Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/557—Immunoassay; Biospecific binding assay; Materials therefor using kinetic measurement, i.e. time rate of progress of an antigen-antibody interaction

Definitions

• the present invention relates to methods for evaluating an effect characteristic of a biochemically active stimulation means, including biochemically active com- pounds .
• the dissociative anaesthetic MK-801 (a dizocilpine maleate developed by Merck, Sharp and Dohme, Inc.) binds to the inside of the channel pore of the N-methyl-D-aspartate (NMDA) receptor complex.
• NMDA N-methyl-D-aspartate
• the non-equilibrium binding of tritiated MK-8C1 is regulated by glutamate and glycine in such a way that botn agonists are required to produce an effect.
• glutamate antagonists or glycine antagonists may prevent the specific binding of subsequently added [ 3 H]MK-801.
• the basic binding level of [ 3 H]MK-801 i.e. when no agonists are added, is dependent on the residual endogenous levels of glutamate and glycine.
• the basic binding level of [ 3 H] K-801 can be decreased by extensive v/ashing of the membranes before the incubation.
• too extensive washing may disintegrate important intramembrane structu- res or wash away other important cofactors.
• the first bias which will lead to a too low estimation of the true EC59 value, is due to the fact that the added amino acid concentrations do not reflect the true amino concentrations present in the test tube.
• the second bias which will lead to a too high estimate of the true EC 50 value, will be present if the basic binding levels are included in the calculations, because these levels are higher than the true baseline (i.e. zero binding) and thus, the EC 50 curve is shifted up and to the right.
• an object of the invention is to provide a method for evaluating an effect characteristic of a bio- chemically active stimulation means, such that a better accommodation of said basic binding levels and the like are obtained.
• a method for evaluating an effect characteristic of a biochemically active stimulation means comprising the steps of: providing a set of measurement pairs, each consisting of an amount of biochemically active stimulation means applied to a biological material and a value related to a biochemically mediated effect associated therewith; selecting a functional relationship, comprising one or more parameters, between said amount of biochemically active stimulation means applied to said biological material and said biochemically mediated effect, wherein said functional relationship is defined so as to accommodate the existence of a non-zero value of biochemically mediated effect at a zero amount of applied biochemically active stimulation means; adjusting said functional relationship to match said set of measurement pairs by adjusting said parameters; and deriving, based upon said functional relationship, a negative, non-zero value of said amount of biochemically active stimulation means at which said value of biochemi- cally mediated effect becomes essentially zero,
• a method for evaluating an effect characteristic of a biochemically active stimulation means comprising the steps of: providing a set of measurement pairs, each consisting of a value related to the amount of a biochemically active stimulation means applied to a biological material and a value related to a biochemi- cally mediated effect associated therewith, said set of measurement pairs together indicating a functional relationship between said amount of biochemically active stimulation means applied to said biological material and said biochemically mediated effect; and extrapolating said set of measurement pairs, indicating said functional relationship, to derive a negative, non-zero value of said amount of applied biochemically active stimulation means at which said value of biochemically mediated effect becomes essentially zero, said negative, non-zero value representing an intrinsic amount of biochemically active stimulation means in said biological material, thereby explaining the existence of a non-zero biochemically mediated effect at a zero amount of applied bio- logically active stimulation means.
• the invention is based upon the accommodation and derivation of an intrinsic or endogenous amount of stimulation means, or an intrinsic representation thereof, present in said biological material and explaining the existence of a non-zero biochemically mediated effect at a zero amount of added biochemically active stimulation means, for example explaining and accommodating basic binding levels discussed above as a result of residual endogenous substance present in the biological material.
• this intrinsic or endogenous stimulation means is taken into account by letting the concentration-response curves discussed above expand into an area of negative added concentration, correspon- ding to a positive amount of intrinsic concentration.
• this intrinsic parameter will occasionally be called the z-value. By taking the presence of this z- value into account, more reliable estimations of E max , EC50 and the Hill coefficient is obtained.
• the invention is preferably performed as a curve- fitting model on a computer and is easily realised by those skilled in the art of curve fitting algorithms using available curve fitting software programs.
• the z-value is obtained by projecting the response-concentration curve to zero binding (or zero effect) , reading the concentration of added biochemically active compound associated therewith, and changing the sign from minus to plus.
• sai ⁇ functional relationship is preferably defined as:
• said amount of biochemically active stimulation means applied to said biological material is reoresented in the form of a concentration of added biochemically active compound, preferably, depending on the application, selected from the group consisting of agonists, antibodies and enzyme substrates.
• biochemically active compound preferably, depending on the application, selected from the group consisting of agonists, antibodies and enzyme substrates.
• such compounds may be any molecule or ion that bind to a receptor
• Agonists may be neurotransmitters, hormones, growth factors and other proteins, enzymes, peptides, nucleotides and nucleic acids, lipids such as arachidonic acid metabolites, metal ions, vitamins, co- enzymes, quinones, cyclitols, steroids, carbohydrates, carotenoids, immunoglubulins, cytokines, folic acid and related compounds, corrinoids, anesthetic compounds, gases such as nitric oxide, and synthesized analogs of these agonists.
• said amount of biochemically active stimulation means applied to said biological material is an amount of physical stimulation, such as a mechanical or electromagnetical stimulation.
• Many, but not all, of such other types of stimulation means causes a release of a biochemically active compound which is thereby added to said biological material.
• said biochemically mediated effect is typically represented as concentration of a chemical substance (which may or may not be the same as the one originating the effect) present in said biological material.
• said effect may be represented as an amount of cells or individuals which show a specific biological change as a result of said applied biochemically active stimulation means.
• the biochemically mediated effect may also be represented as an physiological effect, such as respiratory rate, heart rate, or locomotion, or the like.
• z-analysis An example of where the methods according to the invention, referred to below as z-analysis, is applied is v/hen measuring the binding of the non-competitive NMDA- receptor antagonist MK-801 in response to glutamate, as discussed above, in a membrane preparation of brain tissue.
• MK-801 binds to the inside of the ion channel pore of the NMDA receptor, and its access is dependent of activation of the glutamate binding site.
• the binding of MK-801 may reflect the stimulatory action of glutamate.
• z-analysis of the glutamate concentration-response curve reveal a) the residual, endogenous concentration of glutamate, and b) correct stimulation parameters such as EC 5 0 values, E max values and Hill coefficients, so that the characteristics of glutamate stimulation on NMDA receptors can be compared between different brain areas, in spite of the fact that the respective membrane preparations may contain different concentrations of glutamate.
• the invention will be useful in many types of academic and commercial biochemical, pharmacological and pharmaceutical research and development for properly evaluating the characteristics of substrates interacting with receptors, enzymes or other recognition sites, as do most pharmaceutical agents currently in use and under development.
• a clinical example is challenge tests used for individuals being evaluated for possible or manifest metabolic or endocrine diseases. For example, glucose is given in different doses to an individual to measure insulin production. The resulting dose-response curve may be analyzed using z-analysis to yield estimations of the concentration of endogenous glucose and true stimulation parameters such as EC 50 , E nax and Hill slopes.
• Another example is when thyrotropin-releasing hor- mone is given to an individual, and where serum levels of thyroid-stimulating hormone and thyroid hormone is measured. Endogenous thyrotropin-releasing hormone is released from the hypothalamus and stimulates the release of thyroid-stimulating hormone from the pituitary, which in turn stimulates the formation of thyroid hormones. The resulting dose-response curve may be analyzed using the invention to qud estimations of the concentration of endogenous thyrotropin-releasing hormone and true stimulation parameters such as EC 50 , E max and Hill slopes.
• Another example is when measuring the activity of a purified enzyme, for example from blood, serum, or tissue taken from an individual, in response to an mducer. The invention is then for example used to reveal true affinity parameters for the mducer by compensating for residual, endogenous amounts of mducer.
• Yet another example is when measuring the activity of a receptor, for example from blood, serum, or tissue taken from an individual, m response to an agonist.
• the invention is then for example used to reveal true affinity parameters for the agonist by compensating for residual, endogenous amounts of agonist.
• a clinical example is to use the invention when measuring the serum concentrations of the hormone prolac- t m response to the ⁇ opamine agonist bromocriptme .
• the release of prolactm is under pnysiological conditions inhibited by dopam e through activation of dop- amme D 2 receptors.
• the stimulatory action of the agonist leads to an inhibition of the effect.
• Bromocriptme is used the clinic to inhibit hypersecretion of prolactm from the pituitary .
• the maximal effect is determined m order to use z-analysis.
• the maximal serum concentrations of prolactm is oetermmed by giving a dopamme D 2 receptor antagonist sucn as halo- peridol.
• concentration-response curves of bromocriptme is analyzed according to the invention to yield a) the endogenous concentration of ⁇ opamine, expressed as bromocriptme equivalents (derived from curve fitting extending to the maximal serum concentration of prolactm) after treatment with a dopamme D 2 receptor antagonist, and b) correct inhibition parameters such as IC 50 values, I max values, and Hill coefficients.
• concentration-response curves of bromocriptme is analyzed according to the invention to yield a) the endogenous concentration of ⁇ opamine, expressed as bromocriptme equivalents (derived from curve fitting extending to the maximal serum concentration of prolactm) after treatment with a dopamme D 2 receptor antagonist, and b) correct inhibition parameters such as IC 50 values, I max values, and Hill coefficients.
• Still another example is antibody-antigen assays, where the invention is used for example to determine the concentrations of endogenous antigen or antibody, or to determine true affinity parameters for the antibody- anti
• the invention is preferably used to compare effect characteristics of one biochemically active stimulation means with other biochemically active stimulation means for the selection of stimulation means for the treatment of diseases.
• the invention is preferably used when evaluating saturation or stimulation models for enzymatic reactions or receptor binding.
• the invention should be of value to all kinds of pharmacological and biochemical assays where the presence of an endogenous or intrinsic agonist produces a response baseline above zero but where the addition of an antagonist can block the response completely (at least in theory) .
• FIG. 1 schematically shows a prior art saturation curve diagram of using a linear-linear representation
• Fig. 2 schematically shows a prior art stimulation curve diagram of using a logarithmic-linear representation
• Fig. 3 schematically shows a z-analysis curve diagram according to the invention using a linear-linear representation.
• the measurement pairs provided in Table I may for example have been obtained by studying the binding levels of [ 3 H]MK-801, as discussed above.
• the obtained measurement pairs is inputted manually to the program execution using a computer keyboard connected to said computer or automatically via an input interface from, for example, automated detection equipment used to obtain said measurement pairs .
• Fig. 1 the measurement data were fitted according to a traditional saturation curve model displayed on a linear-linear graph.
• the traditional saturation curve model of Fig. 1 is defined by the functional relationship (1)
• Fig. 2 the same measurement data listed in Table I were fitted according to a traditional stimulation curve model displayed on a logarithmic-linear graph.
• the traditional stimulation curve model of Fig. 2 is defined by the functional relationship (2)
• x is the concentration of added compound
• E is the measured binding level
• E 0 is the basic binding level or effect observed without any added compound
• E max is the maximum binding level not including said non-specific effect
• EC 50 is the concentration, as calculated starting from said non-specific effect level.
• the basic binding level E 0 was found to be 30, the maximal binding level E max , not incorporating the basic binding level, was found to be 81, and the concentration EC 50 of added compound at half the maximal binding level, starting from the basic binding level, was found to be 15 for best curve fitting.
• the selected and derived relationship will accommodate the actual measurement pairs in indicating a basic binding level of about 30 at zero concentration of added compound.
• Fig. 3 the same measurement data listed in Table I were fitted according to a z-analysis curve model according to the present invention and displayed on a linear-linear graph.
• the z-analysis curve model of Fig. 3 according to the invention is defined by the functional relationship (3)
• the maximal binding level E raax was found to be 110 (very similar to the total value of E max +E 0 of Fig. 2), the intrinsic concentration z was found to be 4.0 and the concentration EC 5 0 of added compound at half the maximal binding level, including said intrinsic concentration, was found to be 10.8 for best possible curve fitting.
• the selected and derived relationship will accommodate the actual measurement pairs in indicating a basic binding level of about 30 at zero concentration of added compound.
• the characteristics of the selected functional rela- tionship (3) it is possible to derive said intrinsic representation of an endogenous property.
• an intrinsic or endogenous concentration value corresponding to said z-para- meter, may be calculated from the fitted relationship of Fig. 2, i.e. after having derived the curve fitted para- meters E max , E 0 and EC 50 , by setting the total sum E of the relationship (2) to be zero and deriving the x-value at which such zero effect will be obtained.
• E max the curve fitted para- meters
• E 0 the curve fitted para- meters
• the z-value obtained according to the invention will provide important information in the evaluation of effect characteristics of different biochemically active stimulation means, as discussed above.
• biochemically active stimulation is to be interpreted in a broad sense, such as including an enhanced binding between a compound and a biological material, for instance between an antibody and an antigen.

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• 1997
  • 1997-10-24 SE SE9703893A patent/SE9703893D0/xx unknown
• 1998
  • 1998-10-23 WO PCT/SE1998/001918 patent/WO1999022237A1/en not_active Application Discontinuation
  • 1998-10-23 AU AU97699/98A patent/AU9769998A/en not_active Abandoned
  • 1998-10-23 EP EP98951862A patent/EP1025443A1/de not_active Withdrawn

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