Practical Aspects of Hyaluronan Based Medical Products

by J. W. Kuo, Ph.D.
Table of Contents

1 Overview
    1.2        Primary Structure
    1.3        Molecular Formula
    1.4        Member of Glycosaminoglycans
    1.5        HA Structures of Higher Orders
    1.6        Viscosity, Molecular Weight, and Structures
    1.7        Source and Isolation
    1.8        Distribution and Catabolism
    1.9        Influence on Cell Behavior
    1.10      Chemical Modification
    1.11      HA-Based Medical Products
    1.12      Ever-Growing Interest

2  Chemistry
    2.1     Chemistry in Structural Investigation
    2.1.1         Structure Verification via Chemical Reactions
    2.1.2         Silylation of HA and Its Use in Gas Liquid Chromatography
    2.1.3        Peroxidation Reaction
    2.2     Chromogenic Reactions
    2.3     Chemistry of HA Degradation
    2.3.1         Alkaline Degradation
    2.3.2         Acidic Degradation
    2.3.3         Oxidative Reductive Depolymerization
    2.3.4         Degradation on Freeze Drying
    2.3.5         Radiolysis
    2.3.6        Heat Degradation
    2.3.7         Ultrasonic Depolymerization
    2.4 Chemistry in Isotopic Labeling
    2.4.1         Exchange Reactions
    2.4.2         N-Acetylation
    2.4.3         Aldehyde Reactions
    2.4.4         Hydroxyl Groups Activated by Cyanogen Bromide
    2.5 Synthesis of Fluorescent HA
    2.5.1         Isothiocyanatofluorescein
    2.5.2         Fluorescein Amine in Ugi Reaction
    2.5.3         Fluorescein Amine in Carbodiimide Reaction
    2.5.4         Synthesis of BODIPY-Labeled HA
    2.6 Biotinylated HA
    2.7 HA Derivatives with Various Side Chains
    2.7.1         Acyl-Urea Derivatives
    2.7.2         Water-Insoluble HA Esters
    2.8 HA–Drug Conjugation
    2.8.1         Esterification of HA
    2.8.2         Hydrazide–NHS Ester Reaction
    2.9 Chemical Crosslinking of HA
    2.9.1         Divinylsulfone
    2.9.2         Biscarbodiimide
    2.9.3         Crosslinking via Hydrazide
    2.9.4         Diepoxide
    2.9.5         Self Crosslinking
    2.9.6         Multivalent Cations
    2.9.7         Ugi and Passerini Reactions
    2.9.8         In Situ Crosslinking
    2.10 Preparation of Oligosaccharide
    2.10.1         Enzymatic Degradation
    2.10.2         Chemical Synthesis
    2.10.3         Chemoenzymatic Synthesis
    2.11 Sulfation of HA
    2.12 Summary of Methods of Chemical Modification

3  Rheological Properties of Hyaluronan as Related to Its Structure,
Size, and Concentration
    3.1 Some Basic Concepts of Intrinsic Viscosity
    3.1.1         Intrinsic Viscosity — A Measure of Hydrodynamic Volume
    3.1.2         End-to-End Distance Determines Hydrodynamic Volume
    3.1.3         Extended Conformation Increases Molecular Size
    3.1.4         Structure Features Favoring Extended Conformation
    3.1.5         Mark’s Equation — IV, Rigidity, and MW
    3.2 Hydrodynamic Volume and Molecular Structures
    3.2.1         Hydrodynamic Volume and the Structure of HA
    3.2.2         Hydrodynamic Volume of Carbodiimide-Modified HA
    3.3 Absolute Viscosity Measured at Steady State
    3.3.1         Networking at Higher Concentrations
    3.3.2         Dynamic Viscosity
    3.3.3         Pseudoplasticity
    3.3.4         Zero Shear Viscosity, MW, and Concentration
    3.3.5         Specific Intermolecular Interaction
    3.4 Viscoelasticity
    3.4.1         Elasticity Stores Energy, Viscosity Dissipates Energy
    3.4.2         Viscoelasticity Measured under Oscillating Force
    3.4.3         More Elastic and Less Viscous at Higher Frequencies
    3.4.4        Crosslinked HA Stays Elastic over Wide Frequencies

4  Standards, Tests, and Analytical Methods
    4.1 Standards and Specifications
    4.1.1         European Pharmacopoeia
    4.1.2         Viscoelastics in Ophthalmic Application
    4.1.3         ISO 10993 — Biocompatibility
    4.1.4         U.S. Pharmacopoeia
    4.2 Analyses of HA Structure, Size, and Quantity
    4.2.1         Infrared Spectroscopy
    4.2.2         UV-Visible Spectroscopy
    4.2.3         Nucleic Magnetic Resonance Spectroscopy
    4.2.4         Mass Spectrometry
    4.2.5         Light Scattering
    4.2.6         Liquid Chromatography
    4.2.7         Electrophoresis
    4.2.8         Osmometry
    4.3 Natural Polymeric Impurities
    4.4 Other Bioassays
    4.4.1         Eye Tolerance Assays
    4.4.2         Limulus Amebocyte Lysate Assay
    4.5 Lipoteichoic Acid
    4.6 Chemical Methods of Radiolabeling
    4.6.1         Tritium
    4.6.2         Carbon 14
    4.6.3         Iodine 125
    4.6.4         Carbon 11
5   Safety of Hyaluronan-Based Medical Products in Their Indicated Applications
    5.1 General Safety Considerations
    5.1.1         HA — Natural Component of Human Tissues
    5.1.2         Catabolic Pathways of HA
    5.1.3         Physiological Turnover and Dose Tolerance
    5.1.4         Consensus on Certain Specifications
    5.1.5         Essential Safety Requirement in Preclinical Studies
    5.1.6         Effect of HA Chemical Modification on Its Safety
    5.2 Safety Information from Specific Human Use
    5.2.1         Ophthalmic Surgical Aid
    5.2.2         Intraarticular Injection in the Knee
    5.2.3        Prevention of Postsurgical Adhesions
    5.2.4        Cosmetic Tissue Augmentation

6   Clinical Performance, Mechanism of Action, and Product Characteristics
    6.1 Ophthalmic Surgeries
    6.1.1         Cataract Removal — Intraocular Lens Implantation
    Viscoelasticity and Lubricity - Viscosity and Thickness of Coating – Intraocular
    Pressure, Osmosis, and Corneal Edema - Cohesiveness, Dispersiveness, and
    adaptiveness – Rationale and Benefit of Combination Viscoelastics - Removal
    of Viscoelastics Postsurgery
    6.1.2         Vitreous Substitute
    6.2  Injections for Osteoarthritic Knees
    6.2.1         The Maze of Clinical Data in PMAs
    Strong Placebo Effect of Saline - Synvisc - Hyalgan - Orthovisc - Supartz
    6.2.2         The Big Picture — Metaanalysis
    6.2.3         Recommendations by European League against Rheumatism
    6.2.4         Mechanism of Action
    Local versus Systemic - The Effect of Molecular Weight – Is HA Residence Time
    in the Knee Really Important?
6.3  Adhesion Prevention and Wound Healing
    6.3.1         Design Rationale and Key Product Characteristics
    Mimicking Scarless Wound Healing of Fetus - Minimum Duration of the
    Presenceof a Barrier - HA Prevents Adhesion Better if Applied before Injury
    6.3.2         Products in Solid Forms
    Seprafilm - MeroGel
    6.3.3         Products in Liquid Forms
    Sepracoat – Intergel - HylaSine and Sepragel Sinus
    6.3.4         Challenge in Adhesion Prevention Products
6.4 Tissue Augmentation
    6.4.1         Restylane
    6.4.2         Deflux
    6.4.3         Hylaform
6.5 Dental and Bone Application