Patent Application
20120321640
The invention provides new uses, compositions and methods of administration for specific binding agents to von Willebrand Factor (vWF) in patients with thromboembolic disorders and in particular new combined uses with thrombolytic agents such as tissue plasminogen activator in patients with thromboembolic disorders such as e.g. ischemic stroke. Furthermore, a new group of vWF binding agents and an improved Middle Cerebral Artery Thrombosis Model in guinea pigs to study the effects of stroke such as ischemia (oxygen and glucose depriviation) and hemorrhage (bleeding), in particular hemorrhage, are provided.
A stroke is the rapidly developing loss of brain function(s) due to disturbance in the blood supply to the brain. This can be due to ischemia caused by thrombosis or embolism (80% of all reported cases) or due to hemorrhage (20%). Some hemorrhages develop inside areas of ischemia. As results, the affected area of the brain is unable to function, leading to inability to move one or more limbs, inability to understand or formulate speech, or inability to see one side of the visual field. Stroke is the leading cause of adult disability in the US and Europe. It is the second most common cause of death, the first being heart attacks and third being cancer. The only therapy available is recombinant tissue plasminogen activator (herein also referred to as “rt-PA”), but side effects such as e.g. bleeding and limited beneficial time interval limit its use.
It has recently been suggested that the GPIb-IX-V-von Willebrand factor (herein also referred to as “vWF”) pathway is critically involved in ischemic stroke (Kleinschnitz et al., 2009, Blood, Vol. 113, pages 3600-3603). Moreover, deficiency or reduction of vWF by the vWF cleaving protease ADAMTS13 reduces ischemic brain injury in experimental stroke (Zhao et al., 2009, Blood, Vol. 114, pages 3329-3334). Furthermore, it has been shown that the anti-platelet drug “ALX-0081” (SEQ ID NO: 1) that is a vWF binding agent comprising two identical Nanobodies directed against vWF, interrupts the binding between vWF and platelets, i.e. interrupts binding between the so called A1 domain of vWF and the platelet glycoprotein Ib-IX-V receptor complex (herein also referred to as “GPIb receptor”) of the platelets, and that application of said vWF binding agent prevents thrombus formation in a baboon FOLTS' model (see e.g. Example 18 of WO2006/122825 A2).
It has now been found surprisingly that the combined use of i) a specific anti-platelet drug, i.e. an anti-platelet vWF binding agent, and ii) a thrombolytic drug synergistically reduces thrombus formation in a thromboembolic disorder such as e.g. a myocardial infarction, ischemic stroke, deep vein thrombosis or pulmonary embolism. Indeed the present invention surprisingly provides that thrombolytic drugs, such as rtPA, when combined with an anti-adhesive agent such as e.g. an anti-vWF agent can be used in a broader dose regimen range (lower dose and/or longer treatment window) for the treatment of thromboembolic disorders than the skilled person in the art would have expected.
For example, ALX-0081 (SEQ ID NO: 1) has been found to significantly reduce the ischemic brain damage while no increased intracerebral bleeding was observed in the photochemically induced endothelial damage of the middle cerebral artery (herein also referred to as “MCA”). In contrast to rtPA monotherapy, ALX-0081 monotherapy or in combination with rtPA was able to induce a complete reperfusion of the MCA after injury in the same model.
Accordingly the present invention provides a method for the treatment of a thromboembolic disorder such as e.g. a myocardial infarction, ischemic stroke, deep vein thrombosis or pulmonary embolism, preferably ischemic stroke such as acute ischemic stroke; in patient(s), preferably human(s), in need thereof, wherein said treatment comprises administering
Moreover, the present invention provides a method for the treatment of a thromboembolic disorder such as e.g. a myocardial infarction, ischemic stroke, deep vein thrombosis or pulmonary embolism, preferably ischemic stroke such as acute ischemic stroke; in patient(s), preferably human(s), in need thereof, wherein said treatment comprises the inhibition of reocclusion in said patient(s) treated with a thrombolytic agent, e.g. such as rtPA, by administering to said patient(s) an effective dose regimen of an anti vWF agent, e.g. an A1 vWF binding agent, a vWF binding agent with the epitope of 12a2h1 (SEQ ID NO: 19) or ALX-0081 (SEQ ID NO: 1).
Moreover, the present invention provides a method for the treatment of a thromboembolic disorder such as e.g. a myocardial infarction, ischemic stroke, deep vein thrombosis or pulmonary embolism, preferably ischemic stroke such as acute ischemic stroke; in patient(s), preferably human(s), in need thereof, wherein said patient(s) has rtPA resistant thrombi, and wherein said treatment comprises the administration to said patient(s) of an effective dose regimen of an anti vWF agent, e.g. an A1 vWF binding agent, a vWF binding agent with the epitope of 12a2h1 (SEQ ID NO: 19) or ALX-0081 (SEQ ID NO: 1).
Equivalent uses, combinations and pharmaceutical compositions related to the anti vWF agent and thrombolytic agent as outlined in the method above and herein are also provided.
The invention further provides an anti vWF agent of a particular epitope, wherein the anti vWF agents having identical CDRs from any of the nanobodies 12a2 (SEQ ID NO:20), 12a5 (SEQ ID NO:21), and/or 12b6 (SEQ ID NO:22), are disclaimed; and wherein said binding agent interacts with at least certain specified amino acid residues on the A1 domain of vWF.
The invention yet further provides an in vitro screening method using the epitope information described in this invention.
In the present description, examples and claims:
The present invention provides a method for the treatment or use in the treatment of a thromboembolic disorder such as e.g. a myocardial infarction, ischemic stroke, deep vein thrombosis or pulmonary embolism, preferably ischemic stroke such as acute ischemic stroke; in patients, preferably humans, in need thereof, wherein said treatment comprises administering
An effective dose regimen of an anti vWF agent, e.g. an A1 vWF binding agent, a vWF binding agent with the epitope of 12a2h1, a selected vWF binding agent (any of SEQ ID NO: 1 to 18 or single domain antibody such as e.g. a nanobody having a CDR combination as shown in any of SEQ ID NO: 1 to 18) or ALX-0081 (SEQ ID NO: 1); is a dose regimen that is able to reduce the ex vivo maximum aggregation below 10% measured by RIPA or below 20% RICO activity measured by RICO (RIPA, ristocetin induced platelet aggregation—(Favaloro E J. Clin Haematol 2001; 14: 299-319.), RICO, Ristocetin Cofactor Platelet Agglutination Assay—(Howard M A, Firkin B G. Ristocetin—a new tool in the investigation of platelet aggregation. Thrombosis et Diathesis Haemorrhagica 1971; 26: 362-9) upon administration of compound see also WO 2009/115614. An example for an effective dose regimen for ALX-0081 in humans, such as e.g. humans with acute coronary syndrome, is a multiple dose, intravenous dose of ALX-0081 every 6 h for 24 h starting with 6 mg and 3 times 4 mg but may be also a dose range such as e.g. 2 to 16 mg ALX-0081 every 6 h (e.g. for 24 h) or simply a dose of ALX-0081 (such as e.g. 16 mg of ALX-0081) wherein the interval of application of the next dose is guided by monitoring the RIPA, i.e. RIPA is not higher than 10% or by monitoring RICO, i.e. RICO is not higher than 20%.
A low dose regimen of a thrombolytic agent is a dose regimen that is known to the skilled person in the art. For example, low dose rtPA protocols have been used that utilizes pulse spray injection of rtPA directly into the thrombus in a total amount of 4 mg or less of rtPA each day for thrombolytic therapy (see e.g. Low-Dose rtPA to Treat Blood Clots in Major Arm or Neck Veins (sponsored by NIHCC) clinical trials.gov identifier is NCT00055159). However, a low dose may also be any dose that is a dose per day that is less than the standard or care that is about 1 to 1.5 mg/kg/per day.
The particular dosage regimen may be further influenced by the attending physician taking into account the particulars of the patient, especially age, weight, life style, activity level, and general medical condition as appropriate.
Moreover, the present invention provides a method for the treatment or use in the treatment of a thromboembolic disorder such as e.g. a myocardial infarction, ischemic stroke, deep vein thrombosis or pulmonary embolism, preferably ischemic stroke such as acute ischemic stroke; in patients, preferably humans, in need thereof, wherein said treatment comprises the inhibition of reocclusion in said patients treated with a thrombolytic agent, e.g. such as rtPA, by administering an effective dose regimen of an anti vWF agent, e.g. an A1 vWF binding agent, a vWF binding agent with the epitope of 12a2h1, a selected vWF binding agent (any of SEQ ID NO: 1 to 18 or single domain antibody such as e.g. a nanobody having a CDR combination as shown in any of SEQ ID NO: 1 to 18) or ALX-0081 (SEQ ID NO: 1).
Moreover, the present invention provides a method for the treatment or use in the treatment of a thromboembolic disorder such as e.g. a myocardial infarction, ischemic stroke, deep vein thrombosis or pulmonary embolism, preferably ischemic stroke such as acute ischemic stroke; in patients, preferably humans, in need thereof, wherein said patients has rtPA resistant thrombi, and wherein said treatment comprises the administration of an effective dose regimen of an anti vWF agent, e.g. an A1 vWF binding agent, a vWF binding agent with the epitope of 12a2h1, a selected vWF binding agent (any of SEQ ID NO: 1 to 18 or single domain antibody such as e.g. a nanobody having a CDR combination as shown in any of SEQ ID NO: 1 to 18) or ALX-0081 (SEQ ID NO: 1).
Equivalent uses, combinations and pharmaceutical compositions related to the anti vWF agent and thrombolytic agent as outlined in the method above and herein are also provided.
The invention further provides a vWF binding agent with the epitope of 12a2h1, wherein said agent is not an agent that is a nanobody or comprises a nanobody having identical CDRs from any of the nanobodies 12a2 (SEQ ID NO:20), 12a5 (SEQ ID NO:21), and/or 12b6 (SEQ ID NO:22); and wherein said binding agent with the epitope of 12a2h1 has an epitope on the A1 domain of the vWF that consists of at least 1 atom in a sphere of 3 Angstrom or more around the 12a2h1-vWF binding site, i.e. wherein said binding agent with the epitope of 12a2h1 interacts at least with the following A1-vWF amino acid residues that are at the positions 500, 502, 503, 505-511, 545 and 550 of the A1 domain of the vWF (taking into account the numbering of the A1 domain of vWF as set out in Cruz et al. Journal of Biological Chemistry (2000), 275 (25), 19098-19105), more preferably wherein said binding agent with the epitope of 12a2h1 has an epitope on the A1 domain of the vWF that consists of at least 1 atom in a sphere of 4 Angstrom or more around the 12a2h1-vWF binding site, i.e. wherein said binding agent with the epitope of 12a2h1 interacts at least with the following A1-vWF amino acid residues that are at the positions 498, 500, 502-511, 545, 550, 695 and 701 of the A1 domain of the vWF (taking into account the numbering of the A1 domain of vWF as set out in Cruz et al. Journal of Biological Chemistry (2000), 275 (25), 19098-19105), even more preferably wherein said binding agent with the epitope of 12a2h1 has an epitope on the A1 domain of the vWF that consists of at least 1 atom in a sphere of 5 Angstrom or more around the 12a2h1-vWF binding site, i.e. wherein said binding agent with the epitope of 12a2h1 interacts at least with the following A1-vWF amino acid residues that are at the positions 498, 500-511, 545, 550, 692, 695, 696, 700 and 701 of the A1 domain of the vWF (taking into account the numbering of the A1 domain of vWF as set out in Cruz et al, Journal of Biological Chemistry (2000), 275 (25), 19098-19105), more preferably wherein said binding agent with the epitope of 12a2h1 has an epitope on the A1 domain of the vWF that consists of at least 1 atom in a sphere of 6 Angstrom or more around the 12a2h1-vWF binding site, i.e. wherein said binding agent with the epitope of 12a2h1 interacts at least with the following A1-vWF amino acid residues that are at the positions 498, 500-511, 543, 545, 550, 691, 692, 695, 696, 700 and 701 of the A1 domain of the vWF (taking into account the numbering of the A1 domain of vWF as set out in Cruz et al. Journal of Biological Chemistry (2000), 275 (25), 19098-19105).
The invention yet further provides an in vitro screening method for the generation of the nanobodies of the invention using the epitope information described in this invention. Generally, it should be noted that the term nanobody as used herein in its broadest sense is not limited to a specific biological source or to a specific method of preparation. For example, the nanobodies of the invention can generally be obtained by any of the techniques (1) to (8) mentioned on pages 61 and 62 of WO 08/020,079, or any other suitable technique known per se. One preferred class of nanobodies corresponds to the VHH domains of naturally occurring heavy chain antibodies directed against the epitope of 12a2h1 on vWF as defined herein. Such naturally occurring VHH domains against the epitope of 12a2h1 on vWF as defined herein, can be obtained from naïve libraries of Camelid VHH sequences, for example by screening such a library using the epitope of 12a2h1 on vWF as defined herein, using one or more screening techniques known per se. For example, the invention yet further provides an in vitro screening method by screening such a library using the above described epitope using one or more screening techniques known per se. Such libraries and techniques are for example described in WO 99/37681, WO 01/90190, WO 03/025020 and WO 03/035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naïve VHH libraries may be used, such as VHH libraries obtained from naïve VHH libraries by techniques such as random mutagenesis and/or CDR shuffling, as for example described in WO 00/43507.
Thus, in another aspect, the invention relates to a method for generating nanobodies that are directed against the epitope of 12a2h1 on vWF as defined herein. In one aspect, said method at least comprises the steps of:
In such a method, the set, collection or library of nanobody sequences may be a naïve set, collection or library of nanobody sequences; a synthetic or semi-synthetic set, collection or library of nanobody sequences; and/or a set, collection or library of nanobody sequences that have been subjected to affinity maturation.
In a preferred aspect of this method, the set, collection or library of nanobody sequences may be an immune set, collection or library of nanobody sequences, and in particular an immune set, collection or library of VHH sequences, that have been derived from a species of Camelid that has been suitably immunized with the epitope of 12a2h1 on vWF as defined herein. In one particular aspect, said epitope of 12a2h1 on vWF as defined herein may be embedded in an antigenic determinant region.
In the above methods, the set, collection or library of nanobody or VHH sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) Nanobody sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to WO 03/054016 and to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).
In another aspect, the method for generating Nanobody sequences comprises at least the steps of:
In the method according to this aspect, the collection or sample of cells may for example be a collection or sample of B-cells. Also, in this method, the sample of cells may be derived from a Camelid that has been suitably immunized with the epitope of 12a2h1 on vWF as defined herein.
The above method may be performed in any suitable manner, as will be clear to the skilled person. Reference is for example made to EP 0 542 810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of step b) is preferably performed using a flow cytometry technique such as FACS. For this, reference is for example made to Lieby et al., Blood, Vol. 97, No. 12, 3820. Particular reference is made to the so-called “Nanoclone™” technique described in International application WO 06/079372 by Ablynx N.V.
In another aspect, the method for generating an amino acid sequence directed against the epitope of 12a2h1 on vWF as defined herein may comprise at least the steps of:
In such a method, the set, collection or library of nucleic acid sequences encoding heavy chain antibodies or nanobody sequences may for example be a set, collection or library of nucleic acid sequences encoding a naïve set, collection or library of heavy chain antibodies or VHH sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of nanobody sequences; and/or a set, collection or library of nucleic acid sequences encoding a set, collection or library of nanobody sequences that have been subjected to affinity maturation.
In a preferred aspect of this method, the set, collection or library of nucleic acid sequences may be an immune set, collection or library of nucleic acid sequences encoding heavy chain antibodies or VHH sequences derived from a Camelid that has been suitably immunized with the epitope of 12a2h1 on vWF as defined herein.
In the above methods, the set, collection or library of nucleotide sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to WO 03/054016 and to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).
As will be clear to the skilled person, the screening step of the methods described herein can also be performed as a selection step. Accordingly the term “screening” as used in the present description can comprise selection, screening or any suitable combination of selection and/or screening techniques. Also, when a set, collection or library of sequences is used, it may contain any suitable number of sequences, such as 1, 2, 3 or about 5, 10, 50, 100, 500, 1000, 5000, 104, 105, 106, 107, 108 or more sequences.
Also, one or more or all of the sequences in the above set, collection or library of amino acid sequences may be obtained or defined by rational or semi-empirical approaches such as computer modelling techniques or biostatics or data mining techniques.
Furthermore, such a set, collection or library can comprise one, two or more sequences that are variants from one another (e.g. with designed point mutations or with randomized positions), compromise multiple sequences derived from a diverse set of naturally diversified sequences (e.g. an immune library), or any other source of diverse sequences (as described for example in Hoogenboom et al, Nat Biotechnol 23:1105, 2005 and Binz et al, Nat Biotechnol 2005, 23:1247). Such set, collection or library of sequences can be displayed on the surface of a phage particle, a ribosome, a bacterium, a yeast cell, a mammalian cell, and linked to the nucleotide sequence encoding the amino acid sequence within these carriers. This makes such set, collection or library amenable to selection procedures to isolate the desired amino acid sequences of the invention. More generally, when a sequence is displayed on a suitable host or host cell, it is also possible (and customary) to first isolate from said host or host cell a nucleotide sequence that encodes the desired sequence, and then to obtain the desired sequence by suitably expressing said nucleotide sequence in a suitable host organism. Again, this can be performed in any suitable manner known per se, as will be clear to the skilled person.
Furthermore, such an amino acid sequence such as e.g. a nanobody directed against the epitope of 12a2h1 on vWF as defined herein may not include an agent that is a nanobody or comprises a nanobody having identical CDRs from any of the nanobodies 12a2 (SEQ ID NO:20), 12a5 (SEQ ID NO:21), and/or 12b6 (SEQ ID NO:22).
The uses and methods of the present invention represent an improvement to existing therapy of thromboembolic disorders in which a combination of i) an anti vWF agent, e.g. an A1 vWF binding agent, a vWF binding agent with the epitope of 12a2h1, a selected vWF binding agent (any of SEQ ID NO: 1 to 18 or single domain antibody such as e.g. a nanobody having a CDR combination as shown in any of SEQ ID NO: 1 to 18) or ALX-0081 (SEQ ID NO: 1); and ii) an thrombolytic agent are used to inhibit inappropriate thrombus formation and to reduce the already formed inappropriate thrombus or clot in the blood vessels of patients with said disorders.
Thus in the present description the terms “treatment” or “treat” refer to both prophylactic or preventative treatment as well as curative or palliative treatment of inappropriate thrombus formation under high shear condition and include not only new formation of thrombus but also reduction of the thrombus. The terms “treatment” or “treat” refer especially in the treatment setting in patients with a thromboembolic disorder or having a risk to develop a thromboembolic disorder such as e.g. a myocardial infarction, ischemic stroke, deep vein thrombosis or pulmonary embolism.
Thus in the present description the terms “prevent”, “preventing” and “prevention” (and the like) include, in addition to complete prevention, “reduce”, “reducing”, “reduction”, “inhibit”, “inhibiting” and “inhibition” of inappropriate thrombus formation under high shear condition and reduction of existing clots or thrombi.
Thus in a particular embodiment, the invention provides:
The specific A1 vWF binding agents used in the present invention are typically those which prevent thrombus formation under high shear condition, in particular those which are indicated to have a safe application in patients with a thromboembolic disorder, e.g. a disorder selected from the group consisting of myocardial infarction, ischemic stroke, deep vein thrombosis and pulmonary embolism, preferably ischemic stroke such as acute ischemic stroke.
Thus, for example, suitable agents of specific A1 vWF binders for use in the invention may include the compounds in Table 1 or a compound having 80% or more, more preferably 85% or more, most preferred 90%, 95%, 96%, 97%, 98%, 99% or more, amino acid sequence identity to a compound in Table A-2 (see Definition section for “sequence identity”).
In another preferred selection, suitable agents of specific A1 vWF binders for use in the invention may include agents such as e.g. antibodies that cross-block or are cross-blocked by the compounds of Table 1 (see Definition section for “cross-blocked” and “cross-block”). In another preferred selection, suitable agents of specific A1 vWF binders for use according to the present invention are antibodies, preferably single variable domains, cross-blocking at least 50% of ALX-0081 (SEQ ID NO: 1) binding, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80% of ALX-0081 binding. In another preferred selection, suitable agents of specific A1 vWF binders for use according to the present invention are antibodies, preferably single variable domains, cross-blocked at least 50% by ALX-0081 (SEQ ID NO: 1), more preferably at least 60%, more preferably at least 70%, even more preferably at least 80% by ALX-0081. Said cross-blocking or cross-blocked measurements are e.g. done by BiaCore measurements.
Preferably the specific A1 vWF binders for use in the invention are the 12a2h1-like compounds. For the purposes of the present description a 12a2h1-like compound is a compound which comprises 12a2h1 (i.e. SEQ ID NO: 19) or a compound having 80% or more, more preferably 85% or more, most preferred 90%, 95%, 96%, 97%, 98%, 99% or more, amino acid sequence identity to 12a2h1 (SEQ ID NO: 19): A particularly preferred specific A1 vWF binder is ALX-0081 (SEQ ID NO: 1).
All the specific A1 vWF binders mentioned above are well known from the literature. This includes their manufacture (see in particular e.g. WO 2006/122825 but also WO 2004/062551). For example, ALX-0081 is prepared as described e.g. in WO 2006/122825.
The vWF binding agent with an epitope to 12a2h1 that is identical or overlapping to the nanobody 12a2h1 (SEQ ID NO: 19) is a binding agent that has an epitope on the A1 domain of the vWF that consists of at least 1 atom in a sphere of 3 Angstrom or more around the 12a2h1-vWF binding site, i.e. wherein said binding agent with the epitope of 12a2h1 interacts at least with the following A1-vWF amino acid residues that are at the positions 500, 502, 503, 505-511, 545 and 550 of the A1 domain of the vWF (taking into account the numbering of the A1 domain of vWF as set out in Cruz et al. Journal of Biological Chemistry (2000), 275 (25), 19098-19105), more preferably wherein said binding agent with the epitope of 12a2h1 has an epitope on the A1 domain of the vWF that consists of at least 1 atom in a sphere of 4 Angstrom or more around the 12a2h1-vWF binding site, i.e. wherein said binding agent with the epitope of 12a2h1 interacts at least with the following A1-vWF amino acid residues that are at the positions 498, 500, 502-511, 545, 550, 695 and 701 of the A1 domain of the vWF (taking into account the numbering of the A1 domain of vWF as set out in Cruz et al. Journal of Biological Chemistry (2000), 275 (25), 19098-19105), even more preferably wherein said binding agent with the epitope of 12a2h1 has an epitope on the A1 domain of the vWF that consists of at least 1 atom in a sphere of 5 Angstrom or more around the 12a2h1-vWF binding site, i.e. wherein said binding agent with the epitope of 12a2h1 interacts at least with the following A1-vWF amino acid residues that are at the positions 498, 500-511, 545, 550, 692, 695, 696, 700 and 701 of the A1 domain of the vWF (taking into account the numbering of the A1 domain of vWF as set out in Cruz et al. Journal of Biological Chemistry (2000), 275 (25), 19098-19105), more preferably wherein said binding agent with the epitope of 12a2h1 has an epitope on the A1 domain of the vWF that consists of at least 1 atom in a sphere of 6 Angstrom or more around the 12a2h1-vWF binding site, i.e. wherein said binding agent with the epitope of 12a2h1 interacts at least with the following A1-vWF amino acid residues that are at the positions 498, 500-511, 543, 545, 550, 691, 692, 695, 696, 700 and 701 of the A1 domain of the vWF (taking into account the numbering of the A1 domain of vWF as set out in Cruz et al. Journal of Biological Chemistry (2000), 275 (25), 19098-19105).
The thrombolytic agent may be an agent such as e.g. a tissue plasminogen activator (herein also referred to as “t-PA, rt-PA, rtPA, Alteplase, alteplase activase”), a reteplase (herein also referred to as “retavase”), a tenecteplase (herein also referred to as “TNKase”), an anistreplase (herein also referred to as “Eminase”), a streptokinase (herein also referred to as “Kabikinase, Streptase”), and/or an urokinase (herein also referred to as “Abbokinase”).
The specific vWF agents as disclosed herein and specific thrombolytic agents as disclosed herein (hereinafter referred to also as the Agents of the Invention) may be used in the form of a polypeptide concentrate or ready-to-use solution (hereinafter also referred to as “pharmaceutical composition of the invention”). For example, the Agents of the Invention can be used in a pharmaceutical composition comprising a buffer (such as e.g. citrate, histidine, Tris, PBS, d-PBS), a tonicifier (such as e.g. mannitol, glycine or sodium chloride) and a surfactant (such as e.g. Polysorbate 80 or Polysorbate 20). Additionally, osmolytes and preservatives may be added. The Agents of the Invention may be in a small-volume, high-dose solution such as e.g. in an amount of from 1 mg agent per ml solution up to 100 mg, e.g. 2 to 50 mg agent per ml solution. Other concentrations such as e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 mg per ml solution are also feasible.
A preferred pharmaceutical formulation for ALX-0081 comprises between 1 to 20 mg, e.g. 5 or 10 mg, ALX-0081 per ml solution that comprises a buffer, a tonicifier and a surfactant. A more preferred pharmaceutical composition comprises between 1 to 20 mg, e.g. 5 or 10 mg, ALX-0081 per ml solution that consists of a buffer, e.g. d-PBS, a tonicifier, e.g. glycine, and a surfactant, e.g. Polysorbate 80. An even more preferred pharmaceutical composition comprises 5 (+/−1) mg/ml ALX-0081, suitable d-PBS buffer; suitable amount of glycine; and a suitable amount of Polysorbate 80 pH 7.1. A most preferred pharmaceutical composition comprises 5 (+/−1) mg/ml ALX-0081, 0.137 M NaCl, 3.7 mM KH2 PO4, 9.8 mM Na2 HPO4x2H2O, 2.7 KCl, 0.2 M glycine, 0.02% (volume %) Polysorbate 80 pH 7.1. Said compositions may be in the form of a concentrate and thus e.g. the dose applied to a patient in need thereof may be adopted by diluting the concentrate to the desired dose (see e.g. experimental part for suitable doses).
A preferred pharmaceutical formulation for rtPA (e.g. 100 mg rtPA) comprises L-Arginine (e.g. 3.5 mg per 100 mg rtPA), phosphoric acid (e.g. 1 mg per 100 mg rtPA), polysorbate 80 (approximately 11 mg per 100 mg rtPA) and sterile water.
The Agents of the invention are preferably used in the form of pharmaceutical compositions that contain a therapeutically appropriate (as described herein) amount of active ingredient optionally together with or in admixture with inorganic or organic, solid or liquid, pharmaceutically acceptable carriers which are suitable for administration.
The pharmaceutical compositions may be, for example, compositions for oral, pulmonary, or parenteral administration, more preferably parenteral administration, such as intravenous or subcutaneous administration, or compositions for transdermal administration (e.g. passive or iontophoretic).
Preferably, the pharmaceutical compositions are adapted to parenteral (especially intravenous, intra-arterial or transdermal) administration. Intravenous administration is considered to be of particular importance. Preferably the Agents of the invention are in the form of a parenteral form, most preferably an intravenous or subcutaneous form.
The particular mode of administration and the dosage may be selected by the attending physician taking into account the particulars of the patient, especially age, weight, life style, activity level, and general medical condition as appropriate.
However, in general the dosage of the Agents of the Invention may depend on various factors, such as effectiveness and duration of action of the active ingredient, warm-blooded species, and/or sex, age, weight and individual condition of the warm-blooded animal.
Formulations in single dose unit form contain preferably from about 1 to about 20 mg, e.g. 5 mg/ml and formulations not in single dose unit form contain preferably from also about 1 to about 20 mg, e.g. 5 mg/ml of the active ingredient.
Pharmaceutical preparations for parenteral administration are, for example, those in dosage unit forms, such as ampoules. They are prepared in a manner known per se, for example by means of conventional mixing, dissolving or lyophilising processes.
Parenteral formulations are especially injectable fluids that are effective in various manners, intra-arterially, intramuscularly, intraperitoneally, intranasally, intradermally, subcutaneously or preferably intravenously and subcutaneously. Such fluids are preferably isotonic aqueous solutions or suspensions which can be prepared before use, for example from lyophilised preparations or concentrate which contain the active ingredient alone or together with a pharmaceutically acceptable carrier. The pharmaceutical preparations may be sterilised and/or contain adjuncts, for example preservatives, stabilisers, wetting agents and/or emulsifiers, solubilisers, salts for regulating the osmotic pressure and/or buffers.
Suitable formulations for transdermal application include an effective amount of the active ingredient with carrier. Advantageous carriers include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host. Characteristically, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the active ingredient of the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin.
The following Experimental Part illustrates the invention described hereinbefore.
The aim of this study was to analyze in Hartley-Dunkey guinea pigs the pharmacokinetics (PK) and pharmacodynamics (PD) of ALX-0081. The PD of ALX-0081 can be measured via the ristocetin-induced platelet aggregation (RIPA) technique and its equivalent ristocetin cofactor (RICO) assay. Both techniques are accepted clinically and measure the ability of ristocetin-activated vWF to interact with the platelet receptor GP1b-IX-V. We wanted to have a dosing regimen of ALX-0081 which gives inhibition in the RICO assay for 24 h, without using infusion pumps. Hartley-Dunkey guinea pigs, male (50%) and female (50%), weighing about 400-450 g (Charles River, Italy) were used in this study. The animals were numbered and divided in groups of 3 individuals.
The dosing regimen was simulated based on the results of a previous PK/PD study, where the plasma PK profiles of ALX-0081 were compared after a single intravenous (i.v.) or subcutaneous (s.c.) administration to female guinea pigs (20 mg/kg, 7 mg/kg and 1 mg/kg for both routes). This simulation showed that with a dosing regimen of 0.2 mg/kg i.v.+0.8 mg/kg s.c. on t=0 h and 1.5 mg/kg s.c. on t=6 h (total dose of 2.5 mg/kg), a full inhibition of the RICO could be expected for approximately 24 h. In addition, a dosing scheme with a lower (1.25 mg/kg: 0.1 mg/kg i.v.+0.4 mg/kg s.c. on t=0 h+0.75 mg/kg s.c. on t=6 h) and one with a higher cumulative dose (5 mg/kg: 0.4 mg/kg i.v.+1.6 mg/kg s.c. on t=0 h and 3 mg/kg s.c. on t=8 h) of ALX-0081 was tested (Table A-2).
#First dose will be injected i.v. on t = 0 h, the second and third dose will be injected s.c.
Blood samples were taken at different time points for PK and PD analysis (0.5 ml per time point) through a catheter inserted into the carotid. Blood samples were collected into tubes with citrate (0.32% final concentration) anticoagulant.
The model is according to the method of Moriguchi A et al.1
Briefly, animals were anesthetized with ketamine and xylazine. A catheter for the administration of drugs was inserted into the left jugular vein while a catheter for rose Bengal (RB) infusion was inserted in the femoral artery. After a left temporal incision, the temporal muscle was removed. A subtemporal craniotomy was performed using a dental drill under an operation microscope to open a 6-mm-diameter oval bony window. The main trunk of the MCA was observed without cutting the dura mater. The head of a 3-mm-diameter optic fiber mounted on a micromanipulator was placed on the MCA segment proximal to the olfactory tract for photoirradiation. Blood flow velocity in the MCA was measured by a pen-type pulse-Doppler flow probe (Transonic) positioned on the MCA 2-3 mm distal to the irradiated segment. Photoirradiation was conducted using a xenon lamp (Hamamatsu Photonics, Hamamatsu, Japan) with a heat-absorption filter and a green filter. When a stable baseline blood flow was obtained, rose Bengal infusion and photoirradiation with green light (wavelength 540 nm, intensity 600,000 lux for 15 min) was simultaneously started. In the optimization experiments, different doses of rose Bengal were analyzed, namely 10 mg/kg, 20 mg/kg and 30 mg/kg, all were infused for 6 min. In subsequent experiments, 20 mg/kg rose Bengal infused over a period of 6 min was used.
The probe of the laser Doppler was gently positioned close to the vessel wall to measure the blood movements under its surface (˜1 mm3). Cerebral blood flow (CBF) was measured and results expressed as tissue perfusion units (TPU). In conditions of complete occlusion of the MCA, CBF was expected to be 12±2 TPU, a value expressing blood movements in the examined tissue outside the MCA. This “zero” value was subtracted from the values recorded for each treated animal, to standardize the analysis and to report only TPUs expressing blood flow in the vessel of interest. CBF was measured for 3 hours after the start of the operation, after which animals were allowed to recover from anesthesia. Body temperature was maintained at 36° C. by a heating pad during surgery. At the end of the photoirradiation period, the skin incision was sutured.
Just before administration, ALX-0081 (5.205 mg/mL) was diluted in vehicle buffer (DPBS pH 7.1+0.2M glycine+0.05% Tween-80) to the appropriate concentrations. ALX-0081 was administered before or after the induction of the photochemical damage to the MCA at the lowest dosing regimen capable to inhibit the ex vivo RICO for 24 hours, namely 0.4 mg/kg i.v.+1.6 mg/kg s.c. on t=0 h and 3 mg/kg s.c. on t=8 h.
One vial of rtPA (20 mg, lyophilized powder, Boehringer Ingelheim) was reconstituted with 20 mL sterile water for injection (Boehringer Ingelheim) without preservative to make a 1 mg/mL solution. rtPA was administered after the induction of the photochemical damage to the MCA. Two dosing regimens have been analysed, namely 0.032 mg/kg (bolus)+0.576 mg/kg (infusion over 30 min) and 0.1 mg/kg (bolus)+0.9 mg/kg (infusion over 30 min). Doses of rtPA were chosen based on literature1,2. In one group, ALX-0081 (0.4 mg/kg i.v.+1.6 mg/kg s.c. on t=0 h and 3 mg/kg s.c. on t=8 h) and rtPA (0.032 mg/kg as bolus+0.576 mg/kg as an infusion over 30 minutes) were administered simultaneously. Both test items were prepared as described above. In control animals, PBS was administered.
To evaluate if the administration of the drugs exerts an effect on haemostasis in the guinea pig, the template bleeding time model was assessed. A standardized cut was inflicted on the ventral face of the foot of guinea pigs using a commercial bleeding time template (Surgicutt, ITC, USA). The blood emerging from the cut was blotted every 30 sec with filter paper until the arrest of bleeding and the total time to bleeding arrest was calculated. The bleeding time was measured at baseline, 30 min and 2 hours after the first administration of drugs.
Guinea pigs were sacrificed 24 hrs after the end of photoirradiation by overdose of anesthetic. For ischemic brain damage analysis, the brains were coronally sectioned using Ringer solution in the presence of oxygen (entire striatum was cut in sections of 500 μm) and were stained with 1% of 2,3,5-triphenyltetrazolium chloride (TTC, Sigma) at 37° C. for 10 min. TTC stained sections were photographed and brain damage (indicated by a white area in the damaged hemisphere) was calculated using image analysis software (Image J software) and reported as % of brain area damaged (the calculation was made considering the damaged hemisphere).
For measurement of intracerebral hemorrhage, TTC stained sections were collected and homogenized. Subsequently, supernatants were collected by centrifugation at 10,000×g for 20 min and then treated with Drabkin reagent (Sigma) for 15 min at RT to convert hemoglobin into cyanomethemoglobin. The absorbance of cyanomethemoglobin was measured at 540 nm. After transforming the absorbance data into corresponding hemoglobin levels through use of a standard curve, the degree of hemorrhage was expressed as percent increase of hemoglobin in the damaged hemisphere compared to the undamaged hemisphere.
A PK/PD study with 3 different dosing regimens of ALX-0081 (total doses of 1.25, 2.5 and 5 mg/kg; N=3/dosing regimen) was performed with the aim to find an optimal dose of ALX-0081 which gives inhibition of the RICO for 24 h.
The PK profiles showed an increase in ALX-0081 plasma levels with higher administered doses (
To optimize the model, a more standardized damage of the MCA was desired. In previous experiments, total occlusion of the MCA was not obtained in some of the animals with a 10-min irradiation of the MCA in combination with a 10 mg/kg dose of rose Bengal (RB) infused over 6 min. Therefore, a reduction of the blood flow under 40% of baseline as critical point was suggested, which occurred approximately 30 to 40 min after the beginning of RB infusion. By increasing the amount of RB, the damage and the time to total occlusion (CBF≦12±2 TPU) was standardized. The latter was preferably obtained in all animals in less than 30 min after the beginning of RB infusion.
Nine animals were randomized in three groups (N=3). The amount of RB in the different groups was varied. When a stable baseline blood flow was established, RB mg/kg (Group 1), 30 mg/kg (Group 2) or 50 mg/kg (Group 3) was administered as an infusion over 6 min. The MCA was irradiated for 15 min, starting simultaneously with RB infusion.
Results showed that a faster occlusion of the MCA and less variation in time to occlusion between animals was obtained with a 30 mg/kg and 50 mg/kg dose of RB compared to the 20 mg/kg dose (
By using drabkin reagent (Sigma) to measure hemoglobin content in the brains, we have obtained an objective way of measuring the degree of brain hemorrhage. Compatibility with TTC staining was shown. Some brains, previously injected with a known volume of blood, were first coronally sectioned and stained with 1% of 2,3,5-triphenyltetrazolium chloride (TTC). After staining, all sections were collected, homogenized and treated with Drabkin reagent. Subsequently, hemoglobin concentration was measured (data not shown).
Effect of ALX-0081 and rtPA in the Stroke Model
The aim of the study was to assess the effect of ALX-0081 and rtPA on the photochemically-induced thrombosis in the MCA of guinea pigs by evaluation of the CBF (by continuous laser Doppler measurement of blood flow), assessment of brain damage (by TTC staining) and determination of intracerebral hemorrhage (by measuring hemoglobin content). The effect of ALX-0081 and rtPA on the template bleeding time was also analyzed.
Thirty guinea pigs were randomized in 6 groups (N=5 in each group). Each of the animals was infused i.v. (via femoral artery) for 6 min with 30 mg/kg RB, immediately followed by an irradiation for 10 min. The guinea pigs from group I received vehicle. In group 2, the guinea pigs followed a dosing regimen of ALX-0081 (total dose of 5 mg/kg), starting just before the start of the photoirradiation (pre-injury). In groups 3-6, ALX-0081 and/or rtPA were administered after the induction of the photochemical damage to the MCA, starting from the moment total occlusion was obtained (CBF≦12±2 TPU; post-injury). In group 3, the guinea pigs received the same dosing regimen of ALX-0081 as group 2 (total dose of 5 mg/kg). The guinea pigs of group 4 received a combination therapy of ALX-0081 (total dose of 5 mg/kg) with rtPA (0.032 mg/kg as bolus+0.576 mg/kg as infusion), starting simultaneously from the moment total occlusion was obtained. In group 5, guinea pigs received a bolus of rtPA (0.032 mg/kg), immediately followed by a continuous infusion of rtPA for 30 min (0.576 mg/kg). Based on literature, this would be a suboptimal dose of rtPA1. Guinea pigs of group 6 received a higher and clinically more relevant dose of rtPA, namely 0.1 mg/kg as bolus+0.9 mg/kg as infusion.
After photochemical damage, the MCA was occluded by a platelet-rich thrombus. The time required to have this occlusion of the MCA was measured by a laser Doppler probe positioned on the artery close to the site of damage. The cerebral blood flow (CBF) was measured and expressed in tissue perfusion units (TPUs). The time from the end of the photoirradiation period to occlusion (time to occlusion; CBF≦12±2 TPU) was 19±6 min in vehicle animals (
The analysis of ischemic brain damage and intracerebral hemorrhage was carried out 24 hrs after the induction of ischemia. Results are shown in
In control animals, the ischemic area was 14.6±2.7% and a 13.8±9.7% increase in hemorrhage was measured. ALX-0081 (post-injury) was able to significantly reduce the ischemic area (3.6±4.8%) while no increased intracerebral bleeding was observed (15.3±8.6%). Treatment with rtPA had a dose-dependent effect on hemorrhage and brain damage. Intracerebral bleeding was increased in both rtPA-treated groups. While there was already a 40.4±14.2% increase in hemoglobin content in the low dose rtPA (0.032+0.576 mg/kg) group, this intracerebral bleeding was further increased in the high dose rtPA (0.1+0.9 mg/kg) group to 64.7%±38.8 (
The template bleeding time was carried out before the procedure, 30 min after the i.v. bolus administration and 2 hrs after the beginning of the procedure.
Vehicle and ALX-0081 had no effect on the template bleeding time. rtPA, however, induced a significant and dose-dependent prolongation of the template bleeding time min after bolus administration (
In a first phase of the study an optimal dosing regimen of ALX-0081 was found which gave complete inhibition of the RICO for 24 hours. In this dosing scheme, one i.v. administration of 0.4 mg/kg (on t=0 h) was combined with two s.c. administrations (1.6 mg/kg on t=0 h and 3 mg/kg on t=6 h). With this new dosing regimen, the desired drug levels could be obtained without the use of infusion pumps.
The objective of the second phase of the study was to optimize the model by increasing the dose of Rose Bengal and consequently increasing the damage to the MCA. By doing this, we obtained a more reproducible time to total occlusion of the MCA and the extent of brain damage correlated well with the time to occlusion. Previously, it was also reported that brain damage correlates with the time to reperfusion and the total MCA occlusion time4,5.
In addition, measurement of the hemoglobin content was evaluated as a read-out for the degree of hemorrhage in the brain. It was shown that this is a more objective method to assess intracerebral bleeding compared to macroscopically analysis of the brains. The method is compatible with the brain damage assessment by TTC staining.
Although rtPA is currently the only FDA-approved treatment for acute ischemic stroke, rtPA can only be used in limited cases due to the potential risk of brain hemorrhage and the brief 3 h time window of efficacy from symptom onset to treatment. To validate the optimized MCA thrombosis model in guinea pig and to ensure accurate comparison with ALX-0081, clinical relevant doses of rtPA were analyzed in this model. rtPA reperfused the MCA dose-dependently, suggesting that rtPA effectively lysed the obstructive thrombus in the MCA. However, rtPA also increased the degree of hemorrhage in a dose-dependent manner, leading to brain damage. The effective and safe dosages of rtPA were similar to these previously reported1,2.
When administered before the injury in the optimized stroke model, ALX-0081 was effective in preventing occlusion of the MCA. If administered after the onset of ischemia, ALX-0081, as monotherapy or in combination with rtPA, was able to induce a complete reperfusion of the MCA. As ALX-0081 has no or only limited thrombolytic activity, it most likely prevented the secondary thrombus formation after spontaneous reperfusion of the MCA. Spontaneous reperfusion after the first occlusion and regeneration of occlusive platelet thrombi was already previously observed in this photochemically-induced thrombosis model. Reperfusion in combination with reocclusion has also been observed in human cerebral arteries in some patients treated with rtPA3. Therefore, inhibition of reocclusion and improvement of brain circulation by ALX-0081 is also expected to prevent development of cerebral infarction in humans. ALX-0081 not only improved the blood flow in the MCA but also ameliorated ischemic brain damage. Compared to the vehicle group, brain damage was reduced in the guinea pigs which received ALX-0081 monotherapy. The template bleeding time was also assessed and was only prolongated in the rtPA-treated groups. The hemoglobin content measurement in the brain may represent a more predictive model for the pro-hemorrhagic potential of antithrombotic agents in patients with acute ischemic stroke.
In conclusion, ALX-0081 was found to prevent reocclusion and decrease brain damage in the photochemically-induced MCA thrombosis model in guinea pig and showed a superior efficacy and safety profile in this model compared to rtPA. The fact that ALX-0081 has no effect on the incidence of hemorrhage in this model while the brain damage is reduced favors the view that ALX-0081 is a potentially promising antiplatelet agent for the treatment of acute ischemic stroke, in which intracranial hemorrhage by antithrombotic agents is the most lethal complication. Given that large platelet-rich thrombi contribute to the clinical failure of thrombolysis with rtPA (del Zoppo, 1992), ALX-0081 can be beneficial in the case of rtPA-resistant thrombi. Even if there is successful tysis of the thrombus in the major artery, downstream platelet-rich thrombus formation in the microvasculature may produce ischemic damage for which ALX-0081 therapy may show a benefit over treatment with rtPA.
The A1-vWF domain is part of the multimeric von Willebrand Factor and the complete sequence of the protein is shown in
Crystals were flash-frozen and measured at a temperature of 100K. The X-ray diffraction data of the complex were collected at the SWISS LIGHT SOURCE (SLS, Villigen, Switzerland) using cryogenic conditions. The structure was solved and refined to a final resolution of 1.75 A.
The crystal belongs to space group P 21 21 21 and contains 2 essentially identical A1-vWF:12a2h1 complexes (complex A and B) in the asymmetric unit. The resulting electron density shows an unambiguous binding mode for the Nanobody 12a2h1, including the orientation and conformation of the Nanobody.
The structure of the A1-vWF:12a2h1 complex is shown in
For the A1 domain of vWF (amino-acids 479-717) residues Asp-498 to Ala-701 in complex A and residues Ser-500 to Ala-704 in complex B that are covered by the electron density.
Interactions Between 12a2h and A1-vWF
The interaction pattern between the Nanobody 12a2h1 and A1-VWF can be divided in 4 regions: CDR1, CDR2, CDR3 and “CDR4”.
Five residues of CDR1 show significant interactions with A1-vWF; the main interactions are provided by S30 and Y31:
Three CDR2 residues significantly interact with A1-vWF:
Four residues of CDR 3 are important:
The loop region between residues 73 and 76 in framework 3 is also referred to as CDR4. Two residues in this region interact with A1-vWF:
The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention.
All of the references described herein are incorporated by reference, in particular for the teaching that is referenced hereinabove.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP10/68208 | 11/25/2010 | WO | 00 | 9/5/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61265508 | Dec 2009 | US |
