Protein Internal Dynamics Associated With Pre-System Glass Transition Temperature Endothermic Events: Investigation of Insulin and Human Growth Hormone by Solid State Hydrogen/Deuterium Exchange.

Lyophilized proteins are generally stored below their glass transition temperature (Tg) to maintain long-term stability. Some proteins in the (pure) solid state showed a distinct endotherm at a temperature well below the glass transition, designated as a pre-Tg endotherm. The pre-Tg endothermic event has been linked with a transition in protein internal mobility. The aim of this study was to investigate the internal dynamics of 2 proteins, insulin and human growth hormone (hGH), both of which exhibit the pre-Tg endothermic event with onsets at 50°C-60°C. Solid state hydrogen/deuterium (H/D) exchange of both proteins was characterized by Fourier transform infrared spectroscopy over a temperature range from 30°C to 80°C. A distinct sigmoidal transition in the extent of H/D exchange had a midpoint of 56.1 ± 1.2°C for insulin and 61.7 ± 0.9°C for hGH, suggesting a transition to greater mobility in the protein molecules at these temperatures. The data support the hypothesis that the pre-Tg event is related to a transition in internal protein mobility associated with the protein dynamical temperature. Exceeding the protein dynamical temperature is expected to activate protein internal motion and therefore may have stability consequences.

Using the fluorescence red edge effect to assess the long-term stability of lyophilized protein formulations.

Nanosecond relaxation processes in sugar matrices are causally linked through diffusional processes to protein stability in lyophilized formulations. Long-term protein degradation rates track mean-squared displacement (⟨u(2)⟩) of hydrogen atoms in sugar glasses, a parameter describing dynamics on a time scale of picoseconds to nanoseconds. However, measurements of ⟨u(2)⟩ are usually performed by neutron scattering, which is not conducive to rapid formulation screening in early development. Here, we present a benchtop technique to derive a ⟨u(2)⟩ surrogate based on the fluorescence red edge effect. Glycerol, lyophilized trehalose, and lyophilized sucrose were used as model systems. Samples containing 10(-6) mole fraction of rhodamine 6G, a fluorophore, were excited at either 532 nm (main peak) or 566 nm (red edge), and the ⟨u(2)⟩ surrogate was determined based the corresponding Stokes shifts. Results showed reasonable agreement between ⟨u(2)⟩ from neutron scattering and the surrogate from fluorescence, although deviations were observed at very low temperatures. We discuss the sources of the deviations and suggest technique improvements to ameliorate these. We expect that this method will be a valuable tool to evaluate lyophilized sugar matrices with respect to their ability to protect proteins from diffusion-limited degradation processes during long-term storage. Additionally, the method may have broader applications in amorphous pharmaceutical solids.

Amorphization of itraconazole by inorganic pharmaceutical excipients: comparison of excipients and processing methods.

The aim of this study was to investigate the effects of solid carriers and processing routes on the properties of amorphous solid dispersions of itraconazole. Three solid carriers with a range of surface properties were studied, (1) a mesoporous silicate, magnesium aluminum silicate (Neusilin US2), (2) a nonporous silicate of corresponding composition (Veegum) and (3) a non-silicate, inorganic excipient, calcium phosphate dibasic anhydrous (A-TAB). The drug was incorporated via either solvent-deposition or ball milling. Both the maximum drug deposited by solvent-based method that produced an amorphous composite and the time for complete amorphization by co-milling was determined by X-ray powder diffraction (XRPD). Changes in the drug and excipients were monitored by nitrogen adsorption and wettability of the powder. The ability of the excipients to amorphize the drug and enhance its dissolution was related to the powder characteristics. Neusilin provided the fastest amorphization time in the mill and highest drug loading by solvent-deposition, compared with the other two excipients. Solvent-deposition provided greater dissolution enhancement than milling, due to the reduction in Neusilin porosity during high energy milling.This study confirms that substrates as well as the processing routes have notable influence on the drug deposition, amorphization, physical stability and drug in vitro release.

Protein quantity on the air-solid interface determines degradation rates of human growth hormone in lyophilized samples.

Recombinant human growth hormone (rhGH) was lyophilized with various glass-forming stabilizers, employing cycles that incorporated various freezing and annealing procedures to manipulate glass formation kinetics, associated relaxation processes, and glass-specific surface areas (SSAs). The secondary structure in the cake was monitored by infrared and in reconstituted samples by circular dichroism. The rhGH concentrations on the surface of lyophilized powders were determined from electron spectroscopy for chemical analysis. Glass transition temperature (Tg ), SSAs, and water contents were determined immediately after lyophilization. Lyophilized samples were incubated at 323 K for 16 weeks, and the resulting extents of rhGH aggregation, oxidation, and deamidation were determined after rehydration. Water contents and Tg were independent of lyophilization process parameters. Compared with samples lyophilized after rapid freezing, rhGH in samples that had been annealed in frozen solids prior to drying, or annealed in glassy solids after secondary drying retained more native-like protein secondary structure, had a smaller fraction of the protein on the surface of the cake, and exhibited lower levels of degradation during incubation. A simple kinetic model suggested that the differences in the extent of rhGH degradation during storage in the dried state between different formulations and processing methods could largely be ascribed to the associated levels of rhGH at the solid-air interface after lyophilization.

Pitavastatin, a new HMG-CoA reductase inhibitor, induces phototoxicity in human keratinocytes NCTC-2544 through the formation of benzophenanthridine-like photoproducts.

This study reports the results of an investigation of the phototoxicity mechanism induced by pitavastatin and its photoproducts, namely 6-cyclopropyl-10-fluoro-7,8-dihydrobenzo[k]phenanthridine (PP3) and 6-cyclopropyl-10-fluorobenzo[k]phenanthridine (PP4). The phototoxicity was tested in human keratinocytes cell lines NCTC-2544, and the results proved that under the same conditions, all three compounds exhibited phototoxic effects in the model tested. The reduction in cell viability was found to be both concentration- and UVA dose-dependent. A point of note is that both the photoproducts produced a dramatic decrease in cell viability with GI(50) values one order of magnitude lower compared to the parent compound. In particular, the fully aromatic derivative (PP4) showed the highest antiproliferative activity. Flow cytometric analysis indicated that pitavastatin and the photoproduct PP4 principally induced necrosis, as revealed by the large appearance of propidium iodide-positive cells and also confirmed by the rapid drop in cellular ATP levels. Further studies committed to better understanding of photoinduced cell death mechanism(s) revealed that neither pitavastatin nor PP4 induced mitochondrial depolarization or lysosomal damage, but, interestingly, extensive cell lipid membrane peroxidation along with a significant oxidation of model proteins occurred, suggesting that pitavastatin and PP4 exert their phototoxic effect mainly in the cellular membranes. The present results suggest that the phototoxicity of pitavastatin may be mediated by the formation of benzophenanthridine-like photoproducts that appear to have high potential as photosensitizers.

The phototoxicity of fluvastatin, an HMG-CoA reductase inhibitor, is mediated by the formation of a benzocarbazole-like photoproduct.

In this paper, we have investigated the mechanism of phototoxicity of fluvastatin, an 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, in human keratinocytes cell line NCTC-2544. Fluvastatin underwent rapid photodegradation upon Ultraviolet-A (UVA) irradiation in buffered aqueous solution as shown by the changes in absorption spectra. Interestingly, no isosbestic points were observed but only a fast appearance of a spectral change, indicative of the formation of a new chromophore. The isolation and characterization of the main photoproduct revealed the formation of a polycyclic compound with a benzocarbazole-like structure. This product was also evaluated for its phototoxic potential. Cell phototoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide test after 72 h from the irradiation in the presence of fluvastatin. The results showed a reduction of the cell viability in a concentration and UVA dose-dependent manner. Surprisingly, the photoproduct showed a dramatic decrease of the cell viability that occurred at concentrations of an order of magnitude lower than the parent compound. Flow cytometric analysis indicated that fluvastatin and its main photoproduct induced principally necrosis as revealed by the large appearance of propidium iodide-positive cells and confirmed also by the rapid drop in cellular adenosine triphosphate levels. Interestingly, a rapid increase of intracellular calcium followed by an extensive cell lipid membrane peroxidation and a significant oxidation of model proteins were induced by fluvastatin and its photoproduct, suggesting that these compounds exerted their toxic effect mainly in the cellular membranes. On the basis of our results, the phototoxicity of fluvastatin may be mediated by the formation of benzocarbazole-like photoproduct that acts as strong photosensitizer.

Indigotine, azorubine, and cochineal red as photoprotectors of manidipine.

BACKGROUND: The calcium antagonists from the group of 1,4-dihydropyridine (DHP) derivatives are photolabile, and the products of their photochemical decomposition have no pharmacological activity. Like the other DHP derivatives, after exposure to light, manidipine undergoes decomposition leading to a weakening of the pharmacological activity. METHOD: The protective influence of selected dyes on manidipine has been tested. The photodegradation of manidipine was performed according to the first version of the ICH Guideline for photostability testing. The solutions were illuminated by a high-pressure lamp HBO-200, and the methods used for evaluation of manidipine photodegradation were HPLC, HPLC-MS, and UV-vis spectrophotometry. Quantitative evaluation of photodegradation was made on the basis of the quantum yields of the processes with the use of Reinecke salt as a chemical actinometer. RESULTS: Results have shown that all the dyes studied, indigotine, azorubine, and cochineal red, slow down the process of manidipine photodegradation, and the most effective is azorubine. The role of the dyes has been determined on the basis of the Stern-Volmer dependence. CONCLUSION: The dyes studied, indigotine, azorubine, and cochineal red, can be potential photoprotectors of manidipine as they quench the excited states of the compound.

Photostability of pitavastatin--a novel HMG-CoA reductase inhibitor.

The photostability of pitavastatin, an HMG-CoA reductase inhibitor used in the treatment of hypercholesterolemia, was investigated. The sample solution was exposed to UV-A radiation and the photodegradation process was monitored by means of spectrophotometric method and HPLC-DAD. Pitavastatin was shown to be photolabile and its photodegradation reaction followed the first-order kinetics with the rate constant k=3.54 x 10(-4)+/-9.43 x 10(-6)s(-1). The chromatograms revealed the presence of four major photoproducts (PP-1-PP-4). The separated and isolated photolytic products were identified using a mass spectrometer coupled with a time of flight (TOF) analyzer. The main reaction observed during exposure to radiation of pitavastatin was photocyclisation leading to formation of four-ring photoproducts.

DFT study on hydroxy acid-lactone interconversion of statins: The case of fluvastatin.

Fluvastatin is a member of the HMG-CoA reductase inhibitor family of drugs, commonly referred to as statins. It is generally known that, under physiological conditions, statins are susceptible to pH-dependent interconversion between their active (hydroxy acid) and inactive (lactone) forms. The mechanism of this interconversion, under both acidic and basic conditions, was investigated theoretically using the density functional theory (DFT) method. Regardless of the conditions, the lactone form was always higher in energy by 6-19 kcal mol(-1). However, under basic conditions, the activation barrier for the hydrolysis was significantly lower (9 kcal mol(-1)) than for the reverse reaction (28 kcal mol(-1)), making the lactone form unstable. The activation barriers under acidic conditions were of comparable height in both directions (22 and 28 kcal mol(-1)), making the occurrence of both forms equally probable. Due to the high activation barrier (>40 kcal mol(-1)), a one-step, direct interconversion between the two forms turned out to be unfavourable. Moreover, the potential energy surface of fluvastatin was briefly inspected, revealing relatively small energetic differences (<5 kcal mol(-1)) between the key conformers.

Identification of photoproducts of hexahydroquinoline derivatives by GC-EI-MS and HPLC-ESI-MS.

Photoproducts of hexahydroquinoline derivatives have been analyzed with gas chromatography electro ionization-mass spectrometry (GC-EI-MS) and high performance chromatography electrospray ionization-mass spectrometry (HPLC-ESI-MS). The study was performed on four HHQ derivatives: 2,6,6-trimethyl-3-carbomethoxy-5-oxo-4-(R-phenyl)-1,4,5,6,7,8-hexahydroquinoline; R=2'-Me, 3'-Me, 2'-MeO, and 3'-MeO. The photochemical degradation of each of the HHQ derivatives led to the appearance of one product. The photoproducts were identified as the corresponding tetrahydroquinoline analogues, which were formed by dehydrogenation of dihydropyridine moiety. In GC-mass spectra, the most frequent way of fragmentation was elimination of CH3* or CH3O* radical of the ester group. In the photoproducts substituted at 2'-position of the phenyl ring, elimination of isobutene (C4H8) was observed. In the photoproducts with 3'-position substituents, elimination of COOCH3* radical was noted.

Photoprotection of 1,4-dihydropyridine derivatives by dyes.

The possibility of increasing the photochemical stability of nisoldipine by using indigotine and azorubine as photoprotectors has been studied. The course of the photodegradation was monitored by means of UV-vis spectrophotometry and HPLC. Quantitative assessments of the nisoldipine photodegradation included evaluation of the quantum yields and kinetic parameters. In order to establish the light intensity absorbed by a system, Reinecke salt was used as a chemical actinometer. The values of the quantum yields (phi) of photodegradation decreased with increasing dye concentration and were 0.24-0.15 and 0.27-0.25 for indigotine and azorubine, respectively. Furthermore, our attention was focused on determination the role of the selected dyes during the photodegradation of nisoldipine and the calculations were made to eliminate an inner filter effect. The values obtained were used to construct a Stern-Volmer plot and calculate the Stern-Volmer constants (KSV). On the basis of the KSV and the values of the quenching constant (kq) the exited state lifetime (tauo) of nisoldipine in the presence of indigotine and azorubine were estimated. The calculated values of tauo for NS were 6.66x10(-6)s--in the presence of indigotine and 2.76x10(-6)s--in the presence of azorubine, indicating that the photodegradation of nisoldipine occurs from the lowest triplet excited state.