Drug testing and analysis2024; 16(10); 1182-1194; doi: 10.1002/dta.3642

Comprehensive metabolic investigation of dopamine reuptake inhibitor HDMP-28 in equine liver microsomes and Cunninghamella elegans for doping control.

Muhammed Ajeebsanu, M
·
Subhahar, MB
·
Karakka Kal, AK
·
Philip, M
·
Perwad, Z
·
Karatt, TK
·
Graiban, FM
·
Joseph, M
·
Jose, SV

Abstract: A dopamine reuptake inhibitor is a type of medication or substance that works by blocking the reuptake of dopamine in the brain. Dopamine reuptake inhibitors offer multiple effects, including increased alertness, improved mood, and therapeutic potential for conditions like depression, ADHD, and Parkinson's disease. HDMP-28, or methylnaphthidate, is a potent synthetic stimulant from the phenyltropane class. It surpasses methylphenidate in both dopamine reuptake inhibition and half-life. As a dopamine reuptake inhibitor, it boosts dopamine levels by hindering reuptake into nerve cells, resulting in heightened stimulation and increased energy. In order to comprehensively address both the tangible and potential repercussions of the unauthorized utilization of the aforementioned substance in sports, it is imperative to establish analytical methodologies for the identification of the parent drug and its primary metabolites. Additionally, a comprehensive analysis of the metabolic characteristics of HDMP-28 in both human and animal subjects has yet to be published. This study explores the metabolic conversion of HDMP-28 mediated by equine liver microsomes and Cunninghamella elegans. An extraction and detection method was developed, optimized, and validated for doping assessment in equine urine and plasma. Liquid chromatography-high-resolution mass spectrometry was employed to determine metabolite structures. The study identified 31 (22 phase I and 9 phase II) metabolites of HDMP-28, including hydroxylated, hydrogenated, and hydrolyzed analogs. Glucuronic acid-conjugated metabolites were also detected. This manuscript describes metabolites based on the in vitro studies, which might not be the same in vivo. These findings aid in the detection and understanding of the illicit use of HDMP-28 in equestrian sports.

Get Full Text

Publication Date: 2024-01-15 PubMed ID: 38225724DOI: 10.1002/dta.3642Google Scholar: Lookup

The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.

Facebook X LinkedIn Email Copy

Summary
Cite This
Publication
Affiliations
MeSH
References
Citations

Journal Article
Validation Study

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

This research investigates the metabolic breakdown of the synthetic stimulant HDMP-28, a potent dopamine reuptake inhibitor, using equine liver enzymes and fungal models to help develop detection methods for doping control in horses.

Introduction to HDMP-28 and Its Relevance

HDMP-28, also known as methylnaphthidate, is a synthetic stimulant belonging to the phenyltropane class.
It functions by blocking dopamine reuptake, which increases dopamine levels in the brain, leading to stimulatory effects such as enhanced alertness and energy.
HDMP-28 is more potent than methylphenidate, with stronger dopamine reuptake inhibition and a longer half-life.
Due to its stimulant properties, HDMP-28 has potential for abuse in sports, particularly in equestrian disciplines, where performance enhancement is a concern.

Purpose and Importance of the Study

The study addresses the lack of comprehensive data on how HDMP-28 is metabolized in both humans and animals.
It aims to establish reliable analytical methods to identify both the parent drug and its metabolites, which is essential for doping control and regulatory enforcement in equine sports.
Developing detection methods in biological samples like urine and plasma helps to monitor and prevent illicit use.

Methodology

Metabolic conversion of HDMP-28 was studied using two in vitro systems:
- Equine liver microsomes: to simulate the metabolism as it occurs in horses.
- Cunninghamella elegans: a fungus used as a microbial model to mimic mammalian metabolism.
Extraction, detection, and quantification methods were developed and optimized specifically for equine urine and plasma samples.
Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) was employed
- This enabled the identification and structural characterization of HDMP-28 metabolites with high sensitivity and accuracy.

Key Findings

A total of 31 metabolites of HDMP-28 were identified in the in vitro systems:
- 22 phase I metabolites: including hydroxylated (addition of OH groups), hydrogenated, and hydrolyzed forms, representing initial metabolic transformations.
- 9 phase II metabolites: glucuronic acid-conjugated metabolites indicative of further processing and increased solubility for excretion.
The elucidation of these metabolites provides a metabolic fingerprint of HDMP-28 in equine systems.
These metabolites may serve as biomarkers for detecting HDMP-28 administration in horses.

Implications and Limitations

The metabolic profiles obtained can assist laboratories conducting doping control to identify illicit use of HDMP-28 in horse racing or other equestrian events.
This contributes to fair play and animal welfare by enabling more accurate and sensitive screening assays.
The study results are based on in vitro models, which might not perfectly replicate in vivo metabolism in live animals.
Further in vivo studies are needed to confirm these metabolic pathways and validate the identified biomarkers under physiological conditions.

Conclusion

The research successfully developed a comprehensive analytical framework for detecting HDMP-28 and its metabolites in equine biological samples.
The identification of 31 metabolites provides valuable insight into the drug’s metabolism in horses, facilitating improved doping control measures.
These findings represent an important step toward understanding and regulating the misuse of potent dopamine reuptake inhibitors in equestrian sports.

Cite This Article

APA

Muhammed Ajeebsanu M, Subhahar MB, Karakka Kal AK, Philip M, Perwad Z, Karatt TK, Graiban FM, Joseph M, Jose SV. (2024). Comprehensive metabolic investigation of dopamine reuptake inhibitor HDMP-28 in equine liver microsomes and Cunninghamella elegans for doping control. Drug Test Anal, 16(10), 1182-1194. https://doi.org/10.1002/dta.3642

Publication

Drug testing and analysis

ISSN: 1942-7611

NlmUniqueID: 101483449

Country: England

Language: English

Volume: 16

Issue: 10

Pages: 1182-1194

Researcher Affiliations

Muhammed Ajeebsanu, Meleparappil

Equine Forensic Unit, Central Veterinary Research Laboratory, Dubai, United Arab Emirates.

Subhahar, Michael Benedict

Equine Forensic Unit, Central Veterinary Research Laboratory, Dubai, United Arab Emirates.

Karakka Kal, Abdul Khader

Equine Forensic Unit, Central Veterinary Research Laboratory, Dubai, United Arab Emirates.

Philip, Moses

Equine Forensic Unit, Central Veterinary Research Laboratory, Dubai, United Arab Emirates.

Perwad, Zubair

Equine Forensic Unit, Central Veterinary Research Laboratory, Dubai, United Arab Emirates.

Karatt, Tajudheen Kunhamu

Equine Forensic Unit, Central Veterinary Research Laboratory, Dubai, United Arab Emirates.

Graiban, Fatma Mohammed

Equine Forensic Unit, Central Veterinary Research Laboratory, Dubai, United Arab Emirates.

Joseph, Marina

Department of Bacteriology, Diagnostic Section, Central Veterinary Research Laboratory, Dubai, United Arab Emirates.

Jose, Shantymol V

Department of Bacteriology, Diagnostic Section, Central Veterinary Research Laboratory, Dubai, United Arab Emirates.

MeSH Terms

Horses
Animals
Microsomes, Liver / metabolism
Doping in Sports / prevention & control
Substance Abuse Detection / methods
Cunninghamella / metabolism
Dopamine Uptake Inhibitors / pharmacology
Dopamine Uptake Inhibitors / metabolism
Tandem Mass Spectrometry / methods
Chromatography, High Pressure Liquid / methods

References

This article includes 26 references

Rothman RB, Baumann MH, Dersch CM. Amphetamine‐type central nervous system stimulants release norepinephrine more potently than they release dopamine and serotonin.. Synapse 2001;39(1):32‐41.
doi: 10.1002/1098‐2396(20010101)39:1<32::aid‐syn5>3.0.co;2‐3google scholar: lookup
Carboni E, Silvagni A. Dopamine reuptake by norepinephrine neurons: exception or rule?. Crit Rev Neurobiol 2004;16(1–2):121‐128.
doi: 10.1615/critrevneurobiol.v16.i12.130google scholar: lookup
Volkow ND, Wang GJ, Fowler JS. Dopamine transporter occupancies in the human brain induced by therapeutic doses of oral methylphenidate.. Am J Psychiatry 1998;155(10):1325‐1331.
doi: 10.1176/ajp.155.10.1325google scholar: lookup
Song R, Hai‐Ying Z, Li X, Bi G‐H, Gardner EL, Xi Z‐X. Increased vulnerability to cocaine in mice lacking dopamine D3 receptors.. Neuroscience 2012;109(43):17675‐17680.
Heal DJ, Gosden J, Smith SL. Dopamine reuptake transporter (DAT) “inverse agonism”—a novel hypothesis to explain the enigmatic pharmacology of cocaine.. Neuropharmacology 2014;87:19‐40.
doi: 10.1016/j.neuropharm.2014.06.012google scholar: lookup
Loland CJ, Mereu M, Okunola OM. R‐modafinil (armodafinil): a unique dopamine uptake inhibitor and potential medication for psychostimulant abuse.. Biol Psychiatry 2012;72(5):405‐413.
doi: 10.1016/j.biopsych.2012.03.022google scholar: lookup
Madras BK. History of the discovery of the antipsychotic dopamine D2 receptor: a basis for the dopamine hypothesis of schizophrenia.. J Hist Neurosci 2013;22(1):62‐78.
doi: 10.1080/0964704x.2012.678199google scholar: lookup
Carroll FI, Howard JL, Howell LL, Fox BS, Kuhar MJ. Development of the dopamine transporter selective RTI‐336 as a pharmacotherapy for cocaine abuse.. AAPS J 2006;8(1):E196‐E203.
doi: 10.1208/aapsj080124google scholar: lookup
Kintscher U. Reuptake inhibitors of dopamine, noradrenaline, and serotonin.. Handb Exp Pharmacol 2012;209:339‐347.
doi: 10.1007/978‐3‐642‐24716‐3_15google scholar: lookup
Ashok AH, Mizuno Y, Volkow ND, Howes OD. Association of stimulant use with dopaminergic alterations in users of cocaine, amphetamine, or methamphetamine: a systematic review and meta‐analysis.. JAMA Psychiatry 2017;74(5):511‐519.
doi: 10.1001/jamapsychiatry.2017.0135google scholar: lookup
Matecka D, Rothman RB, Radesca L. Development of novel, potent, and selective dopamine reuptake inhibitors through alteration of the piperazine ring of 1‐[2‐(diphenylmethoxy)ethyl] and 1‐[2‐[bis(4‐fluorophenyl)methoxy]ethyl]‐4‐(3‐phenylpropyl)piperazines (GBR 12935 and GBR 12909).. J Med Chem 1996;39(24):4704‐4716.
doi: 10.1021/jm960305hgoogle scholar: lookup
Schmeichel BE, Zemlan FP, Berridge CW. A selective dopamine reuptake inhibitor improves prefrontal cortex‐dependent cognitive function: potential relevance to attention deficit hyperactivity disorder.. Neuropharmacology 2013;64(1):321‐328.
doi: 10.1016/j.neuropharm.2012.07.005google scholar: lookup
Schreiber R, Campbell U, Quinton MS, Hardy LW, Fang QK, Lew R. In vitro and in vivo pharmacological characterization of dasotraline, a dual dopamine and norepinephrine transporter inhibitor in vivo.. Biomed Pharmacother 2022;153:113359.
doi: 10.1016/j.biopha.2022.113359google scholar: lookup
Davies HM, Hopper DW, Hansen T, Liu Q, Childers SR. Synthesis of methylphenidate analogues and their binding affinities at dopamine and serotonin transport sites.. Bioorg Med Chem Lett 2004;14(7):1799‐1802.
doi: 10.1016/j.bmcl.2003.12.097google scholar: lookup
Volkow ND, Swanson JM. Variables that affect the clinical use and abuse of methylphenidate in the treatment of ADHD.. Am J Psychiatry 2003;160(11):1909‐1918.
doi: 10.1176/appi.ajp.160.11.1909google scholar: lookup
Dinis‐Oliveira RJ. Metabolomics of methylphenidate and ethylphenidate: implications in pharmacological and toxicological effects. Eur J Drug Metab Pharmacokinet 2017;42(1):11‐16.
doi: 10.1007/s13318-016-0362-1google scholar: lookup
Lile JA, Wang Z, Woolverton WL. The reinforcing efficacy of psychostimulants in rhesus monkeys: the role of pharmacokinetics and pharmacodynamics. J Pharmacol Exp Ther 2003;307(1):356‐366.
doi: 10.1124/jpet.103.049825google scholar: lookup
Schweri MM, Deutsch HM, Massey AT, Holtzman SG. Biochemical and behavioral characterization of novel methylphenidate analogs. J Pharmacol Exp Ther 2002;301(2):527‐535.
doi: 10.1124/jpet.301.2.527google scholar: lookup
Deutsch HM, Ye X, Shi Q, Liu Z, Schweri MM. Synthesis and pharmacology of site specific cocaine abuse treatment agents: a new synthetic methodology for methylphenidate analogs based on the Blaise reaction. Eur J Med Chem 2001;36(4):303‐311.
doi: 10.1016/s0223-5234(01)01230-2google scholar: lookup
Negreira N, Erratico C, van Nuijs AL, Covaci A. Identification of in vitro metabolites of ethylphenidate by liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry. J Pharm Biomed Anal 2016;117:474‐484.
doi: 10.1016/j.jpba.2015.09.029google scholar: lookup
Karatt TK, Sathiq MA, Laya S, Philip M, Karakka Kal AK, Subhahar MB. Investigation of in vitro generated metabolites of GLPG0492 using equine liver microsomes for doping control. Drug Test Anal 2023;15(6):1‐24.
doi: 10.1002/dta.3453google scholar: lookup
Philip M, Mathew B, Tajudheen KK, Zubair P, Subhahar MB, Abdul KKK. Metabolic studies of HIF stabilizers IOX2, IOX3, and IOX4 (in vitro) for doping control. Drug Test Anal 2021;13(4):794‐816.
doi: 10.1002/dta.3000google scholar: lookup
Watanabe S, Iwai T, Matsushita R. Comparison between human liver microsomes and the fungus Cunninghamella elegans for biotransformation of the synthetic cannabinoid JWH‐424 having a bromo‐naphthyl moiety analysed by high‐resolution mass spectrometry. Forensic Toxicol 2022;40(2):278‐288.
doi: 10.1007/s11419-022-00612-2google scholar: lookup
Karakka Kal AK, Karatt TK, Nalakath J. Simultaneous analysis of 44 frequently abused corticosteroid drugs using polysaccharide‐based chiral column‐HRMS approach. Anal Sci Adv 2021;2(9‐10):427‐439.
doi: 10.1002/ansa.202000166google scholar: lookup
Ventura E, Gadaj A, Buckley T, Mooney MH. Development of a multi‐residue high‐throughput UHPLC‐MS/MS method for routine monitoring of SARM compounds in equine and bovine blood. Drug Test Anal 2020;12(9):1373‐1379.
doi: 10.1002/dta.2875google scholar: lookup
Karatt TK, Muhammed Ajeebsanu MP, Kal AKK, Subhahar MB, Sathiq MA, Laya S. Electrospray ionisation mass spectrometry (ESI‐MS) adduct formation by mobile phase additives: a case study using nitrile functional groups containing selective androgen receptor modulators (SARMs). Rapid Commun Mass Spectrom 2023;37(14):e9530.
doi: 10.1002/rcm.9530google scholar: lookup