# Photodegradation study of a new activator of the cystic fibrosis chloride channel, the 6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-07)

Jean-Christophe Olivier, Joachim Manceau, Cécile Marivingt-Mounir, Yvette Mettey, Jean-Michel Vierfond, William Couet

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Jean-Christophe Olivier, Joachim Manceau, Cécile Marivingt-Mounir, Yvette Mettey, Jean-Michel Vierfond, et al. Photodegradation study of a new activator of the cystic fibrosis chloride channel, the 6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-07). Journal of Pharmaceutical Sciences, 2002, 91 (2), pp.324-330. 10.1002/jps.10056. hal-05002616

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Photodegradation study of a new activator of the cystic fibrosis chloride channel, the 6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-07)

Jean-Christophe Olivier<fnref n="1" />,<fnref n="3" />, Joachim Manceau<fnref n="1" />, Cécile Marivingt-Mounir<fnref n="2" />,<fnref n="3" />, Yvette Mettey<fnref n="2" />,<fnref n="3" />, Jean-Michel Vierfond<fnref n="2" />,<fnref n="3" />, William Couet<fnref n="1" />,<fnref n="3" />*

Faculté de Médecine et Pharmacie
34, rue du Jardin des Plantes, B.P. 199
86005 Poitiers Cedex, France

Tel.: +33 (0) 5 49 45 43 79

Fax: +33 (0) 5 49 45 43 78

E-mail: William.Couet@univ-poitiers.fr

<footnote n="1">¹Laboratoire de Pharmacie Galénique et Biopharmacie</footnote>

<footnote n="2">²Laboratoire de Chimie Organique</footnote>

<footnote n="3">³Equipe Médicaments et Barrière Hématoencéphalique</footnote>

<footnote>*Corresponding author</footnote>
ABSTRACT

The photodegradation of 6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-07), a new activator of the transmembrane conductance regulator chloride channel, was studied in aqueous solutions exposed to artificial daylight (2300 Lux intensity). Various conditions of pH, concentration and temperature were used. MPB-07 concentration was determined at regular time intervals by reversed-phase HPLC. MPB-07 stability was also studied at pH 7.4 in dark. Results showed that in all the conditions tested MPB-07 underwent rapid photodegradation, apparently following first-order kinetics. Rate constants were dependent on the initial MPB-07 concentration, temperature and pH. At pH 7.4, and for concentrations from 1 to 125 µM, half-lives ranged from 0.681 ± 0.047 to 4.54 ± 0.28 h. Arrhenius plot was linear and activation energy was calculated to be 20.7 kJ.mol⁻¹. Analysis by chemical ionization-mass spectrometry showed that the chlorine atom of the MPB-07 molecule might be replaced by an OH group during the photodegradation process. In dark, MPB-07 in solutions at pH 7.4 was found to be stable over a 6 week period. In conclusion, MPB-07 is a highly photolabile molecule which should be carefully protected from light when used.
INTRODUCTION

Benzo[c]quinolizinium salts constitute a new class of potent activators of the transmembrane conductance regulator (CFTR) chloride channel, the defect of which is involved in the development of cystic fibrosis (CF) (Becq et al., 1999). Such CFTR activators may open new perspectives in the treatment of CF of particular interest, when gene therapy appears to be difficult to develop and probably to use in clinical practice (Davies et al., 1998). The 6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-07) (Figure 1) is the leading compound of this new class of CFTR activators. In order to define conditions for proper handling of MPB-07 solutions, basic stability studies were carried out. These included stability of MPB-07 solutions protected from or exposed to light. Photodegradation was evaluated at light spectrum and intensity recommended by the International Commission for Harmonization in "Photostability testing of new active substances and medicinal products (CPMP/ICH/279M/95)". MPB-07 photodegradation was studied in various conditions of pH, concentration and temperature and the chemical structure of the major photoproduct is proposed in this paper.
# MATERIALS AND METHODS

## Materials

The chemical 6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-07), Mw 266.13, batch n°14, was synthesized as previously described (Becq et al. 1999) by the Laboratory of organic chemistry (Faculty of Medicine and Pharmacy, Poitiers, France). Phosphate solutions were prepared with phosphoric acid 84% from Prolabo (France) and were adjusted to appropriate pH with sodium hydroxide or hydrochloride solutions 1 N from Prolabo. Solvents, including water, were all of analytical grade.

## Stability studies

### Stock Solution

Aliquots of MPB-07 chloride stock solution (10 mM) in purified water were stored at -25°C. All the solutions used in this work were prepared by appropriate dilutions of the stock solution.

### Stability studies of MPB-07 in dark

MPB-07 solutions, 1 to 125 µM, were prepared in 10 mM phosphate buffer, pH 7.4, in glass tubes closed with polyethylene stoppers and were stored at room temperature in an aluminium box to protect them from light. Samples were regularly collected over a period of 6 weeks to determine MPB-07 concentrations by HPLC.

### Photodegradation studies of MPB-07

MPB-07 solutions were prepared as follows, depending on the study:

**Photodegradation in unbuffered aqueous solution** – MPB-07 stock solution was diluted in (unbuffered) water (concentration: 125 µM, initial pH: 4.0).

**Effect of concentration** – 1 to 125µM MPB-07 solutions were prepared in 10mM phosphate buffer, pH 7.4, by dilution of the stock solution.

**Effect of the pH** – 5µM MPB-07 solutions were prepared in 10mM phosphate solutions at pH values ranging from 1 to 7.4. Phosphate salts were either used as a buffering agent or to keep solute composition constant.
*Effect of temperature* – 5µM MPB-07 solutions were prepared in 10 mM phosphate buffer, pH 7.4 and photodegradation kinetics were carried out at temperature ranging from 25 to 72°C.

*General procedure* – MPB-07 solutions (50 ml) were prepared in triplicate in 50 ml conic Pyrex glass vials (8 cm height, 3.5 cm neck diameter) in a dark room. The pH of the solutions was checked with a Knick pH-meter Portamess 751 (Bioblock Scientific, Illkirch, France) and adjusted to the suitable values with sodium hydroxide solution or hydrochloric acid 1N if necessary. Just before light exposure, 200 µl samples were collected from the three vials for accurate determination of the initial MPB-07 concentrations by HPLC. Vials were then stopped with a piece of Parafilm (5 X 5 cm) to prevent evaporation and were exposed to the artificial light emitted by a daylight fluorescent lamp (Mazda Fluor, 120 cm length) hung at 6 cm above the tops of the flasks. The intensity of light at the bottom of the vials was determined to be 2300 Lux using a Gossen Luxmeter (Panlux Electronic) in accordance to the recommendation CPMP/ICH/279M/95 of the International Commission for Harmonization. The bottoms of the vials bathed in 1 cm-deep thermostated water and the solutions were gently stirred with a magnetic spin bar. Samples (200 µl) were collected at appropriate time intervals for MPB-07 determination by HPLC to establish photodegradation kinetic curves. A determination of pH was performed after exposure.

## HPLC analysis

The chromatographic system consisted of a Shimadzu LC-10AT pump, a Waters 717 Plus Autosampler, a Waters 474 Scanning Fluorescence Detector and a Waters 746 Data Module for data recording and processing. MPB-07 was analyzed by isocratic reversed-phase HPLC using a Nucleosil C18 Column 5 µm Interchrom, 150 x 4 mm (Interchim, Montluçon, France), a mobile phase made of 49:51 (v/v) methanol:5 mM acetate buffer pH 4.8 and containing 0.06% (m/v) heptane-sulfonic acid, at a 1 ml/min flow rate. Excitation and emission wavelengths were respectively 390 nm and 460 nm, the gain being set at 100 and the attenuation at 256. Injection volume was 10 µl and MPB-07 concentration range for calibration was from 0.1 to 5 µM. MPB-07 retention time was 8.1 min.

## Mass Spectrometry Analysis

MPB-07 stock solution was diluted in (unbuffered) water (concentration: 125 µM, initial pH: 4.0). After an exposure time sufficient for at least 99% MPB-07 degradation (30 hours), water was evaporated off under vacuum at 30°C using a Büchi Rotavapor® (Bioblock Scientific, Illkirch,
France). The chemical ionization-mass spectroscopy (CI-MS) was performed using an Esquire LC spectrometer (Brucker, Bremen, Germany) with positive electrospray and methane as reagent gas.

## Data analysis

For the kinetics curve, the natural logarithms of concentrations were plotted versus time of light exposure. The rate constants k of degradation were calculated by linear regression analysis and half-lives ($t_{1/2}$) were deduced ($t_{1/2}$ = $\ln(2)/k$).

The activation energy Ea was derived from the slope of the natural logarithm of the mean rate constants $k_{mean}$ (n=3) plotted versus the inverse absolute temperature T (K), using the Arrhenius equation:

$$ \ln (k_{mean}) = \ln (A) - \frac{Ea}{RT} \text{ where R is the gas constant (8.31451 J mol$^{-1}$ K$^{-1}$)} $$
# RESULTS

## Stability in dark

In dark, no degradation was observed at pH 7.4 for concentrations ranging from 1 to 125µM over a period of 6 weeks (data not shown).

## Photodegradation studies

### Photodegradation in unbuffered water and CI-MS analysis of photoproducts

Exposed to light, MPB-07 (125 µM) underwent extensive degradation apparently following first-order kinetics, as shown by the linearity of the Ln-concentration versus exposure time curves (Figure 2). The degradation rate constant was calculated to be 0.159±0.006 h⁻¹ (t½ = 4.36 ±0.15h). During the 24 hour light exposure, the pH slightly decreased from 4.0 to 3.7 and the yellow MPB-07 solutions became slightly turbid. Figure 3 shows chromatograms of the MPB-07 solution before (A) and after a light exposure of 2 hours (B), 4 hours (C) and 10 hours (D). MPB-07 peak (peak1) diminished, while an additional peak (peak 2) of a fluorescent compound with a shorter retention time progressively increased (Figure 3, B, C and D). From 6 hours on, two more (minor) peaks of fluorescent compounds with very low surface areas were visible on chromatograms (peaks 3 and 4 in Figure 3, D). Peak 4 elution time corresponded to the void volume of the column. The CI-MS spectrum of MPB-07 exposed to light for 30 hours showed a major peak at m/z 212 [M+H]⁺, with no chlorine atom associated, as deduced from the absence of isotopic peak at m/z 214 [M+H]⁺ (Figure 4).

### Effect of pH, concentration and temperature on the photodegradation kinetics of MPB-07

For all the conditions tested the Ln-concentration versus exposure time curves were linear, which permitted to calculate the degradation rate constants (Tables 1-3). Photodegradation kinetics of MPB-07 were pH-dependent (Table 1). After a gradual increase from 0.728 to 2.94 h from pH 7.4 to pH 2.5, half-lives dramatically increased beyond pH 2.5, reaching 33.2 h at pH 1. In sodium phosphate solutions, chromatogram profiles (not shown) slightly differed from those obtained in pure water: at pH from 7.4 to 2.5, peaks 2 and 4 appeared gradually, but not peak 3, and at pH 1 and 2, only peak 2 was present. At constant pH (7.4), degradation rate constants were found to be dependent on the concentrations of MPB-07, half-lives increasing more than six times between 1µM and 125µM (Table 2). Degradation rates were also temperature-dependent, with an almost
three-times decrease of half-lives between 25 and 72°C (Table 3). The Arrhenius plot was linear (Figure 5) and the energy of activation was calculated to be 20.7 kJ.mol⁻¹.
DISCUSSION

Stability studies of newly synthesised pharmacologically active molecules are important for their appropriate handling. Insufficient care about the preservation or use conditions of such a potential drug may lead to non-reproducible results or erroneous conclusions due to the loss of active molecule or the presence of unidentified active or inactive degradation products. MPB-07 in solution was found to be stable in dark over a period of 6 weeks. The photostability testings were undertaken according to the ICH guidelines which recommend an accumulated light exposure of 6000 Wh/m² (corresponding to 1.2 MLux hours). By using an artificial daylight fluorescent lamp, with a light intensity of 2300 Lux, approximately 21 days are required to get the total cumulated exposure recommended. No such long exposure was needed for an extensive degradation of MPB-07. MPB-07 photodegradation was indeed a very rapid process, apparently following first-order kinetics (Figure 2). In most conditions tested (except below pH 2.5) a 50% degradation was reached in a few hours (Tables 1-3). In particular, at pH 7.4 and at a concentrations usual for in vitro studies (125 µM, Beck et al. 1999), half-live was 4.54±0.28 h (Table 2).

MPB-07 degradation was of first order, but rate constants were found to be dependent on the initial molecule concentration (Table 2). Similar concentration dependencies have already been reported for other light-sensitive molecules (Teraoka and Matsuda, 1993; Nuñez-Vergara et al., 1992; Tammilehto and Torniainen, 1989) and interpretations of such results state that at constant light intensity, but increasing initial concentration, the same amount of photolytic energy is initially shared by an increasing number of (light-absorbing) molecules, thus explaining the decrease of the degradation rate constants. This explanation is not fully satisfactory, because, in that case, degradation rates should gradually increase in the course of the degradation, due to the decreasing number of reactive molecules. Quenching of the photodegradation reaction or light absorption by photoproducts might therefore be involved. In the case of MPB-07, the main photoproduct absorbed in the same conditions as MPB-07 (Figure 3), which might compensate, in term of photolytic energy available per molecule, the progressive disappearance of the parent molecule.

MPB-07 photodegradation was also temperature dependent with an almost threefold increase in degradation rate constants between 25 and 72°C (Table 3). The value of the activation energy derived from the Arrhenius plot was calculated to be 20.7 kJ.mol⁻¹ at pH 7.4 and was close to values obtained for highly labile drugs: 23 to 28 kJ.mol⁻¹ for promethazine (Stavchansky et al. 1983), 30 kJ.mol⁻¹ for quinine (Christensen et al. 2000).
The studies performed in various conditions of pH showed that the ionization state of the molecule was important for its light-sensitivity (Table 1). MPB-07 is a quaternary ammonium with a pKa value of 3.65 (Marivingt-Mounir, 2000) which characterises the equilibrium between the acid cationic (enol) form and the free base non-ionic (keto) form (Figure 1). Therefore, the free base form seems to be more sensitive to light than the acid form, as shown by the much higher half-lives at pH below than above the pKa value. Chromatograms (Figure 2) obtained by reversed-phase HPLC with fluorimetric detection indicated that a major fluorescent photoproduct (peak 2) rapidly appeared. Additional, but minor, fluorescent products (peak 3 and 4) appeared later on. Analysis by CI-MS showed that after 99% degradation a major photoproduct was formed. This photoproduct did not contain chlorine and had a molecular weight of 211 which would suggest a formula of $C_{13}H_9NO_2$. This formula might result from the loss of chlorine from MPB-07 (MPB-07 free base formula is $C_{13}ClH_8NO$) and its replacement by OH arising from the aqueous solvent. The loss of a chlorine atom and its substitution to a hydroxyl group has been reported for chlorinated molecules, such as amiloride (Li et al. 1999), diclofenac (Encinas et al., 1998), hydrochlorothiazide and frusemide (Moore and Tamat, 1980). Such a photodechlorination might be the first step of MPB-07 photodegradation. The slight pH decrease of the MPB-07 unbuffered solution exposed to light supports this hypothesis, since the chlorine atoms released react with water to form hydrochloric acid (Moore and Tamat, 1980). Further degradation steps probably occurred, as shown by the presence of additional peaks on chromatograms and mass spectrum, and by the slight increase in turbidity of the solutions exposed to light. Other non-fluorescent photoproducts may also have been formed during the degradation process. These were not investigated in this study, as it would have been premature at this step of pharmaceutical development of this potential drug.

## CONCLUSION

MPB-07 was shown to be stable in dark conditions in solution for at least six weeks. However, it is rapidly degraded when exposed to light. Care should therefore be taken when handling MPB-07 to always protect samples from light, particularly in highly lighted cell culture equipment.
## REFERENCES

Becq F, Mettey Y, Gray MA, Galietta LJV, Dormer RL, Merten M, Métayé T, Chappe V, Marivingt-Mounir C, Zegarra-Moran O, Tarran R, Bulteau L, Dérand R, Pereira MMC, McPherson MA, Rogier C, Joffre M, Argent BE, Sarrouilhe D, Kammouni W, Figarella C, Verrier B, Gola M, Vierfond JM 1999. Development of substituted benzo[c]quinolizinium compounds as novel activators of the cystic fibrosis chloride channel. J Biol Chem 274: 27415-27425

Christensen KL, Chistensen JØ, Frokjaer S, Langballe P, Hansen LL 2000. Influence of temperature and storage time after light exposure on the quinine monohydrochloride chemical actinometric system. Eur J Pharm Sci 9: 317-321

Davies JC, Geddes DM, Alton WFW 1998. Prospects for gene therapy for cystic fibrosis. Mol Med Today 4: 292-9

Encinas S, Bosca, F and Moranda MA 1998. Phototoxicity associated with diclofenac: a photophysical, photochemical and photobiological study on the drug and its photoproducts. Chem Res Toxicol 11: 946-952

Li YNB, Moore DE, Tattam BN 1999. Photodegradation of amiloride in aqueous solution. Int J Pharm 183: 109-116.

Nuñez-Vergara LJ, Sunkel C, Squella JA 1992. Photodecomposition of a new 1,4-dihydropyridine: furnidipine. J Pharm Sci 83: 502-507

Marivingt-Mounir C 1997. Synthèse de sels de benzoquinolizinium activateurs de la protéine CFTR. Ph.D thesis, University of Poitiers, France.

Moore DE, Tamat SR 1980. Photosensitization by drugs: photolysis of some chlorine-containing drugs. J Pharm Pharmacol 32: 172-177.
Stavchansky S, Wallace JE, Wu P 1983. Thermal and photolytic degradations studies of promethazine hydrochloride: a stability-indicating assay. J Pharm Sci 72:546-548

Tammilehto S and Torniainen K 1989. Phtochemical stability of dothiepin in aqueous solutions. Int J Pharm 52:123-128

Teraoka R and Matsuda Y 1993. Stabilization-oriented preformulation study of photolabile menatetrone (vitamin K₂). Int J Pharm 93: 85-90
TABLES

<table><caption>Table 1: Effect of pH on the photodegradation kinetic parameters of MPB-07 in solution exposed to 2300 Lux artificial daylight. Conditions: 10 mM phosphate, 25°C, MPB-07 concentration: 5 µM, n=3</caption><thead><tr><th>pH</th><th>k ± SD (h<sup>-1</sup>)</th><th>Half-lives ± SD (h)</th></tr></thead><tbody><tr><td>1.0</td><td>0.0209 ± 0.0005</td><td>33.2 ± 0.1</td></tr><tr><td>2.0</td><td>0.0549 ± 0.0053</td><td>12.7 ± 1.3</td></tr><tr><td>2.5</td><td>0.236 ± 0.010</td><td>2.94 ± 0.12</td></tr><tr><td>3.0</td><td>0.462 ± 0.023</td><td>1.50 ± 0.08</td></tr><tr><td>4.0</td><td>0.571 ± 0.056</td><td>1.22 ± 0.13</td></tr><tr><td>5.0</td><td>0.822 ± 0.018</td><td>0.843 ± 0.019</td></tr><tr><td>6.0</td><td>0.691 ± 0.011</td><td>1.00 ± 0.02</td></tr><tr><td>7.4</td><td>0.962 ± 0.123</td><td>0.728 ± 0.087</td></tr></tbody></table>

<table><caption>Table 2: Effect of concentration on the photodegradation kinetic parameters of MPB-07 in solution exposed to 2300 Lux artificial daylight. Conditions: 10 mM phosphate buffer, pH 7.4, 25°C, n=3</caption><thead><tr><th>MPB-07 concentration (µM)</th><th>k (h<sup>-1</sup>) ± SD</th><th>Half-lives (h) ± SD</th></tr></thead><tbody><tr><td>1</td><td>1.02 ± 0.07</td><td>0.681 ± 0.047</td></tr><tr><td>5</td><td>0.962 ± 0.123</td><td>0.728 ± 0.087</td></tr><tr><td>25</td><td>0.582 ± 0.015</td><td>1.19 ± 0.03</td></tr><tr><td>125</td><td>0.153 ± 0.010</td><td>4.54 ± 0.28</td></tr></tbody></table>
<table><caption>Table 3: Effect of the temperature on the on the photodegradation kinetic parameters of MPB-07 in solution exposed to 2300 Lux artificial daylight. Conditions: 10 mM phosphate buffer, pH 7.4, MPB-07 concentration: 5 µM, n=3</caption><thead><tr><td>Temperature (°C)</td><td>k ± SD (h<sup>-1</sup>)</td><td>Half-lives (h) ± SD</td></tr></thead><tbody><tr><td>25</td><td>0.962 ± 0.123</td><td>0.728 ± 0.087</td></tr><tr><td>37</td><td>1.13 ± 0.07</td><td>0.617 ± 0.039</td></tr><tr><td>51</td><td>1.56 ± 0.08</td><td>0.444 ± 0.021</td></tr><tr><td>60</td><td>2.32 ± 0.07</td><td>0.299 ± 0.009</td></tr><tr><td>72</td><td>2.71 ± 0.04</td><td>0.256 ± 0.004</td></tr></tbody></table>
FIGURE LEGENDS

Figure 1: Chemical structure of MPB-07 chloride. MPB-07 is a quaternary ammonium in equilibrium between its acid enol form and its free base keto form.

Figure 2: First-order plots for the photodegradation of 125 µM MPB-07 unbuffered solution exposed to 2300 Lux artificial daylight (initial pH: 4.0, final pH 3.7).

Figure 3: HPLC analysis. Chromatogram of the 125 µM MPB-07 unbuffered solution (diluted 1/25 prior analysis) (A). Chromatograms of the same solution of MPB-07 after 2300 Lux artificial daylight exposure for 2 hours (B, 1/25 dilution), 4 hours (C, 1/25 dilution) and 10 hours (D, 1/5 dilution). Peak 1 ($R_t$ = 8.06 min) is MPB-07. Peaks 2 ($R_t$ = 3.95 min), 3 ($R_t$ = 3.05 min) and 4 ($R_t$ = 1.62 min) are fluorescent photoproducts. For HPLC conditions see text.

Figure 4: CI-MS spectrum of photoproducts obtained from 125 µM MPB-07 unbuffered aqueous solution exposed to 2300 Lux artificial daylight for 30 hours (99% degradation).

Figure 5: Arrhenius plot of $Ln k$ versus 1/T. Conditions: MPB-07 concentrations: 5 µM, 10 mM phosphate buffer pH 7.4, 2300 Lux artificial daylight exposure.
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