Silk Fibroin Microneedle Patches for the Treatment of Insomnia

As a patient-friendly technology, drug-loaded microneedles can deliver drugs through the skin into the body. This system has broad application prospects and is receiving wide attention. Based on the knowledge acquired in this work, we successfully developed a melatonin-loaded microneedle prepared from proline/melatonin/silk fibroin. The engineered microneedles’ morphological, physical, and chemical properties were characterized to investigate their structural transformation mechanism and transdermal drug-delivery capabilities. The results indicated that the crystal structure of silk fibroin in drug-loaded microneedles was mainly Silk I crystal structure, with a low dissolution rate and suitable swelling property. Melatonin-loaded microneedles showed high mechanical properties, and the breaking strength of a single needle was 1.2 N, which could easily be penetrated the skin. The drug release results in vitro revealed that the effective drug concentration was obtained quickly during the early delivery. The successful drug concentration was maintained through continuous release at the later stage. For in vivo experimentation, the Sprague Dawley (SD) rat model of insomnia was constructed. The outcome exhibited that the melatonin-loaded microneedle released the drug into the body through the skin and maintained a high blood concentration (over 5 ng/mL) for 4–6 h. The maximum blood concentration was above 10 ng/mL, and the peak time was 0.31 h. This system indicates that it achieved the purpose of mimicking physiological release and treating insomnia.

Prevalence of Ketosis Detected by an Electronic Cow Side Test and its Impact to Days Open on Dairy Cows

The study aimed to resolve and confirm the previous results of subclinical and clinical ketosis prevalence in northwestern Croatia detected by an electronic cow side test and its impact on days open in dairy herds. Cows (N=559) 2-8 years old from 96 farms located in northwestern Croatia were included in the study. The average milk yield was 7327.80 ± 968.21 kg. The cows were classified into two separate groups: ketotic cows group KET (n=73) with BHBA blood concentration of ≥1.4 mmol/L, and negative NEG (n=486) group with serum BHBA level of <1.4 mmol/L. One droplet of blood from the caudal vein was collect from randomly selected cows in the period 7 to 15 days after parturition. BHBA level determined with Precision Xceed BHBA devices. The period from parturition to first insemination (days open to the first (artificial) insemination, DOFI) was shorter in NEG than in the POS group (110.56±10.65 days vs.114.82±12.23 days, respectively) such as period from parturition to successful conception (days open to successful conception, DOSC), (139.97±15.18 days vs. 127.99±15.87 days, respectively). The prevalence of clinical and subclinical ketosis was 15.02%. Ketosis significantly prolonged days open to first artificial insemination and days open to successful conception in cows from northwestern Croatia.

Detecting low blood concentrations in joints using T1 and T2 mapping at 1.5, 3, and 7 T: an in vitro study

Background Intra-articular blood causes irreversible joint damage, whilst clinical differentiation between haemorrhagic joint effusion and other effusions can be challenging. An accurate non-invasive method for the detection of joint bleeds is lacking. The aims of this phantom study were to investigate whether magnetic resonance imaging (MRI) T1 and T2 mapping allows for differentiation between simple and haemorrhagic joint effusion and to determine the lowest blood concentration that can be detected. Methods Solutions of synovial fluid with blood concentrations ranging from 0 to 100% were scanned at 1.5, 3, and 7 T. T1 maps were generated with an inversion recovery technique and T2 maps from multi spin-echo sequences. In both cases, the scan acquisition times were below 5 min. Regions of interest were manually drawn by two observers in the obtained T1 and T2 maps for each sample. The lowest detectable blood concentration was determined for all field strengths. Results At all field strengths, T1 and T2 relaxation times decreased with higher blood concentrations. The lowest detectable blood concentrations using T1 mapping were 10% at 1.5 T, 25% at 3 T, and 50% at 7 T. For T2 mapping, the detection limits were 50%, 5%, and 25%, respectively. Conclusions T1 and T2 mapping can detect different blood concentrations in synovial fluid in vitro at clinical field strengths. Especially, T2 measurements at 3 T showed to be highly sensitive. Short acquisition times would make these methods suitable for clinical use and therefore might be promising tools for accurate discrimination between simple and haemorrhagic joint effusion in vivo.

A Method to Measure Voxelotor Exposure in People with Sickle Cell Disease Using Capillary Zone Electrophoresis

Background: Voxelotor (Oxbryta®) is a small molecule that binds to the alpha chain of hemoglobin (Hb) and increases the affinity of Hb for oxygen which reduces sickle Hb polymerization. It was approved by the FDA in 2019 for the treatment of sickle cell disease (SCD). Currently, the only method available to estimate the concentration of voxelotor in the blood is to obtain exposure measurements which are only available in select research laboratories. A method to measure voxelotor at a standard laboratory would allow clinicians to assess compliance and may be useful in determining optimal dosing. Case studies have reported that voxelotor binding to Hb interferes with capillary zone electrophoresis (CZE). As previously reported, in CZE the characteristic peaks of hemoglobin A2 (HbA2) and hemoglobin F (HbF) split in the presence of voxelotor. Interestingly, it does not cause the hemoglobin S peak to split. We posited that we could use this split to estimate the presence of voxelotor and whole blood concentration. Biophysical measurements of voxelotor binding to Hb were also measured in these samples. Methods: Patients were enrolled prospectively in an IRB approved protocol. Voxelotor was initiated at 1500 mg daily on day 0 and samples were taken at day 0 (pre-dose), 14, 30, and 60. Samples had Hb variants quantified by CZE using the Capillarys 2 FlexPiercing Instrument (Sebia, Georgia). Hematology parameters were measured with the Sysmex XN-1000 automated analyzer (Sysmex, Illinois). To determine whole blood concentration of voxelotor, samples were sent to Worldwide Clinical Trials where a validated liquid chromatography-tandem mass spectrometry method was used. Voxelotor's interference with HbA2 on CZE is dependent on the HbF percentage, therefore samples from patients with SCD were combined into three pooled samples (5-10 samples per pool) of HbF percentages spanning 5-30%. Three hundred µL aliquots of each pool were spiked with voxelotor in DMSO in triplicate to different concentrations between 0 and 600 µMol/L and were incubated at room temperature for 1 hour before being tested with CZE. Samples were then analyzed for voxelotor interference resulting in split peaks of HbF and HbA2. HbA2 interference percent (%VarA2) was calculated as the reported value of the HbA2 split peak over the total HbA2 value (both split and parent peak) times 100. F% was used as directly reported by the instrument without consideration of voxelotor interference. Results were then analyzed in Excel (Data Analytics package) using a multiparameter regression to generate a line of best fit. To allow for logarithmic fit when examining the correlation of calculated concentration with increase in Hb due to voxelotor, samples with negative Hb rises were excluded and concentrations which resulted as negative values were changed to 0.01 µM. Results: Of 20 patients which have been enrolled to date, 9 patients have completed the study and their data was used for these analyses. Using the CZE method described above the concentration of voxelotor was quantifiable using the following equation. Equation 1: uM voxelotor = -99.13 + 7.10*%HbF +12.52*%VarA2 The calculated concentrations of voxelotor based on CZE results had a strong correlation with whole blood concentration (R 2 = 0.85, p &lt;0.001). (Figure 1) When calculated concentration was compared to change in Hb at days 14, 30, and 60 there was a significant positive logarithmic correlation between concentration and change in Hb (R 2=.56, p&lt;0.01). (Figure 2) Conclusions: Using equation 1, CZE can be used to detect the presence of voxelotor and estimate its whole blood concentration. This will allow clinicians to have a better understanding of how their patients are using voxelotor. Additionally, higher calculated whole blood concentrations correlated with higher increases in Hb. It was previously shown that patients who receive higher doses of voxelotor have on average larger increases in Hb. If it could be shown that increasing concentration in an individual on voxelotor is associated with an increased Hb for that individual, then our method could also be used to help clinicians select and adjust doses of voxelotor in a similar manner to how HbF is used in hydroxyurea dosing. Figure 1 Figure 1. Disclosures Curtis: GBT: Consultancy. Minniti: CSL Behring: Other: Endpoint adjudicator; Bluebird Bio: Other: Endpoint adjudicator; F. Hoffmann-La Roche: Consultancy; Chiesi: Consultancy; Novo Nordisk: Consultancy; Forma: Consultancy; Novartis: Consultancy; GBT: Consultancy. Ngyuen Dang: GBT: Current Employment. Pochron: GBT: Current Employment. Campbell: GBT: Research Funding; Sebia: Research Funding.

CYP3A-status is associated with blood concentration and dose-requirement of tacrolimus in heart transplant recipients

High inter-individual variability in tacrolimus clearance is attributed to genetic polymorphisms of CYP3A enzymes. However, due to CYP3A phenoconversion induced by non-genetic factors, continuous changes in tacrolimus-metabolizing capacity entail frequent dose-refinement for optimal immunosuppression. In heart transplant recipients, the contribution of patients’ CYP3A-status (CYP3A5 genotype and CYP3A4 expression) to tacrolimus blood concentration and dose-requirement was evaluated in the early and late post-operative period. In low CYP3A4 expressers carrying CYP3A5*3/*3, the dose-corrected tacrolimus level was significantly higher than in normal CYP3A4 expressers or in those with CYP3A5*1. Modification of the initial tacrolimus dose was required for all patients: dose reduction by 20% for low CYP3A4 expressers, a 40% increase for normal expressers and a 2.4-fold increase for CYP3A5*1 carriers. The perioperative high-dose corticosteroid therapy was assumed to ameliorate the low initial tacrolimus-metabolizing capacity during the first month. The fluctuation of CYP3A4 expression and tacrolimus blood concentration (C0/D) was found to be associated with tapering and cessation of corticosteroid in CYP3A5 non-expressers, but not in those carrying CYP3A5*1. Although monitoring of tacrolimus blood concentration cannot be omitted, assaying recipients’ CYP3A-status can guide optimization of the initial tacrolimus dose, and can facilitate personalized tacrolimus therapy during steroid withdrawal in the late post-operative period.

Effect of Level of Concentrate Feeding and Addition of Monensin on Blood Parameters of Awassi Lambs

This study was conducted to investigate the effect of level of concentrate feeding and addition of monensin on blood parameters. Sixteen Awassi lambs were used at 4-6 months of age and mean initial weight of 21.27 kg. Concentrate diet was offered at two levels (2.5 and 3% of body weight, BW) with or without the addition of monensin at a rate of 30 mg/kg DM. Ground wheat straw was offered adlibitum. Results showed that increasing level of concentrate significantly increased (P<0.01) blood concentration of total protein (BTP) from 6.94 to 8.84 g.100 ml-1, urea nitrogen (BUN), and triglyceride (TG) from 15.54 to 18.23 and 22.66 to 25.12 mg.100 ml-1 respectively. The addition of monensin significantly increased (P<0.05) TP concentration from 7.61 to 8.17 g.100 ml-1, whereas, BUN concentration was decreased (P<0.01) from 18.15 to 15.62 mg.100 ml-1. All blood parameters were also affected (P<0.05) by the interaction between the level of concentrate and the addition of monensin. In the study of diurnal changes, blood parameters showed an expected response to the time of withdrawing blood samples from lambs.