Recombinant Therapeutic Proteins I PDF 1120-111 1966

1120-111 Biochemistry and Biotechnology Fundamentals Recombinant Therapeutic Proteins I 1120-111 Biochemistry and...

1120-111 Biochemistry and Biotechnology Fundamentals Recombinant Therapeutic Proteins I 1120-111 Biochemistry and Biotechnology Fundamentals Recombinant Therapeutic Proteins I  1120-111 Biochemistry and Biotechnology Fundamentals Recombinant Contents Therapeutic Proteins I Cystic Fibrosis rhDNase Type 1 diabetes mellitus Insulin Recombinant Insulin  1120-111 Biochemistry and Biotechnology Fundamentals Recombinant Therapeutic Learning Objectives Proteins I By the end of this lecture, you should be able to: Explain the impact of recombinant therapeutic proteins, specifically in treating conditions such as cystic fibrosis and diabetes mellitus. Describe the mechanisms of action for therapeutic proteins like rhDNase and recombinant insulin, including their interactions with disease pathways. Differentiate between types of recombinant insulin and understand their specific therapeutic applications and benefits for diabetes management. Discuss the genetic factors contributing to diseases such as cystic fibrosis and diabetes mellitus and how recombinant proteins address these genetic challenges.  1120-111 Biochemistry and Biotechnology Recombinant proteins Fundamentals Recombinant Therapeutic Proteins I They are proteins that are artificially produced using genetic engineering techniques. They have played a significant role in biomedical biotechnology, being used in research and as drugs in the treatment of various diseases. This is done through inserting a human gene into the genetic material of a common bacterium. This “recombinant” micro-organism could now produce the protein encoded by the human gene. The first recombinant protein used in treatment was recombinant human insulin in 1982. The recombinant protein industry has rapidly grown. To date, more than 130 recombinant proteins are approved by the US FDA for clinical use.  1120-111 Biochemistry and Cystic Fibrosis Biotechnology Fundamentals Recombinant Therapeutic Proteins I Cystic fibrosis is a genetic condition. It's caused by a faulty gene that affects the movement of salt and water in and out of cells. This, along with recurrent infections, can result in a build-up of thick, sticky mucus in the body's tubes and passageways – particularly the lungs and digestive system.  1120-111 Biochemistry and Cystic Fibrosis Biotechnology Fundamentals Recombinant Therapeutic Proteins I Normally: Gene Cystic Fibrosis Transmembrane conductance Regulator (CFTR) This protein regulates the Cl- permeability in exocrine glands, helps to maintain the balance of salt and water on many surfaces in the body, such as the surface of the lung. Cystic Fibrosis: Mutation in the gene missing phenylalanine Defective CFTR  1120-111 Biochemistry and Biotechnology Fundamentals Recombinant Therapeutic Proteins I In cystic fibrosis: Cl- is not secreted, stays with Na+ in the cell, then H2O follows Na + by osmosis. The secretions will be very thick and viscous. This mostly happens in the lungs and pancreas.  1120-111 Biochemistry and Biotechnology Fundamentals Recombinant Therapeutic Proteins I CFTR genetic mutation at birth leads to either reduced or improperly folded CFTR protein. In the airways, abnormal CFTR results in altered secretions and mucociliary clearance, leading to a cycle of obstruction, chronic bacterial infection, and neutrophil-dominated inflammation. This bacterial infection and neutrophil-dominated airway inflammation begins early in the patient’s life. Poorly regulated neutrophil-dominated inflammation damages the airways over time due to necrosis of neutrophils that leads to the accumulation of extracellular DNA and actin, increasing the viscosity of mucous and creating further obstruction, lung damage, loss of lung function, and ultimately premature death.  1120-111 Complications of Biochemistry and Biotechnology Fundamentals Recombinant Therapeutic Cystic Fibrosis Proteins I Worsening lung function, leading to the inability to do daily activities Lung infections Inflammation of the pancreas Vitamin deficiencies (Fat soluble vitamins) Fat in the stools Thick mucus that clogs certain organs such as the lungs and pancreas. This may cause malnutrition, poor growth, frequent respiratory infections, breathing problems, and ongoing (chronic) lung disease. Gallstones  1120-111 Deoxyribonuclease (Dnase) Biochemistry and Biotechnology Fundamentals Recombinant Therapeutic Proteins I A group of glycoprotein endonucleases which are enzymes that catalyze the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. The role of the DNase enzyme in cells includes breaking down extracellular DNA excreted by apoptosis and neutrophil extracellular necrosis to help reduce inflammatory responses that otherwise are elicited. DNase has been applied as a treatment for diseases that are caused by extracellular DNA as in cystic fibrosis.  1120-111 Recombinant Human Biochemistry and Biotechnology Fundamentals Recombinant Therapeutic DNase Proteins I RhDNase is an enzyme that breaks down DNA strands in airway secretions, hydrolyzes the DNA present in sputum/mucus of CF patients, reducing viscosity in the lungs and promoting secretion clearance.  1120-111 Biochemistry and Biotechnology Fundamentals Recombinant Therapeutic Proteins I In the late 1980s, human deoxyribonuclease I gene was cloned, sequenced and expressed recombinantly using mammalian cell culture in Chinese Hamster Ovary (CHO) cells to reevaluate the potential of DNase I as a therapeutic for cystic fibrosis. In vitro incubation of sputum from CF patients with catalytic concentrations of rhDNase I reduced its viscoelasticity. The reduction in viscoelasticity was directly related to both rhDNase I concentration and reduction in the size of the DNA in the samples. Therefore, reduction of high molecular weight DNA into smaller fragments by treatment with aerosolized rhDNase I was proposed as a mechanism to reduce the mucus viscosity and improve mucus clearability from obstructed airways in patients. It was hoped that improved clearance of the viscous mucus would enhance pulmonary function and reduce recurrent exacerbations of respiratory symptoms requiring parenteral antibiotics. This proved to be the case and rhDNase I was approved by the Food and Drug Administration in 1993. Since that time, the clinical use of rhDNase I has continued to increase with over 67% of CF patients receiving chronic therapy.  1120-111 Biochemistry and Biotechnology Fundamentals Recombinant Therapeutic Proteins I rhDNase I is aerosolized into the airways where it degrades DNA to lower molecular weight fragments, thus reducing CF mucus viscosity and allowing expectoration, which improves lung function and reduces bacterial infections.  1120-111 Biochemistry and Type 1 Biotechnology Fundamentals Recombinant Therapeutic diabetes mellitus Proteins I Type 1 diabetes, once known as juvenile diabetes or insulin-dependent diabetes, is a chronic condition. In this condition, the pancreas makes little or no insulin. Insulin is a hormone the body uses to allow sugar (glucose) to enter cells to produce energy. Different factors, such as genetics and some viruses, may cause type 1 diabetes. Although type 1 diabetes usually appears during childhood or adolescence, it can develop in adults. Even after a lot of research, type 1 diabetes has no cure. Treatment is directed toward managing the amount of sugar in the blood using insulin, diet and lifestyle to prevent complications.  1120-111 Biochemistry and Biotechnology Complications Fundamentals Recombinant Therapeutic Proteins I - Diabetic nephropathy - Diabetic peripheral neuropathy - Eye injury - Diabetic retinopathy - Kidney disease - Cardiomyopathy - Foot damage - Heart and blood vessel disease  1120-111 Biochemistry and Biotechnology Insulin Fundamentals Recombinant Therapeutic Proteins I Insulin is a hormone made by the pancreas that helps glucose in your blood enter cells in your muscle, fat, and liver, where it's used for energy. The human insulin protein is composed of 51 amino acids. It is a heterodimer of an A-chain and a B-chain, which are linked together by disulfide bonds. Insulin's structure varies slightly between species of animals. Porcine insulin is especially close to the human version and was widely used to treat type 1 diabetics before human insulin could be produced in large quantities by recombinant DNA technologies. Insulin was the first peptide hormone discovered. Insulin is also the first protein to be chemically synthesized and produced by DNA recombinant technology. It is on the WHO Model List of Essential Medicines, the most important medications needed in a basic health system.  1120-111 Biochemistry and Biotechnology Fundamentals Recombinant Therapeutic Proteins I  1120-111 Insulin Insulin is produced and stored in the body as a hexamer (a unit of six Biochemistry and Biotechnology Fundamentals Recombinant Therapeutic insulin molecules), while the active form is the monomer. The six molecules Proteins I are linked together as three dimeric units to form symmetrical molecule. An important feature is the presence of zinc atoms (Zn2+) on the axis of symmetry, which are surrounded by three water molecules. The hexamer is an inactive form with long-term stability, which serves to keep the highly reactive insulin protected, yet readily available. The hexamer-monomer conversion is one of the central aspects of insulin formulations for injection. The hexamer is far more stable than the monomer, which is desirable for practical reasons; however, the monomer is a much faster-reacting drug because diffusion rate is inversely related to particle size. A fast-reacting drug means insulin injections do not have to precede mealtimes by hours, which in turn gives people with diabetes more flexibility in their daily schedules.  1120-111 Biochemistry and Biotechnology Fundamentals Insulin Therapy Recombinant Therapeutic Proteins I As a medication, insulin is any pharmaceutical preparation of the protein hormone insulin that is used to treat high blood glucose. It is on the World Health Organization's List of Essential Medicines. In 2021, it was the 179th most commonly prescribed medication in the United States, with more than 2 million prescriptions. Insulin can be made from the pancreas of pigs or cows, but when used for treating diabetic patients, may cause allergic reactions. A protein from other animals may not be accepted by the immune system of the human body and recognize it as non-self which may elicit an immune response in the body. Another challenge was the increasing demand and large-scale production. Human versions can be made by recombinant technology using mainly E. coli.  1120-111 Biochemistry and Recombinant Insulin Biotechnology Fundamentals Recombinant Therapeutic Proteins I Insulin is prepared by recombinant DNA (rDNA) technology for medicinal purposes on a large scale. In October 1982, the U.S. Food and Drug Administration (FDA) approved Eli Lilly and Company's Humulin, the first human insulin for diabetes treatment created using recombinant insulin DNA technology. It was first produced in 1983. The trademark name is Humulin®. Genes, which code for functional A and B peptides of insulin, were inserted in the plasmids of non- pathogenic E.coli strains. Both the chains are produced separately and joined afterwards by disulphide linkages.. In fact, it was the first recombinant medicine to be used in the USA. Biosynthetic insulin produced by rDNA technology is purer than animal insulin. It reduces the formation of antibodies against it.  1120-111 Biochemistry and Biotechnology Insulin Absorption Fundamentals Recombinant Therapeutic Proteins I Upon injection into the SC tissue, insulin monomers and dimers are readily absorbed by blood capillaries. Insulin hexamers, however, are not absorbed into the capillaries due to their larger size. For storage purposes, excipients are usually added to the insulin formulation, and this shifts the equilibrium of insulin oligomers towards the hexamers. These excipients include zinc, which is needed to form insulin hexamers, and phenol and/or phenol-like substances that stabilize the hexamers and act as preservatives. When injected into the SC tissue, lipophilic excipients such as phenol and zinc disperse away from the insulin depot into the adipose tissue, allowing for the subsequent dissociation of insulin hexamers into dimers and monomers before transcapillary transport.  1120-111 Biochemistry and Types of Biotechnology Fundamentals Recombinant Therapeutic Recombinant Insulin Proteins I Rapid-acting Insulin: This is used in rapid-acting insulin preparations. In the SC tissue -in the absence of zinc and phenol- these modifications give rise to a reduced self-association of insulin monomers into dimers compared to human insulin thus yield a larger fraction of insulin monomers in the SC tissue. The result is a more rapid absorption from the SC tissue with faster onset of action, a higher maximum plasma concentration (Cmax), and shorter duration of action. Not only does the rapid absorption profile associated with rapid-acting insulin preparations allow for a more effective correction of incidental hyperglycemia but also the preparations can be injected at a shorter time prior to meal intake, increasing the flexibility of their use.  1120-111 Types of Biochemistry and Biotechnology Fundamentals Recombinant Recombinant Insulin Therapeutic Proteins I Intermediate and long-acting insulin: Intermediate- and long-acting insulin preparations exhibit a delayed absorption profile. The Neutral Protamine Hagedorn (NPH) insulin preparation contains human insulin as the active ingredient. It is associated with intermediate onset of action due to the addition of the basic protein protamine that binds to insulin, forming a precipitate that is slow to dissolve, thereby slowing the absorption and action of insulin after injection. The crystals in the NPH suspension are formed by mixing human insulin, protamine, zinc, and phenolic substances. Whereas soluble insulin diffuses in the SC tissue, insulin crystals will remain near the injection site. Before dissociation of hexamers into dimers and monomers, these crystalline structures need to dissolve, and this process prolongs the absorption phase of NPH insulin.  1120-111 Types of Biochemistry and Biotechnology Fundamentals Recombinant Recombinant Insulin Therapeutic Proteins I insulin glargine analogue: Compared to native human insulin, the insulin glargine analogue contains one modification in the amino acid sequence, where asparagine has been replaced by glycine in A chain, and adding two arginines at the end of the B chain. Whereas human insulin has an isoelectric point of pH 5.4, insulin glargine has an isoelectric point of pH 7, which renders the insulin molecule soluble in acidic solution (pH 4). Upon SC injection, the acidic solution is neutralized leading to formation of microprecipitates from which small amounts of insulin glargine are continuously released to the circulation.  1120-111 Biochemistry and Types of Biotechnology Fundamentals Recombinant Therapeutic Recombinant Insulin Proteins I  1120-111 Biochemistry and Biotechnology Fundamentals Recombinant Therapeutic Proteins I