PHARMACEUTICAL PRODUCTS OF RECOMBINANT DNA TECHNOLOGY: AN OVERVIEW

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Drug formulations:

Generic name

Trade name

Interferon alpha 2a

Roferon A

Interferon alpha 2b

Intron A/Reliferon/Uniferon

Human leukocyte Interferon-alpha (HuIFN-alpha-Le)

Multiferon

Interferon beta 1a, liquid form

Rebif

Interferon beta 1a, lyophilized

Avonex

Interferon beta 1a, biogeneric (Iran)

Cinnovex

Interferon beta 1b

Betaseron/ Betaferon

Interferon gamma 1b

Actimmune

PEGylated interferon alpha 2a

Pegasys

PEGylated interferon alpha 2a(Egypt)

Reiferon Retard

PEGylated interferon alpha 2b

PegIntron

PEGylated interferon alpha 2bplus ribavirin(Canada)

Pegetron

Several different types of interferon are now approved for use in humans. By March 10, 2009, Multiferon — a brand name known generically as human leukocyte interferon-alpha (HuIFN-alpha-Le) — was being used in 14 European countries. This drug was approved for treatment of patients with high risk (stage IIb–III) cutaneous melanoma, after two treatment cycles with dacarbazine, following a clinical trial performed in Germany.

In January 2001, the Food and Drug Administration(FDA) approved the use of PEGylated interferon-alpha in the USA; in this formulation, polyethylene glycolis added to make the interferon last longer in the body. Initially used for production of PEGylated interferon-alpha-2b(Pegintron), approval for PEGylated interferon-alpha-2a(Pegasys) followed in October 2002. These PEGylated drugs are injected once weekly, rather than administering three times per week, as is necessary for conventional interferon-alpha. When used with the antiviral drugribavirin, PEGylated interferon is effective in treatment of hepatitis C; at least 75% people with hepatitis C genotypes 2 or 3 benefit from interferon treatment, although this is effective in less than 50% of people infected with genotype 1 (the more common form of hepatitis C virus in both the U.S. and Western Europe).[36][37][38]Recently, 2 new Protease Inhibitors have been approved which improves the outcomes for Genotype 1 hepatitis C - boceprevirand telaprevir. These drugs are used in addition to PEG-IFN / Ribavirin.

Erythropoietin:
It is  also known as erythropoetin or erthropoyetin and or EPO, is a glycoprotein hormone that controls erythropoiesis, or red blood cell production. It is a cytokine(protein signaling molecule) for erythrocyte (red blood cell) precursors in the bone marrow. Human EPO has a molecular weight of 34 kDa.

Also called hematopoietin or hemopoietin, it is produced by interstitial fibroblasts in the kidney in close association with peritubular capillary and tubular epithelial cells. It is also produced in perisinusoidal cells in the liver. While liver production predominates in the fetal and perinatal period, renal production is predominant during adulthood. In addition to erythropoiesis, erythropoietin also has other known biological functions. For example, it plays an important role in the brain's response to neuronal injury. EPO is also involved in the wound healing process.

When exogenous EPO is used as a performance-enhancing drug, it is classified as an erythropoiesis-stimulating agent (ESA). Exogenous EPO can often be detected in blood, due to slight differences from the endogenous protein, for example, in features of posttranslational modification.

Mechanism of action:
Erythropoietin has been shown to exert its effects by binding to the erythropoietin receptor(EpoR).

EPO is highly glycosylated (40% of total molecular weight), with half-life in blood around five hours. EPO's half-life may vary between endogenous and various recombinant versions. Additional glycosylation or other alterations of EPO via recombinant technology have led to the increase of EPO's stability in blood (thus requiring less frequent injections). EPO binds to the erythropoietin receptor on the red cell progenitor surface and activates a JAK2 signaling cascade. Erythropoietin receptor expression is found in a number of tissues, such as bone marrow and peripheral/central nervous tissue. In the bloodstream, red cells themselves do not express erythropoietin receptor, so cannot respond to EPO. However, indirect dependence of red cell longevity in the blood on plasma erythropoietin levels has been reported, a process termed neocytolysis.

Pharmaceutical companies make human recombinanterythropoietin with recombinant technology, in which genes are inserted to create a custom organism. In this particular case, bacteria are modified with recombination so that they will produce human erythropoietin which can be administered to patients. The same technology is used to produce a variety of other human hormones. These hormones are as effective in the body as hormones of human or animal origin, but they are easier and safer to produce.

There are side effects associated with human recombinanterythropoietin, especially in patients who use it for a long time. It can increase the risk of heavy clotting and adverse cardiovascular events, and it can also lead to iron deficiency and high blood pressure. In some young athletes, unexpected death has been linked to EPO usage, which is one of the reasons sports authorities are concerned about blood doping. Recombinant EPO is chemically slightly different from the made in the body version, and this can be used on blood tests to determine whether or not an athlete is doping.

3)  VACCINES:
Vaccination is the phenomenon of preventive immunization. In the modern concept, vaccination involves the administration (injection or oral) of an antigen to elicit an antibody response that will protect the organism against the future infections.

Vaccine is a biological preparation that improves immunity to a particular disease. A vaccine typically contains an agent that resembles a disease-causing microorganism, and is often made from weakened or killed forms of the microbe, its toxins or one of its surface proteins. The agent stimulates the body's immune system to recognize the agent as foreign, destroy it, and "remember" it, so that the immune system can more easily recognize and destroy any of these microorganisms that it later encounters.

Vaccines may be prophylactic (example: to prevent or ameliorate the effects of a future infection by any natural or "wild" pathogen), or therapeutic (e.g. vaccines against cancer are also being investigated; see cancer vaccine).

The term vaccine derives from Edward Jenner's 1796 use of cow pox (Latin variolavaccinia, adapted from the Latin vacc?n-us, from vacca, cow), to inoculate humans, providing them protection against smallpox.

Effectiveness
Vaccines do not guarantee complete protection from a disease.Sometimes, this is because the host's immune system simply does not respond adequately or at all. This may be due to a lowered immunity in general (diabetes, steroid use, HIV infection, age) or because the host's immune system does not have a B cell capable of generating antibodies to that antigen.

Even if the host develops antibodies, the human immune system is not perfect and in any case the immune system might still not be able to defeat the infection immediately. In this case, the infection will be less severe and heal faster.

Adjuvants are typically used to boost immune response. Most often aluminium adjuvants are used, but adjuvants like squalene are also used in some vaccines and more vaccines with squalene and phosphate adjuvants are being tested. Larger doses are used in some cases for older people (50–75 years and up), whose immune response to a given vaccine is not as strong.

Maurice Hilleman's measles vaccine is estimated to prevent 1 million deaths every year.

The efficacy or performance of the vaccine is dependent on a number of factors:

  • the disease itself (for some diseases vaccination performs better than for other diseases)
  • the strain of vaccine (some vaccinations are for different strains of the disease)
  • whether one kept to the timetable for the vaccinations (see Vaccination schedule)
  • some individuals are "non-responders" to certain vaccines, meaning that they do not generate antibodies even after being vaccinated correctly other factors such as ethnicity, age, or genetic predisposition.

Recombinant vaccines:
Biotechnology sector has also played its part in developing vaccines against certain diseases. Such vaccine which makes use of recombinant DNA technology is known as recombinant vaccines. It is also known as subunit vaccines.

Recombinant vaccines can be broadly grouped into two kinds:
(i) Recombinant protein vaccines: This is based on production of recombinant DNA which is expressed to release the specific protein used in vaccine preparation

(ii) DNA vaccines: Here the gene encoding for immunogenic protein is isolated and used to produce recombinant DNA which acts as vaccine to be injected into the individual.

Steps involved:
Production of recombinant vaccines involves the following steps:
(i) First and foremost, it is important that the protein which is crucial to the growth and development of the causative organism be identified.
(ii) The corresponding gene is then isolated applying various techniques. Further to this, an extensive study of the gene explains the gene expression pattern involved in the production of corresponding protein.
(iii) This gene is then integrated into a suitable expression vector to produce a recombinant DNA.
(iv) ThisrDNA is used as vaccines or is introduce into another host organism to produce immunogenic proteins which acts as vaccines.

Recombinant protein vaccines:
A pathogen upon infection produces proteins, vital for its functions, which elicit an immune response from the infected body. The gene encoding such a protein is isolated from the causative organism and used to develop a recombinant DNA. This DNA is expressed in another host organism, like genetically engineered microbes; animal cells; plant cells; insect larvae etc, resulting in the release of the appropriate proteins which are then isolated and purified. These when injected into the body, causes immunogenic response to be active against the corresponding disease providing immunity against future attack of the pathogen.

Based on the proteins involved in evoking immune response recombinant protein vaccines are of two types:
Whole protein vaccines: The whole immunogenic protein is produced in another host organism which is isolated and purified to act as vaccines.

Polypeptide vaccines: It is known that in the immunogenic protein produced, the actual immunogenic property is limited to one or two polypeptides forming the protein. The other parts of the protein may be successful in evoking an immune response but do not actually cause the disease. For eg: in the case of cholera caused by Vibrio cholerae, consists of three polypeptide chains like A1, A2, and B. The A polypeptides are toxic while B is non-toxic. Thus while producing vaccines, the polypeptide B is produced by rDNA technology and used for vaccination.

DNA vaccines:
It refers to the recombinant vaccines in which the DNA is used as a vaccine. The gene responsible for the immunogenic protein is identified, isolated and cloned with corresponding expression vector. Upon introduction into the individuals to be immunized, it produces a recombinant DNA. This DNA when expressed triggers an immune response and the person becomes successfully vaccinated. The mode of delivery of DNA vaccines include: direct injection into muscle; use of vectors like adenovirus, retrovirus etc; invitro transfer of the gene into autologous cells and reimplantation of the same and particle gun delivery of the DNA.

In certain cases, the responsible gene is integrated into live vectors which are introduced into individuals as vaccines. This is known as live recombinant vaccines. Eg: vaccinia virus. Live vaccinia virus vaccine (VV vaccine) with genes corresponding to several diseases, when introduced into the body elicit an immune response but does not actually cause the diseases.

Advantages:
(i) Since it does not involve actual pathogen, recombinant vaccines is considered to be safe than the conventional vaccines.
(ii) It induces both humoral and cellular immune response resulting in effective vaccination.

Risks involved:
(i) High cost of production.
(ii) Have to be stored at low temperature since heat destabilizes protein. Hence storage and transportation is tedious.
(iii) Individuals with immunodeficiency may elicit poor immune response.

CONCLUSION:
Recombinant DNA Technology is playing very important role in revolutionizing medicinei.e., enabling mass production of safe, pure, more effective versions of biochemicals that human body produces naturallyexamples includes a variety of products such as hormones, Therapeutic proteins, Vaccines and various Enzymes and also to create new pharmaceuticals..With sensible regulatory requirements and expeditious product review by regulatory agencies, biotech pharmaceuticals can within decades become unprecedented preventers and relievers of human suffering.

REFERENCES:
1) Johnson, I. S. (1983)."Human insulin from recombinant DNA technology".Science 219 (4585): 632–637. doi:10.1126/science.6337396
2) Ronald Kahn et al. (2005). Joslin's Diabetes Mellitus (14th ed.). Lippincott Williams & Wilkins. ISBN978-8493531836.
3) Bell GI, Pictet RL, Rutter WJ, Cordell B, Tischer E, Goodman HM (March 1980). "Sequence of the human insulin gene". Nature284 (5751): 26–32. doi:10.1038/284026a0. PMID6243748.
4) Sindelar RD. Pharmaceutical biotechnology. In: Foyes WO, Lemke TL, Williams DA, eds. Principles of Medicinal Chemistry. 4th ed. Media, PA: Williams & Wilkins; 1995:637.
5) Kotulak R. A brave, new world emerging at "biopharms." Chicago Tribune, February 8, 1998:12.
6) De Boer H, Blok GJ, Van Der Veen E. 1995 Clinical aspects of growth hormone deficiency in adults. Endocr Rev. 16:63– 86.
7) Bengtsson BA, Staffan A, Lonn L, et al. 1993 Treatment of adults with growth hormone (GH) deficiency with recombinant GH. J ClinEndocrinolMetab.76:309 –317.
8) Paoletti, Enzo, Bernard R. Lipinskas, Carol Samsonoff, Susan Mercer, and Dennis Panicali (1984) "Construction of Live Vaccines Using Genetically Engineered Poxviruses: Biological Activity of Vaccinia Virus Recombinants Expressing the Hepatitis B Virus Surface Antigen and the Herpes Simplex Virus Glycoprotein D" Proc. Natl. Acad. Sci. USA 81:193-197
9) Ishikawa, T. (Oct 2008). "Secondary prevention of recurrence by interferon therapy after ablation therapy for hepatocellular carcinoma in chronic hepatitis C patients" (Free full text).World Journal of Gastroenterology 14 (40): 6140–6144. doi:10.3748/wjg.14.6140
10) Knobler RL, Greenstein JI, Johnson KP, et al. Systemic Recombinant Human Interferon beta treatment of relapsing remitting multiple sclerosis: pilot study analysis and six-year follow up. J Interferon Res. 1993;13:333–340
11) American Society of Health-System Pharmacists (2009-02-01). "Insulin Injection". PubMed Health. National Center for Biotechnology Information, U.S. National Library of Medicine. Retrieved 2012-10-12.
12) Alarcon JB, Waine GW, McManus DP (1999). "DNA vaccines: technology and application as anti-parasite and anti-microbial agents". Adv. Parasitol. 42: 343–410

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