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26.3.1.2.1 N- Alkylation
Pharmaceutical Dosage Forms and Drug Delivery
Peptides often adopt a trans conformation unless a peptide bond is followed by a proline residue. This
Furthermore, reducing the quantity of viable intramolecular and intermolecular H- bonds caused by the
elimination of the NH- group can enhance metabolic stability via conformational regulation or steric
hindrance.
26.3.1.2.2 Substitution of L- Amino Acids With D- Amino Acids
This methodology entails replacing naturally occurring L- amino acids that are particularly vulnerable
to proteolytic degradation and are metabolically unstable with unnatural D- amino acids that function as
three- dimensional (3D) mirror images. Therapeutic peptides that have employed this approach include
agonists and antagonists of gonadotropin- releasing hormone (GnRH), octreotide, ipamorelin, exenatide,
and liraglutide, which are glucagon- like peptide- 1 (GLP- 1) analogs, D- peptides including PIE12- trimer
and rotigaptide, and others.
26.3.1.2.3 α- Carbon Modification
functional group. It makes the peptide more selective in its interactions with its target, a property known
increase the resistance of peptides to proteolytic degradation. 2- aminoisobutyric acid, a widely studied
10 helices. Diethylglycine extends conform10
output transformations.
26.3.1.2.4 Carbonyl Thionation
Researchers focus on isosteric substitution of amide bonds with thioamides in physiologically active
hydrogen acceptor, resulting in longer H- bonds. In comparison, the thioamide C– N bond displays a large
rotational barrier, owing to the lower C= S double bond character. Sulfur is less electronegative than
the proteolytic stability of peptides. For instance, in GLP- 1, a thioamide substitution at the scissile bond
increased the linear peptide’s half- life by up to 100 times.
26.3.1.2.5 Pseudopeptides
atom, are used to create protease inhibitors and hormone analogs, such as inhibitors of cysteine and
C- termini sequences and D- amino acids, reducing proteolytic degradation rates. This substitution
reduces the biological activity of peptides, as the charges on both termini are reversed. Examples
-
ition and NH groups replaced with CH2 groups. The positions of carbonyl groups and side chains
remain unchanged, while CH2 and NH groups exchange places. Peptoids have greater conformational
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from protegrins, melittin, and pexiganan.
26.3.1.3 Side- Chain Modification
degradation.
26.3.1.3.1 Amino Acid Analogs
this approach include GLP- 1 analogs such as liraglutide and liraglutide.
26.3.1.3.2 Peptide Mimicking of α- Helices and Stabilization
cross- links through side chains or replacing hydrogen bonds with covalent bonds (HBS). Stapled peptides
26.3.1.3.3 Peptide Mimicking of β- Strands and β- Sheets
amyloid beta- sheet mimics have also been used in some instances.
26.3.1.4 Cyclization of Peptide
as it can induce structural rigidity and decrease polar atom exposure, increasing oral bioavailability.
26.3.2 Protein Conjugation
can be linked to carbohydrates, lipids, polymers, organic complexes, and other peptides and can also
counterparts as a result of reduced renal clearance, proteolytic degradation, and RES clearance.
26.3.2.1 PEGylation
PEGylation PEGylated protein. PEG consists of a
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Pharmaceutical Dosage Forms and Drug Delivery
-
dynamic diameter of proteins. Either straight chain or branched PEG can be used for PEGylation. The
increase biocompatibility, reduce immune response, increase in vivo stability, delay clearance by the
reticuloendothelial system, and prevent protein adsorption on the surface of the delivery device, such as
a syringe.
26.3.2.1.1 Applications
to branched PEG 40 (i.e., PEG of 40 kDa average molecular weight) provides sustained plasma
clearance rate of proteins include PEG- adenosine deaminase (PEG- ADA), PEG- asparaginase, PEG- rIL2,
and PEG- IFN. Native ADA is not effective due to its short half- life (<30 min) and is immunogenic due
to its bovine source. In contrast, PEGylated ADA (Adagen®) is quite effective, has a long half- life, and
is nonimmunogenic.
26.3.2.1.2 Chemistry
PEG has two hydroxyl groups at each end of the linear chain. PEGylation is often done by creating
a reactive electrophilic intermediate with succinimide (thus producing N- hydroxysuccinimide, NHS),
which undergoes electrophilic substitution by an amine group of the protein (). The NHS
Two hydroxyl groups— one at either end— make the natural PEG bifunctional. To prevent the poten-
polymer can be used. To make PEG monofunctional, one end of the chain is blocked with a methyl
ether (methoxy) group. Such a monofunctional PEG is termed monomethoxyPEG (mPEG). Thus, mPEG
contains only one hydroxyl group per chain, thus limiting activation and coupling to one site.
26.3.2.1.3 Limitations
contains peroxide impurities, which can lead to oxidative protein degradation during shelf- life storage.
26.3.2.2 Glycosylation
to improve their physiological properties. This process has several advantages, such as enhanced biodistribution in tissues, improved membrane permeability, and increased in vivo stability. Peptide– sugar
their enhanced oral bioavailability.
A vast number of naturally occurring sugars can be combined to create a variety of unique glycan
mannose (mannosylation), or lactose (lactosylation), to proteins has been successfully applied. For
example, receptors for carbohydrates, such as the asialoglycoprotein receptor on hepatocytes, and the
bovine serum albumin (Man- BSA) and galactosylated BSA (Gal- BSA) preferentially bind to alveolar
macrophages and hepatocytes, respectively. Galactosylated and mannosylated recombinant human
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FIGURE 26.2 PEGylation of proteins using N- hydroxysuccinimide (NHS) derivative of methoxy PEG.
superoxide dismutase (Gal- SOD, Man- SOD) exhibited inhibitory effects superior to native SOD against
26.3.2.3 Conjugation with Albumin
peptides. Albumin is a good candidate for this purpose because it has a long half- life, low immunogenic
potential, and is widely distributed in the body. Examples of peptide- albumin fusion therapeutics include
tein of exenatide and albumin with a half- life of 8 days, while albiglutide is a GLP- 1 dimer fused with
26.3.2.4 Conjugation with the Fc Portion of Antibodies
activity and prolonged half- life of the antibody. Following interaction with the neonatal Fc receptor
(FcRn), the Fc segment of the antibody is recirculated endosomally. By safeguarding the peptide against
metabolism and elimination, this mechanism effectively prolongs its plasma half- life. For example,
romiplostim received FDA approval in 2008 for the management of chronic idiopathic thrombocytopenic
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Pharmaceutical Dosage Forms and Drug Delivery
polypeptide chain comprising two TPO receptor- binding peptides covalently attached to two human
IgG1 Fc domains.
two examples of this method.
26.3.2.5 Stapled Peptides
Stapled peptides are chains with an external brace that converts them into alpha- helical shapes. This is
cing within the peptide chain. It is essential that the two or more amino acids used in the stapling process
have side chains that can form a brace of the appropriate length and are spaced apart from one another.
There can be more than one stapling bridge inside a single peptide. For lengthy peptides, double stapling
as cell- penetrating and exhibit selectivity similar to antibodies. Stapling gives the peptides resistance to
proteolysis and a longer half- life in the plasma. For example, ALRN- 5281 is a growth hormone- releasing
hormone (GHRH) agonist used to treat orphan diseases.
26.3.2.6 Conjugation with Cell- Penetrating Peptides
Cell- penetrating peptides (CPPs) are peptides with less than 30 residues that can easily pass through
penetratin, and HIV- TAT1 peptides. CPPs have basic residues like arginine and lysine, which promote
interactions with negatively charged cell surface glycosaminoglycans. The uptake of CPPs is mediated
demonstrated by the reduction in permeability across Caco- 2 cells when the parathyroid hormone was
26.3.2.7 Conjugation with Lipids
steroids, and glycerides, forming stable ester or amide bonds. Examples of fatty acids used include
squalenoic acid, stearic acid, palmitic acid, and docosahexaenoic acid. FDA- approved peptide drugs like
exhibited membranotropic behavior toward lipid vesicles and strongly interacted with the membranes
the gastrointestinal lumen. These monoglycerides are absorbed by enterocytes, re- acylated to form
triglycerides, and incorporated into lipoproteins for lymphatic system accumulation. This process
enhances absorption and targeting of the lymphatic system.
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26.3.2.8 Other Protein Conjugation Approaches
-
tion and long in vivo half- life make it suitable for monthly administration. Polysialic acid (PSA) is
genicity of peptides by forming a glycocalyx to prevent recognition by the body’s immune system. PSA
in large doses.
effects on peptides, including increased solubility (for hydrophobic peptides), masked antigenicity for
minimum immune response in the host, and prolonged half- life through reduced renal clearance. PEG is
26.4 Methods of Delivery
26.4.1 Nanotechnology
Advanced peptide- based drugs are being developed using chemistry and nano approaches to overcome
physicochemical constraints in the pharmaceutical industry. Ionic surfactants like cetrimide and sodium
dodecyl- sulfate (SDS) enhance peptide distribution across tissue membranes. Encapsulating peptides
into nanoparticles or combining them with polymers like polyvinylpyrrolidone (PVP) and PEG can also
improve their bioavailability.
degeneration by peptidases, preventing their breakdown into amino acids. They also offer sustained drug
polylactic- co- glycolic acid (PLGA) are also viable substitutes for drug carriers, providing consistent drug
overcome limitations in current peptide- based drug delivery systems by increasing plasma circulation
26.5 Antibody– Drug Conjugates
® and Adcentris®. Most current ADCs are
through a covalent linker to a monoclonal antibody that serves as a targeting moiety. The discovery and
development of ADCs follow unique paradigms that overlap small- and large- molecule drug discovery
and development but have unique distinctions. For example, the attachment of a hydrophobic drug to the
mAb changes mAb surface properties and conformational stability. It can increase protein aggregation
and surface hydrophobicity.
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TABLE 26.2
Pharmaceutical Dosage Forms and Drug Delivery
The ADCs on Market
Product Name Antibody/ target Payload Disease
Mylotarg
Adcetris
Besponsa
Lumoxiti
®
Polivy
Blenrep
Zynlonta
Padcev
Enhertu
Trodelvy
Akalux
®
Aidixi
®
Tivdak
Elahere
®
®
®
®
®
®
®
®
®
®
®
®
Adults with AML,
Brentuximab vedotin (Seagen)/ CD30 MMAE R/ R CD30 positive HL and
systemic ALCL
Adults with R/ R B- cell precursor
ALL
Moxetumomab pasudotox/ CD22 PE38 Adult patients with R/ R HCL
MMAE Patients with R/ R DLBCL
Belantamab mafodotin/ BCMA MMAF Adult patients with R/ R MM
Loncastuximab tesirine/ CD19 PBD dimer R/ R large B- cell lymphoma
DM1 Patients with HER2- positive
early breast cancer
Enfortumab vedotin/ Nectin- 4 MMAE Metastatic urothelial cancer
Metastatic HER2- positive breast
cancer
SN38 Metastatic TNBC
Cetuximab sarotalocan/ EGFR recurrent HNSCC
Disitamab vedotin/ HER2 MMAE metastatic gastric cancer
Tisotumab vedotin/ TF MMAE Metastatic cervical cancer
Mirvetuximab soravtansine- gyxn/ Fra Fr- a Ovarian cancer
selection of mAb, payload, and linker for an effective ADC. Currently, several ADCs are in clinical trials
as monotherapies or in combination with other anticancer drugs.
ADCs on the market.
AML acute myeloid leukemia, MMAE monomethyl auristatin E, MMAF monomethyl auristatin- F, HL
Hodgkin lymphoma, ALCL anaplastic large cell lymphoma, ALL Acute Lymphoblastic Leukemia, PBD
DXd Exatecan derivative, PE38 a 38kD fragment of Pseudomonas exotoxin A,
IRDye700 Infrared dye 700, DM1 derivative of maytansine 1, HER2 human epidermal growth factor
receptor 2, HCL hairy cell leukemia, TNBC triple- negative breast cancer, SN38 active metabolite of
irinotecan, MM multiple myeloma, HNSCC head and neck squamous cell carcinoma, TF tissue factor.
Fr- a folate receptor alpha.
Review Questions
Antibody molecules
A. Are globular proteins found predominantly in the gamma region during electrophoresis
B.
C. Are produced by T cells
D.
The antibody class found at the highest concentrations in serum is
A. IgE
B. IgM
C. IgD
D. IgG
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Antibody Therapeutics
A. Via their hypervariable regions
B.
C.
D.
Monoclonal antibodies currently used clinically
A. Can protect against a wide variety of viruses and bacteria
B.
C. Are derived from the plasma of individuals already immune to these organisms
D.
Monoclonal antibodies are produced by
A. Antibody- producing B cells from a mouse are fused with myeloma cells, and then the cells
are grown in tissue culture.
B.
C. They are produced by the human immune system as a natural response to an infection.
D. They are produced by a mouse’s immune system as a natural response to an infection.
compared to conventional therapies due to:
A. ADCs combine the favorable pharmacokinetics and biodistributions of the drug
B.
C. ADCs allow the immune system to destroy tumor cells.
D. All of the above
FURTHER READINGS
Clin
Diagn Lab Immunol, 10: 587– 595.
proteins: a role for polysialic acids. Int J Pharm, 300: 125– 130.
proteins via neonatal Fc receptor- mediated transcytosis. Hum Reprod, 20: 1805– 1813.
mediated drug disposition (PDMDD) and precursor pool lifespan model for single dose of romiplostim
AAPS J, 12 729– 740.
M.E., Milenic D.E., et al. (1993) An improved linker for single- chain Fv with reduced aggregation and
enhanced proteolytic stability. Protein Eng
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27
Biotechnology- Based Drugs
LEARNING OBJECTIVES
On the completion of this chapter, the students should be able to
1.
2.
3. Describe the three basic components of gene medicines.
4.
5. Discuss the characteristics of viral and nonviral gene therapy.
27.1 Introduction
Almost all human diseases are the result of inappropriate protein production or due to some structural
disorder that impacts protein performance. Traditional small- molecule drugs are designed to interact
with protein molecules that support or cause diseases. Protein drugs seek to replace the defective protein
in cases where missing or defective protein is the cause of the disease. Enzyme replacement therapy is a
hypertension, ischemic heart disease, asthma, Parkinson’s disease, motor neuron disease, and multiple
sclerosis) remain inadequately treated by conventional small molecular weight and protein drugs.
Compared to conventional small molecular weight and protein drugs, nucleic acid medicines
are designed to suppress or generate endogenous proteins by acting on or with the transcription and
translation mechanisms of the formation of proteins from the genetic code. These medicines can be
tion. Several different approaches are used for turning nucleic acids into therapeutics. Among them,
antisense oligonucleotides (ODNs), RNA interference (RNAi) technologies, plasmid deoxyribonucleic
acid (DNA), and virus- based gene therapy approaches are the most widely studied. Antisense ODNs and
small interfering RNAs (siRNAs) aim to inhibit aberrant protein production. In contrast, gene therapy
aims at using the patient’s somatic cells to produce therapeutic proteins needed for treating genetic or
acquired diseases. These nucleic acid drugs promise to allow either the production of therapeutic proteins
27.2 Genes and Gene Expression
The information necessary to produce proteins in cells is encoded in the genetic material— the chromosomal strands, which are made of DNA. A gene
Transcription is a nuclear process whereby information from DNA is transferred to messenger ribonucleic acid (mRNA). In this process, the two complementary strands of the DNA partly uncoil. The
DOI: 10.1201/9781003389378-31
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sense strand separates from the antisense strand. The antisense strand of DNA is used as a template by the
plasm, where ribosomes transfer the encoded information in mRNA’s base sequence in a complementaritytranslation. The long strings of amino acids
(called polypeptide chains) that are thus generated can fold by themselves or assemble with other polypep-
of proteins. These folding and assembly processes happen through multiple noncovalent (such as hydrophobic, hydrogen bond, and ionic) interactions, leading to the secondary, tertiary, and quaternary structure
proteins then migrate to their site of action— which can be membrane, cytoplasmic, intraorgnelle (such as
intranuclear or intramitochondrial), or extracellular (secreted proteins, such as hormones).
27.3 Gene Silencing
Antisense drugs inhibit the existing but abnormally expressed genes by blocking the transcription of
DNA or the translation of mRNA. Figure 27.1 illustrates the different modes of action of antisense
compounds. Overexpression of a particular protein can lead to or contribute to a disease state, such as
drugs work at the genetic level to interrupt the process by which disease- causing proteins are produced.
ciency syndrome [AIDS]).
27.4 Gene Silencing Technologies
27.4.1 Antisense Oligonucleotides
To create antisense drugs, nucleotides are linked together in short chains called ODNs. When
deoxyribonucleotides are linked in small chains, these are called oligodeoxyribonucleotides. The
sequence of nucleotides in the antisense drugs is complementary to small segments of mRNA. Each
FIGURE 27.1 Mode of action of nucleic acids. Gene therapy aims at producing therapeutic proteins, whereas antisense
therapy aims at blocking the production of aberrant proteins.
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