In vivo validation of a cell culture test for biocompatibility testing of urinary catheters.

Biocompatibility tests have been compared for their suitability as routine safety tests for urinary catheters. Latex catheters from five manufacturers were tested by each of the following four methods: (1) a cell culture cytotoxicity assay of catheter extracts, (2) intracutaneous injection of the extracts into rabbits, (3) intramuscular implant of catheter pieces into rabbits, (4) catheterization of sheep (mucous membrane irritation). The rabbit intracutaneous and intramuscular tests are both current pharmacopoeial methods for ascertaining the suitability of polymers for medical use. The four tests each showed a cell or tissue response ranging from no detectable change to severe damage, according to the catheter batch or brand, and they each identified the same samples as most toxic and least toxic. However, they differed in sensitivity. The sheep test and the cell culture assay discriminated between catheters of intermediate toxicity and ranked as toxic catheters not identified as such by the two pharmacopoeial tests. The sheep test most closely approximates clinical usage, but is impractical for routine use. The cell culture assay is a suitable alternative. It also has the advantages of a clearly defined endpoint, good sensitivity, reproducibility, speed, and reduced animal usage.

An in-vitro test for the duration of action of insulin suspensions.

A method is described which measures the prolongation effects of commercial insulin suspensions by monitoring the rate of solution of an insulin suspension into a phosphate buffer, pH 7.4, at 37 degrees C. The method can rapidly categorize an insulin into the clinical classifications of either fast, intermediate or slow acting. It offers advantages of speed and sensitivity over the British Pharmacopoeial test for prolongation of insulin effect in fasted animals. Large differences between the dissolution rates of commercial samples of isophane insulin were observed which suggested a lack of bioequivalence.

Cellular toxicity of urinary catheters.

The cellular compatibility of each of several brands of urinary catheters available on the Australian market was measured by means of cell-culture methods, a rabbit intramuscular implant test, and the traditional mouse systemic toxicity test. Good agreement was obtained between the in-vitro tests and the rabbit implant tests (although the cell-culture tests were simpler, more rapid, more sensitive, and quantitative). The mouse systemic toxicity test was insensitive, and detected no toxic samples. The cell-culture and rabbit-implant test results indicated that some urinary catheters can release substances harmful to mammalian cells. Such substances may contribute to the clinical reactions of urethritis and strictures after urethral catheterization.

An evaluation of the British Pharmacopoeial assay of heparin: a comparison with other methods.

The potencies of six commercially manufactured heparins have been measured by the British Pharmacopoeial (BP) assay and activated partial thromboplastin time (APTT), protamine sulphate, and anti-Xa assays. The APTT/BP potency ratios were found to vary with the preparation but this was not dependent on the tissue source of heparin. For mucosal heparins, the anti-Xa/BP potency ratios were close to unity, but for heparin of lung origin the anti-Xa potency was approximately one-quarter of the BP potency. Four heparin fractions prepared by column gel chromatography of a commercial heparin were similarly examined by all four assays, and there was a wide divergence between the BP potency estimates and those obtained with the other methods. The degree of divergence was found to depend on the molecular size of the fraction.

Relative activities of heparin fractions obtained by gel chromatography.

In order to investigate the reported heterogeneity of commercial heparin injections, lung and mucous preparations were fractionated by gel chromatography according to molecular size. Eluent pools were characterized by measuring electrophoretic mobility, anticoagulant activity using a reactivation assay and an anti-Xa assay, lipolytic activity and the protamine neutralization value. The only biological activity to show a marked relationship with the molecular size of the heparin fraction was the anti-Xa activation activity. This effect was more pronounced with the mucous heparins than with the lung heparin preparation studied.

A in vivo assay for standardizing heparin preparations.

The limitations of currently used in vitro assays of heparin have demonstrated the need for an in vivo method suitable for routine use. The in vivo method which is described in this paper uses, for each heparin preparation, four groups of five mice which are injected intravenously with heparin according to a "2 and 2 dose assay" procedure. The method is relatively rapid, requiring 3 to 4 hours to test five heparin preparations against a standard preparation of heparin. Levels of accuracy and precision acceptable for the requirements of the British Pharmacopoeia are obtained by combining the results of 3 to 4 assays of a heparin preparation. The similarity of results obtained in vivo method and in vitro method of the British Pharmacopoeia for heparin preparations of lung and mucosal origin validates this in vivo method and, conversely, demonstrates that the in vitro method of the British Pharmacopoeia gives a reliable estimation of the in vivo activity of heparin.

Measurement of the heparin neutralizing capacity of protamine.

Several methods of estimating the heparin neutralizing capacity of protamine were investigated for their reliability and practicability. The results from two chemical methods were compared with those from two in vitro biological assays, one of which was the method of the British Pharmacopoeia (1973). An in vivo method using mice was used to assess the accuracy of the in vitro test methods. Three standard heparin preparations were tested against the W.H.O. 1st International Reference Preparation for Protamine. Two of the heparin preparations were of mucosal origin, one of which was the 3rd International Standard, and the third heparin preparation was of lung origin (the 2nd International Standard for Heparin). The mean neutralization values (all methods) of heparin by protamine for a House Reference Preparation and the 3rd and 2nd International Standards for Heparin were 95.8, 109.8 and 89.9 units per mg of Protamine, respectively. All methods read the House Reference Preparation to have a lower value than the 3rd International Standard, which had a higher value than the 2nd International Standard for Heparin. There was a constant relationship between the results of any one method and those of another. The chemical and in vitro biological methods gave results of comparable precision although the latter required a greater degree of technical skill and time to perform.

Stability of oral pancreatin powders.

In-vitro study which duplicated the conditions to which the orally administered pancreatin is exposed in the human stomach showed that the oral administration of pancreatin in uncoated powder form could result in a substantial loss of enzymic activity. Therefore, it may not be an efficacious replacement therapy in pancreatic disorders where pancreatin secretion is reduced.

Pyrrolizidine alkaloids: actions on muscarinic receptors in the guinea-pig ileum.

1. Eleven pyrrolizidine alkaloids have been tested on the isolated guinea-pig ileum preparation.2. Platyphylline, supinine, heleurine and cynaustraline were more potent in antagonizing responses to acetylcholine and carbachol than responses to histamine. Their anticholinergic activity appeared to involve a competitive mechanism.3. Lasiocarpine, monocrotaline, spectabiline, sarracine, 7-angelylheliotridine, heliotrine and senecionine had similar antagonistic potencies against responses to both acetylcholine and histamine.4. The alkaloids had no appreciable activity as antagonists of acetylcholine in the isolated toad rectus abdominis preparation.5. These results are discussed with respect to interactions of the alkaloids at receptor sites involved in anticholinergic activity at the muscarinic receptor.