FR2947728A1 - Pharmaceutical composition, useful for treating male infertility secondary to testicular pathology involving hypoxia and/or tissue ischemia, comprises mixture of polynucleotides extracted from natural or synthetic source - Google Patents

Abstract

Pharmaceutical composition (I) comprises a mixture of polynucleotides extracted from a natural or synthetic source. ACTIVITY : Antiinfertility. MECHANISM OF ACTION : Vascular endothelial growth factor stimulator.

Description

The present invention relates to the therapeutic treatment of male infertility secondary to testicular pathologies involving the treatment of male secondary infertility in patients with Testicular Diseases involving Testicular Hypertension and / or Ischemia, Such as Viscotic and Testicular Torment. hypoxia and / or tissue ischemia such as varicocele and testicular torsion.

Varicocele is a pathology characterized by chronic venous stasis with tissue hypoxia that generates high hydrostatic pressure on the pampiniform plexus. The incidence of varicocele is in the order of 35 to 40% in infertile men and this pathology is the main cause of infertility treated by surgery.

Testicular torsion is a medical emergency that requires immediate diagnosis and treatment to prevent subsequent testicular damage and possible infertility. The main pathophysiological process of testicular torsion is ischemia - reperfusion injury due to spermatic cord rotation and relaxation.

Various mechanisms contribute to the development of the testicular lesion resulting from the twisting and untwisting process. The testis produces various inflammatory cytokines such as tumor necrosis factor alpha (TNF-a) and interleukin-1 beta (IL-1R). In addition, testicular ischemia - reperfusion results in excessive production of reactive oxygen species, activates the family of mitogen - activated protein kinases (MAPKs) and initiates the process of apoptosis. In particular, active MAPKs are responsible for the phosphorylation of a type of effector proteins, among which various transcription factors such as nuclear factor kappa B (NF-KB), which then bind to the inducible gene regulatory elements. This pathological cascade is responsible for testicular atrophy, reduced blood flow and altered spermatogenesis observed at the most advanced stages of the lesion.

To date, there is no coded medical therapy for the treatment of the sequelae of this type of pathology. Hypoxia activates both HIF-1 and VEGF and stimulates angiogenesis and the formation of new capillaries and blood vessels during normal physiological processes. has a protective role in the regulation of testicular and spermatogenic function. VEGF exerts its biological function by binding to two receptors coupled to tyrosine kinases and called VEGFR-1 (or fit-1) and VEGFR-2 (or flk / KDR), which are also present on the surface of Sertoli cells. and Leydig. VEGF is present in the prostatic epithelium as well as in the epithelium of the seminal glands of the male genital system and in sperm. Experimental data have suggested that treatment with VEGF exerts protective effects in an experimental model of testicular torsion. However, therapy with such a recombinant protein faces many difficulties because of the low bioavailability of this protein, its reduced plasma half-life and its potential to induce severe side effects.

However, the inventors have surprisingly discovered that the administration of a composition comprising a mixture of polynucleotides to a patient suffering from one of the abovementioned pathologies makes it possible to exert a therapeutic effect on the tissue lesion which manifests itself in this type of pathologies, by stimulating the physiological production of VEGF under pathological conditions of tissue hypoperfusion and / or hypoxia.

Accordingly, a first object of the present invention is a pharmaceutical composition comprising, as active ingredient, a mixture of polynucleotides extracted from natural or synthetic sources, for use as a medicament in the therapeutic treatment of secondary male infertility. testicular pathology involving hypoxia and / or tissue ischemia such as varicocele or testicular torsion resulting in hypospermia, asthenospermia or azoospermia.

In the context of the present description, the term polynucleotide (PN) denotes a fraction of polymer chains of nucleic acids such as DNA or RNA, but preferably DNA, the molecular weight distribution of which is preferably in the range of 220 Daltons to 2500 kDaltons. Said fraction of polymeric nucleic acid chains is preferably obtained by extraction from a natural source, of animal or plant origin, such as fish sperm or plants, by a process which preferably comprises a step of partial fragmentation of the DNA chains to reduce their molecular weight and a partial depurination step of the nucleic acid to suppress its informational capacity. The preferred percentage of depurination of the nucleic acid is preferably in the range of 1% to 5% of removed purine bases (i.e., apural sites), reduced to the total purine bases initially present. A particularly preferred depurination percentage is 2.5%. As will be seen in more detail below, the length of the polynucleotide chains suitable for the application, object of the present invention, and therefore their molecular weight, varies according to several parameters, among which the chosen route of administration. In certain embodiments of the invention, the polynucleotides are in particular a mixture of polydeoxyribonucleotides (PDRNs). By way of non-limiting example, the polydeoxyribonucleotide mixture (PDRN) has a molecular weight distribution which is in the range of 50 to 1500 kDaltons. A polynucleotide composition usable according to the invention comprises a fraction of polyribonucleotide and / or polydeoxyribonucleotide chains of different molecular weights, obtained by synthesis or obtained from natural sources, which may be of plant or animal origin, for example fish plants or sperm for human consumption, the latter being the preferred source.

Polynucleotide-based compositions are known and commercially available.

FR 2,676,926 discloses pharmaceutical compositions containing highly polymerized polydeoxyribonucleotides derived from fish sperm, which are used for the treatment or prevention of immunodeficiencies; compositions containing polydeoxyribonucleotides in a nonionic solvent are described which can be used parenterally, in particular intramuscularly and / or intravenously.

Processes for preparing

Polydeoxyribonucleotides derived from mammalian placenta are described in EP 0 226 254.

The polynucleotides used in the context of the invention are highly purified, or it is a fraction of polynucleotides which is substantially free of pharmacologically active proteins or peptides. In a preferred embodiment, the polynucleotide fraction preferably has a purity equal to or greater than 95% and more preferably greater than 98%, referred to the dry substance.

The polynucleotides used in the context of the invention preferably have a molecular weight distribution which is in the range of 220 Daltons (0.22 kDaltons) to 2500 kDaltons. The polynucleotides can be obtained from an animal or plant source rich in DNA or RNA, by the methods described in the aforementioned literature.

By way of example, a fraction of polynucleotides that can be used in the context of the invention is obtained by a process that comprises the following steps: 1) enzymatic lysis of the organic matrix

2) clarification by filtration of the resulting solution;

3) precipitation with quaternary ammonium salts; 4) separating the precipitate from the complex;

5) molecular selection by selective precipitation with mixtures of water and alcohol; 6) purification by reprecipitation with alcohol A polynucleotide composition that can be used in the context of the invention is formulated in any dose form adapted to the pathology to be treated. Preferred dosage forms are those which are suitable

for systemic administration, in particular oral or intramuscular, for example in the form of an injectable formulation intramuscularly or in the form of tablets. The choice and preparation of the form of administration and the dosage to be used in each

30, cases fit without problem into the skills of an average person skilled in the art.

In a preferred embodiment, a polynucleotide composition that can be used in the invention is suitable for administering a dose of polynucleotides ranging from 0.01 to 10 mg / kg, more preferably from 0.01 to 5 mg / kg, and even more more preferably from 0.07 to 1.25 mg / kg, based on the body weight of the patient. For a human patient weighing approximately 70 kg, these doses correspond to a quantity by weight of polynucleotides ranging from 0.7 mg to 700 mg, preferably from 0.7 mg to 350 mg and more preferably from 4.9 mg to 87, 5 mg. For oral administrations, lower molecular weights (from 220 Daltons to 1000 KDaltons) are more preferably used, whereas for parenteral administrations preferentially higher molecular weights (from 70 kDaltons to 2500 KDaltons) are used. In the context of the present description, the term "patient" is used to designate any subject, human or non-human, which requires a therapeutic treatment aimed at resolving male infertility secondary to a pathology involving hypoxia and / or ischemia. Testicular tissue such as varicocele or testicular torsion. Preferably, the patient is a human being. The following examples, which describe the animal models of varicocele and torsion of the testicle used by the inventors as well as the therapeutic treatments tested on these models, are given for illustrative purposes only and in no way limit the aim of the invention. invention as defined in the appended claims. In the following examples, reference is made specifically to the accompanying figures, in which: Figure 1 is a microscope view showing the histology of the testicle in a falsely operated animal (pseudo-varicocele); Figure 2 is a histology of the varicocele testis; FIG. 3 is a histology of the testicle seen under a microscope showing in an animal suffering from

microscopic view showing in an animal with varicocele and subjected to one after 7 days); Figure 4 is a histology of the testis varicocele and submitted to one after 14 days); Figure 5 is a varicocele testicle histology treated with varicocelectomy (evaluation

under a microscope showing in an animal suffering from varicocelectomy (evaluation

under a microscope showing in an animal with polynucleotides (PDRN) (evaluation after 7 days); Figure 6 is a microscope view showing testicular histology in a varicocele-treated animal treated with polynucleotides (PDRN) (14-day evaluation); Figure 7 is a microscope view showing testicular histology in an animal with varicocele and treated with polynucleotide combination (PDRN) and surgery (evaluation after 7 days);

Figure 8 is a microscope view showing testicular histology in an animal with varicocele and treated with the combination of polynucleotides (PDRN) and surgery (evaluation after 14 days);

Figure 9 is a bar graph showing the expression of VEGF mRNA in the ischemia-reperfusion (9A) and contralateral testis (9B) testis after 1, 7 and 30 days of the treatments indicated in the legend. ;

Figure 10 is a bar graph showing the expression of mature VEGF protein in the testis subjected to ischemia-reperfusion (10A) and in the contralateral testis (10B) after 1, 7 and 30 days of the treatments indicated in the legend;

FIG. 11 is a series of microscope views of histological preparations showing changes in edema, venous ectasia, and globular coagulation necrosis after ischemia-reperfusion of the testis and treatment with the vehicle alone (11A, 11C, 11E), DMPX + PDRN (11G), the PDRNs alone (11B, 11D and 11F) or the DMPX alone (11H);

Figure 12 is a series of microscope views of histological preparations showing VEGF expression in germinal cells of the homolateral testicle after ischemia-reperfusion and treatment with vehicle alone (12A, 12C, 12E), DMPX + PDRN (12G ), the PDRNs alone (12B, 12D and 12F) or the DMPX alone (12H);

Figure 13 is a series of microscope views of histological preparations showing VEGF receptor expression in Leydig cells of the homolateral testicle after ischemia-reperfusion and treatment with vehicle alone (13A, 13C, 13E), DMPX + PDRN (13G), PDRN alone (13B, 13D and 13F) or DMPX alone (13H). Example 1: animal model of varicocele

Male Sprague-Dawley rats (weighing between 250 and 300 g) underwent partial ligation of the left renal vein to create reflux into the left spermatic vein and thereby cause profound alteration of spermatogenesis by varicocele in the pampiniform plexus (varicocele group) after general pentobarbital sodium anesthesia (50 mg / kg ip). Varicocele was prolonged for 28 days. In the pseudo-varicocele group, the rats underwent the same surgical procedure as that performed in varicocele rats, with the exception of partial occlusion of the renal vein. The animals were randomized to receive, after 28 days, the following treatments: 1) simple varicocelectomy by ligation of the left spermatic vein and sacrifice of the animals after 7 or 14 days; 2) maintenance of varicocele and administration of polydeoxyribonucleic acid

nucleotides (PDRN) (8 mg / kg i.p.) for 7 or 14 days; 3) varicocelectomy and PDRN (8 mg / kg i.p.) after 7 or 14 days. Animals in each group were sacrificed respectively 7 or 14 days after randomization.

The histological results illustrated in FIGS. 1 to 8 demonstrate that, with respect to the pseudo-varicocele animals, the varicocele results in a considerable alteration of the normal architecture of the testis, characterized by edema, an ectasia of the venous and lymphatic vessels, and significant changes in Sertoli and Leydig cells (Figures 1 and 2). Reduced varicocelectomy

significantly these histological changes evaluated 7 and 14 days after surgery (Figures 3 and 4). Surprisingly, the administration of PDRN makes it possible to protect, in a manner quite comparable to the surgical procedure, the testicle against the lesions due to the varicocele at both J7 and J14 (FIGS. 5 and 6). Medical treatment combined with PDRN and varicocelectomy do not appear to provide additional protection against varicocele injury (Figures 7 and 8). On the other hand, varicocele caused impairment of spermatogenesis, as can be seen in Table 1, which summarizes the evaluation of spermatogenesis, expressed by the Johnsen score (P <0.05).

PDRN administration improved spermatogenesis (Table 1).

Overall, these results demonstrate that

the administration of PDRN has a surprising and unexpected therapeutic action in case of varicocele.

Example 2: animal model of testicular torsion

Male Sprague-Dawley rats (weighing 250-300 g) underwent, after general pentobarbital sodium anesthesia (50 mg / kg ip), testicular ischemia-reperfusion (TI / R) or pseudoischemia (pseudo-ischemia). SHOOT). TI / R was induced by 720 ° twisting of the testicle and left spermatic cord to cause complete occlusion of the flow for 1 hour. The same testicle was then untwisted. The pseudo-varicocele animals underwent the same surgical procedure as the TI / R animals, with the exception of testicular occlusion. The animals were then randomized to receive the following treatments, immediately after the detorsion: 1) PDRN (8 mg / kg i.p.); 2) DMPX (3,7-dimethyl-1-propargylxanthine

0.1 mg / kg i.p.); 3) PDRN (8 mg / kg i.p.) + DMPX (0.1 mg / kg i.p.) simultaneously; 4) vehicle (1 ml / kg NaCl 0.9%). DMPX is a selective antagonist of A2A adenosine receptors. The PDRNs, the DMPX and the vehicle were respectively injected intraperitoneally for 30 days. Animals in each group were sacrificed 1, 7 or 30 days after ischemia - reperfusion.

Both mRNA and VEGF-A protein expression were studied 7 and 30 days after injury in both testes. The histological lesion was also analyzed at D1, D7 and D30. Finally, immunohistochemical analysis of VEGF-A and its VEGFR2 receptor, as well as analysis of spermatogenic activity, were performed on days 1, 7 and 30.

Ischemia - reperfusion of the testis produced an increase in messenger RNA or VEGF protein in both testes. PDRN treatment significantly increased both VEGF messenger RNA (FIGS. 9A and 9B) and its mature protein (FIGS. 10A and 10B). In contrast, combined administration with DMPX nullified the protective effect of PDRNs (Figures 9A and 9B, 10A and 10B). No significant changes were found for the parameters evaluated in pseudo-TI / R animals treated with both the vehicle and the different substances.

Ischemia - reperfusion of the testicle caused edema, venous ectasia and globular coagulation necrosis at 1, 7 and 30 days post reperfusion (Figures 11A, 11C, 11E and Table 1). Table 2 shows the histological scores of the ischemia-reperfusion testis (TI / R) of the PDRN-treated rats (8 mg / kg ip) or the DMPX (0.1 mg / kg ip) or the DMPX + PDRN or with the vehicle (1 ml / kg / ip). Each group includes 7 animals. The histological score was scored on the basis of the following criteria: 0 (absent), 1 (low), 2 (moderate), 3 (severe).

PDRN treatment reduced histological changes in the ipsilateral testis (Figures 11B, 11D, 11F and Table 2).

In contrast, combined administration with DMPX suppressed the protective effects of PDRNs

(Figures 11G and H, Table 2). Comparable results were obtained in the contralateral testicle of animals having undergone ischemia - reperfusion and treated either with the vehicle or with the different substances.

In addition, testicular ischemia - reperfusion caused spermatogenesis alteration, especially 30 days after reperfusion, as shown in Table 3, which summarizes the evaluation of spermatogenesis, expressed by the Johnsen score (P < 0.05).

PDRN administration improved spermatogenesis (Table 3).

Immunohistochemical analysis further revealed modest expression of VEGF in germinal cells of the ipsilateral testicle in ischemia-reperfusion and vehicle-treated animals (Figures 12A, 12C, 12E). PDRN treatment showed strong reactivity of VEGF 1, 7 and 30 days after reperfusion (Figures 12B, 12D, 12F). Combined administration with DMPX did not cause any significant changes in VEGF expression (Figures 12G and 12H). Comparable results were obtained in the contralateral testicle of animals having undergone ischemia - reperfusion and treated either with the vehicle or with the different substances.

Finally, immunohistochemical analysis revealed moderate expression of the VEGF receptor in Leydig cells of the ipsilateral testicle 1 day after reperfusion (Figure 13A). PDRN treatment increased the reactivity of the VEGF receptor (Figure 13B). In contrast, strong immunoreactivity for the VEGF receptor was demonstrated 7 days after reperfusion in both vehicle-treated and PDRN-treated animals (Figures 13C and 13D), whereas moderate expression was observed after 30 days (Figures 13E and 13F).

Combined administration with DMPX did not cause any significant changes in VEGF receptor expression (Figures 13G and 13H). Comparable results were obtained in the contralateral testicle of animals having undergone ischemia - reperfusion and treated either with the vehicle or with the different substances.

Example 3: Evaluation of the therapeutic range in humans

To evaluate the therapeutic range of PDRNs in humans, experiments were carried out in the rat using, as an evaluation criterion, the histological lesion 24 hours after reperfusion and testing increasing amounts of PDRN expressed in equivalent human doses ( HED, Table 3 and Figure 14). Table 4, in particular, relates to the dose / range study carried out with PDRNs for the purpose of identifying the therapeutic dose in humans. The criterion used in the study was the histological lesion at 24 hours. The histological score was evaluated in the testes of rats with ischemia - reperfusion (TI / R). The histological score was scored on the basis of the following criteria: 0 (absent), 1 (weak), 2 (moderate), 3 (severe).

To convert these doses, the following formula was used: HED = animal dose in mg / kg x (animal weight in kg / human weight in kg). The results shown in Table 4 and Figure 14 demonstrate that PDRNs exert an optimal protective effect in a dose range that ranges from about 5.4 mg to about 87.3 mg in a 70 kg man.

In conclusion, the results obtained indicate 1) that polynucleotides (PDRNs) protect the testis against hypoxic / ischemic injury through the production of VEGF and 2) that they can represent a new and innovative approach to the treatment of infertility in patients with testicular torsion or varicocele. Table 1: Evaluation of spermatogenesis in the experimental varicocele model Experimental groups Johnsen score Pseudo-varicocele 9.33 0.22 Varicocele 2.63 0.46 Varicocele + surgery 8.22 0.54 (7 days) Varicocele + surgery 8.66 0.33 (14 days) Varicocele + PDRN (7 days) 8.42 0.37 Varicocele + PDRN (14 days) 8.78 0.41 Varicocele + PDRN + surgery 8.48 0.40 (7 days) Varicocele + PDRN + surgery 8.83 0.48 (14 days) Table 2: Histological score in the model of ischemia - testicular reperfusion (TI / R). Groups edema Ectasia Experimental Atrophy Necrosis extravenular globular tubular coagulation Pseudo-Varicocele 0 0 0 0 TI / R + vehicle 3 3 3 0 (1 day) TI / R + PDRN 0 1 1 0 (1 day) TI / R + vehicle 3 3 3 0 (7 days) TI / R + PDRN 1 0 0 0 (7 days) TI / R + vehicle 2 2 2 3 (30 days) TI / R + PDRN 1 0 0 0 (30 days) TI / R + DMPX + PDRN 3 2 3 (30 days) TI / R + DMPX 3 3 2 1 (30 days) Table 3: Evaluation of spermatogenesis during ischemia - testicular reperfusion (TI / R) Experimental groups Score of Johnsen Pseudo-varicocele 9.81 0.15 TI / R + vehicle (1 day) 5.47 1.21 TI / R + PDRN (1 day) 8.83 0.20 TI / R + vehicle (7 days) 1 , 21 0.04 TI / R + PDRN (7 days) 9.00 0 40 TI / R + vehicle (30 days) 1.73 0.08 TI / R + PDRN (30 days) 8.92 0.30 TI / R + DMPX + PDRN (30 days) 1.75 0.06 TI / R + DMPX (30 days) 2.36 1.0 Table 4: Identification of the optimal therapeutic dose in humans (6 animals per experiment ) HED Group of Dose of Ideema Ectasia PDRN PDRN extra-intravenular (mg / day) (mg) tubular tubular PDN-97.3 8 0 0 0 varicocele TI / R + 3 3 3 vehicle TI / R + PDRN 5.4 0.5 2 2 2 TI / R + PDRN 10.9 1 1 1 1 TI / R + PDRN 21.8 2 0 0 1 TI / R + PDRN 43.6 4 0 0 1 RT / R + PDRN 87.3 8 0 0 1

Claims (15)

REVENDICATIONS1. Pharmaceutical composition comprising as active principle, a mixture of polynucleotides, extracted from a natural or synthetic source, intended to be used as a medicament in the therapeutic treatment of male infertility secondary to a testicular pathology involving hypoxia and / or ischemia tissue. 2. The composition of claim 1, wherein the testicular pathology is varicocele or testicular torsion. 3. The composition of claim 1 or 2, wherein said polynucleotide mixture is partially depurinated. 4. A composition according to claim 3, wherein said polynucleotide mixture has a depurination percentage of from 1% to 5% of removed purine bases, reduced to the total purine bases initially present. The composition of any one of claims 1 to 4, wherein said polynucleotide mixture is a fraction of polymeric nucleic acid chains having a molecular weight distribution of from 220 Daltons to 2500 Kdaltons. The composition of claim 5, wherein said polynucleotide mixture is a fraction of polymeric nucleic acid chains whose molecular weight distribution is from 220 Daltons to 1000 KDaltons. The composition of claim 5, wherein said polynucleotide mixture is a fraction of polymeric nucleic acid chains whose molecular weight distribution is from 70 kDaltons to 2500 kDaltons. The composition according to any one of claims 1 to 7, wherein said polynucleotide mixture can be obtained by extraction from a natural source, by a process comprising a step of partial fragmentation of polynucleotide chains of the nucleic acid. and a step of partial depurination of the nucleic acid. The composition of claim 8, wherein said natural source is fish sperm or a plant. 10. A composition according to any one of claims 1 to 9 in a dosage form suitable for oral administration. 11. A composition according to any one of claims 1 to 9 in a dosage form adapted for parenteral administration. The composition of any one of claims 1 to 11, wherein the polynucleotides are polydeoxyribonucleotides (PDRNs). 13. Use of a composition according to any one of claims 1 to 12 for preparing a medicament for the therapeutic treatment of male infertility secondary to a testicular pathology involving hypoxia and / or tissue ischemia. The use of claim 13, wherein the testicular pathology is varicocele or testicular torsion. 15. Use according to claim 13 or 14, wherein the therapeutic treatment is carried out by administering a daily dose of active ingredient ranging from 0.01 to 10 mg / kg, preferably from 0.01 to 5 mg / kg and more preferably from 0.07 to 1.25 mg / kg, based on the body weight of the patient.