The Drug Development Process
(Lec II)
Dr. Anum Gul
(BT-606)
Content
Approaches to Determine Protein Function
Initial Biological Drug Characterization
Patenting
Delivery of biopharmaceuticals
Assessment of Protein Function
In the context of genomics, gene function is assigned a broader
meaning, incorporating not only the isolated biological
function/activity of the gene product, but also relating to:
1) where in the cell that product acts and, in particular, what other
cellular elements does it influence/interact with;
2) how do such influences/interactions contribute to the overall
physiology of the organism.
Approaches to Determine Protein
Function
Approaches to Determine Protein Function
Sequence homology studies DNA array technology (gene
Phylogenetic profiling chips)
Rosetta stone method Proteomics approach
Gene neighbourhood method Structural genomics approach
Knockout animal studies
6 Sequence Homology Studies
Studies depend upon bioinformatic sequence comparison between a
gene of unknown function (or, more accurately, of unknown gene
product function) and genes whose product has previously been
assigned a function.
High homology suggests likely related functional attributes.
Sequence homology studies can assist in assigning a putative function
to 40–60 percent of all new gene sequences.
Pax-6 Mutant Drosophilla
Phylogenetic profiling
Phylogenetic profiling entails establishing a pattern of the presence
or absence of the particular gene coding for a protein of unknown
function across a range of different organisms whose genomes have
been sequenced.
If it displays an identical presence/absence pattern to an already
characterized gene, then in many instances it can be inferred that
both gene products have a related function.
Phylogenetic profiling
Gene A and Gene B are
Functionally Linked
11 Rosetta Stone Method
The Rosetta stone or Domain Fusion method is dependent upon the
observation that sometimes two separate polypeptides (i.e., gene
products X and Y) found in one organism occur in a different
organism as a single fused protein XY.
In such circumstances, the two protein parts (domains), X and Y,
often display linked functions.
Rosetta Stone Method
Therefore, if gene X is recently discovered in a newly sequenced
genome and is of unknown function but gene XY of known function
has been previously discovered in a different genome, then the
function of the unknown X can be deduced.
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Gene Neighborhood Method
The gene neighborhood method is yet another computation-
based method.
It depends upon the observation that two genes are likely to be
functionally linked if they are consistently found side by side in
the genome of several different organisms.
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Knockout Animal Studies
Knockout animal studies are dependent upon phenotype
observation.
The approach entails the generation and study of animals in
which a specific gene has been deleted.
Phenotypic studies can sometimes yield clues as to the
function of the gene knocked out.
Fly with normal Fly with knocked out
gene for controlling gene for controlling
eye development eye development
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Gene Chips
Background
Sequence data provide no information as to which genes are
switched on (transcribed) and, hence, which are functionally active
at any given time/under any given circumstances.
The study of under which circumstances an RNA is expressed/not
expressed in the cell/organism can provide clues as to the
biological function of finally expressed protein product.
Gene Chips
In the context of drug lead/target discovery, the conditions under
which a specific mRNA is produced can also point to drug targets.
For example, if a particular mRNA is only produced by a cancer
cell, that mRNA (or, more commonly, its polypeptide product) may
represent a good target for a novel anti-cancer drug.
Gene Chips
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What are Gene Chips?
Also called DNA micro array or Oligonucleotide arrays.
These are devices which are based on a glass substrate
wafer and contain many tiny cells.
Each holds DNA from a different human gene.
Gene Chips
Gene Chips
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The Technique
The technique is based upon the ability to anchor nucleic acid
sequences (usually DNA based) on plastic/glass surfaces at very
high density.
About 250,000 different short oligonucleotide probes or 10,000
full-length cDNA sequences per square cm of surface.
Gene Chips
Any complementary RNA sequences present will hybridize with
the appropriate immobilized chip sequence.
Hybridization patterns obviously yield critical information
regarding gene expression.
Generalized outline of a gene chip
In this example, short oligonucleotide sequences (AGGCA) are
attached to the anchoring surface.
By incubating the chip with, any mRNA with a complementary
sequence (UCCGU in the case of the probe sequence shown) will
hybridize with the probes.
Generalized outline of a gene chip
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Proteomics
Background
Although virtually all drug targets are protein based, the inference that
protein expression levels can be accurately detected/measured via DNA
array technology is not true, as:
❖mRNA concentrations do not always directly correlate with the
concentration of the mRNA encoded polypeptide;
❖a significant proportion of eukaryote mRNAs undergo differential
splicing and, therefore, can yield more than one polypeptide product.
Production of two polypeptides by differential splicing of a single mRNA
Although most eukaryotic primary transcripts produce only one mature
mRNA (and hence code for a single polypeptide), some can be
differentially spliced, yielding two or more mature mRNAs. The latter can,
therefore, code for two or more polypeptides. E: exon; I: intron
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Background (Continued..)
❖Additionally, the cellular location at which the resultant
polypeptide will function often cannot be predicted from RNA
sequence.
❖No detailed information regarding how the polypeptide product’s
functional activity will be regulated (e.g., via post-translational
mechanisms such as phosphorylation, partial proteolysis, etc.).
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What is ‘Proteomics’?
Protein-based drug leads/targets are often more successfully
identified by direct examination of the expressed protein
complement of the cell, i.e., its proteome.
The proteome is not static, with changes in cellular conditions
triggering changes in cellular protein profiles/concentrations.
This field of study is termed proteomics.
The Proteomics Approach
30 Structural Genomics
It focuses upon the large-scale systematic study of gene product
structure.
The ultimate goal of structural genomics is to provide a complete
three-dimensional description of any gene product.
The basic approach to structural genomics entails the cloning and
recombinant expression of cellular proteins, followed by their
purification and three-dimensional structural analysis.
31
Continued…
Techniques
X-ray crystallography
o Drawback: target protein is required to be in crystalline
form.
NMR
o No crystallization required.
Initial Biological Drug Characterization
32
Initial Biological Drug
Characterization
Patenting
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Patenting
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A patent may be described as
“a monopoly granted by a government to an inventor, such that only
the inventor may exploit the invention/innovation for a fixed period
of time (up to 20 years).
In return, the inventor makes available a detailed technical
description of the invention/innovation so that, when the monopoly
period has expired, it may be exploited by others without the
inventor’s permission”.
What is Patentable
36
In order to be considered patentable, an invention/innovation
must satisfy several criteria, the most important four of which
are:
✓ novelty
✓ non-obviousness
✓ sufficiency of disclosure
✓ utility
What cannot be patented
37
Naturally obtained substance without any modification
The human body;
The cloning of humans;
The use of human embryos for commercial purposes;
Modifying the genetic complement of an animal if the
modifications cause suffering without resultant substantial
medical benefits to the animal/to humans.
38 Delivery of Biopharmaceuticals
Oral Delivery System
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Oral Delivery System
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Most preferred route of delivery. Accompanied with some
drawbacks.
o Inactivation due to stomach acid. Virtually all
biopharmaceuticals are acid labile and are inactivated at
low pH values.
o Inactivation due to digestive proteases. Therapeutic
proteins would represent potential targets for digestive
proteases such as pepsin, trypsin and chymotrypsin.
Oral Delivery System
Their (relatively) large size renders difficult the passage of
intact biopharmaceuticals across the intestinal mucosa.
Orally absorbed drugs are subjected to first-pass
metabolism. Upon entry into the bloodstream, the first
organ encountered is the liver, which usually removes a
significant proportion of absorbed drugs from circulation.
Oral Delivery System
42
Solution
Physically protecting the drug via encapsulation and formulation as
microparticulates or as specialized coated tablets.
Inclusion of protease inhibitors (e.g: aprotinin and ovomucoids) and
permeability enhancers.
‘Mucoadhesive delivery systems’ in which the biopharmaceutical is
formulated with/encapsulated in molecules that interact with the
intestinal mucosa membranes.
Pulmonary Delivery System
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Pulmonary Delivery System
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Pulmonary delivery currently represents the most promising
alternative to parenteral delivery systems for biopharmaceuticals.
Delivery via the pulmonary route moved from concept to reality in
2006 with the approval of Exubera, an inhalable insulin product.
Although the lung is not particularly permeable to solutes of low
molecular mass (e.g., sucrose or urea), macromolecules can be
absorbed into the blood via the lungs surprisingly well.
Pulmonary Delivery System
Many peptides/proteins delivered to the deep lung are detected
in the blood within minutes (upto 500 kDa).
Bioavailabilities approaching/exceeding 50 per cent (relative to
s.c. injection) have been reported for therapeutic proteins.
Pulmonary Delivery System
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High pulmonary bioavailability is due to:
The lung’s very large surface area;
Their low surface fluid volume;
Thin diffusional layer;
Relatively slow cell surface clearance;
The presence of proteolytic inhibitors.
Pulmonary Delivery System
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Benefits of Pulmonary Delivery Systems
the avoidance of first-pass metabolism;
the availability of reliable, metered nebulizer-based delivery
systems capable of accurate dosage delivery, either in powder or
liquid form;
levels of absorption achieved without the need to include
penetration enhancers which are generally too irritating for long-
term use.
Pulmonary Delivery System
Biological barriers
Lung surfactant, and its underlying surface lining fluid, which lies
immediately above the lung epithelial cells
The epithelial cells lining the lung
The interstitium (an extracellular space), and the basement membrane
The vascular endothelium
Transcytosis or paracellular transport
Nasal Delivery System
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Nasal Delivery System
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It is easily accessible and increase patient compliance;
Nasal cavities are serviced by a high density of blood
vessels;
Nasal microvilli generate a large potential absorption surface
area;
Nasal delivery ensures the drug bypasses first-pass
metabolism.
Nasal Delivery System
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Disadvantages
Clearance of a proportion of administered drug occurs due to its
deposition upon the nasal mucous blanket, which is constantly
cleared by ciliary action;
The existence of extracellular nasal proteases/peptidases;
Low uptake rates for larger peptides/polypeptides (molecules
>10kDa).
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