Biotechnology based drugs

clatteringlippsBiotechnology

Dec 5, 2012 (4 years and 8 months ago)

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Biotechnology based drugs


Objectives: Technological advances in drug development
and biological sciences are allowing for the rapid
development of new diagnostic methods and drugs based
on biological molecules, including proteins and nucleic
acids.


Upon completion of these lectures, the student
will know issues associated with the biochemical
mechanisms, stability, use and dispensing of
biotechnology derived drugs, including current and
anticipated applications.


This includes issues that the
practicing pharmacist must be aware of to effectively
dispense such medications.


Groundwork for protein based drugs


Insulin (1922)


Genetic Engineering


clone, express and manipulate proteins on microorganisms


Somatostatin (1977)


Pharmacists

represent a primary point of contact from which the
public can be informed of the nature, efficacy, potential adverse
effects etc. of biotechnology based drugs.


In addition the
pharmacist will be responsible for proper dispensing of use of this
class of medications.


Examples of Classes of Protein
-
based Biotech
drugs


Erythropoietin (EPO)

Blood factors (Factor VII)

Growth Factors: Becaplermin

Human Growth Hormone: HGH, Somatotropin, Sermorelin

Cytokines


i) Interleukins (ILs): Interleukin
-
11 (rhIL
-
11, Neumega)


ii) Interferons


Enzymes: Adenosine deaminase


$$$$$

Monoclonal Antibodies (mAbs)

i)
Specific

and have high affinities for certain antigens or cell types

ii) Attack foreign toxins, viruses or cancer cells

iii) Drug delivery to specific targets (e.g. radioisotopes)

iv) Half
-
life of many "humanized" antibodies is often greater than one week




Basiliximab/Daclizumab

Herceptin (Trastuzumab)

Zevalin

Immunoassays

Issues related to the use of protein based drugs

1) Antigenicity

2) Stability

3) Drug Delivery

Proteins versus low molecular weight drugs

Proper 3D structure required for biological activity

Antigenicity

Foreign proteins may induce allergic reactions

i) anaphylaxis

ii) loss of efficacy

Administer human proteins

Humanized antibodies


i) chimeric antibodies


ii) antibodies produced in transgenic mice


iii) phage display antibodies

Stability

a) Specific amino acids contribute to destabilization


deamidation: Asn, Gln


oxidation: Met


proteolysis: Arg, Lys


racemization and acid labile: Asp


disulfide exchange: Cys, disulfide


aminolysis: Lys


beta
-
elimination: Cys, Ser, Thr, Lys

b) Proteolysis during storage due to enzymes associated with bacterial contamination.

c) Protein often more stable in dry form (lyophilized)

d) Additives to enhance stability

e) Detection of instability


i) Denaturation leads to loss of proper 3
-
D conformation

ii) Covalent bond breaking at high tempertures and low pH

Shelf Life of Recombinant Protein Drugs


Name

Protein

Physical Form

Expiration Dating Period

Humulin

HI

liquid

2 years (2
-
8 C)

Orthoclone

OKT3

MuMAb

liquid

1 year (2
-
8 C)

Roferon
-
A

IFN
-
a2a

solid

3 years (2
-
8 C)

Intron A

IFN
-
a2b

solid

3 years (2
-
8 C)

Activase

TPA

solid

2.5 years (2
-
8 C)

Protropin

hGH

solid

2 years (2
-
8 C)

Use Life of Reconstituted Solutions


Name

Protein

Maximum Hold Time

Bacteriostat

Roferon
-
A

IFN
-

2a

1 month at 2
-
8 C

phenol (0.3 %)

Intron A

IFN
-

2b

1 month at 2
-
8 C

benzyl alcohol (0.9)%

Activase

TPA

14 days at 2
-
8 C

m
-
cresol (0.3%)

Protropin
2

hGH

7 days at 2
-
8 C

benzyl alcohol (0.9)%

Humatrope

hGH

8 hours at 2
-
30 C

none

Stability
-
Indicating Test Methods for
Recombinant Proteins


Method

Change that can be detected

Example of Use

SDS Page

fragmentation

IFN
-


crosslinking

hGH

oligomerization

IFN
-


RP
-
HPLC

deamidation

Insulin

crosslinking

Insulin

methionine oxidation

IL
-
2

disulfide scrambling

IL
-
2

IEF

deamidation

IL
-
1

Potency Determination

disulfide scrambling

IFN
-


Stability of Recombinant TNF (Liquid
Formulation) Stored Under Refrigeration

(2
-
8
°
C)

Time in Storage (months)

Potency

Protein Purity by SDS Page

0

100 %

100 %

3

100 %

100 %

6

70 %

100 %

9

60 %

100 %

12

50 %

99 %

Drug Delivery

Problems

Denaturation/chemical alteration

Rapid liver clearance


Solution: Alternative modes of administration

parenterally

nasal

implants

Sustained delivery via microspheres

Inhalers: Exubera, inhalable insulin


Drug product development

Mutate chemically labile amino acids to other
amino acids, however, antigenicity problems
may occur due to protein becoming non
-
self
and/or loss of biological activity may arise due to
changing the amino acids.


a)

Human protein preferable

b) 2nd generation protein
-
based drugs

c) Protein chemical modifications

Protein chemical modifications:

increase circulating half life


i) Changes in glycosylation

ii) Bind polyethylene glycol (PEG) to proteins



Nanotechnology/Nanomedicine

Drug product selection:

Increase protein half
-
life.



a) Human product preferable

b) Original protein product versus closely related
products

c) Protein chemical modification

Original protein product versus closely
related products

i) Modification or removal of selected amino acids to increase
stability

ii) Production via an alternate source (see below)

iii) Deletion of unessential portion of the protein

iv) Introduction of disulfide bonds

v) Proper phosphorylation required for biological activity

Protein chemical modification

Glycosylation


Asialoglycoprotein
receptor


Polyethylene glycol (PEG)


Asialoglycoprotein receptor

Binds and endocytoses proteins in which the
terminal sialic acid has been removed.

Sources of protein products


E.coli

Yeast

Mammalian cells

Transgenic Animal sources

Transgenic Plant sources

Biogenerics: bioequivalence

Antisense oligodeoxynucleotides (ODNs) and
other nucleic acid related therapeutic agents

Antisense ODNs

Use of small synthetic oligonucletides, resembling single
-
stranded DNA to
inhibit gene expression (production of proteins).



i) Hybridization to coding
(sense strand)

sequences in a specific messenger
RNA or in duplex DNA (the sense strand is that which is copied)

ii) The antisense strand is the "uncopied" strand


Drug specificity: Protein (3D) versus antisense (1D) complementarity

Affinity versus Specificity

i) Increase length to maximize affinity


ii) Increasing length, however, increases binding to sequences
that differ by one or two sites leading to a decreased specificity


iii) Base composition: more G/C greater affinity (3 versus 2
hbonds in A/T)

Antisense Targets

i) proteins in microorganisms to kill invading organism

ii) proteins specific to cancer

iii) any undesired protein

iv) fields of genomics and proteomics will help identify new targets


Vitravene: Cytomegalovirus (CMV) Retinitis

Macugen: Macular degeneration

Genasense: advanced malignant melanoma (adjunct therapy)


Leukemia


i) remove bone marrow from individual


ii) kill only the leukemia cells in that bone marrow via antisense
agent


iii) replace the remaining healthy bone marrow

Hypertension




Mechanisms of Antisense Agents


Block transcription at the DNA
level

(triplex, ss regions)

mRNA level


During synthesis


Intron/exon junctions


Transport


Inhibit protein initiation factors


Block interaction with ribosome


at start codon


overall interactions


Cleavage of mRNA portion of
RNA:ODN duplex by RNase H

DNA triplex

Non
-
antisense mechanisms

i) Interaction of ODN backbone or degradation products
with proteins or cell surface

ii) Polyanion effects

iii) Test for non
-
antisense mechanism using a scrambled
control

ODN


Hurdles to Antisense Development



Permeation/Absorption: Limited ability to cross
cellular membranes

Stability to degradation

Affinity of binding


Methods to enhance uptake/permeation

Coadministration with cationic lipids

Alternate backbones: methylphosphonate

Chimeric molecules

Transport from cytoplasm to nucleus is typically
rapid

Stability

Phosphodiester backbone
modifications


i) Block 3'exonuclease activity

ii) endonuclease activity

phosphorothioate

methylphosphonate

peptide
-
nucleic acid

Additional modifications

Sugar modifications



i) Enhance stability and affinity:

-
anomer at 1' position of
2'deoxyribose


ii) Resistance to nucleases: 2
-
OH modifications of ribose
including 2'methyl,


2'
-
allyl, or

2'
-
fluoro (also enhance
affinity)


Base modifications



Hydrophobic modifications of 5' position of pyrimidines that
enhance affinity for target RNA or DNA

RNA interference (RNAi or siRNA)

Target specific mRNAs for degradation, thereby leading to decreased
expression of the corresponding protein. One interference RNA can
remove large numbers of mRNAs.

Methods of delivery of siRNA

From
DNA Repair
, 2:759
-
63, 2003

A) Synthesized and then
transfected into cells

B) generated by the RNAase
activity of Dicer on short
hairpin RNAs (shRNAs)


Gene therapy methods

C) transcribed in vivo

D) Viral transfection

E) integration of plasmid DNA

All pathways lead to F) the
siRNA binding to the RISC,
which targets complementary
mRNA for degradation.

Applications of siRNA

Genomics

Therapeutic agents

Respiratory Syncytial Virus


HIV proteins


Limitations


Similar to problems with antisense


Mechanism of action not totally known

Ribozymes

RNA molecules that assume tertiary structures and have the
ability to catalyze chemical reactions, making them catalysts.


Target mRNA by synthesizing RNA that

1) contains the sequence to bind specifically with the mRNA
of interest

2) contains a ribozyme to catalyze the hydrolysis of the
targeted mRNA


Ribozymes are found in the ribosome and it is thought that they
may have been involved in catalysis in early forms of life prior
to protein based catalysts


Diagrams of the hammerhead and hairpin
classes of ribozymes interacting with target
mRNA

From Journal of Leukocyte Biology, 66: 361, 1999.

Published by AAAS


W. G. Scott et al., Science 274, 2065
-
2069 (1996)

Diagram of the Hammerhead ribozyme

(requires Mg
+2

for activity)

Application of ribozymes

HIV


TAT, TAR, Rev

CCR5 and CXCR4

Gene therapy: retroviral gene delivery

Mutations still a problem! Also with RNAi

Drug resistance due to MDR

Drug transporters lead to resistance due to transport
of drug out of the cell

Lower MDR expression via ribozymes to overcome
resistance


Ribozyme bioavailability issues

Same as antisense and RNAi.


Same types of chemical modifications


Enhance uptake/permeation

Enhance stability (i.e. increase half life)


Avoid many of the above problems with Gene
Therapy based methods

Others

Lupus treatment:
autoimmune disease due
to self dsDNA antibodies




Aptamers: oligonucleotides that specifically bind proteins
and other molecules.

SELEX: systematic evolution of ligands by exponential
enrichment