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.

The role of occlusion and micro-incontinence in the pathogenesis of penile lichen sclerosus: an observational study of pro-inflammatory cytokines' gene expression.

PURPOSE: To assess the expression of selected cytokines in penile lichen sclerosus (PLS) and associate them with the occurrence of micro-incontinence (MI) in different stages of PLS. METHODS: The skin biopsies from 49 PLS affected, and 13 from nonlesional foreskins (healthy control adult males undergoing circumcision due to phimosis caused by short frenulum) were obtained. All specimens were used for RNA extraction and RT-qPCR. Quantitative assessment of the gene expression of interleukin 1-A (IL-1A), interleukin 1-B (IL-1B), interleukin 1 receptor antagonist (IL-1RN), interleukin 6 (IL-6), transforming growth factor ß1 (TGF-ß1), and interferon-gamma (INF-γ) was performed. To determinate the presence of MI, the patients were asked about voiding patterns, especially leaking tiny drops of urine from the urethral meatus after urination. RESULTS: IL-1A, IL-6, and INF-γ mRNA levels were approximately 150, 16, and 59 times higher in PLS than in control samples, respectively. The highest IL-1A mRNA levels were observed in early PLS (n = 13), INF-γ in moderate PLS (n = 32), while IL-6 in severe PLS (n = 4). MI was noted in 45 PLS patients vs. 0 in control (p < 0.0001). IL-1A and IL-6 vs control ratios were concentration (ca.) 400 and 30 times higher, respectively, in MI PLS samples than in PLS without MI. CONCLUSION: Occlusion and irritating urine effect are associated with the clinical progression of penile LS with increased mRNA expression of IL-1A, INF-γ, and IL-6 pro-inflammatory cytokines in the foreskin.

Decreased systemic IL-7 and soluble IL-7Rα in multiple sclerosis patients.

Polymorphisms (single-nucleotide polymorphism (SNP)) in the interleukin-7 receptor-α (IL-7Rα)/IL-7 pathway are associated with an increased risk to develop multiple sclerosis (MS). The rs6897932 SNP in the IL-7Rα leads to increased soluble IL-7Rα production. Given the functional interaction between sIL-7Rα, membrane-bound IL-7Rα and IL-7, we assessed IL-7, mIL-7Rα and sIL-7Rα levels in MS patients and healthy controls (HCs). One-hundred and twenty eight MS patients had significantly lower sIL-7Rα levels compared with 73 HCs. The levels of sIL-7Rα increased dose-dependent upon rs6897932 [C] risk allele carriership in both HCs and MS. Next, we hypothesized that lower sIL-7Rα could result in a higher mIL-7Rα to soluble IL-7Rα ratio. Indeed, 52 MS patients had significantly increased mIL-7Rα to sIL-7Rα ratio for both CD4 and CD8 T cells compared with 44 HCs. Given the supposed role of IL-7 in autoimmunity, we determined whether sIL-7Rα influences IL-7 levels. IL-7 levels were significantly decreased in 40 MS patients compared with 40 HCs. In conclusion, MS patients had lower free IL-7 and a higher membrane to soluble IL-7Rα ratio. The soluble IL-7Rα levels correlate with the rs6897932 [C] risk allele carriership. The skew at the IL-7 and IL-7Rα level may influence responsiveness of IL-7Rα(+) cells.

Callosal lesion predicts future attacks after clinically isolated syndrome.

BACKGROUND: Current MRI criteria can help predict a second attack after a clinically isolated syndrome (CIS). Given the known association between corpus callosum lesions (CC) and multiple sclerosis (MS), such lesions on MRI could provide additional predictive information. This study assessed whether the presence of CC lesion on MRI could, next to the modified Barkhof criteria, further enhance prediction of conversion from CIS to MS. METHODS: Follow-up study of 158 patients with CIS who underwent MRI after CIS was performed. MRI were scored for the Barkhof criteria and CC lesion. Patients were classified as having MS according to Poser criteria. Cox regression models were used for the time to conversion from CIS to MS. RESULTS: The Barkhof criteria and CC lesion were strongly associated with conversion to MS with hazard ratios (HR), respectively, of 2.6 (95% confidence interval [CI] 1.5-4.3) and 2.7 (95% CI 1.6-4.5). The HRs of CC lesion adjusted for the Barkhof criteria and the Barkhof criteria adjusted for CC lesion were similar (HRs 1.8, not significant). The combined prediction of the Barkhof criteria and CC lesion was 3.3 (95% CI 1.9-5.7). Patients not fulfilling the Barkhof criteria had a fourfold increased risk of MS (HR 3.8, 95% CI 1.5-9.3) when they had a lesion in the CC. CONCLUSIONS: Corpus callosum (CC) lesion and the Barkhof criteria both predicted conversion to multiple sclerosis (MS). When both variables were combined, the association was stronger. The assessment of CC lesion may be a useful additional tool for predicting conversion to MS in patients with clinically isolated syndrome.

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.

Qualitative determination of polyvinylpyrrolidone type by near-infrared spectrometry.

Soluble polyvinylpyrrolidones are very useful and versatile pharmaceutical auxiliaries. The different types of povidone are characterised by their viscosity measured in water, expressed as a K-value. We have developed a rapid, accurate, reliable, and non-destructive near infrared (NIR) spectroscopy method for the determination of PVP type and consequently identification thereof. We have implemented chemometrics onto NIR spectra collected in diffuse reflectance mode using fibre optics to build a qualitative model that enables us to obtain useful analytical information. A principal component analysis and a modelling technique soft independent modelling of class analogy (SIMCA) were applied. An approach to validate the method was developed.

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.