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Pharmaceutical substances : Medicinal therapy Traditional pharmaceutical sectors:
- Chemical-based drugs : chemical synthesis (Table 1.1) - Extraction or isolation from biological sources (Table 1.2)
-Biopharmaceuticals : a class of therapeutic proteins produced by
modern biotechnological techniques, like recombinant DNA, protein engineering, and hybridoma technologies etc. (used in the 1980s) - Nucleic acids used for gene therapy and antisense technology - Proteins for in vivo diagnostics
- A Protein or nucleic acid-based pharmaceutical substances used for
therapeutic or diagnostic purpose, which is produced by modern biotechnological techniques
Form the backbone of medicinal agents in the modern biotech era
History of the pharmaceutical industry
- Before 20th century ; naturally occurring substances : Medicinal herbs Digitalis to stimulate heart muscle Quinine for malaria Pecacuanha for dysentery Mercury to treat syphilis - Chemical synthesis of artificial dyes proved to be therapeutically useful foundation of Bayer and Hoechst - Synthesis of aspirin in 1895 by Bayer - 1930s ; beginning of pharmaceutical industry - landmark discovery and synthesis of sulpha drugs against bacterial infections from the red dye, Prontosil rubrum (Fig. 1.1) - large-scale industrial production of insulin - 1940s : industrial-scale production of penicillin boosting the development of pharmaceutical industry
cf) 1992 : Taxol approved by FDA for ovarian, lung, breast cancer
Taxol from the bark of the Pacific yew tree by Bristol-Myers-Squibb(BMS)
Glaxo. Eli Lilly.000 pharmaceutical companies . Wellcome.Ciba Geigy.000 substances are used routinely in medicine .500 potential drugs under clinical trials .$ 200 billion market .10.5. Foundation of the leading pharmaceutical companies . Roche Current pharmaceutical industry .
Commercialized by Bristol-Myers-Squibb (BMS) -.Used by American Indian for treatment of inflammation Paclitaxel .Story of Taxol -Discovery in 1967 by NCI from the bark of the Pacific Yew tree (Taxus brevifolia) .
000 trees annually 1989 : Cooperative Research & Development Agreement Practical and financial supports from a company 1989 : BMS selected as the partner : Investment of $100 million .200 kg of bark 1979 : Mechanism of action in leukamic mice inhibition of miosis by stabilization of microtubules 1984 : Phase 1 clinical trial problem of supply All the ovarian cancer and melanoma cases in the US : destruction of 360. 1955 : Plant screening plan by NCI to discover new anticancer agents screening of 35.000 plants 1967 : Identification of cytotoxic ingredient from the bark of Pacific yew tree 1969 : 10 g pure compounds from 1.
1992 : FDA approval Five years exclusive marketing rights to BMS : No patent Trade name : TAXOL The name taxol had been used for more than two decades Change of trade name : Paclitaxel Effective for cancers such as ovarian.Annual sales : $ 2 billion 1993 : production of taxol by fungus living in the yew tree Possibility of using Fungus More cost effective process . and lung cancers Currently produced by plant cell culture technology developed by Phyton Biotech. Inc The use of Taxus cell line in a large fermentation tank .. breast.
Inhibiting the formation of peptidoglycan cross-links in the bacterial cell wall : Inhibition of DD-transpeptidase Penicillin G (Benzyl penicillin) R = benzyl group .History of Penicillin .Modern era of antibiotic discovery .
Biosynthesis of Penicillin G in Fungus .
Staphylococcus aureus by growing it on the surface of nutrient at St.Recovery of a tiny quantity of secreted material using the crude extraction methods .The discovery laid essentially dormant for over a decade World War II resurrected the discovery : desperate demand an antibiotic with minimal side effects and broad applicability Howard Florey and Ernst Chain of Oxford : rebuilt on Fleming¶s observation .Secreted material : powerful antimicrobial activity and named ³penicillin´ . : Meaningful finding. not a failed experiment .Discovery and development Alexander Fleming was trying to isolate the bacterium.He thought the cell killing must be due to an antibacterial agent .Identification of foreign particles as common mold of the Penicillium genus (later identified as Penicillium notatum) .He noticed that no bacteria grew near the invading substance in the contaminated plate. Mary¶s Hospital in 1928 : Breakthrough in antibiotic history .
and life scientists . microbiologists.. Pfizer.Approached pharmaceutical companies in the USA like Merck.The supply of penicillin was exhausted .L.Need a process to make large amounts of penicillin .Process development required engineers.They produced enough penicillin to treat some laboratory animals Treat of a London bobby for a blood infection great efficacy against infection . Elder to coordinate the activities of penicillin producers to greatly increase the supply of penicillin in 1943 the commercial production of penicillin by a fermentation process . and to develop the capacity to produce penicillin .Great Britain¶s industrial facilities were totally devoted to the war . Squibb.First attempt : chemical synthesis of penicillin because of a great deal of success with other drugs Chemical synthesis : proved to be extremely difficult Fermentation process : an unproved approach The War Production Board appointed A.
In 1939.001 g/L . the final concentration of penicillin in broth : ~0.fragile and unstable penicillin constraints on recovery and purification methods .Low rate of production per unit volume very large and inefficient fermentors . Problems : very low concentration (titer) of penicillin .very difficult in product recovery and purification .
Mold physiology : productivity vs conditions .growth of the mold on top of a liquid medium . mechanical sealing.First plant for commercial production by Pfizer (100. requires many milk bottles Bottle plant long growing cycle and labor intensive For the required amount. mass transfer ).The other hurdle : Manufacturing process .Reactor design : reactor size and configuration. .000 gal scale) in 1945 .growth of the mold on the surface of moist bran . heat removal. oxygen supply (viscosity. mixing. a row stretching of bottles from NY to SF .Development of a corn steep liquor-lactose based medium ten-fold increased productivity .Nobel prize in 1945 for three scientists . decontamination.Product recovery/purification : pH shift and liquid-liquid extraction . agitator design.Submerged fermentation process : Challegnes .Isolation of a new strain (> few hundreds) : Penicillium chrysogenum . Major contribution to the penicillin program by NRRL .
Directed molecular evolution .in silico design .001. reaching currently ~ 100 g/L Production of penicillin derivatives with greater potency: Antibiotic resistance . metabolic pathway engineering. reactor design: Starting with 0.Protein engineering to design relevant enzymes: More economically feasible process Full enzymatic process -Structure-based rational design . Accomplishment required a high level of multidisciplinary work Ex) Merck assigned a engineer and microbiologist together to each aspect of the problem Continued progress with penicillin fermentation through physiology. process control.Semi-synthetic antibiotics . mold genetics.
III) New antibiotics with greater potency .Penicillin F Penicillin G Enzymatic process Protein Engineering Penicillin nucleus (6-APA) Derivatives (rational design) Animal test Clinical trials (Phase I. II.
Amoxicillin Ampicillin Flucloxacillin Methicillin Carbenicillin Dicloxacillin .
Lessons from the penicillin story Penicillin process established a paradigm for bioprocess development and biochemical engineering The development of biological process requires a high level of multidisciplinary work .
Flavonoids.10 . corticosteriods.Protein-based drugs are produced by recombinant DNA technology . and xanthines . steroids. salicylates.Chemical families of plant-derived medicines : alkaloids. coumarines.Direct chemical synthesis . quinines. prostaglandin are produced synthetically Pharmaceutical substances of plant origin : Table 1. terpens.Directly extracted .Traditional Pharmaceuticals of Biological Origins Pharmaceuticals of animal origin : Table 1.Non-proteinous pharmaceuticals like steroid hormones.an estimated 3 billion people worldwide continue to use traditional plant medicines as their primary form of healthcare .Chemical modification of plant ³ lead drug´ . terpenoids.16 .
Antibiotics : the greatest positive impact on human healthcare low molecular mass microbial secondary metabolite . 1.Inhibits the synthesis of peptidoglycan .More than 100 antibiotics available on the market .Produce a wide variety of secondary metabolites with potential therapeutic applications .17) -Lactams (Fig.000 antibiotic substances isolated and characterized .Major families of antibiotics (Table 1.Characteristic -Lactam core ring structure (Fig.about 10.1.14) : penicillins and cephalosporins . Pharmaceutical substances of microbial origin .14) .Semi-synthetic derivatives from 6-APA and 7-ADCA enzymatic removal of a natural penicillin side chain followed by addition of novel side chains .
.inhibit protein synthesis by binding to 30S and 50S ribosomal subunits .negative and Gram-positive .16) . .cyclic amino alcohol to which amino sugars are attached .inhibit protein synthesis.Derivatives ( Table 1.widespread use due to broad spectrum against Gram.Tetracyclines : characteristic 4-fused-core ring (Fig.19) .exclusively produced by the genus Streptomyces and Micromonospora .1.Aminoglysides : (Table 1.18) .
a core aromatic ring structure .Macrolides: ..core ring structure containing 12 or more carbon atoms .predominantly produced by Streptomyces . effective against Gram-positive and mycobacterium ( Mycobacterium tuberculosis) .produced by Actinomycetals .Ansamycins : .rifamycine .
Widespread medical application was limited due to the tiny quantities and consequently high cost The advent of recombinant DNA technology and biotechnology Mass production and cheap supply Advances in biosciences : understanding of the molecular mechanisms underlying diseases development of new therapeutic proteins with greater efficacy Era of biopharmaceuticals . EPO. insulin etc. interleukins.The age of Biopharmaceuticals Biomedical research since the 1950s : Naturally occurring proteins having therapeutic efficacy: interferons.
and Urokinase .Overcomes problems of product safety : ex) transmission of blood-borne pathogens like hepatitis B and HIV via infected blood products transmission of Creutzfeldt-Jacob disease to persons from receiving human growth hormone preparation from human pituitaries .5) .Overcomes problem of source availability .Facilitates the generation of engineered therapeutic proteins displaying some clinical advantages over the native ones (Table 1. hCG.Provides an alternative to direct extraction from inappropriate sources ex) Purification from urine : Fertility hormone (FSH). Recombinant DNA technology : Four impacts on the production of therapeutic proteins .
Examples of engineered therapeutic proteins by protein engineering technology EPO with longer plasma half-life by incorporation of N-glycosylation Faster-acting insulin by modification of amino acid sequence Slow-acting insulin Faster-acting tissue plasminogen activator(t-PA) by removal of three of the five native domains higher clot-degrading activity Ontak : a fusion protein consisting of the diphtheria toxin linked to IL2 selectively kill cells expressing an IL-2 receptor approved for the treatment of cutaneous T cell lymphoma in 1999 in the US .
Big company : slow to invest in biotech R &D ex) Genentech and Eli Lilly .Merger of biotech capability with pharmaceutical experience > Biotech sector Development of new biopharmaceuticals .High cost : ~ $ 200-500 million . but lack of experience in drug development process and marketing .High Risk (low success rate ) but High Return .Development of recombinant human insulin .High return ~ $ 1-2 billion per single drug for 20 years Major biopharmaceutical companies : Table 1.Clinical trials and marketing by Eli Lilly (Humulin) .Long term : 6 ~ 10 years .6 .Start-up company : Significant technical expertise.Impact of DNA technology on the bio-industries Establishment of start-up biotech companies in 1980s Strategic alliance : Start-up and pharmaceutical companies .
. EPO.Biopharmaceuticals : Current status and future prospects Initial biopharmaceuticals : simple replacement proteins Large-scale production by using recombinant DNA technology The vast majority of recombinant proteins are produced in E coli.) has now come/is coming to an end. interferon. cerevisiae or in animal cell lines (Chinese hamster ovary (CHO) or baby chamster kidney(BHK) cell lines). granulocyte colony stimulating factor etc.Most of these drugs have an annual market value in excess of $ 1 billion. insulin. The use of modern biotechnology such as protein engineering in conjunction with an increased understanding of structure-function relations of protein has facilitated the development of more potent therapeutics Patent protection for many first-generation biopharmaceuticals (human growth hormone.Market for generic drugs : $ 75 Billion in 2015 . S. ..
Alteration of a single amino acid residues at specific site .Alteration or deletion of an entire domain .Protein Engineering .Generation of a novel fusion protein Ex) Site-directed mutagenesis .Insertion or deletion of a single amino acid residue .
Need further improvement : Substrate specificity Binding affinity Stability Catalytic activity Folding/Expression level etc..Protein/Enzyme : Evolved for host itself. . not for human Most proficient catalysts with high specificity .Why Protein Engineering .Goal in protein engineering :Design of protein/enzyme with desired function and property for practical applications Designer proteins/Enzymes Therapeutic proteins Industrial enzymes .
immuno-genecity . MW 36 kDa) produced in kidney : promote the formation of red blood cells(erythrocytes) in the bone marrow . solubility.Carbohydrate content : ~ 40 % .Erythropoietin (EPO) Glycoprotein : growth factor (166 amino acids.About 50 % of EPO¶ secondary structure : a-Helix .Carbohydrate moiety : in vivo activity.Three N-glycosylation sites and one O-glycosylation site . cellular processing and secretion. stability.
protease resistance.About 60 % of therapeutic proteins are glycoprotein .Carbohydrates on cell surface : Cell signaling. and pharmacokinetics . cell attachment.EPO . solubility. recognition.Protein Glycosylation : Glycobiology . immunogenicity. cell adhesion.Therapeutic proteins : 140 approved .Essential roles in in vivo : Biological activity. folding. and inflammation .One of the most important post-translational modifications (PTMs) : N-glycosylation / O-glycosylation in Mammalian / Yeast . signal transduction.
Glycan Profile of Glycoprotein Man Man GlcNAc Gal NANA Man ASN GlcNAc GlcNAc Man Man GlcNAc Gal NANA ASN GlcNAc GlcNAc Man Man Man Man Man Man Human (in Golgi) P. depending on cell type and production conditions : Glycan moiety. length of glycosylation chain .Glycan profile is very complex and varies broadly. its role / function in vivo. occupation number. glycosylation pathway.pastoris (in Golgi) Man Man Man Man Man Man .Therapeutic proteins require proper glycosylation for efficacy Analysis of glycan profile. and property of glycoproteins Glycobiology .
GalNAc C8O6NH15 N-Acetylglucosamine. NAcNeuA C11O9NH19 N-Acetylgalactosamine. GlcNAc C8O6NH15 . Man C6O6H12 Sialic Acid. Gal C6O6H12 Mannose.Monosaccharide Structures Fucose. Fuc C6O5H12 Galactose.
N-Linked Glycan Structures .
Glycosylation pattern of EPO .
5 billion) New version of EPO by protein engineering (Amgen) . First purified from the plasma of anaemic sheep in 1971 Produced by recombinant DNA technology in 1985 Approved by FDA for treatment of anemia resulting from chronic kidney disease and cancer treatment (chemotherapy and radiation) in 1989 Total sales : $ 11 billion (2005) Major brand EPO : generic form .Epogen by Amgen ($ 2.Neorecormon by Boehringer-Mannheim ($ 1.5 billion) .Aranesp : introduction of two additional N-glycosylation sites (where?) a prolonged serum half-life from 4-6 up to 21 hours What benefit? Launched in 2001 Current sale : $ 3.5 billion) .Procrit by Ortho Biotech ($ 3.5 billion .
HW # 1 : Due February 23 1. 2. How to incorporate two additional N-glycosylation sites on EPO? . What curiosity (or question) do you have about the protein glycosylation ? Describe your curiosity (or question) and answer to that.
.Competition among manufacturers .Production at a much lower cost . since these trials have already been conducted by the brand name company . intended use Generics also go through a rigorous scientific review to ensure both safety and efficacy Benefit to consumers and insurance companies : Lower price .Generic drug Produced and distributed without patent protection Bioequivalent to the brand name counterpart with respect to pharmacokinetics and pharmacodynamics Identical in safety. strength. efficacy.Generic manufacturers : no burden of proving the safety and efficacy of the drug through clinical trials. dose.Only need to prove that their preparation is bioequivalent to the original drug to gain regulatory approval. route of administration.
distribution. excretion . metabolism. the mechanisms of drug action. Pharmacodynamics : the study of the biochemical and physiological effects of drugs on the body. and relationship between drug concentration and effect Pharmacokinetics : the fate of substances administered externally to a living organism including absorption.
bioequivalence. Approval and regulation . Brand-name drug companies : a number of strategies to extend the period of market exclusivity on their drug. as some differences exist An applicant files an Abbreviated New Drug Application (ANDA) with demonstration of therapeutic equivalence to a previously approved drug FDA launched the Generic Initiative for Value and Efficiency in 2007 to increase the number and variety of generic drug products available.bioequivalence to the original drug . however. and prevent generic competition : ever-greening ex) EPO . does not mean that generic drugs are exactly the same as their original counterparts.
Small-molecule drugs : generic form can be marketed if their therapeutic equivalence to the original drug is proved pharmaceutical equivalence ( identical active substance) and bioequivalence ( comparable pharmacokinetics) no clinical efficacy and safety test .Bio-similar (Bio-generics) ? .Therapeutic proteins : the generic approach can not be applied to copies of therapeutic proteins because of complexity impossible to prove two protein products to be identical comprehensive clinical data : clinical equivalence approval by regulatory authority marketing .
high throughput screening will have a great impact on the development of bio-drugs (therapeutic proteins) These technologies will identify new drug target and facilitate the bio-drug development .Future prospects in biotech industry Technology development in areas like genomics. proteomics.
Narrow spectrum penicillins benzathine penicillin benzylpenicillin (penicillin G) phenoxymethylpenicillin (penicillin V) procaine penicillin Narrow spectrum penicillinase-resistant penicillins methicillin dicloxacillin Flucloxacillin Narrow spectrum -lactamase-resistant penicillins Temocillin Moderate spectrum penicillins amoxicillin Ampicillin Broad spectrum penicillins co-amoxiclav (amoxicillin+clavulanic acid) Extended Spectrum Penicillins azlocillin carbenicillin ticarcillin mezlocillin piperacillin .
Beta-lactam antibiotics .