Section15:Data Types and Associated Rules for In Vitro Functional Assays

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Antagonists

Normalized Results

Inhibition with a UOM of % should be calculated for responses to individual concentrations of test substances.

Calculation:

Notes

• It’s common for many pharmacologists to apply a curve algorithm that yields a Hill Coefficient with a negative slope. Based on feedback from project teams and other consumers of published data, it is preferred to publish a positive Hill Coefficient as well as an antagonist result curve. This rule along with the use of % Inhibition (and not %Activity) provides more consistency when viewing, analyzing and comparing published results.

Derived Result: Rel IC50

Relative IC50 = the molar concentration of a substance (antagonist) that reduces the efficacy of the reference agonist or the constitutive activity of the biological target by 50% of the antagonist curve (Top-Bottom) for that particular test substance.

Derived Result: Kb

Calculation of Kb by Schild analysis isn’t standard practice due to throughput and cost disadvantages. Consequently, the Cheng-Prusoff equation is typically used to reduce the data and subsequently assigned the label of Kb.

Calculation: Use standard Cheng-Prusoff equation for functional assays.

If the slope of the curve for the reference agonist deviates significantly from 1, the use of the modified Cheng-Prusoff equation (see section VI) is recommended.

Other Derived Result

Schild Kb

Schild Kb is measure of affinity for a competitive antagonist that is calculated using the ratios of equi-active concentrations of a full agonist (most typically EC50 concentrations are used) in the absence and presence of one or more concentrations of the antagonist. Schild Kb offers a true evaluation of a test compound’s ability to mechanistically perform as an antagonist. This process exposes toxic effects and compound precipitation as false positive activity, and therefore, should be used when time and cost are not limitations.

Emin

The maximum activity of an antagonist test substance relative to a reference agonist. This is obtained by first generating a fitted top from a %Inhibition curve and then converting that to the corresponding %Stimulation of the reference agonist curve. The Emin value for antagonist mode should equal the relative efficacy for agonist mode for competitive inhibitors. In order to make use of Emin, the selected agonist concentration (i.e. EC80) should produce an activity above the expected Emin value.

Notes

• Kb carries the same prefix as the IC50 from which it is derived.
• The use of Abs IC50 is discouraged.
• Since partial antagonists exist, a full response curve with defined Top & Bottom can be achieved even if the %Inh doesn’t exceed 50%.
• A concentration response curve for the reference agonist should be determined in each experimental run if a Kb is to be determined. The frequency within the run depends on assay variability. A statistician should be consulted concerning this frequency during the assay validation process. The identity of this “standard” and the frequency of it’s occurrence within a run should be included as part of the written protocol for the assay. In the rare occurrence that an assay run is to use the historical EC50 value for calculation of Kb, a statistician should first be consulted.

Optional Result Types for Data Publication

The following table includes possible result types that can be used when calculating and publishing data.

Agonists

Normalized Data

Stimulation with a UOM of % should be calculated for responses to individual concentrations of test substances.

Calculation:

Derived Results: Relative EC50 and Relative Efficacy

Relative EC50 = the molar concentration of a substance that produces 50% of that test substance's maximum stimulation.

Relative Efficacy = the maximum activity of a test substance relative agonist. The unit of measure (UOM) for Relative efficacy is %.

Calculation:

Other Derived Results

Fold Activity and Fold Activity Max

The fold activity (or fold activity max) result is useful when comparing test compounds evaluated across multiple functional assays because varying levels of efficacy can be observed amongst the different or same reference agonists. The intended use of this calculation is to provide additional information to reduce or define differences between assays so that differences between compounds can be further quantified. For example, a compound run in an assay normalized to a reference agonist with low efficacy would appear to be more efficacious when compared to another compound run in a separate assay normalized to a reference agonist with high efficacy. Comparing folds activities, which looks at the magnitude of compound induced activity relative to baseline, enables a scientist to make a conclusion that is not influenced by differences in reference agonist responsiveness. Also, the fold activity result of a control compound can be useful to quality control chart, tracking changes in assay responsiveness over time.

Calculation:

Relative AUC

Relative AUC (Area Under the Curve) is defined as the ratio of the area under the fitted concentration-response curve for the test compound to the area under the fitted concentration-response curve for the reference compound. Specifically, areas are calculated as the area under the curve that lies above the horizontal line y = 0%. The area calculation corresponds to the shaded region in the figure below, where the contribution to the area as one moves along the concentration axis is proportional to the log of the concentration distance covered, not the linear concentration distance covered. One should calculate the area using an exact formula when it is available, as is the case for the 4PL and 3PL models. Otherwise, one may use an approximation method, such as the trapezoid rule. In either case, for the calculated value of relative AUC to be meaningful, the areas for both the test and reference compounds must be computed with respect to the same concentration range. Likewise, the comparison between two relative AUCs is only meaningful when each is computed with respect to the same concentration range. If the same concentration range was not used for assaying the test and reference compounds, the equations for the fitted curves may be used for extrapolation in order to compute the components of the relative AUC over the same concentration range.

Rel AUC is useful with functional assays in which compounds are measured with varying efficacies (agonists and partial agonists) and potencies. Since Rel AUC measures the area of activity, both efficacy and potency data are essentially combined, generating a value that provides an overall assessment of activity and selectivity between tested compounds. However, Rel AUC should not be a substitute but rather a supplement to individual efficacy and potency data during the analysis process.

Calculation:

Notes

• A four-parameter curve fit must be used for the Ref Agonist.
• The maximum and minimum asymptotes must be defined by the data for the Ref Agonist.
• Calculation of Rel Eff requires that both the test compound and positive control each have a defined Top asymptote.

Optional Result Types for Data Publication

The following table includes possible result types that can be used when calculating and publishing data.

Orphan Receptors – Stimulation Mode

Assays exist for which there are no identified positive control or reference agonist compounds. An example of this situation is an assay that utilizes an “orphan” target as a bio-entity. An “orphan” target is a bio-entity that has a primary sequence suggesting it is a member of one of the super families of biological targets; however, no ligand for this “receptor” has been identified. Generally, it is the aim of the research effort to identify ligands for this “orphan” so that a protocol for a validated assay can be created. Until at least enough data is gathered to identify a ligand for these types of bio-entities, assays utilizing them will be considered “validated” at only the hit to lead level. During this period, responses to individual concentrations of test substances can be normalized by one of the following formulae, which either make use of a known nonspecific activator or simply use basal activity of the constitutive receptor.

Orphan Receptors Normalized to Nonspecific Activator

Stimulation with a UOM of  % should be calculated for responses to individual concentrations of test substances.

Optional Result Types for Data Publication

The following table includes possible result types that can be used when calculating and publishing data.

Orphan Receptors Normalized to Constitutive Receptor

Responses to individual concentrations of test substances that increase the measured activity of the orphan target are normalized to the basal level of activity of the target measured in the absence of the test substance. These responses can be expressed as either a percent of the basal activity or as a fold of the basal activity using one of the following:

Calculation:

Notes

• Results from this equation can generate percents much greater than 100.
• Expression of Fold Act or Fold Act Max should only be determined until either a nonspecific activator or ligand is identified; and should only be used to rank order compounds tested in the same assay.
• The calculated Fold Act or Fold Act Max value is expected to be greater than 1 for an agonist. If the calculated value is less than 1, the test compound could be an inverse agonist.

Optional Result Types for Data Publication

The following table includes possible result types that can be used when calculating and publishing data.

Potentiators

Potentiation assays measure the ability of an inactive test substance to augment the response produced by a relatively low concentration of an active substance in some biological system. Currently, these assays are run in one of two modes. The following paragraphs address the most frequently used mode.

The first mode involves the addition of one or more concentrations of a test substance in the presence of a fixed concentration of the known active substance called the “Reference Agonist”. In this mode, potentiation is the response produced by the combination of substances minus the response produced by the specific concentration of Reference Agonist alone. But, how does one normalize this response?

It is recognized that potentiation assays might be executed when no known potentiator exists. However, no potentiation assay would be run without the existence of a known Reference Agonist. Therefore, the response to the specific concentration of the Reference Agonist plus the test substance (potentiation) should be normalized to the fitted Top of a concentration response curve of the Reference Agonist determined at least once in every run of the assay. The frequency of the determination of the concentration response curve of the Reference Agonist for the purpose of normalizing other responses in any potentiation assay would be dependent upon other factors such as plate variability and run-to-run reproducibility.

Normalized Data

Stimulation with a UOM of % should be calculated for responses to the Reference Agonist.

Potentiation with a UOM of % should be calculated for responses to individual concentrations of test substances.

Calculation:

Notes

• This provides for a Potentiation equal to 0% when the response to the combination of test substance and Reference Agonist is equal to the response to the Reference Agonist alone (e.g. a test substance that is not a potentiator).

Derived Data

Relative EC50= the molar concentration of a substance that produces 50% of that test substance's maximum stimulation.

Relative Potentiator Efficacy: There is little if any discussion in the scientific literature addressing a standard term or calculation of efficacy of a potentiator. It is suggested that this result type be termed Relative Potentiator Efficacy (or Rel Pot Eff) to distinguish it from the Relative Efficacy of an agonist. It is equal to the fitted Top of the potentiation curve minus the normalized response to the specific concentration of Reference Agonist alone divided by 100 minus the normalized response to the specific concentration of Reference Agonist alone. The following cartoon illustrates the above decisions.

Optional Result Types for Data Publication

The following table includes possible result types that can be used when calculating and publishing data.

Inverse Agonists

According to multiple models of drug-receptor interaction, receptors have been demonstrated to exist in equilibrium between two states. These two states are R*, the active form of the receptor, and R, the inactive form.

Agonists exhibit higher affinity for the active form of the receptor. When an agonist binds to a receptor, it stabilizes the active form of the receptor, shifts the equilibrium toward the active state and produces a response in the biological system under investigation. Substances that produce this kind of effect possess positive intrinsic activity.

Antagonists exhibit equal affinity for both forms of the receptor. When an antagonist binds to a receptor, it stabilizes the initial equilibrium between the active and inactive forms of the receptor. Therefore, no observable change in the activity of the biological system occurs. Substances of this type possess zero intrinsic activity.

Inverse agonists exhibit higher affinity for the inactive state of the receptor. When an inverse agonist binds to a receptor, it stabilizes the inactive form of the receptor, shifts the equilibrium toward that state and produces an opposite response in the biological system. These substances possess negative intrinsic activity.

Receptors have been demonstrated to exist in a constitutively active state both in vitro and in vivo. In vitro, the constitutive activity observed in assays utilizing transfected cell lines is generally attributed to the over expression of the receptor at levels hundreds to thousands of times higher than occur in vivo. Under these conditions, the total number of receptors in the active state is sufficiently high to produce a measurable response even when no exogenous substance has been added to the system. The addition of an inverse agonist to the system produces a decrease in the measured response. The magnitude of the decrease is related to the amount of negative intrinsic efficacy of the inverse agonist.

The possibility for confusion exists when one desires to quantify results for potential drug candidates that are inverse agonists. Some of the questions that arise are:

1. Since the measured response is a decreased activity produced by an inverse agonist, is the normalized result type Inhibition or Stimulation?
2. What is the algorithm for normalized results?
3. What is the algorithm for fitting concentration response curves?
4. Is the result type describing potency of a test substance a Relative EC50 or a Relative IC50 or something else?
5. How is the result type describing potency differentiated from the potency result type for an agonist?
6. Is Relative Efficacy a negative number?

There are no absolute answers to these questions provided by the current literature; however, there is a consistent theme.

1. The most frequently used normalized result type is Inhibition with a unit of measure of %.
2. The dynamic range for inverse agonists is the difference between activity in the absence of, or fully inhibited, biological target and the constitutive activity. Use of the “absence” method is preferable in early development of inverse agonist assays because it eliminates the dependency on a pre-existent known inverse agonist to compare responses of test substances to. However, as with other functional assays, as soon as an appropriate inverse agonist has been found, it should be utilized as a positive control in the assay for the purpose of calculating relative efficacies.

Assays Normalizing Data to an Inverse Agonist Control

Normalized Data

Inhibition with a UOM of % should be calculated for responses to individual concentrations of test substances.

Calculation:

Derived Data

Relative EC50 Inverse = the molar concentration of a substance that produces 50% of the range of inverse agonist curve (Top – Bottom) for that particular test substance.

Rel Efficacy Inverse = 100 x (Fitted Top of the test substance expressed as %/Fitted Top of the Positive Control Reference Inverse Agonist expressed as %)

Calculation:

Optional Result Types for Data Publication

The following table includes possible result types that can be used when calculating and publishing data.

Notes

• Because inverse agonist response curve profiles look similar to profiles generated by toxic compounds, it’s advised that a confirmation assay be used to provide more evidence that a given test compound is an inverse agonist.
• Hill Coefficient and Rel Eff Inv values are positive.
• The calculated Fold Act or Fold Act Max value is expected to be greater than 1 for an agonist. If the calculated value is less than 1, the test compound could be an inverse agonist.

Assays Normalizing Data to No Receptor Control (Orphan Receptor)

Normalized Data

Inhibition with a UOM of % should be calculated for responses to individual concentrations of test substances.

Calculation:

Derived Data

Relative EC50 Inverse = the molar concentration of a substance that produces 50% of the range of inverse agonist curve (Top – Bottom) for that particular test substance.

Notes

• Because inverse agonist response curve profiles look similar to profiles generated by toxic compounds, it’s advised that a confirmation assay be used to provide more evidence that a given test compound is an inverse agonist.
• Hill Coefficient and Rel Eff Inv values are positive.
• The calculated Fold Act or Fold Act Max value is expected to be greater than 1 for an agonist. If the calculated value is less than 1, the test compound could be an inverse agonist.

Optional Result Types for Data Publication

The following table includes possible result types that can be used when calculating and publishing data.

Assays Normalizing Data to Reference Agonist

Normalized Data

Stimulation with a UOM of % should be calculated for responses to individual concentrations of test substances.

Calculation:

Notes

• %Stimulation values will be negative for inverse agonist test compounds.

Derived Data

Relative EC50 Inverse = the molar concentration of a substance that produces 50% of that test substance's inverse agonism.

Relative Efficacy = the maximum activity of a test substance relative to a Reference Agonist. The unit of measure (UOM) for Relative efficacy is %.

Calculation:

Notes

• Rel Eff and Hill Coeff values for inverse agonists will be negative.
• Calculation of Rel Eff requires the test compound have a defined Bottom asymptote and Reference Agonist have a defined Top asymptote.
• Because inverse agonist response curve profiles look similar to profiles generated by toxic compounds, it’s advised that a confirmation assay be used to provide more evidence that a given test compound is an inverse agonist.
• The calculated Fold Act or Fold Act Max value is expected to be greater than 1 for an agonist. If the calculated value is less than 1, the test compound could be an inverse agonist.

Optional Result Types for Data Publication

The following table includes possible result types that can be used when calculating and publishing data.