FUNCTIONAL ASSAY (CELL BASED ASSAYS)

Binding assays provide useful information with regard to the ability of compounds to bind to a receptor, essentially no information is provided on the efficacy of the compound, i.e. whether the compound elicits or inhibits a response at that receptor.

In a functional assay, information what we obtain is whether the compound elicits or inhibits a response at that receptor.  Thus, in a this assay functional activity is measured by adding gradually increasing amounts of the drug compound while measuring a particular function of that receptor, for instance.

·         Calcium mobilization assay is the most widely used functional assay for implementation of GPCR HTS. The assay is applicable for GPCRs whose signaling pathway induces the calcium release from its intracellular stores.

ENZYME / RECEPTOR BASED ASSAYS

KINDS OF ASSAYS:  ENZYME / RECEPTOR BASED ASSAYS (ALSO CALLED AS BINDING ASSAYS), CELL BASED ASSAYS (FUNCTIONAL ASSAYS)

https://www.slideshare.net/ankit_2408/radioligand-binding-studies

Compounds interacting with therapeutic targets like enzymes, cell-surface receptors, nuclear receptors, ion channels and signal transduction proteins are usually identified using in vitro biochemical assays.

LIGAND BINDING ASSAY: 

This is an analytical procedure which relies on the binding of ligand molecules to receptors, antibodies or other macromolecules. 

There are numerous types of ligand binding assays, both radioactive and non-radioactive. 

·         There are two typical assay formats used for analysis of receptor-ligand interactions in screening applications, filtration and scintillation proximity assay (SPA).  Both use radiolabeled ligand and a source of receptor.

·         Receptor binding assays using non-radioactive formats include fluorescence polarization, time-resolved fluorescence, fluorescence resonance energy transfer (FRET), and surface Plasmon resonance (SPR).  One of the largest differences between radioactive and non-radioactive ligand assays are in regards of dangers to human health. Radioactive assays are harmful in that they produce radioactive waste; whereas, non-radioactive ligand assays utilize a different method to avoid producing toxic waste.

v  Although binding assays are simple, they fail to provide information on whether or not the compound being tested affects the target's function.  Binding assays provide useful information with regard to the ability of compounds to bind to a receptor, essentially no information is provided on the efficacy of the compound, i.e. whether the compound elicits or inhibits a response at that receptor.

v  In general, these receptor binding assays are used to characterize known drug targets. 

In a typical filter-based separation technology what we obtain is “bound vs. free” fractions for assay validation.  In a filter binding assay, the filters are used to trap cell membranes by sucking the medium through them.  Washing filters with a buffer removes residual unbound ligands and any other ligands present that are capable of being washed away from the binding sites. The receptor-ligand complexes present while the filter is being washed will not dissociate significantly because they will be completely trapped by the filters. 

Competition or displacement binding studies allow determination of binding affinities for non-labelled ligands. These binding studies utilize a fixed concentration of a radiolabel and the affinity of non-labelled ligands are determined by a drugs ability to compete for the same binding site. Increasing concentrations of non-labelled ligand are used to displace or out-compete the fixed concentration of a radiolabel, generating an IC50 for the competing ligand.

Radiolabeled known drugs are used in these competitive binding assays.  Therefore, the assay is designed as a competitive inhibition assay using the radiolabeled known drug or ligand for a receptor to screen for more effective new chemical entities (NCEs).

SCINTILLATION PROXIMITY ASSAY:

SPA TECHNIQUE HAS BEEN WIDELY APPLIED TO RADIOIMMUNOASSAYS, RECEPTOR-LIGAND BINDING ASSAYS, ENZYMATIC ACTIVITY ASSAYS, RNA TRANSCRIPT DETECTION, PROTEIN-PEPTIDE INTERACTIONS, AMONG OTHERS.

Receptor-binding SPA assays are conducted by immobilizing receptors directly to SPA beads via a number of coupling methods.  In the SPA format, cell membrane or receptor is captured onto SPA beads.  The method utilizes scintillant-containing microspheres that are chemically treated to enable the coupling of molecules (e.g., antibodies, receptors, and enzymes) to their surface.

The type of beads that are involved in the SPA are microscopic in size and within the beads itself, there is a scintillant which emits light when it is stimulated. Stimulation occurs when radio-labelled molecules interact and bind to the surface of the bead. This interaction will trigger the bead to emit light, which can be detected using a photometer. 

·         When the radio-labelled molecule is attached or is in proximity to bead, light emission is stimulated.

·         However, if the bead does not become bound to the radio-labelled molecule, the bead will not be stimulated to emit light. This is because the energy released from the unbound molecule is not strong enough to excite the SPA bead which is not stimulated to produce a signal.

SPA reagents are suitable for the detection of a range of isotopes including ³H, ¹²⁵I, ³²P, ³⁵S, and ¹⁴C, when used as radiolabels.  

The path length of the b particle is determined by its energy, and varies for different isotopes.  For example, b particles from ³H have an average path-length of approximately 1.5 mm, which easily meets the distance requirement for SPA.  The Auger electrons emitted by ¹²⁵I (g-emitting isotope) which have energies in the same range as b-particles and travel between 1 and 17.5 mm in aqueous media, also satisfy the distance requirement.  Therefore, ³H and ¹²⁵I are ideal isotopes for labeling ligands in SPA.    

Other isotopes of interest, such as ¹⁴C, ³²P and ³⁵S have longer path-lengths with mean ranges of approximately 58, 126 and 66 mm, respectively, and are less suited to application of the proximity principle.  Although SPA has recently been adapted to these isotopes as well.

The beta particles emitted by ³H and the Auger electrons released from ¹²⁵I by isotopic decay have average energies of 6 and 35 keV, respectively, and thus have short path lengths in water. This property makes them ideal for use with the SPA technology.

·         When ³H, ¹⁴C, ³²P, and ¹²⁵I radioisotopes decay, they release β-particles (or Auger electrons, in the case of ¹²⁵I). The distance these particles travel through an aqueous solution is dependent on the energy of the particle. If a radioactive molecule is held in close enough proximity to a SPA Scintillation Bead or a SPA Imaging Bead, the decay particles stimulate the scintillant within the bead to emit light, which is then detected in a PMT-based scintillation counter or on a CCD-based imager, respectively. 

·         However, if the radioactive molecule does not associate with the SPA bead, the decay particles will not have sufficient energy to reach the bead and no light will be emitted.

The core of this technique is the support material: the SPA bead.  The most commonly used SPA beads are polyvinyltoluene (PVT) beads and yttrium silicate (YSi) beads.  High-efficiency scintillant has been incorporated inside the PVT matrix, which can be excited by the high-energy b-emission generated from the decay of radioactive isotopes when the SPA bead is within the effective path length for energy transference.

The surface of this SPA bead is coated with hydrophilic polyhydroxy film that reduces hydrophobicity of the bead to reduce nonspecific interactions.  This film has been chemically derived to covalently couple capture molecules, which can selectively bind molecules of interest including receptors, proteins or other disease targets. 

SPA beads with the following capture molecules are available commercially:  Protein A, avidin, streptavidin, wheatgerm agglutinin (GA), glutathione and various polyclonal secondary antibodies.

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IN VIVO PK SCREENING - 2

üUnderstanding of in vitro-in vivo correlation and frontloading PK/PD is crucial for the compound selection even at an early stage. 

ü Obviously, an excellent PD data can save a poor PK as long as the PD effect is notable. 

In Vivo ADME studies are mostly custom-designed.  Often these studies are performed in multiple species by administering the test compounds by variety of routes (includes oral, intravenous, dermal, intraperitoneal, inhalation to name a few).  These in-life studies provide information for:

·        mass balance

·        metabolite isolation and characterization

·        dosing frequency

·        routes of administration

·        exposure

·        interspecies comparisons

·        PK/TK

·        tissue distribution studies to determine clearance routes and rates

·        tissue half-life

·        Potential sites of toxicity after systemic exposure.  

The basic assumption in all pre-clinical ADME studies is that the administered NCEs and dose levels are not toxic and do not cause any harm to the welfare of the animals.

In general rats of similar age and weight are chosen for the experiments.  Experimental procedures are kept strictly in accordance with the related ethics regulations.  In many studies, all the subjects are kept in an environmentally controlled breeding room (temperature maintained at about 25 ± 2 oC and with a 12 h light / 12 h dark cycle) for at least once week before starting the experiments and fed with standard laboratory food and water.  Prior to each experiment, the rats are fasted for 12 h with free access to water.

RANK ORDER OF COMPOUND EXPOSURE:  A small cohort of animals (selected number), is administered compound orally (p.o.) or by intraperitoneal injection (i.p.) at a single dose (5 - 50 mg/kg). Blood samples are collected at 20 and 120 minutes. The plasma samples are analyzed which provides a snapshot of compound exposure as the area under the curve (AUC(20-120 min)), providing a rank order of estimated AUC values to prioritize compounds for further investigation. 

Based on the concentration vs. time data, pharmacokinetic parameters such as body clearance, volume of distribution and bioavailability can be calculated, information which is used to refine in vivo experiments with the same series of drugs in the development.

MATERIAL BALANCE STUDY:

• Groups of male and female rats at a single dose level (equal number of male and female rats are used)
• Daily collection of urine and feces for 7 days
• Collect blood and major internal organs, and retain carcass (corpse) upon necropsy
• Total radioactivity level in each sample is assayed and the recovered dose is compared with the administered dose. The percentage of distribution of the administered dose in urine, feces, and each major internal organ and the half-time for excretion are calculated.

A necropsy is a surgical examination of a dead body, most commonly a dead animal, in order to learn why the animal died. A more common word for necropsy is autopsy. Either way, it's the dissection of a corpse performed to learn something about the cause of death or about a particular disease.

PLASMA LEVEL AND BIO-AVAILABLITY STUDY:

The objective of this study is to examine the pharmacokinetic profiles of the chemicals under test. These studies can be conducted by using unlabeled chemicals provided that appropriate analytical procedures (e.g., HPLC) are available.

·         Groups of 5 male and 5 female rats per route of dosing

·         One group is dosed intravenously and the second group is dosed by an extra-vascular route (e.g., oral, percutaneous)

·         The dosages for the 2 groups need not be identical

·         Blood is sampled at frequent intervals (e.g., 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 hr), and plasma levels of the chemical are assayed.

·         The plasma- concentration-time data are analyzed to obtain the pharmacokinetic profile, including the area under the curve (AUC). Comparison of the AUCs after extra-vascular and intravenous dosing, after correcting for the dosage, provides a measure of bioavailability.  Alternatively, if a radioactive chemical is used, comparison of the administered doses excreted in urine can be used to calculate the approximate bioavailability of the orally dosed chemical.

IN VIVO PHARMACOKINETIC STUDY (PLASMA PHARMACOKINETICS):

Depending up on the study, rats were randomly assigned for pharmacokinetic investigation.  Test compound is administered by i.v. injection via the lateral tail vein and orally at the dose of for example, 10 and 20 mg/kg, respectively. At the time points of 0 (pre-dose), 5, 15, 30, 45, 60, 90, 120, 240 and 480 min post injection / administration, blood samples (0.5 mL) are collected in heparinized (add heparin to (blood or a container about to be filled with blood) to prevent it from coagulating) tubes from the orbital vein, and then centrifuged to obtain plasma. The plasma is stored at −70 °C prior to analysis by HPLC.

METABOLISM STUDY IN RAT URINE AND FECES:

Similar to above study, but here feces and urine samples are separately collected at 24 h after administration of test compound.  All the samples were stored at -70 ^oC prior to analysis.

TISSUE DISTRIBUTION STUDIES:

For tissue distribution study, number of rats is first divided into several groups randomly and test compound is administered intravenously through the tail vein at a particular dose, say 20 mg/kg.  After injection, the rats are sacrificed at 0.5, 1.0, 2.0, 4.0 and 8.0 h following administration and the tissue specimens including lung, liver, heart, spleen, stomach, small intestine, brain, thymus, muscle, fat and kidney are collected. Tissue samples are then rinsed in saline and blotted dry with filter paper, and then weighed for wet weight and homogenized in ice-cold physiological saline solution (500 mg/mL). The obtained tissue homogenates are stored at −70 °C until analysis performed.

ALTERNATIVE ROUTES OF ADMINISTRATION:

Alternative routes of administration, typically intraperitoneal (i.p.) or subcutaneous (s.c.), may be used depending on the targeted type of treatment and indication or to avoid first-pass metabolism in the liver occurring after absorption from the gut wall when oral dosing is used.

REPEATED DOSE STUDY:

Other typical experimental set-ups include a repeated dose study, where the drug is administered once a day for up to two weeks. This experimental design can be used to find out not only the drug pharmacokinetics but also pharmacokinetic changes in the drug, for example, in a situation where the drug metabolism is either induced or saturated.

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When the bioanalysis has been completed and the results processed to gain plasma concentrations, plasma concentration vs. time data is then input into a software program for the calculation of PK parameters.

Pharmacokinetic parameters calculated include:

The area under the plasma concentration–time curve (AUC) is first calculated.

C_max: After the drug administration, the highest concentration obtained is considered the peak plasma concentration.

T_max:  Time take for peak plasma concentration is T_max

C₀: Concentration at time zero

V_d: Volume of distribution.  The apparent volume to which the mass of compound is distributed in the body at any given time.

CL: Clearance.  This value describes the tendency of the NCE to disappear from plasma.

T_1/2: Half-life. Time taken for the plasma concentration of an NCE to halve.

BA: Bioavailability.  This is the fraction of the NCE that reaches the systemic circulation unchanged.

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WHAT IS A GOOD PK PROFILE OF A PRECLINICAL CANDIDATE THAT IS BEING CONSIDERED FOR TAKING INTO DEVELOPMENT?

Ideally, preclinical candidate should,

    have acceptable solubility for development; 

    be completely absorbed, preferably via passive absorption; 

    have high bioavailability (e.g., F>50%) for oral drug; 

    have a low plasma clearance CL (e.g, <30% blood flow), long half‐life (t1/2) (e.g., >6 hrs), and  acceptable distribution volume; 

    have a linear kinetics, i.e., exposure proportional to dose and a clear PK/PD correlation; 

    be eliminated by several pathways, i.e., renal excretion and hepatic metabolism, also  metabolised preferably by more than one enzyme for de‐risking DDI; 

    have a simple metabolite profile, with no reactive metabolite; 

    have no obvious CYP and major transporters like P‐gp inhibition or induction or low DDI potential;  and 

    have a sufficient or at least acceptable safety margin (safety margin >10x, depending  on different  therapeutic targets). 

IN VIVO PK SCREENING - 1

v  Prior to clinical trials a new chemical entities’ (NCE’s) pre-clinical pharmacokinetics should be determined.  Once the test compound/NCE/drug molecule enters the body, it will undergo various processes including absorption via different administration routes, distribution into the body including different types of tissue, elimination via metabolic processes, as well as excretion via urine and feces.  It is possible to isolate and study some of these processes, by means of in vitro methods, and these methods will indeed be utilized prior to in vivo testing.  Due to the complexity of the entire body, in vivo tests cannot be completely avoided.

v  Before animal testing, the medicinal products are examined in a comprehensive manner using versatile in vitro methods, and only the most promising pharmaceutical compounds are selected for animal tests.  The basic assumption in all pre-clinical ADME studies is that the administered NCEs and dose levels are not toxic and do not cause any harm to the welfare of the animals.

Before talking about In Vivo ADME, let us re-cap In Vitro ADME and why is it important to perform In Vitro ADME before it’s In Vivo PK profiling.

In Vitro ADME

Prior to actual dosing in animals, a number of relatively rapid and cost effective in vitro assays can serve as surrogates and indicators of the ADME fate of compounds in vivo.

·         Evaluation of the pharmacological properties of Absorption, Distribution, Metabolism and Excretion of a test compound / candidate chemical leads are critical.

·         Once the ADME properties are evaluated then the next duty of a medicinal chemist is lead optimization while preserving the potency and selectivity of the chemical lead.

·         What one should keep in mind is sometimes more efficacious compounds have lower in vitro potencies, but better ADME properties.

Simply designing new analogs and developing a SAR for increased potency against the biological target is inadequate for the development of small molecule drugs.  Evaluation and optimization of structure-pharmacologic property-relationship (SPR) is a critical step for efficacy evaluation.

In addition to evaluating compound properties such as solubility, protein binding, serum stability, medicinal chemists team has to prioritize different structural classes and rank order them not only based on potency but also in relation to potential downstream absorption and metabolism liabilities.

Improvement in these ADME properties is sought prior to actual dosing in animals to assess PK, and certainly for larger compound efficacy studies, since animals are expensive and the ethics of sacrificing animals in poorly designed studies uninformed by pharmacological guidance are indefensible.

Usual physicochemical properties obtained from In Vitro ADME studies include,

Ø  Log D:  Provides lipophilicity information. 

Ø  Aqueous Solubility: Mostly Kinetic Solubility

Ø  Microsome Stability: Stability of the test compounds against liver microsomes, S9 fraction,   hepatocytes. Here we tend to get Human Liver Microsome (HLM), Mouse Liver Microsome (MLM), Rat Liver Microsome (RLM) stablity data

Ø  Plasma Stability:  Compounds stability in plasma.

Ø  Plasma Protein Binding:  How much of compound is bound to plasma protein?

Ø  Permeability:  Passive permeability information is obtained from PAMPA.  Other permeability      assays including Caco-2 and MDCK gives information related to active transport and P-gp efflux information.

Ø  Cytotoxicity: Most often hepatotoxicity information is what we look for.

Ø  CYP450 Inhibition:  Provides information related to possible drug-drug interaction.

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For in vivo PK studies, Association for Assessment and Accreditation of  Laboratory Animal Care (AAALAC)‐accredited animals, such as mice, rats,  dogs, and non‐human primates are employed to generate in vivo PK data like 

drug clearance (CL)

bioavailability (F%)

exposure (AUC)

half life (t1/2)

distribution volume (L)

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v It is highly recommended for PK/PD strategies to be implemented in early research phases of drug discovery projects to enable successful transition to drug development.

v Effective PK/PD study design, analysis, and interpretation can help scientists elucidate the relationship between PK and PD, understand the mechanism of drug action, and identify PK properties for further improvement and optimal compound design.

v Additionally, PK/PD modeling can help increase the translation of in vitro compound potency to the in vivo setting, reduce the number of in vivo animal studies, and improve translation of findings from preclinical species into the clinical setting.

After PK/PD studies are designed and conducted then the conclusions and interpretations are performed by

·        Pharmacology Laboratories – Pharmacodynamic portion of PK/PD studies, e.g., animal dosing and measurement of response

·        DMPK Laboratories – Measurement of concentrations and evaluation of pharmacokinetics

·        Other expert partners include, Formulation Department, Mathematical Modeling Department etcetera.

MOUSE XENOGRAFT MODELS

As discussed earlier, mouse models are more attractive than big animal models because of the low cost, ease-of-handling and known genetic information.

Xenograft = a tissue graft or organ transplant from a donor of a different species from the recipient.  Xenotransplantation = Xenotransplantation is the transplantation of living cells, tissues or organs from one species to another.

One of the most widely used models is the human tumor xenograft. In this model, human tumor cells are transplanted, either under the skin or into the organ type in which the tumor originated, into immunocompromised mice that do not reject human cells.  For example, the xenograft will be readily accepted by athymic nude mice, severely compromised immunodeficient (SCID) mice, or other immunocompromised mice.

After establishment of cancer cell lines for anticancer screening in National Cancer Institute (NCI), xenograft models derived from these cell lines were developed. Sixty cell lines derived from eight organs were used for establishment of xenograft models.

HUMANIZED MOUSE: 

·       This refers to a mouse that has a few human cells, a short strand of human DNA, human tissues, a human tumor, or a humanized immune system.  Mice that are humanized can be powerful models of the human being and thus better for research into the problems of human development and disease.

·        Mouse engrafted with human tumor, known as patient-derived xenograft (PDX).  In general immunocompromised mice are engrafted with PDX tumors.  Tumor from a patient is collected and cut into pieces and put these pieces into multiple mice.  Each fragment grows and can then be divided into pieces and put into additional mice.  Through this process, you can end up with dozens of mice, each with tumors nearly identical to each other and to the original human patient’s tumor.  Now we can treat different groups of PDX mice with alternative drugs, drug combinations, and drug sequences allowing us to determine with approach works best to eliminate a very specific tumor type.

·        Interestingly, a mouse with no immune system can be selected and to it specific human stem cells typically derived either from fetal tissue or cord blood can be injected to obtain mouse with humanized immune system.

·        CRISPR CAS/9 technique can be used to break a strand of DNA at a very precise location and either can eliminate a short stretch of genetic code or add code from another organism.  Using this technique, DNA in a mouse embryo is broken in this way and a human genetic sequence is inserted into its genome.  By doing so, we can determine whether a human gene or a specific genetic variation functions or impacts on human health the way they we think it does.  These mice an be treated with alternative therapies to see if a disease causing genomic abnormality can be stopped or mitigated.

Ectopic tumor xenograft model. Generally, human cancer cells are subcutaneously injected into the hind leg or back of mice. In an ectopic tumor xenograft model (ectopic model), the transplanted site is different from the origin of the cultured cells.  Because an ectopic model can be used to monitor tumorigenicity and tumor growth easily, many researchers have utilized this model for evaluation of anticancer efficacy.

Orthotopic tumor xenograft model. Alternative models for assessment of tumor sensitivity have been developed. The orthotopic tumor xenograft model (orthotopic model) is an advanced tool, but is based on a immunosuppressive murine microenvironment. In the orthotopic model, the human cancer cells are transplanted into the same origin site of the tumor. For instance, lung cancer cells were directly injected into the mouse lung for the orthotopic model.

Patient-derived tumor xenograft model. Xenograft models, despite their advantages, are limited in their ability to demonstrate how a cancer patient would respond to a particular treatment. The reliable prediction of drug response in a clinical trial is needed, and the current models are not sufficient. In an effort to address the shortcomings of these models, a patient-derived tumor xenograft (PDTX) was developed and utilized.

Humanized Mouse Xenograft Models.

ü  Xenograft rodent models and cultured cells have long been used in pre-clinical studies of cancers and other human problems.  There are significant limitations to both of these model systems.

ü  Immune system humanized mice have allowed for researchers to examine cancer xenograft growth in the context of a human-like immune system and resulting tumor microenvironment.

ü  Humanized mouse models are “engineering mouse strains expressing human cytokines or HLA proteins and implanting human bone, liver, and thymus tissues to facilitate immune cell maturation and trafficking.”