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Pharmacodynamics and pharmacokinetics of insulin aspart assessed by use of the isoglycemic clamp method in healthy cats
Accepted Manuscript Pharmacodynamics and pharmacokinetics of insulin aspart assessed by use of the isoglycemic clamp method in healthy cats H.N. Pipe-Martin, J.M. Fletcher, C. Gilor, M.A. Mitchell PII:
S0739-7240(17)30051-6
DOI:
10.1016/j.domaniend.2017.09.002
Reference:
DAE 6281
To appear in:
Domestic Animal Endocrinology
Received Date: 2 March 2017 Revised Date:
7 September 2017
Accepted Date: 7 September 2017
Please cite this article as: Pipe-Martin HN, Fletcher JM, Gilor C, Mitchell MA, Pharmacodynamics and pharmacokinetics of insulin aspart assessed by use of the isoglycemic clamp method in healthy cats, Domestic Animal Endocrinology (2017), doi: 10.1016/j.domaniend.2017.09.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Insulin aspart has a rapid onset of action and short duration of effect in healthy cats.
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Route of administration (intramuscular versus subcutaneous) did not result in significant differences in pharmacodynamics.
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Subcutaneous insulin aspart resulted in a greater maximum plasma concentration (Cmax ).
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Pharmacodynamics and pharmacokinetics of insulin aspart assessed by
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use of the isoglycemic clamp method in healthy cats
3 H.N. Pipe-Martina,1, J.M. Fletchera, C. Gilorb, M.A. Mitchella
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Louisiana State University, Baton Rouge, Louisiana 70803, USA
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b
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University of California, Davis, Davis, California, 95616, USA
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Department of Veterinary Clinical Sciences, School of Veterinary Medicine,
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Department of Medicine and Epidemiology, School of Veterinary Medicine,
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Stafford Heights, Queensland, 4053, Australia
Current address: Queensland Veterinary Specialists, 263 Appleby Road,
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Corresponding author: Jon M. Fletcher
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Mailing address: Department of Veterinary Clinical Sciences, School of
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Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana 70803,
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USA
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Phone: 225-578-9040
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Fax: 225-578-9218
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E-mail: [email protected]
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ABSTRACT
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The objective of this study was to determine the pharmacodynamics (PD) and
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pharmacokinetics (PK) of insulin aspart in healthy cats following intramuscular
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(IM) and subcutaneous (SC) injection. Eight healthy, purpose-bred cats were
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used in a randomized, crossover study design. Each cat had two isoglycemic
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clamps performed, one after receiving 0.25 IU/kg of insulin aspart by IM injection
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and one after receiving the same dose by SC injection. The two isoglycemic
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clamps were performed on different days, at least 48 h apart. The blood glucose,
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plasma endogenous insulin, and plasma insulin aspart concentrations were
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measured and the glucose infusion rate (GIR) was recorded during the clamp.
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The GIR over time was used to create a time-action curve for each clamp which
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was used to describe the PD of insulin aspart. Data that are normally distributed
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are reported as mean ± SD, while data that are not normally distributed are
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reported as median (25 - 75 percentile). When compared to the PD data that
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have been reported for regular insulin in healthy cats, insulin aspart had a more
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rapid onset (IM: 10 min [10 - 21.25 min], SC: 12.5 min [10 - 18.75 min]) and
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shorter duration of action (IM: 182.5 ± 34.33 min, SC: 159.38 ± 41.87 min). The
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onset of action (P = 0.795), time to peak action (P = 0.499), duration of action (P
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= 0.301), and total metabolic effect (P = 0.603) did not differ with route of
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administration; however, SC administration did result in a higher maximum
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plasma insulin aspart concentration (IM: 1265.17 pmol/L [999.69 – 1433.89
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pmol/L], SC: 3278.19 pmol/L [2485.29 – 4132.01 pmol/L], P = 0.000) and larger
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area under the insulin aspart versus time curve (IM: 82662 ± 30565 pmol/L, SC:
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135060 ± 39026 pmol/L, P = 0.010). Insulin aspart has a rapid onset of action
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and short duration of effect in healthy cats when administered by IM and SC
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injection. Although it cannot be assumed that the PD and PK of insulin aspart will
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be the same in cats with diabetic ketoacidosis (DKA), our data supports further
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investigation into the use of SC insulin aspart as an alternative to regular insulin
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for the treatment of DKA in cats.
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Key words: Feline; Diabetes; Insulin analog; Rapid-acting analog
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This information was presented in part at the 2016 Annual American College of
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Veterinary Internal Medicine Forum, Denver, Colorado.
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1. Introduction During the last 20 years, it has become possible to modify the amino acid structure of human insulin to create insulin analogs which have time-action
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profiles that differ from short-acting (i.e., regular) and intermediate-acting
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insulin formulations. This has led to the development of new insulin
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formulations with a more rapid onset of action than regular insulin (i.e., rapid-
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acting analogs) as well as additional long-acting formulations (e.g. insulin
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glargine, insulin detemir, insulin degludec) [1]. For many human diabetics,
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maintenance insulin therapy consists of a combination of a long-acting analog
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which mimics basal insulin secretion and a rapid-acting insulin analog at meal
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time [1]. When combined, the time-action profiles of these insulin analogs more
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closely mimic normal physiologic insulin secretion than do crystalline insulin
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formulations [1]. The improved predictability and consistency of the time-action
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profiles of insulin analogs decreases the risk and occurrence of hypoglycemic
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events in people [1,2].
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The molecular structure of the rapid-acting analogs (e.g., insulin lispro,
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insulin aspart, insulin glulisine) differ from endogenous human insulin by one to
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two amino acids [1,3]. While this alteration affects absorption, it does not interfere
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with binding to the insulin receptor or other biophysiologic properties of the
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insulin molecule [4]. The amino acid substitutions destabilize hexamerization
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producing insulin solutions consisting primarily of monomers and dimers which
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allows rapid absorption following subcutaneous (SC) injection [3]. This is in
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contrast to regular insulin, which has a relatively high tendency to form hexamers
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requiring dissociation following injection and leading to slower onset of action and
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longer duration of effect [1,3]. The process of hexamer dissociation following
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injection also leads to a more variable time-action profile of regular insulin and as
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a result, less predictability. These characteristics of regular insulin lead to
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increased risk of hypoglycemia and were the driving force behind the
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development of rapid-acting analogs [1-6]. In people, rapid-acting analogs have
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an onset of action ranging from 5 - 20 min and duration of effect of 2 - 6 h with
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peak action occurring 30 - 90 min after SC injection [1,3].
Rapid absorption, predictable onset of action, and a short duration of
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effect are the properties that led researchers to also investigate the use of
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subcutaneously administered rapid-acting analogs in the treatment of human
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diabetic ketoacidosis (DKA). When SC insulin aspart and insulin lispro were
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compared to intravenous (IV) regular insulin in the treatment of people with mild
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to moderate DKA, researchers found that intermittent injection of rapid-acting
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analogs was as effective [7,8].
Pharmacodynamic (PD) and pharmacokinetic (PK) data for rapid-acting
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analogs are not available for cats and their use has not been reported in this
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species. In veterinary medicine, the PD of insulin formulations have traditionally
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been determined by analyzing the reduction in blood glucose concentration over
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time following insulin administration in euglycemic, fasted animals [9,10]. An
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alternative and superior technique is the isoglycemic clamp method which uses
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IV glucose to counteract the glucose-lowering effect of the insulin. This method
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avoids hypoglycemia and the effects of counter-regulatory hormone secretion,
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which is a concern when administering insulin to nondiabetic, fasted individuals.
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This technique also minimizes the effects of endogenous insulin secretion due to
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hyperglycemia by attempting to maintain or “clamp” the blood glucose
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concentration within a normal concentration range. The clamp method is
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considered the gold standard in people and uses the curve generated by
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graphing the glucose infusion rate (GIR) over time to describe the PD properties
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of the administered insulin [11,12]. Historically, somatostatin was used by some
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investigators to inhibit endogenous insulin secretion during glucose clamp
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studies evaluating the PD of exogenous insulin. More recently, the use of
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somatostatin has largely been abandoned in people due to side effects and
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potential effects on insulin clearance [13]. The isoglycemic clamp method without
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the administration of somatostatin is an accepted and utilized method to study
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insulin PD in people [12,13] and has been used in cats to study the PD of long-
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acting analogs [14]. Additionally, by measuring both the endogenous and
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exogenous insulin concentrations during the clamp, it is possible to confirm that
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the observed metabolic effect is the result of insulin administration.
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The purpose of this study was to utilize the isoglycemic clamp method to determine the PD and PK of insulin aspart in healthy cats following intramuscular
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(IM) and SC injection.
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2. Materials and Methods
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2.1 Animals
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The Louisiana State University Institutional Animal Care and Use
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Committee approved all animal use, and the experiment was performed in an
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AAALAC accredited facility. Eight healthy, purpose-bred, research cats were
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used in this study. Given the repeated measures crossover design, a sample size
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of eight cats was needed to obtain a desired power of 80%, a Type I error of 5%,
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and a large effect size equal to 1 standard deviation. All cats were well
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acclimated to human interaction and had a physical examination, complete blood
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count, serum biochemistry profile, and serum total thyroxine concentration
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performed prior to inclusion in the study. Body condition score (BCS) was
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recorded for the cats by use of a 9-point system (1 = extremely thin, 5 = optimal,
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and 9 = obese) [15]. Five cats were neutered males and three cats were spayed
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females with a mean age of 5 ± 3 years (range 2 - 9 years). The mean body
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weight and BCS were 5.9 ± 1.4 kg (range: 4.0 - 8.3 kg) and 7/9 (range: 4/9 - 9/9).
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Cats were fed a maintenance laboratory diet (LabDiet 5003-Laboratory Feline
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Diet, LabDiet®, St. Louis, MO, USA) ad libitum and group-housed in two groups
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of four prior to the study. Following IV catheter placement at the beginning of the
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study, the cats were individually housed until completion of the study. The cats
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were not sedated or physically restrained during the clamps, which were
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performed individually in a large metabolic cage (91 cm x 91 cm x 81 cm) that
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contained bedding and a litter box.
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2.2 Study Design
A randomized, crossover study design was used with each cat receiving one IM and one SC injection of insulin aspart (NovoLog, Novo Nordisk Inc.,
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Plainsboro, NJ, USA), on different days, at least 48 h apart. The dose of insulin
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aspart administered was 0.25 IU/kg rounded to the nearest half unit. In order to
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minimize dosing inaccuracies, 0.3 mL U100 insulin syringes were used and a
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single investigator measured all insulin doses. Insulin was injected midway
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between the scapulas for SC administration and in the quadriceps for IM
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administration. Cats were fasted overnight for 12 - 16 h before each clamp. Jugular (Small Animal Long Term Venous Catherization Set, MILA
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International, Inc., Florence, KY, USA) and cephalic (Surflo Intravenous Catheter,
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Terumo Corp., Binan, Laguna, PHL) IV catheters were placed and maintained for
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the duration of the study. Catheter placement was performed under sedation with
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dexmedetomidine (0.002 mg/kg), butorphanol (0.2 mg/kg) and ketamine (5
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mg/kg) a minimum of 36 h before the clamp was performed. Catheters were
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heparinized at the time of placement and following completion of each cat’s first
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clamp. The heparin was removed from the jugular catheters and the cephalic
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catheters were flushed prior to beginning each clamp. The jugular catheter was
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flushed with non-heparinized saline while sampling during the clamp. All
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catheters remained patent and functional during the study with the exception of a
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cephalic catheter in one cat. This catheter was replaced and the clamp was
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performed 48 hours after replacement. The cephalic catheters were used
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exclusively for glucose infusion, while jugular catheters were used exclusively for
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blood sampling. The blood glucose concentration was measured with a
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handheld glucometer (AlphaTrak2, Zoetis, Florham Park, NJ, USA) that has been
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validated in cats [16]. The 20% glucose infusion, made by diluting 50% dextrose
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(50% Dextrose USP, VetOne®, Nova-Tech, Inc., Grand Island, NE, USA) with
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saline, was administered through an extension set (Microbore Extension Set, 36
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inch, Secure Lock, Hospira, Inc., Lake Forest, IL, USA) using a syringe pump
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(Medfusion 2010i, Medexinc, Duluth, GA, USA).
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A small (≤ 0.1 mL) blood sample was collected from the jugular catheter
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every 5 min for blood glucose measurement during the clamp. A total of 1.5 mL
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of blood was collected every 15 minutes into chilled, glass
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ethylenediaminetetraacetic tubes and remained on ice until refrigerated
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centrifugation (4° C, 805 x g) at the end of each clamp. The plasma was frozen in
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glass tubes and stored at - 80° C until analysis. T he plasma insulin aspart
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concentration was measured at time zero, then every 15 min for the duration of
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the clamp. The endogenous insulin concentration was measured at time zero,
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every 60 min during the clamp, at the end of the clamp, and at the time point that
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corresponded to peak insulin action (determined after completion of the clamp).
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2.3 Isoglycemic Clamp Method
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Following insulin administration, an IV glucose infusion was used to maintain or “clamp” the blood glucose concentration in the target isoglycemic
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range. This range was ± 10% of the baseline blood glucose concentration and
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was calculated at the beginning of each clamp. The baseline blood glucose
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concentration was the average of the blood glucose concentration at t = -15 min
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and time zero. Following insulin administration, the GIR was adjusted to maintain
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the blood glucose concentration within the target range. Administration of the
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glucose infusion began when the blood glucose concentration decreased below
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the target range. Based on previous experience performing isoglycemic clamps
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in cats (CG), the initial GIR was approximately 10 mL/h (range 7-12 mL/h) with
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adjustments in rate between 1-3 mL/h, depending on the blood glucose, rate of
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change, and the degree of deviation from the target range. The blood glucose
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concentration and GIR were manually recorded every 5 min. The clamp was
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considered complete when the blood glucose concentration remained within the
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target range for 45 min without the administration of glucose.
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The GIR over time was used to create a time-action curve for each clamp.
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The onset of action was defined as the time from insulin administration (t = 0) to
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initiation of the glucose infusion. The end of action was defined as the time the
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glucose infusion was discontinued. The duration of action was defined as the
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time from the onset of action to the end of action. The peak insulin action was
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defined as the highest GIR during the clamp (GIRmax), and time to peak (tmax GIR)
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was the time elapsed from administration to peak insulin action. The GIR is
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expressed in milligrams of glucose per kilogram body weight per minute. Total
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metabolic effect was calculated as the total amount of glucose infused during the
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clamp divided by body weight ([total milliliters of glucose solution infused x 0.2] /
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body weight in kilograms).
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2.4 Insulin measurement Insulin quantification was performed with commercially available insulin ELISA kits (Mercodia AB, Uppsala, Sweden). The insulin ELISAs were performed
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according to the manufacturer’s protocol and absorbance was read at 450 nm
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with an automated ELISA plate reader (Epoch, BioTek, Winooski, Vermont,
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USA). The Iso-Insulin ELISA (Mercodia AB, Uppsala, Sweden) used to measure
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the plasma insulin aspart concentration has a reported cross-reactivity of 100%
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with insulin aspart [17] and lacks cross-reactivity with feline insulin [18,19]. The
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detection limit is ≤ 7.18 pmol/L and the assay range is 21.53 – 171.50 pmol/L.
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For statistical analysis, sample results with an absorbance greater than the
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negative control but less than standard 1 were reported as 21.53 pmol/L, and
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samples with an absorbance equal to or less than the negative control were
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reported as 0 pmol/L. Samples that exceeded the assay range were diluted 1:10
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per the manufacturer’s recommendations. The intra-assay coefficient of variation
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(CV) was 5.09% and the inter-assay CV was 3.39%.
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The time to detection of insulin aspart was defined as time from insulin
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administration to the first detected increase in the plasma concentration. The
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peak concentration (Cmax) was defined as the highest plasma insulin aspart
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concentration measured and the time to peak concentration (tmax Asp) was the
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time from administration to Cmax. The end of insulin detection was defined as the
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last time point that the insulin aspart concentration was above or equal to the
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concentration of standard 1 (24.90 pmol/L). The duration of insulin aspart
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detection was defined as the time from initial detection until the end of detection.
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The previously validated [20] Feline Insulin ELISA (Mercodia AB, Uppsala, Sweden) used to measure the plasma endogenous insulin concentration has a
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detection limit of 1.60 pmol/L, an assay range of 1.74 – 121.86 pmol/L, and a
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reported 5.9% cross-reactivity with insulin aspart. For statistical analysis, sample
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results with an absorbance greater than the negative control but less than
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standard 1 were recorded as 1.60 pmol/L, and samples with an absorbance
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equal to or less than the negative control were recorded as 0 ng/L. The intra-
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assay CV was 16.48% and the inter-assay CV was 7.65%.
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2.5 Statistical analysis
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Statistical analysis was performed using commercially available computer software (SPSS 23.0, IBM Statistics, Armonk, NY, USA). The distribution of the
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data was evaluated using the Shapiro-Wilk test, skewness, kurtosis, and q-q
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plots. Data that were not normally distributed (onset of action, Cmax, tmax Asp,
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fasting insulin concentration, and blood glucose concentration time zero) were
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log transformed for parametric testing. Data that are normally distributed are
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reported as mean ± SD, while data that are not normally distributed are reported
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as median (25-75 percentile). An independent samples t-test was used to
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determine if body weight or BCS were different by sex. A linear mixed model was
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used to determine if the onset of action, end action, duration of action, GIRmax,
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tmax GIR, area under the GIR versus time curve (AUC GIR), end detection insulin
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aspart, duration detection insulin aspart, Cmax, tmax ASP, area under the aspart
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versus time curve (AUC Asp), fasting insulin concentration, baseline blood
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glucose concentration, and blood glucose concentration 30 min after completion
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of the clamp were affected by route of administration or BCS. When comparing
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BCS, cats were categorized as ideal (BCS 4-6) or increased (BCS 7-9). A paired
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samples t-test was used to determine if CV of blood glucose concentration was
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significantly different during IM and SC clamps. A P value < 0.05 was used to
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determine statistical significance.
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There was not a difference in body weight (P = 0.564) or BCS (P = 0.735)
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between male and female cats. The total dose of insulin aspart administered was
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1, 1.5, or 2 IU depending on body weight, with each cat receiving the same
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insulin dose for the IM and SC clamps. No adverse effects or injection site
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reactions were noted with IM or SC administration of insulin aspart.
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The baseline blood glucose concentration (IM: 94.1 ± 7.4 mg/dL, SC: 95.4 ± 6.2 mg/dL; P = 0.347), blood glucose concentration 30 min after completion of
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the clamp (IM: 94.8 ± 6.1 mg/dL, SC: 96.8 ± 7.2 mg/dL; P = 0.660), and fasting
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endogenous insulin concentration (IM: 14.92 pmol/L [7.2- 69.24 pmol/L]), SC:
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33.95 pmol/L [20.61 – 56.44 pmol/L]; P = 0.332) did not differ with route of
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administration. Although an increased BCS was associated with a higher fasting
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blood glucose concentration (P = 0.004), it was not associated with a higher
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fasting endogenous insulin concentration (P = 0.593).
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There was not a difference (P = 0.904) in CV of blood glucose
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concentrations during IM (17.94% ± 4.5) and SC (17.71% ± 3.9) clamps which
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confirms that the blood glucose concentration was maintained within a similar
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range for all clamps (Figure 1).
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3.1 Pharmacodynamics The PD parameters are summarized in Table 1 and the time-action curves are individually graphed in Figure 1. Route of administration was not associated
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with a difference in onset of action (P = 0.795), time to peak action (P = 0.499),
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duration of action (P = 0.301), AUC GIR (P = 0.736), or total metabolic effect (P =
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0.603). Body condition score was not associated with a difference in PD of insulin
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aspart.
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Endogenous insulin secretion was suppressed within 60 min (IM: 5.42 ± 5.16 pmol/L; SC: 5.80 ± 4.38 pmol/L) of insulin administration and remained
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suppressed to a low concentration at 120 min (IM: 1.86 ± 0.81 pmol/L; SC: 1.57 ±
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0 pmol/L) (Figure 2). The endogenous insulin concentration was less than the
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baseline concentration at the completion of all clamps (P = 0.000).
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The PK data are summarized in Table 2 and the concentration over time curves are individually graphed in Figure 1. Insulin aspart was detected in all cats
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at the first time point sampled (15 min after injection). The maximum plasma
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insulin aspart concentration (Cmax) achieved (IM: 1265 pmol/L [1000-1434
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pmol/L] vs. SC: 3278 pmol/L [2485-4132 pmol/L], P = 0.000) and AUC Asp (P =
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0.010) were greater following SC administration. The time to maximum plasma
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concentration (tmax) and duration of detection did not differ (P = 0.586, P = 0.268,
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respectively) with route of administration. An increased body condition score was
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associated an increased AUC Asp (P = 0.009).
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4. Discussion In this study, both IM and SC administration of insulin aspart resulted in a rapid and predictable onset of action and short duration of effect. By confirming
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suppression of the endogenous insulin concentration during the clamps, it was
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possible to confirm that the glucose lowering effect observed was the result of
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the administered insulin aspart. Route of administration was not associated with
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a significant difference in the PD of insulin aspart. This is consistent with a study
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in people that failed to find a difference between IM and SC administration of
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insulin lispro, a similar rapid-acting analog [21]. Although the PD and total
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metabolic effect were not influenced by the route of administration, a higher peak
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plasma concentration (Cmax) and larger area under the insulin aspart versus time
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curve was seen with SC administration. The cause of this difference has not
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been determined, but potential explanations include differences in absorption
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related to the site of administration (e.g., pelvic limb versus trunk) or a difference
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in SC versus IM absorption that has not been previously reported in people. Site
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of SC injection has been shown to have a significant effect on the PK of rapid-
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acting analogs in people with SC injections in the abdominal region resulting in
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more rapid absorption and greater peak insulin concentration than those in the
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thigh, arm, or buttocks [3,22,23]. Additionally, and similar to our findings,
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differences in PK are not always associated with a significant difference in the
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overall blood glucose lowering effect [22,23].
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In people, the slow onset and long duration of action of regular insulin is
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related to the dissociation of insulin that must occur prior to absorption [24,25],
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and it has been demonstrated that the peak effect of regular insulin occurs more
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rapidly following IM injection [21]. As mentioned previously, IM and SC
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administration were not associated with a difference in glucose lowering effect in
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people receiving insulin lispro [21] or in the cats in our study receiving insulin
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aspart. The lack of difference and rapid absorption regardless of route of
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administration of rapid-acting analogs is likely the result of destabilized
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hexamerization leading to increased concentrations of monomers and dimers in
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solution and rapid dissociation following injection [6]. While dehydration might be
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expected to further slow the absorption of SC regular insulin, it is possible that
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insulin formulations that are mostly monomers and dimers could be less affected
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by hydration status. If this is true and insulin aspart has a consistent and
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predictable time-action profile in the presence of dehydration, it could provide an
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alternative for treating DKA in cats. Although the use of SC insulin aspart for the
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treatment of feline DKA needs to be investigated, it is promising that studies in
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people have found intermittent SC rapid-acting protocols to be as effective as IV
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insulin continuous rate infusion [8,9].
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Both the heterogeneity of BCS and the increased body conditions of the cats were considered when interpreting the results of this study. Although an
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increased BCS was associated with an increase in the fasting blood glucose
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concentration, it is worth noting that the fasting blood glucose was ≤ 100 mg/dL
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in 14/16 clamps and the maximum fasting blood glucose concentration of 103
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mg/dL was in an overweight (BCS 7/9), not obese cat. All fasting blood glucose
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concentrations were below the upper cutpoint (116 mg/dL) for fasting blood
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glucose recently reported for normal cats [26], and below 118 mg/dL which has
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recently been suggested as the cutpoint for impaired fasting glucose [27]. Also,
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BCS was not associated with a difference in fasting insulin concentration. An
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increased BCS was also associated with an increase in AUC Asp, but was not
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associated with differences in the other PK parameters or PD. A possible
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explanation for the increased AUC Asp is decreased insulin clearance which has
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been observed in people with an increased body mass index receiving insulin
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aspart [28]. In that study, the decrease in insulin clearance was found to be
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clinically insignificant. The difference in AUC Asp associated with BCS was also
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insignificant in our study as there was no associated difference in PD.
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The potential impact of stress-induced hyperglycemia was considered when designing and executing this study. All cats used in this study were well
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acclimated to human interaction and the laboratory environment, and no signs of
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stress or anxiety were observed during the clamps. Most importantly, this study
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was designed in such a way that the cats were not physically restrained and only
384
required minimal handling during the clamps. Additionally, the fasting blood
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glucose concentration and blood glucose concentration 30 min after completion
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of the clamps were within the euglycemic range and not consistent with the
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occurrence of stress hyperglycemia [29].
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In conclusion, insulin aspart has a rapid onset of action and short duration
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of effect in healthy cats when administered by IM and SC injection. Although it
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cannot be assumed that the PD and PK of insulin aspart will be the same in cats
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with DKA, our data supports further investigation into the use of SC insulin aspart
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as an alternative to regular insulin for the treatment of DKA in cats.
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Acknowledgements
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The authors thank Claire Webster for her technical assistance, Chin-Chi Liu for
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assistance with sample analysis, and Michael Kearney for help with statistical
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analysis.
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Funding: This work was supported in part by the Louisiana State University
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Veterinary Clinical Sciences Departmental Competitive Organized Research
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Program.
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[7]
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Neumiller J, Odegard P, Wysham C. Update on insulin management in type 2 diabetes. J Fam Pract. 2012;61(5):1-32. Pfutzner A, Kustner E, Forst T, Schulze-Schleppinghoff B, Trautmann ME, Haslbeck M, Schatz H, Beyer J. Intensive insulin therapy with insulin lispro in patients with type 1 diabetes reduces the frequency of hypoglycemic episodes. Exp Clin Endocrinol Diabetes. 1996;104(1):25-30. doi:10.1055/s-0029-1211418. Home PD. The pharmacokinetics and pharmacodynamics of rapid-acting insulin analogues and their clinical consequences. Diabetes, Obes Metab. 2012;14(9):780-788. doi:10.1111/j.1463-1326.2012.01580.x. Setter SM, Corbett CF, Campbell RK, White JR. Insulin aspart: a new rapid-acting insulin analog. Ann Pharmacother. 2000;34(12):1423-1431. Evans M, Schumm-Draeger PM, Vora J, King AB. A review of modern insulin analogue pharmacokinetic and pharmacodynamic profiles in type 2 diabetes: Improvements and limitations. Diabetes, Obes Metab. 2011;13(8):677-684. doi:10.1111/j.1463-1326.2011.01395.x. Berenson DF, Weiss AR, Wan Z, Weiss MA. Insulin analogs for the treatment of diabetes mellitus: therapeutic applications of protein engineering. Ann N Y Acad Sci. 2011;1243:E40-E54. doi:10.1111/j.17496632.2012.06468.x. Umpierrez GE, Cuervo R, Karabell A, Latif K, Freire AX, Kitabchi AE. Treatment of diabetic ketoacidosis with subcutaneous insulin aspart. Diabetes Care. 2004;27(8):1873-1878. Umpierrez GE, Latif K, Stoever J, Cuervo R, Park L, Freire AX, Kitabchi AE. Efficacy of subcutaneous insulin lispro versus continuous intravenous regular insulin for the treatment of patients with diabetic ketoacidosis. Am J Med. 2004;117(5):291-296. doi:10.1016/j.amjmed.2004.05.010. Clark M, Thomaseth K, Heit M, Hoenig M. Pharmacokinetics and pharmacodynamics of protamine zinc recombinant human insulin in healthy dogs. J Vet Pharmacol Ther. 2012:342-350. Marshall R, Rand J. Comparison of the pharmacokinetics and pharmacodynamics of glargine, protamine zinc, and porcine lente insulins in normal cats (abstract). J Vet Intern Med. 2002:358. Heinemann L, Anderson JH. Measurement of insulin absorption and insulin action. Diabetes Technol Ther. 2004;6(5):698-719. Porcellati F, Bolli GB, Fanelli CG. Pharmacokinetics and pharmacodynamics of basal insulins. Diabetes Technol Ther. 2011;13 Suppl 1:S15-S24. doi:10.1089/dia.2011.0038. Heise T, Zijlstra E, Nosek L, Heckermann S, Plum- Mörschel L, Forst T. Euglycaemic glucose clamp: what it can and cannot do, and how to do it. Diabetes, obesity and metabolism. 2016; 18(10):962–972. Gilor C, Ridge TK, Attermeier KJ, Graves TK. Pharmacodynamics of insulin determir and insulin glargine assessed by an isoglycemic clamp method in healthy cats. J Vet Intern Med. 2010;24:870-874.
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[15] Laflamme DP. Development and validation of a body condition score system for cats: a clinical tool. Feline Practice 1997;25:13-18. [16] Zini E, Moretti S, Tschuor F, Reusch CE. Evaluation of a new portable glucose meter designed for the use in cats. Schweiz Arch Tierheilkd. 2009;151(9):448-451. doi:10.1024/0036-7281.151.9.448. [17] Lindström T, Hedman CA, Arnqvist HJ. Use of a novel double-antibody technique to describe the pharmacokinetics of rapid-acting insulin analogs. Diabetes Care. 2002;25(6):1049-1054. [18] Bryan C, Lathan P, Curotto S, Bulla C, Lunsford K, Pruett S. Lack of cross reactivity of feline insulin with a commercial human ELISA (abstract). J Vet Intern Med. 2011;25:632-767. [19] Fletcher J, Pipe-Martin H, Liu C. Using purified feline insulin to evaluate cross-reactivity with a human insulin analog ELISA (abstract). J Vet Intern Med. 2016;30:1407-1519. [20] Strage EM, Holst BS, Nilsson G, Jones B, Lilliehk I. Validation of an enzyme-linked immunosorbent assay for measurement of feline serum insulin. Vet Clin Pathol. 2012;41(4):518-528. doi:10.1111/j.1939165x.2012.00476.x. [21] Rave K, Heise T, Weyer C, Hernberger J, Bender R, Hirschberger S, Heinemann L. Intramuscular versus subcutaneous injection of soluble and lispro insulin: comparison of metabolic effects in healthy subjects. Diabet Med. 1998;15(2):747-751. doi: 10.1002/ (SICI) 1096-9136 (199809)15:9<747::AID-DIA664>3.0.CO:2-V. [22] Mudaliar SR, Lindberg FA, Joyce M, Beerdsen P, Strange P, Lin A, Henry RR. Insulin aspart (B28 Asp-insulin): A fast-acting analog of human insulin: Absorption kinetics and action profile compared with regular human insulin in healthy nondiabetic subjects. Diabetes Care. 1999;22(9):1501-1506. doi:10.2337/diacare.22.9.1501. [23] ter Braak EW, Woodworth JR, Bianchi R, Cerimele B, Erkelens DW, Thijssen JH, Kurtz D. Injection site effects on the pharmacokinetics and glucodynamics of insulin lispro and regular insulin. Diabetes Care. 1996;19(12):1437-1440. doi:10.2337/diacare.19.12.1437. [24] Howey DC, Bowsher RR, Brunelle RL and Woodworth JR. [Lys(B28), Pro(B29)]-Human Insulin: A Rapidly Absorbed Analogue of Human Insulin. Diabetes 1994;43(3):396-402. [25] Hirsch I.B. Insulin analogues. N Engl J Med. 2005;352(2): 174-183. [26] Reeve-Johnson MK, Rand JS, Vankan D, Anderson ST, Marshall R, Morton JM. Cutpoints for screening blood glucose concentrations in healthy senior cats. J Fel Med Surg. 2017 (Epub ahead of print) doi:10.1177/1098612X1668567. [27] Reeve-Johnson M, Rand J, Vankan D, Anderson S, Marshall R, Morton J. Diagnosis of prediabetes in cats: glucose concentration cut points for impaired fasting glucose and impaired glucose tolerance. Domest Anim Endocrinol. 2016;57:55-62.
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[28] Holmes G, Galitz L, Hu P, Lyness W. Pharmacokinetics of insulin aspart in
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obesity, renal impairment, or hepatic impairment. Br J Clin Pharmacol.
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2005;60:469–476. [29] Rand JS, Kinnaird E, Baglioni A, Blackshaw J, Priest J. Acute stress hyperglycemia in cats Is associated with struggling and increased concentrations of lactate and norepinephrine. J Vet Intern Med. 2002;16(2):123–132.
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Table 1. Pharmacodynamic parameters of insulin aspart (0.25 U/kg) in 8 healthy cats after intramuscular and subcutaneous injections. Data are reported as mean ± SD if normally distributed or median (25-75 percentile) if not normally distributed. Subcutaneous
10 (10-21.25) 196.25 ± 38.24 182.50 ± 34.33 8.29 ± 2.18 49.38 ± 35.60 901.13 ± 22.72 0.93 ± 0.24
12.5 (10-18.75) 173.13 ± 40.88 159.38 ± 41.87 7.91 ± 2.51 43.75 ± 33.46 858.50 ± 266.18 0.89 ± 0.30
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Onset of action (min) End of action (min) Duration of action (min) Peak action / GIRmax (mg/kg/min) Time to peak action / tmax GIR (min) AUC GIR Total metabolic effect (g/kg)
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Pharmacodynamic parameters
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0.795 0.281 0.301 0.749 0.499 0.736 0.603
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Table 2. Pharmacokinetics of insulin aspart (0.25 U/kg) in 8 healthy cats after intramuscular and subcutaneous injections. Data are reported as mean ± SD if normally distributed or median (25-75 percentile) if not normally distributed.
15 186.25 ± 56.24 171.25 ± 56.24 1265 (1000-1434) 15 (15-41.25) 82662 ± 30565
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Initial detection (min) End of detection (min) Duration of detection (min) Cmax (pmol/L) tmax Asp (min) AUC Asp
15 161.25 ± 49.91 146.25 ± 49.91 3278 (2485-4132) 15 (15-26.25) 135060 ± 39026
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Pharmacokinetic parameters
Subcutaneous
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P Value
0.268 0.268 0.000 0.586 0.010
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Figure 1. Plasma insulin aspart concentration (solid line), glucose infusion rate (GIR) / time action profile (dashed line), and the blood glucose concentration (dotted line) during the clamp procedure following intramuscular (o) and subcutaneous () injections of 0.25 U/kg insulin aspart in individual cats (A-H).
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S0739-7240(17)30051-6
DOI:
10.1016/j.domaniend.2017.09.002
Reference:
DAE 6281
To appear in:
Domestic Animal Endocrinology
Received Date: 2 March 2017 Revised Date:
7 September 2017
Accepted Date: 7 September 2017
Please cite this article as: Pipe-Martin HN, Fletcher JM, Gilor C, Mitchell MA, Pharmacodynamics and pharmacokinetics of insulin aspart assessed by use of the isoglycemic clamp method in healthy cats, Domestic Animal Endocrinology (2017), doi: 10.1016/j.domaniend.2017.09.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Insulin aspart has a rapid onset of action and short duration of effect in healthy cats.
•
Route of administration (intramuscular versus subcutaneous) did not result in significant differences in pharmacodynamics.
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Subcutaneous insulin aspart resulted in a greater maximum plasma concentration (Cmax ).
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Pharmacodynamics and pharmacokinetics of insulin aspart assessed by
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use of the isoglycemic clamp method in healthy cats
3 H.N. Pipe-Martina,1, J.M. Fletchera, C. Gilorb, M.A. Mitchella
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a
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Louisiana State University, Baton Rouge, Louisiana 70803, USA
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b
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University of California, Davis, Davis, California, 95616, USA
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Department of Veterinary Clinical Sciences, School of Veterinary Medicine,
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Department of Medicine and Epidemiology, School of Veterinary Medicine,
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1
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Stafford Heights, Queensland, 4053, Australia
Current address: Queensland Veterinary Specialists, 263 Appleby Road,
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Corresponding author: Jon M. Fletcher
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Mailing address: Department of Veterinary Clinical Sciences, School of
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Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana 70803,
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USA
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Phone: 225-578-9040
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Fax: 225-578-9218
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E-mail: [email protected]
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ABSTRACT
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The objective of this study was to determine the pharmacodynamics (PD) and
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pharmacokinetics (PK) of insulin aspart in healthy cats following intramuscular
27
(IM) and subcutaneous (SC) injection. Eight healthy, purpose-bred cats were
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used in a randomized, crossover study design. Each cat had two isoglycemic
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clamps performed, one after receiving 0.25 IU/kg of insulin aspart by IM injection
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and one after receiving the same dose by SC injection. The two isoglycemic
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clamps were performed on different days, at least 48 h apart. The blood glucose,
32
plasma endogenous insulin, and plasma insulin aspart concentrations were
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measured and the glucose infusion rate (GIR) was recorded during the clamp.
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The GIR over time was used to create a time-action curve for each clamp which
35
was used to describe the PD of insulin aspart. Data that are normally distributed
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are reported as mean ± SD, while data that are not normally distributed are
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reported as median (25 - 75 percentile). When compared to the PD data that
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have been reported for regular insulin in healthy cats, insulin aspart had a more
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rapid onset (IM: 10 min [10 - 21.25 min], SC: 12.5 min [10 - 18.75 min]) and
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shorter duration of action (IM: 182.5 ± 34.33 min, SC: 159.38 ± 41.87 min). The
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onset of action (P = 0.795), time to peak action (P = 0.499), duration of action (P
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= 0.301), and total metabolic effect (P = 0.603) did not differ with route of
43
administration; however, SC administration did result in a higher maximum
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plasma insulin aspart concentration (IM: 1265.17 pmol/L [999.69 – 1433.89
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pmol/L], SC: 3278.19 pmol/L [2485.29 – 4132.01 pmol/L], P = 0.000) and larger
46
area under the insulin aspart versus time curve (IM: 82662 ± 30565 pmol/L, SC:
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135060 ± 39026 pmol/L, P = 0.010). Insulin aspart has a rapid onset of action
48
and short duration of effect in healthy cats when administered by IM and SC
49
injection. Although it cannot be assumed that the PD and PK of insulin aspart will
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be the same in cats with diabetic ketoacidosis (DKA), our data supports further
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investigation into the use of SC insulin aspart as an alternative to regular insulin
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for the treatment of DKA in cats.
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Key words: Feline; Diabetes; Insulin analog; Rapid-acting analog
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This information was presented in part at the 2016 Annual American College of
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Veterinary Internal Medicine Forum, Denver, Colorado.
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1. Introduction During the last 20 years, it has become possible to modify the amino acid structure of human insulin to create insulin analogs which have time-action
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profiles that differ from short-acting (i.e., regular) and intermediate-acting
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insulin formulations. This has led to the development of new insulin
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formulations with a more rapid onset of action than regular insulin (i.e., rapid-
76
acting analogs) as well as additional long-acting formulations (e.g. insulin
77
glargine, insulin detemir, insulin degludec) [1]. For many human diabetics,
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maintenance insulin therapy consists of a combination of a long-acting analog
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which mimics basal insulin secretion and a rapid-acting insulin analog at meal
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time [1]. When combined, the time-action profiles of these insulin analogs more
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closely mimic normal physiologic insulin secretion than do crystalline insulin
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formulations [1]. The improved predictability and consistency of the time-action
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profiles of insulin analogs decreases the risk and occurrence of hypoglycemic
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events in people [1,2].
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The molecular structure of the rapid-acting analogs (e.g., insulin lispro,
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insulin aspart, insulin glulisine) differ from endogenous human insulin by one to
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two amino acids [1,3]. While this alteration affects absorption, it does not interfere
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with binding to the insulin receptor or other biophysiologic properties of the
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insulin molecule [4]. The amino acid substitutions destabilize hexamerization
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producing insulin solutions consisting primarily of monomers and dimers which
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allows rapid absorption following subcutaneous (SC) injection [3]. This is in
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contrast to regular insulin, which has a relatively high tendency to form hexamers
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requiring dissociation following injection and leading to slower onset of action and
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longer duration of effect [1,3]. The process of hexamer dissociation following
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injection also leads to a more variable time-action profile of regular insulin and as
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a result, less predictability. These characteristics of regular insulin lead to
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increased risk of hypoglycemia and were the driving force behind the
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development of rapid-acting analogs [1-6]. In people, rapid-acting analogs have
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an onset of action ranging from 5 - 20 min and duration of effect of 2 - 6 h with
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peak action occurring 30 - 90 min after SC injection [1,3].
Rapid absorption, predictable onset of action, and a short duration of
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effect are the properties that led researchers to also investigate the use of
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subcutaneously administered rapid-acting analogs in the treatment of human
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diabetic ketoacidosis (DKA). When SC insulin aspart and insulin lispro were
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compared to intravenous (IV) regular insulin in the treatment of people with mild
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to moderate DKA, researchers found that intermittent injection of rapid-acting
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analogs was as effective [7,8].
Pharmacodynamic (PD) and pharmacokinetic (PK) data for rapid-acting
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analogs are not available for cats and their use has not been reported in this
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species. In veterinary medicine, the PD of insulin formulations have traditionally
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been determined by analyzing the reduction in blood glucose concentration over
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time following insulin administration in euglycemic, fasted animals [9,10]. An
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alternative and superior technique is the isoglycemic clamp method which uses
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IV glucose to counteract the glucose-lowering effect of the insulin. This method
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avoids hypoglycemia and the effects of counter-regulatory hormone secretion,
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which is a concern when administering insulin to nondiabetic, fasted individuals.
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This technique also minimizes the effects of endogenous insulin secretion due to
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hyperglycemia by attempting to maintain or “clamp” the blood glucose
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concentration within a normal concentration range. The clamp method is
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considered the gold standard in people and uses the curve generated by
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graphing the glucose infusion rate (GIR) over time to describe the PD properties
122
of the administered insulin [11,12]. Historically, somatostatin was used by some
123
investigators to inhibit endogenous insulin secretion during glucose clamp
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studies evaluating the PD of exogenous insulin. More recently, the use of
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somatostatin has largely been abandoned in people due to side effects and
126
potential effects on insulin clearance [13]. The isoglycemic clamp method without
127
the administration of somatostatin is an accepted and utilized method to study
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insulin PD in people [12,13] and has been used in cats to study the PD of long-
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acting analogs [14]. Additionally, by measuring both the endogenous and
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exogenous insulin concentrations during the clamp, it is possible to confirm that
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the observed metabolic effect is the result of insulin administration.
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The purpose of this study was to utilize the isoglycemic clamp method to determine the PD and PK of insulin aspart in healthy cats following intramuscular
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(IM) and SC injection.
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2. Materials and Methods
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2.1 Animals
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The Louisiana State University Institutional Animal Care and Use
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Committee approved all animal use, and the experiment was performed in an
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AAALAC accredited facility. Eight healthy, purpose-bred, research cats were
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used in this study. Given the repeated measures crossover design, a sample size
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of eight cats was needed to obtain a desired power of 80%, a Type I error of 5%,
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and a large effect size equal to 1 standard deviation. All cats were well
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acclimated to human interaction and had a physical examination, complete blood
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count, serum biochemistry profile, and serum total thyroxine concentration
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performed prior to inclusion in the study. Body condition score (BCS) was
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recorded for the cats by use of a 9-point system (1 = extremely thin, 5 = optimal,
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and 9 = obese) [15]. Five cats were neutered males and three cats were spayed
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females with a mean age of 5 ± 3 years (range 2 - 9 years). The mean body
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weight and BCS were 5.9 ± 1.4 kg (range: 4.0 - 8.3 kg) and 7/9 (range: 4/9 - 9/9).
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Cats were fed a maintenance laboratory diet (LabDiet 5003-Laboratory Feline
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Diet, LabDiet®, St. Louis, MO, USA) ad libitum and group-housed in two groups
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of four prior to the study. Following IV catheter placement at the beginning of the
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study, the cats were individually housed until completion of the study. The cats
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were not sedated or physically restrained during the clamps, which were
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performed individually in a large metabolic cage (91 cm x 91 cm x 81 cm) that
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contained bedding and a litter box.
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2.2 Study Design
A randomized, crossover study design was used with each cat receiving one IM and one SC injection of insulin aspart (NovoLog, Novo Nordisk Inc.,
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Plainsboro, NJ, USA), on different days, at least 48 h apart. The dose of insulin
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aspart administered was 0.25 IU/kg rounded to the nearest half unit. In order to
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minimize dosing inaccuracies, 0.3 mL U100 insulin syringes were used and a
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single investigator measured all insulin doses. Insulin was injected midway
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between the scapulas for SC administration and in the quadriceps for IM
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administration. Cats were fasted overnight for 12 - 16 h before each clamp. Jugular (Small Animal Long Term Venous Catherization Set, MILA
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International, Inc., Florence, KY, USA) and cephalic (Surflo Intravenous Catheter,
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Terumo Corp., Binan, Laguna, PHL) IV catheters were placed and maintained for
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the duration of the study. Catheter placement was performed under sedation with
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dexmedetomidine (0.002 mg/kg), butorphanol (0.2 mg/kg) and ketamine (5
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mg/kg) a minimum of 36 h before the clamp was performed. Catheters were
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heparinized at the time of placement and following completion of each cat’s first
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clamp. The heparin was removed from the jugular catheters and the cephalic
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catheters were flushed prior to beginning each clamp. The jugular catheter was
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flushed with non-heparinized saline while sampling during the clamp. All
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catheters remained patent and functional during the study with the exception of a
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cephalic catheter in one cat. This catheter was replaced and the clamp was
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performed 48 hours after replacement. The cephalic catheters were used
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exclusively for glucose infusion, while jugular catheters were used exclusively for
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blood sampling. The blood glucose concentration was measured with a
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handheld glucometer (AlphaTrak2, Zoetis, Florham Park, NJ, USA) that has been
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validated in cats [16]. The 20% glucose infusion, made by diluting 50% dextrose
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(50% Dextrose USP, VetOne®, Nova-Tech, Inc., Grand Island, NE, USA) with
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saline, was administered through an extension set (Microbore Extension Set, 36
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inch, Secure Lock, Hospira, Inc., Lake Forest, IL, USA) using a syringe pump
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(Medfusion 2010i, Medexinc, Duluth, GA, USA).
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A small (≤ 0.1 mL) blood sample was collected from the jugular catheter
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every 5 min for blood glucose measurement during the clamp. A total of 1.5 mL
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of blood was collected every 15 minutes into chilled, glass
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ethylenediaminetetraacetic tubes and remained on ice until refrigerated
193
centrifugation (4° C, 805 x g) at the end of each clamp. The plasma was frozen in
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glass tubes and stored at - 80° C until analysis. T he plasma insulin aspart
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concentration was measured at time zero, then every 15 min for the duration of
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the clamp. The endogenous insulin concentration was measured at time zero,
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every 60 min during the clamp, at the end of the clamp, and at the time point that
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corresponded to peak insulin action (determined after completion of the clamp).
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2.3 Isoglycemic Clamp Method
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Following insulin administration, an IV glucose infusion was used to maintain or “clamp” the blood glucose concentration in the target isoglycemic
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range. This range was ± 10% of the baseline blood glucose concentration and
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was calculated at the beginning of each clamp. The baseline blood glucose
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concentration was the average of the blood glucose concentration at t = -15 min
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and time zero. Following insulin administration, the GIR was adjusted to maintain
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the blood glucose concentration within the target range. Administration of the
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glucose infusion began when the blood glucose concentration decreased below
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the target range. Based on previous experience performing isoglycemic clamps
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in cats (CG), the initial GIR was approximately 10 mL/h (range 7-12 mL/h) with
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adjustments in rate between 1-3 mL/h, depending on the blood glucose, rate of
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change, and the degree of deviation from the target range. The blood glucose
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concentration and GIR were manually recorded every 5 min. The clamp was
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considered complete when the blood glucose concentration remained within the
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target range for 45 min without the administration of glucose.
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The GIR over time was used to create a time-action curve for each clamp.
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The onset of action was defined as the time from insulin administration (t = 0) to
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initiation of the glucose infusion. The end of action was defined as the time the
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glucose infusion was discontinued. The duration of action was defined as the
220
time from the onset of action to the end of action. The peak insulin action was
221
defined as the highest GIR during the clamp (GIRmax), and time to peak (tmax GIR)
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was the time elapsed from administration to peak insulin action. The GIR is
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expressed in milligrams of glucose per kilogram body weight per minute. Total
224
metabolic effect was calculated as the total amount of glucose infused during the
225
clamp divided by body weight ([total milliliters of glucose solution infused x 0.2] /
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body weight in kilograms).
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2.4 Insulin measurement Insulin quantification was performed with commercially available insulin ELISA kits (Mercodia AB, Uppsala, Sweden). The insulin ELISAs were performed
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according to the manufacturer’s protocol and absorbance was read at 450 nm
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with an automated ELISA plate reader (Epoch, BioTek, Winooski, Vermont,
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USA). The Iso-Insulin ELISA (Mercodia AB, Uppsala, Sweden) used to measure
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the plasma insulin aspart concentration has a reported cross-reactivity of 100%
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with insulin aspart [17] and lacks cross-reactivity with feline insulin [18,19]. The
237
detection limit is ≤ 7.18 pmol/L and the assay range is 21.53 – 171.50 pmol/L.
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For statistical analysis, sample results with an absorbance greater than the
239
negative control but less than standard 1 were reported as 21.53 pmol/L, and
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samples with an absorbance equal to or less than the negative control were
241
reported as 0 pmol/L. Samples that exceeded the assay range were diluted 1:10
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per the manufacturer’s recommendations. The intra-assay coefficient of variation
243
(CV) was 5.09% and the inter-assay CV was 3.39%.
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The time to detection of insulin aspart was defined as time from insulin
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administration to the first detected increase in the plasma concentration. The
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peak concentration (Cmax) was defined as the highest plasma insulin aspart
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concentration measured and the time to peak concentration (tmax Asp) was the
248
time from administration to Cmax. The end of insulin detection was defined as the
249
last time point that the insulin aspart concentration was above or equal to the
250
concentration of standard 1 (24.90 pmol/L). The duration of insulin aspart
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detection was defined as the time from initial detection until the end of detection.
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The previously validated [20] Feline Insulin ELISA (Mercodia AB, Uppsala, Sweden) used to measure the plasma endogenous insulin concentration has a
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detection limit of 1.60 pmol/L, an assay range of 1.74 – 121.86 pmol/L, and a
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reported 5.9% cross-reactivity with insulin aspart. For statistical analysis, sample
256
results with an absorbance greater than the negative control but less than
257
standard 1 were recorded as 1.60 pmol/L, and samples with an absorbance
258
equal to or less than the negative control were recorded as 0 ng/L. The intra-
259
assay CV was 16.48% and the inter-assay CV was 7.65%.
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2.5 Statistical analysis
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Statistical analysis was performed using commercially available computer software (SPSS 23.0, IBM Statistics, Armonk, NY, USA). The distribution of the
263
data was evaluated using the Shapiro-Wilk test, skewness, kurtosis, and q-q
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plots. Data that were not normally distributed (onset of action, Cmax, tmax Asp,
265
fasting insulin concentration, and blood glucose concentration time zero) were
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log transformed for parametric testing. Data that are normally distributed are
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reported as mean ± SD, while data that are not normally distributed are reported
268
as median (25-75 percentile). An independent samples t-test was used to
269
determine if body weight or BCS were different by sex. A linear mixed model was
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used to determine if the onset of action, end action, duration of action, GIRmax,
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tmax GIR, area under the GIR versus time curve (AUC GIR), end detection insulin
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aspart, duration detection insulin aspart, Cmax, tmax ASP, area under the aspart
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versus time curve (AUC Asp), fasting insulin concentration, baseline blood
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glucose concentration, and blood glucose concentration 30 min after completion
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of the clamp were affected by route of administration or BCS. When comparing
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BCS, cats were categorized as ideal (BCS 4-6) or increased (BCS 7-9). A paired
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samples t-test was used to determine if CV of blood glucose concentration was
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significantly different during IM and SC clamps. A P value < 0.05 was used to
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determine statistical significance.
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There was not a difference in body weight (P = 0.564) or BCS (P = 0.735)
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between male and female cats. The total dose of insulin aspart administered was
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1, 1.5, or 2 IU depending on body weight, with each cat receiving the same
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insulin dose for the IM and SC clamps. No adverse effects or injection site
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reactions were noted with IM or SC administration of insulin aspart.
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The baseline blood glucose concentration (IM: 94.1 ± 7.4 mg/dL, SC: 95.4 ± 6.2 mg/dL; P = 0.347), blood glucose concentration 30 min after completion of
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the clamp (IM: 94.8 ± 6.1 mg/dL, SC: 96.8 ± 7.2 mg/dL; P = 0.660), and fasting
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endogenous insulin concentration (IM: 14.92 pmol/L [7.2- 69.24 pmol/L]), SC:
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33.95 pmol/L [20.61 – 56.44 pmol/L]; P = 0.332) did not differ with route of
292
administration. Although an increased BCS was associated with a higher fasting
293
blood glucose concentration (P = 0.004), it was not associated with a higher
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fasting endogenous insulin concentration (P = 0.593).
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There was not a difference (P = 0.904) in CV of blood glucose
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concentrations during IM (17.94% ± 4.5) and SC (17.71% ± 3.9) clamps which
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confirms that the blood glucose concentration was maintained within a similar
298
range for all clamps (Figure 1).
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300 301
3.1 Pharmacodynamics The PD parameters are summarized in Table 1 and the time-action curves are individually graphed in Figure 1. Route of administration was not associated
303
with a difference in onset of action (P = 0.795), time to peak action (P = 0.499),
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duration of action (P = 0.301), AUC GIR (P = 0.736), or total metabolic effect (P =
305
0.603). Body condition score was not associated with a difference in PD of insulin
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aspart.
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Endogenous insulin secretion was suppressed within 60 min (IM: 5.42 ± 5.16 pmol/L; SC: 5.80 ± 4.38 pmol/L) of insulin administration and remained
309
suppressed to a low concentration at 120 min (IM: 1.86 ± 0.81 pmol/L; SC: 1.57 ±
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0 pmol/L) (Figure 2). The endogenous insulin concentration was less than the
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baseline concentration at the completion of all clamps (P = 0.000).
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The PK data are summarized in Table 2 and the concentration over time curves are individually graphed in Figure 1. Insulin aspart was detected in all cats
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at the first time point sampled (15 min after injection). The maximum plasma
317
insulin aspart concentration (Cmax) achieved (IM: 1265 pmol/L [1000-1434
318
pmol/L] vs. SC: 3278 pmol/L [2485-4132 pmol/L], P = 0.000) and AUC Asp (P =
319
0.010) were greater following SC administration. The time to maximum plasma
320
concentration (tmax) and duration of detection did not differ (P = 0.586, P = 0.268,
321
respectively) with route of administration. An increased body condition score was
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associated an increased AUC Asp (P = 0.009).
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4. Discussion In this study, both IM and SC administration of insulin aspart resulted in a rapid and predictable onset of action and short duration of effect. By confirming
326
suppression of the endogenous insulin concentration during the clamps, it was
327
possible to confirm that the glucose lowering effect observed was the result of
328
the administered insulin aspart. Route of administration was not associated with
329
a significant difference in the PD of insulin aspart. This is consistent with a study
330
in people that failed to find a difference between IM and SC administration of
331
insulin lispro, a similar rapid-acting analog [21]. Although the PD and total
332
metabolic effect were not influenced by the route of administration, a higher peak
333
plasma concentration (Cmax) and larger area under the insulin aspart versus time
334
curve was seen with SC administration. The cause of this difference has not
335
been determined, but potential explanations include differences in absorption
336
related to the site of administration (e.g., pelvic limb versus trunk) or a difference
337
in SC versus IM absorption that has not been previously reported in people. Site
338
of SC injection has been shown to have a significant effect on the PK of rapid-
339
acting analogs in people with SC injections in the abdominal region resulting in
340
more rapid absorption and greater peak insulin concentration than those in the
341
thigh, arm, or buttocks [3,22,23]. Additionally, and similar to our findings,
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differences in PK are not always associated with a significant difference in the
343
overall blood glucose lowering effect [22,23].
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In people, the slow onset and long duration of action of regular insulin is
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related to the dissociation of insulin that must occur prior to absorption [24,25],
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and it has been demonstrated that the peak effect of regular insulin occurs more
347
rapidly following IM injection [21]. As mentioned previously, IM and SC
348
administration were not associated with a difference in glucose lowering effect in
349
people receiving insulin lispro [21] or in the cats in our study receiving insulin
350
aspart. The lack of difference and rapid absorption regardless of route of
351
administration of rapid-acting analogs is likely the result of destabilized
352
hexamerization leading to increased concentrations of monomers and dimers in
353
solution and rapid dissociation following injection [6]. While dehydration might be
354
expected to further slow the absorption of SC regular insulin, it is possible that
355
insulin formulations that are mostly monomers and dimers could be less affected
356
by hydration status. If this is true and insulin aspart has a consistent and
357
predictable time-action profile in the presence of dehydration, it could provide an
358
alternative for treating DKA in cats. Although the use of SC insulin aspart for the
359
treatment of feline DKA needs to be investigated, it is promising that studies in
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people have found intermittent SC rapid-acting protocols to be as effective as IV
361
insulin continuous rate infusion [8,9].
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Both the heterogeneity of BCS and the increased body conditions of the cats were considered when interpreting the results of this study. Although an
364
increased BCS was associated with an increase in the fasting blood glucose
365
concentration, it is worth noting that the fasting blood glucose was ≤ 100 mg/dL
366
in 14/16 clamps and the maximum fasting blood glucose concentration of 103
367
mg/dL was in an overweight (BCS 7/9), not obese cat. All fasting blood glucose
368
concentrations were below the upper cutpoint (116 mg/dL) for fasting blood
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glucose recently reported for normal cats [26], and below 118 mg/dL which has
370
recently been suggested as the cutpoint for impaired fasting glucose [27]. Also,
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BCS was not associated with a difference in fasting insulin concentration. An
372
increased BCS was also associated with an increase in AUC Asp, but was not
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associated with differences in the other PK parameters or PD. A possible
374
explanation for the increased AUC Asp is decreased insulin clearance which has
375
been observed in people with an increased body mass index receiving insulin
376
aspart [28]. In that study, the decrease in insulin clearance was found to be
377
clinically insignificant. The difference in AUC Asp associated with BCS was also
378
insignificant in our study as there was no associated difference in PD.
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The potential impact of stress-induced hyperglycemia was considered when designing and executing this study. All cats used in this study were well
381
acclimated to human interaction and the laboratory environment, and no signs of
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stress or anxiety were observed during the clamps. Most importantly, this study
383
was designed in such a way that the cats were not physically restrained and only
384
required minimal handling during the clamps. Additionally, the fasting blood
385
glucose concentration and blood glucose concentration 30 min after completion
386
of the clamps were within the euglycemic range and not consistent with the
387
occurrence of stress hyperglycemia [29].
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In conclusion, insulin aspart has a rapid onset of action and short duration
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of effect in healthy cats when administered by IM and SC injection. Although it
390
cannot be assumed that the PD and PK of insulin aspart will be the same in cats
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with DKA, our data supports further investigation into the use of SC insulin aspart
392
as an alternative to regular insulin for the treatment of DKA in cats.
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Acknowledgements
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The authors thank Claire Webster for her technical assistance, Chin-Chi Liu for
396
assistance with sample analysis, and Michael Kearney for help with statistical
397
analysis.
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Funding: This work was supported in part by the Louisiana State University
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Veterinary Clinical Sciences Departmental Competitive Organized Research
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Program.
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Neumiller J, Odegard P, Wysham C. Update on insulin management in type 2 diabetes. J Fam Pract. 2012;61(5):1-32. Pfutzner A, Kustner E, Forst T, Schulze-Schleppinghoff B, Trautmann ME, Haslbeck M, Schatz H, Beyer J. Intensive insulin therapy with insulin lispro in patients with type 1 diabetes reduces the frequency of hypoglycemic episodes. Exp Clin Endocrinol Diabetes. 1996;104(1):25-30. doi:10.1055/s-0029-1211418. Home PD. The pharmacokinetics and pharmacodynamics of rapid-acting insulin analogues and their clinical consequences. Diabetes, Obes Metab. 2012;14(9):780-788. doi:10.1111/j.1463-1326.2012.01580.x. Setter SM, Corbett CF, Campbell RK, White JR. Insulin aspart: a new rapid-acting insulin analog. Ann Pharmacother. 2000;34(12):1423-1431. Evans M, Schumm-Draeger PM, Vora J, King AB. A review of modern insulin analogue pharmacokinetic and pharmacodynamic profiles in type 2 diabetes: Improvements and limitations. Diabetes, Obes Metab. 2011;13(8):677-684. doi:10.1111/j.1463-1326.2011.01395.x. Berenson DF, Weiss AR, Wan Z, Weiss MA. Insulin analogs for the treatment of diabetes mellitus: therapeutic applications of protein engineering. Ann N Y Acad Sci. 2011;1243:E40-E54. doi:10.1111/j.17496632.2012.06468.x. Umpierrez GE, Cuervo R, Karabell A, Latif K, Freire AX, Kitabchi AE. Treatment of diabetic ketoacidosis with subcutaneous insulin aspart. Diabetes Care. 2004;27(8):1873-1878. Umpierrez GE, Latif K, Stoever J, Cuervo R, Park L, Freire AX, Kitabchi AE. Efficacy of subcutaneous insulin lispro versus continuous intravenous regular insulin for the treatment of patients with diabetic ketoacidosis. Am J Med. 2004;117(5):291-296. doi:10.1016/j.amjmed.2004.05.010. Clark M, Thomaseth K, Heit M, Hoenig M. Pharmacokinetics and pharmacodynamics of protamine zinc recombinant human insulin in healthy dogs. J Vet Pharmacol Ther. 2012:342-350. Marshall R, Rand J. Comparison of the pharmacokinetics and pharmacodynamics of glargine, protamine zinc, and porcine lente insulins in normal cats (abstract). J Vet Intern Med. 2002:358. Heinemann L, Anderson JH. Measurement of insulin absorption and insulin action. Diabetes Technol Ther. 2004;6(5):698-719. Porcellati F, Bolli GB, Fanelli CG. Pharmacokinetics and pharmacodynamics of basal insulins. Diabetes Technol Ther. 2011;13 Suppl 1:S15-S24. doi:10.1089/dia.2011.0038. Heise T, Zijlstra E, Nosek L, Heckermann S, Plum- Mörschel L, Forst T. Euglycaemic glucose clamp: what it can and cannot do, and how to do it. Diabetes, obesity and metabolism. 2016; 18(10):962–972. Gilor C, Ridge TK, Attermeier KJ, Graves TK. Pharmacodynamics of insulin determir and insulin glargine assessed by an isoglycemic clamp method in healthy cats. J Vet Intern Med. 2010;24:870-874.
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[15] Laflamme DP. Development and validation of a body condition score system for cats: a clinical tool. Feline Practice 1997;25:13-18. [16] Zini E, Moretti S, Tschuor F, Reusch CE. Evaluation of a new portable glucose meter designed for the use in cats. Schweiz Arch Tierheilkd. 2009;151(9):448-451. doi:10.1024/0036-7281.151.9.448. [17] Lindström T, Hedman CA, Arnqvist HJ. Use of a novel double-antibody technique to describe the pharmacokinetics of rapid-acting insulin analogs. Diabetes Care. 2002;25(6):1049-1054. [18] Bryan C, Lathan P, Curotto S, Bulla C, Lunsford K, Pruett S. Lack of cross reactivity of feline insulin with a commercial human ELISA (abstract). J Vet Intern Med. 2011;25:632-767. [19] Fletcher J, Pipe-Martin H, Liu C. Using purified feline insulin to evaluate cross-reactivity with a human insulin analog ELISA (abstract). J Vet Intern Med. 2016;30:1407-1519. [20] Strage EM, Holst BS, Nilsson G, Jones B, Lilliehk I. Validation of an enzyme-linked immunosorbent assay for measurement of feline serum insulin. Vet Clin Pathol. 2012;41(4):518-528. doi:10.1111/j.1939165x.2012.00476.x. [21] Rave K, Heise T, Weyer C, Hernberger J, Bender R, Hirschberger S, Heinemann L. Intramuscular versus subcutaneous injection of soluble and lispro insulin: comparison of metabolic effects in healthy subjects. Diabet Med. 1998;15(2):747-751. doi: 10.1002/ (SICI) 1096-9136 (199809)15:9<747::AID-DIA664>3.0.CO:2-V. [22] Mudaliar SR, Lindberg FA, Joyce M, Beerdsen P, Strange P, Lin A, Henry RR. Insulin aspart (B28 Asp-insulin): A fast-acting analog of human insulin: Absorption kinetics and action profile compared with regular human insulin in healthy nondiabetic subjects. Diabetes Care. 1999;22(9):1501-1506. doi:10.2337/diacare.22.9.1501. [23] ter Braak EW, Woodworth JR, Bianchi R, Cerimele B, Erkelens DW, Thijssen JH, Kurtz D. Injection site effects on the pharmacokinetics and glucodynamics of insulin lispro and regular insulin. Diabetes Care. 1996;19(12):1437-1440. doi:10.2337/diacare.19.12.1437. [24] Howey DC, Bowsher RR, Brunelle RL and Woodworth JR. [Lys(B28), Pro(B29)]-Human Insulin: A Rapidly Absorbed Analogue of Human Insulin. Diabetes 1994;43(3):396-402. [25] Hirsch I.B. Insulin analogues. N Engl J Med. 2005;352(2): 174-183. [26] Reeve-Johnson MK, Rand JS, Vankan D, Anderson ST, Marshall R, Morton JM. Cutpoints for screening blood glucose concentrations in healthy senior cats. J Fel Med Surg. 2017 (Epub ahead of print) doi:10.1177/1098612X1668567. [27] Reeve-Johnson M, Rand J, Vankan D, Anderson S, Marshall R, Morton J. Diagnosis of prediabetes in cats: glucose concentration cut points for impaired fasting glucose and impaired glucose tolerance. Domest Anim Endocrinol. 2016;57:55-62.
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[28] Holmes G, Galitz L, Hu P, Lyness W. Pharmacokinetics of insulin aspart in
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obesity, renal impairment, or hepatic impairment. Br J Clin Pharmacol.
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Table 1. Pharmacodynamic parameters of insulin aspart (0.25 U/kg) in 8 healthy cats after intramuscular and subcutaneous injections. Data are reported as mean ± SD if normally distributed or median (25-75 percentile) if not normally distributed. Subcutaneous
10 (10-21.25) 196.25 ± 38.24 182.50 ± 34.33 8.29 ± 2.18 49.38 ± 35.60 901.13 ± 22.72 0.93 ± 0.24
12.5 (10-18.75) 173.13 ± 40.88 159.38 ± 41.87 7.91 ± 2.51 43.75 ± 33.46 858.50 ± 266.18 0.89 ± 0.30
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Onset of action (min) End of action (min) Duration of action (min) Peak action / GIRmax (mg/kg/min) Time to peak action / tmax GIR (min) AUC GIR Total metabolic effect (g/kg)
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Pharmacodynamic parameters
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Intramuscular
P Value
0.795 0.281 0.301 0.749 0.499 0.736 0.603
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Table 2. Pharmacokinetics of insulin aspart (0.25 U/kg) in 8 healthy cats after intramuscular and subcutaneous injections. Data are reported as mean ± SD if normally distributed or median (25-75 percentile) if not normally distributed.
15 186.25 ± 56.24 171.25 ± 56.24 1265 (1000-1434) 15 (15-41.25) 82662 ± 30565
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Initial detection (min) End of detection (min) Duration of detection (min) Cmax (pmol/L) tmax Asp (min) AUC Asp
15 161.25 ± 49.91 146.25 ± 49.91 3278 (2485-4132) 15 (15-26.25) 135060 ± 39026
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Pharmacokinetic parameters
Subcutaneous
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Intramuscular
P Value
0.268 0.268 0.000 0.586 0.010
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Figure 1. Plasma insulin aspart concentration (solid line), glucose infusion rate (GIR) / time action profile (dashed line), and the blood glucose concentration (dotted line) during the clamp procedure following intramuscular (o) and subcutaneous () injections of 0.25 U/kg insulin aspart in individual cats (A-H).
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Figure 2. Plasma endogenous insulin concentrations for individual cats during the clamp procedure following intramuscular and subcutaneous injection of 0.25 U/kg insulin aspart.