Optimised subcutaneous therapeutic agents

Abstract

Methods and dosage formulations are provided for subcutaneous administration in which therapeutic agents are modified to increase the hydrophilicity and molecular dimensions in relation to the native state of the therapeutic agent, in which the C.sub.max:C.sub.average ratio is lower than the C.sub.max:C.sub.average ratio of the agent when delivered intravenously.

Claims

1. A method of treatment of haemophilia A in a human subject in need thereof, comprising: (a) subcutaneously administering a composition comprising 1 IU/kg to 50 IU/kg of a PEGylated Factor VIII, wherein the PEG is directly or indirectly conjugated to the Factor VIII via a serine or threonine residue, or via an amide, N-terminal amino group, or a carboxyl group; and (b) repeating the subcutaneous administration of the composition once-daily, twice-daily, less frequently than once daily, less frequently than twice daily, or before the concentration of the Factor VIII in the blood reduces to sub-therapeutic levels, in order to maintain a consistent therapeutic effect in the human subject, wherein plasma titers of Factor VIII are maintained above the 5% normal level of Factor VIII in a human subject not suffering from haemophilia A following sub-cutaneous administration for at least 48.5 hours after the subcutaneous administration.

2. A method of providing a sustained therapeutic effect of a therapeutic agent to a human subject in need thereof, comprising: (a) subcutaneously administering a composition comprising the therapeutic agent in a PEGylated form, wherein the PEG is directly or indirectly conjugated to the therapeutic agent via a serine or threonine residue, or via an amide, N-terminal amino group, or a carboxyl group, wherein the therapeutic agent is Factor VIII and wherein the composition comprises 1 IU/kg to 50 IU/kg of the therapeutic agent; and (b) repeating the subcutaneous administration of the composition once-daily, twice-daily, less frequently than once daily, less frequently than twice daily, or before the concentration of the therapeutic agent in the blood reduces to sub-therapeutic levels, in order to sustain the therapeutic effect in the human subject, wherein plasma titers of Factor VIII are maintained above the 5% normal level of Factor VIII in a human subject not suffering from haemophilia A following sub-cutaneous administration for at least 48.5 hours after the subcutaneous administration.

3. A method of delivering a consistent infusion of a therapeutic agent into the circulatory system of a human subject in need thereof, comprising: (a) subcutaneously administering a composition comprising the therapeutic agent in a PEGylated form, wherein the PEG is directly or indirectly conjugated to the therapeutic agent via a serine or threonine residue, or via an amide, N-terminal amino group, or a carboxyl group, wherein the therapeutic agent is Factor VIII and wherein the composition comprises 1 IU/kg to 50 IU/kg of the therapeutic agent; and (b) repeating the subcutaneous administration of the composition once-daily, twice-daily, less frequently than once daily, or less frequently than twice daily, without waiting for the concentration of the therapeutic agent in the bloodstream to reduce to sub-therapeutic levels, in order to deliver a consistent infusion of the therapeutic agent into the circulatory system of the human subject, wherein plasma titers of Factor VIII are maintained above the 5% normal level of Factor VIII in a human subject not suffering from haemophilia A following sub-cutaneous administration for at least 48.5 hours after the subcutaneous administration.

4. A method of delivering a prolonged and constant level of a therapeutic agent to the blood of a human subject in need thereof without increasing the incidence of a thrombotic event, comprising subcutaneously administering once-daily, twice-daily, less frequently than once daily, or less frequently than twice daily to the human subject a composition comprising the therapeutic agent in a PEGylated form, wherein the therapeutic agent is Factor VIII, wherein the composition comprises 1 IU/kg to 50 IU/kg of the therapeutic agent and wherein the PEG is directly or indirectly conjugated to the therapeutic agent via a serine or threonine residue, or via an amide, N-terminal amino group, or a carboxyl group, wherein plasma titers of Factor VIII are maintained above the 5% normal level of Factor VIII in a human subject not suffering from haemophilia A following sub-cutaneous administration for at least 48.5 hours after the subcutaneous administration.

5. A method of treatment of haemophilia A in a human subject in need thereof, comprising subcutaneously administering once-daily, twice-daily, less frequently than once daily, or less frequently than twice daily to the human subject a composition comprising a PEGylated therapeutic agent, wherein the PEG is directly or indirectly conjugated to the therapeutic agent via a serine or threonine residue, or via an amide, N-terminal amino group, or a carboxyl group, and wherein the therapeutic agent is Factor VIII, wherein the composition comprises 1 IU/kg to 50 IU/kg of the therapeutic agent, in order to deliver a prolonged and constant level of the therapeutic agent to the blood of the human subject without increasing the ratio C.sub.max:C.sub.average, wherein plasma titers of Factor VIII are maintained above the 5% normal level of Factor VIII in a human subject not suffering from haemophilia A following sub-cutaneous administration for at least 48.5 hours after the subcutaneous administration.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) Reference is also made herein to the following drawings in which:

(2) FIG. 1 shows the blood coagulation cascade. Abbreviations: HMWK—High Molecular Weight Kininogen; PK—Prekallikrein; PL—Phospholipid.

(3) FIG. 2 shows the steps involved in disulphide-specific biopolymer conjugation chemistry with the use of a PEGylation reagent as an example of a conjugation reagent (from Shaunak et al. in Nat. Chem. Biol. 2006; 2(6):312-313).

(4) FIG. 3 shows Whole Blood Clotting Times (WBCT) following subcutaneous (SQ) administration of PEGhrFIX to subject Dog 1 and hrFIX to subject Dog 2.

(5) FIG. 4 shows APTT (Activated Partial Thromboplastin Time) Values with Time Following SQ Administration.

(6) FIG. 5 shows APTT of Retained Plasma Following SQ Administration.

(7) FIG. 6 shows APTT Relative Values to Baseline Following SQ Administration.

(8) FIG. 7 shows subject Dog 9 WBCT following 25 IU/Kg SQ Administration.

(9) FIG. 8 shows PK profiles and parameters of FVIIa following 200 ug/kg rFVIIa.

(10) FIG. 9 shows PK profiles and parameters of FVIIa following 800 ug/kg TheraPEG-rFVIIa.

(11) FIG. 10 shows PK profiles and parameters of FVIIa following 1600 ug/kg TheraPEG-rFVIIa.

(12) FIG. 11 shows concentration of FVIII in Plasma (all dogs).

(13) FIG. 12 shows concentration of FVIII in Plasma (SQ administered dogs only).

(14) FIGS. 13A and 13B shows immune data (Bethesda value) for PEGFVIII administered subcutaneously (SQ) compared to intravenous (IV), number of subjects is given by “n”.

DETAILED DESCRIPTION

(15) The invention will now be further described by way of reference to the following Examples which are included for the purposes of illustration only and should not be construed as being limiting. References to subcutaneous administration of dosage formulations of the invention are given as SQ (s.c.) and intravenous administration as IV (i.v.).

Example 1: Preparation of Dosage Forms and Administration Subcutaneously

(16) The study includes an assessment of the bioavailability and efficacy of hrFIX following subcutaneous administration. Naked (unPEGylated) hrFIX was compared to its PEGylated analogue by both circulating titre and clotting activity.

(17) 10 kDa PEGylated hrFIX was prepared following standard technology whereby 10 kDa, straight-chain, mono-disperse polyethyleneglycol was conjugated via a three carbon bridge to a single disulphide bond.

(18) The test article was prepared for administration by forming a suitable aqueous solution, buffered to pH 6.8 with 10 mM histidine, 40 mM NaCl and 0.005% Tween® 80. 1 mM benzamidine was added as a stabiliser.

(19) On the basis that dilution studies of hrFIX showed comparable clotting times with PEGrFIX at 25% dilution, the allocated potency for this study was 4× protein equivalents. The control article was supplied as a lyophilised powder and prepared for administration following the enclosed instructions for reconstitution. The delivery vehicle is identical to that described above for PEG.

(20) As an adjunct to this study it was decided to explore the possibility of subcutaneous (SQ) administration of rFIX. The prospect of the PEGylated form of rFIX being suitable for SQ administration emerged from the above observation that PEG provided a shielding effect of the protein. Historically the SQ route was considered unavailable for FIX since there was the concern that this would exacerbate the incidence of antibody production and would not translate into meaningful quantities in the blood.

(21) In this part of the study PEGhrFIX 50 IU/Kg was administered subcutaneously to an additional test animal (Dog 1) and compared to 2 SQ administrations of naked hrFIX) to 2 other test subjects, namely Dog 6 and Dog 2 respectively.

(22) In this particular representation, each animal had a slightly different baseline so for ease of comparison, the pre-administration APTT level was normalised to 1. Dog 1 and Dog 2 were the test subjects in the previous PEGrFIX trial in January 2010 from which the recorded plasma titres following intravenous administration were available for comparison.

(23) Blood samples were taken over a regular time course to follow the decay of titre and the effect on blood coagulation. Table 1 is a summary of the titres measured at 22 hours as circulating FIX following intravenous administration.

(24) TABLE-US-00001 TABLE 1 Subject Article Dose (IU/Kg) Titre Dog 1 PEGhrFIX 50 9.9 Dog 2 Benefix ® 50 5.6 Dog 3 hrFIX 50 5.1 Dog 4 PEGhrFIX 50 9.7 Dog 5 PEGhrFIX 100 11.1 Dog 6 PEGhrFIX 100 10.2 Dog 7 PEGhrFIX 150 17.6 Dog 8 PEGhrFIX 150 75.5

(25) Table 2 shows comparison of measured Circulating FIX Titre at 22 Hours Following subcutaneous (SQ) Administration.

(26) TABLE-US-00002 TABLE 2 Subject Article Dose (IU/Kg) Titre Dog 1 PEGhrFIX 50 7.8 Dog 6 hrFIX 50 1.7 Dog 2 Benefix ® 25 ND

(27) It can be seen that 25 IU/Kg of Benefix® by the SQ route was undetectable in circulation and 50 IU/Kg of PEGhrFIX was barely detectable in plasma. In stark contrast the SQ administration of PEGhrFIX was at a level (7.8) approaching that of the IV administered product (9.9).

(28) The effect of these titres on the correction of clotting times was then investigated. In the first instance the whole blood clotting times were recorded. The WBCT following sub-cutaneous administration is displayed in Table 3 and in FIG. 3. In addition, APTT on Hemochron® Junior was also recorded and the values are shown in Table 4 below (Whole blood citrated) and in FIG. 4 after subcutaneous administration. The APTT values on retained plasma samples are displayed in Table 5 and in FIG. 5 also following subcutaneous administration.

(29) TABLE-US-00003 TABLE 3 Whole Blood Clotting Time (minutes)** Following Subcutaneous Administration Hours Dog 1 (PEGhrFIX) Dog 2 (hrFIX) 6 4  45* 22 3.5 45 48 8 45 72 9.5 45 120 4 45 144 4 45 168 45 45 192 45 45 216 45 45 *Note 45 minutes was the time at which monitoring was ceased, due to no clot having been formed, according to standard procedures. **both dogs were naïve dogs, meaning they had not previously been exposed to FIX.

(30) TABLE-US-00004 TABLE 4 Citrated APTT Following Subcutaneous Administration Dog 1 (PEGhrFIX Dog 2 (hrFIX) Pre 279.7 225.1 6 99.4 375.5 22 75.6 328.7 48 88.6 72 90.1 261.7 120 124.4 274.5 144 138.9 301 168 300 372.5 192 254.2 216 300 298.3

(31) TABLE-US-00005 TABLE 5 APTT Following Subcutaneous Administration Dog 1 (PEGhrFIX Dog 2 (hrFIX) Pre 67.2 66.3 6 44.6 71.9 22 40.2 61.1 48 45.3 64.7 72 43.9 60.7 120 49.6 59.6 144 — 58.5 168 55.4 64.9

(32) FIG. 6—This collection of data clearly shows that naked rFIX is (practically) not bioavailable from subcutaneous injection. This is entirely expected from published literature and general knowledge of the art. It is all the more surprising then such high circulating titres of rFIX can be detected following subcutaneous injection of PEGhrFIX. Indeed it can be seen in table 10 that ca 80% of the subcutaneously injected PEGrFIX is available for participation in haemostatic control.

(33) The contrast is starkest in the measured clotting times, both WBCT and APTT for rFIX are barely corrected, whereas PEGhrFIX from subcutaneous injection corrects clotting times immediately. The duration of haemostasis by these measurements is prolonged to approximately 1 week from a single 50 IU/Kg subcutaneous injection.

Example 2: Dog 9 Subcutaneous Administration (SQ) of hrFIX

(34) Given the success of the above SQ studies it was decided to conduct a further single SQ administration of PEGhrFIX and similarly follow haemostatic control over an extended period. The test subject chosen was a naïve subject Dog 9 to explore the influence of neutralising antibodies on the SQ route of administration.

(35) Studies of human blood factors in dogs are confounded by the response of the canine immune system to a human protein. Human rFIX is a xenoprotein therefore in canine studies and neutralising antibodies should be expected at some point following administration of the test article. Indeed when test subjects are reintroduced to human blood factors the production of antibodies is more pronounced and speedier. The subjects Dog 1 and Dog 7 following subcutaneous administration have a shortened haemostasis period as a consequence.

(36) The test subject Dog 9 was a naïve animal and was given a small subcutaneous dose and therefore revealed the true sustained protection that PEGylated blood factors of this invention can provide. Since Dog 9 had no previous exposure to human blood factors the true underlying (and highly surprising) result was observed.

(37) FIG. 7 shows results for WBCT following 25 IU/Kg subcutaneous (SQ) administration of PEGylated IB1001 to Dog 9 of a dose of 1 ml (volume 1 ml)) and also in Table 6 below.

(38) TABLE-US-00006 TABLE 6 Time WBCT FIX Titre APTT (hours) (minutes) (% Normal) (seconds) Pre 45 67.2  6 1.75 0.68 53.8  24 6 2.46 50.9  48 9.5 2.42 49.8  72 3 1.69 55.6  94 9 1.24 57.7 118 5.5 1.13 53.2 142 8 0.66 56.3 168 9 0.28 61.4 189 18 ND 64.3 216 22.5 ND 47.7 240 25 ND 59.7 336 61.4

Example 3: Comparative Example

(39) Comparison of intravenous and subcutaneous administration of FIX and PEG-FIX.

(40) TABLE-US-00007 TABLE 7* Dose IV SQ SQ/IV Animal IU/kg Type C.sub.max ng/ml C.sub.max ng/ml % Cmax Beagle 200 BFIX 4517.5 550.7  12% Haemophilia B 200 BFIX 7916 658.3 8.2% (HB) dog *from McCarthy et al Thromb. Haemost. 87(5) 824-830, (2002).

(41) TABLE-US-00008 TABLE 8 Dose IV SQ SQ/IV Animal IU/kg Type % Normal % Normal % Cmax Dog 1 50 PEGFIX 9.9 7.8 78.8% Dog 7 50 PEGFIX 7.8 Dog 6 50 hrFIX 1.7 Dog 2 25 BFIX ND BFIX = Benefix ® PEGFIX = PEG-hrFIX

(42) Results show a C.sub.max of the subcutaneous dose of 78.8% of the intravenous dose. The percentage values for IV and SQ compared to normal appear to be low but are actually experimental artefacts. It is assumed that the FIX in each case is being spun down with the cells as the samples are prepared. It can be seen that the value of 9.9% for an intravenous dose is actually a representation of a good result. Consequently, the comparison with 7.8% for a subcutaneous dose is favourable as indicated by the calculated C.sub.max value given.

(43) Conclusions:

(44) Administration of hrFIX by subcutaneous injection of both 25 and 50 IU/Kg resulted in a barely detectable circulating titre and did not correct haemophilia in the canine subjects.

(45) In stark contrast to the above, subcutaneous dosing of 50 IU/Kg of PEGhrFIX gave rise to approximately 80% bioavailability and corrected clotting times to be within the normal range for duration of 1 week.

Example 4: Factor VIIa with 20 kDa PEG

(46) This example reports a study on PEGFVIIa Bioavailability of Blood Factor from Subcutaneous Injection. Two haemophilic dogs (HB) were treated with equipotent quantities of PEGFVIIa at time 0; one intravenously (IV), one subcutaneously (SQ). Blood samples were taken and the plasma recovered to be measured for FVIIa protein. The table of results display a bioavailability from subcutaneous administration of 89.5%.

(47) TABLE-US-00009 TABLE 9 PEGylated blood factor VIIa Time plasma titres (hours) IV SQ 0 9.5 9.5 4 167.5 73.9 (max) 12 122.2 62.4 24 45.7 57.5 48 23.1 39.6 72 9.3 22.5 Average 62.88 44.23 Max/ 2.66 1.67 Average

(48) The presence of PEG confers aqueous solubility which facilitates mobility in lymph vessels. The data shows a steady controlled infusion of FVIIa rather than the bolus peak and trough associated with the IV injection.

(49) The area under the curve indicates 89.5% bioavailability for PEGylated FVIIa and a more steady state of the level of FVIIa when delivered subcutaneously.

Example 5: PEGFVIII Drug Products

(50) To make the comparison, reference is made to Kogenate® FS (a commercially available recombinant FVIII). The PEGylated excipient, Tween® 80 is used in large quantity.

(51) Polysorbate, Tween® 80, has a molecular weight of 1310 g/mol, 880 g of which is derived from PEGylation (total monomer units of 20 which each carry 44 g/mol, (CH2-CH2-O)).

(52) The calculation is thus:

(53) Molar PEG Length Equivalent:

(54) Reference: Product Monograph Example taken 250 IU Vial

(55) TABLE-US-00010 FVIII Molecular weight 3.00E+05 g/mol IU/g 4.00+06 IU/g IU/Vial 2.50E+02 IU/Vial Vial volumes 2.50E+00 ml/vial Polysorbate concentration 6.40E−05 g/ml Molecular weight Polysorbate 1.31E+03 g/mol Molecular weight PEG per mol Polysorbate 880 g/mol

(56) TABLE-US-00011 TABLE 10 FVIII Kogenate ® Polysorbate 4.00E+06 IU/g 1.31E+03 g/mol 3.00E+05 g/mol 6.40E−05 g/ml 1.2E+12 IU/Mol 2.50E+00 ml/vial 1.60E−04 g/vial 2.50E+0.2 IU/Vial 2.08E−10 Mol/vial 1.22E−07 mol/vial Ratio of Tween ®/FVIII 5.86E+02 PEG equivalent Mol Wt. 5.16E+05

(57) Therefore, in Kogenate® FS, each FVIII molecule has the equivalent of an associated 516 kDa PEG. By comparison, the PEGFVIII dosage formulation prepared according to the present invention has a single 20 kDa PEG.

(58) Conclusions:

(59) On a dose-for-dose basis there is a 25.8 fold reduction in polyethylene glycol; given the PEG-FVIII dosage formulation of the present invention may be administered once per week versus a prophylactic use of Kogenate® on a three times a week basis, there is a potential overall reduction of ca 80-fold reduction in the administration of PEG; and the amount of PEG administered by the FVIII dosage formulation of the present invention over a dosing period is 1.25% of that administered by Kogenate®.

Example 6: Subcutaneous Administration FVIIa

(60) The objectives of this study were to investigate the pharmacokinetics of TheraPEGylated and non-TheraPEGylated recombinant human FVIIa (TheraPEGrFVIIa and FVIIa respectively) following intravenous and subcutaneous administration in haemophilic B dogs.

(61) TheraPEGylation of transgenic FVIIa (rFVIIa) was carried out according to WO 2011/135308. TheraPEGrFVIIa was supplied to the test site as a lyophile in multiple batches which, on reconstitution with high purity water, resulted in 1 mg/ml TheraPEGrFVIIa in a physiologically acceptable buffer which maintained activity of FVIIa

(62) The experimental animals were Lhasa Apso-Basenji cross dogs with congenital severe haemophilia B caused by a 5-bp deletion and a C.fwdarw.T transition in the F9 gene that results in an early stop codon and unstable FIX transcript. Prior to dosing, all dogs were tested to verify normal health status, including complete blood chemistry, serum chemistry profile fibrinogen, fibrinogen derived peptides (FDPs), thrombin time and urinary analysis. Drugs given intravenously (IV) were given as a bolus injection into the cephalic vein. Subcutaneous (SQ) doses were given between the scapula as a single dose.

(63) Individual batches of TheraPEGrFVIIa were reconstituted and then combined in order to produce a single dose solution used to dose the animals as described in Table 11.

(64) TABLE-US-00012 TABLE 11 Dog Subject Dose and Code Dog Weight Dose Dose Level Amount (Gender) (kg) Drug route (ug/kg) (mg) Dog 9 5.4 TheraPEG- SQ 800 4.32 HB1 rFVIIa (Male) Dog 3 11.4 rFVIIa SQ 200 2.28 HB2 (Male) Dog 5 5.6 TheraPEG- IV 800 4.48 HB3 rFVIIa (female) Dog 7 10.0 rFVIIa IV 200 2.0 HB4 (female) Dog 10 5.5 TheraPEG- IV 1600 8.8 HB5 rFVIIa (female) Dog 11 4.8 TheraPEG- SQ 1600 7.68 HB6 rFVIIa (male)

(65) A 5 ml blood sample was protocolled to be taken from each dog at the following times points:

(66) Pre-drug administration and at 10, 30 minutes, 1, 2, 4, 8, 12, 18, 24, 36, 48, 72, 96, 120, 144, 168, 192, 216 and 240 hours post-dose.

(67) 4 ml of the blood sample was transferred into a tube containing 0.109M tri-sodium citrate anticoagulant (9:1 v/v) on ice. Plasma was prepared by centrifugation of the remaining citrated blood and the resulting plasma samples were stored in aliquots at −80° C. An aliquot of plasma was assayed for FVIIa concentration by ELISA.

(68) The Stago Asserachrom VII:Ag ELISA assay is an enzyme linked immunoassay procedure for the quantitative determination of Factor VII/VIIa concentration in plasma samples. The assay is a sandwich ELISA which comprises of microtitre wells pre-coated with a rabbit anti-human FVII antibody. Because the antibody has a different affinity for FVIIa than for PEG-FVIIa, a standard curve was prepared by dilution of a protein appropriate to the FVIIa that is present in the test plasma, i.e. rFVIIa (0.78 to 50 ng/ml) for assay of plasma from dogs that were administered rFVIIa, or PEG-rFVIIa (0.78 to 50 ng/ml) for assay of plasma from dogs that were administered PEG-rFVIIa.

(69) Plasma samples were diluted to an appropriate concentration to fall within the standard curve. Diluted plasma samples and standards were loaded and incubated at room temperature before washing and subsequent development with a rabbit anti-human FVII HRP conjugate and OPD (a colorimetric HRP substrate). The plate was read at 492 nm and the concentration of the test samples (ng/ml) is read from the standard curve.

(70) TABLE-US-00013 TABLE 12 AUC(0-t) AUC(0-∞) Half- Dose Tmax Cmax (ng .Math. h/ (ng .Math. h/ Rate life Bio. Route (h) (ng/mL) mL) mL) (/h) (h) (%) IV 0.16 1643 2467 2534 0.2994 2.3 100 SQ 7.5 31.3 276 — — — 11

(71) TABLE-US-00014 TABLE 13 Half- Dose Tmax Cmax AUC(0-t) AUC(0-∞) Rate life Bio. Route (h) (ng/mL) (ng .Math. h/mL) (ng .Math. h/mL) (/h) (h) (%) IV 0.5 19372 128305 129646 0.0256 27.0 100 SQ 12.0 1378 84960 87139 0.0262 26.5 67

(72) TABLE-US-00015 TABLE 14 Dose Tmax Cmax AUC(0-t) AUC(0-∞) Rate Half-life Bio. Route (h) (ng/mL) (ng .Math. h/mL) (ng .Math. h/mL) (/h) (h) (%) IV 0.5 26609 236116 240449 0.050 13.8 100 SQ 24 2030 107728 108454 0.038 18.3 45.6
Pharmacokinetics

(73) The IV and SQ profiles and PK parameters for 200 ug/kg FVIIa, 800 ug/kg TheraPEG-rFVIIa and 1600 ug/kg TheraPEG-rFVIIa are shown in FIGS. 8, 9 and 10 (Table 12, Table 13 and Table 14). The half-life of TheraPEG-rFVIIa was found to be between 14 and 27 hours, which is a clear extension over the 2.3 hour half-life of non-PEGylated rFVIIa. The AUC of the 1600 ug/kg IV dose of TheraPEG-rFVIIa was 1.8× higher than that of the 800 ug/kg IV dose. However the 1600 ug/kg SQ dose was only 1.2× higher than that of the 800 ug/kg dose. This is reflected in the bioavailability calculations of 67% and 45% for the 800 ug/kg and 1600 ug/kg doses respectively, which represented a significant increase over the 11% SQ bioavailability observed for non-PEGylated rFVIIa.

(74) The AUC for the 800 ug/kg IV dose of TheraPEG-rFVIIa is 84× that of the AUC following 200 ug/kg IV non-PEGylated rFVIIa and the AUC for the 800 ug/kg SQ dose of TheraPEG-rFVIIa is 300× that of 200 ug/kg SQ non-PEGylated rFVIIa.

Example 7: Subcutaneous Administration FVIII

(75) The objectives of this study were to investigate the pharmacokinetics and pharmacodynamics of TheraPEGylated plasma derived FVIII (TheraPEG-pdFVIII) when administered intravenously and subcutaneously to haemophilic A dogs. TheraPEG-pdFVIII was prepared as described in WO 2011/135307 with a 20 kDa linear PEG and further purified to yield purified TheraPEG-pdFVIII.

(76) The experimental animals were greyhound cross dogs which had congenital severe haemophilia A and had previously been administered canine plasma for the treatment of spontaneous bleeds, but were naïve to treatment with human FVIII. Prior to dosing, all animals were tested to verify normal health status, including complete blood chemistry, serum chemistry profile fibrinogen, fibrinogen derived peptides, thrombin time and urinary analysis.

(77) Table 15 shows the weight of each dog and the FVIII doses that were administered. Each dog received a single dose of either TheraPEG-pdFVIII at a higher (approx. 0.14 mg/kg) or a lower (0.07 mg/kg) dose or non-PEGylated pdFVIII at 0.03 mg/kg. Intravenous (IV) administration was given as a bolus dose via the cephalic vein. Sub cutaneous (SQ) administration was given as a single dose between the scapulae.

(78) TABLE-US-00016 TABLE 15 Dose total Dog subject Dose amount TEST Dose (gender) Weight FVIII Conc. volume FVIII Dose article route and code (kg) (mg/ml) (ml) (mg) (mg FVIII/kg) TheraPEG- SQ Dog 12 (F) 21.8 0.211 14 2.954 0.135 pdFVIII HA1 TheraPEG- SQ Dog 13 (M) 26.6 0.235 16 3.76 0.141 pdFVIII HA2 TheraPEG- IV Dog 14 (F) 20.6 0.211 14 2.954 0.143 pdFVIII HA3 TheraPEG- IV Dog 15 (M) 31 0.235 17.1 4.019 0.130 pdFVIII HA4 TheraPEG- SQ Dog 16 (M) 28 0.273 7.0 1.911 0.068 pdFVIII HA6 (low dose) Non-PEG'd SQ Dog 17 (F) 27.4 0.090* 9.0 0.810 0.030 pdFVIII HA5

(79) A blood sample was protocolled to be taken from each dog at the following times points. Pre-drug administration and at 10, 30 minutes, 1, 2, 4, 8, 12, 18, 24, 36, 48, 72, 96, 120, 144, 168, 192, 216 and 240 hours post-dose. Whole blood (non-citrated) was used for the whole blood clotting assay and the activated clotting time assay. The remaining blood sample was transferred into tubes containing 0.109M tri-sodium citrate anticoagulant (9:1 v/v) on ice. The activated partial thromboplastin time assay was conducted on citrated blood. Plasma was prepared by centrifugation of the citrated blood and the resulting plasma samples were stored in aliquots at −80° C. for the FVIII antigen ELISA.

(80) Whole Blood Clotting Time Assay (WBCT)

(81) Blood samples were divided between 2 vacutubes, (2×0.5 ml), and observed carefully with periodic and judicious levelling of the tube until a clot was determined by interruption of flow in the fully horizontal position. The quality of the clot was then observed by holding the tube in the fully inverted position. The WBCT was recorded as the mean of the total time from sample extraction until visual observation of blood clot for both samples and the quality of the clot in the inverted position was also noted.

(82) Activated Clotting Time (ACT) and Activated Partial Thromboplastin Time (APTT)

(83) The ACT and APTT tests were carried out using a Haemachron Jr coagulation analyzer (International Technidyne Corps.) according to the manufacturer's instructions.

(84) The concentration of FVIII antigen in plasma samples was determined by ELISA using the Visulize FVIII antigen kit from Affinity Biologicals (Ancaster, Ontario, Canada) according to the manufacturer's instructions.

(85) Results

(86) Whole Blood Clotting Time (WBCT)

(87) Haemostasis (WBCT <12 minutes) was maintained in all dogs that had received the higher dose of TheraPEG-pdFVIII (HA1-4) for between 80-100 hours. There appeared to be no difference in the WBCT profile between IV and SQ administration. A lower dose of TheraPEG-pdFVIII (HA6) given SQ maintained haemostasis for between 56-75 hours. In contrast, although non-PEGylated FVIII administered SQ reduced the WBCT, it did not result in a sustained WBCT below 12 minutes.

(88) Activated Clotting Time (ACT)

(89) ACT was reduced into the normal range of less than 200 seconds in all dogs that had received the higher dose of TheraPEG-pdFVIII (HA1-4) for approximately 80 hours post-dose. There was no difference in the ACT profile between IV and SQ administration.

(90) A lower dose of TheraPEG-pdFVIII (HA6) given SQ maintained ACT below 200 seconds for at least 36 h. In contrast, although non-PEGylated FVIII given SQ reduced the ACT, it did not result in a sustained ACT below 200 seconds.

(91) Activated Partial Thromboplastin Time (APTT)

(92) APTT was reduced to less than 60 seconds in all dogs that had received the higher dose of TheraPEG-pdFVIII (HA1-4) for approximately 60 hours post-dose. There was no difference in the APTT profile between IV and SQ administration.

(93) A lower dose of TheraPEG-pdFVIII (HA6) given SQ maintained APTT at less than 60 seconds for 40 h. In contrast, although non-PEGylated FVIII given SQ reduced the APTT, the shortest APTT time was 80 seconds. The reason why the APTT for this individual remained below base-line value for the duration of the study post-dose is obscure, but may be due to dog-to-dog variation.

(94) FVIII Plasma Concentrations and Pharmacokinetics

(95) The FVIII plasma concentration against time in all dogs is shown in FIG. 11. The data for SQ dosed dogs alone is shown in FIG. 12. Raw data are listed in Tables 23-28. Key PK parameters are shown in Table 16.

(96) The half-life of TheraPEG-FVIII administered SQ was 18.3h and 16.6h for HA′ and 2 respectively. When administered IV, half-lives were slightly shorter at 15.2h and 13.9h for HA3 and 4 respectively. Bioavailability was calculated at 32% following SQ administration. The concentrations of FVIII following SQ administration of non-PEGylated FVIII (HA5) were mainly below the level of quantification and therefore no PK parameters could be calculated.

(97) TABLE-US-00017 TABLE 16 Dose Dog T.sub.max C.sub.max AUC.sub.0-t AUC.sub.0-∞ λ.sub.z t.sub.1/2 (mg/kg) Ref. (h) (% Normal) (% Normal .Math. h) (% Normal .Math. h) (/h) (h) 0.135 HA1 (SQ) 8.00 31.20 871.4 1066.8 0.0379 18.3 0.141 HA2 (SQ) 8.00 32.80 1085.3 1171.7 0.0417 16.6 0.143 HA3 (IV) 0.16 176.40 2929.8 3698.0 0.0456 15.2 0.130 HA4 (IV) 0.16 179.20 3302.8 3522.8 0.0500 13.9 0.068 HA6 (SQ) 4.00 9.01 345.2 510.1* 0.0143* 48.4* *Approximate value due to variability of data.
Conclusions

(98) Sub-cutaneous delivery of the higher dose of TheraPEG-FVIII resulted in haemostatic control for 80-100 hours following a single dose as measured by WBCT, APTT and ACT. The profile of SQ in these assays was indistinguishable from the profile of an equivalent dose of TheraPEG-FVIII given IV This clearly demonstrated the feasibility of delivering TheraPEG-FVIII SQ.

(99) The half-life of TheraPEG-pdFVIII ranged from 13.9 to 18.3 h. This demonstrates a clear extension in half-life compared to marketed recombinant FVIII which is reported to be 7-11 h in haemophilia A dogs (Karpf et al., Haemophilia 17, 5 (2011)). Hence, the TheraPEG-FVIII was not only bioavailable SQ but also demonstrated an extended half-life.

(100) The PK profile of FVIII following SQ administration of TheraPEG-pdFVIII had a much reduced C.sub.max and AUC compared to IV administration and bioavailability was determined to be 32%. However, at this dose level, due to the “slow release” nature of the PK curve, exposures were maintained above the 5% normal level following SQ administration for a similar amount of time as after the IV dose which is likely to explain the equivalent functional responses. The decrease in C.sub.max and AUC, coupled to the increase in duration of action for SQ delivered TheraPEG-FVIII highlighted potential, additional safety features of this product and dosing options.

(101) Sub-cutaneous administration of non-PEGylated FVIII resulted in no detectable FVIII in plasma and although clotting times were reduced, there was no sustainable maintenance of haemostasis. This demonstrated that non-PEGylated FVIII had a very low SQ bioavailability, but that very small amounts of FVIII can affect haemostasis. In contrast to non-PEGylated FVIII, a low dose of TheraPEG-pdFVIII resulted in plasma levels of up to 9% normal and haemostasis was maintained for 56-75 hours. Therefore, the addition of TheraPEG to pdFVIII resulted in a greater bioavailability and functional response when administered SQ. In conclusion, this study clearly demonstrated that TheraPEGylation of FVIII resulted in a superior product that can be administered subcutaneously with an extended duration of action.

(102) TABLE-US-00018 TABLE 17 HA1 (SQ PEG-pdFVIII) Dog 12 WBCT APTT ACT ELISA Time (h) (min) (min) (min) (% normal) 0.00 40.00 186.95 378.00 0 0.16 31.00 178.4 400 0 0.50 32.00 168.1 319 0 1.00 8.50 114.1 229 0 2.00 8.75 81.3 241 0 3.50 7.00 57.3 177 7.8 8.00 7.50 40.4 167 31.2 12.00 6.50 51.4 183 29.4 20.00 5.25 33.4 184 23.4 26.50 5.50 37.3 194 21.4 33.50 5.80 40.4 196 15.4 48.50 6.40 81.3 205 7.4 58.00 8.13 58.5 183 3.8 72.00 9.00 71.5 188 0.1 94.00 13.00 119.2 275 0 117.75 29.00 146.4 400 0 145.50 33.00 174.3 386 170.00 31.75 137 360 398.00 32.65 140.8 365 696.00 30.00 133.4 347

(103) TABLE-US-00019 TABLE 18 HA2 (SQ PEG-pdFVIII) Dog 13 WBCT APTT ACT ELISA Time (h) (min) (min) (min) (% normal) 0.00 36.50 117.50 367.00 0 0.16 12.50 79.8 265 0 0.50 7.50 52.5 197 0 1.00 7.50 39.3 181 4.4 2.16 7.00 40.4 179 12.8 4.00 7.75 33.4 172 24.6 8.00 6.00 30.6 168 32.8 11.00 6.75 35.3 177 26.6 21.00 5.00 36.3 185 29.8 24.75 7.50 32.4 173 26.4 32.50 7.75 51.4 186 18.8 46.00 7.25 47.9 183 9.2 52.25 8.00 50.2 185 1.4 72.75 8.00 56.1 213 3.6 97.00 19.75 122.7 314 0 120.50 24.00 174.3 400 143.50 26.00 176.3 371 165.00 33.50 306.4 400

(104) TABLE-US-00020 TABLE 19 HA3 (IV PEG-pdFVIII) Dog 14 WBCT APTT ACT ELISA Time (h) (min) (min) (min) (% normal) 0.00 24.00 239.45 0 0.16 4.00 38.3 174 176.4 0.50 4.00 38.3 158 169.4 1.00 4.50 32.4 174 172.2 2.00 5.50 37.3 180 154.8 3.50 6.50 31.5 179 140.6 8.00 4.50 44.6 160 116.4 12.00 4.90 29.6 166 98.6 20.00 5.00 28.7 176 70.2 26.50 6.00 42.5 171 52.2 33.50 6.75 37.3 163 35 48.50 7.63 43.5 179 13.6 58.00 7.13 44.6 184 4 72.00 6.00 209 0 94.00 9.00 234 0 117.75 19.00 112.4 320 0 145.50 26.25 176.3 347 170.00 29.25 114.1 333 398.00 40.00 142.6 362 696.00 30.00 53.7 294

(105) TABLE-US-00021 TABLE 20 HA4 (IV PEG-pdFVIII) Dog 15 WBCT APTT ACT ELISA Time (h) (min) (min) (min) (% normal) 0.00 26.25 84.2 396 0 0.16 40.00 77 227 179.2 0.50 22.50 62.3 168 170.8 1.00 8.25 33.4 151 166.2 2.16 6.00 24.4 158 151.8 4.00 5.25 30.6 165 129.8 8.00 7.50 29.6 150 115.6 11.00 8.50 31.5 157 100.2 21.00 6.50 35.3 185 68.8 24.75 7.00 34.3 162 55 32.50 7.00 38.3 177 35.8 46.00 6.25 43.5 174 18.4 52.25 6.75 45.7 167 11 72.75 7.00 53.7 203 0 97.00 22.00 79.8 334 0 120.50 20.50 102.5 356 143.50 23.50 178.4 306 165.00 34.50 114.1 371

(106) TABLE-US-00022 TABLE 21 HA5 (SQ pdFVIII) Dog 17 WBCT APTT ACT ELISA Time (h) (min) (min) (min) (% normal) 0.00 31 104.2 0 0.166 21 137 351 0 0.5 31.5 100.9 311 0 1 19.25 105.8 268 0 2 16 98.5 268 0 4 18 84.2 261 0 8 13.25 105.8 238 0.186 12 13 79.8 200 0 18 12.5 93.1 230 0 24 10.25 82.7 226 0 36 15.5 84.2 247 0 48 18.5 122.7 237 0 56 19.25 148.3 278 0 72 23 364 0.489 96 25.5 122.7 347 0 120 109.1 328 0 192 37.00 87.1 334 0 432 40 128 375 0

(107) TABLE-US-00023 TABLE 22 HA6 (SQ PEG-pdFVIII Low Dose) Dog 16 WBCT APTT ACT ELISA Time (h) (min) (min) (min) (% normal) 0.00 31.75 81.3 321 0 0.166 39 104.2 0 0.5 33.5 115.8 298 0 1 12.5 94.2 224 0 2 8.25 82.7 190 0.131 4 9 62.3 168 9.006 8 9 44.5 188 8.621 12 7.5 40.4 156 3.053 18 7 49.1 179 5.148 24 7.75 57.3 168 6.167 36 11.5 62.3 180 6.553 48 8.75 68.8 217 2.419 56 15 74.2 178 5.01 72 10.5 88.6 215 2.364 96 27 110.7 281 0 120 90.1 0 192 31.25 100.9 323 0 432 35.5 93.1 333 0

Example 8: Immune Response to Subcutaneous Administration in Dogs

(108) In the present invention, it has been observed that there is a lower immune response arising from subcutaneous administration. This effect is diametrically opposite to what would be anticipated prior to the present invention by someone of ordinary skill in the art of blood factor administration. It is generally accepted that by administering subcutaneously the existing very high level of immune response (FVIII inhibitor frequency) would be exacerbated.

(109) In the present invention, a very surprising outcome has been found. In order to lower the incidence of immune (inhibitor) responses it is proposed to adopt subcutaneous administration where the level of immune response is directly related to the level of systemic exposure. By providing a subcutaneous delivery, the C.sub.max can be radically lowered and in so doing there is a lowering of immune response.

(110) In the examples of the invention, the PEGylated product is exposed to the most testing of immune environments, namely the dog system. It can be seen that the Bethesda values (units of inhibitor quantities) are highest and earliest when given intravenously. By contrast the subcutaneous deliveries have a very much lower systemic exposure as evidenced by the C.sub.max and a lower and later Bethesda response. Indeed the lowest value of all is the naked FVIII given SQ which has almost no systemic exposure and is never seen to give an inhibitor value. See FIG. 13/Table 23 for a representation of the data obtained.

(111) TABLE-US-00024 TABLE 23 Summary N (no. of Bethesda Units Product & route subjects) Cmax PRE Day 7 Day 14 Day 30 PEGFVIII IV 2 177.8 0 0 20 17.5 PEGFVIII SQ 2 32 0 0 10 17 PEGFVIII SQ (LD) 1 9.0 0 0 0 6 FVIII SQ 1 0.5 0 0 0 0

(112) The plots in FIG. 13 demonstrate how much inhibitor activity has been found in blood plasma over time, as stimulated by the treatments. In the case of the direct IV treatment, there is a more rapid occurrence of a higher level of inhibitors, compared to SQ treatment which leaches into the system more slowly and is less provocative to the immune system.

Example 9: Comparative Studies on Subcutaneous Administration in Rats

(113) This example describes the surprising depot effect encountered with blood factors when conjugated to polymers such as PEG. Moreover, the results show that it is possible to engineer the rate at which blood factors are made available from the subcutaneous space by manipulating the level of hydration imposed on the protein from the size (or amount) of PEG.

(114) The relative pharmacokinetics of Factor VIIa PEGylated via 3 different forms of PEGylation was studied in rat subjects to compare their performance in terms of delivery from the subcutaneous space.

(115) Native, recombinant Factor VIIa was administered to rat subjects, as well as 3 different PEGylated forms of FVIIa, either subcutaneously (SQ) or by intravenous (IV) administration: a) TheraPEGylated FVIIa: FVIIa was mono-PEGylated to a 20 kDa PEG molecule using the “TheraPEG” technology of Polytherics Ltd (as described elsewhere and in WO 2011/135308); b) GlycoPEGylated FVIIa: FVIIa was conjugated to PEG via standard glycoPEGylation technology giving a test product that was dominated by di-conjugated 20 kDa PEG with also some significant amounts of higher PEG products: c) HATU-catalysed PEGylated FVIIa: FVIIa was monoPEGylated to a 20 kDa PEG (using a conjugation method derived from one described in U.S. Pat. No. 5,644,029).
TheraPEG-FVIIa

(116) 20 kDa PEG was dispersed to 10 mg/mL in 5 mM Na phosphate pH8.0, 15 mM NaCl, 2 mM EDTA. It was then incubated at 20° C. for 3 hours. A vial (5.3 mg) of FVIIa was reconstituted to 0.8 mg/mL in 20 mM sodium citrate pH6.0, 0.1M NaCl, 10 mM EDTA. It was incubated at 20° C. for 10 minutes. TCEP, 1.5 Molar Equivalents (ME) of 24 mM and 0.025ME of 0.4 mM SeCM were then added and incubated at 20° C. for 1 hour. 2 ME of activated PEG was then added to the reduced FVIIa. The mixture was incubated at 20° C. for 1 hour, and then at 5° C. for 17 hours. Size Exclusion Chromatography using a Superdex 200 column was then carried out in formulation buffer in order to purify the PEGylated FVIIa.

(117) For analysis of the product, reconstituted rFVIIa, activated PEG, reaction mixture, and selected Superdex fractions (25, 30, 35, 39, 45, 51, 80) were run on non-reduced SDS-PAGE gels. Fractions containing PEGylated FVIIa were pooled and concentrated to approximately 3 mL prior to lyophilisation. The concentrated SEC pool was tested by reduced and non-reduced SDS PAGE, clotting activity and reversed phase HPLC assays both pre- and post-lyophilisation.

(118) GlycoPEGylated FVIIa

(119) A vial (5.3 mg) of rFVIIa was reconstituted in 2.5 mL MOPS buffered saline. The reconstituted rFVIIa was then buffer exchange on a PD10 desalting column into MOPS buffered saline and diluted to 1 mg/mL. The buffer exchanged rFVIIa was placed on ice and 100 mM sodium periodate was added to a final concentration of 2.5 mM. The mixture was incubated in the dark for a maximum of 30 minutes. Glycerol (50%) was the added to a final concentration of 3%. The mixture was then buffer exchanged into 0.1M sodium acetate buffer using a Zeba spin column. A 50 mg/mL stock solution of Amino oxy PEG was made and 10ME of this PEG was added to the desalted FVIIa. The reaction mixture was incubated at Room Temperature for 1-2 hours before further incubation at 4° C. overnight. The GlycoPEGylated FVIIa was then purified by SEC chromatography as described above.

(120) For analysis of the product, selected SEC fractions (23, 27, 32, 35, 40, and 80) were run on non-reduced SDS-PAGE. Fractions containing GlycoPEGylated FVIIa were pooled and concentrated to approximately 3 mL prior to lyophilisation. The concentrated SEC pool was tested by reduced and non-reduced SDS PAGE, clotting activity and reversed phase HPLC assays both pre- and post-lyophilisation.

(121) HATU PEG-FVIIa

(122) A vial (5.3 mg) of rFVIIa was reconstituted in 2.5 mL borate buffer, buffer exchange on a PD10 column into borate buffer and dilute to 0.5 mg/mL. A stock solution of Methoxy-PEG was made up in acetonitrile to 16 mg/mL. The buffer exchanged rFVIIa was activated with 1.0ME of HATU and 2.5ME of DIEA for 10 minutes at room temperature. Following activation 8 ME of Methoxy-PEG was added to the activated rFVIIa over 2-5 minutes. The reaction mixture was then incubated at room temperature for 80-100 minutes. The HATU PEGylated FVIIa was then purified by SEC chromatography as described above.

(123) For analysis of the product, selected SEC fractions were run on non-reduced SDS-PAGE. Fractions containing HATU PEGylated FVIIa were pooled and concentrated to approximately 3 mL prior to lyophilisation. The concentrated SEC pool was tested by reduced and non-reduced SDS PAGE, clotting activity and reversed phase HPLC assays both pre- and post-lyophilisation.

(124) Method

(125) Formulations at a dose of 0.5 mg/kg were administered either IV or SQ to Healthy rat subjects. For IV administration the appropriate volume of test article was injected into the tail vain. For SQ administration the appropriate volume of test article was injected into the scruff of the neck. Following administration of the control test articles (native rFVIIa) blood samples were taken at the following time intervals:

(126) TABLE-US-00025 TABLE 24 Time (h) 0.033 0.25 0.5 1 1.5 2 3 4 6 8 12 18 24 36 48 IV control ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ SQ control ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

(127) Following administration of the test articles blood samples were taken at the following time intervals:

(128) TABLE-US-00026 TABLE 25 Time (h) 0.033 0.25 0.5 1 2 4 6 8 12 18 24 48 72 96 120 IV article ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ SQ article ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

(129) At each time point plasma was prepared from the blood sample and the FVIIa concentration determined using the Stago Asserachrom VII:Ag ELISA assay. This assay is an enzyme linked immunoassay procedure for the quantitative determination of Factor VII/VIIa concentration in plasma samples. The assay is a sandwich ELISA which comprises of microtitre wells pre-coated with a rabbit anti-human FVII antibody. Because the antibody has a different affinity for FVIIa than for PEG-FVIIa, a standard curve was prepared by dilution of a protein appropriate to the FVIIa that is present in the test plasma, i.e. rFVIIa (0.78 to 50 ng/ml) for assay of plasma from rats that were administered rFVIIa, or PEG-rFVIIa (0.78 to 50 ng/ml) for assay of plasma from dogs that were administered PEG-rFVIIa.

(130) Plasma samples were diluted to an appropriate concentration to fall within the standard curve. Diluted plasma samples and standards were loaded and incubated at room temperature before washing and subsequent development with a rabbit anti-human FVII HRP conjugate and OPD (a colorimetric HRP substrate). The plate was read at 492 nm and the concentration of the test samples (ng/ml) is read from the standard curve. Results of the study are as shown in Table 26(a) and (b) where there are two routes of administration: intravenous (IV) and sub-cutaneous (SQ) for each of the PEGylated FVIIa molecules and a control arm which was the native FVIIa.

(131) As shown in Table 26(a) and (b), there are 2 routes of administration, intravenous (IV) and subcutaneous (SQ) for each of the PEGylated FVIIa molecules and a control arm which was the native FVIIa.

(132) This example describes the surprising depot effect encountered with blood factors when conjugated to polymers such as PEG. Moreover, the results show that it is possible to engineer the rate at which blood factors are made available from the subcutaneous space by manipulating the level of hydration imposed on the protein from the size (or amount) of PEG.

(133) From the results shown in Table 26(a) and (b), it can be seen that: All the PEGylated proteins have extended plasma half-lives by comparison to the naked protein

(134) The mono-PEG products, namely TheraPEG and HATU PEGylated proteins have a slower rate of entry to the plasma than the di-PEG conjugate (GlycoPEG) and therefore a more pronounced depot effect. This can be deduced by comparing the differences in the IV and SQ half-lives in each product. For TheraPEG-FVIIa the IV t1/2 was 8.68 hours which compares to 23.2 hours for the same product given by SQ. This represents a 2.7-fold increase implying a very large depot effect for this mono-PEGylated product. Similarly, for the mono-PEGylated HATU PEG-FVIIa the SQ t1/2 has an enhanced depot effect represented by a 1.7-fold increase over the IV t1/2 (24.3/14.07) In contrast, for the heavily PEGylated product, GlycoPEG-FVIIa, the half-lives for both products are closer to parity (22.3/19.34=1.15-fold) implying that the SQ administration of this product has little depot effect compared to IV administration.

(135) In other words, the mono-PEGylated products when administered SQ would appear to have resisted being dispersed through the sub-cutaneous space for longer than the di-PEGylated product, thus providing the enhanced depot effect. The reduced amount of PEG on the mono-PEGylated products would leave more of the protein exposed; the greater PEG coverage on the GlycoPEG product would render it more water dispersible within the subcutaneous space, leading to a faster rate of entry via the lymphatic vessels into the plasma.

(136) Surprisingly therefore, to achieve the longest duration of depot release, a lesser degree of PEGylation is required. Without being bound by theory, this can be rationalised by the lesser PEGylation exposing some of the protein to the subcutaneous tissue which confers a slow rate on the diffusion through the lymph. By contrast the higher degree of PEGylation covers the protein completely leaving the product free to quickly enter the blood circulation.

(137) This supports the teaching that the modification of target molecules, in this case via PEGylation, may be tuned to exquisitely modify the release characteristics and thereby the concentration of the product in the blood over time and its bioavailability.

(138) Overall, there is a very surprising total effect whereby the combination of PEGylation followed by subcutaneous delivery, renders an observed 35-fold increase in apparent half-life (0.66 hours for naked FVIIa to 23.2 hours following subcutaneous (SQ) administration).

(139) Finally, it can be seen overall that the bioavailability favours the higher PEGylated species, namely GlycoPEG, confirming that the higher PEG and hydration levels promote a higher degree of mobility and therefore bioavailability.

(140) TABLE-US-00027 TABLE 26 (a) Dose Cmax AUC0-t AUC0-∞ Test Article route Rat Tmax (h) (ng/ml) (ng .Math. h/ml) (ng .Math. h/ml) t½ (h) FVIIa IV 1 0.03 2251.8 600 624 0.55 2 0.03 2538.3 711 728 0.75 3 0.03 1892.3 533 561 0.66 Mean 0.03 2227.47 615 638 0.65 TheraPEG-FVIIa IV 4 0.03 10024.5 33598 34021 8.97 5 0.03 8181 22051 22435 9.42 6 0.03 10799.7 23251 23449 7.66 Mean 0.03 9668.40 26300 26635 8.68 GlycoPEG-FVIIa IV 7 0.03 6647.2 55987 57138 21.24 8 0.03 5674.9 46702 47609 21.21 9 0.03 6227.3 47188 47663 15.57 Mean 0.03 6183.13 49959 50803 19.34 HATU catalysed IV 10 0.03 8090.5 31172 31775 13.49 PEG-FVIIa 11 0.03 7586.5 30448 31003 13.07 12 0.03 7557.1 35317 35697 15.66 Mean 0.03 7744.70 32312 32825 14.07

(141) TABLE-US-00028 TABLE 26 (b) Bioavailability Dose Tmax Cmax AUC0-t AUC0-∞ t½ (% AUC0-t Test Article route Rat (h) (ng/ml) (ng .Math. h/ml) (ng .Math. h/ml) (h) SQ vs IV) TheraPEG-FVIIa SQ 16 18.0 215.5 8015 9557 23.96 30.5 17 12.0 117.6 3695 4839 21.15 14.0 18 18.0 129.3 4227 5805 24.52 16.1 Mean 16.0 154.13 5312 6734 23.21 20.2 GlycoPEG-FVIIa SQ 19 18.0 297.8 16868 17665 22.27 33.8 20 24.0 234.3 12471 13456 23.55 25.0 21 18.0 407.6 22243 22871 21.16 44.5 Mean 20.0 313.23 17194 17997 22.33 34.4 HATU catalysed SQ 22 18.0 224.8 10234 10996 20.85 31.7 PEG-FVIIa 23 18.0 138.7 5544 6750 28.03 17.2 24 18.0 249.1 11277 12147 24.03 34.9 Mean 18.0 204.20 9018 9964 24.30 27.9