International Journal of Obesity (2001) 25, 1454–1458 ß 2001 Nature Publishing Group All rights reserved 0307–0565/01 $15.00 www.nature.com/ijo PAPER Monoamine oxidase inhibition is unlikely to be relevant to the risks associated with phentermine and fenfluramine: a comparison with their abilities to evoke monoamine release{ IC Kilpatrick1*, M Traut2 and DJ Heal1 1Knoll Limited Research and Development, Nottingham, UK; and 2Knoll GmbH, 50 Knollstrasse, D-67061, Ludwigshafen, Germany OBJECTIVE AND DESIGN: It has been proposed that the anti-obesity agent, phentermine, may act in part via inhibition of monoamine oxidase (MAO). The ability of phentermine to inhibit both MAOA and MAOB in vitro has been examined along with that of the fenfluramine isomers, a range of selective serotonin reuptake inhibitors and sibutramine and its active metabolites. RESULTS: In rat brain, harmaline and lazabemide showed potent and selective inhibition of MAOA and MAOB, their respective target enzymes, with IC50 values of 2.3 and 18 nM. In contrast, all other drugs examined were only weak inhibitors of MAOA and MAOB with IC50 values for each enzyme in the moderate to high micromolar range. For MAOA, the IC50 for phentermine was estimated to be 143 mM, that for S( þ )-fenfluramine, 265 mM and that for sertraline, 31 mM. For MAOB, example IC50s were as follows: phentermine (285 mM), S( þ )-fenfluramine (800 mM) and paroxetine (16 mM). Sibutramine was unable to inhibit either enzyme, even at its limit of solubility. CONCLUSION: We therefore suggest that MAO inhibition is unlikely to play a role in the pharmacodynamic properties of any of the tested drugs, including phentermine. Instead, the lack of potency of these drugs as MAO inhibitors is contrasted with their powerful ability either to inhibit the uptake of one or more monoamines (fluoxetine, paroxetine, sertraline, sibutramine’s active metabolites) or to evoke the release of one or more monoamines (S( þ )-fenfluramine, S( þ )-norfenfluramine, phentermine). These differences in mode of action may be linked to the adverse cardiovascular events experienced with some of the releasing agents. International Journal of Obesity (2001) 25, 1454 – 1458 Keywords: phentermine; fenfluramine; monoamine oxidase; serotonin reuptake; serotonin release; cardiovascular Introduction otonin (5HT) has been proposed on more than one occa- Phentermine is an amphetamine derivative that has been sion3–6 and for valvulopathy, 5HT receptor mechanisms on used to treat obesity for over three decades and from 1992, it valve tissue are also postulated to be relevant.7 Aside from was often used for this purpose in combination with either the known ability of phentermine to promote dopamine fenfluramine or D-fenfluramine (S( þ )-fenfluramine) until an release in the brain,8,9 recent discussion on its mode of association of fenfluramines with both heart-valve disease1,2 action has centred on a proposal that phentermine may and pulmonary hypertension3 led to the withdrawal of inhibit monoamine oxidase (MAO).10 Within this proposal, fenfluramines in 1997. Whilst the pathobiology of both the authors not only inferred that such an action could boost events remains uncertain, a link with elevated plasma ser- free 5HT concentrations in plasma beyond those evoked by 5HT-releasing agents such as fenfluramine and S( þ )-fenflur- *Correspondence: IC Kilpatrick, Knoll Limited, St Nicholas Court, amine, but for the same reason they also warned against the 25 – 27 Castle Gate, Nottingham NG1 7AR, UK. possible combined use of phentermine with selective sero- E-mail: [email protected] tonin reuptake inhibitors (SSRIs) such as fluoxetine.10 How- { This article is dedicated to the late Professor Michael J Stock, colleague ever, such a role of phentermine as an MAO inhibitor and friend. Received 11 September 2000; revised 2 March 2001; (MAOI) has been discounted based on a review of some accepted 27 March 2001 basic and clinical biochemical findings.11 Phentermine and monoamine oxidase inhibition IC Kilpatrick et al 1455 Whilst such warnings undoubtedly generate concern After stopping the reaction, 3.5 ml toluene-based scintil- among physicians and patients alike, neither report provides lant were added and the samples were shaken for 30 min in an unequivocal answer to the fundamental question of order for the MAO-generated non-basic metabolites to be whether MAO inhibition is relevant to the actions of phen- extracted into the organic phase, leaving the unreacted termine. The problem is compounded because until the substrates in the non-scintillating aqueous phase. Once the recent study,10 no published work from within the last two aqueous and organic phases had separated, the vials were decades has examined the interaction between phentermine transferred to a Packard TriCarb 20001 scintillation counter. and MAO. This prompted us to compare the abilities of some anti-obesity agents including phentermine, S( þ )-fenflura- mine and the 5HT and noradrenaline reuptake inhibitor (SNRI), sibutramine, along with a series of SSRIs, to influence Data presentation and statistical analysis MAO activity directly. In an attempt to shed light on the Inhibition of enzyme activity was expressed as IC50 values possible link between elevated 5HT levels and cardiovascular calculated by linear regression of the logit-transformed inhi- disease, comparisons of the potency of these drugs derived bition values vs log concentration values. Since MAOA is here against the isoenzymes of MAO (MAOA, which is chiefly largely responsible for the metabolism of 5HT in rat brain, an responsible for 5HT degradation and MAOB) are then made estimate of the relative potency of each drug to inhibit this with their known abilities to inhibit 5HT reuptake and to isoenzyme was made in comparison to the inhibitory evoke 5HT release. Some of these data have been published potency of phentermine. For this analysis, data were log10- in preliminary form.12 transformed and subjected to one-way ANOVA followed by Dunnett’s test. A P-value lower than 0.05 was taken to be significant. Methods Monoamine oxidase A and B activity and its inhibition We used modifications to a published method13 for MAO Drugs estimation that allowed us to selectively assay MAOA and Fluoxetine hydrochloride, paroxetine hydrochloride, sertra- MAOB in samples of rat brain. Briefly, male Sprague – Dawley line hydrochloride, S( þ )-fenfluramine, R( 7 )-fenfluramine, rats (190 – 200 g) were killed by cervical dislocation, their S( þ )-norfenfluramine, sibutramine (N-(1-[1-(4-chlorophe- brains dissected free and the cerebella removed from them. nyl)cyclobutyl]-3-methylbutyl)-N,N-dimethylamine hydro- Each brain was weighed, suspended in 50 mM potassium chloride monohydrate) and its active metabolites, phosphate buffer (9 mlg71 wet weight; pH 7.4) at 2C and metabolite 1 (BTS 54 354; N-(1-[1-(4-chlorophenyl)cyclobu- homogenised in a Polytron1 PT. The homogenate was fil- tyl]-3-methylbutyl)-N-methylamine hydrochloride) and tered through four layers of gauze and diluted 1 in 40 vol=vol metabolite 2 (BTS 54 505; 1-[1-(4-chlorophenyl)cyclobutyl]- by the same ice-cold buffer. This preparation was appor- 3-methylbutylamine hydrochloride, were synthesised by the tioned into aliquots and stored at 780C until use. One Medicinal Chemistry Department of Knoll Limited Research hundred microlitres of homogenate were pre-incubated with & Development, Nottingham, UK. Lazabemide was synthe- 100 ml of phosphate buffer (containing test compounds as sised by the Medicinal Chemistry Department of Knoll required) for 5 min at 37C. A 100 ml aliquot of substrate GmbH Research & Development, Ludwigshafen, Germany. solution (see below) was then added and the mixture incu- Phentermine hydrochloride and harmaline hydrochloride bated at 37C for 20 min. The reaction was stopped by were purchased from Sigma. All general reagents were adding 1.5 ml of 0.1 M HCl. All assays were performed purchased from Merck, Darmstadt. in triplicate and controls were obtained by adding HCI immediately after adding substrate solution without further incubation. Results 14 The substrate for MAOA was [side chain-2- C]-tryptamine Reference agents (NEN Life Science Products; specific activity 1900 MBq= Under the present conditions of low ( < 1.0 mM) substrate 14 14 mmol). The compound was diluted to obtain 10 000 – concentration ( C-tryptamine for MAOA and C-b-pheny- 30 000 dpm=100 ml corresponding to 0.3 – 1.0 mM final con- lethylamine for MAOB), the high potencies and selectivities centration in the assay mixture. The substrate for MAOB was of reference inhibitors of MAOA and MAOB were confirmed. 2-phenyl-[1-14C]-ethylamine (NEN Life Science Products; Thus, the data in Table 1 show that the reversible and 14 specific activity 1550 MBq=mmol). The compound was selective MAOA inhibitor, harmaline, yielded an IC50 diluted to obtain 10 000 – 30 000 dpm=100 ml corresponding value of 2.3 nM against MAOA compared with 59 000 nM to 0.35 – 1.05 mM final concentration in the assay mixture. against MAOB. On the other hand, the reversible and selec- 15 Under the conditions employed, substrate turnover in con- tive MAOB inhibitor, lazabemide, produced an IC50 value trol assays was in the range of 20 – 30% for MAOA and 30 – of 18 nM against MAOB compared with 125 000 nM against 40% for MAOB, respectively. MAOA. International Journal of Obesity Phentermine and monoamine oxidase inhibition IC Kilpatrick et al 1456 Table 1 Drug action (IC50 values) on MAOA and MAOB activities Phentermine and fenfluramines as putative MAO inhibitors P-values vs The present estimates of the inhibitory potency of phenter- MAOA MAOB phentermine IC50 (mM) IC50 (mM) for MAOAI mine are in accord with IC50 data from nearly three decades ago which ranged from 180 to 1000 mM20,21 obtained from Phentermine 143Æ 6285Æ 22 S( þ )-Fenfluramine 265Æ 16 800Æ 29 < 0.001 studies using rat brain or liver that were unable to differ- S( þ )-Norfenfluramine 36Æ 2160Æ 14 < 0.001 entiate between the isozymes of MAO.