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Efavirenz dissolution enhancement V - A combined top down/bottom up approach on nanocrystals formulation

Abstract

Efavirenz is one of the most commonly used drugs in HIV therapy. However the low water solubility tends to result in low bioavailability. Drug nanocrystals, should enhance the dissolution and consequently bioavailability. The aim of the present study was to obtain EFV nanocrystals prepared by an antisolvent technique and to further observe possible effect, on the resulting material, due to altering crystallization parameters. A solution containing EFV and a suitable solvent was added to an aqueous solution of particle stabilizers, under high shear agitation. Experimental conditions such as solvent/antisolvent ratio; drug load; solvent supersaturation; change of stabilizer; addition of milling step and solvents of different polarities were evaluated. Suspensions were characterized by particle size and zeta potential. After freeze- dried and the resulting powder was characterized by PXRD, infrared spectroscopy and SEM. Also dissolution profiles were obtained. Many alterations were not effective for enhancing EFV dissolution; some changes did not even produced nanosuspensions while other generated a different solid phase from the polymorph of raw material. Nevertheless reducing EFV load produced enhancement on dissolution profile. The most important modification was adding a milling step after precipitation. The resulting suspension was more uniform and the powder presented grater enhancement of dissolution efficacy.

Keywords:
Efavirenz; Particle size; Nanocrystals; Anti-solvent precipitation; Dissolution

INTRODUCTION

Efavirenz (EFV) is a non-nucleoside inhibitor of HIV reverse transcriptase, an antiretroviral drug used widely in anti-AIDS therapy (Burger et al., 2006Burger D, van der Heiden I, la Porte C, van der Ende M, Groeneveld P, Richter C, et al. Interpatient variability in the pharmacokinetics of the hiv non-nucleoside reverse transcriptase inhibitor efavirenz: the effect of gender, race, and CYP2B6 polymorphism. Br J Clin Pharmacol. 2006;61(2):148-54.). The bioavailability of EFV tablets is between 40% and 45%. Clinical studies show that the bioavailability of the liquid form is 20% lower than that of the solid form and has high variability between the starved and fed states (Chiappetta et al., 2010Chiappetta DA, Hocht C, Taira C, Sosnik A. Oral pharmacokinetics of the Anti-HIV efavirenz encapsulated within polymeric micelles. Biomaterials. 2010;32(9):2379- 87.). EFV is a crystalline powder with a low water solubility of 3-9 µg/mL and a low intrinsic dissolution rate (IDR) of 0.037 mg.cm-2.min-1. According to the Biopharmaceutics Classification System (BCS), EFV is defined as a class II drug, meaning it has low solubility and high permeability (Chiappetta et al., 2010Chiappetta DA, Hocht C, Taira C, Sosnik A. Oral pharmacokinetics of the Anti-HIV efavirenz encapsulated within polymeric micelles. Biomaterials. 2010;32(9):2379- 87.; Cristofoletti et al., 2013Cristofoletti R, Nair A, Abrahamsson B, Groot DW, Kopp S, Langguth P, et al. Biowaiver monographs for immediate release solid oral dosage forms: efavirenz. J Pharm Biomed Anal. 2013;102(2):318-29.; Patel et al., 2014Patel GV, Patel VB, Pathak A, Rajput SJ. Nanosuspension of efavirenz for improved oral bioavailability: formulation optimization, in vitro, in situ and in vivo evaluation. drug. Dev Ind Pharm. 2014;40(1):80-91.).

The most common enhancement technique to increase the dissolution rate of a drug is particle size reduction to the micrometric scale (Cho et al., 2010Cho E, Cho W, Cha KH, Park J, Kim MS, Kim JS, et al. Enhanced dissolution of megestrol acetate microcrystals prepared by antisolvent precipitation process using hydrophilic additives. Int J Pharm. 2010;396(1-2):91-98.; Khadka et al., 2014Khadka P, Ro J, Kim H, Kim I, Kim JT, Kim H, et al. ScienceDirect Pharmaceutical Particle Technologies : An Approach to Improve Drug Solubility, Dissolution and Bioavailability. Asian J Pharm. 2014;1-13.; Savjani, Gajjar, Savjani, 2012Savjani KT, Gajjar AK, Savjani JK. Drug solubility : importance and enhancement techniques. ISRN Pharmaceutics. 2012;2012:1-10.). However, for drugs with very low water solubility, such as EFV, it can be very promising to reach the nanometric scale (Fandaruff et al., 2014Fandaruff C, Rauber GS, Araya-sibaja AM, Pereira RN, Campos CEM, Rocha HVA, et al. Polymorphism of Anti- HIV drug efavirenz : investigations on thermodynamic and dissolution properties. Cryst Growth Des. 2014;14:4968-75.; Muller, Keck, 2004Muller RH, Keck CM. Challenges and solutions for the delivery of biotech drugs--a review of drug nanocrystal technology and lipid nanoparticles. J Biotechnol. 2004;113(1-3):151-70.). Drug nanocrystals are more commonly used than other nanotechnology approaches in the pharmaceutical market (Gao et al., 2013Gao L, Liu G, Ma J, Wang X, Zhou L, Li X, et al. Application of drug nanocrystal technologies on oral drug delivery of poorly soluble drugs. Pharm Res. 2013;30(2):307-24.). Drug nanocrystals are crystalline particles dispersed in an organized crystal arrangement with an average diameter smaller than 1000 nm (Patel et al., 2011Patel AP, Patel JK, Patel KS, Deshmukh AB, Mishra BR. A review on drug nanocrystal a carrier free drug delivery. Int J Res Ayurveda Pharm. 2011;2(2):448-58.). Nanocrystals are generally considered a safe structure to enhance the bioavailability of low water- soluble drugs (Gao et al., 2012Gao L, Liu G, Ma J, Wang X, Zhou L, Li X, et al. Drug nanocrystals: in vivo performances. J Control Release. 2012;160(3):418-30.).

The methods used to prepare nanocrystals can be divided into two categories: bottom-up and top-down. The bottom-up techniques consist of dissolving the drug in a solvent system and then transforming this solution into an antisolvent environment (Verma, Gokhale, Burgess, 2009Verma S, Gokhale R, Burgess DJ. A Comparative study of top-down and bottom-up approaches for the preparation of micro/nanosuspensions. Int J Pharm . 2009;380(1-2):216-22.). The driving force of crystal formation is supersaturation, which is why these methods are also known as precipitation methods (de Waard et al., 2009de Waard H, Grasmeijer N, Hinrichs WLJ, Eissens AC, Pfaffenbach PPF, Frijlink HW. Preparation of drug nanocrystals by controlled crystallization: application of a 3-way nozzle to prevent premature crystallization for large scale production. Eur J Pharm Sci . 2009;38(3):224-29.). Of all the available techniques for nanocrystal preparation, antisolvent precipitation was chosen for this study.

A brief review of the published literature about EFV nanocrystals includes a nanosuspension prepared by a modified antisolvent method (Jain et al., 2013Jain S, Sharma JM, Agrawal AK, Mahajan RR. Surface stabilized efavirenz nanoparticles for oral bioavailability enhancement. J Biomed Nanotechnol. 2013;9(11):1862-74.), the preparation of nanocrystals by pearl milling (Patel et al., 2014Patel GV, Patel VB, Pathak A, Rajput SJ. Nanosuspension of efavirenz for improved oral bioavailability: formulation optimization, in vitro, in situ and in vivo evaluation. drug. Dev Ind Pharm. 2014;40(1):80-91.), and a preparation method combining a modified antisolvent precipitation procedure with hot melt extrusion (Ye et al., 2015Ye X, Patil H, Feng X, Tiwari RV, Lu J, Gryczke A, et al. Conjugation of hot-melt extrusion with high-pressure homogenization: a novel method of continuously preparing nanocrystal solid dispersions. AAPS PharmSciTech 2015;17(1):78-88.). Overall, these studies propose complex procedures, with several long steps, which could present some problems during scaling up. Another important issue is that the resulting nanosuspensions have low drug loads. Therefore, the development of an EFV nanocrystal preparation method that combines the simplicity of nanocrystallization with a high drug load is still needed.

Many criteria can affect the outcome of this technique, especially those related to the crystallization kinetics (such as supersaturation degree) and those related to particle size and growth (such as stabilizer concentration) (Sinha, Müller, Möschwitzer, 2013aSinha B, Müller RH, Möschwitzer JP. Bottom-up approaches for preparing drug nanocrystals: formulations and factors affecting particle Size. Int J Pharm . 2013a;453(1):126-41.). Another factor vital to ensuring a uniform size distribution is agitation. The homogenizing process influences the nucleation rate and can lead to a more adequate size distribution of the crystals (Liu et al., 2012Liu G, Zhang D, Jiao Y, Zheng D, Liu Y, Duan C, et al. Comparison of different methods for preparation of a Stable Riccardin D Formulation via nano-technology. Int J Pharm. 2012;422(1-2):516-22.; Matteucci et al., 2006Matteucci ME, Hotze MA, Johnston KP, Williams RO. Drug nanoparticles by antisolvent precipitation: mixing energy versus surfactant stabilization. Langmuir. 2006;22(21):8951-59.).

The present article follows previous work (da Costa et al., 2015da Costa MA, Lione VOF, Rodrigues CR, Cabral LM, Rocha HVA. Efavirenz dissolution enheancement II: aqueous co- spray-drying. Int J Pharm Sci Res. 2015;6(9):3807-20.; da Costa et al., 2013da Costa MA, Seicera RC, Rodrigues CR, Hoffmeister CRD, Cabral LM, Rocha HVA, et al. Efavirenz dissolution enhancement I: Co-Micronization. Pharmaceutics. 2013;5(1):1-22.; Hoffmeister et al., 2017Hoffmeister C, Fandaruff C, Costa M, Cabral L, Pitta L, Bilatto S, et al. Efavirenz dissolution enhancement iii: colloid milling, pharmacokinetics and eletronic tongue evaluation. Eur J Pharm Sci. 2017;99:310-17.; Sartori, Prado, Rocha, 2017Sartori GJ, Prado LD, Rocha HVA. Efavirenz dissolution enhancement iv-antisolvent nanocrystallization by sonication, physical stability, and dissolution. AAPS PharmSciTech. 2017;18(8):3011-3020.) that investigated ways to enhance EFV dissolution by developing a new medicine with greater and more reproducible dissolution/ bioavailability. A previous study has already shown good prospects for the nanocrystal approach, using cavitation as a stirring method (Sartori, Prado, Rocha, 2017Sartori GJ, Prado LD, Rocha HVA. Efavirenz dissolution enhancement iv-antisolvent nanocrystallization by sonication, physical stability, and dissolution. AAPS PharmSciTech. 2017;18(8):3011-3020.). In this same study, several experimental parameters were tested until a promising sample was found.

Nevertheless, when ultrasound waves are applied to a liquid as a stirring method, they result in the formation and collapse of bubbles. This process produces a cyclic succession of expansion and compression phases (Wu et al., 2011Wu L, Zhang J. Watanabe W Physical and chemical stability of drug nanoparticles. Adv Drug Deliv Rev. 2011;63(6):456-69.). The mechanical vibration derived from this phenomenon generates enormous local heating (Flint Suslick, 1991Flint EB, Suslick KS. The Temperature of cavitation. Science. 1991;253(5026):1397-99.); hence, there is a possibility of drug instability or scale-up difficulties. Therefore, there is a need to develop a low-energy method for nanocrystal preparation.

The aim of the present paper is not only to determine the effects of changing the homogenizing technique from the ultrasound technique used in previous studies (Sartori, Prado, Rocha, 2017Sartori GJ, Prado LD, Rocha HVA. Efavirenz dissolution enhancement iv-antisolvent nanocrystallization by sonication, physical stability, and dissolution. AAPS PharmSciTech. 2017;18(8):3011-3020.) to a rotor-stator agitation but also to observe the consequences of modifications of the experimental conditions on the dissolution profile of EFV nanocrystals, especially the addition of a milling step (top-down technique) to prevent particle growth.

MATERIAL AND METHODS

Material

EFV was purchased from two different suppliers, which cannot be disclosed because of confidentiality issues. Ethanol, acetonitrile, acetone and methanol (analytical degree) were purchased from Tedia; hydroxypropyl methylcellulose (HPMC) E5 was purchased from Colorcon; polyvinyl pyrrolidone (PVP) K30 was purchased from Boai NKY; sodium lauryl sulphate (SLS) was purchased from VETEC.

Methods

Nanocrystal preparation

For the formation of EFV nanosuspensions, it was necessary to prepare two different solutions. The first one, the solvent phase, comprised the drug dissolved in methanol. The second solution contained stabilizers dissolved in deionized water. The electrostatic stabilizer used in all preparations was SLS at a concentration of 2% (w/w), and the steric stabilizers used were HPMC and PVP at a concentration of 40% (w/w), related to the EFV mass used, at room temperature. In some samples, different experimental conditions were tested, and the rationale for the preparation of samples and the conditions tested in those experiments are outlined as a workflow in Figure 1. In TX2, the solvent/antisolvent ratio was 1:1 (the ratio in other samples was 1:9); in TX4, the influence of a low-temperature antisolvent solution was evaluated, and sample TX7 was passed through a Meteor model REX 1-K/B90-52 colloid mill (Table I).

FIGURE 1
Workflow for the design of the different formulations.

TABLE I
Experimental parameters used in the preparation of the samples

Efavirenz (EFV) nanocrystallization experiments presented step-by-step. Samples were considered promising or excluded based in the dissolution assay, hence this test was in a diamond

The solvent solution was added to the antisolvent phase under vigorous agitation by an Ultra-turrax IKA model T25 at 20 000 RPM, and this agitation was maintained for one minute. The resulting suspension was freeze-dried using a BETA 1-16 Christ freeze-dryer to obtain a powder.

Particle size and zeta potential analysis

The particle size and zeta potential (ζ) were evaluated by dynamic light scattering (DLS) using a Nano ZS90 Malvern Zetasizer equipped with He-Ne LASER (λ = 633 nm) and a detector fixed at a 90° angle. Aliquots of the suspensions, taken immediately after preparation, were diluted to approximately 0.1% (v/v) in deionized water at room temperature. The zeta potential was measured by determining the electrophoretic mobility of the suspension using the Smoluchowski equation (Sze et al., 2003Sze A, Erickson D, Ren L, Li D. Zeta-potential measurement using the smoluchowski equation and the slope of the current- time relationship in electroosmotic flow. J Colloid Interface Sci. 2003;261(2):402-10.).

Powder X-ray diffraction

The analyses were performed on a D8 Advance Bruker diffractometer equipped with a LYNXEYE XE detector at room temperature using Cu-Kα (λ=1.5418 Å) radiation; the voltage and current during the assay were 40 kV and 40 mA, respectively, with a step size of 0.02° and a step time of 0.01 second. Powder samples, placed in the appropriate support, were scanned from 4° to 40°. Samples TX4, TX6 and TX7 also underwent a second analysis using the same parameters except with a step time of 0.5 second.

Infrared spectroscopy

The samples were analysed with a Nicolet 6700 Thermo-Nicolet infrared spectrometer equipped with OMNIC 7.0 software, with small amounts of the samples deposited directly in the attenuated total reflectance (ATR) accessory. The spectra were registered from 4000 to 600 cm-1 with 4 cm-1 resolution and 32 scans.

Scanning electron microscopy

The samples were spread on a sample holder and then coated with gold by an SCD 050 Sputter BalTech coater. The particle morphology of each sample was observed at several magnifications ranging from 500 to 30000 times using Quanta 400 FEI and TM3030Plus Hitachi scanning electron microscopes.

Sample dosing (drug assay)

The samples were dissolved in methanol to produce a primary solution with a concentration of 1 mg/mL. The primary solution was then diluted to enable the analysis with a UV-1800 Shimadzu spectrophotometer at a λ of 248 nm. The EFV content of each sample was calculated using a previously obtained analytical curve.

Dissolution profile

The dissolution test was conducted according to the Farmacopeia Brasileira 5th edition (Brasil, 2010BRASIL. Farmacopéia Brasileira. 5ª. ed. [s.l.] Agencia Nacional de Vigilância Sanitária - ANVISA, 2010.) paddle method using an Evolution 6000 Distek dissolution instrument. The temperature of the medium was maintained at 37 °C, and the sample was stirred at a constant stirring rate of 50 rpm. A sample with a mass corresponding to 100 mg of efavirenz, calculated based on sample dosing, was dispersed in 900 mL of medium containing an aqueous 0.1% (w/v) SLS solution; 11 mL samples were drawn at 5, 10, 15, 20, 25, 30, 45, 60 and 90 minutes. Sink conditions were maintained during the entire assay. The drug content was determined using a UV-1800 Shimadzu spectrophotometer at 248 nm, based on a previously obtained calibration curve. The dissolution profiles were compared two-by-two using ANOVA with Microsoft Excel® software. According to this statistical test, all dissolution profiles present significant differences from each other, and the dissolution efficiency was also calculated.

RESULTS AND DISCUSSION

Nanocrystal preparation

Initial crystallization conditions

Previous crystallization studies evaluated EFV solubility in different solvents and at several stabilizer concentrations (data not shown). The most promising sample is labelled TX1 and contains 40% HPMC and 2% SLS. Hence, this sample was chosen as the base formulation from which other experiments were derived.

Ultra-turrax samples generate foam during and just after preparation, especially at the top of the beaker, which is possibly related to the high concentration of SLS and to the agitation method. The bottom part of the suspension was similar to a clustered thick paste.

From a production point of view, suspensions were not favourable, since they could hinder nanosuspension handling. Therefore, a less viscous suspension would be more suitable for processing.

Changing the solvent/antisolvent ratio

It is expected that a greater volume difference between the solvent and antisolvent phases will result in a higher nucleation rate and, therefore, a smaller particle size (Sinha, Müller, Möschwitzer, 2013bSinha B, Müller RH, Möschwitzer JP. Systematic investigation of the cavi-precipitation process for the production of ibuprofen nanocrystals. Int J Pharm . 2013b;458(2):315-23.; Zhao et al., 2007Zhao H, Wang JX, Wang QA, Chen JF, Yun J. Controlled liquid antisolvent precipitation of hydrophobic pharmaceutical nanoparticles in a microchannel Reactor. Ind Eng Chem Res. 2007;46(24):8229-35.). TX1 was prepared using a solvent/antisolvent ratio of 1:9; sample TX2 used a ratio of 1:1.

The resulting suspensions were similar to a very thick paste and did not disperse in water. After drying, the resulting powder was characterized.

Drug load reduction

To enhance the mixing efficiency by reducing the system viscosity, two samples were prepared by decreasing the EFV drug load. Sample TX3 maintained the antisolvent at room temperature, while TX4 maintained the antisolvent solution only between 7.0 and 7.5 °C, which was achieved by applying an ice bath.

The TX3 sample was foamy and viscous, while TX4 was milky and fluid. Sample TX4 also presented visible sedimentation, but no visible large particles were observed.

Particle size analysis of sample TX4 shows that the most significant particle population has an average diameter of 222.6 nm. However, a second peak representing micrometric particles was also observed (Table II). This second population could be related to the sediment particles, since this sample had visible sedimentation or even particle aggregation. The polydispersity index (PDI) is lower than 0.5, indicating a uniform distribution. The zeta potential (ζ) indicates the stability of suspensions. The optimum value of the zeta potential for suspensions with steric and electrostatic stabilization is greater than ±20 mV (Liu et al., 2012Liu G, Zhang D, Jiao Y, Zheng D, Liu Y, Duan C, et al. Comparison of different methods for preparation of a Stable Riccardin D Formulation via nano-technology. Int J Pharm. 2012;422(1-2):516-22.). Sample TX4 presented an absolute value of ζ higher than ± 20 mV, which is considered adequate. Both PDI and ζ are parameters related to the physical stability of the suspension (Sawant et al., 2011Sawant SV, Kadam VJ, Jadhav KR, Sankpal SV. Drug Nanocrystals: novel technique for delivery of poorly soluble drugs. I. J Sci Innov Disc. 2011;1(3):1-15.; Wu, Zhang, Watanabe, 2011Wu L, Zhang J. Watanabe W Physical and chemical stability of drug nanoparticles. Adv Drug Deliv Rev. 2011;63(6):456-69.). Because sample TX3 did not disperse in water, DLS analysis was not possible.

TABLE II
Relation of particle size and zeta potential analysis

Solvent supersaturation

There is a relationship between a high degree of saturation and intensification of the nucleation rate (Sinha, Müller, Möschwitzer, 2013aSinha B, Müller RH, Möschwitzer JP. Bottom-up approaches for preparing drug nanocrystals: formulations and factors affecting particle Size. Int J Pharm . 2013a;453(1):126-41.). Hence, sample TX5 was prepared using a supersaturated solution of EFV in methanol as the solvent phase. The suspension had a thick and clustered appearance; however, the TX5 suspension was dispersible in water, so DLS was possible.

Table II presents the particle size distribution of TX5. Three different peaks can be observed. Peak 1 is more intense than the other peaks and is attributed to particles with an average diameter larger than 1 µm; peak 2 indicates a second particle population with an average diameter of 170.6 nm, and peak 3 is attributed to even larger micrometric particles. Accordingly, the PDI obtained was very high, reflecting the low uniformity of particles in the suspension.

Although TX5 presented an adequate ζ, the particle size analysis indicates that the supersaturation condition was not the most favourable for nanocrystal formation. As a result, TX5 was not subjected to the characterization and dissolution tests.

Change in the steric stabilizer

A brief literature review revealed studies using EFV and PVP (Alves et al., 2014Alves LDS, De La Roca Soares MF, Albuquerque CT, Silva ER, Vieira ACC, Fontes DAF, et al. Solid dispersion of efavirenz in pvp k-30 by conventional solvent and kneading methods. Carbohydr Polym. 2014;104:166-74.; Jain et al., 2013Jain S, Sharma JM, Agrawal AK, Mahajan RR. Surface stabilized efavirenz nanoparticles for oral bioavailability enhancement. J Biomed Nanotechnol. 2013;9(11):1862-74.; Patel et al., 2014Patel GV, Patel VB, Pathak A, Rajput SJ. Nanosuspension of efavirenz for improved oral bioavailability: formulation optimization, in vitro, in situ and in vivo evaluation. drug. Dev Ind Pharm. 2014;40(1):80-91.), and since the efficacy of these polymers as steric stabilizers has already been tested in other nanocrystallization methods by this group, sample TX6 was prepared using the same experimental conditions used to prepare TX3, but the polymer used was PVP K30.

The suspension had a fluid nature; however, after some time, it was possible to observe large aggregate formation. DLS analysis (Table II) exhibited peaks 1 and 2 corresponding to particles with average diameters of 182 nm and 550.2 nm, respectively. Since both peaks have nanometric dimensions, the sample PDI was lower than 0.5. The zeta potential of -57.6 mV is considered suitable, indicating good physical stability.

Adding a milling step after crystallization

It has been reported in the literature that a bottom- up preparation can be followed by a top-down technique to prevent particle growth (Salazar et al., 2012Salazar J, Ghanem A, Müller RH, Möschwitzer JP. Nanocrystals: comparison of the size reduction effectiveness of a novel combinative method with conventional top-down approaches. Eur J Pharm Biopharm. 2012;81(1):82-90.; Yang et al., 2016Yang L, Chu D, Wang L, Ge G, Sun H. Facile synthesis of porous flower-like srco3 nanostructures by integrating bottom-up and top-down routes. Mater Lett. 2016;167:4-8.). Sample TX7 was prepared using the same conditions used to prepare TX3; however, a milling step was added immediately after precipitation. The mechanical stress generated by particle collision inside the mill is expected to prevent particle growth (Carstensen, 2001Carstensen JT, Drugs and the Pharmaceutical Sciences: Advanced Pharmaceutical Solids. 1st ed. New York: Marcel Dekker, Inc. 2001.).

After one hour of milling, a reduction in the viscosity was observed. The initial appearance was the same as that of TX3, and the foam, which was initially only on the top of the suspension, was observed throughout the whole suspension.

To verify the suspension stability, a particle size DLS analysis was performed before and after the milling step. Peak 1 in the data for TX7 before and after milling was observed at 210.5 nm and 491.9 nm, respectively (Table II).

There was also a reduction in the PDI after the milling process (Table II), indicating that this step produces more uniform suspensions. Although some growth has been detected, the particles are still within the nanoscale range, indicating that milling is effective in preventing significant particle growth. The zeta potentials of both suspensions were similar and considered adequate for a stable suspension.

Changing the solvent

With drugs of very low solubility, such as EFV, the use of a less polar solvent should result in more effective interaction between the drug and the stabilizers and a higher nucleation rate (Beck, Dalvi, Dave, 2010Beck C, Dalvi SV, Dave RN. Controlled Liquid Antisolvent Precipitation Using a Rapid Mixing Device. Chem Eng Sci. 2010, 65(21):5669-75.; Sinha, Müller, Möschwitzer, 2013aSinha B, Müller RH, Möschwitzer JP. Bottom-up approaches for preparing drug nanocrystals: formulations and factors affecting particle Size. Int J Pharm . 2013a;453(1):126-41.). Thus, smaller particles should be obtained.

Three new samples (TX8, TX9 and TX10) were prepared using ethanol, acetone and acetonitrile, respectively; the conditions used to prepare TX3 were applied. The suspensions were reasonably viscous and presented visible particles; they also presented phase separation, forming one slightly turbid liquid and a dense foam. Only TX9 and TX10 dispersed in water and were subsequently subjected to DLS analysis.

The DLS data for samples TX9 and TX10 exhibited peak 1 related to particles with an average diameter close to 1 µm; peak 2 corresponding to particles larger than 4 µm was also observed. Both samples had PDI values greater than 0.5. Sample TX10 had a ζ potential of -3.74 mV, which is considered low for particle stabilization. Although TX9 had a ζ potential of -29.5 mV, particle analysis proved that this sample was not a nanosuspension. Considering the unsatisfactory results for TX9 and TX10, these samples were discarded.

Solid state characterization

All samples considered promising were freeze-dried and characterized by SEM, infrared spectroscopy and PXRD. The samples were TX1, TX2, TX3, TX4, TX6, TX7 (after milling) and TX8.

Particle morphology

Photomicrographs of the processed samples and raw EFV exhibit a modified particle morphology. The active pharmaceutical ingredient (API) is composed of rough micrometric particles. Overall, the samples exhibit gel formation or long needle-shaped particles (Figure 2).

FIGURE 2
Photomicrographs from SEM analysis.

Pictures of different samples, obtained through microscopy analysis. In general samples presented needle shape particles with nanometric width


Sample TX1 presented more intense gel formation than did TX2, displaying large aggregates formed by smaller elongated particles with a nanometre-scale width. It is possible that the gelation in TX2 is not as intense as that in TX1 because a smaller volume of water was used during the preparation. Another hypothesis is that the great amount of methanol utilized could prevent the formation of the HPMC gel (Nickerson et al., 2009Nickerson B, Joseph R, Palmer C, Opio A, Beresford G. Analytical method development: Challenges and solutions for low-dose oral dosage forms. In: Zheng J, editor. Formulation and Analytical Development for Low-Dose Oral Drug Products 1st ed. Hoboken: Wiley, 2009. p. 241-264.).

Film formation was also observed in samples TX3 and TX4, which both contain the same polymer concentration as TX2. This reinforces the idea that the methanol/water ratio is related to this phenomenon. Evidently, the film in TX3 is more uniform than that in TX4. This may be due to deformations that occurred during the freezing step of the freeze-drying process (Lee, Cheng, 2006Lee J, Cheng Y. Critical Freezing Rate in Freeze Drying Nanocrystal Dispersions. J Controlled Release. 2006;111(1-2):185-92.), which makes clear the need for a specific study of the drying method.

Sample TX6 shows elongated particles of nanometric width, as well as aggregates and thicker particles, in accordance with the DLS analysis. The TX6 morphology demonstrates significant particle growth, indicating that PVP may not be an ideal steric stabilizer for this preparation method.

Sample TX7 also presented intense film formation, and TX8 was composed of large agglomerates of several sizes. With a more accurate analysis, it was observed that the aggregates are formed by long and fine particles. This indicates that the conditions used are not favourable for preparing nanocrystals, in conflict with the literature (Beck et al., 2010Beck C, Dalvi SV, Dave RN. Controlled Liquid Antisolvent Precipitation Using a Rapid Mixing Device. Chem Eng Sci. 2010, 65(21):5669-75.; Sinha, Müller, Möschwitzer, 2013aSinha B, Müller RH, Möschwitzer JP. Bottom-up approaches for preparing drug nanocrystals: formulations and factors affecting particle Size. Int J Pharm . 2013a;453(1):126-41.). Although fine elongated particles were generated, it is shown that they tend to aggregate, implying that the same preparation conditions were not affective for suspension stabilization when the solvent was ethanol.

Infrared spectroscopy.

The infrared spectra of the samples and API (Figure 2) present all the bands associated with the EFV functional groups (Gomes et al., 2013Gomes ECL, Mussel WN, Resende JM, Fialho SL, Barbosa J, Yoshida MI. Chemical interactions study of antiretroviral drugs efavirenz and lamivudine concerning the development of stable fixed-dose combination formulations for AIDS treatment. J Braz Chem Soc. 2013;24(4):573-79.). This indicates not only that EFV is present in the samples but also that no chemical reaction occurs between the drug and the stabilizers, due to some kind of incompatibility.

The high polymer concentration in the samples reduces the EFV proportion in the bulk, leading to a decrease in the signal intensity; this effect is more evident in TX3 than in other samples (arrow in Figure 2). Another possibility is that the lactam portion of the EFV molecule forms hydrogen bonds with the polymer, thereby reducing the vibration and intensity of absorption (Stuart, 2004Stuart B. Infrared Spectroscopy: Fundamentals and Applications. Hoboken: J. Wiley, 2004.). A common effect of particle comminution by this method is the reduction of the crystalline domain, which tends to decrease the infrared absorption of crystalline powders (Shankar, Rhim, 2016Shankar S, Rhim J. Preparation of nanocellulose from micro-crystalline cellulose: the effect on the performance and properties of agar-based composite Films. Carbohydr Polym . 2016;135:18-26.).

Samples TX3 and TX4 presented bands at 2930 cm-1 and 2902 cm-1, respectively, which are not characteristic of EFV molecules. These bands are commonly associated with CH and CH2 (Stuart, 2004Stuart B. Infrared Spectroscopy: Fundamentals and Applications. Hoboken: J. Wiley, 2004.), probably indicating the presence of a polymer. Steric stabilization of TX6 is achieved by PVP, and the infrared spectrum of this sample has a band at 1652 cm-1 associated with the pyrrolic ring of the polymer (Laot, 1997Laot CM. Spectroscopic characterization of molecular interdiffusion at a poly(vinyl pyrrolidone)/vinyl ester interface. [Master’s dissertation] Blacksburg: Virginia Polytechnic Institute and State University, 1997.). Overlapping of the bands in the range between 2860 and 3304 cm-1 was detected.

The presence of hydrogen bonds reduces the vibration of amine, amide and hydroxyl groups, reducing the intensity or wavenumber of the bands related to these functional groups (Theophile, 2012Theophile T. Infrared Spectroscopy - Materials science, engineering and technology. London: InTechOpen. 2012.). The inflexibility of the hydrogen bonds formed between EFV and PVP masks the NH band (arrow in Figure 2). The spectra of samples TX7 and TX8 have a band of 2936 cm-1, usually associated with alkane CH bonds (Stuart, 2004Stuart B. Infrared Spectroscopy: Fundamentals and Applications. Hoboken: J. Wiley, 2004.), which is possibly related to the presence of a high concentration of HPMC.

Powder X-ray diffraction

According to the diffraction patterns presented in Figure 3a, samples TX1, TX2, TX3 and TX8 maintained crystalline characteristics after precipitation. It is possible to identify EFV polymorph I, especially by the peak at 2θ=6.2° (Mahapatra et al., 2010Mahapatra S, Thakur TS, Joseph S, Varughese S, Desiraju GR. New solid state forms of the Anti-HIV drug efavirenz. Conformational flexibility and High Z’ Issues. Cryst Growth Des . 2010;10(7):3191-32.). Moreover, an increase in the peak width can be observed, possibly related to the reduction in the crystalline domain that is usually associated with particle size reduction (Blachére, Brittain, 2008Blachére JR, Brittain HG. X-Ray Diffacttion Methods for the Characterization of Solid Pharmaceutical Materials. In: Adeyeye M, Brittain HG, editor. Drugs and Pharmaceutical Sciences: Preformulation in Solid Dosage Form Development. 1st ed. New York: Informa Healthcare. 2008. p. 229-52.), in accordance with the infrared analysis.

FIGURE 3
Infrared spectra of EFV and processed samples.

Efavirenz (EFV) respective group bands are marked with arrows. Subsequent arrows are marking: 2902 cm-1, 2930 cm-1 and 2936 cm-1 are relative to CH and CH2 bands; 1652 cm-1 associated with the pyrrolic ring of the PVP;


Sample TX7 presents a halo indicating the presence of amorphous EFV resulting from the preparation method (Blachére, Brittain, 2008Blachére JR, Brittain HG. X-Ray Diffacttion Methods for the Characterization of Solid Pharmaceutical Materials. In: Adeyeye M, Brittain HG, editor. Drugs and Pharmaceutical Sciences: Preformulation in Solid Dosage Form Development. 1st ed. New York: Informa Healthcare. 2008. p. 229-52.). For a more meticulous investigation, samples TX4, TX6 and TX7 underwent a new PXRD assay, increasing the step time to enhance the signal intensity (Blachére, Brittain, 2008Blachére JR, Brittain HG. X-Ray Diffacttion Methods for the Characterization of Solid Pharmaceutical Materials. In: Adeyeye M, Brittain HG, editor. Drugs and Pharmaceutical Sciences: Preformulation in Solid Dosage Form Development. 1st ed. New York: Informa Healthcare. 2008. p. 229-52.).

The pattern obtained for samples TX4 was compared with the calculated patterns of other polymorphs of EFV (data not shown); however, no clear resemblance was found. In a study of the thermodynamic relations between several EFV polymorphs (Chadha et al., 2012Chadha R, Poonam A, Anupam S, Jain DS. An Insight into thermodynamic relationship between polymorphic forms of efavirenz. J Pharm Pharm Sci. 2012;15(2):234-51.), a crystalline form was found by slow recrystallization from hexane, with a diffraction pattern similar to that of TX4 (Figure 3b).

In a new PXRD analysis of TX6, it was possible to confirm the presence of EFV form I, but still some preferential orientation can be observed (Blachére, Brittain, 2008Blachére JR, Brittain HG. X-Ray Diffacttion Methods for the Characterization of Solid Pharmaceutical Materials. In: Adeyeye M, Brittain HG, editor. Drugs and Pharmaceutical Sciences: Preformulation in Solid Dosage Form Development. 1st ed. New York: Informa Healthcare. 2008. p. 229-52.). Increasing the step time reduces the noise associated with the diffraction pattern, so it is possible to detect form I with 2θ=6.2° in sample TX7.

Dissolution

Observing the dissolution profiles (Figure 4) and dissolution efficiency calculated for each sample (Table III) suggests that all samples, with the exception of TX2, TX6 and TX8, presented enhanced dissolution profiles. Sample TX2 has the lowest dissolution profile, even in comparison with the drug itself. This result was expected, since modification of the solvent/antisolvent ratio could significantly reduce the saturation degree of the system, favouring crystal growth. Hence, the dissolution profile is in accordance with theory.

FIGURE 4
(a) Diffraction patterns using step time of 0.01 seconds; (b) Diffraction patterns using step time of 0.5 seconds.

Comparison of diffraction patterns of samples with efavirenz (EFV), showing the presence of peaks relative to efavirenz polymorph I.


TABLE III
Drug concentration and dissolution efficiency of each sample in % with the respective standard deviation (SD)

Sample TX6 uses PVP as a steric stabilizer, as mentioned in the EFV-related literature. Nevertheless, the physical characterization and dissolution profile indicated that this modification was not beneficial. The low dissolution profile of sample TX8 is not in agreement with the literature since the use of a less polar solvent, when crystallizing a drug with very low water solubility such as EFV, should increase the nucleation rate (Beck, Dalvi, Dave, 2010Beck C, Dalvi SV, Dave RN. Controlled Liquid Antisolvent Precipitation Using a Rapid Mixing Device. Chem Eng Sci. 2010, 65(21):5669-75.).

Samples TX3 and TX4 have the same formulation, with their main difference being the antisolvent temperature used in their preparation. Due to the higher degree of saturation promoted by the low antisolvent temperature, it was expected that TX4 has a greater dissolution enhancement than TX3 (Sinha, Müller, Möschwitzer, 2013bSinha B, Müller RH, Möschwitzer JP. Systematic investigation of the cavi-precipitation process for the production of ibuprofen nanocrystals. Int J Pharm . 2013b;458(2):315-23.). However, by maintaining the antisolvent at room temperature in conjunction with EFV mass reduction, sample TX3 presented a more positive effect towards dissolution profile enhancement.

As TX4 has a different crystalline structure than TX3, this can affect several physicochemical properties, and thus, a more accurate study may be conducted in the future. Among all samples, the one that achieved the highest dissolution was TX7, proving that the addition of a milling step was able to prevent particle growth and produce a uniform suspension, the particles maintained their stability through freeze-drying, and the obtained EFV nanocrystals presented a high dissolution profile.

Comparison with the sonication method

A comparison of the dissolution profiles of samples TX3 and TX7, the most promising formulations, with the profile of a sonication sample (SN11) from a previous study (Sartori, Prado, Rocha, 2017Sartori GJ, Prado LD, Rocha HVA. Efavirenz dissolution enhancement iv-antisolvent nanocrystallization by sonication, physical stability, and dissolution. AAPS PharmSciTech. 2017;18(8):3011-3020.) with the best improvement and EFV is presented in Figure 5.

FIGURE 5
Dissolution profiles of samples.

Dissolution profiles of samples compared with efavirenz, pointing that the use of the technique was efficient in enhancing dissolution.


In general, the samples prepared by rotor-stator agitation were more viscous, similar to a paste, while the sonicated samples were more fluid. In a supersaturated environment, crystallization tends to occur. It has been reported that mass transfer from solution to the solid phase could be compromised due to the high viscosity of the suspension (Tung et al., 2008Tung HH, Paul E, Midler M, McCauley JA. Crystallization of organic compounds: an industrial perspective. Hoboken: Wiley . 2008.).

However, it is well known that in a suspension, particles are in Brownian motion, which is more intense in the case of more fluid environments, increasing particle collisions with subsequent growth (Comba, Sethi, 2009Comba S, Sethi R. Stabilization of highly concentrated suspensions of iron nanoparticles using shear-thinning gels of xanthan gum. Water Res. 2009;43(15):3717-26.). Therefore, increasing the viscosity is an alternative to improve the stability of a highly concentrated suspension (Peltonen, Hirvonen, 2010Peltonen L, Hirvonen J. Pharmaceutical nanocrystals by nanomilling: critical process parameters, particle fracturing and stabilization methods. J Pharm Pharmacol. 2010;62(11):1569-79.).

FIGURE 6
Comparison between dissolution profiles of samples TX3, TX7, EFV and sonication sample (SN11).

Comparison of dissolution profiles of the most promising samples with efavirenz and sample SN11 (from previous)


Thus, TX3 and TX7 exhibit greater dissolution than SN11. However it is important to emphasize that suspension viscosity is a property to optimize, not maximize, and that the determined level should not be exceeded, which would hinder the crystallization process.

From a production point of view, the higher enhancement of nanocrystals prepared by a high shear-based technique can be considered positive. Since cavitation-based techniques are more expensive and generate intense heat, the use of rotor-stators is more suitable when developing a low-cost and easy-to-scale up process.

CONCLUSION

Early attempts to crystallize TX1 were promising, as confirmed by dissolution enhancement, and they were used to establish a basic formulation. Changing the solvent/antisolvent ratio and the kind of solvent did not produce satisfactory results, and the sample had the lowest dissolution profile. Reducing the antisolvent temperature leads to the formation of a different crystalline state, possibly impairing dissolution. Changing the solvent also resulted in unsatisfactory outcomes.

Reducing the mass of EFV in TX3 produced a sample with one of the highest levels of dissolution. Sample TX7 exhibited higher dissolution than turrax samples and previous samples prepared using sonication, confirming that the addition of a milling step after crystallization is an interesting alternative to prevent growth.

Suspensions prepared by sonication were less viscous than those prepared with high shear. However, the dissolution percentage was higher for the latter samples (when compared with those prepared by sonication). The addition of a milling step produced a suspension that was more viscous than those prepared with sonication but not as pasty as the other samples that did not pass through the colloidal mill.

As a result, the combination of bottom-up (antisolvent precipitation) and top-down (colloid milling) techniques was shown to be the most efficient for producing EFV nanosuspensions. This resulted in dried nanocrystals with high and fast dissolution profiles. Another advantage of this method is that the drug concentration (drug load) was higher than those presented by other studies referenced previously. This is important since the drug load is directly related to the yield of the process.

Performing studies to comprehend the EFV crystallization process could provide insight into the reason why some of the presented results were not in agreement with the literature. Scale-up studies will be required to adapt the viscosity issue of the suspensions to the production point of view. Many aspects are still open for study, such as EFV tablet compression and nanocrystal performance in vivo.

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Publication Dates

  • Publication in this collection
    01 Apr 2022
  • Date of issue
    2022

History

  • Received
    09 Oct 2018
  • Accepted
    18 Apr 2019
Universidade de São Paulo, Faculdade de Ciências Farmacêuticas Av. Prof. Lineu Prestes, n. 580, 05508-000 S. Paulo/SP Brasil, Tel.: (55 11) 3091-3824 - São Paulo - SP - Brazil
E-mail: bjps@usp.br