Biotechnology based drugs

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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.

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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.

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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

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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

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Issues related to the use of protein based drugs
Proteins versus low molecular weight drugsProper
3D structure required for biological activity

• 1) Antigenicity
• 2) Stability
• 3) Drug Delivery

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Antigenicity

• Foreign proteins may induce allergic reactionsi)
  anaphylaxisii) loss of efficacy
• Administer human proteinsHumanized antibodies
  i) chimeric antibodies ii) antibodies
  produced in transgenic mice
• iii) phage display antibodies

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Stability
i) Denaturation leads to loss of proper 3-D
conformation ii) Covalent bond breaking at high
tempertures and low pH

• 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

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Shelf Life of Recombinant Protein Drugs
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Use Life of Reconstituted Solutions
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Stability-Indicating Test Methods for Recombinant
Proteins
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Stability of Recombinant TNF (Liquid Formulation)
Stored Under Refrigeration(2-8C)
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Drug Delivery

• ProblemsDenaturation/chemical alterationRapid
  liver clearance
• Solution Alternative modes of administrationpare
  nterallynasalimplantsSustained delivery via
  microspheres Inhalers Exubera, inhalable
  insulin

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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.
• Human protein preferable
• b) 2nd generation protein-based drugs
• c) Protein chemical modifications

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Protein chemical modificationsincrease
circulating half life

• i) Changes in glycosylation
• ii) Bind polyethylene glycol (PEG) to proteins
• Nanotechnology/Nanomedicine

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Drug product selectionIncrease protein
half-life.

• a) Human product preferable
• b) Original protein product versus closely
  related products
• c) Protein chemical modification

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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

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Protein chemical modification
Glycosylation Asialoglycoprotein
receptor Polyethylene glycol (PEG)
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Asialoglycoprotein receptorBinds and endocytoses
proteins in which the terminal sialic acid has
been removed.
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Sources of protein products

• E.coli
• Yeast
• Mammalian cells
• Transgenic Animal sources
• Transgenic Plant sources

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Biogenerics bioequivalence
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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

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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)

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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

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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
interactionsCleavage of mRNA portion of RNAODN
duplex by RNase H
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DNA triplex
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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

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Hurdles to Antisense Development

• Permeation/Absorption Limited ability to cross
  cellular membranes
• Stability to degradation
• Affinity of binding

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Methods to enhance uptake/permeation

• Coadministration with cationic lipids
• Alternate backbones methylphosphonate
• Chimeric molecules
• Transport from cytoplasm to nucleus is typically
  rapid

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Stability

• Phosphodiester backbone modifications
• i) Block 3'exonuclease activity
• ii) endonuclease activityphosphorothioate
  methylphosphonate peptide-nucleic acid

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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

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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.
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Methods of delivery of siRNA
A) Synthesized and then transfected into cellsB)
generated by the RNAase activity of Dicer on
short hairpin RNAs (shRNAs) Gene therapy
methodsC) transcribed in vivoD) Viral
transfectionE) integration of plasmid DNA All
pathways lead to F) the siRNA binding to the
RISC, which targets complementary mRNA for
degradation.
From DNA Repair, 2759-63, 2003
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Applications of siRNA

• Genomics
• Therapeutic agentsRespiratory Syncytial Virus
• HIV proteins
• Limitations
• Similar to problems with antisense
• Mechanism of action not totally known

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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 interest2) 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

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Diagrams of the hammerhead and hairpin classes of
ribozymes interacting with target mRNA
From Journal of Leukocyte Biology, 66 361, 1999.
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Diagram of the Hammerhead ribozyme(requires Mg2
for activity)
W. G. Scott et al., Science 274, 2065 -2069
(1996)
Published by AAAS
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Application of ribozymes

• HIV
• TAT, TAR, RevCCR5 and CXCR4Gene therapy
  retroviral gene deliveryMutations still a
  problem! Also with RNAi
• Drug resistance due to MDRDrug transporters lead
  to resistance due to transport of drug out of the
  cellLower MDR expression via ribozymes to
  overcome resistance

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Ribozyme bioavailability issues

• Same as antisense and RNAi.
• Same types of chemical modifications
• Enhance uptake/permeationEnhance stability
  (i.e. increase half life)
• Avoid many of the above problems with Gene
  Therapy based methods

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Others
Aptamers oligonucleotides that specifically bind
proteins and other molecules.SELEX systematic
evolution of ligands by exponential enrichment

• Lupus treatment autoimmune disease due to self
  dsDNA antibodies