A frame-shift mutation in COMTD1 is associated with impaired pheomelanin pigmentation in chicken.

The biochemical pathway regulating the synthesis of yellow/red pheomelanin is less well characterized than the synthesis of black/brown eumelanin. Inhibitor of gold (IG phenotype) is a plumage colour variant in chicken that provides an opportunity to further explore this pathway since the recessive allele (IG) at this locus is associated with a defect in the production of pheomelanin. IG/IG homozygotes display a marked dilution of red pheomelanin pigmentation, whilst black pigmentation (eumelanin) is only slightly affected. Here we show that a 2-base pair insertion (frame-shift mutation) in the 5th exon of the Catechol-O-methyltransferase containing domain 1 gene (COMTD1), expected to cause a complete or partial loss-of-function of the COMTD1 enzyme, shows complete concordance with the IG phenotype within and across breeds. We show that the COMTD1 protein is localized to mitochondria in pigment cells. Knockout of Comtd1 in a mouse melanocytic cell line results in a reduction in pheomelanin metabolites and significant alterations in metabolites of glutamate/glutathione, riboflavin, and the tricarboxylic acid cycle. Furthermore, COMTD1 overexpression enhanced cellular proliferation following chemical-induced transfection, a potential inducer of oxidative stress. These observations suggest that COMTD1 plays a protective role for melanocytes against oxidative stress and that this supports their ability to produce pheomelanin.

Genetic architecture and evolution of color variation in American black bears.

Color variation is a frequent evolutionary substrate for camouflage in small mammals, but the underlying genetics and evolutionary forces that drive color variation in natural populations of large mammals are mostly unexplained. The American black bear, Ursus americanus (U. americanus), exhibits a range of colors including the cinnamon morph, which has a similar color to the brown bear, U. arctos, and is found at high frequency in the American southwest. Reflectance and chemical melanin measurements showed little distinction between U. arctos and cinnamon U. americanus individuals. We used a genome-wide association for hair color as a quantitative trait in 151 U. americanus individuals and identified a single major locus (p < 10-13). Additional genomic and functional studies identified a missense alteration (R153C) in Tyrosinase-related protein 1 (TYRP1) that likely affects binding of the zinc cofactor, impairs protein localization, and results in decreased pigment production. Population genetic analyses and demographic modeling indicated that the R153C variant arose 9.36 kya in a southwestern population where it likely provided a selective advantage, spreading both northwards and eastwards by gene flow. A different TYRP1 allele, R114C, contributes to the characteristic brown color of U. arctos but is not fixed across the range.

A BLOC-1-AP-3 super-complex sorts a cis-SNARE complex into endosome-derived tubular transport carriers.

Membrane transport carriers fuse with target membranes through engagement of cognate vSNAREs and tSNAREs on each membrane. How vSNAREs are sorted into transport carriers is incompletely understood. Here we show that VAMP7, the vSNARE for fusing endosome-derived tubular transport carriers with maturing melanosomes in melanocytes, is sorted into transport carriers in complex with the tSNARE component STX13. Sorting requires either recognition of VAMP7 by the AP-3δ subunit of AP-3 or of STX13 by the pallidin subunit of BLOC-1, but not both. Consequently, melanocytes expressing both AP-3δ and pallidin variants that cannot bind their respective SNARE proteins are hypopigmented and fail to sort BLOC-1-dependent cargo, STX13, or VAMP7 into transport carriers. However, SNARE binding does not influence BLOC-1 function in generating tubular transport carriers. These data reveal a novel mechanism of vSNARE sorting by recognition of redundant sorting determinants on a SNARE complex by an AP-3-BLOC-1 super-complex.

Research Techniques Made Simple: Cell Biology Methods for the Analysis of Pigmentation.

Pigmentation of the skin and hair represents the result of melanin biosynthesis within melanosomes of epidermal melanocytes, followed by the transfer of mature melanin granules to adjacent keratinocytes within the basal layer of the epidermis. Natural variation in these processes produces the diversity of skin and hair color among human populations, and defects in these processes lead to diseases such as oculocutaneous albinism. While genetic regulators of pigmentation have been well studied in human and animal models, we are still learning much about the cell biological features that regulate melanogenesis, melanosome maturation, and melanosome motility in melanocytes, and have barely scratched the surface in our understanding of melanin transfer from melanocytes to keratinocytes. Herein, we describe cultured cell model systems and common assays that have been used by investigators to dissect these features and that will hopefully lead to additional advances in the future.

Strategies to Address Unmet Needs and Facilitate Return to Learn Guideline Adoption Following Concussion.

BACKGROUND: Many students do not receive return to learn (RTL) services upon return to academics following a concussion. METHODS: Using a mixed-methods approach, we conducted a survey of RTL practices and experiences in Washington State schools between January 2015 and June 2015. We then held a statewide summit of RTL stakeholders and used a modified Delphi process to develop a consensus-based RTL implementation model and process. RESULTS: Survey participants included 83 educators, 57 school nurses, 14 administrators, and 30 parents, representing 144 schools in rural and urban areas. Unmet need domains and recommendations identified were (1) a current lack of school policies; (2) barriers to providing or receiving accommodations; (3) wide variability in communication patterns; and (4) recommendations shared by all stakeholder groups (including desire for readily available best practices, development of a formal school RTL policy for easy adoption and more training). Using stakeholder input from RTL summit participants and survey responses, we developed an RTL implementation model and checklist for RTL guideline adoption. CONCLUSIONS: Washington State children have unmet needs upon returning to public schools after concussion. The student-centered RTL model and checklist for implementing RTL guidelines can help schools provide timely RTL services following concussion.

Comparative analysis of human ex vivo-generated platelets vs megakaryocyte-generated platelets in mice: a cautionary tale.

Thrombopoiesis is the process by which megakaryocytes release platelets that circulate as uniform small, disc-shaped anucleate cytoplasmic fragments with critical roles in hemostasis and related biology. The exact mechanism of thrombopoiesis and the maturation pathways of platelets released into the circulation remain incompletely understood. We showed that ex vivo-generated murine megakaryocytes infused into mice release platelets within the pulmonary vasculature. Here we now show that infused human megakaryocytes also release platelets within the lungs of recipient mice. In addition, we observed a population of platelet-like particles (PLPs) in the infusate, which include platelets released during ex vivo growth conditions. By comparing these 2 platelet populations to human donor platelets, we found marked differences: platelets derived from infused megakaryocytes closely resembled infused donor platelets in morphology, size, and function. On the other hand, the PLP was a mixture of nonplatelet cellular fragments and nonuniform-sized, preactivated platelets mostly lacking surface CD42b that were rapidly cleared by macrophages. These data raise a cautionary note for the clinical use of human platelets released under standard ex vivo conditions. In contrast, human platelets released by intrapulmonary-entrapped megakaryocytes appear more physiologic in nature and nearly comparable to donor platelets for clinical application.

Defective release of α granule and lysosome contents from platelets in mouse Hermansky-Pudlak syndrome models.

Hermansky-Pudlak syndrome (HPS) is characterized by oculocutaneous albinism, bleeding diathesis, and other variable symptoms. The bleeding diathesis has been attributed to δ storage pool deficiency, reflecting the malformation of platelet dense granules. Here, we analyzed agonist-stimulated secretion from other storage granules in platelets from mouse HPS models that lack adaptor protein (AP)-3 or biogenesis of lysosome-related organelles complex (BLOC)-3 or BLOC-1. We show that α granule secretion elicited by low agonist doses is impaired in all 3 HPS models. High agonist doses or supplemental adenosine 5'-diphosphate (ADP) restored normal α granule secretion, suggesting that the impairment is secondary to absent dense granule content release. Intravital microscopy following laser-induced vascular injury showed that defective hemostatic thrombus formation in HPS mice largely reflected reduced total platelet accumulation and affirmed a reduced area of α granule secretion. Agonist-induced lysosome secretion ex vivo was also impaired in all 3 HPS models but was incompletely rescued by high agonist doses or excess ADP. Our results imply that (1) AP-3, BLOC-1, and BLOC-3 facilitate protein sorting to lysosomes to support ultimate secretion; (2) impaired secretion of α granules in HPS, and to some degree of lysosomes, is secondary to impaired dense granule secretion; and (3) diminished α granule and lysosome secretion might contribute to pathology in HPS.

The PKD domain distinguishes the trafficking and amyloidogenic properties of the pigment cell protein PMEL and its homologue GPNMB.

Proteolytic fragments of the pigment cell-specific glycoprotein, PMEL, form the amyloid fibrillar matrix underlying melanins in melanosomes. The fibrils form within multivesicular endosomes to which PMEL is selectively sorted and that serve as melanosome precursors. GPNMB is a tissue-restricted glycoprotein with substantial sequence homology to PMEL, but no known function, and was proposed to localize to non-fibrillar domains of distinct melanosome subcompartments in melanocytes. Here we confirm that GPNMB localizes to compartments distinct from the PMEL-containing multivesicular premelanosomes or late endosomes in melanocytes and HeLa cells, respectively, and is largely absent from fibrils. Using domain swapping, the unique PMEL localization is ascribed to its polycystic kidney disease (PKD) domain, whereas the homologous PKD domain of GPNMB lacks apparent sorting function. The difference likely reflects extensive modification of the GPNMB PKD domain by N-glycosylation, nullifying its sorting function. These results reveal the molecular basis for the distinct trafficking and morphogenetic properties of PMEL and GPNMB and support a deterministic function of the PMEL PKD domain in both protein sorting and amyloidogenesis.

SLC35D3 delivery from megakaryocyte early endosomes is required for platelet dense granule biogenesis and is differentially defective in Hermansky-Pudlak syndrome models.

Platelet dense granules are members of a family of tissue-specific, lysosome-related organelles that also includes melanosomes in melanocytes. Contents released from dense granules after platelet activation promote coagulation and hemostasis, and dense granule defects such as those seen in Hermansky-Pudlak syndrome (HPS) cause excessive bleeding, but little is known about how dense granules form in megakaryocytes (MKs). In the present study, we used SLC35D3, mutation of which causes a dense granule defect in mice, to show that early endosomes play a direct role in dense granule biogenesis. We show that SLC35D3 expression is up-regulated during mouse MK differentiation and is enriched in platelets. Using immunofluorescence and immunoelectron microscopy and subcellular fractionation in megakaryocytoid cells, we show that epitope-tagged and endogenous SLC35D3 localize predominantly to early endosomes but not to dense granule precursors. Nevertheless, SLC35D3 is depleted in mouse platelets from 2 of 3 HPS models and, when expressed ectopically in melanocytes, SLC35D3 localizes to melanosomes in a manner requiring a HPS-associated protein complex that functions from early endosomal transport intermediates. We conclude that SLC35D3 is either delivered to nascent dense granules from contiguous early endosomes as MKs mature or functions in dense granule biogenesis directly from early endosomes, suggesting that dense granules originate from early endosomes in MKs.

ESCRT-I function is required for Tyrp1 transport from early endosomes to the melanosome limiting membrane.

Melanosomes are lysosome-related organelles that coexist with lysosomes within melanocytes. The pathways by which melanosomal proteins are diverted from endocytic organelles toward melanosomes are incompletely defined. In melanocytes from mouse models of Hermansky-Pudlak syndrome that lack BLOC-1, melanosomal proteins such as tyrosinase-related protein 1 (Tyrp1) accumulate in early endosomes. Whether this accumulation represents an anomalous pathway or an arrested normal intermediate in melanosome protein trafficking is not clear. Here, we show that early endosomes are requisite intermediates in the trafficking of Tyrp1 from the Golgi to late stage melanosomes in normal melanocytic cells. Kinetic analyses show that very little newly synthesized Tyrp1 traverses the cell surface and that internalized Tyrp1 is inefficiently sorted to melanosomes. Nevertheless, nearly all Tyrp1 traverse early endosomes since it becomes trapped within enlarged, modified endosomes upon overexpression of Hrs. Although Tyrp1 localization is not affected by Hrs depletion, depletion of the ESCRT-I component, Tsg101, or inhibition of ESCRT function by dominant-negative approaches results in a dramatic redistribution of Tyrp1 to aberrant endosomal membranes that are largely distinct from those harboring traditional ESCRT-dependent, ubiquitylated cargoes such as MART-1. The lysosomal protein content of some of these membranes and the lack of Tyrp1 recycling to the plasma membrane in Tsg101-depleted cells suggests that ESCRT-I functions downstream of BLOC-1. Our data delineate a novel pathway for Tyrp1 trafficking and illustrate a requirement for ESCRT-I function in controlling protein sorting from vacuolar endosomes to the limiting membrane of a lysosome-related organelle.

Melanoregulin (MREG) modulates lysosome function in pigment epithelial cells.

Melanoregulin (MREG), the product of the Mreg(dsu) gene, is a small highly charged protein, hypothesized to play a role in organelle biogenesis due to its effect on pigmentation in dilute, ashen, and leaden mutant mice. Here we provide evidence that MREG is required in lysosome-dependent phagosome degradation. In the Mreg(-/-) mouse, we show that loss of MREG function results in phagosome accumulation due to delayed degradation of engulfed material. Over time, the Mreg(-/-) mouse retinal pigment epithelial cells accumulate the lipofuscin component, A2E. MREG-deficient human and mouse retinal pigment epithelial cells exhibit diminished activity of the lysosomal hydrolase, cathepsin D, due to defective processing. Moreover, MREG localizes to small intracellular vesicles and associates with the endosomal phosphoinositide, phosphatidylinositol 3,5-biphosphate. Collectively, these studies suggest that MREG is required for lysosome maturation and support a role for MREG in intracellular trafficking.

Localization to mature melanosomes by virtue of cytoplasmic dileucine motifs is required for human OCA2 function.

Oculocutaneous albinism type 2 is caused by defects in the gene OCA2, encoding a pigment cell-specific, 12-transmembrane domain protein with homology to ion permeases. The function of the OCA2 protein remains unknown, and its subcellular localization is under debate. Here, we show that endogenous OCA2 in melanocytic cells rapidly exits the endoplasmic reticulum (ER) and thus does not behave as a resident ER protein. Consistently, exogenously expressed OCA2 localizes within melanocytes to melanosomes, and, like other melanosomal proteins, localizes to lysosomes when expressed in nonpigment cells. Mutagenized OCA2 transgenes stimulate melanin synthesis in OCA2-deficient cells when localized to melanosomes but not when specifically retained in the ER, contradicting a proposed primary function for OCA2 in the ER. Steady-state melanosomal localization requires a conserved consensus acidic dileucine-based sorting motif within the cytoplasmic N-terminal region of OCA2. A second dileucine signal within this region confers steady-state lysosomal localization in melanocytes, suggesting that OCA2 might traverse multiple sequential or parallel trafficking routes. The two dileucine signals physically interact in a differential manner with cytoplasmic adaptors known to function in trafficking other proteins to melanosomes. We conclude that OCA2 is targeted to and functions within melanosomes but that residence within melanosomes may be regulated by secondary or alternative targeting to lysosomes.

Evaluation of a potential tigecycline-warfarin drug interaction.

STUDY OBJECTIVE: To evaluate the potential for a clinically significant drug interaction between tigecycline and warfarin by using pharmacokinetic and anticoagulant assessments. DESIGN: Open-label, nonrandomized study. SETTING: Inpatient clinical pharmacology unit. SUBJECTS: Nineteen healthy male volunteers were enrolled; eight completed all study assessments. INTERVENTION: All subjects received a single oral dose of warfarin 25 mg (day 1). Seven days later (day 8), they received a 100-mg loading dose of intravenous tigecycline, followed by 50 mg every 12 hours for eight additional doses. On day 12, they received another single oral dose of warfarin 25-mg with their last dose of tigecycline. MEASUREMENTS AND MAIN RESULTS: Serum tigecycline and plasma R- and S-warfarin concentrations were determined by high-performance liquid chromatography with tandem mass spectroscopy. Pharmacokinetic parameters were calculated by using noncompartmental methods and analyzed by the two 1-sided tests equivalence procedure. Pharmacodynamic analyses were based on anticoagulant parameters derived from international normalized ratios of prothrombin times. Tigecycline peak concentration, trough concentration, area under the concentration-time curve (AUC) from 0-12 hrs, and clearance were not affected by single-dose warfarin. In contrast, R- and S-warfarin AUC from time zero extrapolated to infinity was increased by 68% and 29%, respectively, and clearance was decreased by 40% and 23%, respectively, when warfarin was administered after eight doses of tigecycline. Nevertheless, tigecycline did not alter the anticoagulant effects of warfarin, which is consistent with a mechanism based only on increased warfarin protein binding. CONCLUSION: These results suggest that a dosage adjustment of either drug is not necessary during coadministration of tigecycline and warfarin. However, consistent with good medical practice, the anticoagulant activity of warfarin should be monitored during coadministration with tigecycline.

Premelanosome amyloid-like fibrils are composed of only golgi-processed forms of Pmel17 that have been proteolytically processed in endosomes.

Melanin pigments are synthesized within specialized organelles called melanosomes and polymerize on intraluminal fibrils that form within melanosome precursors. The fibrils consist of proteolytic fragments derived from Pmel17, a pigment cell-specific integral membrane protein. The intracellular pathways by which Pmel17 accesses melanosome precursors and the identity of the Pmel17 derivatives within fibrillar melanosomes have been a matter of debate. We show here that antibodies that detect Pmel17 within fibrillar melanosomes recognize only the luminal products of proprotein convertase cleavage and not the remaining products linked to the transmembrane domain. Moreover, antibodies to the N and C termini detect only Pmel17 isoforms present in early biosynthetic compartments, which constitute a large fraction of detectable steady state Pmel17 in cell lysates because of slow early biosynthetic transport and rapid consumption by fibril formation. Using an antibody to a luminal epitope that is destroyed upon modification by O-linked oligosaccharides, we show that all post-endoplasmic reticulum Pmel17 isoforms are modified by Golgi-associated oligosaccharide transferases, and that only processed forms contribute to melanosome biogenesis. These data indicate that Pmel17 follows a single biosynthetic route from the endoplasmic reticulum through the Golgi complex and endosomes to melanosomes, and that only fragments encompassing previously described functional luminal determinants are present within the fibrils. These data have important implications for the site and mechanism of fibril formation.

Metabolism, excretion, and pharmacokinetics of [14C]tigecycline, a first-in-class glycylcycline antibiotic, after intravenous infusion to healthy male subjects.

Tigecycline, a novel, first-in-class glycylcycline antibiotic, has been approved for the treatment of complicated intra-abdominal infections and complicated skin and skin structure infections. The pharmacokinetics, metabolism, and excretion of [(14)C]tigecycline were examined in healthy male volunteers. Tigecycline has been shown to bind to bone; thus, to minimize the amount of radioactivity binding to bone and to maximize the recovery of radioactivity, tigecycline was administered intravenously (30-min infusion) as a single 100-mg dose, followed by six 50-mg doses, every 12 h, with the last dose being [(14)C]tigecycline (50 microCi). After the final dose, the pharmacokinetics of tigecycline in serum showed a long half-life (55.8 h) and a large volume of distribution (21.0 l/kg), whereas radioactivity in serum had a shorter half-life (6.9 h) and a smaller volume of distribution (3.3 l/kg). The major route of elimination was feces, containing 59% of the radioactive dose, whereas urine contained 32%. Unchanged tigecycline was the predominant drug-related compound in serum, urine, and feces. The major metabolic pathways identified were glucuronidation of tigecycline and amide hydrolysis followed by N-acetylation to form N-acetyl-9-aminominocycline. The glucuronide metabolites accounted for 5 to 20% of serum radioactivity, and approximately 9% of the dose was excreted as glucuronide conjugates within 48 h. Concentrations of N-acetyl-9-aminominocycline were approximately 6.5% and 11% of the tigecycline concentrations in serum and urine, respectively. Excretion of unchanged tigecycline into feces was the primary route of elimination, and the secondary elimination pathways were renal excretion of unchanged drug and metabolism to glucuronide conjugates and N-acetyl-9-aminominocycline.

Absence of an interaction between tigecycline and digoxin in healthy men.

STUDY OBJECTIVE: To evaluate a potential interaction between tigecycline and digoxin using pharmacokinetic and pharmacodynamic assessments. DESIGN: Open-label, three-period, one-sequence crossover study. SETTING: Hospital-affiliated, inpatient clinical pharmacology unit. SUBJECTS: Twenty healthy men. INTERVENTION: Tigecycline 100 mg was administered intravenously as a single dose on day 1 (period 1). Digoxin was administered as a 0.5-mg oral loading dose on day 7, followed by 0.25 mg/day on days 8-14 (period 2). Digoxin 0.25 mg/day was continued on days 15-19; in addition, on day 15, a loading dose of tigecycline 100 mg was administered intravenously, followed by 50 mg every 12 hours starting on the evening of day 15 through the morning of day 19 (period 3). MEASUREMENTS AND MAIN RESULTS: Pharmacokinetic assessments were performed on days 1 and 19 for tigecycline and on days 14 and 19 for digoxin. Electrocardiographic parameters were measured at baseline and on days 1, 14, and 19 to assess digoxin pharmacodynamics. Serum tigecycline concentrations were determined by liquid chromatography with tandem mass spectrometry detection, and plasma and urine digoxin concentrations were determined by radioimmunoassay. Tigecycline area under the concentration-time curve (AUC), AUC from 0-12 hours (AUC(0-12)), weight-normalized clearance, and mean resistance time were not affected by concomitant multiple-dose digoxin administration, but tigecycline half-life was decreased during period 1, apparently due to fewer detectable terminal concentrations in some subjects. Digoxin steady-state AUC(0-24), weight-normalized oral dose clearance, cumulative amount of drug excreted in urine over 24 hours, renal clearance, and QTc (change from baseline) were not affected by multiple-dose tigecycline administration. CONCLUSION: No significant effects of tigecycline on digoxin pharmacokinetics and pharmacodynamics were noted, but a small effect of digoxin on tigecycline pharmacokinetics cannot be ruled out due to design issues with period 1 of the study.

Dual loss of ER export and endocytic signals with altered melanosome morphology in the silver mutation of Pmel17.

Pmel17 is a pigment cell-specific integral membrane protein that participates in the formation of the intralumenal fibrils upon which melanins are deposited in melanosomes. The Pmel17 cytoplasmic domain is truncated by the mouse silver mutation, which is associated with coat hypopigmentation in certain strain backgrounds. Here, we show that the truncation interferes with at least two steps in Pmel17 intracellular transport, resulting in defects in melanosome biogenesis. Human Pmel17 engineered with the truncation found in the mouse silver mutant (hPmel17si) is inefficiently exported from the endoplasmic reticulum (ER). Localization and metabolic pulse-chase analyses with site-directed mutants and chimeric proteins show that this effect is due to the loss of a conserved C-terminal valine that serves as an ER exit signal. hPmel17si that exits the ER accumulates abnormally at the plasma membrane due to the loss of a di-leucine-based endocytic signal. The combined effects of reduced ER export and endocytosis significantly deplete Pmel17 within endocytic compartments and delay proteolytic maturation required for premelanosome-like fibrillogenesis. The ER export delay and cell surface retention are also observed for endogenous Pmel17si in melanocytes from silver mice, within which Pmel17 accumulation in premelanosomes is dramatically reduced. Mature melanosomes in these cells are larger, rounder, more highly pigmented, and less striated than in control melanocytes. These data reveal a dual sorting defect in a natural mutant of Pmel17 and support a requirement of endocytic trafficking in Pmel17 fibril formation.

A lumenal domain-dependent pathway for sorting to intralumenal vesicles of multivesicular endosomes involved in organelle morphogenesis.

Cargo partitioning into intralumenal vesicles (ILVs) of multivesicular endosomes underlies such cellular processes as receptor downregulation, viral budding, and biogenesis of lysosome-related organelles such as melanosomes. We show that the melanosomal protein Pmel17 is sorted into ILVs by a mechanism that is dependent upon lumenal determinants and conserved in non-pigment cells. Pmel17 targeting to ILVs does not require its native cytoplasmic domain or cytoplasmic residues targeted by ubiquitylation and, unlike sorting of ubiquitylated cargo, is insensitive to functional inhibition of Hrs and ESCRT complexes. Chimeric protein and deletion analyses indicate that two N-terminal lumenal subdomains are necessary and sufficient for ILV targeting. Pmel17 fibril formation, which occurs during melanosome maturation in melanocytes, requires a third lumenal subdomain and proteolytic processing that itself requires ILV localization. These results establish an Hrs- and perhaps ESCRT-independent pathway of ILV sorting by lumenal determinants and a requirement for ILV sorting in fibril formation.

Pharmacokinetics of sirolimus (rapamycin) in subjects with mild to moderate hepatic impairment.

Eighteen adult subjects with mild to moderate hepatic impairment and 18 healthy control subjects were given a single 15-mg dose of sirolimus by oral solution. Mean whole-blood sirolimus weight-normalized oral-dose clearances (CL/F) were significantly decreased (P = .02) in subjects with mild to moderate hepatic impairment by -31.8% and -36.0%, respectively, compared with controls. There were no significant differences in mean sirolimus C(max) and t(max) values among groups. The observed decreases in CL/F may be relevant in renal transplant patients with mild to moderate hepatic impairment, based on the close similarity of sirolimus CL/F in controls and previously studied stable renal transplant patients receiving multiple-dose administration of sirolimus and cyclosporine. There was considerable overlap in the CL/F values of hepatic-impaired subjects and controls, suggesting that whole-blood sirolimus trough concentrations in renal transplant patients exhibiting mild to moderate hepatic impairment be initially monitored to assess the need for dose adjustments.

Pharmacokinetic profile of tigecycline in serum and skin blister fluid of healthy subjects after multiple intravenous administrations.

The pharmacokinetics of tigecycline, when given as a 100-mg loading dose followed by 50 mg every 12 h, were determined in serum and blister fluid. The peak tigecycline concentration and half-life in serum were greater than those in blister fluid. Tigecycline penetrates into blister fluid well, with a mean penetration rate of 74%.