(R)-thionisoxetine, a potent and selective inhibitor of central and peripheral norepinephrine uptake

Life Scicnm, Vol. 56, No. 22 pp. 191549241995 Copy?&@ 0 1995 Elscvier Science Ltd Printed in the USA. All rights rcscrwd al24-32051% $950 t .oo

Pergamon 0024-3205(95)00166-2

(It)-THIONISOXETINE, A POTENT CENTRAL AND PERIPHERAL

AND SELECTIVE NOREPINEPHRINE

INHIBITOR UPTAKE

Donald R. Gehlert, Susan K. Hemrick-Luecke, Douglas A. Schober, Joseph Krushinski, Howbertl, David W. Robertson2, David T. Wong and Ray W. Fuller

OF J. Jeffry

Central Nervous System Research, Lilly Research Laboratories, A Division of Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285 U.S.A. (Received in final form March 16, 1995)

Inhibitors of neuronal norepinephrine (NE) uptake are useful for the treatment of a variety of diseases including depression and urinary incontinence. In the present study, we synthesized and evaluated a novel analog of the potent and selective NE uptake inhibitor, nisoxetine. Thionisoxetine more potently inhibited the uptake of [%+NF into hypothalamic synaptosomes and [3H]-nisoxetine binding to the NE transporter than (R)-nisoxetine. The (R) enantiomer of this compound was significantly more potent than the (S) enantiomer, having a Ki of 0.20 nM in [3H]nisoxetine binding. The (R) enantiomer was approximately 70-fold more potent in inhibiting [3H]-NE uptake when compared to [3H]-5HT uptake. In rats, (R)thionisoxetine prevented hypothalamic NE depletion by 6-hydroxydopamine with an EDso of 0.21 mg/kg. Depletion of NE in peripheral nerves was accomplished by the administration of metaraminol to rats. In this paradigm, (R)-thionisoxetine prevented the depletion of heart NE with an EDSO of 3.4 mg/kg and urethral NE with an ED50 of 1.2 m@g. Thus, (R)-thionisoxetine is a potent and selective inhibitor of NE uptake in both central and peripheral tissues. Key Words: norepinephrine, serotonin, uptake inhibitors, antidepressants

Antidepressants such as imipramine and desipramine are potent inhibitors of the uptake of monoaminergic neurotransmitters. Neurotransmitter uptake is believed to be the primary mechanism in removing the neurotransmitter from the synaptic cleft. The process occurs in the neuron as well as extraneuronally in the surrounding glial cells. In addition, specific norepinephrine (NE) uptake occurs in peripheral tissues such as smooth muscle, salivary gland and the heart (1). Desipramine is an example of a compound that selectively inhibits NE uptake. As a class, NE uptake inhibitors have demonstrated clinical utility in the treatment of depression (2,3) and urinary incontinence (4,5) .

Present addresses: lPathoGenesis Corp., and 2Ligand Pharmaceuticals, 9393 Towne Correspondence: Dr. Donald R. Gehlert, Lilly Research Laboratories, A Division Indianapolis, IN 46285 U.S.A.

201 Elliott Ave. West, Suite 150, Seattle, WA 98119 Centre Drive, Suite 100, San Diego, CA 92121. Mail Code 0510, Central Nervous System Research, of Eli Lilly and Company, Lilly Corporate Center,

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(R)-thionisoxetine as Inhibitor of NE Uptake

Vol. 56, No. 22,1995

Tomoxetine (6) and nisoxetine (7) are phenoxyphenyl propylamines that have high affinity for the NE uptake site with little affinity for other uptake sites or neurotransmitter receptors. Only limited structure activity information is available on these molecules. Recently, we (8) and others (9) found that halogen substitutions in the 2-position resulted in improved potency for the NE uptake site compared to nisoxetine or tomoxetine. This improvement appeared to be maximal with the substitution of an iodine. Since a thiomethyl group has similar electronic properties, we evaluated the enantiomers of 2-thionisoxetine as inhibitors of neuronal NE uptake in vitro. In addition, we evaluated the potency at peripheral and central NE uptake sites in vivo.

Materials Tomoxetine and analogs were synthesized at Eli Lilly and Company. 6-hydroxydopamine (6OHDA) was purchased from Regis Chemical Company (Morton Grove, IL) and metaraminol was purchased from Winthrop Laboratories (New York, NY). [3H]-nisoxetine, [3H]-NE and [3H]serotonin (5HT) were purchased from DuPont-NEN (Boston, MA). Binding Studies Homogenate binding was performed as described by Tejani-Butt et al. (10) with some modification. Briefly, frozen male, Sprague-Dawley rat brains (ABS, Wilmington, DE) were thawed and homogenized in a 50 mM Tris buffer (pH 7.4) using a polytron (Brinkman Instruments, N.J.). The homogenate was centrifuged once at 800 X g and the supematant was transferred to a clean centrifuge tube. The suspension was then pelleted at 20,000 X g, resuspended in fresh buffer and centrifuged again. The final pellet was resuspended in a 50 mM Tris buffer (pH 7.4) containing 300 mM NaCl, 5 mM KCl. Incubations were conducted in an identical buffer containing 0.7 nM [3H]-nisoxetine and various concentrations of inhibitor compounds. Protein concentration was determined using the commassie blue technique with bovine serum albumin as a standard (11). To determine nonspecific binding, 10 PM desipramine (RBI, Natick, MA) was added to some tubes. After a four hour incubation at 4”C, the incubation was terminated by rapid filtration over glass microfiber filters (presoaked in 0.3% polyethyleneimine, Sigma, St. Louis, MO) using a Tomtek cell harvester. The radioactivity retained on the filters was determined using scintillation counting techniques. Ki values were obtained using the Lundon- software. In vitro Uptake Studies Male, Sprague-Dawley rats (loo-15Og, Harlan Industries, Cumberland, IN) were killed by decapitation. Hypothalamus or cortex were homogenized in 9 volumes of a medium containing Crude synaptosomal preparations were isolated after 0.32 M sucrose and 10 mM glucose. differential centrifugation at 1000 x g for 10 minutes and 17,000 x g for 28 minutes. The final pellets were suspended in the same medium and kept on ice until used within the same day. Synaptosomal uptake of [3H]-5HT or [3H]-NE was determined as follows: synaptosomal preparations (equivalent to 0.5 to 1.O mg of protein) were incubated at 37°C for 5 minutes in 1 ml of Krebs bicarbonate medium containing also 10 mM glucose, 0.1 mM iproniazid, 1 mM ascorbic acid, 0.17 mM EDTA, and [3H]-monoamine at a specified concentration. The reaction mixture was immediately diluted with 2 ml of 0.9% saline and filtered using Whatman GF/B filters under vacuum with a cell harvester (Brandel, Gaithersburg, MD). Filters were rinsed twice with approximately 5 ml of ice-chilled 0.9% saline and were transferred to a counting vial containing 10 ml of scintillation fluid (PCS; Amersham, Arlington Heights, IL). Radioactivity was measured by liquid scintillation counting. Accumulation of activity at 4“C represented the background and was subtracted from all samples. Protection against CHydroxydopamine-Znduced Depletion of Hypothalamic NE and Epinephrine (Epi) Male Sprague-Dawley rats (180-200 g) from Charles River Breeding Laboratories (Portage, MI) were used. 6-Hydroxydopamine hydrobromide (6-OHDA) (Regis Chemical Company, Morton Grove, IL) was dissolved in 0.1% ascorbic acid and injected intracerebroventricularly (right ventricle) at a dose of 50 micrograms per rat (under Phenobarbital/chloral hydrate anesthesia) 24 hrs before rats were killed. Control rats received the same volume of 0.1% ascorbic acid into the

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ventricle. (R)-Thionisoxetine was injected 15 min prior to 6-hydroxydopamine, at doses of 0.1, 0.3, 1,3 or 10 mg/kg S.C. in 2% Emulphor. Hypothalamus was rapidly dissected after decapitation of the rats, was frozen on dry ice, and was stored at -70” prior to analysis. After alumina absorption, NB and epinephrine (Epi) were determined by liquid chromatography with electrochemical detection (12,13). Protection against metaraminol-induced depletion of NE from the rat heart and urethra. Male Sprague-Dawley rats (180-200 g) from Charles River Breeding Laboratories (Portage, MI) were used. Metaraminol bitartrate was dissolved in distilled water and injected subcutaneously (s.c.) 4 hours before the rats were killed. WThionisoxetine was injected at doses of 0.1, 0.3, 1, 3 and 10 mg/kg, S.C. in 2% emulphor 15 min prior to metaraminol . Rats were decapitated and hearts and urethras were rapidly dissected, frozen using dry ice and stored frozen at -70°C. Catecholamines were absorbed on to alumina at pH 8.6 and NE concentrations were determined by liquid chromatography with electrochemical detection (12,13).

Table 1. Potency of nisoxetine, tomoxetine and analogs for inhibiting [3H]-5HT uptake into synaptosomes and [3H]-nisoxetine binding.

[3H]-NB and

NHMe

X

X 2-MeS R/S 2-MeS R 2-MeS S 2-M&* R/S 2-Me0 R 2-Me0 S 2-Me** R *Nisoxetine,

It& (nM k S.E.M.) Ki (nM f S.E.M.) 3H-NE Uptake 3H-5HT Uptake 3H-Nisoxetine binding 0.3 lkO.06 3.9rt1.2 2Oti70 0.2OkO.03 1.9kO.5 13Ok30 31+6 85k23 29Ok50 7.2k3.1 23ort40 5.8k1.3 23Ok50 0.3E.15 15fl 18k4 18Ok90 13Ok50 0.63kO. 15 4.7k0.8 **Tomoxetine. N=3-5, ND = not determined.

Three versions of 2-methylthio substituted phenoxyphenyl propylamines were synthesized, the racemic (R/S) and the resolved enantiomers (i? and S). The racemic compound was a potent inhibitor of @II-NE uptake into rat hypothalamic synaptosomes (Table 1). This compound also potently inhibited [3H]-nisoxetine binding to the NE uptake site in rat cortical membranes. (R/S)Thionisoxetine had substantially lower affinity for the [3H]-5HT uptake site. The resolved enantiomers displayed differing affinities for NE uptake sites. The R enantiomer was more potent in the inhibition of [3H]-NB uptake and [3H]-nisoxetine binding than the S-enantiomer . Both enantiomers were much less potent in inhibiting [3H]-5HT uptake relative to their affinities for [3H]-NB uptake. When compared to tomoxetine (Table l), (R)-thionisoxetine had 2.5 X greater potency in [3H]-NE uptake and 3 X higher affinity in [3H]-nisoxetine binding with similar lower potency in [SI-IJ-5HT uptake. In addition, (R)-thionisoxetine was substantially more potent than the

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(R)-thionisoxetine as Inhibitor of NE Uptake

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active enantiomer of nisoxetine, (R)-nisoxetine, exhibiting a three-fold higher affinity in NE uptake and approximately two-fold higher affinity in [3H]-nisoxetine binding. To evaluate the potency of (R)-thionisoxetine at central NE uptake sites in viva, we examined its ability to block 6-OHDA induced depletion of rat hypothalamic NE and Epi. When administered subcutaneously 15 minutes prior to an intracerebroventricular injection of 6-OHDA, (R)thionisoxetine dose-dependently prevented the depletion of NE and Epi with ED50 values of 0.21 mg/kg and 0.18 mg/kg, respectively. The affinity at peripheral NE uptake sites was assessed using metaraminol-induced depletion of heart and urethra NE. Again, the compound was administered subcutaneously 15 minutes prior to injection of metaraminol. (R)-Thionisoxetine was able to prevent the depletion of NE in both the heart (ED50 = 3.4 mg/Kg) and urethra (ED50 = 1.2 mg/kg). Complete protection was accomplished in the urethra with a dose of 10 mg&g, while this dose produced partial protection in the heart. (b) Epinephrine ED= = 0.18 mg/kg

(a) Norepinephrine E%O = 0.21 mg/kg

7,

2oJ .l

1

10

I

--.--I

-....I

.l

1

10

Dose of (R)-thionisoxetine (mglkg, s.c.)

Fig. 1. Antagonism by (R)-thionisoxetine of the 6-OHDA-induced depletion of (a) norepinephrine and (b) epinephrine in rat hypothalamus. 6-OHDA (50 pg) was injected intracerebroventricularly 24 hrs before rats were killed and 15 min after (R)thionisoxetine was injected S.C. at the doses indicated. Values are expressed as nmoles NE/g tissue (a) or pmoles Epi/gm tissue (b). Mean values f. standard errors for 5 rats per group are shown. Asterisks indicate significant difference from the control group (P<.O5; Tukey’s test after analysis of variance) while # indicates significant difference from the 6-OHDA group (P<.O5; Tukey’s test after analysis of variance). Horizontal shaded areas represent standard error range (mean is the center) for the control group and group treated with 6-OHDA alone.

Tomoxetine and nisoxetine are selective NE uptake inhibitors and tomoxetine has shown antidepressant activity in a clinical trial (14). Only limited structure-activity information is available about these molecules. Recently, we demonstrated halogen substitution of the 2-methyl or 2methoxy in tomoxetine or nisoxetine, respectively, resulted in NE uptake inhibitors with equal or greater affinity. While fluorine substitution provided similar potency, bromine and iodine substitution substantially (3-4 X) improved potency (8). To explore this concept further, the 2methoxy of nisoxetine was replaced with a methylthio group. This substitution should have similar electronic properties to iodine and, thus provide a compound with similar or improved affinity and

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selectivity. In accordance with the hypothesis, (Qthionisoxetine had 3-fold higher affinity than tomoxetine in displacing [3H+nisoxetine binding to the NE transporter. When [3H]-NE uptake into (b) Urethra norepinephrine ED9 = 1.2 mgkg

(a) Heart norepinephrine = 3.40 mg/kg Qo 4.0

12

3.0 e : g 2.0 e 1.0

Metaraminol alone

l

0.0 .l

1

10 Dose of (R)-thionisoxetine (mgkg, s.c.)

Fig. 2. Antagonism by (R)-thionisoxetine of the metaraminol-induced depletion of (a) heart and (b) urethra NE. Metaramin ol(5 mg/kg) was injected subcutaneously 4 hrs before rats were killed and 15 min after (R)-thionisoxetine was injected S.C. at the doses indicated. Values are expressed as nmoles NE/g tissue. Mean values + standard errors for 5 rats per group are shown. Asterisks indicate significant difference from the control group (Pc.05; Tukey’s test after analysis of variance) while # indicates significant difference from the 6-OHDA group (Pc.05; Tukey’s test after analysis of variance). Horizontal shaded areas represent standard error range (mean is the center) for the control group and group treated with metaraminol alone. hypothalamic synaptosomes was measured, this compound had 2.5-fold higher potency than tomoxetine. While the affinity improved at the NE uptake site, (R)-thionisoxetine had similar affinity to tomoxetine in the inhibition of [3H]-5HT uptake into cortical synaptosomes. When compared to the active enantiomer of nisoxetine, (R)-nisoxetine, (R)-thionisoxetine had 2-3 times higher affinity in both NE uptake and [3H]-nisoxetine binding. Therefore, (R)-thionisoxetine is a potent and selective inhibitor of NE uptake sites when studied using in vitro preparations. To explore the properties of (R)-thionisoxetine in vivo, the ability of the compound to inhibit the depletion of NE was examined. The first paradigm used was the depletion of brain NE by the central administration of 6-OHDA. This toxin uses the uptake site to gain entrance into noradrenergic and adrenergic neurons where it destroys the neuron and depletes the transmitter. As expected, 6-OHDA administration results in the loss of approximately 50% of the hypothalamic content of NE and Epi. By blocking the uptake site, (R)-thionisoxetine prevented the uptake of 6OHDA into the noradrenergic neurons and the subsequent depletion of NE. Similar to other noradrenergic antidepressant compounds, it was equipotent in protecting noradrenergic and adrenergic neurons (13,15). The activity of (R)-thionisoxetine at peripheral NE uptake sites was also assessed using an in vivo protection paradigm. For these studies, metaraminol was used to deplete the NE from the heart and urethra. This agent reduced NE concentrations by 90% in the heart and over 60% in the urethra. (R)-Thionisoxetine dose-dependently antagonized the depletion of NE in both tissues.

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(R)-thionisoxetine as Inhibitor of NE Uptake

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While the use of NE uptake inhibitors as antidepressants is well documented, this class of compounds is also used clinically in the treatment of urinary incontinence (4,5) These agents can affect the function of the lower urinary tract in several ways. NE uptake inhibition in the bladder results in a relaxation of the bladder via B-adrenergic receptors (4) This allows for increased expansion and capacity. NE can contract the urethra via alpha adrenergic receptors (16) and, thus, may improve urinary retention and bladder filling. NE reuptake inhibitors can potentiate NEinduced contractions of the urethra in vitro (17,18) though this does not appear to occur in vivo (19). Increased noradrenergic and adrenergic activity at the spinal and supraspinal levels may also suppress the micturition reflexes. Therefore, a NE uptake inhibitor with activity at both central and peripheral sites could be valuable for the treatment of urinary incontinence. In conclusion, (R)-thionisoxetine is a potent and selective inhibitor of NE uptake sites both in vitro and in vivo. This compound has activity at both central and peripheral uptake sites and, therefore, may provide a novel treatment for urinary incontinence.

References 1. 2. 3. ::

6.

;:

9.

::* 12: 13. 14. 15. 16. :;* 19:

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