CA2222429A1 - Contrast medium and its use - Google Patents

Abstract

Contrast medium comprising an effective amount of a contrast agent and a protecting agent wherein the protecting agent is active in protecting against possible damaging effects of the contrast medium.

Description

CA 02222429 1997-ll-26 CONTRAST MEDIUM AND ITS USE

FIELD OF THE INVENTION
The present invention is generally in the field of contrast media.
More specifically, the present invention relates to a contrast medium with illlproved patient's tolerance.
s PRIOR ART
The following is a list of references which are believed to be pertinent as prior art to the present invention:

1. Hirshfeld, J.W., N. Engl. J. Med., ~, 482-484, (1989).

2. Sovak M., Handbook of Experimental Pharmacology, ~, 1-19, Springer-Verlag, Berlin (1984).
15 3. Gmelin E., et al., Recent developments in nonionic contrast media, pp.
67-69, Thieme Med. Pub. Inc., N.Y. (1989).
4. Scherberich, J.E., et al., Recent developments in nonionic contrast media, pp. 1-94, Thieme Med. Pub. Inc., N.Y. (1989).
5. Hou S.H., et al., Am. J. Med., ~, 243-248, (1983).
6. Schwab, S.J., et al., N. Engl. J. Med., ~, 149-153, (1989).

W O96/38183 PCT~US96107666 7. Morgan D.M.L. and Bettm~nn, Cardiovasc. Intervent. Radiol., ~, 154-160, (1989).
8. Hoffman E.K. and Simonsen L.O., Physiol. Rev., ~2, 315-382, (1989).
9. Cala, M.C., et al., Comp. Biochem. Physiol., 90A, 551-555, (1988).
10. Busa W.B., Annu. Rev. Cell Physiol., ~, 3~9-402, (1986).
11. Dascalu A., Peer A., Academic Rad., 1 145-150, (1994).
The above references will be referred to herein by indicating, within brackets, the number from the above list.

BACKGROUND OF THE INVENTION
Contrast Media (hereinafter "CM") are routinely used in various im~ging procedures. Such procedures include visll~li7~tion of blood vessels in cardiac angiography, either by x-ray im~ging or by Magnetic Resonance Tm~ging (MRI), intravenous urography (kidney im~ing), computerized tomography and neurologic visll~li7~tion of the spinal cord, the brain, etc. In the U.S.A. alone, there are more than 10 million x-ray radiologic ex~min~tiorls using CM, performed each year. 5 to 10~o of these procedures are accompa-nied by clinical side effects; in 1 out of 1000-2000 of such procedures, there occurs a life threatening complication.
The currently used CM in the x-ray im~gin,, procedure can be grouped, on the basis of their osmolarity, to such which have a low osmolarity (hereinafter "LOCN") and such having a high osmolarity (hereinafter "HOCM"). It should be noted that both LOCM and HOCM have an osmolarity which is above that of the blood. HOCM have a typical osmolarity of about 1500 - 2000 mOsm/kg and LOCM have an osmolarity within the range of 300 - 700 mOsm/kg. Adverse side effects associated with CMs include such which result from the high osmolarity. The introduction of LOCMs, which are the new generation of CMs. was meant to counter some of these side effects. It should be noted that one big disadvantage of LOCMs is their high price-tag (about 5-10 times that of HOCM). Therefore, there is a dispute of the kinds of x-ray CMs which should be used(l).
The visll~li7~tion which is the outcome of CM injection into the blood, results from a local dispersion of the high iodine atom concentration 5 contained in the CM, from the high osmolarity, as well as from an increased viscosity in the blood vessels of the visualized organ(~). In procedures whereinthe CMs are introduced into the blood, this is achieved either by injection intothe blood vessels or by catheterization.
The MRI method for vis~l~li7~tion of blood vessels comprises the 10 injection of a paramagnetic substance dissolved in a hyperosmotic CM to the region to be vicu~li7ed.
Individuals subjected to procedures involving the use of CMs, are exposed to several hazards, depending on the CM used, including:
1. Hyperosmotic damage: Typically 100-200 ml of CM are injected into a 15 total plasma volume of 5 liters within a period of several minlltes Cells such as endothelial cells, red and white blood cells, cells within the kidney, etc., are exposed to a hyperosmotic solution, re~chin~ 200-2000 mOsm/kg at the site of injection, as compared to the osmolarity of the blood with its 300 mOsm/kg, giving rise to a hyperosmotic shock which may elicit related 20 damages. In the following description the term "hyperosmotic CM" will refer to any CM having osmolarity higher than the blood osmolarity which is typically 300 mOsm/kg.
2. Iodine specific toxicity: In an x-ray visu~li7~tion procedure typically 30-40 grams of iodine (included within the contrast media) are injected into the 25 blood within the period of 2-10 minutes. It should be noted that target vis~l~li7~tion requires a minimllm accllm~ tion of 1~-20 mg of iodine/ml in the target tissue(3) and this is the reason that the initial iodine concentration in the CM is relatively high in the range of 300-420 mg iodine/ml.

3. Kidney damage: The iodine load to which the kidney is exposed and 30 which it has to secrete is a potential cause for renal damage(~). It is generally believed today that 12~o of all patients which are injected with an x-ray CM, encounter renal complications(5). A recent study shows that during cardiac catheterization procedure, 9% of low risk and 16~o of high risk patients develop renal failure(6). Various studies made with CMs have shown that exposure of cells to x-ray CM causes the cell damages(' 8 9 l0).

It is an object of the present invention to provide a novel contrast medium composition. It is particularly an object of the invention to provide a contrast medium composition with lower hazardous side effects as compared to hitherto used CM compositions.
It is more particularly the object of the present invention to provide such a composition comprising, in addition to the contrast medium, also other agents which protect tissue against damages which can be in~licted by the CM
by either direct cytotoxic effects or hyperosmotic effects. According to a particular embodiment, it is the object of the present invention to provide 15 such a composition cont~inin~ endothelial and kidney cells protecting agents.It is further the object of the present invention to provide a method for protecting tissue and organs within the body from harrnful effect which may otherwise be inflicted by the CM.
In the following description, the term "contrast agent" or "CA" will 20 be used to denote an agent which absorbs or reacts in another way with the electromagnetic irradiation used in a body visualization procedure. The CA, which is injected into the circulation, may be an agent, such as an iodine containing substance, which blocks x-ray irradiation and can thus be used for the vis11~1i7~tion of blood vessels or other body fluid-cont~inin~ organs or 25 tissue in x-ray vis~-~1i7~tion procedure. The CA may also be a par~m~netic substance used in an MRI vis~1~1i7~tion procedure.
The term "contrast medium" or "CM" will be used to denote a liquid composition comprising a CA which is injected into the circularization prior to visl1~li7~tion process. The CM is typically a hyperosmotic composi-30 tion.
The term "protecting agent" or "PA" ~~ill be used to denote anagent which in accordance with the present invention is incorporated into a CA 02222429 1997-ll-26 W O96/38183 PCT~US96/07666 s CM, for the purpose of protecting tissue or organs, fully or partially, against some or all of the hazardous effects of CM's noted above. The CM in accordance with the present invention thus comprises both a CA and a PA.
In the following the term "effective amount" will be used to S denote an amount of an agent sufficient to achieve the desired effect. In the case of a CA, the effective amount is an amount sufficient to achieve the desired contrast. In the case of a PA, an effective amount is an amount sufficient to achieve a protecting effect.
The present invention provides by a first of its aspects, a contrast medium, comprising an effective amount of a contrast agent, and an effective amount of a protecting agent being an agent active in protecting against possible cl~m~ging effects of the contrast medium, said protecting agent being one or more members selected from the group consisting of (i) non-steroidal anti-infl~mm~tory drugs (NSAID), (ii) agents which induce cells to generate nitrous oxides (NO), (iii) polysaccharides capable of sealing the intercellular spaces which exist between endothelial cells, (iv) direct antico~ ntc of the heparin class and (v) antioxidants which are free radical scavengers.
The present invention further provides, by a second of its aspects, use of said protecting agents in the preparation of a contrast medium.
The invention still further provides, by a third of its aspects, a method for protecting an individual against harrnful effects of a CM, comprising ~(lminictering to the individual, together with the contrast medium, also a protecting agent as defined above.
The non-steroidal anti-infl~mm~tory drugs (NSAID) are compounds which have an anti-infl~mm~tory, and in addition anti-pyretic and analgesic activities. Examples of NSAID are:
- Aniline derivatives such as paracetamol, phenacetin;
- Anthranilic derivatives such as flnf~n~mic acid, mefenamic acid;
- Phenyl alkanoic derivatives such as fenoprofen, flurbiprofen, ibufrofen, ketoprofen;
- Pyrazole derivatives such as amidopyrine. dipyrone, phenazone, phenylbutazone;

W O96/38183 PCTrUS96/07666 - Salicylic acid derivatives such as aspirin, salicylic acid, choline salicylate.
Examples of agents which induce cells to generate nitrous oxide (NO) are nitroglycerine, l~i~op~Llsside, isosorbide dinitrate and others.
Polysaccharides which act in sealing the intercellular spaces between endothelial cells are agents capable of forming a coating over the walls of blood vessels and of forming a barrier between the interior of the blood vessel and the cells. An example of such an agent is dextran.
Examples of direct anti-coagulants of the heparin class are: heparin, low molecular weigbt heparins (such as inoxaprine) and the like.
Anti-oxidants which are free radical scavengers are useful particularly where the CA is of a kind that is capable of inducing the formation of free radicals which cause cellular damage. Examples for such agents are glllt~thione, tocopherols and the like.
The invention will now be illustrated in the following examples with occasional reference to the annexed drawings:

BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows the effect of CA (added at tl and t,) on intracellular calcium endothelial cells in vitro.
Fig. 2 demonstrates the effect of the combination of PA and CA upon the intracellular calcium (CA and PA were added together at tl then washed and then CA was added separately at t~).

EXAMPLES
2~ Cell Culture Preparation Human umbilical vascular endothelial cells (HUVEC) CRL-1730 (American Type Culture Collect) were cultured according to known techniques. Briefly, HUVEC were grown in F 12K medium (Irving Scl. USA) containing 30 llg/ml Endothelial Cell Growth Factor. 100 ~lg/ml heparin, 1OO~G
fetal calf serum and 5G~o neonatal calf serum. Cells were seeded at an initial density of 1-2 x 105 cells/cm' and kept at 370C under an atmosphere of 50~G
CO~ in air. To all such mediums, 4.5 mg/ml glutamine, 50 units/ml penicillin~

200 ,ug/ml of streptomycin and 1250 units/mi nystatin were added. Media were changed twice weekly and cells were grown for about 7-10 days until confluence. Confluent cells were trypsinized and experiments were performed when trypan blue was excluded from 95% or more of the cells.

Measuring Techniques Image analysis procedu~es and calibration [Ca+2] analysis was performed by using an Applied ~m~ging Magical system (TARDIS version 7.3). Cover slips with attached endothelial cells in the epifluorescence mode (oil-immersion objective, X 40) and equipped with a xenon lamp. Intracellular calcium measurements were assayed by ratio im~ging of FURA-2/AM (excitation, 340 and 380 nm; emission, 515 mn). A
temperature-controlled cell perfusion chamber (Applied im~ging) was used to keep cells at a temperature of 32 o C. All solutions were m~int~ined at 32 o C
in a temperature controlled bath. Single cell images (10-40 cells) were collected by an intensified video camera (Photonic Science). The video signal was averaged over 4 video frames and averaged image pairs (340 and 380 nm) were captured every 5-15 seconds. Images were digitized at 256 x 256 pixels and averaged after subtracting the background.
Intracellular calcium calibration: Fura-2/AM signals were calibrated by addition of lonomycin (5 ,uM) to cells kept in a 2 mM Ca+2 solution to obtain m~imllm fluorescence. An addition of 10 mM of [ethylene-bis- (oxyethylenenitrilo)]-tetraacetic acid) (EGTA) followed in order to adjust the pH to > 8.5 in order to obtain the minim~l fluorescence. The intracellular calcium was calculated as previously described employing a Ca+2-Fura 2 dissociation constant of 224 nM.

Tetrazolium salt assay The 3-[4,5-Dimethythiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) colorimetric reaction is based on the uptake of tetrazolium salt exclusively by live cells and its reduction to a soluble violet (formazan) compound. Absorbance of forma~an is proportional to the amount of active CA 02222429 1997-ll-26 W O96/38183 PCTrUS96/07666 mitochondrial enzyme succinate-dehydrogenase of the cells and consequently to cell viability. Both endothelial or kidney cells were seeded at 30000-40000 cells/well in 96-well microliter wells (Corning) and grown until confluence.
The measurements were performed after statistically testing the assumption 5 that endothelial cells as well as the kidney cell line display contact inhibited cell growth, therefore yielding about equal cell numbers in each well. The absorbance spectrum of M~ was determined by a diode array spectropho-tometer (Hewlett Packard, 8452A). Ml-r exhibited peak absorbance at 560 nm and minim~l re~ingc beyond 620 nm, as previously shown. A microplate 10 reader (Thermomax, Molecular Devices) was used to read absorbance at 550 nm with background subtraction at a reference absorbance of 650 nm at 25 o C.
The experiments employed confluent cells which were loaded with 0.15 ~lg/rnl of MlT and dark incubated at 370C. Four hours later the medium was removed by plate flipping and 100 ~g/ml DMSO were added to 15 each well in order to solubilize the formazan crystals. Results are expressedin percentages as compared to control values of untreated cells and each result consisted of four to six repeated measurements in at least two different experiments. HUVEC cells were exposed to CM for either 4 or 24 hours prior to optical density re~-ling~
PRIOR ART REFERENCE E~'ERIMENTS
The cytotoxic effect of CA materials on these cells was hitherto evaluated by Dascalu and Peer (1994)(11), in a series of experiments wherein the viability of the cells was determined in the presence or absence of CA.
25 This evaluation was done by determining the tetrazolium salt (Ml-r) which was reduced to violet formazan as will be further elaborated. This reaction required the activity of mitochondrial dehydrogenase and thus is indicative of cell viability. The results presented there show that both endothelial and kidney cells are damaged when exposed to CA for ~ hours.

Example 1 The effect of using PA in conjunction with CA materials was evaluated by determining the cytotoxic effect and intracellular calcium levels in endothelial cells with or without PA.
S The effect of CA (in this case Telebrix~, (Guerbet, France)) can be seen in Fig. 1. As can be seen, upon addition of 3S ~l of CA into 1 rnl of hepes-buffered saline at t1, there is an imrnediate increase in the intracellular calcium level. The calcium level increase also occurs upon re-addition of the same CA at t2.
When a CA as described above was ~minictered together with indomethacin, as shown in Fig. 2 (at t1), there was no or negligible increase in the intracellular calcium level. Upon re-~-lmini~tration of the CA, at t2, after the CA and indomethacin were washed out, there was a marked increase in the intracellular calcium level. This demonstrates the protective effect 15 which indomethacin has on such harmful effects of the CA.

Example 2 Endothelial cell viability was tested upon 4 hours of exposure to CA with or without a protecting agent (PA). The results are shown in the 20 following Table 1:

W O96/38183 PCTrUS96/07666 Table 1 CA PA : CA only CA+P~
CA1 A 142 + 17 199 + 40 CA1 A 228 + 26 265 + 29 CA1 A 407 + 52 452 + 43 CA1 A 707 + 53 768 + 16 CA2 A 678 + 94 835 + 62 CA2 A 315 + 38 369 + 38 CA3 A 774 + 103929 + 64 CA3 A 602 + 73 752 + 134 CA3 B 884 + 991059 + 205 CA2 B 355 + 18 402 + 56 CA4 B 124 + 24 22~ + 62 CA2 C 678 + 94 800 + 85 CA4 C 244 + 23 292 + 31 CA1 D 856 + 69 934 + 55 CA2 D 678 + 94 825 + 65 CA1 E 707 + 53 812 + 44 CA1 E 583 + 55 769 + 32 CA1 E 228 + 26 297 + 41 CA3 E 602 + 73 802 + 141 CA1 = Ultravist, Schering, AG, Germany CA2 = Hexabrix-320, Guerbet, France CA3 = Omnipaque, Schering AG, Germany CA4 = Conray-60, Malinckrodt, U.S.A.

A = Indomethacin (50 micromolar) B = Heparin (100 units/ml) C = Dextran (50 microgram/ml) D = Nitroprusside (50 micromolar) E = Glutathione (2 mM).

The average and the standard deviations of the absolute optical density readings of forrnazan absorption of light are presented in the above Table. The results given, reflect the rnitochondrial activity of the tested cells, and serve as an indicative tool to evaluate the cells viability.

Claims (3)

CLAIMS:

1. A contrast medium, comprising an effective amount of a contrast agent, and an effective amount of a protecting agent being an agent active in protecting against possible damaging effects of the contrast medium, said protecting agent being one or more members selected from the group consisting of:
(i) non-steroidal anti-inflammatory drugs (NSAID);
(ii) agents which induce cells to generate nitrous oxides (NO);
(iii) polysaccharides capable of sealing the intercellular spaces which exist between endothelial cells;
(iv) direct anticoagulants of the heparin class; and (v) antioxidants which are free radical scavengers.

2. Use of contrast medium in combination with a protecting agent selected from the group consisting of:
(i) non-steroidal anti-inflammatory drugs (NSAID);
(ii) agents which induce cells to generate nitrous oxides (NO);
(iii) polysaccharides capable of sealing the intercellular spaces which exist between endothelial cells;
(iv) direct anticoagulants of the heparin class; and (v) antioxidants which are free radical scavengers.

3. A method for protecting an individual against harmful effects of contrast media in medical procedure involving injection to said individual of a contrast medium, comprising administering to the individual a contrast medium in combination with a protecting agent being a member of the group consisting of:
(i) non-steroidal anti-inflammatory drugs (NSAID);
(ii) agents which induce cells to generate nitrous oxides (NO);
(iii) polysaccharides capable of sealing the intercellular spaces which exist between endothelial cells;

(iv) direct anticoagulants of the heparin class; and (v) antioxidants which are free radical scavengers.