Light imaging contrast agents

The present invention relates to the use of particulate materials as contrast agents in vivo light imaging.

FIELD OF THE INVENTION 
The present invention relates to the use of particulate contrast agents in 
various diagnostic imaging techniques based on light, more particularly to 
particulate light imaging contrast agents. 
BACKGROUND OF THE INVENTION 
Contrast agents are employed to effect image enhancement in a variety of 
fields of diagnostic imaging, the most important of these being X-ray, 
magnetic resonance imaging (MRI), ultrasound imaging and nuclear medicine. 
Other medical imaging modalities in development or in clinical use today 
include magnetic source imaging and applied potential tomography. The 
history of development of X-ray contrast agents is almost 100 years old. 
The X-ray contrast agents in clinical use today include various 
water-soluble iodinated aromatic compounds comprising three or six iodine 
atoms per molecule. The compounds can be charged (in the form of a 
physiologically acceptable salt) or non-ionic. The most popular agents 
today are non-ionic substances because extensive studies have proven that 
non-ionic agents are much safer than ionics. This has to do with the 
osmotic loading of the patient. In addition to water-soluble iodinated 
agents, barium sulphate is still frequently used for X-ray examination of 
the gastrointestinal system. Several water-insoluble or particulate agents 
have been suggested as parenteral X-ray contrast agents, mainly for liver 
or lymphatic system imaging. Typical particulate X-ray contrast agents for 
parenteral administration include for example suspensions of solid 
iodinated particles, suspensions of liposomes containing water-soluble 
iodinated agents or emulsions of iodinated oils. 
The current MRI contrast agents generally comprise paramagnetic substances 
or substances containing particles (hereinafter "magnetic particles") 
exhibiting ferromagnetic, ferrimagnetic or superparamagnetic behaviour. 
Paramagnetic MRI contrast agents can for example be transition metal 
chelates and lanthanide chelates like Mn EDTA and Gd DTPA. Today, several 
gadolinium based agents are in clinical use; including for example Gd DTPA 
(Magnevist.RTM.), Gd DTPA-BMA (Omniscan.RTM.), Gd DOTA (Dotarem.RTM.) and 
Gd HPDO3A (Prohance.RTM.). Several particulate paramagnetic agents have 
been suggested for liver MRI diagnosis; for example suspensions of 
liposomes containing paramagnetic chelates and suspensions of paramagnetic 
solid particles like for example gadolinium starch microspheres. Magnetic 
particles proposed for use as MR contrast agents are water-insoluble 
substances such as Fe.sub.3 O.sub.4 or .delta.-Fe.sub.2 O.sub.3 optionally 
provided with a coating or carrier matrix. Such substances are very active 
MR contrast agents and are administered in the form of a physiologically 
acceptable suspension. 
Contrast agents for ultrasound contrast media generally comprise 
suspensions of free or encapsulated gas bubbles. The gas can be any 
acceptable gas for example air, nitrogen or a perfluorocarbon. Typical 
encapsulation materials are carbohydrate matrices (e.g. Echovist.RTM. and 
Levovist.RTM.), proteins (e.g. Albunex.RTM.), lipid matrials like 
phospholipids (gas-containing liposomes) and synthetic polymers. 
Markers for diagnostic nuclear medicine like scintigraphy generally 
comprise radioactive elements like for example technetium (99m) and indium 
(III), presented in the form of a chelate complex, whilst 
lymphoscintigraphy is carried out with radiolabelled technetium sulphur 
colloids and technetium oxide colloids. 
The term "light imaging" used here includes a wide area of applications, 
all of which utilize an illumination source in the UV, visible or IR 
regions of the electromagnetic spectrum. In light imaging, the light, 
which is transmitted through, scattered by or reflected (or re-emitted in 
the case of fluorescence) from the body, is detected and an image is 
directly or indirectly generated. Light may interact with matter to change 
its direction of propagation without significantly altering its energy. 
This process is called elastic scattering. Elastic scattering of light by 
soft tissues is associated with microscopic variations in the tissue 
dielectric constant. The probability that light of a given wavelength 
(.lambda.) will be scattered per unit length of travel in tissue is termed 
the (linear) scattering coefficient .mu..sub.s. The scattering coefficient 
of soft tissue in an optical window of approx. 600-1300 nm ranges from 
10.sup.1 -10.sup.3 cm.sup.-1 and decreases as 1/.lambda.. In this range 
.mu..sub.s &gt;&gt;.mu..sub.a (the absorption coefficient) and although 
.mu..sub.s (and the total attenuation) is very large, forward scattering 
gives rise to substantial penetration of light into tissue. Ballistic 
light is light that has travelled through a region of tissue without being 
scattered. Quasi-ballistic light ("snake" light) is scattered light that 
has maintained approximately the same direction of travel. The effective 
penetration depth shows a slow increase or is essentially constant with 
increasing wavelengths above 630 nm (although a slight dip is observed at 
the water absorption peak at 975 nm). The scattering coefficient shows 
only a gradual decrease with increasing wavelength. 
Light that is scattered can either be randomly dispersed (isotropic) or can 
scatter in a particular direction with minimum dispersion (anisotropic) 
away from the site of scattering. For convenience and mathematical 
modelling purposes, scattering in tissue is assumed to occur at discrete, 
independent scattering centers ("particles"). In scattering from such 
"particles", the scattering coefficient and the mean cosine of scatter 
(phase function) depend on the difference in refractive index between the 
particle and its surrounding medium and on the ratio of particle size to 
wavelength. Scattering of light by particles that are smaller than the 
wavelength of the incident light is called Rayleigh scattering. This 
scattering varies as 1/.lambda..sup.4 and the scattering is roughly 
isotropic. Scattering of light by particles comparable to or larger than 
the wavelength of light is referred to as Mie scattering. This scattering 
varies as 1/.lambda. and the scattering is anisotropic (forward peaked). 
In the visible/near-IR where most measurements have been made, the 
observed scattering in tissue is consistent with Mie-like scattering by 
particles of micron scale: e.g. cells and major organelles. 
Since the scattering coefficient is so large for light wavelengths in the 
optical window (600-1300 nm), the average distance travelled by a photon 
before a scattering event occurs is only 10-100 .mu.m. This suggests that 
photons that penetrate any significant distance into tissue encounter 
multiple scattering events. The ballistic component of light that has 
travelled several centimeters through tissue is exceedingly small. 
Multiple scattering in tissue means that the true optical path length is 
much greater than the physical distance between the light input and output 
sites. The scattering acts, therefore, to diffuse light in tissue 
(diffuse-transmission and -reflection). The difficulty that multiple 
scattering presents to imaging is three-fold: (i) light that has been 
randomized due to multiple scattering has lost signal information and 
contributes noise to the image (scattering increases noise); (ii) 
scattering keeps light within tissue for a greater period of time, 
increasing the probability for absorption, so less light transmits through 
tissue for detection (scattering decreases signal); and (iii) the 
determination of physical properties of tissue (or contrast media) such as 
concentration that could be obtained from the Beer-Lambert law is 
complicated since the true optical path length due to scattering is 
difficult to determine (scattering complicates the quantification of light 
interactions in tissue). However, although light cannot penetrate more 
than a few tens of microns in tissue without being scattered, the large 
value of the mean cosine of scattering indicates that a significant 
fraction of photons in an incident beam may undergo a large number of 
scatters without being deviated far from the original optical axis, and as 
such can contribute in creating an image. As a result, it can be possible 
to perform imaging on tissue despite the predominance of scatter, if the 
noise component can be rejected and the quasi-ballistic component of the 
light can be detected. 
The most interesting wavelengths for light imaging techniques are in the 
approximate range of 600-1300 nm. These wavelengths have the ability to 
penetrate relatively deeply into living tissue without absorption by 
natural substances and furthermore are harmless to the human body. 
However, for optical analysis of surface structures or diagnosis of 
diseases very close to the body surface or body cavity surfaces or lumens, 
UV light and visible light below 600 nm wavelength can also be used. 
Light can also be used in therapy; thus for example in Photodynamic Therapy 
(PDT) photons are absorbed and the energy is transformed into heat and/or 
photochemical reactions which can be used in cancer therapy. 
The main methods of light imaging today include simple transillumination, 
various tomographic techniques, fluorescence imaging, and hybrid methods 
that involve irradiation with or detection of other forms of radiation or 
energy in conjunction with irradiation with or detection of light (such as 
photoacoustic or acousto-optical). These methods take advantage of either 
transmitted, scattered or emitted (fluorescence) photons or a combination 
of these effects. The present invention relates to contrast agents for any 
of these and further imaging methods based on any form of light. 
There is today great interest in development of new equipment for imaging 
based on light. Interesting methods are especially the various types of 
tomographic techniques in development especially in Japan. As scientific 
references to the use of light in diagnostic medicine and PDT see for 
example Henderson, B. and Dougherty, T. in Photodynamic Therapy. Basic 
Principles and Clinical Application (1992), Gonzalez, E. et al. in 
Psoriasis (1991) 519 and 603, Scrip 1815 (1993) 25, Andersson-Engeles, S. 
et al. in Lasers in Medical Science 4 (1988) 115, Andersson, P. et al. in 
IEEE 23 (1987) 1798, Andersson, P. et al. in Lasers in Medical Science 2 
(1987) 261, Anderson, R. et al. in Applied Optics 28 (1989) 2256, Amato, 
I., in Science 260 (1993) 1234, Alfano, R. et al. in IEEE 20 (1984) 1512, 
Lipowska, M. et al. at ACS Nat Meeting (1991), Clinica 528 (1992) 17, 
Andersson-Engles, S. et al. in Optics Letter 15 (1990) 1179, 
Andersson-Engles, S. et al. in Lasers in Medical Science 4 (1989) 241, 
Andersson-Engles, S. et al. in Lasers in Medical Science 4 (1989) 171, 
Andersson-Engels, S. et al. in IEEE 26 (1990) 2207, Andersson-Engels, S. 
et al. in Photochem and Photobiol 53 (1991) 807, Andersson-Engels, S., et 
al. in SPIE 908 (1988) 116, Andersson-Engels, S. et al. in SPIE 1205 
(1990) 179, Andersson-Engels, S. et al. in Anal. Chem 61 (1989) 1367 and 
in Anal. Chem 62 (1990) 19, Andreoni, A. in Photochem and Photobiol 52 
(1990) 423, Ankerst, J. et al. in Appl. Spectroscopy 38 (1984) 890, 
Anthony, D. et al. in Photochem and Photobiol. 49 (1989) 583, Araki, R. et 
al. in Time Resolved Spectroscopy and Imaging of Tissues 1431 (1991) 321, 
Arnfield, M. et al. in Photochem and Photobiol 51 (1990) 667, Arridge, S. 
et al. in Phys. Med. Biol 37 (1992) 1531, Arridge, S. et al. in SPIE 1431 
(1991) 204, Balzani, S. et al. in Photochem and Photobiol 52 (1990) 409, 
Barabash, R. et al. in IEEE 26 (1990) 2226, Barker, D. et al. in Br. J. 
Exp. Path 51 (1970) 628, Baum, R. in C&EN Oct. 31 (1988) 18, Baumgartner, 
R. et al. in Photochem and Photobiol 46 (1987) 759, Benaron, D. et al. in 
Science 259 (1993) 1463, Benson, R. et al. in J. Chem. Eng. Data 22 (1977) 
379, Bickers, D. in Invest Radiol 21 (1986) 885, Blasdel, G. et al. in 
Nature 321 (1986) 579, Blasse, G. in Photochem and Photobiol 52 (1990) 
417, Bolin, F. et al. in Appl. Optics 28 (1989) 2297, Boulnois, J. in 
Lasers in Medical Science 1 (1985) 47, Brodbeck, K. et al. in Med. Phys 14 
(1987) 637, Carney, J. et al. in Anal. Chem 65 (1993) 1305, Chan, W. et 
al. in Photochem and Photobiol 50 (1989) 617, Chance, B. in SPIE 1641 
(1992) 162, Chance, B. et al. in SPIE 1204 (1992) 481, Cheong, W. et al. 
in IEEE 26 (1990) 2166, Cope, M. et al. in SPIE 1431 (1991) 251, 
Deckelbaum, L. et al. in Lasers in Surgery and Medicine 7 (1987) 330, 
Delpy, D. et al. in Phys Med Biol 33 (1988) 1433, Detty, M. et al. in JACS 
112 (1990) 3845, Detty, M. et al. in J. Med. Chem 33 (1990) 1108, Doiron, 
D. in Prog. in Clin & Biol. Res 170 (1984) 41, Driver, I. in Phys. Med. 
Biol 36 (1991) 805, Feather, J. et al. in Lasers in Medical Science 5 
(1990) 345, Fishkin, J. et al. in SPIE 1431 (1991) 122, Flock, S. in Med. 
Phys. 14 (1987) 835, Gathje, J. et al. in Applied Physiology 29 (1970) 
181, Gomer, C. et al. in Cancer Research 50 (1990) 3985, Grunbaum, F. et 
al. in SPIE 1431 (1991) 1431, Haglund, M. et al. in Nature 358 (1992) 668, 
Hebden, J. et al. in Am. Assoc. Phys. Med. 19 (1992) 1081, Hebden, J. et 
al. in Med. Phys. 17 (1990) 41, Hoek, A. V. et al. in IEEE 23 (1987) 1812, 
Hohla, K. et al. in SPIE 908 (1988) 128, Holbrooke, D. et al. in Proc. 
N.Y. Ac Sci 267 (1976) 295, Hoyt, C. et al. in Lasers in Surgery and 
Medicine 8 (1988) 1, Huang, D. et al. in Science 254 (1991) 1178, Jacson, 
P. et al. in Invest Radiol. 20 (1985) 226, Jacques, S. et al. in Lasers in 
the Life Science 1 (1987) 309, Kittrell, C. et al. in Applied Optics 24 
(1985) 2280, Lakowicz, J. in Biophys J. 46 (1984) 463, Lam, S. et al. in 
Chest 97 (1990) 333, Li, W. in Opthalmology 100 (1982) 484, Lilge, L. et 
al. in SPIE 1203 (1990) 106, Lytle, A. et al. in SPIE 1200 (1990) 466, 
MacVicar, B. et al. in J. Neuroscience 11 (1991) 1458, Maijnissen, J. et 
al. in Lasers in Surgery and Medicine 2 (1987) 235, Marynissen, J. et al. 
in J. Urology 142 (1989) 1351 and Prog. in Clin & BioRes 170 (1984) 133. 
McCormick, P. et al. in Critical Care Medicine 19 (1991) 89, Mcormick, P. 
et al. in J. Neurosurg 76 (1992) 315, McKenzie, A. et al. in Phys. Med. 
Biol. 30 (1985) 455, Moes, C. et al. in Applied Optics 28 (1989) 2292, 
Montan, S. et al. in Optics Letters 10 (1985) 56, Montforts, F. et al. in 
Angev Chem (Int. Ed.) 31 (1992) 1592, Morgan, A. et al. in Photochem and 
Photobiol 52 (1990) 987, Morgan, A. et al. in J. Med. Chem 33 (1990) 1258, 
Morgan, A. et al. in J. Med. Chem 32 (1989) 904, Navarro, G. et al. in 
Med. Phys. 15 (1988) 181, Nelson, J. et al. in Cancer Research 4 (1987) 
4681, Orbach, H. et al. i J. Neuroscience 5 (1985) 1886, Pandey, R. et al. 
in Chem. Lett. 2 (1992) 491, Parker, F. et al. in Analyt. Biochem 18 
(1967) 414, Parrish, J. in J. Derm 17 (1990) 587, Parrish, J. et al. in 
Photochem and Photobiol 53 (1991) 731, Patterson, M. et al. in in Appl. 
Optics 28 (1989) 2331. Patterson, M. et al. in SPIE 1203 (1990) 62, 
Patterson, M. et al. in SPIE 1065 (1989) 115, Patterson, M. et al. in 
Photosenziation 15 (1988) 121, Patterson, M. et al. in Lasers in Medical 
Science 6 (1991) 379, Peters, V. et al. in Phys. Med. Biol 35 (1990) 1317, 
Profio, A. in Photochem. and Photobiol 46 (1987) 591, Profio, A. et al. in 
Med. Phys 11 (1984) 516, Prout, G. et al. in New England Journal of 
Medicine 317 (1987) 1251, Roberts, W. et al. in J. Nat Cancer Inst 80 
(1988) 330, Rosenthal, I in Photochem and Photobiol 53 (1991) 559, 
Sartori, M. et al. in IEEE 23 (1987) 1794, Schmitt, J. in Applied Optics 
31 (1992) 6535, Schmitt, J. et al. in Opt. Soc. Am. 7 (1990) 2141, 
Schneckenburger, H. et al. in Optical Engineering 31 (1992) 1447, Selman, 
S. et al. in SPIE 997 (1988) 12, Selman, S. et al. in J. Urology 143 
(1990) 630, Sevick, E. et al. in Anal. Biochem 195 (1991) 330, Shen, N. et 
al. in Pharmacologica Sinia 4 (1986) 346, Shiner, W. et al. in Photonics 
Spectra September (1992) 109, Spears, K. et al. in IEEE 36 (1989) 1210, 
Spikes, J. in Photochem and Photobiol 43 (1986) 691, Star, W. et al. in 
Lasers in Medical Science 5 (1990) 107, Star, W. et al. in Photochem and 
Photobiol 46 (1987) 619, Steike, J. et al. in J. Opt. Soc. Am. 6 (1988) 
813, Stekeil, W. in J. Physiol 198 (1960) 881, Sullivan, F. et al. in 
Applied Radiology January (1993) 26, Svaasand, L. et al. in Med. Phys 12 
(1985) 455, Svaasand, L. et al. in Lasers in Medical Science 5 (1985) 589, 
Svaasand, L. et al. in Photochem and Photobiol 41 (1985) 73, Svaasand, L. 
et al. in Photochem and Photobiol 38 (1983) 293, Svanberg, K. et al. in 
Cancer Research 46 (1986) 3803, Svanberg, K. et al. in Physica Scripta 26 
(1989) 90, Tam, A. in Ultrasensitive Laser Spectroscopy (1983) 72, Toida, 
M. et al. in Electronics and Communications in Japan 75 (1992) 137, Tsay, 
T. et al. in SPIE 1646 (1992) 213, Tsuchiya, A. et al. in J. Urology 130 
(1983) 79, Unsold, E. et al. in Lasers in Medical Science 5 (1990) 207, 
Vergara, J. et al. in Biophysical Journal 59 (1991) 12, Vitkin, I. et al. 
in J. Photochem Photobiol 16 (1992) 235, Wang, L. et al. in Optics & 
Photonics 2 (1991) 38, Wang, L. et al. in Science 253 (1991) 769, Wang, L. 
et al. in SPIE 1431 (1991) 97, Watmough, D. in Brit. J. Radiology 55 
(1982), Wilson, B. et al. in Phys Med Biol 31 (1986) 327, Wilson, B. et 
al. Lasers in Medical Science 1 (1986) 235, Wyatt, J. et al. in J. Appl. 
Physiol 68 (1990) 1086, Wyatt, J. et al. in Archives of Disease in 
Childhood 64 (1989) 953, Yoo, K. et al. in Optics Letters 16 (1991) 1068, 
Yoo, K. et al. in Optics Letters 15 (1990) 320. 
There are several patent publications which relate to light imaging 
technology and to the use of various dyes in light imaging: a labeling 
fluorescent dye comprising hydroxy aluminium 2,3-pyrido cyanide in JP 
4,320,456 (Hitachi Chem), therapeutic and diagnostic agent for tumors 
containing fluorescent labelled phthalocyanine pigment in JP 4288 022 
(Hitachi Chem), detection of cancer tissue using visible native 
luminescence in U.S. Pat. No. 4,930,516 (Alfano R. et al.), method and 
apparatus for detection of cancer tissue using native fluorescence in U.S. 
Pat. No. 5,131,398 (Alfano, R. et al.), improvements in diagnosis by means 
of fluorescenct light emmision from tissue in WO 90/10219 
(Andersson-Engels, S. et al.), fluorescent porphyrin and fluorescent 
phthalocyanine-polyethylene glycol, polyol, and saccharide derivatives as 
fluorescent probes in WO91/18006 (Diatron Corp), method of imaging a 
random medium in U.S. Pat. No. 5,137,355 (State Univ. of New York), 
tetrapyrrole therapeutic agents in U.S. Pat. No. 5,066,274 (Nippon 
Petrochemicals), tetrapyrrole polyaminomonocarboxylic acid in therapeutic 
agents in U.S. Pat. No. 4,977,177 (Nippon Petrochemicals), tetrapyrrole 
aminocarboxylic acids in U.S. Pat. No. 5,004,811 (Nippon Petrochemicals), 
porphyrins and cancer treatment in U.S. Pat. No. 5,162,519 (Efamol 
Holdings), dihydroporphyrins and method of treating tumors susceptible to 
necrosis in U.S. Pat. No. 4,837,221 (Efamol), parenterally administered 
zinc phthalocyanide compounds in form of liposome dispersion containing 
synthetic phospholipids in EP 451 103 (CIBA Geigy), apparatus and method 
for detecting tumors in U.S. Pat. No. 4,515,165 (Energy Conversion 
Devices), time and frequency domain spectroscopy determining hypoxia in 
WO92/13598 (Nim Inc), phthalocyanatopolyethylene glycol and phthalocyanato 
saccharides as fluorescent digoxin reagent in WO 91/18007 (Diatron), 
fluorometer in U.S. Pat. No. 4,877,965 (Diatron), fiberoptic fluorescence 
spectrometer in WO 90/00035 (Yale Univ.), tissue oxygen measuring system 
in EP 502,270 (Hamamatsu Photonics), method for determining bilirubin 
concentration from skin reflectance in U.S. Pat. No. 4,029,084 (Purdue 
Research Foundation), bacteriochlorophyll-a derivative useful in 
photodynamic therapy in U.S. Pat. No. 5,173,504 (Health Research Inc), 
purified hematoporphyrin dimers and trimers useful in photodynamic therapy 
in U.S. Pat. No. 5,190,966 (Health Research Inc), drugs comprising 
porphyrins in U.S. Pat. No. 5,028,621 (Health Research Inc), hemoporphyrin 
derivatives and process of preparing in U.S. Pat. No. 4,866,168 (Health 
Research Inc), method to destroy or impair target cells in U.S. Pat. No. 
5,145,863 (Health Research Inc), method to diagnose the presence or 
absence of tumor tissue in U.S. Pat. No. 5,015,463 (Health Research Inc), 
photodynamic therapeutic technique in U.S. Pat. No. 4,957,481 (U.S. 
Bioscience), apparatus for examining living tissue in U.S. Pat. No. 
2,437,916 (Philip Morris and Company), transillumination method apparatus 
for the diagnosis of breast tumors and other breast lesions by 
normalization of an electronic image of the breast in U.S. Pat. No. 
5,079,698 (Advanced Light Imaging Technologies), tricarbocyanine infrared 
absorbing dyes in U.S. Pat. No. 2,895,955 (Eastman Kodak), optical imaging 
system for neurosurgery in CA 2,048,697 (Univ. Techn. Int.), new porphyrin 
derivatives and their metallic complexes as photosensitizer for PDT in 
diagnosis and/or treatment of cancer in JP 323,597 (Hogyo,T), light 
receiving system of heterodyne detection and image forming device for 
light transmission image in EP 445,293 (Research Development Corp. of 
Japan), light receiving system of heterodyne detection and image forming 
device for light transmission image using light receiving system in WO 
91/05239 (Research Development Corp. of Japan), storage-stable porphyrin 
compositions and a method for their manufacture in U.S. Pat. No. 4,882,234 
(Healux), method for optically measuring chemical analytes in WO 92/19957 
(Univ. of Maryland at Baltimore), wavelength-specific cytotoxic agents in 
U.S. Pat. No. 4,883,790 (Univ. of British Columbia), 
hydro-monobenzo-porphyrin wavelength-specific cytotoxic agents in U.S. 
Pat. No. 4,920,143 (Univ. of British Columbia), apparatus and method for 
quantitative examination and high-resolution imaging of human tissue in EP 
447,708 (Haidien Longxing Med Co), optical imaging system for neurosurgery 
in U.S. application Ser. No. 7,565,454 (University Technologies Int. 
Inc.), --characterization of specific drug receptors with fluorescent 
ligands in WO 93/03382 (Pharmaceutical Discovery Corp), 
4,7-dichlorofluorescein dyes as molecular probes in U.S. Pat. No. 
5,188,934 (Applied Biosystems), high resolution breast imaging device 
utilizing non-ionizing radiation of narrow spectral bandwith in U.S. Pat. 
No. 4,649,275 (Nelson, R. et al.), 
meso-tetraphenyl-porphyrin-Komplexverbindungen, Verfaren zu ihrer 
Herstellung und Diese Enthaltends Pharmazeutische Mittel in EP 336,879 
(Schering), 13,17-propionsaure und propionsaurederivat Substituerte 
Porphyrin-Komplexverbindungen, Verfahren zu ihrer Herstellung und diese 
Enthaltende Pharmazeutische Mittel in EP 355,041 (Schering), 
photosensitizing agents in U.S. Pat. No. 5,093,349 (Health Research), 
pyropheophorbides and their use in photodynamic therapy in U.S. Pat. No. 
5,198,460 (Health Research), optical histochemical analysis, in vivo 
detection and real-time guidance for ablation of abnormal tissues using 
Raman spectroscopic detection system in WO 93/03672 (Redd, D.), 
tetrabenztriazaporphyrin reagents and kits containing the same in U.S. 
Pat. No. 5,135,717 (British Technology Group), system and method for 
localization of functional activity in the human brain in U.S. Pat. No. 
5,198,977 (Salb, J.). photodynamic activity of sapphyrins in U.S. Pat. No. 
5,120,411 (Board of Regents, University of Texas), process for preparation 
of expanded porphyrins in U.S. Pat. No. 5,152,509 (Board of Regents, 
University of Texas), expanded porphyrins (Board of Regents, University of 
Texas), infrared radiation imaging system and method in WO 88/01485 
(Singer Imaging), imaging using scattered and diffused radiation in WO 
91/07655 (Singer Imaging), diagnostic apparatus for intrinsic fluorescence 
of malignant tumor in U.S. Pat. No. 4,957,114, indacene compounds and 
methods for using the same in U.S. Pat. No. 5,189,029 (Bo-Dekk Ventures), 
method of using 5,10,15,20-tetrakis (carboxy phenyl) porphine for 
detecting cancers of the lung in U.S. Pat. No. 5,162,231 (Cole, D. A. et 
al.), Verfahren zur Abbildung eines Gewebebereiches in DE 4327 798 
(Siemens), chlorophyll and bacteriochlorophyll derivatives, their 
preparation and pharmaceutical compositions comprising them in EPO 584 552 
(Yeda Research and Development Company), wavelength-specific 
photosensitive porphacyanine and expanded porphyrin-like compounds and 
methods for preparation and use thereof in WO 94/10172 (Qudra Logic 
Technologies), method and apparatus for improving the signal to noise 
ratio of an image formed of an object hidden in or behind a semiopaque 
random media in U.S. Pat. No. 5,140,463 (Yoo, K. M. et al.), 
benzoporphyrin derivatives for photodynamic therapy in U.S. Pat. No. 
5,214,036 (University of British Columbia), fluorescence diagnostics of 
cancer using delta-amino levulinic acid in WO 93/13403 (Svanberg et al.), 
Verfahren zum Diagnostizieren von mit fluoreszierenden Substansen 
angereicherten, inbesondere tumorosen Gewebebereichen in DE 4136 769 
(Humboldt Universitat), terpyridine derivatives in WO 90/00550 (Wallac). 
All the light imaging dyes or contrast agents described in the 
state-of-the-art have different properties, but all those agents have an 
effect on the incident light, leading to either absorption and/or 
fluorescence. However none of these contrast agents is used as a 
particulate contrast agent. 
SUMMARY OF THE INVENTION 
We have now found that contrast enhancement may be achieved particularly 
efficiently in light imaging methods by introducing particulate materials 
as scattering contrast agents. For the sake of clarity, the word 
"particle" is used to refer to any physiologically acceptable particulate 
materials. Such particles may be solid (e.g. coated or uncoated 
crystalline materials) or fluid (e.g. liquid particles in an emulsion) or 
may be aggregates (e.g. fluid containing liposomes). Particulate material 
with a particle size smaller than or similar to the incident light 
wavelength are preferred. 
Thus viewed from one aspect the invention provides the use of a 
physiologically tolerable particulate material for the manufacture of a 
particulate-contrast-agent containing contrast medium for use in in vivo 
diagnostic light imaging. 
Viewed from a further aspect the invention also provides a method of 
generating an image of the human or non-human (preferably mammalian, avian 
or reptilian) animal body by light imaging, characterised in that a 
contrast effective amount of a physiologically tolerable particulate 
contrast agent is administered to said body, and an image of at least part 
of said body is generated. In such a method a contrast effective amount of 
the particulate agent is administered, e.g. parenterally or into an 
externally voiding body organ or duct, light emitted, transmitted or 
scattered by the body is detected and an image is generated of at least 
part of the body in which the contrast agent is present. Hybrid methods in 
which light, either alone or in conjunction with other forms of radiation, 
is administered to the body, and light, or some other form of radiation, 
is detected. In particular, the other form of radiation may be ultrasound.

DETAILED DESCRIPTION OF THE INVENTION 
The particles used according to the invention are preferably 
water-insoluble or at least sufficiently poorly soluble as to retain their 
desired particle size (e.g. 600-1300 nm) for at least 2 hours following 
administration into the body under investigation. 
The images generated may be spatial or temporal and mono- or 
multi-dimensional. 
In a further aspect of the invention, the imaging technique may be used to 
determine a value for a parameter characteristic of the body or the part 
of the body under study, e.g. blood flow rate. In this case however, the 
parameter determination should be based on light detected from particles 
studied through the skin or through an endoscopically or surgically 
exposed surface. 
Particularly preferably, the light imaging procedure used is selected from 
confocal scanning laser microscopy (CSLM), optical coherence tomography 
(OCT), laser doppler, laser speckle, and multi-photon microscopy 
techniques (for a description of the latter see for example Denk, W. in 
Photonics Spectra (1997) July 125-130, Denk, W. et al. in Science (1990) 
April 248 73-76, Denk, W. et al. in J. Neurosci.Meth. (1994) 54:2:151-162, 
Denk, W. et al. in Neuron (1997) January 18:351-357, Maiti, S. et al. in 
Science (1997) January 275 530-532 and Denk, W. et al. in Proc. Natl. 
Acad. (1995) August 92:18:8279-8282). 
Confocal scanning laser microscopy (CSLM) is an imaging modality that 
selectively detects a single point within a test object by focusing light 
from a pinhole source onto that point. The light transmitting past or 
reflecting from that point is refocused onto a second pinhole that filters 
out light coming from any other site in the object except the focal point. 
Raster scanning of the focus point through a plane passing through the 
sample generates a full image of that plane of points. Moving the pinholes 
and focusing apparatus back and forth from the sample selects out 
different sample planes. In effect CSLM is a means for "optically" 
sectioning a test sample. It pulls out images of individual sections of 
the sample, but without the necessity that those sections be physically 
separated from the rest of the sample. 
Optical coherence tomography (OCT) accomplishes optical sectioning in a 
related, but somewhat different manner. A collimated beam of light is 
reflected from the sample, then is compared with a reference beam that has 
travelled a precisely known distance. Only the light travelling exactly 
the same distance to the sample and back as the distance the reference 
beam travels from the source to the detector constructively interferes 
with the reference beam and is detected. Thus the light from a single 
plane within the sample is again selected. Varying the distance that the 
reference beam travels before it is compared with the sampling beam 
selects out different sample planes. 
CSLM, OCT, laser doppler and laser speckle are discussed for example by: 
Rajadhyaksha et al. in Laser Focus World, February 1997, pages 119 to 127; 
Sabel et al. in Nature Medicine 3(2): 244-247 (1997); Tearney et al. in 
SPIE 2389: 29-34 (1995); Bonner et al. in "Scattering techniques applied 
to supramolecular and non-equilibrium systems", pages 685-701, Ed. Chen et 
al., Plenum; Ruth in J. Microcirc: Clin Exp 9: 21-45 (1990); Pierard in 
Dermatology 186: 4-5 (1993); and Bonner et al. in "Laser-doppler blood 
flowmetry" pages 17 to 46, Ed. Shepherd et al., Kluwer, 1990. 
CSLM and OCT may be used particularly effectively to study structures and 
events occurring in the skin or within about a millimeter of an accessible 
surface of the body under study, e.g. a surface exposed during surgical 
operation or exposed endoscopically. 
CSLM and OCT can be useful in optically guided tumor resection. For 
example, either device attached to a colonoscope may facilitate 
determination that no residual malignant tissue remains after removal of a 
cancerous colon polyp. Additional applications include, but are not 
limited to, diagnosis and treatment of disease in the rest of the 
digestive tract, surgical treatment of ulcerative colitis, and diagnosis 
and treatment of endometriosis. 
Dynamically, CSLM and OCT can be used to follow the movement of blood cells 
through the capillaries of the skin and other vascularized tissue lying 
within about a millimeter of an exposed surface. Potentially they can also 
be used in conjunction with laser Doppler or speckle inferferometry for 
the measure of blood flow. 
Laser Doppler and speckle interferometry are related, each relying upon the 
fact that the intensity of light detected after a beam of laser light that 
interacts with a collection of moving particles changes with time. 
Mathematical analysis of the changes provides a basis for calculating the 
rate at which the particles are moving. 
The perfusion of tissue that is exposed by surgery is one important 
indicator of the health of that tissue. Blood flow within the skin of the 
breast may be an indicator of internal disease. Blood flow in the skin can 
be detected by laser Doppler blood-flow measurement or laser speckle 
interferometry, either by itself or in conjunction with CSLM or OCT. 
According to the present invention, synthetic particles, capable of 
scattering light of the wavelength used for the imaging procedure, may be 
administered as contrast agents in an in vivo light imaging procedure. 
Typically such scattering particles will be administered in suspension in 
a physiologically tolerable fluid (e.g. water for injections, 
physiological saline, Ringer's solution etc.) into the vasculature or 
musculature or into the tissue or organ of interest. 
A preferred contrast agent for intraoperative CSLM or OCT will have the 
following properties: it will consist of stabilized particles in an 
aqueous or buffered liquid medium. The particle size will preferably be 
around 600 to 1300 nm, more preferably 700 to 1100 nm (i.e. roughly equal 
to the wavelength of the light source). The refractive index of the 
particles will preferably differ from that of body fluids, such as blood 
and lymph, by at least 0.01. Optionally the particles may have fluorescent 
dyes attached to their surfaces or contained within them or the particles 
themselves may be composed of fluorescent dyes. Optionally the particles 
may have suitable surface modifying agents, such as poly(ethylene glycol), 
to slow their uptake by macrophages in the body and to prolong their blood 
circulation lifetimes. 
The particles may be of a material which is transparent or translucent or 
more preferably opaque to light of the wavelength of the light source. 
Particularly preferably, the particles are substantially monodisperse 
polymer particles (with a coefficient of variation of the particle size 
(i.e. 100.times.standard deviation.div.mean particle size by volume of the 
major mode of the detectable particles) as measured by a Coulter LS 130 
particle size analyzer of less than 10%, preferably less than 5%). Such 
particles may be prepared by the SINTEF technique disclosed in U.S. Pat. 
No. 4,336,173 and U.S. Pat. No. 4,459,378. Such polymer particles may be 
simple scatterers or may be modified to carry a chromophore (or 
fluorophore), preferably having characteristic absorption and/or emission 
maxima in the 600 to 1300 nm range. Furthermore they may be modified to 
include or carry a targetting vector, e.g. a species serving to cause the 
particles to accumulate at a desired target site, for example 
superparamagnetic crystals which allow the particle to be accumulated at a 
target site by application of an external magnetic field, or a drug, 
antibody, antibody fragment or peptide (e.g. an oligopeptide or 
polypeptide) which has a binding affinity for sites within the target 
zone, e.g. cell surface receptors. 
The particulate contrast agent can be applied through simple topical 
application or other pharmaceutically acceptable routes. For 
dermatological applications, the contrast agents may be modified to be 
delivered through transdermal patches or by iontophoretesis. Iontophoretic 
delivery is preferred, as one can control the amount of the agent that is 
delivered. 
For intraoperative uses the contrast agent can be injected into the 
vasculature or into the lesion to be removed prior to or during the 
surgery. For detection of lymph nodes it can be injected into a lymph duct 
draining into the surgical area. Alternatively it may be applied during 
surgery as a topical ointment, a liquid, or a spray. For measurement of 
blood flow the agent can be injected intravascularly prior to the 
measurement. 
As indicated above, the particulate agents used according to the invention 
may comprise a chromophore or fluorophore, i.e. may absorb or emit light 
in the wavelength range detected in the imaging procedure or alternatively 
may rely primarily upon light scattering effects. In the latter case, one 
may simply use physiologically tolerable non photo-labelled particles, 
e.g. particles of an inert organic or inorganic material, e.g. an 
insoluble triiodophenyl compound or titanium dioxide, which appears white 
or colourless to the eye. Where the particles comprise a fluorophore or 
chromophore, i.e. are photo-labelled, this may be in a material carried by 
(e.g. bound to, coated on, or contained or deposited within) a particulate 
carrier (e.g. a solid particulate or a liposome). Alternatively the 
carrier itself may have chromophoric or fluorophoric properties. While the 
photolabel may be a black photolabel (i.e. one which absorbs across the 
visible spectrum and thus appears black to the eye) non-black photolabels 
are preferred. 
Scattering contrast agents (and absorbing contrast agents for that matter) 
can have several mechanisms in image enhancement for light imaging 
applications. The first mechanism is a direct image enhancing role similar 
to the effect that x-ray contrast media have in x-ray imaging. In direct 
image enhancement, the contrast medium contributes directly to an 
improvement in image contrast by affecting the signal intensity emanating 
from the tissue containing the contrast medium. In light imaging, 
scattering (and absorbing) agents localized in a tissue can attenuate 
light differently than the surrounding tissue, leading to contrast 
enhancement. 
For near surface methods such as confocal microscopy and optical coherence 
tomography, scattering agents generate contrast primarily by serving as 
reflection centres that selectively direct the incident light to the 
detector. When scattering sites are trapped in a moving fluid, such as 
blood, the extent of the scattering sites' movement can be used as a 
measure of the fluid's flow rate. 
The "speckle" phenomenon results from the interaction of coherent radiation 
(such as that from a laser) with scattering sites. When the scattering 
sites move, the speckle pattern changes with time, and the rate of change 
of the speckle pattern can be used to determine the rate of movement of 
the scattering sites. If the movement of the scattering sites is 
non-random, for example when they are entrained in a moving fluid, the 
rate of fluid flow can be determined by the changes in the speckle pattern 
over time. 
A second mechanism by which a scattering (or absorbing) agent could be used 
is as a noise rejection agent. The contrast agent in this case is not 
directly imaged as described above, but functions to displace a noise 
signal from an imaging signal so that the desired signal is more readily 
detected. Noise in light imaging applications results from multiple 
scattering and results in a degradation of image quality. The origin of 
this noise is as follows: 
As previously mentioned, light propagating through a random medium such as 
tissue undergoes multiple scattering. This scattering splits the incident 
light into three components, the ballistic, quasi-ballistic, and 
incoherent (highly scattered) components. The ballistic and 
quasi-ballistic signals propagate through tissue in the forward direction 
and carry the object information. The incoherent component constitutes 
noise because the light has undergone random scattering in all directions 
and information about the object is lost. When the intensity of the 
ballistic and quasi-ballistic signals are reduced below the intensity of 
the multiply scattered noise, the object becomes invisible. This multiple 
scattering noise can be partially removed by a spatial filter that rejects 
light scattered away from the collinear direction of the incident light. 
However, a substantial portion of noise emerges from the object after 
multiple scattering events by rejoining the original ballistic signal. 
This multiply scattered light can not be removed by spatial filtering due 
to its collinear path with the desired ballistic signal. 
Scattering (and absorbing) agents can aid in the removal of unwanted noise 
component from the desired ballistic and quasi-ballistic signals. This is 
based on the fact that multiply scattered light undergoes a random walk in 
tissue and thus travels over a longer path length than the ballistic 
signal. The distance the ballistic and quasi-ballistic signals traverses 
is essentially the thickness of the tissue (or body part) being imaged. 
Scattered light traveling a longer distance has a greater probability of 
being attenuated. Current technology uses a time-gate (temporal filter) to 
reject the scattered signal (longer traveling=longer residence time in 
tissue) from the ballistic and quasi-ballistic components. 
The introduction of a small isotropic scattering agent greatly increases 
the residence time of the highly scattered signal component while having a 
lesser effect on the ballistic and quasi-ballistic components. This 
effectively provides a longer separation between the ballistic and 
quasi-ballistic signals and the highly scattered component, providing 
improved rejection of the scattered (noise) component and better image 
quality. 
Very little is disclosed in prior art regarding particulate 
scattering-based contrast agents. To our knowledge the only prior art with 
regard to particulate scattering-based contrast agents is U.S. Pat. No. 
5,140,463 (Yoo, K. M. et al.) which discloses a method and apparatus for 
improving the signal to noise ratio of an image formed of an object hidden 
in or behind a semi-opaque medium. The patent in general terms suggests to 
make the random medium less random (so that there will be less scattered 
light) and it is also suggested to increase the time separation between 
ballistic and quasi-ballistic light and the highly scattered light. One of 
many ways to obtain this will, according to the patent, be to introduce 
small scatterers into the random medium. There are no further suggestions 
regarding these small scatterers and no suggestion of in vivo use. 
Particulate materials in the form of liposomes have been suggested; 
liposome or LDL-administered Zn(II)-phthalocyanine has been suggested as 
photodynamic agent for tumors by Reddi, E. et al. in Lasers in Medical 
Science 5 (1990) 339, parenterally administered zinc phtalocyanine 
compositions in form of liposome dispersion containing synthetic 
phopholipid in EP 451 103 (CIBA Geigy) and liposome compositions 
containing benzoporphyrin derivatives used in photodynamic cancer therapy 
or an antiviral agents in CA 2,047,969 (Liposome Company). These 
particulate materials have been suggested as therapeutic agents and have 
nothing to do with scattering light imaging contrast agents. 
In one embodiment of the invention the contrast medium for imaging 
modalities based on light will comprise physiologically tolerable gas 
containing particles. Preferred are e.g. biodegradable gas-containing 
polymer particles, gas-containing liposomes or aerogel particles. 
This embodiment of the invention includes, for example, the use in light 
imaging of particles with gas filled voids (U.S. Pat. No. 4,442,843), 
galactose particles with gas (U.S. Pat. No. 4,681,119), microparticles for 
generation of microbubbles (U.S. Pat. No. 4,657,756 and DE 3313947), 
protein microbubbles (EP 224934), clay particles containing gas (U.S. Pat. 
No. 5,179,955), solid surfactant microparticles and gas bubbles (DE 
3313946), gas-containing microparticles of amylose or polymer (EP 327490), 
gas-containing polymer particles (EP 458079), aerogel particles (U.S. Pat. 
No. 5,086,085), biodegradable polyaldehyde microparticles (EP 441468), gas 
associated with liposomes (WO 9115244), gas-containing liposomes (WO 
9222247), and other gas containing particles (WO 9317718, EP 0398935, EP 
0458745, WO 9218164, EP 0554213, WO 9503835, DE 3834705, WO 9313809, WO 
9112823, EP 586875, WO 9406477, DE 4219723, EP 554213, WO 9313808, WO 
9313802, DE 4219724, WO 9217212, WO 9217213, WO 9300930, U.S. Pat. No. 
5,196,183, WO 9300933, WO 9409703, WO 9409829, EP 535387, WO 9302712, WO 
9401140). The surface or coating of the particle can be any 
physiologically acceptable material and the gas can be any acceptable gas 
or gas mixture. Specially preferred gases are the gases used in ultrasound 
contrast agents like for example air, nitrogen, lower alkanes and lower 
fluoro or perfluoro alkanes (e.g. containing up to 7, especially 4, 5 or 6 
carbons). 
Where gas microbubbles (with or without a liposomal encapsulating membrane) 
are used according to the invention, advantage may be taken of the known 
ability of relatively high intensity bursts of ultrasound to destroy such 
microbubbles. Thus by comparing the detected light signal (or image) 
before and after ultrasound exposure mapping the distribution of the 
contrast agent may be facilitated. 
In another embodiment of the invention the contrast medium for imaging 
modalities based on light will comprise physiologically tolerable 
particles of lipid materials, e.g. emulsions, especially aqueous 
emulsions. Preferred are halogen comprising lipid materials. This 
embodiment of the invention includes, for example, the use in light 
imaging of fat emulsions (JP 5186372), emulsions of fluorocarbons (JP 
2196730, JP 59067229, JP 90035727, JP 92042370, WO 930798, WO 910010, EP 
415263, WO 8910118, U.S. Pat. No. 5,077,036, EP 307087, DE 4127442, U.S. 
Pat. No. 5,114,703), emulsions of brominated perfluorocarbons (JP 
60166626, JP 92061854, JP 5904630, JP 93001245, EP 231070), 
perfluorochloro emulsions (WO 9311868) or other emulsions (EP 321429). 
In yet another embodiment of the invention the contrast medium for imaging 
modalities based on light will comprise physiologically tolerable 
liposomes. Preferred groups of liposomes are phospholipid liposomes and 
multilamelar liposomes. This embodiment of the invention includes, for 
example, the use in light imaging of phospholipid liposomes containing 
cholesterol derivatives (U.S. Pat. No. 4,544,545); liposomes associated 
with compounds containing aldehydes (U.S. Pat. No. 4,590,060); lipid 
matrix carriers (U.S. Pat. No. 4,610,868); liposomes containing 
triiodobenzoic acid derivatives of the type also suitable for X-ray 
examination of liver and spleen (DE-2935195); X-ray contrast liposomes of 
the type also suitable for lymphography (U.S. Pat. No. 4,192,859); 
receptor-targeted liposomes (WO-8707150); immunoactive liposomes 
(EP-307175); liposomes containing antibody specific for antitumor antibody 
(U.S. Pat. No. 4,865,835); liposomes containing oxidants able to restore 
MRI contrast agents (spin labels) which have been reduced (U.S. Pat. No. 
4,863,717); liposomes containing macromolecular bound paramagnetic ions of 
the type also suitable for MRI (GB-2193095); phospholipid liposomes of the 
type also suitable for ultrasound imaging containing sodium bicarbonate or 
aminomalonate as gas precursor (U.S. Pat. No. 4,900,540); stable 
plurilamellar vesicles (U.S. Pat. No. 4,522,803); oil-filled paucilamellar 
liposomes containing non-ionic surfactant as lipid (U.S. Pat. No. 
4,911,928); liposomal phospholipid polymers containing ligands for 
reversible binding with oxygen (U.S. Pat. No. 4,675,310); large 
unilamellar vesicle liposomes containing non-ionic surfactant (U.S. Pat. 
No. 4,853,228); aerosol formulations containing liposomes (U.S. Pat. No. 
4,938,947 and U.S. Pat. No. 5,017,359); liposomes containing amphipathic 
compounds (EP-361894); liposomes produced by adding an aqueous phase to an 
organic lipid solution followed by evaporating the solvent and then adding 
aqueous lipid phase to the concentrate (FR-2561101); stable monophasic 
lipid vesicles of the type also useful for encapsulation of bioactive 
agents at high concentrations (WO-8500751); homogeneous liposome 
preparations (U.S. Pat. No. 4,873,035); stabilized liposome compounds 
comprising suspensions in liquefiable gel (U.S. Pat. No. 5,008,109); 
lipospheres (solid hydrophilic cores coated with phospholipid) of the type 
also suitable for controlled extended release of active compounds 
(WO-9107171); liposomes sequestered in gel (U.S. Pat. No. 4,708,861); 
metal chelates bound to liposomes, also suitable for use as MR contrast 
agents (WO-9114178); lipid complexes of X-ray contrast agents 
(WO-8911272); liposomes which can capture high solute to lipid ratios 
(WO-9110422); liposomes containing covalently bound PEG moieties on 
external surface to improve serum half-life (WO-9004384); contrast agents 
comprising liposomes of specified diameter encapsulating paramagnetic 
and/or superparamagnetic agents (WO-9004943); liposomes of the type also 
suitable for delivering imaging agents to tumours consisting of small 
liposomes prepared from pure phopholipids (EP-179444); encapsulated X-ray 
contrast agents such as iopromide in liposomes (U.S. Pat. No. 5,110,475); 
non-phospholipid liposome compositions (U.S. Pat. No. 5,043,165 and U.S. 
Pat. No. 5,049,389); hepatocyte-directed vesicle delivery systems (U.S. 
Pat. No. 4,603,044); gas-filled liposomes of the type also suitable as 
ultrasound contrast agents for imaging organs (U.S. Pat. No. 5,088,499); 
injectable microbubble suspensions stabilized by liposomes (WO-9115244); 
paramagnetic chelates bound to liposomes (U.S. Pat. No. 5,135,737); 
liposome compositions of the type also suitable for localising compounds 
in solid tumors (WO-9105546); injectable X-ray opacifying liposome 
compositions (WO-8809165); encapsulated iron chelates in liposomes 
(EP-494616); liposomes linked to targeting molecules through disulphide 
bonds (WO-9007924); and compositions consisting of non-radioactive 
crystalline X-ray contrast agents and polymeric surface modifiers with 
reduced particle size (EP-498482). 
Water soluble compounds which, in simple aqueous solution are not 
apparently significant light scatterers or absorbers, may become efficient 
scatterers on incorporation within liposomes. Thus iodixanol (and other 
soluble iodinated X-ray contrast agents that are commercially available) 
provides a clear solution on dissolution in water. However when iodixanol 
is encapsulated in liposomes the resulting particulate product is 
off-white indicating a significant light scattering capability. 
Besides using liposomes as carriers for light imaging contrast agents, it 
is possible to use simple micelles, formed for example from surfactant 
molecules, such as sodium dodecyl sulphate, cetyltrimethylammonium 
halides, pluronics, tetronics etc., as carriers for photolabels which are 
moderately or substantially water insoluble but are solubilised by the 
amphiphilic micelle forming agent, e.g. photolabels such as indocyanine 
green. Similarly peptides such as PEG modified polyaspartic acid (see Kwon 
et al. Pharm. Res. 10: 970 (1993)) which spontaneously aggregate into 
polymeric micelles may be used to carry such photolabels. Likewise 
photolabel carrier aggregate particles can be produced by treatment of 
polycyclic aromatic hydrocarbons with anionic surfactants (e.g. sodium 
dodecyl sulphate or sulphated pluronic F108) and subsequent addition of 
heavy metal ions (e.g. thorium or silver). Such heavy metal treatment 
gives rise to micelles exhibiting phosphorescent behaviour and these can 
be used in the present invention without incorporation of a photolabel, 
especially using a pulsed light source and gated detection of the 
temporally delayed phosphorescent light. 
In a still further embodiment of the invention the contrast medium for 
imaging modalities based on light will comprise physiologically tolerable 
particles containing iodine. These particles may for example be particles 
of a substantially water insoluble solid or liquid iodine-containing 
compound, e.g. an inorganic or organic compound, in the latter case 
preferably a triiodophenyl group containing compound, or alternatively 
they may be aggregate particles (such as liposomes) in which at least one 
of the components is an iodinated compound. In this case the iodinated 
compound may be a membrane forming compound or may be encapsulated by the 
membrane. For example, the use of emulsified iodinated oils (U.S. Pat. No. 
4,404,182), particulate X-ray contrast agents (JP 67025412, SU 227529, DE 
1283439, U.S. Pat. No. 3,368,944, AU 9210145, EP 498482, DE 4111939, U.S. 
Pat. No. 5,318,767), iodinated esters (WO 9007491, EP 300828, EP 543454, 
BE 8161143) and iodinated lipids (EP 294534) are included in this 
embodiment of the invention. 
In a yet still further embodiment of the invention the contrast medium for 
imaging modalities based on light will comprise physiologically tolerable 
magnetic particles. The term "magnetic particle" as used here means any 
particle displaying ferromagnetic, ferrimagnetic or superparamagnetic 
properties and preferred are composite particles comprising magnetic 
particles and a physiologically tolerable polymer matrix or coating 
material, e.g. a carbohydrate and/or a blood residue prolonging polymer 
such as a polyalkyleneoxide (e.g. PEG) as described for example by 
Pilgrimm or Illum in U.S. Pat. No. 5,160,725 and U.S. Pat. No. 4,904,479 
e.g. biodegradable matrix/polymer particles containing magnetic materials. 
This embodiment of the invention includes, for example, the use in light 
imaging of magnetic liquid (SU 1187221), ferrite particles coated with a 
negatively charged colloid (DE 2065532), ferrite particles (U.S. Pat. No. 
3,832,457), liquid microspheres containing magnetically responsive 
substance (EP 42249), magnetic particles with metal oxide core coated with 
silane (EP 125995), magnetic particles based on protein matrix (DE 
3444939), magnetic vesicles (JP 60255728), magnetic particles (SU 106121), 
magnetic particles embedded in inert carrier (JP 62167730), ferromagnetic 
particles loaded with specific antibodies (DE 3744518), superparamagnetic 
particles coated with biologically acceptable carbohydrate polymers (WO 
8903675), polymerized lipid vesicles containing magnetic material (U.S. 
Pat. No. 4,652,257), superparamagnetic materials in biodegradable matrices 
(U.S. Pat. No. 4,849,210), biodegradable matrix particles containing 
paramagnetic or ferromagnetic materials (U.S. Pat. No. 4,675,173), 
ferromagnetic particles with substances for binding affinity for tissue 
(WO 8601112), ferrite particles (JP 47016625, JP 47016624), ferromagnetic 
particles (NL 6805260), magnetic polymer particles (WO 7800005, JP 
62204501, JP 94016444, WO 870263), barium ferrite particles (WO 8805337), 
magnetic iron oxide particles (U.S. Pat. No. 4,452,773), amino acid 
polymer containing magnetic particles (U.S. Pat. No. 4,247,406), complexed 
double metal oxide particles (EP 186616), magnetic particles (GB 2237198), 
encapsulated superparamagnetic particles (WO 8911154), biodegradable 
magnetic particles (WO 8911873), magnetic particles covalently bond to 
proteins (EP 332022), magnetic particles with carbohydrate matrix (WO 
8301768), magnetic particles with silicon matrix (EP 321322), polymer 
coated magnetic particles (WO 9015666), polymer-protected collodial metal 
dispersion (EP 252254), biodegradable superparamagnetic particles (WO 
8800060), coated magnetic particles (WO 9102811), ferrofluid (DE 4130268), 
organometallic coated magnetic particles (WO 9326019) and other magnetic 
particles (EP 125995, EP 284549, U.S. Pat. No. 5,160,726, EP 516252, WO 
9212735, WO 9105807, WO 9112025, WO 922586, U.S. Pat. No. 5,262,176, WO 
9001295, WO 8504330, WO 9403501, WO 9101147, EP 409351, WO 9001899, EP 
600529, WO 9404197). 
The particulate contrast agent used according to the invention may, as 
mentioned above, be non-photo-labelled or photolabelled. In the latter 
case this means that the particle either is an effective photoabsorber at 
the wavelength of the incident light (i.e. carries a chromophore) or is a 
fluorescent material absorbing light of the incident wavelength and 
emitting light at a different wavelength (i.e. carries a fluorophore). 
Examples of suitable fluorophores include fluorescein and fluorescein 
derivatives and analogues, indocyanine green, rhodamine, 
triphenylmethines, polymethines, cyanines, phalocyanines, naphthocyanines, 
merocyanines, lanthanide complexes (e.g. as in U.S. Pat. No. 4,859,777) or 
cryptates, etc. including in particular fluorophores having an emission 
maximum at a wavelength above 600 nm (e.g. fluorophores as described in 
WO-A-92/08722). Other labels include fullerenes, oxatellurazoles (e.g. as 
described in U.S. Pat. No. 4,599,410), LaJolla blue, porphyrins and 
porphyrin analogues (e.g. verdins, purpurins, rhodins, perphycenes, 
texaphyrins, sapphyrins, rubyrins, benzoporphyrins, photofrin, 
metalloporphyrins, etc.) and natural chromophores/fluorophores such as 
chlorophyll, carotenoids, flavonoids, bilins, phytochrome, phycobilins, 
phycoerythrin, phycocyanins, retinoic acid and analogues such as retinoins 
and retinates. 
In general, photolabels which contain chromophores should exhibit a large 
molar absorptivity, e.g. &gt;10.sup.5 cm.sup.-1 M.sup.-1 and an absorption 
maximum in the optical window 600 to 1300 nm. Particulates for use as 
noise rejection agents by virtue of their absorption properties should 
similarly preferably have molar absorptivities in excess of 10.sup.5 
cm.sup.-1 M.sup.-1 and an absorption maximum in the range 600 to 1300 
nm.sup.-1. For fluorescent particles, the quantum yield for fluorescence 
is one of the most important characteristics. This should be as high as 
possible. However the molar absorptivity should also desirably be above 
10.sup.5 cm.sup.-1 M.sup.-1 for the fluorophore and the absorption maximum 
should desirably be in the range 600 to 1300 nm for diffuse reflectance 
studies or 400 to 1300 nm for surface studies. 
These photo-labelled materials may be used as such if substantially 
water-insoluble and physiologically tolerable, e.g. as solid or liquid 
particles, or alternatively may be conjugated to or entrapped within a 
particulate carrier (e.g. an inorganic or organic particle or a liposome). 
Particularly preferred in this are conjugates of formula I 
EQU I.sub.3 Ph-L-C* (I) 
where I.sub.3 Ph is a triiodophenyl moiety, L is a linker moiety and C* is 
a chromophore or fluorophore (e.g. as described above). Such compounds 
form a further aspect of the invention. 
The I.sub.3 Ph moiety is preferably a 2, 4, 6 triiodo moiety having 
carboxyl or amine moieties (or substituted such moieties, e.g. 
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, 
alkoxycarbonylalkoxycarbonyl, or alkylcarbonylamino groups where the alkyl 
or alkylene moieties are optionally hydroxy substituted and preferably 
contain up to 20, particularly 1 to 6, especially 1 to 3 carbons) at the 3 
and 5 positions. The linker group L may be any group capable of linking 
the group C* to the I.sub.3 Ph moiety, e.g. an amide, amine, NHSO.sub.2 or 
carboxyl group or a thio analog thereof; or a C.sub.1-20. alkylene chain 
terminated by such groups and optionally with one or more methylene groups 
replaced by thia or oxa and optionally substituted for example by thio, 
oxo, hydroxy or alkyl moieties. Examples of group L include --NHSO.sub.2 
-- and --CO.sub.2 (CH.sub.2).sub.2 O--CS--NH--. 
Such compounds may be prepared by conjugating a chromophoric or 
fluorophoric molecule to a triiodophenyl compound of the type proposed as 
X-ray contrast agents by Nycomed, Sterling Winthrop, or Bracco in their 
numerous patent publications (by way of example U.S. Pat. No. 5,264,610, 
U.S. Pat. No. 5,328,404, U.S. Pat. No. 5,318,767 and U.S. Pat. No. 
5,145,684). 
In one particular embodiment of the invention, non-photolabelled particles, 
e.g. solid particles of a polymer or an iodinated X-ray contrast agent, 
are provided with a coating or shell of a photolabel, e.g. a fluorescent 
agent, for example by chemically or physiochemically binding the 
photolabel to the particles (e.g. by using oppositely charged photolabel 
and particles). The resulting coated particles, preferably of nano 
particle size (e.g. 5 to 800 nm, especially 10 to 500 nm) if labelled with 
a fluorophore would allow light energy trapped by the core to be 
transferred to the luminescing surface and so enhance light emission by 
the fluorophore. Compositions containing such particles form a further 
aspect of the invention. 
Alternatively the photo-label may be entrapped within a solid polymer 
matrix, e.g. by co-precipitation of polymer and photolabel or by 
precipitation of photo-label within the pores of a porous inorganic or 
organic matrix. 
Suitable organic polymer matrices for use as carriers or cores for 
photolabels are substantially water insoluble physiologically tolerable 
polymers, e.g. polystyrene latex, polylactide coglycolide, 
polyhydroxybutyrate co-valerate etc. 
Other physiologically acceptable particles may be used in contrast media 
for imaging methods based on light in accordance with of the present 
invention. Preferred groups of materials are e.g. biodegradable polymer 
particles, polymer or copolymer particles and particles containing 
paramagnetic materials. The particles can for example be crosslinked 
gelatin particles (JP 60222046), particles coated with hydrophilic 
substances (JP 48019720), brominated perfluorocarbon emulsions (JP 
58110522), perfluorocarbon emulsions (JP 63060943), particles and 
emulsions for oral use (DE 3246386), polymer particles (WO 8601524, DE 
3448010), lipid vesicles (EP 28917), metal oxide particles (JP 1274768), 
metal transferrin dextran particles (U.S. Pat. No. 4,735,796), 
monodisperse magnetic polymer particles (WO 8303920), polymer particles 
(DE 2751867), microparticles containing paramagnetic metal compounds (U.S. 
Pat. No. 4,615,879), porous particles containing paramagnetic materials 
(WO 8911874), hydrophilic polymer particles (CA 1109792), water-swellable 
polymer particles (DE 2510221), polymer particles (WO 8502772), metal 
loaded molecular sieves (WO 9308846), barium sulphate particles (SU 
227529), metal particles (DE 2142442), crosslinked polysaccharide 
particles (NL 7506757), biodegradable polymer particles (BE 869107), 
niobium particles (SU 574205), biodegradable polymer particles (EP 
245820), amphiphilic block copolymers (EP 166596), uniform size particles 
(PT 80494), coloured particles (WO 9108776), polymer particles (U.S. Pat. 
No. 5,041,310, WO 9403269, WO 9318070, EP 520888, DE 4232755), porous 
polymer particles (WO 9104732), polysaccharide particles (EP 184899), 
lipid emulsions (SU 1641280), carbohydrate particles (WO 8400294), 
polycyanoacrylate particles (EP 64967), paramagnetic particles (EP 
275215), polymer nanoparticles (EP 240424), nanoparticles (EP 27596, EP 
499299), nanocapsules (EP 274961), inorganic particles (EP 500023, U.S. 
Pat. No. 5,147,631, WO 9116079), polymer particles ((EP 514790), apatite 
particles (WO 9307905), particulate micro-clusters (EP 546 939), gel 
particles (WO 9310440), hydrophilic colloids (DE 2515426), particulate 
polyelectrolyte complex (EP 454044), copolymer particles (EP 552802), 
paramagnetic polymer particles (WO 9222201), hydrophilic poly-glutamate 
microcapsules (WO 9402106) and other particles (WO 9402122, U.S. Pat. No. 
4,997,454, WO 9407417, EP 28552, WO 8603676, WO 8807870, DE 373809, U.S. 
Pat. No. 5,107,842, EP 502814). 
In general, where the particulate agent is intended for parenteral 
administration (e.g. into the vasculature), it may be desirable to prolong 
the blood residence time for the particles by attaching to these a blood 
residence time prolonging polymer as described for example by Pilgrimm in 
U.S. Pat. No. 5,160,725 or Illum in U.S. Pat. No. 4,904,479. In this way 
imaging of the vascular system may be facilitated by delaying the uptake 
of the particle by the reticuloendothelial system. In the case of 
liposomal particles, the blood residence prolonging polymer may be bound 
to preformed liposomes or, conjugated to liposomal membrane forming 
molecules, may be used as an amphiphilic membrane forming component so 
resulting in liposomes carrying the hydrophilic blood residence polymer 
component on their surfaces. Alternatively or additionally the particles 
may be conjugated to a biotargetting moiety (e.g. as described in 
WO-A-94/21240) so as to cause the particles to distribute preferentially 
to a desired tissue or organ, e.g. to tumor tissue. 
The particle size utilized according to the invention will depend upon 
whether particle administration is parenteral or into an externally 
voiding body cavity and on whether or not the particles are 
photo-labelled. In general particle sizes will be in the range 5 to 10000 
nm, especially 15 to 1500 nm, particularly 50 to 400 nm and for particles 
which are being used for their scattering effect particle size will 
preferably be in the range 1/15.lambda. to 2.lambda., or more preferably 
1/10.lambda. to .lambda., especially .lambda./4.PI. to .lambda./.PI., more 
especially about .lambda./2.PI. (where .lambda. is the wavelength of the 
incident light in the imaging technique). By selecting a particle size 
which scatters effectively at wavelengths above the absorption maxima for 
blood, e.g. in the range 600 to 1000 nm, and by illuminating at a 
wavelength in that range, the contrast efficacy of non-photolabelled 
particles may be enhanced. 
For administration to human or animal subjects, the particles may 
conveniently be formulated together with conventional pharmaceutical or 
veterinary carriers or excipients. The contrast media used according to 
the invention may conveniently contain pharmaceutical or veterinary 
formulation aids, for example stabilizers, antioxidants, osmolality 
adjusting agents, buffers, pH adjusting agents, colorants, flavours, 
viscosity adjusting agents and the like. They may be in forms suitable for 
parenteral or enteral administration, for example, injection or infusion 
or administration directly into a body cavity having an external voidance 
duct, for example the gastrointestinal tract, the bladder and the uterus. 
Thus the media of the invention may be in conventional pharmaceutical 
administration forms such as tablets, coated tablets, capsules, powders, 
solutions, suspensions, dispersions, syrups, suppositories, emulsions, 
liposomes, etc; solutions, suspensions and dispersions in physiologically 
acceptable carrier media, e.g. water for injections, will however 
generally be preferred. Where the medium is formulated for parenteral 
administration, the carrier medium incorporating the particles is 
preferably isotonic or somewhat hypertonic. 
The contrast agents can be used for light imaging in vivo, in particular of 
organs or ducts having external voidance (e.g. GI tract, uterus, bladder, 
etc.), of the vasculature, of phagocytosing organs (e.g. liver, spleen, 
lymph nodes, etc.) or of tumors. The imaging technique may involve 
endoscopic procedures, e.g. inserting light emitter and detector into the 
abdominal cavity, the GI tract etc. and detecting transmitted, scattered 
or reflected light, e.g. from an organ or duct surface. Where appropriate 
monochromatic incident light may be utilized with detection being of 
temporally delayed light emission (e.g. using pulsed light gated 
detection) or of light of wavelengths different from that of the incident 
light (e.g. at the emission maximum of a fluorophore in the contrast 
agent). Similarly images may be temporal images of a selected target 
demonstrating build up or passage of contrast agent at the target site. 
The light used may be monochromatic or polychromatic and continuous or 
pulsed; however monochromatic light will generally be preferred, e.g. 
laser light. The light may be ultraviolet to near infra-red, e.g. 100 to 
1300 nm wavelength however wavelengths above 300 nm and especially 600 to 
1000 nm are preferred. 
The contrast media of the invention should generally have a particle 
concentration of 1 10.sup.6 g/ml to 50 10.sup.-3 g/ml, preferably 5 
10.sup.-6 g/ml to 10 10.sup.-3 g/ml. Dosages of from 1 10.sup.-7 g/kg to 5 
10.sup.-1 g/kg, preferably 1 10.sup.-6 g/kg to 5 10.sup.-2 g/kg will 
generally be sufficient to provide adequate contrast although dosages of 1 
10.sup.-4 g/kg to 1 10.sup.-2 g/kg will normally be preferred. 
The various publications referred to herein are hereby incorporated by 
reference. 
The invention is further illustrated by the following non-limiting 
Examples. Unless otherwise stated percentages and ratios are by weight. 
EXAMPLE 1 
Iodixanol Containing Liposomes 
Liposomes of average diameter 300 to 600 nm are prepared by a modification 
of the "Thin film hydration method" described by A. D. Bangham et al. 
"Methods in Membrane Biology (E. D. Korn, ed), Plenum Press, N.Y., pp 1-68 
(1974). The maximum batch size produced by the process is 2.0 L. The 
hydrogenated phosphatidylcholine (10 g H-PC) and hydrogenated phosphatidyl 
serine (1 g H-PS) are dissolved in chloroform/methanol/water (4:1:0.025, 
volume ratios) by shaking in a water bath at 70.degree. C. The solvents 
are removed by rotary evaporation until a dry mixture of the PLs appear. 
The phospholipid mixture is added to an aqueous, isotonic solution of 
iodixanol and tonicity agent at a temperature of 60-700C., and the mixture 
is homogenised with a homomixer, (6000 rpm for 10 minutes at a temperature 
of 65-70.degree. C.). The liposomes formed are extruded once through three 
polycarbonate filters. 5.0 mL of the liposome suspension are filled in 20 
mL glass bottles, closed with grey rubber stoppers and sealed with 
aluminium capsules. The liposomes are sterilised by autoclaving (at 
121.degree. C. for 20 minutes). 
EXAMPLE 2 
______________________________________ 
Fat emulsion 
______________________________________ 
An oil-in-water emulsion is prepared from 
soybean oil 10 g 
safflower oil 10 g 
egg phosphatides 
1.2 g 
glycerin 2.5 g 
water to osmolarity of 258 mOsm/L and pH of 8.3 to 
______________________________________ 
9.0 
(Such an emulsion is available commercially under the trade name Liposyn II 
from Abbott Laboratories, Chicago, Ill., U.S.A). This can be diluted with 
physiological saline to the desired concentration. 
EXAMPLE 3 
A. Solid Microparticles 
A gas-filled (e.g. air filled) microbubble suspension, with particle size 1 
to 12 .mu.m may be prepared with oleic acid and human serum albumin as the 
microbubble shell material. 
A 216 ml sample of a 0.5% aqueous solution of sodium oleate was titrated 
with 0.1 N HCl so that the final pH was in the range 3.9-4.0. The solution 
had become very turbid due to the formation of an oleic acid suspension. 
The particle size as measured by optical microscopy was in the 0.1 micron 
range. 
The suspension was pressurized to increase the solubility of the gas in the 
oleic acid suspension. The suspension was placed in a 500 ml stirred 
autoclave (Zipperclave manufactured by Autoclave Engineers, Inc.) fitted 
with a 6 blade turbine-type impeller (from Dispersimax). The vessel was 
sealed and charged to 1000 psig air (typical pressure ranges were 900-1100 
psig). The suspension was agitated at 1000 rpm (agitation ranged from 
750-1500 rpm) for one hour at room temperature (23-25.degree. C.). 
Typically the temperature rose 2-3.degree. C. during the run. Agitation 
was stopped, the vessel vented and the suspension was held for 30 minutes 
before use. The particle size as measured by optical microscopy was in the 
0.1 micron range. 
2 g of a 25% aqueous solution of human serum albumin (HSA) was added to 28 
g of water and 20 g of the emulsion described above. The turbid solution 
was heated to 65.degree. C. while oxygen gas was bubbled in. The solution 
was then stirred using an Omni-Stirrer (homogenizer) for 5 minutes at the 
mid-range setting. The foamy mixture was poured into a separatory funnel 
and left to stand for 30 minutes. The liquid was removed from the bottom 
and 10 ml of fresh 1% HSA solution was added to the foam. After 30 minutes 
the liquid was removed and 10 ml fresh 5% HSA solution was added so that 
the foam was resuspended in solution. The liquid was quickly collected 
from the bottom. The particles (microbubbles) had a diameter range of 1-12 
microns with a wall thickness of 1-2 microns. 
B. Gas Filled Microparticles 
Encapsulated gas micropheres may be prepared according to WO-A-95/01187 by 
mixing an aqueous solution of human serum albumin with a water insoluble 
gas such as a perfluoroalkane (e.g. dodecafluoropentane). 
EXAMPLE 4 
Polymer Particles 
A polymer particle suspension may be prepared by dissolving the 
biodegradable polymer polyhydroxybutyrate-co-valerate in a suitable 
organic solvent such as acetone, methylene chloride and the like, 
precipitation in water and removal of the organic solvent by vacuum 
distillation or diafiltration. Particle size may be selected to be within 
the range 0.05 .mu.m to 10 .mu.m by choice of surfactant stabilizers, rate 
of solvent evaporation, agitations as is well known in the art. 
EXAMPLE 5 
Optionally Photolabelled Nanoparticulate Suspensions 
A solution of WIN 70177 (an iodinated contrast agent prepared according to 
Example 24 below) and, optionally fluoroscein in the molar ratio 100:1, 
optimally 50:1, most optimally 25:1, in DMSO (or DMF) is precipitated in 
water. The resulting precipitate is milled as described in U.S. Pat. No. 
5,145,684 together with a surfactant stabilizer (eg. Pluronic F108 or 
Tetronic T-908 or 1508) to a particle size of 0.2 .mu.m and dispersed in 
an aqueous medium to a contrast agent concentration of 0.5 to 25% by 
weight and a surfactant content of 0.1 to 30% by weight. A cloud point 
modifier such as polyethylene glycol 400 (PEG 400) or propylene glycol as 
disclosed in U.S. Pat. No. 5,352,459 may also be included to ensure 
stability on autoclave stabilization. 
EXAMPLE 6 
Photolabelled Nanoparticulate Suspensions 
Phytochrome is added to an aqueous solution of sodium dodecyl sulphate 
(pH&gt;10). The resulting solution is added to a stirred solution of acetic 
acid containing a surfactant (selected from PVP, pluronics and tetronics) 
and the mixture is diafiltered to remove soluble salts, excess acid etc. 
from the suspension yielding a dispersion of 10-100 nm particles. 
EXAMPLE 7 
Photolabelled Micelles 
Indocyanine green (ICG) (0.1 to 10%) is mixed with 3% Pluronic F108 in 
aqueous solution to form a micellar composition which is sterile filtered. 
The ICG content used may be high (&gt;0.5%) to produce mixed micelles or low 
(&lt;0.5%) to produce micellar solutions of ICG. ICG-concentrations of 0.2 to 
0.5% are preferred. 
EXAMPLE 8 
Photo-labelled Liposomes 
A liposome suspension is prepared using a 0.01 M solution of indocyanine 
green and 5 to 10% of a phospholipid (10:1 ratio of lecithin to 
dipalmitoylphosphatidyl serine). Preparation is effected by conventional 
techniques (eg. ultrasound) followed by extrusion through controlled pore 
size filters and diafiltration or microfluidisation. The resulting 
liposomes are steam sterilizable and are sterile filterable and have 
demonstrated physical stability under nitrogen for over six months. 
EXAMPLE 9 
Photo-labelled Emulsions 
An oil in water emulsion is prepared from 10 g safflower oil, 10 g sesame 
oil, 1.2 g egg phosphatides, 2.5 g glycerin, 0.5 to 10 g photo-label (eg. 
fluorescein or indocyanine green) and water to 100 g total. Emulsification 
is effected by conventional means and the resultant emulsion is sterile 
filtered through 0.2 .mu.m sterile filters or steam sterilized using 
conventional means. 
EXAMPLE 10 
Particulate Iodinated Compounds 
WIN 70146 (an iodinated X-ray contrast agent prepared according to Example 
23 below) was added to each of 3.times.1.5 oz brown glass bottles 
containing approximately 12 ml of zirconium silicate, 1.1 mm diameter 
beads in an amount sufficient to be 15% (wt/vol %) of the final 
suspension. Bottle A was also made 3% (wt/vol %) Pluronic F-68 while 
bottle B was made 3% (wt/vol %)) Pluronic F-108 and bottle C was made 3% 
(wt/vol %) Tetronic T-908. The resulting suspensions were milled at approx 
150 rpm for a total of 9 days with estimates of particle size determined 
at various intervals as detailed below. 
______________________________________ 
Average Particle Size 
(nm) 
Days of milling 
F-68 F-108 T-908 
______________________________________ 
2 1939* 158 162 
3 223 161 162 
7 157 158 156 
9 158 159 159 
After 1 week at room temperature 
166 166 161 
After autoclaving at 121 degrees C. for 20 min..sup.+ 
181 190 183 
______________________________________ 
*Dioctylsulfosuccinate sodium (DOSS) was added at this point to aid in 
milling in an amount equal to 0.2% (wt/vol %). 
.sup.+ DOSS was added to the F108 and T908 samples for autoclaving as a 
cloud point modifier (at 0.2%, wt/vol %). 
These data demonstrate the unexpected ease of small particle preparation 
with this agent (ie. WIN 70146) in both F108 as well as excellent 
stability to heat (autoclaving) and time on the shelf. 
EXAMPLE 11 
Preparation and Acute Safety Testing of Nanoparticle Suspensions of WIN 
70146 in Pluronic F108 
WIN 70146 was prepared as in Example 10 and injected into the tail vein of 
mice at doses of 3 ml/kg, 15 ml/kg, and 30 ml/kg (ie. 0.45 gm/kg, 2.25 
gm/kg and 4.5 gm/kg). No untoward effects were noted in any of the mice at 
any dose for a period of 7 days after which time the animals were 
sacrificed. Gross observation of these animals did not reveal any obvious 
lesions or disfigurations. 
Further in depth safety studies in rats have not revealed significant 
safety issues due to a single dose of WIN 70146/F108 at levels up to and 
including 30 ml/kg (4.5 gm/kg). These studies included in-depth 
histopathology, clinical chemistry, and in life observations. 
EXAMPLE 12 
Preparation of WIN 70146 in Pluronic F108 (I-404) 
WIN 70146 was milled with 1.1 mm diameter zirconium silicate beads for 3 
days under aseptic conditions. The concentration of this agent was 15% WIN 
70146 in the presence of 4% Pluronic F-108. No additional salts or 
surfactants were added. The average particle size of the resulting 
nanoparticle suspension was 162 nm as determined by light scattering. 
EXAMPLE 13 
Preparation of an Autoclavable Formulation of WIN 70146 Using Pluronic 
F-108 and PEG 400 
WIN 70146 was milled with 1.1 mm diameter zirconium silicate beads in the 
presence of Pluronic F-108 for 3 days. The final particle size was 
determined to be 235 nm. At this point, sterile PEG 400 was added to the 
suspension such that at completion, the formulation contained 15% (wt/vol 
%) WIN 70146, 3% (wt/vol %) Pluronic F-108 and 10% PEG 400. This 
formulation was then autoclaved under standard conditions (ie. 121 degrees 
C. for 20 min.) resulting in a final particle size of 248 nm. 
EXAMPLE 14 
Demonstration of Light Scattering Above Incident Wavelengths of 600 nm by 
Nanoparticle Suspensions of WIN 70146 
A nanoparticle suspension of WIN 70146 was prepared as in Example 10 using 
4.25% F108/10% PEG 400 which after autoclaving resulted in particles with 
an average diameter of 228 nm. This suspension was then diluted in water 
to various levels listed below. The per cent of incident light transmitted 
was then determined for each suspension at several wavelengths (see 
below). The suspensions were then dissolved by addition of methanol and 
examined for per cent transmitted light against an equivalent solvent 
blank. The results are given below. 
______________________________________ 
Percent Transmission at 632 nm, 700 nm and 820 nm of 
Both NanoParticulate WIN 70146 and Dissolved WIN 70146 
Sample % T suspension % T solution 
Conc 632 nm 700 nm 820 nm 
632 nm 
700 nm 820 nm 
______________________________________ 
0.015% 54.7 64.5 77.0 100.4 100.3 100.5 
0.0375% 
25.4 36.6 53.8 99.9 99.9 99.9 
0.075% 7.7 15.4 31.8 99.9 99.8 99.9 
0.150% 0.5 1.9 8.6 41.4* 51.9* 66.2* 
0.300% 0.0 0.1 0.8 1.2* 4.0* 13.5* 
______________________________________ 
*These samples were not fully dissolved and showed visible turbidity 
These results demonstrate that the suspensions are efficient light 
scattering agents which do not absorb significant amounts of incident 
light in these wavelength regions (ie., dissolved WIN 70146 does not 
absorb light above 600 nm). Additional examination of the absorbance vs 
wavelength for the dissolved agent does not show any evidence of light 
absorbance from 600 to 800 nm while the nanoparticle agent shows a classic 
absorbance decay due to scattering of the incident light. 
EXAMPLE 15 
Preparation of Nanoparticle Suspension of WIN 70177 
A formulation of WIN 70177 (an iodinated X-ray contrast agent prepared 
according to Example 24) was prepared as 15 gm of WIN 70177/100 ml of 
suspension and 4.25 gm of Pluronic F108/100 ml of suspension and 10 gm of 
PEG 400/100 ml of suspension. The suspension was milled for 5 days after 
which the average particle size was determined by light scattering to be 
about 235 nm. Stability testing in fresh rat plasma and simulated gastric 
fluid did not show any aggregation. 
EXAMPLE 16 
Demonstration of Light Scattering above Incident Wavelengths of 600 nm by 
Nanoparticulate WIN 70177 
A nanoparticle suspension of WIN 70177 was prepared as in Example 15 using 
4.25% F108/10% PEG 400 which after autoclaving resulted in particles with 
an average diameter of 236 nm. This suspension was then diluted in water 
to various levels listed below. The per cent of incident light transmitted 
was then determined for each suspension at several wavelengths (see 
below). The suspensions were then dissolved by addition of methanol and 
examined for per cent transmitted light against an equivalent solvent 
blank. The results are given below. 
______________________________________ 
Percent Transmission at 632 nm and 700 nm of 
Both Nanoparticulate WIN 70177 and Dissolved WIN 70177 
Sample % T suspension % T solution 
Conc 632 nm 700 nm 800 nm 
632 nm 
700 nm 800 nm 
______________________________________ 
0.015% 53.3 62.8 73.1 102.2 101.9 101.8 
0.0375% 
34.6 45.7 59.1 102.3 101.9 101.8 
0.075% 25.8 36.8 51.1 100.9 100.8 101.0 
0.150% 6.7 13.6 26.3 59.5* 67.8* 77.0* 
0.300% 0.1 0.6 3.2 7.4* 14.4* 26.8* 
______________________________________ 
*Did not fully dissolve; particles still present. 
These data demonstrate the scattering abilities of the particulate form of 
WIN 70177 while the dissolved material does not absorb any energy over the 
wavelength of light examined. Further, an examination of the absorbance 
due to the particulate WIN 70177 and that due to the dissolved WIN 70177 
shows that the particulate material provides an exponential drop in 
absorbance with wavelength as would be expected for scattering due to 
suspended particles while the soluble material has virtually no absorbance 
at all even at 5 times the concentration. 
EXAMPLE 17 
Preparation of a Nanoparticle Suspension of WIN 67722 
A formulation of WIN 67722 (an iodinated X-ray contrast agent as described 
in U.S. Pat. No. 5,322,679) was prepared as in Example 1 using 3% Pluronic 
F108 and 15% PEG 1450. The suspension was milled for 3 days and achieved a 
particle size of 213 nm (small fraction at 537 nm) as determined by light 
scattering with a Coulter N4MD particle sizer. 
EXAMPLE 18 
Demonstration of Light Scattering above Incident Wavelengths of 600 nm by 
Nanoparticulate WIN 67722 
A nanoparticle suspension of WIN 67722 was prepared as in Example 17 using 
3% Pluronic F108 and 15% PEG 1450 which after autoclaving gave particles 
with an average diameter of 214 nm. This suspension was then diluted in 
water to various levels listed below. The per cent of incident light 
transmitted was then determined for each suspension at several wavelengths 
(see below). The suspensions were then dissolved by addition of methanol 
and examined for per cent transmitted light against an equivalent solvent 
blank. The results are given below. 
______________________________________ 
Percent Transmission at 632 nm and 700 nm of 
Both NanoParticulate WIN 67722 and Dissolved WIN 67722 
Sample % T suspension % T solution 
Conc 632 nm 700 nm 820 nm 
632 nm 
700 nm 820 nm 
______________________________________ 
0.015% 47.9 57.1 69.2 99.9 99.9 100.6 
0.0375% 
20.5 29.9 45.6 100.2 100.2 100.4 
0.075% 4.8 9.9 22.1 100.1 100.2 100.4 
0.150% 0.2 1.0 4.9 48.2* 55.3* 65.5* 
0.300% 0.0 0.0 0.2 1.3* 35* 10.7* 
______________________________________ 
*Did not fully dissolve; particles still present 
These data demonstrate the scattering abilities of the particulate form of 
WIN 67722 while the dissolved material does not absorb any energy over the 
wavelength of light examined. Further, an examination of the absorbance 
due to the particulate WIN 67722 and that due to the dissolved WIN 67722 
shows that the particulate material provides an exponential drop in 
absorbance with wavelength as would be expected for scattering due to 
suspended particles while the soluble material has virtually no absorbance 
at all even at 5 times the concentration. 
EXAMPLE 19 
Preparation of Nanovarticle Suspension of WIN 72115 
Nanoparticle WIN 72115 (a fluorescent iodinated contrast agent as described 
in Example 21 below) was prepared by combining WIN 72115 and Pluronic F108 
(BASF, Parsippany, N.J.) in a glass jar at concentrations of 15 gm/100 ml 
suspension and 3 gm/100 ml suspension. The jar was then half filled with 
1.0 mm diameter zirconium silicate beads and sufficient water added to 
complete the required concentrations of agent/surfactant as noted above. 
Alternatively, the surfactant can be dissolved in the water before 
addition to the jar (with or without sterile filtration through 0.2 micron 
filters). 
The jar is then rolled on its side for not less than 24 hours or more than 
14 days at a rate of rotation sufficient to cause the beads within the jar 
to "cascade" down the walls of the jar as it turns (see U.S. Pat. No. 
5,145,684). At the end of the milling cycle, the material is harvested 
from the jar and separated from the milling beads. 
Nanoparticles of WIN 72115 prepared in this manner have an average particle 
size of 225 nm by light scattering. 
WIN 72115 was designed to be excited with incident radiation from an Argon 
Ion laser (in the green, near 514 nm) and emit light at wavelengths above 
that value. Thus, after injection, illumination of the patient with green 
light would stimulate emission of light of a slightly different wavelength 
that could be used for diagnostic purposes. The key features of this agent 
are that it can be prepared as nanoparticles, remain within the 
vasculature for greater than 15 minutes, provide both scattering and 
fluorescence contrast for light imaging. 
In place of WIN 72115, the photolabelled agent of Example 22 below may be 
used. 
EXAMPLE 20 
Light Scattering from Polymeric Particles--Dependence Upon Particle Size 
and Concentration 
Three samples of polystyrene latex particles were diluted to various 
extents and examined for their effects upon transmitted light at several 
different wavelengths. The results confirm that larger particles and 
higher concentrations result in better scattering of the incident light. 
______________________________________ 
concentration 
Per cent Transmission 
Sample (Wt/vol %) 600 nm 700 nm 
820 nm 
______________________________________ 
170 nm .0025 97.9 98.3 98.7 
.025 94.8 96.3 97.4 
.075 89.3 92.8 95.2 
300 nm .0025 99.3 99.5 99.6 
.025 92.4 94.5 95.8 
.075 83.1 88.3 91.8 
500 nm .0025 98.8 99.1 99.4 
.025 88.1 91.4 93.9 
.075 68.3 76.5 83.0 
______________________________________ 
EXAMPLE 21 
3-(N-Acetyl-N-ethylamino)-5-[(5-dimethylamino-1-naphthylsulfonyl)aminol-2,4 
,6-triiodobenzoic Acid Ethyl Ester (WIN 72115) 
To a stirred solution of ethyl 
3-(N-acetyl-N-ethylamino)-5-amino]-2,4,6-triiodobenzoate (11.6 g, 18.5 
mmol) in pyridine (75 ml) cooled in ice bath is added 60% NaH/oil 
dispersion (1.8 g, 46.3 mmol). After the reaction of NaH with the amino 
group is over, dansyl chloride (5 g, 18.8 mmol) is added. The resulting 
reaction mixture is stirred in ice bath for 4 hours and at room 
temperature for 20 hours. After quenching with acetic acid (10 ml), the 
brown solution is concentrated on a rotary evaporator. The brown residue 
is first washed with hexanes and then slurried in water (200 ml). The 
resulting dirty yellow gummy solid is collected, washed with water, dried, 
and recrystallized from ethanol to provide 5.3 g (33%) of bright yellow 
crystals: mp 238-240.degree. C., ms (FAB) 862 (90%, MH). Anal. Calcd. for 
C.sub.25 H.sub.26 I.sub.3 N.sub.3 O.sub.5 S: C, 34.86; H, 3.05; N, 4.88; 
I, 44.20. Found: C, 34.91; H, 3.02; N, 4.74; I, 44.53. .sup.1 H-NMR and 
.sup.13 C-NMR spectra are consistent with the structure: 
##STR1## 
EXAMPLE 22 
2-(3,5-Bisacetylamino-2,4,6-triiodobenzoyloxy)ethyl 
N-Fluoreceinylthiocarbamate 
A mixture of 2-hydroxyethyl 3,5-(bisacetylamino)-2,4,6-triiodobenzoate 
(0.658 g, 1 mmol), fluorecein isothiocynate (0.389 g, 1 mmol), 60% NaH/oil 
dispersion (0.24 g, 6 mmol) and DMF (25 ml) is stirred at ambient 
temperature for 26 hours and then quenched with 6N HCl (2.5 ml). The 
resulting mixture is concentrated on a rotary evaporator under reduced 
pressure. The yellow solid residue is washed with water and recrystallized 
from DMF to yield yellow crystals of the product in 65% yield. Elemental 
analysis and spectral data are consistent with the structure: 
##STR2## 
EXAMPLE 23 
Benzoic acid, 3.5-bis(acetylamino)-2.4.6-triiodo-1-(ethoxycarbonyl)pentyl 
ester (WIN 70146) 
To a stirred solution of sodium diatrizoate (150 g, 235.2 mmole) in dry DMF 
(1200 ml) at room temperature, was added ethyl 2-bromohexanoate (63.8 g, 
285.8 mmole, 1.09 eq.). The solution was heated overnight at 90.degree. 
C., then cooled to 60.degree. C. The reaction mixture was then poured into 
201 of water with stirring. The resulting white precipitate was collected 
by filtration and dried at 90.degree. C. under high vacuum. The crude 
material was recrystallized from DMF/water to give, after drying, 
analytically pure product; mp 263-265.degree. C. The MS and .sup.1 H-NMR 
(300 MHz) spectral data were consistent with the desired structure. 
Calculated for C.sub.19 H.sub.23 I.sub.3 N.sub.2 O.sub.6 : C, 30.15; H, 
3.04; N, 3.70; I, 50.35. Found: C, 30.22; H, 3.00; N, 3.66; I, 50.19. 
EXAMPLE 24 
Propanedioic acid, 
[[3,5-bis(acetylamino)-2,4,6-triiodobenzoyl]oxy]methyl-bis(1-methylethyl)e 
ster (WIN 70177) 
To a stirred mixture of sodium diatrizoate (393 g, 616 mmole) in 500 ml of 
DMSO at room temperature, was added 173 g (616 mmol) of diisopropyl 
2-bromo-2-methylmalonate and the solution was heated at 90-100.degree. C. 
under an atmosphere of argon for 56 hours. After cooling, the solution was 
slowly added to 101 of water with mechanical overhead stirring. The 
precipitated solid was allowed to settle for 6 hours and then collected by 
filtration. The crude product was washed thoroughly with water (41) and 
dried at room temperature overnight. The solid was digested with a 
solution of potassium bicarbonate (3 g in 700 ml of water containing 15 ml 
of isopropanol), water and then air dried for 12 hours. Recrystalization 
from DMF followed by washing with water and drying under high vacuum gave 
255 g (51%) of analytically pure product; mp 258-260.degree. C. The MS and 
.sup.1 H-NMR (300 MHz) spectral data were consistent with the desired 
structure. 
Calculated for C.sub.21 H.sub.25 I.sub.3 N.sub.2 O.sub.8 : C, 30.98; H, 
3.10; N, 3.44; I, 46.76. Found: C, 30.96; H, 3.00; N, 3.44; I, 46.77. 
EXAMPLE 25 
In vivo Light Imaging Studies 
A. Particulate Scattering Agents 
A suspension of multilamellar liposomes formed in a solution of 40% (wt/vol 
%) iodixanol were injected into white rats which had been implanted with a 
hepatoma 9L tumor on their rear flank. The injection was imaged using a 
time gated diode laser incident at 780 nm with detection of the scattering 
component at 180 degrees to the incident light using fiber optic cables 
and a phase sensitive detection device in the laboratory of Dr. Britton 
Chance at the University of Pennsylvania. The liposome particles enhanced 
scattering in the tumor over the background signal by more than 4.times. 
at the dose administered (i.e. 3 ml/kg). While not optimized, these data 
indicate the feasibility of contrast by scattering agents for light 
imaging. 
B. Fluorescent particles for light imaging contrast 
A suspension of liposomes were prepared in the presence of 0.7 
micrograms/ml of indocyanine green (ICG) and sterilized using steam and 
pressure. The resulting particles had an average diameter of approximately 
120 nm as determined by light scattering using a Horiba 910 particle 
sizing instrument. Upon injection into the rat flank tumor model, these 
liposomes afforded significantly longer residence in the tumor of the 
fluorescent agent (i.e. the ICG) than observed with a homogeneous solution 
of ICG alone. This is useful for imaging in that signal averaging 
techniques can be applied to enhance the image as well as to mark sites of 
leaky vasculature. These studies were also carried out at the University 
of Pennsylvania in the laboratory of Dr. Britton Chance. 
EXAMPLE 26 
Use of Contrast Media for Enhancement of Laser Doppler Measurement of Blood 
Flow in the Skin 
Approximately 0.5 to 1 hour before the measurements are to be made, a 
sterile aqueous suspension containing 5-20 mg of suspended particles of a 
dye (e.g. 3,3'-diethylthiatricarbocyanine iodide) with an absorbing 
maximum between 600 and 1300 nm is administrated by intravenous injection. 
The mean particle size is preferably about 800 nm and as suspension medium 
is preferably used physiological saline. 
The measurement of blood flow is made after the concentration of contrast 
agent particles in the blood has stabilized. Measurement may be made with 
a standard laser Doppler instrument, for example that from Lisca 
Development AB, Kinkoping, Sweden, that optionally may be modified to 
incorporate a laser source operating at 830 or 780 nm (see Abbot et al., 
J. Invest. Dermatol., 107: 882-886 (1996)). 
EXAMPLE 27 
Use of Contrast Media for Enhancement of Measurement of Blood Flow through 
the Skin with Confocal Microscopy 
Approximately 0.5 to 1 hour before the measurements are to be made, a 
sterile aqueous suspension containing 5-20 mg of dye (e.g. 
3,3'-diethylthiatricarbocyanine iodide) with an absorbing maximum between 
600 and 1300 nm is administrated by intravenous injection. The mean 
particle size is preferably about 800 nm and as suspension medium is 
preferably used physiological saline. 
The measurement of blood flow is made by following the movement of the 
particles through the capillaries with the confocal microscope.