The value of bioassays in detecting narcotics

Abstract

This article relates how a biological method was evolved and developed, by means of infections to mice, whereby "doping" of race-horses could be detected in a matter of minutes

Details

Author: James C. Munch, , Aaron B. Sloane, , Albert R. Latven
Pages: 23 to 26
Creation Date: 1952/01/01

TECHNICAL

The value of bioassays in detecting narcotics

James C. Munch,1
Aaron B. Sloane,2
Albert R. Latven2

This article relates how a biological method was evolved and developed, by means of infections to mice, whereby "doping" of race-horses could be detected in a matter of minutes

Narcotics have a recognized and valuable place in drug practice; unfortunately, there has been a great tendency for diversion into improper channels. The chemical procedures developed throughout the world have permitted official standards in the various national pharmacopoeias; standards have been set for opium preparations and their alkaloids, for example, based upon these chemical procedures.

Pharmacological investigations have pointed out that the various alkaloids in opium influence the pharmacodynamic responses of the individual alkaloids, both qualitatively and quantitatively. Certain alkaloids in opium tend to increase the effect of morphine, additively or synergistically; other alkaloids in opium tend to decrease the morphine effect in men or other animals. For this reason, the chemical assurance that a number of lots of opium all contain 10 per cent of morphine is not necessarily a proof that the different lots of opium will have identical clinical effects. With this in mind, investigations have been conducted in various laboratories for the purpose of developing pharmacological habits of evaluating opium. Presumably, similar findings will be encountered in a thorough study of other narcotics.

Some years ago the illegal diversion of heroin and of morphine from legitimate drug channels for the purpose of "stimulating" race-horses was learned as the result of field inspections by agents of the Federal Bureau of Narcotics of the United States Treasury Department. Under the authority of the Harrison Anti-Narcotic Act, investigations were started to determine the extent of such illegal diversion. A series of conferences was called by the Honorable Harry J. Anslinger, United States Commissioner of Narcotics. These were attended by chemists, pharamacologists, and veterinarians, who discussed the broad bases of investigations and arranged for a co-ordinated research programme for the purpose of detecting the "doping" of racehorses (the illegal administration of drugs, such as morphine or heroin).

Medical Director, Strong, Cobb & Co. Inc., Cleveland, Ohio, U.S.A.

From these conferences, and investigations conducted by the members of this group under the general direction of the United States Government, various state and local agencies, state racing commissions and owners of race-horses, substantial research was conducted which led to a series of procedures for the detecting of "doping". Samples of saliva, of urine, of sweat and of blood were obtained from horses before and after the administration of various products. Tests were made on these samples by laboratory workers. The samples were usually submitted in code so that the identity of the animal and any treatment it might have received were unknown to the testing groups. From this emerged a definite chemical technique for extraction and identification of various products. For various reasons it was not feasible to conduct investigations routinely on sweat or on blood, so these methods were directed specifically to analyses of the saliva and of the urine. The methods of determining various poisons in tissues, as set forth in standard toxicologies, were modified to meet requirements for this specific study. Various new solvents were developed for shake-out processes. The limited quantities of saliva or urine which were available, and the peculiar nature of the physiological constituents of these fluids, required special modifications in chemical technique. From these studies, a definite method of procedure evolved.

Since a period of forty-eight hours is often required for complete examination, and since the chemical studies of the end extraction processes could only be used with materials of certain chemical stability, it was thought that certain products, such as hormones, would not be detected by these methods. Definite metabolic changes occur during the passage of these materials through the body of the race-horse or other animal, i.e., the substances excreted in the saliva and the urine may be chemically different from the products which were administered originally. Because of the complicated metabolic pathways for various products in the animal or human system, it becomes very difficult to determine strange or unusual products, or even the usual degradation products of many materials. With this in mind, the pharmacologists in the original conferences thought that it would be worth while to make detailed studies of the pharmacological methods for detection of drugs which might be employed for the purpose of "doping" horses. While these investigations are still under way in our laboratories, it appears helpful to summarize results to date for possible co-operation with interested pharmacologists throughout the world.

Munch Research Laboratories, Inc., 306 S. 69 St., Upper Darby, Pa., U.S.A.

Our investigations have been conducted upon many species of animals but we finally decided that mice are the most satisfactory. The quantities required to produce effects on cats or dogs are much greater than those required to produce effects on mice. Standard strains of mice are readily available in large numbers (subject at times to demands of governments interested in the production of drugs and in their testing).

Furthermore, it was found that mice showed rather uniform pattern of behaviour. Observations of a large number of animals under identical conditions permitted the determination of their activity, or any characteristics of action which might be typical of the groups of mice under investigation. While it is possible to give doses of drugs by mouth, intravenously or by other routes, it was found that intraperitoneal administration was feasible, rapid and satisfactory for this purpose. Studies on horses which had not received any medication within a period of twenty-four hours indicated that 2.0 cc. of saliva or of 1.0 cc. of urine per mouse did not produce any toxicologic effects within 10-15 minutes. Occasional mice showed some sedation as a result of the administration of a quantity of fluid corresponding to more than one-tenth of the body weight, but these effects were readily distinguishable from those following the administration of pharmacologically active drugs. Studies on saliva or urine from several thousand unmedicated horses have failed to reveal any symptoms in mice which could be mistaken for those following administration of drugs.

When drugs were administered to race-horses, samples of saliva and of urine collected within a period of several hours were capable of producing effects in mice. After administering large doses of drugs, positive reactions were observed following intraperitoneal injection into mice of quantities as small as 0.1 cc. per mouse. As a matter of uniformity, it was deemed advisable to develop a method by which one or more mice would receive intraperitoneal injections of 0.5 cc. per mouse while the others would receive 1 cc. each. Depending on the result of these tests, it was always possible to give larger or smaller doses as desired. A further advantage in the use of mice is that the volume of fluid required is small. This is desirable since the quantities of saliva or urine submitted for test are often limited.

The technique to be followed is simple. Groups of white mice of a standard strain from a recognized dealer are offered a standard diet and kept under observation for a period of several days. Animals in good condition are then selected at random from the colony. Either males or females may be used although we have felt that females were somewhat more susceptible. Mice from different sources will differ significantly in their sensitivity. For this reason we have preferred to use CFCW or CF-1 mice furnished by Carworth Farms, Rockland City, New York. When conducting tests at the race-tracks, it is necessary that prior arrangements be made to permit drop shipments of mice at weekly intervals. The diet of the mice is that recommended by the Carworth Farms, although any standard food for animals may be employed.

Mice are removed individually from a colony and intraperitoneal injections of 0.25 cc., of 0.5 cc. and 1.0 cc. given. After injection, the animals receiving the same sample are placed in the same cage and observed continuously for a period of five to ten minutes. Other doses may be used, if desired. Also, observation periods may be extended to two hours if warranted.

In our experience on several thousand samples of saliva and urine obtained from horses which had not been given drugs within twenty-four hours, no changes in behaviour, appearance, or attitude developed in the mice. If the volume administered is 2 cc. or more per mouse, some depression and sluggish gait may develop as a result of the volume effect. The same effects may be produced by administration of heroic volumes of 0.9 per cent sodium chloride solution or other inert solutions. If these animals are observed for several hours, it is rare to note any symptoms which simulate those produced by drugs used for "doping" horses. Since the samples are not sterile or sterilized, intraperitoneal injection usually leads to infections and death after twelve to twenty-four hours, but this is a bacteriological rather than a pharmacodynamic response

When effective drugs have been deliberately added to samples of saliva or urine, or when these fluids were collected from horses which had previously received effective drugs within a period of twenty-four hours or less, changes developed in the test mice. These alterations in behaviour, appearance or condition developed within 5 to 10 minutes and were obvious alterations from the pre-injection state.

The symptoms and signs which develop depend on the nature of the product administered. Following injection of the barbiturates, or sedatives in general, sluggishness, decrease in voluntary travel, and a tendency to sleep are noted. With increasing quantities of administered drug, symptoms develop more rapidly and to greater intensities. On the other hand, strychnine or caffeine tend to produce increased locomotion, increased respiration, and muscular tremors. These symptoms may progress to definite prostration, coma, and death, if the quantity of drug administered is sufficient.

In learning the technique, known samples of materials may be dissolved in saliva, urine, or distilled water. By injecting a series of animals with various doses, it is possible to determine the threshold dose, i.e., that quantity of material which is capable of producing discernible or detectable alterations in mice under the conditions prevailing in the testing laboratory. Passage of a drug through the horse produces metabolites which may cause symptoms differing from those produced by the original drug.

It is recognized that some animals are more sensitive than others. Certain workers are able to detect alterations more readily. For this reason, it is important that each laboratory should standardize itself, i.e., administer a known sample of a test product at various dosage levels to substantial numbers of mice and determine the effects produced at the different levels. It is also possible to determine the character, type and extent of departures from normal behaviour that will be produced by differing quantities of a product under individual laboratory working conditions. Based upon our studies, the threshold doses established for a number of products are recorded in table I . While the absolute values may differ under varying conditions of test, our experience has indicated that there is a proportional relationship between these threshold levels. This emphasizes the previous point that every laboratory should determine its own threshold values.

We have also established the finding that many narcotics are altered in the body during the process of absorption, distribution and elimination. For example, heroin is converted to a large extent to morphine in the body as suggested by differences in threshold doses and symptoms produced. After completing a series of studies with known drugs, it is possible to develop criteria by which many of the narcotics used in the "doping" of race-horses may be identified qualitatively. A semi-quantitative estimate may also be made by determining the smallest volume of test solution capable of producing the recognized reaction following the established threshold dose.

Of course, it has been desirable to conduct chemical examinations on those samples of saliva or urine which have proven positive by bioassay, for the purpose of identification of the drug or drugs present. The bioassay is very sensitive and is able to detect threshold quantities which are usually less than those required for definite chemical identification. Furthermore, the biological responses will follow administration of impure materials and their metabolites, as distinguished from definitive chemical methods which require substantially pure test material. If several products are present in the saliva or urine the animals usually show mixed responses, which indicate the nature of the several drugs present. For example, strychnine tends to produce increased respiration and muscle tension. Heroin or morphine tend to produce a characteristic S-curve of the tail of the mouse. If a combination of both products is administered, both types of effects may be observed.

We have extended our investigations to include trials of saliva and urine from animals other than race-horses and find equal satisfaction in the testing process. We have done some work on the saliva and urine of humans and find that the biological method permits identification of "dope addicts", as well as malingerers.

Surviving animals can be re-used after an interval of several days if test solutions have been prepared in distilled water. On the other hand, if solutions of saliva or urine have been administered, in our opinion the animals should not be re-used. While the cost of animals varies, arrangements can usually be made by which the cost is around $0.30 per mouse. Using a total of four mice on a single test, this makes the cost of animals about one dollar. No other expenses are incurred, with the exception of the feed and care of the test animals. Results of tests on a preliminary group may be confirmed on additional mice at a slight increase in cost. The amount of the saliva or urine used is relatively small, and the results of the biological assay will be helpful in orienting chemical tests on the remainder for identification of the suspected drug or drugs. In our experience, we have never had positive chemical tests on saliva or urine samples which were negative by the biological test. The converse is not true; we have had positive biological tests following administration of many materials for which adequate chemical methods were not available.

A further advantage of this biological procedure may be pointed out, namely, that the positive results will be obtained in ten minutes or less if tests are made on saliva samples from a race-horse. This permits pre-race testing of horses almost immediately before they are led to the track. This is an advantage which has not been established for the chemical assay since the process requires several hours, even up to two days for separation and purification of extracts before tests for chemical identification can be conducted. Of course, it is recognized that both biological and chemical tests have their peculiar and distinctive values. Before legal hearings which might lead to disbarment of accused persons are held it may be desirable to have both biological and chemical support for the charge of "doping".

After establishing the method of procedure in our laboratory, we worked with the Maryland State Racing Commission and Mr. Mahoney in the development of a mobile laboratory unit for use at the race-track. This proved an advantage in that it permitted cooperation between the pharmacologist and the chemist at all times; it permitted specific leads from pharmacological behaviour to orient special chemical extraction processes; and it made it unnecessary for the chemist to make examinations on samples that were negative by biological test. This saved time and the chemist could concentrate on the positive samples. It was possible to get saliva samples from all of the horses entered in every race during the course of a day (which might be as many as 80 or 90 samples), instead of examining only samples from the eight which had won their respective races. The "doping" of horses which finished in the second, third, or fourth places was more frequent than in the winning horses.

Summarizing, a biological method has been developed for the detection of narcotics in the saliva or urine of "doped" race-horses, which has also been valuable in detecting the use of these drugs by humans. It consists of the intraperitoneal injection of 0.25 to 1.0 cc. of test solution to each of a number of white mice and observation of any changes in behaviour which may develop within a period of ten minutes. Saliva or urine samples from several hundred horses which were not given drugs consistently produced negative results. Samples from horses deliberately "doped" (unknown to the bioassayist) were consistently identified by the biological method. Depending on the nature of the product administered, chemical tests may or may not be correct; we have never obtained positive chemical findings on saliva or urine samples which were negative by the mouse test. Following administration of certain products to race horses, we have obtained positive biological responses but negative chemical tests. Using the biological test, it is possible to make pre-race investigations of all the horses entered in races. By using a mobile field laboratory unit, both chemical and pharmacological tests may be made at the race track. These tests have been conducted under the direc- tion of a number of the state racing commissions in the United States. Positive biological tests should be confirmed by chemical examinations ,before legal steps are instituted. The possibilities of the biological method are being investigated further.

The authors wish to express their appreciation to the Honorable Harry J. Anslinger, United States Commissioner of Narcotics, and to the various officials of the Racing Commissions of the States of Maryland and Washington, particularly Mr. George R. Mahoney, former chairman of the Maryland State Racing Commission, for assistance in arranging for these studies. They also wish to acknowledge the support and cooperation of the various members of the National Horsemen's Benevolent and Protective Association.

TABLE

Thresholds for detection by mice; intraperitoneal injection

Drug

Micrograms/20 gram mouse

Amphetamine
80
Caffeine
600
Cocaine HCl
400
Codeine
60
Coramine
100
Pethidine
400
Desoxyephedrine
40
Dihydromorphinone
12
Ephedrine
200
Heroin
1
Morphine
60-80
Picrotoxin
40
Strychnine
1(?)

BIBLIOGRAPHY

  1. James C. Munch. Bioassays, A Handbook of Quantitative Pharmacology. Publ. Williams & Wilkins, Baltimore, 1931.

  2. James C. Munch, Saliva Tests. I. Morphine. J A.Ph A., 23, 766-773 (1934).

  3. James C. Munch. Saliva Tests. II. Heroin. Ibid., 23, 1185-1187 (1934).

  4. James C. Munch. Saliva Tests. III. Detecting the Administration of Some Opium Derivatives to Horses. Ibid.,24, 557-560 (1935).

  5. James C. Munch, Aaron B. Sloane and Albert R. Latven. Saliva Tests. IV. A Mobile Laboratory for Saliva Testing. In press.

  6. Albert R. Latven and Hans Molitor. Comparison of the Toxic, Hypnotic and Irritating Properties of Eight Organic Solvents. J. Pharmacol. Exper. Therap., 65:89-94 (1939).

  7. Albert R. Latven. Of Mice and Thoroughbreds. The Washington Horse, 2-3 (August. 1946).

  8. Albert R. Latven. Checks and Balances on Stimulation. Ibid., 28-29 (September, 1946).