Christopher Thomas is a retired musician. On several visits over a period of a year you note his blood pressure is 142/97, 138/98, 147/99, and 135/95. He has a history of smoking and has chronic bronchitis. He also has elevated lipid and cholesterol levels. You wish to begin therapy with an antihypertensive medication. There are several choices of drugs which can be prescribed in this situation. Two of the potential choices are prazosin, which is a competitive alpha-receptor blocker, and propranolol, which is a competitive beta-receptor antagonist. You choose to prescribe prazosin 1 mg b.i.d. The next day Mr. Thomas calls and complains that he took his medication and shortly thereafter became dizzy and passed out.

Consider the following questions which you will be able to answer after completion of the lecture sequences listed above:

• What are the various drug classes which can be used to treat hypertension?
  
• What are the pharmacologic and nonpharmacologic approaches to the therapy of hypertension?
  
• What is the agonist blocked by prazosin?
  
• Where are the receptors prazosin blocks located and why does this cause hypotension?
  
• What factors in this case made prazosin a better choice than propranolol?
  
• Why did Mr. Thomas faint and should this be a cause for concern?

Drug:A chemical substance that interacts with a biological system to produce a physiologic effect.

Many chemicals are inert with regard to altering physiologic responses. However, molecules which alter human, animal or plant physiology are referred to as drugs. Most drugs interact with receptors to produce these effects.

The binding of a drug to a receptor is determined by the following forces that allow interaction between functional groups on the drug and complementary binding surfaces in the receptor:

1. Hydrogen bonds
2. Ionic bonds
3. Van der Waals forces
4. Covalent bonds

There are different types of receptors with which endogenous ligands and exogenously administered drugs can interact, including:

1. Hormone receptors
2. Tyrosine kinase linked receptors
3. G-protein coupled receptors
4. Receptors linked to ion channels

Drugs that interact with receptors can be classified as being either agonists or antagonists.

An agonist is a drug that once bound to the receptor, initiates a change in cellular activity. Agonist binding induces a conformational change in the receptor such that cellular signaling pathways are activated. An example of an agonist action is a drug that binds to the myocardial beta adrenergic receptor and increases the force of contraction. The binding of the agonist triggers a series of biochemical events which ultimately lead to the alteration in function. The biochemicals which initiate these changes are referred to as second messengers.

Some important second messenger systems activated by the binding of agonists to cell surface receptors include:

1. The cyclic AMP and GMP systems
2. Calcium and calmodulin
3. Phosphoinositides and diacylglycerol

 

Antagonists molecules have chemical substituents that allow the drug to bind to receptors. However, once bound, the drug cannot alter the receptor in a manner that can initiate a change in cellular function. However, occupation of the receptor can prevent the binding and actions of agonists. Antagonists are also referred to as blockers.

Many chemical substances have the potential to be drugs. Two factors which determine whether a chemical will have drug effects are affinity and intrinsic activity.

• Affinity is the measure of the tightness with which a drug binds to a receptor.
  
• Intrinsic activity is the measure of the ability of a drug, once bound to the receptor, to alter receptor function such that cellular signaling is activated resulting in a measurable physiologic effect.

Affinity and intrinsic activity are independent properties of drugs. Agonists have both affinity and intrinsic activity while antagonists have only affinity for the receptor.

The binding of a drug,D, to the receptor, R, can be described by this expression.

where k₁ is the rate of association of the drug with receptor and k_-1 is the rate of DR complex dissociation. At equilibrium the amount of DR formation is equal to the amount of DR dissociation and thus the steady state amount of [DR] is constant.

Affinity is an important concept in pharmacology. It describes the strength at which a drug binds to the receptor and is equal to the ratio of k₁ and k_-1. It is unique for each drug in each receptor system and can be used to identify receptors. A high affinity drug has a much greater tendency to bind to the receptor ( a large value for k₁) relative to dissociation from the receptor (a small value for k_-1). Kd is the equilibrium dissociation constant and is the reciprocal of the affinity. This is a term widely used to describe the binding of drugs to receptors. The units of the dissociation constant are some measure of concentration such as molar, millimolar, micromolar, or nanomolar. Dissociation constants are small numbers, significantly less than 1, such as 1 x 10^-8M or 10 nanomolar. There is an inverse relationship between the Kd and affinity. The smaller the Kd, the greater the affinity. A drug which has a dissociation constant of 1 nanomolar is said to have higher affinity than a drug which has a dissociation constant of 1 micromolar. This is because 1 nanomolar is much smaller than 1 micromolar.

By appropriate substitution of the equations above we can write:

This equation describes the binding of drugs to pharmacologic receptors and states that the amount of drug bound to the receptor is dependent on the drug concentration and K_d.

This points out that when a drug is given at a concentration equal to its dissociation constant, 50% of the receptors will be occupied. The greater the affinity, the less drug will be required to occupy 50% of the receptors.

where E_max is the maximum obtainable in the receptor system being studied. We can now write:

This equation states that the effect observed, E/Emax, is determined by the concentration of the drug and its affinity (Kd) for the receptor. In other words, the effect is related to the degree of receptor occupancy. This helps us to understand the extreme potency of some drugs. A drug with very high affinity will achieve a large degree of receptor saturation at very low concentrations.

Thus far the effect (E) of a drug has only been related to receptor occupancy. However, drugs once bound to a receptor differ in their ability to initiate a change in receptor conformation and physiologic activity. This is a more difficult parameter to conceptualize. Drug binding to receptors can be measured quite easily and is governed by relatively straightforward biochemical principles. The ability to activate the receptor and induce an effect encompasses much more than the simple chemical process of drug-receptor binding. Let us use the symbol, e to define intrinsic activity. Intrinsic activity describes the ability of a drug induce changes in receptor structure leading to alterations in cellular activity. We can now write:

Therefore, the ability of a drug to produce a physiologic effect is dependent on receptor occupancy (which is in turn governed by [D] and Kd) and the propensity of the drug to activate the receptor (e). While similar, you should understand that equations #1 and #2 calculate different parameters. 

• Equation #1 determines the degree of receptor occupancy. 
  
• Equation #2 (with the presence of e) calculates the effect of a drug on a functional response.

Question: At what concentration of agonist will the effect (E/E_max) be equal to 50 % of the maximal response?    Help  Solution:

This concentration is also referred to as the Effective Dose-50 or ED₅₀.

While the precise mechanism is not known, agonists have the ability to impart a stimulus to the receptor such that cellular signaling is activated. Agonists differ in their propensity to deliver an activating stimulus to receptors. As a result, agonists can be further divided into full and partial agonists:

• Full Agonists: Compounds that are able to elicit a maximal response following receptor occupation and activation.
  
• Partial Agonists: Compounds that can activate receptors but are unable to elicit the maximal response of the receptor system.

Drugs which are full agonists are arbitrarily assigned an intrinsic activity value of 1. Partial agonists, which cannot produce the same maximal effect as full agonists will have intrinsic activity values less than 1. The effect of partial and full agonists on equation # 2 is apparent. Because partial agonists have e values less than 1, the value of E/Emax will be some fraction of the value obtained with a full agonist.

Dose-response curves are used as a graphical method to present data describing the ability of a drug to produce a given physiologic effect. For example, a clinical study may examine the effect of increasing amounts of an analgesic on pain threshold. To present the data, the concentration of the drug would be plotted on the x-axis and the effect on pain threshold would be presented on the y-axis. A plot of drug concentration versus effect is a rectangular hyperbola. Notice how the effect increases until a maximum is achieved. This is a cumbersome graph because drug concentrations often vary over 100 to 1000-fold, necessitating a long X-axis. To overcome this problem, the log of the drug concentration versus the effect is plotted. A plot of the log of [D] versus effect is a sigmoid curve.

As illustrated below, the position and shape of the log-dose response curve is dependent on the affinity of the ligand for the receptor and its intrinsic activity. Affinity determines the position of the dose-response curve on the X-axis, while intrinsic activity affects the magnitude of the response.

	Norepinephrine and phenylephrine are full agonists with intrinsic activity values of 1. However, Norepinephrine has a higher affinity for the receptor. As is illustrated, affinity affects the position of the dose-response curve on the x-axis.
	Clonidine and Methoxamine are partial agonists. Clonidine has a higher affinity but a lower intrinsic activity than does Methoxamine. Intrinsic activity affects the magnitude of the response.

Selectivity versus Specificity

A drug would be considered specific if it produces one and only one effect. For example a drug that interacts with one and only one receptor to lowered blood pressure would be considered specific. Few drugs are specific. Most drugs interact with multiple receptors. However, they do so with different affinities. These drugs are said to be selective. The biological response is determined by the affinity the drug exhibits for these different receptors and the total response of a patient to such a drug will be the sum of effects at each receptor. This can be illustrated with the following example. A hypothetical drug can interact at 2 distinct receptor systems. At each of these receptors, the ligand has a different affinity, intrinsic activity, as well as pharmacologic effect as indicated;

Receptor System # 1:
K_D#1 = 0.4 nM, intrinsic activity 0.65, effect- lowering of systemic arterial blood pressure.

Receptor System # 2:
K_D#2 = 40.0, intrinsic activity 1.0,effect- lethal venticular arrhythmias.

Two important points are to be taken from this example- " a little drug is good, a lot may be quite bad." Secondly, the difference between a chemical being a drug or a poison is the concentration.

Antagonists drugs have a chemical structure that premits binding to the receptor (affinity for the receptor) but are not capable of triggering receptor activation (they do not have intrinsic activity). An antagonist which binds to the receptor in a reversible manner is referred to as a competitive antagonist. This has important implications regarding the effect on the response of agonists. This is illustrated with the equations below :

The antagonist [B] and agonist [D] are competing for the same limited number of receptors [R]. The drug which binds to the receptor in the highest concentration will be determined by two factors, the affinities the agonist and antagonist have for the receptor and their relative concentrations. The agonist [D] effect is determined by the level of [DR]. Therefore, the more R complexed with the antagonist ([B]), the less will be available for a productive interaction with D. In the presence of a competitive antagonist equation #2 is modified as follows:

Where [B] is the concentration of antagonist and Kb is the equilibrium dissociation constant exhibited by the antagonist for the receptor. Inspection of this equation will reveal that the affinity of the agonist, Kd, is modified by the term (1+[B]/Kb). If the concentration of antagonist [B] is large in relation to its affinity Kb, the term (1+[B]/Kb) will be large. This will have the same effect on the dose-response curve for the agonist as does increasing the value of Kd. Therefore, the major effect of a competitive antagonist is to shift the dose-response curve for an agonist to the right. As illustrated below, the dose-response curve obtained in the presence of a competitive antagonist is also parallel to the curve obtained in the absence of antagonist.

Prazosin is a competitive antagonist at the same receptor where phenylephrine acts.

1. Reversible binding to the receptor
2. Blockade can be overcome by increasing the agonist concentration
3. Does not decrease the maximal response of the agonist
4. Shifts the agonist dose-response curve to the right in a parallel manner

Irreversible Receptor Antagonists

Another type of antagonist is an irreversible receptor antagonist. The properties of irreversible antagonists are markedly different from competitive antagonists. Irreversible receptor antagonists are chemically reactive compounds. These ligands first bind to the receptor. Following this binding step, the ligand reacts with the functional groups of the receptor. The consequence of this chemical reaction is that the ligand becomes covalently bound to the receptor. Due to the fact that a chemical bond is formed, an irreversible ligand does not freely dissociate from the receptor. The synthesis of new receptor protein may be required to generate a receptor free of an irreversible blocker. Because an irreversible receptor antagonist reduces the total number of active receptors, [RT], the maximal pharmacologic effect Emax is also decreased. Furthermore, the blocking activity of irreversible receptor antagonists can not be overcome by increasing the agonist concentration.

To summarize, the properties of irreversible receptor blockers are:

1. Chemically reactive compounds
2. Form covalent bonds with the receptor
3. Irreversibly inactivate the receptor
4. Insurmountable blockade
5. Shifts the agonist dose-response curve to the right and depresses maximal responsiveness

Examine the following interaction of two drugs at a single receptor:

Norepinephrine is an endogenous neurotransmitter. Phenoxybenzamine is an irreversible antagonist at the same receptor that norepinephrine interacts.

An experiment is carried out in the following manner with phenoxybenzamine and norepinephrine. A control norepinephrine dose-response curve is generated. This is curve A in Figure 1.

In another experiment, the preparation is treated with 1 x 10^-9M phenoxybenzamine for 3 min. Phenoxybenamine is extensively washed out of the preparation and the effect on the norepinephrine dose-response curve is determined. Dose-response curve B in Figure 1 shows the results of this experiment.

In the final experiment, the tissue is first incubated with 1 x 10^-4M norepinephrine for 15 min. 1 x 10^-9M phenoxybenzamine is added in the presence of this norepinephrine and both drugs are incubated for 3 min. All drugs are then washed extensively out of the preparation. After the washout, another norepinephrine log dose-response curve is generated. The results of this experiment are shown in dose-response curve C.

Thus far we have made the assumption that the relationship between receptor occupancy [DR]/[RT] and response E/Emax is linear. This linear relationship can be expressed by equation # 2 and is shown in the graph below. In this type of response system, all receptors must be occupied to produce a maximal response.

In most physiological systems in which drugs will be administered, the relationship between receptor occupancy and response is not linear but some unknown function f of receptor occupancy. In the graph, this unknown function is presented as being hyperbolic. As the graph depicts in this type of system, all receptors do not have to be occupied to produce a full response. Because of this hyperbolic relationship between occupancy and response, maximal responses are elicited at less than maximal receptor occupancy. A certain number of receptors are "spare." Spare receptors are receptors which exist in excess of those required to produce a full effect. There is nothing different about spare receptors. They are not hidden or in any way different from other receptors.

A = Large Receptor Reserve
B = Intermediate Receptor Reserve
C = No Receptor Reserve

In a non-spare receptor system, the ED₅₀ = Kd. In a spare receptor system, the pharmacologic ED₅₀ and the Kd are not equal. The larger the number of spare receptors, the greater the difference between the ED₅₀ and Kd.

Partial agonists have lower intrinsic activities than full agonists but values greater than competitive antagonists. At certain concentrations partial agonists actually can be antagonists.

This curve shows the % Maximal Response vs Log Dose (magenta) as well as the  % Receptor Occupancy vs Log Dose (green) of a partial agonist.  Note that the response is only 10% (e=0.10) even when 100% of the receptors are occupied.  Click on A or B to visualize the occupancy at that point of the curve.

Reasons for the Nonlinear Relationship Between Receptor Occupancy and Physiologic Response

To understand how the relationship between occupancy and response can be non linear let us analyze the components which contribute to the response.

G-Protein Coupled Receptors

G-protein coupled receptors are a large family of receptors that serve as the site of action for many drugs. The name reflects the fact that the activity of these receptors is regulated by interaction with guanine nucleotide regulatory proteins (hence G-proteins). Despite major differences in the physiologic responses they activate and the variety of second messengers involved, the structure of all G-protein coupled receptors is similar. G-protein coupled receptors have a single polypeptide chain which passes through the cell membrane seven times. This arrangement results in the formation of loops on both the extracellular and intracellular sides of the membrane. Seven clusters of hydrophobic amino acids make up the membrane spanning domains of the receptor. The membrane spanning regions also form a binding pocket with which agonists and antagonists interact. The intracellular loops are thought to be necessary for the interaction with G-proteins and second messenger systems.

In cellular signaling pathways involving G-proteins, the receptor/agonist complex does not interact directly with the enzyme which generates the second messenger. Rather, an intermediate or transducing protein couples the receptor to the second messenger generating system. This is the role of the G-protein . There is not a single G-protein, but a family of G-proteins which functions to regulate second messenger systems. G-proteins consist of three subunits: alpha, beta and gamma. In the resting state the receptor is not occupied by an agonist and the G-protein exists as trimer of the alpha, beta and gamma subunits with GDP bound to the alpha subunit. In this state, G-proteins are poor activators of intracellular signaling systems. Agonist binding to the receptor promotes the dissociation of the GDP and binding of GTP. GTP binding promotes the dissociation of the alpha subunit from the beta and gamma subunits. It is the GTP bound alpha subunit that activates effector enzyme systems. The alpha subunit is also a GTPase and is thus able to hydrolyze the GTP. The hydrolysis of GTP to GDP deactivates the alpha subunit and terminates the activation effector systems. The alpha subunit/GDP complex is then re-associated with the beta and gamma subunits to complete the regulatory cycle. The G-protein heterotrimer is again available for interaction with a receptor and activation of second messenger generating systems. Therefore, the rate at which the GTP is hydrolyzed regulates the time the G-protein is active. The longer the G-protein is active, the more second messenger can be generated.

In a responding system which has a linear relationship between occupancy and physiologic response, there is a direct proportionality between the degree of receptor activation and the generation of second messengers. While this is difficult to conceptualize, it can be thought of as a small amount of receptor occupancy producing a small increase in the level of the second messenger. This small amount of second messenger activates a small increase in physiologic response. In the more realistic nonlinear occupancy versus response system, a small degree of receptor occupancy generates a large increase in second messenger levels which in turn generate an even larger physiologic response. The signal is amplified at every step of the signal transduction process. In this fashion, then, a small degree of receptor occupancy leads to a large physiologic response. Consider the following example. In a given beta-receptor system, 50,000 cAMP molecules are needed to yield a full response. In a linear response relationship, 50,000 receptors would have to be occupied to give a full response. However, in a nonlinear system, only 100 would be required to achieve a full response.

Continuous exposure of an agonist results in a phenomenon referred to as desensitization. The same concentration of agonist becomes less and less effective at producing the same level of effect. When this desensitization occurs very rapidly, it is referred to as tachyphylaxis. Recent evidence has suggested potential mechanisms by which the process of tachyphylaxis and desensitization occur. The receptor becomes phosphorylated in the third cytoplasmic loop and c-terminal tail. The phosphorylated receptor is less efficient at activating G-protein and also exhibits lower affinity for agonists. The receptors can also be removed from and sequestered away from the cell surface. These events indicate that second messengers not only regulate intracellular processes but are also capable of regulating the receptor systems which generate them.

Traditionally, G-protein coupled receptors were thought to be inactive and that agonist occupation was required to allow the receptor to assume an active conformation. Recently, though, it has been suggested that the receptor can be active without the presence of agonist. The term for this is constitutive activity. Constitutively active receptors are thought to be coupled to second messenger pathways in the absence of agonists.

This has led to the postulate that in addition to traditional agonists, drugs can function as inverse agonists. Inverse agonists bind to constitutively active receptors and shift the equilibrium to the formation of the inactive conformer. In this system an inverse agonist would actually reverse receptor activity. The concept of inverse agonism has added a level of complexity to our thinking of drug action. As the diagram below illustrates, the spectrum of drug activity can range from a full agonist to a full inverse agonist.