Characterization of osthenol metabolism in vivo and its pharmacokinetics.

Osthenol, a prenylated coumarin, is a C8-prenylated derivative of umbelliferone isolated from the root of Angelica koreana and Angelica dahurica, an intermediate and is known as a major metabolite of desmethyl-osthole.The various pharmacological effects of osthenol have been reported. In previous studies, we investigated five hydroxylated metabolites by cytochromes P450 (CYP) and glucuronide conjugates of osthenol by uridine diphosphate-glucuronosyltransferases (UGTs). However, osthenol have very few studies have been reported on its pharmacokinetic (PK) profiling, we reported the PK parameters in mouse of osthenol through this study.After oral (5 and 20 mg/kg) and intravenous (5 mg/kg) administration, the concentration of osthenol in plasma was determined by LC-MS/MS. The quantitative method was validated in terms of linearity, accuracy, and precision. When 5 and 20 mg/kg of osthenol were orally administered, the bioavailability (BA) was found to be very low at 0.43 and 0.02%, respectively.In fact, osthenol was mostly metabolized to a two-Phase II conjugates, a sulfonyl and glucuronyl-osthenol, in the blood, which was determined by LC-HR/MS analysis of the blood sample. Because osthenol is rapidly metabolized to two conjugates by first-pass effect the BA of osthenol is low after oral administration.

Comparison of target coverage and dose to organs at risk between simultaneous integrated-boost whole-field intensity-modulated radiation therapy and junctioned intensity-modulated radiation therapy with a conventional radiotherapy field in treatment of nasopharyngeal carcinoma.

We have retrospectively investigated 15 nasopharyngeal carcinoma patients treated at our institution between March 2007 and August 2009. We used simultaneous integrated-boost whole field intensity-modulated radiation therapy (SIB WF-IMRT) to treat the entire planning target volume in the head and neck cancer. All of the SIB WF-IMRT plans were replanned by use of the junctioned intensity modulated radiation therapy (J-IMRT) technique for comparison. The effect on target coverage and sparing of organs at risk, including laryngeal sparing in the optimal SIB WF-IMRT plan was compared with that achieved with use of the J-IMRT technique. The mean larynx dose and standard deviation was 25.2 ± 5.8 Gy for SIB WF-IMRT and 19.8 ± 16.8 Gy for J-IMRT. A comparison between SIB WF-IMRT and the J-IMRT technique demonstrated that the larynx dose was increased in SIB WF-IMRT. However, when the strong dose constraint was applied to the larynx and the pseudo-volume was used for a steep dose fall-off immediately outside the target, the SIB WF-IMRT technique would have led to a larynx dose comparable to that achieved with J-IMRT. Therefore, in our current practice we use the SIB WF-IMRT technique, which does not have the problem of setup error at the match line for treatment of nasopharyngeal carcinoma.

Proton range uncertainty due to bone cement injected into the vertebra in radiation therapy planning.

We wanted to evaluate the influence of bone cement on the proton range and to derive a conversion factor predicting the range shift by correcting distorted computed tomography (CT) data as a reference to determine whether the correction is needed. Two CT datasets were obtained with and without a bone cement disk placed in a water phantom. Treatment planning was performed on a set of uncorrected CT images with the bone cement disk, and the verification plan was applied to the same set of CT images with an effective CT number for the bone cement disk. The effective CT number was determined by measuring the actual proton range with the bone cement disk. The effects of CT number, thicknesses, and position of bone cement on the proton range were evaluated in the treatment planning system (TPS) to draw a conversion factor predicting the range shift by correcting the CT number of bone cement. The effective CT number of bone cement was 260 Hounsfield units (HU). The calculated proton range for native CT data was significantly shorter than the measured proton range. However, the calculated range for the corrected CT data with the effective CT number coincided exactly with the measured range. The conversion factor was 209.6 [HU · cm/mm] for bone cement and predicted the range shift by approximately correcting the CT number. We found that the heterogeneity of bone cement could cause incorrect proton ranges in treatment plans using CT images. With an effective CT number of bone cement derived from the proton range and relative stopping power, a more actual proton range could be calculated in the TPS. The conversion factor could predict the necessity for CT data correction with sufficient accuracy.

Simultaneous integrated boost intensity-modulated radiotherapy in patients with high-grade gliomas.

PURPOSE: We analyzed outcomes of simultaneous integrated boost (SIB) intensity-modulated radiotherapy (IMRT) in patients with high-grade gliomas, compared with a literature review. METHODS AND MATERIALS: Forty consecutive patients (WHO grade III, 14 patients; grade IV, 26 patients) treated with SIB-IMRT were analyzed. A dose of 2.0 Gy was delivered to the planning target volume with a SIB of 0.4 Gy to the gross tumor volume with a total dose of 60 Gy to the gross tumor volume and 50 Gy to the planning target volume in 25 fractions during 5 weeks. Twenty patients received temozolomide chemotherapy. RESULTS: At a median follow-up of 13.4 months (range, 3.7-55.9 months), median survival was 14.8 months. One- and 2-year survival rates were 78% and 65%, respectively, for patients with grade III tumors and 56% and 31%, respectively, for patients with grade IV tumors. Age (≤50 vs. >50), grade (III vs. IV), subtype (astrocytoma vs. oligodendroglioma or mixed), and a Zubrod performance score (0-1 vs. >2) were predictive of survival. Of 25 (63%) patients who had recurrences, 17 patients had local failure, 9 patients had regional failure, and 1 patient had distant metastasis. Toxicities were acceptable. CONCLUSIONS: SIB-IMRT with the dose/fractionation used in this study is feasible and safe, with a survival outcome similar to the historical control. The shortening of treatment time by using SIB-IMRT may be of value, although further investigation is warranted to prove its survival advantage.

Dosimetric comparison of four different external beam partial breast irradiation techniques: three-dimensional conformal radiotherapy, intensity-modulated radiotherapy, helical tomotherapy, and proton beam therapy.

BACKGROUND AND PURPOSE: As an alternative to whole breast irradiation in early breast cancer, a variety of accelerated partial breast irradiation (APBI) techniques have been investigated. The purpose of our study is to compare the dosimetry of four different external beam APBI (EB-APBI) plans: three-dimensional conformal radiation therapy (3D-CRT), intensity-modulated radiation therapy (IMRT), helical tomotherapy (TOMO), and proton beam therapy (PBT). METHODS AND MATERIALS: Thirty patients were included in the study, and plans for four techniques were developed for each patient. A total dose of 30Gy in 6Gy fractions once daily was prescribed in all treatment plans. RESULTS: In the analysis of the non-PTV breast volume that was delivered 50% of the prescribed dose (PD), PBT (mean: 16.5%) was superior to TOMO (mean: 22.8%), IMRT (mean: 33.3%), and 3D-CRT (mean: 40.9%) (p<0.001). The average ipsilateral lung volume percentage receiving 20% of the PD was significantly lower in PBT (0.4%) and IMRT (2.3%) compared with 3D-CRT (6.0%) and TOMO (14.2%) (p<0.001). The average heart volume percentage receiving 20% and 10% of the PD in left-sided breast cancer (N=19) was significantly larger with TOMO (8.0%, 19.4%) compared to 3D-CRT (1.5%, 3.1%), IMRT (1.2%, 4.0%), and PBT (0%, 0%) (p<0.001). CONCLUSIONS: All four EB-APBI techniques showed acceptable coverage of the PTV. However, effective non-PTV breast sparing was achieved at the cost of considerable dose exposure to the lung and heart in TOMO.

Performance evaluation of field-in-field technique for tangential breast irradiation.

Conventional hard or dynamic wedge systems are commonly applied to reduce the dose inhomogeneity associated with whole breast irradiation. We evaluated the dosimetric benefits of the field-in-field (FIF) technique by comparing it with the electronic compensator (EC), Varian enhanced dynamic wedge (EW) and conventional hard wedge (HW) techniques. Data were obtained from 12 patients who had undergone breast-conserving surgery (six left-sided and six right-sided). For these patients, the average breast planning target volume (PTV) was 447.4 cm(3) (range, 211.6-711.8 cm(3)). For the experiments, a 6 MV photon beam from a Varian 21 EX was used, the HW and EW angles were applied from 15 to 45 degrees, while 40-50% isodose values were chosen to achieve the best dose distribution for electronic compensation. In applying the FIF technique, we used two or three subfields for each portal. To evaluate the performance for each planning technique, we analysed a dose-volume histogram (DVH) for the PTV and organs-at-risk (OARs). To evaluate the effects of these techniques on dose inhomogeneity, we defined the PTV Dose Improvement (PDI) index, which was derived from a PTV volume between 97-103% of the differential DVHs. In addition, we compared the average monitor units (MUs) for each technique. The average PDI index with FIF is 76.4%, while the PDI indices for other treatments were 65.8, 41.8 and 50.9% for EC, EW and HW, respectively. This study demonstrated an improved performance using the FIF technique compared with the conventional HW/EW system, as well as a new modality for EC. We demonstrated that FIF is a very useful technique for improving PTV conformity, while protecting the OARs from breast tangential irradiation.

Carbonic anhydrase 9 (CA9) expression in tumor cells enhances sensitivity to tirapazamine.

PURPOSE: Carbonic anhydrase 9 (CA9) is over-expressed in many human solid tumors under conditions of low oxygen concentration and can be associated with a low probability of survival. In this study, stable CA9-expressing cell lines were established using the CA9 gene-defective human C33a cell line and the HeLa cell line to investigate the role of CA9 in response to ionizing radiation and hypoxia-selective cytotoxin, Tirapazamine (TPZ). METHODS AND MATERIALS: Human CA9 cDNA or an empty vector was transfected into the C33a and HeLa cell lines and C33a-vector, C33a-CA9, HeLa-vector, and HeLa-CA9 cell lines were produced accordingly. Sensitivity of the C33a-vector/C33a-CA9 cells to ionizing radiation and TPZ was measured using clonogenic assays. The alkaline comet assay was used to measure single strand DNA breaks caused by TPZ in the C33a-vector, C33a-CA9, HeLa-vector, and HeLa-CA9 cell lines. RESULTS: Radiation sensitivity, as determined with clonogenic survival assays, of C33a-vector/C33a-CA9 cells did not differ under either normoxic or hypoxic conditions. However, increased clonogenic sensitivity to TPZ was observed in C33a-CA9 cells under the hypoxic condition by 26% (95% CI 14-39%, P = 0.02 in comparison to the C33a-vector cells). The comet assay showed significantly greater DNA damage in the C33a-CA9 cells compared with that of the C33a-vector cells with the same treatment under hypoxic conditions, supporting the results of the clonogenic survival data. Because this difference in the amount of DNA damage was not observed for the hypoxic HeLa-CA9/HeLa-vector cell lines, both of which have induced CA9 expression by hypoxia, the enhanced sensitivity of C33a-CA9 cells to TPZ is considered to be due to the specific condition of CA9 over-expression. CONCLUSION: Our results suggest the possibility that CA9 over-expression in tumors might be exploited to increase the treatment effects of TPZ.

Effect of radiation scattering on dose uniformity in open and closed cell culture vessels.

PURPOSE: Dose uniformity in cell culture vessels such as Petri dishes and anoxic irradiation chambers is very important in radiobiological work as dose uniformity affects cell survival probabilities. In this study, we investigated X-ray dose inhomogeneity, caused by scattering, in typical culture vessels. MATERIALS AND METHODS: Three different cubic cell culture vessels, with side lengths of 10 cm, 15 cm and 20 cm, were designed and irradiated by X-rays of 6 MV and 15 MV at a source-to-surface distance (SSD) of 100 cm using a Varian 2,100CD linear accelerator. RESULTS: The relative X-ray dose distribution in a cell culture vessel depended strongly on whether the vessel had a lid. The percentage of the cell culture surface with the dose differing by more than 10% from the mean value of the dose was 43.4% in lidless vessels and 9.7% in lidded vessels. CONCLUSIONS: In radiobiological work, X-ray dose inhomogeneity within a cell culture vessel is not negligible and the placement of cells in the vessel should be carefully considered.

Accuracy of inhomogeneity correction algorithm in intensity-modulated radiotherapy of head-and-neck tumors.

We examined the degree of calculated-to-measured dose difference for nasopharyngeal target volume in intensity-modulated radiotherapy (IMRT) based on the observed/expected ratio using patient anatomy with humanoid head-and-neck phantom. The plans were designed with a clinical treatment planning system that uses a measurement-based pencil beam dose-calculation algorithm. Two kinds of IMRT plans, which give a direct indication of the error introduced in routine treatment planning, were categorized and evaluated. The experimental results show that when the beams pass through the oral cavity in anthropomorphic head-and-neck phantom, the average dose difference becomes significant, revealing about 10% dose difference to prescribed dose at isocenter. To investigate both the physical reasons of the dose discrepancy and the inhomogeneity effect, we performed the 10 cases of IMRT quality assurance (QA) with plastic and humanoid phantoms. Our result suggests that the transient electronic disequilibrium with the increased lateral electron range may cause the inaccuracy of dose calculation algorithm, and the effectiveness of the inhomogeneity corrections used in IMRT plans should be evaluated to ensure meaningful quality assurance and delivery.

Effects of static dosimetric leaf gap on MLC-based small-beam dose distribution for intensity-modulated radiosurgery.

The aim of the present study was to evaluate the effect of various specific dosimetric leaf gaps on the multileaf collimator (MLC)-based small-beam dose distribution. The dosimetric static leaf gap was determined by comparing the profiles of small MLC-based beams with those of small collimated fields (square fields of 1, 2, 3, and 4 cm). The results showed that an approximately 2-mm gap was optimal with the Millennium 120-leaf MLC (Varian Medical Systems, Palo Alto, CA) and a Varian 21EX 6-MV photon beam. We also investigated how much the leaf gap affects the planning results and the actual dose distribution. A doughnut-shaped planning target volume (PTV, 6.1 cm3) and inner organ at risk (OAR, 0.3 cm3) were delineated for delicate intensity-modulated radiosurgery test planning. The applied leaf gaps were 0, 1, and 2 mm. The measured dose distributions were compared with the dose distribution in the treatment planning system. The maximum dose differences at inside PTV, outside PTV, and inner OAR were, respectively, 22.3%, 20.2%, and 35.2% for the 0-mm leaf gap; 17.8%, 22.8%, and 30.8% for the 1-mm leaf gap; and 5.5%, 8.5%, and 6.3% for the 2-mm leaf gap. In a human head phantom (model 605: CIRS, Norfolk, VA) study, large dose differences of 1.3%-12.7% were noted for the measurements made using the MLC files generated by the three different leaf gaps. The planned results were similar, and measurements showed a large dose difference associated with the various leaf gaps. These results strongly suggest that plans generated by a commercial inverse planning system commissioned using general collimated field data will probably demonstrate discrepancies between the planned treatments and the measured results.

Novel glycine transporter type-2 reuptake inhibitors. Part 1: alpha-amino acid derivatives.

A variety of alpha-amino acid derivatives were prepared as glycine transport inhibitors and their ability to block the uptake of [(14)C]-glycine in COS7 cells transfected with human glycine transporter-2 (hGlyT-2) was evaluated. An array of substituents at the chiral center was studied and overall, L-phenylalanine was identified as the preferred amino acid residue. Compounds prepared from l-amino acids were more potent GlyT-2 inhibitors than analogs derived from the corresponding d-amino acids. Introducing an achiral amino acid such as glycine, or incorporating geminal substitution in the alpha-position, led to a significant reduction in GlyT-2 inhibitory properties.

Novel glycine transporter type-2 reuptake inhibitors. Part 2: beta- and gamma-amino acid derivatives.

Several beta- and gamma-amino acid derivatives were prepared as glycine transport inhibitors and their ability to block the uptake of [(14)C]-glycine in COS7 cells transfected with human glycine transporter-2 (hGlyT-2) were evaluated. A range of lipophilic side chains were tolerated in the beta-amino acid series (i.e., Ph, CH(2)Ph, CH(CH(3))(2), and CH(2)CH(CH(3))(2)). In the gamma-amino acid series, minimal differences in potency were observed between the alpha,beta-unsaturated analogs and the corresponding saturated derivatives. In both series, a 4-biphenyl or 4-phenoxyphenyl substituent appended to the urea or cyanogunaidine moiety was necessary for in vitro activity.

Neuronal hyperpolarization-activated pacemaker channels drive neuropathic pain.

Neuropathic pain is a common and often incapacitating clinical problem for which little useful therapy is presently available. Painful peripheral neuropathies can have many etiologies, among which are trauma, viral infections, exposure to radiation or chemotherapy, and metabolic or autoimmune diseases. Sufferers generally experience both pain at rest and exaggerated, painful sensitivity to light touch. Spontaneous firing of injured nerves is believed to play a critical role in the induction and maintenance of neuropathic pain syndromes. Using a well characterized nerve ligation model in the rat, we demonstrate that hyperpolarization-activated, cyclic nucleotide-modulated (HCN) "pacemaker" channels play a previously unrecognized role in both touch-related pain and spontaneous neuronal discharge originating in the damaged dorsal root ganglion. HCN channels, particularly HCN1, are abundantly expressed in rat primary afferent somata. Nerve injury markedly increases pacemaker currents in large-diameter dorsal root ganglion neurons and results in pacemaker-driven spontaneous action potentials in the ligated nerve. Pharmacological blockade of HCN activity using the specific inhibitor ZD7288 reverses abnormal hypersensitivity to light touch and decreases the firing frequency of ectopic discharges originating in Abeta and Adelta fibers by 90 and 40%, respectively, without conduction blockade. These findings suggest novel insights into the molecular basis of pain and the possibility of new, specific, effective pharmacological therapies.

Behavioral characterization of mice lacking histamine H(3) receptors.

Brain histamine H(3) receptors are predominantly presynaptic and serve an important autoregulatory function for the release of histamine and other neurotransmitters. They have been implicated in a variety of brain functions, including arousal, locomotor activity, thermoregulation, food intake, and memory. The recent cloning of the H(3) receptor in our laboratory has made it possible to create a transgenic line of mice devoid of H(3) receptors. This paper provides the first description of the H(3) receptor-deficient mouse (H(3)(-/-)), including molecular and pharmacologic verification of the receptor deletion as well as phenotypic screens. The H(3)(-/-) mice showed a decrease in overall locomotion, wheel-running behavior, and body temperature during the dark phase but maintained normal circadian rhythmicity. H(3)(-/-) mice were insensitive to the wake-promoting effects of the H(3) receptor antagonist thioperamide. We also observed a slightly decreased stereotypic response to the dopamine releaser, methamphetamine, and an insensitivity to the amnesic effects of the cholinergic receptor antagonist, scopolamine. These data indicate that the H(3) receptor-deficient mouse represents a valuable model for studying histaminergic regulation of a variety of behaviors and neurotransmitter systems, including dopamine and acetylcholine.

Non-noxious A fiber afferent input enhances capsaicin-induced mechanical hyperalgesia in the rat.

Intradermal injection of capsaicin induces primary hyperalgesia at the injection site and secondary hyperalgesia in the surrounding undamaged skin. The secondary hyperalgesia is thought to be due to central sensitization of the dorsal horn neurons while primary hyperalgesia is caused by sensitization of nociceptors in the damaged skin. In this study, we asked if additional non-noxious afferent input from the undamaged skin influences the already developed secondary hyperalgesia, which follows an intradermal injection of capsaicin. Capsaicin dissolved in olive oil was injected into the middle of the hind paw of male Sprague-Dawley rats (250-300 g) under gaseous anesthesia. This produced a decrease in the mechanical threshold at the base of the toes for hind limb withdrawals lasting for 1-2h, thus showing a short-lasting (hours) secondary hyperalgesia. When the capsaicin injection was immediately followed by repeated non-noxious mechanical stimuli or weak electrical stimuli (A fiber strength) applied to the area of secondary hyperalgesia (toes) for 30 min, the reduction of the mechanical threshold lasted longer than 24h. These results suggest that non-noxious A fiber afferent input can powerfully modulate central sensitization in the spinal dorsal horn, causing the duration of the secondary hyperalgesia to be greatly extended.

Adrenergic sensitivity of the sensory receptors modulating mechanical allodynia in a rat neuropathic pain model.

This study focuses on changes in adrenergic sensitivity in untransected sensory axons that innervate an area of skin made neuropathic by transection of neighboring nerves. The segmental nerve injury model is favorable for this since all axons in the L5 and L6 nerves are transected whereas the L4 axons are intact. Earlier findings are that pain behaviors develop after this injury and that these behaviors are ameliorated by sympathectomy. The present study shows that behavior indicating mechanical allodynia can be rekindled after sympathectomy by intradermal norepinephrine and alpha-2 but not alpha-1 adrenergic ligands and the rekindling can be blocked by alpha-2 but not alpha-1 adrenergic antagonists. By contrast neither intradermal norepinephrine nor other adrenergic agonists or antagonists have any demonstrable effects in the normal or after either neuropathic surgery or sympathectomy alone. These data suggest that the combination of neuropathic surgery and sympathectomy results in an upregulation of active alpha-2 adrenergic receptors on the undamaged sensory axons that provide the remaining sensory innervation to a neuropathic area partially denervated by segmental nerve lesions. These changes on undamaged axons presumably compliment similar changes on the transected axons and, thus play a role in the development of neuropathic pain.