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Freeze-Drying of Nanosuspensions, Part 3: Investigation of Factors Compromising Storage Stability of Highly Concentrated Drug Nanosuspensions
Freeze-Drying of Nanosuspensions, Part 3: Investigation of Factors Compromising Storage Stability of Highly Concentrated Drug Nanosuspensions JAKOB BEIROWSKI,1 SABINE INGHELBRECHT,2 ALBERTINA ARIEN,2 HENNING GIESELER1 1
Division of Pharmaceutics, Freeze-Drying Focus Group, University of Erlangen Cauerstraße, 91058 Erlangen, Germany
2
Janssen Pharmaceutical Research and Development, A Division of Janssen Pharmaceutical NV, 2340 Beerse, Belgium
Received 20 June 2011; revised 3 August 2011; accepted 10 August 2011 Published online 8 September 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22745 ABSTRACT: On the basis of a previously developed formulation and process guideline for lyophilized, highly concentrated drug nanosuspensions for parenteral use, it was the purpose of this study to demonstrate that the original nanoparticle size distribution can be preserved over a minimum period of 3 months, even if aggressive primary drying conditions are used. Critical factors were evaluated that were originally believed to affect storage stability of freeze-dried drug nanoparticles. It was found that the nature and concentration of the steric stabilizer, such as Poloxamer 338 and Cremophor EL, are the most important factors for long-term stability of such formulations, independent of the used drug compound. The rational choice of an adequate steric stabilizer, namely Poloxamer 338, in combination with various lyoprotectants seems crucial to prevent physical instabilities of the lyophilized drug nanoparticles during short-term stability experiments at ambient and accelerated conditions. A 200 mg/mL concentration of nanoparticles could successfully be stabilized over the investigated time interval. In the course of the present experiments, polyvinylpyrrolidone, type K15 was found superior to trehalose or sucrose in preserving the original particle size distribution, presumably based on its surface-active properties. Lastly, it was demonstrated that lower water contents are generally beneficial to stabilize such systems. © 2011 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:354–362, 2012 Keywords: freeze-drying; lyophilization; stability; milling; nanosuspensions; nanoparticles; nanotechnology; calorimetry; parenterals
INTRODUCTION The general purpose of pharmaceutical freeze-drying is to achieve long-term stability of heat labile active compounds in a formulation.1–4 When freezedrying colloidal systems, the ultimate goal is to avoid physical instabilities such as aggregation, agglomeration, or particle fusion.5,6 However, literature dealing with long- or short-term stability of freeze-dried drug nanosuspensions is generally quite limited, and specific information on storage stability of such systems is not available. Formulations of drug nanosuspensions typically consist of suspended (pure) drug nanoparticles in Correspondence to: Henning Gieseler (Telephone: +499131-8529556; Fax: +49-9131-8529545; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 101, 354–362 (2012) © 2011 Wiley Periodicals, Inc. and the American Pharmacists Association
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water that, in turn, are conventionally stabilized by steric stabilizers.7,8 The main benefit of creating an active pharmaceutical ingredient (API) in nanometer range is to improve its solubility and dissolution velocity.9 It has been recommended that drug nanosuspensions should have an average mean particle size below 1 :m and a d99 value below 5 :m to obtain the above-mentioned properties.10 Note that d99 is the volume diameter 99% which means 99% of the particles are below the given size. However, this recommendation turns into an important specification once such drug systems are intended for parenteral application. Otherwise, negative side effects such as embolisms might be observed.10 Previous research in this field of interest has shown that the concentration as well as the nature of the steric stabilizer plays an important role in stabilizing drug nanoparticles during freezing and freeze-drying.6,11–14 Moreover, it has been
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demonstrated that the combination of steric stabilizer and typical freeze-drying stabilizing excipient is a critical factor in formulation development when freezing and freeze-drying highly concentrated drug nanosuspensions.6,12–14 In other words, when the nature and the concentration of a steric stabilizer is inappropriate for freeze-drying, even excessive amounts of a cryoprotectant or lyoprotectant are unable to prevent the system from freezing and drying stresses.6 Even if several steric stabilizers such as Poloxamer 338, Tween 80, and Cremophor EL were discussed in the literature to successfully stabilize drug nanoparticles during their manufacturing step (e.g., by media milling), the key question on what characteristics a steric stabilizer must exhibit to impart nanoparticle stability after freeze-drying and during storage still needs to be addressed.14–20 As a general rule, all stabilizing agents in a formulation should be converted into the solid state where the API is “arrested” and “mobility” is reduced to a minimum.2,3 In this context, glass formation and glass-forming excipients play an important role in this discussion.2,5 Amorphous components remain, however, only vitrified after freeze-drying in case that the storage temperature (Ts ) remains well below the glass transition temperature (Tg ) of the glass. As a rule of thumb, a temperature difference “Tg − Ts ” of, at least, 50◦ C was proposed. In line with this, an important question immediately emerges when using high concentrations of, for example, Cremophor EL and Tween 80 when freeze-drying drug nanosuspensions. Both materials are naturally highly viscous liquids and have, as pure substances, low Tg values. Thus, it is questionable if a formulation containing such materials can be converted in the solid state after freeze-drying and subsequently stored at ambient conditions. From that point of view, one may expect that even at ambient conditions nanoparticles in those lyophilized formulations are mobile and not completely immobilized. This argument applies specifically to cases where a steric stabilizer is the only protecting agent present.11 Another point of consideration is that Poloxamer 338 does crystallize during freeze-drying, exhibiting a melting point beyond conventional storage temperature conditions. For that reason, it is believed that Poloxamer 338 might be superior as a stabilizing agent in the lyophilized form. In contrast to Cremophor EL or Tween 80, Poloxamer 338 remains as a solid after freeze-drying, which might be a prerequisite for long-term stability of highly concentrated lyophilized nanoparticles. Lastly, recent work revealed that vitrification of lyoprotectants during actual freeze-drying of drug nanosuspensions is not a prerequisite for stability.12–14 As a consequence, drug nanosuspensions could be dried at extreme product temperatures, resulting in an above-average reduction in freezedrying process time.13,14 It must be noted, however, DOI 10.1002/jps
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that this observation was only accessible with an “optimized” formulation design. “Optimized” in this context refers to (1) a lyoprotectant concentration, which is as low as possible to prevent shrinkage or collapse but sufficiently high to assure stabilization of the nanoparticles; (2) a minimum steric stabilizer concentration, which is typically higher than the minimum concentration to achieve initial nanoparticle stability right after milling; and (3) an elevated nanoparticle concentration to improve mechanical stability of the cake.14 On the basis of the above-mentioned findings, it seems intuitive to investigate if storage stability of highly concentrated drug nanoparticles can be achieved after freeze-drying, even if aggressive cycle conditions were used. Again, the higher the concentration of the drug nanosuspension, the higher the expected instability of such a drug formulation. The purpose of this study is therefore to examine the factors compromising storage stability of lyophilized drug nanosuspensions. Cremophor EL and Poloxamer 338 were used as steric stabilizers and used either alone or in combination with the lyoprotectants trehalose, sucrose, and polyvinylpyrrolidone, type K15 (PVP K15). Short-term (3 months) stability testing was performed at 25◦ C and 40◦ C to investigate the influence of the storage conditions on the particle size distribution, the key parameter for parenteral delivery of drug nanosuspensions. Also, the influence of elevated residual moisture contents on nanoparticle stability (between 1% and 2%) was included in the present evaluation. Lastly, a practical consideration for accelerated stability testing protocols at 40◦ C/ 75% relative humidity (RH) for such systems according to International Conference on Harmonization guidelines21 will be critically discussed.
MATERIALS AND METHODS Two poorly water-soluble crystalline APIs with a molar mass of 705 g/mol (Model API I) and 366 g/ mol (Model API II) and 0.3 mm yttrium-stabilized zirconia beads were kindly provided by Janssen Pharmaceutical Research and Development (Beerse, Belgium). Lutrol F108 Prill (Poloxamer 338) and Cremophor EL were provided by BASF (Ludwigshafen, Germany). PVP K15, trehalose, and sucrose of analytical grade were purchased from Sigma (Sigma Chemical Company, Munich, Germany) and used as received. Water for injection was purchased from Braun (Braun Melsungen AG, Melsungen, Germany) for the production of the nanosuspensions. Demineralized water was filtered through Sartobran PH2 O membrane filters (Sartorius Stedim Biotech, Goettingen, Germany) and used for laser diffraction analysis. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
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Primary Packing Material 15R Vials from Lutz GmbH (Wertheim, Germany) and R 20 mm West Flurotec lyophilization stoppers from West Pharmaceutical Services, Inc. (Lionville, Pennsylvania) were used as received. All vials were placed onto the shelf using a bottomless tray. Production of Nanosuspensions Model API I (100 mg/mL) was mixed with either 50 mg/mL of Poloxamer 338 or 50 mg/mL of Cremophor EL and prepared by wet bead milling in a R , Netzschhigh-shear media mill (Netzsch MiniCer ¨ Geratebau GmbH, Selb, Germany). For milling, 0.3 mm yttrium-stabilized zirconia beads were used. For data comparison purpose, 100 mg/mL of Model API II was stabilized with 50 mg/mL Poloxamer 338 and milled using the same procedure as mentioned above. The Model API II concentration was then further increased to 200 mg/mL and stabilized again with 50 mg/mL Poloxamer 338. The resulting stock nanosuspensions were mixed with 50 mg/mL PVP K15, trehalose, or sucrose. Laser Diffraction Analysis R (Malvern Instruments A Malvern MasterSizer GmbH, Herrenberg, Germany) was used to allow a determination of the particle size distribution over a range in which even agglomerates can be found. Dependent on the steric stabilizer present in the formulation, an aqueous solution of 5 mg/mL Poloxamer 338 or Cremophor EL was used as a medium because particle stability was not sufficient in pure water during a measurement. Basis for particle size calculation was the Mie theory with a product refractive index of 1.65 and product absorption of 0.001. The R 2000 obtained data fit reported by the MasterSizer Software indicated validity of the optical model (residual value <1%). Background and measurement integration time were kept at 60 s, measurements were conducted in triplicate (n = 3) per unit sample.
Freeze-Drying Procedure One milliliter of each formulation was filled in 15R vials. Lyophilization was then performed on a laboratory scale freeze-dryer (VirTis Advantage Plus, SP Scientific, Gardiner, New York). Freezing was conducted at −40◦ C (shelf inlet temperature) for 60 min, including equilibrating steps at +5◦ C and −5◦ C for 15 min each. To facilitate crystallization of the Poloxamer 338 in the formulation, an annealing step at −20◦ C (shelf inlet temperature) for 90 min was implemented. The shelf temperature ramp rates from the freezing set point to the primary drying shelf temperature setting were 1◦ C/min throughout the study. The shelf inlet temperature set point during primary and secondary drying was 40◦ C, which is further deJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
noted as “aggressive” freeze-drying. The holding time (soak period) of this step was either 60 or 600 min to allow a modulation in water content in the samples. The chamber pressure during primary and secondary drying was controlled at 100 mTorr throughout the experiments. Note that the freeze-drying cycles were performed in duplicate (n = 2) per formulation. Product temperatures during freeze-drying were measured using calibrated 30 gauge T-type copper/ constantan thermocouples from Omega (Omega Engineering, Stamford, Connecticut). Each thermocouple was introduced through a stopper and positioned bottom center of the vial to obtain both a representative temperature monitoring in the product and an accurate endpoint detection of the ice sublimation phase. Differential Scanning Calorimetry Determination of thermal transitions in all formulation mixtures was performed using a MettlerDSC822e (Mettler Toledo, Greifensee, Switzerland). Data acquisition was performed in the temperature range between 5◦ C and 150◦ C. The applied heating rate was 5◦ C/min. Temperature calibration of the differential scanning calorimetry (DSC) instrument was performed on a weekly basis using the melting point of Indium (156.5◦ C) as a reference. Karl Fischer Residual Moisture Measurements Residual moisture of the lyophilized samples was measured using a Metrohm Karl Fischer 831 KF Coulometer combined with a Metrohm Thermoprep 832 oven unit. About 50 mg of product was weighed into a custom glass vial and then inserted into the oven after purging the sample vial with dry nitrogen. The product was heated to 140◦ C for a defined time period and the moisture was accumulated in the titration solvent. Residual moisture content was reported in percent (%) as a function of the sample weight. Stability Testing Procedure Immediately after freeze-drying, the products (n = 4) were sealed and stored at 25◦ C and 40◦ C/75 RH, respectively. After 1, 2, and 3 months, samples were analyzed in terms of mean particle size and particle size distribution. DSC and KF measurements were performed after freeze-drying as well as after 3 months of storage.
RESULTS AND DISCUSSION Selection of an Adequate Steric Stabilizer to Achieve Storage Stability To prove the concept that Poloxamer 338 is superior to Cremophor EL as a steric stabilizer in terms of storage stability of lyophilized drug nanosuspensions, Poloxamer 338 and Cremophor EL drug nanosuspensions were freeze-dried without any addition of a DOI 10.1002/jps
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Figure 1. Comparison of the particle size distributions of a stock nanosuspension (100 mg/mL Model API I) stabilized with 50 mg/mL Cremophor EL directly after freeze-drying and 3 months of storage at ambient conditions (dashed line). The 5 :m boundary represents the upper particle size limit for a parenteral drug nanosuspension.
Figure 3. Comparison of the particle size distributions of a stock nanosuspension (100 mg/mL Model API I) stabilized with 50 mg/mL Cremophor EL after aggressive process conditions (solid line, shelf temperature: 40◦ C) and conservative process conditions (dashed line, shelf temperature 0◦ C).
lyoprotectant (if possible). However, preliminary experiments revealed for the model formulation containing Model API I that for drug nanoparticle concentrations of 100 mg/mL, a minimum concentration of 50 mg/mL Cremophor EL or 50 mg/mL Poloxamer 338 is beneficial to assure that the desired particle size after lyophilization is consistently obtained below the critical upper limit (Figure 1 and 2).10 Note that the critical formulation temperature (CFT), the temperature which represents the maximum allowable product temperature during primary drying, was determined to be about −73◦ C for the pure drug nanosuspension stabilized with solely Cre-
mophor EL.14 There is no doubt that controlling product temperature during primary drying at or even below the CFT is literally impossible. Surprisingly, the particle size distribution after lyophilization met the particle size prerequisites (cf. Fig. 3), even though shelf temperatures during primary drying were preprogrammed at 40◦ C and resulted in a product temperature for the Cremophor EL drug nanosuspension of about −18◦ C. Indeed, the product temperature exceeded the CFT of roughly 55◦ C, suggesting that vitrification is apparently not mandatory for drug nanoparticle stabilization during primary drying. The higher the observed product temperature during primary drying the smaller the aggregates which were detected (cf. Fig. 3). The observation that a more aggressive drying protocol results in a better particle size distribution may be explained if aggregation is considered as a time-dependent process where the nanoparticles have less time to show physical instabilities during a short than during a long cycle time. This, in turn, would suggest that immobilization is not necessary during this process step. This result confirms a previous study from our laboratory, which revealed that drug nanosuspensions can be aggressively freeze-dried well above the CFT.14 In the course of this study, it was found that nanoparticle and cake stability are not related to a rigid amorphous glassy excipient matrix during an actual freezedrying run but dependent on the combination of the used nanoparticle, steric stabilizer, and lyoprotectant concentration. In contrast to this, we would speculate that months or even years of storage would be sufficient time for particle growth in cases in which diffusion processes are accelerated. Thus, immobilization
Figure 2. Comparison of the particle size distributions of a stock nanosuspension (100 mg/mL Model API I) stabilized with 50 mg/mL Poloxamer 338 directly after freeze-drying and 3 months of storage at ambient conditions (dashed line). DOI 10.1002/jps
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Figure 4. DSC plots of the pure lyophilized drug nanosuspension stabilized only either with Cremophor EL (upper dashed curve) or with Poloxamer 338 (lower solid curve), which are equivalent to Model API I and II. The upper dashed curve illustrates a broad endotherm between 10◦ C and 25◦ C, lower solid curve indicates a melting peak at approximately 58◦ C that originates from the crystalline Poloxamer 338.
during storage should be compulsory to avoid physical instabilities. On the basis of this line of argumentation, Cremophor EL is expected to show a poor long-term stabilization performance. DSC analysis for Cremophor EL-stabilized drug nanosuspensions revealed a broad endothermic signal between 10◦ C and 25◦ C (Fig. 4). Recall that this steric stabilizer was thought to be a reasonable excipient to stabilize drug nanoparticles throughout milling and freeze-drying experiments because particle size distributions measured right after processing were found acceptable. Cremophor EL was found insufficient to impart stability during storage at 25◦ C and, even more distinct, at 40◦ C (Fig. 1). After 3 months of storage at ambient conditions, the nanoparticle size distribution turned into a more excessive bimodal distribution, indicating that strong aggregates have been formed during this period of time. In addition, residual moisture contents above 1% were found to further deteriorate aggregation tendency. The simple explanation for this observation might be grounded in the plasticizing effect of water in the formulation, which results in a decrease of Tg . The DSC plot in Figure 4 illustrates, however, that the melting point of the lyophilized Poloxamer 338 formulation can be found at about 58◦ C, assuming that nanoparticle mobility must be more constrained in this formulation. Figure 2 also confirms this postulation and shows that the measured particle size distribution of the drug nanosuspension stabilized with Poloxamer 338 after 3 months at 25◦ C is rather unimodal. Nevertheless, particle growth could not be fully arrested at such harsh process conditions. In this case, the plasticizing effect of water is not expected to JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
Figure 5. Comparison of the redispersibility indices (RDIs) for the stock nanosuspensions (100 mg/mL Model API I) after 3 months of storage at ambient conditions stabilized with either Cremophor EL (Cr EL) or Poloxamer 338 (P338) as a function of a high or low residual moisture (RM) content. Note that RM did not exceed 2% for the corresponding formulations.
be critical for the (fully) crystalline material. However, calculations of the redispersibility index (RDI) showed that slightly higher residual moisture contents (i.e., above 1% but below 2%) can, at least to some extent, destabilize the system (Fig. 5). The RDI is defined as D0 /D, where D0 is the volume-weighed mean particle size of the reconstituted nanosuspension directly after freeze-drying (time mark = T0 ) and D is the corresponding value after 3 months of storage (time mark = T3 ). An RDI of 100% would therefore mean that the stored freeze-dried drug nanosuspension can be completely transformed to the original particle size at T0 after rehydration.22 Figure 5 confirms the above-described observations obtained from Figures 1 and 2, and additionally underlines that the factor “residual moisture” should be considered as a critical parameter. For the Poloxamer 338 formulations, a water content above 1% led to a significant 3% decrease of the RDI, while the drug nanosuspension stabilized with Cremophor EL suffered a 7% decrease in the RDI when the residual moisture was high (Fig. 5). For Poloxamer 338 drug nanosuspensions, the distinct shift of the unimodal distribution toward larger particles accompanied by a decrease of the volume of the nanoparticle amount (Fig. 2) and a maximum achievable RDI of 93% implies that further formulation work might be necessary for nanoparticle stabilization. In theory, the addition of a lyoprotectant may further immobilize and isolate individual particles. However, when dealing with highly concentrated drug nanosuspensions, excessive concentrations of lyoprotectant would be necessary to achieve the desired effect.5 Experiments showed that mixing commonly used lyoprotectants (e.g., sucrose and trehalose) with DOI 10.1002/jps
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Table 1. d50 , d95 , and RDI Values Obtained Directly After Completion of Freeze-Drying (residual moisture <1%) and After 3 Months of Storage at 25◦ C and 40◦ C, Respectively
Without Protectant
Tr
Su
PVP
Figure 6. Comparison of the particle size distributions of a drug nanosuspension (100 mg/mL Model API I) stabilized with 50 mg/mL Cremophor EL and 50 mg/mL trehalose as a lyoprotectant directly after freeze-drying and 3 months of storage at ambient conditions as a function of a high or low residual moisture content. Note that initial nanoparticle stability was independent of the water content (solid versus dotted line) but after storage residual moisture below 1% (dashed line) revealed better particle stability compared with a water content above 1% (dashed dotted line).
those drug nanosuspensions containing Cremophor EL was not acceptable for storage stability, irrespective of both the nanoparticle concentration and model API used. The final particle size was found to be higher than the specified target in every investigated case and an RDI beyond 80% was never achieved (data not shown), even though the residual moisture content was below 1% (cf. Fig. 6). Note that similar observations were also made with other steric stabilizers, such as Tween 80 (data not shown), and even more importantly with the second drug compound Model API II. It may be concluded from the present set of freezedrying experiments that the physicochemical characteristic of the steric stabilizer plays an important role for subsequent storage conditions. From that point of view, it seems recommendable to preferentially use those steric stabilizers that remain in the solid state after freeze-drying. As only Poloxamer 338 fulfils this prerequisite, all of the subsequent storage stability experiments were performed with Poloxamer 338. As a side note, DSC experiments have demonstrated to be indispensable to evaluate storage conditions when freeze-drying drug nanosuspensions.23 Selection of Adequate Lyoprotectants to Achieve Storage Stability The preliminary experiments mentioned above have indicated that lyoprotectants might improve the longterm stability of drug nanoparticles during storage, DOI 10.1002/jps
T0 T3 at 25◦ C T3 at 40◦ C T0 T3 at 25◦ C T3 at 40◦ C T0 T3 at 25◦ C T3 at 40◦ C T0 T3 at 25◦ C T3 at 40◦ C
d50 Value (:m)
d95 Value (:m)
RDI (%)
0.185 0.185 0.199 0.166 0.168 0.177 0.168 0.172 0.195 0.165 0.159 0.161
1.18 1.89 3.01 0.82 0.85 0.95 0.90 0.94 1.48 0.80 0.78 0.85
– 75% 42% – 98% 94% – 96% 65% – 101% 98%
Note that the calculated standard deviations were small (σ < 0.045 for n = 4) for all formulations, indicating very consistent d50 and d95 . For the RDI values, standard deviations were calculated in the range of 1.27–1.93. Formulation: 200 mg/mL nanoparticle concentration (Model API II) stabilized with 50 mg/mL Poloxamer 338 alone or in combination with 50 mg/mL Polyvinylpyrrolidone (PVP type K15), 50 mg/mL trehalose, or 50 mg/mL sucrose. P338, Poloxamer 338; Tr, trehalose; Su, sucrose; PVP, PVP K15. T0 = d50 , d95 , and RDI values determined directly after freeze-drying, time mark T3 = d50 , d95 , and RDI values after 3 months of storage, time mark T0 = d50 and d95 values measured directly after freeze-drying of the reconstituted formulations.
even in cases when the desired physical stability right after completion of freeze-drying can be achieved without the addition of any lyoprotectant. Table 1 compares the d50 , d95 , and the RDI values after aggressive freeze-drying of formulations containing 200 mg/mL Model API II. Note that the abbreviations d50 and d95 stand for 50% and 95%, respectively, of the particles being smaller in size than the reported value. The purpose of using “aggressive” cycle conditions was to illustrate that even such considerably high particle concentrations do not require vitrification in a lyoprotectant matrix during primary drying for stabilization.24 Product temperatures were constantly maintained well above the CFT in every case (data not shown).14 Results demonstrated that the addition of the stabilizers PVP K15, trehalose, or sucrose in a 50 mg/mL concentration to the drug nanosuspensions further improved nanoparticle stability, relative to the pure lyophilized drug nanosuspension stabilized with Poloxamer 338. Fifty milligram per milliliter was found as the highest possible lyoprotectant concentration to obtain a good compromise between (a) sufficient mechanical cake stability when using aggressive drying conditions and (b) preservation of the original particle size distribution. The rationale behind the use of such materials as “lyoprotectants” might be given by the particle isolation hypothesis. Recall that this hypothesis claims that the colloidal particles represent an emulsion in which the stability is governed by the surface tension of the dispersion medium.25 PVP K15 was found superior JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
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over trehalose or sucrose in providing stability. This might be explained due to its distinct surface-active nature. As a conclusion, drug nanoparticles are sufficiently dispersed in the PVP K15, trehalose, or sucrose solution to overcome aggregation or agglomeration tendencies, as these materials possess relatively low surface tensions compared with other protectants such as starch derivatives.25 The Poloxamer 338 concentration provided in Table 1 reflects the highest possible particle concentration, keeping in mind that this material may cause toxic side effects for the patient. Higher steric stabilizer concentrations were always associated with a better nanoparticle size distribution throughout the present study, which is in good agreement to previous investigations.6,12–14 A set of shortterm stability tests with every single steric stabilizer used in this study confirmed this observation (data not shown). Table 1 also demonstrates that the examined nanoparticle stability right after completion of freeze-drying perfectly matched the anticipated particle size distribution.
Figure 8. Comparison of the DSC plots of the highly concentrated drug nanosuspension (Model API II) stabilized with Poloxamer 338 as a function of PVP K15 (top curve), sucrose (middle curve), or trehalose (bottom curve) in the formulation. Note that only sucrose caused a glass transition before the melting peak of Poloxamer 338 at about 58◦ C.
Storage Stability Investigations of Freeze-Dried Drug Nanosuspensions Table 1 illustrates that after 3 months of storage at ambient conditions, all lyoprotectants used in this study showed an efficient stabilizing effect when the water content was below 1%. In other words, no matter if PVP K15, trehalose, or sucrose were used, an RDI of at least 96% was obtained, relative to a maximally achievable RDI of 75% after storage of the pure drug nanosuspension stabilized only with Poloxamer 338 (Table 1). Note that these results are also in good
Figure 7. Comparison of the particle size distributions of a highly concentrated drug nanosuspension (200 mg/mL Model API II) stabilized with 50 mg/mL Poloxamer 338 and 50 mg/mL PVP K15 as a lyoprotectant showing that the original particle size distribution after freeze-drying (solid line) was completely preserved after 3 months of storage as well as at 25◦ C (dashed line) and 40◦ C (dotted line). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
agreement to previous studies performed with a lower drug to steric stabilizer ratio. For comparison, the data presented in the previous chapter revealed an RDI of 93% after 3 months of storage at 25◦ C for the pure drug nanosuspension stabilized with only Poloxamer 338. Here, the effective amount of Poloxamer 338 was twice as high related to the nanoparticle concentration. Accelerated storage stability testing at 40◦ C showed after 3 months that solely PVP K15 was suitable to prevent nearly completely particle growth (average RDI value of 98%). In addition, Figure 7 shows that for those formulations containing 50 mg/mL PVP K15, similar particle size distributions were obtained directly after freeze-drying and storage at 40◦ C for 3 months. To better explain these results, thermal analysis by DSC was included into the data interpretation (Fig. 8). For the drug nanosuspension containing sucrose, a weak glass transition at about 30◦ C is noticeable prior to the melting peak of Poloxamer 338, which is consistently reproducible. It would be expected that the (amorphous) sucrose phase influences the crystallization tendency of Poloxamer 338 during freezing. Early experiments showed that this is indeed the case which, in turn, suggests that at least a part of the Poloxamer 338 in the formulation remains amorphous.14 The observed glass transition, which reflects a contribution of both amorphous sucrose and Poloxamer 338 might then induce the particle growth due to enhanced nanoparticle mobility during storage at high temperatures. A decrease of the corresponding RDI from 96% to 65% might prove this hypothesis (cf. Table 1). A higher residual moisture content in the sucrose formulation was found, DOI 10.1002/jps
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which further deteriorates particle stability because water inevitably decreases the Tg below storage temperature (data not shown). The reason for PVP K15 being superior in providing stability over, for example, trehalose at 40◦ C storage conditions could not be pinpointed by DSC. Both plots illustrate a melting event between 55◦ C and 58◦ C, which originates from the Poloxamer 338. However, no glass transition or other thermal event can be detected prior to the Poloxamer 338 melt (Fig. 8). It could be speculated that the rather early “onset” of the melting endotherm at about 40◦ C might influence the particle growth at 40◦ C, but the same course in transition is also found in the PVP K15 formulation. Both lyoprotectants cause an endothermic signal of more than 100◦ C (data not shown), which is conform to the described Tg s in the literature for PVP and trehalose, respectively.2 For these reasons, DSC analysis seems to be insufficient to depict the significant differences in terms of improved physical stability stabilization when using PVP K15 instead of trehalose. One approach to find an appropriate explanation for these findings might be related to the surface-active properties of the two excipients. The drug nanoparticles seem to remain better dispersed in a rigid PVP K15 matrix, resulting in a higher degree of dispersity and hence more isolated colloidal particles, relative to the trehalose matrix. Moreover, as the measured glass transitions of PVP K15 and trehalose are high, one may assume that the degree of immobilization of both matrices after freeze-drying is comparable, even at 40◦ C storage temperature conditions. Against this background, the explanation of the superior stabilization capacity of PVP K15 by its lower surface tension seems reasonable which, in turn, is claimed by the particle isolation hypothesis.25 In addition, the relatively low melting point of Poloxamer 338 seems critical for accelerated stability tests at 40◦ C. Although not always desirable in practice, stability tests at 25◦ C over a period of 1 year (or longer) appear to be more reliable to evaluate the storage stability of drug nanosuspensions. As a consequence, not only PVP K15 but also trehalose and sucrose appear to be suitable lyoprotectants to prevent physical instabilities of lyophilized drug nanosuspensions. As a proof of concept, it should be mentioned that at least trehalose completely inhibited particle growth over 6 months at ambient temperature in the course of the present study (data not shown). To be more accurate, the corresponding particle size distribution and RDI value did not indicate a significant deterioration in particle size. Note that this result is in contradiction to the results obtained from accelerated stability testing. It is well known that in this case, stability is derived from the Arrhenius equation.21 This model describes the relation between a specific temperature and its corresponding reaction rate constant for homogeneous chemical reactions. However, the validity DOI 10.1002/jps
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of Arrhenius law is only strictly given when the relation between the logarithmic reaction rate is constant and the reciprocal temperature is linear.21 Only in such a case, it is possible to extrapolate from higher storage to lower storage temperatures. Therefore, it is strongly recommendable to critically interpret stability data obtained from storage stability testing at higher temperatures and related to physical stability of lyophilized nanoparticles. Again, DSC plots in this set of experiments revealed that at 40◦ C storage temperature, nanoparticle stability could be compromised disproportionately because the drug nanoparticles could remain in a partly melted environment. In contrast, at 25◦ C, the drug nanoparticles are expected to be fully arrested. As a consequence, misleading conclusions regarding the choice of freeze-drying stabilizing agents for nanoparticles and long-term stability of highly concentrated drug nanosuspensions could be drawn. DSC analysis must be used to evaluate adequate storage conditions after freeze-drying of drug nanosuspensions, specifically if accelerated stability testing at 40◦ C and 75% RH is expected to be predictive.
CONCLUSION In the present study, preservation of the original particle size distribution of freeze-dried highly concentrated drug nanosuspensions was achieved during short-term stability experiments at ambient and accelerated stability testing conditions (40◦ C/75% RH). It could be demonstrated for the first time that the nature of the steric stabilizer is of particular importance for storage stability of lyophilized drug nanoparticles. Cremophor EL was found inappropriate to stabilize the drug nanosuspensions. Even addition of the lyoprotectants PVP K15, trehalose, or sucrose was found to fail in the preservation of the initial particle size distribution during storage when Cremophor EL was used. In contrast, Poloxamer 338 was clearly identified as the steric stabilizer of choice. This further underlines the relevance of the role of the steric stabilizer for long-term stability. In addition, higher residual moisture contents were found to compromise the aggregation tendency, presumably due to the plasticizing effect. In case of a rational choice of lyoprotectants, even nanoparticle concentrations of 200 mg/ mL could be stabilized over a time period of at least 3 months, even when the formulation was freeze-dried using rather aggressive cycle conditions. According to the surface-active properties and based on its high Tg in the solid state, PVP K15 was identified to be superior over trehalose and sucrose as a freeze-drying stabilizer. Lastly, DSC analysis has proved again to be an indispensable tool when evaluating storage conditions and stability for drug nanosuspensions. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
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REFERENCES 1. Franks F. 1998. Freeze-drying of bioproducts: Putting principles into practice. Eur J Pharm Biopharm 45:221–229. 2. Wang W. 2000. Lyophilization and development of solid protein pharmaceuticals. Int J Pharm 203:1–60. 3. Pikal MJ, Costantino MR. 2004. Lyophilization of biopharmaceuticals. In Biotechnology: Pharmaceutical aspects; Borchardt RT, Middaugh CR, Eds. Arlington: AAPS, pp 1–75. 4. Tang XC, Pikal MJ. 2004. Design of freeze-drying processes for pharmaceuticals: Practical advice. Pharm Res 21(2):191– 200. 5. Abdelwahed W, Degobert G, Stainmesse S, Fessi H. 2006. Freeze-drying of nanoparticles: Formulation, process and storage considerations. Adv Drug Deliv Rev 58(15):1688–1713. 6. Beirowski J, Inghelbrecht S, Arien A, Gieseler H. 2011. Freezedrying of nanosuspensions, 1: Freezing rate versus formulation design as critical factors to preserve the original particle size distribution. J Pharm Sci 100(5):1958–1968. 7. Merisko-Liversidge E, Liversidge GG, Cooper ER. 2003. Nanosizing: A formulation approach for poorly-water-soluble compounds. Eur J Pharm Sci 18(2):113–120. 8. Gao L, Zhang D, Chen M. 2008. Drug nanocrystals for the formulation of poorly soluble drugs and its application as a potential drug delivery system. J Nanopart Res 10:845–862. 9. Kesisoglou F, Panmai S, Wu Y. 2007. Nanosizing—Oral formulation development and biopharmaceutical evaluation. Adv Drug Deliv Rev 59:631–644. 10. Wong J, Brugger A, Khare A, Chaubal M, Papadopoulos P, Rabinow B, Kipp J, Ning J. 2008. Suspensions for intravenous (IV) injection: A review of development, clinical and preclinical aspects. Adv Drug Deliv Rev 60:939–954. 11. Beirowski J, Inghelbrecht S, Arien A, Gieseler H. 2009. Investigation of various Poloxamers as freeze-drying stabilizers in freeze-drying of nanosuspensions. Frankfurt/Main, Germany: Proc. Parent Drug Association. 12. Beirowski J, Inghelbrecht S, Arien A, van Assche I, Gieseler H. 2010. Stabilization of nanosuspensions during freeze-drying: The role of vitrification (Part 1). Valetta, Malta, March 8–11: Proc. 7th World Meeting on Pharmaceutics, Biopharmaceutics and Pharmaceutical Technology. 13. Beirowski J, Inghelbrecht S, Arien A, van Assche I, Gieseler H. 2010. Stabilization of nanosuspensions during freeze-drying: The role of vitrification and its practical implications (Part 2). Valetta, Malta, March 8–11: Proc. 7th World Meeting on Pharmaceutics, Biopharmaceutics and Pharmaceutical Technology. 14. Beirowski J, Inghelbrecht S, Arien A, Gieseler H. 2011. FreezeDrying of Nanosuspensions, 2: The Role of the Critical Formu-
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lation Temperature on Stability of Drug-Nanosuspensions and Its Practical Implication on Process Design. J Pharm Sci [Article first published online: 23 MAY 2011] Merisko-Liversidge E, Sarpotdar P, Bruno J, Hajj S, Wei L, Peltier N, Rake J, Shaw J M, Pugh S, Polin L Jones J, Corbett T, Cooper E, Liversidge GG. 1996. Formulation and antitumor activity evaluation of nanocrystalline suspensions of poorly soluble anticancer drugs. Pharm Res 13:272–278. Mouton JW, van Peer A, de Beule K, van Vliet A, Donnelly JP, Soons PA. 2006. Pharmacokinetics of itraconazole and hydroxyitraconazole in healthy subjects after single and multiple doses of a novel formulation. Antimicrob Agents Chemother 50:4096–4102. Wahlstrom JL, Chiang PC, Ghosh S, Warren CJ, Wene SP, Albin LA, Smith ME, Roberds SL. 2007. Pharmacokinetic evaluation of a 1,3-dicyclohexylurea nanosuspension formulation to support early efficacy assessment. Nanoscale Res Lett 2:291–296. Chiang PC, Wahlstrom JL, Selbo JG, Zhou S, Wene SP, Albin LA, Warren CJ, Smith ME, Roberds SL, Ghosh S, Zhang LL, Pretzer DK. 2007. 1,3-dicyclohexylurea nanosuspension for intraveneous steady-state delivery in rats. J Exp Nanosci 2:239–250. Kumar MP, Rao YM, Apte S. 2007. Improved bioavailability of albendazole following oral administration of nanosuspension in rats. Curr Nanosci 3:191–194. Kumar MP, Rao YM, Apte S. 2008. Formulations of nanosuspensions of albendazole for oral administration. Curr Nanosci 4:53–58. Grimm W. 1998. Extension of the International Conference on Harmonization tripartite guideline for stability testing of new drug substances and products to countries of climatic zones III and IV. Drug Dev Ind Pharm 24(4):313–325. Lee J, Cheng Y. 2006. Critical freezing rate in freeze drying nanocrystal dispersions. J Control Release 111:185–192. Beirowski J, Gieseler H. 2008. Application of DSC and MDSC in the development of freeze-dried pharmaceuticals. Eur Pharm Rev 13(6):63–70. Pikal MJ. 1998. Mechanisms of protein stabilization during freeze-drying and storage: The relative importance of thermodynamic relaxation and glassy state relaxation dynamics. In Freeze-drying/lyophilization of pharmaceuticals and biological products 96; Rey L, May LC, Eds. New York: Marcel Dekker, pp 161–198. Allison SD, Molina MDC, Anchordoquy TJ. 2000. Stabilization of lipid/DNA complexes during the freezing step of the lyophilization process: The particle isolation hypothesis. Biochim Biophys Acta 1468:127–138.
DOI 10.1002/jps
Division of Pharmaceutics, Freeze-Drying Focus Group, University of Erlangen Cauerstraße, 91058 Erlangen, Germany
2
Janssen Pharmaceutical Research and Development, A Division of Janssen Pharmaceutical NV, 2340 Beerse, Belgium
Received 20 June 2011; revised 3 August 2011; accepted 10 August 2011 Published online 8 September 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22745 ABSTRACT: On the basis of a previously developed formulation and process guideline for lyophilized, highly concentrated drug nanosuspensions for parenteral use, it was the purpose of this study to demonstrate that the original nanoparticle size distribution can be preserved over a minimum period of 3 months, even if aggressive primary drying conditions are used. Critical factors were evaluated that were originally believed to affect storage stability of freeze-dried drug nanoparticles. It was found that the nature and concentration of the steric stabilizer, such as Poloxamer 338 and Cremophor EL, are the most important factors for long-term stability of such formulations, independent of the used drug compound. The rational choice of an adequate steric stabilizer, namely Poloxamer 338, in combination with various lyoprotectants seems crucial to prevent physical instabilities of the lyophilized drug nanoparticles during short-term stability experiments at ambient and accelerated conditions. A 200 mg/mL concentration of nanoparticles could successfully be stabilized over the investigated time interval. In the course of the present experiments, polyvinylpyrrolidone, type K15 was found superior to trehalose or sucrose in preserving the original particle size distribution, presumably based on its surface-active properties. Lastly, it was demonstrated that lower water contents are generally beneficial to stabilize such systems. © 2011 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:354–362, 2012 Keywords: freeze-drying; lyophilization; stability; milling; nanosuspensions; nanoparticles; nanotechnology; calorimetry; parenterals
INTRODUCTION The general purpose of pharmaceutical freeze-drying is to achieve long-term stability of heat labile active compounds in a formulation.1–4 When freezedrying colloidal systems, the ultimate goal is to avoid physical instabilities such as aggregation, agglomeration, or particle fusion.5,6 However, literature dealing with long- or short-term stability of freeze-dried drug nanosuspensions is generally quite limited, and specific information on storage stability of such systems is not available. Formulations of drug nanosuspensions typically consist of suspended (pure) drug nanoparticles in Correspondence to: Henning Gieseler (Telephone: +499131-8529556; Fax: +49-9131-8529545; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 101, 354–362 (2012) © 2011 Wiley Periodicals, Inc. and the American Pharmacists Association
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water that, in turn, are conventionally stabilized by steric stabilizers.7,8 The main benefit of creating an active pharmaceutical ingredient (API) in nanometer range is to improve its solubility and dissolution velocity.9 It has been recommended that drug nanosuspensions should have an average mean particle size below 1 :m and a d99 value below 5 :m to obtain the above-mentioned properties.10 Note that d99 is the volume diameter 99% which means 99% of the particles are below the given size. However, this recommendation turns into an important specification once such drug systems are intended for parenteral application. Otherwise, negative side effects such as embolisms might be observed.10 Previous research in this field of interest has shown that the concentration as well as the nature of the steric stabilizer plays an important role in stabilizing drug nanoparticles during freezing and freeze-drying.6,11–14 Moreover, it has been
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demonstrated that the combination of steric stabilizer and typical freeze-drying stabilizing excipient is a critical factor in formulation development when freezing and freeze-drying highly concentrated drug nanosuspensions.6,12–14 In other words, when the nature and the concentration of a steric stabilizer is inappropriate for freeze-drying, even excessive amounts of a cryoprotectant or lyoprotectant are unable to prevent the system from freezing and drying stresses.6 Even if several steric stabilizers such as Poloxamer 338, Tween 80, and Cremophor EL were discussed in the literature to successfully stabilize drug nanoparticles during their manufacturing step (e.g., by media milling), the key question on what characteristics a steric stabilizer must exhibit to impart nanoparticle stability after freeze-drying and during storage still needs to be addressed.14–20 As a general rule, all stabilizing agents in a formulation should be converted into the solid state where the API is “arrested” and “mobility” is reduced to a minimum.2,3 In this context, glass formation and glass-forming excipients play an important role in this discussion.2,5 Amorphous components remain, however, only vitrified after freeze-drying in case that the storage temperature (Ts ) remains well below the glass transition temperature (Tg ) of the glass. As a rule of thumb, a temperature difference “Tg − Ts ” of, at least, 50◦ C was proposed. In line with this, an important question immediately emerges when using high concentrations of, for example, Cremophor EL and Tween 80 when freeze-drying drug nanosuspensions. Both materials are naturally highly viscous liquids and have, as pure substances, low Tg values. Thus, it is questionable if a formulation containing such materials can be converted in the solid state after freeze-drying and subsequently stored at ambient conditions. From that point of view, one may expect that even at ambient conditions nanoparticles in those lyophilized formulations are mobile and not completely immobilized. This argument applies specifically to cases where a steric stabilizer is the only protecting agent present.11 Another point of consideration is that Poloxamer 338 does crystallize during freeze-drying, exhibiting a melting point beyond conventional storage temperature conditions. For that reason, it is believed that Poloxamer 338 might be superior as a stabilizing agent in the lyophilized form. In contrast to Cremophor EL or Tween 80, Poloxamer 338 remains as a solid after freeze-drying, which might be a prerequisite for long-term stability of highly concentrated lyophilized nanoparticles. Lastly, recent work revealed that vitrification of lyoprotectants during actual freeze-drying of drug nanosuspensions is not a prerequisite for stability.12–14 As a consequence, drug nanosuspensions could be dried at extreme product temperatures, resulting in an above-average reduction in freezedrying process time.13,14 It must be noted, however, DOI 10.1002/jps
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that this observation was only accessible with an “optimized” formulation design. “Optimized” in this context refers to (1) a lyoprotectant concentration, which is as low as possible to prevent shrinkage or collapse but sufficiently high to assure stabilization of the nanoparticles; (2) a minimum steric stabilizer concentration, which is typically higher than the minimum concentration to achieve initial nanoparticle stability right after milling; and (3) an elevated nanoparticle concentration to improve mechanical stability of the cake.14 On the basis of the above-mentioned findings, it seems intuitive to investigate if storage stability of highly concentrated drug nanoparticles can be achieved after freeze-drying, even if aggressive cycle conditions were used. Again, the higher the concentration of the drug nanosuspension, the higher the expected instability of such a drug formulation. The purpose of this study is therefore to examine the factors compromising storage stability of lyophilized drug nanosuspensions. Cremophor EL and Poloxamer 338 were used as steric stabilizers and used either alone or in combination with the lyoprotectants trehalose, sucrose, and polyvinylpyrrolidone, type K15 (PVP K15). Short-term (3 months) stability testing was performed at 25◦ C and 40◦ C to investigate the influence of the storage conditions on the particle size distribution, the key parameter for parenteral delivery of drug nanosuspensions. Also, the influence of elevated residual moisture contents on nanoparticle stability (between 1% and 2%) was included in the present evaluation. Lastly, a practical consideration for accelerated stability testing protocols at 40◦ C/ 75% relative humidity (RH) for such systems according to International Conference on Harmonization guidelines21 will be critically discussed.
MATERIALS AND METHODS Two poorly water-soluble crystalline APIs with a molar mass of 705 g/mol (Model API I) and 366 g/ mol (Model API II) and 0.3 mm yttrium-stabilized zirconia beads were kindly provided by Janssen Pharmaceutical Research and Development (Beerse, Belgium). Lutrol F108 Prill (Poloxamer 338) and Cremophor EL were provided by BASF (Ludwigshafen, Germany). PVP K15, trehalose, and sucrose of analytical grade were purchased from Sigma (Sigma Chemical Company, Munich, Germany) and used as received. Water for injection was purchased from Braun (Braun Melsungen AG, Melsungen, Germany) for the production of the nanosuspensions. Demineralized water was filtered through Sartobran PH2 O membrane filters (Sartorius Stedim Biotech, Goettingen, Germany) and used for laser diffraction analysis. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
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Primary Packing Material 15R Vials from Lutz GmbH (Wertheim, Germany) and R 20 mm West Flurotec lyophilization stoppers from West Pharmaceutical Services, Inc. (Lionville, Pennsylvania) were used as received. All vials were placed onto the shelf using a bottomless tray. Production of Nanosuspensions Model API I (100 mg/mL) was mixed with either 50 mg/mL of Poloxamer 338 or 50 mg/mL of Cremophor EL and prepared by wet bead milling in a R , Netzschhigh-shear media mill (Netzsch MiniCer ¨ Geratebau GmbH, Selb, Germany). For milling, 0.3 mm yttrium-stabilized zirconia beads were used. For data comparison purpose, 100 mg/mL of Model API II was stabilized with 50 mg/mL Poloxamer 338 and milled using the same procedure as mentioned above. The Model API II concentration was then further increased to 200 mg/mL and stabilized again with 50 mg/mL Poloxamer 338. The resulting stock nanosuspensions were mixed with 50 mg/mL PVP K15, trehalose, or sucrose. Laser Diffraction Analysis R (Malvern Instruments A Malvern MasterSizer GmbH, Herrenberg, Germany) was used to allow a determination of the particle size distribution over a range in which even agglomerates can be found. Dependent on the steric stabilizer present in the formulation, an aqueous solution of 5 mg/mL Poloxamer 338 or Cremophor EL was used as a medium because particle stability was not sufficient in pure water during a measurement. Basis for particle size calculation was the Mie theory with a product refractive index of 1.65 and product absorption of 0.001. The R 2000 obtained data fit reported by the MasterSizer Software indicated validity of the optical model (residual value <1%). Background and measurement integration time were kept at 60 s, measurements were conducted in triplicate (n = 3) per unit sample.
Freeze-Drying Procedure One milliliter of each formulation was filled in 15R vials. Lyophilization was then performed on a laboratory scale freeze-dryer (VirTis Advantage Plus, SP Scientific, Gardiner, New York). Freezing was conducted at −40◦ C (shelf inlet temperature) for 60 min, including equilibrating steps at +5◦ C and −5◦ C for 15 min each. To facilitate crystallization of the Poloxamer 338 in the formulation, an annealing step at −20◦ C (shelf inlet temperature) for 90 min was implemented. The shelf temperature ramp rates from the freezing set point to the primary drying shelf temperature setting were 1◦ C/min throughout the study. The shelf inlet temperature set point during primary and secondary drying was 40◦ C, which is further deJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
noted as “aggressive” freeze-drying. The holding time (soak period) of this step was either 60 or 600 min to allow a modulation in water content in the samples. The chamber pressure during primary and secondary drying was controlled at 100 mTorr throughout the experiments. Note that the freeze-drying cycles were performed in duplicate (n = 2) per formulation. Product temperatures during freeze-drying were measured using calibrated 30 gauge T-type copper/ constantan thermocouples from Omega (Omega Engineering, Stamford, Connecticut). Each thermocouple was introduced through a stopper and positioned bottom center of the vial to obtain both a representative temperature monitoring in the product and an accurate endpoint detection of the ice sublimation phase. Differential Scanning Calorimetry Determination of thermal transitions in all formulation mixtures was performed using a MettlerDSC822e (Mettler Toledo, Greifensee, Switzerland). Data acquisition was performed in the temperature range between 5◦ C and 150◦ C. The applied heating rate was 5◦ C/min. Temperature calibration of the differential scanning calorimetry (DSC) instrument was performed on a weekly basis using the melting point of Indium (156.5◦ C) as a reference. Karl Fischer Residual Moisture Measurements Residual moisture of the lyophilized samples was measured using a Metrohm Karl Fischer 831 KF Coulometer combined with a Metrohm Thermoprep 832 oven unit. About 50 mg of product was weighed into a custom glass vial and then inserted into the oven after purging the sample vial with dry nitrogen. The product was heated to 140◦ C for a defined time period and the moisture was accumulated in the titration solvent. Residual moisture content was reported in percent (%) as a function of the sample weight. Stability Testing Procedure Immediately after freeze-drying, the products (n = 4) were sealed and stored at 25◦ C and 40◦ C/75 RH, respectively. After 1, 2, and 3 months, samples were analyzed in terms of mean particle size and particle size distribution. DSC and KF measurements were performed after freeze-drying as well as after 3 months of storage.
RESULTS AND DISCUSSION Selection of an Adequate Steric Stabilizer to Achieve Storage Stability To prove the concept that Poloxamer 338 is superior to Cremophor EL as a steric stabilizer in terms of storage stability of lyophilized drug nanosuspensions, Poloxamer 338 and Cremophor EL drug nanosuspensions were freeze-dried without any addition of a DOI 10.1002/jps
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Figure 1. Comparison of the particle size distributions of a stock nanosuspension (100 mg/mL Model API I) stabilized with 50 mg/mL Cremophor EL directly after freeze-drying and 3 months of storage at ambient conditions (dashed line). The 5 :m boundary represents the upper particle size limit for a parenteral drug nanosuspension.
Figure 3. Comparison of the particle size distributions of a stock nanosuspension (100 mg/mL Model API I) stabilized with 50 mg/mL Cremophor EL after aggressive process conditions (solid line, shelf temperature: 40◦ C) and conservative process conditions (dashed line, shelf temperature 0◦ C).
lyoprotectant (if possible). However, preliminary experiments revealed for the model formulation containing Model API I that for drug nanoparticle concentrations of 100 mg/mL, a minimum concentration of 50 mg/mL Cremophor EL or 50 mg/mL Poloxamer 338 is beneficial to assure that the desired particle size after lyophilization is consistently obtained below the critical upper limit (Figure 1 and 2).10 Note that the critical formulation temperature (CFT), the temperature which represents the maximum allowable product temperature during primary drying, was determined to be about −73◦ C for the pure drug nanosuspension stabilized with solely Cre-
mophor EL.14 There is no doubt that controlling product temperature during primary drying at or even below the CFT is literally impossible. Surprisingly, the particle size distribution after lyophilization met the particle size prerequisites (cf. Fig. 3), even though shelf temperatures during primary drying were preprogrammed at 40◦ C and resulted in a product temperature for the Cremophor EL drug nanosuspension of about −18◦ C. Indeed, the product temperature exceeded the CFT of roughly 55◦ C, suggesting that vitrification is apparently not mandatory for drug nanoparticle stabilization during primary drying. The higher the observed product temperature during primary drying the smaller the aggregates which were detected (cf. Fig. 3). The observation that a more aggressive drying protocol results in a better particle size distribution may be explained if aggregation is considered as a time-dependent process where the nanoparticles have less time to show physical instabilities during a short than during a long cycle time. This, in turn, would suggest that immobilization is not necessary during this process step. This result confirms a previous study from our laboratory, which revealed that drug nanosuspensions can be aggressively freeze-dried well above the CFT.14 In the course of this study, it was found that nanoparticle and cake stability are not related to a rigid amorphous glassy excipient matrix during an actual freezedrying run but dependent on the combination of the used nanoparticle, steric stabilizer, and lyoprotectant concentration. In contrast to this, we would speculate that months or even years of storage would be sufficient time for particle growth in cases in which diffusion processes are accelerated. Thus, immobilization
Figure 2. Comparison of the particle size distributions of a stock nanosuspension (100 mg/mL Model API I) stabilized with 50 mg/mL Poloxamer 338 directly after freeze-drying and 3 months of storage at ambient conditions (dashed line). DOI 10.1002/jps
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Figure 4. DSC plots of the pure lyophilized drug nanosuspension stabilized only either with Cremophor EL (upper dashed curve) or with Poloxamer 338 (lower solid curve), which are equivalent to Model API I and II. The upper dashed curve illustrates a broad endotherm between 10◦ C and 25◦ C, lower solid curve indicates a melting peak at approximately 58◦ C that originates from the crystalline Poloxamer 338.
during storage should be compulsory to avoid physical instabilities. On the basis of this line of argumentation, Cremophor EL is expected to show a poor long-term stabilization performance. DSC analysis for Cremophor EL-stabilized drug nanosuspensions revealed a broad endothermic signal between 10◦ C and 25◦ C (Fig. 4). Recall that this steric stabilizer was thought to be a reasonable excipient to stabilize drug nanoparticles throughout milling and freeze-drying experiments because particle size distributions measured right after processing were found acceptable. Cremophor EL was found insufficient to impart stability during storage at 25◦ C and, even more distinct, at 40◦ C (Fig. 1). After 3 months of storage at ambient conditions, the nanoparticle size distribution turned into a more excessive bimodal distribution, indicating that strong aggregates have been formed during this period of time. In addition, residual moisture contents above 1% were found to further deteriorate aggregation tendency. The simple explanation for this observation might be grounded in the plasticizing effect of water in the formulation, which results in a decrease of Tg . The DSC plot in Figure 4 illustrates, however, that the melting point of the lyophilized Poloxamer 338 formulation can be found at about 58◦ C, assuming that nanoparticle mobility must be more constrained in this formulation. Figure 2 also confirms this postulation and shows that the measured particle size distribution of the drug nanosuspension stabilized with Poloxamer 338 after 3 months at 25◦ C is rather unimodal. Nevertheless, particle growth could not be fully arrested at such harsh process conditions. In this case, the plasticizing effect of water is not expected to JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
Figure 5. Comparison of the redispersibility indices (RDIs) for the stock nanosuspensions (100 mg/mL Model API I) after 3 months of storage at ambient conditions stabilized with either Cremophor EL (Cr EL) or Poloxamer 338 (P338) as a function of a high or low residual moisture (RM) content. Note that RM did not exceed 2% for the corresponding formulations.
be critical for the (fully) crystalline material. However, calculations of the redispersibility index (RDI) showed that slightly higher residual moisture contents (i.e., above 1% but below 2%) can, at least to some extent, destabilize the system (Fig. 5). The RDI is defined as D0 /D, where D0 is the volume-weighed mean particle size of the reconstituted nanosuspension directly after freeze-drying (time mark = T0 ) and D is the corresponding value after 3 months of storage (time mark = T3 ). An RDI of 100% would therefore mean that the stored freeze-dried drug nanosuspension can be completely transformed to the original particle size at T0 after rehydration.22 Figure 5 confirms the above-described observations obtained from Figures 1 and 2, and additionally underlines that the factor “residual moisture” should be considered as a critical parameter. For the Poloxamer 338 formulations, a water content above 1% led to a significant 3% decrease of the RDI, while the drug nanosuspension stabilized with Cremophor EL suffered a 7% decrease in the RDI when the residual moisture was high (Fig. 5). For Poloxamer 338 drug nanosuspensions, the distinct shift of the unimodal distribution toward larger particles accompanied by a decrease of the volume of the nanoparticle amount (Fig. 2) and a maximum achievable RDI of 93% implies that further formulation work might be necessary for nanoparticle stabilization. In theory, the addition of a lyoprotectant may further immobilize and isolate individual particles. However, when dealing with highly concentrated drug nanosuspensions, excessive concentrations of lyoprotectant would be necessary to achieve the desired effect.5 Experiments showed that mixing commonly used lyoprotectants (e.g., sucrose and trehalose) with DOI 10.1002/jps
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Table 1. d50 , d95 , and RDI Values Obtained Directly After Completion of Freeze-Drying (residual moisture <1%) and After 3 Months of Storage at 25◦ C and 40◦ C, Respectively
Without Protectant
Tr
Su
PVP
Figure 6. Comparison of the particle size distributions of a drug nanosuspension (100 mg/mL Model API I) stabilized with 50 mg/mL Cremophor EL and 50 mg/mL trehalose as a lyoprotectant directly after freeze-drying and 3 months of storage at ambient conditions as a function of a high or low residual moisture content. Note that initial nanoparticle stability was independent of the water content (solid versus dotted line) but after storage residual moisture below 1% (dashed line) revealed better particle stability compared with a water content above 1% (dashed dotted line).
those drug nanosuspensions containing Cremophor EL was not acceptable for storage stability, irrespective of both the nanoparticle concentration and model API used. The final particle size was found to be higher than the specified target in every investigated case and an RDI beyond 80% was never achieved (data not shown), even though the residual moisture content was below 1% (cf. Fig. 6). Note that similar observations were also made with other steric stabilizers, such as Tween 80 (data not shown), and even more importantly with the second drug compound Model API II. It may be concluded from the present set of freezedrying experiments that the physicochemical characteristic of the steric stabilizer plays an important role for subsequent storage conditions. From that point of view, it seems recommendable to preferentially use those steric stabilizers that remain in the solid state after freeze-drying. As only Poloxamer 338 fulfils this prerequisite, all of the subsequent storage stability experiments were performed with Poloxamer 338. As a side note, DSC experiments have demonstrated to be indispensable to evaluate storage conditions when freeze-drying drug nanosuspensions.23 Selection of Adequate Lyoprotectants to Achieve Storage Stability The preliminary experiments mentioned above have indicated that lyoprotectants might improve the longterm stability of drug nanoparticles during storage, DOI 10.1002/jps
T0 T3 at 25◦ C T3 at 40◦ C T0 T3 at 25◦ C T3 at 40◦ C T0 T3 at 25◦ C T3 at 40◦ C T0 T3 at 25◦ C T3 at 40◦ C
d50 Value (:m)
d95 Value (:m)
RDI (%)
0.185 0.185 0.199 0.166 0.168 0.177 0.168 0.172 0.195 0.165 0.159 0.161
1.18 1.89 3.01 0.82 0.85 0.95 0.90 0.94 1.48 0.80 0.78 0.85
– 75% 42% – 98% 94% – 96% 65% – 101% 98%
Note that the calculated standard deviations were small (σ < 0.045 for n = 4) for all formulations, indicating very consistent d50 and d95 . For the RDI values, standard deviations were calculated in the range of 1.27–1.93. Formulation: 200 mg/mL nanoparticle concentration (Model API II) stabilized with 50 mg/mL Poloxamer 338 alone or in combination with 50 mg/mL Polyvinylpyrrolidone (PVP type K15), 50 mg/mL trehalose, or 50 mg/mL sucrose. P338, Poloxamer 338; Tr, trehalose; Su, sucrose; PVP, PVP K15. T0 = d50 , d95 , and RDI values determined directly after freeze-drying, time mark T3 = d50 , d95 , and RDI values after 3 months of storage, time mark T0 = d50 and d95 values measured directly after freeze-drying of the reconstituted formulations.
even in cases when the desired physical stability right after completion of freeze-drying can be achieved without the addition of any lyoprotectant. Table 1 compares the d50 , d95 , and the RDI values after aggressive freeze-drying of formulations containing 200 mg/mL Model API II. Note that the abbreviations d50 and d95 stand for 50% and 95%, respectively, of the particles being smaller in size than the reported value. The purpose of using “aggressive” cycle conditions was to illustrate that even such considerably high particle concentrations do not require vitrification in a lyoprotectant matrix during primary drying for stabilization.24 Product temperatures were constantly maintained well above the CFT in every case (data not shown).14 Results demonstrated that the addition of the stabilizers PVP K15, trehalose, or sucrose in a 50 mg/mL concentration to the drug nanosuspensions further improved nanoparticle stability, relative to the pure lyophilized drug nanosuspension stabilized with Poloxamer 338. Fifty milligram per milliliter was found as the highest possible lyoprotectant concentration to obtain a good compromise between (a) sufficient mechanical cake stability when using aggressive drying conditions and (b) preservation of the original particle size distribution. The rationale behind the use of such materials as “lyoprotectants” might be given by the particle isolation hypothesis. Recall that this hypothesis claims that the colloidal particles represent an emulsion in which the stability is governed by the surface tension of the dispersion medium.25 PVP K15 was found superior JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
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over trehalose or sucrose in providing stability. This might be explained due to its distinct surface-active nature. As a conclusion, drug nanoparticles are sufficiently dispersed in the PVP K15, trehalose, or sucrose solution to overcome aggregation or agglomeration tendencies, as these materials possess relatively low surface tensions compared with other protectants such as starch derivatives.25 The Poloxamer 338 concentration provided in Table 1 reflects the highest possible particle concentration, keeping in mind that this material may cause toxic side effects for the patient. Higher steric stabilizer concentrations were always associated with a better nanoparticle size distribution throughout the present study, which is in good agreement to previous investigations.6,12–14 A set of shortterm stability tests with every single steric stabilizer used in this study confirmed this observation (data not shown). Table 1 also demonstrates that the examined nanoparticle stability right after completion of freeze-drying perfectly matched the anticipated particle size distribution.
Figure 8. Comparison of the DSC plots of the highly concentrated drug nanosuspension (Model API II) stabilized with Poloxamer 338 as a function of PVP K15 (top curve), sucrose (middle curve), or trehalose (bottom curve) in the formulation. Note that only sucrose caused a glass transition before the melting peak of Poloxamer 338 at about 58◦ C.
Storage Stability Investigations of Freeze-Dried Drug Nanosuspensions Table 1 illustrates that after 3 months of storage at ambient conditions, all lyoprotectants used in this study showed an efficient stabilizing effect when the water content was below 1%. In other words, no matter if PVP K15, trehalose, or sucrose were used, an RDI of at least 96% was obtained, relative to a maximally achievable RDI of 75% after storage of the pure drug nanosuspension stabilized only with Poloxamer 338 (Table 1). Note that these results are also in good
Figure 7. Comparison of the particle size distributions of a highly concentrated drug nanosuspension (200 mg/mL Model API II) stabilized with 50 mg/mL Poloxamer 338 and 50 mg/mL PVP K15 as a lyoprotectant showing that the original particle size distribution after freeze-drying (solid line) was completely preserved after 3 months of storage as well as at 25◦ C (dashed line) and 40◦ C (dotted line). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
agreement to previous studies performed with a lower drug to steric stabilizer ratio. For comparison, the data presented in the previous chapter revealed an RDI of 93% after 3 months of storage at 25◦ C for the pure drug nanosuspension stabilized with only Poloxamer 338. Here, the effective amount of Poloxamer 338 was twice as high related to the nanoparticle concentration. Accelerated storage stability testing at 40◦ C showed after 3 months that solely PVP K15 was suitable to prevent nearly completely particle growth (average RDI value of 98%). In addition, Figure 7 shows that for those formulations containing 50 mg/mL PVP K15, similar particle size distributions were obtained directly after freeze-drying and storage at 40◦ C for 3 months. To better explain these results, thermal analysis by DSC was included into the data interpretation (Fig. 8). For the drug nanosuspension containing sucrose, a weak glass transition at about 30◦ C is noticeable prior to the melting peak of Poloxamer 338, which is consistently reproducible. It would be expected that the (amorphous) sucrose phase influences the crystallization tendency of Poloxamer 338 during freezing. Early experiments showed that this is indeed the case which, in turn, suggests that at least a part of the Poloxamer 338 in the formulation remains amorphous.14 The observed glass transition, which reflects a contribution of both amorphous sucrose and Poloxamer 338 might then induce the particle growth due to enhanced nanoparticle mobility during storage at high temperatures. A decrease of the corresponding RDI from 96% to 65% might prove this hypothesis (cf. Table 1). A higher residual moisture content in the sucrose formulation was found, DOI 10.1002/jps
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which further deteriorates particle stability because water inevitably decreases the Tg below storage temperature (data not shown). The reason for PVP K15 being superior in providing stability over, for example, trehalose at 40◦ C storage conditions could not be pinpointed by DSC. Both plots illustrate a melting event between 55◦ C and 58◦ C, which originates from the Poloxamer 338. However, no glass transition or other thermal event can be detected prior to the Poloxamer 338 melt (Fig. 8). It could be speculated that the rather early “onset” of the melting endotherm at about 40◦ C might influence the particle growth at 40◦ C, but the same course in transition is also found in the PVP K15 formulation. Both lyoprotectants cause an endothermic signal of more than 100◦ C (data not shown), which is conform to the described Tg s in the literature for PVP and trehalose, respectively.2 For these reasons, DSC analysis seems to be insufficient to depict the significant differences in terms of improved physical stability stabilization when using PVP K15 instead of trehalose. One approach to find an appropriate explanation for these findings might be related to the surface-active properties of the two excipients. The drug nanoparticles seem to remain better dispersed in a rigid PVP K15 matrix, resulting in a higher degree of dispersity and hence more isolated colloidal particles, relative to the trehalose matrix. Moreover, as the measured glass transitions of PVP K15 and trehalose are high, one may assume that the degree of immobilization of both matrices after freeze-drying is comparable, even at 40◦ C storage temperature conditions. Against this background, the explanation of the superior stabilization capacity of PVP K15 by its lower surface tension seems reasonable which, in turn, is claimed by the particle isolation hypothesis.25 In addition, the relatively low melting point of Poloxamer 338 seems critical for accelerated stability tests at 40◦ C. Although not always desirable in practice, stability tests at 25◦ C over a period of 1 year (or longer) appear to be more reliable to evaluate the storage stability of drug nanosuspensions. As a consequence, not only PVP K15 but also trehalose and sucrose appear to be suitable lyoprotectants to prevent physical instabilities of lyophilized drug nanosuspensions. As a proof of concept, it should be mentioned that at least trehalose completely inhibited particle growth over 6 months at ambient temperature in the course of the present study (data not shown). To be more accurate, the corresponding particle size distribution and RDI value did not indicate a significant deterioration in particle size. Note that this result is in contradiction to the results obtained from accelerated stability testing. It is well known that in this case, stability is derived from the Arrhenius equation.21 This model describes the relation between a specific temperature and its corresponding reaction rate constant for homogeneous chemical reactions. However, the validity DOI 10.1002/jps
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of Arrhenius law is only strictly given when the relation between the logarithmic reaction rate is constant and the reciprocal temperature is linear.21 Only in such a case, it is possible to extrapolate from higher storage to lower storage temperatures. Therefore, it is strongly recommendable to critically interpret stability data obtained from storage stability testing at higher temperatures and related to physical stability of lyophilized nanoparticles. Again, DSC plots in this set of experiments revealed that at 40◦ C storage temperature, nanoparticle stability could be compromised disproportionately because the drug nanoparticles could remain in a partly melted environment. In contrast, at 25◦ C, the drug nanoparticles are expected to be fully arrested. As a consequence, misleading conclusions regarding the choice of freeze-drying stabilizing agents for nanoparticles and long-term stability of highly concentrated drug nanosuspensions could be drawn. DSC analysis must be used to evaluate adequate storage conditions after freeze-drying of drug nanosuspensions, specifically if accelerated stability testing at 40◦ C and 75% RH is expected to be predictive.
CONCLUSION In the present study, preservation of the original particle size distribution of freeze-dried highly concentrated drug nanosuspensions was achieved during short-term stability experiments at ambient and accelerated stability testing conditions (40◦ C/75% RH). It could be demonstrated for the first time that the nature of the steric stabilizer is of particular importance for storage stability of lyophilized drug nanoparticles. Cremophor EL was found inappropriate to stabilize the drug nanosuspensions. Even addition of the lyoprotectants PVP K15, trehalose, or sucrose was found to fail in the preservation of the initial particle size distribution during storage when Cremophor EL was used. In contrast, Poloxamer 338 was clearly identified as the steric stabilizer of choice. This further underlines the relevance of the role of the steric stabilizer for long-term stability. In addition, higher residual moisture contents were found to compromise the aggregation tendency, presumably due to the plasticizing effect. In case of a rational choice of lyoprotectants, even nanoparticle concentrations of 200 mg/ mL could be stabilized over a time period of at least 3 months, even when the formulation was freeze-dried using rather aggressive cycle conditions. According to the surface-active properties and based on its high Tg in the solid state, PVP K15 was identified to be superior over trehalose and sucrose as a freeze-drying stabilizer. Lastly, DSC analysis has proved again to be an indispensable tool when evaluating storage conditions and stability for drug nanosuspensions. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
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DOI 10.1002/jps