Different Potencies of Biosynthetic Human and Purified Porcine Insulin

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Authors: K.J. Schlüter, F. Enxmann* and L. Kerp

Zentrum für Innere Medizin, Abteilung für Klinische Endokrinologie, Universität Freiburg, and *Eli Lilly, Bad Homburg, Germany

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Different Potencies of Biosynthetic Human and Purified Porcine Insulin

  1. 1. Fasting and Skeletal Muscle Enzymes in Obese Men Horm. metabol. Res. 15 (1983) 271 Rath, R., K. Vondra: Short-term fasting in the treatment of obesity. Vondra, K., R. Rath: Obesity and thyroid function. 2. The effect Nahrung 21: 193-197 (1977) of prolonged caloric restriction on Achilles tendon reflex values. Rath, R., Z. Slabochova, K. Vondra: Immunoreactive insulin in Endokrinologie 66: 332-336 (1975) obesity of adult women. Intemat J. of Obesity 1: 279-286 Vondra, K., R. Rath, A. Bass: Skeletal muscle HK activity and (1977) fasting FFA blood level in man. Horm. Metab. Res. 8: 323 (1976) Vondra, K., R. Rath: Obesity and thyroid function. 1. Values of Vondra, K., R. Rath, A. Bass, L. Kukla, Z. Slabochova: Effect of the Achilles tendon reflex. Endokrinologie 62: 310-320 (1973) protracted intermittent fasting on the activities of enzymes in- Vondra, K., R. Rath, Z. Kroupa: Improved needle for muscle volved in energy metabolism, and on the concentration of glyco- biopsy. Klin. Wschr. 52: 747-748 (1974) gen, protein a DNA in skeletal muscle of obese women. Nutr. Vondra, K., R. Rath, A. Bass: Activity of some enzymes of energy Metab. 20: 329-337 (1976) metabolism in striated muscle of obese subjects with respect Vondra, K., A. Bass, V. Brodan, E. Kuhn, M. Andel, A. Veselkova, to body composition. Horm. Metab. Res. 7: 475-480 (1975) V. Vitek: Activities of muscle energy supplying enzymes after 5 days complete fasting in young men. Physiol. Bohemoslov. (1982)31:311-314(1982) Requests for reprints should be addressed to: MUDr. K. Vondra, CSc, Department of Medicine I of the Institute for Clinical and Experimental Medicine, Videnska 800, 146 22 Prague 4 (Czechoslovakia) Horm. metabol. Res. 15 (1983) 271-274 ©Georg Thieme Verlag Stuttgart • New York Different Potencies of Biosynthetic Human and Purified Porcine Insulin K.J. Schliiter, F. Enzmann* and L. Kerp Zentrum fiir Innere Medizin, Abteilung fiir Klinische Endokrinologie, Universitat Freiburg, and * E l i Lilly, Bad Homburg, Germany Summary Introduction The glucose clamp technique was used to compare the biological The biological activity of insulin has been assessed by intra- activity of purified porcine insulin and Biosynthetic Human venous bolus injection, continuous infusion, and applica- Insulin (BHI). An intravenous bolus of 0.1 U/kg BW was tion of a single subcutaneous dose. Studies show that injected in eight male volunteers, and the glucose was intravenous bolus injection produce counterregulatory clamped at baseline values (euglycemic clamp). hormonal responses which modify the metabolism of Serum insulin, serum C-peptide and plasma glucose did not glucose. Since differences in the hormonal responses to differ between porcine and human insulin. The insulin in- duced glucose consumption differed significantly ( p < 0.007) human and porcine insulin have been reported, namely that between purified porcine insulin (50.5 ± 5.2 [SEM] g/2h) and human insulin produced a lower output of growth hormone, Biosynthetic Human Insulin (63.5 ± 4 . 5 g / 2 h ) . Purified Cortisol, and epinephrine (SchViter, Petersen, Borsche, porcine Insulin induced a hormonal response w i t h significantly Hobitz and Kerp 1981; SchViter, Petersen and Kerp 1982), ( p < 0.05) elevated concentrations of serum growth hormone (12.1 ± 0.25 ng/ml) and serum Cortisol (161.4 ± 28.6 ng/ml), it is difficult to compare the effects on glucose metabolism. which were not observed following Biosynthetic Human With the glucose clamp technique, essentially normal fasting Insulin (serum growth hormone: 2.6 ± 0.2 n g / m l ; serum plasma glucose concentrations can be maintained (Pfeiffer, Cortisol: 117.3 ± 14.8 ng/ml). The data confirm earlier results indicating hormonal and metabolic differences between Thum and Clemens 1974; DeFronzo, Tobin and Andres human and porcine insulin. 1979;Nosadini, Noy, Kurtz and Alberti 1981). Hormonal responses are minimal under these experimental conditions. Key-Words: Biosynthetic Human Insulin — Glucose Clamp The effect of Biosynthetic Human Insulin (BHI) and puri- Technique — Glucose Requirement — Growth Hormone — fied porcine insulin (PPI) on glucose metabolism were meas- Cortisol ured using the euglycemic clamp technique to avoid inter- ference of other hormones. Subjects and Methods Informed consent was obtained from 8 male volunteers (age: 24.2 ±1.3 years [mean ± SEM], body weight: 75.1 ± 1.3 kg; height: 185.6 Received: 15 March 1982 Accepted: 25 Aug. 1982 ±3.9 cm) without family or personal history of diabetes. Laboratory
  2. 2. 272 Horm. metabol. Res. 15 (1983) K.J. Schliiter, F. Enzmann and L. Kerp chemistry, ECG, physical examination and oral glucose tolerance tests (100 g) were normal. In a randomized study, each subject received both Biosynthetic Human Insulin (BHI) (Eli Lilly, Indianapolis, CT 4969-1B) and identical formulated purified pork insulin (PPI) (Eli Lilly, Indiana- polis, CT 4970-OA) intravenously (bolus: 0.1 U/kg BW). The com- pound of BHI used in this experiment was identical to the BHI (Lot 615-70N-174-9) used in the USP-rabbit hypoglycemia assay. The biological activity in rabbits was 27.5 ±1.7 units/mg, which was 160 nmoles/mg for BHI and 159 nmoles/mg for PPI. The tests were performed at one week intervals in this study. One hour before the administration of insulin, three catheters were in- serted into antecubital veins. The glucose-controlled insulin infusion system (Biostator, Life Science Instruments, Miinchen, FRG) was calibrated for continuous blood glucose monitoring and glucose in- fusion. The plasma glucose concentration was clamped at individual fasting baseline levels (± 0.25 mMol/1). The Biostator was pro- grammed to maintain the individual (76—96 mg/dl) euglycemia of the subjects. Glucose (40 g/100 ml) was infused through the three channels (saline-, glucose-, and optional channel) of the Biostator. The maximum glucose infusion by the Biostator was 2.4 g/min. After an hour's rest, a bolus of insulin (0.1 U/kg BW) was injected intravenously. Venous blood samples were obtained at-15, 0, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105 and 120 minutes. Plasma glucose was determined by the glucose oxidase technique with a Beckmann glucose analyzer (Beckmann Instruments, Inc., Fullerton, Calif.). The following radioimmunoassays were carried out: serum insulin (Phadebas-Insulintest, Pharmacia-Diagnostics, AB, Uppsala, Sweden) (the cross-reaction of PPI and BHI with the antibody used in this RIA was identical in the range from 10 MU/ml to 320 MU/ml), serum Fig. 1 Plasma glucose (mg/ml), serum insulin (MU/ml), serum C-peptide (Riamat, C-peptide assay, Byk-Mallinckrodt, Diezenbach, C-peptide (ng/ml) after an i.v. bolus (0.1 U/kg BW, given at 0 FRG), serum Cortisol (Cortisol-Ria, Travenol, Cambridge, Mass.), minutes) of Biosynthetic Human Insulin (• •) and purified and serum growth hormone (Serono, Freiburg, FRG). pork insulin (o—o) during euglycemic clamp in eight male Interassay variations were reduced by using the same immunoassay volunteers. The values at each time interval represent the mean for all samples of an individual subject. Intra-assay error, measured ± SEM of eight samples. There was no statistical significance of as coefficient of variation, was below 5% in all cases. differences between the porcine and human group. The data are expressed as mean ± SEM. Areas under the concentra- tion-time curves were calculated according to the equation: *f ( x n+l- x n> (vn+l + yn) L Wilcoxons test for paired differences was used. Results Eight male volunteers received either Biosynthetic Human Insulin (BHI) (0.1 U/kg BW) or purified porcine insulin (PPI) during an euglycemic clamp study. Plasma glucose, serum insulin, serum C-peptide, serum growth hormone, and serum Cortisol concentrations obtained from the venous blood samples are shown in Fig. 1 and Fig. 2. The individual fasting plasma glucose values (BHI: 89.1 ± 1.6 mg/dl;PPI: 90.1 ± 2.7 mg/dl) were maintained through- out the clamping procedure, but those following Bio- synthetic Human Insulin were slightly lower than those following purified porcine insulin. Throughout the experi- ment the maximum blood glucose fluctuation was 20 mg/dl and the coefficient of variation of the plasma glucose values Fig. 2 Serum growth hormone and serum Cortisol after an i.v. was below 15%. bolus (0.1 U/kg BW, given at 0 minutes) of Biosynthetic Human Insulin (• • ) and purified porcine insulin (o—o) during Serum insulin concentrations did not differ during the test euglycemic clamp in eight male volunteers. The values at each time period. Following the intravenous administration identical interval represent the mean ± SEM of eight samples. X, values that differ significantly from the human insulin group at peak values (BHI: 309.3 ± 61.0 juU/ml; PPI: 335.2 ± 64.2 that time interval (p< 0.05 by paired Wilcoxon's test). /xU/ml) of insulin were obtained.
  3. 3. Potencies of Biosynthetic Human and Purified Porcine Insulin Horm. metabol. Res. 15 (1983) 273 Serum C-peptide levels following Biosynthetic Human Chiasson, Keller and Rubenstein 1978; Beischer, Schmid, Insulin and purified porcine insulin did not differ signifi- Kerner, Keller and Pfeiffer 1978) may be due to this tech- cantly. A suppression of endogenous insulin secretion by nical difference. exogenous insulin was not observed (Fig. 1). A significant A small but significant rise in serum growth hormone and (p< 0.05) elevation of serum growth hormone concentra- serum Cortisol concentrations was observed following the tion (from 2.8 ± 1.7 ng/ml at 0 minutes to 12.1 ± 6.2 ng/ml injection of a bolus of purified porcine insulin, which was at 30 minutes) occurred after the administration of puri- not observed after Biosynthetic Human Insulin. This can- fied porcine insulin. BHI did not produce any fluctuation not be attributed to differences in plasma glucose or to of serum growth hormone (2.5 ± 1.9 ng/ml at 0 minutes plasma glucose concentrations because plasma glucose was and 2.5 ± 2.0 ng/ml at 30 minutes). clamped at individual fasting glucose levels. The secretion Serum Cortisol levels were also elevated (A serum Cortisol of growth hormone and of Cortisol appears to be a response 38.8 ± 8.1 ng/ml) after purified porcine insulin but not to the heterologous insulin. after human insulin (A serum Cortisol 23.1 ± 4.5 ng/ml) Significantly more exogenous glucose was required to (p<0.05). compensate the hypoglycemic effect of Biosynthetic Human The amount of glucose required to compensate the effect Insulin in comparison to purified pork insulin (+ 30.6 ± 7.6 of exogenous insulin was significantly (2p< 0.007) higher percent). In five insulin-dependent diabetic subjects BHI after Biosynthetic Human Insulin (63.5 ± 4.5 g/2h) than was more (but not significantly) effective than natural pork after purified porcine insulin (50.4 ±5.2 g/2h). insulin (Klier, Kerner, Torres and Pfeiffer 1981). This dif- Table 1 shows the individual glucose requirements over the ference which is in contrast to a previous study (Massi- two-hour periods for each subject. Benedetti, Burrin, Capaldo and Alberti 1981) with the insulin infusion technique, can be explained by the small in- crements of endogenous Cortisol and growth hormone ob- served after porcine insulin. Cortisol and growth hormone produced hyperglycemia by decreasing both hepatic and Discussion extrahepatic sensitivity to insulin (Rizza, Mandarino and The glucose clamp technique has been used to assess the Gerich 1981; Rizza, Mandarino, Westland and Gerich biological activity of insulin by continuous infusion (De 1981). Fronzo, Tobin and Andres 919;Pfeiffer, Thum and Clemens 1974;Nasadini, Noy, Kurtz and Alberti 1981; On the other hand a significantly inhibited hepatic glucose Massi-Benedetti, Burrin, Capaldo and Alberti 1981). In this production has been observed after porcine insulin in com- study, however, a bolus injection of insulin was used. The parison to semisynthetic human insulin (Mutter, Keller and peak serum insulin concentrations (about 300 /iU/ml in Berger 1982). The weaker suppression of hepatic glucose comparison to values obtained by continuous infusion of production following porcine insulin, caused by Cortisol about 50—100 ^U/ml) (Massi-Benedetti, Burrin, Capaldo and growth hormone secretion, might explain the comparab- and Alberti 19Sl;Dobeme, Schulz and Reaven 1981) per- ly low glucose requirement following porcine insulin in- sisted for only a short period while continuous infusion jection in comparison to human insulin. The results indi- results in prolonged elevated levels. The fact that we have cate that homologous insulin produces effects which are not observed a suppression of endogenous insulin secretion different from those produced by heterologous insulin in as judged by serum C-peptide (Liljenquist, Horwitz, Jennings, man. Acknowledgements Table 1 Glucose requirement (g/2h) after a bolus injection (0.1 U/kg The authors gratefully acknowledge the skilled technical assistance BW) of Biosynthetic Human Insulin (BHI) and purified pork insulin of Mrs. Heike Vorwerck and thank Prof. Dr. John A. Galloway (PPI) during euglycemic clamp. (University of Indianapolis) for his comments on the manuscript. glucose requirement g/2h Subject BHI PPI References No. Beischer, W., M. Schmid, W. Kerner, L. Keller, E.F. Pfeiffer: Does insulin play a role in the regulation of its own secretion? Horm. Metab. Res. 10: 168-169 (1978) De Fronzo, R.A., J.D. Tobin, R. Andres: Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am. J. Physiol. 237 (3): E214-E223 (1979) Doberne, L., M.S. Greenfield, B. Schulz, M. Reaven: Enhanced glucose utilization during prolonged glucose clamp studies. Diabetes 30: 829-835 (1981) Klier, M., W. Kerner, A.A. Torres, E.F. Pfeiffer: Comparison of the biologic activity of Biosynthetic Human Insulin and natural pork insulin in juvenile-onset diabetic subjects assessed by the glucose controlled insulin infusion system. Diabetes Care 4: *Significance of mean differences, p< 0.007. 193-195 (1981)
  4. 4. 274 Horm. metabol. Res. 15 (1983) K J. Schliiter, F. Enzmann and L. Kerp Liljenquist, J.E., D.L. Horwitz, A.S. Jennings, J.-L. Chiasson, Rizza, R., L. Mandarino, J. Gerich: Dose-response characteristics U. Keller, A.H. Rubenstein: Inhibition of insulin secretion by for the effects of insulin on production and utilization of exogenous insulin in normal man as demonstrated by C-peptide glucose in man. Am. J. Physiol. 240: 630-639 (1981) assay. Diabetes 27: 563-570 (1978) Rizza, R., L. Mandarino, R. Westland, J. Gerich: Growth hormone Massi-Benedetti, M., J.M.Burrin, B. Capaldo, K.GM.M. Alberti: induced insulin resistance in man: Postreceptor impairment in A comparative study of the activity of biosynthetic human hepatic and peripheral tissue sensitivity to insulin. Diabetes 30: insulin and pork insulin using the glucose clamp technique in 38(1981) normal subjects. Diabetes Care 4:163-167 (1981) Schliiter, K.J., K.-G. Petersen, A. Borsche, H. Hobitz, L. Kerp: Effects Miiller, R., U. Keller, W. Berger: Comparison of semisynthetic and of fully synthetic human insulin in comparison to porcine insulin porcine insulin in man. J. Clin. Invest. 12: 281 (1982) in normal subjects. Horm. Metab. Res. 13: 657-659 (1981) Nosadini, R., G. Noy, A.B. Kurtz, K.G.M.M. Alberti: Differential Schliiter, K.J., K.-G. Petersen, A., L. Kerp: Unterschiedliche Wir- response to infusions of highly purified and conventional bovine kung von Human- und Schweineinsulin. In: Neue Insuline, eds. and porcine insulins. Diabetes 30: 650-655 (1981) Petersen, K.-G., K.J. Schliiter, L. Kerp. Freiburg, Freiburger Pfeiffer, E.F., Ch. Thum, A.H. Clemens: The artificial beta cell - a Graphische Betriebe, 86-92 (1982) continuous control of blood sugar by external regulation of insulin infusion (glucose controlled insulin infusion system). Horm. Metab. Res. 487: 339-342 (1974) Requests for reprints should be addressed to: K.J. Schliiter, M.D., Zentrum fur Innere Medizin, Universitat Freiburg, Hugstetter Str. 55, D-7800 Freiburg (Germany) Horm. metabol. Res. 15 (1983) 274-278 ©Georg Thieme Verlag Stuttgart • New York 125 Clinical Factors Influencing the Absorption of I-NPH Insulin in Diabetic Patients K. Kdlendorf1, J. Bojsen2 and T. Deckert1 1 Steno Memorial Hospital, Gentofte, and The Finsen Laboratory, The Finsen Institute, Copenhagen, Denmark Summary Introduction Clinical factors which might influence the absorption of sub- Earlier studies have shown great inter- and intrasubject cutaneously injected 125 I-NPH insulin were studied in 101 variations for absorption of intermediate-acting insulins diabetics. The disappearance curve was monoexponential (Binder 1969; Galloway, Spradlin, Nelson, Wentworth, after a delay period of 1.5±0.8 h (mean ± SD). Lipohyper- Davidson and Swamer 1981; Lauritzen, Faber and Binder trophy significantly prolonged insulin absorption (half life ( T 1 / 2 ) = 11.2±3.1 h, p = 0.0001 >. Low bicarbonate levels 1979). However, only the influence of age and diabetes increased the absorption ( T 1 / 2 3.9±2.3 h , p < 0.05). Lean duration on NPH insulin absorption have been systematical- diabetics had a faster absorption (6.2 ± 1.9 h) than normal ly studied (Dobson, Robbins, Johnson, Mdalel, Odem, Corn- weight diabetics (7.5 ± 2.0 h, p < 0.02). Sex, age, diabetes wall and Davis 1967). The aim of this study was to assess duration and injection depth d i d not influence T 1 / 2 . The some clinical factors which might influence the absorption half life was significantly inversely correlated to the resting subcutaneous blood f l o w (r= - 0 . 8 8 2 , p < 0.01). The overall of I25I-NPH insulin from the subcutaneous tissue in diabetic interindividual coefficient of variation for insulin absorption patients by using the biotelemetric technique. The absorp- in nonketotic diabetics was 27.4%. Also considerable intra- tion of insulin was measured to determine the influence patient day-to-day variation was found (24.5%), and between of lipohypertrophy, ketosis, body weight, sex, age, dia- different injection sites (30.2%). These variations emphasize betes duration and inter- and intrapatient variation. In some the drawbacks of conventional insulin therapy in the manage- ment of insulin-requiring diabetics. diabetics, also the effect of injection depth and subcutane- ous bloodflow(SBF) was evaluated. Key-Words: Insulin Absorption — NPH Insulin — Diabetes Mellitus — Lipohypertrophy — Ketosis — Body Weight — Sex — Age — Diabetes: Duration — Subcutaneous Blood-Flow — Material and Methods 125 l-NPH Insulin One hundred and one diabetic patients were investigated as inpatients in 194 studies after informed consent was obtained. Group 1 com- prised 20 patients with insulin-dependent diabetes mellitus (IDDM) Received: IS March 1982 Accepted: 31 July 1982 with palpable lipohypertrophy at the injection site. Group 2 included

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