DIAGNOSTIC COMPOSITIONS FOR ASSESSMENT OF PANCREATIC INSUFFICIENCY

Abstract: Fluorescein dilaurate is dissolved in coconut oil and converted into an oil-in-water emulsion in the presence of a mannitol. The composition is administered orally. Fluorescein and mannitol are excreted in the urine. The ratio of recovered fluorescein and mannitol allows for the detection of pancreatic insufficiency without the need for control experiments.

Description: DIAGNOSTIC COMPOSITIONS FOR ASSESSMENT OF PANCREATIC INSUFFICIENCY This invention relates to diagnostic compositions and method for detecting pancreatic insufficiency. Identification of pancreatic insufficiency is a useful tool in diagnosis of a variety of diseases, including pancreatitis and cystic fibrosis. One test which has been widely used for routine detection of pancreatic insufficiency involves the administration of fluorescein dilaurate (herein referred to as FDL) When this compound comes into contact with pancreatic juices it is broken down by pancreas-specific cholesterol ester hydrolase into fluorescein and two molecules of lauric acid. The coloured, water-soluble fluorescein is absorbed and excreted in the urine. If the patient's urine iε collected over a ten hour period, and the fluorescein content determined, this provides an indirect measure of glandular output of the exocrine pancreas. This procedure is described, for example in Barry et al, The Lancet 1982, Vol. 2, pages 742 to 744. One disadvantage which iε inherent in the . above- described pancreolauryl test is that it requires to be carried out over a two day period, since the amount of fluorescein which is excreted must be compared with the amount excreted when an equivalent amount of non- esterified fluorescein is administered to the patient. This control test enables the physician to compensate for any individual absorption or excretion characteristics which could interfere with the result. Thus, while effective and suitable for routine diagnostic testing, the pancreolauryl test does have two disadvantages, namely, that it iε necessary to carry out a control test and secondly the whole of the urine excreted by the patient after administration of the diagnostic composition must be carefully collected. One aspect of the present invention iε based upon the discovery that by incorporating a particular type of permeability marker in the diagnostic composition, it becomes no longer necessary to carry out a control test, since determination of the marker simultaneously with determination of the.fluorescein enables the technician or physician to compensate for differences in absorption of the active materials and excretion of the fluorescein and permeability marker by individual patients. According to one aspect of the present invention there is provided a diagnostic composition which comprises a di-fatty acid, eεter of fluorescein and a non-toxic water-soluble substance, such as a saccharide, which iε resistant to break-down within the stomach and does not take part in any active transport system across the intestinal mucoεa. Preferably, the di-fatty acid ester is fluorescein dilaurate (hereinafter referred to as FD ), since this iε readily broken down by the enzyme present in pancreatic juices produced by a healthy pancreas. Preferably the saccharide iε a saccharide such as mannitol, lactulose, rhamnose, xylose or fructose of which mannitol is most preferred. Mannitol and lactulose have the particular property that they are highly resistant to break down in the stomach and absorption through the intestinal mucoεa seems to be substantially passive. In other words, the transport of the saccharide through the intestinal wall does not seem to involve any reaction with any other material in the digestive system. Preferably, the saccharide, e.g. the mannitol, iε present in the diagnostic composition in a molar excess compared with the FDL. Preferably, the molar ratio of FDL to mannitol is from 1:5 to 1:100, more preferably 1:10 to 1:60, e.g. 1:50. Although various dosage forms of the diagnostic test kits may be used, e.g. encapsulated compositions or tabletted solid compositions, such dosage forms are usually unsuitable for children (including neonates) or indeed for adult patients who have swallowing difficulties. However, because the fluorescein fatty acid ester is water-insoluble, addition of the marker compound to a liquid feed, such aε milk, or a watery drink iε not possible. According to a further aspect of the present invention, there is provided an emulsified diagnostic composition for detecting pancreatic insufficiency which comprises an oil phase dispersed in a continuous aqueous phase, the oil phase having dissolved therein a di-fatty acid ester of fluorescein. In a preferred form of the invention as applied to liquid formulations, there is provided an emulsified diagnostic composition for detecting pancreatic insufficiency which comprises an oil phase dispersed in an aqueous phase, the oil phase having a di-fatty acid ester of fluorescein dissolved therein and the composition including a saccharide which is resistant to breakdown within the stomach and which does not take part in any active transport system across the intestinal mucosa. Emulsions prepared in accordance with the invention may be readily added to an infant's drink ana an accurate pancreatic insufficiency assessment carried out. In the case of emulsionε which additionally contain a saccharide or other metabolic resistant second marker, no control test need, of course, be carried out. The emulsions are prepared by dissolving the fluorescein di-fatty acid ester (preferably the dilaurate) in a suitable oil phase. Triglycerides have been found suitable, especially those of medium chain length. One specific example is a fractionated coconut oil obtainable under the trade name 'Miglyol 812' from Dynamit Nobel. These dissolve the fluorescein ester without presenting absorption problems to patients suffering from cystic f ibrosis . Having dissolved the fluorescein di-ester in the triglyceride, an oil-in-water emulsion iε formed by disperεing the triglyceride aε fine droplets in . the aqueous phase. Where a saccharide is present, this may be pre-disεolved in the water. An emulεion is preferably formed by use of ultrasonic energy, although other disperεing methods may be used to obtain emulεion homogeniεation. ϋltraεonic diεpersion has the advantage that an extremely fine dispersion of oil droplets iε formed in the aqueouε phase. Thiε 'micro-emulεion' system enhances the physical stability of the emulsion, important in paediatric formulations, where the choice of acceptable emulsifiers is limited. Moreover, rnicro- emulεionε require little effort to form an homogenous mixture when added to an infant drink. Suitable stabilising surfactantε include lecithin, e.g. εoya bean lecithins which are obtainable from Lipoid K.G. and Lucas Meyer under the trade names 'Lipoid 505' and 'Topcithin', reεpectively. The emulsions are prepared in dosage unit forms so that a single dosage unit can be added to a standard infant feed, e.g. a 150 ml feed. The fluorescein di-fatty acid ester may be present in an amount of from about 0.1 to 0.5 m.moles, preferably about 0.125 m.moles. 1 6 In the case of the emulsionε containing a εaccharide aε the εecond marker compound, the εaccharide iε, of course, present to provide a means of measuring gastro¬ intestinal permeability. The saccharide should be non- metaboliεable and the preferred saccharide iε mannitol. Alternatives include lactulose, xylose and rhamnoεe. The saccharide should be present in molar excess compared with the fluorescein ester. Typically, the εaccharide is present in the emulsion in concentrations of from 1 to 10 m.moles, e.g. about 6.25 m.moles. The resulting emulsions are filled into boro-silicate vials in the desired unit doseε, sealed and autoclaved to sterilise them. The following specific formulations of emulεion foru.ul&tionε will illustrate the invention. In all cases, the FDL was pre-diεsolved in the Miglyol 812, the remaining components were added and emulsified using an ultrasonic disperser. Example I Single Marker System Component mσ/άose % w/w Fluorescein Dilaurate 87.00 0.87 Miglyol 812 1250.00 12.50 (coconut oil triglyceride) Lipoid S05 250.00 2.50 (soya bean lecithin) Water (mains, boiled) 8413.00 84.13 10000.00 100.00 Component Fluoreεcein Dilaura Mannitol B.P. Miglyol 812 Lipoid S05 Water (mainε, boile As εtated above, the diagnoεtic composition may be administered as an encapsulated εolid composition or a tabletted composition which may be administered in ' one or more tablets or capsules. Typically, a dosage unit of the FDL iε from 0.2 to 0.8 mmolε and the mannitol from about 20 to 30 mols with FDL and mannitol being typically in the moler ratio of 1:50. The FDL and Mannitol may be intimately mixed to form a homogeneous powder and filled into hard gelatin capsuleε. Suitable flow and compaction aids, commonly known to those familiar to capsule technology, may be added to asεist production. An example of an encapεulated formulation is as followε: The powder mix may be filled into two/three size 00 hard gelatin capεuleε. The formulation illustrated above iε intended for infantε and iε one-quarter of the standard adult does. A standard adult dose would therefore be 0.5m Moles (348.5 mg) of FDL and 25m Moles (4552.0 mg) of Mannitol contained in the appropriate number of capsules. It has been found that after administration of the diagnostic composition a steady state is reached after about 4 to 6 hours from administration, at which time a sample may be taken and analyεed for fluorescein and mannitol contents. The ratio of fluorescein to mannitol in the sample gives a clear discrimination between normal •pancreatic activity and pancreatic insufficiency. Fluorescein may be conveniently analysed quantitatively in a sample using a εpectrophotometer. The evaluation can be carried out directly by meaεuring the abεorption . of a sample of urine containing the excreted fluorescein and mannitol. In the case of mannitol determination, this iε conveniently carried out by the method described in the paper by Lunn et al, published in Clinica Chimica Acta, 1989, CCA04504. In this method, mannitol is treated with mannitol dehydrogenase which iε extracted from a culture of a bacterium NCIB NCTC 6992, obtainable from NCIB Torry Reεearch Station, Aberdeen Scotland. This particular enzyme converts mannitol into fructose and NADH which can be determined by optical density measurements at 340 nm. The relationship between the original mannitol concentration and the optical density of a sample treated with the bacterium is almost exactly linear, so that the optical density can be calibrated in mmols of mannitol. Thus, samples of the excreted urine after administration of the diagnostic composition can be determined for fluorescein and mannitol concentrations by a simple spectrophotometric analysis before and after addition of the enzyme which converts the mannitol into fructose. In order to demonstrate the operation of the diagnostic method, the following study was carried out of 6 healthy adults (four male and two female) aged 29 to 40 yfcάiε which constituted group- 1. Six patients diagnosed as having cystic fibroεiε (five male and one female aged 7 to 21 years) were tested both before having been supplied with pancreatic enzyme supplement (group 2) and after having been supplied with such a supplement {group 3). After an overnight fast, each of the patients ingested an encapsulated composition comprising 0.5 mmolε of FDL and 25 mmols of mannitol at time zero followed by a standardised breakfast consisting of 50 gms of bread spread with 20 gms of butter and 1 cup of liquid. Urine was collected for ten hours in 2 hourly aliquots and urinary fluorescein was meaεured photometrically and mannitol waε meaεured enzymatically and photometrically as described above. There was no significant difference in mean (standard deviation) ten hour urine volume which were 1475 (278 mmls) group 1, 1008 (398 mmlε) group 2 and 849 (536 mmls) for group 3. There was also no significant difference in percentage mannitol recovery 35.0 (11.1%) group 1, 40.1 (18.1%) group and 28.6 (7.0%) group 3. However, there was a pronounced difference in percent fluorescein recoveries between the three groups, i.e. 18.7 (3.8%) group 1, 1.2 (0.5 %) group 2 and 0.8 (0.3%) group 3, resulting in a significant difference in ten hourly fluorescein to mannitol . ratios 57.3 (18.2). 3.4 (1.4), 3.1 (1.9) respectively. These resultε are εhown schematically in the attached bar charts. When the resultε were analysed in two hour urine aliquotε it was seen that there waε a delay in • urinary recovery of fluorescein versuε mannitol which is due to the pancreatic digestive phase in both controls and cystic fibrosiε groups. Differences in urinary fluorescein to mannitol ratios between controls and cystic fibroεiε patients (both those with and without pancreatic supplements) achieved biological and εtatiεtical significance from 2 to 8 hours post ingestion of the diagnoεtic compoεition. Theεe resultε show that the addition of mannitol to FDL permits clear distinction between subjects with normal pancreatic function and those with cystic fibrosiε whether receiving pancreatic enzyme supplements or not. Thus, by using the new diagnostic test it is possible to detect pancreatic insufficiency by taking samples between 2 and 8 hours after ingestion of the composition without the need to run a further control .

Claims: CLAIMS 1. A diagnostic composition for detecting pancreatic insufficiency which comprises a fatty acid ester of fluorescein and a non-toxic substance which iε resistant to breakdown within the stomach and does not take part in any active transport system across the intestinal mucosa. 2. A composition according to claim 1 wherein the non-toxic subεtance iε a εaccharide. 3. A composition according to claim 2 wherein the saccharide iε mannitol, lactulose, xylose or rhamnose. 4. A composition according to any one of the preceding claims wherein the fatty acid ester is fluorescein dilaurate. 5. A composition according to any one of the preceding claims wherein the fluorescein ester and the saccharide are present in a molar proportion of about 1:5 to about 1:100. 6. A composition according to any one of the preceding claims which is in the form of an emulεion. 7. An emulsified diagnostic composition for detecting pancreatic insufficiency which comprises an oil phase dispersed in a continuous aqueous phase, the oil phase having dissolved therein a di-fatty acid ester of luorescein. 8. A composition according to claim 7 which also contains a saccharide which is resistant to breakdown in the stomach and does not take part in any active transport system acrosε the intestinal mucosa. 9. A compoεition according to claim 8 in which the fluoreεcein eεter and the saccharide are present in a molar proportion of about 1:5 to about 1:100, preferably from about 1:10 to about 1:60. 10. A composition according to any one of claims 7 to 9 which iε in the form of a concentrate intended for addition to a liquid feed and wherein the fluorescein eεter in present in the emulsion in a concentration of from about 0:1 to 0.5 m.moles. 11. A composition according to any one of claims 7 to 10 in which the saccharide iε present in a concentration of from about 1 to 10 m.moles. 12. A composition according to any one of claims 7 to 10 in which the fluorescein eεter iε fluoreεcein dilaurate. 13. A compoεition according to any one of claims 7 to 12 in which the saccharide iε mannitol, lactuloεe, xylose or rhamnose. 14. A composition according to any one of claims 7 to 13 in which the fluorescein iε dissolved in a triglyceride. 15. A composition according to claim 14 in which the triglyceride is a medium chain length triglyceride. 16. Use of a diagnostic compoεition compriεing a di- fatty acid ester of fluorescein and a εaccharide which iε resiεtant to breakdown within the stomach and .does not take part in any active transport system acrosε the intestinal mucosa, in the assessment of exocrine pancreatic function.

Inventors: JOLLIFEE STEPHEN RONALD, WEAVER LAWRENCE TREVELYAN, JOLLIFEE, STEPHEN, RONALD, WEAVER, LAWRENCE, TREVELYAN
Applicants: CHARWELL PHARMA, CHARWELL PHARMACEUTICALS LIMITED
Application year: 1991
Main classification: A61K49
IPC codes: A61K49