Study of Drugs Produced by Biotechnology Insulin, Activase, Somatotropin, Hyaluronidase, Penicillinase, Streptokinases, Amylases, Proteases

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.

The pharmaceutical industry produces a wide range of products using recombinant DNA techniques.
Human proteins can be cloned : Gene recovered from human genome (as genomic DNA or cDNA) Placed in an expression vector using ligation techniques and restriction endonucleases. Transform bacteria (e.g. Escherichia coli or Bacillus spp.) Grow in batch culture in large industrial scale. Purify protein product Human proteins produce fewer side effects than proteins from other animals (e.g. pork insulin vs. human insulin)

Insulin
Made by pancreatic beta cells Enables cells to take up glucose from the bloodstream to use in production of ATP Insufficient insulin causes diabetes (insulin dependent diabetes mellitus IDDM)

Must inject insulin to avoid physiological complications Cells cannot take up glucose Insufficient ATP is made Glucose spills into urine (excreted by kidneys; kidney tries to dilute glucose by excreting large amounts of water) Insulin is made up of 2 chains = 51 amino acids total A chain = 21 amino acids . B chain = 30 amino acids

S - S __l____________l____ A S S

___S___________S____________________ B Two disulfide bonds hold A and B together (interchain disulfide bond). One disulfide bond within the A chian (intrachai disulfide bond) Insulin processing Preproinsulin Contains a signal sequence + 3 sections of amino acids Signal sequence is removed after targeting to RER Translation continues on RER > forming proinsulin Removal of 33 amino acids at Golgi Apparatus, and joining of A and B chains to form insulin Preproinsulin too difficult for E. coli to produce Before recombinant insulin was available, insulin was obtained from cows or pigs pancreases (7-10 lb pancreatic tissue per patient per year) Cow (Bovine) = 3 amino acid differences Pig (Porcine) = 1 amino acid difference Amino acid differences can stimulate allergic responses Therefore human insulin is preferred

Humulin Production:
Recombinant DNA used to produce human insulin Ever since Banting and Best isolated a protein they called "isletin" from the Islets of Langerhans from a pancreas and injected it into diabetic dogs, insulin has been used to help diabetics live out their lives. However, this protein had its problems. For many years, insulin was extracted and purified from either porcine or bovine pancreases, and this carried with it two main difficulties. The first was that though the animal insulin was chemically similar to human insulin, there were some differences, and these differences led to antibody attack and inactivation as well as inflammation in many patients. Also, there was the problem that this method of extracting insulin from animal organs made it difficult to obtain large amounts of pure insulin. Nowadays, it is a well known fact that DNA contains genes which encode all the different proteins that an organism can produce. But back then, the composition of proteins and their relation to DNA was unknown. It wasn't until Sanger, in 1955, determined the sequence of insulin and determined that proteins are composed of specific amino acids linked to each other in a peptide chain. And even then, it wasn't until 1979 that human insulin was able to be produced in large quantities. This was done by the use of recombinant DNA.

Recombinant DNA, or rDNA, is DNA which specifically encodes a protein.

This is cut from genomic DNA by a restriction enzyme which cuts DNA at specific sequences along the chain. These pieces are then analyzed and the DNA needed to make the protein is extracted and purified. Since insulin contains two polypeptide chains linked by disulfide bonds, two pieces of DNA are extracted. These DNA strands are then placed into two different plasmids, as shown in the figure below

These plasmids are also cut with the same restriction enzymes as the DNA,which will allow the DNA to be fixed into the plasmid by DNA ligase. The plasmids are then incubated with a weakened strain of E. coli. Since only some of the bacteria will take up the plasmid,a gene encoding an enzyme which breaks down a certain antibiotic is also included in the plasmid, which allows bacteria with the plasmid to grow on a plate containing the antibiotic while the other bacteria die. These bacteria are then allowed to grow and replicate, which allows the plasmid and the insulin gene to replicate millions of times. Then the bacteria are given a signal to produce the protein, and insulin identical to that of humans can be produced and purified. This insulin, which is abtly named humulin, can then be used to treat many people with type I diabetes without the worry of allergic reaction.

Human Growth Hormone (HGH)

New method Gene production for HGH synthesis Protropin Genentech Humatrope Eli Lilly HGH promotes overall body growth by increasing: amino acid uptake by cells, protein synthesis and fat utilization for energy Dwarfism caused by insufficient production of HGH by the pituitary gland Growth retardation Chubby face Baby fat around waist Unusual body properties as an adult ~ 4 feet tall only IQ = Normal HGH can treat dwarfism to help undersized children reach their normal height and size

Old method:
Purification of HGH from cadaver pituitary glands 8 cadavers/year for 8 10 years per patient Risky to use brain tissue Prion disease transmission: Creutzfeldt-Jacob Disease (CJD) Muscle wasting Convulsions Tremors Dementia 24 cases reported by 1993 in France from cadaver HGH

Recombinant DNA technology is widely used in the production of therapeutic agents such as hormones, cytokines, growth factors, antibiotics, vaccines, blood products like albumin, thrombolytics, fibrynolytics, clotting factors such as factor VII, factor IX, tissue plasminogen activator and many more. All these therapeutic agents can be produced in a large quantity and also more economically by using recombinant DNA technology.

Growth hormones are produced in the pitutary gland, and are involved in the overall growth of the body by increasing the production of protein, thus increase the amino acid uptake by the body cells. Growth hormone also increases the use of fat as body fuel.

Recombinant human growth hormone Somatotropin Production:

Recombinant human growth hormone is generally produced by inserting the human growth hormone gene into plasmids of E.Coli bacteria. Recombinant bacterial cells are cultured and human growth hormones produced by these bacteria are extracted from the extracellular media.

Human growth hormones are also produced and extracted using animal cell culture are now a days are used in treating patients with renal carcinoma and also to treat children who are suffering from human growth deficiency New Approach: 1. To resolve the interruption of signal peptides in the production of human growth hormone, the base sequence coding for signal peptides of around 26 amino acids along with neighbouring 24 amino acids is cut in the coding cDNA using restriction enzyme EcoR1. 2. Deleted 24 amino acid sequence of human growth hormone cDNA is freshly prepared and ligated to the remaining human growth hormone cDNA. 3. This coding cDNA is then integrated into the plasmid, then inserted into E.Coli bacterial cell.

4. These recombinant bacterial cells are cultured, and then human growth hormones are extracted from the media. Production of Recombinant Human Growth Hormone in Chinese Hamster Ovary (CHO) Cells:

This approach has got some advantages such as recombinant human growth hormone is secreted directly into the protein free media. Hence hormone can be extracted and purified using simple methods, and this also avoids resolubilization of inclusion bodies and refolding of proteins.

Extraction of Somatotropin:
1. Somatotropin is a growth hormone contains 191 amino acids and produced by the anterior pitutary or in other words front section of the pitutary gland. This hormone is activated by another hormone produced in the liver known as somatomedin. Thereby somatotropin causes growth.

2. In the case of human growth hormone somatotropin, the protein is sequestered in refractile bodies within the cytoplasm of the body cells.

3. Refractile bodies can be recovered from the cell culture by disrupting the cell to release the refractile bodies. These refractile bodies are then collected as pellet by using differential centrifugation method. 4. These refractile bodies are solubilized using suitable chotropic agents, such as urea or guanidine hydrochloride at pH 9-12 or alkaline pH.

5. Solubilized proteins are then treated with oxidizing agent, and this forms intramolecular disulfide bondsand protein is refolded to form its biologically active structure.

6. Biologically active somatotropins are used to enhance the growth of animals, and also animal productivity by administering these hormones by subcutaneous injection or intramuscular injection. They can also be admistered as implants.

Uses:
Somatotropin is also used to treat patients with Turner's syndrome and also in chronic renal insufficiency. Bovine somatotropin is used effectively to increase milk production in lactating cow. Porcine somatotropin is used effectively to improve feed efficiency and to increase meat content, by administering it to finishing hogs. Some of the examples of recombinant human growth hormone are Protropin produced by Genetech Company Humatrope produced or manufactured by Eli Lilly Company

Tissue Plasminogen Activator (TPA)

Activase 1. A thrombolytic agent which works by activating the bodys own fibrinolytic system by activating the production of plasmin from plasminogen. Plasmin is an enzyme which degrades fibrin clots and fibrinogen, as well as several other protein clotting factors. FDA approves Activase Genetech 1987 Clot-Dissolving Agent Fewer side effects than streptokinase and urokinase TPA is used to treat coronary thrombosis (thrombolytic agent) Protease that attaches to blood clots and induces other blood components to break down clot ** Plasminogen Fibrin Plasmin ---------- Degradation of fibrin

Streptokinase is an extra cellular protein, extracted from certain strains of beta hemolytic streptococcus. It is a non-protease plasminogen activator that activates plasminogen to plasmin, the enzyme that degrades fibrin cloth through its specific lysine binding site; it is used therefore as a drug in thrombolytic therapy (Mohammad et al., 2009).

Streptokinase is currently used in clinical medicine as a therapeutic agent in the treatment of thromboembolic blockages, including coronary thrombosis (Banerjee et al., 2004; Endrogan et al., 2006). Streptokinase (SK) a group of extracellular proteins produced by a variety of streptococci betahemolytic strains, and is a plasminogen activator composed of 414 amino acids with a molecular mass of 47 kDa. Unlike urokinase or tissue-type plasminogen activator that performs direct proteolysis, SK forms a high affinity equimolar complex with a plasminogen (Kim et al., 2000).

-haemolytic streptococci was grown on medium containing corn steep liquor 8% and 12% serelose, 7% KH2 PO4, 0.33% K2HPO4 , 0.2% cysteine, 0.01% Glycine 0.01% tryptone, 0.01% Uracil, 0.001% adenine sulfate, 0.001% nicotinic acid, 0.001% pyridoxine- HCl, 0.0018% calcium phosphate, 0.005% thiamine-HCl, 0.002% riboflavin, 0.001% and salt mixture 2 g/lit. The pH was adjusted to 7.0 with 1 M HCl and 1M NaOH. Medium was sterilized and cooled at room temperature. One ml of culture was used as inoculum; incubated at 37C and 170 rpm in orbital shaker. After 75 h of fermentation, cells were removed by centrifugation. Streptokinase once used for this purpose Derived from Streptococcal bacteria Must be delivered to blood vessel directly Urokinase = alternative, but has risk of hemorrhage TPA does not compromise blood clotting elsewhere therefore reduces risk of internal hemorrhaging Administered IV transported via circulation to affected area Produced in mammalian cell culture (not E.coli)

Early 1980s Plasmid constructed

Shuttle vector had characteristics for maintenance in E. coli and expression in mammalian cells cDNA for TPA TPA signal sequence TPA promoter TPA termination sites Antibiotic resistance + ORI for prokaryotic cells Methotrexate resistance marker for mammalian cells Mammalian cells transfected Stable integration into genome of mammalian host cell High levels of secreted TPA produced in large scale culture conditions