Impact of Diabetic Nephropathy on Pharmacodynamic and Pharmacokinetic Properties of Insulin in Type 1 Diabetic Patients

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1 Emerging Treatments and Technologies O R I G I N A L A R T I C L E Impact of Diabetic Nephropathy on Pharmacodynamic and Pharmacokinetic Properties of Insulin in Type 1 Diabetic Patients KLAUS RAVE 1 TIM HEISE 1 ANDREAS PFÜTZNER 2 1 LUTZ HEINEMANN PETER T. SAWICKI 3 OBJECTIVE To quantify properties of regular insulin and insulin lispro in type 1 diabetic patients with and without overt diabetic. RESEARCH DESIGN AND METHODS In this double-blind, two-way cross-over, euglycemic (5 mmol/l) glucose clamp study, we investigated the metabolic response to subcutaneous injections of regular insulin and insulin lispro (0.2 U/kg) in 12 type 1 diabetic patients with overt diabetic (proteinuria 500 mg/24 h and/or serum creatinine 1.5 mg/dl; NP group) and in a control group of 12 type 1 diabetic patients with normal renal function (DC group). RESULTS Peak plasma free insulin levels with insulin lispro (359 [NP] vs. 254 pmol/l [DC]) were higher and time to maximal insulin concentrations (85 [NP] vs. 99 min [DC]) shorter than with regular insulin (213 [NP] vs. 144 pmol/l [DC]; 118 [NP] vs. 153 min [DC]) in both patient groups. Overall insulin levels for regular insulin and for insulin lispro were higher in patients with overt diabetic compared with control patients. Time to maximal metabolic effect was shorter with insulin lispro than with regular insulin in both patient groups (102 vs. 191 min [NP]; 105 vs. 172 min [DC]). The overall metabolic effect of regular insulin but not of insulin lispro was lower in patients with diabetic than in diabetic control patients (967 vs. 1,510 mg/kg, respectively). CONCLUSIONS Although insulin levels are higher in patients with overt diabetic, the metabolic response to regular insulin is reduced. Insulin lispro maintains its characteristic properties in patients with overt diabetic. Insulin therapy in type 1 diabetic patients with overt diabetic is characterized by two features: while suffering from a considerably increased risk of severe hypoglycemic episodes (1,2), they show poorer metabolic control than diabetic patients with normal renal Diabetes Care 24: , 2001 function (3). The increased hypoglycemic risk may be partially explained by the decline in renal insulin clearance, as 30 80% of systemically circulating insulin is removed by the kidneys (4). A reduction of insulin clearance has been described in nondiabetic patients with end-stage renal From the 1 Profil Institute for Metabolic Research, Neuss; 2 Institut für Klinische Forschung und Entwicklung, Mainz; and 3 St. Franziskus Hospital, Cologne, Germany. Address correspondence and reprint requests to Dr. Klaus Rave, Profil Institute for Metabolic Research GmbH, Stresemannallee 6, 41460, Neuss, Germany. klaus.rave@profil-research.de. Received for publication 31 October 2000 and accepted in revised form 18 January Abbreviations: AUC, area under the curve; GIR, glucose infusion rate; INS, insulin. A table elsewhere in this issue shows conventional and Système International (SI) units and conversion factors for many substances. failure (5,6) and mild to moderate renal impairment (7); however, until now, it has not been studied in patients with diabetic. Insulin sensitivity, on the other hand, is attenuated in patients with renal impairment, not only in those with endstage renal failure, but also in patients in earlier stages of renal impairment (7 9). However, to date the metabolic consequences of reduced insulin clearance and diminished insulin sensitivity on pharmacokinetic and pharmacodynamic properties of regular insulin or rapid acting insulin analogues have never been studied in type 1 diabetic patients. In view of the diminished clearance of insulin in patients with altered renal function, the use of a rapid-acting insulin analogue might theoretically be advantageous because of its shorter duration of action, leading to a reduced incidence of hypoglycemic episodes (10,11). Therefore, the aim of this study was to quantify summary measures for regular insulin and insulin lispro in type 1 diabetic patients with and without overt diabetic. RESEARCH DESIGN AND METHODS Patients For this study, 12 type 1 diabetic patients fulfilling the following inclusion criteria were recruited: age years; GHb 10%; overt diabetic (defined according to the modified Mogensen criteria: proteinuria 500 mg/24 h on repeated measurements and/or serum creatinine 1.5 mg/dl [122 mol/l] with no clinical or laboratory evidence of renal or urinary tract disease other than diabetic glomerulosclerosis) (12); negative insulin-antibody screening; and a negative pregnancy test in women with childbearing potential. The control group consisted of 12 type 1 diabetic patients 886 DIABETES CARE, VOLUME 24, NUMBER 5, MAY 2001

2 Rave and Associates Table 1 Baseline characteristics of type 1 diabetic patients with and without overt diabetic Characteristic Patients with without ; that is, their protein excretion and endogenous creatinine clearance were within the normal range (Table 1). Patients with overt diabetic were older and had a longer duration of diabetes than patients without (29 vs. 18 years). The mean HbA 1c was higher in patients with diabetic, whereas the mean daily insulin dosage was comparable for both groups of patients. According to the inclusion criteria, patients with overt diabetic showed higher serum creatinine levels and lower glomerular filtration rates. This randomized study was performed in a double-blind fashion (study medication was applied by a person otherwise not involved in the study). The study protocol was approved by the local ethical committee and was carried out according to the guidelines of Good Clinical Practice. After detailed oral and written explanation of the study objectives, all patients gave their written informed consent to participate in the study. After a fasting interval of 12 h, patients arrived at our institute in the morning ( 8:00 A.M.) and were connected to a Biostator (Life Science Instruments, Elkhart, IN). The left hand was placed in a box that was warmed to an air temperature of 55 C to allow the sampling of arterialized venous blood. Fasting blood glucose of the patients was lowered during the first 60 min of a 4-h run-in period by means of an insulin infusion to a target level of 5.0 mmol/l (90 mg/dl). After having reached this target level, blood glucose was kept constant Patients without Sex (female/male, n) 6/6 3/ Age (years) Weight (kg) BMI (kg/m 2 ) Duration of diabetes (years) HbA 1c (%) Serum creatinine ( mol/l) GFR (ml/min/[1.73 m 2 ]) Proteinuria (mg/24 h) Daily insulin dosage (U/day) Data are means SD or frequencies. GFR, glomerular filtration rate. over the next 180 min by adapting the dosage of a continuous intravenous insulin infusion. At the end of the run-in period ( 12:00 P.M.), patients received subcutaneous injections of 0.2 U/kg body wt of either regular human insulin (Humulin U 100; Lilly Deutschland, Bad Homburg, Germany) or insulin lispro (Humalog U 100; Lilly Deutschland); patients with received U (mean SD) and diabetic control subjects received U into a lifted skinfold in the abdominal region. Thereafter, an 8-h euglycemic glucose clamp was established, as described in detail elsewhere (13). Mean blood glucose levels during glucose clamps in patients with overt diabetic were mmol/l with regular insulin and mmol/l with insulin lispro. For diabetic control patients, mean blood glucose was mmol/l with regular insulin and mmol/l with insulin lispro during the glucose clamp experiments. All patients were on an intensified insulin therapy before the study and were instructed to take their last short-acting insulin injection in the evening and their long-acting insulin injection at the latest 24 h before the glucose clamp experiments. Insulin pump treated patients were asked to stop the insulin infusion at 6:00 A.M. on the day of the glucose clamp. To exclude ongoing carbohydrate absorption during the time of the glucose clamp, which may occur in patients with severely impaired gastric emptying, a modified 13 C-labeled octanoid acid breath test was performed before every P glucose clamp (14). In brief, patients gave a baseline breath sample in an aluminumcoated bag in the evening preceding the glucose clamp. Thereafter, they ingested a test meal including a scrambled egg with the yolk supplemented with 0.1 ml 13 C-octanoid acid (Wagner Analysen Technik, Bremen, Germany) at 8:00 P.M. Additional breath samples were collected the next morning and after completion of the glucose clamp run-in period. All three samples were analyzed for their 13 C/ 12 C ratio using an infrared isotope analyzer (Iris Infrared Isotope Analyzer; Wagner Analysen Technik). A 13 C/ 12 C ratio 2.5% above baseline would be regarded as a sign of an ongoing carbohydrate absorption attributable to delayed gastric emptying. In those cases, that particular experiment would have to be excluded from further analysis. However, this was never necessary. Blood glucose was measured by a glucose oxidase method (Super G Ambulance, RLT Möhnesee, Germany). HbA 1c was measured with high-performance liquid chromatography (Diamat, Munich, Germany; reference range, %). Proteinuria was determined by means of a laser turbidimetric method (Laserphotometer pm4, Lasermed GmbH, Cologne, Germany) in 24-h urine samples. The glomerular filtration rate was assessed by endogenous creatinine clearance in 24-h urine samples, corrected to 1.73 m 2 body surface. Plasma concentrations of free regular insulin and free insulin lispro were determined after polyethylene glycol precipitation of antibody bound insulin by means of a commercial radioimmunoassay kit (DPC Coat-A-Count Insulin Radioimmunoassay; DPC, Bad Nauheim, Germany; inter- and intraassay coefficients of variation 15.0 and 6.6%, respectively). This assay has recently been validated for the determination of free human insulin and free insulin lispro with similar sensitivity (15). Insulin-antibodies were determined by means of a radioligand assay (CentAK, Medipan Diagnostika, Selchow, Germany). Statistics A polynomial function of sixth order was fitted to the individual glucose infusion rate (GIR) and free insulin profiles, allowing the calculation of pharmacokinetic and pharmacodynamic summary measures. The area under the curve (AUC) for DIABETES CARE, VOLUME 24, NUMBER 5, MAY

3 Diabetic and insulin Figure 1 A: Plasma free insulin levels after subcutaneous injection of 0.2 U/kg regular insulin ( ) and insulin lispro (F) in type 1 diabetic patients with overt diabetic. Thick lines indicate free insulin concentrations; thin lines indicate fitted polynomial function of sixth order. Data represented are means SE. B: Plasma free insulin levels after injection of 0.2 U/kg regular insulin (f) and insulin lispro (E) in type 1 diabetic patients without diabetic. Thick lines indicate free insulin concentrations; thin lines indicate fitted polynomial function of sixth order. C: Fitted polynomial function of sixth order to glucose infusion rate after subcutaneous injection of 0.2 U/kg regular insulin (heavy black line) and insulin lispro (dotted line) in type 1 diabetic patients with overt diabetic. Thin lines indicate glucose infusion rate original registration. D: Fitted polynomial function of sixth order to glucose infusion rate after subcutaneous injection of 0.2 U/kg regular insulin (heavy black line) and insulin lispro (dotted line) in type 1 diabetic patients without diabetic. Thin lines indicate glucose infusion rate original registration. GIR and free insulin was calculated by the trapezoid rule. Wilcoxon s signed-rank test was used for within-group statistical analyses, and the Mann-Whitney U test was applied for between-group comparisons. Multiple regression analyses were performed by using the summary measures as response variables and status, diabetes duration, and HbA 1c as explanatory factors. The aim of these analyses was to assess the impact of diabetic adjusted to diabetes duration and HbA 1c on the response variables. Because of the exploratory character of the study, no adjustments for multiple comparisons were performed. With multiple regression analyses, only the overall metabolic activity for regular insulin in patients without diabetic was significantly associated with a parameter other than the status (i.e., HbA 1c ). For statistical calculations, StatView 5.0 (SAS Institute, Cary, NC) was used. P 0.05 was considered statistically significant. Results are given as means SD throughout the text and as means SE in the figure. RESULTS Peak free plasma insulin levels (INS max ) were higher and time to maximal insulin concentration (t INS max ) was shorter after subcutaneous administration with insulin lispro than after injection of regular insulin in diabetic patients with (P for both) and without (P 0.02 and P 0.006, respectively) (Fig. 1A and B; Table 2). Accordingly, insulin AUCs for the first 3 h after injection were higher with insulin lispro than with regular insulin in both patient groups (P and P 0.005, respectively). When comparing nephropathic patients with the diabetic control subjects, both with regular insulin and with insulin lispro, peak free plasma insulin levels and the insulin AUCs for the intervals 0 3 h and 0 8 h were higher in patients with overt diabetic (regular insulin: P for free plasma insulin, P for 0 3 h AUC, and P for 0 8 h AUC; insulin lispro: P for free plasma insulin, P for 0 3 h AUC, and P for 0 8 h AUC). In contrast, time to late half maximal insulin concentration (late t INS 50% ) for regular insulin and insulin lispro did not differ 888 DIABETES CARE, VOLUME 24, NUMBER 5, MAY 2001

4 Rave and Associates Table 2 Pharmacokinetic and pharmacodynamic summary measures in 12 type 1 diabetic patients with overt diabetic and 12 diabetic patients without diabetic Regular insulin significantly between the group of patients with and without. Comparing the metabolic activity of regular insulin with insulin lispro, the maximal metabolic effect of subcutaneously injected insulin lispro was higher (P 0.003) than the effect of regular insulin in patients with overt diabetic, and tended to be higher (P 0.078) in patients without (Fig. 1C and D; Table 2). Time to maximal metabolic effect (t GIR max ), time to early half maximal activity (early t GIR 50% ), and time to late half maximal activity (late t GIR 50% ) were significantly shorter with insulin lispro than with regular insulin in both patient groups (patients with : P for t GIR max, P 0.03 for early t GIR 50%, and P for late t GIR 50% ; diabetic control subjects: P for t GIR max, P 0.02 for early t GIR 50%, and P for late t GIR 50% ). The metabolic activity during the first 3 h after injection (AUC GIR 0 3 h ) with insulin lispro was higher than with regular insulin in patients with overt diabetic (P 0.004). However, the metabolic activity 4 8 h after administration of insulin (AUC GIR 4 8 h ) was higher for regular insulin than for insulin lispro Patients with Insulin lispro Regular insulin Patients without Insulin lispro INS max (pmol/l) * * t INS max (min) * * Early t INS 50% (min) * Late t INS 50% (min) * * AUC INS 0 3 h (nmol/l) * * AUC INS 0 8 h (nmol/l) * GIR max (mg kg 1 min 1 ) t GIR max (min) * * Early t GIR 50% (min) * * Late t GIR 50% (min) * * AUC GIR 0 3 h (mg/kg) * AUC GIR 0 8 h (mg/kg) AUC GIR 4 8 h (mg/kg) * * AUC GIR 0 8 h /AUC INS 0 8 h *P 0.05 for within-group comparison (patients with overt diabetic or diabetic patients without ) between insulin lispro and regular insulin; P 0.05 for between-group comparison for regular insulin or for insulin lispro. INS max, maximal plasma free insulin concentration; t INSmax, time to maximal free insulin concentration; early/late t INS 50%, time to early/late half-maximal insulin concentration; AUC INS 0 3 h, AUC for free insulin concentration 0 3 h; GIR max, maximal glucose infusion rate; t GIR max, time to GIR max ; early/late t GIR 50%, time to early/late half-maximal glucose infusion rate; AUC GIR 0 3 h, AUC for glucose infusion rate 0 3 h. in both patient groups (P 0.02 for patients with vs for diabetic control subjects). When comparing patients with overt diabetic with diabetic control subjects, the maximal metabolic effect (GIR max ) for regular insulin was lower (P 0.03) and metabolic activity 3 and 8 h after administration of insulin was smaller (P 0.05 and P 0.05, respectively) in patients with overt diabetic than in patients without renal impairment. It is noteworthy that the lower metabolic effect of regular insulin in patients with overt diabetic was in contrast to the higher insulin levels observed in these patients. Glucose consumption per plasma insulin level (Table 2) was significantly lower in diabetic patients with overt than in control subjects with insulin lispro or regular insulin (P and P 0.03, respectively). CONCLUSIONS This study demonstrates for the first time that plasma insulin levels the maximal insulin concentration and insulin levels during 8 h after subcutaneous injection of regular insulin and insulin lispro were higher in patients with diabetic than in diabetic control patients without. This finding suggests a 30 40% reduction of insulin clearance in patients with overt diabetic compared with diabetic patients without. Paradoxically, in spite of higher circulating insulin levels, both the peak metabolic effect and the overall metabolic activity of regular insulin were diminished in patients with overt diabetic when compared with diabetic patients without renal impairment. Because of the lower metabolic response to subcutaneous regular insulin, patients with overt diabetic would have to inject a higher dosage of regular insulin to achieve a metabolic effect comparable with that of nonnephropathic patients. Based on the metabolic activity of regular insulin observed in this study, it can be calculated that a 50% higher dosage is necessary for patients with to achieve the same metabolic effect as diabetic patients with normal renal function. With increasing dosages of regular insulin, however, it has been shown that not only the maximal metabolic response but also the duration of action (i.e., the overall blood glucose lowering effect) increases (16). The prolongation of action with higher insulin dosages, in turn, may increase the risk for late postprandial hypoglycemic episodes. Therefore, the necessity for higher dosages of regular insulin with the resulting prolonged effect may partially explain the substantially higher risk for severe hypoglycemia in patients with diabetic (2). In our study, patients with overt diabetic had a slightly, but not significantly, lower daily insulin dosage compared with diabetic patients without. At first glance, this observation contradicts our finding that diabetic patients with overt diabetic need higher dosages of insulin to achieve the same metabolic effect as patients without renal impairment. However, patients with overt in this study not only injected less insulin, but at the same time exhibited a metabolic control significantly worse than that of diabetic control patients, thus confirming the reduced response to insulin. In the group of patients with overt diabetic, we included subjects exhibiting macroproteinuria and/or elevated serum creatinine. Hence, in pa- DIABETES CARE, VOLUME 24, NUMBER 5, MAY

5 Diabetic and insulin tients classified as having, 10 of 12 subjects showed proteinuria 500 mg/24 h and 7 of 12 patients exhibited serum creatinine 1.5 mg/dl (122 mol/l). One may speculate that in nephropathic patients with moderate to severe renal impairment (serum creatinine 1.5 mg/dl), insulin clearance and/or insulin action that is, pharmacokinetic and pharmacodynamic parameters may have been different compared with patients with but with normal glomerular filtration rate or only mild renal impairment. Therefore, we analyzed the results for these two subgroups of patients in our study separately and interestingly did not find any statistically significant differences between both patient groups. An important finding of this study is that insulin lispro, in comparison to regular insulin, maintained its characteristic properties in patients with overt diabetic. Given that it has been demonstrated that the duration of action of insulin lispro, in contrast to regular insulin, only moderately increases with higher dosages (16), it seems possible that fastacting insulin analogues like insulin lispro and insulin aspart could be of particular advantage in patients with diabetic in reducing the risk of hypoglycemia (17). However, this remains to be shown in adequate clinical studies. The increased risk of patients with overt diabetic for severe hypoglycemic episodes cannot be explained simply by a prolonged duration of insulin action attributable to reduced insulin clearance. In fact, the time to late half maximal insulin concentrations for both regular insulin and insulin lispro was not higher in patients with. More important, the duration of metabolic activity in terms of time to late t GIR 50% was similar for either insulin formulation in patients with and without overt diabetic. Apart from the altered metabolism of insulin and the reduced insulin action, the contribution of the kidneys to gluconeogenesis in the postabsorptive state has to be taken into account in patients with renal impairment (18). Results of two studies in type 1 and type 2 diabetic patients indicate that renal glucose production in the diabetic state per se is increased and accounts for 30% of the overall gluconeogenetic capacity (19, 20). It seems sensible to assume that renal gluconeogenetic capacity decreases with declining renal function, which, consequently, would increase the risk of hypoglycemia in these patients. In summary, we have shown that peak insulin concentrations and overall insulin levels were higher in patients with overt diabetic than in diabetic patients with normal renal function. In contrast to the higher insulin levels, the overall metabolic response to regular insulin but not to insulin lispro was lower in patients with diabetic as in diabetic control patients. The promising results of insulin lispro in patients with overt diabetic should promote clinical studies aimed at reducing the hypoglycemic risk and improvement of metabolic control in these patients (11). Acknowledgments This study was partly supported by a research grant from Lilly Deutschland, Bad Homburg, Germany. We gratefully acknowledge the excellent technical assistance of Uta Eckers, Till Schroer, Andre Feldmann, and Claudia Gottschalk. Our special thanks go to Dr. Ralf Bender for his valuable advice in statistical questions. References 1. Arem R: Hypoglycemia associated with renal failure. Endocrinol Metab Clin North Am 18: , Mühlhauser I, Toth G, Sawicki PT, Berger M: Severe hypoglycemia in type 1 diabetic patients with impaired kidney function. Diabetes Care 14: , Bending JJ, Pickup JC, Viberti GC, Keen H: Glycaemic control in diabetic. BMJ 288: , Rabkin R, Ryan MP, Duckworth WC: The renal metabolism of insulin. 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