High-Performance Liquid Chromatography Method for Rich Pharmacokinetic Sampling Schemes in Translational Rat Toxicity Models With Vancomycin.

A translational need exists to understand and predict vancomycin-induced kidney toxicity. We describe: (i) a vancomycin high-performance liquid chromatography (HPLC) method for rat plasma and kidney tissue homogenate; (ii) a rat pharmacokinetic (PK) study to demonstrate utility; and (iii) a catheter retention study to enable future preclinical studies. Rat plasma and pup kidney tissue homogenate were analyzed via HPLC for vancomycin concentrations ranging from 3-75 and 15.1-75.5 µg/mL, respectively, using a Kinetex Biphenyl column and gradient elution of water with 0.1% formic acid: acetonitrile (70:30 v/v). Sprague-Dawley rats (n = 10) receiving 150 mg/kg of vancomycin intraperitoneally had plasma sampled for PK. Finally, a catheter retention study was performed on polyurethane catheters to assess adsorption. Precision was <6.1% for all intra-assay and interassay HPLC measurements, with >96.3% analyte recovery. A two-compartment model fit the data well, facilitating PK exposure estimates. Finally, vancomycin was heterogeneously retained by polyurethane catheters.

Endothelin-1 levels and renal function in newborns of various gestational ages.

BACKGROUND: Renal failure is common in the NICU; Acute Kidney Injury (AKI) occurs in 8-24% of admissions. Although AKI is preventable with early diagnosis, no reliable AKI biomarkers exist. Endothelin-1 (ET-1) has been implicated in renal pathogenesis, and elevated urinary ET-1 (uET-1) levels may correlate with progression of renal dysfunction. The study objectives were to determine whether uET-1 levels correlate with renal function parameters and/or fetal growth restriction, and if uET-1 is a potential neonatal AKI biomarker. METHODS: Sixty-three neonates were enrolled and divided into gestational age (GA) groups by weeks: 1) (24-30 6/7; n = 24); 2) (31-36 6/7; n = 26); and 3) (37-42; n = 13). Additional preterm subgroups for fetal growth restriction analysis included: 1) Appropriate for GA (AGA; n = 40), and 2) Small for GA (SGA; n = 10). ET-1 levels, measured using enzyme linked immunosorbent assay, were collected at birth (cord blood) and 24 h ( ± 4) of life (blood/urine). RESULTS: No correlation was found between uET-1 and blood plasma levels at birth (r = 0.15; p > 0.05) or 24 h (r = 0.17; p > 0.05). uET-1 negatively correlated with GA (r = -0.44; p < 0.001) and GFR (r = -0.34; p < 0.01). uET-1 levels did not correlate with creatinine (r = 0.13; p > 0.05), BUN (r = 0.19; p > 0.05), BUN/Cr ratio (r = 0.15; p > 0.05), or urinary output (r = 0.12; p > 0.05). In fetal growth restriction subgroup analyses: uET-1 levels negatively correlated with GFR in the PT-AGA subgroup (r = -0.38; p = 0.017), but not with PT-SGA (r = 0.01; p > 0.05). CONCLUSION: Plasma and uET-1 levels did not correlate; therefore, renal ET-1 excretion may reflect renal ET-1 production. uET-1 levels correlated negatively with GA and GFR. uET-1 may be a marker of impaired neonatal circulatory regulation and consequent renal injury.

Evaluation of liposomal nanocarriers loaded with ETB receptor agonist, IRL-1620, using cell-based assays.

One common feature of most neurodegenerative diseases, including Alzheimer's disease (AD) and stroke, is the death of neuronal cells. Neuronal cell death is associated with apoptosis, generation of reactive oxygen species and oxidative stress. Neuronal cell death pathways can be reversed by endothelin B receptor agonist, IRL-1620, which was found to enhance neuroprotection by promoting vascular and neuronal growth in a rodent stroke model. Previous studies conducted at our institution indicated that the treatment with IRL-1620 significantly improved neurological and motor function while reducing oxidative stress and overall infarct area. IRL-1620 is a hydrophilic, 15 amino acid peptide and has a molecular weight of 1820Da. In this study, we have encapsulated IRL-1620 in PEGylated liposomes in order to enhance its efficacy. Each batch of liposomes encapsulating IRL-1620 was evaluated for particle size, polydispersity index, and charge (zeta potential) over a period of time to determine their stability. A dose-response bar graph was plotted based on the effect of neuroprotection by free IRL-1620 on differentiated neuronal PC-12 cells. The 1nM concentration was found to have the highest cell viability. The liposomes loaded with IRL-1620 were tested on differentiated neuronal PC-12 cells for their neuroprotective ability against apoptosis caused by removal of nerve growth factor (NGF) against free (non-encapsulated) IRL-1620. The liposomal IRL-1620 was found to proliferate the growth of serum-deprived differentiated PC-12 cells significantly (p<0.0001). In the western blot analysis, the expression of the anti-apoptotic marker, BCL-2 was found to be increased, and that of pro-apoptotic marker, BAX was found to be decreased with liposomal IRL-1620. The effects were found to be independent of the NGF levels. Finally the free IRL-1620 was found to cause neuronal outgrowth equivalent to the 75ng/ml NGF treatment.

Utilization of a peptide lead for the discovery of a novel PTP1B-binding motif.

Examination of the PTP1B inhibitory potency of an extensive series of phosphotyrosyl (pTyr) mimetics (Xxx) expressed in the EGFr-derived hexapeptide platform Ac-Asp-Ala-Asp-Xxx-Leu-amide previously led to the finding of high inhibitory potency when Xxx = 4-(phosphonodifluoromethyl)phenylalanyl (F2Pmp) (K(i) = 0.2 microM) and when Xxx = 3-carboxy-4-carboxymethyloxyphenylalanyl (K(i) = 3.6 microM). In the first instance, further work led from the F2Pmp-containing peptide to monomeric inhibitor, 6-(phosphonodifluoromethyl)-2-naphthoic acid (K(i) = 22 microM), and to the pseudo-dipeptide mimetic, N-[6-(phosphonodifluoromethyl)-2-naphthoyl]-glutamic acid (K(i) = 12 microM). In the current study, a similar approach was applied to the 3-carboxy-4-carboxymethyloxyphenylalanyl-containing peptide, which led to the preparation of monomeric 5-carboxy-6-carboxymethyloxy-2-naphthoic acid (K(i) = 900 microM). However, contrary to expectations based on the aforementioned F2Pmp work, incorporation of this putative pTyr mimetic into the pseudo-dipeptide, N-[5-carboxy-6-carboxymethyloxy-2-naphthoyl]-glutamic acid, resulted in a substantial loss of binding affinity. A reevaluation of binding orientation for 5-carboxy-6-carboxymethyloxy-2-naphthoic acid was therefore undertaken, which indicated a 180 degrees reversal of the binding orientation within the PTP1B catalytic site. In the new orientation, the naphthyl 2-carboxyl group, and not the o-carboxy carboxymethyloxy groups, mimics a phosphoryl group. Indeed, when 5-carboxy-2-naphthoic acid itself was examined at neutral pH for inhibitory potency, it was found to have K(i) = 31 +/- 7 microM, which is lower than parent 5-carboxy-6-carboxymethyloxy-2-naphthoic acid. In this fashion, 5-carboxy-2-naphthoic acid (or more appropriately, 6-carboxy-1-naphthoic acid) has been identified as a novel PTP1B binding motif.