WO2005015181A1

WO2005015181A1 - Method and kit for determining the metabolic stability of test substances

Info

Publication number: WO2005015181A1
Application number: PCT/EP2004/008597
Authority: WO
Inventors: Niels KREBSFÄNGER; Johannes DÖHMER
Original assignee: Genpharmtox Biotech Ag
Priority date: 2003-07-31
Filing date: 2004-07-30
Publication date: 2005-02-17
Also published as: DE10335114A1

Abstract

The invention relates to a method for measuring the enzymatic oxidation of test substance by oxygen (metabolic stability) by time-resolved measurement of the oxygen concentration in a solution which, besides the enzyme, includes the test substance and oxygen at the start of the measurement, by measuring the luminescent light signal of two or more different luminescent materials, wherein at least the light intensity of a first luminescent material is influenced by the oxygen concentration, and the light intensity or the light intensity and the decay time of a second luminescent material is not influenced by the oxygen concentration, and wherein the oxygen concentration is determined from the intensities of the light signals of the first and second luminescent materials, said signals being measured either separately or in the form of a total signal by one or more detectors. The invention furthermore relates to a kit comprising a sensor means comprising said at least two luminescent materials and a biological sample containing an enzyme suitable for the oxidation of the test substance by oxygen.

Description

Method and kit for determining the metabolic stability of test substances

Description

The invention relates to a method for determining the metabolic stability of a test substance, to a device and to the use of a device for the purposes of this method.

The development of novel medicaments, herbicides or insecticides and of many other substances includes pharmacokinetic studies in order to determine the residence time in the human and animal body and in order to draw conclusions therefrom about the activity and toxicity of the particular test substance on the human body.

Among the enzyme systems involved in the conversion of substances for the purpose of excretion (detoxication) are those subsumed under the name cytochrome P450 oxidase (CYP; EC 1.14.14.1). These enzyme systems catalyse the funtionalization of a test substance with consumption of oxygen e.g. according to the following reaction:

Test substance + O₂ + NADPH+H⁺ -» test substance-OH + NADP⁺+H₂O

To date, in this connection the enzymatic degradation (the so-called "metabolic stability" and derived

"species differences" and "CYP profiling") of the particular test substance (substrate) is determined by direct measurement of the substance by means of LC-MS (mass spectrometry) ("loss of parent compound"). This method requires an LC-MS analytical method which is substance-specific in each case and which limits the throughput in screening in terms of time and costs, and use is not regarded as practicable for larger substance libraries (millions of compounds) . Since, however, NADPH and oxygen are consumed in the CYP-dependent metabolism of every substance, it is possible by measuring one or both of these parameters to avoid the limitation of the substance-specific analytical method.

In practice, the problems of this approach derive both from the not always stoichiometric reaction of the CYPs (a certain proportion of H₂0₂ and water is produced in blank/parallel reactions) and the reliable detection of the NADPH or 0₂, which is why it has not been possible to obtain results acceptable in practice with the methods tested to date.

For example, determination of the metabolic stability of diclofenac with the aid of CYP2C9, a recombinantly expressed human CYP enzyme, has been described (Laura E. Dike, Richard Guarino, Darwin Asa, Mark Timmins : "Development of an Automatible, High Throughput Method for Rapid Analysis of Drug Metabolism" , experimental data were shown at the ISSX in Nashville, TN, USA on 19.10.1999). In this case, a microtitre plate from BD Vias-Sante® which has an oxygen-sensitive fluorescent dye, namely tris-4 , 7-diphenyl-l, 10-phenanthroline- ruthenium(II) chloride ( or tris-1, 7-diphenyl-l, 10- phenanthroline- ruthenium(II) chloride ?) , incorporated in a gas-permeable and hydrophobic matrix on the bottom of the microtitre plate, was used, the oxygen in the neighbourhood of the dye being in equilibrium with that in liquid phase. The fluorescent light which is reduced by the oxygen quenching processes is measured.

The inventors have found that the described measurement system does not provide usable results at any rate on comparison of substrates other than that diclofenac, or on use of other cytochrome P450 systems than that mentioned above (CYP2C9) , and in particular on use of cellular systems or on use of (liver) microsomes as source of CYP enzyme systems. It is true that cellular systems are also mentioned in the state of the art, however, this does not take place in connection with measurement of the conversion by means of the reaction equation mentioned above, but takes place in another connection, namely for cytotoxic tests in which the toxicity-related reduction in oxygen consumption by the cells, and not the metabolism-related reduction in oxygen consumption by the xenobiotic-metabolizing cytochromes P450, is detected.

The inventor's object was therefore to provide a method for measuring the enzymatic oxidation of a test substance by oxygen, • which can be applied universally, in which the enzyme source can be either an isolated, possibly recombinant, enzyme, a microsome preparation, an S9 fraction or a homogenate from a cell culture or from an animal organ or else a cell suspension, • which is suitable for investigating a large number of test substances,

• which avoids the elaborate apparatus and the costs associated therewith for an LC-MS method,

• which is easy to carry out and reliable, • which is suitable for automated high-throughput methods .

It has been found that the method described in WO 99/06821, and the device described therein, is very suitable for achieving the object, for which reason

WO 99/06821 is incorporated in this application by reference .

The invention described therein relates to a method and to a device which can be used in a spectrophotometer for the fluorometric determination of a biological, chemical or physical parameter of a sample using at least two different luminescent materials, the first of which responds to the parameter at least in the luminescence intensity, and the second of which does not respond to the parameter at least in the luminescence intensity and the decay time.

In particular WO 99/06821 relates to a novel concept for the optical detection of chemical parameters with the aid of optical sensors based on phase-shift and time-resolved measurements. The modulation frequencies used are between 0.5 and 5 MHz and can be detected with low-cost optical semiconductor components.

It is known from the literature and practice of optical sensors that in luminescence measurements there are crucial practical advantages in determining the decay time, instead of the intensity, as measured quantity. This quantity is only inconsiderably or, in the best case, absolutely not influenced by fluctuations in the optical system. Both changes in the intensity of the light source and in the sensitivity of the photodetector and signal losses through curvature of fibres or influencing the signal intensity through changing the geometry of the sensor have no effects on the measured signal. This also applies to undefined optical properties of the sample (such as turbidity, intrinsic colour and refractive index) which may lead to problems in intensity measurements . There is furthermore in many cases a substantial independence of the measured signal from the concentration of the indicator in the sensitive layer. For this reason, photodecompositions and leaching are critical to only a small degree.

A large number of measurement principles for measuring chemical parameters on the basis of the decay time have been proposed in the literature. One of the methods most frequently used is dynamic luminescence quenching, in which case the excited state of the luminescent indicator undergoes radiationless deactivation by the analyte. This is the basis for the optical measurement of molecular oxygen, and the detection of halide and heavy metal ions (1) .

A further possibility of deactivation makes use of the photoinduced electron transfer in a single indicator molecule. In this effect (called PET for short), the luminescent indicator is present in various forms, only one of which (acidic form or with bound metal ion) is highly luminescent and has a long lifetime. In the second form (basic form or without bound metal ion) , the indicator has a free electron pair capable of radiationless deactivation of the excited state. The consequence is a reduction both in the decay time and in the luminescence quantum yield. This principle can be applied to the optical determination of the pH or in optical ion sensors (2) .

A further possibility proposed for measuring the decay time consists in that certain pH indicators luminesce with differing intensity and differing but defined decay time in the protonated and deprotonated state. In this case, an average decay time results from the particular (pH-dependent) ratio of the two intensities, and can be measured (3) . It is a prerequisite for this method that both forms of an indicator luminesce, and their absorption and emission spectra show distinct regions of overlap .

It is important to note that the decay times measured in most of the measurement principles cited hitherto are ordinarily in the region of few nanoseconds. However, accurate measurement of decay times in the lower nanosecond range requires very elaborate instruments and, besides very fast circuits and high modulation frequencies, also requires fast light sources and detectors. The development of low-cost measuring equipment for this type of sensors, based on optical semiconductor components such as light-emitting diodes and photodiodes, therefore appears to be largely precluded in the near future. Low-cost measuring equipment is, however, indispensable for use of optical sensors on a wide scale. For this reason there is a great interest in decay sensors whose measurement range varies in the region of microseconds or even milliseconds. Such sensors have to date been developed as far as the practical stage almost exclusively for optical oxygen measurement, employing indicators with decay times ranging to a few milliseconds.

A route which has recently been followed for developing novel long-lived decay time sensors makes use of radiationless energy transfer between a luminescent donor molecule, whose photophysical properties are not influenced by the analyte, to a colour indicator which is sensitive to the analyte and is referred to as acceptor. The absorption spectrum thereof must overlap to different extents, depending on the particular analyte concentration, with the emission spectrum of the donor. It is possible to employ as luminescent donor transition metal complexes with ruthenium (II) , ruthenium (I) or osmium and iridium as central atom. These compounds are distinguished by long lifetimes (a few 100 nsec to a few microseconds) and high quantum yields. This route was proposed for the first time by Lakowicz and has recently been applied to optical pH sensors, it being possible in principle for optodes for pC0₂, NH₃ and ion detection to be implemented analogously (4,5).

A serious problem on practical use of such sensors is that the rate of energy transfer, and thus the measurable average decay time, depends on the one hand significantly on the distance and the positioning of donor and acceptor molecule, but also on the other hand on the concentration of the acceptor in the matrix. For this reason, every change in the distribution and distance of the indicators in the matrix leads to changes in the characteristics of the sensors. Swelling of the matrix in particular represents a considerable problem.

A further problem is the influence of oxygen on the sensors . Since the luminescence of the employed long- lived donors is in some cases considerably quenched by oxygen, the oxygen concentration must also be measured and the measured signal must be corrected. In addition, in this process there is production in the membrane of reactive singlet oxygen which speeds up the photo- decomposition of the immobilized indicators. This reduces both the storage and the long-term stability of the sensors. This is also, of course, associated with loss of one of the classical advantages of measuring the decay time.

US 5 102 625 discloses a device of the type mentioned at the outset. In this case, two separate measuring channels are used to measure the intensities of two luminescent materials separately. The intensity ratio thereof is used as final signal for measuring the parameter. The decay times of the luminescent materials are not included in the measurement . The two luminescent materials have different spectral ranges.

The object of WO 99/06821 is therefore to indicate a method and a device for fluorometric determination of the parameter of a sample, which method answers the purpose with less elaborate apparatus but with high measurement accuracy.

WO 99/06821 describes a new measurement principle which makes fluorometric determination possible for various chemical, physical and biological parameters with the aid of time-resolved and phase modulation techniques. The invention permits the intensity signal of most fluorescence sensors described in the literature to be referenced very efficiently by admixing a long-lived luminescent material. For this purpose, two different luminescent materials are co-immobilized together in the sensor. The total of a luminescence signal with constant long-lived decay time (min. a few hundred nanoseconds) and of a short-lived fluorescence signal is measured. Whereas the long-lived luminescence is not influenced in its parameters by analytes, the intensity of the co-immobilized short-lived luminescent material changes depending on the particular analyte concentration. Since the phase shift Φm ascertained by phase-modulation techniques depends only on the ratio of the intensity contributions of the two individual luminescent materials, this parameter directly reflects the intensity of the luminescent material which responds to the parameter. The invention thus comprises a novel method for internal referencing of the signal intensity of fluorophores without the need for a second light source or a second photodetector. Provided that the distribution of the two luminescent materials is kept constant during the production process, Φm depends exclusively on physical or chemical parameters to be determined, whereas fluctuations in the optoelectronic system, losses in the light guides and the optical properties of the sample do not influence the signal .

It is preferred for both luminescent materials to absorb light in the same wavelength range and thus to be capable of luminescence excitation with the same light source. The emission spectra are preferably in the same spectral range. Thus, it is possible for example to excite both luminescent materials with blue light at a wavelength of 450 nm, one luminescent material emitting green light at 520 nm and the second emitting red light at 600 nm, because both signals can nevertheless be measured with the same detector. However, simultaneous measurement of the luminescence of two luminescent materials which differ distinctly from one another in their emission spectra is also possible . The described measurement method has the advantage that the long-lived luminescent material need not show any analyte-specific reaction, but functions solely as carrier of a constant background signal which has a long decay time and which is modulated by the shortlived luminescent material. For this reason, a large number of phosphorescent compounds described in the literature are suitable for this purpose.

The long-lived luminescent material does not need to interact with the sample, the analyte and the fluorescent indicator, and can therefore be immobilized in a form in which it is inert for all components of the sample, and thus potential interferences by chemical parameters are precluded from the outset.

WO 99/06821 is explained below by means of exemplary embodiments .

Figure 1 shows the dependence of the measured phase angle Φm on the ratio of the intensity of the fluorescent indicator and of the reference luminescent material: A high fluorescence signal, B lower fluorescence signal. The meanings are: flu = variable fluorescence signal; ref = constant reference signal; tot = total measured signal;

Figure 2 shows a calculated relationship between the measured phase angle Φm, and cot (Φm) , and the amplitude ratio R of the two luminescent materials;

Figure 3 shows spectral properties of a suitable pair of fluorescent indicator and reference luminescent material . The optimal spectral windows for excitation of the luminescence signal and measurement of the emitted light are shaded;

Figure 4 shows a time-resolved measurement of the ratio of the signal intensity during the excitation pulse (II) and during the decay of the luminescence (12) , where the ratio R depends on the total height of the signal and represents only one function of the chemical parameter to be determined;

Figure 5 shows pH calibration plots of a pH sensor according to Example 1 with varying amount of HPTS (A: little HPTS; B: much HPTS), measured as phase shift at a modulation frequency of 880 kHz. The light source used here is a blue LED and the detector is a photodiode; and

Figure 6 shows four possible combinations of shortlived chemically sensitive luminescent material (A) and of inert long-lived luminescent material (B) in an optical sensor.

Examples of suitable luminescent materials which are inert for the analyte and have long decay times are:

- transition metal complexes with ruthenium (II) , rhenium (I) or osmium and iridium as central atom and diimine ligands,

- phosphorescent porphyrins with platinum, palladium, lutetium or tin as central atom;

- phosphorescent complexes of the rare earths such as europium, dysprosium or terbium;

- phosphorescent crystals such ruby, Cr-YAG, alexandrite or phosphorescent mixed oxides such as magnesium fluorogermanate .

Suitable short-lived fluorescent luminescent materials are all dyes whose excitation and emission spectrum overlaps with one of the long-lived luminescent materials quoted above and whose fluorescence intensity depends on the parameter to be determined.

Examples of potentially possible luminescent material pairs or luminophore/fluorophore pairs are: ruthenium(II) (tris-4, 7-diphenyl-l, 10-phenanthroline) /-

HPTS ruthenium (11) (tris-4, 7-diphenyl-l, 10-phenanthroline) /- fluorescein ruthenium(II) (tris-4, 7-diphenyl-l, 10-phenanthroline) -

/rhodamine B ruthenium(II) (tris-4, 7-diphenyl-l, 10-phenanthroline) -

/rhodamine B octadecyl ester ruthenium(II) (tris-4, 7-diphenyl-l, 10-phenanthroline) -

/hexadecyl acridine orange europium (III) tristhenoyltrifluoroacetonate/hydroxyl- methylcoumarin platinum (II) tetraphenylporphyrin/rhodamine B platinum (II) tetraphenylporphyrin/sulphorhodamine 101 platinum (II) octaethylporphyrin/eosin platinum(II) octaethylporphyrin/thionin platinum (II) octaethylketoporphyrin/Nile blue

The long-lived luminescent material can be integrated into the sensor in various ways (Fig. 6) :

- by directly dissolving the luminescent material in the analyte-sensitive layer (Fig. 6, Example 3) - by incorporating into a polymer which serves as primer for the sensor layer (Fig 6, Example 1)

- by incorporating into a polymer which is dispersed in micro- or nanoparticles in the sensitive layer (Fig. 6, Example 2) - by incorporating luminescent dyes into sol-gel glass with subsequent sintering, powdering and dispersion of the glass in the sensor layer (Fig. 6, Example 2)

- by using powdered phosphors which are dispersed in the sensitive layer (Fig. 6; Example 2) - by coating the outside with a sensor sheet without contact with the sample (Fig. 6; Example 4)

- by covalent or electrostatic linkage of the fluorescent indicator to the surface of luminescent material particles which are either dispersed in a polymer layer or directly dispersed in the sample - by dispersing particles in the sample in which the fluorescent indicator is present in dissolved form.

It is important to mention that it is possible with the aid of phase modulation techniques at frequencies in the kHz range with this type of sensors always only to measure an average phase shift. Although splitting in the two decay time components is possible in principle, the high frequencies required make the measurement techniques complicated. The distinct differences in their decay times of the two co-immobilized indicators results in the time-resolved measurement in which, after an excitation light pulse, exclusively the decay behaviour of the luminescence signal after switching off the excitation pulse is ascertained, in many cases not a useful parameter because the short-lived component decays too quickly and can be detected only with very elaborate measurement techniques.

Separate time-resolved measurement of the signal intensity during the excitation pulse and in the decay phase, and calculation of the ratio of these two signals R, are also possible. As is evident from Figure 4, this ratio depends exclusively on the intensity ratio R of the two luminescent components and is entirely independent of the total intensity of the signal .

It is furthermore possible with phase modulation techniques to measure the average phase shift of the luminescence signal . The measurement frequency in this case is adapted to the decay time of the luminescent material and is between 0.5 and 100 kHz. As is shown below, it is evident from equation (1) that the measured phase angle Φm depends only on the ratio of the two signal intensities but not on the absolute height of the signal, and thus enables referencing of the intensity of the short-lived fluorescence contribution.

There is an additive overlap of the signals (index ref = reference signal, index flu = fluorescence signal; index m = measured quantity) .

A_m -cøsΦ_m ^« A„_f -eos ^ ÷A_flu .cosΦ_flu A_ffl . sin _m * Ar-tf . sϊn Φr._f + A_u - sin Φ

General conditions No.l

The longer decay time is very much larger than the shorter decay time

If the modulation frequency is chosen to be optimal for

''-ref i .e. t _tmi «2w-f_lβarf.τ_Λ,«*1 the following applies for Φfi_u

with the condition applying for the angle Φfi_u: tanΦ 'flu %t_te«τ_m flu " Q∑& fl-u - ' t

No. 2

The decay time of the dye with the longer decay time is constant for the measurement of interest: χ_nt - constant =-> tanΦ _f ~ constant s> φref = constant

The additive equations thus simplify to: A_m * CQSΦ_ro sr Artf ' CO _r#f ÷ A_fla A_I7, sinΦ-_n = A_Mr . slnΦ _#f

Dividing the first equation by a second one results in

A_M-s-nΦ_m ^■_ AøH.alnΦ* ~ A_nf rTS 'r«f

Since a plot of cot Φm linearly reflects the amplitude ratio, a linear relation applies between the cotangent of the measured phase angle Φm and the amplitude ratio R (and thus also intensity ratio) of the two fluorescent materials (see Figure 2) .

cot Φm expresses an intensity ratio without the need to measure two intensity signals separately.

The essential advantages of WO 99/06821 are:

- A very small expenditure on synthesis and optimization in the production of novel sensors.

- Simple changeover of previously optimized fluorescence sensors to decay time measurements through simple admixture of the long-lived luminescent material . - It is possible without difficulty always to use the same long-lived luminescent material for a field of sensors for different analytes . It is thus possible to evaluate different sensors with the same optoelectronic system. - Since the shape of the calibration plot depends only on the ratio of the two intensities, the sensitivity range of a single sensor can be optimized solely by changing the added amount of luminescent material . - The same change can be achieved by an optimal selection of the spectral windows both on the side of excitation and on that of emission.

- The cross-sensitivity of long-lived luminescence to oxygen can be eliminated by incorporating the indicators in materials which are not accessible to gases .

- If phosphorescent solids, or luminescent materials incorporated into glass, are used, every influencing of the signal by chemical luminescent parameters in the sample is completely precluded.

- Because oxygen cannot quench luminescence, no reactive singlet oxygen is produced in the membrane. As a consequence, the photodecomposition is reduced and the stability of the sensors is improved.

- Incorporation of the long-lived luminescent materials into a glass matrix or as solid completely prevents leaching thereof. In addition, their photostability is exceptionally high. - Whereas with measurement principles in which the analyte deactivates the excited state of the long- lived luminescent material (such as PET effect, dynamic quenching or energy transfer) , the decrease in the average decay time is always associated with a parallel decrease in the signal intensity, leading to a deterioration of the signal/noise ratio, the signal intensity becomes larger as the decay time decreases with this type of sensors. The consequence is a considerably better signal/noise ratio over the entire measurement range.

Since the characteristics of these sensors depend solely on the ratio of the signal contributions of the two indicators, however, the following conditions should be met:

1) neither of the indicators may leach during the measurement ;

2) the rates of decomposition of the two indicators due to the action of light during the measurement should not differ;

3) the concentration ratio of the two indicators must be kept constant in the production of the membranes;

4) the decay time of the long-lived luminescent material must always be constant .

These conditions can be met without difficulty for many sensors.

The oxygen sensor to be used according to the invention has a similar structure to the following systems, which are indicated for illustration:

pH sensor

- HPTS absorbed onto cellulose with quaternary ammonium groups and incorporated in poly (hydroxyethyl methacrylate) (HEMA) hydrogel

- Ru(phen)₃Cl₂ in sol-gel (sintered, ground and dispersed in the hydrogel) Figure 5 shows the calibration plot of this sensor measured as phase shift at a frequency of 80 kHz.

pH sensor

- Aminofluorescein covalently coupled to sol -gel particles with incorporated Ru (phen) ₃C1₂ (sintered, ground and dispersed in the hydrogel)

C0₂ sensor

- HPTS-CTA₃ ion pair dissolved in ethylcellulose with tetraoctylammonium hydroxide as buffer (according to 6)

NH₃ sensor - rhodamine B dissolved in PVC with NPOE (according to 7)

- Pt(II) tetrapentafluorophenylporphyrin in PVC as outer coating of a sensor sheet

Potassium sensor

- lipophilized Nile blue dissolved in PVC with plasticizers (according to 8) - Pt(II) octaethylketoporphyrin in PVC as outer coating

WO 99/06821 thus permits optical determination of a chemical, biological or physical parameter of a sample with the aid of an optical sensor. Two luminescent indicators are co-immobilized in the sensor, of which one indicator functions as carrier of a background luminescence signal which is characterized by a long luminescence lifetime (preferably in the range from at least 100 nanoseconds up to some milliseconds) , and which is influenced neither in its intensity nor in its decay time by the parameter to be measured. The second indicator has a short-lived fluorescence, preferably in the region of a few nanoseconds, which overlaps the long-lived luminescence signal and whose intensity is a function of the parameter to be measured. Phase modulation or time-resolved measurement methods are used in this case to determine a reference quantity which expresses the ratio of the two luminescence intensity contributions, this reference quantity being independent of the total intensity of the luminescence signal, and thus enabling the referencing of the shortlived analyte-dependent fluorescence component.

References

O.S. Wolfbeis, Fiber Optic Chemical Sensors and Biosensors Vol. II CRC press 1991 S. Draxler, M.E. Lippitsch, Sens. Actuators B29, 199, 1995 3. J.R. Lakowicz, H. Szmacinski, Sens. Actuators Bll, 133, 1993

4. J.R. Lakowicz, H. Szmacinski, M. Karakelle Anal. Chim. Acta 272, 197, 1993 5. J. Sipior, S. Bambot, M. Romauld, G.M. Carter, J.R. Lakowicz, G. Rao, Anal. Biochem 227, 309, 1995

6. A. Mills, Q. Chang, Analyst, 118, 839, 1993

7. C. Preininger, G.J. Mohr, I. Klimant, O.S. Wolfbeis, Anal. Chim. Acta 334, 113, 1996

8. U.E. Spichinger, D. Freiner, E. Bakker, T. Rosatzin, W. Simon, Sens. Actuators Bll, 262, 1993

Achievement of the object of the present invention is based on a method according to Claim 1, a use according to Claim 24 and on a kit according to Claim 29.

The achievement of the object is a method for measuring the enzymatic oxidation of a first reactant, of a test substance by a second reactant, namely oxygen, by time- resolved measurement of the oxygen concentration in a solution which, besides the enzyme, includes both reactants at the start of the measurement, by measuring the luminescent light signal of two or more different luminescent materials (flu, ref) , where, under the chosen measurement conditions, (a) at least the light intensity of the first luminescent material (flu) is influenced by the oxygen concentration, and where furthermore (b) the light intensity or the light intensity and the decay time of the second luminescent material (ref) is not influenced, or is influenced only inconsiderably within the scope of the accuracy of measurement, by the oxygen concentration set up during the measurement, and where furthermore (c) the intensity of the light signals of the two luminescent materials is measured either separately in each case or in the form of a total signal by one or more detectors, and the oxygen concentration is determined therefrom.

In this connection, the expression "under the chosen measurement conditions" means that the properties of the luminescent materials may be different under different conditions, and it is sufficient for the purposes of the invention that they satisfy the abovementioned criteria under the measurement conditions . A time-resolved measurement of the oxygen concentration is the determination of the oxygen concentration (or of a parameter proportional thereto) as a function of time . One modification of the method consists in that the decay time of the first luminescent material (flu) is longer than the decay time of the second luminescent material (ref) .

One modification of the method consists in that, in step (c) , the time or phase behaviour is measured, and the second luminescent material is selected so that both the light intensity and the decay time of the second luminescent material (ref) is not influenced, or is influenced only inconsiderably within the scope of the accuracy of measurement, by the oxygen concentration set up during the measurement.

One modification of the method consists in that a reference quantity, which is independent of the total intensity of the two luminescent materials in the particular solution, is obtained from the measured time or phase behaviour, and the oxygen concentration is determined using the reference quantity.

One modification of the method consists in that the decay time of the second luminescent material (ref) is longer than that of the first luminescent material (flu) . One modification of the method consists in that the excitation or/and emission spectra of the luminescent materials (flu, ref) overlap one another.

One modification of the method consists in that the luminescent materials (flu, ref) are excited jointly by a single light source.

One modification of the method consists in that the luminescent materials (flu, ref) are excited simultaneously, in particular with simultaneous start of excitation and same duration of excitation.

One modification of the method consists in that a measured phase shift (Φm) of the phases of the total signal (tot) of the two luminescent materials is used as reference quantity.

One modification of the method consists in that a ratio of the two intensities 11 and 12 over time is used as reference quantity, where 11 is the measured intensity over time of the emitted light from the two luminescent materials (flu, ref) with the light source switched on, and 12 is the measured intensity over time of the emitted light of the reference dye (ref) after the illumination is switched off.

One modification of the method consists in that the short-lived luminescent material is immobilized on the surface of particles which contain the long-lived luminescent material, and the particles are introduced without additional carrier directly into the sample.

One modification of the method consists in that the enzyme is a cytochrome P450 oxidase, including all families, subfamilies and isoenzymes, and variants of all species, and all artificial recombinant variants.

One modification of the method consists in that the enzyme is EC 1 . 14 . 14 . 1 .

One modification of the method consists in that the enzyme is added to the solution in a form enclosed in animal cells, in which case mitochondrial respiration is suppressed.

One modification of the method consists in that the enzyme is made available in the form of subcellular systems, e.g. a protein, a microsome fraction, an S9 mix or a homogenate, the preparations being derived from animal cell cultures or from animal organs such as the liver, or from microorganisms, including bacteria or yeasts.

One modification of the method consists in that entry of oxygen from the surroundings into the solution is avoided.

One modification of the method consists in that the avoidance is brought about by a protective gas or by sealing with a liquid in which oxygen dissolves only in traces .

One modification of the method consists in that the solution is covered with a layer of silicone oil.

One modification of the method consists in that the measurement error in measuring the oxygen decrease is reduced by determining the hydrogen peroxide formed during the enzymatic oxidation, and by correcting the stoichiometry of the enzymatic oxidation for the side reaction yielding the hydrogen peroxide.

One modification of the method consists in that catalase or an enzyme with the same catalytic activity is added to the solution.

The invention also relates further to methods for determining one or more of the pharmacokinetic characteristics of a test substance, including: - the metabolic stability, meaning measuring the metabolism-related decrease in the amount of substance, - the species dependence of the metabolic stability of a test substance on comparison of the same test system from different species, - the kinetics of the metabolizing enzymes, meaning investigation of the substrate concentration and time dependence of the reaction rate of an enzymatic reaction relating to metabolization of the test substance, - determination of CYP inhibition/CYP inhibition constant, meaning the CYP inhibition/CYP inhibition constant of inhibition of CYP activity in relation to the metabolism of a test substance by reference inhibitors or of reference substrates by other (new) substrates. - CYP profiling, meaning the identification of which CYP metabolizes a particular substrate, where it includes the features of one or more of the preceding methods .

The invention also relates further to methods where one luminescent material is a platinum porphyrin, in particular a C1-, C2-, C3-, C4-, C5-, C6-, to C12- tetra to octa alkyl derivative of platinum (II) porphyrin, in particular platinum(II) tetraphenyl- porphyrin or platinum (II) octaethylporphyrin.

One modification of the method consists in that two of the luminescent materials are platinum(II) tetraphenyl- porphyrin/rhodamine B or platinum (II) tetraphenyl- porphyrin/sulphorhodamine 101.

The invention also relates to the use of a device comprising a sensor means which has at least two different luminescent materials (flu, ref) , where • at least the light intensity of the first luminescent material (flu) is influenced by the oxygen concentration, and where further • the light intensity or the light intensity and the decay time of the second luminescent material (ref) is not influenced, or is influenced only inconsiderably within the scope of the accuracy of measurement, by the oxygen concentration set up during the measurement , for measuring the enzymatic oxidation of a first reactant, of a test substance by a second reactant, namely oxygen in a solution.

The purpose of use may also relate to the determination of the metabolic stability of a test substance.

The purpose of use may also relate to the determination of one or more of the pharmacokinetic characteristics of a test substance, including: - the metabolic stability, meaning measuring the metabolism-related decrease in the amount of substance, - the species dependence of the metabolic stability of a test substance on comparison of the same test system from different species, - the kinetics of the metabolizing enzymes, meaning investigation of the substrate concentration and time dependence of the reaction rate of an enzymatic reaction relating to metabolization of the test substance, - determination of CYP inhibition/CYP inhibition constant, meaning the CYP inhibition/CYP inhibition constant of inhibition of CYP activity in relation to the metabolism of a test substance by reference inhibitors or of reference substrates by other (new) substrates. - CYP profiling, meaning the identification of which CYP metabolizes a particular substrate, in particular by time-resolved measurement of the oxygen concentration.

One modification of the use comprises one luminescent material comprising a platinum porphyrin, in particular a C1-, C2-, C3-, C4-, C5-, C6-, to C12- tetra to octa alkyl derivative of platinum (II) porphyrin, in particular platinum (II) tetraphenylporphyrin or platinum(II) octaethylporphyrin, even more preferably platinum (II) tetraphenylporphyrin/rhodamine B or platinum(II) tetraphenylporphyrin/sulphorhodamine 101.

The invention also relates to a kit which is intended to be used in one of the abovementioned methods. This kit includes as first constituent a sensor means which has at least two different luminescent materials (flu, ref) , where • at least the light intensity of the first luminescent material (flu) is influenced by the oxygen concentration, and where further • the light intensity or the light intensity and the decay time of the second luminescent material (ref) is not influenced, or is influenced only inconsiderably within the scope of the accuracy of measurement, by the oxygen concentration set up during the measurement, and as second constituent a biological sample containing an enzyme suitable for the oxidation of a first reactant, of a test substance by a second reactant, namely oxygen, with the proviso that no actively respiring plant or animal cell or actively respiring microorganism is involved.

The second constituent is preferably a subcellular system, e.g. a protein, a microsome fraction, an S9 mix or a homogenate, the preparations being derived from animal cell cultures or from animal organs such as the liver, or from microorganisms, including bacteria or yeasts. Subcellular systems are generally produced from cells which have been lysed. They accordingly do not represent a living cell.

In a preferred embodiment of the kit, the second constituent is lyophilized. In a preferred embodiment of the kit, both constituents represent one unit. For example, the first constituent may represent a microtitre plate which has an incorporated sensor and in the wells of which the second constituent is present, in particular preferably in lyophilized form, so that both constituents form a unit .

Oxygen sensor

An oxygen sensor which makes it possible to measure the oxygen concentration even without time-resolved measurement of the luminescent light signal is described below.

An oxygen sensor may be provided in the form of a microtitre plate in 96-well or other (e.g. 384-well) format, each well containing an integrated sensor. The latter is advantageously immobilized on the bottom of each plate as described in Figure 7. The sensor Θ can, as described in Figure 7, just like the source for the excitation light ®, be disposed on the bottom. This disposition is implemented or can be implemented in a commercially available fluorescence reader able to measure luminescence signals. The sensor contains two different dyes. One dye/luminescent material is an oxygen indicator. Its luminescent light intensity I_IN, which preferably comprises phosphorescence, depends on the concentration of oxygen in the liquid present in the well. The oxygen sensor is preferably a platinum porphyrin, in particular selected from platinum (II) tetraphenylporphyrin, platinum (II) octaethylporphyrin, platinum (II) octaethylketoporphyrin.

The other luminescent material/dye represents the reference. Its luminescent light intensity I_RF, which is preferably fluorescence, does not depend on the oxygen concentration. The luminescent material in this case is preferably a rhodamine derivative, preferably rhodamine, in particular selected from rhodamine B, rhodamine B octadecyl ester, sulphorhodamine 101.

It is possible, starting from these two luminescent light signals, that is to say luminescence intensities, to calculate the following ratio I_R. This signal I_R correlates with the concentration of oxygen. The response times are usually very short if the sensor is also permeable for non-gaseous substances, which is particularly preferred.

Calibration of the reader, which may be a conventional luminescence reader, usually takes place by determining two instrument constants through measurement of the luminescence of 8 wells. The subsequent measurement with the aid of other ' sensor plates is calibration- free. It may be necessary, as a consequence of the drift of the optical system of the fluorescence reader, to recalibrate the reader from time to time.

A commercially available luminescence reader which determines the fluorescence intensity from the bottom of the sensor plate can be used for measuring the sensor plate. Because the sensor of the plate described above has two different dyes whose light signals are to be measured without recourse to time-resolved measurements, the reader must be able to operate in the dual kinetic mode. This means that two measurements can be recorded with the aid of two different filter pairs or gratings per relevant time point (measurement point) . In this case, usually two filter pairs (4 filters) are required to measure the oxygen concentration in a well of the sensor plate .

The following system is preferably employed: filter pair 1: 544/650 nm filter pair 2: 544/590 nm

However, it is also, of course, possible to use equivalent means such as gratings, as known to the skilled person. The first filter pair (filter pair 1) makes it possible to measure the luminescence of luminescent material Iι_N, and the second filter pair (filter pair 2) measures the luminescence of the reference dye I_RF. Although these combinations are suitable, other filter combinations are conceivable.

Calibration of the fluorescence reader

The ratio I_R correlates with the oxygen concentration in each well. Because the absolute light intensity of the fluorescence reader is unknown, it is determined for example with the aid of a two-point calibration of the reader.

Preparation of the calibration standards

Calibration of the fluorescence reader used is carried out for example by a conventional two-point calibration using oxygen-free water ("cal 0") and air-saturated water ("cal 100") .

Preparation of the cal 0 solution (oxygen-free water) For example, one gram of sodium sulphite (Na₂S0₃) or half a gram of sodium dithionite (Na₂S₂0₄) is put into a suitable vessel and dissolved in 100 ml of water. The vessel is closed after the calibration in order to miminize the entry of oxygen. After addition of the sodium sulphite, a minute is allowed to elapse until all the sodium sulphite or sodium dithionite has dissolved, in order to ensure that the water is oxygen- free. Provided that the vessel in which the cal 0 solution has been stored was closed, it is possible to use the standard solution for 24 hours. Preparation of the calibration solution cal 100 (oxygen-saturated water)

For example, 100 ml of water are put into a suitable vessel. This is closed and vigorously shaken for two minutes until the contents are oxygen-saturated. The calibration solution "cal 100" can be used for 3 days, provided that the temperature is kept constant and the vessel has been tightly closed.

Since the oxygen saturation is salt- and temperature- dependent, it is advantageous to take care that the calibration is carried out at the temperatures and ionic strength conditions at which the measurement is also intended to take place later.

Two-point calibration of the measuring instrument

The signal I_R for solution cal 0 and solution cal 100 which has been introduced into the well of the sensor plate must be determined in order to obtain the instrument constants k₀ and kioo which are defined below. If the calibration has been carried out once using 8 wells in the sensor plate, it is possible to carry out further sensor plates without recalibration, i.e. they are then calibration-free. However, as a consequence of drift phenomena, repeated calibration may be necessary for example each week. For the purpose of calibration, the solution cal 0 can be introduced into well Al-Dl. Solution cal 100 can be introduced into well El-Hl.

For a microtitre plate in 96-well format, each time 100-200 microlitres of the solution are necessary. In order to avoid further entry of oxygen, it may be necessary to cover with a layer of a sealing solution such as, for example, oil or to cover with a film or to work under a protective gas atmosphere (e.g. nitrogen) . The signal I_R for each well is determined using the equation

The instrument constant k₁₀o is determined by the following equation:

kioo = ¹/4*[I_R(A1)+ I_R(BI)+ I_R(C1)+ I_R(D1)]

The instrument constant k₀ is ascertained after determining the respective values of I_R from the following equation:

k₀ = %*[I_R(E1)+ I_R(FI)+ I_R(G1)+ I_R(H1)]

The oxygen partial pressure or the oxygen concentration in a measurement solution is derived from the following equation, using the previously determined instrument constants :

p0₂ = 100*(ko/l_R-l)/(ko/k₁₀o-l)

This value can be converted into other units such as Torr, hPa, g/1, mol/1 or ppm at a given temperature and atmospheric pressure, as is known to the skilled person.

Other measurement methods are also conceivable. If only the total intensity of the luminescent light signal is measured, it is, of course, possible to dispense with a complicated optical system to separate the light signals of the two luminescent materials (double filter pair or grating pair) . Time-resolved determination of the total intensity then takes place, either after regular excitation indirectly via the phase angle with the aid of phase modulation techniques, as described above, or by direct (real) time measurement, which makes determination of the oxygen concentration possible, even with a single or irregular excitation. Systems which are particularly suitable as source of the enzyme which converts the test substance are described below.

(I) Non-recombinant enzymes: subcellular preparations from microbiological cultures (e.g. bacteria or yeast preparations), cell cultures (e.g. Sf9 insect cells or mammalian cells such as V79 cells) or animal organs, such as, in particular, the liver, which contain these enzymes. The subcellular preparations may be Supersomes®, Bactosomes®, microsomes, cell homogenates or an S9 mix. It is additionally possible also to employ cellular systems as long as mitochondrial respiration is at least suppressed by appropriate inhibitors, so that the oxygen consumption resulting from ^' oxidation of the test substance remains measurable . Concerning the term "S9 mix" : S9 mix from the liver in particular is employed to determine the metabolic stability of substances. This comprises the 9000 x g fraction after differential centrifugation of homogenized (liver) tissue, see, for example, Maron, D. and Ames, B. (1983) Revised methods for the Salmonella mutagenicity test. u at. Res . , 113, 173-215. http://mutage.oupjournals.org/cgi/external ref?access n um=6341825&link type=MED;

Atsushi Hakura, Satoshi Suzuki, Shigeki Sawada, Satoru Motooka, Tet^'suo Satoh, An improvement of the Ames test using a modified human liver S9 preparation, Journal of Pharmacological and Toxicological Methods 46(3) ; (2001) pp. 169-172; http : //www. elsevier . com/cdweb/journals/10568719/viewe r.htt?vol=46&viewtype=issue&iss=3#S105687190200186. Concerning the term microsomes : microsomes, in particular liver microsomes, are conventionally employed for determining the microsomal metabolic stability of substances. This comprises the 100 000 x g fraction after differential centri- fugation of homogenized (liver) tissue, see, for example, Gill HJ, Tingle MD and Park BK (1995) N-hydroxylation of dapsone by multiple enzymes of cytochrome P450: Implications for inhibition of haemotoxicity . Br. J. Clin Pharmacol 40: 531-538; http: //dmd. aspetjournals .org/cgi/external ref?access num=8703658&link type=MED;

R.J. Riley, D. Howbrook, In vitro Analysis of the Activity of the Major Human Hepatic CYP Enzyme (CYP3A4) Using [N-Methyl-¹⁴C] -Erythromycin, Journal of Pharmacological and Toxicological Methods 38 (4) (1997) pp. 189-193; htt : //www. elsevier . com/cdweb/journals/10568719/viewe r.htt?vol=38&viewtype=issue&iss=4#S105687199700103; (II) Recombinant enzymes, especially CYPs, which have been expressed recombinantly in mammalian or insect cells, can likewise be used. It is possible to employ in the method subcellular preparations of these expression systems such as Supersomen®, microsomes, cell homogenates, S9 mix as well as the cellular systems described above .

Recombinant and non-recombinant enzymes, for example also in the form of microsomes and of S9 mix, can preferably be obtained from animal species and organs. Particularly important for the method is human, monkey, dog, pig, rat, mouse, rabbit and hamster liver. Appropriate enzyme preparations can moreover also be obtained from microbiological cultures or plant cell cultures or plants.

Example A

Test protocol for determining the metabolic stability

"Mastermix" preparation on ice: Aqueous buffer (50 mM potassium phosphate, pH 7.4), test system (human liver microsomes, 1.25 mg of microsomal protein/ml) , electron donor (1 mM NADPH) - pipetting of controls oxidized control: water; reduced control: 1% sodium sulphite in water or 0.5% sodium dithionite in water - distribution of the mastermix on the microtitre plate in 96 -well format, described above, 100 μl per well - addition of the substances to be tested: as lOOx concentrated stock solution in DMSO - covering with a layer of silicone oil (about 2 drops per well) reduces the oxygen resaturation by entry of air; an alternative possibility is to work under a nitrogen atmosphere - reading the plate with a fluorescence microtitre plate reader from Labsystems by measuring the fluorescence from the direction of the base of the plate; duration for example 20-40 min - data processing with instrument control software and Excel (Microsoft) .

The example is described below by Fig. 7b to 17. These show:

Fig. 7b: the most important reaction equation and possible applications,

Fig. 8a: the raw data obtained after oxygen measurement for 60 minutes,

Fig. 8b: the data obtained after two-point calibration with oxygen-saturated control (Ox. Control) , and oxygen-free control (Red. Control) , control without test substance (TI blank) , with 100 μM coumarin, midazolam, testosterone, paclitaxel, bufuralol, tamoxifen and verapamil from the raw data from Fig. 8a, Fig. 9: a summary of parameters to be considered,

Fig. 10: a further summary of parameters to be considered,

Fig. 11: a demonstration of the effect of oil on the oxygen resaturation with 10 μm bufuralol with and without oil,

Fig. 12: the optimum found for the measured parameters, where the microsome concentration relates to the corresponding protein concentration, the test substance concentration is optimal at 100 μM when different substances are to be compared, and the optimal incubation volume relates to a 96-well plate, Fig. 13: a state of the art screening method with LC-MS/MS, Fig. 14: a measurement of the oxygen consumption for 100 μM coumarin, midazolam, testosterone, paclitaxel, bufuralol, tamoxifen, verapamil and grouping into fast metabolism (fast), moderately fast (i.m.), slow metabolism (slow) , Fig. 15: a comparison of state of the art LC-MS/MS with oxygen consumption according to the invention (good agreement) ;

Fig. 16: an example of the use ' of recombinant enzymes, in this case human CYP2D6, for demonstrating the slow metabolism of tamoxifen and the fast metabolism of bufuralol, in each case 30 μM, compared with the measurements obtained by HPLC-FLD/LC-MS (good agreement) ;

Fig. 17: measures for improving the accuracy of measurement .

Claims

1. Method for measuring the enzymatic oxidation of a first reactant, of a test substance by a second reactant, namely oxygen, by time-resolved measurement of the oxygen concentration in a solution which, besides the enzyme, includes both reactants at the start of the measurement, by measuring the luminescent light signal of two or more different luminescent materials (flu, ref) , where, under the chosen measurement conditions, (a) at least the light intensity of the first luminescent material (flu) is influenced by the oxygen concentration, and where furthermore (b) the light intensity or the light intensity and the decay time of the second luminescent material (ref) is not influenced, or is influenced only inconsiderably within the scope of the accuracy of measurement, by the oxygen concentration set up during the measurement , and where furthermore (c) the intensity of the light signals of the two luminescent materials is measured either separately in each case or in the form of a total signal by one or more detectors, and the oxygen concentration is determined therefrom.

2. Method according to Claim 1, characterized in that the decay time of the first luminescent material (flu) is longer than the decay time of the second luminescent material (ref) .

3. Method according to Claim 1, characterized in that in step (c) , the time or phase behaviour is measured, and the second luminescent material is selected so that both the light intensity and the decay time of the second luminescent material (ref) is not influenced, or is influenced only inconsiderably within the scope of the accuracy of measurement, by the oxygen concentration set up during the measurement .

4. Method according to Claim 3, characterized in that a reference quantity, which is independent of the total intensity of the two luminescent materials in the particular solution, is obtained from the measured time or phase behaviour, and the oxygen concentration is determined using the reference quantity.

5. Method according to either of Claims 3 and 4, characterized in that the decay time of the second luminescent material (ref) is longer than that of the first luminescent material (flu) .

6. Method according to any of the preceding claims, characterized in that the excitation or/and emission spectra of the luminescent materials (flu, ref) overlap one another .

7. Method according to any of the preceding claims, characterized in that the luminescent materials (flu, ref) are excited jointly by a single light source. .

8. Method according to any of the preceding claims, characterized in that the luminescent materials (flu, ref) are excited simultaneously, in particular with simultaneous start of excitation and same duration of excitation.

9. Method according to any of Claims 3 to 8, characterized in that a measured phase shift (Φm) of the phases of the total signal (tot) of the two luminescent materials is used as reference quantity.

10. Method according to any of Claims 3 to 9, characterized in that a ratio of the two intensities 11 and 12 over time is used as reference quantity, where 11 is the measured intensity over time of the emitted light from the two luminescent materials (flu, ref) with the light source switched on, and 12 is the measured intensity over time of the emitted light of the reference dye (ref) after the illumination is switched off.

11. Method according to any of Claims 1 to 10, characterized in that the short-lived luminescent material is immobilized on the surface of particles which contain the long-lived luminescent material, and the particles are introduced without additional carrier directly into the sample.

12. Method according to any of Claims 1 to 11, characterized in that the enzyme is a cytochrome P450 oxidase, including all families, subfamilies and isoenzymes, and variants of all species, and all artificial recombinant variants.

13. Method according to any of Claims 1 to 11, characterized in that the enzyme is EC 1.14.14.1.

14. Method according to any of Claims 1 to 13, characterized in that the enzyme is added to the solution in a form enclosed in animal cells, in which case mitochondrial respiration is suppressed.

15. Method according to any of Claims 1 to 13, characterized in that the enzyme is made available in the form of subcellular systems, such as a protein, a microsome fraction, an S9 mix or a homogenate, the preparations being derived from animal cell cultures or from animal organs, such as the liver, or from microorganisms, including bacteria or yeasts.

16. Method according to any of Claims 1 to 13, characterized in that entry of oxygen from the surroundings into the solution is avoided.

17. Method according to Claim 16, characterized in that the avoidance is brought about by a protective gas or by sealing with a liquid in which oxygen dissolves only in traces .

18. Method according to Claim 17, characterized in that the solution is covered with a layer of silicone oil.

19. Method according to any of the preceding claims, characterized in that the measurement error in measuring the oxygen decrease is reduced by determining the hydrogen peroxide formed during the enzymatic oxidation, and by correcting the stoichiometry of the enzymatic oxidation for the side reaction yielding the hydrogen peroxide .

20. Method according to any of Claims 1 to 18, characterized in that catalase or an enzyme with the same catalytic activity is added to the solution.

21. Method for determining one or more of the phar acokinetic characteristics of a test substance, including: - the metabolic stability, meaning measuring the metabolism-related decrease in the amount of substance, - the species dependence of the metabolic stability of a test substance on comparison of the same test system from different species, - the kinetics of the metabolizing enzymes, meaning investigation of the substrate concentration and time dependence of the reaction rate of an enzymatic reaction relating to metabolization of the test substance, - determination of CYP inhibition/CYP inhibition constant, meaning the CYP inhibition/CYP inhibition constant of inhibition of CYP activity in relation to the metabolism of a test substance by reference inhibitors or of reference substrates by other (new) substrates. - CYP profiling, meaning the identification of which CYP metabolizes a particular substrate, characterized in that it includes the features of one or more of the preceding claims.

22. Method according to any of Claims 1 to 21, where one luminescent material is a platinum porphyrin, in particular a C1-, C2-, C3-, C4-, C5-, C6-, to C12- tetra to octa alkyl derivative of platinum(II) porphyrin, in particular platinum(II) tetraphenylporphyrin or platinum (II) octaethylporphyrin.

23. Method according to any of Claims 1 to 22, characterized in that two of the luminescent materials are platinum (II) tetraphenylporphyrin/rhodamine B or platinum(II) tetraphenylporphyrin/sulphorhodamine 101.

24. Use of a device comprising a sensor means which has at least two different luminescent materials (flu, ref) , where • at least the light intensity of the first luminescent material (flu) is influenced by the oxygen concentration, and where further • the light intensity or the light intensity and the decay time of the second luminescent material (ref) is not influenced, or is influenced only inconsiderably within the scope of the accuracy of measurement, by the oxygen concentration set up during the measurement , for measuring the enzymatic oxidation of a first reactant, of a test substance by a second reactant, namely oxygen in a solution.

25. Use according to Claim 24 for determining the metabolic stability of a test substance.

26. Use according to Claim 24 for determining one or more of the pharmacokinetic characteristics of a test substance, including: - the metabolic stability, meaning measuring the metabolism-related decrease in the amount of substance, - the species dependence of the metabolic stability of a test substance on comparison of the same test system from different species, - the kinetics of the metabolizing enzymes, meaning investigation of the substrate concentration and time dependence of the reaction rate of an enzymatic reaction relating to metabolization of the test substance, - determination of CYP inhibition/CYP inhibition constant, meaning the CYP inhibition/CYP inhibition constant of inhibition of CYP activity in relation to the metabolism of a test substance by reference inhibitors or of reference substrates by other (new) substrates. - CYP profiling, meaning the identification of which CYP metabolizes a particular substrate, in particular by time-resolved measurement of the oxygen concentration.

27. Use according to any of Claims 24 to 26, where one luminescent material is a platinum porphyrin, in particular a C1-, C2-, C3-, C4-, C5-, C6-, to C12- tetra to octa alkyl derivative of platinum (II) porphyrin, in particular platinum (II) tetraphenylporphyrin or platinum (II) octaethylporphyrin.

28. Use according to any of Claims 24 to 27, characterized in that two of the luminescent materials are platinum (II) tetraphenylporphyrin/rhodamine B or platinum(II) tetraphenylporphyrin/sulphorhodamine 101.

29. A kit comprising as first constituent a sensor means which has at least two different luminescent materials (flu, ref) , where • at least the light intensity of the first luminescent material (flu) is influenced by the oxygen concentration, and where further • the light intensity or the light intensity and the decay time of the second luminescent material (ref) is not influenced, or is influenced only inconsiderably within the scope of the accuracy of measurement, by the oxygen concentration set up during the measurement , and as second constituent a biological sample containing an enzyme suitable for the oxidation of a first reactant, of a test substance by a second reactant, namely oxygen, with the proviso that no actively respiring plant or animal cell or actively respiring microorganism is involved.

30. Kit according to Claim 29, where the second constituent is a subcellular system, e.g. a protein, a microsome fraction, an S9 mix or a homogenate, the preparations being derived from animal cell cultures or from animal organs such as the liver, or from microorganisms, including bacteria or yeasts.

31. Kit according to Claim 30, where the second constituent is in lyophilized form.

32. Kit according to Claim 29 or 30, in which the two constituents form a unit.