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«Date:_ Approved: _ Lori A. Setton, Supervisor _ Ashutosh Chilkoti _ Stephen L. Craig _ Virginia B. Kraus _ Fan Yuan Dissertation submitted in partial ...»

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Development of Depot Forming Elastin-Like Polypeptide-Curcumin Drug Conjugates

for Sustained Drug Delivery to Treat Neuroinflammatory Pathologies

by

Steven Michael Sinclair

Department of Biomedical Engineering

Duke University

Date:_______________________

Approved:

___________________________

Lori A. Setton, Supervisor

___________________________

Ashutosh Chilkoti

___________________________

Stephen L. Craig

___________________________

Virginia B. Kraus ___________________________

Fan Yuan Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biomedical Engineering in the Graduate School of Duke University 2013 i v

ABSTRACT

Development of Depot Forming Elastin-Like Polypeptide-Curcumin Drug Conjugates for Sustained Drug Delivery to Treat Neuroinflammatory Pathologies by Steven Michael Sinclair Department of Biomedical Engineering Duke University Date:_______________________

Approved:

___________________________

Lori A. Setton, Supervisor ___________________________

Ashutosh Chilkoti ___________________________

Stephen L. Craig ___________________________

Virginia B. Kraus ___________________________

Fan Yuan An

Abstract

of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biomedical Engineering in the Graduate School of Duke University 2013 Copyright by Steven Michael Sinclair 2013 Abstract Neuroinflammation associated with lumbar radiculopathy and peripheral nerve injury is characterized by locally increased levels of the pro-inflammatory cytokine tumor necrosis factor alpha (TNF Systemic administration of TNF antagonists for radiculopathy in the clinic has shown mixed results, and there is growing interest in local delivery of anti-inflammatory drugs to treat this pathology, as well as similar inflammatory events of peripheral nerve injury. Curcumin, a known antagonist of TNF in multiple cell types and tissues, was chemically modified and conjugated to a thermally responsive elastin-like polypeptide (ELP) to create an injectable depot for sustained, local delivery of curcumin to treat neuroinflammation.

ELPs are biopolymers capable of thermally-triggered in situ depot formation that have been successfully employed as drug carriers and biomaterials in several applications. For this work, a library of ELP-curcumin conjugates were synthesized and characterized. One lead conjugate was shown to display high drug loading, rapidly release curcumin in vitro via degradable carbamate bonds, and retain in vitro bioactivity against TNF and NF-B with near-equivalent potency compared to free curcumin.

When injected into the perineural space via intramuscular (i.m.) injection proximal to the sciatic nerve in mice, ELP-curcumin conjugates underwent a thermally triggered

–  –  –

curcumin over 4 days post-injection.

The results of this dissertation support the use of ELP as a drug carrier for local perineural drug delivery, and the strategy presented here for drug conjugate development and local injection of depot-forming ELP-curcumin conjugates represents a novel means of providing sustained treatment of neuroinflammation and pain associated with radiculopathy and peripheral nerve injury.

–  –  –

I would like to dedicate this work to my family who have all encouraged me and supported me on this journey. I want to thank my parents for supporting me throughout my many years of schooling and helping me to make the decision to become an engineer. I want to thank my mother for sharing her passion for medicine and patient care with me, which is still the root of my own passion for biomedical research. I want to thank my father for passing on his common sense, personable nature, and project manager mentality, which have helped me succeed and focus throughout my dissertation work.

Finally, I want to thank my wife, Jill, for her constant support, willingness to listen to my latest research report, and understanding of my hectic schedule. Not only has she been an extraordinary friend and partner who helps me stay the course, she has worked tirelessly these last 13 months to raise our happy, healthy baby girl. I could not have succeeded without her support, care, and love. Hopefully, Evelyn reads this someday and realizes how important her mother was to its completion.

–  –  –

Abstract

List of Tables

List of Figures

List of Abbreviations

Acknowledgements

1. Introduction

1.1 Neuroinflammatory pathologies

1.1.1 Disc herniation induced radiculopathy

1.1.1.1 Structure of the IVD and DRG

1.1.1.2 Pathophysiology of disc herniation induced radiculopathy

1.1.1.3 Inflammatory and immunogenic properties of the NP

1.1.2 Peripheral nerve injury

1.2 Role of TNF in neuroinflammation

1.3 Clinical interest in TNF antagonists for radiculopathy

1.4 Preclinical investigations of local antagonism of neuroinflammation for radiculopathy





1.5 Curcumin

1.5.1 Background and anti-inflammatory properties

1.5.2 Curcumin in the clinic

1.5.3 Curcumin and neuroinflammation

1.5.4 Strategies for delivering curcumin

–  –  –

1.6.1 Thermally responsive depot-forming behavior

1.6.2 ELP as a Drug Carrier

1.6.2.1 ELP fusion proteins

1.6.2.2 Small molecule coupling to ELP

1.7 Central hypothesis and aims

2. Curcumin conjugate synthesis and characterization

2.1 Introduction

2.2 Methods

2.2.1 Materials and Reagents

2.2.2 Chemical synthesis of Monofunctional Curcumin Carbamate (MCC).............. 40 2.2.3 UV-Vis characterization of curcumin and MCC

2.2.4 ELP expression, purification, and amine blocking

2.2.5 Conjugate synthesis and purification

2.2.6 Drug-to-carrier ratio characterization

2.2.7 Thermal characterization and particle formation of conjugates

2.3 Results

2.3.1 Synthesis of MCC

2.3.2 Absorbance properties of curcumin and MCC

2.3.3 Blocking of ELP N-terminal amines

2.3.4 Characterization of ELP-MCC drug-to-carrier ratios

2.3.5 Thermal transition properties of ELP-MCC conjugates

–  –  –

2.5 Conclusion

3. In vitro characterization of MCC80 release kinetics and bioactivity

3.1 Introduction

3.2 Methods

3.2.1 Quantitation of curcumin and MCC80 with HPLC

3.2.2 In vitro drug release

3.2.3 Release kinetics analysis and modeling

3.2.4 L929 cytoprotection assay of anti-TNF bioactivity

3.2.5 NF-B p65 phosphorylation assays

3.3 Results

3.3.1 Characterization of curcumin and MCC80 with HPLC

3.3.2 Anti-TNF L929 cytoprotection with curcumin and MCC80

3.3.3 Inhibition of NF-B p65 phosphorylation with curcumin and MCC80............ 87

3.4 Discussion

3.5 Conclusion

4. In vivo pharmacokinetics of locally delivered curcumin and MCC80

4.1 Introduction

4.2 Background

4.3 Methods

4.3.1 Local perineural delivery of curcumin and MCC80 to the sciatic nerve in mice

–  –  –

4.3 Results

4.3.1 Fluorescence assay optimization

4.3.2 Fluorescence quantitation of curcumin in mouse plasma

4.4 Discussion

4.5 Conclusion

5. In vivo clearance kinetics of locally delivered curcumin and MCC80

5.1 Introduction

5.2 Background

5.3 Methods

5.3.1 Local perineural delivery of curcumin and MCC80

5.3.2 Tissue solubilization and preparation

5.3.3 Fluorescence detection assay and standards for tissue samples

5.3.4 Quantitation of mass ratio of free and ELP-bound curcumin in tissue samples

5.3.5 Statistical analysis of tissue samples

5.4 Results

5.5 Discussion

5.6 Conclusion

6. Conclusions and future directions

References

Biography

x List of Tables Table 1: Moleculer inflammatory mediators in NP tissue and known activity of curcumin against each mediator

Table 2: Conjugation and entrapment strategies for curcumin

Table 3: Summary of ELP and ELP-MCC conjugate characterization

Table 4: Hydrodynamic radii of AcELP and MCC80 as measured by DynaProTM DLS... 54 Table 5: List of HPLC elution times for curcumin, MCC, and MCC80.

Table 6: Preclinical pharmacokinetic studies of curcumin

Table 7: Fluorescent quantum yield of curcumin in various organic solvents................ 102 Table 8: Naïve mouse plasma autofluorescence and calculated LLD.

Table 9: Final fluorescence assay parameters to detect curcumin in mouse plasma....... 112 Table 10: Summary of PK values for local delivery of curcumin and MCC80................. 117 Table 11: Local clearance rates of soluble and depot-forming ELPs

Table 12: Tissue clearance rates for curcumin and MCC80

–  –  –

Figure 2: Peripheral nerve injury inflammatory responses.

Figure 3: Role of glial cell NF-B activation in painful hypersensitivities following CCI.

Figure 4: Local application of TNF to the DRG reduces nerve conduction velocities...... 15 Figure 5: Changes in animal gait, weight bearing, and mechanical allodynia following NP-induced radiculopathy

Figure 6: Clinical trial results for epidural delivery of etanercept to treat radiculopathy.

Figure 7: Inflammatory responses of rat DRG explants to TNF.

Figure 8: Chemical structure of curcumin

Figure 9: Illustration of conjugated systems like curcumin with overlapping π-bonds... 23 Figure 10: Canonical NF-B signaling pathway

Figure 11: Antihyperalgesic effect of curcumin in a TRPV1-mediated model of pawinflammation

Figure 12: Efficacy of twice daily oral delivery of curcumin in the CCI model................. 28 Figure 13: Synthesis and structure of PEGylated curcumin.

Figure 14: Molecular representation of inverse phase transition of ELPs.

Figure 15: Design of ELP-doxorubicin micelle-forming conjugates

Figure 16: Graphical summary of ELP-curcumin depot development and perineural delivery.

Figure 17: Graphical summary of synthesis and characterization of ELP-Curcumin conjugates.

–  –  –

Figure 19: Chemical coupling schematic of ELP-MCC conjugates

Figure 20: Absorbance spectrum of curcumin and MCC in UV-Vis buffer

Figure 21: Confirmation of ELP amine blocking with sulfo-NHS-acetate

Figure 22: Thermal transition properties of MCC80 and AcELPL=80..

Figure 23: Particle diameters of MCC80 conjugates measured with a Zetasizer NanoZS..

Figure 24: Screening criteria for conjugate library.

Figure 25: Graphical summary of in vitro characterization of MCC80 release kinetics and bioactivity.

Figure 26: MCC80 carbamate cleavage mechanisms

Figure 27: Sustained release of curcumin from PEG-poly(curcumin) hydrogels.............. 69 Figure 28: HPLC standard curves for curcumin and MCC80

Figure 29: HPLC chromatograms of curcumin and MCC

Figure 30: HPLC chromatograms of MCC80 release study at early time points............... 82 Figure 31: HPLC chromatograms of MCC80 release study at late time points................. 83 Figure 32: Validation of stabilizing in vitro release buffer with HPLC

Figure 33: In vitro release kinetics of curcumin from MCC80..

Figure 34: Dose-dependent anti-TNF bioactivity of curcumin, MCC, and MCC80 in the L929 cytoprotection assay.

Figure 35: Kinetics of NF-B p65 phosphorylation in U937 and S16 cells.

Figure 36: Attenuation of TNF-induced NF-B p65 phosphorylation in U937 monocytes

xiii Figure 37: Attenuation of TNF-induced NF-B p65 phosphorylation in S16 Schwann cells.

Figure 38: Graphical summary of studies of in vivo pharmacokinetics and clearance kinetics for MCC80 depots

Figure 39: Cartoon of MCC80 depot and curcumin kinetics following local injection... 104 Figure 40: In vivo PK and clearance rates experimental design

Figure 41: Absorbance and fluorescence spectra of curcumin and naïve plasma........... 110 Figure 42: Standard curves of curcumin in acetonitrile with varying well volumes and flash numbers.

Figure 43: Final fluorescence assay standard curve for curcumin quantitation in plasma

Figure 44: Pharmacokinetics of curcumin and MCC80 following perineural placement

Figure 45: Pharmacokinetics of detectable levels of curcumin and MCC80 following perineural placement

Figure 46: Plasma pharmacokinetics of curcumin and MCC80 delivered via i.m injection.

Figure 47: Fluorescent tomography of NIR-dye-labeled ELP depots

Figure 48: Images of sciatic nerve exposure and perineural drug delivery

Figure 49: Clearance kinetics of curcumin and MCC80 depots from the perineural space.

Figure 50: Clearance kinetics of curcumin and MCC80 depots following i.m. injection proximal to the sciatic nerve

Figure 51: Standard curves of curcumin and MCC80 in tissue buffer with and without muscle buffer

Figure 52: Images of curcumin and MCC80 localized to the nerve 2 h after i.m. injection.

–  –  –

CCI chronic constriction injury CCL2 chemokine ligand 2 (a.k.a. MCP-1) CD68 cluster of differentiation 68 CGRP calcitonin gene-related peptide

–  –  –

HSD honestly significant difference HOBt hydroxybenzyltriazole HPLC high-performance liquid chromatography Iba1 ionized calcium-binding adapter molecule 1 IC50 half-maximal inhibitory concentration ICAM-1 intercellular adhesion molecule 1

–  –  –



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