Target Site Delivery and Residence of Nanomedicines: Application of Quantitative Systems Pharmacology.

Quantitative systems pharmacology (QSP), an emerging field that entails using modeling and computation to interpret, interrogate, and integrate drug effects spanning from the molecule to the whole organism to forecast treatment outcomes, is expected to enhance the efficiency of drug development. Since late 2017, the U.S. Food and Drug Administration has advocated the use of an analogous approach of model-informed drug development. This review focuses on issues pertaining to nanosized medicines (NP) and the potential utility of QSP to determine NP delivery and residence at extracellular or intracellular targets in vivo. The kinetic processes governing NP disposition and transport, interactions with biologic matrix components, binding and internalization in cells, and intracellular trafficking are determined, sometimes jointly, by NP properties (e.g., dimension, materials, surface charge and modifications, shape, and geometry) and target tissue properties (e.g., perfusion status, vessel pore size and wall thickness, vessel and cell density, composition of extracellular matrix, and void volume fraction). These various determinants, together with the heterogeneous tissue structures and microenvironment factors in solid tumors, lead to environment-, spatial-, and time-dependent changes in NP concentrations that are difficult to predict. Adding to the complexity is the recent discovery that NP surface-coating protein corona, whose composition depends on NP properties and which undergoes continuous evolution with time and local protein environments, is yet another unpredictable variable. Examples are provided to demonstrate the potential utility of QSP-based multiscale modeling to capture the physicochemical and biologic processes in equations to enable computational studies of the key kinetic processes in cancer treatments.

Observational study of compliance with infection control practices among healthcare workers in subsidized and private residential care homes.

BACKGROUND: The elderly population in Hong Kong is rapidly growing, and the need for residential care homes (RCHs) is increasing. The risk of being infected with micro-organisms increases among the frail and the vulnerable elderly population as their immunity system begins to deteriorate. Furthermore, the residents in RCHs are at high risk of healthcare-associated infections (HAIs) due to the confined living environments and individual co-morbidities. In relation to this, infection control practice (ICP) is considered a crucial and effective approach in preventing HAIs. This study aimed to observe the daily ICP of healthcare workers in RCH settings. METHODS: An observational study was conducted to observe daily ICP among healthcare workers in private and subsidized RCHs. Each RCH was separated into different units based on the location (common area and bedroom area) and nature of residents for successive days. The ICP episodes were observed until 200 opportunities in each unit. The ICP episodes were recorded by an electronic tool called "eRub," which is an ICP checklist based on international guidelines. RESULTS: The most frequent observed ICP episodes were hand hygiene (n = 1053), the use of gloves (n = 1053) and respiratory protection (n = 1053). The overall compliance of hand hygiene was poor, with only 15% of participants performing this during the "five moments for hand hygiene." Furthermore, the observations showed that 77.9% improperly performed the use of gloves, and 31.8% failed to wear a mask during the care provision for the elderly. However, the results showed that most healthcare workers can wear the mask in a proper way when they should. Generally, the personal care workers were the worst in terms of hand hygiene and use of gloves compared with the other types of healthcare workers. CONCLUSIONS: Despite the fact that the practice of hand hygiene, the use of gloves, and respiratory protection were the important elements of ICP, overall compliance to these elements was still poor. Personal care workers had the most frequent contact with the residents, but they had the worst compliance rate. Hence, continued monitoring and training among healthcare workers is needed, particularly personal care workers, in this healthcare service setting.

Intravenous siRNA Silencing of Survivin Enhances Activity of Mitomycin C in Human Bladder RT4 Xenografts.

PURPOSE: Survivin inhibits apoptosis and enables tumor cells to escape from therapy induced senescence. High survivin expression is associated with bladder cancer aggressiveness and recurrence. We evaluated whether survivin expression is reduced by siRNA and whether survivin silencing would enhance mitomycin C activity in human RT4 bladder transitional cell tumors in vitro and in vivo. MATERIALS AND METHODS: We assessed the effectiveness of siRNA therapy using 2 newly developed pegylated cationic liposome carriers, PCat and PPCat. Each has a fusogenic lipid to destabilize the endosomal membrane. PPCat further contains paclitaxel to enhance in vivo delivery and transfection of survivin siRNA. In vitro antitumor activity was evaluated by short-term MTT and long-term clonogenicity cytotoxicity assays. In vivo intravenous therapy was assessed in mice bearing subcutaneous tumors. RESULTS: Nontarget siRNA showed no antitumor activity in vitro or in vivo. Treatment of cultured cells with mitomycin C at a 50% cytotoxic concentration enhanced survivin mRNA and protein levels. Adding PPCat or PCat containing survivin siRNA reversed survivin induction and enhanced mitomycin C activity (p <0.05). In tumor bearing mice single agent mitomycin C delayed tumor growth and almost tripled the survivin protein level in residual tumors. Adding PPCat-survivin siRNA, which alone resulted in a minor survivin decrease of less than 10%, completely reversed mitomycin C induced survivin and enhanced mitomycin C activity (p <0.05). CONCLUSIONS: Results indicate that there is effective in vivo survivin silencing and synergism between mitomycin C and PPCat-survivin siRNA. This combination represents a potentially useful chemo-gene therapy for bladder cancer.

From Leaf Metabolome to In Vivo Testing: Identifying Antifeedant Compounds for Ecological Studies of Marsupial Diets.

Identifying specific plant secondary metabolites that influence feeding behavior can be challenging, but a solid understanding of animal preferences can guide efforts. Common brushtail possums (Trichosurus vulpecula) predominantly eat Eucalyptus species belonging to the subgenus Symphyomyrtus, and avoid eating those belonging to the Monocalyptus subgenus (also called subgenus Eucalyptus). Using an unbiased (1)H NMR metabolomics approach, a previous study identified unsubstituted B ring flavanones in most species of monocalypts examined, whereas these compounds were absent from symphyomyrtles. We hypothesised that unsubstituted B ring flavanones act as feeding deterrents for common brushtail possums. In the current study, we tested this hypothesis by comparing how much possums ate of a basal diet, with diets containing one of four structurally related compounds; pinocembrin, flavanone (unsubstituted B ring flavanones), chrysin (the flavone analogue of pinocembrin), and naringenin (a flavanone with B ring substitution). We found that pinocembrin and flavanone deterred feeding relative to the basal diet, but that chrysin and naringenin did not at equivalent concentrations. Thus, unsubstituted B-ring flavanones may explain why brushtail possums avoid eating monocalypt species. Furthermore, small differences in the structure of secondary compounds can have a large impact on antifeedant properties. These results demonstrate that metabolomics can be a valuable tool for ecologists seeking to understand herbivore feeding preferences.

Postoperative adjuvant combination therapy with doxorubicin and noncytotoxic suramin in dogs with appendicular osteosarcoma.

Although conventional treatment of dogs with osteosarcoma (OSA) by amputation and chemotherapy results in reported survival times (STs) of 262-413 days, no major improvements in STs have occurred in the past 2 decades. Suramin is a polysulfonated napthylurea, which at noncytotoxic concentrations in vitro, increases tumor sensitivity to chemotherapy, including doxorubicin. The study authors evaluated the combination of noncytotoxic suramin and doxorubicin after amputation in dogs with OSA. The hypothesis was that treatment of dogs with appendicular OSA with amputation, adjuvant doxorubicin, and noncytotoxic suramin would be well tolerated and result in STs at least comparable to those of doxorubicin alone. Forty-seven dogs received 6.75 mg/kg of suramin IV followed by 30 mg/m(2) of doxorubicin IV 4 hr later. Treatment was repeated q 2 wk for five doses. The median disease free time (DFI) was 203 days (range, 42-1,580+ days) and the median ST for all dogs was 369 days (range, 92-1,616+ days). There was no statistical difference in ST and DFI between greyhounds and nonngreyhounds. Adjuvant doxorubicin and noncytotoxic suramin was well tolerated in dogs with OSA following amputation. Additional studies are needed to determine if this combination treatment protocol provides additional clinical benefit compared with doxorubicin alone.

Quantitative image analysis of intracellular protein translocation in 3-dimensional tissues for pharmacodynamic studies of immunogenic cell death.

A recent development in cancer chemotherapy is to use cytotoxics to induce tumor-specific immune response through immunogenic cell death (ICD). In ICD, calreticulin is translocated from endoplasmic reticulum to cell membrane (ecto-CRT) which serves as the 'eat-me-signal' to antigen-presenting cells. Ecto-CRT measurements, e.g., by ecto-CRT immunostaining plus flow cytometry, can be used to study the pharmacodynamics of ICD in single cells, whereas ICD studies in intact 3-dimensional tissues such as human tumors require different approaches. The present study described a method that used (a) immunostaining with fluorescent antibodies followed by confocal microscopy to obtain the spatial locations of two molecules-of-interest (CRT and a marker protein WGA), and (b) machine-learning (trainable WEKA segmentation) and additional image processing tools to locate the target molecules, remove the interfering signals in the nucleus, cytosol and extracellular space, enable the distinction of the inner and outer edges of the cell membrane and thereby identify the cells with ecto-CRT. This method, when applied to 3-dimensional human bladder cancer cell spheroids, yielded drug-induced ecto-CRT measurements that were qualitatively comparable to the flow cytometry results obtained with single cells disaggregated from spheroids. This new method was applied to study drug-induced ICD in short-term cultures of surgical specimens of human patient bladder tumors.

Surgical management of peritoneal metastasis: Opportunities for pharmaceutical research.

Cytoreductive surgery (CRS) has emerged as a survival-extending treatment of peritoneal metastasis (PM); recent advances include using intraperitoneal chemotherapy (IPC) at normothermic or hyperthermic temperatures, or under pressure (CRS + IPC). Clinical CRS + IPC research has established its highly variable efficacy and suggested tumor size, tumor locations and presence of ascites as potential determinants. On the other hand, there is limited knowledge on the effects of pharmaceutical properties on treatment outcomes. The present study investigated the inter-subject variability of paclitaxel binding to proteins in patient ascites because some PM patients show accumulation of ascites and because activity and transport of highly protein-bound drugs such as paclitaxel are affected by protein binding. Ascites samples were collected from 26 patients and investigated for their protein contents using LC/MS/MS proteomics analysis and for the concentrations of total proteins and two major paclitaxel-binding proteins (human serum albumin or HSA and α-1-acid glycoprotein or AAG). The association constants of paclitaxel to HSA and AAG and the extent of protein binding of paclitaxel in patient ascites were studied using equilibrium dialysis. Proteomic analysis of four randomly selected samples revealed 288 proteins, >90% of which are also present in human plasma. Between 72% - 94% of paclitaxel was bound to proteins in patient ascites. The concentrations of HSA and AAG in ascites showed substantial inter-subject variations, ranging from 14.7 - 46.3 mg/mL and 0.13-2.56 mg/mL, respectively. The respective paclitaxel association constants to commercially available HSA and AAG were âˆ¼ 3.5 and âˆ¼ 120 mM. Calculation using these constants and the HSA and AAG concentrations in individual patient ascites indicated that these two proteins accounted for >85% of the total protein-binding of paclitaxel in ascites. The extensive drug binding to ascites proteins, by reducing the pharmacologically active free fraction, may lead to the diminished CRS efficacy in PM patients with ascites. Clinical advances in CRS + IPC have outpaced current knowledge of pharmaceutical properties in this setting. IPC, as a locally acting therapy, is subjected to processes different from those governing systemic treatments. This study, to our knowledge, is the first to illustrate the implications of drug properties in the CRS + IPC efficacy against PM. While drugs are now an integral part of PM patient management, there is limited pharmaceutical research in this treatment setting (e.g., effects of hyperthermia or pressure on drug transport or release from delivery systems, pharmacokinetics, pharmacodynamics). Hence, CRS + IPC of PM represents an area where additional pharmaceutical research can assist further development and optimization.

Tracking canopy chlorophyll fluorescence with a low-cost light emitting diode platform.

Chlorophyll fluorescence measured at the leaf scale through pulse amplitude modulation (PAM) has provided valuable insight into photosynthesis. At the canopy- and satellite-scale, solar-induced fluorescence (SIF) provides a method to estimate the photosynthetic activity of plants across spatiotemporal scales. However, retrieving SIF signal remotely requires instruments with high spectral resolution, making it difficult and often expensive to measure canopy-level steady-state chlorophyll fluorescence under natural sunlight. Considering this, we built a novel low-cost photodiode system that retrieves far-red chlorophyll fluorescence emission induced by a blue light emitting diode (LED) light source, for 2 h at night, above the canopy. Our objective was to determine if an active remote sensing-based night-time photodiode method could track changes in canopy-scale LED-induced chlorophyll fluorescence (LEDIF) during an imposed drought on a broadleaf evergreen shrub, Polygala myrtifolia. Far-red LEDIF (720-740 nm) was retrieved using low-cost photodiodes (LEDIFphotodiode) and validated against measurements from a hyperspectral spectroradiometer (LEDIFhyperspectral). To link the LEDIF signal with physiological drought response, we tracked stomatal conductance (gsw) using a porometer, two leaf-level vegetation indices-photochemical reflectance index and normalized difference vegetation index-to represent xanthophyll and chlorophyll pigment dynamics, respectively, and a PAM fluorimeter to measure photochemical and non-photochemical dynamics. Our results demonstrate a similar performance between the photodiode and hyperspectral retrievals of LEDIF (R2 = 0.77). Furthermore, LEDIFphotodiode closely tracked drought responses associated with a decrease in photochemical quenching (R2 = 0.69), Fv/Fm (R2 = 0.59) and leaf-level photochemical reflectance index (R2 = 0.59). Therefore, the low-cost LEDIFphotodiode approach has the potential to be a meaningful indicator of photosynthetic activity at spatial scales greater than an individual leaf and over time.

Paclitaxel gelatin nanoparticles for intravesical bladder cancer therapy.

PURPOSE: We have noted that inadequate drug delivery to tumor cells is a major cause of failed intravesical therapy for nonmuscle invading bladder cancer, partly due to the dilution of drug concentration by urine production during treatment. To address this problem we developed gelatin nanoparticles of paclitaxel designed to yield constant drug concentrations. The hypothesis that a constant, therapeutic concentration in urine, bladder tissue and tumors can be attained was evaluated in dogs. MATERIALS AND METHODS: We studied drug release from paclitaxel gelatin nanoparticles in culture medium in vitro. In vivo studies were performed in tumor-free dogs and in pet dogs with naturally occurring transitional cell carcinoma, in which the pharmacokinetics of paclitaxel gelatin nanoparticles were determined in plasma, urine and tumors. RESULTS: Paclitaxel release from paclitaxel gelatin nanoparticles in vitro and in vivo was rate limited by the drug solubility in aqueous medium. This property yielded constant drug concentrations independent of changes in urine volume during the 2-hour treatment. Intravesical paclitaxel gelatin nanoparticles showed low systemic absorption, and favorable bladder tissue/tumor targeting and retention properties with pharmacologically active concentrations retained in tumors for at least 1 week. CONCLUSIONS: Constant drug release from paclitaxel gelatin nanoparticles may overcome the problem of drug dilution by newly produced urine and the sustained drug levels in tumors may decrease treatment frequency.

Paclitaxel tumor-priming enhances siRNA delivery and transfection in 3-dimensional tumor cultures.

The clinical development of siRNA cancer therapeutics is limited by the poor interstitial transport and inefficient transfection in solid tumors. We have shown that paclitaxel pretreatment, by inducing apoptosis, causes expansion of the interstitial space and thereby improves nanoparticle delivery and transport in tumor interstitium (referred to as paclitaxel tumor priming) and efficacy of nanomedicines in tumor-bearing animals. The present study evaluated whether paclitaxel tumor priming improves the delivery and transfection of siRNA in 2- and 3-dimensional cultures of human oropharyngeal carcinoma FaDu cells. We used the fluorescent siGLO and confocal microcopy to monitor transport, and used survivin siRNA and immunostaining and immunoblotting to monitor transfection. Survivin is a chemoresistance gene/protein, inducible by chemotherapy. siRNA was loaded in cationic liposomes. The results showed that pretreatment with 50-200 nM paclitaxel (24 or 48 h before siRNA) enhanced the total uptake of siGLO into monolayers (∼15%, p < 0.05), and the depth of penetration into 3-dimensional spheroids and tumor fragment histocultures (2.1- to 2.5-times greater area under the penetration-depth curve). In both monolayer cells and histocultures, paclitaxel pretreatment induced survivin upregulation (p < 0.05). Survivin siRNA alone decreased the survivin levels in a dose-dependent manner, and applying survivin siRNA after paclitaxel pretreatment completely abolished the paclitaxel-induced survivin increases. These findings indicate that paclitaxel tumor priming did not compromise the siRNA functionality. In summary, paclitaxel tumor priming improved the penetration, transfection and functionality of siRNA in tumors, thus offering a promising and practical means to develop chemo-siRNA cancer gene therapy.

Is It Time to Use Modeling of Cellular Transporter Homeostasis to Inform Drug-Drug Interaction Studies: Theoretical Considerations.

Mathematical modeling has been an important tool in pharmaceutical research for 50 + years and there is increased emphasis over the last decade on using modeling to improve the efficiency and effectiveness of drug development. In an earlier commentary, we applied a multiscale model linking 6 scales (whole body, tumor, vasculature, cell, spatial location, time), together with literature data on nanoparticle and tumor properties, to demonstrate the effects of nanoparticle particles on systemic disposition. The current commentary used a 4-scale model (cell membrane, intracellular organelles, spatial location, time) together with literature data on the intracellular processing of membrane receptors and transporters to demonstrate disruption of transporter homeostasis can lead to drug-drug interaction (DDI) between victim drug (VD) and perpetrator drug (PD), including changes in the area-under-concentration-time-curve of VD in cells that are considered significant by the US Food and Drug Administration (FDA). The model comprised 3 computational components: (a) intracellular transporter homeostasis, (b) pharmacokinetics of extracellular and intracellular VD/PD concentrations, and (c) pharmacodynamics of PD-induced stimulation or inhibition of an intracellular kinetic process. Model-based simulations showed that (a) among the five major endocytic processes, perturbation of transporter internalization or recycling led to the highest incidence and most extensive DDI, with minor DDI for perturbing transporter synthesis and early-to-late endosome and no DDI for perturbing transporter degradation and (b) three experimental conditions (spatial transporter distribution in cells, VD/PD co-incubation time, extracellular PD concentrations) were determinants of DDI detection. We propose modeling is a useful tool for hypothesis generation and for designing experiments to identify potential DDI; its application further aligns with the model-informed drug development paradigm advocated by FDA.

Bioluminescence-based hygiene evaluation of public washroom environment: Repeated measurement of posthandwashing facilities on baseline and before and after cleaning schedule.

BACKGROUND: The risk of hand recontamination is often neglected after using hand washing facilities, which can increase the spread of pathogens. The study aimed to evaluate the hygienic condition of posthandwashing facilities in public washrooms at different timeslots, particularly those near food courts and restaurants located in shopping malls. METHODS: This observational study was conducted in 12 public washrooms that ranged from low-end, middle-end, to high-end category on 3 different timeslots including baseline, T1 (immediate postcleaning) and T2 (1-hour postcleaning, with counting the footfall). Hand-touch surfaces with a high risk of recontamination after handwashing, which included paper tower dispensers, air drying outlets, and exit door handles, were evaluated by the surface adenosine triphosphate (ATP) bioluminescence method (ATP-value). ATP-values <500 relative light units (RLUs) were considered a good hygiene. Cleaning schedules and footfalls of each sampled washroom were obtained by direct observations. RESULTS: The overall mean ATP value of washroom was 785 RLU (N = 108); the ATP values of female and male washrooms at T2 were 203 and 3,718 RLUs, respectively. The highest ATP value was found on the exit door handles of male washrooms (range = 13-26,695 RLUs, mean = 3,229 RLU). Regarding passed/failed hygiene conditions, there were significant differences in the proportion of exit door handles between genders (P = .018) and timeslots (P = .007) as well as that of paper towel button/screw between timeslots (P= .025). CONCLUSION: Attention should be paid at the exit door handles of male washrooms, where are high risks of cross and re-contamination.

A Quantitative Pharmacology Model of Exosome-Mediated Drug Efflux and Perturbation-Induced Synergy.

Exosomes, naturally occurring vesicles secreted by cells, are undergoing development as drug carriers. We used experimental and computational studies to investigate the kinetics of intracellular exosome processing and exosome-mediated drug efflux and the effects of exosome inhibition. The experiments used four human-breast or ovarian cancer cells, a cytotoxic drug paclitaxel (PTX), two exosome inhibitors (omeprazole (OME), which inhibits exosome release, and GW4869 (GW), which inhibits synthesis of sphingolipid ceramide required for exosome formation), LC-MS/MS analysis of PTX levels in exosomes, and confocal microscopic study of endocytic transport (monitored using fluorescent nanoparticles and endocytic organelle markers). In all four cells, exosome production was enhanced by PTX but diminished by OME or GW (p < 0.05); the PTX enhancement was completely reversed by OME or GW. Co-treatment with OME or GW simultaneously reduced PTX amount in exosomes and increased PTX amount and cytotoxicity in exosome-donor cells (corresponding to >2-fold synergy as indicated by curve shift and uncertainty envelope analyses). This synergy is consistent with the previous reports that OME co-administration significantly enhances the taxane activity in tumor-bearing mice and in patients with triple negative metastatic breast cancer. The experimental results were used to develop a quantitative pharmacology model; model simulations revealed the different effects of the two exosome inhibitors on intracellular PTX processing and subcellular distribution.

Intraperitoneal therapy for peritoneal cancer.

Cancers originating from organs in the peritoneal cavity (e.g., ovarian, pancreatic, colorectal, gastric and liver) account for approximately 250,000 new cancer cases annually in the USA. Peritoneal metastases are common owing to locoregional spread and distant metastases of extraperitoneal cancers. A logical treatment is intraperitoneal therapy, as multiple studies have shown significant targeting advantage for this treatment, including significant survival benefits in stage III, surgically debulked ovarian cancer patients. However, the clinical use of intraperitoneal therapy has been limited, in part, by toxicity, owing to the use of indwelling catheters or high drug exposure, by inadequate drug penetration into bulky tumors (>1 cm) and by the lack of products specifically designed and approved for intraperitoneal treatments. This article provides an overview on the background of peritoneal metastasis, clinical research on intraperitoneal therapy, the pharmacokinetic basis of drug delivery in intraperitoneal therapy and our development of drug-loaded tumor-penetrating microparticles.

Systemic Bioequivalence Is Unlikely to Equal Target Site Bioequivalence for Nanotechnology Oncologic Products.

Approval of generic drugs by the US Food and Drug Administration (FDA) requires the product to be pharmaceutically equivalent to the reference listed drug (RLD) and demonstrate bioequivalence (BE) in effectiveness when administered to patients under the conditions in the RLD product labeling. Effectiveness is determined by drug exposure at the target sites. However, since such measurement is usually unavailable, systemic exposure is assumed to equal target site exposure and systemic BE to equal target site BE. This assumption, while it often applies to small molecule drug products that are readily dissolved in biological fluids and systemically absorbed, is unlikely to apply to nanotechnology products (NP) that exist as heterogeneous systems and are subjected to dimension- and material-dependent changes. This commentary provides an overview of the intersecting and spatial-dependent processes and variables governing the delivery and residence of oncologic NP in solid tumors. In order to provide a quantitative perspective of the collective effects of these processes, we used quantitative systems pharmacology (QSP) multi-scale modeling to capture the physicochemical and biological events on several scales (whole-body, organ/suborgan, cell/subcellular, spatial locations, time). QSP is an emerging field that entails using modeling and computation to facilitate drug development; an analogous approach (i.e., model-informed drug development) is advocated by to FDA. The QSP model-based simulations illustrated that small changes in NP attributes (e.g., size variations during manufacturing, interactions with proteins in biological milieu) could lead to disproportionately large differences in target site exposure, rending systemic BE unlikely to equal target site BE.

Tumor-penetrating microparticles for intraperitoneal therapy of ovarian cancer.

Intraperitoneal chemotherapy prolongs survival of ovarian cancer patients, but its utility is limited by treatment-related complications and inadequate drug penetration in larger tumors. Previous intraperitoneal therapy used the paclitaxel/Cremophor EL (polyethoxylated castor oil) formulation designed for intravenous use. The present report describes the development of paclitaxel-loaded microparticles designed for intraperitoneal treatment (referred to as tumor-penetrating microparticles or TPM). Evaluation of TPM was performed using intraperitoneal metastatic, human ovarian SKOV3 xenograft tumor models in mice. TPM were retained in the peritoneal cavity and adhered to tumor surface. TPM consisted of two biocompatible and biodegradable polymeric components with different drug release rates; one component released the drug load rapidly to induce tumor priming, whereas the second component provided sustained drug release. Tumor priming, by expanding interstitial space, promoted transport and penetration of particulates in tumors. These combined features resulted in the following advantages over paclitaxel/Cremophor EL: greater tumor targeting (16-times higher and more sustained concentration in omental tumors), lower toxicity to intestinal crypts and less body weight loss, greater therapeutic efficacy (longer survival and higher cure rate), and greater convenience (less frequent dosing). TPM may overcome the toxicities and compliance-related problems that have limited the utility of intraperitoneal therapy.

Quantitative image analysis of intra-tumoral bFGF level as a molecular marker of paclitaxel resistance.

BACKGROUND: The role of basic fibroblast growth factor (bFGF) in chemoresistance is controversial; some studies showed a relationship between higher bFGF level and chemoresistance while other studies showed the opposite finding. The goal of the present study was to quantify bFGF levels in archived tumor tissues, and to determine its relationship with chemosensitivity. METHODS: We established an image analysis-based method to quantify and convert the immunostaining intensity of intra-tumor bFGF to concentrations; this was accomplished by generating standard curves using human xenograft tumors as the renewable tissue source for simultaneous image analysis and ELISA. The relationships between bFGF concentrations and tumor chemosensitivity of patient tumors (n = 87) to paclitaxel were evaluated using linear regression analysis. RESULTS: The image analysis results were compared to our previous results obtained using a conventional, semi-quantitative visual scoring method. While both analyses indicated an inverse relationship between bFGF level and tumor sensitivity to paclitaxel, the image analysis method, by providing bFGF levels in individual tumors and therefore more data points (87 numerical values as opposed to four groups of staining intensities), further enabled the quantitative analysis of the relationship in subgroups of tumors with different pathobiological properties. The results show significant correlation between bFGF level and tumor sensitivity to the antiproliferation effect, but not the apoptotic effect, of paclitaxel. We further found stronger correlations of bFGF level and paclitaxel sensitivity in four tumor subgroups (high stage, positive p53 staining, negative aFGF staining, containing higher-than-median bFGF level), compared to all other groups. These findings suggest that the relationship between intra-tumoral bFGF level and paclitaxel sensitivity was context-dependent, which may explain the previous contradictory findings on the merit of using plasma or urine bFGF level as a prognostic indicator. CONCLUSION: The present study established a quantitative image analysis method that enabled the measurement of intratumoral bFGF level in archived tissues. The ability to quantify a potential biomarker provided the opportunity to study the relationship between the biomarker and chemosensitivity in tumor subgroups and thereby enabled hypothesis generation for additional translational research.

Phase I/II trial of 5-fluorouracil and a noncytotoxic dose level of suramin in patients with metastatic renal cell carcinoma.

BACKGROUND: Renal cell carcinoma (RCC) is recognized as a neoplasm resistant to chemotherapy. In vitro experiments demonstrated that suramin, at noncytotoxic doses, enhanced the activity of chemotherapy including 5-fluorouracil (5-FU) in xenograft models. PATIENTS AND METHODS: A phase I/II trial of noncytotoxic suramin in combination with weekly 5-FU in patients with metastatic RCC was conducted. The treatment consisted of intravenous (i.v.) suramin followed by a 500 mg/m2 i.v. bolus of 5-FU given 4.5 hours after starting suramin. In the phase I portion, a cohort of 6 patients received a suramin dose calculated to achieve a plasma level of 10-50 micromol/L. Therapy was administered once weekly for 6 doses, followed by 2 weeks off. This was followed by a phase II portion in which the primary goal was to determine the objective response rate. RESULTS: Twenty-three patients were enrolled in the study: 6 in the phase I portion and 17 in phase II. Seventy-eight percent of patients were men, the mean age was 58.8 years, 96% had previous nephrectomy, and 70% had received previous systemic therapy. Histologic subtype was clear cell in 91%. Dose-limiting toxicity was observed in 1 of 6 patients (grade 3 hypersensitivity related to suramin infusion). The suramin dosing nomogram used in phase I and II portions of the trial yielded the desired plasma level of 10-50 micromol/L from 4.5 hours to 48 hours after infusion in 94 of 115 treatments. No objective responses were noted, and the median time to treatment failure was 2.5 months. The major toxicities (all grades) were fatigue (83%), nausea/vomiting (78%), diarrhea (61%), and chills (61%). CONCLUSION: Suramin levels expected to reverse fibroblast growth factor-induced resistance can be achieved with the dosing regimen used in this study. The toxicity observed with suramin and 5-FU was acceptable. The combination does not have clinical activity in patients with metastatic RCC.

Computational modeling to predict effect of treatment schedule on drug delivery to prostate in humans.

PURPOSE: To evaluate a computational approach that incorporates experimental data in preclinical models to depict doxorubicin human tissue pharmacokinetics. EXPERIMENTAL DESIGN: Beagle dogs were given 2 mg/kg doxorubicin as i.v. bolus, 4-h infusion, or 96-h infusion. Concentrations in plasma, prostate (target tissue), heart (toxicity), and major tissues for disposition were determined and modeled. Model parameters were obtained after the bolus injection with model validation based on the 4-h and 96-h infusion data. Clinical pharmacokinetic data and scale-up gave doxorubicin profiles in human prostate and heart. RESULTS: In agreement with in vitro results, tissues were best modeled with two compartments, one rapidly and one slowly equilibrating. The developed tissue distribution model predicted concentrations for all three administration regimens well, with an average deviation of 34% (median, 29%). Interspecies scale-up to humans showed that the change from a bolus injection to a slow, 96-h infusion (a) had different effects on the drug partition and accumulation in heart and prostate, and (b) lowered the peak concentration in the plasma by approximately 100-fold but had relatively little effect on maximal heart concentration ( approximately 33% lower). The simulated drug exposure in a human prostate was above the exposure required to inhibit tumor proliferation but was 30 to 50 times below that needed for cell death. CONCLUSION: The present study shows a computation-based paradigm for translating in vitro and in vivo preclinical data and to estimate and compare the drug delivery and pharmacokinetics in target tissues after different treatment schedules.

Quantitative contributions of processes by which polyanion drugs reduce intracellular bioavailability and transfection efficiency of cationic siRNA lipoplex.

RNA Interference (RNAi) is a potentially useful tool to correct the detrimental effects of faulty genes; several RNAi are undergoing clinical evaluation in various diseases. The present study identified the relative contributions of three mechanisms by which polyanion drugs reduced the gene silencing activity of Lipoplex, a complex of small interfering RNA (siRNA) and cationic liposomes. The study used a siRNA against the chemoresistance gene survivin and two model polyanion drugs (suramin, heparin). Products of Lipoplex destabilization were separated, identified, and/or quantified using ultrafiltration, gel electrophoresis, and RT-qPCR (quantitative reverse transcription polymerase chain reaction). Cell binding and endocytosis of fluorescence-labeled Lipoplex and the amount of siRNA at its site of action RISC (RNA-induced silencing complex) were evaluated using endocytosis markers, confocal microscopy, quantitative image analysis, immunoprecipitation, and RT-qPCR. The results show suramin and heparin exerted multiple concentration-dependent effects. First, these agents altered several Lipoplex properties (i.e., reduced particle size, changed surface charge, modified composition of protein biocorona). Second, both caused Lipoplex destabilization to release double- and single-strand siRNA and/or smaller siRNA-lipid complexes with reduced siRNA cargo. Third, both prevented the cell surface binding and internalization of Lipoplex, diminished the siRNA concentration in RISC, and retarded the mRNA knockdown. Suramin and heparin yielded qualitatively and quantitatively different results. Analysis of the experimental results of suramin using quantitative pharmacology (QP) modeling indicated the major cause of gene silencing activity loss depended on drug concentration, changing from inhibition of endocytosis at lower concentration (accounting for 60% loss at ~9µM) to inhibition of cell surface binding and loss of siRNA cargo at higher concentrations (accounting for 64% and 27%, respectively, at 70µM). In summary, the present study demonstrates the complex and dynamic interactions between polyanions and Lipoplex, and the use of QP modeling to delineate the contributions of three mechanisms to the eventual loss of gene silencing activity.