- Email: [email protected]
Solubilization of Cyclosporine in Topical Ophthalmic Formulations: Preformulation Risk Assessment on a New Solid Form
Journal of Pharmaceutical Sciences 108 (2019) 3233-3239
Contents lists available at ScienceDirect
Journal of Pharmaceutical Sciences journal homepage: www.jpharmsci.org
Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Solubilization of Cyclosporine in Topical Ophthalmic Formulations: Preformulation Risk Assessment on a New Solid Form Ke Wu*, Anu Gore, Richard Graham, Richard Meller Pharmaceutical Development Department, Allergan Plc, Irvine, California 92612
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 January 2019 Revised 2 May 2019 Accepted 7 June 2019 Available online 20 June 2019
Owing to the discovery of a less soluble crystalline form (form 2) of cyclosporine (CsA), risks in solubility and physical stability of these formulations need to be revisited. This work focused on understanding the solubility behavior of various CsA forms in different media, including water, castor oil, and selected cosolvent micellar systems. In water, form 2 was approximately 8-9 times less soluble than form 1 (aka. tetragonal dihydrate). In neat nonaqueous solvent, for example, castor oil, form 3 (aka. orthorhombic hydrate) was found to have the lowest solubility and therefore the most stable form. In addition, the solubility-temperature relationship of CsA is complex and solvent-dependent. In aqueous vehicles, retrograde temperature dependence of solubility was observed in aqueous vehicles, that is, the solubility of CsA decreased with temperature, which was attributed to the effect of temperature on the strength of hydrogen bonding interactions; conversely, the solubility of CsA increased with temperature in nonaqueous solvents. In addition, the solubility of these CsA forms was very sensitive to temperature. Temperature-dependent form transformation was also observed in the media studied, with faster form conversion occurring at elevated temperatures. These studies provided key information to support the risk assessment for topical ophthalmic formulation development of CsA. © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
Keywords: solubilization preformulation ophthalmic drug delivery solubility formulation vehicle micelle(s) physical stability phase transformation(s)
Introduction Solid forms, such as polymorphs, solvates and hydrates, amorphous phases, and salts or cocrystals, may have great impact on the bioavailability and stability of drug products.1 Indeed, choosing a thermodynamically higher energy form may facilitate solubility enhancement; however, metastable forms tend to convert to more stable forms, thus posing great risks in physical stability. Owing to the significance of polymorphism of the active pharmaceutical ingredient (API) on quality and performance of drug product, solid form studies are an integral part of drug development program. However, unwanted form change still lurks during scaleup, stability studies, or when the API is in excursive conditions. The catastrophe of a late polymorph identification was exemplified by ritonavir, an antiretroviral drug of the protease inhibitor class used to treat HIV-1 infections.2 Two years after the launch of the Conflicts of interest: Ke Wu, Anu Gore, and Richard Graham are employees of Allergan and own Allergan stock. Richard Meller was an Allergan employee when the research was conducted. Allergan provided funding for the research. This article contains supplementary material available from the authors by request or via the Internet at https://doi.org/10.1016/j.xphs.2019.06.008. * Correspondence to: Ke Wu (Telephone: þ1-714-246-2372). E-mail address: [email protected] (K. Wu).
soft gel capsule product, several batches failed dissolution specifications. It turned out that a new thermodynamically stable form II precipitated out of solution. This new form was about less than 50% as soluble as the original form (form I). The manufacturer had to recall the original formulation from the market and reformulate it in an oily vehicle, resulting in huge loss in investment. Therefore, continuous efforts should be made in understanding the polymorphism and physicochemical properties of any emerging forms during formulation and process development. Solubility is a critical physicochemical property that directly impacts the bioavailability of a drug molecule.3 The solubility ratio between a metastable form and a lower energy form is a good indicator for evaluating the opportunity of bioavailability enhancement if supersaturation can be maintained. Thermodynamically higher energy forms, or metastable forms, are more soluble than their more stable counterparts. The solubility ratio between polymorphs is typically within twofold. In addition, the aqueous solubility ratios of anhydrate versus hydrate or between hydrates appear to be more variable and higher than the typical ratio for nonsolvated polymorphs.4 However, greater solubility ratio implies higher risk of product failure when the thermodynamically more stable form wins. In any regard, it is of great value to compare the solubility of relevant forms of the API. This is particularly important
https://doi.org/10.1016/j.xphs.2019.06.008 0022-3549/© 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
3234
K. Wu et al. / Journal of Pharmaceutical Sciences 108 (2019) 3233-3239
for ophthalmic formulation development because typically ophthalmic products are aqueous-based formulations and their physical stability is governed by the thermodynamically most stable form in the vehicle. For a topical ophthalmic product, form conversion could lead to loss of potency, as well as unexpected changes in viscosity, osmolality, surface tension, contact angle, pH, and particulate matter.5 Subsequently, this often cascades into assay variability which correlates to fluctuation in ocular bioavailability to tissues because of small window of absorption, and tolerability risk due to particles in the drug products. The discovery of medical use of cyclosporine, or cyclosporin A (CsA, Fig. 1), triggered a fundamental innovation in immunology. CsA works by acting on the immune system to reduce antigen sensitization and subsequent proliferation of immunocompetent cells via inhibition of calcineurin.6,7 When it comes to ophthalmic indications, CsA is well known for topical treatment of ocular surface disorders, particularly dry eye disease and chronic allergic keratoconjunctivitis.8,9 CsA is currently marketed as a topical ophthalmic emulsion10 because of the limitation of its aqueous solubility. Ideally, an aqueous solution is considered more desirable for ophthalmic dosing. Therefore, a variety of strategies have been tried to solubilize CsA, such as cosolvents, micelles, complexation, liposomes, and prodrugs, etc.11-19 Because CsA was often fully solubilized in those ophthalmic formulations, formulators seemed to overlook the characterization of its physical form, presumably believing that solid form did not seem to matter in this scenario. However, the discovery of a new crystalline hydrate (form 2)20 of CsA raises the concern whether the reported solubility values in the literature represent thermodynamic solubility or simply apparent solubility of a metastable form. The preliminary solubility study in water suggested that form 2 was the lower energy form than the 2 well-known hydrates of CsA, tetragonal dihydrate (form 1),21 and orthorhombic hydrate (form 3).22 Because the discovery of form 2 is relatively recent, it is conceivable that many of the formulations reported in the literature might be supersaturated systems prepared with those known CsA forms, which may lead to risk of precipitation and physical instability. Given the therapeutic significance of this molecule, preformulation efforts involving the solubility of CsA and physical stability of its formulations need to be revisited with urgency. In addition, CsA represents a special type of molecules that showed inverse solubility-temperature relationship in aqueous media, and temperature appeared to dramatically impact its solubility.23 The retrograde solubility behavior of CsA is in sharp contrast to most organic APIs whose solubility increases with temperature.24 Therefore, it is also critical to compare the effect of temperature on the solubility of different CsA forms. The objective of this study was to understand the solubility behavior of the newly discovered CsA form 2 in comparison to the other known solid forms in vehicles typically used for ophthalmic formulation development. This includes solubility behavior in water and oils as well as more complex aqueous formulation prototypes such as micellar systems containing 1 or more surfactant as solubilizer. Comparison in the solubility-temperature relationship among different crystalline forms was also made. These preformulation efforts were intended to support the development of commercially viable ophthalmic formulations of CsA.
into form 2 in a mixture of ethanol, polyethylene glycol 400 (PEG 400), and water (v/v/v ¼ 1: 5: 4) by seeded cooling from 65 C to 25 C. Final form 2 solids were obtained after filtration, washing with deionized water, and subsequent overnight air-drying. In addition, CsA form 3 were recrystallized by slowly cooling the solution of form 1 in polysorbate 80 (PS80) from 50 C to 25 C. The solids were washed and dried in the similar procedure for form 2 as described previously. In addition, amorphous CsA was purchased from Fujian Kerui Pharmaceutical Co., Ltd. (PR China). All the solvents used for the recrystallization studies were of analytical grade. All the reagents were used as received without further purification.
Solubility Determination Shake flask method was used to determine the solubility of CsA in various vehicles in duplicate. Briefly, excess amounts of CsA forms were placed into various vehicles. The solubility samples were rotated end-over-end at 8 rpm under predefined experimental conditions. For the solubility study in water, the equilibrium time was 72 h for form 1 and amorphous CsA, and 7 days for form 2 and form 3. For the studies in other aqueous vehicles, at each predetermined time points, the resulting suspensions in aqueous vehicles were filtered through 0.22 mm PTFE syringe filters. To minimize the drug adsorption by the filter, the first 1.0 mL of the filtrate was discarded, and the remaining portions were diluted at appropriate dilution factors for HPLC assay. For the studies in nonaqueous systems, centrifugation was used for solid-liquid separation because of the higher viscosity of the solvents. All the parts in contact with solubility samples during solid-liquid separation were pre-equilibrated at respective solubility equilibration temperatures. The diluent was water/acetonitrile mixture (v/v ¼ 1:1) unless otherwise specified.
Dissolution Study Dissolution of form 1 and form 2 were determined in 250 mL 0.5% sodium dodecyl sulfate solution at 37 ± 1 C in triplicate. A CE 7smart system (SOTAX, Westborough, MA) was used for the experiment. Twenty milligrams of CsA powders were predispersed in the dissolution media and loaded in 22.6 mm diameter cells with semisolid adapters. The pump delivery flow rate of the system was maintained at 16 mL/min. At predetermined time points, CsA dissolved in the dissolution medium was sampled and analyzed by HPLC.
Experimental Materials Tetragonal cyclosporine (form 1) was purchased from the AbbVie, Inc (North Chicago, IL). Preparation procedure of CsA form 2 was described elsewhere.20 Briefly, CsA form 1 was recrystallized
Figure 1. Molecular structure of cyclosporine.
K. Wu et al. / Journal of Pharmaceutical Sciences 108 (2019) 3233-3239
HPLC Assay The HPLC system consisted of an isocratic pump (Waters 600 pump, Milford, MA) and a UVeVis detector (Waters 2487 UV detector, Milford, MA) set at 210 nm. For the solubility study assay, the chromatographic column used was a Gemini C18 (5 mm in 4.6 mm 250 mm, Phenomenex, Torrance, CA). The mobile phase consisted of 90% acetonitrile and 10% DI water. The flow rate was set at 1.0 mL/min and the column was maintained at 50 C. For the dissolution sample assay, the column was a Halo C18 (2.7 mm, 3.0 mm 150 mm, Advanced Materials Technology, Wilmington, DE). The mobile phase was 30/70 (water/acetonitrile, v/v) containing 0.1% phosphoric acid. The flow rate was 0.5 mL/min, and the column temperature was 55 ± 2 C. Empower 2 software was used for chromatogram analysis. X-Ray Powder Diffractometry (XRPD) The XRPD diffractograms were recorded on a Rigaku (The Woodlands, TX) MiniFlex X-ray diffractometer (Cu Ka radiation, l ¼ 1.54 Å, 30 kV/15 mA). The instrument was calibrated with a silicon standard with a reference peak at 28.44 (2q). Powder samples were prepared in a low background Si holder by applying gentle pressure to keep the sample surface flat and level with the reference surface of the sample holder. Each sample was analyzed from 3 to 45 (2q) using a continuous scan of 2 or 10 (2q) per minute with a step size of 0.05 (2q). The samples were rotated during data collection to minimize preferred orientation. PDXL 2.4.2.0 software was used for the data analysis. Karl Fischer Water Content Titration Water content of the CsA samples (30-50 mg) was determined in dry methanol (HYDRANAL™, Fluka) by using a C30 coulometric titrator (Mettler-Toledo, Columbus, OH). AQUASTAR® water standard (1.0%) from EMD was used to verify the performance of the apparatus. Samples were analyzed in duplicate and average values were reported. Results and Discussion Form 2 was comprehensively characterized and found to be a new crystalline form of CsA.20 Figure 2 overlays the XRPD patterns of CsA solid forms of our interest in comparison to that of an amorphous CsA material. The characteristic peaks are about 6.9 , 7.8 , 8.6 , 9.4 , 10.8 , 15.9 (2q) for form 1, 5.3 , 5.9 , 14.4 , 15.5 ,
3235
17.5 (2q) for form 2, and 8.5 , 9.3 , 11.6 , 14.2 , 20.3 (2q) for form 3. Form 1 and form 3 were substantiated to be the tetragonal and orthorhombic forms reported in the literature.21,22 The representative peaks of these forms were used as references to monitor the physical stability of CsA in this work. In addition, water contents of form 1, 2, and 3 were determined to be 1.3%, 2.1%, and 1.3%, respectively.
Solubility of CsA in Water The solubility of CsA forms in water was measured at different temperatures. As Table 1 shows, form 2 was the least soluble form between 5 C and 40 C. This indicates that form 2 of CsA is the thermodynamically most stable form in water among these crystalline forms. Interestingly, form 1 was about 8 to tenfold more soluble than form 2, in contrast to the “2- to 5-fold” solubility ratio normally observed in polymorphs.4 This indicates that the driving force of physical instability is relatively high because a higher degree of supersaturation may be achieved with form 1. Next, the van’t Hoff plots (Fig. 3) show the effect of temperature on the solubility of CsA forms. This type of inverse temperaturesolubility profile has been observed with relatively large molecules with many H-bond acceptor groups such as clarithromycin, erythromycin, and ivermectin, etc.25 Temperature seemed to affect both the intra- and inter-molecular H-bonding and hence the conformation of CsA, as proposed by Loosli et al.21 Specifically, intramolecular H-bonds are stronger at higher temperature. This causes the CsA molecule to favor adopting an isolated conformation over a solvated one, which results in decrease in solubility. This solubility behavior of a molecule with complex intramolecular and intermolecular interactions was previously observed with some peptides or proteins.26,27 As the modalities of active pharmaceutical ingredients are trending toward peptides and proteins, it is not surprising that pharmaceutical scientists may encounter more examples such as CsA. Although the trend of the van’t Hoff plots in Figure 3 are generally compatible with the literature,23 there are some discrepancies in the linearity of the plots and solubility values. This may be attributed to (1) the difference in equilibrium time, (2) variability in the incubation temperature, and (3) unknown physical form of the CsA reported previously. Nonetheless, the solubility of CsA at 5 C was approximately 50 times higher than that at 40 C in this study for all these forms. Such a sensitive temperature effect on solubility is uncommon, and obviously this can cause issue in physical stability of aqueous drug products if temperature excursion occurs.
Figure 2. XRPD patterns of cyclosporine forms.
3236
K. Wu et al. / Journal of Pharmaceutical Sciences 108 (2019) 3233-3239
Table 1 Solubility of CsA Forms in Water at Various Temperatures Temp. ( C)
5±3 25 ± 1 40 ± 1 a
Concentration (mg/mL) Form 1a
Form 2
Form 3
Amorphous
399.1 ± 7.4 21.8 ± 0.7 8.8 ± 1.0
42.4 ± 3.7 2.5 ± 0.1 0.8 ± 0.0
43.5 ± 4.1 2.7 ± 0.3 1.4 ± 0.5
336.0 ± 1.3 22.3 ± 0.8 7.8 ± 2.8
Apparent solubility due to form conversion.
During the solubility studies, undissolved form 1 gradually lost its crystallinity in water and converted to an amorphous phase. Although it seemed counter-intuitive at first sight, this could be explained as the formation of a noncrystalline transition state when form 1 was on its way to converting to the more stable form, that is, form 2. This was manifested by the observation that form 1 gradually converted to form 2 at elevated temperatures. For example, form 2 was detected after 4 days of equilibration at 50 C. At 70 C, the conversion occurred just within several hours. At lower temperatures, the form conversion was slower despite of relatively higher solubility of CsA at these temperatures. For example, formation of form 2 was not observed after 7 months of equilibration at 25 C. For another instance, CsA form 1 did not convert to form 2 after at least 1 year at refrigerated temperatures. This temperaturedependent form conversion kinetics may be because the H-bonding interactions that form 2 assumed were not thermodynamically favored at lower temperatures. Therefore, the nucleation of form 2, the rate-limiting step, was retarded. Because of this form 1 / amorphous / form 2 conversion, the solubility of form 1 is not the equilibrium solubility in essence, but an apparent solubility. Nonetheless, the apparent value is still of practical value from risk assessment perspectives. In summary, the solubility studies of CsA in water revealed a number of risks in physical instability. First, aqueous formulations prepared with CsA form 1 may result in supersaturated systems with respect to form 2 which is the much less soluble form in the vehicles. Second, the dramatic effect of temperature on solubility implies that temperature excursion could cause large fluctuation in the solubility. In a worst-case scenario, once the low energy form, form 2, crystallizes, it may not readily redissolve into solution because of its lower solubility and dissolution rate. This could occur anywhere from drug product manufacturing and storage to postadministration. For example, as Figure 4 shows, it took
Figure 4. Dissolution time courses of cyclosporine form 1 and form 2 in 0.1% SDS solution at 37 C.
approximately 2 h to completely dissolve form 2 while only 30 min for form 1 in 0.1% sodium dodecyl sulfate solution. Finally, the solubility of CsA form 2 seems to be much more sensitive to temperature than that of other forms. Therefore, extra caution is needed when designing formulation of CsA in aqueous vehicles. Solubility of CsA in Castor Oil Figure 5 presents the concentration time courses of CsA forms in castor oil at 40 C. The profiles of form 1 and form 2 gradually approached to the same level as form 3, indicating that these 2 forms were transforming into form 3. This was confirmed by the XRPD analysis of the residual solids isolated from the solubility experiments (Figs. S1 and S2). By contrast, no form transformation was observed at 25 C over the same period, which is probably due to the slower kinetics at lower temperature. Form 3 is a lower hydrate compared to form 1 and form 221,22; it is the favorite form in a nonaqueous medium such as castor oil. More importantly, solvent environment also impacts the conformation of CsA.28-32 Because the polarity of castor oil is similar to chloroform, CsA might favor adopting a more “closed” state with intramolecular hydrogen bonds that could hide the H-bond donor and acceptor groups of CsA from solvent.21,33 In practice, risks exist with the dissolution and solubility behaviors of CsA forms in castor oil, especially during manufacturing process. For example, for lipid-based formulations, CsA drug substance (form 1) is usually first dissolved in the oil or lipid components under heating followed by dilution with other excipients. Should form conversion occur during the dissolution step, the resulting drug substance may not have adequate time to completely dissolve in castor oil, resulting in issues for subsequent steps. Therefore, solubility behavior of CsA form 3 as a function of temperature should also be thoroughly studied to support process development. Solubility of CsA in Polysorbate 80 Solutions
Figure 3. Van’t Hoff plots of CsA forms in water.
Nonionic surfactants are the preferred type of surface active agent used in ophthalmic formulations due to their advantages in compatibility, stability, and toxicity.34 In an earlier study, solubility enhancement capacity of PS80 on CsA was determined to be
K. Wu et al. / Journal of Pharmaceutical Sciences 108 (2019) 3233-3239
3237
Table 2 Compositions of Prototype Formulation Vehicles Components
Polysorbate 80 Cremophor RH40 Triton X-100 Octoxynol-40 Sodium chloride PVP-K90 Buffer pH WFI
Figure 5. Dissolution profiles of cyclosporine forms in castor oil at 40 C.
Vehicle #1
Vehicle #2
Vehicle #3
(%w/w)
(%w/w)
(%w/w)
1.0 0 0 0 0 0 NA NA q.s.
0 1 0.05 0 0.1 0.6 NA NA q.s.
0 1 0 0.05 0.05 0.3 Phosphate 7 q.s.
solubility behavior, physical stability is often less of a concern because high temperatures usually facilitate maintaining the supersaturation. Unfortunately, for APIs with inverse solubilitytemperature behavior such as CsA, higher temperatures may cause precipitation due to decreased solubility. Therefore, it is critical to explore the solubility-temperature relationship of an API during solution or colloidal formulation development.
between that of polysorbate 20 and Cremophor EL.35 Figure 6 shows the concentration-time profiles of form 1 and form 2 in 1.0% (w/v) PS80 solution (prototype vehicle 1, Table 2) over an extended period. At room temperature, the supersaturation ratio, that is, apparent solubility of form 1 over solubility of form 2, reached a maximum of about 7-8 folds after 1 week. Subsequently, the concentration of form 1 started declining and approached the level of form 2 after about 10 weeks. The concentration time course of CsA form 1 is typical of a form interconversion, as confirmed by XRPD (Fig. S3). In addition, as Figure 7 shows, temperature had a significant effect on the solubility of CsA in the PS80 solution, similar to the abovementioned observations in water. Because of such a dramatic impact of temperature on the conversion kinetics of CsA, it is not surprising to observe variability in solution concentration and particulate matter count depending on where the system was on the concentration time course. For liquid formulations containing a metastable API form, accelerated stability studies at higher temperatures are frequently conducted to assess risks in chemical stability. For APIs with normal
Solubility of CsA in Complex Micellar Systems
Figure 6. Dissolution profiles of cyclosporine form 1 and form 2 in prototype ophthalmic formulation vehicle #1 at RT (22 ± 2 C).
Figure 7. Dissolution profiles of CsA form 2 in prototype ophthalmic formulation vehicle (#1) at various temperatures.
Polymeric nanomicelles provide a promising strategy for ocular delivery of CsA because of their potential in enhancing bioavailability, improving tolerability, and reducing systemic side effects.36 Two mixed micellar systems used in this study were referred to as prototype vehicle 2 and vehicle 3 which were prepared according to the literature.37 The compositions of the vehicles are provided in Table 2. The examples cited in the published patents showed that the cyclosporine concentration in these formulations was 0.1%, which is higher than the target dose (0.09%) in the drug product. It is therefore of interest to evaluate whether these formulations with mixtures of surfactants and polymers would prevent the conversion of the metastable forms to the thermodynamically stable form 2. Figure 8 presents the concentration-time profile of CsA forms in prototype vehicle 2 at 25 C. The inverse solubility behavior of CsA in this vehicle was similar to that in water and aqueous solution containing 1% PS80. At 25 C, form conversion of either form 1 or amorphous CsA to form 2 occurred after about 2-3 weeks, as
3238
K. Wu et al. / Journal of Pharmaceutical Sciences 108 (2019) 3233-3239 Table 4 Solubility of CsA Form 1 and Form 2 in Prototype Vehicles CsA
Form 1
Form 2
Temp. ( C)
5±3 25 ± 1 40 ± 1 5±3 25 ± 1 40 ± 1
Solubility (mg/mL) Vehicle #1
Vehicle #2
Vehicle #3
ND 128 ND ND 127 ND
2432 275 83 781 183 87
2004 258 151 797 182 109
ND, not determined.
Figure 8. Concentration time course of cyclosporine forms in prototype ophthalmic formulation vehicle (#2) at 5 ± 3 C.
evidenced by the XRPD results (Fig. S4). At 40 C, however, similar conversion took place only after 1 day (Figs. S5 and S6). By contrast, the conversion to form 2 did not occur even after 8 months at 5 C (Figs. S7 and S8). This may be due to decreased supersaturation as driving force along with slower nucleation and growth at lower temperature. In addition, the form transformation rate of CsA seemed to be determined by a balance of solubility and strength of the solvent-solute interactions such as hydrogen bonding. The transformation rate is generally higher in stronger solubilizers, as was observed here in comparison with water. Solid form interconversion in presence of solvent typically involves 3 essential steps: (1) dissolution of metastable solid, (2) self-recognition of the molecular units to nucleate a more stable solid phase, and (3) growth of the stable phase.38 Because form 1 started losing crystallinity shortly after it was in contact with water even at lower temperatures, step 1 does not seem to be the rate-determining step. Therefore, higher temperatures appear to facilitate faster crystal growth in this case. Moreover, CsA is a relatively large
molecule and hence higher temperature is required to enable enough molecular mobility to initiate crystallization. In summary, the lower solubility of CsA form 2 in aqueous vehicle suggests great risks and challenges to its drug product development. Both type of solubilizer and temperature have great impacts on the solubility and stability of CsA in the vehicles studied through affecting the conformation of CsA and crystallization kinetics of CsA, as summarized in Tables 3 and 4. The effect of solubilizer was compatible with the observation by El Tayar et al.28 They found that both intra- and inter-molecular hydrogenbonding capacity of CsA was observed to be dependent on the solvent environment. Recently, Chennell et al.39 observed increased assay variability when they studied the physical stability of CsA formulations at 25 C. The authors raised the alarm of instability risk of the formulations at higher temperatures. Based on their studies, storage temperature was recommended to be no higher than 25 C. Therefore, understanding of the solubility behavior of form 2 is recommended to support the formulation development. Conclusion This work studied the solubilization of various crystalline cyclosporine forms in a number of vehicles that are relevant to ophthalmic formulation development and assessed the associated risks in physical stability. Because commercial sources of CsA only provide materials that are form 1, amorphous form, or their mixtures, most solubility studies reported in the literature probably utilized those forms. The newly discovered form of CsA, form 2, has
Table 3 Solid Form Summary in CsA Solubility Studies Starting Material
Vehicle
Temp. ( C)
Final Form
Time Point Conversion Observed
Duration of Study
Form 1
Water
5±3 25 ± 1 40 ± 1 5±3 25 ± 1 40 ± 1 5±3 25 ± 1 40 ± 1 25 ± 1 40 ± 1 25 ± 1 40 ± 1 25 ± 1 40 ± 1 22 ± 2 22 ± 2 5±3 25 ± 1 40 ± 1 5±3 25 ± 1 40 ± 1
Form 1 Partially crystalline form 1 Mainly amorphous with form 1 Form 2
NA
3d
No form change
7d
Form 3
No form change
7d
No form change Form 3 No form change Form 3 Form 3
NA Month 3 NA Month 3 No form change
3 mo
Form 2 Form 2 Partially form 1 and amorphous Form 2 Form 2 Form 2
Week 10 NA NA Week 2 Day 1 No form change
Form 2
Form 3
Form 1
Castor oil
Form 2 Form 3 Form 1 Form 2 Form 1
Form 2
Prototype Vehicle #1 Prototype Vehicle #2
3 mo 24 wk 8 mo
K. Wu et al. / Journal of Pharmaceutical Sciences 108 (2019) 3233-3239
aqueous solubility which is 8-9 times lower than form 1 or amorphous CsA, which suggested that the reported equilibrium solubility of CsA in aqueous vehicles with high water activity may be grossly overestimated. This may be further exacerbated by the inverse solubility behavior of CsA in aqueous systems which shows at least 4-fold difference in solubility over temperature range of 5 C40 C commonly encountered by commercial products. A solution drug product that is supersaturated with respect to form 2 may show crystallization over time and on exposure to elevated temperatures even for short intervals. This study was undertaken to thoroughly investigate the impact of this newly discovered form of form 2 on liquid-based formulations of CsA, focusing on compositions suitable for ophthalmic delivery. Results of this study show that form 2 was about 5 times less soluble than form 1 in these vehicles. Therefore, caution should be taken to guarantee the drug stability over the shelf life of the drug product and to withstand temperature excursions. The solubility behavior of form 2 in the vehicles is key to any commercially viable solution formulations. Acknowledgments The authors are grateful to the technical supports from Duan Su, Catherine Agbayani, Matthew McInnes, and Drs. Thomas Karami, Sumit Kumar, Yuwei Wang, and Yumna Shabaik, as well as support and advice from the management team of the Pharmaceutical Development Department at Allergan. References 1. Brittain H, ed. Polymorphism in Pharmaceutical Solids. New York, NY: Marcel Decker; 1999. 2. Chemburkar SR, Bauer J, Deming K, et al. Dealing with the impact of ritonavir polymorphs on the late stages of bulk drug process development. Org Process Res Dev. 2000;4(5):413-417. 3. Augustijns P, Brewster ME, eds. Solvent Systems and Their Selection in Pharmaceutics and Biopharmaceutics. 1st ed. New York, NY: Springer-Verlag; 2007. 4. Pudipeddi M, Serajuddin ATM. Trends in solubility of polymorphs. J Pharm Sci. 2005;94(5):929-939. 5. Rahman Z, Xu X, Katragadda U, Krishnaiah YSR, Yu L, Khan MA. Quality by design approach for understanding the critical quality attributes of cyclosporine ophthalmic emulsion. Mol Pharm. 2014;11(3):787-799. 6. Ptachcinski RJ, Venkataramanan R, Burckart GJ. Clinical pharmacokinetics of cyclosporin. Clin Pharmacokinet. 1986;11(2):107-132. 7. Freeman DJ. Pharmacology and pharmacokinetics of cyclosporine. Clin Biochem. 1991;24(1):9-14. 8. Pflugfelder SC. Antiinflammatory therapy for dry eye. Am J Ophthalmol. 2004;137(2):337-342. 9. Utine CA, Stern M, Akpek EK. Clinical review: topical ophthalmic use of cyclosporin A. Ocul Immunol Inflamm. 2010;18(5):352-361. 10. RESTASIS® (Cyclosporine Ophthalmic Emulsion 0.05%) [Packaging Insert]. Irvine, CA: Allergan, Inc.; 2012. 11. Aliabadi HM, Mahmud A, Sharifabadi AD, Lavasanifar A. Micelles of methoxy poly(ethylene oxide)-b-poly(ε-caprolactone) as vehicles for the solubilization and controlled delivery of cyclosporine A. J Control Release. 2005;104(2):301311. 12. Kang H, Cha K-H, Cho W, et al. Cyclosporine A micellar delivery system for dry eyes. Int J Nanomed. 2016;11:2921-2933. 13. Bas¸aran E, Demirel M, Sirmagül B, Yazan Y. Cyclosporine-A incorporated cationic solid lipid nanoparticles for ocular delivery. J Microencapsul. 2010;27(1):37-47.
3239
hannsdo ttir S, Jansook P, Stefa nsson E, Loftsson T. Development of a 14. Jo cyclodextrin-based aqueous cyclosporin A eye drop formulations. Int J Pharm. 2015;493(1-2):86-95. 15. Lallemand F, Furrer P, Felt-Baeyens O, et al. A novel water-soluble cyclosporine A prodrug: ocular tolerance and in vivo kinetics. Int J Pharm. 2005;295(1-2):714. 16. Dutescu RM, Panfil C, Merkel OM, Schrage N. Semifluorinated alkanes as a liquid drug carrier system for topical ocular drug delivery. Eur J Pharm Biopharm. 2014;88(1):123-128. 17. Malaekeh-Nikouei B, Nassirli H, Davies N. Enhancement of cyclosporine aqueous solubility using a- and hydroxypropyl b-cyclodextrin mixtures. J Incl Phenom Macrocycl Chem. 2007;59(3-4):245-250. 18. Di Tommaso C, Bourges JL, Valamanesh F, et al. Novel micelle carriers for cyclosporin A topical ocular delivery: in vivo cornea penetration, ocular distribution and efficacy studies. Eur J Pharm Biopharm. 2012;81(2):257264. 19. Luschmann C, Tessmar J, Schoeberl S, Straub O, Luschmann K, Goepferich A. Self-assembling colloidal system for the ocular administration of cyclosporine A. Cornea. 2014;33(1):77-81. 20. Wu K, Gore A, Graham R. A new hydrate of cyclosporine: strutural and physicochemical characterization. J Pharm Sci. 2018;107(12):3070-3079. 21. Loosli H-R, Kessler H, Oschkinat H, Weber H-P, Petcher TJ, Widmer A. Peptide conformations. Part 31. The conformation of cyclosporin A in the crystal and in solution. Helv Chim Acta. 1985;68(3):682-704. 22. Johnas SKJ, Dittrich B, Meents A, Messerschmidt M, Weckert EF. Charge-density study on cyclosporine A. Acta Crystallogr D Biol Crystallogr. 2009;65(3):284293. 23. Ismailos G, Reppas C, Dressman JB, Macheras P. Unusual solubility behaviour of cyclosporin A in aqueous media. J Pharm Pharmacol. 1991;43(4):287-289. 24. Grant DJW, Higuchi T, eds. Solubility Behavior of Organic Compounds. New York, NY: Wiley; 1990. 25. Avdeef A. Solubility temperature dependence predicted from 2D structure. ADMET DMPK. 2015;3(4):298-344. 26. Vekilov PG, Feeling-Taylor AR, Petsev DN, Galkin O, Nagel RL, Hirsch RE. Intermolecular interactions, nucleation, and thermodynamics of crystallization of hemoglobin C. Biophys J. 2002;83(2):1147-1156. 27. Christopher GK, Phipps AG, Gray RJ. Temperature-dependent solubility of selected proteins. J Cryst Growth. 1998;191(4):820-826. 28. El Tayar N, Mark AE, Vallat P, Brunne RM, Testa B, van Gunsteren WF. Solventdependent conformation and hydrogen-bonding capacity of cyclosporin A: evidence from partition coefficients and molecular dynamics simulations. J Med Chem. 1993;36(24):3757-3764. 29. Shaw RA, Mantsch HH, Chowdhry BZ. Solvent influence on the conformation of cyclosporin. An FT-IR study. Can J Chem. 1993;71(9):1334-1339. 30. Ko SY, Dalvit C. Conformation of cyclosporin A in polar solvents. Int J Pept Protein Res. 1992;40(5):380-382. 31. Wang CK, Swedberg JE, Harvey PJ, Kaas Q, Craik DJ. Conformational flexibility is a determinant of permeability for cyclosporin. J Phys Chem B. 2018;122(8): 2261-2276. €ck M, Seebach D, Thaler A. Complexation and 32. Kessler H, Gehrke M, Lautz J, Ko medium effects on the conformation of cyclosporin A studied by NMR spectroscopy and molecular dynamics calculations. Biochem Pharmacol. 1990;40(1): 169-173. €ck M, Wein T, Gehrke M. Reinvestigation of the conformation of 33. Kessler H, Ko cyclosporin A in chloroform. Helv Chim Acta. 1990;73(7):1818-1832. 34. Jiao J. Polyoxyethylated nonionic surfactants and their applications in topical ocular drug delivery. Adv Drug Deliv Rev. 2008;60(15):1663-1673. 35. Ran Y, Zhao L, Xu Q, Yalkowsky SH. Solubilization of cyclosporin A. AAPS PharmSciTech. 2001;2(1):E2. 36. Guo C, Zhang Y, Yang Z, et al. Nanomicelle formulation for topical delivery of cyclosporine A into the cornea: in vitro mechanism and in vivo permeation evaluation. Sci Rep. 2015;5:12968. 37. Mitra A, Weiss S. Ocular Technologies SARL, Assignee. Topical Aqueous Nanomicellar, Ophthalmic Solutions and Uses Thereof. US 8980839 B2. 2015. 38. Gu C-H, Young Jr V, Grant DJW. Polymorph screening: influence of solvents on the rate of solvent-mediated polymorphic transformation. J Pharm Sci. 2001;90(11):1878-1890. 39. Chennell P, Delaborde L, Wasiak M, et al. Stability of an ophthalmic micellar formulation of cyclosporine A in unopened multidose eyedroppers and in simulated use conditions. Eur J Pharm Sci. 2017;100:230-237.
Contents lists available at ScienceDirect
Journal of Pharmaceutical Sciences journal homepage: www.jpharmsci.org
Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Solubilization of Cyclosporine in Topical Ophthalmic Formulations: Preformulation Risk Assessment on a New Solid Form Ke Wu*, Anu Gore, Richard Graham, Richard Meller Pharmaceutical Development Department, Allergan Plc, Irvine, California 92612
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 January 2019 Revised 2 May 2019 Accepted 7 June 2019 Available online 20 June 2019
Owing to the discovery of a less soluble crystalline form (form 2) of cyclosporine (CsA), risks in solubility and physical stability of these formulations need to be revisited. This work focused on understanding the solubility behavior of various CsA forms in different media, including water, castor oil, and selected cosolvent micellar systems. In water, form 2 was approximately 8-9 times less soluble than form 1 (aka. tetragonal dihydrate). In neat nonaqueous solvent, for example, castor oil, form 3 (aka. orthorhombic hydrate) was found to have the lowest solubility and therefore the most stable form. In addition, the solubility-temperature relationship of CsA is complex and solvent-dependent. In aqueous vehicles, retrograde temperature dependence of solubility was observed in aqueous vehicles, that is, the solubility of CsA decreased with temperature, which was attributed to the effect of temperature on the strength of hydrogen bonding interactions; conversely, the solubility of CsA increased with temperature in nonaqueous solvents. In addition, the solubility of these CsA forms was very sensitive to temperature. Temperature-dependent form transformation was also observed in the media studied, with faster form conversion occurring at elevated temperatures. These studies provided key information to support the risk assessment for topical ophthalmic formulation development of CsA. © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
Keywords: solubilization preformulation ophthalmic drug delivery solubility formulation vehicle micelle(s) physical stability phase transformation(s)
Introduction Solid forms, such as polymorphs, solvates and hydrates, amorphous phases, and salts or cocrystals, may have great impact on the bioavailability and stability of drug products.1 Indeed, choosing a thermodynamically higher energy form may facilitate solubility enhancement; however, metastable forms tend to convert to more stable forms, thus posing great risks in physical stability. Owing to the significance of polymorphism of the active pharmaceutical ingredient (API) on quality and performance of drug product, solid form studies are an integral part of drug development program. However, unwanted form change still lurks during scaleup, stability studies, or when the API is in excursive conditions. The catastrophe of a late polymorph identification was exemplified by ritonavir, an antiretroviral drug of the protease inhibitor class used to treat HIV-1 infections.2 Two years after the launch of the Conflicts of interest: Ke Wu, Anu Gore, and Richard Graham are employees of Allergan and own Allergan stock. Richard Meller was an Allergan employee when the research was conducted. Allergan provided funding for the research. This article contains supplementary material available from the authors by request or via the Internet at https://doi.org/10.1016/j.xphs.2019.06.008. * Correspondence to: Ke Wu (Telephone: þ1-714-246-2372). E-mail address: [email protected] (K. Wu).
soft gel capsule product, several batches failed dissolution specifications. It turned out that a new thermodynamically stable form II precipitated out of solution. This new form was about less than 50% as soluble as the original form (form I). The manufacturer had to recall the original formulation from the market and reformulate it in an oily vehicle, resulting in huge loss in investment. Therefore, continuous efforts should be made in understanding the polymorphism and physicochemical properties of any emerging forms during formulation and process development. Solubility is a critical physicochemical property that directly impacts the bioavailability of a drug molecule.3 The solubility ratio between a metastable form and a lower energy form is a good indicator for evaluating the opportunity of bioavailability enhancement if supersaturation can be maintained. Thermodynamically higher energy forms, or metastable forms, are more soluble than their more stable counterparts. The solubility ratio between polymorphs is typically within twofold. In addition, the aqueous solubility ratios of anhydrate versus hydrate or between hydrates appear to be more variable and higher than the typical ratio for nonsolvated polymorphs.4 However, greater solubility ratio implies higher risk of product failure when the thermodynamically more stable form wins. In any regard, it is of great value to compare the solubility of relevant forms of the API. This is particularly important
https://doi.org/10.1016/j.xphs.2019.06.008 0022-3549/© 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
3234
K. Wu et al. / Journal of Pharmaceutical Sciences 108 (2019) 3233-3239
for ophthalmic formulation development because typically ophthalmic products are aqueous-based formulations and their physical stability is governed by the thermodynamically most stable form in the vehicle. For a topical ophthalmic product, form conversion could lead to loss of potency, as well as unexpected changes in viscosity, osmolality, surface tension, contact angle, pH, and particulate matter.5 Subsequently, this often cascades into assay variability which correlates to fluctuation in ocular bioavailability to tissues because of small window of absorption, and tolerability risk due to particles in the drug products. The discovery of medical use of cyclosporine, or cyclosporin A (CsA, Fig. 1), triggered a fundamental innovation in immunology. CsA works by acting on the immune system to reduce antigen sensitization and subsequent proliferation of immunocompetent cells via inhibition of calcineurin.6,7 When it comes to ophthalmic indications, CsA is well known for topical treatment of ocular surface disorders, particularly dry eye disease and chronic allergic keratoconjunctivitis.8,9 CsA is currently marketed as a topical ophthalmic emulsion10 because of the limitation of its aqueous solubility. Ideally, an aqueous solution is considered more desirable for ophthalmic dosing. Therefore, a variety of strategies have been tried to solubilize CsA, such as cosolvents, micelles, complexation, liposomes, and prodrugs, etc.11-19 Because CsA was often fully solubilized in those ophthalmic formulations, formulators seemed to overlook the characterization of its physical form, presumably believing that solid form did not seem to matter in this scenario. However, the discovery of a new crystalline hydrate (form 2)20 of CsA raises the concern whether the reported solubility values in the literature represent thermodynamic solubility or simply apparent solubility of a metastable form. The preliminary solubility study in water suggested that form 2 was the lower energy form than the 2 well-known hydrates of CsA, tetragonal dihydrate (form 1),21 and orthorhombic hydrate (form 3).22 Because the discovery of form 2 is relatively recent, it is conceivable that many of the formulations reported in the literature might be supersaturated systems prepared with those known CsA forms, which may lead to risk of precipitation and physical instability. Given the therapeutic significance of this molecule, preformulation efforts involving the solubility of CsA and physical stability of its formulations need to be revisited with urgency. In addition, CsA represents a special type of molecules that showed inverse solubility-temperature relationship in aqueous media, and temperature appeared to dramatically impact its solubility.23 The retrograde solubility behavior of CsA is in sharp contrast to most organic APIs whose solubility increases with temperature.24 Therefore, it is also critical to compare the effect of temperature on the solubility of different CsA forms. The objective of this study was to understand the solubility behavior of the newly discovered CsA form 2 in comparison to the other known solid forms in vehicles typically used for ophthalmic formulation development. This includes solubility behavior in water and oils as well as more complex aqueous formulation prototypes such as micellar systems containing 1 or more surfactant as solubilizer. Comparison in the solubility-temperature relationship among different crystalline forms was also made. These preformulation efforts were intended to support the development of commercially viable ophthalmic formulations of CsA.
into form 2 in a mixture of ethanol, polyethylene glycol 400 (PEG 400), and water (v/v/v ¼ 1: 5: 4) by seeded cooling from 65 C to 25 C. Final form 2 solids were obtained after filtration, washing with deionized water, and subsequent overnight air-drying. In addition, CsA form 3 were recrystallized by slowly cooling the solution of form 1 in polysorbate 80 (PS80) from 50 C to 25 C. The solids were washed and dried in the similar procedure for form 2 as described previously. In addition, amorphous CsA was purchased from Fujian Kerui Pharmaceutical Co., Ltd. (PR China). All the solvents used for the recrystallization studies were of analytical grade. All the reagents were used as received without further purification.
Solubility Determination Shake flask method was used to determine the solubility of CsA in various vehicles in duplicate. Briefly, excess amounts of CsA forms were placed into various vehicles. The solubility samples were rotated end-over-end at 8 rpm under predefined experimental conditions. For the solubility study in water, the equilibrium time was 72 h for form 1 and amorphous CsA, and 7 days for form 2 and form 3. For the studies in other aqueous vehicles, at each predetermined time points, the resulting suspensions in aqueous vehicles were filtered through 0.22 mm PTFE syringe filters. To minimize the drug adsorption by the filter, the first 1.0 mL of the filtrate was discarded, and the remaining portions were diluted at appropriate dilution factors for HPLC assay. For the studies in nonaqueous systems, centrifugation was used for solid-liquid separation because of the higher viscosity of the solvents. All the parts in contact with solubility samples during solid-liquid separation were pre-equilibrated at respective solubility equilibration temperatures. The diluent was water/acetonitrile mixture (v/v ¼ 1:1) unless otherwise specified.
Dissolution Study Dissolution of form 1 and form 2 were determined in 250 mL 0.5% sodium dodecyl sulfate solution at 37 ± 1 C in triplicate. A CE 7smart system (SOTAX, Westborough, MA) was used for the experiment. Twenty milligrams of CsA powders were predispersed in the dissolution media and loaded in 22.6 mm diameter cells with semisolid adapters. The pump delivery flow rate of the system was maintained at 16 mL/min. At predetermined time points, CsA dissolved in the dissolution medium was sampled and analyzed by HPLC.
Experimental Materials Tetragonal cyclosporine (form 1) was purchased from the AbbVie, Inc (North Chicago, IL). Preparation procedure of CsA form 2 was described elsewhere.20 Briefly, CsA form 1 was recrystallized
Figure 1. Molecular structure of cyclosporine.
K. Wu et al. / Journal of Pharmaceutical Sciences 108 (2019) 3233-3239
HPLC Assay The HPLC system consisted of an isocratic pump (Waters 600 pump, Milford, MA) and a UVeVis detector (Waters 2487 UV detector, Milford, MA) set at 210 nm. For the solubility study assay, the chromatographic column used was a Gemini C18 (5 mm in 4.6 mm 250 mm, Phenomenex, Torrance, CA). The mobile phase consisted of 90% acetonitrile and 10% DI water. The flow rate was set at 1.0 mL/min and the column was maintained at 50 C. For the dissolution sample assay, the column was a Halo C18 (2.7 mm, 3.0 mm 150 mm, Advanced Materials Technology, Wilmington, DE). The mobile phase was 30/70 (water/acetonitrile, v/v) containing 0.1% phosphoric acid. The flow rate was 0.5 mL/min, and the column temperature was 55 ± 2 C. Empower 2 software was used for chromatogram analysis. X-Ray Powder Diffractometry (XRPD) The XRPD diffractograms were recorded on a Rigaku (The Woodlands, TX) MiniFlex X-ray diffractometer (Cu Ka radiation, l ¼ 1.54 Å, 30 kV/15 mA). The instrument was calibrated with a silicon standard with a reference peak at 28.44 (2q). Powder samples were prepared in a low background Si holder by applying gentle pressure to keep the sample surface flat and level with the reference surface of the sample holder. Each sample was analyzed from 3 to 45 (2q) using a continuous scan of 2 or 10 (2q) per minute with a step size of 0.05 (2q). The samples were rotated during data collection to minimize preferred orientation. PDXL 2.4.2.0 software was used for the data analysis. Karl Fischer Water Content Titration Water content of the CsA samples (30-50 mg) was determined in dry methanol (HYDRANAL™, Fluka) by using a C30 coulometric titrator (Mettler-Toledo, Columbus, OH). AQUASTAR® water standard (1.0%) from EMD was used to verify the performance of the apparatus. Samples were analyzed in duplicate and average values were reported. Results and Discussion Form 2 was comprehensively characterized and found to be a new crystalline form of CsA.20 Figure 2 overlays the XRPD patterns of CsA solid forms of our interest in comparison to that of an amorphous CsA material. The characteristic peaks are about 6.9 , 7.8 , 8.6 , 9.4 , 10.8 , 15.9 (2q) for form 1, 5.3 , 5.9 , 14.4 , 15.5 ,
3235
17.5 (2q) for form 2, and 8.5 , 9.3 , 11.6 , 14.2 , 20.3 (2q) for form 3. Form 1 and form 3 were substantiated to be the tetragonal and orthorhombic forms reported in the literature.21,22 The representative peaks of these forms were used as references to monitor the physical stability of CsA in this work. In addition, water contents of form 1, 2, and 3 were determined to be 1.3%, 2.1%, and 1.3%, respectively.
Solubility of CsA in Water The solubility of CsA forms in water was measured at different temperatures. As Table 1 shows, form 2 was the least soluble form between 5 C and 40 C. This indicates that form 2 of CsA is the thermodynamically most stable form in water among these crystalline forms. Interestingly, form 1 was about 8 to tenfold more soluble than form 2, in contrast to the “2- to 5-fold” solubility ratio normally observed in polymorphs.4 This indicates that the driving force of physical instability is relatively high because a higher degree of supersaturation may be achieved with form 1. Next, the van’t Hoff plots (Fig. 3) show the effect of temperature on the solubility of CsA forms. This type of inverse temperaturesolubility profile has been observed with relatively large molecules with many H-bond acceptor groups such as clarithromycin, erythromycin, and ivermectin, etc.25 Temperature seemed to affect both the intra- and inter-molecular H-bonding and hence the conformation of CsA, as proposed by Loosli et al.21 Specifically, intramolecular H-bonds are stronger at higher temperature. This causes the CsA molecule to favor adopting an isolated conformation over a solvated one, which results in decrease in solubility. This solubility behavior of a molecule with complex intramolecular and intermolecular interactions was previously observed with some peptides or proteins.26,27 As the modalities of active pharmaceutical ingredients are trending toward peptides and proteins, it is not surprising that pharmaceutical scientists may encounter more examples such as CsA. Although the trend of the van’t Hoff plots in Figure 3 are generally compatible with the literature,23 there are some discrepancies in the linearity of the plots and solubility values. This may be attributed to (1) the difference in equilibrium time, (2) variability in the incubation temperature, and (3) unknown physical form of the CsA reported previously. Nonetheless, the solubility of CsA at 5 C was approximately 50 times higher than that at 40 C in this study for all these forms. Such a sensitive temperature effect on solubility is uncommon, and obviously this can cause issue in physical stability of aqueous drug products if temperature excursion occurs.
Figure 2. XRPD patterns of cyclosporine forms.
3236
K. Wu et al. / Journal of Pharmaceutical Sciences 108 (2019) 3233-3239
Table 1 Solubility of CsA Forms in Water at Various Temperatures Temp. ( C)
5±3 25 ± 1 40 ± 1 a
Concentration (mg/mL) Form 1a
Form 2
Form 3
Amorphous
399.1 ± 7.4 21.8 ± 0.7 8.8 ± 1.0
42.4 ± 3.7 2.5 ± 0.1 0.8 ± 0.0
43.5 ± 4.1 2.7 ± 0.3 1.4 ± 0.5
336.0 ± 1.3 22.3 ± 0.8 7.8 ± 2.8
Apparent solubility due to form conversion.
During the solubility studies, undissolved form 1 gradually lost its crystallinity in water and converted to an amorphous phase. Although it seemed counter-intuitive at first sight, this could be explained as the formation of a noncrystalline transition state when form 1 was on its way to converting to the more stable form, that is, form 2. This was manifested by the observation that form 1 gradually converted to form 2 at elevated temperatures. For example, form 2 was detected after 4 days of equilibration at 50 C. At 70 C, the conversion occurred just within several hours. At lower temperatures, the form conversion was slower despite of relatively higher solubility of CsA at these temperatures. For example, formation of form 2 was not observed after 7 months of equilibration at 25 C. For another instance, CsA form 1 did not convert to form 2 after at least 1 year at refrigerated temperatures. This temperaturedependent form conversion kinetics may be because the H-bonding interactions that form 2 assumed were not thermodynamically favored at lower temperatures. Therefore, the nucleation of form 2, the rate-limiting step, was retarded. Because of this form 1 / amorphous / form 2 conversion, the solubility of form 1 is not the equilibrium solubility in essence, but an apparent solubility. Nonetheless, the apparent value is still of practical value from risk assessment perspectives. In summary, the solubility studies of CsA in water revealed a number of risks in physical instability. First, aqueous formulations prepared with CsA form 1 may result in supersaturated systems with respect to form 2 which is the much less soluble form in the vehicles. Second, the dramatic effect of temperature on solubility implies that temperature excursion could cause large fluctuation in the solubility. In a worst-case scenario, once the low energy form, form 2, crystallizes, it may not readily redissolve into solution because of its lower solubility and dissolution rate. This could occur anywhere from drug product manufacturing and storage to postadministration. For example, as Figure 4 shows, it took
Figure 4. Dissolution time courses of cyclosporine form 1 and form 2 in 0.1% SDS solution at 37 C.
approximately 2 h to completely dissolve form 2 while only 30 min for form 1 in 0.1% sodium dodecyl sulfate solution. Finally, the solubility of CsA form 2 seems to be much more sensitive to temperature than that of other forms. Therefore, extra caution is needed when designing formulation of CsA in aqueous vehicles. Solubility of CsA in Castor Oil Figure 5 presents the concentration time courses of CsA forms in castor oil at 40 C. The profiles of form 1 and form 2 gradually approached to the same level as form 3, indicating that these 2 forms were transforming into form 3. This was confirmed by the XRPD analysis of the residual solids isolated from the solubility experiments (Figs. S1 and S2). By contrast, no form transformation was observed at 25 C over the same period, which is probably due to the slower kinetics at lower temperature. Form 3 is a lower hydrate compared to form 1 and form 221,22; it is the favorite form in a nonaqueous medium such as castor oil. More importantly, solvent environment also impacts the conformation of CsA.28-32 Because the polarity of castor oil is similar to chloroform, CsA might favor adopting a more “closed” state with intramolecular hydrogen bonds that could hide the H-bond donor and acceptor groups of CsA from solvent.21,33 In practice, risks exist with the dissolution and solubility behaviors of CsA forms in castor oil, especially during manufacturing process. For example, for lipid-based formulations, CsA drug substance (form 1) is usually first dissolved in the oil or lipid components under heating followed by dilution with other excipients. Should form conversion occur during the dissolution step, the resulting drug substance may not have adequate time to completely dissolve in castor oil, resulting in issues for subsequent steps. Therefore, solubility behavior of CsA form 3 as a function of temperature should also be thoroughly studied to support process development. Solubility of CsA in Polysorbate 80 Solutions
Figure 3. Van’t Hoff plots of CsA forms in water.
Nonionic surfactants are the preferred type of surface active agent used in ophthalmic formulations due to their advantages in compatibility, stability, and toxicity.34 In an earlier study, solubility enhancement capacity of PS80 on CsA was determined to be
K. Wu et al. / Journal of Pharmaceutical Sciences 108 (2019) 3233-3239
3237
Table 2 Compositions of Prototype Formulation Vehicles Components
Polysorbate 80 Cremophor RH40 Triton X-100 Octoxynol-40 Sodium chloride PVP-K90 Buffer pH WFI
Figure 5. Dissolution profiles of cyclosporine forms in castor oil at 40 C.
Vehicle #1
Vehicle #2
Vehicle #3
(%w/w)
(%w/w)
(%w/w)
1.0 0 0 0 0 0 NA NA q.s.
0 1 0.05 0 0.1 0.6 NA NA q.s.
0 1 0 0.05 0.05 0.3 Phosphate 7 q.s.
solubility behavior, physical stability is often less of a concern because high temperatures usually facilitate maintaining the supersaturation. Unfortunately, for APIs with inverse solubilitytemperature behavior such as CsA, higher temperatures may cause precipitation due to decreased solubility. Therefore, it is critical to explore the solubility-temperature relationship of an API during solution or colloidal formulation development.
between that of polysorbate 20 and Cremophor EL.35 Figure 6 shows the concentration-time profiles of form 1 and form 2 in 1.0% (w/v) PS80 solution (prototype vehicle 1, Table 2) over an extended period. At room temperature, the supersaturation ratio, that is, apparent solubility of form 1 over solubility of form 2, reached a maximum of about 7-8 folds after 1 week. Subsequently, the concentration of form 1 started declining and approached the level of form 2 after about 10 weeks. The concentration time course of CsA form 1 is typical of a form interconversion, as confirmed by XRPD (Fig. S3). In addition, as Figure 7 shows, temperature had a significant effect on the solubility of CsA in the PS80 solution, similar to the abovementioned observations in water. Because of such a dramatic impact of temperature on the conversion kinetics of CsA, it is not surprising to observe variability in solution concentration and particulate matter count depending on where the system was on the concentration time course. For liquid formulations containing a metastable API form, accelerated stability studies at higher temperatures are frequently conducted to assess risks in chemical stability. For APIs with normal
Solubility of CsA in Complex Micellar Systems
Figure 6. Dissolution profiles of cyclosporine form 1 and form 2 in prototype ophthalmic formulation vehicle #1 at RT (22 ± 2 C).
Figure 7. Dissolution profiles of CsA form 2 in prototype ophthalmic formulation vehicle (#1) at various temperatures.
Polymeric nanomicelles provide a promising strategy for ocular delivery of CsA because of their potential in enhancing bioavailability, improving tolerability, and reducing systemic side effects.36 Two mixed micellar systems used in this study were referred to as prototype vehicle 2 and vehicle 3 which were prepared according to the literature.37 The compositions of the vehicles are provided in Table 2. The examples cited in the published patents showed that the cyclosporine concentration in these formulations was 0.1%, which is higher than the target dose (0.09%) in the drug product. It is therefore of interest to evaluate whether these formulations with mixtures of surfactants and polymers would prevent the conversion of the metastable forms to the thermodynamically stable form 2. Figure 8 presents the concentration-time profile of CsA forms in prototype vehicle 2 at 25 C. The inverse solubility behavior of CsA in this vehicle was similar to that in water and aqueous solution containing 1% PS80. At 25 C, form conversion of either form 1 or amorphous CsA to form 2 occurred after about 2-3 weeks, as
3238
K. Wu et al. / Journal of Pharmaceutical Sciences 108 (2019) 3233-3239 Table 4 Solubility of CsA Form 1 and Form 2 in Prototype Vehicles CsA
Form 1
Form 2
Temp. ( C)
5±3 25 ± 1 40 ± 1 5±3 25 ± 1 40 ± 1
Solubility (mg/mL) Vehicle #1
Vehicle #2
Vehicle #3
ND 128 ND ND 127 ND
2432 275 83 781 183 87
2004 258 151 797 182 109
ND, not determined.
Figure 8. Concentration time course of cyclosporine forms in prototype ophthalmic formulation vehicle (#2) at 5 ± 3 C.
evidenced by the XRPD results (Fig. S4). At 40 C, however, similar conversion took place only after 1 day (Figs. S5 and S6). By contrast, the conversion to form 2 did not occur even after 8 months at 5 C (Figs. S7 and S8). This may be due to decreased supersaturation as driving force along with slower nucleation and growth at lower temperature. In addition, the form transformation rate of CsA seemed to be determined by a balance of solubility and strength of the solvent-solute interactions such as hydrogen bonding. The transformation rate is generally higher in stronger solubilizers, as was observed here in comparison with water. Solid form interconversion in presence of solvent typically involves 3 essential steps: (1) dissolution of metastable solid, (2) self-recognition of the molecular units to nucleate a more stable solid phase, and (3) growth of the stable phase.38 Because form 1 started losing crystallinity shortly after it was in contact with water even at lower temperatures, step 1 does not seem to be the rate-determining step. Therefore, higher temperatures appear to facilitate faster crystal growth in this case. Moreover, CsA is a relatively large
molecule and hence higher temperature is required to enable enough molecular mobility to initiate crystallization. In summary, the lower solubility of CsA form 2 in aqueous vehicle suggests great risks and challenges to its drug product development. Both type of solubilizer and temperature have great impacts on the solubility and stability of CsA in the vehicles studied through affecting the conformation of CsA and crystallization kinetics of CsA, as summarized in Tables 3 and 4. The effect of solubilizer was compatible with the observation by El Tayar et al.28 They found that both intra- and inter-molecular hydrogenbonding capacity of CsA was observed to be dependent on the solvent environment. Recently, Chennell et al.39 observed increased assay variability when they studied the physical stability of CsA formulations at 25 C. The authors raised the alarm of instability risk of the formulations at higher temperatures. Based on their studies, storage temperature was recommended to be no higher than 25 C. Therefore, understanding of the solubility behavior of form 2 is recommended to support the formulation development. Conclusion This work studied the solubilization of various crystalline cyclosporine forms in a number of vehicles that are relevant to ophthalmic formulation development and assessed the associated risks in physical stability. Because commercial sources of CsA only provide materials that are form 1, amorphous form, or their mixtures, most solubility studies reported in the literature probably utilized those forms. The newly discovered form of CsA, form 2, has
Table 3 Solid Form Summary in CsA Solubility Studies Starting Material
Vehicle
Temp. ( C)
Final Form
Time Point Conversion Observed
Duration of Study
Form 1
Water
5±3 25 ± 1 40 ± 1 5±3 25 ± 1 40 ± 1 5±3 25 ± 1 40 ± 1 25 ± 1 40 ± 1 25 ± 1 40 ± 1 25 ± 1 40 ± 1 22 ± 2 22 ± 2 5±3 25 ± 1 40 ± 1 5±3 25 ± 1 40 ± 1
Form 1 Partially crystalline form 1 Mainly amorphous with form 1 Form 2
NA
3d
No form change
7d
Form 3
No form change
7d
No form change Form 3 No form change Form 3 Form 3
NA Month 3 NA Month 3 No form change
3 mo
Form 2 Form 2 Partially form 1 and amorphous Form 2 Form 2 Form 2
Week 10 NA NA Week 2 Day 1 No form change
Form 2
Form 3
Form 1
Castor oil
Form 2 Form 3 Form 1 Form 2 Form 1
Form 2
Prototype Vehicle #1 Prototype Vehicle #2
3 mo 24 wk 8 mo
K. Wu et al. / Journal of Pharmaceutical Sciences 108 (2019) 3233-3239
aqueous solubility which is 8-9 times lower than form 1 or amorphous CsA, which suggested that the reported equilibrium solubility of CsA in aqueous vehicles with high water activity may be grossly overestimated. This may be further exacerbated by the inverse solubility behavior of CsA in aqueous systems which shows at least 4-fold difference in solubility over temperature range of 5 C40 C commonly encountered by commercial products. A solution drug product that is supersaturated with respect to form 2 may show crystallization over time and on exposure to elevated temperatures even for short intervals. This study was undertaken to thoroughly investigate the impact of this newly discovered form of form 2 on liquid-based formulations of CsA, focusing on compositions suitable for ophthalmic delivery. Results of this study show that form 2 was about 5 times less soluble than form 1 in these vehicles. Therefore, caution should be taken to guarantee the drug stability over the shelf life of the drug product and to withstand temperature excursions. The solubility behavior of form 2 in the vehicles is key to any commercially viable solution formulations. Acknowledgments The authors are grateful to the technical supports from Duan Su, Catherine Agbayani, Matthew McInnes, and Drs. Thomas Karami, Sumit Kumar, Yuwei Wang, and Yumna Shabaik, as well as support and advice from the management team of the Pharmaceutical Development Department at Allergan. References 1. Brittain H, ed. Polymorphism in Pharmaceutical Solids. New York, NY: Marcel Decker; 1999. 2. Chemburkar SR, Bauer J, Deming K, et al. Dealing with the impact of ritonavir polymorphs on the late stages of bulk drug process development. Org Process Res Dev. 2000;4(5):413-417. 3. Augustijns P, Brewster ME, eds. Solvent Systems and Their Selection in Pharmaceutics and Biopharmaceutics. 1st ed. New York, NY: Springer-Verlag; 2007. 4. Pudipeddi M, Serajuddin ATM. Trends in solubility of polymorphs. J Pharm Sci. 2005;94(5):929-939. 5. Rahman Z, Xu X, Katragadda U, Krishnaiah YSR, Yu L, Khan MA. Quality by design approach for understanding the critical quality attributes of cyclosporine ophthalmic emulsion. Mol Pharm. 2014;11(3):787-799. 6. Ptachcinski RJ, Venkataramanan R, Burckart GJ. Clinical pharmacokinetics of cyclosporin. Clin Pharmacokinet. 1986;11(2):107-132. 7. Freeman DJ. Pharmacology and pharmacokinetics of cyclosporine. Clin Biochem. 1991;24(1):9-14. 8. Pflugfelder SC. Antiinflammatory therapy for dry eye. Am J Ophthalmol. 2004;137(2):337-342. 9. Utine CA, Stern M, Akpek EK. Clinical review: topical ophthalmic use of cyclosporin A. Ocul Immunol Inflamm. 2010;18(5):352-361. 10. RESTASIS® (Cyclosporine Ophthalmic Emulsion 0.05%) [Packaging Insert]. Irvine, CA: Allergan, Inc.; 2012. 11. Aliabadi HM, Mahmud A, Sharifabadi AD, Lavasanifar A. Micelles of methoxy poly(ethylene oxide)-b-poly(ε-caprolactone) as vehicles for the solubilization and controlled delivery of cyclosporine A. J Control Release. 2005;104(2):301311. 12. Kang H, Cha K-H, Cho W, et al. Cyclosporine A micellar delivery system for dry eyes. Int J Nanomed. 2016;11:2921-2933. 13. Bas¸aran E, Demirel M, Sirmagül B, Yazan Y. Cyclosporine-A incorporated cationic solid lipid nanoparticles for ocular delivery. J Microencapsul. 2010;27(1):37-47.
3239
hannsdo ttir S, Jansook P, Stefa nsson E, Loftsson T. Development of a 14. Jo cyclodextrin-based aqueous cyclosporin A eye drop formulations. Int J Pharm. 2015;493(1-2):86-95. 15. Lallemand F, Furrer P, Felt-Baeyens O, et al. A novel water-soluble cyclosporine A prodrug: ocular tolerance and in vivo kinetics. Int J Pharm. 2005;295(1-2):714. 16. Dutescu RM, Panfil C, Merkel OM, Schrage N. Semifluorinated alkanes as a liquid drug carrier system for topical ocular drug delivery. Eur J Pharm Biopharm. 2014;88(1):123-128. 17. Malaekeh-Nikouei B, Nassirli H, Davies N. Enhancement of cyclosporine aqueous solubility using a- and hydroxypropyl b-cyclodextrin mixtures. J Incl Phenom Macrocycl Chem. 2007;59(3-4):245-250. 18. Di Tommaso C, Bourges JL, Valamanesh F, et al. Novel micelle carriers for cyclosporin A topical ocular delivery: in vivo cornea penetration, ocular distribution and efficacy studies. Eur J Pharm Biopharm. 2012;81(2):257264. 19. Luschmann C, Tessmar J, Schoeberl S, Straub O, Luschmann K, Goepferich A. Self-assembling colloidal system for the ocular administration of cyclosporine A. Cornea. 2014;33(1):77-81. 20. Wu K, Gore A, Graham R. A new hydrate of cyclosporine: strutural and physicochemical characterization. J Pharm Sci. 2018;107(12):3070-3079. 21. Loosli H-R, Kessler H, Oschkinat H, Weber H-P, Petcher TJ, Widmer A. Peptide conformations. Part 31. The conformation of cyclosporin A in the crystal and in solution. Helv Chim Acta. 1985;68(3):682-704. 22. Johnas SKJ, Dittrich B, Meents A, Messerschmidt M, Weckert EF. Charge-density study on cyclosporine A. Acta Crystallogr D Biol Crystallogr. 2009;65(3):284293. 23. Ismailos G, Reppas C, Dressman JB, Macheras P. Unusual solubility behaviour of cyclosporin A in aqueous media. J Pharm Pharmacol. 1991;43(4):287-289. 24. Grant DJW, Higuchi T, eds. Solubility Behavior of Organic Compounds. New York, NY: Wiley; 1990. 25. Avdeef A. Solubility temperature dependence predicted from 2D structure. ADMET DMPK. 2015;3(4):298-344. 26. Vekilov PG, Feeling-Taylor AR, Petsev DN, Galkin O, Nagel RL, Hirsch RE. Intermolecular interactions, nucleation, and thermodynamics of crystallization of hemoglobin C. Biophys J. 2002;83(2):1147-1156. 27. Christopher GK, Phipps AG, Gray RJ. Temperature-dependent solubility of selected proteins. J Cryst Growth. 1998;191(4):820-826. 28. El Tayar N, Mark AE, Vallat P, Brunne RM, Testa B, van Gunsteren WF. Solventdependent conformation and hydrogen-bonding capacity of cyclosporin A: evidence from partition coefficients and molecular dynamics simulations. J Med Chem. 1993;36(24):3757-3764. 29. Shaw RA, Mantsch HH, Chowdhry BZ. Solvent influence on the conformation of cyclosporin. An FT-IR study. Can J Chem. 1993;71(9):1334-1339. 30. Ko SY, Dalvit C. Conformation of cyclosporin A in polar solvents. Int J Pept Protein Res. 1992;40(5):380-382. 31. Wang CK, Swedberg JE, Harvey PJ, Kaas Q, Craik DJ. Conformational flexibility is a determinant of permeability for cyclosporin. J Phys Chem B. 2018;122(8): 2261-2276. €ck M, Seebach D, Thaler A. Complexation and 32. Kessler H, Gehrke M, Lautz J, Ko medium effects on the conformation of cyclosporin A studied by NMR spectroscopy and molecular dynamics calculations. Biochem Pharmacol. 1990;40(1): 169-173. €ck M, Wein T, Gehrke M. Reinvestigation of the conformation of 33. Kessler H, Ko cyclosporin A in chloroform. Helv Chim Acta. 1990;73(7):1818-1832. 34. Jiao J. Polyoxyethylated nonionic surfactants and their applications in topical ocular drug delivery. Adv Drug Deliv Rev. 2008;60(15):1663-1673. 35. Ran Y, Zhao L, Xu Q, Yalkowsky SH. Solubilization of cyclosporin A. AAPS PharmSciTech. 2001;2(1):E2. 36. Guo C, Zhang Y, Yang Z, et al. Nanomicelle formulation for topical delivery of cyclosporine A into the cornea: in vitro mechanism and in vivo permeation evaluation. Sci Rep. 2015;5:12968. 37. Mitra A, Weiss S. Ocular Technologies SARL, Assignee. Topical Aqueous Nanomicellar, Ophthalmic Solutions and Uses Thereof. US 8980839 B2. 2015. 38. Gu C-H, Young Jr V, Grant DJW. Polymorph screening: influence of solvents on the rate of solvent-mediated polymorphic transformation. J Pharm Sci. 2001;90(11):1878-1890. 39. Chennell P, Delaborde L, Wasiak M, et al. Stability of an ophthalmic micellar formulation of cyclosporine A in unopened multidose eyedroppers and in simulated use conditions. Eur J Pharm Sci. 2017;100:230-237.