Injectable viscoelastic gel containing polydeoxyribonucleotides

Abstract

The present invention refers to an injectable viscoelastic gel containing polydeoxyribonucleotides (PN / PDRN) extracted from natural sources, whether animal or plant, in an amount between 3 and 8% and to uses thereof for aesthetic and orthopaedic purposes. The polydeoxyribonucleotides have an average molecular weight between 50 and 500 bp and in aqueous solution, and at the appropriate concentrations, thanks to intermolecular bridges, naturally form a gel.

Description

[0001] Title

[0002] Injectable viscoelastic gel containing polydeoxyribonucleotides

[0003] The present invention refers to an injectable viscoelastic gel containing polydeoxyribonucleotides (PN / PDRN) extracted from natural sources, whether animal or plant and to uses thereof for aesthetic and orthopaedic purposes.

[0004] The polydeoxyribonucleotides consist of polymer chains of nucleotides of different lengths which, in aqueous solution and at the appropriate concentrations, thanks to intermolecular bridges, naturally form a gel.

[0005] The gel under study, despite not having undergone any chemical crosslinking process, showed rheological characteristics comparable to products based on cross-linked hyaluronic acid. The fillers currently on the market, in fact, have a more or less high degree of cross-linking thanks to the reaction of the linear hyaluronic acid with a cross-linking agent, generally an epoxide, such as 1,4-butanediol diglycidyl ether (BDDE): the cross-linking makes the polymer more resistant to the action of the tissue hyaluronidases, prolonging its filling effect at the intradermal level. However, these synthetic processes, implemented through the use of chemical cross-linkers, can leave reactive residues that often cause adverse reactions in the clinical setting.

[0006] Prior art

[0007] The use of polymer chains of nucleotides in anti-aging treatments to achieve orthoderma stems from over 50 years of experience with polynucleotides. The polynucleotides are considered important biostimulating agents due to their action on many different cell types, including dermal fibroblasts, as evidenced by the long list of international literature references on their use. Many in vitro studies demonstrate that the nucleotides and nucleosides, derived from the physiological degradation of PN / PDRN by tissue enzymes (ubiquitous DNAases) stimulate cell growth and the secretory activity of both collagen and other extracellular matrix proteins, also have a marked trophic action on human fibroblasts in primary cultures.

[0008] In light of scientific evidence, the clinical experiences in aesthetic medicine with PN / PDRNs have focused on skin bio-revitalization.

[0009] Intra-dermal infiltrations are able to build the extracellular matrix, with a rapid increase in elasticity, tone and firmness of the skin.

[0010] The PN / PDRNs have also shown a strong anti-radical action: during their biodegradation by enzymes located in the extracellular space, many metabolites (mono- and oligo-nucleotides) that are produced are available to increase the protective action against free radicals.

[0011] Prolonged iso-osmotic hydration and anti-radical action help to recreate the most favourable physiological conditions in the dermal matrix by stimulating the metabolic activity of fibroblasts and optimising their vitality and secretory activity.

[0012] Thanks to their unique characteristics, the PN / PDRN-based gels can be used as an anti-aging therapy to improve skin quality; recent market surveys show that the new trends in aesthetic medicine are increasingly focused on a new way of understanding facial fillers: abandoning excessive volumes in favour of a more natural effect. The PN / PDRNs with extraordinary bio-revitalizing abilities not only meet this new market demand, but also ensure patient rejuvenation without distortion by recovering the elasticity, tonicity and firmness of the treated skin.

[0013] These new gels can be used not only in aesthetic medicine, but also in orthopaedics.

[0014] The trophic-regenerating effects of PN / PDRNs have also been studied on primary cultures of cartilage and numerous clinical studies have shown their effectiveness in the treatment of diseases of the osteo-articular system.

[0015] The synovial fluid, like most biological fluids, has particular rheological characteristics thanks to the macromolecular component contained therein. It has been demonstrated that PN / PDRNs are capable of stimulating the deposition of extracellular matrix so that, at the intra-articular level, they would contribute not only to the improvement of the mechanical function, but also of the regenerative function, favouring the physiological recovery of the joint.

[0016] The present study, in fact, demonstrates that the high concentration PN / PDRN-based gel has completely unique visco-elastic characteristics and that, thanks to its ability to flow at high frequencies (comparable to the joint in motion), it is able to lubricate the joint, while, thanks to the elastic component measured at low frequencies, it is able to cushion shocks and act as a “bearing” at rest.

[0017] Formulations at PN / PDRN concentrations between 1,875 and 25 mg / ml are currently available on the market with applications in both the aesthetic and orthopaedic fields. At these concentrations the rheological parameters are not comparable to those of the cross-linked hyaluronic acid-based fillers and far this reason formulations containing higher concentrations of PN / PDRN have been investigated.

[0018] Below is a list of the main products available on the market in both the aesthetic (Tab.1) and orthopaedic fields (Tab, 2)

[0019] Table 1: Aesthetic medicine

[0020] Table 2: Orthopaedics From the study of the published bibliography on the rheological behaviour of DNA in aqueous solution (Bravo-Anaya et al. Polymers 2016, 8, 51; doi: 10.3390 / polym8020051; e Bravo-Anaya et al. Polymers 2016, 8, 279; doi: 10.3390 / polym8080279) it emerged that rheological analyses were carried out on samples at high concentrations and low molecular weight and at low concentrations and high molecular weight.

[0021] EP2358373B1 describes a composition based on polynucleotides extracted from natural sources for use in the treatment of degenerative diseases of the joints, in particular osteoarthritis; the polynucleotides have a molecular weight between 70 and 240 kDa and are present in an amount of 2% by weight, relative to the weight of the composition.

[0022] WO 2022 / 162556 describes a process for the production of a viscous gel based on polynucleotides, intra-articularly injectable, wherein said polynucleotides have an average molecular weight between 2600 and 3500 kDa, which finds application for the treatment of osteoarticular diseases in the medical and veterinary field.

[0023] The PN / PDRNs (extracted from trout gonads) have specific chemical, physical and biological characteristics thanks to their peculiar composition in polynucleotide fragments of different length which, in aqueous solution, tend to form a three-dimensional network such as to guarantee completely peculiar rheological characteristics.

[0024] It was therefore decided to investigate the rheological behaviour of the PN / PDRNs in order to assess how the presence of DNA fractions with different chain lengths within the same product affects the viscoelastic characteristics of the formulation. Since the PN / PDRNs have a wide distribution of molecular weights (between 50 and 2000 bp), we decided to investigate a range of concentrations starting from the one currently on the market up to the highest possible concentration for the PN / PDRNs under study, i.e. 100 mg / ml, beyond which the PN / PDRNs solubilise with difficulty, resulting in a gel that is too consistent to be adequately distributed and treated.

[0025] Definitions

[0026] Unless otherwise defined, all the terms of the art, notations and other scientific terms used herein are intended to have the meanings commonly understood by those who are skilled in the art to which this description pertains. In some cases, terms with commonly understood meanings are defined herein for clarity’s sake and / or ready reference; the insertion of such definitions in the present description must therefore not be interpreted as representative of a substantial difference with respect to what is generally understood in the art.

[0027] The term polydeoxyribonucleotides (PDRN) refers to a mixture of doublestranded deoxyribonucleotide polymers of different lengths, such as DNA, having a molecular weight preferably between 45 and 10000 base pairs; mixtures of partially or totally purified deoxyribonucleotide polymers also fall within this definition. Polydeoxyribonucleotides (PDRNs) are also often called polynucleotides (PNs).

[0028] The base pair or base pairs (base pair = BP) is the unit of measurement used to define the length of the double-stranded deoxyribonucleotide polymers, such as DNA chains; it is possible to convert this value into Kilodaltons (kDa) by means of the equivalence: IBP = 0.66 kDa

[0029] The term “average molecular weight (MW)”, both expressed in BP and in kDa, refers to the number average molecular weight (Mn).

[0030] The term “physiological solution” refers to a solution of one or more water- soluble compounds, such as salts, having osmolality values equal to the physiological one.

[0031] The term “buffered physiological solution” refers to a solution of one or more water-soluble compounds, such as salts, having osmolality and pH values equal to physiological ones.

[0032] The term “physiologically acceptable excipient” refers to a substance which lacks any specific pharmacological effect and which does not produce adverse reactions when administered to a mammal, preferably a human being. Physiologically acceptable excipients are well known in the art and are described, for example, in the Handbook of Pharmaceutical Excipients, sixth edition 2009, incorporated herein for reference.

[0033] The term “arthrosis" or “osteoarthritis” refers to arthritic or osteoarthritic disease in all its classifications, that is, in its primary form (i.e. idiopathic, caused by genetic factors), secondary form (caused by trauma, surgery, mechanical or septic problems), localized form (monoarticular) as well as generalized form (pluriarticular).

[0034] The term “aesthetic medicine” refers to a medical branch that deals with improving the quality of life of those who experience discomfort due to an imperfection.

[0035] The terms “comprising”, “having”, “including” and “containing” are to be understood as open terms (i.e. the meaning “comprising, but not limited to”) and are to be considered as a support also for terms such as “essentially consist of", “essentially consisting of", “to consist of’ or "consisting of".

[0036] The terms “consists essentially of", “consisting essentially of" are to be understood as semi-closed terms, which means that no other ingredient that affects the new characteristics of the invention is included (optional excipients can therefore be included).

[0037] The terms “consists of”, “consisting of” are to be understood as closed terms.

[0038] Except where expressly indicated, the percentages by weight are to be understood relative to the total weight of the composition.

[0039] Description of the invention

[0040] The object of the present invention is represented by an injectable gel containing polydeoxyribonucleotides having an average molecular weight between 50 and 500 bp and present in an amount between 3 and 8% by weight, relative to the weight of the injectable gel.

[0041] According to one aspect of the invention, the polydeoxyribonucleotides have an average molecular weight between 100 and 370 bp, preferably between 190 and 290 bp.

[0042] According to a further aspect of the invention the average molecular weight is preferably determined by electrophoresis on the agarose gel.

[0043] According to one aspect of the invention, the polydeoxyribonucleotides are present in an amount between 3.1 and 6% by weight, preferably between 3.2 and 5%, even more preferably between 3.3 and 4.5%, relative to the weight of the injectable gel. According to one aspect of the invention, the injectable gel has a complex viscosity between 20 and 200 Pa-s, preferably between 25 and 100 Pa.s, at a temperature of 25 °C.

[0044] According to one aspect of the invention, the injectable gel has a viscosity between 5 and 20 Pa-s at a shear rate of 10s'1, preferably between 6 and 18 Pa-s, at a temperature of 25 °C.

[0045] According to one aspect of the invention, the injectable gel has a viscosity between 1 and 10 Pa-s at a shear rate of 100s'1, preferably between 1 and 8 Pa-s, more preferably between 1 and 4 Pa-s, at a temperature of 25 °C.

[0046] According to one aspect of the invention, the injectable gel has a residual % elastic modulus (G!% residual), between 70% and 100%, preferably between 70 and 75%, at a temperature of 25CC.

[0047] According to one aspect of the invention, the injectable gel has a residual % complex modulus (G* % residual), between 70% and 100%, preferably between 70 and 75%, at a temperature of 25 °C.

[0048] According to one aspect of the invention, the injectable gel has a residual % viscous modulus (G” % residual), between 65% and 100%, preferably between 75% and 100%, at a temperature of 25 °C.

[0049] According to one aspect of the invention, the residual % elastic modulus, the residual % complex modulus and / or the residual % viscous modulus are preferably determined by rotational rheometry.

[0050] According to one aspect of the invention, the injectable gel contains one or more physiologically acceptable excipients.

[0051] According to one aspect of the invention, the injectable gel contains saline solution, preferably buffered, more preferably buffered with phosphate buffer; according to a further aspect of the invention, the injectable gel contains from 92 to 97% by weight of the aforesaid saline solution, preferably from 94 to 96.9%, more preferably from 95 to 96.8%, even more preferably from 95.5 to 96.7%.

[0052] According to one aspect of the invention, the injectable gel contains one or more additional active ingredients, such as for example amino acids, bone and tissue substitutes, hyaluronic acid, hyaluronates and / or anaesthetics, preferably lidocaine; according to a further aspect of the invention, the injectable gel does not contain chitosan and / or its salts and / or its derivatives.

[0053] According to one aspect of the invention, polydeoxyribonucleotides contained in the injectable gel are extracted from natural sources, preferably from fish sperm or gonads.

[0054] According to one aspect of the invention, the injectable gel is administered intra-articularly.

[0055] According to a further aspect of the invention, the injectable gel is used in the treatment of degenerative or post-traumatic diseases of the joints, in particular in the treatment of arthrosis or osteoarthritis, or in aesthetic medicine.

[0056] Experimental section

[0057] The polymeric chains of nucleotides have a high hydration capacity and, in aqueous solution, form hydrogels with different visco-elastic characteristics depending on the length of the chains and the dissolution conditions (pH, solvent, osmolarity and concentration). Several PN / PDRN-based formulations having broad molecular weight distribution (50-2000 bp with higher concentration between 100 and 1000 bp) in physiological saline were studied.

[0058] This particular and wide distribution of the molecular weights makes it possible to obtain highly interesting formulation characteristics. The preparations based on PN / PDRN, described below, have rheological properties similar to the cross-linked hyaluronic acid (hereafter HAc) gels, but without having to perform synthetic chemical modifications with crosslinking agents. These synthetic processes implemented through the use of chemical cross-linkers can leave reactive residues and cause adverse reactions in the clinical setting.

[0059] Most of the cross-linked hyaluronic acid-based gels currently on the market have high visco-elasticity values (as shown in Table 3 and 4) thanks to the cross-linking process capable of originating a stable network given by the intermolecular covalent bonds that make it more resistant to hyaluronidases. As is known, in fact, the cross-linking of hyaluronic acid allows obtaining a filler product (dermal filler) that is reabsorbed more slowly than the linear molecule.

[0060] Table 3 (Data reported in Fagien et al)

[0061] The same filters were analysed in the Mastelii R&D laboratory, using a different instrumentation than that used as a bibliographic reference (1. Fagien S, Bertucci V, von Grote E, Mashbum JH. Rheologic and Physicochemical Properties Used to Differentiate Injectable Hyaluronic Acid Filler Products. Plast Reconstr Surg. 2019 Apr; 143(4) :707e-720e. DOI: 10. 1097 / PRS.0000000000005429. PMID: 30921116; PMCID: PMC7597953)

[0062] Table 4 (Data obtained from the Lab. R&D Mastelii)

[0063] Similarly, a higher concentration of PN / PDRN in the injected product (compared to those currently on the market), despite having weak intermolecular bonds, could prolong the stay at the tissue level, as the DNases would have more substrate to digest, thus obtaining prolonged efficacy in situ.

[0064] In preparing the various formulations at increasing concentrations of PN / PDRN, several parameters including visco-elastic ones were analysed and, unexpectedly, quite peculiar rheological behaviours emerged. Outlines of rheology and main definitions

[0065] Rheology studies the deformation characteristics of a material under the action of external forces. The rheological analysis methods can be divided into two clearly distinct classes:

[0066] - The methods that allow assessing the flowing capacity (viscosity) of the material;

[0067] - The methods that define the visco-elastic and deformability properties of the material.

[0068] Viscosity measurement

[0069] To evaluate the viscosity of a sample, a mechanical action is applied with constant direction and sense, forcing the material to flow in that direction and sense. The measure of the resistance with which the material will oppose this flowing will allow to evaluate its degree of flowability.

[0070] The parameters that are used are:

[0071] » Viscosity q (Pa x s): indicates the degree of resistance to flow, the higher its value, the lower the flowing capacity of the material.

[0072] • Shear Rate (s-1): indicates the speed with which the material is made to flow.

[0073] Measurement of the Visco-Elasticity

[0074] The mechanical action applied in the Visco-Elasticity measurements is of an oscillatory nature: while the direction of the mechanical stress is constant, the sense follows a frequency-controlled sinusoidal oscillation.

[0075] Two types of measurement can be distinguished:

[0076] * the first in which an increasing deformation is applied with a constant frequency oscillation. This measurement allows to determine the maximum deformability of the material (LVER: Linear Visco-Elastic Region) (Figure 1);

[0077] * the second provides for the application of a constant deformation while the frequency oscillation varies. The deformation applied during this measurement must be chosen in such a way that the mechanical structure of the sample is not compromised, i.e. the test must be performed in compliance with LVER.

[0078] The main parameters used during Visco-Elasticity measurements are:

[0079] The term “residual % modulus” referring to the elastic modulus (G’)> the viscous modulus (G”) and the complex modulus (G*), indicates the percentage of the rheological parameters maintained by the formulation following a stress, whether thermal or other nature, which may cause a decrease; it is calculated by means of the equation reported below. 100

[0080] The samples analysed were prepared as described below.

[0081] Sample preparation and analytical technique Seven formulations with different concentration of polynucleotides having the following weight percent compositions (summarized in Tab.5) were prepared in the laboratory.

[0082] Table 5

[0083] Example 1 (PN20):

[0084] 2 g of Polynucleotides and 98 g of buffered saline solution were transferred into a calibrated flask; the buffered aqueous solution used in both this and subsequent examples was water containing phosphate buffer and sodium chloride. The mixture is kept under stirring until it is completely dissolved. To facilitate solubilisation, it is possible to heat in a water bath while maintaining the temperature at around 65 °C. Upon complete dissolution, the final weight is checked again, if necessary it is returned to 100 g with water for injectable preparations (WFI).

[0085] This example represents the current state of the art of the commercially available polynucleotide formulations. Compared to this sample, it is possible to identify the differences with the new formulations.

[0086] Example 2 (PN30):

[0087] 3 g of Polynucleotides and 97 g of buffered saline solution were transferred into a calibrated flask. The mixture is kept under stirring until it is completely dissolved. To facilitate solubilisation, it is possible to heat in a water bath white maintaining the temperature at around 65 °C. Upon comptete dissolution, the final weight is checked again, if necessary it is returned to 100 g with WFI water.

[0088] Example 3 (PN40):

[0089] 4 g of Polynuoteotides and 96 g of buffered saline solution were transferred into a calibrated flask. The mixture is kept under stirring until it is completely dissolved. To facilitate solubilisation, it is possible to heat in a water bath while maintaining the temperature at around 65 °C. Upon comptete dissolution, the final weight is checked again, if necessary it is returned to 100 g with WFI water.

[0090] Example 4 (PN50):

[0091] 5 g of Polynucleotides and 95 g of buffered saline solution were transferred into a calibrated flask. The mixture is kept under stirring until it is completely dissolved. To facilitate solubilisation, it is possible to heat in a water bath while maintaining the temperature at around 65 °C. Upon complete dissolution, the final weight is checked again, if necessary it is returned to 100 g with WFI water.

[0092] Example 5 (PN60):

[0093] 6 g of Polynucleotides and 94 g of buffered saline solution were transferred into a calibrated flask. The mixture is kept under stirring until it is completely dissolved. To facilitate solubilisation, it is possible to heat in a water bath white maintaining the temperature at around 65 °C. Upon comptete dissolution, the final weight is checked again, if necessary it is returned to 100 g with WFI water.

[0094] Example 6 (PN80): 8 g of Polynucleotides and 92 g of buffered saline solution were transferred into a calibrated flask. The mixture is kept under stirring until it is completely dissolved. To facilitate solubilisation, it is possible to heat in a water bath while maintaining the temperature at around 65 °C. Upon complete dissolution, the final weight is checked again, if necessary it is returned to 100 g with WFl water.

[0095] Example 7 (PN100):

[0096] 10 g of Polynucleotides and 90 g of buffered saline solution were transferred into a calibrated flask. The mixture is kept under stirring until it is completely dissolved. To facilitate solubilisation, it is possible to heat in a water bath while maintaining the temperature at around 65 °C. Upon complete dissolution, the final weight is checked again, if necessary it is returned to 100 g with WFl water.

[0097] The preparations were made with techniques similar to those implemented in the production department.

[0098] At the end of the preparation, each formulation was then divided into 3 ml syringe vials. For each batch, some samples were subjected to thermal cycling (TT), white others were not subjected to it (NTT). The formulations were subsequently analysed to study their rheological and extrusion characteristics and to evaluate their molecular size.

[0099] The tools used to perform the analyses are:

[0100] - Rheological Analysis: performed using a Kinexus Ultra+ Rheometer at a controlled temperature of 25 °C with PU20 geometry. Evaluating first the LVER of the sample and then performing the Amplitude table test (0.1-100% Shear Strain at 1Hz) and the Viscometry Shear rate Tabie test (0.1-1000s '1).

[0101] - Molecular weight assessment: performed by electrophoresis on 1% Agarose Gel, using a DNA Standard that covers at least a distribution between 50 and 4000bp using Gel Green or Gel Red as an electrophoretic tracer and analysing the results by trans illuminator.

[0102] - Extrusion test: performed with digital dynamometer (100N) on Mecmesin motorized stand, extruding 10mm at 12mm / min with 1ml BD syringe (Hylok™ WILL) and BD needle (microlance™ 3) 27g%” and with 3ml BD syringe (Hypak™ 3ML) with BD needle (microlance ™ 3) 30G7Z.

[0103] Assessing the results

[0104] It was noted that, under particular conditions, related to the molecular weight distribution of the PN / PDRNs and their concentration in solution, it is possible to obtain an unexpected and substantial increase in rheological parameters. The different molecular weights of the PN / PDRN mixture used for the preparation of the formulations allow an optimal intercalation of the chains, as the smaller ones (50-200 bp) manage to insert into and interact with the longer chains (200-2000 bp) forming countless intermolecular bonds mainly represented by ionic bonds and hydrogen bonds (also referred to as cross-bridges). The concentration, on the other hand, affects the number of possible interactions between the polymer chains. At too low PN / PDRN concentrations, in fact, there is not a sufficient number of molecular interactions to obtain the desired highly elastic consistency. On the contrary, excessively high concentrations of PN / PDRN give rise to overly consistent formulations that can cause considerable difficulties in their use in clinical practice with consequent increased risks for the patient.

[0105] In the experiments performed, taking as reference the formulations already known at 20 mg / ml, it was possible to increase the elastic (G’) and viscous (G”) moduli by almost 200% by adding only 50% of PN / PDRN in solution. Unexpectedly, by doubling the amount of PN / PDRN, thus taking into account the 40 mg / ml formulation, the rheological parameters increase by more than 400% (Tab. 6. 7 and 8).

[0106] Tab.6 Values of Elastic Modulus (G!) Tab.7 Values of Viscose Modulus (G”)

[0107] Tab.8 Values of Complex Viscosity (q*)

[0108] The high viscoelasticity, therefore, would make it possible to obtain a product that could last longer if injected intradermally or intra-articularly, prolonging the therapeutic effect of bio-rivitalization even without chemical crosslinking.

[0109] The particular mixture of molecular weights, an intrinsic and exclusive characteristic of Mastelli PN / PDRN, in fact, allows at the appropriate concentrations, an optimal intercalation of the chains that are able to form intermolecular bonds capable of stabilizing the gel making it as consistent as a cross-linked gel, without the need for a chemical synthesis to stabilize the polymers. The bonds, which are established between the PN / PDRN chains, unlike the covalent ones obtained synthetically, are able to break and form again, making these formulations also suitable for intra-articular use. In the data reported below (Tab. 9), inherent to the viscosity evaluation, the gels were subjected to different Shear Rate conditions to measure the viscosity of the material (defined as resistance to flow). For moderate stresses, i.e. low Shear Rate, the gels must have a good viscosity, in order to offer protection of the articular cartilage. When the joint is in motion, it is important that the formulation offers a lower viscosity, i.e. less resistance to flow, to ensure optimal lubrication of the articular joints. However, to ensure adequate protection of the articular surfaces, it is preferable to maintain viscosity values that are not too low (not lower than I Pa s) at high shear rates. The gels at high PN / PDRN concentration according to the present invention have in fact good viscosity values at low shear rates, compared to gels currently on the market having a PN / PDRN concentration of 20 mg / ml; which viscosity values decrease by increasing the deformation rate, while always remaining above 1 Pa s.

[0110] Table 9 Viscosity Assessment

[0111] Making the parallelism with the cross-linked HA gels, these are characterized by a high viscosity at rest and a good protection capacity of the joint, but at high Shear Rate they often excessively oppose to flowing.

[0112] This is because the covalent molecular bridges of HAc do not have the ability to break and recreate themselves as is the case with the weak bonds that characterize the PN / PDRN-based formulations.

[0113] This chemical characteristic causes HAc gels to deform and move under stress, without actually flowing and lubricating.

[0114] Another interesting and unexpected aspect that emerged from the study of the formulations based on PN / PDRN at high concentration concerns the greater resistance to the heat treatments exhibited by these formulations.

[0115] By treating the rheological data of the formulations before and after undergoing heating cycles, in particular by making the ratio of the moduli G*, G' and G" of the formulations after heat treatment (TT) and pre-heat treatment (NTT), it is possible to monitor the residue that can be found after the action of the heat, i.e. to detect the % of the rheological properties maintained after the heat treatment.

[0116] We have therefore unexpectedly noticed that, at higher PN / PDRN concentrations, the gel suffers less negative influence from the heat treatment, with better preservation of the rheological characteristics of the same.

[0117] In fact, there is a significant increase in the residual rheological characteristics relating to the elastic modulus (G’), going from 65% of the reference formulations (20 mg / ml) to more than 90% of the most concentrated solutions (Tab.10, 11 and 12).

[0118] Tais.10 Complex Modulus Values IG"; Tab.11 values of Elastic Modulus

[0119] The viscous modulus (G"), on the other hand, seems to be more sensitive to the concentration, highlighting how excessive concentrations (>50mg / ml) lead to a gradual decrease in the protective effect towards rheological parameters, although they are maintained higher than those of reference.

[0120] In order to investigate this aspect, we performed a series of molecular weight (M.W.) analyses of the TT and NTT formulations using Electrophoresis on 1% Agarose Gel in TBE 1X with direct Gel Green staining. The purpose of the analysis was to assess whether the rheological differences found in the post-treatment (TT) were attributable to a different impact of the heat cycle on the molecular weights, or if it was a characteristic of the formulation (Tab, 13),

[0121] Tabte 13

[0122] Analysing the data it is clear that in the formulations that have undergone a heat treatment (TT in Figure 2) the molecular weights are absolutely superimposable, demonstrating that the thermal stress acts uniformly on the DNA regardless of the concentration of dissolution and that the greater heat resistance is a characteristic of the formulation. This unexpected but very interesting aspect makes it possible to obtain a product that is less affected by heat treatments such as tindalization and sterilization, making it possible to proceed with production cycles with higher safety, without drastically affecting the characteristics of the formulation.

[0123] A last evaluation parameter taken into account was the extrusion force.

[0124] In this test, the force necessary to extrude the product contained in the syringe vial through a certain needle is measured. The purpose of the test is to simulate the administration by the doctor and evaluate the force required to perform it. For this reason, the standard analysis conditions are selected. It was decided to use an extrusion speed of 12mm / min and as a reference the 1ml syringe vials with 27 G Vz needle usually used for the HAc fillers available on the market.

[0125] The degree of injectability is then defined based on the recorded extrusion force (Robinson T.E. et al. - Filling the gap: A correlation between objective and subjective measures of injectabiiity Advanced Healthcare Materials (2020), 9, 1901521).

[0126] For this type of test, 38N is considered as the limit of acceptability.

[0127] The measured results for the formulations from PN20 TT to PN100 TT are reported below in Tab. 14

[0128] Table 14

[0129] Under these conditions all formulations proved usable and generated extrusion forces comparable to the normal HAc fillers (measured under the same conditions) while having much higher concentrations (Tab. 15).

[0130] Table 15

[0131] We therefore wanted to evaluate the usability of the same formulations even under conditions that increase the effort required for administration, using a larger diameter syringe (3 ml BD) and a narrower needle (30G7z"). These conditions are not usually used with the HAc fillers, but are used for less viscous and less consistent formulations such as bio-revitalizers (Tab. 16). Under these conditions, the concentrations that had demonstrated the best elastic modulus values (30 mg / ml and 40 mg / ml) proved to be administrable and fully usable.

[0132] Table 16 Conclusions

[0133] The new PN / PDRN formulations highlighted interesting characteristics both when compared to the reference formulations (20 mg / ml) and compared to the HAc gels.

[0134] Regarding the use for aesthetic purposes, we were able to obtain polymeric hydrogels with rheological parameters comparable to the fillers but with the advantage of not intervening on the molecules with synthetic steps that involve the use of crosslinking agents. The peculiar rheological characteristics of the gel make it a promising candidate for intradermal and subcutaneous use with interesting volumetric characteristics, but capable of allowing modelling, making it applicable even in areas subjected to stresses such as the lips and perioral area.

[0135] As regards the possible use in the orthopaedic field of the formulations subject-matter of the invention, despite the high consistency at rest, they, under stress, are able to flow without clearly opposing the movement, a very important characteristic for visco-supplementation.

[0136] However, the higher concentration would allow the treatments to be split over time, minimising discomfort for the patient.

[0137] For both possible applications (Aesthetic Medicine and Orthopaedics), the high flowability at high Shear Rate allows the force required for extrusion to be kept low, guaranteeing the doctor an easy and controllable administration. These particular formulations have also demonstrated beter resistance to the heat cycles, which translates into greater preservation of the rheological characteristics of the product even if subjected, for example, to terminal sterilization, allowing the maximum safety profile and maintaining high quality parameters.

Claims

Claims1. Injectable gel containing polydeoxyribonucieotides, characterized in that said polydeoxyribonucieotides have an average molecular weight between 50 and 500 bp and are in an amount between 3 and 8% by weight, relative to the weight of the injectable gel.

2. injectable gel according to claim 1, characterized in that said polydeoxyribonucleotides have an average molecular weight between 100 and 370 bp, preferably between 190 and 290 bp.

3. Injectable gel according to any one of the preceding claims, characterized in that said polydeoxyribonucleotides are present in an amount between 3.1 and 6% by weight, preferably between 3.2 and 5%, even more preferably between 3.3 and 4.5%.

4. Injectable gel according to any one of the preceding claims, characterized in that it has a complex viscosity between 20 and 200 Pa-s, preferably between 25 and 100 Pa s, at a temperature of 25 °C.

5. Injectable gel according to any one of the preceding claims, characterized in that it has a viscosity between 5 and 20 Pa s at a shear rate of 10s'1, preferably between 6 and 18 Pa-s, at a temperature of 25CC.

6. Injectable gel according to any one of the preceding claims, characterized in that it has a viscosity between 1 and 10 Pa-s at a shear rate of 100s'1, preferably between 1 and 8 Pa-s, more preferably between 1 and 4 Pa s, at a temperature of 25 °C.

7. Injectable gel according to any one of the preceding claims, characterized in that it has a residual % elastic modulus (G* % residual), between 70% and 100%, preferably between 70 and 75%, at a temperature of 25 °C.

8. Injectable gel according to any one of the preceding claims, characterized in that it has a residual % complex modulus (G* % residual), between 70% and 100%, preferably between 70 and 75%, at a temperature of 25 °C.

9. Injectable gel according to any one of the preceding claims, characterized in that it has a residual % viscous modulus (G” % residual), between 65% and 100%, preferably between 75% and 100%, at a temperature of 25 °C.

10. Injectable gel according to any one of claims 7-9, characterized in that said residual % elastic modulus, said residual % complex modulus and / or said residual % viscous modulus are determined by rotational rheometry.

11. Injectable gel according to any one of the preceding claims, characterized in that it contains one or more physiologically acceptable excipients.

12. Injectable gel according to any one of the preceding claims, characterized in that it contains saline solution.

13. Injectable gel according to any one of the preceding claims, characterized in that it contains from 92 to 97% by weight of saline solution, preferably from 94 to 96.9%, more preferably from 95 to 96.8%, even more preferably from 95.5 to 96.7%.

14. Injectable gel according to any one of the preceding claims, characterized in that it contains one or more additional active ingredients, such as for example amino acids, bone and tissue substitutes, hyaluronic acid, hyaluronates and / or anaesthetics, preferably lidocaine.

15. Injectable gel according to any one of the preceding claims consisting essentially of said polydeoxyribonucleotides and said saline solution and, optionally, amino acids, bone and tissue substitutes, hyaluronic acid, hyaluronates and / or anaesthetics, preferably lidocaine.

16. Injectabie gel according to any one of the preceding claims, characterized in that it does not contain chitosan and / or its salts and / or its derivatives.

17. Injectable gel according to any one of the preceding claims, consisting of 3 to 8% by weight of said polydeoxyribonucleotides and 92 to 97% by weight of saline, preferably 3.1 to 6% of said polydeoxyribonucleotides and 94 to 96.9% of saline solution, more preferably 3.2 to 5% of said polydeoxyribonucleotides and 95 to 96.8% of saline solution, still more preferably 3.3 to 4.5% of said polydeoxyribonucleotides and 95.5 to 96.7% of saline solution.

18. Injectable gel according to any one of claims 12-17, characterized in that said saline solution is buffered, preferably with phosphate buffer.

19. Injectable gel according to any one of the preceding claims, characterized in that said polydeoxyribonucleotides are extracted from natural sources, preferably from fish sperm or gonads.

20. Injectable gel according to any one of the preceding claims, for use as a medicament.21 . Injectable gel according to claim 14, for use in the treatment of degenerative or post-traumatic diseases of the joints, in particular in the treatment arthrosis or osteoarthritis.

22. Injectable gel according to claim 14, for use in aesthetic medicine.

23. Injectable gel according to any one of claims 14 to 16, characterized in that it is administered intra-articularly.

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