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Free sialic acid storage disorder (FSASD) is a rare, autosomal recessive, neurodegenerative disorder caused by biallelic mutations in SLC17A5, encoding the lysosomal transmembrane sialic acid exporter, SLC17A5. Defects in SLC17A5 lead to lysosomal accumulation of free sialic acid and other acid hexoses. The clinical spectrum of FSASD ranges from mild (Salla disease) to severe infantile forms. The pathobiology underlying FSASD remains elusive. In this study, two induced pluripotent stem cell (iPSC) lines were generated from a mild and an intermediate FSASD patient and characterized to provide much-needed additional models for basic and translational studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Purpose: To evaluate the safety and efficacy of N-acetylmannosamine (ManNAc) in GNE myopathy, a genetic muscle disease caused by deficiency of the rate-limiting enzyme in N-acetylneuraminic acid (Neu5Ac) biosynthesis., Methods: We conducted an open-label, phase 2, single-center (NIH, USA) study to evaluate oral ManNAc in 12 patients with GNE myopathy (ClinicalTrials.gov NCT02346461). Primary endpoints were safety and biochemical efficacy as determined by change in plasma Neu5Ac and sarcolemmal sialylation. Clinical efficacy was evaluated using secondary outcome measures as part of study extensions, and a disease progression model (GNE-DPM) was tested as an efficacy analysis method., Results: Most drug-related adverse events were gastrointestinal, and there were no serious adverse events. Increased plasma Neu5Ac (+2,159 nmol/L, p < 0.0001) and sarcolemmal sialylation (p = 0.0090) were observed at day 90 compared to baseline. A slower rate of decline was observed for upper extremity strength (p = 0.0139), lower extremity strength (p = 0.0006), and the Adult Myopathy Assessment Tool (p = 0.0453), compared to natural history. Decreased disease progression was estimated at 12 (γ = 0.61 [95% CI: 0.09, 1.27]) and 18 months (γ = 0.55 [95% CI: 0.12, 1.02]) using the GNE-DPM., Conclusion: ManNAc showed long-term safety, biochemical efficacy consistent with the intended mechanism of action, and preliminary evidence clinical efficacy in patients with GNE myopathy., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Background: Cancer patients are at a higher risk of developing severe coronavirus disease 2019 (COVID-19). However, the safety and efficacy of COVID-19 vaccination in cancer patients undergoing treatment remain unclear., Patients and Methods: In this interventional prospective multicohort study, priming and booster doses of the BNT162b2 COVID-19 vaccine were administered 21 days apart to solid tumor patients receiving chemotherapy, immunotherapy, targeted or hormonal therapy, and patients with a hematologic malignancy receiving rituximab or after allogeneic hematopoietic stem cell transplantation. Vaccine safety and efficacy (until 3 months post-booster) were assessed. Anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) antibody levels were followed over time (until 28 days after the booster) and in vitro SARS-CoV-2 50% neutralization titers (NT50) toward the wild-type Wuhan strain were analyzed 28 days after the booster., Results: Local and systemic adverse events (AEs) were mostly mild to moderate (only 1%-3% of patients experienced severe AEs). Local, but not systemic, AEs occurred more frequently after the booster dose. Twenty-eight days after the booster vaccination of 197 cancer patients, RBD-binding antibody titers and NT50 were lower in the chemotherapy group {234.05 IU/ml [95% confidence interval (CI) 122.10-448.66] and 24.54 (95% CI 14.50-41.52), respectively} compared with healthy individuals [1844.93 IU/ml (95% CI 1383.57-2460.14) and 122.63 (95% CI 76.85-195.67), respectively], irrespective of timing of vaccination during chemotherapy cycles. Extremely low antibody responses were seen in hematology patients receiving rituximab; only two patients had RBD-binding antibody titers necessary for 50% protection against symptomatic SARS-CoV-2 infection (<200 IU/ml) and only one had NT50 above the limit of detection. During the study period, five cancer patients tested positive for SARS-CoV-2 infection, including a case of severe COVID-19 in a patient receiving rituximab, resulting in a 2-week hospital admission., Conclusion: The BNT162b2 vaccine is well-tolerated in cancer patients under active treatment. However, the antibody response of immunized cancer patients was delayed and diminished, mainly in patients receiving chemotherapy or rituximab, resulting in breakthrough infections., Competing Interests: Disclosure MP declares to have an advisory role within Remedus. All other authors have declared no conflicts of interest., (Copyright © 2021. Published by Elsevier Ltd.)

Disorders caused by defects in lysosomal membrane transporters form a distinct subgroup of lysosomal storage disorders (LSDs). To date, defects in only 10 lysosomal membrane transporters have been associated with inherited disorders. The clinical presentations of these diseases resemble the phenotypes of other LSDs; they are heterogeneous and often present in children with neurodegenerative manifestations. However, for pathomechanistic and therapeutic studies, lysosomal membrane transport defects should be distinguished from LSDs caused by defective hydrolytic enzymes. The involved proteins differ in function, localization, and lysosomal targeting, and the diseases themselves differ in their stored material and therapeutic approaches. We provide an overview of the small group of disorders of lysosomal membrane transporters, emphasizing discovery, pathomechanism, clinical features, diagnostic methods and therapeutic aspects. We discuss common aspects of lysosomal membrane transporter defects that can provide the basis for preclinical research into these disorders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

The outbreak of the SARS-CoV-2 pandemic has overwhelmed health care systems in many countries. The clinical presentation of the SARS-CoV-2 varies between a subclinical or flu-like syndrome to that of severe pneumonia with multi-organ failure and death. Initial reports have suggested that cancer patients may have a higher susceptibility to get infected by the SARS-CoV-2 virus but current evidence remains poor as it is biased by important confounders. Patients with ongoing or recent cancer treatment for advanced active disease, metastatic solid tumors and hematological malignancies are at higher risk of developing severe COVID-19 respiratory disease that requires hospitalization and have a poorer disease outcome compared to individuals without cancer. However it is not clear whether these are independent risk factors, or mainly driven by male gender, age, obesity, performance status, uncontrolled diabetes, cardiovascular disease and various other medical conditions. These often have a greater influence on the probability to die due to SARS-CoV-2 then cancer. Delayed diagnosis and suboptimal cancer management due to the pandemic results in disease upstaging and has considerable impact cancer on specific death rates. Surgery during the peak of the pandemic seems to increase mortality, but there is no convincing evidence that adjuvant systemic cancer therapy and radiotherapy are contraindicated, implicating that cancer treatment can be provided safely after individual risk/benefit assessment and some adaptive measures. Underlying immunosuppression, elevated cytokine levels, altered expression of the angiotensin converting enzyme (ACE-2) and TMPRSS2, and a prothrombotic status may fuel the effects of a SARS-CoV-2 in some cancer patients, but have the potential to be used as biomarkers for severe disease and therapeutic targets. The rapidly expanding literature on COVID-19 should be interpreted with care as it is often hampered by methodological and statistical flaws., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Introduction: Sialic acids are important contributors to the polyanionic component of the glomerular filtration barrier, which regulates permeability selectivity. Pathologic glomerular hyposialylation, associated with podocyte effacement, has been implicated in human and mouse glomerulopathies. Oral treatment with N -acetylmannosamine (ManNAc), the uncharged precursor of sialic acid, ameliorates glomerular pathology in different models of glomerular disease., Methods: Here we explore the sialylation status of kidney biopsies obtained from 27 subjects with various glomerular diseases using lectin histochemistry., Results: We identified severe glomerular hyposialylation in 26% of the biopsies. These preliminary findings suggest that this condition may occur relatively frequently and may be a novel target for therapy. We describe the background, rationale, and design of a phase 1 study to test safety, tolerability, and pharmacokinetics of ManNAc in subjects with primary podocyte diseases., Conclusion: We recently demonstrated that ManNAc was safe and well tolerated in a first-in-human phase 1 study in subjects with UDP- N -acetylglucosamine (GlcNAc) 2-epimerase/ManNAc kinase (GNE) myopathy, a disorder of impaired sialic acid synthesis. Using previous preclinical and clinical data, we propose to test ManNAc therapy for subjects with primary glomerular diseases. Even though the exact mechanisms, affected cell types, and pathologic consequences of glomerular hyposialylation need further study, treatment with this physiological monosaccharide could potentially replace or supplement existing glomerular diseases therapies.

Background: Breast cancer has, due its high incidence, the highest mortality of cancer in women. The most common molecular variety of breast cancer is luminal subtype that expresses estrogen and progesterone receptors. Estrogen receptor alpha (ERα), encoded by the estrogen receptor1 (ESR1) gene, is expressed in approximately 70% of all breast cancers, and hormonal therapy represents a major treatment modality in all stages of ER positive breast cancers. Acquired mutations in the ligand-binding domain (LBD) of ERα, referred as ESR1 mutation, result in resistance to different endocrine therapies leading to disease progression or recurrence. Recent studies reviled that these ESR1 mutations lead to constitutive activity of the estrogen receptor ER, meaning that the receptor is active in absence of its ligand conferring resistance against endocrine therapy and tumor growth. Published studies have not yet been able to determine the exact prevalence rate of ESR1 mutations, but set the outer boundaries between 11-55%., Purpose: The goal of the present study is to determine the frequency rate of ESR1 mutations in ER positive recurrent breast cancer by using digital droplet PCR (ddPCR) technique., Materials and Methods: This retrospective study was conducted in the Multidisciplinary Breast Clinic of Antwerp University Hospital. The seven most common ESR1mutations (c.1138G>C (p. (E380Q)), c.1610A>G (p.(Y537C)), c.1613A>G (p.(p.D538G)), c.1607T>G (p.(L536R)), c.1387T>C (p.S463R)), c.16410A>C (p.(Y537S)), c.609T>A (p.(Y537N)) were assessed in available baseline plasma samples of 21 patients with ER positive recurrent breast cancer. Inclusion criteria for study participation were: female, age above 18 years, ER positive breast cancer, 5years adjuvant hormonal therapy of primary disease, and disease recurrence or metastasis during or after stop of endocrine therapy. ESR1 mutations were analyzed in cell-free DNA (cfDNA) by using digital droplet PCR (ddPCR)., Results: cfDNA was obtained from 21 patients with recurrent breast cancer. ESR1 mutations were found in 4/21 (19%; 95% CI, 5%-42%). The test sensitivity was lower than the targeted value <0.1% in 29% of patients (6/21). No significant statistical difference in baseline clinical characteristics was observed in patients with wild-type and mutant ER (p>0.05). Adjuvant endocrine therapy for primary disease was Tamoxifen (TAM) for 57% of patients (12 of 21) of whom 8 patients had received aromatase inhibitor (AI) after two years, while 43% of patients (9 of 21) had received AI as first line adjuvant hormonal therapy. All the patients had received aromatase inhibitor AI therapy in first or second line therapy with initially a variable period of good response., Conclusion: ESR1 mutation analysis could be determined in archived plasma samples using simple non-invasive methods. In the future, screening for mutation status could improve the therapeutic strategies in controlling ER signaling before the occurrence of wide spread disease metastasis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Approximately 40% of hormone receptor-positive, HER2-negative breast cancers (BCs) are associated with activating mutations of the phosphatidylinositol 3-kinase (PI3K) pathway. Pictilisib, a potent and highly specific class I pan-PI3K inhibitor, demonstrated preclinical activity in BC cell lines and may potentiate the effect of taxanes, benefiting patients with or without aberrant activation of the PI3K pathway. PEGGY (NCT01740336), a randomised, placebo-controlled phase II trial, examined whether pictilisib augments the anti-tumour activity of paclitaxel in patients with hormone receptor-positive, HER2-negative locally recurrent or metastatic BC (mBC). We report results from the protocol-specified interim analysis., Patients and Methods: One hundred and eighty-three eligible patients were randomised (1:1) to receive paclitaxel (90 mg/m 2 weekly for 3 weeks in every 28-day cycle) with either 260 mg pictilisib or placebo (daily on days 1-5 every week). The primary end point was progression-free survival (PFS) in the intention-to-treat (ITT) population and patients with PIK3CA-mutated tumours. Secondary end points included overall response rate (ORR), duration of response, and safety., Results: In the ITT population, the median PFS was 8.2 months with pictilisib (n = 91) versus 7.8 months with placebo (n = 92) [hazard ratio (HR) for progression or death, 0.95; 95% confidence interval (CI) 0.62-1.46; P = 0.83]. In patients with PIK3CA-mutated tumours, the median PFS was 7.3 months for pictilisib (n = 32) versus 5.8 months with placebo (n = 30) (HR, 1.06; 95% CI 0.52-2.12; P = 0.88). ORR was similar between treatment arms. The safety profile of pictilisib was consistent with previous reports, with no new safety signals. Proportions of patients with grade ≥3 adverse events (AEs), serious AEs, and dose reductions/discontinuations due to AEs were higher with pictilisib., Conclusions: PEGGY did not meet its primary end point, revealing no significant benefit from adding pictilisib to paclitaxel for patients with hormone receptor-positive, HER2-negative locally recurrent or mBC., Clinical Trial Number: NCT01740336., (© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Data on CA-125 as a predictor of disease progression (PD) in ovarian cancer come predominantly from patients with platinum-sensitive disease receiving chemotherapy alone. We assessed concordance between CA-125-defined and RECIST-defined PD using data from the Gynecologic Cancer InterGroup (GCIG) randomized phase III AURELIA trial in platinum-resistant ovarian cancer (PROC)., Patients and Methods: Patients with PROC were randomized to receive single-agent chemotherapy with or without bevacizumab. PD by CA-125 was defined according to GCIG criteria (except that confirmatory CA-125 measurement was not required). This exploratory analysis included patients with RECIST PD and a CA-125 reading ≤28 days before and ≤21 days after RECIST-defined PD., Results: Of 218 eligible patients, only 94 (43%, 95% confidence interval 36% to 50%) had concordant RECIST and CA-125 PD status (42% in the chemotherapy-alone arm; 45% in the bevacizumab combination arm, P = 0.6). There was no evidence of CA-125-defined PD in the remaining 124 patients despite PD according to imaging. There were no significant differences in baseline characteristics between patients with PD defined by both RECIST and CA-125 and those with RECIST-only PD. CA-125 was even less sensitive in detecting PD in patients with early (<8 weeks after randomization) compared with later RECIST-defined PD (69% versus 53%, respectively, not meeting CA-125 criteria; P = 0.053). There was no significant difference in survival after PD in patients with concordant PD by RECIST and CA-125 versus those with PD only by RECIST. We validated our findings in an independent study population of PROC., Conclusions: In this platinum-resistant population, PD was typically detected earlier by imaging than by CA-125, irrespective of bevacizumab treatment. Disease status by CA-125 at the time of PD was not prognostic for overall survival. Regular radiologic assessment as well as symptom benefit assessment should be considered during PROC follow-up., (© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Only human epidermal growth factor receptor (HER)2 status determined by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) has been validated to predict efficacy of HER2-targeting antibody-drug-conjugate trastuzumab emtansine (T-DM1). We propose molecular imaging to explore intra-/interpatient heterogeneity in HER2 mapping of metastatic disease and to identify patients unlikely to benefit from T-DM1., Patients and Methods: HER2-positive mBC patients with IHC3+ or FISH ≥ 2.2 scheduled for T-DM1 underwent a pretreatment HER2-positron emission tomography (PET)/computed tomography (CT) with (89)Zr-trastuzumab. [(18)F]2-fluoro-2-deoxy-D-glucose (FDG)-PET/CT was performed at baseline and before T-DM1 cycle 2. Patients were grouped into four HER2-PET/CT patterns according to the proportion of FDG-avid tumor load showing relevant (89)Zr-trastuzumab uptake (>blood pool activity): patterns A and B were considered positive (>50% or all of the tumor load 'positive'); patterns C and D were considered negative (>50% or all of the tumor load 'negative'). Early FDG-PET/CT was defined as nonresponding when >50% of the tumor load showed no significant reduction of FDG uptake (<15%). Negative (NPV) and positive predictive values (PPV) of HER2-PET/CT, early FDG response and their combination were assessed to predict morphological response (RECIST 1.1) after three T-DM1 cycles and time-to-treatment failure (TTF)., Results: In the 56 patients analyzed, 29% had negative HER2-PET/CT while intrapatient heterogeneity (patterns B and C) was found in 46% of patients. Compared with RECIST1.1, respective NPV/PPV for HER2-PET/CT were 88%/72% and 83%/96% for early FDG-PET/CT. Combining HER2-PET/CT and FDG-PET/CT accurately predicted morphological response (PPV and NPV: 100%) and discriminated patients with a median TTF of only 2.8 months [n = 12, 95% confidence interval (CI) 1.4-7.6] from those with a TTF of 15 months (n = 25, 95% CI 9.7-not calculable)., Conclusions: Pretreatment imaging of HER2 targeting, combined with early metabolic response assessment holds great promise for improving the understanding of tumor heterogeneity in mBC and for selecting patients who will/will not benefit from T-DM1., Clinicaltrialsgov Identifier: NCT01565200., (© The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

N-acetylneuraminic acid (Neu5Ac or NANA) is the most predominant sialic acid in mammals. As a terminal component in many glycoproteins and glycolipids, sialic acid is believed to be an important biomarker related to various diseases. Its precursor, N-acetylmannosamine (ManNAc), is being investigated as a potential treatment for GNE myopathy. In this work, we developed two highly sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the quantitation of ManNAc and free Neu5Ac in human plasma. A fit-for-purpose approach was adopted during method validation and sample analysis. To measure the endogenous compounds and overcome the interference from plasma samples, a surrogate matrix that contained 5% bovine serum albumin (BSA) was used for the preparation of calibration standards and certain levels of quality control (QC) samples. QC samples at higher concentrations were prepared in the authentic matrix (human plasma) to best mimic incurred samples. For both methods, an Ostro 96-well phospholipid removal plate was used for sample extraction, which efficiently removed the phospholipids from the plasma samples prior to LC injection, eliminated matrix effect, and improved sensitivity. Chromatographic separation was achieved using hydrophilic interaction chromatography (HILIC) and gradient elution in order to retain the two polar compounds. The lower limit of quantitation (LLOQ) for ManNAc and Neu5Ac was 10.0 and 25.0ng/mL, respectively. The overall accuracy of the two assays was within 100%±8.3% based on three levels of QC samples. Inter- and intra-run precision (coefficient of variation (%CV)) across three analytical runs was less than 6.7% for ManNAc and less than 10.8% for Neu5Ac. These methods have been validated to support clinical studies., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Pathological glomerular hyposialylation has been implicated in certain unexplained glomerulopathies, including minimal change nephrosis, membranous glomerulonephritis, and IgA nephropathy. We studied our previously established mouse model carrying a homozygous mutation in the key enzyme of sialic acid biosynthesis, N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Mutant mice died before postnatal day 3 (P3) from severe glomerulopathy with podocyte effacement and segmental glomerular basement membrane splitting due to hyposialylation. Administration of the sialic acid precursor N-acetylmannosamine (ManNAc) led to improved sialylation and survival of mutant pups beyond P3. We determined the onset of the glomerulopathy in the embryonic stage. A lectin panel, distinguishing normally sialylated from hyposialylated glycans, used WGA, SNA, PNA, Jacalin, HPA, and VVA, indicating glomerular hyposialylation of predominantly O-linked glycoproteins in mutant mice. The glomerular glycoproteins nephrin and podocalyxin were hyposialylated in this unique murine model. ManNAc treatment appeared to ameliorate the hyposialylation status of mutant mice, indicated by a lectin histochemistry pattern similar to that of wild-type mice, with improved sialylation of both nephrin and podocalyxin, as well as reduced albuminuria compared with untreated mutant mice. These findings suggest application of our lectin panel for categorizing human kidney specimens based on glomerular sialylation status. Moreover, the partial restoration of glomerular architecture in ManNAc-treated mice highlights ManNAc as a potential treatment for humans affected with disorders of glomerular hyposialylation., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive condition characterized by a bleeding diathesis and hypopigmentation of the skin, hair, and eyes. Some HPS patients develop other complications such as granulomatous colitis and/or fatal pulmonary fibrosis. Eight genes have been associated with this condition, resulting in subtypes HPS-1 through HPS-8. The HPS gene products are involved in the biogenesis of specialized lysosome-related organelles such as melanosomes and platelet delta granules. HPS1 and HPS4 form a stable complex named biogenesis of lysosome-related organelles complex (BLOC)-3, and patients with BLOC-3 or AP-3 deficiency develop pulmonary fibrosis. Therefore, it is important to subtype each HPS patient. HPS type 1 (HPS-1) occurs frequently on the island of Puerto Rico because of a founder mutation. Here, we describe seven mutations, six of which, to our knowledge, are previously unreported in the HPS1, HPS4, and HPS5 genes among patients of Mexican, Uruguayan, Honduran, Cuban, Venezuelan, and Salvadoran ancestries. Our findings demonstrate that the diagnosis of HPS should be considered in Hispanic patients with oculocutaneous albinism and bleeding symptoms. Moreover, such patients should not be assumed to have the HPS-1 subtype typical of northwest Puerto Rican patients. We recommend molecular HPS subtyping in such cases, as it may have significant implications for prognosis and intervention.

We evaluated a 32-year-old woman whose oculocutaneous albinism (OCA), bleeding diathesis, neutropenia, and history of recurrent infections prompted consideration of the diagnosis of Hermansky-Pudlak syndrome type 2. This was ruled out because of the presence of platelet δ-granules and absence of AP3B1 mutations. As parental consanguinity suggested an autosomal recessive mode of inheritance, we employed homozygosity mapping, followed by whole-exome sequencing, to identify two candidate disease-causing genes, SLC45A2 and G6PC3. Conventional dideoxy sequencing confirmed pathogenic mutations in SLC45A2, associated with OCA type 4 (OCA-4), and G6PC3, associated with neutropenia. The substantial reduction of SLC45A2 protein in the patient's melanocytes caused the mislocalization of tyrosinase from melanosomes to the plasma membrane and also led to the incorporation of tyrosinase into exosomes and secretion into the culture medium, explaining the hypopigmentation in OCA-4. Our patient's G6PC3 mRNA expression level was also reduced, leading to increased apoptosis of her fibroblasts under endoplasmic reticulum stress. To our knowledge, this report describes the first North American patient with OCA-4, the first culture of human OCA-4 melanocytes, and the use of homozygosity mapping, followed by whole-exome sequencing, to identify disease-causing mutations in multiple genes in a single affected individual.

Gray platelet syndrome (GPS) is an inherited bleeding disorder characterized by macrothrombocytopenia and absence of platelet α-granules resulting in typical gray platelets on peripheral smears. GPS is associated with a bleeding tendency, myelofibrosis, and splenomegaly. Reports on GPS are limited to case presentations. The causative gene and underlying pathophysiology are largely unknown. We present the results of molecular genetic analysis of 116 individuals including 25 GPS patients from 14 independent families as well as novel clinical data on the natural history of the disease. The mode of inheritance was autosomal recessive (AR) in 11 and indeterminate in 3 families. Using genome-wide linkage analysis, we mapped the AR-GPS gene to a 9.4-Mb interval on 3p21.1-3p22.1, containing 197 protein-coding genes. Sequencing of 1423 (69%) of the 2075 exons in the interval did not identify the GPS gene. Long-term follow-up data demonstrated the progressive nature of the thrombocytopenia and myelofibrosis of GPS resulting in fatal hemorrhages in some patients. We identified high serum vitamin B(12) as a consistent, novel finding in GPS. Chromosome 3p21.1-3p22.1 has not been previously linked to a platelet disorder; identification of the GPS gene will likely lead to the discovery of novel components of platelet organelle biogenesis. This study is registered at www.clinicaltrials.gov as NCT00069680 and NCT00369421.

Background: Overall survival (OS) and outcome of cancer patients with bisphosphonate-associated osteonecrosis of the jaw (ONJ) using conservative treatment (chlorhexidine 0.12% rinse, intermittent antibiotics, and careful sequestrectomy) are unknown., Design: In all, 33 ONJ patients were studied for OS and ONJ outcome., Results: Median duration of bisphosphonate treatment was 27 months (range 4-115) and was stopped in 25 (76%) patients. Nine (27%) cases presented with stage 1, 21 (64%) with stage 2, and 3 (9%) with stage 3 disease. During median follow-up of 23 months, 11 patients (33%) died (median survival 39 months), with no ONJ-related fatalities. Out of 30 assessable patients, 53% no longer had exposed bone, 37% had stable lesions, and 10% showed progressive necrosis. The hazard ratio for healing with doubling of bisphosphonate exposure was 0.419 [95% confidence interval (CI) 0.178-0.982; P = 0.045], stage 2 versus stage 1 disease 0.216 (95% CI 0.063-0.738; P = 0.015), and stage 3 versus stage 1 disease 0.084 (95% CI 0.008-0.913; P = 0.042). Cessation of bisphosphonate treatment did not influence outcome., Conclusions: Conservative treatment of ONJ leads to mucosal closure in 53% of patients. Doubling the exposure time to bisphosphonates and higher stages of ONJ significantly reduce ONJ healing rates.

Recently aromatase inhibitors have become a standard care as an adjuvant treatment for many postmenopausal patients with hormone receptor positive early breast cancer. Adjuvant letrozole was made available either immediately postoperative, after 2-3 years of tamoxifen, or as an extended treatment after 5 years of tamoxifen. Between October 2003 and October 2005, we analyzed the subjective tolerance in 185 postoperative early breast cancer patients receiving letrozole outside of a clinical trial. The most prominent toxicity was musculoskeletal pain. In addition hot flushes, increased fatigue, nausea, vomiting, anorexia, mood disturbances, vaginal dryness, hair loss and rash were also recorded. In contrast to the prospective randomized clinical trials, a high drop-out rate of 20% was documented, mainly due to aromatase inhibitor-associated arthralgia syndrome interfering significantly with the daily life of our patients. Although adjuvant aromatase inhibitors have proven to be generally superior to tamoxifen in the adjuvant setting, it is important to focus attention on the tolerance during the adjuvant therapy and to balance this against the potential benefit in individual patients. Alternative options including switching to tamoxifen remain available.

Background: Radiotherapy (RT) with concurrent chemotherapy is the current standard of care for patients with unresectable locally advanced squamous cell carcinoma of the head and neck (SCCHN). Gemcitabine (GEM) is a potent radiosensitizer and in addition has activity as an anticancer agent in SCCHN., Patients and Methods: Twenty-six patients with locally far advanced SCCHN were enrolled in a chemoradiation feasibility study between November 1998 and September 2003. Use was made of conventionally fractionated RT and GEM 100 mg/m(2), which was given within 2 h prior to radiotherapy on a weekly basis starting on day 1 of RT. Response was assessed according to WHO criteria, toxicity according to NCI-CTC version 2., Results: The patients received a median of 7 (2-8) weekly cycles of gemcitabine and a median cumulative RT dose of 70 Gy (66-84.75). Hematologic toxicity was mild, but non-hematologic toxicity was severe: grade 3-4 stomatitis occurred in 85% of patients, dermatitis in 69%, pharyngitis/esophagitis in 81% and 80% of the patients needed a feeding tube during treatment. All 22 evaluable patients responded (50% complete, 50% partial). Median follow up of the surviving patients is 46 months. Median disease-free and overall survival is 13 months and 19 months, respectively; 27% of the patients are alive without evidence of recurrence beyond 3 years., Conclusions: Conventionally fractionated RT in combination with GEM 100 mg/m(2) weekly is feasible and highly active in the treatment of locally advanced SCCHN. In particular, long-term local control rate is promising. Acute mucosal toxicities are significant but manageable. Long-term toxicity interferes with normal food intake.

Hermansky-Pudlak syndrome (HPS) is a disorder of lysosome-related organelle biogenesis resulting in melanosome dysfunction and absent platelet dense bodies. HPS patients have oculocutaneous albinism, bruising, and bleeding. HPS-5 results from deficiency of the HPS5 protein, a component of the biogenesis of lysosome-related organelles complex-2 (BLOC-2). HPS5 has an unknown function and lacks homology to known proteins. We performed ultrastructural studies of HPS-5 melanocytes revealing predominantly early-stage melanosomes with many small 3,4(OH)2-phenylalanine-positive vesicles throughout the cell body and dendrites. These findings resemble the distinct ultrastructural features of HPS-3 melanocytes; HPS3 is also a BLOC-2 component. Immunofluorescence and immunoEM studies showed decreased TYRP1 labeling in the dendrites of HPS-5 melanocytes, and the overall abundance of TYRP1 was reduced. No substantial differences were observed in the distribution or abundance of Pmel17 in HPS-5 melanocytes. In normal melanocytes, endogenous tyrosinase colocalized with Pmel17 and TYRP1 in the perinuclear area and dendritic tips; this was much reduced in HPS-5 melanocytes, particularly in the tips. We conclude that early stage melanosome formation and Pmel17 trafficking are preserved in HPS5-deficient cells. Tyrosinase and TYRP1 are mistrafficked, however, and fail to be efficiently delivered to melanosomes of HPS-5 melanocytes.

Platelet dense granules are lysosome-related organelles which contain high concentrations of several biologically important low-molecular-weight molecules. These include calcium, serotonin, adenine nucleotides, pyrophosphate, and polyphosphate, which are necessary for normal blood hemostasis. The synthesis of dense granules and other lysosome-related organelles is defective in inherited diseases such as Hermansky-Pudlak syndrome (HPS) and Chediak-Higashi syndrome (CHS). HPS and CHS mutations in 8 human and at least 16 murine genes have been identified. Previous studies produced contradictory findings for the function of the murine ashen (Rab27a) gene in platelet-dense granules. We have used a positional cloning approach with one line of ashen mutants to establish that a new mutation in a second gene, Slc35d3, on mouse chromosome 10 is the basis of this discrepancy. The platelet-dense granule defect is rescued in BAC transgenic mice containing the normal Slc35d3 gene. Thus, Slc35d3, an orphan member of a nucleotide sugar transporter family, specifically regulates the contents of platelet-dense granules. Unlike HPS or CHS genes, it has no apparent effect on other lysosome-related organelles such as melanosomes or lysosomes. The ash-Roswell mouse mutant is an appropriate model for human congenital-isolated delta-storage pool deficiency.

Background: An increasing amount of reports are being published suggesting a relationship between the use of bisphosphonates (BPs) and the development of osteonecrosis of the jaw (ONJ). We reviewed the currently available evidence and explore the potential mechanisms of action based on the known effects of the concerned BP., Design: The MEDLine, Current Contents and Science Citation Index Expanded databases were queried and the results augmented by analyzing cited references and recent congress proceedings., Results: 22 papers were included detailing 225 patients, all based on retrospective chart review without control groups. The prevalence of ONJ was estimated at 1.5%. The involved BPs were pamidronate, zoledronic acid, alendronate and risedronate, all potent nitrogen-containing agents. The most common symptom was pain (81.7%), although 12.2% of cases were asymptomatic. In 69.3% of patients ONJ was preceded by a dental extraction. At the time of diagnosis, 74.5% of patients were receiving chemotherapy and in 38.2% of cases corticosteroids were administered. Although various conservative and surgical treatment modalities were reported, residual sites of ONJ persisted in 72.5% of cases., Conclusion: Although not enough evidence is available to prove a causal link, it seems that under specific circumstances local defenses can become overwhelmed resulting in ONJ.

Hermansky-Pudlak Syndrome (HPS) is a genetically heterogeneous disorder in which mutations in one of several genes interrupts biogenesis of melanosomes, platelet dense bodies, and lysosomes. Affected patients have oculocutaneous albinism, a bleeding diathesis, and sometimes develop granulomatous colitis or pulmonary fibrosis. In order to assess the role of HPS genes in melanosome biogenesis, melanocytes cultured from patients with HPS subtypes 1, 2, or 3 were assessed for the localization of various melanocyte proteins. Tyrosinase, Tyrp1, and Dct/Tyrp2 were atypically and distinctly expressed in HPS-1 and HPS-3 melanocytes, whereas only tyrosinase showed an atypical distribution in HPS-2 melanocytes. The HPS1 and AP3B1 (i.e., HPS-2) gene products showed no expression in HPS-1 and HPS-2 melanocytes, respectively, whereas HPS-3 melanocytes exhibited normal expression for both proteins. In normal human melanocytes, the HPS1 protein was expressed as an approximately 80 kDa molecule with both granular and reticular intracellular profiles. In HPS-1, lysosome associated membrane protein 1 (LAMP1), and LAMP3 were localized to abnormal large granules; in HPS-2, all LAMPs exhibited a normal granular expression; and in HPS-3, LAMP1, and LAMP3 exhibited a distinct less granular and more floccular pattern. In contrast, the expressions of Rab 27, transferrin, and cKit were unaffected in all three HPS genotypes. These data demonstrate that the three initially identified subtypes of human HPS exhibit distinct defects in the trafficking of various melanocyte-specific proteins.

Hermansky-Pudlak Syndrome-type 3 (HPS-3) is a relatively mild subtype of HPS with minimal cutaneous and ocular depigmentation. The HPS-3 gene encodes a novel protein of unknown function with a predicted molecular weight of 114 kd. To assess the role of the HPS3 protein in melanization, cultured melanocytes developed from HPS-3 patients were evaluated biochemically and histologically for activity and localization of melanocyte-specific proteins. Endogenous tyrosinase activity of HPS-3 melanocytes was substantial, but tyrosinase activity and melanin synthesis was suppressed in intact melanocytes. However, the level of suppression, as well as extent to which up-regulation by isobutylmethylxanthine and cholera toxin was muted, was less that in HPS-1 melanocytes. Ultrastructurally, HPS-3 melanocytes contained morphologically normal melanosomes, predominantly of stage I and II with minimal stage III and few stage IV melanosomes. Dihydroxyphenylalanine (DOPA) histochemistry demonstrated an increase in melanization of melanosomes. Unique to HPS-3 melanocytes were numerous DOPA-positive 50-nm vesicles and tubular elements present throughout the cell body and dendrites. Tyrosinase, tyrosinase-related protein-1 (Tyrp1), dopachrome tautomerase (Dct), and LAMP1 and 3 localization in HPS-3 melanocytes, as evaluated by immunocytochemistry and confocal microscopy, demonstrated a fine, floccular distribution in contrast to the coarse, granular distribution characteristic of control melanocytes. The localization profile of other proteins expressed by melanocytes (ie, Silver/Pmel17, Melan-A/MART-1, LAMP2, Rab 27, transferrin, c-kit, adaptin-3, and the HPS1 protein) appeared normal. These results suggest that a specific subset of melanocyte proteins are aberrantly trafficked throughout the HPS-3 melanocyte and may be responsible for the reduction in melanin synthesis.

Hermansky-Pudlak syndrome (HPS), a disorder of organelle biogenesis, affects lysosomes, melanosomes, and platelet dense bodies. Seven genes cause HPS in humans (HPS1-HPS7) and at least 15 nonallelic mutations cause HPS in mice. Where their function is known, the HPS proteins participate in protein trafficking and vesicle docking/fusion events during organelle biogenesis. HPS-associated genes participate in at least 4 distinct protein complexes: the adaptor complex AP-3; biogenesis of lysosome-related organelles complex 1 (BLOC-1), consisting of 4 HPS proteins (pallidin, muted, cappuccino, HPS7/sandy); BLOC-2, consisting of HPS6/ruby-eye, HPS5/ruby-eye-2, and HPS3/cocoa; and BLOC-3, consisting of HPS1/pale ear and HPS4/light ear. Here, we report the cloning of the mouse HPS mutation reduced pigmentation (rp). We show that the wild-type rp gene encodes a novel, widely expressed 195-amino acid protein that shares 87% amino acid identity with its human orthologue and localizes to punctate cytoplasmic structures. Further, we show that phosphorylated RP is part of the BLOC-1 complex. In mutant rp/rp mice, a premature stop codon truncates the protein after 79 amino acids. Defects in all the 5 known components of BLOC-1, including RP, cause severe HPS in mice, suggesting that the subunits are nonredundant and that BLOC-1 plays a key role in organelle biogenesis.

Purpose: To compare clinically 2 different subtypes of Hermansky-Pudlak syndrome (HPS), type 1 (HPS-1) and type 3 (HPS-3)., Design: Cross-sectional study of a series of patients., Participants: Sixteen patients with HPS-1 and 14 patients with HPS-3 were studied., Methods: Complete eye examination, including best-corrected visual acuity and photographs and photographic grading of iris transillumination and macular transparency using a previously established grading system., Results: Snellen visual acuity was 20/160-2 in the HPS-1 group and 20/125+2 in the HPS-3 group (P = 0.017). Iris grading was statistically significant for less translucence in the HPS-3 patients. The HPS-3 patients also tended to have less transparent maculas, but the difference was not statistically significant., Conclusions: Patients with HPS-3 have less severe ophthalmic manifestations than patients with HPS-1. Ophthalmologists treating patients with albinism should consider HPS in their differential diagnosis even in the case of mild iris and macular hypopigmentation.

Hermansky-Pudlak syndrome (HPS) is a disorder of organelle biogenesis affecting 3 related organelles-melanosomes, platelet dense bodies, and lysosomes. Four genes causing HPS in humans (HPS1-HPS4) are known, and at least 15 nonallelic mutations cause HPS in the mouse. Where their functions are known, the HPS-associated proteins are involved in some aspect of intracellular vesicular trafficking, that is, protein sorting and vesicle docking and fusion. Biochemical and genetic evidence indicates that the HPS-associated genes encode components of at least 3 distinct protein complexes: the adaptor complex AP-3; the HPS1/HPS4 complex; and BLOC-1 (biogenesis of lysosome-related organelles complex-1), consisting of the proteins encoded at 2 mouse HPS loci, pallid (pa) and muted (mu), and at least 3 other unidentified proteins. Here, we report the cloning of the mouse HPS mutation cappuccino (cno). We show that the wild-type cno gene encodes a novel, ubiquitously expressed cytoplasmic protein that coassembles with pallidin and the muted protein in the BLOC-1 complex. Further, we identify a frameshift mutation in mutant cno/cno mice. The C-terminal 81 amino acids are replaced with 72 different amino acids in the mutant CNO protein, and its ability to interact in BLOC-1 is abolished. We performed mutation screening of patients with HPS and failed to identify any CNO defects. Notably, although defects in components of the HPS1/HPS4 and the AP-3 complexes are associated with HPS in humans, no defects in the known components of BLOC-1 have been identified in 142 patients with HPS screened to date, suggesting that BLOC-1 function may be critical in humans.

Objective: To describe and correlate pulmonary function and high-resolution CT (HRCT) scan scores in individuals with a high risk for development of pulmonary fibrosis, ie, Hermansky-Pudlak syndrome (HPS) patients with mutations in the HPS-1 gene., Design: Cross-sectional analysis of consecutive, eligible patients., Patients: Thirty-eight HPS inpatients at the National Institutes of Health Clinical Center with HPS-1 mutations., Results: Thirty-seven patients were Puerto Rican and exhibited the typical 16-base pair (bp) duplication in exon 15 of HPS-1. One non-Puerto Rican was homozygous for a different mutation (intervening sequence 17 -2 A-->C) previously reported in the HPS-1 gene; he died at age 35 of pulmonary insufficiency. For the 23 patients who had pulmonary symptoms, the mean age of onset was 35 years. For all 38 patients (mean age, 37 +/- 2 years), the mean FVC was 71% of predicted; the mean FEV(1), 76%; mean total lung capacity (TLC), 72%; mean vital capacity (VC), 68%; and mean diffusing capacity of the lung for carbon monoxide (DLCO), 72%. When patients were grouped according to the extent of their reduction in FVC, the other four pulmonary function parameters followed the FVC. Seventeen patients had abnormal chest radiographs, and 31 (82%) had abnormal HRCT scans of the chest, for which a scoring system of 0 (normal) to 3 (severe fibrosis) is presented. The mean +/- SEM HRCT score for 38 patients was 1.30 +/- 0.17. HRCT scores correlated inversely with FVC and DLCO., Conclusions: Mutations in the HPS-1 gene, whether or not they involve the typical 16-bp duplication seen in Puerto Rican patients, are associated with fatal pulmonary fibrosis. In affected patients, the FVC, FEV(1), TLC, VC, and DLCO fall in concert, and this functional deficit correlates with HRCT scan evidence of progression of interstitial lung disease.

Eukaryotic porins or VDACs (Voltage-Dependent Anion-selective Channels) are integral membrane proteins forming large hydrophilic pores. Three functioning genes for VDAC isoforms have been detected in mouse and the corresponding cDNAs are known also in humans. Tissue-specific VDAC isoform 1 (HVDAC1) deficiency in human skeletal muscle is responsible of a rare mitochondrial encephalomyopathy, fatal in childhood. Since coding sequences are not affected in the patient, we focused our interest in the gene structure. HVDAC1 and 2 have been previously mapped at chromosomes Xq13-21 and 21, respectively. Screening of an human chromosome X cosmid library resulted only in the isolation of processed pseudogenes, finely mapped at Xq22 and Xp11.2. Here, we report the mapping of HVDAC1 to chromosome 5q31 and HVDAC2 to chromosome 10q22 by FISH. Exon/intron probes, designed on the basis of the mouse gene structures, were obtained by long extension PCR amplification using the whole genomic DNA as a template. The sequence of the probe extremities clearly pointed to a genuine VDAC genomic sequence. Human and mouse regions where VDAC 1 and 2 genes were mapped are known to be synthetic, thus reinforcing the mapping of the human homologues., (Copyright 1999 Academic Press.)

Paclitaxel (Taxol(R)) is an active agent in platinum-refractory ovarian cancer. Since the available pharmacokinetic data of 135 mg m-2 paclitaxel administered by 3-h infusion are scarce and fragmented, we now describe a comprehensive pharmacologic study in a group of 13 patients who were pretreated with platinum for advanced ovarian cancer. The mean paclitaxel AUC was 10.3+/-2.4 h micromol l-1 (range 6.8-13.9 h micromol l-1). Quantification of the two major paclitaxel metabolites, 6alpha-hydroxypaclitaxel and 3'-p-hydroxypaclitaxel yielded AUCs of 0.44+/-0.30 h micromol l-1 and 0.31+/-0.20 h micromol l-1, respectively. The AUC of 3'-p-hydroxypaclitaxel was significantly different from that of patients with an altered hepatic function. The administration of 135 mg m-2 single-paclitaxel was safe, and the toxicities observed at higher doses in earlier studies were absent in this study. This is important, because the schedule and paclitaxel dose of 135 mg m-2 given by a 3-h infusion is expected to be used more frequently in combination with other cytotoxic agents with the aim of improving efficacy., (Copyright 1998 The Italian Pharmacological Society)

For the quantitative determination of paclitaxel in human plasma reversed-phase high-performance liquid chromatographic (HPLC) methods with solid-phase extraction (SPE) as sample pretreatment procedure are frequently used. Recovery problems arose during the quantification of paclitaxel in plasma samples of patients. The major problems were a large batch-to-batch difference in performance of the SPE columns and the effects of the pharmaceutical vehicle Cremophor EL on the performance of the SPE. Cremophor EL concentrations exceeding 1.0% (v/v) had a great impact on the absolute recovery of paclitaxel from human plasma with the SPE procedure. The recoveries decreased approximately 10 to 40% depending on the quality of the batch SPE columns. The problems are avoided by using 2'-methylpaclitaxel as the internal standard. This study points out the importance of including the effects of a pharmaceutical vehicle, like Cremophor EL, in the validation programme of a bioanalytical assay and the use of an internal standard in HPLC paclitaxel assays preceded by SPE as sample pretreatment procedure.

A sensitive and selective reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of polyoxyethyleneglycerol triricinoleate 35 (Cremophor EL; CrEL), which requires only microvolumes (20 microliters) of plasma, has been developed and validated. The procedure is based on saponification of CrEL in alcoholic KOH, followed by extraction of the released fatty acid ricinoleic acid with chloroform and derivatization with 1-naphthylamine. Margaric acid was used as the internal standard. The products are separated using an HPLC system consisting of an analytical column packed with Spherisorb ODS-1 material and a mobile phase of methanol-acetonitrile-10 mM potassium phosphate buffer pH 7.0 (72:13:15, v/v). Detection was executed by UV absorption at 280 nm. The lower limit of quantitation and the lower limit of detection in plasma are 0.01 and 0.005% (v/v) of CrEL, respectively. The percentage deviation and precision of the procedure, over the validated concentration range of 0.01 to 1.0% (v/v) of CrEL in plasma, are < or = 8.0% and < or = 6.6%, respectively. Compared to the previously described bioassay, the presented HPLC method possesses superior sensitivity and reliability. Preliminary pharmacokinetic studies of CrEL in mice and patients receiving paclitaxel formulated in CrEL have demonstrated the applicability of the presented assay.

A reversed-phase high-performance liquid chromatographic (HPLC) method has been validated for the quantitative determination of the three major paclitaxel metabolites (6 alpha-hydroxypaclitaxel, 3'-p-hydroxypaclitaxel, 6 alpha,3'-p-dihydroxypaclitaxel) in human plasma. The HPLC system consists of an APEX-octyl analytical column and acetonitrile-methanol-0.02 M ammonium acetate buffer pH 5 (AMW; 4:1:5, v/v/v) as the mobile phase. Detection is performed by UV absorbance measurement at 227 nm. The sample pretreatment of the plasma samples involves solid-phase extraction (SPE) on Cyano Bond Elut columns. The concentrations of the metabolic products could be determined by using the paclitaxel standard curve with a correction factor of 1.14 for 6 alpha,3'-p-dihydroxypaclitaxel. The recoveries of paclitaxel and the metabolites 6 alpha,3'-p-dihydroxypaclitaxel, 3'-p-hydroxypaclitaxel and 6 alpha-hydroxypaclitaxel in human plasma were 89, 78, 91 and 89%, respectively. The accuracy of the assay for the determination of paclitaxel and its metabolites varied between 95 and 97%, at a 50 ng/ml analyte concentration. The lower limit of quantitation was 10 ng/ml for both the parent drug and its metabolites.

Background: In advanced adenocarcinoma of the pancreas treatment with 5-fluorouracil (5-FU) or ifosfamide results in response rates of approximately 20%. Continuous infusion of these drugs is on many grounds theoretically attractive and may therefore offer advantages over bolus or short-term infusion., Patients and Methods: Sixteen patients with advanced adenocarcinoma of the pancreas with progressive measurable disease and no previous chemotherapy entered the study. After implantation of a subcutaneous infusion chamber patients were treated on days 1-12 with ifosfamide (1.0 g/m2/day) and 5-FU (300 mg/m2/day) as a continuous intravenous infusion using a portable infusion pump. Mesna (1.0 g/m2/day) was added as uroprotective agent from day 1-14. Courses were repeated every 4 weeks., Results: Fifteen of the 16 patients were evaluable for response. One partial response was observed (response rate 7% [95% CI: 0%-32%]). Toxicity occurred in 64% of the courses. Dose limiting toxic effects were grade 3 nausea/vomiting (WHO) in 3 patients, grade 2 mucositis in 1 patient and grade 4 leukopenia in 1 patient., Conclusion: Intermittent continuous infusion with ifosfamide, mesna and 5-FU is feasible on an outpatient basis. Although continuous infusion of ifosfamide may have a more favorable toxicity profile, the combination of 5-FU and ifosfamide in this schedule is no more effective than bolus or short-term infusion.

Background: Hepatic metabolism and biliary clearance play pivotal roles in the disposition of the anticancer drug paclitaxel. 6-alpha-hydroxypaclitaxel, 3'-p-hydroxypaclitaxel and 6-alpha,3'-p-dihydroxypaclitaxel were the major metabolic products of paclitaxel found in human bile. Recently, these metabolic products were detected in human plasma. The pharmacokinetics of paclitaxel and its metabolites were investigated in anthracycline-resistant breast cancer patients treated with high-dose paclitaxel and granulocyte colony-stimulating factor (G-CSF) support., Patients and Methods: Nine patients were entered into this study in which paclitaxel was administered at the relatively high dose of 250 mg/m2 during a 3-hour infusion. G-CSF was administered daily subcutaneously (s.c.)on days 2 to 19 following chemotherapy. Analysis of paclitaxel and metabolite concentrations was performed by a new highly sensitive reversed-phase high performance liquid chromatographic (HPLC) assay., Results: The dose-limiting toxicity in this study was cumulative neurotoxicity. One patient had a partial response and 2 patients had mixed responses of their skin metastases. Relatively low peak plasma concentration (Cmax), with mean values of 6.91 micromol/L (range 3.08 to 8.98) and area under the plasma concentration time curve (AUC), with mean values of 27.04 micromol/L.h (range 14.88 to 40.57), were observed. The total body clearance was 16.99 L/h (range, 10.25 to 27.39). The pharmacokinetic parameter for the prediction of leuko-neutropenia, the duration of the plasma concentration above the threshold of 0.1 micromol/L.h (T > or = 0.1 microM), was 19.72 h (range 10.54 to 26.31). The three major metabolites detected in human plasma were identified as 6-alpha-hydroxypaclitaxel, 3'-p-hydroxypaclitaxel and 6-alpha,3'-p-dihydroxypaclitaxel. Cmax and AUC values of these metabolites are reported., Conclusions: The three main metabolic products of paclitaxel in human plasma are 6-alpha-hydroxypaclitaxel, 3'-p-hydroxypaclitaxel and the dihydroxymetabolite 6-alpha,3'-p-dihydroxypaclitaxel. Two patients with liver function disturbances showed a tendency to higher paclitaxel and 6-alpha-hydroxypaclitaxel AUC levels, with more pronounced neuropathy.

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed and validated for the quantitative determination of paclitaxel in human urine. A comparison is made between solid-phase extraction (SPE) and liquid-liquid extraction (LLE) as sample pretreatment. The HPLC system consists of an APEX octyl analytical column and acetonitrile-methanol-0.2 microM ammonium acetate buffer pH 5 (4:1:5, v/v) as the mobile phase. Detection is performed by UV absorbance measurement at 227 nm. The SPE procedure involves extraction on Cyano Bond Elut columns. n-Butylchloride is the organic extraction fluid used for the LLE. The recoveries of paclitaxel in human urine are 79 and 75% for SPE and LLE, respectively. The accuracy for the LLE and SPE sample pretreatment procedures is 100.4 and 104.9%, respectively, at a 5 micrograms/ml drug concentration. The lower limit of quantitation is 0.01 microgram/ml for SPE and 0.25 microgram/ml for LLE. Stability data of paclitaxel in human urine are also presented.