Novel Polymeric hGH Prodrugs

Abstract

The present invention relates to a polymeric human growth hormone prodrug and dry, liquid and reconstituted pharmaceutical formulations comprising said prodrug. It furthermore relates to their use as medicaments for the treatment of diseases which can be treated with growth hormone and to methods of treatment. It also relates to methods of application of such polymeric human growth hormone prodrug or pharmaceutical formulation.

Claims

1. A polymeric human growth hormone (hGH) prodrug or a pharmaceutically acceptable salt thereof of formula (Ia) or (Ib): ##STR00020## wherein: -D is a hGH moiety connected to the rest of the molecule through an amine functional group; n is 0, 1, 2, 3, or 4; —X— is a chemical bond or a spacer; ═Y.sub.1 is selected from the group consisting of ═O and ═S; —Y.sub.2— is selected from the group consisting of —O— and —S—; —Y.sub.3— and —Y.sub.5— are independently of each other selected from the group consisting of —O— and —S—; —Y.sub.4— is selected from the group consisting of —O—, —NR.sup.5— and —C(R.sup.6R.sup.6a)—; —R.sup.1 is is a water-soluble PEG-based moiety comprising at least 40% PEG having a molecular weight ranging from 30 to 50 kDa; —R.sup.2, —R.sup.3, —R.sup.5, —R.sup.6, and —R.sup.6a are independently of each other selected from the group consisting of —H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl and 3,3-dimethylpropyl; —R.sup.4 is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, text-butyl, n-pentyl, 2-methylbutyl, 2,2-ditnethylpropyl, n-hexyl, 2-tnethylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dirnethylbutyl and 3,3-dimethylpropyl; —W— is selected from the group consisting of C.sub.1-20 alkyl optionally interrupted by one or more groups selected from the group consisting of C.sub.3-10 cycloalkyl, 8- to 30-membered carbopolycyclyl, 3- to 10-membered heterocyclyl, —C(O)—, —C(O)N(R.sup.7)—, —O—, —S—, and —N(R.sup.7)—; —Nu is a nucleophile selected from the group consisting of —N(R.sup.7R.sup.7a), —N(R.sup.7OH), —N(R.sup.7)—N(R.sup.7aR.sup.7b), —S(R.sup.7), —COOH, ##STR00021## —Ar— is selected from the group consisting of ##STR00022## ##STR00023## wherein: dashed lines indicate attachment to the rest of the prodrug, —Z.sup.1— is selected from the group consisting of —O—, —S— and —N(R.sup.7)—, and —Z.sup.2— is —N(R.sup.7)—; and —R.sup.7, —R.sup.7a, and —R.sup.7b are independently of each other selected from the group consisting of —H, C.sub.1-6 alkyl, C.sub.3-6 alkenyl and C.sub.2-6 alkynyl; and wherein the prodrug of formula (Ia) and (Ib) is optionally further substituted.

2. The prodrug of claim 1, wherein —R.sup.1 comprises a moiety of formula (II): ##STR00024## wherein: —BP.sup.1<, —BP.sup.2<, and —BP.sup.3< are independently of each other selected from the group consisting of —N< and —C(R.sup.8)<; R.sup.8 is selected from the group consisting of H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl; —P.sup.1, —P.sup.2, P.sup.3, and P.sup.4 are independently of each other a PEG-based chain comprising at least 40% PEG and having a molecular weight ranging from 8 to 12 kDa; —C.sup.1— and —C.sup.2— are independently of each other selected from the group consisting of C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl, which C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are; optionally substituted with one or more R.sup.9, which are the same or different; and optionally interrupted by one or more groups selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.10)—, —S(O).sub.2N(R.sup.10)—, —S(O)N(R.sup.10)—, —S(O).sub.2—, —S(O)—, —N(R.sup.10)S(O).sub.2N(R.sup.10a), —S—, —N(R.sup.10), OC(OR.sup.10)(R.sup.10a)—, —N(R.sup.10)C(O)N(R.sup.10a)—, and —OC(O)N(R.sup.10)—; each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8-to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl, wherein each T is independently optionally substituted with one or more R.sup.9, which are the same or different; each BY is independently selected from the group consisting of halogen, —CN, oxo (═O), —COOR.sup.11, —OR.sup.11, —C(O).sup.11, —C(O)N(R.sup.11R.sup.11a), —S(O).sub.2N(R.sup.11R.sup.11a), —S(O)N(R.sup.11R.sup.11a), —S(O).sub.2R.sup.11, —S(O)R.sup.11a), —N(R.sup.11)S(O).sub.2N(R.sup.11aR.sup.11b), —SR.sup.11, —N(R.sup.11R.sup.11a), —NO.sub.2, —OC(O)R.sup.11, —N(R.sup.11)C(O)R.sup.11a, —N(R.sup.11)S(O).sub.2R.sup.11a, —N(R.sup.11)S(O)R.sup.11a, 13 N(R.sup.11)C(O)OR.sup.11a, —N(R.sup.11)C(O)N(R.sup.11aR.sup.11b), OC(O)N(R.sup.11R.sup.11a), and C.sub.1-6 alkyl, which C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; and each R.sup.10, R.sup.10a, R.sup.11, R.sup.11a and R.sup.11b is independently selected from the group consisting of —H and C.sub.1-6 alkyl, which C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different.

3. The prodrug of claim 1; wherein —R.sup.1 comprises a moiety of formula (IIc): ##STR00025## wherein: p1, p2, p3, and p4 are independently an integer ranging from 180 to 270.

4. The prodrug of claim 1; wherein the polymeric hGH prodrug of formula (IV): ##STR00026## wherein: D is an hGH moiety connected to the rest of the molecule through an amine functional group; and p1, p2, p3, and p4 are independently an integer ranging from 180 to 270.

5. A pharmaceutical formulation comprising; the prodrug of claim 1; and at least one excipient.

6. The pharmaceutical formulation of claim 5; wherein the pharmaceutical formulation is a liquid formulation and comprises from 3 to 300 mg/mL of the prodrug.

7. The liquid pharmaceutical formulation of claim 6; wherein the liquid formulation comprises 3-300 mg/ml of the polymeric hGH prodrug; 5-50 mM of succinic acid; optionally 25-150 mg/m1 of trehalose dihydrate; and optionally 1-50 mM of methionine; and wherein the liquid formulation has a pH ranging from pH 4.0 to pH 6.0, which is titrated using a suitable buffer.

8. The pharmaceutical formulation of claim 5; wherein the pharmaceutical formulation is a dry formulation and comprises from 1 to 99.9% (w/w) of the prodrug.

9. The dry pharmaceutical formulation of claim 8; wherein the dry formulation is obtained by a process comprising the steps of: (a) providing a liquid formulation comprising.sub.1 3-300 mg/ml or the polymeric hGH prodrug 5-50 mM of succinic acid; and optionally 25-150 mg/ml of trehalose dihydrate; wherein the liquid formulation has a pH ranging from pH 4.0 to pH 6.0, which is titrated using a suitable buffer; and (b) drying the liquid formulation of step (a).

10. The dry pharmaceutical formulation of claim 8; wherein the dry formulation comprises: 14-65% (w/w) of the polymeric hGH prodrug; 0.5-2.5% (w/w) of succinic acid; 31-84% (w/w) of trehalose dihydrate; and 0.4-4% (w/w) of Tris.

11. A method of preparing a reconstituted formulation comprising the prodrug of claim 1, wherein the method comprises the step of: contacting a dry pharmaceutical formulation with a reconstitution solution; wherein the dry pharmaceutical formulation comprises: from 1 to 99.9% (w/w) of the prodrug; and at least one excipient.

12. A reconstituted formulation obtained from the method of claim 11.

13. The reconstituted formulation of claim 12; wherein the reconstituted formulation comprises; 3-300 mg/m1 of the polymeric hGH prodrug; 5-50 mM of succinic acid; 25-150 mg/ml of trehalose dihydrate; and Tris 1-50 mM; and wherein the reconstituted formulation has a pH ranging from pH 4.0 to pH 6.0;

14-17. (canceled)

18. A method of treating, controlling, delaying, or preventing in a mammalian patient in need of the treatment, control, delay, or prevention of at least one diseases which can be treated, controlled, delayed, or prevented with hGH, wherein the method comprises: a step of administering to said patient a therapeutically effective amount of the prodrug of claim 1.

19. The method of claim 18; wherein the administration is via topical, enteral, or parenteral administration, or is by external application, injection, or infusion, direct delivery to the brain via implanted device allowing delivery of the invention or the like to brain tissue or brain fluids, direct intracerebroventricular injection or infusion, injection or infusion into brain or brain associated regions, injection into the subchoroidal space, retro-orbital injection, or ocular instillation.

20. The method of claim 19; wherein the administration is by intraarticular, periarticular, intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, intracapsular, intraorbital, intravitreal, intratympanic, intravesical, intracardiac, transtracheal, subcuticular, subcapsular, subarachnoid, intraspinal, intraventricular, or intrasternal injection or infusion.

21. The method of claim 18; wherein the disease is selected from the group consisting of growth hormone deficiency in children, idiopathic short stature, short stature homeobox gene mutations, Turner syndrome, Noonan syndrome, Prader-Willi syndrome, children born small for gestational age, clironic renal insufficiency, growth hormone deficiency in adults, wasting due to HIV or AIDS or other malignancies, short bowel syndrome, sarcopenia, and frailty.

Description

EXAMPLES

[0377] Cation Exchange Chromatography

[0378] The purification of conjugates by cation exchange chromatography was performed using an ÄKTA Pure system (GE Healthcare) equipped with a Macrocap SP column with a column volume of 279 mL. The respective reaction mixture was applied to the column which was pre-equilibrated in 20 mM sodium acetate, 10 mM L-methionine buffer, pH 4.0 (buffer A). After loading, the column was washed with three column volumes of buffer A to remove any unreacted PEG reagent. Mono-conjugates were eluted using a gradient of 0-30% buffer B (20 mM sodium acetate, 1 M sodium chloride, pH 4.5) over 15 column volumes. A gradient of 30-80% B over three column volumes was used to elute unreacted growth hormone. The column was cleaned with 3 column volumes of 100% buffer B. The flow rate was 20 mL/min for loading and 25 mL/min during the elution. The elution was monitored by detection at 280 nm.

[0379] SDS-PAGE Analysis

[0380] The mPEG-hGH conjugates were analysed by SDS-PAGE using NuPAGE® Novex 4-12% Bis-Tris gels (1.0 mm thick, 12 lanes), NuPAGE MOPS SDS-Running Buffer, HiMark™ Pre-stained High Molecular Weight Protein Standard and Coomassie Colloidal Blue™ Staining Kit (lnvitrogen). In each lane 1 p.g hGH eq. of the conjugate were applied and the electrophoresis and subsequent staining performed according to the supplier's protocol. Images of the gels were generated using a Digi Image System (Kisker Biotech) and a Power Shot G10 camera (Canon).

[0381] Dia-/Ultrafiltration

[0382] Dia- and Ultrafiltration steps were performed using a labscale TFF system (Millipore) equipped with Pellicon XL Biomax membranes with a membrane are of 50 cm.sup.2 and a molecular weight cut-off of 5 or 10 kDa for hGH only, 10 kDa for 4× 10 kDa mPEG-linker-hGH monoconjugate 2 and 50 kDa for 4× 20 kDa mPEG-linker-hGH monoconjugate 1.

[0383] RP—HPLC

[0384] The following RP-HPLC parameters were used:

[0385] Mobile phase A was composed of 0.05% aqueous TFA and mobile phase B was composed of 0.04% TFA in acetonitrile. A Waters UPLC C18 BEH 300Å 1.7 μm 2.1×50mm column was used. Flow rate was set to 0.2-0.4 mL/min, detection was at a wavelength of 215 mn, the column running temperature was 30° C. (+5° C.). The autosampler temperature was set at 4° C. and the sample injection load was 20 μL. For peak separation the gradient shown in Table 1 was used.

TABLE-US-00034 TABLE 1 RP-HPLC gradient Time [min] % B 0 25 1 25 8 40 30 60 30.1 90 30.5 90 30.6 25 35 25

[0386] Buffer Exchange

[0387] Buffer exchange was perfo ined using an ÄKTA explorer system (GE Healthcare) equipped with a HiPrep 26/10 Desalting column or a HiTrap Desalting column.

Example 1

Synthesis of Transient 4× 20 kDa mPEG-linker-hGH Monoconjugate 1 (Reference Substance; not According to the Invention)

[0388] ##STR00018##

[0389] 4× 20 kDa mPEG-linker-hGH monoconjugate 1 was synthesized according to a similar procedure as described in WO2009/133137 A2. The formulations of 4× 20 kDa mPEG-linker-hGH monoconjugate 1 as shown in Table 2 were prepared.

TABLE-US-00035 TABLE 2 Formulations of 4x 20 kDa mPEG-linker-hGH monoconjugate 1 Concentration of 4x 20 kDa Formulation mPEG-linker-hGH monoconjugate Concentration of hGH name: 1 formulation [mg conjugate/mL] eq. [mg hGH eq./mL] 1A 30 6 1B 45 9 1C 75 15

Example 2

Synthesis of High Strength Transient 4× 10 kDa mPEG-linker-hGH Monoconjugate 2

[0390] ##STR00019##

[0391] 4× 10 kDa mPEG-linker-hGH monoconjugatc 2 was synthesized according to a similar procedure as described in WO2009/133137 A2; in detail the manufacturing process was conducted as follows:

[0392] hGH was buffer exchanged to 100 mM sodium borate pH 9 and the concentration of hGH was adjusted to 10 mg/mL. A molar excess of 4-arm branched 40 kDa mPEG-pentafluorophenylcarbonate derivative relative to the amount of hGH was dissolved in water to form a 6% (w/w) reagent solution. The reagent solution was added to the hGH solution in a 1-to-1 ratio (based on weight) and mixed. The reaction mixture was incubated under stirring for 105 min at 12-16° C. and subsequently quenched by adding 4 volumes of a solution comprising 27 mM acetic acid and 12.5 mM L-methionine to 1 volume of the reaction mixture to lower the pH of the solution to 4-4.5. After sterile filtration, the reaction mixture was incubated at room temperature for 16±4 h. 4× 10 kDa mPEG-linker-hGH monoconjugate 2 was purified by cation exchange chromatography.

[0393] Buffer exchange and adjustment to the desired concentration of 4× 10 kDa mPEG-linker-hGH monoconjugate 2 was achieved using a tangential-flow filtration system. Herewith the eluate from the cation exchange chromatography was ultra-filtrated and dia-filtrated to formulation buffer (10 mM succinic acid, 85 g/L trehalose dihydrate, pH 5.0 with 1M Tris-solution). Using the same system the trehalose concentration was lowered to 65 g/L and the concentration of this stock solution adjusted to 105+3 mg/mL of 4× 10 kDa mPEG-linker-hGH monoconjugate 2 (corresponding to 35±1 mg hGH eq./mL). The formulations as shown in Table 3 were prepared based on this stock-solution of compound 2 by diluting the stock solution with high strength formulation buffer (10 mM succinic acid, 89 g/L trehalose dihydrate, adjusted to pH 5.0 with 1M Tris-base).

TABLE-US-00036 TABLE 3 Formulations of 4x 10 kDa mPEG-linker-hGH monoconjugate 2 Concentration of 4x 10 kDa Formulation mPEG-linker-hGH monoconjugate Concentration of hGH name: 2 formulation [mg/mL] eq. [mg hGH eq./mL] 2A 103.8 34.6 2B 95.1 31.7 2C 81.9 27.3 2D 65.1 21.7 2E 47.4 15.8

[0394] Individual batches were analyzed by RP—HPLC, SE—HPLC, peptide mapping and SDS-PAGE. SDS-PAGE showed that all formulation have comparable product qualities which are similar to the reference. During method development it was discovered that the load of the cation exchange chromatography column which is used to purify the 4× 10 kDa mPEG-linker-hGH monoconjugate 2 could be significantly increased compared to the purification procedure of 4× 20 kDa mPEG-linker-hGH monoconjugate 1.

[0395] Conclusion:

[0396] 4× 10 kDa mPEG-linker-hGH monoconjugate 2 could by synthesized by implementing only minor changes to the manufacturing process compared to the manufacturing process described in EP-A 2113256 and showed improved handling and product properties. Loading of the CIEX column for purification could be at least tripled without impairing the separation efficacy and product quality. Additionally, the content of the final product could be increased to above 100 mg/mL of the 4× 10 kDa mPEG-linker-hGH-conjugate 2 which corresponds to approx. 35 mg hGH eq./mL.

Example 3

Syringability of High Strength Formulations of 4× 10 kDa mPEG-iinker-hGI-1 monoconjugate 2 compared to 4× 20 kDa mPEG-linker-hGH monoconjugate 1

[0397] Individual formulations from example 1 & 2 were investigated for their ability of being injected through injection needles with various inner diameters. Tests were performed on a

[0398] Mecmesin ultitest 1-d stand, equipped with measuring device BFG 200N and using the Emperor Lite software (Vers. no. 1.16-015). Tested injection needles comprised a 27G needle 0.4×13 mm 27Gx½″ from BD (Ref 300635, Lot 101009), a 29G needle, 0.33×13 mm from Transcoject, and a 30G needle 0.30×12 mm, 30Gx½″, from Sterican (Lot 2G13258811). The measuring device was setup to measure the force for pushing the plunger down for a given constant plunger speed. The applied plunger speeds which correspond to the applied injection speeds were as follows:

TABLE-US-00037 Injection 688 mm/min  5 sec/mL 12 mL/min  speed 344 mm/min 10 sec/mL 6 mL/min 229 mm/min 15 sec/mL 4 mL/min 172 mm/min 20 sec/mL 3 mL/min 138 mm/min 25 sec/mL 2.4 mL/min   115 mm/min 30 sec/mL 2 mL/min

[0399] Testing was performed using the following steps:

[0400] 1. Charging of a lml Luer-lok Syringe, (BD, Ref 309628) with sample (using a 20G needle, 0.90×40mm , 20Gx1½″ from Sterican)

[0401] 2. Removal of air bubbles

[0402] 3. Attachment of test needle (starting with the largest inner diameter) onto the syringe

[0403] 4. Clamping the syringe into the holder

[0404] 5. Selection of appropriate measuring settings

[0405] 6. Start measurement and collect the sample in a glass vial (placed underneath the syringe)

[0406] 7. Removal of syringe from holder

[0407] 8. Re-charging of the syringe with test material and measuring of subsequent setting −> these steps were repeated for all needles (with descending needle diameter) and for every test sample.

[0408] Formulation buffer without mPEG-linker-hGH monoconjugate 1 or 2 was used as reference solution.

[0409] For all different injection needles and for all injection speeds the injection forces were determined for 4× 10 kDa mPEG-linker-hGH monoconjugate 2 and compared with the results for 4× 20 kDa mPEG-linker-hGH monoconjugate 1. Table 4 shows the comparison of injection forces between 4× 10 kDa mPEG-linker-hGH monoconjugate 2 and 4× 20 kDa mPEG-linker-hGH monoconjugate 1 for the 27G needle 0.4×13 mm 27Gx½″ from BD (Ref 300635, Lot 101009).

TABLE-US-00038 TABLE 4 Injection forces of 4x 10 kDa mPEG-linker-hGH monoconjugate 2 and 4x 20 kDa mPEG-linker- hGH monoconjugate 1 for a 27 G needle (0.4 × 13 mm 27 G × ½″ from BD) Injection Force [N] Injection Injection Formulation of 4x 10 kDa Formulation of 4x 20 kDa speed speed mPEG-linker-hGH monoconjugate 2 mPEG-linker-hGH monoconjugate 1 [sec/mL] [mL/min] 2E 2D 2C 2B 2A 1A 1B 1C 5 12 5.35 7.35 9.65 22.0 30.0 6.6 12..1 20.3 10 6 2.90 4.00 4.90 11.35 16.0 3.6 6.5 10.7 15 4 2.05 2.95 3.75 7.95 10.8 2.7 4.6 7.5 20 3 1.60 2.40 3.15 6.15 8.85 2.2 3.8 5.7 25 2.4 1.45 2.05 2.65 5.05 7.35 1.8 3.2 4.5 30 2 1.30 1.70 2.25 4.45 6.40 n.d. n.d. n.d.

[0410] Table 5 shows the comparison of injection forces between 4× 10 kDa mPEG-linker-hGH monoconjugate 2 and 4× 20 kDa mPEG-linker-hGH monoconjugate 1 for the 29G needle, 0.33× 13mm from Transcoject.

TABLE-US-00039 TABLE 5 Injection forces of 4x 10 kDa mPEG-linker-hGH monoconjugate 2 and 4x 20 kDa mPEG- linker-hGH monoconjugate 1 for a 29 G needle (0.33 × 13 mm from Transcoject) Injection Force [N] Injection injection Formulation of 4x 10 kDa Formulation of 4x 20 kDa speed speed mPEG-linker-hGH monoconjugate 2 mPEG-linker-hGH monoconjugate 1 [sec/mL] [mL/min] 2E 2D 2C 2B 2A 1A 1B 1C 5 12 12.70 20.95 26.70 32.70 n.d. n.d. 27.3 n.d. 10 6 6.40 10.05 13.25 16.90 25.40 12.0  14.9 28.6 15 4 4.40 6.90 9.20 11.50 19.20 8.0 10.6 20.2 20 3 3.70 5.30 6.75 8.95 13.95 6.3 7.9 15.2 25 2.4 2.80 4.40 5.70 7.50 11.50 5.0 6.5 12.3 30 2 2.50 3.70 4.65 6.05 10.05 n.d. n.d. n.d.

[0411] Table 6 shows the comparison of injection forces between 4× 10 kDa mPEG-linker-hGH monoconjugate 2 and 4× 20 kDa mPEG-linker-hGH monoconjugate 1 for the 30G needle 0.30×12mm, 30Gx½″, from Sterican (Lot 2G13258811).

TABLE-US-00040 TABLE 6 Injection forces of 4x 10 kDa mPEG-linker-hGH monoconjugate 2 and 4x 20 kDa mPEG-linker-hGH monoconjugate 1 for a 30 G needle (0.30 × 12 mm, 30 G × ½″, from Sterican) Injection Force [N] Injection injection Formulation of 4x 10 kDa Formulation of 4x 20 kDa speed speed mPEG-linker-hGH monoconjugate 2 mPEG-linker-hGH monoconjugate 1 [sec/mL] [mL/min] 2E 2D 2C 2B 2A 1A 1B 1C 5 12 26.6 28.50 50.90 n.d. n.d. n.d. 45.2 * 10 6 12.95 19.60 26.90 36.50 n.d. 15.0 25.5 51.0 15 4 8.40 13.70 18.90 25.20 34.7 10.3 17.7 37.6 20 3 7.00 10.50 13.90 19.50 28.2  8.2 13.1 28.9 25 2.4 5.50 8.05 11.20 15.70 20.6  7.0 10.5 23.4 30 2 4.75 7.50 9.50 13.15 17.5 n.d. n.d. n.d.

[0412] Conclusion:

[0413] The injectability of 4× 10 kDa mPEG-linker-hGH monoconjugatc 2 was highly improved and the injection force could be reduced 3.5-fold to 4-5 fold compared to 4× 20 kDa mPEG linker-hGH monoconjugate 1.

Example 4

Viscosity Measurements of 4× 10 kDa mPEG-linker-hGH Monoconjugate 2 compared to 4× 20 kDa mPEG-linker-hGH Monoconjugatc 1

[0414] The dynamic viscosity of test samples was determined at lnfrasery Knapsack (now synlab Pharma Institute) using a method according to EP method 2.2.10. All measurements were performed with approx. 1-5 mL of test sample at 23.0 +0.1° C. using a cone/plate measuring system (CP50/1). The shearing rate was in the range of 100 s.sup.−1-10 s.sup.−1.

[0415] All tested formulations of 4× 10 kDa mPEG-linker-hGH monoconjugate 2 and 4× 20 kDa mPEG-linker-hGH monoconjugate 1 were adjusted to an equal osmolality of approx. 290 mOsmol/kg by increasing or decreasing the amount of trehalose in the formulation. The dynamic viscosity values measured for all test samples are summarized in Table 7.

TABLE-US-00041 TABLE 7 Dynamic viscosity values for different formulations of 4x 10 kDa mPEG-linker-hGH monoconjugate 2 and 4x 20 kDa mPEG-linker-hGH monoconjugate 1 which were adjusted to similar osmolalities. Content Conc. trehalose in Formu- [mg/mL formulation Osmo- Viscosity lation: hGH eq.] buffer [g/L] lality [mPa * s] 4x 10 kDa 2A 34.6 65 286 25.6 mPEG-linker- 2B 31.7 68 290 18.9 hGH mono- 2C 27.3 71 286 14.9 conjugate 2 2D 21.7 75 283 9.9 2E 15.8 78 284 6.0 4x 20 kDa 1A 6 85 291 7.4 mPEG-linker- 1B 9 80 293 12.8 hGH mono- 1C 15 70 285 31 conjugate 1

[0416] Conclusion:

[0417] The dynamic viscosity of 4× 10 kDa mPEG-linker-hGH monoconjugate 2 could be significantly reduced about a factor of 4- to 5-fold compared to 4× 20 kDa mPEG linker-hGH monoconjugate 1.

Example 5

Reconstitution Time of Lyophilisates of 4× 10 kDa mPEG-linker-hGH Monoconjugate 2

[0418] 1 mL of 4× 10 kDa mPEG-linker-hGH monoconjugate 2 was lyophilized in a Din2R vial and after lyophilization the lyo cake was dissolved with 1 mL water for injection. The reconstitution time was compared to the dissolution time of a lyophilisate of 4× 20 kDa mPEG-linker-hGH monoconjugate 1. During reconstitution more gas bubbles were detected for 4× 20 kDa mPEG-linker-hGH monoconjugate 1. While the dissolution of the lyo cake itself was quite fast, the time until a clear solution was obtained with only a minimal amount of gas bubbles remaining, was significantly shorter for 4× 10 kDa mPEG-linker-hGH monoconjugate 2. The results of this reconstitution procedure are summarized in Table 8.

TABLE-US-00042 TABLE 8 Reconstitution times of 4x 10 kDa mPEG-linker-hGH monoconjugate 2 and 4x 20 kDa mPEG-linker-hGH monoconjugate 1 4x 10 kDa 4x 20 kDa mPEG-linker-hGH mPEG-linker-hGH monoconjugate 2 monoconjugate 1 Time for dissolution <1 min  <1 min Time until a clear solution is <5 min >15 min obtained Time for disappearance of <5 min >15 min most air bubbles

[0419] Conclusion:

[0420] The time of reconstitution until a clear and virtually bubble free solution is achieved is significantly shorter for 4× 10 kDa mPEG-linker-hGH monoconjugate 2 compared to 4× 20 kDa mPEG linker-hGH monoconjugate 1.

Example 6

In vitro Hydrolysis of 4× 10 kDa mPEG-linker-hGH Monoconjugate 2

[0421] For the determination of in vitro linker cleavage rates of 4× 10 kDa mPEG-linker-hGH monoconjugate 2 or 4× 20 kDa mPEG-linker-hGH monoconjugate 1, the compounds were buffer exchanged to PBST buffer at pH 7.4 and the eluted solutions were filtered through a 0.22 μm filter and incubated at 37° C. for 1 week. Samples were taken at certain time intervals and analyzed by RP-HPLC. All peaks were integrated and allocated and the relevant peak areas were plotted against incubation time. Curve fitting software was applied to determine first-order cleavage rates. Table 9 shows in vitro hydrolysis rates of 4× 10 kDa mPEG-linker-hGH monoconjugate 2 and 4× 20 kDa mPEG-linker-hGH monoconjugate 1 at pH 7.4 and 37° C.

TABLE-US-00043 TABLE 9 In vitro hydrolysis rates of 4x 10 kDa mPEG-linker- hGH monoconjugate 2 or 4x 20 kDa mPEG-linker- hGH monoconjugate 1 at pH 7.4 and 37° C. 95% confidence Half life time [h] interval [h] 4x 10 kDa mPEG-linker-hGH 104.7 90.70-123.8 monoconjugate 2 4x 20 kDa mPEG-linker-hGH 107.2 91.89-128.6 monoconjugate 1

[0422] Conclusion:

[0423] The in vitro hydrolysis rates of conjugates 1 and 2 at pH 7.4 and 37° C. were in the range of 105±5 h. Both half life times were highly comparable and lay within the 95% confidence interval.

Example 7

Quantification of Conjugates 1 and 2 in Serum Samples from Animal Studies

[0424] An ELISA based method was used to quantify conjugates 1 and 2 in serum samples from animal studies. The same sandwich ELISA format was used for both conjugates 1 and 2, which utilized a sheep anti-hGH polyclonal antibody (Abeam, Cat. No. ab64499) as capture antibody and a biotinylated rabbit anti-PEG antibody (Epitomics, Cat. No. 2137-1) as detection antibody. Read-out was done with streptavidin—HRP (Jackson ImmunoResearch, Cat, No. 016-030-084) and a commercial TMB liquid substrate system (Sigma, Cat. No. T0440), Serum standards and samples were diluted 1:50 with a pH 7.0 buffer (50 mM HEPES, 1 mM CaCl.sub.2, 0.05% Tween-20 and 1° A) BSA) prior to measurement. Sample incubation on the ELISA plate was performed under shaking for 2 h at 37° C.

Example 8

Quantification of Total mPEG40 and 80 in Serum Samples from Animal Studies

[0425] An ELISA based method was used to quantify mPEG40 and mPEG80 in serum samples from animal studies. The same sandwich ELISA format was used for both analytes mPEG40 and mPEG80, which utilized an anti-PEG (methoxy group) rabbit monoclonal antibody, (Epitomics, Cat. No. 2061-1) as capture antibody and a biotinylated anti-PEG mouse monoclonal IgM antibody (ANP Tech, Cat. No. 90-1052) as detection antibody. Read-out was done with streptavidin—HRP (Jackson ImmunoResearch, Cat. No. 016-030-084) and a commercial TMB liquid substrate system (Sigma, Cat. No. T0440). Serum standards and samples were diluted 1:50 with a pH 7.0 buffer (50 mM HEPES, 1 mM CaCl.sub.2, 0.05° A Tween-20 and 1% BSA) prior to measurement. Sample incubation on the ELISA plate was performed under shaking for 2 h at 37° C.

Example 9

Comparative Pharmacokinetic Study in Cynomolgus Monkeys Treated with Conjugates 1 and 2

[0426] Two groups of five healthy male non-nave cynomolgus monkeys each received a single subcutaneous administration of conjugate 1 or a single subcutaneous administration of conjugate 2 at a target dose level of 1 mg hGH equivalents per kg (corresponding to 3 mg conjugate 2/kg and 5 mg conjugate 1/kg, respectively). For PK-detelininations blood samples were collected up to 336 hours post dose and serum generated thereof (for mPEG quantification serum samples were collected up to 56 days). Pharmacokinetic analysis according to Example 7 indicated that both compounds effected a comparable maximal conjugate level (9,200 ng hGH equivalents/mL for conjugate 1 and 7,400 ng hGH equivalents/mL for conjugate 2) which was reached around 36 hours post dosing. mPEG concentration levels were determined according to Example 8. Both mPEG PK-profiles had their maximum concentration levels at 48 hours post dosing. Clearance of mPEG40 was faster than for mPEG80 as indicated in the terminal elimination half lifes (300 h for mPEG80 and 260 h for mPEG40). This resulted in an overall significant lower mPEG exposure for conjugate 2 over conjugate 1 in this comparative PK-study.

[0427] Abbreviations:

[0428] AIDS acquired immunodeficiency syndrome

[0429] CR1 chronic renal insufficiency

[0430] DF Diafiltration

[0431] ELISA Enzyme linked immunosorbent assay

[0432] EP European Pharmacopoeia

[0433] eq stoichiometric equivalent

[0434] G gauge

[0435] ED

[0436] HIV human immunodeficiency virus

[0437] ISS idiopathic short stature

[0438] MW molecular weight

[0439] NS Noonan syndrome

[0440] PEG polyethylene glycol

[0441] PWS Prader-Willi syndrome

[0442] PK Phannacokinetic

[0443] RP-HPLC reversed-phase high performance liquid chromatography

[0444] rt room temperature

[0445] SBS short bowel syndrome

[0446] SDS-PAGE sodium dodecyl sulfate polyacrylamid gel electrophoresis

[0447] SEC size exclusion chromatography

[0448] SHOX short stature hoeobox

[0449] SGA small for gestational age

[0450] TFF Tangential flow filtration

[0451] Tris tris(hydroxymethyl)aminomethane

[0452] TS Turner syndrome

[0453] UF Ultrafiltration