ABOUT R DNA AND SOME OF ITS NEWER PHARMACEUTICALS

1. NEW PHARMACEUTICAL
PRODUCT DERIVED FROM
BIOTECHNOLOGY
PRESENTED BY
PRIYADARSHINI.R
M.PHARM 1ST SEMESTER
DEP.OF.PHARM.CHEMISTRY
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2. DEFINITION:
RECOMBINANT DNA
Joining together of DNA molecules
from two different species that are
inserted into a host organism to
produce new genetic combinations.
-Recombinant dna is also called
chimeric dna .
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3. 1970- Hamilton smith-isolates the 1st
restriction enzyme,
1972-stanley cohen and Herbert boyer
combine their efforts to create recombinant
DNA.
1978-Stomatostatin,which regulates human
growth hormones,is the first human protein
made using recombinant technology.
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4. BASIC STEPS:
Isolation of the gene of interest.
Preparation of vector DNA and
DNA to be cloned.
Insertion of the gene to the
vector and ligation.
Introduction of the vector DNA to
the appropriate host cell.
Amplification of the recombinant
DNA molecule in the host cell.
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5. 5

6. Tools for Recombinant DNA
Technology:
(A) Enzymes:
a. Restriction Endonucleases
b. Exonucleases
c. DNA ligases
d. DNA polymerase
(B) Cloning Vector
(C) Host organism
(D) DNA insert or foreign DNA
(E) Linker and adaptor sequences.
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7. ENZYMES USED IN RDNA
TECHNOLOGY
DNA ligase Binds to dna molecule
Restriction endonucleases Cleaves DNA at specific sites
Reverse transcriptase Make DNA copy of RNA
molecule.
Exonuclease from Removes nucleotide residues
the 3’ ends.
Bacteriophage exonuclease Removes nucleotide from the
5’ ends.
Alkaline phosphatase Removes terminal phosphate.
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8. RESTRICTION ENZYME
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9. EXAMPLES
Name Source Recognition
site
Cut pattern
EcoRI Escherichia coli 5′−GAATTC−3′3′−CTTA
AG−3′
5′−G↓AATTC−3′3′−
CTTAA↑G−5′
BamHI Bacillus
amyloliquefaciens
5′−GGATCC−3′3′−CCTA
GG−3′
5′−G↓GATCC−3′3′−
CCTAG↑G−5′
HindIII Haemophilus
influenza
5′−AAGCTT−33′−TTCG
AA−5′
5′−A↓AGCTT−33′−T
TCGA↑A−5′
SmaI Serratia
marcescens
5′−CCCGGG−33′−GGG
CCC−5′
5′−CCC↓GGG−33′−
GGG↑CCC−5′
SacI Streptomyces
achromogenes
5′−GAGCTC−33′−CTCG
AG−5′
5′−GAGCT↓C−33′−
C↑TCGAG−5′
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11. STICKY ENDS BLUNT ENDS
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12. DNA ligase
“STICKY ENDS” Stick
together,but gap remains.
Ligase seals the gaps.
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13. PHARMACEUTICAL PRODUCTS:
SOME OF THE APPROVED RDNA PRODUCTS:
R DNA PRODUCT TRADE NAME APPLICATIONS /USES
Insulin Humulin Diabetes
Growth hormone Protropin/Humatrope Pituitary dwarfism
Interferon Intron A Hairy cell leukemia
Hepatitis B vaccine Recombinax HB Hepatitis B
Tissue
plasminogen
activator
Activase
Myocardial infarction
Factor vIII Kogenate/Recombinate Hemophilia
Dnase Pulmozyme Cystic fibrosis
Erythropoietin Epogen/rocrit
Severe anemia with
kidney damage
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14. TYPES:
The pharmaceutical products of rDNA
technology are broadly divided into following
three types,
1. Human protein replacements eg.insulin
2. Therapeutic agents for human diseases
3. Vaccines eg.hepatitis B etc.,
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15. INSULIN :
Insulin consists of two polypeptide chain,
Chain A-21 amino acids long
Chain B -30 amino acids long
Both chains are joined together by disulphide
bond between two cysteine residue.
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16. FORMS OF INSULIN:
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 Humulin R (Regular insulin human
injection,Rdna origin)
 Humulin N (NPH Human insulin rDNA
origin isophane suspension)
 Humulin 70/30 (70% human insulin
isophane suspension;30% human insulin
injection rDNA origin)

17. PRODUCTION OF INSULIN
For insulin production, two methods are
there,
1.Chains are encoded by separate genes in
plasmids inserted into bacteria.
2.Proinsulin production.
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18. Technique for recombinant insulin
production:
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20. 20

21. PURIFICATION PROCESS:
 Only chromatographic purification
methods are generally used.
 A combination of two to four different
chromatographic techniques is generally
employed.
 Gel-filtration and ion-exchange chro-
matography are the most common
methods.
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23. ACTIVASE
 Produced by the Genentech Inc.
 It is a tissue plasminogen activator (t-PA) and is produced on a
global scale by recombinant DNA technology.
 The (t-PA) is glycoprotein- 527 amino acids.
 The methods uses cDNA corresponding to the natural, human t-
PA derived from melanoma cells.
PREPARATION OF ACTIVASE BY r DNA:
 By introducing the mammalian cell line CHO cells (chinese
hamster ovary cells) into the c DNA for alteplase has been
genetically inserted.
 Fermentation is carried out in a nutrient medium containing the
antibiotics gentamicin,100 mg/L.
 During the breeding, alteplase is excreted into the medium.
 For leveling the pH a phosphoric acid or a sodium hydroxide is
added,-freeze-drying -sterile white powder.
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24. PRODUCTION OF TISSUE PLASMINOGEN
ACTIVATOR:
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25. DARBEPOETIN ALFA
 Synthetic form of erythropoietin.
 Marketed by Amgen under the trade name Aranesp.
 Aranesp (darbepoetin alfa) is an erythropoiesis stimulating
protein that is produced in CHO cells by recombinant
DNA technology.
 Aranesp is a 165-amino acid
 Recombinant human erythropoietin- 5 N-linked
oligosaccharide chains,
 Recombinant human erythropoietin contains 3 chains.
 The 2 additional N-glycosylation sites result from amino
acid substitutions in the erythropoietin peptide backbone.
 The molecular - 37,000 D- given intravenously or
subcutaneously.
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26. LIST OF SOME OTHER NEWER
DRUGS BY R-DNA TECHNOLOGY:
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DRUGS APPLICATIONS
EPOGEN/PROCIT For patients with anemia due to
dialysis/renal failure
NEULASTA For neutropenia
INFERGEN For patients with chronic or relapsing
hepatitis c viral (HCV) infection.
AVONEX Treatment of relapsing forms of ms.
BETASERON Significantly delays the progression of
secondary MS.
FORTEO Treatment of osteoporosisin womwn and
men.
INTRON A Treat different types of leukemia.

27. DRUGS IN CLINICAL
TRIALS:
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28. Advantages of recombinant product:
1.Substantial quantity.
2.No need for natural or organic factors.
3.Cheap.
4.Resistance to natural inhibitors.
Disadvantage :
1.Commercialized and became big source of income for
buissness.
2.Effects on natural immune system of the body.
3.Can destroy natural ecosystem that relies on organic
cycle.
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29. CONCLUSION
 Rdna technology is the new era in the field of biology
which has huge applications and possibilities.
 Currently, more than 30 urologic drugs are in clinical
trials, including 12 small molecule drugs (SMDs), 7
vaccines, and 5 protein drugs.
 RecDNA technology is an important development in
science .
 In recent years, it has advanced strategies for
biomedical applications such as cancer treatment,
genetic diseases, diabetes, and several plants disorders
especially viral and fungal resistance.
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30. REFERENCES:
1. Agnieszka Stryjewska1, Katarzyna
Kiepura1, Tadeusz Librowski2, Stanis³aw
Lochyñski3Biotechnology and genetic
engineering in the new drug development.
Part I. DNA technology and recombinant
proteins,2013,65,1075-1085.
2.Biotechnology by purohit and mathur,agro-
bios,13th edition.
3. Burger’s medicinal chemistry.
4. wikipedia.
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