AS X-RAY CONTRAST AGENTS FOR BLOOD POOL AND LYMPHATIC

SYSTEM IMAGING

This invention relates to methods of x-ray diagnostic imaging the blood pool, liver, spleen,. and/or lymph system of a mammal employing particulate triiodo benzoate derivatives as a contrast agent, and particularly to certain such novel carbonic, carboxylic or carbamic anhydrides useful as contrast agents in x- ray contrast compositions and methods of diagnostic imaging.

X-ray imaging is a well known and extremely valuable tool for the early detection and diagnosis of various disease states in the human body. The use of contrast agents for image enhancement in medical x-ray imaging procedures is widespread. An excellent background on iodinated and other contrast agents for medical imaging is provided by D.P. Swanson et al,

Pharmaceuticals in Medical Imaging. 1990, MacMillan Publishing Company.

Chemical Abstract 149397-07-9 describes an alkyl ester having two acylamino groups having the formula:

N

This compound is not a mixed carbonic anhydride and no mention is made of its potential use as an x-ray contrast agent.

U.S. Patent No. 3,097,228 describes derivatives of 2,4,6-triiodobenzoyloxyalkanoic acids having the structure

wherein R 1 is H or lower alkyl; R*2 is H or lower alkanoyl; R is H or lower alkanoylamino and R is lower alkyl. The agents are useful as x-ray contrast agents for visualizing the gall bladder

(cholecystography) when administered orally, in the free acid form or in the form of a non-toxic salt, or intravenously, in the form of water soluble, non-toxic salt. Example 15 therein describes ethyl 2- (3,5- diacetamido-2,4, 6-triiodobenzoyloxy) hexanoate, i.e.,

C0 ₂ C ₂ H ₅

COOCH

U.S. Patent No. 5,332,679 describes iodinated aroyloxy esters which are useful as contrast agents in x-ray imaging compositions and methods. However, all

of the. compounds thus described feature an ester group linked through a C ₂ or higher alkylene group to another ester group on an iodinated aromatic ring.

EP-A 498,482 describes nanoparticulate x-ray contrast compositions which have proven to be extremely useful in medical imaging. The compositions comprise particles of an organic x-ray contrast agent and a surface modifier absorbed on the surface thereof and have an effective average particle size of less than 400 nm. The agents can be delivered to a specific tissue or fluid site, e.g., the blood pool, liver, spleen, kidney or lymph nodes. Example 8 therein describes a formulation comprising ethyl 2- (3,5- bis (acetylamino) -2,4,6-triiodobenzoyloxy) butyrate, i.e. ,

wherein (Z) -COO is the residue of diatrizoic acid.

However, it has been discovered that ethyl 2- (3, 5-bis (acetylamino) -2,4, 6-triiodobenzoyloxy) butyrate exhibits multiple crystal forms, i.e., polymorphism, e.g., when recrystallized from various solvents. The reasons for this behavior are not completely understood but, in any event, multiple crystal forms are disadvantageous for a variety of reasons. For example, the presence of multiple crystal forms renders scale up problematic due to the lack of reproducibility of the results obtained, including, e.g., in chemical manufacturing and in the milling process. Additionally, it has been found that nanoparticulate formulations of ethyl 2- (3, 5-bis (acetylamino) -2,4, 6- triiodobenzoyloxy) butyrate do not exhibit good stability during autoclaving, i.e., conventional heat sterilization.

Consequently, to be useful as x-ray contrast

agents, the agents must possess stability prior to and during the imaging period and then be cleared from the body rapidly afterward. It would be desirable to provide a poorly soluble x-ray contrast agent having the potential to hydrolyze to safe, tolerated metabolic end products.

Summary of the Invention We have discovered that among the triiodo benzoate derivatives certain such mixed carbonic, carboxylic or carbamic anhydrides exhibit reproducibly consistent crystal morphology during manufacture and purification and hydrolyze to safe, tolerated metabolic end products and thus are particularly amenable to use as particulate contrast agents for use in methods of x- ray diagnostic imaging the blood pool, spleen, liver, and lymphatic system of a mammal. In one aspect of the invention, we have discovered and synthesized such novel mixed carbonic, carboxylic or carbamic anhydrides which are useful as contrast agents in x-ray diagnostic imaging compositions and methods.

More specifically, in accordance with this invention, there is provided a method of medical x-ray diagnostic imaging which comprises administering to the blood pool, liver, spleen, or lymph system of a mammal a contrast-effective amount of a particulate diatrizoxy ester contrast agent having Formula I.

X is H, NR R ² or CCOONNR^-R ³

R ¹ is H or alkyl R ² is COR ³ t ³ is H, alkyl, or aryl; or when Z is OR ³ or

Z can also be hydroxy-substituted alkyl or a steroidally derived moiety

R ⁴ is H, alkyl, aryl or CH(R ⁵ )CO-,R ¹ wherein R ^J is H, methyl or benzyl or the like such that said group is derived from a naturally occurring amino acid Y is N ^-R ² or CONR^-R ³ and Z is OR ³ ,

Y In another aspect, there are provided novel mixed carbonic, carboxylic or carbamic anhydrides having Formula I above. This invention further provides an x-ray contrast composition comprising such novel compounds and a method for medical x-ray diagnostic imaging which comprises administering to a mammal an effective contrast-producing amount of the above-described x-ray contrast composition.

It is an advantageous feature of this invention that methods of x-ray diagnostic imaging the blood pool, liver, spleen, and lymphatic system are provided employing an x-ray contrast composition featuring a mixed carbonic, carboxylic or carbamic anhydride which exhibits a consistent crystal morphology during purification and thus is particularly amenable to reproducible scale up and hydrolyzing to a

safe, tolerated metabolic end product.

It is a particularly advantageous feature of this invention that x-ray contrast compositions are provided for blood pool, liver, spleen, and lymphatic system imaging which exhibit improved safety in that these contrast agents will clear from the body in a very short amount of time, thus giving maximum efficiency with maximum safety features.

Still another advantageous feature of this invention is that novel mixed carbonic, carboxylic or carbamic anhydrides are provided, which find particular utility as particulate x-ray contrast agent.

Description of Preferred Embodiments In Formula I above, X is H, NR^-R ² or CONR ¹ R ³ .

R ¹ is H or a linear or branched alkyl, preferably containing from 1 to 20 carbon atoms, more preferably 1 to 16 carbon atoms, such as methyl, ethyl, propyl, iβopropyl, butyl, pentyl, hexyl and the like, fluoroalkyl, the alkyl portion of which is as defined above and containing from 1 to (2n+l) fluorine atoms (where n is the number of carbon atoms in the alkyl group, such as trifluoromethyl) cycloalkyl, preferably containing 3 to 8 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl and substituted alkyls such as alkylβ substituted with one or more hydroxyls, alkoxyls (OR ¹ ) , or X and the like.

R is an acyl group containing from 2 to 16 carbon atoms such as COCH3, COC ₃ H ₇ , COC ₄ H _g , and including hydroxy substituted acyl groups such as COCH ₂ OH, COCH(OH)CH ₃ , COCH(OH)CH ₂ OH and the like.

R ³ is H, an alkyl preferably containing from 1 to 16 carbon atoms such as described above (when Z has the first two definitions given above) , hydroxy- substituted alkyl containing 1 to 16 carbon atoms and up to (n-1) hydroxyls where n is the number of carbon atoms, aryl, preferably containing from 6 to 10 carbon

atoms such as phenyl and naphthyl or (when Z has the first two definitions as given above) a steroidally derived moiety such as cholesteryl.

X is most preferably NHCOCH3.

Y is NHCOCH3.

NR^-R ² or CONR ¹ R ³ wherein R ¹ , R ² and R ³ are as described above., Y is most preferably NHCOCH3. In one class of compounds (Formula IA)

Z is OR 3, wherein R ^■ "! is as described above or

wherein X and Y are as described above.

In another class of compounds (Formula IB) Z is (linear or branched) alkyl, preferably containing from 1 to 20 carbon atoms, more preferably 6 to 18 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl and the like, fluoroalkyl, the alkyl portion of which is defined above and containing from 1 to (2n+l) fluorine atoms (where n is the number of carbon atoms in the alkyl group) , such as trifluoromethyl, cycloalkyl, preferably containing from 3 to 8 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl; aryl, preferably containing from 6 to 10 carbon atoms, such as phenyl and naphthyl; or aralkyl preferably containing from 7 to 12 carbon atoms; such as benzyl. In another class of compounds (Formula IC) Z is NR*R ⁴ and R ⁴ is H, alkyl, aryl or CH(R ⁵ )C0 ₂ R ¹ where R ⁵ is H, methyl, benzyl, or the like such that the moiety NH(R ⁵ )C0 ₂ R ¹ is derived from one of the 20 naturally occurring amino acids (glycine, alanine, phenylalanine,

and the like) .

The compounds of Formula IA can be prepared by acylation of the iodinated bβnzoate anion by the appropriate chloroformate such as:

wherein M is Na, K, Ca, or NR ⁵ ₄ where R ⁵ is alkyl having 1 to 4 carbon atoms, aralkyl, R is alkyl having from 1 to 4 carbon atoms or mixtures thereof and L is a leaving group such as Cl, Br, F, I or R ⁶ S0 ₃ where R ⁶ is methyl or tolyl.

The compound of Formula IB can be prepared by acylation of the iodinated benzoate having the structure

where X and Y are as defined above and M is selected from the group consisting of Na, R, Ca and NR ₄ wherein R ³ is alkyl having 1 to 4 carbon atoms such as methyl, ethyl, propyl, isopropyl and butyl and the like or aralkyl preferably containing from 7 to 10 carbon atoms and as benzyl, with an acid chloride or anhydride having the structure

0 0 0

Cl—C—Z or Z"^0-^Z wherein Z is as described above. The compound of Formula IC can be prepared

when Z is NR ¹ R ⁴ where both R ¹ and R ⁴ are not H, by acylating an iodinated benzoate having the structure

wherein X and Y are as defined above and M is selected from the group consisting of Na, K, Ca and NR ³ ₄ wherein

₃ R ^J is alkyl having 1 to 4 carbon atoms such as methyl, ethyl, propyl, isopropyl and butyl and the like or aralkyl preferably consisting of 7 to 10 carbon atoms such as benzyl with a carbamyl chloride having the structure

wherein 1 and R4 are as defined above and where both

Alternatively, the compound of Formula IC can be prepared when Z is NR 1R where R1 = H and R4 is not H, by acylating an iodinated benzoate having the structure

₄ with an isocyanate having the structure 0=C=NR' wherein is as defined above.

The above reactions can be run in any suitable non-nucleophilic solvent where both starting components have full or partial solubility. Preferred solvents include N,N-dimethylformamide,

dimethylsulfoxide and N,N-dimethylacetamide. The iodinated benzoate starting material is preferably derived from diatriazoic acid, metrizoic acid, iothalamic acid, iodipamide and the like. The iodinated compounds can contain substituents which do not deleteriously affect the contrast-enhancing capability of the compound. For example, the alkyl, cycloalkyl, aryl, aralkyl and alkoxy groups in Formula IA above can be unsubstituted or substituted with various substituents which do not adversely affect the stability or efficacy of the compounds as x-ray contrast agents such as alkyl, cycloalkyl, aryl, aralkyl, alkoxy, hydroxy, acyloxy, halogen, such as chlorine, bromine and iodine, acylamino, carboalkoxy, carbamyl and the like.

When Z in preparing compounds of Formula IA is

the reaction involves the acylation of benzoates with phosgene or a phosgene equivalent such as carbonyldiimidazole, bis(trichloromethyl)carbonate or the like such as:

Y -» formula IA wherein Z is

This reaction takes place in suitable solvents such as N,N-dimethylacetamide and N,N- dimethylformamide and the like. Applicable temperatures for this reaction scheme are from -60 to 100°C and useful pressures are 1 to 3 atmospheres. When used as an x-ray contrast agent, the compounds of this invention preferably comprise at least about 30%, more preferably at least 35%, and most preferably at least 40% iodine by weight.

In preferred embodiments, the compounds of this invention can be formulated into particulate x-ray contrast compositions, preferably nanoparticulate x-ray contrast compositions, as described in commonly-owned EP-A 498,482. Preferred compounds exhibit a melting point of greater than 150°C. Such nanoparticulate compositions can be prepared by dispersing the compounds of the invention in a liquid dispersion medium, and wet grinding the compound in the presence of rigid grinding media and a surface modifier to form the nanoparticleβ. Alternatively, the surface modifier can be contacted with the compound after attrition. Preferred surface modifiers include nonionic surfactants.

In preferred embodiments, the surface modifier is a high molecular weight nonionic surfactant. Preferred surfactants include poloxamers such as Pluronic™ F68 and F108, which are block copolymers of ethylene oxide and propylene oxide, poloxamines, such as Tetronic™ 908, which is tetrafunctional block copolymer derived from sequential addition of propylene oxide and ethylene oxide to

ethylenediamine, and dialkyl esters of sodium sulfosuccinic acid, such as dioctylsulfosuccinate sodium (DOSS) . The concentrations of the surface modifier can vary from about 0.1-75%, preferably 1-60%, and more preferably 5-25% by weight based on the total combined weight of the contrast agent and surface modifier.

In preferred embodiments, the x-ray contrast compositions in the form of surface modified nanoparticles can be associated with a cloud point modifier to further enhance stability during steam heat autoclaving, i.e., the cloud point modifier can reduce particle aggregation during heat sterilization. Preferred cloud point modifiers include nonionic cloud point modifiers, such as polyethylene glycols such as PEG 400, propylene glycol, ethanol, hydroxypropylcyclodextrin and glycerol; ionic cloud point modifiers, such as those described in U.S. Patent No. 5,298,262 including dialkylesters of sodium sulfosuccinic acid such as the dioctylester of sodium sulfosuccinic acid (DOSS) ; and charged phospholipids, such as diacylphosphatidyl glycerol e.g., dimyristoylphoβphatidyl glycerol. The cloud point modifier can be present in an amount of 0.005-50%, preferably 0.01-30% and more preferably 0.05-20% by weight based on the total weight of the x-ray contrast composition.

The x-ray contrast compositions of this invention comprise the above-described compounds, preferably in the form of particles, and a physiologically acceptable carrier therefor. For example, the particles can be dispersed in an aqueous liquid which serves as the carrier for the x-ray contrast agent. Other suitable carriers include liquid carriers such as mixed aqueous and nonaqueous solvents, such as alcohol; gels; gases, such as air; and powders. The x-ray contrast compositions can comprise

from about 1-99.9%, preferably 2-45% and more preferably 10-30% by weight of the above-described particles, the remainder of the compositions being the carrier, additives and the like. Compositions up to about 100% by weight of the particles are contemplated when the composition is in a lyophilizβd form. The dose of the contrast agent to be administered can be selected according to techniques known to those skilled in the art such that a sufficient contrast enhancing effect is obtained.

Typical doses can range from 20 to 450 mg of iodine per kilogram of body weight of the subject for many imaging applications. For some applications, e.g., lymphography, lower doses, e.g., 0.5-20 mg I/kg, can be effective. For blood pool imaging, the dose can range from 50 to 450 mg of iodine per kilogram of body weight and preferably from 100 to 250 mg of iodine per kilogram of body weight. For liver and spleen, the dose can be from 50 to 300 mg iodine per kg body weight.

The x-ray contrast compositions can contain one or more conventional additives used to control and/or enhance the properties of the x-ray contrast agent. For example, thickening agents such as dextran or human serum albumin, buffers, viscosity regulating agents, suspending agents, peptizing agents, anti- clotting agents, mixing agents, and other drugs and the like can be added. A partial listing of certain specific additives includes gums, sugars such as dextran, human serum albumin, gelatin, sodium alginate, agar, dextrin, pectin and sodium carboxymethyl cellulose. Such additives, surface active agents, preservatives and the like can be incorporated into the compositions of the invention. A method for diagnostic imaging for use in medical procedures in accordance with this invention comprises administering to the body of a test subject

in need of an x-ray an effective contrast producing amount of the above-described x-ray contrast composition. In addition to human patients, the test subject can include mammalian species such as rabbits, dogs, cats, monkeys, sheep, pigs, horses, bovine animals and the like. Thereafter, at least a portion of the body containing the administered contrast agent is exposed to x-rays to produce an x-ray image pattern corresponding to the presence of the contrast agent. The image pattern can then be visualized. For example, any x-ray visualization technique, preferably, a high contrast technique such as computed tomography, can be applied in a convention manner. Alternatively, the image pattern can be observed directly on an x-ray sensitive phosphor screen-silver halide photographic film combination.

The compositions of this invention can be administered by a variety of routes depending on the type of procedure and the anatomical orientation of this tissue being examined. Suitable administration routes include intravascular (arterial or venous) administration by catheter, intravenous injection, rectal administration, subcutaneous administration, intramuscular administration, intralesional administration, intrathecal administration, intracisternal administration, oral administration, administration via inhalation, administration directly into a body cavity, e.g., arthrography, and the like. In addition to preferred applications, i.e., for blood pool and lymph node imaging, the x-ray contrast compositions of this invention are also expected to be useful as contrast agents for any organ or body cavity. For example, the compositions of this invention are expected to be useful as angiographic contrast media, urographic contrast media, myelographic contrast media, gastrointestinal contrast media, cholecystographic and cholangiographic contrast media,

arthrographic contrast media, hysterosalpingographic contrast media, oral contrast media and bronchographic contrast media.

The following examples further illustrate the invention.

The following compounds set forth in Table I are specific illustrative examples of preferred compounds in accordance with this invention that have been prepared. These compounds conform to formula IA above.

Table I

Compound X Y Z

1 NHCOCH3 NHCOCH3 OEt

2 NHCOCH3 NHCOCH3 OiBu

3 NHCOCH3 NHCOCH3 0- (2-ethyl) - hexyl

4 NHCOCH3 NHCOCH3 0- cholesteryl Example 1

Prepi ..ration of Compound 3 from Table I

To a stirred solution of sodium diatrizoate (100 g, 159.3 mmol) in dry DMF (300 ml) was added 33.7 g 2-ethylhexyl chloroformate (174.7 mmol) . The solution was heated overnight at 90°C, then cooled to 60°C. whereupon the reaction mixture was poured slowly into water (3 L) . The resulting white precipitate was concentrated by filtration and dried at 90°C. under high vacuum to give 78 grams of pure compound having a melting point of 229°C. to 232°C. Example 2

Preparation of Compound 4 from Table I To a stirred solution of sodium diatrizoate (100 g, 159.3 mmol) in dry DMF (300 ml) was added cholesteryl chloroformate (71.5 g, 159.3 mmol) and the mixture was then heated at 80°C. overnight. After cooling to 60°C, the mixture was slowly poured into 3 L of water with stirring. The solid formed was

filtered, washed with water and dried at 60°C. under vacuum for 14 hours. The yield was 130 g.

Example 3 Preparation of Compound 2 in TnV.ι«-> T To a stirred solution of sodium diatrizoate

(100 g, 159.3 mmol) in dry DMF (600 ml) was added 21.7 g isobutyl chloroformate (159.3 mmol) slowly over 2 minutes. The reaction mixture was stirred at room temperature overnight and poured into 3 L of water. After filtration, the white precipitate was dried in a high vacuum at a temperature of 90°C. overnight. The product was recrystallized by adding a DMF solution to water, filtered and dried. The final product had a yield of 54 g and a melting point of 237°C. to 240°C. Example 4

Preparation of Compound 1 of »M» T To 300 ml DMF warmed to 35°C. was added 90 g sodium diatrizoate (0.142 mol) and 14.9 ml (0.156 mol) ethyl chloroformate was added over 2 minutes dropwise with stirring. The mixture was warmed to 70°C. and stirred for 10 minutes. It was then cooled to 20°C. in an ice bath and poured into 3 liters of water at 15°C. over 90 seconds with vigorous stirring.

After 5 minutes of additional stirring, the reaction mixture was filtered, washed twice with 250 ml of water and dried overnight under suction and then under vacuum over P ₂ °5 (70°C, 48 hours) . The solids were ground to a fine powder and drying was continued over P ₂ °5 (70 ^β C, 24 hours). The resulting product had a yield of 80.1 gms of white powder. Recryβtallization was performed by dissolving 76.3 grams of the product in 150 ml anhydrous DMF with warming. The mixture was cooled in ice to 20°C. and poured into 1.5 L water (16°C.) with stirring over 60 seconds. After filtering and washing twice with 100 ml water, the mixture was suction-dried then dried under vacuum (70°C./20 hours) over P ₂ 0 ₅ .

The final product yield was 68.7 grams of white solid.

Example 5 General Procedure for preparation of carboxylic anhydrides of Formula IB To a solution of 200 mmol of the iodinated benzoate sodium salt in 350 mL of DMF can be added 220 mmol of the appropriate carboxylic chloride or anhydride. After stirring the mixture at a temperature in the range of -10°C. to 60°C. from one to 24 hour, the mixture can be poured into 3-4 L of water, dried under suction and dried under vacuum over P_>0 ₅ from 20- 70°C. Further purification, if necessary, can be carried out by pouring a 25-50% (w/v) solution of the product in DMF into 10 volumes of water, filtering and drying as before.

Example 6 General procedure for preparation of carbamic anhydrides of Formula IC where Z is NR^'R : and both R 1 and R4 are not H To a solution of 200 mmol of the iodinated benzoate sodium salt in 350 mL of DMF can be added 220 mmol of the appropriate carbamyl chloride. After stirring the mixture at a temperature in the range of

-10°C. to 60 ^β C. from one to 24 hour, the mixture can be poured into 3-4 L of water, dried under suction and dried under vacuum over P ₂ °5 from 20-70°C. Further purification, if necessary, can be carried out by pouring a 25-50% (w/v) solution of the product in DMF into 10 volumes of water, filtering and drying as before.

Example 7

General procedure for preparation of c»τΗ->n.*.<- anhydrides of Formula IC where Z is NR^-R ⁴ ; and R ¹ is H

The preparation of Example 6 can be followed except with use of an isocyanate rather than a carbamyl chloride and the same results should be obtained.

Preparation of Nanoparticulate Compound 2 Contrast Agents with Pluronic F68, Pluronic F108. or Tetronic T-908 Compound 1 can be added to each of 3 x 1.5 oz brown glass bottles containing approximately 12 ml of zirconium silicate (1.1 media.) beads in an amount sufficient to be 15% (w/v) of the final suspension. Bottle A can contain 3% (w/v) Pluronic F-68. Bottle B can contain 3% (w/v) Pluronic F108. Bottle C can contain 3% (w/v) Tetronic T-908. The resulting suspensions were milled on a roller mill at approximately 150 rpm for a total of 9 days.

Examples 11-12 Preparation of Nanoparticulate Compound 2 in Table I Contrast Agent with Pluronic F108 and Blood Pool

Imaging 15% of Compound 2 can be milled with 4% Pluronic F-108 in the presence of zirconium silicate (1.1 mm dia) beads for 3 days under aseptic conditions. No additional salts or surfactants are added.

This sample can be examined for imaging efficacy as follows. The sample can be injected into white New Zealand rabbits at a dose of 3 ml/kg as a slow bolus injection. At times of 5, 15, 30, 60 and 120 min. post injection, the opacification of the liver, spleen, and blood pool as measured in the aorta and within the left ventricle can be determined by computed tomography (CT) using Toshiba 900S Imager CT scanner and associated software. Results from this analysis should indicate that formulation of Compound 2 should show excellent blood pool opacification in excess of 30 min. followed by very good liver and very good spleen opacification. Imaging at 24 hours post injection should show complete clearance from the blood with partial clearance from the liver and spleen.

^• gy-itnpl 0* 13

Preparation of an Autoclavable Formulation of Nanoparticulate Compound 2 in T*^** T Contrast Agent with Pluronic F108 and PEG 400 and Lvmphographv Imaging

Compound 2 can be milled with zirconium silicate (1.1 mm _. dia) beads in the presence of Pluronic F-108 for 3 days. At this point, sterile PEG 400 can be added to the suspension such that at completion, the formulation contained 15% (w/v) WIN 70146, 3% (w/v) Pluronic F-108, and 10% (w/v) PEG 400. This formulation can then be autoclaved under standard conditions, i.e., 121°C. for 20 min., resulting in a final nanoparticulate size of less than 1000 nm. This formulation can be evaluated for both blood pool and lymphographic imaging in New Zealand White Rabbits using the above-described protocol (3 ml/kg) for blood pool imaging and 2 injections (0.25 ml) per paw for lymphography. The results should indicate that Compound 2 is capable of blood pool opacification to at least 30 min. and is an excellent lymphography agent affording high levels of opacification. Scanning can be carried out using a Toshiba 90OS Imager CT scanner and image density calculated from iodinated standards imaged simultaneously with the animals.

The invention has been described in detail with particulate reference to certain preferred embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and scope of the invention.