Patricia Brown-Augsburger

Anabolic and catabolic bone effects of human parathyroid hormone (1-34) are predicted by duration of hormone exposure

Parathyroid hormone (PTH)(1-34), given once daily, increases bone mass in a variety of animal models and humans with osteoporosis. However, continuous PTH infusion has been shown to cause bone loss. To determine the pharmacokinetic profile of PTH(1-34) associated with anabolic and catabolic bone responses, PTH(1-34) pharmacokinetic and serum biochemical profiles were evaluated in young male rats using dosing regimens that resulted in either gain or loss of bone mass. Once-daily PTH(1-34) or 6 PTH(1-34) injections within 1 h, for a total daily dose of 80 microg/kg, induced equivalent increases in proximal tibia bone mass. In contrast, 6 PTH(1-34) injections/day over 6 h for a total dose of 80 microg/kg/day or 3 injections/day over 8 h for a total of 240 microg/kg/day decreased tibia bone mass. The PTH(1-34) pharmacokinetics of the different treatment regimens were distinctive. The magnitude of the maximum serum concentrations (Cmax) of PTH(1-34) and area under the curve (AUC) did not predict the catabolic bone outcome. Compared to the anabolic pharmacokinetic profile of a transient increase in PTH(1-34) with rapid decreases in serum calcium and phosphate, the catabolic regimen was associated with PTH(1-34) concentrations remaining above baseline values during the entire 6-h dosing period with a trend toward an increase in serum calcium and a prolonged decrease in phosphate. The pharmacokinetic profiles suggest that the anabolic or catabolic response of bone to PTH(1-34) is determined primarily by the length of time each day that serum concentrations of PTH(1-34) remain above baseline levels of endogenous PTH and only secondarily by the Cmax or AUC of PTH(1-34) achieved.

Demonstrating the intrinsic ion channel activity of virally encoded proteins

This review summarizes the types of evidence that can be invoked in order to demonstrate that a virally encoded protein possesses ion channel activity that is intrinsic to the life cycle of the virus. Ion channel activity has been proposed to be a key step in the life cycle of influenza virus, and the protein responsible for this activity has been proposed to be the M2 protein encoded by the virus. This review contrasts the evidence supporting the conclusion that the A/M2 protein of influenza A virus has intrinsic ion channel activity with the evidence that the 3AB protein encoded by the human rhinovirus possesses intrinsic ion channel activity.

Bioequivalence and comparative pharmacodynamics of insulin lispro 200 U/mL relative to insulin lispro (Humalog®) 100 U/mL

Insulin lispro 200 U/mL (IL200) is a new strength formulation of insulin lispro (Humalog®, IL100), developed as an option for diabetic patients on higher daily mealtime insulin doses. This phase 1, open‐label, 2‐sequence, 4‐period crossover, randomized, 8‐hour euglycemic clamp study aimed to demonstrate the bioequivalence of IL200 and IL100 after subcutaneous administration of 20 U (U) to healthy subjects (n = 38). Pharmacokinetic (PK) and pharmacodynamic (PD) responses were similar in both formulations. All 90%CIs for the ratios of area under the concentration‐versus‐time curve from time zero to the time of the last measurable concentration (AUC0–tlast) and maximum observed drug concentration (Cmax), as well as the total glucose infused throughout the clamp (Gtot) and the maximum glucose infusion rate (Rmax), were contained within 0.80 and 1.25. Time of maximum observed drug concentration (tmax) was similar between formulations, with a median difference of 15 minutes and a 95%CI of the difference that included zero. Inter‐ and intrasubject variability estimates were similar for both formulations. Both formulations were well tolerated. IL200 was bioequivalent to IL100 after subcutaneous administration of 20‐U single doses, and PD responses were comparable between formulation strengths.

Development of an ion-pair reverse-phase liquid chromatographic/tandem mass spectrometry method for the determination of an 18-mer phosphorothioate oligonucleotide in mouse liver tissue.

A quantitative method for the determination of a partially modified, 2′-ribose alkoxy 18-mer phosphorothioate oligonucleotide in liver tissue has been developed. A liquid:liquid extraction, ion-pair reverse phase chromatographic separation and tandem mass spectrometry were used to achieve a quantitation range of 125 to 10,000 ng g−1 mouse liver tissue. A total cycle time of 5 min was obtained while maintaining separation of three potential impurities. Separations were performed using a Discovery RP-Amide C16, 100 × 2 mm column packed with 5 μm particles. The separation was facilitated by the use of triethylamine (TEA) and hexafluoroisopropanol (HFIP) as ion-pair agents. The method has subsequently been used for the determination of other phosphorothioate oligonucleotides in support of discovery research.

Development of a Sensitive and Specific in Situ Hybridization Technique for the Cellular Localization of Antisense Oligodeoxynucleotide Drugs in Tissue Sections

A sensitive method has been developed for the identification and assessment of phosphorothioate oligonucleotide accumulation in dosed animal tissues using an in situ hybridization approach, which is both sequence specific yet adaptable to every antisense oligonucleotide (ASO), which has been tested to date. Hybridization is accomplished using a digoxigenin-tailed oligonucleotide probe complementary to the ASO target sequence on routinely processed paraffin sections which have been pretreated with a mild target retrieval solution. The DIG-labeled probe is amplified first with an anti-DIG:FITC antibody conjugate followed by an anti:FITC Alexa 488 antibody, then visualized using FITC epifluorescence microscopy. Fluorescent labeling of ASO drug in tissue sections by this method confirms that H&E basophilia previously observed in dosed tissues represents largely intact ASO. However, the fluorescent method enables a wider assessment of tissue distribution in a variety of tissue types due to increased sensitivity and lower signal to noise than can be obtained through an examination of H&E stained tissue sections alone.

Subcutaneous Injection Depth Does Not Affect the Pharmacokinetics or Glucodynamics of Insulin Lispro in Normal Weight or Healthy Obese Subjects

Background: An 8-mm needle length is commonly used for insulin injections; however, recent recommendations suggest shorter needles may help patients avoid intramuscular injections and reduce pain, while maintaining adequate glucose control. The goal of these analyses was to compare the pharmacokinetics (PK) and glucodynamics (GD) of insulin lispro after a 5-mm or an 8-mm injection depth administration in 2 populations: normal weight (study 1) or obese (study 2). Methods: In both open-label, randomized, 2-period crossover euglycemic clamp studies, subjects received single 0.25 U/kg insulin lispro doses on 2 occasions (at 5-mm and 8-mm injection depths); samples for PK and GD analyses were collected up to 6 hours postdose. Noncompartmental PK parameters AUC0-tlast, AUC0-∞, Cmax and GD parameters Gtot, Rmax, tRmax were log-transformed prior to analysis using a mixed effects model. Results: There were no apparent differences between PK profiles at the 5-mm or 8-mm injection depth in either study, demonstrated by the ratios of geometric means of AUC0-tlast, AUC0-∞, and Cmax being close to 1, with 90% confidence intervals (CI) within (0.80, 1.25). There were no apparent differences between GD profiles at either injection depth with the ratios of Gtot and Rmax near unity and 90% CIs that included 1. In both studies, the tRmax values were similar between injection depths, with a small median of pairwise differences and a 90% CI that included zero. Conclusions: Injection depths in the 5-8 mm range did not affect the PK or GD of insulin lispro in normal weight or obese subjects.

Disposition of Basal Insulin Peglispro Compared with 20-kDa Polyethylene Glycol in Rats Following a Single Intravenous or Subcutaneous Dose

Basal insulin peglispro (BIL) comprises insulin lispro covalently bound to a 20-kDa polyethylene glycol (PEG) at lysine B28. The biologic fate of BIL and unconjugated PEG were examined in rats given a single 0.5-mg/kg i.v. or 2-mg/kg s.c. dose of BIL with 14C label in 20-kDa PEG or 125I label in lispro. Unconjugated 14C-labeled 20-kDa PEG was dosed at 10 mg/kg i.v. or s.c. Blood, urine, and feces were collected up to 336 hours after dosing. Radioactivity was measured by scintillation spectrometry, and BIL was quantitated by enzyme-linked immunosorbent assay. Area under the curve and half-life for immunoreactive BIL were lower than those for both 14C and 125I after subcutaneous and intravenous administration. The half-lives of 14C after BIL and PEG dosing were similar. The clearance of immunoreactive BIL was 2.4-fold faster than that of 14C and 1.6-fold faster than 125I. After a subcutaneous dose of BIL, immunoreactive BIL accounted for 31% of the circulating 125I and 16% of the circulating 14C, indicating extensive catabolism of BIL. Subcutaneous bioavailability of BIL was 23%–29%; bioavailability for unconjugated PEG was 78%. For unconjugated PEG, most of the 14C dose was recovered in urine. For BIL, ≥86% of 125I was eliminated in urine and 14C was eliminated about equally in urine and feces. The major 14C-labeled catabolism product of BIL in urine was 20-kDa PEG with lysine attached. The attachment of 20-kDa PEG to lispro in BIL led to a different elimination pathway for PEG compared with unconjugated 20-kDa PEG.

An immunoadsorption strategy to produce specific antisera against analogs of human proteins: development of sensitive and specific radioimmunoassays for two analogs of human leptin.

Immunoassay technology is routinely used to measure concentrations of proteins and polypeptides in biological matrices. Increasingly, research efforts have sought to create analogs of human proteins with the aim of improving efficacy or pharmaceutical properties relative to the native protein. Pharmacokinetic assessment of these polypeptide analogs, however, can be greatly confounded by the presence of endogenous native protein. This report describes an immunization and immunoabsorption strategy that was used to create monospecific polyclonal antibodies against analogs of human leptin (LY355101 and LY396623, one and two amino acid changes relative to native human leptin, respectively). Rabbits were immunized with either LY355101 or LY396623. Antisera were screened to determine if any showed increased specificity for the analog relative to native human leptin. Antisera showing increased specificity for the leptin analog were then treated by immunoabsorption against native human leptin, thus depleting human leptin cross-reactivity. The antibodies developed in this process were used in radioimmunoassays. which were validated for use in clinical studies. Both assays proved to be highly specific for LY355101 or LY396623 in the presence of native human leptin. Use of this procedure permitted the measurement of LY355101 and LY396623 pharmacokinetics that were not confounded by the high levels of endogenous human leptin found in obese subjects. This technique has the potential for broad application in the development of assays capable of specifically measuring protein analogs without cross-reactivity to an endogenous substance.