We evaluated the usefulness of the nuclear matrix protein 22 BladderChek (NMP22BC) test for the screening and follow-up of bladder cancer.
Materials and Methods
From February 2006 to September 2009, we enrolled 1,070 patients who had hematuria or who were being followed up for bladder cancer. We compared the sensitivity and specificity of the NMP22BC test with those of urine cytology.
The sensitivity of the NMP22BC test (77.5%) was significantly higher than that of urine cytology (46.3%). The specificity of the NMP22BC test was 88.8%, compared with 97.9% for urine cytology. The sensitivity of the NMP22BC test (81.8%) in non-muscle-invasive bladder cancer was higher than that of cytology (36.4%). However, the sensitivity of the NMP22BC test and of urine cytology in invasive bladder cancer were 57.1% and 92.9%, respectively. The sensitivity of the NMP22BC test was higher for low-grade bladder cancer (83.9%) than for high-grade (62.5%), and the sensitivity of cytology was higher for high-grade bladder cancer (66.7%) than for low-grade (37.5%). Follow-up bladder cancer was detected in 262 patients. The sensitivity of the NMP22BC test in that group (72.7%) was decreased and the specificity (91.7%) was increased. The sensitivity of cytology (54.5%) in the follow-up group was increased and the specificity (95.6%) was decreased. The presence of pyuria was significantly associated with the lower specificity of the NMP22BC test.
The greater sensitivity of the NMP22BC test may be more useful for the diagnosis of non-muscle-invasive bladder cancer and low-grade bladder cancer than for the diagnosis of invasive or high-grade bladder cancer. If the NMP22BC test is performed in the absence of pyuria, it may play a compensatory role for urine cytology.
Nuclear matrix protein 22; Cytology; Urinary bladder neoplasms
Urinary biomarkers for bladder cancer detection are constrained by inadequate sensitivity or specificity. Here we evaluate the diagnostic accuracy of Mcm5, a novel cell cycle biomarker of aberrant growth, alone and in combination with NMP22.
1677 consecutive patients under investigation for urinary tract malignancy were recruited to a prospective blinded observational study. All patients underwent ultrasound, intravenous urography, cystoscopy, urine culture and cytologic analysis. An immunofluorometric assay was used to measure Mcm5 levels in urine cell sediments. NMP22 urinary levels were determined with the FDA-approved NMP22® Test Kit.
Genito-urinary tract cancers were identified in 210/1564 (13%) patients with an Mcm5 result and in 195/1396 (14%) patients with an NMP22 result. At the assay cut-point where sensitivity and specificity were equal, the Mcm5 test detected primary and recurrent bladder cancers with 69% sensitivity (95% confidence interval = 62–75%) and 93% negative predictive value (95% CI = 92–95%). The area under the receiver operating characteristic curve for Mcm5 was 0.75 (95% CI = 0.71–0.79) and 0.72 (95% CI = 0.67–0.77) for NMP22. Importantly, Mcm5 combined with NMP22 identified 95% (79/83; 95% CI = 88–99%) of potentially life threatening diagnoses (i.e. grade 3 or carcinoma in situ or stage ≥pT1) with high specificity (72%, 95% CI = 69–74%).
The Mcm5 immunoassay is a non-invasive test for identifying patients with urothelial cancers with similar accuracy to the FDA-approved NMP22 ELISA Test Kit. The combination of Mcm5 plus NMP22 improves the detection of UCC and identifies 95% of clinically significant disease. Trials of a commercially developed Mcm5 assay suitable for an end-user laboratory alongside NMP22 are required to assess their potential clinical utility in improving diagnostic and surveillance care pathways.
Difficulty exists in interpreting the significance of atypical urine cytology. This study was performed to assess the diagnostic utility of nuclear matrix protein-22 (NMP-22) testing when atypical cells are detected during urine cytology.
Materials and Methods
Among patients whose urine cytology was reported as atypical between January 2004 and December 2009, a total of 275 who also underwent NMP-22 testing were enrolled in the present study. These patients were further divided into the screening group (143 patients examined as outpatients for hematuria) and the follow-up group (132 patients followed up for previously diagnosed bladder cancer). The sensitivity, specificity, positive and negative predictive values, and accuracy were assessed for atypical cytology alone and in conjunction with NMP-22.
Of the 275 patients exhibiting atypical urine cytology, cancer was confirmed in 85, yielding a positive predictive value of 30.9% (85/275). Of the 96 patients testing positive for NMP-22, 58 were diagnosed with bladder cancer. The positive predictive value in conjunction with NMP-22 was 60.4% (58/96). The sensitivity, specificity, negative predictive value, and accuracy were 68.2% (58/85), 80.0% (152/190), 84.9% (152/179), and 76.2% (210/275), respectively. Testing for NMP-22 in the screening and follow-up groups increased the positive predictive value from 30.0% (43/143) to 64.0% (32/50) and from 31.3% (42/132) to 56.5% (26/46), respectively; there was no significant difference between the screening and follow-up groups (p=0.106).
When only cases with atypical urine cytology were examined, NMP-22 testing increased the detection rate of bladder cancer regardless of whether the test was used in screening hematuria or in following up patients.
Cytology; Nuclear matrix; Urinary bladder neoplasms
Several studies have shown that abnormal levels of nuclear matrix protein 22 (NMP22) are associated with bladder cancer, leading to NMP22 being approved as a urinary biomarker by the FDA. Nonetheless, the clinical significance of NMP22 remains unclear.
To use decision analysis to determine whether NMP22 improves medical decision-making.
Design, Setting, and Participants
The study included 2,222 patients with a history of non–muscle-invasive bladder cancer and current negative cytology. We developed models to predict cancer recurrence or progression to muscle-invasive disease using NMP22 levels, age, and gender.
Voided NMP22 and cystoscopy.
Clinical net benefit was calculated by summing the benefits (true positives) and subtracting the harms (false positives) and weighting these by the threshold probability at which a patient or clinician would opt for cytoscopy.
Results and limitations
After cystoscopy, 581 (26%) patients were found to have cancer. NMP22 level was significantly associated with bladder cancer recurrence and progression (p<0.001 for both). Using NMP22 in a model with age and gender was associated with better patient outcomes than performing cystoscopy on everyone for threshold probabilities above 8% for recurrence and above 3% for progression. Only offering cystoscopy to those with a 15% or greater risk would reduce the number of cystoscopies by 229, while missing only 25 cancer recurrences per 1000 men with a negative cytology. The study was limited by its multicenter design.
For clinicians who would perform a cystoscopy at a threshold of 5% for recurrence or 1% for progression, NMP22 will not aid clinical decision-making. For less risk-averse clinicians who would only perform a cystoscopy at a threshold probability >8% for recurrence or >3% for progression, NMP22 can help determine which patients require cystoscopy and which can be spared this procedure.
nuclear matrix protein 22; bladder cancer; urothelial carcinoma; detection; surveillance
The nuclear matrix protein 22 (NMP22) assay has been shown to have greater sensitivity for the diagnosis and detection of recurrent urothelial carcinoma of the bladder (UCB) over that of traditional urine cytology. We assessed the use of NMP22 to predict which high-risk superficial UCB patients will have recurrence, progression or disease-related death; we compared these results to standard urine cytology.
One hundred consecutive patients with high-risk superficial UCB were enrolled. During surveillance, urine was collected for cytology and NMP22 testing. Patients were followed for at least 6 months. Retrospective chart review was undertaken to collect data on previous tumour history, tumour characteristics, disease recurrences, progression and death. Kaplan-Meier analyses were performed to determine the significance between NMP22-positive and -negative patients in terms of recurrence-free, progression-free and overall survival. Similar analyses were performed for urine cytology.
From 94 eligible patients, 15 and 79 were NMP22 positive and negative, respectively. The baseline characteristics between the 2 groups were not significantly different in terms of patient characteristics, prior tumour history or intravesical therapies received. Mean recurrence-free survival time was significantly lower in the NMP22 positive group (p = 0.038); however, mean progression-free and overall survival were not significantly different between the 2 groups (p = 0.297 and 0.519, respectively). Urine cytology demonstrated no significant predictive power for disease recurrence, progression or survival.
The nuclear matrix protein 22 assay appears to have predictive value for future tumour recurrences, but not progression or overall survival in patients with high-risk superficial UCB.
The solubilization efficiency of N-methyl pyrrolidone (NMP) has been determined and compared to that of ethanol and propylene glycol for 13 poorly soluble drugs. NMP is found to be a more efficient solubilizer for all the drugs studied. The solubility enhancement as high as about 800-fold is obtained in 20% v/v NMP solution as compared to water. The mechanism of drug solubilization by NMP has also been investigated. It is proposed that NMP enhances drug solubility by simultaneously acting as a cosolvent and a complexing agent. A mathematical model is used to estimate the drug solubility in NMP–water mixture, according to which the total solubility enhancement is a sum of the two effects. This model describes the experimental data well and is more accurate than other models. A large and uniform reduction in the surface tension of water as a function of NMP concentration demonstrates its cosolvent effect. The complexation is supported by the fact that it’s strength is affected by the temperature and the polarity of the medium. A strong correlation exists between log Kow of the drugs and the cosolvency coefficients. The correlation between log Kow and the complexation coefficients is weak suggesting that factors such as molecular shape and aromaticity of the drug molecule are significant in determining the complexation strength. This has been confirmed by the absence of a significant complexation between NMP and linear drug-like solutes.
complexing agent; cosolvent; N-methyl pyrrolidone; solubility enhancement
Cancer of the urinary bladder is the fifth most common neoplasm in the industrialized countries. Diagnosis and surveillance are dependent on invasive evaluation with cystoscopy and to some degree cytology as an adjunct analysis. Nomuscle invasive bladder cancer is characterized by frequent recurrences after resection, and up to 30% will develop an aggressive phenotype. The journey towards a noninvasive test for diagnosing bladder cancer, in order to replace or extend time between cystoscopy, has been ongoing for more than a decade. However, only a handful of tests that aid in clinical decision making are commercially available. Recent reports of DNA methylation in urine specimens highlight a possible clinical use of this marker type, as high sensitivities and specificities have been shown. This paper will focus on the currently available markers NMP22, ImmunoCyt, and UroVysion as well as novel DNA methylation markers for diagnosis and surveillance of bladder cancer.
Current urine-based assays for bladder cancer (BCa) diagnosis lack accuracy, so the search for improved biomarkers continues. Through genomic and proteomic profiling of urine, we have identified a panel of biomarkers associated with the presence of BCa. In this study, we evaluated the utility of three of these biomarkers, interleukin 8 (IL-8), Matrix metallopeptidase 9 (MMP-9) and Syndecan in the diagnosis of BCa through urinalysis.
Voided urines from 127 subjects, cancer subjects (n = 64), non-cancer subjects (n = 63) were analyzed. The protein concentrations of IL-8, MMP-9, and Syndecan were assessed by enzyme-linked immunosorbent assay (ELISA). Data were also compared to a commercial ELISA-based BCa detection assay (BTA-Trak©) and urinary cytology. We used the area under the curve of a receiver operating characteristic (AUROC) to compare the performance of each biomarker.
Urinary protein concentrations of IL-8, MMP-9 and BTA were significantly elevated in BCa subjects. Of the experimental markers compared to BTA-Trak©, IL-8 was the most prominent marker (AUC; 0.79; 95% confidence interval [CI], 0.72-0.86). Multivariate regression analysis revealed that only IL-8 (OR; 1.51; 95% CI, 1.16-1.97, p = 0.002) was an independent factor for the detection of BCa.
These results suggest that the measurement of IL-8 in voided urinary samples may have utility for urine-based detection of BCa. These findings need to be confirmed in a larger, prospective cohort.
IL-8; Biomarkers; Diagnosis; Bladder cancer
OBJECTIVES: To study the acute effects of exposure to the increasingly used solvent, N-methyl-2-pyrrolidone (NMP) in male volunteers. Further, to determine the NMP concentration in plasma and urine during and after the exposure. METHODS: Six male volunteers were exposed for eight hours on four different days to 0, 10, 25, and 50 mg/m3 NMP. Plasma was collected and urine was sampled during and after the exposure. Changes in nasal volume were measured by acoustic rhinometry and in airway resistance by spirometry. RESULTS: The eight-hour experimental exposure to 10, 25, and 50 mg/m3 did not induce discomfort to eyes or upper airways. Acute changes in nasal volume were not found, and no changes in the spirometric data could be registered. The elimination curves suggested a non-linear pattern and at the end of exposure showed mean (range) half lifes of NMP in plasma of about 4.0 (2.9-5.8) hours and in urine 4.5 (3.5-6.6) hours. The unmetabolised NMP found in urine samples collected during exposure and at the subsequent 44 hours corresponded to about 2% of the calculated absorbed dose. At the end of the exposure there was a close correlation between exposures and the plasma concentration and urinary excretion of NMP. CONCLUSIONS: NMP was absorbed through the respiratory tract and readily eliminated from the body, mainly by biotransformation to other compounds. Exposure to 10, 25, or 50 mg/m3 NMP did not cause nose, eye, or airway irritation. Thus, NMP is a mild irritant.
To assess the possibility of hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP) as a biological marker for detecting Bladder cancer (BCa), we examined the expression of HIP/PAP in both BCa specimens and BCa cell lines and measured HIP/PAP levels in urine from patients with BCa.
HIP/PAP expression in BCa samples was evaluated by western blot analysis, and urinary levels of HIP/PAP in patients with BCa were measured by enzyme-linked immunosorbent assay. Urine samples were collected from 10 healthy volunteers and 109 with benign urological disorders as controls, and from 101 patients who were diagnosed with BCa.
HIP/PAP was highly expressed in BCa samples as compared with control bladder. Urinary HIP/PAP concentrations were significantly higher in BCa patients than in controls (median value; 3.184 pg/mL vs. 55.200 pg/mL, P <0.0001, by Mann–Whitney U test). Urinary HIP/PAP levels in BCa patients correlated positively with pathological T stages and progression-risk groups among non-muscle invasive BCa (P = 0.0008, by Kruskal-Wallis test). Regarding the recurrence-risk classifications of non-muscle invasive BCa, the urinary levels of HIP/PAP were significantly higher in the intermediate than in the low risk group (P = 0.0002, by Mann–Whitney U test). Based on a cut-off of 8.5 pg/mL, the ability of urinary HIP/PAP levels to detect BCa had a sensitivity of 80.2%, specificity of 78.2%, positive predictive value (PPV) of 75.7%, and negative predictive value (NPV) of 82.3%.
HIP/PAP was abundantly expressed in BCa, and the urinary levels of HIP/PAP could be a novel and potent biomarker for detection of BCa, and also for predicting the risks of recurrence- and progression-risk of non-muscle invasive BCa. A large scale study will be needed to establish the usefulness of this biomarker.
Bladder cancer; Urinary marker; HIP/PAP; ELISA; ROC
The early detection of bladder cancer (BCa) is pivotal for successful patient treatment and management. Through genomic and proteomic studies, we have identified a number of bladder cancer-associated biomarkers that have potential clinical utility. In a case-control study, we examined voided urines from 127 subjects: 64 tumor-bearing subjects and 63 controls. The urine concentrations of the following proteins were assessed by enzyme-linked immunosorbent assay (ELISA); C-C motif chemokine 18 (CCL18), Plasminogen Activator Inhibitor 1 (PAI-1) and CD44. Data were compared to a commercial ELISA-based BCa detection assay (BTA-Trak©) and voided urinary cytology. We used analysis of the area under the curve of receiver operating characteristic curves to compare the ability of CCL18, PAI-1, CD44, and BTA to detect BCa in voided urine samples. Urinary concentrations of CCL18, PAI-1, and BTA were significantly elevated in subjects with BCa. CCL18 was the most accurate biomarker (AUC; 0.919; 95% confidence interval [CI], 0.8704-0.9674). Multivariate regression analysis highlighted CCL18 (OR; 18.31; 95% CI, 4.95-67.70, p<0.0001) and BTA (OR; 6.43; 95% CI, 1.86-22.21, p = 0.0033) as independent predictors of BCa in voided urine samples. The combination of CCL18, PAI-1 and CD44 improved the area under the curve to0.938. Preliminary results indicate that CCL18 was a highly accurate biomarker for BCa detection in this cohort. Monitoring CCL18 in voided urine samples has the potential to improve non-invasive tests for BCa diagnosis. Furthermore using the combination of CCL18, PAI-1 and CD44 may make the model more robust to errors to detect BCa over the individual biomarkers or BTA.
Accurate urinary assays for bladder cancer (BCa) detection would benefit both patients and healthcare systems. Through genomic and proteomic profiling of urine components, we have previously identified a panel of biomarkers that can outperform current urine-based biomarkers for the non-invasive detection of BCa. Herein, we report the diagnostic utility of various multivariate combinations of these biomarkers. We performed a case-controlled validation study in which voided urines from 127 patients (64 tumor bearing subjects) were analyzed. The urinary concentrations of 14 biomarkers (IL-8, MMP-9, MMP-10, SDC1, CCL18, PAI-1, CD44, VEGF, ANG, CA9, A1AT, OPN, PTX3, and APOE) were assessed by enzyme-linked immunosorbent assay (ELISA). Diagnostic performance of each biomarker and multivariate models were compared using receiver operating characteristic curves and the chi-square test. An 8-biomarker model achieved the most accurate BCa diagnosis (sensitivity 92%, specificity 97%), but a combination of 3 of the 8 biomarkers (IL-8, VEGF, and APOE) was also highly accurate (sensitivity 90%, specificity 97%). For comparison, the commercial BTA-Trak ELISA test achieved a sensitivity of 79% and a specificity of 83%, and voided urine cytology detected only 33% of BCa cases in the same cohort. These datashow that a multivariate urine-based assay can markedly improve the accuracy of non-invasive BCa detection. Further validation studies are under way to investigate the clinical utility of this panel of biomarkers for BCa diagnosis and disease monitoring.
Cannabinoid receptors and T-type calcium channels are potential targets for treating pain. Here we report on the design, synthesis and analgesic properties of a new mixed cannabinoid/T-type channel ligand, NMP-181.
NMP-181 action on CB1 and CB2 receptors was characterized in radioligand binding and in vitro GTPγ[35S] functional assays, and block of transiently expressed human Cav3.2 T-type channels by NMP-181 was analyzed by patch clamp. The analgesic effects and in vivo mechanism of action of NMP-181 delivered spinally or systemically were analyzed in formalin and CFA mouse models of pain. NMP-181 inhibited peak CaV3.2 currents with IC50 values in the low micromolar range and acted as a CB2 agonist. Inactivated state dependence further augmented the inhibitory action of NMP-181. NMP-181 produced a dose-dependent antinociceptive effect when administered either spinally or systemically in both phases of the formalin test. Both i.t. and i.p. treatment of mice with NMP-181 reversed the mechanical hyperalgesia induced by CFA injection. NMP-181 showed no antinocieptive effect in CaV3.2 null mice. The antinociceptive effect of intrathecally delivered NMP-181 in the formalin test was reversed by i.t. treatment of mice with AM-630 (CB2 antagonist). In contrast, the NMP-181-induced antinociception was not affected by treatment of mice with AM-281 (CB1 antagonist).
Our work shows that both T-type channels as well as CB2 receptors play a role in the antinociceptive action of NMP-181, and also provides a novel avenue for suppressing chronic pain through novel mixed T-type/cannabinoid receptor ligands.
T-type Channels; Cannabinoid Receptors; Pain; Patch-Clamp; Mice
One of the biggest controversial issues in the research of olfaction has been the mechanism underlying response generation to odorants that have been shown to fail to produce cAMP when tested by biochemical assays with olfactory ciliary preparations. Such observations are actually the original source proposing a possibility for the presence of multiple and parallel transduction pathways. In this study the activity of transduction channels in the olfactory cilia was recorded in cells that retained their abilities of responding to odorants that have been reported to produce InsP3 (instead of producing cAMP, and therefore tentatively termed “InsP3 odorants”). At the same time, the cytoplasmic cNMP concentration ([cNMP]i) was manipulated through the photolysis of caged compounds to examine their real-time interactions with odorant responses. Properties of responses induced by both InsP3 odorants and cytoplasmic cNMP resembled each other in their unique characteristics. Reversal potentials of currents were 2 mV for InsP3 odorant responses and 3 mV for responses induced by cNMP. Current and voltage (I-V) relations showed slight outward rectification. Both responses showed voltage-dependent adaptation when examined with double pulse protocols. When brief pulses of the InsP3 odorant and cytoplasmic cNMP were applied alternatively, responses expressed cross-adaptation with each other. Furthermore, both responses were additive in a manner as predicted quantitatively by the theory that signal transduction is mediated by the increase in cytoplasmic cAMP. With InsP3 odorants, actually, remarkable responses could be detected in a small fraction of cells (∼2%), explaining the observation for a small production of cAMP in ciliary preparations obtained from the entire epithelium. The data will provide evidence showing that olfactory response generation and adaptation are regulated by a uniform mechanism for a wide variety of odorants.
olfactory receptor cell; signal transduction; second messenger; cAMP; caged compound
Although most nanofabrication techniques can control nano/micro particle (NMP) size over a wide range, the majority of NMPs for biomedical applications exhibits a diameter of ~100 nm. Here, the vascular distribution of spherical particles, from 10 to 1,000 nm in diameter, is studied using intravital microscopy and computational modeling. Small NMPs (≤100 nm) are observed to move with Red Blood Cells (RBCs), presenting an uniform radial distribution and limited near-wall accumulation. Larger NMPs tend to preferentially accumulate next to the vessel walls, in a size-dependent manner (~70% for 1,000 nm NMPs). RBC-NMP geometrical interference only is responsible for this behavior. In a capillary flow, the effective radial dispersion coefficient of 1,000 nm particles is ~3-fold larger than Brownian diffusion. This suggests that sub-micron particles could deposit within diseased vascular districts more efficiently than conventional nanoparticles.
To investigate whether elevated urinary levels of vascular endothelial growth factor (VEGF), carbonic anhydrase 9 (CA9) and angiogenin are associated with BCa.
This is a case-control study in which voided urines from 127 patients: control subjects (n = 63) and tumor bearing subjects (n = 64) were analyzed. The urinary concentrations of VEGF, CA9, angiogenin and BTA were assessed by enzyme-linked immunosorbent assay (ELISA). We used the area under the curve (AUC) of receiver operating characteristic curves to determine the ability of VEGF, CA9, and angiogenin to detect BCa in voided urine samples. Data were also compared to a commercial ELISA-based BCa detection assay (BTA-Trak©). Sensitivity, specificity, positive and negative predictive values were calculated.
Urinary concentrations of VEGF, CA9, angiogenin and BTA were significantly elevated in BCa. VEGF was the most accurate urinary biomarker (AUC: 0.886; 95% confidence interval [CI]: 0.8301–0.9418). Furthermore, multivariate regression analysis highlighted VEGF (OR: 5.90; 95% CI: 2.60–13.40, p < 0.0001) as an independent variable. The sensitivities and specificities for VEGF (sensitivity, 83% and specificity, 87%) outperformed BTA (sensitivity, 80% and specificity, 84%).
VEGF may be a valuable addition to voided urine sample analysis for the detection of BCa. Larger, prospective studies are needed to determine the clinical utility of urinary VEGF and angiogenin as biomarkers in the non-invasive evaluation of BCa patients.
angiogenin; bladder cancer; biomarkers; diagnosis; VEGF
Terminal deoxynucleotidyl transferase, which requires a single-stranded DNA primer under the usual assay conditions, can be made to accept double-stranded DNA as primer for the addition of either rNMP or dNMP, if Mg+2 ion is replaced by Co+2 ion. The priming efficiency in the presence of Co+2 ion with respect to initial rate tested with 2 single-stranded primer, is 5-6 fold higher than that observed with Mg+2 ion. In the presence of Co+2 ion, the primer specificity is altered so that all forms of duplex DNA molecules can be labeled at their unique 3'-ends regardless of whether such ends are staggered or even. Thus, using ribonucleotide incorporation, we have for the first time employed this reaction for sequence analysis of duplex DNA fragments generated by restriction endonuclease cleavages. Furthermore, by using Co+2 ion, it is possible to add a long homopolymer tract of deoxyribonucleotides to the 3'-terminus of double-stranded DNA. Therefore, without prior treatment with lambda exonuclease to expose the 3' terminus as single-stranded primer, this reaction now permits insertion of homopolymer tails at the 3'-ends of all types of DNA molecules for the purpose of in vitro construction of recombinant DNA.
Terminal deoxynucleotidyl transferase, which requires a single-stranded DNA primer under the usual assay conditions, can be made to accept double-stranded DNA as primer for the addition of either rNMP or dNMP, if Mg+2 ion is replaced by Co+2 ion. The priming efficiency in the presence of (C leads to) CO+2 ion with respect to initial rate tested with 2 single-stranded primer, is 5-6 fols higher than that observed with Mg+2 ion. In the presence of Co+2 ion, the primer specificity is altered so that all forms of duplex DNA molecules can be labeled at their unique 3' -ends regardless of whether such ends are staggered or even. Thus, using ribonucleotide incorporation, we have for the first time employed this reaction for sequence analysis of duplex DNA fragments generated by restriction endonuclease cleavages. Furthermore, by using Co+2 ion, it is possible to add a long homopolymer tract of deoxyribonucleotides to the 3'-terminus of double-stranded DNA. Therefore, without prior treatment with lambda exonuclease to expose the 3' terminus as single-stranded primer, this reaction now permits insertion of homopolymer tails at the 3'-ends of all types of DNA molecules for the purpose of in vitro construction of recombinant DNA.
The purpose of this study was to investigate the effects of vehicles, enhancers, and polymer membranes on 3-azido-3-deoxythymidine (AZT) permeation across cadaver pig skin. Four binary vehicles (ethanol/water, isopropyl alcohol/water, polyethylene glycol 400/water, and ethanol/isopropyl myristate [IPM] were tested for AZT solubility and permeability across pig skin; ethanol/IPM (50/50, vol/vol) demonstrated the highest AZT flux (185.23 μ/cm2/h). Next, the addition of various concentrations of different enhancers (N-methyl-2-pyrrolidone [NMP], oleic acid, and lauric acid) to different volume ratios of ethanol/IPM was investigated for their effect on AZT solubility and permeability across pig skin. The use of 2 conbinations (ethanol/IPM [20/80] plus 10% NMP and ethanol/IPM [30/70] plus 10% NMP) resulted in increased AZT solubility (42.6 and 56.27 mg/mL, respectively) and also high AZT flux values (284.92 and 460.34 μg/cm2/h, respectively) without appreciable changes in lag times (6.25 and 7.49 hours, respectively) when compared with formulations using only ethanol/IPM at 20/80 and 30/70 volume ratios without addition of the enhancer NMP. Finally, AZT permeation across pig skin covered with a microporous polyethylene (PE) membrane was investigated. The addition of the PE membrane to the pig skin reduced AZT flux values to ∼50% of that seen with pig skin alone. However, the AZT flux value attained with ethanol/IPM (30/70) plus 10% NMP was 215.30 μg/cm2/h, which was greater than the target flux (208 μg/cm2/h) needed to maintain the steady-state plasma concentration in humans. The results obtained from this study will be helpful in the development of an AZT transdermal drug delivery system.
Zidovudine permeation; enhancer; binary vehicles; polymer membrane; transdermal delivery system
The aim of the present work was to investigate the influence of formulation factors including different grades of Carbopol® matrices and penetration enhancers on the percutaneous permeation of tetrahydropalmatine (THP), rheological properties, and in vitro release; and the correlation behind rheological properties, in vitro release, and percutaneous permeation. Transdermal penetration of THP through excised rabbit skin and in vitro release of THP across transparent Cellophane® were performed by vertical Franz diffusion cell. Rheological analyses were proceeded in terms of “steady flow tests”, “oscillation stress sweep”, and “creep recovery”. The result of percutaneous penetration of THP indicated that, the emulgel prepared with Carbopol® 971P (Cp 971P) as the matrix and N-methyl-2-pyrrolidone (NMP) as the penetration enhancer had the highest cumulative permeation amount (118.19 μg/cm2). All the experimental data showed a good fit to the Casson model in viscosimetric studies no matter what the types of matrices or the kinds of penetration enhancers were. The release profile fitted the zero-order release kinetics model with Cp 971P as the matrix without any penetration enhancers. However, when adding penetration enhancers, in vitro release of THP presented anomalous (non-Fickian) release kinetics. Clarifying the relationship behind percutaneous permeation of THP, rheological properties, and in vitro release will provide us with profound insights and facilitate the design of specific emulgel.
Carbopol®; emulgel; in vitro release; rheological properties; THP
Background and the purpose of the study
Solubility of pharmaceuticals is still a challenging subject and solubilization using cosolvents is the most common technique used in the pharmaceutical industry. The purpose of this study was reporting and modeling the experimental molar solubility of pioglitazone hydrochloride (PGZ-HCl) in binary and ternary mixtures of ethanol (EtOH), N-methyl pyrrolidone (NMP), polyethylene glycols (PEGs) 200, 400, 600 and water along with the density of saturated solutions at 298.2 °K.
To provide a computational method, the Jouyban-Acree model was fitted to the solubilities of the binary solvents, and solubilities of the ternary solvents were back-calculated by employing the solubility data in mono-solvents. In the next step, the ternary interaction terms were added to the model and the prediction overall mean percentage deviation (MPD) of the ternary data was reduced. Also a previously proposed version of the model was used to predict the solubility of PGZ-HCl in binary and ternary mixtures employing the experimental solubility data in mono-solvents.
The overall MPD of the model for fitting the binary data and predicted data of ternary solvents were 2.0 % and 50.5 %, respectively. The overall MPD of the predicted solubilities in ternary solvents using the ternary interaction terms in the model was 34.2 %, and by using the proposed version of the Jouyban-Acree model for binary and ternary data the overall correlation and prediction errors were 18.0 and 15.0 %, respectively.
The solubility of PGZ-HCl was increased by addition of EtOH, NMP, PEGs 200, 400 and 600 to aqueous solutions. The reported data extended the available solubility data of pharmaceuticals which are crucial in formulation of liquid dosage forms. The constants of the Jouyban-Acree model using the generated data are also reported which provides the possibility of solubility prediction in other solvent mixtures and temperatures.
Cosolvency; Jouyban-Acree model; Solubilization prediction; Pharmaceuticals
Bladder cancer is one of the most prevalent cancers worldwide. Furthermore, nonmuscle invasive bladder cancer has a 70% rate of recurrence, making it a considerable strain to the healthcare system. Patients with bladder cancer require repeat cystoscopic examinations of the bladder to monitor for tumor recurrence. The reason these patients have to undergo these costly, painful, invasive procedures is owing to the absence of accurate urine-based assays to detect the presence of bladder cancer noninvasively. Consequently, the development of a urine-based test to detect bladder cancer would be of tremendous benefit to both patients and healthcare systems. This article reports some of the more prominent urine markers in use today. In addition, the article will highlight some new technologies that are used to investigate novel urinary markers.
bladder cancer; genomics; marker; NMP 22; proteomics; urinary cytology; urine
In situ forming implants (ISFI) have shown promise in delivering adjuvant chemotherapy following minimally invasive cancer therapies such as thermal ablation of tumors. While ISFI systems have been thoroughly investigated for delivery of high molecular weight (Mw) therapeutics, little research has been conducted to optimize their design for delivery of low Mw drugs. This study examined the effect of varying the formulation components on the low Mw drug release profile from a ISFI consisting of poly(D,L-lactide-co-glycolide), fluorescein (model drug), and excipient dissolved in 1-methyl-2-pyrrolidinone (NMP). Effects of varying PLGA Mw, excipient concentration, and drug loading were studied. Additionally, solubility studies were conducted to determine the critical water concentration required for phase inversion. Results demonstrated that PLGA Mw was the most significant factor in modulating low Mw drug release from the ISFI systems. ISFI formulations comprised of a low Mw (16 kDa) PLGA showed a significantly (p<0.05) lower burst release (after 24 hours), 28.2 ± 0.5%, compared to higher Mw PLGA (60 kDa), 55.1 ± 3.1%. Critical water concentration studies also demonstrated that formulations with lower Mw PLGA had increased solubility in water and may thus require more time to phase invert and release the drug.
In situ forming implant; Phase inversion; Biodegradable polymers; Cancer chemotherapy; Controlled release
Patients presenting with painless hematuria form a large part of the urological patient population. In many cases, especially in younger patients, the cause of hematuria is harmless. Nonetheless, hematuria could be a symptom of malignant disease and hence most patients will be subject to cystoscopy. In this study, we aimed to develop a prediction model based on methylation markers in combination with clinical variables, in order to stratify patients with high risk for bladder cancer.
Material and Methods
Patients (n=169) presenting with painless hematuria were included. 54 patients were diagnosed with bladder cancer. In the remaining 115 patients, the cause of hematuria was non-malignant. Urine samples were collected prior to cystoscopy. Urine DNA was analyzed for methylation of OSR1, SIM2, OTX1, MEIS1 and ONECUT2. Methylation percentages were calculated and were combined with clinical variables into a logistic regression model.
Logistic regression analysis based on the five methylation markers, age, gender and type of hematuria resulted in an area under the curve (AUC) of 0.88 and an optimism corrected AUC of 0.84 after internal validation by bootstrapping. Using a cut-off value of 0.307 allowed stratification of patients in a low-risk and high-risk group, resulting in a sensitivity of 82% (44/54) and a specificity of 82% (94/115). Most aggressive tumors were found in patients in the high-risk group. The addition of cytology to the prediction model, improved the AUC from 0.88 to 0.89, with a sensitivity and specificity of 85% (39/46) and 87% (80/92), retrospectively.
This newly developed prediction model could be a helpful tool in risk stratification of patients presenting with painless hematuria. Accurate risk prediction might result in less extensive examination of low risk patients and thereby, reducing patient burden and costs. Further validation in a large prospective patient cohort is necessary to prove the true clinical value of this model.
How parathyroid hormone (PTH) increases bone mass is unclear but understanding this phenomenon is significant to the improvement of osteoporosis therapy. Nmp4/CIZ is a nucleocytoplasmic shuttling transcriptional repressor that suppresses PTH-induced osteoblast gene expression and hormone-stimulated gains in murine femoral trabecular bone. To further characterize Nmp4/CIZ suppression of hormone-mediated bone growth we treated 10 wk-old Nmp4-knockout (KO) and wild-type (WT) mice with intermittent human PTH (1-34) at 30μg/kg/day or vehicle, 7 days/wk, for 2, 3, or 7 wks. Null mice treated with hormone (7 wks) gained more vertebral and tibial cancellous bone than WT animals paralleling the exaggerated response in the femur. Interestingly, Nmp4/CIZ suppression of this hormone-stimulated bone formation was not apparent during the first 2 wks of treatment. Consistent with the null mice enhanced PTH-stimulated addition of trabecular bone these animals exhibited an augmented hormone-induced increase in serum osteocalcin 3 wks into treatment. Unexpectedly the Nmp4-KO mice displayed an osteoclast phenotype. Serum C-terminal telopeptides, a marker for bone resorption, was elevated in the null mice, irrespective of treatment. Nmp4-KO bone marrow cultures produced more osteoclasts, which exhibited an elevated resorbing activity, compared to WT cultures. The expression of several genes critical to the development of both osteoblasts and osteoclasts were elevated in Nmp4-KO mice at 2 wks but not 3 wks of hormone exposure. We propose that Nmp4/CIZ dampens PTH-induced improvement of trabecular bone throughout the skeleton by transiently suppressing hormone-stimulated increases in the expression of proteins key to the required enhanced activity/number of both osteoblasts and osteoclasts.
c-fos; Fra-2; ephrins; osteoclastogenesis; osteocalcin; osteoporosis