Ketotifen is a Prodrug. Norketotifen is the active metabolite.

Evaluation of the in vitro human liver microsome and hepatocyte metabolism of ketotifen demonstrated that norketotifen (NK) is the major demethylated hepatic metabolite of ketotifen. It is here reported that NK is completely devoid of the severe and dose-limiting sedative effects of ketotifen. Thus, while ketotifen is clinically dose-limited to 1 mg, bid, there are no dose-limiting sedative effects elicited by NK, even after the highest single-dose (16 mg) or after repeat-doses (8 mg × 7 days) in humans or after the highest doses given to dogs in repeat-dose toxicological studies (40 mg/kg × 14 days). In addition, NK-but not ketotifen-was found to express potent and dose-dependent inhibition of the release of the pro-inflammatory cytokine TNFα from activated human buffy coat preparations. Thus, when used as an anti-inflammatory drug, ketotifen is the sedating prodrug which is converted to NK a nonsedating metabolite with anti-inflammatory activity.

Characterization and Validation of a Canine Pruritic Model.

Preclinical Research The mechanisms mediating canine pruritus are poorly understood with few models due to limited methods for inducing pruritus in dogs. Chloroquine (CQ) is a widely used antimalarial drug that causes pruritus in humans and mice. We have developed a canine model of pruritus where CQ reliably induced pruritus in all dogs tested following intravenous administration. This model is presently being used to test antipruritic activity of drug candidate molecules. This publication has been validated in a blinded cross-over study in eight beagle dogs using the reference standards, oclacitinib and prednisolone, and has been used to test a new compound, norketotifen. All compounds reduced CQ-induced pruritus in the dog. The sensitivity of the model was demonstrated using norketotifen, which at three dose levels, dose-dependently, inhibited scratching events compared with placebo.

Relationship between the in vitro efficacy, pharmacokinetics and in vivo efficacy of curcumin.

Considerable interest continues to be focused on the development of curcumin either as an effective stand-alone therapeutic or as an adjunct therapy to established therapies. Curcumin (1, 7-bis (4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3, 5- dione; also called diferuloylmethane) is a polyphenolic phytochemical extracted from the root of curcuma longa, commonly called turmeric. Despite evidence from in vitro (cell culture) and preclinical studies in animals, clinical studies have not provided strong evidence for a therapeutic effect of curcumin. The relevance of curcumin as a drug has been questioned based on its classification as a compound with pan assay interference and invalid metabolic panaceas properties bringing into question the relevance of the therapeutic targets identified for curcumin. To some extent this is due to the lack of a complete understanding of the link between the in vitro (cell culture activity), pharmacokinetics and in vivo activity of curcumin. In this review and using NF-κB as a cellular target for curcumin, we have investigated the relationship between the potency of curcumin as an inhibitor of NF-κB in cell culture, the pharmacokinetics of curcumin and curcumin's anticancer and anti-inflammatory effects in preclinical models of cancer and inflammation. Plausible explanations and rationale are provided to link these activities together and suggest that both curcumin and its more soluble Phase II metabolite curcumin glucuronide may play a key role in the treatment effects of curcumin in vivo mediated at NF-κB.

Intense Uptake of Liposomal Curcumin by Multiple Myeloma Cell Lines: Comparison to Normal Lymphocytes, Red Blood Cells and Chronic Lymphocytic Leukemia Cells.

BACKGROUND/AIM: Curcumin is being widely investigated for its anticancer properties and several studies in the literature suggest that curcumin is distributed to a higher degree in cancer cells compared to normal cells. The goal of this study was to investigate the disposition of curcumin in the form of Lipocurc™ in multiple myeloma (MM)-causing plasma cell lines and B-lymphocytes from healthy individuals and compare the uptake to previously published data for red blood cells (RBCs), peripheral blood mononuclear cells (PBMCs) from healthy individuals and PBMCs from patients with chronic lymphocytic leukemia (CLL-cells). MATERIALS AND METHODS: Two MM-producing cell lines were studied: RPMI-8266, an IgG lambda cell line, and NCL-H929, an IgA kappa line. The distribution of liposomal curcumin and its metabolism to the major stable metabolite tetrahydrocurcumin (THC) were measured in vitro in the cell lines and B-lymphocytes. The cells were incubated in plasma protein-supplemented media with liposomal curcumin (Lipocurc™) for 15 min at 37°C and the levels of curcumin and THC in cells and medium were determined by liquid chromatography tandem mass spectrometry. RESULTS: Extremely intense uptake was seen in both MM lines compared to that in B-lymphocytes and previously published data in RBCs, PBMCs and CLL cells. The levels of curcumin in RPMI-8266 and NCI-H929 cells were 14,225±847 and 12,723±500 pg/106 cells compared to 19±5,587±86 and 3,122±166 pg/106 cells in RBCs, PBMCs and CLL cells, respectively. Conversion of curcumin to THC was greatest in PBMCs, considerably less in CLL cells and minimal or absent in B-lymphocytes and MM cell lines. CONCLUSION: The extremely intense uptake of curcumin (as Lipocurc™) in both MM lines further suggests that Lipocurc™ should be investigated in the treatment of patients with this disease.

Pharmacokinetics of liposomal curcumin (Lipocurc™) infusion: effect of co-medication in cancer patients and comparison with healthy individuals.

PURPOSE: Investigation of the impact of co-medication on the plasma levels of curcumin and tetrahydrocurcumin (THC) in cancer patients and a comparison of the pharmacokinetics of curcumin and plasma levels of THC between cancer patients and healthy individuals following intravenous infusion of Lipocurc™ (liposomal curcumin). METHODS: Correlation analysis was used to determine the impact of co-medication on infusion rate normalized plasma levels of curcumin and THC in cancer patients and to compare the plasma levels of curcumin and THC at different infusion rates between cancer patients and healthy individuals. In vitro hepatocyte and red blood cell distribution experiments were conducted with Lipocurc™ to support clinical findings. Plasma concentration time data were analyzed by the non-compartmental method to determine and compare the pharmacokinetic parameters of curcumin in cancer patients and healthy individuals. RESULTS: Of 44 co-medications studied, three medications targeting the renin-angiotensin system, Lisinopril, Ramipril, and Valsartan elevated plasma levels of curcumin and THC in three cancer patients infused with Lipocurc™. Cell distribution experiments indicated that the disposition of curcumin in red blood cells may be a target for elevation of the plasma levels of curcumin. Plasma levels of curcumin in cancer patients increased to a greater extent with increased infusion rate compared to healthy individuals. Upon termination of infusion, the elimination phase for curcumin was shorter with a shorter terminal half-life and smaller volume of distribution for curcumin in cancer patients compared to healthy individuals. CONCLUSION: Either co-medications or health status, or both, can impact the pharmacokinetics of curcumin infusion (as Lipocurc™) in cancer patients.

Towards an exposure narrative for metals and arsenic in historically contaminated Ni refinery soils: Relationships between speciation, bioavailability, and bioaccessibility.

Archived soils contaminated with Ni, Cu, Co, and As from legacy operations of a nickel refinery at Port Colborne, Ontario, Canada were speciated using mineral liberation analysis. Four Ni minerals were identified as fingerprint compounds of the historical refinery emissions. Cu and Co were present in solid solution in these minerals due to their presence in the refinery's feed. The highest concentrations of Ni, Cu, Co, and As in these soils were 18,553, 1915, 196, and 79mg/kg, respectively, these elevated contaminant concentrations attesting to the importance of incidental soil ingestion to the oral exposure pathway in Port Colborne. The in vitro gastric bioaccessibility (BAc) was determined for these contaminants, as was in vivo oral bioavailability (BAv), using a mass balance approach in male Sprague-Dawley rats. In spite of the elevated soil concentrations of Cu, the BAv of this physiologically important metal could not be distinguished from that in commercial rat chow, suggesting low potential for exposure. Co and As also had low apparent BAv (<2%). For Ni, baseline oral BAv of naturally sourced dietary Ni was found to be approximately 2%, as was the oral BAv of Ni from nickel sulfate hexahydrate. The mass balances of NiSO4·6H2O were fully accounted-for in urine and feces after a single gavage dose, indicating little to no organ incorporation from this highly soluble salt. Therefore, the urinary estimates of Ni BAv for these soils were assumed to represent true BAv despite variable fecal recoveries. The high Ni concentrations enabled BAc-BAv relationships to be developed for these contaminated soils. For absolute bioavailability (ABA) and relative bioavailability (RBA) the relationships were: ABA=0.0116(BAc)-0.0479 and RBA=0.5542(BAc)-2.2817. These findings will advance the development of robust exposure narratives for soil metal contamination in Port Colborne and elsewhere.

Distribution of Curcumin and THC in Peripheral Blood Mononuclear Cells Isolated from Healthy Individuals and Patients with Chronic Lymphocytic Leukemia.

Background/Aim: Curcumin is being widely investigated for its anticancer properties and studies in the literature suggest that curcumin distributes to a higher degree in tumor versus non-tumor cells. In the current study, we report on investigation of the distribution of curcumin and metabolism to THC in PBMC from healthy individuals and chronic lymphocytic leukemia (CLL) patients following exposure to Lipocurc™ (liposomal curcumin). Materials and Methods: The time and temperature-dependent distribution of liposomal curcumin and metabolism to tetrahydrocurcumin (THC) were measured in vitro in human peripheral blood mononuclear cells (PBMC) obtained from healthy individuals, PBMC HI (cryopreserved and freshly isolated PBMC) and CLL patients (cryopreserved PBMC) with lymphocyte counts ranging from 17-58×106 cells/ml (PBMCCLL,Grp 1) and >150×106 cells/ml (PBMCCLL,Grp 2). PBMC were incubated in plasma protein supplemented media with Lipocurc™ for 2-16 min at 37°C and 4°C and the cell and medium levels of curcumin determined by LC-MS/MS. Results: PBMC from CLL patients displayed a 2.2-2.6-fold higher distribution of curcumin compared to PBMC HI Curcumin distribution into PBMCCLL, Grp 1/Grp 2 ranged from 384.75 - 574.50 ng/g w.w. of cell pellet and was greater compared to PBMC HI that ranged from 122.27-220.59 ng/g w.w. of cell pellet following incubation for up to 15-16 min at 37°C. The distribution of curcumin into PBMCCLL,Grp 2 was time-dependent in comparison to PBMC HI which did not display a time-dependence and there was no temperature-dependence for curcumin distribution in either cell type. Curcumin was metabolized to THC in PBMC. The metabolism of curcumin to THC was not markedly different between PBMC HI (range=23.94-42.04 ng/g w.w. cell pellet) and PBMCCLL,Grp 1/Grp 2 (range=23.08-48.22 ng/g. w.w. cell pellet). However, a significantly greater time and temperature-dependence was noted for THC in PBMCCLL,Grp 2 compared to PBMC HI Conclusion: Curcumin distribution into PBMC from CLL patients was higher compared to PBMC from healthy individuals, while metabolism to THC was similar. The potential for a greater distribution of curcumin into PBMC from CLL patients may be of therapeutic benefit.

A phase 1 dose-escalation study on the safety, tolerability and activity of liposomal curcumin (Lipocurc™) in patients with locally advanced or metastatic cancer.

PURPOSE: This study was conducted to investigate the safety and tolerability of increasing doses of liposomal curcumin in patients with metastatic cancer. Investigations of anti-tumor activity and of the pharmacokinetics of curcumin were secondary objectives. METHODS: In this phase I, single-center, open-label study in patients with metastatic tumors, liposomal curcumin was administered as a weekly intravenous infusion for 8 weeks. Dose escalation was started at 100 mg/m2 over 8 h and the dose increased to 300 mg/m2 over 6 h. RESULTS: 32 patients were treated. No dose-limiting toxicity was observed in 26 patients at doses between 100 and 300 mg/m2 over 8 h. Of six patients receiving 300 mg/m2 over 6 h, one patient developed hemolysis, and three other patients experienced hemoglobin decreases > 2 g/dL without signs of hemolysis. Pharmacokinetic analyses revealed stable curcumin plasma concentrations during infusion followed by rapid declines to undetectable levels after the infusion. Anti-tumor activity by RECIST V1.1 was not detected. Significant tumor marker responses and transient clinical benefit were observed in two patients. CONCLUSION: 300 mg/m2 liposomal curcumin over 6 h was the maximum tolerated dose in these heavily pretreated patients, and is the recommended starting dose for anti-cancer trials.

Safety and Biosimilarity of ior®LeukoCIM Compared to Neupogen® Based on Toxicity, Pharmacodynamic, and Pharmacokinetic Studies in the Sprague-Dawley Rat.

This study examined the safety, pharmacodynamic and pharmacokinetic similarity of the human recombinant filgrastim products ior®LeukoCIM and Neupogen® following a 28-day repeated subcutaneous dose administration in male and female Sprague-Dawley rats with a 14-day recovery period. Safety profiling was based on clinical observations, clinical pathology, and pathology findings for control rats dosed with vehicle and rats dosed either with 15, 75, and 150 µg/kg of ior®LeukoCIM or with 150 µg/kg of Neupogen®. The major adverse treatment-related clinical finding was mild to severe swelling of the hock-joint (tarsal joint) and hind limb, alone or accompanied with lameness which was more prominent in males and which had a similar frequency of occurrence for both ior®LeukoCIM and Neupogen®. All adverse findings were fully reversible. As expected, ior®LeukoCIM and Neupogen® both increased white blood cell and neutrophil levels in rats and to a similar extent for high-dose ior®LeukoCIM and Neupogen®. The pharmacokinetics of filgrastim following dosing with ior®LeukoCIM were well behaved and comparable for high-dose ior®LeukoCIM and Neupogen®. The results of this study imply that ior®LeukoCIM and Neupogen® had similar safety profiles, pharmacodynamic responses, and pharmacokinetic profiles that suggest they are biosimilar.

Distribution and Metabolism of Lipocurc™ (Liposomal Curcumin) in Dog and Human Blood Cells: Species Selectivity and Pharmacokinetic Relevance.

BACKGROUND/AIM: The aim of this study was to investigate the distribution of curcumin (in the form of Lipocurc™) and its major metabolite tetrahydrocurcumin (THC) in Beagle dog and human red blood cells, peripheral blood mononuclear cells (PBMC) and hepatocytes. MATERIALS AND METHODS: Lipocurc™ was used as the source of curcumin for the cell distribution assays. In vitro findings with red blood cells were also compared to in vivo pharmacokinetic data available from preclinical studies in dogs and phase I clinical studies in humans. RESULTS: High levels of curcumin were measured in PBMCs (625.5 ng/g w.w. cell pellet or 7,297 pg/106 cells in dog and 353.7 ng/g w.w. cell pellet or 6,809 pg/106 cells in human) and in hepatocytes (414.5 ng/g w.w. cell pellet or 14,005 pg/106 cells in dog and 813.5 ng/g w.w. cell pellet or 13,780 pg/106 cells in human). Lower curcumin levels were measured in red blood cells (dog: 78.4 ng/g w.w. cell pellet or 7.2 pg/106 cells, human: 201.5 ng/g w.w. cell pellet or 18.6 pg/106 cells). A decrease in the medium concentration of curcumin was observed in red blood cells and hepatocytes, but not in PBMCs. Red blood cell levels of THC were ~5-fold higher in dog compared to human and similar between dog and human for hepatocytes and PBMCs. The ratio of THC to curcumin found in the red blood cell medium following incubation was 6.3 for dog compared to 0.006 for human, while for PBMCs and hepatocytes the ratio of THC to curcumin in the medium did not display such marked species differences. CONCLUSION: There was an excellent correlation between the in vitro disposition of curcumin and THC following incubation with red blood cells and in vivo plasma levels of curcumin and THC in dog and human following intravenous infusion. The disposition of curcumin in blood cells is, therefore, species-dependent and of pharmacokinetic relevance.

From B.Sc. to Ph.D., my shuffle off to Buffalo.

From the fall of 1978 until the summer of 1982, I was a graduate student in the Laboratory of Dr. David Triggle in the Department of Biochemical Pharmacology, School of Pharmacy, State University of New York at Buffalo. This contribution permits me the opportunity to take you back in time into David's laboratory and tell you the story of how my early research career was borne and to provide a glimpse into some of the accomplishments that David, I and my fellow graduates students made. The central theme of my research was to bring together the many events that controlled the contraction of guinea-pig ileal longitudinal muscle, from the binding of muscarinic agonists, the movement of mono- and divalent cations that control depolarization to contraction itself and the differences between muscarinic and non-muscarinic mediated contraction and tachyphylaxis. From these studies, we were able to provide concrete data supporting a fluid muscarinic receptor-effector coupling model that challenged the concept of spare receptors. We also were able to develop methods to quantitate the binding sites for dihydropyrine calcium channel antagonists thereby opening the door to a flood of studies that furthered our understanding of these clinically employed drugs, providing a new target to elucidate the mechanism(s) of action of drugs that act outside of and within the central nervous system.

HPMPC therapy of MCMV-induced retinal disease in the SCID mouse measured by electroretinography, a non-invasive technique.

The purpose of these studies was to investigate the use of non-invasive electroretinography for the evaluation of retinal disease and its treatment in an ocular murine cytomegalovirus (MCMV) disease model. While under anesthesia, 10(2.6)plaque forming units (pfu) of salivary gland passaged, Smith strain MCMV was injected in the anterior chamber of 6- to 8-week-old severe combined immunodeficiency (SCID) mice. At various times post-inoculation, bright-flash scotopic electroretinogram, viral titer, and histology were obtained from the injected eye. Antiviral therapy was tested using 0.1 and 5mg/kg/day subcutaneous injections of HPMPC (Cidofovir) once daily for 5 consecutive days. In infected animals, the a- and b-waves of the electroretinographic (ERG) signal were significantly reduced as of 10 days post-inoculation when compared to control animals. Therapy with HPMPC 0.1mg/kg/day subcutaneously (s.c.) once daily for 5 consecutive days was able to delay the decrease in ERG wave amplitude and inhibit viral replication, whereas 5mg/kg/day s.c. significantly protected the ERG, completely inhibited viral replication, and maintained ocular viral titer below the limit of detection for up to 17 days post-infection. The reduction of ERG activity during progression of retinal disease correlated well with reduction of disease pathology. ERG recording represents a valuable non-invasive technique to measure the progression of the retinal disease induced by MCMV and the efficacy of antiviral treatment in the ocular MCMV disease model.

Safety and biosimilarity of ior(®) EPOCIM compared with Eprex(®) based on toxicologic, pharmacodynamic, and pharmacokinetic studies in the Sprague-Dawley rat.

This study examined the safety, pharmacodynamic (PD), and pharmacokinetic (PK) biosimilarity of the human recombinant erythropoietin (EPO) products ior(®) EPOCIM and Eprex(®) following a 28-day repeated intravenous dose administration in male and female Sprague-Dawley rats with a 14-day recovery period. Safety profiling was based on clinical observations, clinical pathology, and pathology findings for control rats dosed with vehicle and rats dosed either with 30, 300, and 600 I.U./kg of ior(®) EPOCIM or 600 I.U. of Eprex(®) . Adverse findings for both ior(®) EPOCIM and Eprex(®) were similar and were a consequence of thrombotic events (ulcerative skin lesions, swollen hock joints/lameness, stomach ulcers) and decreased body weight gains, all known adverse reactions to this class of drug in rats. With the exception of stomach ulcers, all other adverse findings were fully reversible. Neither drug stimulated the production of antidrug antibodies. As expected, ior(®) EPOCIM and Eprex(®) both increased reticulocyte, red blood cell, hemoglobin, and hematocrit levels in rats. The PK of EPO following dosing with ior(®) EPOCIM was well behaved and consistent with the literature. The results of this study imply that ior(®) EPOCIM and Eprex(®) had safety profiles, PD responses, and toxicokinetic profiles that were biosimilar.