An example of how to handle amorphous fractions in API during early pharmaceutical development: SAR114137 – A successful approach

The so-called pharmaceutical solid chain, which encompasses drug substance micronisation to the final tablet production, at pilot plant scale is presented as a case study for a novel, highly potent, pharmaceutical compound: SAR114137. Various solid-state analytical methods, such as solid-state Nuclear Magnetic Resonance (ssNMR), Differential Scanning Calorimetry (DSC), Dynamic Water Vapour Sorption Gravimetry (DWVSG), hot-stage Raman spectroscopy and X-ray Powder Diffraction (XRPD) were applied and evaluated to characterise and quantify amorphous content during the course of the physical treatment of crystalline active pharmaceutical ingredient (API). DSC was successfully used to monitor the changes in amorphous content during micronisation of the API, as well as during stability studies. ¹⁹F solid-state NMR was found to be the method of choice for the detection and quantification of low levels of amorphous API, even in the final drug product (DP), since compaction during tablet manufacture was identified as a further source for the formation of amorphous API. The application of different jet milling techniques was a critical factor with respect to amorphous content formation. In the present case, the change from spiral jet milling to loop jet milling led to a decrease in amorphous API content from 20–30 w/w% to nearly 0 w/w% respectively. The use of loop jet milling also improved the processability of the API. Stability investigations on both the milled API and the DP showed a marked tendency for recrystallisation of the amorphous API content on exposure to elevated levels of relative humidity. No significant impact of amorphous API on either the chemical stability or the dissolution rate of the API in drug formulation was observed. Therefore, the presence of amorphous content in the oral formulation was of no consequence for the clinical trial phases I and II.     

Dielectric Relaxation Study on Tramadol Monohydrate and Its Hydrochloride Salt

Dielectric relaxation measurements as well as differential scanning calorimetry and X-ray diffraction investigations were performed on tramadol monohydrate and its hydrochloride salt. Examined samples do not crystallize during cooling and in consequence they reach the glassy state. In the case of the hydrochloride tramadol we are able to monitor α-relaxation process despite large contribution of dc conductivity to the loss spectra. It is the first such study on the salt of the drug. Up to now the dielectric spectroscopy has been regarded as useless in measuring such kind of API (active pharmaceutical ingredient). In this paper we also made some suggestions about the nature of the secondary relaxations in the amorphous tramadol monohydrate and its salt. The knowledge about the molecular mechanisms, which govern the observed secondary relaxations seems to be the key in predicting the stability of the amorphous form of the examined API. Finally additional dissolving measurements on the amorphous and crystal tramadol hydrochloride were performed. As a result we understood that dissolution properties of the amorphous form of the considered drug are comparable to those of crystalline one. However, we have found out that amorphous tramadol hydrochloride has greater ability to form tablets than its crystalline equivalent. This finding shows that amorphous drugs can be alternative even for the freely solved pharmaceuticals such as tramadol hydrochloride, because the former one has better ability to form tablets. It implies that during tabletting of the amorphous drugs there is no need to use any excipients and chemicals improving compaction properties of the API. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:94–106, 2010     

Measuring the Rate of In-vitro Drug Release From Polymeric Nanoparticles by 19F Solution State NMR Spectroscopy

We report what we believe is the first use of ¹⁹F NMR spectroscopy to directly measure in-vitro release (IVR) from polymeric nanoparticles (PNPs). Using ¹⁹F NMR we selectively measured IVR of AZD2811 from PNPs. Due to rapid nuclear relaxation in solid-like environments only AZD2811 in solution is detected, and physical separation from the PNPs isn't required. The NMR approach and ultra-centrifugation/UHPLC were shown to be equivalent. The selectivity of ¹⁹F NMR means it is readily applied to complex IVR media such as recombinant human serum albumin (rHSA).     

Process Development and Robust Control of Physical Attributes of an Amorphous Drug Substance

Control of physical attributes of amorphous active pharmaceutical ingredients (APIs) can be challenging due to processability issues, their wide variation during processing and the requirement to control them to specific ranges. In this article, we report our efforts to develop a robust isolation process for boceprevir, which delivers specific surface areas between 3.0 and 9.4 m²/g. We developed mechanistic process understanding by utilizing a new method to measure glass transition temperature of API suspensions. Boceprevir processability and surface area are determined by the interplay between the suspension operating conditions and glass transition temperature. Processing time and thermal history also influence API surface area evolution, rendering a dynamic nature to it. A control strategy was developed consisting of 2 elements: a continuous tee mixer precipitation process, which delivers API in the 40-60 m²/g range and 1 of 2 annealing step variants. In the first, surface area was controlled in 4 batches to 6.7-7.5 m²/g by equilibrating the API suspension above its glass transition temperature and a subsequent vacuum distillation. The second annealing variant controlled surface area to 4.5-6.5 m²/g for over 70 commercial batches through a dynamic vacuum distillation step with prescribed temperature and % batch volume distilled profiles versus time.     

Solid-state nuclear magnetic resonance study of the physical stability of electrospun drug and polymer solid solutions

A major challenge in utilizing the amorphous form of an active pharmaceutical ingredient (API) in a final oral dosage form is preventing crystallization over time and ensuring stability. One method to improve stability is lowering the mobility of an API by formulating as a solid solution with an excipient. In this work, we use electrospinning to prepare solid solutions of API, aliskiren (SPP) or indomethacin (IND), and a polymer, polyvinylpyrrolidone (PVP). The stability of the solid solutions over 6-month storage in a desiccator at 40 °C was investigated. Using X-ray diffraction and differential scanning calorimetry, it was determined that no crystals were present in the four formulations tested--1:1 SPP-PVP, 4:1 SPP-PVP, 1:1 IND-PVP, and 2:1 IND-PVP at any time. Solid-state nuclear magnetic resonance relaxation time measurements were used to determine whether phase separation of the API and polymer occurred during the study period. It was found that all formulations remained homogeneous down to at least a 2-10 nm length scale, indicating that for these APIs, electrospinning is an acceptable method for forming stable amorphous solid solutions.     

Evaluation of drug physical form during granulation, tabletting and storage

An active pharmaceutical ingredient (API) was found to dissociate from the highly crystalline hydrochloride form to the amorphous free base form, with consequent alterations to tablet properties. Here, a wet granulation manufacturing process has been investigated using in situ Fourier transform (FT)-Raman spectroscopic analyses of granules and tablets prepared with different granulating fluids and under different manufacturing conditions. Dosage form stability under a range of storage stresses was also investigated. Despite the spectral similarities between the two drug forms, low levels of API dissociation could be quantified in the tablets; the technique allowed discrimination of around 4% of the API content as the amorphous free base (i.e. less than 1% of the tablet compression weight). API dissociation was shown to be promoted by extended exposure to moisture. Aqueous granulating fluids and manufacturing delays between granulation and drying stages and storage of the tablets in open conditions at 40 degrees C/75% relative humidity (RH) led to dissociation. In contrast, non-aqueous granulating fluids, with no delay in processing and storage of the tablets in either sealed containers or at lower temperature/humidity prevented detectable dissociation. It is concluded that appropriate manufacturing process and storage conditions for the finished product involved minimising exposure to moisture of the API. Analysis of the drug using FT-Raman spectroscopy allowed rapid optimisation of the process whilst offering quantitative molecular information concerning the dissociation of the drug salt to the amorphous free base form.     

Raman spectroscopy for the in-line polymer-drug quantification and solid state characterization during a pharmaceutical hot-melt extrusion process

The aim of this study was to evaluate the suitability of Raman spectroscopy as a Process Analytical Technology (PAT) tool for the in-line determination of the active pharmaceutical ingredient (API) concentration and the polymer-drug solid state during a pharmaceutical hot-melt extrusion process.For in-line API quantification, different metoprolol tartrate (MPT) - Eudragit® RL PO mixtures, containing 10%, 20%, 30% and 40% MPT, respectively, were extruded and monitored in-line in the die using Raman spectroscopy. A PLS model, regressing the MPT concentrations versus the in-line collected Raman spectra, was developed and validated, allowing real-time API concentration determination. The correlation between the predicted and real MPT concentrations of the validation samples is acceptable (R² = 0.997). The predictive performance of the calibration model is rated by the root mean square error of prediction (RMSEP), which is 0.59%.Two different polymer-drug mixtures were prepared to evaluate the suitability of Raman spectroscopy for in-line polymer-drug solid state characterization. Mixture 1 contained 90% Eudragit® RS PO and 10% MPT and was extruded at 140 °C, hence producing a solid solution. Mixture 2 contained 60% Eudragit® RS PO and 40% MPT and was extruded at 105 °C, producing a solid dispersion. The Raman spectra collected during these extrusion processes provided two main observations. First, the MPT Raman peaks in the solid solution broadened compared to the corresponding solid dispersion peaks, indicating the presence of amorphous MPT. Second, peak shifts appeared in the spectra of the solid dispersion and solid solution compared to the physical mixtures, suggesting interactions between Eudragit® RS PO and MPT, most likely hydrogen bonds. These shifts were larger in the spectra of the solid solution. DSC analysis confirmed these Raman solid state observations and the interactions seen in the spectra. Raman spectroscopy is a potential PAT-tool for in-line determination of the API concentration and the polymer-drug solid state during pharmaceutical hot-melt extrusion.     

Addition of Cationic Surfactants to Lipid-Based Formulations of Poorly Water-Soluble Acidic Drugs Alters the Phase Distribution and the Solid-State Form of the Precipitate Upon In Vitro Lipolysis

It has been previously shown that the interaction of some weakly basic drugs with oppositely charged fatty acids during digestion can influence the solid-state form of the drug if it precipitates. The present study hypothesized the opposite effect for weakly acidic drugs. Tolfenamic acid (TA) and an oppositely charged cationic surfactant, didodecyldimethylammonium bromide (DDAB) were combined in a model medium chain lipid formulation. The phase distribution upon in vitro lipolysis was determined using HPLC and the solid-state form of precipitated TA was determined using X-ray diffraction and crossed polarized light microscopy. TA precipitated in a different polymorphic crystalline form to the starting reference material in the absence of DDAB but precipitated in an amorphous form when DDAB was included in the same formulation. The solubility of TA upon dispersion and digestion of the formulation was considerably higher in the presence of DDAB. The findings point to ionic interactions between TA and DDAB as the reason for the improved drug solubility throughout digestion, and precipitation of drug in an amorphous salt form, analogous to what has been observed in the past for some poorly water-soluble weakly basic drugs with anionic co-formers.     

In Situ Dehydration of Carbamazepine Dihydrate: A Novel Technique to Prepare Amorphous Anhydrous Carbamazepine

The purposes of this project were to prepare amorphous carbamazepine by dehydration of crystalline carbamazepine dihydrate, and to study the kinetics of crystallization of the prepared amorphous phase. Amorphous carbamazepine was formed and characterized in situ in the sample chamber of a differential scanning calorimeter (DSC), a thermogravimetric analyzer (TGA), and a variable temperature x-ray powder diffractometer (VTXRD). It has a glass transition temperature of 56°C and it is a relatively strong glass with a strength parameter of 37. The kinetics of its crystallization were followed by isothermal XRD, under a controlled water vapor pressure of 23 Torr. The crystallization kinetics are best described by the three-dimensional nuclear growth model with rate constants of 0.014, 0.021, and 0.032 min¹ at 45, 50, and 55°C, respectively. When the Arrhenius equation was used, the activation energy of crystallization was calculated to be 74 kJ/mol in the presence of water vapor (23 Torr). On the basis of the Kissinger plot, the activation energy of crystallization in the absence of water vapor (0 Torr water vapor pressure) was determined to be 157 kJ/mol. Dehydration of the dihydrate is a novel method to prepare amorphous carbamazepine; in comparison with other methods, it is a relatively gentle and effective technique.     

Mechanism of action of AZD0865, a K+-competitive inhibitor of gastric H+,K+-ATPase

AZD0865 is a member of a drug class that inhibits gastric H⁺,K⁺-ATPase by K⁺-competitive binding. The objective of these experiments was to characterize the mechanism of action, selectivity and inhibitory potency of AZD0865 in vitro. In porcine ion-leaky vesicles at pH 7.4, AZD0865 concentration-dependently inhibited K⁺-stimulated H⁺,K⁺-ATPase activity (IC₅₀ 1.0 ± 0.2 μM) but was more potent at pH 6.4 (IC₅₀ 0.13 ± 0.01 μM). The IC₅₀ values for a permanent cation analogue, AR-H070091, were 11 ± 1.2 μM at pH 7.4 and 16 ± 1.8 μM at pH 6.4. These results suggest that the protonated form of AZD0865 inhibits H⁺,K⁺-ATPase. In ion-tight vesicles, AZD0865 inhibited H⁺,K⁺-ATPase more potently (IC₅₀ 6.9 ± 0.4 nM) than in ion-leaky vesicles, suggesting a luminal site of action. AZD0865 inhibited acid formation in histamine- or dibutyryl-cAMP-stimulated rabbit gastric glands (IC₅₀ 0.28 ± 0.01 and 0.26 ± 0.003 μM, respectively). In ion-leaky vesicles at pH 7.4, AZD0865 (3 μM) immediately inhibited H⁺,K⁺-ATPase activity by 88 ± 1%. Immediately after a 10-fold dilution H⁺,K⁺-ATPase inhibition was 41%, indicating reversible binding of AZD0865 to gastric H⁺,K⁺-ATPase. In contrast to omeprazole, AZD0865 inhibited H⁺,K⁺-ATPase activity in a K⁺-competitive manner (K_i 46 ± 3 nM). AZD0865 inhibited the process of cation occlusion concentration-dependently (IC₅₀ 1.7 ± 0.06 μM). At 100 μM, AZD0865 reduced porcine renal Na⁺,K⁺-ATPase activity by 9 ± 2%, demonstrating a high selectivity for H⁺,K⁺-ATPase. Thus, AZD0865 potently, K⁺-competitively, and selectively inhibits gastric H⁺,K⁺-ATPase activity and acid formation in vitro, with a fast onset of effect.     

Exploring the feasibility of the use of biopolymers as a carrier in the formulation of amorphous solid dispersions – Part I: Gelatin

Biopolymers have rarely been used so far as carriers in the formulation of amorphous solid dispersions (ASD) to overcome poor solubility of active pharmaceutical ingredients (APIs). In an attempt to enlarge our knowledge on this topic, gelatin, type 50PS was selected. A screening study was initiated in which twelve structurally different poorly soluble biopharmaceutical classification system (BCS) Class II drugs (carbamazepine, cinnarizine, diazepam, itraconazole, nifedipine, indomethacin, darunavir (ethanolate), ritonavir, fenofibrate, griseofulvin, ketoconazole and naproxen) were selected for evaluation. Solid dispersions of five different drug loadings of these twelve compounds were prepared by lyophilization and evaluated for their solid state properties by mDSC and XR(P)D, and in vitro dissolution performance. Even without any process optimization it was possible to form either fully amorphous or partially amorphous systems, depending on the API and API to carrier ratio. Hence in this respect, gelatin 50PS behaves as any other carrier. Dissolution of the API from the solid dispersions significantly exceeded that of their crystalline counterparts. This study shows the potential of gelatin as a carrier to formulate amorphous solid dispersions.     

Characterization of Hydrochloride and Tannate Salts of Diphenhydramine

Proper characterization is an important aspect of any dosage form design. The objective of this work was to characterize tannate salt and hydrochloride salt of diphenhydramine. As a part of characterization studies, Differential scanning calorimetry was used to investigate thermal effects and nature of salts, supported by X-ray powder diffraction. Scanning electron microphotographs was used to surface topography of salts of diphenhydramine. Fourier-transform infrared spectroscopy, solubility study and flowability studies were carried out as part of characterization. Differential scanning calorimetry and X-ray powder diffraction studies indicated amorphous nature of the tannate while hydrochloride salt has crystalline properties. Scanning electron microphotographs indicated the differences in surface topography between both the salts. Solubility studies at different pH showed pH dependant solubility of both the salts and less solubility of tannate. Stability of bulk drug at accelerated conditions of 40^°/75% RH was determined for both salts. Good stability of both salts was observed.     

Following the surface response of caffeine cocrystals to controlled humidity storage by atomic force microscopy

Active pharmaceutical ingredient (API) stability in solid state tablet formulation is frequently a function of the relative humidity (RH) environment in which the drug is stored. Caffeine is one such problematic API. Previously reported caffeine cocrystals, however, were found to offer increased resistance to caffeine hydrate formation. Here we report on the use of atomic force microscopy (AFM) to image the surface of two caffeine cocrystal systems to look for differences between the surface and bulk response of the cocrystal to storage in controlled humidity environments. Bulk responses have previously been assessed by powder X-ray diffraction. With AFM, pinning sites were identified at step edges on caffeine/oxalic acid, with these sites leading to non-uniform step movement on going from ambient to 0% RH. At RH >75%, areas of fresh crystal growth were seen on the cocrystal surface. In the case of caffeine/malonic acid the cocrystals were observed to absorb water anisotropically after storage at 75% RH for 2 days, affecting the surface topography of the cocrystal. These results show that AFM expands on the data gathered by bulk analytical techniques, such as powder X-ray diffraction, by providing localised surface information. This surface information may be important for better predicting API stability in isolation and at a solid state API–excipient interface.     

Use of a Glutaric Acid Cocrystal to Improve Oral Bioavailability of a Low Solubility API

Purpose The bioavailability of a development candidate active pharmaceutical ingredient (API) was very low after oral dosing in dogs. In order to improve bioavailability, we sought to increase the dissolution rate of the solid form of the API. When traditional methods of forming salts and amorphous material failed to produce a viable solid form for continued development, we turned to the non-traditional approach of cocrystallization.Methods A crystal engineering approach was used to design and execute a cocrystal screen of the API. Hydrogen bonding between the API and pharmaceutically acceptable carboxylic acids was identified as a viable synthon for associating multiple components in the solid state. A number of carboxylic acid guest molecules were tested for cocrystal formation with the API.Results A cocrystal containing the API and glutaric acid in a 1:1 molecular ratio was identified and the single crystal structure is reported. Physical characterization of the cocrystal showed that it is unique regarding thermal, spectroscopic, X-ray, and dissolution properties. The cocrystal solid is nonhygroscopic, and chemically and physically stable to thermal stress. Use of the cocrystal increased the aqueous dissolution rate by 18 times as compared to the homomeric crystalline form of the drug. Single dose dog exposure studies confirmed that the cocrystal increased plasma AUC values by three times at two different dose levels.Conclusions APIs that are non-ionizable or demonstrate poor salt forming ability traditionally present few opportunities for creating crystalline solid forms with desired physical properties. Cocrystals are an additional class of crystalline solid that can provide options for improved properties. In this case, a crystalline molecular complex of glutaric acid and an API was identified and used to demonstrate an improvement in the oral bioavailability of the API in dogs.     

Templated Reactive Crystallization of Active Pharmaceutical Ingredient in Hydrogel Microparticles Enabling Robust Drug Product Processing

Commercialization of most promising active pharmaceutical ingredients (APIs) is impeded either by poor bioavailability or challenging physical properties leading to costly manufacture. Bioavailability of ionizable hydrophobic APIs can be enhanced by its conversion to salt form. While salt form of the API presents higher solution concentration than the non-ionized form, poor physical properties resulting from particle anisotropy or non-ideal morphology (needles) and particle size distribution not meeting dissolution rate targets can still inhibit its commercial translation. In this regard, API physical properties can be improved through addition of non-active components (excipients or carriers) during API manufacture. In this work, a facile method to perform reactive crystallization of an API salt in presence of the microporous environment of a hydrogel microparticle is presented. Specifically, the reaction between acidic antiretroviral API, raltegravir and base potassium hydroxide is performed in the presence of polyethylene glycol diacrylamide hydrogel microparticles. In this bottom-up approach, the spherical template hydrogel microparticles for the reaction lead to monodisperse composites loaded with inherently micronized raltegravir-potassium crystals, thus improving API physical properties without hampering bioavailability. Overall, this technique provides a novel approach to reactive crystallization while maintaining the API polymorph and crystallinity.     

Estimation of the Degree of Crystallinity of Cefazolin Sodium by X-Ray and Infrared Methods

The pentahydrate ( form) of cefazolin sodium (CEZ) exhibited sharp X-ray diffraction peaks, while the dehydrated form showed weak but distinct diffraction peaks. As expected the amorphous form exhibited a diffuse and halo diffraction pattern. The X-ray procedure to estimate the degree of crystallinity of CEZ was based upon the measurement of the total scattering and the scattering from the crystalline region of the drug. The major difference in the infrared (IR) spectra among the three forms of CEZ was the absence of a spectral band at 1542 cm^–1 in the amorphous form. The IR procedure was based upon the measurement of the peak percentage area ratio between the bands at 1542 and 1760 cm^–1, where the latter was used as a normalizing peak. The degree of crystallinity of CEZ samples, obtained by either freeze-drying aqueous CEZ solutions or storing the crystalline forms under different humidity conditions, was determined by these two methods. Although the correlation of results by the two methods was good, the X-ray procedure appears to be superior since it can differentiate among the three solid CEZ forms, whereas IR could distinguish between only crystalline and amorphous CEZ, reproducibly.     

Novel ultra-rapid freezing particle engineering process for enhancement of dissolution rates of poorly water-soluble drugs.

An ultra-rapid freezing (URF) technology has been developed to produce high surface area powders composed of solid solutions of an active pharmaceutical ingredient (API) and a polymer stabilizer. A solution of API and polymer excipient(s) is spread on a cold solid surface to form a thin film that freezes in 50 ms to 1s. This study provides an understanding of how the solvent's physical properties and the thin film geometry influence the freezing rate and consequently the final physico-chemical properties of URF-processed powders. Theoretical calculations of heat transfer rates are shown to be in agreement with infrared images with 10ms resolution. Danazol (DAN)/polyvinylpyrrolidone (PVP) powders, produced from both acetonitrile (ACN) and tert-butanol (T-BUT) as the solvent, were amorphous with high surface areas (approximately 28-30 m2/g) and enhanced dissolution rates. However, differences in surface morphology were observed and attributed to the cooling rate (film thickness) as predicted by the model. Relative to spray-freezing processes that use liquid nitrogen, URF also offers fast heat transfer rates as a result of the intimate contact between the solution and cold solid surface, but without the complexity of cryogen evaporation (Leidenfrost effect). The ability to produce amorphous high surface area powders with submicron primary particles with a simple ultra-rapid freezing process is of practical interest in particle engineering to increase dissolution rates, and ultimately bioavailability.     

Preparation of an amorphous sodium furosemide salt improves solubility and dissolution rate and leads to a faster Tmax after oral dosing to rats

Amorphous forms of furosemide sodium salt and furosemide free acid were prepared by spray drying. For the preparation of the amorphous free acid, methanol was utilised as the solvent, whereas the amorphous sodium salt was formed from a sodium hydroxide-containing aqueous solvent in equimolar amounts of NaOH and furosemide. Information about the structural differences between the two amorphous forms was obtained by Fourier Transform Infrared Spectroscopy (FTIR), and glass transition temperature (T_g) was determined using Differential Scanning Calorimetry (DSC). The stability and devitrification tendency of the two amorphous forms were investigated by X-ray Powder Diffraction (XRPD). The apparent solubility of the two amorphous forms and the crystalline free acid form of furosemide in various gastric and intestinal stimulated media was determined. Moreover, the dissolution characteristics of the two amorphous forms and of crystalline free acid were investigated.FTIR confirmed molecular differences between the amorphous free acid and salt. The amorphous salt showed a T_g of 101.2 °C, whereas the T_g for the amorphous free acid was found to be 61.8 °C. The amorphous free acid was physically stable for 4 days at 22 °C and 33% relative humidity (RH), while the amorphous salt exhibited physical stability for 291 days at the same storage conditions. When storing the amorphous forms at 40 °C and 75% RH both forms converted to crystalline forms after 2 days.The apparent solubility of the amorphous salt form was higher than that of both amorphous and crystalline free acid in all media studied. All three forms of furosemide exhibited a greater solubility in the presence of biorelevant media as compared to buffer, however, an overall trend for a further increase in solubility in relation to an increase in media surfactant concentration was not seen. The amorphous salt demonstrated an 8- and 20-fold higher intrinsic dissolution rate (IDR) when compared to amorphous and crystalline free acid, respectively.The promising properties of the amorphous salt in vitro were further evaluated in an in vivo study, where solid dosage forms of the amorphous salt, amorphous and crystalline free acid and a solution of furosemide were administered orally to rats. The amorphous salt exhibited a significantly faster T_max compared to the solution and amorphous and crystalline free acid. C_max for the solution was significantly higher compared to the three furosemide forms. No significant difference was found in AUC and absolute bioavailability for the solution, crystalline free acid and the two amorphous forms of furosemide. It can be concluded that the higher IDR and higher apparent solubility of the amorphous salt resulted in a faster T_max compared to the amorphous and crystalline free acid.     

Surface Denaturation at Solid-Void Interface—A Possible Pathway by Which Opalescent Participates Form During the Storage of Lyophilized Tissue-Type Plasminogen Activator at High Temperatures

During protein lyophilization, it is common practice to complete the freezing step as fast as possible in order to avoid protein denaturation, as well as to obtain a final product of uniform quality. We report a contradictory observation made during lyophilization of recombinant tissue-type plasminogen activator (t-PA) formulated in arginine. Fast cooling during lyophilization resulted in a lyophilized product that yielded more opalescent particulates upon long term storage at 50 °C, under a 150 mTorr nitrogen seal gas environment. Fast cooling also resulted in a lyophilized cake with a large internal surface area. Studies on lyophilized products containing 1% (w/w) residual moisture and varying cake surface areas (0.22 - 1.78 m²/gm) revealed that all lyophilized cakes were in an amorphous state with similar glass transition temperatures (103 - 105 °C). However, during storage the rate of opalescent particulate formation in the lyophilized product (as determined by UV optical density measurement in the 360 to 340 nm range for the reconstituted solution) was proportional to the cake surface area. We suggest that this is a surface-related phenomenon in which the protein at the solid-void interface of the lyophilized cake denatures during storage at elevated temperatures. Irreversible denaturation at the ice-liquid interface during freezing in lyophilization is unlikely to occur, since repeated freezing/thawing did not show any adverse effect on the protein. Infrared spectroscopic analysis could not determine whether protein, upon lyophilization, at the solid-void interface would still be in a native form.     

Effect of Amorphous Nanoparticle Size on Bioavailability of Anacetrapib in Dogs

Amorphous solid dispersions (ASDs) are used as bioavailability-enhancing formulations on the premise of the increased solubility of the amorphous form over its crystalline counterpart. Recent studies have shown that ASDs can, during dissolution, generate amorphous nanoparticles that were initially postulated to serve as a source of rapidly dissolving compound during absorption. Researchers have proposed that nanoparticles, including crystalline nanoparticles, may provide additional benefits to absorption such as drifting in the mucous layer. However, there are limited published data on the impact of nanoparticle size on bioavailability in vivo and, to our knowledge, there have been no published examples looking at the impact of differential size of in situ–generated nanoparticles from an ASD. Anacetrapib, a highly lipophilic, Biopharmaceutics Classification System IV compound, formulated as an ASD that generates nanoparticles on dissolution, was used in the studies described in this article. A differential response in bioavailability was observed with ∼100 nm or smaller particles, resulting in higher average exposure compared to ∼200 nm or larger particles. This increase in bioavailability could not be fully accounted for by the improvement in dissolution rate and was not as pronounced as that achieved by improving solubilization by coadministration with a high-fat meal.