PHARMACOKINETICS AND PHARMACODYNAMICS OF BIOTECHNOLOGY DRUGS : MONOCLONAL ANTIBODIES, PROTEINS AND PEPTIDE, OLIGO NUCLEOTIDES

1. PHARMACOKINETICS AND
PHARMACODYNAMICS OF BIOTECHNOLOGY
DRUGS : MONOCLONAL ANTIBODIES, PROTEINS
AND PEPTIDE, OLIGO NUCLEOTIDES
Presented By
D. Sai Adiseshu (I/II M.PHARM)

2. Monoclonal Antibodies
An Anitbody is a protein that protect the human body against foreign
substances or invading microorganisms, the immune system has developed several
defense mechanisms, the ultimate goal being to eliminate these potentially harmful
interferences from the body.

3. Pharmacokinetic of mAbs :
Absorption :
The majority of mAbs that have been approved or are currently in clinical
development are administered by intravenous (IV) infusion.
Consequently, extra vascular routes have been chosen as alternatives, including
subcutaneous administration and intramuscular administration.
The mAbs enter the lymphatic system by convective flow of interstitial fluid
into the porous lymphatic vessels. The molecular mass cut-off of these pores is
>100-fold the molecular mass of mAbs. From the lymphatic vessels, the mAbs are
transported uni directionally into the venous system.
It has been shown that antibodies can reach the systemic circulation after oral
administration, but only to a very small extent. The antibodies pass the intestinal
epithelium not by passive transcellular but by receptor-mediated transcellular or
paracellular transport.

4. Distribution :
In general, the distribution of classical mAbs in the body is poor. Limiting
factors are, in particular, the high molecular mass and the hydrophilicity/polarity of
the molecules.
Transport :
Permeation of mAbs across the cells or tissues is accomplished by transcellular
or paracellular transport, involving the processes of diffusion, convection, and
cellular uptake. Due to their physico-chemical properties, the extent of passive
diffusion of classical mAbs across cell membranes in transcellular transport is
minimal.
Endocytosis is an absorptive process of large and polar molecules such as
mAbs, and involves the formation of intracellular vesicles from parts of the cell
membrane.
The mAbs initially distribute into a restricted central volume (Vc) of 3–5 L,
which in humans approximates the serum volume.

5. Elimination :
- Clearence :
As glomerular filtration has an approximate molecular size limit of 20–30
kDa, mAbs do not undergo filtration in the kidneys due to their relatively large size.
The situation is different, however, for low molecular-mass antibody fragments,
which can be filtered.
Tubular secretion has not been reported to occur to any significant extent
for mAbs, and peptides/small proteins are readily reabsorbed in the proximal or
distal tubule of the nephron or are even metabolized.
Thus, renal elimination in total is uncommon or low for mAbs. Biliary
excretion of mAbs has been reported only for IgA molecules, and only to a very
small extent. Therefore, total clearance (CL) does usually not comprise renal or
biliary clearance.

6. -Binding to Antigen
Binding of mAbs not only affects distribution but also reflects another means of
elimination. Binding of the Fab region to the antigen with high affinity must be regarded
as almost irreversible. The antigen–antibody complex, if located on the surface of a cell,
will be internalized and subsequently degraded.
-Binding to Anti-Idiotype Antibodies:
A third elimination pathway occurs if anti-idiotype antibodies are formed as an
immune response of the human body to the administration of mAbs. Following
repeated administration, anti-idiotype antibodies are usually observed after one to
two weeks, with the extent of the adverse reaction strongly depending on several
factors:
1.Dosage regimen: single doses rarely evoke a strong immunological response, whereas
multiple dosing often results in anti-idio type antibody formation.
2. Route of administration: SC injection has a higher incidence of forming anti-idio type
antibodies than IM or IV administration.
3. Patient’s genetics: patients with autoimmune diseases or prior anti-antibody
production are more likely to produce an immune response.

7. Pharmacodynamics of mAbs:
Three different pharmacodynamic principles of action can be distinguished
for mAbs, comprising lysis or apoptotic activity, coating activity, and inactivating
activity, depending on the type of antigen and the antigen–antibody interaction.
In the first two cases, the ideally disease-specific antigen is located on the
surface of a cell. The majority of mAbs exert their pharmacodynamic activity by Fc-
mediated ADCC/ CDC activation or delivery of toxic substance to the targeted cell .
Cell death will be the ultimate result..
In last case are directed against soluble substances as antigens (cytokine,
growth factor, immunoglobulin). The pharmacodynamic principle is to block the
antigen-binding activity to its receptor and thereby inactivate the target antigen
function. The binding of the therapeutic antibody to the target antigen finally leads
to agglutination and neutralization of the antigen.

8. PROTEIN AND PEPTIDES
Protein :- Protein are the large organic compounds made up of amino acids
arranged in a linear chain attached by a peptide chain.
Protein > 50 aminoacids
Peptide :- These are the short polymers formed from the linking, in a
defined order of amino acids.
Peptide < 50 amino acids
The protein and peptides are important in biological cells. Our body requires
many proteins for development of organs and maintaing circadian rhythm.

9. Pharmacokinetics :- Administration Pathways :-
Peptides and proteins, unlike conventional small-molecule drugs, are
generally not therapeutically active upon oral administration.
The lack of systemic bioavailability is mainly caused by two factors:
high gastrointestinal enzyme activity, and low permeability through the
gastrointestinal mucosa.
The substantial peptidase and protease activity in the gastrointestinal
tract makes it the most efficient body compartment for peptide and protein
metabolism & gastrointestinal mucosa presents a major absorption barrier for water
soluble macromolecules such as peptides and proteins.
Due to the lack of activity after oral administration for most peptides
and proteins, administration by injection or infusion – that is, by intravenous (IV),
subcutaneous (SC), or intramuscular (IM) administration – is frequently the
preferred route of delivery for these drug products.

10. Administration by Injection or Infusion :-
Injectable administration of peptides and proteins offers the advantage
by overcoming presystemic degradation, thereby achieving the highest concentration
in the biological system.
Examples of FDA-approved proteins given by the IV route include
the tissue plasminogen activator (t-PA) analogues alteplase , the recombinant human
erythropoietin epoetin – α .
IV administration as either a bolus dose or constant rate infusion may
not always provide the desired concentration– time profile depending on the
biological activity of the product, and IM or SC injections may be more appropriate
alternatives.
For example, luteinizing hormone releasing hormone (LH-RH) in
bursts stimulates the release of follicle-stimulating hormone (FSH) and luteinizing
hormone (LH), whereas a continuous baseline level will suppress the release of
these hormones

11. Inhalational Administration :-
Inhalational delivery of peptides and proteins offers the advantage of ease
of administration, the presence of a large surface area (75 m2) available for
absorption, high vascularity of the administration site, and bypass of hepatic first-
pass metabolism.
Disadvantages of inhalation delivery include the presence of certain
proteases in the lung, potential local side effects of the inhaled agents on the lung
tissues (i. e., growth factors and cytokines), and molecular weight limitations.
The success of inhaled peptide and protein drugs can be exemplified by
inhaled recombinant human insulin products, with Exubera being the first approved
product (2006), and several others in clinical development.

12. Inhaled insulin offers the advantages of ease of administration and
rapid onset with a shorter duration of action for tighter postprandial glucose control
as compared to subcutaneously administered regular insulin.
Dornase-α , which is indicated for the treatment of cystic fibrosis, is
another example of a protein drug successfully administered through the inhalation
route.
Transdermal Administration :-
Transdermal drug delivery offers the advantages of bypassing
metabolic and chemical degradation in the gastrointestinal tract, as well as first-pass
metabolism
by the liver.
Methods frequently used to facilitate transdermal delivery include
sonophoration and iontophoresis. Both methodologies increase skin permeability to
ionic compounds. Therapeutic doses of insulin, interferon-γ, and epoetin-α have all
been successfully delivered transdermally via sonophoresis

13. Distribution :-
Whole-body distribution studies are essential for classical small-molecule
drugs in order to exclude any tissue accumulation of potentially toxic metabolites.
This problem does not exist for protein drugs, where the catabolic
degradation products (amino acids) are recycled in the endogenous amino acid pool.
Therefore, biodistribution studies for peptides and proteins are performed
primarily to assess targeting to specific tissues as well as to identify the major
elimination organs.
The volume of distribution of a peptide or protein drug is determined
largely by its physico-chemical properties (e. g., charge, lipophilicity), protein
binding, and dependency on active transport processes.
After IV application, peptides and proteins usually follow a biexponential
plasma concentration–time profile that can best be described by a two-compartment
pharmacokinetic model.
.

14. The central compartment in this model represents primarily the vascular
space and the interstitial space of well-perfused organs with permeable capillary
walls, especially liver and kidneys, while the peripheral compartment comprises the
interstitial space of poorly perfused tissues such as skin and (inactive) muscle.
Active tissue uptake can substantially increase the volume of distribution
of peptide and protein drugs, as for example observed with atrial natriuretic peptide
(ANP).
Another factor that can influence the distribution of therapeutic peptides
and proteins is binding to endogenous protein structures. Physiologically active
endogenous peptides and proteins frequently interact with specific binding proteins
involved in their transport and regulation. Ex :- growth hormone
Protein binding not only affects whether the peptide or protein drug will
exert any pharmacological activity, but on many occasions it may also have an
inhibitory or stimulatory effect on the biological activity of the agent . Eg :-
Recombinant cytokines.

15. Elimination :-
Proteolysis :- Proteolytic enzymes such as proteases and peptidases are
ubiquitous throughout the body. As proteases and peptidases are also located within
cells, intracellular uptake is seen more an elimination rather than a distribution process.
Gastrointestinal :- For orally administered peptides and proteins, the
gastrointestinal tract is the major site of metabolism. Presystemic metabolism is the
primary reason. Parenterally administered peptides and proteins may also be
metabolized in the intestinal mucosa following intestinal secretion.
Hepatic :- the liver may also contribute substantially to the metabolism of
peptide and protein drugs. Proteolysis usually starts with endopeptidases that attack in
the middle part of the protein, and the resulting oligopeptides are then further degraded
by exopeptidases.
The ultimate metabolites of proteins, amino acids and dipeptides, are finally reutilized
in the endogenous amino acid pool. The rate of hepatic metabolism is largely dependent
on specific amino acid sequences in the protein.

16. Renal :- Renal metabolism of peptides and small proteins is mediated
through three highly effective processes . Consequently, only minuscule amounts of
intact protein are detectable in the urine.
1. The first mechanism involves the glomerular filtration of larger, complex
peptides and proteins, followed by reabsorption into endocytic vesicles in the
proximal tubule and subsequent hydrolysis into small peptide fragments and AA.
2. The second mechanism entails glomerular filtration followed by intra luminal
metabolism, predominantly by exopeptidases in the luminal brush border
membrane of the proximal tubules.
3. The third mechanism is peritubular extraction of peptides and proteins from
post glomerular capillaries and intracellular metabolism.

17. The determining factors for clearance of protein and peptide include molecular
weight as well as a molecule’s physico-chemical properties, including size, overall
charge, lipophilicity, functional groups, secondary and tertiary structure.

18. ANTISENSE OLIGONUCLEOTIDE
Antisense oligo nucleotides are unmodified or chemically modified SS DNA
or their analogues.
They are 13-25 nucleotides long & specifically designed to hybridze to the
corresponding mRNA by Watson Crick binding.
In this technique the short segments of single standard DNA is called as
oligodeoxy nucleotide are complementary to the mRNA &physically bind to it.
DNA mRNA Protein
translationtranscription
oligonucleotide

19. Pharmacokinetics :
Absorption :
Antisense oligonucleotides are administered parenterally in the majority of reported in-
vivo studies: intravenous (IV), intraperitoneal (IP), or subcutaneous .
Parenteral administration has been the preferred route because it allows complete
systemic availability.
Similar to first-generation antisense oligonucleotides (ASOs), the pharmacokinetics of
2-MOE partially modified ASOs are characterized by:
 A plasma concentration–time profile that is poly-phasic with rapid distribution half-
life (1 h) and long elimination half-life reflecting slow elimination from the tissues.
 High binding to plasma proteins (>90% across species).
 Plasma clearance that is dominated by distribution into the tissues.
 Long tissue elimination half-life cleared by nuclease-mediated metabolism.
 Minor urinary or fecal excretion of the intact drug.

20. Distribution :
The highest concentrations of oligonucleotides in all species studied were
found in kidney, liver, spleen, and lymph nodes, but oligonucleotides can be
measured in almost every tissue, except brain, at 24 h after IV administration.
Consistent with the pattern of distribution, liver and kidney were monitored closely
for evidence of toxicity in mouse and monkey toxicology studies.
Elimination:-
While distribution to tissues relies on the mechanism of plasma clearance,
whole body clearance is the result of metabolism and the excretion of low molecular
weight oligonucleotides.
Although tissue metabolism is slow, it is continuous and represents the
primary route of whole-body elimination as oligonucleotide fragments are excreted
from the body in urine.

21. oligonucleotide
5-MOE – Protected terminus + 3- MOE –
protected terminus
endonuclease
exonuclease
Shorter mono oligonucleotide
fragments
Urine excertion catabolic pathways
Purines and pyrimidines
Metabolic pathway of oligonucleotide

22. Pharmacodynamics :-
The mechanism of action for antisense compounds is to inhibit gene
expression sequence-specifically by hybridization to mRNA through Watson–Crick
base pair interactions.
This is followed by degradation of the target mRNA through an RNase H-
dependent terminating mechanism.
Consequently, the ASO prevents translation of the encoded protein product,
or the disease-causing factor in a highly sequence-specific manner.

23. Reference :
1. Applied Biopharmaceutics and Pharmacokinetics by Shargel. Land YuABC,
2ndedition, Connecticut Appleton Century Crofts, 1985
2. Pharmacokinetics and Pharmacodynamics of Biotech Drugs by Bernd
Meibohm,WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ,Germany,2006.