WO2005051303A2 - Screening for synergistic compounds - Google Patents

Abstract

This invention provides a powerful systematic approach to discovering next generation of chemical compounds or formulation that acts synergistically with the low dosage of known drugs. Existing drug or dropped drug candidate is selected. Then a library of either natural products or synthetic chemicals (pure or combinatorial) including macromolecules like nucleic acids and proteins is created. A functional assay including biochemical, cell based assays, animal models or clinical treatments are established, and a sub-optimal dose (10 % - 40 % of the maximum activity) of the selected drug is determined. Under this condition, 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. Finally, 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.

Description

SCREENING FOR SYNERGISTIC COMPOUNDS

This application claims benefit of U.S. Serial No. 60/523,446 Filed on November 19, 2003, the contents of which is hereby incorporated in its entireties by reference into this application.

Throughout this application, references are made to various publications. Disclosures of these publications are hereby incorporated in their entireties by reference into this application to a more fully described the state of the art to which this invention pertains.

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

Even though the investment on research and development is steadily increasing, the pharmaceutical discovery pipeline has declined substantially over the past decade. One of the major limiting factors is the prevalent "one-drug-one-target dogma" in the biotechnology and pharmaceutical industry. One of the major limiting factors in drug discovery industry is that pharmaceuticals have been traditionally designed to target individual factors in a disease system, but diseases are complex in nature and vulnerable at multiple attacks. Therefore, a systematic novel synergistic drug screening approach based on a multifactorial principle is urgently needed. Many drugs could be more effective at a reduced dosage if low dosages of other synergistic compounds are introduced simultaneously. This Many marketed traditional medicines demonstrated great efficacy and safety profiles in the long history. ^' However, when efforts made to purify a single molecule, the activity usually got lost. It is fully possible that several components instead of one molecule are required to achieve the desired activity. It is an object of the present invention to develop a drug discovery approach consonant with the systems biology framework, and complementary to the target-based approach. These synergistic co- drugs from natural products will enable the existing drugs to be more effective and contribute to better understanding of multiple pathways to cure a disease.

Instead of the hit and miss technology of the past, the current biological research and much of drug discovery is often driven by the search for new molecules targeting disease-relevant proteins.

In this approach, 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. For example, the recently FDA-approved drug Avastin, a recombinant humanized antibody designed to bind to and inhibit Vascular Endothelial Growth Factor (VEGF) in tumor angiogenesis .

However, in real physiology, diseases are multigenic. Therefore, to cure diseases, multiple stages along the disease pathway may need to be manipulated simultaneously for an effective treatment. Systems biology has revealed that human cells and tissues are composed of complex, networked systems with redundant, convergent and divergent signaling pathways. For example, the redundant function of proteins involved in cell-cycle regulation has inspired efforts to intervene simultaneously at multiple points in these signaling pathways. Another important note is for natural product based drug discovery. Microbial and plant secondary metabolites doubled our life span during the 20th 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. It is unfortunate that the pharmaceutical industry has downgraded natural products just at the time that new assays are available, and major improvements have been made in detection, characterization and purification of small molecules. In the early to mid-nineties combi-chem companies attempted to fill the void with large numbers of new molecules. Unfortunately, it appears that the chemistry employed did not create sufficiently diverse or pharmacologically active molecules.^' It is clear that the future success of the pharmaceutical industry depends on the combining of complementary technologies such as natural product discovery, high throughput screening, genetics, genomics and proteomics, combinatorial biosynthesis and combinatorial chemistry.

Just like the networked biological systems with redundant, convergent and divergent signaling pathways, a disease symptom is a progressional accumulation of mutations and interventions of different genes and pathways. With the advancement of biotechnology and better understanding of the molecular mechanism of biological systems, it is obvious to use multi-component therapy. 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. On February 2, 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

UJ.UUU pressure and high cholesterol, billed' as the first medicine to treat two different conditions in one pill. By treating both conditions at the same time, physicians can help patients reduce their risk of developing cardiovascular disease. In clinical trials of 1600 subjects, about 57% reached both the blood pressure and cholesterol targets for their age. Eli Lilly recently made a similar move with Symbyax, which entered the U.S. market in January of 2004. The drug pairs Zyprexa and Prozac—the latter of which lost its patent protection several years ago.

Lilly is promoting the drug as the first medicine to treat depression. Another drug that may soon emerge on the U.S. market (it was recently approved in Mexico) brings together not only two medicines but also two companies. Merck and Schering-Plough have submitted a New Drug Application for Vytorin, which contains Zocor, Merck's high-selling cholesterol-lowering statin that faces likely generic competition in 2006, and the newer intestinal with Merck. Zetia has been shown to substantially augment statins' cholesterol lowering activity.

Combination drugs are not a completely new concept. 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. Pharmaceutical companies can infuse new life into drugs facing expiring patents. More importantly, this platform also permits pharmaceutical companies to rescue clinical-phase drug candidates discarded from the drug development pipeline due to toxicity, resistance or clinical bioavailability problems. This allows pharmaceutical companies to revive drug candidates from a larger pool of real, efficient and pre-clinically developed drugs for full development into marketable products.

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. In the vast pharmacopeia of traditional medicine prescriptions, 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. It is unfortunate that the pharmaceutical industry has de-emphasized natural products just at the time that new assays are available, and major improvements have been made in detection, characterization and purification of small molecules. In the early to mid-nineties combi-chem companies attempted to fill the void with large numbers of new molecules. Unfortunately, it appears that the chemistry employed did not create sufficiently diverse or pharmacologically active molecules. It is clear that the future success of the pharmaceutical industry product discovery, high throughput screening, synergy drug identification, combinatorial biosynthesis and a systems biology approach.

Recognition of the potential for multipoint intervention in biology and medicine has a long history. As early as 1928, Loewe observed and quantified effects of combinations of compounds that were different from, and not predicted by, the activities of the constituents. The concepts of synergy, additivism, and antagonism have been explored extensively, particularly in the fields of pharmacology and toxicology. Moreover, patients with infectious diseases and with cancer have benefited from combination chemotherapy, where combination drugs are in many cases the standard of care. This clinical experience has led to the testing of combinations of drugs in patients as an explicit strategy for drug improvement by physicians. However, this clinical mixing, and its in vitro surrogate, has generally been conducted with agents already known to be effective in the therapeutic area of interest, or where there is a clear rationale for the combination. Such limited combination testing samples only a tiny fraction of combination space and is unlikely to have resulted in the selection of optimal combinations among the very large number of possibilities. Therefore, a novel drug screening approach based on the multifactorial principle is in urgent need.

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. They aim at non-obvious combinations and the known mechanism of action of the existing drugs has not been taken r fully advantage of. They have to make many initial decisions and assumptions that affect the research direction and potentially the outcome. Thirdly, two variable factors of two drug dosages are hard to get the optimal ratio and have great potential for undesirable side effects. Lastly, phenotype is not synergy effect focused.

To go one step further, 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.

SUMMARY OF THE INVENTION

Methods and compositions for enhancing the efficacy of existing drugs while decreasing the side effects and other bad properties are provided. More specifically, in the given example, the present invention provides methods and compositions for enhancing Ketoconazole . In particular, the present invention provides a method for identifying an agent in a high throughput assay.

The 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.

A case study was given as a proof-of-concept to find synergistic compound of Beauvericin from natural products with ketoconazole. 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.

DETAILED DESCRIPTION OF THE FIGURES

Figure 1. As shown in 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.

Figure 3. In order to show that the ketoconazole/F101604 combination was not toxic to human cells, 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 C0₂ 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 .

Figure 5. Identified chemical structure of Beauvericin from screening for a synergistic co-drug with ketoconazole.

DETAILED DESCRIPTIONS OF THE INVENTION

Definition

"Drug" is defined as commercially used medicines approved by FDA or equivalent authorities in other countries. "Drug candidates" means that the compounds or proteins have not reached the point for approval, but have promising properties or indication for drugs . "Synergy" refers to the effect of the combination of two or more agents is far beyond the addition of those individual results, e.g. 1+1>2. "Additive" means 1+1=2. "Antagonism" means 1+1<2.

"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 (s) 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.

Minimal Inhibitory Concentration (MIC) is defined as the lowest concentration of a drug that inhibits more than 99% of the bacterial population. Table 1. Typical hit rate of synergistic co-drugs as well as additive and antagonistic samples in the same screening.

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. Under this condition, 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. Finally, 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. In an embodiment, 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. the above description composition and a pharmaceutically acceptable carrier. As used herein, for the purposes of this invention, "pharmaceutically acceptable carriers" 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. Typically 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. Using the synergistic compounds screened from above assays as a great tool to discover what kind of proteins and pathways may contribute to a new understanding of a disease. SiRNA and other approaches could be used to identify the mechanism of the synergistic effect. 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.

Algorithm

A clear understanding of the mode of action of one given existing drug and its therapeutic dosage for dramatically decreasing the dosage to a suboptimal level to sensitize and potentiate the cell for screening a synergy partner is necessary. 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, and 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.

A number of algorithms for quantifying synergy in the screening experiments have been implemented. For example, median effect and isobolographic analyses effectively identify combinations ,in which one drug enhances the potency of the other drug, but these models are not appropriate for combinations in which one drug enhances only the intrinsic activity of the other. Clinically and either a shift in potency or an increase in intrinsic activity.

Three standard reference models of additivism were employed to identify synergies. The highest single agent (HSA) model is the larger of the effects produced by each of the combination's single agents at the same concentrations as in the mixture. In contrast, the Bliss additivism model, sometimes also called check board model, predicts the combined response C for two single compounds with effects A and B is:

C = A + B- A*B

Where each effect is expressed as fractional inhibition between 0 and 1. These effect-based synergy models make no assumptions about the functional form of the dose-response curves, and do not require dose-response information that lies outside the range sampled by each screening matrix. The third model, Loewe additivity, measured by the combination index, is dose-based, and applies only to activity levels achieved by the single agents.

Based on the 3 models discussed, a proprietary algorithm was derived. Compound combinations that show excess inhibition at a chosen significance level over a synergy model are selected for follow-up. Such candidates were confirmed by using repeat assays in 384-well plates at higher density concentration coverage, and by using other in vitro and in vivo assays.

The proprietary algorithm is a modification of the above 3 algorithm. It is the first to combine two or more drugs in the form of a co-drug which generates two active drug compounds with improved pharmaceutical properties. Additive drug compounds were specifically ignored in favor of synergistic compounds. Put another way, in additive developments, 1+1=2._ However, in synergistic developments, 1+1>2. Furthermore, this methodology can be applied to rescue promising drug candidates possessing reduce the undesirable effects, dwindling drug discovery pipelines from existing pharmaceutical companies can be revived.

Secondly, 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. As the library of compounds grows, the knowledge of complementary compounds exponentially grows, reducing the cost and time to development of new drugs . Important developments within the FDA with regard to biotech drugs, whereby new compounds generated from previously approved compounds can rely on the data generated by the original manufacturer, have provided an avenue for more cost effective and rapid development of these new drugs .

For proof-of-concept , 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. Systematic approaches were established to maximize the biodiversity of microorganisms within a natural product library are discussed from the following three perspectives: (1) Isolation and selection of samples from diverse ecosystems and isolation of different kind of microbes based on their morphology and 16S rRNA diversity , (2) Manipulating microbial physiology to activate microbial natural product machinery, and (3) Genetically modifying strains for production of unnatural microbial natural products . extract collection can be maximized. In doing so, the chance of finding a novel compound can be increased. An integrated database linking microbial genetic diversity to metabolite diversity will be built (Figure 4) . Once a hit is identified from screening, it could easily trace back to see the resource of the hit. The database helped on dereplicate the hits from reported profiles and focused our efforts to brand new structures and activities of the natural products.

The reason to use 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.

In addition, a documented fungal infection has been identified as an independent risk factor for in-hospital death. Resistance to existing classes of drugs is also on the rise, and since there are limited classes of antifungal drugs available today and few new drugs in development, there is critical need for the development of new approaches. Within microbes' complex biochemical regulatory systems lay the keys to developing entirely new classes of anti-infectives that selectively cripple microbes without harming human cells. Comparative genomics revealed that handful of genes are unique to fungus and served as essential genes.

Many research groups and companies used the protein derived from those genes as the specific target for novel drug screening. However, microbes have gone through 3.8 billion years of the essential genes are impaired, with the ability to respond with versatility to threats to their survival. Unfortunately, they continue to evolve beyond the capabilities of the existing inventory of anti-infective drugs.

The commercial market for antifungal agents is approaching $5 billion worldwide and is one of the fastest growing. The annual market for drugs that treat systemic fungal diseases is approximately $3 billion worldwide. More than 100 companies are engaged in antifungal R&D worldwide. Most available antifungal agents are only fungistatic and do not eradicate the fungus from the system. This has led to the development of resistant strains of fungi. Treatment of drug resistant fungal infection is much more expensive than that of sensitive fungal pathogens. Those agents that are fungicidal are rather toxic and often lead to kidney damage, liver damage and a range of other side effects. These toxic effects lead to the suspension of treatment. Additionally, most antifungal agents which treat systemic infection can only be delivered parentally since oral bioavailability of these compounds is very poor. Thus, there is tremendous medical need for the development of novel compounds to treat this growing problem.

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. Unfortunately none of the currently available antifungal therapeutics meets the majority of these requirements. For example : 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.

Azoles (fluconazole, itraconazole, ketoconazole)

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

The only member of this group is terminating, and 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

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.

Other commercially available drugs like nystatin, Cancidas, and Voriconazole, all had some limitations.

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.

Instead of using high dosage of an existing drug to achieve maximum efficacy, it only used a sub-optimal dose and screened for synergistic partners to enhance the potency. The partner itself may not have any activity alone at that concentration. For proof-of-concept, 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.

Therefore, 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. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.

EXAMPLE 1

Since the introduction of highly active antiretroviral therapy (HAART) in the mid 1990s, the HIV antifungals market has undergone tremendous change. Prior to the HAART era, the incidence of AIDS and HIV-related opportunistic infections was increasing at an alarming rate. Frequent recurrent fungal infections were common, often requiring chronic prophylactic therapy that contributed to widespread concern among physicians about antifungal drug resistance. Particularly, HIV specialists and researchers were greatly concerned about the increasing prevalence of azoles-resistance Candida. A solution to this problem, they believed, was the most important unmet need in HIV opportunistic fungal therapy. Azoles resistance had increased primarily because of the chronic therapy required to combat recurrent infections in patients with declining immune function. With the advent of HAART, immune reconstitution in HIV-infected patients has increased and the incidence of opportunistic infections has declined by as much as 60-80%. However, it is believed that resistance to HAART is virtually inevitable. Currently, patients who fail one HAART regimen can be successfully treated with second-, third-, and fourth-line regimens .

However, resistance to HAART therapy is common. Clinicians estimate that, in any given year, 10-30% of patients exhibit resistance to antiretroviral therapy that requires a change of regimen or addition of a drug. If the antiretroviral drug pipeline does not maintain a supply of novel agents that can compensate for HAART failure, a resurgence of opportunistic increase will occur, but it is likely to be the single most important factor in the HIV market over the next ten years and will substantially affect demand in the antifungals market. Moreover, this factor will impact the future market shares of emerging antifungals, determining if they occupy niche positions or attain wider use. On the other hand, that among non-HIV hospitalized patients at high risk for fungal infections—e . g. , cancer and transplant patients—Candida resistance is a growing concern.

There is an increasing need for safer and more effective antifungal agents. Some of the more effective antifungal agents, Amphotericin B and the azoles (e.g. Fluconazole, Itraconazole and Ketoconazole) 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. Additionally, 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. In particular, 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. According to the CDC, 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. Taken together, this information demonstrates the significant medical and economic importance of Candida albicans pathogenesis . 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.

The 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.

Two methods were used to generate the crude extracts. For liquid fermentation samples, the cells (about 40 ml volume) were centrifuged and decant off the supernatant from the pellet into a separate 50 ml centrifuge tube. The cell pellet is then extracted with 20 ml of methanol . The supernatant is processed by adding approximately 2 ml of HP20 resin. Both sets of tubes are placed onto a shaker for one hour. The HP20 resin is combined with the methanol supernatant from the pellets and then dried overnight. The HP20 is then rinsed with DI water to remove highly polar compounds and eluted with 9:1 acetone/water. The samples are dried and redissolved in 100% DMSO. This sample is the 20X stock to be diluted for screening. For solid stationary samples, 40 ml of methanol is added and the mixture incubated overnight. 2ml of HP20 resin is mixed with the supernatant. The rest of the steps are identical to the treatment of liquid samples.

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.

Two microliter (2 ul) of 2OX natural extract samples and controls from master plates are added to the cell culture using a 96-pin replicator. All tests were done in duplicate following National Center for Clinical Laboratory Standards recommendations. The assay plates are incubated overnight at 35° C in a moistured chamber and the fluorescence reading is measured at Ex 544 nm and Em 590 nm^', A pilot screen from the natural product samples was performed to look for hits which synergistically increased the potency of ketoconazole, which₍ has no antimicrobial activity by itself .

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 .

The concentration of ketoconazole alone at 0.01 x only gave about 20% inhibition of growth and the mode of action was static. When the F101604 extract was tested alone, no inhibition of the yeast pathogen was observed. When ketoconazole was tested at 1 X, it gave 90 % inhibition of growth. However, 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.

Therefore, 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.

This proof of concept shows the promise and potential for the technology platform of the present invention to work in many therapeutic fields.

EXAMPLE 2

Pharmaceutical Compositions The present invention 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. In additional embodiments, 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.

After 16s rDNA sequencing, the production strain was identified as Fusarium proliferatum. The natural product from the broth was isolated and purified by the following purification procedure:

1. Add 100 ml of Acetone into each flask, total 36 flasks. 2. Stir to break the mass nto pieces and allowed to stand for at least 4 hours.

3. Filter to separate the biomass from acetone solution.

4. Gather all the biomass in a proper size of container and add 2 Liter of Acetone to extract for 2 hours . 5. Filter and combine two Acetone extractive solutions.

6. Remove the acetone by evaporation under vacuum (about 500 ml left)

7. Add 500 ml of distilled water

8. Extract by Liquid-Liquid partition with solvent MEK (1:1) , do twice

9. Concentrate the MEK extractives, respectively, down to dryness.

10. Kept in the refrigerator.

The hit is identified as a cyclic hexadepsipeptide Beauvericin (SZC-101) by LC-MS-MS and NMR study from Fusarium proliferatum broth mixture .

Identifiers: Beauvericin Synonyms cyclo (D-α-Hydroxyisovaleryl-L-N-methyl- A Phe) ₃ i

Molecular C₄₅H_{5 3}θ₉ Formula

Molecular 783.95

Weight Enlarge CAS Number 26048-05-5

MDL number MFCD00056846

See Figure 5. secondary metabolites that in small concentrations are toxic to vertebrates and other animals when introduced via anatural route" . These compounds are usually non-volatile and may be sequestered in spores and vegetative mycelium or secreted into the growth substrate. The mechanism of toxicity of many mycotoxins involves interference with various aspects of cell metabolism, producing neurotoxic, carcinogenic or teratogenic effects. Other toxic fungal metabolites such as the cyclosporins exert potent and specific toxicity on the cellular immune system, which was developed as an immunosuppressant agent .

In order to show that the 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.

It's important to know if the hits profile is universal or specific for one class of anti-fungal drug. Assays for other common anti-fungal drugs, like Fluconazole, Itraconazole, Amphotericin B, Flucytosine, Cancidas, Voriconazole and Terbinafin were tried. It looks like that Beauvericin is specific to azoles drugs. the preliminary data report, other fungal pathogens, including Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida Krusei (Issatchenkia orientalis) ATCC 6258, Aspergillus fumigatus ATCC 46645, Saccaromyces cerevisiae ATCC 2601, and Cryptococcus neofor ans ATCC 14116, were also used. 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. Preliminary data showed that the synergy co-drugs dramatically improved the efficacy of ketoconazole on fluconazole resistant clinical isolate.

Azoles resistance in the pathogenic yeast Candida albicans is an emerging problem in the human immuno-deficiency virus (HIV) - infected population. Statistic data showed that more than 33% of isolates from patients with AIDS have MICs of fluconazole at least 3 fold greater than that of standard susceptible strains. A fluconazole resistant clinical isolate #17 (SZP-17) was purchased from Dr. Theodore C. White's lab in the University of Washington. Bioactive compound SZC-101 was fractionate and purified from natural product mixture F101604 that synergized the activity of ketoconazole on fungal pathogens. 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)

Other secondary assays include checking the serum binding activities, the mammalian cell toxicity assays, etc. Profiling the compounds using the assays is an important step to turn hits to leads . The function of the combination therapy in animal model or clinical trial with a comparable single drug treatment was also tested. The novel compounds isolated could serve as a tool in combination with DNA (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. Thus, the antibiotic is protected e.g. Augmentin (amoxicillin and clavulanic acid).

2) : Membrane Barrier Potentiators

Failure of the antibiotic to reach its target usually reflects the inability of the antibiotic to pass through the cell wall, either to the periplasmic space or into the cytoplasm of the cell. Thus, compounds that increase the permeability of these membrane barriers may synergistically enhance the activity of a drug e.g. gentamicin can increase the permeability of Beta lactam agents to the Enterococcus .

3) : Multidrug Resistance Pumps (MDR) Inhibitors

Pumps in microbial membranes can translocate and pump out antibiotics from the cell. Inhibiting these pumps may also synergistically enhance the activity of a drug. Many MDR inhibitors have been discovered but no MDR inhibitor is on the market. 2. The combination of ketoconazole and SZC-101 prevents dye efflux, as one of the mode of actions for the synergy effect. To test the effect of SZC-101 and ketoconazole on multidrug resistance pumps of strain SZP-17, dye efflux experiments were performed. 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.

4): Hitting Other Synergistical Pathways. It 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.

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.

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.

Although death has historically been used as an end point for studies of this type, this end point is no longer suitable in the current era of animal research. In an attempt to modify the study methodology to contemporary standards, the approach suggested by Dr. T.E. Hamm (Proposed institutional animal care and use committee guidelines for death as an end point in rodent studies, AALAS Contemporary Topics. 1995; 34:69-71) was adopted. Although these experiments may lead to death attempts were made to lessen the duration of pain and suffering by utilizing the following approach:

1) Animals were monitored thrice daily (at approx. 9AM, 12N, and 5PM) by members of the study team which have been trained and are experienced in recognizing the signs of illness and abnormal schedule is in excess of the initial twice-daily approach suggested by Dr. Hamm (1) . In addition, more frequent monitoring may be required if the mice are found to be experiencing pain, distress or death.

2) Animals, which appear to have substantial alterations in, posture (e.g., abnormal posture or head tucked into abdomen), coat, exudate around eyes and/or nose, breathing or movement are be removed from the group-housing situation. Analgesics were not administered to these animals because the possibility of drug- drug interactions and its influence on the outcome of the study is unknown. Instead, these animals were euthanized by C02 exposure followed by cervical dislocation in an area separate from the housing area.

The term mortality has been used as end point for this study; however, it should be clearly understood that when possible every attempt was made to minimize pain/suffering and that the animals would be euthanized prior to naturally succumbing to infection if this is observed. For the purposes of this study whether an animal dies due to the natural infection process or is euthanized, both were considered the same end point for experimental and statistical purposes.

a. Animals: 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.

b. 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.

c. Isolate: A single Candida albicans (ATCC 36082) or a drug- resistant clinical isolate supplied by SynerZ was used to conduct all the studies.

d. Susceptibility Testing: The minimum inhibitory concentration (MIC) of all test compound (s) was determined for the test organism using a standard NCCLS microdilution technique.

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.

i. 5 mice for each of the 5 dosages per test compound (and combination with low dosage of fluconazole) * 5 compounds (125 mice) . ii. 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. iv. 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. v. Study termination: Animals were euthanized on Day 4 (~72 hours post exposure) by a lethal exposure of C02 followed by cervical dislocation in an area separate from the housing area. compound = 130 mice.

f. Confirmation studies: Prior to infection, mice were rendered neutropenic by injecting cyclophosphamide intraperitoneally 4 days (150 mg/kg) and 1 day (100 mg/kg) before inoculation. The test organism was subcultured onto Sabouraud dextrose agar for 24 h at 35°C. Groups of mice (5 per group) were infected with 0.1 ml of a 106 CFU/ml inoculum suspension in warmed saline (35°C) via the lateral tail vein. This inoculum has reproducibly established infection with the proposed methodology in previous studies. Confirmation of infection was determined in duplicate for final isolate candidates. Calculation of mice required: (5 animals/group x 1 inoculum x duplicate) = 10 mice per isolate.

Quantitative efficacy studies

Comparative efficacy as assessed by changes in fungal density in the kidneys of infected mice after treatment was undertaken for each test compound (s) either alone or in combination. Six to eight dosage regimens per compound were studied in groups of 3 mice over a 24 h period. Control animals received synergy co- drug-free vehicle in the same volume as treatments. A group of untreated controls was sacrificed just prior to initiation of treatment, to confirm infection was established, and at the end of the treatment period for each set of treatment groups completed. After sacrifice by C02 inhalation, kidney tissue was removed and homogenized in sterile 0.9% saline. Serial dilutions of the homogenate were plated onto SDA for determination of viable fungal counts after 24 hours of incubation at 35DC.

1. 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.

3. 24 [3 mice per dosage regimen * 8 regimens * 1 comparator] mice were utilized and drug administered by the IP route 2 h after inoculation.

4. 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.

5. 72 [(3 Oh control + 3 24h control) * 12 sets of treatment groups] 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.

6. Total animal requirement: 216 mice h. Data Analysis: Sample size

While no a priori assessment of sample size has been calculated for the acute toxicity studies because these investigations have not been previously conducted, a sample size of n=5 is consistent with industry standards and thus has provided sufficient information for a preliminary evaluation of the test compound.

The sample for the quantitative culture section was calculated as follows: 1) for typical antimicrobial agents optimal dosing regimen usually produces approximately 2-3 log decrease in fungal density with %CV of 40, 2) in order to have an observed mean which deviates from the true mean by no more than 1 SD using a two side 95% confidence interval with 80% probability, n=6 data points are required. Using the proposed methodology of this assessment of the kidney tissue.

Quantitative culture studies , 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.

Claims

1. 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 enhance the efficacy of the known drug.

2. The method of claim 1, further comprising purifying the identified compound.

3. The method of claim 1, wherein the library includes small molecules as well as macromolecules, such as nucleic acids or proteins, including SiRNA libraries.

4. The method of claim 1, wherein the hit rate for synergistic lead compound is 0.1% to 1%.

5. The method of claim 1, wherein the co-drug enhances the efficacy of the known drug by 10% - 40% at low dosage levels.

6. The method of claim 1, wherein the assay is any biochemical binding assay or enzymatic assay.

7. The method of claim 6, the assay is any cell or animal model based biological assay.

8. The method of claim 6, the assay could be clinical trial related to any disease models .

9. The method of claim 1, wherein the screen step (d) can be performed manually or using a robotic.

10. The method of claim 1, wherein the identifying step (e) can be performed by a reporter gene assay, cytoblot assay or microscopic assay.

11. The method of claim 1, wherein the sub-optimal dosage in step (c) is determined using a software and algorithm.

12. The method of claim 10, wherein the sub-optimal dosage is used to guide formulation manipulation.

13. The method of claim 10, wherein 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.

14. The method of any of claims 1-12 wherein the compound was not previously known or not reported as the specific application this method identified.

15. The compound(s) identified by the method of claim 13.

16. A composition comprising the compound identified by the method of any of claims 1-12, the known drug or dropped drug candidate and an acceptable carrier.

17. The suboptimal level of the known drug determined by the method of any of claims 1-12.

18. A composition comprising the suboptimal concentration of the known drug or dropped drug candidate determined by any of claims 1-12 and an acceptable- carrier . identified as a synergistic drug for ketoconazole and other azoles drugs .

20. A pharmaceutical composition or formulation comprising the composition of claim 15.

21. Uses of the compound identified by the method of any of claims 1-3 for identification of a protein or pathway which is related to a disease.