Pharmacokinetics and Tissue Distribution of Aegeline after Oral Administration in Mice

Aegeline is claimed to be a biologically active constituent of Aegle marmelos. Preclinical studies have reported possible therapeutic potential for aegeline against obesity and diabetes. In recent years, aegeline has been added to several weight loss products. However, the consumption of aegeline-containing supplements such as OxyELITE Pro and VERSA-1 has been linked to multiple cases of acute and chronic liver failure. This study was carried out to evaluate the pharmacokinetics and tissue distribution of aegeline in ND4 mice. Two doses of aegeline, a human equivalent dose (1×) 30 mg/kg and a 10× dose (300 mg/kg), were orally administered to the mice, and blood and tissue samples were collected over 8 h. The quantitative analysis of plasma and tissue homogenates (liver, kidney, and brain) was done by UHPLC-QTOF to determine aegeline concentrations. The peak plasma level of aegeline was achieved at a Tmax of 0.5 h, indicating its rapid absorption from the gastrointestinal tract. Aegeline was not detected in the plasma at 8 h after oral administration, with a half-life of 1.4 ± 0.01 and 1.3 ± 0.07 h for the 30 and 300 mg/kg doses, respectively. The half-life of aegeline in the liver was 1.2 h and 1.7 h for 30 and 300 mg/kg doses, respectively, with a Tmax of 1.9 h, which indicates relatively fast elimination of aegeline from the liver.

Apixaban Interacts with Haemoglobin: Effects on Its Plasma Levels

The direct oral anticoagulant apixaban (APX), a strong factor Xa inhibitor, binds also to plasma proteins, especially albumin, and minimally to α1-acid glycoprotein. Although APX can cross the red cell membrane, due to its chemical structure, and could bind to haemoglobin (Hb), no investigation was performed on this possible phenomenon that could affect the APX plasma concentration and thus its pharmacokinetics and pharmacodynamics. We addressed this issue by (1) measuring the levels of APX and haematological/biochemical parameters in 90 patients on APX therapy; (2) assessing the effect of APX on oxygen saturation curves of Hb; (3) testing the direct APX binding to Hb by fluorescence spectroscopy and a zinc-induced precipitation of Hb coupled to a reversed-phase high-performance liquid chromatography (HPLC)-based method; and (4) simulating in silico by molecular docking the APX interaction with human Hb. In a multivariable analysis, Hb was the only independent variable significantly and inversely associated in 90 patients with APX peak plasma level, at variance with patients treated with rivaroxaban (n = 86) and dabigatran (n = 34) therapy. APX causes a progressive left-shift of the oxygen dissociation curve of purified Hb solution, with a Kd ≅300 µM. Fluorescence- and HPLC-based assays concordantly showed that APX binds to Hb with a Kd ≅350 µM. Finally, docking simulations showed that APX can fit into in the central cavity of Hb. These findings support the hypothesis that APX does bind to Hb, which, due to its millimolar concentration in blood, can act as ‘buffer’ for the drug and consequently affect its free plasma level.

Pharmacokinetic and Pharmacodynamic (PKPD) Modeling to Determine the Andexanet Alfa Dose to Reverse the Anticoagulant Activity of Direct Fxa Inhibitors

Introduction Andexanet alfa (AnXa) is a specific antidote under development for direct and indirect factor Xa (fXa) inhibitors. AnXa is a recombinant, engineered version of human fXa that is catalytically inactive but retains high affinity for direct fXa inhibitors. AnXa binds fXa inhibitors, sequestering them in plasma, thereby reducing the free inhibitor concentration, reversing fXa inhibition, and normalizing thrombin generation. The decreased unbound concentrations of the direct fXa inhibitors (apixaban, rivaroxaban and edoxaban) cause a portion of the extravascular fXa inhibitors to move into the vasculature. Reversal of fXa inhibitor-induced anticoagulation requires a molar excess of AnXa relative to the total amount of fXa inhibitor in the blood - i.e., the initial fXa inhibitor plasma concentration plus the redistributed amount. We report a PKPD model that accounts for this extravascular-intravascular redistribution and allows determination of the appropriate AnXa dose to reverse the anti-fXa activity for each approved direct fXa inhibitor. Methods A model for rivaroxaban was constructed first by jointly analyzing AnXa concentrations, total and unbound rivaroxaban concentrations, and anti-fXa activity for 5 different doses of AnXa (each administered at the peak plasma level of 20 mg QD rivaroxaban at steady state). Model parameters were estimated by maximum likelihood using nonlinear regression (NONMEM, v. 7.2.0). The model was used to simulate the potential level of reversal of anti-fXa activity after 2.5-40 mg/day rivaroxaban (QD and BID) for different bolus doses of AnXa and to predict maintenance of reversal by various follow-on infusion rates. A similar model was developed to characterize the interaction between AnXa and apixaban. Learnings from the rivaroxaban and apixaban models were used to develop a universal model to predict the level of reversal of anticoagulation for 60 mg edoxaban after a particular dose of AnXa. The model accurately predicted the degree of reversal of edoxaban anticoagulation. The final model was used to construct a nomogram of AnXa doses that could be used for each fXa inhibitor at each approved dose based on timing of the last dose of the inhibitor. Results The final PKPD model was a three-compartment PK model for the fXa inhibitors, including one central and two tissue compartments. The two tissue compartments included one that rapidly equilibrated with the central compartment and a second that equilibrated slowly. The combined volume of the central and rapidly equilibrating compartments was similar to the volume of central compartment of the fXa inhibitor PK models in the absence of AnXa. The AnXa PK was best described by a two-compartment PK model with an additional saturable binding component. The AnXa-fXa inhibitor complex moved to a "sequestration" compartment. Upon release from AnXa, the fXa inhibitor was able to return to the central compartment. The fXa inhibitor did not clear with AnXa. The binding between AnXa and free fXa inhibitor was characterized by a reversible binding equilibrium. There was a direct PKPD linear relationship between free fXa inhibitor concentrations and anti-fXa activity. Simulations were used to estimate the best dosing regimen to reverse anticoagulation for each direct fXa inhibitor. A bolus dose of 800 mg of AnXa followed by an 8 mg/min infusion was sufficient to fully reverse the peak level of anti-fXa activity after the 20 mg QD dose of rivaroxaban. This represents a >90% decrease in anti-fXa activity compared to pre-AnXa administration. Sustained reversal of peak anti-fXa levels for the 5 mg BID dose of apixaban required a 400 mg bolus AnXa dose with a follow-on infusion of 4 mg/min. The peak anticoagulant activity of the 60 mg dose of edoxaban could be reversed by an 800 mg bolus dose of AnXa followed by an 8 mg/min infusion. Conclusions A PKPD model was constructed that accurately predicted the AnXa dose necessary to reverse coagulation inhibition of each direct fXa inhibitor. The model incorporated the PK of AnXa, the anti-fXa activity, unbound and total levels of the fXa inhibitor, and the redistribution of each fXa inhibitor between the extravascular and intravascular compartments. Simulations were used to predict the AnXa dose necessary to reverse each approved dose of each direct fXa inhibitor and for different times after the last dose taken of the fXa inhibitor. Disclosures Janet: Portola Pharmaceuticals, Inc.: Employment. Lu:Portola Pharmaceuticals, Inc.: Employment. Curnutte:3-V Biosciences: Equity Ownership; Sea Lane Biotechnologies: Consultancy; Portola Pharmaceuticals, Inc.: Employment. Conley:Portola Pharmaceuticals, Inc.: Employment. Mandema:Portola Pharmaceuticals, Inc.: Consultancy.

A Pharmacokinetic Study of Antimalarial 3,5-Diaryl-2-aminopyridine Derivatives

Malaria caused by Plasmodium falciparum is responsible for approximately 80% of the incidence and 90% of deaths which occur in the World Health Organization (WHO) African region, with children and pregnant women having the highest incidence. P. falciparum has developed resistance, and therefore new effective candidate antimalarial drugs need to be developed. Previous studies identified 3,5-diaryl-2-aminopyridines as potential antimalarial drug candidates; therefore, derivatives of these compounds were synthesized in order to improve their desired properties and pharmacokinetic (PK) properties of the derivatives were investigated in a mouse model which was dosed orally and intravenously. Collected blood samples were analyzed using liquid chromatography coupled to mass spectrometer (LC-MS/MS). The mean peak plasma level of 1.9 μM was obtained at 1 hour for compound 1 and 3.3 μM at 0.5 hours for compound 2. A decline in concentration was observed with a half-life of 2.53 and 0.87 hours for compound 1 in mice dosed orally and intravenously, respectively. For compound 2 a half-life of 2.96 and 0.68 hours was recorded. The bioavailability was 69% and 59.7% for compound 1 and compound 2, respectively.

Oral Administration of Carbon-14 Labeled Cyanocobalamin (14C-Cbl) Reveals Variable Degradation of Vitamin B12 in the Gastrointestinal Tract That Impacts Vitamin B12 Absorption and Status.

Abstract 3018 Poster Board II-994 Recent evidence from our laboratory and others suggests that a variable portion of ingested cobalamin (Cbl), either crystalline or from food, is degraded in the gastrointestinal tract. We have developed a biosynthetic method to incorporate 14C into the lower axial ligand of cobalamin that has made it possible to study the fate of this vitamin during its passage through the gastrointestinal tract and to assess the presence of Cbl or its breakdown products in biological samples. Following oral administration of an aqueous physiological tracer dose of 14C-Cbl (1.3 μg, 50 nCi), blood, urine, and feces are analyzed for 14C by accelerator mass spectrometry. In 9 subjects, the plasma response was consistent with the expected behavior of peroral Cbl: 14C-Cbl first appeared in the plasma 3h post-dose reaching a peak level within 6-8h. Confirmation that this dose appears bound to the physiological transport protein transcobalamin (TC) was obtained in a subset of subjects by an immunoaffinity method using anti-human TC antibody-coated magnetic beads which selectively bound 95-98% of plasma 14C. Urinary excretion of 14C was maximal in the first 24h, with 14C first appearing in urine as early as 1.5h after dosing. Fecal excretion occurred variably over several days. The amount of 14C found in the urine (10-50% of the dose) was 100-fold greater than in previous reports using 57Co-labeled cyanocobalamin (0.1-0.5%), and fecal excretion was lower than expected (10-20% vs 30-70%). Urinary excretion of 14C was inversely correlated with the peak plasma level of 14C attained (r2=0.610; p<0.001). The bulk of urinary 14C was not associated with intact Cbl and first appeared in the urine before peak 14C levels were attained in the plasma. The peak plasma level of 14C attained also showed a strong positive correlation with plasma holotranscobalamin concentration measured before administration of the 14C-Cbl (r2=0.571; p<0.001). No such correlation was found with total plasma Cbl. In additional experiments on normal volunteers using eggs endogenously labeled with 14C Cbl following intramuscular injection of hens with 14C Cbl, comparably high urinary excretion of 14C was also observed. We conclude that a variable fraction of ingested Cbl is degraded in the gastrointestinal tract of normal individuals. This may be an important determinant of the amount of Cbl absorbed from food or supplement sources. Additionally, our findings suggest that the concentration of holoTC in the plasma reflects absorptive capacity and may therefore be a good surrogate for Cbl absorptive status. Our findings also have implications regarding the bioavailability of Cbl and may inform pending considerations to fortify food supplies with Cbl in order to mitigate the incidence of Cbl deficiency, particularly among the elderly. Intestinal degradation, either microbial or through the action of digestive enzymes, may also be a source of Cbl analogues that have previously been detected in the plasma and tissues. Cbl analogues may interfere with the physiological function of cobalamin, resulting in some of the manifestations of cobalamin deficiency. Disclosures: Green: Vitalea Science: Research Funding. Miller:Vitalea Science: Research Funding. Lee:Vitalea Science: Research Funding. Sutter:Vitalea Science: Research Funding. Allen:Vitalea Science: Research Funding. Dueker:Vitalea Science: Employment.

Pharmacokinetics of Cannabinoids

Delta-9-tetrahydrocannabinol (Δ-9-THC) is the main psychoactive ingredient of cannabis (marijuana). The present review focuses on the pharmacokinetics of THC, but also includes known information for cannabinol and cannabidiol, as well as the synthetic marketed cannabinoids, dronabinol (synthetic THC) and nabilone. The variability of THC in plant material (0.3% to 30%) leads to variability in tissue THC levels from smoking, which is, in itself, a highly individual process. THC bioavailability averages 30%. With a 3.55% THC cigarette, a peak plasma level of 152±86.3 ng/mL occured approximately 10 min after inhalation. Oral THC, on the other hand, is only 4% to 12% bioavailable and absorption is highly variable. THC is eliminated from plasma in a multiphasic manner, with low amounts detectable for over one week after dosing. A major active 11-hydroxy metabolite is formed after both inhalation and oral dosing (20% and 100% of parent, respectively). THC is widely distributed, particularly to fatty tissues, but less than 1% of an administered dose reaches the brain, while the spleen and body fat are long-term storage sites. The elimination of THC and its many metabolites (from all routes) occurs via the feces and urine. Metabolites persist in the urine and feces for severalweeks. Nabilone is well absorbed and the pharmacokinetics, although variable, appear to be linear from oral doses of 1 mg to 4 mg (these doses show a plasma elimination half-life of approximately 2 h). As with THC, there is a high first-pass effect, and the feces to urine ratio of excretion is similar to other cannabinoids. Pharmacokineticpharmacodynamic modelling with plasma THC versus cardiac and psychotropic effects show that after equilibrium is reached, the intensity of effect is proportional to the plasma THC profile. Clinical trials have found that nabilone produces less tachycardia and less euphoria than THC for a similar antiemetic response.

Activated thrombin-activatable fibrinolysis inhibitor attenuates spontaneous fibrinolysis of batroxobin-induced fibrin deposition in rat lungs

SummaryStudies have shown that inhibition of TAFI by small peptides enhances pharmacological effects of tPA in animal models of thrombosis, suggesting that TAFI modulates the fibrinolytic system. In this study, we investigated the effect of activated human TAFI (TAFIa) on endogenous fibrinolysis in a rat model of intravascular fibrin deposition. 125I-labeled fibrinogen was injected intravenously followed by a bolus injection of batroxo-bin, a thrombin-like enzyme. Batroxobin cleaved fibrinogen to form insoluble fibrin that was deposited in tissues, including the lungs. This was shown by a decrease of radioactivity in the blood as a result of consumption of 125I-labeled fibrinogen and an elevation of radioactivity in the lungs 5 min following batroxobin administration. Endogenous fibrinolysis was detected by a gradual increase in radioactivity in the blood and a decrease in radioactivity in the lungs at 30 min, an indication of radio-labeled fibrin degradation products (FDPs) being released into the circulation from the tissues. Intravenous administration of human TAFIa dose-dependently attenuated the later phase reduction of radioactivity in the lungs. When the dose of TAFIa was 218 μg/kg, giving a peak plasma level of TAFIa 0.9 ± 0.05 μg/ml, the spontaneous fibrinolysis was completely prevented. These results provide direct evidence that an increase in circulating TAFIa impairs endogenous clot lysis in a rat model of fibrin deposition.

Pharmacokinetics of ciprofloxacin in layer chicks

Pharmacokinetics parameters of ciprofloxacin were calculated from constructed plasma disappearing curves (PDC) after oral or i.v. injection of 5mg / kg in two-week old hybird layer · The i.v. PDC indicated a first order biphasic kinetic. The distribution phase was too short to be considered. The parameters of the elimination phase phase indicated that the biological half – life ( t 12) was 3.7 hours and the volume of distribution (Vd) was 1.7 1/kg. The oral PDC indicated a first order kinetic with a peak plasma level (PPL) of 3.0 ug/ml achieved after 1.75 hours , a t 12 of 2.9 hours and a Vd of 1.7 1/kg . Bioavailability value was 84 +9.7% . Binding of ciprofloxacin to chicken plasma protein was estimated to be 25.4 61.3%.

Disposition Kinetics and Dosage Regimen of Ceftriaxone in Crossbred Calves (Short Communication)

Disposition kinetics and urinary excretion of ceftriaxone were investigated in healthy crossbred calves after its single intravenous administration (10 mg kg–1). Based on kinetic parameters, an appropriate dosage regimen of ceftriaxone in calves was calculated. The peak plasma level of ceftriaxone at 1 min was 84.0 ± 1.55 μg ml–1 which declined to 0.43 ± 0.05 μg ml–1 at 8 h. The value of elimination half-life (t1/2α), volume of distribution Vd (area) and total body clearance (ClB) were 4.39 ± 0.63 h, 1.91 ± 0.19 L kg–1 and 0.31 ± 0.01 L kg–1 h–1, respectively. Approximately 41 per cent of total administered drug was recovered in the urine within 24 h of its administration. The plasma protein binding of ceftriaxone was found to be concentration dependent with an overall mean of 38.55 per cent. The binding capacity of ceftriaxone to plasma proteins and the dissociation rate constant of protein-drug complex were 20.1 × 10–8 ± 18.4 × 10–8 mole g–1 and 1.07 × 10–6 ± 0.52 × 10–6 mole, respectively. An appropriate intravenous dosage regimen of ceftriaxone in cattle would be 12 mg kg–1 repeated at 24 h.

Pentoxifylline Promotes Replication of Human Cytomegalovirus In Vivo and In Vitro

OKT3 monoclonal antibody (MoAb) therapy is well established in the prevention and therapy of acute rejection in transplant patients. Unfortunately, this therapy is associated with several short-term (cytokine release syndrome) and long-term (infections, EBV-related lymphoma) side effects. Recently, we were able to demonstrate an association between the TNFα release following the first OKT3 MoAb infusions and the appearance of human cytomegalovirus (HCMV) reactivation several days later. In order to prevent this TNFα associated HCMV reactivation patients were additionally treated with pentoxifylline (PTX), a methylxanthine derivative that has been shown to suppress TNFα induction. Although the TNFα peak plasma level following OKT3 MoAb treatment was markedly reduced, the incidence of HCMV reactivation and HCMV disease was not influenced. In transient transfection experiments using HCMV immediate early enhancer/promoter CAT reporter gene constructs PTX enhanced the promoter activity independently from TNFα in premonocytic cells. Furthermore, PTX acted synergistically with TNFα. In virus-infected human embryonal lung fibroblasts HCMV replication was triggered in the presence of both PTX and TNFα, while either substance alone had only marginal effects. The stimulatory effect of PTX on the immediate early (IE) enhancer/promoter was mediated via CREB/ATF, a eukaryotic transcription factor that binds to the 19 bp sequence motif in the enhancer region, while TNFα stimulation was mediated by activation of the transcription factor NF-kB and its binding to the 18 bp sequence motif in the enhancer. These data suggest a potential side effect of cAMP-elevating drugs such as PTX.