Plasma protein binding of sorafenib, a multi kinase inhibitor: in vitro and in cancer patients

Sorafenib is an orally administered multikinase inhibitor that exhibits antiangiogenic and antitumor activity. Few investigators have been able to correlate cumulative sorafenib dose or total exposure to pharmacodynamic effects. This discrepancy may be in part due to poorly understood protein binding characteristics. Since unbound drug concentrations are believed to be more relevant to pharmacological and toxicological responses than total drug, an equilibrium dialysis method using 96-well microdialysis plates was optimized and validated for determining the fraction unbound (F(u)) sorafenib in human plasma and in isolated protein solutions. Unbound sorafenib concentrations were determined in cancer patients receiving the drug orally at a dose of 400?mg and 600?mg twice daily. Sorafenib was extensively bound with mean F(u) value of 0.3% in both non-cancer and cancer patient's plasma. The binding in plasma was concentration independent, indicating a low-affinity, possibly nonspecific and nonsaturable process. In isolated protein solutions, 99.8% and 79.3% of sorafenib was bound to human serum albumin (HSA) (4?g/dL) and α(1)-acid glycoprotein (AAG) (0.1?g/dL) with binding constants of 1.24?×?10(6)?M(-1) and 1.40?×?10(5)?M(-1), respectively. In cancer patients receiving sorafenib, unbound sorafenib was not correlated with patient characteristics or laboratory values. In conclusion, sorafenib is highly protein bound in human plasma with a higher affinity towards albumin and limited free drug may be partly responsible for its borderline clinical activity.     

Differential protein binding of indinavir and saquinavir in matched maternal and umbilical cord plasma

AIMS: To determine whether lower umbilical cord than maternal binding of indinavir and saquinavir contributed to the low cord : maternal (C : M) total concentration ratios reported previously. METHODS: Indinavir and saquinavir unbound fraction (fu) was determined using equilibrium dialysis. Buffer solutions of human serum albumin (HSA) (20.0, 30.0, 40.0 g l(-1)) and alpha(1)-acid glycoprotein (AAG) (0.20, 0.60, 2.00 g l(-1)) were spiked with indinavir (1.00 and 8.00 mg l(-1)) or saquinavir (0.15 and 1.50 mg l(-1)). Matched maternal and umbilical cord plasma was spiked with 1.00 mg l(-1) indinavir (n = 12) or 0.15 mg l(-1) saquinavir (n = 20). Spiked protein/plasma solutions were dialyzed against isotonic phosphate buffer, at 37 degrees C. At equilibrium, indinavir and saquinavir concentrations were quantified, and the f(u) determined. RESULTS: Indinavir and saquinavir demonstrated protein concentration-dependent binding in buffer solutions of HSA and AAG. Indinavir f(u) was significantly higher in umbilical cord (0.53 +/- 0.12) compared with maternal (0.36 +/- 0.11) plasma (95% CI of the difference -0.26, -0.097). Similarly, saquinavir fu was different between umbilical cord (0.0090 +/- 0.0046) and maternal plasma (0.0066 +/- 0.0039) (95% CI of the difference -0.0032, -0.0016). The transplacental AAG concentration gradient contributed significantly to the binding differential of both drugs. CONCLUSIONS: The differential plasma binding of both drugs, which was largely the result of the transplacental AAG concentration gradient, would contribute to the low C : M total plasma concentration ratios observed previously. Unbound concentrations of indinavir and saquinavir are likely to be substantially lower in umbilical cord than maternal plasma.     

Decreased Biosynthesis of Lung Surfactant Constituent Phosphatidylcholine Due to Inhibition of Choline Transporter by Gefitinib in Lung Alveolar Cells

Purpose We investigated whether gefitinib, an anticancer agent, inhibits phosphatidylcholine (PC) biosynthesis and choline uptake by alveolar epithelial type II cells. Materials and Methods Uptake of choline and PC biosynthesis were examined in vitro, using human alveolar epithelia-derived cell line A549 and rat alveolar type (AT) II cells as models. Results Gefitinib reduced the incorporation of [³H]choline into PC in A549 and rat ATII cells. The uptake of [³H]choline by A549 and rat ATII cells was concentration-dependent, and the Km values were 15.0 and 10–100 μM, respectively. The uptake of [³H]choline by A549 and rat ATII cells was weakly Na⁺-dependent, and inhibited by hemicholinium-3. RT-PCR revealed expression of choline transporter-like protein (CTL)1 and organic cation transporter (OCT)3 mRNAs in both cells. The choline uptake by A549 and rat ATII cells was strongly inhibited by gefitinib with the IC₅₀ value of 6.77 μM and 10.5 μM, respectively. Conclusions Our results demonstrate that gefitinib reduces PC biosynthesis via inhibition of cellular choline uptake by A549 and rat ATII cells, which is mainly mediated by CTL1, resulting in abnormality of lung surfactant that can be one of mechanisms of the interstitial lung disease associated with gefitinib.     

Relationship between alpha 1-acid glycoprotein and plasma binding of disopyramide and mono-N-dealkyldisopyramide.

Highly purified serum albumin did not bind either disopyramide (DP) or mono-N-dealkyldisopyramide (MND). The unbound fraction of DP and MND in highly purified serum alpha 1-acid glycoprotein (AAG) at 0.5 g/l was 57 and 62 and at 2.0 g/l 19 and 30% respectively. Unbound DP and MND were measured in spiked plasma (10 mumol/l of DP or MND), from 60 patients, having AAG concentrations varying from 0.4 to 3.0 g/l. Unbound drug varied from 13 to 58 and from 24 to 62% for DP and MND, respectively, and was inversely related to the plasma concentration of AAG (r = -0.9016, r = -0.9157). A linear relationship was found between the binding ratio (moles bound divided by moles unbound) and the plasma concentration of AAG for both DP (r = 0.9199) and MND (r = 0.9270), whereas no relationship was found between the binding ratios of DP or MND and the plasma concentrations of total protein, albumin, haptoglobin, alpha 1-antitrypsin or the immunoglobulins IgG, IgA or IgM. In patients on DP maintenance therapy, a linear relationship was found between percent unbound DP and the plasma concentration of DP in samples with similar AAG concentrations. Furthermore, a linear relationship was found between the binding ratio of DP and the plasma concentration of AAG in samples with similar DP concentrations. The present findings support the concept that AAG is the major serum protein responsible for the binding of DP and MND.     

Binding of a new multidrug resistance modulator,S9788, to human plasma proteins and erythrocytes

Summary The interactions of S9788 with human plasma proteins have been investigatedin vitro by an erythrocyte partitioning technique that allows an estimation of the plasma proteins and erythrocytes binding parameters. S9788 was 98% bound to plasma and blood. Lipoproteins bound S9788 with high affinities (binding constants of 0.645, 12.8 and 87.0×10⁶M⁻¹ for, HDL, LDL and VLDL, respectively) and accounted for more than 55% of the total circulating S9788. Albumin and alpha₁-acid glycoprotein also bound S9788 with lower binding constants of 0.022 and 0.245×10⁶ M⁻¹. S9788 was mainly distributed in the plasma blood compartment (75–80%) with blood-to-plasma concentrations ratio of 0.6 to 0.7. These results indicate that,in vivo, the fraction of blood S9788 available for tissue diffusion,i.e., the free drug fraction in blood, should depend on lipoprotein concentration in plasma.     

BINDING CHARACTERISTICS OF KNI-272 TO PLASMA PROTEINS,A NEW POTENT TRIPEPTIDE HIV PROTEASE INHIBITOR

The binding characteristics of KNI-272, a potent and selective human immuno-deficiency virus (HIV) protease inhibitor, were evaluated in rat and human plasma, and in solutions of human α₁-acid glycoprotein (AAG) and human serum albumin (HSA). The unbound fractions (F_u ) of KNI-272 were 12·13 and 2·24% in rat and human plasma, respectively, at the drug concentration of 1·0 μg mL⁻¹. Although KNI-272 binds to both AAG and HSA, the F_u of KNI-272 in AAG solution was 1·83%, and only one-quarter of that in HSA solution (F_u6·78%). Binding displacing agents, such as disopyramide, warfarin, diazepam, and digitoxin, were used to determine the binding site of KNI-272 on these plasma proteins. The F_u of KNI-272 in AAG solution increased 14-fold when disopyramide was added to the AAG solution. In addition, warfarin, diazepam, and digitoxin were added to HSA solution as representative drugs bound to distinct binding sites on HSA, namely sites I, II, and III, respectively. The F_u values of KNI-272 in HSA solution significantly increased when warfarin and diazepam were added. In particular, with the addition of warfarin to HSA solution, the F_u of KNI-272 increased to 16%. The modified Scatchard plots of KNI-272 binding to AAG and HSA both showed biphasic curves, and the KNI-272 binding sites at low concentration range on AAG and HSA disappeared with the addition of disopyramide and warfarin, respectively. Therefore, it is considered that KNI-272 binds to the identical site as disopyramide on AAG and site I on HSA in the low KNI-272 concentration range. By comparing the KNI-272 binding parameters obtained in human plasma and these protein solutions, we can assume that KNI-272 binding at low concentration in human plasma is mainly concerned with the binding on AAG. As KNI-272 concentration in plasma increases, HSA becomes concerned with KNI-272 binding.     

Gefitinib

Gefitinib (ZD1839) is an orally active selective inhibitor of epidermal growth factor receptor tyrosine kinase, an enzyme that regulates intracellular signalling pathways implicated in the proliferation and survival of cancer cells. In human non-small cell lung cancer (NSCLC) cell lines and xenografts, gefitinib dose-dependently inhibited cellular proliferation and tumour growth, and potentiated the cytotoxic effects of chemotherapy and/or radiation. Gefitinib is orally bioavailable and is cleared via the cytochrome P450 3A4 pathway. In patients receiving gefitinib (50 to 700 mg/day) in phase I trials, steady-state plasma concentration was reached in 7 to 10 days. In patients with advanced NSCLC who had failed one or two prior chemotherapies, gefitinib 250 or 500mg once daily induced an objective response in approximately 19% of patients in a double-blind trial (n = 210). In another double-blind trial including 216 patients with NSCLC who had failed two or more prior chemotherapies, gefitinib 250 or 500mg once daily induced an objective response in 11.8 and 8.8% of patients, respectively; approximately 40% showed an improvement in disease-related symptoms. Gefitinib was generally well tolerated and the most common adverse events were mild skin rashes and diarrhoea.     

Gefitinib (Iressa) inhibits the CYP3A4-mediated formation of 7-ethyl-10-(4-amino-1-piperidino)carbonyloxycamptothecin but activates that of 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecin from irinotecan.

Gefitinib (Iressa) is an anticancer drug that selectively inhibits tyrosine kinases of epidermal growth factor receptor. Gefitinib might affect CYP3A4-mediated metabolism, since the drug is a substrate of human CYP3A. In this study, we evaluated the effects of gefitinib on drug metabolism catalyzed by human CYP3A4. The effects of gefitinib on the CYP3A4-mediated formation of NPC (7-ethyl-10-(4-amino-1-piperidino)carbonyloxycamptothecin) and that of APC (7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecin) from irinotecan were examined with the use of human liver and small intestinal microsomes. Gefitinib inhibited the formation of NPC in liver and small intestinal microsomes. The apparent intrinsic metabolic clearance (CL(int)) in the presence of 40 microM gefitinib was equivalent to about 26% of control in liver microsomes and 45% of control in small intestinal microsomes. Gefitinib stimulated the formation of APC by CYP3A4. CL(int) in the presence of 20 microM gefitinib with human liver microsomes was about 1.9 times higher than control. In human small intestinal microsomes, APC formation was enhanced by the addition of gefitinib at concentrations 20 microM or higher. CL(int) in the presence of 40 microM gefitinib was 2.8 times higher than control. Thus, we discovered that gefitinib inhibited the formation of NPC but stimulated the formation of APC from irinotecan.     

Binding of a metabolite of triflusal (2-hydroxy-4-trifluoromethylbenzoic acid) to serum proteins in rat and man

Summary 2-hydroxy-4-trifluoromethylbenzoic acid (HTB) is the main active metabolite of the platelet anti-aggregant drug triflusal. Its binding to plasma proteins of rats and healthy volunteers in vitro and in vivo has been studied.Rats were given a single oral dose of 50 mg·kg^–1 triflusal and the healthy volunteers received 300 mg as a single oral dose or a multiple dose regimen of 600 mg every 24 h and 300 mg every 8 h, both for 13 days. Protein-free HTB was obtained by ultrafiltration. Unbound and total HTB concentrations were determined by HPLC.HTB was primarily bound to albumin in plasma. The Scatchard plots suggested two types of binding sites for HTB on the albumin molecule. In rats, the binding constants (K=intrinsic affinity constant, n=number of binding sites) were K₁=1.4×10⁵ l·mol^–1, n₁=1.23, and K₂=4.1×10³ l·mol^–1 and n₂=3.77. The mean plasma concentration in rats after oral administration was 185 (37) g·ml^–1 (protein-free HTB: 2.44 (0.77)%). The binding constants in human plasma were K₁=4.7×10⁵ l·mol^–1, n₁=1.93, K₂=4.3 l·mol^–1 and n₂=4.28.The plasma HTB concentration in man (n=8) was 35 g·ml^–1 (C_max) after a single oral dose of triflusal 300 mg, 172.96 g·ml^–1 (C_max·ss) during the multiple dosage regimen of 300 mg every 8 h, and 131 g·ml^–1 (C_max·ss) during the multiple oral dose regimen of 600 mg every 24 h. Unbound HTB ranged from 0.27 to 0.43%, depending on dose. HTB had high affinity for plasma albumin, which was not saturable after therapeutic doses. It showed linear elimination.     

Plasma Protein Binding of the Experimental Antitumour Agent Acridine-4-carboxamide in Man,Dog, Rat and Rabbit

Abstract— The plasma binding of N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (AC) was investigated in-vitro by equilibrium dialysis for 3 h at 37°C against isotonic phosphate buffer (pH 7·35) using [³H]AC. There were significant species differences with the smallest % free fraction (mean ± s.d.) occurring in human plasma (3·4 ± 0·2), followed by dog (8·1 ±0·4), mouse (14·8 ± 0·8), rat (16·3 ± 0·9) and rabbit (20·2 ± 0·7). In plasma from healthy individuals (n = 5), the % free fraction ranged from 2·7 to 3·8. In physiological solutions of human proteins, the greatest binding was observed for α-acid glycoprotein (AAG) (0·75 g L^?1) with a mean free fraction of 24·1 ± 2·2%, followed by albumin (40 g L^?1) with 31·6 ± 0·7 and 39·8 ± 2·5% for fatty-acid-free and globulin-free, respectively. There was also some binding to globulins (5 g L^?1) with a mean % free fraction of 70·3 ± 1·6 and 84·8 ± 2·2 for Conn's fraction I and IV, respectively. Binding data from the displacement of [³H]AC by increasing concentrations of AC in human AAG (0·75 g L^?1) or albumin solution (40 g L^?1) indicated that AAG had 10-fold greater binding affinity for AC (K_a, 7·8 × 10⁴ m^?1) compared with albumin (K_a, 6·8 × 10³ m^?1). In human plasma enriched with AAG there was a significant negative linear correlation (r = 0·932; P < 0·001) between % AC free fraction and increasing AAG concentration over the range 0·6–4·5 g L^?1. Small but significant (P < 0·05) increases in AC free fraction occurred in the presence of various metabolites (50 and 100 μm) but, of those tested, only N-monomethyl-acridine carboxamide increased the free fraction to the same extent as parent AC.     

Binding of Prazosin to α1-Acid Glycoprotein and Albumin: Effect of Protein Purity and Concentrations

Abstract: Prazosin is extensively bound in human serum/plasma. In the present study a bound fraction of 93–95% was observed at 37° for therapeutic drug concentrations. Both α₁-acid glycoprotein (AAG) and albumin (HSA) are established as transport proteins for prazosin, but their individual contribution to the extent and variability of protein binding in serum/plasma is unclear. The present study showed that AAG possesses one binding site per molecule with high affinity (Kd≈0.8 μM) for prazosin. HSA, essentially globulin-free, bound prazosin with lower affinity (Kd≈30 μM) with an average of 0.3 binding sites per molecule. However, less purified HSA, containing globulins, exhibited apparently higher affinity (Kd≈8 μM), but lower binding capacity (0.07 sites per molecule) for prazosin. In mixtures of highly purified proteins, the concentrations of AAG, and not HSA, determined the extent and variability of prazosin binding.     

Tianeptine binding to human plasma proteins and plasma from patients with hepatic cirrhosis or renal failure.

1. The binding of tianeptine to human plasma, isolated plasma proteins, red blood cells and to plasma from patients with cirrhosis or renal failure was studied in vitro by equilibrium dialysis. 2. Tianeptine is highly bound to plasma (95%) at therapeutic concentrations (0.3-1 microM). No saturation of the binding sites was seen. 3. Human serum albumin (HSA) was shown to be mainly responsible for this binding (94%) with a saturable process characterized by one binding site with a moderate affinity (Ka = 4.2 x 10(4) M-1) and a non-saturable process with a low total affinity (nKa = 1.2 x 10(4) M-1). 4. Like many basic and amphoteric drugs, tianeptine showed a saturable binding to alpha 1-acid glycoprotein (AAG) with one site and a moderate affinity (Ka = 3.7 x 10(4) M-1). Its binding to lipoproteins and red blood cells (RBC) was weak and non-saturable. Over the range of therapeutic drug concentrations (0.3-1 microM), the unbound fraction in blood remains constant (4.5%). 5. Interactions were studied using non-esterified fatty acids (NEFA) at pathological concentrations; they altered tianeptine binding to plasma and to isolated HSA. Tianeptine seems to bind to a HSA site different from sites I (warfarin) and II (diazepam), but close to site II. It also shares the only basic-site on AAG. However, at therapeutic drug concentrations (0.3-1 microM), not all of these interactions occur. 6. The binding of tianeptine varied according to HSA, AAG and NEFA concentrations both in patients and healthy subjects. In patients with chronic renal failure having high NEFA concentrations the unbound fraction of tianeptine (fu) increased from 0.045 to 0.153 compared with normal (P less than 0.001). In cirrhotic patients, with relatively low HSA concentrations, the fu of tianeptine increased from 0.045 to 0.088 compared with normal (P less than 0.01). 7. Multiple regression analysis of all of the data indicated that the fu of tianeptine was related significantly to HSA, NEFA and AAG concentrations, with a particularly strong correlation with NEFA concentrations. Therefore, variation of HSA and NEFA concentrations in patients on maintenance therapy may cause an increase of tianeptine fu.     

In Vitro Binding and Partitioning of Irinotecan (CPT-11) and its Metabolite, SN-38, in Human Blood

The binding of CPT-11 and SN-38 to human plasma proteinswas studied by ultrafiltration at 37°C and pH 7.4. In plasma,CPT-11 was 66–60% bound in the range 100–4000ng/ml and SN-38 was 94–96% bound in the range50–200 ng/ml. At these concentrations the plasma bindingof CPT-11 was slightly saturable, but the plasma binding of SN-38was concentration-independent. Albumin was the main carrier ofCPT-11 and SN-38 in plasma. In blood, the binding of CPT-11 wasmoderate (80%), mainly to plasma proteins (47%) anderythrocytes (33%). The binding of SN-38 was high(99%) and most of SN-38 in blood was located in bloodcells (approximately 66%) The simulation of a grade 3hematotoxicity (according to National Cancer Institute's CommonToxicity Criteria grading) on the SN-38 blood distributionyielded an increase in fu (free fraction of drug in plasma) from1.05 to 2.08 and a decrease in C_Bl/C_P from1.66 to 1.14 (both resulting from a decreased cellbinding).     

Influence of plasma protein binding on the brain uptake of an antifungal agent, terbinafine, in rats

The intracarotid injection technique has been used to determine the unidirectional brain uptake of an antifungal, lipophilic agent, terbinafine (Lamisil, Sandoz Basle), in the rat. Ultrafiltration showed it to be highly bound to human plasma, human serum albumin (HSA), alpha 1-acid glycoprotein (AAG) and lipoproteins (VLDL, LDL, HDL). The effect of plasma protein binding of the drug on brain uptake was also examined with the technique. The lowest brain uptake was observed in the presence of plasma (6%); it varied from 23 to 30% with physiological concentrations of VLDL, LDL and HSA and was significantly higher (43-45%) in the presence of physiological concentrations of AAG and HDL. The free fraction as determined in-vitro and the brain uptake of the drug varied inversely with the plasma protein concentrations; however, the brain uptake was higher than expected from in-vitro measurements. These data indicate that the amount of circulating Lamisil available for brain penetration exceeds its free fraction; they also show that plasma proteins differently reduce the brain transport of the drug.     

Pharmacokinetic and Pharmacodynamic Aspects of Artelinic Acid in Rodents

Abstract— The efficacy of artelinic acid and artemisinin, orally administered at 10 and 50 mg kg^?1 day^?1, was compared in Plasmodium berghei infected mice. Subsequently, the pharmacokinetics of artelinic acid after intravenous, intramuscular, oral and rectal administration of a 20 mg kg^?1 aqueous solution to rabbits were studied in a four-way randomized cross-over experiment. After intravenous administration, artelinic acid concentrations in blood plasma were high (C₀: 76 ± 15 mg L^?1), and the drug was rapidly eliminated from the central compartment, showing linear elimination kinetics with an elimination half-life of 15 ± 3 min. A large inter-subject variation appeared in the absorption rate and the extent of absorption (2–92%) over the 120 min interval after intramuscular administration. Also, a large inter-subject variation in individual rectal bioavailability (17–100%) was shown, which was dependent on the site of absorption in the rectum. The estimated oral bioavailability was low (4·6 ± 1·7%), probably due to a high first-pass effect and possible decomposition in the acidic gastric environment.     

The pharmacokinetics,toxicities, and biologic effects of FK866, a nicotinamide adenine dinucleotide biosynthesis inhibitor

Summary Background FK866 is a potent inhibitor or NAD synthesis. This first-in-human study was performed to determine the maximum-tolerated dose, toxicity profile, and pharmacokinetics on a 96-h continuous infusion schedule. Materials and methods Twenty four patients with advanced solid tumor malignancies refractory to standard therapies were treated with escalating doses of FK866 as a continuous, 96-h infusion given every 28 days. Serial plasma samples were collected to characterize the pharmacokinetics of FK866. Further blood samples were collected for the measurement of plasma VEGF levels. Results There were 12 women and 12 men with a median age of 61 (range 34-78) and a median KPS of 80%, received a 4-day of infusion of FK866 at dose levels of 0.018 mg/m²/h (n = 3), 0.036 mg/m²/h (n = 3), 0.072 mg/m²/h (n = 3), 0.108 mg/m²/h (n = 4), 0.126 mg/m²/h (n = 6), and 0.144 mg/m²/h (n = 5). Thrombocytopenia was the dose limiting toxicity, observed in two patients at the highest dose level and one patient at the recommended phase II dose of 0.126 mg/m2/h No other hematologic toxicities were noted other than mild lymphopenia and anemia. There was mild fatigue and grade 3 nausea; the latter was controlled with antiemetics and was not a DLT. C_ss (the mean of the 72 and 96 h plasma concentrations) increased in relation to the dose escalation. The study drug did not significantly affect plasma concentrations of VEGF. There were no objective responses, although four patients had stable disease (on treatment for 3 months or greater). Conclusions The recommended phase II dose is 0.126 mg/m²/h given as a continuous 96-h infusion every 28 days. The dose limiting toxicity of FK866 is thrombocytopenia. Pharmacokinetic data suggest an increase in the plasma C_ss in relation to the escalation of FK866.     

Kinetic Interaction between Fluoxetine and Imipramine as a Function of Elevated Serum Alpha-1-acid Glycoprotein Levels

The effect of elevated serum alpha-1-acid glycoprotein (AAG) levels on the pharmacokinetic interaction between imipramine and fluoxetine has been examined by utilizing a novel strain of transgenic mice which express serum AAG levels several times greater than normal. Before fluoxetine treatment, serum imipramine levels were approximately three times greater in transgenic mice than in control mice. Despite higher serum imipramine levels in transgenic mice, brain drug levels were lower than those found in control mice. Fluoxetine pre-treatment (20mg kg^?1 for 5 days) resulted in an increase in serum imipramine levels in both groups of mice and the extent of the increase was greater in transgenic mice than in control mice (4.5-fold increase compared with 3.1-fold). Similarly, fluoxetine pre-treatment resulted in an increase in brain levels of imipramine in both groups of mice and the extent of the increase was greater in transgenic mice than in control mice (3.0-fold increase compared with 2.0-fold). Similar trends were observed for levels of desipramine in the serum and brain. Serum imipramine and desipramine levels did not correlate with their respective brain levels in the presence of elevated serum AAG levels before and after pre-treatment. These findings indicate that the extent of increases in imipramine and desipramine serum and brain levels are greater during elevated serum AAG states than during normal AAG states when imipramine is co-administered with fluoxetine.     

Pharmacokinetic properties of YM17E, an inhibitor of acyl coenzyme A: cholesterol acyl transferase, and serum cholesterol levels in healthy volunteers

  We conducted a single and repeat oral dose study of YM17E, a novel inhibitor of acyl coenzyme A (CoA): cholesterol acyltransferase, in healthy male volunteers to evaluate the pharmacokinetic profile, tolerability and effect of the drug on serum cholesterol. In the single administration study, YM17E was administered after a meal to two groups of subjects (each containing six subjects taking the drug and three taking placebo) receiving 3, 60 and 300 mg or 15, 60 and 450 mg YM17E, respectively. Plasma concentrations of unchanged drug following single oral administration at 3–300 mg after a meal increased with increasing dose. In contrast, plasma concentrations after administration of 450 mg were almost the same as after 300 mg. Unchanged YM17E was not detected in urine after single administration, suggesting that it was excreted via the bile or urine after metabolism. Five active metabolites (M1, M2-a, M2-b, M3 and M4) were observed in plasma at concentrations comparable to those of unchanged YM17E. Their plasma concentrations increased in a slightly greater than dose-dependent manner from 3 to 300 mg. The effect of food was studied in an open crossover design with a 1-week washout period. Twelve subjects received 150 mg YM17E in both the fasted and postprandial states. The AUC and C_max after fasting were closely similar to those after a meal, showing that bioavailability was not affected by food intake. In the repeated oral dose study, the subjects received test drug at 150 mg or 300 mg (n = 6 each) or placebo (n = 3) twice a day (after breakfast and after dinner) for 7 days. On days 1 and 7, the subjects received YM17E once a day (after breakfast) for evaluation of pharmacokinetic properties. After repeated oral administration of 150 mg b.d., plasma concentrations reached steady state by day 5 (mean C_min 48.6 ng · ml⁻¹). After repeated administration of 300 mg b.d., plasma concentrations prior to each daily morning dose increased up to the 5th day (mean C_min 166.6 ng · ml⁻¹) and then tended to decrease until the 7th day. No significant signs, symptoms or changes in serum cholesterol levels were observed during the single and repeated oral dose studies at 150 mg b.d. Although statistical analysis was not conducted because of the small number of subjects, all subjects receiving repeated oral administration of 300 mg twice daily showed a 25% decrease in serum cholesterol level on day 7, but also the simultaneous occurrence of diarrhoea. Received: 23 April 1996 / Accepted in revised form: 1 August 1996     

Identification, characterization, and implications of species-dependent plasma protein binding for the oral Hedgehog pathway inhibitor vismodegib (GDC-0449)

Vismodegib (GDC-0449) is is an orally available selective Hedgehog pathway inhibitor in development for cancer treatment. The drug is ≥95% protein bound in plasma at clinically relevant concentrations and has an approximately 200-fold longer single dose half-life in humans than rats. We have identified a strong linear relationship between plasma drug concentrations and α-1-acid glycoprotein (AAG) in a phase I study. Biophysical and cellular techniques have been used to reveal that vismodegib strongly binds to human AAG (K(D) = 13 μM) and binds albumin with lower affinity (K(D) = 120 μM). Additionally, binding to rat AAG is reduced ～20-fold relative to human, whereas the binding affinity to rat and human albumin was similar. Molecular docking studies reveal the reason for the signficiant species dependence on binding. These data highlight the utility of biophysical techniques in creating a comprehensive picture of protein binding across species.