The Relationship Between Protein Aggregation and Molecular Mobility Below the Glass Transition Temperature of Lyophilized Formulations Containing a Monoclonal Antibody

Purpose. To find out if the physical instability of a lyophilized dosage form is related to molecular mobility below the glass transition temperature. Further, to explore if the stability data generated at temperatures below the glass transition temperature can be used to predict the stability of a lyophilized solid under recommended storage conditions. Methods. The temperature dependence of relaxation time constant, , was obtained for sucrose and trehalose formulations of the monoclonal antibody (5 mg protein/vial) from enthalpy relaxation studies using differential scanning calorimetry. The non-exponentiality parameter, , in the relaxation behavior was also obtained using dielectric relaxation spectroscopy. Results. For both sucrose and trehalose formulations, the variation in with temperature could be fitted Vogel-Tammann-Fulcher (VTF) equation. The two formulations exhibited difference sensitivities to temperature. Sucrose formulation was more fragile and exhibited a stronger non-Arrhenius behavior compared to trehalose formulation below glass transition. Both formulations exhibited <2% aggregation at t values <10, where t is the time of storage. Conclusions. Since the relaxation times for sucrose and trehalose formulations at 5°C are on the order of 10⁸ and 10⁶ hrs, it is likely that both formulations would undergo very little (<2%) aggregation in a practical time scale under refrigerated conditions.     

Visible light-mediated photocatalytic oxidative cleavage of activated alkynes via hydroamination: a direct approach to oxamates

The direct oxidative cleavage of activated alkynes via hydroamination has been described using organic photocatalyst under visible-light irradiation at room temperature. In this reaction, the single electron oxidation of an in situ formed enamine followed by radical coupling with an oxidant finally delivers the oxamate. The key features of this photocatalytic reaction are the mild reaction conditions, metal-free organic dye as a photocatalyst, and TBHP playing a dual role as “O” source and for the regeneration of the photocatalyst.

The direct oxidative cleavage of activated alkynes via hydroamination has been described using organic photocatalyst under visible-light irradiation at room temperature.     

Two step,one-pot sequential synthesis of functionalized hybrid polyheterocyclic scaffolds via a solid state melt reaction (SSMR)

A new one pot assembly of highly functionalized benzo[a]phenazinone fused chromene/bicyclic scaffolds via a domino Knoevenagel intramolecular hetero-Diels–Alder (IMHDA) strategy using a solid state melt reaction (SSMR) of 2-hydroxynaphthalene 1,4-dione, o-phenylenediamine, O-allyl salicylaldehyde/O-vinyl salicylaldehyde derivatives is reported. The formation of five new bonds (two C–C bonds and three C–O bonds), three six-membered rings, and three stereogenic centers in a one-pot manner is very attractive. Ease of reaction with short time, good yields with water as the only byproduct and work up free procedure are some of the excellent features of the present protocol.

A new one pot assembly of highly functionalized benzo[a]phenazinone fused chromene/bicyclic scaffolds via a domino Knoevenagel intramolecular hetero-Diels–Alder (IMHDA) strategy using a solid state melt reaction (SSMR) is reported.

Multicomponent reactions (MCRs)¹ are known to construct complex structures from simple starting materials in a rapid and highly efficient manner where the production of wastes is minimized. Those multicomponent reactions which are carried out in a ‘one-pot’ MC sequential manner provide a high degree of reaction mass efficiency, which is crucial in the development of modern synthetic methodology for drug discovery and pharmaceutical programs.² Enormous work has been carried out in the field of multicomponent reactions in the past decade where several MCRs have been developed and extensively used in natural product synthesis and drug discovery. The Ugi reaction is a prime example of a four-component reaction that has been found to be a powerful and efficient method for the preparation of α-amino amides both in academia and industry.³ Therefore, the development of new MCRs for the synthesis of biologically active molecules continues to attract considerable attention for their atom- and step economy features.Heterocycles having nitrogen atom are widely available in nature and possess diverse and important biological activities.⁴ Phenazines are a group of organic compounds known to the mankind since past 150 years and are known to exhibit significant biological activities such as antimalarial, fungicidal, trypanocidal, antiplatelet, etc.⁵ A large number of drug molecules bearing the phenazine scaffold have been designed and evaluated in the recent years. The phenazines motif possess two dentate N atoms with three fused aromatic rings and an electron deficient conjugated system. The presence of the above features in phenazines give them an ability to form hydrogen bonds, ionic bonds, and π–π interactions with relative ease, and their use has also been demonstrated in supramolecular chemistry for molecular recognition (MR), supramolecular self-assembly (MS-A), and organic optic electronics materials.⁶ Few representative examples of phenazine and its derivatives are shown in the Fig. 1.Open in a separate windowFig. 1Some of naturally occurring phenazine derivatives.Chromenes and their derivatives are privileged scaffolds due to their ubiquitous presence in many natural products and synthetic molecules.⁷ Chromenes are also an important class of compounds displaying interesting biological activities against prostate cancer (DU-145) and breast cancer (MCF-7).⁸ Additionally, they have medicinal qualities such as antiviral and antimicrobial activity.⁹ Chromenes have also found their use in medicine, health-promoting agents, and photochromic materials.¹⁰In recent years, the domino-Knoevenagel-hetero-Diels–Alder reaction (DKHDA) which was extensively studied by Tietze''s group constitutes an important process for the preparation of complex compounds having interesting biological properties.¹¹ The Diels–Alder reaction is important since it allows the formation of functionalized rings where there is complete control on regio-, diastereo-, and enantioselectivity. Moreover, the concerted nature of the Diels–Alder reactions allow the selective formation of up to three stereogenic centers in a single reaction step. Majority of the reactions reported on DKHDA have utilized 1,3-dicarbonyl compounds such as dimethyl barbituric acid, Meldrum''s acid and 1,3-indanedione.¹² Additionally, other active methylene compounds like 1-phenyl-3-methyl pyrazolone, 4-hydroxy-coumarin, dihydroindole-2-thione, benzoylacetonitrile, 4-hydroxydithiocoumarin have been also used;¹³ but phenazinones have never been employed in DKHDA reactions. To the best of our knowledge, there are no reports on intramolecular DKHDA reactions using phenazinone as the coupling partner. Due to our interest in the construction of bioactive fused chromenophenazinone derivatives and since this molecule contains a 1,3-dicarbonyl as well as a 1,3-imine moiety, it would be very challenging to control the regioselectivity of the molecule; therefore phenazinone scaffold can act as a model substrate for an intramolecular DKHDA. We envisaged that a series of angular polycyclic chromeno fused phenazinone derivatives could be obtained and it is possible that the final hybrid compounds will result in being more selective and efficient than chromene and phenazinone compounds in biological assays.In this direction, we have decided to develop a new method for the preparation of novel benzo[a]phenazinone fused chromene/bicyclic scaffolds via a domino Knoevenagel intramolecular hetero-Diels–Alder (IMHDA) strategy using a solid state melt reaction (SSMR). As part of our research program for the development of new synthetic methods in heterocyclic chemistry¹⁴ and solid state melt reactions,^15,16 we would like to report a simple and a general domino three-component reaction of 2-hydroxynaphthalene-1,4-dione, o-phenylenediamine and O-allylated/vinylated salicylaldehyde for the synthesis of pentacyclic benzo[a]phenazinone fused chromene/bicyclic scaffolds under environmentally benign reaction conditions (Scheme 1).Open in a separate windowScheme 1Synthetic approach to benzo[a]phenazinone fused chromenes.We have started this study by melting 2-hydroxynaphthalene-1,4-dione (2), o-phenylenediamine (1), and (E)-methyl 2-((2-formylphenoxy)methyl)-3-phenylacrylate (3) at 180 °C for 1 h in solvent free condition. Unfortunately, we could not obtain any desired product and the formation of a complex mixture was observed. To minimize the formation of the side products, the 2-hydroxynaphthalene-1,4-dione (2) and o-phenylenediamine (1) were first melted at 180 °C for 10 minutes to form the intermediate benzo[a]phenazin-5-ol. Subsequently, (E)-methyl 2-((2-formylphenoxy)methyl)-3-phenylacrylate 3a was added and the mixture was melted under the same temperature for 1 h which afforded the desired hybrid product chromene fused benzo[a]phenazinone 4a (ester moiety in ring junction) in 78% yield as shown in

Inhalation of a dry powder tobramycin PulmoSphere formulation in healthy volunteers

STUDY OBJECTIVES: To evaluate the efficiency and reproducibility of pulmonary delivery of an investigational tobramycin PulmoSphere formulation (PStob) [Inhale Therapeutic Systems; San Carlos, CA] by a passive dry powder inhaler, and to compare serum concentrations and whole-lung deposition with a commercial nebulized tobramycin product (TOBI; Chiron Corporation; Seattle, WA). DESIGN: A five-period, open-label, nonrandomized crossover study. PARTICIPANTS: Fourteen healthy volunteers were studied, and 12 completed the study. INTERVENTIONS: PStob powder was manufactured using lipid-based PulmoSphere technology, producing highly dispersible porous particles. PStob was radiolabeled with (99m)Tc, and in vitro experiments confirmed it as a valid drug marker. To identify whole-lung distribution via scintigraphy, subjects inhaled contents of a single capsule (72 L/min) containing 25 mg of (99m)Tc-labeled PStob (13.5 mg of tobramycin free base) in periods 1 to 3. In period 4, subjects received (99m)Tc nebulized tobramycin, approximately 2.5 mL of 300 mg/5 mL. Deposition and blood samples were obtained. In period 5, six 25-mg doses of unlabeled PStob (81 mg of tobramycin base) were inhaled and blood samples were collected. Measurements and results: Mean whole-lung deposition of PStob was 34 +/- 6% and nebulized tobramycin was 5 +/- 2%. Peak tobramycin concentration in serum (Cmax) values were 0.6 microg/mL with PStob and 0.28 microg/mL after nebulized tobramycin. Serum area under the curve was 4.4 microg x h/mL vs 2.1 micro g x h/mL for nebulized tobramycin. Median time to Cmax for PStob was comparable to nebulized tobramycin. CONCLUSIONS: The aerosol doses of PStob (25 mg and 150 mg) were well dispersed and tolerated. Serum drug concentrations matched scintigraphy data and were roughly twice that of the comparator. Intrasubject dose variability for three equivalent periods did not exceed 18% relative SD. PStob Cmax (0.6 microg/mL) was well below the toxic threshold (2 micro g/mL).     

Effect of Glass Transition Temperature on the Stability of Lyophilized Formulations Containing a Chimeric Therapeutic Monoclonal Antibody

Purpose. The purpose of this study is to highlight the importance of knowing the glass transition temperature, T_g, of a lyophilized amorphous solid composed primarily of a sugar and a protein in the interpretation of accelerated stability data. Methods. Glass transition temperatures were measured using DSC and dielectric relaxation spectroscopy. Aggregation of protein in the solid state was monitored using size-exclusion chromatography. Results. Sucrose formulation (T_g ~ 59°C) when stored at 60°C was found to undergo significant aggregation, while the trehalose formulation (T_g ~ 80°C) was stable at 60°C. The instability observed with sucrose formulation at 60°C can be attributed to its T_g (~59°C) being close to the testing temperature. Increase in the protein/sugar ratio was found to increase the T_gs of the formulations containing sucrose or trehalose, but to different degrees. Conclusions. Since the formulations exist in glassy state during their shelf-life, accelerated stability data generated in the glassy state (40°C) is perhaps a better predictor of the relative stability of formulations than the data generated at a higher temperature (60°C) where one formulation is in the glassy state while the other is near or above its T_g.     

Formation of the Racemic Compound of Ephedrine Base from a Physical Mixture of Its Enantiomers in the Solid,Liquid, Solution,or Vapor State

Physical mixtures (conglomerates) of the two enantiomers of ephedrine base, each containing 0.5% (w/w) of water, were observed to be converted to the 1:1 racemic compound in the solid, liquid, solution, or vapor state. From a geometrically mixed racemic conglomerate of particle size 250–300 µm (50–60 mesh), the formation of the racemic compound follows second-order kinetics (first order with respect to each enantiomer), with a rate constant of 392 mol^–1 hr^–1 at 22°C. The reaction appears to proceed via the vapor phase as indicated by the growth of the crystals of the racemic compound between diametrically separated crystals of the two enantiomers in a glass petri dish. The observed kinetics of conversion in the solid state are explained by a homogeneous reaction model via the vapor and/or liquid states. Formation of the racemic compound from the crystals of ephedrine enantiomers in the solution state may explain why Schmidt et al. (Pharm. Res. 5:391–395, 1988) observed a consistently lower aqueous solubility of the mixture than of the pure enantiomers. The solid phase in equilibrium with the solution at the end of the experiment was found to be the racemic compound, whose melting point and heat of fusion are higher than those of the enantiomers. An association reaction, of measurable rate, between the opposite enantiomers in a binary mixture in the solid, liquid, solution, or vapor state to form the racemic compound may be more common than is generally realized.     

Stereoselective Dissolution of Propranolol Hydrochloride from Hydroxypropyl Methylcellulose Matrices

Since many chiral pharmaceutical excipients, such as cellulose polymers and cyclodextrins, are used as stationary phases for the separation of enantiomers by high performance liquid chromatography (HPLC), it is hypothesized that one enantiomer of a chiral drug will be released faster than the other from a pharmaceutical formulation containing a racemic drug and a chiral excipient. The mechanism of such an event may arise from preferential intermolecular interaction between the chiral excipient and one of the enantiomers. To test this hypothesis, the release of the enantiomers of propranolol hydrochloride into water from formulations containing the chiral excipients, hydroxypropyl methylcellulose (HPMC) or -cyclodextrin, was investigated by stereospecific HPLC analysis of the dissolved concentrations of each of the enantiomers from the formulations. The release of the enantiomers of propranolol hydrochloride from the formulations containing HPMC, although variable, was found to be stereoselective. However, the release of propranolol hydrochloride enantiomers from the -cyclodextrin complex was found to be non-stereoselective.     

Prediction of the onset of crystallization of amorphous sucrose below the calorimetric glass transition temperature from correlations with mobility

The objective of the present work is to determine if crystallization onset observed for an amorphous solid correlate with relaxation time at temperatures above and below the calorimetric glass transition (T(g)). Crystallization onset of spray-dried and freeze-dried amorphous sucrose were measured calorimetrically. Relaxation times measured in two temperature ranges by different techniques (isothermal calorimetry, dielectric spectroscopy) followed the expected modified Vogel-Tammann-Fulcher (VTF) behavior when extrapolated to a temperature near T(g). However, the change in slope was more conspicuous for freeze-dried sucrose, indicating that amorphous materials generated using different techniques differ in their mobilities for temperatures below T(g). Dielectric relaxation time values obtained above T(g) were well correlated to onset of crystallization. The model predicted 21 days for crystallization onset for spray-dried samples stored 7 K below T(g), compared to the experimentally observed crystallization onset of 17 days. Onset times versus temperature for freeze-dried sucrose, however, show a change in slope on approaching T(g), with the onsets somewhat decoupling from measured mobility for temperatures below T(g). Molecular mobility in amorphous materials at temperatures both above and below T(g) can be correlated to macroscopic physical change such as crystallization, but prediction of crystallization onset from relaxation time is only qualitatively correct at temperatures well below T(g).     

Ruthenium-catalyzed,site-selective C–H activation: access to C5-substituted azaflavanone

A site-selective ruthenium-catalyzed keto group assisted C–H bond activation of 2-aryl tetrahydroquinoline (azaflavanone) derivatives has been achieved with a variety of alkenes for the first time. A wide range of substrates was utilized for the synthesis of a wide variety of alkenylated azaflavanones. This simple and efficient protocol provides the C5-substituted azaflavanone derivatives in high yields with a broad range of functional group tolerance. Further, the C5-alkenylated products were converted into substituted 2-aryl quinoline derivatives in good yields.

A site-selective ruthenium-catalyzed keto group assisted C–H bond activation of 2-aryl tetrahydroquinoline (azaflavanone) derivatives has been achieved with a variety of alkenes for the first time.