PAPER Monoamine Oxidase Inhibition Is Unlikely to Be Relevant To

Home , Harmaline, Lazabemide

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 )-fenfluramine 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. The current data also R( 7 )-Fenfluramine 115Æ 16 685Æ 57 < 0.001 concur with those from the very recent study of Ulus and Sibutramine > 1000 > 1000 10 colleagues, in which estimates of the IC50 of phentermine BTS 54 354 (M1) 158Æ 10 41Æ 1 < 0.001 BTS 54 505 (M2) 128Æ 634Æ 2 < 0.001 as a MAOAI in brain tissue are 100 – 200 mM although their Fluoxetine 69Æ 322Æ 2 < 0.001 IC50 estimate of 600 – 900 mM for phentermine as a MAOBIin Paroxetine 81Æ 316Æ 2 < 0.001 brain is slightly less potent than that observed in the current Sertraline 31Æ 549Æ 4 < 0.001 study. In comparison with the potency of the reference Harmaline 0.0023Æ 0.0003 59Æ 1 inhibitors used in the present study, phentermine is approxi- Lazabemide 18Æ 1 0.018Æ 0.001 mately 62 000 times less active as an inhibitor of MAOA and Data are meanÆ s.e.m. for four to eight observations. some 16 000 times less active as an inhibitor of MAOB. M1 and M2 are the respective metabolites 1 and 2 of sibutramine. In fact, the inhibitory potencies of harmaline and lazabemide for their respective non-target enzyme are slightly greater than those of phentermine for these same enzymes and are similar to those of the SSRIs. However, moclobemide, Anti-obesity agents and SSRIs an established antidepressant and reversible inhibitor of

Table 1 shows that phentermine is a very weak inhibitor of MAOA, has a relatively weak potency in vitro 22 MAOA and MAOB with IC50 values in the high micromolar (IC50 6 mM ). This potency, which is at least partly range. The two enantiomers of fenfluramine are similarly reflected in the large daily dosing requirements of patients, weak inhibitors of each enzyme, although S( þ )-fenflura- is barely an order of magnitude greater than that displayed mine is slightly but significantly less active than phenter- by some SSRIs, and yet none of these is endowed with

mine as a MAOAI whilst R( 7 )-fenfluramine is slightly but properties of MAO inhibition at therapeutic doses (see significantly more potent than phentermine in this regard. below). As phentermine is less potent still, we would argue The active metabolite of S( þ )-fenfluramine, S( þ )-norfen- that phentermine should not be classed as an MAOI, espe- fluramine, is significantly more active than phentermine as cially in view of its estimated plasma level in man of 0.7 mM 11 a MAOAI, being four times as potent here. following recommended doses. Various SSRIs along with metabolites 1 and 2, the active metabolites of the SNRI, sibutramine, are also weak inhibi-

tors of MAOA and MAOB with IC50 values in the micromolar MAO inhibition as a link with drug-associated range. Of the SSRIs tested, sertraline was the most potent cardiovascular disease 10 MAOAI although each SSRI was significantly more potent One clear omission from the work described by Ulus et al than phentermine as a MAOAI. Whilst neither metabolite of was a failure to consider the potency of the fenfluramines as sibutramine differed significantly from phentermine in its MAOIs. This was surprising because fenfluramine and its

potency as an inhibitor of MAOA, sibutramine itself was isomers were attributed with weak MAO inhibitory proper- 23 ineffective as an inhibitor of either isoenzyme, even at its ties (IC50 1000 mM) 3 y prior to the earliest studies on limit of solubility (Table 1). phentermine and the results of these studies were later reinforced.24 Using the more sophisticated assay conditions here, the present data show that phentermine is barely twice

Discussion as potent as S( þ )-fenfluramine as a MAOAI, whilst a more Methodological considerations striking finding is that the major active metabolite of S( þ )- The assay was validated by the use of the reversible MAO fenfluramine, ie S( þ )-norfenfluramine, is four times as

inhibitors, harmaline and lazabemide, whose selectivity for potent as phentermine as a MAOAI. Thus, coupled with MAOA and MAOB and high inhibitory potency within the earlier data that placed S( þ )-fenfluramine 10-fold more low nanomolar range was confirmed.14,15 Given the reported active than phentermine in this regard,20,21,23,24 there is no differences in assay conditions (eg substrates, substrate con- reason why, if phentermine were to be recognised as a MAOI, centrations, incubation time), the various literature S( þ )-fenfluramine should not be similarly treated. Indeed, values16 – 18 for the potency of fluoxetine as an inhibitor of MAO inhibition has already been proposed21 as a causal link

MAOA and MAOB and that reported here concur well. The between the incidence of pulmonary hypertension and early report19 that sibutramine did not inhibit rat brain MAO treatment with aminorex or chlorphentermine, two early either at its highest tested in vitro concentration of 100 mMor anorectic agents that were withdrawn over 25 y ago. How- in ex vivo studies is also borne out by the current ﬁndings. ever, neither phentermine nor compounds with signiﬁcantly

International Journal of Obesity Phentermine and monoamine oxidase inhibition IC Kilpatrick et al 1457 greater MAO inhibition than phentermine (Table 1) have are 2000 – 162 000-fold less than their IC50 values for MAO been associated with such cardiovascular disease. As dis- inhibition. Unlike the SSRIs, both S( þ )-fenfluramine and its cussed below, it is therefore considered unlikely that MAO metabolite are extremely weak inhibitors of 5HT reuptake, 30 – 32 inhibition contributes to fenfluramine’s pharmacological and phentermine is essentially inactive with a Ki of 11 mM actions. (see Table 2). Importantly, the SSRIs, none of which releases 5HT, are more potent MAO inhibitors than either S( þ )- fenfluramine or phentermine and each is of similar potency Alternative sources of 5HT elevation by phentermine and to S( þ )-norfenfluramine. fenfluramine? These data illustrate two vital points. First, the com- Whether valvulopathy and=or pulmonary hypertension are pounds that are the most potent 5HT reuptake inhibitors, linked to prolonged or even brief rises in circulating 5HT is the SSRIs, also happen to be the most potent MAO unresolved. The acute ability of S( þ )-fenfluramine and its inhibitors. Yet, if MAO inhibition was of major pharmaco- active metabolite to release 5HT from blood platelets25,26 logical relevance to the action of the SSRIs and SNRIs, they could provide a peripheral source of 5HT that is relevant to would never have been developed because of the risks of the pathobiology. However, plasma 5HT changes after drug inducing, for example, the serotonin syndrome. That this treatment are difficult to interpret since it is very likely that does not occur is simple testament to the many orders of repeated fenfluramine treatment will deplete platelets of magnitude between their potency as SSRIs=SNRIs and MAO their 5HT stores27 and may even cause plasma 5HT levels inhibitors. Second, the data stress that 5HT reuptake inhi- to fall, as demonstrated both in rats given S( þ )-fenfluramine bition, as borne out by enormous clinical experience, is for 14 days27 and in patients treated with fenfluramine and not associated with these cardiovascular disorders because phentermine for at least 2 months.28,29 Alternatively, direct they have not been reported even with prolonged use of 5HT receptor stimulation by S( þ )-norfenfluramine7 could the SSRIs. trigger mechanisms that lead to valvulopathy. In any event, it is clear from Tables 1 and 2 that S( þ )-fenfluramine and its active metabolite, S( þ )-norfenfluramine, are more potent Conclusions releasers of 5HT from brain slices30 than they are inhibitors For any drug that has multiple possible targets, relative of MAO and that this 5HT-releasing action is not shared by potency is the most important parameter to consider. On phentermine, the SSRIs, the SNRI, sibutramine, or its two these grounds alone, the hypothesis10 that MAO inhibition active metabolites (Table 2). by phentermine may contribute to cardiovascular disorders However, these comparisons of potency ratios pale into has little to commend it given the many orders of magnitude insignificance when the MAO inhibitory activities of each of that separate the potency of SSRIs and SNRIs as 5HT reuptake the tested SSRIs are placed alongside their ability to inhibit inhibitors vs MAOIs. Whilst none of the SSRIs or SNRIs reuptake of radiolabelled 5HT by rat frontal cortex synapto- releases 5HT, all are very weak MAOIs and some are margin- somes.31 As shown in Table 2, this analysis reveals that the ally more potent than phentermine, the fenfluramine SSRIs inhibit 5HT reuptake by 50% at concentrations which enantiomers or S( þ )-norfenfluramine.

Table 2 Drug action on 5HT release and 5HT uptake inhibition plus ratios for MAOA or MAOB inhibition to 5HT uptake inhibition

5HT release Percentage increase 5HT uptake

above basal (slices) inhibition MAOAI: uptake MAOBI: uptake (Synaptosomes) inhibition inhibition

1 mM10mMKi (mM) ratio ratio

Phentermine 0 0 11.17Æ 1.66 12.8 25.5 S( þ )-Fenfluramine 70Æ 8* 135Æ 19* 0.28Æ 0.018 950 2,867 S( þ )-Norfenfluramine 0 100Æ 35* 0.54Æ 0.08 66.5 296 R( 7 )-Fenfluramine NT NT NT Sibutramine 0 0 3.13Æ 0.19 > 319 > 319 BTS 54 354 (M1) 0 0 0.018Æ 0.002 8681 2253 BTS 54 505 (M2) 0 0 0.026Æ 0.007 4885 1298 Fluoxetine 0 0 0.011Æ 0.001 6,330 2,018 Paroxetine 0 0 0.0005Æ 0.0001 162,000 32,000 Sertraline 0 0 0.0028Æ 0.0003 11,071 17,500

Data are meanÆ s.e.m. for four to eight observations. NT not tested. Synaptosomal uptake data are taken from Cheetham et al.31 Brain slice release data are taken from Lancashire et al.33 *P < 0.001. Signiﬁcantly different from control fractional release values; ANOVA followed by Dunnett’s test.

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