Misoprostol Dehydration Kinetics in Aqueous Solution in the Presence of Hydroxypropyl Methylcellulose DAVIDTOLEDO-VELASQUEZ*, HENRYT. GAUD*, AND KENNETH A.
CONNORS"
r
Received December 7 , 199q, from the 'School of Pharmacy, Universi of Wisconsin, Madison, WI 53706, and *G.D. Searle & Company, Accepted for publication pril 2, 1991. 4901 Searle P a h a y , Skokre, IL 60077. Abstract 0 Misoprostol (Searle),an El-type prostaglandin,is known to
be stabilized in the form of a solid dispersion with hydroxypropyl
methylcellulose (HPMC), yet no evidence has been found for specific intermolecular interactions. In the present study, the dehydration kinetics of this prostaglandinwere studied in aqueous solution in the absence and the presence of HPMC. The dispersion of the drug with HPMC, when dissolved in pH 7.66 aqueous solution, exerted a small but significant stabilizing effect. A possible interpretation of this kinetic result, together with lack of evidence for complex formationin both the solid and solution states, may be that HPMC exerts its stabilizing effect by physically limiting the access of water to the prostaglandinthrough an entanglement of the prostaglandin in the polymer environment, the diffusion of drug away from the polymer being slow on the time scale of the dehydration kinetics.
Scheme I 0
Prostaglandin E l (PGE,, 1) is a Phydroxyketone that is subject to facile dehydration, yielding the a,&unsaturated Mlsoprostol (2) ketone PGA,, which subsequently isomerizes to the resonance-stabilized PGB,. The essential chemistry is shown in Scheme 1.1-3 Variations in the PGE, structure can result in spectroscopy,gmicroscopy, and differential scanning calorimalterations in the pattern of pharmacological activity and in etry.10 No evidence of intermolecular interaction between 2 the chemical stability. Compound 2 (Misoprostol, Searle; and the HPMC was obtained. Beno and Gaud11 and Kararli (*)-methyl(1R ,2R ,3R)-3-hydroxy-2-[(E)-(4RS)-4-hydroxy-4-and Catalan012 have studied the dependence of the degradamethyl-l-octenyl]-5-oxocyclopentaneheptanoate), an El-type tion rate of 2 on the moisture content of the 2-HPMC solid prostaglandin in the methyl ester form, undergoes the reacdispersion. The stabilization of 2 by HPMC was postulated to tions in Scheme I and is the subject of this paper. be controlled by the physical state of the polymer, which in Several pharmaceutical approaches to the stabilization of turn is dependent on the effect of moisture content on the glass E-type prostaglandins have been reported. Hirayama et al.4 transition temperature. It was postulated that above the glass increased prostaglandin stability in solution by formation of transition temperature, the mobility of water and of 2 is inclusion complexes with methylated cyclodextrins. increased within the dispersion. Yalkowsky and Roseman5 described the use of triacetin as a At this time, our understanding of the stabilization effect of solvent to solubilize and stabilize prostaglandins. Vigo and the solid dispersion of 2 in HPMC is incomplete. There is no L a n p improved the stability of prostaglandins by incorpoevidence for specific molecular interactions between the rating them in liposomes. Fung and Cho7 studied the struccomponents, yet there is a profound effect on the rate. This tural requirements for coacervate formation between prostamay be some sort of physical protective effect by which the glandins and polyvinylpyrrolidone (PVP). polymer reduces access of water to the drug molecule. (Note, Sanvordekers developed solid dispersions of 2 with PVP and in this connection, that the decomposition reaction is a with hydroxypropyl methylcellulose (HPMC). Stability studdehydration, not a hydrolysis.) The present study was underies of neat 2 showed 50%degradation within 2 weeks a t 55 "C, taken to establish whether or not HPMC can exert its whereas the 2-HPMC solid dispersion retained >95% of the stabilizing effect in aqueous solution and to seek evidence for initial potency after 8 weeks a t 55 "C. The molecular basis of a possible interaction between 2 and HPMC in aqueous this stabilization effect has been investigated by several solution. techniques, including IR spectroscopy, solid-state "C NMR
Experimental Section
HO
OH Prostaglandin El (1)
OO22-3H9/92/02OO-0745$02.50/0 0 1992, American Pharmaceutical Association
MaterialeMisoprostol (2) was used as supplied by Searle Research and Development Division of G . D. Searle & Company. Hydroxypropyl methylcellulose (Pharmacoat, HPMC) was obtained from Shin-Etsu Chemical (Biddle Sawyer Company, New York). Ethyl alcohol and methylene chloride (J. T. Baker) were spectrophotometric grade. All other chemicals were analytical grade. Water was distilled and then purified with a Barnstead PCS water purification system. The dialysis membrane was a SpectrdPor 1 with a 60008000 molecularweight cutoff or a SpectrdPor 3 with a 3500 molecular weight cutoff (Spectrum Medical Industries, Los Angeles). Journal of Pharmaceutical Sciences I 145 Vol. 81, No. 2, February 1992
A p p a r a t u s T h e pH was measured with an Orion model 701A pH meter. Spectrophotometric measurements were made with a Cary 14, modified by OLIS (On-Line Instrument System, Jefferson, GA). Polymer characterization was carried out on TSK-PW (3000, 4000, 5000, and 6000) GPC columns from Toya Soda Manufacturing Company with a Waters 410 differential refractometer detector. Dialysis studies were performed with 10-mL dialysis cells (H40262) from Scienceware (Bel-Art Products, NJ). Procedures-Spectrophotornetric Analysis-The kinetics of Scheme I were followed spectrophotometrically with a wellestablished procedure.2.3For convenience, we designate PGE,, PGA,, and PGBl as E, A, and B, respectively. The analytical method is baaed on the following observations (a and b) and assumption (c): (a)E does not absorb at either 222 or 288 nm; (b)A does not absorb at 288 nm; (c) at t = 30, all of E initially present has been transformed to B.Then, at any time t, the observed solution absorbances (A) at 222 and 288 nm are shown in eqs 1 and 2, respectively:
(1) = A&
At t =
(2)
a:
G 2 2 = Ai22
(3)
A&=A&
(4)
Dividing eq 3 by eq 4 yields the following correction factor:
Thus, f is determined by means of eq 5. Using eq 5 in eq 1results in the following:
M stock solution of 2 in methylene chloride or mL) of 5.22 x ethanol were added, and the dispersion was stirred for 1 h. The stirring bar was removed from the flask and was rinsed with methylene chloride or ethanol. The flask was placed on a vacuum line, and the organic solvent was removed by flushing with nitrogen gas at 35 "C for 1 h. This provided the 2:HPMC solid dispersions for the kinetic studies. Characterization of Hydroxypropyl Methylcellulose-Gel permeation chromatography (GPC) was used to establish the molecular weight distribution of HPMC. Pullulan standards provided a molecular weight calibration curve. Solutions containing 0.2% (w/v) of pullulan standards (molecular weights from 0.58 x lo4to 10.0 x lo4) were prepared by adding water to the pullulan and allowing polymer hydration to take place for 12-24 h at 25 "C. One hundred milliliters of pullulan solution was injected into four columns (connected in series and containing TSK-PW 3000, 4000, 5000, and 6000 gel that was swollen in water and coupled in order of descending pore size). The refractomer settings were sensitivity 16 and scale factor 30. Two HPMC samples were analyzed by GPC using the same settings. One of these samples was HPMC without further purification; the other was HPMC purified by dialysis as follows. A 6% (w/v)HPMC solution was placed in a dialysis tubing with a 6000-8000 molecular weight cutoff and dialyzed into water for 5 days (the water being changed daily). ARer dialysis, the HPMC solution was lyophilized, and the purified HPMC was analyzed by GPC. This purified HPMC was used in the dialysis binding studies. Dialysis-A dialysis membrane having a 3500 molecular weight cutoff was used. Solid dispersions of 2 and HPMC were prepared from methylene chloride. For dialysis, the solid dispersions were dissolved in pH 4.3 (Na2HP0,:citric acid) buffer to yield a concentration of 2 of 2.6 x M, with HPMC concentrations ranging from 0 to 1g%. The solutions were dialyzed for 120 h at 15 "C on a 20 rpm shaker bath. (The equilibration time was established in a preliminary study.) The prostaglandin concentration was determined by the spectrophotometric method of Stehle and Oesterling.13 Solubility Determination-An amount of 2 in excess of its solubility was placed in each of several 5-mL ampules. To each ampule, 5.0 mL ofpH 4.0 buffer (0.003 M Na2HP0, and 0.002 M citric acid) was added, and the ampules were flushed with nitrogen gas, sealed, and rotated in a 25.0 "C water bath for 72 h. A portion of the aqueous phase was withdrawn for spectrophotometric analysis. 13
Results and Discussion was determined from solutions of E in pH The molar absorptivity ge8 10.6 g1ycine:NaOH buffers held a t 60 "C for 3-30 h, corresponding to t = (see eq 4). The molar absorptivity was found from observations at pH 1.1. (At this pH the concentration of A is not so rapidly depleted by the A+ B step as at high pH, and the spectra were shown to be insensitive to pH.) At any time t , the concentration of B is available from eq 2 and the known gee; subtracting this from the known initial concentration of E gives the concentration of A, and then is calculated with eq 6. In the kinetic studies, AiZ2and A& were measured. The measurement at 288 nm yields the concentration of B (CB), and the concentration of A (CAIwas calculated with eq 6. The concentration of E (CE) was obtained by difference. Kinetic Studies-The kinetics were studied in aqueous solution at 60 "C and pH values of 1.18, 1.26, 4.00, 7.66, and 10.66; the buffer compositions were those used by earlier workers.2-3 Reaction was initiated by adding a stock solution of 2 in methanol to the thermostated buffer such that the diluted concentrations were 5.2 x M for 2 and 2% (v/v) for methanol. Then, 3-mL portions were placed in 5-mL ampules, which were flushed with nitrogen gas and sealed. Ampules were withdrawn from the 60°C water bath at timed intervals, immediately frozen with liquid nitrogen, and stored at -60 "C until analyzed spectrophotometrically after thawing. Kinetic studies in the presence of HPMC (either added separately or as the solid dispersion with 2) were carried out at 60 "C in the pH 1.26 and 7.66 buffers. Preparation of Solid Dispersions-Three solid dispersions of 2:HPMC in the ratios 1:500,1:100, and 1 : l O were prepared as follows: 0.2 g of the polymer was slowly added, with stirring, to a volumetric flask containing either methylene chloride or ethanol. This mixture was stirred at low speed for 0.5 h to ensure polymer solvation and swelling. Appropriate volumes (either 0.2, 1.0, or 10.0
eZ2
146 / Journal of Pharmaceutical Sciences Val. 8 1, NO. 2, February 1992
Solubility-The purpose of the solubility work was to seek evidence of possible complex formation between 2 and HPMC in solution.14 Preliminary results were negative, so the study was not pursued. However, the solubility of 2 in pH 4.0 buffer a t 25.0 "C was determined to be 7.7 x lop4 M (eight determinations, standard deviation 0.2 x M). Dialysis-Possible interaction between 2 and HPMC was studied by dialysis using a 3500 molecular weight cutoff to ensure that the membrane would be impermeable to the HPMC. These studies were carried out a t pH 4.3 and 15 "C to ensure the integrity of 2 throughout the 120-h equilibration period. Percent concentrations of HPMC were 0, 0.00024, 0.00097, 0.0050, 0.010, 0.050, 0.416, 0.50, and 1.00. In no system was a significant difference found in the concentration of 2 between the donor and acceptor compartments after equilibration. Thus, under these conditions, dialysis yielded no evidence for interaction between 2 and HPMC in solution. Spectroscopic Observations-The correction factor f, defined in eq 5, was found to be 0.092 (seven observations, standard deviation 0.011). Molar absorptivities were as follows: = 2.4 ( 2 0.1) x lo4 Umol- cm and E $ ~= 9.6 ~ ( 2 0.1) x lo3 Umol- cm. These results are in good agreement with literature data on closely related compounds.2.3 The spectrophotometric method used in this work assigns all absorption to E, A, or B, and does not permit the detection of other substances, such as epimers. However, in later work in this laboratory, an HPLC method showed negligible interference from alternative reaction routes. Kinetics in Aqueous Solution-The kinetics were studied
in aqueous solution to the extent needed to establish the general pattern of reaction and to provide a reference for the studies in the presence of HPMC. Reaction 1 is shown in Scheme 11, which is a consecutive first-order kinetic scheme. The well-known integrated rate equations are as follows:
The dehydration rate constant, k,, was obtained from plots of In [El against time; these plots were satisfactorily linear. Estimates of the isomerization rate constant, k,, were obtained from the slope of plots of In [A1 against time in the long-time regime, where most of E had been converted to A. The k, value was then refined by nonlinear regression. Figure 1 shows an example of the final fit to the data for one set of conditions. Table I lists values of k, and k, at the several pH values. All of these results are very similar to those observed with other El-type compounds,2~3so further treatment is not required. Kinetics in the Presence of HPMC-The kinetics of dehydration and subsequent isomerization of 2 were studied at pH 1.26 and 7.66 and60 "C in aqueous solutions containing HPMC. In some of these studies. the initial concentration of 2 was held constant and the HPMC concentration was varied, whereas in other studies the concentration of 2 was varied with the HPMC concentration being held constant. In all of these cases, the kinetics followed the same pattern described for aqueous solutions in the preceding section. The solutions were prepared in two different ways. One method simply involved adding 2 and HPMC separately to the reaction solution; in the second method, 2 and HPMC were added together in the form of their solid dispersion. (It was found that the dispersions prepared from ethanol and from methylene chloride gave essentially identical results.) Table II records the dehydration rate constants (k,) measured in these studies. (The isomerization rate constants, k,, were essentially unchanged by the presence of HPMC, so they are not given in Table 11.)The following observations can be made: (1)at pH 1.26, the presence of HPMC has no significant effect on the stability of 2; (2)at pH 7.66, HPMC exerts a small but significant stabilizing effect; and (3)the stabilizing effect by HPMC a t pH 7.66 is greater when the solutes are added together as the solid dispersion than when they are added separately. (The difference in behavior at the two pH conditions is unexplained, but might be related to acid catalysis of the diffusional process noted below.) These observations must be considered together with the earlier resultssJ0 that failed to show evidence of specific 2:HPMC interactions in the solid dispersion and the results in the present paper that likewise yield no evidence for interaction in the solution phase. That is, there appears to be no significant extent of complex formation between 2 and HPMC, yet stabilization of 2 by
---.
kl A k2 E --+ Scheme I1
loo
80
0
25
75
50
100
125
tlmeRlr Flgure l-Concentration-time data (points) and curves (smooth lines) calculated with eqs 7-9 at pH 7.66and 60 "C. Table CDehydratlon (&,) and Isomerlzatlon (6) Rate Constanto
a 6o oCa,b
of
kl x
pH 1.18 1.26 4.00 7.66 10.66
Id,h-' 0.7 (0.01) 0.5 (0.02)
25.0 (0.9) 29.0 (0.7) 0.42(0.005) 7.9 (0.08) 1600 (500)
-
0.2 (0.002) 190 (2)
'Standard deviations in parentheses. In aqueous solution, in the absence of HPMC. Table ICEffect of Hydroxypropyl Methylcellulose on Dehydratlon Rate of 2 at 60 'Cm
HPMC:2 Ratio
k, x
Aqueous solution
pH 1.26
0
100 pH 7.66
0
10 100
500 lo00
29.0 (0.7)
7.9 (0.08)
-
lo2,h-' Mixtureb
Dispersion'
-
-
25.0 (0.9)
27.2(0.8)
7.0(0.1) 7.2(0.08)
-
5.5 (0.2)
4.5(0.3) 4.9(0.2) 4.8(0.2)
-
Standard deviations in parentheses. HPMC and 2 added separately. HPMC and 2 added as a pre-prepared solid dispersion. HPMC is observed, this effect being dramatic in the solid state and modest, though real, in solution. The present results, therefore, appear to lead toward the speculative conclusion that the stabilizing effect of HPMC is a physical protective effect, perhaps acting by decreasing the mobility of 2 and H,O and thus decreasing their encounter frequency. That the effect persists in solution is explicable if the dehydration rate is significantly greater than the diffusional rates controlling the molecular environment of 2 in the presence of HPMC; that is, it is necessary to postulate that Journal of Pharmaceutical Sciences I 147 Vol. 81, No. 2,February 1992
HPMC exerts its solution protective effect on the time scale of the chemical reaction as a consequence of a relatively slow disengagement of 2 from the HPMC environment.
References and Notes 1. Oesterling, T. 0.;Morozowich, W.; Roseman, T. J. J.Pharm. Sci. 1972,61,1861. 2. Monkhouse. D. C.: Van CamDen. A. J. J.Pharm. Sci. . . L.:. Aeuiar. 1973.62.576. 3. Lee,H.K.; Lambert, H. J.; Schowen, R. L. J. Pharm. Sci. 1984, 73,306. 4. Hirayama, F.; Kurihara, M.; Uekama, K. Chem. Phurm. Bull. 1986,34,5093. 5. Yalkowsky, S. H.; Roseman, T. J. J . Pharm. Sci. 1979,68,114. 6. Vigo, C.; Lang, J. Pharm. Res. 1988,5,S-71. 7. Fung, H.-L.; Cho, M. J. J. Pharm. Sci. 1978,67,971. 8. Sanvordeker, D. US. Patent 4 301 146,1981. I
'
148 I Journal of Pharmaceutical Sciences Vol. 8 1, No. 2, February 1992
9. Gaud, H.; Damascus, A.; Seul, C. Searle Research and Development Project Report AMX-861-2002, 1986. 10. Kararli, T.T.;Needham, T. E.; Seul, C. J.; Finnegan, P. M.; Hidvegi, M. I.; Hurlbut, J. Pharm. Res. 1990,7, 1181. 11. Beno, M.F.; Gaud, H. T. Proceedin s, 7th Annual Wisconsin
Update Conference, University of $isconsin, School of Pharmacy, Madison, 1988. 12. Kararli, T. T.;Catalano, T. Pharm. Res. 1990,7,1186. 13. Stehle, R. G.; Oesterling, T. 0. J. Pharm. Sci. 1977,66, 1590. 14. Connors, K. A. Binding Constants: the Measurement ofMolecular Complex Stability; Wiley-Interscience: New York, 1987;Chapter 8.
Acknowledgments Financial support of this work by G. D. Searle & Company is gratefully acknowledged.
CONNORS"
r
Received December 7 , 199q, from the 'School of Pharmacy, Universi of Wisconsin, Madison, WI 53706, and *G.D. Searle & Company, Accepted for publication pril 2, 1991. 4901 Searle P a h a y , Skokre, IL 60077. Abstract 0 Misoprostol (Searle),an El-type prostaglandin,is known to
be stabilized in the form of a solid dispersion with hydroxypropyl
methylcellulose (HPMC), yet no evidence has been found for specific intermolecular interactions. In the present study, the dehydration kinetics of this prostaglandinwere studied in aqueous solution in the absence and the presence of HPMC. The dispersion of the drug with HPMC, when dissolved in pH 7.66 aqueous solution, exerted a small but significant stabilizing effect. A possible interpretation of this kinetic result, together with lack of evidence for complex formationin both the solid and solution states, may be that HPMC exerts its stabilizing effect by physically limiting the access of water to the prostaglandinthrough an entanglement of the prostaglandin in the polymer environment, the diffusion of drug away from the polymer being slow on the time scale of the dehydration kinetics.
Scheme I 0
Prostaglandin E l (PGE,, 1) is a Phydroxyketone that is subject to facile dehydration, yielding the a,&unsaturated Mlsoprostol (2) ketone PGA,, which subsequently isomerizes to the resonance-stabilized PGB,. The essential chemistry is shown in Scheme 1.1-3 Variations in the PGE, structure can result in spectroscopy,gmicroscopy, and differential scanning calorimalterations in the pattern of pharmacological activity and in etry.10 No evidence of intermolecular interaction between 2 the chemical stability. Compound 2 (Misoprostol, Searle; and the HPMC was obtained. Beno and Gaud11 and Kararli (*)-methyl(1R ,2R ,3R)-3-hydroxy-2-[(E)-(4RS)-4-hydroxy-4-and Catalan012 have studied the dependence of the degradamethyl-l-octenyl]-5-oxocyclopentaneheptanoate), an El-type tion rate of 2 on the moisture content of the 2-HPMC solid prostaglandin in the methyl ester form, undergoes the reacdispersion. The stabilization of 2 by HPMC was postulated to tions in Scheme I and is the subject of this paper. be controlled by the physical state of the polymer, which in Several pharmaceutical approaches to the stabilization of turn is dependent on the effect of moisture content on the glass E-type prostaglandins have been reported. Hirayama et al.4 transition temperature. It was postulated that above the glass increased prostaglandin stability in solution by formation of transition temperature, the mobility of water and of 2 is inclusion complexes with methylated cyclodextrins. increased within the dispersion. Yalkowsky and Roseman5 described the use of triacetin as a At this time, our understanding of the stabilization effect of solvent to solubilize and stabilize prostaglandins. Vigo and the solid dispersion of 2 in HPMC is incomplete. There is no L a n p improved the stability of prostaglandins by incorpoevidence for specific molecular interactions between the rating them in liposomes. Fung and Cho7 studied the struccomponents, yet there is a profound effect on the rate. This tural requirements for coacervate formation between prostamay be some sort of physical protective effect by which the glandins and polyvinylpyrrolidone (PVP). polymer reduces access of water to the drug molecule. (Note, Sanvordekers developed solid dispersions of 2 with PVP and in this connection, that the decomposition reaction is a with hydroxypropyl methylcellulose (HPMC). Stability studdehydration, not a hydrolysis.) The present study was underies of neat 2 showed 50%degradation within 2 weeks a t 55 "C, taken to establish whether or not HPMC can exert its whereas the 2-HPMC solid dispersion retained >95% of the stabilizing effect in aqueous solution and to seek evidence for initial potency after 8 weeks a t 55 "C. The molecular basis of a possible interaction between 2 and HPMC in aqueous this stabilization effect has been investigated by several solution. techniques, including IR spectroscopy, solid-state "C NMR
Experimental Section
HO
OH Prostaglandin El (1)
OO22-3H9/92/02OO-0745$02.50/0 0 1992, American Pharmaceutical Association
MaterialeMisoprostol (2) was used as supplied by Searle Research and Development Division of G . D. Searle & Company. Hydroxypropyl methylcellulose (Pharmacoat, HPMC) was obtained from Shin-Etsu Chemical (Biddle Sawyer Company, New York). Ethyl alcohol and methylene chloride (J. T. Baker) were spectrophotometric grade. All other chemicals were analytical grade. Water was distilled and then purified with a Barnstead PCS water purification system. The dialysis membrane was a SpectrdPor 1 with a 60008000 molecularweight cutoff or a SpectrdPor 3 with a 3500 molecular weight cutoff (Spectrum Medical Industries, Los Angeles). Journal of Pharmaceutical Sciences I 145 Vol. 81, No. 2, February 1992
A p p a r a t u s T h e pH was measured with an Orion model 701A pH meter. Spectrophotometric measurements were made with a Cary 14, modified by OLIS (On-Line Instrument System, Jefferson, GA). Polymer characterization was carried out on TSK-PW (3000, 4000, 5000, and 6000) GPC columns from Toya Soda Manufacturing Company with a Waters 410 differential refractometer detector. Dialysis studies were performed with 10-mL dialysis cells (H40262) from Scienceware (Bel-Art Products, NJ). Procedures-Spectrophotornetric Analysis-The kinetics of Scheme I were followed spectrophotometrically with a wellestablished procedure.2.3For convenience, we designate PGE,, PGA,, and PGBl as E, A, and B, respectively. The analytical method is baaed on the following observations (a and b) and assumption (c): (a)E does not absorb at either 222 or 288 nm; (b)A does not absorb at 288 nm; (c) at t = 30, all of E initially present has been transformed to B.Then, at any time t, the observed solution absorbances (A) at 222 and 288 nm are shown in eqs 1 and 2, respectively:
(1) = A&
At t =
(2)
a:
G 2 2 = Ai22
(3)
A&=A&
(4)
Dividing eq 3 by eq 4 yields the following correction factor:
Thus, f is determined by means of eq 5. Using eq 5 in eq 1results in the following:
M stock solution of 2 in methylene chloride or mL) of 5.22 x ethanol were added, and the dispersion was stirred for 1 h. The stirring bar was removed from the flask and was rinsed with methylene chloride or ethanol. The flask was placed on a vacuum line, and the organic solvent was removed by flushing with nitrogen gas at 35 "C for 1 h. This provided the 2:HPMC solid dispersions for the kinetic studies. Characterization of Hydroxypropyl Methylcellulose-Gel permeation chromatography (GPC) was used to establish the molecular weight distribution of HPMC. Pullulan standards provided a molecular weight calibration curve. Solutions containing 0.2% (w/v) of pullulan standards (molecular weights from 0.58 x lo4to 10.0 x lo4) were prepared by adding water to the pullulan and allowing polymer hydration to take place for 12-24 h at 25 "C. One hundred milliliters of pullulan solution was injected into four columns (connected in series and containing TSK-PW 3000, 4000, 5000, and 6000 gel that was swollen in water and coupled in order of descending pore size). The refractomer settings were sensitivity 16 and scale factor 30. Two HPMC samples were analyzed by GPC using the same settings. One of these samples was HPMC without further purification; the other was HPMC purified by dialysis as follows. A 6% (w/v)HPMC solution was placed in a dialysis tubing with a 6000-8000 molecular weight cutoff and dialyzed into water for 5 days (the water being changed daily). ARer dialysis, the HPMC solution was lyophilized, and the purified HPMC was analyzed by GPC. This purified HPMC was used in the dialysis binding studies. Dialysis-A dialysis membrane having a 3500 molecular weight cutoff was used. Solid dispersions of 2 and HPMC were prepared from methylene chloride. For dialysis, the solid dispersions were dissolved in pH 4.3 (Na2HP0,:citric acid) buffer to yield a concentration of 2 of 2.6 x M, with HPMC concentrations ranging from 0 to 1g%. The solutions were dialyzed for 120 h at 15 "C on a 20 rpm shaker bath. (The equilibration time was established in a preliminary study.) The prostaglandin concentration was determined by the spectrophotometric method of Stehle and Oesterling.13 Solubility Determination-An amount of 2 in excess of its solubility was placed in each of several 5-mL ampules. To each ampule, 5.0 mL ofpH 4.0 buffer (0.003 M Na2HP0, and 0.002 M citric acid) was added, and the ampules were flushed with nitrogen gas, sealed, and rotated in a 25.0 "C water bath for 72 h. A portion of the aqueous phase was withdrawn for spectrophotometric analysis. 13
Results and Discussion was determined from solutions of E in pH The molar absorptivity ge8 10.6 g1ycine:NaOH buffers held a t 60 "C for 3-30 h, corresponding to t = (see eq 4). The molar absorptivity was found from observations at pH 1.1. (At this pH the concentration of A is not so rapidly depleted by the A+ B step as at high pH, and the spectra were shown to be insensitive to pH.) At any time t , the concentration of B is available from eq 2 and the known gee; subtracting this from the known initial concentration of E gives the concentration of A, and then is calculated with eq 6. In the kinetic studies, AiZ2and A& were measured. The measurement at 288 nm yields the concentration of B (CB), and the concentration of A (CAIwas calculated with eq 6. The concentration of E (CE) was obtained by difference. Kinetic Studies-The kinetics were studied in aqueous solution at 60 "C and pH values of 1.18, 1.26, 4.00, 7.66, and 10.66; the buffer compositions were those used by earlier workers.2-3 Reaction was initiated by adding a stock solution of 2 in methanol to the thermostated buffer such that the diluted concentrations were 5.2 x M for 2 and 2% (v/v) for methanol. Then, 3-mL portions were placed in 5-mL ampules, which were flushed with nitrogen gas and sealed. Ampules were withdrawn from the 60°C water bath at timed intervals, immediately frozen with liquid nitrogen, and stored at -60 "C until analyzed spectrophotometrically after thawing. Kinetic studies in the presence of HPMC (either added separately or as the solid dispersion with 2) were carried out at 60 "C in the pH 1.26 and 7.66 buffers. Preparation of Solid Dispersions-Three solid dispersions of 2:HPMC in the ratios 1:500,1:100, and 1 : l O were prepared as follows: 0.2 g of the polymer was slowly added, with stirring, to a volumetric flask containing either methylene chloride or ethanol. This mixture was stirred at low speed for 0.5 h to ensure polymer solvation and swelling. Appropriate volumes (either 0.2, 1.0, or 10.0
eZ2
146 / Journal of Pharmaceutical Sciences Val. 8 1, NO. 2, February 1992
Solubility-The purpose of the solubility work was to seek evidence of possible complex formation between 2 and HPMC in solution.14 Preliminary results were negative, so the study was not pursued. However, the solubility of 2 in pH 4.0 buffer a t 25.0 "C was determined to be 7.7 x lop4 M (eight determinations, standard deviation 0.2 x M). Dialysis-Possible interaction between 2 and HPMC was studied by dialysis using a 3500 molecular weight cutoff to ensure that the membrane would be impermeable to the HPMC. These studies were carried out a t pH 4.3 and 15 "C to ensure the integrity of 2 throughout the 120-h equilibration period. Percent concentrations of HPMC were 0, 0.00024, 0.00097, 0.0050, 0.010, 0.050, 0.416, 0.50, and 1.00. In no system was a significant difference found in the concentration of 2 between the donor and acceptor compartments after equilibration. Thus, under these conditions, dialysis yielded no evidence for interaction between 2 and HPMC in solution. Spectroscopic Observations-The correction factor f, defined in eq 5, was found to be 0.092 (seven observations, standard deviation 0.011). Molar absorptivities were as follows: = 2.4 ( 2 0.1) x lo4 Umol- cm and E $ ~= 9.6 ~ ( 2 0.1) x lo3 Umol- cm. These results are in good agreement with literature data on closely related compounds.2.3 The spectrophotometric method used in this work assigns all absorption to E, A, or B, and does not permit the detection of other substances, such as epimers. However, in later work in this laboratory, an HPLC method showed negligible interference from alternative reaction routes. Kinetics in Aqueous Solution-The kinetics were studied
in aqueous solution to the extent needed to establish the general pattern of reaction and to provide a reference for the studies in the presence of HPMC. Reaction 1 is shown in Scheme 11, which is a consecutive first-order kinetic scheme. The well-known integrated rate equations are as follows:
The dehydration rate constant, k,, was obtained from plots of In [El against time; these plots were satisfactorily linear. Estimates of the isomerization rate constant, k,, were obtained from the slope of plots of In [A1 against time in the long-time regime, where most of E had been converted to A. The k, value was then refined by nonlinear regression. Figure 1 shows an example of the final fit to the data for one set of conditions. Table I lists values of k, and k, at the several pH values. All of these results are very similar to those observed with other El-type compounds,2~3so further treatment is not required. Kinetics in the Presence of HPMC-The kinetics of dehydration and subsequent isomerization of 2 were studied at pH 1.26 and 7.66 and60 "C in aqueous solutions containing HPMC. In some of these studies. the initial concentration of 2 was held constant and the HPMC concentration was varied, whereas in other studies the concentration of 2 was varied with the HPMC concentration being held constant. In all of these cases, the kinetics followed the same pattern described for aqueous solutions in the preceding section. The solutions were prepared in two different ways. One method simply involved adding 2 and HPMC separately to the reaction solution; in the second method, 2 and HPMC were added together in the form of their solid dispersion. (It was found that the dispersions prepared from ethanol and from methylene chloride gave essentially identical results.) Table II records the dehydration rate constants (k,) measured in these studies. (The isomerization rate constants, k,, were essentially unchanged by the presence of HPMC, so they are not given in Table 11.)The following observations can be made: (1)at pH 1.26, the presence of HPMC has no significant effect on the stability of 2; (2)at pH 7.66, HPMC exerts a small but significant stabilizing effect; and (3)the stabilizing effect by HPMC a t pH 7.66 is greater when the solutes are added together as the solid dispersion than when they are added separately. (The difference in behavior at the two pH conditions is unexplained, but might be related to acid catalysis of the diffusional process noted below.) These observations must be considered together with the earlier resultssJ0 that failed to show evidence of specific 2:HPMC interactions in the solid dispersion and the results in the present paper that likewise yield no evidence for interaction in the solution phase. That is, there appears to be no significant extent of complex formation between 2 and HPMC, yet stabilization of 2 by
---.
kl A k2 E --+ Scheme I1
loo
80
0
25
75
50
100
125
tlmeRlr Flgure l-Concentration-time data (points) and curves (smooth lines) calculated with eqs 7-9 at pH 7.66and 60 "C. Table CDehydratlon (&,) and Isomerlzatlon (6) Rate Constanto
a 6o oCa,b
of
kl x
pH 1.18 1.26 4.00 7.66 10.66
Id,h-' 0.7 (0.01) 0.5 (0.02)
25.0 (0.9) 29.0 (0.7) 0.42(0.005) 7.9 (0.08) 1600 (500)
-
0.2 (0.002) 190 (2)
'Standard deviations in parentheses. In aqueous solution, in the absence of HPMC. Table ICEffect of Hydroxypropyl Methylcellulose on Dehydratlon Rate of 2 at 60 'Cm
HPMC:2 Ratio
k, x
Aqueous solution
pH 1.26
0
100 pH 7.66
0
10 100
500 lo00
29.0 (0.7)
7.9 (0.08)
-
lo2,h-' Mixtureb
Dispersion'
-
-
25.0 (0.9)
27.2(0.8)
7.0(0.1) 7.2(0.08)
-
5.5 (0.2)
4.5(0.3) 4.9(0.2) 4.8(0.2)
-
Standard deviations in parentheses. HPMC and 2 added separately. HPMC and 2 added as a pre-prepared solid dispersion. HPMC is observed, this effect being dramatic in the solid state and modest, though real, in solution. The present results, therefore, appear to lead toward the speculative conclusion that the stabilizing effect of HPMC is a physical protective effect, perhaps acting by decreasing the mobility of 2 and H,O and thus decreasing their encounter frequency. That the effect persists in solution is explicable if the dehydration rate is significantly greater than the diffusional rates controlling the molecular environment of 2 in the presence of HPMC; that is, it is necessary to postulate that Journal of Pharmaceutical Sciences I 147 Vol. 81, No. 2,February 1992
HPMC exerts its solution protective effect on the time scale of the chemical reaction as a consequence of a relatively slow disengagement of 2 from the HPMC environment.
References and Notes 1. Oesterling, T. 0.;Morozowich, W.; Roseman, T. J. J.Pharm. Sci. 1972,61,1861. 2. Monkhouse. D. C.: Van CamDen. A. J. J.Pharm. Sci. . . L.:. Aeuiar. 1973.62.576. 3. Lee,H.K.; Lambert, H. J.; Schowen, R. L. J. Pharm. Sci. 1984, 73,306. 4. Hirayama, F.; Kurihara, M.; Uekama, K. Chem. Phurm. Bull. 1986,34,5093. 5. Yalkowsky, S. H.; Roseman, T. J. J . Pharm. Sci. 1979,68,114. 6. Vigo, C.; Lang, J. Pharm. Res. 1988,5,S-71. 7. Fung, H.-L.; Cho, M. J. J. Pharm. Sci. 1978,67,971. 8. Sanvordeker, D. US. Patent 4 301 146,1981. I
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148 I Journal of Pharmaceutical Sciences Vol. 8 1, No. 2, February 1992
9. Gaud, H.; Damascus, A.; Seul, C. Searle Research and Development Project Report AMX-861-2002, 1986. 10. Kararli, T.T.;Needham, T. E.; Seul, C. J.; Finnegan, P. M.; Hidvegi, M. I.; Hurlbut, J. Pharm. Res. 1990,7, 1181. 11. Beno, M.F.; Gaud, H. T. Proceedin s, 7th Annual Wisconsin
Update Conference, University of $isconsin, School of Pharmacy, Madison, 1988. 12. Kararli, T. T.;Catalano, T. Pharm. Res. 1990,7,1186. 13. Stehle, R. G.; Oesterling, T. 0. J. Pharm. Sci. 1977,66, 1590. 14. Connors, K. A. Binding Constants: the Measurement ofMolecular Complex Stability; Wiley-Interscience: New York, 1987;Chapter 8.
Acknowledgments Financial support of this work by G. D. Searle & Company is gratefully acknowledged.
