INHIBITION OF BONE RESORPTION WITH RANKL BINDING PEPTIDES

Abstract

Methods are provided for inhibiting bone resorption and/or osteoclast activity. More specifically, methods are provided wherein polypeptides against RANK-L are administered to a subject less frequently and/or at lower dose, while still maintaining effective inhibition of bone resorption and/or osteoclast activity in the subject at unexpectedly prolonged periods of time, particularly in view of the doses administered.

Claims

1. A method for prevention and/or treatment of bone diseases and/or disorders by inhibiting bone resorption or osteoclast activity comprising administering a polypeptide that specifically binds Receptor Activator of Nuclear Factor kappa B Ligand (RANKL) to a subject, wherein the amount of the polypeptide administered is effective to change one or more markers of bone metabolism and/or bone homeostasis selected from cross-linking telopeptide of type I collagen (CTX-1), N-terminal telopeptide of type I collagen (NTX-1), tartrate-resistant acid phosphatase isoform 5b (TRACP5b), N-terminal propeptide of type I procollagen (P1NP) and bone-specific alkaline phosphatase (BAP), for at least 30 days after administration, wherein the polypeptide comprises two or more immunoglobulin single variable domains that specifically bind RANKL and that essentially consist of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which CDR1 is chosen from SEQ ID NO: 1, CDR2 is chosen from SEQ ID NO: 2, and CDR3 is chosen from SEQ ID NO: 3, and wherein the polypeptide is administered in an amount from about 0.003 mg/kg to about 0.03 mg/kg and the serum levels of CTX-1 are reduced by 30% for at least about 30 days after administration; the polypeptide is administered in an amount from about 0.01 mg/kg to about 0.1 mg/kg and the serum levels of CTX-1 are reduced by 30% for at least about 120 days after administration; the polypeptide is administered in an amount from about 0.1 mg/kg to about 1 mg/kg and the serum levels of CTX-1 are reduced by 30% for at least about 270 days after administration; the polypeptide is administered in an amount of less than or equal to 0.03 mg/kg and the serum levels of CTX-1 are reduced by at least 45% for at least about 30 days after administration; the polypeptide is administered in an amount from about 0.01 mg/kg to about 3 mg/kg, preferably in an amount from about 0.01 mg/kg to about 0.1 mg/kg and the serum levels of CTX-1 are reduced by at least 45% for at least about 60 days after administration; the polypeptide is administered in an amount from about 0.01 mg/kg to about 0.3 mg/kg, preferably 0.03 mg/kg to about 0.3 mg/kg and the serum levels of CTX-1 are reduced by at least 45% for about 30 days to 3 months, preferably for at least about 90 days after administration; the polypeptide is administered in an amount from about 0.1 mg/kg to about 1 mg/kg and the serum levels of CTX-1 are reduced by at least 45% for at least about 120 days after administration; the polypeptide is administered in an amount from about 0.3 mg/kg to about 1 mg/kg and the CTX-1 is reduced by at least 45% for about 3 months to 6 months after administration; the polypeptide is administered in an amount of less than or equal to 0.3 mg/kg and the CTX-1 is reduced by at least 45% for about 3 months to 6 months after administration; the polypeptide is administered in an amount of less than or equal to 1 mg/kg and the serum levels of CTX-1 are reduced by at least 45% for about 6 months after administration; the polypeptide is administered in an amount from about 0.03 mg/kg to about 0.3 mg/kg and the serum levels of CTX-1 are reduced by at least 70% for at least about 30 days after administration; the polypeptide is administered in an amount from about 0.1 mg/kg to about 1 mg/kg and the serum levels of CTX-1 are reduced by at least 70% for at least about 120 days after administration; the polypeptide is administered in an amount from about 0.01 mg/kg to about 0.1 mg/kg and the levels of NTX-1 are reduced by at least 30% for at least 60 days after administration; the polypeptide is administered in an amount from about 0.03 mg/kg to about 0.3 mg/kg and the levels of NTX-1 are reduced by at least 30% for at least about 90 days after administration; the polypeptide is administered in an amount from about 0.1 mg/kg to about 1 mg/kg and the levels of NTX-1 are reduced by at least 30% for at least about 120 days after administration; the polypeptide is administered in an amount from about 1 mg/kg to about 10 mg/kg and the levels of NTX-1 are reduced by at least 30% for at least about 180 days (or 6 months), preferably at least 10 months, or even at least 12 months after administration; the polypeptide is administered in an amount from about 0.01 mg/kg to about 3 mg/kg, preferably in an amount from about 0.01 mg/kg to about 0.1 mg/kg and the levels of NTX-1 are reduced by at least 45% for at least about 30 days after administration; the polypeptide is administered in an amount from about 0.03 mg/kg to about 0.3 mg/kg and the levels of NTX-1 are reduced by at least 45% for at least about 60 days after administration; the polypeptide is administered in an amount from about 0.01 mg/kg to about 3 mg/kg, preferably, 0.01 mg/kg to about 0.3 mg/kg, or 0.03 mg/kg to about 0.3 mg/kg and the levels of NTX-1 are reduced by at least 45% for at least about 30 days to 3 months after administration; the polypeptide is administered in an amount of less than or equal to 0.03 mg/kg and the levels of NTX-1 are reduced by at least 45% for at least about 30 days to 3 months after administration; the polypeptide is administered in an amount from about 0.1 mg/kg to about 1 mg/kg and the levels of NTX-1 are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 150 days after administration; the polypeptide is administered in an amount from about 0.3 mg/kg to about 1 mg/kg and the levels of NTX-1 are reduced by at least 45% for about 3 months to 6 months after administration; the polypeptide is administered in an amount of less than or equal to 0.3 mg/kg and the levels of NTX-1 are reduced by at least 45% for about 3 months to 6 months after administration; the polypeptide is administered in an amount from about 1 mg/kg to about 10 mg/kg, preferably in an amount from about 1 mg/kg to about 3 mg/kg the levels of NTX-1 are reduced by at least 45% for at least 6 months to 1 year after administration; the polypeptide is administered in an amount of less than or equal to 1 mg/kg and the levels of NTX-1 are reduced by at least 45% for at least about 180 days (or 6 months) after administration; the polypeptide is administered in an amount from about 0.1 mg/kg to about 1 mg/kg and the levels of NTX-1 are reduced by at least 70% for at least about 30 days after administration; the polypeptide is administered in an amount from about 0.3 mg/kg to about 3 mg/kg and the levels of NTX-1 are reduced by at least 70% for at least about 90 days after administration; the polypeptide is administered in an amount from about 1 mg/kg to about 10 mg/kg and the levels of NTX-1 are reduced by at least 70% for at least about 120 days after administration; the polypeptide is administered in an amount from about 0.1 mg/kg to about 1 mg/kg, preferably in an amount from about 0.3 mg/kg to about 3 mg/kg, more preferably in an amount from about 0.3 mg/kg to about 1 mg/kg and the levels of NTX-1 are reduced by at least 80% for at least about 30 days (to 3 months) after administration; the polypeptide is administered in an amount from about 1 mg/kg to about 10 mg/kg and the levels of NTX-1 are reduced by at least 80%, compared to pre-treatment or normal levels, for at least about 90 days after administration; the polypeptide is administered in an amount from about 0.03 mg/kg to about 0.3 mg/kg and the serum levels of TRACP5b are reduced by at least 30% for at least about 60 days after administration; the polypeptide is administered in an amount from about 0.1 mg/kg to about 1 mg/kg and the serum levels of TRACP5b are reduced by at least 30% for at least about 90 days after administration; the polypeptide is administered in an amount from about 0.3 mg/kg to about 3 mg/kg and the serum levels of TRACP5b are reduced by at least 30% for at least about 120 days after administration; the polypeptide is administered in an amount from about 0.1 mg/kg to about 1 mg/kg and the serum levels of TRACP5b are reduced by at least 45% for at least about 30 days after administration; the polypeptide is administered in an amount from about 0.3 mg/kg to about 3 mg/kg and the serum levels of TRACP5b are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 90 days after administration; the polypeptide is administered in an amount from about 0.003 mg/kg to about 0.03 mg/kg and the serum levels of P1NP are reduced by at least 30% for at least about 90 days after administration; the polypeptide is administered in an amount from about 0.01 mg/kg to about 0.1 mg/kg and the serum levels of P1NP 1 are reduced by at least 30% for at least about 120 days after administration; the polypeptide is administered in an amount from about 0.03 mg/kg to about 0.3 mg/kg and the serum levels of P1NP are reduced by at least 45% for at least about 90 days after administration; or the polypeptide is administered in an amount from about 0.1 mg/kg to about 1 mg/kg and the serum levels of P1NP are reduced by at least 70% for at least about 90 days after administration.

2. (canceled)

3. The method according to claim 1, wherein the polypeptide comprises or consists of SEQ ID NO: 10 or SEQ ID NO: 12.

4. The method according to claim 1, wherein the polypeptide is administered subcutaneously.

5. The method according to claim 1, wherein the subject has osteoporosis.

6.-59. (canceled)

60. The method according to claim 3, wherein the polypeptide is administered subcutaneously.

61. The method according to claim 3, wherein the subject has osteoporosis.

62. The method according to claim 4, wherein the subject has osteoporosis.

63. The method according to claim 60, wherein the subject has osteoporosis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0395] FIG. 1: Open two-compartmental pharmacokinetic model with linear and non-linear clearance from the central compartment. CL.sub.NON-RANKL is the linear non-RANKL mediated clearance, Vc the volume of the central compartment, Vt the volume of the peripheral compartment, CLd the inter-compartmental flow, and CL.sub.RANKL is the non-linear RANKL-mediated clearance (V.sub.max the maximum metabolic rate and K.sub.m the ALX-0141 concentration corresponding to 50% of V.sub.max). The absorption rate constant and the bioavailability after s.c. administration are represented by ka and F, respectively.

[0396] FIG. 2: Open mono-compartmental pharmacokinetic model with parallel first order absorption from the s.c. space and linear and non-linear clearance from the central compartment. CL.sub.NON-RANK/F is the linear non-RANKL mediated clearance corrected for s.c. bioavailability, V/F the volume of the central compartment corrected for s.c. bioavailability, F1 is the faction of the dose absorbed with k.sub.a1, F2 is the fraction of the dose absorbed with k.sub.a2 with k.sub.a1 and k.sub.a2 being the respective fast and slow first order absorption rate constants, and CL.sub.RANKL/F is the non-linear RANKL-mediated clearance corrected for s.c. bioavailability (V.sub.max the maximum metabolic rate and K.sub.m the ALX-0141 concentration corresponding to 50% of V.sub.max).

[0397] FIG. 3: Pharmacokinetics. Geometric mean plasma concentration versus time profiles of ALX-0141. (a) Linear scale; (b) Semi-logarithmic scale.

[0398] FIG. 4: Pharmacodynamics—changes in serum CTX-1. A rapid decrease in serum CTX-1 level was observed within 8 hours post-dose in ALX-0141 treated subjects. A dose-dependent, long-lasting inhibitory effect of ALX-0141 on serum CTX-1 level (% of baseline) is shown. Values are mean±SEM, n=11 for placebo, n=6 for each ALX-0141 treatment group.

[0399] FIG. 5: Pharmacodynamics—changes in urine NTX-1/creatinine. A rapid decrease in urine NTX-1/creatinine level was observed within 8 hours post-dose in ALX-0141 treated subjects. A dose-dependent, long-lasting inhibitory effect of ALX-0141 on urine NTX-1/creatinine level (% of baseline) is shown. Values are mean±SEM, n=11 for placebo, n=6 for each ALX-0141 treatment group.

[0400] FIG. 6: Arithmetic mean CTX-1 serum concentration (% of baseline) after s.c. dosing with ALX-0141 or placebo in post-menopausal healthy female volunteers.

[0401] FIG. 7: Arithmetic mean NTX-1/Creatinine ratios in urine (% of baseline) after s.c. dosing with ALX-0141 or placebo in post menopausal healthy female volunteers.

[0402] FIG. 8: Arithmetic mean TRACP5b serum concentration (% of baseline) after s.c. dosing with ALX-0141 or placebo in post-menopausal healthy female volunteers.

[0403] FIG. 9: Arithmetic mean P1NP serum concentration (% of baseline) after s.c. dosing with ALX-0141 or placebo in post-menopausal healthy female volunteers.

[0404] FIG. 10: Arithmetic mean BAP serum concentration (% of baseline) after s.c. dosing with ALX-0141 or placebo in post-menopausal healthy female volunteers.

[0405] FIG. 11: Observed (lines with data points indicated) and model based simulations (lighter lines, no data points indicated) of ALX-0141 plasma concentration-time profiles in healthy post-menopausal women following a single s.c. dose with ALX-0141 at (a) 0.003, (b) 0.01, (c) 0.03, (d) 0.1, (e) 0.3, and (f) 1 mg/kg.

[0406] FIG. 12: Observed (lines with data points indicated) and model based simulations (lighter lines, no data points indicated) of serum CTX-1-time profiles in healthy post-menopausal women following a single s.c. dose with ALX-0141 at (a) 0.003, (b) 0.01, (c) 0.03, (d) 0.1, (e) 0.3, and (f) 1 mg/kg.

EXAMPLES

List of Abbreviations

[0407]

TABLE-US-00005 Symbol Unit Definition ADA Anti-drug antibody AUC.sub.inf (ug .Math. d/mL) Area under the plasma druc concentration-time curve extrapolated to infinity AUC.sub.last (ug .Math. d/mL) Area from time zero to the last measurable concentration after dosing BAP Bone specific alkaline phsophatase BED mg/kg biological effective dose b.w. — Body weight C Amount/volume ALX-0141 plasma concentration CL Volume/time*kg Apparent total body clearance of the drug from serum CL.sub.d Volume/time*kg Inter-compartmental flow CL.sub.RANKL Volume/time*kg Non-linear RANKL-mediated clearance CL.sub.NON-RANKL Volume/time*kg Linear non-RANKL mediated clearance CTX-1 — C-terminal cross-linked telopeptide of type I collagen D Amount/kg Dose F — s.c. bioavailability i.v. — Intravenous F1 — Fraction of the dose absorbed with k.sub.a1 F2 — Fraction of the dose absorbed with k.sub.a2 I.sub.max % Maximum inhibition of serum CTX-1 (1 < I.sub.max < 0) k.sub.a 1/time Absorption rate constant (s.c. administration) k.sub.in Amount/ Zero-order synthesis rate volume*time k.sub.a1 1/time Fast first order absorption rate constant (s.c. administration) k.sub.a2 1/time Slow order absorption rate constant (s.c. administration) k.sub.out 1/time First order elimination rate constant of serum CTX-1 K.sub.m Amount/volume ALX-0141concentration corresponding to 50% of V.sub.max (CL.sub.RANKL). Lk.sub.in — typical fractional dose effect on k.sub.in at the lower doses (0.003 and 0.01 mg/kg) LLOQ Lower limit of quantification nadir The lowes point in the curve n — Concentration-response shape factor NTX-1 N-terminal telopeptide of type 1 collagen P1NP Procollagen type 1 amino-terminal propeptide RANK(L) — Receptor activator for nuclear factor kappa B (ligand) R Amount/volume (Pharmacodynamic) Response (i.e. serum CTX-1 level) s.c. — Subcutaneous t.sub.1/2, .sub.terminal Time Terminal elimination half-life calculated as ln(2)/λ.sub.z TRACP5b Tartrate-resistant acid phosphatase V.sub.c Volume/kg Apparent volume of the central or plasma compartment in a two- compartment model Vd.sub.ss Volume/kg Apparent volume of distribution at steady state V.sub.max Amout/time Maximum metabolic rate of CL.sub.RANKL V.sub.t Volume/kg Apparent volume of the peripheral compartment in a two-compartment model

Example 1: Toxicology Studies with ALX-0141

[0408] In a single dose toxicity study ALX-0141 was administered to male and female cynomolgus monkeys as single s.c. doses of 0, 0.02, 2, 20, and 50 mg/kg b.w ALX-0141 and a single i.v. bolus dose of 20 mg/kg. The sequence of ALX-0141 is depicted as SEQ ID NO: 12 (SEQ ID NO: 759 in US 2010/0104568).

[0409] Blood samples for pharmacokinetic (PK), anti-drug antibody (ADA), and pharmacodynamic (PD) analysis purposes were collected from all animals at pre-dose and at selected time points post-dose. Samples were analysed for PK, PD and ADA purposes using validated methods, with the exception of the CTX-1 determinations where a human commercial kit was used that was verified to be cross-reactive with cynomolgus monkey CTX-1.

[0410] In a repeated dose toxicity study, ALX-0141 was administered to male and female cynomolgus monkeys as s.c. doses of 0, 0.2, 2, 20, and 50 mg/kg b.w ALX-0141 at 6 occasions during 2 weeks (test days 1, 3, 5, 8, 10, and 12).

[0411] Toxicokinetic samples were taken up to study day 15 (i.e. 14 days after the first dose administration). Three groups (0, 2 and 50 mg/kg) included a recovery period, where additional 2 animals per sex were treated and sampling was performed over 99 days (i.e. up to 98 days post-dose).

[0412] Blood samples for PK, ADA, and PD purposes were collected from all animals at pre-dose and at selected time points post-dose. Samples were analyzed for PK, PD and ADA purposes using validated methods with the exception of the CTX-1 determinations where a human commercial kit was used, which was shown to be cross-reactive with cynomolgus monkey CTX-1.

Example 2: Preclinical Data

[0413] Pharmacokinetic (PK) and pharmacodynamic (PD) modeling was performed on data generated in a single dose toxicity study with ALX-0141 in the cynomolgus monkey as described in Example 1. Data from a 2-week repeated dose toxicity study were used to verify whether the single dose data could accurately predict the repeated dose pharmacokinetics (i.e. to verify time-dependency of ALX-0141 PK).

Pharmacokinetic Modeling of Cynomolgus Monkey ALX-0141 Plasma Concentrations

[0414] The non-linear pharmacokinetic behavior of ALX-0141 in the cynomolgus monkey was captured by fitting the data to an open two-compartmental pharmacokinetic model with linear and a non-linear clearance from the central compartment and first order absorption (s.c. application). The structural model is depicted in FIG. 1.

[0415] The linear clearance mechanism is likely related to the non-saturable, and non-RANKL mediated removal of ALX-0141 and corresponds to the slow and non-specific proteolytic degradation of ALX-0141. The non-linear and RANKL-mediated clearance process is a saturable clearance mechanism; most probably representing binding of ALX-0141 to membrane bound RANKL and subsequent internalization and clearance.

[0416] All available individual plasma concentration data after a single s.c. and i.v. dose with ALX-0141 were fitted simultaneously to the model (WinNonlin Professional Software Version 5.1 (Pharsight Corporation, Mountain View Calif., USA) using iterative re-weighting (1/ŷ*ŷ), where ŷ is the predicted plasma concentration.

[0417] The estimated PK parameters of ALX-0141 in the cynomolgus monkey were estimated with sufficient precision and are listed in Table B-1.

[0418] An open two-compartmental pharmacokinetic model with parallel linear non RANKL-mediated clearance (CL.sub.NON-RANKL) and non-linear saturable RANKL-mediated clearance (CL.sub.RANKL) from the central compartment and first order absorption (s.c. application) was used to adequately describe the non-linear PK of ALX-0141 in the cynomolgus monkey.

[0419] At low ALX-0141 concentrations (C<<<K.sub.m) the contribution of the CL.sub.RANKL predominates, whereas at higher, RANKL-saturating plasma concentrations, the overall CL is mainly governed by the CL.sub.NON-RANKL.

[0420] The estimated values of CL.sub.RANKL and CL.sub.NON-RANKL indicate that clearance via the RANKL-mediated component occurs more efficiently (about 5.6-fold) as compared to the non-RANKL-mediated pathway. The RANKL-mediated clearance is however readily saturated as indicated by the low K.sub.m−value (0.149 μg/mL; 3.6 nM) of the CL.sub.RANKL pathway.

[0421] The estimated volume of distribution at steady-state suggests that ALX-0141 distributes outside the central compartment into the interstitial space of the tissues.

[0422] At ALX-0141 concentrations which saturate the RANKL-mediated pathway, the estimated half-life of ALX-0141 was 8.5 days, i.e. similar to that reported for cynomolgus monkey serum albumin.

[0423] The prominent role of the non-linear RANKL-mediated component of ALX-0141 in overall clearance explains the more than dose-proportional increase in exposure (C.sub.max- and AUC-values) at the lower dose levels. At higher dose levels exposures increase dose-proportionally since the overall clearance is then mainly dictated by the linear non-RANKL mediated pathway.

[0424] ALX-0141 displayed time-independent pharmacokinetics since repeated dose pharmacokinetics could be adequately predicted on the basis of single dose data. In the monkey, the (single dose) s.c. absolute bioavailability of ALX-0141 was virtually complete (96%).

Semi-Mechanistic PK/PD Modeling (Cynomolgus Monkey ALX-0141 Plasma Concentrations and Serum CTX-1 Concentrations)

[0425] The effect of ALX-0141 administration on serum CTX-1 levels is considered indirect in nature since there is no direct relationship between achieved ALX-0141 plasma concentrations and effect on CTX-1 levels. Therefore, an indirect response model was employed to describe the pharmacological effect of i.v. and s.c. administered ALX-0141 on the physiological turnover of serum CTX-1.

[0426] The model is analogous to that previously employed for the anti-RANKL monoclonal antibody denosumab (Peterson M., Stouch B., Chen D. et al. 2004, A PK/PD Model Developed in cynomolgus Monkeys Predicts Concentrations and Effects of AMG 162, A Fully Human Monoclonal Antibody Against RANKL, in Healthy Postmenopausal Women. AAPS Annual Meeting, Baltimore, Md., Nov. 7-11, 2004; Abstract) and describes a drug response that results from the inhibition of the production of serum CTX-1. In this indirect response model, the rate of change of CTX-1 (Response, R) is described by:

[00001]$\frac{\mathrm{dR}}{\mathrm{dt}}=k_{\mathrm{in}}.Math.\left[1-\frac{I_{\max }.Math.C^{.Math.n}}{{\mathrm{IC}}_{50}^{.Math.n}+C^{.Math.n}}\right]-k_{\mathrm{out}}.Math.R$

[0427] With k.sub.in, the zero-order synthesis rate; R, the serum CTX-1 level, I.sub.max, the maximum inhibition (1<I.sub.max<0); C, the concentration of ALX-0141; n, the concentration-response shape factor; and k.sub.out, the first order elimination rate constant of serum CTX-1.

[0428] All available individual serum CTX-1 data obtained after a single i.v. and s.c. dose were fitted simultaneously to the model (WinNonlin Professional Software Version 5.1, Pharsight Corporation, Mountain View Calif., USA) using the pharmacokinetic function as input function for the indirect response PK/PD model.

[0429] The pharmacodynamic effect of i.v. and s.c. administered ALX-0141 on the physiological turnover of serum CTX-1 in the monkey were adequately captured using a semi-mechanistic PK/PD model (indirect response model).

[0430] PK/PD modeling indicated that ALX-0141 was a potent inhibitor of serum CTX-1 production in the cynomolgus monkey (IC.sub.50 of ca 22 ng/mL or 0.54 nM). In addition, ALX-0141 was able to almost completely inhibit the production of serum CTX-1 (I.sub.max≈73%).

Example 3: Scaling to Human: Simulation of ALX-0141 PK/PD in Humans

[0431] The PK/PD model developed to describe the temporal profile of ALX-0141 concentrations and corresponding serum CTX-1 levels in the cynomolgus monkey was scaled to human to assist in the dose selection of the first in human study. A similar approach was previously successfully employed to simulate the human PK/PD of denosumab based on cynomolgus monkey data. (Peterson M., Stouch B., Chen D. et al. 2004, A PK/PD Model Developed in cynomolgus Monkeys Predicts Concentrations and Effects of AMG 162, A Fully Human Monoclonal Antibody Against RANKL, in Healthy Postmenopausal Women. AAPS Annual Meeting, Baltimore, Md., Nov. 7-11, 2004; Abstract).

[0432] To this end, the estimated CL.sub.NON-RANKL, volumes of distribution (Vc and Vt), and absorption rate constant (ka) in the cynomolgus monkey were scaled to human using standard allometric equations (Boxenbaum H. 1984, Interspecies Pharmacokinetic Scaling and the Evolutionary-Comparative Pardigm. Drug Metab. Rev. 15(5&6): 1071-1121). For the CL.sub.RANKL, the K.sub.m-value was assumed to be similar between the cynomolgus monkey and man, whereas the V.sub.max-value in humans was considered to be one third of that in the cynomolgus monkey since the metabolic rate for bone turnover is 3-fold slower in humans (FDA. 1994, Guidelines for preclinical and clinical evaluation of agents used in the prevention or treatment of postmenopausal osteoporosis).

[0433] Since the bioavailability cannot be scaled, predictions were performed using two reasonable values of F, i.e. 0.8 and 1.0. The PK parameters used for the simulation of the ALX-0141 plasma concentration-time profiles are listed in Table B-2.

[0434] To simulate the temporal profile of ALX-0141 concentrations in humans after s.c. administration, the scaled up pharmacokinetic parameters were combined with the previously described open two-compartmental pharmacokinetic model with a parallel linear non-RANKL-mediated clearance (CL.sub.NON-RANKL) and a non-linear RANKL-mediated clearance (CL.sub.RANKL) from the central compartment and first order absorption (s.c. application).

[0435] The predicted human ALX-0141 concentrations were subsequently used as input function for the indirect PK/PD response model (inhibition of synthesis) to predict the temporal serum CTX-1 profile in humans.

[0436] The baseline value of serum CTX-1 (RO) and the first order elimination rate constant (k.sub.out) were taken from the literature (Glover S. J., Garnero P., Naylor K., Rogers A. and Eastell R. 2008, Establishing a reference range for bone turnover markers in young, healthy women. Bone 42: 623-630; Holford N, Pillai G, Kaila N, Collins W, Roy S, Cremers S, Trechsel U, Bouisset F, Steimer J-L. June 2006, PKPD model for cathepsin K inhibition with balicatib and changes in bone turnover biomarkers, in particular NTx. PAGE (Population Approach Group Europe), Bruges (B), 14-16). The maximum effect value (I.sub.max) and the ALX-0141 concentration were assumed to be similar between cynomolgus monkeys and humans, whereas the concentration corresponding to half of the maximum effect (IC.sub.50) was taken six-fold lower in humans relative to monkey. The latter is related to the effective concentration being 3-fold lower in humans compared to monkey as a result of differences in metabolic rate for bone turnover (3-fold slower in humans compared to monkeys) (FDA 1994, Guidelines for preclinical and clinical evaluation of agents used in the prevention or treatment of postmenopausal osteoporosis), in combination with a 2-fold greater affinity of ALX-0141 for binding to human RANKL versus cynomolgus monkey RANKL.

[0437] In Table B-3, the PD parameters used for the simulation of the ALX-0141 serum CTX-1 concentration-time profiles are listed.

[0438] Simulated PD profiles indicated a dose-dependent suppression of serum CTX-1 after a single s.c. dose with ALX-0141, consistent with the indirect response model describing inhibition of serum CTX-1 synthesis. Per this model, the suppression of serum CTX-1 would dependent on the dose of ALX-0141. The maximum degree of serum CTX-1 inhibition would increase with the administered dose up to its maximum effect (I.sub.max)-value; a further increase in dose was not expected to further increase the effect but would prolong the time to return to baseline.

Example 4: Clinical Data

4.1 Methodology

4.1.1 Design

[0439] A double-blind, placebo-controlled, single ascending dose phase I study was performed in 1 cohort of 2 healthy postmenopausal female subjects receiving a single subcutaneous (s.c.) dose of ALX-0141 at 0.003 mg/kg or placebo (1 verum and 1 placebo) and 5 cohorts of 8 healthy postmenopausal female subjects receiving a single s.c. doses of ALX-0141 at 0.01, 0.03, 0.1, 0.3 and 1 mg/kg or placebo (6 verum and 2 placebo for each cohort). The day of treatment was Day 1 for purposes of calculating sample timing.

[0440] Plasma samples were collected at pre-dose, at 8, 12, 24, 48, 72, 96, 120 and 144 hours post-dose, once on Days 14, 30, 60 and at follow-up (Day 90) and, if applicable, at additional monthly follow-up visits to quantify the levels of the drug and of the biomarker CTX-1.

4.1.2 Procedures and Assessments

[0441]

TABLE-US-00006 Screening and each Clinical laboratory (including calcium [Ca] and intact-parathyroid follow up visit: hormone [PTH]), 25-hydroxy Vitamin D in serum, vital signs, physical examination (at screening and first follow-up visit only), weight, 12-lead electrocardiogram (ECG), blood sampling for immunogenicity, PK and PD (blood sampling for immunogenicity, and blood and urine sampling for PD only at follow-up visit[s]); At eligibility Medical history, height, drug and alcohol screen, hepatitis B surface screening only: antigen (HBsAg), anti-hepatitis C virus (HCV) and anti-human immunodeficiency virus 1/2 (HIV 1/2); Repeated upon Clinical laboratory (including Ca and intact-PTH), 25-hydroxy Vitamin D, admission: weight, drug and alcohol screen, vital signs and ECG; Observation One period in clinic from −65 h (Day −3) before up to 144 h after drug period: administration on Day 1 and ambulatory visits on Day 14 ± 2, 30 ± 2, 60 ± 5 and 90 ± 5 (follow-up) and additional follow-up visits if necessary*; Blood sampling: For PK of ALX-0141 in plasma: pre-dose and 8 h and 12 h post-dose on Day 1, once in the morning on Days 2-7 and once on Days 14 ± 2, 30 ± 2, 60 ± 5 and 90 ± 5 (follow-up) and additional follow-up visits if necessary*; For PD of procollagen type I amino-terminal propeptide (P1NP), cross- linking telopeptide of type 1 collagen (CTX-1), bone specific alkaline phosphatase (BAP) and tartrate-resistant acid phosphatase (TRACP5b) in serum: at screening, once in the mornings on Days −2, −1, pre-dose and 8 h and 12 h post-dose on Day 1, and once in the morning on Days 2, 3, 4, 5, 6, 7, 14 ± 2, 30 ± 2, 60 ± 5 and 90 ± 5 (follow-up) and additional follow-up visits if necessary*; Urine sampling: For PD of CTX-1 and N-terminal telopeptide of type 1 collagen (NTX-1) and creatinine (under fasted conditions) in urine: once on Days −2, −1, 1 (pre-dose), 2, 3, 4, 5, 6, 7, 14 ± 2, 30 ± 2, 60 ± 5 and 90 ± 5 (follow-up) and additional follow-up visits if necessary*; Safety Adverse events (AEs) and local tolerability: throughout the study; vital assessments: signs and 12-lead ECG: pre-dose and 3, 6, and 9 h post-dose on Day 1, once in the morning on Days 2-7 and once on Days 14 ± 2, 30 ± 2, 60 ± 5 and 90 ± 5 (follow-up) and additional follow-up visits if necessary*; clinical laboratory (including Ca and intact-PTH), 25-hydroxy Vitamin D, immunophenotyping of white blood cells (WBC) to differentiate between B- and T-lymphocytes: once on Days 1, 2, 4, 6, 14 ± 2, 30 ± 2, 60 ± 5 and 90 ± 5 (follow-up) and additional follow-up visits if necessary*. *a monthly additional follow-up visit was conducted if CTX-1 levels had not returned to 70% of baseline level on Day 90 and were repeated until CTX-1 levels were back to 70% of baseline. On Day 420 CTX-1 levels of all subjects had returned to at least 70% of baseline level, and no additional follow up visits were required for any of the subjects.

4.1.3 Subjects

[0442] A total of 42 subjects were enrolled in this study. All subjects were healthy post-menopausal female volunteers. The post-menopausal female population is an anticipated target population for the medication.

Main Criteria for Inclusion

[0443] Age: maximum of 80 years, inclusive [0444] Body Mass Index (BMI): 18-36 kg/m.sup.2, inclusive [0445] Gender: females [0446] Subjects: healthy post-menopausal female volunteers

Study Medication

[0447] 1. Active substance: ALX-0141, a trimeric construct of 41 kD, having two Nanobody building blocks targeting RANKL linked with a Nanobody targeting human serum albumin (HSA). [0448] Activity: anti-RANKL (anti-receptor activator of NFκB ligand) [0449] Indication: bone loss [0450] Strength: 0.65 mg/mL (Cohort 1), 2.17 mg/mL (Cohort 2), 6.5 mg/mL (Cohort 3) [0451] : 21.7 mg/mL (Cohort 4) and 65 mg/mL (Cohorts 5 and 6) [0452] Dosage form: s.c. injection [0453] 2. Placebo: Visually matching medication

Criteria for Evaluation

[0454] PK: plasma ALX-0141 concentrations, PK parameters [0455] PD: P1NP, CTX-1, BAP and TRACP5b concentrations in serum and creatinine, CTX-1 and NTX-1 concentrations in urine [0456] Safety: AEs, local tolerability, vital signs, 12-lead ECG, clinical laboratory, physical examination, immunogenicity, 25-hydroxy Vitamin D in serum, immunophenotyping of WBC

4.1.4 Analyses

[0457] The PD effects of ALX-0141 were explored by measuring CTX-1, TRACP5b, P1NP and BAP concentrations in serum and NTX-1/creatinine concentration ratios in urine. CTX-1, TRACP5b and NTX-1 are involved in bone resorption, whereas P1NP and BAP are involved in bone formation.

Biomarker assays used were:

TABLE-US-00007 CTX-1 Serum CROSSLAPS ® ELISA (cat. no. AC-02F1; Immunodiagnostic Systems Inc., Boldon, Tyne & Wear, UK) NTX-1 OSTEOMARK NTx Urine ELISA (cat. no. 9006; Inverness Medical (Wampole Laboratories), Princeton, NJ) TRACP5b BONETRAP ® Assay ELISA (cat. no. SB-TR201A; Immunodiagnostic Systems Inc., Boldon, Tyne & Wear, UK) P1NP UNIQ ™ P1NP RIA (cat. no. Q67034; Orion Diagnostica Oy, Espoo, Finland) BAP OSTASE ® BAP Immunoenzymetric Assay (cat. no. AC-20F1; Immunodiagnostic Systems Inc., Boldon, Tyne & Wear, UK) Creatinine creatinine test (cat. No. 03039070; Siemens Medical Solutions Diagnostics, Breda, Netherlands).

4.1.5 Statistics

[0458] Plasma concentrations of ALX-0141 and serum CTX-1 levels were modeled by non linear mixed effects modeling using NONMEM. Inter-individual variability (IIV) was set on each structural parameter and was calculated as the square root of the variance for the respective parameter×100. An exponential error model (additive in the log scale) was assumed for residual variability. The estimation method used within NONMEM was the FOCEI.

[0459] Data from 31 human post-menopausal female volunteers were used for this population analysis.

4.2 Pharmacokinetics

4.2.1 Concentration Data in Plasma

[0460] The drug concentration measurements of ALX-0141 were performed by a validated ELISA method. Geometric mean ALX-0141 plasma concentration-time profiles by treatment (linear and semi-logarithmic scale) are presented in FIG. 3.

[0461] Following s.c. administration of 0.003 mg/kg ALX-0141 (n=1), measurable plasma levels of ALX-0141 were observed from 48 h post-dose. On average, at the dose level of 0.01 mg/kg, ALX-0141 was measurable in plasma within 24 h. At dose levels of 0.03 mg/kg up to 1 mg/kg ALX-0141, ALX-0141 was measurable in plasma within 8 h. The geometric mean ALX-0141 plasma concentrations were found to increase gradually with maximum mean plasma concentrations being reached between 3 to 6 days post-dose at the dose levels ranging from 0.003 mg/kg to 0.03 mg/kg and between 1.5 to 2 days at the higher dose levels (0.1 mg/kg to 1 mg/kg). The mean plasma concentrations of ALX-0141 increased with increasing dose. After reaching the peak plasma concentration, mean plasma concentrations decreased very gradually. On average, at the highest dose levels, mean ALX-0141 plasma concentrations were still above LLOQ (0.0102 μg/mL) up to Day 60 (0.1 mg/kg and 0.3 mg/kg) or Day 90 (1 mg/kg). At the lower dose levels, ALX-0141 plasma concentrations were measurable up to Day 7 (0.003 mg/kg 1, n=1), Day 14 (0.01 mg/kg) or Day 30 (0.03 mg/kg).

4.2.1 Pharmacokinetic Parameters in Plasma

[0462] A summary of the ALX-0141 plasma PK parameters is presented in Table B-4.

[0463] The median t.sub.max ranged between 1.5 to 6 days post-dose and tended to be shorter (1.5 to 2 days) at the higher dose levels ranging from 0.1 mg/kg to 1 mg/kg compared to the lower dose levels ranging from 0.003 mg/kg to 0.03 mg/kg (3 to 6 days). The geometric mean half life could only be calculated reliably for the higher dosing groups (0.1 mg/kg, 0.3 mg/kg and 1 mg/kg ALX-0141) and was between 12.0 and 20.6 days. After a single s.c. dose administration of ALX-0141, the C.sub.max, AUC.sub.0-last and AUC.sub.0-inf values increased with increasing doses. Over the dose range of 0.1 mg/kg up to 1 mg/kg, the C.sub.max and AUC values increased in a dose-proportional manner, whereas at the lower doses ranging from 0.01 mg/kg up to 0.1 mg/kg, it appeared that C.sub.max and AUC.sub.0-last increased somewhat more than dose-proportional.

4.2.3 Pharmacokinetic Analysis

[0464] In healthy post-menopausal women, the pharmacokinetics of ALX-0141 were adequately characterized by an open mono-compartmental model with two first order absorption rates from the subcutaneous compartment, and a linear (non-target related) and non-linear (target related) clearance from the central compartment. The structural model is depicted in FIG. 2 and the PK parameter estimates are listed in Table B-5.

[0465] The target-mediated is ca. 4-fold more efficient than the non-RANKL-mediated pathway, but is readily saturated.

[0466] Body weight was a determining covariate on the volume of distribution.

[0467] Overall all the structural parameters of the final PK model were estimated with good precision (25.2% or less). The inter-Individual variability (IIV) was low to moderate for V (22.2%) and for F1 (36.5%). A larger inter-individual variability was found for k.sub.a1 and k.sub.a2 (170 and 144% respectively), for CL (53%) and for K.sub.m (135%). The standard deviation for residual error was estimated to 0.11 with a good precision (13.7%).

4.3 Pharmacodynamics

[0468] The PD effects of ALX-0141 were explored by measuring the serum concentrations of CTX-1, TRACP5b, P1NP and BAP, and urine NTX-1/creatinine ratios, which are involved in bone resorption and/or bone formation. Mean serum concentrations of CTX-1, TRACP5b, P1NP and BAP expressed as percentage of baseline are presented in FIGS. 4 and 6 (CTX-1), FIG. 8 (TRACP5b), FIG. 9 (P1NP) and FIG. 10 (BAP). Mean urine NTX-1/creatinine concentration ratios are presented in FIGS. 5 and 7. A summary of PD parameters is presented in Table B-6.

4.3.1 CTX-1

[0469] CTX-1 is a bone degradation product released as a result from osteoclast activity, and considered as a biomarker for bone resorption. Mean serum concentrations of CTX-1 (as % of baseline) after s.c. dosing with ALX-0141 or placebo in post-menopausal healthy female volunteers are shown in FIGS. 4 and 6. CTX-1 changes from baseline are shown in Table B-7.

[0470] After s.c. dosing with ALX-0141, a rapid decrease in CTX-1 was observed within 8 h post-dose (the first sampling time point) for all 6 dose levels tested. The mean nadir values for CTX-1 serum concentrations after treatment with ALX-0141 (active treatment) varied between 8.8% (0.3 mg/kg, N=6) and 50.7% (0.003 mg/kg, n=1) of baseline across the dose levels studied and decreased with increasing dose (FIG. 6). However, an almost maximum effect seemed to be reached at a dose level of 0.1 mg/kg or higher. In this study the dose level of 0.003 mg/kg can be considered as the BED. At the lowest 3 dose levels, the CTX-1 serum concentrations started to increase after a nadir had been reached and before Day 90, whereas at the highest 3 dose levels mean CTX-1 serum concentrations remained at a low level close to nadir up to Day 90 (0.1 mg/kg, 0.3 mg/kg, 1 mg/kg) and started to gradually increase thereafter. The ALX-0141 treated subject in Cohort 1 (0.003 mg/kg) showed a CTX-1 serum concentration close to baseline on Day 60, i.e. within the margin of baseline—30%. The mean CTX-1 values for the other ALX-0141 dose groups did not reach the 70% of baseline cut-off within the originally planned 90 days of follow-up. The low CTX-1 levels which persisted for more than 90 days in almost all ALX-0141-treated subjects and lasted for approximately 1 year in several subjects treated with 0.01 mg/kg or more, indicated a long-lasting PD effect upon a single s.c. ALX-0141 injection.

4.3.2 NTX-1

[0471] NTX-1 is a degradation product of type I collagen, which is the organic major component of the extracellular matrix. Therefore NTX-1 is considered as a biomarker for bone resorption as a result of osteoclast activity. Since NTX-1 concentration in urine varies with urine volume, the ratio of NTX-1 to creatinine in urine was determined instead of the urine concentration of NTX-1, to correct for this variation. Mean urine NTX-1/creatinine concentration ratios (as % of baseline) after s.c. dosing with ALX-0141 or placebo in post-menopausal healthy female volunteers are shown in FIGS. 5 and 7. NTX-1/creatine ratio changes from baseline are shown in Table B-8.

[0472] Upon s.c. dosing with ALX-0141, a rapid decrease of NTX-1/creatinine concentration ratios was observed with a nadir on Day 4 (0.003 mg/kg, n=1), Day 6 (0.3 mg/kg, n=6), Day 14 (0.01 mg/kg [n=6] and 0.03 mg/kg [n=6]) and Day 60 (0.1 mg/kg [n=6] and 1 mg/kg [n=6]). The mean nadir values for urine NTX-1/creatinine ratios varied between 64.8% (0.003 mg/kg, n=1) and 16.1% (1 mg/kg, n=6) of baseline across the dose range studied and decreased with increasing doses. After treatment with 0.01 mg/kg and 0.03 mg/kg the mean NTX-1/creatinine concentration ratios remained low up to Day 30 and started to increase thereafter and were approximately 70% of baseline on Day 120. After treatment with 0.1 mg/kg, 0.3 mg/kg or 1 mg/kg the NTX-1/creatinine ratios in urine remained low up to Day 60 (0.1 mg/kg) or Day 90 (0.3 mg/kg and 1 mg/kg). Thereafter the NTX-1/creatinine levels started to increase again, but still remained far below baseline on Day 120. Twenty-two subjects had their NTX-1/creatinine ratios still below 80% of baseline at the time of their individual last follow-up. In the placebo-treated group, levels in NTX-1/creatinine ratios remained close to baseline (80-120%) throughout the period studied.

4.3.3 TRACP5b

[0473] TRACP5b is an enzyme derived from osteoclasts and is a measure for osteoclast numbers, and therefore considered as a biomarker for bone resorption. Arithmetic mean serum concentration of TRACP5b (as percentage of baseline) after s.c. dosing with ALX-0141 or placebo in post-menopausal healthy female volunteers are shown in FIG. 8. TRACP5b serum concentrations showed a similar profile as CTX-1 serum levels.

[0474] Upon s.c. dosing with ALX-0141, the TRACP5b serum concentrations started to decrease within 2 to 4 days post-dose. An almost maximum effect was reached after approximately 14 days with a nadir reached on Day 30 (0.003 mg/kg, 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg and 1 mg/kg) or on Day 60 (0.3 mg/kg). The mean nadir values for TRACP5b serum concentrations varied between 70.1% (0.003 mg/kg, n=1) and 39.1% (0.3 mg/kg, n=6) of baseline across the dose levels studied and decreased with increasing doses. Until Day 60 (0.003 mg/kg, 0.01 mg/kg, 0.03 mg/kg and 0.1 mg/kg) and Day 90 (0.3 mg/kg and 1 mg/kg), the TRACP5b serum concentrations hardly changed and were close to levels that were already reached on Day 14. Thereafter, the TRACP5b levels gradually increased and were back to at least 80% of baseline level at the time of last follow-up for all subjects (reason for using 80% is that within the placebo-treated subjects, lowest values hardly reach values below 80%; this indicates that decreases of 20% are within the normal variations over time). The strongest decrease in TRACP5b serum concentrations were reached with 0.3 mg/kg and 1 mg/kg ALX-0141. A decrease in TRACP5b levels was not observed in subjects treated with placebo. Although TRACP5b serum concentrations showed a similar profile compared to CTX-1 serum concentrations, based on comparison of individual TRACP5b and CTX-1 concentrations there was no indication that TRACP5b concentrations influenced directly the CTX-1 serum concentrations.

4.3.4 P1NP

[0475] P1NP is released upon extracellular processing from newly synthesized pre procollagen prior to the incorporation of collagen into the bone and is considered as a biomarker for bone formation. Arithmetic mean serum concentration of P1NP (as percentage of baseline) after s.c. dosing with ALX-0141 or placebo in post-menopausal healthy female volunteers are shown in FIG. 9.

[0476] After s.c. administration of ALX-0141 the P1NP serum levels started to decrease between Day 14 and 30 onwards up to Day 60 across the dose range studied. Nadir values were reached between Day 60 and 150. In general, the higher the treatment dose, the stronger the decrease in P1NP plasma concentrations and the longer the period of decreased P1NP plasma concentrations. However, an almost maximum effect seemed to be reached at a dose level of 0.1 mg/kg ALX-0141. The mean nadir values for P1NP varied between 60.4% (0.01 mg/kg, n=6) and 21.2% (1 mg/kg, based on n=5) of baseline across the dose levels studied. Most subjects had their P1NP serum concentrations not returned to 80% of the baseline at their last follow-up visit (reason for using 80% is that within the placebo-treated subjects, lowest values hardly reach values below 80%; this indicates that decreases of 20% are within the normal variations over time). These data suggest that the effect on P1NP is strongly delayed compared to that on CTX-1 and TRACP5b. No effect on P1NP was observed in the placebo-treated subjects.

4.3.5 BAP

[0477] BAP is an enzyme that serves as a biomarker for active bone formation. Arithmetic mean serum concentration of BAP (as percentage of baseline) after s.c. dosing with ALX-0141 or placebo in post-menopausal healthy female volunteers are shown in FIG. 10.

[0478] There was no clear change from baseline after treatment with 0.003 mg/kg ALX-0141 (n=1) and BAP serum concentrations tended to increase in placebo-treated subjects. After treatment with 0.01 mg/kg to 1 mg/kg ALX-0141, BAP serum concentrations tended to decrease from Day 30 onwards. This decrease in BAP serum levels was less pronounced than that of P1NP serum levels. A nadir was reached on Day 60 after treatment with 0.01 mg/kg and 0.03 mg/kg, and on Day 90 after treatment with 0.1 mg/kg, 0.3 mg/kg and 1 mg/kg. The mean nadir values for BAP varied between 85.3% (0.003 mg/kg, n=1) and 58.3% (1 mg/kg, n=6) of baseline across the dose range studied and decreased with increasing doses. After treatment with 0.01 mg/kg and 0.03 mg/kg ALX-0141, BAP serum concentrations tended to increase from Day 60 onwards, and were close to baseline levels on Day 90. After treatment with 0.1 mg/kg, 0.3 mg/kg and 1 mg/kg, BAP serum concentrations remained low (0.1 mg/kg and 0.3 mg/kg) or were still decreasing (1 mg/kg) on Day 120. The BAP serum concentrations at the last follow-up visits varied among the subjects, with most subjects showing BAP levels returned to at least 80% of baseline. These data suggest that similar to P1NP, the effect on BAP is strongly delayed compared to that of CTX-1 and TRACP5b.

4.3.6 Conclusion

[0479] Single s.c. administrations of ALX-0141 at dose levels ranging from 0.003 mg/kg to 1 mg/kg resulted in a rapid decrease in CTX-1 and TRACP5b serum concentrations as well as in urine NTX-1/creatinine concentration ratios, which suggests a decrease in bone resorption. The inhibitory effect tended to be dose dependent and was long-lasting.

[0480] An (almost) maximal inhibitory effect appeared to be reached at 0.1 mg/kg ALX-0141 for CTX-1 and at 0.3 mg/kg ALX-0141 for TRACP5b serum concentrations and NTX-1/creatinine concentration ratios.

[0481] Single s.c. administrations of ALX-0141 at dose levels of 0.003 mg/kg to 1 mg/kg ALX-014 resulted in a gradual decrease in P1NP serum concentrations, which indicates a trend towards a decrease in bone formation. The inhibitory effect tended to be dose-dependent, with a nadir reached at approximately Day 90 to Day 150. A maximal inhibitory effect appeared to be reached at 0.1 mg/kg for P1NP serum concentrations.

[0482] Similar to the P1NP profile, but to a lesser extent, a trend towards a dose-dependent, gradual decrease in BAP serum concentrations seemed to occur upon the highest dose levels (0.1 mg/kg, 0.3 mg/kg and 1 mg/kg ALX-0141) tested.

[0483] Overall, the lowest dose administered (0.003 mg/kg) appeared to be biologically effective whereas the biological effect of 0.1 mg/kg seemed to be close to that of 0.3 mg/kg and 1 mg/kg. Therefore, the dose level of 0.003 mg/kg can be considered as the BED. In line with the expected effects of anti-resorptive agents, ALX-0141 exhibits a strong inhibitory effect on bone resorption markers and a modest effect in reduction of markers for bone formation.

4.3.7 Pharmacodynamic Analysis

[0484] An indirect PK/PD model of inhibition of the serum CTX-1 production captured the observed serum CTX-1 time profiles adequately.

[0485] In this indirect response model, the rate of change of CTX-1 (Response, R) is described by:

[00002]$\frac{\mathrm{dR}}{\mathrm{dt}}=k_{\mathrm{in}}.Math.\left[1-\frac{I_{\max }.Math.C^{.Math.n}}{{\mathrm{IC}}_{50}^{.Math.n}+C^{.Math.n}}\right]-k_{\mathrm{out}}.Math.R$

[0486] With k.sub.in, the zero-order synthesis rate; R, the serum CTX-1 level, I.sub.max, the maximum inhibition (1<I.sub.max<0); C, the concentration of ALX-0141; n, the concentration-response shape factor; and k.sub.out, the first order elimination rate constant of serum CTX-1.

[0487] Overall all the structural parameters of the final PK/PD model were estimated with a precision of <88% (see Table B-9). The interindividual variability (IIV) was low to moderate for I.sub.max (6.4%), for baseline (22.1%) and for k.sub.1 (46.4%). The residual variability was estimated to 32.1% with a good precision (12.1%).

[0488] ALX-0141 was determined to be a potent inhibitor of serum CTX-1 production, as evidence by its low IC.sub.50-value of 0.674 ng/ml or 16.4 pM.

[0489] The dose level was identified as a determining covariate on the zero order production rate of the response (serum CTX-1).

[0490] The predictive performance of the models was confirmed by Visual Predictive Checks.

4.4 Discussion

[0491] The clinical pharmacokinetics of ALX-0141 were fairly well predicted based on preclinical data from monkeys, allometrically scaled, and combined with reasonable assumptions regarding differences in bone remodelling (see FIG. 11).

[0492] Unexpectedly, the observed serum CTX-1-time profiles in post-menopausal women turned out to be substantially more sustained in humans than what was predicted based on preclinical information (see FIG. 12).

[0493] This discrepancy between the observed and the model-based simulations of serum CTX-1-time profiles was attributed to an estimated 5.4-fold greater potency of ALX-0141 in vivo (IC.sub.50 of 0.674 ng/ml or 16.4 pM) (see Table B-9) relative to that anticipated based on preclinical data (3.7 ng/ml or 89 pM) (see Table B-3).

4.5 Conclusion

[0494] Single s.c. administration of ALX-0141 up to 1 mg/kg was safe and well tolerated in healthy post-menopausal women. No deaths or treatment-related serious adverse events (SAEs) occurred and maximum tolerated dose (MTD) has not been reached.

[0495] A dose-dependent increase in exposure (C.sub.max and AUC) was observed. The PK model is non-linear, due to saturable target-dependent CL component. Serum half-life between 12.0 and 20.6 days was observed for 0.1-1 mg/kg cohorts.

[0496] ALX-0141 exhibits statistically significant suppression of serum CTX-1 and urine NTX-1/creatinine biomarkers for bone resorption. This effect is unexpectedly prolonged and sustained as compared to what was predicted based on preclinical information.

[0497] Duration of biomarker inhibition following single s.c. ALX-0141 injections increased with dose, reaching maximum duration of 360 days after 1 mg/kg dosing.

[0498] The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention.

[0499] All references disclosed herein are incorporated by reference, in particular for the teaching that is referenced hereinabove, including specific polypeptides and properties of polypeptides that are useful in the methods described herein.

Tables

[0500]

TABLE-US-00008 TABLE B-1 Pharmacokinetic parameters of ALX- 0141 in the cynomolgus monkey. Parameter Estimate CV % V.sub.c (mL/kg) 68.4 10. V.sub.t (mL/kg) 73.4 15. V.sub.dss (mL/kg) 142 CL.sub.NON-RANKL (mL/day/kg) 8.57 5.7 CL.sub.d (mL/day/kg) 36.2 33. V.sub.max (μg/day/kg) 7.096 27 K.sub.m (μg/mL) 0.149 49. CL.sub.RANKL (mL/day/kg) 47.7 ka (day.sup.−1) 0.748 13. Bioavailability (F) 0.965 6.8

TABLE-US-00009 TABLE B-2 Allometrically scaled pharmacokinetic parameters of ALX-0141 in humans. Parameter Units Value V.sub.c (mL/kg 68 V.sub.t (mL/kg 73 V.sub.dss (mL/kg 72 CL.sub.NON-RANKL (mL/d* 3.22 CL.sub.d (mL/d* 14 V.sub.max (μg/d* 2.34 K.sub.m (μg/mL 0.149 CL.sub.RANKL (mL/d* 15.7 ka (1/d) 0.331 Bioavailability (F) — 0.80-1.0

TABLE-US-00010 TABLE B-3 Pharmacodynamic parameters of ALX-0141 in the PK/PD simulations of serum CTX-1 concentrations. Parameter Units Value k.sub.in (nM/d) 0.732 k.sub.out (1/d) 16.6 I.sub.max (%) 0.729 IC.sub.50 (ng/mL) 3.7 (nM) 0.089 n — 0.88

TABLE-US-00011 TABLE B-4 ALX-0141 plasma PK parameters Dose C.sub.max t.sub.max AUC.sub.0-last AUC.sub.0-inf t.sub.1/2 (mg/kg) n (μg/mL) (days) (μg .Math. d/mL) (μg .Math. d/mL) (d) 0.003 1 0.0146.sup.a 4.00.sup.a 0.0612.sup.a —.sup.b —.sup.b 0.01 6 0.0327 6.00 0.321 —.sup.b —.sup.b 0.03 6 0.109 3.00 1.80 1.54.sup.a 8.86.sup.a 0.1 6 0.538 2.00 14.7 16.0.sup.c 12.0.sup.c 0.3 6 2.24 1.50 43.3 44.3 12.4 1 6 5.76 2.00 193 200 20.6 .sup.asingle observation .sup.blimited data, therefore no descriptive statistics are available .sup.cn = 2

TABLE-US-00012 TABLE B-5 PK parameter estimates in healthy post-menopausal women Estimate Precision Parameter [95% CI] (% CV).sup.(a) Absorption k.sub.a1 (1/d) 1.43 [0.722-2.14] 25.2 k.sub.a2 (1/d) 0.648 [0.411-0.885] 18.7 F1 0.268 [0.194-0.342] 14.1 Distribution V/F (L) 10.9 [9.83-12.0] 5.0 WT/V 1.08 [0.819-1.34] 12.3 Elimination CL/F (L/d) 0.326 [0.258-0.394] 10.6 V.sub.max/F (mg/d) 0.0422 [0.0355-0.0489] 8.13 K.sub.m (ng/mL) 33.1 [11.3-54.9] 11.1 Inter-Individual Estimate Precision Variability (IIV) (% CV).sup.(b) (% CV).sup.(a) IIV in k.sub.a1 169.7 37.8 IIV in k.sub.a2 144 19.3 IIV in F1 0.364.sup.(c) 78.2 IIV in V/F 22.2 26.1 IIV in CL/F 53.2 37.8 IIV in V.sub.max/F — — IIV in K.sub.m 135 57.5 Residual variability Estimate Precision in PK (SD) (% CV).sup.(a) 0.11 13.7 .sup.(a)Precision was calculated as the s.e. divided by the parameter estimate × 100. .sup.(b)The % CV for both inter-subject and residual variability is an approximation taken as the square root of the variance × 100. .sup.(c)An additive random effect model was used for F1, therefore the estimated SD is reported.

TABLE-US-00013 TABLE B-6 Summary of PD parameters after single s.c. treatment with ALX-0141 in post-menopausal female subjects Nadir*.sup.,$ Nadir PD as percent of as absolute parameter Treatment N Time of nadir.sup.$ baseline (SD) values.sup.#,$ (SD) CTX-1 Placebo 11 12 h post-dose Day 1 66.7 (12.7) 0.423 (0.094) 0.003 mg/kg 1 Day 7 50.7 (—) 0.296 (—) 0.01 mg/kg 5 Day 14 23.7 (14.2) 0.137 (0.073).sup.†& 0.03 mg/kg 6 Day 14 16.3 (9.5) 0.085 (0.043) 0.1 mg/kg 6 Day 14 and 30 8.8 (3.6) 0.047 (0.000).sup.†& 0.3 mg/kg 6 12 h post-dose on Day 7.8 (3.1) 0.047 (0.000) 1 and Day 5 to Day 60 1 mg/kg 6 Day 6 to Day 60 11.7 (4.7) 0.047 (0.000).sup.†‡ TRACP5b Placebo 11 NA NA NA 0.003 mg/kg 1 Day 30 70.1 (—)  2.59 0.01 mg/kg 6 Day 30 56.0 (10.8) 2.10 (0.32) 0.03 mg/kg 6 Day 30 65.5 (10.9) 1.95 (0.38) 0.1 mg/kg 6 Day 30 50.6 (11.7) 1.62 (0.30) 0.3 mg/kg 6 Day 60 39.1 (6.4) 1.68 (0.30) 1 mg/kg 6 Day 30 40.4 (6.5) 1.52 (0.32) P1NP Placebo 11 NA NA NA 0.003 mg/kg 1 Day 60 59.3 (—) 36.6 0.01 mg/kg 6 Day 60 60.4 (9.1) 36.0 (7.6) 0.03 mg/kg 6 Day 90 44.9 (5.7) 20.8 (4.9) 0.1 mg/kg 6 Day 90 25.7 (5.5) 17.1 (6.8) 0.3 mg/kg 5 Day 120 23.8 (5.3) 17.2 (9.3) 1 mg/kg 5 Day 150 21.2 (3.9) 12.1 (2.9) BAP Placebo 11 NA NA NA 0.003 mg/kg 1 NA NA NA 0.01 mg/kg 6 Day 60 77.7 (12.1) 12.2 (1.3) 0.03 mg/kg 6 Day 60 67.6 (11.3) 12.6 (2.7) 0.1 mg/kg 6 Day 90 71.1 (9.6) 13.8 (0.8) 0.3 mg/kg 6 Day 90 60.0 (11.3) 13.8 (4.1) 1 mg/kg 6 Day 90 58.3 (9.2) 10.5 (2.5) NTX-1 Placebo 11 NA NA NA 0.003 mg/kg 1 Day 4 64.8 (—) 31.3 0.01 mg/kg 6 Day 14 37.5 (10.9) 24.7 (7.5) 0.03 mg/kg 6 Day 14 27.9 (13.5) 12.4 (6.3) 0.1 mg/kg 6 Day 60 19.4 (8.1) 12.9 (4.4) 0.3 mg/kg 6 Day 6 18.2 (4.0) 12.8 (3.6) 1 mg/kg 6 Day 60 16.1 (7.4) 9.8 (4.8) For all data arithmetic mean (SD) are presented, except for the 0.003 mg/kg Cohort, since N = 1 for this cohort. NA: not applicable .sup.$Only given when nadir is below predefined threshold value: 70% of baseline for CTX-1, 80% of baseline for other PD parameters *The lowest mean value compared to baseline. The results of the placebo-treated subjects of all treatment groups were pooled. Except for CTX-1 serum concentrations and the 0.003 mg/kg dose group, this table only presents mean nadir levels based on N ≧ 5. .sup.#CTX-1 levels: ng/mL; TRACP5b: mU/mL; P1NP and BAP: μg/L; NTX-1/creatinine ratios: μmol/mol

TABLE-US-00014 TABLE B-7 CTX-1 change from baseline (%) 0.1 mg/kg 1.0 mg/kg Timepoint Placebo ALX-0141 ALX-0141 (nominal) (n = 11) (n = 6) (n = 6) 1 month −11.6 (4.4) −84.8 (NA.sup.3)*** BLQ*** 4 months 2.5 (6.7) −60.9 (9.1)*** −76.7 (1.1)*** 5 months.sup.1 2.5 (4.7) −45.2 (9.2)** −67.3 (2.8)*** 7 months.sup.2 2.5 (3.8) −31.6 (8.3)** −40.4 (7.4)*** 9 months 2.5 (6.7) −38.0 (7.3)* −47.0 (7.0)**  Values: mean (SE), *p < 0.05, **p < 0.01, ***p < 0.0001; Placebo values: Last Value Carried Forward .sup.1150 d + 180 d, .sup.2210 d + 270 d + 300 d, .sup.3single observation, .sup.4days 300-420

TABLE-US-00015 TABLE B-8 NTX-1/creatine change from baseline (%) 0.1 mg/kg 1 mg/kg Timepoint Placebo ALX-0141 ALX-0141 (nominal) (n = 11) (n = 6) (n = 6) 1 month −5.0 (3.2) −52.9 (10.6)** −57.7 (13.6)*** 3 months −2.3 (4.5) −39.0 (4.7)** −58.9 (12.8)*** 6 months −2.8 (4.8) −25.1 (12.8) −55.5 (17.8)** 9 months −2.8 (4.8) −24.5 (4.4) −28.6 (11.9) ≧10 months.sup.4 −2.8 (2.1) −12.6 (6.1) −33.2 (9.3)** Values: mean (SE), *p < 0.05, **p < 0.01, ***p < 0.0001; Placebo values: Last Value Carried Forward .sup.1 150 d + 180 d, .sup.2 210 d + 270 d + 300 d, .sup.3 single observation, .sup.4days 300-420

TABLE-US-00016 TABLE B-9 PD parameter estimates in healthy post-menopausal women. Estimate Precision Parameter [95% CI] (% CV).sup.(a) I.sub.max 0.743 [0.695-0.791] 3.3 IC.sub.50 (ng/ml) 0.674 [−0.494-1.842] 88.4 Baseline (ng/ml) 0.492 [0.436-0.548] 5.8 k.sub.in (1/d) 3.86 [0.254-7.466] 47.7 N 1.sup.(c) — Lkin.sup.(d) 0.0434 [0.000-0.087] 51.2 Interindividual Estimate Precision Variability (IIV) (% CV).sup.(b) (% CV).sup.(a) IIV in I.sub.max  6.4 128.9 IIV in IC.sub.50 — — IIV in Base 22.1 39.4 IIV in k.sub.in 46.4 76.3 Residual 32.1 12.1 variability in PD .sup.(a)precision was calculated as the s.e. divided by the parameter estimate × 100. .sup.(b)the % CV for both inter-subject and residual variability is an approximation taken as the square root of the variance × 100. .sup.(c)fixed to unity. .sup.(d)typical fractional dose effect on k.sub.in at the lower doses (0.003 and 0.01 mg/kg).

Aspects of the Invention

[0501] Aspect A-1: A method for inhibiting bone resorption or osteoclast activity in a subject comprising administering to the subject a polypeptide that specifically binds Receptor Activator of Nuclear Factor kappa B Ligand (RANKL), wherein the amount of the polypeptide administered is effective to change one or more markers of bone metabolism and/or bone homeostasis for at least 30 days after administration. [0502] Aspect A-2: The method according to Aspect A-1, wherein the polypeptide is administered in an amount from about 0.01 mg/kg to about 10 mg/kg. [0503] Aspect A-3: The method according to any of Aspects A-1 and A-2, wherein the markers of bone metabolism are selected from cross-linking telopeptide of type I collagen (CTX-1), N-terminal telopeptide of type I collagen (NTX-1), tartrate-resistant acid phosphatase isoform 5b (TRACP5b), N-terminal propeptide of type I procollagen (P1NP) and bone-specific alkaline phosphatase (BAP). [0504] Aspect A-4: The method according to Aspect A-3, wherein one or more of the CTX-1, NTX-1, TRACP5b, P1NP and BAP is/are measured using an ELISA assay specific for CTX-1, NTX-1, or TRACP5b; a radioimmunoassay specific for P1NP, or an immunoenzymetric assay specific for BAP, respectively. [0505] Aspect A-5: The method according to any of Aspects A-1 to A-4, wherein the polypeptide comprises one or more immunoglobulin single variable domains that specifically bind RANKL. [0506] Aspect A-6: The method according to any of Aspects A-1 to A-5, wherein the polypeptide has an apparent K.sub.D for binding to recombinant soluble RANKL (sRANKL) of 0.01-0.05 nM, preferably about 0.04 nM, as determined by Biacore. [0507] Aspect A-7: The method according to any of Aspects A-1 to A-6, wherein the immunoglobulin single variable domain(s) comprise(s) or consist(s) of one or more VHH domains, one or more humanized VHH domains and/or one or more camelized VH domains. [0508] Aspect A-8: The method according to any of Aspects A-1 to A-7, wherein the polypeptide comprises one or more domain antibodies that specifically bind RANKL, one or more amino acid sequences that specifically bind RANKL that are suitable for use as a domain antibody, one or more single domain antibodies that specifically bind RANKL, one or more amino acid sequences that specifically bind RANKL that are suitable for use as a single domain antibody, or one or more “dAb”s that specifically bind RANKL. [0509] Aspect A-9: The method according to any of Aspects A-1 to A-8, wherein the polypeptide is a multivalent construct comprising two or more immunoglobulin single variable domains that specifically bind RANKL. [0510] Aspect A-10: The method according to Aspect A-9, wherein the multivalent construct comprises two immunoglobulin single variable domains that specifically bind RANKL. [0511] Aspect A-11: The method according to any of Aspects A-5 to A-10, wherein the immunoglobulin single variable domain essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which CDR1 is chosen from SEQ ID NO: 1, CDR2 is chosen from SEQ ID NO: 2, and CDR3 is chosen from SEQ ID NO: 3. [0512] Aspect A-12: The method according to any of Aspects A-5 to A-11, wherein the multivalent construct comprises or consists of SEQ ID NO: 10 or SEQ ID NO: 12. [0513] Aspect A-13: The method according to any of Aspects A-5 to A-12, wherein the multivalent construct further comprises at least one half-life extension moiety. [0514] Aspect A-14: The method according to Aspect A-13, wherein the at least one half-life extension moiety specifically binds a serum protein. [0515] Aspect A-15: The method according to Aspect A-14, wherein the serum protein is serum albumin, and in particular human serum albumin, thyroxine-binding protein, (human) transferrin, fibrinogen, an immunoglobulin such as IgG, IgE or IgM, or one of the serum proteins listed in WO 04/003019. [0516] Aspect A-16: The method according to any of Aspects A-13 to A-15, wherein at least one half-life extension moiety comprises or consists of an immunoglobulin single variable domain. [0517] Aspect A-17: The method according to Aspect A-16, wherein the immunoglobulin single variable domain comprises or consists of a VHH domain, a humanized VHH domain or a camelized VH domain. [0518] Aspect A-18: The method according to any of Aspects A-16 or A-17, wherein the immunoglobulin single variable domain comprises or consists of SEQ ID NO: 14. [0519] Aspect A-19: The method according to any of Aspects A-13 to A-16, wherein the at least one half-life extension moiety comprises a domain antibody, an amino acid sequence that is suitable for use as a domain antibody, a single domain antibody, an amino acid sequence that is suitable for use as a single domain antibody, or a “dAb”. [0520] Aspect A-20: The method according to Aspect A-13, wherein the at least one half-life extension moiety comprises one or more polyethylene glycol molecules. [0521] Aspect A-21: The method according to any of Aspects A-5 to A-11, wherein the polypeptide cross-blocks the binding of SEQ ID NO: 10 or SEQ ID NO: 12 to RANKL. [0522] Aspect A-22: The method according to any of Aspects A-5 to A-11, wherein the polypeptide is cross-blocked from binding RANKL by SEQ ID NO: 10 or SEQ ID NO: 12. [0523] Aspect A-23: The method according to any of Aspects A-1 to A-22, wherein the polypeptide is administered as a single dose. [0524] Aspect A-24: The method according to any of Aspects A-1 to A-23, wherein the polypeptide is administered subcutaneously. [0525] Aspect A-25: The method according to any of Aspects A-1 to A-24, wherein the subject has osteoporosis. [0526] Aspect B-1: The method according to any of Aspects A-1 to A-25, wherein the marker of bone metabolism is cross-linking telopeptide of type I collagen (CTX-1). [0527] Aspect B-2: The method according to Aspect B-1, wherein the amount of the polypeptide administered is effective to reduce serum levels of CTX-1 by at least 30%, compared to pre-treatment or normal levels, for at least about 30, 60, 90, 120, 150, 180, 210, or 270 days after administration. [0528] Aspect B-3: The method according to Aspect B-2, wherein the polypeptide is administered in an amount from about 0.003 mg/kg to about 0.03 mg/kg. [0529] Aspect B-4: The method according to Aspects B-3, wherein the serum levels of CTX-1 are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 30 days after administration. [0530] Aspect B-5: The method according to Aspect B-2, wherein the polypeptide is administered in an amount from about 0.01 mg/kg to about 0.1 mg/kg. [0531] Aspect B-6: The method according to Aspects B-5, wherein the serum levels of CTX-1 are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 60 days after administration. [0532] Aspect B-7: The method according to Aspect B-6, wherein the serum levels of CTX-1 are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 90 days after administration. [0533] Aspect B-8: The method according to Aspect B-7, wherein the serum levels of CTX-1 are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 120 days after administration. [0534] Aspect B-9: The method according to Aspect B-2, wherein the polypeptide is administered in an amount from about 0.1 mg/kg to about 1 mg/kg. [0535] Aspect B-10: The method according to Aspect B-9, wherein the serum levels of CTX-1 are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 150 days after administration. [0536] Aspect B-11: The method according to Aspect B-10, wherein the serum levels of CTX-1 are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 180 days after administration. [0537] Aspect B-12: The method according to Aspect B-11, wherein the serum levels of CTX-1 are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 210 days after administration. [0538] Aspect B-13: The method according to Aspect B-12, wherein the serum levels of CTX-1 are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 270 days after administration. [0539] Aspect B-14: The method according to Aspect B-1, wherein the amount of the polypeptide administered is effective to reduce serum level of cross-linking telopeptide of type I collagen (CTX-1) by at least 45%, compared to pre-treatment or normal levels, for at least about 30, 60, 90, 120, 150, or 180 days, and/or 30 days to 3 months, 3 months to 6 months, or 6 months to 1 year after administration. [0540] Aspect B-15: The method according to Aspect B-14, wherein the polypeptide is administered in an amount of less than or equal to 0.03 mg/kg. [0541] Aspect B-16: The method according to Aspect B-14, wherein the polypeptide is administered in an amount from about 0.01 mg/kg to about 3 mg/kg. [0542] Aspect B-17: The method according to Aspect B-16, wherein the polypeptide is administered in an amount from about 0.01 mg/kg to about 0.1 mg/kg. [0543] Aspect B-18: The method according to any of Aspects B-15 to B-17, wherein the serum levels of CTX-1 are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 30 days after administration. [0544] Aspect B-19: The method according to any of Aspects B-16 and B-17, wherein the serum levels of CTX-1 are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 60 days after administration. [0545] Aspect B-20: The method according to Aspect B-14, wherein the polypeptide is administered in an amount from about 0.03 mg/kg to about 0.3 mg/kg. [0546] Aspect B-21: The method according to Aspect B-14, wherein the polypeptide is administered in an amount from about 0.01 mg/kg to about 0.3 mg/kg. [0547] Aspect B-22: The method according to any of Aspects B-20 and B-21, wherein the serum levels of CTX-1 are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 90 days after administration. [0548] Aspect B-23: The method according to any of Aspects B-20 and B-21, wherein the CTX-1 is reduced by at least 45%, compared to pre-treatment or normal levels, for about 30 days to 3 months after administration. [0549] Aspect B-24: The method according to Aspect B-14, wherein the polypeptide is administered in an amount from about 0.1 mg/kg to about 1 mg/kg. [0550] Aspect B-25: The method according to Aspect B-24, wherein the serum levels of CTX-1 are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 120 days after administration. [0551] Aspect B-26: The method according to Aspect B-14, wherein the polypeptide is administered in an amount from about 0.3 mg/kg to about 1 mg/kg. [0552] Aspect B-27: The method according to Aspect B-14, wherein the polypeptide is administered in an amount of less than or equal to 0.3 mg/kg. [0553] Aspect B-28: The method according to any of Aspects B-26 and B-27, wherein the CTX-1 is reduced by at least 45%, compared to pre-treatment or normal levels, for about 3 months to 6 months after administration. [0554] Aspect B-29: The method according to Aspect B-14, wherein the polypeptide is administered in an amount from about 1 mg/kg to about 10 mg/kg. [0555] Aspect B-30: The method according to Aspect B-14, wherein the polypeptide is administered in an amount from about 1 mg/kg to about 3 mg/kg. [0556] Aspect B-31: The method according to Aspect B-14, wherein the polypeptide is administered in an amount of less than or equal to 1 mg/kg. [0557] Aspect B-32: The method according to any of Aspects B-29, B-30 and B-31, wherein the serum levels of CTX-1 are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 150 days after administration. [0558] Aspect B-33: The method according to Aspect B-32, wherein the serum levels of CTX-1 are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 180 days (or 6 months) after administration. [0559] Aspect B-34: The method according to any of Aspects B-29, B-30 and B-31, wherein the CTX-1 is reduced by at least 45%, compared to pre-treatment or normal levels, for at least 6 months to 1 year after administration. [0560] Aspect B-35: The method according to Aspect B-1, wherein the amount of the polypeptide administered is effective to reduce serum levels of CTX-1 by at least 70%, compared to pre-treatment or normal levels, for at least about 30, 60, 90, or 120 days after administration. [0561] Aspect B-36: The method according to Aspect B-35, wherein the polypeptide is administered in an amount from about 0.03 mg/kg to about 0.3 mg/kg. [0562] Aspect B-37: The method according to Aspect B-36, wherein the serum levels of CTX-1 are reduced by at least 70%, compared to pre-treatment or normal levels, for at least about 30 days after administration. [0563] Aspect B-38: The method according to Aspect B-35, wherein the polypeptide is administered in an amount from about 0.1 mg/kg to about 1 mg/kg. [0564] Aspect B-39: The method according to Aspect B-38, wherein the serum levels of CTX-1 are reduced by at least 70%, compared to pre-treatment or normal levels, for at least about 60 days after administration. [0565] Aspect B-40: The method according to Aspect B-39, wherein the serum levels of CTX-1 are reduced by at least 70%, compared to pre-treatment or normal levels, for at least about 90 days after administration. [0566] Aspect B-41: The method according to Aspect B-40, wherein the serum levels of CTX-1 are reduced by at least 70%, compared to pre-treatment or normal levels, for at least about 120 days after administration. [0567] Aspect B-42: The method according to Aspect B-1, wherein the amount of the polypeptide administered is effective to reduce serum levels of CTX-1 by at least 80%, compared to pre-treatment or normal levels, for at least about 30, 60, or 90 days after administration. [0568] Aspect B-43: The method according to Aspect B-42, wherein the polypeptide is administered in an amount from about 0.1 mg/kg to about 1 mg/kg. [0569] Aspect B-44: The method according to Aspect B-42, wherein the polypeptide is administered in an amount from about 0.3 mg/kg to about 1 mg/kg. [0570] Aspect B-45: The method according to any of Aspects B-43 and B-44, wherein the serum levels of CTX-1 are reduced by at least 80%, compared to pre-treatment or normal levels, for at least about 60 days after administration. [0571] Aspect B-46: The method according to Aspect B-45 wherein the serum levels of CTX-1 are reduced by at least 80%, compared to pre-treatment or normal levels, for at least about 90 days after administration. [0572] Aspect B-47: The method according to any of Aspects B-1 to B-46, wherein the amount of the polypeptide administered is effective to reduce serum levels of cross-linking telopeptide of type I collagen (CTX-1) by at least 45% by 8 hours after administration. [0573] Aspect B-48: The method according to any of Aspects B-1 to B-47, wherein bone resorption is inhibited in the subject as determined by ELISA assay for CTX-1. [0574] Aspect B-49: The method according to any of Aspects B-1 to B-48, wherein the CTX-1 is reduced by at least 50%, compared to pre-treatment or normal levels, for at least 30 days after administration. [0575] Aspect B-50: The method according to any of Aspects B-1 to B-49, wherein the CTX-1 is reduced by at least 50%, compared to pre-treatment or normal levels, for at least 60 days after administration. [0576] Aspect B-51: The method according to any of Aspects B-1 to B-50, wherein the CTX-1 is reduced by at least 50%, compared to pre-treatment or normal levels, for at least 90 days after administration. [0577] Aspect B-52: The method according to any of Aspects B-1 to B-51, wherein the CTX-1 is reduced by at least 50%, compared to pre-treatment or normal levels, for at least 120 days after administration. [0578] Aspect B-53: The method according to any of Aspects B-1 to B-52, wherein the CTX-1 is reduced by at least 50%, compared to pre-treatment or normal levels, for at least 150 days after administration. [0579] Aspect B-54: The method according to any of Aspects B-1 to B-53, wherein the CTX-1 is reduced by at least 50%, compared to pre-treatment or normal levels, for at least 180 days after administration. [0580] Aspect B-55: The method according to any of Aspects B-1 to B-54, wherein the CTX-1 is reduced by at least 60%, compared to pre-treatment or normal levels, for at least 30 days after administration. [0581] Aspect B-56: The method according to any of Aspects B-1 to B-55, wherein the CTX-1 is reduced by at least 60%, compared to pre-treatment or normal levels, for at least 60 days after administration. [0582] Aspect B-57: The method according to any of Aspects B-1 to B-56, wherein the CTX-1 is reduced by at least 60%, compared to pre-treatment or normal levels, for at least 90 days after administration. [0583] Aspect B-58: The method according to any of Aspects B-1 to B-57, wherein the CTX-1 is reduced by at least 60%, compared to pre-treatment or normal levels, for at least 120 days after administration. [0584] Aspect B-59: The method according to any of Aspects B-1 to B-58, wherein the CTX-1 is reduced by at least 60%, compared to pre-treatment or normal levels, for at least 150 days after administration. [0585] Aspect B-60: The method according to any of Aspects B-1 to B-59, wherein the CTX-1 is reduced by at least 70%, compared to pre-treatment or normal levels, for at least 30 days after administration. [0586] Aspect B-61: The method according to any of Aspects B-1 to B-60, wherein the CTX-1 is reduced by at least 70%, compared to pre-treatment or normal levels, for at least 60 days after administration. [0587] Aspect B-62: The method according to any of Aspects B-1 to B-61, wherein the CTX-1 is reduced by at least 70%, compared to pre-treatment or normal levels, for at least 90 days after administration. [0588] Aspect B-63: The method according to any of Aspects B-1 to B-62, wherein the CTX-1 is reduced by at least 70%, compared to pre-treatment or normal levels, for at least 120 days after administration. [0589] Aspect B-64: The method according to any of Aspects B-1 to B-63, wherein the CTX-1 is reduced by at least 80%, compared to pre-treatment or normal levels, for at least 30 days after administration. [0590] Aspect B-65: The method according to any of Aspects B-1 to B-64, wherein the CTX-1 is reduced by at least 80%, compared to pre-treatment or normal levels, for at least 60 days after administration. [0591] Aspect B-66: The method according to any of Aspects B-1 to B-65, wherein the CTX-1 is reduced by at least 80%, compared to pre-treatment or normal levels, for at least 90 days after administration. [0592] Aspect C-1: The method of any of Aspects A-1 to A-25, wherein the marker of bone metabolism is N-terminal telopeptide of type I collagen (NTX-1). [0593] Aspect C-2: The method according to Aspect C-1, wherein the amount of the polypeptide administered is effective to reduce NTX-1 by at least 30%, determined as ratio of NTX-1 to creatinine in urine, compared to pre-treatment or normal levels, for at least 30, 60, 90, 120, or 180 days, and/or at least 10 months or at least 12 months after administration. [0594] Aspect C-3: The method according to Aspect C-2, wherein the polypeptide is administered in an amount from about 0.01 mg/kg to about 0.1 mg/kg. [0595] Aspect C-4: The method according to Aspect C-3, wherein the levels of NTX-1 are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 30 days after administration. [0596] Aspect C-5: The method according to Aspect C-4, wherein the levels of NTX-1 are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 60 days after administration. [0597] Aspect C-6: The method according to Aspect C-2, wherein the polypeptide is administered in an amount from about 0.03 mg/kg to about 0.3 mg/kg. [0598] Aspect C-7: The method according to Aspect C-6, wherein the levels of NTX-1 are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 90 days after administration. [0599] Aspect C-8: The method according to Aspect C-2, wherein the polypeptide is administered in an amount from about 0.1 mg/kg to about 1 mg/kg. [0600] Aspect C-9: The method according to Aspect C-8, wherein the levels of NTX-1 are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 120 days after administration. [0601] Aspect C-10: The method according to Aspect C-2, wherein the polypeptide is administered in an amount from about 1 mg/kg to about 10 mg/kg. [0602] Aspect C-11: The method according to Aspect C-10, wherein the levels of NTX-1 are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 180 days (or 6 months) after administration. [0603] Aspect C-12: The method according to Aspect C-10, wherein the levels of NTX-1 are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 10 months after administration. [0604] Aspect C-13: The method according to Aspect C-10, wherein the levels of NTX-1 are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 1 year (12 months; 360 days) after administration. [0605] Aspect C-14: The method according to Aspect C-1, wherein the amount of the polypeptide administered is effective to reduce NTX-1 by at least 45%, determined as ratio of NTX-1 to creatinine in urine, compared to pre-treatment or normal levels, for at least 30, 60, 90, 120, 180, or 210 days and/or 30 days to 3 months, 3 months to 6 months, or 6 months to 1 year after administration. [0606] Aspect C-15: The method according to Aspect C-14, wherein the polypeptide is administered in an amount from about 0.01 mg/kg to about 3 mg/kg. [0607] Aspect C-16: The method according to Aspect C-15, wherein the polypeptide is administered in an amount from about 0.01 mg/kg to about 0.1 mg/kg. [0608] Aspect C-17: The method according to any of Aspects C-15 and C-16, wherein the levels of NTX-1 are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 60 days after administration. [0609] Aspect C-18: The method according to Aspect C-14, wherein the polypeptide is administered in an amount from about 0.03 mg/kg to about 0.3 mg/kg. [0610] Aspect C-19: The method according to Aspect C-18, wherein the levels of NTX-1 are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 60 days after administration. [0611] Aspect C-20: The method according to Aspect C-14, wherein the polypeptide is administered in an amount from about 0.01 mg/kg to about 3 mg/kg. [0612] Aspect C-21: The method according to Aspect C-14, wherein the polypeptide is administered in an amount from about 0.01 mg/kg to about 0.3 mg/kg. [0613] Aspect C-22: The method according to Aspect C-14, wherein the polypeptide is administered in an amount from about 0.03 mg/kg to about 0.3 mg/kg. [0614] Aspect C-23: The method according to Aspect C-14, wherein the polypeptide is administered in an amount of less than or equal to 0.03 mg/kg. [0615] Aspect C-24: The method according to any of Aspects C-20, C-21, C-22 and C-23, wherein the levels of NTX-1 are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 30 days to 3 months after administration. [0616] Aspect C-25: The method according to Aspect C-14, wherein the polypeptide is administered in an amount from about 0.1 mg/kg to about 1 mg/kg. [0617] Aspect C-26: The method according to Aspect C-25, wherein the levels of NTX-1 are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 90 days after administration. [0618] Aspect C-27: The method according to Aspect C-26, wherein the levels of NTX-1 are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 120 days after administration. [0619] Aspect C-28: The method according to Aspect C-27, wherein the levels of NTX-1 are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 150 days after administration. [0620] Aspect C-29: The method according to Aspect C-14, wherein the polypeptide is administered in an amount from about 0.01 mg/kg to about 3 mg/kg. [0621] Aspect C-30: The method according to Aspect C-14, wherein the polypeptide is administered in an amount from about 0.3 mg/kg to about 1 mg/kg. [0622] Aspect C-31: The method according to Aspect C-14, wherein the polypeptide is administered in an amount of less than or equal to 0.3 mg/kg. [0623] Aspect C-32: The method of any of Aspects C-29, C-30 and C-31, wherein the NTX-1 is reduced by at least 45%, compared to pre-treatment or normal levels, for about 3 months to 6 months after administration. [0624] Aspect C-33: The method according to Aspect C-14, wherein the polypeptide is administered in an amount from about 1 mg/kg to about 10 mg/kg. [0625] Aspect C-34: The method according to Aspect C-14, wherein the polypeptide is administered in an amount from about 1 mg/kg to about 3 mg/kg. [0626] Aspect C-35: The method according to Aspect C-14, wherein the polypeptide is administered in an amount of less than or equal to 1 mg/kg. [0627] Aspect C-36: The method according to any of Aspects C-33, C-34 and C-35, wherein the levels of NTX-1 are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 180 days (or 6 months) after administration. [0628] Aspect C-37: The method according to any of Aspects C-33, C-34 and C-35, wherein the levels of NTX-1 are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 210 days after administration. [0629] Aspect C-38: The method according to any of Aspects C-33, C-34 and C-35, wherein the levels of NTX-1 are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 6 months to 1 year after administration. [0630] Aspect C-39: The method according to Aspect C-1, wherein the amount of the polypeptide administered is effective to reduce levels of NTX-1 by at least 70%, compared to pre-treatment or normal levels, for at least about 30, 60, 90, or 120 days after administration. [0631] Aspect C-40: The method according to Aspect C-39, wherein the polypeptide is administered in an amount from about 0.1 mg/kg to about 1 mg/kg. [0632] Aspect C-41: The method according to Aspect C-40, wherein the levels of NTX-1 are reduced by at least 70%, compared to pre-treatment or normal levels, for at least about 30 days after administration. [0633] Aspect C-42: The method according to Aspect C-39, wherein the polypeptide is administered in an amount from about 0.3 mg/kg to about 3 mg/kg. [0634] Aspect C-43: The method according to Aspect C-42, wherein the levels of NTX-1 are reduced by at least 70%, compared to pre-treatment or normal levels, for at least about 60 days after administration. [0635] Aspect C-44: The method according to Aspect C-43, wherein the levels of NTX-1 are reduced by at least 70%, compared to pre-treatment or normal levels, for at least about 90 days after administration. [0636] Aspect C-45: The method according to Aspect C-39, wherein the polypeptide is administered in an amount from about 1 mg/kg to about 10 mg/kg. [0637] Aspect C-46: The method according to Aspect C-45, wherein the levels of NTX-1 are reduced by at least 70%, compared to pre-treatment or normal levels, for at least about 120 days after administration. [0638] Aspect C-47: The method according to Aspect C-1, wherein the amount of the polypeptide administered is effective to reduce levels of NTX-1 by at least 80%, compared to pre-treatment or normal levels, for at least about 30, 60, or 90 days after administration. [0639] Aspect C-48: The method according to Aspect C-47, wherein the polypeptide is administered in an amount from about 0.3 mg/kg to about 3 mg/kg. [0640] Aspect C-49: The method according to Aspect C-47, wherein the polypeptide is administered in an amount from about 0.3 mg/kg to about 1 mg/kg. [0641] Aspect C-50: The method according to Aspect C-47, wherein the polypeptide is administered in an amount from about 0.1 mg/kg to about 1 mg/kg. [0642] Aspect C-51: The method according to any of Aspects C-48 and C-49, wherein the levels of NTX-1 are reduced by at least 80%, compared to pre-treatment or normal levels, for at least about 30 days after administration. [0643] Aspect C-52: The method according to any of Aspects C-49 and C-50, wherein the levels of NTX-1 are reduced by at least 80%, compared to pre-treatment or normal levels, for at least about 30 days to 3 months after administration. [0644] Aspect C-53: The method according to Aspect C-47, wherein the polypeptide is administered in an amount from about 1 mg/kg to about 10 mg/kg. [0645] Aspect C-54: The method according to Aspect C-53, wherein the levels of NTX-1 are reduced by at least 80%, compared to pre-treatment or normal levels, for at least about 60 days after administration. [0646] Aspect C-55: The method according to Aspect C-54, wherein the levels of NTX-1 are reduced by at least 80%, compared to pre-treatment or normal levels, for at least about 90 days after administration. [0647] Aspect C-56: The method according to any of Aspects C-1 to C-55, wherein the NTX-1 is reduced by at least 20%, compared to pre-treatment or normal levels, for at least 90 days after administration. [0648] Aspect C-57: The method according to any of Aspects C-1 to C-56, wherein the NTX-1 is reduced by at least 20%, compared to pre-treatment or normal levels, for at least 120 days after administration. [0649] Aspect C-58: The method according to any of Aspects C-1 to C-57, wherein the NTX-1 is reduced by at least 20%, compared to pre-treatment or normal levels, for at least 150 days after administration. [0650] Aspect C-59: The method according to any of Aspects C-1 to C-58, wherein the NTX-1 is reduced by at least 20%, compared to pre-treatment or normal levels, for at least 180 days after administration. [0651] Aspect C-60: The method according to any of Aspects C-1 to C-59, wherein the amount of the polypeptide administered is effective to reduce urine levels of NTX-1 by at least 45% by 8 hours after administration. [0652] Aspect C-61: The method according to any of Aspects C-1 to C-60, wherein the ratio of NTX-1 to creatinine in urine in the subject is determined by ELISA assay for NTX-1.

[0653] The method according to aspects C-1 to C-61 may in particular be a method according to any of the Aspects B-1 to B-66. [0654] Aspect D-1: The method of any of Aspects A-1 to A-25, wherein the marker of bone metabolism is tartrate-resistant acid phosphatase isoform 5b (TRACP5b). [0655] Aspect D-2: The method according to Aspect D-1, wherein the amount of the polypeptide administered is effective to reduce serum levels of TRACP5b by at least 30%, compared to pre-treatment or normal levels, for at least about 30, 60, 90 or 120 days after administration. [0656] Aspect D-3: The method according to Aspect D-2, wherein the polypeptide is administered in an amount from about 0.03 mg/kg to about 0.3 mg/kg. [0657] Aspect D-4: The method according to Aspect D-3, wherein the serum levels of TRACP5b are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 30 days after administration. [0658] Aspect D-5: The method according to Aspect D-4, wherein the serum levels of TRACP5b are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 60 days after administration. [0659] Aspect D-6: The method according to Aspect D-2, wherein the polypeptide is administered in an amount from about 0.1 mg/kg to about 1 mg/kg. [0660] Aspect D-7: The method according to Aspect D-6, wherein the serum levels of TRACP5b are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 90 days after administration. [0661] Aspect D-8: The method according to Aspect D-2, wherein the polypeptide is administered in an amount from about 0.3 mg/kg to about 3 mg/kg. [0662] Aspect D-9: The method according to Aspect D-8, wherein the serum levels of TRACP5b are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 120 days after administration. [0663] Aspect D-10: The method according to Aspect D-1, wherein the amount of the polypeptide administered is effective to reduce serum level of TRACP5b by at least 45%, compared to pre-treatment or normal levels, for at least about 30, 60, or 90 days after administration. [0664] Aspect D-11: The method according to Aspect D-10, wherein the polypeptide is administered in an amount from about 0.1 mg/kg to about 1 mg/kg. [0665] Aspect D-12: The method according to Aspect D-11, wherein the serum levels of TRACP5b are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 30 days after administration. [0666] Aspect D-13: The method according to Aspect D-10, wherein the polypeptide is administered in an amount from about 0.3 mg/kg to about 3 mg/kg. [0667] Aspect D-14: The method according to Aspect D-13, wherein the serum levels of TRACP5b are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 60 days after administration. [0668] Aspect D-15: The method according to Aspect D-14, wherein the serum levels of TRACP5b are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 90 days after administration.

[0669] The method according to aspects D-1 to D-15 may in particular be a method according to any of the Aspects B-1 to B-66 and/or C-1 to C-61. [0670] Aspect E-1: The method of any of Aspects A-1 to A-25, wherein the marker of bone metabolism is N-terminal propeptide of type I procollagen (P1NP). [0671] Aspect E-2: The method according to Aspect E-1, wherein the amount of the polypeptide administered is effective to reduce serum levels of P1NP by at least 30%, compared to pre-treatment or normal levels, for at least about 30, 60, 90, or 120 days after administration. [0672] Aspect E-3: The method according to Aspect E-2, wherein the polypeptide is administered in an amount from about 0.003 mg/kg to about 0.03 mg/kg. [0673] Aspect E-4: The method according to Aspect E-3, wherein the serum levels of P1NP are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 30 days after administration. [0674] Aspect E-5: The method according to Aspect E-4, wherein the serum levels of P1NP are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 60 days after administration. [0675] Aspect E-6: The method according to Aspect E-5, wherein the serum levels of P1NP are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 90 days after administration. [0676] Aspect E-7: The method according to Aspect E-2, wherein the polypeptide is administered in an amount from about 0.01 mg/kg to about 0.1 mg/kg. [0677] Aspect E-8: The method according to Aspect E-7, wherein the serum levels of P1NP 1 are reduced by at least 30%, compared to pre-treatment or normal levels, for at least about 120 days after administration. [0678] Aspect E-9: The method according to Aspect E-1, wherein the amount of the polypeptide administered is effective to reduce serum level of P1NP by at least 45%, compared to pre-treatment or normal levels, for at least about 30, 60, or 90 days after administration. [0679] Aspect E-10: The method according to Aspect E-9, wherein the polypeptide is administered in an amount from about 0.03 mg/kg to about 0.3 mg/kg. [0680] Aspect E-11: The method according to Aspect E-10, wherein the serum levels of P1NP are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 30 days after administration. [0681] Aspect E-12: The method according to Aspect E-11, wherein the serum levels of P1NP are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 60 days after administration. [0682] Aspect E-13: The method according to according to Aspect E-12, wherein the serum levels of P1NP are reduced by at least 45%, compared to pre-treatment or normal levels, for at least about 90 days after administration. [0683] Aspect E-14: The method according to Aspect E-1, wherein the amount of the polypeptide administered is effective to reduce serum levels of P1NP by at least 70%, compared to pre-treatment or normal levels, for at least about 30, 60, or 90 days after administration. [0684] Aspect E-15: The method according to Aspect E-14, wherein the polypeptide is administered in an amount from about 0.1 mg/kg to about 1 mg/kg. [0685] Aspect E-16: The method according to Aspect E-15, wherein the serum levels of P1NP are reduced by at least 70%, compared to pre-treatment or normal levels, for at least about 30 days after administration. [0686] Aspect E-17: The method according to Aspect E-16, wherein the serum levels of P1NP are reduced by at least 70%, compared to pre-treatment or normal levels, for at least about 60 days after administration. [0687] Aspect E-18: The method according to Aspect E-17, wherein the serum levels of P1NP are reduced by at least 70%, compared to pre-treatment or normal levels, for at least about 90 days after administration.

[0688] The method according to aspects E-1 to e-18 may in particular be a method according to any of the Aspects B-1 to B-66, C-1 to C-61 and/or D-1 to D-15.