THE USE OF BILE ACID DERIVATIVES

Abstract: The invention relates to the use of a coloured or fluorescent hepatobiliary targeting compound in the manufacture of an administrable composition for visualising the bile duct and biliary leakage during an operation in the region of the bile duct, and is useful as an aid to a surgeon performing an operation in the region of the gall bladder. The compound is preferably based on a steroid moiety.

Description: THE USE OF BILE ACID DERIVATIVES This invention relates to the novel use of bile acid derivatives, particularly bile acid derivatives carrying a fluorescent or visibly coloured moiety. Fluorescent bile acid probes for use in studying the mechanism of intra¬ cellular transport of bile acids have been disclosed by a number of workers, see for example Sherman, I.A. and Fisher, M.M., (1986) Hepatology 6: p 444-449 which discloses the use of glycocholic acid- FITC where the FITC (fluorescein isothiocyanate) moiety is on the 3α- hydroxyl group of the steroid nucleus; and Crawford et al, Biochim. Biophys. Acta. 1991 ;1085:223-234 which discloses a series of so-called fluorescent bile acids with a dansyl-ethylene diamine precursor condensed with the sulfonyl moiety of taurine, conjugated to intact bile salts via an amide linkage to give dansyl-tauro bile salts, the side chain carboxyl being blocked by the dansyl derivative. Whilst such compounds can assist in establishing the degree of liver function and can thereby assist surgeons in deciding whether to operate on the liver, we are not aware of any previous proposal to use such compounds in the way which is proposed in the present invention. We have now surprisingly discovered that these fluorescent bile acid derivatives can be used to assist surgeons in visualising the gall bladder and in identifying the existence of biliary leaks during operations, notably cholecystectomy operations. Accidental injury to the bile ducts during open cholecystectomy is the most severe complication of this operation with an incidence of one lesion per 300-500 cholecystectomies. The present invention is considered to be particularly useful in laparoscopic operations where the real impact of this complication in laparoscopic procedures is still controversial. Some authors believe that the frequency is comparable with, while others believe that it exceeds by up to tenfold, that of an open cholecystectomy. Bile duct injury necessitates complex and repeated operations with mortality and morbidity much higher than those of the original procedure. A significant long-term morbidity (recurrent stricture, cholangitis, cirrhosis, and premature death) can also follow these injuries, Injuries recognised intraoperatively have much better outcome compared with the extensive morbidity and mortality associates with biliary peritonitis caused by overlooked injuries. Both in open and laparoscopic procedures, bile duct injury usually represents failure to identify the anatomic structures in the triangle of Calot. Inability to identify anatomic structures properly is also the cause for conversion to an open procedure from a laparoscopic one, in almost every case. The length of the operating time, both laparoscopic and traditional, also depends on quick recognition of subhepatic anatomy. It is an object of the present invention to provide a way of facilitating visualisation of the biliary tree (including the gall bladder) and/or facilitating visualisation biliary leakage during operations in the vicinity of the biliary tree. According to one aspect of the present invention, there is provided the use of a coloured or fluorescent hepatobiliary targeting compound in the manufacture of an administrable composition for visualising the bile duct and/or visualising biliary leakage during an operation in the region of the bile duct. According to another aspect of the present invention, there is provided a method of visualising the bile duct and/or visualising biliary leakage, comprising administering to a patient an effective amount of a coloured or fluorescent hepatobiliary targeting compound The coloured or fluorescent hepatobiliary targeting compound is most preferably a fluorescent compound, most preferably a UV light fluorescent compound. The compound preferably comprises a steroid moiety having an unblocked 3-hydroxyl substituent and an unblocked carboxyl group attached by means of an amide linkage to the side chain of the steroid moiety, and an active moiety which is to be targeted to the liver, said active moiety being attached to the α-carbon atom relative to the unblocked carboxyl group. Preferably, said compound has the general formula ( I ):- wherein A is -αOH or -βOH; B is -αH or βH; C is,-H, -αOH or -βOH; or B and C together form a double bond; D is -H, -αOH or -βOH; E is -H, -αOH or β-OH; L is a linking moiety; J is said coloured or fluorescent moiety; and n is 0 or 1. Preferably, J is or includes a fluorescein, rhodamine or other fluorescing moiety. The linking moiety is preferably N-terminated at its end attached to active moiety J, and L may be:- -(CH 2 ) n NH, where n is 3 or 4, -(CH 2 ) 4 N H-(CH 2 ) 3 N HC( = N H)N H-, or -(CH 2 ) 2 -CH(OH)CH 2 NH-. Alternatively, the moiety -NH-CH(COOH)-L- may be derived from S- adenosylhomocysteine, S-adenosyl methionine, S-amino-imadazole-4- carboxamide, asparagine, cadaverine, cystamine, citrulline, diaminopimelic acid, 2, 4-diaminobutyric acid, cysteamine, glutamine, 3-hydroxykynurenine, kynurenine, putrescine or negamycin. Alternatively, acidic amino acids can be used instead of the above where active moiety J has one amino group and/or is hydrophobic. The steroid moiety is preferably a bile acid moiety based on cholic acid, chenodeoxycholic acid, deoxycholic acid, hyodeoxycholic acid, hyocholic acid, α-, β- or w-muricholic acid, nor-bile acids, lithocholic acid, 3β-hydroxycholenoic acid, ursodeoxycholic acid, allocholic acid (5α-cholan-24-oic-acid), or the like, hereinafter simply referred to as "a bile acid moiety". The compounds preferably used in the present invention can be produced by the steps of (a) reacting a steroid moiety having an unblocked 3-hydroxyl group and a side chain carboxyl group, with the α- amino group of an amino acid having a blocked amino group in the side chain, preferably at the δ or ε position relative to the carboxyl group to form an amide, (b) unblocking said blocked amine group, and (c) reacting the unblocked amine group with the active moiety, preferably an isocyanate or isothiocyanate derivative thereof. Alternatively, the active moiety, or preferably an isocyanate or isothiocyanate derivative thereof, is reacted with a side chain amino group of an amino acid having a blocked α-amino group, and the blocked α-amino group is unblocked and then reacted with the side chain carboxyl group of the steroid moiety. As a further alternative, when the active moiety has a carboxyl group and an amino group, the latter is reacted directly with the side chain carboxyl group of the steriod moiety. Such processes of preparation enable the 3-hydroxyl group and also a side chain carboxy group in the compound to remain unblocked. In the case where the bile acid moiety is cholic acid, it will be appreciated that the above described reaction produces a moiety based on glycocholic acid, which is another bile acid. The invention will now be described in further detail in the following description:- Synthesis of Cholyl-Lysyl-Fluoresceiπ A suspension of N-ε-CBZ-L-lysine methyl ester hydrochloride (7mmol) in 70ml ethyl acetate containing triethylamine (1 ml) was stirred at 25°C for 30 minutes. Cholic acid (5mmol) and N-ethoxycarbonyl-2-ethoxy-1,2- dihydroquinoline (EEDQ) were then added to the solution. After stirring at 25°C for 10 minutes, the suspension was refluxed on a steam bath over night. The resulting suspension was cooled to room temperature. The residue was filtered, dissolved in 10 ml of boiling ethanol on a steam bath, which 10 ml of 10% K 2 C0 3 solution was added slowly to the ethanolic solution to maintain a clear solution. The solution was heated on the steam bath for 15 minutes and then evaporated in vacuo to half its original volume. After diluting the solution with 20 ml of water, it was acidified with 0.5m HCl. The precipitate was collected and washed with water to obtain cholyl-N-ε-CBZ-L-lysine. The cholyl-lysine- CBZ was subjected to catalytic transfer hydrogenation by dissolving about 200 mg of cholyl-lysine-CBZ in 10ml of 4.4% formic acid- methanol, adding to a 25ml flask containing 200 mg of palladium black and 10 ml of 4.4% formic acid-methanol, and then stirring the mixture under a nitrogen atmosphere ovemight. The solution was evaporated to dryness at 40 ϋ C and then triturated with ethyl acetate to obtain cholyl- lysine formate. The sodium salt was obtained by dissolving the formate salt in 5ml of a 0.1 M NaOH solution in methanol and precipitating with diethyl ether. 50mg of the sodium salt of cholyl-lysine obtained was then dissolved in 5ml of bicarbonate buffer (pH 9). 28mg of fluorescein solution in bicarbonate buffer (5ml) were added and the reagents stirred at rom temperature for 18 hours. Free fluorescein was removed by percolating cholyl-lysyl-fluorescein through Sep-Pak C 18 cartridges. Further purification was by thin layer chromatography to obtain homogenous cholyl-lysl-fluorescein of the formula:- Rhodamine B can also be attached to a bile salt-lysine conjugate in an analogous manner. The following further fluorescent bile salt derivatives may be prepared using analogous techniques:- R-Lys-Fluorescein R-Lys-Rhodamine B R-Ornithyl-Fluorescein R-Ornithyl-Rhodamine B R-HydroxyLys-Fluorescein R-HydroxyLys-Rhodamine B R-Arg-Fluorescein R-Arg-Rhodamine B where R = cholic acid, chenodeoxycholic acid, deoxycholic acid, hyodeoxycholic acid, hyocholic acid, α-,β- or ω-muricholic acid, nor-bile acids, lithocholic acid, 3β-hydroxy-cholenoic acid, ursodeoxycholic acid, or allocholic acid. Intraoperative imaging of the biliary tree with cholyl-lysyl-fluorescein (CLF) in animals has been studied and found to be extremely easy and simple to perform, involving an ordinary intravenous injection and the exposure of the hepatic region to the light of a Wood lamp (or other source of UV light). The fluorescein intensity was maximal after about 3 minutes and enabled excellent visualization of the entire biliary tree including the gall bladder which shined for the whole time of observation (20 min) and persisted much longer than the experimental observation limit (up to 45 min). The active moieties of cholyl-lysyl-fluorescein (glycocholate and fluorescein) have been used safely for a long time as intravenous injections in humans, and so it is considered that CLF is also safe to use in the manner proposed in the present invention. It is considered that an intravenous injection of between about 0.5 and 2mg of CLF per kilogram body weight is sufficient for effective visualisation of the biliary tree including the gallbladder. A typical method of using a UV light-fluorescent hepatobiliary targeting compound in accordance with the present invention will now be described:- 1. About 20 minutes prior to performing a cholecystectomy operation, inject 0.5 to 2mg of the compound into the arm of the patient. 2. After about 20 minutes, the fluorescent compound has been transported to the liver and from thence to the bile duct in a sufficient quantity to enable it to be readily discerned under UV light through the wall of the bile duct and continues to be visible for at least 45 minutes thereafter. 3. The surgeon illuminates the surgical site with UV light in addition to visible light at least over the critical operation phase when it is important to be able to discern the biliary duct from the surrounding tissue. 4. Any biliary leakage through the wall of the bile ductand/or the gall bladder will become immediately apparent to the surgeon as a much stronger fluorescence because of the lack of the shielding effect of the wall. The location of the leakage in the wall can be quickly identified and repaired. The strongly fluorescing bile fluid which has leaked out can be easily discerned and efficiently removed using swabs.

Claims: CLAIMS 1. The use of a coloured or fluorescent hepatobiliary targeting compound in the manufacture of an administrable composition for visualising the bile duct and/or visualising biliary leakage during an operation in the region of the bile duct. 2. The use as claimed in claim 1, wherein the targeting compound is a fluorescent compound. 3. The use as claimed in claim 2, wherein the fluorescent compound is a UV light fluorescent compound. 4. The use as claimed in any preceding claim, wherein the compound comprises a steroid moiety having an unblocked 3-hydroxyl substituent and an unblocked carboxyl group attached by means of an amide linkage to the side chain of the steroid moiety, and an active moiety which is to be targeted to the liver, said active moiety being attached to the α-carbon atom relative to the unblocked carboxyl group. 5. The use as claimed in claim 1 , wherein said compound has the general formula ( I ):- ( I ) wherein A is -αOH or -βOH; B is -αH or βH; C is.-H , -αOH or -βOH; or B and C together form a double bond; D is -H, -αOH or -βOH; E is -H, -αOH or β-OH; L is a linking moiety; J is said coloured or fluorescent moiety; and n is 0 or 1. 6. The use as claimed in claim 5, wherein J is or includes a fluorescein, rhodamine or other fluorescing moiety. 7. The use as claimed in claim 5 or 6, wherein the linking moiety L is N-terminated at its end attached to active moiety J. 8. The use as claimed in claim 5, 6 or 7, wherein and L is selected from the group consisting of -(CH 2 ) n NH, where n is 3 or 4, -(CH 2 ) 4 NH-(CH 2 ) 3 NHC(= NH)NH-, and -(CH 2 ) 2 -CH(OH)CH 2 NH-. 9. The use as claimed in claim 5, 6 or 7, wherein the moiety -NH- CH (COOH)-L- is derived from S-adenosylhomocysteine, S-adenosyl methionine, S-amino-imadazole-4-carboxamide, asparagine, cadaverine, cystamine, citrulline, diaminopimelic acid, 2, 4-diaminobutyric acid, cysteamine, glutamine, 3-hydroxykynurenine, kynurenine, putrescine or negamycin. 10. A method of visualising the bile duct and/or visualising biliary leakage, comprising administering to a patient an effective amount of a coloured or fluorescent hepatobiliary targeting compound.

Inventors: MILLS CHARLES OSWALD, MILLS, CHARLES, OSWALD
Applicants: MILLS CHARLES OSWALD, UNIV BIRMINGHAM, MILLS, CHARLES, OSWALD, THE UNIVERSITY OF BIRMINGHAM
Application year: 1996
Main classification: A61K49
IPC codes: A61K49, C07J41