Liver complications of haemoglobin H disease in adults.

Haemoglobin H (HbH) disease is a type of non-transfusion-dependent thalassaemia. This cross-sectional study aimed at determining the prevalence and severity of liver iron overload and liver fibrosis in patients with HbH disease. Risk factors for advanced liver fibrosis were also identified. A total of 80 patients were evaluated [median (range) age 53 (24-79) years, male 34%, non-deletional HbH disease 24%]. Patients underwent 'observed' T2-weighted magnetic resonance imaging examination for liver iron concentration (LIC) quantification, and transient elastography for liver stiffness measurement (LSM) and fibrosis staging. In all, 25 patients (31%) had moderate-to-severe liver iron overload (LIC ≥7 mg/g dry weight). The median LIC was higher in non-deletional than in deletional HbH disease (7·8 vs. 2.9 mg/g dry weight, P = 0·002). In all, 16 patients (20%) had advanced liver fibrosis (LSM >7.9 kPa) and seven (9%) out of them had probable cirrhosis (LSM >11.9 kPa). LSM positively correlated with age (R = 0·24, P = 0·03), serum ferritin (R = 0·36, P = 0·001) and LIC (R = 0·28, P = 0·01). In multivariable regression, age ≥65 years [odds ratio (OR) 4·97, 95% confidence interval (CI) 1·52-17·50; P = 0·047] and moderate-to-severe liver iron overload (OR 3·47, 95% CI 1·01-12·14; P = 0·01) were independently associated with advanced liver fibrosis. The findings suggest that regular screening for liver complications should be considered in the management of HbH disease.

Half-life extension of peptidic APJ agonists by N-terminal lipid conjugation.

Agonism of the endothelial receptor APJ (putative receptor protein related to AT1; AT1: angiotensin II receptor type 1) has the potential to ameliorate congestive heart failure by increasing cardiac output without inducing hypertrophy. Although the endogenous agonist, pyr-apelin-13 (1), has shown beneficial APJ-mediated inotropic effects in rats and humans, such effects are short-lived given its extremely short half-life. Here, we report the conjugation of 1 to a fatty acid, providing a lipidated peptide (2) with increased stability that retains inotropic activity in an anesthetized rat myocardial infarction (MI) model. We also report the preparation of a library of 15-mer APJ agonist peptide-lipid conjugates, including adipoyl-γGlu-OEG-OEG-hArg-r-Q-hArg-P-r-NMeLeuSHK-G-Oic-pIPhe-P-DBip-OH (17), a potent APJ agonist with high plasma protein binding and a half-life suitable for once-daily subcutaneous dosing in rats. A correlation between subcutaneous absorption rate and lipid length/type of these conjugates is also reported.

Anti-PCSK9 antibody pharmacokinetics and low-density lipoprotein-cholesterol pharmacodynamics in nonhuman primates are antigen affinity-dependent and exhibit limited sensitivity to neonatal Fc receptor-binding enhancement.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as an attractive therapeutic target for cardiovascular disease. Monoclonal antibodies (mAbs) that bind PCSK9 and prevent PCSK9:low-density lipoprotein receptor complex formation reduce serum low-density lipoprotein-cholesterol (LDL-C) in vivo. PCSK9-mediated lysosomal degradation of bound mAb, however, dramatically reduces mAb exposure and limits duration of effect. Administration of high-affinity mAb1:PCSK9 complex (1:2) to mice resulted in significantly lower mAb1 exposure compared with mAb1 dosed alone in normal mice or in PCSK9 knockout mice lacking antigen. To identify mAb-binding characteristics that minimize lysosomal disposition, the pharmacokinetic behavior of four mAbs representing a diverse range of PCSK9-binding affinities at neutral (serum) and acidic (endosomal) pH was evaluated in cynomolgus monkeys. Results revealed an inverse correlation between affinity and both mAb exposure and duration of LDL-C lowering. High-affinity mAb1 exhibited the lowest exposure and shortest duration of action (6 days), whereas mAb2 displayed prolonged exposure and LDL-C reduction (51 days) as a consequence of lower affinity and pH-sensitive PCSK9 binding. mAbs with shorter endosomal PCSK9:mAb complex dissociation half-lives (<20 seconds) produced optimal exposure-response profiles. Interestingly, incorporation of previously reported Fc-region amino acid substitutions or novel loop-insertion peptides that enhance in vitro neonatal Fc receptor binding, led to only modest pharmacokinetic improvements for mAbs with pH-dependent PCSK9 binding, with only limited augmentation of pharmacodynamic activity relative to native mAbs. A pivotal role for PCSK9 in mAb clearance was demonstrated, more broadly suggesting that therapeutic mAb-binding characteristics require optimization based on target pharmacology.

A proprotein convertase subtilisin/kexin type 9 neutralizing antibody reduces serum cholesterol in mice and nonhuman primates.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates serum LDL cholesterol (LDL-C) by interacting with the LDL receptor (LDLR) and is an attractive therapeutic target for LDL-C lowering. We have generated a neutralizing anti-PCSK9 antibody, mAb1, that binds to an epitope on PCSK9 adjacent to the region required for LDLR interaction. In vitro, mAb1 inhibits PCSK9 binding to the LDLR and attenuates PCSK9-mediated reduction in LDLR protein levels, thereby increasing LDL uptake. A combination of mAb1 with a statin increases LDLR levels in HepG2 cells more than either treatment alone. In wild-type mice, mAb1 increases hepatic LDLR protein levels approximately 2-fold and lowers total serum cholesterol by up to 36%: this effect is not observed in LDLR(-/-) mice. In cynomolgus monkeys, a single injection of mAb1 reduces serum LDL-C by 80%, and a significant decrease is maintained for 10 days. We conclude that anti-PCSK9 antibodies may be effective therapeutics for treating hypercholesterolemia.

Intrathecal chemotherapy for hematologic malignancies: drugs and toxicities.

Intrathecal (IT) chemotherapy is an important component of the prophylaxis or treatment of hematologic malignancies in the central nervous system (CNS), especially in patients with acute lymphoblastic leukemia and aggressive lymphomas. Different regimens of IT chemotherapies have been formulated, often in conjunction with systemic high-dose chemotherapy leading to penetration of the drugs into the cerebrospinal fluid (CSF). The three commonest IT drugs are methotrexate, cytosine arabinoside (Ara-C), and corticosteroids. The CSF half-lives of methotrexate and Ara-C are much prolonged, a factor to be considered if these drugs are also administered systemically in high doses. Neurotoxicities attributed to IT chemotherapy have been reported, including spinal cord lesions, seizures, and encephalopathy. Spinal cord lesions, manifesting as tetraplegia, paraplegia, and cauda equina syndrome, are the commonest neurotoxicity. It is mostly related to combined IT methotrexate and Ara-C, or Ara-C as the sole IT agent when given at high doses or as a slow-release preparation. Cord lesions rarely recover and patients are left with motor deficits, bowel and urinary disabilities. Seizures and encephalopathy are reported in relatively fewer patients, with variable manifestations and prognosis. Knowledge of the pharmacokinetics, dosing schedules and potential toxicities of IT chemotherapeutic drugs is important in the design of CNS prophylaxis and treatment in hematologic malignancies.

4q loss is potentially an important genetic event in MM tumorigenesis: identification of a tumor suppressor gene regulated by promoter methylation at 4q13.3, platelet factor 4.

In this study, we have elucidated the chromosomal imbalances in the multistep pathogenesis and delineated several critical tumor suppressor gene (TSG) loci in multiple myeloma (MM). By using comparative genomic hybridization, allelotyping, and multicolor interphase fluorescence in situ hybridization, 5 MM cell lines and bone marrow CD138+ plasma cells from 88 Chinese patients with monoclonal gammopathy of undetermined significance (MGUS) and early and advanced stages of MM were investigated. In all MGUS and MM samples, chromosome copy number abnormalities were detected. A higher number of chromosomal imbalances and specific genetic alterations are involved in MGUS to MM transition (-6q, +3p, and +1p) and MM progression (+2p and +9q). In addition to -13q, we first found high frequencies (42% to 46%) of -4q involving high percentages (70% to 74%) of clonal plasma cells in both MGUS and MM, suggesting that inactivation of TSG in this region is also a potentially critical genetic event in MM tumorigenesis. By high-resolution allelotyping, we defined a common deletion region on 4q13.3 and found that a candidate TSG, platelet factor 4, was frequently silenced by promoter hypermethylation in MM (15 of 28) and MM cell lines (5 of 5). These data have opened up a new approach in the molecular targeting therapy and provide novel insights into MM tumorigenesis.

Use of the oral chelator deferiprone in the treatment of iron overload in patients with Hb H disease.

Seventeen non-transfusion-dependent Chinese haemoglobin H (Hb H) disease patients (age 29-76 years) with serum ferritin >900 microg/l were treated with deferiprone for up to 18 months. One patient withdrew and data from 16 patients were analysed. Sixteen other Hb H patients with ferritin <900 microg/l, matched for age and genotype, acted as controls. Treatment was well tolerated except for mild arthralgia. Serum ferritin fell with treatment, reaching significance at 6 and 18 months (from 1492.3 +/- 901.4 to 519.4 +/- 405.4 microg/l at 18 months, P = 0.0008). Nine of 16 patients had levels below 397 microg/l before 18 months. Serum ferritin remained stable 6 months after stopping treatment. In contrast, there was no change in ferritin levels in the control group. Magnetic resonance imaging was used for measurement of liver iron content. Spin echo T(1)-signal intensity ratio (T(1)-SIR) and gradient echo T(2)-signal intensity ratio (T(2)-SIR) increased with treatment. T(2)-SIR rose from 0.17 +/- 0.08 pretreatment to 0.58 +/- 0.50 at 2 years (P = 0.0055). Improvement occurred in 12 of 16 patients, reaching normal in three patients. Using echocardiography, peak early diastolic : late diastolic blood flow (E/A) remained unchanged with treatment, but isovolumic relaxation time (IVRT) was prolonged at 2 years indicating mild impairment of diastolic function. All systolic function parameters were normal. A longer treatment period is desirable to demonstrate improvement in cardiac function.

Hypertension in mice lacking the proatrial natriuretic peptide convertase corin.

Atrial natriuretic peptide (ANP) is a cardiac hormone that regulates blood pressure. In cardiomyocytes, the hormone is synthesized as a precursor, proatrial natriuretic peptide (pro-ANP), which is proteolytically converted to active ANP. Corin is a cardiac transmembrane serine protease that has been shown to process pro-ANP in vitro, but its physiological importance had not been established. Here, we show that corin-deficient (Cor-/-) mice develop normally during embryogenesis and survive to postnatal life. Cor-/- mice have elevated levels of pro-ANP but no detectable levels of ANP as compared with WT littermates. Infusion of an active recombinant soluble corin transiently restores pro-ANP conversion, resulting in the release of circulating biologically active ANP. Using radiotelemetry to assess blood pressure, we find that Cor-/- mice have spontaneous hypertension as compared with WT mice, and it is enhanced after dietary salt loading. Pregnant Cor-/- mice demonstrate late-gestation proteinuria and enhanced high blood pressure during pregnancy. In addition, Cor-/- mice exhibit cardiac hypertrophy resulting in a mild decline in cardiac function later in life. Thus, our data establish corin as the physiological pro-ANP convertase and indicate that corin deficiency may contribute to hypertensive heart disease.

Alterations of RAS signalling in Chinese multiple myeloma patients: absent BRAF and rare RAS mutations, but frequent inactivation of RASSF1A by transcriptional silencing or expression of a non-functional variant transcript.

The methylation status, mutation and expression of RASSF1A, and mutations of RAS and BRAF were studied in 52 patients with multiple myeloma (MM), one plasma cell leukaemia (PCL) patient and four MM-derived cell lines. Aberrant methylation of RASSF1A was found in nine of 32 MM patients and in one of four MM cell lines (U266), where the associated loss of transcription was reversible by demethylation treatment. RASSF1A transcription was further investigated on anti-CD138-sorted plasma cell-enriched bone marrow samples from 10 MM, one PCL and three reactive plasmacytosis patients. While the wild-type RASSF1A transcript was detected in all three reactive plasmacytosis and the PCL samples, we found no detectable wild-type transcripts in six of 10 MM samples studied. In two MM samples, only the non-functional variant transcript was detected, whereas the other four showed loss of transcription. In great contrast to western data, RAS mutations were identified in only four of 31 (13%) MM patients. While no RASSF1A or BRAF mutation (V599E) was detected in any of the primary MM studied (n = 21), the latter was found in the U266 cell line. Taken together, these data indicate that alterations of RAS signalling are critical in MM pathogenesis. In our current studies of Chinese MM patients, these alterations involved frequent RASSF1A inactivation (60%) as a result of transcriptional silencing or expression of a non-functional variant transcript.