Drug nanocrystals in the commercial pharmaceutical development process

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ARTICLE IN PRESS International Journal of Pharmaceutics xxx (2012) xxx–xxx

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Drug nanocrystals in the commercial pharmaceutical development process Jan P. Möschwitzer ∗ Pharmaceutical Development, Abbott GmbH & Co. KG, Knollstr. 50, 67061 Ludwigshafen am Rhein, Germany

a r t i c l e

i n f o

Article history: Received 18 July 2012 Received in revised form 13 September 2012 Accepted 14 September 2012 Available online xxx Keywords: Drug nanocrystals Nanosuspension Wet ball milling High pressure homogenization Dissolution rate limited

a b s t r a c t Nanosizing is one of the most important drug delivery platform approaches for the commercial development of poorly soluble drug molecules. The research efforts of many industrial and academic groups have resulted in various particle size reduction techniques. From an industrial point of view, the two most advanced top-down processes used at the commercial scale are wet ball milling and high pressure homogenization. Initial issues such as abrasion, long milling times and other downstream-processing challenges have been solved. With the better understanding of the biopharmaceutical aspects of poorly water-soluble drugs, the in vivo success rate for drug nanocrystals has become more apparent. The clinical effectiveness of nanocrystals is proven by the fact that there are currently six FDA approved nanocrystal products on the market. Alternative approaches such as bottom-up processes or combination technologies have also gained considerable interest. Due to the versatility of nanosizing technology at the milligram scale up to production scale, nanosuspensions are currently used at all stages of commercial drug development, Today, all major pharmaceutical companies have realized the potential of drug nanocrystals and included this universal formulation approach into their decision trees. © 2012 Published by Elsevier B.V.

1. Introduction Drug nanocrystals are without any doubt one of the most discussed drug delivery technology of the past twenty years. In contrast to other nanoparticulate systems, drug nanocrystals consist mainly of pure API. They are often prepared in aqueous media which contain stabilizers for the colloidal state; therefore these systems can also be referred to as nanosuspensions. Often drug nanocrystals are referred to as drug nanoparticles. This leads to confusion with polymeric nanoparticles, which have normally a significantly lower drug load since they consist mainly of a polymer material loaded with API. The pioneering works of many academic as well as industrial researchers have laid the foundation for broad utilization and acceptance of this approach within the field of pharmaceutical sciences. Before the first top-down processes were developed (i.e. techniques reducing the size of larger crystals by means of attrition forces) nanosized drug particles had to be produced by using rather simple precipitation techniques, also referred to as bottomup approaches. However, it is often very difficult to control the particle growth using this technique. Therefore it was suggested to perform the precipitation step in conjunction with immediate lyophilization or spray-drying in order to reduce the risk of crystal growth (Sucker, 1998). Due to the difficulty in controlling the

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process, bottom-up techniques could not gain enough interest to become a standard approach in the pharmaceutical industry at that time. Around the 1990s Gary Liversidge and his colleagues from Sterling Drug Inc./Eastman Kodak have applied a wet media-based milling technique (wet ball milling, WBM), adapted from the paint and photographic industry, to reduce the particle size of poorly water-soluble drugs (Liversidge et al., 1992; Liversidge and Conzentino, 1995). This process has evolved since and eventually became well known as NanoCrystal® technology in the pharmaceutical industry and is to date the most successful nanosizing approach with currently 5 products on the market (see Table 1) (Merisko-Liversidge and Liversidge, 2011). In 1994 Müller and his colleagues have developed an alternative technology based on piston gap high pressure homogenization (HPH) to produce nanosuspensions (Müller). This technology was named as DissoCubesTM , according to the cubic shape of the drug nanocrystals produced with this process (Müller et al., 2003). Later this technology was acquired by SkyePharma PLC and is currently offered in addition to other size reduction technologies such as the insoluble drug delivery microparticle technology (IDD-PTM ) (Keck and Müller, 2006; Shegokar and Müller, 2010). This technology, also referred to as Microfluidizer technology, is a typical top-down process which is based on jet-stream homogenization. The drug is pumped under high pressure of up to 1700 bar through a microfluidizer system (Junghanns and Müller, 2008). In the collision chamber of either Z-type or Y-type it comes to particle collision, shear forces and cavitation forces leading to the desired

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Parenteral, Intramuscular Janssen NanoCrystal® (WBM) Modified after Möschwitzer and Rainer (2011).

Paliperidone palmitate Invega® Sustenna® Xeplion®

2009

Oral Oral Oral Fournier Pharma, Abbott Laboratories Sciele, Shionogi Pharma Inc. PAR Pharmaceuticals NanoCrystal® (WBM) IDD-P® (HPH) NanoCrystal® (WBM) 2004 2005 2005 Fenofibrate Fenofibrate Megestrole acetate Tricor® Lyphantyl® Triglide® Megace® ES

Oral Merck NanoCrystal (WBM) 2003 Aprepitant Emend

Pfizer (Wyeth)

Oral

Reformulation, Patient friendly tablet instead of solution NCE, High bioavailability, no food effects Reformulation, No food effects Reformulation, No food effects Reformulation, No food effects, more patient friendly Reformulation ®

NanoCrystal (WBM) 2000 Sirolimus Rapamune

®

Adminstration route Company Nanosizing technology

®

FDA approval INN name Trade name

Table 1 Marketed products containing drug nanocrystals.

®

Therapeutic benefit

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particle size reduction. The resulting particle size is preserved by the use of various phospholipids or other surfactants and stabilizers. Due to the relatively low power density of the standard equipment, up to 50 or more passes of the suspension are necessary for a sufficient particle size reduction (Mishra et al., 2003). In 1999 the Nanopure® technology was developed, another variant of a piston-gap homogenization process, which is conducted with water-reduced or even water-free liquids as dispersion media (Müller; Radtke, 2001). By reducing the particle size to the nanometer range, the bioavailability of many poorly soluble compounds could be improved significantly, which eventually led to a broader acceptance of the WBM and HPH techniques as enabling technology. Today, the two techniques are by far the most industrial relevant technologies to produce drug nanocrystals. Six different commercial pharmaceutical products based on nanosizing approaches have already been approved (Table 1). Following the success of these two technologies, this has triggered the development of completely new or slightly different technologies. During the first ten years after the invention of the NanoCrystal® technology various groups, mainly specialized drug delivery companies but also academic research groups, have embarked on the “Nano” approach. They have developed their own technology to provide alternative solutions to their customers. Alternative technologies have mainly been developed based on bottom-up approaches, mainly because of more freedom to generate new intellectual property (IP) (Chan and Kwok, 2011; de Waard et al., 2011). Basically, these approaches have the common goal to provide better process control for producing nanoparticulate structures with enhanced dissolution characteristics. Precipitation in the presence of special polymers to prevent crystal growth was successfully applied for some APIs, such as ibuprofen, itraconazole and ketoconazole (Rasenack and Müller, 2002a,b). The precipitation can also be performed at elevated temperatures (Evaporative Precipitation into Aqueous Solution, EPAS) (Chen et al., 2002). Furthermore, organic drug solutions can be sprayed into cryogenic liquids using the SFL technology (SFL: spray freezing into liquid technology) (Hu et al., 2003). Upon contact with the cryogenic liquid (e.g. liquid nitrogen) the droplets are frozen. A subsequent lyophilization step removes the organic solvent. Due to the mild process conditions this technology is suitable for temperature sensitive molecules, such as biological molecules (Yu et al., 2004). Alternatively, precipitation can be performed in conjunction with centrifugation techniques (High gravity precipitation) (Chiou et al., 2007). In recent years many drug delivery companies have started to develop production methods for drug nanocrystals based on supercritical fluid technologies (Fages et al., 2004; York, 1999). In cases where the drug is soluble in supercritical fluids, such as supercritical carbon dioxide, the RESS technology (RESS: Rapid Expansion from Supercritical Solutions) can be applied (Maston et al., 1987). In contrast, in many cases the supercritical gas is used as antisolvent for the drug. Mixing of an organic drug solution with the supercritical antisolvent leads to a precipitation of nanometer-sized drug particles, which are collected in various ways. The general principle is referred to as gas antisolvent technology. Depending on process conditions and mixing types, various process variants exist (e.g. GAS: gas antisolvent process, SAS: supercritical antisolvent process, SEDS: solution enhanced dispersion of solids) (Byrappa et al., 2008). Although the results obtained with these alternative approaches are very promising, they are currently not as widely used in the pharmaceutical industry to produce drug nanocrystals. Most of these approaches require custom-made production equipment and special processing expertise, which limits their applicability mainly to dedicated research groups.

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It is important to mention in this context that the development of drug nanocrystals requires suitable analytical techniques such as microscopic techniques or particle size analysis. These technologies have also evolved over time. Nowadays, the equipment is much more user-friendly than some years ago. Particle characterization can be easily performed on routine basis and the results are available within minutes (Levoguer, 2012). This review will focus mainly on drug nanocrystals produced by applying the industrial relevant and well-established top-down technologies wet ball milling as well as high pressure homogenization. Obviously like many other newly developed technologies the top-down approaches had initially also some drawbacks. The discussion will focus on how the top-down particle size reduction technologies have evolved over the years to mature as established techniques which are now widely accepted and frequently applied by the pharmaceutical industry. In addition, this review will discuss how drug nanocrystals in general can be successfully used as enabling technology for poorly water-soluble drugs.

2. Technological aspects for the production of drug nanocrystals 2.1. Wet ball milling Wet ball milling (also referred to as pearl milling or bead milling) is by far the most frequently used production method for drug nanocrystals in the pharmaceutical industry. The milling procedure itself is rather simple; therefore this process can be basically performed in almost every lab. The easiest way of doing WBM is through low energy ball milling (LE-WBM) using a jar filled with milling media (often just very simple glass beads). This system is charged with coarse drug substance, preferably in micronized form, which is suspended in dispersion medium containing at least one stabilizing agent. By moving the beads either with an electric stirrer (Fig. 1a), e.g. a magnetic stirrer, or by moving the whole jar, e.g. with a roller plate or a mixer (Fig. 1b), the milling beads can interact with the drug particles. At the beginning of the nineties, very similar set-ups were used in order to establish this technology for pharmaceutical purposes. The relatively low energy input leads to very long milling times of several days (Liversidge et al., 1992; Liversidge and Conzentino, 1995; Merisko-Liversidge et al., 1996). The comminution process itself is caused by abrasion, cleavage and fracturing (Hennart et al., 2012). For LE-WBM a combination of cleavage and abrasion can be assumed as the main mechanism of size reduction principles, as the process generally yields very fine particles with a narrow size distribution when it is performed long enough. Alternative milling procedures based on high energy processes had to be developed in order to make this process more desirable for industrial pharmaceutical applications. The NanoCrystalTM process in its current form is based on such a high energy wet ball milling process (HE-WBM) (Merisko-Liversidge and Liversidge, 2008). A necessary prerequisite for HE-WBM is the availability of suitable equipment. The manufacturers for milling equipment had to develop equipment with sufficiently high power densities for the improved processes. Today, HE-WBM can be regarded as a standard procedure to produce nanosuspensions. Due to the much higher power density, the production times are significantly reduced. Normally, the drug needs to be exposed to the high energy for about 30–120 min in order to achieve a nanosuspension of good quality (Merisko-Liversidge and Liversidge, 2011). Agitated ball mills have the advantage that they can be operated in discontinuous mode (often referred to as batch mode) or in continuous mode (often referred to as re-circulation mode). Typically, the current standard for large scale production is often using agitated ball mills in re-circulation mode. These mills have media separators, either

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as separating gap system or as filter cartridge, to hold the milling medium back in the milling chamber, when the nanosuspension is circulating (Kwade, 1999b). The suspension is pumped from the hold tank with a certain velocity through the milling chamber. Only within the relatively short passage period (i.e. the residence time in the chamber) the drug particles are exposed to the energy input and reduced in size. The comminution is a result of shear stresses and compression forces inside the milling chamber (Kwade, 1999a). The drug particles are reduced in size by abrasion and cleavage mechanisms (Hennart et al., 2012). It is obvious that high energy mills require special milling media which has to be properly selected based on the material of the inner surfaces of the mill, the agitator types and other factors. Using just glass beads or zirconium oxide milling beads can lead to significant contamination of the nanosuspension caused by the abrasion either of the milling beads or parts of the milling chamber (Hennart et al., 2010; Juhnke et al., 2012). Initially, impurities caused by abrasion were one of the major obstacles for a broader acceptance of WBM. Therefore, a major milestone for the broad acceptance of the milling process was the introduction of highly crosslinked polystyrene beads as milling media (Bruno, 1992; Kesisoglou et al., 2007; Merisko-Liversidge et al., 2003). This milling media shows elastic deformation, thereby the formation of cracks and abrasion from beads is reduced. Nowadays, the commercial NanoCrystal® process is performed with special PolyMillTM media, i.e. polysterene beads with a diameter of about 0.5 mm (Kesisoglou et al., 2007). This leads to product qualities which allow the usage of nanosuspensions even for parenteral administration (Merisko-Liversidge and Liversidge, 2011). In the early nineties, there was no equipment available to produce nanosuspensions at very small scale. Hence, it was difficult to use this formulation approach for discovery purposes. Initially several grams of API were needed to produce prototype formulations (Liversidge et al., 1992). Today, even high energy mills are available for small scale production of nanosuspensions. Several research groups have reported ways to use existing planetary ball mills with modified sample holders which can be used to process several nanosuspensions at the same time (Juhnke et al., 2010; Van Eerdenbrugh et al., 2009a). Alternatively, agitated ball mills are used for drug quantities starting from 10 mg (Merisko-Liversidge and Liversidge, 2011). Using these mills it is now possible to produce nanosuspensions during the early discovery phase of the formulation development or to perform stabilizer screening studies with a minimal API consumption. With the commercial availability of suitable equipment for small scale production up to the commercial scale production, wet ball milling can be regarded as scalable approach. This aspect has definitely helped for broader acceptance of this rather complex technology (Merisko-Liversidge and Liversidge, 2011). The versatility of wet ball milling is certainly another, if not the most important aspect for the success of this technology. Almost any API can be processed with wet media milling (Cooper, 2010). Additionally, in most cases aqueous solutions of electrostatic surfactants in combination with cellulosic polymers can be used as stabilizing vehicles (Cerdeira et al., 2010; Van Eerdenbrugh et al., 2009b; Wu et al., 2011). Interestingly, most particle sizes reported for nanosuspensions prepared by wet ball milling are in the range between 100 and 300 nm, irrespectively whether LE-WBM or HEWBM was used. Table 2 gives a snapshot of some examples found in the literature. Overall, the reported particle sizes of the various APIs illustrate again the universal applicability of this particle size reduction method. Based on the reported results it can be stated that wet ball milling is in general superior over standard high pressure homogenization in terms of the achievable particle sizes. All these aspects have opened the possibility to use wet ball milling as a platform technology for formulating poorly soluble compounds.

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Fig. 1. Setup for low energy wet ball milling. (A) Vial filled with milling beads, suspension and a magnetic bar placed on a magnetic stirrer plate, the beads are moved by the rotating magnetic bar inside the vial. (B) Plastic bottle (small picture lower right) filled with milling beads and suspension moved by a standard mixer, the whole system is moved.

Table 2 Literature examples for drug nanocrystals prepared by high pressure homogenization or wet ball milling, respectively. No

Drug

Top-down method

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 32 33 34 35 36 37 38 39 40 41 42 44 45 46 47 48 49 50

PX-18 Asulacrine Ubc-35440-3 Indometacin Prednisolone Resveratrol Danazol Hesperitin Celecoxib Ascorbyl palmitate Azithromycin Tarazepide Spironolactone Omeprazole RMKP22 Amphotericin B Hydrocortisone Budenoside Bupravaquone Clofazimine Nimodipine Rutin RMKK98 Oridonin Dexamethasone Diclofenac Itraconazole Candesartan cilexetil Crystalline API Loviride Ketoconazole Cyclosporine Camptothecin Piposulfan Piposulfan Cilostazol Etoposide Griseofulvin Naproxen Paclitaxel Hydrocortisone Cinnarizine 1,3-Dicyclohexylurea

HPH HPH HPH HPH HPH HPH HPH HPH HPH HPH HPH HPH HPH HPH HPH HPH HPH HPH HPH HPH HPH HPH HPH HPH HPH HPH WBM WBM WBM WBM WBM WBM WBM WBM WBM WBM WBM WBM WBM WBM WBM WBM WBM

Smallest reported particle size (nm) 41 133 182 200 211 244 300 300 320 348 400 400 400 500 502 528 539 599 600 601 650 750 800 913 930 <800 128 128 150 156 164 199 202 210 210 220 256 256 270 279 300 366 800

Delivery route

Topical

Oral Oral I.v. injection I.v. injection I.v. injection

Oral I.v. injection I.v. injection Oral

I.v. injection Oral

Oral I.v. injection I.v. injection Oral I.v. injection Oral/I.v. injection I.v. injection Ophthalmic Subcutaneous

Reference Pardeike and Müller (2010) Ganta et al. (2009) Hecq et al. (2006) Sharma et al. (2009) Kassem et al. (2007) Kobierski et al. (2009) Crisp et al. (2007) Mishra et al. (2009) Dolenc et al. (2009) Teeranachaideekul et al. (2008) Zhang et al. (2007) Jacobs et al. (2000) Langguth et al. (2005) Möschwitzer et al. (2004) Grau et al. (2000) Kayser et al. (2003) Kassem et al. (2007) Jacobs and Müller (2002) Jacobs et al. (2001) Peters et al. (2000) Xiong et al. (2008) Mauludin et al. (2009) Krause et al. (2000) Zhang et al. (2010) Kassem et al. (2007) Lai et al. (2009) Beirowski et al. (2011) Nekkanti et al. (2009) Lee (2003) Van Eerdenbrugh et al. (2007) Basa et al. (2008) Nakarani et al. (2010) Merisko-Liversidge et al. (1996) Merisko-Liversidge et al. (1996) Merisko-Liversidge et al. (1996) Jinno et al. (2006) Merisko-Liversidge et al. (1996) Van Eerdenbrugh et al. (2008) Liversidge and Conzentino (1995) Merisko-Liversidge et al. (1996) Ali et al. (2011) Van Eerdenbrugh et al. (2008) Chiang et al. (2011)

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2.2. High pressure homogenization HPH can be regarded as the second most important technique to produce drug nanocrystals. The broad acceptance of this approach is supported by many examples from the literature (e.g. references of Table 2). The application of HPH as particle size reduction method requires the availability of special equipment; it cannot be tested with a system as simple as “beads in a beaker”. Interestingly, high pressure homogenizers were already widely available in the pharmaceutical industry as well as in the food industry at the time the first nanosuspensions based on HPH have been developed. The use of homogenizers was already described for the production of liposomes and emulsion systems (Brandl et al., 1990; Collins-Gold et al., 1990). Today, high pressure homogenizers can also be used for the production of solid lipid nanoparticles or nanostructured lipid carriers (Müller et al., 2000, 2002, 2011). The possibility to employ the production equipment for various formulation approaches (multipurpose production lines) is an important advantage, as it is rather costly to establish production lines in-house. The steps involved in producing nanosuspensions by means of HPH are similar and as simple as for WBM. Normally, a premix of the coarse drug and the dispersion medium is prepared using high speed stirrers. The dispersion medium contains normally similar surfactant and/or stabilizer systems used for the WBM approach (Wu et al., 2011). Subsequently, this coarse suspension (the so called “macro-suspension”) is passed several times through the high pressure homogenizer. Typically, the applied pressure is increased step-wise from 10% to 100% in order to avoid clogging of the narrow homogenization gap. At production pressure, which spans between 1000 and 2000 bar, the gap has an opening of only a few micrometer. This explains the importance of the pre-mixing procedure for de-agglomeration and wetting purposes, especially when relatively coarse material is processed. The particle size reduction itself is caused by cavitation forces, shear forces and collision. In general, several homogenization cycles are needed to reach the minimal particle size. The number of passes (i.e. homogenization cycles) depends on many factors. Thereby, the employed drug delivery technology defines the type of homogenizer as well as the process conditions (e.g. IDDPTM technology, Dissocubes® or the Nanopure® technology, see Section 1) (Keck and Müller, 2006; Shegokar and Müller, 2010). Additional factors determining the process efficiency include size of the starting material, hardness of the drug and maximum pressure that can be reached by the machine. In general, higher pressure leads to faster particle size reduction (Dumay et al., 2012; Fichera et al., 2004; Kluge et al., 2012). The size of the impaction zone and the corresponding volume are important factors, as they determine proportionally the power density of the equipment. The difference in power density of the Microfluidizer technology compared to piston-gap processes is one reason for the different particle size reduction effectiveness of the two types of high pressure homogenizers (Xiong et al., 2008). HPH is less prone in generating process impurities as consequence of abrasion and wearing of the equipment compared to WBM. Although high pressure homogenizers consist mainly of steel parts, the impurity levels found in nanosuspensions prepared via HPH processes are considerably low. A comparative study revealed that a typical nanosuspension after 20 cycles at 1500 bar contained less than 1 ppm iron (Krause et al., 2000). Abrasion and wearing of HPH equipment can occur when extremely hard material is processed in piston-gap homogenizers. In this case, the tip of the homogenization valve posses a relatively small surface compared to the volume of suspension passing through it. Wear and tear tend to happen when only stainless steel parts are used, leading to a reduction in process efficiency. Therefore, modern homogenizers

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have homogenization valves equipped with ceramic tips, which can withstand harsh process conditions (Innings et al., 2011). HPH is a scalable process, which is applied not only in the pharmaceutical but also in the cosmetics and food industry (Dumay et al., 2012). Today high pressure homogenizers are available from ml-scale to large production scale (Keck and Müller, 2006). Some references report an enzyme inactivation and a reduced microorganism load as a result of HPH processes (Diels and Michiels, 2006; Dumay et al., 2012). This can be seen as advantage for large scale production, as the reduced microorganism load increases the shelf life of the nanosuspension intermediate without the need of additional filtration steps, at least if the intended product is administered orally. There are numerous examples in the literature where HPH was applied successfully to produce nanosuspensions. As opposed to WBM, it seems that the particle size reduction effectiveness of the standard processes depends more on the physico-chemical properties of the processed drug. Table 2 shows an overview of mean particle sizes generated by HPH. The results are more scattered than for WBM. There is no general rule, but it seems that HPH is the method of choice for relatively soft materials with tendency to smear when processed with others methods, such as WBM. Table 2 shows that for the lipidic compound PX-18 (2N,N-Bis(oleoyloxyethyl)amino-1-ethanesulfonic acid) the smallest particle size reported in the literature (41 nm) could be obtained by standard HPH. 2.3. Combinative technologies for the production of drug nanocrystals Although the standard technologies WBM and HPH are in the meantime widely accepted and applied, there were still some disadvantages which have been addressed by continuous improvement of these processes. For both, WBM as well as HPH it is suggested to start with micronized starting material. Clogging of the equipment can occur when the process is conducted with too coarse drug particles. In case of agitated ball mills in re-circulation mode this clogging can occur at the media separator; for high pressure homogenizers clogging can occur within the feeding system of the homogenizer or at the narrow homogenization gap as well as the interaction chamber. Relatively long process times are another disadvantage of the standard approaches. This stands in contrast to the abovementioned 30–120 min to produce nanosuspensions by WBM. However, this time is the minimum contact time of a drug inside the milling chamber. When a large scale mill is run in re-circulation mode, the suspension is only exposed to high energy at the time it passes the milling chamber. Therefore, the total production time is significantly longer, depending on the ratio between total batch volume and volume of the milling chamber. A similar situation applies for HPH. Commercially available high pressure homogenizers can process 1000 l of nanosuspension or more within 1 h. However, when 20 homogenization cycles are required to achieve a certain particle size, the total process time can easily go up to 20 h for batch sizes of 1000 l, unless more homogenizers are used in series. In order to address the above-mentioned disadvantages, alternative processes have been developed. Significant reduction of process times can be achieved when the drug is pre-treated before the top-down process step is performed. These relatively recently developed techniques are referred to as combinative particle size reduction methods. The company Baxter developed the first combinative method, the so called NanoedgeTM technology. It consists of a bottom-up step (micro-precipitation) followed by a top-down step (high-pressure homogenization). The drug is dissolved, e.g. in a water-miscible, non-aqueous media and precipitated in form

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of a suspension consisting of brittle drug particles. This suspension is then further processed to a nanosuspension by means of HPH (Kipp et al., 2003; Kipp, 2004; Rabinow, 2004). An alternative method, which is also known as H 69 process, was developed more recently by Müller and colleagues. Ideally, the time between the precipitation and the high pressure homogenization step should be minimized, in order to obtain smaller drug nanocrystals. In this regard it is optimal to conduct the precipitation directly within the dissipation zone of the homogenizer. First results have shown that this method can lead to very small particles (Müller and Möschwitzer, 2005), but more systematical research is needed for a better understanding of all critical process parameters. Obviously, no micronized starting material is needed to perform the two above-mentioned technologies. However, a remaining disadvantage is the presence of the non-aqueous solvent in the final nanosuspension. The non-aqueous solvent can act as a co-solvent which increases the solubility of the drug to an extent which could potentially compromise its physical and chemical stability. In most cases, the non-aqueous solvent has to be removed in order to reduce the risk of Ostwald ripening. To avoid this problem, alternative combinative methods have been developed by Möschwitzer and colleagues, which are referred to as H 42 and H 96 technologies (Shegokar and Müller, 2010). The H 42 technology uses spray drying of organic drug solutions as bottom-up step to produce a modified starting material for a subsequent process step, where the modified drug is very efficiently processed by standard top-down processes, e.g. HPH into nanosuspensions with small particle sizes and narrow size distributions (Möschwitzer, 2005; Möschwitzer and Müller, 2006a). The spray drying process results in a pre-treated, finedispersed starting material which can be directly used for the subsequent high-pressure homogenization step. The spray drying process yields basically solvent-free material. Thus, the second size reduction step can be performed in solvent-free, aqueous media. The risk for particle growth is significantly reduced compared to the combination of precipitation and HPH. The H 96 technology combines freeze-drying as bottom-up step with standard top-down processes (Möschwitzer and Lemke, 2005; Shegokar and Müller, 2010). Pre-treatment with freeze-drying can be used when temperature sensitive material has to be processed or when ultra-small drug nanocrystals of expensive drugs are needed. The freeze-drying process can be controlled to produce extremely brittle starting material. Therefore, the subsequent top-down step yields nanosuspensions with a very small particle size. The H 96 technology was used for the production of ultrasmall nanocrystals of amphotericine B by combining freeze-drying with HPH. The resulting nanosuspensions had a particle size clearly below 100 nm, which enabled their use in specialized red-blood-cell carriers (Staedtke et al., 2010). Since this approach is still relatively new, the factors leading to the improved particle size reduction efficiency are not fully understood yet. It seems that solid state modifications play a significant role. A study using glibenclamide as model compound has shown that smallest particle sizes were obtained with amorphous starting material (Salazar et al., 2012). However, in another study similar improvement of the particle size reduction effectiveness was also seen for modified glibenclamide which was predominantly crystalline (Salazar et al., 2011). The combination technologies nicely illustrate how standard technologies are continuously improved in order to extend their application areas. Fig. 2 compares the particle size evolution of the model compound glibenclamide as a function of process time for standard top-down processes in comparison to the novel combinative techniques. It can be seen that the pre-treatment leads to a significant improvement of the particle size reduction effectiveness. Much smaller particles were obtained already at the second time point, which means after 1 homogenization cycle at 1500 bar or 1 h milling time. Although standard WBM results eventually in the

3500 Standard HPH

3000

Mean particle size [nm]

6

Standard WBM H 96 (FD-HPH)

2500

H 42 (SD-HPH) H 96 (FD-WBM)

2000 1500 1000 500 0 I

II

III

IV

V

VI

Process time

Fig. 2. Mean particle size (PCS z-average) as function of the process time and the particle size reduction technique. All discontinuous lines represent novel combinative methods with modified starting materials; the continuous lines represent the standard methods with unmodified starting material. Point I: after the pre-mixing step (high-speed mixer), points II–VI represent: 1, 5, 10, 15, 20 homogenization cycles for HPH results, or results after 1, 2, 4, 8, 24 h of milling for WBM (using a low energy ball mill). Modified after Salazar (2012).

same final particle size, process time for the conventional process is much longer. All combinative technologies perform distinctly better than the standard HPH process. Pre-treatment of the API material before HPH, makes it possible to obtain the same particle size as with the standard WBM method. In this regard, the combinative methods allow the application of HPH processes also for harder APIs which are more difficult to nanosize. It should be mentioned in this context that any pre-treatment step increases the complexity of the overall process and can add significant costs. Therefore it is obvious that combinative particle size reduction methods will be only used, in case the more established methods, like wet ball milling or standard high pressure homogenization cannot be used to come to the desired results. One example is the production of nanosuspensions from amorphous APIs with particle sizes smaller than 100 nm using the combinative methods. It is very challenging to achieve this in a reasonable time frame with established methods, such as wet ball milling. 3. The formulation selection process for poorly water-soluble compounds The selection of the right formulation approach is one of the key activities of formulators in the pharmaceutical industry. Key factors are the physico-chemical properties of APIs, such as aqueous solubility, melting point and temperature and chemical stability. In addition, the formulator needs information about the potency of the compound and the desired route of administration, as this determines the type of the final dosage form as well as the required drug load. All these factors can be considered in decision trees, which are often used in the industry to guide the formulator. However, there are some biopharmaceutical relevant aspects which need more attention, in order to avoid false negative results. Like any other formulation technology, drug nanocrystals as enabling technology can only be successful when all of these factors are taken into account. It would not be sufficient to assume that the oral bioavailability of any poorly soluble drug can be increased just by formulating it as drug nanocrystal. The well-known BCS system (Biopharmaceutics Classification System) is used very frequently to categorize compounds (Amidon et al., 1995). According to the BCS system poorly soluble compounds can belong to class 2 (low solubility, high permeability) or class 4 (low solubility, low permeability). Therefore, BCS class

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d d s+d (s) + d (s) + d

s+d s+d (s) + d s+d Complex Complex Medium Simple s s s l+s Not all cyclodextrine types are suitable for IV administration. E.g., liquid or semi-solid filled hard or soft gelatine capsules. a

Medium Medium to high High Low

b

Formulation flexibility liquid, solid Suitable for compounds with high melting point Suitable for temperature labile substances Table 3 Formualtion approaches for poorly soluble drugs.

2 and 4 compounds would be theoretically good candidates for nanosizing approaches. This is a widely used and well accepted perception within the pharmaceutical industry. However, using the BCS system as guidance for formulation selection might sometimes oversimplify the complex nature of drug dissolution, solubility and permeability. Poorly water-soluble compounds can possess such a low aqueous solubility that the dissolution rate even from ultra-small drug nanocrystals (e.g. sub 100 nm) is too slow. In this case it is not possible to reach sufficiently high drug concentrations in the gastro-intestinal tract for an effective flux across the epithelial membrane. In addition, other factors such as efflux transport or pre-systemic metabolism can negatively influence the oral bioavailability. Therefore it was recommended to classify compounds into slightly different categories, as they can show dissolution rate limited, solubility or permeability limited oral bioavailability. The result is known as the “Developability Classification System”, which is another way to categorize compounds in a more biorelevant manner (Butler and Dressman, 2010). This system distinguishes between dissolution rate limited compounds (DCS class IIa) and solubility limited compounds (DCS class IIb) (see Fig. 3). In order to select the right formulation approach and to address the compound specific issues with a suitable formulation type it is imperative to first understand the bioavailability limiting factors. It is important to note that there is no one-fits-all formulation approach. Each technology has its own advantages and disadvantages. The main approaches to address poor water-solubility are summarized in Table 3. The better the formulator understands the interplay of the physico-chemical properties of the drug, the special aspects of the various formulation options and the required in vivo performance, the higher the chance that the optimal formulation approach will be chosen. This minimizes the risk of late failures in human clinical trials, e.g. due to insufficient or highly variable drug exposures. Compounds showing dissolution rate limited bioavailability can be referred to as DCS class IIa compounds. Obviously, they represent only one part of the BCS class 2 compounds. The extent of the oral bioavailability of such compounds is directly correlated with their dissolution rate in vivo. The fraction of the dose that dissolves in the lumen is readily absorbed through the intestinal membrane. Consequently, the bioavailability of such compounds can be improved by any technique which increases primarily the dissolution rate. Various formulation approaches are known which lead to an increased dissolution rate, including salt formation, the use of co-crystals or particle size reduction. The formulator has to select the optimal formulation approach according to the properties of the specific drug molecule.

Administration route Oral or IV

Modified after Butler and Dressman (2010).

No Yes Yes Limited

Fig. 3. DCS classification system and relevant formulation approaches for the various compound classes.

No Limited Yes Limited

0.1

Solid state manipulation – HME – SDD Co-crystals Lipid based systemsb

Lead optimization

Lead optimization

Simple Simple

Permeation enhancer + solubilization

Mucoadhesion

Simple Complex

Class IV

Permeation enhancer

High High Low High Low

Class III

Complexity of the process

1

Oral Both Botha Oral Both

Lipid-based systems Solid-state manipulation (ASD)

l + s, l+s l+s s (l: limited) l

Complexation

Yes Yes Yes Yes Yes

Salt formation Co-crystals

Yes, cryogenic Yes Yes Yes Yes

Class IIb

Nanosizing

Micronization Nanosizing Cyclodextrin formulations Salt formation pH adjustment

10

Class IIa

For solubility or dissolution rate limited compounds

Standard approaches

10000

Potential drug load low, medium, high

Class I

250 500

Formulation approach

Predicted Peff in humans cm/sec x 10-4

Dose/solubility ratio

7

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Salt formation is often preferred by pharmaceutical chemists, as crystallization of salts can be used to produce very pure material. However, it can only be applied when the compound is ionizable. Salt formation can be regarded as conventional way to increase the dissolution rate of APIs (Li et al., 2005; Serajuddin, 2007). The increased dissolution rate of a salt can have a positive effect on the bioavailability of poorly soluble compounds. Sometimes, it is difficult to identify pharmaceutical acceptable salts, which can be produced on a large scale. APIs can also be crystallized together with guest molecules, in order to create fast-dissolving co-crystals. Although this approach seems to be very promising it is not frequently used as standard formulation approach for poorly soluble compounds (Schultheiss and Newman, 2009). Particle size reduction is by far the most important approach to address dissolution rate limited bioavailability. According to the well-known Noyes–Whitney equation (Eq. (1)) the dissolution rate depends directly on the surface area (A) of the dissolving particles. Particle size reduction leads to an increase in surface area and hence to an accelerated dissolution rate. dcx D·A = (cs − cx ) dt h

(1)

where dcx /dt is the dissolution rate; D is the diffusion coefficient; A is the surface of drug particle; h is the thickness of diffusional layer; cs is the saturation solubility of the drug; cx is the concentration in surrounding liquid at time x. Two particle size reduction approaches can be distinguished, namely micronization and nanosizing, often also referred to as nanonization. Micronization can be regarded as standard technique, which is used on a routine basis to produce standardized API starting material having a certain particle size distribution. This unit operation is often carried out under the responsibility of the chemical department, which delivers API with a standardized size distribution. Micronization techniques include hammer milling, pin milling or air jet milling. Depending on the technique employed, the mean particle size generally ranges between 1 and 50 ␮m. The fraction of fine particles below 1 ␮m is comparatively low. The dissolution rate of poorly soluble drugs is increased compared to non-micronized material. However, the effect on the bioavailability improvement is limited. Nanosizing is particle size reduction to another dimension. The term nanosizing subsumes the various formulation techniques which generate drug nanocrystals with a mean particle size between 1 and 1000 nm. Due to their small particle size these particles can vary distinctly in their properties from micronized drug particles. Similarly to other colloidal systems drug nanocrystals tend to reduce their energy state by forming larger agglomerates or crystal growth. Thus, they are often stabilized with surfactants, stabilizers or combinations thereof. Reduction of the particle size to the nanometer range results in a substantial increase in surface area (A), thus this factor alone will result in a faster dissolution rate. In addition, the Prandtl equation shows that drug nanocrystals also have a decreased diffusional distance h. This further enhances the dissolution rate. Finally, the concentration gradient (cs − cx ) is also of high importance. There are reports that drug nanocrystals show an increased saturation solubility cs . This can be explained by the Ostwald–Freundlich equation (Kipp, 2004) and by the Kelvin equation (Müller and Böhm, 1998). It is still not clear to what extend the saturation solubility can be increased solely as a function of smaller particle size. Most probably the increased solubility of drug nanocrystals is a combined effect of nanosized drug particles and solid state effects caused by the particle fractionation during the process. Authors have reported effects of 10% increase in saturation solubility up to several folds (Dai et al., 2007; Hecq et al., 2005; Müller and Peters, 1998). In a detailed study a marginal increase of

the solubility has been found for four drug molecules which were processed to drug nanocrystals (Van Eerdenbrugh et al., 2010). It can be stated that the increase of the dissolution rate remains the main effect of nanosizing. For compounds belonging to DCS class IIb and IV the intrinsic solubility and the related achievable intraluminal drug concentration are too low in order to achieve sufficient flux over the epithelial membrane. These compounds possess solubility limited oral bioavailability. In order to achieve sufficient exposure levels they have to be formulated with techniques that increase substantially the apparent solubility of the drug in the lumen. Basically, this can be achieved by solubilization, complexation or solid state manipulation. When formulations based on these principles are ingested orally, drug concentration levels above the thermodynamic equilibrium are reached in the gastrointestinal lumen. This leads to an increased concentration gradient and a higher flux across the membrane (Brouwers et al., 2009). Solubilization using lipid based systems or co-solvent systems is a simple and elegant way to formulate poorly water-soluble compounds (Pouton, 2006; Strickley, 2004). Very often, they are applied as first-choice option to formulate poorly soluble compounds. Solubilized systems can theoretically also be used as formulation approach for dissolution rate limited compounds (DCS class IIa). Lipid based systems can be administered very flexible as liquid formulations in pre-diluted form, or as water-free concentrate filled in hard or soft gelatine capsules. They can be used for oral as well as parenteral applications (Strickley, 2004). Nevertheless, these systems are of limited use when very high drug doses are needed. Often the API is not sufficiently soluble in the available excipients. Another limitation is the quantity of certain solubilizers that can be used, especially for chronic indications, as they can lead to undesired side-effect, e.g. increase in plasma-lipid levels. Alternatively, cyclodextrines, a class of functional excipients, can be used as solubilizers to increase the bioavailability of poorly water-soluble drug molecules (Brewster and Loftsson, 2007). The formation of inclusion and non-inclusion complexes can lead to an increase in the apparent solubility of compounds. Therefore these excipients can be used when a certain degree of supersaturation is required to achieve higher bioavailability. Commercially available cyclodextrin formulations are available for many administration routes. Depending on the molecular type these systems can be used in liquid as well as solid form for oral and with some exceptions also for parenteral use. Similarly to lipid based systems, cyclodextrine formulations require relatively high excipients to drug ratios. Another way to address solubility limited bioavailability is the manipulation of the drug’s solid state. In general, these techniques result in formulations which carry the drug molecules in a higher energy state, e.g. in form of amorphous solid dispersions (ASDs) (Leuner and Dressman, 2000). Various ways including hot-melt extrusion (HME) (Breitenbach, 2002) and spray-drying (spray dried dispersions, SDD) (Friesen et al., 2008) are applied to produce ASDs. These techniques are an elegant way to produce oral dosage forms of poorly soluble compounds at industrial scale. Several marketed products have proven the suitability of this approach as commercial oral dosage forms. However, ASDs are not as flexible as other formulation approaches, e.g. they cannot be easily used in liquid form for parenteral administration of poorly soluble drugs. Ideally, the poorly soluble drug needs to be well soluble in the polymer(–surfactant) systems, which are used as matrix to keep the drug in amorphous form. The required drug-polymer ratio is often a limiting factor in achieving high drug loads in the final solid dosage form. In addition, this method is less suitable for thermolabile compounds, as they are exposed to elevated temperatures during the processing, if HME is used for manufacturing. For the sake of completeness it should be mentioned here that recently a novel technology called NanOsmotic® (Alkermes) has

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been developed which aims to combine the principles of particle size reduction, solubilization and osmotic controlled-release (Liversidge, 2011).

4. Special biopharmaceutical aspects of drug nanocrystals 4.1. Drug nanocrystals for oral dosage forms The previous section discussed the application for drug nanocrystals as compared to other formulation approaches. As a consequence of the very fast dissolution rate and other specific factors drug nanocrystals possess some unique features with regard to biopharmaceutical performance which will be discussed in more detail in the following section. When particle size reduction is used to formulate dissolution rate limited compounds (DCS class IIa), the extend of the oral bioavailability can be described as a function of the particles size. A smaller particle size leads to higher cmax values and proportionally also an increased AUC. Jinno et al. (2006) have reported the relationship between particle size, dissolution velocity in vitro and the in vivo effects for the poorly water-soluble drug cilostazol in a very clear und understandable manner. The particle size of the drug was reduced by using different techniques. Hammermilling resulted in a mean particle size of 13 ␮m, jet-milling in 2.4 ␮m and wet ball milling using the NanoCrystal® technology to a particle size of 0.22 ␮m. The effect of the particle size on the dissolution velocity was first demonstrated with in vitro dissolution tests. The cilostazol nanocrystals dissolved immediately, independently of the dissolution medium. In a study in beagle dogs, this fast dissolution led to a superior performance of the nanocrystalline cilostazol. The exposure was almost a function of the particle size of the drug, with the best performance obtained from cilostazol nanocrystals. In addition, the differences between fed and fasted state were significantly reduced compared to the suspension prepared with jet-milled or hammermilled drug. Meanwhile, the direct relationship between the particle size of the drug and the achievable extend of drug absorption have been reported for many other drugs. All these drugs have benefitted from the nanosizing approach in terms of bioavailability improvement (Kesisoglou and Wu, 2008; Lenhardt et al., 2008; Li et al., 2011; Quan et al., 2011; Shono et al., 2010; Willmann et al., 2010; Xia et al., 2010). Compounds with a pronounced absorption window in the upper intestinal tract do also benefit from fast dissolving formulations. The accelerated dissolution of drug nanocrystals leads to sufficiently high drug concentrations at the absorption site. The drug aprepitant, which is marketed as Emend® by Merck and Co., is an example for a compound with an absorption window in the upper intestinal tract (Wu et al., 2004). Similar results were found for fenofibrate in a regional absorption study. The bioavailablity of fenofibrate formulated as nanosuspension and administered directly into the proximal and distal bowel was approximately 100% relative to the bioavailability when the nanosuspension was administered orally. In contrast, the relative bioavailability was only 32% when the fenofibrate nanosuspension was administered directly into the colon (Zhu et al., 2010). Nanosized fenofibrate dissolves quickly and is already dissolved at the site of preferred absorption, i.e. the upper intestinal tract. In contrast, micronized fenofibrate might not get sufficiently absorbed, because it dissolves too slowly and misses therefore the absorption window in the upper intestinal tract. Furthermore, an increased dissolution rate of poorly watersoluble drugs can lead to faster onset of action. This can be beneficial for compounds, where the pharmacodynamic effect is directly linked with the achievable plasma concentration, e.g. pain treatments like naproxen (Liversidge and Conzentino, 1995;

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Merisko-Liversidge et al., 2003). In this case the short tmax and high cmax levels resulting from using fast-dissolving nano-formulations can lead to a faster pain relief. As briefly mentioned above, the use of nano-formulations can lead to reduced variation between the drug absorption in fasted and fed state. This is another important reason for choosing drug nanocrystals as formulation approach. Many studies have reported reduced food effects when poorly water-soluble drugs were administered as drug nanocrystal formulation. Poorly watersoluble compounds administered as standard formulation based on micronized API show often an enhanced absorption when administered together with food. One potential explanation is that bile salts and food components can have a positive effect on the solubility and consequently on the dissolution rate of micronized drugs. In addition, the dissolution is also prolonged by a reduced gastric emptying rate; this can further enhance the oral absorption. In contrast, nano-formulations show maximum dissolution already in fasted state. Therefore the extent of absorption cannot be further increased for those compounds when administered together with food (Jinno et al., 2006; Sauron et al., 2006; Shono et al., 2010). Special attention is needed when ionizable compounds are formulated as drug nanocrystals. The particle size reduction itself is a rather versatile approach which works for all compounds irrespectively of their chemical nature. When neutral or acidic compounds are administered orally in nanosized form the pH shift from acidic to alkaline conditions works in favor for an increased extend of dissolution in the intestine. An opposite situation exists for basic compounds. When they are formulated as nanosized product, sometimes a decreased bioavailability is found in in vivo studies. The pH shift from acidic to neutral or alkaline conditions can cause a decrease in solubility of these compounds, which can result in uncontrolled precipitation of already dissolved material (Sigfridsson et al., 2011b). The in vivo effect of such a pH shift has to be examined for each compound, before excluding the nanoapproach. There are also examples for basic compounds which have been developed as nano-formulations and tested successfully in vivo (Hecq et al., 2006; Jia et al., 2003). 4.2. Drug nanocrystals for non-oral applications Nanosizing is a versatile formulation approach which can be potentially used for all routes of administration (Cooper, 2010). In the beginning drug nanocrystals were developed as oral dosage forms, nowadays they are also considered for non-oral applications. Literature examples are available for basically all administration routes, including dermal (Al Shaal et al., 2010; Mishra et al., 2009), ophthalmic (Kassem et al., 2007), pulmonary (Shrewsbury et al., 2009; Steckel et al., 2003) or buccal (Rao et al., 2011). Injectable formulations are the most important non-oral application area for drug nanocrystals. The various aspects of nanosuspensions for parenteral administration have already been discussed extensively. For detailed information, the reader is referred to these references (Kipp, 2004; Shi et al., 2009; Wong et al., 2008). In the context of this review only the most important aspects regarding the use of drug nanocrystals for non-oral administration will be discussed below. Nanosuspensions show some advantages over other formulation types which contain the drug in solubilized form. Solutions of poorly water-soluble compounds bear always the risk of precipitation upon administration. This can be avoided when stable nanosuspensions are administered. Furthermore, the injection of large amounts of solubilizers can be associated with side effects, such as pain on the injection site. Therefore, stable nanosuspensions, produced with a minimum amount of safe and well-tolerated stabilizers, can be advantageous. Moreover, in contrast to other injectable formulations, nanosuspensions are highly concentrated

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systems, with a relatively low viscosity. Since the viscosity of nanosuspensions mainly depends on drug concentration and vehicle composition, it can be to some extent adjusted. Sterility is an important requirement of injectable products. It could be shown, that sterile nanosuspensions can be obtained either by aseptic production (Baert et al., 2009; Peters et al., 2000), by sterile filtration (Zheng and Bosch, 1997), heat treatment (Na et al., 1999) or gamma radiation (Wong et al., 2008). In this regard nanosuspensions are compatible with industrial available filling lines and as flexible as other parenteral products. The same holds for the various presentations of drug nanocrystals. The final drug product can be provided either as ready-to-use suspension or as lyophilized powder. In 2009 a first parenteral product was successfully launched on the market. Paliperidone palmitate is marketed as ready-touse pre-filled syringe containing a nanosuspension prepared by the NanoCrystal® technology. The patient-friendly nanosuspension has a low viscosity and a high drug load which results in a low injection volume and low pain levels upon injection. The correct use of nanosuspensions for injectable dosage forms requires some considerations. Depending on the particle size and the aqueous solubility of the drug, nanosuspensions can perform comparable to solutions types (Gao et al., 2008). In this case it can be assumed, that the drug nanocrystals dissolve immediately. However, when a drug is administered as nanosuspension its PK characteristics and the biodistribution profile might be altered compared to a solution (Du et al., 2012; Ganta et al., 2009; Wang et al., 2011). In this case it can be assumed that the particles do not dissolve fast enough. Consequently, they are accumulated as particles in MPS (mononuclear phagocytic system) rich organs, like liver and spleen. This can lead to a prolonged action of the drug. In many cases intravenously administered nanosuspensions showed a better tolerability in patients compared to drug solutions (Kipp, 2004; Merisko-Liversidge et al., 2003; Rabinow et al., 2007).

5. Special aspects of drug nanocrystals as formulation approach for commercial drug product development The value to use drug nanocrystals as enabling technology to improve the performance of poorly water-soluble new chemical entities has been recognized by many companies. They have added this approach to their formulation toolbox and have included it to their formulation decision trees (Branchu et al., 2007; Chaubal, 2004; Ku, 2008; Li and Zhao, 2007; Maas et al., 2007; Möschwitzer and Op’t Land, 2008). Over the years many companies have recognized the need for adopting their development strategies in order to address the increased complexity of their pipeline candidates. Some have even shifted their development efforts to the very early stages, an approach which is often referred to as frontloading. This approach is used to increase the success rate of the drug discovery by enabling a robust and reliable testing of poorly water-soluble compounds very early on (Ku and Dulin, 2012). It has been estimated that the improvement rate of the screening process can be increased significantly when the appropriate techniques for poorly soluble drugs are available (Merisko-Liversidge and Liversidge, 2011). Their scalability is one important factor why drug nanocrystals are included in the formulation decision trees of so many companies. As mentioned earlier, drug nanocrystals can be used at all development stages, since nanometer-sized drug particles can be produced from extremely small scale up to commercial production. The first formulations for animal studies are needed when various lead compounds are tested in early pharmacokinetic studies (PK) as well as in efficacy studies using pharmacological animal models (PD). Such tests are normally performed at relatively low

dose levels. However, already at this stage nano-formulations can offer some advantages: (1) due to the versatility of the nanoapproach almost any substance can be formulated in this way provided it is poorly soluble enough so that a nanosuspension can be made. The only strict prerequisite, like for any other topdown method, is that the drug has to be poorly soluble in the dispersion medium (e.g. an aqueous medium). The solubility limit differs depending on the employed nanosizing technique between 10 ␮g/ml and 100 ␮g/ml (Merisko-Liversidge and Liversidge, 2008). (2) Formulations for very early PK studies should not require extensive development. They are mostly performed in rodents, in order to limit the API requirements. Initial PK formulations have to be rather straight-forward and can be either solutions or suspension-based systems (Li and Zhao, 2007). The purpose is primarily to establish important pharmacokinetic parameters, such as rate/extend of absorption, clearance, and distribution volume. Many approaches, such as cyclodextrin formulations, lipid or surfactant based systems as well as nano-formulations are used (Chaubal, 2004). Nanosuspensions can be seen in this regard as universal platform approach which is the preferred option at this stage. (3) A further advantage of using nanosuspensions already at that stage is the universal route of administration. Properly chosen, nanosuspensions can be administered orally as well as parenterally without the need to adopt the formulation. This allows establishing meaningful data for the absolute oral bioavailability very early on. In contrast to the simple PK formulations, the requirements for pharmacological models are more complex. Depending on the indication and the pharmacological model the selection of a universal formulation can be sometimes very challenging, especially when some frequently used systems are excluded because of their interference with the model read-out (Ghosh et al., 2008). In this case nanosizing is an elegant approach; sometimes it might be even the only technique that can be easily applied to develop the first formulations for pharmacological tests. At this stage of development the available drug amounts are normally extremely limited, therefore only some standard formulations are tested. With an increased understanding on how to develop robust nanosuspensions it is nowadays possible to obtain acceptable formulations in a very short development time without using a lot of scarce drug material (Chaubal, 2004). When the efficacy of the new chemical entities (NCEs) is sufficiently high, one needs to demonstrate and establish the sufficient safety margin for the selected lead compounds. Nanosuspensions are ideal formulations for toxicological studies as they can be relatively easily formulated with safe vehicle compositions that are already established as standard excipients for toxicological testing. In many cases standard cellulose/poloxamer nanosuspensions can be produced which differ only in their distinctly smaller particle size from NCEs formulated into standard suspensions for toxicological studies (Kesisoglou et al., 2007; Maas et al., 2007). Nanosuspensions are superior tox-formulations as their drug load can be very high compared to other systems like surfactant solutions or amorphous solid dispersions. The higher drug load reduces the effects which are potentially associated with excipients of the formulation. The use of nanosuspensions for toxicological studies has been intensively discussed in the literature (Chaubal, 2004; Maas et al., 2007; Sharma et al., 2011; Sigfridsson et al., 2011c). One publication provides a very comprehensive overview about preparation and manufacturing logistics of nanosuspensions for such a purpose (Kesisoglou et al., 2007). Another literature example demonstrated that the safety margin could be raised from 5× to 85× by using a nanosuspension instead of a suspension based on micronized drug. The nanosuspension was prepared in a very simple way by low energy WBM using a simple Eppendorf tube and zirconium beads (Kwong et al., 2011). For the acidic compound UG558 the

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same positive trend was found. The nanosuspension performed about 4.6 times better than the microsupension. In addition, the nanosuspension could be administered intravenously allowing the determination of the absolute bioavailability of the compound (Sigfridsson et al., 2009). When compounds have dissolution rate limited oral bioavailability they should normally show a linear dose/AUC relationship. However, in some toxicological studies a non-linear absorption is seen at very high dose levels. In this case particle size reduction is not sufficient to increase the exposure further as the systems turns from a dissolution rate limited to a solubility limited system (Sigfridsson et al., 2011a). The next stage of development begins when a series of NCEs has been successfully tested in pre-clinical programs and at least one compound could be qualified as clinical candidate. Many companies have implemented formulation ranking or special PK screening studies with the aim to identify the most appropriate formulation approach for the first-in-human (FIH) studies. Several factors do play a role in the selection of the optimal formulation approach for human clinical trials. In most of the cases the desired drug product will be an oral solid dosage form. Nanosuspensions can be transferred into solid dosage form by applying various conventional drying techniques. Spray drying is a straight-forward method for drying of nanosuspensions (Chaubal and Popescu, 2008; Gao et al., 2010; Lee, 2003). It has the advantage of being as scalable as nanosizing itself. Often, it is the first choice at the beginning of the drug product development, because it can be easily performed at bench as well as pilot scale. The spray dried intermediate can be compressed to tablet formulations (Jinno et al., 2008). The tablet composition has to be selected carefully, in order to obtain a fast and complete reconversion to a nanosuspension with a particle size distribution comparable to the non-dried formulation (Heng et al., 2009). In general, spray drying yields powders with rather low densities, which could require additional process steps, such as roller compaction to obtain tablettable intermediates. Fluidized bed granulation is an alternative method to produce a dry intermediate. Due to the available equipment it requires somewhat larger quantities of nanosuspension and is therefore used at a later stage. During fluidized bed granulation the nanosuspension is normally layered onto a core material, e.g. lactose or microcrystalline cellulose (Wang et al., 2012). This method results in a free-flowing granulate which can be easily compressed into tablets in a subsequent process step. Spray-layering of nanosuspensions onto beads is an alternative approach (Kayaert et al., 2011; Möschwitzer and Müller, 2006b). After an optional overcoatingstep the drug nanocrystal-loaded cores can be either filled into capsules or transferred into tablets. Another interesting approach is used for the commercial manufacturing of Rapamune® tablets. The nanosuspension is coated onto inert tablet cores which consist basically of lactose monohydrate, macrogol and talc (EMA, 2004). Energetically nanosuspensions can be regarded as high energy systems, because surface area is significantly increased when the particle size is reduced. Consequently, nanosuspensions tend to reduce their free energy by either aggregation or crystal growth. For a long time this led to the perception that nanosuspensions as such would have a limited shelf life and would not be suitable as readyto-use formulations. The physical stability of a nanosuspension depends on many aspects, e.g. the selection of the right stabilizer principle, the solubility of the API in the liquid phase of the suspension and last but not least also on the employed nanosizing method. But with all parameters selected carefully, nanosuspensions can be physically as well as chemically very stable systems. For both topdown methods, examples have been reported, where the particle sizes of physically stable nanosuspensions remained unchanged for years (Jacobs and Müller, 2002; Merisko-Liversidge and Liversidge, 2011). These data indicate that nanosuspensions can indeed be

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used as ready-to-use suspensions. This is also demonstrated nicely by the example of megestrole acetate; a formulation that was developed with a shelf-life of two years (Merisko-Liversidge and Liversidge, 2011). In view of the flexibility of this approach it is to some extent surprising why not more formulations based on drug nanocrystals have reached the market yet. Many pharmaceutical companies do not have sufficient in-house capabilities both in terms of equipment and know-how to develop drug nanocrystals for clinical studies. Even if all necessary prerequisites would be fulfilled, companies may not have all IP rights to use the different approaches for late-stage development programs (Müller and Keck, 2012). At the time when the different technologies for drug nanocrystals production have been developed most pharmaceutical companies did not put enough effort in developing their own IP portfolio in the nano-area. One of the potential reasons might be that in the nineties of the last century major pharmaceutical companies had not fully realized the potential of this approach (Cooper, 2010). Although poor aqueous solubility was already at that time recognized as an issue for drug product development, it was still possible to select alternative molecules with better developability. The perception was that the risk of developing poorly soluble compounds would be too high and therefore water-soluble alternatives were chosen (Merisko-Liversidge and Liversidge, 2011). Obviously, the situation has changed dramatically. No major pharmaceutical company can afford anymore to exclude molecules with difficult physico-chemical properties from further development. Nowadays, as result of extensive formulation screening during pre-clinical programs, compounds with more challenging properties are frequently selected as clinical candidates. Consequently, enabling technologies are needed to support human clinical studies. However, the selection of an optimal formulation approach with regard to a potential bioavailability enhancement is only one aspect. Other criteria are the availability of suitable equipment for lab scale and pilot scale as well as access to manufacturing equipment for GMP-production. As the development of a robust and reliable clinical trial material based on the nanosizing principle is rather complex in comparison to providing simple pre-clinical formulations, not many companies have built the necessary capabilities to perform this task in-house. Therefore, these development programs need to be outsourced to specialized contractors, who own the IP and have know-how regarding the development of such formulations. In this regard drug nanocrystals have often to compete with other enabling technologies which can be easier performed in-house using already established methods. This could be another reason why after twenty years not more compounds based on drug nanocrystals have reached the market.

6. Conclusion Over the past twenty years nanosizing has become a wellestablished and proven formulation approach for poorly soluble drugs. Extensive research has generated many different techniques to produce drug nanocrystals. Up to now the standard top-down techniques WBM and HPH could sustain a leading position in this area, mainly because these techniques have also evolved with the time. Many open questions and technological disadvantages from their infancy have been addressed and could be solved as a result of continuous and dedicated efforts of many researchers in this area. The great versatility of the nanosizing approach is certainly a main driver for the success. It can be used for almost every compound and for almost every route of administration. Although only a few products have reached the market so far, nanosizing is definitely well established and widely applied in the pharmaceutical industry. Drug nanocrystals can be used for all stages

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of industrial pharmaceutical development, from the very early studies up to commercial manufacturing. In addition to the technological improvement also the understanding of the biopharmaceutical aspects has advanced. Nowadays, it is clear that drug nanocrystals used for oral administration can only lead to better bioavailability when the compounds show dissolution rate limited bioavailability. However, this approach can add tremendous dosing flexibility for other administration routes, especially when highly concentrated formulations are needed. The researchers in this area will continue to use the original idea of making very small drug particles in order to make drugs more effective. The full potential of this approach has not been fully capitalized yet. For the future it can be speculated that more intelligent drug delivery systems based on drug nanocrystals will be developed. It will become possible to guide the nanosized drug particles with special ligands or other surface modifications to their target site in order to further increase the performance of these systems. At the end the drug delivery system can only be a tool to optimize the pharmacodynamic effect of a drug substance in order to treat patients in the most effective way.

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