Investigation of supramolecular structures in various aqueous solutions of an amyloid forming peptide using small-angle X-ray scattering.

Aggregation of peptide molecules into amyloid fibrils is a characteristic feature of several degenerative diseases. However, the details behind amyloid-formation, and other self-assembled peptide aggregates, remain poorly understood. In this study, we have used small-angle X-ray scattering (SAXS), static and dynamic light scattering (SLS and DLS) as well as cryogenic transmission electron microscopy (cryo-TEM) to determine the structural geometry of self-assembled peptide aggregates in various dilute aqueous solutions. Pramlintide was used as a model peptide to assess the aggregation behaviour of an amyloid-forming peptide. The effects of adding sodium chloride (NaCl), sodium thiocyanate (NaSCN), and sodium fluoride (NaF) and the co-solvent dimethyl sulfoxide (DMSO) on the aggregation behaviour were studied. Our scattering data analysis demonstrates that small oligomeric fibrils aggregate to form networks of supramolecular assemblies with fractal dimensions. The choice of anion in small amounts of added salt has a significant impact on the size of the fibrils as well as on the fractal dimensions of supramolecular clusters. In DMSO the fractal dimension decreased with increasing DMSO concentration, indicating the formation of a less compact structure of the supramolecular assemblies.

The In Vivo Fate of Polycatecholamine Coated Nanoparticles Is Determined by a Fibrinogen Enriched Protein Corona.

Polycatecholamine coatings have attracted significant attention in the past 10 years owing to their ability to functionalize a wide range of materials. Here we apply the use of such coatings to drug nanocrystals, made from a poorly soluble drug compound, to postfunctionalize the nanocrystal surface with the aim of providing steric stabilization and extending their circulation time after intravenous injection. We show that both polydopamine and polynorepinephrine can be used to successfully modify drug nanocrystals and subsequently incorporate end-functionalized PEG to the surface. Even though high grafting densities of PEG were achieved, we observed rapid clearance and increased liver uptake for polycatecholamine functionalized drug nanocrystals. Using both surface sensitive model systems and protein corona profiling, we determine that the rapid clearance was correlated with an increase in adsorption of proteins involved in coagulation to the polycatecholamine surface, with fibrinogen being the most abundant. Further analysis of the most abundant proteins revealed a significant increase in thiol-rich proteins on polycatecholamine coated surfaces. The observed interaction with coagulation proteins highlights one of the current challenges using polycatecholamines for drug delivery but might also provide insights to the growing use of these materials in hemostatic applications.

Pharmacokinetic evaluation of poorly soluble compounds formulated as nano- or microcrystals after intraperitoneal injection to mice.

Intraperitonial (i.p.) delivery during initial stages of drug discovery can allow efficacy readouts for compounds which have suboptimal pharmacokinetics (PK) due to poor physiochemical properties and/or oral bioavailability. A major limitation for widespread use of i.p. administration is the paucity of published data and unclear mechanisms of absorption, particularly when using complex formulations. The aim of the present study was to investigate the PK of poorly soluble compounds with low oral bioavailability when administered i.p. as crystalline nano- and microsuspensions. Three compounds, with varying aqueous solubility (2, 7, and 38 µM, at 37 °C), were dosed to mice at 10 and 50 mg/kg. In vitro dissolution confirmed that nanocrystals dissolved faster than microcrystals and hence were expected to result in higher exposure after i.p. dosing. Surprisingly, the increase in dissolution rate with decrease in particle size did not result in higher in vivo exposure. In contrast, the microcrystals showed higher exposure. The potential of smaller particles to promote access to the lymphatic system is hypothesized and discussed as one plausible explanation. The present work demonstrates the importance of understanding physicochemical properties of drug formulations in the context of the microphysiology at the delivery site and how that knowledge can be leveraged to alter systemic PK.

Supersaturated formulations of poorly soluble weak acid drugs evaluated in rodents; a case study.

In the present study we describe a way of working to overcome oral administration challenges in an early preclinical project. As candidate drugs were obtained, the preclinical delivery route was replaced by the intended route of the product and resources were allocated to optimize the oral absorption. Two main approaches were followed in order to formulate a selected weak acid, AZ'403, for oral administration in large scale toxicological studies and the early clinical phases. Both approaches relies on the suppression of precipitation from obtained supersaturated solutions achieved either by amorphous solid dispersions (using hydroxypropyl methylcellulose acetate succinate, HPMC-AS) or crystalline salts (sodium and potassium salts). In vivo studies in rodents were performed to evaluate oral AZ'403 absorption from amorphous and crystalline formulations, using nano- and micro crystalline particles of the neutral form, as references. The oral absorption of AZ'403 formulated using both approaches was significantly higher compared with the references. The improvements in overall exposures were 7-100 times during the investigated conditions. The pharmacokinetic profiles implied that both solid dispersions and crystalline salts of AZ'403 generated supersaturation in the small intestine in rodents and indicated that both approaches may be ways forward for subsequent late stage product development.

A candidate drug administered subcutaneously to rodents as drug particles showing hepatic recirculation which influenced the sustained release process.

The aim of the present study was to evaluate and interpret the pharmacokinetic profiles after subcutaneous (s.c.) administration of crystalline AZ'72 nano- and microsuspensions to rodents. Both formulations were injected at 1.5 and 150 mg/kg to rats. For the lower dose, the profiles were similar after s.c. injection but extended as compared to oral administration. The overall exposure was higher for nanoparticles compared with microparticles during the investigated period. For the higher dose, injection of both suspensions resulted in maintained plateaus caused by the drug depots but, unexpectedly, at similar exposure levels. After addition of a further stabilizer, pluronic F127, nanosuspensions showed improved exposure with dose and higher exposure compared to larger particles in mice. Obviously, a stabilizer mixture that suits one delivery route is not necessarily optimal for another one. The differences in peak concentration (Cmax) between nano- and microparticles were mainly ascribed to differences in dissolution rate. Plasma profiles in mice showed curves with secondary absorption peaks after intravenous and oral administration, suggesting hepatic recirculation following both administration routes. This process, together with the depot formulation, complicates the analysis of absorption from s.c. administration, i.e. multiple processes were driving the plasma profile of AZ'72.

Nano- and microcrystals of griseofulvin subcutaneously administered to rats resulted in improved bioavailability and sustained release.

Griseofulvin is a commonly used antifungal agent which is administered per oral (p.o.). The oral administration route, however, shows rather low bioavailability. The aim of this study was to improve the bioavailability and to evaluate and interpret the pharmacokinetic profiles after subcutaneous (s.c.) administration of crystalline griseofulvin nano- and microsuspensions. Both formulations were injected at 5 and 500 µmol/kg to rats. For the lower concentration, the profiles were similar after s.c. injection but extended as compared to p.o. administration. For the higher concentration, injection of microsuspension resulted in a maintained plateau whereas the nanosuspension resulted in an obvious peak exposure followed by extended elimination. Both suspensions showed improved exposure with dose. The differences in peak exposures between nano- and microparticles, at the high dose, were mainly ascribed to differences in dissolution rate, experimentally determined in vitro, using spectroscopic methods. The extended appearance in the circulation may depend on the physicochemical properties of the compound and the physiological conditions at the injection site. The bioavailability was improved for both formulations compared with an orally administered nanosuspension, suggesting the s.c. route to be a preferred administration option for compounds with low oral bioavailability regarding both overall exposure and extended efficacy.

Sustained release and improved bioavailability in mice after subcutaneous administration of griseofulvin as nano- and microcrystals.

The objective of the study was to evaluate the pharmacokinetic profile after different subcutaneous (s.c.) administrations of nano- and microparticle suspensions of griseofulvin to mice. The solubility of the compound was determined as approximately 40 µM, at 37 °C, independent of particle size, stabilizer mixtures investigated and solvent used for measurement. The present in vivo studies demonstrated non-linear absorption kinetics (in peak concentration, Cmax) for griseofulvin up to 50 mg/kg after s.c. administration of nanocrystals and microsuspensions but linear increase in area under the curve (AUC) at all occasions investigated. Cmax was higher for smaller particles administered. Both investigated suspensions, at 10 and 50 mg/kg, showed significantly sustained plasma profiles compared to i.v. and p.o. administration. Administering 10 and 50 mg/kg of griseofulvin nanocrystals as 10 mL/kg, instead of 2.5 mL/kg, improved Cmax but AUC was unchanged. The present study showed that the bioavailability of griseofulvin, administered as nano- and microparticles, increased significantly after s.c. administration (60-100%) compared with p.o. dosing (17%). The drug is currently orally administered and clearly exposed to a significant first pass metabolism, i.e. an ideal candidate for an alternative administration route, like s.c. injection.

Nanoshells prepared by atomic layer deposition - Long acting depots of indomethacin.

There is a trend in pharmaceutical research and development to develop depot formulations with dosing once weekly, once monthly, or even less frequently. A novel approach to achieve long acting injectable suspensions is to produce dense inorganic nanoshells with atomic layer deposition (ALD) on active pharmaceutical ingredients. Such particles can be suspended in an aqueous vehicle and administered subcutaneously. The purpose of this work was to study the release of a model drug, indomethacin, coated with aluminium oxide nanoshells. Indomethacin was ball-milled to a median particle size of 6 µm. The nanoshells were produced with a proprietary ALD process that is trademarked as PharmaShell® by Nanexa AB. The drug load was determined with HPLC-UV to 82 wt%. The test materials were administered subcutaneously in rats (1, 10, and 100 mg/kg) from which blood samples were collected during 12 weeks. Plasma was generated and analyzed with regards to indomethacin using UPLC-MS/MS. The release rate was dramatically slower for the nanoshell coated indomethacin compared with uncoated indomethacin. Drug was released in vivo during more than 12 weeks for the 10 and 100 mg/kg doses, and during 10 weeks for the 1 mg/kg dose, while uncoated indomethacin was eliminated with a half-life of 15 h, as calculated from the release data by fitting a one phase decay function. The exposure levels were similar as earlier reported for therapeutic indomethacin doses, but significantly sustained in the present study using coated drug particles in rats. In conclusion, this is the first long-term in vivo evaluation of nanoshell depot formulations. The stable plasma concentrations for more than 12 weeks demonstrate that nanoshells can enable long-term depot injections with high drug load.

Promoting intestinal lymphatic transport targets a liver-X receptor (LXR) agonist (WAY-252,623) to lymphocytes and enhances immunomodulation.

Lymphocytes play a central role in the pathology of a range of chronic conditions such as autoimmune disease, transplant rejection, leukemia, lymphoma HIV/AIDs and cardiometabolic diseases such as atherosclerosis. Current treatments for lymphocyte-associated conditions are incompletely effective and/or complicated by a range of off-target toxicities. One major challenge is poor drug access to lymphocytes via the systemic blood and this may be attributed, at least in part, to the fact that lymphocytes are concentrated within lymph fluid and lymphoid tissues, particularly in gut-associated lymphatics. Here we demonstrate that promoting drug uptake into the intestinal lymphatics with a long chain fatty acid, thereby increasing lymphocyte access, enhances the pharmacodynamic effect of a highly lipophilic liver X receptor (LXR) agonist, WAY-252623, that has been suggested as a potential treatment for atherosclerosis. This has been exemplified by: (1) increased mRNA expression of key markers of LXR activation (ABCA1) and regulatory T cells (Foxp3) in local lymphatic lymphocytes and (2) enhanced numbers of CD4+CD25+Foxp3+ regulatory T cells in the systemic circulation, after administration of a 5-fold lower dose with a lymph directing lipid formulation when compared with a non-lipid containing formulation. These data suggest that combining lipophilic, lymphotropic drug candidates such as WAY-252,623, with lymph-directing long chain lipid based formulations can enhance drug targeting to, and activity on, lymphocytes in lymph and that this effect persists through to the systemic circulation. This presents a promising approach to achieve more selective and effective therapeutic outcomes for the treatment of lymphocyte associated diseases.

The crystalline salt form of a selected candidate drug showed photo-, thermal- and humidity induced form transitions.

AZ3411 was selected as a lead compound for the treatment of Inflammatory Bowel Disease (IBD). The present research aimed to perform an early pharmaceutical assessment of this NK antagonist candidate focusing on the challenging solid-state part of the evaluation. X-ray powder diffraction (XRPD), hot stage XRPD and microscopy, differential scanning calorimetry, thermogravimetrical analysis measurements, nuclear magnetic resonance spectroscopy and liquid chromatographic analysis were used to characterize AZ3411. The amorphous, free base form of AZ3411 was transformed to a poorly crystalline material by salt formation using maleic acid. Suspensions of the poorly crystalline form (type A), prepared in various solvents, exhibited phase transformation on storage. Some precipitate was identified as a new, more crystalline form (type B) of the maleate salt of AZ3411. Also, a third crystalline form was observed at high temperatures (type C). AZ3411 maleate type A, maleate type B and amorphous, free base form was stored in 40 °C/75% relative humidity (RH), 60 °C and 80 °C for three months. Form B was found to be the most chemically stable at all conditions. After three months at 40 °C/75%RH, both type A and type B had transformed to the anhydrous type C. Moreover, type B was transformed to form C at 60 °C and 80 °C, while type A remained unchanged. These results, together with the loss of water with temperature, suggest that type B is a hydrate. The relative stability between the hydrate type B and anhydrous type C depend on humidity and temperature. Moreover, the photosensitivity of maleate type A, maleate type B and amorphous free base has been investigated under three different illumination conditions. In similarity to the previous study, Form B was the most chemically stable form. However, after completion the study, at the highest energy conditions (765 W/m2, 250-800 nm), the crystalline type B had transformed to type C, while type A had lost in crystallinity. A similar photostability study was performed on solutions of pH 1 and pH 7. The degradation pattern was similar for the two pHs but appeared different from the unstressed solution stability study performed on different pHs between pH 1 and 7. Neither was there any obvious correlation between the degradation patterns obtained after the stressed thermal- and photostability studies performed on the drug substance in solid-state. The salt of AZ3411 fulfils basic requirements for further development of an oral immediate release (IR) dosage form, although the compound displays signs of light sensitivity and there may be a risk of solid-state transitions during formulation development and long-term storage.

A case study where pharmaceutical salts were used to address the issue of low in vivo exposure.

The present active pharmaceutical ingredient (API) is a lipophilic compound with a significant risk of not achieving therapeutic plasma concentrations due to solubility-limited absorption. The aim of the presented studies was to investigate whether three novel salts of a new selected candidate in the cardiovascular therapy area could be applied to improve intestinal absorption and the subsequent in vivo exposure. Three salts (chloride, hydrogen sulfate, and hemi-1.5-naphtalenedisulphonate) of the compound were manufactured and investigated regarding solubility, dissolution rate, and in vivo exposure in rats. The chemical and physical stability of the salt forms (and the crystalline parent compound) were followed in solid state, when dissolved and when formulated as microsuspensions. All salts showed improved solubility in investigated media, increased dissolution rate, and elevated in vivo exposures compared to a nanocrystal formulation (top-down) of the parent free base of the compound. The chloride- and the hydrogen sulfate salts of the API showed similar patterns regarding the chemical stability in solid state as the crystalline free base, while the salt formed of the hemi-1.5-naphtalenedisulphonic acid showed significantly improved stability. In conclusion, this study showed that three salts of a new selected candidate drug could be used to improve solubility, increase dissolution rate, and enhance oral absorption compared with a more commonly used nanocrystal formulation of the API. However, the identity of the counter ion appeared to be of less importance. On the other hand, only the salt of the hemi-1.5-naphtalenedisulphonic acid seemed to improve chemical stability compared with the API.

Salt formation improved the properties of a candidate drug during early formulation development.

The purpose of this study was to investigate if AZD5329, a dual neurokinin NK1/2 receptor antagonist, is a suitable candidate for further development as an oral immediate release (IR) solid dosage form as a final product. The neutral form of AZD5329 has only been isolated as amorphous material. In order to search for a solid material with improved physical and chemical stability and more suitable solid-state properties, a salt screen was performed. Crystalline material of a maleic acid salt and a fumaric acid salt of AZD5329 were obtained. X-ray powder diffractiometry, thermogravimetric analysis, differential scanning calorimetry and dynamic vapor sorption were used to investigate the physicochemical characteristics of the two salts. The fumarate salt of AZD5329 is anhydrous, the crystallization is reproducible and the hygroscopicity is acceptable. Early polymorphism assessment work using slurry technique did not reveal any better crystal modification or crystallinity for the fumarate salt. For the maleate salt, the form isolated originally was found to be a solvate, but an anhydrous form was found in later experiments; by suspension in water or acetone, by drying of the solvate to 100-120 °C or by subjecting the solvate form to conditions of 40 °C/75%RH for 3 months. The dissolution behavior and the chemical stability (in aqueous solutions, formulations and solid-state) of both salts were also studied and found to be satisfactory. The compound displays sensitivity to low pH, and the salt of the maleic acid, which is the stronger acid, shows more degradation during stability studies, in line with this observation. The presented data indicate that the substance fulfils basic requirements for further development of an IR dosage form, based on the characterization on crystalline salts of AZD5329.

A preformulation evaluation of a photosensitive surface active compound, explaining concentration dependent degradation.

A candidate drug within the cardiovascular area was identified during early research and evaluated for further development. The aim was to understand and explain the degradation mechanisms for the present compound. The stability of the active pharmaceutical ingredient (API) in solution and solid state was studied during different conditions. The bulk compound was exposed to elevated temperatures, increased relative humidity and stressed light conditions. Degradation of the drug in solutions was followed in the presence versus absence of ethylenediaminetetraacetic acid (EDTA), during aerobic versus anaerobic conditions, stored protected from light versus exposed to light and as a function of pH and concentration. It was possible to improve the stability by adding EDTA and completely abolish degradation by storing dissolved compound at anaerobic conditions. Solutions of API were stable between pH3 and 7, with some degradation at pH1, when stored protected from light and at 22°C, but degrade rapidly when exposed to ambient light conditions. The degradation products were identified by mass spectroscopy. Degradation schemes were drawn. There was concentration dependence in the degradation of dissolved drug when exposed to light, showing a titration behavior that concurred with the measured critical micelle/aggregation concentration (CMC/CAC) of the compound. The compound was stable in solution during the investigated time period, at concentrations above CMC/CAC, where the molecule was protected from photodegradation when the compound aggregated. Below CMC/CAC, a significant degradation of the API occurred. This may be a potential explanation why other surface active compounds show concentration dependent degradation. The photosensitivity was also observed for the neutral compound in crystalline and amorphous form, as well as for the crystalline chloride salt of the drug. However, the degradation of amorphous form was faster compared to crystalline material. No difference was observed in the degradation pattern between the neutral form of the compound and the salt form of the drug.

Nanocrystal formulations of a poorly soluble drug. 2. Evaluation of nanocrystal liver uptake and distribution after intravenous administration to mice.

A stabilized high drug load intravenous formulation could allow compounds with less optimal pharmacokinetic profiles to be developed. Polyethylene glycol (PEG)-ylation is a frequently used strategy for particle delivery systems to avoid the liver, thereby extending blood circulation time. The present work reports the mouse in vivo distribution after i.v. administration of a series of nanocrystals prepared with the bead milling technique and PEG-ylated with DSPE-PEG2000 and Pluronic F127, with and without polyvinylpyrrolidone K30 (PVP)/Aerosol OT (AOT) as primary stabilizers. While all formulations were cleared significantly faster than expected from nanocrystal dissolution alone, purely DSPE-PEG2000 PEG-ylated particles displayed prolonged circulation time (particles elimination half-life of 9min) compared to DSPE-PEG2000/PVP/AOT formulation (half-life of 3min). The two Pluronic F127 stabilized formulations displayed similar half-lives (9min with and without PVP/AOT, respectively). Whole tissue kinetics shows that clearance of particles could be attributed to accumulation in the liver. A separate in vivo study addressed the liver cell distribution after administration. Dissolved compound accumulated in hepatocytes only, while particles were distributed between liver sinusoidal endothelial cells and Kupffer cells. More DSPE-PEG2000/PVP/AOT stabilized particles accumulated in the liver, preferably in Kupffer cells, compared to Pluronic F127/PVP/AOT stabilized particles. The present study extends the understanding of PEG-ylation and "stealth" behaviour to also include nanocrystals.

Preformulation investigation and challenges; salt formation, salt disproportionation and hepatic recirculation.

A compound, which is a selective peroxisome proliferator activated receptor (PPAR) agonist, was investigated. The aim of the presented studies was to evaluate the potential of the further development of the compound. Fundamental physicochemical properties and stability of the compound were characterized in solution by liquid chromatography and NMR and in solid-state by various techniques. The drug itself is a lipophilic acid with tendency to form aggregates in solution. The neutral form was only obtained in amorphous form with a glass-transition temperature of approximately 0°C. The intrinsic solubility at room temperature was determined to 0.03mg/mL. Chemical stability studies of the compound in aqueous solutions showed good stability for at least two weeks at room temperature, except at pH1, where a slight degradation was already observed after one day. The chemical stability in the amorphous solid-state was investigated during a period of three months. At 25°C/60% relative humidity (RH) and 40°C/75% RH no significant degradation was observed. At 80°C, however, some degradation was observed after four weeks and approximately 3% after three months. In an accelerated photostability study, degradation of approximately 4% was observed. Attempts to identify a crystalline form of the neutral compound were unsuccessful, however, salt formation with tert-butylamine, resulted in crystalline material. Results from stability tests of the presented crystalline salt form indicated improved chemical stability at conditions whereas the amorphous neutral form degraded. However, the salt form of the drug dissociated under certain conditions. The drug was administered both per oral and intravenously, as amorphous nanoparticles, to conscious dogs. Plasma profiles showed curves with secondary absorption peaks, indicating hepatic recirculation following both administration routes. A similar behavior was observed in rats after oral administration of a pH-adjusted solution. The observed double peaks in plasma exposure and the dissociation tendency of the salt form, were properties that contributed to make further development of the candidate drug challenging. Options for development of solid dosage forms of both amorphous and crystalline material of the compound are discussed.

Nanocrystal formulations of a poorly soluble drug. 1. In vitro characterization of stability, stabilizer adsorption and uptake in liver cells.

In the present work, milled nanocrystals of a poorly soluble compound using different stabilizers were prepared and characterized. The aim of the study was to evaluate a fundamental set of properties of the formulations prior to i.v. injection of the particles. Two polyethylene oxide containing stabilizers; (distearoyl phosphatidylethanol amine (DSPE)) -PEG2000 and the triblock copolymer Pluronic F127, were investigated, with and without polyvinylpyrrolidone K30/Aerosol OT (PVP/AOT) present. The solubility in water was around 10nM for the compound, measured from nanocrystals, but 1000 times higher in 4% human serum albumin. The particles were physically stable during the time investigated. The zeta potential was around -30 and -10mV for DSPE-PEG2000 and Pluronic F127 stabilized particles, respectively, at the conditions selected. The dissolution rate was similar for all four formulations and similar to the theoretically predicted rate. Critical micelle concentrations were determined as 56nM and 1.4µM for DSPE-PEG2000 and Pluronic F127, respectively. The adsorption isotherms for the PEG lipid showed a maximum adsorbed amount of about 1.3mg/m2, with and without PVP/AOT. Pluronic F127 showed a higher maximum amount adsorbed, at around 3.1mg/m2, and marginally lower with PVP/AOT present. Calculated data showed that the layer of Pluronic F127 was thicker than the corresponding DSPE-PEG2000 layer. The total amount of particles distributed mainly to the liver, and the hepatocellular distribution in vitro (Liver sinusoidal endothelial cells and Kupffer cells), differed depending on the stabilizing mixture on the particles. Overall, DSPE-PEG2000 stabilized nanocrystals (with PVP/AOT) accumulated to a larger degree in the liver compared to particles with Pluronic F127 on the surface. A theoretical model was developed to interpret in vivo pharmacokinetic profiles, explaining the balance between dissolution and liver uptake. With the present, fundamental data of the nanocrystal formulations, the platform for forthcoming in vivo studies was settled.

Biopharmaceutic Profiling of Salts to Improve Absorption of Poorly Soluble Basic Drugs.

AZD1175 and AZD2207 are 2 highly lipophilic compounds with a significant risk of not achieving therapeutic plasma concentrations due to solubility-limited absorption. The compounds have the same molecular weight and minimal structural differences. The aim of the present work was to investigate whether salts could be applied to improve the intestinal absorption, and the subsequent in vivo exposure. Drug solubilities, dissolution rates, and degree of supersaturation and precipitation were determined in biorelevant media. Dog studies were performed, in the absence and presence of a precipitation inhibitor (hydroxypropyl methylcellulose). Finally, a human phase I study was performed. For AZD1175, there was a good agreement between dissolution rates, in vivo exposure in dog, and the obtained exposure in human with the selected hemi-1,5-naphthalenedisulfonate of the compound. For AZD2207, the picture was more complex. The same counter ion was selected for the study in man. In addition, the chloride salt of AZD2207 showed promising data in the presence of a precipitation inhibitor in vitro and in dog that, however, could not be repeated in man. The differences in observations between the 2 compounds could be attributed to the difference in solubility and to the degree of supersaturation in the gastric environment rather than in the intestine.

Evaluation of preclinical formulations for a poorly water-soluble compound.

One central aim of the present work was to find a robust oral formulation approach for Compound A, both to achieve reliable pharmacodynamic read outs but also for long time safety assessment studies. The compound has low aqueous solubility (0.4µM at 37°C), is highly lipophilic and has high Caco-2 permeability, i.e. a typical BCS II compound. A nanocrystal formulation, some oil approaches and a fat diet approach were evaluated in vivo in rats. The two latter strategies resulted in significantly higher in vivo exposures after oral administration compared to the nanocrystal approach. For simplicity, and due to the project development program, a food pellet formulation was selected. In addition, tentative data from a subcutaneous study in mice using nanocrystals of the compound are presented, showing extended profiles on the cost of Cmax. Exposure data in monkeys after administration of nanocrystals both intravenously and per oral are presented. When switched from nanocrystals to an oil formulation, the observed oral exposure behavior was similar as observed in rats.

Probing adsorption of DSPE-PEG2000 and DSPE-PEG5000 to the surface of felodipine and griseofulvin nanocrystals.

Nanosized formulations of poorly water-soluble drugs show great potential due to improved bioavailability. In order to retain colloidal stability, the nanocrystals need to be stabilized. Here we explore the use of the poly(ethylene glycol) (PEG) conjugated phospholipids DSPE-PEG2000 and DSPE-PEG5000 as stabilizers of felodipine and griseofulvin nanocrystals. Nanocrystal stability and physicochemical properties were examined and the interaction between the PEGylated lipids and the nanocrystal surface as well as a macroscopic model surface was investigated. Using quartz crystal microbalance with dissipation monitoring both mass adsorption and the thickness of the adsorbed layer were estimated. The results indicate that the PEGylated lipids are adsorbed as flat layers of around 1-3nm, and that DSPE-PEG5000 forms a thicker layer compared with DSPE-PEG2000. In addition, the mass adsorption to the drug crystals and the model surface are seemingly comparable. Furthermore, both DSPE-PEG2000 and DSPE-PEG5000 rendered stable drug nanocrystals, with a somewhat higher surface binding and stability seen for DSPE-PEG2000. These results suggest DSPE-PEG2000 and DSPE-PEG5000 as efficient nanocrystal stabilizers, with DSPE-PEG2000 giving a somewhat higher surface coverage and superior colloidal stability, whereas DSPE-PEG5000 shows a more extended structure that may have advantages for prolongation of circulation time in vivo and facilitation for targeting modifications.

Discovery of AZD6642, an inhibitor of 5-lipoxygenase activating protein (FLAP) for the treatment of inflammatory diseases.

A drug discovery program in search of novel 5-lipoxygenase activating protein (FLAP) inhibitors focused on driving a reduction in lipophilicity with maintained or increased ligand lipophilic efficiency (LLE) compared to previously reported compounds led to the discovery of AZD6642 (15b). Introduction of a hydrophilic tetrahydrofuran (THF) ring at the stereogenic central carbon atom led to a significant shift in physicochemical property space. The structure-activity relationship exploration and optimization of DMPK properties leading to this compound are described in addition to pharmacokinetic analysis and an investigation of the pharmacokinetic (PK)-pharmacodynamic (PD) relationship based on ex vivo leukotriene B4 (LTB4) levels in dog. AZD6642 shows high specific potency and low lipophilicity, resulting in a selective and metabolically stable profile. On the basis of initial PK/PD relation measured, a low dose to human was predicted.