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FURTHER READINGS
Theory and
Practice of Contemporary Pharmaceutics
Invest
Ophthalmol 15: 1008– 1010.
Pharmaceutical Dosage Forms and Drug Delivery
In Meibohm B. (Ed.) Pharmacokinetics and Pharmacodynamics of Biotech Drugs: Principles and Case
Studies in Drug Development
Niven R. (1993) Delivery of biopharmaceutics by inhalation aerosols. Pharm Technol 17: 72– 81.
Nat Rev Drug Discov 5: 371– 372.
J Control
Release 28: 79– 85.
Scheindlin S. (2004) Transdermal drug delivery: Past, present and future. Mol Interv 4: 308– 3122.
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Part IV
Pharmaceutical Biotechnology
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25
Protein and Peptide Drug Delivery
On completion of this chapter, the students should be able to
1. Describe the differences between the primary, secondary, and tertiary structures of proteins.
2. List types of physical instability of proteins and peptides.
3. -
4. Identify key components of protein formulations.
25.1 Introduction
The use of therapeutic proteins to replace or supplement endogenous protein molecules has been a
oncology agents Opdivo®®®
and Adcetris®.
Protein and peptide drugs are either natural in origin or synthetically produced using recombinant
DNA technology or from transgenic animals. Recombinant DNA technology has allowed the large-
macrophage colony- stimulating factor (GM- CSF), erythropoietin (EPO), interleukins, insulin- like
philia), mAbs, and tissue plasminogen activator (t- PA). Protein therapeutics to replace or supplement
endogenous protein molecules are used for several diseases, such as diabetes (insulin), growth hormone
Table 25.1 lists some of the FDA- approved marketed products of therapeutic proteins.
The physical and chemical instabilities of proteins and peptides, arising from their large molecular
weight and complex structure, pose many challenges for pharmaceutical formulation development.
Clinical applications of protein drugs are limited by their inadequate concentration in blood, poor oral
bioavailability, high manufacturing cost, chemical or biological instability, and/ or rapid hepatic metab-
their target cells. These limitations lead to their high dose and/ or need for frequent administration, which
can cause undesirable side effects. Also, proteins can elicit host immune responses following repeated use
-
atic environment, resulting in poor oral bioavailability. Therefore, proteins are primarily administered
powder for reconstitution prior to administration.
LEARNING OBJECTIVES
DOI: 10.1201/9781003389378-29
429
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430
TABLE 25.1
Pharmaceutical Dosage Forms and Drug Delivery
List of Some Commercial Products of Therapeutic Proteins
Protein Type Protein Names Description Indication
Polyclonal antibodies
Polyclonal antibodies
(solution)
Monoclonal antibodies Rituximab
Radioactively tagged
antibodies
Mouse antibodies Tositumomab IgG2 anti- CD20 monoclonal antibody
Chimeric antibodies Monoclonal antibody against TNFa. Psoriasis, Crohn’s disease,
Fusion proteins Abatacept Fusion protein that is composed of human
Sandoglobulin Human immune globulin for intravenous
administration. It is a polyvalent antibody product that contains all IgG
antibodies, which regularly occur in
the donor population in a concentrated
form. It is prepared by fractionation of
the plasma of volunteer donors.
Gammagard Concentrated human IgG antibodies
similar to that of normal plasma. It
is manufactured from pooled human
plasma from donors.
sterile liquid formulation.
lymphoma cells and triggers body’s
immune system.
Ibritumomab
tiuxetan
Monoclonal antibody radioimmuno-
therapy. It is prepared from monoclonal
mouse IgG1 antibody ibritumomab and
uses the chelator tiuxetan, which has
a radioactive isotope (yttrium- 90 or
indium- 111).
of murine origin. Also available as
radioactively labeled
131
I- tositumomab,
which has covalently bound iodine- 131.
subunit of IL- 2 receptor on T- cells.
Ig fused to the extracellular domain
of cytotoxic T- lymphocyte- associated
protein 4 (CTLA- 4), a molecule involved
in T- cell stimulation.
such as severe combined
common variable immunoglobulinemia, and immune
thrombocytopenic purpura
(ITP)
Combination therapy for tumors
such as non- Hodgkin’s
lymphoma (NHL) and chronic
lymphocytic leukemia (CLL),
and autoimmune diseases such
as rheumatoid arthritis
B- cell NHL
Follicular lymphoma
ankylosing spondylitis,
psoriatic arthritis, rheumatoid
arthritis, and ulcerative colitis
transplantation, especially in
kidney transplantation
Rheumatoid arthritis
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Protein and Peptide Drug Delivery
TABLE 25.1 (Continued)
List of Some Commercial Products of Therapeutic Proteins
Protein Type Protein Names Description Indication
Physiological proteins Erythropoietin Glycoprotein hormone that controls
erythropoiesis (red blood cell
production). It is available as a
®
small molecule antimicrotubule drug.
cancer
Breast cancer
431
FIGURE 25.1 Chemical structure of a typical peptide bond. Polypeptides consist of a linear chain of amino acids successively linked via peptide bonds.
25.2 Structure
Proteins and peptides consist of simple building blocks called amino acids, which are linked together
by peptide bonds. A peptide bond is formed by the nucleophilic addition of the primary amine of
one amino acid to the electropositive carboxylate carbon of the other amino acid (Figure 25.1). Two
amino acids. Long chains of amino acids tend to self- associate and fold into three- dimensional
often a function of their unique amino acid sequence and the resulting conformation that makes up
the protein.
As shown in Figure 25.2, a chain of amino acids forming a polypeptide through covalent linkages
constitutes a protein or peptide’s primary structure. The spatial folding of a polypeptide chain through
noncovalent interactions of neighboring amino acids results in the secondary structure, which consists
dis-
tant amino acids, which give the overall structure to one polypeptide chain, called the tertiary structure.
The spatial interaction of more than one polypeptide chain to form a protein is termed the quaternary
structure.
OH
2
−
++
+
+
3
ionizationconstant forthe acid
[][]
[]
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432
FIGURE 25.2
structure (combination of polypeptides).
Pharmaceutical Dosage Forms and Drug Delivery
25.2.1 Amino Acids
There are 20 naturally occurring amino acids that form the structural basis of all the proteins and peptides.
The chemical structures of these amino acids, along with their abbreviated and one- letter designations,
are presented in Figure 25.3. Each amino acid possesses unique physicochemical properties governed by
its chemical structure.
• Nineteen amino acids contain an amino (– NH2) and carboxyl (– COOH) group attached to a carbon
because it is next to the carboxylate group in the structure. The amino acid proline is unusual in
that its side chain forms a direct covalent bond with the nitrogen atom of an amino group. This is
indicated in the higher hydrophobic character of proline (higher log P, Table 25.2) compared to
most other amino acids.
•
This chirality can lead to two optical isomers, - and - amino acids, which would be mirror images
of each other. Natural amino acids are exclusively - amino acids.
Ka, whereas the polarity is indicated
by log P
Table 25.2.
COOH R-CO
R-NH HR-NH
+
R-COOH
=
a
R-COOH
−+
functio
nnal group
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Protein and Peptide Drug Delivery
433
FIGURE 25.3 Chemical structure of the 20 amino acids commonly found in proteins. The amino acids may be subdivided
TABLE 25.2
Hydrophobicity and Acidity of Amino Acids
Three Letter
Amino Acid
Abbreviation
Alanine Ala A 2.35 9.87 —
Arginine Arg R 2.01 9.04 12.48
Asparagine Asn N 2.02 8.80 —
Aspartic acid Asp D 2.10 9.82
Cysteine Cys C 2.05 10.25 8.00
Glutamic acid Glu E 2.10 9.47 4.07
Glutamine Gln 2.17 9.13 —
Glycine Gly G 2.35 9.78 —
Histidine His H 1.77 9.18
Isoleucine Ile I 0.41 2.32 —
Leucine Leu L 2.33 9.74 —
Lysine Lys 2.18 8.95 10.53
Methionine Met M 2.28 9.21 —
Phenylalanine Phe F 2.58 9.24 —
Proline Pro P 2.20 —
Serine Ser S 2.21 9.15 —
Threonine Thr T 2.09 9.10 —
Tryptophan Trp W 2.38 9.39 —
Tyrosine Tyr 2.20 9.11 10.07
Valine Va l V 2.29 9.72 —
One Letter
Designation
Log P
value
pKa Value of
Carboxylate Group
pKa Value of
Amino Group
pKa Value of
Side Chain
ionizationconstant forthebase
[]
+
aa
KK=−
bb
KK=−
abww
KKKK+= =
Water
[]
[]
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434
R-NH
3
=
b
R-NH H
pp wherep is theionizationconstantof water
2
14,
+
Pharmaceutical Dosage Forms and Drug Delivery
func
log
log
ttional group
and
solute
glog
P =
solute
Octanol
Amino acids with low pKa values are acidic, whereas amino acids with high (>7) pKa values (which
would correspond to low pKb values) are basic. Amino acids typically have an acidic carboxylate group
and a basic amino group, which contribute to their acidity or basicity. In addition, the side chain may
Ka values associated with an amino acid. However, in a polypep-
tide chain, the carboxylate and amino groups are covalently bonded to neighboring amino acids (except
different than those of pure amino acids. Aspartic and glutamic amino acids are considered acidic because
The hydrophobic character of amino acids as individual molecules is indicated by their log P value
(Table 25.2
tional groups. In a protein structure, these functional groups are covalently bonded. In the context of the
side chains of amino acids with water (Figure 25.4).
-
chemical properties of amino acid side chains while ignoring the effects of the carboxylate and the amino
groups (scales 1 and 2 in Figure 25.4) or (b) scaling the probability for an amino acid to be found inside
or outside a protein structure by examining three- dimensional structures of known proteins (scales 3
and 4 in Figure 25.4). The scaling criteria inherently result in different predictions. For example, cysteine
hydrophobic interior of a globular protein. Thus, cysteine is relatively more hydrophobic by the scaling
criterion of its location in a protein.
FIGURE 25.4 Relative hydrophobicity of different amino acids is estimated based on either their side- chain sequence
(scales 1 and 2) or their typical location in a globular protein structure (scales 3 and 4).
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Protein and Peptide Drug Delivery
25.2.2 Primary Structure
435
bonds in the constituent polypeptide chain(s) (Figure 25.2). Primary structure determines a protein’s
dict the three- dimensional structure and shape of the proteins in solution.
25.2.3 Secondary Structure
Secondary structure can be described as the local spatial conformation of a polypeptide’s backbone,
Figure 25.2
amino acids with the hydrophobic groups facing inside and the hydrophilic groups facing outside the
the same chain or between two different chains, thus exposing the amino acid functional groups to the
solvent medium. The chain folding of the secondary structures often arises from cross- linking through
25.2.4 Tertiary Structure
The tertiary structure of a protein refers to the exact three- dimensional structure of its constituent
polypeptide chain(s) (Figure 25.2). The spatial proximity of secondary structural elements determines
the tertiary structure of a polypeptide. Spatially close amino acids on the folded (secondary structure) polypeptide chains can form an attractive hydrogen bond, ionic, or hydrophobic interactions,
their distinctive tertiary structure of minimum free energy, which is a prerequisite for their biological
function.
25.2.5 Quaternary Structure
that have more than one noncovalently linked constituent polypeptide chain (Figure 25.2). These
polypeptide chains can associate to form dimers, trimers, and oligomers, which constitute the quaternary structure of a protein. Almost all proteins that are greater than 100 kDa have a quaternary
structure. For example, hemoglobin consists of nonidentical subunits that associate to form a dimer
S- transferase consists of homotetramer (all
heterodimer.
-
ation upon binding of an agonist, and membrane ion channels change conformation to facilitate transport
upon ion binding on their surface.
25.3 Protein Characterization
25.3.1 Biophysical Characterization
Therapeutic applications of proteins require an understanding of fully elucidated structure, pharmacology, and mechanism of action. In addition, protein behavior in solution and the impact of chemical
properties and components of solutions on the physical properties of solutions (termed biophysical char-
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