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Carfilzomib [(2S)-N-[(S)-1-[(S)-4-methyl-1-[(R)-2-methyloxiran-2-yl]-1-oxopentan-2-ylcarbamoyl]-2-phenylethyl]-2-[(S)-2-(2-morpholinoacetamido)-4-phenylbutanamido]-4-methylpentanamide, also known as PR-171] is a selective, irreversible proteasome inhibitor that has shown encouraging results in clinical trials in multiple myeloma. In this study, the pharmacokinetics, pharmacodynamics, metabolism, distribution, and excretion of carfilzomib in Sprague-Dawley rats were characterized. After intravenous administration, the plasma concentration of carfilzomib declined rapidly in a biphasic manner. Carfilzomib displayed high plasma clearance [195–319 ml/(min · kg)], a short-terminal half-life (5–20 min), and rapid and wide tissue distribution in rats. The exposure to carfilzomib (Cmax and area under the curve) increased dose proportionally from 2 to 4 mg/kg but less than dose proportionally from 4 to 8 mg/kg. The high clearance was mediated predominantly by extrahepatic metabolism through peptidase cleavage and epoxide hydrolysis. Carfilzomib was excreted mainly as metabolites resulting from peptidase cleavage. Carfilzomib and its major metabolites in urine and bile accounted for approximately 26 and 31% of the total dose, respectively, for a total of 57% within 24 h postdose. Despite the high systemic clearance, potent proteasome inhibition was observed in blood and a variety of tissues. Together with rapid and irreversible target binding, the high clearance may provide an advantage in that "unnecessary" exposure to the drug is minimized and potential drug-related side effects may be reduced.
The proteasome is a multicatalytic enzyme complex that is the primary means for protein degradation in all cells (Kisselev and Goldberg, 2001; Ciechanover, 2005). Inhibition of the proteasome leads to the accumulation of misfolded proteins and the induction of apoptosis. The proteolytic activities of the proteasome are encoded by distinct, N-terminal threonine-containing enzymes that function as chymotrypsin-like (CT-L), trypsin-like, and caspase-like proteases. Tumor cells are more susceptible than nontransformed cells to the cytotoxic effects of proteasome inhibition, making the proteasome an attractive target for the treatment of malignant diseases (Adams, 2004). The approval of bortezomib (Velcade) by the U.S. Food and Drug Administration for the treatment of multiple myeloma (MM) and mantle cell lymphoma provided clinical validation of the proteasome as a therapeutic target in oncology (Adams et al., 1998; Bross et al., 2004; Chauhan et al., 2005; Goy et al., 2005; O'Connor et al., 2005; Richardson et al., 2005). Bortezomib is a dipeptide boronic acid that reversibly inhibits the 20S proteasome by forming a tetrahedral intermediate with the side chain hydroxyl group of the N-terminal threonine (Borissenko and Groll, 2007). Despite the encouraging clinical success of bortezomib, adverse effects including painful peripheral neuropathy have been reported, and a significant fraction of patients relapse from or are refractory to treatment with bortezomib (Orlowski et al., 2002, 2007a,b; Richardson et al., 2003, 2005; Goy et al., 2005; O'Connor et al., 2005). In addition, bortezomib is metabolized primarily by cytochrome P450 3A4 (Uttamsingh et al., 2005), and coadministration of cytochrome P450 3A4 inhibitors such as ketoconazole causes a significant change in bortezomib plasma levels in humans (Venkatakrishnan et al., 2009). In the past several years, the next generation of proteasome inhibitors has been explored, with the aim to improve safety and efficacy profiles. Three small molecules [carfilzomib, (4R,5S)-4-(2-chloroethyl)-1-((1S)-cyclohex-2-enyl(hydroxy)methyl)-5-methyl-6-oxa-2-azabicyclo[3.2.0]heptane-3,7-dione (NPI-0052), and [(1R)-1-[[(2S,3R)-3-hydroxy-2-[[(6-phenylpyridin-2-yl)carbonyl]amino]-1-oxobutyl]amino]-3-methylbutyl]boronic acid (CEP-18770)] are currently in clinical development (Bennett and Kirk, 2008).
Carfilzomib (Fig. 1) is a tetrapeptide epoxyketone analog of epoxomicin and eponemycin, a pair of related natural products that were discovered initially as antitumor agents in animals and later shown to inhibit the CT-L activity of the proteasome (Hanada et al., 1992; Kim et al., 1999; Meng et al., 1999a,b; Groll et al., 2000). The epoxyketone pharmacophore forms a dual covalent morpholino adduct with the N-terminal threonine. Carfilzomib shows primary selectivity for the CT-L activity and displays little activity against the trypsin-like or caspase-like activities of the proteasome (Demo et al., 2007). Upon the basis of encouraging preclinical data, including the induction of potent proteasome inhibition in blood and tissues in rodents and greater antitumor activity compared with that of bortezomib in mouse models of cancer (Demo et al., 2007; Kuhn et al., 2007), carfilzomib entered clinical testing in MM. Carfilzomib has shown encouraging results in a broad clinical trial program in MM (Alsina et al., 2007; Jakubowiak et al., 2010a,b; Jagannath et al., 2010; Martin et al., 2010; Siegel et al., 2010; Singhal et al., 2010; Vij et al., 2010), including in patients refractory to bortezomib. Carfilzomib is currently being developed by Onyx Pharmaceuticals, Inc. for the treatment of MM and is in phase III clinical trials.
The objective of the present study was to characterize the disposition of carfilzomib in rats, a pharmacological and toxicological species used in preclinical evaluation of the compound. We show here that, after intravenous administration to Sprague-Dawley rats, carfilzomib was cleared rapidly from systemic circulation. In vitro and in vivo studies showed that carfilzomib was metabolized extensively in blood and a variety of tissues. The major metabolites detected in rat plasma, urine, and bile were from peptidase cleavage and epoxide hydrolysis. Elimination occurred mainly via biliary and renal excretion in the form of metabolites. Despite rapid systemic clearance, carfilzomib generated potent proteasome inhibition in a variety of tissues due to irreversible inhibition of its target, demonstrating the wide tissue distribution of carfilzomib upon intravenous administration. We finally show that a 30-min infusion administration resulted in a 28-fold lower maximum plasma concentration (Cmax) but with comparable levels of proteasome inhibition in blood and tissues as those of an intravenous bolus.
Materials and Methods
(2S)-N-[(S)-1-[(S)-4-Methyl-1-[(R)-2-methyloxiran-2-yl]-1-oxopentan-2-ylcarbamoyl]-2-phenylethyl]-2-[(S)-2-(2-morpholinoacetamido)-4-phenylbutanamido]-4-methylpentanamide (carfilzomib, also known as PR-171), N-[(S)-1-[(S)-1-[(S)-4-methyl-1-[(R)-2-methyloxiran-2-yl]-1-oxopentan-2-ylcarbamoyl]-2-phenylethylcarbamoyl]-3-methylbutyl]-5-(morpholinomethyl)isoxazole-3-carboxamide (PR-054591), and (2S)-3-methoxy-2-[(2S)-3-methoxy-2-[(2-methyl-1,3-thiazol-5-yl)formamido] propanamido]-N-[(2S)-1-[(2R)-2-methyloxiran-2-yl]-1-oxo-3-phenylpropan-2-yl]propanamide (ONX 0912), tripeptide analogs of carfilzomib shown in Fig. 1, and authentic standards of carfilzomib metabolites (M14, M15, and M16; Fig. 2 and Table 3) were synthesized at Onyx Pharmaceuticals, Inc. PR-054591 was used as the internal standard (IS) for all of the quantitative analyses. Rat whole blood and rat and monkey plasma in sodium heparin were from Bioreclamation (East Meadow, NY). Tissue homogenates were prepared in house. All of the other reagents were obtained from commercial sources with an appropriate quality.
Pharmacokinetics Studies.
All of the animal studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996). Male Sprague-Dawley rats, weighing approximately 250 to 350 g and containing cannulas in the jugular vein and carotid artery, were obtained from Charles River Laboratories, Inc. (Wilmington, MA) and used for all of the pharmacokinetics (PK) studies. Animals were allowed to acclimate for a minimum of 3 days before experimentation and allowed food and water ad libitum.
Carfilzomib was formulated in 10% (w/v) sulfobutyl ether β-cyclodextrin (Captisol; Cydex Pharmaceuticals Inc., Lenexa, KS) in 10 mM citrate (pH 3.5) at a concentration of 2 mg/ml. For intravenous bolus administration, four to five animals per dose level received a single dose of carfilzomib at 2, 4, or 8 mg/kg, respectively. Blood samples (∼0.4 ml) were collected before and immediately after dosing (within 10 s) and at 1, 2, 5, 15, 30, and 60 min postdose. For intravenous infusion administration, four rats received carfilzomib via a 30-min constant infusion at 8 mg/kg. Blood samples were collected before dosing, at 15 min after the start of infusion, end of infusion, and at 2, 5, 15, 30, 60, and 120 min postdose.
To assess the effects of target binding, the PK profiles of carfilzomib from two groups of male Sprague-Dawley rats (three per group) were compared. One group was pretreated with an intravenous bolus dose of 10 mg/kg ONX 0912 (also known as PR-047), a tripeptide analog of carfilzomib with potent irreversible proteasome inhibition (Zhou et al., 2009), whereas the other group was pretreated with the formulation vehicle. Thirty minutes after pretreatment, carfilzomib was administered via an intravenous bolus at 2 mg/kg. Blood samples were taken before and immediately after the carfilzomib dose and at 1, 2, 5, 15, 30, and 60 min after the carfilzomib dose.
A PK study also was conducted for M16, the diol of carfilzomib derived from epoxide hydrolysis via an intravenous bolus administration to male Sprague-Dawley rats at 5 mg/kg. M16 was formulated at 2 mg/ml in Captisol containing 10 mM citrate (pH 3.5). Blood samples were collected before dosing and at 2, 5, 10, 15, 30, 60, 120, and 360 min postdose.
The PK of carfilzomib also was assessed in non-naive male cynomolgus monkeys with an average weight of 3 kg. The in-life portion of the study was conducted at SNBL USA (Everett, WA). Carfilzomib was formulated in 5% hydroxypropyl-β-cyclodextrin (w/v) in 50 mM sodium citrate (pH 3.35) at a concentration of 1 mg/ml. Three animals received a single intravenous bolus dose of carfilzomib at 1 mg/kg. Blood samples were collected before dosing and at 2, 5, 15, 30, and 60 min postdose.
All of the blood samples were collected in tubes containing sodium heparin as the anticoagulant. Blood samples were kept on ice during sample collection and centrifuged at 3000g for 10 min at 4°C to obtain plasma. Plasma samples were stored in cryotubes at −70°C until analysis.
Calculation of PK Parameters.
PK parameters were calculated by noncompartmental analysis using WinNonlin 5.2 (Pharsight, Mountain View, CA). Plasma concentrations reported as below the lower limit of quantification were treated as zero for PK analysis. Area under plasma concentration-time curve (AUC) values were estimated using the linear trapezoidal method. Plasma clearance (CL) was calculated from dose/AUC extrapolated to infinity (AUCinf). Terminal half-life (t1/2) values were calculated as ln(2)/k, where k represents the terminal elimination rate constant obtained from the slope of the semilogarithmic plot of the concentration-time profile. Mean residence time was estimated by moment analysis. Volume of distribution at steady state (Vss) was calculated from mean residence time × CL. Cmax was recorded as observed. The amount of M16 generated in rats after carfilzomib dosing was calculated by multiplying the AUCinf of M16 by the clearance of M16 obtained in the PK study for M16.
Pharmacodynamic Study.
Blood and tissue (adrenal, heart, liver, and lung) samples were collected from Sprague-Dawley rats receiving 8 mg/kg carfilzomib by either an intravenous bolus or a 30-min infusion administration. Blood samples were collected at predose and 0.25, 2, and 24 h postdose. For intravenous infusion, blood samples also were taken at 0.25 h after the start of infusion. Tissue samples were collected at 2 and 24 h postdose. Proteasome CT-L activity was measured in lysates from cells and blood with Leu-Leu-Val-Tyr-AMC (LLVY-AMC) as the substrate as described previously (Demo et al., 2007).
Excretion Study.
Excretion of carfilzomib was determined in male Sprague-Dawley rats with bile duct cannulation after a single intravenous bolus administration at 2 mg/kg. Bile was collected cumulatively at 0 to 4 and 4 to 8 h postdose, no bile samples were collected at 8 to 24 h postdose, and urine was collectively 0 to 4, 4 to 8, and 8 to 24 h postdose. Volumes of the bile and urine samples were measured. Acetonitrile was added to the original urine sample containers after the first thawing to achieve a final organic content of 20% (v/v). This was done to ensure full recovery of carfilzomib from the container. Plasma samples also were collected in this study before and immediately after dosing and at 1, 2, 5, 15, 30, 60, and 120 min postdose into tubes containing sodium heparin. The urine, bile, and plasma samples were stored at −70°C until analysis.
Blood Plasma Partitioning.
The whole blood to plasma ratio of carfilzomib was determined in vitro. Carfilzomib was spiked into prewarmed rat blood at nominal concentrations of 0.2, 2, or 10 μM. The samples were mixed gently by inverting the tubes several times and then incubated at 37°C. At 15 min, an aliquot of 100 μl of blood sample was taken and immediately quenched into 300 μl of acetonitrile containing 0.5 μM IS. The remaining blood samples were centrifuged at 3000g for 10 min at 4°C to obtain plasma.
Metabolite Profiling in Rat Plasma, Urine, and Bile Samples.
Rat plasma samples collected in a 6-month toxicity study to support toxicokinetic evaluation of carfilzomib were used to search for circulating metabolites. Carfilzomib was given to three groups of rats at dose levels of 1, 2, or 4 mg/kg for six 28-day cycles with intravenous bolus dosing on days 1, 2, 8, 9, 15, and 16. Plasma samples were collected on the first day of cycles 1, 3, and 6 (days 1, 57, and 142, respectively) before dose and at 5, 15, 30, and 60 min postdose. Aliquots of plasma samples were pooled across animals, dose levels, collection days, and collection time points to generate one pooled sample for metabolite profiling. A pooled predose plasma sample was used as a control. The pooled samples were extracted multiple times with three volumes of acetonitrile to recover carfilzomib and metabolites. The supernatants were combined and evaporated to dryness under a gentle stream of nitrogen. The residues were reconstituted with 0.5 ml of acetonitrile/water (1:1, v/v) and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS).
The urine and bile samples used in metabolite profiling were obtained from the excretion study as described in the preceding section. Aliquots of bile samples or acetonitrile-treated urine samples were pooled across animals to generate one pooled bile or urine sample per time interval. The pooled urine and bile samples were subjected directly to LC-MS/MS analyses.
LC-MS/MS analyses were done in positive ionization mode using precursor ion and neutral loss scans on LTQ mass spectrometer (Thermo Fisher Scientific, Waltham, MA) and multiple ion reaction monitoring (MRM) on an API 4000 QTrap LC/MS/MS System (AB SCIEX, Foster City, CA) using a Luna C18(2) column (150 × 4.6 mm, 3-μm particle size; Phenomenex, Torrance, CA) and gradient elution. The mobile phases were 0.4% formic acid in water adjusted to pH 3.2 with ammonium hydroxide (A) and acetonitrile (B). The flow rate was 0.6 ml/min with solvent A held at 100% for 5 min, followed by ramping solvent B from 0 to 70% in 55 min and 70 to 100% B in 2 min. The structures of the metabolites were proposed based on the molecular ion masses and fragmentation patterns generated by product ion scans. Semiquantification was done in MRM mode by comparing the peak area of each metabolite to that of carfilzomib.
Metabolism Study in Rat Blood and Tissue Homogenates from Adrenal, Blood, Heart, Kidney, Liver, and Lung.
The metabolism of carfilzomib was evaluated in rat blood and tissue homogenates (adrenal, heart, kidney, liver, and lung) harvested from untreated male Sprague-Dawley rats. The isolated organs were chopped into small pieces, washed several times with phosphate-buffered saline (pH 7.4), and then weighed into 2-ml Eppendorf tubes. Dulbecco's phosphate-buffered saline (pH 7.4) then was added to the tubes to achieve a 1:5 ratio of tissue weight to Dulbecco's phosphate-buffered saline volume. The tissues were homogenized using a TissueLyser (Retsch, Inc., Newtown, PA) at a frequency of 20 s−1. Aliquots of 300 μl of rat blood or tissue homogenates were preincubated at 37°C for 5 min before the addition of 5 μl of carfilzomib working solution at a concentration of 60 μg/ml in acetonitrile. The final concentration of carfilzomib in the incubation was 1 μg/ml. The mixture was incubated at 37°C with gentle agitation. An aliquot of 40 μl of reaction mixture was taken at 2, 10, 20, 30, 60, and 90 min and quenched with 120 μl of acetonitrile containing 0.5 μM IS. After centrifugation, the supernatants were used to measure the disappearance of carfilzomib and the formation of selected metabolites, M14, M15, and M16. The relative rates of carfilzomib metabolism in rat adrenal and liver were evaluated in a separate experiment under the same experimental conditions described above.
Quantitative Determination of Carfilzomib and Its Metabolites in Blood, Plasma, Urine, and Bile.
Concentrations of carfilzomib and its metabolites (M14, M15, and M16) in blood, plasma, urine, and bile samples were determined by LC-MS/MS. Plasma, urine, and bile samples were stored at −70°C immediately after collection. Stability testing demonstrated that the analytes were stable under the storage condition, during the extraction process, and for at least three freeze/thaw cycles. Carfilzomib and its metabolites were quantified by comparing the peak area ratios of the analyte to IS in study samples to those of the standard calibration samples prepared in the same manner. To determine the concentration of carfilzomib in blood samples from an in vitro partitioning study, the standard calibration samples were prepared by spiking carfilzomib into blood prequenched with three volumes of acetonitrile containing 0.5 μM IS. An aliquot (40 μl) of plasma, acetonitrile-treated urine, or bile sample was mixed with 80 μl of acetonitrile containing 0.5 μM IS to extract carfilzomib and its metabolites. The extracted samples were vortexed and centrifuged. The supernatants were filtered through a 96-well filter plate (AcroPrep 96-well Filter Plate, 0.45 μm; Pall Corporation, East Hills, NY) into a 96-well microplate, and 10 μl was injected for LC-MS/MS analysis.
The LC-MS/MS system comprised either an API 3000 or an API 3200 mass spectrometer (AB SCIEX) equipped with two Shimadzu LC-10ADvp pumps and a Shimadzu SIL-HTc autosampler with controller system (Shimadzu, Kyoto, Japan). Chromatographic separation was achieved on an ACE C18 column (2.1 × 50 mm, 3.5-μm particle size; Advanced Chromatography Technology, Aberdeen, Scotland) using gradient elution. The mobile phases were 0.1% formic acid in water (A) and methanol (B). Mass spectrometric detection was accomplished using the turbo ion spray interface in the positive ionization mode by MRM of the selective m/z transitions: 720.5 → 100 for carfilzomib, 307.3 → 100 for M14, 420.4 → 100 for M15, 738.5 → 100 for M16, and 626.3 → 199.0 for IS. The lower limit of quantification was 2.00 ng/ml with calibration ranges of 2.00 to 3000 ng/ml for blood and plasma samples and 2.00 to 1000 ng/ml for urine and bile samples.
After a single intravenous bolus dose of 2, 4, or 8 mg/kg, carfilzomib plasma concentration-time curves display a very steep initial phase (Fig. 3A). The first plasma sampling was done within 10 s after dosing to accurately measure the PK of carfilzomib in rats; plasma levels at 2 min were <5% of those immediately after dosing (Fig. 3A, inset). The t1/2 value ranged from 5 to 20 min, and CL values were 195 to 296 ml/(min · kg) (Table 1). Both AUC and Cmax increased dose proportionally, and the CL values were constant from 2 to 4 mg/kg. When the dose level was increased to 8 mg/kg, however, AUC and Cmax increased less than dose proportionally, and the CL value increased. Rapid clearance and short half-life also were observed in cynomolgus monkeys after intravenous administration at 1 mg/kg (Supplemental Fig. S1).
Carfilzomib is a rapidly binding and irreversible inhibitor of the proteasome, a ubiquitously expressed target that accounts for approximately 1% of total cellular protein (Ciechanover 2005; Demo et al., 2007). To determine whether the high level of target expression affects its PK, carfilzomib was administered to two groups of rats: one pretreated with an intravenous bolus dose of the vehicle (control group) and the other pretreated with an intravenous bolus dose of ONX 0912 (formerly known as PR-047), a tripeptide analog of carfilzomib that displays potent and irreversible inhibition of the 20S proteasome (Zhou et al., 2009). A dose of 10 mg/kg was chosen for ONX 0912 to completely inhibit the primary target of carfilzomib, the CT-L activity of the proteasome, in blood and available tissues (J. Jiang and C. Kirk, unpublished observations). Thirty minutes after pretreatment, carfilzomib was administered as an intravenous bolus at 2 mg/kg to both groups of animals. No significant differences in the PK parameters between the two groups were noted, suggesting that target binding is not a major mechanism of clearance for carfilzomib (Table 2).
The level of proteasome inhibition could be a function of peak plasma levels or total exposure to carfilzomib. When administered as a 30-min infusion at 8 mg/kg, steady state was achieved essentially within 15 min, which is the first plasma sampling during the infusion (Fig. 3B). The steady-state concentration (Css) of carfilzomib is 28-fold lower than the Cmax in the bolus group at the same dose level (Table 1). The other PK parameters, including AUC, were equivalent. The level of proteasome inhibition in blood and tissues was also equivalent between the two groups (Fig. 3C). These data demonstrate that the level of target inhibition achieved in vivo is a function of the total dose administered but not Cmax. The potent proteasome inhibition in a variety of tissues also demonstrated rapid and wide tissue distribution of carfilzomib after a single intravenous bolus or infusion administration.
In Vitro Blood Partitioning.
The ratios of whole blood concentrations to plasma concentrations were 0.82, 0.74, and 0.63 at the nominal blood concentrations of 0.2, 2, and 10 μM carfilzomib, respectively. Because the calibration standards were prepared by spiking carfilzomib into prequenched whole blood, the measured whole blood levels do not represent the fraction bound to the drug target.
All of the predictable metabolites were searched in rat plasma, urine, and bile using the LC-MS/MS method described under Materials and Methods. A total of 21 metabolites were identified based on molecular ion masses and fragmentation patterns (Table 3). Although a few oxidative metabolites were observed, most metabolites were derived from peptide and epoxide hydrolysis. Semiquantification in MRM mode, assuming that carfilzomib and its metabolites exhibited similar responses under analytical conditions, has shown that morpholino-homophenylalanine (M14), morpholino-homophenylalanine-leucine (M15), and the diol of carfilzomib (M16) were the most abundant metabolites and that the oxidative metabolites were minor. Authentic standards of these three metabolites were synthesized to further confirm their structures and used for quantitative determination of their levels in plasma and excreta. Amino acids, naturally occurring dipeptides, and a low level of the dipeptide epoxyketone (M11) resulting from hydrolysis of carfilzomib also were observed. In an in vitro metabolism study in rat hepatocytes using [3H]carfilzomib with the label present in the aromatic ring of the phenylalanine, radioactive phenylalanine indeed was detected as a relatively abundant metabolite (J. Jiang and C. Kirk, unpublished observations). However, when nonradiolabeled carfilzomib was administrated to rats as conducted in the present study, these amino acids and dipeptides could not be differentiated from endogenous ones. Therefore, they were not assessed quantitatively in this study.
As shown in Fig. 4, all three metabolites, M14, M15, and M16, formed rapidly, with measurable levels immediately postdose and peak levels within 5 min of dosing. M14 is the most abundant circulating metabolite, with a terminal half-life longer than that of the parent drug. The AUC ratios (metabolite/carfilzomib) were 2.7, 0.33, and 0.12 for M14, M15, and M16, respectively. As determined in a separate PK study, plasma clearance of M16 was rapid [110 ml/(min · kg)] (Supplemental Table S1). Using this clearance value and the observed AUCinf of M16 after intravenous bolus administration of carfilzomib, we estimated that approximately 6% of the carfilzomib administered to rats was biotransformed to the diol.
Carfilzomib was also metabolized rapidly in vitro after incubation with rat tissue homogenates, including adrenal, heart, kidney, liver, and lung. The half-life of carfilzomib disappearance was 4 to 39 min in blood and tissues (Fig. 5; Supplemental Fig. S2). Consistent with observations in the in vivo samples, the peptide hydrolysis products M14 and M15 were the abundant metabolites. M16 was also detected, but at a lower level. Taken together, these data suggest that carfilzomib is metabolized rapidly in rats mainly via peptidase cleavage and epoxide hydrolysis. The peptidase cleavage product M14 was the most abundant metabolite in both systemic circulation and excreta. Although M14 can be a secondary metabolite of M16 as depicted in Fig. 2, conversion of M16 to M14 is not likely the main route of M14 formation in rats because only approximately 6% of carfilzomib was converted to M16.
Carfilzomib and its major metabolites were quantified in the collected urine and bile samples. Less than 1% of the intravenously dosed carfilzomib was excreted intact (Table 4). The products of peptide cleavage (M14 and M15) were the main forms of excretion. The majority of the metabolites were excreted within 4 h after dosing of carfilzomib. Renal and biliary excretion of carfilzomib and metabolites accounted for 26 and 31% of the dose, respectively, for a total of 57% recovered within 24 h postdose.
In this study, we characterized the PK, pharmacodynamics (PD), metabolism, distribution, and excretion of carfilzomib, an irreversible proteasome inhibitor, in rats, the rodent species used in pharmacology and toxicology studies of the drug candidate. The results of these studies suggest that carfilzomib displays several properties that may help to explain the favorable clinical safety profile of this agent (Vij et al., 2010).
After intravenous bolus administration, carfilzomib was cleared rapidly from systemic circulation with a t1/2 value of <20 min. The clearance is more rapid than that reported for bortezomib in preclinical and clinical models (Papandreou et al., 2004; Hemeryck et al., 2007). Furthermore, plasma clearance at all of the dose levels was much higher than the reported hepatic blood flow for rats [55 ml/(min · kg)] (Davies and Morris, 1993), suggesting that, unlike bortezomib, carfilzomib clearance is mediated largely by extrahepatic mechanisms. We determined that the rapid clearance of carfilzomib was not due to irreversible binding of the proteasome because pretreatment with another irreversible proteasome inhibitor (ONX 0912) did not affect the clearance of carfilzomib. Furthermore, the clearance of M16, which contains the peptide backbone of carfilzomib but lacks the ability to irreversibly bind to the proteasome, was also higher than the hepatic blood flow in rats. In addition, the high plasma clearance was not due to partitioning of carfilzomib to blood cells because the in vitro whole blood/plasma partitioning ranged from 0.64 to 0.82 across a concentration range from 0.2 to 10 μM. Because carfilzomib is an irreversible inhibitor, target inhibition is a function of proteasome turnover and persists even after the drug is cleared systemically. Indeed, rapid clearance may prevent its ability to inhibit nonproteasomal targets and thus reduce potential idiosyncratic toxicities.
The major metabolites in rat plasma, urine, and bile samples were from peptide and epoxide hydrolysis; oxidative metabolites were observed only at low levels (<0.1% of total dose in urine and bile samples). In vitro, carfilzomib was metabolized rapidly in a variety of tissues and blood also via hydrolysis. Together with the rapid (<10 s) formation of these hydrolysis metabolites in vivo, these data suggest that metabolism of carfilzomib occurs immediately upon administration and likely occurs systemically throughout the body. These metabolites lack the epoxyketone pharmacophore and therefore have no activity as proteasome inhibitors. Their further processing to individual amino acids also suggests that they have little toxicological impact in vivo. A minor metabolite (Phe-Leu-Epoxyketone, M11), resulting from peptidase cleavage, was detected in rat plasma, urine, and bile samples. The low abundance of M11 may be attributed to rapid secondary metabolism to phenylalanine (M7), tyrosine (M5), leucine diol (M8), and a low level of oxidated leucine epoxyketone (M2). We showed recently that dipeptide epoxyketone, morpholino-Phe-Leu-epoxyketone, is not an active proteasome inhibitor and has no activity against serine proteases in an activity-based profiling assay (Zhou et al., 2009; Arastu-Kapur et al., 2011). Therefore, the low level of M11 is unlikely to contribute to PD or induce off-target toxicity. Because peptide epoxyketones are highly selective for proteasome active sites, these findings suggest that the PD effect of carfilzomib is being driven by the parent compound (Mirabella et al., 2011). We also have noted rapid clearance and extrahepatic metabolism in the form of peptide and epoxide hydrolysis in humans, and similar to rats, the peptidase cleavage product M14 is the most abundant circulation and excretion metabolite in humans (Z. Wang, J. Yang, and C. Kirk, manuscript in preparation). Therefore, exposure to carfilzomib is unlikely to be altered in patients with hepatic impairment or taking concomitant medications that alter cytochrome P450 activity.
Carfilzomib also distributes rapidly to tissues after intravenous administration as demonstrated by potent proteasome inhibition in a variety of tissues. Potent proteasome inhibition in different tissues (except brain) after intravenous bolus administration has been described previously (Demo et al., 2007). In this study, we determined that a 30-min infusion of carfilzomib at 8 mg/kg resulted in equivalent levels of proteasome inhibition in blood and tissues despite a 28-fold lower Cmax (1.6 versus 42.9 μM) than that with intravenous bolus administration at the same dose. Therefore, we conclude that the in vivo potency of this agent is a consequence of total dose administered but not Cmax. As has been described with other classes of covalent inhibitors, these data indicate that the time course of target inhibition by carfilzomib is not determined directly by its plasma exposure (Singh et al., 2011). Furthermore, we found that bolus administration of carfilzomib at 8 mg/kg resulted in a mortality rate of approximately 44% (14 of 32 animals), whereas a 30-min infusion of the same dose was well tolerated generally and did not result in mortality (0 of 24 animals; J. Jiang and C. Kirk, unpublished observations). The higher incidence of mortality with bolus administration is likely associated with the high Cmax. Thus, intravenous infusion might be an alternative route of administration to bolus injection to improve the therapeutic window of carfilzomib. We have demonstrated currently that carfilzomib can be administered safely up to 56 mg/m2 in MM patients with intravenous infusion, which is more than 2-fold the maximum tolerated dose for an intravenous bolus administration (Papadopoulos et al., 2010).
Despite the wide tissue distribution of carfilzomib, the apparent Vss values for carfilzomib were relatively small (Table 1), 0.3, 0.3, and 0.6 l/kg at 2, 4, and 8 mg/kg, respectively, when administered via an intravenous bolus. With intravenous infusion administration at 8 mg/kg, the measured Vss was 2 l/kg. It appears that the Vss value was lower at low doses (2 and 4 mg/kg). At 8 mg/kg, Vss obtained from intravenous infusion was higher than that from bolus administration. The specific reason for this observation was not clear. However, the apparent Vss values may not truly reflect the tissue distribution of an irreversible inhibitor. Moreover, as described in the preceding sections, carfilzomib is highly metabolized in a variety of tissues by peptidase and epoxide hydrolase. This portion of carfilzomib was counted as elimination rather than as distribution.
Previously and in this study, we have noted that the liver was relatively insensitive to proteasome inhibition compared with adrenal, blood, heart, and lung (Demo et al., 2007). However, radioactivity in the liver at 0.5 h postdose is quite high (Demo et al., 2007). These disparate findings may be attributed to the competition between drug-metabolizing enzymes and binding to the proteasome within the liver. Indeed, the rate of carfilzomib disappearance in liver was more rapid relative to those in blood and other organs (adrenal, heart, and lung), as demonstrated in the in vitro study (Fig. 5; Supplemental Fig. S2). Therefore, we believe that a large portion of the radioactivity observed in rat livers represents radioactive metabolites.
Less than 1% of carfilzomib was excreted intact in urine and bile within 24 h postdose in rats. Carfilzomib is eliminated mainly in the form of metabolites, particularly peptidase cleavage products M14 and M15. In a previous report, high levels of radioactivity in bile and urine at 0.5 h postdose in the quantitative whole-body autoradiography study were noted (Demo et al., 2007), which likely represents the rapid elimination of radiolabeled metabolites. However, the radioactivity did not represent M14 and M15 because these two major metabolites do not contain the 3H-labeled phenylalanine group. The total recoveries of carfilzomib and monitored metabolites (M14, M15, and M16) in rat bile and urine and bile were 26 and 31% of the dose, respectively, for a total of 57% within 24 h postdose in the present study using nonradiolabeled carfilzomib (Table 4). The incomplete recovery of the administrated carfilzomib may represent the incorporation of the amino acids from the peptide backbone of the drug into biosynthetic pathways; retention of carfilzomib bound to proteasomes in enucleated cells (erythrocytes and platelets), which cannot turnover their proteasomes; and the observed dipeptides, minor metabolites, or undiscovered metabolites that were not quantified in the present study. Further studies are needed to better understand the complete fate of administered carfilzomib.
In summary, carfilzomib was cleared rapidly from systemic circulation after intravenous administration to rats largely due to extrahepatic metabolism via peptidase cleavage and epoxide hydrolysis. Despite rapid clearance, carfilzomib showed potent proteasome inhibition in a variety of tissues, demonstrating widespread tissue distribution. Elimination occurred mainly via biliary and renal excretion in the form of metabolites rather than intact drug. Together with the irreversible and rapid onset of target binding, the high clearance may provide an advantage in that unnecessary exposure to the drug is minimized and potential drug-related side effects may be reduced. Furthermore, the major metabolic pathways of carfilzomib result in toxicologically insignificant metabolites (modified peptides and amino acids). Carfilzomib is currently in late-stage clinical development, with encouraging safety and efficacy profiles demonstrated with intravenous bolus and infusion administrations. The studies presented here help to shed light on the disposition of this novel class of irreversible proteasome inhibitors.
Authorship Contributions
Participated in research design: Yang, Wang, Fang, Jiang, Bennett, Molineaux, and Kirk.
Conducted experiments: Yang, Fang, Wang, Jiang, Zhao, and Kirk.
Performed data analysis: Yang, Wang, Fang, Jiang, Zhao, Wong, and Kirk.
Wrote or contributed to the writing of the manuscript: Yang, Wang, Wong, and Kirk.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
↵ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
- chymotrypsin-like
- multiple myeloma
- [(1R)-1-[[(2S,3R)-3-hydroxy-2-[[(6-phenylpyridin-2-yl)carbonyl]amino]-1-oxobutyl]amino]-3-methylbutyl]boronic acid
- (4R,5S)-4-(2-chloroethyl)-1-((1S)-cyclohex-2-enyl(hydroxy)methyl)-5-methyl-6-oxa-2-azabicyclo[3.2.0]heptane-3,7-dione
- PR-171 (carfilzomib)
- (2S)-N-[(S)-1-[(S)-4-methyl-1-[(R)-2-methyloxiran-2-yl]-1-oxopentan-2-ylcarbamoyl]-2-phenylethyl]-2-[(S)-2-(2-morpholinoacetamido)-4-phenylbutanamido]-4-methylpentanamide
- N-[(S)-1-[(S)-1-[(S)-4-methyl-1-[(R)-2-methyloxiran-2-yl]-1-oxopentan-2-ylcarbamoyl]-2-phenylethylcarbamoyl]-3-methylbutyl]-5-(morpholinomethyl)isoxazole-3-carboxamide
- (2S)-3-methoxy-2-[(2S)-3-methoxy-2-[(2-methyl-1,3-thiazol-5-yl)formamido] propanamido]-N-[(2S)-1-[(2R)-2-methyloxiran-2-yl]-1-oxo-3-phenylpropan-2-yl]propanamide
- internal standard
- pharmacokinetics
- area under plasma concentration-time curve
- plasma clearance
- t1/2
- terminal half-life
- Vss
- volume of distribution at steady state
- liquid chromatography tandem mass spectrometry
- multiple ion reaction monitoring
- AUClast
- area under plasma concentration-time curve calculated to the last measurable concentration
- AUCinf
- area under plasma concentration-time curve extrapolated to infinity
- Css
- steady-state concentration
- rat plasma
- rat urine
- rat bile
- pharmacodynamics.
- Received March 4, 2011.
- Accepted July 12, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics


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a Trajectory for Flourishing
September 19, 2015 § 1 Comment
"To love, and to lose what we love, are equally things appointed for our nature. If we cannot bear the second well, that evil is ours."
C.S. Lewis, Till We Have Faces
I thought it good to reenter into the blogosphere by way of confession, so pardon the meandering – there really is a point.
First the ridiculous. You need to know that I have this propensity to find what I like and then hold on to it – like forever, whether a pair of shoes, a style of pants, or a shirt (our Congregation will tell you that I only wear one shirt on Sunday mornings!). My guess is that it is born of tons of insecurity, control and pride, but it is the way it is. For instance, I have worn the same style of top siders for about ten years, and normally I have a backup pair – in the box for when the initial pair 'dies.' When that backup is gone and there is no other and the shoes are unattainable (because in a sane universe things go out of style), it unnerves me and sends me on a twisted journey to find its replacement (which I would prefer not to have to do – thus the backup!).
Hey, I warned you. Ridiculous, right?
When we moved to the Baltimore region nearly ten years ago I thought my life was over. It wasn't because the people we left hated us or the people we came to were other than welcoming. It was because I held on to the idea of living in my hometown for life (yes, idolatry). But somewhere in that delusion, God stirred our hearts to move. How could He do this and still love me? It was the most disruptive, confusing and dislodging time of my life, and our lives. But the Father's leading was unmistakable. He wanted us here. And we have since discovered that it was out of love that He did.
Baltimore has become home and we are blessed.
If you read Joseph's story, you will find that 'the Lord was with Him,' and prospered him in Egypt, even when as slave, and later when imprisoned on false charges (chapter 39). He continued to thrive and care for people.
I have come to realize that most of us live out of unholy trajectories for how our lives should unfold. If we become slaves to these trajectories, then well, we are just that – slaves. In this pattern regret becomes torturous, forgiveness seems impossible, and the present, intolerably joyless.
But we were redeemed to flourish, and if we buy into the fact that we have a Father who loves us, who sent His only Son on the most dangerous, yet redemptive journey of all – for us – then we have discovered something. We have discovered that our true trajectory is heaven and everything between now and there – is good.
Friends, the gospel is an adventure to be embraced…
March 7, 2015 § Leave a comment
"There are no ordinary people. You have never talked to a mere mortal."
C.S. Lewis, The Weight of Glory
If you have ever lived in the north, then you know that snow is part of the seasonal rhythm of life. Having grown up in the tropics, I still find it to be magical. There is something about watching it fall, and then seeing an entire area that has been turned white by hours of God's carefully placed flakes and flurries upon a landscape. Don't get me wrong. I could do without the cold! Seasonal life has a way of bringing both anticipation of the next season, and weariness with the present.
But then there is the inevitable cleanup. Municipalities constantly attempt to get ahead of the snow, salting roads, positioning plows and alerting citizens.
Unless one can 'dig out,' they are stuck. The other night, after a lovely eight-inch snow, Katherine and I got out and shoveled our entryways, the back of our garage, and then the car we had parked outside. You have to dig out in order to get out.
At our church, a large facility on 62-acres, it is crucial for us to be plowed, cleared and salted, in order for people to enter. A team of unsung heroes, both from our Staff and contracted, work tirelessly, with heavy equipment, doing what is needed for our church to be ready for whatever activity or gathering is scheduled.
This hit me freshly yesterday as I watched our crew work, while at the same time fifty or sixty Middleschoolers sledded and snowboarded down a hill on the property. While the Young People played, the workers plowed. Or to put it another way, the workers plowed so that our Young People could play.
I so love that.
Sometimes our own sense of anonymity can be torturous if we have no greater context. We want to matter, right?
I know what happens – Our tendency is to narrow our influence to the small quarters we work or live in. It is hard to conceive of an influence beyond what we know. But you see, the gospel demonstrates the opposite.
Hey, I don't know what you do in life, but I can tell you this: Whether you are high profile, or all but invisible, you matter, and what you do matters to God. Someone is affected by who you are and what you do. Can your ego enable you to accept this? If not, flee to the Throne! Just read through the scriptures and rediscover the attention God places on those who labored behind the scenes. An unnamed slave girl in Syria comes to mind (2 Kings 5).
What you do may never grab the headlines, but have you read the headlines lately? Besides, in the end, isn't it the sweeter things that matter most when it comes to a meaningful life?
And isn't it stunning that the most meaningful Person in history was born in anonymity, that in Him, what appeared to His contemporaries to be an act of meaningless sacrifice, turned out to be the only hope of the world.
In fact, this is our good news…
November 15, 2014 § 2 Comments
"'I wish I had never been born,' she said. 'What are we born for?' 'For infinite happiness,' said the Spirit. 'You can step out into it at any moment…'"
C.S. Lewis, The Great Divorce
The other night, Katherine and I sat in our courtyard, beside a small fire, reflecting on our lives together, our family and children, our ministry and calling – you name it. There is something about a fire that seems to suspend all urgency, and draw us into such conversation. And yes, we listened to Christmas music as we did, while sipping on hot chocolate.
As we conversed, we agreed that much of what we thought to be important many years ago in our lives was not so important after all. It wasn't so much about regret, but of perspective. The thing is that we would not trade anything for where we are now. Fortunately God's hand has not been thwarted by our stumbles.
'If I had to do it over again…' can be a dangerous statement. So can, 'What if…' Micromanaging our pasts and obsessing over the future only steal from the present.
Unfortunately the Church hasn't always been helpful in this area. Through the years Christians have fixated on the future to the point of madness, parsing events and people, while attempting to fit them into a sensationalized 'end times' blockbuster movie-type scenario. Authors have raked in millions, feeding off the fears and frenzy of 'end times' enthusiasts.
But the gospel teaches that what God will one day do, He is already doing – Right Now. He didn't identify Himself to Moses with the words, 'I was' – It was, 'I am,' and you can hear this in Jesus' prayer, 'Thy kingdom come, thy will be done, on earth as it is in heaven…'
The gospel works because of this. It enables us to find fresh hope in the moment – Today. Only our regrets and fear of the future will rob us of enjoying God's grand design for our lives in the present.
Within weeks we will read and recite the beautiful announcement, 'Today, in the city of David, a Savior has been born…' This is the refrain we find throughout the scriptures – it is all so present tense!
In fact, you would be hard pressed to find a single instance in Jesus' ministry, in which He dwelled on the pasts of the people He encountered. Other than opening one woman's eyes to His ability to see through her defenses, He never drudged up incidents and failures of the people He healed and consoled. He was all about the moment.
This was no accident. It was His announcement that with the Father there is grace to continue on with the journey, in spite of our pasts that are speckled with failures, poor decisions and regrets, as well as with the future unknown.
Settle this in your minds and hearts, friends: Regret over the past and Anxiety over the future are not on the Father's radar.
You are.
Right Now.
'You can step out into it at any moment…'
That's good news…
Brush with an Immortal
March 22, 2014 § 1 Comment
"There are no ordinary people. You have never talked to a mere mortal. Nations, cultures, arts, civilization—these are mortal, and their life is to ours as the life of a gnat. But it is immortals whom we joke with, work with, marry, snub, and exploit—immortal horrors or everlasting splendors… Next to the Blessed Sacrament itself, your neighbor is the holiest object presented to your senses." C.S. Lewis, The Weight of Glory
This past week I was thrilled to meet up with an old friend and one of the most inspiring people I have ever known. When we first met, Jenni Gold was 10 years old and I was an 18 year-old volunteer church youth worker. Jenni has Muscular Dystrophy and was in a full body cast at the time, having had a steel rod surgically placed in her spine. Along with her parents, her two amazing sisters provided an environment of healing. The only thing they wouldn't offer was sympathy, and this produced a will that far surpassed the strength of the rod in her back. We became fast friends, all of us. She fully entered into the life of our Youth Group. The word 'limitation' was not in her vocabulary. It still isn't.
After graduating with a double major from the University of Central Florida in Orlando, Jenni, along with her husband Jeff, moved to California, determined to produce movies. Today she is the co-founder of Gold Pictures in Universal City, CA. On Wednesday we reunited in DC to see a viewing of her Documentary, CinemAbility, a stunningly beautiful film about the history of the entertainment industry in relation to people with disabilities. She was also in DC to accept the 2014 American Association of Persons with Disabilities Image Award, presented by Danny Woodburn ('Mickey' on Seinfeld!).
In accepting the award, Jenni sited Paul's words in Philippians 4:13, thanking Christ who gives her strength, and so revealed the source of her amazing will and character. Somewhere along the way, in all she has endured and overcome, as a little girl, and since, Jenni met Jesus. And He has provided everything she needs to be nothing short of amazing.
She mounts up with wings like eagles. She runs and doesn't grow weary. She walks and doesn't faint.
And she happens to have MD.
It is Lent. The world is broken. Suffering is part of the daily narrative.
But Jesus has come. And in entering into our brokenness by subjecting Himself to temptation, sorrow and pain, even death, by His resurrection He has assured that until He returns, and regardless of our circumstances, we may dance to the song of His redemption.
Friends, there is no greater news…
Taken on Palm Sunday in 1977 when Jenni joined the church
a Christ-Sized Embrace
February 1, 2014 § 1 Comment
"Come on. There has never been a sadness that can't be cured by breakfast food."
Ron Swanson, Parks and Recreation
This morning, upon entering the Mall at Columbia, the site of the unspeakable act of violence that resulted in the taking of three lives last Saturday, we were handed ribbons in order to join in unified sorrow, and to participate in a moment of silence at the time the shootings occurred.
Pain and sorrow exist in a broken world – and they always will until Jesus makes all things new, and everything is restored to what it was always intended to be. This is something we don't naturally understand because pain is not written into our DNA. Deep within, we know that we were created to be whole, and pain threatens wholeness. It is a polluting element in a world that was intended to be filled with justice, flourishing and peace (this is captured in the Hebrew word 'shalom'). We cry when we are sad, and ache when there is distress because pain, suffering and evil will never 'fit' our human impulses.
Unfortunately the Church's response often comes in the form of judgment and isolation, although Jesus did just the opposite. Rather than condemn, He came to heal, and instead of insulating Himself in self-protective seclusion from pain and suffering – even sin – He embraced it to Himself.
So until He comes and makes everything new, sorrow, heartache and tragedy will always be part of the human experience, not because of sins, but because of the first sin, when the beauty, loveliness and glory of the Garden were violated by Adam's rebellion.
But because Jesus (the scriptures call Him 'the last Adam') has come and has submitted Himself to the very rebellion and violence that brokered the fall, the curse has been broken forever. One day every remnant and memory of the fall will be eradicated from the human experience.
In the mean time the Church is called to something higher than self-righteous condemnation and self-preserving isolation. It is called to imitate Jesus, who entered into this world's pain and sorrow, and to embrace the brokenness He embraced, when He came for us.
Can you think of any higher calling?
This is our good news.
Love @ Thanksgiving
November 23, 2013 § 3 Comments
"Love anything and your heart will be wrung and possibly broken. If you want to make sure of keeping it intact you must give it to no one, not even an animal. Wrap it carefully round with hobbies and little luxuries; avoid all entanglements. Lock it up safe in the casket or coffin of your selfishness. But in that casket, safe, dark, motionless, airless, it will change. It will not be broken; it will become unbreakable, impenetrable, irredeemable. To love is to be vulnerable."
C.S. Lewis, The Four Loves
Life is difficult. This is the first line of M. Scott Peck's book, The Road Less Traveled. Life is difficult, and love sometimes seems an unobtainable dream in the disappointments, pitfalls and disparities of life as it wears on.
Broken hearts. Broken promises. Unrealized dreams. Unforeseen calamities. Unbearable trials. Painful seasons. Haunting Regrets. Betrayals. Sedentary Relationships.
All have their ways of extinguishing the dream of love.
The picture posted above is from our basement wall. Katherine and one of our daughters had left the house to me while I painted, and to signal to them that the work had begun, I painted the now-famous heart – the less-than-three sign. Once that went on the wall, the commitment was made. Someone had to slosh the walls with blue.
It is curious to me that the closer he moved to the Cross the more Jesus' teachings were predominated by love, particularly with His disciples who that very evening would betray and abandon Him to His captors
Our instinct is to do the opposite. Whenever disappointment threatens, we tend to insulate ourselves against fresh experiences with pain. We say and do harmful things to protect our fragile hearts. We force ourselves to be cold and uncaring.
Love is the last thing we want to risk our vulnerabilities on because we are never more exposed than when we open our hearts to the possibility of their being broken.
I get that. Life is difficult and Love is terrifying. Who has room for Love and all the mess it brings?
But it is also what we most yearn for. To love and be loved. And someone has to make the first move. Someone has to start painting.
So God did.
Let it be your prompt to love those who may be hiding from you behind their own self-protections, though every bit as terrified. Start painting. Forgiveness, tenderness and vulnerability are scandalously risky. We know. Jesus went there for us. Trust me, Love is the first stroke.
He knew the risks when He opened His mouth to call the disciples His friends. Amazingly, to Him they were worth it.
So are you.
What good news.
Happy Thanksgiving.

subset: commoncrawl


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Code: DB0601
This fantastic large Victorian solid oak door is fully morticed and tenoned, and suitable for internal or external use. The door is constructed with six long panels with a decorative gothic chamfer detail to one face, and decorative mouldings to the other. A rare and beautiful door. The door is in good condition, but is currently painted. We are happy to strip it at an additional cost.
Request a quote for further work (see RESTORATION SERVICES)


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Yun-Hui's (My Mother's) Red-Cooked Pork
Source: Cecilia Chiang, author, The Seventh Daughter: My Culinary Journey from Beijing to San Francisco (Ten Speed Press, 2007)
Recipe Type: Entrees | Seasons: Autumn
This recipe was demonstrated at CUESA's Market to Table programs on September 20, 2008.
I've had dreams about this dish that have been so vivid that I thought I could actually the smell the aroma of the meat as it was being carried from the kitchen into the dining room of our family home in Beijing. My mother was from Shanghai Province, famous for its red-cooked dishes—meat or poultry braised in rich, dark, aromatic mixtures of soy, wine, and spices—and her red-cooked pork was famous throughout our circle of family and friends. For New Year's Eve she would red-cook a whole pork shoulder To this day, I think of my mother every time I cook this dish, and sometimes I think that's why I cook it so often.
Cook's Notes: Pork belly is rich—it's really just a big piece of unsmoked bacon—so small portions are in order. (Five pounds sounds like a lot, but it shrinks at least by half.) You could also substitute pork shoulder, sometimes called pork butt. If there happens to be any leftover it's wonderful diced and tossed in a stir-fry of green beans or fried rice. As with any kind of braise, it's even better made a day or two in advance.
Serves 6 to 8 as part of a Chinese meal or 4 to 6 as a Western-style entrée
5 pounds skin-on (Chinese-style) or skin-off (Western-style)
pork belly, as lean as possible 1 (24.5 ounce)
bottle Shaoxing wine
1 (2-inch) piece ginger, cut into 6 rounds
1 ½ cups regular soy sauce
2 large pieces rock sugar
2 tablespoons dark soy sauce
- Cut the pork into 1 ½-inch cubes. Put them in a casserole and add water to cover the meat by 1-inch. Bring to a boil, decrease the heat to maintain a simmer and cook for 5 minutes or until the foaming diminishes. Transfer the pork to a sieve or colander and rinse well with cold water. Discard the cooking liquid.
- Rinse out the casserole and add the pork, Shaoxing, ginger, and cold water to cover. Bring the liquid to a boil over high heat, decrease the heat to maintain a low simmer and cook until the pork starts to seem tender when pierced with a fork, about 45 minutes. Add 1 cup of the regular soy sauce and if it doesn't cover the meat add the remaining ½ cup. Continue to cook until the meat is completely fork tender, about 30 minutes longer.
- Gently stir in the rock sugar and dark soy until the sugar dissolves. Simmer 5 minutes more or until the sauce is slightly thickened and shiny.
- Serve in a large decorative bowl or family-style from the casserole.
Picture by Kattebelletje


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Annual Breast Milk Letting Activity at the Taguig City University Auditorium
The local government of Taguig, in partnership with the Philippine General Hospital, conduct the annual breast milk letting activity at the Taguig City University Auditorium last August 08.
A number of 250 mothers responded to donate their own milk for the human milk bank that will facilitate the availability of human milk especially during emergency situations.
The city of Taguig will also launch its own human milk bank at the Taguig-Pateros District Hospital that will accommodate mothers that want to help and donate their breast milk to benefit mothers and their infants.
During the event, participants are given lectures and conseling on proper child care and the importance of breastfeeding for babies.
In attendance were Vice Mayor "Ricardo" Ading Cruz Jr., City Councilors Jimmy Labampa, Rodil Marcelino, and Ome Tanyag, together with Taguig City Health Office OIC Dr. Isaias Ramo, City Nutrition Office Head Ms. Julie Bernabe, Luzon Health City Health Program Officer Dr. Alyn Nagi, Dr. Elaine Kho of the PGH-Human Milk Bank,and TPDH-OIC Chief of Hospital Dra. Anna Richie Quilatan.


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LAHORE: The Indian government on Saturday denied visas to six Pakistani chefs to participate in the World Heritage Cuisine and Food Festival scheduled to be held in Amritsar next month.
The decision came amid rising tensions between the two countries after India canceled an earlier announced meeting between Foreign Minister Shah Mehmood Qureshi and his Indian counterpart Sushma Swaraj.
The festival will take place from October 12-14. Chefs from over 40 different countries will take part in the event.
Cancelling the invitation to the Pakistani chefs, the Indian government cited political tension between the neighbours as the reason to not issue visas.
Speaking to Geo News, head of the six-member group S.M Shafiq said the Pakistani chefs have decided to lodge a written complaint against India to the secretary general of WorldChefs — a global network of chefs associations in Paris.
The event is an initiative of the World Cultural Culinary Heritage Committee of the World Association of Chefs Societies (WorldChefs).


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Arnab Saha
Arnab SahaAssistant Professor, Mathematics
- BSc: Chennai Mathematical Institute, 2004
- MSc: Chennai Mathematical Institute, 2006
- PhD: University of New Mexico, 2012
Website : https://sites.google.com/view/arnab-saha
Selected Publications
Arithmetic Jet Spaces (with A. Bertapelle and E. Previato), arXiv:2003:12269 (submitted)
Differential modular forms over totally real fields of non zero integral weights (with D. Banerjee), arXiv:1806.00191 (submitted)
Isocrystals associated to arithmetic jet spaces of abelian schemes (with J. Borger), Advances in Mathematics, 351: 388-428, 2019.
Differential characters of Drinfeld modules and de Rham cohomology (with J. Borger), Algebra and Number Theory, 13(4): 797–837, 2019
Invariant rational forms for correspondences of curves, Journal of Number Theory 143:170-184, 2014.
The first p-jet space of an elliptic curve: global functions and lifts of Frobenius (with A. Buium), Mathematical Research Letters, 21(4):677- 689, 2014.
Hecke operators on differential modular forms mod p (with A. Buium), Journal of Number Theory 132(5):966-997, 2012.
The ring of differential Fourier expansions (with A. Buium), Journal of Number Theory 132(5):896-937, 2012.
Differential Overconvergence (with A. Buium), Algebraic methods in dynamical systems, volume 94 of Banach Center Publications, pages 99-129. Polish Acad. Sci. Inst. Math., Warsaw, 2011.
Work Experience
- Assistant Professor, Indian Institute of Technology Gandhinagar (Jul 2019 to present)
- Visiting Fellow, Max Planck Institute, Bonn, Germany (2018 - 2019)
- Visiting Fellow, Indian Statistical Institute, Bangalore (2018 - 2018)
- Postdoctoral Fellow, Australian National University, Australia (2012 - 2018)
- Teaching Assistant, University of New Mexico, USA (2006 - 2012)


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How Digital Print Solutions Support The Success Of Business
Digital Print Makes Direct Mail Easy
The most popular methods include inkjet or laser printers that deposit pigment or toner onto a wide variety of substrates including paper, photo paper, canvas, glass, metal, marble, and other substances. RIP & Print Management Software assists users in taking full advantage of the company's printers. These software platforms make the printing process much smoother by providing color management to suit the features of the printer, ink and media being used. They also come with page layout functions for enlarging, reducing and repositioning data, as well as advanced capabilities such as the Roland Color System Library for efficient color matching. From the beginning, Roland DG recognized the significance that RIP software plays in the printing industry, and has aggressively devoted resources to improve functionality and user friendliness.
Direct Mail Postcard Marketing: Simple, Effective, And Easier Than You Think!
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John Oliver says his parody Pence rabbit book has sold out
John Oliver's satire book about Vice President Pence's family rabbit has sold out, the late night host said Tuesday.
Oliver said his "Last Week Tonight with John Oliver Presents a Day in the Life of Marlon Bundo" proved to be more popular than he anticipated. The book has sold out in the days since it was announced on his show Sunday night, Oliver told Ellen DeGeneres.
"We were not anticipating people really buying it," Oliver said. "But they're doing a reprint, so you can still buy it. It'll take a few weeks."
The book parodies the Pence family's own book about their pet rabbit, Marlon Bundo. That book is titled "Marlon Bundo's A Day in the Life of the Vice President."
However, the rabbit in Oliver's book is gay. Pence was criticized for his stance on LGBT issues when he was governor of Indiana.
Oliver's book is No. 1 on the Amazon best-sellers list as of Tuesday night. The Pence family's book is at No. 4 on the chart.
Pence's daughter, Charlotte, said Tuesday that she is "all for" Oliver's book.
"I think imitation is the most sincere form of flattery in a way," Pence said during an interview with Fox Business Network's Maria Bartiromo Tuesday.
"But in all seriousness, his book is contributing to charities that I think we can all get behind. We have two books that are giving to charities that are both about bunnies, so I'm all for it, really," she continued.
Proceeds from the Pences' book are going to Tracy's Kids, a group that offers art therapy to young cancer patients.
Funds from Oliver's book will be donated to AIDS United, a group dedicated to ending the AIDS epidemic in the U.S., and the Trevor Project, a suicide prevention organization that provides support to young LGBT community members.


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Year / Date of Issue: 01.2021
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Shorter than short skis
Ski Skates are combination of skis and skates. At 44 cm, they're shorter than short skis and can be used with either snowboard boots or ski boots depending on the model.
Before Skiskates, there was SnowFeet. Made by the same company (called Snowfeet), Ski Skates are about the bringing the "joy of skating to the snow" as written on their Indiegogo campaign. For SnowFeet, on its Kickstarter Campaign it "brings the thrill of skating to the slopes".
Basically SnowFeet and SkiSkates are basically the same thing. SnowFeet was the original and SkiSkates the new and improved version. Both for skating on snow.
SkiSkates are easier to learn than skiing according to many users. Many also claim that it's easier than ice skating. It's also easier to make sharp turns with SkiSkates and easier to control compared to skiing.
One important thing about SkiSkates is that they only work on groomed snow. You'll have difficulty staying afloat on snow with SkiSkates or SnowFeet because there's a lot of surface area to support your weight.
According to SnowFeet, most ski resorts allow "short skis" on their slopes but not all of them. SkiSkates use the same metal ski edge as other skis, which is a common requirement of ski resorts.
The difference between SkiSkates and SnowFeet
- Skiskates are faster than SnowFeet
- There's a version of the SnowFeet that can fit just about any winter boots
- SkiSkates are designed for either snowboard boots or ski boots - there's no guarantee that a normal winter boot will fit
- Heel braking from SnowFeet is now absent on SkiSkates
- SkiSkates have a different bottom surface to SnowFeet
SkiSkates are so small they likely fit in your day pack. They make a great backup to your snowboard or skis on a ski resort. However, not all ski resorts allow the use of "short skis". Be sure to ask your ski resort.


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We asked members of the BuzzFeed Community to tell us their best Disneyland hacks and the responses were pretty damn clever:
1. "Use the inside line for Dole Whips, which is much shorter than the outside line. And then head to the snack shop across from the Jungle Cruise to get some free Tajin to sprinkle on it!"
2. "Ride Big Thunder Mountain Railroad during the fireworks for a perfect view of the show. It's an incredible experience."
3. "Instead of waiting in the long line at the corn dog cart on Main Street, head to Stage Door Cafe in Frontierland. The line is much shorter and you can get fries as a side order for the same price as apples or chips at the cart."
4. "When you enter the park, start your journey from left to right. Most people start on the right side of the park, so you'll avoid crowds that way."
5. "No need to buy those expensive water bottles – you can get cups of ice water for free at any park establishment where there's a soda fountain. All you have to do is ask."
6. "Buy the popcorn in the tall plastic container, and when you're finished, buy a turkey leg. It fits perfectly in the popcorn container, so you aren't stuck carrying a huge, greasy meat leg like a savage!"
7. "If you want a more thrilling rollercoaster experience, always sit in the last car."
8. "Take pictures in front of the blue wall in California Adventure's Hollywood Backlot area – it's a really cute backdrop."
9. "Buy a shirt at the Disney Store during a sale, then ask park characters to sign it with a marker for a souvenir that is less expensive and even more magical."
10. "If you're traveling with kids, have bulky items (diapers, cases of water, etc...) shipped to your hotel before you arrive. Disney will have them waiting for you at check-in, and you won't have to worry about traveling with them."
11. "Use the single-riders line. If you're in a group and you're comfortable with not sitting together, you will have a much shorter wait time."
12. "Always remember where you parked by making it into a fun little song for your kids, and when you get back to the parking lot, ask them who remembers."
13. "Keep your phone in a Ziploc bag for protection during wet rides."
14. "Buy Disney gift cards in bulk from Sam's Club or Costco and use them to buy your tickets. You get bonus gift card money when you buy the multi-pack!"
15. "Get yourself on a meal schedule that's outside of the usual times, so when everyone is sitting down for dinner, you're walking straight onto Pirates of the Caribbean."
16. "Head to the Plaza Inn for kosher food options."
17. "If you don't want to get wet on a boat ride, sit towards the middle and pick your feet up during drops. Then you won't have to walk in squishy shoes!"
18. "Get in a line for a ride right before closing. As long as you are in line by closing time, you can still ride the ride, even if it has a long wait time."
19. "If you want to beat the massive crowd exiting Disneyland after the fireworks show, head to the roof of the parking structure to view it instead. You'll be the first ones out after the fireworks end, and your view will be pretty damn cool, too."
Some responses have been edited for length and clarity.


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Sambusa Recipe
- 1 (14 ounce) package spring roll wrappers
- 2 tablespoons olive oil
- 2 pounds ground beef
- 1 leek, chopped
- 2 teaspoons ground cumin
- 2 teaspoons ground cardamom
- 1 teaspoon salt
- 1 teaspoon pepper
- 1 small onion, finely chopped
- 1 clove garlic, minced
- 1 tablespoon all-purpose flour
- 1 tablespoon water, or as needed
- 1 quart oil for frying
step 1: Heat olive oil in a large skillet over medium heat. Add onions, leek and garlic, and cook, stirring until the onions are transparent. Add ground beef, and cook until about halfway done. Season with cumin, cardamom, salt and pepper. Mix well, and continue cooking until beef has browned.
step 2: In a small dish or cup, mix together the flour and water to make a thin paste. Using one wrapper at a time, fold into the shape of a cone. Fill the cone with the meat mixture, close the top, and seal with the paste. Repeat until wraps or filling are used up.
step 3: Heat the oil to 365 degrees F ( 170 degrees C) in a deep-fryer or deep heavy pot. There should be enough oil to submerge the wraps. Fry the Sambusa a few at a time until golden brown. Remove carefully to drain on paper towels.