PEGylated Recombinant Human Growth Hormone Compounds

Abstract

A chemically modified human Growth Hormone (rhGH) prepared by attaching a transient linker which comprises a polyethylene glycol. The chemically modified protein may have a much longer lasting rhGH activity than that of the unmodified rhGH, enabling reduced dose and scheduling opportunities and the modified rhGH may not cause lipoatrophy. Also includes methods of use for the treatment and/or prevention of diseases or disorders in which use of growth hormone is beneficial.

Claims

1. (canceled)

2. A method of treating a growth hormone (GH) related disease, wherein the method comprises administering once weekly an effective amount of a prodrug conjugate of the human growth hormone (hGH) to a patient having a GH related disease and wherein the prodrug conjugate of the human growth hormone (hGH) is of formula (AA)
hGH-NH-L.sup.a-S.sup.0  (AA), wherein hGH-NH represents the hGH residue; L.sup.a represents a functional group, which is self hydrolysable (auto-cleavable) by an auto-cleavage inducing group G.sup.a; and S.sup.0 is a polymer chain having a molecular weight of at least 5 kDa and comprising an at least first branching structure BS.sup.1, the at least first branching structure BS.sup.1 comprising an at least second polymer chain S.sup.1 having a molecular weight of at least 4 kDa, wherein at least one of S.sup.0, BS.sup.1, S.sup.1 further comprises the auto-cleavage inducing group G.sup.a and wherein the branching structure BS.sup.1 further comprises an at least third polymer chain S.sup.2 having a molecular weight of at least 4 kDa or at least one of S.sup.0, S.sup.1 comprises an at least second branching structure BS.sup.2 comprising the at least third polymer chain S.sup.2 having a molecular weight of at least 4 kDa and wherein the molecular weight of the prodrug conjugate without the hGH-NH is at least 25 kDa and at most 1000 kDa.

3. The method of claim 2, wherein the molecular weight of the prodrug without the hGH-NH is at least 30 kDa and at most 120 kDa.

4. The method of claim 2, wherein the molecular weight of the prodrug without the hGH-NH is at least 40 kDa and at most 100 kDa.

5. The method of claim 2, wherein the molecular weight of the prodrug without the hGH-NH is at least 40 kDa and at most 90 kDa.

6. The method of claim 2, wherein L.sup.a is selected from the group consisting of C(O)—O—, and C(O)—, which form together with the primary amino group of hGH a carbamate or amide group resulting in formula (AA1) or (AA2)
hGH-NH—C(O)O—S.sup.0  (AA1),
hGH-NH—C(O)—S.sup.0  (AA2).

7. The method of claim 2, wherein L.sup.a forms together with the amino group of hGH a carbamate functional group, the cleavage of said group is induced by a hydroxyl or amino group of G.sup.a via 1,4- or 1,6 benzyl elimination of S.sup.0, wherein G.sup.a contains ester, carbonate, carbamate, or amide bonds that undergo rate-limiting transformation.

8. The method of claim 2, wherein G.sup.a is an aromatic ring or fluorenylmethyl directly attached to a carbamate functional group formed by La and the primary amino group of hGH.

9. The method of claim 2, wherein at least one of the branching structures BS.sup.1, BS.sup.2 comprises a further fourth polymer chain S.sup.3 having a molecular weight of at least 4 kDa or one of S.sup.0, S.sup.1, S.sup.2 comprises a third branching structure BS.sup.3 comprising the at least fourth polymer chain S.sup.3 having a molecular weight of at least 4 kDa.

10. The method of claim 2, wherein the at least three chains S.sup.0, S.sup.1, S.sup.2 are independently based on a polymer selected from the group consisting of polyalkyloxy polymers, hyaluronic acid and derivatives thereof, polyvinyl alcohols, polyoxazolines, polyanhydrides, poly(ortho esters), polycarbonates, polyurethanes, polyacrylic acids, polyacrylamides, polyacrylates, polymethacrylates, polyorganophosphazenes, polysiloxanes, polyvinylpyrrolidone, polycyanoacrylates, and polyesters.

11. The method of claim 2, wherein the at least three chains S.sup.0, S.sup.1, S.sup.2 are based on a polyalkoxy polymer.

12. The method of claim 2, wherein the shortest distance between the attachment site of S.sup.0 to L.sup.a and the first branching structure BS.sup.1 measured as connected atoms is less than 50 atoms.

13. The method of claim 12, wherein the shortest distance is less than 20 atoms.

14. The method of claim 2, wherein S.sup.0 is of formula (AAA1) ##STR00059## wherein G.sup.a has the meaning as indicated in claim 1; S.sup.00 is CH.sub.2; or C(O); S.sup.0A is an alkylene chain having from 1 to 20 carbon atoms, which is optionally interrupted or terminated by one or more groups, cycles or heteroatoms selected from the group consisting of optionally substituted heterocycle; O; S; C(O); and NH; BS.sup.1, BS.sup.2, BS.sup.3 are independently selected from the group consisting of N; and CH. S.sup.0B, S.sup.1A are independently an alkylene chain having from 1 to 200 carbon atoms, which is optionally interrupted or terminated by one or more groups, cycles or heteroatoms selected from the group consisting of optionally substituted heterocycle; O; S; C(O); and NH; S.sup.0C, S.sup.1B, are (C(O)).sub.n2(CH.sub.2).sub.n1(OCH.sub.2CH.sub.2).sub.nOCH.sub.3, wherein each n is independently an integer from 100 to 500, each n1 is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8, and n2 is 0 or 1; S.sup.2, S.sup.3 are independently hydrogen; or (C(O)).sub.n2(CH2).sub.n1(OCH.sub.2CH.sub.2).sub.nOCH.sub.3, wherein each n is independently an integer from 100 to 500, each n1 is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8, and n2 is 0 or 1, provided that at least one of S.sup.2, S.sup.3 is other than hydrogen; R.sup.2, R.sup.3 are selected independently from hydrogen, methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, tertiary butyl.

15. The method of claim 2, wherein G.sup.a is OC(O)—R and R is of formula (I), which is ##STR00060## wherein R1, R4, R5 are selected independently from hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl; and n=1 or 2.

16. The method of claim 15, wherein R is selected from the group consisting of: ##STR00061##

17. The method of claim 2, wherein L.sup.a-S.sup.0 is represented by formula (AAA2), ##STR00062## wherein the dashed line indicates the attachment to the primary amino group of hGH so that L.sup.a and the amino group form an amide bond; X is C(R.sup.4R.sup.4a); N(R.sup.4); O; C(R.sup.4R.sup.4a)—C(R.sup.5R.sup.5a); C(R.sup.5R.sup.5a)—C(R.sup.4R.sup.4a); C(R.sup.4R.sup.4a)—N(R.sup.6); N(R.sup.6)—C(R.sup.4R.sup.4a); C(R.sup.4R.sup.4a)—O; or O—C(R.sup.4R.sup.4a); X.sup.1 is C; or S(O); X.sup.2 is C(R.sup.7, R.sup.7a); or C(R.sup.7, R.sup.7a)—C(R.sup.8, R.sup.8a); R.sup.1, R.sup.1a, R.sup.2, R.sup.2a, R.sup.3, R.sup.3a, R.sup.4, R.sup.4a, R.sup.5, R.sup.5a, R.sup.6, R.sup.7, R.sup.7a, R.sup.8, R.sup.8a are independently selected from the group consisting of H; and C.sub.1-4 alkyl; or Optionally, one or more of the pairs R.sup.1a/R.sup.4a, R.sup.1a/R.sup.5a, R.sup.4a/R.sup.5a, R.sup.4a/R.sup.5a, R.sup.7a/R.sup.8a form a chemical bond; Optionally, one or more of the pairs R.sup.1/R.sup.1a, R.sup.2/R.sup.2a, R.sup.4/R.sup.4a, R.sup.5/R.sup.5a, R.sup.7/R.sup.7a, R.sup.8/R.sup.8a are joined together with the atom to which they are attached to form a C.sub.3-7 cycloalkyl; or 4 to 7 membered heterocyclyl; Optionally, one or more of the pairs R.sup.1/R.sup.4, RVR.sup.5, R.sup.1/R.sup.6, R.sup.4/R.sup.5, R.sup.7/R.sup.8, R.sup.2/R.sup.3 are joined together with the atoms to which they are attached to form a ring A; Optionally, R.sup.3/R.sup.3a are joined together with the nitrogen atom to which they are attached to form a 4 to 7 membered heterocycle; A is selected from the group consisting of phenyl; naphthyl; indenyl; indanyl; tetralinyl; C.sub.3-10 cycloalkyl; 4 to 7 membered heterocyclyl; and 9 to 11 membered heterobicyclyl; and wherein S.sup.0 is substituted with one group L.sup.2-Z and optionally further substituted, provided that the hydrogen marked with the asterisk in formula (I) is not replaced by a substituent; wherein L.sup.2 is a single chemical bond or a spacer; and Z is of formula (AAA2a) ##STR00063## wherein S.sup.00, S.sup.0A, S.sup.0B, S.sup.0c, S.sup.1A, S.sup.1B, S.sup.2, S.sup.3, BS.sup.1, BS.sup.2, and BS.sup.3 have the meaning as indicated for formula (AAA1) in claim 14.

18. The method of claim 2, wherein the GH related disease is selected from the group consisting of growth hormone deficiency (“GHD”), adult onset growth hormone deficiency, Turner syndrome, Prader-Willi syndrome, short bowel syndrome, chronic renal insufficiency, small for gestational age (“SGA”), AIDS wasting, anti-ageing, rheumatoid arthritis, idiopathic small stature, short stature homeobox gene, somatopause, Noonan syndrome, skeletal dysplasia, Down syndrome, short stature associated with prolonged steroid use, Aarskog's syndrome, chronic renal disease, juvenile rheumatoid arthritis; cystic fibrosis, HIV-infection in children receiving HAART treatment; short stature in children born with very low birth weight but SGA; skeletal dysplasia; hypochondroplasia; achondroplasia; idiopathic short stature; GHD in adults; fractures in or of long bones, such as tibia, fibula, femur, humerus, radius, ulna, clavicula, matacarpea, matatarsea, and digit; fractures in or of spongious bones, such as the scull, base of hand, and base of food; patients after tendon or ligament surgery in hand, knee, or shoulder; distraction oteogenesis; disorders resulting from hip or discus replacement, meniscus repair, spinal fusions or prosthesis fixation; disorders resulting from fixing of osteosynthesis material; non-union or mal-union of fractures; disorders resulting from osteatomia; disorders resulting from graft implantation; articular cartilage degeneration in knee caused by trauma or arthritis; osteoporosis in patients with Turner syndrome; osteoporosis in men; adult patients in chronic dialysis (“APCD”); malnutritional associated cardiovascular disease in APCD; reversal of cachexia in APCD; cancer in APCD; chronic abstractive pulmonal disease in APCD; HIV in APCD; elderly with APCD; chronic liver disease in APCD, fatigue syndrome in APCD; Crohn's disease; impaired liver function; males with HIV infections; short bowel syndrome; central obesity; HIV-associated lipodystrophy syndrome; male infertility; patients after major elective surgery, alcohol/drug detoxification or neurological trauma; aging; frail elderly; osteo-arthritis; traumatically damaged cartilage; erectile dysfunction; fibromyalgia; memory disorders; depression; traumatic brain injury; subarachnoid haemorrhage; very low birth weight; metabolic syndrome; glucocorticoid myopathy; and short stature due to glucucorticoid treatment in children.

19. The method of claim 2, wherein the GH related disease is growth hormone deficiency.

Description

FIGURES

[0332] In the figures the following is shown.

[0333] FIG. 1 shows SDS-PAGE analysis of purified permanent PEG-hGH conjugates, wherein Lane 1:HiMark™ Pre-stained High Molecular Weight Protein Standard; lane 2: compound 23; lane 3: compound 23; lane 4: compound 25; lane 5: compound 26, lane 6: compound 33; lane 7: compound 32; lane 8: compound 28; lane 9: compound 28; lane 10: compound 28; lane 11: compound 30; lane 12: compound 34; lane 13: compound 34; lane 14: compound 27; lane 15: compound 29.

[0334] FIG. 2 shows size exclusion chromatogram of quenched reaction solution of the synthesis of conjugate 28 and size exclusion chromatogram of purified 28

[0335] FIG. 3 shows cation exchange chromatography purification of conjugate 35 and size exclusion chromatogram of purified 35

[0336] FIG. 4 shows cation exchange chromatography purification of conjugate 36 and size exclusion chromatogram of purified 36

[0337] FIG. 5 shows cation exchange chromatography purification of conjugate 37 and size exclusion chromatogram of purified 37

[0338] FIG. 6 shows size exclusion chromatograms of samples of conjugate 35 incubated in buffer at pH 7.4 and 37° C. at various time points

[0339] FIG. 7 to 10 show preferred prodrug conjugates of the present invention indicating S.sup.0, S.sup.1, S.sup.2, L.sup.a, G.sup.a, BS.sup.1, BS.sup.2, BS.sup.3 and the critical distance.

[0340] FIG. 11 shows pharmacodynamic response curves of conjugate 36.

[0341] In detail, FIGS. 7 and 8 shows exemplary structures 35 and 38 of type (AA1, AAA1), where the at least 5 kDa polymer chain of S.sup.0 comprising G.sup.a and BS.sup.1 and BS.sup.2 is marked as S.sup.0; the carbamate group resulting from L.sup.a and the primary amino group of hGH is marked as L.sup.a; BS.sup.1 comprises the at least 4 kDa polymer chain marked as S.sup.1, wherein S.sup.1 comprises BS.sup.3, which comprises the at least 4 kDa polymer chain marked as S.sup.3. BS.sup.2 comprises the at least 4 kDa polymer chain marked as S.sup.2. The critical distance is given by 18 atoms for FIGS. 7 and 4 atoms for FIG. 8.

[0342] In FIGS. 9 and 10, exemplary structures of the type (AA2, AAA2) are shown, wherein the amide group resulting from L.sup.a and the primary amino group of hGH is marked as L.sup.a and the residue attached to L.sup.a is marked S.sup.0 comprising G.sup.a and “PEG” representing the rest of S.sup.0 comprising at least BS.sup.1, S.sup.1 and S.sup.2 (all not shown).

EXAMPLES

[0343] Methods

[0344] Analytical and Preparative RP-HPLC

[0345] Analytical RP-HPLC/ESI-MS was performed on Waters equipment consisting of a 2695 sample manager, a 2487 Dual Absorbance Detector, and a ZQ 4000 ESI instrument equipped with a 5 μm Reprosil Pur 300 A ODS-3 columns (75×1.5 mm) (Dr. Maisch, Ammerbuch, Germany; flow rate: 350 μL/min, typical gradient: 10-90% acetonitrile in water, 0.05% TFA over 5 min).

[0346] For preparative RP-HPLC a Waters 600 controller and a 2487 Dual Absorbance Detector was used equipped with the following columns (Reprosil Pur 300 A ODS-3)

[0347] A): 100×20 mm, 10 mL/min flow rate, typical gradient: 10-90% acetonitrile in water, 0.1% TFA over 11 min or

[0348] B): 100×40 mm (10 μm particles), 40 mL/min flow rate, typical gradient: 10-90% acetonitrile in water, 0.1% TFA over 11 min.

[0349] Cation Exchange Chromatography

[0350] The purification of conjugates by cation exchange chromatography was performed using an AKTA Explorer system (GE Healthcare) equipped with a Macrocap SP column. The respective conjugate in 20 mM sodium acetate buffer pH 4 was applied to the column that was pre-equilibrated in 20 mM sodium acetate buffer pH4 (buffer A). The column was washed with three column volumes of Buffer A to remove any unreacted PEG reagent. Conjugates were eluted using a gradient of 10-60% buffer B (20 mM sodium acetate, 1 M sodium chloride, pH 4.5) over 20 column volumes or 0-40% buffer B over 20 column volumes and then 40-80% B over three column volumes. The flow rate was 7 ml/min and the eluent was monitored by detection at 280 nm.

[0351] Anion Exchange Chromatography

[0352] The purification of conjugates by anion exchange chromatography was performed using an AKTA Explorer system (GE Healthcare) equipped with a Source Q column. The respective conjugate in 20 mM Tris/HCl buffer pH 7.5 (buffer C) was applied to the column that was pre-equilibrated in buffer C. The column was washed with three column volumes of buffer C to remove any unreacted PEG reagent. Conjugates were eluted using a gradient of 0-20% buffer D (20 mM Tris/HCl, 1 M sodium chloride, pH 7.5) over 25 column volumes. The flow rate was 5 ml/min and the eluent was monitored by UV detection at 280 nm. Alternatively, the buffer system 20 mM bis-tris/HCl, pH 6.5 (buffer E) and 20 mM bis-tris/HCl, 1 M sodium chloride, pH 6.5 (buffer F) was used.

[0353] Analytical Size Exclusion Chromatography

[0354] Analytical size exclusion chromatography analysis was performed on a AKTA Explorer (GE Healthcare) system. Samples were analyzed using a Superdex 200 or a Sepharose 6 column (10×300 mm) and 20 mM sodium phosphate, 135 mM sodium chloride, pH 7.4 was used as mobile phase. The flow rate for the column was 0.75 ml/min and the eluted hGH and polymer-hGH conjugates were detected at 215 nm and 280 nm.

[0355] Activity Determination of pfp-Activated mPEG-Linker Reagents

[0356] A defined amount of pfp-activated mPEG-linker reagent (3-5 mg) was dissolved in 100 μL WATER. 10 μL 0.5 M NaOH were added and the reaction mixture was reacted for 60 min at 40° C. 1.5 μL TFA were added and 10% of this mixture were analyzed by analytical RP-HPLC. The chromatograms were recorded at 260 and 280 nm. The peak corresponding to pentafluorophenol was integrated. Determined values were compared with an appropriate calibration curve generated by analyzing defined amounts of pfp by analytical RP-HPLC and integration of chromatograms recorded at 260 and 280 nm.

[0357] SDS-PAGE Analysis

[0358] The permanent mPEG-hGH conjugates were analysed using NuPAGE® Novex Tris-Acetate gels (1.5 mm thick, 15 lanes), NuPAGE Tris-Acetate SDS-Running Buffer, HiMark™ Pre-stained High Molecular Weight Protein Standard and Simply Blue™ SafeStain (Invitrogen). In each lane 0.2-0.6 μg conjugate were applied and the electrophoresis and subsequent staining performed according to the supplier's protocol.

Example 1: Assay to Measure hGH PEGylated Prodrug and hGH Activity

[0359] The biological activity of hGH is measured by using standard assays known to the skilled person in the art. As described in EP1715887B1 and as also discussed above, the biological activity associated with the native or modified hGH (for example a PEGylated hGH), can be measured using standard FDC-P1 cell proliferation assays, (Clark et al, Journal of Biological Chemistry 271:21969-21977) or receptor binding assay (U.S. Pat. No. 5,057,417).

[0360] On line 8 (page 14) of patent EP1715887B1, it is described that the preferred in vitro activity has to be as high as possible, most preferred the modified hGH has equivalent or improved in vitro biological activity, in current invention, the biological activity has to be as low as possible compared to native hGH. Thus current inventors did the complete opposite compared to the prior art described in EP1 715887B1.

[0361] In vitro Assay

[0362] The in vitro activities of the permanent PEG-hGH conjugates described in the examples below are determined using one or more standard assays for assessing biological activity in vitro. Standard assays that may be employed include cell proliferation assays using, e.g., FDC-PI cells (see, e.g., Clark et al., Journal of Biological Chemistry,

[0363] 271:21969-21977, 1996), or Ba/F3-hGHR cells, which express receptors for hGH, hGH delta 135-146, or Nb2 rat lymphoma cells, which proliferate in response to hGH via the lactogenic receptors (see, e.g., Alam, K. S., et al., J. Biotech 2000, February 28, 78(1), 49-59). Receptor binding assays (see, e.g., U.S. Pat. No. 5,057,417) may also be used.

[0364] Nb2-1 1 is a clone of the Nb-2 rat lymphoma line which was derived from a transplant of a lymphoma that developed in the thymus/lymph node of a male noble (Nb) strain rat following prolonged oestrogen treatment. The cells are of the pre-T cell origin and their proliferation is dependent on mammalian lactogens, such as prolactin. Nb2-1 1 can also be mitogenically stimulated by IL-2. Injection of Nb2 cells into Nb rats gives rise to malignant tumors that are highly sensitive to treatment with vinca alkaloids. Karyotypic analysis has shown that the cell line has only five well developed chromosome abnormalities. The cells do not express surface immunoglobulin, and their lactogens dependency is confirmed. Protocols for the use of Nb2-1 1 cells in bioassays are available from ECACC on request.

[0365] As WO20061 02659 describes on page 74 paragraph 0240 example 7, the biological activity of hGH and the conjugates described herein shall be assessed in vitro using an NB2-1 1 rat lymphoma cell proliferation assay. Briefly, NB2-1 1 cells derived from a rat lymphoma are incubated with hGH, which lead to binding of the hGH molecule to its receptor on the cell surface. Receptor binding induces the signal transduction cascade, which results in proliferation of the cells. Assay results are based on determined protein content, and a 100% bioactivity of unmodified hGH.

[0366] Conclusion:

[0367] Based on detailed instructions of this example 1 it is routine work for the skilled person to measure this residual activity of the prodrug.

Example 2: Assay to Measure Autocleavage Rate of the Transient Linker of the Prodrug

[0368] Determination of In Vitro Half-Life

[0369] For determination of in vitro linker cleavage rate of PEG-linker-hGH conjugates, the compounds are dissolved in buffer at pH 7.4 (e.g. 10 mM sodium phosphate, 140 mM NaCl, 3 mM EDTA) and solution is filtered through a 0.22 μm filter and incubated at 37° C. Samples are taken at time intervals and analyzed by RP-HPLC or size exclusion chromatography at 215 nm. Peaks corresponding to liberated hGH are integrated and plotted against incubation time. Curve fitting software is applied to determine first-order cleavage rates.

[0370] In Vivo Half-Life Determination and In Vitro/In Vivo Half-Life Correlation

[0371] Linker cleavage rates in vivo are determined by comparing the pharmacokinetics of permanent PEG-hGH conjugates with the respective transient PEG-linker-hGH conjugate carrying the same PEG moiety after intravenous injection into rat.

[0372] Firstly, permanent PEG-hGH conjugate is injected intravenously into rats and blood samples are taken at time intervals, plasma prepared, and analyzed for hGH using an ELISA.

[0373] Secondly, transient PEG-hGH conjugate is injected intravenously in rats, blood samples are taken at time intervals, plasma prepared, and analyzed for hGH using an ELISA.

[0374] In vivo half-life is calculated from the ratio of hGH concentration of transient conjugate divided by determined hGH concentration of permanent conjugate at the respective time points and curve fitting. Data are compared to in vitro half-life measurements.

[0375] Conclusion

[0376] Based on detailed instructions of this example 2 it is routine work for the skilled person to measure the in vivo half-life of the hGH-PEGylated prodrug.

Example 3: Assay to Measure Lipoatrophy

[0377] As said above compound PHA-794428 is a PEGylated-hGH and described in patent EP1715887 from the company Pharmacia. According to www.clinicaltrials.gov, the study was terminated on 10 Dec. 2007. Pfizer's (Pharmacia) decision to terminate the program was due to cases of injection-site lipoatrophy that were reported in the clinical Phase 2 studies after a single injection of PH A 794428. Lipoatrophy is the term describing the localized loss of fat tissue and is visible on humans as holes in the skin (visible by the eye).

[0378] Assay

[0379] There are several in vitro methods described in the art in measure lipoatrophy. One proposal is described in publication J. Anim. Sci (1972), 35: 794-800 (L J. Machlin) on page 795. Another description is found in Int. J. Cancer; 80, 444-447 (1999).

[0380] Generally, lipoatrophy can be measured as proposed below.

[0381] Lipolytic effect can be determined using an in vitro assay consisting of isolated mammal adipocytes, preferable murine adipocytes. Samples to be assayed were incubated at physiologically relevant conditions with a predetermined number of adipocytes in Krebs-Ringer bicarbonate buffer containing appropriate nutrients for up to 6 hours. The concentration of released glycerol is determined by standard methods, for example enzymatically or by a radiometric detection. Control samples containing adipocytes alone are analyzed to determine the spontaneous glycerol release.

[0382] The lipolytic effect of native unmodified recombinant human growth hormone and permanently PEGylated recombinant human growth hormone is compared to that of transiently PEGylated recombinant human growth hormone.

[0383] Conclusion

[0384] Based on detailed instructions of this example 3 it is routine work for the skilled person to measure the lipoatrophy effect.

Example 4 Synthesis of Permanent Linker Reagent 12a and Transient Linker Reagents 12b and 12c

[0385] Synthesis of Compound 6

##STR00034##

[0386] 6-Amino-hexan-1-ol (2.85 g, 24.3 mmol) was dissolved in aq. HBr (48%, 10 mL, 89 mmol) and stirred at 80° C. for 3 h. Excess HBr was evaporated at 50-65° C. and 15 Torr and the residue was dried in vacuo.

[0387] 1: Yield 6.07 g (96%)

[0388] MS [M+H].sup.+=180.3 g/mol (MW+H calculated=180.0 g/mol)

[0389] DBU (3.5 mL, 23.2 mmol) was added to a suspension of 6-Bromohexylamine hydro-bromide 1 (3.03 g, 11.6 mmol) and triphenyl-methanethiol (2.14 g, 7.74 mmol) in DCM (25 mL) and DMSO (13 mL) were added. The reaction mixture was stirred for 30 min at room temperature and diluted with water (150 mL).

[0390] The aqueous layer was extracted with ether and the combined organic phase was evaporated. 2 was purified by RP-HPLC.

[0391] 2: Yield 1.17 g (40%)

[0392] MS [M+H].sup.+=376.7 g/mol (MW+H calculated=376.2 g/mol).

[0393] DBU (4.56 mL, 30.0 mmol) was added to 6-bromo-hexanoic acid (3.90 g, 20.0 mmol) and triphenyl-methanethiol (11.1 g, 40.0 mmol) in DCM (40 mL). After stirring at room temperature for 1 h ice cold 1 M H.sub.2SO.sub.4 (50 ml.) was added and the mixture was extracted with DCM. The combined organic phase was dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Compound 3 was purified by silica gel column chromatography (200 ml.) using heptane/ethyl acetate (4/1, R.sub.f=0.2) as mobile phase. 3: Yield 5.83 g (75%)

[0394] DMAP (37 mg, 0.31 mmol) was added to 6-tritylsulfanyl-hexanoic acid 3 (5.83 g, 14.9 mmol), thiazolidine-2-one (3.56 g, 29.9 mmol), and dicyclohexylcarbodiimide (3.08, 14.9 mmol) in DCM (100 mL). After stirring at room temperature for 1 h 1 M HCl (0.6 ml.) was added and the mixture was filtered. The filtrate was concentrated in vacuo and 4 was purified by silica gel column chromatography (180 ml.) using heptane/ethyl acetate (1/1) as mobile phase.

[0395] 4: Yield 7.15 g (97%) as yellow oil

[0396] A solution of 1-(2-thioxo-thiazolidin-3-yl)-6-tritylsulfanyl-hexan-1-one 4(1.53 g, 3.11 mmol) in THF (13 ml.) was added over a period of 2 min to 6-tritylsulfanyl-hexylamine 2 (1.17 g, 3.11 mmol) in DMSO (1 ml.) and THF (5 mL). After addition of triethylamine (435 μL, 3.11 mmol) the reaction mixture was stirred for 90 min at room temperature. Ether (200 ml.) and water (100 ml.) were added and the phases separated. After extraction of the aqueous phase with ether the combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Compound 5 was purified by silica gel column chromatography (150 ml.) using heptane/ethyl acetate (2/1, R.sub.f=0.1) as mobile phase.

[0397] 5: Yield 1.23 g (53%)

[0398] MS [M+Na].sup.+=770.6 g/mol (MW+Na calculated=770.4 g/mol)

[0399] Under nitrogen, a 1M solution of LiAlH.sub.4 in THF (1.2 ml, 4.8 mmol) was placed in a dry flask, and a solution of 5 (509 mg, 0.680 mmol) in 10 ml of THF was added over 4 min. The mixture was stirred under reflux for 2 h, until complete conversion of the starting material was shown by thin layer chromatography (heptanes/ethyl acetate 1:1). The reaction mixture was carefully quenched with a 10:1 suspension of water in diethyl ether until the gas evolution had stopped. The mixture was poured into 50 ml of a saturated solution of sodium potassium tartrate and stirred for 90 min. 90 ml of ethyl acetate were added and the phases were separated. The aqueous phase was extracted with ethyl acetate (4×20 ml), and the combined organic phase was washed with brine (30 ml), dried over MgSO.sub.4, filtered, and concentrated to give a transparent oil. 6 was adsorbed on silica and purified by flash chromatography (30 g silica, CH.sub.2Cl.sub.2/MeOH 20:1 (v/v)+0.1% NEt.sub.3). The product was obtained as an off-white viscous oil.

[0400] 6: Yield 270 mg (54%)

[0401] MS [M+H].sup.+=734.4 g/mol (MW+H calculated=734.4 g/mol)

[0402] R.sub.f=0.28 (CH.sub.2Cl.sub.2/MeOH 19:1)

[0403] Synthesis of Compound 9

##STR00035##

[0404] AlCl.sub.3 (23.0 g, 172.3 mmol) was added to glutaric anhydride (10.0 g, 86.2 mmol) in anisole (85 mL, 781 mmol). The reaction mixture was heated to 110° C. for 2 h, cooled to room temperature, poured on 3 N HCl/ice and extracted with dichloromethane. The aqueous phase was extracted with dichloromethane (4×20 ml), and the combined organic fractions were washed with brine (30 ml), dried over MgSO.sub.4, filtered and concentrated to give a red oil that was recrystallized from toluene. Product 7 was obtained as an off-white solid.

[0405] 7: Yield 5.2 g (48%)

[0406] MS [M+Na].sup.+=245.8 (MW+Na calculated=245.1 g/mol)

[0407] AlCl3 (9.0 mg, 68 mmol) was added to 7 (5.0 g, 23 mmol) in 1,2-dichloroethane. The reaction mixture was stirred for 14 h at 85° C. and subsequently cooled to room temperature. Ice cold 1 N HCl (50 ml.) was added and the mixture was extracted with ethyl acetate (4×30 mL). The combined organic fractions were dried over Na.sub.2SC>.sub.4, filtered and concentrated in vacuo to give a light red solid that was used in the next step without further purification.

[0408] 8: Yield 3 g (62%)

[0409] MS [M+H].sup.+=209.1 (MW+H calculated=209.1 g/mol)

[0410] dicyclohexylcarbodiimide (532 mg, 2.6 mmol), acid 8 (403 mg, 1.9 mmol), HOSu (297 mg, 2.6 mmol), and collidine (1.0 mL, 7.8 mmol) in DCM (10 ml.) were stirred for 90 min at room temperature. After removal of dicyclohexylurea by filtration, amine 6 (947 mg, 1.3 mmol) in DCM (5 ml.) and DIEA (450 μL, 2.6 mmol) were added to the filtrate and the mixture was reacted for 14 h at room temperature. 1 N H.sub.2SO.sub.4 (2×50 ml.) was added and the phases were separated. The aqueous phase was extracted with ethyl acetate (4×20 ml), and the combined organic phase was washed with brine (30 ml), dried over MgSO.sub.4, filtered and concentrated in vacuo. The residues were purified by silica gel column chromatography (150 ml.) using heptane/ethyl acetate (½, R.sub.f=0.66) as mobile phase.

[0411] 9: Yield 819 mg (69%)

[0412] MS [M+Na].sup.+=946.4 (MW+Na calculated=946.4 g/mol)

[0413] Synthesis of Permanent Linker Reagent 12a and Transient Linker Reagent 12b and 12c

##STR00036##

[0414] 9 (1 eq., 175 mg, 0.19 mmol) was dissolved in dry THF (1.5 mL, p-nitrophenylchloroformate (1.1 eq., 42 mg, 0.21 mmol) and DIPEA (2 eq., 66 μl, 0.38 mmol) were added and the mixture was stirred for 60 min at room temperature. Di-ethylamine (R1═R2=Et, 2 eq., 39 gi, 0.38 mmol) was added and stirring was continued for 15 min. The solvent was removed in vacuo, 100 μl of AcOH were added and 10a was purified by RP-HPLC.

[0415] MS [M+Na].sup.+=1045.9 (MW+Na calculated=1045.5 g/mol)

[0416] NaBH.sub.4 (5 eq., 37 mg, 0.95 mmol) was added to 10a containing HPLC fraction (acetonitrile/H20 ˜3/1 (v/v)+0.1% TFA) and the mixture was reacted for 10 min at room temperature. An additional portion of NaBH.sub.4 (5 eq., 37 mg, 0.95 mmol) was added and the reaction mixture was stirred until complete conversion of the starting material was indicated by LC/MS analysis (10 min at room temperature). 11a was purified by RP-HPLC and lyophilized.

[0417] 11a: Yield 95 mg (49% based on 9)

[0418] MS [M+Na+H].sup.+=1047.7 (MW+Na calculated=1047.5 g/mol)

[0419] 9 (1 eq., 175 mg, 0.19 mmol) was dissolved in dry THF (1.5 mL), p-nitrophenylchloroformate (1.1 eq., 42 mg, 0.21 mmol) and DIPEA (2 eq., 66 μl, 0.38 mmol) were added and the mixture was stirred for 60 min at room temperature. N,N,N′-Triethyl-ethane-1,2-diamine (R1=Et, R2=2-(diethylamino)ethyl, 2 eq., 68 μl, 0.38 mmol) was added and stirring was continued for 15 min. 100 μl of AcOH were added, the solvent was removed in vacuo and 10b was purified by RP-HPLC and lyophilized.

[0420] 10b: Yield 147 mg as TFA salt (65%)

[0421] MS [M+Na].sup.+=1116.4 (MW+Na calculated=1116.6 g/mol)

[0422] 10c was synthesized as described above using N,N,N′-trimethyl-propane-1,3-diamine (R1=Me, R2=3-(dimethylamino)propyl, 56 μL, 0.38 mmol) as diamine.

[0423] 10c: Yield 134 mg as TFA salt (59%)

[0424] MS [M+Na].sup.+=1088.4 (MW+Na calculated=1088.6 g/mol)

[0425] NaBH.sub.4 (46 mg, 1.2 mmol) was added to 10b (147 mg, 0.12 mmol) in MeOH/water=95:5 (v/v) (3 mL) in two doses and the mixture was stirred for 1h at room temperature. After addition of AcOH (300 μL) and concentration, product 11b was purified by RP-HPLC and lyophilized.

[0426] 11b: Yield 107 mg as TFA salt (73%)

[0427] MS [M+Na].sup.+=1118.4 (MW+Na calculated=1118.6 g/mol)

[0428] 11c was synthesized according to the same protocol.

[0429] 11c: Yield 65 mg as HCl salt (54%) from 134 mg starting material

[0430] MS [M+Na].sup.+=1090.4 (MW+Na calculated=1090.6 g/mol)

[0431] Under a nitrogen atmosphere bis-pentafluorophenyl-carbonate (2.5 eq., 25 mg, 63 μmol), DMAP (1 mg), and DIEA (5 eq., 22 μL, 127 μmol) were added to 11a (1 eq., 26 mg, 26 μmol) in dry acetonitrile (0.5 mL. The reaction mixture was stirred for 30 min at room temperature, cooled to 0° C., and acidified with AcOH (200 μL. Product

[0432] 12a was purified by RP-HPLC and lyophilized.

[0433] 12a: Yield 13 mg (42%)

[0434] MS [M+Na].sup.+=1258.2 (MW+Na calculated=1257.5 g/mol)

[0435] 12b and 12c were prepared accordingly from 11b (56 mg, 48 μmol) and 11c (88 mg, 73 μmol), respectively.

[0436] 12b: Yield 63 mg as TFA salt (93%)

[0437] MS [M+H].sup.+=1306.3 (MW+H calculated=1306.6 g/mol)

[0438] 12c: Yield 4 1 mg as TFA salt (41%)

[0439] MS [M+H].sup.+=1278.4 (MW+Na calculated=1278.5 g/mol)

Example 5 Synthesis of Permanent Linker Reagent 14a and Transient Linker Reagents 14c

[0440] ##STR00037##

[0441] 13a and 13c were synthesized as described in WO2005/099768A2.

[0442] Under an atmosphere of nitrogen bispentafluorophenylcarbonate (631 mg, 1.6 mmol), DMAP (20 mg, 0.16 mmol), and DIEA (556 μL, 3.2 mmol) were added to 13a (364 mg, 0.64 mmol) in dry acetonitrile (5 mL). The reaction mixture was stirred for 15 min at room temperature, cooled to 0° C., and acidified with acetic acid (1 mL). Product

[0443] 14a was purified by RP-HPLC and lyophilized.

[0444] 14a: Yield 379 mg (77%)

[0445] MS [M+Na].sup.+=800.4 (MW+Na calculated=800.3 g/mol)

[0446] 14c was prepared accordingly from 13c (97 mg, 130 μmol).

[0447] 14c: Yield 114 mg as TFA salt (94%)

[0448] MS [M+H].sup.+=821 0.5 (MW+H calculated=821.3 g/mol)

Example 6 Synthesis of Permanent Linker Reagent 15

[0449] ##STR00038##

[0450] Glutaric anhydride (0.41 mmol), amine 6 (200 mg, 0.27 mmol), DIPEA (72 μL, 0.41 mmol), and DMAP (11 mg, 0.09 mmol) were stirred in acetonitrile (3 ml.) for 2h at 80° C. The mixture was cooled to room temperature and acetic acid (200 μL) was added. Product 15 was purified by RP-HPLC and lyophilized.

[0451] 15: Yield 130 mg (57%)

[0452] MS [M+Na].sup.+=870.2 (MW+Na calculated=870.4 g/mol)

Example 7 Synthesis of Activated mPEG-Linker Reagents

[0453] mPEG-Maleimide Starting Materials:

##STR00039##

[0454] mPEG residues after reacting with thiol group (R3 in structures below):

##STR00040##

[0455] The vertical dashed line denotes the attachment site to the thiol group in the respective structure

[0456] Synthesis of Permanent pfp-Activated mPEG-Linker Reagents 17aa, 17ab, 17ac, 17ad, and Transient pfp-Activated mPEG-Linker Reagents 17b, 17ca, and 17cb

##STR00041##

[0457] Carbonate 12a (13 mg, 10 μmol) was stirred in 10 μL AcOH, 700 μL, HFIP, 1 μL TFA and 2 μL TES for 10 min at room temperature. The volatile components were removed in a nitrogen stream and 16a was purified by RP-HPLC.

[0458] 16a: Yield 3.8 mg (5 g/mol)

[0459] MS [M+H].sup.+=751 0.3 (MW+H calculated=751.3 g/mol)

[0460] 16b and 16c were prepared accordingly from 12b (7.7 mg, 5.4 μmol) and 12c (2 mg, 1.5 μmol), respectively.

[0461] 16b: Yield 2.5 mg (2.7 μmol)

[0462] MS [M+Na].sup.+=845.1 (MW+Na calculated=844.3 g/mol)

[0463] 16c: Yield 0.5 mg (0.6 μmol)

[0464] MS [M+Na].sup.+=816.6 (MW+Na calculated=816.3 g/mol)

[0465] mPEG-maleimide 1B (NOF, Japan) (521 mg, 12.7 μmol) was added to 3.5 mg (3.9 μmol) 16c in 4 ml. 3/1 (v/v) acetonitrile/water+0.1% TFA. 200 μL of 0.5 M phosphate buffer pH 7.4 were added and the mixture was reacted for 10 min at room temperature. 1 μL (13 μmol) mercaptoethanol were added and the reaction mixture was acidified to pH 4-5 by addition of TFA. 17 ca was purified by RP-HPLC and lyophilized.

[0466] 17ca: Yield 220 mg (pfp-carbonate activity 82%)

[0467] 17cb was synthesized as described for 17ca using 16c (3.5 mg, 3.9 μmol) and mPEG-maleimide 2B (656 mg, 16 μmol).

[0468] 17cb: Yield 130 mg (pfp-carbonate activity 85%)

[0469] 184 mg (8.8 μmol) mPEG-maleimide 1A (NOF, Japan) were added to 16a (2.0 mg, 2.7 μmol) in 4 ml. 1/1 (v/v) acetonitrile/water+0.1% TFA. 200 μL of 0.5 M phosphate buffer pH7.4 were added and the mixture was reacted for 10 min at room temperature. 0.2 μL (1.6 μmol) mercaptoethanol were added and the reaction mixture was acidified to pH 2-3 by addition of TFA. 17aa was separated from unreacted PEGs by RP-HPLC and lyophilized.

[0470] 17aa: Yield 90 mg (pfp-carbonate activity 88%)

[0471] 17ab was synthesized as described above using 16a (3.8 mg, 5.0 μmol) and 680 mg (16 μmol) mPEG-maleimide 1B (NOF, Japan).

[0472] 17ab: Yield 250 mg (pfp-carbonate activity 83%)

[0473] 17ac was synthesized as described above using 16a (2.5 mg, 3.3 μmol) and 200 mg (9.5 μmol) mPEG-maleimide 2A (Jenkem, PR China).

[0474] 17ac: Yield 80 mg (pfp-carbonate activity 80%)

[0475] 17ad can be synthesized as described above using 16a and mPEG-maleimide 2B.

[0476] 17b can be synthesized as described for 17cb using 16b and mPEG-maleimide 1B.

Example 8 Synthesis of pfp-Activated Permanent mPEG Linker Reagents 19aa and 19ab and Transient Permanent mPEG-Linker Reagent 19c

[0477] ##STR00042##

[0478] Carbonate 14c (20 mg, 21 μmol) was stirred in 10 μL AcOH, 400 μL HFIP, and 5 μL TES for 10 min at room temperature and cooled to 0° C. Ice cold acetonitrile/water=9/1 (v/v) was added and 18c was separated by RP-HPLC and lyophilized.

[0479] 18c: Yield 5.0 mg as TFA salt (7.2 μmol)

[0480] MS [M+H].sup.+=579.6 (MW+H calculated=579.2 g/mol)

[0481] 18a was synthesized as described above using carbonate 14a (24 mg, 31 μmol).

[0482] 18a: Yield 8.0 mg (15 μmol)

[0483] MS [M+H].sup.+=536.2 (MW+H calculated=536.5 g/mol)

[0484] 205 mg (5 μmol) mPEG-maleimide 3A (NOF, Japan) were added to 18a (4.0 mg, 7.5 μmol) in 2 ml. 1/1 (v/v) acetonitrile/water+0.1% TFA. 100 μL of 0.5 M phosphate buffer (pH7.4) were added and the mixture was reacted for 10 min at room temperature. The reaction mixture was acidified to pH 2-3 by addition of TFA and 19aa was separated from unreacted PEGs by RP-HPLC and lyophilized.

[0485] 19aa: Yield 125 mg (pfp-carbonate activity 85%)

[0486] 19ab was prepared accordingly from 410 mg (5 μmol) mPEG-maleimide 3B (NOF, Japan) and 18a (4.0 mg, 7.5 μmol).

[0487] 19ab: Yield 265 mg (pfp-carbonate activity 87%)

[0488] 19c was prepared accordingly from 205 mg mPEG-maleimide 3A and 18c (5 mg, 7.2 μmol)

[0489] 19c: Yield 120 mg (pfp-carbonate activity 88%)

Example 9: Synthesis of Permanent 4-Arm Branched 80 kDa mPEG-NHS ester Derivative

[0490] ##STR00043## ##STR00044##

[0491] Acid 15 (12 mg, 14 μmol) was stirred in 1 ml. TFA, 1 ml. DCM, and 10 μL TES for 10 min at room temperature. The volatile components were removed in a nitrogen stream and the dithiol 20 was purified by RP-HPLC.

[0492] 20: Yield 2.9 mg (8 μmol)

[0493] MS [M+Na].sup.+=386.8 (MW+Na calculated=386.2 g/mol)

[0494] 20 (1 mg, 2.8 μmol) in 170 μL acetonitrile was added to mPEG-maleimide 1B (NOF, Japan) (380 mg, 9.2 μmol) in 4 ml. 1/1 (v/v) acetonitrile/water+0.1% TFA. 200 μL of 0.5 M phosphate buffer pH7.4 were added and the mixture was reacted for 10 min at room temperature. 0.6 μL (7.8 μmol) mercaptoethanol were added and the reaction mixture was acidified to pH 4-5 by addition of TFA. The buffer was exchanged to 0.005% HCl (HiPREP Desalting column, 26/10 GE healthcare) and 21 was lyophilized without further purification.

[0495] 21: Yield 320 mg

[0496] 21 was dissolved in 50 ml. of toluene and the polymer solution was azeotropically dried for two hours under reflux using a Dean-Stark trap. The polymer solution was then cooled to room temperature. The dried mPEG-linker reagent 21 was precipitated by addition of chilled ether (60 mL).

[0497] dicyclohexylcarbodiimide (1.2 mg, 6 μmol) in DCM was added to a solution of 21 (240 mg, 3 μmol) and N-hydroxy-succinimide (0.7 mg, 6 μmol) in DCM (3 mL). The reaction mixture was stirred for 14 h at room temperature and 22 was precipitated by addition of cold ether (20 mL). Product 22 was dried in vacuo.

[0498] 22: Yield 200 mg

Example 10: Synthesis of Permanent Amide-Linked mPEG-hGH Monoconjugate 23 and mPEG.SUB.2.-hGH Bisconjugate 24 Using Linear 40 kDa mPEG-Succinimidyl Hexanoate Derivative

[0499] ##STR00045##

[0500] hGH was buffer exchanged to 50 mM sodium borate pH 8.5 (alternatively sodium borate pH 8 or sodium borate pH 9 can be used). The concentration of hGH was approximately 2.5 mg/ml. A three-fold molar excess of 40 kDa mPEG-succinimidyl hexanoate derivative (NOF, Japan) relative to the amount of hGH was dissolved in water to form a 20% (w/v) reagent solution (alternatively a four-fold or five-fold molar excess can be used). The reagent solution was added to the hGH solution and mixed. The reaction mixture was incubated for 2h at room temperature and quenched with hydroxylamine at room temperature and pH 7 for two hours. The quenched reaction mixture was analyzed by size exclusion chromatography. The monoconjugate 23 and bisconjugate 24 were purified by cation exchange chromatography. Alternatively, anion exchange chromatography can be used for purification. The purified conjugates were analyzed by SDS-PAGE (FIG. 1).

Example 11: Synthesis of Permanent Amide-Linked mPEG-hGH Monoconjugate 25 and mPEG-hGH Bisconjugate 26 Using Branched 40 kDa mPEG-NHS Ester Derivative

[0501] ##STR00046##

[0502] Permanent mPEG-hGH monoconjugate 25 and bisconjugate 26 were synthesized according to the procedure described in Example 10 using branched 40 kDa mPEG-NHS ester derivative (NOF, Japan). The purified conjugates were analyzed by SDS-PAGE (FIG. 1).

Example 12: Synthesis of Permanent Amide-Linked mPEG-hGH Monoconjugate 27 Using 4-Arm Branched 80 kDa mPEG-NHS Ester Derivative

[0503] ##STR00047##

[0504] Permanent mPEG-hGH monoconjugate 27 was described according to Example 10 using 4-arm branched 80 kDa mPEG-NHS ester derivative 22. Purified 27 was analyzed by SDS-PAGE (FIG. 1).

Example 13: Synthesis of Permanent Carbamate-Linked mPEG-hGH Monoconjugate 28 Using 4-Arm Branched 40 kDa mPEG-Pentafluorophenylcarbonate Derivative 17aa

[0505] ##STR00048##

[0506] hGH was buffer exchanged to 50 mM sodium borate pH 9 (alternatively sodium borate pH 8.5 or sodium borate pH 8 can be used). The concentration of hGH was approximately 2.5 mg/ml. A four-fold molar excess of permanent 4-arm branched 40 kDa mPEG-linker reagent 17aa relative to the amount of hGH was dissolved in water to form a 20% (w/v) reagent solution. The reagent solution was added to the hGH solution and mixed. The reaction mixture was incubated for 1.5 h at room temperature and quenched by incubating in 100 mM hydroxylamine at pH 7 and room temperature for 2 h. The quenched reaction mixture was analyzed by size exclusion chromatography (FIG. 2 top). Permanent mPEG-linker-hGH monoconjugate 28 was purified by anion exchange chromatography at pH 7.5 and analyzed by SDS-PAGE (FIG. 1) and size exclusion chromatography (FIG. 2 bottom).

Example 14: Synthesis of Permanent Carbamate-Linked mPEG-hGH Monoconjugate 29 Using 4-Arm Branched 80 kDa mPEG-Pentafluorophenylcarbonate Derivative

[0507] ##STR00049##

[0508] Permanent carbamate-linked mPEG-hGH monoconjugate 29 was synthesized according to Example 13 using 4-arm branched 80 kDa mPEG-pentafluorophenyl carbonate derivative 17ab. Purified 29 was analyzed by SDS-PAGE (FIG. 1).

Example 15: Synthesis of Permanent Carbamate-Linked mPEG-hGH Monoconjugate 30 Using 4-Arm Branched 40 kDa mPEG-Pentafluorophenylcarbonate Derivative 17ac

[0509] ##STR00050##

[0510] Permanent mPEG-hGH monoconjugate 30 was synthesized according to Example 13 using 4-arm branched 40 kDa mPEG-pentafluorophenyl carbonate derivative 17ac. Purified 30 was analyzed by SDS-PAGE (FIG. 1).

Example 16: Synthesis of Permanent mPEG-hGH Monoconjugate 31 Using 4-Arm Branched 80 kDa mPEG-Pentafluorophenylcarbonate Derivative

[0511] ##STR00051##

[0512] Permanent mPEG-hGH monoconjugate 31 can be synthesized according to Example 13 using 4-arm branched 80 kDa mPEG-pentafluorophenyl carbonate derivative 17ad.

Example 17: Synthesis of Permanent Carbamate-Linked mPEG-hGH Monoconjugate 32 Using 4-Arm Branched 40 kDa mPEG-Pentafluorophenylcarbonate Derivative

[0513] ##STR00052##

[0514] Permanent carbamate-linked mPEG-hGH monoconjugate 32 was synthesized according to Example 13 using 4-arm branched 40 kDa mPEG-pentafluorophenyl carbonate derivative 19aa. Purified 32 was analyzed by SDS-PAGE (FIG. 1).

Example 17: Synthesis of Permanent Carbamate-Linked mPEG-hGH Monoconjugate 33 Using 4-Arm Branched 80 kDa mPEG-Pentafluorophenylcarbonate Derivative

[0515] ##STR00053##

[0516] Permanent mPEG-hGH monoconjugate 33 was synthesized according to Example 13 using 4-arm branched 80 kDa mPEG-pentafluorophenyl carbonate derivative 19ab. [0517] Purified 33 was analyzed by SDS-PAGE (FIG. 1).

Example 19: Synthesis of Permanent Amine-Linked mPEG-hGH Monoconjugate 34 Using Branched 40 kDa mPEG-Propionaldehyde Derivative

[0518] ##STR00054##

[0519] hGH was buffer exchanged to 50 mM MES buffer pH 6 (alternatively HEPES buffer pH 7 was used) and the concentration of hGH was adjusted to 1.5 mg/ml. A three-fold molar excess of 40 kDa mPEG-propionaldehyde (GL2-400AL3, NOF, Japan) relative to the amount of hGH was dissolved in water to form a 25% (w/v) reagent solution. The reagent solution was added to the hGH solution and mixed. An aliquot of a 1M stock solution of sodium cyanoborohydride in water was added to give a final concentration of 25 mM in the reaction mixture. The solution was incubated for 18h at room temperature in the dark. The reaction was quenched by the addition of Tris buffer. The reaction mixture was analyzed by size exclusion chromatography and conjugate 34 was purified by cation exchange chromatography. Purified mPEG-hGH monoconjugate 34 was analyzed by SDS-PAGE (FIG. 1).

Example 20: Synthesis of Transient Carbamate-Linked mPEG-hGH Monoconjugate 35 Using Transient 4-Arm Branched 40 kDa m PEG-Pentafluorophenylcarbonate Derivative 19c

[0520] ##STR00055##

[0521] hGH was buffer exchanged to 50 mM sodium borate pH 9 (alternatively sodium borate pH 8.5 or sodium borate pH 8 can be used) and the concentration of hGH was adjusted to 2.5 mg/ml. A four-fold molar excess of transient mPEG-linker reagent 19c relative to the amount of hGH was dissolved in water to form a 20% (w/v) reagent solution. The reagent solution was added to the hGH solution and mixed. The reaction mixture was incubated for 1 h at room temperature and quenched by incubating in 100 mM hydroxylamine at pH 7 and room temperature for 2 h. mPEG-linker-hGH monoconjugate was purified by anion exchange chromatography at pH 6.5 (FIG. 3 top) and analyzed by size exclusion chromatography (FIG. 3 bottom).

Example 21: Synthesis of Transient mPEG-Linker-hGH Monoconjugate 36 Using 4-Arm Branched 80 kDa mPEG-Pentafluorophenylcarbonate Derivative

[0522] ##STR00056##

[0523] hGH was buffer exchanged to 100 mM sodium borate pH 9 (alternatively sodium borate pH 8.5 or sodium borate pH 8 can be used) and the concentration of hGH was adjusted to 10 mg/ml. A four-fold molar excess of transient 4-arm branched 80 kDa mPEG-linker reagent 17ca relative to the amount of hGH was dissolved in water to form a 25% (w/v) reagent solution. The reagent solution was added to the hGH solution and mixed. The reaction mixture was incubated for 45 min at room temperature and quenched by incubating in 100 mM hydroxylamine at pH 7 and room temperature for 2 h. mPEG-linker-hGH monoconjugate 36 was purified by cation exchange chromatography (FIG. 4 top) and analyzed by size exclusion chromatography (FIG. 4 bottom).

Example 22: Synthesis of Transient mPEG-hGH Monoconjugate 37 Using 4-Arm Branched 80 kDa mPEG-Pentafluorophenylcarbonate Derivative 17cb

[0524] ##STR00057##

[0525] PEG-linker-hGH conjugate 37 was synthesized as described according to the procedure described in Example 21 using activated mPEG-linker reagent 17cb.

[0526] The cation exchange chromatogram and analytical size exclusion chromatogram are shon in FIG. 5 top and bottom, respectively.

Example 23: Synthesis of Transient Carbamate-Linked mPEG-hGH Monoconjugate 38 Using 4-Arm Branched 80 kDa mPEG-Pentafluorophenylcarbonate Derivative 17b

[0527] ##STR00058##

[0528] Transient carbamate-linked mPEG-linker-hGH conjugate 38 can be synthesized as described in Example 21 using transient 4-arm branched 80 kDa mPEG-linker reagent 17b.

Example 24: Assay to Measure hGH PEGylated Prodrug and hGH Activity

[0529] It is routine work for the skilled person to determine the residual activity of the polymeric prodrug as expressed by the activity of the corresponding permanent polymer conjugate using standard assays as described in example 1.

[0530] Specifically, NB2-1 1 cells were grown in serum free media with 100 ng/ml hGH supplement. For the in vitro proliferation assay, cell suspension containing 2×10.sup.5 cells/ml were washed twice with serum free and hGH free medium and dispensed into a 96-well flat bottom microtitre plate (10.sup.4 cells/well). Compounds were tested in triplicate in a series of titration steps (9 steps, using a factor 3 dilution between each step). The cells with compound solutions were incubated for 48 hours followed by incubation for 2.5 hours with cell proliferation reagent WST-1. NB2-1 1 proliferation was determined by optical density reading in an ELISA reader and the response plotted as a function of concentration and EC50 values determined. The results are shown as % residual in vitro bioactivity in relation to unmodified hGH is provided in table 1.

[0531] In the in vitro experiments as described above, native hGH (source Novo Nordisk, Den-mark) was used as reference compound. The same hGH preparation was used for the synthesis of the permanent PEG-hGH conjugates.

TABLE-US-00001 TABLE 1 In vitro bioactivity of permanently PEGylated hGH conjugates as compared to native hGH (Norditropin, Novo Nordisk, Denmark). In vitro characterization: In vitro activity of permanent Compound conjugates Native hGH (hGH, Novo  100% Nordisk, Denmark) 23 10.3% 24  0.4% 25  4.4% 26  0.2% 27  0.7% 28  2.3% 29  0.7% 30  2.0% 32  6.3% 33  2.2% 34  4.8%

[0532] Conclusion:

[0533] As seen from table 1, by conjugation of a suitable PEG molecule to hGH, the in vitro activity of the PEGylated hGH can be reduced to less than 5% of the activity of the native unconjugated hGH. For example, conjugation of a branched PEG 4×20 kDa to hGH reduces the residual activity to 0.7% of the unconjugated hGH standard.

[0534] Furthermore, from these results it was also surprisingly discovered, that the residual activity of the PEGylated growth hormone is related not only to the size of the attached PEG, but also to the degree of branching and the spacing between the hGH and the branching points within the PEG structure.

[0535] Linear PEG

[0536] Specifically, attachment of a 40 kDa linear PEG to hGH results in an in vitro activity of 10.3% (compound 23) compared to native hGH.

[0537] Branched PEG

[0538] When a branched 2×20 kDa PEG is attached (compound 25), the in v/fro activity is further reduced to 4.4% compared to native hGH.

[0539] Further, when a 4×20 kDa PEG with a short spacing between the hGH and the branching points within the PEG reagent is attached (compound 27 and 29) the in vitro activity is even further reduced to respectively 0.7% compared to native hGH.

[0540] Surprisingly, when a 4×20 kDa PEG with a relative long spacer between the human growth hormone and the first branching point within the PEG reagent is attached (e.g. compound 33) the in vitro activity is less reduced (2.2%) showing the importance of the spacer between the hGH functional group and the first branching point within the branched PEG reagent.

[0541] Conjugation of more than one PEG moiety to the hGH to form bisPEG-conjugates reduces the in vitro activity to lower than 0.5%. (e.g. compound 24 and 26).

Example 25: Determination of In Vitro Autocleavage Rate of Conjugate 35, 36, 37, and 38

[0542] The autocleavage rate of conjugate 35, 36 and 37 at pH 7.4 and 37° C. was determined as described in Example 2. Autocleavage half-lives of approximately 75 h were determined for these conjugates. FIG. 6 shows size exclusion chromatograms of samples of incubated 35 analyzed after 0 h, 8 h, 47 h, 97 h, and 168 h showing slow release of hGH from conjugate 35 over time. Autocleavage rate of conjugate 38 can be determined accordingly and give half-lives of ca. 50h.

Example 26: Assay to Measure Terminal In Vivo Half Life of the hGH PEGylated Prodrugs as Expressed by the Half Life of the Corresponding Permanent Conjugate In Vivo

[0543] The pharmacokinetics of the permanent conjugates were determined after intravenous injection of 0.25 mg (hGH equivalents) into rats. In order to select a conjugate suitable for weekly injections in humans, a plasma half life of more than 10 hours in the rat is desirable.

[0544] A single dose of 0.25 mg hGH or 0.25 mg permanent PEG-hGH conjugate (dose based on hGH) per rat was administered intravenously to male Wistar rats (200-250 g). Two groups of two animals each were used for each compound. 200-300 μl whole blood was withdrawn sublingually to obtain 100 μl Ca-Heparin plasma per animal and time point. Samples were collected after 0.5, 3, 24, 48, 72 and 96h for group 1 and after 5, 8, 32, 56, 80 and 168 h for group 2. Plasma samples were stored at −80° C. until assayed.

[0545] hGH and PEG-hGH conjugate concentrations were measured using a hGH ELISA kit (DSL). Plasma concentrations were calculated from a calibration curve of the respective conjugate or hGH and plotted against time, and the terminal half-life (t.sub.1/2) was calculated using a single compartment model. The result of the half life determination is tabulated in table 2.

[0546] In order to select a conjugate suitable for weekly injections in humans pharmacokinetic studies in rats were performed. As the half life of PEGylated conjugates in rats are in the range of 5 times faster than in humans, the half life of a PEGylated hGH in rats should be about 10 hours or longer. In order to obtain an estimate of the half life of the conjugated hGH PEGylated prodrug without linker cleavage, the permanently conjugated corresponding conjugate is injected into rat.

[0547] The results of the in vivo half-life determinations are tabulated in Table 2.

TABLE-US-00002 TABLE 2 Half life of permanent PEG-hGH conjugates in rats In vivo characterization: in vivo Compound half-life of permanent conjugates Native hGH (Novo Nordisk, 20 minutes Denmark) 23 4 hours 25 5 hours 26 11 hours 27 13 hours

[0548] Conclusion:

[0549] From table 1 and table 2 it is obvious that residual activity correlates inversely with half life e.g. a high degree of residual activity causes faster elimination. This is typical for conjugates eliminated by receptor mediated clearance mechanisms.

[0550] Furthermore, in order to obtain a hGH PEGylated prodrug that can be administered once weekly in humans and with a low residual activity, a PEG molecule with one or more branching points and with a molecular weight of 40 kDa or above is preferred. Alternatively, conjugation of PEG to more than one site on hGH to form bisPEG-hGH conjugates results in a long terminal half-life.

Example 27 Pharmacodynamic Study of Transient Carbamate-Linked mPEG-Linker-hGH Conjugate 36 and Human Growth Hormone in Cynomolgus Monkeys

[0551] The objective of this study was to compare the pharmacodynamic response in cynomogus monkeys of one dose of transient carbamate-linked mPEG-linker-hGH conjugate 36 with once-daily human growth hormone dosing for one week.

[0552] The following dosing groups were studied:

TABLE-US-00003 Dosing Dose Test article Dose route occasion Human growth hormone 0.3 mg/kg/day SC Day 1, 2, 3, 4, 5, 6, 7 Transient carbamate-linked   5 mg/kg SC Day 1 mPEG-linker-hGH conjugate 36 Transient carbamate-linked  10 mg/kg SC Day 1 mPEG-linker-hGH conjugate 36 Vehicle (10 mM succinic acid,   0 mg/kg SC Day 1 92 mg/mL trehalose, pH 4.0)

[0553] Since transient carbamate-linked mPEG-linker-hGH conjugate 36 is transiently PEGylated using a 80 kDa PEG group, the hGH amounts in the 5 and 10 mg/kg transient carbamate-linked mPEG-linker-hGH conjugate 36 dosing groups were approximately 1 and 2 mg/kg, respectively. Hence, the hGH amount in the 10 mg/kg group of transient carbamate-linked mPEG-linker-hGH conjugate 36 was equivalent to a daily dose of 0.3 mg/kg hGH.

[0554] Each test article was injected subcutaneously into 2 cynomolgus monkeys (1 male, 1 female) using a dose volume of 1 ml/kg. The age and weight of the animals were 2.5-3 years and 2.0-2.5 kgs, respectively.

[0555] Blood samples were collected from the femoral artery/vein for determination of serum concentrations of IGF-1, a pharmacodynamic marker for human growth hormone. The blood sample were collected at the following timepoints: 0 (predose), 3, 6, 12, 24, 36, 48, 72, 96, 120, and 144 hours after dosing on Day 1

[0556] Blood samples were collected, allowed to clot, and then stored on an ice block or wet ice until centrifuged. After centrifugation, the serum samples were aliquoted into pre-labeled vials and tightly capped. The vials were stored at −70° C. upon aliquoting into vials.

[0557] IGF-1 levels in the serum samples were measured using the Quantikine Human IGF-1 ELISA kit (R&D systems) that had been adapted and validated for use in determining IGF-1 levels in cynomolgus monkey serum.

[0558] The pharmacodynamic response of the test articles is shown on FIG. 11. Both daily hGH administration and one administration of transient carbamate-linked mPEG-Linker-hGH conjugate 36 increased the IGF-1 levels over the levels measured in the vehicle group. One administration of 5 mg/kg transient carbamate-linked mPEG-Linker-hGH conjugate 36 was equivalent to daily hGH administration while one administration of 10 mg/kg transient carbamate-linked mPEG-linker-hGH conjugate 36 was shown to be superior to daily hGH. This clearly indicated that a once-weekly dose of transient carbamate-linked mPEG-linker-hGH conjugate 36 was superior to an equivalent daily dose of hGH.

Abbreviations

[0559] DBU 1,3-diazabicyclo[5.4.0]undecene

[0560] DCM dichloromethane

[0561] DIEA diisopropylethylamine

[0562] DMAP dimethylamino-pyridine

[0563] DMF N,N-dimethylformamide

[0564] DMSO dimethylsulfoxide

[0565] eq stoichiometric equivalent

[0566] fmoc 9-fluorenylmethoxycarbonyl

[0567] HFIP hexafluoroisopropanol

[0568] HOSu N-hydroxysuccinimide

[0569] LCMS mass spectrometry-coupled liquid chromatography

[0570] Mal maleimidopropionyl

[0571] MS mass spectrum

[0572] MW molecular mass

[0573] PEG polyethylene glycol

[0574] RP-HPLC reversed-phase high performance liquid chromatography

[0575] Rt retention factor

[0576] r.t. room temperature

[0577] SEC size exclusion chromatography

[0578] Sue succinimidopropionyl

[0579] TES triethylsilane

[0580] TFA trifluoroacetic acid

[0581] THF tetrahydrofurane

[0582] T rt trityl

REFERENCE LIST

[0583] 1. Büyükgebiz A. et al J. Pediatr. Endocrinol. Metab. 1999 January-February; 12(1):95-7 [0584] 2. Clark et al, 1996, Journal of Biological Chemistry 271:21969-21 977 [0585] 3. Girard, J. Mehls, O., J. Clin Invest. 1994 March; 93(3): 1163-1 171 [0586] 4. Philip Harris et al. Horm. Res. 2006; 65 (suppl. 4): 1-213, CF1-98 GH/IGF Treatment with title “First in-human study of PEGylated recombinant human growth hormone”. [0587] 5. Veronese, F. M. “Enzymes for Human Therapy: Surface Structure Modifications,” Chimica Oggi, 7:53-56 (1989).