Quadrivalent hemagglutinin and adhesion expressed on Saccharomyces cerevisiae induce protective immunity against Mycoplasma gallisepticum infection and improve gut microbiota.

Mycoplasma gallisepticum (MG) infection causes infectious respiratory diseases in poultry, causing economic losses to the poultry industry. Therefore, this study aims to develop a safe, convenient, and effective multivalent recombinant Saccharomyces cerevisiae vaccine candidate and to explore its potential for oral immunization as a subunit vaccine. Mycoplasma gallisepticum Cytadhesin (MGC) and variable lipoprotein and hemagglutinin (vlhA) are associated with the pathogenesis of MG. In this study, a quadrivalent recombinant Saccharomyces cerevisiae (ST1814G-MG) displaying on MGC2, MGC3, VLH5, and VLH3, proteins was innovatively constructed, and its protective efficiency was evaluated in birds. The results showed that oral immunization with ST1814G-MG stimulates specific antibodies in chickens, reshapes the composition of the gut microbiota, reduces the Mycoplasma loading and pulmonary disease injury in the lungs. In addition, we found that oral ST1814G-MG had better protection against MG infection than an inactivated vaccine, and co-administration with the inactivated vaccine was even more effective. The results suggest that ST1814G-MG is a potentially safer and effective agent for controlling MG infection.

Construction of Anthocyanin Biosynthesis System Using Chalcone as a Substrate in Lactococcus lactis NZ9000.

Anthocyanins are high-value natural compounds, but to date, their production still mainly relies on extraction from plants. A five-step metabolic pathway was constructed in probiotic Lactococcus lactis NZ9000 for rapid, stable, and glycosylated anthocyanin biosynthesis using chalcone as a substrate. The genes were cloned from anthocyanin-rich blueberry: chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanin synthase (ANS), and UDPG-flavonoid 3-O-glycosyltransferase (3GT). Using HR, the polysaccharide pellicle (PSP) segment of the cell wall polysaccharide synthesis (cwps) gene cluster from L. lactis NZ9000 was cloned into vector p15A-Cm-repDE. Then, CHI and F3H were placed sequentially under the control of NZProm 3 of this gene cluster in the vector, which was transformed into L. lactis NZ9000 to obtain Strain A. Furthermore, Strain B was constructed by placing F3H-DFR-ANS and 3GT under NZProm 2 and 3, respectively. Using LC-MS/MS analysis, several types of anthocyanins, including callistephin chloride, oenin chloride, malvidin O-hexoside, malvidin 3,5-diglucoside, and pelargonidin 3-O-malonyl-malonylhexoside, increased in the supernatant of the co-culture of Strains A and B compared to that of L. lactis NZ9000. This is the first time that a five-step metabolic pathway has been developed for anthocyanin biosynthesis in probiotic L. lactis NZ9000. This work lays the groundwork for novel anthocyanin production by a process involving the placement of several biosynthesis genes under the control of a gene cluster.

Identification of an Overlooked Halogen-Bond Synthon and Its Application in Designing Fluorescent Materials.

Research on new supramolecular synthons facilitates the progress of materials design. Herein, the ability of sp2 carbonyl oxygen atoms to act as halogen-bond acceptors was established through cocrystallization. Four sets of carbonyl compounds, including aldehydes, ketones, esters, and amides, were selected as halogen-bond acceptors. In the absence of strong hydrogen bonds, 14 out of 16 combinations of halogen-bond donors and acceptors could form cocrystals, whereby the supramolecular synthon C=O⋅⋅⋅X acts as the main interaction. Further, the geometric parameters of the C=O⋅⋅⋅X interaction were statistically revealed on the basis of the crystallographic database. The bifurcated interaction mode that has been observed in other halogen-bond synthons rarely occurs in the case of C=O⋅⋅⋅X. The robustness of C=O⋅⋅⋅X makes its application in crystal engineering possible and opens up new opportunities in designing multicomponent fluorescent materials, as indicated by multicolor emission of cocrystals D through C=O⋅⋅⋅X interactions.

Confocal Raman micro-spectral evidence and physicochemical evaluation of triamterene salts.

Discrimination of active pharmaceutical ingredients (APIs) existing as neutral molecules or salts is essential and complicated. However, the discrimination of pharmaceutical salts by confocal Raman micro-spectroscopy remains relatively poorly understood. In this paper, four new salts of triamterene (Tri) cocrystallized with nicotinic acid (NA), benzoic acid (BA), p-toluenesulfonic acid (TA), or isonicotinic acid (INA) were prepared and characterized comprehensively by powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), polarized light microscopy (PLM), and dynamic vapor sorption (DVS). Ionized pteridine is identified by marker peaks in the confocal Raman micro-spectra that are characteristic of C[double bond, length as m-dash]N. The single crystal structures of Tri-NA·H2O and Tri-TA further demonstrate that a proton transfers from the carboxylic group of NA or TA to the pyrimidine N1 atom of Tri and their salts formation take place. The intrinsic dissolution rate (IDR) and apparent equilibrium solubility of these four salts are improved compared to the pure Tri component, especially for Tri-BA. This study provides a valuable insight into pharmaceutical salt discrimination by vibrational spectroscopy and presents that the combination of Tri with an acid can be a possible and promising alternative formulation of Tri.

Amino acids as co-amorphous excipients for tackling the poor aqueous solubility of valsartan.

Co-amorphization has recently been shown to be a promising approach for stabilizing amorphous drugs and improving the dissolution rate of poorly water-soluble drugs. In this study, three basic amino acids were chosen as small molecular weight excipients to interact with the drug to form co-amorphous combinations. The co-amorphous combinations of valsartan (VAL) with l-histidine, l-arginine, and l-lysine were prepared by vibrational ball milling. Solid-state characterization with X-ray powder diffraction and differential scanning calorimetry (DSC) revealed that all of the co-amorphous mixtures were homogeneous. The molecular interactions of the co-amorphous mixtures were investigated through the glass transition temperature (Tg) in the DSC measurements and Fourier transform infrared spectroscopy. The drug remained chemically stable during the milling process, and the co-amorphous formulations were generally physically stable over at least 3 months at 40 °C under dry conditions. The dissolution rate of all of the co-amorphous mixtures was significantly increased over that of the amorphous VAL alone. The results of this study demonstrated the potential of amino acids as small molecular weight excipients in co-amorphous formulations to improve the drug solubility and dissolution rate.

Thermodynamic and kinetic investigation of agomelatine polymorph transformation.

Thermodynamic properties of polymorphic forms I and II of Agomelatine were investigated and the bimorphism was determined to be monotropically related. The phase transition kinetics from metastable form I to thermodynamically stable form II was studied and a quantification method was developed based on X-ray powder diffraction technique. Various solid-state kinetic models were examined and the results were fit to the experimental data. The nucleation kinetic models were found to be the best fit to describe the experimental data across the temperature range. The activation energy of the form transformation was calculated in the range of 116-122 kJ mol(-1), irrespective of which kinetic model selected.

Improving the dissolution and bioavailability of 6-mercaptopurine via co-crystallization with isonicotinamide.

6-Mercaptopurine (6-MP) is a clinically important antitumor drug. The commercially available form was provided as monohydrate and belongs to BCS class II category. Co-crystallization screening by reaction crystallization method (RCM) and monitored by powder X-ray diffraction led to the discovery of a new co-crystal formed between 6-MP and isonicotinamide (co-crystal 1). Co-crystal 1 was thoroughly characterized by X-ray diffraction, FT-IR and Raman spectroscopy, and thermal analysis. Noticeably, the in vitro and in vivo studies revealed that co-crystal 1 possesses improved dissolution rate and superior bioavailability on animal model.

[Polymorph transformation of solid drugs].

For the minimized phase transtorming risk, the most stable polymorph is generally considered as the desirable solid form for pharmaceutical applications. However, occasionally, the stable form may have some shortcomings such as low solubility, dissolution rate and bioavailability, etc. In that case, the metastable form which is kinetically stable at room or lower temperature could be selected. Using metastable form may result in polymorph transformation in pharmaceutical manufacture and storage. Hence, the knowledge of the transformation between solid forms is essential to the development of the drug materials. In this paper, we will review the recent studies in the area of crystal conversion of polymorphs and hydrates, to illustrate some cases to introduce the types, conditions and mechanisms of the crystalline solid transformation.

[Yuleshu oral mixture combined with conventional therapy for chronic prostatitis].

OBJECTIVE: To evaluate the clinical effects of Yuleshu oral mixture combined with conventional therapy on chronic prostatitis. METHODS: Eighty-eight patients with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) were equally randomized to a control and an experimental group to receive conventional therapy (oral antibiotics, alpha blockers, proprietary Chinese medicine for activating blood circulation and massage of the prostate) and conventional therapy combined with Yuleshu oral mixture respectively. Before and after treatment, the severity of symptoms and sexual function of the patients were evaluated using NIH-CPSI and IIEF-5, their anxiety, depression and other emotional problems assessed with Hamilton Depression Rating Scale (HAMD) and Hamilton Anxiety Scale (HAMA), and the results subjected to statistical analysis. RESULTS: Both the experimental and control groups showed significant improvement in prostatitis symptoms and sexual function after treatment as compared with the baseline (P < 0.01), even more significant in the former than in the latter group, especially in pain symptoms (7.89 +/- 2.82 vs 10.41 +/- 2.55, P < 0.01). Before and after treatment, the HAMA and HAMD score had no significant difference in the control, but there was significant difference in the experimental group. The experimental group exhibited remarkably higher scores after than before treatment on HAMA (24.30 +/- 5.07 vs 13.80 +/- 3.62, P < 0.01) and HAMD (23.81 +/- 5.01 vs 16.23 +/- 5.93, P < 0.01), but not the control group (P > 0.05). CONCLUSION: Yuleshu oral mixture can effectively relieve anxiety, depression and other psychological problems in CP/CPPS patients, and improve their clinical symptoms as well. Therefore, it is an effective drug for chronic prostatitis.

Improving the Dissolution Rate and Bioavailability of Curcumin via Co-Crystallization.

Curcumin (CUR) is a natural polyphenolic compound with various pharmacological activities. Low water solubility and bioavailability limit its clinical application. In this work, to improve the bioavailability of CUR, we prepared a new co-crystal of curcumin and L-carnitine (CUR-L-CN) via liquid-assisted grinding. Both CUR and L-CN have high safe dosages and have a wide range of applications in liver protection and animal nutrition. The co-crystal was fully characterized and the crystal structure was disclosed. Dissolution experiments were conducted in simulated gastric fluids (SGF) and simulated intestinal fluids (SIF). CUR-L-CN exhibited significantly faster dissolution rates than those of pure CUR. Hirshfeld surface analysis and wettability testing indicate that CUR-L-CN has a higher affinity for water and thus exhibits faster dissolution rates. Pharmacokinetic studies were performed in rats and the results showed that compared to pure CUR, CUR-L-CN exhibited 6.3-times-higher AUC0-t and 10.7-times-higher Cmax.

Enhancing physical and chemical stability of hygroscopic hydroxytyrosol by cocrystal formation.

Hydroxytyrosol (HT) is a natural phenolic compound with potent antioxidant activity extracted from olive trees. It is generally a slightly hydrated viscous liquid at ambient conditions, and it is highly susceptible to oxygen due to the presence of catechol moiety. Although encapsulation technique provides HT in powder form, it does not improve its chemical stability. Herein, we propose an efficient solution to the high hygroscopicity and poor stability of HT. Four cocrystals were first reported, and their intermolecular interactions were analyzed in detail. After cocrystallization, the melting point is increased and the hygroscopicity is significantly decreased. HT cocrystals are thus solid at room temperature. Moreover, hydroxytyrosol cocrystals with betaine (HT-BET) and nicotinamide (HT-NIC) demonstrate superior chemical stability than pure HT, olive extract, and HT encapsulation material. Therefore, cocrystallization can be considered as a promising approach to overcome the application obstacles of HT.

Rucaparib cocrystal: Improved solubility and bioavailability over camsylate.

Rucaparib (Ruc) is a drug used to treat advanced ovarian cancer associated with deleterious BRCA mutations. Its commercial form, the camsylate salt (Ruc-Cam), suffers from poor aqueous solubility and thus causes low and erratic oral bioavailability. In this work, we aimed to improve the oral exposure of Ruc through cocrystallization. Liquid-assisted grinding, slurry, and solvent evaporation methods were employed to prepare new solid forms of Ruc. Cocrystals of rucaparib-theophylline monohydrate (Ruc-Thp MH), rucaparib-maltol (Ruc-Mal), and rucaparib-ethyl maltol (Ruc-Emal) were obtained. Powder X-ray diffraction, Fourier transform infrared spectroscopy, thermogravimetric analysis, differential scanning calorimetry, and dynamic vapor sorption were utilized to characterize these multi-component systems. All cocrystals dissolve faster than Ruc-Cam at pH 2.0 and 4.5, and Ruc-Thp MH displays the highest apparent solubility in pH 4.5 and 6.8 buffers. Pharmacokinetic studies in rats show that Ruc-Thp MH exhibits 2.4 times the Cmax and 1.4 times the AUC0-24h at a single dose compared with Ruc-Cam. The enhanced solubility and bioavailability of Ruc-Thp MH showcase the power of cocrystallization in addressing absorption issues in drug development.

The Cocrystal of Ubiquinol: Improved Stability and Bioavailability.

Coenzyme Q10 (CoQ10) exists in two forms, an oxidized form and a reduced form. Ubiquinol is the fully reduced form of CoQ10. Compared to the oxidized form, ubiquinol has a much higher biological absorption and better therapeutic effect. However, ubiquinol has an important stability problem which hampers its storage and formulation. It can be easily transformed into its oxidized form-ubiquinone-even at low temperature. In this work, we designed, synthesized, and characterized a new cocrystal of ubiquinol with vitamin B3 nicotinamide (UQ-NC). Compared to the marketed ubiquinol form, the cocrystal exhibited an excellent stability, improved dissolution properties, and higher bioavailability. The cocrystal remained stable for a long period, even when stored under stressed conditions. In the dissolution experiments, the cocrystal generated 12.6 (in SIF) and 38.3 (in SGF) times greater maximum ubiquinol concentrations above that of the marketed form. In addition, in the PK studies, compared to the marketed form, the cocrystal exhibited a 2.2 times greater maximum total coenzyme Q10 concentration and a 4.5 times greater AUC than that of the marketed form.

Improving the dissolution behaviors and bioavailability of abiraterone acetate via multicomponent crystal forms.

Abiraterone acetate (ABA), the first-line drug for the treatment of metastatic castration resistant prostate cancer (mCRPC), is administered at a high daily dosage of 1000 mg due to its poor solubility, and its fasted absolute oral bioavailability is estimated to be less than 10%. In this work we have focused on developing multicomponent forms with improved dissolution behaviors and bioavailability. Two salts of ABA with malonic acid (ABA-MA) and saccharin (ABA-SAC), and five cocrystals with trans-aconitic acid (ABA-TAA), 1-hydroxy-2-naphthoic acid (ABA-1HNA), pyrocatechol (ABA-PCA), resorcinol (ABA-RES) and hydroquinone (ABA-HDE) were successfully obtained. Their crystal structures were elucidated by single crystal X-ray diffraction, and these multicomponent forms were fully characterized by powder X-ray diffraction, thermal analysis and Fourier Transform Infrared spectra. Among them, ABA-TAA cocrystal shows substantial enhancements both in the solubility and intrinsic dissolution rates in different buffer solutions. In the meantime, we unexpectedly found the gelation of ABA-MA salt and ABA-SAC salt in pH 2.0 buffer solution. The gel-like materials generated on the surface of drug will suppress the release of ABA. Moreover, in vivo pharmacokinetic study on beagle dogs was conducted for ABA-TAA cocrystal preparation and ABA commercial product, and ABA-TAA cocrystal preparation shows enhanced absorption. These advantages in dissolution behaviors and bioavailability demonstrate the potential of ABA-TAA cocrystal to be a better candidate for the treatment of mCRPC compared with ABA.

Dehydroepiandrosterone Cocrystals with Improved Solubility and Bioavailability.

Dehydroepiandrosterone (DHEA) is an FDA-approved food supplement used as an assisted reproductive sex hormone. The bioavailability is severely limited by its poor solubility (23 µg/mL). Herein, we aimed to modulate its solubility through cocrystallization. Eight cocrystals of DHEA with pyrocatechol (CAT), hydroquinone (HQ), resorcinol (RES), phloroglucinol (PG), 1,5-dihydroxy naphthalene (DHN), p-hydroxybenzoic acid (PHBA), gallic acid (GA), and 5-hydroxyisophthalic acid (5HIPA) were designed and synthesized. Some basic characterization tools, including powder X-ray diffraction, thermogravimetric analysis, differential scanning calorimetry, and Fourier transform infrared spectroscopy, were also applied in our work for basic analyses of cocrystals. It is indicated that DHEA-GA exhibits its superiority in dissolution and pharmacokinetic behaviors. While the area under the curve values of DHEA-GA is improved at the ratio of 2.2, the corresponding bioavailability of DHEA is expected to be accordingly increased.

Cocrystal Prediction of Bexarotene by Graph Convolution Network and Bioavailability Improvement.

Bexarotene (BEX) was approved by the FDA in 1999 for the treatment of cutaneous T-cell lymphoma (CTCL). The poor aqueous solubility causes the low bioavailability of the drug and thereby limits the clinical application. In this study, we developed a GCN-based deep learning model (CocrystalGCN) for in-silico screening of the cocrystals of BEX. The results show that our model obtained high performance relative to baseline models. The top 30 of 109 coformer candidates were scored by CocrystalGCN and then validated experimentally. Finally, cocrystals of BEX-pyrazine, BEX-2,5-dimethylpyrazine, BEX-methyl isonicotinate, and BEX-ethyl isonicotinate were successfully obtained. The crystal structures were determined by single-crystal X-ray diffraction. Powder X-ray diffraction, differential scanning calorimetry, and thermogravimetric analysis were utilized to characterize these multi-component forms. All cocrystals present superior solubility and dissolution over the parent drug. The pharmacokinetic studies show that the plasma exposures (AUC0-8h) of BEX-pyrazine and BEX-2,5-dimethylpyrazine are 1.7 and 1.8 times that of the commercially available BEX powder, respectively. This work sets a good example for integrating virtual prediction and experimental screening to discover the new cocrystals of water-insoluble drugs.

Conformational polymorphs of isotretinoin and their impact on physicochemical and biological properties.

Isotretinoin is the first-line drug for treatment of severe acne. Only one polymorph was reported even though it has been launched for nearly 40 years, and its clinic application was however limited by its stability and solubility challenges. In our study, two new polymorphs of isotretinoin were discovered and fully characterized. The transformation relationships between these solid forms were fully discussed, and a visible color change during single-crystal-to-single-crystal phase transition with the conformational change was investigated. Form II is determined to be thermodynamic stable form at room temperature, but metastable form at body temperature. The results show that form II is an ideal solid state possessing both superior thermal stability (60℃, open air) and higher absorption once delivered into body. The thermal stability can be associated with the crystal structure such as torsion angle. The relative bioavailability of form II is higher than form I as expected, and the bioavailability of form II formulation is about 2 times as that of the marketed form I capsule. Therefore, form II formulation could provide an alternative for better performing isotretinoin.

Circular RNA circSETD3 hampers cell growth, migration, and stem cell properties in bladder cancer through sponging miR-641 to upregulate PTEN.

Bladder cancer (BLCA) is a common malignant urothelial cancer in the world. Although circular RNAs (circRNAs) involve in regulating BLCA progression, the role of a novel circular RNA circSETD3 in regulating BLCA pathogenesis has not been studied. The expression of circSETD3, miR-641, PTEN mRNA in BLCA tissues and cell lines were measured using RT-qPCR. The gain-of-function experiments were performed in vitro and in vivo to detect the effects of circSETD3 on cell proliferation, migration, EMT, and stemness maintenance. Besides, rescue experiments were performed to demonstrate the regulatory mechanism of circSETD3/miR-641/PTEN in BLCA cell malignant phenotypes in vitro. CircSETD3 was remarkably downregulated in the cancerous clinical tissues and cell lines, in contrast with their normal counterparts, and circSETD3 tended to be deficient in BLCA patients with larger tumor size, advanced clinical stages, positive lymph metastasis and worse prognosis. In addition, circular isoforms of circSETD3 were more resistant to RNase R+ and actinomycetes D treatment compared to their linear isoforms, and circSETD3 mainly distributed in the cytoplasm of the BLCA cells. Further gain-of-function experiments showed that circSETD3 acted as a tumor suppressor to suppress BLCA cell proliferation, migration, EMT and stemness, and the underlying mechanisms had also been elucidated. Mechanistically, circSETD3 sponged miR-641 to upregulate PTEN, resulting in the blockage of BLCA progression. Our findings indicated that circSETD3 acted as a vital tumor suppressor in BLCA via regulating the miR-641/PTEN axis.

Cocrystals to tune oily vitamin E into crystal vitamin E.

d-α-tocopherol (d-αToc), the most biologically active form of natural Vitamin E, is oily in appearance and unstable to oxygen. Esterification and encapsulation are generally needed to stabilize and solidify d-αToc for the purpose of its expanding applications. In this study, we propose a more effective way to stabilize and solidify d-αToc oil in one step. By cocrystallization, the melting point of d-αToc is significantly increased, such that the oily d-αToc is successfully transformed into solid form at room temperature. The single crystal structure of d-αToc was firstly uncovered and the molecular interaction in cocrystals was revealed. Crystalline Vitamin E shows high stability to light and temperature. Its spherical crystallization affords good powder flowability, which is extremely important as food or feed additives. Moreover, cocrystal Vitamin E remains the original form of tocopherol without esterification and thus has a great advantage on higher bioavailability. Cocrystallization of oily d-αToc spares the use of acetic ester and a mass of excipients, which is of great environmental importance and greatly reduces the production cost.

Systematic review and meta-analysis: α-adrenergic receptor blockers in chronic prostatitis.

BACKGROUND: Prostatitis seriously endangers the health of men. While they have been widely used in recent years, there remains a lack of systematic evaluation of the clinical efficacy of α-receptor blockers (α-RBs)/α-adrenergic receptor blockers (α-ARBs) in its treatment. Based on this, this study was developed to systematically evaluate the clinical effect of α-ARB in the treatment of prostatitis. METHODS: Randomized controlled trials (RCTs) studying α-RBs or α-ARBs, placebos, or other measures to treat prostatitis were searched in Cochrane Library, PubMed, Embase, and CBM databases from establishment to December 2020. The quality of included articles was evaluated using the Cochrane System Review Manual and Jadad tools, and a meta-analysis was performed using Review Manager 5.3 software. RESULTS: A total of six articles meeting the requirements were found and included 450 patients. Meta-analysis showed that the National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI) score [mean difference (MD) =-1.76, 95% confidence interval (CI): (-3.35 to -0.17), and P=0.03], pain score [MD =-2.24, 95% CI: (-3.65 to -0.83), and P=0.002], voiding symptom score [MD =-1.21, 95% CI: (-2.06 to -0.35), and P=0.006], and quality of life score [MD =-1.40, 95% CI: (-1.48 to -1.33), and P<0.00001] for patients in the experimental group were lower in contrast to those in the control group after the treatment. DISCUSSION: The use of α-ARB could significantly improve the treatment effect of patients with prostatitis and improve their quality of life.