WO1991010451A1

WO1991010451A1 - Improvements in or relating to radiotherapeutic agents

Info

Publication number: WO1991010451A1
Application number: PCT/GB1991/000040
Authority: WO
Inventors: Richard Wootton
Original assignee: Royal Postgraduate Medical School
Priority date: 1990-01-12
Filing date: 1991-01-11
Publication date: 1991-07-25
Also published as: GB9000705D0

Abstract

A radiotherapeutic agent consists of microspheres having diameters of no more than a few νm, formed of a denatured protein or other biodegradable (or non-biodegradable) substance and incorporating so as not to be released into a bloodstream both a ferromagnetic substance and a radioactive substance which emits short-range tissue-damaging radiation and having a half-life in the approximate range of 3 to 300 hours. In use the agent is suspended in a physiologically tolerable liquid and injected into the bloodstream of a patient when a magnetic field is applied to the patient to cause the microspheres to collect in the vicinity of a tumour where they are held while the radiation impinges on the tumour.

Description

IMPROVEMENTS IN OR RELATING TO RADIOTHERAPEU IC AGENTS

The invention relates to radiotherapy and, in particular, to a radiotherapeutic agent for delivering therapeutic radiation to a desired target location in a human or animal body.

It is known to use tissue-damaging radiation from a radioactive substance in the treatment of tumours. A difficulty with such treatment is that the radiation will damage healthy tissue as well as tumour tissue and various techniques have been employed to reduce as far as possible the damage to the healthy tissue whilst maintaining the dose of radiation applied to the tumour- One technique that has been used is to employ several sources external to the body to direct radiation in different directions towards the tumour, so that the healthy tissue on which the radiation falls receives only a part of the total dose applied to the tumour.

It has also been proposed to use invasive methods to place a source of short-range tissue-damaging radiation inside a tumour so that little of the radiation reaches the healthy tissue around the tumour. This has the disadvantages that many tumours are not accessible because they are located behind or within vital organs for example, and it is clearly undesirable to impose the strain of an operation on an already sick patient.

Cytotoxic pharmaceuticals have been delivered to a tumour site by slowly releasing them into the bloodstream feeding the tumour from microspheres of a substance which is degradable by enzymes in the blood, the microspheres including a magnetic material which enables them to be collected in a suitable place near the tumour by means of an externally applied magnetic field. It is an object of the present invention to provide an improved technique for applying radiotherapy to a tumour.

According to a first aspect of the present invention there is provided a radiotherapeutic agent including microspheres which are sufficiently small to pass through capillary vessels of a human or animal body, which are degradable over a period of time by the body and which are physiologically tolerable to the body, there being attached to the microspheres a substantially inert ferromagnetic substance and a radioactive substance emitting short-range tissue-damaging radiation which decays over a period of time long enough to produce a required therapeutic radiation dose.

The microspheres may have diameters approximately in the range 0.1 μ to 10 μm.

In use, the microspheres are introduced into the bloodstream, for example by injection of a suspension of them in a liquid carrier, and are caused to be collected by an externally applied magnetic field at a specific location, such as adjacent to or within a tumour, and held there by maintaining the magnetic field at least for an initial period.

The material of the microspheres may be a denatured protein which will be degraded subsequently; for example human serum albumin is suitable for use in human beings. ^' The microspheres should not be completely degraded until the required radiation dose has been delivered, by which time it is preferable that the radioactive material will have decayed substantially. The concentration of the radioactive material by a magnetic field in this way means that a relatively small amount of radioactive material can be sufficient to provide the localised radiation dose required and, when finally released into the bloodstream, provides no more than a safe level of radiation distributed throughout the body. One example of a suitable ferromagnetic material for incorporation in the microspheres is_. magnetic iron oxide or magnetite. Ferrites may also be used.

The manner of attachment of the radioactive material to the microspheres may be by chemical bonding and should such that it is not substantially released into the bloodstream, at least until they become degraded. A chelating agent can, for example, be used to attach the radioactive material to the microspheres. Examples of suitable radioactive materials include yttrium-90, phosphorus-32, silver-Ill and indium-Ill. The radioactiv material may be in elemental or compound form. Another method of labelling the microspheres with a radioactive agent is iodination, which is a standard method for substituting a radioiodine atom in one of the amino acids a protein; in this case it may be necessary to label the protein before making it into microspheres.

According to a second aspect of the present invention there is provided the use of microspheres in the treatment of a tumour in a human or animal body, the microspheres being sufficiently small to pass through capillary vessels of the body, being degradable over a period of time by the body and being physiologicall tolerable to the body, in which the microspheres have attached to them a ferromagnetic substance and a radioactive substance emitting short-range tumour-damaging radiation which decays over a period of time long enough for the radiation to cause significant damage to the tumour, the use including the introduction of the microspheres into the bloodstream of the body and applying a magnetic field to the body to cause the microspheres to collect in the vicinity of the tumour and be held there fo time sufficient to apply a required therapeutic radiation

According to a third aspect of the present invention there is provided a method for the treatment of a tumour in a human or animal body including introducing into the bloodstream a quantity of microspheres of a size able to be carried by the bloodstream through capillary vessels of the body, the microspheres being formed of a denatured protein substance physiologically tolerable to the body and being degradable over a period of time by the body, and there bei attached to the microspheres in a manner so as not to be substantially released into the bloodstream a substantially inert ferromagnetic substance and a radioactive substance emitting short-range tumour cell-damaging radiation for a period of time suitable for causing significant damage to the tumour cells before decaying and applying a magnetic field to the body to collect the microspheres in the vicinity of a tumour and be held there for a time sufficient to apply a required therapeutic radiation dose.

The magnetic field applied to the body may be generated by one or more permanent magnets or electrically powered magnets having suitably shaped pole pieces for producing a magnetic field maximum at a required location. If the required location is one at which it is difficult to be sure that the magnetic field maximum is correctly positioned, one way in which the effect of the magnetic field can be tested before the microspheres are introduced is to use other microspheres containing a radioactive tracer, such as technetium-99m, and then examine the distribution of gamma ray or other relatively harmless but penetrating radiation emitted by the tracer using, for example, a gamma ray camera. The magnetic field may be adjusted until the image received by the camera indicates that the magnetic field maximum is in the desired location. Once the correct magnetic field is established the microspheres with the therapeutic radioactive material can be introduced.

According to a fourth aspect of the present invention there is provided a method for the treatment of a tumour in a human or animal body including introducing a quantity of microspheres of a first kind into the bloodstream of the body, the microspheres of the first kind having bonded thereto in such a way as not to be substantially released into the blood a ferromagnetic substance and a radioactive substance emitting a low level of gamma radiation for a period of time of the order of a few hours, applying an adjustable magnetic field to the body, observing the resulting distribution of gamma radiation from the body, adjusting the magnetic field in order to obtain a distribution of gamma radiation indicating that the microspheres are collected in a required location relative to the tumour, introducing a quantity of microspheres of a second kind into the bloodstream of the body, the microspheres of the second kind having bonded thereto in such a way as not to be substantially released into the blood a magnetic substance and a radioactive substance emitting short-range tumour cell-damaging radiation for a period of time sufficient to produce a required therapeutic radiation dose before decaying, and applying the adjusted magnetic field to the body to collect the microspheres of the second kind at the required location and hold them there long enough to apply a required therapeutic radiation dose to the tumour cells.

Although in the following description the method for treating tumours is described with reference to human beings, it will be understood that it can also be applied to other animals especially mammals. In such a case it will be appreciated that the material of the microspheres should be selected so as to be physiologically tolerable to the particular animal .

An example of the manufacture of examples of a radiotherapeutic agent including microspheres and the use of such an agent in the treatment of tumours will now be described in greater detail with reference to the accompanying drawings, of which:-

Figure 1 is a diagram of one form of apparatus whic can be used for the manufacture of microspheres; and

Figure 2 is a flow diagram of a method for the treatment of tumours in a body.

Microspheres can be produced by dispersing a small volume of an aqueous solution of a suitable protein in a larger volume of an oil, the solution and the oil not being miscible, and heating or otherwise treating the mixture to denature the protein particles dispersed in the oil . Using the apparatus shown in Figure 1 which is suitable for small scale production of microspheres, the protein solution is placed in a syringe 1 and added to a flask 2 containing a larger volume of oil and clamped upright in a heating mantle 3. An electric motor 4, fixed abpve the flask 2, is used to drive an impeller 5 to disperse the protein solution in the oil . A plug 6 in the neck of the flask 2 supports the impeller shaft. The speed of stirring is variable by a rheostat (not shown). The heating of the oil is controlled by adjusment of the proportion of time that the mantle element is on. The method of production may be adapted to microsphere production on a different scale using suitably modified apparatus.

In order to prepare magnetic microspheres, ultrafine particles of ferromagnetic material such as magnetite are incorporated into the denatured protein; the size of the magnetic particles is not critical . Other substantially inert ferromagnetic materials may be used, but suitable ultrafine particles of magnetite (10-20 nm in diameter) are commercially available as a slurry. The magnetite is made into a suspension in water with, for example, about 1 mg magnetite per microlitre. A small volume of the suspension (agitated ultrasonically before use) is added to the protein solution and the mixture agitated ultrasonically for a period. The mixture is then added drop by drop to the continuously stirred oil at room temperature as described above. The resulting microspheres include the magnetite. Other methods may be used to produce magnetic microspheres.

Microspheres incorporating magnetite or some other ferromagnetic material and, for example, produced by the method described above can be used to produce two types of radioactive microspheres, respectively labelled with two different radioactive substances. The first radioactive substance is one which emits predominantly gamma rays which are highly penetrating but cause little, if any, tissue damage; an example of a suitable substance for this purpose is technetium-99m. The second radioactive substance is one which emits short-range tissue-damaging beta particles; examples of suitable substances for this purpose are yttrium-90, phosphorus-32, silver-Ill and indium-Ill. The half-lives of these substances lie in the range of a few tens to a few hundreds of hours. A radioactive isotope of iodine could be used instead of technetium-99m and a different isotope of iodine could be used as the second radioactive substance. A disadvantage with the use of iodine isotopes in either of these ways is that no iodine isotope produces only highly penetrating but non-tissue-damaging radiation or only short-range tissue-damaging radiation.

The attachment to the magnetic microspheres of a radioactive substance of the tissue-damaging type could also be carried out in a manner analogous to that described above so as to produce chemical bonding of the element to the denatured protein of the microspheres. Alternatively, chelating agents could be used to attach radioactive substances of either type to the material of the magnetic microspheres. Iodination could also be used to label the protein of the microspheres with a radioactive agent; this process requires the substitution of a radioiodine atom in an amino acid in the protein and may be performed on the protein before it is made into microspheres.

All methods for protein iodination are based on the conversion of iodide to iodine which substitutes onto the aromatic ring of tyrosyl groups. Examples of suitable methods are as follows:-

1. Simple mixing of the protein with an iodide (of the particular isotope of iodine required) and chloramine T gives high yields of iodinised protein in a rapid reactio time (seconds) .

2. Electrolytic methods in which iodine is liberated from potassium iodide by the anode of an electrolytic system in the presence of the protein. This method produces good yields with moderate specific activities.

3. Enzymatic methods are possible using lactoperoxidase, glucose oxidase or chlorperoxidase to stimulate the iodine substitution into the protein. In these methods iodide is oxidised by hydrogen peroxide and represents a mild approach with high specific activities attainable. A stable iodination of the histidine imidazole ring and reversible iodination of amino and sulphydryl groups also occurs.

4. Iodogen (1,3,4,6-tetrachloro-3 ,6 -dephenyl- glycouril) is insoluble in aqueous solution but can be used for iodination of proteins in a solid phase reaction. Protein iodination, using the above techniques, involves a simple mixing of the protein with a particular isotope of iodine in the presence of the iodinating agent at or above neutral pH.

For proteins which do not contain tyrosine residues, or for which iodination of a tyrosine residue would alter the protein characteristics, a conjunction method could be employed. An example would be the use of the Bolton and Hunter reagent, an acylating agent which combines with iodine and condenses with free amino groups to form a covalent linkage. This approach requires a two-step procedure in which the Bolton and Hunter reagent is first iodinated and purified before reaction with the protein.

Due to the absence of specific metallic binding sites on most proteins, direct complexing of a metallic isotope which could be the second radioactive substance with protein involves weak and unstable interactions. A more stable radiolabel can be obtained by the use of bifunctional chelating agents. These are first attached to the protein (to the amino groups of lysine residues) by the use of anhydrides, glutaraldehyde or carbodiimides, after which the free chelating group is available for complexing metallic radioisotopes . Suitable chelating agents include DTPA and EDTA. The cyclic anhydride of DTPA has also been used for protein labelling. This technique must be conducted in the absence of any other metallic ions. Radioisotopes which can be coupled to proteins using DTPA include indium-Ill, gallium-67, scandium-47 and yttrium-90. Technetium-99m can also be coupled to protein by the use of DTPA.

The need for chelating agents with high stability under physiological conditions has led to the development of macrocyclic structures. A 14-membered ring structure (nitrobenzyl-TETA) , and a 12-membered ring structure (DOTA) possess high formation constants for particular metallic ions which are very stable under physiological conditions.

Microspheres are injected, or otherwise introduced, into the bloodstream of a patient and are carried round the body by the blood flow. Magnetic microspheres can be caused to collect in a specific location in the body by producing a magnetic field maximum at that location. The use of magnetism in that way is convenient because the means producing the magnetic field, which may be one or more permanent magnets, one or more electromagnets or a combination of the two with suitably shaped pole pieces and/or ferromagnetic components for modifying the magnetic field, is generally external to the body.

The purpose of the first type of microspheres, those labelled with the first radioactive substance, is to enable the collecting effect of a particular magnetic field to be ascertained. The microspheres of the first kind are introduced into the patient's bloodstream in a pharmaceutically suitable liquid carrier, for example, physiological saline solution. The magnetic field is then applied to the patient's body and an image obtained from a gamma ray sensitive camera which responds to the radiation from the radioactive material and shows where the microspheres are collected. The magnetic field can be adjusted and the change to the image observed, enabling an optimum concentration of the microspheres, for example in the vicinity of a tumour, to be obtained.

Once the magnetic field configuration required to produce the desired local concentration of microspheres is established, a quantity of the microspheres of the second kind are introduced into the patient's bloodstream and the required magnetic field applied and maintained to collect and hold the microspheres in the desired location. The radiation from the second radioactive substance now damages the tissue within its range, and can be used to apply the required therapeutic radiation dose of the tissue-damaging radiation to a tumour whilst inflicting only a small amount of damage on other tissue. The range of the radiation produced by the second radioactive substance should be greater than half the distance between capillary vessels within a tumour. This treatment is illustratd in the flow diagram shown in Figure 2.

There is some evidence to suggest that, after being held in one place for a few tens of minutes, the microspheres become stuck to the capillaries in which they are held by the magnetic field. This could be of advantage in that after a suitable period of time the magnetic field could be removed, and the patient returned to the ward, although the radiation therapy is required to be maintained for a longer period. The radiation dose delivered in the end will be determined by the decaying of the radioactive material and the disintegration of the protein.

Instead of applying a constant magnetic field to the patient to cause the concentration of the magnetic microspheres in a required location, the microspheres may be collected in a first location under the influence of a first magnetic field and then allowed to flow with the blood to a second location by reducing the first magnetic field and applying a second, differently located magnetic field.

Although the invention is described with reference to specific examples it is not limited to those examples. For example, other biodegradable materials than the denatured protein described may be used for the basis of the microspheres and other ferromagnetic materials than magnetite may be used to make the microspheres magnetically responsive. Similarly, other radioactive substances than technetium-99m which produce penetrating but non-injurious radiation may be used as the first radioactive substance; preferably it has a short half-life so that it does not have to be removed from the blood after use. The second radioactive substance may be any substance emitting short-range tissue-damaging radiation for a suitable period of time.

If desired, at least some of the microspheres may be removed from the blood after use by removing the magnetic field from the body, passing the blood through a tube external to the body with a magnet adjacent to the tube so that the microspheres are collected there.

Example 1

Production of microspheres without magnetic material or radioactive material was effected using apparatus of the kind shown in Figure 1. A small quantity, 0.5 ml, of 20 % aqueous solution of human serum albumin was loaded into the syringe 1 and added drop by drop to 100 ml of cottonseed oil in a 250 ml short-necked round-bottomed flask 2 at room temperature over a period of about 5 minutes. The solution was added through a catheter having its end from 1 to 2 cm above the surface of the oil. While the solution was being added the impeller 5, which was located less than 1 cm above the bottom of the flask, was driven at speeds from 3000 to 4000 r.p.m. The oil was changed from a clear brown to a cloudy dispersion by the addition of the solution.

After from 5 to 20 minutes further stirring of the mixture, the heating mantle 3 was turned on to about 0.7 of maximum power and left with further stirring for a period of 30 minutes. The mixture reached a final temperature of about 150 C. The mixture was then allowed to cool and when cool was filtered through a Whatman No. 5 filter paper using a Buchner funnel and a vacuum pump. Ethyl alcohol was then poured onto the paper three or four times to wash the microspheres free of oil . The microspheres were then washed off the filter paper by distilled water using a syringe, and were centrifuged out of the suspension at 1000 r.p.m. for 2 minutes. The microspheres were then washed in ether, allowed to dry and stored in a refrigerator.

In order to ascertain the size of the microspheres they were suspended in physiological saline solution for about one hour prior to measurement, because microspheres formed of denatured protein tend to swell in physiological saline solution. The diameters were then measured using an eyepiece graticule calibrated against a stage micrometer. Two batches were found to have median diameters of 1 μm and 2 μm.

Example 2

The method described in Example 1 was modified by the addition of ultrafine magnetite particles to the 20 % albumin solution. The magnetite (Fe-,0., 'EMG 1111 ' slurry from Ferrofluidics Ltd., Oxford) consists of particles 10 - 20 nm in diameter suspended in water, with 1 mg magnetite per icrolitre. 30 μl of the magnetite suspension, agitated ultrasonically before use, was added to the 0.5 ml of 20 % human serum albumin solution, and the mixture agitated ultrasonically for 15 minutes. The method of Example 1 was then repeated using the mixture i place of the albumin solution.

The resulting magnetic microspheres were found to have median diameters 2 to 3 times larger than those produced by Example 1, but nevertheless small enough to pass along the capillary blood vessels of a human body. Example 3

Magnetic microspheres produced by the method of Example 2 were labelled with technetium-9 m as the tracer radioactive substance in the following way:-

15 mg of magnetic albumin microspheres were placed in a dry container. One millilitre of phosphate buffer (pH 6.8) was added and the mixture agitated ultrasonically for 10 minutes. 20 mg stannous chloride (SnCl₂.2H₂0) was dissolved in 2.5 ml 2M HC1, with the addition of 5.5 ml distilled water and the solution filtered through a 0.22 μm millipore filter into a container under nitrogen. 0.4 ml of the stannous chloride solution was added to the microspheres under an atmosphere of nitrogen. The pH of the resulting dispersion was 2.5. 0.8 ml of the phosphate buffer was added to bring the pH of the dispersion' up to 3.7. 3 ml of water containing

QQrη technetium-99m obtained from a TC generator was added to the dispersion, which was then held at room temperature, and occasionally agitated, for 30 minutes. The microspheres were then centrifuged out of the dispersion at 1000 r.p. . for 2 minutes. A labelling efficiency of

84 % was obtained. The eluate obtained from the 99mTc generator contained technetium-99m in the form of pertechnetate which was reduced by the action of the stannous chloride to technetium-99m in such a way that the element became chemically bonded to the denatured protein of the microspheres.

Claims

1. A radiotherapeutic agent including microspheres which are sufficiently small to pass through capillary vessels of a human or animal body, which are degradable over a period of time by the body and which ar physiologically tolerable to the body, there being attach to the microspheres a substantially inert ferromagnetic substance and a radioactive substance emitting short-rang tissue-damaging radiation which decays over a period of t long enough to produce a required therapeutic radiation d

2. A radiotherapeutic agent according to claim 1 wherein the microspheres are suspended in a liquid carrie physiologically tolerable to the body, so that the microspheres can be introduced into the bloodstream by injection.

3. A radiotherapeutic agent according to claim claim 2 wherein the microspheres are formed of denatured protein.

4. A radiotherapeutic agent according to claim 3 wherein the protein which is denatured to form the microspheres is human serum albumin.

5. A radiotherapeutic agent according to any preceding claim wherein the microspheres have diameters in the range from 0.1 μm to 10 μm.

6. A radiotherapeutic agent according to any preceding claim wherein the radioactive substance is attached to the material of the microspheres by chemical bonding.

7. A radiotherapeutic agent according to claim 6 wherein the radioactive substance is attached to the material of the microspheres by a chelating agent.

8. A radiotherapeutic agent according to claim 6 wherein the radioactive substance is attached to the material of the microspheres by iodination.

9. A radiotherapeutic agent according to any preceding claim wherein the radioactive substance emits short-range radiation (e.g., alpha of beta) and has a half-life in the range from a few tens of hours to a few hundreds of hours.

10. A radiotherapeutic agent according to claim 9 wherein the radioactive substance is yttrium-90, phosphorus-32, indium-Ill or erbium-169.

11. A radiotherapeutic agent according to any preceding claim wherein the magnetic substance includes ultrafine particles of magnetite, Fe,0..

12. A radiotherapeutic agent as defined in any one of claims 1 to 11 for use in a method of treatment of a tumour in a human or animal body.

13. The use of microspheres in the treatment of a tumour in a human or animal body, the microspheres being sufficiently small to pass through capillary vessels of the body, being degradable over a period of time by the body an being physiologically tolerable to the body, in which the microspheres have attached to them a ferromagnetic substance^'and a radioactive substance emitting short-range tumour-damaging radiation which decays over a period of time long enough for the radiation to cause significant damage to the tumour, the use including the introduction of the microspheres into the bloodstream of the body and applying a magnetic field to the body to cause the microspheres to collect in the vicinity of the tumour and be held there for a time sufficient to apply a required therapeutic radiation dose.

14. A method for the treatment of a tumour in a human or animal body including introducing into the bloodstream a quantity of microspheres of a size able to be carried by the bloodstream through capillary vessels of the body, the microspheres being formed of a substance physiologically tolerable to the body and being degradable over a period of time by the body, and there being attached to the microspheres in a manner so as not to be substantially released into the bloodstream a substantially inert ferromagnetic substance and a radioac substance emitting short-range tumour cell damaging radiation for a period of time suitable for causing significant damage to the tumour cells before decaying and applying a magnetic field to the body to collec the microspheres in the vicinity of a tumour and be held there for a time sufficient to apply a required therapeutic radiation dose.

15. A method according to claim 14, initially including, for the purpose of ascertaining the magnetic field configuration needed to cause the microspheres to collect in the vicinity of the tumour, the steps of introducing into the bloodstream a quantity of similar microspheres to which is attached a radioactive substance emitting only highly penetrating but substantially non-tissue-damaging radiation in place of the radioactive substance emitting short-range tumour cell damaging radiation of the first-mentioned microspheres, applying a adjustable magnetic field configuration to the body, producing an image from the radiation from the microspheres representing their location in the body, and adjusting the magnetic field configuration so as to cause the microspheres to collect in the vicinity of the tumour

16. A method according to claim 14 or 15 wherein the application of the magnetic field to collect the microspheres in the vicinity of the tumour includes the application of a sequence of at least two different magnetic field configurations.

17. A method for the treatment of a tumour in a human or animal body including introducing a quantity of microspheres of a first kind into the bloodstream of the body, the microspheres of the first kind having bonded thereto in such a way as not to be substantially released into the blood a ferromagnetic substance and a radioactive substance emitting a low level of gamma radiation for a period of time of the order of a few hours, applying an adjustable magnetic field to the body, observing the resulting distribution of gamma radiation from the body, adjusting the magnetic field in order to obtain a distribution of gamma radiation indicating that the microspheres are collected in a required location relative to the tumour, introducing a quantity of microspheres of a second kind into the bloodstream of the body, the microspheres of the second kind having bonded thereto in such a way as not to be substantially released into the blood a magnetic substance and a radioactive substance emitting short-range tumour cell damaging radiation for a period of time sufficient to produce a required therapeutic radiatio dose before decaying, and applying the adjusted magnetic field to the body to collect the microspheres of the second kind at the required location and hold them there long enough to apply a required therapeutic radiation dose to the tumour cells.