Lack of tolerance to the anticonvulsant effects of tiagabine following chronic (21 day) treatment

EPILEJ?SY RESW

Epilepsy Research19 (1994) 205-213

Lack of tolerance to the anticonv~lsant effects of tiagabine following chronic ( 21 day) treatment Peter D. Suzdak * De~r~e~t

of Receptor N~~r~kern~t~,

Drug Discovery, P~rm~~e~ti&ais ~iv~~~, M~~~v, Denmark

Nova Nordisk A /S, Nooo Nordisk Park DK-2760

Received9 February 1994; revised 30 May 1994; accepted 1 June 1994

Abstract

The anticonvulsant, and side effect, profile of the y-aminobutyric acid (GABA) uptake inhibitor (RI-IV-(4,4-di-(3methylthien-2-yl)but-3-enyl) nipecotic acid hydrochloride (tiagabine) was examined in mice following chronic (21 day) administration. Twenty-four hours following the discontinuation of the 21 days’ treatment with twice daily administration of vehicle or tiagabine at 15 or 30 mg/kg p.o., an ED,, for tiagabine was determined for the anticonvulsant effect, the rotarod performance, the traction response and the inhibition of locomotor activity in the animals treated with vehicle only, and in the groups previously treated with 15 or 30 mg/kg p.o. of tiagabine. There was no significant decrease in the anti#nv~sant efficacy of acutely administered tiagabine (ED,, for inhibition of methyl 6,7-dime~oxy-4-ethyl-~~arboline-2-car~xylate (DMCM)-induced seizures of 1.7 f 0.4, 1.9 f 0.3, and 2.0 f 0.50 mg/kg i.p., respectively). However, there was a significant decrease in the ability of acutely administered tiagabine to impair rotarod performance (ED,, of 5.9 f 1.2, 14 f 1.9 and 21 f 2.7 mg/kg i.p., respectively), inhibit a traction response (ED,, of 10 f 1.6, 23 f 3.0 and 34 & 4.6 mgfkg i.p., respectively), and to inhibit exploratory locomotor activity (ED, of 13 rfi 2.3, 19 f 2.6 and 28 + 3.8 mg/kg i.p., respectively). Following the dis~ntinuation of chronic tiagabine administration there was no change in ~ntylenetetrazol (PTZ) seizure threshold, animal weight or gross behavior, suggesting the lack of a behavioral withdraws syndrome. The production of tolerance to the sedative and ataxic effects, but not the anticonvulsant effects, of tiagabine suggests that tiagabine may be a useful agent for the long-term treatment of epilepsy. Keywords: Tiagabine; GABA uptake; Anticonvulsant; Chronic treatment

1. Introduction

y-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous systern (CNS) [5,17,18,29]. It has been estimated that GABA is present in approximately 70% of all

* Correspondingauthor. Tel.: + 45 44 44 88 88, ext. 4760; Fax: +4544663939.

synapses within the CNS 161. A reduction in GABAergic neuronal tone has been implicated in a number of neurological disorders, including epilepsy, anxiety, and pain [4,17,19,23,26]. Thus, substantial interest has focused on finding novel mechanisms for enhancing GABAergic neurotransmi~ion. One such approach is to inhibit the neuronal and/or glial uptake of GABA [17,28,30]. Several groups [2,21,31,33,34] have recently described novel series of potent and selective GABA uptake inhibitors that

~ZO-1211/94/$07.~ Q 1994 Elsevier Science B.V. All rights reserved SSDI 0920-1211(94)00048-2

Xh

P.D. Suzdak / Epilepsy Research 19 (19941 ZOS-21.7

possess potent anticonvulsant activity in vivo. These compounds differ from nipecotic acid and similar cyclic amino acid GABA uptake inhibitors in that they readily cross the blood-brain barrier due to increased lipophilicity following substitution of a lipophilic anchor on the amino acid nitrogen atom El]. Tiagabine ((Rl-N-(4,4-di-(3-methylthien-2-yl)but3-enyl) nipecotic acid hydrochloride: previously NO328, NNC-05-0328) is a potent (IC,, for inhibition of f3 HIGABA uptake of 67 nM) and selective GABA uptake inhibitor [2], which is currently in Phase II/III clinical trials for epilepsy. Tiagabine has been shown to produce potent anticonvulsant effects against chemically induced seizures [21,22] and in genetic models of epilepsy 17,211. In clinical trials, as of June 1993, a total of 1600 patients have received tiagabine. Tiagabine has been shown to dose-dependently decrease seizure frequency in patients with complex partial and simple partial seizures [27], and complex partial and secondary generalized tonic-clonic seizures [3,25]. The use of compounds that increase GABAergic neurotransmission (e.g. benzodiazepines) for the treatment of epilepsy has been greatly restricted due to their ability to induce anti~onvulsant tolerance and physical dependence [14]. Thus, the present investigation examined the tolerance and withdrawal liability of tiagabine following chronic (21 days) administration in mice.

described [21]. Briefly, mice were pretreated with tiagabine either p.0. (1 to 17.5 hour pretreatment) or i.p. (30 min pretreatment) prior to the administration of 1.5 mg/kg i.p. DMCM. The mice were then observed for the next 30 min for the presence of clonic seizures and death (N = 10 per dose). 2.3. Pentylenetetrazol

(PTZ) seizure threshold

PTZ seizure threshold was determined by a modification of the method of Gent et al. [12]. Briefly, PTZ (15 mg/ml) was infused into the mouse tail vein at a constant rate of 0.3 ml/min until the end point (clonic convulsion) was reached. The minimum convulsant dose, in mg/kg PTZ, required to elicit the clonic convulsions was calculated for each mouse, and the mean + SEM for each group (N = 101 was calculated. 2.4. Traction test Inhibition of the traction response was determined as previously described [21]. Briefly, the animal was picked up by its tail and swung close to a horizontal rod (30 cm above the table, 2 mm in diameter, 14 cm length). As soon as the animal held onto the rod with its forepaws, it was left hanging in this position. A normal traction response was observed if the animal grasped the rod in less than 5 s with one of its hind-paws while maintaining its grip with the forepaws.

2. Materials and methods

2.5. Rotarod test

2.1. Animals

Inhibition of rotarod performance was determined as previously described [21]. Briefly, the animals were pretrained on the rotarod apparatus @go-Basile, Italy) for 2 min prior to testing (speed: 6 rpm>. The rod diameter was 3 cm. In the test procedure, the animals were placed on the rotating rod. If the animal fell from the rod, the animal was immediately picked up by its tail and again placed on the rod. Testing was stopped when a total of 10 failures was reached or 2 min had elapsed.

Male NMRI mice (Moellegaard, Denmark), weighing 20 + 3 g were used. The animals were housed in conventional group cages and placed in a room at constant temperature and relative humidity. Room lights were on from 07:OO to 19:OO h; food and water were freely available. 2.2. Antagonism of seizures induced by methyl 6,7dimetho~-4-eihyl-~-carbo~ine-2-carbo~late @NfCM~ Antagonism of DMCM-induced clonic convulsions in NMRI mice was determined as previously

2.6. Inhibition

of exploratory

locomotor

behavior

Inhibition of exploratory locomotor behavior was determined as previously described [21]. Briefly, ex-

P.D. Suzdak/Epilepsy

207

Research 19 (1994) 205-213

perimentally naive mice were injected with tiagabine (N = lo/dose), th en placed individually in a plexiglass box (20 X 20 X 38 cm) equipped with two frames of photocells (spaced equidistantly) situated so as to detect locomotor behavior of the animals. The photocell chamber was housed in a sound-insulated, dimly lit, ventilated chest. The number of photocell crossings in a lo-min period was counted with a minicomputer as a measure of exploratory behavior. Testing was done between 1090 and 16:00 h. 2.7. Binding of [3H]tiagabine in viva TIAGABINE

[3Hniagabine in-vivo binding was performed as previously described [32]. Briefly, groups of mice were pretreated with tiagabine. [ 3H]Tiagabine, 4 &i/ mouse, in 0.9% NaCl, was injected via the tail vein, and the mice were decapitated after 30 min. The forebrains were rapidly excised and homogenized in 12 ml of ice-cold 25 nM potassium phosphate buffer, pH 7.1. Aliquots, 1 ml, were immediately filtered through Whatman GF/C glass fiber filters, followed by two lo-ml washings with the same buffer. The amount of tritium bound to filters and in another l-ml aliquot of homogenate was determined by liquid scintillation spectroscopy. Non-specific binding was the binding in mice that had received 30 mg/kg i.p. of unlabelled tiagabine in saline 35 min before decapitation; non-specific binding was subtracted from the total binding on filters to give specific binding.

PRETREATYENT

TIME

(HOURS)

Fig. 1. The effect of pretreatment time on the anticonvulsant efficacy of tiagabine against DMCM-induced clonic seizures in NMRI mice. Mice were pretreated with tiagabine (0.3-60 mg/kg p.0.) for 1 to 17.5 hours prior to the administration of 15 mg/kg i.p. of DMCM. The mice (N = 10 mice per dose) were then observed for the next 30 min for the presence of clonic seizures. ED,, values were obtained from the individual dose response curves. The data represent the mean f SEM of three experiments.

3. Results The effect of pretreatment time (1 to 17.5 hours) on the anticonvulsant activity of tiagabine following p.o. administration is shown in Fig. 1. The ED,, for inhibition of DMCM-induced seizures following a 8 or 17.5 hour pretreatment of tiagabine was 15 and 30 mg/kg p.o., respectively, and these doses were used in the subsequent chronic experiments. The duration of anticonvulsant action of tiagabine (15 or 30 mg/kg p.o.1 during twice daily (8:OO and 16:OO h) administration, and in-vivo receptor occupancy to the central GABA uptake carrier, is shown in Figs. 2 and 3. Tiagabine, at 15 mg/kg p.o., produced a 2 50%

30

l-t-./t 0

TIME

(HOURS)

1

10 FOLLOWING

TIAGABINE

AOYINISTRATION

Fig. 2. The duration of anticonvulsant action of tiagabine. Tiagabine, 15 (v) or 30 (0) mg/kg p.o., was administered at HI0 and 1690 h, and the duration of anticonvulsant effect over 24 hours was determined. Following various pretreatment times, 15 mg/kg i.p. of DMCM was administered, and the mice (N = 10 mice per dose) were observed for the next 30 min for the presence of clonic seizures. The data are expressed as the percent protection against DMCM-induced clonic seizures. The data represent the meanf SEM for three experiments. The SEM was < 20%. The up-arrow denotes when tiagabine was administered.

0

1

20

TIME (HOURS) FOLLObtyNC TIACABINE ADMINISTRATION

Fig. 3. The duration of in-vivo receptor occupancy to the central GADA uptake carrier by tiagabine was determined by [ ‘Hltiagabine in-vivo binding. Tiagabine, 15 (0) or 30 (0) mg/kg p.o., was administered twice daily (8:OO and 14:00 h). At the specified times after tiagabine administration, mice received 4 pCi/mouse of [3H]tiagabine injected in the tail vein, were sacrificed 30 min later, and the forebrain was rapidly excised and homogenized in assay buffer. A l-ml aliquot was filtered through GF/C filters and the amount of radioactivity was determined. Non-specific binding was determined in mice receiving 30 mg/kg i.p. of tiagabine 35 min prior to sacrifice. The data represent the mean It SEM (N = 7 mice per group). The SEM was 5 20%. The up-arrow denotes when tiagabine was administered.

protection against DMCM-induced convulsions for up to 20 hours. Tiagabine, at 30 mg/kg p.o., produced a 2 60% protection against DMCM-induced seizures over the entire 24-hour period. In a parallel set of experiments, [3H]tiagabine in-vivo binding to the central GABA uptake carrier was measured. Tiagabine, at either 15 or 30 mg/kg p.o., produced a 2 30% and 2 45% in-vivo receptor occupancy at the central GABA uptake carrier during the 24 hour period measured. These data are in agreement with a previous study showing that tiagabine, at the EDSo for inhibiting DMCM-induced clonic seizures, occupied 30% of the GABA uptake sites [32]. In chronic experiments, mice were treated twice daily (8:OO and 16:00 h) with vehicle or tiagabine (15 or 30 mg/kg p.o.1 for 21 days. In order to determine if the metabolism of tiagabine was induced during the chronic treatment period, [3H]tiagabine in-vivo binding was measured in separate groups of mice 6 hours after the administration

II i

ii

25

7

2 CHRONIC

14

TREATMENT

J

20

DAY

Fig. 4. The ability of tiagabine, 15 (solid bars) or 30 (open bars) mg/kg p.o., to inhibit i3H]tiagabine in-viva binding following 2, 7, 14, or 20 days of tiagabine administration. Six hours following the first daily injection of tiagabine (8:OO h) on days 2, 7, 14, or 20, separate groups of mice were administered 4 wCi/mouse of [sH]tiagabine, and sacrificed 30 min later. The percent in-viva receptor occupancy to the central GABA uptake carrier was determined as described in Fig. 3 and the methods section. Data represent the mean f SEM (N = 6 mice per group).

of tiagabine (15 or 30 mg/kg p.o.1 on days 2, 7, 14, and 20 (Fig. 4). Tiagabine at 15 mg/kg p.o. produced a 30, 35, 30, and 40% in-vivo receptor occu-

POST

TREATMENT

DAY

Fig. 5. PTZ-seizure threshold was measured in separate groups of mice 1, 2, 3, 4, 7, or 10 days following the discontinuation of chronic (21 days) treatment with either vehicle (solid bars) or tiagabine (15 (cross-hatched bars) or 30 (open bars) mg/kg p.0.). PTZ (1.5 mg/ml) was infused into the tail vein at a constant rate of 0.3 ml/min until the production of a clonic convulsion. In the chronic vehicle treated group, the minimal convulsant dose of PTZ (PTZ seizure threshold) was 56.2 f 1.4 mg/kg PTZ. Data represent the mean k SEM (N = 7 mice per group).

209

P. D. Suzdak / Epilepsy Research 19 (1994) 205-213

panty on days 2, 7, 14, and 20 of chronic treatment, respectively. Tiagabine at 30 mg/kg p.o. produced a 55, 55, 57, and 60% receptor occupancy on days 2, 7, 14, and 20 of chronic treatment, respectively. As shown in Fig. 5, following the discontinuation of chronic administration of tiagabine there was no change in PTZ seizure threshold (measured 1, 2, 3, 4, 7, or 10 days following the discontinuation of

chronic tiagabine treatment), animal weight (data not shown) or gross behavior (data not shown). In order to determine if residual tiagabine was present in the brain following discontinuation of chronic administration of tiagabine, which would interfere with the subsequent behavioral testing, [ 3H]tiagabine in-vivo binding studies were performed at 24, 48, and 72 hours following the discontinuation of chronic

60t

1

TIAGABINE (mg/kg

1.p.)

90

TIAGABINE (mg/rg

1.p.)

TIAGABINE (md;kg

1.p.)

-

80 70 -

1

1

TIAGABINE (mdjkg

Lp.)

Fig. 6. The effect of chronic (21 day) administration of either vehicle (m f or tiagabine at 15 (A ) or 30 (A) mg/kg p.o. on the acute ~tjcon~~nt (A), rotarod impairing (B), ataxic (0, or inhibition of exploratory i~omotor activity (D) effects produced by the acute ad~nistration of tiagabine. Tiagabine was administered to mice 24 hours following the discontinuation of chronic tiagabine treatment. (A) 30 min after the administered tiagabine (0.3-20 mg/kg i.p.1 mice were injected with DMCM (15 mg/kg i.p.) and observed for a subsequent 30 min for the presence of clonic seizures. 30 min after the administration of tiagabine (l-30 mg/kg i.p.) separate groups of mice were placed on either the rotarod (B), traction bar (Cl, or in a plexiglass box for measurement of a exploratory locomotor activity (D). See methods section for experimental details. Data represent the mean of three individual experiments (N = 10 mice per group). The SEM was 5 20%.

tiagabine administration. At either 24, 48. or 72 hours following the discontinuation of chronic tiagabine administration, there was no significant inhibition (4 + 3, 5 k 4, and 5 f 4 percent, respectively) of [3H]tiagabine in-vivo binding. All subsequent behavioral experiments were conducted 24 hours after the discontinuation of chronic tiagabine administration. As shown in Fig. 6 and Table 1, 24 hours following the discontinuation of chronic tiagabine administration (15 or 30 mg/kg p.o.1, there was no significant decrease in the anticonvulsant efficacy of acutely administered tiagabine (ED,, for inhibition of DMCM-induced seizures of 1.7 + 0.4, 1.9 & 0.3, and 2.0 f 0.50 mg/kg i.p., respectively). Following chronic administration of either vehicle or tiagabine (15 or 30 mg/kg p.o> there was a significant decrease in the ability of acutely administered tiagabine to impair rotarod performance (ED,,, of 5.9 F 1.2, 14 + 1.9 and 21 + 2.7 mg/kg i.p., respectively), inhibit a traction response (EDs,, of 10 & 1.6. 23 + 3.0 and 34 f 4.6 mg/kg i.p., respectively), and to inhibit exploratory locomotor activity (ED5(, of 13 Ifr 2.3, 19 + 2.6 and 28 & 3.8 mg/kg i.p., respectively), as compared to vehicle control. Following chronic administration of tiagabine there was a significant increase in the separation between the EDT,) for inhibition of DMCM-induced clonic seizures and the ED,(, values for impairment of rotarod, inhibition of traction, or the impairment of locomotor activity, following the acute administration of tiagabine (see Table 1).

Table 1 Summary table of the anticonvulsant either vehicle or tiagabine Chronic treatment

Vehicle Tiagabine 15 mg/kg 30 mg/kg

d

p.o. p.o.

and motor impairing

Anticonvulsant

’

ED,, (mg/kg

i.p.1

4. Discussion The use of compounds that increase GABAergic neurotransmission (e.g. benzodiazepines and barbiturates) for the long-term treatment of epilepsy has been greatly restricted by their ability to induce anticonvulsant tolerance and dependence [13,14]. Anticonvulsant tolerance has been shown to also develop with a number of other antiepileptic drugs [S-lo], including carbamazepine. The therapeutic effectiveness of traditional antiepileptic agents has also in general been limited by their narrow therapeutic index [24]. This has led to the development of a new generation of potential antiepileptic agents which includes vigabatrin, felbamate, lamatrogen and tiagabine [24]. Tiagabine is a potent and selective GABA uptake inhibitor that is currently in phase II/III clinical trials for epilepsy [3,25,27]. The present study examined the tolerance and withdrawal liability, and therapeutic index, of tiagabine following chronic (21 day) administration in the mouse. In the present study chronic administration of tiagabine did not induce tolerance to the acute anticonvulsant effects of tiagabine. These results are in agreement with the previous reports of Picardo et al. [22] and Judge et al. [15] who demonstrated a lack of anticonvulsant tolerance following the subacute (4 days) administration of tiagabine. In addition, Karbon et al. [16] have also demonstrated a lack of anticonvulsant tolerance following subchronic (14 days) administration of the GABA uptake inhibitors SKF89976A and SKF100330A. These data suggest

effects of acutely administered

Rotarod ED,,, (mg/kg i.p.)

1.7 + 0.4

5.9 + 1.2

1.9 f 0.3 2.0 * 0.5

14.0 & 1.9 * 21.0 + 2.7 *

tiagabine

in NMRI mice chronically

Traction ED,,, (mg/kg i.p.)

Exploratory locomotor activity ED,, (mg/kg i.p.)

10.1 k 1.6

13.2 + 2.3

23.2 at 3.0 * 34.1 + 4.6 *

19.1 + 3.6 28.6 + 3.8 *

treated with

Ratio of rotarod ED,,,/ DMCM ED,,, c 3.4 + 1.1 7.4 f 0.9 * 10.5 f 3.3 *

a Mice were administered vehicle or tiagabine (15 or 30 mg/kg) p.o. twice daily for 21 days. b Thirty minutes after the administration of tiagabine, mice were administered DMCM (15 mg/kg i.p.) and observed for a subsequent 30 minutes for the presence of clonic seizures. * P < 0.05, ANOVA followed by multiple-comparisons test (Dunnett’s test). ’ Therapeutic index is defined as the ED,, for inhibiting rotarod performance/EDsO for inhibition of DMCM-induced clonic seizures.

P.D. Suzdak/ Epilepsy Research 19 (1994) 205-213

that inhibition of GAHA uptake may represent a promising mechanism for the long-term treatment of epilepsy. In order to rule out that the lack of tolerance to the anticonvulsant effects of tiagabine is due to pharmacokinetic changes leading to an insufficient exposure of the GABA uptake carrier to tiagabine, [3H]tiagabine in-vivo binding studies were performed. An acceleration of metabolism by the induction of hepatic enzymes following chronic drug treatment has been reported for anticonvulsant agents such as carbamazepine [ 111. Chronic administration of tiagabine did not result in apparent pharmacokinetic changes as the degree of inhibition of ~3H]tiagabine in-vivo receptor binding to the central GAHA uptake carrier was similar on treatment days 2, 7, 14, or 20 following the administration of tiagabine at 15 or 30 mg/kg p.o. An in-vivo receptor occupancy of the central GAHA uptake carrier (2 45%), and anticonvulsant efficacy (2 60% protection), was maintained by the high dose of tiagabine over the entire study period. In addition, the administration of tiagabine to NMRI mice for 7 days does not result in the induction of liver PdsOenzymes (J.A. Jansen, personal communication). The lack of ~ti~nvulsant tolerance following chronic adminis~ation of tiagabine may be related to the unaltered binding of 13H]tiagabine to the GABA uptake carrier or the binding of 13H]GABA to GABA, and GAHA, receptor sites in the inferior colliculus and substantia nigra (Thomsen and Suzdak, unpublished data). In~bition of GAE3A uptake in the inferior colliculus and substantia nigra is highly correlated to the anticonvulsant potency of GAHA uptake inhibitors [7] (Suzdak et al., unpublished data). The tolerance to the sedative and ataxic effects of tiagabine following chronic administration may be related to a decrease in GABA, and GAHA, receptor sites in the hippocampus, motor cortex, and cerebellum (Thornsen and Suzdak, unpublished data). Inhibition of GAHA uptake in the hippocampus and motor cortex is highly correlated to the sedative and ataxic effects of GAHA uptake inhibitors (Suzdak et al., unpublished data). The production of tolerance to the sedative and ataxic effects, but not to the anticonvulsant effects of tiagabine, represents a distinct clinical advantage to the use of tiagabine for the long-term treatment of seizure disorders. These data

211

do not, however, rule out that higher doses of tiagabine may produce some degree of anticonvulsant tolerance. The narrow therapeutic ratios of traditions antiepileptic agents, such as benzodiazepine, carbamazepine, and valproic acid, has limited their clinical usefulness [21]. Thus, it was important to determine the side effect liability of tiagabine following chronic administration. Following acute a~inistration, tiagabine has been shown to have an improved therapeutic ratio (ED,, for inhibiting rotarod performance/ED,, for inhibiting DMCM-induced clonic seizures) over traditional anti-epileptic agents [21]. In the present study, the therapeutic ratio for tiagabine signific~tly increased following chronic treatment. These data would suggest that tiagabine may possess a lower incidence of side effects during the long-term treatment of epilepsy. In phase II add-on clinical trials with tiagabine, the overall incidence of side-effects seen in patients maintained on tiagabine (at doses which were efficacious against complex partial and simple partial epilepsy and complex partial and secondary generalized tonic-clonic seizures) was similar to that of the corresponding placebo-treated groups [3,25,27]. These data suggest that inhibition of GAHA uptake by compounds such as tiagabine represents a novel and highly promising mechanism of the chronic treatment of seizure disorders. The production of withdrawal has greatly limited the usefulness of benzodiazepine agonists for the treatment of epilepsy [24]. Considering the similar mechanism of action (increase in GABAergic neurotransmission) between compounds that inhibit GABA uptake and benzodiazepine receptor agonists, it was important to determine if withdrawal and dependence would be produced following discontinuation of chronic tiagabine administration. In the present study, following the discontinuation of chronic tiagabine treatment, there were no signs of withdrawal present (change in seizure threshold, changes in body weight, seizures or hyperexcitability). These data would suggest that tiagabine, at the doses used in the present study, does not produce withdrawal in the mouse upon withdrawal from chronic treatment. In conclusion, chronic (21 day) administration of tiagabine at either 15 or 30 mg/kg p.o. did not induce anticonvulsant tolerance or withdrawal. However, tolerance did develop to motor-impairing side

212

P.D. Suzdak/ Epilepsy Research 19 (1994) 205-21.7

effects of tiagabine (as measured by rotarod activity, traction response and inhibition of exploratory locomotor activity). These data suggest that tiagabine may be a useful agent for the long-term treatment of epilepsy.

Acknowledgments The technical assistance of Allan Hansen and Dorte Andersen is greatly appreciated. The author would like to thank E.B. Nielsen and M.D.B. Swedberg for their critical evaluation of the manuscript, and T.K. Hansen for help in preparation of the manuscript.

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