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![14 REVIEW
Table 5. Food Effect
PK Parameter
Fed Fast
Compound NSa Refb NS Ref Reference
ELND-006 Tmax (h) 1.4 3 1.4 1.8 90
Cmax (ng/mL) 365 159 294 49.4
AUC (ng h/mL) 3063 1767 2430 315
Fc 110 63.4 87.2 11.3
Fed Fast
NCd Jetmilled NC Jetmilled
Cilostazole Tmax (h) 1 1 1.3 ± 0.5 1 91
Cmax (ng/mL) 4872 ± 112 2901 ± 314 5371 ± 1173 1029 ± 218
AUC (ng h/mL) 13,589 ± 3895 10,669 ± 3417 17,832 ± 4994 2875 ± 587
F 0.67 ± 0.22 0.53 ± 0.21 0.86 ± 0.29 0.15 ± 0.04
Fed Fast
SNCDe SDDf SNCD SDD
Ziprasidone Cmax (ng/mL) 260 285 416 140 92
AUC (ng h/mL) 1911 1949 2044 879
a
NS, nanosuspensions.
b
Ref, reference sample.
c
Relative bioavailability.
d
NC, nanocrystal.
e
SNCD, solid nanocrystalline dispersion.
f
SDD, solid amorphous spray-dried dispersion.
future success, persistence in oral bioavailability can only be
achieved by eliminating the food effect.89
The presence of bile
salts in the gastric environment also has an impact on the dis-
solution behavior of the molecule. It has been reported that the
presence of food reduces the dissolution behavior of micronized
particles in certain cases as reported in Table 5.90–92
The ratio
of bioavailability of the fed to fast state was observed to be close
to 1 in the case of nanocrystals, while micronized formulation
of the same drug showed a significantly higher ratio (approx.
sixfold) of the fed to fast state by demonstrating the poten-
tial food effect after oral administration to an animal model
with the same dosing. Bile salt secretion usually increases
in the fed state.93
A change in concentration of the bile salt
in the stomach leads to a change in dissolution behavior and
slows down or enhances absorption of the drug as depicted in
Figure 3. This phenomenon causes a large variability from pa-
tient to patient and also from fed state to fast state. Micronized
or larger particles that have shown improved absorption in the
fed state might be because of the micelle formation. For the
nanocrystals though, they are not helping in improving the in-
trinsic solubility, but they provide an advantage by enhancing
the initial dissolution rate because of the larger SA. The higher
rate of dissolution leads to an increased rate of absorption, and
eventually, enhancement in the overall bioavailability, irrespec-
tive of the fed or fast state. Hence, it can be stated that the bile
salts concentrations have significantly less effect on the absorp-
tion behavior of nanoparticles. The same observation has been
reported for cilostazole91
[fed/fast ratio—0.78 (nano vs. 3.53
jet-milled)]. Thombre et al.92
have reported the PK behavior of
an anti-psychotic drug ziprasidone in beagle dogs by conduct-
ing the fed- and fasted-state bioavailability experiment for the
both solid nanocrystalline dispersion and the commercial blend
(amorphous spray dried dispersion). The commercial blend has
shown a twofold improvement in bioavailability in the fed state
as compared with the fasted state, indicating the failure of the
commercial blend to prevent variability in absorption behavior
irrespective of the food state. A similar food effect was observed
with human data. On the other hand, nanocrystals have proven
to be efficient enough to prevent the variability from fed to fast
state by generating a similar bioavailability profile in beagle
dogs. From the reported literature, it can be concluded that vul-
nerability in the variation of oral bioavailability for potential
drugs can be avoided with reduction in particle size to nanome-
ter scale.
Particle Size Effect: Micro Versus Nano
The effect of particle size on in vitro and in vivo performance is
well documented elsewhere.91,94–96
Different particle sizes have
a different effect on the initial dissolution rate, the rate of ab-
sorption, and on oral bioavailability. Moreover, particles with
nanometer size range may have the tendency to pass through
both active and passive diffusion pathway, which increases the
chances for sub-micron particles to reach to peak plasma con-
centration with a reduction in time compared to macro/large
particles. Sun et al.97
have shown the effects of particle size,
ranging from micron to nanometer size, on Cmax, Tmax, and
AUC.98
A large increase in bioavailability was observed for the
both nanoparticles (i.e., 300 and 750 nm) as compared with
the micron size particles and coarse powder, which is reported
in Table 6. The rise in bioavailability can be attributed to in-
creased SA and adhesion behavior of nanosized particles. There
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A Review- Pharmaceutical and Pharmacokinetic Aspect of Nanocrystalline Suspensions
This document reviews pharmaceutical and pharmacokinetic aspects of nanocrystalline suspensions. It discusses how nanocrystals have emerged as a potential formulation strategy to enhance dissolution rate and solubility for poorly soluble drugs. The review provides an in-depth look at processing methods, quantitative assessments of solubility and dissolution rates, and their correlation to pharmacokinetic data. It also discusses the lack of understanding around changes in thermodynamic and kinetic properties like solubility and dissolution rate upon nanosizing, and reviews literature on quantitatively studying the effect of particle size and surface area on initial dissolution rate enhancement.
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A Review- Pharmaceutical and Pharmacokinetic Aspect of Nanocrystalline Suspensions
- 1. REVIEW A Review: Pharmaceuticaland Pharmacokinetic Aspect of Nanocrystalline Suspensions DHAVAL A. SHAH,1 SHARAD B. MURDANDE,2 RUTESH H. DAVE1 1 Arnold & Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn, New York 11201 2 Drug Product Design, Pfizer Worldwide R&D, Groton, Connecticut 06340 Received 7 August 2015; revised 23 September 2015; accepted 25 September 2015 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24694 ABSTRACT: Nanocrystals have emerged as a potential formulation strategy to eliminate the bioavailability-related problems by enhancing the initial dissolution rate and moderately super-saturating the thermodynamic solubility. This review contains an in-depth knowledge of, the processing method for formulation, an accurate quantitative assessment of the solubility and dissolution rates and their correlation to observe pharmacokinetic data. Poor aqueous solubility is considered the major hurdle in the development of pharmaceutical compounds. Because of a lack of understanding with regard to the change in the thermodynamic and kinetic properties (i.e., solubility and dissolution rate) upon nanosizing, we critically reviewed the literatures for solubility determination to understand the significance and accuracy of the implemented analytical method. In the latter part, we reviewed reports that have quantitatively studied the effect of the particle size and the surface area change on the initial dissolution rate enhancement using alternative approaches besides the sink condition dissolution. The lack of an apparent relationship between the dissolution rate enhancement and the observed bioavailability are discussed by reviewing the reported in vivo data on animal models along with the particle size and food effect. The review will provide comprehensive information to the pharmaceutical scientist in the area of nanoparticulate drug delivery. C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci Keywords: nanocrystals; nanosuspensions; nanoparticles; solubility; dissolution; pharmacokinetics; food interactions; bioavailability; particle size reduction INTRODUCTION Recent advances in synthetic, analytical, and purification chemistry, along with the development of specialized tools such as high-throughput screening, combinatorial chemistry, and proteomics, have led to a sharp influx of discovery com- pounds entering into development. Many of these compounds are highly lipophilic, as the in vitro screening techniques place considerable emphasis on the interaction of compounds with de- fined molecular targets. In recent years, it has been estimated that up to 70% of the new drugs discovered by the pharmaceu- tical industry are poorly soluble or lipophilic compounds. Poor aqueous solubility is one of the major hurdles in the develop- ment of new compounds into oral dosage forms, as absorption is limited by dissolution for these compounds.1 The well-known Biopharmaceutics Classification System (BCS) is frequently used to categorize pharmaceutical com- pounds. According to the BCS system, poorly soluble com- pounds belong to Class II (low solubility, high permeability) or Class IV (low solubility, low permeability). In another words, we can also say that Class II and IV compounds provide more opportunities for the development of newer technologies to overcome the solubility- or dissolution-related issues based on chemical and physical properties of the compounds. This per- ception is widely used and well established within the pharma- ceutical industry. However, using the BCS system for guidance in formulation selection may sometimes oversimplify the com- Correspondence to: Rutesh H. Dave (Telephone: +718-488-1660; Fax: +718- 780-4586; E-mail: [email protected]) Journal of Pharmaceutical Sciences C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association plex nature of drug dissolution, solubility, and permeability. Poorly water-soluble compounds can possess such a low aque- ous solubility that the dissolution rate, even from micronized particle, is very slow. In this case, it is not possible to reach suf- ficiently high drug concentrations in the gastrointestinal tract for an effective flux across the epithelial membrane. Other fac- tors, such as efflux transport or pre-systemic metabolism, can also negatively influence oral bioavailability. Therefore, it is recommended to classify compounds into slightly different categories, as they can show dissolution rate-limited, solubility-limited, or permeability-limited oral bioavailability. Butler and Dressman2 designed the “Developa- bility Classification System (DCS),” as another way to catego- rize compounds in a more bio-relevant manner. This system dis- tinguishes between dissolution rate-limited compounds (DCS Class IIa) and solubility-limited compounds (DCS Class IIb). In order to select the right formulation approach and to ad- dress the compound-specific issues with a suitable formulation type, it is imperative to first understand the bioavailability lim- iting factors. Selection of the right formulation approach is one of the key activities for formulators in the pharmaceutical in- dustry. Key factors include the physicochemical properties of active pharmaceutical ingredient (API), such as aqueous solu- bility, the melting point temperature, and chemical stability. In addition, the formulator needs information about the potency of the compound and the desired route of administration to de- termine the type of 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 as- pects that need more attention in order to avoid false nega- tive results. In addition, it is also important to note that there Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES 1
- 2. 2 REVIEW is nouniform approach that solves all the formulation-related problems. Each technology has its own advantages and disad- vantages. Depending on the formulator’s understanding of the interplay between the physicochemical properties of the drug, the special aspects of the various formulation options and the required in vivo performance, the higher the chance that the op- timal formulation approach will be chosen. This minimizes the risk of late failures in the human clinical trials, for example, due to insufficient or highly variable drug exposures. Compounds showing dissolution rate limited bioavailability may be referred to as DCS Class IIa compounds, but they represent only one part of the BCS Class II compounds. The extent of the oral bioavailability of such compounds directly correlates with their dissolution rate in vitro. The fraction of the dose that dissolves in the lumen is readily absorbed through the intestinal mem- brane. Consequently, the bioavailability of such compounds can be improved by any technique that increases the primarily the dissolution rate. Various formulation approaches are known to lead to increased dissolution rate and bioavailability, includ- ing salt formation, the use of cocrystals, particle size reduction, complexing with cyclodextrins,3 microemulsions,4 and solid dis- persion technologies.5,6 The formulator has to select the optimal formulation approach based on the properties of a specific drug molecule. However, all these technologies have certain limita- tions and cannot be used as universal formulation techniques for all the poorly soluble compounds, especially those which are insoluble in both aqueous as well as non-aqueous solvents.7 To prevent the removal of poorly soluble compounds from the pharmaceutical pipeline, a broad-based technology is required for drug molecules that are insoluble or poorly soluble in both aqueous and non-aqueous solvents. This will have the tremen- dous impact in discovery sciences and will improve the perfor- mance of existing molecules suffering from formulation-related issues.8 In the last two decades, after the introduction of Nano crystal R technology, particle-size reduction approaches have grown to a commercial level. Several formulation ap- proaches have been reported to formulate the nanoparticles, such as nanocrystalline suspensions, Poly Lactic-co-Glycolic acid(PLGA)based nanoparticles, nanosphears, and solid-lipid nanoparticles. By the virtue of their large surface area (SA) dc dt = AD(Cs−C) h ln S S0 = 2MY DrRT hH = k √ L/ √ V Noyes–Whitney Equation Ostwald–Freundlich Prandtl Equation dc/dt = Dissolution velocity S = Solubility at Temp T hH = Hydrodynamic boundary layer thickness A = Surface area S0 = Solubility of infinite big particle k = Constant D = Diffusion coefficient M = Molecular weight L = length of surface in flow direction Cs = Saturation solubility D = Density V = relative velocity of flowing liquid C = Drug concentration in U = Interfacial tension Solution at time t R = Gas constant h = Thickness of diffusion layer r = Radius T = Temperature to volume ratio, nanocrystals provide an alternative method to formulate poorly soluble compounds. Nanosizing refers to the reduction of the APIs’ particle size down to the sub-micron range. Nanosuspensions are sub-micron colloidal dispersions of discrete particles that have been stabilized using a surfactant and a polymer or a mixture of both.9 Stabilized sub-micron particles in nanosuspensions can be further processed into standard dosage forms, such as tablets or capsules, which are best suited for oral administration. It has been studied and observed that the reduction in par- ticle size in the micron or nano range have a positive impact on the in vitro dissolution rate, which can be used to predict in vivo enhancement in bioavailability for poorly soluble compounds.10 Compound-specific properties, such as high melting point, high log P value and poor aqueous solubility, are required to consider before the selection of this approach. Therefore, BCS Class II and IV compounds would theoretically be good candidates for the nanosizing approach, along with some exceptions, such as fenofibrate (FBT) (low melting point).11 Drug nanocrystals ex- hibit many advantages, including high efficiency of drug load- ing, easy scale-up for manufacture, relatively low cost for prepa- ration, and applicability to various administration routes, such as oral, parenteral, ocular, and pulmonary delivery (Table 1). All these advantages have led to successful promotion of drug nanocrystals from experimental research to patients’ usage. The availability of several products on the market shows the therapeutic and commercial effectiveness of the approach.12 The pioneering work of many academics and industrial re- searchers has laid the foundation for broad utilization and ac- ceptance of this approach within the field of pharmaceutical sciences. By definition, nanosizing is particle-size reduction to 1 and 1000 nm. Because of their small 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, which is why they are often stabilized with sur- factants, stabilizers, or with a mixture of both. Reduction of the particle size to the nanometer range results in a substantial increase in SA (A), thus, this factor alone will result in a faster dissolution rate as described by Noyes–Whitney.13 In addition, the Prandtl equation shows that the drug nanocrystals showed 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 nanocrys- tals have shown increased saturation/thermodynamic solubil- ity (Cs). This can be explained by the Ostwald–Freundlich equation14 and by the Kelvin equation.15 It is still not clear to what extend the saturation solubility can be increased solely as a function of particle size. Most prob- ably 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. A number of authors have reported improvement from a 10% increase in Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps.24694
- 3. REVIEW 3 Table 1.Advantages of Nanocrystals in Different Route of Administration Route Advantages Oral r Increase bioavailability r Decrease in fed/fast variations r Increase rate of absorption; decrease in Tmax and increase in Cmax r Quick and easy to formulate Parenteral r About 100% bioavailability can be achieved if given as an IV formulation r Targeting drug delivery r Avoidance of organic solvent, surfactants, pH extremes Pulmonary r Used in nebulizer as a liquid solution or dry powder r A single drop can contain many nanoparticles r Increase the concentration and or loading of nanocrystalline dispersion saturation solubility to several folds using different approaches.16–20 Below are the established equations to de- scribe nanocrystals and their physicochemical properties. Advantages of nanocrystals over conventional and special drug delivery systems: 1. Because of high surface enlargement factor in nanocrys- tals, there is an increase in the dissolution rate as well as a modest increase in saturation solubility as compared with micronized particles. 2. With a size range in nanometers, it can be injected as a IV to get 100% bioavailability. 3. Dose reduction and patient compliance. 4. Lessen or eliminate the food effect on bioavailability. 5. Targeted drug delivery either by transcellular or intra- cellular uptake. 6. Molecule can be delivered via a required route with ease in scale up. This review focuses on the various established approaches for the formulation of nanocrystals, the different published an- alytical methods applied for thermodynamic solubility deter- mination, assessment of dissolution properties and dissolution rate enhancement upon nanosizing, the effect on pharmacoki- netic (PK) properties such as bioavailability, the area under curve (AUC), and the half-life due to size reduction as well as future research opportunities. FORMULATION APPROACHES FOR NANOCRYSTALS Before the first top-down processes were developed (i.e., tech- niques reducing the size of larger crystals by means of attrition forces), nanosized drug particles were produced using a sim- ple precipitation approach known as solvent–anti-solvent ad- dition technique. It is also referred as one of the “bottom-up” approaches. However, it is often difficult to control the particle growth/crystal growth using this technique as well as to scale up by maintaining all the parameters constant. Therefore, it was suggested to perform the precipitation step in conjunction with immediate lyophilization, or spray-drying, in order to re- duce the risk of crystal growth. Top-Down Approach There are two basic approaches which are well established for the formulation of nanocrystals: 1. Top down: Involves the mechanical reduction of the par- ticle size by wet media milling or high-pressure homoge- nization (HPH). 2. Bottom up: Involves the generation of nanosized particles from dissolved molecules by means of precipitation.9 Top-down methods can be further divided in to two approaches—homogenization and attrition wet media milling. Attrition Wet Media Milling This technology was developed at the Pharmaceutical Research Division of Eastman Kodak (Sterling Winthrop, Inc.), which was set-up as NanoSystems LLC and later acquired by Elan. An active drug substance is dispersed with an aqueous solution in which the stabilizers were pre-dissolved. As the surface of nanocrystals is highly cohesive and has high surface energy, it should be stabilized by a single or mixture of stabilizers. Stabilizers can be ionic or stearic and can be used as a single and/or in a combination of polymeric as well as surfactant sta- bilizers. This solution is poured in the grinding chamber along with spherical beads/balls while the beads are rotated at very high speed. It is believed that because of the attrition between molecules’ surface and surface of the beads, particle size reduc- tion occurs; the beads/balls serving as a milling media. Beads are available in various sizes and are of different materials, but generally are made of glass, zirconium oxide, or polymeric material. The type of material the beads are made of is a crit- ical factor as they can interact with the active drug substance. There is a fair chance that an impurity related to the material of beads may contaminate the final product. Yttrium-stabilized zirconium oxide is the most widely used type of bead by ma- jor pharmaceutical companies because in most cases, it does not interact with active drug substances. Although expensive, these beads are the best alternative to avoiding impurities in the final formulation.21 The size of the beads has a direct relationship with the de- sired particle size range in the formulation of nanocrystals.22 The usual duration for conventional milling using overhead stirring is somewhere between 3 and 12 h. Certainly, these pa- rameters can change from molecule to molecule. Milling should be stopped once the desired particle size range is achieved. The rotational speed of the milling media is also a critical parame- ter. With the too slow speed, the beads cannot rotate efficiently and milling cannot be performed accurately, and with the too fast speed, the evenly rotating balls may remain at the upper surface of the media and milling does not take place. With a systematic study by trial and error the formulator selects the stabilizers, as well as other milling parameters and optimizes them in order to achieve the desired particle size range and stability. The final product characteristics can vary, depend- ing on the amount of beads, the ratio of active drug substance to the amount of beads, the ratio of concentrations of active substance to the stabilizer, milling time, milling temperature as well as milling duration.23 This method is simple, inexpen- sive, and easily scalable. The only drawback associated with this technology is the contamination related to the beading DOI 10.1002/jps.24694 Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES
- 4. 4 REVIEW material. Thataside, several products have successfully reached the commercial level using this technology. High-Pressure Homogenization There are several established methods for the formulation of nanocrystals using the homogenization approach. The microflu- idization technology (Insoluble Drug Delivery-Particles IDD- PTM Technology), Dissocubes R technology, and Nanopure R technology are examples of the methods that fall under this category. Microfluidizers are known as high shear fluid pro- cessors that are unique in their ability to achieve monomodal particle size reduction. It reduces particle size by a frontal colli- sion of fluid streams under pressure of up to 1700 bar.24 At very high pressure, collision and cavitation occur. The major draw- back associated with this method is that it requires at least 50–75 cycles to achieve the desired nanometer size range. This makes the method more tedious and relatively more time con- suming as compared to milling. Dissocubes R technology works with piston gap homogenizer, which was developed by Muller and his colleagues. In this method, a crude aqueous suspension of active drug substance and stabilizer is forced through a tiny hole, which can reach a pressure of up to 4000 bar. The width of the homogenization gap is adjustable, which is typically in the micrometer range. Compared with wet media ball milling, there are fewer chances to generate impurities with HPH. The negative aspects of using this method are cavitation, which causes mechanical wear, as well as noise, although fragmentation is a beneficial effect associated with cavitation. The main source of impurity comes from the wearing out of equipment parts. Almost all ma- chine parts are made of stainless steel, which leads to a very low impurity level when the nanosuspension is prepared using the HPH. Krause and Muller25 carried out a comparative study and observed a negligible amount of iron impurities in the nanosus- pension formulated with 20 cycles at 1500 bar. Wear and tear occurs only when very hard material is processed through the piston gap. Using stainless steel material can also lead to wear and tear as the new type of homogenization valves used today are made of ceramic tips which are able to withstand the harsh processing conditions.26 Homogenizers vary in size from a small scale to large scale production.27 Many research studies have reported minimal growth of microorganisms as a result of the HPH process.28 These improve the shelf life of the nanosuspen- sion and avoid the need for further studies that are required if it is administered orally. However, it is not a rule of thumb, the HPH is generally used for relatively soft material and bead media mill is used for relatively harder or harsh material. Combinative Approach In order to proceed with both the top down technology (wet me- dia milling, HPH) micronized powder is required as the starting material, which leads to a long process time. In order to over- come this drawback, a combinative formulation approach was developed. The combinative approach was first developed and introduced by Baxter Inc. as NanoedgeTM technology. Today five combinative methods have been successfully developed. 1. NanoedgeTM —microprecipitation + HPH 2. H69—microprecipitation immediately followed by HPH (minimization of time between two steps in order to pro- duce even smaller crystals) 3. H42—drug pre-treatment by means of spray-drying fol- lowed by standard HPH 4. H96—Freeze drying combination with HPH 5. CT—Media milling followed by HPH In the microprecipitation stage, the drug is usually dissolved in a suitable organic solvent that is miscible with water. The drug solution is then added to an aqueous solution in which stabilizers have been pre-dissolved. The drug solution is added in a controlled manner to prevent inadequate crystal growth. After the microprecipitation step, precipitates are converted into more stable crystals in the nanometer size range with the help of top down technologies (i.e., HPH, media milling, and sonication). The amount of residual content in the final prod- uct is the major concern while using a combinational approach during scale up. The presence of organic solvent can alter the physicochemical properties of the active drug substance.29 It may also be responsible for the Ostwald’s ripening. To prevent this from happening, an alternative method was developed by Salazar et al.21 known as H 42 and H 96 technology. H42 uses the spray drying of the microprecipitated solution that was de- veloped with the bottom-up approach, and then followed by HPH. In the case of H96, it employs the freeze drying of the mi- croprecipitated solution, followed by a top-down approach. In- deed, on the one hand, this method has more advantages than any single step conventional method, but on the other hand, any additional steps in the procedure require more careful and more extensive research, and control of additional parameters, which will increase the cost of the end product development. To date, no product has been developed and marketed using this technology, but research papers have been published for the formulation or production of nanocrystals using the combina- tive approach. Among these, the top-down approaches are more convenient because of the ease of being able to govern the parti- cle size range as well as the ease of scaling up. Because of these benefits, several products have been successfully launched to the commercial level. Bottom-Up Approach This method is also known as the precipitation approach. Hydrosols30 and Nanomorph31 techniques are examples of the bottom-up approach. The particles generated by Nanomorph technology are amorphous in nature, which give an advantage of both a higher supersaturation and a higher dissolution rate. It is well known that amorphous systems are high energy sys- tems; therefore, because of their high rate of crystallization, un- controllable crystal growth occurs, which leads to a reduction in solubility and eventually, reduction in bioavailability. Although both technologies are scalable, they require the control of dif- ferent parameters, such as temperature and the stoichiometry of the solute, solvent, and the stabilizer. UNDERSTANDING SOLUBILITY BEHAVIOR AND METHODS OF DETERMINATION Several research papers have discussed the impact of solubility and particle size on the PK performance of nanocrystals. Some of the literature has reported that the generation of surface cur- vature and crystal defects on the particle surface have an enor- mous impact on its solubility behavior. Another possible cause might be the development of high energy surfaces through Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps.24694
- 5. REVIEW 5 attrition duringparticle size reduction. According to the litera- ture, solubility may range from one-fold to several fold, based on particle size.17–19 Bioavailability enhancement associated with nanocrystalline API is attributed to an increase in the dissolu- tion rate because of the enlargement of SA and some increase in solubility based on particle size. This solubility enhance- ment should be in fair agreement with what would be expected based on the Ostwald–Freundlich equation. A change in solu- bility is more significant when particle size is reduced to below 100 nm, which can also be described by the Ostwald–Freundlich equation. Rapid dissolution associated with a nanoparticulate system is clear evidence of a generation of transient super sat- uration of a solution compared with the bulk solubility of a stable crystal form. In the case of crystalline nanoparticles, the degree of supersaturation is low compared with high energy amorphous solids, as particle size has limited impact on satu- ration solubility. Determining accurate solubility is vital to characterize the effectiveness of the formulation. There are several challenges associated with the accurate determination of solubility of any formulation, as it varies case by case. Accurate measurement is significantly more complicated in the case of nanoparticulate systems, as they have the tendency to remain in suspended form in the solution after using conventional approaches. It is almost impossible to visualize the presence of nanoparticles in the filtrate with the naked eye. The intrinsic solubility of poorly soluble compounds is extremely low in number; therefore, the presence of a couple of undissolved particles can lead to a signif- icant error in measurement. While reviewing the literature for determining solubility by a separation-based method, we have found the absence of a validated universal method for accurate solubility determination. This makes it even more challenging when dealing with particle size in the nanometer range, as com- pared with the micronized or bulk particles. The following are the general challenges associated with solubility determination of the nanoparticulate system. 1. No standard method is available in the literature for sol- ubility determination, 2. Difficulty in separation of the dissolved and undissolved nanocrystals/particles because of smaller size. 3. Confirmation that equilibrium is attained or not. 4. Reproducibility of results. 5. Validation of method for accuracy. In addition to the above challenges, one also has to consider other process parameters which vary with the physicochemical properties of the active drug substance for solubility determina- tion. For instance, if the API is weakly acidic or basic, then the pH of the solution plays an important role. It is difficult to deter- mine whether or not equilibrium is attained in this particular case. Several researches have published different approaches for the solubility determination of nanocrystals. Although nu- merous methods for the separation of dissolved and undissolved nanoparticles have been reported in the literature, these are the most common approaches used described in Figure 1. An aque- ous solubility determination by separation-based approach is widely accepted, has been used in industry and academia for many decades, and is the most convenient way to determine solubility. Typically, it is a two-step process: initially, an excess amount of drug is dispersed in an aqueous or buffer solution. The equilibrium is established by shaking or stirring the solu- tion at a specific rpm for a specific time and temperature, at which we want to determine the solubility. Usually the sam- ples are withdrawn after 24 and 48 h. Samples were either cen- trifuged or filtered from syringe filters. A sample analysis was performed using the HPLC and UV. In the case of crystalline nanoparticles, the research articles listed in the Table 2 have reported the solubility determination data for nanosuspensions and nanocrystals by utilizing a separation-based approach as their primary method for solubility determination. The most commonly reported approaches for solubility determination of nanoparticles are by shake-flask method at a specific temper- ature. Most of them have overestimated the thermodynamic solubility associated with nanocrystals, which is why it is im- portant to consider some additional factors during solubility determination when particle size is reduced to nano from mi- cron range. The selection of appropriate pore size filters with respect to the particle size of crystals, and the selection of an appropriate spectroscopic analytical method, is the key factor that needs to be taken in to consideration for accurate solubility determination of crystalline nanoparticles. Bernard Van Eerdenbrugh reported that the UV spectra is a reliable tool to determine the concentration of micronized par- ticles, however, it is not a reliable tool for the determination of the concentration of nanosuspensions, as it is overestimat- ing the actual solubility data. With particles in the nanome- ter range, they itself absorbs the UV light. Therefore, the ab- sorbance data are the mixture of the dissolved and undissolved nanosized particles.32 Today, the measurement of the dissolved drug concentration using an in situ UV probe is the preferred noninvasive method because of its sophistication in terms of contingency and its ability to record data from the start of dis- solution. However, absorption of light from particles is size de- pendent and it is having a great influence on smaller particles. With felodipine as a model drug, an observation has been made that both nanoparticulates of felodipine and free felodipine in the solution absorb light in a similar way, which results in an overestimation of dissolved concentration than what was actu- ally dissolved.33 The results were also dramatic, even for the second derivative of UV spectra. Moreover, the generation of nanoparticles occurs when working with nanosuspensions or supersaturated systems, so caution should be taken. The solubility associated with crystalline nanoparticles is moderately higher (10%–15%)16 compared with what is re- ported in Table 2.34–44 In the case of indomethacin, the reported solubility enhancement was nearly twofold higher. The same is true in the case of oridonin,36 reccardin D,38 and simvastatin39 where reported solubility was substantially higher as compared with bulk crystal solubility. One possible reason behind this misleading data may be the use of an inappropriate separating method and/or analytical method. Nanoparticles with an aver- age size of 200–400 nm can easily pass through 0.22 or 0.45 :m pore sized filters. Determining the concentration of such a fil- tered solution using UV leads to a further over-estimation of actual data because of the mixture of absorbance of both the undissolved and dissolved particles. Moreover, centrifugation at moderate speed of about 10,000–30,000 rpm is not suffi- cient to suspend particles in the nanometer range, therefore, the resultant supernatant contains a mixture of dissolved and undissolved particles. It is noticeable that care should be taken in choosing syringe filters for appropriate pore size. Juene- mann et al.45 were able to show in their study a differentiation DOI 10.1002/jps.24694 Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES
- 6.
- 7.
- 8. 8 REVIEW Figure 1.Reported approaches for the solubility determination. between suspended submicron colloidal particles and molecu- larly dissolved particles. It has been reported that the results of solubility and dissolution of nanocrystals are comparable with each other if the analysis is performed with filters having pore size ࣘ 0.1 :m. Using larger 0.2 and 0.45 :m filters, the nanocrystalline system shows apparent supersaturated behav- ior because of the combinatory effect of dissolved and undis- solved nanocrystalline particles. In other words, it has been demonstrated that filters with a bigger pore size may not be sufficient enough to hold back the colloidal particles. An ex- cellent demonstration has been reported by carrying out the study of a selection of appropriate filters and their impact on observed in vitro results. It was also compared with an in silico model for more precise justification of the experimental data. Overall, the conclusion and recommendation has been made for using filters with smaller pore sizes (i.e., 0.1 and 0.02 :m) when dealing with nanoparticles. Kinetic solubility determination by nephelometric or turbidi- metric methods during early screening of the drug molecule was introduced earlier by Bevan and Lloyd.46 Bernard Van Eerden- brugh critically evaluated different methods for the solubility determination of nanocrystals using four model compounds: itraconazole, loviride, phenytoin, and naproxen. The data ob- tained show that separation-based methodologies were not suf- ficient to determine solubility, as the data were not in fair agree- ment with the Ostwald–Freundlich equation. Noninvasive analytical techniques, such as light scattering and turbidime- try, were found to be more reliable for appropriate understand- ing of the solubility behavior of crystalline nanoparticles. In the case of amorphous nanosuspensions, Lindfors et al.33 have determined the solubility by plotting scattering intensity with the drug concentrations. Presence of excessive stabilizers in nanosystems also tends to generate the scattering of intensity. Hence, the method that Lindfors demonstrated is not accurate enough because of the summation of scattering intensities that are generated by dissolved nanoparticles and micelles. In or- der to eliminate this, Van Eerdenbrugh et al.16 have chosen the point of intersection at which scattering is exclusively due to the dissolved drug concentration and determine the solu- bility. Measures solubility data by scattering, for loviride was comparable to bulk solubility data. It was not feasible to determine solubility for itraconazole from the turbidimetry method, as the method cannot be distinguished because of very low solubility. Solubility determination for phenytoin can be determined precisely with both light scattering and turbidime- try. In the case of naproxen, determined solubility values were slightly less than the unmilled compound but the results were within the standard margin of error. Hence, this shows that both turbidimetry and light scattering methods can be applied to obtain more realistic solubility data for crystalline nanopar- ticles. Scattering can be used more precisely for compounds that have low scattering intensities and turbidimetry can be applied on compounds having higher scattering intensities. Moreover, the solubility enhancement was in fair agreement with the theoretical prediction from the Ostwald–Freundlich equation. For the theoretical calculation, the interfacial ten- sion was estimated (interfacial tension of typical pharmaceu- tical API ranges between 5 and 50 mN/m) as described in the reference literature.47 Experimental data and theoretical pre- dictions were also in agreement with the Ostwald–Freundlich equation, suggesting that solubility enhancement should be marginal in the case of crystalline nanoparticles and both the light scattering and turbidimetry are good noninvasive meth- ods for the solubility determination of nanocrystals. However, there are certain assumptions and limitations associated with these analytical methods. In most cases, the dynamic light scat- tering is used to measure the light scattering intensity. The instrument assumes each particle as a sphere and carries out the determination. When the particles are not spherical ini- tially or if the particles tend to change shape during disso- lution or solubilization, they may have different results than those measured by the instrument. Therefore, the impact of particle shape is a factor that also needs to be considered, as it plays major role during dissolution and solubilization. Anhalt et al.48 has reported a method for the solubility determination by real time measuring the light scattering. The main focus was to determine the equilibrium solubility and dissolution rate of a crystalline nanosuspension with different particle size using FBT as a model compound. The solubility results of the light scattering method were fair enough to justify it as a reliable analytical tool for the solubility measurement. Reported sol- ubility enhancement for nanocrystals was around 10%–15%, Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps.24694
- 9. REVIEW 9 Table 3.Alternative Approaches for Accurate Solubility Determination Analytical Method, Compound and Particle Size Solubility Determination Solubility Enhancement Ratio Reference Light scattering 48 Fenofibrate 8.69 ± 0.78 :g/ml 140 nm 10.38 ± 0.01 :g/ml 1.19 270 nm 8.70 ± 0.24 :g/ml 1.00 1070 nm 9.62 ± 0.50 :g/ml 1.11 Light scattering and turbidimetry 16 Loviride (162 nm) 0.0108 mg/mL 1.10 Scattering – 0.0119 mg/mL Itraconazole (220 nm) 0.0047 mg/mL 1.15 Scattering – 0.0054 mg/mL Phenytoin (406 nm) 0.0667 mg/mL 1.07 Scattering – 0.0711 ± 0.007 mg/mL Turbidity – 0.07 ± 0.0034 mg/mL 1.05 Naproxen (288 nm) 0.2116 mg/mL 0.97 Scattering – 0.2053 ± 0.003 mg/mL Turbidity – 0.2083 ± 0.0024 mg/mL 0.97 Separation-based methodology (ultracentrifugation, filtration—0.1 :m, 0.02 :m, and dissolution) 51 Griseofulvin-micro 7.63 ± 0.89 :g/mL 1.10 362 nm 8.40 ± 0.25 :g/mL 122 nm 9.99 ± 0.15 :g/mL 1.30 Compound X-micro 65.17 ± 1.58 :g/mL 0.97 238 nm 63.41 ± 1.26 :g/mL 93 nm 89.06 ± 6.36 :g/mL 1.36 Fenofibrate-micro 0.74 ± 0.27 :g/mL 1.11 290 nm 0.82 ± 0.26 :g/mL Celecoxib-micro 1.00 ± 0.03 :g/mL 1.11 341 nm 1.11 ± 0.03 :g/mL which justifies the previously reported solubility data by Van Eerdenbrugh.16 However, one should consider certain limita- tions before utilizing this light scattering method. Most impor- tantly, the sample has to be ultra clean; the presence of any dust and debris may lead to the generation of scattering intensity that affects to the observed data. The use of a plastic cuvette is also a potential source of error. Moreover, as light scattering is less sensitive to small particles, the results are slanted towards the larger sized particles as they have a tendency to generate more scattering of light. Besides these methods, other analyti- cal techniques like potentiometry and pulse polarography have also been reported for real time solubility measurement.49,50 However, these methods are not universal and can be used for a fewer number APIs with certain properties (i.e., electroac- tive). In the case of potentiometric measurement, each time a specific electrode has to use for the specific API. In general, separation-based methods are universally ac- cepted as they do not require a high level of experimental skill. Murdande et al.51 has reported three different approaches (ul- tracentrifugation, ultracentrifugation with filter, dissolution) to determine the accurate solubility. Ultracentrifugation was used after optimizing different parameters such as speed, time, and temperature for the satisfactory separation of dissolved and undissolved nanocrystals. Syringe filters were also used (i.e., 0.1 and 0.02 :m) based on the particle size to determine sol- ubility from the supernatant of the ultra-centrifuged sample. In addition, dissolution data at the end of the experiment was also evaluated for the total dissolved concentration based on the theoretical knowledge that nanocrystals should reach the satu- ration level at equilibrium. Results from all three methods were similar and in fair agreement with the Ostwald–Freundlich equation. The results from the solubility determination using the separation-based approach described above are in Table 3. Interfacial solubility (the concentration in a boundary layer of a spherical particle) plays an important role. As the particle size decreases, the SA increases proportionally, along with the in- terfacial tension. Sun et al.52 have determined the equilibrium solubility of coenzyme Q10 nanocrystals and bulk drug in three different dissolution media, which were mixtures of different concentrations of tween 20 and isopropanol. Solubility usually leads via diffusion and the driving force would be the difference between the solubility at the boundary layer and the bulk drug concentration. Based on observations, they had also proposed a solubility model for nanocrystals and bulk drugs.52 Another separation-based approach is the determination of solubility and/or drug release with the application of equi- librium dialysis. The published research has applied this concept, mainly for the nanoparticle-based formulation of large molecules and a few for small molecules, and de- termined the equilibrium solubility and drug release from nanoparticulate formulation. The most common approaches in- clude: sac dialysis,53,54 side by side dialysis,55–57 and reverse dialysis.58,59 In dialysis, separation is achieved by means of a semi-permeable membrane using molecular weight cut-off membrane. Assuming that the dialysis membrane is perme- able to free API only, Frank et al.60 reported the quantification DOI 10.1002/jps.24694 Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES
- 10. 10 REVIEW of molecularlydissolved, poorly soluble drug by using the equi- librium dialysis method and determined the apparent solubility (molecularly dissolved API + miscellany solubilized drug). The solubility of the drug in both the Hanks Balance salt solutions and the supplementary salts (HBSS++) and fasted-state sim- ulated intestinal fluid (FaSSIF) buffer were nearly the same within the margin of error.60 It is well known that nanoparti- cles have a rapid release dissolution kinetics. Efforts have been made to determine the interplay of the diffusion rate, size of the molecular weight cutoff (MWCO) membrane, and concen- tration difference of the donor and receiver compartment by Moreno-Bautista and Tam.61 Decreasing the dialysis cassettes to smaller MWCO showed a reduction in the diffusion rate. The compounds having a molecular weight of around 200–400 Da showed a diffusion rate profile several times higher with 10 kDa membrane compared to 2 kDa membrane MWCO. The cassettes having a pore size smaller than the drug molecule should be suitable for the evaluation of the dissolution rate of nanopar- ticles. Furthermore, the release profile from the dialysis cas- settes failed to catch some necessary events, such as the burst effect and lag time, which leads to a question the suitability of the method for true discrimination of the rapid release kinetics of submicron size colloidal particles. This situation prompted Zambito et al.62 to ask question whether the dialysis is a re- liable method for studying drug release from nanoparticulate systems. Their study concluded that for nanoparticles, the dial- ysis method was insufficient in expressing the discrimination and founded results were deceptive. Because of a reversible in- teraction between the drug and dispersed nanoparticles, the rate of permeation through the dialysis membrane was neg- ligible as compared with the plain drug solution. Hence, the kinetic release was found to be far less and unrealistic. With such a study, the release kinetic is governed by the dialysis membrane (not dependent on the drug release from colloidal particles), which makes it unreliable for the in vitro predictions and means the data may not be very discriminative. DISSOLUTION Dissolution is known as a significant tool for the analysis of pharmaceutical formulations. Indeed, it has somewhat poten- tial to predict the in vivo performance. Prediction of the extent of absorption can be made from the rate of drug release in the gastrointestinal tract.63 A drug candidate has to pass through some pre-requisites steps before being absorbed into the sys- temic circulation. Dissolution is one of the essential steps for effective absorption of a drug candidate and is a key parame- ter for assessing the onset of action of an oral dosage form. As per the well-known BCS, classification compounds belonging to Class II and IV are poorly soluble in nature, especially ones belonging to Class II, which have a dissolution rate of limited absorption because of their low aqueous solubility.64 In another words, the rate of absorption is proportional to the rate of disso- lution in the gastrointestinal tract. For a given drug moiety that dissolves in the gastrointestinal tract, its dissolution can be tai- lored by either generating amorphous systems or reducing the particle size to the micron or submicron level (i.e., nanometer). The motivation for the development of nanosystems has been generated from the increased number of new chemical entities with poor aqueous solubility. For such low aqueous-soluble com- pounds, even micronization is not sufficient enough to eliminate the problem. The reduction of particle size leads to an increase in the SA of the drug particle. The SA enhancement factor from micron to nanoparticle is about 10-fold. Thus, significant en- hancement in SA has a positive impact on the dissolution rate of a drug particle. This positive effect on the dissolution rate can create a higher concentration difference between the gut lu- men and the blood, which increases the absorption via passive diffusion and leads to an improved therapeutic response. Direct proportionality of the rate of dissolution with respect to specific SA has been well documented by the Noyes–Whitney equation. As a follow up to this, Nernst–Brunner and Danckwerts also proposed modified equations to describe the dissolution behav- ior a solid powder. Noyes − Whitney Equation : dM dt = k(Cs − C) (1) Nernst − Brunner Equation : dM dt = S D h (Cs − C) (2) Danckwerts surface − renewal model : dM dt = S √ DP (Cs − C) (3) This section will provide an overview of the different estab- lished methods to assess the dissolution for nanocrystals. As of now, it is well understood that particle size is inversely pro- portional to SA and the dissolution rate has a direct relation- ship with the change in SA. The major challenge is to develop a discriminative dissolution method for poorly water-soluble drugs. Earlier reports have indicated that there is an alteration of wetting behavior with particle size reduction. In the case of nanoparticles, the wetting phenomenon is more prominent, which leads the dissolution rate to increase more quickly. More- over, the reduction in the diffusional distance upon size reduc- tion generates a moderate spring effect that quickly achieves supersaturation level with respect to particle size during dis- solution (Fig. 2). Therefore, the traditional methods are inad- equate to determine the actual dissolution rate enhancement for nanoparticles which intensify the need for an appropriate method to perform the dissolution study of nanoparticles. Sink Condition (Conventional) Dissolution Sink condition dissolution condition is defined as the volume of the dissolution medium which can dissolve more than three times the amount of the dose used in the dissolution study.65 In most cases, the dissolution study is performed using a USP type – I or II apparatus. Usually for a study of a drug release profile, the dissolution is carried out in 900 mL of dissolution media (i.e., in a different pH or in deionized water) at 37 ± 0.5°C temperature and by rotating at a specific rpm (i.e., 100 rpm). Samples are withdrawn at specific time intervals throughout the dissolution study. After each sample withdrawal, the same amount of the fresh dissolution media, which is equilibrated at the same temperature, is introduced to maintain perfect sink condition. In most cases, samples will be filtered prior to anal- ysis. However, for the nanoparticles, sink condition dissolution will provide good qualitative and less quantitative information regarding the dissolution behavior of the formulation. So, the comparison of dissolution profiles between nanosuspension and microsuspension will show the impact of particle size on disso- lution velocity. The literature has reported a higher dissolution Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps.24694
- 11. REVIEW 11 Figure 2.In vitro surface dissolution, (A) surface image dissolution in correspondence with diffusional layer thickness (h), and (B) intrinsic dissolution profile in correspondence with the surface image dissolution. rate for the nanosuspensions,66,67 which dissolve instanta- neously as compared with micronized suspension. Hence, sink condition dissolution is an appropriate tool for QC analysis. However, because of the instantaneous dissolution behavior of nanocrystals during sink condition, it is challenging to quan- titatively discriminate dissolution rate enhancement with re- spect to particle size. In addition, the long sampling interval during conventional dissolution also adds to the difficulty. In response to these problems, few scientists have tried alterna- tive approaches. Flow-Through Cell The flow-through cell dissolution apparatus demonstrated the capability of studying the dissolution of all kinds of formula- tions, such as tablets, capsules, and powders.68,69 Heng et al.70 have carried out dissolution studies and compared the dissolu- tion rate by asking the question, “What is the suitable disso- lution method for drug nanoparticles?” Initial dissolution rates were determined for the paddle, basket, and flow-through ap- paratus. The initial dissolution rate was calculated from the following equation: dM dt Nanoparticles dM dt Unprocessed = slope %dissolved time nanoparticles slope %dissolved Time unprocessed The paddle and basket dissolution apparatus were described as inappropriate for nanoparticles because of their tendency to form aggregates. The ratios obtained for the initial dissolution rate enhancement in both cases did not agree with the model predicted values. Dissolution studies via the dialysis process (membrane with MWCO 12–14 kDa) were found to be very slow and having a limited ability for size discrimination. As a result, there was no significant difference noted in the dissolution pro- files between nanoparticles and the unprocessed powder. Based on the analysis, the flow-through cell set-up was described as appropriate for dissolution of nanoparticulate powder by mini- mizing the wetting problems.71 The experimental data for the dissolution profiles were able to discriminate well with the un- processed powder. The initial reported dissolution rate ratio was the average number of the triplicate study and was in agreement (6.95 vs. 7.97) with model predicted value. The au- thors concluded it as the most suitable analytical process for the dissolution of nanoparticulate powders. However, selection of an appropriate flow rate is required for proper discrimination because a change in the flow rate can generate change in the release behavior. Note that the presence of air can also affect the flow rate during the study, which may alter the dissolu- tion behavior. Therefore, the study should be carried out with caution. Alternative Approaches Nanosuspensions/nanocrystals exhibit instantaneous dissolu- tion behavior, making it difficult to monitor them with conven- tional methods, which requires sample withdrawals, as well as dilution and filtration steps before quantitative analysis. In situ fiber optic probes are not able to provide an accurate quantita- tive determination because of the generation of absorbance by both the dissolved and undissolved nanocrystalline particles.32 Recently, Kayaert et al.72 reported an alternative approach by studying the dissolution behavior of nanosuspensions using a solution calorimeter. The use of a solution calorimeter for dis- solution study has been previously reported for polymer and polymeric blends.73 This dissolution study was examined based on appraising the change in the heat throughout the process. In other words, heat required for the dissolution process is mea- sured. The methodology consists of sealing the breakable glass ampoule along with nanosuspension using bees wax. The glass vial should be kept in the sample cell, surrounded by the me- dia in which the dissolution study is intended to perform. After reaching the required temperature, the glass vial should be broken, and the dissolution process is started. The rate of dis- solution can be then assessed by converting the raw data (i.e., temperature vs. time and cumulative heat vs. time) to percent- age dissolved versus time. The dissolution process for nanosus- pensions was observed to be extremely fast. It takes just 20 s to complete the dissolution process for nanosuspension, whereas in the case of crude suspension, it was found to be between 8 and 15 min For the traditional dissolution apparatus (i.e., basket and paddle), the earliest sample one can withdraw is at 2 min. Hence, the solution calorimeter unquestionably pro- vides the advantage of real time measurement compared with traditional methods. Consumption and/or production of heat can be monitored from the beginning of the dissolution process without disturbing (i.e., sample withdrawal) the system. There are drawbacks with this method, which need to be considered for an accurate evaluation of the data generated. The method DOI 10.1002/jps.24694 Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES
- 12. 12 REVIEW measures thesummation of the total heat change, which in- cludes contributions from the breakage of the glass ampoule, heat change from other excipients in the formulations, and heat change due to the dissolution process. Therefore, a careful eval- uation of the data is warranted by considering all these con- tributing factors. In addition, the reported dissolution time is far shorter (i.e., 20 s to 15 min) using this method, but a single experiment requires additional time for the equilibration before the test and also requires post-test time after the dissolution, making it more time consuming than the conventional dissolu- tion methods. Furthermore, the instrument is very subtle and requires a skilled operator. Recently, another separation-based approach was reported by Shah et al.74 using a modified dial- ysis method known as pulsatile microdialysis (PMD)75 for the characterization of supersaturation and precipitation behavior of poorly soluble drugs. Here, the pore size for the dialysis probe was approximately 18 kDA (ß2 nm). Hence, the final sample should contain only the dissolved drug. This analytical method can be useful for characterization of high energy solid forms (amorphous) or nanoamorphous33 forms because of the advan- tage of quick sampling (i.e., 10 s). As a more convenient direct sampling method, PMD may not be much useful for the study of nanocrystals with the size range of 150–400 nm because of the availability of the syringe filters with pore size 0.1 :m. The use of 0.02 :m (ß20 nm) anotop syringe filters for the dissolution study has also been reported in the literature.45 As a result, it may be of great interest if the comparison of the sample anal- ysis from PMD and the sample analysis from 0.02 :m syringe filters has been carried out to describe the usefulness of the method more precisely. Another separation-based approach has been reported by Murdande et al.51 by modifying the traditional sink dissolu- tion to various non-sink conditions. Non-sink conditions can be generated by pre-dissolving API in the dissolution me- dia prior to perform the dissolution experiment. Recently, an- other literature has also reported the dissolution of nanocrys- talline suspension for poorly soluble molecules using non-sink conditions.76 Because of the lack of discrimination during the initial phase of dissolution by sink dissolution, the goal was to slow down the dissolution velocity of nanocrystals and mi- cronized crystals in such way that the initial dissolution phase becomes more discriminative. Here, the samples were filtered with both 0.02 :m (for nano) and 0.1 :m (for micro) after es- tablishing the filter binding capacity of the each model com- pounds. As the saturation level increases, the rate of disso- lution decreases, creating a more discriminating portrayal of the initial dissolution rate enhancement (i.e., at 75% satura- tion level the initial rate of dissolution, 10 :m:362 nm:122 nm were 1.0:1.8:3.6). The observed data look more promising com- pared with the data obtained under sink conditions. However, filtration from 20 nm pore size of filters should be performed carefully because of the chance of blockage or clogging of the pores by larger size particles. From our knowledge, all these approaches are reported specifically for nanocrystals. One can select any analytical method listed above based on their re- quirements and the availability of resources. PK BEHAVIOR OF CRYSTALLINE NANOPARTICLES Different formulations have different effects on the PK profile of the same drug. Besides chemical properties, a change in the physical property (i.e., particle size reduction) may also have a considerable impact on altering the PK behavior of a molecule. The reduction of the particle size increases the dissolution ve- locity and may have some effect on in vitro solubility. Similar behavior was observed in the case of in vivo parameters, such as AUC (+ve), Cmax (+ve), Tmax (−ve), and fed/fast variability (−ve). A reduction of particle size improves the dissolution rate, which increases the absorption of the drug in the body, and it leads to an increase in PK performance. Compounds belong- ing to the BCS Class II have issues related to poor solubility, dissolution-rate-limited absorption, and bioavailability. Parti- cle size reduction can improve the bioavailability of such com- pounds by improving the dissolution rate. For the nanocrystals, an increase in the oral bioavailability of the compounds can be attributed to an increase in the SA.77 Improvement in Oral Bioavailability An improvement in the dissolution rate and adhesion to the gut wall can be considered the main contributing factors for enhancing bioavailability and overall PK performance. After oral administration, the formulation disintegrates and begins the dissolution process. The dissolution rate resembles the concentration gradient in the physiological environment. As a result, an improvement in the dissolution rate leads to an enhancement in the absorption, and finally, in the bioavail- ability of the compound. Because of the smaller size of the nanoparticles, they tend to adhere to the gut wall.78 Bioavail- ability enhancement can be achieved both actively and pas- sively. The classic example for bioavailability enhancement has been represented in the case of danazol, which is poorly sol- uble (<1 :g/mL) and one of the most challenging compound to work within the pharmaceutical industry.79 In vivo studies of nanosuspensions of danazol (169 nm) in beagle dogs have shown enormous enhancement in Cmax and a 16-fold enhance- ment in relative bioavailability, compared with the micronized suspension.12 Therefore, it is understood that by reducing the particle size, the solubility issue associated with danazol can be solved. Moreover, it can also be delivered at a lower dose by enhancing the therapeutic outcome of the compound. FBT is another classic example of a poorly soluble drug with a high log P (i.e., 5.3) value. FBT is used in hypercholesterolemia and has a solubility and dissolution-rate-limited absorption property. Bioavailability of FBT is attributed to the dissolution behavior of particles in the gastric environment. A comparison of the PK profile of nanocrystals and coarse powder demonstrated signif- icant enhancement in the rate of absorption after oral adminis- tration. The Cmax of nanocrystals was observed to be five times higher than that of the coarse powder, along with a higher AUC and Tmax.80 Zuo et al.40 made an effort to compare formulated nanocrystalline particles with the commercial nanocrystalline formulation LipidilTM -ez with respect to in vitro and in vivo behavior. No significant difference was observed between the two nanocrystalline formulations, suggesting that variability will be less when particle size is reduced to nanometer range. Certainly, the re-dispersion behavior of dried nanocrystals also plays a contributing role by demonstrating reversible or irre- versible aggregation behavior. Bioavailability can be altered by several factors in the gastric environment, such as pH change, food effect, the presence of salts, and so on. In all the reported cases reported in the Table 4,12,40,80–88 a significant enhance- ment in dissolution rate was observed for the nanocrystals, Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps.24694
- 13. REVIEW 13 Table 4.Effect on In Vivo Parameters Compound Animal Model PK Parameters Comment Reference Aripiprazole Beagle dogs NSa (350 nm) Coarse suspension 71% enhancement in relative bioavailability 81 Tmax (h) 1.04 ± 0.24 3.33 ± 1.50 Cmax (ng/mL) 137.37 ± 17.38 43.10 ± 11.68 AUC (ng h/mL) 618.15 ± 81.28 386.06 ± 78.54 Nitrendipine Rat NS (209 nm) Tablet Fivefold enhancement in relative bioavailability 82 Tmax (h) 1 ± 0 2.2 ± 1.17 Cmax (:g/mL) 3.65 ± 0.43 0.60 ± 0.11 AUC (:g h/mL) 17.12 ± 2.59 3.44 ± 0.47 Danazol Beagle dogs NS (169 nm) Coarse suspension 16-fold enhancement in relative bioavailability 12 Tmax (h) 1.5 ± 0.3 1.7 ± 0.4 Cmax (:g/mL) 3.01 ± 0.80 0.20 ± 0.06 AUC (:g h/mL) 16.5 ± 3.2 1.0 ± 0.4 Naproxen Rat NS (270 nm) Unmilled suspension ß1.25-fold enhancement in relative bioavailability 83 Tmax (min) 23.7 ± 5.1 33.5 ± 2.9 Cmax (:g/mL) 187 ± 18 126 ± 4 AUC (:g min/mL) 19,062 ± 573 15228 ± 994 Apigenin Rat NCb (400—800 nm) Coarse powder ß3.5-fold enhancement in relative bioavailability 84 Tmax (min) 90 ± 14 120 ± 16 Cmax (:g/mL) 5.4 ± 0.6 1.5 ± 0.2 AUC (:g min/mL) 1509 ± 196 445 ± 45 Cefpodoxime proxetil Rabbit SDNSc (<300 nm) Microsuspension ß1.6-fold enhancement in relative bioavailability 85 Tmax (h) 0.75 ± 0.11 1.75 ± 0.68 Cmax (:g/mL) 18.36 ± 2.03 10.88 ± 1.01 AUC (mg h/mL) 47.55 ± 4.33 29.78 ± 3.47 Baicalein Rat Baicalin NC (335 nm) Coarse powder ß1.6-fold enhancement in relative bioavailability 86 Tmax (h) 0.92 ± 0.38 1.67 ± 0.29 Cmax (:g/mL) 11.12 ± 1.25 7.18 ± 1.25 AUC (:g h/mL) 119.25 ± 20.26 71.41 ± 4.38 Simvastatin Rat NC (387 nm) Coarse powder ß1.5-fold enhancement in relative bioavailability 87 Tmax (h) 1.99 ± 0.05 2.88 ± 0.08 Cmax (ng/mL) 450.3 ± 140.5 300.2 ± 67.01 AUC (ng h/mL) 1110.3 ± 280.62 770.9 ± 110.3 Fenofibrate Rabbit NC (460 nm) Coarse powder ß4.7-fold enhancement in relative bioavailability 80 Tmax (h) ß0.4 ß1 Cmax (:g/mL) 536.66 ± 35.09 113.46 ± 29.05 AUC (:g h/mL) 134.38 ± 6.47 28.41 ± 5.52 BMS-347070 Beagle dog NC Micronized ß2.7-fold enhancement in relative bioavailability 88 Tmax (h) 2 3 Cmax (ng/mL) 1475 ± 375 483 ± 79 AUC (ng h/mL) 28,613 ± 3850 10,870 ± 1651 Fenofibrate Beagle dog SDNCd LipidilTM-ez No significant difference in relative bioavailability 40 Tmax (h) 2.8 ± 1.8 5.2 ± 9.2 Cmax (ng/mL) 2075.2 ± 1101.1 2349.5 ± 1050.5 AUC (ng h/mL) 30,496 ± 6541 34,035.9 ± 19,286.4 a NS, nanosuspension. b NC, nanocrystal. c SDNS, spray-dried nanosuspension. d SDNC, spray-dried nanocrystals. which caused a significant reduction in the time to reach max- imum concentration, an improvement in the rate of absorp- tion, peak plasma concentration, and enhancement in relative bioavailability. Food Effect In most cases, food increases the bioavailability of the drug by increasing bile secretion and increasing the duration of gas- tric emptying time. For a drug molecule’s clinical efficacy and DOI 10.1002/jps.24694 Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES
- 14. 14 REVIEW Table 5.Food Effect PK Parameter Fed Fast Compound NSa Refb NS Ref Reference ELND-006 Tmax (h) 1.4 3 1.4 1.8 90 Cmax (ng/mL) 365 159 294 49.4 AUC (ng h/mL) 3063 1767 2430 315 Fc 110 63.4 87.2 11.3 Fed Fast NCd Jetmilled NC Jetmilled Cilostazole Tmax (h) 1 1 1.3 ± 0.5 1 91 Cmax (ng/mL) 4872 ± 112 2901 ± 314 5371 ± 1173 1029 ± 218 AUC (ng h/mL) 13,589 ± 3895 10,669 ± 3417 17,832 ± 4994 2875 ± 587 F 0.67 ± 0.22 0.53 ± 0.21 0.86 ± 0.29 0.15 ± 0.04 Fed Fast SNCDe SDDf SNCD SDD Ziprasidone Cmax (ng/mL) 260 285 416 140 92 AUC (ng h/mL) 1911 1949 2044 879 a NS, nanosuspensions. b Ref, reference sample. c Relative bioavailability. d NC, nanocrystal. e SNCD, solid nanocrystalline dispersion. f SDD, solid amorphous spray-dried dispersion. future success, persistence in oral bioavailability can only be achieved by eliminating the food effect.89 The presence of bile salts in the gastric environment also has an impact on the dis- solution behavior of the molecule. It has been reported that the presence of food reduces the dissolution behavior of micronized particles in certain cases as reported in Table 5.90–92 The ratio of bioavailability of the fed to fast state was observed to be close to 1 in the case of nanocrystals, while micronized formulation of the same drug showed a significantly higher ratio (approx. sixfold) of the fed to fast state by demonstrating the poten- tial food effect after oral administration to an animal model with the same dosing. Bile salt secretion usually increases in the fed state.93 A change in concentration of the bile salt in the stomach leads to a change in dissolution behavior and slows down or enhances absorption of the drug as depicted in Figure 3. This phenomenon causes a large variability from pa- tient to patient and also from fed state to fast state. Micronized or larger particles that have shown improved absorption in the fed state might be because of the micelle formation. For the nanocrystals though, they are not helping in improving the in- trinsic solubility, but they provide an advantage by enhancing the initial dissolution rate because of the larger SA. The higher rate of dissolution leads to an increased rate of absorption, and eventually, enhancement in the overall bioavailability, irrespec- tive of the fed or fast state. Hence, it can be stated that the bile salts concentrations have significantly less effect on the absorp- tion behavior of nanoparticles. The same observation has been reported for cilostazole91 [fed/fast ratio—0.78 (nano vs. 3.53 jet-milled)]. Thombre et al.92 have reported the PK behavior of an anti-psychotic drug ziprasidone in beagle dogs by conduct- ing the fed- and fasted-state bioavailability experiment for the both solid nanocrystalline dispersion and the commercial blend (amorphous spray dried dispersion). The commercial blend has shown a twofold improvement in bioavailability in the fed state as compared with the fasted state, indicating the failure of the commercial blend to prevent variability in absorption behavior irrespective of the food state. A similar food effect was observed with human data. On the other hand, nanocrystals have proven to be efficient enough to prevent the variability from fed to fast state by generating a similar bioavailability profile in beagle dogs. From the reported literature, it can be concluded that vul- nerability in the variation of oral bioavailability for potential drugs can be avoided with reduction in particle size to nanome- ter scale. Particle Size Effect: Micro Versus Nano The effect of particle size on in vitro and in vivo performance is well documented elsewhere.91,94–96 Different particle sizes have a different effect on the initial dissolution rate, the rate of ab- sorption, and on oral bioavailability. Moreover, particles with nanometer size range may have the tendency to pass through both active and passive diffusion pathway, which increases the chances for sub-micron particles to reach to peak plasma con- centration with a reduction in time compared to macro/large particles. Sun et al.97 have shown the effects of particle size, ranging from micron to nanometer size, on Cmax, Tmax, and AUC.98 A large increase in bioavailability was observed for the both nanoparticles (i.e., 300 and 750 nm) as compared with the micron size particles and coarse powder, which is reported in Table 6. The rise in bioavailability can be attributed to in- creased SA and adhesion behavior of nanosized particles. There Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps.24694
- 15. REVIEW 15 Figure 3.Effect of food on dissolution of nanocrystals and microcrystals. Table 6. Particle Size Effect: Micro versus Nano PK Parameter Compound Size (nm) Tmax (h) Cmax (ng/mL) AUC (:g h/mL) Reference Itraconazole 300 3 712 ± 121 9967 ± 2527 97 750 4 501 ± 73 8649 ± 1580 5500 3 218 ± 86 1271 ± 398 Powder – 40 ± 9 197 ± 61 Nitrendipine 200 1.0 ± 0.0 3.65 ± 0.43 17.12 ± 2.59 98 620 1.0 ± 0.0 2.75 ± 0.50 14.36 ± 2.75 2700 0.8 ± 0.1 1.74 ± 0.26 8.19 ± 0.85 4100 0.7 ± 0.1 1.19 ± 0.08 7.41 ± 0.46 20,200 3.4 ± 1.2 1.04 ± 0.21 6.89 ± 1.15 Powder 4.1 ± 2.4 0.42 ± 0.08 2.76 ± 0.46 was significant difference observed in the dissolution profiles between both of the nanoparticles (i.e., 300 and 750 nm), but no significant difference has been reported in the PK profiles. The same observation was reported by Xia et al.98 by studying effect of particle size on the oral bioavailability of nitrendipine in the rat model. The overall bioavailability was reported to be a ninefold increase compared with the coarse powder and a threefold increase as compared with the micronized particles. Comparing the PK parameters for both nanoparticles (200 vs. 620 nm), a slight improvement was noted in Cmax, Tmax, and AUC. Based on the literature data, an interesting area for a further research is possible, by lowering the particle size down to the 50 nm range with a narrow size distribution and mea- suring the relative bioavailability. This may have more pro- nounced effect on the bioavailability because of the further im- provement in the thermodynamic solubility suggested by the Ostwald–Freundlich equation. A comparison of adhesion rates, along with the various particle size ranges, starting from 50 nm to micron level, will also be an interesting area to explore for further understanding the particle size effect on the bioavail- ability. SUMMARY Drug nanocrystal formulation is a well-established and suc- cessful approach, and it is described in the initial phase of this review article. Nanocrystal formulation approach is more promising because most of the major pharmaceutical compa- nies have adopted this approach because of its effectiveness and convenience. During the early phase development and during formulation development for pre-clinical studies, it is more con- venient to formulate the nanocrystal for the determination of the exposure and the PK parameters to confirm the candidacy DOI 10.1002/jps.24694 Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES
- 16. 16 REVIEW of thedrug molecule. It is also important to know the thermody- namic and the kinetic behavior with respect to size reduction. Several approaches have been tried by researchers for the accu- rate determination of thermodynamic solubility (Tables 2 and 3). Some have reported suspicious results due to generation of artifacts by the analytical method for the solubility determi- nation. Despite the shortcomings of some of the conventional methods for determining solubility (specifically for nanoparti- cles), others have reported excellent results by adopting newer approaches for the solubility determination. The boost in ther- modynamic solubility can be observed only when the particle size is reduced to approximately 50–80 nm, in the remainder of the cases, only kinetic boost (i.e., the dissolution rate) can be observed without greatly impacting the thermodynamic sol- ubility. As the nanocrystals have a high dissolution velocity, the traditional dissolution method is not suitable to discrimi- nate the quantitative difference in the dissolution behavior for the nanoparticle. However, there are several alternative ap- proaches that have been described in this review article for discriminative analysis of the dissolution rate enhancement upon nanosizing. The initial dissolution rate enhancement as- sociated with particle size is a potential parameter for the esti- mation of bioavailability enhancement. More advanced work is warranted in this area to understand the behavior of nanocrys- tals and to avoid excessive animal studies. The effect of food is found to be negligible in the case of nanocrystals as compared with microcrystals. The particle size is shown to be a dominant characteristic in governing dissolution and bioavailability per- formances. 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