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Amorphous Active Pharmaceutical Ingredients in Preclinical Studies: Preparation, Characterization, and Formulation Karthik Nagapudi* and Janan Jona Small Molecule Process and Product Development, Amgen Inc. Thousand Oaks, CA, USA Abstract: A large number of the new pharmaceutical small molecules under development today are found to have poor water solubility. This in turn may lead to low bioavailability, which can have a significant impact on the development of the compound. Compounds with low bioavailability pose a greater challenge in early preclinical work involving animal studies, where obtaining maximum exposure is the primary goal especially in toxicology studies designed to establish the safe dose. From the standpoint of maximizing exposure, the amorphous phase is of great interest as pharmaceutical materials since it is the most metastable state and as such offers the potential of higher solubility and better bioavailability. However, the amorphous approach is not actively pursued in preclinical work owing to the tendency of the amorphous phase to crystallize thereby neutralizing the solubility advantage. This review focuses on (i) methods to generate the amorphous phase, (ii) methods to estimate the degree of crystallinity of the amorphous phase, (iii) methods to predict the stability of the amorphous phase against crystallization, and (iv) choice of polymers carrier and formulation of the amorphous phase for preclinical studies.
Keywords: Amorphous pharmaceuticals, crystallization, preclinical, solid dispersions. 1. INTRODUCTION Increased use of high throughput methods in drug discovery and lead optimization has lead to compounds with more lipophilic properties that exhibit poor water solubility. The number of poorly soluble compounds range from 4050% of the entire portfolio of new molecules in major pharmaceutical companies. Such compounds under the biopharmaceutical classification system (BCS) fall into class II and IV [1, 2]. Class II compounds have low aqueous solubility and high intestinal permeability while class IV compounds have low aqueous solubility and low intestinal permeability. While low solubility compounds may pose significant development challenge in the clinical arena, these challenges are exacerbated in the preclinical arena where the goal is to increase exposure many fold when compared to the therapeutic dose. For example, preclinical toxicology studies often aim to maximize exposure in order to assess the detrimental effects of the development compound. In these studies it is not uncommon for doses to be as high as 100200 times the predicted ED50 dose in humans [3]. In case of compounds with solubility limited absorption, exposure tends to plateau at a dose beyond which it does not increase. Thus there is a much greater need in the preclinical arena to increase exposure and as such preformulation groups are actively involved in this area. While formulation approaches have minimal impact on permeability, they can play a significant role in improving solubility/dissolution rates. As such increasing importance is being given to devising new and better formulation approaches for poorly water soluble compounds. To this end several approaches such as changing polymorphic form, particle size reduction, salt formation and use of solubility enhancers have been attempted. In this *Address correspondence to this author at the Small Molecule Process and Product Development, Amgen Inc. Thousand Oaks, CA, USA; E-mail:
[email protected] 1573-4072/08 $55.00+.00
context, utilizing the amorphous phase of the compound is gaining relevance for pharmaceutical materials of poor solubility since it is the most metastable state and as such offers the promise of highest solubility and improved dissolution rates. The amorphous phase is usually defined with reference to its crystalline counterpart. Whereas the crystal is defined by specific long range order, the amorphous phase is defined by the lack of such long range order. Hancock et al. have shown that the solubility advantage of the amorphous phase is likely to be several-fold higher than their crystalline counterparts [4]. In spite of the solubility advantage provided by amorphous materials, they are largely avoided during drug development due to physical/chemical stability issues and processing difficulties. The amorphous form tends to be more chemically unstable than their crystalline counterparts. However from a development standpoint, the physical instability is the most problematic. Implicit in the term physical stability is the resistance of the amorphous material towards reversion to the thermodynamically favored crystalline phase. There have been a number of publications in the last few years where issues pertaining to amorphous solids have been discussed [5-9]. Typically, physical stability has been discussed in terms of relaxation times (molecular mobility) that have been measured spectroscopically or calorimetrically. In spite of the progress made, predicting the propensity for amorphous materials to crystallize still remains challenging. While the problem of predicting amorphous stability against crystallization is more acute for clinical development it is less so for preclinical studies. From the preclinical standpoint moderate stability of three to six months for the solid active pharmaceutical ingredient (API) and about a week worth of stability of the API in the formulation is sufficient to progress the amorphous compound through all the required preclinical studies.
© 2008 Bentham Science Publishers Ltd.
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are widely available techniques which require very little in terms of training. The final product of milling among other things will depend on the milling temperature and its relationship to glass transition temperature of the API and the strength of the crystal. Descamp et al. have shown that the milling product will be amorphous if the milling temperature is below the glass transition temperature of the API [15]. Thus cryogrinding that is performed at liquid nitrogen temperatures is more likely to produce amorphous material than ball milling but is more laborious to operate. Desolvation is a specialized technique that is also described in literature [16, 17] for those organic solvates that desolvate to produce an amorphous phase. Depending on the desolvation temperature this can be a useful technique to produce gram quantities of amorphous phase. However of the techniques mentioned so far, desolvation is the only one that cannot be used to make ASD. Also of note is that the methods that are not scalable are dry methods of making the amorphous material i.e. methods without involvement of solvent in the process.
Thus, the issues pertaining to the development of amorphous materials for preclinical studies are quite different from that for clinical studies. With the development of increasingly potent compounds where the therapeutic doses are low (0.15 to 1.5 μg/Kg) the real advantage of the amorphous materials for low solubility compounds are expected to be in the preclinical area rather than the clinical area. While there are many papers that deal with amorphous materials in general, there are no papers that exclusively focus on development of amorphous materials for use in the preclinical area. This review aims to fill this gap in the literature. This paper focuses on (i) methods to generate the amorphous phase, (ii) methods to estimate the degree of crystallinity of the amorphous phase, (iii) methods to predict the stability of the amorphous phase against crystallization, and (iv) choice of polymer carriers and formulation of the amorphous phase for preclinical studies. 2. METHODS TO GENERATE THE AMORPHOUS PHASE In order to assess the suitability of the amorphous phase for preclinical work a number of studies have to be performed. To facilitate these studies, the amorphous active pharmaceutical ingredient (AAPI) or the amorphous API in combination with a pharmaceutically acceptable polymer must be generated. API-polymer combinations that are amorphous are denoted as amorphous solid dispersions (ASD). Table 1 lists the typical lab scale techniques that can be used to make amorphous materials. While a number of these lab-scale techniques are suitable for making a few hundred milligrams to a gram of AAPI/ASD, they cannot be scaled up. Melt quenching is a useful technique to produce small quantities of amorphous phase for preliminary evaluation. Melt quenching can only be applied to API’s that are thermally stable. There are some reports in literature of using melt quenching to produce the AAPI/ASD [10-12]. However, the materials produced have been predominantly used to gauge amorphous stability against crystallization. Milling techniques such as ball milling and cryogrinding [13, 14] are also useful to make small quantities of amorphous materials. Their primary advantage lies in the fact that they Table 1.
1
The remaining three methods namely: Lyophilization, spray drying and precipitation are capable of being run on scale. Lyophilization is highly limiting in its scope when applied to organic small molecules with poor aqueous solubility. Although lyophilization can be performed with organic solvents it is not a preferred technique since complete removal of solvent from the system may be challenging. Spray drying [18] and precipitation techniques have both been used to make large quantities of AAPI and ASD material. While both techniques are comparable in the quality of AAPI they produce, they differ in the quality of the ASD they produce. For making ASD materials spray drying is generally a better method than precipitation as it gives more phase mixed material. Phase separation between the API and the polymer is not desired while making the ASD’s since this can cause crystallization of the API [19]. Precipitation technique can also be optimized to yield ASD that are better phase mixed, however this may take more time and effort. While spray drying and precipitation remain preferred methods of generating AAPI and ASD, the yields from these processes are different. Yield is an important
Methods of Preparing Amorphous API and Amorphous Solid Dispersions Technique
AAPI1
ASD2
Yield3
Scalability
Melt quenching
Y
Y
60-70%
N
Ball Milling
Y
Y
50-70%
N
Cryogrinding
Y
Y
40-50%
N
Lyophilization
Y
Y
75-85%
Y
Spray drying
Y
Y
40-75%
Y
Desolvation4
Y
N
-
N
Precipitation
Y
Y
75-85%
Y
AAPI: Amorphous active pharmaceutical ingredient 2 ASD: Amorphous solid dispersions (API + Polymer) 3 Low end represents yields at small scale ( 1g). 4 Applicable only when amorphous phase is formed upon desolvation. Y: Yes; N: No
Amorphous Active Pharmaceutical Ingredients in Preclinical Studies
consideration especially in preclinical studies, where the quantity of material available for evaluation is limited. Precipitation in general has higher yields than spray drying. Yields for spray drying can be improved at larger scale (>1 gram). Another important technique that has been used in the past to generate large quantities of AAPI or ASD is hot melt extrusion [20]. Hot melt extrusion has not been included in the list in Table 1 since it requires a minimum of 2 to 5 grams to operate and as such is not a useful technique for preclinical evaluation studies. 3. METHODS TO QUANTIFY THE AMOUNT OF CRYSTALLINITY IN THE AMORPHOUS PHASE Even small amounts of crystalline material can profoundly affect the in-vivo performance of the amorphous material [4]. Thus, once the AAPI or ASD is generated, it is important to have a good analytical tool to monitor the crystallinity in the samples. There are numerous analytical tools that are available to quantify the crystallinity based on differences in physico-chemical properties between the crystalline and amorphous phases (e.g. density, viscosity, xray diffraction and so forth) [21]. X-ray powder diffraction (XRPD) [22-25] and differential scanning calorimetry (DSC) [26-28] have been widely used for this purpose as these techniques are usually available in most pharmaceutical labs. Limit of detection (LOD) and the limit of quantitation (LOQ) of crystallinity often tend to be better with DSC. DSC also requires much less material than XRPD. DSC in both standard and modulated modes has been used to quantify extent of crystallinity [29, 30]. An example of crystallinity quantitation for Lactose using modulated DSC is shown in Fig. (1). The specific heat capacity change across glass transition is used to build the calibration curve. Use of spectroscopic tools such as Raman, IR, and NIR has also been reported in literature for crystallinity quanti-
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tation [31-34]. With the advent of chemometric techniques, spectroscopic methods have become attractive tools for phase quantitation. They provide chemically resolved information with sparse material requirements. However, data interpretation from spectroscopic methods can run into problems because of their inability to unambiguously separate peaks from different phases in the sample. Solid state NMR (SSNMR) is another powerful technique that has not been that widely used [27, 35]. SSNMR’s primary advantage lies in its selectivity and its ability to probe a variety of nuclei. Among the methods mentioned so far SSNMR is the only technique that does not require a pure reference standard for phase quantitation. LOQ down to 0.25% can be achieved when 1H, 31P, or 19F nuclei are used for quantitation while LOQ of about 3% can be achieved when using 13C. In spite of its inherent advantages SSNMR usage has been limited owing to instrument availability and large sample size requirements (60-200 mgs). An example of crystallinity quantitation for Sulindac using 19F SSNMR is shown in Fig. (2). The differences in 19F T1 relaxation rates can be used to quantify the extent of crystallinity. Solution calorimetry is another popular technique to determine extent of crystallinity, as the amorphous phase has in general a different heat of dissolution than the crystalline phase. Over the last few years a host of other techniques such as thermally stimulated depolarization current (TSDC) [36], dielectric (DES) and dynamic mechanical analysis (DMA) have also been used to quantify crystallinity. While a number of techniques are available crystallinity quantitation, XRPD, DSC, and SSNMR remain the primary tools. The quantitation of the crystallinity in ASD can be more challenging than in the AAPI. The amount of the API in the ASD material could be as low as 10% and as such interference from the polymer becomes the major challenge
(a) (b) Fig. (1). Amorphous quantitation of spray dried lactose using modulated DSC. (a) Modulated DSC thermogram of spray dried lactose showing total heat flow and reversing heat flow. Glass transition temoperature (Tg): 81.2 ºC and specific heat capacity change across glass transition (Cp) = 0.5 J/g/ºC. (b) Calibration curve for determining the weight percentage amorphous lactose in crystalline lactose showing the specific heat capacity change across glass transition temperature as a function of amorphous lactose content. R2 =0.99.
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(a) (b) Fig. (2). (a) 19F SSNMR spectra of crystalline and amorphous Sulindac. The crystalline line shape is narrower than the amorphous line shape. (b) 19F T1 relaxation data for crystalline and amorphous material. T1(crystal) = 72 seconds, T1(amorphous) = 2.1 seconds. The difference in relaxation rates can be used to quantify extent of crystallinity.
in crystallinity quantitation. While, IR and Raman spectroscopies may work in some instances, in the majority of the cases DSC and SSNMR are the primary tools. SSNMR owing to its chemical selectivity can separate excipient peaks from the API peaks. Moreover, if 19F nuclei are present in the API, the API can be independently monitored from that of the polymer [37]. Summary of the methods used to estimate the extent of crystallinity is provided in Table 2. It must also be borne in mind that the methods used to quantify crytsallinity require AAPI or ASD relatively free of chemical impurities. Presence of chemical impurities can confound phase quantitation.
when the amorphous is used solely for the purpose of preclinical development, stability in the range of few months is adequate. The shorter period of stability requirement makes the amorphous phase a more viable candidate for preclinical development. Thus from a preclinical perspective, the best way of gauging amorphous stability remains stressing the amorphous phase of known particle size under typical accelerated conditions (25 ºC/60% relative humidity or 40 ºC/75% relative humidity) for the required length of time. A stability program is then established where the amorphous material is withdrawn periodically and the degree of crystallinity and glass transition are monitored using the preferred analytical technique. Such stability programs take time to implement and the therefore it is preferable to have methods to predict the physical stability of the amorphous phase.
4. METHODS TO MEASURE AND PREDICT THE PHYSICAL STABILITY OF THE AMORPHOUS PHASE 4.1. Solid State Stability of AAPI
Considerable progress has been in the last decade in understanding the factors that influence crystallization from the amorphous phase. In spite of the progress, prediction of the physical stability of the amorphous phase remains
When amorphous phase is considered as a candidate for clinical development, physical stability under normal storage conditions is expected to be in the order of years. However, Table 2.
1
Methods of Quantifying Crystallinity in Amorphous API and Amorphous Solid Dispersions Technique
AAPI
ASD
LOQ1
LOD2
X-ray
Y
N
5-10%
2-5%
DSC
Y
M
1-5 %
