WO2022051060A1 - Réticulation d'acide hyaluronique non dérivé d'animal avec de la divinylsulfone - Google Patents

Réticulation d'acide hyaluronique non dérivé d'animal avec de la divinylsulfone Download PDF

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WO2022051060A1
WO2022051060A1 PCT/US2021/045150 US2021045150W WO2022051060A1 WO 2022051060 A1 WO2022051060 A1 WO 2022051060A1 US 2021045150 W US2021045150 W US 2021045150W WO 2022051060 A1 WO2022051060 A1 WO 2022051060A1
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linked
cross
derived
kda
process according
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PCT/US2021/045150
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James J. Mencel
David Toledo-Velasquez
Michael Joseph DALEY
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Orthogenrx, Inc.
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Priority to TW110130122A priority Critical patent/TW202227103A/zh
Publication of WO2022051060A1 publication Critical patent/WO2022051060A1/fr
Priority to US18/224,200 priority patent/US20230359066A1/en

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions

Definitions

  • HA is ubiquitously found in soft and hard tissues such as hyaline cartilage, skin and organ tissues, and bone, as well as synovial fluid (SF) of mammalian joints. In the latter location, it plays a role in joint lubrication, cushioning and resistance to shear forces, taking advantage of its unique viscoelastic physical properties.
  • HA is biocompatible, biodegradable, and hydrophilic. Due to the abundant hydrophilic carboxyl groups that are part of its chemical structure, HA allows the influx and retention of large amounts of water to the biopolymer. (Allemaan & Baumann, 2008) Naturally-occurring HA has been reported to have a molecular weight ranging from 5 oligo, less than 6x10 Da, to low molecular weight, 0.8 to 8x10 Da, and a high molecular weight of 1x10 ⁇ Da (Girish & Kemparaju, 2007, Kogan, et al., 2007; Stern, Kogan, Jedzejas & Soltes, 2007;Day and Sheenan, 2001, Fouissac, Milas, Rinaudo & Borsali, 2002; Lapcik, Lapcik, DeSmedt, Demeester & Charbrecek, 1998; Laurent & Fraser, 1992).
  • the larger polymers apparently are the result of the hydrophilic interactions between the aforementioned LINK proteins, e.g. Aggrecan.
  • the hyaluronan synthase genes (HAS) that synthesize HA seem to be limited to polymers of 2x 10 ⁇ in MW with the dominant gene expressed in adults being HAS-3 synthesizing 2x 10 ⁇ MW polymers (Galloway, et al. 2013).
  • HA fragments with specific uniform sizes may be achieved by controlling the degradation of high molecular weight HA using acidic, alkaline, ultrasonic and/or thermal degradation (Stem et al., 2007).
  • HA of defined length has been prepared by chemo enzymatic synthesis, as well (DeAngelis, 2008).
  • HA Native non-cross-linked HA has a short half-life after it is injected to a mammalian skeletal joint due to degradation by hyaluronidases and reactive oxygen species (ROS) that can limit the HA utility
  • ROS reactive oxygen species
  • Some HA preparations used for cosmetic, medical and other human in vivo applications comprise a form of HA that has been cross linked to achieve a higher average molecular weight resulting in HA “hydrogels” offering longer residence time after injection (Carbohydrate Polymers, June, 2011).
  • Cross linking may be achieved by physical entanglement of the HA chains, and by the formation of covalent bonds involving functional groups appended to the two monosaccharides comprising the basic HA disaccharide.
  • non-covalent associations such as those due to Van der Waals forces between the saccharide units may be the basis of cross linking.
  • covalent cross linking Choante, Zuber, Vandamme
  • Carbohydrate Polymers June, 2011; Khnmanee, Joeng, Park: Journal of tissue Engineering, 8, 2017, 1-16
  • linking agent between functional groups on HA, whether within a single polysaccharide HA strand or between such strands.
  • the glucuronic acid carboxylic acid may be used in a variety of chemistries, both as an electrophile via various carboxyl activating methodologies, or as a nucleophile in the form of its carboxylate anion.
  • the deacetylated amine of N-acetyl glucosamine may be used in a variety of ways including reductive amination with aldehyde terminal linking agents and amidation with carboxylic acid crosslinking agents.
  • the hydroxymethylene of N-acetyl glucosamine is the focus of many covalent crosslinking methodologies.
  • esters may involve esters, acetals, for example with glutaraldehyde (Tomahita & Ikada (1977b); Collins & Birkishaw, 2007; Crescenzi et al, 2003a, 2003b) and formaldehyde (US 3713448) , or ethers derived by attack on epoxides such as 1,4-butanediol diglycidyl ether.( US 6921819), and Michael additions to acrylates and addition to vinyl sulfonates (Balazs & Leshchiner (1968); Collins & Birkinshaw (2007); Ramasmurthi & Vesely (2002)).
  • glutaraldehyde Tomahita & Ikada (1977b); Collins & Birkishaw, 2007; Crescenzi et al, 2003a, 2003b
  • formaldehyde US 3713448
  • ethers derived by attack on epoxides such as 1,4-but
  • hemiacetals are reversible under the low pH conditions required to form them, and the glutaraldehyde cross-linked material is found to be unstable unless the material is swelled in a buffer.
  • the formaldehyde treatment medium is removed, the treated rooster combs are dried, then washed with water to extract a water-soluble limitedly formaldehyde cross-linked HA with an average molecular weight in the 3000 to 6000 kDa range.
  • the cross-linked HA may be isolated as a solid material by pH adjustment and precipitation from the aqueous mixture with acetone.
  • Proteins associated with natural HA may be able to play a more direct role in altered rheology of HA.
  • the apparent crosslinking observed for rooster comb-derived HA following formaldehyde treatment may not be due to formaldehyde alone but may include covalent bonds to functional groups on proteins native to rooster comb. Those proteins may serve as a bridge between two or more molecules of HA (See Figure 3).
  • the crosslinking would comprise, as an example, an acetal bond between a hydroxyethyl moiety on N-acetyl glucosamine with formaldehyde, with the same molecule of formaldehyde bonded to an amine or hydroxyl group on a protein.
  • the present invention thus provides a benefit in the search to provide a stable, non-animal -derived, cross-linked HA of an average molecular weight which confers properties of water solubility and that is useful in therapeutic administration.
  • High molecular weight crosslinked HAs are usually not soluble and are usually in gel form.
  • the HA of this invention is greater than 500 kDa to less than 10000 kDa and is water soluble and non-avian derived. It is preferably about 500 kDa to about 6000 kDa, and even more preferred of about 3000 kDa to about 6000 kDa.
  • the HA of the invention is less prone to inducing immunogenic reactions when administered to a subject because of its lack of avian - derived proteins in the formulation.
  • the subject can be an animal or human being.
  • the HA of the present invention is therefore used to treat musculoskeletal ailments such as to be used for viscosupplementation.
  • the product is dissolved in water and lyophilized to form a white powder.
  • FIGURE 1 Disaccharide Core for Hyaluronic Acid (HA) shows repeating disaccharide molecular core of hyaluronic acid (HA).
  • FIGURE 2 Illustration of Acetal Cross Linking represents chemical cross linking of HA via acetal covalent bonding.
  • FIGURE 3 Illustration of Formaldehyde Cross Linking Via Protein represents a cross linking between HA subunits via bridging between HA subunits involving formaldehyde and a protein.
  • FIGURE 6 Graphic Depiction of the Relationship Between Mn, Mw, Mz and Mv. This generic plot shows the relationship between the various molecular moments used to evaluate different aspects of molecular weight in a polydisperse material such as a biopolymer like HA.
  • FIGURE 7 Illustration of Formaldehyde Acetal Cross Linking. This is an illustration of the cross linking of HA via acetal covalent bonding with formaldehyde.
  • FIGURE 8 Chemical Structure of Spermine that can be used as a polyamine bridge to aid cross linking via formaldehyde.
  • FIGURE 9 Chemical Structure of poly-L-lysine that can be used as a polyamine bridge to aid cross linking via formaldehyde.
  • FIGURE 10 Illustration of Protein Bridge Cross Link via Poly-L-lysine Hemiaminal. This chemical representation is of HA cross linking as mediated by formaldehyde with a poly-L- lysine bridge. Illustrated are hemiaminal covalent bond linkages between HA, formaldehyde and poly-L-lysine.
  • FIGURE 12 Rheological Comparison of Microbiologically-derived HA with Various Cross Linking Systems. These are comparative plots of G’ and G” for HTL Biotechnology microbiologically-derived HA, vs the untreated HA control, as treated with formalin alone, or with formalin is in the presence of poly-L-lysine (the protein) or in the presence of spermine. Solid black line depicts G’ for HA control in water; Solid grey curve depicts G” for HA control in water. Black short-dashes curve depicts G’ for HA treated with formalin in water (5: 1 w/w, vs.
  • FIGURE 13 GPC Molecular Weight Distributions for Various Treatments of Microbiologically-derived HA. These over lay traces are for HTL Biotechnology microbiologically-derived HA, vs untreated HA control, as treated with formalin, formalin in the presence of poly-L-lysine, and formalin in the presence of spermine, thus enabling a comparison of the relative molecular weights on the basis of retention times. Dashed vertical grey lines denote center points for individual GPC peaks. Grey solid curve depicts formaldehyde cross-linked rooster comb HA.
  • Black short-dashes curve depicts the untreated HA control
  • Black long-dashes curve depicts HA treated with formalin (5: 1 wt/wt ratio vs. HA).
  • Black solid curve depicts HA treated with formalin (5 : 1 wt/wt ratio vs HA) and spermine in water.
  • Grey long-dashes curve depicts HA treated with formalin (5: 1 wt/wt ratio vs HA) and poly-L-lysine in water.
  • FIGURE 14 Microbiologically-derived HA Treated with a Series of Weight/Weight Ratios of Formalin (50% Solution in Water), vs. the Untreated HA Control.
  • This figure provides comparative plots of G’ and G” for HTL Biotechnology microbiologically- derived HA, vs. the untreated HA control, as treated with various wt/wt ratios of formaline to HA, thus enabling a comparison of relative crossover frequencies in view of treatment conditions.
  • Black sequential long-dashes short-dashes curve depicts G’ for untreated HA control.
  • Grey sequential long-dashes short-dashes curve depicts G” for untreated 750-1000 kDa linear control.
  • Solid black curve depicts G’ for HA treated with 10: 1 wt/wt formalin to HA.
  • Solid gray curve depicts G” for HA treated with 10: 1 wt/wt formalin to HA.
  • Black short-dashes curve depicts G’ for HA treated with 7.5: 1 wt/wt formalin to HA.
  • Grey short-dashes curve depicts G” for HA treated with 7.5:1 wt/wt formalin to HA
  • Black long-dashes curve depicts G’ for HA treated with 5 : 1 wt/wt formalin to HA.
  • Grey long-dashes curve depicts G” for HA treated with 5:1 wt/wt formalin to HA.
  • FIGURE 15 Viscosity Regression Behavior for Mi crobiologically -Derived HA Cross-Linked with Formaldehyde in Water. This provides the comparative viscosities for formaldehyde cross-linked HTL Biotechnology microbiologically-derived HA after exposure to water for various lengths of time thus showing regression in viscosity. This suggests an apparent reversal of cross linking and return to the untreated (native) state.
  • Black sequential long-dashes short dashes curve represents change in viscosity after 24 h.
  • Black short-dashes curve represents change in viscosity after 28 h.
  • Grey long-dashes curve represents change in viscosity after 33 h.
  • Black long-dashes curve represents change in viscosity after 47 h.
  • Grey solid curve represents change in viscosity after 52 h.
  • Black solid curve represents change in viscosity of 73 h.
  • Grey short dashes curve represents change in viscosity after 114 h.
  • FIGURE 16 Frequency Sweep Data for Cross Linked Rooster Comb HA With and Without Enzyme.
  • the comparative plot of G’ and G” highlight the increase in crossover frequency for formaldehyde cross-linked rooster comb HA in water after exposure to proteinase K vs. the untreated material in the absence of proteinase. This suggests that formaldehyde cross linking of rooster comb HA is aided by linkages with proteins, the disruption of which leads to degradation of cross linking.
  • Black solid curve depicts G’ for formaldehyde cross-linked rooster comb HA without enzyme.
  • Grey solid curve depicts G” for formaldehyde cross-linked rooster comb HA without enzyme.
  • Black dashed curve depicts G’ for formaldehyde cross-linked rooster comb HA with enzyme.
  • Grey dashes curve depicts G” for formaldehyde cross-linked rooster comb HA
  • FIGURE 17 Shear Rate Sweep Data for Formaldehyde Cross-Linked Rooster Comb HA with and without Enzyme. This is a comparison of viscosity that highlights the differences in viscosity for formaldehyde cross-linked rooster comb HA that has and has not been treated with proteinase K. This illustrates the lower viscosity, and by implication, the reduced cross linking and average reduced molecular after treatment with the proteinase. Grey curve represents cross-linked rooster comb HA without enzyme. Black curve represents cross-linked rooster comb HA with enzyme.
  • FIGURE 18 Illustration of HA Cross-Linked via Divinyl Sulfone (DVS) This figure depicts the covalent bonding of the N-acetyl glucosamine hydroxymethyl locus with DVS, wherein DVS crosslinks two subunits of HA.
  • DVS Divinyl Sulfone
  • FIGURE 19 GPC Data for Untreated HA and for HA Treated with DVS under a Variety of Conditions, Fitted for Average Molecular Weights.
  • Solid grey curve denotes untreated Lifecore Biomedical 750-1000 kDa HA control.
  • Grey dashes curve denotes untreated HTL Biotechnology HA.
  • Black short-dashes curve denotes HTL Biotechnology HA at 0.75 weight % in aqueous media after treatment with 10 wt% DVS (vs. HA) at pH 8.
  • Solid black curve denotes HTL Biotechnology HA at 0.65 weight % in aqueous media after treatment with 10 wt% DVS (vs. HA) at pH 11.5.
  • Black long-dashes curve denotes the reanalysis for HTL Biotechnology HA after treatment with 10 wt% DVS (vs HA) in aqueous media at pH 11.5.
  • FIGURE 20 GPC Overlay of DVS Treated Microbiologically-derived HA with Untreated HA Control. These overlay GPC traces are arranged left to right for increasing molecular weight for untreated HTL Biotechnology HA exposed to the reaction medium in the absence of DVS and for HTL Biotechnology HA at 0.65 wt% in aqueous media after treatment with DVS at a 10: 1 HA/DVS wt/wt ratio at pH 11.5. This shows an increase in average molecular weight after treatment. Black curve denotes 2400-3600 kDa HA exposed to reaction media in the absence of DVS. Grey curve denotes treated HA treated with DVS.
  • FIGURE 21 GPC Overlay for DVS Treated Microbiologically-derived HA with Formaldehyde Cross-Linked Rooster Comb HA (MW 3000-6000 kDa), the Untreated Lifecore Biomedical 750-1000 kDa HA GPC chromatography Standard, and the Untreated HTL Biotechnology microbiologically-derived HA (2400-3600 kDa), fitted for Molecular Weight.
  • overlays are arranged from left to right for increasing molecular weight for untreated Lifecore Biomedical 750-1000 kDa HA (black long- dashes curve), untreated HTL Biotechnology 2400-3600 kDa HA (black short-dashes curve), and for the HTL Biotechnology HA at 0.69 wt% in aqueous media after treatment with 10: 1 wt/wt HA to DVS at pH 11.5 (grey long-dashes curve) and grey curve; reinjected sample for 10: 1 wt/wt HA to DVS at pH 11.5.
  • a sample polymer of HA may be comprised of a mixture of materials of differing molecular weights, and thus may be thought of as polydispersable material.
  • their viscoelastic properties are studied and reviewed. For purely elastic solids, the stress is directly proportional to the strain (not the strain rate) and deformation is reversible. In contrast, for purely viscous liquids, the stress is directly proportional to the strain rate (not the strain), and deformation is not reversible.
  • SAOS small amplitude oscillatory shear
  • the average molecular weights of HA and cross-linked varieties may be evaluated using gel permeation chromatography (GPC).
  • GPC curves represent the qualitative distribution of molecular weights within a material, and they provide a view of the average molecular weight within a selected number of standard deviations from the apex of the curve.
  • GPC also shows bimodal or further distributions of average molecular weight subpopulations. This methodology is particularly useful for less extensively cross-linked materials, and a standard may thus be used as a comparative measurement (See Figure 5).
  • poly dispersity analysis estimates polymer molecular weight by measuring averages associated with categories of mass distributions that contribute to the overall average molecular weight.
  • Each “average” is sensitive to different aspects or “moments” of distribution (RJ Young and PA Lovell, Introduction to Polymers, 1991; Septo, RFT; Gilbert, RG; Hess, M; Jenkins, AD, Jones, RG, Kratochvil, P, 2009) “Disparity inPolymer Science” Pure Appl. Chem. 81(2): 351- 353). This average includes the following:
  • Mn the number average molecular weight moment (derived form the number of molecules above and below the molecular weight in the distribution);
  • Mv the viscosity-average molecular weight
  • Mw the viscosity-average molecular weight
  • Mv is most closely related to the Intrinsic Viscosity (IV) of the system, which is a measure of the contribution of the solute to the viscosity of the system.
  • IV Intrinsic Viscosity
  • the ratio of the weight average molecular weight and the number average molecular weight provides the poly dispersity index, a value which represents the breadth of molecular weight distribution. The larger the PDI, the broader the distribution.
  • a graphic representation of the range of weights represented by each average is provide in Figure 6.
  • Example 1 isolates a 3000 to 6000 kDa cross-linked HA from commercially available (Genzyme Biosurgery) rooster comb derived Synvisc-One.
  • Synvisc-One consists of an aqueous mixture of 80%, by weight, of a limited formaldehyde cross-linked water-soluble HA and 20%, by weight, of a more extensively cross-linked, water-insoluble HA component.
  • the more extensively crosslinked component is prepared by initial cross-link with formaldehyde, followed by treatment with DVS.
  • the formaldehyde-only cross-linked HA and the formaldehyde then DVS crosslinked HA are formulated in a combination that provides 8 mg/ml of total HA in aqueous media.
  • the recovered water-soluble cross-linked HA when compared using gel filtration chromatography (GPC) against Lifecore Biomedical 750-1000 kDa HA GPC standard is shown to have a relatively higher average molecular weight (See Figure 5).
  • An illustration of HA as cross-linked only by formaldehyde is shown in Figure 7.
  • HA’s in the 3000 to 6000 kDa MW range confer certain benefits over lower MW HA’s.
  • marketed products that utilize HAs in this MW range in their manufacturing process such as Synvisc-One, utilize avian derived HA as its starting material.
  • avian derived HA as its starting material.
  • the intent of using microbiologically-derived or other non-animal derived HA as a starting material is to avoid incorporation of avian antigens into the final product and consequently reduce the likelihood of triggering a Severe Acute Immune Response (SAIR) to the HA product.
  • SAIR Severe Acute Immune Response
  • Example 2 - This example is a formaldehyde treated microbiologically-derived HA with and without, separately, poly-L-lysine and spermine.
  • HTL Biotechnology microbiologically-derived HA is exposed to formaldehyde (50% concentrated formalin in a 10: 1 ratio formaldehyde to HA) in water and acetone at ambient temperature (4- 35°C). Then, it is treated with dilute aqueous sodium hydroxide, followed by dilution with acetone to precipitate the treated material in an attempt to achieve limited cross linking of this microbiologically- derived HA.
  • This procedure is also conducted in the presence of spermine ( Figure 8), and separately, it is conducted in the presence of poly- L-lysine ( Figure 9).
  • spermine Figure 8
  • Figure 9 poly- L-lysine
  • HTL Biotechnology microbiologically-derived HA (average MW of 2400-3600) (10 mg) is treated with 50% wt/wt concentrated formalin (200 volumes 10: 1 ratio formaldehyde vs. HA) in water (1000 volumes) and acetone (1200 volumes) at ambient temperature (ca. 4-35°C) for 24 hours. It is then treated with 0.04 M aqueous sodium hydroxide, followed by acetone (10 volumes vs. the volume of the HA solution in water) to precipitate the treated material. This procedure also is conducted in the presence of, separately, approximately 5 mg spermine and approximately 6.5 mg of poly-L-lysine.
  • the HA in this example treated with formaldehyde alone or in combination with spermine or poly-L-lysine is exposed to frequency sweeps at 1% stain from 100 Hz to 0.01 Hz oscillatory shear (logarithmic 10 points per decade).
  • G remains above G”, indicating a predominantly elastic fluid and highly cross-linked material (See Figure 11) (i.e., a gel).
  • a crossover point is observed for the untreated HA control (see Figure 11).
  • Example 3 Cross linking of HA using reduced weight/weight ratios of formalin, spermine or poly-L-lysine is studied:
  • Example 4 In this example HTL Biotechnology microbiologically-derived HA treated with formalin at pH 7, 8, 9 and 10 is compared with formaldehyde cross-linked rooster comb HA (See Table 1). Among the pH levels tested, the crossover frequency is lowest at pH 7, at 0.07084 HZ, vs. the untreated HA control, at 0.1699 Hz. However, the crossover data do not closely approach that for the rooster comb material, at 0.0155 Hz. The crossover modulus increases with decreasing pH, which is consistent with an increasing degree of crosslinking.
  • crossover frequency may be correlated with average molecular weight
  • these conditions for preparing the HA using formaldehyde crosslinking do not produce cross-linked HA with an average molecular weight comparable to the cross-linked rooster comb derived HA.
  • HTL Biotechnology microbiologically-derived HA prepared as a solution in 2.5 mL water, (ie - concentration of 10 mg/mL) is treated with 0.5 mL Formalin Fixx (50% weight /weight formaldehyde in water) and 3 mL acetone, agitated, and then allowed to incubate overnight at ambient temperature (ca. 4-35°C). After overnight incubation, each sample is then individually treated with dilute aq. NaOH to achieve a pH of 10 (0.4 M aq. NaOH), 9 (0.004 M aq. NaOH), 8 (0.0004 M aq.
  • Example 5 In yet this example HTL Biotechnology microbiologically-derived HA is treated with formalin (50% formaldehyde weigjht/weight in aq solution) alone in a weight/weight ratio to HA of 10: 1, 7.5: 1 and 5:1.
  • HTL Biotechnology microbiologically-derived HA (average MW of 2400-3600) in water (10 volumes) and acetone (120 volumes) is treated with Formalin Fixx (50% wt/wt formaldehyde in water) alone with ratios by volume of Formalin Fixx to HA of 10:1, 7.5: 1 and 5: 1..
  • Formalin Fixx 50% wt/wt formaldehyde in water
  • HA 7.5: 1 and 5: 1.
  • Each vial is stirred 25 hours at ambient temperature (ca. 4-35°C), then individually treated with 0.04 M aqueous sodium hydroxide (120 volumes), followed with 0.1 M sodium acetate (20 volumes).
  • the resulting materials in each vial is then precipitated with acetone (10 volume vs. the starting volume of HA in water), and the isolated materials are vacuum dried without applying heat.
  • the resulting materials after this treatment in each case, are dissolved in water at a concentration of 10 mg/mL in IX PBS, and then are examined
  • Ratios of 10: 1 and 7.5: 1 yield gels, as indicated by the rheology (no crossover point), and the ratio of 5: 1 yields a material that is Theologically similar to the untreated HA control ( Figure 14).
  • Example 6 In addition to failing to achieve a cross-linked microbiologically-derived HA comparable with formaldehyde cross-linked rooster comb HA, upon further examination it is apparent that formaldehyde treated microbiologically-derived HA stored as a solution in aqueous media is not physically stable over time and appears to revert to the untreated state. Examples of data demonstrating this behavior are shown in Figure 15, which illustrates that an aqueous solution of formaldehyde cross-linked microbiologically-derived HA undergoes a decrease in viscosity over 24-144 hours. In Table 2, it is seen that a decrease in the modulus and an increase in crossover frequency occurs over the same period, both indicative of a degradation in crosslinking.
  • This example evaluates the physical stability of microbiologically derive HA treated with formaldehyde.
  • HTL Biotechnology microbiologically-derived HA treated with Formalin Fixx as in Example 5, above, at the 10: 1 ratio is stored unstirred at ambient temperature in water at about pH 7.
  • the mixture is evaluated at intervals of 28, 33, 47, 73 and 144 hours.
  • the aqueous mixture is exposed to frequency sweeps at 1% strain from 100 Hz to 0.01 Hz oscillatory shear (logarithmic 10 points per decade).
  • Viscosity regression behavior data are plotted in Figure 15.
  • Data for crossover frequency, crossover modulus and zero sheer rate viscosity are provided in Table 2 and demonstrate changes over time consistent with a degradation of crosslinking. TABLE 2
  • Example 8 Microbiologically-derived HA is treated with aqueous DVS.
  • Solutions of HTL Biotechnology microbiologically-derived HA (2400-3600 kDa) are prepared in in 1 X PBS in the concentrations shown Table 3 in 20 mL scintillation vials. Aqueous NaOH (IN) and aqueous HC1 (IN) are added to adjust pH as per Table 3. Then, DVS dissolved in one volume IX PBS (vs DVS) is added to give the HA DVS concentrations shown in Table 3. The mixtures are briefly vortexed then incubated for 24 hours at ambient temperature (4-35°C).
  • the mixtures are treated with dilute aqueous HC1 (IN) to adjust pH to neutral at ambient temperature (4- 35°C).
  • the resultant mixtures are diluted with acetone (10 volume vs. the starting volume of HA in 1 X PBS) to precipitate the materials.
  • the precipitated materials are re-dissolved in IX PBS at a concentration of 10 mg/mL and compared by GPC (See Figures 19 and 20). They are tested for their various comparable molecular moments by using the Agilent GPC/SEC Software (v A.02.01).
  • FIG. 19 provides GPC overlays for DVS-treated microbiologically-derived HTL Biotechnology HA according to conditions in Table 3, along with the substrate (2400-3600 kDa HA, HTL Biotechnology) exposed to the cross coupling media in the absence of DVS (Control 1) , untreated substrate as purchased, (Control 2) and untreated Lifecore Biomedical 750-1000 kDa HA .
  • Example 9 This example further evaluates favorable cross linking conditions for microbiologically-derived HA using DVS from the previous example (Example 8) and compares the product with Lifecore HA (750-1000 kDa), untreated HTL Biotechnology microbiologically derived HA (2400-3600 kDa) and formaldehyde cross-linked rooster comb HA.
  • the resulting cross-linked material is precipitated as a solid by adding approximately 1 liter of acetone and isolated by filtration.
  • the resulting material is re-dissolved in water at approximately 10 mg/mL and lyophilized to afford a white powder.
  • the cross-linked HA obtained by this procedure is re-dissolved in water at a concentration of 10 mg/mL and analyzed by GPC (See Figure 21).
  • the GPC analysis for thusly treated HA shows a higher average molecular weight vs. the Lifecore Biomedical 750-1000 kDa GPC standard and vs. the untreated HTL Biotechnology HA control, and co-elutes with the formaldehyde crosslinked rooster comb HA isolated from commercial product (See Example 1 above).
  • the GPC maxima for the material prepared after approximately 4 days of incubation in Example 9 is comparable with the GPC maxima for HA treated under the same conditions for approximately a day in Example 8 (see Tables 3 and 4, RB2- 9-4; see Figure 19).
  • HA in both cases is used in vast molar excess to DVS. Because the GPC maxima are comparable in both cases, no further crosslinking is observed after the extended incubation to approximately four days, indicating the reaction between HA and DVS reaches completion within about a day and that DVS is substantially depleted in that period, with little or none remaining for further reaction with HA.
  • the approximately 4-day incubation period extends well beyond this apparent depletion of DVS.
  • This DVS cross-linked material thus is useful for medical uses in a manner that provides HA without the immunogenic liability and reactions caused by animal proteins in the HA materials currently used.
  • Example - HTL Biotechnology microbiologically derived solid powdered HA average molecular weight 2400-3600 was used for cross linking examples.
  • Lifecore Biomedical microbiologically derived, solid powdered HA with average molecular weight of 750-1000 kDa is used as the linear HA GPC chromatography standard.
  • Poly-L- lysine is obtained from Sigma Aldrich.
  • Spermine is obtained from Sigma Aldrich.
  • Formalin Fixx is obtained as a 50% wt/wt solution in water from Fisher Scientific.
  • DVS is obtained from Van Waters, and Rogers (VWR).
  • a standard reference fluid (Cannon S600 lot# 13301) with a nominal viscosity at 37.78 degrees C of 486.4 mPas.s is run.
  • the viscosity standard is run under the same conditions as the example samples.
  • the selected geometry is chosen to provide a balance between sensitivity (a function of diameter) and sample volume under the plate. This geometry requires 0.69 ml of fluid and therefore uses approximately half of the total volume available in the smallest samples.
  • SAOS Small Amplitude Oscillatory Shear
  • the liquid is then expelled directly from the syringe by depressing the plunger until the mark is reached.
  • the geometry is immediately lowered into place and rotated slowly to distribute the liquid evenly around the geometry. Then, the example experiment is started. During the initial equilibrium phase, excess fluid is scraped away if required, and a thin layer of 5 mPa.s silicone oil (Sigma Aldrich PN 317667) is deposited onto the exposed fluid surface to prevent evaporation.
  • the column chosen for separation is the Waters Ultrahydrogel Linear Column 10 micron, 7.8 mm X 300 mm. This column has a nominal molecular weight separation range from about 500 g/mol to 10,000,000 g/mol. A single column is used to minimize band broadening and overall run time.
  • the Ultrahydrogel column is packed with a stationary phase consisting of about 10-micron gel particles composed of a cross-linked hydroxylate polymethacrylate, that contains some residual carboxyl functionality.
  • the Ultrahydrogel column provides stability in a pH range of 2 to 12 and a temperature range of 10°C to 8°C.
  • Compatible mobile phases include pure aqueous phases, as well as aqueous solutions of organic solvents, such as methanol, ethanol, acetonitrile, formic acid and dimethylsulfoxide.
  • sample concentrations and method flow rate are reduced until no adverse chromatographic behavior is observe (poor peak shape, evidence of poor material diffusion through the mobile phase, light scattering date suggesting prolong elution of high molecular weight material, and the like).
  • Samples are ultimately analyzed at a concentration of approximately 0.028 mg/mL Samples are allowed to dissolve overnight in the mobile phase prior to loading on the instrument auto sampler.
  • the mobile phase selected consists of 0.1 NaNO3 with 0.02% sodium azide. The sodium nitrate is added to mitigate aggregation effects while the sodium azide is added to mitigate bacterial growth.
  • Samples are analyzed in triplicate using the conditions described in Table 4.
  • An injections volume of 200 pL is used to maximize signal to noise under the dilute sample concentrations employed.
  • a slow draw up speed of 52 pL/sec is used to improve injection reproducibility and mitigate the potential for sample shear degradation.
  • samples are not filtered prior to analysis, and the flow rate through the column is minimized to prevent potential shear degradation of the high molecular weight material.
  • the mobile phase is triple filtered through a 0.02 um inorganic membrane filter, and a 0.2 um post column PES (polyethersulfone) filter is used to reduce background light scattering noise.
  • PES polyethersulfone

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Abstract

La présente invention concerne un acide hyaluronique (HA) non dérivé d'un animal, tel que non dérivé d'oiseaux, qui a un poids moléculaire d'environ 500 à environ 16 000 kD, de préférence d'environ 500 kDa à environ 10 000 kDa. Les HA idéalement préférés ont un poids moléculaire d'environ 3 000 kDa à environ 6 000 kDa. Ce HA est réticulé avec de la DVS, est hydrosoluble et est stable dans le temps. Le HA selon la présente invention est exempt des protéines immunogènes qui sont trouvées dans des HA dérivés d'animaux. En outre, les HA de la présente invention sont microbiologiquement dérivés ou chimiquement synthétisés, mais encore réticulés pour demeurer hydrosolubles et stables sur une certaine période de temps. La préparation de HA réticulés de l'invention à l'aide d'un HA non dérivé d'un animal évite les réactions immunologiques négatives observées pour des HA réticulés dérivés d'animal préalablement décrits.
PCT/US2021/045150 2020-09-01 2021-08-09 Réticulation d'acide hyaluronique non dérivé d'animal avec de la divinylsulfone WO2022051060A1 (fr)

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US18/224,200 US20230359066A1 (en) 2021-01-28 2023-07-20 Eyeglass lens design device, eyeglass lens design method, and program

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6521223B1 (en) * 2000-02-14 2003-02-18 Genzyme Corporation Single phase gels for the prevention of adhesions
US20050142152A1 (en) * 2003-12-30 2005-06-30 Leshchiner Adelya K. Polymeric materials, their preparation and use
US20060148755A1 (en) * 2004-12-30 2006-07-06 Genzyme Corporation Regimens for intra-articular viscosupplementation
US20100266512A1 (en) * 2007-12-19 2010-10-21 Evonik Goldschmidt Gmbh Crosslinked hyaluronic acid in emulsion
US20150045887A1 (en) * 2012-03-22 2015-02-12 Trb Chemedica International S.A. Method for repair of ligament or tendon
US20150190469A1 (en) * 2010-12-28 2015-07-09 Depuy Mitek, Llc Methods for Forming Compositions for Treating Joints Comprising Bone Morphogenetic Protein and Hyaluronic Acid

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6521223B1 (en) * 2000-02-14 2003-02-18 Genzyme Corporation Single phase gels for the prevention of adhesions
US20050142152A1 (en) * 2003-12-30 2005-06-30 Leshchiner Adelya K. Polymeric materials, their preparation and use
US20060148755A1 (en) * 2004-12-30 2006-07-06 Genzyme Corporation Regimens for intra-articular viscosupplementation
US20100266512A1 (en) * 2007-12-19 2010-10-21 Evonik Goldschmidt Gmbh Crosslinked hyaluronic acid in emulsion
US20150190469A1 (en) * 2010-12-28 2015-07-09 Depuy Mitek, Llc Methods for Forming Compositions for Treating Joints Comprising Bone Morphogenetic Protein and Hyaluronic Acid
US20150045887A1 (en) * 2012-03-22 2015-02-12 Trb Chemedica International S.A. Method for repair of ligament or tendon

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