Semi-Mechanism-Based Population Pharmacokinetic Modeling of the Hedgehog Pathway Inhibitor Vismodegib.

Vismodegib, approved for the treatment of advanced basal cell carcinoma, has shown unique pharmacokinetic (PK) nonlinearity and binding to α1-acid glycoprotein (AAG) in humans. A semi-mechanism-based population pharmacokinetic (PopPK) model was developed from a meta-dataset of 225 subjects enrolled in five clinical studies to quantitatively describe the clinical PK of vismodegib and identify sources of interindividual variability. Total and unbound vismodegib were analyzed simultaneously, together with time-varying AAG data. The PK of vismodegib was adequately described by a one-compartment model with first-order absorption, first-order elimination of unbound drug, and saturable binding to AAG with fast-equilibrium. The variability of total vismodegib concentration at steady-state was predominantly explained by the range of AAG level. The impact of AAG on unbound concentration was clinically insignificant. Various approaches were evaluated for model validation. The semi-mechanism-based PopPK model described herein provided insightful information on the nonlinear PK and has been utilized for various clinical applications.

Determination of GDC-0449, a small-molecule inhibitor of the Hedgehog signaling pathway, in human plasma by solid phase extraction-liquid chromatographic-tandem mass spectrometry.

To support clinical development, a solid phase extraction (SPE) liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method for the determination of GDC-0449 concentrations in human plasma has been developed and validated. Samples (200 microl) were extracted using an Oasis MCX 10 mg 96-well SPE plate and the resulting extracts were analyzed using reverse-phase chromatography coupled with a turbo-ionspray interface. The method was validated over calibration curve range 5-5000 ng/mL. Quadratic regression and 1/x(2) weighing were used. Within-run relative standard deviation (%RSD) was within 10.1% and accuracy ranged from 88.6% to 109.0% of nominal. Between-run %RSD was within 8.6% and accuracy ranged from 92.4% to 105.3% of nominal. Extraction recovery of GDC-0449 was between 88.3% and 91.2% as assessed using quality control sample concentrations. GDC-0449 was stable in plasma for 315 days when stored at -70 degrees C and stable in reconstituted sample extracts for 117 h when stored at room temperature. Quantitative matrix effect/ion suppression experiment was performed and no significant matrix ion suppression was observed. This assay allows for the determination of GDC-0449 plasma concentrations over a sufficient time period to determine pharmacokinetic parameters at relevant clinical doses.

Metabolic activation of pioglitazone identified from rat and human liver microsomes and freshly isolated hepatocytes.

Pioglitazone is in the class of compounds known as the thiazolidinediones and is used to treat type 2 diabetes mellitus. The first in its class compound, troglitazone, was withdrawn from the U.S. market in 2000 due to a high incidence of hepatotoxicity and drug-induced liver failure. Reactive ring-opened products of troglitazone have been identified and evidence suggests that these reactive intermediates might be a potential cause of hepatotoxicity. The present work shows that pioglitazone has a reactive ring-opened product which was trapped by glutathione and positively identified by high performance liquid chromatography with tandem mass spectrometry accurate mass measurements. The novel thiazolidinedione ring-opened products of pioglitazone were identified in rat and human liver microsomes and in freshly isolated rat but not human hepatocytes.

Up-regulation of MUC1 in mammary tumors generated in a double-transgenic mouse expressing human MUC1 cDNA, under the control of 1.4-kb 5' MUC1 promoter sequence and the middle T oncogene, expressed from the MMTV promoter.

In this study we examined the regulation of expression of the human MUC1 gene in vivo, by developing MUC1 transgenic mice. The data showed that epithelial-specific expression of MUC1 can be directed by just 1.4 kb of 5' flanking sequence using MUC1 cDNA as a reporter gene in vivo. Furthermore, high levels of MUC1 expression were seen in the lactating mammary gland and in spontaneous mammary tumors generated by crossing the MUC1 transgenics with mice transgenic for the polyoma middle T oncogene under the control of the mouse mammary tumor virus promoter. This pattern of expression in epithelial tissues is comparable to the expression of MUC1 in humans and also to the expression pattern in another transgenic mouse line developed with a 10.6-kb genomic MUC1 fragment. This study confirmed that MUC1 is a compact gene and demonstrated that the 1.4-kb 5' sequence not only directs epithelial-specific expression of MUC1 in vivo but also contains the elements governing the up-regulation observed during lactation and in malignancy.

Metallic fragments on mammography after intraoperative deployment of radiopaque clips.

OBJECTIVE: Our objective was to report the occurrence and determine the frequency of metallic fragments in the breast after placement of surgical clips that are used to delineate the margins of the biopsy cavity. CONCLUSION: Metallic fragments are commonly present in patients who have surgical clips placed during breast biopsy for both benign and malignant disease. Awareness of this phenomenon may prevent the misidentification of these fragments as microcalcifications and thus avert unnecessary concern or biopsy.

Nectarin I is a novel, soluble germin-like protein expressed in the nectar of Nicotiana sp.

We have identified a limited number of proteins secreted into the nectar of tobacco plants. Nectarin I is the most highly expressed nectar protein and has a monomer molecular mass of 29 kDa. The other major nectar proteins are expressed at lower levels and have monomer molecular masses of 41, 54, and 65 kDa respectively. Nectarin I was purified and antiserum was raised against the protein. Under nondenaturing conditions, Nectarin I has an apparent molecular mass of > 120 kDa. The expression of Nectarin I was restricted to nectary tissues and to a much lower level in the ovary. No Nectarin I was found in petals, stems, leaves, or roots or other floral tissues. The expression of Nectarin I was also developmentally regulated. It is expressed in nectary tissues only while nectar is being actively secreted. Subsequently, the N-terminus of purified Nectarin I was sequenced. Sequence identity showed Nectarin I is related to wheat germin. Although hydrogen peroxide is readily detectable in tobacco floral nectar, we were unable to demonstrate any oxalate oxidase activity for Nectarin I. A partial cDNA encoding the mature Nectarin I N-terminus was isolated and used to probe a Nicotiana plumbaginifolia genomic library. The Nectarin I gene was isolated and the translated sequence was consistent with both N-terminal and internal cyanogen bromide-derived amino acid sequence. The gene contains a single 386 nt intron and encodes a mature protein of 197 amino acids.

Local excision of rectal carcinoma: a safe alternative for more advanced tumors?

BACKGROUND AND OBJECTIVES: Local excision of rectal carcinoma has primarily been limited to patients with small (< or =3 cm), early rectal carcinoma. We wanted to determine whether local excision (transanal or transacral), when combined with selective chemoradiation therapy, would be adequate treatment for patients with larger (>3 cm) and more advanced T3 and N1 tumors. METHODS: A prospective study of 20 patients with clinical T1-T3, N0-N1 rectal carcinoma was initiated in 1990. Local excision (transanal or transacral) was performed on all patients. Sixteen patients were treated with postoperative 5-fluorouracil (5-FU) and leucovorin (LV) combined with radiation therapy; six high-risk patients (T3 or N1) received an additional 6 months of 5-FU and LV. All patients were followed for a minimum of 4 years. RESULTS: Tumor size ranged from 2 to 5.5 cm (mean, 3.6 cm). Histology revealed well or moderate differentiation (19/20), gross or microscopic ulceration (14/20), and vessel invasion (5/20). Mucosal margins were 3-12 mm (mean, 8.3 mm); radial margins were clear in all patients except one (microscopically positive). Five patients had T3 tumors; two had node positive tumors (N1). With a median follow-up of 56 months (48-71), there have been no local or regional failures and two patients have died from metastatic disease. CONCLUSIONS: Local excision, when combined with selective chemoradiation therapy, can be safely applied to patients with large (>3 cm) and more advanced T3 and N1 rectal carcinomas.

Arabidopsis thaliana contains a large family of germin-like proteins: characterization of cDNA and genomic sequences encoding 12 unique family members.

We have identified 39 Arabidopsis thaliana ESTs encoding germin-like proteins (GLPs) and have completely sequenced 25 of these cDNAs. Our analysis demonstrates that the Arabidopsis genome contains a gene family with at least 12 GLP genes. Comparisons with other known germins and germin-like proteins indicate that these Arabidopsis GLP subfamilies are unique from wheat germin. All other known GLPs fall into one of these subfamilies. The translated GLPs show approximately 35% amino acid identity with other GLPs outside of their subfamily and significantly higher levels of identity within their respective subfamily. The 3' ends of many of the GLP cDNAs are heterogeneous and several sites of polyadenylation are used. Ten of the GLPs have N-terminal signal sequences and most appear to be exported from the cell. Structurally, the GLPs are predicted to have a high content of beta-pleated sheet. Seven conserved regions of beta-sheet were found in each of the GLP proteins along with alpha-helices located at both N- and C-termini. These same structural elements are also conserved in wheat germin. With one exception, all GLP family members contain at least one N-glycosylation site. All of these sites are conserved in an unstructured loop between beta-1 and beta-2. Genes for two of these GLPs were identified in genomic sequences previously deposited in the GenBank. The GLP3b gene is physically linked to the polyubiquitin 4 gene. The 3' end of the GLP3b mRNA is only 0.5 kb from the ubq4 start of transcription. Analysis of the GLP3b promoter shows the presence of a single putative auxin-response sequence located at -124 to -111 upstream from the 5' end of the GLP3b mRNA. The GLP9 gene was identified in an Arabidopsis contig from Chromosome 4.

Postsurgical surveillance of colon cancer: preliminary cost analysis of physician examination, carcinoembryonic antigen testing, chest x-ray, and colonoscopy.

OBJECTIVE: This study is the first to examine the relative and absolute costs of physician examination, carcinoembryonic antigen (CEA) assessment, chest x-ray, and colonoscopy in detecting recurrent disease in patients who have undergone surgical resection for primary colon carcinoma. METHODS: Of the 1356 Eastern Cooperative Oncology Group patients in Intergroup Protocol 0089 who underwent surgical resection for Dukes' B2 and C colon carcinoma, 421 patients who developed recurrent disease were reviewed. Follow-up testing was performed according to protocol guidelines, with the cost of each test equal to 1995 Medicare reimbursement. Follow-up was defined as the time to recurrence for the 421 patients in whom disease recurred (mean 18.6 months) or up to 5 years for the additional 930 patients in whom disease did not recur (mean 38.6 months). Patients were divided into three categories: nonrecurrent, recurrent but not resectable, and recurrent but resectable with curative intent. The estimated mean cost of each test in detecting group 3 (recurrent but resectable) patients was calculated. RESULTS: Of the 421 patients who developed recurrent disease, 96 underwent surgical resection of their disease with curative intent (group 3). For group 3 patients, the first indication of recurrent disease was CEA testing (30), chest x-ray (12), colonoscopy (14), and other (40). Of the 40 "other" patients, 24 presented with symptoms. Routine physician examination, however, failed to identify a single resectable recurrence, and the total cost for physician examination was $418,615. The detection rate for CEA testing was 2.2%, the total cost was $170,880, and the cost per recurrence was $5,696. The detection rate for chest x-ray was 0.9%, the total cost was $120,934, and the cost per recurrence was $10,078. The detection rate of colonoscopy was 1%, the total cost was $641,344, and the cost per recurrence was $45,810. CONCLUSIONS: CEA measurement was the most cost-effective test in detecting potentially curable recurrent disease. Physician visits were useful only in the evaluation of symptoms; a routine physician examination had no added benefit.

Human endometrial MUC1 carries keratan sulfate: characteristic glycoforms in the luminal epithelium at receptivity.

MUC1 is a high molecular mass, highly glycosylated epithelial apical glycoprotein that has been shown to exhibit both adhesive and anti-adhesive properties. Its expression in human glandular endometrial epithelium is transcriptionally regulated with the highest levels in the mid secretory phase, the "receptive" period during which implantation occurs. We demonstrate that endometrial MUC1 carries highly sulfated lactosaminoglycan chains recognized by monoclonal antibody (Mab) 5D4, and the sialokeratan sulfate epitope recognized by Mab D9B1. These glycans are hormonally regulated in endometrium, and show increased abundance in the secretory phase, but detailed evaluation of their distribution shows important differences. The 5D4 epitope is abundant at the luminal epithelial surface until the implantation phase, when it disappears, first from patches of cells, then altogether. D9B1 binding sites are retained in the luminal epithelium at receptivity. These data show that endometrial MUC1 carries sulfated lactosaminoglycans. They identify the luminal epithelial compartment as a site of unique MUC1 glycosylation and independent regulation. Glycosylation and the negative charge associated with sialo- and sulfoglycans may be important in the regulation of embryo attachment.

Intramuscular immunisation with MUC1 cDNA can protect C57 mice challenged with MUC1-expressing syngeneic mouse tumour cells.

Much interest is currently being shown in immunotherapy as a treatment for cancer since several tumour-associated antigens have been identified and the genes encoding them cloned. One such molecule is the tumour-associated human MUC1 gene product. In this report we describe tumour rejection studies in a C57B1 murine model system with syngeneic MUC1-expressing tumour cells designed to examine the efficacy of MUC1 cDNA as an immunogen. Intra-muscular immunisation with 100 microgram MUC1 cDNA 3 times at 3-weekly intervals resulted in tumour protection in approximately 80% of mice. Tumour protection was dose-dependent, with 50-100 microgram being the most effective dose. Both humoral and cell-mediated MUC1-specific immune responses were detected. Anti-MUC1 antibodies were detected after immunisation with DNA alone, indicating that the injected DNA was expressed. Humoral immune responses did not correlate with tumour rejection. Tumour challenge with syngeneic tumour cells expressing MUC1 appeared to be a pre-requisite for the generation of MUC1-specific cytotoxic T lymphocytes.

The polymorphic epithelial mucin: potential as an immunogen for a cancer vaccine.

The identification and cloning of several tumour antigens together with an improvement in the understanding of the mechanisms involved in antigen presentation and immune recognition has opened up the possibility of using active specific immunotherapy as a treatment for certain cancers. This review discusses the tumour-associated MUC1 gene product of the polymorphic epithelial mucin (PEM), as a potential target molecule for cancer treatment. PEM is both over-expressed and aberrantly glycosylated in many carcinomas resulting in an antigenically distinct molecule. Furthermore, immune responses specific for PEM have been detected in cancer patients. Both syngeneic and transgenic murine model systems have been developed in order to compare the efficacy and toxicity of various PEM-based immunogens in tumour rejection studies, and to further improve the understanding of antigen presentation and the mechanisms underlying tumour rejection. Such models also allow the examination of MUC1-based immunogens as a treatment for existing tumours. Clinical trials in progress using immunogens based on the MUC1 gene product are briefly discussed.

MUC1 in secretory phase endometrium: expression in precisely dated biopsies and flushings from normal and recurrent miscarriage patients.

MUC1 is a cell-surface and secretory product of endometrial epithelium. Immunohistochemical studies carried out using two different antibodies to the mucin-type tandem repeat region of MUC1 indicate a cell-surface location in proliferative phase glands, with intracellular deposits accumulating in the early secretory phase. Commencing 3-4 days after the luteinizing hormone (LH) peak and continuing into the late secretory phase, secretory MUC1 appears in gland lumens. Uterine flushings were collected as a function of time after the LH peak and were analysed using a two-site enzyme-linked immunosorbent assay for MUC1. Low but measurable concentrations were observed up to day 7, while on days 7-13 much higher values were obtained. In women suffering from recurrent spontaneous miscarriage, the concentration of MUC1 in flushings was significantly lower than in the controls on day LH + 10. Lower values were observed on days 7 and 13. Reduced epithelial secretory function and a resultant change in uterine fluid composition are features of endometrium from recurrent miscarriage patients.

Endometrial differentiation in the peri-implantation phase of women with recurrent miscarriage: a morphological and immunohistochemical study.

OBJECTIVES: To study endometrial differentiation in the peri-implantation phase of women with recurrent miscarriage and to compare the results with endometrium of normal fertile women. DESIGN: A prospective study of endometrial specimens precisely timed from the LH surge, using traditional histologic dating (Noyes' criteria), quantitative histologic measurement (morphometric analysis), and immunohistochemical techniques. RESULTS: Fifteen of 25 (60%) subjects in the recurrent miscarriage group had retarded endometrial development in the peri-implantation period as monitored by morphometry. The recurrent miscarriage group showed reduced levels of four mucin-related secretory epitopes, and greater reductions were associated with morphological retardation. Normal differentiation was observed in all of the 14 subjects in the control group. CONCLUSIONS: Women with idiopathic recurrent pregnancy loss may be divided into two distinct subgroups on the basis of their endometrial response in the peri-implantation period. Precisely timed endometrial biopsy should be incorporated in the investigation of recurrent miscarriage.

The endometrial cell surface and implantation. Expression of the polymorphic mucin MUC-1 and adhesion molecules during the endometrial cycle.

The cell surface mucin MUC-1 is present in endometrial epithelial cells and their associated apical glycocalyx and is also released into gland lumens as a secretory product. MUC-1 mRNA and core protein are found at low levels in the proliferative phase of the cycle, but their abundance increases after ovulation. Endometrial MUC-1 has been found to carry sialokeratan sulphate chains and these show a dramatically increased abundance in cells and secretions in the post-ovulatory phase of the cycle, reaching a maximum in secretions 6-7 days after the LH peak. The apical epithelium also contains adhesion receptor molecules of the integrin and CD44 families. MUC-1 is large and highly glycosylated and probably extends farther from the cell surface than these 'conventional' glycoprotein receptors. It has the potential to inhibit sterically receptor-mediated cell-cell adhesion. However, it is also possible that MUC-1 displays specific (e.g., glycan) recognition structures for the initial attachment of the blastocyst or that the embryo may create a specialised microenvironment in which to implant.

Endometrial responses to three different progestins in artificial cycles: a prospective, crossover study.

We found that there was no significant difference in endometrial responses to vaginal or IM P. But the response was suboptimal in cycles treated with dydrogesterone, suggesting that dydrogesterone treatment is not the preferred progestin for use in artificial cycles.

Keratan sulphate as a secretory product of human endometrium: cyclic expression in normal women.

The distribution of keratan sulphate (KS) in normal endometrium has been studied by immunohistochemistry using monoclonal antibody 5D4. KS is present in association with glandular epithelium throughout the normal cycle. Production is hormonally regulated, as indicated by the significant increase observed in the secretory phase of the cycle. Intracellular immunoreactivity increases to a maximum 3 days after the luteinizing hormone (LH) peak. This is followed by an increase in secreted KS from 4 days after the LH peak, and essentially all endometrial glands showed immunoreactive deposits by 5 days after the LH peak. Beginning on day 7 after the LH peak, a fraction of glands was observed to have lost the epitope. Nevertheless, KS remained in a significant proportion of the gland lumens 9 days after the LH peak. The data suggest that analysis of secretory KS in women gives an index of hormonally regulated epithelial differentiation in the peri-implantation phase.

Local excision of rectal carcinoma. Early results with combined chemoradiation therapy using 5-fluorouracil and leucovorin.

PURPOSE: This study was undertaken to evaluate the treatment morbidity, functional outcome, and recurrence risk of patients undergoing local excision and combined chemoradiation therapy for rectal carcinoma. METHODS: Eighteen patients underwent local excision of their rectal carcinoma. Four patients underwent local excision alone (T1-2, N0-X, low risk), 10 patients underwent local excision with postoperative chemoradiation therapy using 5-fluorouracil and leucovorin (T1-2, N0-X, high risk), and 4 patients underwent local excision, chemoradiation therapy, and six months of additional 5-fluorouracil and leucovorin (T3 or N1). RESULTS: Of the four patients undergoing local excision alone, there was no treatment morbidity or alteration in functional outcome. Of the 14 patients receiving chemoradiation therapy, three reported early Grade 3-4 toxicity manifested by cystitis, proctitis, or perineal skin desquamation. At six months, two patients reported persistent rectal urgency and occasional fecal incontinence, and 11 patients reported increasing stool frequency (average, 3: range, 2-8). The six months of additional 5-fluorouracil and leucovorin were well tolerated and did not appear to further affect functional outcome. There were no local recurrences, although one patient developed distant metastatic disease. CONCLUSION: This treatment regimen, while generally well tolerated, is associated with significant acute toxicity in certain patients. We have identified specific causative factors which can be modified to decrease acute morbidity, including the elimination of leucovorin during the combined chemoradiation therapy.

Fine-needle aspiration biopsy of pancreatic ductal adenocarcinoma: loss of diagnostic accuracy with small tumors.

Eighty-three patients underwent CT-directed fine-needle aspiration biopsies (FNAB) for pancreatic ductal adenocarcinoma. Five factors that might have influenced the diagnostic sensitivity of FNAB were analyzed: clinical history, the number of passes for each FNAB, and three radiologic criteria including tumor size, tumor location, and the presence or absence of suspected tumor necrosis by CT scan. Sixty-three patients had a diagnosis of pancreatic carcinoma confirmed by FNAB (overall sensitivity = 76%). Tumor size was the only factor that correlated with the diagnostic sensitivity of FNAB. [table: see text] Of 12 patients whose FNAB was negative but suspicious for malignancy, 10 had a repeat FNAB and 4 were positive for carcinoma. We conclude that the diagnostic sensitivity of FNAB decreases significantly with decreasing tumor size and that a repeat FNAB for suspicious biopsies should be done to increase the diagnostic yield.