Equilibrium Dialysis Method Research Articles

Preclinical pharmacokinetic studies and prediction of human PK profiles for Deg-AZM, a clinical-stage new transgelin agonist.

Deglycosylated azithromycin (Deg-AZM), a newly developed Class I drug with good therapeutic effects on slow transit constipation, is a small-molecule transgelin agonist that has been approved for clinical trials in 2024. The preclinical pharmacokinetic profile of Deg-AZM was investigated to support further development. A LC-MS/MS method was established and validated to detected the concentration of Deg-AZM in various biological samples. In vivo tests such as pharmacokinetic studies in rats and dogs, tissue distribution studies in rats, and extraction studies in rats were conducted to investigated the preclinical pharmacokinetic behaviors of Deg-AZM comprehensively. The plasma protein rate of Deg-AZM was determined by rapid equilibrium dialysis method in vitro. The metabolic stability and metabolite profile of Deg-AZM was assessed using pooled mice, rats, dogs, monkeys and humans microsomes in vitro. The PK profiles of Deg-AZM in human was predicted based on physiologically based pharmacokinetic (PBPK) models. The plasma protein binding rates of Deg-AZM were lower in mice and rats, higher in dogs, and moderate in humans. The metabolic process of Deg-AZM was similar in rat and human liver microsomes. From Pharmacokinetic studies in rats and dogs, Deg-AZM was rapidly absorbed into the blood and then quickly eliminated. Plasma exposure of Deg-AZM was dose dependent with no accumulation after continuous gavage administration. In addition, there is no significant gender difference in the pharmacokinetic behavior of Deg-AZM. Deg-AZM was widely distributed in the tissues without obvious accumulation, and mainly excreted from the urinary excretion pathway. Furthermore, the pharmacokinetic profiles of Deg-AZM in humans showed dose dependency. The pharmacokinetic profiles of Deg-AZM was fully explored, these results could provide valuable information to support the first-in-human dosage prediction and phase I clinical design.

Comparative evaluation of bioavailability of Botulinum toxin A complexed with Tizol (titanium glycerosolvate aquacomplex) versus pure Botulinum toxin A solution for bladder mucosa: an experimental study

Introduction. For the treatment of overactive bladder syndrome (OAB), injection of botulinum toxin A (BoNT-A) has been shown to be effective. However, there is a need for a less invasive method for administering BoNT-A, which could significantly expand the treatment options for OAB.Objective. To assess the impact of tizol on the absorption of BoNT-A by the bladder mucosa and compare it to the individual absorption of BoNT-A.Materials & Methods. Dialysis through the mucous membrane of the сalf bladder was used as an experimental model to study changes in bioavailability of BoNT-A complexed with tisol (BoNT-A + T) and pure BoNT-A solution during in vitro experiment. After dialysis, the BoNT-A concentration in both samples was determined using a spectrophotometer. Dialysis curves were plotted according to the data obtained. Kruvchinsky equilibrium dialysis method was used to determine botulinum toxin A bioavailability. The UV spectrophotometry method was used to determine the concentration of BoNT-A in the acceptor medium by reaction of BoNT-A with Benedict's reagent.Results. It was established that the maximum concentration of BoNT-A diffused into the acceptor medium from the blend of the test substance with tizol after nine hours. The area under the curve for dialysis of BoNT-A + T exceeds the area under the curve of pure BoNT-A by almost 20%, suggesting an improvement in the drug's bioavailability when blended with tizol.Conclusion. Based on our experiment, it was found out that the BoNT-A + T has greater bioavailability than a solution of pure BoNT-A. However, the diffusion rate of the component mixture is sufficiently low.

Species-Specific Unbound Fraction Differences in Highly Bound PFAS: A Comparative Study across Human, Rat, and Mouse Plasma and Albumin.

Per- and polyfluoroalkyl substances (PFAS) are a diverse group of fluorinated compounds which have yet to undergo comprehensive investigation regarding potential adverse health effects and bioaccumulative properties. With long half-lives and accumulative properties, PFAS have been linked to several toxic effects in both non-clinical species such as rat and mouse as well as human. Although biological impacts and specific protein binding of PFAS have been examined, there is no study focusing on the species-specific fraction unbound (fu) in plasma and related toxicokinetics. Herein, a presaturation equilibrium dialysis method was used to measure and validate the binding of 14 individual PFAS with carbon chains containing 4 to 12 perfluorinated carbon atoms and several functional head-groups to albumin and plasma of mouse (C57BL/6 and CD-1), rat, and human. Equivalence testing between each species-matrix combination showed positive correlation between rat and human when comparing fu in plasma and binding to albumin. Similar trends in binding were also observed for mouse plasma and albumin. Relatively high Spearman correlations for all combinations indicate high concordance of PFAS binding regardless of matrix. Physiochemical properties of PFAS such as molecular weight, chain length, and lipophilicity were found to have important roles in plasma protein binding of PFAS.

Methods of mathematical planning of the experiment and their use in the development of the composition of the ointment with essential oil thymus vulgaris l. For vaginal application

One of the most important pharmaceutical factors is excipients, their nature and quantity, which increase the pharmacological activity and safety of APIs by creating dosage forms that are optimal in composition, properties, and type, and/or by changing the chemical modification of the molecule responsible for the pharmacological effect of the active substance. Variability or variation in the number of excipients and the use of different excipients with the same intended functionality can complicate the release rate and bioavailability of drugs. The use of mathematical methods at the stage of the experiment planning reduces the total number of experiments and at the same time provides reliable results. Aim. To study the most used types of mathematical planning of the experiment, and select the most acceptable model for developing the optimal composition of a vaginal ointment with the thyme (Thymus vulgaris L.) essential oil. Results and discussions. To develop the optimal composition of the ointment, 10 bases containing various excipients (factor A) and 6 excipients as an emulsifier (factor B) were used. The concentration of the thyme essential oil was 5 % in all samples. The degree of thymol release in the in vitro study using the equilibrium dialysis method was chosen as the criterion for choosing the optimal composition. The selection of excipients for the development of a soft dosage form was carried out using the method of the a priori ranking since both bases and emulsifiers were selected as the most widely used in the pharmaceutical technology, with known characteristics, safety profile, application experience. Conclusions. The degree of influence of each factor has been determined, and the series of Duncan’s test have been constructed for each factor – by factor a: а3 > а5 > а4 > а6 > а2 > а8 > а7 > а10 > а9> а1, and by factor b: b2 > b3 > b5 > b4 > b6 > b1. Based on the mathematical planning of the experiment, an ointment base and an emulsifier for developing the composition of a vaginal gel with the thyme oil have been determine.

Determination of plasma protein binding rate of Shuganning Injection using equilibrium dialysis and UPLC-MS/MS

Traditional Chinese medicine(TCM) compound preparations have complex compositions. As a widely used TCM injection, Shuganning Injection, its in vivo processes are not yet fully understood. Determining the plasma protein binding rate is of great significance for pharmacokinetic and pharmacodynamic studies. In this experiment, the equilibrium dialysis method combined with UPLC-MS/MS technology was used to determine the plasma protein binding rates of 10 components, including p-hydroxyacetophenone, caffeic acid, baicalein, oroxylin A, geniposide, baicalin, cynaroside, oroxylin A-7-O-β-D-glucuronide, scutellarin, and hyperoside, in Shuganning Injection in rat and human plasma to provide a theoretical basis for further elucidating the in vivo processes of Shuganning Injection and guiding clinical medication. The results showed that, except for baicalein and geniposide, the plasma protein binding rates of the other eight components were higher in human plasma than in rat plasma, and there were interspecies differences. In human plasma, except for geniposide, caffeic acid, and baicalin, the plasma protein binding rates of the remaining seven components were above 80%, with baicalein and oroxylin A exceeding 90%. All components exhibit a high level of binding to plasma proteins, with the exception of geniposide.

Formulation and evaluation of controlled-release, carrageenan-based powder formulations filled into hard gelatin capsules

Hard gelatin capsules (HGCs) are typically used as immediate release dosage forms, however, controlled drug release can from HGCs be obtained by the application of film coating onto the capsule shells or by filling them with controlled release multi-particulates (e.g. pellets and mini-tablets).
 The filling of a hydrophilic gelling polymer into the capsule is an alternative approach to the time-consuming preparation and filling of multi-particulates. Carrageenan is a linear, sulfated polysaccharide that is commonly used as a thickener in pharmaceutical formulations.
 The release behavior of propranolol HCl – carrageenan powder mixtures filled into hard gelatin capsules was investigated as a function of drug – to – polymer ratio, the capsule fill weight, grade of carrageenan used. In addition, the effect of the drug release testing method (USP apparatus I or II), rotation speed, pH of the release medium and ionic strength of the release medium on drug release were investigated.
 The electrostatic interaction of propranolol HCl with carrageenan was also investigated using equilibrium dialysis method to determine the binding capacity and the stability constant of the complex formed.
 Viscarin GP 109 was found to provide a fast-gelling behavior with excellent controlled release properties. Drug loading and ionic strength of the medium significantly influenced drug release. Release in USP apparatus 1 was slower than that in USP apparatus 2. Propranolol HCl formed an insoluble complex with carrageenan with a high binding capacity and stability

Development and research of ointment with essential oil of thyme for the treatment of the scalp and hair parts of the head

Dermatological diseases are the most widespread in the whole world. According to WHO, 20–30% of the world's population suffers from fungal skin diseases and 25–35% from pustular diseases. Pustular diseases (pyoderma) are the result of pathogenic staphylococci and streptococci, and fungal diseases (dermatomycoses) are caused by various types of fungi. Today, these diseases are treated with various types of drugs, but the active ingredient in almost all of them is synthetic compounds (antibiotics, sulfonamides, imidazole derivatives, etc.). Medicines of natural origin are scarce. In order to increase the number of phytoremedies for external use (ointments), which have medicinal plant active components, a study was conducted using standardized essential oil of common thyme.
 The aim of the work was to study the effect of excipients on the ointment base by the method of mathematical planning of the experiment for the development of the optimal composition of the dermatological ointment with the essential oil of common thyme.
 Excipiennts for ointment bases of hydrophobic and emulsion composition were used to develop the optimal composition of the ointment. The concentration of thyme essential oil was 5% in all samples. The degree of thymol release in an in vitro study using the equilibrium dialysis method was chosen as the criterion for choosing the optimal composition. Excipiennts allowed in pharmacy, with known characteristics, safety profile, and application experience were used as auxiliary substances. Using Duncan's multiple criterion, a number of advantages were created: а7 > а8 > а9 > а10 > а6 >а2 > а5 > а3 > а1 > а4.
 On the basis of the conducted studies, the optimal composition of the ointment was chosen for the treatment of lesions of the scalp and hair. The studied organoleptic and technological properties and quality indicators (homogeneity, colloidal stability, thermal stability, microbiological purity) of ointment with thyme essential oil during long-term storage at a temperature of +2–+8 °C established the stability of the developed composition of the ointment during two years of observation. The creation of a new local remedy for the treatment of lesions of the scalp and its hair part will allow to expand the arsenal of safe and effective remedies.

Doxycycline serum protein binding in pigs reveals a relatively high free fraction.

Doxycycline is an antibiotic widely used in pig farming. As with all antibiotics, only the free concentrations are considered to be bacteriologically active. Historically, the free fraction (fu) in pig plasma has been estimated at 7%, which, given the effective dosage regime used in pigs, leads to free plasma concentrations of doxycycline largely lower than the minimum inhibitory concentrations of the target pathogens. This apparent inconsistency led us to reassess plasma protein binding of doxycycline in pigs. Using an equilibrium dialysis method, the extent of doxycycline binding was measured individually in 26 pigs for total doxycycline concentration ranging from 10 to 1000 μmol/L. Analysis of the data using a non-linear mixed-effects model demonstrated linearity of plasma protein binding with a mean fu value of 31% and a relatively low inter-subject variability of approximately 10%. This new data showing that the free fraction is four times greater than what could have been anticipated from historical data is discussed in particular for the calculation of the PK/PD cut-offs, which are used to establish the clinical breakpoints for antimicrobial susceptibility testing.

Development of Cinnarizine Liposome Technology

Introduction. Liposomal preparations have a number of advantages: they protect the cells of the body from the toxic effects of drugs; prolong the action of the drug introduced into the body; protect medicinal substances from degradation; contribute to the manifestation of targeted specificity due to selective penetration from the blood into tissues; change the pharmacokinetics of drugs, increasing their pharmacological efficacy; allow you to create a water-soluble form of a number of medicinal substances, thereby increasing their bioavailability.In this work, studies were carried out to develop a methodology for determining the degree of inclusion in liposomes from soy lecithin of cinnarizine, which has found wide application as a corrector of cerebral circulation disorders.Purpose. The purpose of the study is to determine the amount of adsorption of cinnarizine with liposomes from soy lecithin.Materials and methods. Cinnarizine liposomes from soy lecithin were prepared by the hydration/rehydration method. To study the characteristics of the degree of incorporation of cinnarizine into liposomes, the method of equilibrium dialysis was used. The choice of this method is due to the fact that the quantitative analysis of the equilibrium concentration of cinnarizine in the dispersion medium, which is necessary to determine the amount of adsorption, is hampered by the presence of a dispersed phase, liposomes. A semipermeable membrane with a pore diameter sufficient for the penetration of cinnarizine molecules, but impermeable to liposomes, makes it possible to obtain a cinnarizine solution with a concentration close enough to the concentration in the dispersion medium of liposomes. The solution thus obtained can be subjected to quantitative analysis using spectrophotometry.Results. A graph of the dependence of the value of adsorption of cinnarizine on liposomes on the equilibrium concentration was plotted. It was found that the value of adsorption of cinnarizine during the treatment of liposomes with ultrasound is less for all the studied concentrations. At an equilibrium concentration of cinnarizine of more than 0.0003 mol/l, the proportion of the prearat associated with liposomes stabilizes. Without sonication at the level of 24.83 ± 1.15%, with sonication at the level of 18.4 ± 1.20%.Conclusion. It has been established that ultrasonic treatment of liposomes is expedient when cinnarizine is added to a dry lipid film, since it is a factor that increases bioavailability

Circulating dihydrotestosterone, testosterone, and free testosterone levels and dihydrotestosterone-to-testosterone ratios in healthy women across the menstrual cycle

To characterize the circulating androgen levels across the menstrual cycle in healthy women using highly sensitive and accurate methods and report sex differences in the relative levels of dihydrotestosterone (DHT) to testosterone (T) levels. Prospective cohort study. Research clinic, academic teaching hospital. Twenty-one healthy premenopausal women, aged 19-40 years, with regular menstrual cycles. Not applicable. Serum total T and DHT levels measured using liquid chromatography-tandem mass spectrometry, free T levels measured using a standardized equilibrium dialysis method coupled with measurement of the T levels in the dialysate using liquid chromatography-tandem mass spectrometry, and comparison of the DHT-to-T ratio between healthy women and age-matched healthy men. The serum total and free T levels increased across the follicular phase and peaked at midcycle (total T, 43.6 ± 16.2 ng/dL; free T, 15.6 ± 11.9 pg/mL) and gradually declined in the luteal phase. The DHT level did not significantly change across the menstrual cycle. The DHT-to-T ratios were 1:4 and 1:13 in women and men, respectively. In healthy premenopausal women, the total and free T levels varied significantly across the menstrual cycle, whereas the DHT levels did not change; the peak total and free T levels in the midcycle period were higher than previously reported, underscoring the importance of establishing menstrual phase-specific reference ranges to avoid misdiagnosis of hyperandrogenism. Women have significantly higher DHT levels relative to total T than men; the significance of this sex difference in the DHT-to-T ratio needs further investigation.

PSAT329 A Delayed Diagnosis of Subclinical Hypothyroidism Due to Interferences with Thyroid Function Immunoassays

Abstract Introduction Automated immunoassays used to evaluate thyroid function tests (TFTs) are susceptible to various forms of interference that can lead to inaccurate results and misdiagnosis. Case Description A 55-year-old male was diagnosed with abnormal TFTs since 2017 [TSH 7.32 mIU/L (range 0.4-4.5), FT3 5.1 pg/mL (range 2.3-4.2), and FT4 1.2 ng/dL (range 0.9-2.2)]. Thyroid peroxidase antibody and thyroglobulin antibody were positive. He was on levothyroxine 50 mcg daily since 2017, and he did not feel significantly different despite treatment. He was referred to endocrine clinic in 2021. He had no symptoms of hypothyroidism or hyperthyroidism except occasional palpitations and headache. He reported blurred vision due to cataracts. He denied thyroid diseases in first degree relatives. He denied taking biotin or desiccated thyroid extract. Physical examination was unremarkable. Differentials included TSH-producing adenoma, assay interference, exogenous T3 use, T3 thyrotoxicosis with interference of TSH assay, and thyroid hormone resistance. Levothyroxine was discontinued in May 2021. Other pituitary hormones including alpha-subunit and MRI pituitary were unremarkable, which ruled out TSH-producing adenoma. Human anti mouse antibody (HAMA) was normal. In June 2021, prior to seeing genetics, FT4, FT3 and Total T3 were checked with equilibrium dialysis method and values were 1.2 ng/dL (range 0.9-2.2), 238 pg/dL (range 210-440) and 105 ng/dL (range 76-181), respectively and TSH 9.13 mIU/L (range 0.40-4.50), thyroglobulin 0.6 ng/ml (range 2.8-40.9), reverse T3 LC/MS/MS 12 ng/dL (range 8-25). Labs were therefore consistent with subclinical hypothyroidism, and he was started back on levothyroxine 75 mcg every morning. Discussion TFTs are usually measured by automated immunoassays, and this will have accurate results in most cases. However, immunoassays are vulnerable to interferences that can affect clinical decisions. It is important to consider assay interference such as this and to confirm with equilibrium dialysis method before pursuing further aggressive workup. Conclusion Potential interferences in TFTs should always be suspected whenever discrepancies arise. Awareness of these conditions is critical to the institution of appropriate therapy and preventing misdiagnosis. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

Reference intervals for free testosterone in adult men measured using a standardized equilibrium dialysis procedure.

Free testosterone (FT) determination may be helpful in evaluating men suspected of testosterone deficiency especially in conditions with altered binding-protein concentrations. However, methods for measuring FT by equilibrium dialysis and reference intervals vary among laboratories. To determine reference intervals for FT in healthy, nonobese men by age groups as well as in healthy young men, 19-39 years, using a standardized equilibrium dialysis procedure METHODS: We measured FT in 145 healthy, nonobese men, 19 years or older, using a standardized equilibrium dialysis method performed for 16-h at 37°C using undiluted serum and dialysis buffer that mimicked the ionic composition of human plasma. FT in dialysate was measured using a CDC-certified liquid chromatography tandem mass spectrometry assay. In healthy nonobese men, the 2.5th, 10th, 50th, 90th, and 97.5th percentile values for FT were 66, 91, 141, 240, and 309 pg/ml, respectively; corresponding values for men, 19-39 years, were 120, 128, 190, 274, and 368 pg/ml, respectively. FT levels by age groups exhibit the expected age-related decline. FT levels were negatively associated with body mass index, age, and sex hormone-binding globulin (SHBG) levels. Percent FT was lower in middle-aged and older men than young men adjusting for SHBG level. Further studies are needed to determine how these reference intervals apply to the diagnosis of androgen deficiency in clinical populations and in men of different races and ethnicities in different geographic regions. Reference intervals for free FT levels (normative range 66-309 pg/ml [229-1072 pmol/L] in all men and 120-368 pg/ml [415-1274 pmol/L] in men, 19-39 years), measured using a standardized equilibrium dialysis method in healthy nonobese men, provide a rational basis for categorizing FT levels. These intervals require further validation in other populations, in relation to outcomes, and in randomized trials.

IN-VITRO STUDIES OF THE EFFECT OF ARSENIC ON THE BINDING OF METRONIDAZOLE AT THE BINDING SITES OF BOVINE SERUM ALBUMIN

The effect of arsenic on the binding of metronidazole (an anti-amoebic agent) to bovine serum albumin (BSA) was studied by equilibrium dialysis (ED) method in order to justify the nature of binding capability of the drug and arsenic to BSA. There was decrease in metronidazole concentration due to addition of arsenic. But the free metronidazole concentration was increased with increasing the metronidazole concentration when only the BSA was present. The result obtained when no arsenic was added the free concentration of metronidazole was 41% whereas this release was only 7% when arsenic was added with an increasing concentration from 0.5x10-5 to 10x10-5M. But the free concentration of metronidazole was 36% to 71% when only metronidazole was added to BSA from 0.5x10-5 M to12x10-5M. This result suggests that in presence of arsenic, metronidazole shows a greater binding to BSA and results in a decreased serum drug concentration. It could be suggested that in the presence of arsenic, metronidazole may form complex with arsenic, arsenic may increases the binding affinity to its sites or may form complex to BSA and metronidazole.

Study the interactions between multiple flavonoids and bovine serum albumin by the developed equilibrium dialysis

The therapeutic function of traditional Chinese medicine (TCM) is based on the combination effect of multiple active ingredients. However, the current pharmacological studies mainly focus on the protein binding of the single component from TCM, which is difficult to explain the overall therapeutic mechanism. Thus in this work the equilibrium dialysis method combined with high performance liquid chromatography (HPLC) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed to study the interactions between multi-components and protein. Firstly, the binding constants of seven different structural types of flavonoids with bovine serum albumin (BSA) were determined. The results showed that the binding affinity of flavones and flavonols with BSA was stronger than that of dihydroflavonoids, and the substitution of glycosides would reduce the binding affinity with BSA. The results of competitive displacement experiment showed that there existed competitive interactions among the four flavonoids (rutin, luteolin, hesperetin and kaempferol). The binding constants of flavonoids to BSA were significantly changed under the condition of multi-components coexistence. Especially, the binding constant of hesperetin to BSA increased from 9.46 × 104 L/mol to 1.49 × 106 L/mol under the coexistence of rutin. The results of fluorescence spectroscopy showed that the reason for competitive binding was that the four flavonoids were mainly bound to the IIA region of BSA. Finally, the method was successfully applied to study the binding of multiple components in Radix Scutellariae (RS) extract with BSA. Five flavonoids in RS extract were identified by UPLC-MS/MS, they had different degrees of binding to BSA, among which oroxylin A had the strongest binding degree. In conclusion, the equilibrium dialysis was reliable and sufficiently accurate for study of the interaction between multi-components or TCM extract and protein, which can provided a theoretical basis for the scientific explanation of the overall treatment mechanism of TCM.

A validated UHPLC-MS/MS method for quantification of total and free tedizolid concentrations in human plasma

Tedizolid (TZD) is a novel oxazolidinone antibiotic. Although TZD efficacy correlates with area under the concentration-time curve/minimum inhibitory concentration, there is no report of pharmacokinetic/pharmacodynamic analysis using plasma free TZD concentrations. Several methods have been developed for measuring total TZD concentration, but not for free TZD concentration. We aimed to develop a high-throughput simultaneous quantification method for total and free TZD concentrations using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). The equilibrium dialysis method was used for separation of the free fraction. Pretreatment was conducted by solid-phase extraction using 96-well HLB µElution plate. Chromatographic separation of the analytes was conducted using a C18 column. MS/MS transitions were monitored in the positive ion mode. Full validation was performed in accordance with the bioanalytical method validation guidance prepared by the US Food and Drug Administration (FDA). The assay showed good linearity over wide ranges of 5–5000 (r2 = 0.9964) and 1.5–1500 (r2 = 0.9990) ng/mL for total and free TZD concentrations, respectively. Within-batch accuracy and precision as well as batch-to-batch accuracy and precision for total and free concentrations fulfilled the criteria of the FDA guidance. The recovery rates were higher than 92.3% and higher than 85.3% for total and free concentrations. Matrix effect showed no remarkable differences among three quality control levels for total and free concentrations. In vitro protein binding rates of TZD ranged from 71.6% to 76.9%, indicating no concentration-dependent difference within the calibration ranges. The total and free concentrations in five patients who received TZD were within the ranges of the calibration curves, demonstrating the feasibility of clinical application of the novel method. In conclusion, we have succeeded to develop for the first time a method for simultaneous quantification of total and free TZD concentrations.

STUDYING THE OSMOTIC ACTIVITY OF A POLYCOMPONENT REPARATIVE GEL

Abstract. Hydrogel-based wound dressings are currently in demand due to their positive effect on reparative processes. The range of such is quite diverse and is represented by gelling agents of both synthetic and natural origin. The latter are most often biopolymers, among which collagen deserves special attention. The most important properties of collagen are its biocompatibility, low level of immunogenicity, ability to biodegrade, which leads to the formation of degradation products that stimulate cell proliferation and the biosynthesis of native collagen. Collagen has optimal physical characteristics, which is important for ensuring the structural and mechanical properties of the compositions. In addition, collagen has been successfully used as a carrier for other pharmacologically active substances. In this regard, the expediency of developing a multicomponent gel with a predictable reparative effect based on collagen and a thick extract of maize columns with stigmas was shown. This medicinal plant raw material has anti-inflammatory, antioxidant, immunotropic effects, which suggests its positive effect on tissue healing processes. One of the important properties of the reparative gel is its osmotic activity, which determines the ability to absorb exudate and cleanse the surface of the wound from purulent secretions, reduce tissue hyperthermia and inflammatory edema. The purpose of this study is to study the osmotic activity of a multicomponent gel of reparative action based on collagen and a thick extract of maize columns with stigmas. The object of the study was experimental samples of a polycomponent gel. Osmotic activity was studied by the method of equilibrium dialysis according to Kruvchinsky. For the purpose of a comparative assessment of the osmotic activity of a multicomponent gel, a control study was carried out in parallel under the same conditions for a 0.9% sodium chloride solution. The results obtained demonstrated the presence of hyperosmolar characteristics in the experimental samples of the polycomponent gel during the first four hours of dialysis, which allows us to predict its positive effect on the processes of cleansing damaged tissues from pathological secretions and their effective repair.

Precooked sorghum flour as proper vehicle of ACE‐I and DPP‐IV inhibitory sorghum peptides

AbstractSorghum flour was heat treated for producing an instant dispersion ingredient. The precooked sorghum flour was added with ACE‐I and DPP‐IV inhibitory sorghum peptides (3.0 g peptide 100 g−1). The product was reconstituted in water, and peptide bioaccessibility was evaluated by equilibrium dialysis method after simulated gastrointestinal digestion. Total peptide dialysability of precooked sorghum flour added with sorghum peptides was higher than those obtained for precooked sorghum flour (315.9 ± 14.8 vs. 45.2 ± 5.6 µmol, respectively) (P < 0.05). The ACE‐I and DPP‐IV‐IC50 values of the bioaccessible peptides from the bioactive product were lower than those obtained for precooked sorghum flour ingredient (1.04 ± 0.12 vs. 1.82 ± 0.09 and 0.86 ± 0.02 vs. 2.12 ± 0.08 mg protein mL−1, for ACE‐I and DPP‐IV, respectively) indicating a higher activity. Precooked sorghum flour was a good vehicle since it did not affect the bioaccessibility of ACE‐I and DPP‐IV inhibitory peptides provided by sorghum protein hydrolysate.

Optimizing a High-Throughput Solid-Phase Microextraction System to Determine the Plasma Protein Binding of Drugs in Human Plasma.

Plasma protein binding refers to the binding of a drug to plasma proteins after entering the body. The measurement of plasma protein binding is essential during drug development and in clinical practice, as it provides a more detailed understanding of the available free concentration of a drug in the blood, which is in turn critical for pharmacokinetics and pharmacodynamics studies. In addition, the accurate determination of the free concentration of a drug in the blood is also highly important for therapeutic drug monitoring and in personalized medicine. The present study uses C18-coated solid-phase microextraction 96-pin devices to determine the free concentrations of a set of drugs in plasma, as well as the plasma protein binding of drugs with a wide range of physicochemical properties. It should be noted that the extracted amounts used to calculate the binding constants and plasma protein bindings should be measured at respective equilibrium for plasma and phosphate buffer. Therefore, special attention is placed on properly determining the equilibration times required to correctly estimate the free concentrations of drugs in the investigated systems. The plasma protein binding values obtained with the 96-pin devices are consistent with those reported in the literature. The 96-pin device used in this research can be easily coupled with a Concept96 or other automated robotic systems to create an automated plasma protein binding determination protocol that is both more time and labor efficient compared to conventional equilibrium dialysis and ultrafiltration methods.

Coexistence of Thyroid Hormone Resistance and Autoimmune Thyroid Disease: Not a Mere Coincidence

Introduction: The coexistence of thyroid hormone resistance and autoimmune thyroiditis was initially thought to be a chance event. In a large cohort study, Barcoff et al. demonstrated an increased likelihood of thyroid autoantibodies in patients with thyroid hormone resistance (RTH). We report a unique case to epitomize the coexistence of these two conditions and discuss the postulated mechanisms.Clinical Case: A 22-year-old woman with a history of Hashimoto’s thyroiditis, attention deficit hyperactivity disorder, and migraine presented to the endocrinology clinic with symptoms of weight loss, fatigue, decreased appetite, and heat intolerance for 4 months. She was diagnosed with Hashimoto’s thyroiditis at age 12 with elevated TSH only and had been on levothyroxine 25 mcg since diagnosis. Physical exam demonstrated a body mass index of 34.14 kg/m2, blood pressure of 138/91 mmHg, pulse of 77 bpm, and an enlarged palpable thyroid gland. Laboratory investigations revealed elevated thyroid peroxidase (TPO) antibody at 234 IU/mL (<9) and thyroglobulin Antibody at 3 IU/mL (<1) with elevated free T4 of 3.76 ng/dL (0.61-1.36), elevated total T4 of 21.58 mcg/dL (6.09-12.23), increased T3 uptake of 51.2% uptake (32-48.4), and upper normal TSH of 3.99 uIU/mL (0.44-5.33). Elevated free T4 was confirmed by the equilibrium dialysis method at 6.2 ng/dL (0.9-2.2) with upper normal TSH at 3.77 uIU/mL. Thyroid ultrasound demonstrated thyromegaly and hypervascularity of the gland. TSH secreting pituitary adenoma was ruled out with a normal alpha subunit of 0.2 ng/ml (0.1-0.6) and molar ratio of the alpha subunit to TSH ratio <1 along with a normal pituitary MRI. She was not taking other medications or supplements. She was diagnosed with thyroid hormone resistance and is undergoing genetic testing to differentiate the THRB genetic mutations from Non-TR-RTH. Her symptoms improved after discontinuing her levothyroxine.Clinical Lesson: Our case highlights the importance of evaluating thyroid disorders properly before starting treatment and illustrates the coexistence of autoimmune thyroiditis with thyroid hormone resistance. Barcoff et al. demonstrated that there was an increased likelihood of thyroid autoantibodies with odds ratio = 2.36 (p = 0.002) in a large patient cohort with RTH, compared to their unaffected first-degree relatives. However, since there was no correlation in increased antibody with increased age and the duration of the disease, the proposed hypothesis did not substantiate chronic TSH stimulation inducing autoimmune response. The proposed mechanism is that the elevated TSH in RTH stimulates the immune system at the TRα level which was demonstrated in murine models with increased activation of thymic function in correlation with TSH level. More research is needed to understand the underlying mechanism of their coexistence.

Elucidation of plasma protein binding, blood partitioning, permeability, CYP phenotyping and CYP inhibition studies of Withanone using validated UPLC method: An active constituent of neuroprotective herb Ashwagandha

Ethnopharmacological relevanceWithanone (WN), an active constituent of Withania somnifera commonly called Ashwagandha has remarkable pharmacological responses along with neurological activities. However, for a better understanding of the pharmacokinetic and pharmacodynamic behavior of WN, a comprehensive in-vitro ADME (absorption, distribution, metabolism, and excretion) studies are necessary. Aim of the studyA precise, accurate, and sensitive reverse-phase ultra-performance liquid chromatographic method of WN was developed and validated in rat plasma for the first time. The developed method was successfully applied to the in-vitro ADME investigation of WN. Material and methodsThe passive permeability of WN was assayed using PAMPA plates and the plasma protein binding (PPB) was performed using the equilibrium dialysis method. Pooled liver microsomes of rat (RLM) and human (HLM) were used for the microsomal stability, CYP phenotyping, and inhibition studies. CYP phenotyping was evaluated using the specific inhibitors. CYP inhibition study was performed using specific probe substrates along with WN or specific inhibitors. ResultsWN was found to be stable in the simulated gastric and intestinal environment and has a high passive permeability at pH 4.0 and 7.0 in PAMPA assay. The PPB of WN at 5 and 20 μg/mL concentrations were found to be high i.e. 82.01 ± 1.44 and 88.02 ± 1.15%, respectively. The in vitro half-life of WN in RLM and HLM was found to be 59.63 ± 2.50 and 68.42 ± 2.19 min, respectively. CYP phenotyping results showed that WN was extensively metabolized by CYP 3A4 and1A2 enzymes in RLM and HLM. However, the results of CYP Inhibition studies showed that none of the CYP isoenzymes were potentially inhibited by WN in RLM and HLM. ConclusionThe in vitro results of pH-dependent stability, plasma stability, permeability, PPB, blood partitioning, microsomal stability, CYP phenotyping, and CYP inhibition studies demonstrated that WN could be a better phytochemical for neurological disorders.