Overcoming drug impurity challenges in amorphous solid dispersion with rational development of biorelevant dissolution-permeation method.

Hot-melt extrusion is often used to prepare amorphous solid dispersion to overcome low drug solubility and enhance bio-performance of the formulation. Due to the uniqueness of each drug - polymer combination and its physico-chemical properties, setting the appropriate HME barrel temperature, feed rate and screw speed ensures drug amorphization, absence of residual crystallinity, absence of water, and a suitable drug release profile. In this research, samples with BCS II/IV model drug and PVP/VA polymer were prepared to evaluate the impact of HME process parameters, incoming drug form (anhydrous vs. hydrate), and drug supplier (i.e., impurity profile), on biorelevant drug release. This study provides a relationship between observed in vitro supersaturation and precipitation behavior of amorphous solid dispersion formulation with in vivo results, on patients, by using the acceptor profile of side-by-side dissolution-permeation apparatus. An in vitro dissolution method, in small volumes, in an apparatus with paddles and dissolution-permeation side-by-side method was developed on the MicroFlux™ apparatus to assess if the differences observed in vitro bears relevance to the bioequivalence outcome in vivo. The former was used to guide the generic drug product development due to high discriminatory strength, while the latter was biorelevant, due to the inclusion of the second compartment assuring absorptive environment to capture the impact of supersaturation and subsequent precipitation on bioavailability. Bio-relevancy of the in vitro method was confirmed with the in vivo dog study and clinical study on patients, and an in vitro - in vivo correlation was established. For the investigated BCS II/IV drug, this research highlights the importance of considering supersaturation and formation of colloidal species during amorphous solid dispersion release testing to assure product quality, safety and efficacy.

Nonradioactive northern blotting for the determination of acetylcholinesterase mRNA. Comparison to the radioactive technique.

A sensitive nonradioactive northern blotting for the detection of acetylcholinesterase mRNA in mammalian tissues is described and compared to its radioactive version. Best results were obtained if digoxigenin labeled RNA probe was used for hybridization and CDP-Star, a chemiluminescent alkaline phosphatase substrate, for detection. The described nonradioactive technique for acetylcholinesterase mRNA determination is as sensitive as the radioactive one, but requires no protection against radiation and is less time consuming. Because of higher stability of the labeled probe, nonradioactive technique is also more convenient from the standpoint of experimental planning.

Control levels of acetylcholinesterase expression in the mammalian skeletal muscle.

Protein expression can be controled at different levels. Understanding acetylcholinesterase (EC. 3.1.1.7, AChE) expression in the living organisms therefore necessitates: (1) determination and mapping of control levels of AChE metabolism; (2) identification of the regulatory factors acting at these levels; and (3) detailed insight into the mechanisms of action of these factors. Here we summarize the results of our studies on the regulation of AChE expression in the mammalian skeletal muscle. Three experimental models were employed: in vitro innervated human muscle, mechanically denervated adult fast rat muscle, and the glucocorticoid treated fast rat muscle. In situ hybridization of AChE mRNA, combined with AChE histochemistry, revealed that different distribution patterns of AChE, observed during in vitro ontogenesis and synaptogenesis of human skeletal muscle, reflect alterations in the distribution of AChE mRNA (Z. Grubic, R. Komel, W.F. Walker, A.F. Miranda, Myoblast fusion and innervation with rat motor nerve alter the distribution of acetylcholinesterase and its mRNA in human muscle cultures, Neuron 14 (1995) 317-327). To study the mechanisms of AChE mRNA loss in denervated adult rat skeletal muscle, we exposed deproteinated AChE mRNA to various subcellular fractions in vitro. Fractions were isolated from the normal and denervated rat sternomastoideus muscle. We found significantly increased, but non-specific AChE mRNA degradation capacities in the three fractions studied, suggesting that increased susceptibility of muscle mRNA to degradation might be at least partly responsible for the decreased AChE mRNA observed under such conditions (K. Zajc-Kreft, S. Kreft, Z. Grubic, Degradation of AChE mRNA in the normal and denervated rat skeletal muscle, Book of Abstracts, The Sixth International Meeting on Cholinesterases, La Jolla, CA, March 20-24, 1998, p. A3.). In adult fast rat muscle, treated chronically with glucocorticoids, we found the fraction of early synthesized AChE molecular forms to be reduced and AChE mRNA unchanged. This observation is consistent with the explanation that translation and/or early post-translational processes are impaired under such conditions (M. Brank, K. Zajc-Kreft, S. Kreft, R. Komel, Z. Grubic, Biogenesis of acetylcholinesterase is impaired, although its mRNA level remains normal, in the glucocorticoid-treated rat skeletal muscle, Eur. J. Biochem. 251 (1998) 374-381). The AChE mRNA level is therefore important but not the only control level of AChE expression in the mammalian skeletal muscle.

Localization of cells expressing AChE mRNA in rat striatum using nonradioactive in situ hybridization.

A better understanding of the role of AChE in mammalian brain requires knowledge of the distribution of AChE synthesizing cells in this tissue. The aim of the present study is to test a nonradioactive approach for the localization of AChE mRNA positive cells in rat striatum. Nonradioactive in situ hybridization has not been used before for the localization of this mRNA in mammalian brain. In order to find optimal conditions for localization, we employed both RNA and oligonucleotide probes. We also examined various prehybridization protocols and approaches. The total number of cells in brain sections was determined by subsequent fluorescent staining of the nuclei. Optimal AChE mRNA localization was obtained with a digoxigenine-labeled RNA probe. We were not able to localize AChE mRNA with nonradioactively 3' end-labeled oligonucleotides. An acetylation step prior to hybridization was found to be essential for optimal signal/background ratios; high nonspecific staining was observed, if this step was omitted. In accordance with reports of other authors, who used radioactive in situ hybridization, we found very low percentages of AChE mRNA-positive cells in striatum, although this area exhibits very high AChE staining. In comparison to radioactive techniques, the nonradioactive approach avoids the risks of radioactivity, and is much less time consuming. In our experiments AChE mRNA localization in striatum was practically the same as that demonstrated previously by radioactive approaches.

Biogenesis of acetylcholinesterase is impaired, although its mRNA level remains normal, in the glucocorticoid-treated rat skeletal muscle.

Acetylcholinesterase (AChE) is responsible for the hydrolysis of acetylcholine in the neuromuscular junction and other cholinergic synapses. Insight into the mechanisms controlling AChE expression in skeletal muscle is important for understanding formation, plasticity, and various dysfunctions of the neuromuscular junction. We have investigated the mechanisms responsible for the decreased AChE activity in the fast rat sternomastoideus muscle after chronic glucocorticoid treatment. Under such conditions fast skeletal muscles become atrophic and loose 30-40% of their AChE activity. In order to establish at which level synthesis of AChE is affected by glucocorticoids, we studied the effects of chronic dexamethasone treatment at both AChE mRNA and mature enzyme levels. Reduced rate of AChE recovery after subtotal irreversible AChE inhibition was observed during the first week of dexamethasone treatment, but not later. Statistical analyses of four independent northern blots revealed unchanged AChE mRNA levels. At the same time, we observed more than 60% decrease in the (G1+G2)/A12 ratio of molecular forms at the expense of G forms. It has been generally accepted that globular G1 and G2 molecular forms are synthesized in the rough endoplasmic reticulum as precursors of asymmetric (A) AChE forms, assembled in the Golgi apparatus. Reduced levels of G1 and G2 AChE forms, in combination with unchanged AChE mRNA, are therefore consistent with the reports demonstrating that glucocorticoids downregulate muscle protein synthesis at the translational level. Our findings support but not entirely prove the concept that impaired translation and/or posttranslational control are the primary cause of decreased AChE activity in the glucocorticoid-treated muscle.

Application of the nonradioactive in situ hybridization for the localization of acetylcholinesterase mRNA in the central nervous system of the rat; comparison to the radioactive technique.

In this preliminary report nonradioactive digoxigenine-based and radioactive in situ hybridization procedures for the localization of acetylcholinesterase mRNA were tested and compared in rat brain. General patterns of Ache mRNA localization observed by both techniques did not differ significantly and were practically the same as reported in previous in situ studies on the mammalian brain. Shorter procedure time and avoidance of precautions necessary at work with radioactive materials are major advantages of nonradioactive technique. Under- and over- staining can be prevented by direct examination of coloring reaction. Faint staining in the control experiment with heterologous DNA suggests that proper stringency is essential for the specificity of staining.