7
Pharmacokinetics and Pharmacodynamics of NPH Insulin in Type 1 Diabetes Mellitus: The Importance of Appropriate Resuspension Before Subcutaneous Injection DOI: 10.2337/dc15-0801 OBJECTIVE Crystalline NPH insulin comes in a two-phase solution with either a solvent or a rapid-acting insulin (in premixed formulations) and needs adequate mixing for complete resuspension before injection. The aim of this study was to establish pharmacokinetics (PK) and pharmacodynamics (PD) after injection of appropri- ately resuspended versus nonresuspended NPH insulin. RESEARCH DESIGN AND METHODS PK and PD were assessed after subcutaneous injection of NPH insulin 0.35 units/kg at steady-state by pen either resuspended (R+, tipping of insulin pen 20 times) or nonresuspended (pen maintained in xed position either horizontally [R- horizontal] or vertically with tip up [R- up] or tip down [R- down]). Eleven subjects with type 1 di- abetes (age 31.5 6 12 years, diabetes duration 17.5 6 7.7 years, BMI 22.9 6 1.5 kg/m 2 , A1C 7.2 6 0.4% [55.2 6 4.4 mmol/mol]) were studied (euglycemic clamp) with a randomized crossover design. RESULTS Compared with resuspended NPH insulin (R+), nonresuspended NPH insulin resulted in profound PK/PD differences with either reduced (R- horizontal and R- up) or increased (R- down) plasma insulin concentrations [FIRI_AUC (0end of study) (free immunoreactive insulin area under the concentration-time curve between 0 and end of study)] and PD activity [glucose infusion rate (GIR)_AUC (0end of study) ] (all P < 0.05). Duration of NPH insulin action was shorter in R- up (9.4 6 1.7 h) but longer in R- down (15.4 6 2.3 h) compared with R+ (11.8 6 2.6 h) (P < 0.05). Within-subject variability (percent coefcient of variation) among studies was as high as 23% for PK [FIRI_AUC (0end of study) ] and 62% for PD [GIR_AUC (0end of study) ]. CONCLUSIONS Compared with resuspended NPH insulin, lack of resuspension profoundly alters PK/PD and may importantly contribute to day-to-day glycemic variability of type 1 diabetes. Department of Medicine, Section of Internal Medicine, Endocrinology and Metabolism, Perugia University School of Medicine, Perugia, Italy Corresponding author: Geremia B. Bolli, geremia. [email protected]. Received 15 April 2015 and accepted 13 August 2015. This article contains Supplementary Data online at http://care.diabetesjournals.org/lookup/ suppl/doi:10.2337/dc15-0801/-/DC1. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for prot, and the work is not altered. Paola Lucidi, Francesca Porcellati, Anna Marinelli Andreoli, Ilaria Carriero, Paola Candeloro, Patrizia Cioli, Geremia B. Bolli, and Carmine G. Fanelli Diabetes Care 1 EMERGING TECHNOLOGIES AND THERAPEUTICS Diabetes Care Publish Ahead of Print, published online September 10, 2015

Pharmacokinetics and Paola Lucidi, Francesca Porcellati ... · Paola Lucidi, Francesca Porcellati, Anna Marinelli Andreoli, Ilaria Carriero, Paola Candeloro, Patrizia Cioli, Geremia

  • Upload
    others

  • View
    12

  • Download
    0

Embed Size (px)

Citation preview

Page 1: Pharmacokinetics and Paola Lucidi, Francesca Porcellati ... · Paola Lucidi, Francesca Porcellati, Anna Marinelli Andreoli, Ilaria Carriero, Paola Candeloro, Patrizia Cioli, Geremia

Pharmacokinetics andPharmacodynamics of NPHInsulin in Type 1 DiabetesMellitus:The Importance of AppropriateResuspension BeforeSubcutaneous InjectionDOI: 10.2337/dc15-0801

OBJECTIVE

Crystalline NPH insulin comes in a two-phase solution with either a solvent or arapid-acting insulin (in premixed formulations) and needs adequate mixing forcomplete resuspension before injection. The aim of this study was to establishpharmacokinetics (PK) and pharmacodynamics (PD) after injection of appropri-ately resuspended versus nonresuspended NPH insulin.

RESEARCH DESIGN AND METHODS

PK and PDwere assessed after subcutaneous injection of NPH insulin 0.35 units/kgat steady-state by pen either resuspended (R+, tipping of insulin pen 20 times) ornonresuspended (penmaintained in fixed position either horizontally [R- horizontal]or vertically with tip up [R- up] or tip down [R- down]). Eleven subjectswith type 1 di-abetes (age 31.56 12 years, diabetes duration 17.56 7.7 years, BMI 22.96 1.5 kg/m2,A1C 7.2 6 0.4% [55.2 6 4.4 mmol/mol]) were studied (euglycemic clamp) with arandomized crossover design.

RESULTS

Compared with resuspended NPH insulin (R+), nonresuspended NPH insulinresulted in profound PK/PD differences with either reduced (R- horizontal andR- up) or increased (R- down) plasma insulin concentrations [FIRI_AUC(0–end of study)

(free immunoreactive insulin area under the concentration-time curve between0 and end of study)] and PD activity [glucose infusion rate (GIR)_AUC(0–end of study)](all P < 0.05). Duration of NPH insulin action was shorter in R- up (9.4 6 1.7 h)but longer in R- down (15.4 6 2.3 h) compared with R+ (11.8 6 2.6 h) (P < 0.05).Within-subject variability (percent coefficient of variation) among studies was ashigh as 23% for PK [FIRI_AUC(0–end of study)] and 62% for PD [GIR_AUC(0–end of study)].

CONCLUSIONS

Compared with resuspended NPH insulin, lack of resuspension profoundly altersPK/PD andmay importantly contribute to day-to-day glycemic variability of type 1diabetes.

Department of Medicine, Section of InternalMedicine, Endocrinology andMetabolism, PerugiaUniversity School of Medicine, Perugia, Italy

Corresponding author: Geremia B. Bolli, [email protected].

Received 15 April 2015 and accepted 13 August2015.

This article contains Supplementary Data onlineat http://care.diabetesjournals.org/lookup/suppl/doi:10.2337/dc15-0801/-/DC1.

© 2015 by the American Diabetes Association.Readersmayuse this article as longas thework isproperly cited, the use is educational and not forprofit, and the work is not altered.

Paola Lucidi, Francesca Porcellati,

Anna Marinelli Andreoli, Ilaria Carriero,

Paola Candeloro, Patrizia Cioli,

Geremia B. Bolli, and Carmine G. Fanelli

Diabetes Care 1

EMER

GINGTEC

HNOLO

GIES

ANDTH

ERAPEU

TICS

Diabetes Care Publish Ahead of Print, published online September 10, 2015

Page 2: Pharmacokinetics and Paola Lucidi, Francesca Porcellati ... · Paola Lucidi, Francesca Porcellati, Anna Marinelli Andreoli, Ilaria Carriero, Paola Candeloro, Patrizia Cioli, Geremia

Because of their more physiological phar-macokinetics/pharmacodynamics (PK/PD) (1) at present, the long-acting insulinanalogs glargine and detemir and themore recent basal insulin degludec (2)[along with glargine U300 soon to come(3)] have largely replaced NPH insulin inthe treatment of type 1 diabetes mellitus(T1DM). However, NPH remains a popularbasal insulin in type 2 diabetes mellitus(T2DM).The PK/PD of NPH insulin (4) appears

to vary considerably from day to day(5–7). The quite large variability mightbe related primarily to the formulation ofNPH insulin per se, the lack of its appro-priate resuspension before injection bypatients with diabetes, or both. NPH in-sulin is insoluble, obtained from cocrys-tallization of insulin with zinc in thepresence of the basic polyarginine pep-tide protamine at isophane ratio andneutral pH. The exact binding mode ofthe insulin-protamine complex is notknown (8). Divalent ions like zinc andother additive ligands like phenolic de-rivatives and protamine, along with thetype and morphology of the precipitate,play a role in the slow release of insulinfrom the precipitated NPH insulin crys-tals after subcutaneous injection (9).However, the exact mechanisms of NPHinsulin dissociation are not known. Thus,variability of NPH insulin PK/PD in hu-mans might be due to the variable mech-anisms of precipitation of crystals in thesubcutaneous tissue and/or variable re-lease of insulin from the precipitate.However, variability of NPH insulin

might also be due to a lack of or insuffi-cient resuspension before injection. Infact, NPH insulin is a two-phase solution,and the insulin of the insoluble, cloudypart has to be resuspended in the soluble,clear part by tipping the insulin vial orcartridge pen several times until ahomogeneous suspension is obtained.Jehle et al. (10) reported that themajorityof patients with diabetes do not optimallyresuspend NPH insulin and NPH insulin–based premixed formulations, causing ad-verse effects on blood glucose control.However, with the exception of a pilotstudy in which a few subjects were exam-ined in an incomplete crossover design(11), at present, the effects of lack of re-suspension of NPH insulin before subcu-taneous injection on its PK/PD in T1DMhave not been systematically established.The aimof the current studywas to assess

the differences in PK/PD of a therapeuticdose of NPH insulin injected after appro-priate resuspension compared with otheroccasions without resuspension in sub-jects with T1DM.

RESEARCH DESIGN AND METHODS

SubjectsAfter approval by the local ethics commit-tee andwritten informed consent, 11 sub-jects with T1DM (Supplementary Table 1)were studied according to Declaration ofHelsinki and Good Clinical Practice re-quirements. Subjects were free of anydetectable micro- and macroangiopathiccomplications and of any major illnessother than diabetes as indicated by med-ical history, physical examination, electro-cardiogram, or routine laboratory tests.All were on an intensified basal-bolus in-sulin regimen (glargine once a day withthe evening meal, rapid-acting insulinanalog at mealtime).

Study DesignThe study was a randomized, open-label, crossover design. During 1 weekof run-in, subjects were switched fromtheir basal insulin glargine to NPH insu-lin (four daily injections: breakfast,lunch, dinner, and bedtime) as previ-ously described (12,13). Prandial insulinwas continued. Subjects were subse-quently studied on four different occa-sions at 1–3-week intervals with theeuglycemic glucose clamp techniqueafter subcutaneous injection of 0.35units/kg NPH insulin resuspended (R+)or nonresuspended (R- horizontal, R- up,and R- down) (Protaphane FlexPen, NovoNordisk, Bagsværd, Denmark). In allstudies, the NPH insulin cartridge(pen) was maintained in a refrigerator(108C) until 8 h before use, when it wasmoved to room temperature (18–208C).In R+, the NPH pen was gently tipped 20times over 1 min and 30 s immediatelybefore subcutaneous injection (2 cmright or left to the umbilicus, skinfoldtechnique). In R- horizontal, R- up, andR- down, NPH insulin was not resuspendedbefore injection. In R- horizontal, the penwas maintained in the horizontal posi-tion. In R- up and R- down, the pen wasmaintained in the vertical position withthe tip either up or down, respectively,for 8 h until time of injection (Fig. 1).Subjects were randomly assigned to thetreatments using a Latin square, four-way, crossover design.

Clamp StudyThe clamp technique has been de-scribed in detail elsewhere (4,14). Inbrief, subjects had their last NPH insulininjection on the evening of the day be-fore study and the last subcutaneous in-jection of rapid-acting insulin analog atlunch on study day. They were admittedto the Clinical Study Unit of the Depart-ment of Medicine, Perugia UniversitySchool of Medicine, between 1500 and1600 h on study day and put on bed rest.Two venous lines were started (a super-ficial vein of one forearm for insulin andglucose infusion and a dorsal vein of thecontralateral hand incannulated retro-gradely with a G-20 butterfly needlekept at 658C in a hot box for intermittentsampling of arterialized venous blood)and kept patent with 0.9% NaCl infusion(20 mL/h). A feedback insulin infusionwas started to maintain plasma glucosebetween 90 and 100 mg/dL until thetime of subcutaneous NPH insulin injec-tion (2000 h). Thereafter, the rate of in-travenous insulin infusion was taperedas soon as plasma glucose decreased by5 mg/dL below baseline and eventuallydiscontinued. Infusion of glucose wasinitiated after subcutaneous injectionof NPH insulin to maintain plasma glu-cose at the target of 100 mg/dL (4,14).Studies were planned for 24 h but wereended any time plasma glucose consis-tently exceeded 150 mg/dL in the ab-sence of glucose infusion.

Analytical MethodsPlasma glucose concentration was mea-sured at the bedside using the YSI 2300STAT glucose analyzer (YSI Inc., YellowSprings, OH). Plasma free insulin con-centration was measured by radioim-munoassay after polyethylene glycolextraction of antibodies (15) using acommercial kit specific for human in-sulin (Human Insulin-Specific RIA, DRGDiagnostics GmbH,Marburg, Germany).

Calculations and Statistical AnalysisOnset of action was defined as the timeat which infusion of glucose was initi-ated in response to a decrease in plasmaglucose concentration of 5 mg/dL belowbaseline. End of action was defined asthe time at which plasma glucoseconcentration consistently exceeded118 mg/dL in the absence of glucose in-fusion. Duration of action was calculatedas the difference between end and onsetof action. The end of study was at 24 h

2 PK/PD of Resuspended Versus Nonresuspended NPH Diabetes Care

Page 3: Pharmacokinetics and Paola Lucidi, Francesca Porcellati ... · Paola Lucidi, Francesca Porcellati, Anna Marinelli Andreoli, Ilaria Carriero, Paola Candeloro, Patrizia Cioli, Geremia

or any time plasma glucose consistentlyexceeded 150 mg/dL in the absence ofglucose infusion. The linear trapezoidalrule was used to calculate the area underthe concentration-time curve between0 and 24 h (AUC0–24h) or 0 and endof study if terminated before 24 h(AUC0–end of study) for plasma free insulinconcentration and glucose infusion rate(GIR). Incremental AUC for plasma glucosewas calculated by subtracting the basalvalue from the sample points before inte-grating AUC (16). Maximum plasma con-centration (Cmax) and the time to reachCmax(Tmax) for the same variables were read di-rectly from the plasma concentration-timedata for each subject. The determinationof Cmax and Tmax for GIR were derivedfrom a smoothed three-point runningaverage GIR curve for each subject toprovide reliable data for calculation.The primary analysis of the PK/PD pa-

rameters was performed using ANOVAwith subject, treatment, sequencegroup, and period effects on naturallog–transformed data. Within-subjectvariability was assessed as intraindividualcoefficient of variation (CV) values

calculated using the root mean squareapproach described by Bland (17).

The primary end point of the studywasduration of action. Secondary end pointswere plasma insulin concentration andGIR (AUC0–end of study). A sample size of11 subjects was chosen to achieve 91%power to detect a mean of paired differ-ences of 2.0 hwith an SD of differences of2.0 h and with a significance level (a) of0.05 using a two-sided paired z test. Alltests of statistical hypothesis were carriedout at the 5% level of significance, andcomparisons were two-sided. Data inthe text are expressed as mean 6 SDand median with 25th and 75th per-centiles as appropriate; in figures, dataare expressed as mean 6 SE. Statisticswere performed using NCSS PASS soft-ware (Kaysville, UT; www.ncss.com).

RESULTS

Plasma Insulin and GlucoseConcentrations and GIRsAfter subcutaneous NPH insulin injection,plasma insulin concentration initiallyincreased for 4–5 h and subsequently de-creased in all four studies but to a different

extent (Fig. 2). Compared with resus-pendedNPH insulin (R+), injection of non-resuspended NPH insulin resulted ineither lower (R- horizontal and R- up) orhigher (R- down) plasma insulin concen-tration (Fig. 2), AUC(0–end of study), and Cmax

(Table 1). In contrast, Tmax was no differ-ent among the four studies.

Plasma glucose concentration wasmaintained at the target in all four stud-ies in the initial 4 h but subsequentlyincreased by various rates in the differ-ent studies. Comparedwith resuspendedNPH insulin (R+), plasma glucose in-creased earlier to .110 mg/dL inR- horizontal and R- up (Fig. 2), althoughthe difference in Tmax was statisticallysignificant only in R- up (Table 1). In con-trast, in R- down, Tmax occurred laterthan in the other studies (Table 1). Com-pared with R+, hyperglycemia (calcu-lated as incremental AUC) was morepronounced in R- horizontal and R- up,but reduced in R- down, the latter dif-ference being statistically significant(Table 1).

Onsetof actionofNPH insulinwasearlierin R- down versus R+; end of action tended

Figure 1—How the NPH insulin pen appears immediately before its use for subcutaneous injection. In the R+ study, NPH insulin was appropriatelyresuspended before injection. In the R- horizontal, R- up, and R- down studies, NPH insulin was injected without prior resuspension (R- horizontal,pen maintained horizontally; R- up and R- down, pen maintained vertically with R- up tip up and R- down tip down).

care.diabetesjournals.org Lucidi and Associates 3

Page 4: Pharmacokinetics and Paola Lucidi, Francesca Porcellati ... · Paola Lucidi, Francesca Porcellati, Anna Marinelli Andreoli, Ilaria Carriero, Paola Candeloro, Patrizia Cioli, Geremia

to be earlier in R- horizontal and R- up,but the difference was not statisticallysignificant. In contrast, in R- down, end ofaction occurred later than in the otherstudies (Table 1). Duration of action wasshorter in R- horizontal and R- up than inR+ (the difference statistically significantonly in R- up), whereas it was longer inR- down than in theother studies (Table 1).

TheGIR required tomaintain euglycemiawas lower in R- horizontal and R- upthan in R+ (with only the difference inR- up being statistically significant),whereas it was higher in R- down than inthe other studies. GIR Cmax was lower inR- horizontal and R- up (again, the differ-ence statistically significant only in R- up),whereas it was higher in R- down than inthe other studies. GIR Tmax did not differamong the four studies (Table 1).

Subjects in StudyAll subjects in all four studies ended be-fore the planned conclusion of 24 h be-cause plasma glucose increased to .150mg/dL (threshold for definition of end ofstudy). Consequently, there were fewersubjects in R- horizontal and R- up thanin R+ and, particularly, than in R- downafter the 11th hour (Fig. 3).

Intraindividual CVs of Plasma InsulinConcentrations and GIRsThe intraindividual CVs, ameasureof PK/PDvariability, were calculated in all fourstudies, comparing R+with R- horizontal,R- up, and R- down. In the latter threestudies, the greatest variability for freeimmunoreactive insulin (FIRI) Cmax andGIR Cmax occurred between R+ andR- down. This was also the case for FIRI_AUC(0–end of study). TheGIR_AUC(0–end of study)had the highest CV in all comparisons, themore elevated values being between R+and R- horizontal (Table 2).

CONCLUSIONS

The current study demonstrates that atherapeutic dose of NPH insulin (0.35units/kg) commonly used in patientswith T1DM results in quite differentPK/PD depending on whether NPH insu-lin is appropriately resuspended beforesubcutaneous injection. Compared withresuspended NPH insulin, nonresus-pended NPH insulin may result in eitherpotentiated or reduced insulin PK/PDdepending on the position of the penbefore injection (held horizontally orvertically with tip either up or down).At one extreme, when the pen is held

Figure 2—Plasma free insulin and glucose concentrations and GIRs tomaintain euglycemia in thefour studies after subcutaneous injection of 0.35 units/kg NPH insulin in 11 subjects with T1DM.In the R+ study, NPH insulin was properly resuspended before injection. In the R- horizontal,R- up, and R- down studies, NPH insulin was injected without prior resuspension (R- horizontal,pen maintained horizontally; R- up and R- down, pen maintained vertically with R- up tip up andR- down tip down).

4 PK/PD of Resuspended Versus Nonresuspended NPH Diabetes Care

Page 5: Pharmacokinetics and Paola Lucidi, Francesca Porcellati ... · Paola Lucidi, Francesca Porcellati, Anna Marinelli Andreoli, Ilaria Carriero, Paola Candeloro, Patrizia Cioli, Geremia

vertically tip down with no resuspensionbefore injection (R- down), the injectedNPH insulin is predominantly the cloudypart (Fig. 1), which is rich in insulin crys-tals. In this case, plasma insulin concen-tration, insulin effect (GIR), and durationof action are potentiated compared withthe other extreme of nonresuspendedNPH insulin, with the predominant in-jected part being clear (theoretically noor very few insulin crystals [R- horizontaland R- up]). Within-subject variability ofPK/PD of NPH insulin is elevated bothwhen all four suspension conditions areconsidered and when the resuspendedNPH insulin is compared with one occa-sion of nonresuspension (Table 2). In con-trast to these characteristics of theinsoluble NPH insulin, the modern long-acting insulin analogs (1,2) are solubleand, therefore, do not need resuspensionbefore injection. For this reason, they areexpected to be less variable than NPHinsulin.The present studies examined three

extreme situations of nonresuspension(pen held horizontally or vertically withtip either up or down) and not the morecommon condition where patients in-ject NPH insulin after only partial, in-complete resuspension. Still, the resultsemphasize how critical resuspension isto obtain the expected PK/PD of NPHinsulin. The wide fluctuations in plasmaglucose control reported in subjectswith T1DM who do not adequately

resuspend NPH insulin before injection(10) are likely explained by the PK/PDresults of the current study. A largepart of the reported variability of NPHinsulin (5–7,10,18) is possibly explainedby a lack of or insufficient resuspensionbefore injection, as shown in the currentstudy; thus, the variability of NPH insulinmight be substantially decreased in theeveryday life of patients with diabetes bysimply adhering to the long-well-knownrecommendation of resuspending NPHinsulin before injection, no matter the

manufacturer (19). In fact, the major in-sulin companies all recommend injectingNPH insulin at room temperature afterfull resuspension obtained with at least10–20 times of tipping the pen to allowfull movement of the small glass ballpresent in the Novo Nordisk FlexPenand Lilly KwikPen or the three smallmetal balls of the Sanofi Lantus SoloSTARpen. However, in the current study, ap-propriate resuspension of NPH insulinrequired up to 1 min and 30 s, which isquite long for preparation of an insulin

Table 1—PK/PD parameters

Study

Parameter R+ R- horizontal R- up R- down P value

PKFIRI_AUC(0–end of study) (mU/mL z h21) (*P , 0.05 vs. R+,

R- horizontal, R- up; §P , 0.05 vs. R+) 248 6 88 206 6 41§ 188 6 35§ 323 6 52* 0.001FIRI Cmax (mU/mL) (*Cmax P, 0.05 vs. R- horizontal, R- up) 24 6 9.3* 19 6 3.5 17 6 2.9 27 6 3.1* 0.001FIRI Tmax (h) 4.7 6 0.6 3.6 6 2.2 4.6 6 1.6 4.7 6 0.6 0.143

PDAUC plasma glucose (mg/dL z h21) (*P , 0.05 vs. R- up) 171 6 61 205 6 55 208 6 53 164 6 44* 0.049Plasma glucose Tmax (h) (*P , 0.05 vs. R+, R- horizontal,

R- up; §P , 0.05 vs. R+) 17.3 6 2.6 16.5 6 1.8 15.4 6 2.2§ 19.7 6 2.2* 0.001Onset of action† (h) (*P , 0.05 vs. R+) 2.2 6 1.2 1.7 6 1.2 1.7 6 1 1 6 0* 0.008End of action† (h) (*P, 0.05 vs. R+, R- horizontal, R- up) 13.9 6 2.3 11.9 6 2.1 11.1 6 2.1 16.4 6 2.3* 0.001Duration of action† (h) (*P , 0.05 vs. R+, R- horizontal,

R- up; §P , 0.05 vs. R+) 11.8 6 2.6 10.1 6 1.8 9.4 6 1.7§ 15.4 6 2.3* 0.001

GIRCmax (mg/kg/min) (*P , 0.05 vs. R- horizontal, R- up;

§P , 0.05 vs. R+) 3.8 6 2.8 2.6 6 1.7 1.7 6 1.2§ 5.1 6 3.0* 0.001Tmax (h) 6.2 6 1.4 6.3 6 1.2 5.4 6 0.9 5.1 6 0.7 0.206AUC0–end of study (mg/kg) (*P, 0.05 vs. R+, R- horizontal,

R- up; §P , 0.05 vs. R+) 1,342 6 1,129 774 6 637 481 6 322§ 1,957 6 1,253* 0.001

Data are mean 6 SD. Boldface indicates significant at P , 0.05. †See RESEARCH DESIGN AND METHODS for definition.

Figure 3—Time-course of subjects in the four studies. Studies were terminated at the end of NPHaction (plasma glucose .150 mg/dL) or at 24h. As compared to study R+, in studies R- eitherfewer (R- horizontal, R- up) or more subjects (R- down) remained in study.

care.diabetesjournals.org Lucidi and Associates 5

Page 6: Pharmacokinetics and Paola Lucidi, Francesca Porcellati ... · Paola Lucidi, Francesca Porcellati, Anna Marinelli Andreoli, Ilaria Carriero, Paola Candeloro, Patrizia Cioli, Geremia

injection and a key step that some patientsmight totally or partially skip.The two conditions of nonresuspended

NPH insulin in the current study (R- up andR- down) should establish the PK/PD of ei-ther theclear (R-up)or thecloudy (R-down)part of the injectant. In fact, the cloudy part,which is rich in insulin crystals, exhibitsthe greatest insulin activity (R- down).Of note, however, the clear part, whichshould contain no or very few insulin crys-tals, has a definite insulin activity (Fig. 2)and, thus,may indicate an important pres-ence of insulin. However, it should be ob-served that in R- up, the clear part waslikely enriched with insulin crystals fromthe cloudy part just before injectionwhenthe pen had to be inverted from the initialtip-up position to the tip-down position.The variability in PK/PD of resus-

pended or nonresuspended NPH insulin,reported as CVs in the present study, isnot directly comparable with that of an-other study where NPH insulin was re-suspended before injection (7). In thatstudy, the variability of NPH insulin washigher than glargine and detemir in sub-jects with T1DM. However, the studyexamined parallel groups of subjects,not the same subjects receiving differ-ent basal insulins in a crossover compar-ison as in the present study. In addition,the clamp technique used (automaticBiostator) (7) is not comparable withthat of the current study (manual clamp)(4,14). One limitation of the presentstudy is that calculating within-subjectvariability for resuspended NPH insulin

was not possible because the subjectswere studied only once in the R+ condi-tion. Only within-subject variability of re-suspended and nonresuspended NPHinsulin was calculated (Table 2). As ex-pected, the highest variability occurredbetween the two extreme situations ofinjection of the clear and cloudy parts ofNPH insulin (R- up and R- down).

As mentioned, NPH insulin is used lessand less in T1DM but continues to be pop-ular in the treatment of T2DM either asbasal insulin or as part of premixed formu-lations (20,21). Although PK/PD of NPH in-sulin in T2DM treatment (22) differs fromthat in T1DM treatment (4), the resultsobserved with NPH insulin in the currentstudy in T1DM could be largely applicableto T2DM. The high variability of NPH in-sulin observed in the present study is ex-pected to occur also with the premixedformulations where NPH insulin is com-bined in variable percentages with rapid-acting insulin. Such a variability of theNPHinsulin component might contribute tothe elevated risk for hypoglycemia ofNPH insulin–based premixed formula-tions (23) in addition to its PK/PD (20).

The current study has some limita-tions in addition to those already men-tioned. The number of subjects studiedwas small. However, the fact that differ-ences are quite evident with so few sub-jects speaks in favor of the importanceof the resuspension process as a mainfactor in determining PK/PD of injectedNPH insulin. The study examined NPHinsulin from only one manufacturer

(Novo Nordisk), but it is assumed thatNPH insulin from other manufacturerswould result in similar characteristics(19) and action profiles at least in sub-jects without diabetes (24). In the pres-ent study, the effect of NPH insulin wasexamined for 24 h after administrationof a 0.35 units/kg dose, mimicking thesubjects’ prior daily basal insulin need(Supplementary Table 1). Although NPHinsulin should have been studied in theclamp according to the more commonclinical use of two (or more) dailyadministrations, we speculate that theeffects of resuspension versus nonresus-pension of NPH insulin given in two (ormore) doses compared with the onedose used in the current study wouldbe qualitatively similar. Finally, this studyexamined NPH insulin injected with pensrather than syringes from NPH insulinvials. Although we would anticipate sim-ilar findings between pen and syringeinjections, some differences might existdepending on the vial size, effects ofneedle aspiration, presence/absence ofrolling balls, and so forth.

In conclusion, nonresuspended NPHinsulin before injection may result inan approximately twofold difference inglucodynamic effect (either higher orlower) compared with resuspendedNPH insulin, depending on the positionof the pen. For real-life insulin-treatedpatients with T1DM, and likely T2DM,this may translate to a risk of either hy-perglycemia or hypoglycemia. Addi-tional studies are required to establishthe comparative within-subject variabil-ity of properly resuspended NPH insulinwith that of modern long-acting insulinanalogs, such as glargine, detemir, de-gludec, and the new glargine U300 soonto come (2), to finally answer the long-discussed question of whether solublebasal insulins are less variable than insol-uble NPH insulin.

Acknowledgments. The authors thank MauroLepore, Sanofi Italy, for long-term intellectualcontribution to the research in this topic.Duality of Interest. This is an independent re-search study not supported by pharmaceuticalcompanies. P.L. has received travel grants forscientific meetings from Sanofi, Medtronic, andTheras Group. F.P. has received honoraria forspeaker fees and/or travel grants from Sanofi, EliLilly & Co., Bristol-Myers Squibb, and Merck & Co.G.B.B. received honoraria from sanofi-aventis andEli Lilly & Co. for scientific advising and consulting.C.G.F. has served on scientific advisory panels for

Table 2—Intraindividual CVs of plasma insulin concentrations and GIRs in thefour studies

FIRI_AUC(0–end of study) GIR_AUC(0–end of study)

Studies R+, R- horizontal, R- up, R- down 16 (14–19) 45 (38–53)

Studies R+, R- horizontal 14 (8–21) 62 (51–73)

Studies R+, R- up 20 (11–29) 54 (45–64)

Studies R+, R- down 23 (13–34) 48 (40–56)

FIRI Tmax GIR Tmax

Studies R+, R- horizontal, R- up, R- down 63 (52–75) 9 (8–10)

Studies R+, R- horizontal 39 (33–46) 7 (6–8)

Studies R+, R- up 68 (56–81) 10 (8–11)

Studies R+, R- down 47 (40–55) 9 (8–11)

FIRI Cmax FIRI Tmax

Studies R+, R- horizontal, R- up, R- down 18 (16–21) 28 (24–33)

Studies R+, R- horizontal 20 (17–23) 29 (16–44)

Studies R+, R- up 20 (17–23) 30 (16–44)

Studies R+, R- down 28 (24–33) 31 (17–47)

Data are mean (95% CI).

6 PK/PD of Resuspended Versus Nonresuspended NPH Diabetes Care

Page 7: Pharmacokinetics and Paola Lucidi, Francesca Porcellati ... · Paola Lucidi, Francesca Porcellati, Anna Marinelli Andreoli, Ilaria Carriero, Paola Candeloro, Patrizia Cioli, Geremia

Sanofiandreceivedspeaker feesand/or travel grantsfrom Bristol-Myers Squibb, Merck & Co., and TheMenarini Group. No other potential conflicts of in-terest relevant to this article were reported.Author Contributions. P.L. enrolled patients,performed clamps, analyzed data, and reviewedand edited the manuscript. F.P. enrolled pa-tients, performed clamps, and reviewed andedited the manuscript. A.M.A., I.C., and P.Ci.performed clamps and reviewed and edited themanuscript. P.Ca. performed clamps and labo-ratory assays and reviewed and edited themanuscript. G.B.B. provided the study conceptand design, supervised the protocol develop-ment and research, and wrote the manuscript.C.G.F. performed clamps, analyzed data, per-formed the statistical analysis, and reviewedand edited the manuscript. P.L. is the guarantorof this work and, as such, had full access to allthe data in the study and takes responsibility forthe integrity of the data and the accuracy of thedata analysis.Prior Presentation. Parts of this study werepresented in poster form at the 74th ScientificSessions of the American Diabetes Association,San Francisco, CA, 13–17 June 2014.

References1. Bolli GB, Di Marchi RD, Park GD, Pramming S,Koivisto VA. Insulin analogues and their poten-tial in the management of diabetes mellitus.Diabetologia 1999;42:1151–11672. Gough SC, Harris S, Woo V, Davies M. Insulindegludec: overviewofanovel ultra long-actingbasalinsulin. Diabetes Obes Metab 2013;15:301–3093. Bolli GB, DeVries JH. New long-acting insulinanalogs: from clamp studies to clinical practice.Diabetes Care 2015;38:541–5434. LeporeM, Pampanelli S, Fanelli C, et al. Phar-macokinetics and pharmacodynamics of subcu-taneous injection of long-acting human insulinanalog glargine, NPH insulin, and ultralentehuman insulin and continuous subcutaneousinfusion of insulin lispro. Diabetes 2000;49:2142–21485. Galloway JA, Spradlin CT, Nelson RL,Wentworth SM, Davidson JA, Swarner JL. Factors

influencing the absorption, serum insulin concen-tration, and blood glucose responses after injec-tions of regular insulin and various insulinmixtures. Diabetes Care 1981;4:366–3766. Heinemann L. Variability of insulin absorp-tion and insulin action. Diabetes Technol Ther2002;4:673–6827. Heise T, Nosek L, Rønn BB, et al. Lower within-subject variability of insulin detemir in comparisonto NPH insulin and insulin glargine in people withtype 1 diabetes. Diabetes 2004;53:1614–16208. Norrman M, Hubalek F, Schluckebier G.Structural characterization of insulin NPH for-mulations. Eur J Pharm Sci 2007;30:414–4239. Brange J.Galenics of Insulin. Berlin, Heidelberg,Springer-Verlag, 198710. Jehle PM, Micheler C, Jehle DR, Breitig D,Boehm BO. Inadequate suspension of neutralprotamine Hagendorn (NPH) insulin in pens.Lancet 1999;354:1604–160711. LeporeM, Bartocci L, Fanelli CG, et al. Effectof adequate vs inadequate resuspension of NPHinsulin prior to s.c. injection on pharmacokineticsand –dynamics in T1DM (Abstract). Diabetes2001;50(Suppl. 2):A7012. Lalli C, Ciofetta M, Del Sindaco P, et al.Long-term intensive treatment of type 1 diabe-tes with the short-acting insulin analog lispro invariable combination with NPH insulin at meal-time. Diabetes Care 1999;22:468–47713. Rossetti P, Pampanelli S, Fanelli C, et al. In-tensive replacement of basal insulin in patientswith type 1 diabetes mellitus given rapid-actinginsulin analog at mealtime: a 3-month compar-ison between administration of NPH four timesdaily and glargine insulin at dinner or bedtime.Diabetes Care 2003;26:1490–149614. Porcellati F, Rossetti P, Busciantella NR,et al. Comparison of pharmacokinetics and dy-namics of the long-acting insulin analogsglargine and detemir at steady state in type 1diabetes: a double-blind, randomized, cross-over study. Diabetes Care 2007;30:2447–245215. Kuzuya H, Blix PM, Horwitz DL, Steiner DF,Rubenstein AH. Determination of free and totalinsulin and C-peptide in insulin-treated dia-betics. Diabetes 1977;26:22–29

16. Le Floch JP, Escuyer P, Baudin E, Baudon D,Perlemuter L. Blood glucose area under thecurve. Methodological aspects. Diabetes Care1990;13:172–17517. Bland M. How should I calculate a within-subject coefficient of variation? [article online],2006. Available from http://www-users.york.ac.uk/;mb55/meas/cv.htm. Accessed 5 March201518. Binder C, Lauritzen T, Faber O, Pramming S.Insulin pharmacokinetics. Diabetes Care 1984;7:188–19919. Kaiser P, Maxeiner S, Weise A, et al. Assess-ment of the mixing efficiency of neutral prot-amine Hagedorn cartridges. J Diabetes SciTechnol 2010;4:652–65720. Luzio S, Dunseath G, Peter R, Pauvaday V,Owens DR. Comparison of the pharmacokinet-ics and pharmacodynamics of biphasic insulinaspart and insulin glargine in people withtype 2 diabetes. Diabetologia 2006;49:1163–116821. Inzucchi SE, Bergenstal RM, Buse JB, et al.Management of hyperglycemia in type 2 diabe-tes, 2015: a patient-centered approach: updateto a position statement of the American Diabe-tes Association and the European Associationfor the Study of Diabetes. Diabetes Care 2015;38:140–14922. Lucidi P, Porcellati F, Rossetti P, et al. Phar-macokinetics and pharmacodynamics of thera-peutic doses of basal insulins NPH, glargine, anddetemir after 1 week of daily administration atbedtime in type 2 diabetic subjects: a random-ized cross-over study. Diabetes Care 2011;34:1312–131423. Rys P,Wojciechowski P, Rogoz-Sitek A, et al.Systematic review and meta-analysis of ran-domized clinical trials comparing efficacy andsafety outcomes of insulin glargine with NPHinsulin, premixed insulin preparations or withinsulin detemir in type 2 diabetes mellitus.Acta Diabetol 2015;52:649–66224. Starke AAR, Heinemann L, Hohmann A,Berger M. The action profiles of human NPHinsulin preparations. Diabet Med 1989;6:239–244

care.diabetesjournals.org Lucidi and Associates 7