Voluntary running attenuates behavioural signs of low back pain: dimorphic regulation of intervertebral disc inflammation in male and female SPARC-null mice.

OBJECTIVE: To examine the effect of running exercise on behavioral measures of pain and intervertebral disc (IVD) inflammation in the SPARC-null mouse model. METHODS: Male and female 8-month old SPARC-null and age-matched control mice received a home cage running wheel or a control, fixed wheel for 6 months. Behavioral assays were performed to assess axial discomfort (grip test) and radiating leg pain (von Frey, acetone tests) and voluntary running was confirmed. Expression of inflammatory mediators (TNF-α, IL-1ß, IL-2, IL-10, CCL5, CXCL1, CXCL5, RANKL, M-CSF, and VEGF) in IVDs was determined. Additional inflammatory (IL-1ß, IL-1Ra, CXCR1, CXCR2) and macrophage phenotypic markers (ITGAM, CD80, CD86, CD206, Arg1) in IVDs were investigated by qPCR. RESULTS: Voluntary running attenuated behavioral measures of pain in male and female SPARC-null mice. Increases in mediators including IL-1ß, CXCL1 and CXCL5 were observed in SPARC-null compared to control IVDs. After 6 months of running, increases in M-CSF and VEGF were observed in male SPARC-null IVDs. In females, pro-inflammatory mediators, including CXCL1 and CXCL5 were downregulated by running in SPARC-null mice. qPCR analysis further confirmed the anti-inflammatory effect of running in female IVDs with increased IL-1Ra mRNA. Running induced upregulation of the macrophage marker ITGAM mRNA in males. CONCLUSIONS: Voluntary running reversed behavioral signs of pain in male and female mice and reduced inflammatory mediators in females, but not males. Thus, the therapeutic mechanism of action may be sex-specific.

The influence of the earthworm Lampito mauritii (Kinberg) on the activity of selected soil enzymes in cadmium-amended soil.

The effects of cadmium (CdCl2·7H2O) on cellulase, urease, amylase, invertase and phosphatase were assessed for a period of 45 days in the presence and absence of earthworms [Lampito mauritii (Kinberg)] in alfisol soil. The activities of all enzymes significantly increased with longer incubation times (45 days) under laboratory conditions in both control and Cd-amended soils (both with and without earthworm incubation). However, the activities of all enzymes decreased with increasing Cd concentrations under laboratory conditions, both in the presence and absence of earthworms. In the presence of earthworms, cellulase, urease, invertase and amylase activities increased. However, phosphatase activity was lower in most of the Cd-amended soils in the presence of earthworms compared to its activity levels in soils lacking earthworms. These results show that earthworms modulated the stress imposed by Cd by providing suitable substrates, which in turn acted as stimulants for extracellular enzyme secretion by microbes, and by removing Cd through its accumulation in the tissues of the earthworms.

Diffusion tensor imaging-demonstrated differences between hemiplegic and diplegic cerebral palsy with symmetric periventricular leukomalacia.

BACKGROUND AND PURPOSE: Patients with cerebral palsy have variable clinical presentations such as hemiplegic, diplegic, or quadriplegic patterns though they have PVL on conventional MR images. The authors investigated whether DTT can differentiate between hemiplegic and diplegic CP in patients presenting with symmetric PVL on conventional MR images. MATERIALS AND METHODS: One hundred thirteen consecutive pediatric patients with definite hemiplegic (59 patients; 30 boys, 29 girls; mean age, 34.19 months; range, 24-52 months) or diplegic (54 patients; 27 boys, 27 girls; mean age, 31.07 months; range, 24-48 months) symptoms and bilateral symmetric PVL on conventional brain MR imaging were recruited. The states of CSTs were examined by using DTT, and the asymmetries of right and left CSTs in the hemiplegic and diplegic groups were compared by using asymmetric anisotropy indexes and asymmetric mean diffusivity indexes. RESULTS: All patients in the hemiplegic group with asymmetric results exhibited disrupted integrities of more affected CSTs and sparing of less affected CSTs. However, diplegic patients revealed symmetric disrupted findings of the right and left CSTs at the upper periventricular level. Asymmetric anisotropy index and asymmetric mean diffusivity index values were significantly higher in the hemiplegic group than in the diplegic group (P < .05), and these results of DTT significantly corresponded with their typical clinical manifestation. CONCLUSIONS: DTT may be very useful for the detailed estimation of the CST state in patients with bilateral symmetric PVL.

Proteins involved in DNA damage response pathways and survival of stage I non-small-cell lung cancer patients.

BACKGROUND: Biological complexity leads to significant variation in the survival of patients with stage I non-small-cell lung cancer (NSCLC). DNA damage response (DDR) pathways play a critical role in maintaining genomic stability and in the progression of NSCLC. Therefore, the development of a prognostic biomarker focusing on DDR pathways is an intriguing issue. PATIENTS AND METHODS: Expression of several proteins (ATM, ATMpS1981, γH2AX, 53BP1, 53BP1pS25, Chk2, Chk2pT68, MDC1, MDC1pS964, BRCA1pS1423, and ERCC1) and overall survival were investigated in 889 pathological stage I NSCLC patients. RESULTS: Low expression of BRCA1pS1423 or ERCC1 was significantly associated with worse survival in the whole cohort of patients. Analysis performed based on histology revealed that low expression of γH2AX, Chk2pT68, or ERCC1 was a poor prognostic factor in squamous cell carcinoma patients [adjusted hazard ratio (aHR), Cox P: 1.544, 0.012 for γH2AX; 1.624, 0.010 for Chk2pT68; 1.569, 0.011 for ERCC1]. The analysis of the interaction between two proteins showed that this effect was more pronounced in squamous cell carcinoma patients. However, these effects were not detected in adenocarcinoma patients. CONCLUSIONS: The proteins involved in DDR pathways exhibited differential expression between squamous cell carcinoma and adenocarcinoma and were important determinants of survival in stage I squamous cell carcinoma patients.

Clinical characteristics and outcomes of H1N1-associated pneumonia among adults in South Korea.

BACKGROUND: Pneumonia has been reported to be the most life-threatening complication of influenza virus infection. OBJECTIVE: to describe clinical characteristics and determine risk factors for death among patients with H1N1-associated pneumonia. DESIGN: A retrospective cohort study included all adult patients diagnosed and treated with H1N1-associated pneumonia in 14 participating institutions between 1 May 2009 and 28 February 2010 in South Korea. Clinical outcomes were summarised and predictors for death evaluated through univariate and multivariate analysis. RESULTS: A total of 269 adult patients with H1N1-associated pneumonia were diagnosed and treated. Hospital visits or admissions peaked in November 2009, coinciding with the peak in the 2009 H1N1 epidemic in South Korea. The patients' median age was 48 years; 143 were male. Most (n = 266, 98.9%) were admitted for treatment: 97 (36.1%) required intensive care and 28 (10.4%) needed mechanical ventilation. Despite the use of antiviral and antibacterial agents, 19 patients (7.1%) died. Risk factors predictive of death included presence of malignancy (aOR 12.0, 95%CI 2.8-51.5), and pneumonia severity index (PSI) score (aOR 1.03, 95%CI 1.01-1.04). CONCLUSION: Deaths among adult patients with H1N1-associated pneumonia were not rare. Clinicians should be aware of the possibility of a poor prognosis among H1N1-associated pneumonia patients with underlying malignancy or high PSI score.

Exposure-response of posaconazole used for prophylaxis against invasive fungal infections: evaluating the need to adjust doses based on drug concentrations in plasma.

The purpose of this article is to report the exposure-response (E-R) relationship of posaconazole oral suspension (POS) for prophylaxis against invasive fungal infections (IFIs), on the basis of the US Food and Drug Administration (FDA) clinical pharmacology review of two randomized, active-controlled clinical studies. Posaconazole average steady state plasma concentrations (C(avg)) ranged from 22 to 3,650 ng/ml after administration of POS 200 mg three times daily (t.i.d.). In a double-blind, randomized clinical trial, the quartile ranges of C(avg) with midpoint values of 289, 736, 1,239, and 2,607 ng/ml had clinical failure rates of 44, 21, 18, and 18%, respectively, indicating an inverse association between C(avg) and clinical failure rate. There were no significant relationships between C(avg) and posaconazole-related major adverse events. Determining posaconazole concentrations in plasma will aid in assessing the need for either POS dose adjustment (e.g., increasing the POS dose) or switching to another systemic antifungal drug, thereby improving the effectiveness of prophylaxis against IFIs.

The effect of cationic polymer treatment on adhesion of iron oxide to eyelashes.

The aim of this study was to investigate the effect of iron oxide application on improving the volume of eyelashes. Iron oxide, having a negative surface charge in its natural form, was coated with commercial cationic polymers to increase its adhesion. The iron oxides coated with different types and concentrations of these polymers were incorporated into a basic mascara formula to test their volume effects by means of the weight difference of eyelashes.The results indicated that the type and concentration of coating materials affect the surface zeta potential and particle cluster size of iron oxides. The type of cationic polymer, especially, was shown to modify both factors of iron oxide. The obtained results also suggested that the volume effect of mascara increases with a higher positive surface zeta potential and a smaller particle cluster size of the coated iron oxides.

Beta-carotene inhibits Helicobacter pylori-induced expression of inducible nitric oxide synthase and cyclooxygenase-2 in human gastric epithelial AGS cells.

UNLABELLED: Reactive oxygen species (ROS) play critical roles in Helicobacter pylori (H. pylori)-associated gastric ulceration and carcinogenesis. Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are involved in H. pylori-induced gastric diseases. Previously we demonstrated that H. pylori in Korean isolates induced the activation of mitogen-activated protein kinases (MAPK) and oxidant-sensitive transcription factors NF-kappaB and AP-1 which mediates the expression of iNOS and COX-2 in gastric epithelial AGS cells. beta-Carotene shows antioxidant activity and inhibits NF-kappaB-dependent gene expression in various cells. Present study aims to investigate whether beta-carotene inhibits H. pylori-induced expression of iNOS and COX-2 by suppressing the activation of MAPK, NF-kappaB, and AP-1 in gastric epithelial AGS cells. HP99 (H. pylori in Korean isolates) was added to AGS cells at the ratio of bacterium/cell, 300/1. beta-carotene inhibited H. pylori-induced increase in ROS level, the activation of MAPK (p38, the c-Jun NH2-terminal protein kinases, the extracellular signal-regulated kinases), NF-kappaB, and AP-1 and the expression of iNOS and COX-2 in AGS cells. CONCLUSION: beta-carotene inhibits oxidant-mediated activation of inflammatory signaling and suppresses the expression of iNOS and COX-2 in gastric epithelial AGS cells infected with H. pylori.

Id-1 activates Akt-mediated Wnt signaling and p27(Kip1) phosphorylation through PTEN inhibition.

Inhibitor of differentiation-1 (Id-1) has been accepted as a putative oncogene to promote oncogenic processes through inactivation of tumor suppressors and activation of growth promoting pathways. Here, we show that Id-1 activates the Akt pathway by inhibition of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) transcription through downregulation of p53. Id-1 negatively regulated both p53 and PTEN at the transcriptional level. In promoter assay with serial deletion and chromatin immunoprecipitation assay, the binding of p53 to the PTEN promoter was reduced by Id-1, suggesting that Id-1 regulates PTEN transcription through its p53 modulation. This led to Akt phosphorylation at Ser473 and the activation of the Akt-mediated canonical Wnt signaling pathway. The glycogen synthase kinase-3beta phosphorylation at Ser9, stabilization and nuclear localization of beta-catenin, T-cell factor (TCF)/lymphoid enhancer factor transactivation activity and cyclin D1 expression were enhanced by Id-1. On the other hand, Akt-mediated p27(Kip1) phosphorylation at Thr157 and its cytosolic localization were also increased in Id-1 overexpressing MCF7 cells. In conclusion, our results disclose Id-1 as a novel PTEN inhibitor that could activate the Akt pathway and its downstream effectors, the Wnt/TCF pathway and p27(Kip1) phosphorylation and suggest that the oncogenic function of Id-1 may be partly attributed to its PTEN inhibition in human breast carcinogenesis.

Atomic structures of benzene and pyridine on Si(5 5 12)-2 x 1.

The adsorption structures of benzene and pyridine on Si(5 5 12)-2 x 1 were studied at 80 K by using a low-temperature scanning tunneling microscope and density functional theory calculations. These structures are different from those observed on low-index Si surfaces: benzene molecules exclusively bind to two adatoms, that is, with di-sigma bonds between carbon atoms and silicon adatoms, leading to the loss of benzene aromaticity; in contrast, pyridine molecules interact with adatom(s) through either Si-N dative bonding or di-sigma bonds. Dative bonding configurations with pyridine aromaticity are the dominant adsorption features and are more stable than di-sigma bonding configurations. Thus the dative bonding of nitrogen-containing heteroaromatic molecules provides a strategy for the controlled attachment of aromatic molecules to high-index surfaces.

Transduction of human catalase mediated by an HIV-1 TAT protein basic domain and arginine-rich peptides into mammalian cells.

Antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) have been considered to have a beneficial effect against various diseases mediated by reactive oxygen species (ROS). Although a variety of modified recombinant antioxidant enzymes have been generated to protect against the oxidative stresses, the lack of their transduction ability into cells resulted in limited ability to detoxify intracellular ROS. To render the catalase enzyme capable of detoxifying intracellular ROS when added extracellularly, cell-permeable recombinant catalase proteins were generated. A human liver catalase gene was cloned and fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) and arginine-rich peptides (RRRRRRRRR) in a bacterial expression vector to produce genetic in-frame Tat-CAT and 9Arg-CAT fusion proteins, respectively. The expressed and purified fusion proteins can be transduced into mammalian cells (HeLa and PC12 cells) in a time- and dose-dependent manner when added exogenously in culture medium, and transduced fusion proteins were enzymatically active and stable for 60 h. When exposed to H(2)O(2), the viability of HeLa cells transduced with Tat-CAT or 9Arg-CAT fusion proteins was significantly increased. In combination with transduced SOD, transduced catalase also resulted in a cooperative increase in cell viability when the cells were treated with paraquat, an intracellular antioxide anion generator. We then evaluated the ability of the catalase fusion proteins to transduce into animal skin. This analysis showed that Tat-CAT and 9Arg-CAT fusion proteins efficiently penetrated the epidermis as well as the dermis of the subcutaneous layer when sprayed on animal skin, as judged by immunohistochemistry and specific enzyme activities. These results suggest that Tat-CAT and 9Arg-CAT fusion proteins can be used in protein therapy for various disorders related to this antioxidant enzyme.

Kinetics of P-glycoprotein-mediated efflux of paclitaxel.

Paclitaxel is a substrate of the mdr1 P-glycoprotein (Pgp). The objective of the present study was to determine the kinetics of the Pgp-mediated efflux and its contribution to the overall efflux of paclitaxel at the clinically achievable concentration range of 1 to 1500 nM. Human breast carcinoma BC19 cells that were derived from MCF7 cells by mdr1 transfection and show a >10-fold higher level of the Pgp protein were used to measure the uptake and efflux of [(3)H]paclitaxel. A computational model of intracellular paclitaxel pharmacokinetics was developed to analyze for the Pgp efflux parameters. The results show a saturable Pgp-mediated efflux in BC19 cells; the dissociation constant was 14 nM, and the maximal efflux rate was 2.8 x 10(-4) pmol/h/cell. The contribution of Pgp-mediated efflux to the total efflux decreased with increasing extracellular drug concentrations; the Pgp efflux accounted for 86 and 34% of total efflux at 1 and 1500 nM, respectively. The validity of the model was confirmed by the close agreement between the model-predicted data and the experimentally obtained data (approximately 6% deviation) describing the effect of cell density and intracellular-to-extracellular concentration gradient on the kinetics of drug accumulation and efflux. In conclusion, our results indicate that the Pgp-mediated efflux represents a major efflux mechanism of paclitaxel at the low end of the clinically observed drug concentration range, but accounts for only a minor part of the efflux at higher concentrations in BC19 cells.

Determinants of drug delivery and transport to solid tumors.

This presentation addresses the barriers and determinants and the importance of drug-induced apoptosis in drug transport and delivery to organs and solid tumors. In particular, we examined the roles of interstitial space, drug removal by capillaries, tissue structure and tissue composition on drug distribution. Drug transport in bladder tissues is described by the distributed model which combined monodimensional Fickian diffusion and first order removal of drug by the perfusing blood. Microscopic evaluation of the spatial drug distribution in bladder, prostate and tongue indicates heterogeneous drug distribution with large and erratic concentration gradient. In general, drug distribution favors interstitial space and vasculature, with little penetration in muscles. Drug penetration into 3-dimensional solid tumors is typically 5- to 10-fold slower than in monolayer cultures. The transport of highly protein-bound drugs such as paclitaxel and doxorubicin in a solid tumor is retarded by a high tumor cell density and enhanced by drug-induced apoptosis. Accordingly, the delivery of a highly protein-bound drug to cells in a solid tumor is affected by its apoptotic effects and is therefore determined by the drug concentration and the treatment duration, i.e. treatment schedule. Under in vitro and in vivo conditions, the delivery of highly protein-bound drugs to tumor can be enhanced by using a pretreatment that induces apoptosis and reduction in cell density, and by using treatment schedules designed to take advantage of these drug-induced changes in tumor tissue composition. In conclusion, in addition to the usual processes involved in drug transport such as distribution through vascular space, transport across microvessel walls, and diffusion through interstitial space in tumor tissue, other factors including tissue structure and composition and alteration by drug-induced apoptosis are important determinants of drug distribution in organs and solid tumors.

Determinants of paclitaxel uptake, accumulation and retention in solid tumors.

This report addresses the determinants of the rate and extent of paclitaxel accumulation in tumors. In a 2-dimensional system such as monolayers where the drug is directly in contact with tumor cells, drug accumulation is determined by the extracellular-to-intracellular concentration gradient, the drug binding to extracellular and intracellular macromolecules, the presence of the mdrl p-glycoprotein (Pgp). and the time-dependent and drug concentration-dependent changes in tubulins and cell density. Intracellular pharmacokinetic models were developed to depict the effects of these parameters. Computer simulation results indicate that at the clinically relevant concentration range of 1 to 1,000 nM, (a) the binding affinity and the number of intracellular saturable drug binding sites are important for drug accumulation at low and high extracellular concentrations, respectively, (b) saturation in the drug binding to the high affinity intracellular binding sites (e.g., tubulin/microtubule) occurs at extracellular drug concentration above 100 nM, (c) treatment with 1,000 nM paclitaxel for >4 hr results in increased levels of tubulin/microtubule and consequently increased intracellular drug accumulation, whereas the continued cell proliferation after treatment with low drug concentrations results in reduced intracellular accumulation, and (d) saturation of Pgp in mdr1-transfected cells occurs at the high end of the clinically relevant concentration range. In a 3-dimensional system such as the solid tumor histocultures, which contain tumor cells as well as stromal cells, the drug accumulation into the inner cell layers is determined by the unique properties of solid tumors, including tumor cell density and spatial arrangement of tumor and stromal tissues. Most interestingly, drug penetration is modulated by the drug-induced apoptosis; the reduced cell density due to apoptosis results in an enhancement of the rate of drug penetration into the inner cell layers of solid tumors. In conclusion, the uptake, accumulation, and retention of paclitaxel in solid tumors are determined by (a) factors that are independent of biological changes in tumor cells induced by paclitaxel, i.e., ratio of extracellular and intracellular concentrations, and drug binding to extracellular and intracellular macromolecules, and (b) factors that are dependent on the time- and drug concentration-dependent biological changes induced by paclitaxel, i.e., induction of apoptosis, enhancement of tubulin/microtubule production, and induction of Pgp expression.

Enhancement of paclitaxel delivery to solid tumors by apoptosis-inducing pretreatment: effect of treatment schedule.

The limited penetration of paclitaxel into solid tumors may limit its therapeutic efficacy. We recently showed a correlation between an increase in interstitial space and an enhancement of drug delivery in solid tumors. The present study evaluated whether this observation can be used to develop a treatment strategy, where an apoptosis-inducing pretreatment with paclitaxel is used to enhance its own delivery to solid tumors. In histocultures of human pharynx FaDu xenograft tumors, pretreatment with 1 microM nonradiolabeled paclitaxel, which resulted in approximately 25% apoptosis and a 25% reduction in cell density, enhanced the penetration rate of [(3)H]paclitaxel. Likewise, dividing a total drug exposure to two treatments, separated by an interval to allow apoptosis to occur, resulted in higher drug penetration rate and accumulation compared with giving the same drug exposure continuously. Similar results were obtained in rats bearing subcutaneously implanted prostate MAT-LyLu tumors; fractionation of the dose, to include 1) a pretreatment that yielded sufficient and clinically relevant plasma concentration to induce apoptosis and 2) a second dose given at an interval selected to allow apoptosis and reduction in tumor cell density to occur, resulted in higher tumor concentration compared with other treatments using the same total dose but either did not include an apoptosis-inducing pretreatment or did not allow for apoptosis to occur. We conclude that the pharmacological effect of paclitaxel affects its own delivery to solid tumors and that modifications of the paclitaxel treatment schedule can enhance drug delivery in solid tumors.

Radiation therapy for heterotopic ossification in a patient with traumatic brain injury.

Radiation therapy has been known to have a prophylactic effect for heterotopic ossification (HO), but until now it has not been known to have a therapeutic effect for established HO. We report a case of established HO compounded with a sudden increase in activity, that was improved with radiation therapy. A patient with traumatic brain injury had HO in both hips and thighs two months after the initial trauma. The existing level of HO activity suddenly increased seven months after the initial trauma, and was accompanied by severe pain that was refractory to indomethacin. We assumed that the pain was caused by the increased activity of HO on the basis of clinical symptoms and laboratory results. Initially, the patient received radiation therapy to the left lower extremity, with a total dose of 20 Gy in ten fractions. Next, the patient received radiation therapy at the same dosage to the right lower extremity, after which the pain and level of serum alkaline phosphatase significantly decreased. The patient experienced a mild pancytopenia as a side effect of the radiation therapy, but it was not severe enough to stop the radiation therapy, given the patient's suffering from the increased HO activity.

Identifying a core RNA polymerase surface critical for interactions with a sigma-like specificity factor.

Cyclic interactions occurring between a core RNA polymerase (RNAP) and its initiation factors are critical for transcription initiation, but little is known about subunit interaction. In this work we have identified regions of the single-subunit yeast mitochondrial RNAP (Rpo41p) important for interaction with its sigma-like specificity factor (Mtf1p). Previously we found that the whole folded structure of both polypeptides as well as specific amino acids in at least three regions of Mtf1p are required for interaction. In this work we started with an interaction-defective point mutant in Mtf1p (V135A) and used a two-hybrid selection to isolate suppressing mutations in the core polymerase. We identified suppressors in three separate regions of the RNAP which, when modeled on the structure of the closely related phage T7 RNAP, appear to lie on one surface of the protein. Additional point mutations and biochemical assays were used to confirm the importance of each region for Rpo41p-Mtf1p interactions. Remarkably, two of the three suppressors are found in regions required by T7 RNAP for DNA sequence recognition and promoter melting. Although these essential regions of the phage RNAP are poorly conserved with the mitochondrial RNAPs, they are conserved among the mitochondrial enzymes. The organellar RNAPs appear to use this surface in an alternative way for interactions with their separate sigma-like specificity factor, which, like its bacterial counterpart, provides promoter recognition and DNA melting functions to the holoenzyme.

Computational model of intracellular pharmacokinetics of paclitaxel.

The intracellular pharmacokinetics of paclitaxel is closely related to its pharmacodynamics. Although drug transport across the cell membrane and extracellular and intracellular drug binding have been shown to affect intracellular drug accumulation, their quantitative relationship is unknown. This study was designed to establish a mathematical model for computing the intracellular paclitaxel pharmacokinetics. As a starting point, the model assumes drug transport into and out of cells via passive diffusion. Experimental data on the intracellular pharmacokinetics of [(3)H]paclitaxel were obtained using monolayer cultures of human breast MCF7 tumor cells, which have negligible expression of the mdr1 P-glycoprotein. The results indicate that, in addition to drug binding and microtubule concentration, changes in cell number due to cell growth and drug effects also affected intracellular drug accumulation. A kinetic model was developed to describe several concomitant processes: 1) saturable drug binding to extracellular proteins, 2) saturable and nonsaturable drug binding to intracellular components, 3) time- and concentration-dependent drug depletion from culture medium, 4) cell density-dependent drug accumulation, and 5) time- and drug concentration-dependent enhancement of tubulin concentration. The model was validated by the close prediction (<7% deviation) of the effects of extracellular-to-intracellular concentration gradient and cell density on the kinetics of drug accumulation and efflux. This model was used to predict the effects of changing several parameters (number and binding affinity of intracellular binding sites, free fraction, and concentration of drug in extracellular fluid) on intracellular drug accumulation. In conclusion, the computational model of intracellular paclitaxel pharmacokinetics provides the means to predict drug concentration in cells.

Determinants of paclitaxel penetration and accumulation in human solid tumor.

The present study examined the determinants of the penetration and accumulation of [(3)H]paclitaxel (12-12,000 nM) in three-dimensional histocultures of patient tumors and of a human xenograft tumor in mice. The results showed 1) significant and saturable drug accumulation in tumors, 2) extensive drug retention in tumors, and 3) a slower penetration but a more extensive accumulation in the xenograft tumor compared with patient tumors. Drug penetration was not rate-limited by drug diffusion from medium through the matrix supporting the histocultures. The difference in the expression of the mdr1 P-glycoprotein did not fully account for the difference in the drug accumulation in xenograft and patient tumors. Autoradiography and imaging were used to evaluate the spatial relationship between tumor architecture, tumor cell distribution, and drug distribution as a function of time and initial drug concentration in culture medium. The tumor cell density and the kinetics of drug-induced apoptosis were also evaluated. The results indicate that a high tumor cell density is a barrier to paclitaxel penetration and that the apoptotic effect of paclitaxel enhances its penetration in solid tumor. These factors are responsible for the time- and concentration-dependent drug penetration rate, with drug penetration confined to the periphery until apoptosis and reduction of epithelial cell density occurred at 24 h, after which time paclitaxel penetrated the inner parts of the tumor.