PROCESS FOR PRODUCING A POLYNUCLEOTIDE-BASED GEL AND USE THEREOF IN THE TREATMENT OF OSTEOARTICULAR DISEASES

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DESCRIPTION

FIELD OF THE INVENTION

The present invention concerns a process for producing a polynucleotide-based gel and the use thereof in the treatment of osteoarticular diseases.

The present invention originates in the pharmaceutical field, in particular in the field of medicaments for orthopedic use and of industrial processes for the production thereof.

Specifically, the present invention relates to a process for producing an intraarticularly injectable viscous gel containing polynucleotides and the use thereof in the orthopedic field in reducing joint inflammation or pain and stimulating repair processes in the joints of a human being or animal.

PRIOR ART

Formulations based on polynucleotides (PN) are known and the cosmetic or medical use thereof in humans is also known.

From the scientific literature it is known that the administration, by intra-articular injection, of aqueous PN-based solutions enriches the synovial fluid with nucleotides, purine and pyrimidine bases which support cellular metabolism and help to restore physiological conditions in the environment into which they are injected.

The publication in the name of Saggini, J Biol Regul Homeost Agents 2013;27:543-9. shows how the infiltration of polynucleotide-based products results in a metabolic effect and hydration of the joint, and also an improvement in the synovial fluid viscoelasticity.

Giarratana et al., Knee 2014;21:661-8 reported how the treatment with intraarticular injections of PN results in a reduction in pain perceived by the individual treated and supports the cartilage healing process. In the publication in the name of Vanelli et al., Knee Surg Sports Traumatol Arthrosc 2010;18:901-7 the results of clinical studies with intra-articular PN injections are reported. The experimental data show that the results achieved with the treatment are stable.

Mun. et al., Medicine (Baltimore); 2017 Dec; 96(49): e9127 show how the use of intra-articular injections of PN have proven effective in reducing pain, as an alternative to hyaluronic acid injections, in patients with knee osteoarthritis.

The use of injectable formulations based on hyaluronic acid in the treatment of osteoarticular diseases of humans and animals, in particular dogs and horses, is also known. Literature data have shown that the administration of these formulations results in a decrease in pain, joint inflammation and lameness, and a reduction in cartilage degradation in the subject treated with hyaluronic acid.

In particular, the literature reports that the infiltration of hyaluronic acid results in an improvement of the clinical conditions in subjects suffering from an osteoarticular disease, in particular with traumatic joint injuries and/or osteoarthritis.

However, to obtain a good clinical result and consolidate it over time, it is necessary to provide for a series of 4-5 intra-articular infiltrations with hyaluronic acid, at intervals of 7-14 days. The high costs of such a therapy often favor treatments with alternative medications to hyaluronic acid, such as non-steroidal anti-inflammatory drugs (NSAIDs).

However, the use of NSAIDs is not free of side effects, including important ones that generally occur in the gastrointestinal mucosa.

Furthermore, when treating animals, for example in the horse-riding sector, these drugs are labeled as doping and their use is prohibited in the context of sports competitions.

In addition, the prolonged use of NSAIDs in cases of chronic osteoarticular diseases may be associated with the onset of serious side effects, in particular gastroduodenal ulcers, and gastrointestinal system bleeding.

Therefore, there is a need to have medicaments for intra-articular use as an alternative to those based on hyaluronic acid currently used.

One of the objects of the invention therefore consists in providing a process for producing an injectable formulation for intra-articular administration that is suitable for the treatment of osteoarticular diseases.

Another object is to provide a gel for intra-articular injectable use whose infiltration increases the synovial fluid viscoelasticity, helping to exert a mechanical effect within a mammalian joint.

SUMMARY OF THE INVENTION

As part of the research activity towards medicaments for the treatment of osteoarticular diseases, a process has been developed to produce a highly viscous injectable preparation based on polynucleotides which allows to keep their chemical-physical properties almost unchanged, and to obtain a product with stable and reproducible rheological properties.

In particular, the inventors have identified specific process conditions that make it possible to obtain a sterile polynucleotide-based gel that, when injected intraarticularly, increases the synovial fluid viscoelasticity and exerts a combined metabolic and mechanical effect on mammalian joints, resulting in a rapid improvement of osteoarticular diseases and related symptoms.

The inventors also observed that an injectable gel based on polynucleotides or polydeoxyribonucleotides (PDRN), preferably in the form of a salt, such as a sodium salt, with average molecular weight from 2600 to 3500 kDalton, specifically selected in the range from 2800 to 3300 kDalton, has viscoelastic characteristics that compensate for the loss of viscosity of the synovial fluid in cases of degenerative or traumatic change, thus reducing joint friction, mechanical stress on them, and the associated pain.

Furthermore, the intra-articular administration of a therapeutically effective amount of the PN gel described herein increases the mobility of the treated joint.

The viscoelastic characteristics of the gel are mainly linked to the selected weight average molecular weight of the polynucleotide component (PN/PDRN), and partly with the preparation process, in particular with the neutralization step using HCI up to a neutral pH value at which the solution viscosity increases and the transition from liquid to viscoelastic gel occurs.

Advantageously, the gel of the invention finds application in the medical sector, in particular in the treatment or prevention of osteoarticular diseases of an individual. It was also observed that the gel described herein restores the physiology of the intra-articular environment by providing PN as additional functional nourishment for cartilage homeostasis and has a moisturizing action.

According to one aspect, a process for producing a sterile injectable gel based on polynucleotides is therefore provided, characterized in that it comprises the following steps:

- providing a sodium hydroxide aqueous solution with molarity comprised in the range from 0.1 M to 0.4M.

- dissolving deoxyribonucleotides sodium salt (DNA sodium salt), typically highly polymerized (HPDR-Na), at a concentration comprised in the range from 0.5 to 8.5%, from 1 % to 8%, preferably from 2 to 6% by weight, in said aqueous sodium hydroxide solution until a clear solution having basic pH is obtained.

- filtering the basic solution of HPDR-Na obtained, preferably using filters with a sieve lower than 0.5pm, preferably lower than 0.3 pm.

- adding a HCI aqueous solution with a molarity comprised between 0.1 M and 0.4M, preferably filtered using filters with a sieve lower than 0.5 pm, preferably lower than 0.3 pm, for example 0.2 pm, to the basic solution of polymerized deoxyribonucleotide sodium salt (HPDR-Na) preferably in an amount such as to bring the pH of the final solution from 5.0 to 10, 8.5-9.5, in particular 9.0, with an increase in the viscosity of the solution which turns from a liquid into a viscoelastic gel.

Preferably the gel/hydrogel obtained from the process described herein is placed in an autoclave and heated, for example at 85°C for 5 minutes. At the end of the cycle, a product in the form of a gel is obtained which does not undergo any changes from a chemical-physical and rheological point of view.

In some embodiments, in the process described herein or as defined in claim 1 , the concentration of polynucleotides or polydeoxyribonucleotides is comprised in the range from 3% to 5% w/w, and more preferably the osmolarity is comprised in the range from 250 to 350 mOsm/Kg.

Advantageously, the product obtained from the process described herein is in the form of a viscoelastic gel or hydrogel, preferably having a dynamic viscosity comprised in the range from 28 to 35 Pa*s.

According to some embodiments, the starting polynucleotides or polydeoxynucleotides (PDRN) are obtained starting from biological DNA of plant or animal origin, for example from placenta, preferably from fish, for example trout or salmon, in particular from sperm or gonads of salmon (Oncorhynchus keta) or salmon trout (Oncorhynchus mykiss).

For example, a process for preparing suitable polydeoxyribonucleotides of animal origin, for example from mammary placenta, is described in patent EP 0 226 254, whose content is incorporated herein by reference.

According to one aspect, the invention relates to a composition, preferably in the form of a gel or hydrogel, comprising polynucleotides, preferably polydeoxyribonucleotide (PDRN), preferably with a weight average MW selected in the range from 2600 to 3500 kDalton, more preferably from 2800 to 3300 kDalton for use in the prevention or treatment of a mammalian osteoarticular disease, or for use in repairing or restoring or hastening the healing process of a tendon or ligament injury in a mammalian limb.

According to a preferred embodiment, the composition, gel or hydrogel described herein have a dynamic viscosity of 28 - 35 Pa*s, preferably combined with a PN or PDRN concentration from 3 to 5% by weight. Preferably in this embodiment the osmolarity is comprised in the range from 250 to 350 mOsm/Kg.

It has been observed that compositions or gels of the invention, in particular with dynamic viscosity of 28 - 35 Pa*s, preferably combined with a PN or PDRN concentration comprised in the range from 0.5 to 8.8%, from 1 to 8%, from 3 to 5%, preferably from 2 to 6% by weight, advantageously with osmolarity 250 - 350 mOsm/Kg, when injected into a joint or close to the musculoskeletal junctions, lead to the double effect of increasing the viscoelastic properties of the synovial fluid and providing biological stimulation on tissues and matrices, in particular of the osteoarticular structures.

The increase in the synovial fluid viscosity also results in a mechanical support effect on the structures that form the joints by distributing the forces that interact in the joints during the movement of the body limbs on the various structural components.

These combined effects make it possible to achieve a faster recovery of the physiological functions of the joints, for example in subjects affected by traumatic events or osteoarticular degenerative processes typical of the individual’s advancing age, such as osteoarthritis.

It was also observed that PN or PDRN attract or absorb, and complex water molecules, thus helping to form a viscoelastic solution suitable for protecting the anatomical structures that form joints in humans or animals from mechanical stress.

According to some embodiments, the PN nucleotide polymeric chain is at least partially purified, in particular to eliminate its genetic information capacity.

In some embodiments, the polynucleotide is polydeoxyribonucleotide (PDRN), i.e. polymerized deoxyribonucleotide, wherein the nucleotide unit substantially contains deoxyribose as a sugar. Suitable polydeoxyribonucleotides (PDRNs) are DNA-based polymers comprising deoxyribonucleotides, typically double-stranded, preferably having a chain length from 50 to 2400 base pairs. Typically, the bases can be adenosine, guanosine, uridine, cytidine.

Advantageously, the polynucleotide-based gel obtained by the process described herein is for use in the veterinary field, in particular for intra-articular use in the treatment of an osteoarticular disease, possibly associated with pain.

Advantageously, the gel described herein is a gel based on sterile polynucleotides and can be injected into a joint of a mammal, for example a dog or an equid, typically horse.

The infiltration of the sterile injectable gel based on polynucleotides/PDRN described herein into a joint of a mammal, typically a human or an animal, such as a horse, exerts a combined physical and mechanical action and contributes to restoration of joint homeostasis.

According to certain embodiments, the osteoarticular disease treated with the gel obtained by the process of the invention is a degenerative or post-traumatic disease, for example osteoarthritis, or a degenerative or traumatic disease of the joints associated with pain.

In certain embodiments, the gel described herein finds application in repairing or restoring or hastening the healing process of a tendon or ligament injury of a human or animal limb, such as a horse or dog.

Treatment with PN, compared to intra-articular treatments known in the art, such as treatment with hyaluronic acid, has the additional advantage of providing lower production costs compared to those of hyaluronic acid-based formulations available on the market.

According to one embodiment, the gel obtained by the process described herein comprises a nucleic acid polymeric chain fraction with an average molecular weight distribution comprised in the range from 2800 to 3300 kDalton.

According to a second aspect, a composition is provided for use in the treatment of an osteoarticular disease comprising polynucleotides as described herein and a pharmaceutically acceptable vehicle, preferably water and optionally sodium, for example an aqueous solution containing NaCI.

In one embodiment, the composition is in the form of a gel containing polynucleotides with an average molecular weight distribution comprised in the range from 2600 to 3500 kDalton, from 2800 to 3300 kDalton, water and sodium chloride.

BRIEF DESCRIPTION OF THE FIGURES

Some aspects and advantages of the invention are illustrated by referring to the attached figures wherein:

Figure 1 illustrates a graphical representation of curves relating to viscosity (mPa s) of a gel with a PDRN content of 2% by weight, as a function of the shear rate y.

Figure 2 illustrates a graphical representation of curves relating to viscosity of a gel with a PDRN content of 2.50% by weight, as a function of the shear rate y.

Figure 3 illustrates a graphical representation of curves relating to viscosity of a gel with a PDRN content of 3% by weight, as a function of the shear rate y.

The curves of the viscosity graphs as a function of the shear rate show how, in the gels analyzed, the shear rate increases as the concentration of polynucleotides increases, from 2 to 3% by weight.

In particular, for the gel with 2% PDRN concentration and for shear rate values below 5 s’ ¹ , the gap between downward curve and upward curve is of about 15 Pa*s; for the gel with 2.5% concentration, the gap rises to about 25 Pa*s; for the gel with 3% concentration, it rises to 30 Pa*s.

These graphs show how, as the concentration increases, the restoration of the constant state (steady state), after being subjected to a stress in the form of tangential shear force, leads to conditions different from the conditions of the gel in a steady state, when it is not subjected to stress. This is more apparent as the concentration increases, thus denoting a prevalence of the viscous modulus to the detriment of the elastic modulus, as the concentration increases.

By examining the graphs, it is also observed that at high shear rates, 50 s ^-1 , the viscosity values recorded are almost identical and tend to be lower than 1 Pa*s even at different concentrations. This means that gels subjected to very high shear force values assume the behavior of a pseudoliquid (or liquid-like).

DETAILED DESCRIPTION OF THE INVENTION

The present invention originates from having identified a combination of process conditions that allow to transform a solution based on polynucleotides (PN), in particular PDNR, into a viscoelastic gel whose administration by intra-articular route results in a combined mechanical and therapeutic effect, a reduction in the healing times of an osteoarticular disease and a rapid improvement of the related symptoms.

According to one aspect, the object of the present invention is a process for producing a sterile injectable polynucleotide-based gel, preferably in the form of a pharmaceutically acceptable salt as defined in the attached claim 1.

Advantageously, the use of selected amounts or concentrations of process reagents allows to obtain a gel with viscoelastic properties that increase the viscoelasticity of the synovial fluid causing an unexpected mechanical effect in a mammalian joint.

Advantageously, the following conditions are used in the process described herein, preferably in combination:

- polynucleotides I sodium salt of polymerized deoxyribonucleotide (PDR-Na) in a concentration comprised in the range from 2 to 6%, from 3% to 5%;

- HCI aqueous solution with molarity comprised in the range from 0.15M to 0.25M

- pH of the final solution selected in the range from 6.80 to 7.20.

According to some embodiments, the concentration in the composition or gel of the HPDR-Na (High Polymerized Deoxyribonucleotides Sodium Salt) used in the process is comprised in the range from 3% to 5% by weight.

According to some embodiments of the process, the HCI aqueous solution has a molarity comprised in the range from 0.15M to 0.25M.

Preferably, the starting polynucleotides (PN) of the process described herein are polydeoxyribonucleotides (PDRN), in particular in the form of a sodium salt.

According to a preferred embodiment, the process for producing a gel according to the invention comprises the following steps:

- Providing a sodium hydroxide aqueous solution with molarity comprised in the range from 0.15M to 0.25M.

- Dissolving HPDR-Na (High Polymerized Deoxyribonucleotides Sodium Salt) at a concentration comprised in the range from 2 to 6%, preferably from 3% to 5% in the sodium hydroxide solution of the previous step, stirring until completely dissolved, and obtaining a clear solution;

- Filtering the basic HPDR Na solution obtained with 0.2pm sieve filters.

- Adding, under stirring, a HCI aqueous solution with a molarity comprised in the range from 0.15M to 0.25M and filtered with 0.2pm filters, to the basic solution of HPDR Na until the pH of the final solution is brought from 6.80 to 7.20 resulting in an increase of viscosity of the solution and transition from liquid to viscoelastic gel, preferably

- place the viscoelastic gel in an autoclave at 85°C for 5 minutes.

At the end of the process, a product is obtained that does not undergo any changes from a chemical-physical and rheological point of view.

Advantageously, the sterile PN/PDNR Na gel is obtained by filtration sterilization, developed to keep the chemical-physical properties of the PNs intact and to obtain a product with stable and reproducible rheological properties.

The gel of the invention has hydrophilic and polyanionic characteristics and binds water molecules.

In some embodiments, the gel obtained by the process according to an embodiment described herein contains polynucleotides with an average molecular weight distribution greater than or equal to 1000 kDalton, from 2600 kDalton to 3500 kDalton. By way of example, the polynucleotides or PDRNs have a number average molecular weight of 3000 kDa. The object of the invention is also a sterile gel preparation based on polynucleotides or PDRN as described herein for use in the treatment of osteoarticular diseases and lesions in an animal, for example a dog or horse.

The PN or PDRN gel component stimulates the natural tissue regeneration process of the anatomical structures that form the joints, such as cartilage, tendons, ligaments and bone tissue.

According to some embodiments, the injectable gel obtained by the process described herein is a medicine or a medical device.

According to certain aspects, the object of the invention is an ampoule, a vial or a pre-filled syringe containing a gel as described herein.

Preferably the dosage unit comprises a polynucleotide-based gel in an amount comprised in the range from 1 mL to 4 mL, preferably 2 mL, preferably having a concentration comprised in the range from 1 mg/mL to 100 mg/mL, preferably 5- 30, for example of about 20 mg/mL, having a pH from 6.8 to 7.5 and with a viscous consistency having a wetting, lubricating and trophic function.

In some embodiments, the gel described herein has an osmolarity of 270 - 330 mOsm/kg.

Advantageously, the injectable gel described herein has a dynamic viscosity at 20°C comprised in the range from 23 to 40 Pa*s, preferably from 28 to 35 Pa*s, more preferably from 30 to 32 Pa*s, for example of 31 Pa*s, preferably with an osmolarity ranging from 250 to 350 mOsm/Kg, preferably with a concentration ranging from 0.5 to 8.5, preferably from 2 to 6% by weight.

One embodiment of the PN/PDRN-based gel exhibits the following characteristics:

- molecular weight: from 2800 to 3300 kDa

- appearance: transparent gel

- color: colorless

- pH: 6.8 - 7.5

- osmolarity: 250 - 350 mOsm/kg

- polynucleotides UV identification: maximum absorption @260 nm

- polynucleotides UV assay (three concentrations): 7.5 mg/mL ± 0.75 mg/mL; 20 mg/mL ± 2 mg/mL; 25 mg/mL ± 2.5 mg/mL

- sterility: sterile - bacterial endotoxins: < 0.5 Ell/mL.

According to some embodiments, the heterogeneous compositions object of the invention may optionally include buffers, isotonizers and preservatives.

The present invention also provides a new use for this gel, or composition containing it, as a treatment of osteoarticular diseases in the veterinary field, with examples reported on horses and dogs.

Typically, the molecular weights of polynucleotides/polydeoxyribonucleotides (PDRNs) as described herein are expressed as weight average molecular weight (Mw).

The weight average molecular weight of the PN/PDRN described herein can be determined using conventional methods, known to a person skilled in the art, for example as described in Ueno et al., 1988. Chem Pharm Bull. 36, 4971-4975; Wyatt 1993, Anal Chim Acta 272: 1-40; Watt Technologies 1999 “Light scattering University Dawn Course Manual” e “Dawn Eos Manual” Wyatt Technology Corp. Santa Barbara CA (USA).

Typically, the molecular weight of the polymers described herein, such as hyaluronic acid, is a weight average molecular weight (Mw), which can preferably be determined by SEC-MALLS technique or multi-angle laser light scattering - size exclusion chromatography.

The viscosity of the composition or gel described herein can be detected or measured using traditional methods, for example with Bohlin-VOR strain- controlled rotational rheometer, or using the method described in the publication Bothner, H.;Wik, 0. Rheology of hyaluronate. Acta Oto-Laryngol. 1987, 104, 25- 30.

In particular, the viscosity of the composition or gel described and/or claimed herein is measured using the following conditions and devices:

- Anton Paar MCR 302 Rheometer, in particular with a 25 mm parallel plate

- Temperature 20°C;

- Linear downward ramp with shear rate from 2 1/s to 50 1/s;

- Constant shear rate at 50 1/s for 50 s;

- Linear upward ramp with shear rate from 50 1/s to 2 1/s.

Advantageously, the gel described herein is used as a thickening substance for synovial fluid, particularly suitable in the treatment of osteoarthritis, chronic joint disease with cartilage degeneration, changes in the chemical-physical properties of the synovial fluid, and macroscopic changes in the joint.

The intra-articular injection mechanically protects the articular surfaces where cartilage is present and restores the physiological microenvironment of the chondrocytes through an antioxidant action.

In the context of the invention, the term “polynucleotides” means a biopolymer made by polymerization of the nucleotide monomer catalyzed by enzymes, typically belonging to the polymerase class.

Suitable nucleotides that make up PN or PDRN are monomers constituting RNA and DNA nucleic acids, in particular the latter. Each nucleotide contains a purine or pyrimidine nitrogen base, a five-carbon sugar, typically pentose, at least one phosphate group and sodium. Preferably the sugar is deoxyribose and/or ribose.

In some embodiments, the polynucleotides comprise monomeric units of DNA and are based on polydeoxyribonucleotides (PDRN).

A suitable polynucleotide, in the form of sodium salt, is identified by CAS number 100403-24-5.

According to certain embodiments, the polynucleotides can be obtained by extraction from natural sources of natural, plant or animal origin, such as for example algae, plankton, crustaceans, or fish, by means of conventional technologies.

In some embodiments, the polynucleotides that make up the gel of the invention are obtained by fractionation of polymeric DNA fractions of greater length, optionally purified.

In some embodiments, the polynucleotides are fractions of DNA polymeric strands or chains obtained by fragmentation of a DNA strand or chain, fragmented to reduce the molecular weight, and partial purification to eliminate the ability to carry genetic information.

According to some embodiments, the DNA-based polynucleotides are purified by removing from 1 % to 5%, from 2% to 2.5% by weight of the original total content of purine bases.

According to some embodiments, the polynucleotides are sterilized, preferably by filtration.

The following examples are provided for illustrative purposes of the present invention and to provide experimental data in support to the use of the PN-based gel described herein in the veterinary field.

EXAMPLE 1

Below are three sterile compositions based on the gel obtained by the process of the invention:

Polymerized polynucleotides (PDRN) 7.5 mg/ml

Aqueous saline solution q.s.

Polymerized polynucleotides (PDRN) 20 mg/ml

Aqueous saline solution q.s.

Polymerized polynucleotides (PDRN) 25 mg/ml

Aqueous saline solution q.s.

Each of the compositions may be packaged in a sterile syringe.

EXAMPLE 2

Clinical tests were carried out on animals with osteoarticular diseases to verify the activity and effects of the PN-based gel of Example 1. The gel was administered by intra-articular injection of polynucleotides.

The clinical study was carried out on 6 dogs and 2 horses, all of which were affected by lameness.

The clinical outputs examined were the correct joint motility (motility index - M.I.: 5 = Excellent; 4 = Good; 3 = Acceptable; 2 = Incomplete; 1 = None) and the skin temperature at the same joint (T [°C]), the latter as an index of inflammation of the affected part.

The results collected show an improvement in joint motility and a reduction in inflammation, in the entire population examined after 3 weeks of treatment. In some cases, the improvement is already noticeable after just 2 injections. Results remain constant over time even 3 weeks after stopping the treatment. Only in one case, a return to the pretreatment condition was observed. EXAMPLE 3

Aqueous-based gel compositions having the following characteristics: Polydeoxyribonucleotide sodium salt with average molecular weight of 2600 - 3500 kDalton; pH: 6.8 - 7.5;

Osmolarity: 250 - 350 mOsm/kg;

Dynamic viscosity: 28 - 35 Pa*s.

EXAMPLE 4

An analysis was carried out on the viscosity of the gel of Example 3 in relation to the concentration of the polymer present in the gel. The results are illustrated in the attached figures

1 . Materials and methods

Anton Paar Rheometer MCR 302

Parallel plate: 25 mm

Temperature: 20°C

Linear downward ramp with shear rate from 2 1/s to 50 1/s

Constant shear rate at 50 1/s for 50 s

Linear upward ramp with shear rate from 50 1/s to 2 1/s

2. Results

From the data obtained it is deduced that the viscosity increases as the concentration increases.

The product having a dynamic viscosity comprised in the range from 23 to 40 Pa*s, preferably 28 - 35 Pa*s, and an osmolarity of 250 - 350 mOsm/Kg, preferably with a PN or PDRN concentration from 2 to 6% by weight, is particularly valuable in the intra-articular application, this is due to the gel intrinsic characteristics which help viscosupplementation thanks to the gel mechanical properties.

The high viscosity value of the gel optimizes the viscoelastic characteristics and ensures greater durability over time. The thixotropic properties of the gel make it highly flowing when subjected to physical stresses due to movement.