Anti-miRNA for treating leiomyoma

Abstract

The present invention relates to miRNA inhibitors for treating leiomyomas. In particular, it relates to inhibitors of one or more miRNAs selected from miR-148a-3p, miR-199a-5p and miR-33b-3p, which are used for treating uterine leiomyomas. The inhibitor is preferably an LNA-based oligonucleotide. A pharmaceutical composition comprising the inhibitor for treating uterine leiomyomas is also within the scope of the present invention.

Description

Field of the Invention

[0001] The present invention relates to the fields of pharmacy and biotechnology.

[0002] Specifically, the present invention relates to anti-miRNAs for the treatment of leiomyomas. Background of the Invention

[0004] Uterine leiomyomas, commonly known as fibroids, affect 20 - 50% of women of reproductive age and are the most common benign gynecological tumors (Purohit P, Vigneswaran K: Fibroids and Infertility, Curr Obstet Gynecol Rep 2016, 5: 81 - 88).

[0005] Although the impact of fibroids on female reproductive function and fertility has not been fully established, it is generally believed that their presence seriously interferes with the ability to successfully initiate pregnancy (Pritts EA, Parker WH, Olive DL: Fibroids and infertility: an updated systematic review of the evidence, Fertil Steril 2009, 91: 1215 - 1223). Although they are benign tumors and may even be asymptomatic, in many cases they cause important dysfunctions such as bleeding, pelvic pressure, and impaired fertility as described above, thus requiring therapeutic intervention.

[0006] Currently, the treatment of fibroids is based on the use of drugs that interfere with the hormonal stimuli behind their growth and sometimes manage to control symptoms, as well as surgical resection.

[0007] Hysterectomy, i.e., surgical removal of the uterus, is the only definitive solution to the problem. However, in many cases, this solution is not feasible, for example, if the woman plans to become pregnant or for purely psychological reasons. In these cases, local excision of the tumor mass has been the main treatment option for the past 100 years, initially by laparotomy and more recently in a less invasive manner by laparoscopy or hysteroscopy (El-Balat A, DeWilde RL, Schmeil I, Tahmasbi-Rad M, Bogdanyova S, Fathi A, Becker S: Modern Myoma Treatment in the Last 20 Years: A Review of the Literature, Biomed Res Int 2018, 2018: 4593875).

[0008] Any surgical intervention is always associated with a limited but real risk of complications, such as bleeding (which often requires blood transfusion and thus has a risk of transmission of infectious pathogens), bladder, bowel or ureter lesions, adhesion formation, and complications related to anesthesia and hospitalization.

[0009] Current medical therapies, including oral contraceptives, medical intrauterine devices, GnRH agonists, and selective progesterone receptor modulators (SPRM), are mainly used to control symptoms (bleeding and dysmenorrhea) without affecting the size of the leiomyoma mass (Senol T, Kahramanoglu I, Dogan Y, Baktiroglu M, Karateke A, Suer N: Levonorgestrel-releasing intrauterine device use as an alternative to surgical therapy for uterine leiomyoma, Clin Exp Obstet Gynecol 2015, 42: 224-227; Singh SS, Belland L: Contemporary management of uterine fibroids: focus on emerging medical treatments, Curr Med Res Opin 2015, 31: 1-12).

[0010] Even the latest generation of drugs, such as ulipristal acetate, can control bleeding without significant side effects, but they cannot reduce the size of the lesions (Donnez J, Tatarchuk TF, Bouchard P, Puscasiu L, Zakharenko NF, Ivanova T, Ugocsai G, Mara M, Jilla MP, Bestel E, Terrill P, Osterloh I, Loumaye E, PIS group: Ulipristal acetate versus placebo for fibroid treatment before surgery, N Engl J Med 2012, 366: 409-420; Donnez J, Tomaszewski J, Vazquez F, Bouchard P, Lemieszczuk B, Baro F, Nouri K, Selvaggi L, Sodowski K, Bestel E, Terrill P, Osterloh I, Loumaye E, PIS group: Ulipristal acetate versus leuprolide acetate for uterine fibroids, N Engl J Med 2012, 366: 421-432).

[0011] Currently, there is no drug therapy that can reduce the size of leiomyomas.

[0012] Therefore, it is necessary to develop new conservative therapies that can prevent or reverse the growth of leiomyomas.

[0013] In particular, it is desirable to develop a drug that can reduce the size of leiomyomas without disturbing the female ovulation cycle and fertility.

[0014] From a morphological perspective, leiomyomas are benign proliferative lesions of uterine smooth muscle cells. Despite their high frequency, the cause of the abnormal proliferation of smooth muscle cells remains unknown.

[0015] Several theories have been proposed to try to explain the etiology of these lesions, which suggest the involvement of estrogen and progesterone, as well as several other growth factors, cytokines, chemokines, genes, and microRNAs (miRNAs).

[0016] In particular, miRNA is a small RNA molecule, about 21 nucleotides in length, which can regulate the expression of different genes and binds to the untranslated region at the 3' end of the corresponding mRNA.

[0017] MiRNA can be inhibited using specific LNA (locked nucleic acid)-based oligonucleotides or antagomiRs, i.e., oligonucleotides having a sequence complementary to the miRNA. These oligonucleotides usually contain chemical modifications that increase their affinity for the target mRNA molecule and trap them in an inactive conformation or promote degradation.

[0018] Generally, miRNA-based biopharmaceuticals or their inhibitors are useful tools for interfering with the underlying processes of several diseases. Summary of the Invention

[0020] It has now been found that inhibiting any one of the following miRNAs can block the proliferation of primary leiomyoma cells: miR-148a-3p, miR-199a-5p, miR-33b-3p.

[0021] Accordingly, an object of the present invention is an inhibitor of one or more miRNAs selected from miR-148a-3p, miR-199a-5p, and miR-33b-3p, for use in the treatment of uterine leiomyoma.

[0022] The inhibitor is preferably selected between LNA (locked nucleic acid)-based oligonucleotides and antagomiRs. Preferably, it is an LNA-based oligonucleotide.

[0023] Also within the scope of the present invention is a pharmaceutical composition for the treatment of uterine leiomyoma, comprising the inhibitor and at least one pharmaceutically acceptable carrier and / or excipient. Detailed Description

[0024] Definitions

[0025] In the context of the present invention, the term "microRNA" or "miRNA" refers to short ribonucleic acid (RNA) present in eukaryotic cells.

[0026] In the context of the present invention, the term "inhibitor" refers to a reagent capable of reducing or blocking the activity of one or more target miRNAs. Specifically, it refers to a reagent capable of sufficiently reducing the activity of one or more target miRNAs. In one specific embodiment, the inhibitor according to the present invention is an oligonucleotide comprising at least 5 nucleotides, which oligonucleotide can bind to the target miRNA by complementary base pairing, thereby reducing or blocking the function of the miRNA. For "complementary base pairing", it means that some or all of the bases of the oligonucleotide pair with some or all of the bases of the miRNA target. In particular, the inhibitor can pair at least 40% of its bases with the bases of the target miRNA; for example, the inhibitor can pair at least 40%, 50%, 60%, 70%, 80%, 90% or 100% of its bases with the bases of the target miRNA. The term "base" refers to a nucleobase, i.e., the nitrogen-containing part of a nucleoside, which in turn is a component of a nucleotide.

[0027] In the context of the present invention, the term "anti-miR" or "antagomiR" refers to an oligonucleotide molecule that prevents other molecules from binding to one or more specific miRNAs. Generally, it is a small synthetic RNA that is at least partially complementary to the target miRNA. In particular, it is sufficiently complementary to bind and block the target miRNA.

[0028] In the context of the present invention, the term "locked nucleic acid" or "LNA" refers to a nucleotide in which the ribose ring is closed in an N-type conformation by a 2'-O,4'-C bridge. LNA is described, for example, in WO 99 / 14226, WO 00 / 56746, WO 00 / 56748, WO 01 / 25248, WO 02 / 28875, WO 03 / 006475 and WO 03 / 095467 and the references cited therein.

[0029] In the context of the present invention, the term "LNA-based oligonucleotide" refers to an oligonucleotide comprising at least one locked nucleic acid (LNA) as defined above. The oligonucleotide is generally a small synthetic RNA that is at least partially complementary to the target miRNA, particularly sufficiently complementary to bind and block the target miRNA and includes at least one locked nucleic acid.

[0030] In the context of the present invention, the term "uterine leiomyoma" refers to a benign tumor of the uterus. "Uterine fibroid" is a synonym. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1: Proliferation of human smooth muscle cells untreated or transfected with control miRNA or LNA-based oligonucleotides capable of inhibiting miR-148a-3p, miR-199a-5p, and miR-33b-3p (*p ≤ 0.05; ***p ≤ 0.001).

[0032] Figure 2 : Proliferation of human fibroblasts untreated or transfected with control miRNA or LNA-based oligonucleotides capable of inhibiting miR-148a-3p, miR-199a-5p, and miR-33b-3p (ns, not significant).

[0033] Figure 3 : Proliferation of human smooth muscle cells untreated, or treated with control miRNA or LNA-based oligonucleotides capable of inhibiting miR148a-3p, miR-199a-5p, and miR-33b-3p under gymnosis conditions (in the absence of a transfection agent) (*p ≤ 0.05). Detailed Description of the Invention

[0035] The sequences of miRNA miR-148a-3p, miR-199a-5p, and miR-33b-3p are known to experts in the field and can be found, for example, in the miRbase database (www.mirbase.org, version 22).

[0036] According to the present invention, at least one miRNA selected from miR-148a-3p, miR-199a-5p, and miR-33b-3p is inhibited.

[0037] The inhibition of these miRNAs is particularly advantageous because it shows cell specificity. In fact, it induces a decrease in the proliferation of uterine smooth muscle cells without affecting the proliferation of fibroblasts, thereby reducing possible side effects.

[0038] Even more preferably, miR-148a-3p is inhibited. In fact, it has been found that inhibiting this miRNA is particularly effective in reducing the proliferation of uterine myocytes.

[0039] According to the present invention, one or more inhibitors of the said miRNAs are used.

[0040] Any inhibitor capable of reducing or blocking the activity of one or more of the above miRNAs can be used according to the present invention for the treatment of uterine leiomyomas.

[0041] In a specific embodiment, the inhibitor is an oligonucleotide comprising at least 5 nucleotides and capable of binding to the target miRNA by complementary base pairing. For example, the oligonucleotide may comprise 5 to 27 nucleotides, preferably 10 to 21 nucleotides.

[0042] The oligonucleotide is capable of pairing some or all of its bases with some or all of the bases of the miRNA target. In particular, the inhibitor is capable of pairing at least 40% of its bases with the bases of the target miRNA; for example, it pairs at least 40%, 50%, 60%, 70%, 80%, 90% or 100% of its bases with the bases of the target miRNA.

[0043] In a preferred embodiment, the inhibitor is preferably selected between locked nucleic acid (LNA)-based oligonucleotides and anti-miRs.

[0044] In one embodiment, the inhibitor is an anti-miR, wherein the anti-miR is an RNA oligonucleotide that is at least partially complementary to the target miRNA. In particular, it is sufficiently complementary to bind and at least partially block the target miRNA. Preferably, the anti-miR comprises 5 to 27 nucleotides and has at least 40% of its bases complementary to the bases of the target miRNA.

[0045] In a preferred embodiment, the inhibitor is an LNA-based oligonucleotide as defined above. Preferably, it comprises 5 - 27 nucleotides and comprises at least one locked nucleic acid. It may also comprise more than one locked nucleic acid. The LNA-based oligonucleotide is at least partially complementary to the miRNA target, in particular it may be at least 40%, 50%, 60%, 70%, 80%, 90% or 100% complementary to the miRNA target.

[0046] LNA-based oligonucleotides are commonly used inhibitors in the art and are commercially available. For example, they are available from the following companies: QIAgen, Affymetrix, PerkinElmer.

[0047] In a specific embodiment of the present invention, the LNA-based oligonucleotide is an oligonucleotide, in particular an antisense oligonucleotide, having a perfect sequence complementary to the miRNA target. The LNA-based oligonucleotide can be 100% complementary to the miRNA target. For example, it can be an LNA-based oligonucleotide having a perfect sequence complementary to a miRNA target of the type miRCURY LNA miRNA PowerInhibitor (commercially available from QIAgen).

[0048] Experts in the field can find inhibitors suitable for use according to the present invention based on the general knowledge in the field.

[0049] In fact, how to identify reagents capable of inhibiting the activity of known miRNAs is well-known in the art. In particular, anti-miRs and LNA-based oligonucleotides are commonly used to inhibit the activity of target miRNAs. A person skilled in the art can easily design or purchase anti-miRs or LNA-based oligonucleotides capable of inhibiting the activity of at least one miRNA selected from miR-148a-3p, miR-199a-5p, and miR-33b-3p.

[0050] The inhibitor used as described in the present invention can be administered to a subject in need thereof in any form.

[0051] In particular, it can be administered by conventional methods for administering small RNAs.

[0052] In one embodiment, it is administered without using a transfection agent.

[0053] In another embodiment, it is administered in the form of a pharmaceutical composition.

[0054] The pharmaceutical composition can be in the form of a preparation for parenteral or intrauterine administration, but other forms are equally suitable for practicing the present invention.

[0055] An expert in the field will determine the effective time of administration based on the condition of the patient, the severity of the pathology, the patient's response, and any other clinical parameters included in the general knowledge in the field.

[0056] The pharmaceutical composition contains at least one inhibitor of the present invention and a pharmaceutically acceptable carrier and / or excipient. The excipient can be a particularly useful formulation aid, such as a solubilizer, dispersant, suspending agent, or emulsifying agent.

[0057] According to the present invention, and according to the prior art, the inhibitor can be administered together with a lipid molecule, such as a cationic lipid that can facilitate its transport. Another method of administering such an inhibitor is through a suitable vector known for administering RNA or DNA. A preferred vector is an adeno-associated vector (AAV), a well-known viral vector for in vivo administration of DNA (Mingozzi F, High KA: Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nature reviews genetics. 2011 May;12(5):341).

[0058] Injection is the preferred route of administration. The injection is preferably systemic. Intrauterine administration is also advantageous. An expert in the field can decide to administer the inhibitor by any conventional route.

[0059] For the general knowledge in this field, reference can be made to Remington's Pharmaceutical Sciences, latest edition.

[0060] Another possible method of administration involves using gene transfer technology with non-viral ultrasound (sonoporation), which can be used to locally transfect uterine cells with the inhibitor according to the present invention. This technique is well-known and reference can be made, for example, to Acoustic Cavitation-Mediated Delivery of Small Interfering Ribonucleic Acids with Phase-Shift Nano-Emulsions; Ultrasound Med Biol. 2015 Aug;41(8):2191-201.

[0061] Another mode of administration is by releasing the inhibitor through a medicated intrauterine device. For example, the intrauterine device can be medicated with at least one locked nucleic acid (LNA)-based oligonucleotide or at least one anti-miR capable of inhibiting the one or more miRNAs as defined above. Such medicated intrauterine devices disclosed herein are within the scope of the present invention.

[0062] All of these methods and formulations are conventional and well-known in the art and do not require further explanation.

[0063] Gene therapy is another form of delivery, in which nucleic acids are delivered into the cells of a subject in need. According to the present invention, gene therapy can be used to introduce the oligonucleotide inhibitors as defined above into the cells of a subject, for example into uterine cells, for the uses according to the present invention.

[0064] The following examples further illustrate the present invention.

[0065] Example 1

[0066] Selection of miRNA candidates

[0067] A library of 2000 miRNAs was used for screening to identify miRNAs capable of regulating the proliferation of smooth muscle cell lines. In particular, the screening was aimed at identifying miRNAs capable of stimulating smooth muscle cell proliferation.

[0068] The screening results are shown in Table 1 below.

[0069] Among the miRNAs considered in the screening, 1913 did not interfere with cell viability and 37 increased proliferation by more than 2.5-fold.

[0070] By cross-referencing these data with the data available in the published gene expression profile databases of leiomyoma and normal myometrium samples (Chuang TD, Khorram O: Expression Profiling of lncRNAs, miRNAs, and mRNAs and Their Differential Expression in Leiomyoma Using Next-Generation RNA Sequencing, Reprod Sci 2018, 25: 246-255), 14 miRNAs were selected (Table 1).

[0071] Table 1

[0072]

[0073] Among the 14 selected miRNAs, only 7 "seed sequences" were represented, indicating that their pro-proliferative effects may be mediated by common molecular mechanisms. In addition, two miRNAs (miR-20a-5p and miR-17-5p) belong to the same cluster and are located on chromosome 13, so their expression is expected to be co-regulated.

[0074] From the same selection, the following miRNAs were considered for further validation: miR-148a-3p, miR-199a-5p, miR-20a-5p, miR-17-5p, and miR-33b-3p. MiR-148a-3p and miR-199a-5p were selected because they were ranked second and fifth in the screening, were the most expressed in leiomyomas, and were also overexpressed in leiomyomas compared to healthy myometrium. Two other miRNAs (miR-20a-5p and miR-17-5p) are known to stimulate the proliferation of other cell types and were selected as potential positive controls. Finally, miR-33b-3p was found to be relatively poorly expressed in leiomyomas but completely absent in healthy myometrium and was thus included among the potential candidates of interest.

[0075] Example 2

[0076] In vitro validation

[0077] Based on the results reported in Example 1, we tested the ability of five candidate LNA-based oligonucleotides (miRCURY LNA miRNA Power Inhibitor, Qiagen) to block the proliferation of primary leiomyoma cells, which were obtained through an existing collaboration with the Burlo Garofolo Maternal and Child Hospital in Trieste.

[0078] LNA-based oligonucleotides were tested with cationic lipids using standard transfection protocols or under gymnosis conditions (without transfection agents) to better mimic possible in vivo applications (for the procedures used, see, for example, Single-Dose Intracardiac Injection of Pro-Regenerative MicroRNAs Improves Cardiac Function After Myocardial Infarction. Lesizza P, Prosdocimo G, Martinelli V, Sinagra G, Zacchigna S, Giacca M. Circ Res. 2017 Apr 14;120(8):1298-1304; Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents. Stein CA, Hansen JB, Lai J, Wu S, Voskresenskiy A, A, Worm J, M, Souleimanian N, Miller P, Soifer HS, Castanotto D, Benimetskaya L, H, Koch T. Nucleic Acids Res. 2010 Jan;38(1):e3). Proliferation was analyzed by incorporation of EdU and subsequently quantified by fluorescence microscopy (ImageXpress Micro, Molecular Devices) to visualize smooth muscle cells labeled with an antibody that recognizes an actin isoform specific for smooth muscle. The nuclei of all cells were stained with DAPI.

[0079] Inhibitors (3 candidates plus 2 positive controls) were tested in leiomyoma cells and primary fibroblasts using 17-5p LNA and 20a-5p LNA as positive controls to verify their specificity of action.

[0080] These experiments were able to select three LNA-based oligonucleotides that were highly effective in inhibiting the proliferation of leiomyoma cells and were inactive in fibroblasts: LNA 148a-3p, LNA 199a-5p, and LNA 33b-3p( Figure 1 and 2 ).

[0081] Using the gymnosis method, we verified that the use of the following miRNA-specific inhibitors significantly reduced cell proliferation: 148a-3p LNA, 199a-5p LNA, and 33b-3p LNA. All three inhibitors strongly reduced the proliferation of smooth muscle cells( Figure 3 ).

Claims

1. Use of an inhibitor of one or more miRNAs selected from miR-148a-3p, miR-199a-5p, and miR-33b-3p in the preparation of a medicament for treating uterine leiomyoma, wherein, The inhibitor is a locked nucleic acid-based oligonucleotide, i.e., LNA.

2. The use according to claim 1, wherein The miRNA is miR-148a-3p.

3. The use according to claim 1 or 2, wherein The inhibitor is an oligonucleotide comprising at least 5 nucleotides and capable of binding to the one or more miRNAs by complementary base pairing.

4. The use according to claim 3, wherein the inhibitor is capable of base pairing at least 40% of its bases with the bases of the one or more miRNAs.

5. The use according to claim 1 or 2, wherein the LNA-based oligonucleotide is at least partially complementary to the miRNA.

6. The use according to claim 5, wherein the LNA-based oligonucleotide is at least 40%, 50%, 60%, 70%, 80%, 90% or 100% complementary to the miRNA.

7. The use according to claim 5, wherein, The LNA-based oligonucleotide is an antisense oligonucleotide with a perfect sequence complementary to the miRNA target.

8. The use according to claim 5, wherein, The LNA-based oligonucleotide comprises 5 to 27 nucleotides and contains at least one locked nucleic acid.

9. The use according to claim 1 or 2, wherein The inhibitor is an anti-miR.

10. The use according to claim 9, wherein, The anti-miR comprises 5 to 27 nucleotides and has at least 40% of its bases complementary to the bases of the miRNA.

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