WO2005051303A2 - Criblage de composes synergiques - Google Patents

Criblage de composes synergiques Download PDF

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WO2005051303A2
WO2005051303A2 PCT/US2004/039086 US2004039086W WO2005051303A2 WO 2005051303 A2 WO2005051303 A2 WO 2005051303A2 US 2004039086 W US2004039086 W US 2004039086W WO 2005051303 A2 WO2005051303 A2 WO 2005051303A2
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drug
assay
drugs
compound
identified
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WO2005051303A3 (fr
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Lixin Zhang
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Lixin Zhang
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors

Definitions

  • the present invention is directed to a method for discovering synergistic combinations of therapeutic agents in a systematic high throughput manner.
  • the invention is further directed to methods of identifying novel mechanisms of synergistic therapy to cure diseases.
  • a specific protein is studied in vitro, in cells and in whole organisms, and evaluated as a drug target for a specific therapeutic indication.
  • the historical paradigm, "one- drug-one-target dogma” has resulted in the identification of many effective chemical molecules that affect specific proteins, providing valuable reagents for both biology and medicine.
  • Avastin a recombinant humanized antibody designed to bind to and inhibit Vascular Endothelial Growth Factor (VEGF) in tumor angiogenesis .
  • VEGF Vascular Endothelial Growth Factor
  • a disease symptom is a progressional accumulation of mutations and interventions of different genes and pathways.
  • a paradigm shift is underway in the drug discovery industry towards combination therapies.
  • Genentech's Avastin - received approval from the US FDA in colorectal cancer with combination with intravenous 5-Fluorouracil-based chemotherapy on February 27, 2004.
  • Pfizer received approval from the US FDA for _its combination product CADUET, which combines atorvastatin, a cholesterol-lowering agent, and amlodipine besilate, an antihypertensive .
  • the product is a O 2005/051303
  • Multi- component therapies originating through deliberate mixing of drugs in a clinical setting, through happenstance, and through rational design, have a successful history in a number of areas of medicine, including cancer, infectious diseases, CNS disorders and HIV cocktail therapy. According to a recent report from Cutting Edge Information, $80 billion in blockbuster medicines will face patent expiration and generic competition by 2007. Seeking opportune drug pairings may be a new weapon in the arsenal to combat this threat. Since the establishment of the US Food and Drug Administration (FDA) in 1938, there are about 5000 single molecule drugs have had to be proven safe and efficient for their intended use to gain FDA pre-market approval (unless they had been grandfathered' as old drugs) . accelerates new drug discovery and novel compounds.
  • FDA US Food and Drug Administration
  • a systems biology approach is used to identify novel protein targets as well as novel and alternative pathways for further therapeutic intervention.
  • Many marketed traditional medicines can benefit from the approach of this invention.
  • the active ingredient in the traditional medicine could be identified by utilizing the power of the systems biology approach of this invention.
  • the disclosed technology can validate traditional medicines using a mechanism-based system.
  • the above platform may also benefit drug discovery based on products occurring in nature. Microbial and plant metabolites doubled human life span during the twentieth century, reduced pain and suffering, and revolutionized medicine. Over the years, natural products have accounted for the majority of major therapeutic modalities. This owes in large part to their structural complexity and clinical specificity.
  • CombinatoRx Inc. www.combinatorx.com, used a collection of 2,000 available drug compounds yielding about 2,000,000 pairwise combinations and recently reported that a combination of antipsychotic and antiprotozoal agent prevented the growth of tumors in mice while neither exhibited significant antitumor activity alone. It also provided a perfect example that using the one-drug-one-target dogma would never find such non-obvious but effective combinatorial therapy. limitations. Firstly, their compounds pool only came from about 2000 drugs. Their chemical diversity is therefore limited. Their targeted therapeutic proteins are also limited. All drugs today hit only 120 targets and the top 100 drugs hit only 43 targets. But genomics and proteomics revealed many more disease-relevant protein targets. Secondly, their pairing process is blind and biased.
  • the disclosed invention takes advantage of existing drugs at low dosage concentration, .screen and look for those synergistic partners to compensate and enhance the efficacy.
  • This invention has a much larger pool with unlimited chemical diversity to do screening. Instead of looking for non- obvious combination, established molecular mechanism of known drugs is searched or used. Instead of using the reported therapeutic dosage, the disclosed, platform which may be implemented by a proprietary software and algorithm to determine the sub-optimal dosage, could be used to guide formulation manipulation. This invention started with low dosage so that it is unlikely to have dramatic side effect. And only the synergy phenotype is investigated.
  • the present invention provides methods and compositions for enhancing Ketoconazole .
  • the present invention provides a method for identifying an agent in a high throughput assay.
  • synergy may come from sensitization, mutual induction or potentiation.
  • Synergistic co-drugs also have other advantages. When current drugs targeted only to one protein are used, the required high dosages for efficacy often produce unwanted side effects, and drug-resistant problems may also emerge. If focus is on multiple targets in a pathway through the use of co-drugs, high dosages of single drugs will not be necessary.
  • This invention provides a strategy to screen for novel co-drugs that enhance the activity of existing drugs to combat serious and life-threatening diseases.
  • Synergistic drug discovery approach of the present invention was tried in both anti-cancer and anti-infective study.
  • the co-drug compounds are applied to two-component or higher-order screening, and an efficient experimental strategy and analytical methods to determine ⁇ whether a beneficial interaction occurs between compounds was devised.
  • Systematic testing of all pairwise combinations for a compound set were began by defining the activity of each compound as a single agent in the assay system, and then by testing in two groups (active agents and inactive agents) all pairwise combinations ' of these compounds. Separating the testing of active and inactive compounds makes an efficient and complete search of all pairwise combinations tractable, when combined with automated robotic screenings and informatic systems.
  • Inactive compounds showing no detectable activity as single agents were tested in pools initially (four compounds per pool) and active pools were then be deconvoluted to identify the specific pairwise combination with activity of interest. Because many of these compounds were inactive on their own - and since active combinations comprising two inactive compounds are infrequent., higher efficiency can be obtained by pooling, without show detectable activity on their own (active compounds) are more difficult to assess in pools at a single concentration and are best tested at a range of concentrations to identify potency shifts as well as increases in intrinsic activity. Each active compound was tested against all other compounds (both active and inactive) in dose matrices comprising more than 5 concentrations (including zero) for each compound.
  • Figure 1 the technology platform of the present invention tries to help drug discovery pipeline on drugs either would-be out of patent or abandoned because of failed safety profiles. Instead of using the established therapeutic dosage of drug A, algorithms could be used to quickly figure out a much-reduced dosage. This condition coupling the power of high throughput screening, would enable us to find synergistic co- drugs, which return efficacy to the existing drug A but with an equal or improved performance profile. Co-drug could be screened from not only_ known _drug pools_, but also from abandoned drug candidates, natural products and synthetic chemical libraries. ketoconazole (X is the therapeutic concentration which inhibit 90% of the cell growth) . Samples are treated as labeled on top in duplicate and are reproducible for more than 3 batches.
  • Top panel showed the assay plates after incubated overnight at 35° C in a moistured chamber; Regrowth of top panel samples in fresh MHB media was shown at the middle panel. Fluorescence reading of top panel is measured at Ex 544 nm and Em 590 nm, and converted as percentage of growth inhibition at the bottom panel.
  • N Negative color control, DMSO.
  • P Positive color control, Amphotericin B.
  • HepG2 cells are used as a surrogate system to mimic potential therapeutic side effect in human body. Same amount of HepG2 cells are seeded in each of the 96 wells. After 24 hours incubation at 37 °C C0 2 incubator with a humidified chamber, colors were developed based on the cell viability.
  • Figure 4 An integrated database linking microbial genetic diversity to metabolite diversity for better dereplication purpose .
  • Hit means a synergistic co-drug candidate that generates 70% - 100% of the maximum activity by combination with the sub-optimal concentration of existing drug, while itself alone may have very little effect at the test concentration. Typical hit rate is shown in Table 1.
  • Co-drug means one or more chemical compound (s) that could be used with a low-dosage of a known drug to achieve therapeutic or preventive effect or cure diseases.
  • MIC Minimal Inhibitory Concentration
  • a Powerful Systemic Approach for Screening and identification of Synergistic Compounds The invention described is a systematic approach to discovering next generation of chemical compounds or formulation that act synergistically with the low dosage of known drugs. It started with an existing drug or dropped drug candidate X that may have toxicity, solubility, efficacy or drug resistance problems. This drug could have been used in any of the therapeutic areas, such as cancer, infectious diseases, inflammation, diabetes, CNS disorders and etc. Then a library of either natural products or macromolecules like nucleic acids and proteins, should be created. Thirdly, a functional assay including biochemical, cell based assays, animal models or clinical treatments should be established, and a sub-optimal dose (10% - 40% of the maximum activity) of drug X would be determined.
  • the library at different titration would be screened in a high throughput manner that should give a 0.1% to 1% hit rate.
  • the synergistic co-drug hit should generate 70% - 100% of the maximum activity by combination with the sub-optimal concentration of existing drug, while itself alone may have very little effect at the useful concentration.
  • the co-drug hits would be purified and identified.
  • the co-drug could be a pure synthetic molecule, a compound from a combinatorial synthetic library or a mixture from nature or synthetic resources.
  • This invention provides a method for screening compounds which enhances the efficacy of known drugs at low dosage, comprising: (a) providing one or more known drugs or dropped drug candidates; (b) obtaining libraries of either natural products or synthetic chemicals which contain different compounds; (c) establishing a functional assay for determining the sub-optimal dose of the known drug or dropped during candidates; (d) screening the libraries of step (b) at different titration using the functional assay of step (c) ; and (e) identifying one or more compounds in the libraries which enhances the efficacy of the known drug.
  • the invention provides a method, but is not limited to further comprising purifying the identified compound.
  • the libraries include but are not limited to macromolecules, nucleic acids or protein libraries.
  • This invention provides a method wherein the hit rate for synergistic lead compound is 0.1% to 1%.
  • This invention also provides a method wherein the co-drug enhances the efficacy of the known drug by 10% - 40% at low dosage levels .
  • This invention provides a method wherein the assay is any biochemical binding assay or enzymatic assay.
  • the assay may be cell or animal model based biological assay.
  • This invention provides a method wherein the screen step (d) can be performed manually or using a robotic.
  • This invention provides a method that embodies the identifying step (e) may be performed by a reporter gene assay, cytoblot assay or microscopic assay.
  • This invention provides a sub-optimal dosage in step (c) is determined using a software and algorithm.
  • This invention provides a method wherein the sub-optimal dosage is used to guide formulation manipulation.
  • This invention provides that the dosage of the known drug is decreased to a suboptimal level to sensitize and potentiate the cell for screening a synergy partner or lead compound.
  • This invention provides the above described method wherein the compound was not previously known.
  • This invention provides a compound identified by the method of embodiment.
  • This invention provides a composition comprises the compound identified by the above described method, the known drug or dropped drug candidate and an acceptable carrier.
  • This invention provides the suboptimal level of the known drug determined by one of the above described method.
  • This invention provides a composition comprising the suboptimal concentration of the known drug or dropped drug candidate determined by one of the above-described method and an acceptable carrier.
  • This invention provides a cyclic hexadepsipeptide Beauvericin (SZC-101) is identified as a synergistic drug for ketoconazole.
  • the cyclic hexadepsipeptide Beauvericin (SZC-101) maybe identified by LC-MS-MS and NMR study as a synergistic drug for ketoconazole.
  • the structure of the compound is also disclosed.
  • composition and a pharmaceutically acceptable carrier means any of the standard pharmaceutical carriers. Examples of suitable carriers are well known in the art and may include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution and various wetting agents.
  • Other carriers may include additives , used in tablets, granules and capsules, etc.
  • Such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gum, glycols or other known excipients.
  • Such carriers may also include flavor and color additives or other ingredients. Compositions comprising such carriers are formulated by well-known conventional methods.
  • This invention provides the uses of the compound identified by the method of any of the embodiment for identification of a protein or pathway which is related to a disease.
  • This invention provides the understanding the mechanisms of the synergistic effect: 1) Using systems biology approach including DNA or protein micro-array methods to compare what genetic biomarkers, genes or proteins are dramatically altered in the presence and absence of the synergistic drugs. 2) Using small interference RNA (siRNA) technology and transfer a siRNA library of fungal pathogen into the test fungi in the presence and absence of low concentration of one drug, e.g. ketoconazole. The effected gene will be dead in the presence of ketoconazole.
  • siRNA small interference RNA
  • agent through the described co-drug synergism is merely to demonstrate the power of the method of co-drug synergism in screening for and identification of drug or drug candidates, but does not limit the invention to the example in any way or form.
  • the screening and identification method is useful in identifying drug candidates for any human, plant or animal diseases or conditions using a proper assay and a synergism of a known drug for the disease or condition and the candidate from a library or other source to be screened.
  • This invention discloses an algorithm and high throughput screening process which rapidly determines two key factors for successful new drug discovery.
  • the first factor is the appropriate reduced dosage of an existing drug
  • the second factor is a suitable synergistic partner, or partners, which return efficacy to the existing drug but with an equal or improved performance profile.
  • the compound pool used is a library of either natural products or synthetic chemicals (pure or combinatorial) including macromolecules like nucleic acids and proteins. The chemical diversity is virtually unlimited.
  • the high throughput screening process is a systematic, non-biased approach.
  • HSA single agent
  • Bliss additivism model sometimes also called check board model, predicts the combined response C for two single compounds with effects A and B is:
  • the methodology of this invention is designed to produce low cost medicines, with one of the key tenets being affordability, for both producers and consumers.
  • the library of compounds grows, the knowledge of complementary compounds exponentially grows, reducing the cost and time to development of new drugs .
  • ketoconazole an effective azoles antifungal drug and a natural product crude extract of F101604 from the natural extract library was used as an example.
  • a lead compound was also identified. Lead compounds were identified from other nature products as well as from synthetic libraries. Known drug collection which contains about 3000 single molecule drugs have proven safe and efficient for their intended use to gain FDA pre-market approval will be tested. Since the hits from this pool may not be potent enough, we then tried our natural product library.
  • antifungal drug as example was as follows. Fungi have emerged as the fourth most common pathogens isolated in nosocomial bloodstream infections. There are approximately 90,000 cases of severe systemic fungal infections in the US annually, with nearly 40% of those infections proving fatal. The demand for effective antifungal drugs is increasing in parallel with the growing populations of the immunocompromised patients most affected by invasive fungal infections and the widespread use of broad-spectrum antibacterial therapy.
  • the characteristics of an ideal antifungal agent should include; availability in both an oral and intravenous dosage form, have a broad-spectrum of activity covering both yeast and filamentous fungi, demonstrate fungicidal activity in vitro, display a good pharmacokinetic profile with minimal drug-drug interactions, be stable to resistance, have good tissue penetration, including the Central Nervous System (CNS) , display limited side-effects and be cost-effective.
  • CNS Central Nervous System
  • Mipnotericin B is a polyene macrolide introduced in 1956 and has been the gold standard for antifungal therapy since it was the only agent effective against systemic fungal infection.
  • the mode of action is due, in part, to its selective binding to ergosterol, the major fungal sterol, in the cell membrane. This induces changes in membrane permeability and leakage of cell components leading to cell death.
  • Amphotericin B is highly effective against a wide range of fungi however it must be administered intravenously and is rather toxic, producing a range of side effects. Nephrotoxicity is the most serious side effect and necessitates discontinuation of treatment. High fevers, nausea, vomiting, anemia and myalgia occur in greater that 50% of patients as well. New lipid-based delivery formulations are now available which greatly minimize toxic side effects however these formulations carry exorbitant prices, limiting their use.
  • the azoles interfere with the biosynthesis of sterols and other membrane lipids that comprise the fungal cell membrane by inhibiting a cytochrome P450 enzyme responsible for converting landosterol to ergosterol.
  • the lack of ergosterol in the cell membrane leads to cell permeability and death.
  • Each of the azoles has a different spectrum of effectiveness and defined limitations. For example, fluconazole is ineffective against Aspergillus species with limited effectiveness against certain Candida species but is highly effective against Cryptococcus a common and serious infection in AIDs patients.
  • Itraconazole has unpredictable bioavailability, varying between patients and frequent drug interactions but it has the broadest range of antifungal activities among all the azoles and the fewest side effects.
  • Ketoconazole is associated with more clinically important toxic effects including hepatitis, but is the most effective azoles against chronic, indolent forms of endemic fungal infections. Allylamines
  • This agent acts by inhibiting squalene epoxidase.
  • This is another enzyme in the pathway that leads to synthesis of ergosterol, so this agent is conceptually related to the azoles antifungal agents. It is highly lipophilic in nature and tends to accumulate in skin, nails, and fatty tissues. Terbinafine has oral and topical (cream) formulations. Oral preparation has been first introduced in 1991 in United Kingdom and approved for clinical use in 1996 in USA
  • Flucytosine is a pyrimidine analog that interferes with DNA synthesis in the fungus. Its spectrum of activity is fairly limited and drug resistance develops readily if flucytosine is used alone; for that reason it is always used in combination with amphotericin B. This combination is effective against crytococcal meningitis, a rather difficult disease to treat given the fact that most antifungal agents have poor bioavailability in the CNS. Toxicities however are frequent and include mucositis and myelosuppression, which is very serious in patients whose already immunocompromised status, led to the infection in the first place.
  • Multi-component therapies originating through deliberate mixing of drugs in a clinical setting, through happenstance, and through rational design, have a successful history in a number of areas of medicine, including cancer, infectious diseases, CNS disorders and HIV cocktail therapy. _ The next generation of chemical compounds that act synergistically with the low dosage of known drugs was researched. The application of low dosage is also some drugs. It could also be used to rescue dropped drugs because of bad safety profiles.
  • the invention includes steps of: (a) providing an existing drug or dropped drug candidate X which may have toxicity, solubility, efficacy or drug resistance problems; (b) creating a library of either natural products or synthetic chemicals , (pure or combinatorial) including macromolecules like nucleic acids and proteins; (c) establishing a functional assay and figuring out the sub-optimal dose of drug X; (d) under this condition, screening the above libraries at different titration which gave 0.1% to 1% hit rate for synergistic effect; (e) detecting or measuring a property of the test element (generate 70% - 100% of the maximum activity by combination, while the synergistic co-drugs alone may have very little effect at the useful concentration) ; (f) purifying and identifying the partner co-drugs.
  • the co-drugs could be more than two components.
  • ketoconazole an effective azoles antifungal drug and a natural product crude extract of F101604 screened and isolated from the natural extract library was used as an example. A lead compound was also identified from subsequent fractionation and purification.
  • this invention discloses a drug discovery approach consonant with the systems biology framework, and complementary to the target-based approach.
  • These synergistic co-drugs have enabled the existing drugs to be more effective and contribute to better understanding of multiple pathways to cure disease.
  • An example was given to use novel natural product together with low dosage of Ketoconazole for better antifungal drug discovery. follow.
  • novel natural product together with low dosage of Ketoconazole for better antifungal drug discovery.
  • Amphotericin B and the azoles have toxicity problems because their cellular targets have homologues in mammalian cells.
  • the azoles inhibit lanosterol 14- demethylase, a cytochrome P450 enzyme critical for sterol synthesis in fungi and mammals; the azoles are also effective inhibitors of many cytochrome P450 reactions and because of this are useful tools in mammalian cell biology.
  • Amphotericin B targets plasma membrane sterols and is nephrotoxic.
  • Candida albicans strains resistant to the azoles have been on the increase in recent years .
  • Candida albicans is the single most important fungal pathogen in humans.
  • Candida albicans causes oral and systemic candidiasis in immunocompromised patients and vulvovaginal candiadiasis (WC) in women.
  • Candidiasis is an extremely important problem in HIV infected patients, 84 % of whom exhibited oropharyngeal colonization by Candida spp.
  • WC is extremely widespread and a significant medical problem.
  • some 75 % of women in the USA will have at least one episode of WC in their lives, 40 % will have two, and a smaller number ( ⁇ 5 %) will have the recurrent form.
  • Ketoconazole is commonly used to treat Candida infections. However, at clinical doses, ketoconazole is associated with important toxic side effects including hepatitis. In addition, resistant strains often emerge during long-term or prophylactic treatment as a result of the necessarily high concentrations of drug required.
  • test fungal strain used is Candida parapsilosis ATCC 22019, an opportunistic human pathogen that causes severe infections in immunocompromised individuals.
  • the natural sample used is a microbial fermentation crude extract. Microbes from a variety of ecosystems all over the world were collected and grown under different physiological media to generate a diversified natural product library.
  • the master plates are prepared for screening by diluting the natural extract stocks 100 fold.
  • the test strain Candida parapsilosis ATCC 22019 is cultured in Mueller-Hinton (MH) broth. (Biosource catalog number DAL1100) in the presence and absence of a sub-clinical concentration of 0.01 X ketoconazole (X is the physiological concentration which inhibit 90% of the cell growth), and dispensed at 0.08 ml/well in 96-well microtiter assay plates.
  • FIG. 1A A crude extract F101604 was identified as one of the potent hit.
  • Figure 1A top panel showed the assay plates after incubated overnight at 35° C in a moistured chamber. Equal amount of Candida parapsilosis cells, media and Alamar Blue Dye were in each well. Treatment as indicated on the top of duplicated samples. Positive color control (P) contained antibiotic Amphotericin B and killed all the cells, which remained blue color. The fluorescence reading was converted as 100% of growth inhibition. To test if the fungal pathogen were killed or just growth inhibited, 2 ul of the overnight culture was transferred to fresh MH broth in excess with Alamar Blue Dye . The new plates were incubated again overnight at 35° C in a moistured chamber. The results are shown as Figure 1 top panel. If the color turned red, it meant the pathogen were still alive. The mode of action is static. If the color remained blue, it meant the pathogen was eradicated. The mode of action is called cidal . Negative color def ined
  • ketoconazole alone at 0.01 x only gave about 20% inhibition of growth and the mode of action was static.
  • F101604 extract was tested alone, no inhibition of the yeast pathogen was observed.
  • ketoconazole was tested at 1 X, it gave 90 % inhibition of growth.
  • the combination of ketoconazole at 0.01 X with F101604 achieved about 95% inhibition (better than 100 fold of ketoconazole amount) and the mode of action is cidal, showing the synergistic effect of the two components rather than additive effect.
  • this invention discloses a drug discovery approach consonant with the systems biology framework, and complementary to the target-based approach.
  • These synergistic co-drugs have enabled the existing drugs to be more effective and contribute to better understanding of multiple pathways to cure disease.
  • An example was given to use novel natural product together with low dosage of Ketoconazole for better antifungal drug discovery.
  • the synergy therapy dramatically improved efficacy of Ketoconazole as well as reduced its side effects and drug resistant problems.
  • compositions also provides pharmaceutical formulations or compositions, both for veterinary and for human medical use, which comprise a known drug in combination with one or more pharmaceutically acceptable carriers.
  • the pharmaceutical compositions used less dosage but achieved higher efficacy while decreasing side effects.
  • the carrier (s) compatible with the other ingredients of the formulation and not unduly deleterious to the recipient thereof.
  • the hit is identified as a cyclic hexadepsipeptide Beauvericin (SZC-101) by LC-MS-MS and NMR study from Fusarium proliferatum broth mixture .
  • ketoconazole/F101604 combination was not toxic to human cells
  • HepG2 cells were used as a surrogate system to mimic potential therapeutic side effect in human body.
  • Ketoconazole is toxic at 100 and 50 ug/ml as reported clinically, while F101604 and up to 25 ug/ml ketoconazole did not kill the human liver cell line (Fig 2) .
  • the fungal strain F101604 was identified genetically and morphologically. Upon activity-guided fractionation and purification, two distinct compounds were isolated from F101604 crude extract mixture. Either of them showed great synergistic effect with 0.01 X ketoconazole in inhibiting the growth of Candida parapsilosis. The activity was confirmed by commercial available product ordered from Sigma. The co-drug antifungal activity of those was not reported before in the literature.
  • Candida albicans ATCC 90028 Candida glabrata ATCC 90030
  • Candida Krusei Issatchenkia orientalis ATCC 6258
  • Aspergillus fumigatus ATCC 46645 Aspergillus fumigatus ATCC 46645
  • Saccaromyces cerevisiae ATCC 2601 and Cryptococcus neofor ans ATCC 14116.
  • the combination of Beauvericin and ketoconazole increased the spectrum of ketoconazole to diverse fungal pathogens.
  • Another important aspect is to test the effect of synergistic co- drugs on drug resistant fungal pathogens.
  • Drug resistant clinical isolates from ATCC as well as from other Biomedical Research Institute, including the wild types and mutants were used.
  • the beauty of some strains is that they have been well characterized of the mechanism of resistance, including overexpression of two types of efflux pumps, the major facilitator MDR1 and ABC transporters (CDR1 and CDR2) and the overexpression or mutation * of the target enzyme ERG11.
  • CDR1 and CDR2 the major facilitator MDR1 and ABC transporters
  • ERG11 overexpression or mutation * of the target enzyme
  • ketoconazole MICs of ketoconazole were determined by broth microdilution anti-fungal assay previously described in the absence and presence of SZC-101 with ketoconazole. Table II shows the dramatic synergistic effect of adding SZC-101 to ketoconazole. As reported before, SZP-17 showed cross- resistance to ketoconazole and fluconazole but not amphotericin B (data not shown) . With the synergy co-drug candidate SZC-101, ketoconazole becomes a highly effective agent for the clinical drug resistant strain SZP-17. At 2 ug/ml of SZC-101, the activity of ketoconazole was potentiated 200 fold. Table II. Effect of SZC-101 on the minimum inhibitory concentration (MIC) of ketoconazole against clinical drug resistant strain SZP-17. (Unit: ug/ml)
  • RNA-arrays or protein arrays for identifying novel genes and pathways, for the purpose of deciphering the complex genetic circuitry governing the disease process. Mapping the circuitry of microbial cells will provide potent cellular models for better treatment. The following points suggest possible modes of action for the synergism: 1) : Resistant Enzyme Inhibitors rendering them inactive, are a principal mechanism of resistance.
  • Enzymes can be secreted into the environment e.g. Stap aureus secretes penicillinase, which inactivates Penicillin G; or in the periplasmic space, e.g. Pseudomonas aeruginosa secretes cephalosporinase which degrades ceftazidime.
  • This resistance can be overcome by providing an inhibitor to the degrading enzyme in combination with the antibiotic.
  • the antibiotic is protected e.g. Augmentin (amoxicillin and clavulanic acid).
  • SZP-17 cultures were pretreated with compound alone or combination of 2 ug/ml of SZC-101 and 0.04 ug/ml of ketoconazole for 30 min at 32 °C and then treated with the fluorescent dye rhodamine G for 1 h at 32°C. Cultures were washed, and allowed to recover without compound (s) present. The ability of compound (s) remaining fluorescence in fungal cells. Whereas SZC-101 or ketoconazole alone had little effect on dye efflux, the combination efficiently prevented dye efflux (data not shown) . This result demonstrates that the two compounds together affect membrane pump activity, even though neither agent on its own has such an effect.
  • the synergistic screening provides an end point of the best ratio of several compounds.
  • Several approaches may be used to test and understand the mechanisms of the synergistic effect: 1) Using systems biology approach including DNA or protein micro-array methods to compare what genetic biomarkers, genes or proteins are dramatically altered in the presence and absence of the synergistic drugs. 2) Using small interference RNA (siRNA) technology and transfer a siRNA library of fungal pathogen into the test fungi in the presence and absence of low concentration of one drug, e.g. ketoconazole. The effected gene will be dead in the presence of ketoconazole.
  • siRNA small interference RNA
  • An existing drug was selected to improve its therapeutic value by decreasing its dosage and combining another partner.
  • the existing drug has clear approved drug profile, well-characterized mode of action. It also makes FDA's job easier to evaluate the combinator.ial therapy.
  • These synergistic co-drugs of the present invention contributed to the mapping of the wiring diagrams of fungi. It will enable the existing drugs to be more effective and contribute to the understanding of multiple pathways to cure disease . drugs for treating cancers, cardiovascular diseases, inflammations, diabetes, and other disorders were tested.
  • This invention provides novel approaches to identify co-drugs in nature product or synthetic compound library that may work by the mechanisms described above or by completely novel mechanisms.
  • This example of identifying a natural product as a fungicidal agent through the described co-drug synergism is merely to demonstrate the power of the method of co-drug synergism in screening for and identification of drug or drug candidates, but does not limit the invention to the example in any way or form.
  • the screening and identification method is useful in identifying drug candidates for any human, plant or animal diseases or conditions using a proper assay and a synergism of a known drug for the disease or condition and the candidate from a library or other source to be screened.
  • Pre-clinical trials are conducted using a mouse disseminated candidiasis model of infection in an immuno-compromised host.
  • mice Specific-pathogen-free, female ICR [CD-I] mice weighing approximately 23-27 grams were obtained from a single institutional vendor and utilized throughout the experiment. The animals, fed with standard rodent chow, were allowed to acclimate for 1 week before active experimentation.
  • Antifungals Agents were supplied by SynerZ Pharmaceuticals, Inc. in quantities sufficient to complete the experiments as outlined. In addition, SynerZ provided suitable stability, dissolution and formulation data prior to the preparation of this comparator antifungal, fluconazole, was obtained directly from the manufacturer. The antifungal compounds were administered by intraperitoneal (IP) injection.
  • IP intraperitoneal
  • Isolate A single Candida albicans (ATCC 36082) or a drug- resistant clinical isolate supplied by SynerZ was used to conduct all the studies.
  • MIC minimum inhibitory concentration
  • test compound e.
  • Acute Toxicity Studies Drug Administration and Evaluation: A series of two-fold dilutions of the test compounds were prepared in a suitable vehicle such that administration of the dilutions in 0.2 ml volumes will yield doses that span a wide range of concentrations. Five dosages were evaluated for each test compound. Final dosage selection for the test compound (s) was determined based on further consultation with other scientists.
  • mice for each of the 5 dosages per test compound (and combination with low dosage of fluconazole) * 5 compounds (125 mice) .
  • Test compound (s) were administered by IP route.
  • iii. A control group (5 mice) received 0.2 ml of therapeutic and suboptimal dosage of fluconazole in the vehicle only by the same route as the active treatment regimens.
  • Clinical Observation Animals were observed thrice daily for signs of drug related morbidity or morality post injection until the 96 hour termination point of the study.
  • Infection Mice weighing 23-27 grams were infected by lateral tail vein injection (0.1 ml) of the inoculum suspension prepared from an overnight culture of the test organism as previously described. maximum tolerated dose based on preliminary toxicity studies. Six to eight treatment regimens each for the comparator and test compounds were tested. Final dosage regimens were determined based on consultation with the sponsor.
  • mice were utilized and drug administered by the IP route 2 h after inoculation.
  • mice 120 [3 mice per dosage regimen * 8 regimens * 5 test compounds] mice were utilized and drug administered by the IP route 2 h after inoculation.
  • mice were utilized as control. Sets of treatment groups are based on ability to process a certain volume of samples timely for each experiment. Based on these numbers 8 treatment regimens were divided into 2 sets (4 regimens each) for each compound.
  • Efficacy was calculated as the change in fungal density obtained in treated mice after 24 hours compared with the numbers in the starting control animals.
  • the change in fungal density in tissues, expressed as change in loglO CFU, for both treated and untreated animals were reported using descriptive statistics.
  • the loglO CFU versus antimicrobial dosage curve was constructed for each compound including the comparator. Data was fitted using the Emax model to determine the 50% effective dose. Effectiveness (change in fungal density) of the agents alone vs. the combination was evaluated with appropriate statistical tests.

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Abstract

L'invention concerne une approche systématique efficace associée à la découverte de nouvelle génération de formulation ou de composés chimiques agissant de manière synergique avec un faible dosage de médicaments connus. Un médicament existant ou un médicament candidat abandonné est sélectionné. Une banque de produits naturels ou chimiques synthétiques (purs ou combinés) comprenant des macromolécules de type acides nucléiques et protéines est créée. Une analyse fonctionnelle comprenant des analyses biochimiques fondées sur des cellules, des modèles animaux ou des traitements cliniques sont établis, et une dose sous-optimale (entre 10 % et 40 % de l'activité maximum) du médicament sélectionné est déterminée. Dans cette condition, la banque à un titrage différent devrait être criblée à un rendement élevé, ce qui permettrait d'obtenir un taux de résultat compris entre 0,1 % et 1 %. Le co-médicament synergique obtenu devrait générer entre 70 % et 100 % de l'activité maximum par combinaison avec la concentration sous-optimale du médicament existant. Enfin, les co-médicaments obtenus devraient être purifiés et identifiés. Le co-médicament pourrait être une molécule synthétique pure, un composé issu d'une banque synthétique combinatoire ou d'un mélange de ressources naturelles et synthétiques.
PCT/US2004/039086 2003-11-19 2004-11-19 Criblage de composes synergiques WO2005051303A2 (fr)

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
WO2008104124A1 (fr) * 2007-02-27 2008-09-04 Aixian Qiao Procédé de criblage de produits pharmaceutiques combinés, produits pharmaceutiques obtenus par le procédé et leurs utilisations
CN102636626A (zh) * 2008-02-26 2012-08-15 程宇镳 一种复方药物筛选方法，获得的药物及其应用
US20130231264A1 (en) * 2010-09-21 2013-09-05 Lankenau Institute for Medical Research Chemical Genomics Center Ultra-High Throughput Screening Methods to Detect Synergistic Drug Interactions
US11049590B1 (en) 2020-02-12 2021-06-29 Peptilogics, Inc. Artificial intelligence engine architecture for generating candidate drugs

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US6117668A (en) * 1996-11-22 2000-09-12 Pioneer Hi-Bred International, Inc. Beauvericin detoxification compositions and methods
US20020028463A1 (en) * 2000-08-14 2002-03-07 David Duffy Biomolecule arrays
US6355628B1 (en) * 1999-07-29 2002-03-12 Tularik Inc. Combination therapy using pentafluorobenzenesulfonamides
US20030147945A1 (en) * 2001-10-03 2003-08-07 Paul Tardi Compositions for delivery of drug combinations

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6117668A (en) * 1996-11-22 2000-09-12 Pioneer Hi-Bred International, Inc. Beauvericin detoxification compositions and methods
US6355628B1 (en) * 1999-07-29 2002-03-12 Tularik Inc. Combination therapy using pentafluorobenzenesulfonamides
US20020028463A1 (en) * 2000-08-14 2002-03-07 David Duffy Biomolecule arrays
US20030147945A1 (en) * 2001-10-03 2003-08-07 Paul Tardi Compositions for delivery of drug combinations

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008104124A1 (fr) * 2007-02-27 2008-09-04 Aixian Qiao Procédé de criblage de produits pharmaceutiques combinés, produits pharmaceutiques obtenus par le procédé et leurs utilisations
CN102636626A (zh) * 2008-02-26 2012-08-15 程宇镳 一种复方药物筛选方法，获得的药物及其应用
US20130231264A1 (en) * 2010-09-21 2013-09-05 Lankenau Institute for Medical Research Chemical Genomics Center Ultra-High Throughput Screening Methods to Detect Synergistic Drug Interactions
US8993486B2 (en) * 2010-09-21 2015-03-31 Lankenau Institute for Medical Research Chemical Genomics Center Ultra-high throughput screening methods to detect synergistic drug interactions
US11049590B1 (en) 2020-02-12 2021-06-29 Peptilogics, Inc. Artificial intelligence engine architecture for generating candidate drugs
WO2021162874A1 (fr) * 2020-02-12 2021-08-19 Peptilogics, Inc. Moteur d'intelligence artificielle pour générer des médicaments candidats
US11462304B2 (en) 2020-02-12 2022-10-04 Peptilogics, Inc. Artificial intelligence engine architecture for generating candidate drugs

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