WO2008046045A1 - Drug biovailability screens - Google Patents

Abstract

Provided herein are methods for ranking compounds by predicted bioavailability, methods for predicting bioavailability, and kits for predicting bioavailability of compounds.

Description

DRUG BIOAVAILABILITY SCREENS

Field of Invention

The invention is in general directed to methods for ranking compounds by predicted bioavailability, methods for predicting bioavailability, and kits for predicting bioavailability of compounds.

Background of Information

Discovering and developing new drugs is a complex and expensive process, which often ends in failure when a once promising drug candidate does not perform as expected in clinical trials, or is rejected by regulatory agencies. One reason these drug candidates fail is because of inappropriate bioavailability. That is, although the compound may perform in in vitro experiments, or in vivo at high dosages, the amount of the compound that is able to reach the site of action may be too low. The half life of the compound also may be too short, requiring multiple daily dosing, or too long, which can result in toxicity. In the early stages of drug discovery, hundreds to thousands of compounds may be identified as possible drug candidates based on their binding and/or enzymatic activity on a drug target. Early screening of these compounds for bioavailability allows researchers to select the proper compounds to develop and eventually use in clinical trials.

The term "bioavailability" refers to the extent to which, and sometimes the rate at which, the active moiety of a drug or metabolite enters systemic circulation, thereby gaining access to the site of action. Medications that are administered intravenously are considered to have 100 percent bioavailability, that is, the complete dose of the medication reaches the systemic circulation. But drugs that are administered through other routes, such as oral, subcutaneous, nasal, and rectal routes, generally do not have 100 percent bioavailability because drugs have various degrees of absorption. Routes other than intravenous routes, such as oral delivery, may result in incomplete absorption of drugs. When a drug is administered orally, before reaching the vena cava, it must move down the gastrointestinal tract and pass through the gut wall and liver, which are common sites of drug metabolism. This "first-pass metabolism" may metabolize the drug before it can be measured in the systemic circulation. Drugs such as isoproterenol, norepinephrine, and testosterone have extensive first-pass metabolism, and their bioavailability through the oral route is virtually zero. Another cause of low bioavailability is insufficient time in the gastrointestinal (GI) tract.

If a drug does not dissolve readily, or cannot penetrate the epithelial membrane, the time at the absorption site may be insufficient. Other reactions that compete with absorption can reduce bioavailability. These include complex formation (e.g., between tetracycline and polyvalent metal ions), hydrolysis by gastric acid or digestive enzymes (e.g., penicillin and chloramphenicol palmitate hydrolysis), conjugation in the gut wall (e.g., sulfoconjugation of isoproterenol), adsorption to other drugs (e.g., digoxin and cholestyramine), and metabolism by luminal microflora.

Traditionally, bioavailability determination from plasma concentration-time data usually involves administering the compound to a human or other animal, withdrawing blood at certain times, and determining the maximum (peak) plasma drug concentration, the time at which maximum plasma drug concentration occurs (peak time), and the area under the plasma concentration- time curve (AUC). The plasma drug concentration increases with the extent of absorption; the peak is reached when the drug elimination rate equals the absorption rate. Because drug elimination begins once the drug enters the bloodstream, however, determining bioavailability solely based on peak plasma concentration may be misleading. Peak time is another method used to determine absorption rate, and slower absorption rates have later peak times. Therefore, researchers often select AUC as a more reliable measure of bioavailability. For general information about bioavailability, see The Merck Manual of Diagnosis and Therapy 17^th Ed. (1995) (Section 22, Chapter 298, "bioavailability" entry).

Summary

There is a need for reliable in vitro methods that (a) more closely predict bioavailability, and (b) can be utilized readily to rank various compounds based on bioavailability, such as in a high- or medium-throughput screening process. An in vitro method for predicting bioavailability, and ranking compounds based on bioavailability, allows for (a) more efficient selection of compounds for clinical development and (b) more rapid introduction of new drugs to patients. The present invention satisfies these needs and provides additional advantages. Provided herein are methods for ranking compounds by predicted bioavailability, methods for predicting bioavailability, and kits for predicting bioavailability of compounds. The methods and kits of the present invention are useful for selecting compounds for clinical studies. Thus, featured herein are methods for ranking compounds by predicted bioavailability, comprising determining membrane permeability of a set of compounds in vitro, determining stability of the compounds in vitro, and ranking the compounds for predicted bioavailability according to the permeability and stability. Also featured are methods for predicting bioavailability of a compound, comprising determining membrane permeability of a compound in vitro, determining stability of the compound in vitro, and predicting the bioavailability of the compound according to the membrane permeability and stability determined for the compound. Also featured are methods for ranking compounds, and predicting bioavailability, across the blood-brain barrier. Bioavailability, for example, may be oral bioavailability, it may also, for example, refer to bioavailability after other modes of administration. Membrane permeability, may be, for example, determined by detecting the amount of compound on one or both sides of a membrane. In some examples, the membrane is a lipid membrane. In certain examples, the lipid membrane is a monolayer. In other examples the lipid membrane is selected from the group consisting of a bilayer, a micelle and a liposome. The membrane may, for example, comprise hexadecane. The membrane may, for example, comprise a lipid selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidic acid or a combination of the foregoing. The membrane may, for example, comprise a combination of lipids in ratios present in a plasma membrane of a cell. Where the bioavailability of the compound in the brain is to be analyzed, the membrane may, for example, comprise a combination of lipids in ratios present in a blood-brain barrier (e.g., Di et al. , Eur. Journal of Medicinal Chemistry (2003) 38: 223-232 and Bickel, /. Am. Soc. Experimental N euroTherapeutic s (2005) 2: 15-26). The membrane may be, for example, associated with a solid support.

In some examples, membrane permeability and stability of a compound are determined in parallel, in other examples, these are determined in series. In some illustrative examples, membrane permeability and stability of a compound are determined in a single vessel. That is, for example, in one portion of the vessel, the "donor" portion, the compound is added. If the compound crosses over a membrane barrier into the "acceptor" portion of the vessel, which comprises other assay components (e.g., metabolic stability components), the compound is thus assayed for metabolic stability and permeability, and bioavailability can be assessed.

In some examples, the membrane may comprise a protein pump, for example, a P-glycoprotein pump, to assess permeability properties of the compound other than passive diffusion.

To measure metabolic stability, in some examples, the compound is contacted with acidic pH conditions or basic pH conditions. In some examples, the stability of a compound is determined by the amount of modified compound and/or unmodified compound present after the compound is contacted with a substance selected from the group consisting of a cell, a cell component, blood, a blood fraction, a blood component and a digestive fluid, or a combination of the foregoing. The substance may be, for example, from a mammal, for example, from a human, rat, mouse, dog, or primate. Examples of such substances include, for example, microsomes, for example liver microsomes, liver cells, plasma, isolate liver cytosol, and enzymes. One or more isolated enzymes, for example, may be included in the stability assay. The enzymes may, for example, comprise one or more liver enzymes (e.g., liver cytosolic enzymes). The one or more enzymes may, for example, by cytochrome p450 enzymes, such as, for example, a CYP3A4 enzyme, CYP2D6 enzyme, CYP2C9 enzyme and CYP 1A2 enzyme, or a combination of the foregoing. Also included are oxidase enzymes, such as aldehyde and/or xanthine oxidases, for example. Where the stability assay includes a digestive fluid, the fluid may, for example, be a simulated digestive fluid, such as, for example, a simulated gastric fluid or a simulated intestinal fluid.

In certain examples, the method comprises assessing the protein-binding capability of the compound. Thus, in these examples, the method comprises contacting the compound with a protein, determining the amount of compound bound to the protein, and predicting the bioavailability of the compound according to the membrane permeability and stability determined for the compound and the amount of compound bound to the protein. In some examples, the protein is in association with a solid support, such as, for example, in association with a solid support via a biotin/avidin or streptavidin binding pair. In certain examples, the method comprises determining the oral bioavailability of the compound in vivo. For example, oral bioavailability may be determined in a mammal, for example, in a human, rat, mouse, dog, or primate. In certain examples, the compound tested is a cytotoxic agent.

Kits also are provided in the present invention. For example, provided is a kit that comprises a porous solid support, one or more membrane-forming components, and a substance selected from the group consisting of a cell, a cell component, blood, a blood fraction, a blood component, a simulated digestive fluid, a protein, a nucleic acid that encodes a protein, a compound having a known bioavailability and a liquid-dispenser, or a combination of the foregoing. Examples of such substances include, for example, microsomes, for example liver microsomes, liver cells, plasma, isolate liver cytosol, and enzymes. One or more isolated enzymes, for example, may be included in the kit. The enzymes may, for example, comprise one or more liver enzymes. The one or more enzymes may, for example, by cytochrome p450 enzymes, such as, for example, a CYP3A4 enzyme, CYP2D6 enzyme, CYP2C9 enzyme and CYP1A2 enzyme, or a combination of the foregoing. The substance may be, for example, from a mammal, for example, from a human, rat, mouse, dog, or primate. Where the kit comprises a digestive fluid, the fluid may, for example, be a simulated digestive fluid, such as, for example, a simulated gastric fluid or a simulated intestinal fluid. In some examples, the substance is frozen. The kit may comprise a protein pump, such as, for example, a P-glycoprotein pump. The kit may further comprise instructions for predicting or ranking bioavailability of a compound in vitro. The kit may further comprise reference compounds that may be used to compare results with the assay compounds.

Also provided are compositions, methods and kits for readily determining logP, logD and pKa parameters of multiple compounds. These parameters can be determined for multiple compounds at a time and the parameters can be determined simultaneously from one data set. For example, provided are methods for simultaneously determining partition coefficient, distribution coefficient and ionization constant values for one or more compounds, which comprise: (a) contacting one or more compounds with multiple solutions, where: the pH of each of the solutions is different, and each of the solutions comprises a first phase and second phase immiscible with the first phase; and (b) determining the distribution coefficient for each of the one or more compounds in each of the solutions; whereby the partition coefficient and ionization constant values for each of the one or more compounds is determined from the distribution coefficient. These and other embodiments are described further in the following Detailed Description, Claims and Drawings.

Brief Description of the Drawings

Figure 1 shows the relationship between the Distribution coefficient (D) and pH for a particular compound.

Figure 2 shows an equation that can be used to determine permeability rates (Pe) in a lipid-PAMPA assay. Log Pe can be calculated from the equation as reported by Faller et al. (Wohnsland, F. and Faller, B., J. Med. Chem., 2001; 44, p. 923-930).

Detailed Description Determining the bioavailability of a drug is important at various stages of drug development and administration. Bioavailability often is determined when selecting compounds for clinical studies, for submission to government regulatory agencies, for determining dosage in patients, when comparing new drugs to known drugs, as well as generic drugs to known drugs, for developing formulations, and when switching a patient from one drug or drug formulation to another. Bioavailability often is determined when assessing the possible toxicity of a drug: if a compound is absorbed too quickly, or administered at a dose too high for its absorption level, it could reach toxic levels. Conversely, if a drug is absorbed too slowly, or given at too small a dose relative to its absorption level, it could be ineffective.

Developing new compounds to the stage where they are entered into preclinical studies involves screening numerous test compounds, hundreds, thousands, millions, for certain biological and pharmaceutical properties. Bioavailability is one important factor for determining which compounds to take even farther into the clinic. If a drug has an inappropriate bioavailability, either too low or too high, the investment in developing this particular compound may then be lost. To decrease the risk that a compound that passes the initial stages of drug discovery, later fails in the clinic, researchers seek in vitro methods of determining bioavailability. These methods, including computational prediction based on compound chemical structure, and various in vitro and animal studies, provide additional information researchers can use to select optimal candidates for clinical studies.

Thus, provided herein are methods for ranking compounds by predicted bioavailability, methods for predicting bioavailability, and kits for predicting bioavailability of compounds. As used herein, the term "bioavailability" refers to the availability, amount (e.g., concentration), or pharmacological activity of a drug in a biological fluid, cell, or tissue, e.g., blood, serum, cerebrospinal fluid, or brain in a mammal, e.g., a human.

Certain terms utilized herein are defined hereafter.

ADME Screening

The term "ADME," for "Absorption, Distribution, Metabolism and Excretion," refers to preclinical studies of compounds to help select promising compounds for the clinic. The term refers to assays that measure the absorption, distribution, metabolism, and excretion of these compounds. A screening strategy based upon ADME properties may be utilized to reduce the number of compound candidates for use in clinical studies by selecting those that have the most promising bioavailability. For example, more than 500 compounds can be selected for their binding and/or enzymatic activity against a drug target. These compounds then may be subjected to solubility screening, for example, using an HPLC-UV assay, and are screened for permeability, for example, using a PAMPA assay. This methodology will reduce the number of appropriate compounds (e.g., less than 50). These compounds then can be screened for metabolic stability, for example, using rodent or human liver microsomes, LC-MS/MS, followed by screening for CYP3A4, CYP2D6, and CYP2C9 inhibition, for example, using cDNA- expressed enzymes and/or human liver microsomes, LC-MS/MS. This next level of assays may, for example, reduce the number of promising compounds to less than 5. This reduced number of compounds then may be subjected to pharmacokinetic (PK) studies in rodents. Reducing the number of promising compounds to less than 5 in this example may significantly reduce the cost and time involved in these animal studies. Methods and kits provided herein are useful for ADME studies.

Bioavailability Bioavailability of a compound generally is predicted as a function of membrane permeability and one or more stability factors. For example, bioavailability sometimes is estimated by the product of the membrane permeability and a stability factor. Bioavailability sometimes is predicted by comparing the membrane permeability and stability determined for the compound to the membrane permeability and stability determined for another compound for which bioavailability has been determined in vivo. Solubility Screening

Solubility screening may be performed under different pH conditions. The pH selected can be the pH of a particular region of the digestive tract. For example, the pH in the ileum ranges from about pH 6.8 to about pH 9.5 in a human fasted state and a fed state. Compounds selected in solubility screening assays sometimes are those soluble at the pH of the ileum.

Permeability Screening

The absorption of compounds and permeability through membranes of the gastrointestinal tract, and the blood-brain barrier may be measured using permeability screening. Various assays are designed to measure different pathways of permeation. The majority of drugs enter the bloodstream via passive diffusion, therefore it is desirable to use permeability assays that are designed to measure passive diffusion. These permeability assays include, for example, use of lipid membranes. One membrane based passive diffusion assay uses a hexadecane filled membrane, another, the Parallel Artificial Membrane Permeability Assay (PAMPA) measures the in vitro permeability of compounds across artificial phospholipids membrane barriers, this artificial barrier is supported by a high porosity microfilter.

Permeability may, for example, be represented as "effective permeability," also known as "Pe" and sometimes expressed as "logPe," which can be determined by a time course assessment of the amount of compound that crosses the membrane as a function of time. Permeability often is a determination based on passive diffusion across a membrane. In certain embodiments, permeability is reflected by the fraction or percentage of compound that crosses the membrane in vitro.

The lipid membrane often is a monolayer, and can be a bilayer, micelle or liposome in certain embodiments. In micelle and liposome applications, the amount of compound may be detected inside the lipid structure to determine permeability. The lipid membranes often are artificial, and are prepared in vitro by combining isolated preparations of one or more membrane-forming molecules. Examples of membrane forming molecules include alkyl molecules (e.g., C-10 to C-24 alkyl molecules (e.g., hexadecane)) and lipids (e.g., phospholipids, sphingolipids and the like). A hexadecane method is a basic method that does not include specific lipids (5% hexadecane in hexane). Briefly, a hexadecane mixture is transferred onto a 96-well plate having supported membranes, the membrane is allowed to dry for 1 hr in a fume hood to ensure complete evaporation of hexane, which results in a uniform layer of hexadecane on the membrane. A Lipid-PAMPA method can include depositing a lipid (e.g., L-a- phosphatidylcholine (lecithin)) or a lipid mixture. Briefly, a 4% solution of lecithin in dodecane is sonicated to ensure complete dissolution and the sonicated mixture is carefully pipetted (5 uL) into each donor plate well. A drug/buffer solution is added almost immediately to the well with an artificial membrane to avoid oxidation of the lipid monolayer or bilayer. These hexadecane and lipid-PAMPA methods are designed to assess drug passive transport, mimicking plasma membranes of intestinal enterocytes. In addition to studying permeability in the gastrointestinal tract, membrane permeability may be used to study permeability across the blood-brain barrier or simulated blood-brain barrier. For studying blood-brain barrier passive permeability, a porcine polar brain lipid (PBL) composition may be utilized. One example of a PBL composition includes the following: phosphatidylethanolamine (e.g., about 33.1 %), phosphatidylserine (e.g., about 18.5 %), phosphatidylcholine (e.g., about 12.6 %), phosphatidic acid (e.g., about 0.8 %), and phosphatidylinositol (e.g., about 4.1 %).

Suitable membranes for these assays are often formed on a porous solid support. The solid support sometimes is referenced herein as a "membrane" and can be constructed of any suitable material, such as PVDF. For example, a polycarbonate membrane having about 20% porosity can be utilized as a support for such membranes as a hexodecane/hexane PAMPA membrane. Additional information on permeability assays and PAMPA may be found on the Worldwide Web at, for example, Millipore.com/publications/nsf/docs/anl729en00, application note AN1729EN00.

As the pH in the gastrointestinal tract varies, in order to more closely mimic in vivo absorption, permeability assays (e.g., PAMPA assays) sometimes are performed at the pH found in the ileum, between 6.8 and 9.5.

A comparison of various permeation assays, and examples, may be found in, for example, Balimane, et al., AAPS Journal (2006) 8(1) Article 1 El.

An in vitro cultured cell assay may be utilized to measure permeation via efflux, and may, for example, be used as a secondary assay after, for example, a membrane based assay. For example, a secondary assay can assess permeability in Mardin-Darby canine kidney (MDCK) cells as known in the art (e.g., Balimane et al, AAPS J. (2006)

8(1): Article 1 (http address www.aapsj.org) and Youdim et al, Drug Discovery Today (2003)

8(21):997-1003).

Active Transport While assays such as PAMPA measure passive diffusion across lipid bilayers, active transporters also can be important in selective accumulation and distribution of drugs into target organs. Therefore, in certain embodiments, the membrane includes an efflux pump. An example of an efflux pump is a P-glycoprotein pump. P-glycoproteins (P-gp) are active plasma membrane transporters involved in drug pharmacokinetics and cellular detoxification. P-gp is known to impact the ADMET characteristics of drug molecules. P-gp exhibits a high drug-dependent ATP hydrolysis activity that is a reflection of its drug transport ability. The test of drug stimulation or inhibition of ATPase activity may be used to screen the potential drug interaction with P-gp.

P-gps are present on the apical surface of the enterocytes, the canicular membrane of hepatocytes, and on the apical surface of kidney, placenta, and endothelial cells of the brain membrane. Oral bioavailability of certain drugs may increase when P-gp inhibitors are coadministered, which implicates P-gp as a permeability barrier. Also, P-gp at the blood-brain barrier limits the entry of drugs into the brain. P-gp membranes can be isolated as P-gp-enriched vesicle preparations. P-gp ATPase activity can be measured by a spectophotometric method based on continuous monitoring of ADP formation, in the vesicle suspension medium (e.g., a P-gp kit from SPI-BIO, Catalog no. 789201 (France) can be utilized).

Because PAMPA assays may underestimate the absorption of compounds absorbed by active transporters such as, for example, P-gp, in certain embodiments, the effect of active transport is measured along with permeability.

Metabolic Stability

Orally administered drugs are transported to the liver, where they then are metabolized and excreted either as bile or through the kidneys. Early studies on metabolic stability of compounds allow selection of the compounds with the desired half lives for future clinical trials. If a compound has too short a half life, then it may need to be administered many times each day, or administered using continuous infusion. If a compound has too long of a half life, however, the chances of toxic side effects increases. Because the liver is the site of major drug metabolism, many in vitro assays involve liver tissue, for example, liver microsomes or intact hepatocytes. In certain embodiments, metabolic screening assays involve incubation of the compound with liver microsomes or intact hepatocytes, typically in a multi-well, for example 96- well, plate, followed by analysis of the amount of compound that remains intact after incubation (e.g., LC-MS quantification).

Metabolic stability also is a factor before a compound reaches the liver. Thus, a compound may be incubated with digestive fluids, or simulated digestive fluids, followed by analysis of the amount of compound that remains intact after incubation (e.g., Fu et ah, /. Agricultural Food Chem. (2002) 50(24): 7154-60).

Examples of metabolic stability assays are presented in the Examples section hereafter and in Bjornsson et ah, Drug Metabolism Disposition (2003) 31(7): 815-832 and Walsky & Obach, Drug Metabolism Disposition (2004) 32(6): 647-660.

Cytochrome P450 System The cytochrome P450 family of enzymes is a set of enzymes that metabolize drugs and toxins. They are responsible for Phase I oxidative metabolism of these compounds, mainly in the bowel wall and the liver. Each of the enzymes is primarily responsible for metabolizing certain drugs and toxins. Substrates, inhibitors, inducers, and other components and properties of the cytochrome P450 system are known to the person of ordinary skill in the art. To determine whether a compound has the desired stability, and neither too short nor too long a half-life, it is useful to assay stability in the presence of one or more cytochrome P450 enzymes.

In selecting compounds for clinical studies, it often is informative to examine how the compound will be metabolized, and to identify the P450 enzyme that may be responsible for metabolizing the compound. Also, some individuals have deficiencies in one or more of the enzymes, and a treatment plan for that individual could be modified if it is known that a certain compound is targeted by that deficient enzyme. For example, a drug may be ineffective in an individual that lacks enzymatic activity needed to convert the drug into an active form. Or, a drug may be toxic in an individual that has a defect in one of the detoxification enzymes. Thus, in certain embodiments, compound stability is determined by the amount of modified compound and/or unmodified compound present after the compound is contacted with one or more enzymes selected from the group consisting of a CYP3A4 enzyme, CYP2D6 enzyme, CYP2C9 enzyme and CYPl A2 enzyme, or a combination of the foregoing.

Predicting Bioavailability Using Membrane Permeability and Metabolic Stability Assays In certain embodiments of the present invention, compounds are tested for both membrane permeability and stability, and the compounds then are ranked for predicted bioavailability according to the permeability and stability. In certain embodiments of the present invention, the compounds are tested for membrane permeability and stability sequentially, either being tested first for permeability, and second for stability, or the reverse. For example, the compounds may be first tested for permeability using a PAMPA assay, and then tested for stability in liver hepatocytes. Or, multiple permeability assays, or multiple stability assays may be conducted. The permeability assay membrane may, for example, comprise a protein pump, for example, a P-glycoprotein pump. In other examples, both a hepatocyte stability assay, and a simulated gastric fluid stability assay may be conducted. Different aliquots of compound preparation may, for example, be used for each of the assays. In certain illustrative embodiments of the present invention, the permeability and stability assays are performed simultaneously, in the same vessel, well, tube, container, or the like. For example, in a multiwell filter plate that may be used for a membrane permeability assay, such as, for example, a PAMPA assay, compounds are added to a donor side of the membrane, and diffusion across the membrane is measured by detecting the compound on the acceptor side of the membrane. In this illustrative embodiment, reagents used for a stability assay are present on the acceptor side of the membrane, thus allowing simultaneous determination of membrane permeability and metabolic stability of a compound. The membrane may, for example comprise a protein pump, such as, for example, a P-gp pump. The acceptor side of the membrane may, for example, comprise liver microsomes, or simulated gastric fluids (e.g., the latter also may be present on the donor side). In some examples, the acceptor side of the membrane may comprise one or more cytochrome p450 enzymes. Thus, provided herein are compositions and kits comprising a membrane, a solid support in association with the membrane and one or more other components described herein, including but not limited to a cell, a cell component, blood, a blood fraction, a blood component, a simulated digestive fluid, a protein, a nucleic acid that encodes a protein, a compound having a known bioavailability and a liquid-dispenser, or a combination of the foregoing. Such compositions often are contained in one or more containers (e.g., vessel, well, tube or the like), and the membrane often is in association with a permeable solid support in a container. Kits include such components in one or more containers, and sometimes include instructions, or directions to obtain instructions (e.g., from a site on the World Wide Web), for using the components (e.g., instructions for performing a method described herein).

The results of the membrane permeability and metabolic stability assays may be used to rank compounds by predicted bioavailability. For example, compounds having more desired membrane permeability characteristics, for example, greater membrane permeability than other compounds, may be assigned a higher number than those having less membrane permeability. A 1-100 scale, for example, may be used. Or, P_e or Log P_e may be used to compare different compounds for ranking. Similarly, a 1-100 scale, for example, may be used to rank compounds based on desired metabolic stability characteristics, those compounds having the most desired half lives for oral dosing being assigned the higher numbers. These two or more numbers, obtained from permeability and stability assays, may be combined to achieve an overall predicted bioavailability score, allowing the ranking of the compounds. The two or more numbers, for example, may be averaged to obtain the predicted bioavailability score.

For ranking of compounds in combined assays, in certain embodiments each 96-well plate PAMPA can contain several compounds or drugs with known oral bioavailability in human or rodents. Such compounds can be used to rank test compounds for their possible membrane permeability calculated based on effective permeability. Highly ranked compounds can be tested for their metabolic stability in the presence of human (or rodent) liver microsomes. If a compound is membrane permeable and metabolically stable over at least about 1.5 to about 2 hours it often will be tested for in vivo bioavailability.

The results of the membrane permeability and metabolic stability assays also, for example, may be used to predict bioavailability. Bioavailability may be predicted based on assigned scores such as, for example, those listed above. Or, bioavailability may be predicted by comparing assay results with those of compounds having known bioavailability. For example, certain compounds, having known F (%) values may be used as reference compounds. The F values of certain reference compounds have been compared to the permeability assay results of these compounds (Balimane, P. V., et al, AAPS Journal 2006: 8(1) Article 1 El). Thus, in predicting bioavailability, reference compounds may be, for example, included in the permeability and stability assays, or the results of permeability and stability assays may be compared to those obtained from reference compounds at a different time. By comparing these results, a predicted F value may be obtained.

Enhanced Methods for Determining Partition Coefficient, Distribution Coefficient and pKa

Parameters for Multiple Compounds

The aqueous ionization constant known as the pKa is defined as the negative logarithm of the equilibrium coefficient of the neutral and charged species of a compound. The degree to which a compound is ionized at a given pH can be calculated from the following equation: % ionized = 100/l+10^(charge(pH-^pKa))

This relationship between the degree of ionization and pH can be represented for a basic compound. The sigmoidal curve of a typical plot of percent ionized versus pH depicts how full ionization is reached at low pH and vice versa. The pKa corresponds to the pH at which the concentration of ionized molecule is equal to the concentration of the neutral molecule. The partition coefficient known as P is a constant for a given molecule and solvent.

Coefficient P often is referred to as log P, which is defined as the logarithm of the ratio of the concentration of neutral compound in aqueous phase to the concentration in a particular solvent. In practical terms the neutral molecule exists for bases great than 2 pKa units above the pKa and for acids greater than 2 pKa units below. The distribution coefficient known as D is dependant upon the pH at which it is measured. Coefficient D often is referred to as log D, which is the logarithm of the apparent ratio of concentration of compound in aqueous phase to the concentration in a particular solvent. As the proportion of molecule ionized changes with pH so does log D. Further description of log D, log P and pKa values is at http address www.raell.demon.co.uk/chem/logp/logppka.htm. The three quantities log P, log D and pKa are all related by the following equations. For a weak monoprotic acid that is partially ionized: logD = log P_XH - log(l + 10fp^a-pHjj and for a weak monoprotic base that is partially protonated in the aqueous phase logD = log P_x - logfl + lO(pH-pKa)) where XH and X refer to the neutral form of the weak acid and base, respectively. For polyprotic compounds the equations become more complicated. If log P and the pKa of a compound are known, log D can be calculated at any pH. Conversely, by measuring the apparent distribution at a range of pH values, a curve showing the differing values of D as the pH varies can be derived. The shape of this curve is ultimately determined by the degree to which the compound is ionized at each pH (e.g., Figure 1). For a basic compound, as the pH increases the degree of ionization decreases, there is less ionized compound in the aqueous phase and D→ P. As the pH decreases the compound becomes fully ionized, is unable to penetrate the organic solvent layer and D→ 0. The same is true for an acidic compound however full ionization is reached at low pHs and the neutral form predominates at higher pH ranges. The top of the sigmoidal slope is the distribution coefficient of the fully neutral molecule and hence can be regarded as P, the partition coefficient. Halfway between the top slope and the bottom slope if the point at which 50% of the molecule is ionized. The pH corresponding to this point is also known the pKa of a compound.

Based on the above formula associated measured log D values can be calculated from a series of distribution coefficients at different pHs. By fitting these points to a sigmoidal curve, P (and hence log P) and also the pKa can be derived for the molecule of interest.

Thus, lipophilicity often is expressed by the octanol/water partition coefficient (Log P) or distribution coefficient (Log D). A classical measurement of LogD is by shake-flask analysis. In this approach, a sample is dissolved in flask containing both aqueous buffer solution and water immiscible partition solvent (e.g., octanol). The flask is shaken to equilibrate the sample between the two phases, and phases then are allowed to separate. The pH of the aqueous phase is established by various commercially available certified strong buffers. The relative concentration (UV peak area) of sample is measured in each phase, generally using HPLC with UV detection. From these values the LogD at the experimental pH can be calculated. A lipophilicity profile can be obtained by measuring shake-flask determined LogD values at several different pH values without knowing the pKa values.

This shake-flask method has certain limitations, however. For example, obtaining unambiguous UV peak data requires that only one compound a time can be partitioned and analyzed. If several compounds are subjected to simultaneous analysis, long (20-30 min), shallow gradient HPLC runs are required for complete separation of individual UV peaks at high peak resolution and minimal peak tailing. This latter approach requires pre- validation of HPLC methods to assign specific retention time to the specific UV peaks. Also, the upper and lower range of LogD values are limited by problems of low HPLC-UV sensitivity. A compound having a high LogD value results in a significantly higher solubility in octanol as compared to the aqueous phase, and the resulting concentration of the compound in the aqueous phase is very low and difficult to measure by UV detection (e.g., a compound having a LogD of 4 is 10,000 times more soluble in octanol).

Simultaneous distribution coefficient (e.g., D, Log D, apparent distribution coefficient), partition coefficient (e.g., P, Log P) and pKa determination methods provided herein directly measure an solvent/aqueous phase distribution coefficient of multiple test compounds in a pH range. The methods are cell-free and can be carried out in relatively high throughput formats (e.g., 96-well plates or 46-vial plates (Waters Corporation, Milford, MA)). The methods allow measurement of the relative amounts of compounds in solvent and aqueous phases after incubation (e.g., overnight incubation) by sensitive and selective detection procedures, such as LC-MS/MS. These methods provide rapid, specific, highly sensitive and automated procedures to directly measure experimental Log D and calculate Log P values and pKa for multiple compounds in a short period of time.

Thus, provided herein are methods for simultaneously determining partition coefficient, distribution coefficient and ionization constant values for one or more compounds, which comprise: (a) contacting one or more compounds with multiple solutions, where: the pH of each of the solutions is different, and each of the solutions comprises a first phase and second phase immiscible with the first phase; and (b) determining the distribution coefficient for each of the one or more compounds in each of the solutions; whereby the partition coefficient and ionization constant values for each of the one or more compounds is determined from the distribution coefficient. The partition coefficient generally is the ratio of the concentration or amount of a compound in the first phase as compared to the second phase. The distribution coefficient sometimes is referred to as coefficient D, log D or an apparent partition coefficient. The partition coefficient sometimes is referred to as coefficient P or log P. The ionization constant sometimes is referred to as Ka, Kb, pKa or pKb. The first phase can be any aqueous liquid (e.g., water) and the second phase can be any liquid immiscible with the first phase, such as an organic solvent. Any organic solvent immiscible with water can be utilized, such as octanol or methanol, for example. The number of solutions, the pH range of the solutions, and the average incremental difference in pH of the solutions are selected such that the partition coefficient, distribution coefficient and ionization constant values can be simultaneously determined. Each solution often comprises a buffer agent suitable for stabilizing the particular pH of the solution, and such buffer reagents are readily available to and selected by the person of ordinary skill in the art. About four or more solutions, each having different pHs, often are utilized in the methods, and about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more solutions can be utilized.

The pH range, the difference between the pH of the solution having the lowest pH and the pH of the solution having the highest pH, often is about 4 pH units or greater, and can be about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 or greater pH units. Often, a pH range between (i) about two or more pH units below a measured or calculated pKa of a compound and (ii) about two or more pH units above a measured or calculated pKa of a compound is utilized. In some embodiments, the pH range is from (i) about pH 5 to about pH 10, (ii) about pH 5 to about pH 11, (iii) about pH 5 to about pH 12, (iv) about pH 4 to about pH 12, (v) about pH 3 to about pH 12; (vi) about pH 2 to about pH 12, (vii) about pH 1 to about pH 12, (viii) about pH 1 to about pH 13, and (ix) about pH 0 to about pH 14.

The term "incremental pH difference" as used herein refers to the difference between the pH of a solution and the pH of another solution in the set having the next closest pH value. The incremental pH difference can be expressed as an average across all of the solutions, or a subset thereof, in a given pH range. For example, for a set of eight solutions having the following pHs: Solution A is pH 4.0, Solution B is pH 4.8, Solution C is pH 5.8, Solution D is pH 6.8, Solution E is pH 7.8, Solution F is pH 8.8, Solution H is pH 10.0, (i) the incremental pH difference between Solution A and Solution B is 0.8 pH units, (ii) the incremental pH difference between Solution F and Solution H is 1.2 pH units, and (iii) the average incremental pH difference for all of the solutions in the set is one pH unit. The average incremental difference in pH of the solutions in a set sometimes is between about 0.3 to about 1.5 pH units. In certain embodiments, a set of five or more solutions having pHs from the group consisting of about 4.0, about 5.0, about 6.0, about 7.0, about 7.4, about 8.0, about 9.0, about 10.0, about 11.0, about 12.0, about 13.0 and about 14.0, is utilized. In the methods described above, the distribution coefficient for each of the one or more compounds in each of the solutions can be determined by any suitable method, such as mass spectrometry. Any mass spectrometry method suitable for quantifying the concentration or amount of a compound in each of the two phases can be utilized, and mass spectrometry methods suitable for quantifying the concentration or amount of multiple compounds

(e.g., 5-150 compounds in each phase) can be selected. Mass spectrometric analysis allows for detection of multiple compounds in a sample in a concentration independent manner, and can be utilized to detect compounds present in each solution at a level of about 0.01 micromolar to about 10 micromolar (e.g., about one micromolar concentration). In certain embodiments, a mass spectrometer capable of performing mass spectrometry/mass spectrometry (MS/MS) analysis is utilized. Such mass spectrometers sometimes are utilized in conjunction with a liquid chromatography (LC) device, such as an high performance liquid chromatography (HPLC) device, for conducting LC-MS/MS analysis. In some embodiments, a MS/MS mass spectrometer is utilized in conjunction with an electrospray ionization (ESI) device, and sometimes such devices are utilized for LC-ESI-MS/MS analysis. Such mass spectrometers, LC and ESI devices are known and can be appropriately selected by the person of ordinary skill in the art for use in methods described herein.

Also provided is a kit comprising multiple aqueous solutions, where: each solution comprises a different buffer agent and the pH of each solution is different; the pH of the solutions ranges between about pH 5 or less and about pH 12 or more; and the average incremental pH difference of the solutions is between about 0.3 to about 1.5 pH units. Any of the numbers of solutions, pH ranges, incremental pH differences and average incremental pH differences described above can be incorporated into such kits. Provided also herein is a system comprising such a kit in conjunction with a mass spectrometer, where any of the mass spectrometers described above can be utilized.

The examples set forth below illustrate but do not limit the invention.

Example 1 Lipid-PAMPA Assay with the MultiScreen^® Filter Plates

The following Parallel Artificial Membrane Permeation Assay (PAMPA) is a non-cell based assay designed to predict passive, transcellular permeability of drugs in early drug discovery. (Kansy, M. et al, J. Med. Chem. (1998) 41:1007-1010). The assay is carried out in a 96- well MultiScreen® PAMPA filter plate (Millipore Corporation, Billerica, MA) and measures the ability of compounds to diffuse from a Donor compartment to an Acceptor compartment separated by a polyvinylidene fluoride (PVDF) membrane filter pretreated with a lipid-containing organic solvent. The assay provides rapid, low cost and automation friendly methods to measure passive permeability of a compound. The following protocol details steps required to determine compound permeability rates across the artificial membrane.

Reagents

Lipid or lipid mixture such as L-a-phosphatidylcholine (lecithin) (for example: # P3556; Sigma Chemical Co., St. Louis, MO)

Dodecane (for example: # 112403; Sigma)

Phosphate buffered saline (for example: # P-3813; Sigma)

Equipment

MultiScreen filter plate for PAMPA assay with underdrain removed (# MAIPN45XX; Millipore Corporation, Billerica, MA)

PTFE Acceptor plate (# MSSACCEPTOR; Millipore)

Bioanalytical apparatus such as HPLC-MS, or Spectramax® Plus microtiter plate reader (Molecular Devices, Sunnyvale, CA), or similar UV- Vis device

Polypropylene reagent reservoirs (for example: # 175-RB AS-000; ELKay laboratory consumables Shrewsbury, MA)

Multichannel pipettes.

Methods

1. Prepare a 1 to 4% solution (w/v) of lecithin in dodecane (-500 μL/plate) and sonicate the mixture to ensure complete dissolution. Although this protocol specifically recommends a lecithin solution in dodecane, many alternative lipid mixtures are also compatible with the membrane and protocol. For a particular target barrier, it is critical to choose the appropriate lipid mixture and lipid concentration to ensure accurate permeability ranking and correlation with human absorption data. (MultiScreen Filter Plates for PAMPA: Evaluation of the reproducibility of Parallel Artificial Membrane Permeation Assays (PAMPA); Application Note Millipore Lit. No. AN1728EN00). Typically it is necessary to sonicate the lipid mixture until the solution approaches the clarity of water (using a probe typically used for cell lysis takes about 2 minutes). The lecithin solution will clarify greatly but will never be completely clear. This solution should be used immediately. The lecithin will begin to aggregate and become turbid again after a few minutes.

2. Carefully pipette 5 μL of the lecithin/dodecane mixture into each Donor plate well, avoiding pipette tip contact with the membrane. It is best to add the lecithin/dodecane solution to the membrane while the donor plate is sitting in a single well tray so that the underside of the membrane does not make contact with any surfaces. When the mixture is ejected from the pipette tip it will form a pendant drop suspended from the tip. The pendant drop should be placed close enough to the membrane so that it will wick off, while insuring that the plastic pipette tip itself does not make contact with the membrane.

3. Immediately after the application of the artificial membrane (within 10 minutes maximum), add 150 μL of drug-containing donor solutions (drugs dissolved in 5% DMSO, PBS) to each well of the Donor plate.

4. Add 300 μL of aqueous buffer to each well of the PTFE Acceptor plate (MSACCEPTOR).

5. Slowly and carefully place the drug-filled Donor plate into the Acceptor plate, making sure the underside of the membrane is in contact with the buffer in all wells. The donor plate must be carefully placed into the acceptor plate. Any excess downward pressure will cause the buffer in the acceptor plate to be squeezed up and out of the wells resulting in cross talk.

6. Replace the plate lid and incubate at room temperature for 16 hours. To avoid evaporation, the plate should be placed in humidity controlled environment such as a sealed container with wet paper towels during incubation. 7. After incubation, analyze the acceptor plate for compound concentration. Generally sample analysis using a 96 well UV/Vis plate reader is recommended. Sample quantification techniques such as scanning over a broad absorbance range (e.g. 250-500 nm), a single wavelength (λmax) or a summation of pre- selected fixed wavelengths are all suitable for analysis. HPLC-UV or LC-MS/MS are also alternative means of detection. 8. Make up drug solutions at the theoretical equilibrium (i.e., the resulting concentration if the Donor and Acceptor solutions were simply combined) and similarly analyze. 9. The equation used to determine permeability rates (Pe) is displayed in Figure 2 and calculates log Pe. Variables for the equation are defined in Table 1.

Table 1 Description of variables used to calculate Log P_e

Term Definition Notes

V_D Volume of donor compartment Expressed in cm³, 150 L = 0.15 cm³ VA Volume of acceptor compartment Expressed in cm³, 300 L = 0.30 cm³ Area Active surface area of membrane Defined as membrane area x porosity. For the membrane in MultiScreen Permeability Filter Plate, area = .24 cm² x 100%; or 0.24 cm2

Time Incubation time for the assay Expressed in seconds, 1 hr = 3600 s

[drug] acceptor Concentration of compound in the Measured by LC-UV or LC-MS/MS acceptor compartment at the completion of the assay

[drug] equilibrium Concentration of compound at Measured by LC-UV or LC-MS/MS theoretical equilibrium from step 9 above

Example 2 Example 2: Metabolic Stability Assays of Chemical Entity in Human Liver Microsomes

The following microsomal stability assay is designed to determine the stability of a chemical entity in the presence of human liver microsomes containing various CYP450 enzymes. Metabolism controls are run with the test articles. The controls include tolbutamide, desipramine and testosterone, which are low, moderate and high clearance compounds in vivo. Comparison with these metabolism controls provides a reference for comparison of half-lives.

General Assay Procedure

Prepare pre-warmed 96-well (2 mL) plate with 100 μL of a solution containing 50 mM potassium phosphate, pH 7.4, 2.6 mM NADP⁺, 6.6 mM glucose 6-phosphate, 0.8 U/mL of glucose 6-phosphate dehydrogenase and 1, 10 or 50 μM of the test compound. Run similar reactions with metabolic controls representing low (tolbutamide), moderate (desipramine), and high (testosterone) clearance compounds with the same enzyme solution. Initiate reactions by adding 100 μL of a pre-warmed enzyme solution and incubate at 37°C. Prepare a zero time-point reaction by adding 50 μL of acetonitrile (containing internal standard) to the test compound/cofactor solution prior to adding the enzyme solution. After 15, 30, 60, 90 and 120 minutes, remove reaction plate from the incubator and terminate reaction terminated with 50 μL of acetonitrile containing internal standard. Analyze samples for the parent form of the test compound with MS/MS detection. Perform each assay in duplicate.

Cof actor/Test Compound Solution Concentrations: Prepare a stock solution of 3 mM a chemical entity in wateπacetonitrile (90: 10). For all assays, prepare 10 μM solution of the test article in 50 mM potassium phosphate, pH 7.4, 2.6 mM NADP⁺, 6.6 mM glucose 6-phosphate and 0.8 U/mL of glucose 6-phosphate dehydrogenase (cofactor solution).

Cofactor/Metabolism Control Solution Concentrations: Prepare 10 μM stock solutions of the metabolism controls (tolbutamide, desipramine, and testosterone) in cofactor solution described in step 2.2.

Enzyme Solution Concentrations: Prepare the enzyme solutions by adding human liver microsomes to 50 mM potassium phosphate, pH 7.4, to a final concentration of 1 mg/mL. Microsomes can be purchased from CellzDirect (Austin, TX-USA)

Initiating the Reactions: Warm the reaction plate at 37°C in an incubator for about 3-5 minutes. Prepare the zero time-point control reaction for each replicate by adding 50 μL of acetonitrile containing internal standard to 100 μL of cofactor solution to inactivate the enzymes, and then vortex mixing. Initiate reactions by adding 100 μL of the enzyme solution to each well and vortex mixing. Incubate samples, including the zero time-point control, in an incubator at 37°C .

The final concentrations of all components after initiating the reactions will be 50 mM potassium phosphate, pH 7.4, 1.3 mM NADP⁺, 3.3 mM glucose 6-phosphate, 0.4 U/mL of glucose 6-phosphate dehydrogenase, 0.5 mg/mL liver microsomes and 5 μM test article.

Terminating and Extracting the Reactions: Terminate reactions after 15, 30, 60, 90 and 120 minutes at 37°C by the addition of 50 μL of acetonitrile containing internal standard. Remove the zero time -point control from the water bath after 120 minutes.

Extract reactions by adding 1.0 mL of acetonitrile, vortex mixing for 5 minutes, centrifuging at 16,000 g for 3 minutes, transfer the supernatants into fresh plate, evaporate the solvent to dryness under a stream of nitrogen at 40⁰C, and then reconstitute the samples in 150 μL of water :acetonitrile:formic acid (95:5:0.1 v/v).

Analysis of Reactions

Analyze the reaction mixtures for the chemical entity for the parent form according to the following HPLC conditions:

Column: Phenomenex Synergi Polar RP, 150.0 x 2.0 mm, 4 μM

Guard Column: Phenomenex Cl 8, 4.0 x 2.0 mm

Flow Rate: 0.25 mL/min

Column Temperature: 4O⁰C

Sample Temperature: Room temperature

Injection Volume: 10 μL

Run Time: 7.5 min

Gradient Solvent System

The composition of the solvents and the gradient method for the analysis are:

Solvent A: 0.1% Formic Acid in Water

Solvent B: 0.1% Formic Acid in Acetonitrile

Table 2 Solvent Analysis

Detection: Detect the parent chemical entity compound and metabolism controls using appropriate multiple reaction monitoring (MRM) parameters. Table 3 Mass Transition of Compounds

Example 3 Metabolic Stability Assay of Chemical Entity in Simulated Gastro-intestinal Digestion Fluids

The following assay is designed to determine the stability of a chemical entity in the presence of simulated gastric and intestinal fluids. Metabolism controls are run with the test articles. The controls will include Nα-Benzoyl-L-arginine ethyl ester Hydrochloride (BAEE), which is known prototype substrate for digestive enzymes. Comparison with these metabolism controls provides a reference for comparison of the half-lives.

General Assay Procedure

Simulated gastric fluid (SGF): Prepare pre- warmed 96-well (2 mL) plate with 100 μL of a solution containing 0.03 M sodium chloride, pH 1.2, and 6.4 mg/mL Pepsin (Sigma Cat # P6887). Initiate reactions by adding 100 μL of a pre-warmed chemical entity and/or metabolism control solution and incubate at 37°C. Prepare zero time-point reaction by adding 50 μL of acetonitrile (containing internal standard) to the test compound solution prior to adding the enzyme solution. After 15, 30, 60, 90 and 120 minutes, remove reaction plate from the incubator and terminate reaction terminated with 50 μL of acetonitrile containing internal standard. Analyze samples for the parent form of the test compound with MS/MS detection. Perform each assay in duplicate.

Simulated intestinal fluid (SIF): Prepare pre-warmed 96-well (2 mL) plate with 100 μL of a solution containing 0.05 M KH2PO4, pH 7.5, and 20.0 mg/mL Pancreatin (Sigma Cat # P7545). Initiate reactions by adding 100 μL of a pre-warmed chemical entity and/or metabolism control solution and incubate at 37°C. Prepare zero time-point reaction by adding 50 μL of acetonitrile (containing internal standard) to the test compound solution prior to adding the enzyme solution. After 15, 30, 60, 90 and 120 minutes, remove reaction plate from the incubator and terminate reaction terminated with 50 μL of acetonitrile containing internal standard. Analyze samples for the parent form of the test compound with MS/MS detection. Perform each assay in duplicate.

Cofactor/Test Compound Solution Concentrations

Prepare a stock solution of 1.0 mM chemical entity in DMSO. For SGF stability assays, prepare 10 μM solution of the test article in 0.03 M sodium chloride, pH 1.2. For SIF stability assays, prepare 10 μM solution of the test article in 0.05 M KH2PO4, pH 7.5.

Terminating and Extracting the Reactions Terminate reactions after 15, 30, 60, 90 and 120 minutes at 37°C by the addition of 50 μL of acetonitrile containing internal standard. Remove the zero time-point control from the water bath after 120 minutes.

Extract reactions by adding 1.0 mL of acetonitrile, vortex mixing for 5 minutes, centrifuging at 16,000 g for 3 minutes, transfer the supernatants into fresh plate, evaporate the solvent to dryness under a stream of nitrogen at 40⁰C, and then reconstitute the samples in 150 μL of water: acetonitrile: formic acid (95:5:0.1 v/v).

Analysis of Reactions

Analyze the reaction mixtures for chemical entity for the parent form according to the following HPLC conditions: Column: Phenomenex Synergi Polar RP, 150.0 x 2.0 mm, 4 μM

Guard Column: Phenomenex C18, 4.0 x 2.0 mm

Flow Rate: 0.25 mL/min

Column Temperature: 4O⁰C

Sample Temperature: Room temperature Injection Volume: 10 μL

Run Time: 7.5 min

Gradient Solvent System The composition of the solvents and the gradient method for the analysis are: Solvent A: 0.1% Formic Acid in Water Solvent B: 0.1% Formic Acid in Acetonitrile

Table 4 Solvent Analysis

Detection: Detect the parent compound and metabolism controls using appropriate MRM parameters.

Example 4 Rapid Determination of LogP, LogD and pKa of Compounds

A liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS) method was utilized to determine experimental LogD, LogP and pKa values for a set of twenty (20) unique small molecules. This method combines ease of operation of octanol/water phase partitioning and the high sensitivity and accuracy afforded by LC-MS/MS measurements. General features of the method are: (1) only small amounts of each compound are required (e.g., only 50 μL of a 100 μg/mL solution needed); (2) parameters are simultaneously determined from a combined set compounds (e.g., up to 10 compounds per sample); parameters are determined rapidly (e.g., 3.5 minute generic LC-MS/MS run is suitable for wide range of chemical compounds); (4) high selectivity and sensitivity (e.g., about 0.5 ng/mL to about 30,000 ng/mL); (5) high accuracy and excellent reproducibility of parameters assessed by the method; (6) experimental logD (e.g., pH ranges 4-13) and logP determinations; (7) accurate pKa calculation; (8) wide lipophilicity range; and (9) precise information primarily obtained from a shake-flask logD determination rather than less accurate assumed models based on HPLC retention times of test LogP markers and test compounds.

Preparation of stock solutions of test compounds

Prepare 100 μL of 1 mg/mL stock solution (in DMSO or acetonitrile) of 10 test compounds. Dilute a stock solution 1,000 times by transferring 1 μL of the stock solution into 999 μL acetonitrile containing 0.1 % formic acid. Use obtained solution for mass spectrometric method development based on direct infusion. Determine parent/daughter mass spectra (multiple reaction monitoring - MRM) of each compounds (method described below). Compare parent/daughter mass spectra of each compound to assure their uniqueness in order to avoid any cross-reaction interference during LC-MS/MS measurements. Prepare 0.5 mL cassette of 10 test compounds by mixing of 50 μL of each stock solutions (1 mg/mL) to reach a final concentration of 100 μg/mL of each test compound per cassette.

Octanol/aqueous phase partition

Prepare eleven 2 mL HPLC vials in a 46-well HPLC plate (Waters Corporation) and label them according to pH values: 4, 5, 6, 7, 7.4, 8, 9, 10, 11, 12 and 13. Add 0.5 mL octanol to each vial. Transfer 0.5 mL certified pH buffer solution to the corresponding vial. Transfer 30 μL of cassette solution to each vial with formed two phase system. Place a plate on an orbital shaker and incubate overnight at room temperature under constant shaking to assure complete partition of test compounds between octanol and aqueous phases. After incubation, centrifuge plate for 10 min at 10,000 g and carefully transfer 200 μL samples from each phase into prelabeled HPLC plates for subsequent LC-MS/MS analysis.

LC-MS/MS conditions:

Analyze the reaction mixtures for chemical entity for the parent form according to the following HPLC conditions: Column: Phenomenex Synergi Polar RP, 20.0 x 2.0 mm, 3 μM

Guard Column: Phenomenex C18, 4.0 x 2.0 mm Flow Rate: 0.25 mL/min Column Temperature: 4O⁰C Sample Temperature: Room temperature Injection Volume: 10 μL Run Time: 3.5 min Gradient Solvent System

The composition of the solvents and the gradient method for the analysis are: Solvent A: 0.1% Formic Acid in Water

Solvent B : 0.1% Formic Acid in Acetonitrile

Solvent Analysis

Mass Spectrometry Parameters MS Mode: ESI (+)

Capillary: 4.0 kV

Cone: 40 V

Extractor: 3 V

RF Lens: 0.2 V Source T: 120 OC

Desolvation T: 300 OC

Gas_Desolvation: 450 L/h

Gas_Cone: 72 L/h

LM Resolution: 15.0 HM Resolution: 15.0

Ion Energy: 0.5

Multiplier: 650 Methodology described above was utilized to determine experimental values of pKa and logP for particular compounds. These experimental values were consistent with published pKa and logP values derived using different procedures, as shown in the following table.

a. Data from Wiczling et al. (2006). "Simultaneous determination of pKa and lipophilicity by gradient RP HPLC." Anal Chem 78(1): 239-49. b. Determined by methods and compositions described in the present disclosure.

Example 5 Representative Embodiments

Provided hereafter are representative embodiments of the invention:

1. A method for ranking compounds by predicted bioavailability, which comprises: determining membrane permeability of a set of compounds in vitro; determining stability of the compounds in vitro; and ranking the compounds for predicted bioavailability according to the permeability and stability.

2. A method for predicting bioavailability of a compound, which comprises: determining membrane permeability of a compound in vitro; determining stability of the compound in vitro; and predicting the bioavailability of the compound according to the membrane permeability and stability determined for the compound.

3. The method of embodiments 1 or 2, wherein the bioavailability is oral bioavailability. 4. The method of embodiments 1 or 2, wherein the membrane permeability is determined by detecting the amount of compound on one or both sides of a membrane.

5. The method of embodiment 4, wherein the membrane is a lipid membrane.

6. The method of embodiment 5, wherein the lipid membrane is a monolayer. 7. The method of embodiment 6, wherein the lipid membrane is selected from the group consisting of a bilayer, a micelle and a liposome.

8. The method of embodiment 4, wherein the membrane is in association with a solid support.

9. The method of embodiment 8, wherein the membrane comprises hexadecane.

10. The method of embodiment 8, wherein the membrane comprises a lipid selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidic acid or a combination of the foregoing.

11. The method of embodiment 10, wherein the membrane comprises a combination of lipids in ratios present in a blood-brain barrier.

12. The method of embodiment 10, wherein the membrane comprises a combination of lipids in ratios present in a plasma membrane of a cell.

13. The method of embodiments 1 or 2, wherein the membrane permeability and the stability are determined simultaneously.

14. The method of embodiment 13, wherein the membrane permeability and the stability are determined in a single vessel. 15. The method of embodiments 1 or 2, wherein the compound is contacted with acidic pH conditions.

16. The method of embodiments 1 or 2, wherein the compound is contacted with basic pH conditions.

17. The method of embodiments 1 or 2, wherein the stability is determined by the amount of modified compound and/or unmodified compound present after the compound is contacted with a substance selected from the group consisting of a cell, a cell component, blood, a blood fraction, a blood component and a digestive fluid, or a combination of the foregoing.

18. The method of embodiment 17, wherein the cell component is a microsome. 19. The method of embodiment 18, wherein the microsome is a liver microsome.

20. The method of embodiment 17, wherein the cell is a liver cell. 21. The method of embodiment 17, wherein the blood fraction is plasma.

22. The method of embodiment 17, wherein the cell component is one or more enzymes.

23. The method of embodiment 22, wherein the one or more enzymes comprise one or more isolated enzymes. 24. The method of embodiment 22, wherein the one or more enzymes comprise one or more liver enzymes.

25. The method of embodiment 22, wherein the one or more enzymes are selected from the group consisting of a CYP3A4 enzyme, CYP2D6 enzyme, CYP2C9 enzyme and CYPl A2 enzyme, or a combination of the foregoing. 26. The method of embodiment 17, wherein the digestive fluid is a simulated digestive fluid.

27. The method of embodiment 26, wherein the digestive fluid is a simulated gastric fluid or a simulated intestinal fluid.

28. The method of embodiment 17, wherein the cell component is an isolated liver cytosol.

29. The method of embodiment 17, wherein the substance is from a mammal. 30. The method of embodiment 29, wherein the substance is from a human, rat, mouse, dog or primate.

31. The method of embodiments 1 or 2, which comprises contacting the compound with a protein, determining the amount of compound bound to the protein, and predicting the bioavailability of the compound according to the membrane permeability and stability determined for the compound and the amount of compound bound to the protein.

32. The method of embodiment 31, wherein the protein is in association with a solid support.

33. The method of embodiment 32, wherein the protein is in association with a solid support via a biotin/avidin or streptavidin binding pair.

34. The method of embodiments 1 or 2, which comprises determining the oral bioavailability of the compound in vivo.

35. The method of embodiments 1 or 2, wherein the compound is a cytotoxic agent.

36. The method of embodiments 1 or 2, wherein the membrane permeability and stability are determined in series.

37. The method of embodiments 1 or 2, wherein the membrane permeability and stability are determined in parallel.

38. The method of embodiment 4, wherein the membrane comprises a protein pump. 39. The method of embodiment 38, wherein the protein pump is a P-glycoprotein pump.

40. A method for ranking compounds by predicted bioavailability across a blood-brain barrier, which comprises: determining membrane permeability of a set of compounds in vitro; determining stability of the compounds in vitro; and ranking the compounds according to the membrane permeability and stability.

41. A method for predicting bioavailability of a compound across a blood/brain barrier, which comprises: determining membrane permeability of a compound in vitro; determining stability of the compound in vitro; and predicting the bioavailability of the compound according to the membrane permeability and stability determined for the compound.

42. The method of embodiments 40 or 41, wherein the membrane permeability is determined by detecting the amount of compound on one or both sides of a membrane. 43. The method of embodiment 42, wherein the membrane is a lipid membrane.

44. The method of embodiment 43, wherein the lipid membrane is a monolayer.

45. The method of embodiment 44, wherein the lipid membrane is selected from the group consisting of a bilayer, a micelle and a liposome.

46. The method of embodiment 45, wherein the membrane is in association with a solid support.

47. The method of embodiment 43, wherein the membrane comprises a lipid selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, or phosphatidylinositol.

48. The method of embodiment 43, wherein the membrane comprises a combination of lipids in ratios present in a blood-brain barrier.

49. The method of embodiment 43, wherein the membrane comprises about 33.1

% phosphatidylethanolamine, about 18.5 % phosphatidylserine, about 12.6 % phosphatidylcholine, about 0.8 % phosphatidic acid, and about 4.1 % phosphatidylinositol. 50. The method of embodiments 40 or 41, wherein the membrane permeability and the stability are determined simultaneously. 51. The method of embodiment 50, wherein the membrane permeability and the stability are determined in a single vessel.

52. The method of embodiments 40 or 41, wherein the compound is contacted with acidic pH conditions. 53. The method of embodiments 40 or 41, wherein the compound is contacted with basic pH conditions.

54. The method of embodiments 40 or 41, wherein the stability is determined by the amount of modified compound and/or unmodified compound present after the compound is contacted with a substance selected from the group consisting of a cell, a cell component, blood, a blood fraction, a blood component and a digestive fluid, or a combination of the foregoing.

55. The method of embodiment 54, wherein the cell component is a microsome.

56. The method of embodiment 54, wherein the microsome is a liver microsome.

57. The method of embodiment 54, wherein the cell is a liver cell. 58. The method of embodiment 54, wherein the blood fraction is plasma.

59. The method of embodiment 54, wherein the cell component is one or more enzymes.

60. The method of embodiment 59, wherein the one or more enzymes comprise one or more isolated enzymes.

61. The method of embodiment 59, wherein the one or more enzymes comprise one or more liver enzymes.

62. The method of embodiment 59, wherein the one or more enzymes are selected from the group consisting of a CYP3A4 enzyme, CYP2D6 enzyme, CYP2C9 enzyme and CYP1A2 enzyme, or a combination of the foregoing.

63. The method of embodiment 54, wherein the digestive fluid is a simulated digestive fluid. 64. The method of embodiment 63, wherein the digestive fluid is a simulated gastric fluid or a simulated intestinal fluid.

65. The method of embodiment 54, wherein the cell component is an isolated liver cytosol.

66. The method of embodiment 54, wherein the substance is from a mammal.

67. The method of embodiment 66, wherein the substance is from a human, rat, mouse, dog or primate. 68. The method of embodiments 40 or 41, which comprises contacting the compound with a protein, determining the amount of compound bound to the protein, and predicting the bioavailability of the compound according to the membrane permeability and stability determined for the compound and the amount of compound bound to the protein. 69. The method of embodiment 68, wherein the protein is in association with a solid support.

70. The method of embodiment 69, wherein the protein is in association with a solid support via a biotin/avidin or streptavidin binding pair.

71. The method of embodiments 40 or 41, which comprises determining the oral bioavailability of the compound in vivo. 72. The method of embodiments 40 or 41, wherein the compound is a cytotoxic agent.

73. The method of embodiments 40 or 41, wherein the membrane permeability and stability are determined in series.

74. The method of embodiments 40 or 41, wherein the membrane permeability and stability are determined in parallel. 75. The method of embodiment 43, wherein the membrane comprises a protein pump.

76. The method of embodiment 75, wherein the protein pump is a P-glycoprotein pump.

77. A kit which comprises a porous solid support; one or more membrane-forming components; and a substance selected from the group consisting of a cell, a cell component, blood, a blood fraction, a blood component, a simulated digestive fluid, a protein, a nucleic acid that encodes a protein, a compound having a known bioavailability and a liquid-dispenser, or a combination of the foregoing.

78. The kit of embodiment 77, wherein the substance is frozen. 79. The kit of embodiment 77, wherein the cell component is a microsome.

80. The kit of embodiment 79, wherein the microsome is a liver microsome.

81. The kit of embodiment 77, wherein the cell is a liver cell.

82. The kit of embodiment 77, wherein the blood fraction is plasma.

83. The kit of embodiment 77, wherein the cell component is one or more enzymes. 84. The kit of embodiment 77, wherein the one or more enzymes comprise one or more isolated enzymes. 85. The kit of embodiment 77, wherein the one or more enzymes comprise one or more liver e nzymes.

86. The kit of embodiment 77, wherein the one or more enzymes are selected from the group consisting of a CYP3A4 enzyme, CYP2D6 enzyme, CYP2C9 enzyme and CYP1A2 enzyme, or a combination of the foregoing.

87. The kit of embodiment 77, wherein the cell component is an isolated liver cytosol.

88. The kit of embodiment 77, wherein the substance is from a mammal.

89. The kit of embodiment 88, wherein the substance is from a human, rat, mouse, dog or primate. 90. The kit of embodiment 77, which comprises instructions for predicting or ranking bioavailability of a compound in vitro.

91. The kit of embodiment 77, wherein the simulated digestive fluid is a simulated gastric fluid or a simulated intestinal fluid.

92. The kit of embodiment 77, wherein the protein is a protein pump. 93. The kit of embodiment 92, wherein the protein pump is a P-glycoprotein pump.

94. A composition comprising a membrane, a solid support in association with the membrane and a components selected from the group consisting of a cell, a cell component, blood, a blood fraction, a blood component, a simulated digestive fluid, a protein, a nucleic acid that encodes a protein, a compound having a known bioavailability and a liquid- dispenser, or a combination of the foregoing.

95. A method for simultaneously determining partition coefficient, distribution coefficient and ionization constant values for one or more compounds, which comprises:

(a) contacting one or more compounds with multiple solutions, wherein: the pH of each of the solutions is different, and each of the solutions comprises a first phase and second phase immiscible with the first phase; and

(b) determining the distribution coefficient for each of the one or more compounds in each of the solutions; whereby the partition coefficient and ionization constant values for each of the one or more compounds is determined from the distribution coefficient. 96. The method of embodiment 95, wherein the partition coefficient is the ratio of the concentration or amount of a compound in the first phase as compared to the second phase.

97. The method of embodiment 95, wherein the first phase is an aqueous liquid.

98. The method of embodiment 97, wherein the first phase is water. 99. The method of embodiment 95, wherein the second phase is an organic solvent.

100. The method of embodiment 99, wherein the organic solvent is octanol.

101. The method of embodiment 95, wherein four or more solutions are utilized.

102. The method of embodiment 95, wherein the pH of each solution is in a range of about four or more pH units. 103. The method of embodiment 95, wherein the average incremental difference in pH of the solutions is between about 0.3 to about 1.5 pH units.

104. The method of embodiment 95, wherein the distribution coefficient is log D.

105. The method of embodiment 95, wherein the partition coefficient is log P.

106. The method of embodiment 95, wherein the ionization constant is pKa. 107. The method of embodiment 95, wherein step (b) is performed by mass spectrometry.

108. The method of embodiment 107, wherein the mass spectrometry is a mass spectrometry/mass spectrometry (MS/MS) method.

109. The method of embodiment 95, wherein each solution comprises a buffer agent.

110. The method of embodiment 108, wherein the MS/MS method comprises liquid chromatography.

111. The method of embodiment 108, wherein the MS/MS method comprises electrospray ionization.

112. A kit comprising multiple aqueous solutions, wherein: each solution comprises a different buffer agent and the pH of each solution is different; the pH of the solutions ranges between about pH 5 or less and about pH 12 or more; the average incremental difference in pH of the solutions is between about 0.3 to about 1.5 pH units.

113. The kit of embodiment 112, wherein the pH of the solutions ranges at least between about pH 5 and about pH 10. 114. A system comprising the kit of embodiment 112 and a mass spectrometer. The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

Singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a subset" includes a plurality of such subsets, reference to "a nucleic acid" includes one or more nucleic acids and equivalents thereof known to those skilled in the art, and so forth. The term "or" is not meant to be exclusive to one or the terms it designates. For example, as it is used in a phrase of the structure "A or B" may denote A alone, B alone, or both A and B.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and systems similar or equivalent to those described herein can be used in the practice or testing of the present invention, the methods, devices, and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the processes, systems and methodologies which are reported in the publications which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and yet these modifications and improvements are within the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of, and "consisting of may be replaced with either of the other two terms. Thus, the terms and expressions which have been employed are used as terms of description and not of limitation, equivalents of the features shown and described, or portions thereof, are not excluded, and it is recognized that various modifications are possible within the scope of the invention. Embodiments of the invention are set forth in the following claims.

Claims

Claims

1. A method for ranking compounds by predicted bioavailability, which comprises: determining membrane permeability of a set of compounds in vitro; determining stability of the compounds in vitro; and ranking the compounds for predicted bioavailability according to the permeability and stability.

2. A method for predicting bioavailability of a compound, which comprises: determining membrane permeability of a compound in vitro; determining stability of the compound in vitro; and predicting the bioavailability of the compound according to the membrane permeability and stability determined for the compound.

3. The method of claim 1 or 2, wherein the bioavailability is oral bioavailability.

4. The method of claim 1 or 2, wherein the membrane permeability is determined by detecting the amount of compound on one or both sides of a membrane.

5. The method of claim 1 or 2, wherein the membrane is a blood-brain barrier.

6. The method of any of claims 1-5, wherein the membrane comprises a lipid selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidic acid or a combination of the foregoing.

7. The method of claim 1 or 2, wherein the stability is determined by the amount of modified compound and/or unmodified compound present after the compound is contacted with a substance selected from the group consisting of a cell, a cell component, blood, a blood fraction, a blood component and a digestive fluid, or a combination of the foregoing.

8. The method of claim 7, wherein the digestive fluid is a simulated digestive fluid.

9. The method of claim 1 or 2, which comprises contacting the compound with a protein, determining the amount of compound bound to the protein, and predicting the bioavailability of the compound according to the membrane permeability and stability determined for the compound and the amount of compound bound to the protein.

10. The method of claim 4, wherein the membrane comprises a protein pump.

11. The method of claim 10, wherein the protein pump is a P-glycoprotein pump.

12. The method of claim 5, wherein the membrane comprises a combination of lipids in ratios present in a blood-brain barrier.

13. The method of claim 12, wherein the membrane comprises about 33.1% phosphatidylethanolamine, about 18.5 % phosphatidylserine, about 12.6 % phosphatidylcholine, about 0.8 % phosphatidic acid, and about 4.1 % phosphatidylinositol.

14. A kit which comprises a porous solid support; one or more membrane-forming components; and a substance selected from the group consisting of a cell, a cell component, blood, a blood fraction, a blood component, a simulated digestive fluid, a protein, a nucleic acid that encodes a protein, a compound having a known bioavailability and a liquid- dispenser, or a combination of the foregoing.

15. The kit of claim 14, wherein the substance is a cell component which comprises one or more enzymes selected from the group consisting of a CYP3A4 enzyme, CYP2D6 enzyme, CYP2C9 enzyme and CYP1A2 enzyme, or a combination of the foregoing.

16. The kit of claim 15, wherein the substance is a protein that is a protein pump.

17. A composition comprising a membrane, a solid support in association with the membrane and a components selected from the group consisting of a cell, a cell component, blood, a blood fraction, a blood component, a simulated digestive fluid, a protein, a nucleic acid that encodes a protein, a compound having a known bioavailability and a liquid-dispenser, or a combination of the foregoing.

18. A method for simultaneously determining partition coefficient, distribution coefficient and ionization constant values for one or more compounds, which comprises:

(a) contacting one or more compounds with multiple solutions, wherein: the pH of each of the solutions is different, and each of the solutions comprises a first phase and second phase immiscible with the first phase; and

(b) determining the distribution coefficient for each of the one or more compounds in each of the solutions; whereby the partition coefficient and ionization constant values for each of the one or more compounds is determined from the distribution coefficient.

19. The method of claim 18, wherein step (b) is performed by mass spectrometry.

20. The method of claim 19, wherein the mass spectrometry is a mass spectrometry/mass spectrometry (MS/MS) method.

21. The method of claim 20, wherein the MS/MS method comprises electrospray ionization.

22. A kit comprising multiple aqueous solutions, wherein: each solution comprises a different buffer agent and the pH of each solution is different; the pH of the solutions ranges between about pH 5 or less and about pH 12 or more; the average incremental difference in pH of the solutions is between about 0.3 to about 1.5 pH units.

23. The kit of claim 22, wherein the pH of the solutions ranges at least between about pH 5 and about pH 10.

24. A system comprising the kit of claim 22 and a mass spectrometer.