1 THERAPEUTIC AGENTS 1.Introduction 2.Recombinant Proteins 3.Nucleic Acids

Click here to load reader

  • date post

    22-Dec-2015
  • Category

    Documents

  • view

    214
  • download

    1

Embed Size (px)

Transcript of 1 THERAPEUTIC AGENTS 1.Introduction 2.Recombinant Proteins 3.Nucleic Acids

  • Slide 1
  • 1 THERAPEUTIC AGENTS 1.Introduction 2.Recombinant Proteins 3.Nucleic Acids
  • Slide 2
  • 2 Therapeutic Agents Before the advent of molecular biotechnology most human proteins were available in only small (limited) quantities. Today hundreds of genes (~1000) for human proteins have been cloned, sequenced, expressed in the host cells and are being tested as therapeutic agents (drugs) in humans. Over 140 biopharmaceuticals on the market; over 400 in clinical trials Biopharmaceuticals include: Proteins (made in bacterial, fungal or mammalian cell culture) erythropoietin (EPO) insulin interferon (Intron A) granulocyte-colony stimulating factor (G-CSF) human growth hormone (HGH, human somatotropin) tissue plasminogen activator (tPA) Monoclonal antibodies (made in mammalian cell culture) Vaccines live and inactivated viruses and bacteria subunit vaccines recombinant vaccines Gene Therapy Products (viral and non-viral) Introduction
  • Slide 3
  • 3 Process of Drug Production Cells and plasmid + Cell line TransfectionCell culturePurification Drug substance (crude) Drug substance (pure) Drug product - (sterile) Formulation/ Filling Cell line manufacture Medium development Bioreactor process development & scale-up Downstream purification Analytical characterization Introduction Therapeutic Agents
  • Slide 4
  • 4 Development of the Process Cell Line/ Viral Vector/ Construction Cell/Virus Culture Development/ Media Optimization Purification Process Dev. Formulation Design/ Drug Delivery Design Biological Assay Development and Support Facility/Equipment Design Technology Transfer Process Validation Pilot-Scale cGMP Production Commercial-Scale Production Therapeutic Agents Introduction
  • Slide 5
  • 5 Therapeutic Agents Recombinant Proteins Human Interferons -> to fight viral infections -> Scientists discovered an antiviral protein in 1957 that inhibited growth of influenza virus in chicken embryos. It was named interferon because it interfered with the growth of influenza virus. Anti viral proteins released by host cells (part of the immune system) Interfere with viral multiplication Host cell specific but not virus specific Different types of cells in animals produce different interferons
  • Slide 6
  • 6 Therapeutic Agents Recombinant Proteins Human Interferons -> to fight viral infections 3 types of human interferon: alpha interferon (13 genes) beta interferon (2 genes) gamma interferon (1 gene) Alpha & beta usually produced early in viral infections (viruses or viral RNA) - Gamma appears later -> Presence of double-stranded RNA indicates cell is infected -> Viral infected cells release alpha and beta interferons Diffuse to neighboring cells -> Virus cant replicate
  • Slide 7
  • 7 Therapeutic Agents Recombinant Proteins Human Interferons -> to fight viral infections Antiviral Treatment: Interferon therapy Limited lifetime, short lasting effect Recombinant interferons Pure and fast Hybrid genes for enhanced/new activity Oral administration
  • Slide 8
  • 8 Therapeutic Agents Recombinant Proteins Human Interferons -> to fight viral infections Why are Side Effects Common and Severe for Injectable Interferon? Injectable interferon (beta) is approved world-wide (FDA) for the treatment of various cancers and viral diseases. Interferon is a protein readily eliminated from the blood by the kidney. To counteract the kidneys clearance of interferon from the blood injectable interferon must be given in doses much higher than what occur naturally. Side effects include flu-like symptoms, poor results on liver function tests, and blood cell abnormalities. More serious side effects include depression, epileptic seizures, or liver problems. Why is Oral Interferon Different? Low-dose oral interferon is given in doses 10 thousand times less than injectable interferon. Therefore, side effects are dramatically reduced. Oral interferon is human interferon alpha administered in a small tablet (lozenge) to humans or in powder to animals. Oral interferon binds to surface (mucosal) cells in the mouth and throat resulting in stimulation of white blood cells and activates hundreds of genes affecting the immune system in the peripheral blood of man, cattle and mice. Studies show oral interferon is effective against disorders such as cancer, viral diseases and autoimmunity.
  • Slide 9
  • 9 Mechanism of Action Interferon placed in the mouth binds to receptors in the mucosal lining and initiates systemic effects on the immune system in animals and man. These immunomodulatory effects are safe and effective in helping control viral and autoimmune diseases and cancer. IFN Oral Epithelial Cells Tonsil Mandibular Lymph Nodes Activation of Cell Mediated Immunity Virus Activation of Humoral Immunity (Antibody) Activation of Perioral Lymphoid Cells and Peripheral Lymphoid Tissues Therapeutic Agents Recombinant Proteins Human Interferons -> to fight viral infections
  • Slide 10
  • 10 Therapeutic Agents Recombinant Proteins Human Interferons -> to fight viral infections Manufacturing Steps for Interferon:
  • Slide 11
  • 11 Therapeutic Agents Recombinant Proteins Human Interferons -> to fight viral infections Human diseases in which oral interferon has been tested and reported to be safe
  • Slide 12
  • 12 Therapeutic Agents Recombinant Proteins Human Interferons -> to fight viral infections 14,000 people participated in controlled studies of placebo versus interferon treatment during a natural outbreak of Hong Kong influenza. Interferon (about 128 units) or placebo was dripped into the nose daily for 5 days starting about the time of the first reported influenza cases. Interferon significantly (P
  • 14 Therapeutic Agents Recombinant Proteins Enzymes Treating Cystic Fibrosis Cystic fibrosis (CF) -> ~30,000 cases in the US and 23,000 in Canada. Europe: it occurs in 1 in 2,500 live birth and 1 in 25 are carriers. Caused by more than 500 different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Individuals with CF are highly susceptible to bacterial infection and antibiotic treatment often results in resistant strains. Symptoms: -> small ducts (special channels) become clogged with a thick mucus Clogging and infection of lungs plugging of small bile ducts in liver (impedes digestion) plugging of ducts of pancreas (impedes digestion) obstruction of small intestine males are infertile malfunctioning sweat glands
  • Slide 15
  • 15 Therapeutic Agents Recombinant Proteins Enzymes Treating Cystic Fibrosis Gene responsible for disease: CF is mostly caused by a 3 base pair deletion in Cystic Fibrosis Transmembrane Regulator (CTFR) -> F508 deleted CTFR -> ABC transporter coupled to a channel
  • Slide 16
  • 16 Therapeutic Agents Recombinant Proteins Enzymes Treating Cystic Fibrosis -> It transports Cl- ions after being phosphorylated and binding two ATP molecules -> It has a large regulatory domain that is phosphorylated by a cAMP dependent protein kinase
  • Slide 17
  • 17 Therapeutic Agents Recombinant Proteins Enzymes Treating Cystic Fibrosis A normal lung Chloride into airway; sodium out - keeps mucus moist and thin Normal CFTR regulates the sodium channel (inactivates it) A CF lung Chloride does not get into airway; more sodium leaves; More salt in cell -> water comes in ->This makes the mucus thick
  • Slide 18
  • 18 Therapeutic Agents Recombinant Proteins Enzymes Treating Cystic Fibrosis Treatments: Chloride delivery - activate other chloride carriers Viscous mucus - pounding, DNase treatment, gelosin recurrent infections antibiotics tissue damage due to immune response - anti-inflammatory drugs (ibuprofen)
  • Slide 19
  • 19 Therapeutic Agents Recombinant Proteins Enzymes Treating Cystic Fibrosis Alginate Lyase Alginate is a polysaccharide polymer that is produced by bacteria. The excretion of alginate by Pseudomonas aeruginosa of patients with CF contributes to the viscosity in the lung. The enzyme alginate lyase can liquefy bacteria alginate. Alginate lyase was isolate from Flavobacterium sp. and cloned into E. coli. -> Combined with DNase1, alginate lyse is able to reduce the mucus in the lungs of patients with CF. A thick mucus which is a results of: Alignate produced by bacteria DNA from lysed cells Leucocytes which accumulate due to the infection Makes breathing difficult. Scientist at Genentech isolated the gene for DNase1 The purified enzyme was delivered as an aerosol to the lung where it hydrolysed the DNA into short oligonucleotides. This decrease the viscosity in the lungs and made breathing easier. DNase 1 (GeneTech)
  • Slide 20
  • 20 Therapeutic Agents Recombinant Proteins Monoclonal Antibodies Clinical Applications Transplantation muronomab (OKT3) 1986, basiliximab 1998 Cardiovascular disease abciximab 1994 Cancer rituximab 1997, trastuzumab 1998 Viral infection palivizumab 1998 Inflammatory diseases infliximab 1998, etanercept 1999 Side effects: Transfusion reactions ( any adverse event which occurs because of a blood transfusion) Infections, immunosuppression Cardiac, respiratory arrest ( discontinuation of breathing) Pharmacological toxicity
  • Slide 21
  • 21 Therapeutic Agents Recombinant Proteins Monoclonal Antibodies Production Monoclonal antibodies results from a clone of a B lymphocyte producing a single antibody which will bind to a specific epitope of an a