Mechanism of Action Studies

Mechanism of Action Studies are provided either as individual or multiplexed with other studies. These, in turn, are provided either as a  Complete Service, Full Report that can be used for an IND application or as a Rapidy Study to obtain information as rapidly as possible.

1. Complete Service, Full Report Study: Fully customized study that includes the Study Plan, Draft Text and Final Text Report with QA audit.
2. Rapid Study: A customized study that includes the Study Plan and full protocol, raw results and graphical data in a single Excel Workbook. No formal text report and no QA audit is performed. This type of study is designed for early drug development. No interpretation or conclusions are provided.

• Flow Cytometry: Membrane expression marker detection, cell cycle, ploidy, apoptosis (depends on the species and cell markers available)
• Membrane integrity: LDH and/or PI assays
• Micronucleus assay
• Soft agar assay
• Chemotaxis/ migration assays
• Growth factor/cytokine production/release
• Phospodiesterase assays
• Kinase assays
• Protease assay
• P-Glycoprotein assay
• Monoamine oxidate assay
• UDPglucuronosytransferase (UGT) assay
• Mitochondrial dysfunction assay
• Glutathione Assay: Oxidative stress
• Nitric oxide (NO) assay
• Lipid peroxidation assay
• Reactive oxygen species assays
• OxyFLOW™: A flow cytometric oxidative DNA damage assay
• Biochemical apoptosis assays: CaspaseGlo™
• Cytochrome P450 assays
• Cellular drug-drug interaction studies

Flow cytometry is performed on a Beckman Coulter Cytoflex instrument with 96-well plate capability. Flow cytometry is the most requested add-on for toxicity studies and is used for multi-color membrane expression markers as well as intracellular markers.

Cell cycle and cell ploidy analysis (important for mekaryopoiesis) is usually determined using the proprium iodide (PI) marker.

Apoptosis can be determined using multiple end-points, notably Annexin-5/Propidium iodide (PI) and caspases (see also Biochemical Apoptosis Assays below). 

Membrane leakiness or membrane integrity is often the sign of onset of cell death due to toxicity. Membrane integrity assays are usually performed by measuring lactate dehydrogenase (LDH) or propidium iodide (PI). HemoGenix^® recommends the LDH assay as an add-on to it's own standardized and validated ATP bioluminescence assays. HemoGenix® uses LDH-Glo™ from Promega for this assay. 

A micronucleus assay is used for genotoxicity studies/chromosomal damage. HemoGenix^® uses flow cytometry to perform the micronucleus assay on human and rodent cells. HemoGenix^® has developed it's own human stem cell micronucleus assay.    

The soft agar assay is another form of genotoxicity assay. This assay uses two different concentrations of agar between which the cells of interests are suspended. The cells proliferate and grow to form colonies which can be counted. The addition of potentially toxic test articles administered in a dose-dependent manner, reduces the number of colonies as an indication of toxicity. It should be noted that the Soft Agar Assay is not only difficult and costly to perform, but also produces questionable results.

A chemotaxis migration assay is used to test chemokines, the ability of cells to produce and release chemokines and the ability of cells to home to other cell types. 

HemoGenix^® usually uses 24- or 96-well inserts with filters that allow either cells or small molecules to pass through. Cells placed in the upper part of the insert are labeled with calcein-AM. Migration or chemotaxis from the top to the bottom of the well is detected in real time using a fluorescence plate reader where the fluorescence is read from both the top and bottom of the plate.

The production and release of growth factors, cytokines and/or chemokines is important in many aspects of disease and drug mechanism of action. It is particularly important to ensure that a new drug does not result in a Cytokine Storm that can be lethal to the patient. Traditional ELISAs can be used, but multiplexing LUMINEX Assay Methodology provides a more sensitive, accurate and reliable readout and offered by HemoGenix^®.

Phosphodiesterase assays are used in second-messenger signaling using cyclic AMP and GMP. HemoGenix® incorporates PDE-Glo™ from Promega as an add-on for in standardized ATP bioluminescence toxicity assays that can also be provided for high-throughput screening.   

There are multiple kinases involved in the cell signaling process. Detection of kinases can be immunological, cell-based or biochemical. Please contact HemoGenix^® to discuss the type of kineases to be detected. 

Protease activity can be detected in cultured cells, or specific proteases can be detected in the same assay. The latter may include proteases such as chymotrypsin, trypsin or caspase or calpain. HemoGenix® usually tries to use luminescent assays since they are the most sensitive, but other readouts are also available.

P-Glycoprotein, also known as MDR1 and ABCB1 are plasma membrane proteins that use an ATP-dependent effluc pump. They are involved in multi-drug resistant and drug-drug interactions. Interactions can result in activation or inhibition of ATPase activity. HemoGenix® usually employs Pgp-Glo™ from Promega to detect and measure P-glycoprotein. Used during ADME/Tox screening. Navigate to the HemoGenix^® Drug Interaction Page.  

Monoamine oxidase dysfunction is thought to be associated with neurological diseases. There are two enzymes, A and B. Inhibitors of the enzyme are used to treat depression. Detection of the enzymes can be multiplexed with NeuroGlo™-Tox HT when screening or testing compounds that affect neural cells. Used during ADME/Tox screening.

These transferases play a role in the Phase II metabolism of drugs. Glucuronidation is a mechanism for drug clearance. Regulatory agencies recommend UGT inhibition as part of drug-drug interaction studies. For more information of drug interaction studies from HemoGenix®, please click here.

Environmental agents or xenobiotics can result in the dysfunction of mitochondria. Both membrane integrity and changes in intracellular ATP concentrations can occur. To detect mitochondrial damage, HemoGenix® utilized the Promega Mitochondrial ToxGlo™ assay that complements its standardized and validated ATP bioluminescence toxicity assay platforms. This is used during ADME/Tox screening.

Oxidative stress can take multiple forms and be determined in a number of ways.

1. Mitochondrial dysfunction described above is used as an oxidative stress assay.

2. Glutathione assay. Like many other assays used by HemoGenix^®, measurement of cellular glutathione levels as a measure of oxidate stress is usually determined by GSH-Glo™ from Promega. This is also used during ADME/Tox screening.

3. Nitric oxide. Nitric oxide is a reactive nitrogen species (RNS, as opposed to a reactive oxygen species, ROS), produced by nitric oxide synthase. It can be detected intracellularly by a fluorescence plate reader, or in cell lysates, superanants and biological fluids either by fluorescence or absorbance readouts.

4. Reactive oxygen species (ROS). HemoGenix^® usually uses ROS-Glo™ from Promega for this test, which measures the production of hydrogen peroxide (H₂O₂) using a bioluminescence signal.

5. Lipid peroxidation. Like, RNS and ROS production, lipid peroxidation causes oxidative stress and can be determined using a number of different methods dependent upon the type of lipid peroxidation being caused. Many of the assays used for lipid peroxidation analysis can also be employed during ADME/Tox screening. 

Apoptosis can be measured using several different methodologies. One is by flow cytometry, described above. The other is at a biochemical level in which different caspases are determined: Caspase 3, 7, 8 and 9. These can be incorporated into high-throughput screening.

Drug-drug interaction is usually defined by the cytochrome P450 (CYP450) system in hepatocytes. These enzymes can be induced or inhibited by different drugs and other compounds. Detection of CYP450 enzymes is usually part of ADME/Tox screening. HemoGenix^® usually employes Promega luminescence CYP450 assays for these MOA studies.

In addition, HemoGenix^® also specializes in specific drug-drug interaction and combination studies at the cellular level. More information can be found on the Drug Interaction Page.