ANTIMICROBIAL AND RADIOPROTECTIVE COMPOUNDS
专利汇可以提供ANTIMICROBIAL AND RADIOPROTECTIVE COMPOUNDS专利检索，专利查询，专利分析的服务。并且The present invention relates to a method of treatment and/or prophylaxis of a microbial infection, comprising the step of administering an effective amount of a compound of formula (I), in which X and Y are either the same or different and selected from a heteroatom; is a double or single bond depending on the heteroatoms X and Y; R1 to R5 are either the same or different and selected from hydrogen or a non-deleterious substituent; and R6 and R7 are either the same or different and selected from hydrogen and a non-deleterious substituent or one of R6 and R7 are absent when there is a double bond present, pharmaceutically acceptable salts or derivatives, pro-drugs, tautomers and/or isomers thereof. The present invention also relates to a method for protecting a subject from radiation damage, a method of cancer radiotherapy and use as an antimicrobial or radioprotective agent of the compound of formula (I) defined above. Some of the compounds of formula (I) are novel and are also described in the present invention, together with pharmaceutical or veterinary compositions containing them.，下面是ANTIMICROBIAL AND RADIOPROTECTIVE COMPOUNDS专利的具体信息内容。
The present application is a continuation of U.S. patent application Ser. No. 14/026,641, filed Sep. 13, 2013 (now U.S. Pat. No. 9,045,452; Atty. Dkt. No. 36672.29); which was a continuation of U.S. patent application Ser. No. 13/425,054, filed Mar. 20, 2012 (now U.S. Pat. No. 8,569,363; Atty. Dkt. No. 36672.27); which was a continuation of U.S. patent application Ser. No. 12/776,896, filed May 10, 2010 (now U.S. Pat. No. 8,158,664; Atty. Dkt. No. 36672.25); which was a continuation of U.S. patent application Ser. No. 11/923,404, filed Oct. 24, 2007 (now U.S. Pat. No. 7,825,145; Atty. Dkt. No. 36672.19); which was a continuation of U.S. national-phase § 371 Application No. 10/481,667, filed Aug. 26, 2004 (abandoned; Atty. Dkt. No. 36672.16); which claimed priority to PCT International Patent Application No. PCT/AU2002/000783, filed Jun. 14, 2002 (expired), and Russian Patent Application No. 2001/117033, filed Jun. 18, 2001 (now Russian Patent No. 2,259,825), the contents of each of which is specifically incorporated herein in its entirety by express reference thereto.
Not Applicable.
Not Applicable.
This invention relates to compounds that have antimicrobial and radioprotective activity. In particular, the invention relates to substituted nitrostyrene compounds, which have activity against a wide spectrum of organisms including bacteria, fungi, and protozoa. The compounds of the invention also have the ability to provide protection from radiation damage.
All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia, or in any other country.
Bacterial, fungal, and protozoal pathogens are responsible for a very wide variety of infections, ranging from minor respiratory ailments to fulminant systemic infections and chronic illnesses. Food poisoning caused by organisms such as Salmonella or Campylobacter is common, and is often associated with endemic infection in livestock or poultry raised using intensive animal husbandry techniques.
Despite the wide availability of antibiotics, control of infection is difficult, and many organisms have the ability to develop resistance. Many microorganisms cause problems which have hitherto proved to be quite intractable, such as multi-drug-resistant Staphylococcus aureus infection in hospitals, drug-resistant Enterococcus infections, bacterial, fungal, and protozoal infection in HIV patients, tuberculosis, and malaria and other endemic infections in underdeveloped countries.
Currently, there are only very few agents which have a wide spectrum of activity against pathogens of bacterial, fungal and protozoal origin. Antibiotics are the most widely used agents in the fight against pathogenic microorganisms. However, most antibiotics have narrow specificity. Even broad-spectrum antibacterial antibiotics are not very effective against fungi and protozoa. Most antibiotics belong to a restricted range of classes of compounds; although improved semi-synthetic derivatives of these have developed, only a few new antibiotic compound classes have become available in the last twenty years.
The choice of agents for protection of living organisms against radioactive radiation is also quite limited. Among the radiation protectors, the most effective are sulfur-containing compounds (Kuna, 1989). For example, cystamine is approved for use as a radiation-protective agent (Vladimirov et al., 1989). The index of protection of this preparation does not exceed 1.45, and has the disadvantage that it causes diarrhea. Another known radiation-protective preparation is mercamine (β-mercaptoethylamine) (Mashkovskiy, 1986).
This has a low therapeutic index, short period of action (0.5-1 h), and short duration of radiation protecting activity (15-30 min).
It is known that β-nitrostyrene and some of its derivatives demonstrate biological, and partly fungicidal activity (Foyer, 1973). Russian Patent No. 2145215 showed that certain derivatives of arylnitroalkenes have antimicrobial, antifungal, antiprotozoal activity, and are able to provide protection from radiation damage. These compounds have the following formula:
in which R1′ is H or CH3; and R2′ and R3′, are the same or different and are selected from H, OCH3, OH, NO2 and (CH3)2N.
The activities of these compounds are satisfactory, but there is a need for low cost, low-toxicity agents with a wide spectrum of antimicrobial activities.
The inventors have now found that certain substituted nitrostyrene compounds have excellent activity against very wide spectrum of organisms, including bacteria, fungi, and protozoa, and also have the ability to provide protection from radiation damage.
The invention provides a method of treatment and/or prophylaxis of a microbial infection, comprising the step of administering an effective amount of a compound of Formula I:
in which
X and Y are either the same or different and selected from a heteroatom;
is a double or single bond depending on the heteroatoms X and Y;
R1 to R5 are either the same or different and selected from hydrogen or a non-deleterious substituent; and
R6 and R7 are either the same or different, and selected from hydrogen and a non-deleterious substituent, or one of R6 and R7 are absent when there is a double bond present, pharmaceutically-acceptable salts or derivatives, pro-drugs, tautomers, and/or isomers thereof.
The invention also provides use of the compound of Formula I in the manufacture of a medicament for the treatment and/or prophylaxis of a microbial infection.
The invention further provides use of the compound of Formula I for the treatment and/or prophylaxis of a microbial infection.
The invention still further provides a method for protecting a subject from radiation damage which comprises administering an effective amount of the compound of Formula I to a subject in need thereof.
In another aspect, the invention provides a method of cancer radiotherapy which comprises administering to a subject in need of such therapy an effective amount of the compound of Formula I and subjecting the locus of a tumor in the subject to a radiation source.
In a further aspect, the invention provides use of the compound of Formula I as an antimicrobial or radioprotective agent.
Preferably, X and Y are either the same or different, and selected from O and N; more preferably, both X and Y are oxygen.
Preferably, R1 and R2 are either the same or different and selected from hydrogen, hydroxy, halogen, or optionally substituted C1-6 alkyl.
R3 to R5 are preferably either the same or different and selected from hydrogen, hydroxy, halogen, nitro, C1-6 alkoxy, or optionally substituted C1-6 alkyl.
Preferably, halogen is chlorine or bromine.
The E isomer of the compounds of Formula I is preferred.
Particularly preferred are compounds of Formula I in which X, Y, R6 and R7 are as defined above; R1 and R2 are either the same or different and selected from hydrogen, hydroxy, Cl, Br and C1-4 alkyl; and R3 to R5 are either the same or different and selected from hydrogen, hydroxy, Cl, Br, nitro, C1-4 alkoxy or C1-4 alkyl.
Specific examples of the compounds of the present invention are as follows:
(1) X and Y are O, R1 is methyl and R2 and R3 are hydrogen (3, 4-methylenedioxy-β-methyl-3-nitrostyrene).
(2) X and Y are O and R1 to R3 are hydrogen (3,4-methylenedioxy-β-nitrostyrene).
(3) X is N, Y is NH, R1 is methyl and R2 and R3 are hydrogen (benzimidazole-5-β-nitropropylene)
(4) X is N, Y is NH, R1 is hydrogen, R2 is methyl and R3 is absent (2-methyl benzimidazole-5-β-nitroethylene)
(5) X is O, Y is N, R1 and R2 are hydrogen and R3 is absent (benzoxazole-5-β-nitroethylene)
(6) X is N, Y is O, R1 and R2 are methyl and R3 is absent (2-methyl benzoxazole-5-β-nitropropylene)
Some of the compounds of the Formula I are novel, per se.
Accordingly, the invention also provides a compound of Formula Ia:
in which X, Y, and R1 to R7 are as defined in Formula I above, with the provisos that when both X and Y are O and R2 to R7 are hydrogen, then R1 is not hydrogen, C1-4 alkyl or CO2Et or when both X and Y are O, then R1 to R7 are not hydrogen.
The invention also provides a process for the preparation of the compound of Formula Ia defined above which comprises condensing a compound of Formula II:
in which X, Y, , R3 to R7 are as defined in Formula Ia above with a compound of Formula III:
R1R2CHNO2 III
in which R1 and R2 are as defined in Formula Ia above.
The invention further provides a process for the preparation of the compound of Formula Ia defined above which comprises reacting a compound of Formula IV:
in which X, Y, , R1 to R7 are as defined in Formula Ia above with C(NO3)4.
The processes are preferably performed in the presence of a catalyst, such as, an amine or an alkali metal hydroxide, for example, NaOH or KOH.
In a further aspect, the invention provides a pharmaceutical or veterinary composition comprising the compound of Formula Ia defined above together with a pharmaceutically- or veterinarily-acceptable carrier.
Preferably, the pharmaceutical or veterinary composition is a topical, oral or parenteral composition.
The pharmaceutically- or veterinarily-acceptable carrier is preferably an organic solvent such as acetone, benzene, acetonitrile, DMSO or an alcohol, for example, methanol or ethanol. While the compounds of the present invention show a poor solubility in water, when water is combined with an organic solvent a stable mixture is formed.
For the purposes of this specification it will be clearly understood that the word “comprising” means “including but not limited to,” and that the word “comprises” has a corresponding meaning.
The term “heteroatom” denotes O, N, or S.
The term “non-deleterious substituent” is used herein in its broadest sense and refers to a substituent which does not have a deleterious effect on the antimicrobial or radioprotective properties of the compound. Examples include alkyl, alkenyl, alkynyl, aryl, halo, haloalkyl, haloalkenyl, haloalkynyl, haloaryl, hydroxy, alkoxy, alkenyloxy, aryloxy, benzyloxy, haloalkoxy, haloalkenyloxy, haloaryloxy, nitro, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroaryl, nitroheterocyclyl, amino, alkylamino, dialkylamino, alkenylamino, alkynylamino, arylamino, diarylamino, benzylamino, dibenzylamino, acyl, alkenylacyl, alkynylacyl, arylacyl, acylamino, diacylamino, acyloxy, alkylsulphonyloxy, arylsulphenyloxy, heterocyclyl, heterocycloxy, heterocyclamino, haloheterocyclyl, alkylsulphenyl, arylsulphenyl, carboalkoxy, carboaryloxy mercapto, alkylthio, arylthio, acylthio and phosphorus-containing groups.
Particularly suitable non-deleterious substituents are alkyl, alkenyl, alkynyl, halo, haloalkyl, haloalkenyl, haloalkynyl, hydroxy, alkoxy, alkenyloxy, haloalkoxy, haloalkenyloxy, nitro, nitroalkyl, nitroalkenyl and nitroalkynyl.
In a preferred embodiment, the non-deleterious substituents are C1-6 alkyl, halo, hydroxy, alkoxy and nitro.
The term “optionally substituted” means that a group may or may not be further substituted with, for example, the groups specified above under the definition of non-deleterious substituent.
The term “halogen” refers to fluorine, chlorine, bromine and iodine, preferably chlorine and bromine.
The term “alkoxy” is used herein in its broadest sense and refers to straight chain, branched chain or cyclic oxy-containing radicals each having alkyl portions, preferably C1-6 alkyl, more preferably C1-4 alkyl. Examples of such alkoxy groups are methoxy, ethoxy, propoxy, butoxy and t-butoxy.
The terms “C1-4 alkyl” or “C1-6 alkyl” used either alone or in compound words such as “optionally substituted C1-4 or C1-6 alkyl” refer to straight-chain, branched-chain, or cyclic hydrocarbon groups having from 1 to 6 carbon atoms. Illustrative of such alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tent-butyl, pentyl, neopentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
The salts of the compounds of Formula I or Formula Ia are preferably pharmaceutically-acceptable, but it will be appreciated that non-pharmaceutically-acceptable salts also fall within the scope of the present invention, since these are useful as intermediates in the preparation of pharmaceutically-acceptable salts. Examples of pharmaceutically-acceptable salts include salts of pharmaceutically-acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium; acid addition salts of pharmaceutically-acceptable inorganic acids such as hydrochloric, orthophosphoric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids; or salts of pharmaceutically-acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, trihalomethanesulphonic, toluenesulphonic, benzenesulphonic, salicylic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic, and valeric acids.
In addition, some of the compounds of the present invention may form solvates with water or common organic solvents. Such solvates are encompassed within the scope of the invention.
By “pharmaceutically-acceptable derivative” is meant any pharmaceutically acceptable salt, hydrate or any other compound which, upon administration to the subject, is capable of providing (directly or indirectly) a compound of Formula I or Ia or an antimicrobial or radioprotective active metabolite or residue thereof.
The term “pro-drug” is used herein in its broadest sense to include those compounds that are converted in vivo to compounds of Formula I or Formula Ia.
The term “tautomer” is used herein in its broadest sense to include compounds of Formula I or Ia that are capable of existing in a state of equilibrium between two isomeric forms. Such compounds may differ in the bond connecting two atoms or groups and the position of these atoms or groups in the compound.
The term “isomer” is used herein in its broadest sense and includes structural, geometric and stereo isomers. As the compound of Formula I or Formula Ia may have one or more chiral centers, it is capable of existing in enantiomeric forms.
The term “microbial infection” is used herein in its broadest sense and refers to any infection caused by a microorganism and includes bacterial infections, fungal infections, yeast infections, and protozoal infections.
The term “microorganism” includes any microscopic organism or taxonomically related macroscopic organism within the categories algae, bacteria, fungi, yeast and protozoa or the like.
Bacterial infections include, but are not limited to, infections caused by Bacillus cereus, Bacillus anthracis, Clostridium botulinum, Clostridium difficile, Clostridium tetani, Clostridium perfringens, Corynebacteria diphtheriae, Enterococcus (Streptococcus D), Listeria monocytogenes, pneumoccocci (e.g., Streptococcus pneumoniae), Staphylococci and Streptococci; Gram-negative genera (including, e.g., Bacteroides, Bordetella pertussis, Brucella, Campylobacter, enterohaemorrhagic Escherichia coli (EHEC/E. coli 0157: H7) enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC), Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella spp., Moraxella catarrhalis, Neisseria gonnorrhoeae, Neisseria meningitidis, Proteus spp., Pseudomonas aeruginosa, Salmonella spp., Shigella spp., Vibrio cholerae and Yersinia; acid-fast bacteria (including e.g., Mycobacterium tuberculosis, Mycobacterium avium-intracellulare, Myobacterium johnei, and Mycobacterium leprae), atypical bacteria, Chlamydia, Mycoplasma, Rickettsia, Spirochetes, Treponema pallidum, Borrelia recurrentis, Borrelia burgdorfii, Leptospira icterohemorrhagiae, as well as other miscellaneous bacteria (including, e.g., Actinomyces and Nocardia).
Fungal infections include, but are not limited to, infections caused by Alternaria alternate, Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus versicolor, Blastomyces dermatiditis, Candida albicans, Candida dubliensis, Candida krusei, Candida parapsilosis, Candida tropicalis, Candida glabrata, Coccidioides immitis, Cryptococcus neoformans, Epidermophyton floccosum, Histoplasma capsulatum, Malassezia furfur, Microsporum canis, Mucor spp., Paracoccidioides brasiliensis, Penicillium marneffei, Pityrosporum ovale, Pneumocystis carinii, Rhodotorula spp., Sporothrix schenkii, Trichophyton rubrum, Trichophyton interdigitale, and Trichosporon beigelii.
Yeast infections include, but are not limited to, infections caused by Brettanomyces claussenii, Brettanomyces custerii, Brettanomyces anomalus, Brettanomyces naardenensis, Candida humilis, Candida intermedia, Candida sake, Candida solani, Candida tropicalis, Candida versatilis, Candida bechii, Candida famata, Candida lipolytica, Candida stellata, Candida vini, Debaromyces hansenii, Dekkera intermedia, Dekkera bruxellensis, Geotrichium candidum, Hansenula anomala, Hansenula fabianii, Hanseniaspora uvarum, Hanseniaspora guilliermondii Hanseniaspora vinae, Kloeckera apiculata, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces fragilis, Metschikowia pulcherrima, Pichia guilliermodii, Pichia orientalis, Pichia fermentans, Pichia membranefaciens, Rhodotorula, Saccharomyces bayanus, Saccharomyces boulardii, Saccharomyces cerevisiae, Saccharomyces dairenensis Saccharomyces exigus, Saccharomycodes ludwigii, Saccharomyces oleaginosus, Saccharomyces uinsporus, Saccharomyces uvarum, Schizosaccharomyces pombe, Torulaspora delbrueckii, Torulopsis stellata, Zygosaccharomyces bailii, and Zygosaccharomyces rouxii.
Protozoal infections include, but are not limited to, infections caused by Leishmania, Toxoplasma, Plasmodia, Theileria, Anaplasma, Giardia, Trichomonas, Trypanosoma, Coccidia, and Babesia. Specific examples include Trypanosoma cruzi, Eimeria tenella, Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale.
Preferably, the microbial infection is an infection caused by either a Gram-positive or a Gram-negative bacterium, for example, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Salmonella typhimurium, or pseudotuberculosis, Acinetobacter, Pseudomonas aeruginosa, Clostridium perfringens, Clostridium difficile, Campylobacter jejuni, or Bacteroides fragilis; a fungal or yeast infection, for example, Trichophyton interdigitale, Aspergillus fumigatus, or Candida albicans; or a protozoal infection, for example Plasmodium falciparum or Trichomonas vaginalis.
Examples of microbial infections include bacterial or fungal wound infections, mucosal infections, enteric infections, septic conditions, pneumonia, trachoma, ornithosis, trichomoniasis, fungal infections, and salmonellosis, especially in veterinary practice. The compounds of the invention may also be used for the treatment of resistant microbial species or in various fields where antiseptic treatment or disinfection of materials is required, for example, surface disinfection.
The term “subject” as used herein refers to any animal having a disease or condition that requires treatment with a pharmaceutically-active agent. The subject may be a mammal, preferably a human, or may be a domestic or companion animal. While it is particularly contemplated that the compounds of the invention are suitable for use in medical treatment of humans, it is also applicable to veterinary treatment, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, ponies, donkeys, mules, llama, alpaca, pigs, cattle and sheep, or zoo animals such as primates, felids, canids, bovids, and ungulates.
Suitable mammals include members of the orders Primates, Rodentia, Lagomorpha, Cetacea, Carnivora, Perissodactyla and Artiodactyla. Members of the orders Perissodactyla and Artiodactyla are particularly preferred because of their similar biology and economic importance.
For example, Artiodactyla comprises approximately 150 living species distributed through nine families: pigs (Suidae), peccaries (Tayassuidae), hippopotamuses (Hippopotamidae), camels (Camelidae), chevrotains (Tragulidae), giraffes and okapi (Giraffidae), deer (Cervidae), pronghorn (Antilocapridae), and cattle, sheep, goats and antelope (Bovidae). Many of these animals are used as feed animals in various countries. More importantly, many of the economically important animals such as goats, sheep, cattle and pigs have very similar biology and share high degrees of genomic homology.
The order Perissodactyla comprises horses and donkeys, which are both economically important and closely related. Indeed, it is well known that horses and donkeys interbreed.
As used herein, the term “effective amount” is meant an amount of a compound of the present invention effective to yield a desired antimicrobial or radioprotective activity.
The specific “effective amount” will, obviously, vary with such factors as the particular condition being treated, the physical condition of the subject, the type of subject being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compound or its derivatives.
The term “radiation damage” is used herein in its broadest sense and refers to damage resulting from exposure to a radiation source, such as, ionizing radiation. The term “ionizing radiation” as used herein refers to photons having enough energy to ionize a bond, such as, α, β, and γ rays from radioactive nuclei and x-rays.
The term “cancer radiotherapy” is used herein in its broadest sense and includes radiotherapy involving tumors, which may be either benign or malignant.
The primary application of the radioprotector of the present invention is in cancer radiotherapy. Many of the normal tissues that are a problem in radiotherapy such as the skin, oral mucosa, esophageal mucosa, rectal mucosa, vaginal mucosa, and bladder epithelium can be protected by the radioprotectors of the present invention.
Outside the context of cancer radiotherapy, the radioprotectors of the present invention could be used prophylactly in high-risk radiation situations.
The compounds of the present invention may additionally be combined with other medicaments to provide an operative combination. It is intended to include any chemically compatible combination of pharmaceutically active agents, as long as the combination does not eliminate the activity of the compound of Formula I or Ia. It will be appreciated that the compound of the invention and the other medicament may be administered separately, sequentially or simultaneously.
Other medicaments that may be used when treating microbial infections include other anti-infective agents such as antibiotics.
When the compounds are used as radioprotectors, the other medicaments may include chemotherapeutic agents, for example, radiomimetic agents that are cytotoxic agents that damage DNA in such a way that the lesions produced in DNA are similar to those resulting from ionizing radiation. Examples of radiomimetic agents that cause DNA strand breaks include bleomycin, doxorubicin, adriamycin, SFU, neocarcinostatin, alkylating agents, and other agents that produce DNA adducts. It is anticipated that the radioprotectors of the present invention will protect DNA from damage by some of these agents, in the same way as they protect against the effects of ionizing radiation. In clinical applications, it is unlikely that the radioprotector would be administered systemically together with the chemotherapeutic agent, since this could compromise the action of this agent on the tumor. However, there are circumstances where topical application to problem tissues could be advantageous. For example, oral mucositis is problem side effect for cytotoxic agents, such as, doxorubicin and administration of the present radioprotector as a mouthwash before administration of the chemotherapeutic agent could ameliorate this side effect without compromising the action of this agent on a tumor not located in the oral cavity. Similarly, the gastrointestinal tract could be protected by oral administration, the lungs by aerosol inhalation, or the bladder by intravesical delivery, for example, via a catheter of the radioprotector. Hence, a preferred method in accordance with the present invention utilizes the compound of Formula I or Formula Ia in conjunction with another medicament, such as, a radiomimetic agent.
The compounds of the invention may be conjugated to agents, for example, via the interactive group, which will specifically deliver them to a desired tumor site. Suitable agents may include antibodies or proteins, growth factors, for example, haemopoietic growth factor which will enable preferential radioprotection of haemopoietic stem cells to occur in the context of total body irradiation and bone marrow transplantation.
There is also an ex vivo application of the conjugates of the compounds of the invention in the context of bone marrow transplantation. Bone marrow transplantation generally involves obtaining and storing bone marrow samples from a subject in anticipation of a deterioration of their condition. A rather drastic form of chemotherapy (i.e., a high dose) is then administered. This chemotherapy is such that it would normally be lethal due to the destruction of normal stem cells, but the subject is rescued by the administration of their own hematopoietic stem cells. The problem with this procedure is that the initial sample of stem cells is likely to be contaminated with tumor cells and various procedures are use therefore to purge the bone marrow preparations of the tumor cells. Radioprotectors conjugated to a hematopoietic growth factor could be used in this context by being added to a suspension of bone marrow cells. The suspension could then be irradiated in the expectation that the normal bone marrow cells, but not the tumor cells, would be preferentially protected from the cell-killing effects of the radiation.
As used herein, a “pharmaceutical carrier” is a pharmaceutically-acceptable solvent, suspending agent, or vehicle for delivering the compound of Formula I or Formula Ia to the subject. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. Each carrier must be pharmaceutically “acceptable” in the sense of being compatible with other ingredients of the composition, and non-injurious to the subject.
The compound of Formula I or Formula Ia may be administered orally, topically, or parenterally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral as used herein includes subcutaneous injections, aerosol for administration to lungs or nasal cavity, intravenous, intramuscular, intrathecal, intracranial, injection or infusion techniques.
The present invention also provides suitable topical, oral, and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention. The compounds of the present invention may be administered orally as tablets, aqueous or oily suspensions, lozenges, troches, powders, granules, emulsions, capsules, syrups, or elixirs. The composition for oral use may contain one or more agents selected from the group of sweetening agents, flavoring agents, coloring agents, and preserving agents in order to produce pharmaceutically elegant and palatable preparations. Suitable sweeteners include sucrose, lactose, glucose, aspartame, or saccharin. Suitable disintegrating agents include cornstarch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid, or agar. Suitable flavoring agents include peppermint oil, oil of wintergreen, cherry, orange, or raspberry flavoring. Suitable preservatives include sodium benzoate, vitamin E, α-tocopherol, ascorbic acid, methyl paraben, propyl paraben, or sodium bisulfite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride, or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate. The tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients, which are suitable for the manufacture of tablets.
These excipients may be, for example, (1) inert diluents, such as calcium carbonate, lactose, calcium phosphate, or sodium phosphate; (2) granulating and disintegrating agents, such as cornstarch or alginic acid; (3) binding agents, such as starch, gelatin, or acacia; and (4) lubricating agents, such as magnesium stearate, stearic acid or talc. These tablets may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. Coating may also be performed using techniques described in the U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 (each of which is specifically incorporated herein in its entirety by express reference thereto) to form osmotic therapeutic tablets for control release.
The compound of Formula I or Formula Ia as well as the pharmaceutically-active agent useful in the method of the invention can be administered, for in vivo application, parenterally by injection or by gradual perfusion over time independently or together. Administration may be intravenously, intraarterially, intraperitoneally, intramuscularly, subcutaneously, intracavital, transdermally, or infusion by, for example, an osmotic pump. For in vitro studies, the agents may be added or dissolved in an appropriate biologically-acceptable solvent or buffer, and then added to a cell or a tissue.
Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, growth factors and inert gases and the like.
Generally, the terms “treating,” “treatment,” and the like are used herein to mean affecting a subject, tissue, or cell to obtain a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or sign or symptom thereof, and/or may be therapeutic in terms of a partial or complete cure of a disease. “Treating,” as used herein, covers any treatment of, or prevention of disease in a vertebrate, a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject that may be predisposed to the disease, but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving or ameliorating the effects of the disease, i.e., cause regression of the effects of the disease.
The invention includes various pharmaceutical compositions useful for ameliorating disease. The pharmaceutical compositions according to one embodiment of the invention are prepared by bringing a compound of Formula I or Formula Ia, analogs, derivatives or salts thereof, or combinations of compound of Formula I or Formula Ia and one or more pharmaceutically-active agents into a form suitable for administration to a subject using carriers, excipients, additives, or auxiliaries. Frequently used carriers or auxiliaries include magnesium carbonate, titanium dioxide, lactose, mannitol, and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol and polyhydric alcohols. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial, anti-oxidants, chelating agents, and inert gases. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in Remington's Pharmaceutical Sciences, 20th Ed. Williams & Williams (2000), the British National Formulary, 43rd edition (British Medical Association and Royal Pharmaceutical Society of Great Britain, 2000), the contents of each of which is specifically incorporated herein in its entirety by express reference thereto. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art. See Goodman and Gilman's The Pharmacological Basis for Therapeutics (7th Ed., 1985).
The pharmaceutical compositions are preferably prepared and administered in dose units. Solid dose units may be tablets, capsules, and suppositories. For treatment of a subject, depending on activity of the compound, manner of administration, nature and severity of the disorder, age and body weight of the subject, different daily doses can be used. Under certain circumstances, however, higher or lower daily doses may be appropriate. The administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units, and also by multiple administration of subdivided doses at specific intervals.
The pharmaceutical compositions according to the invention may be administered locally or systemically in a therapeutically effective dose. Amounts effective for this use will, of course, depend on the severity of the disease and the weight and general state of the subject. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of the microbial infections. Various considerations are described, e.g., in Langer (1990). Formulations for oral use may be in the form of hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, or kaolin. They may also be in the form of soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin, or olive oil.
Aqueous suspensions normally contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspension. Such excipients may be (1) suspending agent such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; (2) dispersing or wetting agents which may be (a) naturally occurring phosphatide such as lecithin; (b) a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate; (c) a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example, heptadecaethylenoxycetanol; (d) a condensation product of ethylene oxide with a partial ester derived from a fatty acid and hexitol such as polyoxyethylene sorbitol monooleate, or (e) a condensation product of ethylene oxide with a partial ester derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate.
The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents, which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
The compound of Formula I or Formula Ia may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.
The compound of Formula I or Formula Ia may also be presented for use in the form of veterinary compositions, which may be prepared, for example, by methods that are conventional in the art. Examples of such veterinary compositions include those adapted for:
(a) oral administration, external application, for example drenches (e.g., aqueous or non-aqueous solutions or suspensions); tablets or boluses; powders, granules or pellets for admixture with feed stuffs; pastes for application to the tongue;
(b) parenteral administration for example by subcutaneous, intramuscular or intravenous injection, e.g., as a sterile solution or suspension; or (when appropriate) by intramammary injection, wherein a suspension or solution is introduced in the udder via the teat;
(c) topical applications, e.g., as a cream, ointment or spray applied to the skin; or
(d) intravaginally, e.g., as a pessary, cream, or foam.
Dosage levels of the compound of Formula I or Ia of the present invention may be of the order of up to about 1 g per kg body weight. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for oral administration to humans may contain up to about 1 g of an active compound with an appropriate and convenient amount of carrier material, which may vary from about 5 to about 95 percent of the total composition. Dosage unit forms will generally contain between from about 5 mg to about 500 mg of active ingredient.
Optionally the compounds of the invention are administered in a divided dose schedule, such that there are at least two administrations in total in the schedule. Administrations are given preferably at least every two hours for up to four hours or longer; for example the compound may be administered every hour or every half hour. In one preferred embodiment, the divided-dose regimen comprises a second administration of the compound of the invention after an interval from the first administration sufficiently long that the level of active compound in the blood has decreased to approximately from 5-30% of the maximum plasma level reached after the first administration, so as to maintain an effective content of active agent in the blood. Optionally one or more subsequent administrations may be given at a corresponding interval from each preceding administration, preferably when the plasma level has decreased to approximately 10-50% of the immediately-preceding maximum.
It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
The invention will now be described in detail by way of reference only to the following non-limiting examples and drawings.
Benzdioxols are described in the literature (Perekalkin, 1982a). The synthesis of benzoimidazole and benzoxazole may also be carried out using standard condensation methods 1 and 2 (Perekalkin, 1966; Perekalkin, 1982b) as shown below.
Method 1
Method 2
in which X, Y, , and R1 to R7 are as defined in Formula I above.
In Method 2, equimolecular quantities of benzaldehyde and nitroalkane were mixed in an Erlenmeyer flask and dissolved in equal volume of alcohol. Freshly-distillated ethylenediamine was added to the obtained solution in catalytic quantities (usually 1:10 in relation to aldehyde and nitroalkane) and was left in the dark at room temperature for several days (from 3 up to 10 days). During this time, the compound crystallized. After the cooling up to about 0° C., the crystals were filtrated and washed with cold alcohol and then dried. When the yield is small, the mother liquids can be joined together and evaporated in rotary evaporator. After cooling, the additional quantity of impure product is obtained. The product was purified by dissolving in a minimal quantity of boiling alcohol. It was then treated with activated carbon, filtered hot and while the cooling was in progress, fine yellow needles crystallized. The yield was about 80-85%, the compound being chromatographically homogeneous.
The infrared spectra of the compounds obtained are in accordance with those described in the literature (Hamlin and Weston, 1949; Knoevenagel and Walter, 1904; Burton and Duffield, 1949).
The compounds were soluble in organic solvents such as ethanol, acetone, benzene, methanol, acetonitrile, chloroform, and DMSO, but showed very poor solubility in water (0.1%). When an alcoholic solution was added to water, a stable colloidal mixture was formed.
Compound (1) was prepared using Method 1 described in Example 1 above. The reaction scheme is shown below.
A mixture of 9.8 g of tetranitromethane (1 mole) and 10 cm3 of acetone was cooled by ice and added dropwise to 8.1 g of distilled isosafrole (1 mole) and 4.8 g of pyridine (1.2 mole) dissolved in 20 cm3 of acetone. The very first drops caused darkening of the reaction mixture and the liquid turned non-transparent and murky red when the entire portion of tetranitromethane was added. The smell of tetranitromethane disappeared quickly and in approximately 2 hr, the dark-red solution (which had become transparent) was poured into 100 cm3 of water in a stoppered bottle. The mixture was thoroughly shaken, covered with a layer of ether and a mixture of 6.7 cm3 of 33% solution of caustic potassium (1.03 mole) and 50 cm3 of water was added in small portions. The mixture was shaken after each addition and once the entire amount of alkali was added, the shaking was continued until the entire salt of pyridine and nitroform, which was present as a dark red oil, disappeared. The water layer was then separated and again extracted with ether. Combined ether extracts were first rinsed with water and then with water acidified with sulfuric acid and finally once again with pure water. After distillation of the ether in the vacuum, a sediment of β-nitroisosafrole was found in the form of yellow needles, which were re-crystallized from approximately 65 cm3 of alcohol. Compound (1) was obtained with a melting point of 98° C., and a yield of 7 g. Once the solvent had evaporated, another 0.5 g of Compound (1) was obtained. The total product amounted to 72.5% of the theoretical yield.
Compound (1) was prepared using Method 2 described in Example 1 above. The reaction scheme is shown below.
900 g piperonal was dissolved in 1000 cc alcohol with constant shaking and 450 mL nitroethane was added slowly followed by 10 mL ethyldiamine. After 17 hrs stirring, the mixture was placed in the dark at room temperature for 5-7 days. The resulting yellow crystals were filtrated in a Buchner funnel until dried and then washed twice with 150 mL alcohol. This yielded 1200 g of Compound (1) with a melting point of 95° C. After further crystallization from ethanol, 1000 g of light yellow crystals were obtained with a melting point of 98° C. (˜80% yield).
Molecular Formula: C10H9NO4, molecular weight-207.5
IR Spectrum:
1. Aromatic ring-above 3000 wave number & associate aromatics 1470-1630 region
2. β-methylstyrene-additional groups over styrene 1442 aliphatic-C-+900-1000 peaks
3. Nitrogroup at low wave number e.g. 747, 673 and β-nitrostyrene has 1520.
4. Aromatic Ether Group—1312 (1258) 1138, 1030 Nevertheless a fingerprint of this compound is provided by the IR spectrum (q.v.). This has been done on the recrystallized material in order to reduce peaks due to contaminants.
IR Spectrum
Impurities of molecules weight 303.4 and 331.4. Confirmation of molecular weight of main species 207.1.
NMR Spectrum
Hydrogen NMR (200 MHz) shows:
Aromatic ring with three remaining Hs, 3 Hs as part of a CH3, another attached to the side chain and 2 Hs as part of another ring.
Carbon NMR (50 MHz) shows: —CH3, CH—, —CH2 (as methylenedioxy)
Values of chemical shifts support the structure given and a likelihood of the E-stereoisomer rather than the Z-stereoisomer favored by the synthesis used. A strong-withdrawing group (NO2) is indicated.
UV/Visible Spectrum
Recrystallized material has peaks (broad) at 250-270 mm and 360-370 mm with high absorbance below 210 mm.
Compound (2) was prepared using Method 2 described in Example 1 above. The reaction scheme is shown below.
3,4-methylenedioxybenzaldehyde was condensed with nitromethane using fresh distillated ethylenediamine NH2-CH2-CH2-NH2 as a catalyst. The reaction was conducted in alcohol, darkness and at room temperature for 5 days. The resultant crystals were separated by filtration and washed with cold alcohol. After being dried in air, the yield was 80%, m.p. 158-159° C., and after recrystallization, the m.p. was 162-163° C. Compound (2) was non-soluble in water, soluble in acetone, alcohol, acetic acid and in a majority of organic solvents.
In the experiments described herein, museum strains of pathogens obtained from the museum of the Microbiology chair of the Military Medical Academy (designated by the index “M”) and strains selected from pathological material (designated by the index “B”), taken from patients and having gone through no more than three laboratory passages were used. For each type of pathogen, the corresponding optimal nutrient media was used. For the impregnation method, compounds (1) and (2) were added to solid nutrient media at doses from 0.03% to 2.0%. An agar diffusion assay analogous to the standard method of determining sensitivity to antibiotics was used.
The agar diffusion assay was performed as follows.
Meat peptone agar was prepared and impregnated with the test compounds at concentrations from 0.01 to 2.0%. The medium was poured into Petri dishes and allowed to set. Agar plugs of 10 mm diameter were cut out and placed on the surface of Petri dishes containing the same medium immediately after they were inoculated with the microorganisms to be investigated (at least six plugs per culture). After one day of incubation at 37° C., the diameter of the zone of retardation of growth of the culture around the plugs was measured. The results were evaluated in accordance with official standards of testing sensitivity to antibiotics; a diameter 20 mm corresponded to a stable culture, 21-28 mm to moderate stability and 29 mm to sensitivity.
In parallel to this, the sensitivity of pathogens to 15 antibiotics was tested according to the official protocol of the Russian Supervisory Authority for the Introduction of New Medicinal Substances and Medical Technology (disc method).
Several pathogen types and strains were used to show the limits of sensitivity of the strains and types to the test substances, in order to evaluate their probable overall breadth of performance.
Table 1 below shows the results of experiments with Compound (1) at a concentration of 1.0%, at which it suppressed the propagation of 5×105 to 5×107 organisms/mL, and comparative results of sensitivity experiments using the following 15 antibiotics:
1-penicillin,
2-ampicillin,
3 -gentamycin,
4-carbenicillin,
5-kanamycin,
6-lincomycin,
7-levomicethin,
8-oxacillin,
9-polymixin,
10-rifampicin,
11-ristomycin,
12-streptomycin,
13-tetracycline,
14-erythromycin, and
15-cephalosporin.
Table 2 below shows the results of experiments on sensitivity of some microorganisms to Compound (1) using the agar diffusion method described above.
Table 3 shows the results of experiments of sensitivity of pathogenic fungi to compound (1).
The antimicrobial activity of Compound (1) was tested, using the agar diffusion method.
Due to the very low solubility of the compounds, the studies could not be carried out in the liquid phase. For this reason an initial mother impregnate, containing 0.5% of test compound, was prepared on meat peptone agar. From this mother impregnate, regenerated to the liquid state in a water bath, serial double dilutions were prepared by addition of the agar base. The dilutions thus obtained, containing the compounds in concentrations of 0.5%, 0.25%, 0.12%, 0.06%, 0.03%, 0.015% were poured into Petri dishes, on which six bacterial and two fungal test cultures were inoculated.
The following test cultures were used:
1) Staphylococcus aureus strain 674, isolated from a patient, sensitive to gentamycin, oxacillin, tetracycline, erythromycin and cephalothin, slightly sensitive to streptomycin.
2) Enterococcus faecalis museum strain, sensitive to ampicillin, rifampicin, and streptomycin.
3) Klebsiella pneumoniae strain 312, isolated from a patient, sensitive to gentamycin and polymyxin.
4) Salmonella typhimurium museum strain 727, sensitive to ampicillin, gentamycin, carbenicillin, canamicillin, polymyxin, and cephalothin.
5) Acinetobacter strain 681, isolated from a patient, slightly sensitive to polymyxin.
6) Pseudonomas aeruginosa strain 328, isolated from a patient, slightly sensitive to polymyxin.
7) Trichophyton interdigitale.
8) Candida albicans.
Each of the eight test cultures was sown on sterile meat peptone agar in a Petri dish, and then a standard plug of agar, impregnated with one of the 9 compounds at a concentration of 0.5%, was placed on the agar surface. The retardation zone was measured around the plug after 24 h and 48 h growth at 37° C. For the fungal cultures, the results were assessed after 7-10 days of incubation at 30° C.
The results of the action of the compound on the impregnated agar are summarized in Table 4:
Table 5 below shows the results of the agar diffusion experiments.
In equal concentration, Compound (1) inhibited the growth of ampicillin-resistant Staphylococcus, and the growth of Enterococcus, which was sensitive to this antibiotic. Similar differences could be observed with other pathogens and other preparations.
Anti-TB Effects of Compound (1)
Anti-TB effect was checked by standard method of serial double dilutive in synthetic liquid medium (SOTON) with 10% normal equine serum. The solution was prepared in Tween-80®.
Test culture was Mycobact tub. H.37RV sensitive to anti TB medication.
Mycobacterial suspension (density 5×107 cells/m1) was spread onto a special liquid medium (Vischnevsky, B. I.)
Results were calculated for 10-14 days incubation at 37° C. MIC (totally inhibited M tuberculosis)
The results are shown in Table 6 below.
The effect of Compounds (1) and (2) on trichomonas was also investigated. Trichomonas vaginalis isolated from patients was used. The trichomonads were cultured at pH=5.8-6.5 and 37° C. in medium 199 containing 5.0% native fetal calf serum, carbohydrates, and antibiotics to suppress the accompanying flora. Vaseline was applied to the surface of the medium in the culture tube. The experimental specimens contained the test compounds at a concentration of 0.3%.
7 specimens were investigated, containing motile forms of the parasite in a quantity of 5-8 cells in 1.0 cm3. In the control, parasites were cultured successfully over 3-4 passages (each passage 5-6 days). In contrast, culture of motile forms in medium containing the test compounds was unsuccessful in every case. After only one passage in the presence of the test compounds, motile forms did not propagate.
The therapeutic and prophylactic effect of the substances was determined in experiments in vivo on mice, infected intraperitoneally or intranasally with Corynebacterium paratuberculosis.
Compounds (1) and (2) were administered intraperitoneally, intramuscularly and orally at a dose of <20% LD50/0.2 at different periods from the day of infection, i.e., 2 and 1 days before the infection (schedules 2, 1), on the day of infection (schedule 0) and 1, 2, 3, etc. days after the infection (schedules +1, +2, +3, etc.).
The daily mortality rate was measured, their cumulative variations were calculated and based on this the results of the performance of the preparation was determined by the formula
AI=[(B−A):B]×100
where AI=activity index of the preparation (%),
A=cumulative mortality in the experimental group,
B=cumulative mortality in the control group.
The results are shown in Table 7 below, and indicated that Compounds (1) and (2) showed therapeutic and prophylactic activity in mice infected with Corynebacterium paratuberculosis.
For intramuscular administration, the clinical prophylactic performance in the form of reduced mortality of animals was 52.53%. The clinical performance for salmonellosis varied within the limits of 50.0-20.0%. For pseudo-tuberculosis, the prophylactic performance was 50.0%.
The radiation protective performance of Compounds (1) and (2) investigated was tested on male white mice weighing 18-20 g.
Dosages: 2, 4, 6, 8, 10, 15, 20 Gr (1 hR=100 Rōntgen).
Period of observation=25 days.
Method of reporting: dynamic mortality, calculation of cumulative mortality and factual changes of dosage (FCD).
Schedule of introduction: 50 mg/kg at 0.2 mL in mice.
Group 1—control,
Group 2—2 and 1 days up to irradiation.
Table 8 shows the results of the experiments on radiation performance of the compounds investigated:
The results show that for all irradiation dosages prophylactic administration of the test compounds considerably decreases the mortality of irradiated animals in comparison with the control group. FCD (LD50 contr.)=1.39-1.43, which shows a high radiation protective effect of the compounds indicated, the performances of which do not yield to the reported media.
Human volunteers suffering from skin wounds infected by Streptococcus and Staphylococcus were treated with 0.1% Compounds (1) and (2) in an ointment base of vaseline, sheep fat, and sulfoxide. Application of 0.1% ointment for 3 days cleared the wound completely of pus, with subsequent healing of the wound.
In 3 cases, extensive damage to the skin caused by fungal infection was treated; the type of the fungus was not identified. The damaged area was smeared twice per day with 0.1% ointment. The skin was cleared of fungal growth within a week.
While the pharmacokinetics of the compounds of the invention have not been investigated in detail, in experiments carried out in mice to which Compounds (1) and (2) were administered intraperitoneally, and intragastrically, it has been established that the compound will remain in a biologically active concentration in the blood for longer than 24 hrs.
The average lethal dose for mice when administered intragastrically was 1500 mg/kg body weight; when administered intraperitoneally, the average lethal dose was 575 mg/kg body weight. Thus, the compounds of the invention have low toxicity.
Compound (1) is relatively insoluble in water but is soluble in 10% DMSO at 1 mg/mL (0.1%), 10% ethanol at 2 mg/mL and 10% acetone at 2 mg/mL.
The highest concentration testable is either 512 tg/mL at 5% solvent or 256 tg/mL at 2.5% solvent. It does not require specific chemical neutralization, dilution being sufficient to neutralize residual activity in microbicidal testing.
DMSO was selected as the solvent for testing because it has the lowest toxicity against test strains.
Compound (1) was at least 8 times more active against E. coli when formulated in ethanol, giving an MIC of 128 μg/mL (0.06% ETON) compared to >512 μg/mL (2.5% DMSO). Ethanol is toxic to E. coli at concentrations >2.5%.
NCCLS-USA Standard Method—Broth microdilution (or macrodilution) (Mueller-Hinton).
Inoculum 1-4×104 cfu (or −4×105 cfu). Ciprofloxacin test control. Chlorhexidine values added for disinfectant and antiseptic activity comparison.
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) as 3-log reduction (99.9% kill) at 35° C., 24 hours, aerobically (unless otherwise indicated). 48-hr titers were not significantly different.
Results are shown in Table 9:
Compound (1) is a relatively broad spectrum, antibacterial agent with bactericidal activity within an acceptable concentration range in vitro for a representative selection of Gram-positive and Gram-negative bacteria. Compound (1) is broadly effective against aerobic Gram-positive and Gram-negative cocci, and aerobic Gram-positive rods of clinical significance.
Clinical isolates (multiple-antibiotic-resistant) of S. aureus and E. faecalis were as susceptible as the standard strains. Although not active at the low concentrations c.f. current treatment drugs, there may be a potential use for Compound (1) as a treatment for multiply resistant staphylococci and enterococci not responding to the current drugs of choice.
Compound (1) is active against clinically significant anaerobes, Clostridium perfringens, Clostridium difficile, and Bacteroides fragilis. C. difficile causes enterocolitis in hospitalized patients and is currently treated with vancomycin as the drug of choice. Induction of resistance is a potential problem with vancomycin. There could be a market for an oral drug for C. difficile enterocolitis as an alternative to vancomycin.
Anaerobic infections are generally mixed infections of one or more anaerobes with facultative bacteria, usually enteric Gram-negative rods. The most common anaerobic pathogens are Clostridium difficile, and Bacteroides fragilis. Current treatment with metronidazole in combination with other antibacterial drugs is generally efficacious. Given Compound (1)'s broad spectrum against both aerobic and anaerobic bacteria this could possibly treat these infections.
Compound (1) is very active against Campylobacter jejuni. Campylobacter is currently the greatest cause of enteric infections worldwide and is often treated because of its severity in some patients and the tendency for infection to predispose to development of Guillain-Barre syndrome, a serious CNS disease.
The relative resistance of enteric Gram-negative bacteria could be a function of solubility and ability of Compound (1) to penetrate cells. The successful treatment of recalcitrant enteric infections and the successful treatment of Salmonella infections in animals have been shown.
Compound (1) is active against Neisseria gonorrhoeae. Vulvo-vaginitis is caused by Candida albicans, N. gonorrhoeae, Chlamydia trachomatis, and Trichomonas vaginalis (singly, not as co-infections). Compound (1) is active against two of these agents.
Formulating Compound (1) for greater solubility (and therefore probably greater absorption) could improve both its activity and its distribution in vivo.
NCCLS-USA Broth macro-dilution method (RPMI medium).
Inoculum ˜5×104 hyphal fragments/mL (haemocytometer). Miconazole control.
Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC 2 log reduction-99% kill) at 30° C., aerobically, 2, 7 and 10 days for yeasts and 4, 7 or 14 days for filamentous fungi.
Results are shown in Table 10.
Compound (1) is fungicidal at relatively low concentrations against a broad range of clinically significant yeasts and filamentous fungi (Table 10).
Compound (1) shows good activity against three major causes of skin, hair and nail infections in humans and animals. Superficial fungal infections are the most common fungal infections worldwide. Treatment is prolonged, over months (and years for nail infections). Superficial treatments with antifungal lotions and creams were only partially effective. Current treatments, although generally low-cost, have poor efficacy, and frequent relapse rates. Oral systemic agents (terbinafine and itraconazole) are preferred for superficial infections in compromised patients and nail infections.
Serious fungal infections in compromised patients have increased worldwide in prevalence and severity. Fungal infections are generally long term, with a high therapeutic failure rate, frequent relapse, and development of resistance by fungi. Candidiasis (Candida albicans) and aspergillosis (Aspergillus fumigatus) are the major fungal infections. Severe, invasive infections have a high mortality rate. Very few effective drugs are available (cf antibacterial drugs). Long term therapy makes safety and failure to induce resistance important considerations. Amphotericin B is the main drug of choice for many serious mycoses. It is fungicidal, with poor solubility and low bioavailability and is limited by toxicity and delivery problems and high therapy failure. The azoles and triazoles are fungistatic drugs (e.g., fluconazole and itraconazole) which have low toxicity, good pharmacokinetic characteristics but are often ineffective due to development of resistance on long-term therapy.
Compound (1) is broad-spectrum, fungicidal, and has failed to induce resistance in C. albicans and A. fumigatus (see below).
Bacillus subtilis endospores
Aspergillus fumigatus asexual exospores
Compound (1) in water+Tween-20® was tested for sporicidal activity up to 24 hrs.
Compound (1) 512 μg/mL did not kill B. subtilis endospores in 24 hrs.
Compound (1) 512 μg/mL killed A. fumigatus exospores at 24 hrs but not 6 hrs.
Inhalation of Aspergillus spores is the major mechanism of transmission. Activity against spores could be significant in prophylaxis of compromised individuals. Since the spore must germinate to infect, however, ultimately it is activity against vegetative forms of fungi that determine efficacy.
Bacterial and fungal strains were exposed to Compound (1) in sub-inhibitory concentrations continuously for 12 weeks and monitored for a rise in MIC indicating development of resistance mechanisms. A heavy and variable inoculum is used for weekly subculture so inhibitory concentrations each week vary. The standardized MIC is measured at the beginning and end of exposure. A greater than 4-fold variation of standardized MIC is indicative of increased resistance, or a rising trend in weekly MIC. The genera selected are known to develop resistance readily to many antibiotics and to be a major clinical problem.
Bacterial species and C. albicans, known to develop resistance to many current drugs, did not develop resistance to Compound (1) after 12-weeks' continuous exposure (Table 11). Strains were scanned for abnormal microscopic and macroscopic changes. Proteus vulgaris lost the ability to swarm, indicating an effect on flagella. Other strains appeared normal. Tests are not yet complete for R. rubra and three molds. Failure to induce the development of resistance in these strains is a significant attribute of Compound (1).
The activity of Compound (1) and ciprofloxacin was determined in the presence of plasma and whole blood (horse), by macrodilution method in Mueller-Hinton broth to 48 hrs.
Compound (1) appeared to be relatively unaffected by the presence of 10% plasma and to be more active in the presence of 5% whole blood. The slightly improved inhibitory activity of drugs in the presence of blood sometimes occurs with antibacterial agents and is probably due to natural antibacterial factors present in blood. Further increasing the concentration of plasma and blood reduced the bactericidal activity of Compound (1) against S. aureus as shown in Table 12. Ciprofloxacin showed respectively a 4-fold and 2-fold decrease in activity against S aureus in the presence of 10% plasma and whole blood.
The MIC of Compound (1) was determined in increasing concentrations of plasma. Compound (1) has been shown to bind to human serum albumin and to agarose. Serum binding is significant in drug distribution and bioavailability.
The MIC of Compound (1) is significantly increased with increasing plasma concentrations. Bactericidal activity is much more affected than inhibitory activity.
Bioavailability of Compound (1) is significantly decreased in the presence of plasma proteins (Table 13). Compound (1) is reversibly bound to proteins.
Test strains were inoculated into Compound (1) solutions in water and sampled immediately and at 1, 2, 4, 6, and 9 hours. Survivors were estimated by viable counts on MHA (35° C., 48 h).
Measure of kill: reduction in viable count (log) expressed as log reduction factors.
(e.g., a 1-log reduction=90% kill, 2-log=99% kill, 3-log=99.99% kill, etc.)
Compound (1) showed rapid kill only against Candida albicans, with greater than a 99.999% reduction within 2 hrs at 512 μg/mL and within 4 hours at 256 μg/mL as shown in
Rate of kill was much slower against bacteria. The kill rate at 512 μg/mL was 99.99% within 2 hrs for B. subtilis and 99.99% within 9 hrs for S. aureus.
Ciprofloxacin was not tested.
The aim of this example was to establish absorption and blood levels of Compound (1) in the rat after a single-dose, oral administration.
Test Protocol
Sprague-Dawley rats (6 w/o, delivered 30 Jan, 2001) were acclimatized for 6 days in the Animal Facility under standardized environmental conditions (22° C. ±3° C., rel hum 30-70%, artificial light, 12 hrs light/12 hrs dark). Rats were fed a conventional laboratory diet with food and water ad lib and caged 5 rats per cage.
Test Substance
Compound (1) was prepared as an aqueous suspension in sterile LPW. At higher concentrations the suspension was sonicated to reduce particle size sufficiently to pass through the gavage needle.
Compound (1) was tested at 1250, 1000, 500 and 100 mg/kg.
Test Method
Rats were randomly assigned to treatment groups, identified by numbering on tails. Doses were tested sequentially from the lowest dose.
Compound (1) suspensions and the water control were administered at approx 100 mL/kgbw, in a single. One control and five treatment rats were weighed immediately before each dose administration, the dose volume calculated and the dose delivered by gavage (22 gauge stainless steel, smooth-balled end attached to a syringe).
Approximately 100-200 μL of blood (microfuge tube) was removed from the tail at 4 and 8 hours. Tails were prewarmed using a heat lamp and snipped at the tip with a large scalpel. Blood was massaged into a microfuge tube. Twenty four hour blood samples were not attempted because of the difficulty of snipping scarred tails and the distress caused to rats.
Blood was allowed to clot, centrifuged in a microfuge for 3 minutes (speed 14) and the serum separated and stored at −20° C.
Animals were observed twice daily for 7 days and all observations recorded individually for each animal. Animals were not weighed after the initial weighing. Sacrifice and necropsy was performed at 7 days.
Animals were euthanized by carbon dioxide.
Gross pathology was recorded and samples of heart, lung, liver, kidney, stomach, spleen, duodenum and colon removed (10% formalin) for histology.
Bioassays
Bioassay for Compound (1) levels in blood was not possible because of the interference due to strong binding of Compound (1) to blood proteins and to agar.
Agar Diffusion
An agar diffusion assay for Compound (1) was not possible because Compound (1) bound so strongly to agar that no zones of inhibition were produced at any concentration from 1 to 512 μg/mL with susceptible strains of S. aureus or Streptococcus pyogenes. Both well diffusion and disk diffusion assays were attempted.
Broth Dilution
Dilution of serum in MHB and testing with a low inoculum of S. pyogenes (inhibited at 1 μg/mL of Compound (1) in MHB) was not possible because strong binding to plasma at high concentrations caused a significant prozone.
Assay by UV Spectroscopy
There was insufficient serum for assay of individual rat samples.
Samples for treatment groups and for controls were thus pooled and a mean level of Compound (1) for each treatment group determined.
Test Method
Compound (1) was extracted (x2) from serum by toluene and absorbance measured at 370 nm (Hitachi U2000). A spiked control using 100 μg/mL Compound (1) in 50% methanol/water (V/V) and untreated controls were also assayed.
Blood Levels
Absorption of Compound (1) from the gastrointestinal tract is very low, approximately 2% of the oral dose reaching the blood. Blood levels increased with dose level. Eight-hour levels were generally higher than 4-hour levels. The small difference between 4- and 8-hr levels suggests a slow absorption.
Antifungal Activity
Filamentous Fungi
NCCLS-USA Broth Macrodilution method (RPMI medium) - draft. Inoculum 1-4×105 cfu. Miconazole control. This test was used for initial activity spectrum evaluations.
The MIC and MFC of filamentous fungi tested previously with the NCCLS-USA Broth Macrodilution Method were repeated using the new proposed standard microdilution method for testing fungi M38-P NCCLS-USA.
Results are of 2 or 3 replicates on different days.
Results were not significantly different from results obtained with the older method for all fungi previously tested. Amphotericin B was substituted for Miconazole as control for some tests.
Yeasts
NCCLS-M27-A Method for broth macro dilution antifungal susceptibility testing of yeasts; approved standard.
M38-P microdilution method for filmentous fungi also used.
Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC-2 log reduction-99% kill) at 3520 C., 48 hours. Results of 2 or 3 replicates on different days for each method. Results were not significantly different for the two methods. M38P only reported for yeasts and filamentous fungi.
Bacteria
Methods
NCCLS-M7-A5 Standard Method—Broth microdilution (Mueller-Hinton). Inoculum 1-4.times.104 cfu. Ciprofloxacin test control. Chlorhexidine and cetyl trimethyl ammonium bromide (CTAB) for disinfectant and antiseptic activity comparison.
NCCLS-M7-A5 Standard Method—macrodilution (Mueller Hinton ±specified enrichments) was also used. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) as 3 log reduction (99.9% kill) at 35° C., 24 hours, aerobically. 48 h titres were not significantly different and are not reported. Micro and macro dilution methods did not give significantly different MIC/MMC.
Campylobacter
Compound (1) was tested against a range of clinical strains of Campylobacter spp. isolated from humans.
Campylobacter is the most common cause of gastroenteritis infection worldwide (bloody diarrhoea, abdominal pain, vomiting, headache, fever, lasting about 1 week). Sequelae are arthritis and Guillain-Barre syndrome (0.1%). It is acquired mainly from eating poultry. Incidence is about 2.5 million persons/year in USA. C. jejuni accounts for 99% of cases. It can vary from sub-clinical to severe in compromised patients. It is usually untreated with only fluid replacement or, if the disease is severe or threatening, with antibiotics (Erythromycin, tetracycline or fluoroquinolone).
The above are significant human pathogens, all of which have successful treatment regimens with antibiotics. N. gonorrhoeae is a cause of vaginitis in women. Compound (1) is thus active at low concentrations against two causes, Candida albicans and N. gonorrhoeae.
Trichomonas Vaginalis
Method
The MIC of clinical isolate of Trichomonas vaginalis was determined by macrobroth dilution in Diamond's complete medium, modified by Klass (Modified TYM) as described by Garcia, L. Cultures were contained in 5 mL glass, screw-capped bottles without air-spaces. Volumes of 5 mL of log 2 dilutions, from 512 tg/mL Compound (1) in 5% DMSO to 0.25 μg/mL Compound (1) in 0.002% DMSO in modified TYM, were inoculated with 0.5 mL volume of cells in log phase of growth giving a final inoculum density of 1×104 to 3×104 cell/mL. Bottles were incubated aerobically at 3720 C. for 24 h before microscopic examination of motility. MIC was determined as the lowest concentration showing no motility. Aliquots of 0.5 mL from all tubes showing no motility were subcultured into further 5 mL volumes of modified TYM and incubated aerobically at 37° C. for up to 5 days to confirm non-viability.
Tests were validated by growth controls in TYM, modified TYM with 2.5% DMSO and modified TYM with 5% DMSO.
Tests were performed as 3 replicates on different days.
Results
Selected resistant strains from the 12 week resistance testing were retested simultaneously with parent strains using the new microdilution method.
Fungi exhibit up to a four-fold difference in MIC on repeat testing. Significant increases in MIC are ≧8-fold.
There is no significant development of resistance by the filamentous mould strains tested.
The effect of formulation in DMSO and ethanol on the MIC of Compound (1) was compared for Candida albicans, Salmonella Typhimurium and Escherichia coli using both the macrodilution and microdilution tests.
Stock solutions in ethanol were allowed to stand for 48 h before use to improve solubility. Compound (1) is not as soluble in ethanol as in DMSO. The concentration of ethanol and DMSO was kept constant at 2% and compared to a decreasing concentration of solvent on normal dilution of the stock solution. There was no difference between the two test methods. There was no difference in MIC between constant and decreasing levels of DMSO. Only E. coli showed an enhanced susceptibility to ethanol in the presence of a constant 2% ethanol.
The synergistic effect of ethanol on. E. coli was noted previously when solvents were being tested for selection of an appropriate solvent for the drug. Ethanol has no enhancing effect on Staphylococcus, Salmonella or Candida. DMSO is a better solvent for the drug in in-vitro tests. Ethanol will be used for animal studies.
Stock solutions of Compound (1) at 512 μg/mL in water+5% DMSO were stored at room temperature (RT ˜18-21° C.), 4° C. and −20° C. for up to 12 weeks and the potency tested by measurement of MIC for Bacillus subtilis at 2-weekly intervals.
1. Solution changed from light yellow to dark yellow after 8 weeks.
2. Solution changed from light yellow to dark yellow after 2 weeks.
Compound (1) is very stable, dilute solutions retaining potency for 12 weeks on storage at 4° C. and −20° C. Twofold loss of potency at room temperature after 6 weeks is very low compared to working solutions of antibiotics. It is also within the allowed variation range for MIC measurements for bacteria (2-fold). Control antifungals were not tested.
The aim of this example was to quantify the effect of Compound (1) on invasion and growth of the human malaria parasite Plasmodium falciparum in human red blood cells in vitro.
Methods
Malaria Parasites
3D7 is a well characterised in vitro culture-adapted line of P. falciparum that was used for these experiments. The parasite undergoes repeating cycles of growth and replication within human red blood cells. The duration of each complete cycle is 48 hours, beginning with young ring-stage parasites which mature through pigmented trophozoites during the first 24 hours of the cycle to segmented schizonts which burst to release infectious merozoites which rapidly invade red blood cells. Newly invaded merozoites become ring forms, and the cycle continues.
Parasite Culture and Growth Inhibition Assays
P. falciparum parasites were maintained in synchronous in vitro culture in freshly collected human red blood cells, using well-established techniques. For invasion assays, red blood cells containing stage-synchronized mature, pigmented trophozoites were purified and resuspended in fresh human red blood cells, so that approximately two in every 100 red blood cells was parasitised (2% parasitaemia). Fresh culture media was added to give a final red blood cell concentration of 2×108 red cells/ml.
Aliquots of the red blood cell suspension containing either the test compound, the vehicle alone (in this case EtOH) or PBS (control) were incubated at 37° C. in an atmosphere of reduced oxygen tension (1% O2, 3% CO2, 96% N2). Thin blood smears were made immediately (time=0) then subsequently after 24, 48 and 72 hrs of culture. For each smear, parasitaemia and stage of parasite maturation was quantified by microscopic examination after staining with Giemsa at pH 7.2. This allowed invasion, parasite development, and subsequent re-invasion to be quantified. At each sampling time point, the culture medium (±compound/vehicle) in all samples was completely replaced with fresh medium.
Compound (1) was tested as aqueous solutions of 100, 400 and 1000 μg/mL each containing 10% EtOH. Stock solutions were stored at 4° C. until required. For the assay, each solution was further diluted 1:40 in complete parasite culture medium (pH 7.2) to the desired working concentration (5, 10 and 25 μg/mL), then sterile filtered (0.22 μm) before being added to the parasitised red blood cell suspension. Stock solutions were stored at 4° C. throughout the duration of the assay, and diluted appropriately in parasite culture medium when required. It should be noted that at 1000 μg/mL, the compound was incompletely soluble, even after warming to 37° C. and vigorous vortexing. Thus the tests performed at a putative concentration of 25 μg/mL, may in fact have been performed at a lower effective concentration.
Results
The effect of Compound (1) on parasite growth was tested at final concentrations of 5, 10 and 25 μg/mL. Results are presented graphically in
At 25 μg/mL no parasites were observed and at 10 μg/mL only very few were found, suggesting that the compound actually killed the parasites.
It will be apparent to the person skilled in the art that while invention of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.
References cited herein are listed on the following pages, and are incorporated herein by this reference
Burton, H, and Duffield, G, J. Chem. Soc., 1949, 78
Denisenko P. P., Tarasenko A. A., Russian Patent No. 2145215, “Substances having antimicrobial, antifungal, antiprotozoal activity,” published 10th February 2000
Foyer, G, “Chemistry of nitro and nitroso groups,” Moscow, 1973, Pt. 2, pp. 194-195
Garcia, L, “Parasite culture: Trichomonas vaginalis,” Clinical Microbiology Procedures Handbook, H. D. Isenberg, (ed.), volume. 2, American Society for Microbiology, Washington, USA, 7.9.3.1 to 7.9.3.6.
Hamlin, K. and Weston, A., J. Am. Chem. Soc., 71: 2210 (1949)
Knoevenagel, E., Walter, L., Ber., 37, 4502 (1904).
Kuna, P, Chemical radiation protection, Moscow, 1989, pp. 25-28
Mashkovskiy, M. D., Clinical agents,” Pt. 2, Moscow, 1986, pp. 25-28.
Perekalkin, V. V., Unlimited nitrocompounds,” Leningrad, 1982, pp. 55, 59, 61, 71, 73, 88, 89, 91, and 95
Perekalkin, V. V., “Unlimited nitrocompounds,” Leningrad, 1982, p. 67
Perekalkin, V. V., “Unlimited nitrocompounds,” Moscow, 1966, p. 119
Vladimirov, V. G. et al., “Radiation protectors, structure and operation,” Kiev, 1989, p. 139.
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