Pharmacological Plasma Concentrations Research Articles

Gut-Selective Design of Orally Administered Izencitinib (TD-1473) Limits Systemic Exposure and Effects of Janus Kinase Inhibition in Nonclinical Species.

Izencitinib (TD-1473), an oral, gut-selective pan-Janus kinase (JAK) inhibitor under investigation for treatment of inflammatory bowel diseases, was designed for optimal efficacy in the gastrointestinal tract while minimizing systemic exposures and JAK-related safety findings. The nonclinical safety of izencitinib was evaluated in rat and dog repeat-dose and rat and rabbit reproductive and developmental toxicity studies. Systemic exposures were compared with JAK inhibitory potency to determine effects at or above pharmacologic plasma concentrations (≥1× plasma average plasma concentration [Cave]:JAK 50% inhibitory concentration [IC50] ratio). In rats and dogs, 1000 and 30 mg/kg/day izencitinib, respectively, produced minimal systemic findings (ie, red/white cell changes) and low systemic concentrations (approximately 1× plasma Cave:JAK IC50 ratio) with an 8× nonclinical:clinical systemic area under the curve (AUC) margin compared with exposures at the highest clinically tested dose (300 mg, quaque die, once daily, phase 1 study in healthy volunteers). In dogs, it was possible to attain sufficient systemic exposures to result in immunosuppression characteristic of systemic JAK inhibition, but at high AUC margins (43×) compared with systemic exposures observed at the highest tested dose in humans. No adverse findings were observed in the gastrointestinal tract or systemic tissues. Izencitinib did not affect male or female fertility. Izencitinib did not affect embryonic development in rats and rabbits as commonly reported with systemic JAK inhibition, consistent with low maternal systemic concentrations (2–6× plasma Cave:JAK IC50 ratio, 10–33× nonclinical:clinical AUC margin) and negligible fetal exposures. In conclusion, the izencitinib gut-selective approach resulted in minimal systemic findings in nonclinical species at pharmacologic, clinically relevant systemic exposures, highlighting the impact of organ-selectivity in reducing systemic safety findings.

Effect of Peptide YY3–36 on Food Intake in Humans

Studies in animals and humans suggest a role for peptide YY (PYY3-36) in regulating satiety. The physiologic role of PYY3-36, however, has not been investigated in detail. The present study was designed to examine PYY release in response to 2 meals differing in their calorie content and to relate the plasma levels to those obtained after exogenous infusion. In a second step, the effect of graded intravenous doses (0, 0.2, 0.4, and 0.8 pmol.kg(-1).min(-1)) of synthetic human PYY3-36 on food intake was investigated in healthy male volunteers in a double-blind, placebo-controlled fashion. Plasma PYY concentrations increased in response to food intake reflecting the size of the calorie load. Graded PYY3-36 infusions resulted in a dose-dependent reduction in food intake (maximal inhibition, 35%; P < .001 vs control) and a similar reduction in calorie intake (32%; P < .001). Fluid ingestion was also reduced by PYY (18% reduction; P < .01). Nausea and fullness were the most common side effects produced by PYY, especially at the highest dose. Furthermore, subjects experienced less hunger and early fullness in the premeal period during PYY3-36 infusion at the highest dose (P < .05). This study shows that intravenous infusions of PYY3-36 decrease spontaneous food intake; the inhibition is, however, only significant at pharmacologic plasma concentrations. Whether PYY3-36 has a physiologic role in the regulation of satiety in humans remains to be defined.

Riboflavin Deficiency Impairs Oxidative Folding and Secretion of Apolipoprotein B-100 in HepG2 Cells, Triggering Stress Response Systems

Secretory proteins such as apolipoprotein B-100 (apoB) undergo oxidative folding (formation of disulfide bonds) in the endoplasmic reticulum (ER) before secretion. Oxidative folding depends on flavoproteins in eukaryotes. Here, human liver (HepG2) cells were used to model effects of riboflavin concentrations in culture media on folding and secretion of apoB. Cells were cultured in media containing 3.1, 12.6, and 300 nmol/L of riboflavin, representing moderately deficient, physiological, and pharmacological plasma concentrations in humans, respectively. When cells were cultured in riboflavin-deficient medium, secretion of apoB decreased by >80% compared with controls cultured in physiological medium. The nuclear translocation of the transcription factor ATF-6 increased by >180% in riboflavin-deficient cells compared with physiological controls; this is consistent with ER stress. Nuclear translocation of ATF-6 was associated with activation of the unfolded protein response. Expression of stress-response genes coding for ubiquitin-activating enzyme 1, growth arrest and DNA damage inducible gene, and glucose regulated protein of 78 kDa was greater in riboflavin-deficient cells compared with other treatment groups. Finally, phosphorylation of the eukaryotic initiation factor (eukaryotic initiation factor 2alpha) increased in riboflavin-deficient cells, consistent with decreased translational activity. We conclude 1) that riboflavin deficiency causes ER stress and activation of unfolded protein response in HepG2 cells, and 2) that riboflavin deficiency decreases protein secretion in HepG2 cells. Decreased secretion of apoB in riboflavin-deficient cells might interfere with lipid homeostasis in vivo.

Oxidative Folding of Interleukin-2 Is Impaired in Flavin-Deficient Jurkat Cells, Causing Intracellular Accumulation of Interleukin-2 and Increased Expression of Stress Response Genes

Secretory proteins such as interleukin (IL)-2 undergo oxidative folding (disulfide formation) in the endoplasmic reticulum (ER) before secretion. Studies in yeast have suggested that oxidative folding depends on the flavoprotein Ero1p; unfolded proteins accumulate in the ER, triggering cellular stress response. Here, human lymphoid cells (Jurkat cells) were used to model effects of cellular flavin supply on secretion of IL-2 (containing one disulfide bond) and cellular stress response. Cells were cultured in media containing 0.85, 3.1, 12.6 or 300.6 nmol/L riboflavin for 5 wk, representing severely deficient, moderately deficient, physiologic and pharmacologic plasma concentrations in humans, respectively. Transport rates of riboflavin were increased in severely and moderately deficient cells compared with cells cultured in physiologic medium; this increase was not sufficient to prevent intracellular depletion of riboflavin, as judged by glutathione reductase activity and intracellular concentrations of glutathione. Intracellular accumulation of IL-2 was greater in severely deficient cells than in other groups. Nevertheless, severely deficient cells secreted normal amounts of IL-2 into the extracellular space, mediated by increased transcriptional activity of the IL-2 gene. Riboflavin-deficient cells responded to intracellular accumulation of IL-2 with increased expression of genes encoding ubiquitin-activating enzyme E1 and X box-binding protein, consistent with cellular stress. These findings are consistent with the hypothesis that flavin deficiency may cause cellular stress by accumulation of unfolded proteins in human cells.

Involvement of nuclear factor-kappa B (NF-κB) activation in mitogen-induced lymphocyte proliferation: inhibitory effects of lymphoproliferation by salicylates acting as NF-κB inhibitors

The transcription factor nuclear factor-kappa B (NF-κB) is involved in the production of inflammatory cytokines and in the control of the inflammatory response. Some nonsteroidal anti-inflammatory drugs such as acetylsalicylic acid (ASA) or salicylate are known to exert some of their anti-inflammatory pharmacological properties independently of cyclooxygenase inhibition. For ASA and salicylate, an NF-κB inhibitory effect at mM concentrations (pharmacological plasma concentrations reached in vivo) has been shown. We studied the action of ASA, salicylate, and several NF-κB inhibitors on the mitogen-induced activation of peripheral blood lymphocytes (PBL) and purified T cells. We showed that ASA and salicylate (1–3 mM) (but not indomethacin, a specific cyclooxygenase inhibitor) as well as a group of chemically unrelated inhibitors of NF-κB (including the sesquiterpene lactone parthenolide, Bay 11-7082, sulfasalazine, the proteasome inhibitor MG-132 and the peptide SN-50, an inhibitor of the nuclear transfer of the p50 subunit of NF-κB), were potent inhibitors of phytohemoagglutinin-activated PBL and T cell proliferation. At the same concentrations, they inhibited NF-κB binding to DNA in nuclear extracts. The inhibition of proliferation was not relieved by exogenous interleukin (IL)-2. We concluded that NF-κB activation has a fundamental role in T cell proliferation independently of IL-2 production. Some pharmacological actions of ASA may be ascribed to the inhibition of immune cell proliferation via the inhibition of the transcription factor NF-κB.

Cardiovascular and endocrine effects of endothelin-1 at pathophysiological and pharmacological plasma concentrations in conscious dogs.

Increased plasma concentrations of endothelin-1, a potent vasoconstrictor produced by the endothelium, have been reported in various pathological conditions. This study was conducted to evaluate effects of endothelin-1 at pathophysiological and pharmacological plasma concentrations. Endothelin-1 was infused at increasing doses (2.5, 5, 10, and 20 ng/kg.min for 1 hour each) in nine conscious dogs. During endothelin-1 infusion, plasma endothelin-1 rose from a basal value of 1.8 +/- 0.4 pmol/l to 5.8 +/- 1.1 (pathophysiological), 20.8 +/- 3.9 (pathophysiological), 85.4 +/- 18.9 (pharmacological), and 311.4 +/- 55.7 (pharmacological) pmol/l at each dose, respectively. Heart rate increased at 2.5 ng/kg.min (from 129 +/- 7 to 146 +/- 12 beats/min) but decreased at 20 ng/kg.min (97 +/- 7 beats/min) (p less than 0.001). Such a biphasic response was also observed for peak (+)dP/dt and (dP/dt)/DP40 (p less than 0.005). Left ventricular systolic pressures, mean aortic pressure, and left atrial pressure increased over time (p less than 0.05, p less than 0.005, and p less than 0.001, respectively). The time constant of early isovolumic relaxation rose progressively (p less than 0.001). The percent systolic shortening decreased at 10 and 20 ng/kg.min (p less than 0.005). Pressure-segment length loops showed a reduction in systolic shortening associated with an increase in left ventricular systolic pressure at 20 ng/kg.min. Atrial natriuretic factor rose after 5 ng/kg.min from 28.5 +/- 6.5 to 92.0 +/- 18.2 pmol/l (p less than 0.005). Angiotensin II and catecholamines did not change significantly. Serum urea and creatinine rose progressively (p less than 0.05), whereas glucose decreased (p less than 0.05). The above results differed significantly from measurements obtained in a time-control group of six dogs. A fourfold increase of plasma endothelin-1 obtained after doubling the infusion rate suggests a reduction in endothelin-1 clearance or endothelin-1 endogenous production. The biphasic response of heart rate is consistent with baroreflex-mediated effects resulting from vasodilation at the pathophysiological level and vasoconstriction at the pharmacological level. Hemodynamic data suggest an increase followed by a decrease in contractility at both levels, respectively. Finally, endothelin-1 is a stimulator of atrial natriuretic factor.

Splanchnic disposal of human atrial natriuretic peptide in humans

In healthy men (n = 6), the splanchnic fractional extraction of human atrial natriuretic peptide (hANP), as determined by the hepatic venous catheter technique, was 75% under basal conditions resulting in a splanchnic uptake of hANP of 8.5 ± 5.0 pmol/min. In spite of a drop ( P < .05) in splanchnic fractional extraction to about 50%, splanchnic uptake of hANP rose to 56 to 99 pmol/min ( P < .01) when pharmacologic plasma concentrations of hANP were induced during a bolus (100 μg)-primed intravenous (IV) infusion (100 μg/h; time, one hour) of hANP. This was accompanied by a fall in estimated hepatic blood flow ( P < .05), in pulmonary arterial pressure ( P < .01), and, in each individual, in systemic BP. Total metabolic clearance rates, splanchnic clearance rates, and production rates of hANP were 4.5 ± 2.2 L/min, 0.4 ± 0.1 L/min, and 46.1 ± 20.1 pmol/min, respectively. Thus, in healthy men, the splanchnic area accounts for approximately 10% of total hANP clearance.

Effect of lower body positive pressure on blood pressure, plasma atrial natriuretic factor concentration, and sodium and water excretion in healthy volunteers and cardiac transplant recipients.

The effect of one hour (40 mmHg) lower body positive pressure on blood pressure, plasma atrial natriuretic factor concentration, and urinary sodium and water excretion was studied in 10 healthy volunteers and seven cardiac transplant recipients. Both groups showed a sustained rise in blood pressure and plasma atrial natriuretic factor concentration. The healthy volunteers had a diuresis during the period of lower body positive pressure and a small natriuresis in the subsequent hour. In contrast, lower body positive pressure had no significant effect on urinary sodium and water excretion in the cardiac transplant group. The data suggest that cardiac innervation is not important as a mediator of the haemodynamic response to lower body positive pressure but is necessary for the renal response. Furthermore, small physiological rises in plasma atrial natriuretic factor concentrations do not cause a brisk natriuresis as has been reported with pharmacological plasma concentrations.

Direct thin-layer chromatography—fluorimetric quantification of pharmacological plasma concentrations of an antiarrhythmic steroid (Org-6001)

Direct thin-layer chromatography—fluorimetric quantification of pharmacological plasma concentrations of an antiarrhythmic steroid (Org-6001)