# Patent

## Title
Bifunctional detection agents having an optical dye linked to an MRI

## Abstract

The invention relates to MR contrast media containing composite nanoparticles, preferably comprising a superparamagnetic iron oxide core provided with a coating comprising an oxidatively cleaved starch coating optionally together with a functionalized polyalkyleneoxide which serves to prolong blood residence.

## Background
This invention relates to superparamagnetic particulate contrast agents, in particular for use in magnetic resonance imaging, especially of the vasculature, and to their preparation.

In diagnostic imaging modalities, such as X-ray, ultrasound and magnetic resonance (MR) imaging for example, the use of contrast agents to enhance contrast between different tissues or organs or between healthy and damaged tissue is a well established technique. For the different imaging modalities, contrast enhancement by the contrast agent is achieved in different ways. Thus in proton MR imaging for example contrast agents generally achieve their contrast enhancing effect by modifying the characteristic relaxation times (T.sub.1 and T.sub.2) of the imaging nuclei (generally water protons) from which the MR signal which is used to generate the image derives.

When injected into a living being, materials with magnetic properties such as paramagnetism, superparamagnetism, ferromagnetism and ferrimagnetism can cause a reduction in the T.sub.1 and T.sub.2 (or T.sub.2 *) values of tissue water protons. Although a reduction in T.sub.1 cannot occur without a reduction in T.sub.2 (or T.sub.2 *), the fractional decrease in T.sub.1 can be different from that in T.sub.2 (or T.sub.2 *). If the fractional decrease in T.sub.1 is greater than that in T.sub.2 (or T.sub.2 *) then the intensity of the MR image increases, and the material is referred to as a T.sub.1, or positive, contrast agent. If the fractional decrease in T.sub.1 is less than that in T.sub.2 (or T.sub.2 *) then the intensity of the MR image decreases, and the material is referred to as a T.sub.2 (or T.sub.2 *), or negative, contrast agent.

Particles with the magnetic properties of superparamagnetism, ferrimagnetism and ferromagnetism are referred to herein as magnetic particles.

The first suggestion in the literature for the use of magnetic materials as MR contrast agents was the proposal in 1978 by Lauterbur that manganese salts might be used in this regard. The first proposal in the patent literature was the suggestion by Schering in EP-A-71564 (and its equivalent U.S. Pat. No. 4,647,447) that chelate complexes of paramagnetic metal ions, such as the lanthanide ion Gd (III), might be used.

The early commercial contrast agents for MR imaging, such as GdDTPA, GdDTPA-BMA and GdHP-D03A available from Schering, Nycomed and Bracco under the trade marks MAGNEVIST, OMNISCAN and PROHANCE, are all soluble chelate complexes of paramagnetic lanthanide ions and in use are positive contrast agents which enhance image intensity from the regions to which they distribute.

Subsequently, particulate ferromagnetic, ferrimagnetic and superparamagnetic agents were proposed for use as negative MR contrast agents. Oral formulations of such particulate agents, generally referred to herein as magnetic particles, have become available commercially for imaging of the gastrointestinal tract, e.g. the product ABDOSCAN available from Nycomed Imaging. However parenteral administration of such particulate agents has also been widely proposed for imaging of the liver and spleen as these organs act to remove foreign particulate matter from the blood relatively rapidly. Thus, by way of example, lever and spleen imaging using such agents is proposed by Widder in U.S. Pat. No. 4,859,210.

More recently it has been proposed, for example by Pilgrimm in U.S. Pat. No. 5,160,725 and WO-94/21240, that the uptake of parenterally administered magnetic particles from the blood by the reticuloendothelial system may be hindered, and thus blood residence time prolonged, by chemically binding a stabilizer substance to the magnetic particle surface.

Examples of materials which may be used in this way as stabilizers include carbohydrates such as oligo- and polysaccharides, as well as polyamino acids, oligo- and polynucleotides and polyalkylene oxides (including poloxamers and poloxamines) and other materials proposed by Pilgrimm in U.S. Pat. No. 5,160,725 and WO-94/21240, by Nycomed in PCT/GB94/02097, by Bracco in U.S. Pat. No. 5,464,696 and by Illum in U.S. Pat. No. 4,904,479.

Magnetic particles coated in this fashion can then be used as blood pool agents (i.e. for imaging the vasculature) or for lymph node imaging, or alternatively they may be conjugated to biotargeting agents and used for imaging the targeted tissues or organs.

When administered as blood pool agents, it has been found with magnetic particles that the fractional reduction in T.sub.1 of the blood protons can be greater than the fractional decrease in T.sub.2 (or T.sub.2 *) and thus such agents can be used as positive MR agents for the vasculature.

For parenteral use, the size and size distribution of the composite particles and the chemical nature of the surface of the overall particle are of great importance in determining the contrast generation efficacy, the blood half-life, and the biodistribution and biodegradation of the contrast agent. Ideally the magnetic particle size (i.e. the crystal size of the magnetic material) is within the single domain size range (such that the particles are superparamagnetic and thus have no hysteresis and a reduced tendency to aggregate) and the overall particle size distribution is narrow so that the particles have uniform biodistribution, bioelimination and contrast effects. Preferably, the magnetic particles should be provided with a surface coating of a material which modifies particle biodistribution, e.g. by prolonging blood half-life, or by increasing stability, or which acts as a targeting vector causing preferential distribution to a target site, such as a tumour site.

Mean crystal sizes, i.e. of the magnetic core material, should generally be in the range 1 to 50 nm, preferably 1 to 20 nm and especially preferably 2 to 15 nm and, for use as blood pool agents, the mean overall particle size including any coating material should preferably be below 30 nm. Producing superparamagnetic crystals or composite particles having such sizes is not in itself particularly problematical. However producing particles with the desired size, acceptable size distribution and without undue crystal aggregation does represent a problem and it is to the solution of this problem that one aspect of the present invention is directed.

Typically, the magnetic crystals are produced by liquid phase precipitation, generally in a solution of a polymeric coating agent (e.g. using a co-precipitation technique such as that described by Molday in U.S. Pat. No. 4,452,773). This technique results in the generation of relatively polydisperse particles which require a subsequent size fractionation step, e.g. by centrifugation or chromatography. By way of example it is by such a technique that the product AMI 227 of Advanced Magnetics is produced.

We have now found that magnetic particles with particularly advantageous properties can be produced by precipitation in a branched polymer containing aqueous medium and subsequently cleaving the polymer to release composite particles comprising magnetic particles and a cleaved polymer coating.

Thus viewed from one aspect the invention provides a process for producing composite magnetic particles, said process comprising: a hydrophilic branched organic polymer containing aqueous medium; and majority preferably containing a single magnetic particle.

FIGS. 1, 2 and 3 show pre- and post contrast T.sub.1 -weighted MR images of the rabbit following administration of a contrast medium according to the invention.

The hydrophilic branched organic polymer used in the process of the invention may be any natural, synthetic or semi-synthetic branched polymer and may if desired be produced by grafting to or cross-linking a linear hydrophilic polymer.

Where the branched hydrophilic polymer is an extensively cross-linked polymer, the cleavage of the polymer in the process of the invention will result in a breakdown of the cross-linked polymer matrix. It is preferred therefore that the cross-linking bonds should be susceptible to chemical or biochemical cleavage under conditions which do not cause significant degradation of the magnetic particles. Ester-cross-linked hydrogel-forming polymers (for example formed by cross-linking hydroxy group bearing polymers such as linear carbohydrates (e.g. dextran) or polyvinylalcohol) are thus particularly suitable candidates for such cross-linked polymers. If necessary, the polymers may be appropriately substituted to allow control over the ester cleavage rate. Ester cleavage in this regard may be by base treatment, e.g. with ammonia or an alkali metal hydroxide.

Nonetheless, in the process of the invention, the hydrophilic branched polymer is especially preferably a natural, synthetic or semi-synthetic carbohydrate, particularly a material capable of forming an aqueous gel and more preferably a polysaccharide material, e.g. a glycogen, or even more preferably a starch. If desired, in the process of the invention one may use a mixture of hydrophilic polymers at least one of which is branched. Where a starch is used as the polymer this may be a native starch or alternatively a processed starch, e.g. an acid-treated, enzyme-treated or solubilized starch. Natural starches, especially plant-derived starches such as corn, potato, rice or wheat starch, are particularly preferred.

Natural starches are a combination of the generally linear amylose and the branched amylopectin polysaccharides. For the practice of the invention, while amylose content is acceptable it is preferably not so high as to cause retrogradation, the largely irreversible transition from dispersed state to essentially insoluble microcrystalline state. For this reason the use of the amylopectin rich potato starch is preferred to maize or wheat starches.

Where the polymer material used according to the invention is a material which is affected by dissolution in an aqueous medium and heat treatment therein, e.g. a material such as starch which from a granular state first swells and partially dissolves, the thermal history of the reaction medium may affect the properties of the final product. In such cases, a thermal history which leads to greater control of the viscosity and structure of the medium may be preferable. Thus for starch in particular it is preferred that the reaction medium is created, cooled and then reheated, e.g. by formation of an aqueous dispersion at 60-95.degree. C., cooling to between 5 and 60.degree. C. and reheating to 45 to 80.degree. C. In this way, an advantageous structure for the precipitation medium may be achieved.

It is believed that the branched hydrophilic organic polymer provides dispersed precipitation seeding sites within the aqueous medium allowing uniform small precipitate particles to form. The use of non-branched hydrophilic polymers, such as gelatin or dextran, or of inorganic gels such as silica gel, is not effective.

The hydrophilic branched polymer is preferably present at a concentration sufficient to cause the precipitation medium to be in gel form at ambient or periambient temperatures, e.g. 0 to 60.degree. C. The concentration used is also conveniently such as to produce a gel at temperatures up to 80.degree. C. For starches, concentrations of 1 to 200 g/L, especially 2 to 150 g/L, particularly 20 to 100 g/L, and more particularly 40 to 90 g/L, are preferred.

While the precipitation medium is aqueous, it may also contain mixtures of water and co-solvents such as water-miscible alcohols, ethers or ketones.

As indicated above, it is particularly preferred that the precipitation medium used according to the invention be in the colloidal state of matter referred to as a gel. In such a medium polymeric gel forming material forms a network which is interpenetrated by the aqueous dispersion medium. Such a gel will generally be more viscous than the aqueous dispersion medium alone but for the purposes of the invention the gel need not be so viscous as to be in a rigid or semi-rigid self-supporting state. Indeed the gel structure may even be so weak that the precipitation medium may seem simply to be a free flowing solution. However in that event, the gel nature of the medium may readily be verified by its thixotropic properties, namely that the viscosity of the medium is reduced by agitation.

While the precipitation medium must contain a branched polymer as mentioned above, it may contain other gel forming polymers which may for example be linear. Examples of suitable polymers for use in this regard include proteins, polysaccharides, proteoglycans and gel forming surfactants, for example block copolymer surfactants of the Pluronic and Tetronic series such as F-127, F-108 and F-68. These surfactants form gels in aqueous media at the elevated temperatures and pH levels useful in the process of the present invention. Thus by way of example a precipitation medium in gel form may be prepared using an aqueous dispersion of a gel-forming linear block copolymer surfactant and a branched organic polymer such as amylopectin.

The gel matrix of the precipitation medium however should be such as to allow chemical (or biochemical) scission of at least one of the polymer components, preferably at least the branched polymer, so as to release the magnetic particles. For this reason, the use of heavily cross-linked polymers is not desirable.

Particle precipitation may be effected with the aqueous medium in rigid or semi-rigid gel form, e.g. by permeating the gel with a base, but most preferably precipitation is effected in a heated, agitated (e.g. stirred) aqueous medium which may or may not exhibit noticeably enhanced viscosity relative to water. Particularly preferably precipitation is effected at temperatures in the range 40 to 95.degree. C., and with gentle stirring.

By manipulating the thixotropic properties of the aqueous precipitation medium, composite particles according to the invention having a range of sizes can be produced.

It is believed that magnetic particle formation in the process of the invention may be a two-stage process with domains of amorphous material forming and then transforming into magnetic particles, e.g. at an elevated temperature, for example 40 to 95.degree. C., preferably 50 to 93.degree. C. Thus it has been found that the development of the magnetic properties of the particles can be monitored over a period of minutes to hours, e.g. up to 3 hours. However, in practice it is found that development of magnetic properties is substantially complete within two hours and that significant magnetization is developed within twenty minutes.

The magnetic particles which form in the aqueous medium may be of any precipitable magnetic metal oxide or oxide hydroxide, including mixed metal compounds, for example compounds as discussed in U.S. Pat. No. 4,827,945 (Groman), EP-A-525189 (Meito Sangyo), EP-A-580878 (BASF) and PCT/GB94/02097 (Nycomed) or by Pilgrimm (supra). Particular mention in this regard may be made of magnetic iron oxide compounds of formula

where M.sup.II and M.sup.III are transition or lanthanide metals in the II or II valence state, at least one of which is Fe, and n is zero or a positive number, or more particularly of formula

where M.sup.II is a divalent metal such as Fe, Mg, Be, Mn, Zn, Co, Ba, Sr, and Cu, M.sup.III is a trivalent metal such as Al, Yb, Y, Mn, Cr or a lanthanide, and n and m are each zero or a positive number.

Preferably the magnetic particles are iron oxides of formula (FeO).sub.n Fe.sub.2 O.sub.3 where N is in the range 0 to 1, typified by maghemite (.gamma.--Fe.sub.2 O.sub.3) and magnetite (Fe.sub.3 O.sub.4) or are mixtures of such magnetic iron oxides.

A wide range of iron salts may be used as the source of Fe.sup.III and Fe.sup.II ions, e.g. FeCl.sub.2, FeCl.sub.3, Fe.sup.III citrate, Fe.sup.II gluconate, FeSO.sub.4, Fe.sub.2 (SO.sub.4).sub.3, Fe.sup.II oxalate, Fe(NO.sub.3).sub.3, Fe.sup.II acetylacetonate, Fe.sup.II ethyldiammonium sulphate, Fe.sup.II fumarate, Fe.sup.III phosphate, Fe.sup.III pyrophosphate, ammonium Fe.sup.III citrate, ammonium Fe.sup.II sulphate, ammonium Fe.sup.III sulphate, and ammonium Fe.sup.II oxalate. The ratio of Fe.sup.II and Fe.sup.III ions should preferably be in the range 1:5 to 5:1.

Precipitation is initiated by setting the pH of the aqueous medium above the precipitation initiation threshold, generally by addition of a base, preferably an aqueous base such as an alkali metal hydroxide (e.g. sodium hydroxide, potassium hydroxide or lithium hydroxide) or ammonium hydroxide, particularly preferably concentrated ammonium hydroxide. The base added should have a pKr sufficient to bring the pH of the aqueous medium above the precipitation initiation threshold, e.g. to above 10.

The base is preferably added to an aqueous medium which contains the metal ions and the polymer. Alternatively, the base and polymer may be combined and the metal ions subsequently added. These additions may conveniently be effected by mixing aqueous solutions of the components at an elevated temperature, e.g. 40 to 95.degree. C., preferably 50 to 60.degree. C., with stirring.

It is believed that following initiation of precipitation, seed magnetic particle crystals form within amorphous paramagnetic domains of metal ions or metal hydroxides delimited by the branched polymer and that these amorphous domains transform into the fully formed magnetic particles. Thus particle formation may be allowed to proceed for a selected period of minutes to days, e.g. 1 minute to 24 hours, preferably 20 minutes to 10 hours, especially 1 to 5 hours.

Where the particle formation is effected at a temperature towards the upper end of the ranges specified herein, e.g. at 90.degree. C., the medium is preferably kept at this elevated temperature for only a relatively short period, e.g. up to 2 hours.

If the base, precipitable metal ions and polymer are combined at a lower temperature and the medium is then heated to a higher temperature for magnetic particle formation to occur, then the rate of temperature increase should be carefully controlled, e.g. at 10-100 C..degree./hour. Thus during particle formation the temperature of the aqueous medium is preferably raised in a controlled manner (e.g. with a temperature rise which is substantially linear with time), for example rising from a mixing temperature of 55.degree. C. to a final temperature of 90.degree. C. over a period of two hours.

After the particle formation period, it is particularly preferred to neutralize the reaction medium bringing it to a pH of for example 6.0 to 8.5. This can be done for example by cooling the medium to produce a set gel which can be washed until neutral, by neutralizing the medium with acid (acceptable acids include for example hydrochloric, sulphuric and nitric acids) or solid carbon dioxide or by applying a vacuum to remove the base if it has a high vapour pressure (eg. if it is ammonium hydroxide). The neutralized medium can be procesed further immediately.

For this neutralization step washing is particularly effective where the medium has been allowed to form a set gel and serves to remove excess metal salts and base. Washing may conveniently be effected with deionized water, preferably pre-cooled to 3 to 15.degree. C., and is preferably continued until the pH is approximately neutral.

While one can simply sonicate the washed get to break down the gel matrix without cleaving the polymer while still releasing polymer coated magnetic particles, it has been found that the particulate product has particularly beneficial properties if the polymer is cleaved to release the coated particles, e.g. by breaking down the polymer's molecular structure. Chemical breakdown may be effected using active chemical agents such as oxidants or bases but may also be effected using biochemical agents such as enzymes. For carbohydrate polymers such as starch one may conveniently cleave the polymer enzymatically using an enzyme such as an amylase, e.g. .alpha.-amylase. However it is especially preferred that polymer cleavage be effected using an oxidant. Oxidants which at trace levels are themselves biotolerable, or whose reduction products are similarly biotolerable, such as halogen oxyanions (e.g. alkali metal hypochlorites (such as sodium hypochlorite and calcium hypochlorite), periodates (such as sodium periodate), permanganates (such as KMnO.sub.4), peroxides (such as H.sub.2 O.sub.2), and enzymatic oxidants are preferred. Any excess oxidant that is used is preferably subsequently inactivated, e.g. by the addition of urea when hypochlorite is used as the oxidant. When polymer cleavage is effected using an oxidant, the magnetic particles thereby released possess a negative surface charge and particle agglomeration is further reduced.

The chemical agents used for polymer cleavage should preferably not be agents which will erode the magnetic particles or cause those particles to loose their magnetic properties. Accordingly, the use of acidic agent sis generally undesirable.

The extent to which polymer cleavage occurs can be varied as desired in order to leave a smaller or larger amount of the residue of the polymer as a coating on the particles. It should also be noted that a coating is generally desirable and that as a result the overall particle size of the composite particles is larger than that of the magnetic particle cores. According to the selected cleavage technique, the residue left as a coating on the particles may be polymeric, oligomeric or even monomeric.

After polymer cleavage, the product is preferably washed free of contaminants using membrane filtration techniques, e.g. ultrafiltration or diafiltration.

The resultant overall particle size is generally in the range 1 to 300 nm, preferably 4 to 30 nm and especially preferably 8 to 20 nm. In this regard it may be noted that the preferred oxidant cleavage of the polymer tends to give smaller overall particle sizes than does the enzymatic cleavage procedure and that both give smaller sizes than does simple sonication of a gel matrix.

In one preferred embodiment, the composite particles can be prepared in a one-pot reaction which reduces overall reaction time, avoids work up and handling of intermediates and reduces heat stress on the core magnetic particles. In this embodiment, iron III and iron II salts (eg. chlorides) are dissolved in a starch-water solution and iron oxide particles are precipitated by the addition of a base (eg. aqueous ammonia). The reaction is allowed to run for 1 to 3 hours at 70-90.degree. C. whereafter the quantity of excess base is reduced (eg. by applying a vacuum and/or nitrogen flushing over the hot reaction mixture where ammonia has been used ). The pH is then reduced to below 8.2 or to the point where the reaction mixture loses its buffer capacity. Then the oxidation agent (eg. sodium hypochlorite) is added while the mixture is still hot (eg. 70-90.degree. C.) and the oxidation is allowed to run at 70 to 90.degree. C. until an acceptable composite particle size is reached. For a composite particle size in the range 10 to 20 nm the reaction time will be about 30 to 120 minutes. At 0.47 tesla and 40.degree. C., the magnetic saturation moment should desirably be above 50 EMU/g FeOx, r.sub.1 greater than 15 mM.sup.-1 .multidot.s.sup.-1 and r.sub.2 /r.sub.1 less than 2.3. The reaction mixture is then quenched with urea and filtered, eg. through 0.2 .mu.m filters. The starch residues can be removed by diafiltration, eg. using a UF membrane with a molecular cut off of 20 to 200 kD.

The "core" magnetic particles preferably have particle sizes characteristic of single domain particles, e.g. a particle size of 1 to 50 nm, especially 1 to 20 nm, particularly 2 to 15 nm, and most preferably 4 to 12 nm. Indeed, in the composite particles produced by the process of the invention, the core crystals are generally substantially mono-sized, frequently being in the range 4 to 12 nm.

The process of the invention can be used to produce composite particles (i.e. cleaved polymer coated magnetic particles) having a sufficiently narrow size distribution to make subsequent size fractionation unnecessary, e.g. with at least 90% having particle sizes within 10 nm, preferably within 5 nm and especially preferably within 2 nm, of the intensity average particle size as measured by photon correlation spectroscopy. However, the particles will generally be filtered through a relatively large diameter filter, e.g. a 0.1 to 0.2 .mu.m filter, to remove any occasionally occurring large polymer fragments or any biological or particulate contamination.

The composite particles produced by the process of the invention are themselves novel and form a further aspect of the invention. Viewed from this aspect the invention provides composite particles, preferably charged particles, having a mean overall particle size of 4 to 30 nm and comprising superparamagnetic inorganic core particles with a cleaved hydrophilic polymer coating material, preferably an oxidized carbohydrate material, especially a cleaved starch. Preferably, the majority of the composite particles will contain a single superparamagnetic core crystal.

Viewed from a further aspect the invention provides a contrast medium composition comprising a contrast effective amount of composite particles comprising superparamagnetic metal oxide core crystals and an organic coating, said core crystals having a mean diameter of 2 to 10 nm, preferably 4 to 8 nm, said particles having a mean diameter of up to 30 nm, preferably up to 15 nm, and said coating comprising oxidatively cleaved starch, preferably together with a functionalized polyalkylene oxide, eg. a phosphorus oxide terminal polyethylene glycol such as methoxy PEG phosphate.

The process of the invention can be effected in two discrete stages, with composite particles first being prepared for example by the conventional co-precipitation technique used for preparation of magnetic starch microspheres (MSMs) (see for example Schroder WO-89/03675), and with the polymer subsequently being cleaved to produce the composite particles according to the invention. The composite particles treated in this way may contain a plurality of magnetic crystals in each composite particle, with the polymer cleavage step generally serving to release coated mono-crystals. Such a process forms a further aspect of the invention.

After polymer cleavage, and any desired washing and filtering of the released particles, the particles are preferably provided with a coating of a second material, e.g. in order to enhance biotolerability by reducing complement activation effects, to extend blood pool residue time, to provide a tissue targeting ability, to enhance shelf stability or to improve autoclavability.

Alternatively the second coating material may be introduced at an earlier stage, e.g. before magnetic particle formation or post magnetic particle formation and before polymer cleavage.

Particularly preferably, the second coating material is a coating of a natural or synthetic structural-type polysaccharide, a synthetic polyaminoacid or a physiologically tolerable synthetic polymer as described in PCT/GB94/02097 or of a stabilizer substance as described by Pilgrimm or Illum (supra). Particularly preferably the second coating material is a polyalkyleneoxide (e.g. a poloxamer, poloxamine, a polyethyleneglycol, etc.) or a heparinoid, and especially preferably such a material carrying a functional group, e.g. an oxyacid (e.g. sulphur, carbon or phosphorus oxyacid) function, which permits the coating material to bind chemically or adsorb to the composite particles and especially to the core magnetic particles. In this regard particular mention may be made of methoxy-PEG-phosphate (MPP) and other polyalkyleneoxide materials described by Pilgrimm in U.S. Pat. No. 5,160,725 and WO-94/21240. The beneficial effects of MPP can also be realized using hydrophilic polymers terminally functionalized with other siderophiles than the phosphate groups of MPP. One such group is salicylate. PEG can be functionalized with this by conjugation to 4-amino-salicylic acid or 5-amino-salicylic acid, both of which are essentially innocuous with a long history of biological use.

A further suitable second coating agent is a 3-hydroxy-4-pyridinone carrying a hydrophilic polymer such as PEG at the pyridine nitrogen. Simple analogs, such as 1,2-dimethyl-3-hydroxy-4-pyridinone, have been used clinically to void the human body of excess iron, for instance in people having received excessive red cell transfusions. These species have some of the largest known binding constants for Fe.sup.III, namely a log-beta(3) on the order of 35. There are several synthetic pathways that may conveniently be applied here. The polymer (PEG) could be attached by alkylation of the nitrogen of 2-methyl-3,4-dihydroxypyridine. PEG could also be attached by alkylation of 2-methyl-3-hydroxypyridine, followed by oxidation of the resultant product in the 4-position. PEG could also be attached by reacting a PEG that bears a primary amino group with 3-hydroxy-2-methyl-4-pyrone, therein replacing the ring oxygen atom by a nitrogen and forming the desired 3-hydroxy-4-pyridionone in one step. ##STR1##

Further options for attaching PEG polymers to iron oxide surfaces would be to link them to one of the large group of bacterial siderophores, exemplified by ferredoxin/ferrioxamine. Ferrioxamine has an amino terminal function that may be used to attach a suitable PEG derivative by acylation or alkylation.

The molecular weight of the second coating material has been found not to be particularly critical and may conveniently be in the range 0.1 to 100 kD, but materials having molecular weights of 0.3 to 20 kD, especially 0.5 to 10 kD and most especially 1 to 5 kD, are preferred, for example polyalkylene oxide materials having at least 60 alkylene oxide repeat units.

The weight ratio of the second coating material to the inorganic core particles is preferably in the range 0.02 to 25 g/g, especially 0.4 to 10 g/g and particularly 0.5 to 8 g/g.

One particular advantage that has been found for the doubly coated composite particles produced according to the process of the invention is that they may be sterilized by autoclaving (e.g. at 121.degree. C. for 15 minutes) without unacceptable deterioration in particle size or size distribution. This is of particular importance since it means that earlier process steps need not be carried out under aseptic conditions and thus greatly improves production economics.

Besides providing a coating of a second coating material, other post-production modifications may be made to the composite particles produced according to the invention. Thus in particular the particles may be treated with a functional agent to conjugate or couple the residual carbohydrate coating, and biotargeting moieties (such as antibodies, antibody fragments, peptides or biomolecules such as insulin) may be conjugated to one of the coating materials using conventional chemical techniques. Further appropriate targeting vectors or immunoreactive groups are described for example in WO-93/21957.

Thus one preferred embodiment of the process of the invention comprises the following sequential steps: and a base; to set; being performed before step (ii); cleave the starch and release said particles; polyalkyleneoxide derivative to bind said derivative to said particles; and

Step (iii) can be performed for example by removing excess water and base (eg. ammonium hydroxide) under reduced pressure before step (ii) or by addition of acid (eg. HCl) to adjust for excess base before step (iv).

It has been found that composite particles, comprising inorganic core particles provided with a cleaved hydrophilic polymer coating and with a blood-lifetime-prolonging hydrophilic polymer bound to the inorganic particle surface, have significantly enhanced blood-lifetimes relative to comparable particles having only one of the two coatings. It is believed that this arises as a result of the cleaved polymer serving to shield the particle surface binding site of the blood-lifetime-prolonging polymer and so delaying in vivo cleavage of the blood lifetime-prolonging polymer from the inorganic particle. Such particles form a further aspect of the invention.

Viewed from this aspect the invention provides an injectable composite particulate agent comprising an inorganic particle core (preferably a metal or mixed metal oxide, especially a superparamagnetic iron oxide particle) having chemically or physically (but preferably chemically) bonded to the surface thereof a hydrophilic blood-lifetime-prolonging polymer (preferably a functionalized polyalkyleneoxide such as methoxy-PEG-phosphate, particularly preferably a terminally functionalized linear polymer, and preferably a polymer having a molecular weight below the kidney threshold, e.g. a molecular weight of 0.2 to 30 kD, especially 1 to 10 kD) and provided with a binding site shielding hydrophilic organic polymer coating, for example a cleaved branched carbohydrate, e.g. oxidized starch.

Such particles may be used as blood pool agents or alternatively may be conjugated to a biotargeting vector as described above. While the inorganic core of these particles is preferably a superparamagnetic iron oxide, particles of other metal compounds may be used if desired e.g. compounds incorporating a radionuclide or another therapeutically or diagnostically effective metal.

The relaxivity of the superparamagnetic crystal containing particles of the invention will vary with the size and composition of the core and of the coated particle (as well as with temperature and applied magnetic field). The T.sub.1 relaxivity (r.sub.1) may be as low as 5 and as high as 200, while T.sub.2 relaxivity (r.sub.2) may vary between 5 and 500 at 0.5 T (relaxivity given as (mMFe).sup.-1 (sec).sup.-1). The r.sub.2 /r.sub.1 ratio may vary from 1 to above 100, e.g. from 1 to 10, particularly 1.2 to 3 at 0.47 T and 40.degree. C. Small single crystal particles will have r.sub.2 /r.sub.1 ratios in the lower range while large particles and multicrystal particles will show higher ratios. If the particles show superparamagnetic behavior, the magnetization of the particles in the range 0 T to about 1 T will depend on crystal size, with larger crystals having significantly larger magnetization. At 1 T the magnetization is about 20-100, preferably 30-90 emu/g iron oxide.

Where the superparamagnetic particles are produced by base precipitation of Fe.sup.II and Fe.sup.III, the r.sub.2 /r.sub.1 ratio at 0.47 T and 40.degree. C. will generally be less than 3. As a result, in T.sub.1 -weighted imaging, the particles produced according to the invention are efficient as positive contrast agents. Furthermore, the magnetization curves for suspensions of such particles show that even at field strengths as high as 4 T the particles are not fully magnetized. The incomplete magnetization of the particles at the field strengths conventionally used in MR imaging apparatus means that the magnitude of any susceptibility effects that may be present in the MR images is reduced. Moreover the relaxivity of the particles of the invention doe snot decrease as rapidly with increasing field strength.

In conventional MR imaging, the drive for many years was towards the use of high field machines having primary magnetic field strengths of 1 to 1.5 T. However the use of lower field machines is increasing and there is a developing need for positive MR contrast agents useful at the low field strengths of some commercial imagers, e.g. at 0.1 to 0.3 T. This need is met by the particles of the invention which are at least three times more efficaceous at these field strengths than the conventional metal chelate based positive MR contrast agents such as Magnevist and thus viewed from a further aspect the invention provides a method of contrast enhanced magnetic resonance imaging of a subject wherein a positive contrast agent is administered to a subject and an image of at least part of said subject is generated using an MR imaging apparatus, characterised in that said apparatus has a primary magnet having a field strength of below 0.3 T, in that said positive agent comprises magnetic particles having a physiologically tolerable carbohydrate coating and in that said particles are incompletely magnetized at said field strength (e.g. magnetized to up to 90% of their maximum possible magnetization) and preferably are incompletely magnetized at field strengths of up to 2 T, especially up to 4 T.

Viewed from a further aspect, the invention provides diagnostic compositions comprising the particles of or produced according to the invention together with at least one physiologically acceptable carrier or excipient, e.g. water for injections.

The compositions of the invention may be in any conventional pharmaceutical form, e.g. suspension, emulsion, powder etc. and may contain aqueous vehicles (such as water for injections) and/or ingredients to adjust osmolality, pH, viscosity, and stability. Ideally, the composition is in suspension form with the suspension being isotonic and isohydric with blood. For example, an isotonic suspension can be prepared by the addition of salts like sodium chloride, low-molecular weight sugars like glucose (dextrose), lactose, maltose, or mannitol or a soluble fraction of the coating agent or a mixture of these. Isohydricity can be achieved by the addition of acids like hydrochloric acid or bases like sodium hydroxide if only a minor adjustment of pH is required. Buffers such as citrate, acetate, borate, tartrate, and gluconate may also be used. The chemical stability of the particle suspension can be modified by the addition of antioxidants like ascorbic acid or sodium pyrosulphite. Excipients may also be added to improve the physical stability of the preparation. Most frequently used excipients for parenteral suspensions are surfactants like polysorbates, lecithin or sorbitan esters, viscosity modifiers like glycerol, propyleneglycol and polyethylene glycols (macrogols), or cloud point modifiers, preferably non-ionic surfactants.

The compositions of the invention will advantageously contain the magnetic metal oxide at a diagnostically effective metal concentration, generally 0.1 to 250 mg Fe/ml, preferably 0.5 to 100 mg Fe/ml, and especially preferably 1 to 75 Fe/ml.

The invention further provides a method of generating a contrast enhanced image of a human or non-human, preferably mammalian, body said method comprising administering to said body, preferably parenterally and especially preferably intravascularly, a suspension of a contrast agent according to the invention and generating an image of a last part of said body into which said agent distributes, e.g. by MR or magnetometry (e.g. using a SQUID detector or an array of SQUID detectors).

In an alternative aspect the invention also provides a method of determining distribution of a contrast agent according to the invention within a human or non-human, preferably mammalian, body, said method comprising administering a said agent to said body, preferably parenterally, and detecting a signal from said body emitted or modified by said agent, e.g. a radio active decay emission, a magnetic field distortion or a magnetic resonance signal.

Particularly preferably, the method of the invention involves imaging the vasculature, especially using T.sub.1 -weighted MR imaging. Image generation may be effected before any significant particle uptake by the liver or spleen occurs. With particles provided with a blood-lifetime-prolonging polymer coating e.g. of methoxy PEG phosphate, image generation may conveniently take place within 24 hours, preferably within 4 hours and more preferably within 1 hour, of intravascular administration. In alternative embodiments of the method, following localized injection T.sub.2 weighted images of lymphatic system may be generated or following injection into the vasculature T.sub.2 -weighted studies of liver or spleen or T.sub.2 -weighted diffusion studies may be effected.

For the method of the invention, the dosage used will be a contrast effective dosage for the imaging modality used. Generally this will lie in the region 0.05 to 30 mg Fe/kg bodyweight, preferably 0.1 to 15 mg Fe/kg and especially preferably 0.25 to 8 mg Fe/kg.

Besides being used as contrast agents, the composite particles of the invention may also be used in localized thermal treatments or hyperthermia applications--using their magnetic properties, energy may be transferred to the particles in vivo (eg. by exposure to a magnetic field of changing direction or field strength) and the energy loss from the particle to the surrounding tissue may be used to therapeutic effect, eg. to achieve a cytotoxic effect. This is of particular importance where the particles are conjugated to a targetting vector, eg. via a bifunctional linker such as a bifunctional polyalkylene oxide.

Similarly the particles may be used in iron therapy--in this case it is not necessary to develop the magnetic properties of the core crystals and these may be paramagnetic iron oxides provided with the cleaved polymer coating and optionally a second coating, eg. of MPP.

Various iron oxide preparations prepared according to the prior art are known to give significant adverse effects when administered intravascularly. The most frequently reported findings are suppression of systemic blood pressure and acute platelet depletion. It appears that these side effects may be physiological and haematological responses to particle induced activation of the complement system. While conventional iron oxide particles, such as magnetic starch microspheres (MSM), may strongly activate the complement cascade, the particles of the present invention have no or only minor influence on the number of circulating platelets while conventional preparations cause an acute marked and transient thrombocytopenia.

The particles of the present invention, whether with or without a second coating material (eg. MPP), have surprisingly been shown to have no effect on the complement system or on complement related parameters such as the blood pressure and platelet count. The selected coating material gives rise to a particle surface that will not trigger complement activation in a similar manner to the conventional particles.

The particles can easily be covered with a second coating material, e.g. a polymer which chemically or physically associates with the iron oxide (FeOx) surface (for example MPP). The particles are highly suitable for further surface modification or coating due to their large surface area and to the thin carbohydrate coating layer which permits terminally functionalized hydrophilic polymers such as MPP to penetrate and bind to or adsorb onto the surface of the core magnetic particle.

The FeOx particles have a lower magnetization than conventional iron oxide agents and the magnetization is not fully saturated within the imaging field range. This feature will reduce the appearance of susceptibility artifacts at high magnetic field strengths.

The particles have been found to have a significantly longer blood half-life (by a factor of 2 or more) in mice than conventional MSM particles produced by coprecipitation of starch and FeOx.

The blood kinetics of the particles can be further modified by adding the second coating material. Thus MPP coated particles, i.e. doubly coated particles, have been shown to have significantly longer blood half-lives (by a factor of 2 or more) in mice than particles without MPP coating or with methoxy-PEG (MeO-PEG) as an added excipient (MeO-PEG acts as an excipient and does not interact with the FeOx surface).

The MPP coated particles have been shown to have a significantly longer blood half-life (by a factor of 2 or more) in mice than nano-sized FeOx particles coated with MPP but without the primary coat of the cleaved polymer.

Both the singly coated and doubly coated particles according to the invention have been found to have no haematological effects in rats whereas conventional polysaccharide-FeOx preparations give rise to significant thrombocytopenia.

Moreover both the singly coated and doubly coated particles according to the invention have been found to have no effect on human complement whereas conventional starch-FeOx (MSM) particles are a potent complement activator.

The single crystal cored particles of the invention are particularly advantageous since they have a reduced tendency to aggregate so reducing the need for stabilizers (such as dextran) in the compositions of the invention and thereby reducing the possibility of toxicity problems.

To minimize storage and transportation problems, the particulate contrast agent produced according to the invention may conveniently be produced as a dry powder, eg. by spray drying or freeze drying, preferably under aseptic conditions. The dried agent forms a further aspect of the invention.

The various patent publications referred to herein are incorporated herein by reference.

The present invention will now be described in further detail in with reference to the following non-limiting Examples:

The gel preparation steps are: starch solution preparation and heating to 55.degree. C., addition of iron chloride to starch solution, addition of ammonium hydroxide to iron/starch solution, heating of reaction mixture to 87-90.degree. C., and product cooling/gel neutralization.

A. Preparation of Starch Solution

1. Suspend 50 grams of soluble potato starch (CAS No. 9005-84-9) in 850 grams of boiling deionized water and mix.

2. Bring to the boil and immediately on boiling place the starch solution in a 55.degree. C. water bath.

B. Addition of Iron and Ammonium Hydroxide to Starch

1. Dissolve 9.0 grams of FeCl.sub.3.6H.sub.2 O and 3.3 grams of FeCl.sub.2.4H.sub.2 O (2:1 molar ratio FeIII to FeII) in a total volume of 50 mL of deionized water.

2. After starch solution has cooled to a steady 55.degree. C., pour the iron solution into the starch solution, mix thoroughly and add 50 ml of 30% (conc.) NH.sub.4 OH.

3. Heat the resulting solution so as to increase the temperature to 89.degree. C. over 2 hours and maintain at 89.degree. C. for a further 50 minutes.

4. After the 170 minute heating on the water bath, a) chill overnight at 4.degree. C. to set gel, or b) cool to ambient temperature and neutralize with acid (see below).

C. Gel Washing Procedure (where gel is not acid neutralized)

Wash set gel by pumping cold deionized water through settled gel suspension until pH is less than 8.5.

D. Alternative Neutralization Process

Cool mixture to below 40.degree. C., neutralize with acid.

E. Gel Oxidative Cleavage with Sodium Hypochlorite

A dose titration of the amount of sodium hypochlorite (hypo) per gram of gel can be done on a new lot to optimize production. Magnetic particle production is assessed by photon correlation spectroscopy (PCS) for size and dispersity, and by determination of water proton relaxation rates. Adjust volume of hypochlorite for concentration of available chlorine and mgs Fe in 5 grams of gel. for 45 minutes. hypochlorite. is removed.

F. Analysis

Samples are then subjected to analysis. Material prepared in this way has the characteristics outlined in Table 1: ______________________________________ Analysis     Result         Scale up*** ______________________________________ Composition Iron (Fe) 6.4 mg/ml up to 95 mg/mL Mossbauer spectroscopy predominantly nano crystals of gamma-Fe.sub.2 O.sub.3 Carbobydrate (CHO) 3.6 mg/ml CHO:Fe (weight ratio) 0.57 0.2-0.3 PURITY (Supematant from (Gel Permeation particles centrifuged on Chromatography) CsCl density gradient of 1.4 g/ml) % Free Fe 0% % Free CHO 0% 2% of area SIZE (Iron Oxide Core) Predicted from r.sub.2 /r.sub.1 9 nm 8 nm NMRD* 8 nm 8 nm LFI** 6.24 .+-. 0.74 nm Calculated from 5.7-5.8 nm Magnetization SIZE (Whole Particle) Photon Correlation 11.5 nm 10-12 nm Spectrascopy Sedimentation velocity 42.6 Svedberg Units RELAXIVITY (at 40.degree. C. and 0.47 T) r.sub.1 16.34 (mM .multidot. sec).sup.-1 20-23 (mM .multidot. s).sup.-1 r.sub.2 28.04 (mM .multidot. sec).sup.-1 34-38 (mM .multidot. s).sup.-1 r.sub.2 /r.sub.1 1.72 1.6-1.7 STABILITY.sup.++  at 4.degree. C. <6 months Saturation Magnetization 60 emu/gm FeOx.sup.+++ 80-90 emu/g Fe ______________________________________ .sup.+++ 1 g FeOx is approximately 70% weight Fe *Nuclear Magnetic Relaxation Dispersion **Lattice Fringe Imaging ***Produced in larger scale .sup.++ Less than 5% sediments down on centrifugation at 12000 .times. g for 5 minutes. % area = % total area under HPLC trace

With NMRD, the longitudinal relaxation rate (1/T.sub.1) is measured as a function of magnetic field strength in the range 2.35 Gauss to 1.2 Tesla. See for example Koenig et al. NMR Spectroscopy of Cells and Organisms, Vol. II, page 75, R. K. Gupta (Ed), CRC Press, 1987 and Koenig et al. Progress in NMR Spectroscopy 22: 487-567 (1990).

Magnetic particles were prepared according to Example 1 except that corn starch was substituted for potato starch.

It should be noted that use of materials other than soluble potato starch may require modification of the quantities of materials used for gel formation and degradation.

Magnetic particles were prepared according to Example 1 except that rice starch was substituted for potato starch.

Magnetic particles were prepared according to Example 1 except that they were not treated with hypochlorite (section D). Instead, samples of the gel (4, 8, 12 grams) were diluted with a phosphate buffered saline solution and the starch of the gel was subjected to enzymatic hydrolysis using 100 .mu.g (at 0.7-1.4 units of activity per .mu.g) of the enzyme alpha-amylase (EC 3.2.1.1) at ambient temperature for 16 hours. The resulting released particles were centrifuged at low speed to remove large aggregates and filtered through a 0.45 .mu.m filter. This process produced particles with much larger overall sizes (range 10-110 nm) but with iron oxide cores of 6 nm.

Magnetic particles were prepared according to Example 1 except that they were not treated with hypochlorite (section D). Instead, 10 grams of the gel was subjected to sonication using a Branson sonifier fitted with a 1/4 inch probe. Sonication was carried out continuously for 15 minutes. After being centrifuged at low speed to remove large aggregates and filtered through a 0.45 .mu.m filter, the resulting released particles were also found to have a very much larger overall size (range 30-800 nm) but with similar sized iron oxide cores to those of Example 4.

Methoxy PEG phosphate (MPP) (mol. wt. 5 kD) was added to an aqueous suspension of particles produced according to Example 1 at the desired ratio of MPP to iron oxide (FeOx) (typically 1 to 2 gms MPP/gm Fe), incubated for 15 hrs at 37.degree. C. with constant rotation and then stored at 4.degree. C. until used.

If desired the particles can be autoclave sterilised at 121.degree. C. for 15 minutes.

The particles may be coated analogously using chondroitin sulphate.

Alternatively, the MPP coated particles can be incubated at 75.degree. C. for 12 hours or may be directly autoclaved at 121.degree. C. for 10-20 minutes.

MPP coated particles were produced analogously to Example 6 using different molecular weight MPP (1.1, 2.1, 5.0 and 10.0 kD) and different coating ratios (0.02, 0.2, 0.4, 0.5, 0.8, 1.0, 1.2, 1.5, 1.6, 2, 2.5, 3, 4 and 8 g MPP/g FeOx). 6 and 7

Mouse blood half-life (T.sub.1/2) was determined for particles according to the invention having a range of MPP coating densities. Mice were injected via tail vein with 100 .mu.L samples at 1 mg Fe/mL of the preparations of Examples 1, 6 and 7. At timed intervals, animals were euthanized, blood samples were collected and pooled from two mice and 1/T.sub.1 was measured. From 1/T.sub.1 values the half lives (T.sub.1/2) were determined. The results are set out in Table 2 which includes for comparison the results for uncoated particles and conventional MSM particles: ______________________________________ gm MPP*/gm FeOx T.sub.1/2 (min) ______________________________________ 0               27.9 0.02 28.0 .+-. 3.2# 0.2 31.9 .+-. 3.3 0.4 39.1 .+-. 14.1 0.8 28.5 .+-. 1.3 1.6 69.3 .+-. 27.7 2 48.9 .+-. 3.8 4 45.6 .+-. 3.0 8 62.0 .+-. 5.6 MSM  3.8 ______________________________________ *: Mol. wt. 5 kD. #: Mean .+-. an estimated standard error in the linearity of the T.sub.1/ curve fitting. MSM: Conventional coprecipitated magnetic starch particles.

From Table 2 it is clear that MPP coating prolongs blood half-life significantly but that even uncoated particles according to the invention have much greater blood half-lives than the conventional particles.

Where a non-associating secondary coating agent was used in place of MPP, there was found to be no significant increase in blood half-life in mice. This was shown by a comparison of detected mouse blood half-lives for uncoated and MPP coated particles and for particles treated with methoxy PEG (mol. wt. 5 kD) and with a starch derivative Hetastarch. Unlike MPP, methoxy PEG does not bind chemically to or associate with the particles and so is simply present as an excipient. The results of the comparison are set out in Table 3: ______________________________________ Secondary Coating Agent/Excipient T.sub.1/2 (min) ______________________________________ MPP mol. wt. 5 kD, 8 g/g FeOx 62.0 MeO-PEG mol. wt. 5000, 8 gms/gm FeOx 26.3 HETASTARCH .RTM., 6% solution 28.1 None 27.9 ______________________________________

MPP coated and non-MPP coated particles were tested in rats for hematological effects.

Male rats were prepared with indwelling dosing and sampling catheters inserted in the right jugular vein. Approximately 24 hours prior to, and at approximately 3, 10 and 60 minutes and/or 24 hours following dosing, bloods samples were collected. Hematology parameters measured included: white blood cell and platelet count.

Unlike with dextran magnetite or MSM particles no incidence of transient thrombocytopenia was observed with the MPP-coated and MPP uncoated particles of the invention.

The MPP coated and non-MPP coated particles and MSM were tested on human plasma to determine the extent of activation of the terminal complement complex. Unlike MSM, the MPP coated and non-MPP coated particles did not cause activation.

MPP coated particles produced according to Example 6 were administered into the rabbit at a dosage of 1 mg Fe/kg and fifteen minutes later at a dosage of 2 mg Fe/kg. Pre and post contrast T.sub.1 -weighted MR images were recorded at 1.5 T, 3 D TOF, TR/TE 25/5.6, flop angle 60.degree., and appear as FIGS. 1, 2 and 3 of the accompanying drawings. The enhancement of the vasculature is clear in both post-contrast images and, despite the combination of high dosage and high field, the image of FIG. 3 shows high vessel to tissue contrast without susceptibility artifacts.

(a) Synthesis of MSM

Starch (3 g, Reppe Glycose, Sweden) having a mean molecular weight of 70 kD was dissolved in water (10 mL). At a temperature of 60.degree. C., FeCl.sub.3 6H.sub.2 O (2.7 g) and FeCl.sub.2 4H.sub.2 O (4.5 g) was dissolved in the carbohydrate solution whereafter the mixture was slowly precipitated into 1.2M NaOH (50 mL) at 60.degree. C. while sonicating. The sonication was allowed to continue for another 10 minutes followed by a centrifugation at 5000 rpm for five minutes. The supernatant was collected and dialyzed against 0.9% NaCl. A magnetization curve revealed that the resultant starch particles were superparamagnetic and they exhibit a mean hydrodynamic diameter of 400 nm.

(b) Treatment of MSM With Sodium Hypochlorite

MSM particles (15.5 mgFe/mL, 1.70 mL) having a mean particle size of 400 nm were added to sodium hypochlorite (Fluka #71696, 13.8% free chloride). The container was sealed tightly and heated at 70.degree. C. for 45 minutes. The reaction mixture was cooled, 8M urea (0.17 mL) and added and the suspension was filtered (0.2 .mu.m). The particles were purified with water using a Macrosep centrifugal concentrator (cutoff 100K) at 3000 rpm. The recorded particle size given as a function of the amount of sodium hypochlorite added is given in Table 4 below. ______________________________________ Particle size of MSM before and after treatment with sodium hypochlorite Amount of NaOCl Particle Size (mL) (mean, nm) ______________________________________ 0               400 0.063 88 0.100 76 0.300 70 0.500 68 1.000 Aggregated 1) ______________________________________ 1): All starch cleaved, aggregation of naked iron oxide crystals

Table 5 presents details of properties of particles produced according to Example 1 (Composition A) and (1.2 g MPP (2 kD)/g FeOx) (Composition B) and formulated with excipients (Tris (50 mM), mannitol (2.5%) and sodium hydroxide to pH 6-8) for administration. ______________________________________ ANALYSIS       COMPOSITION A COMPOSITION B ______________________________________ COMPOSITION Iron (Fe) 30 mg/mL 30 mg/mL Carbohydrate (CHO):Fe ratio 0.8 1.2 MPP (2 kD) 0 60 mg/mL PURITY (Gel Permeation (Gel Permeation Chromatography) Chromatography) % Free CHO 10-12% of area 10-12% of area SIZE (Iron Oxide Core) predicted from r2/r1 7 nm 7 nm NMRD 7 nm 7 nm SIZE (whole particle) Photon Correlated 9-11 nm 11-13 nm Spectroscopy RELAXIVITY (at 40.degree. C. and 0.47 Tesla r1 19-20 (mM .multidot. sec).sup.-1 19-21 (mM .multidot. sec).sup.-1 r2 30-32 (mM .multidot. sec).sup.-1 30-33 (mM .multidot. sec).sup.-1 r2/r1 1.5-1.6 1.5-1.6 Saturation Magnetization 90-100 emu/gm Fe 90-100 emu/gm Fe 0.45 Tesla STABILITY >3 months at 40.degree. C. >3 months at 40.degree. C. ______________________________________ Notes: NMRD = Nuclear Magnetic Relaxation Dispersion % area = % total of area under HPLC trace

(Best mode for preparing particles before MPP coating)

(A) In a 22 L three necked flask equipped with an overhead mechanical stirrer and a condenser, 12.8 L of deionized water was heated to 95.degree. C. whereafter a slurry of 800 g of soluble potato starch (Sigma, No. S-2630) in 1.6 L of deionized water was added with an agitation rate of 80-100 rpm. The resulting slightly cloudy solution was stirred for 10 minutes at ambient temperature and then cooled to 55.degree. C. over a 30 minute period. A solution of 144 g iron(III) chloride hexahydrate and 52.8 g of iron(II) chloride tetrahydrate in 1.2 L deionized water and added, and then 3 minutes later 800 mL of 28% ammonium hydroxide was added in one portion. The agitation speed was lowered to about 60 rpm, and the black reaction mixture was stirred at ambient temperature for 15 minutes before heating gradually to 92.degree. C. over a 60 minutes period. The temperature was maintained at 92-94.degree. C. for three hours and progress of the reaction was measured by magnetic susceptibility measurements during and after the reaction. The excess ammonium hydroxide was removed by vacuum distillation. The concentration was cooled to about 20.degree. C. and refrigerated to form a gel.

In all, four batches of gels were prepared with the largest batch using 800 g of potato starch. __________________________________________________________________________ Preparation of Gel M.sub.sus FeCl.sub.3 FeCl.sub.2  Reaction Gel Fe, by M (.times.10.sup.-4 Starch 6H.sub.2 O 4H.sub.2 O NH.sub.4 OH Volume Weight XRF* (emu/ emu/ Batch (g) (g) (g) (mL) (L) (kg) (g/kg) mg Fe) Oe .multidot. cm.sup.3) __________________________________________________________________________ 1  600 108 39.6 600 12   9.8 3.33 0.0932 0.69 2 600 108 39.6 600 12 9.8 3.35 0.0954 0.71 3 600 108 39.6 600 12 9.8 3.36 0.0924 0.69 4 800 144 39.6 800 16 13.7 3.30 0.0955 0.70 __________________________________________________________________________ *XRF = Xray fluorescence spectroscopy

(B) The gel was washed with cold water to remove ammonium chloride obtained in the iron oxide formation plus residual ammonium hydroxide. These compounds would otherwise react with sodium hypochlorite used to digest the starch in step C, requiring a much greater amount of hypochlorite for the hypochlorite step.

Gel washing was accomplished with repetitive water addition/removals in a stirred reactor which was kept near 5.degree. C. to minimize gel dissolution. The gel was stirred (briefly and slowly) in about 2 volumes of cold deionized water and then allowed to settle (about 1 hour). A dark supernatant layer separated from a very dark gel layer was removed by suction (visual observation of the layers). Addition of an equal amount of water, brief slow agitation, settling and separation was repeated until a conductivity of 0.5 mmho was obtained. A total of about 8 volumes of water was necessary to wash the gels, with the recovery of iron about 80%. __________________________________________________________________________ Washed Gel Weight Water    Final  Fe   Conductivity Conductivity charged total  Weight Fe Recovery initial final Batch (kg) (L) Turnovers (kg) (g) (%) (mmho) (mmho) __________________________________________________________________________ 1  1.7 15  7    (centri- 6.1 78   4.8   0.3 fuged) 2 9.5 80 5 14.5 26.5 81 5.2 0.5 3 9.5 80 4 11.2 24.5 77 4.7 0.5 4 13.3 106 5 16.4 36.9 83 4.8 0.3 __________________________________________________________________________

(C) This step involves conversion of washed gel to particle dispersion by oxidation of the starch matrix. This was accomplished by treatment of the gel with sodium hypochlorite. The amount of hypochlorite used for the oxidation was determined by carrying out small scale (50-100 g of gel) oxidation experiments and measuring relaxivity (r1, r2 and r2/r1) and particle sizes.

The washed gel was warmed to 45.degree. C. and treated with 12% sodium hypochlorite solution. The temperature of the reaction mixture dropped by few degrees (because of addition of refrigerated hypochlorite). The temperature of the reaction mixture was allowed to increase to about 45.degree. C. The reaction was heated to 55.degree. C. over 30 minutes, at which point an exothermic reaction was observed, with temperature increasing by about 15.degree. C. over a 15 minute period. After the exotherm subsided, the temperature of the reaction was adjusted to 70.degree. C. and maintained for 45 minutes. The hazard evaluation of this step using RCI calorimeter confirmed a modest (15.degree. C.) but controllable exotherm for this reaction. The reaction mixture was allowed to cool to room temperature and filtered through a Millipore 0.2 micron nominal filter cartridge. The largest oxidation was carried out on 15 kg of gel (34 g of Fe) and recovery was quantitative. __________________________________________________________________________ Particle Dispersion Weight 12% Urea Final  Brook Charged NaOCl Quench Volume Fe haven r1 r2 Batch (kg) (L) (g) (L) (g) (nm) (mM .multidot. s).sup.-1 (mM .multidot. s).sup.-1 r2/r1 __________________________________________________________________________ 1  3.0 0.63 20  3.5 5.5 11.1 13.37 23.1 1.68 2 6.5 1.83 42 8 11.8 11.8 14.5 24.8 1.68 3 10 2.25 112 12 22 12.4 14.9 25.0 1.68 4 15 3.00 80 18 34 12.7 21.0 36.2 1.71 __________________________________________________________________________

(D) This step involves removal of residual starch, free iron and other reactants after the hypochlorite oxidation. Millipore, prescale TM TFF 100K regenerated cellulose membrane cartridge filters were used to achieve the desired results. The purity of the particles was monitored by GPC. The purity of the final product after ultrafiltration was in the range of 97-99% (GPC). __________________________________________________________________________ Final Product Volume         Water, Final Fe   Fe charged  total Weight by ICP Recovery Batch (L) Filter Media (L) (kg) (g) (%) __________________________________________________________________________ 1   2    Millipore 100K, 1 ft.sup.2 25   670  2.2  67 regenerated cellulose 2 6 Millipore 100K, 6 ft.sup.2 45 671 8.8 70 regenerated cellulose 3 12 Millipore 100K, 6 ft.sup.2 60 980 13.7 73 regenerated cellulose 4 18 Millipore 100K, 6 ft.sup.2 100  1674  27.7 82 regenerated cellulose __________________________________________________________________________

The final analytical date for all four batches are summarized in Table 10 below.

(E) Subsequent PEG coating

The particles produced by steps (A) to (D) above may if desired be PEG coated, preferably with methoxy PEG phosphate (MPP) of molecular weight 2000 D at a coating ratio of 1.2 g MPP per gram FeOx.

The particles, with or without such a PEG coating may be formulated for administration with 50 mM Tris buffer, and 2.5% mannitol with pH adjusted to 6 to 8 with sodium hydroxide. ______________________________________ Analytical Results Batch      1         2       3       4 ______________________________________ Purity   97.2      98.1      99.0    99.2 (%, GPC) Iron (mg/ 3.3 13.1 13.7 16.5 ml, (ICP)) r2/r1 1.67 1.66 1.65 1.64 r1 19.6 18.9 20.0 21.3 (mM .multidot. s).sup.-1 r2, 32.7 31.3 33.0 35.0 (mM .multidot. s).sup.-1 Magne- 0.0762 0.0795 0.0801 0.0926 tization (emu/mg Fe) Particle 12 10 10 11 size (nm, (Brookhaven) Mobility -3.02 -3.02 -3.04 -2.92 (mV.sup.-1 s.sup.-2 .times. E10) pH 4.1 4.0 4.0 4.0 Carbon/Iron 0.52 0.28 0.30 0.28 ratio Appearance Non- Non- Non- Non- viscous, viscous, viscous, viscous, dark red dark brown dark brown dark brown to brown ______________________________________

## Claims

1. A process for producing composite magnetic particles, said process comprising: polyalkyleneoxide; greater than 50 EMU/g FeOx, an r.sub.1 value greater than 15 mM.sup.-1 s.sup.-1 (measured at 0.47 tesla and 40.degree. C.) and wherein the overall particle size of said composite particles is in the range 4 to 30 nm.

2. A process as claimed in claim 1 wherein said medium is a gel.

3. A process as claimed in claim 1 wherein said magnetic particles are iron oxide particles.

4. A process as claimed in claim 1 wherein said magnetic particles are superparamagnetic.

5. A process as claimed in claim 1 wherein said starch is oxidatively cleaved.

6. A process as claimed in claim 1 wherein cleavage of said starch is effected by treatment of particles containing a plurality of said magnetic particles within a matrix of said starch.

7. A process as claimed in claim 1 wherein said starch is cleaved following formation of magnetic particle-starch composite particles.

8. A process as claimed in claim 1 which comprises the following sequential steps: salts, and a base; gel to set; being performed before step (ii); particles; derivative to said particles; and

9. A process as claimed in claim 1 wherein the majority of composite particles so released contain a single magnetic particle.

10. Composite particles having a mean overall particle size of 4 to 30 nm, a magnetic saturation moment greater than 50 EMU/g FeOx, and an r.sub.1 value greater than 15 mM.sup.-1 s.sup.-1 (measured at 0.47 tesla and 40.degree. C.) and comprising superparamagnetic iron oxide core particles with a cleaved starch coating material, and further comprising a coating of a polyalkyleneoxide.

11. A injectable composite particulate agent comprising composite particles having a mean overall particle size of 4 to 30 nm, a magnetic saturation moment greater than 50 EMU/g FeOx, and an r.sub.1 value greater than 15 mM.sup.-1 s.sup.-1 (measured at 0.47 tesla and 40.degree. C.), wherein said agent comprises a superparamagnetic particle core having chemically or physically bonded to the surface thereof a polyalkyleneoxide and provided with an oxidized starch fragment coating.

12. A diagnostic composition comprising particles of or produced according to claim 1 together with at least one physiologically acceptable carrier or excipient.

13. A method of generating a contrast enhanced image of a human or non-human body, said method comprising administering to said body a composition according to claim 11 and generating an image of a least part of said body into which said agent distributes.

14. A method of determining distribution of a contrast agent within a human or non-human, said method comprising administering to said body a composition according to claim 12 and detecting a signal from said body emitted or modified by said particles.

15. A contrast medium composition comprising a contrast effective amount of composite particles comprising a superparamagnetic metal oxide core crystals and an organic coating, said core crystals having a mean diameter of 2 to 10 nm, said particles having a mean diameter of up to 30 nm, a magnetic saturation moment greater than 50 EMU/g FeOx, and an r.sub.1 value greater than 15 mM.sup.-1 s.sup.-1 (measured at 0.47 tesla and 40.degree. C.), and said coating comprising oxidatively cleaved starch, and wherein said coating further comprises a polyalkyleneoxide.

16. A composition as claimed in claim 15 wherein said core crystals have a mean diameter of from 4 to 8 nm and said particles have a mean diameter of up to 15 nm.

17. A process as claimed in claim 8 wherein step (iii) is performed by removing excess water and base under reduced pressure before step (ii).

18. A process as claimed in claim 17 wherein said base is ammonium hydroxide.

19. A process as claimed in claim 8 wherein step (iii) is performed by addition of acid to adjust for excess base before step (iv).

20. A process as claimed in claim 19 wherein said acid is HCl.

21. A diagnostic composition comprising particles of or produced according to claim 8 together with at least one physiologically acceptable carrier or excipient.

22. A process as claimed in claim 1, wherein the r.sub.2 /r.sub.1 ratio of said composite particles is in the range from 1 to 100.

23. A process as claimed in claim 1, wherein the r.sub.2 /r.sub.1 ratio of said composite particles is in the range from 1 to 10.

24. A process as claimed in claim 1, wherein the r.sub.2 /r.sub.1 ratio of said composite particles is less than 3.

25. A process as claimed in claim 1, wherein the r.sub.2 /r.sub.1 ratio of said composite particles is in the range from 1.2 to 3.

26. A process as claimed in claim 1 wherein said polyalkyleneoxide is present in said medium after step (ii).

27. Composite particles as claimed in claim 1 wherein the r.sub.2 /r.sub.1 ratio of said composite particles is in the range from 1 to 100.

28. Composite particles as claimed in claim 1, wherein the r.sub.2 /r.sub.1 ratio of said composite particles is in the range from 1 to 10.

29. Composite particles as claimed in claim 1, wherein the r.sub.2 /r.sub.1 ratio of said composite particles is less than 3.

30. Composite particles as claimed in claim 1, wherein the r.sub.2 /r.sub.1 ratio of said composite particles is in the range from 1.2 to 3.

31. An agent as claimed in claim 11, wherein the r.sub.2 /r.sub.1 ratio of said composite particles is in the range from 1 to 100.

32. An agent as claimed in claim 11, wherein the r.sub.2 /r.sub.1 ratio of said composite particles is in the range from 1 to 10.

33. An agent as claimed in claim 11, wherein the r.sub.2 /r.sub.1 ratio of said composite particles is less than 3.

34. An agent as claimed in claim 11, wherein the r.sub.2 /r.sub.1 ratio of said composite particles is in the range from 1.2 to 3.

35. A composition as claimed in claim 15, wherein the r.sub.2 /r.sub.1 ratio of said composite particles is in the range from 1 to 100.

36. A composition as claimed in claim 15, wherein the r.sub.2 /r.sub.1 ratio of said composite particles is in the range from 1 to 10.

37. A composition as claimed in claim 15, wherein the r.sub.2 /r.sub.1 ratio of said composite particles is less than 3.

38. A composition as claimed in claim 15, wherein the r.sub.2 /r.sub.1 ratio of said composite particles is in the range from 1.2 to 3.

39. A method as claimed in claim 14, wherein the r.sub.2 /r.sub.1 ratio of said composite particles is in the range from 1 to 100.

40. A method as claimed in claim 14, wherein the r.sub.2 /r.sub.1 ratio of said composite particles is in the range from 1 to 10.

41. A method as claimed in claim 14, wherein the r.sub.2 /r.sub.1 ratio of said composite particles is less than 3.

42. A method as claimed in claim 14, wherein the r.sub.2 /r.sub.1 ratio of said composite particles is in the range from 1.2 to 3.