Recombinant DNA (rDNA) technology has been applied in the manufacture of insulin analogs through the modification of amino acid sequence in the peptide (A or B) chains of human insulin. The insulin analogs exhibit similar modes of action with native human insulin, but insulin analogs with altered pharmacokinetic properties show improved glycaemic control in diabetic patients than native human insulin. Vora et al. study compares the rate of absorption of subcutaneous monomeric insulin analog derived from rDNA to that of native human insulin (1236).
Objectives of the Study
The main objective of this study was to compare the absorption rates of soluble human insulin (labeled by radioactive iodine-125) and a rDNA derived monomeric human insulin. rDNA technology allows the alteration of the pharmacokinetic properties of insulin analogs to reduce hyperglycemia and achieve improved glycemic control (Kurtzhals 127). Strategies to prevent insulin self-association or formation of insulin hexamers in solution to enhance absorption from subcutaneous injection sites can be achieved through rDNA.
The low absorption of soluble human insulin injected into the subcutaneous tissue is attributed to the low dissociation rate of hexameric units into monomeric insulin. However, through rDNA technology amino acid substitution at the interfaces of the hexamer to produce monomeric insulin with improved pharmacokinetic properties such as enhanced absorption is possible. This study aimed to compare the rate of absorption of equimolar amounts of 125-Iodine labeled insulin monomeric analog with soluble human insulin also labeled with iodine-125.
Background of the Work
rDNA technology has made a synthesis of insulin analogs with improved pharmacokinetic properties possible. Normally, the regular soluble human insulin injected subcutaneously takes two to three hours for a peak effect to be felt. This delay in action is because of the slow rate of dissociation of regular insulin hexameric units into easily absorbable monomeric units. The lower rate of dissociation is associated with the self-association of hexameric insulin caused by steric reactions or metal-binding into the monomer-monomer interfaces (Heinemann 675). Thus, the rDNA technology modifies the enzymatic properties of insulin by steric hindrance and deletion of the metal-binding sites through amino acid substitutions. In this way, monomeric insulin analogs can be synthesized. These analogs have the advantage over native human insulin; they exhibit a rapid absorption rate when injected subcutaneously.
This study identifies the amino acid substitution at the monomer-monomer interface in the structure of hexameric insulin dimer through rDNA technology as potentially yielding analogues with high absorption rates. In particular, the study considers a monomeric insulin analog resulting from amino acid substitutions; substitution of B9 serine residue with aspartate and B27 threonine residue with glutamate in the human insulin dimer. The results from earlier studies involving animal experiments demonstrate significantly higher absorption rates, for insulin monomeric analogs, relative to soluble human insulin injected subcutaneously. This study extrapolates on these findings; it involves a comparison of the absorption rates of the insulin analogue and soluble human insulin subcutaneously injected to normal humans. Equimolar concentrations of both the insulin analogue and the soluble human insulin (labeled with Iodine-125 isotope) were used.
Methods Used
In this study, the subjects involved seven healthy individuals (male volunteers) of between 20 and 39 years. All these individuals had no familial or genetic predisposition to diabetes mellitus, weighed within 10% range of appropriate body weight. They also exhibited normal tolerance to glucose, and none had a history of drug use or allergy. The study gained the approval of the ethical committee at the local level and the subjects provided a written informed consent before participating in this study. The study involved continuous monitoring of the subjects.
The subjects received a random administration of either the soluble human insulin (125I-labelled) or the 125I labeled insulin analogue in equimolar doses four times a week. The procedure involved a premedication procedure where 100mg potassium iodide was administered orally to prevent the uptake of 125-Iodine by the thyroid glands. Prior to the treatment, the subjects underwent a 12-hour fast after which venous blood sampling from intravenous cannula were obtained under isotonic saline infusion (15mol/l). Then the 125-I labeled preparations were administered through subcutaneous injection at the abdominal section using an insulin syringe. Venous blood sampling was done after every 10 minutes in the initial hour following the injection, then at a15-minute interval after two hours, thereafter a half-hourly for 3 hours and finally hourly for the next six hours. A first batch of aliquots of the blood samples and fluoride were used for glucose content estimation with the glucose analyzer. The second batch of aliquots comprised of the blood samples and lithium-heparin, for the estimation of active insulin and glucagon content.
Subsequently, the aliquots were centrifuged before storage at -20°C for five minutes for hormone assay. The plasma concentration of the insulin analogue was determined by use of a standard curve. In this study, the variation coefficients for insulin analogue assay were 7-9%, 8-5% for glucagon assay, and 4-2% for native insulin. Then an assessment of the subcutaneous injection site for residual radioactivity was conducted using a radioactive detector fixed on the skin surface. This assessment was continuous; occurring repeatedly after the injection of the 125-I labeled samples. The residual activity measurements were corrected for background activity effects. The entire experiment occurred at a constant temperature of 22°C.
Data Analysis
Data analysis in this study involved comparative analysis of the three treatments (insulin assay, insulin analogue assay and glucagon assay) expressed in means and standard error measures. The Wilcoxon paired rank test was used to compare the three treatments. The results indicated that the insulin analogue injected subcutaneously disappeared faster than the soluble human insulin. The lower levels (p<0.05) of residual activity in the injection site for insulin analogue after two hours for a 0.05 U/kg dose and three hours for a 0.1U/kg dose indicated the faster rate of analogue insulin absorption compared to normal human insulin.
In particular, the half-life of the initial injection (for the 0.05U/kg dose) was 135 minutes for soluble human insulin compared to 61minutes for the insulin analogue preparation. For a higher dose (0.1U/kg), again the rate of disappearance of the initial radioactivity for the insulin analogue was higher (67 minutes for the initial radioactivity to decrease by half, compared to 145 minutes for the soluble human insulin preparations. In the blood sampling, the concentration of insulin increased following subcutaneous injections to a peak of 90pmol/l for the plasma insulin analogue. In comparison, the plasma concentration for the soluble human insulin stood at 50pm0l/l at between 10minutes and 1.5 hours. Overall, the level of plasma concentrations for plasma insulin analogue was significantly higher than the soluble human insulin in the basal period of 10 minutes to 1.5 hours. This implies that the absorption rate for the insulin analogue is greater compared to that of the soluble human insulin. Consequently, the study established that the hypoglycemic effect occurred faster for the insulin analogue than with the soluble human insulin preparations.
Conclusions of the Study
The study made the finding that, the absorption of monomeric insulin analogue, injected subcutaneously, is higher relative to that of soluble insulin, and thus, occurs at greater concentrations in the blood samples. From this finding, the authors concluded that monomeric insulin analogue is potentially useful for a faster insulin delivery through subcutaneous injection. This eliminates the requirement for the interval between subcutaneous injection and meals.
Critical Analysis of the Experiments
The results of this study indicate that monomeric insulin analogues are relatively effective when injected subcutaneously than normal human insulin. In particular, the researchers used monomeric insulin analogue with substituted amino acid residues (B9 serine and B27 threonine of the dimer replaced with aspartate and glutamate respectively). However, the authors did not describe how these substitutions affect the pharmacokinetic properties of the analogue. Normally, substitution of amino acids through rDNA technology result to rapid-acting insulin analogues, which explains the high rate of absorption of the monomeric insulin analogues. In this regard, the high rate of absorption of insulin analogues relative to soluble human insulin implies that monomeric insulin analogues induce physiological changes in the body. However, in the study, the high absorption rate is attributed to improved effectiveness of insulin analogues resulting from substitution.
Duration of Activity
The study did not address the shorter duration of action of monomeric insulin analogues relative to the normal human insulin. According to Kurtzhals, rapid-acting monomeric insulin analogues (with substituted amino acids residues at the monomer-monomer interfaces) exhibit a shorter duration of action (125). Additionally, for intermediate-acting insulin analogues, a small dose should be administered when the interval between meals is more than four hours. This implies that although insulin analogues exhibit a rapid absorption rates, their level of activity is dependent on the nature of residue substitution involving rDNA technology.
By contrast, the study made two observations; insulin analogues have higher absorption rates as indicated by low radioactivity within the basal period and higher plasma concentrations of insulin analogues relative to the normal human insulin preparations. Based on these observations, the authors conclude that subcutaneous injections of insulin analogues can achieve improved glycaemic control compared to the normal human insulin preparations. However, the study did not involve an examination of the efficacy of insulin analogues with regard to glycemic control for patients to support the assertion that, the faster absorption rates result to faster activity and reduced interval between dose administration and meals.
Additionally, the study involved healthy individuals with no genetic or familial predisposition to type one diabetes mellitus. According to Heinemann, the failure to replenish the basal insulin regularly for patients with type one diabetes mellitus during intensive insulin therapy (insulin lispro therapy) can result to insulin deficiency (673). Additionally, failure to replace the basal insulin lispro is more likely lead to hyperketonaemia than when normal human insulin preparation is used. These findings suggest that insulin analogues may not necessarily achieve sustained glycaemic control as therapeutic agents despite their high absorption rates. Therefore, the study could have explored the effectiveness of the insulin analogue with regard to reduction of elevated blood glucose level in type one diabetes mellitus patients after every meal. In this way, the activity of the insulin analogue with regard to glycaemic control can be compared to that of the normal human insulin. Further, the use of patient subjects can allow the assessment of glycaemic conditions that may result from a given insulin analogue therapy.
The Interval between Injection and Meals
In Vora et al study, the subjects underwent an overnight fast with an allowance of one hour (basal period) between the two subcutaneous injections (0.05 and 0.1U/kg doses) of either soluble human insulin or similar doses of the insulin analogue (1237). Of significance, however, is the study’s failure to address the interval between injection and the meals. Although evidence indicate a reduction in the interval between injections and meals due to the higher absorption rates of insulin analogues, the study did not compare the insulin analogue and normal human insulin with regard to glycaemic control for diabetics before and after meals. The interval between injections and meals is of profound significance to diabetic patients. As a result, the patients face a greater risk of hypoglycemia and poor glycaemic control.
Accordingly, the study could have explored the effect of the insulin analogue on the interval between injection and meals (whether it increases or decreases the basal period) relative to the normal human insulin preparations. Heinemann examined the effect of insulin analogue (rapid-acting rhI) on glycaemia following a carbohydrate-rich meal for patients with type one diabetes mellitus versus human insulin preparation (insulin lispro). The researchers demonstrated that, insulin lispro is more effective in controlling the blood glucose level after carbohydrate-rich meals for diabetic patients. This implies that insulin lispro can reduce the interval between injections and meals.
Additionally, the faster absorption rate for the insulin analogues as established in the article can produce adverse effects on some patients. The study failed to examine whether the insulin analogue’s mode of activity (whether long-acting, intermediate or rapid-acting) or binding mechanism. According to Kurtzhals, faster absorption coupled by shorter period of activity that is characteristic of most insulin analogues can be potentially dangerous (123). In particular, C-peptide negative diabetic patients under insulin analogue therapy face a greater risk of unstable glycaemic control as it requires that the basal insulin analogue be replaced regularly.
Summary and Conclusions
The rDNA technology has been used to modify the structure of insulin dimer through substitution of amino acids resulting to altered physiochemical properties such as self-dissociation of hexameric insulin to monomers, improved solubility, and increased affinity for human serum (Brange, and Volund 307). These properties enhance the plasma concentrations of the insulin analogues relative to the soluble human insulin, and therefore, provide better glycaemic control than soluble human insulin. Additionally, insulin analogues exhibit higher absorption rates resulting from improved dissociation of the hexamer to monomeric units. Vora et al study investigated an insulin analogue (B9 serine residue substituted with aspartate and B27 threonine residue substituted with glutamate) injected subcutaneously. They established that, the insulin analogue exhibited high absorption rate and a higher plasma concentration was achieved at a shorter period implying that, insulin analogues are better in glycemic control relative to soluble human insulin. However, the study failed to determine the effectiveness of the insulin analogue relative to human insulin and the stability of the plasma concentrations of the analogue compared to that of the normal insulin.
References
Brange, Jens, and Volund, Aage. “Insulin Analogs With Improved Pharmacokinetic Profiles.” Adv Drug Deliv Rev 35.4 (1999): 307-335.
Heinemann, Lutz. “Variability Of Insulin Absorption and Insulin Action.” Diabetes Technology & Therapeutics 4.5 (2004): 673-682.
Kurtzhals, Paulas. “Engineering Predictability and Protraction an a Basal Insulin Analogue: The Pharmacology Of Insulin Detemir.” Int J Obes 28.2 (2004):123-128.
Vora, Jiten, Owens, David, Dolben, John, Atiea, Jameel, Dean, John, Kang, Steven, Burch, Anna, and Brange, Jens. “Recombinant DNA Derived Monomeric Insulin Analogue: Comparison With Soluble Human Insulin In Normal Subjects.” BMJ 297.6658 (1988): 1236-1239.