.............. 84 6. Preparation of Samples...................................................................84 7. Assay Validation.............................................................................85 1. Linearity....................................................................................85 2. Accuracy and Precision............................................................85 5. Pharmacokinetic Analysis....................................................................86 6. Acute Toxicity Study............................................................................86 ALB. R esults......................................................................................................87 1. Liposomes.............................................................................................87 2. Chromatographic Results for In V ivo..................................................87 1. Linearity..........................................................................................89 2. Precision and Accuracy..................................................................90 3. Pharmacokinetics................................................................................91 4. Acute Toxicity Study............................................................................94 IV. DISCUSSION.......................................................................................... 96 IV_A. HPLC Analysis of AG2034 In V itro................................................... 97 IVJ3. UV-Vis Analysis of Phospholipids....................................................... 97 IV.C. Preparation of AG2034 Solution............................................. 97 vii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IV.D. Liposomal Delivery System ............................................................. 98 IV.E. Development of A Stealth Liposome Delivery System ......................100 L Effect of Phospholipids and Cholesterol on Encapsulation Efficiency________________________________ 100 2. Influence of PEG(2k)-DPPE Loading on Encapsulation Efficiency............................................................................................. 101 3. Impact of Manufacturing Method of Liposomes on Encapsulation Efficiency.................... 103 4. Effect of Phospholipids Loading on Encapsulation Efficiency 103 5. Effect of AG2034 Loading on Encapsulation Efficiency.............. 104 IV P. Leakage Study........................................................................................ 105 IV .G. In Vitro Release Study..........................................................................105 IV.H. Efficacy Study of Liposome-Associated AG2034 Treated with Human Serum ...................................................................................... 106 IV.I. HPLC Analysis of AG2034 In V iv o .....................................................106 IV J . Pharmacokinetic Analysis.....................................................................107 IV.K. Acute Toxicity.......................................................................................109 1V.L. Mechanism.............................................................................................109 V. CONCLUSIONS........................................................................................... 112 VI. SIGNIFICANCE OF WORK AND FUTURE PROSPECTS.................... 115 VII. BIBLIOGRAPHY......................................................................................... 118 viii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UST OF TABLES TABLES PAGE Table I: Some common saturated and unsaturated fatty acids found in lipids used to form liposomes............. 10 Table 2: Assay validation for AG2034 by HPLC.............................................51 Table 3: Assay validation for D PPC .................................................................. 53 Table 4: Assay validation for DSPC...................................................................55 Table 5: Assay validation for DSPC/PEG(2k)-DPPE with the molar ratio of 15.2:1 standards................................................................................58 Table 6: Assay validation for DSPC/PEG(2k)-DPPE with the molar ratio of 7.2:1 standards................................................................................. 60 Table 7: AG2034 pH-solubility study at ambient temperature......................... 61 Table 8: Solvent selection for AG2034 extraction from liposomal formulations......................................................................................... 63 Table 9: Effect of phospholipids and cholesterol on encapsulation efficiency.............................................................................................. 66 Table 10: Influence of PEG(2k)-DPPE loading on encapsulation efficiency.............................................................................................. 67 Table 11: Impact of manufacturing method of liposomes on encapsulation efficiency.............................................................................................. 68 Table 12: Effect of phospholipid loading on encapsulation efficiency...............69 Table 13: Effect of AG2034 loading on encapsulation efficiency......................69 Table 14: Accuracy of the method for the determination of AG2034 in mouse plasma................................................................................... 90 IX Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 15: Table 16: Table 17: Precision of the method for the determination of AG2034 in mouse plasma......................................................................... Kinetic parameters for free AG2034, conventional liposomes and stealth liposomes................................................................. Toxicity of free AG2034 and stealth liposomes in C3H mice . Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. FIGURE LIST OF FIGURES PAGE Figure I: Structure of AG2034 ................................................................................6 Figure 2: Structure of liposomes: large multilamellar (MLV), large unilamellar (LUV) and small unilamellar (SUV) vesicles...................9 Figure 3: Chemical structure of lipids commonly used to prepare liposomes. PE, PC and cholesterol behave as neutral molecules, whereas PA, PS, PG, and PI have a net negative charge.................. 1 1 Figure 4: Chemical structure of two representative phospholipids showing their polar head groups and nonpolar hydrocarbon chains............... 12 Figure 5: Conventional liposome......................................................................... 15 Figure 6: Stealth liposome..................................................................................... 25 Figure 7: Mini extruder from Avanti Polar Lipids Inc........................................41 Figure 8: Typical chromatogram of AG2034 ...................................................... 49 Figure 9: Linearity for AG2034 standards by HPLC. Each point represents the mean ± s.d. (n=3).............................................................................5 1 Figure 10: Standard curve for DPPC assay. Each point represents the mean ± s.d. (n=3)................................................................................................. 54 Figure 11: Standard curve for DSPC assay. Each point represents the mean ± s.d. (n=3).................................................................................................56 Figure 12: Standard curve for DSPC/PEG(2k)-DPPE with the molar ratio of 15.2:1 assay. Each point represents the mean ± s.d. (n=3)._______ 58 Figure 13: Standard curve for DSPC/PEG(2k)-DPPE with the molar ratio of 7.2:1 assay. Each point represents the mean ± s.d. (n=3)...................61 Figure 14: AG2034 pH-soIubility profile at ambient temperature........................62 Figure 15: Effect of heating time on stability of AG2034 solution at 60°C Each point represents the mean ± s.d. (n=3).___________________ 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 16: Elution profile of liposome-associated AG2034 from gel filtration chromatography on a Sephadex™ G-50 column 65 Figure 17: Figure 18: Figure 19: Figure 20: Figure 21: Figure 22: Figure 23: Figure 24: Figure 25: Figure 26: Figure 27: Figure 28: Figure 29: Particle size distribution for formulations 1-6. Each point represents the mean ± s.d. (n=3).______________ ____________ 71 Particle size distribution for formulations 5. 7 and 8. Each point represents the mean ± s.d. (n=3)........................................................7 1 Particle size distribution for formulations 7, 10, 11 and 15. Each point represents the mean ± s.d. (n=3)................................................ 72 DSC thermograph of formulation 7 .................................................... 73 DSC thermograph of formulation 8 .................................................... 73 Leakage profile of AG2034 from liposomal formulation 15. stored at 5°C and ambient condition for up to 30 Days. Each point represents the mean ± s.d. (n=3)................................................ 74 Comparative analysis of the effect of control, empty liposomes, AG2034 free drug and AG2034 stealth liposomes on proliferation of A549 c ells.................................................................. 75 Irt vitro release study of formulation 15. Each point represents the mean ± s.d. (n=3)................................................................................. 76 Comparative analysis of efficacy study of liposome-associated AG2034 (formulation 15) with and without treatment with human serum on proliferation of A549 cells. Each point represents the mean ± s.d. (n=3)......................................................... 78 Chromatogram of blank mouse plasma.............................................. 88 Chromatogram of AG2034 and AG2037 (I.S.) standard solution........................... 88 Chromatogram of in vivo mouse plasma sam ple ............ 89 Plasma profiles of free AG2034 in mice. Each point represents the mean ± s.d. (n=5).___________________________ 92 X ll Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 30: Figure 31: Plasma profiles of AG2034 conventional liposomes. Each point represents the mean ± s.d. (n=5).____________________________ 93 Plasma profiles of AG2034 stealth liposomes. Each point represents the mean ± s.d. (n=5)............... 93 xiii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. L INTRODUCTION Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LA. Specific Aims: The objective of this proposal is to design and formulate a sterically stabilized liposome delivery system which can feasibly reduce the in vivo toxicity of AG2034, a novel inhibitor of glycinamide ribonucleotide formyltransferase. In order to accomplish this goal, the following five specific aims will be pursued. 1. Development and optimization of a stealth AG2034 liposome formulation 2. Determination of efficiency of encapsulation for AG2034 in liposomal formulation 3. Characterization of the in vitro lipid complex formulations 4. Evaluation of plasma clearance and biodistribution for the optimized stealth AG2034 liposome formulation 5. Investigation of the in vivo toxicity of optimized stealth AG2034 lipid complex delivery system Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 3 . Historical Perspective of Glycinamide Ribonucleotide Formyltransferase Inhibitors Glycinamide ribonucleotide formyltransferase (GARFT) is a 21,800 Da cytosolic protein present in cells as part of a trifunctional enzyme consisting of glycinamide ribonucleotide synthetase (GARS), phosphoribosyi-aminoimidazole synthetase (AIRS), and GARFT. Starting from phosphoribosyl pyrophosphate (PRPP), GARFT is the third enzyme in the sequential, ten step pathway of de novo purine synthesis. (Smith et al., 1980). Inhibition of GARFT causes depletion of cellular guanosine triphosphate (GTP) and adenosine triphosphate (ATP) resulting in inhibition of both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis. Hypoxanthine (Hx), the predominant purine in human plasma, reverses the cytotoxic effects of GARFT inhibitors by entering the purine pathway at a point past the GARFT step via the purine salvage pathway. GARFT inhibition can, therefore, be exploited since purines are limiting in cells requiring the de novo pathway (such as tumors) while most normal tissues are spared through salvage of a physiologically available purine base. It has been postulated that human tissues, with the exception of the liver, obtain purines via the salvage pathway and tumors, due to a high demand for nucleic acid synthesis and genetic alterations in salvage pathway enzymes, tend to make purines by the de novo pathway (Varney et al., 1998a). Inhibitors of de novo purine synthesis, therefore, should show some degree of selectivity for the more rapidly proliferating tumor cells. Inhibitors of GARFT are the first class of compounds 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. whose biological activity can be attributed solely to depletion of cellular purine nucleotides. Azaserine inhibits purine synthesis, but also inhibits other glutamine- requiring reactions in cells. Unlike earlier purine base analog inhibitors of purine synthesis (e.g. 6-methylmercaptopurine), antifolate GARFT inhibitors are not incorporated into nucleic acid bases. Dihydrofolate reductase (DHFR) inhibitors cause a similar perturbation of purine pools to that resulting from GARFT inhibition, but function by depleting pools of tetrahydrofolate cofactors and the resulting accumulation of dihydrofolate polyglutamate inhibitors of GARFT and aminoimidazole carboxamide ribonucleotide formyltransferase (AICARFT). Unlike antifolate inhibitors of DHFR and thymidylate synthase (TS), GARFT inhibition of nucleic acid synthesis does not directly cause DNA strand break formation due to uracil misincorporation into DNA. demonstrating a unique cytotoxic mode of action for this class of antimetabolites (Varney et al., 1998b). In recent years, clinical trials have been conducted on three GARFT inhibitors including 5,10-dideazatetrahydrofoIic acid (DDATHF, Lometrexol), LY309887 (GARFT-IT), and AG2034 (Varney et al.. 1998c: Varney et al., 1997a: Vamey et al., 1997b). Preclinically, these compounds all displayed excellent antitumor activity against a number of murine and human xenograft tumor models that were unresponsive to methotrexate (Vamey et al., 1998b: Vamey et al., 1997c: Vamey et al., I997d). In clinical trials, Lometrexol appeared to exhibit a broad range of anti-tumor activity in murine and human tumor xenograft models and sporadic activity against a variety of human cancers in Phase I trials, including non-small cell 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. lung cancer, head and neck, ovarian, melanoma, and metastatic breast cancers (Vamey et al., 1998c) (Vamey et al., 1997d). One view is that these findings suggest that inhibition of purine synthesis does indeed represent a clinically useful target for cancer chemotherapy. I.C. AG2034 AG2034 (4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5.4-6] [1,41 thiazin-6-yl)-(S)-ethyI]-ethyl]-2,5-thienoyI-L glutamic acid) is a novel inhibitor of GARFT that was designed using knowledge gained from the X-ray crystallographic structure of the enzyme from E. coli and from the GARFT domain of the human trifunctional enzyme (Almassy et al., 1992). It was demonstrated that AG2034 showed in vivo anti-tumor activity against the 6C3HED. C3HBA. and B-16 murine tumors and in the HxGC3, KM20L2, LK-l, and H460 human xenograft models, and was selected for preclinical development (Boritzki et al., 1996: Vamey et al., I997e). Analysis of the GARFT active site, using the computer program. GRID, (Goodford, 1985) suggested that sulfur atoms should have an affinity for two regions of the folate cofactor binding site. The design of AG2034 attempts to satisfy this condition, while retaining substrate activity for the reduced folate carrier (RFC) and for folylpolyglutamate synthetase (FPGS). AG2034 inhibits human GARFT (K, = 28 nM), has a high affinity for the folate receptor (Kj = 0.0042 nM), and is a substrate for rat liver FPGS (K« = 6.4 pM, V ^ = 0.48 nmole/hr/mg). The IC50 for growth inhibition was 4 nM against L1210 cells and 2.9 nM for CCRF-CEM cells in 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. culture. In vitro growth inhibition can be reversed by addition of either Hx or 5- aminoimidazoIe-4-carboxamide (AICA) to the culture medium (Boritzki et al., 1996). AG2034 (Figure I) is highly soluble in water at neutral to high pH (pH>6), with a molecular weight of 467. Preformulation studies indicated that this compound was unstable. As demonstrated in preformulation studies, hydrolysis and oxidation were the two major degradation pathways of AG2034 (Li et al., 1998). Due to the instability of AG2034 in aqueous solution, a disodium salt Iyophilized powder for injection dosage form has been developed and is currently under investigation in a phase I clinical study. .COOH H N ' CQ,H Figure I. Structure of AG2034 I.D. Acute Toxicity in Mice and Dogs Toxicology studies of AG2034 were conducted in mice and dogs. Clinical signs that preceded death included lethargy, weight loss, and reduced food consumption. Macroscopic and microscopic examination of various tissues and 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. organs suggested that the gastrointestinal tract was a target organ for toxicity. Hematological changes such as the reduction in red blood cells count, hemoglobin and absolute lymphocyte count were observed. I.E. Liposomes LE.1. Background of Liposomes In the early I970’s, Bangham A.D. (1974) described how molecules such as phospholipids interact with water to form unique structures, which are now designated as liposomes. Today, liposomes are established as a useful model membrane system, and they have demonstrated potential as a drug delivery system. A wide variety of amphiphathic molecules have been used to form liposomes, and the method of preparation can be tailored to control their size and morphology. The medicinal materials can either be encapsulated in the aqueous space or intercalated into the lipid bilayer. The exact location of a drug in the liposome depends upon its physicochemical characteristics and the composition of the lipids. Because of their high degree of biocompatibility, liposomes were initially conceived of as systems for intravenous delivery. It has become apparent that liposomes can also be useful for delivery of drugs by other routes of administration such as intramuscular injection, topical application, pulmonary delivery and intraocular delivery. The pharmaceutical scientists can use various approaches to design liposomes for specific purposes, thereby improving the therapeutic index of a 7 | Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. drug by increasing the percent of drug that reaches the target organ or decreasing the percent of drug that reaches sites of toxicity. A few of the materials that have been encapsulated into liposomes include anticancer agents such as doxorubicin (Mayer et al., 1994; Park ea al., 1995; Zeisig et al., 1995), vaccines such as cytokines (Nohria and Rubin, 1994), antifimgal agents such as amphotericin B (New et al., 1981) and antibiotics such as vancomycin (Onyeji et al., 1994). These microscopic lipid systems tend to accumulate within organs of the reticuloendothelial system and also at disease sites, including tumors (Morgan et al., 1985; Ogihara et al., 1986; Presant et al., 1986: Williams et al.. 1986). As a result, liposomes have the potential to modify the pharmacokinetics as well as the biodistribution of entrapped drugs. Liposomes are man-made biological membranes that are composed of lipid layers surrounding aqueous layers. They are classified according to the number of bilayers and the size of the vesicle. Liposomes can be multilamellar, oligolamellar, or unilamellar (Figure 2). Multilamellar liposomes contain several lipid layers separated by aqueous layers, and have a diameter ranging between 300 nm to 15 (im. Oligolamellar vesicles contain two to three lipid bilayers with a diameter of less than 200 nm. Unilamellar vesicles contain only one bilayer and their size can vary between 20 and 2000 nm depending upon their classification as small or large unilamellar vesicles. Giant vesicles are multilamellar vesicles, by definition, with diameters larger than 10 fim. 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2. Structure of liposomes: large multilamellar (MLV), large unilamellar (LUV) and small unilamellar (SUV) vesicles. From D.D. Lasic, Liposomes in Gene Delivery, CRC Press (1997) The lipids most commonly used to prepare liposomes are shown in Figure 3. The glycerol-containing phospholipids are the most commonly used component of liposomal formulations and represent more than 50% of the weight of lipid present in biological membranes. The general chemical structure of these types of lipids is exemplified by phosphatidic acid. As indicated in Figure 4, the backbone of the molecule resides in the glycerol moiety. At position number 3 of the glycerol molecule, the hydroxyl is esterified to phosphoric acid. The hydroxyls at positions I and 2 are usually esterified with long chain fatty acids giving rise to the lipidic nature of these molecules. One of the remaining hydroxyls of phosphoric acids can be further esterified with a wide range of organic alcohols including glycerol, choline. 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ethanolamine, serine, and inositol. Therefore, the parent compound is a phosphoric ester of glycerol. In addition, fatty acids are important constituents of glycerol phosphatides and are abundant as building block components of other saponifiable lipids. A number of different fatty acids have been isolated from phospholipids, differing in the number of carbons and degree of unsaturation. Table I lists the common saturated and unsaturated fatty acids that have been found in phospholipids that are used to form liposomes. Table 1. Some common saturated and unsaturated fatty acids found in lipids used to form liposomes Molecular Formula Common Name Structural Formula Saturated fatty acids C 15H 32O2 Palmitic CH3(CH2)[4COOH C 18H 36O2 Stearic CH3(CH2)t6COOH Unsaturated fatty acids C 16H 30O2 Palmitoleic CH3(CH2)5CH=CH(CH2)7COOH C 18H 34O2 Oleic CH3(CH2> 7CH=CH(CH2)7COOH C 18H 32O2 Linoleic CH3(CH2> 4CH=CHCH2CH=CH(CH2)7COOH 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. r RV R , C O CH 1 1 I I O HjC------ O------ P - 1 "C H ^ « M W j* « t----- C O CH, O C H 0 1 ^ 1 I I O HjC------ 0 -------P - -C H j CH, HCCHjI Phosphatidylcthanolaminc (PE) ■CH, CH, CH, CH C H , CH, CH, Cholesterol .,JU T ' T T Phosphatidylcholine (PC) NH^ COO- Phosphatidylserine (PS) o R,—J L — - o — CH, *z 1 o PH P o o- , - i - c T T J j C o P OH O - Phosphaddic acid (PA) OH OH HjC- •CHj Cl Phosphaudylglycerol (PG) HjC O P------- O H O H H ok i t H / ' -- - - - O H Phosphatidylinositol (PI) O H H Figure 3. Chemical structures of lipids commonly used to prepare liposomes. Phosphatidylethanolamine (PE), phosphatidylcholine (PC), and cholesterol behave as neutral molecules, whereas phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylglycerol (PG), and phosphatidylinositol (PI) have a net negative charge. From Pharmaceutical Dosage Forms: Disperse Systems. Marcel Dekker, Inc., (1998) 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A Phosphatidylcholine A Sphingomyelin Figure 4. Chemical structures of two representative phospholipids showing their polar head groups and nonpolar hydrocarbon chains. From Pharmaceutical Dosage Forms: Disperse Systems. Marcel Dekker. Inc., (1998) 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In order to improve the stability of lipids, several synthetic phospholipids were synthesized to form liposomes. The most commonly used saturated phospholipids include dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylserine (DPPS), dipalmitoylphosphatidic acid (DPPA) and dipalmitoylphosphatidylglycerol (DPPG). Several unsaturated phospholipids have also been used for preparing liposomes; these include dioleoyllphosphatidylcholine (DOPQ and dioleoylphosphatidylglycerol (DOPG). Cholesterol and its derivatives are often included as components in liposomal membranes. The inclusion of cholesterol in liposomal membranes has three recognized effects: decreasing the fluidity or microviscosity of the bilayer, reducing the permeability of the membrane to prevent leakage of water-soluble drugs (Mezei, 1993), stabilizing the membrane in the presence of biological fluids such as plasma. The latter effect has been proven to be useful in preparing liposomes for drug delivery applications that use the intravenous route of administration. Liposomes without cholesterol are known to interact rapidly with plasma proteins such as albumin, transferrin, and macroglobulins. These proteins tend to extract bulk phospholipids from liposomes, thereby depleting the outer monolayer of the vesicles leading to physical instability. Cholesterol appears to substantially reduce this type of interaction. (Lasic et al., 1998) The types of drugs (i.e.. hydrophilic or lipophilic) being encapsulated will dictate the most appropriate type of vesicle that is required. Unilamellar vesicles 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. consist of a single bilayer entrapping an aqueous core resulting in a small encapsulation volume for hydrophilic medications. In this instance, it would be best to use a hydrophobic drug for encapsulation due to the miscibility of hydrophobic drug and phospholipids. Either hydrophilic or hydrophobic medications can be successfully enveloped by multilamellar vesicles. However, multilamellar vesicles have been noted to be most useful for hydrophilic drugs because they have a very high encapsulation efficiency for water (Mezei, 1993). It must be recognized that different applications require different types of liposomes, when considering the preparative methodology of liposomes. Variation of the lipid composition and electrical charge of the liposomes was shown to alter the in vivo distribution and clearance rates of otherwise similar liposomes (Gregoriadis et al., 1981; Fidler et al.. 1980). It is possible to distinguish two main types of liposomes, namely, large uni- or multilamellar liposomes, which are rapidly cleared from circulation after intravenous administration and small unilamellar liposomes, which remain in circulation for long periods of time. The previous studies have also shown that conventional liposomes tend to clear from the circulation by phagocytic cells within the liver and spleen (Chang et al., 1997). Liposome recognition and subsequent uptake is believed to be triggered by protein binding to the vesicle surface (Chonn et al., 1992; Funalo et al.. 1992). This protein binding can be inhibited by introducing into the liposomal membrane lipid derivatives possessing long, hydrophilic poly (ethylene glycol) chains. These sterically stabilized liposomes exhibit much longer blood circulation time than 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. equivalent conventional liposomes (Figure 5) (Klibanov et al., 1990; Allen et al., 1991) and when used as drug carriers can enhance delivery to tumor sites, resulting in improved anti-cancer efficacy (Huang et al., 1992; Vaage et al., 1992). A wide variety of techniques has been developed and adapted to suit the individual requirements in terms of size, size distribution, electrical charge, number of lamellae, and lipid composition of the liposomes. Figure 5. Conventional Liposome. From Pharmaceutical Dosage Forms: Disperse Systems. Marcel Dekker, Inc, (1998) A few strategies are available to improve the therapeutic index of antitumor drugs. Among various approaches to develop a more effective and less toxic drug preparation, the incorporation of the drugs into liposomes represents a promising possibility. With the objective of enhancing the therapeutic index and reducing unexpected toxic side effects, some antitumor drags have been incorporated into 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. liposomes. Promising results were found using doxorubicin, whose entrapment into liposomes reduced its cardiotoxicity and increased its antitumor activity (Balazsovits et al., 1989; Hoesel et al., 1984; Rahman et al., 1986). Following the incorporation of other antitumor compounds into liposomes, such as methotrexate (Heath et al., 1986), arabinosyl cytosine (ara-C) (Patel and Baldeschwieler, 1984), and cis- diamminedichloroplatinum (II) (Steerenberg et al., 1988) reductions in toxicity and preservation of or increases in antitumor activity have been observed. The pharmacokinetic profile of a drug may be completely altered by entrapping the drug into liposomes. In addition, the pharmacokinetics of the liposomes themselves are different depending on the particle size, presence of charge, or attachment of hydrophilic moieties on the surface of liposomes (Crommelin and Schreier, 1994). When administered intravenously liposomes are rapidly taken up by the organs of the reticuloendothelial system (RES) and for liposomes to act as circulating reservoirs, they must avoid clearance by the RES (Allen, 1988). Approaches adopted for this purpose include the use of glycolipids. polysaccharides, proteins and polymers (Torchilin et al., 1980; Sunamoto and Iwamoto, 1986: Allen and Chonn. 1987; Gabizon and Papahadjopoulos, 1988; Litzinger et al., 1994). The use of polyethylene glycol-derivatized phosphatidylethanolamine in recent times has further improved the circulation time and avoidance of the RES system of these liposomes. 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I.E.2. Techniques for Preparation of Liposomes LE.2.L Large Liposomes IJE.2.I.1 Multilamellar Vesicles Multilamellar liposomes were first described by Bangham et al. (1965). The procedure involves the deposition of a thin film of phospholipid from an organic solvent on the walls of a container, followed by agitation with an aqueous solution of the material to be encapsulated. The degree of agitation required to form a uniform dispersion of multilamellar vesicles will depend upon the thickness of the lipid film. With thicker films produced by more concentrated lipid dispersions, more vigorous agitation is required. The degree of agitation includes gentle hand-shaking, vortex mixing with or without glass beads, and sonication. I.E.2.1.2 Large Uni- or Oligolamellar Vesicles In general, there are two methods for preparation of large uni- or oligolamellar vesicles. The first method, the diethyl ether infusion technique, uses the basic principle of injection of a solution of lipids dissolved in diethyl ether into an aqueous solution of the material to be encapsulated at an elevated temperature for rapid evaporation of the solvent (Deamer and Bangham, 1976; Schieren et al., 1978). The second method, calcium-induced fusion, developed by Papahadjopoulos et al. (1975) relies on the effect of calcium ion on acidic phospholipids to initially form 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. co-chelate cylindrical structures, which are then transformed into large unilamellar vesicles by the addition of the chelating agent EDTA. l.E.2.2. Small Unilamellar Liposomes The conventional methods of dispersing phospholipids in water to form optically clear dispersions with a particle weight of about 2 x 1 0 6 daltons involve various mechanical means. The sonication method reported by Saunders L. (1962) followed by refinements introduced by Hamilton and Guo (1984) and Barenholz et al. (1977) who employed a high-pressure device to produce the same effect in larger volumes. These types of small unilamellar liposome dispersions have been rigorously characterized by Huang C.H. (1969) and others and shown to consist of uniform closed bilayer vesicles of about 25-50 nm diameter. Solvent injection methods have also been devised to produce small unilamellar vesicles. These typically involve the slow injection of a lipid solution in either diethyl ether or ethanol into warm water containing a drug to be entrapped. I.E3. Formation of Liposomes Lipids that are capable of forming liposomes exhibit a dual chemical nature. Their head groups are hydrophilic and their fatty acyl chains are hydrophobic. It has been estimated that each zwitterionic head group of phosphatidylcholine has on the order of 15 molecules of water weakly bound to it, which explains its overwhelming preference for the aqueous phase. The hydrocarbon fatty acyl chains, on the other 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. hand, vastly prefer each other’s company to that of water. This phenomenon can be understood in quantitative terms by considering the critical micelle concentration (CMC) of PC in water. The CMC is defined as the concentration of the lipid in water above which the lipid forms either micelles or bilayer structures rather than remaining in the solution as monomers. Based on the knowledge of the total concentration of a lipid-like phosphatidylcholine and its CMC in aqueous systems, it is possible to drive the free energy for transfer of one mole of the lipid from water to micelle using the simple expression AF = -RT In(CMC) as described by Shinoda et al. (1963). Applying this equation, a free energy of transfer from water to micelle of = 15.3 kcal/moi is obtained in the case of dipalmitoylphosphatidylcholine and = 13.0 kcal/moi for dimyristoylphosphatidylcholine. The free-energy change between a water and a hydrophobic environment explains the overwhelming preference of typical lipids to assemble in bilayer structures excluding water as much as possible from the hydrophobic core in order to achieve the lowest free energy level and hence the highest stability for the aggregate structure (Lasic et al., 1998). I.E.4. Phase Behavior of Liposomes An important feature of membrane lipids is the existence of a temperature- dependent reversible phase transition, where the hydrocarbon chains of the phospholipid undergo a transformation from an ordered (gel) state to a more disordered fluid (liquid crystalline) state. These changes have been documented by 19 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. freeze-fracture electron microscopy but are most easily demonstrated by differential scanning calorimetric (DSC) analysis. The physical state of the bilayer profoundly affects the permeability, leakage rates, and overall stability of the liposomes. The phase transition temperature Tm is a function of the phospholipid content of the bilayer. The phase transition temperature can be altered by using phospholipid mixtures or by adding sterols such as cholesterol. The T m value can give important clues as to liposomal stability and permeability (Lasic et al., 1998). In general, liposomes with higher Tm values should have better stability properties in vitro and in vivo. I.E.5. Background of Stealth Liposomes Medical applications of liposomal formulations distinguish several types of liposomes based on their reactivity with surrounding media. Conventional liposomes are characterized by nonspecific reactivity. One of the main disadvantages of drug delivery using conventional liposomes is the instability of liposomes in biological systems, such as in blood circulation. However, a major breakthrough in liposomes application was the realization that external steric stabilization can increase liposomes stability in biological environments. Steric stabilization can be induced by surface attachment of polymers as a result of steric repulsion to reduce particle to particle interaction. Therefore, the adsorption of various opsonins (substances that stimulate phagocytosis) onto the surface of liposomes is inhibited. It was discovered that covering liposomes with hydrophilic, nonionic polymers greatly increased their 2 0 I Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. stability in blood circulation. They must be inert, well solvated, and compatible with the solvent, and have polarizability close to that of water. Steric stabilization can be induced by the surface attachment of various natural or synthetic polymers, either by adsorption, hydrophobic insertion, electrostatic binding, or, preferably, by grafting via covalent bonds. Nonionic water compatible, flexible, and well-hydrated polymers are preferred. The repulsion between surfaces with attached polymers was shown to be dependent on the grafting density and degree of polymerization. (Lasic et al., 1998) Qualitatively, one can explain the enhanced stability of such sterically stabilized liposomes in biological environments by their ability to prevent adsorption of various blood components (Lasic and Papahadjopoulos, 1995). It was proposed that the major mechanism of liposome uptake and disintegration in plasma is reaction with proteins of the immune system (Scherphof et al., 1978; Tall et al.. 1993: Gregoriadis, 1994), which adsorb onto foreign colloidal particles and tag them for subsequent macrophage uptake (Lasic and Papahadjopoulos, 1995: Papahadjopoulos, 1991). It is assumed that the adsorption of immunoglobulins or proteins onto liposomes is reduced in the presence of surface-attached polymer. Lipid exchange interactions, which deplete liposome lipids, are also minimized in the presence of surface-attached polymer. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I.E.6. Interactions of Liposomes with Cells Liposomes can interact with ceils in several ways to cause liposomal contents to become associated with cells. I.E.6.1. Intermembrane Transfer Intermembrane transfer of lipid contents can occur upon “close” approach of the phospholipid bilayers of the liposome without disruption of the liposome integrity. Such transfer may occur with complete retention of the contents of the liposome’s aqueous compartment. Cholesterol transfers very readily between bilayers of liposome so that its molar concentration is equalized throughout all membranes present. The mechanism of lipid or protein transfer from cell membrane to liposome is not clear. Phospholipid transfer may occur when liposomes collide with the cell surface (Blumenthal et al., 1982). Special lipid transfer proteins (LTP) on the cell surface could mediate this process (Margolis et al., 1988). I.E.6.2. Contact Release Contact release of aqueous contents of liposomes is poorly understood. Liposome contact with the cell causes an increase in permeability of the liposome membrane. This leads to release of water-soluble solutes in high concentration in the close vicinity of the cell membrane through which these solutes may, under certain circumstances, then pass (Van Renswoude and Hoekstra. 1981). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I.E.6.3. Adsorption Adsorption of liposomes to the cell surface can often occur with little or no internalization of either aqueous or lipid components (Poste, 1980). Large liposomes can be adsorbed on the cell surface in such a manner (Finkelstein and Weissmann, 1978; Pagano and Huang, 1975; Huang et al., 1978). Adsorption may be due to physical attractive forces, or binding by specific receptors to ligands on the liposome membrane. Physical adsorption of liposomes may occur through binding to a specific cell surface protein (Pagano and Takeichi, 1977). Adsorption is an essential prerequisite for liposome ingestion by cells, but factors that determine whether or not a liposome is “consumed” thereafter are not fully understood. Attachment of liposomes to cell membranes via certain surface proteins, but not others, results in rapid uptake into the cell (New et al.. 1990; Leserman et al., 1980). LE.6.4. Fusion Close approach of liposome and cell membrane can lead to fusion. Fusion can result in complete mixing of liposomal and cell plasma membrane lipids and release of liposomal contents into the cytoplasm. Fusion was once considered the dominant mechanism of cell interaction with liposomes in a fluid state (Weinstein et al., 1977). However, spontaneous fusion between liposomes and the plasma membrane is rare (Poste, 1980; Straubinger et al., 1983; Szoka. 1980a). 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I.E.6.5. Phagocytosis/Endocytosis Cells with phagocytic activity take liposomes into endosomes, which have a pH of 5 to 5.5. Endosomes then fuse with lysosomes to form secondary lysosomes where cellular digestion occurs in a milieu of approximately pH 4.5. Lysosomal enzymes break open the liposomes, hydrolyzing the phospholipids to fatty acids, which can be recycled into host phospholipid. (Voet and Voet, 1990) During liposome breakdown in lysosomes, the contents of the aqueous compartment are released. These contents will either remain sequestered in the lysosomes until exocytosis, or they will slowly leak out of the lysosome into the cell. In addition to phagocytosis, liposomes may also be taken up by receptor mediated endocytosis. Endocytosis of liposomes depends on their size. For example, multilamellar liposomes are endocytosed by various cells. Large unilamellar liposomes are endocytosed by Kupffer cells in vitro (Finkeistein and Weissmann. 1978). I.E.7. Rationale for the Development of a Stealth AG2034 Liposome Formulation During the last two decades, considerable attention has been given to the development of novel drug delivery systems. The rationale for controlled drug delivery is to alter the pharmacokinetics and pharmacodynamics of drug substances in order to improve the therapeutic efficacy and safety through the use of novel drug delivery systems. Besides more traditional matrix or reservoir drug delivery systems, 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the liposome delivery system has gained in popularity. However, one of the main setbacks of drug delivery using conventional liposomes is, when administered intravenously, liposomes are rapidly taken up by phagocytic cells. A major breakthrough in liposome applications was the invention of stealth liposomes that are nonreactive or polymorphic. Steric stabilization can be induced by the surface attachment of natural or synthetic polymers which can cause the steric repulsion to reduce particle-particle interaction. Therefore, encapsulation of AG2034 inside small-sized, sterically stabilized liposomes may have the advantage of prolonged circulation time in vivo, reduced systemic toxicity, increased uptake of encapsulated AG2034 into extracellular space within tumors, and gradual release of their payload. Based on this hypothesis, AG2034 entrapped in stealth liposomes (Figure 6 ) can reduce the free drug in circulation and alter bioavailability, biodistribution and thus its biological activity. Figure 6 . Stealth Liposome. From Pharmaceutical Dosage Forms: Disperse Systems. Marcel Dekker, Inc, (1998) 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. H. DEVELOPMENT AND CHARACTERIZATION OF A STEALTH AG2034 LIPOSOME DELIVERY SYSTEM 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. II.A. Abstract AG2034, an anticancer agent, was developed and optimized to be encapsulated in a stealth liposome delivery system. Five parameters were considered to play important roles in the developing a stealth liposome delivery system with maximum drug encapsulated and stability in vitro and in vivo. The effect of cholesterol encapsulation efficiency study showed a DSPC/CHOL ratio of 1:0.25 formulation had higher drug encapsulated. Upon the addition of 5% PEG(2k)-DPPE, the encapsulation efficiency was increased from 5% to 15% compared to formulation without PEG(2k)-DPPE and from 7% to 15% compared to a formulation with 10% PEG(2k)-DPPE loading. Differential scanning calorimetry thermgrams showed that the phase transition temperature shifted from 52.9°C for a formulation with 5% PEG(2k)-DPPE to 52.4°C for a formulation with 10% PEG(2k)-DPPE. These results indicated the gradual phase transition from the bilayer state to a mixed micelles state in the delivery system. The impact of manufacturing method on encapsulation efficiency indicated that the extrusion method had a much higher efficiency than remote loading method in terms of drug encapsulation. The encapsulation efficiency was significantly increased up to 21% as a result of increasing the lipid from 40 jimoIe/mL to 60 |imoIe/mL, with only a slight increase occurring when further increasing the lipid to 80 JimoIe/mL. The effect of AG2034 loading on encapsulation efficiency showed that there were no significant differences in encapsulation efficiency when increasing the AG2034 loading in formulations. This finding is illustrated by the consistent size distribution profiles of the liposome delivery 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. systems. The results of the leakage study indicated that refrigerated temperature was found to be the most suitable environment for storage of AG2034 stealth liposome formulations. The results of the in vitro release study showed that there was an initial rapid leakage for 2 hours followed by a slower release for 1 2 hours and very slow leakage for up to 24 hours. The results of the in vitro cell growth study showed similar antiproliferative activity for free AG2034 and AG2034 stealth liposome delivery system. The pharmacokinetic study showed that the half-lives for free AG2034, conventional liposomes and stealth liposomes were tt^a = 0.2 hours and t[/2p = 2.7 hours, tiaCi = 1.6 hours and ti/2 p = 34.5 hours, and ti^a = 9.7 hours and ti/^P = 57.0 hours, respectively. The results indicated that in contrast to the rapid blood clearance observed for AG2034. the liposomal delivery systems extended AG2034 existence in blood. In the case of sterically stabilized liposomes containing a PEG-lipid conjugate. 50% of AG2034 was present in the blood 57.0 hours after i.v. injection. Administration of free AG2034 was found to be more toxic, resulting in mortality at earlier time points than seen for that of AG2034 encapsulated in the stealth liposome delivery system. The survival rate for mice treated with free AG2034 and stealth liposomal delivery system was 5/30 and 16/30, respectively. The acute toxicity study demonstrated the feasibility of using a stealth liposome delivery system of AG2034 to reduce the toxicity in vivo. The reduction in toxicity should be credited to the slower and controlled release of AG2034 from stealth liposomes. 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ILB. Background AG2034 is a novel glycinamide ribonucleotide formyltransferase (GARFT) inhibitor which was designed using the three dimensional structure of the GARFT domain of the human trifunctional enzyme as determined by X-ray crystallography. Inhibition of GARFT results in a selective blockade of de novo purine biosynthesis, a process that is critical for tumor cell growth (Jackson et al., 1981). A cell line with impaired transport of reduced folates, L1210/CI920 (Fry et al., 1984), was resistant to AG2034 indicating that the compound can enter cells by utilizing the reduced folate carrier. AG2034 showed in vivo anti tumor activity against the 6C3HED, C3HBA, and B-16 murine tumors and in the HxGC3, KM20L2, LX-I and H460 human xenograft models, and has been selected for preclinical development towards clinical trials (Boritzki et al., 1996). Encapsulation of AG2034 in a liposome carrier could provide an ideal drug delivery system to reduce the free drug in circulation and alter bioavailability, biodistribution and thus its biological activity. Attachment of polyethylene glycol- derivatized phosphatidylethanolamine to the surface of these liposomes will further enhance the effects by reducing the RES uptake and further increasing the circulation time. The objective of the present study was to prepare a stable and long circulating formulation of AG2034 in liposomes. The influence of chain length of phospholipid, amount of cholesterol, concentration of polyethylene glycol-derivatized phosphatidylethanolamine, loading of phospholipid, concentration of AG2034 and 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. preparation method of liposomes was studied. A formulation with optim um encapsulation and stability was selected to prepare long circulating liposomes. In vitro stability studies on the retention of encapsulated drug at two storage temperatures, and on exposure to serum were performed. The in vitro cell growth studies for the stealth liposomes with and without treatment with human serum were investigated. Throughout the study the particle size of the liposomes was monitored. n .C . Experimental H.C.1. Materials n.C. I. I. Chemicals AG2034 was synthesized at Agouron Pharmaceuticals. Inc.. Dipalmitoyl-L- a-phosphatidylcholine (DPPC), distearoyl- L-a-phosphatidylcholine (DSPC). cholesterol, and l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethyIene glycol)-2000] (PEG(2k)-DPPE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Polycarbonate films were obtained from Avestin Inc. (Ottawa, ON. Canada). Ferric chloride hexahydrate (FeCL^FLO) and ammonium thiocyanate (NH4SCN) were analytical grade and purchased from J. T. Baker (Phillipsburg, NJ, USA). Ammonium phosphate was obtained from Fisher Scientific (Fair Lawn, NJ, USA). Sodium lauryl sulfate was purchased from Spectrum Chemical Co (Gardena, CA, USA). Tween 20 was obtained from Fisher Scientific (Fair Lawn, NJ, USA). Triton X-100 was obtained from Labchem Inc. 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Pittsburgh, PA, USA). Trichloroacetic acid, acetic acid, SRB solution, Tris buffer and all tissue culture media were purchased from Sigma (St. Louis, MO. USA). All other materials and solvents of HPLC grade were purchased from EM Science (Gibbstown, NJ, USA). II.C.2. HPLC Analysis of AG2034 n.C.2.1. HPLC System A Hewlett-Packard 1100 HPLC equipped with a diode array detector, vacuum degasser. quaternary pump, and auto sampler, was used for AG2034 analysis. The chromatographic separation was accomplished on a Symmetry™ C18 column (Waters, Milford. MA. USA), 5 p. 250 x 4.6 mm. at a flow rate of 1.0 mL/minute at ambient temperature, using an injection volume of 2 pL. The analytical wavelength for peak detection was set at 280 nm. H.C.2.2. Mobile Phase The mobile phase consisted of 50% 25 mM ammonium phosphate buffer adjusted to pH = 3.0 and 50% HPLC grade methanol. H.C.2.3. Preparation of 25 mM Ammonium Phosphate, pH = 3.0 This buffer was prepared by weighing 2.87 grams of ammonium phosphate into a I-liter volumetric flask and made up to final volume with HPLC grade water. The pH was adjusted to 3.0 using 85% o-phosphoric acid. 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. II.C.2.4. Preparation of 0.1 M Phosphate Buffer, pH = 8.0 This buffer was prepared by weighing 13.44 grams of dibasic sodium phosphate (anhydrous) and 0.73 grams of monobasic sodium phosphate into a I-liter volumetric flask and brought up to final volume with HPLC grade water. The pH of 0.1 M phosphate buffer was measured at 8.0, without adjustment. fl.C.2.5. Preparation of Standards Five milligrams of AG2034 were accurately weighed to the nearest 0.01 mg into a 10 mL volumetric flask. Phosphate buffer with the concentration of 0.1 M, pH = 8.0 . was added to dissolve the AG2034 with sonication. A series of dilutions were made from this solution to obtain a range of concentrations down to 0 . 0 1 mg/mL. II.C.2.6. Preparation of Samples The liposome suspension was passed through a gel filtration Sephadex™ G- 50 column (1.0 cm diameter, 30 cm length) (Amersham Pharmacia Biotech AB.. Uppsala, Sweden) and the peak fractions containing the liposome-associated AG2034 were collected. One hundred microliter aliquots of the peak fractions were pipetted into four individual 1.5-mL flat snap cap microcentrifuge tubes. A volume of 100 pi- of 5% (w/v) Triton XlOO solution was added into each of the four tubes. These tubes were vortexed and placed into a water bath at 60°C for 30 minutes. The 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. tubes were then centrifuged at 16,000 g for 10 minutes. The supemantents were transferred into HPLC vials for AG2034 analysis. n.CJ. Assay VaUdation for AG2034 n.C3.1. Linearity Each of the standard solutions was injected three times in succession from the lowest to highest concentration. The data were analyzed by the least squares method to determine the correlation coefficient, slope, and y-intercept. The relative standard deviation (RSD) for each standard concentration level was calculated to demonstrate reproducibility. n.C.3.2. Accuracy and Precision The average peak area for each concentration used for determination of linearity, accuracy and precision was subjected to linear regression analysis to obtain found concentrations. The accuracy and precision of the assay method were calculated as follows. % Accuracy = (found concentration / actual concentration) x 100 % Precision = | (mean found concentration-actual concentration) | x 100 / actual concentration 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. n.C.4. UV-Vis Spectrophotometric Analysis of Phospholipids A number of methods have been previously reported to quantitate phospholipids (Bartlett, 1959; Stewart, 1980; Freeari et al„ 1998). The analysis of phospholipid content used in the present study was a modification of Stewart assay method (Stewart, 1980). In the Stewart assay for phospholipids, the ability of phospholipids to form a complex with ammonium ferrothiocyanate in organic solution is utilized. II.C.4.1. UV-Vis Spectrophotometric System A Hewlett-Packard spectrophotometer 8453 equipped with a diode array detector was used for phospholipids analysis. Quartz cuvettes with a path length of one centimeter were used. The optical density of the complex of phospholipids and ammonium ferrothiocyanate was assayed at 488 nm. n.C.4.2. Preparation of Ammonium Ferrothiocyanate Solution This solution was prepared by weighing 27.0 grams of ferric chloride hexahydrate and 30.4 grams ammonium thiocyanate into a I-liter volumetric flask and made up to final volume with HPLC grade water. n.C .4.3. Preparation of Standards n.C.4.3.1 Preparation of DPPC Standards A stock solution of 11.05 mg DPPC in 100 mL chloroform was prepared. The following volumes of stock solution were pipetted into five separate test tubes: 0.4 34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. mL, 0.8 mL, 1.6 mL, 3.2 mL and 4.0 mL. A 2.0 mL aliquot of ammonium ferrothiocyanate solution was added to each test tube. The following volumes of chloroform were added to the respective tubes to bring the final volume to 6 mL: 3.6 mL, 3.2 mL, 2.4 mL, 0.8 mL and 0.0 mL. n.C.4.3.2 Preparation of DSPC Standards A stock solution of 9.75 mg DSPC in 100 mL chloroform was prepared. The following volumes of stock solution were pipetted into five separate test tubes: 0.4 mL. 0.8 mL, 1.6 mL, 3.2 mL and 4.0 mL. A 2.0 mL aliquot of ammonium ferrothiocyanate solution was added to each test tube. The following volumes of chloroform were added to the respective tubes to bring the final volume to 6 mL: 3.6 mL, 3.2 mL, 2.4 mL. 0.8 mL and 0.0 mL. II.C.4.3.3 Preparation of DSPC/PEG(2K)-DPPE with the Molar Ratio of 15.2:1 Standards A stock solution of 7.43 mg DSPC and 1.69 mg PEG(2k)-DPPE in 100 mL chloroform was prepared. The following volumes of stock solution were pipetted into five separate test tubes: 0.4 mL. 0.8 mL, 1.6 mL, 3.2 mL and 4.0 mL. A 4.0 mL aliquot of ammonium ferrothiocyanate solution was added to each test tube. The following volumes of chloroform were added to the respective tubes to bring the final volume to 8 mL: 3.6 mL. 3.2 mL, 2.4 mL. 0.8 mL and 0.0 mL. 35 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. H.C.4.3.4 Preparation of DSPC/PEG(2K)-DPPE with a Molar Ratio of 7.2:1 Standards A stock solution of 7.11 mg DSPC and 3.38 mg PEG(2k)-DPPE in 100 mL chloroform was prepared. The following volumes of stock solution were pipetted into five separate test tubes: 0.4 mL, 0.8 mL, 1.6 mL, 3.2 mL and 4.0 mL. A 4.0 mL aliquot of ammonium ferrothiocyanate solution was added to each test tube. The following volumes of chloroform were added to respective tubes to bring the final volume to 8 mL: 3.6 mL, 3.2 mL, 2.4 mL, 0.8 mL and 0.0 mL. H.C.4.4. Preparation of Samples The liposome suspension was passed through a gel filtration Sephadex™ G- 50 column (1.0 cm diameter, 30 cm length) (Amersham Pharmacia Biotech AB.. Uppsala, Sweden) and the peak fractions containing the liposome-associated AG2034 were collected. One hundred microliter aliquots of the peak fractions were pipetted into four individual I.5-mL flat snap cap microcentrifuge tubes. Next, 4.0 mL of ammonium ferrothiocyanate solution and 4.0 mL chloroform were pipetted into these polypropylene conical tubes. These biphasic systems were vortexed for one minute and then centrifuged at 300 g for 5 minutes. The lower layer was transferred into a quartz cuvette with a path length of one centimeter and assayed by UV-Vis spectrophotometry at 488 nm. 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. II.C.5. Assay Validation for Phospholipids n.C.5.1. Linearity Each of the standard solutions was vigorously vortexed for one minute and then centrifuged at 300 g for 5 minutes. The lower layer solution was transferred into a quartz cuvette with a path length of one centimeter and the absorbance at 488 nm was obtained three times in succession from lowest to highest concentration. The data were analyzed by the least squares method to determine the correlation coefficient, slope, and y-intercept. The relative standard deviation (RSD) for each standard concentration level was calculated to demonstrate reproducibility (< 2 %). O.C.5.2. Accuracy and Precision The average absorbance at 488 nm for each concentration used for determination of linearity, accuracy and precision was subjected to linear regression analysis to obtain found concentrations. The accuracy and precision of the assay method were calculated as follows. % Accuracy = (found concentration / actual concentration) x 100 % Precision = | (mean found concentration-actual concentration) | x 100 / actual concentration 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. H.C.6. pH-SoIubility Profile AG2034 is insoluble in water and ethanol. It is freely soluble in sodium hydroxide due to ionization of two glutamate carboxylic groups and formation of a sodium salt (Wang et al., 1997). In order to optimize AG2034 loading in liposomal formulations, a pH-solubility study was conducted. The pH-solubility profile of AG2034 was generated by titrating an aqueous suspension of AG2034 in 0.1 N HC1 with 0.1 N NaOH at ambient temperature. All samples were sonicated at ambient temperature overnight. Sample suspensions were filtered through polyvinylidene fluoride (PVDF), 0.45 pm, syringe filters. In order to prepare appropriate concentrations of AG2034 solutions for analytical testing, the predetermined volumes of filtrate were pipetted into various appropriate volumetric flasks and samples were diluted with a 50:50 mixture of 25 mM phosphate buffer, pH = 3.0 and HPLC grade methanol. The AG2034 concentration was determined by HPLC assay. O.C.7. Heating Time Effect on Stability of AG2034 during Preparation of Liposomal Formulations To determine the stability of AG2034 during the formulation of liposomes, the manufacturing procedure for AG2034 liposomal formulations was evaluated, especially heating time in the preparation process. A AG2034 solution with a concentration of 0.6 mg/mL was prepared in 0.1 M sodium phosphate buffer, pH = 8.0. The AG2034 solution was stored in a capped 20 mL scintillation vial and placed into a 60°C water bath with shaking speed of 50 rpm for 120 minutes. Samples of the 38 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. AG2034 solution were collected at sampling time intervals of 30, 60, 90, and 120 minutes. Samples were cooled to ambient temperature. Each sample was injected three times on the HPLC to investigate the stability of AG2034. O.C.8 . Preparation of Liposomes n.C.8.1. Extrusion Method Liposomes were prepared by a thin film hydration method. Phospholipid and cholesterol were dissolved in I mL of chloroform in a stoppered 25 mL round- bottom flask. The organic solvent was slowly evaporated at a pressure of 250 mm Hg, using a rotary evaporator with rotation speed of 180 rpm (Jain and Jain, 1994) at 40°C, such that a thin film of the dry lipid was deposited on the inner wall of the flask. The flask was then left overnight at ambient laboratory conditions in a fume hood to remove any solvent traces remaining in the lipid film. A solution of AG2034 in 0 .1 M phosphate buffer, pH = 8.0, prepared at a predetermined concentration, was added to the flask. The film was hydrated at 60°C for 2 hours with occasional vortexing to form multilamellar liposomes (MLVs). The 15-pass process was used to extrude MLVs through two stacked 0.2 jim polycarbonate filters using a stainless steel extruder (Figure 7) (Avestine Inc.. Ottawa. ON, Canada) to form large unilamellar liposomes (LUVs). 39 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ILC.8.2. Remote Loading Method AG2034 was Loaded into the aqueous inner compartment of preformed liposomes using a gradient of 6 pH units. Liposomes with the composition 76% DSPC : 19% cholesterol: 5% PEG(2k)-DPPE were dissolved in L mL of chloroform in a stoppered 25 mL round-bottom flask. The organic solvent was slowly evaporated at the pressure of 250 mm Hg, using a rotary evaporator with a rotation speed of 180 rpm (Jain and Jain, 1994) at 40°C, such that a thin film of the dry lipid was deposited on the inner wall of the flask. The flask was then left at ambient temperature overnight in a fume hood to remove any solvent traces remaining in the lipid film. Sodium phosphate solution, 0.1 M, adjusted to pH 12 using sodium hydroxide was added to the flask and the film hydrated at 60°C for 2 hours with occasional vortexing to form empty multilamellar liposomes (MLVs). The 15-pass process was used to extrude MLVs through two stacked 0.2 (im polycarbonate filters using a stainless steel extruder (Avestine Inc., Ottawa, ON, Canada) to form empty large unilamellar liposomes (LUVs). The pH of the external medium was then decreased by elution of empty liposomes on a gel filtration Sephadex™ G-50 column (Amersham Pharmacia Biotech AB., Uppsala, Sweden) (1.0 cm diameter, 30 cm length) pre-equilibrated at pH 6.5. Fractions 9. 10, and 11 were collected. The collected empty liposomes were then incubated with 6 mg/mL AG2034 solution at pH 6.5 overnight. Unencapsulated AG2034 was removed by elution of liposomes on a gel filtration Sephadex™ G-50 column (Amersham Pharmacia Biotech AB., Uppsala, Sweden) (1.0 cm diameter, 30 cm length) pre-equilibrated at pH 8.0. 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ILC.8.3. Extraction of AG2034 from Liposomal Formulations One hundred microliters each of ethanol, 1% (w/v) SLS, 5% (w/v) SLS, 10% (w/v) SLS, 1% (w/v) Tween 20, 5% (w/v) Tween 20, 10% (w/v) Tween 20, 1% (w/v) Triton X100, 5% (w/v) Triton X100 and 10% (w/v) X100 were mixed separately with 100 pL of liposome solution. To destroy the structure of liposomes, the mixed solutions were vortexed and then placed into a 60°C water bath for 10, 20 and 30 minute intervals. The sample solutions were centrifuged at 16000 g for 10 minutes. Oring Channel Filter Teflon Bearing Oring Supports Oring dlilos t t t Extruder Outer Casing Internal Membrane Support Polycarbonate Internal Membrane Membrane Support Retainer Nut Figure 7. Mini Extruder from Avanti Polar Lipids Inc. 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. II.C.9. Encapsulation Efficiency Encapsulation efficiency is defined as the percent fraction of the total input drug encapsulated in the liposomes. In order to determine the encapsulation efficiency of AG2034 liposomes, gel filtration chromatography was used. The liposomal suspension was loaded on a gel filtration Sephadex™ G-50 column (Amersham Pharmacia Biotech AB. Uppsala, Sweden) (1.0 cm diameter, 30 cm length) pre-equilibrated and eluted with 0.1 M sodium phosphate buffer, pH = 8.0. The void volume peak fractions containing the liposome-associated AG2034 were collected and quantitated for liposomes and AG2034 content. Each fraction from the Sephadex™ G-50 column was checked for phospholipid content by Stewart assay (Stewart. 1980) with modification and for AG2034 by HPLC analysis. The percent encapsulation efficiency for AG2034 incorporated in liposomal formulations will be calculated by the following equation: Cde / C le Encapsulation % = ------------------------- x 100 C di / C u C d e : Concentration of AG2034 in the fraction of AG2034-lipid complex C le * Concentration of total lipids in the fraction of AG2034-Iipid complex Cdi: Initial concentration of AG2034 Cu: Initial concentration of total lipids Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. II.C.10. Development of a Stealth Liposome Delivery System In order to prepare a stable stealth delivery system with optimum encapsulation efficiency, the following four optimization studies were carried out to develop the ideal liposome composition. II.C.10.1. Effect of Phospholipids and Cholesterol on Encapsulation Efficiency Six formulations were prepared using the extrusion method. Four formulations, formulations 1-4, with the ratios of 1:0, 1:0.25, 1:0.5 and 1:1 for DPPC/CHOL and another two formulations with the ratios of 1:0.25 and 1:0.5 for DSPC/CHOL were prepared at a concentration of 6 pmole/mL AG2034. Based on the results of encapsulation efficiency and stability, one formulation was selected for further optimization. n.C.10.2. Influence of PEG(2k)-DPPE Loading on Encapsulation Efficiency Two formulations, formulations 7 and 8, were prepared with total phospholipids concentration of 40 jimole/mL and at AG2034 concentration of 6 (imole/mL using extrusion method. Formulation 7 was composed of 76% DSPC : 19%CHOL : 5% PEG(2k)-DPPE. Formulation 8 consisted of 72% DSPC : 18% CHOL : 10% PEG(2k)-DPPE. One formulation was selected for the next stage study due to better results of encapsulation efficiency and stability. 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. n.C .10.3. Impact of Manufacturing Method of Liposomes on Encapsulation Efficiency Some drugs, that are weak acids or weak bases, can be loaded into liposomes by the use of a pH gradient (Deamer, 1972). Two formulations with identical composition were prepared at total phospholipids concentration of 40 [imole/mL and at AG2034 concentration of 6 pmole/mL using extrusion method and remote loading method. The results of this study helped select a single manufacturing method for the fourth stage optimization study. n.C.I0.4. Effect of Phospholipids Loading on Encapsulation Efficiency Formulation 10 and formulation 11 were prepared in the same lipids composition. 76% DSPC : 19% CHOL : 5% PEG(2k)-DPPE, with the AG2034 concentration of 6 jimoie/mL. The concentrations of phospholipids for formulation 10 and formulation 11 were 60 [imole/mL and 80 pmoIe/mL, respectively. A single formulation with sufficient encapsulation efficiency was chosen for the final optimization of the formulation. H.C.10.5. Effect of AG2034 Loading on Encapsulation Efficiency Formulation 12, 13, 14, and 15 were prepared in the same lipid composition. 76% DSPC : 19% CHOL: 5% PEG(2k)-DPPE, with the phospholipids concentration of 60 (imole/mL. The concentrations of AG2034 for formulations 12. 13. 14 and 15 were 12,25,35 and 58 pmole/mL, respectively. 44 ' Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. II.C .ll. Determination of Liposome Size A wide variety of techniques are available to size liposomes based on light scattering. The popularity of this method depends on its ease of operation and the speed by which one can obtain data. The vesicle size of the liposomes was determined by light scattering based on laser diffraction using the Malvern Mastersizer (Malvern, Model S, Ver. 2.15, UK). The apparatus consisted of a He-Ne laser (5 mW) and a small volume sample holding cell. The sample was diluted to the appropriate concentration with DI water and stirred using a magnetic stir bar to keep the sample in suspension. O.C.12. Determination of the Phase Transition Temperature An important feature of membrane lipids is the existence of a temperature- dependent reversible phase transition, where the hydrocarbon chains of the phospholipids undergo a transformation from an ordered (gel) state to more disordered fluid (liquid crystalline) state. These changes have been documented by freeze-fracture electron microscopy but are most easily demonstrated by differential scanning calorimetry. Therefore, the phase transition of the different liposome compositions was determined using differential scanning calorimetry (DSC). The DSC scans were performed on a DSC 821 calorimeter (Mettler Toledo, IL, USA) with a TSO 801RO sample robot (Mettler Toledo, IL, USA), at a heating rate of 0.5°C/min between the temperatures of 30 to 65°C. The measured Tm values for 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. vesicles of different compositions are useful for prediction of formulation stability in vitro and in vivo. H.C.13. Leakage Study The optimum formulation was sealed in 20 mL scintillation vials and stored at refrigeration temperature (2-8°Q and 25°C/60% RH for a one month period, and AG2034 leakage from the liposomes was evaluated at pre-determined time intervals. Samples were withdrawn from each storage condition at I day, 2 days, 3 days, I week, 2 weeks and one month. An aliquot of 25 |iL from each sample was loaded onto a gel filtration Sephadex™ G-50 column (1.0 cm diameter, 30 cm length) (Amersham Pharmacia Biotech AB., Uppsala, Sweden) pre-equilibrated and eluted with 0.1 M phosphate buffer. The void volume peak fractions containing the liposome-associated AG2034 were collected and quantitated for phospholipid and AG2034 content. Each fraction from the Sephadex™ G-50 column was checked for phospholipid content by a modified Stewart assay (Stewart, 1980). The quantitation of AG2034 was performed by HPLC analysis. n.C.14. Cell Growth Study The effects of free AG2034, empty liposomes and liposome-associated AG2034 on antiproliferative activity were determined using in vitro cell cultured human lung A549 cells. The sulforhodamine B assay (Skehan et al.. 1990) was used for the determination of cell growth study. The adherent cell cultures were fixed in 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. situ by adding 50 jiL of cold 50% (w/v) trichloroacetic acid (TCA), final concentration, 10% TCA, and incubating for 60 minutes at 4°C. The supernatant was discarded, and the plates were washed five times with deionized water and dried. One hundred microliters of SRB solution (0.4% w/v in 1% acetic acid) were added to each microtiter well, and the culture was incubated for 10 minutes at room temperature. Unbound SRB was removed by washing five times with 1% acetic acid. The plates were dried by air. Bound stain was solubilized with Tris buffer and the optical densities were read on the Victor 1420 multilabel counter, (Gaithersburg, MD. USA) at a single wavelength of 515 nm. O.C.15. In Vitro Release Study The in vitro release of AG2034 from liposomal complexes was studied in human serum. Equal volumes of AG2034 liposome preparations and human serum were mixed in a test tube and placed in an incubator for predetermined time intervals. To separate liposomes from serum, the samples were loaded onto a gel filtration Sephadex™ G-50 column (1.0 cm diameter, 30 cm length) (Amersham Pharmacia Biotech AB., Uppsala, Sweden) pre-equilibrated and eluted with 0.1 M phosphate buffer. The void volume peak fractions containing the liposome- associated AG2034 were collected and quantitated for phospholipid and AG2034 content. Each fraction from Sephadex™ G-50 column was checked for phospholipid content by a modified Stewart assay (Stewart, 1980). The amount of AG2034 47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. released in the serum was determined by HPLC analysis. Triplicate experiments were performed and the average values with standard deviations were plotted. H.C.16. Efficacy Study of Liposome-Associated AG2034 Treated with Human Serum The present study does not attempt to prove the efficacy of the optimal stealth liposomes delivery system in vivo. Therefore, the efficacy of Liposome-associated AG2034 collected from the in vitro release study on antiproliferative activity was determined using in vitro cell cultured human lung A549 cells. The sulforhodamine B assay (Skehan et al., 1990) was used for the determination of cell growth study. The adherent cell cultures were fixed in situ by adding 50 |iL of cold 50% (w/v) trichloroacetic acid (TCA), final concentration, 10% TCA, and incubating for 60 minutes at 4°C. The supernatant was discarded, and the plates were washed five times with deionized water and dried. One hundred microliters of SRB solution (0.4% w/v in 1% acetic acid) were added to each microtiter well, and the culture was incubated for 10 minutes at room temperature. Unbound SRB was removed by washing five times with 1% acetic acid. The plates were dried by air. Bound stain was solubilized with Tris buffer and the optical densities were read on the Victor 1420 multilabel counter, (Gaithersburg, MD, USA) at a single wavelength of 515 nm. 48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. H.D. RESULTS H.D.1. HPLC Analysis of AG2034 Under the chromatographic conditions described in the experimental section, the retention time o f AG2034 was 5.09 minutes. Figure 8 shows a typical chromatogram o f AG2034. mAU 3 5 304 2 5 4 20 151 101 54 0 S 10 Figure 8. Typical chromatogram o f AG2034. 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. n.D.1.1. Linearity A stock standard of AG2034 was prepared at 443 jig/mL and a series of dilutions were made from this solution to obtain a range of concentrations down to 8.85 pg/mL. Each of five concentrations was injected three times in succession from lowest to highest. The peak area versus concentration was statistically analyzed. The linearity of the method was determined by simple least squares analysis. A correlation coefficient of 1.0000 was obtained over the concentration range. The relative standard deviations demonstrated reproducibility, with RSDs ranging from 0.03 to 0.37%. II.D.L2. Accuracy and Precision The average peak area for each concentration used for accuracy and precision was subjected to the linear regression data to obtain found concentrations. The found concentrations for the actual concentrations of 8.85 pg/mL. 17.7 pg/mL, 35.4 pg/mL, 88.6 pg/mL, and 443 pg/mL were 9.07 pg/mL. 17.8 pg/mL, 35.3 pg/mL. 88.2 pg/mL. and 444 pg/mL, respectively. Values for precision were within ± 3% indicating that the method was reproducible. linear, accurate and precise. Results of the study are presented in Table 2. A plot of the regression line for the linearity study is illustrated in Figure 9. The plot includes the error bar for each point. However, the error bars do not show on the plot because the standard deviations are smaller than symbols. 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Peak A re a (m A U x s) Table 2. Assay validation for AG2034 by HPLC Actual Cone. (Ug/mL) Found Cone. (mg/mL) % Accuracy % Precision (N=3) 8.85 9.07 102.49 2.49 17.7 17.8 100.56 0.55 35.4 35.3 99.72 0.28 88.6 88.2 99.55 0.40 443 444 100.23 0.23 2400 2000 1600 1200 800 400 0.0 100.0 200.0 300.0 400.0 500.0 Concentration (ug/mL) Figure 9. Linearity for AG2034 standards by HPLC. Each point represents the mean ± s.d. (n=3). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. II.D.2 UV-Vis Spectrophotometric Analysis of DPPC A modified Stewart assay (Stewart, 1980) was used for DPPC analysis. In the Stewart assay for phospholipids (Stewart, 1980), the ability of phospholipids to form a complex with ammonium ferrothiocyanate in organic solution is utilized. The advantage of this method is that the presence of inorganic phosphate does not interfere with the assay. n.D.2.1. Linearity A stock standard of DPPC was prepared at 0.151 (imole/mL and a series of dilutions were made from this solution to obtain a range of concentrations down to 0.0151 jimole/mL. The absorbance value at 488 nm was obtained for each concentration three times in succession from lowest to highest concentration. The absorbance at 488 nm versus concentration was statistically analyzed. The linearity of the method was determined by simple least squares analysis. A correlation coefficient of 0.9994 was obtained over the concentration range. The relative standard deviations demonstrated reproducibility, with RSDs ranging from 0.20 to 0.40 %. II.D.2.2. Accuracy and Precision The average absorbance for each concentration used for accuracy and precision was subjected to the linear regression data to obtain found concentrations. The found concentrations for the actual concentrations of 0.0151 pmole/mL, 0.0301 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. jimole/mL, 0.0603 jimole/mL, 0.121 |imoIe/mL and 0.151 |imole/mL were 0.0153 jimole/mL, 0.0311 Jimole/mL 0.0596 {imole/mL 0.118 (imole/mL and 0.153 limoIe/mL respectively. Values for precision were within ± 5% indicating that the method was reproducible, linear, accurate and precise. Results of the study are presented in Table 3. A plot of the regression line for the linearity study is illustrated in Figure 10. The plot includes the error bar for each point. However, the error bars do not show on the plot because the standard deviations are smaller than symbols. Table 3. Assay validation for DPPC Actual Cone. Found Cone. % % Cfimole/mL) (pmoIe/mL) Accuracy Precision (N== 3) 0.0151 0.0153 101.32 1.50 0.0301 0.0311 103.32 3.36 0.0603 0.0596 98.84 1.09 0.I2I 0.118 97.52 2.48 0.151 0.153 101.32 1.32 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.60 1.20 E c CO CO 0.80 to 0.40 0.00 0.00 0.04 0.08 0.12 0.16 Concentration (umole/mL) Figure 10. Standard curve for DPPC assay. Each point represents the mean ± s.d. (n=3). II.D.3. UV-Vis Spectrophotometric Analysis of DSPC A modified Stewart assay (Stewart. 1980) was used for DSPC analysis. H.D.3.1. Linearity A stock standard of DSPC was prepared at 0.123 [imole/mL and a series of dilutions were made from this solution to obtain a range of concentrations down to 0.0123 [imole/mL. The absorbance at 488 nm was obtained for each concentration three times in succession from lowest to highest concentration. The absorbance at 488 nm versus concentration was statistically analyzed. The linearity of the method was determined by simple least squares analysis. A correlation coefficient of 0.9987 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. was obtained over the concentration range. The relative standard deviations demonstrated reproducibility, with RSDs ranging from 0.24 to 0.42%. H.D.3.2. Accuracy and Precision The average absorbance for each concentration used for accuracy and precision was subjected to the linear regression analysis to obtain found concentrations. The found concentrations for the actual concentrations of 0.0123 Jimole/mL, 0.0247 [imole/mL, 0.0493 Jimole/mL, 0.0986 pmole/mL and 0.123 (imole/mL were 0.0128 (imole/mL. 0.0241 [imole/mL 0.0478 [imole/mL 0.101 (imole/mL and 0.123 [imole/mL respectively. Values for precision were within ± 5% indicating that the method was reproducible. linear, accurate and precise. Results of the study are presented in Table 4. A plot of the regression line for the linearity study is shown in Figure 11. The plot includes the error bar for each point. However, the error bars do not show on the plot because the standard deviations are smaller than symbols. Table 4. Assay validation for DSPC Actual Cone. Found Cone. % % (pmoIe/mL) (pmoIe/mL) Accuracy Precision (N=3) 0.0123 0.0128 104.07 3.84 0.0247 0.0241 97.57 2.11 0.0493 0.0478 96.96 3.06 0.0986 0.101 102.43 2.43 0.123 0.123 100.00 0.00 55 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 . 6 0 _ 120 E < 0 .a < 0.80 0.40 0.00 ---------------------------------------------------------------------------------------- 0.00 0.04 0.08 0.12 0.16 Concentration (umole/mL) Figure 11. Standard curve for DSPC assay. Each point represents the mean ± s.d. (n=3). ILD.4. UV-Vis Spectrophotometric Analysis of DSPC/PEG(2K)-DPPE with a Molar Ratio of 15.2:1 Standards A modified Stewart assay (Stewart. 1980) was used for determination of DSPC:PEG(2K)-DPPE with a molar ratio of 15.2:1 analysis. H.D.4.1. Linearity A stock standard of DSPC/PEG(2K)-DPPE with a ratio of 15.2:1 was prepared at 0.0501 (imole/mL and a series of dilutions were made from this solution to obtain a range of concentrations down to 0.00501 (imole/mL. The absorbance 56 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. value at 488 run was obtained for each concentration three times in succession from lowest to highest concentration. The absorbance at 488 nm versus concentration was statistically analyzed. The linearity of the method was determined by simple least squares analysis. A correlation coefficient of 0.9999 was obtained over the concentration range. The relative standard deviations showed acceptable reproducibility, with RSDs ranging from 0.34 to 0.54%. II.D.4.2. Accuracy and Precision The average absorbance for each concentration used for accuracy and precision was subjected to the linear regression analysis to obtain found concentrations. The found concentrations for the actual concentrations of 0.00501 pmoIe/mL, 0.0100 pmole/mL. 0.0200 (imoIe/mL, 0.0401 (imoIe/mL and 0.0501 jimole/mL were 0.00527 (imole/mL, 0.00985 |imoIe/mL. 0.0198 jimoIe/mL. 0.0402 (imoIe/mL and 0.0501 [imole/mL respectively. Values for precision were within ± 5% indicating that the method was reproducible. linear, accurate and precise. Results of the study are presented in Table 5. A plot of the regression line for the linearity study is illustrated in Figure 12. The plot includes the error bar for each point. However, the error bars do not show on the plot because the standard deviations are smaller than symbols. 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A b s ( 4 8 8 nm ) Table 5. Assay validation for DSPC/PEG(2K)-DPPE with a molar ratio of 15.2:1 standards Actual Cone. Found Cone. % % (pmoIe/mL) (pmole/mL) Accuracy Precision (N=3) 0.00501 0.00527 105.19 5.26 0.0100 0.00985 98.50 1.71 0.0200 0.0198 99.00 1.07 0.0401 0.0402 100.25 0.35 0.0501 0.0501 100.00 0.04 0.40 i 0.30 0.20 0.10 0.00 0.00 0.02 0.04 0.06 Concentration (umole/mL) Figure 12. Standard curve for DSPC/PEG(2K)-DPPE with a molar ratio of 15.2:1 assay. Each point represents the mean ± s.d. (n=3). 58 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. II.D.5. UV-Vis Spectrophotometric Analysis of DSPC7PEG(2K)-DPPE with a Molar Ratio of 7.2:1 Standards A modified Stewart assay (Stewart, 1980) was used for determination of DSPC/PEG(2K)-DPPE with a molar ratio of 7.2:1 analysis. U.D.5.1. Linearity A stock standard of DSPC/PEG(2K)-DPPE with a molar ratio of 7.2:1 was prepared at 0.1025 pmoIe/mL and a series of dilutions were made from this solution to obtain a range of concentrations down to 0.01025 [imole/mL. The absorbance value at 488 nm was obtained for each concentration three times in succession from lowest to highest concentration. The absorbance at 488 nm versus concentration was statistically analyzed. The linearity of the method was determined by simple least squares analysis. A correlation coefficient of 0.9996 was obtained over the concentration range. The relative standard deviations demonstrated reproducibility, with RSDs ranging from 0.30 to 0.54%. IIJD.5.2. Accuracy and Precision The average absorbance for each concentration used for accuracy and precision was subjected to the linear regression data to obtain found concentrations. The found concentrations for the actual concentrations of 0.01025 pmole/mL, 0.02050 [imole/mL, 0.04100 [imole/mL. 0.08200 [imole/mL and 0.1025 [imole/mL were 0.01078 [imole/mL, 0.01994 [imole/mL. 0.04148 [imole/mL. 0.08044 59 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. {imole/mL and 0.1036 (imole/mL, respectively. Values for precision were within ± 5% indicating that the method was reproducible, linear, accurate and precise. Results of the study are presented in Table 6. A plot of the regression line for the linearity study is shown in Figure 13. The plot includes the error bar for each point. However, the error bars do not show on the plot because the standard deviations are smaller than symbols. Table 6. Assay validation for DSPC/PEG(2K)-DPPE with a molar ratio of 7.2:1 standards Actual Cone. (pmole/mL) Found Cone. (limole/mL) % Accuracy % Precision 0.01025 0.01078 105.17 5.13 0.02050 0.01994 97.27 2.74 0.04100 0.04148 101.17 1.17 0.08200 0.08044 98.10 1.90 0.1025 0.1036 101.07 1.09 60 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Abs(488nm) 1.20 0.90 0.60 0.30 0.00 0.00 0.03 0.06 0.09 0.12 Conce ntration (um ole/m L) Figure 13. Standard curve for DSPC/PEG(2K)-DPPE with a molar ratio of 7.2:1 assay. Each point represents the mean ± s.d. (n=3). II.D.6. pH-Solubility Profile A pH-soIubility profile was obtained over the pH range from 1.5 to 6.7 at ambient temperature. At pH > 6, the solubility increased dramatically. Results of this study are summarized in Table 7. A plot of the pH-soIubility profile is shown in Figure 14. Table 7. AG2034 pH-soIubility study at ambient temperature pH Value Solubility (tng/inL) 1.52 0.02 2.12 0.03 2.98 0.03 4.98 0.05 6.01 0.65 6.48 7.52 6.71 12.03 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 12 - f 1 0 E 8 - ■ » £ e C /) 0 1 2 3 4 5 pH Figure 14. AG2034 pH-soIubility profile at ambient temperature II.D.7. Extraction of AG2034 from Liposomal Formulations Either 5% Triton XlOO or 10% Triton X100 were capable of extracting AG2034 from liposomal formulations in liposome-associated AG2034 by shaking the mixture in a water bath at 60°C for 30 minutes followed by centrifuging at 16000 g for 10 minutes. However, to prevent interference of AG2034 with high concentrations of surfactant in the HPLC assay, 5% Triton XlOO was chosen as the extraction solvent for sample preparation. The results of this study are presented in Table 8. 62 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 69 Table 8. Solvent selection for AG2034 extraction from liposomal formulations Solvent Shaking Time (minutes) at 60°C 10 20 30 Ethanol Cloudy Cloudy Cloudy 1% SLS Cloudy Cloudy Cloudy 5% SLS Cloudy Cloudy Cloudy 10% SLS Cloudy Cloudy Cloudy 1% Tween 20 Cloudy Cloudy Cloudy 5% Tween 20 Cloudy Cloudy Cloudy 10% Tween 20 Cloudy Cloudy Cloudy 1% Triton XlOO Cloudy Cloudy Cloudy 5% Triton XlOO Cloudy Cloudy Clear 10% Triton XlOO Cloudy Cloudy Clear Heating Time Effect on Stability of AG2034 during Preparation of Liposomal Formulations Preformulation studies indicated that AG2034 was unstable and hydrolysis and oxidation were the two major degradation pathways for this compound (Wang et al., 1997). To prevent the occurrence of degradation of AG2034 during the manufacturing process of liposomes, thermal degradation of AG2034 was investigated. The study showed that AG2034 solution with a concentration of 0.6 mg/mL in 0.1 M sodium phosphate buffer, pH = 8.0, was stable for up to 120 minutes when the solution was stored in a water bath at 60°C with shaking speed of 50 rpm. The results of this study are shown in Figure 15. The plot includes the error bar for each point. However, the error bars do not show on the plot because the standard deviations are smaller than symbols. 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. %Recovery II.D.9. Liposomes Figure 16 shows the elution profile of the gel filtration chromatography performed on a Sephadex™ G-50 column at 255 nm in order to separate liposome- associated AG2034 from free AG2034. As clearly appreciable, two major peaks with different relative heights were present in the chromatogram. The first larger peak was the elution of liposome-associated AG2034. The second smaller peak was the elution of free AG2034. 150.0 130.0 110.0 90.0 70.0 50.0 30 0 60 90 120 Tim e (m inutes) Figure 15. Effect of heating time on stability of AG2034 solution at 60°C. Each point represents the mean ± s.d. (n=3). 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.5 2.5 IO CM CM \ .................. i 10 15 20 25 30 35 40 0 5 Fraction N o. Figure 16. Elution profile of liposome-associated AG2034 from gel filtration chromatography on a Sephadex G-50 column (1.0 cm internal diameter: 30 cm length: 1 mL/fraction). Q.D.10. Development of a Stealth Liposome Delivery System II.D.I0.1. Effect of Phospholipids and Cholesterol on the Encapsulation Efficiency Saturated phospholipids such as DMPC, DPPC and DSPC are more chemically stable than lipids containing unsaturated fatty acids such as egg phosphatidylcholine. Therefore, DPPC and DSPC were selected for formulation development. Also, cholesterol is added to strengthen liposomes in that it reduces leakage of entrapped drug on storage as well as in biological environments. Generally, the mole percent cholesterol used in liposomes for pharmaceutical 65 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. purposes ranges from 25 to 50%. To investigate the influence of cholesterol on the encapsulation efficiency in large unilamellar vesicles (LUVs), liposomes with mole ratios of DPPC to cholesterol of 1:0, 1:0.25, 1:0.5 and 1:1 and with mole ratios of DSPC to cholesterol of 1:0.25 and 1:0.5 were prepared. In the present study, an increase in encapsulation efficiency was observed for liposomal formulations when the phospholipid/cholesterol ratios of 1:0.25 were used with each of the two phospholipids. Formulation I, with the DPPC/CHOL ratio of 1:0, was not able to pass through the two stacked 0.2 |im polycarbonate filters. Therefore, formulation I was not considered for the remainder of the preparation process and encapsulation efficiency testing. The results of percent encapsulation efficiency for formulations 2 to 6 are presented in Table 9. The results indicated that formulation 2 had higher percent encapsulation efficiency than formulation 5. However, formulation 5 was more stable than formulation 2 in terms of the smaller change in particle size distribution during storage. Hence, for all further studies the DSPC/CHOL with the ratio of 1:0.25 was used. Table 9. Effect of phospholipids and cholesterol on encapsulation efficiency Formulation ID Phospholipid/Cholesterol (mole ratio) % Encapsulation Efficiency Mean (s.cL) DPPC DSPC CHOL I 1 0 N/A 2 I 0.25 6.6 (0.5) 3 1 0.50 4.2 (0.5) 4 I I 3.8 (0.3) 5 1 0.25 5.0 (0.4) 6 I 0.50 3.0 (0.3) 66 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. n.D. 10.2. Influence of PEG (2k)-DPPE Loading on Encapsulation Efficiency In order to form stealth liposomes, polyethylene glycol-derivatized phosphatidylethanolamine was incorporated into the formulations. The PEG(2k)- DPPE was chosen for preparing the long circulating liposomes formulations. Two formulations, formulations 7 and 8, were prepared at total phospholipids concentration of 40 [imole/mL with AG2034 concentration of 6 pmole/mL using the extrusion method due to the fact that formulation 5 had the higher percent encapsulation efficiency and higher relative stability. Formulation 7 was composed of 76% DSPC. 19% CHOL, and 5% PEG(2k)-DPPE. Formulation 8 consisted of 72% DSPC, 18% CHOL and 10% PEG(2k)-DPPE. The results of percent encapsulation efficiency for formulation 7 and 8 are summarized in Table 10. The results showed the formulation with 5% mole ratio of PEG(2k)-DPPE had a much higher percent encapsulation efficiency compared to formulation without PEG (2k)- DPPE and formulation with 10% mole ratio of PEG(2k)-DPPE. Therefore, formulation 7 was selected for the next optimization study. Table 10. Influence of PEG(2k)-DPPE loading on encapsulation efficiency Formulation ID Phospholipid/Cholesterol (mole ratio) % Encapsulation Efficiency Mean (s.cL) DSPC CHOL PEG(2k)-DPPE 7 76 19 5 15.1 (1.2) 8 72 18 10 6.9 (0.6) 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ELD. 10.3. Impact of Manufacturing Method of Liposomes on Encapsulation Efficiency To evaluate the impact of the liposomes manufacturing method on percent encapsulation efficiency, the formulation with composition of 76% DSPC. 19% CHOL, and 5% PEG(2k)-DPPE with total phospholipids concentration of 40 (imole/mL and AG2034 concentration of 6 pmole/mL was prepared using both extrusion method and remote loading method. The percent encapsulation efficiency for the formulation using the remote loading method was negligible, whereas the extrusion method yielded a percent encapsulation efficiency of 15.1%. The comparison of percent encapsulation efficiency for these two methods is listed in Table 11. Table 11. Impact of manufacturing method of liposomes on encapsulation efficiency Formulation ID PhosphoIipid/CHOL/PEG(2k)-DPPE 76%:19%:5% % Encapsulation Efficiency Mean (s.d.) Manufacturing Method 7 Extrusion method 15.1 (1.2) 9 Remote loading method negligible n.D. 10.4. Effect of Phospholipids Loading on Encapsulation Efficiency The percent encapsulation efficiency for formulation 7. 10 and 11 was 15.1%, 20.5% and 21.6%, respectively. Formulation 10 with the phospholipids concentration of 60 jimole/mL was chosen for next stage optimization due to the potential toxicity concerns in vivo for high level of phospholipid loading, although formulation 11 with the phospholipids concentration of 80 (imole/mL had slightly 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. higher % encapsulation efficiency than formulation 10. The results of percent encapsulation efficiency for these three formulations are summarized in Table 12. Table 12. Effect of phospholipids loading on encapsulation efficiency Formulation ID DSPC/CHOL/PEG(2k)-DPPE 76%:19%:5% % Encapsulation Efficiency Mean (s.d.) Phospholipids loading 7 40 (imole/mL 15.1 (1.2) 10 60 (imole/mL 20.5(1.2) 11 80 (imole/mL 21.6(1.5) O.D.IO.5. Effect of AG2034 Loading on Encapsulation Efficiency To investigate the effect of AG2034 loading on percent encapsulation efficiency, the AG2034 loading was increased while maintaining the phospholipids concentration at 60 |imoIe/mL during preparation of liposomes. Similar results of percent encapsulation efficiency were observed among these five formulations. The results of percent encapsulation efficiency of formulations 10. 12. 13. 14 and 15 are presented in Table 13. Table 13. Effect of AG2034 loading on encapsulation efficiency Formulation ID DSPC/CHOL/PEG(2k)-DPPE=76%: 19%:5% Phospholipids concentration at 60 (imole/mL % Encapsulation Efficiency Mean (s.d.) AG2034 loading 10 6.3 (imole/mL 20.5(1.2) 12 12.6 (imole/mL 21.6(1.4) 13 25.1 (imole/mL 20.9(1.8) 14 35.0 (imole/mL 24.0(1.5) 15 58.1 (imole/mL 24.3(1.7) 69 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ILD.11. Determination of Liposomes Size The average size and size distribution of liposomes are important parameters with respect to physical properties and biological fate of the liposomes and their entrapped substances. The mean size of liposomes was evaluated to assist in determining the stability of liposomal formations. Formulations with smaller size distributions were more physically stable. The results of particle size profiles are summarized and plotted in Figure 17, 18 and 19. The study indicated that formulation with the composition of 76% DSPC: 19% CHOL: 5% PEG(2k)-DPPE at the various phospholipid concentrations prepared by using the extrusion method had good physical stability in terms of changes in size of liposomes during storage at ambient conditions for 72 hours. Also, an approximately 10% decrease in the size distribution for formulation 11 was observed, which had higher phospholipid loading, compared to formulations 7. 10 and 15. 70 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. M ean Particle Size (urn) M ean parttc|e sjze (um) 72 0 24 48 -#— F1 | -■— F2 -A— F3 - H - F 4 - 3 K — F5 F6 Storage Tim e (Hours) Figure 17. Particle size distribution for formulations I - 6. Each point represents the mean ± s.d. (n=3). 12 10 72 24 48 0 F5 F7 F8 Storage Tim e (Hours) Figure 18. Particle size distribution for formulations 5 .7 and 8. Each point represents the mean ± s.d. (n=3). 71 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0.200 J- 0 24 48 Storage Time (Hours) 72 Figure 19. Particle size distribution for formulations 7, 10. 11 and 15. Each point represents the mean ± s.d. (n=3). II.D.12. Differential Scanning Calorimetric Analysis Calorimetric analysis plays a fundamental role in determining both the type and strength of interaction between lipid and drug molecules. The interaction is reflected in the change of the thermotropic behavior of the phospholipid with shifting in the transition temperature (Tm ). An endothermic peak was observed at 53 °C for formulation 7 with 76% DSPC: 19% CHOL: 5% PEG(2k)-DPPE and 52°C for formulation 8 with 72% DSPC: 18% CHOL: 10% PEG(2k)-DPPE, respectively. Figures 20 and 21 show the DSC thermographs for these two compositions. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. * M D DSPC:CHOX.:P£G(2JC) — 76%;19% :5%, 14.11.1999 18:02:39 DSPC: CHOI.: PEG (2K)*76%: 19%:5%, 30.0000 rag >12.42 i«J 52.88 *C 0.50 •C a in ''-! Integral P«alc Beating Rate 3 0 45 55 60 40 50 60 1 0 20 30 4 0 Lab: PO - DSC METTLER TOLEDO STAR* System Rgure 20. DSC thermograph of formulation 7 A ax o D S PC rC H O L :PE SC 2K )* 7 2 % :1 8 % :1 0 % , 1 4 . 1 1 . 1 9 9 9 1 9 : 2 1 : 0 6 D S P C :C H O L :P E G 1 2 K )-7 2 % :1 8 % :1 0 % , 3 0 . 0 0 0 0 m q I n t e g r a l Peak Heating Rate - 9 . 0 7 a J 5 2 . 2 4 *C 0 . 5 0 * c m in — t a * 3 0 3 5 5 0 5 5 60 40 4 5 50 10 20 30 4 0 6 0 Lab: PD - DSC METTLER TOLEDO START System Rgure 21. DSC thermograph of formulation 8 73 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. H.D.13. Leakage Study Formulation 15 was subjected to a leakage study at refrigerated temperatures (2-8°C) and ambient temperature for a 30-day period, and drug leakage from the liposomes was evaluated at specific time intervals. Figure 22 shows the leakage profile of AG2034 for formulation 15. As expected, the leakage rate increased with increasing temperature. Refrigerated temperature was found to be the most suitable environment for the AG2034 liposomal formulation, since the leakage rate was negligible after storage for 30 days. The results of the leakage studies at ambient temperature demonstrated that the liposomes were stable for 14 days, after which leakage of AG2034 from liposomes was observed. The leakage of AG2034 from the formulation may be associated with the rupture of liposomal membrane at ambient temperature. 110 e ioo o 3 1 a. s e ui 90 ■ a s 80 e 70 - Ambient Conditions 5 “C 60 28 7 14 21 0 Storage Time (Days) Figure 22. Leakage profile of AG2034 from liposomal formulation 15, stored at 5°C and ambient condition for up to 30 days. Each point represents the mean ± s.d. (n=3). 74 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. O.D.14. Cell Growth Study In vitro antiproliferative activity of AG2034 in liposomes was determined and compared with the control (phosphate buffer), empty liposomes (placebo) and free drug. Human lung A549 cells were treated with the same concentrations, in terms of AG2034 molarity, of free or formulated AG2034. After 5 days of cell culture, cells were read on the Victor 1420 multilabel counter. Figure 23 presents the results of these experiments, showing that AG2034 delivered by liposomes showed a similar antiproliferative activity compared with free AG2034. The antiproliferative effects of phosphate buffer and the empty liposomes on A549 cells demonstrated that the controls do not cause inhibition of cell growth. e o o % 2 o > s O 100 8 0 6 0 Control Empty Liposomes AG2034 free drug AG2034 liposomes 4 0 20 0 10 100 AG2034 (nM) 1000 10000 Figure 23. Comparative analysis of the effect of control, empty liposomes, AG2034 free drug and AG2034 stealth liposomes on proliferation of A549 cells. Each point represents the mean ± s.d. (n=3). 75 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. II.D.15. In Vitro Release Study The release of AG2034 from the optimal stealth liposome formulation, formulation 15, was studied in the presence of human serum and the results are shown in Figure 24. There was an initial rapid leakage of AG2034 from the stealth liposomal delivery system for 2 hours followed by a slower release up to 12 hours and then followed by a very slow leakage up to 24 hours. The time for 25% of AG2034 to be released from this formulation was found to be 12 hours. At the end of 24 hours, no more than 30% of the initial amount of entrapped AG2034 had leaked and there was also no significant change in the particle size of the stealth liposomes in the serum. Thus, formulation 15 can be considered stable for administration for in vivo studies. 40 35 I « 3 0 s ® 25 o c 3 20 CM 2 15 0 4 8 12 16 20 24 Time (hours) Figure 24. In vitro release study of formulation 15. Each point represents the mean ± s.d. (n=3). 76 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. II.D.16. Efficacy Study of Liposome-Associated AG2034 Treated with Human Serum In order to evaluate the anti cancer efficacy of formulation 15 after being incubated with human serum, the in vitro antiproliferative activity of AG2034 in liposomes collected from gel filtration Sephadex™ G-50 column from the in vitro release study was performed and compared with that of formulation 15 without treatment of human serum. Human lung A549 cells were treated with the same concentrations in terms of AG2034 molarity for formulation 15 treated with and without human serum. After 5 days of cell culture, cells were read on the Victor 1420 multilabel counter. Rgure 25 reports the results of this experiment. The data demonstrated that formulation 15 incubated with human serum does not reduce inhibition of cell growth for A549 cells. 77 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 0 0 | 80 8 60 £ i 40 0 20 1 10 1 0 0 1000 10000 _____________AG2034 (nM)_______________ AG2034 liposomes * — AG2034 liposomes incubated w ith human serumi Figure 25. Comparative analysis of efficacy study of liposome-associated AG2034 (formulation 15) with and without treatment with human serum on proliferation of A549 cells. Each point represents the mean ± s.d. (n=3). 78 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. HI. EVALUATION OF PLASMA CLEARANCE AND TOXICITY OF THE STEALTH AG2034 LIPOSOME DELIVERY SYSTEM 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ni.A. Experimental IILA.l. Materials HI. A. 1.1. Chemicals AG2037 was synthesized at Agouron Pharmaceuticals, Inc.. Formic acid (HCOOH) was purchased from EM Science (Gibbstown, NJ, USA). Ammonium hydroxide (NH4OH) was obtained from VWR (So. Plainfield, NJ, USA). Mouse plasma was obtained from Sigma (St. Louis, MO, USA). The folic acid deficient chow was from Harland Teklad (Madison, WI, USA) HI. A. 1.2. Animals Hairless mice (C3H), ages 8-10 weeks, were obtained from (Harland Sprague Daw ley Inc.. Indianapolis, IN) and used for in vivo studies. The body weight of the animals ranged between 22 to 25 grams. HI.A. 1.3. Equipment Slide-A-Lyzer® dialysis cassette, Slide-A-Lyzer® concentrating solution and Slide-A-Lyzer® syringe with 18-gauge needles were obtained from Pierce (Rockford, IL, USA). Disposable syringes (1.0 mL), 27G(l/2) used for dosing, and 24G(1) used for blood withdrawal, disposable needles and Vacutainer containing sodium heparin were purchased from Becton Dickinson and Co. (Rutherford, NJ. USA). ODS Solid Phase Extraction (SPE) cartridges (100 mg packing, 3 mL cartridge volume) were from Whatman LabSales, Inc. (Hillsboro, OR, USA). 80 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. m .A .2. Preparation of Formulations HI-A.2.1. Preparation of 5 mg/mL and 15 mg/mL of AG2034 solution Fifty milligrams and one hundred and fifty milligrams of AG2034 were weighed into two separate 10-mL volumetric flasks and made up to final volume with 0 .1 M phosphate buffer, pH=8.0. m.A.2.2. Preparation of Liposomal Formulation of AG2034 Conventional liposomes, formulation 2 with a total phospholipid concentration of 60 jimoIe/mL, and stealth liposomes, formulation 15, were selected for in vivo studies. In order to separate the liposome-associated AG2034 from the free AG2034, the large unilamellar liposomal suspension was loaded on a gel filtration Sephadex™ G-50 column (Amersham Pharmacia Biotech AB. Uppsala. Sweden) (1.0 cm diameter, 30 cm length) pre-equilibrated and eluted with 0.1 M sodium phosphate buffer, pH = 8.0. The void volume peak fractions containing the liposome-associated AG2034 were collected and injected into a Slide-A-Lyzer® dialysis cassette using a Slide-A-Lyzer® syringe with 18-gauge needles. To obtain the desired level of AG2034 concentration, this dialysis cassette was then placed into a small plastic bag containing Slide-A-Lyzer® concentrating solution at 5°C for 12 hours. The dialysis cassette was removed from the plastic bag and the concentrated liposomal suspension was drawn from the dialysis bag using a Slide-A-Lyzer® syringe with an 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 18-gauge needle. The content of AG2034 in the concentrated liposomal suspension was determined by HPLC analysis. m A 3 . Pharmacokinetics C3H mice, weighing between 22-25 grams, were housed in a constant temperature environment throughout this study. A commercial pelleted maintenance diet and tap water were freely available to the animal. The mice were randomly assigned to three treatment groups. The first group intravenously received AG2034 at a dose of 20 mg/kg of AG2034 in phosphate buffer. The second group received conventional liposomes, formulation 2 with a total lipid concentration of 60 (Xmole/mL, at a dose of 20 mg/kg of AG2034. The third group received stealth liposomes, formulation 15, at a dose of 20 mg/kg of AG2034. The dose was administered through the tail vein. For each pharmaceutical formulation and each time point of measurement, five mice were used. The required amount of blood was withdrawn from animals by means of a cardiac puncture under ether anesthesia at sampling times of 0.5,1, 2,4 ,8 ,2 4 .4 8 , 72,96 and 168 hours after dosing. The blood samples were transferred into evacuated sodium heparin-containing Vacutainer® test tubes to prevent coagulation. The plasma samples were harvested by centrifugation at 300 g for 10 minutes and frozen at — 20°C prior to analysis. 82 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. m .A .4. HPLC Analysis o f AG2034 In-Vivo III.A.4.1. Liquid Chromatography A Hewlett-Packard L100 HPLC equipped with a diode array detector, vacuum degasser, quaternary pump, and auto sampler, was used for AG2034 analysis. m.A.4.2. Chromatographic Conditions The chromatographic separation was accomplished on a Waters Symmetry™ C18 column (Milford, MA, USA), 5 ji. 150 x 4.6 mm, at a flow rate of 0.6 mL/minute at ambient temperature, with an injection volume of 100 |iL. The analytical wavelength for peak detection was set at 226 nm. A gradient reverse-phase high performance liquid chromatographic method was employed with an initial composition of 70% 25 mM ammonium phosphate, pH =3.0, and 30% methanol for 8.0 minutes; then ramp up to 50% methanol in 7 minutes; 50% 25 mM ammonium phosphate, pH =3.0, and 50% methanol from 15.0 to 20.0 minutes. III.A.4.3. Preparation of 1% (v/v) Formic Acid One milliliter of formic acid was pipetted into a 100-mL volumetric flask and brought to final volume with HPLC grade water. 83 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IH.A.4.4. Preparation of 2% (v/v) Ammonium Hydroxide Two milliliters of ammonium hydroxide were pipetted into a 100-mL volumetric flask and made up to final volume with HPLC grade water. HLA.4.5. Preparation of Standards and Quality Control Samples Fresh AG2034 stock solution was prepared for each validation run. Stock solution with the concentration of 100 pg/mL in 0.1 M phosphate buffer. pH = 8.0, was prepared and the stock solution was further diluted with 0.1 M phosphate buffer, pH = 8.0, to prepare working standards: 200, 400, 1000. 3000 and 6000 ng/mL. A 500-pL aliquot of each working standard was added to 500 (iL of blank mouse plasma to prepare calibration standards for validation runs. Mouse plasma quality control samples containing 200, 1000 and 3000 ng/mL AG2034 were prepared by diluting 20, 100 and 300-|iL aliquots of 100 fig/mL stock into 10 mL of control mouse plasma. Quality control samples were subdivided into I-mL aliquots, stored frozen, and used for up to I month. Internal standard, AG2037. was prepared by diluting a 430 pg/mL stock solution in 0.1 M phosphate buffer, pH = 8.0, to 1.72 pg/mL in HPLC grade water. m.A.4.6. Preparation of Samples ODS Solid Phase Extraction (SPE) cartridges (100 mg packing, 3 mL cartridge volume) were conditioned, under vacuum, with 2 x 1 mL methanol and 2 x 1 mL 1% formic acid taking care not to dry cartridges. One milliliter of 1% formic 84 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. acid, 0.5 mL of plasma standards, quality control samples or unknown samples and 0.5 mL of AG2037 internal standard were applied to cartridges and drawn through under low vacuum (10-15 in Hg). Cartridges were then washed 5 x 1 mL 1% formic acid. Cartridges were eluted with 3 x 0.5 mL 2% ammonium hydroxide and evaporated to dryness under vacuum. The residues were reconstituted in I mL mobile phase which consisted of 70% 25 mM ammonium phosphate, pH = 3.0, and 30% methanol. The solution was then transferred into an HPLC vial and 100 jxL was injected into the HPLC. Runs were accepted if the means of the quality control samples at each concentration fell within ± 20% of the expected values. m.A.4.7. Assay Validation HI.A.4.7.1 Linearity The linearity was evaluated from three calibration curves with five standard points prepared. Each standard solution was injected three times in succession from lowest to highest. These were run on 3 different days in the concentration range of 101.2 to 3036 ng/mL plasma. III.A.4.7.2 Accuracy and Precision The precision and accuracy were evaluated by repeated analyses of AG2034 at three concentrations in three replicate samples analyzed on 3 different days. All 85 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. chromatograms obtained were evaluated by peak area measurement. The theoretical concentrations for quality control samples were calculated using the calibration curve generated each day by linear regression of the AG2034/AG2037 (internal standard) peak area ratio against their concentration ratio in plasma. IIIA i. Pharmacokinetic Analysis Pharmacokinetic calculations were performed for the three groups: free drug, conventional liposomes, and stealth liposomes. The computer program, WinNonlin Professional Edition (Scientific Consulting, Inc.), was used for these calculations. The pharmacokinetic evaluations were fitted to a two compartment open model for these three groups. III.A.6. Acute Toxicity Study The development of GARFT inhibitors is complicated by toxicity that is often delayed, cumulative, and significantly enhanced in low folate model conditions in rodents. Therefore, the evaluation of acute toxicity was performed on the C3H mice, with the weight between 22 to 25 grams, housed in a constant temperature environment and maintained on folic acid deficient diet throughout this study. The mice were treated with low folate diet for two weeks prior to dosing and they were randomly assigned to two treatment groups. Groups of 30 animals per dose were treated with a single i.v. injection of either free drug in phosphate buffer at a dose of 30 mg/kg of AG2034 or stealth liposomes, formulation 15, at a dose of 30 mg/kg of 86 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. AG2034 through the tail vein. The animals were observed for a period of 21 days after drug administration. Drug-induced changes in body weight were recorded at 5- day intervals. The statistical analysis using the Yates-Corrected Chi-Square test was applied for comparing the survival rate for the acute toxicity experiment. m.B. Results ffl.B.1. Liposomes The preparation of liposome-associated AG2034 was conducted by a two- step method based on an evaporation followed by extrusion of liposomes through polycarbonate filters. Liposome extrusion was performed using two stacked 200 nm pore membranes. The concentrations of both conventional and stealth liposomes were condensed by using Slide-A-Lyzer® dialysis cassettes. The final AG2034 concentration of 5 mg/mL was obtained for these two liposome formulations by using the HPLC analysis of AG2034 in vitro method. ra.B.2. Chromatographic Results for In Vivo AG2034 was extracted from mouse plasma using an ODS Solid Phase Extraction (SPE) cartridge. No interference from plasma constituents was seen for AG2034 (analyte) and AG2037 (internal standard) when samples were tested by the HPLC method. The chromatograms obtained from blank mouse plasma, from a 87 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. standard solution of AG2034 and AG2037 (internal standard), and from in vivo mouse plasma sample are shown in Figures 26, 27 and 28. The retention rime*; of AG2034 and AG2037 were 13.3 and 17.1 minutes, respectively. Figure 26. Chromatogram of blank mouse plasma CO o CM o < cn f x * CO o CM O < O ) to o p x ^ o CO CO 8 10 12 14 16 18 Figure 27. Chromatogram of AG2034 and AG2037 (I.S.) standard solution 88 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2 4 6 8 10 12 14 16 1 8 Figure 28. Chromatogram of in vivo mouse plasma sample OLB.2.1. Linearity The linearity of this HPLC assay was evaluated from three calibration curves run on different days over the concentration range of 101.2 to 3036 ng/mL of AG2034 in mouse plasma. A linear regression analysis of the peak area ratio (AG2034/AG2037) against the concentration ratio (AG2034/AG2037) showed linearity over the range of concentrations tested. The mean calibration curve obtained was described by the equation y = 0.0005 x + 0.0323 (slope RSD = 5.2%, n=3). Back-calculated concentrations of AG2034 had RSDs less than 12.0%. Correlation coefficients ranged from 0.9994 to 0.9997. 89 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. III.B.2.2. Precision and Accuracy The inter-day precision for concentrations between 101.2 and 3036 ng/mL of AG2034 in mouse plasma expressed as RSD ranged from 2.72 to 7.24%. The intra day precision RSDs ranged from 1.97 to 9.51%. The intra-day accuracy, evaluated on the same plasma samples and expressed as percentage ratio of the mean amount found to the amount added to plasma, range from 101.0 to 106.9%. The pooled accuracy (inter-day) over the 3-day validation period ranged from 104.0 to 105.9%. The results of accuracy and precision for this method are summarized in Tables 14 and 15, respectively. Table 14. Accuracy of the method for the determination of AG2034 in mouse plasma Standards Day N Accuracy Theo. Mean Mean Pooled (ng/mL) Calc. AG2034 Recovery Recovery (ng/mL) (intra-day) (inter-day) (%)_________________ (%) 101.2 I 3 102.2 101.0 2 3 105.3 104.1 3 3 108.2 106.9 104.0 506.0 I 3 529.0 104.6 2 3 539.3 106.6 3 3 538.7 106.5 105.9 3036 I 3 3241 106.7 2 3 3153 103.8 3 3 3142 103.5 104.7 90 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 15. Precision of the method for the determination of AG2034 in mouse plasma Standards Theo. (ng/mL) Day N Precision Mean Calc. AG2034 (ns/mL) RSD (intra-day) (%) Pooled 1 (inter-di (%) 101.2 I 3 102.2 9.33 2 3 105.3 9.51 3 3 108.2 3.47 7.24 506.0 1 3 529.0 5.45 2 3 539.3 4.28 3 3 538.7 5.01 4.37 3036 L 3 3241 3.52 2 3 3153 1.97 3 3 3142 2.07 2.72 m.B«3. Pharmacokinetics The behavior of free AG2034 and two liposome delivery systems was then examined using a murine model. As seen in Figures 29, 30 and 31. these three delivery systems after i.v. bolus followed a two compartment model. In contrast to the rapid clearance seen for the free AG2034. about 50% of the administered AG2034 remained in circulation at 57 hours when given in a stealth liposome delivery system. Comparison of the two liposomal formulations indicated, as expected, that the conventional systems were cleared from circulation more rapidly than those containing PEG(2k)-DPPE. The half-lives for free AG2034, conventional liposomes and stealth liposomes were t ^ a = 0.2 hours and t^ P = 2.7 hours, tm<x = 1.6 hours and t^ P = 34.5 hours, and t ^ a = 9.7 hours and t^ P = 57.0 hours, 91 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. respectively. As expected, the free drug had a shorter half-life and the two liposomal delivery systems substantially prolonged the half-life for AG2034. Comparison of the two liposomal formulations indicated that the conventional system was cleared from circulation more rapidly than stealth liposomes containing PEG(2k)-DPPE. A brief summary of the kinetic parameters using the computer program of WinNonlin Professional Edition is given in Table 16. Table 16. Kinetic Parameters for free AG2034, conventional liposomes and stealth liposomes Half-live (hr) iis o t Jagg------------- Free AG2034 0.2 2.7 DSPC/CHOL 1.6 34.5 DSPC/CHOL/PEG(2k)-DPPE 9.7 57.0 1 0 0 .0 0 10.00 I ,0 0 e 3 0.10 ■ 0 . 0 1 24 48 0 72 96 120 144 168 Time (H ours) Figure 29. Plasma profiles of free AG2034 in mice. Each point represents the mean ± s.d. (n=5). 92 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 0 0 0 .0 0 100.00 ■ - [ I. 3 c o 10.00 2 e • o c o o 1.00 0.10 0 24 48 72 96 120 144 168 Tim e (H ours) Figure 30. Plasma profiles of AG2034 conventional liposomes. Each point represents the mean ± s.d. (n=5). 1000.00 100.00 - i f 3 C O 10.00 ■ 5 c • o e o o 1.00 0. 10 0 24 48 72 96 120 144 168 Tim e (H ours) Figure 31. Plasma profiles of AG2034 stealth liposomes. Each point represents the mean ± s.d. (n=5). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. m.B.4. Acute Toxicity Study The stealth liposomal formulation prepared for the acute toxicity study consisted of 25% encapsulated and 75% free AG2034 with the total AG2034 concentration of 15 mg/mL. The nonencapsulated drug was not separated because of lower encapsulation efficiency of AG2034 in the stealth liposomal delivery system. If the free drug was removed by using gel filtration chromatography that would have resulted in dilution of liposome-associated AG2034. Therefore, the large quantity of lipids was administered into mice which may lead to the severe toxicity from phospholipids. Administration of free AG2034 was found to be more toxic, resulting in mortality at earlier time points than seen for that of AG2034 encapsulated in the stealth liposome delivery system. At the end of study, the mortality for mice treated with free AG2034 and stealth liposomal delivery system was 25/30 and 14/30. respectively. Stealth liposome delivery system increases the probability of survival rate (p<0.05). When AG2034 was administered within stealth liposomes, its toxicity was significantly ameliorated. The loss of weight in mice treated with free AG2034 was most prominent at 10 days after drug treatment. At 30 mg/kg, free AG2034 caused a weight loss of about 26% at day 10 and an equivalent dose of AG2034 in stealth liposome delivery system resulted in about 11% weight loss. Results of acute toxicity study are presented in Table 17. 94 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 17. Toxicity of free AG2034 and stealth liposomes in C3H mice Treatment Mortality_______ % Weight loss Group N Day 12 Day 14 Day 21 Day 5 Day 10 Free AG2034 30 14/30 23/30 25/30 11% 26% Stealth liposomes 30 6/30 13/30 14/30 5% 11% 95 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IV. DISCUSSION Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IV.A. HPLC Analysis of AG2034 In Vitro The isocratic reverse phase HPLC method developed and validated for quantitation of AG2034 in vitro was sensitive and selective for separation of AG2034 from other impurities. It proved to be linear, precise and capable of accurately quantitating AG2034 in the concentration range of 8.85 (ig/mL to 443 pg/mL. IV.B. UV-Vis Spectrophotometric Analysis of Phospholipids In the Stewart assay (Stewart, 1980) with modification for phospholipids, the ability of phospholipids to form a complex with ammonium ferrothiocyanate in organic solution was utilized. The advantage of this method was that the presence of inorganic phosphate does not interfere with the assay. Therefore, this UV-Vis method for analysis of either DPPC or SDSPC alone or the combination of DSPC and PEG(2k)-DPPE with different ratios was shown to be linear, precise and accurate. IV.C. Preparation of AG2034 Solution The AG2034 pH-solubility profile was evaluated. The solubility of AG2034 substantially increased above pH 6. AG2034 is soluble in alkaline solutions due to ionization of two carboxylic groups of glutamate and formation of soluble salt. Wang et al. (1997) demonstrated that AG2034 solution was much less stable in strong alkaline solutions when they compared the stability of AG2034 in sodium phosphate, 97 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. sodium hydroxide and sodium bicarbonate solutions. Hence, 0.1 M sodium phosphate solution with mild alkaline characteristics (pH = 8) was chosen as a medium for preparing AG2034 disodium solution to prevent the instability of AG2034 due to oxidation and hydrolysis reactions. IV.D. Liposomal Delivery System One of the main objectives for the present study was to develop a stealth liposomal delivery system with maximum drug entrapment and stability. In order to achieve this goal, the process parameters of liposome formation such as vacuum, hydrating medium, hydration time, speed of rotation of the flask, method of size reduction and method of separation of unentrapped drug from liposomes were carefully selected based on the previous studies or optimized. A vacuum of 250 mm Hg was applied for drying the film at 40°C. A vacuum of 130 mm Hg was found to be inefficient for the complete removal of the solvent mixture, resulting in aggregation of the liposomes on hydration. A vacuum of 500 mm Hg resulted in the rapid evaporation of the solvent, leading to crystallization of the drug (Patel and Misra, 1999). AG2034 in 0.1 M sodium phosphate buffer with pH = 8.0 was used to hydrate the dry lipid film due to the high solubility of AG2034 in this medium. Patel and Misra (1999) observed that increasing the hydration time beyond 2 hours resulted in fragmentation of liposomes upon sonication due to the increased fragility of the liposomal membrane, whereas a lower hydration time led to a non-uniform shape and size of the liposomes. Hence, a hydration time of 2 hours was used for the 98 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. preparation of AG2034-carrying liposomes. Rapid rotation of the flask at 180 rpm was found to be adequate in increasing the surface area for evaporation to give thin, uniform and completely dry lipid films which resulted in uniformly sized multilamellar liposomes (Jain and Jain, 1994). Therefore, a speed of 180 rpm of the rotary flash evaporator was used for preparing stealth liposome formulations. The 15-pass process was used to extrude ML Vs through two stacked 200 nm polycarbonate filters for ULVs formulation. Gel filtration chromatography was used to separate the untrapped AG2034 from liposomal suspension with no difference in efficiency when compared to the ultracentrifugation method (Patel and Misra, 1999). As stated in the results section, the liposome-associated AG2034 peak and the unencapsulated AG2034 peak with different relative heights were present in all chromatograms. The identical elution volume obtained for all the liposome preparations suggested that the liposome size was similar for each lipid composition used. The presence of liposomes in the first peak was indicated by the turbidity of the solution and shown by the modified Stewart assay (Stewart. 1980). The presence of AG2034 in both peaks was shown by HPLC analysis of the corresponding fractions. From the results of the modified Stewart assay (Stewart. 1980) experiments, we could conclude that the second peak was free of phospholipids. 99 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IV.E. Development of a Stealth Liposome Delivery System IV.E.1. Effect of phospholipids and Cholesterol on the Encapsulation Efficiency The selection of ingredients and formulation compositions play an important role in developing a stealth liposomal delivery system with maximum drug encapsulated and stability in vitro and in vivo. Synthetic phospholipids such as DPPC and DSPC were used for development of a liposome delivery system due to the fact that the saturated phospholipids were more stable than lipids containing unsaturated fatty acids. Also, addition of cholesterol to phospholipid bilayers was known to cause changes in membrane molecular order and dynamics in both the fluid and gel phases (Davis, L993). Gregoriadis and Davis (1979) have shown that inclusion of cholesterol in the liposomal formulation remarkably improved the stability of the liposome delivery system both in vitro and in vivo. Also, Betageri (1993) and Ryan et al. (1983) have reported an increase in encapsulation of drug in MLVs and LUVs. respectively, with an increase in the concentration of cholesterol in the lipid bilayer. However, the present study showed only an increase in percent encapsulation efficiency when the DPPC/CHOL ratio was increased from 1:0 to 1:0.25. A decrease in percent encapsulation efficiency for DPPC/CHOL ratios of 1:0.5 and 1:1 were obtained. The same results were observed for formulations with DSPC/CHOL ratios of 1:0.25 and 1:0.5 as well. This phenomenon can be explained by the DSC thermograms. Cholesterol is known to cause a characteristic shift to higher temperature for Tm of phospholipids. However, the addition of cholesterol to 100 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. formulations with either DPPC/CHOL ratios of 1:0.5 and 1:1 or DSPC/CHOL ratio of 1:0.5 did not give any phase transition within the range of temperatures investigated. This indicated that the original bilayer state was converted to a mixed micellar state. Particle size analysis indicated that the formulations with ratio of 1:0.25 either for DPPC/CHOL or DSPC/CHOL had less change in particle size distribution than those formulations with ratios of 1:0.5 and 1:1. Based on these findings, the formulations with the ratio of 1:0.25 for phosphoIipid/CHOL have higher percent encapsulation efficiency due to better stability and less leakage of AG2034 from the liposome delivery systems. Upon further analysis of the particle size profile, the formulation with ratio of 1:0.25 for DSPC/CHOL showed better stability than that of 1:0.25 for DPPC/CHOL. Therefore, the mixture of DSPC and cholesterol with molar ratio of 1:0.25 was selected to be used for the development of a long circulating liposome delivery system. [V.E.2. Influence of PEG(2k)-DPPE Loading on Encapsulation Efficiency LUVs have the advantage of entrapping a large volume of drug solution in comparison with small unilamellar vesicles. However, it is well known that larger liposomes are rapidly removed from the bloodstream following i.v. injection, primarily by uptake by Kupffer cells of the liver and macrophages of the spleen. Thus, it is important to develop LUVs which resist uptake by the RES, and thus show extended circulation half-lives in vivo, for the sustained release of entrapped drugs. Moribe et al. (1998) have found that the presence of polyethylene glycol 101 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. derivatives, which were added to acquire the prolonged circulation characteristics, also significantly increased the encapsulation efficiency of drug into liposomes. In the present study, the two different loadings of PEG(2k)-DPPE were incorporated into the formulation with DSPC/CHOL ratio of 1:0.25. The results showed the formulation with 5% molar ratio of PEG(2k)-DPPE increased the percent encapsulation efficiency of AG2034 from 5% to 15% compared to the formulation without PEG(2k)-DPPE. The formulation with 10% molar ratio of PEG(2k)-DPPE only increased the percent encapsulation efficiency of AG2034 from 5% to 7% compared to the formulation without PEG(2k)-DPPE. Bedu-Addo et ai. (1996) reported that mixtures of PEG-phosphoIipid conjugates and phosphatidylcholine existed in three different physical states: a lamellar phase with components exhibiting some miscibility, a lamellar phase with components phase separated, and mixed micelles. Beyond 7% molar ratio of PEG( 1000-3000)-DPPE. a strong tendency towards mixed micelles formation was observed. The DSC thermograms for the present study showed that the phase transition temperature shifted from 53°C for the formulation with 5% molar ratio of PEG(2k)-DPPE to 52°C for formulation with 10% molar ratio of PEG(2k)-DPPE. These results indicated the gradual phase transition from the bilayer state for 5% molar ratio PEG(2k)-DPPE loading to mixed micelles for 10% molar ratio loading in the delivery system. The particle size distribution indicated that the formulation with 5% molar ratio PEG(2k)-DPPE has better stability than that with 10% molar ratio PEG(2k)-DPPE due to less particle size change in the delivery system with 5% molar ratio PEG(2k)-DPPE. This also 102 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. suggested that the Tm shift to a lower temperature indicated bilayer solubilization to a mixed micellar state. This study demonstrated that the most stable and longest circulating liposomes formulations consist of low concentrations, < 5% molar ratio, of short chain PEG-DPPE. IV.E.3. Impact of Manufacturing Method of Liposomes on Encapsulation Efficiency Schwendener et ai. (1994) has reported that mitoxantrone was successfully loaded into the aqueous inner compartment of preformed liposomes using a gradient of 10 pH units. However, the percent encapsulation efficiency of AG2034 was negligible when the formulation was prepared by using the remote loading method. This result indicated that a pH gradient of 6 units was not strong enough to induce AG2034 from outside empty liposomes to inner compartments. Due to the low solubility of AG2034 at pH below 6.7, the remote loading method was not practical for encapsulating AG2034 into the liposomes. IV.E.4. Effect of Phospholipid Loading on Encapsulation Efficiency The issue of dependence of drug entrapment on the phospholipids concentration has been investigated by many authors (Morgan and Williams, 1980: Pidgeon et ai., 1987; Selzer et al., 1988) who have shown that increasing the lipid concentration increases entrapment. The formation of the classic, tightly packed onion skin-like arrangement of concentric lipid bilayers in MLVs on hydration of 103 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. thin lipid films has been well documented (Mayer et al., 1985; Szoka and Papahadjopoulos, 1980b). Repeated freezing and thawing of these MLVs disrupt this packing and new structures are formed that have a much larger interlamellar space and, hence, higher encapsulation efficiency (Mayer et al., 1985). The present study showed that formulations with increased phospholipid loading gave higher percent encapsulation efficiency. This confirms that the tight onion skin-like structures are formed in MLVs only at higher lipid concentrations. The results of this study also indicated that the percent encapsulation efficiency was significantly increased up to 21% when increasing the lipid to 60 jimole/mL followed by a slight increase when further increasing the lipid to 80 |imole/mL. Mayer et al. (1985) have reported similar results of encapsulation efficiency increases when increasing the lipid to 100 mg/mL lipid and further increases in the lipid concentration do not increase the encapsulation efficiency. IV.E.5. Effect of AG2034 Loading on Encapsulation Efficiency The results of the effect of AG2034 loading on encapsulation efficiency showed minimal differences in percent encapsulation efficiency when AG2034 loading increased from 6 |imoIe/mL to 58 pmole/mL. This finding can be shown by the size distribution profiles. Figure 19 showed that the liposomal particles were consistent in size among these long circulating liposome delivery systems. 104 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IV.F. Leakage Study The release of drug from the liposomal vesicles was found to be dependent on both the liposomal composition and the amount of added cholesterol. Al-Angary et al. (1996) has reported that the increase in the length of the lipid acyl chain was accompanied by a decrease in the release rate of drug from the liposomes. Also, the increase of cholesterol in the bilayer composition would lead to a decrease in the leakage rate of drug. As expected, the leakage rate increased with increasing temperature. The results of the present study found that this optimized liposome delivery system was quite stable when stored at refrigerated condition (2-8°C). This finding might result from the incorporation of cholesterol into the liposomes that led to a strong reduction in the permeability of the liposome system and a more ordered structural bilayer. IV.G. In Vitro Release Study Scherphof et al. (1978) has reported that the instability of liposomes in plasma appeared to be the result of the transfer of bilayer lipids to albumin and high- density lipoproteins (HDLs). Additionally, some of the protein was transferred from the lipoprotein to the liposomes. Ferdous et al. (1996) reported that the PEG-DSPE in the liposomal formulations lowered the opsonization of serum proteins and degradation of liposomes in serum. To evaluate the ability of the optimized stealth liposome delivery system to withstand the disruptive effects of HDLs. the liposomes were equilibrated with human serum. The results of the present study indicated that 105 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. at the end of 24 hours, no more than 30% of the initial amount of encapsulated AG2034 had leaked and there was also no significant change in the particle size of the stealth liposomes in the serum. There were indications that the optimum stealth liposome delivery system would be stable in vivo for pharmacokinetic analysis and acute toxicity study. IV.H. Efficacy Study of Liposome-Associated AG2034 Treated with Human Serum In the present study, there was no attempt to evaluate the anti-cancer efficacy for this stealth liposome delivery system in vivo. Therefore, the efficacy study of this delivery system treated with human serum was conducted on the A549 cancer cells. The results demonstrated that this delivery system incubated with human serum did not reduce inhibition of cell growth for A549 cells. This finding indicated that this optimum stealth liposome delivery system should have anti-cancer activity in vivo. IVJ. HPLC Analysis of AG2034 In Vivo The gradient reverse phase high performance liquid chromatography method for the quantitation of AG2034 in mouse plasma was developed and validated. AG2037, the analog of AG2034, was used as an internal standard because it has a structure comparable to AG2034 and its retention time was similar to that of AG2034. The analytical wavelength for AG2034 detection was set at 226 nm to minimize the large unknown peaks which eluted early in the run and maximize the 106 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. sensitivity of AG2034 in the chromatograms of mouse plasma samples. No interference from plasma constituents for this assay system indicated that the HPLC method was selective for AG2034. The results of the validation studies demonstrated that the method was linear, precise and capable of accurately quantitating AG2034 in the concentration range of 101.2 ng/mL to 3036 ng/mL. IVJ. Pharmacokinetic Analysis Morgan et al. (1985) and Ogihara et al. (1986) have demonstrated that liposomes can selectively accumulate at sites of infection, inflammation and neoplastic disease providing both a rationale for their use as drug carriers and an explanation for any observed changes in drug toxicity or efficacy. The therapeutic index of the highly cytotoxic antineoplastic agents is narrow, with severe toxic side effects occurring within the dose range required to mediate effective therapy. A variety of experimental strategies have been developed to improve the therapeutic index and reduce the toxicity of anticancer drugs. In this regard, the stealth liposome delivery systems have been optimized with respect to maximizing the amount of drug incorporated into liposomes, increasing drug retention characteristics (Mayer et al., 1989; Boman et al., 1994) and prolonging the circulation time of the drug- associated carrier (Gabizon, 1992; Wu et al., 1993). Gabizon et al. (1982), Olson et al. (1982), Mayer et al. (1989) and Schwendener et al. (1991) have reported the results from animal studies showing that the toxicities of doxorubicin, vincristine and mitoxantrone can be significantly reduced by administration in a liposome delivery 107 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. system. Batist et al. (1992) and Northfield et al. (1993) have documented similar benefits in human clinical trials, most remarkably a noted reduction in cardiotoxicity for liposomal doxorubicin. Drugs that are liposome bound have to be released from their vehicle to distribute to either plasma proteins, blood cells or tissues. This complicates the comparison of the pharmacokinetic data in whole blood after the administration of the free AG2034, conventional liposome delivery system and long circulating liposome delivery system. Because of analytical difficulties, it was not possible to separate free AG2034 from the liposome-associated drug in the plasma samples. In the present study, the method of residuals was used to calculate the various pharmacokinetic parameters. The results showed that in contrast to the rapid blood clearance observed for free AG2034. the liposomal delivery systems extended AG2034 in blood residency time. In the case of sterically stabilized liposomes containing a PEG-lipid conjugate, 50% of AG2034 was present in the blood 57.0 hours after i.v. injection. In agreement with a previous report from Chang et al. (1997), longer plasma half-lives were observed for sterically stabilized liposomes than for conventional systems. Allen (1994) reported that enhanced circulation time in vivo may result in greater tumor sequestration of liposomal drugs. This does not necessarily result in a corresponding increase in drug efficacy. Mayer et al. (1994) brought up the fact that several factors influence the activity of liposomal drugs in vivo. Following localization at the tumor site, for example, it appears unlikely that the drug carrier is 108 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. directly internalized by tumor cells. Instead it is believed that drug release occurs via passive diffusion across the liposomal bilayer or as the result of carrier processing by macrophages. Free drug would then be taken up into tumor cells via normal transport mechanisms. This suggests that the free drug release from the carrier may represent the limiting factor in determining drug efficacy. Thus, it is not sufficient to develop drug carriers that accumulate at the disease site in high levels; one must also engineer appropriate drug release rates to improve the therapeutic activity. IV.K. Acute Toxicity The stealth liposome delivery system consisting of 25% encapsulated and 75% free AG2034 also significantly reduced AG2034 toxicity (p < 0.05). In a murine model, for example, only 15% survivors were seen in the animal groups treated with free drug, whereas at the dose of 30 mg/kg of liposomal AG2034 survival rates of 50% were observed. This result indicated controlled release of AG2034 from the stealth liposome carriers may reduce the first burst effect in the circulation system. Similar results were observed and reported by Chang et al. (1997). IV.L. Mechanism The exact mechanisms that govern the ways that liposomes are both detected as foreign particles in vivo and cleared from the blood by phagocytic cells of the reticuloendothelial system are presently unsolved (Senior, 1987). Undoubtedly, the clearance rate of intravenously injected liposomes from the blood by phagocytic cells 109 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. of the liver and spleen and their eventual intracellular degradation is dependent upon the vesicular lipid composition and cholesterol content (Patel et al., 1983; Dave and Patel, 1986; Derksen et al., 1988; Roerdink et al., 1989; Patel, 1992; Semple et al., 1996). Furthermore, cholesterol-poor egg PC vesicles (vesicles with 5% mol cholesterol) are cleared faster than their cholesterol-rich counterparts (vesicles with 40% mol cholesterol). Cholesterol-free and cholesterol-poor egg PC vesicles are predominantly localized to the liver; only a small proportion of the injected dose is deposited in other organs of the reticuloendothelial system. For cholesterol-rich vesicles, hepatic sequestration is rather poor when compared to cholesterol-free and cholesterol-poor vesicles; such vesicles tend to localize more effectively in the spleen and to some extent in bone marrow (Patel et al.. 1983; Senior et al.. 1985). Cholesterol can tighten the packing of phospholipids with low transition temperature, thus solidifying the liposomal membrane (Senior et al., 1985; Gregoriadis. 1994). Accordingly, such changes can diminish high density lipoprotein attack and complete liposomes disintegration (Scherphof et al., 1978; Tall et al.. 1993; Gregoriadis, 1994), and minimize susceptibility of the lipid interface to perturbation or penetration, either by opsonizing plasma proteins (e.g. components of complement system, pentraxins) or by Kupffer cell surface proteins serving to establish the initial contact between liposomes and cells. Because of prolonged residence in blood, cholesterol-rich liposomes are believed to encounter the spleen more readily than cholesterol-free and cholesterol-poor vesicles and, hence, are filtered (Moghimi, 1995). A recent study also showed that an increase in the membrane cholesterol 110 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. content in DSPC vesicles was accompanied by a marked decrease in the total amount of plasma protein which bound to liposomes (Semple et al., 1996). It is also interesting to note that cholesterol-rich vesicles are substantially more resistant toward intracellular degradation in Kupffer cells than cholesterol-poor and cholesterol-free liposomes (Roerdink et al., 1989). The inclusion of PEG-PE conjugates in liposomes results in drastically prolonged circulation time (Klibanov et al., 1990; Papahadjopoulos et al., 1991). A number of mechanisms have been proposed to explain the evasion of the MPS by these novel liposomes, and the resulting prolonged circulation. Steric stabilization of colloids, which has been described for inorganic particles (Napper, 1983), is probably the most plausible hypothesis to date (Papahadjopoulos et al., 1991). Steric repulsion resulting from polymer coating leads to reduced particle-particle interaction, therefore causing an inhibition of the adsorption of various opsonins such as complement components (Chonn et al., 1991) onto the liposome surface resulting in increased biological stability. Both the flexibility of short PEG chains forming a dense “polymeric cloud” (Torchilin and Papisov, 1994), and the surface charge and hydrophilicity of PEG may play an important role in imparting this long circulating effect (Gabizon and Papahadjopoulos, 1992). Evidently, all the above described mechanisms are based upon a hindered release of AG2034 from liposomes. This slower and controlled release of AG2034 from liposomes resulted in a reduction of toxicity. I l l Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. V. CONCLUSIONS 112 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The conclusions of this project are: (1) AG2034 was successfully incorporated into a stealth liposome delivery system- The most important factor for increasing encapsulated AG2034 in stealth liposome delivery system was the addition of PEG(2k)-DPPE. (2) The optimum stealth liposome delivery system was composed of molar ratios of 76% DSPC : 19% CHOL : 5% PEG(2k)-DPPE and prepared using the extrusion method. (3) The characterization studies demonstrated that the optimized stealth liposome delivery system has reasonable stability to reduce leakage of entrapped AG2034 on storage as well as in biological environments. (4) In vitro antiproliferative activity indicated that the stealth AG2034 liposome delivery system had similar inhibition for A549 human lung cancer cell growth compared with free AG2034. The results also demonstrated that the control and empty liposomes did not cause any inhibition in cancer cell growth. (5) The pharmacokinetic study showed that the free AG2034 and the conventional liposome delivery system were cleared from circulation more rapidly than the stealth liposome delivery system. In the case of the sterically stabilized liposome delivery system containing a PEG-lipid conjugate, 50% of AG2034 was present in circulation 57 hours after i.v. injection. (6) Administration of free AG2034 was found to be more toxic, resulting in mortality at earlier time points than that found for AG2034 encapsulated in the stealth liposome delivery system. The acute toxicity study demonstrated that the 113 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. stealth liposome delivery system significantly reduced the in vivo toxicity when compared to free AG2034 114 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VL SIGNIFICANCE OF WORK AND FUTURE PROSPECTS 115 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (1) This was the first detailed and systematic study to evaluate the effects of various parameters on the encapsulation efficiency of AG2034 in a stealth liposome delivery system. (2) This study demonstrated the feasibility of a stealth liposome delivery system for AG2034 which has been optimized with respect to maximizing the amount of AG2034 incorporated into liposomes, increasing AG2034 retention characteristics and prolonging the circulation time of the AG2034-associated carrier. (3) This optimized stealth liposome delivery system showed similar in vitro antiproliferative activity for A549 lung cancer cells when compared with free AG2034. (4) This study showed the capability of the sterically stabilized delivery system in altering pharmacokinetic profiles and slowing release of AG2034 from the drug carrier to reduce in vivo toxicity. Prior to the development and availability of a therapeutic stealth liposome delivery system, the following additional studies must be conducted. ( 1) Long term stability of the optimized stealth liposome delivery system must be evaluated. (2) In vivo efficacy of a stealth liposome delivery system against a panel of murine tumors and human xenografts must be determined. (3) The in vivo toxicity of empty sterically stabilized liposome carriers must be investigated. 116 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (4) A more relevant animal species should be used for further evaluation of toxicity. 117 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VII. BIBLIOGRAPHY Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Al-Angary, A.A., Al-Meshal, M.A., Bayomi, M.A. and Khidr, S.H.: Evaluation of Liposomal Formulations Containing the Antimalarial Agent, Arteether, International J. of Pharmaceutics 128: 163-168, 1996. 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Cost -efficient design of main cohort and calibration studies where one or more exposure variables are measured with error

Asset Metadata

Creator Tsai, Wen-Chin (author)

Core Title Design of a stealth liposome delivery system for a novel glycinamide ribonucleotide formyltransferase inhibitor

School Graduate School

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Health Sciences, Pharmacy,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Koda, Robert T. (committee chair), [illegible] (committee member), Azen, Stanley (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-213339

Unique identifier UC11335100

Identifier 3065858.pdf (filename),usctheses-c16-213339 (legacy record id)

Legacy Identifier 3065858.pdf

Dmrecord 213339

Document Type Dissertation

Rights Tsai, Wen-Chin

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA