The development of an inline Raman spectroscopic analysis method as a quality control tool for hot melt extruded ramipril fixed-dose combination products.

Currently in the pharmaceutical industry, continuous manufacturing is an area of significant interest. In particular, hot-melt extrusion (HME) offers many advantages and has been shown to significantly reduce the number of processing steps relative to a conventional product manufacturing line. To control product quality during HME without process interruption, integration of inline analytical technology is critical. Vibrational spectroscopy (Raman, NIR and FT-IR) is often employed and used for real-time measurements because of the non-destructive and rapid nature of these analytical techniques. However, the establishment of reliable Process Analytical Technology (PAT) tools for HME of thermolabile drugs is challenging. Indeed, the Raman effect is inherently weak and might be subject to interference. Moreover, during HME, heating and photodecomposition can occur and disrupt spectra acquisition. The aim of this research article was to explore the use of inline Raman spectroscopy to characterise a thermolabile drug, ramipril (RMP), during continuous HME processing. Offline measurements by HPLC, LC-MS and Raman spectroscopy were used to characterise RMP and its main degradation product, ramipril-diketopiperazine (RMP-DKP, impurity K). A set of HME experiments together with inline Raman spectroscopic analyses were performed. The feasibility of implementing inline Raman spectroscopic analysis to quantify the level of RMP and RMP-DKP in the extrudate was addressed. Two regions in the Raman spectrum were selected to differentiate RMP and RMP-DKP. When regions were combined, a principle component analysis (PCA) model defined by these two main components (PC 1 = 50.1% and PC 2 = 45%) was established. Using HPLC analyses, we were able to confirm that the PC 1 score was attributed to the level of RMP-DKP, and the PC 2 score was related to the RMP drug content. Investigation of the PCA scatterplot indicated that HME processing temperature was not the only factor causing RMP degradation. Additionally, the plasticiser content, feeding speed and screw rotating speed contributed to RMP degradation during HME processing.

Exploring the potential of neutron imaging for life sciences on IMAT.

Neutron imaging has been employed in life sciences in recent years and has proven to be a viable technique for studying internal features without compromising integrity and internal structure of samples in addition to being complementary to other methods such as X-ray or magnetic resonance imaging. Within the last decade, a neutron imaging beamline, IMAT, was designed and built at the ISIS Neutron and Muon Source, UK, to meet the increasing demand for neutron imaging applications in various fields spanning from materials engineering to biology. In this paper, we present the first neutron imaging experiments on different biological samples during the scientific commissioning of the IMAT beamline mainly intended to explore the beamline's capabilities and its potential as a noninvasive investigation tool in fields such as agriculture (soil-plants systems), palaeontology and dentistry. LAY DESCRIPTION: Neutrons form a highly penetrating radiation passing through matter without damaging or structurally modifying it, a property that makes them the ideal tool for many kinds of complementary material investigations. Moreover, the strong interaction of neutrons with hydrogen and their ability to distinguish between hydrogen and deuterium with no radiation damage make neutrons a good probe for imaging biological specimens. The recent technological developments of sources and detectors improved the capabilities of neutron imaging instruments and also have facilitated the use of neutron imaging on a much wider scale than before. Neutron imaging is proving its advantages as being complementary to other known methods of investigation such as X-ray imaging or magnetic resonance imaging and it is no surprise that it is not only employed in engineering or archaeology, but also in life sciences. This definitely opens new perspectives for a more interdisciplinary approach in contemporary science. Within the last decade a neutron imaging beamline, IMAT, was designed and built at the ISIS Neutron and Muon Source, UK, to meet the increasing demands of researchers from different fields, spanning from materials engineering to biology. The results presented here, acquired from first measurements on different biological samples during the scientific commissioning of IMAT beamline show the instrument capability and its suitability to palaeontology, agriculture (soil-plants systems) or dentistry applications.

A comparative study between hot-melt extrusion and spray-drying for the manufacture of anti-hypertension compatible monolithic fixed-dose combination products.

The purpose of this work was to investigate the application of different advanced continuous processing techniques (hot melt extrusion and spray drying) to the production of fixed-dose combination (FDC) monolithic systems comprising of hydrochlorothiazide and ramipril for the treatment of hypertension. Identical FDC formulations were manufactured by the two different methods and were characterised using powder X-ray diffraction (PXRD) and modulated differential scanning calorimetry (mDSC). Drug dissolution rates were investigated using a Wood's apparatus, while physical stability was assessed on storage under controlled temperature and humidity conditions. Interestingly both drugs were transformed into their amorphous forms when spray dried, however, hydrochlorothiazide was determined, by PXRD, to be partially crystalline when hot melt extruded with either polymer carrier (Kollidon® VA 64 or Soluplus®). Hot melt extrusion was found to result in significant degradation of ramipril, however, this could be mitigated by the inclusion of the plasticizer, polyethylene glycol 3350, in the formulation and appropriate adjustment of processing temperature. The results of intrinsic dissolution rate studies showed that hot-melt extruded samples were found to release both drugs faster than identical formulations produced via spray drying. However, the differences were attributable to the surface roughness of the compressed discs in the Wood's apparatus, rather than solid state differences between samples. After a 60-day stability study spray dried samples exhibited a greater physical stability than the equivalent hot melt extruded samples.

Sample environment for neutron scattering measurements of internal stresses in engineering materials in the temperature range of 6 K to 300 K.

Internal stresses in materials have a considerable effect on material properties including strength, fracture toughness, and fatigue resistance. The ENGIN-X beamline is an engineering science facility at ISIS optimized for the measurement of strain and stress using the atomic lattice planes as a strain gauge. Nowadays, the rapidly rising interest in the mechanical properties of engineering materials at low temperatures has been stimulated by the dynamic development of the cryogenic industry and the advanced applications of the superconductor technology. Here we present the design and discuss the test results of a new cryogenic sample environment system for neutron scattering measurements of internal stresses in engineering materials under a load of up to 100 kN and in the temperature range of 6 K to 300 K. Complete cooling of the system starting from the room temperature down to the base temperature takes around 90 min. Understanding of internal stresses in engineering materials at cryogenic temperatures is vital for the modelling and designing of cutting-edge superconducting magnets and other superconductor based applications.

Successful treatment of disseminated relapsed medulloblastoma in an infant by primary radiotherapy.

An infant who experienced disseminated relapse of medulloblastoma while receiving chemotherapy is described. He was subsequently treated with radiation therapy. Seven and one-half years from diagnosis, he is currently disease-free and enjoys a relatively normal life. We emphasize the importance of considering radiation as one of the treatment modalities for young children with relapsed medulloblastoma.

Hepatic dysfunction as the presenting feature of acute lymphoblastic leukemia.

PURPOSE: Hepatic dysfunction is a rare presentation of leukemia in children. Because most chemotherapy agents are metabolized by the liver, this complication may have major adverse consequences and effective treatment could be compromised. PATIENTS AND METHODS: The MEDLINE database and current management guidelines from the United States Pediatric Cooperative Cancer Groups were reviewed and analyzed. Data from two institutional cases are described. RESULTS: Although previous literature is not informative, our experience suggests that children with leukemia and moderate hepatic dysfunction may tolerate aggressive chemotherapy. CONCLUSION: Current protocol guidelines for dose modification for liver disease may be overly stringent and modification may be beneficial.

Bilateral breast relapse in acute myelogenous leukemia.

We present the case of an 11.5-year-old girl with M1 acute myelogenous leukemia (AML) who had isolated extramedullary relapse develop in both breasts 12 months after diagnosis and 7 months off chemotherapy. She received further chemotherapy, focal radiation therapy, then underwent a matched, unrelated bone marrow transplant and continues in remission 37 months later. Review of the literature revealed 10 cases in other children younger than 21-years-old with AML and breast involvement. These cases are summarized, and potential pathophysiologic mechanisms of spread are discussed. Breast involvement in AML is rare in children. However, regular breast examinations should be performed as part of routine follow-up in all girls with AML.

Myosin VI is required for asymmetric segregation of cellular components during C. elegans spermatogenesis.

BACKGROUND: The asymmetric division of cells and unequal allocation of cell contents is essential for correct development. This process of active segregation is poorly understood but in many instances has been shown to depend on the cytoskeleton. Motor proteins moving along actin filaments and microtubules are logical candidates to provide the motive force for asymmetric sorting of cell contents. The role of myosins in such processes has been suggested, but few examples of their involvement are known. RESULTS: Analysis of a Caenorhabditis elegans class VI myosin deletion mutant reveals a role for this motor protein in the segregation of cell components during spermatogenesis. Mutant spermatocytes cannot efficiently deliver mitochondria and endoplasmic reticulum/Golgi-derived fibrous-body membranous organelle complexes to budding spermatids, and fail to remove actin filaments and microtubules from the spermatids. The segregation defects are not due to a global sorting failure as nuclear inheritance is unaffected. CONCLUSIONS: C. elegans myosin VI has an important role in the unequal partitioning of both organelles and cytoskeletal components, a novel role for this class of motor protein.

Intracellular motility: how can we all work together?

Recent results reinforce the view that actin-based and microtubule-based motility systems do not operate independently, but are used in coordinated fashion to determine intracellular localization of cargo such as organelles.

Arp2/3 complex from Acanthamoeba binds profilin and cross-links actin filaments.

The Arp2/3 complex was first purified from Acanthamoeba castellanii by profilin affinity chromatography. The mechanism of interaction with profilin was unknown but was hypothesized to be mediated by either Arp2 or Arp3. Here we show that the Arp2 subunit of the complex can be chemically cross-linked to the actin-binding site of profilin. By analytical ultracentrifugation, rhodamine-labeled profilin binds Arp2/3 complex with a Kd of 7 microM, an affinity intermediate between the low affinity of profilin for barbed ends of actin filaments and its high affinity for actin monomers. These data suggest the barbed end of Arp2 is exposed, but Arp2 and Arp3 are not packed together in the complex exactly like two actin monomers in a filament. Arp2/3 complex also cross-links actin filaments into small bundles and isotropic networks, which are mechanically stiffer than solutions of actin filaments alone. Arp2/3 complex is concentrated at the leading edge of motile Acanthamoeba, and its localization is distinct from that of alpha-actinin, another filament cross-linking protein. Based on localization and actin filament nucleation and cross-linking activities, we propose a role for Arp2/3 in determining the structure of the actin filament network at the leading edge of motile cells.

Phase I/II study of idarubicin given with continuous infusion fludarabine followed by continuous infusion cytarabine in children with acute leukemia: a report from the Children's Cancer Group.

PURPOSE: The Children's Cancer Group (CCG) undertook a phase I study (CCG-0922) to determine a tolerable dose of idarubicin given with fludarabine and cytarabine in children with relapsed or refractory leukemia. The phase I study was extended to a limited phase II study to assess the activity of this combination in children with acute myelogenous leukemia (AML). PATIENTS AND METHODS: This was a multiinstitutional study within the CCG. Eleven patients were entered onto the phase I study: seven with AML, three with acute lymphoblastic leukemia (ALL), and one with chronic myelogenous leukemia (CML). The maximal-tolerated dose (MTD) of fludarabine and cytarabine determined in a previous study was a fludarabine loading dose (LD) of 10.5 mg/m2 followed by a continuous infusion (CI) of 30.5 mg/m2/24 hours for 48 hours, followed by cytarabine LD 390 mg/m2, then CI 101 mg/m2/h for 72 hours. Idarubicin was given at three dose levels: 6, 9, and 12 mg/m2 intravenously (I.V.) on days 0, 1, and 2. The phase II portion of the trial included 10 additional patients with relapsed or refractory AML. RESULTS: A dose of idarubicin 12 mg/m2/d for 3 days given in combination with fludarabine and cytarabine was tolerated. The major toxicity encountered was hematologic. Nonhematologic toxicities included transaminase elevations, hyperbilirubinemia, and infections. Eight of 10 patients with AML in the phase II portion (12 mg/m2 idarubicin) achieved a complete remission (CR). CONCLUSION: This combination is active in patients with relapsed or refractory AML. The major toxicity encountered is hematologic. This regimen may be useful therapy for AML and should be compared with standard induction therapy in children with newly diagnosed AML.

Actin' like actin?

The most biologically significant property of actin is its ability to self-associate and form two-stranded polymeric microfilaments. In living cells, these micro filaments form the actin cytoskeleton, essential for maintenance of the shape, passive mechanical properties and active motility of eukaryotic cells. Recently discovered actin-related proteins (ARPs) appear to share a common ancestor with conventional actin. At present, six classes of ARPs have been discovered, three of which have representatives in diverse species across eukaryotic phyla and may share functional characteristics with conventional actin. The three most ubiquitous ARPs are predicted to share a common core structure with actin and contain all the residues required for ATP binding. Surface residues involved in protein protein interactions, however, have diverged. Models of these proteins based on the atomic structure of actin provide some clues about how ARPs interact with each other, with conventional actin and with conventional actin-binding proteins.

Desmoplastic small round-cell tumor: prolonged progression-free survival with aggressive multimodality therapy.

PURPOSE: To test intensive alkylator-based therapy in desmoplastic small round-cell tumor (DSRCT). PATIENTS AND METHODS: Patients received the P6 protocol, which has seven courses of chemotherapy. Courses 1, 2, 3, and 6 included cyclophosphamide 4,200 mg/m2, doxorubicin 75 mg/m2, and vincristine (HD-CAV). Courses 4, 5, and 7 consisted of ifosfamide 9 g/m2 and etoposide 500 mg/m2 for previously untreated patients, or ifosfamide 12 g/m2 and etoposide 1,000 mg/m2 for previously treated patients. Courses started after neutrophil counts reached 500/microL and platelet counts reached 100,000/microL. Tumor resection was attempted. Post-P6 treatment options included radiotherapy and a myeloablative regimen of thiotepa (900 mg/m2) plus carboplatin (1,500 mg/m2), with stem-cell rescue. RESULTS: Ten previously untreated and two previously treated patients have completed therapy. The male-to-female ratio was 11:1. Ages were 7 to 22 years (median, 14). The largest masses were infradiaphragmatic (n = 11) or intrathoracic (n = 1). Other findings included serosal implants (n = 11), regional lymph node invasion (n = 8), ascites or pleural effusion (n = 7), and metastases to liver (n = 5), lungs (n = 4), distant lymph nodes (n = 3), spleen (n = 2), and skeleton (n = 2). Tumors uniformly responded to HD-CAV, but there were no complete pathologic responses. One patient died at 1 month from tumor-related Budd-Chiari syndrome. Of seven patients who achieved a complete remission (CR), five remain in CR 9, 12, 13, 33, and 38 months from the start of P6, one patient died of infection at 12 months (autopsy-confirmed CR), and one patient relapsed 4 months off therapy. Of four patients who achieved a partial remission (PR), one remains progression-free at 34 months and three developed progressive disease. Five patients received local radiotherapy: three were not assessable for response, but in two patients, antitumor effect was evident. Four patients received thiotepa/carboplatin: two were in CR and remain so, and two patients had measurable disease that did not respond. CONCLUSION: For control of DSRCT, our experience supports intensive use of HD-CAV, aggressive surgery to resect visible disease, radiotherapy to high-risk sites, and myeloablative chemotherapy with stem-cell rescue in selected cases.

Abscisic Acid-Induced Phosphoinositide Turnover in Guard Cell Protoplasts of Vicia faba.

Guard cell protoplasts of Vicia faba treated with 10 [mu]M (+)abscisic acid (ABA) in the light exhibited a 20% decrease in diameter within 1.5 h, from 24.1 to 19.6 [mu]m. Within 10 s of administration of ABA, a 90% increase in levels of inositol 1,4,5-trisphosphate was observed, provided that cells were treated with Li+, an inhibitor of inositol phosphatase activity, prior to incubation. Concomitantly, levels of 32P-labeled phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate decreased 20% compared to levels in control cells; levels of label in the membrane lipids phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol did not change significantly in response to ABA treatment. These results show that phosphoinositide turnover is activated in response to ABA in guard cells. We conclude that phosphoinositide signaling is likely to be a step in the biochemical cascade that couples ABA to guard cell shrinking and stomatal closure.

Sequences, structural models, and cellular localization of the actin-related proteins Arp2 and Arp3 from Acanthamoeba.

We cloned and sequenced the two actin-related proteins (Arps) present in the profilin-binding complex of Acanthamoeba (Machesky, L. M., S. J. Atkinson, C. Ampe, J. Vandekerckhove, and T. D. Pollard. 1994, J. Cell Biol. 127:107-115). The sequence of Arp2 is more similar to other Arp2s than to actin, while the sequence of Arp3 is more similar to other Arp3s than to actin. Phylogenetic analysis of all known Arps demonstrates that most group into three major families, which are likely to be shared across all eukaryotic phyla. Together with conventional actins, the Arps form a larger family distinct from structurally related ATPases such as Hsp70's and sugar kinases. Atomic models of the Arps based on their sequences and the structure of actin provide some clues about function. Both Arps have atoms appropriately placed to bind ATP and divalent cation. Arp2, but not Arp3, has a conserved profilin-binding site. Neither Arp has the residues required to copolymerize with actin, but an Arp heterodimer present in the profilin-binding complex might serve as a pointed end nucleus for actin polymerization. Both Acanthamoeba Arps are soluble in cell homogenates, and both are concentrated in the cortex of Acanthamoeba. The cellular concentrations are 1.9 microM Arp2 and 5.1 microM Arp3, substoichiometric to actin (200 microM) but comparable to many actin-binding proteins.

Prevalence of antibodies to hepatitis C virus in transfused children with cancer.

PURPOSE: Hepatitis C virus (HCV) transmission is a well-documented complication of blood transfusions, although data on transfused children with cancer is sparse. Using a newer assay for anti-HCV antibodies, the prevalence of HCV infection was determined in a population of children with cancer in the United States. PATIENTS AND METHODS: Forty-five transfused children with cancer were studied for evidence of HCV infection. Patients had not received chemotherapy for a mean of 2.3 years or transfusions for a mean of 3.1 years before being evaluated. Levels of serum aminotransferases [aspartate aminotransferase and alanine aminotransferase (ALT)], hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (HBsAb), and hepatitis B core antibody (HBcAb) were assessed. A second-generation enzyme immunoassay (EIA) was used to screen for anti-HCV antibodies. Positive EIAs were supplemented by a radioimmunoblot assay (RIBA-2). RESULTS: No patient tested positively for HBsAg, HBsAb, or HBcAb; four of 45 (8.9%) were positive for HCV antibodies by EIA. Three of the four (6.7% of the total) were also positive by RIBA-2 testing. The mean number of donor exposures was not significantly different between HCV-negative versus RIBA-2-positive patients (23.1 vs. 61.7, p = 0.16). ALT levels off therapy and peak ALT levels during therapy were significantly higher in the RIBA-2-positive group versus the HCV-negative group, although 36% of all patients (16 of 45) had at least one elevation in ALT greater than twice the upper limit of normal. All three RIBA-2-positive patients were transfused before institution of universal screening of blood donors for HCV in 1990 and had hepatomegaly noted at least once. CONCLUSIONS: We have identified a small group of children who may be at high risk for developing chronic active hepatitis and cirrhosis. Testing for HCV should be a routine part of long-term follow-up in children treated for cancer.