WO1998048847A1 - Nanoparticle composition containing iodinated x-ray contrast agent as stabilizer for thermal sterilization - Google Patents

Abstract

Nanoparticle compositions can be stabilised for thermal sterilisation by soluble, iodinated X-ray contrast agents. These soluble, iodinated X-ray contrast agents afford particle size stability during heat sterilisation of nanoparticle compositions and both process and safety advantages over conventional cloud point modifiers.

Description

NANOPARTICLE COMPOSITION CONTAINING IODINATED X-RAY CONTRAST AGENT AS STABILIZER FOR THERMAL STERILIZATION

This invention relates to a new class of stabiliser for therapeutic or diagnostic compositions comprising nanoparticles and in particular to new formulations which facilitate the heat sterilisation of such compositions .

Nanoparticles of both therapeutic and diagnostic agents have demonstrable advantages over conventional drug formulations or delivery systems for enhancing the bioavailability of sparingly soluble or insoluble agents .

Nanoparticles are particles of approximately 1-1000 nm in size, generally comprising a sparingly soluble therapeutic or diagnostic agent and conventionally having a surface modifier adsorbed onto the particle surface. These particles are generally solid, crystalline and rigid particles having high melting points (i.e. in excess of 150°C) . Usually, insoluble agents with melting points below 150°C cannot be formulated into nanoparticles or do not survive the elevated temperatures and pressures required for thermal sterilisation. Nanoparticles generally have an average particle size of less than about 400 nanometres and for use in humans should be of physiologically tolerable materials .

In the development of nanoparticles for parenteral use, product sterilisation represents a major challenge owing to the sizes of the particles. The two most common sterilisation techniques are sterile filtration and thermal sterilisation, e.g. autoclaving or steam sterilisation. Sterile filtration is sufficient to remove most viruses and bacteria but the nanoparticles, due to their size and/or shape, generally cannot be_. sterile filtered. Thermal sterilisation is also problematical as it often leads to significant increases in particle size as a result of heat induced aggregation and/or particle growth, rendering the resulting particles unusable.

Nanoparticle compositions for parenteral administration generally require the use of a surfactant as a surface modifier. In US Patent No. 5,352,459 (Hollister et al . ) a purified polymeric surfactant is employed as the surface modifier for adsorbing onto the surface of the nanoparticles .

In US Patent No. 5,569,448 (Wong et al) a block copolymer linked to at least one anionic group is suggested as a surface modifier for adsorbing onto the surface of the nanoparticles .

Since the presence of ionic surfactants can cause particle agglomeration, and may cause toxicity problems and since particle agglomeration is clearly undesirable for parenterally administered compositions, it is clearly desirable to use non-ionic surfactants. The use of non-ionic surfactants is further desirable since they impart steric stability. Unfortunately, where a nonionic surfactant is used as a surface modifier for such nanoparticles, the problems of thermal sterilisation are exacerbated as at elevated temperatures such non-ionic surfactants undergo a well-known phase separation referred to as a cloud point, which may cause the compositions to flocculate or coalesce.

The aggregation of the nanoparticles upon heating is directly related to the precipitation and/or phase separation of the surface modifier at temperatures above the cloud point of the surfactant where the bound surfactant molecules are likely to dissociate from the nanoparticles and precipitate and/or phase separate, leaving the nanoparticles unprotected. The unprotected nanoparticles can then aggregate into clusters of particles. Upon cooling, the surfactant redissolves into the solution which then coats the aggregated particles and prevents them from dissociating into smaller ones .

In order to prevent this aggregation, cloud point modifiers are currently the preferred means for stabilising nanoparticle suspensions during heat sterilisation. The cloud point modifier is added in an amount sufficient to increase the cloud point of the surface modifier to a temperature greater than that required for heat sterilisation.

In US Patent Application No. 5,346,702 (Na et al . ) a composition is disclosed comprising nanoparticles having a surface modifier adsorbed on the surface thereof and a non- ionic cloud point modifier which is preferably a polyethylene oxide polymer with a molecular weight range of 400 to 10,000.

In US Patent No. 5,336,507 a nanoparticle composition is disclosed in which a charged phospholipid is utilised as the cloud point modifier. This modifier is preferably dimyristoyl phosphatidyl glycerol (DMPG) .

In US Patent No. 5,298,262 (Na et al) an anionic or cationic surfactant is suggested as a cloud point modifier. A preferred anionic cloud point modifier is stated to be dioctylsulfosuccinate .

Cloud point modifiers do not work for every nanoparticle suspension. Also, ionic cloud point modifiers such as dimyristoyl phosphatidyl glycerol, are not preferred due to both safety and efficacy concerns. For example, the cloud point modifier dioctylsulfosuccinate is hemolytic to red blood cells.

Moreover, one of the most preferred classes of cloud point modifiers, the polyethyleneglycols, whilst functioning well, must be used at elevated concentrations where their relative safety in large volume parenteral administrations remains untested and polyoxyethyleneglycol polymers suffer from chemical instability in the presence of oxygen and light. Furthermore, trace amounts of common metal impurities can act as catalysts for the degradation reactions of the polyethyleneglycol molecules . These problems are also encountered in the lyophilised state and thus PEG formulations require to be degassed with an inert gas and stored in metal free containers in the dark.

We have now surprisingly found, however, that nanoparticle compositions can be stabilised for thermal sterilisation by soluble, iodinated X-ray contrast agents. These soluble, iodinated X-ray contrast agents afford particle size stability during heat sterilisation of nanoparticle compositions and both process and safety advantages over conventional cloud point modifiers . Although it is not known what the operative mechanism of protection is for these agents, it is known that they are not cloud point modifiers for the commonly used nonionic surfactants such as Pluronic F-108.

Thus viewed from one aspect the invention provides a diagnostic or therapeutic composition comprising therapeutically or diagnostically effective nanoparticles, a physiologically tolerable surfactant and a physiologically tolerable water-soluble, iodinated X-ray contrast agent. Viewed from a further aspect the invention provides a process for the preparation of a sterile therapeutic or diagnostic composition, said process comprising thermally sterilising (e.g. by steam heat sterilizing, for example by autoclaving) a composition comprising therapeutically or diagnostically effective nanoparticles, a physiologically tolerable surfactant and a physiologically tolerable water-soluble, iodinated X-ray contrast agent.

Viewed from a yet further aspect the invention provides the use of a physiologically tolerable water-soluble iodinated X-ray contrast agent for the manufacture of a therapeutic or diagnostic composition comprising therapeutically or diagnostically effective nanoparticles, a physiologically tolerable surfactant and said X-ray contrast agent, for use in a method of therapy or diagnosis, e.g. in a diagnostic imaging procedure .

Viewed from a yet further aspect the invention provides a method of therapy or diagnosis of a human or non-human animal (preferably a vascularised animal, e.g. a mammalian or avian animal) body, said method comprising administering to said body a therapeutically or diagnostically effective amount of a composition comprising therapeutically or diagnostically effective nanoparticles, a physiologically tolerable surfactant and a stabiliser, the improvement comprising using as said stabiliser a physiologically tolerable water- soluble iodinated X-ray contrast agent.

Viewed from a yet still further aspect the invention provides a method of generating an image of a human or non-human animal (preferably a vascularised animal, e.g. a mammalian or avian animal) body, said method comprising administering to said body a diagnostically effective amount of a composition comprising diagnostically effective nanoparticles, a physiologically tolerable surfactant and a stabiliser, and generating an image of at least part of said body (e.g. using an imaging modality such as MR imaging, X- ray imaging (e.g. CT imaging), ultrasound imaging, magnetotomography, electrical impedance tomography, SPECT, PET and scintigraphy) , characterised in that as said stabiliser is used a physiologically tolerable water-soluble iodinated X-ray contrast agent.

In the present invention, the water-soluble iodinated X- ray contrast agent is used sufficient to achieve a stabilising effect, e.g. a concentration sufficient to stabilise said composition for thermal sterilisation or a concentration sufficient to increase the physical stability of said composition to settling during storage.

The water-soluble iodinated X-ray contrast agents used according to the invention will generally contain iodophenyl moieties, e.g. diiodophenyl or more preferably triiodophenyl moieties and may contain one or more, e.g. 1, 2 or 3 such iodophenyl groups per molecule . The iodophenyl rings will in such an event generally be substituted by solubilizing groups, e.g. ionic groups or more preferably non-ionic groups, for example groups containing mono or polyhydroxy-Ci-io-alkyl moieties optionally linked to the phenyl ring via an amide function. A wide variety of such iodophenyl agents are known from the literature and many are available commercially (see Martindale "The Extra Pharmacopoiea" , 30th Edition, 1993, The Pharmaceutical Press, London, pages 707 to 711) . Examples include iohexol, ioversol, iopamidol, iopentol, iodixanol , iopromide, iomeprol, iosimide, metrizamide, iotasol and iotrolan. The use of water-soluble, iodinated X-ray contrast agents as stabilizers for the heat sterilization of nanoparticle suspensions will provide advantages such as not requiring oxygen free formulations, minimising the requirement for metal ion free processes, and excellent safety at the high concentrations currently used in large volume parenteral administrations . These agents are also readily available and advantageously, have low viscosities at the concentrations required for thermal sterilisation of nanoparticles.

The water-soluble, iodinated X-ray contrast agents of the present invention have a well documented chemical stability and their formulations can be lyophilized and may possibly provide completely GRAS (generally regarded as safe) formulations for the novel nanoparticle agents of interest .

Moreover, in some cases where cloud point modifiers such as PEG 1450 were ineffective in the stabilization of nanoparticle size after thermal sterilisation, the water-soluble, iodinated X-ray contrast agents of the invention were successful at maintaining particle size. Thus, in addition to the process and (shelf) stability advantages, the use of these novel agents for particle size stability during heat sterilization will open this technology to additional agents which would otherwise have failed to meet sterilization requirements for success .

The water-soluble, iodinated X-ray contrast agents of the invention also increase the density of the solution phase thus enhancing the physical stability of the compositions to settling during storage.

The addition of increasing amounts of iohexol to nanoparticle suspensions of iodinated, insoluble X-ray contrast agents is expected to provide improved physical stability to settling over time in the container. Settling of the nanoparticles to the bottom of their respective container is a density driven process; i.e., the solution phase is near the density of water while the iodinated nanoparticles are much more dense with density values > 2.0 g/cc. Thus, addition of iohexol to the solution phase should increase the density of the solution phase such that the density difference between the particles and the solution will decrease.

In view of the increase in density imparted to the solutions of the present invention by water-soluble, iodinated X-ray contrast agents it is envisaged that such agents may be also useful in the stabilisation of any suspension which would benefit from increased density, for example, agricultural formulations where particle settling and aggregation both on the shelf and during administration is a concern.

Other potential applications may include the use of soluble iodinated X-ray contrast agents in the stabilization of suspensions to heat. For example, any use of particle suspensions which might experience either temporary heat extremes or continued elevated temperature such as suspensions exposed to diurnal cycles of sunlight and darkness. Also envisaged is their use for physical and chemical stabilization for liquid crystal displays in flat panel displays, CRT displays, watch displays, etc. The same properties which make these agents useful for nanoparticle stabilization may well extend the utility of these devices past their current useful lifetimes.

Water-soluble iodinated X-ray contrast agents particularly useful according to the invention are preferably of low molecular weight and include iohexol (Omnipaque^®) , iopa idol (Isovue™) , iopentol, ioxilan and iodixanol . The compositions according to the invention may contain a single water-soluble iodinated X-ray contrast agent or a mixture of two or more water- soluble, iodinated X-ray contrast agents.

For thermal sterilisation, the quantity of water-soluble iodinated X-ray contrast agent used will depend upon the nature and quantity of the other excipients present in the composition but will be a quantity sufficient to stabilise the composition for thermal sterilisation. The necessary amount may readily be determined by the person of ordinary skill in pharmaceutical science. Generally, the amount will be in the range 5-40% by weight (relative to the weight of the aqueous phase of the composition) , preferably in excess of 15%, most preferably in excess of 20% by weight.

For improving physical stability to settling, the quantity of water-soluble, iodinated X-ray contrast agent used will depend upon the nature and quantity of the other excipients present in the composition but will be a quantity sufficient to increase the physical stability to settling of the composition. The necessary amount may readily be determined by the person of ordinary skill in pharmaceutical science. Generally, the amount will be in the range 5-40% by weight (relative to the weight of the aqueous phase of the composition), preferably in excess of 15%, most preferably in excess of 20% by weight.

The water-soluble, iodinated X-ray contrast agents useful according to the invention can be prepared according to methods well-known in the art, for example the methods disclosed in SE-7706792-4, by Gulbrandsen in Kjemi No. 6/90, pages 6-8, NO-160918, GB 9618056.7 and GB 9618055.9. The manufacture of such contrast agents includes the production of the chemical drug substance followed by formulation to a drug product . Present multistage preparations of certain contrast agents of the invention require successive known acylation and N- alkylation reactions.

The surfactants used in the compositions of the invention are preferably non-ionic and may be for example alkaline oxide polymers or copolymers, e.g. poloxamers such as the pluronics (e.g. Pluronic F68 and 108 which are block copolymers of ethylene oxide and propylene oxide) or poloxamines such as the tetronics (e.g. Tetronic 908) and the carbowaxes (which are polyethyleneglycols (PEGs) ) , tyloxapol, polyvinylpyrrolidone, P-79, and PEG-modified phospholipids . These will generally be used in relatively minor quantities e.g. 0.1-10% by weight and are normally used at quantities sufficient to ensure that a stable composition can be formed.

The nanoparticles useful according to the invention can be any nanoparticle with the desired diagnostic or therapeutic properties . Thus the particles may be of a substantially water insoluble drug substance (e.g. itraconazole) , a porous inert material (e.g. a zeolite) which is serving as a carrier for a drug or a contrast agent, an inorganic material having desired contrast enhancing properties (e.g. a superparamagnetic iron oxide) , an insoluble or poorly soluble iodinated X-ray contrast agent, etc.

Magnetic iron oxides have been examined for many years as possible MRI contrast agents. These materials are often produced in very small sizes (e.g. 5 to 30 nm) such that their dispersions appear clear to the human eye even though particles in the size range of 10 to 20 nm are present. Nonetheless, water soluble iodinated X- ray contrast agents may be used in accordance with the invention to provide more physically stable materials which are better able to withstand heat sterilization without measurable increases in particle size due to either aggregation or particle growth.

Similarly water soluble X-ray contrast agents may be used in accordance with the present invention in the preparation of particulate ultrasound contrast agents .

The nanoparticles can be prepared for example according to the methods disclosed in US Patent No. 5,145,684 (Na et al . ) whose disclosure is incorporated herein by reference. Thus nanoparticles may be prepared by dispersing a sparingly soluble therapeutic or diagnostic agent in a liquid dispersion medium and wet-grinding the agent in the presence of grinding media to reduce the particle size of the contrast agent to an effective average particle of less than about 400 nanometers.

The quantity of nanoparticles used in the composition of the invention will depend on the nature and quantity of the other excipients present, the use of the composition e.g. whether for therapeutic or diagnostic use, the nature of the active agent, the particular animal species and the size of the animal to be treated but the necessary amount may readily be determined by the person skilled in pharmaceutical science. Generally, the amount of nanoparticles in the compositions of the invention will be in the range 0.1 to 45% by weight, preferably 1 to 30% by weight.

Heat sterilisation in the process of the invention may be carried out in conventional fashion e.g. by autoclaving (steam or moist heat sterilisation) . Sterilisation is preferably effected for at least 15 minutes, preferably 20 minutes or more at a temperature of 121°C or slightly higher. In some cases, sterilisation is performed at lower temperatures for longer times e.g. 110°C for 90 minutes. The compositions of the invention are useful in such conditions .

The compositions of the invention can be lyophilised and the water-soluble, iodinated X-ray contrast agents described herein are suitable for the stabilisation of such compositions.

The compositions of the invention preferably have an aqueous carrier medium, or if in dried form are preferably suspended in such a medium prior to administration. For example in a physiologically tolerable saline medium or in water for injection.

The publications referred to herein are hereby incorporated by reference .

The invention will now be described further by the following non- limiting Examples and the Figures 1 to 3.

Figure 1 shows particle size determination for a nanoparticle suspension of 22.5% NC 68183 after milling in the presence of 6% Pluronic F-87 but before heat sterilisation .

Figure 2 shows particle size determination for a nanoparticle suspension of 15% NC 68183 after both milling in the presence of 4% Pluronic F-87 then autoclaving in the presence of 15% PEG 1450 (wt/vol%) .

Figure 3 shows particle size determination for a nanoparticle suspension of 15% NC 68183 after both milling in the presence of 4% Pluronic F-87 then autoclaving in the presence of 25% Isovue.

SUBSTITUTE SHEET (RULE 2B Example 1. The Effect of Added Iohexol on the Stability to Heat Sterilization of Nanosuspensions of NC 67722.

A nanosuspension of 6-ethoxy-6-oxohexyl-3 , 5-bis (acetyl- amino) -2,4, 6-triiodobenzoate (NC 67722) as prepared in US 5,466,440, Example 10 was supplied at 36% NC 67722 and 7.2% F108P (wt/vol %) after media milling where F108P indicates F108 purified by diafiltration. This suspension was then diluted with stock solutions of either polyethylene glycol 1450 (PEG 1450) or iohexol so that the following formulations were prepared.

Sample %PFG 1450 % Iohexol

1 0 0

2 15 0

2a 15 15

3 15 15

4 0 5

5 0 10

6 0 15

7 0 20

8 0 30

9 0 40

All samples were diluted such that they were 15% NC 67722 and 3% F-108P in addition to the above concentrations of PEG 1450 and Iohexol. These samples were then heat sterilized by autoclaving under standard conditions (i.e., 121.1 °C, 15 min.) followed by assessment of the average particle size using the Horiba LA 910 (Irvine, CA) particle sizer and a volume weighted distribution.

The particle size of each suspension is listed below with the relevant concentrations of Iohexol and PEG 1450. It is clear that either the addition of Iohexol to the 15% PEG 1450 suspensions or the addition of Iohexol alone at or above 10% provides the necessary stability to the suspension so that heat sterilization can be survived.

Sample % PEG 1450 % Iohexol Osmolalitv Average Particle

(mOsm/kg) Size (nm)

Post Autoclave fstd dev

1 0 0 200 1648 (1502)

2 15 0 — 279 (53)

2a 15 15 603 260 (51)

3 15 20 705 254 (52)

4 0 5 72 437 (355)

5 0 10 163 272 (86)

6 0 15 160 302 (59)

7 0 20 230 253 (51)

7a 0 23 J 253 259 (49)

8 0 30 317 264 (50)

9 0 40 423 271 (51)

Example 2. Effect of the Substitution of Iohexol for PEG 1450 upon Lyophilization of Nanoparticle Suspensions .

A nanosuspension of NC 67722 was supplied at 36% NC 67722 and 7.2% F108 (wt/vol %) after media milling. This suspension was then diluted with stock solutions of either polyethylene glycol 1450 (PEG 1450) or iohexol so that the following formulations were prepared. % PEG 1450 Osmolality Average Particle Size(nm

Sample % Iohexol Post Autoclave Post LγQDhilizatiotr fstd dev) Cstd dev

1 0 0 100 1648 (1502) 21.8 um

2 15 0 289 276 (50) 239 (51)

3 15 15 603 260 (51) 2211 (3509)

3 15 20 705 254 (52) 282 (124)

4 0 5 72 437 (355) 4644 (9629)

5 0 10 163 272 (86) 767 (1146)

6 0 15 160 302 (59) 305 (100)

7 0 30 317 264 (50) 321 (128)

8 0 40 423 271 (51) 273 (101)

* Samples lyophilized under standard nanoparticle parameters. See text for details.

All samples were diluted such that they were 15% NC 67722 and 3% F-108P in addition to the above concentrations of PEG 1450 and Iohexol. These samples were then heat sterilized by autoclaving under standard conditions (i.e., 121°C, 15 min.) followed by assessment of the average particle size using the Horiba LA910 (Irvine, CA) particle sizer and a volume weighted distribution. Sample suspensions were then submitted for lyophilization using the same conditions originally optimized for NC 67722 (F-108P/PEG 1450) . The suspension of NC 67722 was frozen to -35°C at atmospheric pressure. This was then followed by warming to -18°C for 30 minutes which was followed by warming to -13°C under 0.6 mm of mercury of vacuum. These conditions were held for 40 hours. The material was then warmed at 0.5°C/hr to -5°C under 0.2 mm of mercury of vacuum followed by a rapid warming at l°C/hr to 0°C. Finally, the product was warmed to 25°C at 25°C/hr and released from the lyophilizer. The volume of water lost was determined by weight (i.e., starting vial weight - final lyophilized weight = weight water lost) . When lyophilization was completed, the samples were reconstituted by adding the weight of water lost back to the individual samples and vortexed until the suspension looked smooth and white. The average particle size obtained after reconstitution is listed above.

The data suggest that the amount of Iohexol required for successful lyophilization should preferably be greater than about 15%. Both average particle size and the standard deviation of the particle size distribution are impacted by increasing iohexol concentration. Further optimization will be required to result in an easily reconstituted formulation which can be stored as a lyophilized suspension, but the initial results support the ability of iohexol to afford this approach to formulation stabilization.

Example 3. Illustration of the Added Physical Stability due to the Presence of Iohexol in Nanoparticle Suspensions .

A series of nanoparticle suspensions of NC 67722 were prepared as 15% NC 67722, 3% F-108 and varying concentrations of iohexol . These suspensions were then centrifuged at 15,000 rpm (15,850 g) in a Beckman Microfuge E in 1.5 ml plastic centrifuge tubes for increasing amounts of time to see their relative resistance to settling. As table 1 shows below, increasing amounts of iohexol add physical stability to the suspensions in terms of retarding pellet formation during centrifugation at all time points considered. Table 1. Relative Sediment Height with Time at Various Concentrations of Iohexol for NC 67722 Nanoparticle Suspensions*

% Iohexol Time (sec)

Ii 30 45 60 120

0 0.80 0.50 0.33 0.20 0.20

5 0.80 0.60 0.45 0.30 0.20

10 0.85 0.70 0.60 0.30 0.20

30 0.95 0.90 0.85 0.75 0.45

40 1.00 1.00 0.90 0.80 0.60

* Fraction of height of sediment to total fluid height after centrifugation,- i.e., 1.00 = no sedimentation, 0.20 is near total sedimentation.

While not exactly duplicative of shelf stability processes, this aggressive sedimentation testing is predicted to mirror relative stability to settling upon the shelf. Thus, in addition to providing particle size stability during heat sterilization and affording lyophilizable suspensions, added iohexol also provides enhanced physical stability to settling upon prolonged standing.

Example 4. The Impact of Nonionic, Iodinated X-Ray Contrast Agents on the Heat Sterilization of NC 70146 Nanoparticles .

Enough benzoic acid, 3 , 5-bis (acetylamino) -2 , 4, 6-triiodo- 1- (ethoxy-carbonyl) pentyl ester (NC 70146) as prepared in Example 3 of US 5,525,328 was added to a 2 oz amber glass bottle such that the final concentration of agent in the suspension was 22.5% (wt/vol%) . 1 oz of 0.7 mm zirconium silicate milling media, having been preconditioned by rolling for 7 days in a dilute sodium dodecylsulfate solution followed by rinsing and was added to the bottle along with enough Pluronic F-108 and nanopure water to make the resulting suspension 4.5% (wt/vol%) in F-108. The Pluronic F-108 used in this suspension had been purified to remove the low molecular weight impurities often found in commercial samples of this material. After rolling at 100 to 108 rpm on a three tiered roller mill (US Stoneware, East Palestine, OH) for a total of 7 days, the suspension exhibited the following properties:

pH = 6.23

Mean Particle Size = 106 nm (std dev = 23 nm)

The effect of the addition of nonionic, iodinated x-ray contrast agents (XCAs) to the suspension in order to provide protection upon steam sterilization is given in the table below. Final concentrations of NC 70146 and F108 are 15% and 3% respectively (wt/vol%)

After Autoclaving

XCAs Concentration Avg Particle Size (nm) Std Dev fnm

Iohexol 23.3% 1 19 34 534

Iopamidol 25.0% 120 37 7.02

Iodixanol 16.7% 125 42 4.92

These data indicate that the nonionic, iodinated x-ray contrast agents examined in this case are very effective at stabilizing the average particle size of nanoparticle NC 70146 to heat sterilization. Example 5. The Impact of Nonionic, Iodinated X-Ray Contrast Agents on the Heat Sterilization of NC 68183 Nanoparticles .

Enough bis- [1- (ethoxycarbonyl) propyl] -2,4, 6-triiodo-5- acetylamino-isopthalate (NC 68183) as prepared in Example 6 of US 5,466,440 was added to a 2 oz amber glass bottle such that the final concentration of agent in the suspension was 22.5% (wt/vol%) . 1 oz of 0.7 mm zirconium silicate milling media, having been preconditioned by rolling for 7 days in a dilute sodium dodecylsulfate solution followed by rinsing, was added to the bottle along with enough Pluronic F108 and nanopure water to make the resulting suspension 4.5% (wt/vol%) in F-108. The Pluronic F-108 used in this suspension had been purified to remove the low molecular weight impurities often found in commercial samples of this material. After rolling at 100 to 108 rpm on a three tiered roller mill (US Stoneware, East Palestine, OH) for a total of 7 days, the suspension exhibited the following properties:

pH = 6.96

Mean Particle Size 103 nm (std dev = 24 nm)

The effect of the addition of nonionic, iodinated x-ray contrast agents (XCAs) to the suspension before steam sterilization is given in the table below:

After Autoclaving

XCAP. Concentration Avg Particle Size (nrn) Std Dev (m (wt./vol. %)

Iohexol 23.3% 309 153 6.25

Iopamidol 25.0% 110 35 7J7

Iodixanol 16.7% 41 um 37 um 5.86 These data indicate that one of the nonionic, iodinated x-ray contrast agents examined in this case is very effective at stabilizing the average particle size of nanoparticle NC 68183 to heat sterilization. It is interesting that the dimeric nonionic agent did not stabilize this nanoparticle suspension to any extent and indeed, may have accelerated the aggregation/particle growth of this agent during heat sterilization.

Example 6. The Impact of Nonionic, Iodinated X-Ray Contrast Agents on the Heat Sterilization of NC 70177 Nanoparticles .

Enough propane dioic acid, [ [3 , 5-bis (acetylamino) -2 , 4, 6- triiodobenzoyl] oxy] methyl-bis (1-methylethyl) ester (NC 70177) as prepared in Example 24 of WO 96/23524 was added to a 2 oz amber glass bottle such that the final concentration of agent in the suspension was 22.5% (wt/vol%) . 1 oz of 0.7 mm zirconium silicate milling media, having been preconditioned by rolling for 7 days in a dilute sodium dodecylsulfate solution followed by rinsing, was added to the bottle along with enough . Pluronic F108 and nanopure water to make the resulting suspension 4.5% (wt/vol%) in F-108. The Pluronic F-108 used in this suspension had been purified to remove the low molecular weight impurities often found in commercial samples of this material . After rolling at 100 to 108 rpm on a three tiered roller mill (US Stoneware, East Palestine, OH) for a total of 7 days, the suspension exhibited the following properties :

pH = 9.08

Mean Particle Size = 109 nm (std dev = 29 nm) The effect of the addition of nonionic, iodinated x-ray contrast agents (XCAs) to the suspension before steam sterilization is given in the table below.-

Avg Particle Size (nm)

XCAs Concentration After Autoclaving Std Dev Cnm

(wt./vol. %)

Iohexol 23.3% 127 49 7.38

Iopamidol 25.0% 125 48 7.55

Iodixanol 16.7% 141 59 6.98

These data indicate that the nonionic, iodinated x-ray contrast agents examined in this case are very effective at stabilizing the average particle size of nanoparticle NC 70177 to heat sterilization.

Example 7. The Impact of Nonionic, Iodinated X-Ray Contrast Agents on the Heat Sterilization of NC 8883 Nanoparticles .

Enough 3 , 5-bis (acetylamino) -2 , 4, 6 -triiodobenzoate (NC 8883) as prepared in Example 5 of US 5,466,440 was added to a 2 oz amber glass bottle such that the final concentration of agent in the suspension was 22.5% (wt/vol%) . 1 oz of 0.7 mm zirconium silicate milling media, having been preconditioned by rolling for 7 days in a dilute sodium dodecylsulfate solution followed by rinsing, was added to the bottle along with enough . Pluronic F108 and nanopure water to make the resulting suspension 4.5% (wt/vol%) in F-108. The Pluronic F-108 used in this suspension had been purified to remove the low molecular weight impurities often found in commercial samples of this material. After rolling at 100 to 108 rpm on a three tiered roller mill (US Stoneware, East Palestine, OH) for a total of 7 days, the suspension exhibited the following properties: pH = 6.85

Mean Particle Size = 101 nm (std dev = 18 nm)

The effect of the addition of nonionic, iodinated x-ray contrast agents (XCAs) to the suspension before steam sterilization is given in the table below:

After Autoclaving

XCAs Concentration Avg Particle Size (nm) Std Dev Cnm pH (wt./vol. %)

Iohexol 23.3% 150 77 6.67

Iopamidol 25.0% 206 102 7.13

Iodixanol 16.7% 209 107 6.22

These data indicate that the nonionic, iodinated X-ray contrast agents examined in this case are very effective at stabilizing the average particle size of nanoparticle NC 8883 to heat sterilization.

Example 8. The Impact of Nonionic, Iodinated X-Ray Contrast Agents on the Heat Sterilization of NC 67722 Nanoparticles .

A NC67722 nanoparticle suspension was received having been media milled at 36% (wt/vol %) in the presence of 6.6% Pluronic F-108. This Pluronic F-108 had been purified to remove the low molecular weight impurities often associated with commercially available material. Solutions of Iohexol, Iopamidol, and Iodixanol were either purchased or prepared to use as diluents for the NC 67722 suspension such that the final concentration of NC 67722 was 15% (wt/vol%) and the final concentration of Pluronic F-108 was 3% (wt/vol%) .

Mean Particle Size = 129, std dev. = 49, pH = 9.23 The effect of the addition of nonionic, iodinated x-ray contrast agents (XCAs) to the suspension before steam sterilization is given in the table below:

After Autoclaving

XCAs Concentration Av Particle Size (nm Std Dev (nm pH (wt./vol. %)

Iohexol 23.3% 259 77 6.44

Iopamidol 25.0% 259 49 7.39

Iodixanol 16.7% 294 98 6.60

These data indicate that the nonionic, iodinated x-ray contrast agents examined in this case are very effective at stabilizing the average particle size of nanoparticle NC 67722 to heat sterilization.

Example 9. The Impact of Nonionic, Iodinated X-Ray Contrast Agents on the Heat Sterilization of NC 68183 Nanoparticles Prepared with Various Pluronic Surfactants .

Suspensions of NC 68183 were prepared by adding enough NC 68183 to a 2 oz amber glass bottle such that the final concentration of agent in the suspension was 22.5% (wt/vol%) . 15 ml of 0.7 mm zirconium silicate milling media, having been preconditioned by rolling for 7 days in a dilute sodium dodecylsulfate solution followed by rinsing, was added to the bottle along with enough Pluronic surfactant and nanopure water (see below) to make the resulting suspension 6.0% (wt/vol%) in surfactant. Each suspension was rolled at 100 to 108 rpm on a three tiered roller mill (US Stoneware, East Palestine, OH) for a total of 4 days. The effect of adding nonionic, iodinated x-ray contrast agents to the suspension to provide particle size protection after autoclaving is given in the table below. The concentration of NC 68183 and Pluronic surfactant is 15% and 3% respectively in each case.

Suspension Particle Size (nm) After Autoclaving

Initial 15% PEG 1450 23% Iohexol 25% Iopamidol

(wt./vol. %) (wt./vol. %)

F-87 106 >4 microns 194 114

F-88 108 120 117 117

F-98 110 119 116 118

It would appear from these data that the iodinated x-ray contrast agents are as efficacious at preserving particle size as is PEG 1450 and in the case of F-87, even more so suggesting that the use of these agents may enable the use of F-87 as a stabilizer for this nanoparticle .

Example 10. The Impact of Nonionic, Iodinated X-Ray Contrast Agents on the Observed Cloud Point of Pluronic F-108.

A stock solution of Pluronic F-108 was prepared at a concentration of 3% (wt/vol%) . This was then placed into 10 ml crimp topped vials as 2 ml aliquots . Enough nonionic, iodinated x-ray contrast agent (i.e., iohexol, iodixanol, iopamidol) was added to the vials such that a concentration range of 10, 20, and 30% (wt/vol%) was achieved for each soluble contrast agent in the presence of 3% F-108. Each vial was crimped and heated in a bath of polyethylene glycol 400 while monitoring the temperature of a blank vial. The results show a moderate effect upon the cloud point of F-108 (Table 2 below) ,- however, in no case was the cloud point raised above the temperature conventionally used for steam sterilization (i.e., 121°c) . Thus, it does not appear that this mechanism is operative for these soluble, iodinated x-ray contrast agents as nanoparticle stabilizers for particle size during steam sterilization.

Table 2. Impact of Soluble, Iodinated X-ray Contrast

Agents upon the Cloud Point of Pluronic F-108

Concentration (wt./vol. %) Modifier 10% 20% 30%

Iohexol 96* 105 112

Iopamidol 90 104 104

Iodixanol 93 101 106

* cloud point in °C.

While there is some increase in cloud point, the increase never approaches that required for steam sterilization under conventional conditions of 121°c for 15 min. Thus, it does not appear that these soluble iodinated contrast agents protect the particle size of nanoparticle suspensions by modification of the suspension cloud point.

Example 11. Impact of the substitution of Iopamidol for PEG 1450 on the subcutaneous safety and imaging efficacy of a nanoparticle suspension of NC 70146 as an indirect lymphography agent .

Enough NC 70146 was added to a 2 oz amber glass bottle such that the final concentration of agent in the suspension was 22.5% (wt/vol%) . 1 oz of 0.7 mm zirconium silicate milling media, having been preconditioned by rolling for 7 days in a dilute sodium dodecylsulfate solution followed by rinsing and was added to the bottle along with enough Pluronic F-108 and nanopure water to make the resulting suspension 4.5% (wt/vol%) in F-108. The Pluronic F-108 used in this suspension had been purified to remove the low molecular weight impurities often found in commercial samples of this material. After rolling at 100 to 108 rpm on a three tiered roller mill (US Stoneware, East Palestine, OH) for a total of 7 days, the suspension exhibited the following properties:

pH = 6.23

Mean Particle Size = 106 nm (std dev = 23 nm)

The effect of the addition of nonionic, iodinated x-ray contrast agents (XCAs) to the suspension in order to provide protection upon steam sterilization is given in the table below. Final concentrations of NC 70146 and F108 are 15% and 3% respectively (wt/vol%) .

The nanoparticle suspension of NC 70146 in Pluronic F108 (Purified) with iopamidol as the sterilization stabilizer was tested subcutaneously in rats at a dose of 5 ml/kg injected bilaterally in the scapular region. The injection site was monitored for edema and erythema and animals were sacrificed at days 3 and 15 post injection to assess effects at the injection site itself. While modest acute inflammation associated with the normal response of the body to foreign objects was observed at day 3 post injection, the test article and any histopathological effects were cleared by day 15 post injection. This compares very favorably with conventional formulations for this indication (i.e., NC 67722/F108/PEG 1450) and indicates that the use of iopamidol as the sterilization stabilizer is not associated with any significant safety liabilities relative to the use of PEG 1450. The nanoparticle suspension of NC 70146 with Pluronic F108 (purified) was submitted for an imaging study in rabbits at the Center for Imaging and Pharmaceutical Research (CIPR) , Massachusetts General Hospital, Charlestown Navy Yard, Charlestown, MA, 02129. Using a standard imaging protocol, the suspension was injected as 2 x .25 ml per paw of the rabbit with CT imaging at 2.5 hr, 24 hr, and 7 days post injection. Quantitative assessments of the imaging results are shown in the table below.

CT Lymphography in rabbits with NC 70146 and PEG1450 vs. NC 70146 and Iopamidol

Formulation Time Post Injection

2.5 hr. 4 hr. 7 days

15% PEG1450

Axillary 4.2(0.4)* 4.9(0.3) 4.4(0.4) Popliteal 4.0(0.7) 4.5(0.5) 3.6(0.3)

15% Iopamidol

Axillary 5.3(1.0) 6.9(1.0) 5.2(0.8) Popliteal 4.6(0.2) 7.4(1.4) 5.3(0.6)

* Values given in Hounsfield Units (HU) , indicating the X-ray enhancement of the node expressed as mg I/ml in the lymph node vs . Nal standards .

Thus, the use of iopamidol rather than PEG 1450 does not alter the imaging response thereby confirming that the use of iopamidol does not impact the efficacy of these nanoparticle agents. In addition, the visual estimation of node to node transport remains excellent with the iopamidol; further supporting the notion that these sterilization stabilizers do not impact the particles per se (i.e., either efficacy or safety) but rather modify the solution properties such that the particles survive autoclaving.

These data indicate that the nonionic, iodinated x-ray contrast agents examined in this case are very effective at stabilizing the average particle size of nanoparticle NC 70146 to heat sterilization.

Claims

Claims

1. A diagnostic or therapeutic composition comprising therapeutically or diagnostically effective nanoparticles, a physiologically tolerable surfactant and a physiologically tolerable water-soluble, iodinated X-ray contrast agent.

2. A composition as claimed in claim 1 wherein said X- ray contrast agent is iohexol, ioversol, iopamidol, iopentol, iodixanol, iopromide, iomeprol , ioxilan, iosimide, metrizamide, iotasol and iotrolan.

3. A composition as claimed in claim 2 wherein said X- ray contrast agent is iohexol, iopamidol, iopentol, ioxilan and iodixanol .

4. A composition as claimed in claim 3 wherein said X- ray contrast agent is iohexol .

5. A composition as claimed in any one of claims 1 to

4 wherein the amount of X-ray contrast agent present in the composition is in the range 5 to 40% by weight relative to the weight of the aqueous phase.

6. A composition as claimed in claim 5 wherein the amount of X-ray contrast agent present in the composition is in excess of 20% by weight relative to the weight of the aqueous phase .

7. A composition as claimed in any one of claims 1 to

6 wherein said surfactant is a poloxamer, poloxamine, tyloxapol, polyvinylpyrrolidone, P-79 or PEG-modified phospholipid.

8. A composition as claimed in any one of claims 1 to

7 wherein the amount of surfactant present in the composition is in the range 0.1 to 10% by weight.

9. A composition as claimed in any one of claims 1 to

8 wherein said nanoparticle is a sparingly soluble or insoluble iodinated X-ray contrast agent.

10. A composition as claimed in any one of claims 1 to

9 wherein said nanoparticles are present in an amount of from 0.1 to 45% by weight.

11. A composition as claimed in any one of claims 1 to

12 wherein said nanoparticles are present in an amount of from 1 to 30% by weight.

12. A process for the preparation of a sterile therapeutic or diagnostic composition as claimed in any one of claims 1 to 11, said process comprising thermally sterilising a composition comprising therapeutically or diagnostically effective nanoparticles, a physiologically tolerable surfactant and a physiologically tolerable water-soluble, iodinated X-ray contrast agent.

13. The process as claimed in claim 12 wherein said thermal sterilisation is carried out by steam sterilisation at at least 121┬░C.

14. The use of a physiologically tolerable water- soluble iodinated X-ray contrast agent for the manufacture of a therapeutic or diagnostic composition as claimed in any one of claims 1 to 11 for use in a method of therapy or diagnosis.

15. A method of therapy or diagnosis of a human or non- human animal body, said method comprising administering to said body a therapeutically or diagnostically effective amount of a composition as claimed in any one of claims 1 to 11 .

16. A method of generating an image of a human or non- human animal body, said method comprising administering to said body a diagnostically effective amount of a composition as claimed in any one of claims 1 to 11.

17. A method of enhancing the physical stability of a diagnostic or therapeutic composition, comprising therapeutically or diagnostically effective nanoparticles, to settling during storage comprising administering to said composition a physiologically tolerable water-soluble, iodinated X-ray contrast agent