Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• the 1 present invention relates to a radiolabelled metal • ⁇ transport protein and use thereof, the radiolabelled metal transport protein being for use particularly, but not exclusively, in imaging tumour sites within a mammalian body.
• Tc technetium
• the isotope 99 Tc is important not in its slowly ⁇ -decaying ground state but in a metastable, nuclear excited state, i.e. as exclusively ⁇ -emitting 99m Tc with a diagnostically useful half-life of six hours.
• One of the major reasons for the popularity of this radioisotope in radiodiagnostics is the availability of an easily operable technetium 'reactor' or 'generator', which allows the convenient preparation of applicable solutions in a normal clinical environment.
• Tc v complexes linked to bisamidedithiol proligands such as ethylenecysteine diester for use in imaging cerebral blood flow in the brain
• 99m Tc-teborqxime and 99m TcN-NOET for imaging the heart disease
• 99m Tc-HIDA, 99m Tc-Lidofenein, 99m Tc-Mebrofenin for imaging the hepatobilary system
• 99m Tc-diethylenetriaminepentaacetic acid for imaging kidney disease
• 99m Tc complexes of phosphonate ligands for imaging bone disease.
• these compounds are tissue specific none of them is specific for tumour detection.
• a 99m Tc labelled imaging agent that is cell selective, inexpensive and simple to make would offer immediate advantage over the prior art.
• tumour cells can be distinguished from normal cells by their rapid rate of proliferation.
• a rapid rate of cellular proliferation creates a high energy requirement in tumour cells for most cellular processes, including a high demand on metal transportation into the cell by metal transport proteins.
• transferrins which includes lactoferrin and, being naturally occurring proteins within the body with a transport function, transferrins inherently transport across all membranes, including the blood- brain barrier.
• the mechanism by which transferrins enter the cells and release iron into the cells is as follows. Circulating transferrin is bound to a specific receptor on the cell surface and is subsequently taken up as a receptor/transferrin complex by endosomes into the cytosol. Once the receptor/transferrin complex is in the cytosol the receptor/transferrin complex releases the Fe 3+ or "demetallates" and the apotransferrin and receptor are released back out through the cytosol to the cell surface where they may be degraded, or recycled.
• the present invention in one aspect, provides tumour-specific imaging agents which will assist the clinician to malce an early clinical diagnosis without the need for invasive exploratory investigation.
• the present invention relates to a 99m Tc labelled metal transport protein and uses thereof in imaging and especially detecting the presence of a tumour within a mammalian body.
• a 99m Tc labelled metal transport protein complex According to a first aspect of the invention' there is provided a 99m Tc labelled metal transport protein complex.
• the metal transport protein is an iron transport protein and more preferably is a transferrin preferably selected from the group comprising lactoferrin, ovotransferrin and/or serum transferrin.
• a transferrin preferably selected from the group comprising lactoferrin, ovotransferrin and/or serum transferrin.
• transferrin is intended to include lactoferrin, ovotransferrin and/or serum transferrin in their apoprotein or metal-loaded states.
• the labelled metal transport protein complex of the present invention carries 99m Tc in the sites normally occupied by the metal ions.
• the metal transport protein is a transferrin
• the protein carries 99m Tc + in place of Fe 3+ ions and encapsulates or folds around the two binding sites occupied by 99m ⁇ c 3+ in a similar way as the transferrin glycoprotein would accommodate Fe 3+ ions in the natural state. Accordingly, the shape/configuration of the Tc-transferrin is not dramatically distorted or substantially altered from that of the naturally occurring Fe-transferrin complex. It is because the 99m Tc-labelled transferrin complex is similar in structure to endogenous Fe 3+ carrying transferrins that the 99m Tc labelled transferrin complex is likely to be recognised by cells and taken up and processed as an endogenous transferrin.
• Rapidly dividing cells such as tumour cells have a high energy and nutrient requirements and have increased demands on a number of normal cellular metabolic processes/activities. Amongst these increased requirements is a demand for iron.
• the iron metal transport proteins as carriers for 99m Tc, one embodiment of the present invention offers a tumour imaging agent that will naturally be attracted to areas within the body that have high iron demand.
• other metal transport proteins can be used for selectively targeting tissues/sites within the body.
• the metal transport protein is derived from mammalian tissue or blood and more preferably is a recombinant protein.
• Recombinant protein is of particular advantage in that the risk of cross haematological infection from other factors/agents present in whole blood, such as by hepatitis virus or HIN, is obviated. Moreover, there is a current abundance of recombinant lactoferrin that is commercially available so that a further advantage of the invention resides in the reduced cost of the complex compared to other prior art compounds/complexes.
• the recombinant protein may be modified as compared with the natural protein, provided that it retains functional metal transport and receptor binding properties.
• a 99m Tc labelled metal transport protein as an imaging agent especially but not exclusively, in detecting the presence of a tumour within a mammalian body.
• the 99m Tc labelled metal transport protein further includes any one or more of the preferred features hereinbefore described.
• the present invention therefore provides an alternative imaging agent from the prior art.
• a product or composition comprising a metal transport protein and a reducing agent, the function of the reducing agent being to convert Tc as the pertechnetate (TcO 4 " ) to Tc so that it may bind to the protein.
• the reducing agent may comprise any agent that is capable of performing the reduction step.
• the metal transport protein is as hereinbefore described.
• the product comprises an amount of metal transport protein in the range of 2-60 mg.
• the product or composition further includes a 99m Tc source, more preferably the source is pertechnetate i.e. TcO " .
• the pertechnetate source can be provided with the product or composition in a suitable vial or vessel or it may be provided separately therefrom and added to the metal transport protein and reducing agent shortly before use.
• the pertechnetate source is provided as a solution; typically it is generated at the site where the investigation treatment is to take place.
• the amount of 99m Tc in the product when labelled with 99m Tc, is in the range of 6-8 GBq and more preferably is about 7.4 GBq (200 mCi).
• the reducing agent is selected from the group comprising a tin(II) salt for example chloride, nitrite and/or sulphite.
• a tin(II) salt for example chloride, nitrite and/or sulphite.
• Another prepared reducing agent is ascorbic acid/ ascorbate.
• the product comprises an amount of reducing agent in the range of 0.2-0.3 mg.
• the product or composition further includes a solubilising agent and/or an isotonic agent.
• references herein to an isotonic agent is intended to mean any agent which is capable of rendering the composition to an isotonic state in solution with respect to the pH and ionic strength of blood, so that upon introduction of the product or composition into a body the recipient does not enter a state of shock or suffer any adverse effect to the pH and ionic strength of the composition in solution.
• the solubilising agent is gentisic acid.
• the purpose of the solubilising agent is to enable solubilisation of the Tc 3+ so as to facilitate formation of the metal/protein complex, and therefore the example provided merely illustrates one suitable compound and is not intended to limit the scope of the application.
• the product comprises an amount of solubilising agent in the range of 0.7- 0.9 mg.
• the isotonic agent comprises a salt and more preferably is sodium chloride.
• the product comprises an amount of the isotonic agent in the range of 20- 40 mg.
• the product or composition is lyophilised, that is to say it is freeze dried.
• the product or composition is pyrogen-free.
• the product or composition is provided in powder form.
• a method of making a m Tc labelled metal transport protein complex comprising mixing a metal transport protein with a 99m TcO " source in the presence of a reducing agent.
• the metal transport protein and reducing agent as hereinbefore described are provided in a sealed vial or vessel which optionally further includes any one or more of the additives hereinbefore described.
• a suitable volume of an appropriate aqueous solution is added to vial containing the metal transport protein and reducing agent.
• the vial is subsequently agitated and allowed to stand for a short period, to ensure that the components have dissolved into the aqueous medium.
• the 9m TcO 4 " source is introduced into the resultant aqueous medium as an appropriate aliquot.
• the 99m Tc source may be provided with the metal transport protein and/or reducing agent and the aqueous medium as well as any necessary additional components which can be added thereto.
• the reducing agent converts the pertechnetate into a form of 99m Tc that is taken up by the metal transport protein so that the resultant complex is labelled.
• a metal transport protein for the manufacture of a 99m Tc labelled imaging agent for imaging and especially for diagnosing the presence of a tumour site within a mammalian body.
• a metal transport protein and a reducing agent for the manufacture of an imaging agent for imaging and especially for diagnosing the presence of a tumour site within a mammalian body.
• pertechnetate for the manufacture of a 99m Tc labelled metal transport protein complex imaging agent for imaging and especially for diagnosing the presence of a tumour site within a mammalian body.
• a method of detecting the presence of a tumour within a mammalian body comprising the steps of: (i) mixing a metal transport protein with an effective amount of pertechnetate in the presence of a reducing agent and an aqueous medium so as to produce a 99m Tc labelled metal transport protein complex, (ii) introducing the resultant aqueous medium into a recipient under investigation, and
• the mixture contains any one or more of the additives and/or features hereinbefore described.
• the composition in solution is injected into the recipient's body, more preferably it is injected by the intravenous route. Alternatively the composition can be taken orally.
• a method for detecting a tumour comprising observing images of 99m Tc introduced into a patient as a labelled metal transport protein complex.
• Reference herein to brain tumour is intended to include any type of tumour or growth which occurs within the blood-brain barrier.
• the product or composition and methods of the present invention which use transferrins are particularly well suited to the detection of the presence of a brain tumour in an individual because of the natural uptake and recognition of the transferrin complex and the inherent ability of transferrins to cross the blood-brain barrier.
• the present invention provides significant advantages over the prior art by provision of a specific tumour-selective imaging agent that is able to access all areas of brain tissue.
• 9m Tc labelled metal transport protein complexes provide a convenient and cost effective means of detecting the presence of tumours within a mammalian body.
• the present invention in addition provides a method of treating the tumours once they have been located/identified by using the metal transport protein to carry a radionuclide.
• a method of treating a tumour detected using a method or product of the invention comprising the steps of: (i) labelling a metal transport protein with a radionuclide,
• the method further includes the step of, prior to step (i), detecting the presence of a tumour with the m Tc labelled metal transport protein complex of the present invention.
• the radionuclide labelled metal transport protein complex further includes any one or more of the features hereinbefore described.
• the aqueous formulation of the radionuclide labelled metal transport protein further includes any one or more of the additives hereinbefore described.
• the method thus provides a means of firstly locating a tumour and subsequently ensuring that a further dose of a metal transport protein labelled with a radionuclide is directed to the same location.
• the radionuclide labelled metal transport protein is thus able to attack/target tumour cells specifically.
• the combined therapy can act as a locator and destroyer treatment for tumour cells.
• this combined therapy is envisaged to be of particular importance in the diagnosis and treatment of brain tumours because transferrins are able to cross the blood-brain barrier.
• the radionuclide is selected from the group comprising 57 Co, 67 Cu, 67 Ga, 90 Y, 97 Ru, 169 Yb, 186 Re, 188 Re, 203 Pb, 153 Sm and/or 212 Bi.
• kits comprising the product or composition of the invention in a suitably sealed vial or vessel and, optionally, a set of written instructions.
• a kit comprising the product or composition of the invention in a suitably sealed vial or vessel and a further product or composition comprising a radionuclide labelled metal transport protein complex in a suitably sealed vial or vessel and optionally, a set of written instructions.
• Figure 1 illustrates the UN difference spectra for the titration of apotransferrin with
• Figure 2 illustrates a graph representing a titration curve for the addition of Fe(NTA) to apotransferrin.
• Figure 3 illustrates the dependence of absorbance on time for a solution containing apotransferrin and 2 Molar equivalents Re(NTA).
• Figure 4 illustrates a graph representing the dependence of A 35 on time for apotransferrin containing 2 Molar equivalents Re(NTA).
• Figure 5 illustrates the UN difference spectra for titration of apotransferrin with
• Figure 6 illustrates a graph representing the reaction of apo-lactoferrin with Re/NTA
• Fig. 6 A showing lxl 0 "5 M apo-lactoferrin and 5mM sodium bicarbonate (black) then with 5-fold excess Re/NTA added after 0 mins, 15, 30, 90, 180, 240, 300, 420 min.
• Fig. 6B illustrates the absorption peak at 231nm
• Fig. 6C illustrates the 500nm region in more detail, X representing absorption peak at 0 and 15 minutes, Y representing maximum absorption wavelength at the end of the time course.
• Figure 7 illustrates a graph representing the reaction of apolactoferrin and sodium bicarbonate with excess Re/NTA, monitored by measuring absorbance at 23 lnm with time.
• Figure 8 illustrates a graph representing the uptake of the control Solution 1 (TcO " and reducing solution) and Solution 2 (TcO " and reducing solution and Tf).
• Iron containing proteins are known to consist of a single polypeptide chain of around 700 amino acid residues. Both lactoferrin and transferrin consist of two lobes, the N- lobe and the C-lobe which have about 40% internal sequence homology' between them and very similar tertiary structure. The folding is identical in the two lobes but the C terminus lobe contains three extra disulfide bridges, giving it extra stability. Each lobe has a cleft in which the iron atom sits, and binding induces a conformational change causing the lobe to close over the metal. Coordination to the metal is by two tyrosine residues, a histidine and an asparagine.
• UV/vis spectroscopy therefore provides a method to distinguish between binding at the iron site compared to other sites such as platinum binding. UV/vis spectroscopy has also been used in conjunction with titration experiments to study the kinetics of binding and to obtain binding constants for a variety of metal transferrin complexes. Herein we have used the same method to determine the binding of rhenium and technetium to transferrin and lactoferrin.
• Tumour cells are known to uptake transferrin at a higher rate than healthy cells, and so incorporation of Tc into transferrin and lactoferrin advantageously provides a new method for imaging cancers.
• Our initial experiments with rhenium whose chemistry is similar to technetium have led us to the present invention.
• Bovine apotransferrin was washed three times with 0.1 M KC1 using Centricon 30 ultra-filters to remove low molecular mass impurities. It was dissolved in 10 cm 3 of 10 mM Hepes buffer (pH 7.4) and stored in a refrigerator. Apolactoferrin was prepared from Fe-lactoferrin using standard procedures. All glassware was acid soaked for several hours before use to remove any traces of heavy metals and ultrapure water was used in all experiments. Due to its hygroscopic nature, Re 2 0 was stored and weighed in a glove box, to ensure accuracy in measurements.
• NTA refers to nitrilotriacetate, and n is amount of NTA present in solution relative to rhenium.
• Re(NTA) where n is equal to 1, was prepared by adding 4.17 cm 3 of NTA solution (0.006 M) to 2.30 cm 3 of Re atomic absorption standard solution (1010 ⁇ g/ml in 1% HN0 ). Slow addition of microlitre amounts of 1 M NaOH was carried out to give a pH of between 5 and 6. This solution was then made up to 25 cm with 10 mM Hepes buffer (pH 7.4), to give an overall rhenium concentration of 0.5 mM.
• Re(NTA) 8 and Re(NTA) 20 were made in the same way but using 8.34 cm 3 and 20.85 cm 3 of NTA (0.024 M) respectively.
• Figure 1 shows the UV/vis difference spectra obtained after each addition of Fe(NTA) 2 during the titration experiment.
• the base line of all the UV difference spectra corresponds to apotransferrin before addition of any metal.
• the increase in absorbance at 240 and 295 nm is directly related to the percentage saturation of transferrin binding sites.
• the increase in absorbance at 240 nm was monitored and the change in molar extinction coefficient.
• the ratio, r, of iron to transferrin was. calculated (Table 1) and plotted against the change in extinction coefficient ( Figure 2).
• Figure 7 represents the reaction of apo-lactoferrin and sodium bicarbonate with excess Re/NTA. This reaction was monitored by measuring absorbance at 231nm with time.
• Example 2 Each solution from Example 2 was incubated with cells as follows.
• Cell culture RTl 12 cells seeded into four 25 cm 2 flasks were grown to confluency in Dulbecco's
• Tc is taken-up by the tumour cells. Since the cells show phagocytotic behaviour, some non-specific uptake is expected. However, as Figure 8 shows there is a clear enhancement of Tc uptake (by almost 50%) once it is complexed to the Tf.
• a product comprising the following components is made up as a lyophilised, pyrogen free powder:
• the pertechnetate source is either sodium pertechnetate ( 99m Tc) injection (Fission) or sodium pertechnetate ( 99m Tc) (Non-Fission), maximum 7.4 GBq 200 mCi.
• An amount of the pertechnate is aseptically added to a volume of 3-6 ml aqueous solution in a vial and shaken and left for several minutes (2-5 minutes).
• the vial containing the lactoferrin and other additives is reconstituted in 3-6 ml saline solution and shaken for 2-5 minutes so as to allow the lyophilised material to reconstitute in solution. All but approximately 1 ml of the solution is withdrawn and added to the required amount of sodium pertechnetate ( 99m Tc) injection (Fission) or sodium pertechnetate ( 99m Tc) (Non- Fission). The mixture is then shaken to ensure a high labelling yield of lactoferrin.
• 99m Tc sodium pertechnetate
• 99m Tc sodium pertechnetate
• 99m Tc Non- Fission
• a single dose of the 99m Tc labelled lactroferrin complex is administered by the i.v route and ⁇ radiation is observed by conventional means over a selected period of time.
• the 9m Tc is found to concentrate in specific areas within the body, the area is found to correspond to a tumour site.

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• 2000
  • 2000-08-08 GB GBGB0019412.6A patent/GB0019412D0/en not_active Ceased
• 2001
  • 2001-08-07 CA CA002418383A patent/CA2418383A1/en not_active Abandoned
  • 2001-08-07 US US10/344,079 patent/US7160536B2/en not_active Expired - Fee Related
  • 2001-08-07 EP EP01956661A patent/EP1307240A2/en not_active Withdrawn
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• 2006
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