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Endocrinology Vol. 148, No. 3 1376-1383
Copyright © 2007 by The Endocrine Society

Alleviation of Thermoregulatory Dysfunction with the New Serotonin and Norepinephrine Reuptake Inhibitor Desvenlafaxine Succinate in Ovariectomized Rodent Models

Darlene C. Deecher, Peter D. Alfinito, Liza Leventhal, Scott Cosmi, Grace H. Johnston, Istvan Merchenthaler and Richard Winneker

Women’s Health and Musculoskeletal Biology, Wyeth Research, Collegeville, Pennsylvania 19426

Address all correspondence and requests for reprints to: Darlene C. Deecher, Ph.D., Women’s Health and Musculoskeletal Biology, Wyeth Research N3164, 500 Arcola Road, Collegeville, Pennsylvania 19426. E-mail: deeched{at}wyeth.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hot flushes and night sweats, referred to as vasomotor symptoms (VMS), are presumed to be a result of declining hormone levels and are the principal menopausal symptoms for which women seek medical treatment. To date, estrogens and/or some progestins are the most effective therapeutics for alleviating VMS; however, these therapies may not be appropriate for all women. Therefore, nonhormonal therapies are being evaluated. The present study investigated a new reuptake inhibitor, desvenlafaxine succinate (DVS), in animal models of temperature dysfunction. Both models used are based on measuring changes in tail-skin temperature (TST) in ovariectomized (OVX) rats. The first relies on naloxone-induced withdrawal in morphine-dependent (MD) OVX rats, resulting in an acute rise in TST. The second depends on an OVX-induced loss of TST decreases during the dark phase as measured by telemetry. An initial evaluation demonstrated abatement of the rise in TST with long-term administration of ethinyl estradiol or with a single oral dose of DVS (130 mg/kg) in the MD model. Further evaluation showed that orally administered DVS acutely and dose dependently (10–100 mg/kg) abated a naloxone-induced rise in TST of MD rats and alleviated OVX-induced temperature dysfunction in the telemetry model. Oral administration of DVS to OVX rats caused significant increases in serotonin and norepinephrine levels in the preoptic area of the hypothalamus, a key region of the brain involved in temperature regulation. These preclinical studies provide evidence that DVS directly impacts thermoregulatory dysfunction in OVX rats and may have utility in alleviating VMS associated with menopause.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THERMOREGULATION IS AN integrated network of neuroendocrine, autonomic, and somatosensory responses. The goal of the thermoregulatory circuitry is maintenance of core body temperature (CBT) regulated through direct and indirect inputs to the hypothalamus (1). Tight regulation of CBT is maintained by means of vasoconstriction, vasodilation, sweating, and shivering. Examples of alterations in temperature regulation are fever, which is caused by increases in peripherally or centrally produced pyrogenic cytokines; hyperthermia, which may be caused by exogenous heat exposure or endogenous heat production; and vasomotor symptoms (VMS), also referred to as hot flushes and night sweats, all regulated by the central nervous system (CNS).

Fever is a common occurrence that usually indicates infection or disease, and hyperthermia can result in rapidly fatal high CBT. In contrast, VMS, although common, are non-life-threatening alterations in homeostatic temperature regulation. They occur in up to 80% of all women after natural or surgically induced menopause (2) and are characterized by a warming sensation in the chest and face accompanied by sweating, vasodilatation (flushing), and, in some instances, feelings of nausea or anxiety and other symptoms. There is little doubt that estrogens play a key role in mediating VMS associated with menopause (3, 4). Consequently, the majority of therapeutic strategies to date have focused primarily on restoring declining hormone levels.

There is supportive evidence for the role of the neurotransmitters norepinephrine (NE) and serotonin (5-HT) in temperature regulation. Furthermore, estrogens have been shown to modulate both 5-HT and NE, which in turn may affect thermoregulatory responses (5, 6). In light of this knowledge, nonhormonal therapies, including the selective 5-HT reuptake inhibitors fluoxetine (7), paroxetine (8, 9, 10), and citalopram (11) and the 5-HT/NE reuptake inhibitor (SNRI) venlafaxine (12, 13), have been evaluated clinically for the treatment of VMS, with varying results. SNRIs may be especially effective in alleviating VMS because of their ability to regulate both NE and 5-HT. It has been speculated that improvements in VMS seen with these compounds, generally thought of as antidepressants, are due to indirect effects on mood such that patients perceive VMS as less bothersome. Because these compounds have not been studied objectively in preclinical models of thermoregulatory dysfunction, this hypothesis has not been effectively supported or refuted.

Desvenlafaxine succinate (DVS) is a novel SNRI (14, 15) in clinical development for the treatment of VMS associated with menopause and, separately, for the treatment of major depressive disorder. It has been well documented that the serotoninergic and noradrenergic systems are involved in depression, and therapies have been developed that modulate these neurotransmitters (16); however, the role of these neurotransmitters in thermoregulatory dysfunction remains unclear. The primary goal of the present studies was to evaluate the effect of DVS on altering thermoregulatory processes in ovariectomized (OVX) rodent models (17, 18, 19, 20, 21). It is presumed that the aberrant signaling in the medial preoptic area of the hypothalamus of the CNS, a region that mediates homeostatic variables including thermoregulation, is the likely site of origin (1). Thus, to elucidate the biochemical and neurochemical effects mediating the action of DVS in thermoregulatory dysfunction, we used microdialysis techniques to monitor changes in extracellular NE and 5-HT concentrations in the medial preoptic area of the hypothalamus in OVX rats. Additionally, the ability of DVS to alleviate temperature dysfunction in two rodent models of temperature regulation, including the effect of DVS on CBT in OVX rats, was investigated. We hypothesized that DVS restores temperature regulation by specifically modifying 5-HT and NE levels in the hypothalamus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by Wyeth’s Institutional Animal Care and Use Committee. OVX females (Sprague Dawley rats, 180–220 g) were purchased from Taconic (Germantown, NY) and individually housed on a 12-h light, 12-h dark cycle with standard rat chow and water available ad libitum. The animals were rested a minimum of 7 d after arrival before any experimentation. For the morphine-dependent (MD) rat model, OVX rats were housed in a room maintained at 25 C, and experimental procedures were performed in a room maintained at 21 C. For the OVX-induced thermoregulatory dysfunction telemetry model, rats were housed and tested in a room maintained at 21 C. The room temperature selected was experimentally chosen because these animals are specifically being tested for changes in temperature during experimentation. In all experiments measuring temperature, 10–16 rats per group were used.

Test compounds
DVS was synthesized by the Discovery Medicinal Chemistry group of Wyeth Research. WAY-100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate salt), a 5-HT1A antagonist, and 17-{alpha}-ethinyl estradiol (EE) were purchased from Sigma Chemical Co. (St. Louis, MO).

MD OVX rat model
This model is based on an established MD naloxone-induced flush paradigm (17, 22) with modifications made during model optimization and validation (20). For testing of long-term administration, rats were injected once daily for 8 d with vehicle or EE (1 mg/kg). To minimize stress responses to handling and injection for acute drug administration, rats were injected once daily for 8 d with vehicle and then on test day administered a single oral dose of either vehicle or DVS (3–130 mg/kg). Rats were food deprived a minimum of 16 h before oral administration of DVS to optimize and reduce variability of drug absorption between animals. For both treatment paradigms, morphine dependence was induced by sc implantation of two slow-release morphine pellets (75 mg/pellet; Murty Pharmaceuticals, Lexington, KY) in the dorsal scapular region on d 4 of dosing. Initial evaluation of morphine dependence was performed starting on d 3 (post-pellet implantation), and testing continued through d 8 using the classical method of opiate-induced analgesia determined by measurement of tail-flick hot plate latency (17, 23, 24, 25). This evaluation was done to define the time period that consistent morphine dependence was achieved and maintained over time. Once identification of consistent morphine dependence was determined (d 5 through 7) assessment of maximum naloxone-induced changes in tail-skin temperature (TST) were performed. The maximal changes in TST induced by naloxone administration directly correlated with the days where consistent morphine dependence was noted. Each test day has a vehicle control group run for comparison of all drugs being evaluated. Data reported in this study were taken on d 5 and 6 after morphine pellet implantation, and the mean change in TST from baseline noted in the vehicles was 4.5 + 0.7 C. On d 5, 6, or 7 after implantation, morphine withdrawal was induced with the general opioid antagonist naloxone (1.0 mg/kg sc; Research Biochemicals International, St. Louis, MO). These test days were selected because of the maximal changes in TST previously defined. Four to six days after implantation, MD withdrawal was induced with the general opioid antagonist naloxone (1.0 mg/kg sc; Research Biochemicals International). For oral-dosing paradigms, DVS was administered 90 min before naloxone injection. Rats were injected with ketamine (Ketaject; Phoenix Pharmaceuticals, Belmont, CA) at a dosage (40 mg/kg im) previously determined to be mildly sedative without causing a significant change in TST (data not shown). The mild sedation is necessary to reduce stress associated with thermistor probe attachment, mild restriction of movement, and naloxone-induced withdrawal that can cause variability in the baseline temperatures. All experiments were internally controlled. All drug-related effects were compared with a vehicle control from the same test day, and this control group also received ketamine. After ketamine administration, a thermistor connected to a MacLab data acquisition system (CB Sciences, Dover, NH) was taped to the base of the rat’s tail. TST was then monitored continuously for 35 min to establish baseline temperatures. Naloxone was subsequently administered and TST measured for an additional 40 min (total recording time, 75 min). The peak of the maximal change of TST occurs 15 min after naloxone administration.

OVX rat telemetry models
The OXV-induced thermoregulatory dysfunction model is based on a previously reported protocol describing estrogen regulation of diurnal TST patterns (18) with modifications (20). In brief, during a 24-h period, intact cycling rats decrease TST during the active (dark) phase, and TST remains elevated during the inactive (light) phase. In an OVX rat, TST is elevated over the entire 24-h period; thus, the usual decrease in TST during the dark phase is lost. This model is used to evaluate a compound’s ability to restore the lowering of TST during the dark phase. A temperature and physical activity transmitter (PhysioTel TA10TA-F40; Data Sciences International) was implanted sc in the dorsal scapular region, and the tip of the temperature probe was tunneled sc 2.5 cm beyond the base of the tail. After a 7-d recovery period, TST and physical activity readings were recorded continuously for the remainder of the study. TST and physical activity readings were collected from each animal every 5 min, with values obtained over a 10-sec sampling period. The day before test day, an average baseline TST and physical activity value for each animal was completed by averaging the readings recorded during the 12-h dark phase. For TST experiments, the mean ± SEM for the vehicles days were 30.6 ± 1.0, 30.0 ± 0.04, and 29.5 ± 0.04 C, and the overall mean for all days was 30.0 ± 0.3 C. Rats were then administered a single oral dose of DVS (10–100 mg/kg). In these studies, animals were dosed approximately 60 (30–100 mg/kg) or 30 min (10 mg/kg) before the onset of the dark phase. For the telemetric measurement of CBT, a 3- to 4-cm-long skin incision was made in the midline of the abdomen and extended through the abdominal musculature. A transmitter (PhysioTel TA-F20; Data Sciences International) was placed in the abdominal cavity. The abdomen was closed with 4-0 absorbable simple interrupted sutures in the muscle layer, and autoclips were used to close the skin. Rats were allowed to recover a minimum of 7 d after surgery before use in these studies. The day before test day, an average baseline CBT value for each animal is completed by averaging temperature readings recorded during the 12-h dark phase. The means ± SEM for the vehicle days were 37.8 ± 0.003 and 37.8 ± 0.004 C, and the overall mean for the studies reported was 37.8 ± 0.001 C. The means and SD for both TST and CBT experiments represent normal physiological variation consistent with historical studies in our laboratory (20, 21, 26, 27). The physical activity measured by the telemetry system (PhysioTel TA10TA-F40; Data Sciences International) used in this report is strictly a relative measure of locomotor activity. The activity monitoring is done to ensure that compounds being evaluated in the telemetry models did not demonstrate enhanced or reduced physical activity when compared with their vehicle control run. No obvious behavioral effects were observed in rats receiving DVS, nor were there changes in locomotor activities when compared with the d-1 vehicle control activities.

Data analysis of thermoregulatory dysfunction models
MD model.
To analyze changes in TST induced by naloxone in the MD model, we analyzed data using a two-factor repeated-measure treatment x time ANOVA model. The model was fit to test whether there were significant differences in the responses between treatment groups. Naloxone administration was designated as time zero, and data were then analyzed at 5-min intervals. The first three readings (35, 30, and 25 min before naloxone administration) were averaged and used as baseline TST scores. All data were analyzed as change ({Delta}) in TST (TST for each time point minus baseline). Multiple comparisons (least significant difference P values) between the treatment groups at each time point were used for the analysis. Hot flush abatement was calculated by evaluating statistical differences at the peak response time (15 min post naloxone), when the typical maximal change in TST is observed. A logistic dose transformation was performed on the {Delta}TST. Maximum flush ({Delta}TST at 15 min post naloxone) was used in the analysis and the minimum locked at zero. A customized SAS-Excel (SAS Institute, Cary, NC) application was used to apply a four-parameter logistic model to determine ED50 value. The ED50 value is defined as the dose of test compound that abates 50% of the naloxone-induced flush. Statisticians in the Biometrics Department of Wyeth Research, Collegeville, PA, developed a customized JMP statistical software application used for data analysis.

Telemetry model.
Evaluation of the ability of the test compound to restore normal lowering of TST or to affect CBT in the telemetric models was analyzed using a 2-d paradigm in which animals are dosed with vehicle 24 h before administration of each test compound run. This 2-d within-group paradigm is used to normalize for differences between groups of animals and for any potential drift in temperature over time within a group. For both vehicle and compound testing, an average temperature was calculated for every 30-min time point. On the vehicle day, an overall average baseline temperature for each animal was determined by taking the mean over the 12-h observation period. Analysis of vehicle-day data at each 30-min time point indicated that there were no significant changes in TST or CBT over the 12-h dark phase (data not shown). All data were analyzed as change in temperature [average temperature for each 30-min time point on compound dosing d 2 minus average baseline temperature (12 h) on vehicle dosing d 1]. The average absolute TST of vehicle-treated rats range from 30–33 C. The means and SD for TST experiments represent normal physiological variation consistent with historical studies in our laboratory. A one-way ANOVA was performed on temperature changes at each 30-min time point over the 12-h dark phase to obtain the average SD. For each 30-min time point, a one-sample t test was done to determine whether the average change in temperature was statistically (P ≤ 0.05) different from zero. The onset of a treatment effect was defined as the first of two consecutive statistically significant half-hour intervals after any number of nonsignificant half-hour intervals. The treatment effect was considered to have ended when two consecutive nonsignificant (P > 0.05) half-hour intervals followed any number of significant half-hour intervals. Data are presented as onset of treatment, duration of treatment, and mean and maximum temperature change during the dark phase.

Stereotaxic surgery
CMA/12 guide cannulas (CMA Microdialysis, Solna, Sweden) were surgically implanted 3 d before drug treatment using a David Kopf model 900 stereotaxic apparatus as described previously (15). Rats were anesthetized by inhalation of 3% isoflurane in 100% oxygen and given sc injections of buprenorphine (0.1 mg/kg). Cannulas were positioned according to the rat atlas of Paxinos and Watson (28) so that they resided just above the left medial preoptic area of the hypothalamus. Cannula placement corresponded to the following coordinates relative to bregma: anteroposterior –0.4, lateral 0.8, dorsoventral –6.2. Microdialysis probe tips extended into hypothalamic tissue approximately 2.0 mm beyond the cannula edge.

Microdialysis procedures
Microdialysis was performed in freely moving, awake animals as described previously (15). Animals were placed in custom-made microdialysis boxes the night before the experiment. On the day of the experiment, CMA/12 2-mm probes (CMA Microdialysis) were implanted into the brain and equilibrated for 3 h before sample collection. Artificial cerebrospinal fluid was composed of NaCl (141 mM), KCl (3 mM), CaCl2 (1.5 mM), NaH2PO4 (0.07 mM), and NaHPO4 (0.30 mM), pH 7.4. The flow rate was 1.0 µl/min, and samples were collected every 30 min. Drug treatment was initiated after three baseline samples were collected. We visually verified probe placement in each animal the day after the experiment using a HM 500 M cryomicrotome (Microm, Walldorf, Germany).

HPLC analysis of dialysate
We determined neurotransmitter levels in microdialysis samples using HPLC with electrochemical detection as described previously (15). We calculated the concentrations (pg/µl) of NE, 5-HT, and dopamine (DA) in microdialysis samples against standard curves for each compound using peak heights. Raw data at each time point were converted to a percent change relative to the average of three baseline samples. Reported data represent the average percent change.

Analysis of microdialysis data
Statistically significant differences between DVS and vehicle (excluding preinjection baseline values) were determined using a two-way ANOVA (treatment x time) for repeated measures. Statistical calculations were performed using SAS Software version 8.02.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DVS vs. EE in the MD rat model
Chronic administration (≥5 d) of ovarian hormones is required to abate a naloxone-induced rise in TST in the MD OVX rat model (17, 19, 20, 21). Preliminary experiments were carried out to determine whether a single oral high dose of DVS (130 mg/kg) could abate a naloxone-induced rise in TST when compared with long-term (8-d) sc administration of EE (1 mg/kg) in the MD OVX rat model. At maximal flush time (15 min post naloxone injection), both DVS and EE significantly (P < 0.00001) abated the naloxone-induced flush (Fig. 1Go). Statistically significant changes in TST were observed in the treatment groups (F12,24 = 9.12; P < 0.0001) compared with the vehicle-treated group. DVS and EE abated the flush to similar magnitudes, 70 and 79%, respectively. No statistically significant increases in TST were observed before naloxone administration with DVS compared with TST of vehicle-treated animals.


Figure 1
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  		FIG. 1. Comparison of efficacy of single dose DVS and long-term (8-d) sc EE in the MD rat model. At maximal flush (15 min post naloxone; {Delta}TST, mean ± SEM), both DVS (130 mg/kg oral) and EE (1.0 mg/kg sc) significantly abated naloxone-induced flush to a similar magnitude. *, P < 0.00001 vs. vehicle.

 
DVS in the MD rat model
After determination that a high single oral dose of DVS could abate a naloxone-induced rise in TST, an ascending half-log dosing strategy was completed to assess whether DVS could dose-dependently abate a naloxone-induced flush in the MD model using a single oral dose paradigm. At maximal flush time (15 min post naloxone injection), acute oral administration of DVS significantly and dose-dependently abated the naloxone-induced flush at 10, 30, and 100 mg/kg (Fig. 2Go). Statistically significant changes in TST were observed among treatment groups (F11,44 = 4.75; P < 0.0001) compared with the vehicle-treated group. In contrast, treatment with either vehicle or 3 mg/kg DVS failed to decrease the naloxone-induced flush. The estimated ED50 value for the experiment graphically represented was 18.2 mg/kg (Fig. 2Go). The ED50 value of DVS when averaging the data from three independent experiments was 19.8 mg/kg. At the highest dose tested (100 mg/kg), DVS produced a 76% abatement (P < 0.0005) of the naloxone-induced flush relative to vehicle.


Figure 2
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  		FIG. 2. Oral administration of DVS dose-dependently abates naloxone-induced flush in the MD OVX rat model. At maximal flush (15 min post naloxone; {Delta}TST, mean ± SEM), DVS significantly abated naloxone-induced flush at 10, 30, and 100 mg/kg but had no effect at 3 mg/kg. *, P < 0.0005 vs. vehicle. The estimated ED50 value for DVS in this experiment was 18.2 mg/kg.

 
DVS in the TST telemetry model
A second model of temperature dysfunction was used to evaluate the ability of DVS to alleviate this OVX-induced dysfunction. The same doses evaluated in the MD model were assessed in this telemetry model. Acute oral administration of DVS (30 and 100 mg/kg) resulted in statistically significant decreases in TST over time (Fig. 3Go). Acute oral administration of the lowest dose of DVS (10 mg/kg) produced no statistically significant changes in TST. The 30-mg/kg DVS dose caused an immediate decrease in TST, with a significant mean change of –2.2 C lasting for 4.5 h (P < 0.05). The maximum decrease in TST was –3.1 C) compared with vehicle. The highest dose of DVS (100 mg/kg) significantly (P < 0.05) decreased TST (–2.4 C) relative to vehicle, with an immediate onset of activity that lasted 7 h. The maximum decrease in TST observed was –3.2 C relative to vehicle (P < 0.05). The highest dose of DVS produced the largest magnitude of effect on all parameters measured (Table 1Go).


Figure 3
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  		FIG. 3. Efficacy of DVS in the OVX-induced thermoregulatory dysfunction telemetry model. A single oral dose of DVS (30 or 100 mg/kg) resulted in statistically significant (P < 0.05 vs. vehicle baseline) decreases in mean and maximum TST (mean/maximum ± SEM) over time compared with vehicle baseline. Specifically, DVS rapidly and transiently decreased TST post injection. Error bars represent the SEM of each data point. Brackets mark periods of statistical significance (P < 0.05). No significant changes were noted at the lowest dose tested (10 mg/kg).

 

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  		TABLE 1. DVS in the TST telemetry model

 
DVS in the CBT telemetry model
Large changes in CBT can have detrimental effects on animals. Therefore, DVS was administered orally to OVX rats, and CBT was measured over time with telemetry. The doses selected for testing in the CBT telemetry model were based on efficacy noted in the TST telemetry model. Acute oral administration of DVS (30–60 mg/kg) resulted in small statistically significant changes in CBT over time (Fig. 4Go). Administration of the lowest dose of DVS (30 mg/kg) resulted in a small decrease in CBT (–0.43 C) relative to vehicle, with a statistically significant difference noted at 3.5 h and significance lasting only 1 h (Table 2Go). The 60-mg/kg dose of DVS produced a similar decrease in CBT, with a mean change of –0.53 C relative to vehicle, onset of action occurring at 5 h post dosing, and the decrease in CBT effect lasting for 2 h (P < 0.05). The decreases in TST with the 30-mg/kg dose of DVS do not appear to correlate with the changes in CBT at this same dose (Fig. 4Go).


Figure 4
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  		FIG. 4. Evaluation of oral administered DVS on CBT compared with effects on TST in the telemetry OVX rat model. Changes in CBT ({Delta}CBT, mean/maximum) measured over time in OVX rats (n = 10 rats per group). Oral administration of DVS at 30 and 60 mg/kg resulted in a small statistically significant decrease (P < 0.05) in CBT relative to vehicle baseline. Error bars represent the SEM of each data point. Brackets mark periods of statistical significance. Data for the measurement of decreases in TST at 30 mg/kg were taken from Fig. 3Go for comparison.

 

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  		TABLE 2. DVS is the CBT telemetry model

 
Neurochemical effects in the medial preoptic area of the hypothalamus after a single oral dose of DVS
Microdialysis techniques were used to determine the effect of DVS (30 mg/kg) on extracellular levels of 5-HT, NE, and DA in the medial preoptic area of the hypothalamus, a key region of the brain involved in temperature regulation. This dose of DVS was selected for these experiments based on the efficacy observed in the MD and TST telemetry models. Because acute DVS treatment does not alter 5-HT levels without first blocking 5-HT1A receptors (15, 23), WAY-100635 was administered 20 min before DVS. We previously reported that WAY-100635 does not alter the effect of DVS on NE or DA levels in the medial preoptic area of the hypothalamus in OVX rats (15). In animals pretreated with WAY-100635 (0.3 mg/kg, ip), a single dose of DVS rapidly increased extracellular levels of 5-HT (F6,27 = 9.55; P < 0.0001; Fig. 5AGo) and NE (F6,27 = 3.82; P < 0.01; Fig. 5BGo) compared with WAY-100635 plus vehicle. Maximal increases in 5-HT and NE occurred 60 and 90 min post dosing and reached 115.6 ± 10.8 and 60.9 ± 11.0% above baseline, respectively. In the same animals, DVS administration had no effect on DA levels (Fig. 5CGo). Baseline levels of 5-HT, NE, and DA in the microdialysate were 2.37 ± 1.25, 1.73 ± 0.48, and 0.44 ± 0.11 pg/µl (mean ± SEM), respectively.


Figure 5
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  		FIG. 5. Oral DVS administration increases 5-HT and NE levels in the hypothalamus of OVX rats. A single dose of DVS induced a rapid and statistically significant increase in both 5-HT and NE levels. A, 5-HT levels increased to a maximum of 116% above baseline by 60 min post dosing; B, NE levels increased to a maximum of 61% above baseline by 90 min post dosing; C, DA levels were unchanged after DVS dosing. *, P < 0.05 vs. vehicle at the same time point (n = 3–5 rats per time point).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
DVS is a novel succinate salt monohydrate of desvenlafaxine, the major active metabolite of venlafaxine (14). The results of these in vivo experiments demonstrate that DVS increases extracellular levels of both NE and 5-HT without increasing DA levels in the hypothalamus of OVX rats. In addition, oral administration of DVS alleviated thermoregulatory dysfunction in two OVX rat models without significantly impacting changes in CBT. These data further support our earlier reports that DVS is a selective SNRI (14, 15). Additionally, the activity of DVS in alleviating temperature dysfunction seen in preclinical models suggests that DVS may have utility in treating VMS associated with menopause. These preclinical models measure the ability of a compound to alleviate physiological aspects of temperature dysregulation independent of behavior measures, such as anxiety or depression.

Further evaluation of DVS, using the acute dosing regimen, demonstrated that DVS dose-dependently abated a naloxone-induced increase in TST. In the telemetry model, single oral DVS doses at varying levels alleviated OVX-induced changes in TST, with an immediate onset of action. Pharmacologically induced acute increases in TST (artificial flush) and the physiologically (OVX) induced dual model approach were used, because skepticism exists regarding the classic MD model (17, 22) and its relevance to menopausal hot flushes and vasomotor instability (18). Taken together, the data from both rodent models suggest that the activity of DVS in the MD model may not be a result of unrelated interactions with morphine or alterations of dependence but rather is a true alleviation of vasomotor instability.

Importantly, under normal conditions, DVS showed only small changes in CBT, which are not believed to be significant enough to induce clinical hypothermia if rat measures of CBT translate to human CBT. Other agents have been tested in the same models used in this study and have demonstrated larger changes in CBT that directly impacted changes in TST (26, 27). Additionally, these data are consistent with the hypothesis that the ability of DVS to alleviate OVX-induced thermoregulatory dysfunction is not due solely to an effect on CBT and likely involves correction of a neurochemical imbalance in the brain. Therefore, the changes in TST in rodent models observed in the present studies are not a result of nonspecific alterations of CBT but rather of a reversal of thermoregulatory dysfunction that is likely due to changes in neurotransmitter activity in the CNS, more specifically the hypothalamus. SNRIs have been studied primarily in models of mood disorders such as depression and anxiety (reviewed in Refs. 29, 30, 31, 32), with little research to assess physiological outcomes, in areas such as thermoregulation, pain sensation, sexual function, and various aspects of endocrine function related to homeostasis that 5-HT and NE are known to regulate. The present studies demonstrate that DVS, an SNRI, works specifically to affect temperature regulation without significant impact on changes in CBT and independent of behavioral measurements of depression or anxiety.

The effective use of estrogens to reduce the number and severity of hot flushes in menopausal women is a clear indicator that estrogen plays a role in the pathophysiology of VMS. Research has shown that estrogen can modulate both the 5-HT (33, 34) and NE systems (35, 36, 37); however, it should be noted that the activity of DVS in both rodent models in these experiments is distinct from that of estrogen (18, 19). Previous studies evaluating estrogen’s action in rodent models of vasomotor instability have determined that chronic dosing is required to restore thermoregulatory function (18, 22). The present findings demonstrate a rapid onset of DVS action with one single dose and may suggest a more rapid onset of action clinically. Although these results suggest that estrogen and DVS are equally effective, one should be cautious about assuming that DVS will achieve the same level of efficacy as hormone-based therapies; clinical studies to date using other nonhormonal therapies have not demonstrated equal efficacy (12, 13).

To investigate a possible mechanism mediating the efficacy of DVS in thermoregulatory dysfunction, we confirmed and extended the pharmacological in vitro profile of DVS (14) by performing a microdialysis study. We evaluated the neurochemical effects produced in the preoptic area of the hypothalamus after oral DVS administration. Neurochemical analysis determined that DVS, in combination with WAY-100635, produced a robust and rapid increase in extracellular NE and 5-HT concentrations in the preoptic area. In these studies, rats were pretreated with a selective 5-HT1A receptor antagonist, because previous reports demonstrated a lack of effect on central 5-HT levels after acute DVS treatment (14), an effect postulated to result from activation of somatodendritic 5-HT1A autoreceptors (38, 39). Given the purported role of the preoptic area of the hypothalamus in temperature regulation, the present data suggest possible serotonergic and/or noradrenergic mechanisms in mediating the efficacy of DVS in preclinical models of thermoregulation. Both 5-HT and NE have been reported to be involved in temperature regulation (40).

It has been suggested that estrogen may stimulate the activity of both the NE (35) and 5-HT systems (41). Therefore, we hypothesized that estrogen may modulate levels of NE and 5-HT activity, providing homeostasis in the hypothalamus. During menopause, when estrogen levels are diminishing or fluctuating, the levels of 5-HT and NE may become unstable. Restoring levels of these key neurotransmitters with DVS may alleviate VMS, a hypothesis supported by the results of the present studies. Additionally, 5-HT reuptake inhibitors and NE reuptake inhibitors are known to act in both the CNS and the peripheral nervous system, and clinical data suggest that the efficacy of DVS in treating VMS may also be a peripheral action based on its ability to potentiate vasoconstrictor responses under certain conditions (42).

In summary, the present findings demonstrate that DVS alleviates thermoregulatory dysfunction in preclinical rodent models of temperature regulation. In addition, these studies confirm earlier reports (14, 15) of DVS as a dual-acting SNRI. Immediate alleviation of temperature dysfunction was noted after a single DVS dose, whereas with EE, chronic (more than 5 d) administration (20, 21) is required to show activity. The models used are specific to thermoregulatory function; therefore, the action of DVS is directly related to the physiological events contributing to thermoregulation rather than to antidepressant mechanisms or nonspecific effects on mood. Thus, these experiments support the hypothesis that DVS may have utility in treating VMS associated with menopause.


   Acknowledgments
 
We thank Drs. Derrick Jansen and Tianhui Zhou for their statistical advisement and generation of the statistical programs used to analyze the data generated from the rat models of thermoregulatory dysfunction. We also thank Dr. Mary E. Hanson from Cardinal Health, Wayne, NJ, for assistance in the preparation and critical review of this manuscript.


   Footnotes
 
Disclosure Statement: All authors were employees of Wyeth Research when data reported within this manuscript were generated.

First Published Online November 22, 2006

Abbreviations: CBT, Core body temperature; CNS, central nervous system; DA, dopamine; DVS, desvenlafaxine succinate; EE, 17{alpha}-ethinyl estradiol; 5-HT, serotonin; MD, morphine-dependent; NE, norepinephrine; OVX, ovariectomized; SNRI, serotonin/norepinephrine reuptake inhibitor; TST, tail-skin temperature; VMS, vasomotor symptoms.

Received August 23, 2006.

Accepted for publication November 14, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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