N-(Pyridin-2-yl) arylsulfonamide inhibitors of 11ß-hydroxysteroid dehydrogenase type 1: strategies to eliminate reactive metabolites.

N-(Pyridin-2-yl) arylsulfonamides 1 and 2 (PF-915275) were identified as potent inhibitors of 11ß-hydroxysteroid dehydrogenase type 1. A screen for bioactivation revealed that these compounds formed glutathione conjugates. This communication presents the results of a risk benefit analysis carried out to progress 2 (PF-915275) to a clinical study and the strategies used to eliminate reactive metabolites in this series of inhibitors. Based on the proposed mechanism of bioactivation and structure-activity relationships, design efforts led to N-(pyridin-2-yl) arylsulfonamides such as 18 and 20 that maintained potent 11ß-hydroxysteroid dehydrogenase type 1 activity, showed exquisite pharmacokinetic profiles, and were negative in the reactive metabolite assay.

Interspecies variation in the metabolism of zoniporide by aldehyde oxidase.

1. Aldehyde oxidase (AO) is a cytosolic enzyme that contributes to the Phase I metabolism of xenobiotics in human and preclinical species. 2. Current studies explored in vitro metabolism of zoniporide in various animal species and humans using S9 fractions. The animal species included commonly used pharmacology and toxicology models and domestic animals such as the cat, cow or bull, pig and horse. 3. In addition, gender and strain differences in some species were also explored. 4. All animals except the dog and cat converted zoniporide to 2-oxozoniporide (M1). 5. Michael-Menten kinetic studies were conducted in species that turned over zoniporide to M1. 6. Marked differences in KM, Vmax and Clint were observed in the oxidation of zoniporide. 7. Although the KM and Vmax of zoniporide oxidation in male and female human S9 was similar, some gender difference was observed in animals especially, in Vmax. 8. The domestic animals also showed marked species differences in the AO activity and affinity toward zoniporide.

Selective mechanism-based inactivation of CYP3A4 by CYP3cide (PF-04981517) and its utility as an in vitro tool for delineating the relative roles of CYP3A4 versus CYP3A5 in the metabolism of drugs.

CYP3cide (PF-4981517; 1-methyl-3-[1-methyl-5-(4-methylphenyl)-1H-pyrazol-4-yl]-4-[(3S)-3-piperidin-1-ylpyrrolidin-1-yl]-1H-pyrazolo[3,4-d]pyrimidine) is a potent, efficient, and specific time-dependent inactivator of human CYP3A4. When investigating its inhibitory properties, an extreme metabolic inactivation efficiency (k(inact)/K(I)) of 3300 to 3800 ml · min⁻¹ · µmol⁻¹ was observed using human liver microsomes from donors of nonfunctioning CYP3A5 (CYP3A5 *3/*3). This observed efficiency equated to an apparent K(I) between 420 and 480 nM with a maximal inactivation rate (k(inact)) equal to 1.6 min⁻¹. Similar results were achieved with testosterone, another CYP3A substrate, and other sources of the CYP3A4 enzyme. To further illustrate the abilities of CYP3cide, its partition ratio of inactivation was determined with recombinant CYP3A4. These studies produced a partition ratio approaching unity, thus underscoring the inactivation capacity of CYP3cide. When CYP3cide was tested at a concentration and preincubation time to completely inhibit CYP3A4 in a library of genotyped polymorphic CYP3A5 microsomes, the correlation of the remaining midazolam 1'-hydroxylase activity to CYP3A5 abundance was significant (R² value equal to 0.51, p value of <0.0001). The work presented here supports these findings by fully characterizing the inhibitory properties and exploring CYP3cide's mechanism of action. To aid the researcher, multiple commercially available sources of CYP3cide were established, and a protocol was developed to quantitatively determine CYP3A4 contribution to the metabolism of an investigational compound. Through the establishment of this protocol and the evidence provided here, we believe that CYP3cide is a very useful tool for understanding the relative roles of CYP3A4 versus CYP3A5 and the impact of CYP3A5 genetic polymorphism on a compound's pharmacokinetics.

Magnetic resonance imaging of abdominal versus vaginal prolapse surgery with mesh.

INTRODUCTION AND HYPOTHESIS: We compared two surgical approaches in patients with symptomatic prolapse of the vaginal apex with normal controls by analyzing pelvic landmark relationships measured using magnetic resonance imaging (MRI) before and after surgery. METHODS: In this prospective multicenter pilot study involving 16 participants, nulliparous controls (n = 6) were compared with ten parous (3.0 ± 1.0) women with uterine apical prolapse equal to or greater than stage 2. Group A (n = 5) underwent abdominal sacral colpopexy with monofilament polypropylene mesh and group B (n = 5) with vaginal mesh kit repair (Total ProLift). Subtotal hysterectomy was performed in all group A and no group B women. All patients underwent preoperative and 3-month postoperative Pelvic Organ Prolapse Quantification (POP-Q) and dynamic MRI. Comparison of MRI pelvic angles and distances was performed and analyzed by Mann-Whitney rank sum test and chi-square test. RESULTS: Vaginal apical support is similar at 3 months for abdominal sacral colpopexy (ASCP) and ProLift by POP-Q examination and MRI analysis. In both treatment groups, the postoperative POP-Q point C and MRI parameters were similar to nulliparous controls at 3 months. CONCLUSIONS: Anatomic outcomes for ASCP compared with ProLift were similar at 3 months in terms of vaginal apical support by POP-Q and MRI analysis. Continued comparative analysis of postoperative support with objective imaging seems warranted.

MRI pelvic landmark angles in the assessment of apical pelvic organ prolapse.

PURPOSE: The aim of the study was to evaluate the utility of magnetic resonance imaging (MRI) pelvic landmark angles and lines in the assessment of apical vault prolapse. METHODS: Seventeen women were evaluated as part of a prospective surgical trial. Baseline data are presented as a pilot study of the utility of MRI in addition to this evaluation of 6 nulliparous volunteers without prolapse and 11 parous women with symptomatic ≥ stage II uterine prolapse. Each patient underwent assessment for pelvic organ prolapse quantification (POPQ) and pelvic MRI. Pelvic landmark angles and lines were measured. Mann-Whitney Rank sum test and Spearman's Rank order correlation test were used to assess agreement. RESULTS: Women with prolapse had a significantly larger h angle, g angle, and e angle at rest than those without prolapse. Correlation between apical vault descent was measured clinically by POPQ point C with MRI measurements: h angle (r = 0.61, p = 0.01), g angle (r = 0.64, p = 0.005), and e angle (r = 0.62, p = 0.007). CONCLUSION: MRI measurements of pelvic landmark angles reliably differentiate between women with and without uterine prolapse and correlate best with POPQ point C.

Outpatient ocular brachytherapy: The USC Experience.

PURPOSE: Ocular brachytherapy is a standard-of-care surgical procedure for globe salvage in the treatment of uveal melanoma. The procedure involves the placement and subsequent removal of a radioactive plaque several days later. At many locations, patients are admitted on an inpatient basis until plaque removal due to radiation safety concerns. However, patients may be discharged to home after plaque insertion, and subsequently return to the medical facility for plaque removal. This study aimed to evaluate the safety and systematic financial benefit of the outpatient ocular brachytherapy program at "?>the University of Southern California (USC) Roski Eye Institute for 30 years. METHODS AND MATERIALS: A single-institution retrospective record review was performed on all 275 patients who underwent brachytherapy for ocular tumors between January 1, 1989 and December 31, 2019 to assess for occurrences of reportable radiation and/or patients safety events. The treatment protocols at our institution are described. Data on hospital-adjusted expenses per inpatient day from the American Hospital Association's 2018 Annual Survey were used as a proxy for costs to patients and the health care system to perform a cost-benefit analysis comparing outpatient versus inpatient brachytherapy. RESULTS: Of the 275 plaque procedures over a 30-year period that were reviewed, there were no internally or externally reportable patient or radiation safety events. There were no adverse events related to patient transportation to the hospital, the patient not returning for plaque removal, operative issues in removing the plaque on time due to cancelled or delayed cases, or loss of radioactive material. Additionally, our cost-benefit analysis estimates that outpatient brachytherapy reduced costs for USC's patients in 2018 by an average of $24,722 per patient treated with ocular brachytherapy. CONCLUSIONS: With appropriate measures, outpatient ocular brachytherapy allows patients to safely return home with the added benefit of decreased financial burden for both patients and the broader health care system.

Characterization of human corneal epithelial cell model as a surrogate for corneal permeability assessment: metabolism and transport.

The recently introduced Clonetics human corneal epithelium (cHCE) cell line is considered a promising in vitro permeability model, replacing excised animal cornea to predict corneal permeability of topically administered compounds. The purpose of this study was to further characterize cHCE as a corneal permeability model from both drug metabolism and transport aspects. First, good correlation was found in the permeability values (P(app)) obtained from cHCE and rabbit corneas for various ophthalmic drugs and permeability markers. Second, a previously established real-time quantitative polymerase chain reaction method was used to profile mRNA expression of drug-metabolizing enzymes (major cytochromes P450 and UDP glucuronosyltransferase 1A1) and transporters in cHCE in comparison with human cornea. Findings indicated that 1) the mRNA expression of most metabolizing enzymes tested was lower in cHCE than in excised human cornea, 2) the mRNA expression of efflux transporters [multidrug resistant-associated protein (MRP) 1, MRP2, MRP3, and breast cancer resistance protein], peptide transporters (PEPT1 and PEPT2), and organic cation transporters (OCTN1, OCTN2, OCT1, and OCT3) could be detected in cHCE as in human cornea. However, multidrug resistance (MDR) 1 and organic anion transporting polypeptide 2B1 was not detected in cHCE; 3) cHCE was demonstrated to possess both esterase and ketone reductase activities known to be present in human cornea; and 4) transport studies using probe substrates suggested that both active efflux and uptake transport may be limited in cHCE. As the first detailed report to delineate drug metabolism and transport characteristics of cHCE, this work shed light on the usefulness and potential limitations of cHCE in predicting the corneal permeability of ophthalmic drugs, including ester prodrugs, and transporter substrates.

Metabolism distribution and excretion of a matrix metalloproteinase-13 inhibitor, 4-[4-(4-fluorophenoxy)-benzenesulfonylamino]tetrahydropyran-4-carboxylic acid hydroxyamide (CP-544439), in rats and dogs: assessment of the metabolic profile of CP-544439 in plasma and urine of humans.

The metabolism and disposition of 4-[4-(4-fluorophenoxy)-benzenesulfonylamino]tetrahydropyran-4-carboxylic acid hydroxyamide (CP-544439), a selective inhibitor of matrix metalloproteinase-13, was investigated in rats and dogs following oral administration of [(14)C]CP-544439. Both species showed quantitative recovery of the radiolabel, and feces was the major route of excretion. Whole-body autoradioluminography study in rats suggested distribution of CP-544439 in all tissues except central nervous system. The radiolabel was rapidly eliminated from most tissues except the periodontal ligament. Metabolism of CP-544439 was extensive in both species. Only 8.4 and 1.5% of the total dose constituted unchanged CP-544439 in the rat and dog, respectively. Similarly, pharmacokinetic analysis of [(14)C]CP-544439 and unchanged CP-544439 indicated that the exposure of the parent drug was 16 and 6.5% of the total radioequivalents in rat and dog, respectively. Metabolic profiling revealed that CP-544439 was primarily metabolized via glucuronidation, reduction, and hydrolysis. Glucuronidation was the primary route of metabolism in dogs, whereas reduction of the hydroxamate moiety was the major pathway in rats. Human plasma and urine obtained from a dose escalation study in healthy human volunteers were also analyzed in this study to assess the metabolism of CP-544439 in humans and ensure that selected animal species were exposed to all major metabolites formed in humans. Analysis suggested that CP-544439 was metabolized via all three pathways in humans consistent with rat and dog; however, the glucuronide conjugate M1 was the major circulating and excretory metabolite in humans. Preliminary in vitro phenotyping studies indicated that glucuronide formation is primarily catalyzed by UGT1A1, 1A3, and 1A9.

Bioactivation of flutamide metabolites by human liver microsomes.

Flutamide, a widely used nonsteroidal antiandrogen drug for the treatment of prostate cancer, has been associated with rare incidences of hepatotoxicity in patients. It is believed that bioactivation of flutamide and subsequent covalent binding to cellular proteins is responsible for its toxicity. A novel N-S glutathione adduct has been identified in a previous bioactivation study of flutamide (Kang et al., 2007). Due to the extensive first pass metabolism, flutamide metabolites such as 2-hydroxyflutamide and 4-nitro-3-(trifluoromethyl)phenylamine (Flu-1) have achieved plasma concentrations higher than the parent in prostate cancer patients. In vitro studies in human liver microsomes were conducted to probe the cytochrome P450 (P450)-mediated bioactivation of flutamide metabolites and identify the possible reactive species using reduced glutathione (GSH) as a trapping agent. Several GSH adducts (G1, Flu-1-G1, Flu-1-G2, Flu-6-Gs) derived from the metabolites of flutamide were identified and characterized. A comprehensive bioactivation mechanism was proposed to account for the formation of the observed GSH adducts. Of interest were the formation of a reactive intermediate by the desaturation of the isopropyl group of M5 and the unusual bioactivation of Flu-1. Studies using recombinant P450s suggested that the major P450 isozymes involved in the bioactivation of flutamide and its metabolites were CYP1A2, CYP3A4, and CYP2C19. These findings suggested that, in addition to the direct bioactivation of flutamide, the metabolites of flutamide could also be bioactivated and contribute to flutamide-induced hepatotoxicity.

Outcomes of choroidal melanomas treated with eye physics plaques: A 25-year review.

PURPOSE: To review long-term outcomes of the University of Southern California Plaque Simulator (PS) software and Eye Physics (EP) plaques. We hypothesize that the PS/EP system delivers lower doses to critical ocular structures, resulting in lower rates of radiation toxicity and favorable visual outcomes compared to Collaborative Ocular Melanoma Study plaques, while maintaining adequate local tumor control. METHODS AND MATERIALS: Retrospective review of 133 patients treated for choroidal melanoma with 125I brachytherapy, using PS software and EP plaques, from 1990 through 2015. A dose of 85 Gy at a rate of 0.6 Gy/h was prescribed to the tumor apex (with a typical margin of 2 mm) over 7 days. Primary outcomes were local tumor recurrence, globe salvage, and metastasis. Secondary outcomes were changes in visual acuity and radiation complications. RESULTS: With median followup of 42 months, 5-year Kaplan-Meier estimated rates for tumor control, globe salvage, and metastatic-free survival were 98.3%, 96.4%, and 88.2%, respectively. Median doses to the macula and optic nerve were 39.9 Gy and 30.0 Gy, respectively. Forty-three percent of patients developed radiation retinopathy, and 20% developed optic neuropathy; 39% lost ≥6 Snellen lines of vision. CONCLUSIONS: The PS/EP system is designed to improve the accuracy and conformality of the radiation dose, creating a steep dose gradient outside the melanoma to decrease radiation to surrounding ocular structures. We report favorable rates of local tumor control, globe salvage, metastases, and radiation complications when compared to the Collaborative Ocular Melanoma Study and other studies. Overall, the PS/EP system results in excellent tumor control and appears to optimize long-term visual and radiation-related outcomes after brachytherapy.

Discovery of a novel class of targeted kinase inhibitors that blocks protein kinase C signaling and ameliorates retinal vascular leakage in a diabetic rat model.

Protein kinase C (PKC) family members such as PKCbetaII may become activated in the hyperglycemic state associated with diabetes. Preclinical and clinical data implicate aberrant PKC activity in the development of diabetic microvasculature abnormalities. Based on this potential etiological role for PKC in diabetic complications, several therapeutic PKC inhibitors have been investigated in clinical trials for the treatment of diabetic patients. In this report, we present the discovery and preclinical evaluation of a novel class of 3-amino-pyrrolo[3,4-c]pyrazole derivatives as inhibitors of PKC that are structurally distinct from the prototypical indolocarbazole and bisindolylmaleimide PKC inhibitors. From this pyrrolo-pyrazole series, several compounds were identified from biochemical assays as potent, ATP-competitive inhibitors of PKC activity with high specificity for PKC over other protein kinases. These compounds were also found to block PKC signaling activity in multiple cellular functional assays. PF-04577806, a representative from this series, inhibited PKC activity in retinal lysates from diabetic rats stimulated with phorbol myristate acetate. When orally administered, PF-04577806 showed good exposure in the retina of diabetic Long-Evans rats and ameliorated retinal vascular leakage in a streptozotocin-induced diabetic rat model. These novel PKC inhibitors represent a promising new class of targeted protein kinase inhibitors with potential as therapeutic agents for the treatment of patients with diabetic microvascular complications.

Effect of intestinal glucuronidation in limiting hepatic exposure and bioactivation of raloxifene in humans and rats.

Raloxifene (Evista) is a second generation selective estrogen receptor modulator used in the treatment of osteoporosis and for chemoprevention of breast cancer. It is bioactivated to reactive intermediates, which covalently bind to proteins and form GSH conjugates upon incubation with NADPH and GSH-supplemented human and rat liver microsomes. Despite these in vitro findings, no major raloxifene-related toxic events have been reported upon its oral administration to humans. This disconnect between safety of raloxifene and its in vitro bioactivation is attributed to its presystemic metabolism via glucuronidation. Current studies investigated the effect of hepatic and intestinal glucuronidation in modulating hepatic availability of raloxifene and its subsequent bioactivation, in vitro. The study design involved preincubation of raloxifene with intestinal microsomes followed by a sequential incubation with liver microsomes. The degree of bioactivation of raloxifene was assessed from the percentage of GSH conjugate formed in liver microsomal incubations or the amount of covalent binding of raloxifene-related material to liver microsomal proteins. The results indicated that human intestinal glucuronidation limited the hepatic exposure of raloxifene that underwent bioactivation in the liver. Similar experiments with rat microsomal preparations showed very little effect of intestinal glucuronidation. This effect of intestinal glucuronidation and the observed species difference were explained by comparing the efficiency (Cl(int)) of glucuronidation and oxidation in the two species. These findings suggested that even though the rate of bioactivation in the two species was similar, the Cl(int) of glucuronidation was 7.5-fold higher in the human intestine as compared to rats. These results support the hypothesis that intestinal glucuronidation modulates the amount of raloxifene undergoing bioactivation by liver and corroborate the importance of assessing other competitive metabolic pathways and species differences in metabolism prior to extrapolation of bioactivation results from rats to humans.

Identification of a novel glutathione conjugate of flutamide in incubations with human liver microsomes.

Flutamide, a nonsteroidal antiandrogen drug widely used in the treatment of prostate cancer, has been associated with rare incidences of hepatotoxicity in patients. It is believed that bioactivation of flutamide and subsequent covalent binding to cellular proteins is responsible for its toxicity. Current in vitro studies were undertaken to probe the cytochrome P450 (P450)-mediated bioactivation of flutamide and identify the possible reactive species using reduced glutathione (GSH) as a trapping agent. NADPH- and GSH-supplemented human liver microsomal incubations of flutamide gave rise to a novel GSH conjugate where GSH moiety was conjugated to the flutamide molecule via the amide nitrogen, resulting in a sulfenamide. The structure of the conjugate was characterized by liquid chromatography-tandem mass spectrometry and NMR experiments. The conjugate formation was primarily catalyzed by heterologously expressed CYP2C19, CYP1A2, and, to a lesser extent, CYP3A4 and CYP3A5. The mechanism for the formation of this conjugate is unknown; however, a tentative bioactivation mechanism involving a P450-catalyzed abstraction of hydrogen atom from the amide nitrogen of flutamide and the subsequent trapping of the nitrogen-centered radical by GSH or oxidized glutathione (GSSG) was proposed. Interestingly, the same adduct was formed when flutamide was incubated with human liver microsomes in the presence of GSSG and NADPH. This finding suggests that P450-mediated oxidation of flutamide via a nitrogen-centered free radical could be one of the several bioactivation pathways of flutamide. Even though the relationship of the GSH conjugate to flutamide-induced toxicity is unknown, the results have revealed the formation of a novel, hitherto unknown, GSH adduct of flutamide.

Influence of lipophilicity on the interactions of N-alkyl-4-phenyl-1,2,3,6-tetrahydropyridines and their positively charged N-alkyl-4-phenylpyridinium metabolites with cytochrome P450 2D6.

The relationship between lipophilicity and CYP2D6 affinity of cyclic tertiary (N-alkyl-4-phenyl-1,2,3,6-tetrahydropyridines) and quaternary (N-alkyl-4-phenylpyridinium) amines was examined. The 1,2,3,6-tetrahydropyridine scaffold was chosen due to its common occurrence in the structures of CYP2D6 ligands such as the Parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the dehydrated haloperidol metabolite N-[4-(4-fluorophenyl)-4-oxobutyl]-4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridine (HPTP). Likewise, the pyridinium framework is found in and 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]pyridinium and N-methyl-4-phenylpyridinium (MPP(+)), the positively charged metabolites of MPTP and haloperidol. The lack of CYP2D6 inhibition by MPTP and its pyridinium metabolite MPP(+) was due to their hydrophilic nature since higher N-alkyl homologs revealed substantial increases in inhibitory potency against recombinant CYP2D6-mediated bufuralol-1'-hydroxylation. The reasonable correlation between lipophilicity and CYP2D6 inhibition by pyridiniums and 1,2,3,6-tetrahydropyridines was only limited to straight chain N-alkyl analogs, since certain N-alkylaryl analogs of lower lipophilicity were better CYP2D6 inhibitors. CYP2D6 substrate properties of straight chain N-alkyltetrahydropyridines were also governed by lipophilicity, and N-heptyl-4-phenyl-1,2,3,6-tetrahydropyridine was the optimal substrate (K(mapp) = 0.63 microM). Metabolism studies indicated that the N-heptyl analog underwent monohydroxylation on the aromatic ring and on the N-heptyl group suggesting that 1,2,3,6-tetrahydropyridines can bind in more than one conformation in the CYP2D6 active site. Increased lipophilicity of haloperidol metabolites did not correlate with inhibitory potency since the more lipophilic HPTP metabolite was less potent as an inhibitor than reduced-haloperidol and reduced-HPTP. Furthermore, HPTP and reduced-HPTP, of comparable lipophilicity to the N-heptyltetrahydropyridine analog were inactive as CYP2D6 substrates. This observation suggests that steric constraints rather than lipophilicity are responsible for the lack of CYP2D6 substrate properties of cyclic tertiary amines tethered to bulky N-substituents. This phenomenon appears to be a common theme among several cyclic tertiary amine-containing anti-depressants and should be taken into consideration when designing central nervous system agents devoid of CYP2D6 substrate properties.