HK1261592A1 - Purification method - Google Patents

Description

Purification method

Technical Field

The present invention relates to the purification of components for radiopharmaceuticals. In particular, the present invention relates to a method for the purification of complexed thorium-227 for internal (endo) radionuclide therapy, in particular where the purification is performed shortly before drug administration to a human subject.

Background

Specific cell killing may be essential for successful treatment of a variety of diseases in mammalian subjects. Typical examples thereof are the treatment of malignant diseases such as sarcomas and carcinomas. However, selective elimination of certain cell types may also play a key role in the treatment of many other diseases, particularly immunological, proliferative and/or other neoplastic diseases.

however, the most common radiopharmaceutical forms currently approved for use in humans employ beta-emitting and/or gamma-emitting radionuclides²²³Ra), has proven to be very effective, particularly for the treatment of diseases associated with bone and bone surfaces additional α -emitters are also being actively investigated, and one particularly interesting isotope is the α -emitter thorium-227.

in a physiological environment, the radiation range of typical α emitters is typically less than 100 microns, corresponding to only a few cell diameters, making these nuclei well suited for the treatment of tumors, including micrometastases, because little radiation energy can exceed the target cells and thus damage to surrounding healthy tissue may be minimized (see Feinenegen et al, radial Res 148:195-201 (1997)). conversely, β particles have a range of 1mm or more in water (see Wilbur, antibody Immunocon radiopharmarm 4: 85-96 (1991)).

this deposition of large amounts of energy over very short distances provides α -radiation with particularly high Linear Energy Transfer (LET), high Relative Biological Efficiency (RBE), and low Oxygen Enhancement Ratio (OER) compared to gamma and β radiation (see Hall, "radio biology for the radiologic", fifth edition, Lippincott Williams & Williams, Philadelphia PA, USA, 2000.) these properties explain the specific cytotoxicity of α -emitting radionuclides and also place stringent requirements on the purity levels required when the isotope is to be administered in vivo.

From²²⁷Ac initiated production of radioactive decay chains²²⁷Th and then lead to²²³Ra and other radioisotopes. The first three isotopes in this chain are shown below. Watch shows²²⁷Th and the elements of the isotopes preceding and following it, molecular weight (Mw), decay pattern (mode), and half-life (year (y) or day (d)).²²⁷Th can be prepared from²²⁷The onset of Ac, which is itself present in trace amounts in uranium ores, originates from²³⁵Part of the natural decay chain of U. A ton of uranium ore contains about one tenth of a gram of actinium and therefore although²²⁷Ac is naturally occurring, but it more commonly passes through in nuclear reactors²²⁶Neutron irradiation of Ra.

From this explanation it can be seen that the preparation of a compound from the above decay chain for pharmaceutical use²²⁷Th having a half-life of more than 20 years²⁷Ac is a very dangerous potential contaminant. But even once it is ready to use²²⁷Ac is removed or reduced to a safe level,²²⁷th will continue to declineHaving a half-life of just below 19 days²²³And Ra. Due to the fact that²²³Ra is an alkaline earth metal that is not readily coordinated by ligands designed for thorium or other actinides. The²²³Ra then forms the onset of a potentially uncontrolled (untargeted) decay chain (comprising 4 α -decays and 2 β -decays) and then reaches a stable²⁰⁷And Pb. These are shown in the following table:

。

as is apparent from the above two decay tables,²²³ra can not be selected from²²⁷Any preparation of Th is completely eliminated as the latter will decay constantly and produce the former. However, it is clear that each is administered to the patient²²³Decay of the Ra nucleus releases more than 25MeV of radiant energy, and the nucleus then reaches the stable isotope. Such that²²³Ra may not be designed to²²⁷Systemic binding and targeting of chelation and specific binding of Th trafficking to its site of action is due to the different chemical nature of the two elements. Therefore, for the purpose of targeting cell killing, maximizing therapeutic effect, and minimizing side effects, it is important to control any²²⁷Th preparations before application²²³Ra level. Even if the product must be stored or transported for a period of time (e.g. 12 to 96 hours, such as up to 48 hours) before administration, it is still important to start with a very pure isotope in order to still minimise contamination by the sub-isotopes.

Separation of²²⁷Th and²²³ra can be performed quickly and conveniently in a radiology laboratory, such as by²²⁷The site where Ac decays. However, this is not always possible and may not effectively achieve the desired results because of the resulting purified²²⁷Th must then be transported to the site of application. If the application site is far away from²²⁷The origin of Th will occur during storage and transportation²²³Further accumulation of Ra.

In addition to this, the present invention is,²²⁷th can be in²²⁷Complexation of Th to form a drug (e.g. targeted radiopharmaceutical complex) either before or after²²³And Ra purification. If the process is performed prior to complexation, purification may be simpler, particularly if the ligand is conjugated to a targeting molecule such as an antibody, since large conjugates may be difficult to handle. However, if a purification method can be devised, the method can be used at or near the point of care and can be used for medical purposes²²⁷Separation of Th complexes (e.g. targeting complexes) undesired²²³Ra, then this would provide a considerable advantage since no delay between purification and application is required for the generation of the complex.

In view of the above, a method for treating a contaminated object is provided²²³Ra purification²²⁷A Th process which can be carried out centrally, purified, would be an important advantage²²⁷Th can reach the site of administration from this location significantly faster than the half-life of the isotope. In case the purified isotope is to be stored for some time, e.g. 12-96 hours, then the method should provide²²³A very high degree of removal of Ra, so that only radium generated by the inevitable growth (in-growth) is administered to the subject. Alternatively, purification can occur at or near the point of care at or shortly before the time of administration using a simple method that can be accomplished without extensive training and experience. In any embodiment, the method should be robust, reliable and efficient, since the resulting purified product is²²⁷Th (²²⁷Th complexes) may be used directly in pharmaceutical preparations.

If this method is available for complexation²²⁷It would be another advantage to do on Th, preferably conjugated to a targeting moiety. From a safety and handling point of view it would be an advantage if the use of strong mineral acids and/or strong bases could be avoided (as well as avoiding possible degradation of sensitive components such as the targeting moiety of the targeting complex) and also because this avoids the necessity of separating these materials from the final product. If used, theThe agent is suitable for direct use in the final pharmaceutical product, which is particularly suitable since the speed is thereby maximised. It would also be an advantage if a small volume could be used to simplify handling and reduce the volume of contaminated waste. It would be a further advantage if the method could be carried out with a simple set of reagents and equipment items that could be provided for such simultaneous preparation, optionally in the form of a kit.

Previously known²²⁷Preparations of Th are typically used for laboratory applications and/or have not been tested for standard purity of pharmaceuticals. In WO2004/091668, for example, preparation from a single column by anion exchange²²⁷Th and used for experimental purposes without confirmation of purity. In that²²⁷The main purpose of the separation in most preparative processes for Th is to eliminate longevity²²⁷Parent isotope of Ac. Has not previously been²²³Ra (which has been previously derived from²²⁷Purification of Ac²²⁷Growth in Th samples) removal design or optimization method。

Summary of The Invention

The inventors of the present invention have now established that a rapid and simple purification operation can be used to remove the impurities from the crude oil²²⁷Th product removal²²³And Ra. The method allows²²⁷Th is in complexed form and even complexed and conjugated to a targeting moiety such as an antibody. The method may use a single purification step. In this way, very high radiochemical purity can be produced²²⁷Th solutions, while providing many desirable advantages in the method, particularly with respect to reduction of delay between purification and application and less product handling at the site of application.

Thus, in a first aspect, the present invention provides a method for complexing from a composition comprising²²⁷Th and²²³ra (complexed or in solution) mixtures for purifying complexed²²⁷A method of Th, the method comprising:

i) preparing a first solution comprisingComplexed in a first aqueous buffer²²⁷Th ion and²²³a mixture of Ra ions;

ii) loading the first solution onto a separation material, such as a strong cation exchange resin;

iii) eluting the complex from the separation material²²⁷Th, thereby producing a complex-containing²²⁷A second solution of Th;

iv) optionally rinsing the separation material with a first aqueous washing medium;

typically, steps i) to iv) will be performed in the order given above, although other steps and processes may obviously be performed in or between the listed steps.

The process optionally and preferably further comprises the following steps before step i) above:

x) making²²⁷Contacting the Th ions with at least one complexing agent in solution, thereby forming complexed²²⁷At least one aqueous solution of Th. Preferably, the complexing agent is a chelating moiety conjugated (e.g., covalently conjugated) to a targeting moiety, such as those described herein.

The process optionally also comprises at least one of the following further steps, each typically performed after steps i) to iv) above:

v) determining the second solution²²⁷The content of Th;

vi) evaporating liquid from the second solution;

vii) complexed by the solution contained in the second solution²²⁷At least a portion of Th forms at least one radiopharmaceutical formulation;

viii) sterile filtering the radiopharmaceutical.

Step vii) forms a particularly preferred further step.

In another aspectThe invention provides²²⁷Solutions or other samples of Th comprising per 1MBq²²⁷Th less than 50KBq²²³Ra, preferably per 1MBq²²⁷Th less than 10KBq²²³And Ra. Such solutions are optionally formed or formable by any of the methods described herein, and preferably formed or formable by the preferred methods described herein. Accordingly, the method of the present invention is preferably used to form²²⁷A solution of Th comprising per 1MBq²²⁷Th less than 50KBq²²³Ra, preferably per 1MBq²²⁷Th less than 10KBq²²³And Ra. Also provided is a corresponding pharmaceutical formulation, which may be sterile, and which may comprise at least one complexing agent (particularly for²²⁷Th), at least one targeting agent (e.g. conjugated to the complexing agent) and optionally at least one pharmaceutically acceptable carrier or diluent.

In yet another aspect, the invention also provides a kit (a kit typically used to carry out the methods of the invention) comprising²²⁷Th and²²³a mixture of Ra, a first aqueous buffer, a chelating agent (preferably conjugated or conjugatible to a targeting moiety), and a separation material (e.g. a cation exchange resin).²²⁷Th and²²³mixtures of Ra (as with the first solution in other aspects of the invention) will generally also contain additional²²³And Ra sub-product. Such mixtures may be purified or partially purified during storage and/or transport²²⁷The result of radioactive decay of Th (optionally complexed or in solution).

Detailed Description

the administration of α -emitting radionuclides to a subject's body requires all of these considerations, but additionally increases the need for high radiochemical purity.

However, in the case of the decay of the target radionuclide to other radioisotopes, another level of radiochemical purification may be necessary. The generation of radioactive daughter isotopes may significantly contribute to the toxicity of radionuclide therapy and may be dose limiting. In that²²⁷this means that any chelation or complexation that may have been suitable for binding thorium will likely not be chemically suitable for retaining daughter radium as a result of conservation of momentum, after the α particles are ejected at very high velocities, the α decay additionally imparts very significant "recoil" energy to the daughter nuclei.

Due to the passing of²²⁷Th decay and in vivo generation²²³The presence of Ra and its progeny potentially limits the dosage, importantly, there is no additional, unnecessary²²³Ra administration to a subject to further limit²²⁷Acceptable therapeutic dose of Th or increased side effects.

In view of²²⁷In Th samples²²³Inevitable growth of Ra and enabling delivery to a subject²²³The need to minimize Ra (as long as there is a reasonable probability), the present invention has been developed. Due to the fact that²²³Ra initially grows at a rate of about 0.2% total activity per hour, and the method should be performed no more than a few hours (e.g., within 72 hours or within 48 hours) prior to administration in order to minimize unnecessary doses. Similarly, if²²⁷Th may be used within 2-4 hours of preparation, then the process should preferably be provided as such²²³Ra (as prepared) has a radiochemical purity of about 99% (e.g. 95% to 99.9%)²²⁷Th. Higher purity may be futile and/or meaningless, as growth prior to use would undermine any benefit of the more rigorous purification processLower purity (e.g., less than 90% or less than 95% radiochemical purity) is undesirable because²²³The dose of Ra (and hence toxicity) may suitably be further limited while allowing realistic administration times.

In one embodiment, for use in the present invention²²⁷Th and²²³mixtures of Ra will not contain significant amounts of compounds that do not begin at²²⁷Radioisotopes in the decay chain of Th. In particular, for any aspect of the invention²²⁷Th and²²³mixtures of Ra will preferably comprise per 100MBq²²⁷Th is less than 20 Bq²²⁷Ac, preferably per 100MBq²²⁷Th less than 5 Bq²²⁷Ac。

The present invention provides a method of producing at a purity level suitable for use in internal radionuclide therapy²²⁷Method of Th. An additional benefit of the method of the invention is that it may be carried out after having been complexed with a chelating agent, preferably a chelating agent conjugated to a targeting moiety²²⁷Th is performed. By isolating the pre-complexed sample, the method reduces the number of further steps required to produce the pharmaceutical formulation, thus allowing for faster administration of the isotope following purification. Since the radioisotope continues to decay under all storage conditions, purification shortly before administration allows for a drug with higher isotopic purity. In the present invention, complexed²²⁷Th, typically in the form of an ion complexed with a ligand conjugated to a targeting moiety (known as "targeted thorium conjugate" -TTC), is purified directly, preferably shortly before administration.

A number of preferred features of the system are indicated below, each of which may be used in combination with any other feature when technically feasible, unless explicitly indicated otherwise.

The method of the invention and all corresponding embodiments will preferably be performed on a scale suitable for administration to a patient. The scale may be a single therapeutic dose, or may be suitable for use in a number of subjects, each subject receiving a dose. Typically, the process will be suitable for use in 1-5 hoursFor internal application on a scale such as²²⁷About 1-10 typical doses of Th. Single dose purification forms a preferred embodiment. Obviously, a typical dosage will depend on the application, but it is envisioned that a typical dosage may be 0.5-200 MBq, preferably 1-25 MBq, and most preferably about 1.2-10 MBq. Combined dose purifications were carried out where possible, with up to 20, preferably up to 10 or up to 5 typical doses being used. Purification can thus be carried out with up to 200 MBq, preferably up to 100MBq, and where appropriate divided into individual doses after purification.

Step i) of the process of the invention is directed to a process comprising²²⁷Th and²²³solutions of Ra (and usually also including²²³Ra sub-isotopes-see those in the table above). Such a mixture will inherently pass through²²⁷The gradual decay of the Th sample forms but for use in the present invention it also preferably has one or more of the following characteristics, either alone or in any feasible combination:

a)²²⁷the Th radioactivity may be at least 0.5 MBq (e.g. 0.5 MBq to 100 MBq), preferably at least 0.5 MBq, more preferably at least 1.4 MBq;

b) the solution may be formed in a first aqueous buffer solution;

c) the solution may have a volume of no more than 50ml (e.g. 0.1-20 ml or 0.1-10 ml), preferably no more than 10ml or 5 ml, more preferably between 3 and 7 ml.

d) The first aqueous buffer solution may be at a pH between 3 and 6.5, preferably between 3.5 and 6, and in particular between 4 and 6.

e) The first aqueous buffer solution may be used at a concentration of 0.01 to 0.5M, such as 0.03-0.05M or 0.1-0.2M.

f) The first aqueous buffer solution may comprise, consist essentially of, or consist of at least one organic acid buffer.

g) The first aqueous buffer solution may comprise, consist essentially of, or consist of at least one organic acid buffer selected from the group consisting of citrate buffers, acetate buffers, and mixtures thereof.

h) The first aqueous buffer solution may optionally additionally comprise at least one radical scavenger and/or at least one chelating agent (in particular a non-buffering chelating agent). Many of which are known in the art and include pABA (scavenger) and EDTA (chelator).

i) The first aqueous buffer solution may optionally additionally comprise other additives, including salts, such as NaCl.

Step ii) of the process of the present invention involves loading the first solution onto a separation material, such as a cation exchange resin. This step and the entities mentioned therein may have the following preferred features, alone or in any feasible combination, and optionally in any feasible combination with any features of the other steps described herein:

a) the separation material may be a cation exchange resin or hydroxyapatite, preferably a strong cation exchange resin.

b) The resin (e.g., cation exchange resin) may be a silica-based resin;

c) the cation exchange resin may comprise one or more acid functional groups;

d) the cation exchange resin may comprise at least one acid moiety and preferably at least one carboxylic or sulfonic acid moiety, such as an alkyl sulfonic acid resin such as Propyl Sulfonic Acid (PSA) resin;

e) the resin (e.g., strong cation exchange resin) may have an average particle size of 5 to 500 μm, preferably 10 to 200 μm.

f) The separation material (e.g., cation exchange resin) may be used in the form of a column.

g) The amount of separation material (e.g. resin) used (e.g. packed in a column) may be 100mg or less (e.g. 2-50mg), preferably 10-50 mg.

h) The separation material (e.g., resin) may be preconditioned by washing with one or more volumes of aqueous medium prior to loading with the first solution. Typically, a buffer solution, more preferably a first aqueous buffer, is used for preconditioning.

Step iii) of the process of the invention involves elution of the complexed from the separation material (e.g. strong cation exchange resin)²²⁷Th, thereby producing a complex-containing²²⁷A second solution of Th. This step and the entities mentioned therein may have the following preferred features, alone or in any feasible combination, and optionally in any feasible combination with any features of the other steps described herein:

a) the elution can be by means of an eluent solution or by means of a "dry" elution, such as by elution under gravity, under centrifugal force or under gas pressure from above and/or vacuum from below;

b) where elution is by means of an eluent solution, this may be an aqueous buffer solution, such as any of those described herein, including organic acid buffer solutions;

c) the elution may be by "dry" means, preferably under gravity or centrifugal force, such as spinning in a centrifuge.

d) Elution by centrifugal force may be for a period of 10 seconds to 10 minutes, preferably 20 seconds to 5 minutes at a "relative centrifugal force" (RCF) of at least 1000 times, preferably at least 2000 times or at least 5000 times the gravity force (e.g. RCF of 1000-50000 g).

Step iv) of the process of the present invention involves an optional step of rinsing the separation material (e.g. strong cation exchange resin) with a first aqueous washing medium. This step and the entities mentioned therein may have the following preferred features, alone or in any feasible combination, and optionally in any feasible combination with any features of the other steps described herein:

a) the first aqueous washing medium may be water, such as distilled, deionized or water for injection, or may be a buffer such as an organic acid buffer as described herein;

b) the first aqueous washing medium may comprise the same buffer as the first buffer solution;

c) the optional washing step may be omitted;

d) the optional washing step may comprise: adding a first wash medium to the resin after the "dry" elution described herein, and then "dry" eluting the wash medium, such as by gravity or centrifugation; or under gas pressure from above and/or vacuum from below.

e) The solution eluted in the washing step may be mixed with a solvent comprising²²⁷A second solution combination of Th.

After step iv) of the process of the invention, the separation material (e.g. resin) is typically disposed of as radioactive waste. Since the amount of resin required is typically very small (e.g., less than 50mg), this does not represent a significant disposal problem. However, if it is desired to reuse the resin or recover it for determination or any other reason²²³Ra, can be eluted using any suitable medium²²³And Ra. Suitable media for such recovery include buffer solutions, such as those described herein, and aqueous mineral acids, such as HCl and H₂SO₄. If the resin is to be reused, it is typically regenerated with several volumes of the first buffer solution before reuse.

The process of the invention may comprise a number of optional steps, each of which may be present or absent independently, as long as technically feasible.

Prior to the separation steps i) to iv), an optional preparation step X) is preferably included. Step by stepStep X) comprises from²²⁷Complexing Th ions with chelating agents²²⁷Th. Preferably, the complexing agent comprises a chelating agent conjugated (e.g. by covalent linkage) to the targeting moiety. This step and the entities involved therein may have the following preferred features, either alone or in any feasible combination, and optionally in any feasible combination with any features of the other steps described herein. Furthermore, all features of the radiopharmaceutical agents described herein form preferred features of the pharmaceutical aspects of the invention, particularly where the agent is formed or formable by the methods of the invention:

a) in contact with chelating agents²²⁷The amount of Th may be 1 to 100MBq, preferably 1 to 10 MBq.

b) The complexing agent may comprise an octadentate ligand.

c) The complexing agent may comprise a hydroxypyridone such as a Hydroxypyridone (HOPO) ligand, preferably an octadentate 3, 2-hydroxypyridone (3, 2-HOPO).

d) The complexing agent may comprise a targeting moiety, which is preferably conjugated with an octadentate ligand, such as a HOPO ligand (e.g. 3, 2-HOPO).

e) The targeting moiety may be an antibody, an antibody construct (construct), an antibody fragment (e.g., a FAB or F (AB)' 2 fragment or any fragment comprising at least one antigen binding region), or a construct of such fragments.

f) The targeting moiety may be a receptor or receptor binding agent (e.g. a hormone, vitamin, folate or folate analogue), a bisphosphonate or a nanoparticle.

g) The targeting moiety may be specific for at least one disease-associated antigen such as a "cluster of differentiation" (CD) cell surface molecule (e.g., CD22, CD33, CD34, CD44, CD45, CD166, etc.).

h) The targeting moiety may be linked to the ligand by a covalent linker, thereby forming a complexing agent in the form of a targeting conjugate.

j) The contacting may comprise contacting in solution²²⁷Th ions are incubated with complexing agents, especially targeting conjugates. Such incubation may be at a temperature below 50 ℃, preferably 10-40 ℃, such as 20-30 ℃. Such incubation may be for a period of less than 2 hours, such as 1 minute to 60 minutes (e.g., 1-15 minutes), preferably 15-45 minutes.

k) The contacting may be in a buffer solution, preferably in said first buffer solution.

Step v) of the method of the invention involves optionally determining the second solution²²⁷The content of Th. This step and the entities mentioned therein may have the following preferred features, alone or in any feasible combination, and optionally in any feasible combination with any features of the other steps described herein:

a) determination can be made by gamma detection/spectroscopy, such as by using a germanium semiconductor detector (high purity germanium detector — HPGe)²²⁷Th；

b) Can be combined with²²⁷Th levels were compared to the expected drug dose and diluted to standard concentrations, or appropriate doses were withdrawn for administration.

Step vi) of the method of the invention involves an optional step of evaporating liquid from the second solution. This step may be desirable where the final pharmaceutical composition has a low volume. Typically, the first aqueous buffer is selected such that it is compatible with the labeling reaction (as described herein) and physiologically tolerable (i.e., suitable for injection at the concentrations and amounts used). In this way, multiple operations and changes of solvent, such as those involving the concentration step vi), are preferably avoided. This step may be included, where necessary, and the entities mentioned therein may have the following preferred features, alone or in any feasible combination, and optionally in any feasible combination with any features of the other steps described herein:

a) the evaporation may be carried out under reduced pressure (e.g. 1-500 mbar).

b) The evaporation may be carried out at elevated temperatures (e.g. 50-200 c, preferably 80-110 c).

Step vii) of the process of the invention relates to purification by means of steps i) to iv)²²⁷At least a portion of Th forms an optional step of at least one radiopharmaceutical. This step and the entities mentioned therein may have the following preferred features, alone or in any feasible combination, and optionally in any feasible combination with any features of the other steps described herein. Furthermore, all features of the radiopharmaceutical agents indicated herein form preferred features of the pharmaceutical aspect of the invention, in particular where the agent is formed or formable by the method of the invention:

a) from complexation of the second solution²²⁷The portion of Th (purified by means of steps i) to iv)) may be from 1MBq to 100MBq, preferably from 1 to 10 MBq.

b) Pharmaceutically acceptable carriers, diluents, buffers, salts, preservatives and the like can be added to form injectable radiopharmaceuticals.

c) Complexing from the second solution may be based on the activity measurement obtained in step v)²²⁷Th is diluted to standard activity, optionally corrected for the time period between preparation (or measurement) and administration.

d) The radiopharmaceutical may be prepared at or near the point of care and/or may be administered within a short period of time (e.g., within 96 hours from purification to injection, preferably within 48 hours or 36 hours of purification).

The radiopharmaceuticals formed or formable in the various aspects of the present invention may be used to treat any suitable disease, such as a tumor or a proliferative disease (e.g. a carcinoma, sarcoma, melanoma, lymphoma or leukemia). The pharmaceutical preparation as such and for such use as well as the corresponding method of treatment of a subject form further aspects of the invention. Such subjects are often in need thereof, such as subjects suffering from a tumor or proliferative disease (e.g., those described herein). The present invention will further provide a method of administering a radiopharmaceutical to a subject (e.g. a subject in need thereof), said method comprising forming said radiopharmaceutical by any one of steps i) to iv), vii) and optionally steps v), vi) and/or viii) and administering said radiopharmaceutical to said subject (e.g. by intravenous injection or directly to a specific tissue or site).

Step viii) of the method of the invention is an optional step comprising sterilizing the solution or drug, in particular those formed in step vii). This step and the entities mentioned therein may have the following preferred features, alone or in any feasible combination, and optionally in any feasible combination with any features of the other steps described herein:

a) the sterilization may be by heating, by irradiation or by filtration.

b) The filtration may be through a suitable membrane, such as a 0.22 μm (or less) membrane.

c) The filtration may be by syringe through a suitable syringe filter.

In addition to the above steps, the method and all corresponding aspects of the invention may comprise additional steps, e.g. to verify for pharmaceutical purposes²²⁷Purity of Th, exchange of counter ions, concentration or dilution of the solution or control factors such as pH and ionic strength. Each of these steps thus forms an optional, but preferred, additional step in various aspects of the invention.

Preferably, the process of the invention provides for complexation²²⁷High yield of Th product. This is not only because it is desirable to avoid waste or valuable products, but also because all lost radioactive material forms radioactive waste that must then be safely disposed of. Thus, in one embodiment, the loaded in step ii) is eluted in step iv)²²⁷At least 50% (e.g., 50-90% or 50% to 98%) of Th. This will preferably be at least 70%, more preferably at least 80% and most preferably at least 85% yield. In a related aspect, eluted in step iv)²²⁷At least 50% of Th²²⁷The form of the Th complex is eluted (the remainder is eluted as uncomplexed ions in solution). This will preferably be at least 70%, preferably at least 80% and more preferably at least 90%. In a preferred embodiment, eluted in step iv)²²⁷Substantially 100% (e.g., at least 95%) of Th²²⁷The form of the Th complex is eluted.

In a corresponding aspect of the invention, there is additionally provided a pharmaceutical composition comprising complexed²²⁷Th (especially purified as described herein) and optionally at least one pharmaceutically acceptable diluent. Such pharmaceutical compositions may comprise a purity as indicated herein²²⁷Th, optionally formed or formable by the methods of the invention. Suitable carriers and diluents are well known to those skilled in the art and include water for injection, pH adjusting and buffering agents, salts (e.g., NaCl), and other suitable materials.

The pharmaceutical composition will comprise a complexed compound as described herein²²⁷Th, which is generally an ion such as Th⁴⁺A complex of ions. Such compositions comprise the invention²²⁷Complexes of Th with at least one ligand, such as an octadentate 3, 2-hydroxypyridinone (2,3-HOPO) ligand. Suitable ligands are disclosed in WO2011/098611, which is hereby incorporated by reference, in particular with reference to the formulae I to IX disclosed therein, which represent typical suitable HOPO ligands. Such ligands may be used alone or conjugated to at least one targeting moiety, such as an antibody. Most commonly, the ligand is conjugated to the targeting moiety prior to steps i) to iv) as described herein. Antibodies, antibody constructs, antibody fragments (e.g., FAB or F (AB)' 2 fragments or any fragment comprising at least one antigen binding region), constructs of fragments (e.g., single chain antibodies), or mixtures thereof are particularly preferred targeting moieties. The pharmaceutical composition of the invention may thus comprise Th⁴⁺Ions (which react with 3,2-A conjugate of a hydroxypyridone (3,2-HOPO) ligand and at least one antibody, antibody fragment, or antibody construct (purified as described herein), and optionally a pharmaceutically acceptable carrier and/or diluent. Embodiments described herein with respect to pharmaceutical compositions also form embodiments of corresponding methods when feasible, and vice versa。

The term "comprising" as used herein is given an open meaning such that additional components may optionally be present (thus "open" and "closed" forms are disclosed). Rather, the term "consisting of … …" is only given a closed meaning such that only those indicated (including any optional substances where appropriate) will be present (to an effective, measurable and/or absolute degree). Accordingly, a mixture or substance described as "consisting essentially of … …" will generally consist of the recited components such that any additional components do not affect the basic properties to any significant extent. Such mixtures may, for example, contain less than 5% (e.g. 0-5%) of other components, preferably less than 1% and more preferably less than 0.25% of other components. Similarly, where the term is given as "substantially", "about" or "approximately" a given value, this allows for the exact given value and independently allows for small deviations (variabilities), particularly where this does not affect the substance of the property. Such a deviation may be, for example, ± 5% (e.g. ± 0.001% to 5%), preferably ± 1%, more preferably ± 0.25%.

The invention will now be further explained with reference to the following non-limiting examples and the accompanying drawings, in which:

FIG. 1 shows²²⁷Decay of Th over time and²²³corresponding growth of Ra and the daughter isotopes during 28 days.

FIG. 2 shows²²⁷Th via²²³Ra to stable²⁰⁷Radioactive decay chain of Pb.

FIG. 3 shows purification of the complex on a micro spin column²²⁷Schematic representation of experimental procedure for samples of Th, wherein²²⁷Partial decay of ThIs changed to²²³Ra。

FIG. 4 shows pH (x-axis) vs. pH in citrate buffered formulations²²⁷Effect of radiochemical purity (y-axis) of Th complexes. Panel a) contains NAP5 purity data. Panel b) is iTLC purity data. Data series without pABA + EDTA (triangle data series) and with pABA + EDTA (square data series) are shown on each figure.

FIG. 5 SDS-PAGE chromatograms, samples 1 to 4 and the application Point and TTC (bound)²²⁷Th) and front line (free)²²⁷Th)。

Examples

Material

Sodium acetate trihydrate (≧ 99.0%), trisodium citrate dihydrate (≧ 99.0%), sodium 4-aminobenzoate (pABA, ≧ 99%), disodium edetate (EDTA, meeting USP test specifications) and sodium hydroxide (98.0-100.5%) were purchased from Sigma-Aldrich (Oslo, Norway). Metal free water (TraceSELECT) was purchased from FLUKA (Buchs, Switzerland). Sodium chloride (for analysis) and hydrochloric acid (fuming, 37% for analysis) were purchased from Merck Millipore (Darmstadt, germany). Citric acid monohydrate (analytical reagent) was purchased from VWR (West Chester, USA). Acetic acid (glacial acetic acid, 100% anhydrous for analysis) was purchased from Merck (Darmstadt, germany).

silica-based PSA (propylsulfonic acid) cation exchange resins are available from Macherey Nagel (Duren, Germany). NAP5 column is available from GE Healthcare Bio-Sciences AB (Uppsala, Sweden.) Pierce micro spin column is available from Thermo Scientific Pierce (product No. 89879 (Rockford, USA).

Trastuzumab from Herceptin ® (150.0 mg powder, concentrate of solution for infusion) was used and is a trademark of Roche Registration Limited (Welwyn Garden City, UK). To prepare the conjugate, a self-made chelator has been attached to the antibody. The resulting colloidal suspension of conjugate was 5.0 mg/ml conjugate in sodium citrate buffer 0.10M pH 5.1 and 0.90% (w/w) sodium chloride.

To be used in 0.05M hydrochloric acid and metal-free water (self-made product)²²⁷Th (as thorium (IV)) was used as the source of radioactivity. To accumulate to approximately 1:1²²⁷Th and²²³ra (as radium (II)) ratio, storage²²⁷Th decays with a half-life of approximately 1 day 19.

For the Instant Thin Layer Chromatography (iTLC) analysis, iTLC-SG chromatography paper impregnated with silica gel from Agilent Technologies was used (Santa Clara, Calif.).

The following materials were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), LDS (4X) sample buffer and NuPage 10% tris-bis gel from Novex (Carlsbad, Calif.). MES (20X) buffer was from NuPage (Carlsbad, Calif.). The Instant blue is from Expedeon (Cambridelshire, UK) and the Precision Plus Protein two-color standard is from BioRad (Hercules, Calif.).

EXAMPLE 1 preparation of buffered formulations

Stock citrate buffers (0.10M pH 4.0, 0.05M pH 5.0 and 0.07M pH4.8) and stock acetate buffers (0.10M pH 4.0, 0.10M pH 6.0 and 0.10M pH 5.0) were prepared in metal-free water and diluted (if necessary) with the metal-free water to the respective buffer concentrations used in the range of DOEs. pABA (2.0 mg/ml) + EDTA (2.0 mM) and sodium chloride were then added to the respective buffer formulations containing these excipients.

The pH of the stock and final formulation was thoroughly controlled at ambient temperature using a calibrated sevenMulti pH meter from Mettler Toledo (Oslo, norway).

A sevenMulti pH meter calibrated from Mettler Toledo (Oslo, norway) was used to measure the pH of the stock and final formulation at ambient temperature.

EXAMPLE 2 preparation of a micro spin column containing PSA cation exchange resin

A100.0 mg/ml suspension of PSA resin was prepared in metal-free water. To ensure homogeneity of the suspension, a vortex mixer was used and the required volumes of 15.0, 30.0 and 22.5mg of resin were added to the column.

To condition the filled resin, 300 μ Ι of the respective buffer formulation was added to the column and then rotated on an Eppendorf comfort thermostatic mixer (Hamburg, germany) (n = 1, 2 or 3 for DOE samples, and n =2 for the center point) for 1 minute at 10000 rcf, resulting in a dry resin bed before further use.

The columns were conditioned with 300 μ l of each buffer formulation. Excess volume was removed by spinning on a thermostatic mixer at 10000 rcf for 1 minute, resulting in a dry column (n =2 for test sample and center point).

Example 3 complexation and purification

The amount of radioactivity added to each sample was about 250 kBq²²⁷Th (as TTC) and 250 kBq 223 Ra. Prior to use, the frozen trastuzumab-chelate conjugate colloidal suspension is equilibrated to ambient temperature. 50 μ l of the conjugate was added to a solution of 500kBq in 0.05M hydrochloric acid (1-5 μ l, depending on the radioactive concentration)²²⁷Th and 500kBq²²³Ra in Eppendorf tubes and mixed with 50. mu.l of the respective buffer preparation. The sample was then shaken on an Eppendorf comfortable thermostatic mixer for 30 minutes (22 ℃, 750rpm, 10s cycles) to mark samples with decay²²⁷Conjugates of Th and formation of TTC. Subsequently 250 μ l of buffer formulation was added and mixed with labeled conjugate (TTC), then 170 μ l of this sample was added to each micro spin column (n = 1, 2 or 3 for test samples, n =2 for center point). For samples with one or three replicates, the radioactivity and volume were adjusted as needed to maintain the same conditions as the two replicates described herein. The column was spun on a thermostatic mixer at 10000 rcf for 1 minute to elute the column, and the column was washedThe purified material was collected in eppendorf tubes.

Example 4 radioactivity determination

Measurement of the concentration of radionuclide on the cation exchange column and in the eluate after the separation method of example 3 before calculation of the distribution of radionuclide between the column and the eluate²²³Ra and²²⁷the amount of Th. Using HPGe spectra from a high purity germanium (HPGe) -detector (GEM (15)) from Ortec (Oak Ridge, TN.) this detector identifies and quantifies radionuclides with gamma energies in the range of about 30-1400 keV²²⁷Th and²²³ra and the distribution of the radionuclide between the column and the eluate is calculated by means of HPGe-detector spectroscopy. The method may be used to determine the concentration of the radioisotope in the eluate prior to preparation of the radiopharmaceutical to ensure standard activity and to verify radiochemical (radioisotope) purity.

Example 5-stability study: radiochemical purity of TTC

The radiochemical purity (RCP) of the radiopharmaceutical is (in the present case) present in bound form (i.e. as TTC)²²⁷Th with free²²⁷The relationship between Th. Since only the radionuclide is measured on the high-purity germanium (HPGe) detector GEM (15)²²⁷Th and²²³ra, so TTC data cannot exclude free²²⁷Th. Thus, TTC and TTC for on-column and in eluate were also analyzed (on the same day) at ambient temperature using the same detector after measuring the separation of the radionuclide²²³Isolation of Ra some samples were analysed for RCP (gel filtration, iTLC, SDS-PAGE).

5.1 gel filtration on NAP5 column

To analyze the radiochemical purity of TTC, a NAP5 column (gel filtration with size exclusion) was used. The manufacturer's standard procedure was followed and a sample volume of 200. mu.l was added to the column. HPGe-detector spectra were recorded to analyze the amount of TTC on NAP5 column (n = 2). See fig. 4 a).

5.2 real-time thin layer chromatography

The iTLC-SG chromatography paper was cut and dried by heating in an incubator at 110-120 ℃ for 20-30 minutes to activate it. The beaker was filled with about 0.5cm of 0.10M citrate buffer pH5.5 (mobile phase) containing 0.90% (w/w) sodium chloride. 1-8 μ Ι of sample (TTC purified on a micro spin column) was applied to the origin line of the paper strip (n = 2). The strips were placed vertically in a beaker, taking care to avoid any damage to the surface. When the solvent reached the solvent front, the bars were removed from the beaker and allowed to dry. The strip was divided into upper and lower portions by cutting the strip in half, and then each portion of the strip was placed into a counting tube. The activity in each half was measured for 5 minutes separately and calculated at the front line and application point by means of Auto HPGe, Ortec gamma spectrometer (Oak Ridge, TN) with HPGe-detector²²⁷Percentage of Th. For decay at front line²²⁷Of Th²²³The presence of Ra (which has accumulated during the time of analysis) corrects the results. See fig. 4 b).

5.3 gel electrophoresis (SDS-PAGE)

The citrate buffered samples in table 1 (below) were analyzed by SDS-PAGE (n = 2).

Standard procedures of the manufacturer NuPAGE Bis-Tris Mini Gels were followed. MES run buffer was prepared by mixing 950ml Milli Q water and 50ml MES buffer. Samples were prepared by dilution with LDS sample buffer, Milli Q water and MES buffer to a conjugate concentration of 1.0mg/ml (in 0.03M citrate buffer ph5.5 and 0.90% w/w sodium chloride). The samples were then mixed and stored on ice until use. 5 μ g of conjugate was loaded into each well (n = 2). Gel electrophoresis was performed manually at 200V constant voltage on an XCell SureLock Mini-Cell (Invitrogen, Carlsbad, Calif.) with Power Pac Adapter, 4mm and Power Pac Basic (BioRad, Hercules, Calif.). The gel was stained with Instant Blue and incubated at ambient temperature for 60 minutes. The staining reaction was terminated by washing the gel with water. The gel was then transferred to a clear film and a photograph taken. See fig. 5.

Example 6-separation optimization.

Design of experiment (DOE) was conceived to study and optimize separations on silica/PSA micro-spin columns²²³Ra and²²⁷condition of Th. For each buffer (citrate and acetate), the following variables were studied:

。

each DoE variable was studied using the isolation and analysis methods indicated in examples 1-5. The results are shown in table 3, which explains the effect of different parameters on the uptake of radioisotopes on PSA resins.

¹The boundary line is significant in that,²high pH and low resin quality contributed to the predictions with pABA/EDTA runs²²³Increased uncertainty in Ra.

Some examples of conditions under which efficient separation can be found are:

。

wherein the predicted% TTC is the predicted absorption of TTC on the resin, and predicted²²³Ra% is²²³Predicted absorption of Ra on resin. High separation efficiency should combine low TTC absorption with high TTC²²³Ra is absorbed.

Claims (28)

1. For complexing from inclusion²²⁷Th and²²³ra (complexed or in solution) mixtures for purifying complexed²²⁷A method of Th, the method comprising:

i) preparing a first solution comprising complexed in a first aqueous buffer²²⁷Th ion and²²³a mixture of Ra ions;

ii) loading the first solution onto a separation material;

iii) eluting the complex from the separation material²²⁷Th, whereby production involves complexationIs/are as follows²²⁷A second solution of Th;

iv) optionally rinsing the separation material with a first aqueous washing medium.

2. The method of claim 1, further comprising the following step X) before step i)

X) making²²⁷Contacting the Th ions with at least one complexing agent in solution, thereby forming complexed²²⁷At least one aqueous solution of Th.

3. The method of claim 2, wherein the complexing agent is a chelating moiety conjugated to a targeting moiety.

4. The method of any one of claims 1-3, further comprising at least one of the following optional steps:

v) determining the second solution²²⁷The content of Th;

vi) evaporating liquid from the second solution;

vii) complexed by the solution contained in the second solution²²⁷At least a portion of Th forms at least one radiopharmaceutical formulation;

viii) sterile filtering the radiopharmaceutical.

5. The method of any one of the preceding claims, wherein the first aqueous buffer solution is at a pH between 3 and 6.5.

6. The process of any preceding claim, wherein the first aqueous buffer solution comprises at least one organic acid buffer selected from the group consisting of citrate buffers, acetate buffers, and mixtures thereof.

7. The method of any one of the preceding claims, wherein the first aqueous buffer solution further comprises at least one radical scavenger and/or at least one chelating agent.

8. The method of any one of the preceding claims, wherein the separation material is a silica-based cation exchange resin.

9. The process of any preceding claim, wherein the cation exchange resin comprises at least one sulfonic acid moiety.

10. The method according to any of the preceding claims, wherein the elution is by means of "drying", preferably under gravity, centrifugal force or under gas pressure from above and/or vacuum from below.

11. The method of claim 10, wherein the elution is by centrifugal force at a "relative centrifugal force" (RCF) of at least 5000 times gravity.

12. The method of claim 10 or claim 11, wherein the elution is by centrifugal force for a period of time from 10 seconds to 10 minutes.

13. The method of any preceding claim, which does not comprise any additional washing steps.

14. The method of claims 1-12, comprising washing the separation material with an aqueous washing medium.

15. The method of any one of the preceding claims, additionally comprising determining of the second solution by gamma detection or gamma spectroscopy, such as by using a germanium semiconductor detector²²⁷The content of Th.

16. The method of any of the preceding claims, further comprising a step of treating the substrate with a composition comprising²²⁷Second solution of Th²²⁷At least a portion of Th forms at least one radiopharmaceutical.

17. The method of claim 16, wherein the moiety is between 0.1 MBq and 20 MBq²²⁷Th.

18. The method of any one of claims 2-17, wherein the method is performed by said²²⁷Th ions and at least one octadentate complexing agent forming said complex²²⁷Th。

19. The method of claim 18, wherein the octadentate complexing agent is conjugated to a targeting moiety selected from an antibody, an antibody construct, an antibody fragment, or a construct of an antibody fragment.

20. The method of claim 18, wherein the octadentate complexing agent is conjugated to a targeting moiety specific for at least one target selected from the group consisting of "cluster of differentiation" (CD) cell surface markers.

21. The method of any one of claims 2-20, wherein said contacting of step X) comprises contacting said²²⁷The Th ions are incubated with a targeting conjugate comprising a complexing agent linked to a targeting moiety, wherein such incubation is performed at a temperature below 50 ℃.

22. The method of claim 21, wherein the incubation is performed for a period of less than 2 hours.

23. The method of claim 22, wherein the incubating is in a first aqueous buffer.

24. Complexed²²⁷A solution of Th comprising per 1MBq²²⁷Th less than 50KBq²²³Ra。

25. Complexed as claimed in claim 24²²⁷A solution of Th formed or formable by a method as claimed in any one of claims 1 to 23.

26. Pharmaceutical composition comprising a complexed as claimed in any one of claims 24 to 25²²⁷A solution of Th and optionally at least one pharmaceutically acceptable diluent.

27. Kit comprising²²⁷Th and²²³a mixture of Ra, a complexing agent, a first aqueous buffer solution, and a strong cation exchange resin.

28. The kit as claimed in claim 27, further comprising at least one of the following optional items:

at least one sterile filter;

at least one heat resistant container;

at least one heating device;

at least one pharmaceutically acceptable excipient or diluent.