drug nanocrystals in the commercial pharmaceutical development process

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Nanosizing is one of the most important drug delivery platform approaches for the commercial developmentof poorly soluble drug molecules. The research efforts of many industrial and academic groupshave resulted in various particle size reduction techniques. From an industrial point of view, the twomost advanced top-down processes used at the commercial scale are wet ball milling and high pressurehomogenization. Initial issues such as abrasion, long milling times and other downstream-processingchallenges have been solved. With the better understanding of the biopharmaceutical aspects of poorlywater-soluble drugs, the in vivo success rate for drug nanocrystals has become more apparent. Theclinical effectiveness of nanocrystals is proven by the fact that there are currently six FDA approvednanocrystal products on the market. Alternative approaches such as bottom-up processes or combinationtechnologies have also gained considerable interest. Due to the versatility of nanosizing technologyat the milligram scale up to production scale, nanosuspensions are currently used at all stages of commercialdrug development, Today, all major pharmaceutical companies have realized the potential ofdrug nanocrystals and included this universal formulation approach into their decision trees.

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 J.P. Möschwitzer / International Journal of Pharmaceutics 453 (2013) 142–156 143

1998). Due to the difficulty in controlling the process, bottom-up

techniques could not gain enough interest to become a standard

approach in thepharmaceutical industry at that time.

Aroundthe 1990sGary Liversidge andhiscolleagues from Ster-

ling 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 

poorlywater-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 pharma-

ceutical 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 alterna-

tivetechnologybasedonpiston gaphighpressurehomogenization

(HPH) to produce nanosuspensions (Müller). This technology was

named as DissoCubesTM, according to the cubic shape of the drug

nanocrystalsproducedwiththisprocess (Mülleret al., 2003). Later

this technologywas 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 tech-

nology, also referred to as Microfluidizer technology, is a typical

top-down process which is based on jet-stream homogenization.The drug is pumpedunder highpressure of up to1700bar through

a microfluidizer system ( Junghanns and Müller, 2008). In the col-

lision chamber of either Z-type or Y-type it comes to particle

collision, shear forces and cavitation forces leading to the desired

particle size reduction. The resulting particle size is preserved by

the use of various phospholipids or other surfactants and stabi-

lizers. Due to the relatively low power density of the standard

equipment, upto50 ormorepassesof thesuspensionarenecessary

for a sufficient particle size reduction (Mishra et al., 2003).

In1999theNanopure® technologywasdeveloped, anothervari-

ant of a piston-gap homogenization process, which is conducted

withwater-reducedor evenwater-free liquids as dispersionmedia

(Müller; Radtke, 2001).

By reducing the particle size to the nanometer range, thebioavailability of many poorly soluble compounds could be

improved significantly, which eventually led to a broader accep-

tance 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 commer-

cialpharmaceuticalproductsbasedon nanosizing approaches have

already been approved (Table 1).

Following the success of these two technologies, this has trig-

gered 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

embarkedon the “Nano” approach. They have developedtheirown

technology to provide alternative solutions to their customers.Alternative technologies have mainlybeen developedbased on

bottom-up approaches,mainly because ofmore freedom to gener-

atenewintellectualproperty (IP) (ChanandKwok,2011; deWaard

et al., 2011). Basically, these approaches have the common goal to

providebetter processcontrol forproducingnanoparticulatestruc-

tures 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 precipita-

tion can also be performed at elevated temperatures (Evaporative

Precipitation into Aqueous Solution, EPAS) (Chen et al., 2002). Fur-

thermore, 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  T

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144   J.P.Möschwitzer / International Journal of Pharmaceutics 453 (2013) 142–156

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 sen-

sitive molecules, such as biological molecules (Yu et al., 2004).

Alternatively, precipitation can be performed in conjunction with

centrifugation techniques (High gravityprecipitation) (Chiouet al.,

2007).

In recent years many drug delivery companies have started to

developproductionmethodsfordrugnanocrystals basedon super-

critical fluid technologies (Fages et al., 2004; York, 1999). In cases

where the drug is soluble in supercritical fluids, such as supercrit-

ical carbon dioxide, the RESS technology (RESS: Rapid Expansion

from Supercritical Solutions) can be applied (Maston et al., 1987).

Incontrast,inmanycasesthesupercriticalgas isusedas antisolvent

for the drug.Mixingof an organic drug solutionwith the supercrit-

ical antisolvent leads to a precipitation of nanometer-sized drug

particles,whicharecollectedin variousways. Thegeneralprinciple

is referred to as gas antisolvent technology. Dependingon process

conditions and mixing types, various process variants exist (e.g.

GAS: gas antisolvent process, SAS: supercritical antisolvent pro-

cess, SEDS: solution enhanced dispersion of solids) (Byrappa et al.,

2008).

Althoughthe resultsobtainedwiththesealternativeapproaches

are very promising, they are currently not as widely used in thepharmaceutical industry to produce drug nanocrystals. Most of 

these approachesrequire custom-madeproductionequipmentand

specialprocessingexpertise,which limits theirapplicabilitymainly

to dedicated research groups.

It is important tomention in this context that the development

of drug nanocrystals requires suitable analytical techniques such

as microscopic techniques or particle size analysis. These tech-

nologies have also evolved over time. Nowadays, the equipment

is much more user-friendly than some years ago. Particle charac-

terization canbe easily performedon routine basis and the results

are available withinminutes (Levoguer, 2012).

This review will focus mainly on drug nanocrystals produced

by applyingthe industrial relevant andwell-established top-down

technologies wet ball milling as well as high pressure homoge-nization. Obviously likemanyother 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 theyears tomature as established

techniqueswhicharenowwidely accepted andfrequently applied

by the pharmaceutical industry. In addition, this review will dis-

cuss 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 

Wetballmilling(alsoreferredtoaspearlmillingor beadmilling)

is by far the most frequently used production method for drug

nanocrystals in the pharmaceutical industry. The milling proce-

dure 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

chargedwithcoarsedrugsubstance,preferablyinmicronizedform,

which is suspended in dispersion medium containing at least one

stabilizing agent. By moving thebeads eitherwith an electric stir-

rer (Fig. 1a), e.g. a magnetic stirrer, or bymoving the whole jar, e.g.

witha rollerplateor amixer (Fig.1b), themilling beadscaninteract

withthedrugparticles.At thebeginningof thenineties, verysimilar

set-upswereused inorder toestablish this technology forpharma-

ceuticalpurposes. Therelatively lowenergyinput leadsto verylong

milling timesof several days (Liversidge et al.,1992; Liversidge and

Conzentino, 1995; Merisko-Liversidge et al., 1996). The comminu-

tion process itself is caused by abrasion, cleavage and fracturing

(Hennart et al., 2012). For LE-WBM a combination of cleavage and

abrasioncanbe assumed as the main mechanism of size reduction

principles, as theprocess generallyyieldsvery fine particleswith a

narrow size distributionwhen it is performed long enough.

Alternativemilling procedures based on high energy processes

had to be developed in order to make this process more desir-

ablefor industrial pharmaceutical applications. TheNanoCrystalTM

process in its current form is based on such a high energy wet

ballmillingprocess(HE-WBM)(Merisko-LiversidgeandLiversidge,

2008). A necessary prerequisite for HE-WBM is the availability of 

suitable equipment. Themanufacturers formilling equipmenthadtodevelopequipmentwith sufficientlyhighpowerdensitiesfor the

improvedprocesses. Today,HE-WBMcanbe regardedas a standard

procedure to produce nanosuspensions. Due to the much higher

powerdensity, theproduction times aresignificantlyreduced.Nor-

mally, the drug needs to be exposed to the high energy for about

Fig. 1. Setup forlow energywet ball milling. (A)Vial filledwith milling beads,suspension anda magnetic barplacedon a magnetic stirrer plate,the beads aremovedby the

rotatingmagnetic bar inside the vial. (B)Plastic bottle (small picture lower right) filled with milling beads and suspension moved by a standardmixer, thewhole system is

moved.

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 J.P. Möschwitzer / International Journal of Pharmaceutics 453 (2013) 142–156 145

30–120min in order to achieve a nanosuspension of good qual-

ity (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 forlargescale production is oftenusingagitatedballmills

in re-circulation mode. These mills have media separators, either

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

holdtankwith a certainvelocity throughthemillingchamber.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

andcompressionforces inside themillingchamber (Kwade,1999a).

The drug particles are reduced in size by abrasion and cleavage

mechanisms (Hennart et al., 2012).

It isobvious that high energymills require specialmillingmedia

whichhas tobeproperly selectedbasedonthematerialof theinner

surfaces of themill, the agitator types andother factors. Using just

glassbeadsor zirconiumoxidemilling beads can lead to significant

contaminationof thenanosuspensioncausedbytheabrasioneither

of themilling beads orparts of themilling chamber (Hennart et al.,

2010; Juhnke et al., 2012). Initially, impurities caused by abrasionwere one of the major obstacles for a broader acceptance ofWBM.

Therefore,a majormilestonefor thebroadacceptanceof themilling

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® pro-

cess is performed with special PolyMillTM media, i.e. polysterene

beads with a diameter of about 0.5mm (Kesisoglou et al., 2007).

This leads to product qualities which allow the usage of nanosus-

pensions even for parenteral administration (Merisko-Liversidge

and Liversidge, 2011).

In the early nineties, there was no equipment available to pro-

duce nanosuspensions at very small scale. Hence, it was difficultto use this formulation approach for discovery purposes. Initially

several grams of API were needed to produce prototype formula-

tions (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 nanosuspensionsat thesame time ( Juhnkeet al., 2010;Van

Eerdenbrugh et al., 2009a). Alternatively, agitated ball mills are

used for drug quantities starting from 10mg (Merisko-Liversidge

and Liversidge, 2011). Using these mills it is now possible to pro-

duce nanosuspensions during the early discovery phase of the

formulationdevelopmentortoperformstabilizerscreeningstudies

with a minimal API consumption.

Withthecommercialavailabilityof suitableequipmentfor smallscale 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 ofwet ballmilling 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 sur-

factants 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 bywet ball milling are in the range

between 100 and 300nm, irrespectively whether LE-WBM or HE-

WBMwas 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 standardhigh pres-

sure homogenization in terms of the achievable particle sizes. All

these aspects have opened the possibility to usewetball milling as

a platform technology for formulating poorly soluble compounds.

 2.2. High pressure homogenization

HPHcanberegardedas the secondmostimportant technique to

produce drug nanocrystals. Thebroad acceptance of this approach

is supportedbymany 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 homogenizerswere already widely available in thephar-

maceutical industry aswell as in the food industry at the time the

first nanosuspensions based onHPHhave been developed. Theuse

of homogenizerswas already described for theproduction of lipo-

somesandemulsionsystems(Brandlet al.,1990;Collins-Goldetal.,

1990). Today, high pressure homogenizers canalso be used for the

production of solid lipid nanoparticles or nanostructured lipid car-riers (Mülleret al., 2000,2002,2011). Thepossibility to employthe

production equipment for various formulation approaches (multi-

purpose 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 pre-

mix of the coarse drug and the dispersion medium is prepared

using high speed stirrers. The dispersion medium contains nor-

mally similarsurfactantand/orstabilizer systemsusedfor theWBM

approach (Wu et al., 2011). Subsequently, this coarse suspension

(theso 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 thenarrow homogenization gap. At production pressure,whichspans between 1000 and 2000bar, the gap has an opening of only

a fewmicrometer. This explains the importance of thepre-mixing

procedure for de-agglomeration and wetting purposes, especially

when relatively coarsematerial 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 onmany factors.

Thereby, the employed drug delivery technology defines the

type of homogenizer as well as the process conditions (e.g. IDD-

PTM 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 startingmaterial, hardness of the drug and maximum pres-sure thatcanbereachedbythemachine.In general,higherpressure

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

thecorrespondingvolumeareimportant factors,as theydetermine

proportionally the power density of the equipment. Thedifference

in power density of the Microfluidizer technology compared to

piston-gap processes is one reason for the different particle size

reductioneffectiveness of the two types of high pressure homoge-

nizers (Xiong et al., 2008).

HPH is less prone in generating process impurities as conse-

quence of abrasion and wearing of the equipment compared to

WBM.Althoughhighpressurehomogenizersconsistmainlyof steel

parts, the impurity levels found in nanosuspensions prepared via

HPHprocessesare considerably low.A comparative studyrevealed

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146   J.P.Möschwitzer / International Journal of Pharmaceutics 453 (2013) 142–156

that a typical nanosuspensionafter 20cyclesat 1500bar contained

less than 1ppm 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 thevolume of suspension passing through it.Wear andtear tend

to happen when only stainless steel parts are used, leading to a

reduction in process efficiency. Therefore, modern homogenizers

havehomogenizationvalvesequippedwithceramic tips,whichcan

withstand harsh process conditions (Innings et al., 2011).

HPH is a scalable process,which is applied not only in the phar-

maceutical but also in the cosmetics and food industry (Dumay

et al., 2012). Today high pressure homogenizers areavailable 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 advan-

tage for large scale production, as the reducedmicroorganism load

increases the shelf life of the nanosuspension intermediate with-

out the need of additional filtration steps, at least if the intended

product is administered orally.

There arenumerous examples in the literature whereHPHwas

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 scat-

tered 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 (2-

N,N-Bis(oleoyloxyethyl)amino-1-ethanesulfonic acid) the smallest

particle size reported in the literature (41nm) could be obtained

by standardHPH.

 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

disadvantageswhich havebeenaddressedbycontinuous improve-

ment of these processes.

For both, WBM as well as HPH it is suggested to start with

micronized startingmaterial. Cloggingof 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 themedia 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 interactionchamber.

 Table 2

Literatureexamples for drugnanocrystals prepared by high pressure homogenization or wet ball milling, respectively.

No Drug Top-down method Smallest reported particle size (nm) Delivery route Reference

1 PX-18 HPH 41 Pardeike andMüller (2010)

2 Asulacrine HPH 133 Ganta et al. (2009)

3 Ubc-35440-3 HPH 182 Hecq et al. (2006)

4 Indometacin HPH 200 Sharmaet al. (2009)

5 Prednisolone HPH 211 Kassemet al. (2007)

6 Resveratrol HPH 244 Topical Kobierski et al. (2009)

7 Danazol HPH 300 Crisp et al. (2007)

8 Hesperitin HPH 300 Mishraet al. (2009)

9 Celecoxib HPH 320 Oral Dolencet al. (2009)10 Ascorbyl palmitate HPH 348 Teeranachaideekul et al. (2008)

11 Azithromycin HPH 400 Oral Zhang et al. (2007)

12 Tarazepide HPH 400 I.v. injection  Jacobs et al. (2000)

13 Spironolactone HPH 400 I.v. injection Langguth et al. (2005)

14 Omeprazole HPH 500 I.v. injection Möschwitzer et al. (2004)

15 RMKP22 HPH 502 Grau et al. (2000)

16 Amphotericin B HPH 528 Kayseret al. (2003)

17 Hydrocortisone HPH 539 Kassemet al. (2007)

18 Budenoside HPH 599  Jacobs andMüller (2002)

19 Bupravaquone HPH 600 Oral  Jacobs et al. (2001)

20 Clofazimine HPH 601 I.v. injection Peterset al. (2000)

21 Nimodipine HPH 650 I.v. injection Xiong et al. (2008)

22 Rutin HPH 750 Oral Mauludin et al. (2009)

23 RMKK98 HPH 800 Krause et al. (2000)

24 Oridonin HPH 913 Zhang et al. (2010)

25 Dexamethasone HPH 930 Kassemet al. (2007)

32 Diclofenac HPH <800 Lai et al. (2009)33 Itraconazole WBM 128 I.v. injection Beirowski et al. (2011)

34 Candesartan cilexetil WBM 128 Oral Nekkanti et al. (2009)

35 Crystalline API WBM 150 Lee (2003)

36 Loviride WBM 156 VanEerdenbrugh et al. (2007)

37 Ketoconazole WBM 164 Oral Basa etal. (2008)

38 Cyclosporine WBM 199 Nakarani et al. (2010)

39 Camptothecin WBM 202 I.v. injection Merisko-Liversidge et al. (1996)

40 Piposulfan WBM 210 I.v. injection Merisko-Liversidge et al. (1996)

41 Piposulfan WBM 210 Merisko-Liversidge et al. (1996)

42 Cilostazol WBM 220 Oral  Jinno et al. (2006)

44 Etoposide WBM 256 I.v. injection Merisko-Liversidge et al. (1996)

45 Griseofulvin WBM 256 VanEerdenbrugh et al. (2008)

46 Naproxen WBM 270 Oral/I.v. injection Liversidge and Conzentino (1995)

47 Paclitaxel WBM 279 I.v. injection Merisko-Liversidge et al. (1996)

48 Hydrocortisone WBM 300 Ophthalmic Ali et al. (2011)

49 Cinnarizine WBM 366 VanEerdenbrugh et al. (2008)

50 1,3-Dicyclohexylurea WBM 800 Subcutaneous Chianget al. (2011)

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 J.P. Möschwitzer / International Journal of Pharmaceutics 453 (2013) 142–156 147

Relatively long process times are another disadvantage of 

the standard approaches. This stands in contrast to the above-

mentioned 30–120min to produce nanosuspensions by WBM.

However, this time is the minimum contact time of a drug inside

themilling chamber.Whena largescalemill is runin re-circulation

mode, the suspension is only exposed to high energy at the time it

passes themilling 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 homoge-

nizers can process 1000l of nanosuspension or more within 1h.

However, when 20 homogenization cycles are required to achieve

a certain particle size, the total process time can easily go up to

20h for batch sizes of 1000l, unless more homogenizers are used

in series.

In order to address the above-mentioned disadvantages, alter-

native processes have been developed. Significant reduction of 

process times canbe achievedwhen 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 com-

binativemethod, 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

of a suspension consisting of brittle drug particles. This suspen-

sion is then further processed to a nanosuspension by means of 

HPH (Kipp et al., 2003; Kipp, 2004; Rabinow, 2004). An alternative

method, which is alsoknown asH 69process,was developedmore

recently by Müller and colleagues. Ideally, the time between the

precipitation and the high pressure homogenization step should

beminimized, 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), butmore systematical research is needed for

a betterunderstanding of all critical process parameters.

Obviously, nomicronized startingmaterialis needed toperformthetwoabove-mentioned technologies.However,a remainingdis-

advantage 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 extentwhichcould

potentially compromise itsphysicalandchemicalstability. Inmost

cases, the non-aqueous solvent has to be removed in order to

reduce the risk of Ostwald ripening. To avoid this problem, alter-

native combinativemethodshave beendevelopedbyMöschwitzer

and colleagues, which are referred to as H 42 and H 96 technolo-

gies (Shegokar and Müller, 2010). The H 42 technology uses spray

drying of organic drug solutions as bottom-up step to produce a

modifiedstartingmaterial fora subsequentprocessstep,where the

modified drug is very efficiently processed by standard top-down

processes, e.g. HPH into nanosuspensions with small particle sizesand narrow size distributions (Möschwitzer, 2005; Möschwitzer

and Müller, 2006a). The spray drying process results in a pre-

treated, fine-dispersed 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,

aqueousmedia.The risk forparticlegrowth 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 andMüller, 2010). Pre-treatmentwith freeze-drying can

beusedwhen temperaturesensitivematerial hasto beprocessedor

when ultra-small drugnanocrystalsof expensivedrugs areneeded.

The freeze-drying process can be controlled to produce extremely

0

500

1000

1500

2000

2500

3000

3500

I II III IV V VI

  Process time

   M  e

  a  n  p  a  r   t   i  c   l  e  s   i  z  e   [  n  m   ]

Standard HPH

Standard WBM

H 96 (FD-HPH)

 H 42 (SD-HPH)

H 96 (FD-WBM)

Fig. 2. Mean particle size (PCS  z -average) as function of the process time and the

particle size reduction technique.All discontinuous linesrepresent novelcombina-

tive methods with modified starting materials; the continuous lines represent the

standardmethods with unmodified starting material. Point I: after the pre-mixing

step (high-speed mixer), points II–VI represent: 1, 5, 10, 15, 20 homogenization

cyclesforHPHresults, orresultsafter 1,2, 4,8, 24h ofmilling forWBM (usinga low

energy ball mill).

Modified afterSalazar (2012).

brittle startingmaterial. 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 nanocrys-

tals of amphotericine B by combining freeze-drying with HPH.

The resulting nanosuspensions had a particle size clearly below

100nm, 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 improvementof theparticlesizereduction effectiveness was also seen for modified glibenclamide

which was predominantly crystalline (Salazar et al., 2011). The

combination technologies nicely illustrate howstandardtechnolo-

giesarecontinuously improvedin orderto extend their application

areas.

Fig. 2 compares the particle size evolution of the model com-

pound glibenclamide as a function of process time for standard

top-downprocesses in comparison to the novel combinative tech-

niques. 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 1500bar or 1h

milling time. Although standard WBM results eventually in the

same final particle size, process time for the conventional processis 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 sameparticle

size as with the standard WBM method. In this regard, the com-

binative methods allow the application of HPH processes also for

harder APIs which aremore difficult to nanosize.

It should be mentioned in this context that any pre-treatment

stepincreases thecomplexityof theoverallprocessandcan addsig-

nificant 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 homoge-

nizationcannotbe used tocometo thedesired results.Oneexample

is the production of nanosuspensions from amorphous APIs with

particle sizes smaller than 100nmusing thecombinativemethods.

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148   J.P.Möschwitzer / International Journal of Pharmaceutics 453 (2013) 142–156

Itis very challengingto achievethis ina reasonable time framewith

establishedmethods, such aswet ball milling.

3. The formulation selection process for poorly 

 water-soluble compounds

Theselectionof therightformulationapproach isoneof thekey

activities of formulators in the pharmaceutical industry. Key fac-

tors are the physico-chemical properties of APIs, such as aqueous

solubility,meltingpoint andtemperature andchemicalstability. In

addition, theformulatorneeds informationabout thepotencyof the

compound and the desired route of administration, as this deter-

mines the typeof the finaldosage formaswell as the requireddrug

load.Allthesefactorscanbeconsidered indecisiontrees,which are

often used in the industry to guide the formulator. However, there

are some biopharmaceutical relevant aspects which need more

attention, inordertoavoidfalsenegative results. Like anyother for-

mulationtechnology,drugnanocrystalsas enablingtechnologycan

only be successfulwhen 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

drugnanocrystal.

The well-known BCS system (Biopharmaceutics ClassificationSystem) is used very frequently to categorize compounds (Amidon

et al., 1995). According to the BCS system poorly soluble com-

pounds can belong to class 2 (low solubility, high permeability)

or class 4 (low solubility, low permeability). Therefore, BCS class

2 and 4 compounds would be theoretically good candidates for

nanosizing approaches. This is a widely used and well accepted

perceptionwithin thepharmaceutical industry.However, usingthe

BCSsystemas guidance for formulation selectionmight 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 100nm) is too slow. In this

case it is not possible to reach sufficiently high drug concentra-

tions in the gastro-intestinal tract for an effective flux across theepithelialmembrane. Inaddition,other factors suchas effluxtrans-

port or pre-systemicmetabolism cannegatively 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 isknownasthe“DevelopabilityClassificationSystem”,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 theright formulation approachandto address

the compound specific issues with a suitable formulation type it

is imperative to first understand the bioavailability limiting fac-tors. It is important to note that there is no one-fits-all formulation

approach. Each technology has its own advantages and disadvan-

tages. 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 thedrug, the special aspects of the

various formulation options and the required in vivo performance,

the higher the chance that the optimal formulation approachwill

be chosen. This minimizestherisk of late failures inhumanclinical

trials, e.g. due to insufficient or highly variable drug exposures.

Compoundsshowingdissolution ratelimitedbioavailability can

bereferredtoasDCSclass IIacompounds.Obviously, theyrepresent

only one part of the BCS class 2 compounds. The extent of the oral

bioavailability of such compounds is directly correlatedwith their  T

     a       b       l     e

       3

    F

   o   r   m   u   a    l   t    i   o   n   a   p   p   r   o   a   c    h   e   s    f   o   r   p   o   o   r    l   y   s   o    l   u    b    l   e    d   r   u   g   s .

    F   o   r   m   u    l   a   t    i   o   n   a   p   p   r   o   a   c    h

    P   o   t   e   n   t    i   a    l    d   r   u   g    l   o   a    d

    l   o   w ,   m   e    d    i   u   m ,    h

    i   g    h

    S   u    i   t   a    b    l   e    f   o   r   t   e   m   p   e   r   a   t   u   r   e

    l   a    b    i    l   e   s   u    b   s   t   a   n   c   e   s

    S   u    i   t   a    b    l   e

    f   o   r   c   o   m   p   o   u   n    d   s

   w    i   t    h    h    i   g    h   m   e    l   t    i   n   g   p   o    i   n   t

    F   o   r   m   u    l   a   t    i   o   n    fl   e   x    i    b    i    l    i   t   y

    l    i   q   u    i    d ,   s   o    l    i    d

    A    d   m    i   n    i   s   t   r   a   t    i   o   n

   r   o   u   t   e    O   r   a    l   o   r    I    V

    C   o   m   p    l   e   x    i   t   y   o    f

   t    h   e   p   r   o   c   e   s   s

    F   o

   r   s   o    l   u    b    i    l    i   t   y   o   r    d    i   s   s   o    l   u   t    i   o   n

   r   a

   t   e    l    i   m    i   t   e    d   c   o   m   p   o   u   n    d   s

    M    i   c   r   o   n    i   z   a   t    i   o   n

    H    i   g    h

    Y   e   s ,   c   r   y   o   g   e   n    i   c

    Y   e   s

    l   +   s ,

    O   r   a    l

    S    i   m   p    l   e

    d

    N   a   n   o   s    i   z    i   n   g

    H    i   g    h

    Y   e   s

    Y   e   s

    l   +   s

    B   o   t    h

    C   o   m   p    l   e   x

    d

    C   y   c    l   o    d   e   x   t   r    i   n    f   o   r   m   u    l   a   t    i   o   n   s

    L   o   w

    Y   e   s

    Y   e   s

    l   +   s

    B   o   t    h   a

   s   +    d

    S   a    l   t    f   o   r   m   a   t    i   o   n

    H    i   g    h

    Y   e   s

    Y   e   s

   s    (    l   :    l    i   m    i   t   e    d    )

    O   r   a    l

    S    i   m   p    l   e

    (   s    )   +    d

   p    H   a    d    j   u   s   t   m   e   n   t

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    Y   e   s

    Y   e   s

    l

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    S    i   m   p    l   e

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  –    H    M    E

    M   e    d    i   u   m

    N   o

    N   o

   s

    C   o   m   p    l   e   x

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  –    S    D    D

    M   e    d    i   u   m   t   o    h    i   g    h

    L    i   m    i   t   e    d

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   s

    C   o   m   p    l   e   x

   s   +    d

    C   o  -   c   r   y   s   t   a    l   s

    H    i   g    h

    Y   e   s

    Y   e   s

   s

    M   e    d    i   u   m

    (   s    )   +    d

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    L   o   w

    L    i   m    i   t   e    d

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    b    E .   g . ,    l    i   q   u    i    d   o   r   s   e   m    i  -   s   o    l    i    d    fi    l    l   e    d    h   a   r    d   o   r   s   o    f   t   g   e    l   a   t    i   n   e   c   a   p   s   u    l   e   s .

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 J.P. Möschwitzer / International Journal of Pharmaceutics 453 (2013) 142–156 149

250 500 10000

Class IV

   P  r  e   d   i  c   t  e   d   P  e   f   f   i  n   h  u  m  a  n  s  c  m   /  s  e  c  x   1   0  -   4

10

1

0.1

Class I

Class III

Dose/solubility ratio

Class IIb

Solid-state manipulation (ASD)

Lipid-based systems

Complexation

Class IIa

Nanosizing

Salt formation

Co-crystals

Standard approaches

Permeation enhancer 

Mucoadhesion

Permeation enhancer + solubilization

Lead optimization

Lead optimization

Class IV

  -

10

1

0.1

Class I

Class III

Class IIb

Solid-state manipulation (ASD)

Lipid-based systems

Complexation

Class IIa

Nanosizing

Salt formation

Co-crystals

Permeation enhancer + solubilization

Lead optimization

Fig. 3. DCS classification systemand relevant formulation approachesfor the vari-

ous compound classes.

Modified afterButler and Dressman (2010).

dissolutionrate invivo.Thefractionof thedosethatdissolves in the

lumenis readilyabsorbedthroughthe intestinalmembrane. Conse-

quently, the bioavailabilityof such compounds canbe improvedby

any techniquewhich increasesprimarily thedissolution rate.Vari-

ous formulation approaches areknownwhich lead to an increased

dissolution rate, including salt formation, the use of co-crystals or

particlesizereduction. Theformulator hastoselect theoptimal for-

mulation approach according to theproperties of the specific drug

molecule.

Salt formationis oftenpreferredbypharmaceutical 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, inorder 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

andNewman, 2009).

Particle size reduction is by far the most important approach

to address dissolution rate limited bioavailability. According to the

well-knownNoyes–Whitney equation (Eq. (1)) thedissolution rate

depends directly on the surface area ( A) of thedissolving particles.

Particlesizereduction leadsto anincreaseinsurfaceareaandhence

to an accelerated dissolution rate.

dc  x

dt   =

D ·  A

h  (c s − c  x) (1)

where dc  x/dt is thedissolution rate;D is thediffusioncoefficient; Ais thesurfaceofdrug particle;h is the thicknessofdiffusional layer;

c s is the saturation solubility of the drug; c  x 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 tech-

nique, which is used on a routine basis to produce standardized

APIstartingmaterialhavinga certainparticle sizedistribution.This

unit operation is often carried out under the responsibility of the

chemical department, whichdelivers API with a standardized size

distribution. Micronization techniques include hammer milling,

pinmillingor air jetmilling.Dependingon thetechniqueemployed,

the meanparticle size generally ranges between 1 and 50m. The

fraction of fine particles below 1m is comparatively low. The

dissolution rate of poorly soluble drugs is increased compared to

non-micronizedmaterial.However, theeffectonthebioavailability

improvement is limited.

Nanosizing is particle size reduction to another dimension.

The term nanosizing subsumes the various formulation tech-

niqueswhichgenerate drug nanocrystalswith amean particle size

between 1 and 1000nm.Due to their small particle size these par-

ticles 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 factoralonewill resultin a fasterdissolutionrate.

Inaddition, the Prandtl equation showsthat drug nanocrystals also

have a decreased diffusional distance h. This further enhances the

dissolution rate. Finally, the concentration gradient (c s− c  x) is also

of high importance. There are reports that drug nanocrystals show

an increased saturation solubility c s. This can be explained by the

Ostwald–Freundlich equation (Kipp, 2004) and by the Kelvinequa-

tion (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.Mostprobablythe increasedsolubilityof drug

nanocrystals is a combined effect of nanosized drug particles andsolid state effects caused by the particle fractionation during the

process. Authors have reported effects of 10% increase in satura-

tion solubility up to several folds (Dai et al., 2007; Hecqet al., 2005;

Müller and Peters, 1998). In a detailed studya 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 concen-

tration are too low in order to achieve sufficient flux over the

epithelialmembrane. These compounds possess solubility limited

oral bioavailability. In order to achieve sufficient exposure levels

they have to be formulatedwith techniques that increase substan-tially 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 thermody-

namic 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).

Solubilizationusing lipidbasedsystemsorco-solvent systems is

a simple and elegant way to formulate poorlywater-soluble com-

pounds (Pouton,2006; Strickley,2004). Veryoften, theyare applied

as first-choice option to formulatepoorly soluble compounds. Sol-

ubilized systems can theoretically also be used as formulation

approach for dissolution rate limited compounds (DCS class IIa).

Lipidbased systems canbe administeredvery flexible as liquid for-mulations in pre-diluted form, or as water-free concentrate filled

inhard or soft gelatinecapsules. Theycanbe used for oralas wellas

parenteral applications (Strickley, 2004). Nevertheless, these sys-

tems are of limited use when very high drug doses are needed.

Often the API is not sufficiently soluble in the available excipi-

ents. 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,

canbe used as solubilizers to increase the bioavailability of poorly

water-soluble drug molecules (Brewster and Loftsson, 2007). The

formationof inclusion andnon-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

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150   J.P.Möschwitzer / International Journal of Pharmaceutics 453 (2013) 142–156

required to achieve higher bioavailability. Commercially available

cyclodextrin formulations are available for many administration

routes. Dependingon themolecular type thesesystemscanbeused

inliquid aswell assolidformfororalandwithsomeexceptions 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

manipulationof thedrug’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) andspray-drying(spraydried

dispersions, SDD) (Friesen et al., 2008) are applied to produce

ASDs. These techniques are an elegant way to produce oral dosage

forms ofpoorly soluble compounds at industrial scale. Severalmar-

keted products have proven the suitability of this approach as

commercial oral dosage forms. However, ASDs are not as flexi-

ble as other formulation approaches, e.g. they cannot be easily

used in liquid form for parenteral administration of poorly solu-

ble drugs. Ideally, the poorly soluble drug needs to bewell 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 suit-able for thermolabile compounds, as they are exposed to elevated

temperatures during the processing, if HME is used for manufac-

turing.

For the sake of completeness it should bementioned here that

recently a novel technology called NanOsmotic® (Alkermes) has

been developed which aims to combine the principles of parti-

cle 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

consequenceof the very fast dissolution rate andotherspecific fac-

tors drug nanocrystals possess some unique features with regard

tobiopharmaceuticalperformancewhichwillbediscussedinmore

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 c max   values and propor-

tionally also an increased AUC.  Jinno et al. (2006) have reported

the relationshipbetween particle size, dissolution velocity in vitro

and the in vivo effects for the poorly water-soluble drug cilosta-

zol in a very clear und understandable manner. The particle sizeof the drug was reduced by using different techniques. Hammer-

milling resulted in a mean particle size of 13m,  jet-milling in

2.4m and wet ball milling using the NanoCrystal® technology

to a particle size of 0.22m. The effect of the particle size on

the dissolution velocity was first demonstrated with in vitro dis-

solution 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

suspensionpreparedwithjet-milledorhammermilleddrug.Mean-

while, the direct relationship between the particle size of the

drug and the achievable extend of drug absorption have been

reportedformanyotherdrugs.All thesedrugs havebenefitted 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).

Compoundswitha pronouncedabsorptionwindowin theupper

intestinal tract do also benefit from fast dissolving formulations.

The accelerated dissolution of drug nanocrystals leads to suffi-

ciently 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 nanosuspensionwas

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

fenofibratemight notgetsufficiently absorbed,becauseitdissolves

too slowly and misses therefore the absorption window in theupper intestinal tract.

Furthermore, an increased dissolution rate of poorly water-

soluble 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;

Merisko-Liversidgeet al.,2003). Inthis case the short t max and high

c max levels resulting fromusing 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 drugswereadministered as drugnanocrystal formulation. Poorly water-

solublecompounds administeredasstandardformulationbasedon

micronized API show often an enhanced absorption when admin-

istered together with food. One potential explanation is that bile

salts and food components can have a positive effect on the solu-

bilityandconsequentlyonthedissolution rateofmicronizeddrugs.

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 fur-

ther increased for those compounds when administered together

with food ( Jinno et al., 2006; Sauron et al., 2006; Shono et al.,

2010).

Special attention is neededwhen ionizable compounds are for-mulated as drug nanocrystals. The particle size reduction itself 

is a rather versatile approach which works for all compounds

irrespectivelyof theirchemicalnature.Whenneutralor acidiccom-

pounds 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 nano-

approach. There are also examples for basic compounds which

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 J.P. Möschwitzer / International Journal of Pharmaceutics 453 (2013) 142–156 151

havebeendevelopedas nano-formulations and tested successfully

invivo (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,nowadaystheyarealsoconsidered fornon-oralapplications.

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 impor-

tant aspects regarding the use of drug nanocrystals for non-oral

administration will be discussed below.

Nanosuspensions show some advantages over other formula-

tion types which contain the drug in solubilized form. Solutionsof poorly water-soluble compounds bear always the risk of pre-

cipitation 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 nanosuspen-

sions, producedwitha minimumamountof safeandwell-tolerated

stabilizers, can be advantageous. Moreover, in contrast to other

injectable formulations, nanosuspensions are highly concentrated

systems, with a relatively low viscosity. Since the viscosity of 

nanosuspensionsmainly depends on drugconcentrationandvehi-

cle 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

eitherby aseptic production (Baert et al., 2009; Peters et al., 2000),by sterile filtration (Zheng and Bosch, 1997), heat treatment (Na

et al., 1999) or gammaradiation (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 thevarious presentations of drug nanocrys-

tals. Thefinal drug product canbe 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-to-

use pre-filled syringe containing a nanosuspension prepared by

theNanoCrystal® technology.The patient-friendlynanosuspension

has a low viscosity and a high drug load which results in a low

injection volumeand lowpain levels upon injection.

Thecorrect use of nanosuspensions for injectable dosage formsrequires 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 beassumed that the particles donot

dissolve fast enough. Consequently, they areaccumulatedas parti-

cles inMPS(mononuclearphagocytic system) richorgans, like liver

and spleen. This can leadto a prolonged actionof the drug. Inmany

cases intravenously administered nanosuspensions showed a bet-

ter 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 bymany companies. They haveadded

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

approachwhichis oftenreferredto asfrontloading.Thisapproachis

used to increase the success rate of the drug discovery byenabling

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 screeningprocess canbe increasedsignif-

icantly when the appropriate techniques for poorly soluble drugs

are available (Merisko-Liversidge and Liversidge, 2011).

Their scalability is one important factorwhy drug nanocrystals

are included in the formulation decision trees of so many com-

panies. As mentioned earlier, drug nanocrystals can be used at alldevelopment stages, since nanometer-sized drug particles can be

producedfromextremelysmall scale uptocommercial production.

Thefirst formulations for animal studies are neededwhen var-

ious 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 nano-

approach 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 top-

down method, is that the drug has to be poorly soluble in the

dispersion medium (e.g. an aqueous medium). The solubility limit

differs dependingon the employed nanosizing technique between10g/mland100g/ml(Merisko-Liversidgeand Liversidge,2008).

(2)FormulationsforveryearlyPKstudies shouldnot require exten-

sivedevelopment.Theyaremostlyperformedinrodents,inorderto

limittheAPIrequirements. InitialPK formulationshaveto berather

straight-forward and can be either solutions or suspension-based

systems (Li and Zhao, 2007). The purpose is primarily to estab-

lish important pharmacokineticparameters, suchas rate/extend of 

absorption, clearance, and distribution volume. Many approaches,

suchas cyclodextrinformulations, lipidor surfactantbasedsystems

as well as nano-formulations are used (Chaubal, 2004). Nanosus-

pensions can be seen in this regardas universalplatformapproach

which isthepreferredoptionat thisstage.(3) A furtheradvantageof 

usingnanosuspensionsalreadyat thatstageistheuniversal routeof 

administration. Properly chosen, nanosuspensions can be admin-istered 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

pharmacologicalmodels aremorecomplex.Dependingon theindi-

cation and the pharmacologicalmodel the selection of a universal

formulation can be sometimes very challenging, especially when

some frequently used systems areexcluded because of their inter-

ference 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 develop-

ment the available drug amounts are normally extremely limited,

therefore only some standard formulations are tested. With an

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152   J.P.Möschwitzer / International Journal of Pharmaceutics 453 (2013) 142–156

increased understanding on how to develop robust nanosuspen-

sions 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 suffi-

ciently high, one needs to demonstrate andestablish thesufficient

safety margin for the selected lead compounds. Nanosuspensions

are ideal formulations for toxicological studies as they can be rel-

atively easily formulated with safe vehicle compositions that are

already established as standard excipients for toxicological test-

ing. In manycases standard cellulose/poloxamernanosuspensions

can be produced which differ only in their distinctly smaller par-

ticle 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 solu-

tionsor amorphoussoliddispersions.The higherdrug 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 publica-

tion provides a very comprehensive overview about preparation

andmanufacturing logisticsof nanosuspensions for sucha purpose(Kesisoglou et al., 2007). Another literatureexample 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 zir-

conium beads (Kwong et al., 2011). For the acidic compound

UG558 the same positive trend was found. The nanosuspension

performed about 4.6 times better than the microsupension. In

addition, thenanosuspension could be administered intravenously

allowing the determination of the absolute bioavailability of the

compound (Sigfridsson et al., 2009). When compounds have dis-

solution rate limited oral bioavailability they should normally

show a linear dose/AUC relationship. However, in some toxico-

logical studies a non-linear absorption is seen at very high doselevels. In this case particle size reduction is not sufficient to

increase the exposure further as the systems turns from a disso-

lution rate limited to a solubility limited system (Sigfridsson et al.,

2011a).

Thenext stageofdevelopmentbeginswhena seriesofNCEs has

been successfully tested in pre-clinical programs and at least one

compound could be qualified as clinical candidate. Many compa-

nieshave implementedformulationrankingorspecialPKscreening

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 theselectionof theoptimal 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 soliddosage form by applying various conventional drying techniques.

Spray drying is a straight-forward method for drying of nanosus-

pensions(Chaubal andPopescu,2008;Gaoet al., 2010;Lee, 2003). It

has the advantage of being as scalable as nanosizing itself. Often, it

is thefirst choiceatthebeginningof thedrugproductdevelopment,

because it can be easily performed at bench as well as pilot scale.

Thespraydried intermediatecanbe compressed to tablet formula-

tions ( Jinno et al., 2008). The tablet compositionhas to be selected

carefully, in order to obtain a fast and complete reconversion to a

nanosuspensionwitha particle sizedistribution comparable to the

non-dried formulation (Heng et al., 2009). In general, spray drying

yieldspowderswithratherlowdensities,whichcould requireaddi-

tional process steps, suchas roller compaction toobtain tablettable

intermediates.

Fluidizedbedgranulation is an alternativemethod to produce a

dryintermediate.Dueto theavailable equipmentit requires some-

what larger quantities of nanosuspension and is therefore used

at a later stage. During fluidized bed granulation the nanosus-

pension 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

tabletsina subsequentprocessstep.Spray-layeringofnanosuspen-

sions onto beads is an alternative approach (Kayaert et al., 2011;

Möschwitzer and Müller, 2006b). After an optional overcoating-

step 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.

Thenanosuspension is coatedonto inert tablet coreswhichconsist

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

reducetheir freeenergybyeitheraggregationorcrystalgrowth.For

a long time this ledto theperception that nanosuspensions as such

would have a limited shelf life and would not be suitable as ready-

to-use formulations. The physical stability of a nanosuspension

depends on many aspects, e.g. the selection of the right stabilizer

principle, thesolubility of theAPI in the liquidphaseof the suspen-sion and last butnot leastalso onthe employednanosizingmethod.

Butwithall parameters selectedcarefully, nanosuspensions canbe

physically aswell as chemically very stable systems. For both top-

downmethods, examples have been reported, where the particle

sizesof physically stablenanosuspensionsremainedunchanged for

years ( JacobsandMüller, 2002;Merisko-Liversidgeand Liversidge,

2011). These data indicate that nanosuspensions can indeed be

used as ready-to-use suspensions.This is also demonstratednicely

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 notmore formulationsbased on drug nanocrystals

have reached themarket yet. Many pharmaceutical companies donot have sufficient in-house capabilities both in terms of equip-

ment and know-how to develop drug nanocrystals for clinical

studies. Even if all necessaryprerequisites would be fulfilled, com-

panies 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 developedmost 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 devel-opability. The perception was that the risk of developing poorly

soluble compoundswould be toohighandthereforewater-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 fur-

ther development. Nowadays, as result of extensive formulation

screening during pre-clinical programs, compounds with more

challenging properties are frequently selected as clinical candi-

dates. 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 aswell as access

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