Small-Scale Physicochemical and Biophysical Characterization to Enable Peptide Delivery in Discovery

Transcription

1 Small-Scale Physicochemical and Biophysical Characterization to Enable Peptide Delivery in Discovery Candice Alleyne, Andrew Leithead, and Ellen Minnihan Senior Scientists, Discovery Pharmaceutical Sciences Merck & Co., Inc., Kenilworth, NJ, USA

2 Outline What makes peptides promising drug candidates? Challenges of peptide delivery Peptide characterization tool kit Case studies Solubility Aggregation Fibrillation Summary 2

3 Why the Move to More Peptide Drugs? Involved in various physiological and pathological processes Play important roles in regulating cells process Low tissue accumulation High potency; high selectivity Potential for fewer side effects lower toxicity and immunogenicity Small molecule Peptide Protein, mabs Broad range of targets May provide the best of small molecules and biologic approaches Craik DJ, et al. Chem Biol Drug Des. 2013;81:

4 General Challenges to Peptide Delivery Challenging Peptide Properties Molecular Weight Solubility Permeability Chemical Stability Physical Stability Biophysical Stability Metabolic Stability 500-5,000 Daltons (2-50 amino acid residues) Variable, dependent on amino acid residues, secondary structure, ph, and pi (isoelectric point) Variable, but generally poor for linear peptides Sensitive to hydrolysis, oxidation, deamidation Solution state or lyophilized solid stability required Adsorption to solid surfaces Risk of aggregation, fibrillation, denaturation Secondary and tertiary structure can affect activity Sensitive to enzymatic degradation (eg, peptidases) Short plasma half-life Endotoxin and bioburden How can we assess these liabilities earlier in discovery with limited material: ie, <5 mg? 4

5 Peptide Characterization Tool Kit Purpose Characterization Technique Output Solubility Reversed-Phase Ultra Performance Liquid Chromatography (RP-UPLC) Conventionally used to evaluate solubility in biorelevant media and formulation vehicles Circular Dichroism (CD) alpha-helix beta-sheet Secondary Structure Changes Particle Size Fourier Transform Infrared (FTIR) Diffusion Ordered Spectroscopy (DOSY-NMR) Fluorescence Size Exclusion Chromatography (SEC) Transmission Electron Microscopy (Cryo-TEM) Dynamic Light Scattering (DLS) EPIC Fibrils and alpha-helix beta-sheet Diffusion coefficient of various species in the formulation Assay used to monitor fibrillation in the presence of dye Ideally suited to monitor irreversible aggregation Particle morphology Particle size distribution (PSD) in sub-micron range Critical Micelle Concentration CMC Analytical Ultracentrifugation (AUC) Separation of sub-visible species 5

6 Case Study 1: Small-scale Solubility Assessment of a Peptide/Small Molecule Conjugate The Compound: Highly charged peptide (pi ~9) Very hydrophobic small molecule payload Biochemically conjugated via stable linker Total MW ~4k Da The Challenge: Limited material: 1 mg of compound available Requirements: 0.1 mg/ml solubility in a very mild vehicle to enable in vivo preclinical studies for immunology Peptide freely soluble in PBS, ph 7.5 (>0.5 mg/ml) peptide conjugate (<0.005 mg/ml) The Strategy: Explore the effect of ph, buffer composition, salt, and surfactant on the solubility of the peptide conjugate 18 conditions x 80 μl sample/condition x 0.5 mg/ml max target solubility 0.72 mg total compound 6

7 Case Study 1: Small-scale Solubility Assessment of a Peptide/Small Molecule Conjugate (continued) The Results: Conjugate solubility strongly dependent on buffer composition and ionic strength ph sensitivity was less pronounced Addition of surfactant gave solubility boost + NaCl Solubility (mg/ml) Buffer Buffer + NaCl Buffer + Tw80 Water ph Water ACN Citrate ph 4.4 <LOQ <LOQ <LOQ Acetate ph <LOQ 0.5 Succinate ph 5.1 <LOQ <LOQ <LOQ MES ph <LOQ 0.18 Water ph 2 Imidazole ph <LOQ 0.5 Hepes ph <LOQ 0.5 RP-UPLC solubility measurement + Tween 80 HEPES ph 6.6 Imidazole ph 6.2 MES ph 5.6 Succinate ph 5.1 Acetate ph 4.9 Citrate ph 4.4 Water: ACN Ability to generate a uniformly disperse suspension of the peptide allowed for low-volume liquid handling Scouting broad formulation conditions revealed strong dependence on buffer composition and ionic strength Use of reverse-phase UPLC allowed for rapid, quantitative detection on sub-microgram peptide samples 7

8 Case Study 2: Aggregation Assessment via SEC Stage: Discovery Objective: Monitor stability of a peptide in ph 7 buffer at 40 C for 4 weeks The Challenge: Limited amount of material available Can aggregates of the peptide be detected? 1 week The Strategy: 2 weeks Physical stability via SEC SEC method SEC allows for separation as a function of size 3 weeks Good qualitative using porous first stationary pass, material phase and sparing an isocratic approach to detect and quantify soluble molecular aggregates mobile phase Blank For 0.5 mg/ml sample concentration, 2 µl When coupled with MALS, can provide greater details on molecular weight; however, requires injection volume: only 1 µg/injection required more material ( µg/sample) Insoluble aggregates/fibrils not detected via this method due to column loading limitation Condition Parent 4.3 % Area Agg % Area Initial Aggregation SEC Deg % Area Deg % Area Deg % Area Deg % Area 40 C Initial C 1 wk C 2 wk C 3 wk C 4 wk

9 Absorbance Case Study 3 Fibrillation Assessment via UV and Fluorescence Stage: Early Discovery Objective: Develop method of detecting fibrillation on small scale with minimum material requirements The Challenge: Current method Thio T is quite sensitive and quantitative Requires 3-5 ml of sample/time 3 mg/ml up to 15 mg Minimization of assay to plated version resulted in loss in sensitivity The Strategy: Develop 96-well plate based Congo Red assay utilizing UV absorbance Requires: 100 μl sample/time 3 mg/ml 0.3 mg Medium to High Throughput Biophysical Stability: Fibrillation (Congo Red Assay) Wavelength (nm) Initial Day 1 Day 3 Day 6 Peptide Z was exposed to shear stress 300 rpm) at room temperature Shift in peak absorption wavelength is indicative of fibril formulation after 6 days 9

10 Fluorescence Response [AU] Case Study 3 Fibrillation Assessment via UV and Fluorescence (continued) Fluorescence [AU] vs Fibril Concentration AU = * Conc, R^2 = Congo Red assay provides quick qualitative read on conditions under which fibrils form Fibril Content [μg/ml] Experiment 1 Experiment 2 Experiment 3 Standard Curve Fit Lower 95% CL AU Upper 95% CL AU Lower 95% PL AU Upper 95% PL AU Conditions AU1 AU2 AU3 Average AU Fibril Content μg/ml Can be used for screening purposes where peptide quantity is limited Should be replaced with more quantitative assay like Thio T once peptide availability no longer an issue Initial Wk 4-80 C Wk 4-20 C Wk 4 5 C Wk 4 40 C AU=abritrary units. 10 Slight increase in fibril concentration under temp stressed conditions!

11 Summary and Conclusion Peptide delivery comes with its own inherent liabilities Working with limited API to de-risk these liabilities at the early discovery stage adds another complication to the mix Miniaturization of analytical methods (ie, solubility, fibrillation) to cope with limited material is not trivial, but creative experimental design can frequently enable small-scale studies that may also be amenable to high throughput approaches 11

12 Acknowledgements Many thanks to my colleagues at Merck who provided data and guidance that made this presentation possible! Annette Bak Erika Bartholomew Andrew Leithead Dennis Leung Sachin Lohani Caroline McGregor Ellen Minnihan Grace Okoh Nathalie Toussaint 12

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15 Routes of Delivery Route Pros Cons Oral Easy access, convenient to dose Good patient compliance High-dose delivery Epithelial barrier Chemical and enzymatic degradation First pass gut and hepatic metabolism Transdermal Pulmonary/ inhalation Intranasal Ocular Easy access, convenient to dose Good patient compliance Large surface area for treatment Large surface area for absorption Thin epithelial barrier moderate permeability Very well perfused Highly permeable epithelia Convenient dosing Commercially available devices Easy access, convenient to dose (eye drops) Good patient compliance (eye drops) Low-dose delivery Tough barrier to penetrate Toxicity/irritation at site of application Limited dose, dose volume Reproducible deposition Safety and lung function Taste liability Device development required Limited dose, dose volume Rapid clearance Taste liability Irritation potential Limited dose, dose volume Limited to local delivery Irritation potential Low permeability barrier May require surgery (injections or implants) Source: Adapted from Mathias NR, et al. J Pharm Sci. 2010;99:

16 Why the Move to More Peptide Drugs? Advantages High potency High selectivity Broad range of targets Potentially lower toxicity and immunogenicity Low accumulation in tissues High chemical and biological diversity Peptide opportunities may provide best of small molecule and biologics approaches Can we optimize peptide design and delivery to minimize challenges with peptides? How do we tailor physicochemical and formulation risk assessment approaches to peptides? Source: Adapted from Craik DJ, et al. Chem Biol Drug Des. 2013;81:

17 Energy Fibrillation Peptides and proteins can assume a variety of possible conformations, the relative free energies of which are sensitive to various environmental conditions Concentration, shear, ph, ionic strength, and temperature Certain environmental conditions can lead to formation of aggregates/fibrils, which can lead to Loss of potency Aggregation Manufacturing issues Increased immunogenicity Folding Aggregate Native-state 17

18 Aggregation vs Fibrillation Aggregation Process is an equilibrium between the native and denatured states Irreversible changes to the denatured species can result in formations of fibrils Fibrillation A hydrophobic driven process where nonpolar amino acid residues of peptides, when exposed to water in unfolded state, can lead to nucleation of fibrils Not bio-active; however, can induce immune response Facilitated by air-water interface and hydrophobic surfaces, eg, Teflon Jorgensen L, et al. Expert Opin Drug Deliv. 2009;6(11): Jansen, et al. Biophys J. 2005;88:

19 Techniques for Detecting Fibrils CD FTIR Bouchard M, et al. Protein Sci. 2000;9:

20 Thioflavin T Assay for Detecting Fibrils Thio T assay Fibril-bound Thio T: rotation around is restricted, leading to increase in fluorescence quantum yield This assay is low throughput using 3 ml cuvette Amendable to detect fibrils, monitor kinetics of fibril formation, and investigate effect of excipients Courtesy: Sachin Lohani. 20

21 Congo Red Fibril Screening Assay Congo Red Assay When CR binds to fibrils, a change in color from orange-red to rose is induced that corresponds to a shift in the characteristic absorbance spectrum of CR This assay is medium throughput and uses 96 well plate Amendable to detect fibrils, monitor kinetics of fibril formation, and investigate effect of excipients Spectra shift observed with CR and glucagon peptide Green line is non-stressed glucagon Red line is stressed glucagon Courtesy: Nathalie Toussaint and Erika Bartholomew. 21

22 What Are Peptides? Peptide Secondary Structures Peptide = chain of amino acids polypeptidechain 22

23 A Business Case for Peptides: Projected Global Peptide Therapeutics Revenues Application CAGR% Parenteral 11,537 12,201 13,141 14,152 15,239 16,408 17,666 19,017 20, Oral ,098 1,233 1,382 1,548 1,733 1, Pulmonary , Mucosal ,130 1, Others (intradermal and nasal) Total 13,200 14,138 15,374 16,719 18,183 19,775 21,504 23,386 25, Source: Clinical trials.gov; Primary Interviews; Cancer Journals; TMR Analysis. (Data in USD Million) 23