Recombinant Human Insulin - Research Paper Example

Recombinant Human Insulin Insulin is a hormone that is secreted by a group of cells called as islets of langerhans which is located at the pancreas. The pancreas produces many digestive enzymes and hormones. After a good meal, the stomach receives a variety of nutrients like carbohydrates, fats, proteins and minerals. Among them the carbohydrates are absorbed in the intestines and these carbohydrate moves into the blood stream as glucose. The insulin after the secretion from the pancreas moves into the blood stream and gets attached to the insulin receptors that are present in most of the cells. Thus insulin circulates in the blood stream. The cells containing the insulin receptors attain the capacity to absorb the glucose molecules in the blood into the cells for their energy and metabolism. Thus insulin helps to reduce the blood glucose level. This continuous absorption provides energy for the normal metabolism of the cells and the person feels very energetic after having a carbohydrate rich food. If the glucose molecule present in the blood is not absorbed then the person will not receive the energy from the glucose and the metabolic products produced by the catabolism and anabolism of the glucose molecule. As glucose molecule is the basic energy giving molecule, the metabolic balance will collapse. Similarly the concentration of the insulin molecules must increase after a meal automatically, if this does not happen then the person is prone to diabetes mellitus. (Colwell 2003). Role of insulin and liver: Sugars such as glucose enter the liver from the gut by the hepatic portal vein, the only blood vessel in the body having an extremely variable sugar concentration. Thus it proves that liver is the important organ as it maintains the blood glucose level. The liver maintains a standard glucose level irrespective of how long or recent the food was taken. When the glucose is in excess the glucose and other hexose sugars such as fructose and galactose are converted into the insoluble glycogen and stored in the liver. When there comes arises a demand for the glucose, the glycogen is converted back into glucose and utilized by the body. Types of Diabetes mellitus: The diabetes is caused because of two reasons. One is due to the lack of insulin and the other is due to the failure of the cell’s receptors to attach the insulin molecules at its receptor sites. The first type is called as type 1 diabetes mellitus and the second type is called as type 2 diabetes mellitus. Sometimes the genetic defects may cause a lack in the secretion of the insulin; this type of diabetes is called as congenital diabetes. Type I diabetes: Type I diabetes mellitus is a lifelong chronic disease. Type I diabetes is also called as juvenile or insulin-dependent diabetes. Type I diabetes occurs at any age. This is more predominant in the children, young adults and adolescents. This disease occurs when the pancreas are not able to produce enough amount of the insulin. (Colwell 2003). Though the exact cause for this disease is not known, it is widely said that this disease is related genetically for the people who are much prone for a viral or environmental trigger. Here the failure of the beta cells of the pancreas to produce insulin is the major cause of this disease, as a result of which they feel tired, hungry, thirsty, frequent urination, losing weight and blurry eyesight. If insulin is not taken at regular intervals then the condition of the person will become risky. To diagnose the type I diabetes fasting glucose level, non fasting glucose level, ketone test and oral glucose tolerance tests are conducted. As previously mentioned the only treatment for this disease is taking insulin every day. This insulin is usually given under the skin. The injections are given one to four times a day depending on the complications of the disease. The future possible complications of this disease include stroke, heart attack, high blood glucose level, diabetic retinopathy, diabetic neuropathy, cataracts and glaucoma. Type II diabetes mellitus: Type II diabetes mellitus is a chronic life long disease caused due to the high blood glucose level. This is the most common form of diabetes found among the people. In type II diabetes, the body and the cells does not respond to the concentrations of the insulin. The fat, liver and the muscle cells does not respond to insulin. This condition is called as insulin resistance. If the glucose does not move into the cells, the persons will not receive the energy required for the normal metabolism. As a result the blood glucose concentration will increase heavily. This condition is called as hyperglycemia. Though high levels of the glucose trigger the beta cells to secrete more amount of insulin, this amount will not be enough for the glucose metabolism. As people with more weight are prone for insulin resistance, this disease affects the people who are having more weight. The fat interferes in the insulin synthesis of the body. (Colwell 2003). The family and the genetic history are also found to influence the occurrence of the type II diabetes mellitus. The risk actors of this disease includes high blood pressure, persons of age greater than 45 years have more risk, similarly if the HDL cholesterol level is less than 35mg/dl and triglyceride level more than 250mg/dl they are prone for higher risks. The most important factor to be noted is that the race and the ethnicity are also found to have a greater role in the occurrence of this disease. The Native American, Hispanic American and African American are more prone for diabetes than any other race people. The noticeable symptoms of this disease includes Erectile dysfunction, Blurred Vision, fatigue, increased Thirst, frequent Urination and high appetite and very slow healing infections. The type II diabetes is diagnosed by the fasting glucose levels, non fasting glucose tests and the oral glucose tolerance tests. The possible complications of this Type II diabetes includes High blood pressure, Stroke, High Cholesterol, Macular Edema, cataracts, glaucoma , Kidney disease, diabetic retinopathy and skin, genital and urinary tract infections. The symptoms such as the pressure, chest pain, unconsciousness and seizure must be noted immediately and given immediate attention if not it may result in severe complications. The best way to prevent this disease is to reduce the over weight at both childhood and adulthood. Gestational diabetes: The presence of high blood sugar during the pregnancy is termed as Gestational diabetes. This disease is also called as Glucose intolerance during pregnancy. The African and Hispanic ancestry are prone to this type of diabetes. This is also found to occur in a family in a continuous manner. The risk factors of this disease include obesity, recurrent infections, giving birth to overweight babies and unexplained miscarriage. The major symptoms of this disease includes blurred vision, increased thirst, increased urination, nausea, vomiting, weight loss and increased appetite and frequent urinary infections. The possible complication of this disease includes increased new born death, complications during delivery because of the infant’s large size. Low blood sugar and development of diabetes in the later part of life are other complications. Risk factors of Diabetes: Viral infection that occurs during the childhood, during pregnancy and early adulthood triggers the Type I diabetes. The scientists believe that the viral infections may alter the immune system and produce antibodies that are able to attack the insulin producing beta cells of the islets of langerhans at the pancreas. Similarly hereditary is found to play a small role on the Type I and important role on the Type II diabetes. From the studies it is clearly stated that the two third of the type II diabetes mellitus patients is mainly due to the family history. The next risk factor is based on the race and ethnicity on the type II diabetes. It is found that the blacks, Asians and Native American s are prone for Type II diabetes than the whites. Metabolic syndrome is another important risk factor that may cause Type II diabetes. Abnormal obesity associated with the insulin resistance, high blood pressure (Hyper tension), high blood glucose levels, elevated triglycerides, low levels of the high density lipoprotein and cholesterol are the abnormalities clustered and called as metabolic syndrome. (Saudek 2007). Complications of Diabetes: The diabetes without any proper treatment will lead to many complications. Some of them include hypoglycemia, diabetic ketoacidosis, cardiovascular disease, retinal damage (diabetic nephropathy), atherosclerosis, stroke, hyperlipidemia, hypertension, urinary tract infection, skin infection and chronic renal failure (Diabetic retinopathy). The maintenance of the body weight, blood pressure are some of the important factors that need to be taken care of. Discovery of insulin as treatment for diabetes: The diabetes mellitus became a treatable disease after the year 1921, when Banting and Best first isolated this hormone from the sheep. They extracted this hormone from the pancreas. They also proved that the pancreas have a particular type of cell called as islets of langerhans that produces this hormone in large concentration than any other organ in the body. Now more than millions of people are using insulin for the treatment of diabetes. The dosage level varies for every patient depending upon the nature of the deficiency of insulin in the body. The insulin is now produced by many companies world wide. (Balasbramaniam, 2004). Treatment for diabetes: The standard treatment recommended for diabetes is the daily injection of insulin. The insulin is obtained from the pancreas of the slaughtered pigs and cattle. This insulin obtained from the pigs and cattle are very less in quantity and they have fewer amounts of impurities and traces. Sometimes it is showed that some damaging side effects are found as a result of the injection. The cattle insulin is sometimes not well accepted by the body. The bovine insulin and the porcine insulin though are similar to the human insulin; they vary little in their composition. The patient’s immune system also sometimes refuses to accept this insulin and produces antibodies against them. These antibodies act on the insulin and try to neutralize it and cause inflammation in the body. The prolonged use of bovine insulin and porcine insulin may also cause long term complications. These factors made the researchers to look for alternative methods for the production of insulin at large scale. As the structure of the insulin was decoded in the 1950s, biotechnology was brought into the light for the synthesis of insulin. In the early 1980s biotechnology revolutionized the insulin synthesis. Before that only the cattle pancreas derived insulin were used for the treatment and the quality of the bovine and porcine insulin was improved by the scientists. Why genetic engineering in insulin? Genetic engineering has been applied by the industry to increase the production of the natural products such as enzymes, antibiotics and vitamins. Specific genes can now be isolated from virtually any biological source and cloned into a bacterium or other host system. The insertion or cloning a gene does not ensure its expression and production. Many factors must be considered to ensure the viable levels of production and the biological activity of the products. The choice of both the host and vector system determines the strategies used for the cloning and expression. Recombinant DNA technology for insulin production (RDNA): The RDNA technology opened up the possibility to clone the gene responsible for the particular product and to produce it in unlimited amounts in a bacterium such as E.coli. The eukaryotic genes such as insulin, human growth hormone and interferon are cloned and produced in a large scale with the help of the RDNA technology now. The first commercial health care product derived from the Recombinant DNA technology is the production of the human insulin. Insulin gene is located at the top of the chromosome11 in the human. The short arm of the chromosome level has insulin gene located at the p13 region leads to pter. (Owerbach 1981). It has 153 nitrogen bases. This insulin gene is isolated from the chromosome and inserted into the bacterium E.coli for the large scale production. Insulin: Insulin is a 5808 Dalton polypeptide hormone. It consists of 2 peptide chains, an A chain composed of 21 amino acids and a B chain containing 30 amino acids. The two chains are linked by 2 disulfide bridges. Of the 153 nitrogen bridges 63 are in A chain and 90 are in the B chain. Hosts for Recombinant protein production: A wide variety of living organisms were tried for the production of the recombinant proteins. The selection of the host depends upon the level of the expression level, the quality of bio molecules, safety and rate of manipulation of the genetic system and at the last and the most important cost of the production of the recombinant protein. Most of the recombinant molecules are produced using the yeast, E.coli and the mammalian CHO cells. The choice of the E. coli was better because for the production of the simple bio molecules that does not require post translational modifications, the bacterial system is good. Hosts for recombinant protein production S.no Hosts 1 Escherichia coli 2 Bacillus subtilis 3 Streptomyces 4 Saccharomyces cerevisiae 5 Pichia pastoris 6 Aspergillus 7 Animal cells (CHO, SP20/NSO) 9 Insect cells: Baculovirus system 10 Transgenic Animals and plants E.coli (Vector): Escherichia coli is a gram negative rod shaped bacterium that is found on the lower intestine of the warm blooded animals. The bacteria can be grown easily as it is ubiquitous and its genetics is very simple and can be easily manipulated. This makes E.coli as an important species in microbiology and biotechnology. Most of the E.coli strains are harmless to the humans; this increases the importance of this gram negative bacterium in the research. They also have an extra chromosomal DNA called Plasmid. The modification and manipulations in the plasmid will not affect the metabolic activities of the bacterium and hence the genetic manipulation is done in the plasmid. Production of the recombinant insulin: The human insulin is produced in a mass by the following steps using the geneti engineering. They are: 1. Isolate gene. 2. Prepare Target DNA. 3. Insert DNA into the plasmid. 4. Insert plasmid back into the cell. 5. Plasmid multiply. 6. Target cells reproduce. 7. Cells produce proteins. (Ratledge and Kristiansen 2002). The recombinant insulin can be produced in two ways. The genes for the mature A ands B chains must be synthesized separately in invitro manner. The A and B chains are cloned into the E.coli β- galactosidase gene separately and are over expressed as β- galactosidase fusion proteins. 18 small oligonucleotides were synthesized and joined together to form a long nucleotide chain (Chain A). In this way the assembly of the small polynucleotides yielded the Chain B. These two single stranded polynucloetides were made into double-stranded DNAs. The long DNA fragment was inserted into the plasmid vector especially at the upstream of its β- galactosidase gene. In the same way, the small DNA fragment was inserted into another vector to make rDNA. The two rDNAs were introduced into E.coli cells separately. Recombinant E.coli was screened from these two E.coli cultures. One E.coli culture produced long polypeptide of insulin bound with the β- galactosidase enzyme and the other produced a short polypeptide bound with the β- galactosidase. The insulin genes are inserted such that they have a methionine codon in between the β- galactosidase gene and the insulin gene. As a result, when the cyanogens bromide treatment is given to the peptides the insulin chains are released into the solution. Thus the β- galactosidase is removed from the insulin polypeptides. The methionine residue is absent in the insulin polypeptide; hence the cleavage with the cyanogens bromide will yield only the insulin chains. The genetically manipulated E.coli strain can produce 1 x 105 insulin molecules in the culture within a week. After the 2 chains of insulin are produced separately, they are fused together by the chemical method. The reduction and re-oxidation method with an oxidizing agent is done for the formation of the disulfide bond. The chemical cross linking of the A and B chain produces the active insulin. The purification of the insulin is done by using the various chromatography techniques. Ion-exchange column, reverse-phase high performance liquid chromatography, and gel filtration chromatography column are used for the purification of the insulin. This is the first method of recombinant insulin production. The second method of recombinant insulin production is done by the over expression of the entire pro-insulin gene in E.coli. After the expression, the pro-insulin gene is purified using the downstream processing techniques. After the complete purification the C-peptide of the pro-insulin is removed. Now the pure insulin is produced. The C-peptide removal is done by either specific hydrolysis technique or by chemical hydrolysis. Production of human insulin for therapy (Owens 1986). Goeddel etal ; Chance etal; 1979-1981 Biosynthetic human insulin derived via recombinant DNA technology. Markussen 1980 Semi synthetic human insulin from porcine insulin Genetic Engineering for the production of Rat Insulin: In 1980, Gilbert and Villa-komaroff isolated the poly (A) containing RNA from the transplantable rat insulinoma. This RNA contained the sequence required for the synthesis of the proinsulin. This proinsulin was identified by the precipitation reaction with the anti-insulin antibody. As this amount of RNA was not sufficient, the cDNA preparation from the given strand was done with an extension of 15 nucleotide short oligo dC tail. A total of 500 base pair was selected and inserted into the PstI site of pBR322 after the elongation of the C terminal. The scientists were able to produce 20% positive clones and about 2.5% of the clones were confirmed top have the insulin gene. The pI19 probe was used to confirm the presence of the insulin gene. To detect the concentration of the insulin in the coned colony, the solid phase radio immunoassay was carried out. It was found from the experiments that the discs coated with the anti-penicillanase antibody and exposed to the I 125 – labeled anti insulin to detect the presence of a fused protein. The genetically manipulated E.coli produced proinsulin together with the penicillinase. The penicillinase and the C-polypeptide present in the proinsulin were digested with trypsin. As a result, biologically active rat insulin molecule was produced. The rat insulin was previously used to treat diabetes but some patients are found to have allergic to the rat insulin.( Komaroff, 1978). (Crea 1978). Preparations of Insulins: The insulin comes in three different forms to the market as vials, prefilled syringes, and cartridges. The human recombinant insulin, insulin lispro, insulin aspart, insulin glargine are the commonly found insulins in the market. Mirsky etal 1963 Isolation and crystallization of human insulin Kung etal 1966 Chemical synthesis of bovine insulin Sieber etal 1974 Chemical synthesis of human insulin Obermeier and Geiger 1976 “Semi synthesis” of human from porcine insulin. Gattner and Morihara etal 1979 Semi synthesis” of human from porcine insulin. (Owens 1986). Types of insulins: Now there are many types of insulin. These vary according to the onset (The time required for the insulin to start working), peak time (The time at which the insulin is working at the best way) and duration (The amount of time the insulin will work).The different types of insulin analogue and insulin are identified based upon their speed of absorption under the skin into the blood and by the time that is taken for the complete absorption of the insulin by the body. Insulin analogues: In order to improve the activity of the insulin, various methods were tried. In connection to it, the production of the insulin analogues was also tried and some simple analogues with the increased pharmacokinetic efficiency were produced. These analogues varied in some amino acids from the original insulin. A number of analogues were prepared based upon the requirements of the patients. The insulin analogues had varied acting levels starting from meal times to the basal requirements. Rapid acting insulin analogue: The onset of this type of insulin occurs in 10-20 minutes with the maximum effect in 1-3 hours. The total duration of this drug inside the body is 3-5 hours. a) Long acting insulin analogue: The onset time for this drug is one hour and the concentration of the insulin will be seen for 24 ours after which an injection of the same dose is required. These long acting analogues were prepared to meet the basal requirements of the patients. To establish an overall glyceamic control , maintenance of the low level plasma insulin during the fasting and the interprandial conditions. These long lasting analogues are produced either by changing the isoelectric points of the protein or by changing the physiological pH of the protein by acylation. The Insulin glargine is a type of long lasting insulin analogue that was produced by the addition of the two arginine amino acid molecules at the end of the B chain to alter the isoelectric point of the insulin. The two arginine amino acids are named as Arg B 31 and Arg B 32. When two amino acids are added to any protein the first thing that is to be checked for is the stability of the molecule. Here the addition of the glycerin at the Gly A 21 position is done for the stability of the molecule. So after a subcutaneous injection, the plasma level insulin rises slowly for up to 8 hours and remains in the body for about 24 hours. The Insulin glargine is suitable for once daily administration only. In the second type acylation of the amino group Lys B 29 is done. This acylation promotes the reversible binding of the insulin to albumin. This type of insulin is still under the development. b) Biphasic insulin analogue: The onset time is very short of 10-15 minutes and the maximum effect of this insulin will last for 24 hours. Hence this is widely used for the diabetes treatment. Human insulins: a) Short acting insulin: This is soluble human insulin that is to be given within 30 minutes of a meal. The onset of this drug takes 30 minutes with its maximum effect for 3 hours and the insulin action will be inside the body for about 8 hours. b) Intermediate acting insulin: This intermediate acting insulin is also called as isophane human insulin. This insulin formulation contains a delaying substance that delays the absorption of insulin by the body. The insulin crystals are homogenized before the injection of this drug. This intermediate insulin drug takes about 90 minutes to have some effect on the concentration of the glucose. The heavy effect of this drug is between the 4-12 hours of injection. The drug is completely absorbed by the body in 24 hours. c) Premixed insulin: As the name suggests it contains short term acting and long term acting insulin in the standard preparations. The maximum effect of this drug will be at about 8th hour after the injection and the complete absorption of the drug occurs in 24 hours. d) Soluble insulins: This is unmodified insulin. Here after the injection, the rise of insulin levels at the plasma occurs after the 3 hours and its complete absorption into the body requires duration of 6 – 8 hours. e) Basal Insulins: These types of insulins act for a long time in the body. They are absorbed at a slow rate by the subcutaneous layer in the body. The use of the protoamine with the insulin reduces the absorbing capacity of the insulin by the subcutaneous tissue. The engineering of the insulin molecules with the help of the isoelectric point’s concept and covalent acylation enhances the reversible binding of the insulin with the albumin and thus making the albumin to act as the depots for insulin. Thus the absorption rate gets lowered. Risks associated with the recombinant bio molecules: In al the expression systems used so far for the production of the recombinant molecules some common risks are found associated with them. They include the safety of the expression, quality, stability of the molecule, species specificity and dependency, vector and transgene toxicity, immunogenicity, toxicity related pharmaco dynamic effects, biological toxicity, local tolerance of the molecule and the oncogenecity. The safety of the recombinant molecules depends upon the above factors. The quality, safety and efficacy are the most important safety factors that require critical evaluation. These three parameters influence the toxicity and the immunogenicity of the final product. These three factors are crucial in all the steps of the manufacturing process to ensure that the right medicine, at the right concentration and at the right method is prepared and brought into the market. Similarly the host bacterium that is used for this expression must be killed at proper sterilization technique to prevent the environmental hazards; similarly the waste products out of this process must be properly disposed as per the regulations for the Recombinant product disposal procedures. (Panda 2007). Factors influencing the absorption of the Human Insulin: The pharmaceutical formulation of human insulin is highly based on the rate of absorption of the insulin by the subcutaneous tissue. Based on the rate of absorption of the insulin, the effect of the insulin on the blood glucose level will vary. So apart from the pharmaceutical formulation, the additional factors such as insulin concentration, site of the injection, aprotinin, mixing of the intermediate and the short term acting preparations must be considered. (Owens 1986). So in order to determine the best route for administration of the insulin a comparative study on the intravenous and intramuscular administration of the human insulin was carried out. It was found from the trails with six male volunteers that the intravenous administration led to a rapid fall in the glucose level and the duration was 210 minutes. Whereas the intramuscular injection of the human insulin had a gradual and steady decrease of the glucose level in the blood and the duration was 360 minutes. So it was concluded that the intramuscular injection is the moist preferred one for the once daily injection mode. The dose-response study of the human, bovine and porcine to explore the time –dependent variation possibilities among the three insulin types used. The glucose level and the insulin response to the subcutaneous administration of the three insulin types were examined. It was found that the human insulin has a stronger hypoglycemic effect than the porcine and bovine insulin. On comparison with the other two types, the porcine insulin was found to have less potent in lowering the plasma level. And also at lower dosage levels of 0.05U/kg the human and the porcine insulins were found to have similar effects and at higher dosage levels the human insulin was found to have more biological activity than the other two types. (Owens 1986). Adverse effects of human insulin: The continuous usage of insulin as resulted in the too high and too low blood glucose, increased lethargy, behavioral changes including aggression and violence, memory loss, joint pains, weight increase, confusion, depression and sudden changes in the menstrual cycle (improper menstrual cycle) etc., As the too high and too low blood pressure cannot be handled by the self it requires the assistance of the family members and friends. If not found at the right time, the hyperglycemia and hypoglycemia can lead to seizure or coma. (Jenny 1997). Other side effects of the human insulin includes bruises after the injection, fatty lumps (they are caused by the stimulating effect of the insulin on the fat tissue, this is a common problem as the fatty lumps have fibrous and fat tissues), lipoatrophy (a cavity in the subcutaneous tissue caused due to frequent injection at the same site), Insulin oedema and remission phase. (Hanas 2006). Why insulin is targeted by the pharma industries? Insulin is the lead molecule in many pharmaceutical companies because of the increase in the demand for insulin and the increase in the diabetic patients every year. The world health organization has estimated that the requirement for the insulin will increase in the next 25 years with 170 million diabetic patients around the world. Even if we consider 10% of the diabetic patients are suffering from the type I diabetes, then an estimated quantity of 5 million tons of insulin will be required. This statistics about the insulin has made the pharmaceutical companies in the world to look for better insulin to compete with the other companies. As inject able insulin was first produced by the companies, now the interest has passed onto to invent oral insulins and nasal spray insulins. The nasal spray insulins are concentrated more by the pharmaceutical companies because of the easy absorption by the liver cells and quick communication to the blood cells for the emergency requirement s and to overcome the painful injection method. Exubera was the first non – inject able insulin therapy to be approved by the WHO and the sales of this product was estimated as US $ 0.6-1 billion in the year 2008. Similarly air insulin and the Technosphere insulin system are under the clinical trials. Dry insulin Powder: In order to improve the stability and the absorbability of the dry insulin powder for the inhalation the insulin was mixed with the pulmonary absorption enhancers. The effect of the pulmonary enhancers on the stability of the active ingredients in the insulin was also checked up in the inhalation therapy. Citric acid, Bacitracin and Span 85 were tested for the inhalation therapy. These pulmonary enhancers proved best only in the solution form and not in the powder form. It was found that the citric acid insulin formulation (MIC SD) improved the hypoglycemic effect but with less stability. So the ratio of the citric acid concentration was varied with the insulin powder and the stability was looked up. This formulation was named as MIC Mix. The MIC Mix showed equivalent hypoglycemic effect as that of the MIC SD and was better stable at the 60 °C/dry condition. So for greater results with the inhalation therapy it was suggested from the above experiment to use a package that prevents the moisture absorption. (Todo et. al 2004). References: Balasubramaniam, D 2004, Concepts in Biotechnology, Universities press. Ratledge, C and Kristiansen, B 2002, Basic Biotechnology, Cambridge press. Colwell JA 2003, Diabetes, Elsevier Health Sciences. Crea, R., Kraszewski, A., Hirose, T and Itakura, K 1978, Chemical synthesis of genes for human insulin, Proceedings of the National Academy of Science, USA vol. 75, no. 12, pp. 5765-5769. Jenny, H 1997, Adverse effects of human insulin, viewed on May 30, 2010 http://www.health-science-spirit.com/insulin.html. Komaroff, LV et. al 1978, A bacterial clone synthesizing proinsulin Proceedings of the National Academy of Science, USA vol. 75, no. 8, pp. 3727-3731. Owens DR 1986, Human insulin: clinical pharmacological studies in normal man, Springer publications. Owerbach, D., Bell, GI., Rutter, WJ., Brown, JA and Shows, TB 1981, The insulin gene is located on the short arm of the chromosome 11 in humans, American Diabetes Association, vol. 30, no. 3, pp 267-270. Panda AK, 2007, Recombinant DNA technology and biotechnology, viewed on May 30, 2010 http://nsdl.niscair.res.in/bitstream/123456789/608/1/BiotechnolohyApplications.pdf. Hanas, R 2006, Type 1 diabetes in children, adolescents, and young adults, Class publishing, London. Saudek, CD 2007, Diabetes 2007, John Hopkins Health press. Todo, H Okamoto, H., Iida, K and Danjo, K 2004, Improvement of the stability and absorbability of insulin dry powder for inhalation by powder combination technique, International Journal of Pharmaceutics, vol.271, no.1-2, pp.41-52 Read More