WO2005004865A1

WO2005004865A1 - Use of 5-ht4(a)-serotonin receptor agonists

Info

Publication number: WO2005004865A1
Application number: PCT/EP2004/007427
Authority: WO
Inventors: Diethelm W. Richter
Original assignee: Georg-August-Universität Göttingen
Priority date: 2003-07-08
Filing date: 2004-07-07
Publication date: 2005-01-20

Abstract

This invention pertains to the use of a 5-HT4(a)-serotonin agonist or a pharmaceutically acceptable salt or ester thereof for the preparation of a pharmaceutical composition for treating or preventing drug induced respiratory depression.

Description

Use of 5-HT_4(a).serotonin receptor agonists

The present application relates to the use of 5-HT_4(a).serotonin receptor agonists or a pharmaceutically acceptable salt or ester thereof for the preparation of a pharmaceutical composition for treating or preventing drug induced respiratory depression or for the stabililzation of spontaneous breathing.

The inventors of the present application have found that 5-HT_4(a)_serotonin receptor agonists have a very strong stimulatory effect on the respiratory centre stabilizing spontaneous breathing. The 5-HT_4(a).serotonin receptor agonists also counteract the side effects of opioids and can be used for treating or preventing drug, preferably opioid, induced respiratory depression without blocking their analgetic effect.

According to the present invention 5-HT_4(a) serotonin receptor agonists may be used for the preparation of a pharmaceutical composition which is to be applied during or after anaesthesia to allow controlled recovery of spontaneous breathing if the respiration is depressed by the anaesthetic and/or opioids. Moreover the pharmaceutical composition of the present application may also by co-administrated with e.g. opioids to protect spontaneous breathing in acute, chronic pain and/or cancer patients.

A further preferred embodiment of the present application is the use of a 5HT_4(a)serotonin receptor agonist for the preparation of a pharmaceutical composition which may be applied in any situation when spontaneous breathing should be stabilized because the patient is treated with other drugs which might dimish the cAMP production within the respiratory neurons such as drugs acting on dopamine D_{2 3 or 4} receptors, adenosine A1 receptors or β-adrenergic receptors. Moreover the pharmaceutical composition may be applied in any situation when spontaneous breathing is unstable.

Any 5-HT_4(a).serotonin receptor agonist may be used, whereby the selective agonists are preferred. Preferred compounds according to the invention are derivatives of benzamidine, benzimidazole, benzothiazole, bezofuran, aminoguanidine indole and piperidinyl propanone. More preferred compounds are the benzoyl, phenylsulfonyl and benzylsulfonyl derivatives of benzamidine. Particularly preferred compounds according to the invention are 4-amino-5-chloro-2-methoxy-N-(piperidin-4-ylmethyl) benzamide with a polar substituent at the 1 -position of the pipridine ring such as benzoyl, phenylsulfonyl and benzylsulfonyl derivatives. Even more preferred compound are compounds of the general formula (I)

wherein R-j is methyl, ethyl, iso-propyl or n-propyl; R2 is halogen selected from CI, Br, F or I;

R3 is selected from H, OH, methyl, ethyl, iso-propyl, n-propyl and halogen selected from CI, Br, F and I; and may be in 0-, p- or m-position; R4 is selected from H, methyl, ethyl, iso-propyl, n-propyl and halogen selected from CI, Br, F and I; n is an integer selected from 0, 1 , 2 and 3; m is an integer selected from 0, 1 , 2 and 3; and Z is CO, SO2 or SO and their pharmaceutically acceptable salts.

More preferred are compounds wherein: Rl is methyl;and/or

R2 is CI or F, and/or

R4 is H and/or n is 1 and/or m is 0 or 1.

In a further embodiment of the present invention the 5-HT4(_a) receptor agonists are preferably selected from the group consisting of metoclopramide, mosapiride citrate, renzapride, prucalopride, 2-[1-(4-piperonyl)piperazinyl]bezothiazole, R 51619, 4-amino- N-[1-[3-(benzylsulfonyl<9propyl]piperidin-4-ylmethyl]-5-chloro-2-methoxybenzamidine (13a, Y-36912), tegaserod maleate, BIMU 8 (endo-N-(8-methyl-8-azabicyclo-[3.2.1]oct- 3-yl)-2,3,-dihydo-(1-methyl)ethyl-2-oxo-1 H-benzimidazole-1-carboxamide, HCI), BIMU 1 (endo-N-(8-methyl-8-azabicyclo-[3.2.1 )oct-3-yl-2,3-dihydro-3-ethyl-2-oxo-1 H- benzimidazole-1-carboxamide, HCI), (R,S)-zacopride ((R,S)-4-amino-N-(1- azabicylo[2.2.2]oct-3-yl)-5-chloro-2-methoxybenzamide, HCI) and its enantiomers, cisapride, HTF-919, R-093877, SL65.0115, RS 67333 ((1-(4-amino-5-chloro-2- methoxyphenyl)-3-(1-n-butyl-4-piperidinyl)-1-propanone), RS 67506 ((1-(4-amino-5- chloro-2-methoxyphenyl)-3-[1-[2-[(methylsulfonyl)amino]ethyl]-4-piperidinyl]-1- propanone) and SC 53116 ((1.(s)-1-exo-4-amino-5-chloro-N-[(hexahydro-1 H-pyrrolizin- 1 -yl)methyl]-2-methoxybenzamide, HCI). However, they are not limited to the above.

Drugs which elicit repiratory depression or which make the stabilization of spontaneous breathing necessary are e.g. barbiturates, narcotics, hypnotics, analgesics, anesthetics and opioids. Of particular interest are fentanyl, morphine, codeine, hydromorphon, oxycodon, buprenorphin, alfentanil, sufentanil, levomethadon, piritramide, pethidine, pentazocine and trifupromacine and salts and derivatives thereof, however, they are not limited thereto.

Background of the invention Opiates are widely used analgesics in anaesthesiology, but they have serious adverse effects such as depression of breathing. This is caused by direct inhibition of rhythm generating respiratory neurons in the Pre-Boetzinger complex (PBC) of the brainstem. The inventors have found that serotonin 4(a) receptors (5-HT4(_a)) are strongly expressed in respiratory PBC neurons and that their selective activation protects spontaneous respiratory activity. Treatment of rats with a 5-HT4 receptor specific agonist overcomes fentanyl-induced respiratory depression and re-establishes stable respiratory rhythm without loss of its analgesic effect. The findings imply the prospect of a fine-tuned recovery from opioid-induced respiratory depression by adjustment of intracellular cAMP levels through the convergent signalling pathways in neurons.

Serotonin (5-hydroxytryptamine, 5-HT) is an important neurotransmitter that is involved in a wide range of neuromodulatory processes in the central nervous system by acting on a number of different 5-HT receptor isoforms (1-2). The 5-HT4 receptor is a recently identified subtype that is widely and abundantly expressed as alternatively spliced variants in various brain regions (3-5). The receptor exerts excitatory effects through its positive coupling to heterotrimeric Gs proteins to activate adenylyl cyclases and to induce robust increases of intracellular cAMP levels (6-7). The receptor also couples to G13 proteins to activate small GTPases of the Rho family (8) (Fig. 1 A). Recent cloning of the 5-HT4 receptor (9) initiated the development of 5-HT receptor subtype specific immunocytochemistry and pharmacology. We produced a specific antibody against a synthetic peptide corresponding to the C-terminal sequence of the 5-HT4(_a) receptors isoform [amino acids His364 to Pro380] ( 10) (Fig. 1 B) which allowed us to specifically identify the spatial expression of the 5-HT4 receptors in the central nervous system including the brainstem ( 11). We found that 5-HT4 receptors are abundantly expressed in the Pre-Boetzinger Complex (PBC), a region in the lower brainstem, which is known to generate and control spontaneous breathing movements (12). This specific antibody was also used to analyse the co-expression of the 5-HT4(_a) receptor with / -opioid receptors and Substance P reactive NK-1 receptors, which have been suggested as potential immunocytochemical markers for respiratory neurons (13- 15) . Medullary motoneurons were visualized by choline acetyl transferase (ChAT) staining and excluded from the analysis ( 16). In multiple-labelling experiments (NK-1 receptors, 5-HT4(_a) receptors and ChAT), we identified three different types of immunoreactive medullary interneurons: 35.5% of interneurons displayed intense NK-1 and 5-HT4(_a) receptor co-immunoreactivities, 34.1% of interneurons revealed only 5-HT4(_a) receptor immunoreactivity, whereas 30.4% of interneurons revealed only a NK-1 receptor immunoreactivity (Fig. 2A and 2B). In a similar study, the brainstem was analysed for co-immunoreactivities of /-opioid and 5-HT4(_a) receptors within the PBC region, which is a region essential for respiratory rhythm generation (12). We found positive staining in 46.8% of interneurons for both //-opioid and 5-HT4(_a) receptors, while a population of 53.2% of interneurons exhibited only /-/-opioid receptor immunoreactivity (Fig. 2C). These data suggest that approximately one half of all 5-HT4(_a) receptor positive interneurons in the PBC region co-express both NK-1 receptors and //-opioid receptors. This was confirmed by multiple staining of 5-HT4(_a), NK-1 and /-opioid receptors in the same slice (Fig. 5) To verify that the 5-HT4(_a) receptor immunoreactive interneurons indeed represent respiratory neurons, single-cell RT-PCR analysis was performed on the cytosol of identified inspiratory neurons in the rhythmically active slice preparation (Fig. 3A) (17). We found that 95.2% of neurons analysed expressed 5-HT4 receptors (Fig. 3B). In view of the large number of 5-HT4 receptor isoforms, it was of particular interest to analyse the expression of the different 5-HT4 receptor splice variants. RT-PCR analysis of the PBC region and of individual respiratory neurons proved the expression of 5-HT4(_a), 5- HT4(_b) and 5- HT4(_f) receptor, but not 5-HT4(_e) receptor mRNAs in the whole PBC region (Fig. 3B) (16), while defined inspiratory neurons expressed only the 5-HT4(_a) mRNA (Fig. 3B). Immunocytochemical studies confirmed these observations demonstrating a strong 5-HT4(_a) receptors immunoreactivity of biocytin-labelled inspiratory neurons (Fig. 3C). We also determined by single-cell RT-PCR the expression profile for NK-1 receptors as well as for the //-opioid receptors mRNAs (16). The expression of the NK-1 receptor mRNA was found in only 30.7% of defined inspiratory neurons, while immunohistochemical data from the PBC region indicated NK-1 receptor expression in 65.9% of PBC neurons (Fig. 2). This difference revealed the presence of non- respiratory neurons expressing NK-1 (18). In contrast to NK-1 , / -opioid receptor mRNA was detected in all inspiratory neurons analysed as reported by other laboratories (13).

Our data demonstrate that 5-HT4(a) and /-opioid receptor-mediated signalling pathways are co-existent in inspiratory neurons (Fig. 1A) and therefore are capable to interact in an antagonistic manner; / -opioid receptors operating via Gi/o proteins to decrease the cAMP levels (19), and 5-HT4(a) receptors counteract by activation of Gs to raise cAMP concentration (7). The physiological significance of such potential interaction between 5-HT4(_a) and / -opioid receptor mediated signalling in the regulation of respiration was further explored in the in-vivo like perfused rat brainstem-spinal cord preparation ( 16, 20), which contains the fully intact respiratory network, and finally verified in the in-vivo rat. First, we tested the effects of the 5-HT4(_a) receptor specific agonistic drug BIMU8 ( 10) on ongoing respiratory activity and found that vascular application of this drug significantly increased phrenic nerve activity at all doses tested (concentration range: 0.3 - 10 //M) (Fig. 4A). The whole animal experiments verified that application of BIMU8 (1-2mg/kg) significantly increase respiratory minute volume (RMV) also in-vivo. This stimulatory effect was 5-HT4 receptor specific, because it was blocked by the specific antagonist GR 113808 in both experimental approaches. Involvement of 5-HT4/GS signalling in the regulation of respiratory activity was confirmed by the findings that application of dibutyryl-cAMP increased, while the AC blocker SQ 22,536 decreased phrenic nerve activity. Co- activation of 5-HT3 receptors by BIMU8 could be excluded, because this receptor is not functionally expressed in respiratory neurons (21-22) The physiological consequences of / -opioid receptor activation was tested with specific agonist fentanyl. Fentanyl is a synthetic opioid widely used for anaesthesia and for the relief of acute and chronic pain, although it produces the serious adverse reaction, such as hypoventilation (19, 23-24). Application of fentanyl to the perfused rat brainstem-spinal cord preparation induced the expected anti-nociceptive effects as seen by a 60.9 ± 6.5% (n = 8; p <0.01) reduction of the C-fiber reflexes (CFR) (Fig. 4B). At the same time, however, respiratory activity was almost completely suppressed (Fig. 4B). In

3 cases, exposure to fentanyl even led to apnea that would be lethal under normal conditions. The effects obtained in in-vivo animals were even more pronounced.

Here, fentanyl produced strong anti-nociceptive effects resulting in a complete abolishment of the tail flick response (TFR) (Fig. 4C). However, similarly to the brainstem preparation, spontaneous respiratory movements were completely blocked (fig. AC).

Therefore, we tested whether activation of the 5-HT4 receptor mediated signalling pathway is effective in overcoming fentanyl-induced respiratory depression and apnea (Fig. 4B) ( 19, 25). To verify the power of 5-HT4 receptors in restoring respiratory activity, we performed successive applications of fentanyl and the 5-HT4 receptor agonistic drug BIMU8. The crucial result was that consecutive applications of BIMU8 indeed reestablished a stable respiratory activity within 3 min in the perfused brainstem preparation (Fig. 4B). This effect was fully reproduced in-vivo. In the latter cases, subsequent application of BIMU8 (1-2mg/kg) overcame the fentanyl induced apnoea and restored stable breathing with RMV recovered to 70.6 ± 18.1% within 3 min (Fig. 4C). Lastly we investigated whether 5-HT4 receptor stimulation obliterates the nociceptive function of opioids. We tested C-fiber reflexes (CFR) in the brainstem-spinal cord preparation and tail flick response (TFR) in-vivo (16). Application of BIMU8 after fentanyl treatment was sufficient to re-establish stable respiration in both test systems without any significant effects on the CFR (Fig. 4B) or TFR (Fig. 4C). Additional application of naloxone (1 mg/kg) immediately re-established the TFR (Fig. 4C). The absence of BIMU8 induced effects on nociception found its explanation by the finding that dorsal horn spinal interneurons reveal abundant / -opiod, but no significant 5-HT4(_a) receptor immunoreactivity (Fig. 6).

The present study provides evidence that activation of 5-HT4 receptors in neurons of the medullary respiratory center represents a novel way for the treatment of respiratory depression induced by opioids. Stimulation of 5-HT4 receptors effectively counteracts fentanyl-induced respiratory depression without compromising its anti-nociceptive potency. An inspiring possibility is that application of 5-HT4 receptor agonists could be used for the treatment of critical respiratory events caused by fentanyl in post-operative situations and for the treatment of pain patients against overdose of opioids (23). In essence, the rationality of a straight forward therapy targeting of convergent intracellular signal pathways by means of a receptor-specific pharmacology (26) might open novel strategies for effective treatment in a wide spectrum of critical clinical situations.

Experiments:

Slice preparation. Experiments were performed on brainstem slice preparations from postnatal Sprague-Dawley rats at postnatal days P5-P8. The brainstem-spinal cord was isolated and a single 700 //m thick transverse slice containing the PBC was cut from the brainstem and transferred into a recording chamber perfused with artificial cerebrospinal fluid. The respiratory rhythm was stabilized by activating the respiratory network by increasing [K+]_θ levels to 8 mM. The hypoglossal (XII) rootlet was sucked into a glass pipette for extracellular recording of rhythmic respiratory discharges.

Patch-clamp recordings. Neurons of the PBC were patched under visual control or 'blindly' by advancing the patch pipette slowly through the tissue until an increase in series resistance indicated contact with a cell. Following the formation of a gigaohm- seal, brief suction was applied to establish a whole-cell recording configuration. Respiratory drive currents or potentials were measured in voltage clamp or current clamp modes, respectively. For immunostaining identified inspiratory neurons were filled with the fluorescent dye biocytin.

Single-cell RT-PCR. In order to collect cytosol for RT-PCR analysis, superficially located cells were patched under visual control using IR microscopy and identified as inspiratory neurons by their ongoing rhythmic activity occurring synchronously with hypoglossal burst discharges. Subsequently, constant suction was applied for 1 - 2 min to harvest the cytoplasm. Reverse transcription was carried out at 25°C for 10 min

followed by reverse transcription (incubation for 50 min at 42°C; Superscript II reverse transcriptase). For all amplification reactions, the primer concentration was 0.2 μWΛ and all primers were tested on plasmid (5-HT4(_a) receptor) as well as on total brain cDNA (5- HT4(_a), //-opioid and NK-1 receptors). The cycling conditions for amplification with AmpliTag Gold polymerase were a hot start at 95 °C for 10 min, 40 cycles at 94 °C for 30 sec, 53 °C (5-HT4(_a) receptor) or 56 °C ( /-opioid and NK-1 receptors) for 30 sec, 72 °C for 1 min and an elongation step at 72 °C for 10 min. In parallel, PCR was also performed with the AccuPrime Taq DNA polymerase system, 94 °C for 2 min followed by 39 cycles of denaturation (94 °C for 30 sec), annealing (55 °C for 30 sec) and elongation (68 °C for 1 min). The following primers were used for specific amplification of the 5-HT4 receptor: sense 5^'-CTGTAATGGACAGACTTGA-3^' and antisense 5^'- GGCAAAACATCTCCCCATAA-3^'. For the evaluation of different 5-HT4 receptor isoform expression, one sense primer in the common part of the variant sequences, 5^'- CCAAGGCAGCCAAGACT-3^' and two antisense primers: 5'-

TAGTAACCTGTTCATGCAGACACA-3^' for amplification of specific (a), (e), (f) isoforms and 5^'- TTGCCTCATGCTCTTGGAA-3^' for amplification of the (b) isoform were employed. The specific amplification of NK-1 receptors was Superscript II reverse transcriptase). For all amplification reactions, the primer concentration was 0.2 //M and all primers were tested on plasmid (5-HT4(_a) receptor) as well as on total brain cDNA (5- HT4(_a), //-opioid and NK-1 receptors). The cycling conditions for amplification with AmpliTag Gold polymerase were a hot start at 95 °C for 10 min, 40 cycles at 94 °C for 30 sec, 53 °C (5-HT4(_a) receptor) or 56 °C ( /-opioid and NK-1 receptors) for 30 sec, 72 °C for 1 min and an elongation step at 72 °C for 10 min. In parallel, PCR was also performed with the AccuPrime Taq DNA polymerase system, 94 °C for 2 min followed by 39 cycles of denaturation (94 °C for 30 sec), annealing (55 °C for 30 sec) and elongation (68 °C for 1 min). The following primers were used for specific amplification of the 5-HT4 receptor: sense 5^'-CTGTAATGGACAGACTTGA-3^' and antisense 5'- GGCAAAACATCTCCCCATAA-3^'. For the evaluation of different 5-HT4performed using the primers: sense 5^'-CAGGACTTATGAGAAAGCGT-3^' and antisense 5^'- AGATCTGGGTTGATGTAGGG-3^'. For amplification of the //-opioid receptor, we used

the sense primer 5^'-TTCTGCATTGCTTTGGGTTACACG-3^' and the antisense primer ^-CTGACAGCAACCTGATTCCACGTA-S^'. All RT-PCR products were evaluated by using direct DNA sequencing.

Preparation of brain tissue for immunohistochemistry. Juvenile rats (P21 - 28) were anaesthetised with halothane before the animals were thoracotomized and transcardially perfused with 50 ml 0.9 % NaCI and 200 ml 4% formaldehyde. The brainstem was removed, post-fixed, cryoprotected in 25 % sucrose overnight at 4°C and then frozen at - 25°C. Series of 40 //m thick transverse sections were cut at the level of the Pre- Boetzinger region using a cryoslicer. Immunofluorescence. Sections were permeabilized at room temperature with 0.2 % Triton X-100 for 30 min followed by incubation at 4 °C for 48 to 72 hrs with one, two or three of the following antibodies: antibody AS9459 (7, 10) against 5-HT4(_a) receptors; guinea pig anti-NK-1 receptors; guinea pig anti-/ opioid receptors; or goat antiserum against ChAT. The sections were then washed and incubated for 2 h with appropriate secondary antibodies conjugated with different fluorochromes. Immunofluorescence analysis was performed using a confocal laser scanning microscope LSM 510.

Perfused Rat Brainstem-Spinal Cord Preparation. This preparation was performed according to the description given by Paton (20, 27). It preserves an in v/Vo-like respiratory activity pattern (20, 28) and allows functional studies of spontaneous respiration and nociceptive reactions. Sprague-Dawley rats (P20-32) were anaesthetised, thoracotomized, decerebrated at the pre-coliicular level and cerebellectomized. Norcuronium-bromide 0.5 mg x 200 ml-i was given for muscle relaxation. The upper body was placed into a recording chamber and perfused retrogradely through the thoracic aorta with artificial cerebrospinal fluid. All larger thoracic vessels were ligated, and heart and lungs were removed. Arterial perfusion pressure, measured at the tip of the perfusion catheter, was set to 45 to 65 mmHg, and guaranteed proper brainstem perfusion and stable phrenic nerve activity (20, 28-29).

Respiratory activity was recorded from the phrenic nerve (PN) with a suction electrode and integrated activity per minute (phrenic nerve minute activity; PNAm-n) was calculated. For studying nociceptive responses, we recorded the C-fiber reflex (CFR) from a branch of the thoracolateral nerve (as separated from the latissimus dorsi muscle) following

electrical stimulation (0.2 ms, 5-15V, 0.1 -Hz) of the contralatral median nerve. In the unrestrained animal, such stimulation caused a stretch reflex of the contrallateral extremity. The averaged amplitudes of CFR were calculated as percent change of control levels. Functional in-vivo tests. The in-vivo studies were performed in accordance with the ethical guidelines for the care and use of animals (Recommendation of the European Commission No L 358, ISSN 0378-6978) and was approved by the local council for animal care. Ten Sprague Dawly rats of either sex (250-350g) were anaesthetised by intraperitoneal injection of pentobarbital (60mg/kg). The femoral vein was cannulated with a polyethylene tubing for drug applications and fluid injections. The rats were tracheotomised and intubated. The tracheal tube was connected to a pressure transducer to record respiratory airflow. When fentanyl application induced apnoea, the animals were artificially ventilated at low frequencies (10-15 breath/min) to prevent hypoxia. Artificial ventilation was stopped when spontaneous breathing movements became visible after BIMU8 application.

Nociceptive behavioural responses were assessed by the tail flick responses (TFRs). The tail was marked with ink (2-3 cm from tip, 3 spots at an interval of 1 cm). High intensity light was applied to such marked spots until the animal responded with a tail flick. The TFRs were quantified by measuring the latency between onset of the heat stimulus and evoked withdrawal response. The average TFR latency values of 3 consecutive trails before drug application were used as baseline. To avoid severe tissue damage, heat stimuli ended when the TFR latency exceeded 300% of control. In these case, we concluded that the TFR was greatly diminished. After experiments, animals were sacrificed by an overdose of pentobarbital.

Immunocytochemistry. Triple-labelling experiments (NK-1 receptors, 5-HT4(_a) receptors and ChAT) were performed on 28 transverse brainstem sections of five animals. We positively identified three different types of non-ChAT immunoreactive cells: (#) 35.5% of cells (245 of 691) within the PBC displayed intense NK-1 and 5-HT4(a) receptor co- immunoreactivity, (ii) 34.1 % of cells (236 of 691) revealed only a 5-HT4(a) receptor immunoreactivity, (Hi) 30.4% of labelled cells (210 of 691) showed only NK-1 receptor immunoreactivity. In a comparable study, 18 brainstem slices of 3 rats were analysed for the co-immunoreactivity of /-opioid receptors and 5-HT4(a) receptors within the PBC region. In these cases, we obtained only two non-ChAT cell populations: (i) 53.2% of cells analysed (438 of 824) exhibited onl /-opioid receptor immunoreactivity, (ii) 46.8% of cells (386 of 824) were positively stained for both //-opioid receptors and 5-HT4(a) receptors.

Single-cell RT-PCR. Using the set of primers, which was common for all known splice variants of 5-HT4 receptors, we found that 95.2% of cells analysed (20 of 21 ) expressed 5-HT4 receptors. The expression of NK-1 receptors was found only in 30.7% of cells analysed (4 of 13 cells). In contrast to the expression patterns obtained for NK-1 receptors, //-opioid receptor mRNA was detected in all neurons analysed (16 of 16 cells).

Functional tests in the perfused brainstem preparation. Systemic application of 0.3/M BIMU8 significantly increased phrenic nerve minute activity (PNAmin) by 34.8 ± 13.3 % (p < 0.05; n = 6). Higher doses of BIMU8 resulted in an even stronger acceleration of PNAmin (p < 0.05 for all measurements) ranging at + 59.9 ± 9.7% with 1 / M BIMU8 (n = 9), + 67.4 ± 19.5 % with 3 //M BIMU8 (n = 8), and + 64.9 ± 19.5 % with 10 / M BIMU8 (n = 9; (Fig. 4A).

Fentanyl (5 to 20 nM) caused a severe depression of respiratory activity to 8.9 ± 4.2% of control level (n = 8; p < 0.05) (Fig. 4B). In 3 cases, exposure to fentanyl even led to

apnoea. There was some variation in fentanyl sensitivity between experimental animals, and different concentrations of fentanyl (5 to 20 nM) were needed to achieve the comparable effects of respiratory depression and antinociceptive effects. Consecutive applications of BIMU8 following fentanyl treatment evidently re-established stable respiratory activity after a latency of approximately 3 min. In a dose-dependent manner, PNAmin recovered to 54,9 ± 24.0% (n = 8) of PNAmin after 1 μU BIMU8, and to 68,4 ± 15.1% (n = 7) after 3 //M BIMU8 and to 74,6 ± 11.2% (n = 8) after 10 / M BIMU8 (Fig. 4B). ( , p < 0.05). Anti-nociceptive effects. Application of low concentrations of fentanyl (5 to 20 nM) induced efficient anti-nociceptive effects as demonstrated by a 60.9 ± 6.5% (n = 8; p < 0.01) diminution of the spinal C-fiber response (CFR) (Fig. 4B). Subsequent application of BIMU8 did not affect opioid-induced anti-nociception, as seen in a firm depression of the CFR by -54.3 ± 9.4 % (1/ M BIMU8; n = 7), -51.8 ± 9.2% (3//M BIMU8; n = 6) and - 65.6 ± 7.2 % (10//M BIMU8; n = 8). At the same time, PNAmin became strongly reinforced.

Behavioural tests in-vivo. Systemic application of BIMU8 (1-2mg/kg) significantly increased respiratory minute volume (RMV) by 52.9 ± 10.4 % (p< 0.05, n=3) of controls. Application of 5HT4 receptor antagonist GR 113808 (3 - 4mg/kg) prior to BIMU8 did not significantly change resting RMV (-8.4% ± 5%), but diminished the BIMU8-evoked increase of RMV to 12.4 ± 10.9 % (p< 0.05, n = 3). Application of fentanyl (10-15//g/kg) induced a significant reduction of RMV to 3.9 ± 8.5 % of control (p< 0.001 , n=5). Subsequent application of BIMU8 (1 -2mg/kg) prevailed over the effect of fentanyl and restored stable breathing, RMV recovering to 70.6 ± 18.1% of control (p< 0.001 , n=5) within 3 min. Additional application of naloxone further increased RMV above control levels to 149,7 6 ± 14,3% (p< 0.001 , n = 4). To investigate drug effects on nociception, we performed tail flick response (TFR) tests. Under control condition (60/ g/kg pentobarbital), the latency of TFR ranged between 4-7 s. Fentanyl (10-15//g/kg) induced its powerful anti-nociceptive action as seen by complete abolishment of the TFR (p< 0.001 n=5) within a maximal stimulation period of

20 s. The abolishment of the TFR was fully maintained after BIMU8 application (TFR latency 300% of baseline, p< 0.001 , n=5). Additional systemic application of naloxone (1 mg/kg) re-established the TFR immediately and induced even a decrease in latency below controls (- 16.5 ± 15.5 %, n=5).

Thus the present invention for the first time provides the use of 5HT4(_a) serotonin receptor agonists for the preparation of a pharmaceutical composition for treating or preventing drug induced respiratory depression or for the stabilization of spontaneous breathing.

The above mentioned 5HT4(_a) serotonin receptor agonists may be formulated with excipients generally known in the field of pharmaceutical formulation to dosage forms for oral, parenteral, rectal, intracerebral or intranasal application.

The figures show: Fig. 1.

(A) Schematic illustration of the signal transduction pathways mediated by 5-HT4 and μ- opioid receptors. Whereas 5-HT4 receptors stimulate adenylyl cyclases (AC) through both Gs and G13 proteins, //-opioid receptors inhibit AC activities through a Gi/o mediated inhibitory pathway.

(B) Western blot analysis of the brainstem lysate with a polyclonal antibody raised against the C-terminal part of the 5-HT4(_a) receptor isoform. (Left pane!) Model of the 5- HT4(_a) receptor with the amino acids CHSGHHQELEKLPIHNDP (underlined with red) used for the production of an antibody. (Right panel) Membrane (m) and cytosolic (c) fractions prepared from the lysate of the rat brainstem were separated by SDS-PAGE and then subjected to Western blotting with an anti-5-HT4<_a) receptor antibody.

Fig. 2. Distribution of 5-HT4(a), NK-1, /-opioid receptors and ChAT immunoreactivities within the ventrolateral region of the brainstem containing the Pre-Boetzinger region (PBC).

(A) Triple-labelling of 5-HT4(_a) receptors (green, Alexa 488), NK-1 receptors (red, Alexa 546) and ChAT (blue, Alexa 647) with appropriate antibodies of a 40 μm thick transversal slice cut at the level of the PBC. NA - Nucleus ambiguus. Scale bar = 50 μ .

(B) Single neuron from the PBC region stained for 5-HT4(_a) (green) and NK-1 receptors (red). 5-HT4(_a) receptors are predominantly expressed on the soma and proximal dendrites. Scale bar =10 μm.

(C) Triple-labelling of 5-HT4{_a) (green, Alexa 488), /-opioid receptors (red, Alexa 546) and ChAT (blue, Alexa 647) at the level of PBC. Scale bar = 50 μm. All images were obtained by confocal laser-scan microscopy.

Fig. 3. Expression of 5-HT4 receptors in functionally identified inspiratory neurons.

(A) Site of electrophysiological recordings (top, left) with an inspiratory neuron on the tip of the patch pipette (top, right). Integrated hypoglossal nerve (Nxn) activity (middle trace) corresponds to rhythmic inward currents in a single inspiratory neuron recorded in the

whole-cell configuration (bottom trace).

(B) (Upper panel) Single-cell RT-PCR analysis of inspiratory neurons. Gel electrophoresis was carried out for RT-PCR products amplified with 5-HT4 primers. The control reaction without reverse transcription is shown in the first line. (Lower anel) RT- PCR analysis of 5-HT4 receptor splice variants in the PBC region (left) and in an individual inspiratory neuron (right). Lane 1 , primers amplifying the (a), (e) and (f) isoforms. Lane 2, primers amplifying the (b) isoform. All RT-PCR products were evaluated by using direct DNA sequencing.

(C) Example of an inspiratory PBC neuron labelled intracellularly with biocytin (big arrow) exhibiting strong 5-HT4(_a) receptor immunoreactivity (red, Alexa 546). The neuron is surrounded by 5-HT4(_a) receptor immunoreactivities on somatic profiles (small arrows) within the PBC. NA - Nucleus ambiguus. Scale bar = 50 μm.

Fig. 4. Stimulation of 5-HT4 receptors by the selective agonist BIMU8 removes opioid- induced respiratory depression without loss of the anti-nociceptive effect of opioids in the perfused brainstem preparation (A,B) as well as in the intact in-vivo rat (C).

(A) Dose-dependent effect of BIMU8 on phrenic nerve minute activity (PNAmin) (16). Statistically significant changes from untreated control are indicated by asterisks (p < 0.05).

(B) (Left) Application of fentanyl (blue line) caused a marked depression of PNAmin by 91.2 ± 4.2% (n = 8; p < 0.05) as compared with a control (black line). In 3 cases, fentanyl even led to apnea. Respiratory activity was re-established by a subsequent application of BIMU8 (red line) in a dose-dependent manner ( 16). (Right) Responses of the spinal C-fiber reflex (CFR). Black trace represents the CFR of the untreated control. Application of fentanyl (blue line) suppressed the CFR by 60.9 ± 6.5% (n = 8; p < 0.01). Subsequent administrations of BIMU8 (red line) did not affect opioid induced depression of the CFR (16).

(C) (Left) Respiratory airflow obtain in anaesthetised spontaneous breathing in-vivo rat (black line). Application of 10-15/ g/kg fentanyl (blue line) induced a dramatic reduction

in respiratory minute volume (RMV) to 3.9 ± 8.5 % of control (p< 0.001 , n=5). Consecutive application of 1-2mg/kg BIMU8 (red line) prevailed over this effect of fentanyl and restored stable breathing with RMV recovering to 70.6 ± 18.1 % (p< 0.001 , n=5) of control. (16). Asterisks indicate transient periods of artificial ventilation, which was necessary to rescue the animal during genuine fentanyl treatment. (Right) Analysis of nociception by the tail flick response (TFR). A quick TFR was obtained in control conditions (black line), which was completely abolished after application of fentanyl (blue line). Such absence of analgetic responses remained unchanged after subsequent administration of BIMU8 (red line). Note that application of naloxone (1 mg/kg) immediately re-established the TFR.

Fig.5: (A) Triple-labelling of 5-HT4(_a) (green, Alexa 488), NK-1 (red, Alexa 546) and μ- opioid (blue, Alexa 647) receptors with appropriate antibodies in a 40 μm thick transverse slice at the level of the PBC. (Arrows) Triple-labelled neurons. NA - Nucleus ambiguus. Scale bar = 50 μm.

(B) Single neuron from the PBC region stained for 5-HT4(_a) (green), NK-1 (red) and //- opioid (blue) receptors. Scale bar =10 μm.

Fig. 6: In contrast to the intense //-opioid receptor immunoreactivity, clear 5-HT4(_a) receptor immunoreactivity is missing in dorsal horn interneurons. (A) Double-labeling of 5-HT4(_a) receptors (green, Alexa 488) and //-opiate receptors (red, Alexa 546) with appropriate antibodies in a dorsal horn region (40 μm slice) at the C5- C6 level. Scale bar = 50 μm. (B) As a control, double-labelling of 5-HT4(_a) receptors (green, Alexa 488) and //- opiate receptors (red, Alexa 546) was analysed in the ventral horn region (40 μm slice) of the spinal segments C5-C6. Note that ventral horn motoneurons show a strong co- immunoreactivity for both / -opioid and 5-HT4(a) receptors. Scale bar = 50 μm.

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Claims

1. Use of a 5-HT_4la)-receptor agonist or a pharmaceutical acceptable salt or ester thereof for the preparation of a pharmaceutical composition for treating or preventing drug induced respiratory depression or for the stabilization of spontaneous breathing.

2. Use according to claim 1 , wherein the drug is selected from barbiturates, narcotics, hypnotics, analgesics, anaesthetics and opioids

3. Use according to claim 1 or 2 for treating or preventing drug induced respiratory depression induced by substance abuse.

4. Use according to claim any one of claims 1-3, wherein the drug is selected from fentanyl, morphine, codeine, hydromorhon, oxycodon, buprenorphin, alfentanil, sufentanil, levomethadon, piritramide, pethidine, pentazocine and trifupromacine and salts and derivatives thereof.

5. Use of according to any one of claims 1 to 4 for treating or preventing drug induced respiratory depression without blocking the drugs^' analgetic effect.

6. Use according to any one of claims 1 to 5, wherein the 5-HT(4a) serotonin receptor agonist is a compound of general formula (I)

wherein R-| is methyl, ethyl, iso-propyl or n-propyl; R2 is halogen selected from CI, Br, F or I;

R3 is selected from H, OH, methyl, ethyl, iso-propyl, n-propyl and halogen selected from CI, Br, F and I; and may be in o-, p- or m-position; R4 is selected from H, methyl, ethyl, iso-propyl, n-propyl and halogen selected from CI, Br, F and I; n is an integer selected from 0, 1 , 2 and 3; m is an integer selected from 0, 1 , 2 and 3; and Z is CO, SO2 or SO and pharmaceutically acceptable salts thereof.

7. Use according to claim 6, wherein R₂ is CI or F

8. Use according to Claim 6 or 7, wherein Z is CO, Ri is methyl, R2 is CI, R4 is H, n is 1 and m is 0

9. Use according to claim 6 or 7, wherein Z is SO2, Ri is methyl, R2 is CI; R4 is H, n is 1 and m is 0.

10. Use according to claim 6 or 7, wherein Z is SO2, Ri is methyl, R2 is CI, R4 is H, n is1 and m is 1.

11. Use according to any one of claims 1 to 10, wherein the 5-HT_4(a) serotonin receptor agonist is selected from metoclopramide, sulpiride, mosapiride citrate, renzapride, prucalopride, 2-[1-(4-piperonyl)piperazinyl]benzothiazole, R 51619, 4- amino-N-[1-[3-(benzylsulfonyl<9propyl]piperidin-4-ylmethyl]-5-chloro-2- methoxybenzamidine (13a, Y-36912), tegaserod maleate, BIMU 1 , BIMU 8, SL65.0155, RS 67333, RS 67506, SC 53116, cisapride, zacopride, HTF-919 and R-093877.

12. Use according to any one of claims 1 , 2, 4-10, wherein the composition is to be applied during or after anaesthesia to allow controlled recovery of spontaneous breathing.

13. Use according to any one of claims 1 , 2, 4-10, wherein the composition is to be co-administered with opioids to protect spontaneous breathing in pain patients.

14. Use according to any one of claims 1 to 10, for stabilizing spontaneous breathing when the patient is treated with drugs that dimish the cAMP production within the respiratory neurons such as drugs acting on dopamine D_{2ι 3 or} receptors, adenosine A1 receptors or β-adrenergic receptors.