Experimental verification of a non-invasive method to monitor the lateral pencil beam position in an anthropomorphic phantom for carbon-ion radiotherapy.

The dose conformation and the sparing of neighboring critical healthy structures are improved in carbon-ion beam radiotherapy in comparison to conventional photon radiotherapy. Inter and intrafractional plan adaptation strategies may preclude the quality assurance (QA) of the actually applied treatment plan before the treatment starts. Therefore, independent measurements of the positions of scanned pencil 12C ion beams are of interest in order to monitor the beam application during the treatment and the beam in the isocenter. In this work, secondary ions outgoing from a patient-like phantom are exploited for the assessment of the lateral pencil beam position in a clinic-like 12C treatment fraction. The experiment was performed at the Heidelberg Ion-Beam Therapy Center (HIT) in Germany. A carbon-ion treatment plan was used to treat a 100 cm3 tumor volume in the center of an Alderson head phantom. Two silicon pixel detectors based on the Timepix3 technology developed at CERN were operated in synchronization to detect and to track outgoing secondary ions. We established an analysis of the measured secondary ion track distribution which enabled us to follow the beam scanning movement of the carbon-ion pencil beam by assessing the lateral position of the single beam spots. The precision of the developed method was found to range from 0.84 mm to 2.59 mm. For beam energies greater than 197.58 MeV/n, the mean of absolute distances of the measured lateral pencil beam positions with respect to the pencil beam positions measured by the beam application system (averaged over each energy layer) were smaller than 2 mm. We conclude that the presented method has shown capabilities of monitoring the lateral pencil beam positions by means of secondary ions with precision and sensitivity of clinical interest.

Co-extruded solid solutions as immediate release fixed-dose combinations.

The aim of this study was to develop by means of co-extrusion a multilayer fixed-dose combination solid dosage form for oral application characterized by immediate release for both layers, the layers containing different drugs with different water-solubility. In this study polymers were selected which can be combined in a co-extruded dosage form. Several polymers were screened on the basis of their processability via hot-melt extrusion, macroscopic properties, acetylsalicylic acid (ASA) decomposition and in vitro drug release. ASA and fenofibrate (FF) were incorporated as hydrophilic and hydrophobic model drugs, respectively. Based on the polymer screening experiments Kollidon® PF 12 and Kollidon® VA 64 were identified as useful ASA carriers (core), while Soluplus®, Kollidon® VA 64 and Kollidon® 30 were applicable as FF carriers (coat). The combination of Kollidon® 30 (coat) with Kollidon® PF 12 or Kollidon® VA 64 (core) failed in terms of processability via co-extrusion. All other combinations (containing 20% ASA in the core and 20% FF in the coat) were successfully co-extruded (diameter core: 2mm/thickness coat: 1mm). All formulations showed good adhesion between core and coat. ASA release from the core was complete within 15-30 min (Kollidon® PF 12) or 30-60 min (Kollidon® VA 64), while FF release was complete within 20-30 min (Kollidon® VA 64) or 60 min (Soluplus®). Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) revealed that both drugs were molecularly dispersed in the carriers. Raman mapping exposed very little intermigration of both drugs at the interface. Fixed-dose combinations with good in vitro performance were successfully developed by means of co-extrusion, both layers providing immediate release.

Hot-melt co-extrusion for the production of fixed-dose combination products with a controlled release ethylcellulose matrix core.

In this study, hot-melt co-extrusion was evaluated as a technique for the production of fixed-dose combination products, using ethylcellulose as a core matrix former to control the release of metoprolol tartrate and a polyethylene oxide-based coat formulation to obtain immediate release of hydrochlorothiazide. By lowering the concentration of the hydrophilic additive polyethylene oxide in the plasticized ethylcellulose matrix or by lowering the drug load, the in vitro metoprolol tartrate release from the core was substantially sustained. The in vitro release of hydrochlorothiazide from the polyethylene oxide/polyethylene glycol coat was completed within 45 min for all formulations. Tensile testing of the core/coat mini-matrices revealed an adequate adhesion between the two layers. Raman mapping showed no migration of active substances. Solid state characterization indicated that the crystalline state of metoprolol tartrate was not affected by thermal processing via hot-melt extrusion, while hydrochlorothiazide was amorphous in the coat. These solid state characteristics were confirmed during the stability study. Considering the bioavailability of metoprolol tartrate after oral administration to dogs, the different co-extruded formulations offered a range of sustained release characteristics. Moreover, high metoprolol tartrate plasma concentrations were reached in dogs allowing the administered dose to be halved.

Co-extrusion as manufacturing technique for multilayer mini-matrices with dual drug release.

The aim of this work was to develop by means of co-extrusion a multilayered dosage form characterized by a dual release profile of the same drug. Co-extrudates consisted of two concentric polymer matrices: a core having a lipophilic character and a coat with a hydrophilic character. Diclofenac sodium (DS) was incorporated as model drug in both layers. Several polymers were screened on the basis of their processability via hot melt extrusion (HME) and in vitro drug release. Polymer combinations with suitable properties (i.e., similar extrusion temperature, appropriate drug release profile) were processed via co-extrusion. (Co-) extruded samples were characterized in terms of solid state (XRD, SEM), in vitro drug release, core/coat adhesion, and bioavailability. Based on the polymer screening, two polymer combinations were selected for co-extrusion: ethylcellulose (core) combined with Soluplus® (coat) and polycaprolactone (core) with PEO (coat). These combinations were successfully co-extruded. XRD revealed that DS remained crystalline during extrusion in ethylcellulose, Soluplus®, polycaprolactone, and PEO. The polycaprolactone/PEO combination could be processed at a lower temperature (70 °C), vs. 140 °C for ethylcellulose/Soluplus®. The maximum drug load in core and coat depended on the extrusion temperature and the die dimensions, while adhesion between core and coat was mainly determined by the drug load and by the extrusion temperature. In vitro drug release from the co-extruded formulations was reflected in the in vivo behavior: formulations with a higher DS content in the coat (i.e., faster drug release) resulted in higher Cmax and higher AUC values. Co-extrusion is a viable method to produce in a single step a multilayer dosage form with dual drug release.

The retrospective binning method improves the consistency of phase binning in respiratory-gated PET/CT.

This study assesses the accuracy of prospective phase-gated PET/CT data binning and presents a retrospective data binning method that improves image quality and consistency. Respiratory signals from 17 patients who underwent 4D PET/CT were analysed to evaluate the reproducibility of temporal triggers used for the standard phase-based gating method. Breathing signals were reprocessed to implement retrospective PET data binning. The mean and standard deviation of time lags between automatic triggers provided by the Real-time Position Management (RPM, Varian) gating device and inhalation peaks derived from respiratory curves were computed for each patient. The total number of respiratory cycles available for 4D PET/CT according to the binning mode (prospective versus retrospective) was compared. The maximum standardized uptake value (SUV(max)), biological tumour volume (BTV) and tumour trajectory measures were determined from the PET/CT images of five patients. Compared to retrospective binning (RB), prospective gating approach led to (i) a significant loss in breathing cycles (15%) and (ii) the inconsistency of data binning due to temporal dispersion of triggers (average 396 ms). Consequently, tumour characterization could be impacted. In retrospective mode, SUV(max) was up to 27% higher, where no significant difference appeared in BTV. In addition, prospective mode gave an inconsistent spatial location of the tumour throughout the bins. Improved consistency with breathing patterns and greater motion amplitude of the tumour centroid were observed with retrospective mode. The detection of the tumour motion and trajectory was improved also for small temporal dispersion of triggers. This study shows that the binning mode could have a significant impact on 4D PET images. The consistency of triggers with breathing signals should be checked before clinical use of gated PET/CT images, and our RB method improves 4D PET/CT image quantification.

Co-extrusion as manufacturing technique for fixed-dose combination mini-matrices.

The aim of this study was to develop a multilayer (core/coat) dosage form via co-extrusion, the core providing sustained drug release and the coat immediate drug release. In this study polymers were selected which can be combined in a co-extruded dosage form. Several thermoplastic polymers were hot-melt extruded and evaluated for processability and macroscopic properties (surface smoothness, die swell). Metoprolol tartrate (MPT) and hydrochlorothiazide (HCT) were incorporated as sustained and immediate release model drugs, respectively. Based on the polymer screening experiments a combination of polycaprolactone (core) and polyethylene oxide (coat) was selected for co-extrusion trials, taking into account their drug release profiles and extrusion temperature (70 °C). This combination (containing 10% HCT in the coat and 45% MPT in the core) was successfully co-extruded (diameter core: 3 mm/thickness coat: 0.5 mm). Adhesion between the two polymer layers was good. HCT release from the coat was complete within 30 min, while MPT release was sustained over 24 h (55%, 70%, 85% and 100% after 4, 8, 12 and 2 4h, respectively). DSC, XRD and Raman spectroscopy revealed that MPT remained crystalline during extrusion, whereas HCT was dissolved in the polyethylene oxide matrix. The in vivo study revealed no significant differences between the experimental formulation and the reference formulation (Zok-Zid tablet). Fixed-dose combination mini-tablets with good in vitro and in vivo performance were successfully developed by means of co-extrusion, using a combination of polycaprolactone and polyethylene oxide.

Raman spectroscopy for the in-line polymer-drug quantification and solid state characterization during a pharmaceutical hot-melt extrusion process.

The aim of this study was to evaluate the suitability of Raman spectroscopy as a Process Analytical Technology (PAT) tool for the in-line determination of the active pharmaceutical ingredient (API) concentration and the polymer-drug solid state during a pharmaceutical hot-melt extrusion process. For in-line API quantification, different metoprolol tartrate (MPT)--Eudragit® RL PO mixtures, containing 10%, 20%, 30% and 40% MPT, respectively, were extruded and monitored in-line in the die using Raman spectroscopy. A PLS model, regressing the MPT concentrations versus the in-line collected Raman spectra, was developed and validated, allowing real-time API concentration determination. The correlation between the predicted and real MPT concentrations of the validation samples is acceptable (R(2)=0.997). The predictive performance of the calibration model is rated by the root mean square error of prediction (RMSEP), which is 0.59%. Two different polymer-drug mixtures were prepared to evaluate the suitability of Raman spectroscopy for in-line polymer-drug solid state characterization. Mixture 1 contained 90% Eudragit® RS PO and 10% MPT and was extruded at 140°C, hence producing a solid solution. Mixture 2 contained 60% Eudragit® RS PO and 40% MPT and was extruded at 105°C, producing a solid dispersion. The Raman spectra collected during these extrusion processes provided two main observations. First, the MPT Raman peaks in the solid solution broadened compared to the corresponding solid dispersion peaks, indicating the presence of amorphous MPT. Second, peak shifts appeared in the spectra of the solid dispersion and solid solution compared to the physical mixtures, suggesting interactions between Eudragit® RS PO and MPT, most likely hydrogen bonds. These shifts were larger in the spectra of the solid solution. DSC analysis confirmed these Raman solid state observations and the interactions seen in the spectra. Raman spectroscopy is a potential PAT-tool for in-line determination of the API concentration and the polymer-drug solid state during pharmaceutical hot-melt extrusion.

Evaluation of the response to therapy of head and neck squamous cell carcinoma by using 3-[123I]iodo-L-alpha-methyl tyrosine and single photon emission tomography.

Early detection of residual tumour tissue offers the possibility for rapid administration of adjuvant treatment. Single photon emission tomography (SPET) with 3-[123I]iodo-L-alpha-methyl tyrosine (IMT) offers the ability to detect recurrence. The aim of this study was to carry out a prospective evaluation of sequential IMT SPET before and after primary therapy and to determine the best timing for scanning in order to establish the response to treatment. Sixteen consecutive patients with histologically proven head and neck cancer (HNC), who underwent IMT SPET before therapy, within 1 week of therapy, and 1 and 3 months after completion of primary therapy were included. Images were classified, according to clinical evaluation, as indicating a high likelihood (HL), intermediate likelihood (IL) and low likelihood (LL) that residual tumoural tissue was present. The definitive clinicopathological diagnosis and follow-up was considered as the 'gold standard'. Based on the definitive clinicopathological outcome, 10 of 16 patients were diagnosed with evidence of local tumour and six without. Nine of 10 patients with evidence of local tumour presented with an HL IMT SPET image after 3 months, seven of whom were from within the first week. In this group, 1/10 patients was considered clinically HS the first week and eventually 4/10 patients became HL, of which there were three at 3 months. Of the six patients diagnosed without local evidence of tumour, with an average follow-up of 15 months, 6/6 were clinically LL in the first week. Three of six had a consistently LL IMT SPET from within the first week. The three other patients had an HL scan the first week, of which one became IL. It is concluded that IMT SPET assessed the response to primary therapy most accurately 3 months after completion of therapy. An IMT SPET image that indicates a high likelihood of residual tumoural tissue may allow earlier stratification of the patients for secondary treatment. If negative, an IMT SPET can exclude residual tumoural tissue from within the first week after completion of therapy.

Diagnosis of recurrent head and neck squamous cell carcinoma with 3-[123I]iodo-L-alpha-methyltyrosine SPET.

The distribution of 3-[123I]iodo-L-alpha-methyltyrosine (123I-3-IMT) in the tumour region of 21 patients with clinically suspected recurrent squamous cell head and neck carcinoma was studied. Single-photon emission tomography (SPET) imaging of the head and neck region was performed 10 min after the injection of 130-170 MBq 123I-3-IMT using a dual-detector gamma camera. Images were interpreted visually and classified as positive or negative for recurrent disease. In addition, target to background ratios (T/B) were measured using semi-automated region of interest analysis. IMT-SPET results were compared with the data derived from clinicopathological follow-up. IMT-SPET detected recurrent disease in 14 of 15 patients (sensitivity 93%). T/B ratios ranged between 1.5 and 2.4 (mean 1.88). One patient with a small tumour (1.2 cm) had a false-negative result. This is attributed to the limited spatial resolution of the SPET system. Five of six patients were correctly diagnosed to be negative for tumour recurrence. T/B ratios ranged between 1.2 and 1.4 (mean 1.30). In one patient IMT-SPET was positive without evidence of recurrence based on clinicopathological follow up. This finding was probably due to uptake into inflammatory tissue. IMT-SPET appears to be a sensitive tool (93%) for the detection of recurrent head and neck squamous cell carcinoma. Further studies with 123I-3-IMT as a metabolic tracer for the detection of head and neck cancer recurrence using SPET are recommended.

In vitro characterization of the influx of 3-[125I]iodo-L-alpha-methyltyrosine and 2-[125I]iodo-L-tyrosine into U266 human myeloma cells: evidence for system T transport.

The aim of this study was to investigate the cellular uptake mechanisms responsible for the accumulation of 3-[(125)I]iodo-L-alpha-methyltyrosine ((125)I-3-IMT) and 2-[(125)I]iodo-L-tyrosine ((125)I-2-IT), two radiotracers for metabolic tumor imaging, using single-photon emission tomography, into U266 human myeloma cancer cells. Time course and concentration dependency of (125)I-3-IMT uptake was assessed. Kinetic parameters were calculated using an Eadie Hofstee plot. A set of competitive inhibitors of the main amino acid transport systems was used for the discrimination of the transporters responsible for the uptake of (125)I-3-IMT and (125)I-2-IT. Protein incorporation of both tracers was determined using acid precipitation. The measured maximum velocity for (125)I-3-IMT transport was 4.199 nmol per mg protein 20 s(-1), and the Michaelis constant was 107.9 microM. Addition of 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH), a competitive inhibitor of System L, reduced the influx by 39.0+/-3.3% for (125)I-3-IMT and 66.3+/-0.9% for (125)I-2-IT. The BCH-insensitive influx was further reduced by Tryptophan (Trp) by 43.8+/-3.5% for (125)I-3-IMT and 15.3+/-1.3% for (125)I-2-IT. This suggests involvement of System T transport. We measured <2% of radioactivity in the acid precipitable fractions of both tracers with no increase in time. We conclude that the influx of (125)I-3-IMT and (125)I-2-IT into U266 human myeloma cells is mediated by both System L and System T amino acid transporters. The kinetic parameters suggest that elevated plasma levels of aromatic amino acids will reduce (123)I-3-IMT uptake in myeloma patients. Both tracers do not enter protein synthesis significantly.

Fascial Tc-99m MDP uptake in eosinophilic fasciitis as demonstrated by SPECT.

BACKGROUND: The presence of bone-seeking radiopharmaceutical uptake in extraskeletal tissues of the lower or upper extremities may indirectly reflect the presence of active inflammatory lesions in patients in whom systemic disease is suspected. MATERIALS AND METHODS: The authors present the case of a 26-year-old woman who had mixed signs of scleroderma and cosinophilic fasciitis, in whom misleading findings on planar bone scintigraphy suggested diffuse muscular tracer uptake in the lower extremities. RESULTS: However, using additional SPECT imaging of the pelvis and thighs, it was shown that the soft tissue radioactivity was clearly restricted to the fascia overlying the muscles. The fascial localization of the inflammation was confirmed by biopsy. CONCLUSION: SPECT imaging was proven useful in indicating the exact localization of an active inflammatory process in the muscle fascia.

Purification and properties of the exocellular beta-lactamase of Actinomadura strain R39.

The exocellular beta-lactamase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) of Actinomadura R39 consists of one single polypeptide chain of molecular weight about 15 200. It exhibits a highly asymmetrical shape, has a low isoelectric point (at pH 5.0) and contains about 9.3% (w/w) of a polydeoxyribonucleotide with which it forms a rather stable complex. Removal of a substantial amount of this deoxyribonucleotide by treatment with DNAase I has no effect on the enzyme activity. The beta-lactamase has a wide spectrum of activity. Penicillins and delta 3-cephalosporins can be either good or poor substrates. Oxacillin, which is a poor substrate of most beta-lactamases from Gram-positive bacteria, is a good substrate of the beta-lactamase of Actinomadura R39. Its best substrate, however, is nitrocefin (kcat/Km: 2300 000 M-1.s-1; catalytic centre activity: 210 s-1). The kcat/Km values observed with some penicillins and delta 3-cephalosporins are similar to the values of the bimolecular rate constants that govern the formation of the acyl-enzyme intermediates between these antibiotics and the serine D-alanyl-D-alanine peptidase that is also secreted by the same strain Actinomadura R39. Such a relationship, however, is not observed with all the beta-lactam compounds tested.

The exocellular beta-lactamase of Streptomyces albus G. Purification, properties and comparison with the exocellular DD-carboxypeptidase.

The exocellular beta-lactamase of Streptomyces albus G has been purified to near protein homogeneity. It consists of one single polypeptide chain of mol.wt. 30 000-31 000, has a rather low isoelectric point (at pH 6.0) and contains less lysine (2.1%) and more half-cystine residues than most beta-lactamases from other Gram-positive bacteria. Penicillins are much better substrates than delta 3-cephalosporins; the catalytic-centre activity of good penicillin substrates is 333-500 s-1. The exocellular, mol.wt. 17 000 DD-carboxypeptidase of S. albus G [previously purified to protein homogeneity; Duez, Frère, Geurts, Ghuysen, Dierickx & Delcambe (1978) Biochem. J. 175, 793-800] behaves as an exceedingly poor beta-lactamase, hydrolysing benzylpenicillin into benzylpenicilloate 5 x 10(-6)-fold less rapidly than does the exocellular beta-lactamase. To all appearances, the beta-lactamase has no bivalent cation requirement whereas, as shown elsewhere [Dideberg, Charlier, Dupont, Vermeire, Frère & Ghuysen (1980) FEBS Lett. 117, 212-214, and Dideberg, Joris, Frère, Ghuysen, Weber, Robaye, Delbrouck & Roelands (1980) FEBS Lett. 117, 215-218], the DD-carboxypeptidase possesses one essential Zn2+ ion per molecule. Peptide 'mapping' and immunological studies suggest that the two Streptomyces enzymes probably have very different structural and mechanistic properties.

Characterization of a new antibiotic of iturin group: bacillomycin D.

The characterization of an antibiotic isolated from a strain of Bacillus subtilis revealed that this compound is a new antifungal antibiotic of the iturin group. It contains a lipid moiety which is a mixture of 3-amino 12-methyl tridecanoic acid (40%) and 3-amino 12-methyl tetradecanoic acid (60%) and a peptide moiety: L-Asp1, D-Asp1, L-Glu1, L-Pro1, D-Ser1, L-Thr1 and D-Tyr1. These two moieties are joined by a threonyl-beta-aminoacid linkage.

The exocellular DD-carboxypeptidase-endopeptidase from Streptomyces albus G. Purification and chemical properties.

The exocellular DD-carboxypeptidase-endopeptidase of Streptomyces albus G was purified to protein homogeneity and compared with the exocellular DD-carboxypeptidases-transpeptidases of Streptomyces R61 and Actinomadura R39. The S. albus G enzyme, as it is isolated, occurs in two forms. Enzyme I (30% of the total amount) and enzyme II (70% of the total amount) are identical in all respects, except that, by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, enzyme I has an apparent mol. wt. (9000) that is half of that found by molecular-sieve filtration under non-denaturing conditions. Irrespective of the technique used, enzyme II has an apparent mol. wt. of about 18500.

Impairment of the M-protein and unmasking of a superficial type-specific antigen by proteolytic treatment of influenza A virions with preservation of host-specific antigenicity.

Influenza PR8 particles resulting from strong treatment with caseinase C are spikeless, devoid of neuraminidase and hemagglutinin 1 and 2 glycopeptides, and contain a Schiff-negative polypeptide of about 13,000 molecular weight which exists as traces in intact virions. Their M-protein polypeptide content is reduced to 50% of its original value, but there is no evidence of particle disruption nor of lipid release. They fix complement in the presence of both anti-M-protein antiserum and antiserum raised against a host polysaccharide. During exposure to caseinase C, an antigen is unmasked. It is type-specific and its identity with the M-protein is discussed.