Characterization, in vitro dissolution, and pharmacokinetics of different batches of efavirenz raw materials.

OBJECTIVE: To perform the solid-state characterization and the in vitro-in vivo correlation (IVIVC) of three batches of efavirenz (EFV) active pharmaceutical ingredients. SIGNIFICANCE: EFV is an effective anti-HIV drug. Due to the poor aqueous solubility, the rate and extent of EFV absorption deeply depend on its dissolution characteristics. METHODS: Thermal analyses, x-ray diffraction, and particle size distribution were performed. The saturation solubility and dissolution profiles were assessed in 0.5% (w/v) sodium lauryl sulfate (SLS), fasted-state simulated intestinal fluid (FaSSIF), and fed-state simulated intestinal fluid (FeSSIF) using a flow-through cell. Each batch was orally administered to Wistar rats and the pharmacokinetic parameters were correlated with those obtained from in vitro dissolution. RESULTS: All batches of EFV consisted polymorph I. EFV-A presented the lowest particle size distribution [d(v,0.5) = 197.8 µm; d(v,0.9) = 444.6 µm] followed by EFV-B [d(v,0.5) = 223.9 µm; d(v,0.9) = 481.1 µm], and EFV-C [d(v,0.5) = 240.8 µm; d(v,0.9) = 497.3 µm]. The saturated solubility in FaSSIF was 36% and 40% of that in FeSSIF and SLS, respectively. EFV-A presented the fastest rate and largest extension of dissolution than EFV-B and C (79.15%, 69.93% and 54.22%, respectively, as well as the highest maximum plasma concentration. Levels B, C, and multiple-C of IVIVC models were achieved. CONCLUSION: The FaSSIF medium discriminated the dissolution profiles of EFV APIs. Small differences in particle size distribution had a significant impact on the biopharmaceutical parameters of EFV, suggesting that strict control of such parameter is an important aspect during API development and drug formulation.

Validation of a HPLC method for quantification of midazolam in rat plasma: Application during a Maytenus ilicifolia-drug interaction study.

Midazolam (MDZ) is routinely employed as a marker compound of cytochrome P450 3A (CYP3A) activity. Despite the many HPLC-UV methods described to quantify MDZ in plasma, all of them use acetonitrile (ACN) or a mixture of methanol-isopropanol as organic solvent of the mobile phase. Since the ACN shortage in 2008, efforts have been made to replace this solvent during HPLC analysis. A simple, sensitive, accurate and repeatable HPLC-UV method (220 nm) was developed and validated to quantify MDZ in rat plasma using methanol instead. The method was applied during a herb-drug interaction study involving Maytenus ilicifolia, a Brazilian folk medicine used to treat gastric disorders. Plasma samples were alkalinized and MDZ plus alprazolam (internal standard) were extracted with diethyl ether. After solvent removal, the residue was reconstituted with methanol-water (1:1). The analyte was eluted throughout a C18 column using sodium acetate buffer (10 mm, pH 7.4)-methanol (40:60, v/v). The precision at the lower limit of quantification never exceeded 19.40%, and 13.86% at the higher levels of quality control standards, whereas the accuracy ranged from -19.81 to 14.33%. The analytical curve was linear from 50 to 2,000 ng/ml. The activity of the hepatic CYP3A enzymes was not affected by the extract.

Anti-inflammatory and antinociceptive activities of a phenylpropanoid-enriched fraction of Duguetia furfuracea.

A previous study reported the in vivo anti-inflammatory and antinociceptive activities of essential oil of the underground stem bark of Duguetia furfuracea, termed EODf. This study aimed to obtain a phenylpropanoid-enriched fraction from the D. furfuracea (EFDf) essential oil and to investigate its anti-inflammatory and antinociceptive effects. The chemical composition of the EFDf was determined by gas chromatography-mass spectrometry (GC-MS). The in vivo anti-inflammatory activity was evaluated with a lipopolysaccharide (LPS)-induced paw oedema model. The effects of the EFDf on the polymorphonuclear leukocyte recruitment and the inducible nitric oxide synthase (iNOS) expression were evaluated in mice footpads. Moreover, the in vivo antinociceptive effect was assayed using the formalin test and the LPS-induced thermal hyperalgesia model. In the EFDf, 8 major compounds were identified, with α-asarone (36.4%) and 2,4,5-trimethoxystyrene (27.8%) the main constituents. A higher concentration of phenylpropanoid derivatives was found in the EFDf, 64.2% compared to the EODf (38%). The oral (p.o.) treatment with the EFDf at a dose of 3 mg/kg significantly attenuated the paw oedema, polymorphonuclear leukocyte migration, iNOS expression, and tumour necrosis factor alpha (TNF-α) production. The EFDf (10 and 30 mg/kg) also inhibited both phases of the formalin test and caused a significant increase in the reaction time in the LPS-induced thermal hyperalgesia model. Finally, EFDf-treated animals did not show any alteration of motor coordination. The results suggest that the enrichment of 2,4,5-trimethoxystyrene and α-asarone enhances the anti-inflammatory activity of the EFDf compared to the EODf. In contrast, the antinociception promoted by the EFDf was similar to the EODf and was mediated via activation of adenosinergic and opioidergic receptors.

Evaluation of the Effects of Maytenus ilicifolia on the Activities of Cytochrome P450 3A and P-glycoprotein.

BACKGROUND: Maytenus ilicifolia is a Brazilian popular medicine commonly used to treat ulcer and gastritis. Despite the absence of toxicity regarding its consumption, possible interactions when co-administrated with conventional drugs, are unknown. OBJECTIVE: This study aimed to evaluate the effects of M. ilicifolia extracts on Cytochrome P450 3A (CYP3A) and P-glycoprotein (P-gp) activities. METHODS: The extracts were obtained by infusion (MI) or turbo-extraction using hydro-acetonic solvent (MT70). The content of polyphenols in each extract was determined. To assess the modulation of M. ilicifolia on P-gp activity, the uptake of fexofenadine (FEX) by Caco-2 cells was investigated in the absence or presence of MI or MT70. The effect on CYP3A activity was evaluated by the co-administration of midazolam (MDZ) with each extract in male Wistar rats. The pharmacokinetic parameters of the drug were determined and compared with those from the control group. The content of total phenolic compounds, tannins, and flavonoids on MT70 extract was about double of that found in MI. RESULTS: In the presence of the extracts, the uptake of the P-gp marker (FEX) by Caco-2 cells increased from 1.7 ± 0.4 ng.mg-1 protein (control) to 3.5 ± 0.2 ng.mg-1 protein (MI) and 4.4 ± 0.5 ng.mg-1 protein (MT70), respectively. When orally co-administrated with MDZ (substrate of CYP3A), the extracts augmented the AUC(0-∞) (Control: 911.7 ± 215.7 ng.h.mL-1; MI: 1947 ± 554.3 ng.h.mL-1; MT70: 2219.0 ± 506.3 ng.h.mL-1) and the Cmax (Control: 407.7 ± 90.4 ng.mL-1; MI: 1770.5 ± 764.5 ng.mL-1; MT70: 1987.2 ± 544.9 ng.mL-1) of the drug in rats indicating a 50% reduction of the oral Cl. No effect was observed when midazolam was given intravenously. CONCLUSION: The results suggest that M. ilicifolia can inhibit the intestinal metabolism and transport of drugs mediated by CYP3A and P-gp, respectively, however, the involvement of other transporters and the clinical relevance of such interaction still need to be clarified.

Anti-inflammatory and central and peripheral anti-nociceptive activities of α-asarone through the inhibition of TNF-α production, leukocyte recruitment and iNOS expression, and participation of the adenosinergic and opioidergic systems.

Alpha-asarone has been found to possess many pharmacological activities, which can improve cognitive function and exert anti-oxidant, anxiolytic, anti-epileptic and protective effects against endothelial cell injury. The anti-inflammatory activity of α-asarone was evaluated using lipopolysaccharide (LPS)-induced paw oedema. Moreover, leukocyte migration, inducible nitric oxide synthase (iNOS) expression and tumour necrosis factor-alpha (TNF-α) levels were quantified in footpads. Formalin and LPS-induced thermal hyperalgesia models were generated using adenosinergic, opioidergic, serotonergic and muscarinic receptor antagonists. The effects on motor coordination were evaluated by means of the rota-rod test. Oral treatment (p.o.) with α-asarone (3 mg/kg) significantly inhibited paw oedema by 62.12 and 72.22%, 2 and 4 h post LPS injection, respectively. Alpha-asarone (3 mg/kg, p.o.) attenuated the inflammatory infiltrate 1, 3 and 6 h after LPS injection. Furthermore, α-asarone (3 mg/kg, p.o.) suppressed iNOS expression and TNF-α production, 6 and 1 h after inflammatory stimulus, respectively. Alpha-asarone (3, 10 and 30 mg/kg, p.o.) inhibited both phases of formalin-induced licking. In the hot-plate test, α-asarone (10 and 30 mg/kg, p.o.) increased the latency to response 3 and 5 h post LPS stimulus. Caffeine and naloxone abolished the central anti-nociceptive effect of α-asarone (neurogenic phase of formalin and hot plate tests), suggesting the participation of the adenosinergic and opioidergic systems. Furthermore, naloxone reversed the peripheral activity of α-asarone (inflammatory phase of formalin test), indicating the possible involvement of the opioidergic pathway. In the rota-rod test, α-asarone did not change motor coordination. These findings suggest that α-asarone has anti-inflammatory, peripheral and central anti-nociceptive effects and could represent a promising agent for future research.

Systematic evaluation of the impact of solid-state polymorphism on the bioavailability of thalidomide.

Thalidomide (TLD) is used to treat erythema nodosum leprosum (ENL), multiple myeloma, aphthous ulceration and wasting syndrome in HIV patients. The API can be found in two crystalline habits known as α-TLD and ß-TLD. The saturation solubility (Cs) and the dissolution profiles under non-sink and sink conditions of both polymorphs were assessed. In addition, mini-capsules containing α-TLD or ß-TLD without excipients were orally given (10 mg/kg) to Wistar rats. An intravenous (i.v.) dose was also administrated (5 mg/kg). The Cs values for α-TLD and ß-TLD were not significantly different (α = 56.2 ±â€¯0.5 µg·mL-1; ß = 55.2 ±â€¯0.2 µg·mL-1). However, the dissolution profile of α-TLD presented the fastest rate and the largest extension of drug dissolution than that from ß-TLD (80% in 4 h versus 55% in 4 h). The α-TLD provided a more favorable pharmacokinetic than the ß-TLD (maximum plasma concentration - Cmax: 5.4 ±â€¯0.90 µg·mL-1versus 2.6 ±â€¯0.2 µg·mL-1; area under the curve of the concentration-time profile from time zero to infinity - AUC0-∞: 44.3 ±â€¯8.8 µg·h·mL-1versus 33.9 ±â€¯4.7 µg·h·mL-1; absolute bioavailability - F: 92.2 ±â€¯18.5% versus 70.5 ±â€¯9.9%, respectively). Drug suppliers and pharmaceutical companies should strictly control the technological processes involved in the TLD API synthesis as well as in the production of the pharmaceutical dosage form in order to guarantee the inter-batch homogeneity and therefore, product compliance.

Rational selection of immunodominant and preserved epitope Sm043300e from Schistosoma mansoni and design of a chimeric molecule for biotechnological purposes.

Human schistosomiasis is a neglected tropical disease of great importance in public health. A large number of people are infected with schistosomiasis, making vaccine development and effective diagnosis important control strategies. A rational epitope prediction workflow using Schistosoma mansoni hypothetical proteins was previously presented by our group, and an improvement to that approach is presented here. Briefly, immunodominant epitopes from parasite membrane proteins were predicted by reverse vaccinology strategy with additional in silico analysis. Furthermore, epitope recognition was evaluated using sera of individuals infected with S. mansoni. The epitope that stood out in both in silico and in vitro assays was used to compose a rational chimeric molecule to improve immune response activation. Out of 2185 transmembrane proteins, four epitopes with high binding affinities for human and mouse MHCII molecules were selected through computational screening. These epitopes were synthesized to evaluate their ability to induce TCD4+ lymphocyte proliferation in mice. Sm204830e and Sm043300e induced significant TCD4+ proliferation. Both epitopes were submitted to enzyme-linked immunosorbent assay to evaluate their recognition by IgG antibodies from the sera of infected individuals, and epitope Sm043300 was significantly recognized in most sera samples. Epitope Sm043300 also showed good affinity for human MHCII molecules in molecular docking, and its sequence is curiously highly conserved in four S. mansoni proteins, all of which are described as G-protein-coupled receptors. In addition, we have demonstrated the feasibility of incorporating this epitope, which showed low similarity to human sequences, into a chimeric molecule. The stability of the molecule was evaluated by molecular modeling aimed at future molecule production for use in diagnosis and vaccination trials.

Anti-inflammatory effects of the butanolic fraction of Byrsonima verbascifolia leaves: Mechanisms involving inhibition of tumor necrosis factor alpha, prostaglandin E(2) production and migration of polymorphonuclear leucocyte in vivo experimentation.

The leaves of Byrsonima verbascifolia (Malpighiaceae) are traditionally used to treat various diseases including inflammatory conditions. The main goal of this study was to evaluate the in vivo anti-inflammatory activity of the polar constituents from the butanolic fraction of B. verbascifolia leaves (BvBF), as well as to investigate the mechanisms involved in the anti-inflammatory activity. The polar constituents were identified by liquid chromatography coupled to diode array detector and mass spectrometry (LC-DAD­MS) and matrix-assisted laser desorption/ionization ­ time-of-flight mass spectrometry (MALDI-TOF MS) to obtain a complete chemical profile of the fraction. Forty-five compounds were detected in the BvBF by LC-DAD­MS/MS, including condensed tannins, phenolic acids, flavonoids (flavones and flavonols) and other compounds. In addition, several condensed tannins were identified by MALDI-MS/MS, which are composed predominantly by procyanidin units (PCY) and up to six flavan-3-ol units. The BvBF exhibited significant antioxidant and anti-inflammatory activities. The BvBF inhibited paw edema and polymorphonuclear (PMN) leukocyte migration to the footpad and pleural cavity induced by carrageenan. Furthermore, a minor dose (12.50 mg/kg) of BvBF effectively decreased tumor necrosis factor alpha (TNF-α) and prostaglandin E2 (PGE2) levels in the footpad. These findings suggest that the mechanism of the anti-inflammatory action in the BvBF is linked to the inhibition of the production of inflammatory mediators such as TNF-α and PGE2 and the PMN cell migration.

Identification of a vaccine against schistosomiasis using bioinformatics and molecular modeling tools.

Schistosomiasis is a serious public health problem in Brazil and worldwide. Although the drugs used to treatment schistosomiasis are effective, the disease continues to expand in all endemic countries due to constant reinfection, poor sanitation, and the lack of effective programs for disease control. However, advances generated through genome projects have provided important information that has improved the understanding of the biology of this parasite. These advances, associated with the advent of bioinformatic analysis, are becoming an important tool in reverse vaccinology. Through database access to the DNA and protein sequences of Schistosoma mansoni and the use of bioinformatics programs, fourteen epitopes were identified. Five epitopes were obtained from proteins whose immunogenic potential had already been assessed in other studies (KP), and nine whose immunogenic potential is unknown (UP). To improve stimulation of the host immune system, the selected epitopes were modeled with a sugar moiety. After this addition, all of the epitopes showed structures similar to those observed in the native proteins, but only eleven of the peptides presented thermodynamically stable structures. Prediction analysis and molecular modeling showed that the glycopeptides presented here are important targets in the search for a vaccine against schistosomiasis. Additionally, they suggest that these molecules may be used in immunological assays to evaluate the level of protection, the effect on pathology reduction and the profile of cytokines and antibodies induced by them.