Omaveloxolone and TX63682 are hepatoprotective in the STAM mouse model of nonalcoholic steatohepatitis.

Omaveloxolone is a potent activator of Nrf2, a master transcriptional regulator of a multitude of cytoprotective functions, including antioxidative, anti-inflammatory, and mitochondrial bioenergetic effects. Some of the most potent known effects of Nrf2 involve hepatoprotective functions. The purpose of this study was to evaluate the effects of omaveloxolone and TX63682, a closely related structural analog with similar oral bioavailability, in the STAM mouse model of nonalcoholic steatohepatitis (NASH). C57Bl/6 mice received a single subcutaneous injection of streptozotocin two days after birth and were fed a high-fat diet from 4 to 9 weeks of age. Omaveloxolone and TX63682 were orally administered at doses of 1, 3, and 10 mg/kg/d from 6 to 9 weeks of age. Consistent with the beneficial effects of Nrf2 on hepatoprotection and improved lipid handling, both omaveloxolone and TX63682 decreased hepatic fat deposition, hepatocellular ballooning, inflammatory cell infiltration, and collagen deposition. Omaveloxolone and TX63682 also improved blood glucose control, as evidenced by reductions in nonfasting blood glucose and glycated hemoglobin A1C concentrations. Reductions in liver and serum triglycerides with omaveloxolone and TX63682 treatment were also observed. Both omaveloxolone and TX63682 decreased leptin and increased adiponectin in serum, which is consistent with the anti-inflammatory and antifibrotic effects observed in the liver. These results were associated with significant induction of Nrf2 target gene expression in the liver, including NAD(P)H:quinone oxidoreductase 1, sulfiredoxin 1, and ferritin heavy chain 1. Overall, these data suggest that omaveloxolone and related Nrf2 activators may be useful for the treatment of NASH.

Bardoxolone methyl analog attenuates proteinuria-induced tubular damage by modulating mitochondrial function.

Multiple clinical studies have shown that bardoxolone methyl, a potent activator of nuclear factor erythroid 2-related factor 2 (Nrf2), is effective in increasing glomerular filtration rate in patients with chronic kidney disease. However, whether an Nrf2 activator can protect tubules from proteinuria-induced tubular damage via anti-inflammatory and antioxidative stress mechanisms is unknown. Using an Institute of Cancer Research-derived glomerulonephritis (ICGN) mouse model of nephrosis, we examined the effects of dihydro-CDDO-trifluoroethyl amide (dh404), a rodent-tolerable bardoxolone methyl analog, in protecting the tubulointerstitium; dh404 markedly suppressed tubular epithelial cell damage in the renal interstitium of ICGN mice. The tubular epithelial cells of ICGN mice showed a decrease in the size and number of mitochondria, as well as the breakdown of the crista structure, whereas the number and ultrastructure of mitochondria were maintained by the dh404 treatment. To further determine the effect of dh404 on mitochondrial function, we used human proximal tubular cells in vitro. Stimulation with albumin and free fatty acid increased mitochondrial reactive oxygen species (ROS). However, dh404 administration diminished mitochondrial ROS. Our data show that dh404 significantly reduced proteinuria-induced tubular cell mitochondrial damage, suggesting that improved redox balance and mitochondrial function and suppression of inflammation underlie the cytoprotective mechanism of Nrf2 activators, including bardoxolone methyl, in diabetic kidney disease.-Nagasu, H., Sogawa, Y., Kidokoro, K., Itano, S., Yamamoto, T., Satoh, M., Sasaki, T., Suzuki, T., Yamamoto, M., Wigley, W. C., Proksch, J. W., Meyer, C. J., Kashihara, N. Bardoxolone methyl analog attenuates proteinuria-induced tubular damage by modulating mitochondrial function.

Pharmacokinetics and pharmacodynamics of the novel Nrf2 activator omaveloxolone in primates.

BACKGROUND: Omaveloxolone is a synthetic oleanane triterpenoid that pharmacologically activates Nrf2, a master transcription factor that regulates genes with antioxidative, anti-inflammatory, and mitochondrial bioenergetic properties, and is being evaluated in patients with Friedreich's ataxia. METHODS: The present study evaluated the pharmacokinetics (PK) and tissue distribution of omaveloxolone in monkeys after single and multiple oral doses, and then compared these data to initial results in Friedreich's ataxia patients. Pharmacodynamic (PD) evaluations in monkeys consisted of Nrf2 target gene mRNA expression in peripheral blood mononuclear cells (PBMCs), liver, lung, and brain. A PK/PD model was generated with the monkey data, and used to further evaluate the Friedreich's ataxia patient PK profile. RESULTS: Oral administration of omaveloxolone to monkeys was associated with dose-linear plasma PK and readily measureable and dose-proportional concentrations in liver, lung, and brain. Dose-dependent induction of Nrf2 target genes in PBMCs and tissues was also observed. Clinically, oral administration of omaveloxolone to Friedreich's ataxia patients at incremental doses from 2.5 to 300 mg produced dose-proportional systemic exposures. Clinical doses of at least 80 mg were associated with meaningful improvements in neurological function in patients and generated plasma omaveloxolone concentrations consistent with those significantly inducing Nrf2 target genes in monkeys, as shown with the monkey PK/PD model. CONCLUSION: Overall, the monkey data demonstrate a well-characterized and dose-proportional PK and tissue distribution profile after oral administration of omaveloxolone, which was associated with Nrf2 activation. Further, systemic exposures to omaveloxolone that produce Nrf2 activation in monkeys were readily achievable in Friedreich's ataxia patients after oral administration.

Safety, pharmacokinetics, and pharmacodynamics of oral omaveloxolone (RTA 408), a synthetic triterpenoid, in a first-in-human trial of patients with advanced solid tumors.

BACKGROUND: Omaveloxolone is a semisynthetic oleanane triterpenoid that potently activates Nrf2 with subsequent antioxidant function. We conducted a first-in-human Phase I clinical trial (NCT02029729) with the primary objectives to determine the appropriate dose for Phase II studies, characterize pharmacokinetic and pharmacodynamic parameters, and assess antitumor activity. METHODS: Omaveloxolone was administered orally once daily continuously in a 28-day cycle for patients with stage 4 relapsed/refractory melanoma or non-small cell lung cancer. An accelerated titration design was employed until a grade 2-related adverse event (AE) occurred. A standard 3+3 dose escalation was employed. Single-dose and steady-state plasma pharmacokinetics of the drug were characterized. Downstream Nrf2 activation was assessed in peripheral blood mononuclear cells by quantification of target gene mRNA expression. RESULTS: Omaveloxolone was tested at four dose levels up to 15 mg given orally once daily. No dose-limiting toxicities were detected, and the maximum tolerated dose was not determined. All drug-related AEs were either grade 1 or 2 in severity, and none required clinical action. The most common drug-related AEs were elevated alkaline phosphatase (18%) and anemia (18%). No drug interruptions or reductions were required. Omaveloxolone was rapidly absorbed and exhibited proportional increases in exposure across dose levels. With some exceptions, an overall trend toward time-dependent and dose-dependent activation of Nrf2 antioxidant genes was observed. No confirmed radiologic responses were seen, although one lung cancer subject did have stable disease exceeding 1 year. CONCLUSIONS: Omaveloxolone has favorable tolerability at biologically active doses, although this trial had a small sample size which limits definitive conclusions. These findings support further investigation of omaveloxolone in cancer.

Topical application of RTA 408 lotion activates Nrf2 in human skin and is well-tolerated by healthy human volunteers.

BACKGROUND: Topical application of the synthetic triterpenoid RTA 408 to rodents elicits a potent dermal cytoprotective phenotype through activation of the transcription factor Nrf2. Therefore, studies were conducted to investigate if such cytoprotective properties translate to human dermal cells, and a topical lotion formulation was developed and evaluated clinically. METHODS: In vitro, RTA 408 (3-1000 nM) was incubated with primary human keratinocytes for 16 h. Ex vivo, RTA 408 (0.03, 0.3, or 3 %) was applied to healthy human skin explants twice daily for 3 days. A Phase 1 healthy volunteer clinical study with RTA 408 Lotion (NCT02029716) consisted of 3 sequential parts. In Part A, RTA 408 Lotion (0.5 %, 1 %, and 3 %) and lotion vehicle were applied to individual 4-cm(2) sites twice daily for 14 days. In Parts B and C, separate groups of subjects had 3 % RTA 408 Lotion applied twice daily to a 100-cm(2) site for 14 days or a 500-cm(2) site for 28 days. RESULTS: RTA 408 was well-tolerated in both in vitro and ex vivo settings up to the highest concentrations tested. Further, RTA 408 significantly and dose-dependently induced a variety of Nrf2 target genes. Clinically, RTA 408 Lotion was also well-tolerated up to the highest concentration, largest surface area, and longest duration tested. Moreover, significant increases in expression of the prototypical Nrf2 target gene NQO1 were observed in skin biopsies, suggesting robust activation of the pharmacological target. CONCLUSIONS: Overall, these data suggest RTA 408 Lotion is well-tolerated, activates Nrf2 in human skin, and appears suitable for continued clinical development.

Topical application of the synthetic triterpenoid RTA 408 protects mice from radiation-induced dermatitis.

Free radicals produced during cancer radiotherapy often leads to dermatitis, with the insult ranging from mild erythema to moist desquamation and ulceration. This toxicity can be dose limiting and promote chronic complications, such as fibrosis and wound recurrence. The purpose of this study was to evaluate if RTA 408, a synthetic triterpenoid that potently activates the antioxidative transcription factor Nrf2 and inhibits the proinflammatory transcription factor nuclear factor-kappa b (NF-κB), could protect skin from radiation-induced dermatitis. Mice were irradiated (10 Gy/day) on days 0-2 and 5-7, and RTA 408 (0.01%, 0.1% and 1.0%) was topically applied once daily starting on day 5 or up to day 40. Dermatitis severity was evaluated using a scale ranging from 0 (normal) to 5 (frank ulceration), as well as histologically. The mRNA expression of Nrf2 and NF-κB target genes in skin was also evaluated. RTA 408 (0.01%, 0.1% and 1.0%) reduced the percentage of animal-days with scores ≥2 by 11%, 31% and 55% and scores ≥3 by 16%, 60% and 80%, respectively. Dose-dependent improvements in the appearance of skin were also manifestly visible, with RTA 408 at 1.0% eliciting a normal macroscopic appearance by the end of the treatment period on day 40, including substantial hair regrowth. Moreover, 1.0% RTA 408 markedly reduced epidermal and collagen thickening, prevented dermal necrosis and completely alleviated skin ulcers. These improvements were associated with significant increases in Nrf2 target genes and significant decreases in NF-κB target genes. Together, these data indicate that RTA 408 represents a potentially promising new therapy for the treatment of radiation-induced dermatitis.

Topical application of the synthetic triterpenoid RTA 408 activates Nrf2 and induces cytoprotective genes in rat skin.

RTA 408 is a member of the synthetic oleanane triterpenoid class of compounds known to potently activate the cytoprotective transcription factor Nrf2. Because skin is constantly exposed to external oxidative stress, such as that from ultraviolet radiation, from chemical exposure, during improper wound healing, and throughout the course of cancer radiation therapy, it may benefit from activation of Nrf2. This study was conducted to evaluate the transdermal penetration properties and Nrf2 activation potential of RTA 408 in normal rat skin. RTA 408 (0.1, 1.0, or 3.0%) was applied topically to the shaved skin of male Sprague-Dawley rats twice daily for 4 days and once on Day 5. Topical application of RTA 408 resulted in transdermal penetration, with low but dose-dependent plasma exposure with AUC(0-24 h) values of 3.6, 26.0, and 41.1 h ng/mL for the 0.1, 1.0, and 3.0% doses, respectively. Further, topical application of RTA 408 resulted in increased translocation of Nrf2 to the nucleus, dose-dependent mRNA induction of Nrf2 target genes (e.g. Nqo1, Srxn1, Gclc, and Gclm), and induction of the protein expression of the prototypical Nrf2 target gene Nqo1 and increased total glutathione (GSH) in normal rat skin. Immunohistochemistry demonstrated that increased staining for Nqo1 and total GSH of structures in both the epidermis and dermis was consistent with the full transdermal penetration of RTA 408. Finally, topically administered RTA 408 was well tolerated with no adverse in-life observations and normal skin histology. Thus, the data support the further development of RTA 408 for the potential treatment of skin diseases.

The use of the African green monkey as a preclinical model for ocular pharmacokinetic studies.

PURPOSE: This investigation evaluated the ocular and systemic pharmacokinetics of besifloxacin in African green monkeys compared with cynomolgus monkeys following topical ocular dosing. METHODS: A suspension formulation containing 0.6% besifloxacin was administered to African green and cynomolgus monkeys. Animals were euthanized at predetermined time intervals, and ocular tissue and systemic blood samples were collected and analyzed by LC/MS/MS. RESULTS: In both African green and cynomolgus monkeys, high concentrations of besifloxacin were detected in anterior segment tissues, while levels in posterior segment tissues and plasma were low. Mean concentration versus time profiles of besifloxacin were generally similar between species, with rapid absorption into ocular tissues after a single dose. In anterior segment tissues, concentrations of besifloxacin were measurable throughout the 24-h sampling period in both species. Quantitatively, concentrations were consistently higher in the conjunctiva of African green monkeys compared with cynomolgus monkeys. Besifloxacin levels were also higher during the first 3 h following dosing in the tear fluid of African green monkeys, but lower in the iris/ciliary body during this timeframe. However after the 3-h time point, concentrations in the tear fluid and iris/ciliary body were similar between species. Exposure in cornea tended to be higher in African green monkeys, but the difference was less pronounced than for conjunctiva. Exposure in aqueous humor was comparable between species. In posterior segment tissues, exposure to besifloxacin tended to be higher in cynomolgus monkeys. Systemic exposure also tended to be higher in cynomolgus monkeys, but measurable levels were present in the plasma of both species throughout the 24-h sampling period. With the exception of iris/ciliary body and vitreous humor, mean ocular tissue weights were generally similar between species although a small, but statistically significant, difference was also observed in the choroid. CONCLUSIONS: African green monkeys may be a suitable model for preclinical ocular pharmacokinetic studies. Additional studies using a variety of compounds would be useful in determining whether the quantitative differences in ocular exposures and ocular tissue weights observed in the present investigation reflect slight variations in the procedures used in these separate experiments, or true physiological and anatomical differences between species.

Human aqueous humor concentrations of besifloxacin, moxifloxacin, and gatifloxacin after topical ocular application.

PURPOSE: To determine the concentrations of besifloxacin, moxifloxacin, and gatifloxacin in human aqueous humor after topical instillation of commercially available besifloxacin ophthalmic suspension 0.6%, moxifloxacin ophthalmic solution 0.5%, and gatifloxacin ophthalmic solution 0.3%, and to assess these concentrations relative to the minimum inhibitory concentration for 90% of strains (MIC(90)) for each drug against bacterial pathogens identified in recent cases of postoperative endophthalmitis. SETTING: Six clinical sites, United States. DESIGN: Randomized open-label controlled clinical trial. METHODS: The aqueous humor drug concentrations were compared 60 minutes ± 5 minutes after instillation of 1 topical drop to patients aged 18 years or older having uncomplicated cataract surgery. Concentrations of besifloxacin, moxifloxacin, and gatifloxacin were determined using a validated liquid chromatography with tandem mass spectrometry method. RESULTS: A total of 105 patients were randomized, and aqueous humor samples were analyzed for 103 patients. Mean aqueous humor concentrations were 0.13 µg/mL ± 0.58 (SD), 0.67 ± 0.50 µg/mL, and 0.13 ± 0.08 µg/mL for besifloxacin, moxifloxacin, and gatifloxacin, respectively. Both besifloxacin and moxifloxacin achieved aqueous humor concentrations equal to or slightly higher than their respective MIC(90) for methicillin-resistant and methicillin-susceptible Staphylococcus aureus and Staphylococcus epidermidis; none of the fluoroquinolones achieved concentrations above their MIC(90) for ciprofloxacin-resistant strains of S aureus and S epidermidis. CONCLUSIONS: Based on the aqueous humor drug concentrations measured in this study, it is unlikely that any of the fluoroquinolones tested would be therapeutically effective in the aqueous humor against the most frequently identified drug-resistant staphylococcal isolates from recent cases of postoperative endophthalmitis. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned. Additional disclosures are found in the footnotes.

Ocular pharmacokinetics of mapracorat, a novel, selective glucocorticoid receptor agonist, in rabbits and monkeys.

Mapracorat is a selective glucocorticoid receptor agonist in development for the treatment of a variety of ocular diseases. The purpose of this investigation was to evaluate the ocular pharmacokinetics of mapracorat after topical dosing over a range of dose levels in rabbits and monkeys. Mapracorat was administered over a range of doses from 0.01 to 3000 µg/eye (rabbit) or 50 to 3000 µg/eye (monkey). All animals received a single instillation, and monkeys also received repeated (three times per day for 4 days) instillations. At predetermined intervals through at least 24 h after dosing, ocular tissues and plasma were collected and analyzed for mapracorat by liquid chromatography-tandem mass spectrometry. Mapracorat was rapidly absorbed and widely distributed into ocular tissues after topical ocular administration, with measurable levels sustained through ≥24 h. In both species, mapracorat concentrations were highest in tears followed by conjunctiva and cornea, with lower levels observed in iris/ciliary body and aqueous humor. Mapracorat concentrations in conjunctiva, cornea, and iris/ciliary body increased linearly with increasing dose levels. Ocular exposure was higher after repeated dosing to monkeys than after a single dose. Systemic exposure to mapracorat was low after a single administration, with an average maximal concentration of ≤2.0 ng/ml at the highest dose tested (3000 µg/eye). In comparison with the traditional glucocorticoids, dexamethasone (0.1%) and prednisolone acetate (1%), mapracorat (3%) demonstrated similar or higher levels in ocular tissues with lower systemic exposure. The favorable pharmacokinetic profile of mapracorat supports further clinical investigation and suggests that a convenient daily dosing regimen may be efficacious for this novel ophthalmic anti-inflammatory therapy.

Ocular pharmacokinetics/pharmacodynamics of besifloxacin, moxifloxacin, and gatifloxacin following topical administration to pigmented rabbits.

PURPOSE: The purpose of this investigation was to evaluate the ocular pharmacokinetic/pharmacodynamic (PK/PD) relationship for besifloxacin, moxifloxacin, and gatifloxacin using rabbit ocular PK data, along with in vitro minimum inhibitory concentration (MIC90) values against methicillin- and ciprofloxacin-resistant Staphylococcus aureus (MRSA-CR) and Staphylococcus epidermidis (MRSE-CR). METHODS: Rabbits received a topical instillation of Besivance™ (besifloxacin ophthalmic suspension, 0.6%), Vigamox (moxifloxacin hydrochloride ophthalmic solution, 0.5% as base), or Zymar (gatifloxacin ophthalmic solution, 0.3%), and ocular tissues and plasma were collected from 4 animals/treatment/collection time at 8 predetermined time intervals during the 24h after dosing. Ocular levels of each agent were measured by LC/MS/MS, and PK parameters (Cmax, Tmax, and AUC0₋24) were determined. AUC0₋24/MIC90 ratios were calculated for tears, conjunctiva, cornea, and aqueous humor using previously reported MIC90values for MRSA-CR and MRSE-CR. RESULTS: All of the fluoroquinolones tested demonstrated rapid penetration into ocular tissues after a single instillation. Besifloxacin demonstrated the highest exposure in tear fluid, while exposure in conjunctiva was comparable for all 3 compounds. Peak concentrations of all fluoroquinolones in aqueous humor were at or below ~1g/mL. In comparison with their MIC90values against MRSE-CR and MRSA-CR, besifloxacin achieved an AUC0₋24/MIC90 ratio of ~800 in tears, compared with values of ≤10 for moxifloxacin and gatifloxacin. In cornea, conjunctiva, and aqueous humor, the AUC0₋24/MIC90 ratios were <10 for all compounds. However, in these tissues AUC0₋24/MIC90 ratios for besifloxacin were 1.5- to 38-fold higher than moxifloxacin and gatifloxacin. CONCLUSIONS: In rabbits, besifloxacin demonstrates a nonclinical ocular PK profile characterized by high and sustained concentrations in tear fluid, resulting in AUC0₋24/MIC90 ratios of ~800 for ciprofloxacin-resistant MRSE and MRSA after a single administration. Although besifloxacin had the highest AUC0₋24/MIC90ratios for intraocular tissues, the ratios for all of the drugs were below the target values needed for effective bacterial killing of ciprofloxacin-resistant MRSE and MRSA. Taken together, these nonclinical data indicate that besifloxacin has a favorable ocular PK/PD profile, consistent with the reported clinical efficacy of besifloxacin in the treatment of bacterial conjunctivitis, and consistent with the profile needed for ocular surface sterilization.

Concentrations of besifloxacin, gatifloxacin, and moxifloxacin in human conjunctiva after topical ocular administration.

PURPOSE: To evaluate the pharmacokinetic properties of besifloxacin, gatifloxacin, and moxifloxacin in the conjunctival tissue of healthy volunteers after topical application. METHODS: One-hundred eight (108) subjects were randomly assigned to receive one drop of besifloxacin (0.6% suspension), gatifloxacin (0.3% solution), or moxifloxacin (0.5% solution) ophthalmic formulations in one eye prior to conjunctival biopsy. Conjunctival samples were taken from subjects at either 15 minutes, 30 minutes, 2 hours, 6 hours, 12 hours, or 24 hours after dosing. RESULTS: All three fluoroquinolones reached a peak mean concentration 15 minutes after dosing. The mean concentrations of besifloxacin, gatifloxacin, and moxifloxacin at 15 minutes were 2.30 +/- 1.42 mug/g, 4.03 +/- 3.84 mug/g, and 10.7 +/- 5.89 mug/g, respectively. Concentrations decreased with each subsequent time point. At 24 hours after dosing, concentrations of besifloxacin were measurable in 4 of 6 subjects, compared with 3 of 6 subjects for gatifloxacin and 2 of 6 subjects for moxifloxacin. Besifloxacin had the greatest mean residence time (4.7 hours) in the conjunctival tissue. With regard to methicillin-resistant strains of Staphylococcus aureus and Staphylococcus epidermidis, besifloxacin had the greatest area-under-the-curve (AUC) to MIC(90) ratio. Nine percent (9%) of study subjects (N = 7) experienced a transient reduction in visual acuity. CONCLUSION: All three fluoroquinolones were well tolerated and reached levels in the conjunctiva above the MIC(90)s of methicillin-sensitive S. aureus and S. epidermidis for at least 2 hours.

Ocular pharmacokinetics of besifloxacin following topical administration to rabbits, monkeys, and humans.

PURPOSE: Studies were conducted to evaluate the ocular penetration and systemic exposure to besifloxacin, a fluoroquinolone antibiotic, following topical ocular administration to animals and humans. METHODS: Besifloxacin ophthalmic suspension (0.6%) was administered as a topical ocular instillation to pigmented rabbits, cynomolgus monkeys, and human subjects. At predetermined intervals after dosing, samples of ocular tissues and plasma were collected and analyzed for besifloxacin levels using HPLC/MS/MS methods. RESULTS: Besifloxacin demonstrated good ocular penetration in rabbits and monkeys, with rapid absorption and sustained concentrations observed in anterior ocular tissues through 24 h after a single administration. Maximum besifloxacin concentrations in conjunctiva, cornea, and aqueous humor of monkeys were 6.43 microg/g, 2.10 microg/g, and 0.796 microg/mL, respectively, after a single topical dose, and concentrations declined in these tissues with an apparent half-life of 5-14 h. Following a single topical ocular administration to humans, the maximum besifloxacin concentration in tears was 610 microg/g with concentrations decreasing to approximately 1.6 microg/g at 24 h. The resulting pharmacokinetic parameters for besifloxacin in human tears were evaluated relative to the MIC(90) values (microg/mL) for besifloxacin against Streptococcus pneumoniae (0.125), Staphylococcus aureus (0.25), Staphylococcus epidermidis (0.5), and Haemophilus influenzae (0.06). Following a single topical administration, the C(max)/MIC(90) ratios for besifloxacin in human tears were > or =1,220, and the AUC((0-24))/MIC(90) ratios were > or =2,500 for these relevant ocular pathogens. Following repeated 3-times daily (TID) topical ocular administration to human subjects with clinically diagnosed bacterial conjunctivitis, maximum besifloxacin concentrations in plasma were less than 0.5 ng/mL, on average. CONCLUSIONS: Taken together, the results of the current investigation provide a PK/PD-based rationale that supports the use of besifloxacin for the safe and effective treatment of ocular infections.

A rapid and sensitive LC/MS/MS assay for the quantitation of brimonidine in ocular fluids and tissues.

We report here the development and validation of an LC/MS/MS method for the rapid and accurate quantitation of brimonidine in ocular tissues and fluids using brimonidine-d(4) as an internal standard (IS). Brimonidine was extracted from retina, iris/ciliary body, and vitreous humor samples with an acetonitrile:water (1:1) solution followed by sonication and vortexing. Aliquots of aqueous humor, iris/ciliary body, retina, and vitreous humor samples were diluted with acetonitrile containing IS and were separated on a reverse-phase HPLC column under isocratic conditions. Brimonidine (m/z transition: 292-->212) and the internal standard (m/z transition: 296-->216) were analyzed via multiple-reaction monitoring (MRM) in the positive electrospray mode on a 4000 Q TRAP instrument. The total analysis time for each sample was less than 2.0 min. The calibration curves for brimonidine (1-1000 ng/mL) were constructed using a linear regression with 1/x(2) weighing. The lower limit of quantitation for brimonidine was 1.0 ng/mL for aqueous humor, 10 ng/g for iris/ciliary body, 12.5 ng/g for retina, and 1.6 ng/g for vitreous humor. Intra-day and inter-day estimates of accuracy and precision were within 15% of their nominal values indicating that the method is reliable for quantitation of brimonidine in ocular tissues and fluids.

Nonclinical safety and pharmacokinetics of intravitreally administered human-derived plasmin in rabbits and minipigs.

The use of plasmin for pharmacologic vitreolysis and the creation of a posterior vitreous detachment offers several potential advantages over surgery. The nonclinical pharmacokinetics and safety of human-derived plasmin was evaluated following single or multiple intravitreal injections to rabbits and minipigs. Single intravitreal injections of plasmin at 45-900 microg resulted in a no adverse effect level (NOAEL) of 45 microg in both species; effects at higher doses included chemosis, mucopurulent discharge, mononuclear cell infiltrates in the iris-ciliary body, and reversible changes in electroretinogram waveforms and parameters. No retinal histopathology abnormalities were observed. Following 4 weekly intravitreal injections at 4-423 microg, a NOAEL of 4 microg was identified. Effects at the higher doses included myosis, iritis, iridolenticular synechiae, and changes in electroretinogram waveforms and parameters that were generally not reversible in the present investigation. Vitreal plasmin concentrations were highest at 30 min after dosing and decreased rapidly; measurable concentrations remained, in some animals, at 24 h. Intravitreal plasmin exposure increased in a less-than-dose-proportional manner and tended to be lower in minipigs than in rabbits. The current findings demonstrate acceptable nonclinical safety and pharmacokinetics of intravitreal human plasmin in rabbits and minipigs and support the clinical development of plasmin for ocular diseases.

Quantitative determination of besifloxacin, a novel fluoroquinolone antimicrobial agent, in human tears by liquid chromatography-tandem mass spectrometry.

A rapid and sensitive method was developed using high-performance liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the quantification of besifloxacin in human tears using sparfloxacin as the internal standard (IS). Besifloxacin was extracted from human tear samples using an ammonium formate buffer at pH 3.25. The method was validated over a concentration range of 2-2000 ng/mL, with a total run time of less than 4 min. The overall intra- and inter-day precision for this method was less than 6%. The method was used to measure besifloxacin concentrations in tear samples collected after topical ocular administration to humans; besifloxacin concentrations were 610+/-540 microg/g (15 min) and 1.60+/-2.28 microg/g (24h).

Cassette dosing pharmacokinetic studies for evaluation of ophthalmic drugs for posterior ocular diseases.

The purpose of this investigation was to evaluate the utility of cassette dosing as a means for increasing throughput and decreasing animal usage for intravitreal ocular pharmacokinetic studies. Pigmented rabbits received a single intravitreal injection of test article containing either a single compound or a mixture of up to five compounds. Samples of vitreous, choroid and retina were collected at predetermined intervals through 7 or 28 days after dosing. Concentrations of each compound were determined by LC/MS/MS, with subsequent pharmacokinetic data analysis. The ocular pharmacokinetic properties of four test compounds administered as a cassette were in agreement with the ocular pharmacokinetics of each compound when administered as a single entity. Cassette dosing was subsequently used to screen an additional 15 compounds, with injection of 5 compounds per study. Based on the results from these cassette-dosing studies, some compounds demonstrated favorable ocular pharmacokinetics, with sustained concentrations above 300 ng/g in retina for at least 1 week after dosing while other compounds showed either considerably less penetration into retina or a shorter residence time in the retina. These findings suggest that the cassette dosing approach can be used in evaluating the intravitreal ocular pharmacokinetic properties of compounds intended for ocular use.

Development of an in vivo preclinical screen model to estimate absorption and first-pass hepatic extraction of xenobiotics. II. Use of ketoconazole to identify P-glycoprotein/CYP3A-limited bioavailability in the monkey.

The effect of P-glycoprotein (Pgp) and/or CYP3A on the disposition of xenobiotics has been extensively investigated and is often of interest during drug discovery lead optimization. We have previously described a monkey pharmacokinetic screen to rapidly estimate absorption and first-pass extraction. In the present work, this monkey screen has been expanded to include an assessment of Pgp/CYP3A effects on absorption and first-pass extraction, using ketoconazole as a prototypic dual Pgp/CYP3A inhibitor. To generate a ketoconazole dosing regimen, the pharmacokinetics of ketoconazole were first determined in the monkey and were found to be consistent with that previously described in the rat, dog, and human. Dose-ranging experiments demonstrated that a single 10-mg/kg intraduodenal ketoconazole dose would provide an appropriate exposure; this dose was used throughout subsequent interaction experiments. Next, erythromycin and propranolol were explored as positive and negative control substrates for Pgp/CYP3A interactions, respectively. As anticipated, ketoconazole produced no change in the absorption or first-pass extraction of propranolol but resulted in a substantial increase in absorption and decrease in first-pass extraction of erythromycin. Finally, this ketoconazole-based monkey screen was deployed in a drug discovery setting, and examples of such use are presented. These experiments have allowed a more complete characterization of ketoconazole as a prototypic dual Pgp/CYP3A inhibitor and its use as a tool in a preclinical setting and further demonstrate the use of the monkey to investigate the role of Pgp/CYP3A in limiting the oral bioavailability of new drug candidates.

A single dose of monoclonal anti-phencyclidine IgG offers long-term reductions in phencyclidine behavioral effects in rats.

These studies tested the hypothesis that a single dose of high-affinity anti-phencyclidine monoclonal antibody (anti-PCP mAb) provides long-term protection against behavioral effects of repeated PCP administration in rats. Rats were treated with saline, nonspecific bovine IgG (NS-IgG), or anti-PCP mAb (1.0 g/kg). The next morning, the rats were challenged with escalating i.v. doses of PCP (0.32, 0.56, and 1.0 mg/kg) at 90-min intervals. This regimen was repeated every 3 days for 2 weeks. In the saline and NS-IgG control groups, PCP yielded reproducible and linear dose-dependent effects that remained constant during the experiment. In contrast, the anti-PCP mAb treatment blocked PCP effects on day 1, and sustained significant (P < 0.05) reductions in drug effects for the entire 2-week experiment. Brain PCP concentrations (determined at study termination) were reduced by ~55%, whereas serum concentrations were increased over 4000% compared with controls. Thus, a single dose of antibody medication provided long-term reductions in drug effects and brain concentrations, beyond the expected capacity of the drug-antibody interaction. These data challenge current concepts about in vivo dose dependence and unimolecular interaction between antibody binding sites and small molecules and establish that neuroprotection by mAbs may have an unique mechanism of action.