intracellular receptors, cell signaling by intracellular receptors, drug interaction and plasma protein binding

2. ◦ an organ or cell able to respond to light, heat, or
other external stimulus and transmit a signal to a
sensory nerve.
◦ "the retina of the octopus has up to 20 million
light receptors"
◦ a region of tissue, or a molecule in a cell
membrane, which responds specifically to a
particular neurotransmitter, hormone, antigen, or
other substance.
◦ "when viruses succeed in binding to cell
membrane receptors they still have to enter the
cell before they can replicate“

3. ◦ Receptors are proteins or glycoprotein that bind signaling molecules known as first
messengers, or ligands. They can initiate a signaling cascade, or chemical response,
that induces cell growth, division, and death or opens membrane channels.
◦ Adequate Stimulus
Sensory receptors with corresponding stimuli to which they respond.
Receptorbaq Stimulus
Apmullae of Lorenzini
(primarily function as
electroreceptors
Electric fields, salinity, and
temperature
Baroreceptor Pressure in blood vessels
Chemo receptor Chemical stimuli

4. ◦ Intracellular receptors are located in the cytoplasm of the cell
and are activated by hydrophobic ligand molecules that can
pass through the plasma membrane. Cell-surface receptors
bind to an external ligand molecule and convert an
extracellular signal into an intracellular signal.

5. ◦ Drug receptors. Receptor is a macromolecule in the membrane or inside the cell that
specifically (chemically) bind a ligand (drug). The binding of a drug to receptor
depends on types of chemical bounds that can be established between drug and
receptor.
◦ Intracellular receptors are receptors located inside the cell rather than on its cell
membrane. Classic hormones that use intracellular receptors include thyroid and
steroid hormones.
◦ Intracellular receptors are located within a cell and bind to molecules that cross
directly through the membrane. Membrane receptors are located in the membrane,
bind to molecules that cannot cross it, and transmit the signal to the cell interior by
changing shape.

6. ◦ Intracellular (nuclear) receptors
◦ Many hormones act at intracellular receptors to produce long-term changes in cellular
activity by altering the genetic expression of enzymes, cytokines or receptor proteins.
Such hormones are lipophilic to facilitate their movement across the cell membrane.
◦ Classic hormones that use intracellular receptors include thyroid and steroid
hormones. Examples are the class of nuclear receptors located in the cell nucleus and
cytoplasm and the IP3 receptor located on the endoplasmic reticulum.
◦ Many signaling pathways, involving both intracellular and cell surface receptors,
cause changes in the transcription of genes. However, intracellular receptors are
unique because they cause these changes very directly, binding to the DNA and
altering transcription themselves.

7. .
◦ The amino acid-derived hormones epinephrine and norepinephrine bind
to beta-adrenergic receptors on the plasma membrane of cells.
Hormone binding to receptor activates a G-protein, which in turn
activates adenylyl cyclase, converting ATP to cAMP. cAMP is a second
messenger that mediates a cell-specific response.
◦ Steroid receptors are intracellular. The aldosterone mineralocorticoid
receptor (MR) complex binds on the DNA to specific hormone response
element, which leads to gene specific transcription.

8. .
◦ Insulin binds outside the cell to the extracellular domain of its
receptor and induces a structural change that is propagated
across the membrane to the intracellular kinase domains
inside the cell, causing them to activate each other, thus
initiating signaling cascades.

9. Intracellular domains
◦ The intracellular (or cytoplasmic) domain of the receptor interacts with the interior of
the cell or organelle, relaying the signal. ... The intracellular domain communicates via
protein-protein interactions against effector proteins, which in turn pass a signal to the
destination.
Is insulin intracellular or extracellular?
◦ The Insulin Receptor and Mechanism of Action
◦ The insulin receptor is composed of two alpha subunits and two beta subunits linked
by disulfide bonds. The alpha chains are entirely extracellular and house insulin
binding domains, while the linked beta chains penetrate through the plasma
membrane.

10. INTRACELLULAR DOMAIN

11. ◦ Steroid and thyroid hormone receptors are members of a large group ("superfamily")
of transcription factors. In some cases, multiple forms of a given receptor are
expressed in cells, adding to the complexity of the response. All of these receptors are
composed of a single polypeptide chain that has, in the simplist analysis, three distinct
domains:
◦ The amino-terminus: In most cases, this region is involved in activating or stimulating
transcription by interacting with other components of the transcriptional machinery.
The sequence is highly variable among different receptors.
◦ DNA binding domain: Amino acids in this region are responsible for binding of the
receptor to specific sequences of DNA.

12. .
◦ The carboxy-terminus or ligand-binding domain: This is the region that
binds hormone.
◦ In addition to these three core domains, two other important regions of
the receptor protein are a nuclear localization sequence, which targets
the the protein to nucleus, and a dimerization domain, which is
responsible for latching two receptors together in a form capable of
binding DNA.

13. .
In most cases, the ligands of intracellular receptors are small, hydrophobic
(water-hating) molecules, since they must be able to cross the plasma
membrane in order to reach their receptors.

14. ◦ Cell signaling is the process of cellular communication within the body driven by cells
releasing and receiving hormones and other signaling molecules. As a process, cell
signaling refers to a vast network of communication between, and within, each cell of our
body. Cell signaling enables coordination within multicellular organisms.
◦ Cell signaling can occur through a number of different pathways, but the overall theme is that the
actions of one cell influence the function of another. Cell signaling is needed by multicellular
organisms to coordinate a wide variety of functions. Nerve cells must communicate with muscle cells
to create movement, immune cells must avoid destroying cells of the body, and cells must organize
during the development of a baby.

15. ◦ Some forms of cell signaling are intracellular, while others are intercellular. Intracellular signals are
produced by the same cell that receives the signal. On the other hand, intercellular signals can
travel all throughout the body. This allows certain glands within the body to produce signals which
take action on many different tissues across the body. Each target cell will have the required
receptors, as in the image below:

16. At its core cell signaling can simply be described as the production of a “signal” by one cell. This signal is
then received by a “target” cell. In effect, signal transduction is said to have three stages:
First, reception, whereby the signal molecule binds the receptor
Then, signal transduction, which is where the chemical signal results in a series of enzyme activations
Finally, the response, which is the resulting cellular responses.


18. ◦ Cell signaling serves a vital purpose in allowing our cells to carry out life as
we know it. Moreover, thanks to the concerted efforts of our cells via their
signaling molecules, our body is able to orchestrate the many complexities
that maintain life. These complexities, in effect, demand a diverse collection
of receptor-mediated pathways that execute their unique functions.
◦ In general, a ligand will activate a receptor and cause a specific response.
Receptors are typically protein molecules, as seen in blue below. The
orange ligand can be many different types of molecules, but it forms an
induced fit with the receptor that is very specific.

20. ◦ Intracellular receptor, which is located within the cytoplasm of the cell and
generally includes two types. In addition to cytoplasmic receptors, nuclear
receptors are a special class of protein with diverse DNA binding domains that
when bound to steroid or thyroid hormones form a complex that enters the
nucleus and modulates the transcription of a gene. IP3 receptors are another class,
which are located in the endoplasmic reticulum and carry out important functions
like the release of Ca2+ that is so crucial for the contraction of our muscles and
plasticity of our neural cells.

21. ◦ Spanning our plasma membranes are another type of receptor called Ligand-gated ion channels that
allow hydrophilic ions to cross the thick fatty membranes of our cells and organelles. When bound to
a neurotransmitter like acetylcholine, ions (commonly K+, Na+, Ca2+, or Cl–) are allowed to flow
through the membrane to allow the life-sustaining function of neural firing to take place, among many
other functions!
◦ Comparatively, G-protein coupled receptors (GPCRs) remain the largest and most diverse group of
membrane receptors in eukaryotes. In fact, they are special in that they receive input from a diverse
group of signals ranging from light energy to peptides and sugars. In effect, their mechanism of
action also starts with a ligand binding to its receptor. However, the demarcation is that ligand binding
results in the activation of a G protein that is then able to transmit an entire cascade of enzyme and
second messenger activations that carry out an incredible array of functions like sight, sensation,
inflammation, and growth.

22. ◦ Likewise, receptor tyrosine kinases (RTKs) are another class of receptors revealed to show
diversity in their actions and mechanisms of activation. For example, the general method of
activation follows a ligand binding to the receptor tyrosine kinase, which allows their kinase
domains to dimerize. Then, this dimerization invites the phosphorylation of their tyrosine kinase
domains that, in turn, allow intracellular proteins to bind the phosphorylated sites and become
“active.” An important function of receptor tyrosine kinases is their roles in mediating growth
pathways. Of course, the downside of having complex signaling networks lies in the unforeseen
ways in which any alteration can produce disease or unregulated growth – cancer. Still, much is
yet to be understood about cell signaling pathways, but one appreciable fact is that the
importance they carry is nothing short of monumental.

23. ◦ Typically, cell signaling is either mechanical or biochemical and can occur locally. Additionally,
categories of cell signaling are determined by the distance a ligand must travel. Likewise,
hydrophobic ligands have fatty properties and include steroid hormones and vitamin D3. These
molecules are able to diffuse across the target cell’s plasma membrane to bind intracellular
receptors inside.
◦ On the other hand, hydrophilic ligands are often amino-acid derived. Instead, these molecules
will bind to receptors on the surface of the cell. Comparatively, these polar molecules allow the
signal to travel through the aqueous environment of our bodies without assistance.

24. ◦ Signaling molecules are currently assigned one of five classifications.
1- Intracrine ligands are produced by the target cell. Then, they bind to a receptor within the cell.
2 -Autocrine ligands are distinct in that they function internally and on other target cells (ex.
Immune cells).
3- Juxtacrine ligands target adjacent cells (often called “contact-dependent” signaling).
4- Paracrine ligands target cells only in the vicinity of the original emitting cell (ex.
Neurotransmitters).
5- Lastly, Endocrine cells produce hormones that have the important task of targeting distant cells
and often travel through our circulatory system.

25. ◦ A great (and well-used) example of a cell signaling pathway is seen in the balancing actions of
insulin. Insulin, a small protein produced by the pancreas, is released when glucose levels in
the blood get far too high.
◦ First, the high glucose levels in the pancreas stimulate the release of insulin into the
bloodstream. Insulin finds its way to the cells of the body, where it attaches to the insulin
receptors. This sets off a signal transduction pathway within each cell that causes the glucose
channels to open, as seen in this graphic:

27. 1.PHOSPHORYLATION
2.SECOND MESSENGERS

28. Phosphorylation
◦ One of the most common chemical modifications that occurs in signaling pathways is the
addition of a phosphate group (PO4–3) to a molecule such as a protein in a process called
phosphorylation. The phosphate can be added to a nucleotide such as GMP to form GDP or
GTP. Phosphates are also often added to serine, threonine, and tyrosine residues of proteins,
where they replace the hydroxyl group of the amino acid (Figure 1). The transfer of the
phosphate is catalyzed by an enzyme called a kinase. Various kinases are named for the
substrate they phosphorylate. Phosphorylation of serine and threonine residues often activates
enzymes. Phosphorylation of tyrosine residues can either affect the activity of an enzyme or
create a binding site that interacts with downstream components in the signaling cascade.
Phosphorylation may activate or inactivate enzymes, and the reversal of phosphorylation,
dephosphorylation by a phosphatase, will reverse the effect.

30. ◦ Second messengers are small molecules that propagate a signal after it has been initiated by the
binding of the signaling molecule to the receptor. These molecules help to spread a signal through
the cytoplasm by altering the behavior of certain cellular proteins.
◦ Calcium ion is a widely used second messenger. The free concentration of calcium ions (Ca2+)
within a cell is very low because ion pumps in the plasma membrane continuously use adenosine-5′-
triphosphate (ATP) to remove it. For signaling purposes, Ca2+ is stored in cytoplasmic vesicles, such
as the endoplasmic reticulum, or accessed from outside the cell. When signaling occurs, ligand-
gated calcium ion channels allow the higher levels of Ca2+ that are present outside the cell (or in
intracellular storage compartments) to flow into the cytoplasm, which raises the concentration of
cytoplasmic Ca2+. The response to the increase in Ca2+ varies, depending on the cell type involved.
For example, in the β-cells of the pancreas, Ca2+ signaling leads to the release of insulin, and in
muscle cells, an increase in Ca2+ leads to muscle contractions.

31. ◦ Another second messenger utilized in many different cell types is cyclic AMP (cAMP). Cyclic
AMP is synthesized by the enzyme adenylyl cyclase from ATP (Figure 2). The main role of
cAMP in cells is to bind to and activate an enzyme called cAMP-dependent kinase (A-kinase).
A-kinase regulates many vital metabolic pathways: It phosphorylates serine and threonine
residues of its target proteins, activating them in the process. A-kinase is found in many
different types of cells, and the target proteins in each kind of cell are different. Differences give
rise to the variation of the responses to cAMP in different cells.

32. ◦ Present in small concentrations in the plasma membrane, inositol phospholipids are lipids that
can also be converted into second messengers. Because these molecules are membrane
components, they are located near membrane-bound receptors and can easily interact with
them. Phosphatidylinositol (PI) is the main phospholipid that plays a role in cellular signaling.
Enzymes known as kinases phosphorylate PI to form PI-phosphate (PIP) and PI-bisphosphate
(PIP2).
◦ The enzyme phospholipase C cleaves PIP2 to form diacylglycerol (DAG) and inositol
triphosphate (IP3) (Figure 3). These products of the cleavage of PIP2 serve as second
messengers. Diacylglycerol (DAG) remains in the plasma membrane and activates protein
kinase C (PKC), which then phosphorylates serine and threonine residues in its target proteins.
IP3 diffuses into the cytoplasm and binds to ligand-gated calcium channels in the endoplasmic
reticulum to release Ca2+ that continues the signal cascade.

34.  A drug interaction is a change in the action or side effects of a drug caused by concomitant administration
with a food, beverage, supplement, or another drug. There are many causes of drug interactions. For
example, one drug may alter the pharmacokinetics of another.
OR
 A drug interaction is a reaction between two (or more) drugs or between a drug and a food, beverage, or
supplement. Taking a drug while having certain medical conditions can also cause a drug interaction. ...
A drug interaction can affect how a drug works or cause unwanted side effects.
OR
 A drug interaction is a reaction between two (or more) drugs or between adrug and a food, beverage, or
supplement. Taking a drug while having certain medical conditions can also cause a drug interaction. For
example, taking a nasal decongestant if you have high blood pressure may cause an unwanted reaction.

35. DRUG INTERACTION

36. Drug interactions can be categorized into 3 groups:
 Interactions of drugs with other drugs (drug-drug interactions)
 Drugs with food (drug-food interactions)
 Drug with disease condition (drug-disease interactions).
 The effect a drug has on a person may be different than expected because that drug interacts with
 Another drug the person is taking (drug-drug interaction).
 Food, beverages, or supplements the person is consuming (drug-nutrient interaction).
 Another disease the person has (drug-disease interaction).
 The effects of drug interactions are usually unwanted and sometimes harmful. Interactions may
 Increase the actions of one or more drugs, resulting in side effects or toxicity.
 Decrease the actions of one or more drugs, resulting in failed treatment.

37. • Drug-drug interactions can involve prescription or nonprescription (over-the-counter) drugs.
• Types of drug-drug interactions include:
1. duplication
2. opposition (antagonism)
3. alteration of what the body does to one or both drugs.

39. When two drugs with the same effect are taken, their side effects may be intensified. Duplication may occur
when people inadvertently take two drugs (often at least one is an over-the-counter drug) that have the same
active ingredient. For example, people may take a cold remedy and a sleep aid, both of which
contain diphenhydramine, or a cold remedy and a pain reliever, both of which contain acetaminophen. This
type of duplication is particularly likely with the use of drugs that contain multiple ingredients or that are sold
under brand names (thus appearing to be different but actually containing the same ingredients).
◦ Awareness of drug ingredients is important, as is checking each new drug to avoid duplication. For
example, many prescription-strength pain relievers contain an opioid plus acetaminophen. People taking
such a product who do not know its ingredients might take over-the-counter acetaminophen for extra relief,
risking toxicity.
◦ People can reduce the risk of this kind of duplication by keeping each doctor informed about all drugs being
taken and by using one pharmacy to obtain all prescriptions. Also, people should not take previously
prescribed drugs (such as a sleeping pill or pain reliever) without checking with the doctor or pharmacist
because that drug may duplicate or otherwise interact with one of their current drugs.

40. ◦ Two drugs with opposing actions can interact, thereby reducing the effectiveness of one or both.
For example, nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, which are
taken to relieve pain, may cause the body to retain salt and fluid. Diuretics, such
as hydrochlorothiazide and furosemide, help rid the body of excess salt and fluid. If a person
takes both types of drug, the NSAID may reduce the diuretic's effectiveness. Certain beta-
blockers (such as propranolol), taken to control high blood pressure and heart disease,
counteract beta-adrenergic stimulants, such as albuterol, taken to manage asthma. Both types
of drugs target the same cell receptors—beta-2 receptors —but one type blocks them, and the
other stimulates them.

41. ◦ One drug may alter how the body absorbs, distributes, metabolizes, or excretes another drug .
◦ Acid-blocking drugs, such as histamine-2 (H2) blockers and proton pump inhibitors, raise the
pH of the stomach and decrease absorption of some drugs, such as ketoconazole, a drug for
fungal infections.
◦ Many drugs are broken down and inactivated (metabolized) by certain enzymes in the liver.
Some drugs affect these liver enzymes, either increasing or decreasing their activity, and may
cause another drug to be inactivated more quickly or more slowly than usual. For example, by
increasing the activity of liver enzymes, barbiturates such as phenobarbital cause the
anticoagulant warfarin to be inactivated more quickly and thus to be less effective when taken
during the same time period. Conversely, by decreasing the activity of the enzyme system,
drugs such as erythromycin and ciprofloxacin can increase the activity of warfarin, risking
bleeding.

42. Nutrients include:
 food
 beverages (including alcohol)
 dietary supplements.
Consumption of these substances may alter the effects of drugs the person takes.

44. Food
◦ Like food, drugs taken by mouth must be absorbed through the lining of the stomach or the small intestine.
Consequently, the presence of food in the digestive tract may reduce absorption of a drug. Often, such
interactions can be avoided by taking the drug 1 hour before or 2 hours after eating.
Dietary supplements
◦ Dietary supplements including medicinal herbs, are products (besides tobacco) that contain a vitamin,
mineral, herb, or amino acid and that are intended as a supplement to the normal diet. Supplements are
regulated as foods, not as drugs, so they are not tested as comprehensively. However, they may interact
with prescription or over-the-counter drugs. People who take dietary supplements should tell their doctors
and pharmacists, so that interactions can be avoided.
Alcohol
◦ Although many people do not consider alcohol a nutrient, it affects body processes and interacts with many
drugs. For example, taking alcohol with the antibiotic metronidazole can cause flushing, headache,
palpitations, and nausea and vomiting. Doctors or pharmacists can answer questions about possible
alcohol and drug interactions.

45. •Certain benzodiazepines
(such astriazolam)
•Calcium channel blockers
(such as felodipine,nifedipine,
andnisoldipine)
•Cyclosporine
•Estrogen and oral
contraceptives
•Certain statins (such
asatorvastatin,lovastatin,
andsimvastatin)
Grapefruit juice
Grapefruit juice inhibits
enzymes involved in drug
metabolism and thereby
intensifies the effect of certain
drugs, including many that
are not listed here.
Digoxin Oatmeal
The fiber in oatmeal and other
cereals, when consumed in
large amounts, can interfere
with the absorption of digoxin.

46. ◦ Sometimes, drugs that are helpful in one disease are harmful in another disorder. For
example, some beta-blockers taken for heart disease or high blood pressure can
worsen asthma and make it hard for people with diabetes to tell when their blood
sugar is too low. Some drugs taken to treat a cold may worsen glaucoma. People
should tell their doctor all of the diseases they have before the doctor prescribes a
new drug. Diabetes, high or low blood pressure, an ulcer, glaucoma, an enlarged
prostate, poor bladder control, and insomnia are particularly important, because
people with such diseases are more likely to have a drug-disease interaction.

47. ◦ Drug-disease interactions can occur in any age group but are common among older people,
who tend to have more diseases .
 Liver disease
 Renal disease
 Cardiac disease ( hepatic blood flow)
 Acute myocardial infarction?
 Acute viral infection?
 Hypothyroidism or hyperthyroidism?

48. ◦ Blood proteins, also termed plasma proteins.
◦ Plasma proteins are proteins present in blood plasma.
◦ They serve many different functions, including:
1. transport of lipids, hormones, vitamins and minerals in activity
2. functioning of the immune system.

49. ◦ Plasma protein binding refers to the degree
to which medications attach to proteins
within the blood. A drug's efficiency may be
affected by the degree to which it binds.
The less bound a drug is, the more
efficiently it can traverse cell membranes
or diffuse. Common blood proteins that
drugs bind to are human serum albumin,
lipoprotein, glycoprotein, and α, β‚ and γ
globulins.

50. ◦ A drug in blood exists in two forms: bound and unbound. Depending on a specific drug's affinity for plasma
protein, a proportion of the drug may become bound to plasma proteins, with the remainder being unbound.
If the protein binding is reversible, then a chemical equilibrium will exist between the bound and unbound
states, such that:
◦ Protein + drug ⇌ Protein-drug complex
◦ Notably, it is the unbound fraction which exhibits pharmacologic effects. It is also the fraction that may be
metabolized and/or excreted. For example, the "fraction bound" of the anticoagulant warfarin is 97%. This
means that of the amount of warfarin in the blood, 97% is bound to plasma proteins. The remaining 3% (the
fraction unbound) is the fraction that is actually active and may be excreted.
◦ Protein binding can influence the drug's biological half-life. The bound portion may act as a reservoir or
depot from which the drug is slowly released as the unbound form. Since the unbound form is being
metabolized and/or excreted from the body, the bound fraction will be released in order to maintain
equilibrium.
◦ Since albumin is alkalotic, acidic and neutral drugs will primarily bind to albumin. If albumin becomes
saturated, then these drugs will bind to lipoprotein. Basic drugs will bind to the acidic alpha-1 acid
glycoprotein. This is significant because various medical conditions may affect the levels of albumin, alpha-
1 acid glycoprotein, and lipoproteins.

51. ◦ Only the unbound fraction of the drug undergoes metabolism in the liver and other tissues. As
the drug dissociates from the protein, more and more drug undergoes metabolism. Changes in
the levels of free drug change the volume of distribution because free drug may distribute into
the tissues leading to a decrease in plasma concentration profile. For the drugs which rapidly
undergo metabolism, clearance is dependent on the hepatic blood flow. For drugs which slowly
undergo metabolism, changes in the unbound fraction of the drug directly change the clearance
of the drug.
◦ The fraction unbound can be altered by a number of variables, such as the concentration of
drug in the body, the amount and quality of plasma protein, and other drugs that bind to plasma
proteins. Higher drug concentrations would lead to a higher fraction unbound, because the
plasma protein would be saturated with drug and any excess drug would be unbound. If the
amount of plasma protein is decreased (such as in catabolism, malnutrition, liver disease, renal
disease), there would also be a higher fraction unbound. Additionally, the quality of the plasma
protein may affect how many drug-binding sites there are on the protein.

52. EFFECT OF PLASMA PROTEIN
BINDING ON FIRST-PASS
METABOLISM
◦ Plasma protein binding plays a key role in drug therapy that affects pharmacokinetics and
pharmacodynamics of drugs and may affect the metabolism of drugs (Fasano et al., 2005).
Human serum albumin (HSA) is one of the most widely examined proteins in plasma. HSA is
well known for its huge ligand binding capacity, providing a depot for a wide range of ligands
that may exist in quantities beyond their plasma solubility. Optimization of drug-plasma protein
binding showed to be very beneficial in the development of dipeptidyl peptidase IV(DPP-IV)
inhibitors as well as for the treatment of type 2 diabetes mellitus. For example, studies showed
that compound A (Fig. 8.4) exhibited a 32-fold activity shift demonstrating very high plasma
protein binding. Meanwhile, introduction of fused heterocycles as in compound B enhanced the
plasma protein binding, as observed via the 11-fold in vitro activity shift in presence of serum.
Moreover, introduction of extra fluorine, as in compound C, produced potent orally active DPP-
IV inhibitor with low plasma protein binding (Edmondson et al., 2006).

53. EFFECT OF PLASMA PROTEIN
BINDING ON FIRST-PASS
METABOLISM
◦ One more therapy, directed towards the treatment of type 2 diabetes mellitus is
through the activation of the hepatic and pancreatic enzyme glucokinase.
Increasing activity of the lead compound GKA22 (Fig. 8.4), through lowering
plasma protein binding and enhancing its in vivo potency by decreasing clearance
of unbound drug to produce higher exposure, was the lead optimization method
implemented by the research group. The most active glucokinase activator,
GKA50,13 with an approximately twofold reduction in plasma protein binding was
selected for future development (Mckerrecher et al., 2006).

54. STRUCTURE OF INHIBITORS AND
GLUCOKINASE ACTIVATORS

55. ◦ Plasma protein binding is generally reduced in children compared with adults, leading
to increased proportions of unbound drug. This in turn influences hepatic clearance,
particularly of drugs with low-to-intermediate extraction ratios. The increase in
unbound drug fractions in children can be mainly attributed to lower concentrations of
serum albumin and alpha-1-glycoprotein (AGP). Neonates and young infants have
albumin and AGP levels that are 20% and 60% lower than adults, respectively;
although AGP levels vary considerably under various pathophysiological conditions.
The constitutive concentrations of both plasma proteins increase linearly with age until
adulthood (McNamara and Alcorn, 2002).

56. ◦ In addition to differences in plasma protein abundance, distinct differences in binding
affinity of drugs for plasma proteins have been demonstrated between adults and
neonates (Windorfer et al., 1974). These differences generally reduce plasma protein
binding further in early life. Moreover, drugs bound to neonatal serum proteins appear
to be more vulnerable to displacement by endogenous compounds like bilirubin and
free fatty acids, both of which are present in higher concentrations in neonates
compared with adults (Fredholm et al., 1975; Windorfer et al., 1974).

57. PLASMA PROTEINS BINDING

58. ◦ Plasma protein binding (PPB) is an important parameter for a drug’s efficacy and safety that
needs to be investigated during each drug-development program. Even though regulatory
guidance exists to study the extent of PPB before initiating clinical studies, there are no detailed
instructions on how to perform and validate such studies. To explore how PPB studies involving
bioanalysis are currently executed in the industry, the European Bioanalysis Forum (EBF) has
conducted three surveys among their member companies: PPB studies in drug discovery (Part
I); in vitro PPB studies in drug development (Part II); and in vivo PPB studies in drug
development. This paper reflects the outcome of the three surveys, which, together with the
team discussions, formed the basis of the EBF recommendation. The EBF recommends a
tiered approach to the design of PPB studies and the bioanalysis of PPB samples: ‘PPB
screening’ experiments in (early) drug discovery versus qualified/validated procedures in drug
development.

59. ◦ RESULTS OF THE SURVEY
◦ OVERVIEW:
In drug discovery, PPB studies are conducted by drug metabolism PK (DMPK)
scientists (70% of responders), bioanalytical scientists or in cooperation between DMPK and
bioanalytical scientists. They are usually performed for PK/PD evaluation, for compound selection
and for allometric scaling. Typically, plasma from different species, such as humans, rats, dogs,
mice, rabbits, monkeys and guinea pigs, is investigated. Generally, PPB is investigated using an
in vitro setting (i.e., spike compound to blank plasma), however, some members also perform
PPB from in vivo studies (i.e., plasma from dosed animals). In drug discovery, the majority of the
EBF member companies do not have standard operating procedures (SOPs) in place, either for
separating bound and unbound fractions or for the bioanalysis of the free drug.

60. ◦ In drug discovery, equilibrium dialysis, ultrafiltration, ultracentrifugation and Transil partitioning
are all used for separation of bound and unbound drugs. Although preliminary experiments,
such as investigation of nonspecific binding, drug stability in plasma and buffer, in silico
evaluation and assessment of the time to reach equilibrium (equilibrium dialysis) were reported,
a majority of EBF member companies do not perform such experiments at the drug-discovery
phase. In general, PPB studies are conducted in triplicate at one standard drug concentration
ranging from approximately 0.3 to 10 µM. Some companies perform more elaborate
experiments, using two or three different drug concentration levels with one to five replicates at
each level. In very early drug discovery ‘PPB screening’ is conducted by approximately 50% of
the responders. A minority of the responders evaluate the mass balance/recovery using an
acceptance criterion of 80%.

61. ◦ The report usually includes a short description of the separation experiment
(equipment, materials and conditions), a description of the bioanalytical method, the
results of the preliminary experiments, the unbound fraction (average and individual
results) at all concentration levels (most companies do not correct for nonspecific
binding when calculating the unbound fraction), and the average and individual
nonspecific binding results.

62. ◦ Equilibrium dialysis is recommended as the first choice for assessment of PPB in drug
discovery as this technique can be used for nearly all small-molecule compounds. In
RED, cycle times are limited to approximately 4 h, thus offering advantages for
increased sample throughput. For compounds with limited stability, ultrafiltration might
be the method of choice.

63. ◦ It is recommended to perform PPB experiments at least in duplicate. Fresh or frozen pooled plasma may be
used. Equilibrium dialysis should be performed at 37°C. For RED, the dialysis time of approximately 4 h
should be applied (if not optimized in a preliminary experiment). A nonspecific binding test of the drug during
the PPB experiment (acceptance criterion ≤30%) as well as a stability test of the drug in plasma and buffer
at 37°C (4 h) can be performed as preliminary experiments; however, instead of these tests, the mass
balance (recovery) of the drug can be evaluated at the end of the PPB study.
◦ Generally, it is not required to perform a prestudy qualification of the bioanalytical method during this phase.
For the analysis of study samples after dialysis it is recommended to perform limited in-study qualification
by including a limited number of calibration samples. Although not critical at this stage, QC samples may
also be included. For efficiency and to enhance simplicity of the analysis, the PPB samples should be
matrix-matched (e.g., buffer added to the plasma and plasma added to the buffer samples, at a ratio of
50:50 v/v). In a screening setup, relative quantification can also be applied but the linearity of the analyte
response should be investigated and confirmed.

64. ◦ Raw data of the analysis should be available upon request. In some
cases, for instance when the data are used in later stages of drug
development, it may add value to write a bioanalytical report that
includes the key features of the analytical method used.