Lymphatic transport and catabolism of therapeutic proteins after subcutaneous administration to rats and dogs.

The mechanism underlying subcutaneous absorption of macromolecules and factors that can influence this process were studied in rats using PEGylated erythropoietins (EPOs) as model compounds. Using a thoracic lymph duct cannulation (LDC) model, we showed that PEGylated EPO was absorbed from the subcutaneous injection site mainly via the lymphatic system in rats, which is similar to previous reports in sheep. After subcutaneous administration, the serum exposure was reduced by ∼70% in LDC animals compared with that in the control animals, and most of the systemically available dose was recovered in the lymph. In both LDC and intact rats, the total radioactivity recoveries in excreta after subcutaneous administration were high (70-80%), indicating that catabolism, not poor absorption, was the main cause for the observed low bioavailability (30-40%). Moreover, catabolism of PEGylated EPO was found with both rat subcutaneous tissue homogenate and lymph node cell suspensions, and a significant amount of dose-related breakdown fragments was found in the lymph of LDC rats. In addition, the bioavailability of PEGylated EPOs was shown to be 2- to 4-fold lower in "fat rats," indicating that physiologic features pertinent to lymphatic transport can have a profound impact on subcutaneous absorption. Limited studies in dogs also suggested similar subcutaneous absorption mechanisms. Collectively, our results suggest that the lymphatic absorption mechanism for macromolecules is probably conserved among commonly used preclinical species, e.g., rats and dogs, and that mechanistic understanding of the subcutaneous absorption mechanism and associated determinants should be helpful in biologic drug discovery and development.

An in vivo microdialysis coupled with liquid chromatography/tandem mass spectrometry study of cortisol metabolism in monkey adipose tissue.

It is postulated that elevated tissue concentrations of cortisol may be associated with the development of metabolic syndrome, obesity, and type 2 diabetes. The 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) enzyme regenerates cortisol from inactive cortisone in tissues such as liver and adipose. To better understand the pivotal role of 11beta-HSD1 in disease development, an in vivo microdialysis assay coupled with liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis using stable isotope-labeled (SIL) cortisone as a substrate was developed. This assay overcomes the limitations of existing methodologies that suffer from radioactivity exposure and analytical assay sensitivity and specificity concerns. Analyte extraction efficiencies (E(d)) were evaluated by retrodialysis. The conversion of SIL-cortisone to SIL-cortisol in rhesus monkey adipose tissue was studied. Solutions containing 100, 500, and 1000 ng/mL SIL-cortisone were locally delivered through an implanted 30-mm microdialysis probe in adipose tissue. At the delivery rate of 1.0 and 0.5 microL/min, E(d) values for SIL-cortisone were between 58.7+/-5.6% (n=4) and 72.7+/-1.3% (n=4), whereas at 0.3 microL/min E(d) reached nearly 100%. The presence of 11beta-HSD1 activities in adipose tissue was demonstrated by production of SIL-cortisol during SIL-cortisone infusion. This methodology could be applied to cortisol metabolism studies in tissues of other mammalian species.

Population pharmacokinetic modeling for enterohepatic recirculation in Rhesus monkey.

Enterohepatic recirculation (EHR) occurs via biliary excretion and intestinal reabsorption of a drug. Drug recycling through EHR can lead to a change in pharmacokinetic (PK) properties, such as reduced clearance (CL), extended half-life (T(1/2)) and increased plasma exposure (AUC). As a result, EHR may prolong the pharmacological effect of drugs. In the present study, the compound (Cpd A) was found to exhibit EHR in Rhesus monkeys associated with a reduction in CL (from 3.8 to 0.33 Lh(-1), IV; from 2.3 to 0.4 Lh(-1), PO), and an increase in T(1/2) (from 0.9 to 18 h, IV) and in AUC (from 1.5 to 17.4 microg h/mL, IV; from 2.8 to 16.3 microg h/mL, PO), by comparing the PK in the monkeys via the interruption of EHR (bile-duct cannulation) with that in the intact monkeys. A population four-compartment model was constructed based on recirculation loops incorporating all possible inputs (bile secretion, a lag-time model for gall bladder emptying, routes and amounts of a single dose administration) to fully evaluate the EHR of Cpd A. The plasma concentrations versus time profiles predicted from the model had a good fit to the values observed in the subjects and were further simulated with 90% confidence interval to demonstrate its utility. Thus, the model could be applied as a useful tool to evaluate the drugs or compounds that undergo EHR in different species.

Cardiac implantable electronic device infections: incidence, risk factors, and the effect of the AigisRx antibacterial envelope.

BACKGROUND: Cardiac implantable electronic device (CIED) infection is associated with morbidity and mortality. OBJECTIVES: To determine the incidence and risk factors for CIED infection, to develop a scoring index for risk stratification, and to analyze the effect of the AIGISRx envelope on infection rates. METHODS: Consecutive patients who underwent a CIED procedure were identified and surveyed for 6 months for the development of an infection necessitating removal of all implanted hardware. RESULTS: In the pre-envelope era, an infection occurred in 25 (1.5%) of 1651 patients. After its availability, an envelope was used in 275 (22%) of 1240 patients; an infection occurred in 8 (0.6%) patients in this era (P = .029 vs pre-envelope). In the overall cohort of 2891 patients, the infection rate was 1.2% and 3.5% in patients with an implantable cardioverter-defibrillator device and those with a cardiac resynchronization therapy defibrillator device, respectively (P = .018); in these patients, 7 independent risk factors predicted infection: early pocket re-exploration, male sex, diabetes, upgrade procedure, heart failure, hypertension, and glomerular filtration rate < 60 mL/min. A composite risk score (0-25; C index 0.72; 95% confidence interval 0.61-0.83) was created by weight, adjusting these 7 factors: 3 groups emerged-low risk (score 0-7; 1% infection), medium risk (score 8-14; 3.4% infection), and high risk (score ≥15; 11.1% infection). The envelope reduced infections by 79% and 100% in the medium- and high-risk groups, respectively. CONCLUSIONS: CIED infection most commonly occurred in patients with an implantable cardioverter-defibrillator device and those with a cardiac resynchronization therapy defibrillator device. A composite score based on clinical variables appeared feasible for infection risk stratification. The AIGISRx envelope significantly lowered the risk of CIED infection. Randomized clinical data are warranted.

Characterization of ischemic index using ultra-widefield fluorescein angiography in patients with focal and diffuse recalcitrant diabetic macular edema.

PURPOSE: To explore the association of angiographic nonperfusion in focal and diffuse recalcitrant diabetic macular edema (DME) in diabetic retinopathy (DR). DESIGN: A retrospective, observational case series of patients with the diagnosis of recalcitrant DME for at least 2 years placed into 1 of 4 cohorts based on the degree of DR. METHODS: A total of 148 eyes of 76 patients met the inclusion criteria at 1 academic institution. Ultra-widefield fluorescein angiography (FA) images and spectral-domain optical coherence tomography (SD OCT) images were obtained on all patients. Ultra-widefield FA images were graded for quantity of nonperfusion, which was used to calculate ischemic index. Main outcome measures were mean ischemic index, mean change in central macular thickness (CMT), and mean number of macular photocoagulation treatments over the 2-year study period. RESULTS: The mean ischemic index was 47% (SD 25%; range 0%-99%). The mean ischemic index of eyes within Cohorts 1, 2, 3, and 4 was 0%, 34% (range 16%-51%), 53% (range 32%-89%), and 65% (range 47%-99%), respectively. The mean percentage decrease in CMT in Cohorts 1, 2, 3, and 4 were 25.2%, 19.1%, 11.6%, and 7.2%, respectively. The mean number of macular photocoagulation treatments in Cohorts 1, 2, 3, and 4 was 2.3, 4.8, 5.3, and 5.7, respectively. CONCLUSIONS: Eyes with larger areas of retinal nonperfusion and greater severity of DR were found to have the most recalcitrant DME, as evidenced by a greater number of macular photocoagulation treatments and less reduction in SD OCT CMT compared with eyes without retinal nonperfusion. Areas of untreated retinal nonperfusion may generate biochemical mediators that promote ischemia and recalcitrant DME.

Interconversion pharmacokinetics of simvastatin and its hydroxy acid in dogs: effects of gemfibrozil.

PURPOSE: To characterize the pharmacokinetics of simvastatin (SV) and simvastatin acid (SVA), a lactone-acid pair known to undergo reversible metabolism, and to better understand mechanisms underlying pharmacokinetic interactions observed between SV and gemfibrozil. METHODS: Pharmacokinetic studies were conducted after intravenous administration of SV and SVA to dogs pretreated with a vehicle or gemfibrozil. In vitro metabolism of SVA in dog hepatocytes as well as in vitro hepatic and plasma conversion of SV/SVA were investigated in the absence and presence of gemfibrozil. RESULTS: In control animals, the irreversible elimination clearances of SV (CL10) and SVA (CL20) were 10.5 and 18.6 ml min(-1) kg(-1), respectively. The formation clearance of SVA from SV (CL12 = 4.8 ml min(-1) kg(-1)) was 8-fold greater than that of SV from SVA (CL21 = 0.6 ml min(-1) kg(-1)), and the recycled fraction was relatively minor (0.009). In gemfibrozil-treated animals, CL10 was essentially unchanged, whereas CL12, CL20, CL21, and recycled fraction were significantly decreased to 2.9, 9, 0.14 ml min(-1) kg(-1), and 0.003, respectively. In control dogs, values for real volume of distribution at steady state (Vss,real) of SV (2.3 L kg(-1)) were much larger than the corresponding values of SVA (0.3 L kg(-1)). Gemfibrozil treatment did not affect Vss,real of either SV or SVA. In dog hepatocytes, gemfibrozil modestly affected the formation of CYP3A-mediated oxidative metabolites (IC50 > 200 microM) and beta-oxidative products (IC5) approximately 100 microM), but markedly inhibited the glucuronidation-mediated lactonization of SVA and the glucuronidation of an SVA beta-oxidation product (IC50 = 18 microM). In in vitro dog and human liver S9 and plasma, hydrolysis of SV to SVA was much faster than that of SVA to SV. Gemfibrozil (250 microM) had a minimal inhibitory effect on the hydrolysis of either SV to SVA or SVA to SV in dog and human liver S9, but had a significant ( approximately 60%) inhibitory effect on the SV to SVA hydrolysis in both dog and human plasma. CONCLUSIONS: In dogs, the interconversion process favored the formation of SVA and was less efficient than the irreversible elimination processes of SV and SVA. Treatment with gemfibrozil did not affect the distribution of SV/SVA, but rather affected the elimination of SVA and the SV/SVA interconversion processes. Gemfibrozil decreased CL20 and CL21 likely via its inhibitory effect on the glucuronidation of SVA, and not on the CYP3A-mediated oxidative metabolism of SV or SVA, the beta-oxidation of SVA, nor the SVA to SV hydrolysis. The decrease in CL12 might be due in part to the inhibitory effect of gemfibrozil on SV to SVA hydrolysis in plasma. Similar rationales may also be applicable to studies in humans and/or other statin lactone-acid pairs.

Induction of CYP1A in the beagle dog by an inhibitor of kinase insert domain-containing receptor: differential effects in vitro and in vivo on mRNA and functional activity.

Compound I [3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one] is a potent inhibitor of human kinase insert domain-containing receptor (KDR kinase), which is under investigation for the treatment of cancer. Bile duct-cannulated male beagle dogs were administered 6 mg/kg compound I q.d. for 14 days. There was an approximately 2.5-fold decrease in the mean plasma area under the curve of I on days 7 and 14 (approximately 11.3 microM . h), relative to day 1 (28.2 microM . h). In the dog, compound I was eliminated by metabolism, with a major pathway being aromatic hydroxylation and subsequent sulfation to form the metabolite M3. Metabolic profiling suggested that the pathway leading to the formation of the sulfated conjugate M3 was induced upon multiple dosing of I. Studies conducted in vitro suggested that CYP1A1/2 was responsible for the formation of the hydroxylated metabolite, which is sulfated to yield M3. Additional studies confirmed induction of CYP1A protein and activity in the livers of dogs treated with I. However, studies in a dog hepatocyte model of induction showed a surprising decrease both in CYP1A mRNA and enzymatic activity in the presence of I, emphasizing the need to consider the results from a variety of in vitro and in vivo studies in deriving an understanding of the metabolic fate of a drug candidate. It is concluded that the autoinduction observed after multiple treatments with compound I occurs since compound I is both an inducer and a substrate for dog CYP1A.