Millisecond Self Assembly of Stable Nano-Dispersed Drug

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Millisecond Self Assembly of Stable Nano-Dispersed Drug Formulations VIKRAM J PANSARE, Aditya Rawal, AARON GOODWIN, RON BEYERINCK, Robert K Prud'homme, Dwayne T. Friesen, Michael Grass, ANNIE MUSKE-DUKES, and David T. Vodak Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00866 • Publication Date (Web): 15 Dec 2017 Downloaded from http://pubs.acs.org on December 17, 2017

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Molecular Pharmaceutics

Millisecond Self Assembly of Stable Nano-Dispersed Drug Formulations Vikram J. Pansare1, Aditya Rawal2, Aaron Goodwin3, Ron Beyerinck3, Robert K. Prud’homme1, Dwayne T. Friesen3, Michael Grass3, Annie Muske-Dukes3, David T. Vodak3 1) Department of Chemical and Biological Engineering, Princeton University, Princeton NJ 08544 2) Mark Wainwright Analytical Centre, University of New South Wales, Sydney, Australia, NSW 2032 3) Bend Research, a Division of Capsugel’s Dosage Form Solutions, Bend Oregon 97701

Abstract We report the development of a new Spray-drying and Nanoparticle Assembly Process (SNAP), that enables the formation of stable, yet rapidly dissolving, sub-200 nm nano-crystalline particles within a high Tg glassy matrix. SNAP expands the class of drugs that Spray Dried Dispersion (SDD) processing can address to encompass highly crystalline, but modestly hydrophobic drugs that are difficult to process by conventional SDD. The process integrates rapid precipitation and spray drying within a custom designed nozzle to produce high supersaturations and precipitation of the drug and high Tg glassy polymer. Keeping the time between precipitation and drying to tens of milliseconds allows for kinetic trapping of drug nano-crystals in the polymer matrix. Powder X-ray diffraction, solid state 2D NMR, and SEM imaging shows that adding an amphiphilic block copolymer (BCP) to the solvent gives essentially complete crystallization of the active pharmaceutical ingredient (API) with sub-200 nm domains. In contrast, the absence of the block copolymer results in the API being partially dispersed in the matrix as an amorphous phase, which can be sensitive to changes in bioavailability over time. Quantification of the APIexcipient interactions by 2D 13C-1H NMR correlation spectroscopy shows that the mechanism of enhanced nanocrystal formation is not due to interactions between the drug and the BCP, but rather the BCP masks interactions between the drug and hydrophobic regions of the matrix polymers. BCP-facilitated SNAP samples show improved stability during aging studies and rapid dissolution and release of API in vitro. Keywords: spray drying, nanoparticle, phenytoin, nanocrystals, solubility enhancement, solid state NMR, 2D NMR

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Introduction Over 40% of new drug candidates have poor water solubility, which causes poor oral bioavailability if dosed in a conventional bulk crystalline form.1 Many of these drugs have the potential to be safe and efficacious (despite not following the ‘rule of five’),2 but can only be effectively delivered using advanced formulation and processing techniques. To increase bioavailability, various approaches have been pursued to increase the rate and extent of dissolution. Fahr, et al.3 and Friesen, et al.4, reviewed (i) lipid –based solubilization (including solvents, co-solvents and micelles), (ii) top down particle production by size reduction via milling, (iii) amorphous solid dispersions, (iv) the formation of salts for ionizable compounds, (v) complexation, and (vi) the formation of prodrugs.5-7 In cases where improved dissolution is required for a highly crystalline active pharmaceutical ingredient (API), a particle size reduction approach, such as nanocrystalline formulation, may be preferred as the crystal form is typically more chemically stable than the solvated or amorphous states.

The goal of this work was to develop a “bottom-up” process from the solution state for producing solid nano-crystalline dispersions with the following properties: crystal domain sizes below 200 nm; rapid dissolution in simulated intestinal media (80% of saturation within 5 minutes); generally applicable to low aqueous solubility, high melting point active compounds; and excellent solid state physical stability upon storage by having a high degree of crystallinity and minimal amorphous drug.

Amorphous solid dispersions composed of low-solubility actives dispersed in an amphiphilic but aqueous-soluble polymer can be made either by heat- or solvent-based processes. In the solvent-


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based spray dried dispersion (SDD) process, a solid amorphous dispersion is produced by the atomization and rapid drying of a volatile solvent solution of the active and amphiphilic matrix polymer such as hydroxypropyl-methylcellulose (HPMC) or hydroxypropyl-methylcelluloseacetate-succinate (HPMCAS). When the resulting solid amorphous dispersion powder is delivered to an aqueous medium, the active dissolves to a highly supersaturated concentration that is maintained, at a high level, by the interaction of the polymer and active to stabilize the amorphous state and inhibit crystallization of the active.

Despite the broad utility of SDDs, it can be challenging to obtain SDD powders with the desired stable amorphous state using drugs with relatively low hydrophobicity (i.e. low logP) and high crystallinity (defined by a high Tm/Tg ratio). In some cases, the amorphous form of the active can be chemically unstable, and recrystallization of the amorphous phase can cause a decrease in bioavailability. In such cases, it may be preferable to formulate the active in the nano-crystalline state to ensure rapid dissolution and consistent bioavailability. The bioavailability enhancement technology map for low solubility actives has been reviewed by Williams5 and is shown in Figure 1. For highly hydrophobic drugs we have shown that nanoparticles of the drug can be formed using a block-copolymer-directed, rapid precipitation process (Flash NanoPrecipitation8), and then the subsequent nanoparticle dispersion can be spray dried with a matrix forming material.9 This corresponds to processing in the ‘Amorphous’ or ‘Lipid Based’ regions of the phase map in Figure 1. For drugs at lower logP values, SDD processing results in amorphous active pharmaceutical ingredient (API) powders with high supersaturations. The ‘Nanocrystal’ region has been challenging, because the strong tendency of the API to crystallize competes with the drug association with the hydrophobic sites on the polymer, and either the initially



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amorphous material may undergo physical instability towards crystallinity over time, or desired nanocrystalline materials cannot be stably formed in the first place.

In this paper we demonstrate the coupling of the rapid precipitation process (Flash NanoPrecipitation) with spray drying technology to produce a new process we term the Spraydrying and Nanoparticle Assembly Process (SNAP). First we demonstrate by calculations that the low API solubility in the ‘Nanocrystal’ region leads to rapid Ostwald ripening of nanoparticles, and particle size increases beyond the desired 200 nm size. Therefore, it is not possible to pre-form nanoparticles and then to spray dry them to form nanocrystalline powders. Then we demonstrate that integration of the nanoprecipitation with block copolymers and the spray drying, which results in nanoparticle formation and drying under 100 ms, allows the desired nanocrystalline powders to be produced. Powder diffraction x-ray diffraction (XRD), and 2D 13C-1H NMR correlation spectroscopy show that the physicochemical interactions leading to this successful nanocrystalline powder formation are completely different than we had supposed. The solid state 2D NMR provides the crucial information that shows the nature of the block copolymer interaction with the HPMC and with the model API, phenytoin.

The process and formulation approach developed to achieve nanocrystalline formulations of compounds in the ‘Nanocrystal’ region is termed the Spray-drying and Nanoparticle Assembly Process (SNAP).10 The SNAP process and formulation approach consists of the following elements: •

Step 1: Forming a solution of API and an amphiphilic block copolymer (BCP) in a solvent, S1;


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•

Step 2: Forming a solution of a high Tg aqueous soluble matrix material in a second solvent, S2, where S2 is miscible in S1, but S2 is a non-solvent for the API;

•

Step 3: Generating a high degree of supersaturation of the API by vigorously micro-mixing the solvent and non-solvent solutions to induce nucleation and growth of API-rich domains;

•

Step 4: Rapid removal of solvent and non-solvent via spray drying to trap the crystalline domains and deliver the formulation in dry powder form.

For the process to yield the desired solid nano-crystalline dispersion, it is critical that the starting API solution be miscible with the chosen API non-solvent, and that the non-solvent solution contains a high glass transition temperature matrix material that remains dissolved upon mixing the solvent and non-solvent (Step 3). Importantly, we found in this work, in the context of a phenytoin/BCP system, that the presence of the amphiphilic block copolymer is required to promote complete phase separation and crystallization of the API. It is also necessary for the intense micro-mixing of the solvent and non-solvent (Step 3) to be rapid, and subsequent solidification via spray drying (Step 4) to also be rapid, in order for the API domains to remain small (less than 200 nm). To achieve these short time scales, a special spray nozzle was designed and built so that solvent/non-solvent mixing occurred only milliseconds prior to spraying of the supersaturated suspension.

In this study, we used phenytoin as a model high melting point, crystalline, and low aqueous solubility active pharmaceutical ingredient (API), hydroxypropylmethylcellulose (HPMC) as a high Tg, aqueous-soluble matrix material, and poly(lactic acid)-poly(ethylene glycol) block copolymer (PLA-b-PEG) as the amphiphilic block copolymer (BCP). The physicochemical



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properties are presented in Table 1. The schematic of SNAP process and jet precipitation nozzle geometry enabling rapid mixing of the solvent and non-solvent streams within the nozzle just milliseconds prior to atomization and initiation of spray drying is presented in Figure 2.


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Experimental section Materials Phenytoin (D0894) was obtained from TCI Chemical and used as received. PLA4.2k-b-PEG5k (100 DL mPEG 5000) was obtained from Evonik Industries. HPMC E3 (METHOCEL E3 Premium LV) was obtained from Dow Chemical Company and used as received. Tetrahydrofuran (THF, 34865) and acetone (270725) were obtained from Sigma Aldrich. Ultrapure water was obtained from a Barnstead E-Pure Ultrapure Water Purification System (1818.2 MΩ-cm). SIF (Simulated Intestinal Fluid) powder used to make fasted state simulated intestinal fluid (FaSSIF) was purchased from Biorelevant.com (Croydon, Surrey, United Kingdom). Sodium phosphate dibasic was purchased from EM Science (Gibbstown, NJ). Hydrochloric acid (1 N) was purchased from J.T. Baker (Phillipsburg, NJ). Water was purified by Milli-Q UV plus systems (Millipore Co., Bedford, MA). All other chemicals were of analytical grade and purchased from commercial vendors.

Methods Measurement of solubility Stock mixtures of acetone/water and THF/water were created at 0%, 25%, 40%, 50%, 70% and 100 v/v% solvent. 10 mL of each mixture were loaded into two 20 mL vials with sufficient phenytoin in each vial to ensure saturation with drug. One set of vials was held at 37°C, the other at ambient temperature (~23°C). After overnight stirring the mixtures were centrifuged at 50,000g and the supernatant fluids extracted. The supernatant solutions were diluted in either acetone or THF and loaded into 2 mL HPLC vials and the concentration of free drug measured by HPLC on an Agilent 1100 system. HPLC method: Isocratic 57.5% H2O/42.5% ACN



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(acetonitrile), Total flow rate 1 mL/min, 10uL injection, ~3 min elution time, 30°C column temperature, Waters symmetry C8 column, 5 µm beads, 3.9 x 150 mm, detection wavelength λ= 242 nm.

It is evident by inspection of Figure 3 the solubility of phenytoin follows a power law relationship with respect to organic solvent/water mixtures for both THF and acetone at RT and at 37°C. The solubility of phenytoin in THF was found to be about 4.5 times higher compared to phenytoin in acetone. Interestingly, we observed that 50% and 70% by volume THF water mixtures saturated with phenytoin showed phase separation into two liquid phases, which made HPLC quantification of dissolved drug difficult, hence, the lack of data at those concentrations. THF-water phase separation in the absence of drug has previously been reported11, 12 at higher temperatures. Since our drug formulations never had a final concentration of THF higher than 25% by volume this phenomenon was not a concern.

Nanoparticle formation by rapid precipitation and block copolymer-directed assembly: Flash NanoPrecipitation (FNP): In previously reported work, nano-crystalline suspensions were made by FNP13. This is a scalable, continuous process for producing nanoparticles of a hydrophobic drug or API between 20 – 400 nm wherein a solvent stream containing dissolved API and an amphiphilic block copolymer (BCP), such as poly(styrene)-b-poly(ethylene glycol) or poly(lactic acid)-b-poly(ethylene glycol), is rapidly mixed with a large volume of an anti-solvent stream, usually water, in a defined mixing geometry. The two most commonly used geometries are a two-jet mixing geometry where mixing is driven by turbulence as the two impinging fluid streams collide14 , and a four-jet mixer, the Multi Inlet Vortex Mixer (MIVM), in which mixing


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is driven by turbulent mixing of the four entering jets.15 The attainment of high supersaturations in millisecond time scales13, 16 causes rapid nucleation and growth of the API particles. The API remains in the nanoparticle size range as determined by the nucleation and growth rates and the rate of adsorption of the hydrophobic block of the block copolymer onto the surface of the growing drug particles. Polymer adsorption onto the growing drug particle surface and depletion of dissolved drug from the solution arrests further growth and the adsorbed BCP provides steric stabilization. In this way, uniform sub-micron size distributions have been obtained.

Spray Drying and Nanoparticle Assembly Process (SNAP) Nano-crystalline dispersions of a crystalline drug (termed active pharmaceutical ingredient (API)) were formed by sequential non-solvent precipitation followed by spray drying to rapidly form solid glassy particles approximately 5-10 microns in diameter. Spray drying was used to solidify the suspensions immediately following non-solvent precipitation to arrest crystal growth and trap and stabilize the drug nano-crystals within the glassy matrix that comprises the 5-10 micron particles. Spray drying was done using a custom two-fluid atomizer at the inlet of a custom spray dryer manufactured by Bend Research. The spray dryer outlet temperature was held constant at 45°C, the inlet temperature was a dependent process parameter and averaged 105°C during steady state. The total liquid feed rate was 10 g min-1, and the flow of nitrogen was 470 g min-1. The atomization gas pressure was regulated to 20 psi. A total of approximately 10 g dried material was sprayed for each formulation. This translated to spraying times between 17 min and 40 min depending on the formulation as reported in Table 2. The initial feed solutions were prepared simply by dissolving the pure drug and block copolymer (where necessary) in



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THF and the HPMC in ultrapure water at room temperature. The feed solutions were pumped into the custom mixing and spray dry nozzle by means of a syringe pump.

Scanning Electron Microscope (SEM) Measurements Spray dried powder samples were thinly spread on an adhesive coated SEM stage and tapped to remove excess powder. The samples were sputter coated and imaged with a Hitachi S-3400N SEM. SEM images were obtained at up to 30,000x magnification.

Powder X-Ray Diffraction Powder samples were scanned on a Bruker D8 Advance from 5 to 40°, at 0.017 seconds per step using a Cu Kα radiation source (λ = 1.54 nm). Only data from 2θ = 5 to 30° are shown. To estimate the size of the crystalline domains,17 the peak full width half maxima (FWHM) were extracted from key peaks in the spectrum and analyzed by the Scherrer equation18 to determine the characteristic crystal size: ‫ܦ‬௛௞௟ =

௄ఒ ஻೓ೖ೗ ୡ୭ୱ ఏ

(1)

Here Dhkl is the characteristic crystal size for the Miller indices being analyzed, K is a numerical factor often referred to as a crystalline shape factor (usually 0.9 in the absence of other information), λ is the x-ray wavelength (1.54 nm), Bhkl is the FWHM in radians after subtracting instrumental line broadening, and θ is the Bragg angle. The Scherrer equation was developed for the ideal case of an infinitely narrow and perfectly parallel, monochromatic X-ray beam incident on a monodisperse powder of cube-shaped crystallites.19 Important limitations are that the equation becomes inaccurate for crystal domains below 200 nm and that peak line broadening can result from a number of different factors, including the inherent crystal habit, instrument, 10 ACS Paragon Plus Environment

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and crystal defects.19, 20 We, therefore, present the crystal size as an approximation. The degree of crystallinity was estimated from the crystalline peak area relative to the total peak area of the sample.

Solid-state 13C Nuclear Magnetic Resonance All NMR experiments were carried out on a Bruker Biospin Avance III 300 MHz spectrometer with a wide bore 7 Tesla superconducting magnet operating at frequencies of 75 MHz and 300 MHz for the 13C and 1H Nuclei respectively. Approximately 80 mg of each sample was packed into 4 mm o.d. zirconia rotors with Kel-F® caps and spun to 8 kHz at the magic angle for measurement. The 13C NMR spectra were acquired with a 1H- 13C Hartman-Hahn crosspolarization with a 1 ms contact pulse ramped from 70-100 % for polarization transfer. The 13C and 1H 90-degree pulse lengths were 4 µs and 3.5 µs respectively. Total Suppression of Spinning Sidebands (TOSS) scheme to ensure that the no overlap of the spinning sideband signals with the isotropic peaks, and Spinal-64 1H decoupling scheme with a 75 kHz decoupling field strength were used during acquisition. 1H saturation recovery experiments and 13C CPMAS experiments with varying recycle delays (SI-Figure 1) were conducted to determine the recycle delay sufficient for the full recovery of the 1H signal ( in particular of the crystalline phenytoin), which was found to be 30 s. Accordingly recycle delays of 30 s were used and 256 to 1k transients were signal averaged for sufficient signal-to-noise for the 13C CPMAS experiments. The 13C spectra were deconvoluted and integrated using DMfit software21 to yield the relative fractions of the amorphous and crystalline phenytoin in the samples. The 2D 13C{1H} Heteronuclear – Correlation spectra were acquired with Frequency-Switch Lee -Goldberg (FSLG) scheme with a field strength of 86 kHz, during the 1H evolution time for homonuclear decoupling. The 2D



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spectra were acquired with 96 t1 increments of 43 µs each, 384 transients, and using a recycle delay of 1.5 s that resulted in an experimental time of 15 h for each 2D experiment. The 13C chemical shifts were referenced to Tetramethylsilane (TMS) using glycine as a secondary reference.

Non-Sink Dissolution Tests Fasted State Simulated Intestinal Fluid (FaSSIF) was prepared by adding 0.5% w/w SIF powder into 66 mM phosphate buffered saline (PBS) and stirring at 37˚C for 1 hr. The pH of the media was 6.5.

The SNAP powder material was weighed into 150 mL glass vessels to achieve a loading of 60 µg/mL of phenytoin. The vessels were placed in a USP 2 dissolution apparatus (Agilent Technologies), equipped with small paddles, and eight UV-Vis fiber optic probes with 5 mm path length tips (Pion Rainbow). At the start of the experiment, 100 mL of FaSSIF was added to each vessel and the media was stirred at 200 rpm. The concentration was calculated from a standard curve prepared in FaSSIF, and analyzed from the peak intensity of the second derivative of the UV spectrum at multiple wavelengths between 274 – 278 nm. Calculations were performed in AuPro (Pion). After 120 minutes, 1 mL was removed from each vessel and centrifuged at 300,000g (Beckman Coulter Ultracentrifuge) for 8 minutes. The fiber optic probes were then placed in the supernatant after thorough washing to determine the drug concentration.

Aging stability studies


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SNAP samples were placed in a temperature and humidity controlled environment at 40°C and 50% relative humidity (RH) after the secondary drying sequence (40°C and 40% RH overnight) following production. Prior to this, the samples were stored at 4°C, well below the Tg of phenytoin and HPMC. Powder X-ray diffraction measurements were then taken on the samples after 2, 7 and 22 hours exposure to 40°C and 50% RH aging conditions to assess the physical stability of the materials by measuring physical changes such as crystal size growth.



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Results and Discussion

Nanoparticle formation followed by spray drying: Initial experiments used a two-step process. In step (1), the non-solvent precipitation was conducted by mixing a solution of the phenytoin in THF or acetone with the aqueous non-solvent containing a matrix polymer and in a Multi-Inlet Vortex Mixer (MIVM) (see Liu et al.15 for design specifications). The outlet stream from the MIVM was the feed to the inlet of a spray dryer. In step (2), the suspension of precipitated particles in a solution of matrix polymer was atomized and rapidly dried. In this process, two syringe pumps (1000HL, Isco Teledyne, Lincoln, NE) were connected to the multi inlet vortex mixer. The output from the MIVM was connected to a two-fluid atomizer spray dry nozzle at the inlet of the custom spray dryer manufactured by Bend Research.

These experiments were ultimately unsuccessful in producing nanocrystals less than 200nm. Using this two-step process, the time between precipitation and drying could not be reduced below about 4 seconds and, as a result, the phenytoin crystals in the materials produced were on the order of 1 to 10 microns in size. The growth in particle size arises from Ostwald ripening of the phenytoin nano-crystals during the time between precipitation and spraying. The time required (td) for growth of crystal of radius r can be estimated from the LSW theory:22, 23

td ≈

ℜTr 3 γυ 2 Dc ∞

(2)

where γ is the interfacial energy, υ the molecular volume, D the diffusion coefficient, and c ∞ is the equilibrium solubility in the solvent/non-solvent mixture formed by the precipitation step. The constants for phenytoin are γ = 5.43 × 10-2 N/m, υ = 1.35 × 10-4 m3/mol, and D =2.98 × 10-5 14 ACS Paragon Plus Environment

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cm2/s.15, 24 Using Eq. (2), the characteristic time for particle instability (i.e. disappearance of small particles and growth of larger particles) for a 100 nm particle would be about 0.7 s. The primary reason for such rapid Ostwald ripening is that phenytoin is relatively soluble in the solvent/non-solvent mixture formed by the precipitation process. For example, precipitation of phenytoin (SNAP-BCP-2) was conducted by mixing a solution of 100 mg/ml phenytoin and 100 mg/ml BCP in THF with an aqueous solution of 25 mg/ml HPMC in a 4:1 volume ratio (1:1:1 phenytoin:BCP:HPMC mass ratio). The solubility of crystalline phenytoin in the resulting 20% THF solution is on the order of 0.8 mg/ml. This high solubility, along with the high total phenytoin concentration of 20 mg/ml (overall supersaturation ratio S = Cbulk/Cs = ~20), drives rapid crystal growth.

Integrated nanoparticle formation and spray drying: Therefore, we integrated the mixing chamber into the spray nozzle device as shown in Figure 2. The nozzle was fabricated such that the jet could be moved vertically changing the residence time between mixing the solvent and anti-solvent streams and atomization. However, investigating the impact of this residence time on crystal size and habit was beyond the scope of the current study. For all SNAP experiments, the total flow rate through the nozzle (solvent plus non-solvent flows) was held constant such that the time between mixing and atomization was on the order of approximately 90 ms, calculated from the volumetric flow rate and linear dimensions of the flow path. As we show below, reducing the time between precipitation and drying in this way resulted in solid HPMC based microparticles containing BCP and crystalline phenytoin domains in the sub-200 nm size range, as confirmed by SEM, powder XRD, and magic angle spinning NMR.



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As shown in Figure 2, the solvent stream was introduced into the nozzle head via a 100 µm inner diameter tube while the non-solvent flowed in through the annular space and mixing took place within the nozzle head. The spray dry nozzle was placed at the inlet of the spray dryer. The spray dryer process parameters are described in the Experimental section. Complete spray drying conditions for all experiments conducted are presented in the Supplemental Information (SI-Table 1). The process conditions and compositions for the 4 SNAP materials described herein are presented in Table 2. The supersaturation ratio was varied by changing the flow rate ratio of organic and non-solvent (aqueous) streams fed to the combined mixing and spray head. The total flow rate was held constant to maintain similar mixing dynamics across experiments. The mass ratio of HPMC:API was kept at 1:1 for all 4 SNAP materials and the ratio of block copolymer (BCP) to API was kept at 1:1 for the two materials that contained block copolymer (SNAP-BCP-1 & SNAP-BCP-2).

This process was successful in producing stable spray dried dispersions due to the custom atomizer that brought transfer time below 100 ms and allowed for rapid quenching of crystal growth. Process yields were greater than 50% in all cases, would improve with larger batch sizes and larger scale equipment. For the purposes of this proof-of-concept study, 50% was deemed sufficient.

Importance of BCP to promote complete crystallization As the various characterization results shown below demonstrate, the physical properties of the solid materials made by the SNAP process were quite different when BCP was present versus


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not present in the formulation. When BCP was present (SNAP-BCP-1 and -2), essentially all of the drug (phenytoin) in the resulting materials was present as sub-micron crystals dispersed in larger 2-10 micron HPMC particles. When BCP was not present (SNAP-1 and -2), the majority of the drug remained molecularly dispersed at the nanometer scale in an amorphous form in the HPMC phase.

Results of PXRD and imaging studies The phenytoin XRD data are shown in Figure 4A for SNAP materials made at the lower phenytoin supersaturation ratio (S = 15) and in Figure 4B for SNAP materials made at the higher supersaturation ratio (S = 25). The spectra are displaced vertically for clarity. In each figure, 4A and 4B, the bottom trace is for bulk crystalline phenytoin powder, as received, showing sharp diffraction peaks between 8 and 37°. Comparison of the traces for the SNAP materials in Figure 4A and 4B indicate significant and similar peak broadening for all SNAP materials. This suggests that the crystalline domains are small and relatively insensitive to the supersaturation ratio within the narrow range studied. However, close examination of the intensity of the diffraction peaks as well as the SEM images (Figure 5) indicate that the SNAP materials without BCP made at the higher supersaturation ratio are somewhat more crystalline than those made at the lower supersaturation ratio. No significant dependence of domain size on angle was seen.

Comparison of the SNAP materials with and without BCP (middle XRD trace versus top trace in each Figure 4A and 4B) shows that the phenytoin in the materials is in a very different state.



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Although all of the peaks in the SNAP materials are quite broad relative to those for phenytoin alone, the magnitude of the crystalline peaks is much stronger for the SNAP materials that contain BCP. The weak crystalline diffraction intensity from SNAP materials without BCP relative to those with BCP suggests that a smaller fraction of phenytoin is crystalline in samples prepared without BCP. The diffraction traces for all four SNAP materials also show 2 broad peaks – centered at approximately 10 and 20 degrees 2θ - associated with the amorphous phase of the materials. From the relative intensity of the broad peaks in the traces, we estimate that the fraction of phenytoin that is crystalline in the SNAP materials that do not contain BCP is only about 20-30% of that for the SNAP materials containing BCP.

The diffraction peaks associated with the crystalline state of phenytoin for all 4 SNAP materials are much broader than those for bulk crystalline phenytoin, indicating that the phenytoin crystal domains are at least sub-micron in size, and most likely sub-200 nm. As described in the Methods section, the phenytoin crystal domain size was estimated using the Scherrer equation.18 Specifically, we used the full width half maximum (FWHM) – a measure of peak width – for this estimate. Although this value varies for the different scattering peaks and has significant error in the estimate, the FWHM values for the peaks at 11.4° and 16.5° indicate crystal domain sizes on the order of 100 nm. This estimate is consistent with the size of the rod-like surface crystals with a diameter on the order of 100 nm and a variable length up to approximately one micron seen in the SEM images in Figure 5. Although it’s not clear if these surface crystals represent the habit of all the phenytoin crystals in the sample, close examination of the SEM images in Figure 5 shows that the SNAP materials with BCP appear to have far more of these surface crystals visible on the particle surfaces than the SNAP materials without BCP. This observation is


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consistent with the percent crystallinity of the samples determined by analysis of the NMR and PXRD data, providing additional evidence that these surface crystals are composed of phenytoin. In addition, comparison of the SEM micrographs for the two BCP-containing materials shows more visible crystals for the material made at a higher supersaturation level.

Results of Solid-state NMR studies. As discussed above, with XRD it is difficult to quantify crystallinity if the crystallites are of subten nanometer sizes. 1D and 2D solid state NMR (ssNMR), in contrast, are powerful techniques that have enabled elucidation of the crystallinity,25-27 and molecular interactions leading to nanocrystal inclusions in the SNAP materials. 1D 13C ssNMR provides quantitative information about the amorphous and crystalline fractions of the API in the formulations. The 13C NMR spectra of phenytoin, HPMC and the BCP are shown in Figure 6(a)-(c), respectively, while the assignments of the peaks to the specific carbon sites are shown as insets in Figure 6. An important aspect to note is that all the phenytoin peaks in the neat material (other than the 3 types of protonated aromatic carbons, D,) are sharp and well resolved (Figure 6(a)). In particular the peaks labeled C and C’, representing the aromatic carbons bonded to a common carbon in the phenytoin molecule, are well resolved and can be used for determining the degree of crystallinity of the phenytoin in the nano-crystalline materials. The 13C NMR spectra of the nano-crystalline materials made by SNAP are shown in Figure 6(d)-(g). A close analysis of the phenytoin signal in the spectra indicates that all the distinct phenytoin peaks for the SNAP materials containing no BCP have both a broad and a narrow component, while the phenytoin peaks for the SNAP materials with BCP have only the narrow component. The narrow component corresponds to the crystalline fraction, while the broad component corresponds to the non-crystalline fraction. This



Page 20 of 45

is consistent with the presence of only well-ordered crystalline phenytoin in the BCP SNAP materials, while the phenytoin in the non-BCP SNAP materials is in two states – crystalline and non-crystalline. The broad peaks of the non-crystalline phenytoin are consistent with those of the amorphous state. While all of the phenytoin peaks (A, B, C, C’ and D) of the two-state nonSNAP materials each have both a broad and narrow component, and could be used to quantify the fraction of the crystalline and non-crystalline phenytoin domains, the C/C’ peaks offer the best signal to noise ratio and resolution. We, therefore, fit the spectra with weighted broad and narrow components for the C/C’ peaks as is shown in SI-Figure 2. To ensure accurate results for the fitting, first an exact fit to the C/C’ peaks in the neat phenytoin was carried out. The values for the peak positions and peak widths extracted from the neat phenytoin were held constant for all subsequent fits to the SNAP materials – with and without BCP. For fitting the non-BCP containing materials, a single, broad Gaussian line shape was used to simulate the broad signal of the C/C’ carbons for the non-crystalline (presumably amorphous) phenytoin; and two sharp peaks for the C/C’ carbons obtained for neat crystalline phenytoin were used to simulate the crystalline phenytoin in the SNAP material. The C/C’ peaks in the BCP-containing materials fit well with no added broad component. The best fit to the spectra of the non-BCP materials was obtained with approximately a 30%-70% peak area ratio of crystalline (narrow) to noncrystalline or amorphous (broad) weighting which is consistent with the PXRD measurements. For the BCP SNAP materials, the spectra were best fit with no non-crystalline component added and so we conclude the phenytoin was essentially completely crystalline in these materials. . SIFigure 1(c) also clearly shows with high signal-to-noise 13C NMR that no detectable amorphous phenytoin present in the sample.


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The solid state NMR clearly shows that the presence of the block copolymer promotes the formation of purely crystalline phenytoin domains – known to be sub-micron in size, based on PXRD data and imaging. Two dimensional (2D) 13C-1H HETCOR spectroscopy was used to elucidate the physical proximity and specific molecular interactions present in the SNAP materials. 2D 13C-1H HETCOR provides a significantly increased resolution of the 13C and 1H NMR signals by spreading them into a 2D frequency map. This map separates overlapping signals and enables correlations and nanometer-scale proximities between different specific chemical species to be measured. For example, Figure 7(a) and (b) show the 2D 13C-1H HETCOR contour plots of two SNAP materials, one without BCP (a) and one with BCP (b), with a separately acquired 1D CP-MAS 13C spectrum plotted along the top horizontal axis, and a projection of the 1H signal plotted along the right vertical axis, to aid interpretation of the 2D data. Both of the 2D spectra were acquired under identical conditions, with 2 ms 1H to 13C crosspolarization time, which enables intermolecular correlation of 13C and 1H moieties within a subnanometer scale.

In Figure 7(a), a material without BCP, correlated signal intensity in the 2D spectrum is observed for the intra-molecular contacts from the 1H to 13C species, within the phenytoin (dashed square “I”) and the HPMC (dashed square “II”), as expected. However additional strong signals are observed for intermolecular 1H to 13C contacts, from 1H species of HPMC to the 13C species of the phenytoin (dashed square “III”), and from 1H species of phenytoin to 13C species of the HPMC (dashed square “IV”). We note that the intermolecular and intra-molecular correlation signals have similar intensities, which points to a molecular level dispersion of the phenytoin molecules within the HPMC matrix, as opposed to a non-crystalline or amorphous



Page 22 of 45

phase composed of only phenytoin. In contrast to the material without BCP, the SNAP material with BCP (Figure 7(b)) shows a different pattern for the correlation intensity. First, we note that the same intra-molecular correlation signals for the HPMC and phenytoin are present in Figure 7(b) as observed in Figure 7(a) (the relevant dashed squares have been omitted for clarity). Secondly, we also see the intra-molecular correlation signal for the block copolymer (dashed squares “V”) as expected. Inter-molecular correlation signals from the 1H species of HPMC to the 13C species of BCP (dashed squares “VI”), and from the 1H species of BCP to the 13C species of HPMC (dashed square “VII”) indicate nanometer scale proximity of the BCP and the HPMC. Additionally, a relatively weak intermolecular correlation signal from the 1H species of HPMC to the 13C species of phenytoin (dashed square “VIII”), and from the 1H species of phenytoin to the 13C species of HPMC (dashed square “IX”), also indicate a nanometer scale proximity of the phenytoin and the HPMC. Importantly, no correlation signal between the BCP and phenytoin is observed. SI-Figure 4, which shows a zoomed in plot of the BCP and HPMC sections of Figure 7(b) further illustrates this point.

The physical state of the SNAP materials can be inferred from: 1a) the absence or presence and 1b) the intensity of the intermolecular correlation signals from the 2D NMR and 2) the degree of phenytoin crystallinity indicated by 2a) ssNMR peak shape, 2b) PXRD, and 2c) imaging. As shown in Figures 7(c) and (d). In the SNAP materials without BCP, approximately 70% of the phenytoin remains dispersed at the nanometer or molecular scale within the HPMC matrix. This molecularly dispersed state is the typical structure obtained when a solution of drug and a high Tg polymer are spray dried. This implies that the mixing of the phenytoin solution with a nonsolvent (within the spray head) just prior to atomization allows only about 30% of the phenytoin


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to nucleate and crystallize prior to solidification of the droplets in the spray dryer, and the reduced mobility of un-crystallized phenytoin (as indicated by the high Tg of the HPMC matrix) prevented further crystallization.

The presence of the BCP in the solvent dramatically changes the resulting material, causing essentially all the phenytoin to crystallize as shown in Figure 7(d). The BCP in the materials shows indications in the NMR spectra of strong association with the HPMC. The phenytoin in the BCP-containing materials (essentially completely existing as phase separated nano-crystals) shows no BCP-phenytoin intermolecular correlation signals, which indicates that there is a depletion of the BCP concentration at the phenytoin nanocrystal surface. However, weak intermolecular HPMC-phenytoin correlation signals are observed which are attributed to the preferential interaction of the phenytoin nano-crystal surface with the HPMC. Compared to Figure 7(a), the strength of the correlation signals is weaker as the formation of nanocrystals increases the effective intermolecular distance between the phenytoin and the HPMC, resulting in a reduced intensity of the correlation peaks. A detailed 1H-T1 relaxation analysis of the phenytoin (SI-Fig. 1 and 5) indicate a bimodal distribution of phenytoin crystals in the ~10 nm to 100 nm regime similar to the sizes deduced from the scattering peak widths, with no evidence of any large (micron scale) crystals forming. Importantly, the smaller ~10 nm sized nanocrystals constitute the major fraction of the phenytoin, with sufficient surface area to yield a correlation signal to the HPMC.

It is clear that the end state of the materials is dictated by both thermodynamics (intermolecular interactions and the resulting equilibrium phase behavior) and kinetics (molecular mixing and



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diffusion in the milliseconds after mixing and prior to solidification via spray drying) to form the immobile end state of the material. Characterization of the end product of the SNAP process allows us to develop plausible mechanistic hypotheses about the formation of the material. For SNAP materials that do not contain BCP, the very strong intermolecular correlation signals between HPMC and phenytoin (Figure 7 (a)) point to the phenytoin being molecularly dispersed in the HPMC, which is promoted by a strong interaction mediated by both hydrogen-bonding and hydrophobic interactions between the HPMC and phenytoin. The NMR results indicate a molecular level dispersion of the phenytoin, in a predominantly amorphous state. This means that in the rapid mixing and drying process there is insufficient time for nucleation and crystallization to occur. However, with the addition of BCP in the same spray head essentially complete crystallization occurs, so the molecular interactions between the BCP, API, and HPMC control crystallization even at very short tens of milliseconds time scales.

The explanation of the phenomena is found by considering the NMR data. For the BCPcontaining SNAP materials there are at least two possible scenarios. In the first scenario, the phenytoin partitions into the hydrophobic domains of the BCP micelles following mixing, where it concentrates and has sufficient mobility during drying to nucleate and form nanocrystals. The crystals are sub-micron in size, due to the enhanced rate of nucleation. They remain that size due to essentially all of the phenytoin having been removed from solution by crystallization (no remaining dissolved or dispersed phenytoin). The ultimate stability of the formulation comes from the nanocrystals being encapsulated in the glassy, high Tg HPMC. In this scenario, as the phenytoin crystals would nucleate within the BCP phase, it would be expected that the phenytoin nanocrystals in the final material would be in close proximity to the BCP. Therefore, we would


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expect to see a correlation signal between phenytoin and BCP in the 2D solid state NMR. The absence of this correlation signal argues against this first scenario.

What we actually observe is the second scenario, where the BCP interacts preferentially with the HPMC in a way that reduces the phenytoin-HPMC interaction. This enables phenytoin crystallization, and HPMC stabilization of those entrapped nano-crystals. The evidence for this preferential interaction is given by the 2D NMR (Figure 7 (b)), where strong intermolecular correlation signals between the BCP and HPMC are, indeed, observed. The suppression of the phenytoin-HPMC interactions allows the phenytoin to nucleate crystals more rapidly and fully crystallize prior to solidification.

Finally, the stability of the BCP-containing materials, relative to conventional SDD materials, is likely due to the absence of any remaining amorphous phenytoin (unlike the non-BCP samples which contain 70% amorphous phenytoin). Our experience is that once a compound like phenytoin is completely in the crystalline state and the crystals are dispersed in a matrix in which they have very little solubility and mobility, that they have little tendency to grow in size via Ostwald ripening. However, in the case of the non-BCP materials, where a large amount of amorphous phenytoin remains dispersed in the surrounding matrix, this dispersed material more readily diffuses and crystallizes on the crystals present, in order to further reduce the free energy of the system. The absence of amorphous phenytoin is proven further by SI-Figures 1(c) and 3.

Results of aging or stability studies



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A major benefit from utilizing a formulation containing nano-crystalline phenytoin would be that physical stability for a given formulation would be expected to be improved, relative to the same formulation in an amorphous state, when held at or near the Tg of the amorphous phase. To assess the physical stability of the SNAP materials, they were subjected to elevated temperature (40 °C) and moisture (50% relative humidity (RH)). This was to show the propensity of the drug to Ostwald ripen or change states over time. The conditions and duration imposed are intended to assess formulation risk and do not indicate 2-year shelf stability. A longer accelerated study that is indicative of 2-year shelf stability that investigates both chemical and physical stability is beyond the scope of this study. Under these conditions, the Tg of phenytoin is not expected to change appreciably from 71°C, given its logP of 2.1, while the Tg of HPMC drops from 140°C to approximately 84°C (see Table 1). PXRD traces for the 4 SNAP materials are shown in Figure 8 as a function of the time of exposure to 40°C and 50% RH. Little change in the PXRD traces is observed for the 2 SNAP materials containing BCP. However, for the two materials that do not contain BCP, the scattering peaks associated with crystalline phenytoin become sharper and grow in intensity as a function of exposure time. This suggests that exposure to these conditions causes any amorphous phenytoin present in the sample to slowly crystallize. As essentially all of the phenytoin in the BCP-containing formulations was already crystalline, no change was observed and indeed, the materials would be expected to have good shelf life stability.


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Results of dissolution studies A critical property for nano-crystalline dispersions to perform well in vivo is their rate of dissolution. In general, the maximum relative absorption rate constant for a high permeability drug* is about 0.10 min-1.28-31 Thus, to be assured that the dissolution rate does not limit drug absorption, the drug should dissolve up to about 80% of its solubility limit in about 5 minutes or less. Figure 9 shows the dissolved phenytoin concentration as a function of time in the dissolution media. Clearly the nano-crystalline SNAP materials containing BCP dissolve very rapidly – reaching greater than 80% of their limiting concentration in only 2 to 3 minutes. The other materials dissolve much more slowly. The somewhat higher final concentration of dissolved phenytoin for the samples containing BCP can be attributed to phenytoin partitioning into the BCP micelles – the phenytoin in the micelles effectively adding to the truly dissolved phenytoin.

Discussion The goal of integrating nanoparticle formation with spray drying is to provide a way to form a stable solid form of nano-crystals of a highly crystalline and somewhat aqueous soluble drug like phenytoin. The aqueous solubility and logP of phenytoin place it in the ‘Nanocrystal’ or ‘Amorphous’ regions of the oral delivery formulation map in Figure 1, where nanocrystalline or amorphous formulations are likely to be applicable to maximize stability and absorption potential. This is achieved by initiating crystal nucleation, followed by rapid quenching of crystal

*

The references provided calculate the maximum absorbable dose (MAD) in two different ways. Curatolo (1998) define it as MAD = S × KA × V × T while Butler and Dressman (2010) define it as MAD = Peff × S × A × T where Peff is the effective human jejunal permeability (cm/s), S is the solubility (mg/cm3), KA is the absorption rate constant (min-1), V is the fluid volume (250 mL), A is the absorption surface area and T is the small intestine transit time (value varies - approx. 199 mins from Dressman and 270 mins from Curatolo). This allows conversion between Peff and KA as: KA = Peff × A/V × 60 [s/min].



Page 28 of 45

growth, to trap the crystal at a sub-micron in size by encapsulation in the polymer matrix. The final solid nano-crystalline powder would be shelf stable, and would allow the nano-crystals of drug to rapidly disperse back to a nano-crystalline dispersion and ultimately rapidly dissolve, when dosed in an aqueous medium, such as actual or simulated intestinal fluid. We chose to promote powder stability and dispersability by adding a water soluble, glassy polymer to the formulation, such that in the dry particles formed, the drug nano-crystals would be dispersed in the glassy, water soluble polymer matrix. We note that although we did not study dispersability directly in Figure 9, the dispersability was sufficient such that differences in dissolution rate from increased crystal surface area were detected.

We succeeded in making such nano-crystalline dispersions of the drug phenytoin via the SNAP process by (1) keeping the time between precipitation and atomization on the order of 100 ms, (2) including the block copolymer (BCP) poly(lactic acid)-b-poly(ethylene glycol) in the formulation and (3) including the glassy water soluble matrix material hydroxypropylmethyl cellulose (HPMC) in the formulation. This process results in a spray dried powder that can be integrated into an oral solid dosage formulation such as a tablet or capsule.

Conclusions The SNAP process succeeded in forming stable and rapidly dissolving nano-crystalline dispersions of phenytoin. Key to successfully making these dispersions was: 1) Having a water soluble high Tg matrix material in the formulation; 2) Having an amphiphilic block copolymer present in the formulation; and


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3) Keeping the time between non-solvent precipitation and solidification via spray drying extremely short – on the order of 100 ms. A special nozzle that combines precipitation and suspension atomization was critical to achieving this short time. The SNAP approach enables the production spray dried dispersions of relatively soluble, but strongly crystalline drug APIs, which was not previously possible. This enables formulations of compounds in Region 3 of the spray dried dispersion map in Figure 1.

The key techniques that enable interpretation of the experimental results are 1D and 2D solidstate NMR. 1D solid state NMR allows quantification of amorphous and crystalline fractions in the formulations when the nano-crystalline domains may be smaller than 10 nm. The 2D solid state NMR is able to identify the molecular interactions between the drug, the block copolymer, and the HPMC. In the surprising result, the block copolymer does not associate with the API, as it would in normal Flash NanoPrecipitaiton nanoparticle formation. Rather, the block copolymer is specifically associating with the HPMC through hydrophobic association. This permits API crystallization, uninhibited by the HPMC. The result is stable nano-crystals embedded in the high Tg HPMC matrix. Though the results shown here are very encouraging, further studies will be required to: 1) Understand the generality of the results for other API’s, block copolymers, and HPMCs 2) Understand the range of Tc / Tg values and logP values for which SNAP enables nanocrystallization of APIs 3) Understand the impact of process variables such as total solid concentration, supersaturation ratio and drug solubility in the post-precipitation solvent/non-solvent mixture on the resulting materials.



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Supporting Information: SI-Table 1: Complete set of formulation conditions. SI-Figure 1: (a) The normalized 1H NMR intensity plotted as a function of recovery time. (b) A series of 13C CPMAS experiments measured with increasing recycle delays upto 30 s (bold line in red) measured for the HPMC-BCP-Phenytoin, zoomed in on the C/C’ region of the Phenytoin, showing full signal after 30 s. (c) The high signal to noise of the 13C spectrum zoomed into C/C’ region (SINO of 148:1 for the C/C’ peaks) of the HPMC-BCP-Phenytoin, with fits (thin lines are the individual components of the fits, dashed line is the overall fit) demonstrating that there is no detectable amorphous phenytoin in the system. SI-Figure 2: Fits to the C and C’ peaks (according to assignment in Figure 6) of the 13C solid state NMR of samples tested. SI-Figure 3: (a) High resolution 2D 13C{1H}HETCOR of HPMC-BCP-Phenytoin, with the VIII and IX correlation peaks identified. (b) and (c) are the 1D 13C slices extracted at VIII and XI which show the 13C signals associated with the respective correlation peaks. (d) Red spectrum is a zoomed in view of the C/C’ region of part (c) showing two distinct signals identifying that the correlation signal is indeed from the HPMC to the crystallinePhenytoin. SI-Figure 4: Zoomed in view of the 2D 13C-1H HETCOR of the HPMC-BCPPhenytoin system. SI-Figure 5 - Recovery of the phenytoin 13C signal as a function of the 1H saturationrecovery times. The stacked spectra are the 13C signals of the phenytoin labelled with the 1 H recovery time. Acknowledgements: We would like to acknowledge financial support from the Princeton University Innovation Fund and the Princeton University IP Accelerator Fund.


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Figures and Tables

Figure 1 - Concept bioavailability enhancement technology map based on drug physicochemical properties at a fixed dose. The graphic plots drug aqueous solubility with respect to log P for a range of drug compounds previously evaluated at Capsugel, with subsequent overlay of the optimal space(s) for nanocrystal, amorphous (including spray-dried dispersions and hot-melt extrusion) and lipid-based technologies at a standardized 100 mg dose (per dosage unit) of active.



Table 1: Drug and excipient properties Compound MW Tm (°C) Phenytoin HPMC E3 PLA-b-PEG

252.3 -4.2k-5k

295-298 ---

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Tg (°C)

Tm/Tg

logP

71 140 --

1.7 ---

2.5 ---

Aqueous Solubility (mg/L at 22°C) 32 ---


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Step 2

Step 1

Step 3

Step 4

Figure 2 – SNAP process schematic and nozzle geometry. The spray nozzle used was a twofluid atomizer modified by inserting a 0.25 mm inner diameter tubing into the central fluid delivery space. A THF mixture containing the API and block copolymer was delivered through the inner tube while an aqueous stream was delivered through the annular space. Steps 1-4 from the Introduction are represented visually here (steps 1 and 2 described in the Figure, step 3: mixing of solvent and anti-solvent streams, step 4: atomization of mixed stream).



3.5x10

4

3.0x10

4

2.5x10

4

2.0x10

4

1.5x10

4

1.0x10

4

5.0x10

3

B 4

10

3

10

2

10

10

LOG

100

0.0 0

20

40

60

80

100

1.6x10

5 5

10

5

1.4x10 1.2x10

5

10

4

1.0x10

5

10

3

8.0x10

4

10

2

6.0x10

4

4.0x10

4

2.0x10

4

LOG

4

Phenytoin Solubility (µg/mL)

4.0x10

LOG

A Phenytoin Solubility (µg/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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10

LOG

100

0.0 0

20

Acetone vol%

40

60

80

100

THF vol%

Figure 3 – Solubility data for phenytoin in mixtures of (A) acetone-water and (B) THF-water at room temperature (■) and 37°C (●). Excluding the pure water data point, the data fit power law relationships: log([phenytoin]) = 3.39log([THF]) – 1.51, R2 = 0.98 and log([phenytoin]) = 2.79log([Acetone]) – 1.11, R2 = 0.99 (both curves at room temperature). The temperature dependence of the solubility curves is insignificant for THF while there is discernible difference in solubility at RT vs 37°C for phenytoin in acetone (approximately a 2.6% difference in slopes). Phenytoin shows a 4.5x higher solubility in pure THF than pure acetone.


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Table 2: Process conditions and compositions for SNAP materials. Name

SNAP-1

Solvent Composition PTN* BCP (mg/ml) (mg/ml) 100 0

Non-solvent Composition HPMC (mg/ml) 33.3

Solvent:No n-solvent THF:water (by volume)

Supersaturatio n Ratio

1:3

15

Solid State Composition (by mass) PTN* HPMC BCP (wt %) (wt %) (wt %) 50 50 0

SNAP-2

100

0

25

1:4

25

50

50

0

SNAPBCP-1 SNAPBCP-2

100

100

33.3

1:3

15

33.3

33.3

33.3

100

100

25

1:4

25

33.3

33.3

33.3

*PTN = phenytoin



140000

A

120000

Counts

100000 80000 60000 40000 20000 0 4

8

12

16

20

24

28

32

36

40

24

28

32

36

40

2θ 140000

B

120000 100000

Counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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80000 60000 40000 20000 0 4

8

12

16

20

2θ Figure 4 – Powder X-ray diffractograms of phenytoin SNAP with and without block copolymer (A: in order from bottom to top: ▬ phenytoin, ▬ SNAP-1, ▬ SNAP-BCP-1, B: in order from bottom to top: ▬ phenytoin, ▬ SNAP-2, ▬ SNAP-BCP-2).


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A

C

B

50 um

D

10 um

F

E

A

10 um

C

10 um

10 um

H

G

1 um

I

B

J

L

K

10 um

M

1 um

3 um

10 um

5 um

2 um

N

30 um

10 um

Figure 5 – SEM micrographs of SNAP-1 (A-C), SNAP-BCP-1 (D-F), SNAP-2 (G-I), SNAPBCP-2 (J-L) and control samples pure phenytoin (M) and pure spray dried HPMC (N). Scale bars as indicated.



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Figure 6 – 13C Solid state NMR. – 13C CPMAS solid-state NMR with total suppression of spinning sidebands (TOSS) of (a) phenytoin (with an inset of its structure), (b) HPMC (with an inset of its structure) (c) PLA-PEG block co-polymer (with an inset of its structure), (d) SNAP1, (e) SNAP-2, (f) SNAP-BCP-1 and (g) SNAP-BCP-2. The peaks are labelled according to the assignment in the insets. The peaks labelled with a subscript ‘a’ correspond to the signal of the amorphous phenytoin in the mixture. Inset in (a): chemical structures of phenytoin, inset in (b) HPMC (where R = H or CH3 or CH3CH(OH)CH3), and inset in (c) PLAx-b-PEGy block copolymer. Labels on the molecular structures from A-N correspond to the peak assignment in the 13C NMR spectra. 38 ACS Paragon Plus Environment

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Figure 7 – 2D 13C{1H} HETCOR NMR of (a) SNAP without BCP (phenytoin:HPMC; 80:20) and (b) SNAP with BCP (Phenytoin:BCP:HPMC; 43:43:14). The dashed squares labelled I, II and V indicate the intramolecular correlation signals while the dashed squares labelled III, IV, VI, VII, VIII and IX identify the intermolecular correlation signals. III: H-HPMC/C-Phenytoin; IV: H-Phenytoin/C-HPMC; VI: H-BCP/C-HPMC; VII: H-HPMC/C-BCP; VIII: H-HPMC/CPhenytoin; IX: H-Phenytoin/C-HPMC. Inset 7(c) and (d) are cartoon representations of the structures of the materials without and with the BCP respectively. In color version of the figure (available in the online version) the green dots represent the molecularly dispersed phenytoin, the blue hexagon represents the crystalline phenytoin, the red lines represent the HPMC and the two toned lines represent the block copolymer.



A

14000 12000

CPS

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B

12000 10000 8000

CPS

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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18000

C

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CPS

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D

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CPS

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10000 8000 6000 4000 2000 0 5

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30

2θ

Figure 8 – Impact of elevated temperature and humidity on the PXRD traces for the four SNAP materials: SNAP-1 (A) and SNAP-2 (B) have no block copolymer, and SNAP-BCP-1 (C) and SNAP-BCP-2 (D) have PLA-b-PEG block copolymer. In each graph, the bottom-most spectrum is that of pure phenytoin and the curves in ascending order are: t = 0 (no exposure to heat and humidity), t = 2 hrs, t = 7 hrs, and t = 22 hrs at 40°C and 50% RH.



50

Concentration (µg/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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40 30 20 10 0 0

20

40

60

80

100

120

Time (mins) Figure 9 – Optic fiber probe dissolution test results: phenytoin concentration versus time for SNAP and control materials (● phenytoin API | ○ 50/50 physical mixture of phenytoin and HPMC | ■ SNAP-1 | □ SNAP-BCP-1 | ▲ SNAP-2 | ∆ SNAP-BCP-2). SNAP-BCP samples show fast burst release (80% dissolution and release within 3 minutes) and higher sustained concentrations compared to SNAP-no BCP and control samples, due to the formation of BCP micelles. Gap in data to improve symbol readability. Starting concentration of phenytoin 60 µg/mL in all cases. .


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TOC graphic: SNAP materials with block copolymer show increased surface crystallinity 82x44mm (256 x 256 DPI)

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Millisecond Self Assembly of Stable Nano-Dispersed Drug

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