Reduction in Intrasubject Variability in the Pharmacokinetic Response to Insulin After Subcutaneous Co-Administration with Recombinant Human Hyaluronidase in Healthy Volunteers

Abstract

Background:

This study was designed to test the hypothesis that co-administration of recombinant human hyaluronidase (rHuPH20) with regular insulin or insulin lispro will reduce intrasubject variability in pharmacokinetic end points compared with lispro alone.

Methods:

Healthy adult volunteers (18–55 years old) were enrolled in this phase 1, randomized, double-blind, crossover study. Subjects were administered two injections, each on a separate occasion, of three treatments during six euglycemic clamps. Treatments were 0.15 U/kg insulin lispro, 0.15 U/kg insulin lispro with 5 μg/mL rHuPH20, and 0.15 IU/kg regular insulin with 5 μg/mL rHuPH20. Insulin formulations were administered at a concentration of 40 U/mL. Serum immunoreactive insulin levels, blood glucose concentration, and glucose infusion rate determinations were made at baseline and for approximately 8 h after study drug administration. Intrasubject variability was assessed using a general linear mixed model with a fixed effect for treatment using a compound symmetric covariance matrix.

Results:

Co-injection of rHuPH20 with lispro significantly reduced intrasubject root mean square differences in time to peak serum insulin, time to early 50% peak serum insulin (t _50%), and time to late t _50% levels compared with lispro alone. Also, the intrasubject coefficient of variation for percentage of total area under the plasma concentration-versus-time curve for early time intervals compared with lispro alone was reduced. Intrasubject variability for regular insulin with rHuPH20 for most pharmacokinetic parameters was similar to the variability of lispro alone, although variability in early exposure was significantly reduced.

Conclusions:

Co-administration of rHuPH20 with lispro significantly reduced the variability of insulin pharmacokinetics relative to insulin lispro alone.

Introduction

O ne of the goals of prandial insulin therapy is to reproduce a physiologic insulin time–action profile, which helps to reduce postprandial glycemic excursions. Current rapid-acting prandial insulin analogs have demonstrated an improved time–action profile compared with regular human insulin^1

–5 and have resulted in small but significant improvement in glycosylated hemoglobin values in adults with type 1 diabetes mellitus.⁶ Despite these improvements, most patients are still unable to reach glycemic goals, even with currently available rapid-acting analog insulins.⁷

Hyaluronidase is a locally and transiently acting enzyme that catalyzes the depolymerization of the hyaluronan long-chain polymer^8,9 and acts as a spreading factor. Dispersion of the injected material into a larger tissue volume may allow access to a larger capillary bed, accelerating absorption.⁹ Hyaluronidase is known to accelerate the rate of absorption of subcutaneously injected drugs.^10,11 Recombinant human hyaluronidase (rHuPH20) is a soluble recombinant human form of hyaluronidase approved for use as “an adjuvant to increase the dispersion and absorption of other injected drugs.”¹² One limitation of all insulin analogs is the erratic absorption because of intrinsic within-subject variability.^13,14 Lower day-to-day variability in drug absorption might be achieved by reducing effects of focal inhomogeneity of subcutaneous tissue on drug absorption efficiency.

Vaughn et al.¹⁵ reported a proof-of-concept glucose clamp study demonstrating that co-administration of rHuPH20 accelerated pharmacokinetics (PK) and glucodynamics (GD) of both regular insulin and insulin lispro in healthy volunteers and reduced time to peak serum insulin concentration (t _max) and increased peak concentration (C _max) for both insulin formulations. Decreased intersubject variability in numerous PK parameters was also observed with rHuPH20 co-injection. These variability results were interpreted to suggest that co-administration of rHuPH20 with insulin also may reduce intrasubject variability, although the study was not designed to assess intrasubject variability per se. The present study was designed to directly test the hypothesis that co-administration of rHuPH20 with insulin reduces intrasubject variability in PK end points by assessing the reproducibility of insulin exposure and action in a euglycemic glucose clamp test of insulin lispro,¹⁶ insulin lispro+rHuPH20, and regular insulin+rHuPH20. It also provides the first direct comparison of PK and GD parameters for insulin lispro alone with regular insulin+rHuPH20 using identical doses of insulin in the same subjects.

Subjects and Methods

This single-center, phase 1, randomized, double-blind, six-way crossover study was designed to determine the reproducibility of insulin PK and GD and the safety and tolerability of three insulin formulations in healthy volunteers. The study was conducted in a U.S. clinical research center according to the U.S. Food and Drug Administration Code of Federal Regulations (21 CFR, Parts 50 and 56) and was consistent with Good Clinical Practice and applicable regulatory requirements. The study protocol received institutional review board approval before the study began, and written informed consent was obtained from all subjects before their enrollment.

Subjects

Subjects were healthy adults (18–55 years of age) with no clinically relevant abnormalities. Eligible subjects had a body mass index of 18–27 kg/m², total body weight of >65 kg for men and >46 kg for women, and fasting blood glucose levels of <100 mg/dL at screening. Eligible women of childbearing potential had a negative serum pregnancy test at screening. Subjects were excluded if they had a known history of diabetes mellitus (type 1 or type 2) or gestational diabetes, a history or evidence of use of any tobacco- or nicotine-containing product within 6 months of screening, or a screening quantitative urine nicotine concentration of >50 ng/mL.

Study design

Subjects were administered two injections, each on a separate occasion, of three treatments in a double-blind, randomized sequence during six euglycemic clamps. The three study treatments were 0.15 U/kg insulin lispro (40 U/mL), 0.15 U/kg insulin lispro (40 U/mL) co-injected with 5 μg/mL rHuPH20, and 0.15 U/kg regular insulin (40 IU/mL) co-injected with 5 μg/mL rHuPH20. The euglycemic glucose clamp procedure used semiautomated equipment (Biostator, Ames Division of Miles Laboratories, Elkhart, IN) according to published methods,¹⁷ with the exception that a basal intravenous insulin infusion was not used during the study period. Blood glucose was clamped at a level 10% below baseline glucose concentration, and glucose levels were maintained with a variable rate intravenous infusion of 20% glucose in water. Study drugs were administered subcutaneously in the lower abdominal area using a standard insulin syringe, alternating left and right side for each subsequent injection.

Serum immunoreactive insulin levels, blood glucose concentration, glucose infusion rate (GIR), and serum C-peptide determinations were made at baseline and at appropriately frequent intervals for approximately 8 h after study drug administration. No food intake was allowed during that period; water only was provided. Subjects received a meal at the end of each visit. Immunoreactive insulin and C-peptide were assayed by a validated radioimmunoassay by Millipore Corp. (now a Merck KGaA affiliate) (St. Charles, MO). Clinical laboratory samples for screening and safety assessments (hematology, blood chemistry, and urinalysis) were analyzed by Laboratory Corp. of America, San Diego, CA.

Statistical methods

For each PK and GD parameter, the intrasubject SD and the intrasubject coefficient of variation (CV) (percentage) were calculated directly from the two replications of each treatment. Intrasubject variability was evaluated using a general linear mixed model with a fixed effect for treatment using a compound symmetric covariance matrix. Comparison of PK and GD parameters among treatment groups was performed using a mixed model with fixed effects for treatment, averaging the two values obtained from each subject for each treatment and log transforming C _max and absolute area under the plasma concentration-versus-time curve (AUC) for insulin, time to peak GIR (GIR_max), and GIR total prior to analysis. The primary analyses were conducted on the per-protocol population, which included all subjects who completed all six periods of the study. Intrasubject variability in PK and GD parameters was compared among treatment groups using descriptive statistics only.

Results

This study was conducted from March 2009 through June 2009 and enrolled 22 subjects (12 men and 10 women); the mean age was 36±9 years, mean weight was 73±12 kg, and mean body mass index was 24±2 kg/m². Subjects' race or ethnicity was divided among 14 non-Hispanic whites, five Hispanics, two blacks, and one Asian. All 22 were exposed to at least one dose of study drug and were included in the safety population. Two subjects discontinued early: one subject withdrew consent, and one was discontinued by the investigator. The per-protocol evaluable population included 20 completers.

PK

Table 1 displays the variability in PK parameters after injection with lispro alone, lispro+rHuPH20, and regular insulin+rHuPH20. Intrasubject variability with lispro was significantly reduced with co-injection with rHuPH20 for several PK parameters related to early insulin exposure. Co-injection of rHuPH20 with lispro significantly reduced intrasubject root mean square differences in t _max and time to early and late 50% peak serum insulin concentration (t _50%) compared with lispro alone. Co-administration of rHuPH20 with lispro also significantly reduced intrasubject CV% for percentage of total AUC achieved for time intervals from 0 to 15 min through 0 to 120 min compared with lispro alone. The intrasubject variability of C _max and total insulin exposure (AUC), however, were not meaningfully different for lispro with or without rHuPH20. Intrasubject variability for regular insulin+rHuPH20 was generally similar to the variability of lispro alone, although the variability in early exposure (percentage of total AUC in the 0–15-min and 0–30-min intervals) was significantly reduced (Table 1). Intersubject variability for co-injection of rHuPH20 was consistently numerically smaller for the early PK parameters that showed significantly reduced intrasubject variability; however, the study was not designed to assess statistical significance of differences in the intersubject variability.

Table 1.

Intrasubject and Intersubject Variability of Insulin Pharmacokinetic Parameters

	Intrasubject variability (n=20)			Intersubject variability (n=20)
	Lispro+rHuPH20	RI+rHuPH20	Lispro alone	Lispro+rHuPH20	RI+rHuPH20	Lispro alone
Early t _50% (min)	2^a	4	5	3	5	5
t _max (min)	4^b	11	11	10	16	15
Late t _50% (min)	8^a	19	17	17	27	32
C _max (pmol/L)	15%	16%	13%	26%	25%	25%
Percentage of AUC in interval
0–15 min	18%^c	28%^c	66%	36%	42%	54%
0–30 min	14%^c	24%^b	41%	32%	40%	41%
0–60 min	9%^b	16%	19%	22%	29%	27%
0–120 min	5%^a	8%	10%	10%	15%	16%
Total AUC (pM·min)	12%	14%	13%	13%	14%	15%

Data are mean values. Exposure parameters are expressed as percentages of mean (coefficient of variance), and time is given as absolute values (root mean square differences).

Analysis of variance model with compound symmetric covariance matrix among repeated measures is used to assess the significance of changes in intrasubject variability; descriptive statistics for intersubject variability are reported without analysis for significance: ^a P<0.05, ^b P<0.01, ^c P<0.001.

AUC, area under the plasma concentration-versus-time curve; C _max, peak serum insulin concentration; early t _50%, time to early 50% peak serum insulin concentration; late t _50%, time to late 50% peak serum insulin concentration; rHuPH20, recombinant human hyaluronidase; RI, regular insulin; t _max, time to maximum serum insulin concentration.

Co-injection of rHuPH20 significantly accelerated exposure to lispro compared with lispro administered alone. Subjects had a larger and earlier peak exposure with co-administration of rHuPH20: after co-injection of lispro+rHuPH20, t _max, early t _50%, and late t _50% were 72%, 57%, and 61% of the respective values for lispro alone (all P<0.0001; Fig. 1A and Table 2). Exposure to insulin after co-administration of regular insulin+rHuPH20 also was accelerated compared with lispro alone. Early t _50% was significantly reduced by 13% (P=0.007) after administration of regular insulin+rHuPH20 compared with lispro alone. However, t _max was significantly increased by 14% (P=0.03). C _max was significantly increased with both lispro+rHuPH20 (+62%; P<0.0001) and regular insulin+rHuPH20 (+16%; P=0.001) compared with lispro alone.

FIG. 1.

(A) Serum insulin levels and (B) glucose infusion rate values over the 8-h period following subcutaneous administration of human insulin plus recombinant human hyaluronidase (rHuPH20) and lispro plus rHuPH20 compared with lispro alone in healthy adults. Data are mean±SEM values.

Table 2.

Insulin Pharmacokinetic Parameters Following Administration of Regular Human Insulin or Insulin Lispro Co-Injected with Recombinant Human Hyaluronidase Compared with Lispro Alone, by Evaluable Population and Compound Symmetric Covariance

	Lispro+rHuPH20 (n=20)			RI+rHuPH20 (n =20)
	Mean±SD	% of lispro	P value vs. lispro	Mean±SD	% of lispro	P value vs. lispro	Lispro (n=20) (mean±SD)
Early t _50% (min)	14±3	57%	<0.0001	22±5	87%	0.007	25±5
t _max (min)	40±10	72%	<0.0001	63±16	114%	0.03	55±15
Late t _50% (min)	88±17	61%	<0.0001	137±27	95%	0.12	144±32
C _max (pmol/L)	783±200	162%	<0.0001	562±142	116%	0.001	482±120
Percentage of total exposure in time window
0–15 min	5±2%	554%	<0.0001	2±1%	242%	<0.0001	1±0.5%
0–30 min	19±6%	291%	<0.0001	9±4%	141%	0.004	6±3%
0–60 min	50±11%	184%	<0.0001	30±9%	111%	0.04	27±7%
0–120 min	83±8%	130%	<0.0001	68±10%	108%	0.003	64±10%
120–480 min	17±8%	47%	<0.0001	32±10%	87%	0.004	36±10%
Total AUC (min·nmol/L)	66±9	109%	0.007	67±9	112%	0.0007	60±9

AUC, area under the plasma concentration-versus-time curve; C _max, peak serum insulin concentration; early t _50%, time to early 50% peak serum insulin concentration; late t _50%, time to late 50% peak serum insulin concentration; RI, regular insulin; t _max, time to maximum serum insulin concentration.

There was a small but statistically significantly increase in mean (±SD) total AUC over the 8-h period for lispro+rHuPH20 (66±9 min·nmol/L; 109%; P=0.007) and regular insulin+rHuPH20 (67±9 min·nmol/L; 112%; P=0.0007) compared with lispro alone (60±9 min·nmol/L). The total exposure was shifted to earlier and away from later times: the percentage of AUC was significantly greater for intervals of 0–15 min up to 0–120 min for both treatments coinjected with rHuPH20 compared with lispro alone (Table 2). For the 120–480-min interval, the percentage of AUC was significantly reduced for lispro+rHuPH20 (47%; P<0.0001) and regular insulin+rHuPH20 (87%; P=0.004) compared with lispro alone.

GD

For most GD parameters, intrasubject variability was numerically lower after co-injection of lispro+rHuPH20, compared with lispro alone, but only the variability (CV%) in percentage of total glucose infused during the first 4 h reached statistical significance (lispro+rHuPH20, 4%; lispro alone, 7%; overall treatment effect, P<0.05; Table 3).

Table 3.

Intrasubject and Intersubject Variability of Glucodynamic Parameters

	Intrasubject			Intersubject
	Lispro+rHuPH20	RI+rHuPH20	Lispro alone	Lispro+rHuPH20	RI+rHuPH20	Lispro alone
Time to percentage of total glucose infused by percentage (min)
10%	4.0	5.5	6.6	7.2	11.4	12.4
25%	6.5	9.3	8.8	12.5	15.8	19.3
50%	11.4	13.4	12.6	22.9	21.3	30.4
75%	16.5	23.0	20.5	40.7	31.1	44.4
90%	26.5	36.8	28.7	64.5	44.7	59.6
GIR_max	14%	12%	13%	33%	39%	37%
Percentage of total glucose infused by interval
0–2 h	11%	14%	12%	21%	23%	30%
0–3 h	6%	12%	9%	16%	15%	22%
0–4 h	4%^a	8%	7%	10%	9%	14%
Total glucose	15%	20%	18%	39%	47%	47%

Data are mean values. Exposure parameters are expressed as percentages of the mean (coefficient of variance), and time is given as absolute values (root mean square differences).

Analysis of variance model with compound symmetric covariance matrix among repeated measures: ^a P<0.05.

GIR_max, time to peak glucose infusion rate; rHuPH20, recombinant human hyaluronidase; RI, regular insulin.

GD results reflected the significant increase in early exposure to insulin with rHuPH20 co-injection (Fig. 1B and Table 4). Times to 10%, 25%, 50%, and 75% of total glucose infused were significantly reduced for both lispro+rHuPH20 (71%, 73%, 76%, and 81% of control, respectively; all P<0.0001) and insulin+rHuPH20 (92%, 92%, 91%, and 91% of control, respectively; all P≤0.02) compared with lispro alone. Time to 90% of total glucose infused was also significantly reduced for lispro+rHuPH20 (86% of control; P=0.0002). Percentages of total glucose infused over the 0–2-h, 0–3-h, and 0–4-h intervals compared with the control were significantly increased for rHuPH20 co-injection with either formulation compared with lispro alone (lispro+rHuPH20, from 111% to 147%; insulin+rHuPH20, from 107% to 111%; all P≤0.009). Mean GIR_max was significantly increased for lispro+rHuPH20 (13.3±4.4 mg/kg·min; 118% of control) compared with lispro alone (11.5±4.3 mg/kg·min; P=0.002) but not for regular insulin+rHuPH20 (12.5±4.9 mg/kg·min; 109% of control). There was no statistically significant difference between groups in total glucose infused.

Table 4.

Glucodynamic Parameters Following Administration of Human Insulin or Insulin Lispro Co-Injected with Recombinant Human Hyaluronidase Compared with Lispro Alone

	Lispro+rHuPH20 (n=20)			RI+rHuPH20 (n=20)
	Mean±SD	% of lispro	P value vs. lispro	Mean±SD	% of lispro	P value vs. lispro	Lispro (n=20) (mean±SD)
Time to percentage of total glucose infused by percentage (min)
10%	43±7	71%	<0.0001	56±11	92%	0.02	61±12
25%	72±12	73%	<0.0001	92±16	92%	0.005	99±19
50%	119±23	76%	<0.0001	143±21	91%	0.001	157±30
75%	179±41	81%	<0.0001	203±31	91%	0.004	222±44
90%	247±64	86%	0.0002	269±45	93%	0.06	288±60
GIR_max (mg/kg·min)	13.3±4.4	116%	0.001	12.5±4.9	109%	0.06	11.5±4.3
Percentage of total glucose infused by interval
0–2 h	53±11%	147%	<0.0001	41±9%	111%	0.009	36±11%
0–3 h	77±12%	125%	<0.0001	68±10%	110%	0.003	62±14%
0–4 h	89±9%	111%	<0.0001	85±8%	107%	0.003	80±11%
Total glucose infused (g/kg)	1.6±0.6	94%	0.31	1.7±0.8	98%	0.72	1.7±0.8

GIR_max, time to peak glucose infusion rate; rHuPH20, recombinant human hyaluronidase; RI, regular insulin.

Safety

Overall, 13 (59.1%) subjects reported one or more adverse events during the study. No severe or serious adverse events or deaths occurred in this study. Four subjects reported four adverse events that the investigator identified as related, probably related, or possibly related to study treatment; these adverse events included injection site pain (lispro+rHuPH20), injection site irritation (lispro alone), injection site hemorrhage (lispro alone), and headache and vomiting (lispro alone); all were mild in severity. All resolved without treatment or sequelae. No hypoglycemic excursions occurred during the study.

Discussion

Insulin absorption variability is a major hurdle in mimicking physiologic insulin response.^13,18 Insulin analogs accelerate the metabolic activity of insulin, but variability in response to insulin administration remains problematic.¹⁹ In general, most studies of intrasubject PK variability of rapid-acting insulin analogs compared with regular human insulin have demonstrated modest reductions in variability, but the coefficients of variability for indices of insulin exposure are still typically in the range of 30% (reviewed by Guerci and Sauvanet²⁰). This degree of PK variability contributes to glycemic variability²⁰ and makes tight control of diabetes all the more challenging and potentially risky because of the possibility of inadvertent overdosing. Any strategy that can meaningfully reduce this variability would be most welcome.

Soluble human insulin absorption is limited by diffusion and dissociation in the subcutaneous space;¹⁸ rapid-acting insulin analogs speed absorption by facilitating dissociation into dimers and monomers, forms that can be absorbed through capillary barriers.²¹ rHuPH20 enhances dispersion and absorption of co-injected insulin by increasing the surface area of exposed capillaries while also effectively reducing tissue insulin concentrations to promote dissociation.^8,9,15 Long-term use of injected medication may contribute to inhomogeneity of subcutaneous tissue structure, and rHuPH20 may act to reduce this variability in medication delivery, allowing medication absorption to occur over a larger volume of subcutaneous tissue, thus diminishing the impact of focal pocket tissue inhomogeneity.²²

Results of the current study show improvements in the variability of both time-related PK measures (e.g., early and late t _50%, t _max) and magnitude-related measures (e.g., percentage of total exposure during intervals up to 2 h after dosage administration) when rHuPH20 is co-administered with short-acting insulins. The decrease in variability was most marked for lispro+rHuPH20 compared with lispro alone but also was demonstrable when comparing regular human insulin+rHuPH20 with lispro alone. Intrasubject variability in PK results mirrored intrasubject results; however, the study was not designed to test for statistical significance of reductions in intersubject variability. GD parameters showed a reduction in variability corresponding to the PK results, but owing to the intrinsically greater variability in these measures, they were not generally statistically significant.

The changes observed in PK and GD parameters in this study also confirm and extend the previous observation that rHuPH20 co-administration accelerates insulin PK and GD in healthy subjects.¹⁵ In that study, healthy male subjects (n=26) were administered either human insulin with or without co-injected rHuPH20, or lispro with or without rHuPH20, in a crossover design. Co-administration of rHuPH20 accelerated the PK response for both insulin and lispro in the glucose clamp, with a reduction in t _max (42% and 49% of control for insulin and lispro, respectively; both P<0.001) and an increase in C _max (242% and 190% of control, respectively; both P<0.001). AUC was increased for early time intervals (AUC_0–1h, 422% and 255% of control, respectively; both P<0.001) and reduced for late time intervals (AUC_4–6h, 52% and 59% of control, respectively; both P<0.001).¹⁵

Safety results from the current study indicate that insulin injections with rHuPH20 are well tolerated. As with the previous rHuPH20 trial,¹⁵ adverse events were generally mild and relatively infrequent. The majority of adverse events were not considered related to study treatment but were primarily general disorders or related to the study procedure (for example, injection site irritation). No severe or serious adverse events were reported.

Early insulin action is required to quickly suppress endogenous hepatic glucose production after a meal has been ingested, thus tempering the degree of hyperglycemia that might otherwise occur.²³ By making these critical, early postprandial metabolic alterations more predictable, reduction in variability in early insulin exposure in particular may have a beneficial effect on diabetes treatment by allowing the use of fully efficacious insulin doses to reduce both postprandial hyperglycemic excursions and later postprandial hypoglycemia risks. Additional studies of rHuPH20 co-administration in patients with diabetes will be necessary to confirm this hypothesis.

Several study limitations should be considered. First, healthy volunteers enrolled in the study may have different subcutaneous tissue architecture compared with patients who have been injecting insulin over several years²² and thus may show differences in response to co-administration of rHuPH20 compared with patients with diabetes receiving long-term insulin therapy. Also, the concentration of insulin in each of these study drugs was 40 U/mL, whereas most prandial products are 100 U/mL. In addition, the reductions in intrasubject variability observed were statistically significant, but the clinical significance of changes of the magnitude observed is unknown. The clinical significance of insulin variability, although intrinsically important, may be small compared with patient-related or other environmental factors in the treatment of diabetes. Finally, in this study, subjects were exposed to six or fewer injections of rHuPH20. Long-term safety of repeat-dose administration of rHuPH20 remains unknown.

Conclusions

Co-administration of rHuPH20 with lispro significantly reduced the variability of early insulin absorption PK relative to insulin alone. This study also confirms and extends the previous observation that rHuPH20 co-administration accelerates insulin exposure and insulin action relative to lispro alone, with a greater magnitude of difference for lispro+rHuPH20 than for regular insulin+rHuPH20. Coupled with acceleration of insulin absorption, reduction in insulin dosing delivery variability conferred by the addition of rHuPH20 should provide an additional aid for the successful use of insulin in the treatment of diabetes.

Footnotes

Acknowledgments

The preparation of this manuscript was sponsored by Halozyme Therapeutics, Inc., San Diego, CA. Professional medical writing support for this manuscript was provided by Kathleen Dorries, Ph.D., of Embryon and was funded by Halozyme Therapeutics.

Author Disclosure Statement

D.E.V. and D.B.M. are employees of and stockholders in Halozyme Therapeutics, the sponsor of this study. At the time of this study E.A.L. was a consultant for Halozyme Therapeutics. M.H. and L.M. have no disclosures.

References

Becker

, Frick

. Clinical pharmacokinetics and pharmacodynamics of insulin glulisine. Clin Pharmacokinet, 2008; 47:7–20.

Lindholm

, McEwen

, Riis

. Improved postprandial glycemic control with insulin aspart. A randomized double-blind cross-over trial in type 1 diabetes. Diabetes Care, 1999; 22:801–805.

Ulrich

, Snyder

, Garg

. Combining insulins for optimal blood glucose control in type I and 2 diabetes: focus on insulin glulisine. Vasc Health Risk Manag, 2007; 3:245–254.

Rossetti

, Porcellati

, Bolli

, Fanelli

. Prevention of hypoglycemia while achieving good glycemic control in type 1 diabetes: the role of insulin analogs. Diabetes Care, 2008; 31,Suppl 2:S113–S120.

Eisenbarth

. Update in type 1 diabetes. J Clin Endocrinol Metab, 2007; 92:2403–2407.

Zinman

, Tildesley

, Chiasson

, Tsui

, Strack

. Insulin lispro in CSII: results of a double-blind crossover study. Diabetes, 1997; 46:440–443.

Davies

. The reality of glycaemic control in insulin treated diabetes: defining the clinical challenges. Int J Obes Relat Metab Disord, 2004; 28,Suppl 2:S14–S22.

Bookbinder

, Hofer

, Haller

, Zepeda

, Keller

, Lim

, Edgington

, Shepard

, Patton

, Frost

. A recombinant human enzyme for enhanced interstitial transport of therapeutics. J Control Release, 2006; 114:230–241.

Frost

. Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration. Expert Opin Drug Deliv, 2007; 4:427–440.

10.

Chain

, Duthie

. Identity of hyaluronidase and spreading factor. Br J Exp Pathol, 1941; 21:324–338.

11.

Forbes

, Deisher

. Effect of hyaluronidase on the subcutaneous absorption of electrolytes in humans. Science, 1950; 111:177–179.

12.

Hylenex Recombinant (Hyaluronidase Human Injection) 150 USP Units/mL [prescribing information]. Deerfield, IL: Baxter Healthcare Corp., 2009.

13.

DeWitt

, Hirsch

. Outpatient insulin therapy in type 1 and type 2 diabetes mellitus: scientific review. JAMA, 2003; 289:2254–2264.

14.

McAulay

, Frier

. Insulin analogues and other developments in insulin therapy for diabetes. Expert Opin Pharmacother, 2003; 4:1141–1156.

15.

Vaughn

, Yocum

, Muchmore

, Sugarman

, Vick

, Bilinsky

, Frost

. Accelerated pharmacokinetics and glucodynamics of prandial insulins injected with recombinant human hyaluronidase. Diabetes Technol Ther, 2009; 11:345–352.

16.

Howey

, Bowsher

, Brunelle

, Woodworth

. [Lys(B28), Pro(B29)]-human insulin: a rapidly absorbed analogue of human insulin. Diabetes, 1994; 43:396–402.

17.

Heinemann

, Anderson

Jr . Measurement of insulin absorption and insulin action. Diabetes Technol Ther, 2004; 6:698–718.

18.

Soeborg

, Rasmussen

, Mosekilde

, Colding-Jorgensen

. Absorption kinetics of insulin after subcutaneous administration. Eur J Pharm Sci, 2009; 36:78–90.

19.

Heinemann

. Variability of insulin absorption and insulin action. Diabetes Technol Ther, 2002; 4:673–682.

20.

Guerci

, Sauvanet

. Subcutaneous insulin: pharmacokinetic variability and glycemic variability. Diabetes Metab, 2005; 31:4S7–4S24.

21.

Brange

, Ribel

, Hansen

, Dodson

, Hansen

, Havelund

, Melberg

, Norris

, Snel

, Sørensen

, Voigt

. Monomeric insulins obtained by protein engineering and their medical implications. Nature, 1988; 333:679–682.

22.

Richardson

, Kerr

. Skin-related complications of insulin therapy: epidemiology and emerging management strategies. Am J Clin Dermatol, 2003; 4:661–667.

23.

Dalla

, Rizza

, Cobelli

. Meal simulation model of the glucose-insulin system. IEEE Trans Biomed Eng, 2007; 54:1740–1749.