DE102021100227A1 - Pharmaceutical composition containing a PARP inhibitor and/or a genotoxic substance that interferes with the synthesis phase (S phase) of cell replication, in combination with an inhibitor of the RECQ5 signaling pathway - Google Patents

Abstract

The present invention relates to novel pharmaceutical compositions for use in the treatment and/or prevention of efficient homologous recombination (HR) tumors in humans.

Description

introduction

The present invention relates to a pharmaceutical composition for use in the treatment and/or prevention of tumors with efficient homologous recombination (HR) in humans.

Homologous recombination (HR) occurs in all organisms, especially when errors occur during replication, resulting in double-strand breaks. Prerequisite for an HR are homologous, double-stranded DNA segments. Homologous means that there are large similarities in the nucleotide sequence. In the case of double-strand breaks, homologous recombination can repair the damage by using the information on the undamaged chromatid as a template. Thus, the HR in healthy cells serves to repair the DNA.

In some tumors, the HR is no longer functional and, at the same time, tumors replicate much more frequently than most healthy cells, so that the lack of this repair mechanism can be exploited in tumor therapy.

A very promising approach is the treatment of patients with so-called PARP inhibitors.

PARP inhibitors are a relatively new, still small group of drugs used to treat various types of cancer (e.g. ovarian cancer). PARP inhibitors inhibit the enzyme poly-ADP-ribose polymerase (PARP) and prevent cancer cells from repairing single-strand breaks, such as those that occur naturally during replication.

By administering a PARP inhibitor, the repair of spontaneously occurring single-strand breaks is inhibited, which in dividing cells become double-strand breaks. These double-strand breaks are repaired via the HR repair pathway. As a result, the DNA double-strand breaks in HR-defective tumor cells remain unrepaired and lead to cell death.

The most prominent examples of this are tumors with defects in the BRCA1 or BRCA2 gene, which are often present in familial breast cancer.

A similar approach to treatment with PARP inhibitors is treatment with genotoxic substances that interfere with the S-phase of cell replication, such as alkylating substances or topoisomerase I inhibitors.

These genotoxic substances provoke single-strand breaks, which become double-strand breaks in dividing cells. Because tumors replicate much more frequently than most benign cells, tumors are much more susceptible to double-strand breaks after treatment with genotoxic agents that interfere with the S-phase of cell replication. If the tumor is then HR-deficient, it can no longer repair the double-strand break and the cells die.

However, the approach of a PARP inhibitor treatment or treatment with genotoxic substances that interfere with the S-phase of cell replication is limited to tumors with defective HR, i.e. to tumors with BRCA 1/2 mutations or mutations in others for the HR essential genes.

Up to now, tumors with a profitable HR (i.e. functioning HR) could not be treated with PARP inhibitors or with genotoxic substances that interfere with the synthesis phase (S phase) of cell replication. Although homologous recombination can also be switched off in cells by chemical inhibitors, this would affect all cells in the body and would therefore not allow selective inactivation of the tumor cells. Therefore, until now, the extremely effective PARP inhibitor treatment, or treatment with genotoxic substances that interfere with the synthesis phase (S-phase) of cell replication, has only been limited to tumors with defective HR.

The present invention now describes a novel composition and method for PARP treatment and/or treatment with genotoxic substances of tumors with efficient homologous recombination (HR) in humans.

object of the invention

Up to now, tumors with a profitable HR (i.e. functioning HR) could not be treated with PARP inhibitors or with genotoxic substances that interfere with the S phase of cell replication.

The present invention relates to a pharmaceutical composition containing a PARP inhibitor and/or a genotoxic substance that interferes with the S phase of cell replication and an inhibitor of the RECQ5 signaling pathway for use in the treatment and/or prevention of tumors efficient homologous recom Bination (HR), preferentially from ATRX-deficient tumors and/or ALT tumors, in humans.

This solves the task.

Description of the invention

Surprisingly, the inventors of the present invention found that homologous recombination (HR) is composed of two independent, parallel sub-signalling pathways and about 10% of all tumors with profitable homologous recombination (HR) have a defect in one of these two sub-signalling pathways.

The two independent signal transduction pathways are ATRX-dependent in one case and RECQ5-dependent in the other case.

ATRX stands for the transcription regulator ATRX, also known as ATP-dependent helicase ATRX, X-linked helicase II or X-linked core protein (XNP). A protein encoded by the ATRX gene in humans.

RECQ5 (often also referred to as “RECQL5”) stands for ATP-dependent DNA helicase Q5. It is an enzyme encoded by the RECQL5 gene in humans.

The two signaling pathways were so named because the inhibition of one of the two factors leads to the inhibition of one of the two sub-signalling pathways. If both signaling pathways are disrupted, homologous recombination no longer takes place, but if only one pathway is disrupted, the cell can still carry out complete homologous recombination using the other pathway and repair DNA damage using HR.

The inventors were able to find out for the first time that about 10-15% of all tumors have defects in the ATRX gene and can therefore only use the RECQ5 sub-signal pathway of the HR to repair DNA damage.

Thus, in ATRX-deficient tumor cells, inhibition of RECQ5 leads to a loss of overall HR function, while in ATRX-proficient normal tissue cells, inhibition of RECQ5 does not adversely affect HR.

The idea of the invention is to treat ATRX-defective tumors with a pharmaceutical composition that contains both an inhibitor of the RECQ5 signaling pathway and also a PARP inhibitor and/or a genotoxic substance that interferes with the S phase of cell replication. contains.

Such an inhibitor of the RECQ5 signaling pathway can be a chemical inhibitor such as 2,3-Butanedione 2-Monoxime or a molecular biologically produced inhibitor such as an antibody.

Those skilled in the art will also recognize that by "an inhibition of RECQ5" or an "inhibitor of the RECQ5 pathway" is meant herein an inhibitor that inhibits any molecule in the RECQ5 pathway. This can be done by direct inhibition of the RECQ5 protein itself or by blocking another factor in the RECQ5 signaling pathway, such as RECQ1 inhibition.

Through the inactivation of the RECQ5 signal path, according to the invention, selective PARP inhibitor treatment and/or treatment with a gene-toxic substance that interferes with the S-phase of cell replication is now also possible in tumor cells with efficient HR.

In principle, all ATRX-defective tumor cells can be treated with the method according to the invention. Thus, in one embodiment, tumors are included that have been shown to be positive for an ATRX defect in a screening. However, tumors with a known ATRX defect are often so-called ALT tumors, such as glioblastomas. Therefore, in a preferred embodiment, the treatment of ALT tumors is the aim of the present invention.

composition

The present invention represents a pharmaceutical composition containing at least one PARP inhibitor and/or a substance that interferes with the S-phase of cell replication and at least one inhibitor of the RECQ5 signaling pathway for use in treatment and/or prevention of tumors with efficient homologous recombination (HR) in humans.

Here, the “genotoxic substance that interferes with the synthesis phase (S phase) of cell replication” means a substance that preferably triggers single-strand breaks in the DNA during the synthesis phase (S phase) of replication. In one embodiment, the “genotoxic substance” is selected from the group containing topoisomerase I inhibitor, DNA ligase inhibitor, primase inhibitor, helicase inhibitor, DNA polymerase inhibitor and/or the combination thereof. Known topoisomerase I inhibitors are e.g. camptothecin, topotecan, irinotecan. The definition of "genotoxic substance" also includes alkylating agents such as methyl methanesulfonate (MMS) for the treatment of HR-defective tumors.

Here, the PARP inhibitor is preferably selected from the group consisting of olaparib, niraparib, rucaparib, talazoparib, veliparib, pamiparib, iniparib, amelparib, and/or the combination thereof. However, those skilled in the art will understand that other PARP inhibitors are also useful, particularly where their use has been approved for the particular tissue type or tumor type.

The RECQ5 signaling pathway inhibitor is preferably selected from the group consisting of a chemical inhibitor such as 2,3-butanedione 2-monoxime, and/or doxycycline; a molecular biology engineered inhibitor such as an antagonistic protein (e.g. antagonistic antibody or anticalin) inhibiting RNA (RNAi); an inactivated RECQ5 protein (e.g. a RECQL5 without helicase function) and/or RECQ1 protein, a proteolysis targeting chimera (PROTAC) with a selective binding site for the RECQ5 protein, and/or the combination thereof .

In this application, the term "an inhibitor of the RECQ5 signaling pathway" means at least one molecule selected from the list of chemical inhibitors, antagonist proteins, inhibitory RNA, inactivated protein, PROTAC, and/or a combination of two, three or four molecules from this list , such as chemical inhibitor and antagonist protein, chemical inhibitor and inhibitory RNA, chemical inhibitor and inactivated protein, chemical inhibitor and PROTAC, antagonist protein and inhibitory RNA, antagonist protein and inactivated protein, antagonist protein and PROTAC; inhibitory RNA and inactivated protein, inhibitory RNA and PROTAC; inactivated protein and inhibitory RNA, and/or inactivated protein and PROTAC. A “chemical inhibitor” is any small organic and/or inorganic compound, but also small peptides with less than 20 amino acids that have an inhibitory effect on the RECQ5 signaling pathway. Examples include 2,3-butanediones, 2-monoximes and/or doxocycline.

All peptides with up to 20 amino acids and proteins with more than 20 amino acids that are able to have an inhibiting effect on the RECQ5 signaling pathway are referred to as “antagonistic protein”. An example of antagonistic proteins are e.g. antagonistic antibodies.

“Antibodies” (immunoglobulins) are proteins from the class of globulins that are formed (synthesized) in vertebrates as a reaction product of special body cells (plasma cells) to certain substances (substances known as antigens).

Each antibody consists of two identical heavy chains (H) and two identical light chains (L) linked together by covalent interchain disulfide bonds (called interchain disulfides) to form a Y-shaped structure are linked. The light chains (also: light chains) each consist of a variable and a constant domain. These are referred to as V _L and C _L . The heavy chains (also: heavy chains), on the other hand, each have one variable and three (IgG, IgA) or four (IgM, IgE) constant domains. IgG type antibodies are preferred. These are referred to analogously as V _H and C _H 1, C _H 2, C _H 3. In a further embodiment, antigen-binding fragments are also included, such as Fab fragments (from engl. fragment antigen binding), which the antigen represent a binding fragment of an antibody which can be obtained by cleavage with the enzyme papain. Likewise, for example, scFv fragments, also scFv antibodies (single chain variable fragment) are to be included, which represent artificially produced antibody fragments in which the antigen-recognizing variable domains of the light and heavy chain of a classic antibody have been covalently linked . Derivatives of scFv are e.g. B. SMIPs. These smaller antigen-binding fragments have the advantage of being able to penetrate certain tissues more easily and thus have a better effect on the tissue.

“Inhibitory antibodies” are understood to mean antibodies and antigen-binding fragments that prevent signal transduction in the RECQ5 signaling pathway either by binding to RECQ5 itself and/or one or more binding partners of RECQ5. In a further embodiment, the inhibiting antibodies prevent signal transduction in the RECQ5 signaling pathway by binding to another protein which plays a role in the RECQ5 signaling pathway.

Here, “anticalins” refer to artificial proteins that have been artificially made capable of binding antigens through protein design. Anticalins are structurally derived from naturally occurring lipocalins. They consist of around 180 amino acids and, with a molecular mass of less than 20 kDa, are around eight times smaller than antibodies of the IgG type. Anticalins are characterized by a superior tissue penetration compared to antibodies. In addition, they have increased heat stability up to temperatures of over 70 °C.

“Inhibitory anticalins” are understood to mean those that prevent signal transduction in the RECQ5 signaling pathway either by binding to RECQ5 itself and/or one or more binding partners of RECQ5. In another In one embodiment, the inhibiting anticalins prevent signal transduction in the RECQ5 signaling pathway by binding to another protein which plays a role in the RECQ5 signaling pathway.

“Inhibitory RNA (RNAi)” is understood to mean those RNA molecules that prevent the production of RECQ5 itself and/or another molecule that is necessary for signal transduction in the RECQ5 signaling pathway. By stopping the production, the RECQ5 signaling pathway then comes to a standstill in a similar way as if there were a defect in the RECQ5 gene (or the gene coding for one of the other proteins involved in the RECQ5 signaling pathway). In one embodiment, the “inhibitory RNA” is a small interfering RNA (siRNA) molecule, in another embodiment it is a small hairpin RNA (shRNA).

“siRNA” (small interfering RNA) refers to short, single or double-stranded ribonucleic acid molecules with a length of 20 to 25 base pairs. They do not encode proteins, but bind to complementary, single-stranded ribonucleic acid molecules, thereby preventing their normal function.

A “small hairpin RNA” (shRNA for short) is an RNA molecule that forms a hairpin structure and can be used to artificially silence genes using RNA interference (RNAi). The expression of shRNA is mostly mediated by plasmids or viral vectors. The choice of promoter is crucial for reliable shRNA expression. Preferred promoters are those of polymerase II.

shRNA is an advantageous mediator of RNAi because it has a relatively low rate of degradation. However, it requires the use of expression vectors. Introduction of plasmids into cells by transfection can be accomplished in vitro using commercially available reagents. However, this method cannot be used in vivo and is therefore limited.

The use of bacterial vectors for shRNA expression in cells in vivo is therefore the preferred method. When fed to mice, recombinant bacteria of the species Escherichia coli containing shRNA plasmids induce silencing of target genes in the gut.

In a further embodiment, constructs based on adeno-associated viruses (AAV), adenoviruses or lentiviruses can be used as viral vectors. Adeno-associated and adenoviruses do not mutate the target cell genome; however, the transgene is lost after cell division. Lentiviruses integrate into the genome and are thus passed on after cell division; mutate the genome of the target cell. This problem can be circumvented by using integrase-deficient lentiviruses.

The siRNA and/or small hairpin RNA (shRNA) can be stabilized by chemical modifications, for example the incorporation of chemically modified nucleotides, so that degradation in the body is slowed down and the effect lasts longer.

As "PROTAC (proteolysis-targeting-chimera) is meant herein a heterobifunctional small molecule consisting of two active domains and a linker, which can remove specific undesired proteins. Instead of acting as a traditional enzyme inhibitor, a PROTAC induces selective intracellular proteolysis. PROTACs consist of two covalently linked protein-binding molecules: one that can bind an E3 ubiquitin ligase and one that binds to a target protein for degradation, e.g. RECQ5. Recruitment of the E3 ligase into the target protein leads to ubiquitination and subsequent degradation of the target protein by the proteasome. PROTACs need only bind their targets with high selectivity rather than inhibiting the enzymatic activity of the target protein. They are therefore also suitable for molecules that specifically recognize RECQ5 (or another protein from the RECQ5 signaling pathway) but do not inhibit it enzymatically.

The term "inhibition" here means at least the reduction in the functionality of a molecule and/or the entire RECQ5 signaling pathway in which the molecule acts by at least 50% compared to the state without inhibition. In preferred embodiments, the inhibition is a reduction of 50% or more, 60% or more, 70% or more, 80% or more, and up to 85%, up to 90%, up to 95%, up to 99%, up to 99.9% or up to 100%, with a reduction of 100% corresponding to complete inhibition, i.e. no function of the inhibited molecule within the RECQ5 signaling pathway is detectable.

The "functionality of a molecule" refers to any function within the RECQ5 signaling pathway, such as binding to a target protein, enzymatic activity, channel and/or transport activity, and/or structure-forming activity.

The inhibition described in each case can in principle be reversible or irreversible, competitive or allosteric, partial or complete be. Reversible, complete, competitive and/or allosteric inhibition is preferred. In the case of some specific tumors, however, even a reversible, partial inhibition can be sufficient to achieve a positive effect.

Inhibitors that prevent the production of RECQ5 and/or RECQ1 are also included in further embodiments. Those skilled in the art will also recognize that "an inhibition of RECQ5" or an "inhibitor of the RECQ5 signaling pathway" is meant herein any inhibitor that inhibits the RECQ5 sub-pathway. This can take place via direct inhibition of the RECQ5 protein itself and/or via blockade of another protein or factor in the RECQ5 signaling pathway. In one embodiment, the goal is selective inhibition of RECQ1, another factor involved in the RECQ5 signaling pathway. In a further embodiment, several factors in the RECQ5 signaling pathway can also be inhibited in order to improve the overall inhibition, such as an inhibitor for RECQ5 and for RECQ1.

Successful PARP inhibition with combined RECQ5 inactivation is characterized in that at least 2 times, preferably at least 3 times, more preferably at least 4 times, particularly preferably at least 5 times more double-strand breaks occur than in the control group.

A successful genotoxic treatment of the S phase of cell division (e.g. with topoisomerase I inhibitor) with combined RECQ5 inactivation is characterized in that at least 2 times, preferably at least 3 times, more preferably at least 4 times, particularly preferably at least 5 times more double-strand breaks than in the control group.

A successful cell treatment is characterized by the fact that after PARP inhibition and/or genotoxic treatment, the ability to form colonies and/or the viability measured in an ATP consumption test in cells with defective ATRX after inactivation of RECQ5 and/or RECQ1 has taken place at least 2-fold, preferably at least 3-fold, more preferably at least 4-fold, particularly preferably at least 5-fold compared to the comparison group of healthy cells with intact ATRX.

It follows that, within the meaning of this invention, successful inactivation of RECQ5 in tumors with defective ATRX is considered to have been achieved if at least 50%, preferably at least 60%, more preferably at least 70%, and up to 80%, up to 90% , and particularly preferably up to 100% of the tumors with defective ATRX are no longer detectable after 14 days.

The pH of the composition is in a physiologically tolerable range from 5.0 to 10.0. The composition also has an osmolarity ranging from 100 to 500 mOsm/l.

Preferably the composition has an osmolarity in the range 100 to 400 mOsm/l. Even more preferably an osmolarity in the range of 125 to 330 mOsm/l. An osmolarity in the range of 125 to 280 mOsm/l is most preferred. Good tolerability is achieved by adjusting the osmolarity accordingly.

Good tolerability means that when the composition described here is administered orally, there is generally no significant irritation of the gastrointestinal tract and/or systemic irritation (e.g. in the case of i.v. administration).

The pH value of the composition also plays a role in ensuring good tolerability. Compositions with pH values in the range from 6.6 to 10.5 are considered to be physiologically tolerable. Compositions with pH values above 10.5 or below 6.6 are typically not well tolerated in the gastrointestinal tract and/or systemically (e.g. with IV administration) and may result in pain or irritation or both.

Preferably the present composition has a pH ranging from 6.6 to 9.7. More preferably, the present composition has a pH ranging from 7.0 to 9.0.

The composition preferably further comprises at least one inorganic salt. This can advantageously be used to adjust the osmolarity mentioned above.

The composition particularly preferably comprises halide salts of the alkali and/or alkaline earth metals, or mixtures thereof. Most preferably, the composition includes sodium chloride, potassium chloride, magnesium chloride, calcium chloride, or mixtures thereof. Sodium chloride or mixtures of sodium chloride with other inorganic salts described above are most preferred. The preferred salts mentioned, in particular sodium chloride, are advantageous since they are extremely well tolerated salts which are well suited for adjusting the osmolarity.

The composition preferably comprises the inorganic salt in a concentration of 0.7 to 1.4% by weight based on the total weight weight of the composition. More preferably, the composition has the inorganic salt at a concentration of from 0.7% to 1.2% and most preferably from 0.8% to 1.0% by weight based on the total weight of the composition. Most preferably, the composition comprises sodium chloride at a concentration of 0.7% to 1.2% by weight based on the total weight of the composition.

The salt concentrations mentioned above are advantageous because they have an osmolarity adapted to the osmotic pressure of the target cells. Adjusted osmolarity can advantageously help minimize pain and irritation that may be caused by ingestion of the composition.

The composition preferably includes a buffer system. The addition of a buffer system is advantageous in order to ensure physiological compatibility. Most preferably the composition comprises a phosphate or citrate buffer system. Phosphate or citrate buffer systems are generally well tolerated physiologically and are therefore advantageous. Other buffers that are used in some embodiments are sodium dihydrogen phosphate, sodium hydrogen carbonate, calcium hydrogen phosphate and/or trometamol.

Preferably, the composition includes other adjuvants. Preferred auxiliaries are, for example, fillers, solvents, emulsifiers, wetting agents, thickeners and binders, coating agents, disintegration accelerators (disintegrants), lubricants (mould release agents), flow regulators, antioxidants, preservatives, taste correctors and/or absorption accelerators.

Preferred fillers, especially in the case of tabletting, are lactose, cellulose, starches, sucrose, polyethylene glycols (macrogols, PEG) and/or polyethylene oxides (PEO).

Preferred solvents, particularly in the case of granulation, are water, ethanol and/or isopropanol.

In some embodiments, preferred emulsifiers can be cetylstearyl alcohol, glycerol monostearate, lecithin, fatty acid esters of sorbitan, polyoxyethylene sorbitan (polysorbates), polyoxyethylene and/or polyoxyethylene fatty alcohol ethers.

Particularly in the case of suspensions, preferred wetting agents are polyethylene glycols (PEG, macrogols), polyethylene oxides (PEO, PolyOx), and/or polysorbates.

Preferred thickeners and binders are, especially for tablets and granules, starches, guar gum, xanthan, alginate, carrageenan, pectin, tragacanth, polyacrylic acids, polyvinylpyrrolidone (granules, tablets), highly disperse silicon dioxide and/or substituted cellulose ethers (methyl cellulose, ethyl cellulose, hydroxypropyl cellulose , Hydroxypropylmethylcellulose, Carboxymethylcellulose).

Preferred coating agents are, especially in the case of capsules and film tablets, sucrose, gelatin, gelatin polysuccinate, polyacrylates, ethyl cellulose and/or methyl cellulose.

Preferred disintegrating agents are, particularly in the case of tableting or effervescent tablets, starches, croscarmellose and/or sodium bicarbonate in combination with citric acid.

Preferred glidants and lubricants are, in particular for tabletting, polyethylene glycols (PEG, macrogols), polyethylene oxides (PEO), talc and/or magnesium stearate.

A preferred flow regulator, particularly in the case of powders and/or granules, is, for example, highly disperse silicon dioxide.

Preferred antioxidants are, for example, butylated hydroxytoluene and/or all-rac-α-tocopherol.

Preferred preservatives include PHB esters (parabens), benzalkonium chloride, benzyl alcohol, and/or thiomersal.

Preferred sweeteners are, for example, sucrose, sorbitol, sweeteners such as sodium saccharin and cyclamate, and/or flavorings.

A preferred absorption accelerator is, for example, a liposomal preparation, optionally in combination with a cell-penetrating peptide (CPP).

Preferably, the osmolarity of the composition is not, or only minimally, affected by the addition of the excipients.

In one embodiment, the composition is in the form of a tablet, granules, caplets and/or film-coated tablets. In another embodiment, as a liquid that can be administered intravenously (IV), or intraarterially (IA). Such administration may be by syringe or infusion, for example.

The manner in which the application forms mentioned are prepared is known to the person skilled in the art.

The composition is sterilized by processes and methods known to those skilled in the art, such as autoclaving or sterile filtration.

Type of treatment/application

The composition according to the invention is suitable for the treatment of all tumors which have a defect in the ATRX sub-signal path of homologous recombination.

This applies in particular to so-called ALT tumors. The abbreviation "ALT" stands for the English term "Alternative lengthening of telomeres". ALT tumors are characterized by heterogeneous, variable telomere lengths, high levels of telomeric sister chromatid exchanges (t-SCEs), frequent extrachromosomal telomeric DNA repeats (ECTR), and often a specialized telomeric DNA core structure (known as ALT-associated promyelytic leukemia -Body (PML-APBs)).

Tumors that can preferably be treated with the present composition are therefore ALT tumors with the following tissue origin: adrenal/peripheral nervous system (e.g. neuroblastoma), bone (e.g. osteosarcoma), breast tissue, central nervous system (e.g. glioblastoma), colorectal, hematopoietic , kidney tissue, liver tissue, lung tissue, neuroendocrine (e.g. pankeraeatic neuroendocrine tumor (PanNET)), ovaries, pancreas, prostate, skin, soft tissues (e.g. leiomyosarcoma, malignant fibrous histiocytoma (MFH), liposarcoma), stomach, testes, thyroid, urinary bladder and /or uterus.

Therefore, in one embodiment, at least the following tumors are included: glioblastoma, neuroblastoma, osteosarcoma, leiomyosarcoma, malignant fibrous histiocytoma (MFH), liposarcoma, pankeraeatic neuroendocrine tumor (PanNET), tumors of the breast tissue, the central nervous system, the peripheral nervous system, the kidney tissue, the liver tissue, lung tissue, ovaries, pancreas, prostate, skin, soft tissue, stomach, testes, thyroid, urinary bladder, uterus; colorectal, hematopoietic, neuroendocrine, adrenal tumors, and/or combinations thereof.

In addition to acute treatment, preventive treatment is also conceivable, e.g. if there is a genetic disposition and/or a previous illness that can result in an ALT tumor. For example, a patient with t(15;17) translocation could be treated preventively with the present composition to prevent the development of acute promyelocytic leukemia (PML).

Accordingly, the group of patients to be treated is characterized in that they have a defect in the ATRX signal path and/or a prevalence of ALT tumors and/or have already developed such a tumor.

• Das Ziel der Behandlung ist jeweils eine möglichst gleichzeitige Anflutung, Plateauphase und Abflutung der beiden Inhibitoren im Zielgewebe zu erreichen, damit die gleichzeitige synergistische Wirkung stattfinden kann.

A typical administration schedule looks like this:

• The aim of the treatment is to achieve the most simultaneous possible flooding, plateau phase and draining of the two inhibitors in the target tissue so that the simultaneous synergistic effect can take place.

Kinetics that are as similar as possible can be adjusted by suitable choice of the inhibitors and/or by means of suitable auxiliaries.

In one embodiment, a combination composition is preferred that contains all inhibitors in one dosage form. In a further embodiment, separate administration of the two inhibitors is also included, e.g. at different times or in different administration forms, e.g. as a tablet and infusion.

For the purposes of the present invention, separate administrations should therefore also be understood as being encompassed by the invention if their aim is to induce the synergistic effect according to the invention of RECQ5 inhibition with simultaneous PARP treatment in the patient.

The administration of the pharmaceutical composition can also preferably include a PARP inhibitor and/or genotoxic substance that interferes with (S-phase) cell replication in combination with another drug (such as alkylating agents such as methyl methanesulfonate ( MMS)) take place, which then causes single-strand breaks or, in the event of replication, double-strand breaks in the target cell, preferably in the target tumor cells, and thus increases the need for homologous recombination of the cells.

In one aspect, the present invention relates to a pharmaceutical composition containing at least one genotoxic substance that intervenes in the synthesis phase (S phase) of cell replication and at least one inhibitor of the RECQ5 signaling pathway.

In a special aspect, the genotoxic substance of the pharmaceutical composition according to the invention is selected from the group containing poly-ADP-ribose polymerase (PARP) inhibitor, topoisomerase I inhibitor, DNA ligase inhibitor, primase inhibitor, helicase inhibitor, DNA - polymerase inhibitor and/or the combination thereof.

In a further aspect of the invention, the PARP inhibitor is selected from the group consisting of olaparib, niraparib, rucaparib, talazoparib, veliparib, pamiparib, iniparib, amelparib, and/or the combination thereof.

In a further aspect of the invention, the inhibitor of the RECQ5 signaling pathway is selected from the group containing a chemical inhibitor, preferably 2,3-butanedione 2-monoxime and/or doxycycline; an antagonistic protein, preferably an antagonistic antibody and/or an antagonistic anticalin; interfering RNA, preferably siRNA and/or shRNA; an inactivated RECQ5 protein, preferably inactivated RECQ5 protein and/or RECQ1 protein; a chimeric proteolysis-inducing substance (PROTAC) having a selective binding site for RECQ5 protein and/or RECQ1 protein, and/or a combination thereof.

In a further aspect of the invention, the composition contains at least one auxiliary selected from the group consisting of buffers, salts, fillers, solvents, emulsifiers, wetting agents, thickeners and binders, encapsulating agents, disintegrants (disintegrants), glidants and lubricants (mould release agents), flow regulators, Antioxidants, preservatives, taste correctors, absorption accelerators and/or combinations thereof.

A further aspect of the invention also includes uses of the pharmaceutical composition for the treatment and/or prevention of tumors with efficient homologous recombination (HR).

In a further aspect, the invention comprises a pharmaceutical composition containing at least one genotoxic substance which interferes with the S-phase of cell replication, for use in the treatment and/or prevention of tumors with efficient homologous recombination (HR) in a patient, wherein the patient has previously been treated with a pharmaceutical composition containing at least one inhibitor of the RECQ5 signaling pathway. Here, the treatment with the inhibitor of the RECQ5 signaling pathway has preferably up to 12 hours, up to 6 hours, up to 3 hours, up to 2 hours, up to 1 hour, up to 30 minutes before the treatment with the inhibitor of the S- phase of cell replication has taken place.

In a further aspect, the invention comprises a pharmaceutical composition containing at least one inhibitor of the RECQ5 signaling pathway for use in the treatment and/or prevention of tumors with profitable homologous recombination (HR) in a patient, the patient having previously been treated with a pharmaceutical composition containing at least a genotoxic substance that interferes with the S-phase of cell replication. Here, the treatment with the genotoxic substance has preferably taken place up to 12 hours, up to 6 hours, up to 3 hours, up to 2 hours, up to 1 hour, up to 30 minutes before the treatment with the inhibitor of the RECQ5 signaling pathway.

According to the present invention, a pharmaceutical composition containing both inhibitors can be used, but also a pharmaceutical composition containing only one of the two inhibitors if the patient has been pretreated with another pharmaceutical composition containing the second of the two inhibitors. The three different forms of use are to clarify that according to the invention all three forms of treatment (combination preparation, first treatment with RECQ5 inhibitor followed by second treatment with PARP inhibitor, first treatment with PARP inhibitor followed by second treatment with inhibitor of the RECQ5 signaling pathway) are included.

In another aspect, the invention includes a pharmaceutical composition for one of the uses mentioned, wherein the genotoxic substance that interferes with the S phase of cell replication is a PARP inhibitor selected from the group containing olaparib, niraparib, rucaparib, talazoparib , veliparib, pamiparib, iniparib, amelparib, and/or the combination thereof.

In a further aspect, the invention comprises a pharmaceutical composition for one of the uses mentioned, wherein the inhibitor of the RECQ5 signaling pathway is selected from the group consisting of a chemical inhibitor, preferably 2,3-butanedione 2-monoxime and/or doxycycline; an antagonistic protein, preferably an antagonistic antibody and/or an antagonistic anticalin; interfering RNA, preferably siRNA and/or shRNA; an inactivated RECQ5 protein, preferably inactivated RECQ5 protein and/or RECQ1 protein; a chimeric proteolysis-inducing substance (PROTAC) having a selective binding site for RECQ5 protein and/or RECQ1 protein, and/or a combination thereof.

In a further aspect, the invention comprises a pharmaceutical composition for one of the uses mentioned, the composition containing at least one excipient selected from the group consisting of buffers, salts, fillers, solvents, emulsifiers, wetting agents, thickeners and binders, coating agents, disintegrants (disintegrants) , glidants and lubricants (mould release agents), flow regulators, antioxidants, preservatives, taste correctors, absorption accelerators and/or combinations thereof.

In a further aspect, the invention comprises a pharmaceutical composition for one of the uses mentioned, wherein the tumor to be treated is selected from the group containing a tumor with a deficiency in the ATRX signaling pathway and/or an ALT tumor.

In another aspect, the invention includes a pharmaceutical composition for one of the uses mentioned, wherein the ATRX-deficient tumor and/or ALT tumor is selected from the group consisting of glioblastomas, neuroblastomas, osteosarcomas, leiomyosarcomas, malignant fibrous histiocytoma (MFH), liposarcomas , pankeraeatic neuroendocrine tumor (PanNET), tumors of breast tissue, central nervous system, peripheral nervous system, kidney tissue, liver tissue, lung tissue, ovaries, pancreas, prostate, skin, soft tissue, stomach, testes , the thyroid gland, the urinary bladder, the uterus; colorectal, hematopoietic, neuroendocrine, adrenal tumors, and/or combinations thereof.

1. 1. Herstellen des PARP-Inhibitors und des Inhibitor des RECQ5-Signalweges,
2. 2. optional Hinzufügen von mindestens einem Hilfsstoff,
3. 3. Zusammenfügen der Inhibitoren und des optionalen Hilfsstoffes zu der pharmazeutischen Zusammensetzung.

In further aspects, the present invention also includes a method for producing one of the pharmaceutical compositions mentioned, with the steps:

1. 1. Manufacturing the PARP inhibitor and the inhibitor of the RECQ5 signaling pathway,
2. 2. optional addition of at least one excipient,
3. 3. Assembling the inhibitors and the optional excipient into the pharmaceutical composition.

examples

example 1

The effect of the approach of a PARP inhibition with combined RECQ5 and/or RECQ1 inactivation is demonstrated via the occurrence of double-strand breaks that cause cell death.

For this purpose, ATRX-defective tumor cells (e.g. U2OS osteosarcoma cells) are treated with siRNA against RECQ5 and/or RECQ1, about 1-2 days later a PARP inhibitor treatment (e.g. with 1 µM olaparib) for about one day (e.g. 16 and 24 h) exposed and then subjected to an analysis of double-strand breaks that have occurred using immunofluorescence.

Nondegenerate fibroblasts in which ATRX is intact are used as reference cells. The mode of action of RECQ5 and/or RECQ1 inhibition will also be compared with that of BRCA2 inhibition, since the effect of PARP inhibition has already been described for the latter and has been used clinically.

In ATRX-defective cells with inactivated RECQ5 and/or RECQ1, a similar number of double-strand breaks occur as after inactivated BRCA2. In the non-degenerate comparison cells with intact ATRX, double-strand breaks occur after BRCA2 inactivation, but not after RECQ5 and/or RECQ1 inactivation.

Successful PARP inhibition with combined RECQ5 inactivation is characterized by 2-5 times more double-strand breaks than in the control group.

example 2

As an alternative and in addition to the PARP inhibitor treatment, treatment with the topoisomerase I inhibitor camptothecin is applied at a concentration of 20 nM for 1 hour.

16 hours after the treatment, the double-strand breaks that have occurred are again quantified using immunofluorescence, particularly in the cells that were in the replication phase during the treatment with camptothecin.

In ATRX-defective cells with inactivated RECQ5 and/or RECQ1, a similar number of double-strand breaks occur as after inactivated BRCA2. In the non-degenerate comparison cells with intact ATRX, double-strand breaks occur after BRCA2 inactivation, but not after RECQ5 and/or RECQ1 inactivation.

A successful genotoxic treatment of the S phase of cell division with combined RECQ5 inactivation is characterized by the fact that 2-5 times more double-strand breaks occur than in the control group.

Example 3

Furthermore, the proof of the effect of the approach takes place directly via the determination of the cell death.

For this purpose, cells are treated with a PARP inhibitor (e.g. olaparib in a concentration range of 10 nM to 1 µM) or a genotoxic substance (camptothecin in a concentration range of up to 20 nM or methyl methanesulfonate in a concentration range of up to 1 mM) for 5-10 days and then their survival is tested will.

For this purpose, the ability of the cells to form colonies and/or their viability is measured using a test based on ATP consumption measurements (e.g., CellTiter-GIo® 2.0 Cell Viability Assay, Promega; scientific reference: https://doi.org/10.1016/j.molcel .2020.12.006).

The same genetic dependencies as described in the examples above are examined, but with the technical difference that instead of RECQ5 and/or RECQ1 inactivation using siRNA, inactivation is carried out using a small hairpin RNA (shRNA) approach.

For this purpose, stable cell lines are produced in which the expression of the shRNA against RECQ5 with subsequent depletion of the protein takes place by means of the addition of doxycycline.

A successful treatment is characterized by the fact that, after PARP inhibition or genotoxic treatment, the ability to form colonies and/or viability measured in an ATP consumption test (see above) in cells with defective ATRX after inactivation of RECQ5 and/or RECQ1 2- is 5-fold higher compared to the comparison group of healthy cells with intact ATRX.

example 4

Evidence of the effect of the approach is also shown in the mouse tumor model.

For this purpose, the prepared shRNA cell lines against RECQ5 with either ATRX-deficient or ATRX-proficient background are injected into mice and allowed to grow into tumors.

Thereafter, doxycycline-mediated RECQ5 is inactivated and the mice are treated with PARP inhibitors.

Inactivation of RECQ5 in tumors with defective ATRX causes tumor death after PARP inhibitor treatment, while the normal tissue cells of mice with intact ATRX show no abnormalities.

Successful inactivation of RECQ5 in tumors with defective ATRX is considered to have been achieved if at least 50% and up to 100% of the tumors with defective ATRX are no longer detectable after 14 days.

Claims (14)

Pharmaceutical composition containing at least one PARP inhibitor and/or a genotoxic substance that interferes with the synthesis phase (S phase) of cell replication and at least one inhibitor of the RECQ5 signaling pathway. Pharmaceutical composition according to claim 1 , wherein the genotoxic substance which interferes with the S phase of cell replication is selected from the group consisting of topoisomerase I inhibitor, DNA ligase inhibitor, primase inhibitor, helicase inhibitor, DNA polymerase inhibitor and/or or the combination thereof. The pharmaceutical composition according to any one of Claims 1 or 2 , wherein the PARP inhibitor is selected from the group consisting of olaparib, niraparib, rucaparib, talazoparib, veliparib, pamiparib, iniparib, amelparib, and/or the combination thereof. The pharmaceutical composition according to any one of Claims 1 until 3 , wherein the inhibitor of the RECQ5 signaling pathway is selected from the group containing a chemical inhibitor, preferably 2,3-butanedione 2-monoxime and/or doxycycline; an antagonistic protein, preferably an antagonistic antibody and/or an antagonistic anticalin; interfering RNA, preferably siRNA and/or shRNA; an inactivated RECQ5 protein, preferably inactivated RECQ5 protein and/or RECQ1 protein; a chimeric proteolysis-inducing substance (PROTAC) having a selective binding site for RECQ5 protein and/or RECQ1 protein, and/or a combination thereof. The pharmaceutical composition according to any one of the preceding claims, wherein the composition contains at least one excipient selected from the group consisting of buffers, salts, fillers, solvents, emulsifiers, wetting agents, thickeners and binders, coating agents, disintegrants (disintegrants), glidants and lubricants (mould release agents ), flow regulators, antioxidants, preservatives, Taste correctors, absorption accelerators and/or combinations thereof. Pharmaceutical composition according to any one of the preceding claims for use in the treatment and/or prevention of tumors with profitable homologous recombination (HR). Pharmaceutical composition containing at least a PARP inhibitor and / or a genotoxic substance that interferes with the S phase of cell replication, for use in the treatment and / or prevention of tumors with profitable homologous recombination (HR), the patient previously with a pharmaceutical composition containing at least one inhibitor of the RECQ5 signaling pathway. Pharmaceutical composition containing at least one inhibitor of the RECQ5 signaling pathway for use in the treatment and/or prevention of tumors with profitable homologous recombination (HR), the patient being previously treated with a pharmaceutical composition containing at least one PARP inhibitor and/or a genotoxic substance, which interferes with the S phase of cell replication. The pharmaceutical composition for use according to any one of Claims 7 or 8th , wherein the PARP inhibitor is selected from the group consisting of olaparib, niraparib, rucaparib, talazoparib, veliparib, pamiparib, iniparib, amelparib, and/or the combination thereof. The pharmaceutical composition for use according to any one of Claims 7 or 8th , wherein the inhibitor of the RECQ5 signaling pathway is selected from the group containing a chemical inhibitor, preferably 2,3-butanedione 2-monoxime and/or doxycycline; an antagonistic protein, preferably an antagonistic antibody and/or an antagonistic anticalin; interfering RNA, preferably siRNA and/or shRNA; an inactivated RECQ5 protein, preferably inactivated RECQ5 protein and/or RECQ1 protein; a chimeric proteolysis-inducing substance (PROTAC) having a selective binding site for RECQ5 protein and/or RECQ1 protein, and/or a combination thereof. The pharmaceutical composition for use according to any one of Claims 6 until 10 , wherein the composition contains at least one auxiliary selected from the group of buffers, salts, fillers, solvents, emulsifiers, wetting agents, thickeners and binders, encapsulating agents, disintegration accelerators (explosive agents), lubricants (mould release agents), flow regulators, antioxidants, preservatives, Taste correctors, absorption accelerators and/or combinations thereof. The pharmaceutical composition for use according to any one of Claims 6 until 11 , wherein the tumor to be treated is selected from the group containing a tumor with a deficiency in the ATRX signaling pathway and/or an ALT tumor. The pharmaceutical composition for use according to claim 12 , wherein the ATRX-deficient tumor and/or ALT tumor is selected from the group consisting of glioblastomas, neuroblastomas, osteosarcomas, leiomyosarcomas, malignant fibrous histiocytoma (MFH), liposarcomas, pankeraeatic neuroendocrine tumors (PanNET), tumors of breast tissue, of the central nervous system , peripheral nervous system, kidney tissue, liver tissue, lung tissue, ovaries, pancreas, prostate, skin, soft tissue, stomach, testes, thyroid, urinary bladder, uterus; colorectal, hematopoietic, neuroendocrine, adrenal tumors, and/or combinations thereof. A process for preparing a pharmaceutical composition as claimed in any one of the preceding claims, comprising the steps of a. Production of the PARP inhibitor and/or a genotoxic substance that interferes with the S phase of cell replication and the inhibitor of the RECQ5 signaling pathway, b. optionally adding at least one excipient, c. combining the inhibitors and the optional excipient into the pharmaceutical composition.