CALCUTTA INSTITUTE OF PHARMACEUTICAL TECHNOLOGY AND ALLIED HEALTH SCIENCES

NEWER DRUGS DEVELOPED BY R-DNA TECHNOLOGY

PREPARED BY-DEEPANJAN DUTTA

GUIDED BY – MANAS CHAKRABORTY

FACTS-DNA and rDNA

RECOMBINANT DNA(r DNA) ROLES OF ENZYMES IN rDNA

HUMULIN(INSULIN) AND ITS MANUFACTURE

ACTIVASE HEPATITIS B VACCINE HUMAN GROWTH HORMONE

LIST OF SOME OTHER DRUGS

ADVANTAGES AND DISADVANTAGES OF rDNA APPLICATIONS OF r DNA

CONTENTS

FACTS- DNA and RDNA

DNA is the keeper of the all the information needed to recreate an organism. All DNA is made up of a base consisting of sugar,Phosphate and nitrogen base.There are four nitrogen bases- adenine (A),thymine (T),guanine (G),andcytosine (C).The nitrogen bases are found in pairs with A & T and G & C paired together. The sequence of the nitrogen bases can be arranged in an infinite ways, and their structure is known as the famous "double helix" as shown in the image.The sugar used in DNA is deoxyribose. THE DNA MOLECULE HAS TWO IMPORTANT PROPERTIES: It can make copies of itself. It can carry information.

RECOMBINANT DNA (r DNA)

Recombinant DNA(rDNA) is a form of artificial DNA that is created by combining two or more sequences.It is made possible by two important enzymes. Restriction enzymes and DNA Ligase are the two principal tools, first used by Paul Berg in 1972,employed to alter DNA

METHODS BY WHICH RECOMBINANT DNA IS MADE ARE:

TRANSFORMATION

TRANSDUCTION

CONJUGATION

TYPE 2 RESTRICTION ENDONUCLEASE: cleaves DNA at a specific base sequenceDNA LIGASE: binds two DNA molecules or fragmentsDNA POLYMERASE 1: fills single-stranded gaps in duplex DNA by stepwise addition of nucleotides to 3’ends ( removes rna primer)REVERSE TRANSCRIPTASE: makes a DNA copy of an rna moleculePOLYNUCLEOTIDE KINASE : adds a phosphate to the 5’-OH end of a polynucleotide, to label it or permit ligation.TERMINAL TRANSFERASE: adds homopolymer tails to the 3’- OH ends of a linear duplexEXONUCLEASE 3: removes nucleotide residues from the 3’ ends of a DNA strand.BACTERIOPHAGE(LAMDA)EXONULEASE: removes nucleotides from the 5’ ends of a duplex to expose 3’ single – stranded ends.ALKALINE PHOSPHATASE: removes terminal phosphates from the 5’ end, the 3’ end , or both.

ROLE OF ENZYMES IN rDNA TECHNOLOGY:

1.HUMULIN(INSULIN) Insulin is one of the smallest protein .The total nunber of amino acid used for synthesis of insulin is 51 in 2 polypeptide chains(Chain A and Chain B).

Manufactured by combining the genes of interest with plasmid, carrying it to the E.coli cells and then by applying the process of cutting and rejoining by specific enzymes and subsequently purifying the product for marketing.

FORMS OF HUMULIN:

Humulin R (Regular insulin human injection ,USP rDna origin)

Humulin N (NPH Human insulin rDna origin isophane suspension)

Humulin70/30 (70% human insulin isophane suspension; 30% human insulin injection-rDna origin)

Plasmid + Achain Plasmid + Bchain

Cleaved with cyanogen bromide (CNBr)

E.Coli lysis purification insulin

PREPARATION OF HUMULIN (INSULIN)

2. ACTIVASE (ALTEPLASE)

is a tissue plasminogen activator produced by recombinant DNA technology.

It is a sterile, purified glycoprotein of 527 amino acids.

It is synthesized using the complementary DNA(cDNA) for natural human tissue-type plasminogen activator obtained from a human melanoma  cell line PREPARATION OF ACTIVASE BY rDNA

The manufacturing process involves the secretion of the enzyme alteplase into the culture medium by an established mammalian cell line (Chinese Hamster Ovary cells) into which the cDNA for alteplase has been genetically inserted.

Fermentation is carried out in a nutrient medium containing the antibiotics gentamicin,100 mg/L.

Hepatitis B vaccine (rDNA) is produced by the expression of the viral gene coding for HBsAg in yeast (Saccharomyces cerevisiae) or

mammalian cells (Chinese hamster ovary (CHO) cells or other suitable cell lines).

3..HEPATITIS B VACCINE(HB)

HEPATITIS B is an infectious inflammatory illness of the liver caused by the hepatitis B virus(HBV). HEPATITIS B VACCINE:

is a sterile solution of Immunoglobin containing antibody to hepatitisB surface antigen.Kept at p H 6.2Formulated in 0.075M NaCl,0.15M glycine,0.01% polysorbate 80.Used by intramuscular route as a vaccine for hepatitis B.

PREPARATION OF HEPATITIS B VACCINE BY rDNA

4..HUMAN GROWTH HORMONE(SOMATOTROPIN/ HUMATOTROPIN) Polypeptide hormone of rdna origin,with 191 amino acids, molecular weight-2-115 dalton.It is a pituitary hormone. Humatroph,a sterilised lauphilised powder sub-cutaneous or i.v. injection. Phosphoric acid or NaOH added to adjust pH upto 7.5 and oxygen sensitive. Recombinant human growth hormone is generally produced by inserting the human growth hormone gene into plasmids of E.coli bacteria. Recombinant bacteria cells are cultured and human growth hormones produced by these bacteria are extracted from the extracellular media. During the production of the human growth hormone in the body it is attached with signal peptides with 26 amino acids. This signal peptide is cleaved in the body before releasing the hGH to do their biological function During the production of recombinant human growth hormone the signal peptides interrupts their production.First cDNA is produced using m RNA encoding for hGH. Then cDNA are integrated into the plasmid and are inserted into the E.coli bacteria,then they are cultured and HGH are extracted along with the signal peptide chains.

PREPARATION OF HGH by rDNA

LIST OF SOME OTHER NEWER DRUGS PREPARED BY RECOMBINANT TECHNOLOGY::

DRUGS APPLICATIONS

Epogen/Procrit For patients with anemia due to Dialysis/ Chronic Kidney Disease / Renal Failure / Chemo / HIV 

Neulasta For Neutropenia: low WBC count febrile neutropenia (low WBC count with fever/ infection) due to chemo, BMT, AML

Infergen For patients with Chronic, non-responding, or relapsing hepatitis C viral (HCV) infection.

Avonex Treatment of relapsing forms of MS. Slows the progression of MS by regulating the body's immune response against myelin. 

Betaseron Multiple Sclerosis: Significantly delays the progression of secondary MS, including relapsing-remitting MS

Forteo Treatment of osteoporosis in women and men

IntronA treat different types of leukemia,malignant melanoma, multiple myeloma,basal cell carcinoma.

DRUGS APPLICATIONS

HepatitisB surface antigen

Vaccination (now in market) 

Interleukin-2 Treatment of cancer

Tissue Plasminogen Activator

Thrombosis

Erythropoetin Treatment of Anemia 

LIST OF SOME OTHER NEWER DRUGS PREPARED BY RECOMBINANT TECHNOLOGY::

ADVANTAGES OF RECOMBINANT TECHNOLOGY:•Provide substantial quantity• No need for natural or organic factors• Unlimited utilizations• Cheap• Resistant to natural inhibitors

DISADVANTAGES OF RECOMBINANT TECHNOLOGY::• Commercialized and became big source of income for businessmen• Effects natural immune system of the body• Can destroy natural ecosystem that relies on organic cycle• Prone to cause mutation that could have harmful effects• Major international concern: manufacturing of biological weapons such as Botulism & anthrax to target humans with specific genotype• Concerns of creating super‐human race

APPLICATIONS OF RECOMBINANT DNA TECHNOLOGY:Insulin for diabeticsFactor VIII for males suffering from haemophilia AFactorIX for haemophilia BHuman growth hormone(HGH)Erythropoietin(EPO) for treating anemiaSeveral types of InterferonsSeveral InterleukinsGranulocyte-macrophage colony-stimulating factor(GSM-CSF) for stimulating the bone marrow after a bone marrow transplantGranulocyte colony stimulating factor(G-CSF) for stimulating neutrophil production(e.g., after chemotherapy) and for mobilizing hematopoetic stem cells from the bone marrow into the blood.Parathyroid hormone Tissue plasminogen activator(TPA)

Many monoclonal antibodiesC1 inhibitor(C1 INH) used to treat hereditary angiodema

 

REFERENCES:

1.PHARMACEUTICAL BIOTECHNOLOGY- FUNDAMENTALS AND APPLICATIONS -S S KORI & M A HALKAI

2.BIOCHEMISTRY- SATYANARAYAN

3.A TEXT BOOK OF BIOTECHNOLOGY – R.C.DUBEY

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