WO2009071114A1

WO2009071114A1 - In situ transformation and analysis platform for high-throughput metabolic stability research making possible determination of in vivo clearance

Info

Publication number: WO2009071114A1
Application number: PCT/EP2007/063190
Authority: WO
Inventors: Iwo Gatlik
Original assignee: Gatlik Ltd
Priority date: 2007-12-03
Filing date: 2007-12-03
Publication date: 2009-06-11

Abstract

Assessing the metabolic stability of novel pharmacologically active substances is at the center of drug-discovery processes. Accordingly, systems able to provide straightforward modeling of in vivo hepatic clearance are of particular interest. The present invention serves as an innovative and efficient high-throughput system for the screening of drugs. It allows to perform controlled multiple in situ (in the reaction mixture) reactions of screened compounds in terms of their stability and to determine in vivo hepatic clearance. The determinations are based on electrochemical parameters of tested compounds and do not require any biological material. The invented system has a screening rate and capacity that are several orders of magnitude higher than any currently available techniques. This invention includes a multi reaction chamber plate connected to a multifunctional apparatus for the analysis and the control of the elapsing reactions. Targeted analysis of the transformations of the screened compounds is obtained.

Description

In situ TRANSFORMATION AND ANALYSIS PLATFORM FOR HIGH-THROUGHPUT METABOLIC STABILITY RESEARCH MAKING POSSIBLE DETERMINATION of in vivo CLEARANCE

TECHNICAL FIELD

The presented invention relates to a multifunctional apparatus for the controlled multiple in situ production and analysis of compounds and methods composed of repeating structural units formed by chemical processes e.g. oxidation, detection, data examination, reduction and coupling. More specifically, the invention is directed to a high-throughput system assessing the stability of compounds, profiling and predicting the in vivo hepatic clearance. All transformations are in situ, using appropriate electrodes.

BACKGROUND OF THE INVENTION

Although the assessment of pharmacologically active molecules has undergone improvements, still substantial progress has to be achieved. This is well illustrated by the following statement (citation from a review by J. H. Ansede and D. R. Thakker entitled High- throughput screening for stability and inhibitory activity of compounds toward cytochrome P450-mediated metabolism. J Pharm ScL (2004) 93(2):239-55) "With the advent of combinatorial chemistry and high-throughput screening technology, thousands of molecules can now be rapidly synthesized and screened for biological activity against large numbers of protein targets, greatly increasing the speed with which lead compounds are identified during the early stages of drug discovery. However, rapid optimization of parameters that determine whether a high-affinity ligand or a potent inhibitor will become a successful drug remains a challenge in improving the efficiency of the drug discovery process. Parameters that define absorption, distribution, metabolism, and excretion properties of drug candidates are important determinants of therapeutic efficacy, and thus should be optimized during early stages of drug discovery. Although the speed with which drugs are screened for properties such as absorption, cytochrome P450 (CYP) inhibition, and metabolic stability has increased over the past several years, the screening rate/capacity is still several orders of magnitude lower than those for high-throughput methods used in lead identification, resulting in a bottleneck in the drug discovery process."

Parameters like in vivo hepatic clearance characterizing the metabolic stability of a compound present an efficient selection of the best lead compounds among hundreds of candidates. Improved medications, thus, can enter the market sooner. At present, in vitro hepatic stability screening methods are commonly used to assess the metabolic stability parameters enabling early estimation of in vivo hepatic clearance (P. Baranczewski et. all, Pharmacol Rep. (2006) 58(4):453-72). A variety of in silico mathematical models of metabolism (Ewing T. J. et. all WO02075609) and in vitro hepatic stability screening assays based on enzymes (D. Thakker and C. Chen US6312917, J. H. Ansede and D. Thakker US20060046278, C. J. James et. all US20040171099), microsomes (M. H. Lerche and K. Golman EP1544634) or hepatocytes in a 96-well format for LC-MS-MS determinations has been designed, constructed and patented (E. L. LeCluyse et. all US6780580, S. Campbell et. all US20030215941, J. K. Leach and S. R. Tannenbaum US 20060110826, T. Lave et. all Pharmaceutical Research, (1997) 14:152- 155; D. F. McGinnity et. all, Drug Metabolism and Disposition, (2004) 32(11): 1247-53; S. J. Griffin and J. B. Houston Drug Metabolism and Disposition, (2005) 33(1): 115-20). These assays are capable to perform stability screening and predict in vivo metabolic clearance with the screening rate/capacity about 1000 compounds per week (K. M. Jenkins et. all, J Pharm Biomed Anal, (2004), 34, 989-1004). However, they are expensive, time consuming and the screening rate/capacity is several orders of magnitude lower then required. Additionally, these assays require enzymes, cellular organelles or freshly isolated cells like hepatocytes. Moreover, they are often imprecise like microsomal assays, and generate lots of organic wastes. These drawbacks become more significant when the number of samples to be extracted and analyzed is large and/or the specimen size is small. Furthermore, these assays are completely inappropriate for the stability screening of leads, because of their low level of sophistication and minor screening capacity (C. M. Masimirembwa et. all, Clin Pharmacokinet. (2003) 42(6):515-28).

Modern high throughput screening technologies use usually chromatographic, spectroscopic or spectrometric detection schemes, or their combinations. In recent years, these technologies were extended by electrochemical measurements, acoustic measurements and active control of the redox environment in conduction with another detection scheme for diverse screening application (A. Heller WO02095355, 1. Gatlik WO2005105292 and J. Johnson US07182853). Theses extended technologies explore very interesting high throughput screening field with huge development potential. However, theses technologies cannot be used to form preferred derivatives, induce transformation, assess metabolic stability, or to predict the in vivo hepatic clearance of screened compounds. The determination of metabolic stability is still a bottleneck in the drug discovery process. A high-throughput assessment for metabolic stability with a screening rate/capacity above 100'0OO compounds per week would, thus represents a substantial progress.

Even more, there is a need for a metabolic stability screening system operating in a high- throughput mode based on in vitro methods but being compatible an early estimation and prediction of in vivo clearance of leads.

SUMMARY OF THE INVENTION

This novel invention in high-throughput technology fulfills the need of a rapid, efficient, and reproducible method for the evaluation of drug candidates. This need is met by the present invention wherein system and method for the in situ transformation and analysis of the samples for controlled formation of derivatives, assessment of the stability of samples and determination of the clearance of drugs is disclosed. This system and method can be combined with optional analysis systems or methods to provide further information about biochemical properties of drugs. The in situ analysis and optional analysis detection scheme can be electrochemical, electroacoustic, chromatographic, spectroscopic, spectrometric, or based on transducers. The invented system and method are especially useful for high- throughput transformation and analysis of drug candidates.

The invented system is based on the results revealing that metabolic stability and in vivo clearance of drugs and their reduction and oxidation potentials possess good correlation. These findings allow the construction of a preferred embodiment of the invention comprising a multi reaction chamber plate, a multifunctional apparatus, and procedures of targeted transformation of screened compounds into desired derivatives and their detection and analysis. The invented reaction chamber of the plate operates with well-established physicochemical parameters. Chemical reactions are induced and controlled to produce distinct derivatives and analyzed at specially prepared electrodes. The composition of these parameters in a study protocol provides an emulation of real cellular metabolic processes in a reaction chamber and production of derivatives compatible with metabolites from hepatic cells. The emulation of cellular metabolic processes in the reaction chamber excludes artefacts from highly parametrized screening systems like enzymes, hepatocytes and microsomes where handling and control are difficult. In accordance with the first basic embodiment of the present invention, a method of controlled in situ electron-transfer induced transformation reactions of a sample in a reaction region is provided. Transformation reactions can be initiated by an appropriate activation in the reaction region and are combined with simultaneous in situ analysis of the sample and the formed sample derivatives. This method can be used to control the formation of derivatives and to assess the stability of samples especially in the in situ transformation and analysis assays.

In accordance with the second basic embodiment of the present invention, a method of controlled in situ electron-transfer induced transformation reactions combined with the simultaneous in situ analysis of the samples is provided. The method comprises providing a sample in the reaction region, providing an appropriate activation in the reaction region in order to induce the transformation reactions and permitting the sample to react in the reaction region in order to form derivatives, operating the control in the reaction region to control the transformation reactions, the environment of transformations and the formation of derivatives, and analyzing the sample together with the formed sample derivatives.

In accordance with the third basic embodiment of the present invention, a system for controlled in situ electron-transfer induced transformation reactions of a sample in a reaction region is provided. Transformation reactions can be initiated by an appropriate activation in the reaction region and are combined with simultaneous in situ analysis of the sample and the formed sample derivatives. This system can be used to control the formation of derivatives and to assess the stability of samples especially in the in situ transformation and analysis assays.

In accordance with the fourth basic embodiment of the present invention, a system for controlled in situ electron-transfer induced transformation reactions combined with the simultaneous in situ analysis of the sample is provided. The system comprises a reaction region for the sample, an appropriate activation in the reaction region in order to induce the transformation reactions, reaction region for the formation of derivatives, a control in the reaction region to control the transformation reactions, the environment of transformations and the formation of derivatives, and in situ analysis for the sample together with the formed sample derivatives.

The in situ electron-transfer process in all four basic embodiments can be induced by an inhomogeneous reaction between at least one electrode and the sample in solution or a homogeneous reaction between at least one mediator and the sample in solution. Preferably, the in situ electron-transfer in all embodiments is performed in a buffered solution under inert gas atmosphere. The transformation reactions in all four basic embodiments can comprise: combination, decomposition, oxidation, reduction, hydrolysis, and/or conjugation.

The appropriate activation or the control in the reaction region in all four basic embodiments can be of various nature e.g. biological, chemical, electrical, electrochemical, thermal, light, electromagnetic, transverse waves, or longitudinal waves.

The sample in all four basic embodiments can contain various molecules like: solvent, water, cosolvent, dimethyl sulfoxide (DMSO), chloride anions, supporting electrolyte, mediator, drug, drug candidate, lead, chemically active species, biologically active species, antioxidant, or oxygen. The sample in all preferred embodiments does not need to contain any biologically originated components like: enzymes, proteins, microsomes, hepatocytes, cells organelles, or cells. However, the sample in all embodiments needs to fulfill several requirements like: the pH of a sample from 3 to 5, the pH of a sample from 6 to 8, the pH of a sample from 8 to 10, the dimethyl sulfoxide (DMSO) concentration up to 12%, the chloride anions concentration up to 5%, the investigated compound concentration up to 1 mM, the total volume of a sample up to 70 micro liters, or the dissolved oxygen concentration up to 8.5 mg/L. The in situ analysis in all four basic embodiments can follow one or more optional analyses. One or more optional analyses or in situ analysis can use various detection schemes like: electrochemical, electroacoustic, chromatographic, spectroscopic, spectrometric, or based on transducers. The detection scheme can be based on a screening template and can use measurement like: amperometry, voltammetry, capacitance, impedance, fluorescence, absorbance, infra red, phosphorescence, chemiluminescence, electroluminescence, Raman, electron spin resonance, refractive index, mass spectrometry (MS), tandem mass spectrometry (MS/MS), liquid chromatography (LC), gas chromatography (GC), or high performance liquid chromatography (HPLC).

The analysis in all embodiments can be used to diverse proposes like: assessment of a resistance, assessment of a impedance, assessment of a capacitance, assessment of a chemical stability, assessment of a biological stability, determination of a susceptibility to metabolism, determination of a susceptibility to oxidative stress, determination of the quantities of the formed derivatives, determination of the qualities of the formed derivatives, determination of the kinetics of the transformations, determination of a susceptibility to reductive stress, the determination of the clearance, determination of the hepatic clearance, or determination of the in vivo hepatic clearance of a sample. The assessments or determinations can be based on various sample parameters, correlations, statistical relations, mathematical description like equation, function, etc. The assessments or determinations can be based on following: (a) sample parameters: pH, concentration, diffusivity, potential, standard potential, oxidation potential, reduction potential, redox potential, based on the tendency of a chemical species to gain electrons, or based on the tendency of a chemical species to loss electrons;

(b) correlations between at least one described in (a) sample parameter and at least one of the following characteristic of a sample: stability, chemical stability, biological stability, susceptibility to metabolism, susceptibility to oxidative stress, formed derivatives quantities, formed derivatives qualities, susceptibility to reductive stress, clearance, hepatic clearance, or in vivo hepatic clearance;

(c) statistical relations between at least one described in (a) sample parameters and at least one described in (b) sample characteristic;

(d) mathematical description of the relations between at least one described in (a) sample parameters and at least one described in (b) sample characteristic; or

(e) filtration, modelling or transformation of at least one described in (a), (b), (c) or (d) parameters, correlation, relation or description of the relation.

The transformation, the control, and the analysis in all four basic embodiments can be performed by the use of at least two electrodes. The transformation, the control, and the analysis in all preferred embodiments can be based on various methods like: amperometry, voltammetry, capacitance, or impedance. The transformation, the control, and the analysis in all embodiments can be operated in at least one of a multiplicity of reaction regions and in at least one of the following modes: separately, collectively, consecutively, simultaneously, or combinations thereof.

The reaction regions in all four basic embodiments may be any structure that can hold a sample and allow the transformation, the control and the analysis to be performed. Preferably, the reaction regions in all embodiments are each of a multiplicity of e.g. chambers, spheres, cubes, wells, tubes, or structure of their combinations and can be composed in the form of a plate, a microplate, an integrated form, or a chip. However, the structures that can hold a sample like: chambers, spheres, cubes, wells, tubes, or structures of their combinations need to have the total cubic capacity below 100 micro liters, need to operate: in whole or in at least one part or in at least one point of the DC-potential range form -2.5 V to 2.5 V, in whole or in at least one part or in at least one point of the compliance voltage range from -5.0 V to 5.0 V, with whole or in at least one part or in at least one point of the AC -potential amplitude from 1 mV to 250 mV, or a current flow through the reaction region is from 1 nA to 10 mA. Additionally, chambers, spheres, cubes, wells, tubes, or structures of their combinations need to be resistant to dimethyl sulfoxide (DMSO) and can be made partially from materials like: metal, semiconductor, platinum, gold, silver, or graphite.

The methods in all four basic embodiments can be used in diversity of modes and for various purposes like: throughput mode, high-throughput mode, fast high-throughput mode, ultra high-throughput mode, automated mode, robotized mode, computerized mode, equipped with peripheral equipment mode, screening, multiple synthesis, parallel synthesis, or combinatorial synthesis.

The systems in all four basic embodiments can be used in diversity of modes and for various purposes like: throughput mode, high-throughput mode, fast high-throughput mode, ultra high-throughput mode, automated mode, robotized mode, computerized mode, equipped with peripheral equipment mode, screening, multiple synthesis, parallel synthesis, or combinatorial synthesis.

This invention further provides a high-throughput system in which the screening rate/capacity is several orders of magnitude higher than in currently available systems. The newly constructed system and invented methods are intended especially for hepatic stability screening and in vivo clearance prediction of leads.

This marked, verified and substantial progress in high-throughput technology can be used in a construction of an integrated miniaturized chemical chip for metabolic stability studies and for prediction of drugs in vivo clearance.

Accordingly, it is an object of the present invention to provide methods and systems for controlled in situ electron-transfer induced transformation reactions, initiated by an appropriate activation, and combined with a simultaneous detection and analysis of the sample and the formed sample derivatives in the reaction region for controlled formation of derivatives or an assessment of the stability of a sample. A further object of the present invention is to provide methods and systems for individually performed transformation of a multiplicity of samples for controlled formation of derivatives and increased throughput analysis. Other objects of the present invention will be apparent in light of the description of the invention embodied herein.

BRIEF DESCRIPTION OF DRAWINGS

A more complete understanding of the invention may be obtained by reading the following detailed description in conjunction with the attached drawing in which: FIG. 1 is a perspective view of a multi reaction chamber plate in accordance with the principles of the invention.

FIG. 2 is a block diagram of a synthesis and analysis system in accordance with the principles of the invention.

FIG. 3 is a XY scatter of drugs in vivo clearance values and oxidation potential for drugs with one and more oxidation steps.

FIG. 4 is a XY scatter of drugs in vivo clearance values and oxidation potential for drugs with one and more oxidation steps with oxidation steps recognition.

FIG. 5 is a linear fit of in vivo clearance of drugs with their oxidation potential with only one oxidation step.

FIG. 6 is a linear fit of in vivo clearance of drugs with their oxidation potential with one and more oxidation steps where oxidations potentials from multiple oxidation steps were averaged.

DETAILED DESCRIPTION OF THE INVENTION

Metabolic stability and clearance of drugs are mainly driven by biotransformation reactions like oxidation, reduction, hydrolysis and conjugation in the liver and extraheptic tissues. The oxidative metabolism is the most common biotransformation reaction and is catalyzed by a family of mixed- function oxidases. In order to accelerate studies on drug metabolism, especially the oxidation process, a multi reaction chamber plate shown on a FIG. 1 together with an analyzing-controlling apparatus (FIG. 2) was designed and constructed. The innovative system allows to test simultaneously up to 98 drugs and to determine conditions in which well-defined derivatives are produced from the investigated compounds. The derivatives are formed in processes similar to natural biotransformations by in situ electron- transfer induced oxidation, reduction and following on hydrolysis and conjugation. These targeted in situ transformations of screened compounds in to desired derivatives and their in situ detection and analysis are based on optimal sample composition and optimized instrumental parameters. Consideration of these parameters and sample requirements in an assay protocol ensures finest emulation of real cellular metabolic processes in a reaction chamber and their in situ detection. The protocol facilitates the production of well-defined derivatives similar to the biochemical decomposition to metabolites by hepatic cells. The emulation of cellular metabolic processes in the reaction chamber allows exclusion of enzymes, hepatocytes and microsomes, which are difficult to handle and in a screening system. The derivatives of drugs formed in a reaction chamber plate are similar to cellular metabolites and can be furthermore transferred to additional analysis systems (electrochemical, electroacoustic, chromatographic, spectroscopic, spectrometric, or based on transducers methods) by robotic, automatic procedures. The outcome of these studies together with information from in situ detection provides valuable data about metabolites formed from the investigated drugs and their metabolic conversions. Markedly, the data enable the correlation with the in vivo clearance of drugs and their reduction and/or oxidation potential. This correlation is very important, because enables the prediction of the in vivo hepatic clearance of drugs from well defined, purely chemical experiments without the use of biological material. To illustrate this finding a set 50 commercially available drugs (Sigma: 5- Fluorouracil, Acyclovir, Allopurinol, Amiloride, Amitriptyline, Bupropion, Chlorothiazide, Cimetidine, Clozapine, Desipramine, Diclofenac, Dobutamine, Doxepin, Enalapril, Felodipine, Flumazenil, Haloperidol, Hydralazine, Imipramine, Isosorbide dinitrate, Itraconazole, Labetalol, Nimodipine, Nortriptyline, Protryptilline, Sertraline, Spironolactone, Tamoxifen, Terfenadine, Timolol; Fluka: Acetaminophen, Amoxicillin, Captopril, Chlorpromazine, Cephalexin, Chloroquine, Dapsone, Dextromethorphan, Diltiazem, Dopamine, Erythromycin, Naloxone, Phenacetin, Procainamide, Quinidine, Theophylline, Verapamil; Aldrich: Propranolol; GlaxoSmithKline: Sumatriptan; Riedel-de-Haen: Nicotine) was chosen and investigated in the invented high-throughput system. The list of 50 known drugs together whit human in vivo metabolic clearance values (ml/min/kg) derived from published data and determined in the invented system by the invented method (Amiloride, Desipramine, Dopamine, Phenacetin and Terfenadine), and their oxidation potential (V) measured during oxidative formation of derivatives in multi reaction chamber plate are presented in the Table 1.

TABLE 1

List of commercially available compounds/drugs with their in vivo hepatic clearance* and oxidation potential values measured in invented system based on multi reaction chamber plate. *Clearance of compounds/drugs derived from: Hardman JG, Limbird LE, and Gilman AG (2001) Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11th edition, McGraw-Hill, New York; (a) http://www.clinicaldruguse.com; (b) R. J. Riley et. all, Drug Metabolism and Disposition, (2005) 33(9): 1304-11; Poster, A. Koganti et. all, In Vitro Technologies, Inc.; (d) clearance values determined in the invented system by the invented method. In vivo hepatic clearance values above 20 ml/min/kg represents CUF calculated from oral pharmacokinetics, where F is absolute bioavailability.

At first glance a correlation between in vivo hepatic clearance of drugs and their oxidation potentials seems not straightforward as it is shown in FIG. 3. However, the same data after a filtering process, based on multiple oxidation steps in drugs, show signs of correlation as shows FIG. 4. If only in vivo clearance of drugs with one oxidation step versus oxidation potentials are selected and presented on a graph as shows FIG. 5, the good correlation can be easy recognized. Finally, if in vivo clearance of drugs, showing plurality of oxidation potentials versus a calculated average values from their oxidation potential is plotted as shows FIG. 6 the good correlation is very well recognized.

The high-throughput systems and methods of the invention include several important features, which markedly improve the transformation and screening process of samples. One important feature is that the screening rate/capacity is several orders of magnitude higher than in currently available systems. The fully automated and computerized system equipped with a multi reaction chamber plate, a connecting and switching module, electrochemical analyzers- controllers, analyzers, plate storage system, baskets, pumps, delivery cups, garbage cans, liquid waste outputs computer and industrial robot as is shown in a FIG. 2 is able to screen above 100 OOO compounds per week. Another important feature is that the multi reaction chamber plate construction prevents cross-contamination of samples and guarantee identical starting conditions in all reaction chambers of the plate. This feature has an enormous impact on the speed and quality of the transformation processes and on the value of the screening results.

Another important feature of the invention is that the system and method of construction allow the hepatic stability screening and in vivo clearance prediction of leads. The invention provides also a validated technology which can be used in a construction of an integrated miniaturized chemical chip for metabolic stability studies and for prediction of drugs in vivo clearance.

The invented high-throughput system is realized by employing a fully automated robot that operates with a multi reaction chamber plate, special detection and controlling systems with novel protocols for handling multiple synthetic and analytic tasks. The multi reaction chamber plate is constituted by a micro array plate comprising multiple reaction chambers, and specially prepared electrodes to induce and control oxidation, reduction, hydrolysis and conjugation of compounds. Electrodes are connected to a controlling, analyzing and detecting system. The invented multi reaction chamber plate operates with scrupulously selected composition of the samples, physicochemical parameters and electrical parameters determined in an extensive research program and partially presented in the Tables 2, 3 and 4.

TABLE 2

TABLE 3

TABLE 4

Composition of these parameters in a study protocol affords the emulation of real cellular metabolic processes in a reaction chamber and their in situ detection. The invented study protocol is realized by different steps and cycles with feedback control in order to transform samples in due time in to desired derivatives in the reaction chambers. The steps and cycles are realized by a various screening templates. Employment of templates enables automatic and widespread in situ transformation of diverse samples and in situ detection of the freshly formed various species. All electrochemically active compounds (e.g, carbohydrates, molecules with thiol and amine moieties, aromatic structures containing hydroxyl, methoxy or amino groups) are directly detected in the reaction chamber by the use of screening templates based on various detection schemes. These screening templates of selected detection scheme are constructed from scrupulously determined scan ranges and electrical parameters by experiments like continuous-current or pulsed amperometry. Examples of such electrochemical screening templates for a Linear Sweep Voltammetry (LSV) and Cyclic Chronovoltammetry (CC) are shown in Tables 5 and 6. Numbers 1, 2, 3 etc. or Y , 2', 3' etc. in Tables 5 and 6 indicate the steps order in a fragmentary protocol. TABLE 5

The electrochemical screening templates based on various electrochemical methods are applied directly in research chambers to all screened samples and to their derivatives. Electrochemically inactive samples together with those selected in template research are further investigated outside the reaction chamber in screening templates based on electrochemical, chromatographic, spectroscopic or spectrometric techniques. The novel protocol enables combination of various detection techniques enables also to collect data of the sample stability and redox values enabling prediction in vivo clearance.

Advantageously, this novel protocol provides the flexibility of adding prioritary candidates to the list of compounds to be oxidized and/or reduced and/or analyzed. Moreover the order in which the compounds are analyzed or processed can be freely modified. More importantly, this protocol flexibility shortens the respite time, accordingly new syntheses and analyses can be performed at a significantly higher efficiency. The respite time in which the next compound is processed and/or analyzed is minimized due to the possibility to perform several different analysis steps simultaneously; thus, even before a complete sequence of analytic steps is completed for all reaction chambers, a new sequence can already be started allowing parallel processing. In an exemplary embodiment, the novel system for determination and evaluation of metabolic stability of compounds and prediction of clearance comprises the following elements: a multi reaction chamber plate, a connecting and switching module, electrochemical analyzers- controllers, analyzers, plate storage system, baskets, pumps, delivery cups, garbage cans and liquid waste outputs computer and industrial robot. System elements are fully automated operating under computer control for performing the production and analysis steps within each cycle of the selected compounds in desired protocol. Each production and analysis cycle comprises different steps: washing, adding compounds, incubation, adding coupling reagents, electrically forced coupling, in situ detection and analysis, collection and injection, additional analysis, determination and evaluation of drugs metabolic stability and clearance. Because incubation, forced coupling, in situ detection and analysis, additional analysis, determination and evaluation of drugs metabolic stability and clearance do not involve any robot action they are passive protocol steps, although are chemically or analytically active, the other steps are active production and analysis steps. Importantly, the novel protocol is realized by having the robot system, during the passive production and analysis steps perform active steps for the production and analysis cycle of the production and analysis of the next compound.

In a preferred embodiment of the invention, a system for determination and evaluation of metabolic stability and prediction of drugs clearance is provided, which contains seven major components: i) a multi reaction chamber plate comprising electrodes, ii) a connecting and switching module, iii) electrochemical analyzers-controllers, iv) analyzers, v) a plate storage system with heated incubators and integrated shakers, vi) a multifunctional robotic station, vii) a main computer with software managing data and controlling all components. The multi reaction chamber with electrodes is additionally equipped with photo-, temperature-, humidity- active electronic elements with memory or serial number. The connecting and switching component is switched sequentially or in a parallel mode, selectively or in another selected scheme to perform most effectively production and analysis steps in desired protocol. The analyzers use electrochemical, electroacoustic, chromatographic, spectroscopic, spectrometric, or based on transducers detection scheme. The robotic station includes a fully automated robotic manipulation arm, pipeting-mixing unit, irradiation unit, baskets, pumps, delivery cups, garbage cans and liquid waste outputs. The system is configured to prepare, incubate, irradiate, analyze, store, re-prepare, re-incubate, re-irradiate, and reanalyze compounds to perform most effectively in situ production and analysis steps with desired protocol and data acquisition. The system is computerized and fully integrated by the use of software managing data and controlling all components. The main computer is used as a data storing, analyzing, processing, and reporting device and also to control, communicate and integrate all production and analysis processes.

The present invention has been described and illustrated in detail with reference to the preferred embodiments by way of example only, and not by way of limitation. Those skilled in the art would recognize that various modifications may be made without departing from the scope of the invention. Therefore, the present invention is not intended to be limited to what is described in the specification. Consequently, it is deliberate that the invention be limited only to the scope of the enclosed claims.

Claims

1. A method of controlled in situ electron-transfer induced transformation reactions of a sample in a reaction region, initiated by at least one appropriate activation in the reaction region, and combined with a simultaneous in situ analysis of the sample and the formed sample derivatives in the reaction region for at least one of the following purposes: controlled formation of derivatives; or an assessment of the stability of a sample.

2. A method of controlled in situ electron-transfer induced transformation reactions combined with the simultaneous in situ analysis of the sample comprising: providing sample in the reaction region; providing at least one appropriate activation in the reaction region in order to induce the transformation reactions; permitting the sample to react in the reaction region in order to form derivatives; operating the control in the reaction region to control the transformation reactions, the environment of transformations and the formation of derivatives; and analyzing the sample together with the formed sample derivatives.

3. The method as claimed in claims 1 or 2 wherein the in situ electron-transfer is induced by at least one of the following: an inhomogeneous reaction between at least one electrode and the sample in solution; or a homogeneous reaction between at least one mediator and the sample in solution.

4. The method as claimed in claims 1 or 2 wherein the in situ electron-transfer is performed in buffered solution under inert gas atmosphere.

5. The method as claimed in claims 1 or 2 wherein the transformation reactions comprising at least one of the following: combination; decomposition; oxidation; reduction; hydrolysis; or conjugation.

6. The method as claimed in claims 1 or 2 wherein at least one of the following: appropriate activation or control in the reaction region has nature selected from at least one of the following: biological; chemical; electrical; electrochemical; thermal; light; electromagnetic; transverse waves; or longitudinal waves.

7. The method as claimed in claims 1 or 2 wherein the sample contain at least one of the following molecules: solvent; water; cosolvent; DMSO; chloride anions; supporting electrolyte; mediator; drug; drug candidate; lead; chemically active species; biologically active species; antioxidant; or oxygen.

8. The method as claimed in claims 1 or 2 wherein the sample do not contain any: enzymes; proteins; microsomes; hepatocytes; cells organelles; or cells.

9. The method as claimed in claims 1 or 2 wherein the sample fulfills at least one of the following requirements: the pH of a sample from 3 to 5; the pH of a sample from 6 to 8; the pH of a sample from 8 to 10; the dimethyl sulfoxide (DMSO) concentration up to 12%; the chloride anions concentration up to 5%; the investigated compound concentration up to 1 mM; the total volume of a sample up to 70 micro liters; or the dissolved oxygen concentration up to 8.5 mg/L.

10. The method as claimed in claims 1 or 2 wherein the in situ analysis follows at least one optional analysis, and where at least one optional or in situ analysis is selected from at least one of the following detection schemes: electrochemical; electroacoustic; chromatographic; spectroscopic; spectrometric; or based on transducers.

11. The methods as claimed in claim 10 wherein the detection scheme is based on a screening template and selected from at least one of the following: amperometry; voltammetry; capacitance; impedance; fluorescence; absorbance; infra red; phosphorescence; chemiluminescence; electroluminescence; Raman; electron spin resonance; refractive index; mass spectrometry (MS); tandem mass spectrometry (MS/MS); liquid chromatography (LC); gas chromatography (GC); or high performance liquid chromatography (HPLC) measurement.

12. The method as claimed in claims 1, 2 or 10 wherein at least one analysis is used for at least one of the following: assessment of a resistance; assessment of a impedance; assessment of a capacitance; assessment of a chemical stability; assessment of a biological stability; determination of a susceptibility to metabolism; determination of a susceptibility to oxidative stress; determination of the quantities of the formed derivatives; determination of the qualities of the formed derivatives; determination of the kinetics of the transformations; determination of a susceptibility to reductive stress; the determination of the clearance; determination of the hepatic clearance; or determination of the in vivo hepatic clearance of a sample.

13. The methods as claimed in claim 12 wherein assessments or determinations are based on at least one of the following:

(a) sample parameters: pH; concentration; diffusivity; potential; standard potential; oxidation potential; reduction potential; redox potential; based on the tendency of a chemical species to gain electrons; or based on the tendency of a chemical species to loss electrons; (b) correlations between at least one described in (a) sample parameters and at least one of the following characteristic of a sample: stability; chemical stability; biological stability; susceptibility to metabolism; susceptibility to oxidative stress; formed derivatives quantities; formed derivatives qualities; susceptibility to reductive stress; clearance; hepatic clearance; or in vivo hepatic clearance;

14. The method as claimed in claims 1 or 2 wherein at least one of the following: the transformation; the control; or the analysis is performed by the use of at least two electrodes.

15. The method as claimed in claims 1 or 2 wherein at least one of the following: the transformation; the control; or the analysis is based on at least one of the following: amperometry; voltammetry; capacitance; or impedance method.

16. The method as claimed in claims 1 or 2 wherein at least one of the following: the transformation; the control; or the analysis is operated in at least one of a multiplicity of reaction regions and in at least one of the following modes: separately; collectively; consecutively; simultaneously; or combinations thereof.

17. The method as claimed in claims 1 or 2 wherein the reaction regions are each of a multiplicity of: chambers; spheres; cubes; wells; tubes; or structures of their combinations and are composed in at least one of the following forms: plate; microplate; an integrated form; or chip.

18. The methods as claimed in claim 17 wherein at least one of described: chambers; spheres; cubes; wells; tubes; or structures of their combinations have the total cubic capacity below 100 micro liters.

19. The methods as claimed in claim 17 wherein at least one of described: chambers; spheres; cubes; wells; tubes; or structures of their combinations operate: in whole or in at least one part or in at least one point of the DC -potential range form -2.5 V to 2.5 V; in whole or in at least one part or in at least one point of the compliance voltage range from -5.0 V to 5.0 V; with whole or in at least one part or in at least one point of the AC-potential amplitude from 1 mV to 250 mV; or a current flow through the reaction region is from 1 nA to 10 mA.

20. The methods as claimed in claim 17 wherein at least one of described: chambers; spheres; cubes; wells; tubes; or structures of their combinations are resistant to dimethyl sulfoxide (DMSO) and are made partially from at least one of the following materials: metal; semiconductor; platinum; gold; silver; or graphite.

21. The method as claimed in claims 1 or 2 wherein the methods are used in at least one of the following: throughput mode; high-throughput mode; fast high- throughput mode; ultra high-throughput mode; automated mode; robotized mode; computerized mode; equipped with peripheral equipment mode; screening; multiple synthesis; parallel synthesis; or combinatorial synthesis.

22. A system for controlled in situ electron-transfer induced transformation reactions of a sample in a reaction region, initiated by at least one appropriate activation in the reaction region, and combined with a simultaneous in situ analysis of the sample and the formed sample derivatives in the reaction region for at least one of the following purposes: controlled formation of derivatives; or an assessment of the stability of a sample.

23. A system for controlled in situ electron-transfer induced transformation reactions combined with the simultaneous in situ analysis of the sample comprising: a reaction region for the sample; at least one appropriate activation in the reaction region in order to induce the transformation reactions; reaction region for the formation of derivatives; a control in the reaction region to control the transformation reactions, the environment of transformations and the formation of derivatives; and in situ analysis for the sample together with the formed sample derivatives.

24. The system as claimed in claims 22 or 23 wherein the in situ electron-transfer is induced by at least one of the following: an inhomogeneous reaction between at least one electrode and the sample in solution; or a homogeneous reaction between at least one mediator and the sample in solution.

25. The system as claimed in claims 22 or 23 wherein the in situ electron-transfer is performed in buffered solution under inert gas atmosphere.

26. The system as claimed in claims 22 or 23 wherein the transformation reactions comprising at least one of the following: combination; decomposition; oxidation; reduction; hydrolysis; or conjugation.

27. The system as claimed in claims 22 or 23 wherein at least one of the following: appropriate activation or control in the reaction region have nature selected from at least one of the following: biological; chemical; electrical; electrochemical; thermal; light; electromagnetic; transverse waves; or longitudinal waves.

28. The system as claimed in claims 22 or 23 wherein the sample contain at least one of the following molecules: solvent; water; cosolvent; DMSO; chloride anions; supporting electrolyte; mediator; drug; drug candidate; lead; chemically active species; biologically active species; antioxidant; or oxygen.

29. The system as claimed in claims 22 or 23 wherein the sample do not contain any: enzymes; proteins; microsomes; hepatocytes; cells organelles; or cells.

30. The system as claimed in claims 22 or 23 wherein the sample fulfills at least one of the following requirements: the pH of a sample from 3 to 5; the pH of a sample from 6 to 8; the pH of a sample from 8 to 10; the dimethyl sulfoxide (DMSO) concentration up to 12%; the chloride anions concentration up to 5%; the investigated compound concentration up to 1 mM; the total volume of a sample up to 70 micro liters; or the dissolved oxygen concentration up to 8.5 mg/L.

31. The system as claimed in claims 22 or 23 wherein the in situ analysis follows at least one optional analysis, and where at least one optional or in situ analysis is selected from at least one of the following detection schemes: electrochemical; electroacoustic; chromatographic; spectroscopic; spectrometric; or based on transducers.

32. The systems as claimed in claim 31 wherein the detection scheme is based on a screening template and selected from at least one of the following: amperometry; voltammetry; capacitance; impedance; fluorescence; absorbance; infra red; phosphorescence; chemiluminescence; electroluminescence; Raman; electron spin resonance; refractive index; mass spectrometry (MS); tandem mass spectrometry (MS/MS); liquid chromatography (LC); gas chromatography (GC); or high performance liquid chromatography (HPLC) measurement.

33. The system as claimed in claims 22, 23 or 31 wherein at least one analysis is used for at least one of the following: assessment of a resistance; assessment of a impedance; assessment of a capacitance; assessment of a chemical stability; assessment of a biological stability; determination of a susceptibility to metabolism; determination of a susceptibility to oxidative stress; determination of the quantities of the formed derivatives; determination of the qualities of the formed derivatives; determination of the kinetics of the transformations; determination of a susceptibility to reductive stress; the determination of the clearance; determination of the hepatic clearance; or determination of the in vivo hepatic clearance of a sample.

34. The systems as claimed in claim 33 wherein assessments or determinations are based on at least one of the following:

(a) sample parameters: pH; concentration; diffusivity; potential; standard potential; oxidation potential; reduction potential; redox potential; based on the tendency of a chemical species to gain electrons; or based on the tendency of a chemical species to loss electrons;

(b) correlations between at least one described in (a) sample parameters and at least one of the following characteristic of a sample: stability; chemical stability; biological stability; susceptibility to metabolism; susceptibility to oxidative stress; formed derivatives quantities; formed derivatives qualities; susceptibility to reductive stress; clearance; hepatic clearance; or in vivo hepatic clearance;

(c) statistical relations between at least one described in (a) sample parameters and at least one described in (b) sample characteristic; or

35. The system as claimed in claims 22 or 23 wherein at least one of the following: the transformation; the control; or the analysis is performed by the use of at least two electrodes.

36. The system as claimed in claims 22 or 23 wherein at least one of the following: the transformation; the control; or the analysis is based on at least one of the following: amperometry; voltammetry; capacitance; or impedance method.

37. The system as claimed in claims 22 or 23 wherein at least one of the following: the transformation; the control; or the analysis is operated in at least one of a multiplicity of reaction regions and in at least one of the following modes: separately; collectively; consecutively; simultaneously; or combinations thereof.

38. The system as claimed in claims 22 or 23 wherein the reaction regions are each of a multiplicity of: chambers; spheres; cubes; wells; tubes; or structures of their combinations and are composed in at least one of the following forms: plate; microplate; an integrated form; or chip.

39. The systems as claimed in claim 38 wherein at least one of described: chambers; spheres; cubes; wells; tubes; or structures of their combinations have the total cubic capacity below 100 micro liters.

40. The systems as claimed in claim 38 wherein at least one of described: chambers; spheres; cubes; wells; tubes; or structures of their combinations operate: in whole or in at least one part or in at least one point of the DC-potential range form -2.5 V to 2.5 V; in whole or in at least one part or in at least one point of the compliance voltage range from -5.0 V to 5.0 V; with whole or in at least one part or in at least one point of the AC-potential amplitude from 1 mV to 250 mV; or a current flow through the reaction region is from 1 nA to 10 mA.

41. The systems as claimed in claim 38 wherein at least one of described: chambers; spheres; cubes; wells; tubes; or structures of their combinations are resistant to dimethyl sulfoxide (DMSO) and are made partially from at least one of the following materials: metal; semiconductor; platinum; gold; silver; or graphite.

42. The system as claimed in claims 22 or 23 wherein the systems are used in at least one of the following: throughput mode; high-throughput mode; fast high- throughput mode; ultra high-throughput mode; automated mode; robotized mode; computerized mode; equipped with peripheral equipment mode; screening; multiple synthesis; parallel synthesis; or combinatorial synthesis.