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A Reversible Albumin-Binding Growth Hormone Derivative is Well Tolerated and Possesses a Potential Once-Weekly Treatment Profile Michael Højby Rasmussen, Minna W. Brændholt Olsen, Lene Alifrangis, Søren Klim, and Mette Suntum Departments of Medical and Science (M.H.R.), Clinical Pharmacology (M.W.B.O.), Development Drug Metabolism and Pharmacokinetics (L.A.), Quantitative Clinical Pharmacology (S.K.), and Biostatistics (M.S.), Novo Nordisk A/S, DK 2880 Bagsværd, Denmark

Context: Human growth hormone (hGH) replacement therapy currently requires daily sc injections for years/lifetime, which may be both inconvenient and distressing for patients. NNC0195– 0092 is a novel hGH derivative intended for once-weekly treatment of GH deficiency. A noncovalent albumin binding moiety is attached to the hGH backbone. Clearance is reduced as a consequence of a reversible binding to circulating serum albumin, which prolongs the pharmacodynamic (PD) effect. Objective: To evaluate safety, local tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of a single dose (SD) and multiple doses (MD) of NNC0195– 0092. Setting and Design: Randomized, single-center, placebo-controlled, double-blind, SD/MD, doseescalation trial of 105 healthy male subjects. NNC0195– 0092 sc administration: Five cohorts of eight subjects received one dose of NNC0195– 0092 (0.01– 0.32 mg/kg) (n ⫽ 6) or placebo (n ⫽ 2). Sixteen subjects (equal numbers of Japanese and non-Asian) received once-weekly doses of NNC0195– 0092 (0.02– 0.24 mg/kg; n⫽12) or placebo (n⫽4) for 4 weeks. Blood samples were drawn for assessment of safety, PK, IGF-1, and IGF binding protein 3 profiles and anti-drug antibodies. Results: SD and MD of NNC0195– 0092 were well tolerated at all dose levels. No safety concerns or local tolerability issues were identified. A dose-dependent IGF-1 response was observed. IGF-1 profiles suggest that NNC0195– 0092 may be suitable for once-weekly dosing, with a clinically relevant dose ⱕ0.08 mg/kg/week. No differences in PK and PD were observed between Japanese and non-Asian subjects. Conclusions: SD and MD of NNC0195– 0092 administered to healthy Japanese and non-Asian male subjects were well tolerated at all doses. The present trial suggests that NNC0195– 0092 has the potential for an efficacious, well-tolerated, once-weekly GH treatment. (J Clin Endocrinol Metab 99: E1819 –E1829, 2014)

G

H treatment with daily injections for years poses a challenge for both patients and parents. Studies investigating compliance have shown that approximately 25% of children receiving GH treatment miss more than two injections per week (1–2) and that low compliance can

partly be attributed to difficulties with injections (1). A long-acting GH preparation allowing for reduced injection frequency is likely to improve treatment adherence and reduce the inconvenience and distress associated with daily injections (3).

ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received March 12, 2014. Accepted July 1, 2014. First Published Online July 11, 2014

Abbreviations: AGHD, adults with growth hormone deficiency; ANCOVA, analysis of covariance; AUC, area under the curve; CI, confidence interval; Cmax, maximum concentration; CV, coefficient of variation; ECG, electrocardiogram; GHD, growth hormone deficiency; hGH, human growth hormone; IGFBP-3, IGF binding protein 3; MD, multiple dose; PD, pharmacodynamics; PK, pharmacokinetics; Racc, accumulation ratio/index; SDS, standard deviation scores; tmax, time to maximum concentration.

doi: 10.1210/jc.2014-1702

J Clin Endocrinol Metab, October 2014, 99(10):E1819 –E1829

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Rasmussen et al

Dosing of a Long-Acting GH Derivative

NNC0195– 0092 is a novel human growth hormone (hGH) derivative, designed for once-weekly treatment of growth hormone deficiency (GHD) in children and adults (AGHD). NNC0195– 0092 consists of a single-point mutation in the hGH backbone, to which a side chain with a terminal fatty acid with noncovalent albumin-binding properties has been attached. This is expected to prolong the absorption rate upon sc injection to some extent, and the noncovalent binding to albumin in the blood significantly prolongs the in vivo half-life because clearance is reduced. NNC0195– 0092 is being developed with the aim of reducing clearance and thereby prolonging the exposure, potentially enabling once-weekly sc administration. It is anticipated that fewer injections with onceweekly therapy will result in greater convenience and potentially treatment adherence compared with daily hGH treatment. The preclinical safety evaluation program of NNC0195– 0092 included in vivo toxicity studies in two pharmacologically responsive species, the Han Wistar rat and the Cynomolgus monkey. The genotoxic potential of the compound was assessed in two in vitro genotoxicity studies: Ames test and Chromosome Aberration Test in human lymphocytes, and one in vivo study, induction of micronuclei in the bone marrow of NNC0195– 0092treated rats. The comprehensive preclinical studies have not identified any safety issues that preclude administration of NNC0195– 0092 to humans. We report the results from the first human trial with NNC0195– 0092 in healthy subjects (single-dose (SD) and multiple-dose (MD) administrations). Safety, tolerability, and local tolerability (ie, injection site reactions) of NNC0195– 0092 were assessed and compared with placebo. Pharmacokinetics (PK) was assessed after both SD and MD administrations and pharmacodynamics (PD) of NNC0195– 0092 were determined by assessment of IGF-1 and IGF binding protein 3 (IGFBP-3).

Subjects and Methods Subjects Healthy, male volunteers who fulfilled the following criteria were recruited (age, 20 – 45 years; body weight, 50 –100 kg; body mass index, 18 –28 kg/m2). All subjects signed informed consent before enrolment. Non-Asian and Japanese subjects were enrolled in the MD arm into separate cohorts. The two populations were compared to exclude unforeseen differences in PK and IGF-1 response between non-Asian and Japanese subjects so as to be able to include Japanese subjects alongside non-Asian subjects in a longer-term clinical trial. Japanese subjects were defined as subjects with a Japanese passport and Japanese-born parents. Only male subjects were included, because adult males are considered a less vulnerable population than females in a first

J Clin Endocrinol Metab, October 2014, 99(10):E1819 –E1829

human dose study, but females will be included in future clinical trials.

Trial design and procedures The trial was conducted at the Profil Institut für Stoffwechselforschung GmbH (Germany) in accordance with the Declaration of Helsinki (4), the International Conference on Harmonization and Good Clinical Practice (5). Health authorities and independent ethics committee/institutional review board approvals were obtained according to local regulations. The trial was a single-center, randomized, double-blind, placebo-controlled, SD/MD, dose-escalation trial. The trial assessed safety, tolerability, local tolerability, PK, and PD of SD and MD administrations of NNC0195– 0092 compared with placebo in healthy subjects. PD was assessed by IGF-1 and IGF binding protein 3 (IGFBP-3) serum concentrations. All MD doses of NNC0195– 0092 were administered once weekly for 4 weeks by sc injections into the thigh (via syringe). The maximal volume per injection was 2 mL. For dosing volumes greater than 2 mL, the dose was divided into equal portions not exceeding 2 mL. NNC0195– 0092 was delivered as freeze-dried powder in single-use vials (6.7 mg/vial), to be reconstituted in sterile water.

SD arm Five cohorts of eight healthy subjects were evaluated, with six subjects receiving active treatment (NNC0195– 0092: 0.01, 0.04, 0.08, 0.16, and 0.32 mg/kg) and two subjects receiving placebo. All subjects received one dose. After dosing, the subjects were closely monitored for clinical and laboratory safety assessments at the trial site for 8 days followed by daily visits to the clinic for 3 days.

MD arm Eight cohorts of eight healthy subjects were evaluated, with six subjects receiving active treatment (NNC0195– 0092: 0.02, 0.08, 0.16, and 0.24, mg/kg) and two subjects receiving placebo. The eight cohorts were split into four cohorts of Japanese subjects and four cohorts of non-Asian subjects (equal numbers of Japanese and non-Asian subjects). NNC0195– 0092 was administered once weekly for 4 consecutive weeks (Days 1, 8, 15, and 22). After the first and the fourth doses the subjects were closely monitored at the trial site for 3 days, followed by daily visits to the clinic for 3 days. The second and third doses were followed by daily visits to the clinic for 2 days. Blood samples were taken for assessment of safety (alanine aminotransferase, albumin, alkaline phosphatase, AST, bilirubin, calcium, chloride, creatinine, gamma glutamyltransferase, potassium, sodium, total protein, urea, uric acid, phosphate, creatine kinase, erythrocytes, hematocrit, hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, leukocytes, c-reactive protein, thrombocytes, thyroid parameters, glucose, and insulin), PK, and PD response (IGF-1 and IGFBP-3) profiles.

Dose escalation After completion of each cohort, a blinded safety group at Novo Nordisk A/S reviewed safety, PK, and PD data to allow progression to the next, higher dose, level. The dose escalations in Japanese and non-Asian subjects were conducted in parallel.

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doi: 10.1210/jc.2014-1702

Trial visits Trial visits included both in-house stays after dosing as well as ambulatory visits. NNC0195– 0092 administration took place in the morning after overnight fasting.

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(SD, MD-Japanese, and MD-non-Asian), irrespective of the allocated dose level.

Pharmacokinetics

Blood samples for PK assessments were taken at baseline and up to 10 days (240 hours) in the SD part and up to 7 days (168 hours) in the MD part of the trial. Analysis of serum concentrations of NNC0195– 0092 were conducted at Novo Nordisk A/S, Diabetes Pharmacology and Bioanalysis using an NNC0195– 0092-specific luminescent oxygen channelling immunoassay developed by Novo Nordisk A/S and validated according to United States Food and Drug Administration guidelines.

PK endpoints were derived from NNC0195– 0092 serum concentration vs time profiles and included peak serum concentration (Cmax), time to maximum serum concentration (tmax), terminal half-life (t1/2), and area under the curve (AUC0-inf and AUC0 –168hrs). The PK endpoints were calculated using standard noncompartmental methods. To investigate whether accumulation took place, the accumulation index (Racc) was calculated for the MD arm (AUC(0 –168h) of fourth dose/AUC(0 –168h) of first dose). All PK endpoints were evaluated using descriptive statistics.

Pharmacodynamics

Dose proportionality

Blood samples for PD assessments were collected at baseline and up to 10 days (240 hours) in the SD arm and up to 7 days (168 hours) in the MD arm of the trial. Analysis of serum concentrations of IGF-1 and IGFBP-3 were conducted at Analytical laboratory Laboratorium für Klinische Forschung GmbH using commercially available assay kits (Immuno Diagnostic Systems immunoassay [IDS-iSYS assay] and Siemens Immulite 2000 immunoassay analyzer; Siemens Healthcare Diagnostics, respectively). Normal reference ranges for the calculation of standard deviation scores (SDS) were provided by the assay kit manufacturer. Reference centiles were obtained using the lambda-musigma method (6).

Dose proportionality was investigated by estimating the slope in a linear regression model with the log-transformed endpoint (SD, AUC and Cmax; MD, AUC(0 –168h) and Cmax of fourth dose) as the dependent variable and log-transformed dose as covariate. The analysis of dose proportionality was repeated as an exploratory analysis, including only the dose levels expected to be clinically relevant (ⱕ0.08 mg/kg).

Pharmacokinetics

Antibodies Serum samples for measurement of antibodies against NNC0195– 0092 were collected at baseline (prior to initiation of treatment), at the end of treatment (168 hours post last dose), and at follow-up (after 4 weeks of drug washout). Determination of antibodies against NNC0195– 0092 was performed using a validated antibody assay developed by Novo Nordisk A/S, Department of Drug Metabolism & Pharmacokinetics, Cell and Antibody Analysis. The assay was a bridging ELISA developed to specifically measure antibody levels against NNC0195– 0092.

Safety The safety of NNC0195– 0092 was assessed from adverse events (incidence of adverse events), safety laboratory assessments, physical examinations, vital signs, electrocardiogram (ECG), and injection site tolerability. Blood sampling was performed up to 10 days (240 hours) after SD and up to 7 days (168 hours) after MD. Blood samples for clinical laboratory assessments were analyzed at Medical Labs Mönchengladbach GmbH.

Pharmacodynamics (IGF-1 and IGFBP-3) PD endpoints included AUC0 –168h, Cmax, and tmax derived from IGF-1 and IGFBP-3 serum concentration vs time profiles. Exploratory statistical comparison between dose levels of NNC0195– 0092 and between NNC0195– 0092 and placebo was performed using an analysis of covariance (ANCOVA) model with treatment as a factor and the predose value as a covariate. Mean ratios of NNC0195– 0092 vs placebo and mean ratios between dose levels of NNC0195– 0092 were estimated with corresponding 95% confidence interval (CI) and P values. In addition, IGF-1 SDS and IGFBP-3 SDS were calculated and presented using descriptive statistics.

Safety The primary endpoint was safety, assessed as the incidence of adverse events from first dose of NNC0195– 0092/placebo until Day 40 (SD) or Day 49 (MD) after dosing. Adverse events with onset after the first dose and up until Day 40 (SD) or Day 49 (MD) were summarized by trial arm, dose, MedDRA system, organ class, and preferred term. Physical examination, changes from baseline in vital signs, ECG, and laboratory safety parameters (hematology, biochemistry and urinalysis) were considered secondary safety endpoints and were evaluated by descriptive statistics. Local tolerability was assessed as the number of injection site reactions from the first dose and up until Day 40 (SD) or Day 49 (MD).

Statistical analysis The significance level was 5% with no adjustment for multiple testing. All tests were two-sided superiority tests. Dose comparisons (including dose proportionality) were evaluated by statistical analyses. The comparisons between dose levels were conducted separately within each trial arm. Otherwise, endpoints were mainly evaluated by descriptive statistics. For descriptive statistics and formal statistical analyses, subjects receiving placebo were pooled into one dose group per trial arm

Results Subjects A total of 105 healthy subjects were enrolled and 102 completed the trial. A total of 40 non-Asian subjects enrolled and completed the SD arm; 33 non-Asian and 32

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Dosing of a Long-Acting GH Derivative

Japanese subjects enrolled in the MD arm, of whom 30 non-Asian and 32 Japanese subjects completed. Three subjects withdrew from MD (0.16 mg/kg, one subject withdrew consent; 0.24 mg/kg, two subjects withdrew because of adverse events, see Safety Results). The results and conclusions are based on the results for all the 105 enrolled subjects.

J Clin Endocrinol Metab, October 2014, 99(10):E1819 –E1829

ity. However, the increase in Cmax with further increasing dose was not consistent with dose proportionality. There were no statistically significant differences in key PK parameters (AUC(0 –168hrs) and Cmax) between non-Asian and Japanese subjects. Limited or no significant degree of accumulation occurred and there was no consistent relation to the

Baseline characteristics Baseline characteristics were similar across groups and are summarized in Table 1. Pharmacokinetics The PK mean profiles (log scale) after SD and MD administration of NNC0195– 0092 are presented in Figure 1. PK endpoints are summarized in Table 2. The NNC0195– 0092 mean serum concentrations, as measured by AUC, AUC(0 –168h), and Cmax increased in a dose-dependent manner after administration to healthy subjects. When including all dose levels in the statistical analyses, the test for dose proportionality was rejected. In the test, the effect of doubling a dose was estimated to result in approximately 3- to 3.5-fold increase in AUC, AUC(0 –168h), or Cmax. When restricting data to the expected clinically relevant dose levels (ⱕ0.08 mg/kg) based on IGF-1 response, the increase in NNC0195– 0092 AUC and AUC(0 –168h) was consistent with dose proportionalTable 1.

Summary of Baseline Characteristics

Dose, mg/kg Single dose, non-Asian 0.00 (placebo) 0.01 0.04 0.08 0.16 0.32 Multiple dose, non-Asian 0.00 (placebo) 0.02 0.08 0.16 0.24 Multiple dose, Japanese 0.00 (placebo) 0.02 0.08 0.16 0.24

n

Age, y

Body Weight, kg BMI, kg/m2

10 6 6 6 6 6

37 (26 – 42) 29 (25–39) 36 (25– 40) 37 (31– 41) 33 (22– 43) 30 (23– 43)

79 (63–91) 74 (65– 83) 83 (66 – 86) 85 (75–100) 80 (63–94) 81 (62– 87)

25 (20 –27) 24 (21–28) 26 (20 –27) 25 (24 –28) 24 (23–29)a 25 (20 –27)

8 6 6 6 7

34 (23– 43) 33 (22– 45) 44 (36 – 45) 35 (26 – 44) 38 (26 – 42)

77 (57–97) 81 (64 –100) 74 (63–94) 80 (67–95) 85 (64 –92)

24 (20 –27) 25 (21–27) 25 (19 –27) 25 (23–28) 26 (20 –27)

8 6 6 6 6

29 (23–38) 30 (26 –37) 35 (30 –38) 31 (24 –38) 33 (23–35)

66 (55–78) 61 (60 –73) 64 (59 –72) 60 (55–72) 64 (56 –76)

22 (19 –27) 21 (20 –24) 21 (19 –26) 20 (20 –24) 22 (19 –25)

Abbreviation: BMI, body mass index. Data are presented as median (range). BMI ⬎28 in one subject was the result of an increase in body weight from screening to baseline assessment (up to 3 weeks). a

Figure 1. Mean serum concentrations (⫾SEM) of NNC0126 – 0092 (ng/mL) after single dose and multiple doses in non-Asian and Japanese subjects.

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doi: 10.1210/jc.2014-1702

Table 2.

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Summary of PK Endpoints - NNC0195– 0092

Dose, mg/kg Single dose, non-Asian 0.01 0.04 0.08 0.16 0.32 Multiple dose, non-Asian 0.02 0.08 0.16 0.24 Multiple dose, Japanese 0.02 0.08 0.16 0.24

n

AUC0 –t, ngⴛh/mL

Cmax, ng/mL

tmax n, h

Racc

6 6 6 6 6

130 (49.9) 624 (51.9) 1737 (39.3) 9594 (110) 49347 (71.9)

1.8 (54.8) 13.7 (89.0) 45.3 (76.0) 254.6 (69.5) 846.9 (62.5)

33.3 (31.7) 8.3 (4.6) 13.3 (11.4) 16.0 (9.8) 35.3 (8.5)

NA NA NA NA NA

6 6 5 5

429 (75.8) 2623 (90.4) 11245 (60.9) 21638 (73.1)

5.6 (79.8) 69.6 (118.1) 347.5 (35.5) 454.4 (30.1)

11.2 (12.7) 11.3 (6.0) 16.4 (5.7) 26.0 (11.3)

1.2 (0.2) 1.1 (0.4) 1.7 (0.9) 1.3 (0.5)

6 6 6 6

445 (45.2) 2,862 (61.2) 16,238 (33.9) 18,087 (63.5)

11.5 (63.9) 102.70 (62.0) 439.0 (45.5) 390.0 (52.4)

8.0 (2.5) 13.0 (3.9) 17.0 (4.7) 18.3 (6.2)

1.3 (0.3) 1.9 (1.0) 2.9 (2.3) 1.6 (1.0)

AUC, Cmax, and Racc are presented as geometric mean (CV%). tmax is presented as mean (SD). Single dose, AUC0-inf; Multiple dose, AUC0 –168 h, ng ⫻ h/mL. Multiple-dose data are presented for the fourth dose. Racc ⫽ AUCdose 4/AUCdose 1.

dose level. The accumulation index (Racc) ranged from 1.1–2.9 across the four dose groups (for details, see Table 2). Pharmacodynamics The mean IGF-1 profiles by dose after NNC0195– 0092 administration are presented in Figure 2 and the IGF-1 parameters and statistical evaluations are listed in Tables 3 and 4. A dose-dependent PD response was observed, with elevated IGF-1 levels with four of five SD administered (0.04, 0.08, 0.16, and 0.32 mg/kg). At the lowest dose level of 0.01 mg/kg the IGF-1 response was comparable to placebo. A dose-dependent IGF-1 response was also observed after MD, with elevated IGF-1 levels at all doses administered (0.02, 0.08, 0.16, and 0.24 mg/kg). The increase in IGF-1 SDS was maintained throughout the dosing interval of 7 days, without significant accumulation for all doses, making it suitable for once-weekly dosing. There were no apparent differences in PD responses (IGF-1 and IGFBP-3) between non-Asian and Japanese subjects. The dose response was reflected in AUC(0 –168h) and Cmax. At the highest dose (0.24 mg/kg), the estimated mean IGF-1 AUC(0 –168 hours) increased up to 2.7 times (95% CI, 2.25–3.27) compared with placebo and the estimated mean IGF-1 Cmax increased up to 2.8 times (95% CI, 2.31– 3.42) compared with placebo. The mean IGF-1 tmax was 3.3 days (78.9 hours), where an average peak of 854.4 (18.3) ng/mL was reached (geometric mean (CV)). The baseline mean (SD) level was 275.9 (22.7) ng/mL. The IGF-1 elevation was maintained for more than 1 week at the highest doses.

The mean IGFBP-3 profiles by dose after NNC0195– 0092 administrations are presented in Figure 3. A dosedependent response for IGFBP-3 was also observed, although this was not as consistent as that observed for IGF-1. Safety NNC0195– 0092 was well tolerated and no safety concerns were identified after administration to healthy subjects. No serious adverse events were reported. A total of 112 adverse events were reported (SD, 44 events in 21 (70%) non-Asian subjects; MD, 59 events in 21 (84%) non-Asian subjects; nine events in five (21%) Japanese subjects). All adverse events were graded mild (85 events, 76%) or moderate (27 events, 24%). The most frequent adverse events were considered typical for this class of compounds and included headaches, myalgia, and peripheral edema. The number of events was greatest with the highest NNC0195– 0092 dose levels and similar to placebo for the lowest NNC0195– 0092 dose levels. All events classified as possibly or probably related to NNC0195– 0092 treatment had resolved by the end of the trial. Two subjects (receiving 0.24 mg/kg, non-Asian) withdrew due to adverse events (abnormal ECG and nasopharyngitis/common cold with fever). Both subjects recovered fully from their event(s). There was no apparent influence of NNC0195– 0092 on fasting glucose. In a few subjects transient increase in fasting insulin was observed but within expectations as observed for daily hGH administration. In two placebo subjects insulin values were also noted to be above the reference range. These values

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Dosing of a Long-Acting GH Derivative

J Clin Endocrinol Metab, October 2014, 99(10):E1819 –E1829

Figure 2. Mean serum concentrations (⫾SEM) of IGF-1 (ng/mL) and IGF-1 standard deviation scores after single dose and multiple doses in nonAsian and Japanese subjects.

were solitary observations as the insulin values were normalized at the next visit and may be ascribed to subjects not being in compliance with fasting conditions at these occasions. There were no clinically significant changes from screening in physical examination, blood pressure, or ECG during exposure to NNC0195– 0092.

Local tolerability No clinically significant local tolerability issues were identified. Transient injection site reactions were reported in 20 of 79 subjects treated with NNC0195– 0092. Injection site reactions were primarily observed in the highest dose groups and all were judged mild except for four injection site reactions judged moderate/

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doi: 10.1210/jc.2014-1702

Table 3.

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Statistical Analysis: IGF-1 and IGFBP-3 After Single Dose of NNC0195– 0092 Compared With Placebo

Dose, mg/kg IGF-I 0.01 vs placebo 0.04 vs placebo 0.08 vs placebo 0.16 vs placebo 0.32 vs placebo IGFBP-3 0.01 vs placebo 0.04 vs placebo 0.08 vs placebo 0.16 vs placebo 0.32 vs placebo

Estimated Mean Ratio AUC0 –168 h

95% CI

P Value

Estimated Mean Ratio Cmax

95% CI

P Value

1.16 1.53 1.98 2.56 3.13

1.07–1.27 1.40 –1.66 1.82–2.16 2.33–2.82 2.87–3.41

.0008 ⬍.0001 ⬍.0001 ⬍.0001 ⬍.0001

1.11 1.51 2.00 2.78 3.53

0.98 –1.26 1.32–1.73 1.76 –2.27 2.41–3.22 3.09 – 4.02

.1053 ⬍.0001 ⬍.0001 ⬍.0001 ⬍.0001

0.98 1.20 1.15 1.30 1.27

0.89 –1.08 1.09 –1.31 1.05;1.25 1.19 –1.43 1.16 –1.39

.7104 .0003 .0040 ⬍.0001 ⬍.0001

0.93 1.33 1.13 1.33 1.35

0.82–1.06 1.17–1.52 0.99 –1.29 1.16 –1.53 1.18 –1.53

.2633 ⬍.0001 .0655 .0002 ⬍.0001

Estimated mean ratio IGF-1 or IGFBP-3 AUC0 –168 h (h⫻ng/mL)/Placebo AUC0 –168 h (h⫻ng/mL). Estimated mean ratio Cmax/Placebo Cmax (ng /mL). Analysis using an ANCOVA model on log-transformed data with ln (baseline value) as covariate and dose as a factor. P value for test of ratio ⫽ 1. Non-Asian subjects.

severe. Injection site reactions were reported in three of 26 placebo subjects. Antibodies No antibodies against NNC0195– 0092 were detected in NNC0195– 0092-treated subjects.

Discussion NNC0195– 0092 is a novel reversible albumin-binding GH derivative intended for once-weekly treatment of Table 4.

GHD. NNC0195– 0092 was well tolerated at all doses when administered as SD and MD (four once-weekly doses) to healthy non-Asian and Japanese subjects. Although GH treatment has proved both efficacious and safe, one major drawback of the treatment has been the need for daily sc injections, which is a burden for the children and their parents, resulting in very low compliance in some cases (7). Development of liquid GH (Norditropin) and convenient pen devices has simplified the administration process; however, daily GH injections are still required. Thus, the need for a treatment with a lower frequency of injections when administering GH is present.

Statistical analysis: IGF-1 and IGFBP-3 After Multiple Doses of NNC0195– 0092 Compared With Placebo

Dose, mg/kg Multiple dose, non-Asian (dose 4) IGF-I 0.02 vs placebo 0.08 vs placebo 0.16 vs placebo 0.24 vs placebo IGFBP-3 0.02 vs placebo 0.08 vs placebo 0.16 vs placebo 0.24 vs placebo Multiple dose, Japanese (dose 4) IGF-I 0.02 vs placebo 0.08 vs placebo 0.16 vs placebo 0.24 vs placebo IGFBP-3 0.02 vs placebo 0.08 vs placebo 0.16 vs placebo 0.24 vs placebo

Estimated Mean Ratio AUC0 –168 h

95% CI

P Value

Estimated Mean Ratio Cmax

95% CI

P Value

1.28 1.91 2.44 2.71

1.08 –1.52 1.61–2.27 2.03–2.92 2.25–3.27

0.0067 ⬍.0001 ⬍.0001 ⬍.0001

1.27 1.9 2.59 2.81

1.06 –1.52 1.59 –2.28 2.14 –3.13 2.31–3.42

.0127 ⬍.0001 ⬍.0001 ⬍.0001

1.15 1.24 1.25 1.32

1.07–1.24 1.15–1.34 1.15–1.35 1.21–1.43

0.0008 ⬍.0001 ⬍.0001 ⬍.0001

1.19 1.32 1.23 1.31

1.06 –1.33 1.18 –1.48 1.09 –1.38 1.16 –1.48

.0049 ⬍.0001 0.002 0.0001

1.36 2.11 3.03 3.36

1.19 –1.56 1.83–2.43 2.65–3.46 2.94 –3.84

⬍.0001 ⬍.0001 ⬍.0001 ⬍.0001

1.39 2.16 3.48 3.65

1.22–1.59 1.89 –2.47 3.06 –3.95 3.21– 4.14

⬍.0001 ⬍.0001 ⬍.0001 ⬍.0001

1.14 1.29 1.34 1.41

1.04 –1.24 1.18 –1.40 1.23–1.46 1.28 –1.55

.0055 ⬍.0001 ⬍.0001 ⬍.0001

1.15 1.32 1.33 1.42

1.02–1.29 1.17–1.49 1.18 –1.49 1.25–1.62

.0219 ⬍.0001 ⬍.0001 ⬍.0001

Estimated mean ratio IGF-1 or IGFBP-3 AUC0 –168 h/Placebo AUC0 –240 h (h⫻ng/mL). Estimated mean ratio Cmax/Placebo Cmax (ng/mL). Analysis using an ANCOVA model on log-transformed data with ln (baseline value) as covariate and dose as a factor. The P value for test of ratio ⫽ 1.

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Dosing of a Long-Acting GH Derivative

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Figure 3. Mean serum concentrations (⫾SEM) of IGF binding protein 3 (IGFBP-3, ng/mL) and IGFBP-3 standard deviation scores by dose and subgroup.

The serum concentration of NNC0195– 0092 after sc injection increased with dose, as seen in Figure 1. As illustrated in Table 2, the PK parameters for both SD and MD were not dose-proportional. This is also visible from the PK profiles in Figure 2. The overall effect of doubling a dose within the dose range of 0.01 to 0.32 mg/kg was

estimated by log-linear regression to result in approximately 3- to 3.5-fold increase in AUC(0 –168h) or Cmax. The deviation from dose proportionality was primarily seen with high doses (ⱖ0.08 mg protein/kg) ie, at doses higher than the expected normal therapeutic doses as judged by the IGF-1 profiles. When restricting data to the expected

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doi: 10.1210/jc.2014-1702

clinically relevant dose levels (ⱕ0.08 mg/kg) based on IGF-1 response, the increase in NNC0195– 0092 AUC and AUC(0 –168h) was consistent with dose proportionality. A high interindividual variation in exposure was observed (Table 2). The larger-than-dose-proportional increase in AUC0-t and Cmax is believed to be caused by a partly saturable (receptor-mediated) mechanism of elimination for GH. This may potentially lead to some degree of accumulation. However, due to the nonlinear PK with faster elimination at lower serum concentrations, the trough concentrations between doses were low for all dose levels (Figure 1). Hence, limited or no significant accumulation occurred with no consistent relation to the dose level. A clear, dose-dependent increase in IGF-1 levels was induced with all MDs of NNC0195– 0092 tested. The IGF-1 profiles suggest that NNC0195– 0092 may be suitable for once-weekly dosing, with a clinically relevant adult dose (ⱕ0.08 mg/kg), and it could be speculated that even a twice-monthly dosing could be feasible for doses ⱖ0.08 mg/kg. No differences in key PK and PD parameters were observed between Japanese and non-Asian subjects. IGF-1 SDS ⬎2 was observed in most dose groups except for the two lowest dose levels administered both able to maintain serum IGF-1 levels within the age-related normal range. However, this would be expected, both due to that the healthy subjects studied had their baseline IGF-1 levels well within the normal range and due to the steep dose escalation applied to assess safety, tolerability, and IGF-1 response. In longer-term clinical trials comprising AGHD subjects, individual dose titration toward IGF-1 should be applied in accordance with the Endocrine Society’s clinical practice guideline to achieve serum IGF-1 levels within the age-related normal range. Given that the safety and local tolerability of Norditropin as a once daily GH treatment is well established, local tolerability (injection site reactions) is an important safety consideration. There was no clinically relevant difference in local tolerability between NNC0195– 0092 and placebo, with reactions comparable to the clinical experience with daily hGH injections. No positive test results for antibodies against NNC0195– 0092 were detected in NNC0195– 0092-treated subjects. There are many years of safety experience with recombinant human (rh) GH, and its safety, efficacy, and local tolerability are well established. In studies of long-acting GH depot formulation, a large number of injection site reactions have been reported (8 –11), and for one pegylated formulation of GH, lipoatrophy was observed (12). It is likely that both these observations are due to a long sc residence time, thus local tolerability must be carefully examined in all long-acting GH preparations.

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The plasma half-life of therapeutic proteins, such as GH, can be extended through binding to serum albumin. Serum albumin has a high affinity and high binding capacity for fatty acids. Acylation of fatty acids to therapeutic proteins has been used to facilitate binding of these molecules to serum albumin. Combined with a high concentration of serum albumin in extracellular fluids, this has been used to extend the PK of insulin (13). NNC0195– 0092 is an hGH derivative to which fatty acids with noncovalent albumin-binding properties have been attached. The noncovalent binding to albumin in the blood significantly prolongs the in vivo half-life as clearance is reduced. The observed PK profile for NNC0195– 0092 and the typical PK profile for daily GH injection, with characteristic peak and trough GH levels over 24 hours (14) are both very different from the general profile of normal physiological pulsatile GH secretion (15). However, the efficacy of daily GH injections when treating adults and children with GHD has been confirmed in several clinical studies, with normalization of growth rate (16), body composition (17–18), bone metabolism (19), and other metabolic abnormalities associated with GHD. Studies in rats have reported the importance of GH pulsatility for various markers of GH action, including growth (20), and a limited number of short-term studies on humans comparing continuous infusion vs bolus injections of GH have shown some differential effects on very low-density lipoprotein, cholesterol, free fatty acids, and lipoprotein(a) (14, 21– 23)., The vast majority of these differences were not confirmed in a longer-term study of continuous GH infusion vs daily injections in adults with GHD (24) and a 6-month study in children with GHD did not observe any difference in growth when comparing continuous GH infusion vs daily injection (25), indicating that GH replacement may be efficacious regardless of the administration mode in humans (24 –26). Sustained release GH preparations have been evaluated in longer-term studies in both adults (9 – 10) and children (8, 11, 27–28) and were assessed as being efficacious and well tolerated, with no significant alterations in lipid metabolism and insulin resistance, but with a larger number of injection site reactions (11). However, previous reported long-term effects from sustained GH preparations does not guarantee that long-term effects of NNC0195– 0092 in GHD subjects will be comparable to daily hGH as reported for sustained release formulations (9 –11). The shape of the NNC0195– 0092 PK is different compared with spontaneous GH release and compared with daily hGH, whereas the IGF-1 profile in the clinical relevant dose range seems more similar. Furthermore, clinical trials over more extensive time periods are necessary

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Rasmussen et al

Dosing of a Long-Acting GH Derivative

to determine whether long-term response to NNC0195– 0092 is comparably efficacious and safe in GHD subjects. In conclusion, SD and MD of NNC0195– 0092 administered sc to healthy Japanese and non-Asian male subjects, were well tolerated at all doses investigated. Importantly, there were no serious safety concerns or significant local tolerability issues identified. A clear dose-dependent increase in IGF-1 levels was induced with all MD of NNC0195– 0092, with no differences in key PK and PD parameters observed between Japanese and non-Asian subjects. Future clinical trials are necessary to determine whether long-term administration with NNC0195– 0092 is efficacious and safe in GHD adults and children. The present trial suggests that NNC0195– 0092 has the potential for an efficacious, well-tolerated, once-weekly GH treatment.

Acknowledgments We thank Nicoline Videbæk, Cell and Antibody Analysis, Novo Nordisk A/S for the antibody analyses and for scientific advice in the interpretation of the results, and Nina Worm White, who provided medical writing services on behalf of Novo Nordisk A/S. Address all correspondence and requests for reprints to: Michael Højby Rasmussen, MD, PhD, MSc, Novo Nordisk A/S, Vandtårnsvej 110 –112, DK 2860 Søborg, Denmark. E-mail: [email protected]. The trial was financially supported by Novo Nordisk A/S. This study was registered in Clinical Trials.gov as trial number NCT01514500. Disclosure Summary: Michael Højby Rasmussen, Minna W. Brændholt Olsen, Lene Alifrangis, Søren Klim, and Mette Suntum are employees of Novo Nordisk A/S.

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7. Smith SL, Hindmarsh PC, Brook CGD. Compliance with growth hormone treatment–are they getting it? Arch Dis Childhood. 1993; 68:91–93 8. Silverman BL, Blethen SL, Reiter EO, Attie KM, Neuwirth RB, Ford KM. A long-acting human growth hormone (Nutropin Depot): Efficacy and safety following two years of treatment in children with growth hormone deficiency. J Pediatr Endocrinol Metab. 2002;15: 715–722 9. Hoffman AR, Biller BM, Cook D, Baptista J, Silverman BL, Dao L, et al. Efficacy of a long-acting growth hormone (GH) preparation in patients with adult GH deficiency. J Clin Endocrinol Metab. 2005; 90:6431– 6440 10. Biller BMK, Ji HJ, Ahn H, Savoy C, Siepl EC, Popovic V, et al. 12-Month effects of once-weekly sustained-release growth hormone treatment in adults with GH deficiency. Pituitary. 2013;16:311–318 11. Khadilkar V, Radjuk KA, Bolshova E, Khadgawat R, El Kholy M, Desai M, et al. 24-month use of once-weekly GH, LB3002, in prepubertal children with GH deficiency. J Clin Endocrinol Metab. 2014;99:126 –132 12. Touraine P, D’Souza GA, Kourides I, Abs R, Barclay P, Xie R, et al. GH Lipoatrophy Study Group. Lipoatrophy in GH deficient patients treated with a long-acting pegylated GH. Eur J Endocrinol. 2009;161:533–540 13. Danne T, Lüpke K, Walte K, Von Schuetz W, Gall MA. Insulin detemir is characterised by a consistent pharmacokinetic profile across age-groups in children, adolescents, and adults with type 1 diabetes. Diabetes Care. 2003;26:3087–3092 14. Laursen T, Jørgensen JO, Jakobsen G, Hansen BL, Christiansen JS. Continuous infusion versus daily injections of growth hormone (GH) for 4 weeks in GH-deficient patients. J Clin Endocrinol Metab. 1995;80:2410 –2418 15. Iranmanesh A, Grisso B, Veldhuis JD. Low basal and persistent pulsatile growth hormone secretion are revealed in normal and hyposomatotropic men studied with a new ultrasensitive chemiluminescence assay. J Clin Endocrinol Metab. 1994;78:526 –535 16. Reiter EO, Price DA, Wilton P, Albertsson-Wikland K, Ranke MB. Effect of Growth Hormone (GH) Treatment on the Near-Final Height of 1258 Patients with Idiopathic GH Deficiency: Analysis of a Large International Database. J Clin Endocrinol Metab. 2006;91: 2047–2054 17. Jørgensen JOL, Pedersen SA, Thuesen L, et al. Beneficial effects of growth hormone treatment in GH-deficient adults. Lancet. 1989; 1:1221–1225 18. van der Klaauw AA, Romijn JA, Biermasz NR, et al. Sustained effects of recombinant GH replacement after 7 years of treatment in adults with GH deficiency. Eur J Endocrinol. 2006;155:701–708 19. Bex M, Abs R, Maiter D, Beckers A, Lamberigts G, Bouillon R. The effects of growth hormone replacement therapy on bone metabolism in adult-onset growth hormone deficiency: a 2-year open randomized controlled multicenter trial. J Bone Miner Res. 2002;17:1081– 1094 20. Laursen T. Clinical pharmacological aspects of growth hormone administration. Growth Horm IGF Res. 2004;14:16 – 44 21. Johansson JO, Oscarsson J, Bjarnason R, Bengtsson BA. Two weeks of daily injections and continuous infusion of recombinant human growth hormone (GH) in GH-deficient adults I. Effects on insulinlike growth-factor-I (IGF-1), GH and IGF-binding proteins and glucose homeostasis. Metabolism. 1996;45:362–369 22. Oscarsson J, Ottosson M, Johansson JO, Wiklund O, Mårin P, Björntorp P, Bengtsson BA. Two weeks of daily injections and continuous infusion of recombinant human growth hormone (GH) in GH-deficient adults II. Effects on serum lipoproteins and lipoprotein and hepatic lipase activity. Metabolism. 1996;45:370 –377 23. Laursen T, Lemming L, Jørgensen JO, Klausen IC, Christiansen JS. Different effects of continuous and intermittent patterns of growth hormone administration on lipoprotein levels in growth hormonedeficient patients. Horm Res. 1998;50:284 –291 24. Laursen T, Gravholt CH, Heickendorff L, et al. Long-term effects of

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