Radiotracer compositions and methods

Abstract

The present invention relates to the field of radiopharmaceuticals for in vivo imaging, in particular to radiotracer compositions which comprises .sup.18F-labelled c-Met binding peptides. The invention provides said compositions, as well as associated automated methods of preparation and cassettes.

Claims

1. A method of preparation of the radiopharmaceutical composition comprising: (A) reacting a first non-radioactive precursor with [.sup.18F]-fluoride to give [.sup.18F]-fluorobenzaldehyde in a single-use cassette comprising: (i) a first non-radioactive precursor, that reacts with [.sup.18F]-fluoride to give [.sup.18F]-fluorobenzaldehyde, the first non-radioactive precursor having the formula I: ##STR00010## (ii) a second non-radioactive precursor of Formula III: ##STR00011## wherein C.sup.a, C.sup.b, C.sup.c, and C.sup.d in Formula III are each cysteine residues and wherein C.sup.a and C.sup.b together and C.sup.c and C.sup.d together are cyclized to form two separate disulfide bonds; (iii) a reverse phase solid phase extraction column; (iv) a supply of the radioprotectant, wherein the radioprotectant is ascorbic acid, para-aminobenzoic acid, or gentisic acid; (v) a reaction vessel; (vi) at least one suitable solvent, wherein the at least one suitable solvent comprises aqueous acetonitrile of 5 to 25% v/v acetonitrile; (B) reacting the [.sup.18F]-fluorobenzaldehyde from step (A) with the second non-radioactive precursor to give a radiotracer of Formula II in a crude radiotracer solution: ##STR00012##  wherein C.sup.a, C.sup.b, C.sup.c, C.sup.d in Formula II are each cysteine residues such that residues C.sup.a and C.sup.b as well as C.sup.c and C.sup.d are cyclized to form two separate disulfide bonds; (C) adding said radioprotectant to the crude radiotracer solution from step (B) to form a second radiotracer solution, wherein the second radiotracer solution comprises said radiotracer of Formula II and said radioprotectant in aqueous acetonitrile of 5 to 25% v/v acetonitrile; (D) purifying the second radiotracer solution from step (C) using said reverse phase solid phase extraction column to give a purified [.sup.18F]-radiotracer solution; (E) optionally diluting the purified [.sup.18F]-radiotracer from step (D) with a biocompatible carrier; (F) filtering the optionally diluted purified [.sup.18F]-radiotracer solution from step (E) to give said radiopharmaceutical composition; wherein said cassette fits removably and interchangeably onto an automated synthesizer apparatus and at least steps (A)-(D) are carried out in the automated synthesizer apparatus.

2. The method of claim 1, where the steps (A)-(F) are carried out in the automated synthesizer apparatus.

3. The method of claim 1, where the second radiotracer solution of step (C) further comprises 0.5 to 5% v/v ethanol.

4. The method of claim 1, where the purification step (D) is carried out as follows: (i) passing the purified [.sup.18F]-radiotracer solution from step (D) through said reverse phase SPE cartridge, wherein the [.sup.18F]-radiotracer of Formula II is retained on said SPE cartridge; (ii) washing the SPE cartridge from step (i) one or more times with a wash solution which comprises an aqueous acetonitrile solution of said radioprotectant of 15 to 25% v/v acetonitrile content; (iii) washing the SPE cartridge from step (ii) one or more times with water or aqueous buffer solution; (iv) eluting the washed SPE cartridge of step (ii) or (iii) with an elution solvent which comprises said radioprotectant in an aqueous ethanol solution having an ethanol content of 35 to 80% v/v, wherein the eluent comprises purified radiotracer in said elution solvent.

5. The method of claim 1, wherein the SPE cartridge is a C.sup.18 SPE cartridge.

6. The method of claim 5, where the elution solvent of step (iv) comprises 40-60% v/v aqueous ethanol.

7. The method of claim 5, which further comprises: (G) dispensing the [.sup.18F]-radiotracer radiopharmaceutical composition of step (F) into one or more syringes.

8. A single use cassette comprising: a first non-radioactive precursor, that reacts with [.sup.18F]-fluoride to give [.sup.18F]-fluorobenzaldehyde, the first non-radioactive precursor having the formula I: ##STR00013## a second non-radioactive precursor of Formula III: ##STR00014##  wherein C.sup.a, C.sup.b, C.sup.c, and C.sup.d in Formula III are each cyssteine residues and wherein C.sup.a and C.sup.b together and C.sup.c and C.sup.d together are cyclized to form two separate disulfide bonds; a reverse phase solid phase extraction column; a supply of radioprotectant, wherein the radioprotectant is ascorbic acid, para-aminobenzoic acid, or gentisic acid; a reaction vessel; and at least one suitable solvent, wherein the at least one suitable solvent comprises aqueous acetonitrile of 5 to 25% v/v acetonitrile.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the radiation elution profile of Compound 3 through an SPE column, during the loading, washing and elution process using six radioactivity detectors positioned at various places on the FASTlab.

(2) FIG. 2 shows a FastLab cassette configuration for the automated radiosynthesis and automated purification of Compound 3.

(3) The invention is illustrated by the non-limiting Examples detailed below. Example 1 provides the synthesis of a c-Met targeting peptide of the invention (“Peptide 1”). Example 2 provides the synthesis of an aminoxy-functionalised Peptide 1 (“Compound 1”), wherein the aminoxy functional group is protected with a protecting group (Eei), and subsequent deprotection to give Compound 2. Example 3 provides the radiosynthesis of [.sup.18F]-fluorobenzaldehyde. Example 4 is a comparative Example, which provides the radiosynthesis of the .sup.18F-labelled conjugate Compound 3, without the methodology of the present invention. The RCP in this case is relatively low (79%) at the end of synthesis.

(4) Example 5 provides an analysis of the identity and time-course of the radiochemical impurities in low RCP Compound 3 purified according to Example 4. This provides evidence that in-process radiolysis during attempted chromatographic purification was responsible for the low RCP. Example 5 provides information on the movement of radioactivity through the SPE column during SPE purification, demonstrating that the RAC is over 45 GBq/mL during the SPE process. This very high, but time-bound RAC, is also indicative of in-process radiolysis.

(5) Example 7 provides an automated synthesis and purification of Compound 3, using an automated synthesizer and cassette. A significant increase in EOS yield was achieved by making up a MeCN/PBS purification solution containing 2.5 mg/Na-pABA, but the EOS RCP was still low, and vulnerable to high RAC (RCP=89% when a RAC of 660 MBq/mL, RCP=85% when a RAC of 844 MBq/ml in a 25 mL formulation). A high RAC at EOS indicates yet higher RAC on the cartridge during the later stages of the purification process. The RCP was improved further by increasing the Na-pABA content of the radiotracer solution to 5 mg/mL to 89-91%. Yet further improvements in RCP were obtained by adding HCl to the pABA wash solution to make it pH 4. The addition of ethanol (2%) to the radiotracer solution and wash solution resulted in a further improvement in RCP to >92%. The process of Example 7 removes 85% of peptide-related impurities and essentially all of the aniline (20 μg remaining out of 100,000 μg aniline present in the crude product before purification). The addition of the pABA and ethanol did not adversely affect the performance of the SPE purification with respect to the removal of chemical impurities.

Compounds of the Invention

(6) TABLE-US-00001 Name Structure Peptide 1 Disulfide bridges at Cys4-16 and Cys6-14; Ac-Ala-Gly-Ser-Cys-Tyr-Cys-Ser-Gly-Pro-Pro-Arg-Phe-Glu- Cys-Trp-Cys-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys-NH.sub.2 or Ac-AGSCYCSGPPRFECWCYETEGTGGGK-NH.sub.2 Com- pound 1 Com- pound 2 Com- pound 3 Pre- cursor 1 where: Compounds 1, 2 and 3 are functionalised at the epsilon amine group of the carboxy terminal Lys of Peptide 1; alternative stereochemistry at the oxime bond is possible, but not shown.

Abbreviations

(7) Conventional single letter or 3-letter amino acid abbreviations are used. Ac: Acetyl Acm: Acetamidomethyl ACN or MeCN: Acetonitrile. AcOH: Acetic acid. Boc: tert-Butyloxycarbonyl. BTM: biological targeting moiety. tBu: tertiary-butyl DCM: Dichloromethane DIPEA: N,N-Diisopropylethyl amine DMF: Dimethylformamide DMSO: Dimethylsulfoxide Eei: ethoxyethylidine; Eei-AOAc-OSu: N-(1-Ethoxyethylidene)-2-aminoxyacetic acid N-hydroxysuccinimidyl ester; EOS: end of synthesis; FBA: 4-fluorobenzaldehyde; Fmoc: 9-Fluorenylmethoxycarbonyl; HBTU: O-Benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate; HPLC: High performance liquid chromatography; MW: molecular weight; NHS: N-hydroxy-succinimide; NMM: N-Methylmorpho line; NMP: 1-Methyl-2-pyrrolidinone; Pbf: 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl; RAC: radioactive concentration. RCP: radiochemical purity. RP-HPLC: reverse-phase high performance liquid chromatography; tBu: tert-butyl; TFA: Trifluoroacetic acid; THF: Tetrahydrofuran; TIS: Triisopropylsilane; Trt: Trityl.

EXAMPLE 1

Synthesis of Peptide 1

(8) Step (a): Synthesis of Protected Precursor Linear Peptide.

(9) The precursor linear peptide has the structure:

(10) 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-NH.sub.2

(11) The peptidyl resin H-Ala-Gly-Ser(tBu)-Cys(Trt)-Tyr(tBu)-Cys(Acm)-Ser(tBu)-Gly-Pro-Pro-Arg(Pbf)-Phe-Glu(OtBu)-Cys(Acm)-Trp(Boc)-Cys(Trt)-Tyr(tBu)-Glu(OtBu)-Thr(ψ.sup.Me,Mepro)-Glu(OtBu)-Gly-Thr(tBu)-Gly-Gly-Gly-Lys(Boc)-Polymer was assembled on an Applied Biosystems 433A peptide synthesizer using Fmoc chemistry starting with 0.1 mmol Rink Amide Novagel resin. An excess of 1 mmol pre-activated amino acids (using HBTU) was applied in the coupling steps. Glu-Thr pseudoproline (Novabiochem 05-20-1122) was incorporated in the sequence. The resin was transferred to a nitrogen bubbler apparatus and treated with a solution of acetic anhydride (1 mmol) and NMM (1 mmol) dissolved in DCM (5 mL) for 60 min. The anhydride solution was removed by filtration and the resin washed with DCM and dried under a stream of nitrogen.

(12) The simultaneous removal of the side-chain protecting groups and cleavage of the peptide from the resin was carried out in TFA (10 mL) containing 2.5% TIS, 2.5% 4-thiocresol and 2.5% water for 2 hours and 30 min. The resin was removed by filtration, TFA removed in vacuo and diethyl ether added to the residue. The formed precipitate was washed with diethyl ether and air-dried affording 264 mg of crude peptide.

(13) Purification by preparative HPLC (gradient: 20-30% B over 40 min where A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 10 mL/min, column: Phenomenex Luna 5μ C18 (2) 250×21.20 mm, detection: UV 214 nm, product retention time: 30 min) of the crude peptide afforded 100 mg of pure Peptide 1 linear precursor. The pure product was analysed by analytical HPLC (gradient: 10-40% B over 10 min where A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 0.3 mL/min, column: Phenomenex Luna 3μ C18 (2) 50×2 mm, detection: UV 214 nm, product retention time: 6.54 min). Further product characterisation was carried out using electrospray mass spectrometry (MH.sub.2.sup.2+ calculated: 1464.6, MH.sub.2.sup.2+ found: 1465.1).

(14) Step (b): Formation of Monocyclic Cys4-16 Disulfide Bridge.

(15) 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-NH.sub.2.

(16) The linear precursor from step (a) (100 mg) was dissolved in 5% DMSO/water (200 mL) and the solution 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 removed by evaporation in vacuo. The residue (40 mL) was injected in portions onto a preparative HPLC column for product purification.

(17) Purification by preparative HPLC (gradient: 0% B for 10 min, then 0-40% B over 40 min where A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 10 mL/min, column: Phenomenex Luna 5μ C18 (2) 250×21.20 mm, detection: UV 214 nm, product retention time: 44 min) of the residue afforded 72 mg of pure Peptide 1 monocyclic precursor. The pure product (as a mixture of isomers P1 to P3) was analysed by analytical HPLC (gradient: 10-40% B over 10 min where A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 0.3 mL/min, column: Phenomenex Luna 3μ C18 (2) 50×2 mm, detection: UV 214 nm, product retention time: 5.37 min (P1); 5.61 min (P2); 6.05 min (P3)). Further product characterisation was carried out using electrospray mass spectrometry (MH.sub.2.sup.2+ calculated: 1463.6, MH.sub.2.sup.2+ found: 1464.1 (P1); 1464.4 (P2); 1464.3 (P3)).

(18) Step (c): Formation of Second Cys6-14 Disulfide Bridge (Peptide 1).

(19) The monocyclic precursor from step (b) (72 mg) was dissolved in 75% AcOH/water (72 mL) under a blanket of nitrogen. 1 M HCl (7.2 mL) and 0.05 M 1.sub.2 in AcOH (4.8 mL) were added in that order and the mixture stirred for 45 min. 1 M ascorbic acid (1 mL) was added giving a colourless mixture. Most of the solvents were evaporated in vacuo and the residue (18 mL) diluted with water/0.1 TFA (4 mL) and the product purified using preparative HPLC. Purification by preparative HPLC (gradient: 0% B for 10 min, then 20-30% B over 40 min where A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 10 mL/min, column: Phenomenex Luna 5μ C18 (2) 250×21.20 mm, detection: UV 214 nm, product retention time: 43-53 min) of the residue afforded 52 mg of pure Peptide 1. The pure product was analysed by analytical HPLC (gradient: 10-40% B over 10 min where A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 0.3 mL/min, column: Phenomenex Luna 3μ C18 (2) 50×2 mm, detection: UV 214 nm, product retention time: 6.54 min). Further product characterisation was carried out using electrospray mass spectrometry (MH.sub.2.sup.2+ calculated: 1391.5, MH.sub.2.sup.2+ found: 1392.5).

EXAMPLE 2

Synthesis, Purification and Lyophilization of Compound 2

(20) Peptide 1 (0.797 g) and Eei-AOAc-OSu (IRIS Biotech; 127 mg) were dissolved in DMF (12 mL). DIPEA (100 μL) was added and the reaction mixture shaken for 26 min. A second aliquot of DIPEA (80 μL) was added and the reaction mixture shaken for 2 hr. The reaction mixture was then diluted with 10% ACN/water/0.1% ammonium acetate (40 mL), and the product purified by preparative HPLC using A=0.1% TFA/water and B=ACN with gradient elution of 20-40% B over 40 min. The fractions containing pure products (these are a mixture of Compound 1 and Compound 2) were pooled in a flask and the flask flushed with argon. The solution was stirred overnight to afford complete removal of Eei protecting groups. The deprotected product was lyophilised affording 550 mg (69% yield) of Compound 2.

(21) The pure product was analysed by analytical LC-MS (gradient: 10-40% B over 5 min where A=H.sub.2O/0.1% TFA and B=ACN TFA, flow rate: 0.6 mL/min, column: Phenomenex Luna 3μ C18 (2) 20×2 mm, detection: UV 214 nm, product retention time: 3.00 min), MH.sub.2.sup.2+ calculated: 1428.1, MH.sub.2.sup.2+ found: 1427.9).

EXAMPLE 3

Radiosynthesis of [.SUP.18.F]-Fluorobenzaldehyde (.SUP.18.F-FBA)

(22) [.sup.18F]-fluoride was produced using a GEMS PETtrace cyclotron with a silver target via the [.sup.18O](p,n) [.sup.18F] nuclear reaction. Total target volumes of 3.2-4.8 mL were used. The radio fluoride was trapped on a Waters QMA cartridge (pre-conditioned with carbonate), and the fluoride is eluted with a solution of Kryptofix.sub.2.2.2. (5.14 mg) and potassium bicarbonate (1.40 mg) in water (800 μL) and acetonitrile (200 μL). Nitrogen was used to drive the solution off the QMA cartridge to the reaction vessel. The [.sup.18F]-fluoride was dried for 9 minutes at 120° C. 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), in DMSO (2.0 mL) was added to the dried [.sup.18F]-fluoride, and the mixture heated to 80° C. for 2 minutes to produce 4-[.sup.18F]-fluorobenzaldehyde.

EXAMPLE 4

Radiosynthesis of Compound 3 (Comparative Example)

(23) Compound 2 from Example 2 was radiolabelled with .sup.18F using .sup.18F-FBA from Example 3, then purified using a MCX+ SPE column, without the in-process radiostabilisation of the present invention, giving Compound 3 with an RCP of 79%.

EXAMPLE 5

Radiochemical Impurities in Low RCP Compound 3

(24) The RCP of Compound 3 prepared as per Example 3 was studied as a function of time. The RCP did not drop further over time (up to 8 hours), showing that: (i) Compound 3 is relatively radiostable at the RAC conditions existing at the end of the SPE process; (ii) the RCP must already have been low at the end of the SPE process.

(25) Analysis of Compound 3 from Example 4, i.e. without using the radiostabilisation methodology of the present invention and exhibiting low RCP (79%), found two radiolysis products to be the main contributors to the low RCP. These were identified by retention time on analytical HPLC, and comparison with the retention time of authentic samples of the non-radioactive analogues. These two radiolysis products are [.sup.18F]4-fluorobenzaldehyde (FBA) and [.sup.18F]4-fluorobenzonitrile (FPhCN), which together represented 12% of the radioactivity present in the low RCP Compound 3 preparation from Example 4.

(26) These principal radiochemical impurities, which do not increase significantly with time, represent radiodegradation products of Compound 3, and in turn, in-process radiolysis.

EXAMPLE 6

SPE Elution Profile in the Purification of Compound 3

(27) Six radioactivity detectors were positioned along a FastLab cassette, with Detector #6 positioned towards the bottom of the SPE column of a preparation according to Example 7. The movement of radioactivity during the loading, washing, and elution steps of the SPE purification process was followed in this way.

(28) The results are shown in FIG. 1. The crude product was shown to be trapped on the top of the SPE cartridge. As the purification proceeds, the radioactivity moved down the cartridge and towards the higher-numbered detectors, increasing their signal. This demonstrated that the radioactivity is not spread out over the entire cartridge, but concentrated in a tight band. During the purification, all the activity was concentrated into a volume of less than 1 mL, giving a RAC during purification (12-14 min) of 45,000 MBq/mL, i.e. 45 GBq/mL.

EXAMPLE 7

Automated Synthesis and Purification of Compound 3

(29) A FastLab automated synthesizer (GE Healthcare Ltd) with cassette was used. The tC18 cartridge was obtained from Waters Limited (address as above). Precursor 1 was reacted with [.sup.18F]-fluoride on the Fastlab according to Example 3 to give [.sup.18F]-FBA. The [.sup.18F]-FBA was reacted subsequently on the FastLab with Compound 2 (aminoxy derivative of Peptide 1), to give crude Compound 3.

(30) Purification.

(31) The cassette configuration is given in FIG. 2. Three external solvent vials are used on the cassette for the SPE purification: Position 17=Anhydrous ethanol; Position 18=Wash Solution of 5 mg/mL Na-pABA 2% EtOH in 79 g PBS/16.5 g MeCN, and HCl to bring to pH 4; Position 20=Formulation Buffer of 34 mL PBS containing 80 mg Na-pABA.

(32) Other Cassette Positions: Position 21: Tubing to the tC18 cartridge in Position 22; Position 22: tC18 cartridge (900 mg); Position 23: Sterile filter.

(33) FASTlab Procedure.

(34) In the following, P17 etc refers to Position 17 of the cassette. S2 and S3 refer to syringe 2 and syringe 3: (i) the first part of the purification process was conditioning with full S2 fill with ethanol from P17, followed by a full S2 fill of MeCN/PBS solution from P18. (ii) crude Compound 3 in the aqueous ethanol solution from the conjugation step was diluted 1:1 with the formulation buffer from P20. This was done in two portions: half of the content of the crude volume from the reaction vessel was transferred to S2, and thereafter mixed with the same volume of formulation buffer from P20. This mixture was then slowly trapped onto the tC18 cartridge. After the first trapping, the same procedure was repeated with the remaining half of the crude. (iii) S2 was rinsed with water and thereafter a full fill of S2 with the MeCN wash solution from P18. The MeCN wash solution was slowly pushed through the tC18 cartridge and to waste. This was repeated 5 more times—six such washes in total (but note that with Eei protecting group synthesis only a total of three washes are needed). (iv) the MeCN on the tC18 cartridge was removed by solvent exchange: first 2× full fill of S2 with the formulation buffer from P20 followed by one full fill of S2 with water from the water bag. (v) the eluent was made by mixing of 3 mL ethanol from P17 and 3 mL of formulation buffer from P20 in S2. The first 1 mL of the eluent was passed through the tC18 and to waste, the following 4 mL eluent was passed through the tC18 and the purified Compound 3 product collected in S3. After elution the product was transferred out from the FASTlab and into the product vial through P19.

(35) The radiochemical purity (RCP) in this case was 92%.