Patent Application
20220387369
The present invention relates to prooxidative chain-transfer agents (CTAs) for use in the treatment or prevention of a malignant tumour disease, or infectious disease in humans or animals.
The currently known treatment strategies for tumour diseases or infectious diseases that are based on a more detailed understanding of the underlying biology are expected to improve the treatment outcome for patients suffering from these diseases. However, many of these strategies still have one common and critical problem, being their limited specificity for tumour cells or parasitic cells.
The three major types of cancer treatment, i.e. surgery, chemotherapy and radiotherapy, are powerful, but are associated with risks of injury or toxicity to healthy tissues. Therefore, novel treatment methods having the ability to kill tumour cells without exhibiting these side-effects undergo extensive research and clinical studies. Among them, photodynamic therapy (PDT), sonodynamic therapy (SDT), T-cell immunotherapy and oncolytic virotherapy are expected to improve the treatment outcomes, because they are based on novel target cell killing mechanisms.
A number of medical treatments are available for the treatment of malignant tumours that yielded in a dramatic improvement in cancer survivorship around the world (Pardee and Stein, 2009). The most common active substances that are used in general pharmacotherapy of tumours are cytostatic and cytotoxic substances such as alkylating or intercalating agents, platinum compounds, antimetabolites of the nucleotide metabolism, or topoisomerase and mitose inhibitors that act on the genomic DNA and the cytoskeleton, respectively (Sessa et al., 2012; Aktories et al., 2017). In addition, low molecular weight or antibody-based antagonists of angiogenesis and hormonal stimulation and growth factor stimulation of tumour cell division are also known classes of agents that are frequently used in cancer therapy. In addition, there are a number of very specific therapies available for cases of individual degenerations, for example a degeneration of the haematological system (Sessa et al., 2012; Aktories et al., 2017).
Parasites are microorganisms that live on or inside another organism known as the host organism and benefit at the expense of their host organism. Parasites are responsible for billions of human infections, including malaria. Parasitic infections are especially prevalent in tropical areas, but they also occur in subtropical and temperate regions, where they tend to infect immigrants and travelers. While parasites can include a diverse array of microorganisms, including fungi and bacteria, medically-relevant parasites known to cause disease in humans are protozoa, helminths, and ectoparasites.
The protozoa that are infectious to humans can be classified into four groups based on their mode of movement Sarcodina, Mastigophora, Ciliophora and Sporozoa. The three main groups of helminths that are human parasites are flatworms (platyhelminths) including trematodes (flukes) and cestodes (tapeworms), thorny-headed worms (acanthocephalans) and roundworms (nematodes). Ectoparasites comprise blood-sucking arthropods such as mosquitoes, but also other organisms such as ticks, fleas, lice, and mites that attach or burrow into the skin and remain there for a few weeks or months.
Parasitic nematodes are responsible for widespread morbidity in humans and animals. It is estimated that approximately 1.5 billion people are infected with one or more of these organisms (Hotez et al., 2008; World Health Organization, 2017) which also pose a considerable burden for animal production (Eijck and Borgsteede, 2005; Nganga et al., 2008). The extraordinary success of these parasites is to a large extent based on their ability to withstand a multitude of stresses such as host immune pressures and infectious challenges from microbes. Most parasitic nematodes inhabit the intestines of their hosts, co-existing with numerous microbial species. In studying these dynamics, research was mainly focused on the host-parasite relationship and only recently, the role of the microbiota as a major third party in said relationship is better appreciated due to diverse and far-reaching influences in health and disease (Donaldson et al., 2016). Much attention was given to host immune mechanisms while the interactions between nematodes and their microbial environments were largely overlooked. Due to many technical and biological challenges associated with studying parasites, these questions still remain difficult to address.
The roundworm Caenorhabditis elegans (C. elegans) has risen to the status of a top model organism for biological research in the last fifty years (Frézal and Félix, 2015). C. elegans has been very well characterized and its interactions with bacteria have been studied in considerable detail. As such, these findings might be conveyed to parasitic nematodes in general, and greatly inform our understanding of how parasites interact with the host-microbiota, as many immune-related pathways and responses may be conserved (Tarr, 2012; Rosso et al., 2013).
Antiparasitic drugs have been developed to manage infections caused by various protozoa, helminths, and ectoparasites. The individual treatment options vary, and they mainly depend on the specific causative organism within each group. However, there are several drugs that are commonly used in the treatment of different types of parasite infections in both humans and animals. For example, metronidazole has been found as an efficient drug that is active both against parasites and bacteria (Löfmark et al., 2010). Chloroquin and Artemisinin have been found to be efficient against plasmodia (Tse et al., 2019), albendazole has been found to be effective against roundworms and praziquantel against tapeworms (Albonico et al., 2015; Aktories et al., 2017). Benznidazole is a nitroimidazole antiparasitic with good activity against acute infection with Trypanosoma cruzi, commonly referred to as Chagas disease.
Overall, the pharmaceutical options available for the treatment of parasitic infections that are caused by either single-cellular or multi-cellular organisms is limited. Pharmaceutically active compounds are often based on a few known lead structures, which are sometimes only moderately specific in their effect (Löfmark et al., 2010; Albonico et al., 2015). In addition, the development of resistances against new drugs in the years following their introduction is often responsible for a reduction of their efficacy. Reduced efficacy has been observed in patients suffering from malaria (Greenwood, 2014; Kavishe et al., 2017), or worm infections in humans and animals (Krücken et al., 2017; Lanusse et al., 2018). The undesirable side effects of antiparasitic drugs are a major drawback in their clinical application, and hence are a frequent cause for forcing the physician to stop treatment. The most frequent adverse effects observed are anorexia, psychic alterations, loss of weight, excitability, sleepiness, digestive manifestations such as nausea or vomiting, and occasionally diarrhoea and intestinal colic. In the case of benznidazole, skin manifestations are the most notorious (e.g., hypersensitivity, dermatitis with cutaneous eruptions, generalized oedema, fever, lymphoadenopathy, articular and muscular pain), with depression of bone marrow, thrombocytopenic purpura and agranulocytosis being the more severe manifestations.
Many parasites are known for a weakly expressed anti-oxidative defense that goes along with similar or higher prooxidative activity as compared to normal human cells (Mehlotra, 1996; Turrens, 2004). Accordingly, if subjected to prooxidative amplification, parasitic cells are supposed to be more severely damaged than the body's own cells.
The known prooxidative therapies for treating parasitic infections apply substances such as peroxides (artemisinin) or nitroimidazoles (metronidazole). However, the specificity of those drugs depends on the binding of specific proteins or the activation of specific reductases (Li et al., 2005; Löfmark et al., 2010). Similar drawbacks have also been reported for chloroquine (Kavishe et al., 2017). There is no known drug which is able to exhibit a local prooxidative activity by reversible activation. Many drugs that have been developed to treat neurodegenerative diseases failed to gain approval for clinical use because they are not well tolerated in humans. As a result, there are strategies that are based on the principle that drugs are activated by the pathological state that they are intended to inhibit (Lipton, 2007). Examples of this approach are the potentially neuroprotective drug and glutamate receptor antagonist memantine.
Similar problems also occur in tumour therapy that faces side effects of their anti-cancer drugs that are manifesting as toxic reactions due to alkylation of proteins, or their lack of selectivity if it comes to differentiate between tumour cells and healthy cells (Sessa et al., 2012; Aktories et al., 2017). The specificity of most tumour drugs is still only based on tumour cell division or tumour antigen presentation (Pardee and Stein, 2009; Trachootham et al., 2009). Antibody-therapies that exhibit no cytostatic or only little side effects have been established for only a few tumour types, and they are extremely cost-intense (Sessa et al., 2012; Dolgin, 2018). With the development of nanotechnology, nano-drug systems offer longer blood circulation times and lower systemic toxicity of anticancer drugs (Collins, 2006).
In spite of the availability of complex therapeutic approaches, it is still not possible to ensure a satisfactory five-year survival rate for many tumour types (Siegel et al., 2019). Therefore, there is a need in tumour therapy for low-molecular weight pharmaceuticals that have a fundamentally new mechanism of action.
Tumour cells have also been known for a long time for their high prooxidative activity if compared with normal body cells (Szatrowski and Nathan, 1991). Said activity usually remains below the deadly result for tumour cells (Trachootham et al., 2009; Gorrini et al., 2013; Sosa et al., 2013). A prooxidative therapy of tumours has essentially been proven to be successful in certain cases such as classical radiotherapy (Moss, 2007; Barker et al., 2015), photodynamic therapy (Dolmans et al., 2003) or upon application of low-molecular weight pharmaceuticals (Trachootham et al., 2009; Gorrini et al., 2013; Galadari et al., 2017). More recent work also addressed the sensitization of tumour cells to prooxidants (Toler et al., 2006; Cui et al., 2017; Kubli et al., 2019).
There are also pharmacochemical approaches. For example EP1478357 B1 describes tricyclic pyrazole derivatives, process for their preparation and their use as antitumour agents. EP1124810 B9 describes 2-amino-thiazole derivatives for treating cell proliferative disorders associated with an altered cell cycle dependent kinase activity.
There are also approaches to treat both tumour diseases and parasitic infections using one class of compounds. For example, U.S. Pat. No. 7,247,715 B2 describes ricin-like toxin variants for treatment of cancer, viral or parasitic infections.
Also chain-transfer agents were used in the synthesis of active polymeric compounds. CN106995516 A describes PHPMA and PEG polymers that have a long body circulation time, and are enriched at a tumour. The polymer can carry a drug through a covalent bond and/or a non-covalent bond, and the obtained product can further be coupled to a targeting molecule and/or a labelled molecule for preparing a drug or a detection reagent. KR101389695 B1 describes a method of killing or inhibiting growth of a microorganism in a mammal other than a human infected with the microorganism, wherein alkoxycarbonylalkylthiol is used as a chain transfer agent. One example of such a microorganism is a parasite.
WO 2019/204233 A1 describes agents that are useful for modulating an immune response in a subject and for treating diseases, such as autoimmune diseases, cardiovascular diseases, infectious diseases, and cancer. These agents may comprise an olfactory receptor (OLFR), an OLFR ligand or a protein involved in the trafficking of an OLFR to the plasma membrane of a cell.
US 2013/0041042 A1 describes polymeric compositions for enhanced wound our burn treatment characteristics. U.S. Pat. No. 9,988,348 B1 describes a method for preparing a trithiocarbonate compound, which can be used as anti-microbial agent, for example for use as an anti-bacterial or anti-fungal drug.
However, no specific drug-based intervention has been reported so far to efficiently exploit the redox differences between tumour cells and healthy cells.
It is therefore the object of the present invention to provide new compounds that have a high selectivity and specificity for the target cells and that are suitable for use in the treatment or prevention of a malignant tumour disease or infectious disease, in particular a parasitic disease in humans or animals.
This object is solved by a prooxidative chain-transfer agent with the features of claim 1. Preferred embodiments of the invention are claimed in the sub-claims.
The present invention is based on chain-transfer agents (CTAs) that are also called modifiers or regulators that have at least one weak chemical bond. These compounds react with the free-radical side of a growing polymer chain and interrupt chain growth. In the process of chain transfer, the radical is temporarily transferred to the regulating agent, which re-initiates growth by transferring the radical to another polymer or monomer.
Chain-transfer agents are often added to control the chain length during polymer synthesis to achieve certain mechanical and processing properties. Preferred chain transfer agents comprise halogen compounds, some aromatic hydrocarbons, and thiols (mercaptans).
The invention shows for the first time that certain compounds of polymer chemistry are highly potent, and act as highly selective cytotoxins for therapeutic applications in humans and other mammals as well as in animals. The specific effect of these substances on killing parasitic cells is based on the fact that parasites are often less fortified with antioxidant defenses than human or other mammalian cells.
The prooxidative chain-transfer agents according to the invention are of low molecular weight and can be administered as part of a single or combined pharmacotherapy for treating malignant tumours or parasitic infections in humans or animals.
The inventive compounds overcome the known problem of drug resistance, are highly efficient, and at the same time are of low toxicity for the treated humans or animals. That said, the inventive compounds can be applied in a selective prooxidative therapy involving a prooxidative chain transfer activity mediated by the inventive compounds. The inventive prooxidative chain-transfer agents can be grouped into four chemical classes: lipophilic thiols, lipophilic trithiocarbonates, lipophilic aromatic dithioesters, and lipophilic, aromatic thiols. These compounds are able to exhibit their prooxidative activity directly at the site of action, i.e. at the target cells. In the absence of endogenous prooxidative activity, the substances have no effect, because they are not initiating oxidation by themselves. As demonstrated herein, the effectiveness of the compounds was not compromised by hypoxic conditions. As further shown by the present invention, tumour cells and parasitic cells are more severely damaged than the body's own cells as a result of the prooxidative amplification of the inventive chain-transfer agents, thus resulting in a specific cell death of the targeted parasitic cells or tumour cells.
The inventive lipophilic thiols, lipophilic trithiocarbonates and lipophilic, aromatic dithioesters, and lipophilic, aromatic thiols can be described by the following formula (I), (II) (III), and (IV), respectively:
The inventive compounds falling under the general formulas of (I), (II), (III), (IV) have in common that they have the capability to build or produce thiyl radicals or sulfur radicals in the cell membrane. This property makes them unique for the therapeutic and preventive applications described herein. The therapeutic effects of the inventive compounds are based on their ability to produce radicals, thus resulting in the high efficiency of the compounds.
The carbon atoms within the general structure (I) are part of an aromatic ring system, preferably part of a benzene ring.
The term “alkyl” as used herein has 1 to 24 carbons, such as, for example, methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl. It further comprises straight and branched chain aliphatic hydrocarbon groups such as isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl and the like.
The term “alkoxy”, as used herein has 1 to 24 carbon atoms, such as, for example methoxy or ethoxy. It also comprises straight or branched chain aliphatic hydrocarbon groups including n-propoxy, isopropoxy and the like. Preferably, the alkoxy chain is 1 to 24 carbon atoms in length, more preferably 6 to 20 carbon atoms, and more preferably 12 to 18 carbon atoms.
As used herein, the term “aryl” refers to an aromatic carbocyclic group comprising 6 to 12 carbons in the ring portion, preferably 6 to 10 carbons in the ring portion. It comprises both monocyclic or bicyclic aromatic groups such as naphthyl and tetrahydronaphthyl.
In a preferred embodiment, each R1-R4 substituent comprises a hydrophobic group comprising one or more S, O or N heteroatoms in the general structure I of the lipophilic thiols. Each R1-R4 substituent may be hydrophobic, but preferably includes one or more heteroatoms (S, O, N) by having a sufficient number of carbon atoms attached thereto to form a hydrophobic group. The hydrophobic group is preferably branched, substituted or unsubstituted. In a preferred embodiment, the branching occurs at the heteroatom.
Under the condition set forth above (R1+R2+R3+R4≥C6), each R1-R4 may be selected from the group consisting of hydrogen, hydroxyl, a substituted or unsubstituted (C1-C24) alkyl, (C1-C24) hydroxyalkyl, (C1-C24) alkyloxy-(C1-C24) alkyl, (C1-C24) alkylsulfo-(C1-C24) alkyl, (C1-C24) alkylcarboxy-(C1-C24) alkyl, (C1-C24) alkylamino-(C1-C24) alkyl, (C1-C24) alkylamino-(C1-C24) alkylamino, (C1-C24) alkylamino-(C1-C24) alkylamino-(C1-C24) alkylamino, a substituted or unsubstituted (C1-C24) aminoalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted arylamino-(C1-C24) alkyl, (C1-C24) haloalkyl, C2-C24 alkenyl, C2-C24 alkynyl, oxo, sulfo.
An aliphatic group is a branched or unbranched hydrocarbon that may be substituted or unsubstituted. Examples of branched alkyl groups include isopropyl, sec-butyl, isobutyl, tert-butyl, sec-pentyl, isopentyl, tert-pentyl, isohexyl. Substituted aliphatic groups may have one, two, three or more substituents, which may be the same or different, each replacing a hydrogen atom. Substituents are halogen (e.g., F, Cl, Br, and I), hydroxyl, protected hydroxyl, amino, protected amino, carboxy, protected carboxy, cyan, methylsulfonylamino, alkoxy, acyloxy, nitro, and haloalkyl.
The term “substituted” in regard of R2 used in formula (I) refers to a methyl group having one, two, or three substituents, which may be the same or different, each replacing a hydrogen atom. Examples of substituents include but are not limited to halogen (e.g., F, Cl, Br, or I), hydroxyl, protected hydroxyl, amino, protected amino, carboxy, protected carboxy, cyan, methylsulfonylamino, alkoxy, alkyl, aryl, arylalkyl, acyloxy, and haloalkyl.
In an alternative embodiment relating to the general structure (II) of the lipophilic trithiocarbonates R1 is selected from the group consisting of a substituted or unsubstituted (C6-C24) alkyl, (C6-C24) hydroxyalkyl, (C6-C24) alkyloxy, (C6-C24) alkylsulfo, (C6-C24) alkyloxy-(C6-C24) alkyl, (C6-C24) alkylsulfo-(C6-C24) alkyl, (C6-C24) alkylcarboxy-(C6-C24) alkyl, (C6-C24) alkylamino-(C6-C24) alkyl, (C6-C24) alkylamino-(C6-C24) alkylamino, (C6-C24) alkylamino-(C6-C24) alkylamino-(C6-C24) alkylamino, a substituted or unsubstituted (C6-C24) aminoalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted arylamino-(C6-C24) alkyl, (C6-C24) haloalkyl, (C6-C24) alkenyl, (C6-C24) alkynyl, and R2 refers to a hydrogen or a methyl group having one, two, or three substituents, which may be the same or different, each replacing a hydrogen atom.
The term “substituted” in regard of R2 used in formula (II) refers to a methyl group having one, two, or three substituents, which may be the same or different, each replacing a hydrogen atom.
Examples of substituents include but are not limited to halogen (e.g., F, Cl, Br, and I), hydroxyl, protected hydroxyl, amino, protected amino, carboxy, protected carboxy, cyan, methylsulfonylamino, alkoxy, alkyl, aryl, arylalkyl, acyloxy, and lower haloalkyl.
An aliphatic group is a branched or unbranched hydrocarbon that may be substituted or unsubstituted. Examples of branched alkyl groups include isopropyl, sec-butyl, isobutyl, tert-butyl, sec-pentyl, isopentyl, tert-pentyl, isohexyl. Substituted aliphatic groups may have one, two, three or more substituents, which may be the same or different, each replacing a hydrogen atom. Preferred substituents are halogen (e.g., F, Cl, Br, or I), hydroxyl, protected hydroxyl, amino, protected amino, carboxy, protected carboxy, cyan, methylsulfonylamino, alkoxy, acyloxy, nitro, and (lower) haloalkyl.
In the embodiment relating to the general structure (III) of the lipophilic, aromatic dithioesters, R1 is selected from the group consisting of a substituted or unsubstituted (C6-C24) alkyl, (C6-C24) hydroxyalkyl, (C6-C24) alkyloxy, (C6-C24) alkylsulfo, (C6-C24) alkyloxy-(C6-C24) alkyl, (C6-C24) alkylsulfo-(C6-C24) alkyl, (C6-C24) alkylcarboxy-(C6-C24) alkyl, (C6-C24) alkylamino-(C6-C24) alkyl, (C6-C24) alkylamino-(C6-C24) alkylamino, (C6-C24) alkylamino-(C6-C24) alkylamino-(C6-C24) alkylamino, a substituted or unsubstituted (C6-C24) aminoalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted arylamino-(C6-C24) alkyl, (C6-C24) haloalkyl, C6-C24 alkenyl, C6-C24 alkynyl, and R2 refers to a hydrogen or a methyl group having one, two, or three substituents, which may be the same or different, each replacing a hydrogen atom.
The term “substituted” in regard of R2 used in formula (III) refers to a methyl group having one, two, or three substituents, which may be the same or different, each replacing a hydrogen atom. Examples of substituents include but are not limited to halogen (e.g., F, Cl, Br, or I), hydroxyl, protected hydroxyl, amino, protected amino, carboxy, protected carboxy, cyan, methylsulfonylamino, alkoxy, alkyl, aryl, arylalkyl, acyloxy, and lower haloalkyl.
An aliphatic group according to the invention is a branched or unbranched hydrocarbon that may be substituted or unsubstituted. Examples of branched alkyl groups include isopropyl, sec-butyl, isobutyl, tert-butyl, sec-pentyl, isopentyl, tert-pentyl, isohexyl. Substituted aliphatic groups may have one, two, three or more substituents, which may be the same or different, each replacing a hydrogen atom. Substituents are halogen (e.g., F, Cl, Br, or I), hydroxyl, protected hydroxyl, amino, protected amino, carboxy, protected carboxy, cyan, methylsulfonylamino, alkoxy, acyloxy, nitro, and haloalkyl.
Preferred lipophilic thiols falling under the general structure (I) that are suitable for the treatment of a malignant tumour disease or infectious disease are compounds in which R1+R2+R3+R4=C6. Examples of such compounds are n-octyithiol or t-octyithiol. Examples in which R1+R2+R3+R4=C10 are n-dodecylthiol, t-dodecyithiol and t-dodecyithiol isomer. t-dodecyithiol isomers are usually branched and composed as a mixture in a composition. Alternative variants of the inventive compounds in which R1+R2+R3+R4>C10 are n-hexadecylthiol or n-octadecylthiol.
Preferred lipophilic trithiocarbonates falling under the general structure (II) that are suitable for the treatment of a malignant tumour disease or infectious disease are compounds in which R1=C8 are S-octyl-S′[dimethyl-cyanomethyl]-trithiocarbonate, S-octyl-S′[methyl-hydroxypropyl-cyanomethyl]-trithiocarbonate, S-octyl-S′[methyl-carboxyethyl-cyanomethyl]-trithiocarbonate. Examples in which R1=C12 are S-dodecyl-S′[dimethyl-cyanomethyl]-trithiocarbonate, S-dodecyl-S′[methyl-hydroxypropyl-cyanomethyl]-trithiocarbonate, S-dodecyl-S′[methyl-carboxyethyl-cyanomethyl]-trithiocarbonate.
Preferred lipophilic, aromatic dithioesters falling under the general structure (III) that are suitable for the treatment of a malignant tumour disease or infectious disease are compounds in which R1=C12. Examples of compounds in which R1=C12 are S-[dimethyl-cyanomethyl]-dodecylbenzodithioate, S-[methyl-hydroxypropyl-cyanomethyl]-dodecylbenzodithioate, or S-[methyl-carboxyethyl-cyanomethyl]-dodecylbenzodithioate. In alternative compounds, R1=O—C12. Examples in which R1=O—C12 are S-[dimethyl-cyanomethyl]-dodecanoxy-benzodithioate, S-[methyl-hydroxypropyl-cyanomethyl]-dodecanoxy-benzodithioate, or S-[methyl-carboxyethyl-cyanomethyl]-dodecanoxy-benzodithioate.
In the embodiment relating to the general structure (IV) of the lipophilic, aromatic thiols, R1 is selected from the group consisting of a substituted or unsubstituted (C6-C24) alkyl, (C6-C24) hydroxyalkyl, (C6-C24) alkyloxy, (C6-C24) alkylsulfo, (C6-C24) alkyloxy-(C6-C24) alkyl, (C6-C24) alkylsulfo-(C6-C24) alkyl, (C6-C24) alkylcarboxy-(C6-C24) alkyl, (C6-C24) alkylamino-(C6-C24) alkyl, (C6-C24) alkylamino-(C6-C24) alkylamino, (C6-C24) alkylamino-(C6-C24) alkylamino-(C6-C24) alkylamino, a substituted or unsubstituted (C6-C24) aminoalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted arylamino-(C6-C24) alkyl, (C6-C24) haloalkyl, C6-C24 alkenyl, C6-C24 alkynyl.
Preferred lipophilic, aromatic thiols comprising the general structure (IV) in which R1=C12 or R1=X—C12 are 4-dodecyithiophenol, O-dodecyl-4-hydroxythiophenol, S-dodecyl-1,4-benzenedithiol, or N-dodecyl-4-aminothiophenol. Preferred examples of compounds in which R1=C18 or R1=X—C18 are 4-octadecyithiophenol, O-octadecyl-4-hydroxythiophenol, S-octadecyl-1,4-benzenedithiol, or N-octadecyl-4-aminothiophenol.
The most efficient compounds of the invention can be summarized as comprising
a prooxidative chain-transfer agent selected from the group consisting of
for use in the treatment or prevention of a malignant tumour disease, or infectious parasitic disease in humans or animals.
The invention also covers methods of treatment of cancer or a parasitic infectious disease that comprises administering to a human or animal patient one or more compounds falling under the general formulas (I), (II), (III), or (IV). For the treatment of cancer or parasitic diseases, compounds of the structures (1) and (IV) showed the greatest effect as compared to compounds falling under the structures (II) and (III).
The inventive compounds falling under the general formulas (I), (II), (III), or (IV) can be used for the treatment of a variety of infectious diseases, preferably for the treatment of parasitic diseases that are caused by a number of parasites, including both single cell parasites and parasitic animals. Preferably, the parasitic infection is caused by parasites such as Acanthamoeba, Anisakis, Ascaris lumbricoides, Botfly, Balantidium coli, Bedbug, Brugia spp., Cestoda (tapeworm), Chiggers, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, Hookworm, Leishmania spp., Linguatula serrata, Liver fluke, Loa spp., Onchocerca spp., Paragonimus—lung fluke, Pinworm, Plasmodium spp., Schistosoma spp., Strongyloides stercoralis, Mite, Tapeworm, Toxoplasma gondii, Trypanosoma spp., Whipworm, or Wuchereria bancrofti. The invention, however, is not restricted to these particular parasites. Preferred diseases that can be treated by the present invention are causes by Leishmania spp., Plasmodium spp., Schistosoma spp., Trypanosoma spp. Preferred Trypanosoma species are Trypanosoma cruzi and Trypanosoma brucei. Preferred Plasmodium species are Plasmodium falciparum or Plasmodium malariae.
As apparent, the invention can be applied for the therapy or prevention of parasitic diseases in humans or animals, in particular pets, farm animals or breeding animals. In a first aspect, the inventive compounds can be applied to any infected host animal that shows clinical symptoms or suffers from a parasitic disease. Such diseases to be treated in humans or animals are, for instance, different forms of filariasis, lymphatic filariasis, elephantiasis, chocercosis, malaria, leishmaniasis, trypanosomiasis. In a second aspect, the inventive compounds can also be applied to humans or animals that serve as a carrier for the parasite, i.e. third organisms that transmit the parasites to the host, but that do not suffer from the disease. As such, the inventive compounds are suitable for an eradication of an infection. Examples of trematodiasis and nematodiasis diseases that may be successfully treated are paragonimiasis, fasciolopsiasis, clonorchiasis and opisthorchiasis, fascioliasis, angiostrongyliasis, schistosomiasis. Preferred parasites to be treated by the inventive compounds in animals are selected from Leishmania spp., Schistosoma spp., Trypanosoma spp.
The following examples show the potential of the inventive compounds to treat parasitic infections that are caused by Caenorhabditis elegans. This organism is also used as a model organism for Wuchereria bancrofti (causes lymphatic filariasis), Brugia spp. (cause filariasis, specifically elephantiasis), Loa loa (causes a form of filariasis), Onchocerca spp. (cause onchocercosis). As such the invention may also be useful in the therapy of neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases. However, the invention is not restricted to nematode diseases but also includes non-nematode parasitic diseases.
Due to the specific cytostatic and cytotoxic potential, the inventive compounds are suitable for tumour therapy, in particular for the treatment of malignant tumours. Preferably, the malignant tumour disease is selected from the group consisting of breast cancer, leukemia, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colon cancer, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma or head and neck cancer.
The substances falling under the general formula (I), (IV), (II), or (III) are preferred embodiments of the present invention. All substances have in common that they can build thiyl radicals or sulfur radicals in the cell membrane, which is the underlying unifying inventive concept of the compounds of the present invention. Preferably, the compounds contain sulfur and are lipophilic. They can be distinguished in the way how the thiyl radical is stabilised, which can be aliphatic (class I) or aromatic (class IV). They can also be distinguished by the sulfur end groups such as a TTC group (class II) or a DTE group (class III).
The invention is further explained in the following examples.
Native lipid membranes from rat brain were examined on the extent of lipid peroxidation (as malondialdehyde) after treatment with the indicated concentrations of n-dodecylthiol (12-SH).
Graph (a) shows the absence of any effect in the absence of any radical initiation (“dead membranes”); in graph (b), a low level of free radical initiator (here: iron/ascorbate) was added, as generally found in living cells depending on the cell type. The added amount of initiator alone did not yet have any measurable oxidizing effect (control in (b)); however, its hidden effect was massively amplified by the concomitant addition of a Chain-Transfer Agent.
The data demonstrate the biochemical activity of the inventive compounds in biological membranes. As such they seem to act as initiator-dependent prooxidants.
The data confirm the basic toxicity of the compounds pursuant to the invention in living (tumour) cells. It is also shown that despite the postulated pro-oxidative mechanism of action, this toxicity is not dependent on the oxygen partial pressure within the (patho)physiological range of 1% to 20% oxygen, which is relevant for its potential use in hypoxic tumours.
HT22 cells were treated for 3 days with the indicated concentrations of n-octylthiol (a) or n-dodecylthiol (b, c). The dotted line indicates the survival of control cultures treated with vehicle.
HT22 cells were treated for 1 day (a) or 3 days (b) with the indicated concentrations of n-dodecylthiol under normal oxygen conditions (20% O2) or under highly hypoxic conditions (1% O2). Such hypoxic conditions may prevail in solid tumours and can complicate treatment. However, the effectiveness of the chain-transfer agents was not compromised by hypoxic conditions.
The data recapitulate the data from HT22 cells in normal fibroblasts. Representatives of lipophilic thiols falling under the general structure (I) and lipophilic trithiocarbonates falling under the general structure (II) have been used for experimentation. Furthermore, various analyses of the basic mechanism of action of the Chain-Transfer Agents in living cells were conducted. This includes analyses in regard of cellular lipid, protein and DNA damage as well as the physiological response of the cell to this damage. The observed types of damage (e.g. an induction of DNA double-strand breaks) indicate a particular efficacy of Chain-Transfer Agents in tumour cells.
Primary human fibroblasts under cell division-stimulating culture conditions (medium with 10% fetal calf serum) were treated for 3 days with the indicated concentrations of n-decylthiol (a), n-dodecylthiol (b), or n-tetradecylthiol (c).
Primary human fibroblasts under cell division-stimulating culture conditions (medium with 10% fetal calf serum) were incubated for 3 days with the indicated concentrations of S-dodecyl-S′-cyanomethyl trithiocarbonate (D-CM-TTC) (a), S-dodecyl-S′-[dimethyl-cyanomethyl] trithiocarbonate (D-DMCM-TTC) (b), or S-dodecyl-S′-[methyl-hydroxypropyl cyanomethyl] trithiocarbonate (D-MHCM-TTC) (c). The dotted line indicates the survival of control cultures treated with vehicle.
Primary human fibroblasts under cell division-stimulating culture conditions (medium with 10% fetal calf serum) were treated with 100 μM (a) and 500 μM (b) n-dodecylthiol (12-SH), respectively, over the indicated time period. Graph (a) shows the concentration of released lipid peroxidation marker 8-isoprostane, graph (b) shows the amount of polyubiquitinated proteins in the cell, which are a marker of protein damage. Both pro-oxidative markers increased significantly within a few hours.
Primary human fibroblasts under cell division-stimulating culture conditions (medium with 10% fetal calf serum) were treated with 500 μM n-dodecylthiol (12-SH) for the indicated time. Panel (a) shows the protein expression of Ser-140 phosphorylated histone 2AX, a marker for DNA double strand breaks and general laboratory proxy for DNA damage. Graph (b) shows the expression of caspase 3 and its active fragment “cleaved” caspase 3. Cleaved caspase 3 is the most important executing caspase in the apoptosis cascade.
Nematodes (C. elegans) were treated with 0.5 mM n-dodecylthiol in the minimum diet supplemented with varying amounts of coliform bacteria as adjunct feed. At the times indicated, the number of worms killed (a) was counted. Panel (b) shows the concentration of the lipid peroxidation marker 8-isoprostane in homogenates of worms treated with 0.1 mM or 0.5 mM n-dodecylthiol (12-SH). This biochemical marker of toxicity increased to a constant maximum after only 6 h, which then led to death after a few days (a).
The data confirm the general cytotoxic activity of the substances according to the invention in vivo, after oral administration. Also, their pro-oxidative mechanism of action in vivo is evidenced biochemically.
The data demonstrate the effectiveness of chain-transfer agents in vivo in a second, higher organism, in fruit flies (Drosophila). They also show directly the predicted mitochondrial specificity of the cytotoxic effect of these agents.
Fruit flies were treated with 1 mM n-dodecylthiol in the feed. The two electron micrographs show intramitochondrial, spiral multilamellar lesions. In tumour cells, such lesions would be expected to lead to apoptosis. The myofibrillar structure, on the other hand, is intact. The black line corresponds to approximately 1 μm.
Fruit flies were treated with 1 mM n-dodecylthiol in the feed. The electron micrographs show spiral multilamellar lesions of mitochondrial origin in the nervous system (a, b) and in photoreceptor cells (c). Other damage phenotypes are the vacuolization and electron-dense aggregation in (b) as well as the bright lipofuscin accumulation in (c). The black line corresponds to approximately 1 μm.
Material and Methods:
Prooxidative Activity of Chemical Chain-Transfer Agents (CTAs) in Biological Membranes (
Native biological membranes from adult rat brain were prepared by differential centrifugation as described (Moosmann and Behl, Proc Nati Acad Sci USA 96:8867-8872, 1999). Samples containing 0.5 mg/ml total protein (as per bicinchoninic acid assay from Pierce, Rockford, Ill., USA) were solubilized by brief sonication in PBS (phosphate-buffered saline) and administered with the indicated concentrations of n-dodecyl thiol (12-SH; from Sigma-Aldrich, St. Louis, Mo., USA) dissolved in ethanol (final concentration: 0.1%). Subsequently, 10 μM Fe2+/200 μM ascorbate were added as radical-initiating mix; controls received vehicle (water). After the indicated time, the reaction was stopped by adding 2.5 volumes of 5% trichloroacetic acid in 1 M acetic acid, followed by centrifugation (10,000 g for 10 min). Subsequently, thiobarbituric acid-reactive substances (TBARS) as marker of lipid peroxidation were quantified fluorimetrically as detailed before (Hajieva et al., J Neurochem 110:118-132, 2009).
Cytotoxic Activity of Chain-Transfer Agents (CTAs) in Cultivated HT22 Cells (
(
For cell survival experiments, HT22 cells were seeded into 96-well plates at a density of ˜5000 cells per well in 0.1 mL medium. After 24 h cultivation, the cells were administered with the indicated concentrations of the tested compounds (n-octyl thiol (8-SH) or n-dodecyl thiol (12-SH)) dissolved in ethanol (final concentration: 1%). After 1 day or 3 days of incubation as indicated, cell survival was analyzed by colorimetric MTT reduction tests (MTT is 3-(4,5-dimethylthiazol-2-yl-)-2,5 diphenyltetrazolium bromide) which were performed exactly as described (Hajieva et al., J Neurochem 110:118-132, 2009).
(
Activities of Chain-Transfer Agents (CTAs) in Cultivated Diploid Human Fibroblasts (
(
For cell survival experiments, cells cultivated in 96-well plates for 24 h were administered with the indicated concentrations of the tested compounds (n-decyl thiol (10-SH), n-dodecyl thiol (12-SH), n-tetradecyl thiol (14-SH)) dissolved in ethanol (final concentration: 1%). After 3 days of incubation, cell survival was analyzed by colorimetric MTT reduction tests (MTT is 3-(4,5-dimethylthiazol-2-yl-)-2,5 diphenyltetrazolium bromide) which were performed exactly as described (Hajieva et al., J Neurochem 110:118-132, 2009).
(
(
Protein polyubiquitination. Cells cultivated in 100 mm dishes for 24 h were administered with 500 μM n-dodecyl thiol (12-SH) for the indicated time before harvesting of the cells in lysis buffer (50 mM Tris-HCl, pH 7.4, 10% sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM NaF, 1× protease inhibitor cocktail from Sigma-Aldrich, St. Louis, Mo., USA) followed by brief sonication and denaturation at 95° C. for 2 min. For the specific analysis of protein ubiquitination, Western immunoblotting was performed as described (Hajieva et al., J Neurochem 110:118-132, 2009). In brief, equal amounts of total protein (as per bicinchoninic acid assay from Pierce, Rockford, Ill., USA) were separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membranes by electroblotting. Blocking of was carried out by incubation with Tris-buffered saline/Tween-20 (TBST) containing 2% fat-free dry milk for 60 min at 20° C., followed by incubation with the specific primary antibodies at 4° C. overnight. The primary antibodies were: Rabbit anti-polyubiquitin antibody (dilution 1:2000; from Agilent Dako, Santa Clara, Calif., USA); mouse anti-α-tubulin antibody (dilution 1:1000; from Sigma-Aldrich, St. Louis, Mo., USA); both diluted in TBST. The next day, the membranes were treated with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies (1:5000; from Jackson Immunoresearch, West Grove, Pa., USA) for 90 min at 20° C. Subsequently, the membranes were washed 3×15 min with TBST. Immunoreactive bands were developed using commercial peroxidase substrate kits (Enhanced Chemiluminescence Plus from Amersham Pharmacia Biotech, Piscataway, N.J., USA), and scanned with a digital chemiluminescent imaging system. Densitometric quantification was performed using automated image analysis software.
(
Prooxidative Toxicity of Chemical Chain-Transfer Agents (CTAs) in Nematodes In Vivo (
Caenorhabditis elegans N2 Bristol strain animals were expanded and cultivated at 20° C. on nematode growth medium (NGM) plates in the presence of Escherichia coli strain HB101 as food source following standard protocols (Mocko et al., Neurobiol Dis 40:120-129, 2010). For the toxicity experiments, synchronized adult worms aged 2 days were maintained in liquid culture medium (S-Basal medium supplemented with 5 mg/L cholesterol, 100 mg/L streptomycin, 100 mg/L fluorodeoxyuridine (FUDR)) to which varying amounts of Escherichia coli were added (measured as optical density (OD) at 600 nm wavelength) as described (Mair et al., PLoS One 4:e4535, 2009). Worms distributed in 48-well plates were administered with 500 μM n-dodecyl thiol (12-SH) dissolved in ethanol (final concentration: 1%) and analyzed, after the indicated time, for survival by visual inspection and mechanical stimulation (nose-touch assay) as detailed (Mocko et al., Neurobiol Dis 40:120-129, 2010).
8-Isoprostanes. Synchronized, 4-day-old adult worms distributed in cell culture flasks were administered with the indicated concentration of n-dodecyl thiol (12-SH) dissolved in ethanol (final concentration: 1%) and cultivated for 6 h or 48 has indicated. The worms were collected by centrifugation at 1200 g, washed twice with S-Basal medium, and once with DMEM medium containing 100 μM butylated hydroxytoluene (BHT). The worms were then homogenized in DMEM/BHT by sonication (3×20 s at 30 kHz on ice). Equal amounts of protein of the resulting homogenate (as per bicinchoninic acid assay from Pierce, Rockford, Ill., USA) were probed for the presence of 8-isoprostane by a commercial enzyme immunoassay (Cayman Chemicals, Ann Arbor, Mich., USA) following the manufacturer's instructions.
Ultrastructural Effects of Chain-Transfer Agents (CTAs) in Insects In Vivo (
Male Drosophila melanogaster (strain Oregon-R) were maintained at 25° C. in plastic vials covered with air-permeable lids and received standard food (50 g/L refined household sugar, 50 g/L baker's yeast, and 20 g/L agar powder). The medium was boiled under stirring, adding the following supplements at approximately 70° C.: 3 g/L methylparabene (dissolved in ethanol, final concentration: 0.15%), 3 mL/L propionic acid and the appropriate amount of n-dodecyl thiol (12-SH) dissolved in ethanol (final concentration: 0.1%). Synchronized male flies were transferred into new vials and scored for survival every other day. Treatment started on day 2 of adulthood.
Electron microscopy. Flies harvested after 50 days of treatment were cryofixed by plunge freezing, cut into head, thorax and abdomen before chemical fixation for 90 min with 3% glutaraldehyde and 3% formaldehyde in PBS. The tissues were washed, fixed with 2% OsO4, washed again, dehydrated with rising concentrations of ethanol, transitionally stabilized with propylene oxide and embedded into epoxy resin essentially as described (Bozzola and Russel, Electron Microscopy: Jones and Bartlett Publishers, Inc., Sudbury, M A, 1999). The polymerized blocks were mounted, trimmed, and sectioned in an ultramicrotome before transfer onto electron microscopy grids. Images were acquired under standard conditions in a Tecnai Transmission Electron Microscope (FEI Company, Hillsboro, Oreg., USA).
Tumour Therapy
Data for the anti-cancer effect are shown for HT22 cells (see
The cytotoxic activity of Chain-Transfer Agents (CTAs) in cultivated MCF7 breast cancer cells was analysed in the experiments conducted in
The cytotoxic activity of Chain-Transfer Agents (CTAs) in cultivated SY5Y neuroblastoma cells was analysed in the experiments conducted in
Szatrowski T P, Nathan C F (1991). Production of large amounts of hydrogen peroxide by human tumor cells. Cancer Res 51, 794-798.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19212199.4 | Nov 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/083744 | 11/27/2020 | WO |
