PHARMACEUTICAL PRODUCTS OF RECOMBINANT DNA TECHNOLOGY: AN OVERVIEW

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Unlike many medicines, insulin currently cannot be taken orally because, like nearly all other proteins introduced into the gastrointestinal tract, it is reduced to fragments (even single amino acid components), whereupon all activity is lost. There has been some research into ways to protect insulin from the digestive tract, so that it can be administered orally or sublingually. While experimental, several companies now have various formulations in human clinical trials, and one, the India-based Biocon, has formed an agreement with BMS to produce an oral-insulin alternative.

HUMAN GROWTH HORMONE:
Growth hormone is produced by the pituitary gland. It regulates the growth and development. Growth hormone stimulates overall body growth by increasing the cellular uptake of amino acids , and protein synthesis, and promoting the use of fat as body fuel.insufficient human growth hormone  (HGH) in young children results in retarded growth, clinically referred to as pituitary dwarfism.

Growth hormone is used as a prescription drug in medicine to treat children's growth disorders and adult growth hormone deficiency. In the United States, it is only available legally from pharmacies, by prescription from a doctor. In recent years in the United States, some doctors have started to prescribe growth hormone in GH-deficient older patients (but not on healthy people) to increase vitality. While legal, the efficacy and safety of this use for HGH has not been tested in a clinical trial.

Function
Effects of growth hormone on the tissues of the body can generally be described as anabolic (building up). Like most other protein hormones, GH acts by interacting with a specific receptor on the surface of cells.

Increased height during childhood is the most widely known effect of GH. Height appears to be stimulated by at least two mechanisms:
1. Because polypeptide hormones are not fat-soluble, they cannot penetrate cell membranes. Thus, GH exerts some of its effects by binding to receptors on target cells, where it activates the MAPK/ERK pathway. Through this mechanism GH directly stimulates division and multiplication of chondrocytes of cartilage.

2. GH also stimulates, through the JAK-STAT signaling pathway,[30] the production of insulin-like growth factor 1(IGF-1, formerly known as somatomedin C), a hormone homologous to proinsulin The liver is a major target organ of GH for this process and is the principal site of IGF-1 production. IGF-1 has growth-stimulating effects on a wide variety of tissues. Additional IGF-1 is generated within target tissues, making it what appears to be both an endocrine and an autocrine/paracrine hormone. IGF-1 also has stimulatory effects on osteoblast and chondrocyte activity to promote bone growth.

Main pathways in endocrine regulation of growth.

In addition to increasing height in children and adolescents, growth hormone has many other effects on the body:
* Increases calciumretention, and strengthens and increases the mineralization of bone
* Increases muscle mass through sarcomerehypertrophy
* Promotes lipolysis
* Increases protein synthesis
* Stimulates the growth of all internal organs excluding the brain
* Plays a role in homeostasis
* Reduces liveruptake of glucose
* Promotes gluconeogenesisin the liver
* Contributes to the maintenance and function of pancreatic islets
* Stimulates the immune system

Production of recombinant  HGH
Biotechnologists can now produce HGH by genetic engineering. The technique adopted is quite comparable with that of insulin production. The procedure essentially consists of inserting HGH gene into E.coli plasmid, culturing the cells and isolation of the HGH from the extracellular medium.

Limitation in HGHproduction : The HGH is a protein comprised of 191 amino acids. During the course of its natural synthesis in the body.,HGH is tagged with a single peptide  (with 26 amino acids) The signal peptide is removed during secretion to release the active HGH for biological functions. The entire process of HGH synthesis goes on in an orderly fashion in the body. However, signal peptide interrupts HGH production by recombinant technology. The complementary DNA (cDNA) synthesized from the mRNA encoding HGH is inserted into the plasmid. The plasmid containing E.coli when cultured, produces full length HGH along with signal peptide.ButE.coli cannot remove the signal peptide. Further, it is also quite difficultto get rid of signal peptide by various other means. Theoretically, cDNA encoding signal peptide can be cut to solve these problems. Unfortunately, there is no restriction endonuclease to do this job, hence this is not possible.

HGH Vial

A novel approach for HGH production:
Biotechnologists  have resolved the problem of signal peptide interruption by a novel approach. The base sequence in cDNA encoding signal peptide ( 26 amino acids ) plus the neighbouring 24 amino acids is cut by restriction endonuclease ECoRI. Now a gene (cDNA ) for 24 amino acid sequence of HGH is freshly synthesized and ligated to the remaining HGHcDNA. The so constituted cDNA , attached to a vector, is inserted into a bacterium such as E. coli for culture and production of HGH. In this manner, the biologically functional HGH  can be produced by DNA technology.

2)   THERAPEUTIC AGENTS FOR HUMAN DISEASES
Biotechnology is very useful for the production of several therapeutic products for treating human diseases. A selected list of rDNAderived therapeutic agents along with trade names and their uses in human are given below…..

rDNA Product

Trade name

Application / Uses

Insulin

Humulin

Diabetes

Growth hormone

Protropin/Humatrope

Pituitary dwarfism

Interferon

Intron A

Hairy cell leukemia

Hepatitis B vaccine

Recombinax HB/ Engerix

Hepatitis B

TissuEplasminogen activator

Activase

Myocardial  infarction

Factor vIII

Kogenate/Recombinate

Hemophilia

Dnase

Pulmozyme

Cystic fibrosis

Erythropoietin

Epogen/rocrit

Severe anemia with kidney damage

INTERFERONS:

Interferons(IFNs) are proteins made and released by host cells in response to the presence of pathogenssuch as viruses, bacteria, parasites or tumor cells. They allow for communication between cells to trigger the protective defenses of the immune system that eradicate pathogens or tumors.

IFNs belong to the large class of glycoproteins known as cytokines. Interferons are named after their ability to "interfere" with viral replication within host cells. IFNs have other functions: they activate immune cells, such as natural killer cells and macrophages; they increase recognition of infection or tumor cells by up-regulating antigen presentation to T lymphocytes; and they increase the ability of uninfected host cells to resist new infection by virus. Certain symptoms, such as aching muscles and fever, are related to the production of IFNs during infection.

Functions
All interferons share several common effects; they are antiviral agents and can fight tumors. As an infected cell dies from a cytolytic virus, viral particles are released that can infect nearby cells. However, the infected cell can warn neighboring cells of a viral presence by releasing interferon. The neighboring cells, in response to interferon, produce large amounts of an enzymeknown as protein kinase R(PKR). This enzyme phosphorylates a protein known as eIF-2 in response to new viral infections; the phosphorylated eIF-2 forms an inactive complex with another protein, called eIF2B, to reduce protein synthesis within the cell. Another cellular enzyme, RNAse L— also induced following PKR activation—destroys RNA within the cells to further reduce protein synthesis of both viral and host genes. Inhibited protein synthesis destroys both the virus and infected host cells. In addition, interferons induce production of hundreds of other proteins—known collectively as interferon-stimulated genes (ISGs)—that have roles in combating viruses. They also limit viral spread by increasing p53 activity, which kills virus-infected cells by promoting apoptosis. The effect of IFN on p53 is also linked to its protective role against certain cancers.

Another function of interferons is to upregulate major histocompatibility complex molecules, MHC Iand MHC II, and increase immunoproteasomeactivity. Higher MHC I expression increases presentation of viral peptides to cytotoxic T cells, while the immunoproteasome processes viral peptides for loading onto the MHC I molecule, thereby increasing the recognition and killing of infected cells. Higher MHC II expression increases presentation of viral peptides to helper T cells; these cells release cytokines (such as more interferons and interleukins, among others) that signal to and co-ordinate the activity of other immune cells.

Interferons, such as interferon gamma, directly activate other immune cells, such as macrophages and natural killer cells. Interferons can inflame the tongue and cause dysfunction in taste bud cells, restructuring or killing taste buds entirely.

Interferon therapy

Three vials filled with human leukocyte interferon

The immune effects of interferons have been exploited to treat several diseases. Agents that activate the immune system, such as small imidazoquinoline molecules that activate TLR7, can induce IFN-α. Imidazoquinoline is the main ingredient of Aldara (Imiquimod) cream, a treatment approved in the United States by the Food and Drug Administration (FDA) for actinic keratosis, superficial basal cell carcinoma, papilloma and external genital warts. Synthetic IFNs are also made, and administered as antiviral, antiseptic and anticarcinogenic drugs, and to treat some autoimmune diseases.

New research has shown that imiquimod's anti-proliferative effect is totally independent of immune system activation or function. Imiquimod exerts its effect by increasing levels of the opioid growth factor receptor (OGFr). Blocking OGFr function with siRNA technology resulted in loss of any antiproliferative effect of imiquimod.

Interferon beta-1a and interferon beta-1b are used to treat and control multiple sclerosis, an autoimmune disorder. This treatment is effective for slowing disease progression and activity in relapsing-remitting multiple sclerosis and reducing attacks in secondary progressive multiple sclerosis.

Interferon therapy is used (in combination with chemotherapy and radiation) as a treatment for many cancers. This treatment is most effective for treating hematological malignancy; leukemia and lymphomas including hairy cell leukemia, chronic myeloid leukemia, nodular lymphoma, cutaneous T-cell lymphoma.[21]Patients with recurrent melanomas receive recombinant IFN-α2b. Type I IFNs have a therapeutic potential for the treatment of a wide variety of leukemias and solid tumors due to their antiproliferative and apoptotic effects, their anti-angiogenic effects and their ability to modulate an immune response specifically activating dendritic cells, cytolytic T cells and NK cells. Research in this area is receiving intensive investigation. Interferon a 2b is also being used for treatment of ocular surface squamous neoplasia (OSSN) in the form of perilesional injection followed by topical interferon a 2b drops at Lahore General Hospital Eye unit II.[

Both hepatitis B and hepatitis C are treated with IFN-α, often in combination with other antiviral drugs. Some of those treated with interferon have a sustained virological response and can eliminate hepatitis virus. The most harmful strain—hepatitis C genotype I virus—can be treated with a 60-80% success rate with the current standard-of-care treatment of interferon-α, ribavirin and recently approved protease inhibitors such as Telaprevir (Incivek) or Boceprevir (Victrelis). Biopsies of patients given the treatment show reductions in liver damage and cirrhosis. Some evidence shows giving interferon immediately following infection can prevent chronic hepatitis C, although diagnosis early in infection is difficult since physical symptoms are sparse in early hepatitis C infection. Control of chronic hepatitis C by IFN is associated with reduced hepatocellular carcinoma.

Administered intranasally in very low doses, interferon is extensively used in Eastern Europe and Russia as a method to prevent and treat viral respiratory diseases such as cold and flu. However, mechanisms of such action of interferon are not well understood; it is thought that doses must be larger by several orders of magnitude to have any effect on the virus. Although most scientists are skeptical of any claims of good efficacy recent findings suggest that interferon applied to mucosa may act as an adjuvant against influenza virus, boosting the specific immune system response against the virus. A flu vaccine that uses interferon as adjuvant is currently under clinical trials in the US.

When used in the systemic therapy, IFNs are mostly administered by an intramuscular injection. The injection of IFNs in the muscle, in the vein, or under skin is generally well tolerated. The most frequent adverse effects are flu-like symptoms: increased body temperature, feeling ill, fatigue, headache, muscle pain, convulsion, dizziness, hair thinning, and depression. Erythema, pain and hardness on the spot of injection are also frequently observed. IFN therapy causes immunosuppression, in particular through neutropenia and can result in some infections manifesting in unusual ways.

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