Using Inhibitors In Vivo
Before using inhibitors in vivo, there are several questions to ask to adequately prepare for your next experiment. These include determining the route of administration, how much inhibitor to inject, questions to ask before injecting inhibitors, and factors that can affect inhibitor use. Find the answers to these critical questions below.
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Determining Route of Administration
The route of inhibitor administration is dependent on its properties and the objective of the experiment. Two major routes of inhibitor administration are (a) enteral, which includes oral, sublingual, and rectal, and (b) parenteral, which includes intravenous, intraperitoneal, intramuscular, and subcutaneous. Sometimes topical and transdermal methods are also used. Oral administration is the most common route for therapeutic use; however, it is also the most complicated pathway for the drug to reach tissues due to the impact of the gastrointestinal tract on bioavailability. Parenteral administration is used for those inhibitors that are poorly absorbed from the gastrointestinal tract.
How Much Inhibitor Should You Inject?
For small animals, due to practical difficulties in oral administration, the intravenous route is the most commonly used method. When injecting an inhibitor into an animal, one important question comes to mind: how much of an inhibitor to inject? There is no simple answer to this question. You must optimize the dose empirically by performing a few preliminary experiments. First, determine if the compound in question is cell-permeable. Also, survey the literature for any reported IC50, ED50, or EC50 values. You can follow the sample calculation given below as a general guide.
Sample Calculation
H-89, dihydrochloride, a cell-permeable protein kinase A inhibitor, has an IC50 of 48 nM. It has a molecular weight of 519.3. If we follow dosing guidelines, then 5 to 10 times the IC50 value should be ideal for inhibition. Hence, for H-89, dihydrochloride, the 240 to 480 nM range is sufficient to cause maximal inactivation of protein kinase A.
To use it in vivo we have to make a few assumptions. Let us assume that we want to inject an inhibitor into a rat weighing about 200 g. We also assume that 70% of its body weight is water; the volume of distribution will be approximately 140 mL. In this case, 240 nM = 240 nmoles/L = 124.63 µg/L. Because the volume of distribution is about 140 mL, 124.63 x 0.140 = 17.45 µg would be the required amount for injection into the rat.
Learn more about these calculations in our article about common enzyme inhibitor terms and calculations.
Questions to Ask Before Injecting Inhibitors
It is important to note that the drug distribution will vary depending on the mode of injection (intravenous, intramuscular, or intraperitoneal), bioavailability, half-life, rates of hepatic and renal clearance, binding to proteins, and tissue-specific distribution and accumulation. The specific tissue uptake may also be limited in whole organs or tissues as compared to isolated cell preparations. In whole animal studies, sometimes a loading dose is required to achieve the target concentration. This may then be followed by a sustained infusion to maintain the drug level in the blood. One must always exercise caution and not overdose the animal.
Here are some basic issues to consider when injecting an inhibitor/drug into an animal.
Is the compound cell-permeable?
If yes, proceed with your experiment.
What is the reported or calculated IC50 or Ki values?
If Ki, IC50, or EC50 values are known, then follow the steps below. Otherwise, search the literature or experimentally determine IC50 or EC50 value.
What is the volume of distribution?
This is an approximate value based on the body weight of the animal. Newborn and younger animals have greater volumes of distribution.
What is the proper injection technique?
Proper injection technique depends on the site of injection. For example, when injecting a substance into the tail vein of a small animal (e.g., rat) it is best to submerge the tail in slightly warm water (37 °C) for a minute and then wipe with ethanol or isopropyl alcohol. This will cause the blood vessel to swell and become visible. Use a smaller gauge needle (e.g., 22-gauge needle) and inject with a slight pressure.
Uncover more answers to common questions in our FAQs on preparing inhibitors article.
Factors That Affect Inhibitor Use
Inhibitor use will also be affected by:
Mode of action
Mode of action is the specific biochemical interaction through which a drug/inhibitor produces its pharmacological effect. A drug may inhibit an enzyme or block a receptor. It could be a reversible effect or an irreversible effect. If the drug and substrate are competing, then the excess availability of substrate may reduce the effect of the drug.
Bioavailability
Not everything presented to a cell or an organism is used. Bioavailability refers to the amount of drug that actually reaches the circulation and is considered to be active in the body.
- For example, if a drug/inhibitor is injected into the bloodstream via injection, most of it is available to the body (i.e. it has high bioavailability).
- However, when it is given orally, some of it may be destroyed by the acid of the stomach or alkalinity of the intestine. Or, due to physical barriers in the digestive system, only a fraction of it may reach the site of action (i.e. it has low bioavailability).
Half-life
The half-life is the time frame in which 50% of the drug/inhibitor is destroyed in the cell or in the body. A shorter half-life means that you have to take it administer it more frequently, because it may be being destroyed in the liver as it circulates through. Half-life can give a good indication of the time required to reach steady-state after a dosing regimen is initiated.
Loading dose (Priming dose)
A loading dose (or priming dose) is one or more doses given initially to rapidly achieve target concentration. However, exercise caution that the loading dose does not cause toxicity.
Inhibitor structure
The inhibitor structure is the chemical nature of the inhibitor that strongly influences its ability to cross membranes.
- Hydrophobic molecules with no net charge readily cross most biological membranes.
- By contrast, hydrophilic drugs that have a nonuniform electron distribution or have a positive or negative charge do not cross membranes.
Hepatic and renal clearance
Hepatic clearance tells us how quickly or slowly the drug/inhibitor is being metabolized in the liver.
- If the drug is rapidly metabolized by the liver, the amount of drug available for systemic circulation is significantly decreased.
- If the liver enzymes that metabolize a drug are already activated, then the drug will clear out faster and may not generate its full biological or clinical effects.
- For example, prior consumption of alcohol will activate the liver enzymes that will cause a faster clearance of some drugs through the system. This will reduce the effectiveness of the drug or, in some cases, may cause toxic effects.
Similarly, renal clearance is the volume of plasma from which a substance is completely removed by the kidney in a given amount of time. This may also be affected by drugs that affect glomerular filtration rates.
Binding to proteins
When circulating in the bloodstream, several drugs/inhibitors can be bound to albumin proteins in the blood, which carry them to the liver for metabolism and clearance. However, if the binding is too tight and the inhibitor or drug is not easily unloaded for further metabolism, then the effect could be delayed. Albumin has a strong affinity for anionic and hydrophobic inhibitors. Most neutral and hydrophilic molecules do not bind to albumin.
Tissue-specific distribution
Most drugs/inhibitors exert their effects not within the plasma compartment, but in defined target tissues into which drugs have to be distributed from the central compartment (blood). Target site drug levels may substantially differ from corresponding plasma levels and a 100% equilibration between blood and tissue cannot always be assumed. Drug distribution processes may be characterized by high inter-tissue variability. Suboptimal target site concentrations may also have important clinical implications and may indicate therapeutic failure in some cases. Therefore, the determination of drug tissue penetration plays an important role in clinical drug development.
Drug/Inhibitor toxicity
The therapeutic benefits of a drug usually outweigh its risks. However, all drugs are likely to have some side effects. Drug/inhibitor toxicity results when too much of it has accumulated in the bloodstream. Toxicity may result when the dose is too high or when the liver or kidneys are unable to remove the drug from the bloodstream in an effective manner.
Discover more characteristics to think about before your next experiment in our Critical Inhibitor Characteristics to Consider article.
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