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Mechanism of Action in Dogs of Slow-Acting Insulin Analog O346

Department of Physiology and Biophysics (M.E., M.H.-W., S.P.K., M.K.D., E.K., A.P., J.M., R.N.B.), Keck School of Medicine, University of Southern California, Los Angeles, California 90033; and Novo Research Institute (J.M.), Novo Nordisk A/S, DK-2880 Bagsvaerd, Denmark

Address all correspondence and requests for reprints to: Richard N. Bergman, Ph.D., Department of Physiology and Biophysics, University of Southern California School of Medicine, 1333 San Pablo Street, MMR 626, Los Angeles, California 90033. E-mail: rbergman{at}usc.edu.

	Abstract

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We compared metabolic effects as well as plasma and interstital fluid kinetics of fatty acid-acylated insulin, Lys^B29(N--carboxynonadecanoyl)-des(B30) human insulin (O346), with previously determined kinetics of native insulin and insulin detemir. Euglycemic clamps with iv injection of O346 (90 pmol/kg) or saline control were performed in 10 male mongrel dogs under inhalent anesthesia. The t_1/2 for the clearance of O346 from plasma was 375.7 ± 26.7 min; the t_1/2 for the appearance of O346 in interstital fluid was 137 ± 20 min (mean ± SEM). Glucose disposal with O346 injection was increased 4-fold (t = 480 min, 8.3 ± 1.42 mg/min/kg) compared with preinjection (t = 0 min, 2.1 ± 0.13 mg/min/kg; P < 0.05) or saline control (t = 480 min, 2.09 ± 0.22 mg/min/kg; P < 0.05). O346 plasma elimination and transendothelial transport were 0.3% and 3.5% of regular insulin and 3% and 50% of insulin detemir, respectively. Combination of in vivo results and compartmental modeling suggests that the duration of action of O346 after iv injection is about 25-fold and 10-fold longer compared with regular human insulin and insulin detemir, respectively. This study demonstrates that O346 stimulates glucose disposal very slowly, but when injected iv, its effect may be maintained for as long as 48 h as estimated from simulation analysis. The data suggest that O346 bound to albumin in plasma acts as a storage compartment for O346 from which the analog is slowly released to insulin-sensitive tissues. Reduced liver clearance of O346 is suggested to be the major mechanism for the protracted action.

	Introduction

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OVER THE LAST decade, human insulin analogs have been introduced with metabolic properties differing from the native hormone. Short-acting insulin analogs with a rapid onset of action have been developed to improve 1-h and 2-h postprandial glucose levels (1). Replacement of basal insulin also must be optimized to improve long-term glycemic control. This has been approached by continuous sc insulin infusion as well as multiple daily administration of neutral protamine Hagedorn (NPH). The development of a peakless long-lasting insulin preparation that mimics the flat interprandial insulin secretion necessary to provide stable overnight and between-meal glycemia would benefit diabetic patients by relieving them of multiple dosing to achieve basal insulinemia. One approach to slowing the metabolic effects of insulin was proposed by Kurtzhals and colleagues (2, 3), who showed that insulin acylated with fatty acids at the -amino group of Lys B29 has prolonged action due to its affinity for human serum albumin. Results from rabbits and pigs using several fatty acid-acylated analogs that differ in their binding affinity for human plasma albumin was recently confirmed by extensive studies in dogs and humans using Lys B29-tetradecanoyl des-(B30) insulin (insulin detemir), which showed the most protracted action of all fatty acid-acylated insulin analogs so far examined (4, 5).

It is well known that human insulin acts rapidly in vitro (6); however, once secreted or exogenously infused into plasma, it stimulates glucose uptake slowly (7). Transendothelial transport of insulin from plasma to the interstitial fluid of insulin-sensitive tissues has been suggested to be responsible for this protracted in vivo action of insulin (8, 9). Similar to the mechanism reported for human insulin, it has recently been shown that transendothelial transport and clearance by the liver are rate-limiting steps for insulin detemir action in vivo (10). However, the kinetics and metabolic effects of analogs with higher affinity for albumin have not been examined in vivo.

The objective of the present study was to investigate pharmacokinetic and pharmacodynamic properties of a novel long-acting fatty acid-acylated insulin analog, Lys^B29(N -carboxynonadecanoyl) des(B30) human insulin (O346) and to compare these results with previously determined kinetics of native insulin and insulin detemir. O346, with 30- to 40-fold increased binding affinity for human serum albumin compared with insulin detemir, indicated a 9-fold increase of the disappearance halftime from plasma after sc injection in pigs compared with human insulin. These data suggest that O346 might reveal substantially protracted pharmacokinetic and pharmacodynamic properties in comparison to human insulin. It is unknown whether the extremely prolonged onset of action is due to receptor binding competition at the level of the target cells or whether it is due to the sluggish transendothelial transport and clearance kinetics of the analog. To be able to compare transport and clearance parameters of O346 with insulin detemir and human insulin, we used compartmental modeling of plasma and interstitial fluid (represented by the hindlimb lymph) concentration profiles. Because of the extremely slow kinetics of O346 in plasma and interstitial fluid as determined from pilot experiments, iv injection of the analog rather than iv infusion was used to identify the model. A simulation approach was used to estimate the overall metabolic profile of the analog and to calculate and compare the biological efficacy (i.e. the total glucose uptake per picomole of iv injected insulin) for human insulin, insulin detemir, and O346.

	Materials and Methods

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Animals

The experimental protocols were approved by the University Institutional Animal Care and Use Committee. Experiments were conducted on 10 healthy male mongrel dogs (27.9 ± 1.9 kg; mean ± SEM) under anesthesia to allow sampling of hindlimb lymph. Dogs were housed under controlled kennel conditions (12 h light, 12 h dark) in the University of Southern California School of Medicine Vivarium. Animals had free access to water and standard chow (24% protein, 9% fat, 49% carbohydrate, 17% fiber; Wayne Dog Chow, Alfred Mills, Chicago, IL). Food was withdrawn 18 h before experiments. Dogs were used for experiments only if judged to be in good health as determined by visual observation, weight stability, body temperature, and hematocrit.

Surgical preparation

Surgery was performed at about 0700 h. Dogs were preanesthetized with acepromazine maleate (Prom-Ace; Aueco, Fort Dodge, IA; 0.22 mg/kg) and atropine sulfate (Western Medical, Arcadia, CA; 0.11 cc/kg). Anesthesia was induced with sodium pentobarbital (Nembutal, Abbott Laboratories, Chicago, IL; 0.44 cc/kg) and maintained with halothane and nitrous oxide. Indwelling catheters were implanted in the carotid artery (sampling) and jugular vein (saline drip). Left and right cepahlic vein intracatheters were inserted for various infusions as detailed below. A perivascular ultrasonic flowprobe (2-mm diameter; Transonic, Ithaca, NY) was placed around the right femoral artery for measurement of blood flow. Hindlimb muscle lymphatic fluid (11, 12) was sampled via a small polyethylene catheter (PE10 to PE90, predominantly PE50) inserted into a deep lymph vessel as described previously (13). To insert the lymph catheter, a longitudinal incision was made in the left hindlimb, distal to the femoral triangle, and the hindlimb lymphatic vessels were carefully exposed. The catheter was inserted through a pinhole and advanced 1–2 cm beyond the insertion point and secured with a silk suture. Flow was induced by gently massaging the limb muscle. Incisions were kept moist with saline-soaked gauze, and body temperature was maintained with heating pads. Blood pressure, heart rate, and respiratory CO₂ were continuously monitored. Dogs received a saline drip throughout both the surgery and the experiment to improve stability (1 liter was administered during the first 90 min of surgery, and a slow drip thereafter). After experiments, animals were killed by an overdose of sodium pentobarbital (Eutha-6, Western Medical; 65 mg/kg).

Experimental protocol

Dogs were maintained under inhalant anesthesia throughout the experimental protocol. At -180 min, a primed (25 µCi) tracer infusion of HPLC-purified, [3-³H]-D-glucose (0.25 µCi/min; NEN Life Science Products, Boston, MA) was started and maintained throughout the study to assess glucose turnover. At -60 min, a continuous infusion of somatostatin (0.8 µg/min/kg; Bachem, Torrance, CA) was started to suppress endogenous insulin release. A basal replacement infusion of regular human insulin (1.2 pmol/min/kg; Novolin-R, Novo Nordisk A/S, Bagsvaerd, Denmark) was initiated at -60 min as well. Both the somatostatin and the replacement insulin infusion were maintained throughout the study. From -60 min until the end of the experiment, arterial glucose was clamped at basal levels by exogenous infusion of 50% glucose labeled with [3-³H]-D-glucose (2.7 µCi/g glucose; Ref. 14). At time 0 min, an iv injection of either insulin analog O346 (90 pmol/kg; Novo Nordisk A/S; n = 7) or saline (n = 3) was administered. Arterial sampling (3 ml blood) was coupled with hindlimb lymph sampling (continuously from 2 min before to 2 min after arterial sample time, 100–900 µl lymphatic fluid). Arterial blood and lymph samples were drawn according to the following schedule: -90, -75, -60, -40, -20, -10, 0, 1, 5, 10, 20, 30, 40, 50, 60 min and in 20-min intervals from 60 min to the end of the experiment at 480 min. At hourly intervals, a portion of the arterial sample was taken for nonesterified fatty acid (NEFA) measurement.

Assays

Arterial blood samples for assay of glucose, insulin, and O346, as well as hindlimb lymph samples for assay of O346 were collected in microtubes precoated with lithium-heparin (Becton, Dickinson and Co., Franklin Lakes, NJ). Arterial sample tubes were additionally precoated with EDTA (Sigma, St. Louis, MO). Arterial blood samples for assay of NEFA were collected with EDTA and Paraoxon (Sigma) to inhibit lipoprotein lipase (15). Arterial blood samples were centrifuged immediately; the supernatant was transferred and, after measurement of plasma glucose, stored at -20 C until further assay. Hindlimb lymph samples were stored at -20 C immediately after sampling. On-line plasma glucose was assayed using glucose oxidase on an automated analyzer (model 23A; YSI, Inc., Yellow Springs, OH). Porcine insulin was measured in plasma with an enzyme-linked immunospecific assay (ELISA). Total (bound plus unbound) concentrations of O346 were measured in plasma and lymph using a specific ELISA method developed by Novo Nordisk and adapted in our laboratory. The ELISA for O346 uses a monoclonal antibody against human insulin as the catching antibody and a biotinylated specific monoclonal antibody against O346 as the detecting antibody. The O346 assay did not exhibit cross-reactivity with dog insulin, and the detection limit in dog plasma was estimated to be 50 pM.

Mathematical modeling

A two-compartment catenary model (Fig. 1) was used to describe transport and degradation kinetics of O346. Compartment 1 represents the distribution volume of O346 in plasma, and compartment 2 represents the distribution space of O346 in interstitial fluid (represented by the hindlimb lymph concentration, a surrogate for interstitial fluid from hindlimb skeletal muscle and fat). The mass equations for the model can be written as:

M₁ and M₂ represent the masses of O346 in plasma and in the interstitial fluid compartment, respectively. k₂₁ represents the rate constant for transport of O346 from plasma to the interstitial fluid compartment (k₁₂ is the flux in reverse direction), k₀₁ represents the rate constant for hepatic clearance of O346, and k₀₂ is the rate constant for degradation of O346 from the interstitial fluid compartment. All fractional rate constants are expressed as minutes^-1. Movement of O346 across the capillary endothelium was assumed to be driven by the concentration gradient, as in diffusion:

with V₁ and V₂ as the distribution volumes of plasma and interstitial fluid, respectively (16). To achieve parameter identifiability, V₂ was assumed to be 17% of body weight (10, 17, 18). The parameters to be identified by the iterative curve fitting of plasma and lymph O346 concentration profiles were k₂₁, k₀₁, k₀₂, and V₁.

View larger version (18K): [in this window] [in a new window]   Figure 1. Two-compartmental model describing distribution kinetics of O346, insulin detemir, and human insulin in plasma and interstitial fluid (isf) after iv injection.

Data are reported as mean ± SEM. Paired and independent Student’s t tests have been used to calculate statistical significance within and between subsets of data, respectively. P values are reported, with values less than 0.05 considered significant. Statistical data analysis has been performed using Minitab (State College, PA) and Excel (Microsoft Corp., Redmond, WA) software. Modeling analysis was performed using MLAB (Civilized Software, Inc., Bethesda, MD) implemented on an IBM-compatible computer. Parameter identification was obtained by nonlinear least-squares using a Marquardt-Levenburg algorithm with inverse-variance weights. Estimated transport and clearance parameters were used to predict the pharmacological response to iv injection of human insulin, insulin detemir, and O346 for a period of 48 h. The corresponding pharmacodynamic response (i.e. the glucose uptake profile) was also predicted for 48 h, applying the known linear relationship between hindlimb lymph insulin and glucose uptake (13). Biological efficacy was calculated as area under the curve of the model-predicted glucose uptake profile divided by the injected insulin dose (grams of glucose uptake per picomole of injected insulin).

	Results

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Glucose

Basal plasma glucose levels (-90 to -60 min) were not statistically different between the O346 and saline groups (O346, 107.5 ± 6.0 mg/dl; saline control, 113.4 ± 8.9 mg/dl; P > 0.6). Plasma glucose was clamped at basal values from -60 min until the end of the experiment (Fig. 2A). The mean coefficient of variation for the clamped glucose was 2.1% for O346 and 2.8% for saline control experiments.

View larger version (49K): [in this window] [in a new window]   Figure 2. Time profiles for glucose (A), blood flow (B), exogenous GINF (C), and glucose turnover (D) for saline control (triangles) and O346 (circles) injection experiments. D, Glucose uptake is expressed as open symbols and EGP as filled symbols. Data are expressed as mean ± SEM; n = 7 for O346, n = 3 for saline control experiments.

No change in FABF was observed after iv injection of O346, and FABF was not different between O346 and the control group (basal O346, 193.4 ± 36.3 ml/min; basal control, 223.7 ± 60.7 ml/min; P > 0.69; Fig. 2B). In the O346 group, MAP remained unchanged throughout the experimental protocol (-60 to 0 min, 60 ± 6 mm Hg; 420 to 480 min, 58 ± 4 mm Hg), and MAP was not different between groups (P > 0.3).

Exogenous glucose infusion (GINF) and glucose turnover

In control experiments, GINF remained stable but nonzero throughout the clamp period (1.1 ± 0.03 mg/min/kg; coefficient of variation = 15.5%; Fig. 2C). Glucose uptake was significantly increased from 60 to 480 min in O346 (-20 to 0 min, 2.1 ± 0.13 mg/min/kg; 460 to 480 min, 8.3 ± 1.42 mg/min/kg; P < 0.05) compared with saline control experiments (460 to 480 min, 2.1 ± 0.22 mg/min/kg; P < 0.05; Fig. 2D). Basal (-20 to 0 min) endogenous glucose production (EGP) was not different between O346 and saline control (O346, 1.5 ± 0.15 mg/min/kg; control, 1.3 ± 0.13 mg/min/kg; P = 0.50). Possibly due to slight overreplacement of basal insulin, EGP was suppressed by half in control animals. Area under the curve (0–480 min) for the relative suppression of EGP between O346 and control experiments was not statistically different (P > 0.46).

O346 and porcine insulin

Basal replacement porcine insulin concentrations (O346, 54.6 ± 7.3 pM; control, 68.9 ± 23.1 pM) were not significantly different between groups (P > 0.6). The rapid increase of O346 to pharmacological concentrations after iv injection and the extremely slow clearance from plasma (t_1/2 = 375.7 ± 26.7 min) is depicted in Fig. 3A. Also note in Fig. 3, A and B, the protracted and slow increase of the hindlimb lymph O346 concentration (t_1/2 = 137 ± 20 min) compared with the plasma concentration, reflecting extremely sluggish transport of the analog across the capillary endothelium. Glucose uptake was strongly correlated with the hindlimb lymph O346 concentration (r = 0.85; range, 0.59–0.96; P < 0.0002; Fig. 3C) as previously reported for native insulin (13) and analogs with less affinity for albumin than O346 (e.g. insulin detemir; Ref. 4).

View larger version (36K): [in this window] [in a new window]   Figure 3. Time profiles for O346 kinetics in plasma (open circles) and hindlimb lymph (filled circles; A), O346 in lymph (B), and comparison between O346 in lymph (open circles) and glucose uptake (filled circles; C). Data are expressed as mean ± SEM; n = 7.

Suppression of NEFA in O346 experiments was not significantly different from saline control (calculated as area under the curve; P = 0.22), suggesting that the analog could not suppress lipolysis more than it was already suppressed by basal insulin replacement.

Modeling analysis

Model fits were based on the 8 h of collected data, and parameter estimates are shown in Table 1. The transendothelial transport for O346 from plasma to interstitial fluid (as represented by parameter k₂₁) was only 3% of transport of human insulin reported previously (10) using the identical kinetic model structure (1.3 ± 0.3 x 10^-3 vs. 43 ± 5 x 10^-3 min^-1). Clearance of O346 by the liver (k₀₁) reached only 0.3% of the liver clearance of human insulin (0.8 ± 0.2 x 10^-3 vs. 280 ± 20 x 10^-3 min^-1). Rate of degradation of O346 by insulin-sensitive tissues (k₀₂) was estimated to be 16% of peripheral degradation rate of regular human insulin (2.3 ± 0.9 x 10^-3 vs. 14 ± 2 x 10^-3 min^-1). Comparison of transport parameters for human insulin, insulin detemir, and O346 are summarized in Table 2.

Because precise parameter estimates were available for O346, insulin detemir, and human insulin (Table 2), it was possible to predict the expected pharmacokinetic response for each peptide from 8–48 h (Fig. 4, A and B). Predicted time to reach maximal stimulation of glucose uptake (t_max) after iv injection of O346 was 7.8 ± 1.1 h, which is approximately 9 times the duration for insulin detemir and approximately 60 times the duration for human insulin. The time to reach 95% of the total action of O346 to stimulate glucose uptake after iv injection (T_95%) was estimated to be 45.4 ± 6.5 h, which is approximately 10 times the duration for insulin detemir, and approximately 25 times the duration for human insulin (Table 3).

View larger version (27K): [in this window] [in a new window]   Figure 4. Time profiles for simulated O346 kinetics of plasma and lymph (A) and hindlimb lymph only (B). Experimental data are expressed as open circles for plasma and filled circles for lymph; simulated data are expressed as mean ± SEM (line/dotted line). All data are expressed as mean ± SEM; n = 7.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Analog O346 is des(B30) human insulin acylated in the amino group of Lys B29 by one of the carboxyl groups of ,-dicarboxy-octadecane. The free fatty acid moiety binds the molecule to albumin, thus slowing its insulin-like action. O346 was designed to have much higher binding affinity for albumin compared with detemir, which is a similar analog but with the tetradecanoyl moiety bound to the same loci. In vitro experiments indicated 30- to 40-fold higher binding affinity for plasma albumin for O346 compared with insulin detemir (19). Like insulin detemir, O346 remains active at the level of the interstitial fluid. Although the efficacy of free fatty acid-bound insulin analogs at the cellular level is not totally understood (3), it appears that interstitial albumin does not interfere with the ability of the analog to bind to and activate skeletal muscle insulin receptors. This robust efficacy of interstitial molecule was previously reported for insulin detemir (9, 10).

The disappearance from plasma after sc injection of [¹²⁵I]-labeled insulin in pigs was studied for human insulin, insulin detemir (3), and O346 (Markussen, J., unpublished observations). Compared with human insulin, which showed a disappearance halftime of approximately 2 h, the halftime was much higher for both insulin detemir (7-fold) and O346 (9-fold). The absorption of O346 from the subcutis after injection is an important mechanism of slowed action for the clinical setting. For insulin detemir, stabilization of hexameric subunits and sc binding have been proposed as mechanisms for this delayed absorption (3). It might be of interest to examine the overall kinetics of O346, including the sc absorption as well as the kinetics once it enters the plasma compartment. That was not done in the present study for two reasons. First, because of the very slow kinetics of action of O346, it is very difficult to dissect out the slow kinetics of absorption from those of action. Second, it is much less accurate to perform kinetic modeling when the input to the plasma O346 is not precisely known and is variable (as in sc absorption). Therefore, we chose to examine kinetics under conditions of iv injection, when the appearance of O346 into plasma is clearly defined.

Pharmacokinetic and pharmacodynamic properties of O346 as well as transport and clearance parameters of O346 were studied using compartmental modeling. Because O346 is retained in the blood compartment for a long period of time due to high affinity for albumin, it was predictable that the analog would act very slowly to prolong insulin action. In fact, from pilot experiments with continuous infusion of O346, we found the action of the molecule to be so prolonged as to preclude traditional approaches such as the constant infusion method with euglycemic clamping to establish its pharmacokinetics and bioactivity. In the present study, we chose instead iv injection of a relatively high dose (90 pmol/kg) of O346. The assumption underlying this altered approach is linearity of distribution kinetics for O346. Disappearance kinetics of similar compounds has proven to be linear. If so, either injection or infusion can be used to achieve accurate distribution parameter estimates. In fact, monitoring plasma and lymph concentrations of O346 for a manageable period of 8 h allowed us to estimate with precision all the parameters of the distribution model for the peptide. From this model, it was straightforward to predict the dynamics of the compound for an extended period (e.g. Fig. 4). Our analysis indicated that a single injection of the compound would yield an extended period of elevated concentration in the blood (Fig. 4A) even at 48 h. Thus, O346 is a long-acting insulin analog indeed. In principle, insulin action could be maintained even if the compound were injected only every 2 d.

The results of the present study suggest that the extremely prolonged action of O346 (95% of total action to stimulate glucose uptake after iv injection was achieved after 45 h) is due to protection from metabolic clearance of the analog by the liver and other tissues while allowing for transport of the analog to the interstitial fluid compartment. In fact, liver clearance of O346 was only 1/500 that of native insulin, whereas transendothelial transport rate was 3% of native insulin. That transendothelial transport was reduced proportionately less than liver degradation accounts for the result that the analog can act physiologically on glucose uptake, although its binding to the liver is essentially nil.

The importance of transendothelial transport to the action of insulin was previously demonstrated for native insulin, as well as insulin detemir. For both compounds, the rate of glucose use has been demonstrated to be proportional to its measured concentration in the interstitial fluid both at steady state and during dynamic changes of the euglycemic clamp (4, 13). Similarly, in the present studies good correlation was observed between hindlimb lymph O346 concentration and glucose uptake (Fig. 3C). In a manner similar to other insulin analogs, this equilibrium between interstitial O346 concentration and glucose uptake supports transport of O346 across the capillary endothelium as the rate limiting step for O346 action in vivo.

Because of the extremely slow kinetics of O346, only a fraction of the postinjection metabolic profile of the analog could be measured during the present 8-h experiments (Fig. 3, A and B). We did observe that 8 h after the iv injection of O346, exogenous GINF was still required to clamp the plasma glucose, confirming a prolonged action of the peptide (Fig. 2C). From the 8-h data alone, it was straightforward to estimate transport parameters precisely (Table 1). Using these parameters, we predicted the complete kinetic profile for O346. The linear correlation between hindlimb lymph O346 concentration and glucose uptake was likewise exploited to calculate the time profile for glucose uptake for a period of 48 h. The biological efficacy of O346 was calculated as area under the curve of the calculated glucose uptake profile over the injected O346 dose. Using this approach, we estimated the biological efficacy of O346 in comparison to human insulin and insulin detemir (Table 3). The biological efficacy of O346 was estimated to be approximately 18% of human insulin. Also, the rate constant describing elimination from the peripheral compartment was clearly lower for O346 (16%) in comparison to human insulin. Because we were not able to determine the in vivo affinity of O346 to the insulin receptor in the present study, we can only speculate that competitive insulin receptor binding at the level of the target cell, which is most likely explained by the relatively higher binding affinity to albumin in comparison to insulin detemir, might have a contribution for the relatively low biological efficacy of O346. Surprisingly, our results suggest almost a doubling of the biological efficacy of insulin detemir in comparison to human insulin in the dog. An explanation for this result might be the longer availability of insulin detemir to the peripheral insulin receptor in response to the slow clearance from the plasma compartment.

Several assumptions were necessary in the development of the two-compartmental model to describe O346 kinetics in plasma and interstitial fluid. Although the two-compartment model yielded estimates for the plasma distribution volume of O346 that did not significantly differ from estimates for human insulin and insulin detemir, with the present two-compartmental model it was not possible to predict the extracellular distribution volume for O346. Therefore, the same distribution volume (17% of body weight) assumed for human insulin and insulin detemir (10) was assumed in the present study. Whether the modified structure of O346 or the higher binding affinity of O346 for plasma albumin might change its extracellular distribution volume is not known. However, any reasonable assumption of the extracellular distribution volume would not alter the extreme differences of transport and clearance parameters that were reported for O346 in comparison to human insulin and insulin detemir.

Hemodynamic actions of insulin are still a controversial issue. Several groups report significant increase of blood flow with insulin stimulation (20). With the methodology of measuring the flow of the femoral artery using an ultrasonic flow probe, neither previous studies using regular human insulin (16) nor the present study with pharmacologically high concentrations of O346 demonstrated any effect of insulin on femoral artery blood flow. Mean arterial pressure remained stable during the experiments and was not different between O346 and control groups.

We conclude that the concept of acylation of insulin using ,-dicarboxylic acid protracts the action on glucose uptake in dogs and that both the duration of action and the biological efficacy are dependent on the affinity of the analog for plasma albumin. We suggest that the high affinity of O346 for albumin at the plasma level affects liver clearance and transendothelial transport of the analog and is responsible for the very prolonged duration of action of O346. The high affinity of the analog for albumin at the interstitial fluid level reduces insulin receptor binding and therefore determines the relatively low biological efficacy of the analog compared with human insulin.

	Acknowledgments

 
We thank Elza Demirchyan and Doug Davis for technical assistance.

	Footnotes

 
This work was supported by grants from Novo Nordisk A/S and the National Institutes of Health (DK-27619 and DK-29867). S.P.K. was supported by a predoctoral training grant from the National Institute of Aging (T32-AG-00093).

Abbreviations: EGP, Endogenous glucose production; FABF, femoral artery blood flow; GINF, glucose infusion; MAP, mean arterial pressure; NEFA, nonesterified fatty acid.

Received July 30, 2002.

Accepted February 6, 2003.

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