HK1226050A1 - Purification method and compositions - Google Patents

Description

Purification methods and compositions

Technical Field

The present invention relates to the field of radiopharmaceuticals for in vivo imaging, in particular to the purification of radiopharmaceuticals comprising¹⁸A method of F-labeling an aminooxy-functionalized biological targeting moiety radiotracer. The present invention provides radiopharmaceutical compositions of tracers containing radioprotectants and related automated methods and cassettes.

Background

WO 2004/080492 a1 discloses a method for radiofluorination of a biological targeting vector, which method comprises reacting a compound of formula (I) with a compound of formula (II):

or the like, or, alternatively,

reacting a compound of formula (III) with a compound of formula (IV)

Wherein:

r1 is an aldehyde moiety, a ketone moiety, a protected aldehyde such as an acetal, a protected ketone such as a ketal, or a functional group that can be rapidly and efficiently oxidized to an aldehyde or ketone using an oxidizing agent, such as a diol or an N-terminal serine residue;

r2 is a group selected from primary amine, secondary amine, hydroxylamine, hydrazine, hydrazide, aminoxy, phenylhydrazine, semicarbazide, and thiosemicarbazide;

r3 is a group selected from primary amine, secondary amine, hydroxylamine, hydrazine, hydrazide, aminoxy, phenylhydrazine, semicarbazide, or thiosemicarbazide;

r4 is an aldehyde moiety, a ketone moiety, a protected aldehyde such as an acetal, a protected ketone such as a ketal, or a functional group that can be rapidly and efficiently oxidized to an aldehyde or ketone using an oxidizing agent, such as a diol or an N-terminal serine residue;

to give a conjugate of formula (V) or (VI), respectively:

wherein X is-CO-NH-, -O-, -NHCONH-, or-NHCSNH-, and is preferably-CO-NH-, or-O-; y is H, alkyl or aryl substituent; and

the linker group in the compounds of formulae (II), (IV), (V) and (VI) is selected from:

wherein:

n is an integer of 0 to 20;

m is an integer of 1 to 10;

p is an integer of 0 or 1;

z is O or S.

Poethko et al [ j. nuclear. med.,45(5), 892-902 (2004)]using radioactive isotopes¹⁸F A method of radiolabeling a peptide, wherein the peptide is functionalized with an aminoxy group¹⁸F]-fluorobenzaldehyde condensation to give a labelled peptide with the following oxime ether bond:

schottelius et al [ Bioconj. chem.,19(6), 1256-1268 (2008)]the method of Poethko et al was further developed. Schottelius et al used aminooxy functionalized peptides in which the amine of the aminooxy group was protected with an N-Boc (Boc = tert-butoxycarbonyl) protecting group. In [ 2 ]¹⁸F]Deprotection of the N-Boc group at acidic pH (pH = 2), 75 ℃ in the presence of fluorobenzaldehyde yields the desired aminooxy-functionalized peptide in situ. Schottelius et al used a 5-fold molar excess of Boc protected precursor, since deprotection was not quantitative under this reaction condition.

Mezo et al [ J. Pept. Sci., 17, 39-46 (2010)]Some of the problems associated with the above oxime ligation chemistry of Boc protected aminoxy functionalized peptides are described. Thus, it is known that Boc-aminoxy reagents can acylate the Boc-protected aminoxy-peptide formed, producing unwanted by-products. It is also known that the free aminoxy group of functionalized peptides is highly reactive towards carbonyl compounds. Thus, it may occur without need with any extraneous aldehyde or ketone present in the reaction mixture or any subsequent purification stepThe desired condensation. Such aldehydes or ketones may be traces of acetone present in the solvent used, or formaldehyde (e.g. from plasticizers). To solve the problem, Mezo et al have used an anticancer drug and¹⁸F]-conjugation of fluorobenzaldehyde to peptides both of interest. Mezo et al solved this problem by performing deprotection of the Boc-aminooxy peptide in the presence of a 10-fold molar excess of free (aminooxy) acetic acid (Aoa) as a "carbonyl capture reagent". The deprotected aminoxy-peptide and excess Aoa were then lyophilized and stored at 4 ℃. Just before the oxime ligation reaction, the lyophilized mixture was redissolved and the excess Aoa was separated by HPLC or Sep-Pak plus C18 column. Mezo et al provide a technique in which the technique is used to render nonradioactive (i.e.¹⁹F) An example of conjugation of 4-fluorobenzaldehyde with an aminooxy-functionalized somatostatin peptide. Mezo et al do not provide any reference¹⁸F-any data of radiolabeling.

WO 2012/022676 discloses a composition comprising formula I¹⁸Imaging agent for F-radiolabelled 18-30 mer c-Met binding cyclic peptides:

Z¹-[cMBP]-Z²　　(I)

wherein:

cMBP is of formula II below:

-(A)_x-Q-(A＇)_y-　　(II)

wherein Q is an amino acid sequence (SEQ-1):

-Cys^a-X¹-Cys^c-X²-Gly-Pro-Pro-X³-Phe-Glu-Cys^d-Trp-Cys^b-Tyr-X⁴-X⁵-X⁶-

wherein X¹Asn, His or Tyr;

X²gly, Ser, Thr or Asn;

X³is Thr or Arg;

X⁴is Ala, Asp, Glu, Gly or Ser;

X⁵ser or Thr;

X⁶asp or Glu;

and Cys^a-dEach a cysteine residue such that residues a and b and c and d are cyclised to form 2 separate disulphide bonds;

a and A 'are independently any amino acid other than Cys, provided that at least one of A and A' is present and Lys;

x and y are independently integers having values of 0-13, and are selected such that [ x + y ] = 1-13;

Z¹is connected with the N end of cMBP and is H or M^IG；

Z²Is connected with the C end of cMBP and is OH or OB^cOr M^IG，

Wherein B is^cIs a biocompatible cation;

M^IGeach independently a metabolism inhibiting group which is a biocompatible group that inhibits or prevents in vivo metabolism of the cMBP peptide;

wherein cMBP is substituted at the Lys residue of the A or A' group¹⁸And F, marking.

WO 2012/022676 also discloses that the imaging agent may be used as a pharmaceutical composition, wherein the composition preferably comprises one or more radioprotectants preferably selected from the group consisting of: ethanol, ascorbic acid, p-aminobenzoic acid (i.e., 4-aminobenzoic acid or pABA), gentisic acid (i.e., 2, 5-dihydroxybenzoic acid), and salts of the acids with biocompatible cations.

WO 2012/072736 discloses the use of alternative protecting group chemistry to functionalize aminoxy groups of biomolecules. The protected aminoxy group has the formula:

wherein:

R¹and R²Is independently selected from C_1-3Alkyl radical, C_1-3Fluoroalkyl or C_4-6And (4) an aryl group.

Lemailre et al [ J. Lab. Comp. radiopharm.,42, 63-75 (1999)]describe¹⁸Solid Phase Extraction (SPE) purification of F-atatanserin:

¹⁸f-astaryrin

They notice that¹⁸F-atatanserin was very sensitive to radiation decomposition and the solution was adjusted using a column containing 0.1% ascorbic acid in ethanol and saline. Column washing was performed with a brine/ascorbic acid mixture. Will be provided with¹⁸F-atrasenterin was eluted in pure ethanol and subsequently diluted with column conditioning solution.

US 2013/0209358A 1 discloses the use of a high radioactive concentration¹⁸F-fluciclatide can be subjected to radiolysis:

¹⁸F-fluciclatide

US 2013/0209358A 1 reports¹⁸F-fluciclatide is not stabilized by ethanol and ascorbic acid is not ideal for automated radiosynthesis, but 4-aminobenzoic acid (or a salt thereof) is effective. US 2013/0209358A 1 teaches the preparation of a¹⁸F-fluciclatide, and then adding a radioprotectant. US 2013/0209358A 1 also teaches a composition comprising¹⁸A radiopharmaceutical composition of F-fluciclatide and the radioprotectant 4-aminobenzoic acid (pABA).

WO 2013/174909A 1 provides purification using SPE (solid phase extraction)¹⁸Methods of F-fluciclatide. WO 2013/174909A 1 teaches that once eluted from SPE cartridges, the same may be used¹⁸The F-fluciclatide is eluted with a biocompatible carrier which may comprise 4-aminobenzoic acid.

There is therefore still a need for improved methods of preparing and purifying aminooxy-functionalized biological targeting moieties to obtain high purity compositions suitable for in vivo radiopharmaceutical applications.

The invention

The present inventors have identified and analyzed the presence of¹⁸F-labeled aminooxy-functionalized biological targeting moieties and how the level of radiochemical impurities in the targeting moieties changes over time. This study led to the recognition that in-process radiolysis during attempts to purify was the root cause of the RCP (radiochemical purity) problem.

This can also be understood as follows. Thus, when purified and formulated for in vivo use, the radiotracer of the invention is present at a radioactive concentration (RAC) of about 500-700 MBq/mL (0.5-0.7 GBq/mL). The radiotracer may show satisfactory stability or minimal degradation under the RAC conditions. However, it was obtained from a crude reaction mixture in which the RAC ranged from about 10-15 GBq/mL. Furthermore, the inventors demonstrated that during purification (i.e. at the end of the radiosynthesis), radioactivity of about 45GBq was concentrated in a tight band on the column in a volume of less than 1 mL. This resulted in an in-process RAC of >45 GBq/mL during purification. Since purification can take up to 20 minutes, the risk of in-process radiolysis is high.

The present invention provides methods for stabilizing radiotracers while performing purification, thus also providing improved radioactive yields and compositions. The present invention provides a process for purifying a protein comprising¹⁸A method of F-aminooxy functionalizing a radioactive tracer of a biological targeting moiety. In fact, it is both a purification method and a method that is radiostabilizing (i.e., stable against radioactive degradation). Thus, by preventing in-process radioactive degradation during purification, the present invention also improves radiochemical purity, as inhibition may otherwise resultAnd impurities produced. The method also provides improved radiochemical yield, since losses in progress during chromatography are minimized. Thus, the present invention provides a yield increase of up to 65% compared to a radiostabilizing purification by HPLC without in-process.

Detailed Description

In a first aspect, the present invention provides a method of purifying a radiotracer, the method comprising the steps of:

(a) providing a composition comprising¹⁸An F-labeled aminooxy-functionalized biological targeting moiety radiotracer;

(b) adding a radioprotectant to the radiotracer to give a radiotracer solution comprising the radiotracer in one or more aqueous water-miscible organic solvents with an organic solvent content of 5-25% v/v;

(c) passing the radiotracer solution of step (b) through a reversed-phase SPE cartridge, wherein radiotracer is retained on the SPE cartridge;

(d) washing the SPE cartridge of step (c) one or more times with a wash solution comprising a radioprotectant that is an aqueous water-miscible organic solvent solution having an organic solvent content of 15-25% v/v;

(e) washing the SPE cartridge of step (d) one or more times with water or an aqueous buffer solution;

(f) eluting the washed SPE cartridge of step (d) or (e) with an elution solvent comprising a radioprotectant in an aqueous ethanol solution having an ethanol content of 35-80% v/v, wherein the elution solution comprises purified radiotracer in the elution solvent;

wherein each radioprotectant independently comprises one or more of the following: ascorbic acid, p-aminobenzoic acid and gentisic acid and salts thereof with biocompatible cations.

The term "radiotracer" has its conventional meaning and refers to a radiopharmaceutical used to follow a physiological or biological process without affecting the process. The term "radiopharmaceutical" has its conventional meaning and refers to a radiolabeled compound administered into the body of a mammal for imaging or therapeutic purposes. During chromatographic purification, e.g., when loaded and thus concentrated to a small volume at the top of an SPE column, the radiotracer can be transiently exposed to extremely high radioactive concentrations ("RAC"). Thus, radiotracers that otherwise appear to be radio-stable may exhibit instability during attempted purification, with subsequent loss of radiochemical purity ("RCP") and/or radiochemical yield.

The term "aminooxy-functionalized" means a biological targeting moiety functionalized with an aminooxy group. The term "aminooxy" has its conventional meaning and refers to the formula-O-NH₂preferably-CH₂-O-NH₂A substituent of (1).

By the term "biological targeting moiety" (BTM) is meant a compound that is selectively absorbed or localized in vivo to a specific site in the mammalian body following administration. The site may, for example, be involved in a particular disease state or indicate how an organ or metabolic process is functioning.

The term "reverse phase" refers to "reverse phase chromatography purification", which has its conventional meaning, referring to chromatography wherein the stationary phase is lipophilic and the mobile phase is hydrophilic (typically including aqueous media). The chromatographic technique of the present invention is Solid Phase Extraction (SPE) using SPE columns (sometimes referred to as "SPE cartridges"). Such SPE columns have the advantage that they are single-use (i.e. disposable) and therefore do not risk cross-contamination with other radiotracers.

The term "aqueous water-miscible organic solvent of 5-25% v/v organic solvent content" refers to an aqueous solution (e.g., water itself, saline, aqueous buffer, or mixtures thereof) mixed with at least one (i.e., possibly two or more different) water-miscible organic solvent. The 5-25% v/v organic solvent content may thus be derived from a single organic solvent, or two or more such solvents. Water-miscible organic solvents are known in the artThe method comprises the following steps: acetonitrile, C_1-4Alcohols, DMF, DMSO, dioxane, acetone, 2-methoxyethanol, THF, and ethylene glycol.

By the term "radioprotectant" is meant a compound that inhibits degradation reactions (e.g., redox processes) by trapping highly reactive free radicals (e.g., oxygen-containing free radicals generated from the radiolysis of water). The radioprotectants according to the invention are suitably selected from: ascorbic acid, para-aminobenzoic acid (i.e., 4-aminobenzoic acid), gentisic acid (i.e., 2, 5-dihydroxybenzoic acid), and salts thereof with biocompatible cations. By the term "biocompatible cation" is meant a positively charged counter ion that forms a salt with an ionized negatively charged group, wherein the positively charged counter ion is also non-toxic and thus suitable for administration to a mammalian body, especially a human body. Examples of suitable biocompatible cations include: alkali metal sodium or potassium; alkaline earth metals calcium and magnesium; and ammonium ions. Preferred biocompatible cations are sodium and potassium, most preferably sodium.

The method of the present invention removes species that remain bound to the SPE column-such as very lipophilic species or any particles. The method of the present invention also minimizes or removes hydrophilic impurities that exhibit low affinity for the SPE column stationary phase-and are therefore removed during the loading and washing steps. The hydrophilic impurities include any salt or ionic species (e.g., fluoride ion); catalysts (e.g., aniline or Kryptofix); and a water-miscible organic solvent from the radiotracer solution.

Radiotracers are generally produced from¹⁸Conjugation of F-fluorobenzaldehyde (or similar) to an aminooxy functionalized biological targeting moiety precursor, the conjugated fluorobenzaldehyde moiety conferring additional lipophilicity to the conjugate. Thus, the radiotracer will tend to remain in the reversed-phase SPE column, whereas the non-radioactive precursor itself (which is more hydrophilic) tends to be removed in the loading step (c) and the washing steps (d) and (e) of the first aspect. Washing steps (d) and (e) are also important to remove the water-miscible organic solvent of step (b). In this manner, the radiotracer solution is purified to remove unwanted bio-based speciesTargeting moieties that, if present, compete in vivo with the radiotracer for the target biological site. Any more hydrophilic 18F-labeled radioactive impurities will tend to be removed in a similar manner. Thus, the initial level of compound 2 in the preparation was 5 mg, which was reduced to about 200 μ g (0.2 mg) in the purified radiotracer.

Preferred features

In the method of the first aspect, the water-miscible organic solvent of the wash solution and/or the radiotracer solution is preferably selected from: acetonitrile, C_2-4Alcohol, DMF, 2-methoxyethanol, THF and ethylene glycol. More preferably, the water-miscible organic solvent is selected from acetonitrile and ethanol.

Acetonitrile has the following advantages: it is a good solvent for many radiotracers, is neither acidic nor basic, is relatively unreactive and therefore compatible with a wide variety of functional groups, and is very miscible with water, allowing it to be easily removed by washing the SPE column in method steps (d) and (e). Acetonitrile was found to be the most efficient solvent for the removal of impurities including aniline from radioactive tracers. The acetonitrile content of the radiotracer solution and wash solution preferably does not exceed 25% v/v, since higher levels risk loss of radiotracer product by elution from the SPE column. The acetonitrile content of the washing solution is preferably 18 to 22% v/v, more preferably 20 to 21% v/v.

The pH of the aqueous components of the radiotracer solution, the wash solution and the elution solvent is preferably 7.5-8.5. This is preferably achieved using a buffer solution, more preferably a phosphate buffer.

The radiotracer solution of step (b) preferably comprises ethanol, more preferably comprises both acetonitrile and ethanol. Ethanol has potentially multiple effects, as it can be used as: a water-miscible organic solvent (as defined above); radioprotectants or radiostabilizers (radiostabilisers); "biocompatible carrier" (defined below); and as an antimicrobial preservative (as defined below). The combination of "radioprotectant" (as defined above) and ethanol found by the present inventors is most effective in stabilizing the radiotracers of the present invention against radiolysis. The ethanol content of the radiotracer solution is preferably 0.5-5% v/v ethanol.

The addition of step (b) is preferably effected by adding the washing solution defined in step (d).

The radiotracer provided in step (a) and/or the radiotracer solution of step (b) is preferably cooled to a temperature in the range of 12-30 ℃, more preferably 15-25 ℃, most preferably 16-22 ℃ prior to use, particularly prior to loading into the SPE column.

The wash solution of step (d) preferably comprises ethanol, more preferably comprises both acetonitrile and ethanol. The ethanol content of the radiotracer solution is preferably 1.0-3%, more preferably 1.5-2.5%, most preferably 2% v/v ethanol.

The reversed-phase SPE cartridge of the first aspect preferably has a carbon loading of 2.7-17% and is more preferably a C8 or C18 SPE cartridge, most preferably a tC18 SPE cartridge.

The elution solvent of step (f) preferably comprises 35-70% v/v aqueous ethanol, more preferably 40-60%, most preferably 48-52% aqueous ethanol, and especially preferably 50% aqueous ethanol.

In the method of the first aspect, the radioprotectants used for the radiotracer solution, the wash solution and the elution solvent may be the same or different, or the same but used in different concentrations. Preferably the radioprotectants used in the radiotracer solution, the wash solution and the elution solvent are the same. In this way, the presence of multiple different non-volatile components in the purified radiotracer is avoided, making it more suitable for in vivo applications. The radioprotectant of the first aspect preferably comprises 4-aminobenzoic acid or a salt thereof with a biocompatible cation, more preferably sodium 4-aminobenzoate.

When the radioprotectant is sodium 4-aminobenzoate, the preferred concentration in the radiotracer solution is 5 mg/mL, while the preferred concentration in the wash solution and elution solvent is 2.5 mg/mL. A particularly preferred radiotracer solution comprises 2% ethanol, 5 mg/mL sodium 4-aminobenzoate in a mixture of 79 g phosphate buffered saline (pH 6-9, preferably 7-8.5) and 16.5g acetonitrile. A particularly preferred wash solution comprises 5 mg/mL sodium 4-aminobenzoate in phosphate buffered saline (pH 6-9, preferably 7-8.5).

When the radioprotectant is sodium 4-aminobenzoate, it is preferred to purge the SPE cartridge with air, rather than nitrogen, between purification steps. Thus, the inventors have found that the radioprotectant 4-aminobenzoic acid functions more efficiently in the presence of air, as opposed to the situation where oxygen is excluded.

The purified radiotracer of step (f) thus preferably contains 4-aminobenzoic acid or a salt thereof with a biocompatible cation as radioprotectant in 35-70% aqueous ethanol. For radiopharmaceutical applications, it is preferably diluted with an aqueous biocompatible carrier to give a final ethanol content of 0.1-10% v/v, as described in the second and third aspects (below).

The SPE cartridge of the first aspect is preferably conditioned by prior treatment with a water-miscible organic solvent, followed by treatment with a conditioning solution. The water-miscible organic solvent is preferably ethanol and the conditioning solution is suitably an aqueous/water-miscible organic solvent mixture, preferably the wash solution of step (d).

The method of the first aspect is preferably carried out as described in the third and fifth aspects (below) using an automated synthesizer apparatus.

In the method of the first aspect, the BTM preferably comprises a single amino acid, a 3-100 mer peptide, an enzyme substrate, an enzyme antagonist, an enzyme agonist, an enzyme inhibitor, or a receptor binding compound. BTMs can be of synthetic or natural origin, but are preferably synthetic. The term "synthetic" has its conventional meaning, i.e., man-made as opposed to isolated from a natural source (e.g., from a mammalian body). Such compounds have the advantage that their production and impurity profile can be fully controlled. Monoclonal antibodies and fragments thereof of natural origin are therefore beyond the scope of the term "synthetic" as used herein. The molecular weight of the BTM is preferably at most 30,000 daltons. More preferably, the molecular weight is in the range of 200-20,000 daltons, most preferably 300-18,000 daltons, with 400-16,000 daltons being especially preferred. When the BTM is non-peptide, the molecular weight of the BTM is preferably at most 3,000 daltons, more preferably 200-.

More preferably, the BTM comprises Affinibody^TMOr a single amino acid, a 3-100 mer peptide, an enzyme substrate, an enzyme antagonist, an enzyme agonist, an enzyme inhibitor, or a receptor binding compound.

By the term "peptide" is meant a compound comprising two or more amino acids as defined below linked by peptide bonds, i.e. amide bonds linking the amine of one amino acid to the carboxyl group of another amino acid. The term "peptidomimetic" or "mimetic" refers to a biologically active compound that mimics the biological activity of a peptide or protein but is no longer chemically peptidic, i.e., they no longer contain any peptide bonds (i.e., amide bonds between amino acids). The term peptidomimetic is used herein in a broader sense to include molecules that are no longer completely peptidic in nature, such as pseudopeptides, semi-peptides, and peptoids. The term "peptide analog" refers to a peptide comprising one or more amino acid analogs as described below. See also Synthesis of Peptides and Peptides, M.Goodman et al, Houben-Weyl E22c, Thieme.

By the term "amino acid" is meant an L-or D-amino acid, amino acid analogue (e.g. naphthylalanine) or amino acid mimetic that may be naturally occurring or of pure synthetic origin and may be optically pure, i.e. a single enantiomer and thus chiral or a mixture of enantiomers. The conventional 3-letter or single-letter abbreviations for amino acids are used herein. Preferably, the amino acids of the invention are optically pure. By the term "amino acid mimetic" is meant a synthetic analog of a naturally occurring amino acid that is an isostere (i.e., designed to mimic the steric and electronic structure of a natural compound). Such isosteres are well known to those skilled in the art and include, but are not limited to, depsipeptides, retro-inverso peptides, thioamides, cycloalkanes, or 1, 5-disubstituted tetrazoles [ see m. Goodman, Biopolymers,24, 137, (1985)]. Radiolabeled amino acids such as tyrosine, histidine or proline are known to be useful in vivo imaging agents.

Affibody^TMThe molecule is based on a domain of 58 amino acid residues derived from one of the IgG binding domains of staphylococcal protein a. Affibodies (Affibodies) can be used in monomeric or dimeric form, as reviewed in Nygren [ febsj.,275, 2668-2676 (2008)]and Nilsson et al [ curr. Opin. drug. disc. Dev.,10, 167-175 (2007)]. The relatively small size of these affibodies allows for better target tissue penetration and blood clearance compared to 10-20 times larger (-150 kDa) antibodies. Affibodies also have the advantage of being stable under various pH conditions (pH 5.5-11). Preferred affibodies of the invention target HER 2. Preferred HER2 targeting affibodies include affibody 1 as described below.

When the BTM is an enzyme substrate, enzyme antagonist, enzyme agonist, enzyme inhibitor or receptor binding compound, it is preferably non-peptide, and more preferably synthetic. By the term "non-peptide" is meant a compound that does not comprise any peptide bonds (i.e. amide bonds between 2 amino acid residues). Suitable enzyme substrates, antagonists, agonists or inhibitors include glucose and glucose analogs; fatty acids or elastase, angiotensin II or metalloproteinase inhibitors. Suitable synthetic receptor binding compounds include estradiol, estrogen, progestin (progestin), progesterone, and other steroid hormones; a ligand for a dopamine D-1 or D-2 receptor, or a dopamine transporter such as tropane; and a ligand for the 5-hydroxytryptamine receptor.

BTMs are most preferably 3-100 mer peptides or peptide analogs. When the BTM is a peptide, it is preferably a 4-30 mer peptide, most preferably a 5-28 mer peptide.

When the BTM is an enzyme substrate, an enzyme antagonist, an enzyme agonist or an enzyme inhibitor, preferred such biological targeting moieties of the invention are synthetic drug-like small molecules, i.e., drug molecules. Preferred dopamine transporter ligands such as tropanes; a fatty acid; dopamine D-2 receptor ligands; a benzamide; amphetamine (amphetamine); benzylguanidine, ioxinil (iomazenil), benzofuran (IBF) or hippuric acid.

When the BTM is a peptide, preferred such peptides include peptide A, peptide B, peptide C and peptide D as defined below, and:

-somatostatin, octreotide and analogues;

-peptides binding to the ST receptor, wherein ST refers to heat-stable toxins produced by escherichia coli (e. coli) and other microorganisms;

-bombesin;

-a vasomotor intestinal peptide;

-neurotensin;

laminin fragments such as YIGSR, PDSGR, IKVAV, LRE and KCQAGTFALRGDPQG;

-an N-formyl chemotactic peptide for targeting sites of leukocyte accumulation;

-platelet factor 4 (PF4) and fragments thereof;

-α₂anti-plasmin, fibronectin or β -peptide fragments of casein, fibrinogen or thrombospondin α₂Antiplasmin, fibronectin, β -casein, fibrinogen and

the amino acid sequence of thrombospondin can be found in α₂Antiplasmin precursor [ M. Tone et al, J. Biochem,102, 1033, (1987)]β -Casein [ L. Hansson et al, Gene,139, 193,(1994)](ii) a Fibronectin [ A. Gutman et al, FEBS Lett.,207, 145, (1996)](ii) a Thrombospondin-1 precursor [ V. Dixit et al, Proc. Natl. Acad. Sci., USA,83, 5449, (1986)]；R. F.Doolittle, Ann. Rev. Biochem.,53, 195, (1984)；

-peptides that are substrates or inhibitors of angiotensin, such as:

angiotensin II Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (E.C. Jorge)nsen et al, J. Med. chem., 1979, Vol22, 9, 1038-1044)

[Sar，Ile]Angiotensin II: Sar-Arg-Val-Tyr-Ile-His-Pro-Ile (R.K. Turker et al, Science, 1972,177, 1203)。

-angiotensin I: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu.

More preferred BTM peptides are selected from peptide A, peptide B, peptide C and peptide D as defined below:

(i) peptide a = Arg-Gly-Asp peptide;

(ii) peptide B = Arg-Gly-Asp peptide comprising the following fragment

(iii) Peptide C = C-Met binding cyclic peptide comprising the amino acid sequence:

-Cys^a-X¹-Cys^c-X²-Gly-Pro-Pro-X³-Phe-Glu-Cys^d-Trp-Cys^b-Tyr-X⁴-X⁵-X⁶-

wherein X¹Asn, His or Tyr;

X²gly, Ser, Thr or Asn;

X³is Thr or Arg;

X⁴is Ala, Asp, Glu, Gly or Ser;

X⁵ser or Thr;

X⁶asp or Glu;

and Cys^a-dEach a cysteine residue such that residues a and b and c and d are cyclised to form 2 separate disulphide bonds;

(i) peptide D = a lantibiotic peptide of formula:

Cys^a-Xaa-Gln-Ser^b-Cys^c-Ser^d-Phe-Gly-Pro-Phe-Thr^c-Phe-Val-Cys^b-(HO-Asp)-Gly-Asn-Thr^a-Lys^d

wherein Xaa is Arg or Lys;

Cys^a-Thr^a、Ser^b-Cys^band Cys^c-Thr^cCovalent linkage through a thioether bond;

Ser^d-Lys^dcovalently linked by a lysine linkage;

HO-Asp is beta-hydroxy aspartic acid.

By the term "lysinoalanine linkage" is meant that the amine group of the Lys residue is attached to the amine bond of the indicated Ser residue by dehydration of the Ser hydroxy function resulting in a- (CH) of 2 α -carbon atoms linking the amino acid residues₂)-NH-(CH₂)₄-a bond.

Particularly preferred BTM peptides are peptide B, peptide C and peptide D.

Most preferred such peptide B peptides have the following formula (a):

wherein X¹is-NH₂Or

Wherein a is an integer of 1 to 10.

In formula a, a is preferably 1.

Preferred c-Met binding cyclic peptides have the following sequence:

Ala-Gly-Ser-Cys^a-Tyr-Cys^c-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys^d-Trp-Cys^b-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys。

when the BTM is a peptide, one or both ends, preferably both ends, of the peptide have a metabolism-inhibiting group (M) conjugated thereto^IG). Having two peptide ends protected in this way is important for in vivo imaging applications, since otherwise rapid metabolism would be expected, with consequent loss of selective binding affinity of the BTM peptide. By the term "metabolism-inhibiting group" (M)^IG) Means a biocompatible group that inhibits or prevents the metabolism of enzymes, particularly peptidases (e.g., carboxypeptidases), of the BTM at the amino-or carboxy-terminus. Such groups are particularly important for in vivo applications and are well known to those skilled in the art and are suitably selected for the peptide amine terminus from:

n-acylating group-NH (C = O) R^GWherein acyl- (C = O) R^GHaving R selected from the group consisting of^G：C_1-6Alkyl radical, C_3-10Aryl groups or contain polyethylene glycol (PEG) building blocks. Preferred amino-terminal moieties M of this type^IGThe group is acetyl, benzyloxycarbonyl or trifluoroacetyl, most preferably acetyl.

Suitable metabolic inhibiting groups for the carboxy terminus of a peptide include: carboxamide, tert-butyl ester, benzyl ester, cyclohexyl ester, amino alcohol or polyethylene glycol (PEG) building blocks. Suitable M for the carboxy-terminal amino acid residue of BTM peptides^IGThe radical being in which the terminal amine of the amino acid residue is substituted by C_1-4Where alkyl (preferably methyl) is N-alkylated. Preferred such M^IGThe group is carboxamide or PEG, most preferably such group is carboxamide.

Suitable reversed-phase SPE cartridges for use in the present invention are available from Waters Limited (730-.

Aminooxy-functionalized peptides can be prepared by the following method: poethko et al [ j. nuclear. med.,45, 892-902(2004)](ii) a Schirmacher et al [ Bioconj. chem.,18, 2085-2089 (2007)](ii) a Indenovoll et al [ bioorg. med. chem. Lett,16, 6190-6193 (2006)](ii) a Glaser et al [ Bioconj. chem.,19,951-957 (2008)]or Dall' Angelo et al [ org. biomol. chem.,11, 4551-4558 (2013)]. Aminooxy groups can optionally be conjugated in two steps. First, an N-protected aminooxy carboxylic acid or N-protected aminooxy active ester is conjugated to a peptide (e.g., by conjugation to an amine group of a Lys residue or by conventional solid phase synthesis). Second, deprotection of the intermediate N-protected aminoxy-functionalized peptide to give the desired product [ see Solbakken and Glaser paper cited above]. N-protected aminooxy carboxylic acids, e.g. Boc-aminooxy acetic acid [ Boc-NH-O-CH₂(C=O)OH]And Eei-N-O-CH₂(C = O) OH is commercially available from, for example, Sigma-Aldrich, Novabiochem, and IRIS.

The term "protected" refers to the use of a protecting group. By the term "protecting group" is meant a group that inhibits or prevents unwanted chemical reactions, but is designed to be sufficiently reactive that it can be cleaved from the functional group in question under sufficiently mild conditions that the remainder of the molecule is not modified. After deprotection, the desired product is obtained. Amine protecting groups are well known to those skilled in the art and are suitably selected from: boc (wherein Boc is tert-butoxycarbonyl); eei (where Eei is ethoxyethylene); fmoc (wherein Fmoc is fluorenylmethoxycarbonyl); trifluoroacetyl group; allyloxycarbonyl; dde [ i.e., 1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) ethyl ] or Npys (i.e., 3-nitro-2-pyridinesulfinyl). The use of additional protecting Groups is described in Protective Groups in organic Synthesis, 4 th edition, Theoroda W. Green and Peter G.M. Wuts, [ Wiley Blackwell, (2006) ]. Preferred amine protecting groups are Boc and Eei, most preferably Eei.

In addition, Padilla de Jesus et al [ US 7,902,332 and mol. Imaging biol.,10, 177-181(2008)]aminooxy-functionalized maleimides Mal-AO are described:

Mal-AO

a bifunctional linker Mal-AO can be used to link the aminooxy function with the thiol-containing BTM by selective reaction of the thiol group with the maleimide function of Mal-AO. Padilla de Jesus (cited above) applied this to the conjugation of Mal-AO to HER2 selective affibody.

In a second aspect, the present invention provides a method of preparing a radiopharmaceutical composition in a form suitable for mammalian administration, which composition comprises:

(i) a radiotracer as defined in the first aspect;

(ii) at least one radioprotectant as defined in the first aspect;

(iii) a biocompatible carrier comprising an aqueous ethanol solution having an ethanol content of 0.1-10% v/v;

wherein the preparation method comprises the following steps:

● carrying out the radiotracer purification method of steps (a) to (f) as defined in the first aspect;

(g) optionally diluting the purified of step (f) with a biocompatible carrier¹⁸F]-a radioactive tracer;

(h) subjecting the optionally diluted solution of step (g) to sterile filtration to obtain the [ alpha ], [¹⁸F]A radiopharmaceutical composition.

Preferred embodiments of the radiotracer and radioprotectant in the second aspect are as described in the first aspect (above).

A "biocompatible carrier" is a fluid, especially a liquid, in which the radioactive conjugate can be suspended or preferably dissolved, such that the composition is physiologically tolerable, i.e., can be administered to a mammalian body without toxicity or excessive discomfort. The biocompatible carrier is suitably an injectable carrier liquid, for example sterile pyrogen-free water for injection; aqueous solutions, such as saline (which may be advantageously balanced so that the final injectable product is isotonic); an aqueous buffer solution (e.g., phosphate buffer) comprising a biocompatible buffer; aqueous solutions of one or more tonicity-adjusting substances (e.g., salts of plasma cations with biocompatible counterions), sugars (e.g., glucose or sucrose), sugar alcohols (e.g., sorbitol or mannitol), glycols (e.g., glycerol), or other non-ionic polyol substances (e.g., polyethylene glycol, propylene glycol, etc.). Preferably, the biocompatible carrier is pyrogen-free water for injection, isotonic saline or phosphate buffer.

By the phrase "in a form suitable for mammalian administration" is meant a composition that is sterile, pyrogen-free, free of toxic or adverse effects, and formulated at a biocompatible pH (about pH 4.0-10.5). The composition is free of particles that may risk causing emboli in vivo and is formulated so that precipitation does not occur upon contact with biological fluids (e.g., blood). The composition also contains only biocompatible excipients and is preferably isotonic.

The radiotracer and biocompatible carrier are supplied in suitable vials or containers, including sealed containers that allow for maintaining sterile integrity and/or radioactive safety, optionally with addition of an inert headspace gas (e.g., nitrogen or argon), while allowing for addition and withdrawal of solutions via syringes or cannulas. A preferred container of this type is a septum-sealed vial in which the gas-tight lid is crimped with a top seal, typically an aluminium top seal. The cap is adapted for single or multiple punctures (e.g., crimping a septum-sealed cap) with a hypodermic needle while maintaining sterile integrity. Such containers have the additional advantage that the lid can withstand vacuum (e.g., replacement of headspace gas or degassing of solution) if desired, and withstand pressure changes, e.g., pressure decay, without allowing external atmospheric gases (e.g., oxygen or water vapor) to enter.

Preferred multi-dose containers comprise single volume vials containing multiple patient doses, so that a single patient dose can be drawn therefrom into a clinical grade syringe at different time intervals over the viable life of the article to suit the clinical situation. Prefilled syringes are designed to contain a single use dose or "unit dose" and are therefore preferably disposable or other syringes suitable for clinical use.

The radiopharmaceutical composition may contain other optional excipients, for example: antimicrobial preservatives, pH adjusting agents, fillers, solubilizers or osmolality adjusting agents.

By the term "bulking agent" is meant a pharmaceutically acceptable bulking agent that facilitates handling of the material during manufacture and lyophilization. Suitable bulking agents include inorganic salts such as sodium chloride and water soluble sugars or sugar alcohols such as sucrose, maltose, mannitol or trehalose.

By the term "solubilizer" is meant an additive present in the composition that increases the solubility of the agent of interest in the solvent. Preferred solvents of this type are aqueous media, and thus the solubilizer preferably improves solubility in water. Suitable such solubilizers include: c_1-4An alcohol; glycerol; polyethylene glycol (PEG); propylene glycol; polyoxyethylene sorbitan monooleate; sorbitan monooleate; a polysorbate; polyoxyethylene polyoxypropylene polyoxyethylene block copolymers (Pluronics)^TM) Cyclodextrins (e.g., α, β or gamma cyclodextrin, hydroxypropyl- β -cyclodextrin or hydroxypropyl-gamma-cyclodextrin) and lecithin.

By the term "antimicrobial preservative" is meant an agent that inhibits the growth of potentially harmful microorganisms (e.g., bacteria, yeast, or mold). Antimicrobial preservatives may also exhibit some bactericidal properties depending on the dosage used. The primary effect of the antimicrobial preservative of the present invention is to inhibit the growth of any such microorganisms in the pharmaceutical composition. However, antimicrobial preservatives may also optionally be used to inhibit the growth of potentially harmful microorganisms in one or more components of a kit used to prepare the composition prior to administration. Suitable antimicrobial preservatives include: parabens, i.e. methyl, ethyl, propyl or butyl paraben or mixtures thereof; benzyl alcohol; phenol; cresol; cetyltrimethylammonium bromide and thimerosal. A preferred antimicrobial preservative is paraben.

The term "pH adjusting agent" means a compound or mixture of compounds that can be used to ensure that the pH of the composition is within acceptable limits (about pH 4.0-10.5) for human or mammalian administration. Suitable such pH adjusting agents include pharmaceutically acceptable buffers such as tricine, phosphate or TRIS [ i.e. TRIS (hydroxymethyl) aminomethane ], and pharmaceutically acceptable bases such as sodium carbonate, sodium bicarbonate or mixtures thereof. When the composition is used in the form of a kit, the pH adjusting agent may optionally be provided in a separate vial or container, such that the user of the kit can adjust the pH as part of a multi-step procedure.

The method of the second aspect may be implemented in various ways:

1) aseptic production techniques, wherein the steps are carried out in an absolutely clean room environment;

2) end-point sterilization, wherein steps (a) - (g) of the first aspect are performed without aseptic manufacture, followed by sterilization in the final step [ e.g., by gamma irradiation, autoclaving, dry heat or chemical treatment (e.g., with ethylene oxide) ];

3) an aseptic production technique, wherein the steps are performed using an automated synthesizer apparatus.

The method (3) is preferred. Thus, in the method of the third aspect, at least one of steps (b) - (f) or (b) - (h) is preferably automated. More preferably, automation is performed using an automated synthesizer apparatus. Most preferably, the automated synthesizer apparatus comprises a single use cassette.

By the term "automated synthesizer" is meant an automated module based on the principle of unit operation described by Satyamurthy et al [ Clin. Positr. Imag., 2(5), 233-. The term "unit operation" means that a complex process is simplified into a series of simple operations or reactions, which can be applied to various materials. Such automated synthesizers are preferred for the method of the invention especially when a radiopharmaceutical composition is required. They are commercially available from a number of suppliers [ Satyamurthy et al, supra ], including: GE Healthcare; CTI Inc; ion Beam Applications S.A. (Chemin DuCyclinon 3, B-1348 Louvain-La-Neuve, Belgium); raytest (Germany) and bioscan (USA).

Commercial automated synthesizers also provide suitable containers for liquid radioactive waste resulting from radiopharmaceutical preparation. Automated synthesizers with radiation shielding are not typically provided because they are designed for use with appropriately configured radioactive work cells. The radioactive work cell provides suitable radiation shielding to protect the operator from potential radiation doses, and ventilation to remove chemical and/or radioactive vapors. The automated synthesizer preferably comprises a cassette.

By the term "cartridge" is meant a unit component of the apparatus designed to control the operation of the cartridge from outside (i.e., from the outside) of the cartridge in such a way that the entire unit is removably and replaceably mounted on an automated synthesizer apparatus (as defined below) with the mechanical movement of the movable parts of the synthesizer. Suitable cassettes contain linear array valves each connected to an access port where reagents or vials can be connected by needle piercing inverted septum sealed vials or by gas tight synthetic junctions (marrying junctions). Each valve has a male-female connection that interfaces with a corresponding moving arm of the automated synthesizer. When the cartridge is connected to the automated synthesizer, the external rotation of the arm thus controls the opening or closing of the valve. Other moving parts of the auto synthesizer are designed to clip onto the syringe plunger end, thus lifting or depressing the syringe barrel.

The cartridge is versatile, typically having several positions to which reagents can be attached and several positions suitable for attaching a syringe vial or chromatography cartridge (e.g. solid phase extraction or SPE) of reagents. The cartridge always contains a reaction vessel. The volume of the reaction vessel is preferably 1 to 10 cm³Most preferably 2-5 cm³And is configured such that 3 or more ports of the cassette are connected thereto to allow reagents or solvents to be transferred from different ports on the cassette. Preferably the cartridge has 15 to 40 valves in a linear array, most preferably 20 to 30, especially preferably 25. The valves of the cartridges are preferably identical each, most preferably 3-way valves. The cassette is designed to be suitable for radiopharmaceutical production, and thereforeIs itself pharmaceutical grade and is also ideally a material resistant to radiolysis.

Preferred automated synthesizers of the present invention comprise disposable or single use cassettes containing all the reagents, reaction vessels and apparatus necessary to carry out the preparation of a defined batch of radiofluorinated radiopharmaceutical. The cassette means that the automated synthesizer has the flexibility to be able to prepare a variety of different radiopharmaceuticals with minimal risk of cross-contamination by simply replacing the cassette. The cassette approach also has the following advantages: simplifying the setup so that the risk of operator error is reduced; GMP (Good Manufacturing Practice) compliance improvement; (ii) a multi-tracer capacity; rapid changes between production runs; pre-run automated diagnostic checks of cassettes and reagents; repeatedly checking the chemical reagent with an automatic bar code of the synthesis to be carried out; reagent traceability; single use and thus without the risk of cross-contamination, tampering and abuse resistance.

The single-use cassette method of the third aspect preferably comprises:

(i) comprising the [ alpha ] peptide to be purified as defined in the first aspect¹⁸F]-a container of a radiotracer solution;

(ii) one or more reversed-phase SPE cartridges;

(iii) supplying a wash solution as defined in the first aspect;

(iv) supplying an elution solution as defined in the first aspect.

Preferred embodiments of the radiotracer, SPE cartridge, wash solution and elution solvent in the cartridge are as described in the first aspect (above).

The method of the second aspect preferably further comprises:

(j) subjecting the solution of step (h)¹⁸F]-dispensing the radiotracer radiopharmaceutical composition into one or more syringes.

The sequence of steps following step (h) is intentionally chosen here to be "(j)' -to avoid possible confusion with roman numeral 1.

In a third aspect, the present invention provides a single-use cassette for carrying out the definition of the second aspect of the automated method described herein, said cassette comprising:

(i) is suitable for a composition as defined in the first aspect comprising the [ alpha ], [ alpha ] peptide to be purified¹⁸F]-a container of a radiotracer solution;

(ii) one or more reversed-phase SPE cartridges as defined in the first aspect;

(iii) supplying a wash solution as defined in the first aspect;

(iv) supplying an elution solution as defined in the first aspect.

Preferred embodiments of the radiotracer, SPE cartridge, wash solution and elution solvent in the cartridge are as described in the first aspect (above).

In a fourth aspect, the present invention provides the use of an automated synthesizer apparatus for carrying out the method of preparing of the first aspect or the method of radiolabelling of the third aspect.

Preferred embodiments of the automated synthesizer of the fourth aspect are as described in the second aspect (above).

Description of the drawings

Figure 1 shows the radiation elution profile of compound 3 through the SPE column during loading, washing and elution using a radioactivity detector located throughout the FastLab cassette (including through the side of the SPE column).

Figure 2 shows the FastLab cassette configuration for automated radiosynthesis and automated purification of compound 3.

The invention is illustrated by the following detailed non-limiting examples. Example 1 provides the synthesis of a c-Met targeting peptide of the invention ("peptide 1"). Example 2 provides the Synthesis of aminoxy-functionalized peptide 1 ("Compound 1"), in which the aminoxy functionality is presentProtection with a protecting group (Eei) followed by deprotection affords compound 2. Example 3 provides the term¹⁸F]-radiosynthesis of fluorobenzaldehyde. Example 4 is a comparative example which providesNeed not useIn the case of the method of the invention¹⁸Radiosynthesis of F-labeled conjugated Compound 3. In this case, the RCP is relatively low at the end of the synthesis (79%).

Example 5 provides an analysis of the identity and time course of radiochemical impurities in compound 3 purified according to example 4. This provides evidence that in-process radiolysis during attempted chromatographic purification is responsible for low RCP. Example 5 provides information on the movement of radioactivity through the SPE column during SPE purification, indicating that RAC exceeded 45 GBq/mL during SPE. This rather high, but time-limited RAC also indicates an in-process radiolysis.

Example 7 provides automated synthesis and purification of compound 3 using an automated synthesizer and cassette. By formulating MeCN/PBS purification solution containing 2.5 mg/Na-pABA, a significant improvement in EOS yield was achieved, but EOS RCP was still low and sensitive to high RAC (RCP = 89% when RAC was 660 MBq/mL, RCP =85% when RAC was 844 MBq/mL in 25 mL formulation). A high RAC at EOS indicates a still higher RAC on the cartridge at a later stage of the purification process. RCP was further improved to 89-91% by increasing the Na-pABA content of the radiotracer solution to 5 mg/mL. Addition of ethanol (2%) to the radiotracer solution and wash solution resulted in further improvement of RCP to > 92%. The procedure of example 7 removed 85% of the peptide related impurities and substantially all of the aniline (20 μ g of aniline remaining in 100,000 μ g of aniline present in the crude product prior to purification). For chemical impurity removal, the addition of pABA and ethanol did not adversely affect the performance of SPE purification.

Example 8 provides the synthesis of a bifunctional aminooxymethylmaleimide linker (compound 4).

Compounds of the invention

Wherein:

compounds 1, 2 and 3 were functionalized at the amine group of the carboxy-terminal Lys of peptide 1;

affibody 1 is selective for HER 2.

Abbreviations

Conventional single letter or 3-letter amino acid abbreviations are used.

Ac: acetyl;

acm: an acetamidomethyl group;

ACN or MeCN: acetonitrile;

AcOH: acetic acid;

boc: a tert-butoxycarbonyl group;

BTM (basic BTM): a biological targeting moiety;

tBu: a tertiary butyl group;

DCM: dichloromethane;

DIPEA: n, N-diisopropylethylamine;

DMF: dimethylformamide;

DMSO, DMSO: dimethyl sulfoxide;

and Eei: an ethoxyethylene group;

Eei-AOAc-OSu: n- (1-ethoxyethylene) -2-aminoxyacetic acid N-hydroxy

Succinimidyl esters;

EOS: finishing the synthesis;

FBA: 4-fluorobenzaldehyde;

fmoc: 9-fluorenylmethoxycarbonyl;

HBTU: O-benzotriazol-1-yl-N, N, N ', N' -tetramethyluronium hexafluorophosphate；

HPLC: high performance liquid chromatography;

MW: a molecular weight;

NHS: n-hydroxy-succinimide;

NMM: n-methylmorpholine;

NMP: 1-methyl-2-pyrrolidone;

PBS: phosphate buffered saline;

pbf: 2,2,4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl;

RAC: the concentration of radioactivity;

RCP: radiochemical purity;

RP-HPLC: reversed phase high performance liquid chromatography;

tBu: a tertiary butyl group;

TFA: trifluoroacetic acid;

THF: tetrahydrofuran;

and (3) TIS: triisopropylsilane;

trt: a trityl group.

Example 1: synthesis of peptide 1

Step (a): synthesis of protected precursor linear peptides

The precursor linear peptide has the following structure:

Ac-Ala-Gly-Ser-Cys-Tyr-Cys(Acm)-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys(Acm)-Trp-Cys-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys-NH₂

starting with 0.1 mmol of Rink Amide Novagel resin using Fmoc chemistry, peptidyl resin H-Ala-Gly-Ser (tBu) -Cys (Trt) -Tyr (tBu) -Cys (Acm) -Ser (tBu) -Gly-Pro-Arg (Pbf) -Phe-Glu (OtBu) -Cys (Acm) -Trp (Boc) -Cys (Trt) -Tyr (tBu) -Glu (OtBu) -Thr (psi) was assembled on an Applied Biosystems433A peptide synthesizer^Me,Mepro) -glu (otbu) -Gly-thr (tbu) -Gly-lys (boc) -polymer. An excess of 1 mmol of pre-activated amino acid (using HBTU) was used in the coupling step. Glu-Thr pseudoproline (pseudoproline) (Novabiochem 05-20-1122) was incorporated into the sequence. The resin was transferred to a nitrogen bubbler and treated with a solution of acetic anhydride (1 mmol) and NMM (1 mmol) in DCM (5 mL) for 60 min. The anhydride solution was removed by filtration, the resin washed with DCM and dried under a stream of nitrogen.

While removing the side chain protecting groups and cleaving the peptide from the resin, it was performed in TFA (10 mL) containing 2.5% TIS, 2.5% 4-thiocresol and 2.5% water for 2 hours and 30 minutes. The resin was removed by filtration, TFA was removed in vacuo, and diethyl ether was added to the residue. The resulting precipitate was washed with ether and air dried to yield 264 mg of crude peptide.

The crude peptide was purified by preparative HPLC (gradient: 20-30% B in 40 min, where A = H₂O/0.1% TFA, B = ACN/0.1% TFA, flow rate: 10 mL/min, column: phenomenex Luna 5 mu C18 (2)250 x21.20 mm, detection: UV 214nm, product retention time: 30 minutes) to yield 100 mg of pure peptide 1 linear precursor. The pure product was analyzed by analytical HPLC (gradient: 10-40% B in 10 min, where A = H₂O/0.1% TFA, B = ACN/0.1% TFA, flow rate: 0.3 mL/min, column: phenomenex Luna 3 mu C18 (2) 50 x 2mm, detection: UV 214nm, product retention time: 6.54 minutes). Further product characterization was performed using electrospray mass spectrometry (MH)₂ ²⁺Calculated values: 1464.6, MH₂ ²⁺Measured value: 1465.1).

Step (b): sheetFormation of the disulfide bridge of Cys4-16

Cys4-16；Ac-Ala-Gly-Ser-Cys-Tyr-Cys(Acm)-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys(Acm)-Trp-Cys-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys-NH₂。

The linear precursor of step (a) (100 mg) was dissolved in 5% DMSO/water (200 mL) and the solution was adjusted to pH 6 using ammonia. The reaction mixture was stirred for 5 days. The solution was then adjusted to pH 2 using TFA and most of the solvent was removed by evaporation in vacuo. The residue (40 mL) was injected portionwise into a preparative HPLC column for product purification.

The residue was purified by preparative HPLC (gradient: 0% B10 min, then 0-40% B over 40 min, where a = H₂O/0.1% TFA, B = ACN/0.1% TFA, flow rate: 10 mL/min, column: phenomenex Luna 5 mu C18 (2)250 x21.20 mm, detection: UV 214nm, product retention time: 44 minutes) to yield 72 mg of pure peptide 1 monocyclic precursor. The pure product (as a mixture of isomers P1-P3) was analyzed by analytical HPLC (gradient: 10-40% B in 10 min, where A = H₂O/0.1% TFA, B = ACN/0.1% TFA, flow rate: 0.3 mL/min, column: phenomenex Luna 3 mu C18 (2) 50 x 2mm, detection: UV 214nm, product retention time: 5.37 minutes (P1); 5.61 minutes (P2); 6.05 minutes (P3)). Further product characterization was performed using electrospray mass spectrometry (MH)₂ ²⁺Calculated values: 1463.6, MH₂ ²⁺Measured value: 1464.1 (P1); 1464.4 (P2); 1464.3 (P3)).

Step (c): formation of the second Cys6-14 disulfide bridge (peptide 1)

The monocyclic precursor of step (b) (72 mg) was dissolved in 75% AcOH/water (72 mL) under a nitrogen blanket (blanket of nitrogen). 1M HCl (7.2 mL) and a solution containing 0.05M I were added sequentially₂AcOH (4.8 mL) and the mixture was stirred for 45 minutes. 1M ascorbic acid (1 mL) was added to give a colorless mixture. Most of the solvent was evaporated in vacuo, the residue (18mL) was diluted with water/0.1% TFA (4 mL), and the product was purified by preparative HPLC. The residue was purified by preparative HPLC (gradient: 0% B10 min)Clock, then 20-30% B over 40 minutes, where a = H₂O/0.1% TFA, B = ACN/0.1% TFA, flow rate: 10 mL/min, column: phenomenex Luna 5 mu C18 (2)250 x21.20 mm, detection: UV 214nm, product retention time: 43-53 min) to yield 52 mg of pure peptide 1. The pure product was analyzed by analytical HPLC (gradient: 10-40% B in 10 min, where A = H₂O/0.1% TFA, B = ACN/0.1% TFA, flow rate: 0.3 mL/min, column: phenomenex Luna 3 mu C18 (2) 50 x 2mm, detection: UV 214nm, product retention time: 6.54 minutes). Further product characterization was performed using electrospray mass spectrometry (MH)₂ ²⁺Calculated values: 1391.5, MH₂ ²⁺Measured value: 1392.5).

Example 2: synthesis, purification and lyophilization of Compound 2

Peptide 1 (0.797 g) and Eei-AOAc-OSu (IRIS Biotech; 127 mg) were dissolved in DMF (12 mL). DIPEA (100 μ L) was added and the reaction mixture was shaken for 26 minutes. A second aliquot of DIPEA (80 µ L) was added and the reaction mixture was shaken for 2 hours. The reaction mixture was then diluted with 10% ACN/water/0. l% ammonium acetate (40 mL) and the product was purified by preparative HPLC using a =0.1% TFA/water, B = ACN, eluting with a gradient of 20-40% B over 40 minutes. Fractions containing pure product (these were mixtures of compound 1 and compound 2) were combined in a flask, which was purged with argon. The solution was stirred overnight to allow complete removal of the Eei protecting group. The deprotected product was lyophilized to give 550 mg (69% yield) of compound 2.

The pure product was analyzed by analytical LC-MS (gradient: 10-40% B in 5 min, where A = H₂O/0.1% TFA, B = ACN TFA, flow rate: 0.6 mL/min, column: phenomenex Luna 3 mu C18 (2) 20 x 2mm, detection: UV 214nm, product retention time: 3.00 min), MH₂ ²⁺Calculated values: 1428.1, MH₂ ²⁺Measured value: 1427.9).

Example 3: [ ¹⁸ F]-fluorobenzaldehyde (f) ¹⁸ Radiosynthesis of F-FBA)

Using a GEMS PETTrace cyclotron of [ pass ], [ solution of ] having a silver target¹⁸O](p,n) [¹⁸F]Nuclear reaction to produce [ 2 ]¹⁸F]-a fluoride. Total target volumes of 3.2-4.8 mL were used. The radioactive fluoride was trapped on a Waters QMA cartridge (preconditioned with carbonate) and the fluoride was purified with Kryptofix_2.2.2.(5.14 mg) and potassium bicarbonate (1.40 mg) in water (800. mu.L) and acetonitrile (200. mu.L). Nitrogen was used to drive the solution from the QMA cartridge to the reaction vessel. Will 2¹⁸F]Fluoride was dried at 120 ℃ for 9 minutes under a steady stream of nitrogen and vacuum. Trimethylammonium benzaldehyde triflate [ precursor 1; haka et al, j. lab. comp. radiopharm,27, 823-833 (1989)](3.7 mg) of DMSO (2.0 mL) was added to the dried [ solution ]¹⁸F]-fluoride, heating the mixture to 80 ℃ for 2 minutes, yielding 4-, [ 2 ], [¹⁸F]-fluorobenzaldehyde.

Example 4: radiosynthesis of Compound 3 (comparative example)

Using example 3¹⁸F-FBA, with¹⁸Compound 2 from example 2 was radiolabeled and then purified using an MCX + SPE column without radiostabilization in the course of the present invention to give compound 3 with an RCP of 79%.

Example 5: radiochemical impurities in Low RCP Compound 3

The RCP of compound 3 prepared according to example 3 was studied as a function of time. RCP did not decline further with time (up to 8 hours), indicating:

(i) compound 3 is relatively radio stable under RAC conditions present at the end of the SPE process;

(ii) the RCP must always be low at the end of the SPE process.

Analysis of compound 3 of example 4, i.e. without using the radiostabilisation method of the invention and showing low RCP (79%), found that two radiolysis products are the main contributors to low RCP. These were identified by comparison of retention times in analytical HPLC with retention times of authentic samples of non-radioactive analogs. The two radiolysis products are¹⁸F]4-Fluorobenzaldehyde (FBA) and¹⁸F]4-fluorobenzonitrile (FPhCN), which together represent 12% of the radioactivity present in the low RCP compound 3 preparation from example 4.

These major radiochemical impurities, which do not increase significantly over time, represent radioactive degradation products of compound 3 and thus represent ongoing radiolysis.

Example 6: SPE elution profile in purification of compound 3

Along the FastLab cassette, 6 radioactivity detectors were placed, with detector #6 placed towards the bottom of the SPE column configured according to example 7. The movement of radioactivity during the loading, washing and elution steps of the SPE purification process was tracked in this manner.

The results are shown in FIG. 1. Showing the crude product trapped at the top of the SPE cartridge. As purification proceeds, the radioactivity moves down the column and towards the detector 6, causing the signal to increase. This indicates that the radioactivity does not diffuse throughout the column, but is concentrated in the dense band. During purification, all activity was concentrated in a volume of less than 1 mL, resulting in a RAC of 45,000 MBq/mL, i.e., 45 GBq/mL, during purification (up to 20 minutes).

Example 7: automated Synthesis and purification of Compound 3

Using a cassetteFastLab Autosynthesizer (GE Healthcare). the tC18 column was obtained from Waters Limited (addresses as above). Precursor 1 is contacted with Fastlab according to example 3¹⁸F]-fluoride reaction to give [ alpha ], [ beta¹⁸F]-FBA. Then [ 2 ]¹⁸F]FBA is reacted with Compound 2 (aminoxy derivative of peptide 1) at FastLab to give crude Compound 3.

Purification of

The box configuration is given in fig. 2. 3 external solvent vials were used in the cassette for SPE purification:

position 17 = anhydrous ethanol;

position 18 = wash solution of 79 g PBS/16.5g mecn with 5 mg/mL Na-pABA 2% EtOH;

position 20 = 34 mL formulation buffer containing 80 mg Na-pABA in PBS.

Other cartridge positions:

position 21: tubing to the tC18 column at position 22;

position 22: a tC18 column;

position 23: and (5) a sterilizing filter.

FASTlab program

In the following, P17 etc. refers to position 17 of the cartridge. S2 and S3 refer to syringe 2 and syringe 3:

(i) the first part of the purification process was adjusted with a full load of S2 filled with ethanol from P17, followed by a full load of S2 filled with MeCN/PBS solution from P18.

(ii) Crude compound 3 in aqueous ethanol from the conjugation step was diluted 1:1 with formulation buffer from P20. This is done in two parts: half of the volume contents of the crude product from the reaction vessel were transferred to S2 before mixing with the same volume of formulation buffer from P20. The mixture was then slowly trapped in a tC18 cartridge. After the first cut-off, the same procedure was repeated with the remaining half of the crude product.

(iii) S2 was rinsed with water, followed by a full S2 rinse with MeCN wash solution from P18. The MeCN wash solution was pushed slowly through the tC18 cartridge and into the waste zone (waste). This was repeated 5 additional times — a total of 6 washes (but similar results were obtained with a total of 3 washes for the procedure).

(iv) MeCN was removed from the tC18 cartridge by solvent exchange: s2 was completely filled 2 times with formulation buffer from P20 followed by 1 time with water from the water bag S2.

(v) The eluate was prepared by mixing 3 mL ethanol from P17 and 3 mL formulation buffer from P20 in S2. First 1 mL of the eluate was passed through tC18 and to the waste zone, then 4 mL of the eluate was passed through tC18 and the purified compound 3 product was collected in S3. After elution, the product was transferred from FASTlab via P19 and into product vials.

In this case, the radiochemical purity (RCP) is 92%.

Example 8: synthesis of bifunctional linker (Compound 4)

Compound 4

N- (2-aminoethyl) maleimide TFA-salt (Sigma-Aldrich; 151 mg) and Eei-AOAc-OSu (IRIS Biotech; 77 mg) were stirred in NMP (2 mL) at ambient temperature. Collidine (80 μ L) was added and the reaction mixture was stirred at ambient temperature for 70 min. The reaction was quenched by dilution with 0.1% acetic acid (7 mL). The product was purified by preparative HPLC as follows:

detection of	UV at 214nm and 254 nm
		Column type and size	Luna C-18 (2), 5 μm, 100 Å, 20 x 250 mm from Phenomenex
Eluent A	0.1% v/v acetic acid/water, 1 mL/L
		Eluent B	Acetonitrile (Lichrosolv)
Gradient of gradient	15-30% B in 40 min
		Flow rate of flow	Gradient elution period 10 ml/min

The purified compound 4 was lyophilized. Yield 43 mg (75%), purity >97% by area.

The pure product was analyzed by analytical LC-MS (gradient: 10-40% B in 5 min, where A = H₂O/0.1% TFA, B = ACN/0.1% TFA, flow rate: 0.6 mL/min, column: phenomenex Luna 3 mu C18 (2) 20 x 2mm, detection: UV 214nm, product retention time: 1.93 minutes), MH⁺Calculated values: 284.1, MH⁺Measured value: 284.1).

Claims (19)

1. A method of purifying a radiotracer, the method comprising the steps of:

(a) providing a composition comprising¹⁸An F-labeled aminooxy-functionalized biological targeting moiety radiotracer;

(b) adding a radioprotectant to the radiotracer to give a radiotracer solution comprising the radiotracer in one or more aqueous water-miscible organic solvents with an organic solvent content of 5-25% v/v;

(c) passing the radiotracer solution of step (b) through a reversed-phase SPE cartridge, wherein the radiotracer is retained on the SPE cartridge;

(d) washing the SPE cartridge of step (c) one or more times with a wash solution comprising a radioprotectant that is an aqueous water-miscible organic solvent solution having an organic solvent content of 15-25% v/v;

(e) washing the SPE cartridge of step (d) one or more times with water or an aqueous buffer solution;

(f) eluting the washed SPE cartridge of step (d) or (e) with an elution solvent comprising a radioprotectant in an aqueous ethanol solution having an ethanol content of 35-80% v/v, wherein the elution solution comprises purified radiotracer in the elution solvent;

wherein each radioprotectant independently comprises one or more of the following: ascorbic acid, p-aminobenzoic acid and gentisic acid and salts thereof with biocompatible cations.

2. The method of claim 1, wherein the water-miscible organic solvent of the radiotracer solution comprises acetonitrile.

3. The method of claim 1 or claim 2, wherein the radiotracer solution of step (b) comprises 0.5-5% v/v ethanol.

4. The method of any one of claims 1-3, wherein said SPE cartridge is a C18 SPE cartridge.

5. The method of any one of claims 1-4, wherein the elution solvent of step (f) comprises 35-70% v/v ethanol in water.

6. The method of any one of claims 1-4, wherein the elution solvent of step (f) comprises 40-60% v/v ethanol in water.

7. The method of any one of claims 1-6, wherein the radioprotectant used for the radiotracer solution, the wash solution and the elution solvent is the same.

8. The method of any one of claims 1-7, wherein the radioprotectant comprises 4-aminobenzoic acid or a salt thereof with a biocompatible cation.

9. The method of any of claims 1-8, wherein the BTM comprises a single amino acid, a 3-100 mer peptide, an enzyme substrate, an enzyme antagonist, an enzyme agonist, an enzyme inhibitor, or a receptor binding compound.

10. The method of claim 9, wherein the BTM comprises Affibody^TM。

11. The method of claim 9, wherein the BTM comprises a 3-100 mer peptide selected from the group consisting of peptide a, peptide B, peptide C, and peptide D as defined below:

(i) peptide a ═ Arg-Gly-Asp peptide;

(ii) peptide B ═ Arg-Gly-Asp peptide comprising the following fragment

(iii) Peptide C ═ C-Met binding cyclic peptide comprising the amino acid sequence:

-Cys^a-X¹-Cys^c-X²-Gly-Pro-Pro-X³-Phe-Glu-Cys^d-Trp-Cys^b-Tyr-X⁴-X⁵-X⁶-

wherein X¹Asn, His or Tyr;

X²gly, Ser, Thr or Asn;

X³is Thr or Arg;

X⁴is Ala, Asp, Glu, Gly or Ser;

X⁵ser or Thr;

X⁶asp or Glu;

and Cys^a-dEach a cysteine residue such that residues a and b and c and d are cyclised to form 2 separate disulphide bonds;

(iv) peptide D ═ lantibiotic peptide of formula:

Cys^a-Xaa-Gln-Ser^b-Cys^c-Ser^d-Phe-Gly-Pro-Phe-Thr^c-Phe-Val-Cys^b-(HO-Asp)-Gly-Asn-Thr^a-Lys^d

wherein Xaa is Arg or Lys;

Cys^a-Thr^a、Ser^b-Cys^band Cys^c-Thr^cCovalent linkage through a thioether bond;

Ser^d-Lys^dcovalently linked by a lysine linkage;

HO-Asp is beta-hydroxy aspartic acid.

12. A method of preparing a radiopharmaceutical composition in a form suitable for mammalian administration, which composition comprises:

(i) a radiotracer as defined in any one of claims 1 or 9 to 11;

(ii) at least one radioprotectant as defined in claim 1 or 8;

(iii) a biocompatible carrier comprising an aqueous ethanol solution having an ethanol content of 0.1-10% v/v;

wherein the preparation method comprises the following steps:

a method of purifying a radiotracer which implements steps (a) to (f) as defined in any one of claims 1 to 11;

(g) optionally diluting the purified of step (f) with a biocompatible carrier¹⁸F]-a radioactive tracer;

(h) sterilizing and filtering the optionally diluted solution of step (g) to obtain said [ alpha ], [ beta¹⁸F]A radiopharmaceutical composition.

13. The method of claim 12, wherein at least one of steps (b) - (f) or (b) - (h) is automated.

14. The method of claim 13, wherein the automation is performed using an automated synthesizer apparatus.

15. The method of claim 14, wherein the automated synthesizer apparatus comprises a single use cassette.

16. The method of claim 15, wherein the single-use cassette comprises:

(i) containing the [ 2 ], [¹⁸F]-a container of a radiotracer solution;

(ii) one or more reversed-phase SPE cartridges;

(iii) supplying a wash solution as defined in claim 1;

(iv) supplying an elution solvent as defined in claim 1 or claim 6.

17. The method of any one of claims 11-15, further comprising:

(j) subjecting the solution of step (h)¹⁸F]-dispensing the radiotracer radiopharmaceutical composition into one or more syringes.

18. A single-use cassette as defined in claim 15 for carrying out the automated method of claim 14 or claim 15, comprising:

(i) is suitable for the polypeptide containing the polypeptide to be purified¹⁸F]-a container of a radiotracer solution;

(ii) one or more reversed-phase SPE cartridges;

(iii) supplying a wash solution as defined in claim 1;

(iv) supplying an elution solvent as defined in claim 1 or claim 7.

19. Use of an automated synthesizer apparatus as defined in any one of claims 14 to 16 to carry out a purification method according to any one of claims 1 to 11 or a preparation method according to any one of claims 12 to 17.