COMPOSITION FOR THE PROTECTION AND REPAIR OF THE BLOOD BRAIN BARRIER

Abstract

The present invention relates to a pharmaceutical composition for its application as a drug, in particular for use in the protection of the blood-brain barrier and/or the repair and/or the restoration of the blood brain barrier. The present invention finds an application in particular in the therapeutic, pharmaceutical and veterinary fields.

Claims

1. A method for the protection and/or repair/restoration of the blood-brain barrier, comprising administering a pharmaceutical composition comprising: a biocompatible polymer of the following general formula (I)
AaXxYy   (I) in which: A represents a monomer, X represents an R.sub.1COOR.sub.2 group, or —R.sub.9(C═O)R.sub.10 Y represents an O or N-sulfonate group responding to one of the following formulas —R.sub.3OSO.sub.3R.sub.4, —R.sub.5NSO.sub.3R.sub.6, —R.sub.7SO.sub.3R.sub.8 in which: R1, R.sub.3, R.sub.5 and R.sub.9 independently represent an aliphatic hydrocarbon chain, optionally branched and/or unsaturated and which, optionally, contains one or more aromatic rings with the exception of benzylamine and benzylamine sulfonate, R2, R.sub.4, R.sub.6 and R.sub.8 independently represent a hydrogen atom or an M.sup.+ cation, and R.sub.7 and R.sub.10 independently represent a bond, an aliphatic hydrocarbon chain, optionally branched and/or unsaturated, a represents the number of monomers, x represents the degree of substitution of monomers A by X groups, y represents the degree of substitution of monomers A by Y groups to an individual in need thereof.

2. The method of claim 1, wherein the pharmaceutical composition further comprises hyaluronic acid.

3. The method of claim 1, wherein identical or different monomers A are selected among sugars, esters, alcohols, amino acids, nucleotides, nucleic acids, proteins or derivatives thereof.

4. The method of claim 1, wherein identical or different monomers A are selected among sugars or derivatives thereof.

5. The method of claim 1, wherein the number of monomer “a” is such that the mass of said polymers of formula (I) is greater than or equal to 2,000 daltons.

6. The method of claim 1, wherein x is between 10 and 150%.

7. The method of claim 1, wherein the degree of substitution “y” is between 10 and 170%.

8. The method of claim 1, wherein said biocompatible polymer further comprises functional chemical Z groups, different from X and Y, capable of imparting to said polymer additional biological or physicochemical properties.

9. The method of claim 8, wherein the degree of substitution of all the monomers A by Z groups represented by “z” is between 1 and 50%.

10. The method of claim 8, wherein the Z group is a substance capable of imparting to said polymers better solubility or lipophilicity.

11. The method of claim 8, the Z groups are identical or different and are selected among the group comprising amino acids, fatty acids, fatty alcohols, ceramides, or derivatives thereof, or even nucleotide sequences for addressing.

12. The method of claim 8, in which the R.sub.9 and R.sub.10 groups are independently and optionally substituted by a Z group.

13. The method of claim 1, wherein said pharmaceutical composition is administered parenterally at a dose of 0.1 to 5 mg/kg of body weight, and/or orally at a dose of 0.1 to 5 mg/kg of body weight, and/or intracranially at a dose of 0.1 to 100 μg.Math.ml.sup.−1.

14. The method of claim 2, wherein the concentration of hyaluronic acid is from 1 to 10 mg/ml.

15. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0114] FIG. 1 represents the evolution of the permeability of the blood-brain barrier (BBB) after an ischemic vascular accident according to time; the ordinate corresponds to the permeability and the abscissa to the time in hours.

[0115] FIG. 2 represents an example of the structure of a biocompatible polymer, for example the structure of the compound OTR4132.

[0116] FIG. 3 is a bar graph showing the evolution of BBB permeability in regions of interest studied by MRI. In this figure, the abscissa corresponds to the time in hours or days after cerebral ischemia: 1 h, 3 h, 24 h, 48 h, and 7 days after ischemia. The values obtained correspond to the mean +/−standard deviation. In this figure, the ordinate corresponds to the volume of modification of the integrity of the BBB in mm.sup.3. The values obtained for rats for which a composition comprising a biocompatible polymer (OTR4132) was administered are represented by white bars, the values obtained for rats for which a control composition was administered are represented by black bars.

[0117] FIG. 4 is a bar graph showing the permeability of the BBB after cerebral ischemia by Evans blue staining. In this figure, the ordinate represents the quantity of Evans blue in μg/g of cerebral tissue according to the ischemic region of the central nervous system, namely ipsilateral or contralateral. The values obtained for rats for which a composition comprising a biocompatible polymer (OTR4132) was administered are represented by gray bars, the values obtained for rats for which a control composition was administered are represented by black bars.

[0118] Other advantages may also appear to a person skilled in the art on reading the examples below, illustrated by the appended figures, given by way of illustration.

EXAMPLES

Example 1: Use of a Biocompatible Polymer for the Treatment of an Impaired Blood-Brain Barrier and Functional Restoration of the Blood-Brain Barrier

A/Preparation of Biocompatible Polymers.

[0119] The synthesis of biocompatible polymers, RGTA, is widely described in the prior art, for example in U.S. Pat. No. 7,396,923 entitled “Process for the sulfonation of compounds comprising free hydroxyl (OH) groups or primary or secondary amines” and also in the bibliographic reference Yasunori I. et al., Biomaterials 2011, 32: 769e776) and Petit E. et al., Biomacromolecules. 2004 March-April; 5(2):445-52 [28].

[0120] Several RGTAs are known and have been described, including the OTR4120 describes numerous preclinical and clinical publications (RGTA®-based matrix therapy—A new branch of regenerative medicine in locomotion. Barritault D, Desgranges P, Meddahi-Pellé A, Denoix J M, Saffar J L. Joint Bone Spine. 2017 May; 84(3):283-292. DOI: 10.1016/j.jbspin.2016.06.012 [29], RGTA® or ReGeneraTing Agents mimic heparan sulfate in regenerative medicine: from concept to curing patients Barritault D, Gilbert-Sirieix M, Rice K L, Sineriz F, Papy-Garcia D, Baudouin C, Desgranges P, Zakine G, Saffar J L, van Neck J. Glycoconj J. 2017 June; 34 (3): 325- 338. DOI: 10.1007/s10719-016-9744-5 [2]. The compound OTR4131 is a compound comprising a radical Z which is a fatty acid, namely acetic acid as described in Frescaline G. et al., Tissue Eng Part A 2013 July; 19 (13-14): 1641-53. DOI: 10.1089/ten.TEA.2012.0377 [30]), Randomized controlled trial demonstrates the benefit of RGTA® based matrix therapy to treat tendinopathies in racing horses. Jacquet-Guibon S, Dupays A G, Caudry V, Crevier-Denoix N, Leroy S, Sineriz F, Chiappini F, Barritault D, Denoix J M. PLoS One. 2018 Mar. 9; 13 (3): e0191796. DOI: 10.1371/journal.pone.0191796 [31]. Other compounds also described in patent documents US066897 41, US2014301972A 1 in which Z is an amino acid such as phenylalanine (Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds. Holmes B B, DeVos S L, Kfoury N, Li M, Jacks R, Yanamandra K, Ouidja M O, Brodsky F M, Marasa J, Bagchi D P, Kotzbauer P T, Miller T M, Papy-Garcia D, Diamond M I. Proc Natl Acad Sci US A 2013 Aug. 13; 110 (33): E3138-47. DOI: 10.1073/pnas.1301440110 [32]) or other hydrophobic compound (Structure-activity studies of heparan mimetic polyanions for anti-prion therapies. Ouidja M O, Petit E, Kerros M E, Ikeda Y, Morin C, Carpentier G, Barritault D, Brugere-Picoux J, Deslys J P, Adjou K, Papy-Garcia D. Biochem Biophys Res Commun. 2007 Nov 9; 363 (1): 95-100 [33]).

B/Functional Restoration of the Blood-Brain Barrier with a Biocompatible Polymer

[0121] In the present example, an evaluation of the effects of the biocompatible polymer according to the invention, RGTA, on the permeability of the BBB after alteration, for example following a cerebrovascular accident (CVA).

[0122] In this example, a rat CVA model was used. It was a cerebral ischemia of 1 hour, obtained by occlusion of the cerebral artery by intraluminal route followed by reperfusion. The rats used were male Sprague Dawley rats with an average weight of 300-350 g. The number of rats used was four to six rats per group per time. In this model, it is well known that the permeability of the BBB gradually increases to reach a peak at 24-48 h after the induction of cerebral ischemia (Garrigue et al. 2016 [6]; Sharif et al., 2018 [16]). FIG. 1 represents the change in permeability according to time in the model used as described in Abdullahi et al., 2018.

[0123] The BBB permeability evaluation was carried out by MRI after an injection of a contrast agent, Dotarem®, at different times: 1 h, 3 h, 24 h, 48 h, and 7 days after the cerebral ischemia. This contrast agent does not cross the BBB under physiological conditions. The contrast agent was injected intravenously, through the femoral vein. The amount of contrast agent administered by injection was 200 μmol/kg (Dotarem (registered trademark), Guerbet S.A).

[0124] Rats, namely four to five animals per group and per time, were treated with a biocompatible polymer, namely RGTA OTR4132 with a molecular weight of 100,000 to 150,000 Da. FIG. 2 represents the structure of said polymer. The biocompatible polymer OTR4132 was administered 1 hour after cerebral ischemia, the volume of composition administered comprising a concentration of 0.5 mg/kg of OTR4132 was 300 μl per tail vein.

[0125] Rats, namely four to five animals per group and per time were treated with a control solution i.e., physiological serum (0.9% NaCl saline solution). The control solution was administered in an identical manner to the composition comprising the compound OTR4132, namely 1 hour after cerebral ischemia, the volume of composition administered being 250 μl administered by the femoral vein.

[0126] The observation of the permeability and the diffusion of the contrast agent was carried out by observation on images obtained by MRI. The regions of the central nervous system observed were located in the cerebral hemisphere affected by ischemia as well as in the healthy contralateral hemisphere. The determination of the diffusion of the contrast agent on the images obtained was carried out by MRI analysis using appropriate software (Image J (trademark) (Wayne Rasband, NIMH, Maryland, USA)). Diffusion of the contrast agent and/or permeability of the blood-brain barrier are shown in FIG. 3.

[0127] As shown in the diagram of FIG. 3, the rats given the contrast agent and the control solution show an increased permeability of the blood-brain barrier 24 hours, 48 hours and 7 days after the CVA (black bars); these results were in agreement with those obtained in the state of the art (Garrigue et al. 2016 [6]). This diagram also clearly and unexpectedly demonstrates that the treatment of the rats with the biocompatible polymer OTR4132 makes it possible to significantly reduce the permeability of the BBB at 24 and 48 h post-ischemia in the group of rats treated with RGTA compared to the rats of the ischemic group that received the control solution. In particular, the results demonstrated a statistically significant difference between the rats treated with a control solution compared to the rats treated with a composition comprising a biocompatible polymer according to the invention (ANOVA followed by a post-hoc HSD test by Tukey p<0.05).

[0128] The results obtained and illustrated in FIG. 3 clearly demonstrate that the use of biocompatible polymers according to the invention makes it possible to preserve the integrity of the BBB after a CVA. In particular, these results clearly demonstrate that the use of a biocompatible polymer according to the invention makes it possible both to protect the BBB, to promote its repair and, in the case where the physiological properties of the BBB are altered/modified, to restore physiological properties and/or reduce their changes.

[0129] In addition to the results obtained by MRI, the permeability of the BBB was measured by staining with Evans blue after induction of cerebral ischemia according to the method described in the document Hone et al., 2018 [7]. The rats used were male Sprague Dawley rats with an average weight of 300-350 g, the experiment was carried out on 11 rats including five rats for which Evans blue, which does not cross the BBB under physiological conditions, was injected intravenously at a concentration of 2%, 72 h after cerebral ischemia, the volume injected being 4 ml/kg, i.e., 1.2 to 1.4 ml for a rat weighing 300 to 350 g respectively.

[0130] Six rats had been treated with a biocompatible polymer, namely RGTA OTR4132 with a molecular weight of 100,000 to 150,000 Da, administered 1 hour after cerebral ischemia, the volume of composition administered comprising a dose of 2.22 μg of OTR4132 were 50 μl intraarterially through the internal carotid.

[0131] Five rats had been treated with a control solution, namely physiological serum (0.9% NaCl saline solution) administered in an identical manner to the composition comprising the compound OTR4132, namely 1 hour after the cerebral ischemia, the volume of composition administered being 50 μl intraarterially through the internal carotid.

[0132] Thirty minutes after the administration of Evans blue, an intracardiac infusion with physiological saline was performed on the animal, the brain was removed and the hemispheres separated. The samples were then ground in phosphate buffered saline and then placed at 4° C. in the presence of 60% trichloroacetic acid. The samples were then centrifuged (1,000 g for 30 minutes) and the supernatant collected for a spectrophotometer reading at 610 nm. In parallel, an ascending concentration range of Evans blue was prepared.

[0133] The tissue Evans blue present in the collected supernatant was then quantified by spectrophotometry with a measurement at 610 nm.

[0134] FIG. 4 represents the results obtained according to the individuals. The results obtained in the group of control animals show a significant alteration in the permeability of the BBB in the ipsilateral hemisphere compared to the contralateral hemisphere (2-way ANOVA (p group=0.1582; p hemisphere=0.0933; p group*hemisphere=0.0175) followed by a Tukey HSD post hoc test p=0.0374). Unexpectedly, no difference was observed between the ipsilateral and contralateral hemispheres in the group of animals treated with the biocompatible polymer OTR4132 (2-way ANOVA (p group=0.1582; p hemisphere=0.0933; p group *hemisphere=0.0175) followed by a Tukey HSD post hoc test p=0.9965). In addition, the analyzes also demonstrate a statistically significant decrease in the alteration of the BBB in the ipsilateral hemisphere of the animals treated with the biocompatible polymer OTR4132 compared to the control animals (2-way ANOVA (p group=0.1582; p hemisphere=0.0933; p group*hemisphere=0.0175) followed by a Tukey HSD post hoc test p=0.0439).

[0135] This example clearly demonstrates that examples of a composition according to the invention comprising a polymer of formula AaXxYy or AaXxYyZz advantageously makes it possible to protect the BBB and/or to restore the physiological properties of the BBB. In particular, this example clearly demonstrates that examples of a composition according to the invention comprising a polymer of formula AaXxYy or AaXxYyZz make it possible to preserve the integrity of the BBB after a CVA. In particular, these results clearly demonstrate that the use of a biocompatible polymer according to the invention makes it possible both to protect the BBB, to promote its repair, and, in the case where the physiological properties of the BBB are altered/modified, to restore physiological properties and/or reduce their changes.

Example 2: Use of a Biocompatible Polymer for the Treatment of an Altered Blood-Brain Barrier and Functional Restoration of the Blood-Brain Barrier

[0136] An individual, a 75-year-old man (75 kg) suffering from neurological disorders, in particular cognitive disorders, attributed to several CVAs as noted by neurologists, which altered the blood-brain barrier, was treated with a biocompatible polymer, namely the compound OTR4120, a daily intake of 30 ml of a 100 μg/ml aqueous solution of OTR4120 over 45 days. The dose administered was 3 mg/day/for 75 kg or 40 μg/kg/day. Following the administration, an improvement in cognitive performance was observed by neurologists and also by the attending or referring physician and the individual's family.

[0137] Another individual, an 85-year-old woman with major memory problems, in particular difficulty in reading and recognizing individuals, particularly close relatives (families), inability to write, etc. The individual (weighing about 60 kg), diagnosed as having Alzheimer's with handicap coefficient thus implicating an alteration of the blood-brain barrier, was treated by twice-weekly sublingual intake of a dose of OTR4120 from 300 microL to 100 μg/ml or 0.5 μg (0.5 μg/kg twice weekly). After treatment for 6 months, the individual showed an improvement in cognitive functions, social relations, for example with her environment, especially relatives and medical personnel, was able to telephone, go out, see friends, play scrabble, etc. These improvements were in particular linked to an improvement and recovery of the functions of the blood-brain barrier.

References

[0138] 1. Abdullahi W, Tripathi D, Ronaldson PT. Blood-Brain Barrier Dysfunction in Ischemic Stroke: Targeting Tight Junctions and Transporters for Vascular Protection. Am J Physiol Cell Physiol. 2018 Jun. 27. doi: 10.1152/ajpce11.00095.2018. [Epub ahead of print] PubMed PMID: 29949404 [0139] 2. Barritault D, Gilbert-Sirieix M, Rice K L, Siñeriz F, Papy-Garcia D, Baudouin C, Desgranges P, Zakine G, Saffar J L, van Neck J. RGTA(®) or ReGeneraTing Agents mimic heparan sulfate in regenerative medicine: from concept to curing patients. Glycoconj J. 2017 June; 34(3):325-338. doi: 10.1007/s10719-016-9744-5. Epub 2016 Dec. 7. Review. PubMed PMID: 27924424; PubMed Central PMCID: PMC5487810. [0140] 3. Blaney, S. M., Balis, F. M., Berg, S., Arndt, C. A., Heideman, R., Geyer, J. R., & Aikin, A. (2005). Intrathecal mafosfamide: a preclinical pharmacology and phase I trial. Journal of clinical oncology, 23(7), 15551563. [0141] 4. Cardoso, F. L., Brites, D., & Brito, M. A. (2010). Looking at the blood—brain barrier: molecular anatomy and possible investigation approaches. Brain research reviews, 64(2), 328-363. [0142] 5. Erickson M A. and Banks W A. Neuroimmune Axes of the Blood—Brain Barriers and Blood—Brain Interfaces: Bases for Physiological Regulation, Disease States, and Pharmacological Interventions. Pharmacol Rev. 2018 April; 70(2):278-314. doi: 10.1124/pr.117.014647. Review. [0143] 6. Garrigue P, Giacomino L, Bucci C, Muzio V, Filannino M A, Sabatier F, Dignat-George F, Pisano P, Guillet B. Single photon emission computed tomography imaging of cerebral blood flow, blood-brain barrier disruption, and apoptosis time course after focal cerebral ischemia in rats. Int J Stroke. 2016 Jan;11(1):117-26. [0144] 7. Hone E A, Hu H, Sprowls S A, Farooqi I, Grasmick K, Lockman P R, et al. Biphasic Blood-Brain Barrier Openings after Stroke. Neurol Disord Stroke Int. 2018; 1(2): 1011. [0145] 8. Hynes R O, Naba A. Overview of the matrisome—an inventory of extracellular matrix constituents and functions. Cold Spring Harb Perspect Biol. 2012 Jan. 1; 4(1):a004903. doi: 10.1101/cshperspect.a004903. Review. PubMed PMID: 21937732; PubMed Central PMCID: PMC3249625. [0146] 9. Khelif, Y., Toutain, J., Quittet, M. S., Chantepie, S., Laffray, X., Valable, S., & Barritault, D. (2018). A heparan sulfate-based matrix therapy reduces brain damage and enhances functional recovery following stroke. Theranostics, 8(21), 5814. [0147] 10 Marks Jr, W. J., Ostrem, J. L., Verhagen, L., Starr, P. A., Larson, P. S., Bakay, R. A., . . . & Bartus, R. T. (2008). Safety and tolerability of intraputaminal delivery of CERE-120 (adeno-associated virus serotype 2—neurturin) to patients with idiopathic Parkinson's disease: an open-label, phase I trial. The Lancet Neurology, 7(5), 400-408. [0148] 11. Möbius-Winkler, S., Linke, A., Adams, V., Schuler, G., & Erbs, S. (2010). How to improve endothelial repair mechanisms: the lifestyle approach. Expert review of cardiovascular therapy, 8(4), 573-580. [0149] 12. Peschillo S, Diana F, Berge J, Missori P. A comparison of acute vascular damage caused by ADAPT versus a stent retriever device after thrombectomy in acute ischemic stroke: a histological and ultrastructural study in an animal model. J Neurointery Surg. 2017 August; 9(8):743-749. doi: 10.1136/neurintsurg-2016-012533. Epub 2016 Jul. 7. PubMed PMID: 27387708. PMID: 11152991. [0150] 13. Rafii, M. S., Baumann, T. L., Bakay, R. A., Ostrove, J. M., Siffert, J., Fleisher, A. S., & Chu, Y. (2014). A phasel study of stereotactic gene delivery of AAV2-NGF for Alzheimer's disease. Alzheimer's & Dementia, 10(5), 571-581. [0151] 14. Rajendran P, Rengarajan T, Thangavel J, Nishigaki Y, Sakthisekaran D, Sethi G,Nishigaki I. The vascular endothelium and human diseases. Int J Biol Sci. 2013 Nov. 9; 9(10):1057-69. doi: 10.7150/ijbs.7502. eCollection 2013. Review. PubMed PMID: 24250251; PubMed Central PMCID: PMC3831119. [0152] 15. Ronaldson, P. T., & Davis, T. P. (2015). Targeting transporters: promoting blood—brain barrier repair in response to oxidative stress injury. Brain research, 1623, 39-52. [0153] 16. Sharif Y, Jumah F, Coplan L, Krosser A, Sharif K, Tubbs R S. The Blood Brain Barrier: A Review of its Anatomy and Physiology in Health and Disease. Clin Anat. 2018 Apr. 10. doi: 10.1002/ca.23083. [Epub ahead of print] PubMed PMID: 29637627. [0154] 17. Sifat, A. E., Vaidya, B., & Abbruscato, T. J. (2017). Blood-brain barrier protection as a therapeutic strategy for acute ischemic stroke. The AAPS journal, 19(4), 957-972. [0155] 18. Steiner E, Enzmann G U, Lyck R, Lin S, Ruegg M A, Kroger S, and Engelhardt B. The heparan sulfate proteoglycan agrin contributes to barrier properties of mouse brain endothelial cells by stabilizing adherens junctions. Cell Tissue Res 358: 465-479, 2014. [0156] 19. Sweeney M D, Sagare A P, Zlokovic B V. Blood-brain barrier breakdown in Alzheimer disease and other neurodegenerative disorders. Nat Rev Neurol. 2018 March; 14(3):133-150. doi: 10.1038/nrneuro1.2017.188. Epub 2018 Jan. 29. Review. PubMed PMID: 29377008; PubMed Central PMCID: PMC5829048. [0157] 20. Teng D, Pannell J S, Rennert R C, Li J, Li Y S, Wong V W, Chien S, Khalessi A A. Endothelial trauma from mechanical thrombectomy in acute stroke: in vitro live-cell platform with animal validation. Stroke. 2015 April; 46(4):1099-106. doi: 10.1161/STROKEAHA.114.007494. Epub 2015 Feb. 24. PubMed PMID: 25712942. [0158] 21. Thomsen M S, Routhe L J, Moos T. The vascular basement membrane in the healthy and pathological brain. J Cereb Blood Flow Metab. 2017 October; 37(10):3300-3317. doi: 10.1177/0271678X17722436. Epub 2017 Jul. 28. Review. [0159] 22. Warren, K, E Beyond the Blood: Brain Barrier: The Importance of Central Nervous System (CNS) Pharmacokinetics for the Treatment of CNS Tumors, Including Diffuse Intrinsic Pontine Glioma Front. Oncol., 3 Jul. 2018 I https://doi.org/10.3389/fonc.20. Review [0160] 23. Wilgus A. 2012 Growth Factor—Extracellular Matrix Interactions Regulate Wound Repair. Advances in Wound Care, 1 (6): 249-254. doi: 10.1089/wound.2011.0344. [0161] 24. Vladimir A. Kornev et al: Hydrogel-assisted neuroregeneration approaches towards brain injury therapy: A state-of-the-art review. Computational and Structural Biotechnology Journal 16 j.csbj.2018.10.011. [0162] 25. Gopalakrishnan A, Shankarappa S A, Rajanikant G K. Hydrogel Scaffolds: Towards Restitution of Ischemic Stroke-Injured Brain 2019 February; 10(1):1-18. [0163] 26. Tammi R., Agren U M., Tuhkanen A L., Tammi M. Hyaluronan metabolism in skin. Progress in Histochemistry & Cytochemistry. 29(2):1-81, 1994 [0164] 27. R. Stern et al., European Journal of Cell Biology 58 (2006) 699-715. [0165] 28. Yasunori I. et al., Biomaterials 2011, 32 :769e776) et Petit E. et al,. Biomacromolecules. 2004 March-April; 5(2):445-52 [0166] 29. RGTA®-based matrix therapy—A new branch of regenerative medicine in locomotion. Barritault D, Desgranges P, Meddahi-Pellé A, Denoix J M, Saffar J L. Joint Bone Spine. 2017 May; 84(3):283-292. doi: 10.1016/j.jbspin.2016.06.012 [0167] 30. Frescaline G. et al., Tissue Eng Part A. 2013 July; 19(13-14):1641-53. doi: 10.1089/ten.TEA.2012.0377 [0168] 31. Randomized controlled trial demonstrates the benefit of RGTA® based matrix therapy to treat tendinopathies in racing horses. Jacquet-Guibon S, Dupays A G, Coudry V, Crevier-Denoix N, Leroy S, Siñeriz F, Chiappini F, Barritault D, Denoix J M. PLoS One. 2018 Mar. 9; 13(3):e0191796. doi: 10.1371/journal.pone.0191796 [0169] 32. Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds. Holmes B B, DeVos S L, Kfoury N, Li M, Jacks R, Yanamandra K, Ouidja M O, Brodsky F M, Marasa J, Bagchi D P, Kotzbauer P T, Miller T M, Papy-Garcia D, Diamond M I. Proc Natl Acad Sci U S A. 2013 Aug. 13; 110(33):E3138-47. doi: 10.1073/pnas.1301440110 [0170] 33. Structure-activity studies of heparan mimetic polyanions for anti-prion therapies. Ouidja M O, Petit E, Kerros M E, Ikeda Y, Morin C, Carpentier G, Barritault D, Brugere-Picoux J, Deslys J P, Adjou K, Papy-Garcia D. Biocshem Biophys Res Commun. 2007 Nov. 9; 363(1):95-100