Single-Step Co-Crystallization and Lipid Dispersion by Supercritical

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Single-step Co-crystallization and Lipid Dispersion by Supercritical Enhanced Atomization João M. Tiago, Luis M. Padrela, Henrique A. Matos, Miguel A. Rodrigues, Antonio Almeida, and Edmundo Gomes Azevedo Cryst. Growth Des., Just Accepted Manuscript • Publication Date (Web): 27 Sep 2013 Downloaded from http://pubs.acs.org on September 29, 2013

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Crystal Growth & Design

Single-step Co-crystallization and Lipid Dispersion by Supercritical Enhanced Atomization João M. Tiago§, Luís Padrela§, Miguel A. Rodrigues§, Henrique A. Matos§, António J. Almeida¥, Edmundo Gomes de Azevedo§* §

Department of Chemical Engineering and Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal ¥

School of Pharmacy, Universidade de Lisboa, Lisboa, Portugal

* E-mail: [email protected] ABSTRACT

This work evaluates the feasibility of the supercritical enhanced atomization (SEA) process to improve stability and delivery of active pharmaceutical ingredients (APIs). This process was used to generate distinct microcomposites of a model API – theophylline (TPL) – namely pure TPL, theophylline-saccharin (TPL-SAC) co-crystal, and dispersions of each crystalline form in hydrogenated palm oil (HPO) – TPL-HPO and TPL-SAC-HPO. The formation of TPL-SAC co-crystal within the HPO suggests that the co-crystallization step anticipates the lipid dispersion during the formation of the microcomposites. The TPL-SAC co-crystal extended the TPL stability at 92% relative humidity by over 6 months, contrarily to that of raw TPL, which converted into a monohydrate after a few days only, even when dispersed into HPO. The TPL-SAC co-crystal slowed the TPL release from the lipid particles, which is explained by its higher stability towards hydration. The feasibility of the co-crystal microcomposites for

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therapeutic application was evaluated by estimating the plasmatic concentration of TPL using a pharmacokinetic model (one compartment approach). This model revealed that the small therapeutic concentration window and high elimination rate of TPL raises serious limitations to control the TPL release. The microcomposites were able to attenuate the TPL burst effect and improve stability towards hydration, but could not extend significantly its deliver.

INTRODUCTION Over the past years, the modification and optimization of physicochemical properties of active pharmaceutical ingredients (APIs) has been considered a challenge in the pharmaceutical industry. Undesired solubility, inadequate dissolution rate and instability are common issues that make many drugs unusable in spite of their well-documented therapeutic properties.1, 2 Pharmaceutical co-crystallization has been presented as a potentially reliable technique for improving the physicochemical properties of APIs, including solubility, dissolution, stability, bioavailability, moisture sorption and compressibility.3 In general, co-crystals may be defined as substances produced by joining a molecular or ionic API with a coformer that is a solid at room temperature, containing therefore two or more discrete molecular entities in the crystal lattice.4-7 APIs’ structures are composed by functional groups, such as amides and carboxylic acids, which allow their bonding in a supramolecular event with co-crystal formers that have complementary hydrogen bond donor and acceptor sites.5 Co-crystallization may therefore stabilize the APIs’ crystallinity consequently preventing any adverse reactions during formulation

and

storage.

Essentially,

co-crystallization

may

enhance

the

APIs’

pharmacokinetics, enabling novel and better therapeutics - reasons why co-crystals have been a field of intense research. The use of dense fluids, such as supercritical CO2, has been proposed as an alternative to conventional solvent-based methods in the field of materials processing. There are numerous


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reports regarding the use of supercritical fluid processes to produce micro and nanoparticles, including polymers, semiconductors, metals, and pharmaceutics, with application in different areas such as medicine, pharmaceutics, plastics, and electronics.8-14 In this work we address the formation of microcomposites containing an API (theophylline, TPL), a co-crystal former (saccharin, SAC) and/or a lipid excipient (hydrogenated palm oil, HPO), by using a process wherein the atomization is enhanced by supercritical CO2. This is a supercritical enhanced atomization (SEA) process that consists essentially in atomization of a solution by using a nozzle that enables the liquid to be mixed and co-dispersed with a large flow of CO2 at supercritical conditions. The increase in momentum and shear forces causes the liquid jet to breakup into small droplets that are rapidly dried by normal pressure spray-drying. Several processes have been described that take advantage of this atomization enhancement promoted by CO2 to improve the jet-breakup in spray-drying being an example the CO2-Assisted Nebulization with a Bubble Dryer process (CAN-BD).15-17 Herein ‘‘Supercritical Enhanced Atomization’’ (SEA) may be regarded as a generalization that includes all methods that use the atomization enhancement caused by dense fluids applied to normal pressure spray-drying. Under this scope, SEA has been used as a single-step process either for the co-crystallization or the encapsulation of APIs.5,

18

However, it has never been used to promote both effects simultaneously. The

simultaneous co-crystallization and lipid dispersion of APIs would be highly desirable because stability issues, such as polymorphism, and bioavailability concerns could be overcome by cocrystallization and enhanced further by dispersion in a lipid or polymer. Yet, this desirable goal raises some questions that must be fully addressed: will the API and the conformer co-crystallize in a supersaturating media that is richer in a dispersing excipient? How will the co-crystallization influence the stability of the API and its dissolution from the microcomposites? These questions are the guidelines for this work. Herein, we address the production of TPL microcomposites. TPL is a dimethylxanthine drug used in therapy for respiratory diseases such as asthma, chronic



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bronchitis and other lung diseases that require extended treatment. It is characterized by an undesirable burst effect, a tight therapeutic window and pharmacokinetic variability (especially in children) — factors that have been confining its clinical applications.19,20 Moreover, TPL instability may result in the formation of novel crystalline forms, such as polymorphs or hydrates.19, 21 Consequently, many efforts have been directed to overcome these disadvantages. Several co-crystals of TPL prepared by classical techniques have demonstrated improved stability under relative humidity (RH) conditions compared with that of raw TPL.21 For instance, TPL-SAC co-crystal, is known to form strong hydrogen bonds as Fig. 1 schematically shows.22

Figure 1. Molecular structure of theophylline (TPL), saccharin (SAC) and of the TPL-SAC cocrystal in 1:1 molar proportion. Previously, our research team had prepared this co-crystal by grinding and SEA processing techniques.5 Also, other authors produced TPL-SAC co-crystal but using distinct processes as described elsewhere.22, 23 Furthermore, disperse TPL in HPO using a supercritical CO2 technique (Particles from Gas Saturated Solutions) improve its dissolution kinetics.24 However, most of these studies don’t address how the produced microcomposites may enhance the pharmacokinetic properties of the formulations. Pharmacologically, we intended to obtain a formulation for oral administration with an extended-release behavior of interest to the asthma treatment. The HPO excipient was chosen as 4 ACS Paragon Plus Environment

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carrier because of its natural oil properties and ability in delaying the drug release, as has been reported previously.24 Herein we use a pharmacokinetic modeling approach to estimate plasmatic concentration curves. The knowledge of the required doses of microcomposites based on the drug’s dissolution kinetics and latency period provides a more realistic perspective regarding its feasibility as controlled release formulations.

EXPERIMENTAL SECTION

Materials and Methods. TPL, SAC and THF (tetrahydrofuran) were purchased from SigmaAldrich, Stockholm, Sweden (minimum 99% purity). The polymer HPO (also known as GV-60) has a normal melting point of 60 ºC and was obtained from Microlithe SA, Marignane, France. Absolute ethanol (99.5%) and copper (II) sulfate pentahydrate (99%) were supplied by Panreac, Barcelona, Spain, and carbon dioxide (99.95%) by Air Liquide, Lisbon, Portugal. Acetonitrile was of HPLC-grade from Merck, Darmstadt, Germany.

Solution preparation for SEA processing. For the production of TPL-SAC, TPL-HPO or TPLSAC-HPO particles, the respective components of each formulation were dissolved into 100 g of THF. The solutions for each sample were prepared according to Table 1.

Table 1. Composition of the solutions used in the production of microcomposites by SEA. The indicated masses of each component (TPL, SAC or HPO) were dissolved in 100 g of THF. Samples TPL-SAC TPL-HPO 1 TPL-HPO 2 TPL-HPO 3 TPL-SAC-HPO 1 TPL-SAC-HPO 2 TPL-SAC-HPO 3

Mass proportion 1:1 1:2 1:10 1:20 1:1:2 1:1:10 1:1:20

TPL (mg) 250.0 250.0 250.0 250.0 250.0 250.0 250.0

SAC (mg) 254.2 254.2 254.2 254.2

HPO (g) 0.50 2.5 5.0 0.50 2.5 5.0



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Microcomposites production by the SEA technique. Particles of TPL-SAC, TPL-HPO and TPL-SAC-HPO were produced using the SEA setup schematically shown in Figure 2.

Figure 2. Schematic diagram of the SEA apparatus. 1: Gas cylinder. 2: Gas compressor. 3: Back-pressure controller. 4: Temperature-controlled gas storage cylinder. 5: Piston liquid pump. 6: Coaxial nozzle (described in detail elsewhere18). 7: Jacketed wall precipitator. 8: Filter. T, P, and F refer to the location of temperature, pressure, and mass flow indicators. By using the gas compressor (Newport, model 46-13421-2), carbon dioxide stored in the cylinder was compressed until the desired pressure was achieved by regulating the back-pressure controller. The temperature of CO2 was controlled in a temperature-controlled gas storage cylinder. The THF solution containing TPL and SAC, or TPL and HPO, or TPL, SAC and HPO was pumped by a TSP metering pump (model 2396-74) and mixed with the CO2 in the coaxial nozzle before depressurization into the precipitator vessel. The nozzle consisted of three sections: the inlet (where the feed lines of each phase meet), the mixer, and the orifice disk. The mixer was prepared from a 0.5 cm long and 1/16 in. diameter tubing where the mixing of the phases occurs. The orifice disk was 0.25 mm thick with a centered orifice of 100 µm diameter. The droplets formed during the atomization were dried inside a temperature-controlled precipitator (50±1 ºC) near atmospheric pressure (0.1 to 0.5 MPa). Table 2 presents the experimental conditions for the production of each microcomposite.


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The nozzle liquid/gas mass flow ratio was controlled to be below 50% THF saturation at atmospheric CO2 at 50oC. At this temperature, the saturation vapor pressure of THF is 0.0586 MPa and the mole fraction solubility of THF in CO2 was determined to be 0.57. The nozzle flow was obtained by using a mass flowmeter (Rheonik, model RHM007) and pressures were measured by a transducer (Omega, model PX603). Temperatures were controlled in the air chamber or in the water bath by T-type thermocouples and Ero Electronic controllers (model LDS). The particles were collected in the precipitator walls and from a filter at the precipitator exit. Samples were stored in a desiccator before their morphological, stability and dissolution analysis.5, 18

Table 2. Experimental conditions for the production of microcomposites by SEA processing at 50 ± 1 ºC (temperature in the mixing chamber). P is the pressure before the nozzle and R is the mass flow-rate ratio of the aqueous feed to the supercritical fluid Sample TPL TPL-SAC TPL-HPO 1 TPL-HPO 2 TPL-HPO 3 TPL-SAC-HPO 1 TPL-SAC-HPO 2 TPL-SAC-HPO 3

P (MPa) 8.0 ± 0.1 8.0 ± 0.1 8.1 ± 0.1 8.1 ± 0.1 8.0 ± 0.1 8.0 ± 0.1 8.1 ± 0.1 8.0 ± 0.1

R (g/g) 0.08 0.28 0.07 0.12 0.08 0.08 0.12 0.16

The temperature selected (50 ºC) ensures that the boiling point of THF (66 ºC) was not exceeded and that the precipitator contained one gaseous phase only. Moreover, a pressure above 7.4 MPa and a temperature above 31.2 ºC are required for CO2 to be in the supercritical state.

Microcomposites characterization: morphology and solid state. The morphology was studied by using a Hitachi S2400 scanning electron microscope (SEM). Particles of different



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microcomposites were coated prior to measurement with a gold film by electrodeposition in vacuum. X-ray powder data were collected in a D8 Advance Bruker AXS θ-2θ diffractometer, with copper radiation (Cu Kα, λ = 1.5406 Å) and a secondary monochromator. The tube voltage and current were 40 kV and 40 mA, respectively. Each sample was scanned with 2θ between 5º and 35º with a step size of 0.01º and 0.5 s at each step.

Relative humidity (RH) stability experiments. A sealed glass desiccator jar containing a solution of CuSO4.5H2O was used to create a saturated humidity atmosphere (92% RH) at ambient room temperature (about 20 ºC). Humidity-indicator cards (Sigma-Aldrich) were used to indicate RH conditions. Stability of the particles containing TPL-SAC, TPL-HPO and TPLSAC-HPO were evaluated and compared to the raw TPL for the time periods of 1, 3, 6, 10 days and 6 months. Open paper dishes containing 120 mg of powder were stored in the RH chamber at ambient temperature. A dish was removed for every co-crystal material at each selected time. Upon removal from the chamber, the samples crystallinity was promptly evaluated by PXRD.

Drug dissolution testing. Drug dissolution studies were performed according to the USP Apparatus 2 paddle method using a dissolution apparatus Erewka, DT 626. The paddle rotation speed was kept at 50 rpm, and the temperature was maintained at 37±0.5 ºC. Each test was carried out by loading 20 mg of the sample inside hydroxypropylmethylcellulose capsules (water-soluble), in 500 mL of 0.05 M phosphate buffer (pH 7.4). Aliquots of 500 µL were then withdrawn from the dissolution medium at predetermined time intervals. Drug concentrations were determined by HPLC after filtration of the samples through a 0.20 µm membrane (Nylon). The HPLC system consisted of a Jasco HPLC-system provided with a Kromasil 2.1×150 mm


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C18 column and equipped with a variable UV detector (set at 254 nm). The column was kept at room temperature. The mobile phase consisted of acetate buffer, pH 4: acetonitrile (95:5), delivered at a flow-rate of 0.3 mL/min. The mobile phase was filtered (0.45 µm PVDF membrane) and degassed by sonication prior to use. The injection volume was 10 µL. A running time of 14 minutes was sufficient for samples analysis. The retention time was, respectively, 6 and 11 minutes for SAC and TPL. Dissolution curves were determined from triplicate runs.

Pharmacokinetics modeling. Drug dissolution from solid dosage forms can be described by kinetic models in which the dissolved amount of drug is a function of time (t).25 Selected kinetic models commonly used, namely the models of Higuchi,26 Korsmeyer-Peppas,27 and Weibull28 were fitted to the experimental data obtained. The latter is a general empirical equation that was adapted to a drug-release process.28 Hereupon, a one-compartment physiological based pharmacokinetic model was used to estimate the dose of the TPL for oral administration, taken into consideration the reported29 therapeutic window between 10 mg/L (target therapeutic concentration) and 20 mg/L (toxic concentration), and the volume of distribution V (that relates the amount of drug in the body to the concentration of drug in blood) of 35 L (assuming a standard adult weight of 70 kg).29 In the one-compartment modeling approach, the variation in TPL plasmatic concentration,

dX/dt, is described by30

dX dXa dXe = + dt dt dt

(1)

where dXa/dt and dXe/dt are, respectively, the absorption and the elimination rates. For simplicity, we assume that the absorption rate is equal to the dissolution rate, given by the experimental dissolution data. This assumption is reasonable for a one-compartment model, considering also that the available pharmacokinetic data of TPL, which shows that it is absorbed promptly after oral administration because its oral bioavailability is F ≥ 96%.29 9 ACS Paragon Plus Environment


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The elimination rate is typically assumed to be first order, dXe = − ke X dt

(2)

TPL half-life is 8.1 h,29 which corresponds to an elimination rate constant (ke) of 0.086 h-1.

RESULTS

Microcomposites

characterization:

morphology

and

solid

state.

Morphological

characteristics of particles produced by SEA were analyzed by SEM. Figure 3 shows irregular shape particles of micrometric range (1 to 5 microns). There were no significant differences in morphology and size between HPO microcomposites with TPL (Figure 3a, sample TPL-HPO 1) or TPL-SAC co-crystal (Figure 3b, sample TPL-SAC-HPO 1).

(a)

(b) 10 ACS Paragon Plus Environment

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Figure 3. SEM images of microcomposites produced by SEA: (a) TPL in HPO (sample TPLHPO 1); (b) co-crystal in HPO (sample TPL-SAC-HPO 1).

Crystallinity was evaluated by comparing PXRD diffractograms of the microcomposites (TPLHPO and TPL-SAC-HPO) with the diffractograms of the pure materials. Theophylline diffraction peaks were present in all TPL-HPO microcomposites, revealing that TPL is in its crystalline form, as Fig. 4 shows (for TPL-HPO 1 only). All samples containing SAC showed the characteristic peaks of the TPL-SAC co-crystal revealing that TPL successfully co-crystallized with saccharin, despite being dispersed in the HPO matrix during the SEA processing, as Fig. 5 shows for the sample TPL-SAC-HPO 1. The diffraction peaks of the TPLSAC co-crystal are shown in Fig. 5. This figure also reveals that the co-crystallization was extensive, because the characteristic peaks of pure TPL and SAC are not present in the composite’s diffractogram, which shows that the fraction of unreacted components is small. All other formulations richer in HPO had similar diffractograms (results not shown), except for the intensity of the co-crystal diffraction peaks, which was obviously lower. Although TPL and TPL-SAC diffractograms (shown in Figs. 4a and 5c, respectively) share common peaks, the peak at 25.9 (2 theta) in the TPL-SAC-HPO 1 diffractogram (Fig. 5d) is unique and characteristic of the TPL-SAC co-crystal, as it is clear when its diffractrogram is compared with those of TPLSAC and TPL.

Figure 4. PXRD diffractograms: (a) TPL; (b) HPO; (c) TPL-HPO 1.



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Figure 5. PXRD diffractograms: (a) physical mixture of TPL with SAC; (b) HPO; (c) TPL-SAC; (d) TPL-SAC-HPO 1.

Relative Humidity (RH) stability analysis. Relative humidity (RH) stability analyses were performed to evaluate the stability of TPL at room temperature, under controlled humidity conditions (92% RH). The samples analyzed were: raw TPL, TPL-SAC, TPL-HPO 1, TPL-HPO 2, TPL-HPO 3, TPL-SAC-HPO 1, TPL-SAC-HPO 2 and TPL-SAC-HPO 3. Table 3 presents the evolution with time of the TPL conversion into other polymorphs, essentially its hydrate, at 92% RH. The PXRD method was used to evaluate any solid-state phase transition of TPL or TPL-SAC co-crystal.

Table 3. Stability of samples under 92% of relative humidity (RH) analyzed by powder X-ray diffraction (PXRD). Time period Molar 1 day 3 days 6 days 10 days 6 months proportion TPL 1 TPL-SAC 1:1 + + + + + TPL-HPO 1 1:2 + / TPL-HPO 2 1:10 + + / TPL-HPO 3 1:20 + + + / TPL-SAC-HPO 1 1:1:2 + + + + + TPL-SAC-HPO 2 1:1:10 + + + + + TPL-SAC-HPO 3 1:1:20 + + + + + (+) stable; (-) physically instable (monohydrate formation); (/) TPL conversion into a different polymorphic form. (Diffractograms used to prepare this table are presented in the supporting information). Sample


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As Table 3 shows, TPL converts into a monohydrate form (diffractogram peaks of this sample corresponds to those described elsewhere21) in less than 1 day at 92% RH. However, when encapsulated in HPO, its stability towards hydration is slightly improved: the TPL-HPO 1 formulation shows 1 day stability at 92% RH. Yet, after 3 days, TPL has already converted into another polymorph, whereas after 6 days TPL is in the monohydrate form despite being within the HPO matrix. TPL hydration is delayed by a couple of days when the composition in HPO was higher. Theophylline in the TPL-HPO 2 sample shows a 3 day stability whereas the TPLHPO 3 sample shows a 6 day stability at 92% RH. Remarkably, none of the TPL-SAC co-crystal samples converted into TPL monohydrate or any other polymorph during the 6 months of this experiment, regardless of the HPO composition.

Dissolution profiles analysis. In vitro dissolution studies were performed with raw TPL, TPLSAC, TPL-HPO and TPL-SAC-HPO particles. Samples without HPO (raw TPL and TPL-SAC) achieve maximum dissolution within a few minutes, as Fig. 6 illustrates.

Figure 6. Dissolution rate of theophylline from raw TPL ( ) and TPL-SAC co-crystal ( ) in phosphate-buffered saline pH 7.4 at 37 ºC during 10 minutes. (n = 3±SD).



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Raw TPL dissolution rate is slightly higher than TPL-SAC co-crystal; however, both samples are completely dissolved in less than 3 minutes. Figure 7 shows the cumulative percentage of dissolved TPL from several TPL-HPO and TPLSAC-HPO formulations during 72 hours (3 days).

Figure 7. Experimental data from the dissolution rate of TPL from different samples (n = 3±SD) in phosphate-buffered saline pH 7.4 at 37 ºC during 72 hours, ( ) TPL-HPO 1 (1:2 stoichiometry); (

) TPL-HPO 2 (1:10 stoichiometry); (

stoichiometry); (

) TPL-SAC-HPO 1 (1:1:2 stoichiometry); (

(1:1:10 stoichiometry); (

) TPL-HPO 3 (1:20 ) TPL-SAC-HPO 2

) TPL-SAC-HPO 3 (1:1:20 stoichiometry).

The dissolution rate of TPL increases as HPO composition decreases. However, the formulations that contain the co-crystal show a substantially slower dissolution rate compared to that of pure crystalline TPL (for the corresponding HPO fraction).

Modeling dissolution kinetics. The models of Higuchi, Korsmeyer-Peppas and Weibull were fitted to the experimental dissolution data obtained for the formulations TPL-SAC-HPO (samples 1, 2 and 3). Table 4 summarizes the parameters calculated by each mathematical model and the corresponding coefficients of linear regression (R2).


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The Korsmeyer-Peppas model and the Weibull model fitted the experimental data better than Higuchi´s model, with relatively good regression coefficients. Nonetheless, the KorsmeyerPeppas low n values (n < 0.5) are below what is acceptable for this model.27 The Weibull equation fitted particularly well the experimental drug dissolution data for the TPL-SAC-HPO formulations (Table 4). The models and their kinetic parameters (listed in Table 4) are fully described elsewhere25 and for clarity and conciseness the same nomenclature was used here.

Table 4. Model parameters and corresponding coefficients of linear regression for different TPLSAC-HPO samples. Higuchi model Samples

Korsmeyer-Peppas model Kk n R2

Weibull model a

b

R2

0.91

0.076

0.50

1.00

30.45

0.98

9.590

0.44

0.98

15.17

0.98

39.719

0.45

0.99

KH

R2

TPL-SAC-HPO 1

149.98

0.73

0.31

110.83

TPL-SAC-HPO 2

41.68

0.66

0.30

TPL-SAC-HPO 3

13.22

0.68

0.37

KH: kinetic Higuchi constant; n: exponent characterizing the diffusion mechanism; Kk: kinetic Korsmeyer-Peppas constant; a: scale factor representing the time when 63.2% of the material is dissolved; b: shape factor for the dissolution curve.

Estimation of TPL plasmatic concentration curves. To analyze the feasibility of the cocrystal microcomposites for controlled release purposes, the one-compartment pharmacokinetic model was used to estimate the TPL dose (D) and the corresponding quantity of particles required for a therapeutic formulation. For simplicity and taking into consideration TPL´s high oral bioavailability, we assumed the absorption rate dXa/dt as the dissolution rate, which is defined by

dXa F D = × f (t ) dt V

(3)

where the ratio FD/V ratio is the concentration of TPL that changes with time t being this dependence taken as the Weibull probability density function.31 Therefore, Eq. (3) takes the form b−1

dXa F D b  t  = × dt V a  a 

e−(t / a)

b

(4)



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Substitution of Eq. (4) in Eq. (1) gives dX F D b  t  = dt V a  a 

b−1

e−(t / a) − ke X b

(5)

The integration of Eq. (5) gives Eq. (6), which allows the prediction of TPL concentration in plasmatic conditions:   b   − ( t / a ) FD e − ket  − X= + e  V  1 − ke   b − 1 kat  

for 10 < X < 20 mg/L

(6)

where ka is given by b/(ab). The dose (D) may then be adjusted so that the predicted plasmatic concentrations do not exceed the toxic concentration limit. The calculated dose was 800 mg of TPL for the TPL-SACHPO (1:1:2) formulation, which corresponds to approximately 3 g of microcomposite particles. This model cannot be applied whenever the dissolution rate is slower than the elimination rate. For this reason the one-compartment model was applicable only to the formulation TPL-SACHPO (1:1:2). Figure 8 compares the predicted TPL plasmatic concentration curves of raw TPL with that of the TPL-SAC-HPO (1:1:2) formulation.

Figure 8. Prediction of TPL plasmatic concentration (mg/L) as a function to time (h) for raw TPL ( ) and for the sample TPL-SAC-HPO (1:1:2) ( ). Light dotted lines show the borders of the therapeutic window.


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Raw TPL dissolves instantaneously causing a burst peak after the drug intake (t = 0) followed by an exponential decrease. This burst effect is greatly attenuated in the TPL-SAC-HPO (1:1:2) sample, though its concentration decay is very similar to that of the raw TPL. The microcomposites increased slightly the therapeutic action by 1 hour: 8 h (for raw TPL) and 9 hours for the encapsulated TPL co-crystal.

DISCUSSION The experimental results obtained in this work evidence that SEA is capable of providing a single-step approach for micronization, co-crystallization and encapsulation of APIs. The results show that TPL-SAC co-crystal was formed regardless of being precipitating in a solution with high composition of a lipid (HPO). This may result from a faster supersaturation of the TPL-SAC co-crystal compared to HPO. TPL and SAC were closer to saturation in the initial solution (TPL and SAC solubility in THF are approximately 5 mg/g and 100 mg/g, respectively) than HPO, which is very soluble in THF. For example Li et al.32 reported that the HPO solubility in THF is 365 mg/g at 37 ºC, and therefore at the working temperature used here (50 ºC) it is expected to be considerably higher. Consequently, the co-crystal may start nucleating and crystalizing earlier than the lipid. This differential precipitation may be amplified by the fact that crystallization of small molecules (TPL and SAC) is also expected to be faster than triglycerides such as HPO. This sequential crystallization was crucial for the release kinetics of the microcomposites because it enabled the microdispersion of the API’s in the lipid. The API (TPL) physical stability was enhanced by the co-crystal form, i.e., no transition in the crystalline structure occurred during 6 months at 92% RH (as Table 3 shows). This evidence suggests that there is a competition between SAC and water to form hydrogen bonding with TPL, thereby preventing its hydration. Under this scope, the stability conferred by the co-crystal was much more effective than the hydrophobic barrier posed by HPO. The influence of the co17 ACS Paragon Plus Environment


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crystal was also significant in the dissolution studies (Fig. 7). As expected, HPO hindered substantially the TPL release, delaying therefore its dissolution kinetics, a fact that was previously reported by Rodrigues et al.24 The higher the proportion of HPO in the formulation, the slower will be the dissolution rate kinetics of TPL. However, the encapsulated co-crystal was able to decrease further the dissolution rate for the equivalent composition in TPL-HPO microcomposite (Fig. 7). This effect may be related to saccharin’s ability to hinder TPL hydration and consequently slow down its dissolution. This hindrance is observed regardless of the presence of HPO. Figure 4 shows that the co-crystal dissolves slower than the pure TPL, even though in this case the dissolution time is a few minutes only. The release kinetics of the microcomposite particles didn’t fall into any of the classical dissolution models. The kinetic Higuchi constant (KH) is inversely proportional to the HPO content (Table 4); consequently a high amount of HPO implies an extended release mechanism. However, the correlation was not acceptable (Table 4), most likely the degradation of HPO polymer by erosion or relaxation of its structure invalidates this model, which establishes that the matrix should be rigid and water-insoluble.26 In Korsmeyer-Peppas model, the low n values (n < 0.5) suggest anomalous transport kinetics, i.e., a combined mechanism of pure diffusion and initial fast release for TPL-SAC co-crystals, which should be located closer to the surface of the microcomposites. Therefore, even though the Weibull distribution is an empiric model, i.e. it is not obtained from any kinetic fundament25 it could be adjusted accurately to the drug dissolution kinetics, as presented in Table 4. The good correlation obtained with the Weibull equation was very convenient for the prediction of the plasmatic concentrations curves using a onecompartment model. It is difficult to evaluate the impact of microcomposite particles for controlled release purposes without considering its practical constraints. This is particularly evident in this work. Even though the TPL dissolution could be substantially retarded by co-crystallization and


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encapsulation, the pharmacokinetic model puts that into perspective by taking into account the high TPL elimination rate and narrow therapeutic concentration window. The model revealed that TPL dissolution rate from most of the formulations is substantially smaller than the body’s elimination rate. In this situation, the therapeutic window could not be achieved, reason why the model could not be solved. Exceptionally, the TPL-SAC-HPO 1 formulation enables to reach therapeutic levels, with the advantage of providing a mild initial release compared to raw TPL. Still, it requires 3 g of microcomposites to deliver a dose of 800 mg of TPL, which is difficult to administer. The modeled formulation could not extend significantly the therapeutic time (only 1 hour); however, this was a simple approach to formulation that must be regarded as an extended critical interpretation of the dissolution kinetics. Obviously, the predicted plasmatic concentration curve can only be regarded as a rough estimate of what should occur in the body; however we consider that it provides a preliminary tool to access the practical feasibility of this particular approach – TPL-SAC-HPO microcomposites. Overall, we realize that the major advantage of these particular microcomposite particles was the substantial improvement in the TPL stability caused by its co-crystallization. This is relevant for TPL because it has several known polymorphs. This work also demonstrates the SEA ability to produce co-crystal microcomposites in a single-step. In this regard, the precipitation sequence of the solutes may determine the efficacy of the single-step process to produce the desired effect. The spray cooling caused by CO2 depressurization may also favor the crystallization of the solutes (by temperature drop) compared to conventional spray-drying. The Joule-Thompson cooling effect is particularly strong in CO2 compared to N2, which is used in conventional spray-drying. Nonetheless, TPL is known to co-crystallize easily, which must be taken into account in any analogy concerning how these results may apply to other APIs. The clarification of these mechanisms would require substantial comparative work, perhaps between SEA and conventional spray-drying, also using



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other API systems. We therefore anticipate that single-step generation of lipid dispersed cocrystal microcomposites may open further interesting questions for future research.

CONCLUSIONS The SEA process was able to improve stability and delivery of TPL by single-step micronization, co-crystallization and encapsulation. Therefore, this process has the potential to simplify manufacturing of controlled release formulations, that otherwise would require several steps. The TPL-SAC co-crystal was physically stable towards hydration and TPL dissolution kinetics was improved. Despite TPL and TPL-SAC dissolve promptly in water, the co-crystal had a significantly slower release from the HPO particles, which may be related to its higher stability towards hydration. The TPL-SAC-HPO formulations were evaluated in terms of its feasibility for therapeutic application using a one-compartment pharmacokinetic model. The microcomposite particles were able to attenuate the TPL burst effect and extended slightly the time within therapeutic concentration range.

ASSOCIATED CONTENT Supporting Information. PXRD diffractograms used to construct Table 3 in relative humidity stability analysis. This information is available free of charge via the Internet at http://pubs.acs.org/

AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected]. Fax: +351 21 841 9595. Tel: +351 21 841 9394.

Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENTS For financial support the authors are grateful to Fundação para a Ciência e Tecnologia, FCT, Lisbon, for Grants SFRH/BD/39836/2007 and PTDC/EQUFTT/099912/2008.

REFERENCES (1) Jones, W.; Motherwell, S.; Trask, A. V., MRS Bulletin 2006, 31, 875-879. (2) Trask, A. V., Mol. Pharm. 2007, 4, 301-309. (3) Basavoju, S.; Bostrom, D.; Velaga, S. P., Crys. Growth Des. 2006, 6, 2699-2708. (4) Fleischman, S. G.; Kuduva, S. S.; McMahon, J. A.; Moulton, B.; Walsh, R. D. B.; Rodriguez-Hornedo, N.; Zaworotko, M. J., Crys. Growth Des. 2003, 3, 909-919. (5) Padrela, L.; Rodrigues, M. A.; Velaga, S. P.; Fernandes, A. C.; Matos, H. A.; Azevedo, E. G., J. Supercrit. Fluids 2010, 53, 156-164. (6) Padrela, L.; Rodrigues, M. A.; Velaga, S. R.; Matos, H. A.; Azevedo, E. G., Eur. J. Pharm. Sci. 2009, 38, 9-17. (7) Shan, N.; Zaworotko, M. J., Drug Disc. Today 2008, 13, 440-446. (8) Anitescu, G.; Bruno, T. J., J. Supercrit. Fluids 2012, 63, 133-149. (9) Cansell, F.; Chevalier, B.; Demourgues, A.; Etourneau, J.; Even, C.; Garrabos, Y.; Pessey, V.; Petit, S.; Tressaud, A.; Weill, F., J. Mater. Chem. 1999, 9, 67-75. (10) Foerster, T.; Fabry, B.; Hollenbrock, M.; Kropf, C. Use of nanoscale sterols and sterol esters for producing cosmetic and/or pharmaceutical preparations. Patent WO2000021490, 2000. (11) Perez, Y.; Wubbolts, F. E.; Witkamp, G. J.; Jansens, P. J.; de Loos, T. W., AIChE J. 2004, 50, 2408-2417. (12) Reverchon, E.; Della Porta, C.; Di Trolio, A.; Pace, S., Ind. Eng. Chem. Res. 1998, 37, 952-958. (13) Van Hoa, N.; Kim, B.-K.; Jo, Y.-L.; Shim, J.-J., J. Supercrit. Fluids 2012, 72, 28-35. (14) Yeo, S. D.; Kiran, E., J. Supercrit. Fluids 2005, 34, 287-308. (15) Burger, J. L.; Cape, S. P.; Braun, C. S.; McAdams, D. H.; Best, J. A.; Bhagwat, P.; Pathak, P.; Rebits, L. G.; Sievers, R. E., J. Aerosol Med. Pulm. D. 2008, 21, 25-34. (16) Sievers, R. E.; Milewsky, P. D.; Sellers, S. P.; Kusek, K. D.; Kleutz, P. G.; Miles, B. A., J. Aerosol Sci. 1998, 29, 1271-1272. (17) Sievers, R. E.; Quinn, B. P.; Cape, S. P.; Searles, J. A.; Braun, C. S.; Bhagwat, P.; Rebits, L. G.; McAdams, D. H.; Burger, J. L.; Best, J. A.; Lindsay, L.; Hernandez, M. T.; Kisich, K. O.; Iacovangelo, T.; Kristensen, D.; Chen, D., J. Supercrit. Fluids 2007, 42, 385-391. (18) Rodrigues, M.; Figueiredo, L.; Padrela, L.; Cadete, A.; Tiago, J.; Matos, H.; Azevedo, E. G.; Goncalves, L. M. D.; Almeida, A. J., Eur. J. Pharm. Biopharm. 2012, 82, 392-400. (19) Rodrigues, M.; Padrela, L.; Geraldes, V.; Matos, H.; Azevedo, E. G. In Converting spray dryers into supercritical machines by atomization of supercritical antisolvent induced suspensions, 13th European Meeting on Supercritical Fluids. The Hague, The Netherlands, 2011. (20) Weinberger, M.; Hendeles, L., New Engl. J. Med. 1983, 308, 760-764. (21) Trask, A. V.; Motherwell, W. D. S.; Jones, W., Int. J. Pharm. 2006, 320, 114-123. (22) Fernandez-Ronco, M. P.; Kluge, J.; Mazzotti, M., Crys. Growth Des. 2013, 13, 20132024. 21 ACS Paragon Plus Environment


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(23) Alhalaweh, A.; Kaialy, W.; Buckton, G.; Gill, H.; Nokhodchi, A.; Velaga, S. P., AAPS PharmSciTech 2013, 14, 265-276. (24) Rodrigues, M.; Peirico, N.; Matos, H.; Azevedo, E. G.; Lobato, M. R.; Almeida, A. J., J. Supercrit. Fluids 2004, 29, 175-184. (25) Costa, P.; Sousa Lobo, J. M., Eur. J. Pharm. Sci. 2001, 13, 123-133. (26) Higuchi, T., J. Pharm. Sci. 1963, 52, 1145-1148. (27) Korsmeyer, R. W.; Peppas, N. A., J. Membrane. Sci. 1981, 9, 211-227. (28) Langenbucher, F., J. Pharm. Pharmacol. 1972, 24, 979-981. (29) Katzung, B. G., Basic & Clinical Pharmacology, 12th ed. McGraw-Hill Medical: New York, 2012. (30) Dhillon, S.; Kostrzewski, A., Clinical Pharmacokinetics. Pharmaceutical Press: Grayslake, 2006. (31) Papoulis, A.; Pillai, S. U., Probability, Random Variables, and Stochastic Processes, 4th ed. McGraw-Hill: 2001. (32) Li, J.; Rodrigues, M.; Paiva, A.; Matos, H. A.; Azevedo, E. G., J. Supercrit. Fluids 2007, 41, 343-351.


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For Table of Contents Use Only

Single-step Co-crystallization and Lipid Dispersion by Supercritical Enhanced Atomization

João M. Tiago, Luís Padrela, Miguel A. Rodrigues, Henrique A. Matos, António J. Almeida, Edmundo Gomes de Azevedo

Synopsis This work presents an experimental study of the co-precipitation of teophylline with saccharine and hydrogenated palm oil carriers using a supercritical CO2-assisted technique. A structural characterization of co-precipitates by SEM and XRD techniques is presented and the characterization of the stability and drug release characteristics of the products were obtained.


Single-Step Co-Crystallization and Lipid Dispersion by Supercritical

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