Drug Development
and
Evaluation
Phase II
May 2010
Protein-based Therapeutics
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Today protein therapeutics are obtained from
two sources:
Purified from a wide variety of different
organisms or
Produced by genetic engineering using
recombinant DNA technology
Protein-based Therapeutics
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Nonrecombinant proteins are purified from their native
source, such as pancreatic enzymes from pig pancreas
and alpha-1-proteinase inhibitor from pooled human
plasma.
Production systems for recombinant proteins include
bacteria, yeast, insect cells, mammalian cells, and
transgenic animals and plants.
The system of choice can be dictated by the cost of
production or the modifications of the protein (e.g.,
glycosylation, phosphorylation, or proteolytic cleavage)
that are required for biological activity.
For example, bacteria do not perform glycosylation
reactions, and each of the other biological systems listed
previously produces a different type or pattern of
glycosylation.
Protein-based Therapeutics
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Protein glycosylation patterns exert dramatic effects on
the activity, half-life, and immunogenicity of the
recombinant protein in the body.
As one example, the half-life of native erythropoietin, a
growth factor important in erythrocyte production, can be
lengthened by increasing the glycosylation of the protein.
Darbepoetin is an erythropoietin analogue engineered
to contain two additional amino acids that are substrates
for N-linked glycosylation reactions.
When expressed in Chinese hamster ovary (CHO) cells,
the analogue is synthesized with five rather than three Nlinked carbohydrate chains; this modification causes the
half-life of darbepoetin to be threefold longer than that of
erythropoietin.
Recombinant Protein Drugs
ADVANTAGES
 Transcription and translation of the exact human
gene lead to a higher specific activity of the
protein and a decreased chance of immunologic
rejection by the patient ( the example of bovine insulin
compared to recombinant human insulin).

Recombinant proteins are often produced more
efficiently and inexpensively and in potentially
limitless quantity. For example, therapy for Gaucher's
disease, a chronic congenital disorder of lipid metabolism caused by
a deficiency of the enzyme beta-glucocerebrosidase. Most patients
with this disease have an enlarged liver and spleen, increased skin
pigmentation, and painful bone lesions. Although patients can be
treated with beta-glucocerebrosidase purified from human
placenta, this treatment requires purification of protein from 50,000
placentas per patient per year. This requirement obviously places a
practical limit on the amount of purified protein available for patients
with the disease. A recombinant form of beta-glucocerebrosidase is
now available.
Recombinant Protein Drugs
ADVANTAGES (cont.)
 The recombinant protein eliminates the risk of
transmissible (e.g., viral or prion) diseases
associated with purifying the protein from human
placentas.
 Recombinant technology allows the modification
of a protein to improve function or specificity. For
example, recombinant beta-glucocerebrosidase provides an
interesting example. When this protein is made recombinantly, a
change of amino acid arginine-495 to histidine allows the addition of
mannose residues to the protein. The mannose is recognized by
endocytic carbohydrate receptors on macrophages and many other
cell types, allowing the enzyme to enter these cells more efficiently
and to cleave the intracellular lipid that has accumulated in
pathologic amounts. This results in an improved therapeutic
outcome.
Recombinant Protein Drugs in Medical Use
erythropoietin, a protein hormone secreted by the
kidney that stimulates erythrocyte production in
the bone marrow.
 In patients with chemotherapy-induced anemia
or myelodysplastic syndrome, recombinant
erythropoietin is used to increase erythrocyte
production and thereby improve the anemia.
 In patients with chronic kidney disease, whose
levels of endogenous erythropoietin are below
normal, recombinant protein is administered to
correct this deficiency.
Recombinant Protein Drugs in Medical Use
Darbepoetin alfa is a recombinant variant of erythropoietin with a
longer half-life.
 Protein glycosylation patterns exert dramatic effects on the activity,
half-life, and immunogenicity of the recombinant protein in the body.
 As one example, the half-life of native erythropoietin, a growth factor
important in erythrocyte production can be extended by increasing
the glycosylation of the protein.
 Darbepoetin is an erythropoietin analogue engineered to contain
two additional amino acids that are substrates for N-linked
glycosylation reactions.
 When expressed in Chinese hamster ovary (CHO) cells, the
analogue is synthesized with five rather than three N-linked
carbohydrate chains; this modification causes the half-life of
darbepoetin to be threefold longer than that of erythropoietin.
EPO
Darbepoetin
Darbepoetin alfa has:
Two additional sialic acid–containing carbohydrates (red)
Up to 8 additional sialic acids
Increased molecular weight (~37,100 daltons)
Classes of Approved Recombinant
Protein Drugs
Ref: Scientific and Legal Viability of Follow-on Protein Drugs. David M Dudzinski, Aaron S Kesselheim. The New England Journal of Medicine
Vol. 358, Iss. 8; pg. 843, 2008.
Recombinant Protein Drugs

In 2006, the recombinant proteins
darbepoetin alfa (Aranesp), epoetin alfa
(Epogen, Procrit), and etanercept (Enbrel)
were among the year's top 10-selling
pharmaceuticals on the basis of dollar
value and accounted for almost 4% of the
$275 billion annual U.S. pharmaceutical
market.
Recombinant Protein Drugs in Medical Use
INSULIN
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insulin in the treatment of diabetes mellitus type I (DM-I) and type II
(DM-II).
Untreated, DM-I is a disease that leads to severe wasting and death
due to lack of the protein hormone, insulin, which signals cells to
perform a number of functions related to glucose homeostasis and
intermediary metabolism.
In 1922, insulin was first purified from bovine and porcine (pig)
pancreas and used as a lifesaving daily injection in patients with
DM-I.
At least three challenges prevented widespread use of this protein
therapy:
(1) requirement of big number of animal pancreas for purification of
insulin;
(2 the cost of insulin purification from animal pancreas was high;
(3) existing immunologic reactions of some patients to animal insulin.
Recombinant Protein Drugs in Medical Use
INSULIN (cont.)
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These problems were addressed by isolating the human insulin gene,
“recombining” the gene with bacterial DNA, and engineering Escherichia coli
using this recombinant DNA technology to express human insulin.
By growing vast quantities of these bacteria, large-scale production of
human insulin was achieved.
The resulting insulin was abundant, inexpensive, of low immunogenicity, and
free from other animal pancreatic substances.
Recombinant insulin was the first commercially available recombinant
protein therapeutic; it was approved by the US FDA in 1982, and has been
the major therapy for DM-I (and a major therapy for DM-II) ever since.
The 25 years since the approval of insulin by the FDA have seen a
remarkable expansion of proteins in the pharmacologic therapy used by
physicians to treat disease.
Currently, more than 70 different proteins (over 40 of which are produced
recombinantly) are approved by the FDA for clinical use.
Protein-based therapies are, and will continue to be, a mainstay in treating
human disease.
ABD’de Bir İlacın Geliştirilme Süreci
In vitro
Çalışmalar
Pazarlama
dönemi
NDA
Klinik İlaç Geliştirme
Çalışmaları
Hayvan
Çalışmaları
Biyolojik
Ürünler
FAZ I
F
D
A
İ
N
C
E
L
E
M
E
S
İ
çalışmaları
Etkililik
Seçicilik
Seçilen Toksisite
Molekül Etki
Mekanizması
FAZ II
çalışmaları
FAZ III
çalışmaları
Kimyasal
Sentez
Ürünleri
0
YILLAR
2
4
IND Başvurusu
(Araştırılacak Yeni İlaç Başvurusu)
5000 molekül
5 molekül
8
NDA
FAZ IV
çalışmaları
9
(FDA Onayı)
20
(Patent Bitimi)
(Yeni İlaç Başvurusu)
1 molekül
Ref: Goodman and Gilman’s, The Pharmacological Basis of Therapeutics, Mc GrawHill, 1996.
Katzung, B.G., Basic and Clinical Pharmacology, Appleton & Lange, 1998
FDA report: From Test Tube to Patient 1999.
May 2004
Drug Development Process
Clinical Drug Testing in Humans
NUMBER OF SUBJECTS
LENGTH OF PHASE
PURPOSE
Phase I
20–100
Several months
Mainly safety
Phase II
Up to several hundred
Several months to 2
years
Effectiveness, dosage and shortterm safety
Phase
III
Several hundred to several
thousand
1–4 years
Safety, dosage, effectiveness
Drug Development Process
Phase I Studies
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Phase I studies generally involve between 20 and 100
healthy normal subjects and are intended to establish
the safety and tolerability of a drug.
If high levels of toxicity are expected, such as with many
cancer drugs, patients with the target condition may be
used in place of healthy volunteers.
The focus of phase I investigation is the drug's overall
effect and kinetics in the body, including maximum
tolerated dose, absorption, distribution, metabolism,
and excretion.
To determine the effect of varying doses, subjects are
started on a dose anticipated to have little effect, and
receive increasing doses thereafter.
Drug Development Process
Phase I Studies (cont.)
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The primary goal of phase I studies is to establish safety,
toxicity, kinetics, and major adverse effects.
Phase I studies may involve nonblinded trials, in which
subject and investigator are both aware of what is being
administered.
Phase I studies must yield sufficient information about a
drug's pharmacokinetics to inform the design of
scientifically valid phase II studies.
For example, knowing the drug's volume of distribution
and clearance enables study designers to determine an
appropriate maintenance dose and dosing frequency for
phase II and III trials.
Drug Development Process
Phase II Studies
 Phase II studies may involve up to several hundred patients with
the medical condition of interest.
 Phase II clinical trials have multiple objectives, including the
acquisition of preliminary data regarding the effectiveness of the
drug for treatment of a particular condition.
 Like phase I trials, phase II trials continue to monitor safety.
Because phase II studies enroll more patients, they are capable of
detecting less common adverse events.
 Phase II studies also evaluate dose-response and dosing
regimens, which are critically important in establishing the optimum
dose or doses and frequency of administration of the drug.
 The trial usually compares several dosing regimens to obtain
optimum dose range and toxicity information.
 The results of phase II studies are critically important in establishing
a specific protocol for phase III studies.
Drug Development Process
Phase III Studies
 Phase III studies involve several hundred to several
thousand patients and are conducted at multiple sites
and in settings similar to those in which the drug would
ultimately be used.
 Safety, dosage and efficacy properties obtained from
Phase II study is tested in a larger patient population.
 Because of the large number of patients under study,
phase III trials typically provide an adequate basis for
extrapolating the results to the general population.