Dose Response of Inhaled Dry-PowderInsulin and Dose Equivalence toSubcutaneous Insulin LisproKLAUS M. RAVE, MD1

LESZEK NOSEK, MD1

AMPARO DE LA PENA, PHD2

MARY SEGER, MS2

CHARLES S. ERNEST II, BS2

LUTZ HEINEMANN, PHD1

RICHARD P. BATYCKY, PHD3

DOUGLAS B. MUCHMORE, MD2

OBJECTIVE To determine the pharmacokinetic (PK) and glucodynamic (GD) dose re-sponse of human insulin inhalation powder (HIIP) delivered via AIR particle technology anddose equivalence to subcutaneous (SC) insulin lispro.

RESEARCH DESIGN AND METHODS Twenty healthy, nonsmoking, male or fe-male subjects (aged 29.6 6.9 years, BMI 23.2 2.3 kg/m2, means SD) with normal forcedvital capacity and forced expiratory volume were enrolled in an open-label, randomized, seven-period, euglycemic glucose clamp, cross-over trial. Each subject received up to four single dosesof HIIP (2.6, 3.6, 5.2, or 7.8 mg) and three doses of SC lispro (6, 12, or 18 units) from 5 to 18days apart.

RESULTS HIIP demonstrated a similar rapid onset but an extended time exposure and aprolonged duration of effect (late t50% 412 vs. 236 min, P 0.001) compared with SC lispro. TheHIIP versus SC lispro doses of 2.6 mg vs. 6 units, 5.2 mg vs. 12 units, and 7.8 mg vs. 18 unitsachieved similar PK area under the serum immunoreactive insulin (IRI) concentration-versus-time curve from time zero until the serum IRI concentrations returned to the predose baselinevalue [AUC(0-t)] and GD (Gtot) responses. The median insulin (tmax) was not different betweenHIIP and SC lispro (45 min for both), although the median time of return to baseline for PK wasapparently longer for HIIP compared with SC lispro (480 vs. 360 min). Relative bioavailabilityand relative biopotency of HIIP were consistent across doses (8 and 9%).

CONCLUSIONS While the time-action profile was longer for HIIP than for SC lispro,both treatments showed rapid initial absorption and similar overall PK exposure and GD effect.HIIP was as well tolerated as SC lispro, thereby offering a promising alternative to injectableinsulin therapy.

Diabetes Care 28:24002405, 2005

The disease burden of diabetes con-tinues to grow and currently affects18 million Americans and theirfamilies (1). Despite increased use of dia-betes medications (without, however, in-creased utilization of insulin), overalldiabetes control A1C among individualsdiagnosed with diabetes in the U.S. hasnot improved, with A1C rising from 7.7to 7.9% during the final decade of the last

century (2). These data emphasize a needfor alternative diabetes therapies with ear-lier more physiologic use of insulin.

The delivery of insulin by the lungmay provide an attractive alternative formany patients with diabetes (38). How-ever, alternative insulin delivery systemsmust meet certain pharmacokinetic (PK)and glucodynamic (GD) requirements toreach maximum utility (9). Specifically,

the dose-response characteristics of in-haled insulin should be similar to those ofinjectable insulins, like human regular in-sulin or fast-acting insulin analogs such asinsulin lispro. Moreover, inhaled insulinshould demonstrate satisfactory dose re-producibility; that is, intrasubject vari-ability of inhaled insulin should be similarto or better than that of injectable insulin.Finally, the ratio of dose equivalence be-tween inhaled insulin and injectable insu-lin ought to be consistent across a range ofdoses. The present study evaluated thekey performance features of a novel in-haled insulin delivery system based onAIR particle technology (10 12) (Alk-ermes, Cambridge, MA) (including timeexposure and time-action profiles, PK andGD dose-response relationships, dose re-producibility, relative bioavailability, andrelative biopotency compared with sub-cutaneous [SC] insulin lispro).

RESEARCH DESIGN ANDMETHODS Twenty-two healthy,nonsmoking, male (10) or female (12)subjects with normal pulmonary function(at least 75% of predicted forced expira-tory volume [FEV1] and forced vital ca-pacity [FVC]) participated in the study.The age of the subjects was (means SD)29.6 6.9 years, and BMI was 23.2 2.3kg/m2. Subjects were ineligible if they hada history of asthma or a recent upper re-spiratory infection or if they had a medicalhistory of diabetes, impaired glucose tol-erance, allergy to insulin, or a fastingblood glucose 5.6 mmol/l. Pregnantwomen, nursing mothers, and subjectswith serious medical conditions were alsoexcluded. The local ethics committee ap-proved the protocol. This phase I clinicaltrial was conducted according to the prin-ciples outlined in the Declaration of Hel-sinki and the International Conference onHarmonization Guideline to Good Clini-cal Practice. All subjects were given a fullexplanation of study procedures at thescreening visit, and a signed informedconsent was obtained for every subject.

This open-label, randomized, seven-period, incomplete block, cross-over, eu-glycemic glucose clamp study wasconducted at one center (Profil Institut

From the 1Profil Institut fur Stoffwechselforschung, Neuss, Germany; 2Eli Lilly and Company, Indianapolis,Indiana; and 3Alkermes, Cambridge, Massachusetts.

Address correspondence and reprint requests to Klaus Rave, MD, Profil Institut fur Stoffwechselfor-schung, Hellersbergstr. 9, D-41460 Neuss, Germany. E-mail: klaus.rave@profil-research.de.

Received for publication 31 March 2005 and accepted in revised form 11 July 2005.Abbreviations: AUC, area under the curve; FEV, forced expiratory volume; FVC, forced vital capacity;

GD, glucodynamic; GIR, glucose infusion rate; HIIP, human insulin inhalation powder; IRI, immunoreactiveinsulin; PK, pharmacokinetic; SC, subcutaneous.

A table elsewhere in this issue shows conventional and Syste`me International (SI) units and conversionfactors for many substances.

2005 by the American Diabetes Association.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby

marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

E m e r g i n g T r e a t m e n t s a n d T e c h n o l o g i e sO R I G I N A L A R T I C L E

2400 DIABETES CARE, VOLUME 28, NUMBER 10, OCTOBER 2005

fur Stoffwechselforschung, Neuss, Ger-many). Each subject received up to foursingle doses of human insulin inhalationpowder (HIIP) (Eli Lilly and Company,Indianapolis, IN) (2.6, 3.6, 5.2, and 7.8mg) and three single doses of SC lispro(Humalog; Eli Lilly and Company) sup-plied in 10-ml vials (100 units/ml; 6, 12,or 18 units),518 days apart. The orderof doses was randomized in accordancewith an incomplete block study design,where one-third of the subjects receivedall seven of the possible treatment condi-tions specified above and two-thirds of thesubjects received five of these treatments,with two treatments being performed inreplicate. Two different formulations ofHIIP were used in this study. The firstformulation comprised the 2.6-, 5.2-, and7.8-mg dose, while the secondary HIIPformulation was represented by the3.6-mg dose. The latter was administeredto determine bioequivalence to the 2.6-mgprimary formulation dose. Because the fo-cus of this report is dose response anddose equivalence compared with SC insu-lin lispro of the HIIP primary formulation,data for the 3.6-mg dose are not presentedhere.

There was a total of nine visits, in-cluding an initial screening visit and a fi-nal visit for a medical examination. Ateach visit in which a glucose clamp pro-cedure was performed (visits 28), insu-lin was administered by inhalation or SClispro injection90 min after the start ofthe baseline glucose clamp procedure.The investigative staff trained each sub-ject on the proper inhalation techniquebefore pulmonary administration. Sub-jects were trained to inhale at 30 l/minfor the duration of 5 s using a real-timerecording spirometer attached to the in-haler device.

System descriptionThe inhaled insulin system used in thisstudy included a developmental versionof the inhaler (Fig. 1) and AIR particletechnology (1012) (Alkermes) to deliverHIIP to the lung. The AIR powder is for-mulated in capsules that are puncturedbefore inhalation. Each individual parti-cle contains both drug and excipientwithin a large, low-density matrix (10,11). HIIP formulated in this manner haslow cohesive forces but retains small ef-fective aerodynamic diameter and canthus be aerosolized and delivered to thedeep lung using a simple breath-actuatedinhaler (12).

Safety measurementsRoutine physical examinations and elec-trocardiograms were performed atscreening and at visit 9 (or early termina-tion from the study). Flow-volume spi-rometry (FEV1 and FVC) was performedat screening and at visit 9 to assess safety.For visits that included HIIP dosing, FEV1and FVC were measured using a portablespirometer within 90 min before dosingand within 60 min after the end of theglucose clamp procedure.

PK analysesSerum immunoreactive insulin (IRI) con-centrations following administration ofHIIP and SC lispro (Humalog) were mea-sured using a conventional, competitiveradioimmunoassay method validatedover the range of 202,500 pmol/l (MDSPharma Services, St. Laurent QC, Can-ada). The antibody used had comparablecross-reactivity with both native humaninsulin and insulin lispro and therebyprovided relevant IRI concentrations foreach treatment.

Noncompartmental PK parameters,computed using WinNonlin (Professionaledition, version 3.1; Pharsight, Cary,NC), included the maximum serum IRIconcentration (Cmax), the time of maxi-mum serum IRI concentration (tmax), andthe area under the serum IRI concentra-tion-versus-time curve from time zero un-til the serum IRI concentrations returnedto the predose baseline value [AUC(0-t)].An assessment of relative bioavailability(F) for HIIP compared with SC lispro wasconducted based on AUC(0-t) accordingto equation 1:

F DSC AUC0-tHIIPDHIIP AUC0-tSC

(1)

where D is dose and AUC(0-t) is the IRIAUC from zero to return to baseline forHIIP and SC lispro treatments.The SAS system for Windows (version8.2) was used for the statistical analyses ofboth PK and GD parameters. The primaryanalysis variable AUC(0-t), secondaryvariable, Cmax, and the dose (milligrams)were log transformed before statisticalanalysis. The dose-response relationshipwas described separately for HIIP and SClispro using a mixed-effects ANCOVAwith fixed factors for sequence, log (dose)used as a covariate and subject as a ran-dom factor, and was performed separatelyfor HIIP and SC lispro. No direct compar-isons of AUC(0-t) or Cmax were made be-tween HIIP and SC lispro. Rather, theHIIP dose expected to yield the same PKresponse as the corresponding SC lisprodose was estimated by back calculationfrom the ANCOVA results. The 95% CIsfor the predicted HIIP doses were con-structed based on Fiellers theorem (13)and incorporated variance estimates fromboth HIIP and SC lispro ANCOVA analyses.

GD analysesThe euglycemic glucose clamp procedurewas used to measure the effect of HIIP orSC lispro treatments. Baseline blood glu-cose concentrations were obtained beforeexogenous insulin administration. Theglucose clamp procedure was designed tomaintain blood glucose at a level 5%below baseline for up to 10 h after insulinadministration (14). Blood glucose con-centrations were monitored on a minute-to-minute basis by the Biostator (glucose-controlled insulin infusion system;Medizintechnik, Ulm, Germany), whichautomatically adjusted a 20% intravenousglucose infusion to maintain euglycemia.

Figure 1Breath-actuated inhaler.

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A LOESS smoothing function was fit-ted to the glucose infusion rate (GIR) databy means of S-plus (version 2000). Themaximum GIR (Rmax) and the time ofmaximum GIR (tRmax) were identifiedfrom the individual LOESS-fitted data.Other parameters, such as the times of50% of maximum GIR before and afterRmax (early and late t50%), were also cal-culated based on the individual LOESS-fitted data. The total amount of glucoseinfused from time 0 to 10 h (Gtot) and thetime of the first change in the GIR (tonset)were calculated from the raw GIR data. Anadditional LOESS fit was performed,where all GIR data from all subjects werefitted simultaneously in order to obtainpredicted values per dose group. The rel-ative biopotency (F) of HIIP comparedwith SC insulin lispro was calculatedbased on Gtot, according to equation 2:

FDSC GtotHIIPDHIIP GtotSC

(2)

where D is dose and Gtot is the totalamount of glucose infused from time 0to 10 h for HIIP and SC insulin lisprotreatments.The primary analysis variable Gtot, thesecondary variable Rmax, and the dose(milligrams) were log transformed beforestatistical analysis. A mixed-effects AN-

COVA was performed as described for thePK measures to determine the dose-response relationship for both HIIP andSC insulin lispro. The GD time variableswere directly compared between HIIPand SC insulin lispro using a mixed-effectANOVA with fixed factors for insulin,dose, and sequence and a random factorfor subject.

RESULTS

Subject dispositionTwenty of 22 subjects who entered thestudy proceeded to the first euglycemicclamp visit and received a study drug onat least one occasion. Eighteen subjectscompleted all study visits. None of thesubjects discontinued due to an adverseevent. Safety data from all 22 subjectswere included in the safety analyses. Sub-jects who completed at least one glucoseclamp procedure were included in the GDanalyses. Those who completed one glu-cose clamp and had measurable IRI con-centrations were included in the PKanalyses. All available PK and GD datawere included in the statistical analyses.

PK and GD resultsA positive trend in the exposure versustime and the effect versus time profilesconfirmed the PK and GD dose-response

relationships for HIIP (Fig. 2). In its earlyPK profile, HIIP demonstrated a rapid ini-tial absorption comparable with that ofSC lispro (Fig. 2A and B). The rapid initialabsorption of HIIP was reflected in its GDprofile, that is, a slightly but significantlyearlier onset of action compared with SClispro (P 0.005). Following this accel-erated onset, HIIP demonstrated a pro-longed insulin time-concentration profilethat was correlated with a longer durationof effect compared with SC lispro (Table 1).

For doses of HIIP comparable withSC lispro (2.6 mg to 6 units, 5.2 mg to 12units, and 7.8 mg to 18 units, respec-tively), mean AUC(0-t) values were simi-lar, while mean Cmax values appeared tobe lower for HIIP. Thus, even though theinsulin concentration versus time profileappeared to be prolonged for HIIP com-pared with SC lispro, the total insulinexposure was comparable for corre-sponding doses. Both the PK and GD pro-files were flatter for HIIP than for SClispro (Fig. 2AD), and at doses that pro-vided similar overall exposure, total GDeffects were also comparable (Table 1).

Based on AUC(0-t), intersubject vari-ability appeared to be larger for HIIP(42%) than for SC lispro (15%), while in-trasubject variability was comparable be-tween both (30%). Based on G tot,intersubject variability was comparable

Table 1PK and GD results following administration of HIIP or SC insulin lispro

HIIP SC lispro

2.6 mg 5.2 mg 7.8 mg 6 units 12 units 18 units

PK parametersPK predicted HIIP dose

(mg) (95% CI) 3.4 (2.64.4) 5.3 (4.36.6) 6.9 (5.59.0) AUC(0-t)

(pmol min1 l1) 28,500 (42.8) 59,000 (51.1) 105,000 (46.8) 37,100 (25.2) 65,800 (33.2) 78,700 (39.3)Cmax (pmol/l) 161 (50.3) 287 (58.3) 512 (60.1) 311 (22.4) 414 (34.6) 523 (43.3)NPK 12 31 13 8 32 12tmax (min) 45 (10120) 30 (10120) 45 (20120) 45 (3090) 45 (20180) 45 (3090)Percent F 7.35 (62.8) 7.39 (61.9) 8.90 (58.5)

GD parametersGD predicted HIIP dose

(mg) (95% CI) 2.6 (1.83.6) 5.0 (3.86.7) 7.4 (5.610.5) Gtot (0600) (mg) 87,200 (48.5) 137,000 (37.1) 175,000 (40.6) 82,500 (42.4) 132,000 (35.3) 161,000 (29.8)Rmax (mg/min)* 267 (55.7) 425 (38.1) 499 (34.1) 423 (44.3) 563 (37.8) 672 (34.6)NGD 12 31 13 12 32 14tRmax (min)* 260 (44489) 191 (65315) 211 (66374) 85 (63188) 112 (64211) 136 (84211)tonset (min) 13 (2031) 13 (234) 15 (427) 24 (229) 16 (433) 22 (741)Early t50% (min)* 40 (10185) 43 (21139) 33 (16139) 35 (2555) 38 (1553) 38 (2761)Late t50% (min)* 458 (252575) 371 (242568) 404 (280592) 184 (125308) 248 (158326) 273 (199360)Percent F 9.74 (52.1) 8.28 (47.4) 8.32 (43.0)

Data are geometric mean (% CV) and median (range) unless otherwise indicated. *Observed from Loess smoothed data; P 0.001 for HIIP vs. SC lispro; P 0.005for HIIP vs. SC lispro; P 0.013 for HIIP vs. SC lispro; P 0.001 for HIIP vs. SC lispro. F, relative bioavailability; F, relative biopotency.

Dose response of inhaled insulin

2402 DIABETES CARE, VOLUME 28, NUMBER 10, OCTOBER 2005

between HIIP and SC lispro (27%), andintrasubject variability was comparablebetween both as well (28%). The pre-dicted doses of HIIP that would be inter-changeable with 6, 12, and 18 units of SClispro were in excellent agreement withinhalation of the 2.6-, 5.2-, or 7.8-mgdoses of HIIP (Table 1). The overall meanrelative bioavailability and relative biopo-tency of HIIP compared with SC lisproacross all doses tested were 8 and 9%,respectively.

SafetyReview of the laboratory, vital signs, elec-trocardiogram, and pulmonary functiondata revealed no safety findings of clinicalrelevance. The postdose mean FVC in-creased in magnitude compared with thepredose FVC; however, the increase wasslight and was not considered clinicallysignificant (predose means SE:110.7 2.5% of predicted value; post-dose means SE: 113.3 2.5%; P 0.003). For FEV1, there was no statisti-cally significant difference between thepredose and postdose mean values (P 0.134). A single adverse event was re-ported that entailed a case of mild, non-

serious influenza but was not consideredby the investigator to be study related.

CONCLUSIONS This open-label,cross-over study in healthy subjectsshowed that inhalation of the HIIP formu-lation was safe and produced a similar ini-tial rate of absorption, an extendedinsulin exposure, and a prolonged dura-tion of effect when compared with SC lis-pro PK and GD profiles. Although Cmaxvalues appeared to be lower with HIIP,the overall exposure and metabolic effectat similar doses was comparable betweenHIIP and SC lispro, as evidenced by boththe PK and GD profiles being flatter forHIIP. This observation of comparableoverall exposure and metabolic effect,coupled with the comparable intrasubjectvariability estimates for HIIP and SC lis-pro, suggests that patients may be able totransition between inhaled and SC lisprotreatment with predictable results.

In a clinical setting, predictable doseequivalence is important for successfullyswitching patients between different in-sulin formulations and routes of adminis-tration. This is the first study investigatingdose equivalence of inhaled insulin versus

an SC injected rapid-acting insulin analoglike insulin lispro. With this study, wewere able to demonstrate that the pre-dicted doses were in good agreement withthe actual HIIP doses used in the study, asconfirmed by the narrow 95% CI ob-served for the predicted doses.

The safety and tolerability of all HIIPdoses was assessed as excellent, as no pul-monary-related adverse events were re-ported. This aligns with other studiesusing different insulin delivery systems inhealthy subjects (1517) and patientswith type 1 (18,19) and type 2 (2022)diabetes, during which pulmonary func-tion testing was performed using spirom-etry. However, when measuring carbonmonoxide lung diffusing capacity (DLco)small decreases were noted following in-halation of dry-powder insulin in severalphase 2 and 3 studies of Exubera(18,22,23). The decreases in DLco occurquickly, are not progressive, and have notbeen associated with any clinical manifes-tations to date (18,22,23). As the currentstudy focused on dose response and doseequivalence of HIIP in comparison to SCinsulin lispro, DLco was not measured,which represents a limitation of the study.

Figure 2PK and GD results after inhalation of HIIP and SC lispro injection. A: Mean serum insulin concentration versus time profiles of threedifferent doses of HIIP. B: Mean serum insulin concentration versus time profiles of three different doses of SC lispro.C: Plots of simultaneous LOESSfits of GIR versus time data of three different doses of HIIP.D: Plots of simultaneous LOESS fits of GIR versus time data of three different doses of SClispro.

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The most common respiratory ad-verse event in studies of inhaled Exuberais mild-to-moderate cough following in-halation of dry-powder insulin (18,22,23). The incidence of cough decreasesover time and has not been associatedwith declines in lung function (18). It isremarkable that in the current study not asingle episode of cough was registered,suggesting an excellent tolerability of theinhaled insulin when formulated by theAIR particle technology. This result, how-ever, needs to be confirmed in muchlarger phase 3 trials.

The administration of HIIP by meansof a simple, handheld, breath-actuated in-haler in healthy subjects in this glucose-clamp study exhibited a slightly morerapid initial absorption of insulin com-pared with SC lispro coupled with alonger duration of metabolic activity thanseen with SC lispro. These findings agreewith other studies (24,25) comparing in-haled insulin with SC insulin lispro. Inaddition, the intrasubject variability foroverall insulin exposure and GD effectwas comparable with SC lispro. This find-ing also agrees with previous studies(21,26,27) investigating a number of dif-ferent technologies for the delivery of in-haled insulin. One inhaled insulin systemthat demonstrates a different PK and GDprofile is that of Technosphere (Mann-Kind, Valencia, CA). This formulation ex-hibits a very rapid onset and very short (3h) duration of insulin exposure and actionwhen compared with regular SC insulin(16,28,29).

Taken together, these results supportthe potential utility of inhaled insulin asan alternative to insulin injections. Be-cause the fear of injections is a frequentcause for delaying appropriate care in pa-tients with type 2 diabetes, inhaled insu-lin may provide a treatment that deliverseffective doses of insulin in a less threat-ening, more satisfying manner, poten-tially resulting in improved patientcompliance and better outcomes.

Acknowledgments This study was fundedby Eli Lilly and Company in collaboration withAlkermes.

The authors acknowledge Drs. Bernard Sil-verman and Jen Schmitke from Alkermes fortheir collaboration on this project. Apprecia-tion is also expressed to Kathryn Gilmore forpreparation of the manuscript and to PeggyCampbell and Todd Cravens for editorial as-sistance.

Parts of this study have been presented inabstract form at the 65th annual meeting of

the American Diabetes Association, San Diego,California, 10-14 June 2005 [Rave K, NosekL, de la Penna A, Segar M, Ernest C 2nd,Heinemann L, Batycky R, Muchmore D: Doseresponse and dose equivalency of human in-sulin inhalation powder (HIIP) using the Lilly/Alkermes Inhaled Insulin System compared tosubcutaneous (SC) insulin lispro (Abstract).Diabetes 54 (Suppl. 1):A89, 2005].

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