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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

 Proteins versus low molecular weight drugs
 Proper 3D structure required for biological activity


             1) Antigenicity
             2) Stability
             3) Drug Delivery
                 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
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
Shelf Life of Recombinant Protein Drugs

Name         Protein   Physical Form   Expiration Dating Period

Humulin      HI        liquid          2 years (2-8 C)

Orthoclone
             MuMAb     liquid          1 year (2-8 C)
OKT3
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%)

Protropin2    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.
                                                              terminal sialic acid removed due
                                                              to mechanical damage
                     Sia

                     Gal                             Gal

                    GlcNAc                        GlcNAc

                Core Sugars                     Core Sugars

              Sialoglycoprotein              Asialoglycoprotein




         glycoprotein does NOT bind     glycoprotein binds to the
         to the asialoglycoprotein      asialoglycoprotein receptor and
         receptor                       is internalized and destroyed

                             H

                                      O                            H      C    OH
        CH3     C     NH                   COO-
                             R                                     H      C    OH
                O
                             H         H
                                                                          CH2OH
                      H                    OH

                                                                          R
                             OH        H


                           Sialic acid (N-Acetylneuraminate (Sia))
   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
                                                          Base                           Base                O        Base
Phosphodiester backbone                          O                              O

   modifications
                                                     2'                             2'                           2'
                                         O                              O                            O
                                   (-)                            (-)
                                  O      P       O                S     P       O                CH3 P       O
i) Block 3'exonuclease activity
                                         O                              O                            O
ii) endonuclease activity                                                                                              Base
                                                 O         Base                 O         Base               O
    phosphorothioate
    methylphosphonate
                                                                                                         O
    peptide-nucleic acid                     O                              O


                                   Phosphodiester                 Phosphorothioate                Methylphosphonate
           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
                               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.

    From DNA Repair, 2:759-63, 2003
        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



                                        Q      c     m
                                             u i k T i   e ™     a n d       a
                                            d e c o m   p r e s s o r
                                a r e       n e e d e d   t o   s e e    t         s
                                                                                 h i     c
                                                                                       p i t u r e .




      From Journal of Leukocyte Biology, 66: 361, 1999.
              Diagram of the Hammerhead ribozyme
                   (requires Mg+2 for activity)




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

Published by AAAS
        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
 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

				
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