Correlation of mRNA delivery timing and protein expression in lipid-based transfection.

Non-viral gene delivery is constrained by the dwell time that most synthetic nucleic acid nanocarriers spend inside endosomal compartments. In order to overcome this endosomal-release bottleneck, methods are required that measure nanocarrier uptake kinetics and transfection efficiency simultaneously. Here, we employ live-cell imaging on single-cell arrays (LISCA) to study the delivery-time distribution of lipid-based mRNA complexes under varied serum conditions. By fitting a translation-maturation model to hundreds of individual eGFP reporter fluorescence time courses, the protein expression onset times and the expression rates after transfection are determined. Using this approach, we find that delivery timing and protein expression rates are not intrinsically correlated at the single-cell level, even though population-averaged values of both parameters conjointly change as a function of increasing external serum protein fraction. Lipofectamine-mediated delivery showed decreased transfection efficiency and longer delivery times with increasing serum protein concentration. This is in contrast to ionizable lipid nanoparticle (i-LNP)-mediated transfer, which showed increased efficiency and faster uptake in the presence of serum. In conclusion, the interdependences of single-cell expression rates and onset timing provide additional clues on uptake and release mechanisms, which are useful for improving nucleic acid delivery.

Optimizing oncology therapeutics through quantitative translational and clinical pharmacology: challenges and opportunities.

Despite advances in biomedical research that have deepened our understanding of cancer hallmarks, resulting in the discovery and development of targeted therapies, the success rates of oncology drug development remain low. Opportunities remain for objective dose selection informed by exposure-response understanding to optimize the benefit-risk balance of novel therapies for cancer patients. This review article discusses the principles and applications of modeling and simulation approaches across the lifecycle of development of oncology therapeutics. Illustrative examples are used to convey the value gained from integration of quantitative clinical pharmacology strategies from the preclinical-translational phase through confirmatory clinical evaluation of efficacy and safety.

Exposure-response relationship of T-DM1: insight into dose optimization for patients with HER2-positive metastatic breast cancer.

Exposure-response (E-R) analyses for ado-trastuzumab emtansine (T-DM1, Kadcyla) were performed using data from a randomized, active control (lapatinib plus capecitabine) trial in patients with human epidermal growth factor 2-positive metastatic breast cancer. Kaplan-Meier survival analyses stratified by T-DM1 trough concentration on day 21 of cycle 1 (Cmin,C1D21) were performed for overall survival (OS) and progression-free survival (PFS). E-R analyses indicated that after adjusting for baseline risk factors, higher T-DM1 exposure is associated with improved efficacy. T-DM1-treated patients with Cmin,C1D21 lower than the median value had values of OS and PFS comparable to those of the active control arm. The percentage of patients who received T-DM1 dose adjustments was similar across the exposure range and was lower than that of the active control arm. Our findings suggest that there may be an opportunity to optimize Kadcyla dose in the patient subgroup with low T-DM1 exposure for improved efficacy with acceptable tolerability.

Is this the dose for you?: the role of modeling.

To make an informed benefit-risk evaluation of a drug, a range of doses needs to be evaluated and its dose-response and exposure-response relationships for safety and effectiveness assessed during drug development (International Conference on Harmonisation E4). Once a safe and effective population dose (or doses) has been determined for indicated use(s), the individual patient dose can then be adjusted based on patient-specific factors (e.g., age, race, genetics, organ functions, concomitant medications).

Effect of copper on growth, yield and concentration of Fe, Mn, Zn and Cu in wheat plants (Triticum aestivum L.).

A pot experiment was conducted at six graded levels of copper (Cu) viz., 0, 0.5, 1.0, 1.5, 2.0 and 2.5 mg kg(-1) to test the response of wheat plants grown in a copper-responsive alluvial soil (entisol) under glass house conditions. The growth attributes like plant height, fresh and dry matter yield, percent dry matter enhanced with increasing Cu levels and was maximum at 1.5 mg kg(-1) Cu while the number of tillers was minimum at this level. The grain yield at 1.5 mg kg(-1) Cu was enhanced by 62.9% from the control. The increase in weight of 1000 grains ranged from 33.93 to 41.35 g in comparison to control (32.58 g). Harvest index (%) also increased and ranged from 39.42 to 47.73 in different treatments in comparison to control (35.92). Both 1000 grain weight and harvest index were maximum in the plants at 1.5 mg kg(-1) copper. Cu concentrations in leaves, grain and straw enhanced with increasing levels of Cu application. The Fe concentration in leaves was significantly reduced by Cu application and the reduction was 10.3% at 2.5 mg kg(-1) Cu and was not influenced in by Cu application in grain and straw. The Mn concentration was not affected by Cu application in any of the plant part studied. However, Zn concentration decreased significantly at higher levels of Cu (2.0 and 2.5 mg kg(-1)) in leaves and remained unaffected in the grain and straw.

Giant vestibular schwannomas: focusing on the differences between the solid and the cystic variants.

In vestibular schwannomas (VS), the tumour size, as well as the size of the cystic component, have a considerable bearing on the outcome. This study addresses the differences between the cystic and solid variants of giant vestibular schwannomas. The study included 62 patients with giant VSs, of which 40 were solid and 22 were cystic (those in which cystic component greater or equal to 30% of the total tumour volume). The cystic tumour group was further divided into type A (31-60% volume of the cyst within tumour), type B (61-90% intra-tumoural cyst volume) and type C (more than 90% volume of the cyst). The clinicoradiological features, operative findings, histopathological characteristics and outcome of surgery of the two groups were compared. The mean duration of symptoms for the solid and cystic tumours were 21.1 and 26.2 months, respectively. However, six patients with cystic tumours showed recent and rapid neurological deterioration after a protracted existence. Papilloedema, lower cranial nerve involvement, facial paraesthesias and preoperative hydrocephalus were significantly more in cystic tumours. Total excision was achieved in 38 of the solid and 18 of the cystic tumours. VIIth nerve preservation was higher in the cystic lesions [solid 33/40 (82.5%), cystic 21/22 (95.4%)]. Myxoid degeneration, lobular growth patterns and cellular atypia were more prominent in the cystic variants. The giant vestibular schwannomas were associated with a higher incidence of cystic degeneration than has been reported for smaller tumours in literature. In cystic lesions, VIIth nerve preservation was higher due to early decompression of the lesion that facilitated in early identification of the VIIth nerve, except in patients with type C cystic tumour.

Management strategies for bilateral vestibular schwannomas.

BACKGROUND: Bilateral vestibular schwannomas (VS) are rare. Most patients in India present late in the course of illness with large tumors and disabling deafness. Clinical presentation and management goals are different from that of unilateral VS. AIMS: To highlight the differences in clinical presentations and surgical results of bilateral VS compared to unilateral VS; and, to propose a management strategy for these tumors with reference to tumor size, extent of growth and the presence or absence of hearing impairment. METHOD: This is a retrospective study of 16 patients with bilateral VS treated over 10 years in a tertiary referral hospital. Assessment of VIIth and VIIIth cranial nerve function, tumor size, volume and extent of growth was performed in all patients. The management strategy was based on Samii's classification of tumor extent. All patients were operated using a retromastoid suboccipital approach. Postoperative results were analyzed and compared with those of unilateral VS. RESULTS: The mean age of presentation was 25.7 years. Hearing impairment was the commonest symptom. Headache with features of raised intracranial pressure were present in 10 (62.5%) patients. Giant tumors were present in seven (43.7%) patients; large tumors in eight (50%) and a medium-sized tumor in one (6.3%). Total tumor resection was achieved in 13 patients and subtotal resection in two. One patient was managed conservatively and followed up with serial CT scans. On the contralateral side, one large tumor required total excision. One medium sized tumor underwent sub-capsular excision in an attempt to preserve hearing. The facial nerve was anatomically preserved in seven (46.7%) patients and in one, the cochlear nerve was anatomically preserved. There was no peri-operative mortality. CONCLUSIONS: Patients with bilateral schwannomas are younger, have larger lesions, poorer preoperative hearing and are more likely to lose either auditory and/or facial nerve function during attempted total resection of the tumor. Classifying the tumors into two groups by extent, that is, tumors extending to the cerebellopontine angle cistern (T1-T3a) and, tumors extending to or compressing the brainstem (T3b to T4b), allows the surgical strategy to be defined.

The role of pharmacokinetics and pharmacodynamics in phosphodiesterase-5 inhibitor therapy.

Differences in clinical pharmacology of the currently marketed phosphodiesterase (PDE)5 inhibitors sildenafil, vardenafil and tadalafil are largely determined by their pharmacokinetic (PK) properties and their PDE5 inhibitory activity profile. This review outlines the basic concepts of pharmacokinetics and pharmacokinetic pharmacodynamic (PK/PD) relationships and their relevance to dose selection and applied pharmacotherapy. It is followed by a detailed comparative discussion on the pharmacokinetics and exposure-response relationship of the currently available PDE5 inhibitors, including known drug-drug interactions and dosage adjustments in special populations. The review is aimed at providing a critical assessment of the pharmacokinetics of PDE5 inhibitors, which may assist clinicians in tailoring drug and/or treatment regimens to the unique needs of each individual patient with erectile dysfunction.

Preoperative ultrasound-guided fine-needle aspiration cytology for axillary staging in breast carcinoma.

Axillary lymph node (ALN) status is considered to be the single most important prognostic indicator in patients with breast cancer. It can be assessed by various radiological, pathological and surgical techniques, the most accurate being histological examination of lymph nodes after axillary lymph node dissection (ALND). This prospective study was conducted to assess the feasibility and diagnostic accuracy of preoperative ultrasound (US) and ultrasound-guided fine-needle aspiration cytology (USG-FNAC) of ALN in patients with breast cancer. Thirty patients with FNAC-proven breast cancer, planned for definitive surgery with axillary clearance, were included in this study. Ultrasonographic evaluation of the axillae of these patients was conducted for alterations in size, shape, contour and cortical morphology of lymph nodes that could reflect presence of underlying metastases. Ultrasound-guided fine-needle aspiration cytology of the ALN was done in 24 of these patients. These findings were evaluated, with the ALN status determined by histological examination after ALND. Out of the 30 patients, eight had T(1), 16 had T(2), five had T(3), and one had T(4) lesions. Ultrasound evaluation of the ALN had a sensitivity of 86.3%, a specificity of 41.6%, a positive predictive value of 79%, a negative predictive value of 50% and a diagnostic accuracy of 73.3%. Sensitivity of USG-FNAC was 78.95%, specificity was 100%, positive predictive value was 100%, negative predictive value was 55.56% and diagnostic accuracy was 83.33%. Our study concludes that preoperative USG-FNAC of ALN is a simple, minimally invasive, easily available and reliable technique for the initial determination of ALN status in patients with breast cancer. Those who are USG-FNAC positive can be directed towards ALND straight away, and only those who are USG-FNAC negative should be considered for sentinel lymph node biopsy. This will save considerable operating time, especially where facilities for sentinel lymph node biopsy (costly dye, gamma camera, nuclear medicine facilities) are restricted or not available.

Sonographic findings in renal parenchymal malacoplakia.

Malacoplakia of the kidney is a rare inflammatory disorder. We describe a case of bilateral renal malacoplakia in which the patient had diffuse involvement of the left kidney and a focal lesion in the right kidney. Sonography showed a diffusely echogenic, enlarged left kidney with loss of corticomedullary differentiation and a single anechoic lesion measuring 1.8 x 1.6 x 1.3 cm in the right kidney. A left nephrectomy was performed, and the right kidney was managed conservatively with sonographic monitoring.

Evaluation of carcinogenic and co-carcinogenic potential Quinalphos in mouse skin.

Quinalphos [O,O-diethyl-O-quinoxalinyl-phosphorothidate] is an organophosphorus pesticide with tremendous utility in mixed pest control due to its insecticidal and acaricidal properties. Apart from its pesticidal property, Quinalphos is known to induce various toxic effects in nontarget species and experimental animals. No studies have been conducted to evaluate the carcinogenic/co-carcinogenic hazards associated with Quinalphos exposure. In the present set of investigations, the tumorigenic potential of Quinalphos has been evaluated following topical exposure in Swiss albino mice. Long-term animal bioassays conducted for the evaluation of complete carcinogenic, tumour-initiating and tumour-promoting potential of Quinalphos revealed that it has only tumour-initiating potential at a dose of 10 mg/kg body weight (b.wt.), in the two-stage mouse skin model of carcinogenesis. Quinalphos exposure failed to produce neoplasia when tested for complete carcinogenic activity at all three tested dose levels or tumour promoting activity.

Carcinogenicity and co-carcinogenicity studies on propoxur in mouse skin.

Propoxur (2-isopropoxyphenyl methylcarbamate) is a widely used broad spectrum carbamate insecticide mainly used to control household pests. Propoxur exposure is reported to inhibit cholinesterase activity in rodents. Apart from other toxic effects, propoxur was found to possess tumorigenic activity in rats after oral administration. Propoxur does not produce tumours in mice or hamsters, or bladder hyperplasia in dogs and monkeys following oral feeding. In this set of investigations the complete carcinogenic, tumour initiating and promoting potential of propoxur was evaluated in male and female Swiss albino mice, since no information was available following dermal exposure of propoxur. The animals were exposed to propoxur through topical painting on the interscapular region at a dose of 100 mg/kg body weight. The results revealed that propoxur has tumour promoting potential on mouse skin following a two-stage initiation-promotion protocol, but it failed to induce the tumour(s) at a significant level, when tested for tumour initiating and complete carcinogenic property.

Chemopreventive effects of black tea polyphenols in mouse skin model of carcinogenesis.

In the present investigations, the antitumorigenic effect of black tea polyphenols (BTP) in two-stage mouse skin model of carcinogenesis was studied. The animals were initiated with a single "subcarcinogenic" topical dose (52 micrograms/200 microliters acetone) of 7, 12-dimethylbenzanthracene (DMBA). To evaluate the anti-tumour initiating activity, BTP was topically applied twice a week for three weeks prior to DMBA application, followed by topical treatment with 12-o-tetradecanoyl phorbol-13-acetate (TPA) (5 micrograms/200 microliters acetone, 2x/wk.) as promoter. For evaluation of antitumor promoting activity, BTP was applied prior to each treatment of TPA. BTP application showed marked inhibitory effect as antitumour initiator as well as antitumour promoter in mouse skin model of two-stage carcinogenesis. Since initiation involves genetic pathway and tumour promotion involves epigenetic pathway, it seems that BTP exerts its antitumorigenic effect by altering both genetic and epigenetic pathways.

Effect of some essential oils on microorganisms.

The essential oils tested viz., Cardiaca Oil, Mentha Oil, Artemisia Oil and Geranium Oil against pathogenic bacterial and fungal strains. Significant activity of all oils was found against all microorganisms. All the four oils showed a higher inhibition against all the microorganisms, except S. epidermidis at different level of concentrations used (1000 ppm and 500 ppm). It is clearly indicated that inhibition activity increased approximately 1 to 2 folds at 1000 ppm concentration as compared to 500 ppm. S. epidermidis has shown resistant towards all the oils at both the concentration while M. smegmatis and S. mutans have shown higher inhibition as compared to S. epidermidis.

Assessment of mutagenic potential of thiram.

Thiram is a widely used dithiocarbamate fungicide. In this study, the mutagenicity of thiram was investigated using the micronucleus and dominant lethal tests in Swiss albino mice. A single ip injection of 100 mg thiram/kg body weight, which is the maximum tolerated dose (MTD), significantly induced micronucleus formation in bone marrow cells after 30 and 48 hr of exposure; 50% and 25% of the MTD also induced micronucleus formation after the above time periods. A significant number of dead implants were induced when thiram was given to male mice in the diet at 10% of the oral LD50 during the whole spermatogenesis cycle (8 wk); this post-implantation loss indicates a dominant lethal mutation.

Congestive heart failure caused by Wilms' tumor.

A 2-year-old child with unilateral Wilms' tumor presented with congestive heart failure (CHF). The heart failure was secondary to severe hypertension from hyperreninemia. After surgical removal of the tumor the CHF disappeared. This case is the first one reported of a child presenting with CHF due to unilateral Wilms' tumor.

Differential effects of butyric acid on mouse skin tumorigenesis.

We studied the effects of butyric acid (BA) on mouse skin tumorigenesis using chronic animal bioassays. Topical application of BA immediately after each treatment with 12-0-tetradecanoylphorbol-13-acetate (TPA) promoter-inhibited skin tumors. The effect was dependent on the dose of BA applied. BA showed no marked inhibitory effect on either skin tumor initiation or complete tumorigenesis induced by dimethylbenzanthracene (DMBA). Since tumor promotion reportedly involves epigenetic events whereas tumor initiation or complete tumorigenesis takes place through genetic pathways, it is possible that BA exerts its antitumorigenic effects mainly by altering the epigenetic events responsible for tumor promotion. The results of the study could further be used to study the mechanism of action and modification of antitumorigenic effects of BA in combination with other substances.

Assessment of mutagenic potential of propoxur and its modulation by indole-3-carbinol.

Propoxur is a widely used dithiocarbamate pesticide. In the present set of investigations, mutagenicity of propoxur (in formulation) was studied using the micronucleus assay in bone marrow of Swiss mice. Single intraperitoneal (i.p.) administration of 25 mg/kg body weight dose of propoxur, which is a maximum tolerated dose (MTD), significantly induced the micronucleus formation in bone marrow cells after a 24- and 48-hr exposure. A half and a quarter of the MTD (12.5 and 6.25 mg/kg) were found ineffective to induce the micronuclei formation after 24- and 48-hr time periods by the i.p. route. However, the PCE:NCE ratio was inhibited significantly with all the dose levels at both time periods. Oral administration of propoxur at different dose levels also induced micronuclei formation. A single application of 50 and 25 mg/kg dose levels of propoxur, which are MTD and 50% of MTD, also significantly induced micronuclei formation after 24- and 48-hr time periods in bone marrow cells of Swiss mice as compared with solvent control group, whereas a 12.5 mg/kg dose of propoxur was ineffective in inducing micronuclei formation. Single application of indole-3-carbinol (I3C), a glucobrassicin derivative present in cruciferous vegetables, significantly inhibited the propoxur-induced micronuclei formation when it was given at the dose level of 500 mg/kg body weight 48 hr before the single application of propoxur. Therefore, it seems that propoxur is mutagenic in the above test systems and I3C inhibited the mutagenicity of propoxur significantly.

Micronucleus induction by diuron in mouse bone marrow.

Diuron, a widely used substituted urea herbicide, induced the formation of micronuclei in bone marrow cells of Swiss mice. A single i.p. dose of 340 mg/kg b.w. diuron which is maximum tolerated dose (MTD) increased significantly the number of micronuclei at 30 h and 48 h time period. The dose of 170 mg/kg b.w. also induced the micronuclei formation in the above time period. However, a dose of 85 mg/kg b.w. was ineffective at the time periods studied. No induction of micronuclei was observed at 72 h time period after all the doses of diuron studied as compared to the solvent control. The diuron-induced frequency of micronucleated erythrocytes was independent of the sex of the test animals.