Extending INR testing intervals in warfarin patients at a multi-center anticoagulation clinic.

Warfarin has been used as an anticoagulant by millions of patients due to its effectiveness, availability, and low cost. Evidence on the safe extension of international normalized ratio (INR) testing frequency remains an area of interest, especially during the recent COVID-19 pandemic. The purpose of this study is to safely extend INR testing intervals in patients throughout a multisite, system-wide anticoagulation clinic. Updates were made to the pharmacist's collaborative practice agreement (CPA) and nurse protocol to optimize practice and allow INR testing interval extension up to a maximum of 8-weeks. The primary outcome was the change in duration between INR tests (INR testing interval) measured before and after providing staff education on clinic updates. The mean duration between INR tests (SD) was 23.69 days (11.29) in the pre-intervention period and 25.58 days (13.91) in the post-intervention period. During the COVID-19 pandemic (post2), intervals were extended further to 27.81 days (14.96), demonstrating a statistically significant increase in INR testing interval from pre-intervention to post-intervention and to post2 (p < 0.001 and p < 0.001, respectively). A secondary outcome indicated the mean time in therapeutic range (SD) showed no significant difference in pre-intervention 70.11% (25.95) versus post-intervention of 69.76% (25.69) with a difference of - 0.35% (29.93) (p = 0.956) or versus the post2 of 68.82% (27.20) with a difference of - 1.29% (33.20) (p = 0.120). This study showed that changes to the CPA and protocol allowed for a significant increase in INR testing interval while simultaneously maintaining a mean time in therapeutic range > 60% for the clinic population.

2,4-Diaminopyrimidine MK2 inhibitors. Part I: Observation of an unexpected inhibitor binding mode.

MK2 is a Ser/Thr kinase of significant interest as an anti-inflammatory drug discovery target. Here we describe the development of in vitro tools for the identification and characterization of MK2 inhibitors, including validation of inhibitor interactions with the crystallography construct and determination of the unique binding mode of 2,4-diaminopyrimidine inhibitors in the MK2 active site. Use of these tools in the optimization of a potent and selective inhibitor lead series is described in the accompanying Letter.

Rational mutagenesis to support structure-based drug design: MAPKAP kinase 2 as a case study.

BACKGROUND: Structure-based drug design (SBDD) can provide valuable guidance to drug discovery programs. Robust construct design and expression, protein purification and characterization, protein crystallization, and high-resolution diffraction are all needed for rapid, iterative inhibitor design. We describe here robust methods to support SBDD on an oral anti-cytokine drug target, human MAPKAP kinase 2 (MK2). Our goal was to obtain useful diffraction data with a large number of chemically diverse lead compounds. Although MK2 structures and structural methods have been reported previously, reproducibility was low and improved methods were needed. RESULTS: Our construct design strategy had four tactics: N- and C-terminal variations; entropy-reducing surface mutations; activation loop deletions; and pseudoactivation mutations. Generic, high-throughput methods for cloning and expression were coupled with automated liquid dispensing for the rapid testing of crystallization conditions with minimal sample requirements. Initial results led to development of a novel, customized robotic crystallization screen that yielded MK2/inhibitor complex crystals under many conditions in seven crystal forms. In all, 44 MK2 constructs were generated, ~500 crystals were tested for diffraction, and ~30 structures were determined, delivering high-impact structural data to support our MK2 drug design effort. CONCLUSION: Key lessons included setting reasonable criteria for construct performance and prioritization, a willingness to design and use customized crystallization screens, and, crucially, initiation of high-throughput construct exploration very early in the drug discovery process.

CD40/anti-CD40 antibody complexes which illustrate agonist and antagonist structural switches.

BACKGROUND: CD40 is a 48 kDa type I transmembrane protein that is constitutively expressed on hematopoietic cells such as dendritic cells, macrophages, and B cells. Engagement of CD40 by CD40L expressed on T cells results in the production of proinflammatory cytokines, induces T helper cell function, and promotes macrophage activation. The involvement of CD40 in chronic immune activation has resulted in CD40 being proposed as a therapeutic target for a range of chronic inflammatory diseases. CD40 antagonists are currently being explored for the treatment of autoimmune diseases and several anti-CD40 agonist mAbs have entered clinical development for oncological indications. RESULTS: To better understand the mode of action of anti-CD40 mAbs, we have determined the x-ray crystal structures of the ABBV-323 (anti-CD40 antagonist, ravagalimab) Fab alone, ABBV-323 Fab complexed to human CD40 and FAB516 (anti-CD40 agonist) complexed to human CD40. These three crystals structures 1) identify the conformational CD40 epitope for ABBV-323 recognition 2) illustrate conformational changes which occur in the CDRs of ABBV-323 Fab upon CD40 binding and 3) develop a structural hypothesis for an agonist/antagonist switch in the LCDR1 of this proprietary class of CD40 antibodies. CONCLUSIONS: The structure of ABBV-323 Fab demonstrates a unique method for antagonism by stabilizing the proposed functional antiparallel dimer for CD40 receptor via novel contacts to LCDR1, namely residue position R32 which is further supported by a closely related agonist antibody FAB516 which shows only monomeric recognition and no contacts with LCDR1 due to a mutation to L32 on LCDR1. These data provide a structural basis for the full antagonist activity of ABBV-323.

Outcomes of oral anticoagulant therapy managed by telephone vs in-office visits in an anticoagulation clinic setting.

BACKGROUND: Anticoagulation management by a dedicated anticoagulation clinic improves patient outcomes compared to routine medical care. Telephone-based anticoagulation management has been described but has not been compared to management with traditional office-based visits. The objective of this study was to compare warfarin-related monitoring outcomes, clinical end points, and the use of health-care resources as a result of warfarin-related complications in anticoagulation clinic patients whose management was conducted by telephone or in-office-based visits. SETTING: Two university-affiliated anticoagulation clinics in Seattle, WA, and Chicago, IL. METHODS: A retrospective, observational cohort design was used to investigate anticoagulation clinic patients who were managed by telephone encounters compared to those managed during face-to-face in-office encounters. RESULTS: A total of 234 patients were evaluated; 117 patients managed by telephone were compared to 117 patients managed in office-based clinic visits. Monitoring outcomes (ie, time in therapeutic range and clinic visits per patient-year) were similar between groups. Differences in major bleeding (5.67% vs 5.62% per patient-year, respectively) and thromboembolic events (1.42% vs 2.81% per patient-year, respectively) between telephone-managed and face-to-face-managed patients did not reach statistical significance. The same was true for differences in the frequency of emergency department visits and hospital admissions to manage complications of warfarin therapy. CONCLUSIONS: Telephone-based management of oral anticoagulation through a pharmacist-staffed anticoagulation clinic yielded clinical outcomes that were at least as favorable as those associated with traditional office-based visits. Telephone follow-up can be successfully used to manage warfarin therapy in patients who are unable to present in person to an anticoagulation clinic.

Resprouting and survival of guinea pig cochlear neurons in response to the administration of the neurotrophins brain-derived neurotrophic factor and neurotrophin-3.

Degeneration of auditory neurons occurs after deafening and is associated with damage to the organ of Corti. The administration of neurotrophins can protect auditory neurons against degeneration if given shortly after deafening. However, it is not known whether the delayed administration of neurotrophins, when significant degeneration has already occurred, will provide similar protection. Furthermore, little is known about the effects of neurotrophins on the peripheral processes of the auditory neurons or whether these neurons can resprout. This study examined the morphological effects on auditory neurons following deafening and the administration of brain-derived neurotrophic factor and neurotrophin-3. Results showed that neurotrophins were effective in preventing death of auditory neurons if administered 5 days after deafening and were also effective in preventing the continued loss of neurons if the administration was delayed by 33 days. The peripheral processes of auditory neurons in cochleae that received neurotrophins were in greater number and had larger diameters compared with the untreated cochleae. Localized regions of resprouting peripheral processes were observed in deafened cochleae and were enhanced in response to neurotrophin treatment, occurring across wider regions of the cochlea. These findings have significant implications for an improvement in the performance of the cochlear implant and for future therapies to restore hearing to the deaf.

Survival of partially differentiated mouse embryonic stem cells in the scala media of the guinea pig cochlea.

The low regenerative capacity of the hair cells of the mammalian inner ear is a major obstacle for functional recovery following sensorineural hearing loss. A potential treatment is to replace damaged tissue by transplantation of stem cells. To test this approach, undifferentiated and partially differentiated mouse embryonic stem (ES) cells were delivered into the scala media of the deafened guinea pig cochlea. Transplanted cells survived in the scala media for a postoperative period of at least nine weeks, evidenced by histochemical and direct fluorescent detection of enhanced green fluorescent protein (EGFP). Transplanted cells were discovered near the spiral ligament and stria vascularis in the endolymph fluid of the scala media. In some cases, cells were observed close to the damaged organ of Corti structure. There was no evidence of significant immunological rejection of the implanted ES cells despite the absence of immunosuppression. Our surgical approach allowed efficient delivery of ES cells to the scala media while preserving the delicate structures of the cochlea. This is the first report of the survival of partially differentiated ES cells in the scala media of the mammalian cochlea, and it provides support for the potential of cell-based therapies for sensorineural hearing impairment.

A single dose of neurotrophin-3 to the cochlea surrounds spiral ganglion neurons and provides trophic support.

Degeneration of spiral ganglion neurons (SGNs) in the cochlea following sensorineural hearing loss is preventable by the infusion of neurotrophins into the scala tympani. This study investigates the trophic effects and distribution of a single bolus infusion of neurotrophin-3 (NT3) into the scala tympani of the cochlea. The left cochleae of 28-day deafened guinea pigs were infused with 0, 100 or 140 ng 125I NT3 via a cochleostomy in the scala tympani of the basal turn. Seven days post-infusion, cochlear sections were processed for measurements of trophic effects on SGNs and autoradiography. A single infusion of NT3 increased the soma size of SGNs in a dose-dependent and significant manner but did not contribute to SGN survival. Following infusion of 140 ng 125I NT3 into the cochlea, 0.31% of the total 125I NT3 signal in the basal turn was detected in Rosenthal's canal, 2.4% was in peripheral processes and 0.35% was in the modiolar auditory nerve. Despite influencing SGN soma size, 125I NT3 was not observed to accumulate in SGN cell bodies. The data suggest that only a small proportion of neurotrophins infused into the scala tympani diffuses to the SGNs and their processes and produces trophic effects on SGN cell bodies.

Delivery of neurotrophin-3 to the cochlea using alginate beads.

OBJECTIVE: The aim of this study was to design a novel cochlear neurotrophin (NT) delivery system for the rescue of auditory neurons after ototoxicity-induced deafening. BACKGROUND: NT-3 is a trophic growth factor that promotes the survival of the auditory nerve and may have a potential therapeutic role in slowing neuron loss in progressive deafness, especially as an adjunct to the current cochlear implant. Beads made from alginate are biodegradable, slow release substances that can be placed at the round window or inside the cochlea. This study investigated the loading properties, release kinetics, and implantation potential of alginate beads loaded with NT-3. METHODS: Alginate beads were prepared using an ionic gelation technique and postloaded with NT-3. Release of NT-3 was measured using enzyme-linked immunosorbent assay over 5 days. Alginate beads were implanted into deafened guinea pigs for 28 days, after which survival of auditory neurons was assessed. RESULTS: Enzyme-linked immunosorbent assay studies demonstrated a 98% to 99% loading of NT-3 with a slow, partial release over 5 days in Ringer's solution. Furthermore, the addition of heparin to the delivery system modulated NT-3 release to a steadier pattern. Implantation of alginate-heparin beads in guinea pig cochleae produced minimal local tissue reaction. NT-3 loaded beads implanted at both the round window and within the scala tympani of the basal turn provided auditory neurons significant protection from degradation and apoptosis compared with unloaded beads or untreated cochleae. CONCLUSIONS: This study demonstrates alginate beads to be a safe, biodegradable and effective delivery system for NT-3 to the cochlea.

Tracing neurotrophin-3 diffusion and uptake in the guinea pig cochlea.

Neurotrophin therapy in the cochlea can potentially slow or reverse the degeneration of the auditory nerve that occurs during progressive deafness. Studies were performed to trace the diffusion and uptake of neurotrophin-3 (NT-3) following infusion into the cochlea. NT-3 labeled with (125)I or coated onto fluorescent microspheres was introduced into the basal turn of normal hearing and deafened guinea pig cochleae via a single slow-rate injection. Cochleae were examined between 2 h and 28 days post-infusion by autoradiography or fluorescent microscopy to determine the number of turns labeled by NT-3, identify individual cells and tissues receiving NT-3 and quantify the proportion of signal in each tissue. In general, long-term infusions were required for all cochlear turns to receive NT-3. (125)I NT-3 signal was strongest in cells lining the perilymphatic space of the scala tympani, basilar membrane, osseous spiral lamina and spiral ligament. Signal in the peripheral nerve tract and Rosenthal's canal was only 1.3-2.1 times background levels of radiation. NT-3 microspheres were detected within neural areas of the cochlea (nerve tract and Rosenthal's canal) in all cases, but not within neuronal cell bodies. NT-3 microspheres remained in the cochlea for at least 28 days, suggesting a low clearance rate within cochlear tissues.

Postmenopausal hormone therapy: a concise guide to therapeutic uses, formulations, risks, and alternatives.

Postmenopausal hormone replacement therapy is helpful in relieving menopausal vasomotor symptoms and vaginal atrophy and can prevent osteoporosis; however, attendant risks include breast cancer, thromboembolism, gallbladder disease, stroke, CHD, dementia, and hypertriglyceridemia. Decision making must weigh these risks and benefits and also include potential benefits on mood, colorectal cancer prevention, and hip fracture reduction. Some areas, such as ovarian cancer risk and the impact of combination estrogen-progestin versus unopposed estrogen on risk, remain unclear. The physician and patient need to carefully assess, discuss, and monitor the individual's symptoms and risks when considering HT use. For those with contraindications or concerns about HT, there are alternative therapies of variable efficacy for vasomotor symptoms and vaginal atrophy.

Influence of gestational age and body weight on the pharmacokinetics of labetalol in pregnancy.

BACKGROUND AND OBJECTIVES: Labetalol is frequently prescribed for the treatment of hypertension during pregnancy; however, the influence of pregnancy on labetalol pharmacokinetics is uncertain, with inconsistent findings reported by previous studies. This study examined the population pharmacokinetics of oral labetalol during and after pregnancy in women receiving labetalol for hypertension. METHODS: Data were collected from 57 women receiving the drug for hypertension from the 12th week of pregnancy through 12 weeks postpartum using a prospective, longitudinal design. A sparse sampling strategy guided collection of plasma samples. Samples were assayed for labetalol by high-performance liquid chromatography. Estimation of population pharmacokinetic parameters and covariate effects was performed by nonlinear mixed effects modeling using NONMEM. The final population model was validated by bootstrap analysis and visual predictive check. Simulations were performed with the final model to evaluate the appropriate body weight to guide labetalol dosing. RESULTS: Lean body weight (LBW) and gestational age, i.e. weeks of pregnancy, were identified as significantly influencing oral clearance (CL/F) of labetalol, with CL/F ranging from 1.4-fold greater than postpartum values at 12 weeks' gestational age to 1.6-fold greater at 40 weeks. Doses adjusted for LBW provide more consistent drug exposure than doses adjusted for total body weight. The apparent volumes of distribution for the central compartment and at steady-state were 1.9-fold higher during pregnancy. CONCLUSIONS: Gestational age and LBW impact the pharmacokinetics of labetalol during pregnancy and have clinical implications for adjusting labetalol doses in these women.

Purification and kinetic characterization of recombinant human mitogen-activated protein kinase kinase kinase COT and the complexes with its cellular partner NF-kappa B1 p105.

Cancer osaka thyroid (COT), a human MAP 3 K, is essential for lipopolysaccharide activation of the Erk MAPK cascade in macrophages. COT 30--467 is insoluble, whereas low levels of COT 30--397 can be expressed, but this protein is unstable. However, both COT 30--467 and COT 30--397 are expressed in a soluble and stable form when produced in complex with the C-terminal half of p105. The k(cat) of COT 30--397 is reduced approximately 47--fold in the COT 30--467/p105 Delta N complex. COT prefers Mn(2+) to Mg(2+) as the ATP metal cofactor, exhibiting an unusually high ATP K(m) in the presence of Mg(2+). When using Mn(2+) as the cofactor, the ATP K(m) is reduced to a level typical of most kinases. In contrast, the binding affinity of COT for its other substrate MEK is cofactor independent. Our results using purified proteins indicate that p105 binding improves COT solubility and stability while down-regulating kinase activity, consistent with cellular data showing that p105 functions as an inhibitor of COT.