WO2011033118A1

WO2011033118A1 - Preparation and use of 68ga-comprising particles for lung ventilation/perfusion and pet imaging and quantification

Info

Publication number: WO2011033118A1
Application number: PCT/EP2010/063860
Authority: WO
Inventors: Bengt Langstrom; Hans-Olof Sandberg; Irina Velikyan
Original assignee: Ge Healthcare Limited; Hammersmith Imanet Limited
Priority date: 2009-09-21
Filing date: 2010-09-21
Publication date: 2011-03-24
Also published as: GB0922497D0

Abstract

The invention provides a preparation of a ventilation/perfusion imaging agent comprising positron emitting ⁶⁸Ga, produced, from a ⁶⁸Ge/⁶⁸Ga generator. The agent provides high quality images and quantification that make PET examinations available to improve the management of patients under critical conditions such as pulmonary embolism. Assessment of the ventilation distribution is part of the evaluation of lung function prior to lung resection and is also part of the diagnosis of pulmonary emboli.

Description

Preparation and Use of Ga-comprising Particles for Lung Ventilation/perfusion and

PET Imaging and Quantification-

Field of the Invention.

The invention provides a preparation of a ventilation/perfusion imaging agent comprising positron emitting ⁶⁸Ga, produced, from a ⁶⁸Ge/⁶⁸Ga generator. The agent provides high quality images and quantification that make PET examinations available to improve the management of patients under critical conditions such as for example pulmonary embolism. Assessment of the ventilation distribution is part of the evaluation of lung function prior to lung resection and is also part of the diagnosis of pulmonary emboli.

Background to the Invention.

Radiopharmaceuticals for lung ventilation/perfusion imaging in vivo are known [Chilton and Ball, Chapter 12 pages 394-418 in "Pharmaceuticals in Medical Imaging" D.P.Swanson, H.M.Chilton & J.H.Thrall, MacMillan Publishing (1990)]. These typically comprise the radioisotope ^99mTc, which is a gamma-emitting isotope suitable for SPECT imaging. One such ^99mTc agent is an ultra-fine suspension of carbon nanoparticles called Technegas™ [Suga, Annals.Nucl.Med., 16(5), 303-310 (2002); and James et al [Nucl.Med.Comm., 16(10), 802-810 (1995)]. US 5,064,634 discloses the preparation of an inhalable radioactive metal vapour suitable for lung imaging, which comprises heating technetium in a carbon crucible at a temperatures of at least 1900 °C. US 5,064,634 teaches that ^99mTc is the preferred radioisotope, but that ¹²⁵L ^113mIn, ¹³¹I or ^{1 1} 'in could also be used.

Nozaki et al [Appl.Rad.Isotop., 46(3), 157-165 (1995)] disclose the preparation of radioactive aerosols by sublimation from a graphite boat. Nozaki et al study the radioisotopes ¹⁸F, ⁴³ , ^Cu, ⁶⁸Ga and ^99mTc. They conclude that the ^99mTc is associated with carbon nanoparticles, and that ^MCu is mainly in particulate form but partly cationic. The ⁶⁸Ga was found to be mainly in cationic form, i.e. not associated with the carbon nanoparticles. Nozaki et al suggest that either the ⁶⁸Ga forms condensates other than the carbon nanoparticle, or is deposited on the surface of the particle so that it can be washed off. WO 99/04826 discloses that biological macromolecules (eg. fibrin) can be radiolabelled using "nano-encapsulates" which comprise particles comprising radioisotopes encapsulated in multiple layers of graphite carbon. The radioisotope can be ^mIn, ⁶⁷Ga, ⁶⁸Ga or ^99mTc, and is preferably ^99mTc. WO 99/04826 teaches that the graphite crucible is heated at 2250-2900 °C, for 0.1 to 5 seconds, typically 1 to 3 seconds. WO 99/04826 does not provide any specific teaching on how to prepare ⁶⁸Ga nano-encapsulates.

A disadvantage of ventilation/perfusion imaging using SPECT is that it is not amenable to quantification. There is therefore still a need for alternative and/or improved ventilation/perfusion imaging radiopharmaceuticals.

The Present Invention.

The present invention provides ⁶⁸Ga- labelled agents useful in the preparation of high quality ventilation/perfusion agents that may improve management of critical care patients when accurate diagnosis and quantification is required. The present ⁶⁸Ga ventilation/perfusion tracers offer higher sensitivity, better image quality, more accurate quantification and less radiation burden to patients and personnel. They are particularly useful as radiopharmaceuticals in the imaging and quantification of pulmonary embolism.

The radionuclide ⁶⁸Ga has the advantage that, although it is a positron emitter, it is readily available on demand from a ⁶⁸Ge/⁶⁸Ga generator - i.e. without the need for a local cyclotron. In addition, it decays by 89% through positron emission resulting in PET (Positron Emission Tomography) images with high resolution and the potential for accurate quantification. Its half-life (ti_/2 = 68 min) is sufficient for production and application of radiotracers and it minimizes the radiation dose to the patient. It also allows repetitive examinations - e.g. when two scans are needed, one for perfusion and the other for ventilation. Detailed Description of the Invention.

In a first aspect, the present invention provides a radioactive agent which comprises carbon nanoparticles radiolabeled with ⁶⁸Ga, wherein said ⁶⁸Ga is encapsulated within multiple layers of said carbon.

The term "nanoparticle" has its conventional meaning, i.e. a particle of diameter of the order of a nanometre (1 x 10^~9 m). That is to be contrasted with a 'microparticle' with a diameter of the order of a micrometre (1 x 10^~6 m). The nanoparticles of the invention suitably are unconjugated, i.e. do not have attached thereto a biological targeting molecule (such as a peptide, protein or antibody).

The term "radio labelled with ⁶⁸Ga" means that the ⁶⁸Ga radioisotope is incorporated in the nanoparticle in a manner which means that the radioisotope remains attached in vivo. The ⁶⁸Ga radioisotope is suitably incorporated as a core within the nanoparticles, encapsulated within multiple layers of said carbon. Consequently, the gallium metal is encapsulated within the nanoparticles. Once encapsulated, the ⁶⁸Ga does not exhibit its usual chemistry, because it is insulated from contact with the environment by the encapsulating layers of carbon. Preferred aspects.

The radioactive carbon nanoparticles preferably have a particle size of 0.005 - 0.2 microns (5 to 200 nm), more preferably 10 to 100 nm.

The nanoparticles of the invention are preferably covered by multiple layers (2 to 10 layers) of carbon atoms, which isolate the radiometal from the external environment of the nanoparticle. The carbon is preferably graphite.

In a second aspect, the present invention provides a radioactive aerosol composition, which comprises the radioactive agent of the first aspect, as a suspension in a carrier gas.

Preferred aspects of the radioactive agent in the second aspect are as described in the first aspect (above). The term "aerosol" has its conventional meaning, i.e. a composition in which colloidal particles are dispersed with in a gas such that the composition behaves like a gas. By the term "carrier gas" is meant a chemically unreactive gas in which the colloidal radioactive particles are suspended. By the term "chemically unreactive gas" is meant a gas which would be used in chemistry to provide an "inert atmosphere" as is known in the art. Such a gas does not undergo facile oxidation or reduction reactions (eg. as would oxygen and hydrogen respectively), or other chemical reactions with organic compounds (as would eg. chlorine), and is hence compatible with a wide range of chemical compounds without reacting, even on prolonged storage over many hours or even weeks in contact with the gas. Suitable such gases include nitrogen or the inert gases such as helium or argon. Preferably the chemically unreactive gas is an inert gas, most preferably argon. Most preferably, the chemically unreactive gas is heavier than air. Hence, a preferred chemically unreactive gas is argon. Pharmaceutical grade chemically unreactive gases are commercially available.

The carrier gas is preferably biocompatible. By the term "biocompatible" is meant nontoxic and hence suitable for administration to the mammalian body, especially the human body, without adverse reaction, or pain or discomfort on administration.

Preferred chemically unreactive gases which are also biocompatible are nitrogen and argon (or mixtures thereof), more preferably argon.

In a third aspect, the present invention provides radiopharmaceutical composition which comprises the radioactive aerosol of the second aspect in sterile form suitable for human administration.

Preferred aspects of the aerosol in the third aspect are as described in the second aspect (above). The ⁶⁸Ga radiopharmaceuticals of the present invention have the advantage of lower radiation dose to the patients compared to CT or ^99mTc SPECT. Also, gallium-68 is readily available upon demand and the preparation of the imaging agent is performed within 10-15 min. In a fourth aspect, the present invention a method of preparation of the radioactive aerosol of the second aspect, which comprises:

(i) provision of a supply of ⁶⁸Ga eluate from a suitable generator;

(ii) transfer of said eluate to a graphite crucible and evaporation to dryness via:

(a) transfer of an aliquot of said eluate to said crucible, wherein each aliquot has a volume of ca. 100 to 400 μί;

(b) for each aliquot in turn from step (a), evaporation to dryness via heating the crucible at 50-70 °C;

(c) repeating steps (a) and (b) until the desired amount of eluate has been transferred to the crucible, and the transferred ⁶⁸Ga eluate has been reduced to dryness;

(iii) heating the ⁶⁸Ga-containing crucible from step (ii)(c) at 2500-2700 °C for 15-20 seconds, to give reduced ⁶⁸Ga encapsulated by carbon composites. Preferred aspects of the aerosol in the fourth aspect are as described in the second aspect (above).

The method of the fourth aspect is preferably carried out using an automated synthesizer apparatus. By the term "automated synthesizer" is meant an automated module based on the principle of unit operations as described by Satyamurthy et al [Clin.Positr.Imag., 2(5), 233-253 (1999)]. The term 'unit operations' means that complex processes are reduced to a series of simple operations or reactions, which can be applied to a range of materials. Such automated synthesizers are preferred for the method of the present invention especially when a radiopharmaceutical product is desired. They are commercially available from a range of suppliers [Satyamurthy et al, above], including: GE Healthcare; CTI Inc; Ion Beam Applications S.A.(Chemin du Cyclotron 3, B-1348 Louvain-La-Neuve, Belgium); Raytest (Germany) and Bioscan (USA). Commercial automated synthesizers also provide suitable containers for the liquid radioactive waste generated as a result of the radiopharmaceutical preparation. Automated synthesizers are not typically provided with radiation shielding, since they are designed to be employed in a suitably configured radioactive work cell. The radioactive work cell provides suitable radiation shielding to protect the operator from potential radiation dose, as well as ventilation to remove chemical and/or radioactive vapours. The automated synthesizer preferably comprises a cassette. By the term "cassette" is meant a piece of apparatus designed to fit removably and interchangeably onto an automated synthesizer apparatus, in such a way that mechanical movement of moving parts of the synthesizer controls the operation of the cassette from outside the cassette, i.e. externally. Suitable cassettes comprise a linear array of valves, each linked to a port where reagents or vials can be attached, by either needle puncture of an inverted septum-sealed vial, or by gas-tight, marrying joints. Each valve has a male- female joint which interfaces with a corresponding moving arm of the automated synthesizer. External rotation of the arm thus controls the opening or closing of the valve when the cassette is attached to the automated synthesizer. Additional moving parts of the automated synthesizer are designed to clip onto syringe plunger tips, and thus raise or depress syringe barrels. The cassette is versatile, typically having several positions where reagents can be attached, and several suitable for attachment of syringe vials of reagents or chromatography cartridges (eg. SPE). The cassette always comprises a reaction vessel. Such reaction vessels are preferably 1 to 10 cm³, most preferably 2 to 5 cm³ in volume and are configured such that 3 or more ports of the cassette are connected thereto, to permit transfer of reagents or solvents from various ports on the cassette. Preferably the cassette has 15 to 40 valves in a linear array, most preferably 20 to 30, with 25 being especially preferred. The valves of the cassette are preferably each identical, and most preferably are 3-way valves. The cassettes are designed to be suitable for radiopharmaceutical manufacture and are therefore manufactured from materials which are of pharmaceutical grade and ideally also are resistant to radiolysis.

The method of the fourth aspect preferably further comprises a pre-concentration step. Thus, the ⁶⁸Ga eluate from step (i) is concentrated prior to transfer using an anion- exchange cartridge, so that in step (ii)(b) only a single transfer and evaporation to dryness is necessary.

The method of the fourth aspect, is preferably either carried out in a sterile manner throughout under aseptic manufacture conditions, or subjected to terminal sterilisation, such that the product is the radiopharmaceutical composition of the third aspect. Alternatively, the radiopharmaceuticals may also be prepared under non-sterile conditions, followed by terminal sterilisation using e.g. gamma-irradiation, autoclaving, dry heat or chemical treatment (e.g. with ethylene oxide).

In a fifth aspect, the present invention provides a method of diagnostic imaging in vivo of a mammalian subject, wherein said method comprises:

(i) provision of a mammalian subject to which the ⁶⁸Ga radiopharmaceutical aerosol composition of the third aspect had been previously administered;

(ii) waiting a suitable time until the administered ⁶⁸Ga aerosol of step (i) has localised to a site of interest within said subject;

(iii) imaging the subject using the radioactive emissions from the localised ⁶⁸Ga of step (ii) using Positron Emission Tomography (PET). Preferred aspects of the aerosol and radiopharmaceutical composition and in the fifth aspect are as described in the second and third aspects respectively.

"PET" imaging is a tomographic nuclear imaging technique that uses radioactive tracer molecules that emit positrons. When a positron meets an electron, both are annihilated and the result is a release of energy in form of gamma rays, which are detected by the PET scanner. Gamma radiation produced from the positron-emitting radioisotope is detected by the PET scanner and reflects eg. the accumulation of the tracer in certain areas or tissues of the body, e.g. in the brain or the heart. Generally, a tracer is chosen that will accumulate in the area of interest, or be selectively taken up by a certain type of tissue, e.g. cancer cells. Scanning consists of either a dynamic series or a static image obtained after an interval during which the radioactive tracer molecule enters the biochemical process of interest. The scanner detects the spatial and temporal distribution of the tracer molecule. PET also is a quantitative imaging method allowing the measurement of regional concentrations of the radioactive tracer molecule.

Preferably the site of interest is the lung, and the imaging of step (iii) is

ventilation/perfusion imaging (also known as V/Q imaging). The term

"ventilation/perfusion imaging" has its conventional meaning in radiopharmaceutical imaging. Such lung imaging of ventilation distribution is part of the evaluation of lung function prior to lung resection and is also part of the diagnosis of pulmonary emboli. A preferred application for the imaging method of the fifth aspect is to assist in the diagnosis of pulmonary embolism. The ⁶⁸Ga PET agent provides high quality images, with quantification making the PET examination available to improve the management of patients with critical conditions such as for pulmonary embolism.

Access to high quality ventilation/perfusion agents would improve drastically the management of patients under critical condition when time counts and the accurate diagnosis and quantification are required. Thus, it is recognized that pulmonary embolism requires urgent and correct diagnosis which in turn improves the treatment. PET imaging is a sensitive technique with high diagnostic accuracy and possibility for quantification. High resolution of PET images leads to more definitive diagnosis of smaller lesions that cannot be captured by other techniques as for example, CT. PET imaging offers higher sensitivity compared to that of SPECT. In addition, the shorter half- life of ⁶⁸Ga (68 min) compared to that of ^99mTc (6 hours) allows collection of data at a higher count rate, thus shortening the imaging time and consequently improving the image quality, in particular resolution, due to less organ movement.

The imaging agent is used for patient examinations via inhalation immediately and within 10 min in order to avoid the risk of aggregation and enlargement of the particles in vitro prior to patient administration.

Included in the fifth aspect is a method of diagnosis of the mammalian body, which comprises the imaging method described. Preferred aspect of the imaging methods in said method of diagnosis are as described for the imaging method.

In a sixth aspect, the present invention provides the use of the radioactive aerosol of the second aspect, or the radiopharmaceutical composition of the third aspect in the method of diagnostic imaging of the fifth aspect.

Preferred aspects of the aerosol and radiopharmaceutical composition and in the sixth aspect are as described in the second and third aspects respectively. The invention is illustrated by the following Examples. Example 1 provides the preparation of a ⁶⁸Ga aerosol of the invention. Example 2 compares the biodistribution properties of a ⁶⁸Ga aerosol of the invention ("GallGas") using (PET/CT) with the prior art agent TechneGas™ (SPECT). The production of the ⁶⁸Ga-aerosl was found to be feasible. The high quality of the PET images in pigs demonstrates the superiority of the approach over the prior art ^99mTc agent.

Example 1: Preparation of a Ga aerosol of the invention ("Gallgas").

6⁸Ga was eluted from a ⁶⁸Ge/⁶⁸Ga generator using 0.1 M HC1 (6 ml). The volume of eluate was pre-concentrated to 200 using the method of Velikyan et al

[Bioconj.Chem., 15(3), 554-560 (2004)]. The pre-concentrated ⁶⁸Ga solution (140 μΐ,) was transferred to the crucible of a commercial TechnegasPlus Generator, and evaporated to dryness in the furnace at 50-70 °C. The encapsulated ⁶⁸Ga carbon nanoparticles (pseudogas) were obtained by heating the crucible to 2500-2700 °C for 15-20 seconds. The decay-corrected radiochemical yield was 10-15%.

Example 2: Comparison of a Ga aerosol of the invention with TechneGas .

Both agents were tested in 12 healthy piglets (2 -month old, mean body weight 28 ± 2 kg). The piglets were anaesthetized by intramuscular injection of xylazine prior to imaging, and ventilated mechanically using an ID 7.0 mm endotracheal tune

(Mallinckrodt, Athlone, Ireland). Four animals each were imaged in 3 groups:

(i) healthy lungs (control group);

(ii) lobar obstruction group;

(iii) diffuse obstruction group.

The lobar obstruction was via a pulmonary artery catheter induced into trachea, and advance to the left or right lower branch (confirmed by CT). The balloon at the catheter tip was inflated with air (5 ml) to completely occlude the main bronchus. A

metacholine infusion (0.1 mg/ml; mean infusion rate 0.2 mg/min) was used to cause a 50% decrease in respiratory resistance. The animals in each group were imaged by first PET (⁶⁸Ga), then SPECT (^99mTc) nanoparticles. The control group showed an even distribution of radioactivity. The absence of ventilation in the lower lobe was clearly visible by both PET and SPECT for the lobar group. For the diffuse obstruction group, the SPECT image showed an even distribution of radioactivity, whereas the PET image showed more varied activity over the lung filed - indicating inhomogeneity of the ventilation.

The results are shown in Figures 1 and 2. Both agents were also tested in a pig with induced bronchial occlusion - the results are shown in Figure 3.

Claims

CLAIMS.

1. A radioactive agent which comprises carbon nanoparticles radio labelled with ⁶⁸Ga, wherein said ⁶⁸Ga is encapsulated within multiple layers of said carbon.

2. The radioactive agent of claim 1, where the carbon nanoparticles have a particle size of 0.005-0.2 microns (5 to 200 nm).

3. A radioactive aerosol composition, which comprises the radioactive agent of claim 1 or claim 2, as a suspension in a carrier gas.

4. The radioactive aerosol of claim 3, where the carrier gas comprises a chemically unreactive gas.

5. A radiopharmaceutical composition which comprises the radioactive aerosol of claim 3 or claim 4 in sterile form suitable for human administration.

6. A method of preparation of the radioactive aerosol of claim 3 or claim 4, which comprises:

(i) provision of a supply of ⁶⁸Ga eluate from a suitable generator;

(iii) heating the ⁶⁸Ga-containing crucible from step (ii)(c) at 2500-2700 °C for

15-20 seconds, to give reduced ⁶⁸Ga encapsulated by carbon composites.

7. The method of claim 6, where the ⁶⁸Ga eluate from step (i) is concentrated prior to transfer using an anion-exchange cartridge, so that in step (ii)(b) only a single transfer and evaporation to dryness is necessary.

8. The method of claim 6 or claim 7, which is either carried out in a sterile manner throughout, or subjected to terminal sterilisation, such that the product is the radiopharmaceutical composition of claim 5.

9. A method of diagnostic imaging in vivo of a mammalian subject, wherein said method comprises:

(i) provision of a mammalian subject to which the ⁶⁸Ga radiopharmaceutical aerosol composition of claim 5 had been previously administered;

(iii) imaging the subject using the radioactive emissions from the localised ⁶⁸Ga of step (ii) using Positron Emission Tomography (PET).

10. The method of diagnostic imaging of claim 9, where the site of interest is the lung, and the imaging of step (iii) is ventilation/perfusion imaging.

11. Use of the radioactive aerosol of claim 3 or claim 4, or the radiopharmaceutical composition of claim 5 in the method of diagnostic imaging of claim 9 or claim 10.