Lyophilized Compositions Comprising Rhannexin V-128, Process for Their Preparation and Their Use for Preparing Formulations Containing 99MTc-Rhannexin V-128

Abstract

It is described a composition comprising lyophilized rhAnnexin V-128 suitable for the preparation of 99mTc-rhAnnexin V-128 formulations suitable for intravenous administration.

Claims

1. A lyophilized composition suitable for intravenous administration comprising rhAnnexin V-128 in combination with an antioxidant agent, in a pH range of 5.0-6.6.

2. The lyophilized composition according to claim 1 wherein the antioxidant agent is chosen among: sodium metabisulfite, nicotinamide, pyridoxine hydrochloride, a-tocopherol acetate, monothioglycerol.

3. The lyophilized composition according to claim 1, comprising also a buffer, chosen among lactate buffer, succinate buffer, glycolic buffer, TRIS, histidine buffer.

4. A lyophilized composition according to claim 1, suitable for intravenous administration, comprising: an antioxidant agent, a buffer having a pH comprised between 5.0-6.6; a reducing agent; a transchelating agent; a lyoprotectant and cake-forming agent; and possibly a radiation stability enhancer and/or a solubilizer.

5. The lyophilized composition according to claim 4 wherein said components are present in the following quantities: the antioxidant agent in a quantity above 0.005 mg/vial the buffer having a pH comprised between 5.0-6.6 with a concentration above 10 mM; the reducing agent, in a quantity above 0.005 mg/vial; the transchelating agent, in a quantity above 0.02 mg/vial; the lyoprotectant and cake-forming agent, in a quantity above 10 mg/vial the radiation stability enhancer and the solubilizer, if present, in a quantity above 0.005 mg/vial and above 1 mg/vial respectively.

6. A process for preparing a lyophilized formulation according to claim 1 comprising the following steps: thawing of the frozen rhAnnexin V-128; adding an antioxidant/reducing agent, buffer exchange by tangential flow filtration, to substitute the buffer in which the rhAnnexin V-128 is supplied with a buffer chosen among lactate buffer, succinate buffer, glycolic buffer, TRIS, and histidine buffer; preparation of the excipient bulk solution including: transchelating agent, radiation stability enhancer, solubilizer, antioxidant agent, lyoprotectant and cake-forming agent; addition of the required volume of rhAnnexin V-128 solution to the excipient bulk solution; dispensation of the bulk solution into vials and lyophilization.

7. A formulation suitable for intravenous administration comprising a .sup.99mTc-rhAnnexin V-128 obtained by reacting a single-vial lyophilized rhAnnexin V-128 formulation according to claim 1 with an eluate from a commercial .sup.99mTc04-generator.

8. A process for obtaining a .sup.99mTc-rhAnnexin V-128 according to claim 7 comprising the following steps: adding a suitable volume of eluate from a commercial .sup.99mTc04-generator containing up to 740 MBq of radioactivity to the vial containing the lyophilized formulation; and rotating the vial for 90 minutes at room temperature.

9-13. (canceled)

14. A method for monitoring treatment efficacy of a disease in a subject, comprising the administration of the formulation according to claim 7 to the subject, wherein the disease is selected in the group consisting of rheumatic diseases, cardiovascular diseases, oncology, transplant rejection, autoimmune diseases, neurologic diseases and atherosclerosis.

15. A method of diagnosing a disease in a subject comprising the administration of the formulation according to claim 7, wherein said disease is selected in the group consisting of rheumatic diseases, cardiovascular diseases, oncology, transplant rejection, autoimmune diseases, neurologic diseases and atherosclerosis.

16. The method according to claim 15, wherein said cardiovascular diseases are selected from the group consisting of aortic aneurysm, chemotherapy cardiotoxicity, endocarditis and myocarditis.

17. The diagnostic method according to claim 15, wherein said rheumatic diseases are selected from the group consisting of rheumatoid arthritis and Axial Spondyloarthritis.

18. The diagnostic method according to claim 15, wherein said disease is atherosclerosis, and said diagnostic method detects and stages atherosclerotic plaque.

19. The method of claim 14, wherein the subject is a human subject.

20. The method of claim 15, wherein the subject is a human subject.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0025] FIG. 1 reports a schematic view of the process for the preparation of the lyophilized rhAnnexin V-128 according to the invention

[0026] FIG. 2 shows SPECT images of .sup.99mTc-rhAnnexin V-128 uptake in front paws of a healthy mouse (a) and in a mouse model of collagen induced arthritis (b); (c) shows the uptake in the CIA mouse after treatment with an anti-inflammatory drug (no .sup.99mTc-rhAnnexin V-128 uptake detectable anymore).

SUMMARY OF THE INVENTION

[0027] It is described a composition comprising lyophilized rhAnnexin V-128 suitable for the preparation of .sup.99mTechnetium formulation for intravenous administration.

DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention allows to overcome the above said problems thanks to a lyophilized composition suitable for intravenous administration comprising Annexin V-128 in combination with suitable excipients, including particularly an antioxidant agent, in a pH range of 5.0-6.6.

[0029] Moreover the present application refers also to a formulation obtained by adding to the above said composition a suitable volume of eluate from a commercial .sup.99mTcO.sub.4— generator.

[0030] Preferably the lyophilized composition as above defined includes also a specific buffer.

[0031] The antioxidant is included with the purpose of decreasing the Annexin V-128 dimer content both during the long-term storage of the lyophilized composition and also in the .sup.99mTc-rhAnnexin V-128 preparation after labeling. In the absence of the antioxidant the dimer formation was not under control, leading to a non-adequate chemical purity.

[0032] The addition of the antioxidant agent allowed to limit the dimer formation and ensure a dimer content below 10% during the long-term storage of the lyophilized composition and also for at least 6 h after radiolabeling.

[0033] Therefore, according to the present invention, the antioxidant has a key role in maintain a good level of chemical purity and it is not added with the purpose of preventing the re-oxidation of reduced technetium to pertechnetate. Different antioxidants, such as sodium metabisulfite, nicotinamide, pyridoxine hydrochloride, a-tocopherol acetate, monothioglycerol, were evaluated.

[0034] Besides the use of the antioxidant, the control of dimerization and therefore of the chemical purity, is also accomplished by keeping the pH within the range 5.0-6.6. Higher pH values favor the dimer formation, while at lower pH values opalescence of the solution was observed, probably due to a decrease in protein solubility.

[0035] Regarding the choice of the buffer, initially the use of citrate buffer was attempted, as this is the buffer in which Annexin V-128 is currently stored. However, a radiochemical purity around 85-90% could only be achieved. With the purpose of shifting the radiochemical purity value above 90%, which is the commonly accepted lower limit for radiopharmaceutical preparations, different buffers, such as lactate, succinate, glycolic, TRIS and histidine, were evaluated. The lactate buffer was chosen, as it allowed to reach radiochemical purity values consistently around 94-96%.

[0036] The use of the antioxidant and of the specific buffer allowed also to obtain a single-vial lyophilized composition with a long-term stability of at least 18 months. As a reference, the lyophilized composition described in the previous art (Lu et al., 2015) is declared to be stable for 210 days.

[0037] According to the present invention, the antioxidant and the specific buffer mentioned above are included in lyophilized composition comprising also: [0038] a reducing agent; [0039] a transchelating agent; [0040] a lyoprotectant and cake-forming agent

[0041] Optionally, the composition can also include a radiation stability enhancer and/or a solubilizer.

[0042] In the lyophilized composition according to the invention the above said components are normally present in the following quantities: [0043] antioxidant agent (with the purpose of achieving a high chemical purity): above 0.005 mg/vial [0044] buffer: pH comprised between 5.0 and 6.6, with a concentration above 10 mM—reducing agent: above 0.005 mg/vial; [0045] transchelating agent: above 0.02 mg/vial; [0046] lyoprotectant and cake-forming agent: above 10 mg/vial

[0047] Optionally, the composition can also include a radiation stability enhancer in a quantity above 0.005 mg/vial and/or a solubilizer in a quantity above 1 mg/vial Several tests were performed during the development in order to define the above-described composition. Initially, the rhAnnexin V-128 lyophilized composition was prepared in presence of citrate buffer, including the following components: [0048] rhAnnexin V-128 (active pharmaceutical ingredient) [0049] stannous chloride (reducing agent) [0050] sodium a-D-Glucoheptonate dihydrate (transchelating agent) [0051] gentisic acid sodium salt hydrate (radiation stability enhancer) [0052] hydroxyproplyl-p-cyclodextrin (solubilizer) [0053] trehalose dihydrate (lyoprotectant and cake-forming agent) [0054] pH=5.4

[0055] The quantities of each component were slightly modified with the purpose of optimizing the formulation. The various batches of this lyophilized composition, after radiolabeling, gave a radiochemical purity that was never above 90% (as determined by both ITLC and HPLC) and a stability not longer than 1.5 hours.

[0056] In a second step, the rhAnnexin V-128 lyophilized composition was prepared in presence of lactate buffer, instead of citrate, at different pH values (up to 6.4), in the absence of antioxidant agent. The so-obtained composition, after radiolabeling, gave an improved radiochemical purity (well above 90% by both ITLC and HPLC). However, in these tests it was also observed that pH increase favors dimer formation, the dimer percentage increasing more rapidly at higher pH, after radiolabeling. In a third step, the presence antioxidant agent and lactate buffer were tested. A lyophilized composition similar to the previous one was prepared, at a pH of 5.8, including also an antioxidant (sodium metabisulfite). After radiolabeling, the radiochemical purity was confirmed to be well above 90% and the chemical purity was remarkably improved, giving values around 97-98% (by SEC-HPLC and RP-HPLC) for at least 6 h after radiolabeling.

[0057] A lyophilized composition according to the present invention can be prepared according to a process comprising the following steps (see also FIG. 1): [0058] Thawing of the frozen rhAnnexin V-128 at a controlled temperature (5° C.±3° C.)—Reduction by the addition of an antioxidant/reducing agent, [0059] Buffer exchange by tangential flow filtration, to substitute the buffer in which the rhAnnexin V-128 is supplied (normally citrate buffer) with the buffer as above defined; [0060] Preparation of the excipient bulk solution (consisting of: antioxidant agent, transchelating agent, radiation stability enhancer, solubilizer, lyoprotectant/cake-forming agent as above defined) [0061] addition of the required amount of rhAnnexin V-128 solution to the excipient bulk solution [0062] Dispensation of the bulk solution into vials and lyophilization.

[0063] The final .sup.99mTc-rhAnnexin V-128 formulation suitable for intravenous administration can be prepared by adding to the above said composition a suitable volume of eluate from a commercial .sup.99mTcO.sub.4— generator containing up to 740 MBq of radioactivity and keeping the vial under slight rotation for 90 min at room temperature.

[0064] It is worth noticing that the so-prepared .sup.99mTc-rhAnnexin V-128 formulation maintains a high chemical and radiochemical purity, as determined by ITLC, SEC-HPLC and RP-HPLC for at least 6 hours.

[0065] The essential features of the composition/formulation according to the invention as well as of their process of preparation as above described are the use of an antioxidant agent and the use of a specific buffer, in the pH range 5.0-6.6.

[0066] These features allow to obtain an injectable formulation with a high radiochemical purity (.sup.99mTc-rhAnnexin V-128 monomer by SEC-HPLC and ITLC 90%) and high chemical purity (rhAnnexin V-128 monomer by SEC-HPLC and RP-HPLC 90%, dimers 10%), which are maintained for at least 6 h after radiolabelling.

[0067] The lyophilized composition according to the invention has a shelf life of at least 18 months at 2-8° C., and can be radiolabeled at room temperature, giving a high chemical and radiochemical purity and good stability for at least 6 hours after labeling, with a controlled dimer percentage.

[0068] The composition according to the invention makes the rhAnnexin V-128 available as a lyophilized single vial product that needs just to be reconstituted with a .sup.99mTcO.sub.4— solution eluted from a commercially available generator without the need for any final purification.

[0069] Moreover, thanks to the fact that the radiolabeling procedure has been validated, only a limited quality control check (radiochemical purity analysis by ITLC) is required at the hospital level prior injection. The formulation according to the invention was tested for its diagnostic performance in several animal models (liver apoptosis, collagen induced arthritis model, endocarditis/myocarditis, inflammatory bowel disease, and others).

[0070] An example of SPECT images in a collagen induced arthritis model is shown as FIG. 2.

[0071] Annexin V-128 was also tested for its toxicity in a complete preclinical toxicology package (designed in accordance with the relevant guidelines and with the input received from regulatory agencies), in 15 days repeated dose toxicology studies in rodent and non-rodent species. A cytokine release assay was also included in the preclinical package. The outcome of these studies showed that Annexin V-128 has a very favorable safety profile (data can be provided if needed).

[0072] The formulation was tested for its safety and biodistribution in a Phase I study in human volunteers and is currently being tested in Phase II studies in Rheumatology and Cardiovascular indications.

Example 1

[0073] Preparation of a Lyophilized rhAnnexin V-128 Composition Suitable for the Preparation of .sup.99mTc-rhAnnexin V-128 Formulation for Intravenous Administration

Composition:

[0074] rhAnnexin V-128 (0.4 mg);
stannous chloride (0.01 mg);
sodium a-D-Glucoheptonate dihydrate (3 mg)
gentisic acid sodium salt hydrate (0.02 mg)
hydroxypropyl-p-cyclodextrin (5 mg)
sodium metabisulfite (0.02 mg)
trehalose dihydrate (50 mg)
lactate buffer 150 mM, pH 5.8

Preparation:

[0075] rhAnnexin V-128 was thawed and introduced into a tangential flow filtration system in order to exchange the buffer. Metabisulfite was also introduced at this step. This filtration procedure was carried on until at least 7 diavolumes of formulation buffer have been exchanged. The dimer content was checked by SEC-HPLC (maximum acceptable value=5%), and the protein concentration was also assessed (acceptable range 1-2 mg/mL). At the end of the filtration procedure, the solution was brought to a final concentration of 1 mg/mL of protein.

[0076] The other excipients were all dissolved in water for injection, in appropriate amounts: [0077] D(+)-Trehalose dehydrate (powder): an appropriate amount was weighed to obtain a concentration of 50 mg/ml in the final bulk solution; [0078] Sodium α-D-Glucoheptonate dihydrate (stock solution of 60 mg/ml): appropriate volume is added to obtain a concentration of 3 mg/ml in the final bulk solution; [0079] Gentisic acid sodium salt hydrate (stock solution of 0.1 mg/ml): appropriate volume is added to obtain a concentration of 0.02 mg/ml in the final bulk solution; [0080] Stannous Chloride dehydrate (stock solution of 1 mg/ml): appropriate volume is added to obtain a concentration of 0.01 mg/ml in the final bulk solution.

[0081] The final bulk solution was prepared adding an appropriate volume of rhAnnexin V-128 solution to an appropriate volume of excipient bulk solution.

[0082] The final bulk solution was filtered (0.22 μm filter) and automatically filled into the vials (1 mL/vial) and lyophilized.

Example 2

[0083] Preparation of .sup.99mTc-rhAnnexin V-128 Starting from Lyophilized Composition Described in Example 1 [0084] The vial cap was flipped off and the vial was placed in a suitable radiation shield; [0085] 2 mL of Sodium Pertechnetate Tc-99m solution containing 740 MBq of radioactivity were aseptically added to the vial in the lead shield; [0086] The vial was removed from the lead shield and placed in an appropriately shielded roller. The vial was kept under slight rotation for 90 min at room temperature: [0087] The vial was removed from the shielded roller and placed again in a lead shield; A sample of the so-obtained solution was withdrawn and analyzed for its chemical and radiochemical purity (SEC-HPLC, ITLC) immediately after radiolabelling and also after 6 h, and the following results were obtained. These data have been obtained from three different batches.

TABLE-US-00002 STANDARD ACCEPTANCE AVERAGE DEVIATION TEST METHOD CRITERIA (n = 3) (n = 3) Radiochemical Purity (% .sup.99mTc- ITLC ≥90.0% 98.6 0.6 rhAnnexin V-128) T = 0 Radiochemical Purity (% .sup.99mTcO.sub.4 + ≤8.0% 0.9 0.9 .sup.99mTcO.sub.2) Radiochemical Purity (% .sup.99mTc- ≤10.0% 0.5 0.2 glucoheptonate) Radiochemical Purity (% .sup.99mTc- ITLC ≥90.0% 98.2 0.5 rhAnnexin V-128) T = 0 + 6 h Radiochemical Purity (% .sup.99mTcO.sub.4 + ≤8.0% 1.4 0.6 .sup.99mTcO.sub.2) Radiochemical Purity (% .sup.99mTc- ≤10.0% 0.4 0.1 glucoheptonate) Radiochemical Purity (% .sup.99mTc- SEC-HPLC ≥90.0% 96.8 0.5 rhAnnexin V-128) T = 0 Radiochemical Purity (% .sup.99mTc- SEC-HPLC ≥90.0% 96.6 0.4 rhAnnexin V-128) T = 0 + 6 h

[0088] The above said formulation can be used as a diagnostic tool and also for selecting the best treatment as well as for monitoring medical treatment efficacy in rheumatology (for example rheumatoid arthritis, Axial Spondyloarthritis), cardiovascular diseases (as for example aortic aneurysm, chemotherapy cardiotoxicity, endocarditis and myocarditis) atherosclerosis (in particular for the detection and staging of atherosclerotic plaque), oncology, transplant rejection, autoimmune diseases, neurology, and other pathologies having as a hallmark the process of apoptosis and/or as marker of treatment response for treatment-induced apoptosis.