1	ELISA TESTING OF RACING HORSES: A REGULATORY REVOLUTION By Thomas Tobin University of Kentucky Maxwell H. Gluck Equine Research Center for the California Association of Toxicologists Tempe, Arizona May 18th, 2002 Last Modified: May 30th, 2002 3:00 pm
2	HISTORY OF DRUGS IN HORSES The less than proper medication of racing horses has a long history, with an absolutely impeccable starting point, Greek mythology
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8	How do Some Horsemen try to Win Races? Any way they can, including but not limited to the creative use of drugs and medications
9	Acute Stimulant Medication Classic stimulants: cocaine, caffeine, amphetamine, methylphenidate, methamphetamine, mazindol Opiates: etorphine, fentanyl, morphine, buprenorphine Tranquilizers: “washy horses”: acepromazine, promazine, fluphenazine, detomidine, etc. Bronchodilators: clenbuterol, terbutaline, atropine, ipratropium bromide Always illegal and usually an “inside job”
10	Narcotic Analgesics Morphine and synthetic analgesics Locomotor stimulant Suppress pain Prolongs endurance Potentiated by stimulants Reliable, classic stimulant medication: widely used pre-ELISA
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12	Bronchodilators Beta-2 agonist bronchodilators: clenbuterol, albuterol, terbutaline, etc. Highly potent agents Presumably improve oxygen delivery to blood and musculature Widely used close to post prior to ELISA Post-ELISA: use cut back dramatically
13	Tranquilizers as Stimulants Used to treat “washy” horses (“running” race in paddock) Allows jockey to “rate” horse in the race Detomidine, reserpine, acepromazine, promazine All legitimate therapeutic agents: rate of improper use?
14	Chronic Stimulant Medication Anabolic steroids, growth hormone, erythropoietin (EPO) Special food supplements Vitamins By definition an “inside job” With the exception of anabolic steroids, not currently a very active area
15	“Stopping” Horses “Nobbling” the English term Horses heavily backed “ante-post” Administer tranquilizer or depressant to slow or stop a horse Oldest form of illegal medication Usually considered an “outside” job
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17	Medication to Lose The oldest form of illegal medication In principle, always possible to medicate a horse so that it will lose Challenge is to treat a horse such that he goes to post but does not win his race More likely where there is heavy ante-post betting Also possible to medicate a horse to lose so that a favored horse will win Always illegal, and usually considered an “outside” job
18	Medication to Restore “Normal” Performance Thought is that you can medicate a horse to restore “normal” performance No stimulant or depressant actions Non-steroidal anti-inflammatory agents (NSAIDs) Prophylaxis of EIPH: furosemide Corticosteroids?
19	Medication to “Mask” Other Agents Administer agents that will interfere with testing As testing improves, a less likely scenario Furosemide: dilution Polyethylene glycol: smear TLC plates Thiamine: interfere with UV analysis
20	Medication to Dilute Out Other Drugs Administer a drug that dilutes drugs/drug metabolites and makes them more difficult to detect Diuretics generally do not affect blood levels of drugs Dilute out water soluble drugs/drug metabolites in equine urine Effect is generally up to but not greater than the diluting effect Rules on furosemide in the US specify dose and time of administration
21	Can Drugs/Medications Influence Performance?
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26	Equine Drug Testing Equine drug testing has been in place longer (since 1910), and is more rigorous and extensive (250-2,500 agents) than any other ongoing human drug testing endeavor WHAT HAPPENS Horsemen use drugs ® Chemists develop tests ® Horsemen switch to new drugs ® Chemists develop new tests; one outcomes is that: Neogen has a large and developing panel of Forensic Drug Tests ad infinitum?
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28	Equine Drug Testing Based on post-race urine samples About 10% of runners sampled/500,000 samples/year worldwide Winners, beaten favorites, selected horses that the stewards nominate Usually a central university-based laboratory, testing 10-40K samples/year Scope of testing very broad: 250-2,500 agents
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30	High Potency Agents Any drug given to a horse < 10 mg/horse Etorphine, Fentanyl, Acepromazine, Mazindol, Terbutaline, Buprenorphine, Cocaine, Oxymorphone, Detomidine Not being reliably detected pre-ELISA Being used at will by some horsemen
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32	The 1980’s: ELISA Testing Immunoassay program starts; Illinois Racing Board Laboratory, IDS, a Biotech Company, and the University of Kentucky Sensitivity of testing increased 100-1,000 fold, virtually overnight, for 20+ drugs Multiple identifications of narcotics, stimulants, tranquilizers, bronchodilators
33	ELISA Testing Enzyme Linked ImmunoSorbent Assay (ELISA) Simple, fast, high sensitivity testing ~1,000 times more sensitive than thin layer chromatography In 1985, UK started a research project that rapidly evolved into the ELISA testing program
34	Enzyme-Linked ImmunoSorbent Assay (ELISA) Testing Thomas Tobin, Daniel Tai, and David Watt, 1985-1992 1985: Kentucky Racing Commission requests control of high potency narcotics 1986: RIA tests for fentanyl and etorphine (“Elephant juice”) 1987: Program develops ELISA tests 1988: ELISA tests dramatically effective: POLITICS 1989: Program reorganized – development of tests continues 1992: 50 ELISA tests for high potency drugs, yielding ELISA Technologies, a division of Neogen Corporation
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37	How to make an ELISA test Obtain drug or drug structure Derivatize drug: add COOH or NH₂group: hapten Link derivatized drug (hapten) to BSA Inject into rabbit, re-inject, wait Link hapten to Horse Radish Peroxidase (HRP) Check rabbit serum for antibodies Construct and validate ELISA test
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45	ELISA Screening of Post-Race Urine Samples Followed by Mass Spectral Analysis
46	Efficacy of PCFIA/ELISA Tests, 1987-1988
47	Fall 1987 ELISA testing of frozen post-race samples in New Mexico First panel of ELISA tests puts down 40 plus trainers in New Mexico A problem that had been in place, in one form or another for 100, years had been controlled
48	The 1980’s: Impact of ELISA Testing Probably the biggest single “across the board” increase in the sensitivity of equine drug screening Stopped patterns of medication that had been in place in one form or another for the better part of 100 years Also greatly increased the sensitivity of testing for therapeutic medications
49	ELISA Testing Offers virtually complete control of the abuse of high potency medications Also offers super-sensitive testing for therapeutic medications Has accentuated a long-standing problem in equine medication control: Detection of trace residues of therapeutic medications and environmental and endogenous substances
50	Where To Go, 1987? 1987 -- Five ELISA tests (but 100’s of drugs) 2002 -- Neogen’s nearly 100 ELISAs, can screen for more than 250 agents Many current ELISA tests are of interest in HUMAN FORENSICS and the FOOD SAFETY INDUSTRIES
51	Neogen’s ELISA Tests for Use in Racing (May 1st, 2002) Alfentanil, amphetamine, amphetamine ultra, anileridine, apomorphine, azaperone, barbiturate group, benzodiazepine group, boldenone, bronchodilator group, bumetanide, buprenorphine, butorphanol, caffeine/pentoxifylline, carfentanil, clenbuterol, cocaine/benzoylecgonine, corticosteroid group, cromoglycate, dantrolene, detomidine, dexamethasone, diprenorphine, doxapram, droperidol, ethacrynic acid, etorphine, fentanil group, fentanyl, flunitrazepam, flunixin, fluoxetine, fluphenazine, furosemide, glycopyrrolate, guanabenz, haloperidol…
52	Neogen’s ELISA Tests, cont’d Haloperidol metabolite, hydromorphone, isoxsuprine, ketamine, ketorolac, levallorphan, lidocaine, lofentanil, mazindol, mazindol metabolite, meperidine, mephentermine, mepivacaine, methadone, methamphetamine/MDMA, methocarbamol, methotrexate, methylphenidate, methylprednisolone, nalbuphine, nandrolone, opiate group, oxymorphone/oxycodone, pentazocine, phenylbutazone, procaine, promazine group, propanolol, pyrilamine, reserpine, stanozolol, sufentanil, sulfamethazine, THC metabolite, theophylline, trenbolone, triamcinolone acetonide, tricyclics group, zomepirac
53	Neogen’s Forensic Market ELISA Tests Alfentanil, Amphetamine, Barbiturate group, Benzodiazepine group, Boldenone, Buprenorphine, Butorphanol,Carfentanil, Cocaine/BZE, Etorphine, Fentanil group, Flunitrazepam, Hydromorphone, Lofentanil, Ketamine, LSD, Meperidine, Methadone, Methylphenidate, Methamphetamine/MDMA, Nandrolone, Opiate group, Oxymorphone/Oxycodone, Sufentanil, THC Metabolite
54	NEOGEN AND FORENSIC CHEMISTRY Neogen has available a large and expanding range of high quality forensic tests Largest selection of high quality forensic ELISA tests available worldwide Available to Equine and Human Forensic Labs. -AD INFINITUM?-
55	Efficacy of Equine Drug Testing Rate of detection of performance altering agents in racing is relatively low “Positives” in about 1-1,000 samples tested. All identifications confirmed by mass spectrometry in the primary and the referee laboratory. “Top-end” of racing is actually very clean Thanks in part to the technology I have just described
56	SUMMARY ELISA testing technology is highly effective: the Worldwide Standard in Racing Simple,fast,inexpensive and highly sensitive Technology is highly adaptable Single, portable “Horse Side” tests Inexpensive automated systems High end, high volume, high throughput completely automated systems
57	THREE CUSTOMIZED LEVELS OF ELISA TEST UTILIZATION Limited Throughput: 10K samples/year 5-10 tests/sample Medium Throughput: 20K samples/year 10-15 tests/sample High Throughput: 100K samples/year 25-30 tests/sample
58	LIMITED VOLUME THROUGHPUT Contract to buy a certain annual volume of ELISA tests (100K tests/year, or so) Make available fully automated $50K Thermolabsystems ‘DSX’ Analyzer to perform the ELISA analyses Information: scottcongleton@Neogen.com
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62	MEDIUM VOLUME THROUGHPUT Contract to buy a certain annual volume of ELISA tests (300K tests/year, or so) Make available fully automated $100K Tecan Genesis System to perform the ELISA analyses Information: scottcongleton@Neogen.com
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64	HIGH VOLUME THROUGHPUT Contract to buy a certain annual volume of ELISA tests (3,000K tests/year, or so) Make available fully automated $200K Beckman Coulter Orca System to perform the ELISA analyses Information: scottcongleton@Neogen.com
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71	THANKS TO THE FOLLOWING Thanks to my longtime colleagues at Neogen Corp, for their ongoing support in expanding and commercializing this technology To all the members of my research group,and especially Drs. W. Karpiesiuk, S. Kwiatkowski and Mr. W. E. Woods. The Kentucky Racing Commission and the Kentucky Equine Drug Council for support To the Director of the Gluck Equine Research Center and to my colleagues there and at UK for their support over the years
72	Finally, in closing, thank you very much,Ladies and Gentleman, for your kind attention this morning
73	LITERATURE REFERENCES 1/ Remifentanil in the Horse: Identification and Detection of its Major Urinary Metabolite; Lehner et al., J. Anal. Tox. Vol 24, July/August, 2000 2/ “Kentucky Laboratory Takes Lead Against Horse Dopers,” Tobin, Clinical Chemistry News, Vol. 18, Issue 1, January 1992 3/ Non-isotopic Immunoassay Drug Tests in Racing Horses: A Review of their Application to Pre- and Post-Race Testing, Drug Quantitation and Human Testing; Tobin et al., Res. Comm. Chem. Path. Pharmacol. Vol 62, #3, P371-396, 1988
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