MXPA00006545A - Bipapcitide-based pharmaceutical compositions for imaging and treating thrombi - Google Patents

Abstract

The invention provides novel precursor reagents used in production of imaging agents derived from apcitide. Imaging agents made using the precursor reagents of the invention are useful for i(in vivo) detection and diagnosis of thrombi. The precursor reagents of the invention may also be used in production of antithrombotic agents derived from apcitide. The presence of the free carboxylate groups affords the precursor reagents greater solubility than bibapcitide in aqueous media.

Description

Pharmaceutical compositions based on bibapsitide for the formation of images and the treatment of thrombi.

The present invention relates to the field of imaging for the diagnosis of thrombosis. More particularly, this invention relates to pharmaceutical compositions for obtaining thrombus images. The invention also relates to the field of thrombosis treatment, using drugs produced from novel precursor reagents.

BACKGROUND OF THE INVENTION Thrombosis and thrombosis, particularly deep vein thrombosis (DVT) and pulmonary embolism (PE), are common clinical conditions that are associated with significant morbidity and mortality. It has been estimated that, in the United States, approximately five million patients experience one or more episodes of DVT per year and that more than 500,000 cases of pulmonary embolism per year resulting in 100,000 deaths. It is also estimated that more than 90% of all pulmonary embolisms arise from DVT in the lower extremities. Anticoagulant therapy can effectively treat these conditions, if it is applied early enough. However, such treatment carries risks with it (for example, internal bleeding) that unnecessarily hampers the application REF .: 121079 prophylactic. In acute cases, more advanced techniques of thrombolytic intervention (such as the supply of recombinant in vivo tissue plasminogen activator or streptokinase) may be used, but these techniques involve an even greater risk. In addition, the effective clinical application of these techniques requires that the location of the thrombus aggressor be identified, in order to monitor the effect of the treatment.

For these reasons, a rapid means for locating thrombi in vivo is highly desirable, preferably using non-invasive methods. In the past, contrast venography and B-type compression ultrasound were used to identify the location of thrombosis in deep veins; the choice of technique used depended on the location the thrombus was expected to have. However, the first technique is invasive, and both techniques produce discomfort for the patient. In addition, in many cases these methods are either inadequate or give inaccurate results. Current methods used to diagnose PE include chest radiographs, electrocardiograms (EKG), arterial oxygen tension, pulmonary perfusion and ventilation scans, and pulmonary angiography. Apart from the aforementioned invasive procedure, none of these methods has the capacity to provide an unequivocal diagnosis.

Recently, clinical trials for the formation of acute DVT 99mTc scintigraphic imaging, which are platelets, a component of thrombi, were completed, providing an image-forming agent specifically targeted to thrombi. A kit to manufacture radiolabelled apcitide with 99m Tc, ACUTEC®, is in the process of obtaining authorization for its sale as a radiopharmaceutical product. ACUTEC® is formulated with bibapcitide, the chemical structure of which is presented below: The bibapcitide and the labeled radio that is made with it are described in U.S. Patent Nos. 5,508,020; 5,645,815, common transferee, in US Act No. 08 / 253,317; and in WO 93/23085; WO 93/25244; WO 94/23758 and WO 95/33496. WO 94/07918 of common assignee discloses that bibapcitide can also be used in an unlabelled form with radioactive isotopes, as an antithrombotical agent.

Bibapcitide is a dimer of the monomeric apcitide that is also disclosed in US patents and in the above-mentioned international applications and patent applications. The bibapcitide dimer is formed through a bismaleimide bond of the carboxy-terminated cysteines of the apcytid monomers. The monomeric apcystide is complexed with 99m TcO, and the apcitid / 99mTc complex was characterized in Zheng et al, Compendium 366, Meeting 213a of the American Chemical Society, April 13-17, 1997.

BRIEF DESCRIPTION OF THE INVENTION The present inventors have discovered two novel dimers of apcitide, bibapcitide monocarboxylate and bibapcitide dicarboxylate, which are present in aqueous solutions at a pH greater than about 5. These novel dimers can be used as precursors for the production of radiolabelled apcytid with 99m Tc.

In one embodiment, the invention provides a precursor reagent comprising bibapcitide monocarboxylate. In another embodiment, the invention provides a precursor reagent comprising bibapcitide dicarboxylate. In another embodiment, the invention provides a composition comprising bibapcitide monocarboxylate. In another embodiment, the invention provides a composition comprising bibapcitide dicarboxylate. In still another embodiment, the invention provides a pharmaceutical composition comprising bibapcitide monocarboxylate and a pharmaceutically acceptable carrier. In another embodiment, the invention provides a pharmaceutical composition comprising bibapcitide dicarboxylate and a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION The patent and scientific literature referred to herein establishes the knowledge available to experts in this technology. Patents and granted applications of US patents are incorporated herein by reference.

The pharmaceutical compositions object of the invention provide novel precursor reagents, bibapcitide monocarboxylate and bibapcitide dicarboxylate, both for producing imaging agents, and antithrombotic agents, bibapcitide derivatives.

The chemical structure of bibapcitide monocarboxylate The chemical structure of bibapcitide dicarboxylate is illustrated below The presence of carboxyl-free groups allows the precursor reagents greater solubility in aqueous media, than a1 blbapcitide. For example, in Table 1, 1 below, the comparison between the solubility of bibapcitide and bibapcitide dicarboxylate in 0.1 M phosphate buffer, at various pH values, at room temperature is shown: Table 1 Solubilities Bibapcitide is obtained from Diatide, Inc., Londonberry, NH, USA. Bibapcitide can be produced, for example, by using peptide solid base synthesis, as set forth in U.S. Patent Nos. 5,508,020, 5,645,815; in the U.S. patent application Act No. 08 / 253,317 and in WO 93/23085; WO 93/25244; WO 94/23758; WO 94/07918 and WO 95/33496. Preferably, the bibapcitide is produced at a pH of less than about 4, and is isolated as the trifluoroacetate salt. The bibapcitide trifluoroacetate is solubilized using acetonitrile or ethanol and water, or an aqueous solution, before the formulation is made. For use in mammals such as humans, solubilization with ethanol and water or an aqueous solution is preferred.

The bibapcitide and bibapcitide dicarboxylate monocarboxylate are preferably produced from bibapcitide by raising the pH of the solubilized bibapcitide by using a suitable buffer, such as a phosphate buffer adjusted to the pH desired, such as exemplified by Example 1, or a bicarbonate buffer as disclosed in Example 2. Most preferably, the bibapcitide monocarboxylate and bibapcitide dicarboxylate are produced by the reconstitution of lyophilized trifluoroacetate of bibapcitide with a buffer in physiological pH. Any buffer can be used to adjust the pH of bibapcitide to produce bibapcitide monocarboxylate and / or bibapcitide dicarboxylate. For example, phosphate buffer, bicarbonate buffer, borate buffer, citrate buffer, sulfate buffer, and the like can be used to produce the precursor reagents of the invention. Alternatively, the bibapcitide monocarboxylate and / or bibapcitide dicarboxylate can be produced in enzymatic form using, for example, a hydrolase. The bibapcitide monocarboxylate and bibapcitide dicarboxylate can be isolated and purified using known methods, such as HPLC, as shown in Examples 1 and 2.

The stability of the bibappedide, bibapcitide monocarboxylate and bibapcitide dicarboxylate in a variety of pH values is shown in Table 2, below. In Table 2, the stability is expressed in terms of a stability time of 95% at room temperature.

Table 2 Solubilities The precursor reagents of the invention can be provided in the form of a pharmaceutical composition. Preferably, the pharmaceutical composition object of the invention consists of bibapcitide monocarboxylate or bibapcitide dicarboxylate. More preferably, the pharmaceutical composition of the invention consists of bibapcitide monocarboxylate and bibapcitide dicarboxylate. More preferably, the pharmaceutical composition of the invention consists of bibapcitide monocarboxylate, bibapcitide dicarboxylate, and bibapcitide. The amount of bibapcitide monocarboxylate, bibapcitide dicarboxylate and bibapcitide in the pharmaceutical composition may vary in accordance with this practical embodiment of the invention. The commercially formulated bibapcitide, to be sold as ACUTEC®, typically contains between about 10% and about 50% bibapcitide monocarboxylate and between about 3% and about 12% bibapcitide dicarboxylate.

The pharmaceutical composition of the invention may further comprise a pharmaceutically acceptable diluent or a carrier such as albumin suitable for the species. As used herein, a "pharmaceutically acceptable diluent or carrier" can comprise any and all solvents, dispersion media, antibacterial and antifungal agents, isotonic agents, enzyme inhibitors, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. For example, the injection of Sodium Chloride and the injection of Ringer's Solution are commonly used as diluents. The precursor reagent is formulated as a sterile, pyrogen-free, parenterally acceptable aqueous solution which can optionally be provided in lyophilized form and can be reconstituted by the user. The preparation of such parenterally acceptable solutions, with due regard to pH, isotonicity, stability and the like, is well within the capabilities of this technology.

The novel precursor reagents of the invention can be used to produce diagnostic or therapeutic agents derived from bibapcitide. Such agents include agents for scintigraphic imaging for the detection and diagnosis of thrombi, as described in more detail in U.S. Patent Nos. 5,508,020; 5,645,815, in US Act No. 08 / 253,317 and in WO 93/23085; WO 93/25244; WO 94/23758; and WO 95/33496. The bibapcitide monocarboxylate and / or the bibapcitide dicarboxylate can be used to produce antithrombotic agents, as disclosed in WO94 / 07918. The precursor reagents of the invention can be used to produce an antithromotic agent consisting of a target-seeking peptide, derived from bibapcitide, to which it covalently binds with a thrombolytic proteinase, as described in detail in the applications co-pending patent No. 08 / 753,781 and act No. 08 / 982,981.

When a precursor reagent of the invention is used to produce a radiolabelled therapeutic or diagnostic agent derived from bibapcitide, any marker that generates signals can be used. Such labels can be incorporated into or complexed with a precursor reagent of the invention in any manner suitable for that particular marker, or by direct covalent or non-direct covalent chemical linkage with the precursor reagent or by indirect covalent or non-covalent chemical linkage to it. Suitable markers include radioactive labels, fluorescent labels, paramagnetic markers, heavy element or rare earth ions, suitable for use in computed tomography and similar systems. Radioactive labels are preferred. More preferably, radiation-emitting radionuclides 3, such as 123I, 67Ga are used in the methods according to the invention; l?: LIn, and 99mnpTc. More preferably, the 9"9" mTpe is used to label the precursor reagents of the present invention.

When 99m, Tc is used as a marker, 99m? Tc is added to a pharmaceutical composition comprising bibapcitide monocarboxylate and / or bibapcitide dicarboxylate at a pH greater than about 5, and the resulting mixture is mixed for a time and at a temperature sufficient to allow the formation of apcytid monomer and the radiolabelling of said monomer. Preferably, the mixture of the pharmaceutical composition consisting of bibapcitide monocarboxylate and / or bibapcitide dicarboxylate and 99mtc is heated for about 15 minutes in a boiling water bath, to form an imaging agent for scintigraphy comprising labeled apcytide with 99mTc.

Agents labeled or unlabeled for thrombus imaging or the antithrombotics produced by means of the precursor reagents of the invention are preferably administered intravenously, in combination with a pharmaceutically acceptable carrier, to a living mammal. According to the teachings of the invention, the antithrombotic or imaging agents produced from the pharmaceutical compositions containing bibapcitide monocarboxylate and / or bibapcitide dicarboxylate are preferably administered in an injectable dose in a single unit, in any medium conventional for intravenous injection, such as an aqueous saline medium, or in blood plasma medium. The amount of solution to be injected in the unit dosage ranges from about 0.01 ml to about 10 ml.

Diagnostic and therapeutic agents produced from pharmaceutical compositions containing bibapcitide monocarboxylate and / or bibapcitide dicarboxylate are delivered in a diagnostic or therapeutically effective amount to a mammal that is potentially at risk of a disease state related to a thrombus. or who suffers from such a disease state.

As used herein, the term "diagnostically effective amount" means the total amount of each active component of the pharmaceutical composition of the diagnostic agent produced from the bibapcitide monocarboxylate and / or bibapcitide dicarboxylate or the total amount of such composition that is administered according to a method employing the diagnostic agent, which is sufficient to produce an edible signal located at the site of a thrombus in vivo. As used herein, the term "therapeutically effective amount" means the total amount of each active component of the therapeutic composition of the therapeutic agent produced from the bibapcitide monocarboxylate and / or bibapcitide dicarboxylate or the total amount of such a composition that is administered according to a method employing the therapeutic agent, which is sufficient to exhibit a significant benefit to the patient, that is, a reduction in the incidence and severity of the thrombi, compared to that expected for a patient. comparable group of patients who do not receive the therapeutic agent, as established by the attending physician. When the active ingredient administered alone is applied to an individual, the terms refer to an ingredient alone. When applied in a combination, the terms refer to combined amounts of the active ingredients that are in effect diagnostic or therapeutic, whether they are supplied in combination, serially or simultaneously. For example, imaging or therapeutic agents produced from bicapcitide monocarboxylate and / or bicapcitide dicarboxylate can be delivered in a dose ranging from about 0.1 to about 10 mg / kg of body weight, administered intravenously and in its entirety as a bolus to partially as a bolus followed by infusion for 1-2 hours. When radio-labeled agents, for diagnostic or therapeutic purposes they are produced from bicapcitide monocarboxylate and / or bicapcitide dicarboxylate, the unit dose to be administered has a radioactivity ranging from about 0.01 mCi to about 100 mCi, preferably around 1 mCi to about of 20 mCi. After intravenous administration, the thrombus site is monitored, in certain embodiments, by the formation of radioimaging in vivo.

Methods for manufacturing bibapcitide monocarboxylate and / or bicapcitide dicarboxylate are illustrated in more detail in the following examples, which are given by way of illustration and not by way of limitation.

EXAMPLE 1 SYNTHESIS OF BIBAPCITIDE MONOCARBOXYLATE The bibapcitide trifluoroacetate (100 mg) was placed in suspension in 10 ml of acetonitrile (CH3N) sonicated for one minute and then diluted with 40 ml of water (H20). The peptide was completely dissolved when water (H2 O) was added. To this solution was added 40 ml of 0.05 M sodium phosphate at a pH of 7 causing the solution to become slightly cloudy. The peptide solution had a pH of .2. The solution was incubated in a boiling water bath for three minutes, which resulted in a clear solution. The HPLC analysis indicated the presence of bibapcitide dicarboxylate, bibapcitide monocarboxylate and bibapcitide, in approximate amounts of 26%, 54% and 14% respectively. The reaction solution was directly loaded in a Delta-Pak C18 column of 47 x 300 mm, equilibrated in 10 mM of ammonium bicarbonate (NH4HCO3) and adjusted to a pH of 6-6.5 with solid C02 (Mobile Phase C). The column was washed by irrigation with Mobile Phase C for five minutes followed by a gradient of 100/0 C / D at 90/10 C / D for five minutes, and then 90/10 C / D at 80/20 C / D. for 30 minutes (Mobile Phase D = 10 mM NH4HCO3 in 75/25 CH3 CN / H20 at pH 6-6.5). The shocks for HPLC were continuously maintained at a pH of 6-6.5 by liquid C02. Fractions were collected on the basis of monitoring the effluent at 220 nm. The fractions were then analyzed by analytical HPLC and those found to contain pure bibapcitide monocarboxylate (> 98%) were pooled and lyophilized to obtain approximately 30 mg of bibapcitide monocarboxylate (30% yield) as the ammonium carbonate salt, a white powder. The NMR analysis of bibapcitide monocarboxylate (20% CD3CN / 80% H20, pH 6, T = 20 ° C) thus produced is shown in Table 3 below Table 3 Data on Chemical shift - ^ H NMR (L, ppm) for Bibapcitide monocarboxylate * Dystereomeric resonances EXAMPLE 2 SYNTHESIS OF BIBAPCITIDE DICARBOXYLATE The bibapcitide trifluoroacetate (100 mg) was placed in suspension in 5 ml of CH3CN, sonicated for one minute and then diluted with 25 ml of H20. The peptide was completely dissolved when H20 was added. To this solution was added one ml of saturated sodium bicarbonate (NaHCO3) and 0.5 ml of 1 M potassium carbonate (K2C03). With pH paper it was estimated that the peptide solution was estimated to have a pH of 8.5. This solution became cloudy with the addition of K2C03 but slowly cleared after two hours at room temperature. After three hours it was found that the reaction contained 84% bibapcitide dicarboxylate as measured by analytical HPLC. The reaction solution was directly loaded on a 47 x 300 mm Delta-Pak C18 column equilibrated in 10 mM ammonium bicarbonate (NH4 HC03) and adjusted to a pH of 6-6.5 with solid C02 (Mobile Phase C) . The column was washed by irrigation with 100% Mobile Phase C for 5 minutes followed by a gradient of 100/0 C / D at 90/10 C / D for 5 minutes, and then 90/10 C / D at 70 / 30 C / D for 30 minutes. The dampers for HPLC were continuously maintained at a pH of 6-6.5 by solid C02. The fractions were collected on the monitoring of the effluent at 220 nm. The fractions were then analyzed by analytical HPLC, and those found to contain pure bibapcitide dicarboxylate (> 98%) were pooled and lyophilized to obtain approximately 54 mg of bibapcitide dicarboxylate (86% peptide content). isolated yield 53%) like the ammonium carbonate salt, a white powder. The NMR analysis of bibapcitide dicarboxylate (20% CD3CN / 80% H2O, pH 6, T = 20 ° C) thus produced is set forth in Table 4 below.

Table 4 Data about! the chemical shift 1H NMR (L, ppm) I for the Bibapcitide Dicarboxylate ^ Distereomeric Resonances It is to be understood that the foregoing disclosure emphasizes certain specific embodiments of the present invention, and that all modifications, or activities equivalent thereto, fall within the spirit and general scope of the present invention, as set forth in the appended claims. .

It is noted that in relation to this date the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Having described the invention as above, the contents of the following are declared as property:

Claims (10)

1. A precursor reagent characterized in that it comprises bibapcitide monocarboxylate.

2. A precursor reagent characterized in that it comprises bibapcitide dicarboxylate.

3. A composition characterized in that it comprises bibapcitide monocarboxylate.

4. The composition of claim 3, characterized in that it also comprises bibapcitide dicarboxylate.

5. The composition of claim 4, characterized in that it also comprises bibapcitide.

6. A composition characterized in that it comprises bibapcitide dicarboxylate.

7. A pharmaceutical composition characterized in that it comprises bibapcitide monocarboxylate and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition of claim 7, characterized in that it further comprises bibapcitide dicarboxylate.

9. The pharmaceutical composition of claim 8, characterized in that it further comprises bibapcitide.

10. A pharmaceutical composition characterized in that it comprises bibapcitide dicarboxylate and a pharmaceutically acceptable carrier.