Cellular Uptake and Efflux of Palbociclib In Vitro in Single Cell and Spheroid Models.

Adequate drug distribution through tumors is essential for treatment to be effective. Palbociclib is a cyclin-dependent kinase 4/6 inhibitor approved for use in patients with hormone receptor positive, human epidermal growth factor receptor 2 negative metastatic breast cancer. It has unusual physicochemical properties, which may significantly influence its distribution in tumor tissue. We studied the penetration and distribution of palbociclib in vitro, including the use of multicellular three-dimensional models and mathematical modeling. MCF-7 and DLD-1 cell lines were grown as single cell suspensions (SCS) and spheroids; palbociclib uptake and efflux were studied using liquid chromatography-tandem mass spectrometry. Intracellular concentrations of palbociclib for MCF-7 SCS (C max 3.22 µM) and spheroids (C max 2.91 µM) were 32- and 29-fold higher and in DLD-1, 13- and 7-fold higher, respectively, than the media concentration (0.1 µM). Total palbociclib uptake was lower in DLD-1 cells than MCF-7 cells in both SCS and spheroids. Both uptake and efflux of palbociclib were slower in spheroids than SCS. These data were used to develop a mathematical model of palbociclib transport that quantifies key parameters determining drug penetration and distribution. The model reproduced qualitatively most features of the experimental data and distinguished between SCS and spheroids, providing additional support for hypotheses derived from the experimental data. Mathematical modeling has the potential for translating in vitro data into clinically relevant estimates of tumor drug concentrations. SIGNIFICANCE STATEMENT: This study explores palbociclib uptake and efflux in single cell suspension and spheroid models of cancer. Large intracellular concentrations of palbociclib are found after drug exposure. The data from this study may aid understanding of the intratumoural pharmacokinetics of palbociclib, which is useful in understanding how drug distributes within tumor tissue and optimizing drug efficacy. Biomathematical modelling has the potential to derive intratumoural drug concentrations from plasma pharmacokinetics in patients.

Plasma and rectal mucosal oxylipin levels during aspirin and eicosapentaenoic acid treatment in the seAFOod polyp prevention trial.

BACKGROUND: Aspirin and eicosapentaenoic acid (EPA) have colorectal polyp prevention activity, alone and in combination. This study measured levels of plasma and rectal mucosal oxylipins in participants of the seAFOod 2 × 2 factorial, randomised, placebo-controlled trial, who received aspirin 300 mg daily and EPA 2000 mg free fatty acid, alone and in combination, for 12 months. METHODS: Resolvin (Rv) E1, 15-epi-lipoxin (LX) A4 and respective precursors 18-HEPE and 15-HETE (with chiral separation) were measured by ultra-high performance liquid chromatography-tandem mass spectrometry in plasma taken at baseline, 6 months and 12 months, as well as rectal mucosa obtained at trial exit colonoscopy at 12 months, in 401 trial participants. RESULTS: Despite detection of S- and R- enantiomers of 18-HEPE and 15-HETE in ng/ml concentrations, RvE1 or 15­epi-LXA4 were not detected above a limit of detection of 20 pg/ml in plasma or rectal mucosa, even in individuals randomised to both aspirin and EPA. We have confirmed in a large clinical trial cohort that prolonged (12 months) treatment with EPA is associated with increased plasma 18-HEPE concentrations (median [inter-quartile range] total 18-HEPE 0.51 [0.21-1.95] ng/ml at baseline versus 0.95 [0.46-4.06] ng/ml at 6 months [P<0.0001] in those randomised to EPA alone), which correlate strongly with respective rectal mucosal 18-HEPE levels (r = 0.82; P<0.001), but which do not predict polyp prevention efficacy by EPA or aspirin. CONCLUSION: Analysis of seAFOod trial plasma and rectal mucosal samples has not provided evidence of synthesis of the EPA-derived specialised pro-resolving mediator RvE1 or aspirin-trigged lipoxin 15­epi-LXA4. We cannot rule out degradation of individual oxylipins during sample collection and storage but readily measurable precursor oxylipins argues against widespread degradation.

A mathematical model of doxorubicin penetration through multicellular layers.

Inadequate drug delivery to tumours is now recognised as a key factor that limits the efficacy of anticancer drugs. Extravasation and penetration of therapeutic agents through avascular tissue are critically important processes if sufficient drug is to be delivered to be therapeutic. The purpose of this study is to develop an in silico model that will simulate the transport of the clinically used cytotoxic drug doxorubicin across multicell layers (MCLs) in vitro. Three cell lines were employed: DLD1 (human colon carcinoma), MCF7 (human breast carcinoma) and NCI/ADR-Res (doxorubicin resistant and P-glycoprotein [Pgp] overexpressing ovarian cell line). Cells were cultured on transwell culture inserts to various thicknesses and doxorubicin at various concentrations (100 or 50 microM) was added to the top chamber. The concentration of drug appearing in the bottom chamber was determined as a function of time by HPLC-MS/MS. The rate of drug penetration was inversely proportional to the thickness of the MCL. The rate and extent of doxorubicin penetration was no different in the presence of NCI/ADR-Res cells expressing Pgp compared to MCF7 cells. A mathematical model based upon the premise that the transport of doxorubicin across cell membrane bilayers occurs by a passive "flip-flop" mechanism of the drug between two membrane leaflets was constructed. The mathematical model treats the transwell apparatus as a series of compartments and the MCL is treated as a series of cell layers, separated by small intercellular spaces. This model demonstrates good agreement between predicted and actual drug penetration in vitro and may be applied to the prediction of drug transport in vivo, potentially becoming a useful tool in the study of optimal chemotherapy regimes.

Membrane type matrix metalloproteinases (MMPs) show differential expression in non-small cell lung cancer (NSCLC) compared to normal lung: correlation of MMP-14 mRNA expression and proteolytic activity.

Improved understanding of the involvement of matrix metalloproteinases (MMPs), including membrane-type MMPs (MT-MMPs), in human tumours has potential diagnostic, prognostic and therapeutic implications. We assessed the relationship between MT-MMP expression and clinicopathological parameters in human non-small cell lung cancer (NSCLC) and histologically normal lung tissue by quantitative Real Time PCR (qRT-PCR). All MT-MMPs (MMPs 14-17, 24 and 25) were detected by qRT-PCR with significantly higher MMP-14, -15 and -17 expression observed in tumour relative to normal lung specimens. MMP-16 was undetectable in normal lung but expressed in 8% tumours. MMP-15 demonstrated significant overexpression in adenocarcinomas relative to squamous cell carcinomas and normal lung tissue. MMP-14 mRNA expression strongly correlated to MMP-14 proteolytic activity in preclinical tumour models, indicating that qRT-PCR may predict MMP-14 activity levels in NSCLC. These data suggest that MMP-14, -15 and -17 may be good markers of disease, or therapeutic targets for treatment of human NSCLC.

Preclinical pharmacology of the pyrrolobenzodiazepine (PBD) monomer DRH-417 (NSC 709119).

The pyrrolobenzodiazepine monomer DRH-417 is a member of the anthramycin group of anti-tumor antibiotics that bind covalently to the N2 of guanine within the minor groove of DNA. DRH-417 emerged from the EORTC-Drug Discovery Committee and NCI 60 cell line in vitro screening programs as a potent antiproliferative agent with differential sensitivity towards certain cancer types such as melanoma, breast and renal cell carcinoma (mean IC(50) = 3 nM). DRH-417 was therefore tested for in vivo activity. The maximum tolerated dose (MTD) was established as 0.5 mg/kg given i.p. Marked anti-tumor activity was seen in two human renal cell cancers, one breast cancer and a murine colon tumor model (p<0.01). A selective HPLC (LC/MS) analytical method was developed and plasma pharmacokinetics determined. At a dose of 0.5 mg kg(-1), the plasma AUC was 540 nM h (197.1 ng h ml(-1)) and the peak plasma concentration (171 nM [62.4 ng ml(-1)]) occurred at 30 min., reaching doses levels well above those needed for in vitro antiproliferative activity. Genomic profiling of in vivo sensitive tumors revealed that the latter have an activated insulin-like growth factor signaling pathway.

Drug delivery in a tumour cord model: a computational simulation.

The tumour vasculature and microenvironment is complex and heterogeneous, contributing to reduced delivery of cancer drugs to the tumour. We have developed an in silico model of drug transport in a tumour cord to explore the effect of different drug regimes over a 72 h period and how changes in pharmacokinetic parameters affect tumour exposure to the cytotoxic drug doxorubicin. We used the model to describe the radial and axial distribution of drug in the tumour cord as a function of changes in the transport rate across the cell membrane, blood vessel and intercellular permeability, flow rate, and the binding and unbinding ratio of drug within the cancer cells. We explored how changes in these parameters may affect cellular exposure to drug. The model demonstrates the extent to which distance from the supplying vessel influences drug levels and the effect of dosing schedule in relation to saturation of drug-binding sites. It also shows the likely impact on drug distribution of the aberrant vasculature seen within tumours. The model can be adapted for other drugs and extended to include other parameters. The analysis confirms that computational models can play a role in understanding novel cancer therapies to optimize drug administration and delivery.

Reduction of tumor blood flow by flavone acetic acid: a possible component of therapy.

Flavone acetic acid (FAA) is active against normally refractory murine sc tumors. Clinical studies are disappointing despite achievement of plasma profiles associated with the antitumor murine activity in man. To clarify the mechanism of action, we have followed histologic changes, tumor blood volume, and drug concentrations in a well-differentiated, slow-growing cystic adenocarcinoma in mice. FAA causes massive tumor necrosis beginning 2 hours after treatment. Tumor plasma volumes are reduced by 2 hours after treatment and tumor blood vessels are shutdown, which suggests that tumor vasculature plays a role in the dramatic response of sc tumors in pure-strain male NMRI mice.

Angiogenesis in the hollow fiber tumor model influences drug delivery to tumor cells: implications for anticancer drug screening programs.

The National Cancer Institute uses the hollow fiber assay as part of its screening program for anticancer drug discovery. Angiogenesis to hollow fibers implanted s.c. has not been reported, thereby raising concerns about the efficiency of drug delivery and its subsequent effects on chemosensitivity. By extending postimplantation times beyond the 6-day period presently used, extensive vascular networks develop, resulting in both increased delivery and chemosensitivity to doxorubicin. This study suggests that present protocols used to evaluate compounds may produce false negative results, and additional studies to determine the predictive value of the assay are required.

Predicting tumor responses to mitomycin C on the basis of DT-diaphorase activity or drug metabolism by tumor homogenates: implications for enzyme-directed bioreductive drug development.

Mitomycin C (MMC) is a clinically used anticancer drug that is reduced to cytotoxic metabolites by cellular reductases via a process known as bioreductive drug activation. The identification of key enzymes responsible for drug activation has been investigated extensively with the ultimate aim of tailoring drug administration to patients whose tumors possess the biochemical machinery required for drug activation. In the case of MMC, considerable interest has been centered upon the enzyme DT-diaphorase (DTD) although conflicting reports of good and poor correlations between enzyme activity and response in vitro and in vivo have been published. The principle aim of this study was to provide a definitive answer to the question of whether tumor response to MMC could be predicted on the basis of DTD activity in a large panel of human tumor xenografts. DTD levels were measured in 45 human tumor xenografts that had been characterized previously in terms of their sensitivity to MMC in vitro and in vivo (the in vivo response profile to MMC was taken from work published previously). A poor correlation between DTD activity and antitumor activity in vitro as well as in vivo was obtained. This study also assessed the predictive value of an alternative approach based upon the ability of tumor homogenates to metabolize MMC. This approach is based on the premise that the overall rate of MMC metabolism may provide a better indicator of response than single enzyme measurements. MMC metabolism was evaluated in tumor homogenates (clarified by centrifugation at 1000 x g for 1 min) by measuring the disappearance of the parent compound by HPLC. In responsive [T/C <10% (T/C defined as the relative size of treated and control tumors)] and resistant (T/C >50%) tumors, the mean half life of MMC was 75+/-48.3 and 280+/-129.6 min, respectively. The difference between the two groups was statistically significant (P < 0.005). In conclusion, these results unequivocally demonstrate that response to MMC in vivo cannot be predicted on the basis of DTD activity. Measurement of MMC metabolism by tumor homogenates on the other hand may provide a better indicator of tumor response, and further studies are required to determine whether this approach has real clinical potential in terms of individualizing patient chemotherapy.

Pharmacokinetics of PK1 and doxorubicin in experimental colon tumor models with differing responses to PK1.

PK1 is a synthetic N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin (dox) conjugate currently undergoing Phase II evaluation in the United Kingdom. We have studied the activity of PK1 in three murine colon tumor models that differ in terms of morphology and vascularization in an attempt to determine which factors are most important in the tumor response to PK1. Vascular permeability was evaluated with Evans Blue, and pharmacokinetic studies in MAC15A and MAC26 used high-performance liquid chromatography to monitor both PK1 uptake and dox release in the tumors. Cathepsin B activity was assessed using a specific substrate. PK1 (40 mg x kg(-1) dox equivalent) was significantly more effective than dox alone (10 mg x kg(-1)) was against MAC15A tumors, which possess enhanced perfusion and retention, but not against MAC26 tumors, although MAC15A was also responsive to PK1 when grown as avascular micrometastatic deposits in the lung. Pharmacokinetic studies showed similar levels of PK1 in both tumors. Peak tumor levels of released dox were 7-fold greater in the responsive MAC15A tumor (53 microg x ml(-1)) compared with the less responsive MAC26 tumor (7.7 microg x ml(-1)) and more than 18-fold greater in MAC15A than when free dox was given. These differences in response correlated also with an increased lysosomal activity of cathepsin B. Calculated AUCs for intratumoral dox released were 431 microg x h x g(-1) and 775 microg x h x g(-1) for MAC15A and MAC26, respectively. These AUCs are 4-fold and 7-fold higher, respectively, than when dox is given alone. This study has shown that activity and the pharmacokinetics of PK1 and released dox are dependent on both the vascular properties and enzyme content of the tumors. These studies are likely to have clinical implications as aggressive tumors are known to have increased protease activity.

Potentiation of EO9 anti-tumour activity by hydralazine.

EO9[3-hydroxy-5-aziridinyl-1-methyl-2(1H-indole-4,7-dione)prop-bet a-en-alpha-ol] has been selected for phase I evaluation in Europe. Activity has been seen previously in a highly refractory, necrotic mouse adenocarcinoma (MAC 16) but EO9 is shown here to be inactive against early tumours (MAC 15A and MAC 13) and a well vascularised, well-differentiated established adenocarcinoma (MAC 26). EO9 becomes active against MAC 26 tumours when hydralazine (10 mg/kg) is administered 1 min after EO9. Co-administration of hydralazine decreases EO9 plasma clearance and increases plasma area under the curve values (0.053 to 0.115 micrograms h/ml). These pharmacokinetic changes are accompanied by anti-tumour activity but no increase in bone marrow toxicity so this therapeutic gain may be due, at least in part, to microenvironmental changes resulting from hydralazine induced tumour vascular shutdown.

Pharmacokinetic drug interactions with anticancer drugs.

Drug dosage is of paramount importance in the treatment of cancer, the aim being to optimise drug exposure with a view to maximising antitumour effect and minimising normal tissue toxicity. Pharmacokinetic parameters of anticancer drugs vary considerably from patient to patient. Most clinically useful drug regimens consist of a cocktail of drugs with different mechanisms of action and hence different toxicity profiles. Therefore, it is even more difficult to optimise drug dosage for individual patients. Variability in the pharmacokinetic profile of anticancer agents in individual patients can be further complicated by pharmacokinetically based drug interactions between different anticancer drugs or anticancer drugs and other concomitant medication. Most of the reported studies provide useful information and identify major interactions, but many also demonstrate the difficulty in identifying therapeutically important drug interactions in patients. Even with all the problems associated with acquiring suitable data from cancer patients it is clear that drug interactions do occur and that these can be clinically significant. It is important that potential interactions are identified early in the drug development of new anticancer drugs. This may be made possible by the rapid improvements in analytical techniques and the availability of more appropriate clinically relevant model systems. Therefore, the therapeutic significance of any detected interactions may be assessed, and steps to avoid them may be established, before the drug is under clinical investigation.

Pharmacological approach towards the development of indolequinone bioreductive drugs based on the clinically inactive agent EO9.

The bioreductive drug EO9 (3-hydroxy-5-aziridinyl-1-methyl-2[indole-4,7-dione]-prop-beta-en-alpha-ol) has good pharmacodynamic properties in vitro, modest anti-tumour activity in experimental tumour models, but failed to show activity in clinical trials. Understanding the reasons for its poor efficacy in vivo is important in terms of progressing second generation analogues into the clinic. In two human tumour xenografts, direct intra-tumoural injection resulted in improved anti-tumour activity compared with intravenous administration suggesting that drug delivery to tumours is suboptimal. Compared with Mitomycin C (MMC) and the experimental agent MeDZQ, EO9 was rapidly cleared from the systemic circulation (t1/2=1.8 min) whereas MMC and MeDZQ had significantly increased plasma t1/2 values (14 and 22 min respectively). These three compounds demonstrated similar pharmacodynamic properties in terms of potency towards the NQO1 (NAD(P)H:Quinone oxidoreductase) rich H460 cell line in vitro but differed significantly in their in vivo activity with growth delays of 17.7, 4.5 and 1.0 days for MMC, MeDZQ and EO9 respectively. EO9 was rapidly metabolized by red blood cells in vitro (t1/2=14.5 min) which must contribute to its rapid pharmacokinetic elimination in vivo whereas MMC and MeDZQ were metabolized at comparatively slower rates (t1/2>120 min and 77.0 min respectively). In conclusion, the development of second generation EO9 analogues should address the issue of drug delivery and analysis of drug metabolism by murine whole blood in vitro could be utilized as a preliminary screen to identify lead compounds that are likely to have improved pharmacokinetic profiles in vivo.

Pharmacological properties of a new aziridinylbenzoquinone, RH1 (2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), in mice.

RH1 (2,5-diaziridinyl-3-(hydroxylmethyl)-6-methyl-1,4-benzoquinone) has shown preferential activity against human tumour cell lines which express high levels of DTD (EC 1.6.99.2; NAD(P)H:quinone oxidoreductase, NQO1, DT-diaphorase) and is a candidate for clinical trials. EO9 (3-hydroxy-5-aziridinyl-1-methyl-2-[1H indole-4,7-dione]prop-beta-en-alpha-ol) is a known substrate for DTD but clinical trials were disappointing, as a result of rapid plasma clearance and reversible dose-limiting kidney toxicity. It is an obvious concern that RH1 does not exhibit the same limitations. We therefore describe the antitumour activity and pharmacology of RH1 in mice and compare its pharmacological characteristics to those of EO9. Significant antitumour activity (P = 0.01) was seen for RH1 (0.5 mg/kg, i.p.) against the high DTD-expressing H460 human lung carcinoma. Pharmacokinetic analysis of RH1 in mice showed a t1/2 of 23 min with an area under the curve of 43.0 ng hr mL(-1) resulting in a calculated clearance of 5.1 mL min(-1), 10-fold slower than EO9. RH1 was also more stable than EO9 in murine blood, where the breakdown was thought to be DTD-related. NADH-dependent microsomal metabolism of RH1 and EO9 in both liver and kidney was slow (<100 pmol/min/g tissue), reflecting the low microsomal DTD expression (<35 nmol/mg/min). Liver cytosol metabolism was rapid for both compounds (>4500 pmol/min/g tissue), although DTD levels were low (21.4+/-0.6 nmol/mg/min). DTD activity in the kidney cytosol was high (125+/-8.2 nmol/mg/min) and EO9 was rapidly metabolised (4396+/-1678 pmol/min/g), but the metabolic rate for RH1 was 7-fold slower (608+/-86 pmol/min/g), even though RH1 was shown to be an excellent substrate for DTD (Vmax = 800 micromol/min/mg and a Km of 11.8 microM). The two DTD substrates RH1 and EO9 are clearly metabolised differently, suggesting that RH1 may have different pharmacological properties to those of EO9 in the clinic.

Pharmacokinetics and tissue distribution of the imidazoacridinone C1311 in tumour-bearing mice.

C1311 is the most active member of a new series of rationally designed anti-cancer agents, the imidazoacridinones, which has shown promising pre-clinical anti-tumour activity in vitro and in vivo against a variety of human colon cancers and is a strong candidate for clinical trials. Data are not available on the pharmacokinetic properties of this compound; therefore, the main aim of this project was to study the plasma pharmacokinetics and tissue and tumour distribution of C1311 in mice and to assess, prior to potential clinical application, whether these pharmacokinetics were linear with respect to the dose. The distribution of C1311 in whole blood was also studied. NMRI or NCR-Nu mice were used throughout the study. C1311 was given i.p. at doses of 15, 50, 100 and (the maximum tolerated dose, (MTD) 150 mg kg(-l) i.p. Plasma, tissue and tumour levels were monitored over a 24-h period using high-performance liquid chromatography (HPLC) with fluorescence detection. The distribution of C1311 in murine and human whole blood was studied using both HPLC and fluorescence microscopy. C1311 was quickly cleared from the plasma (47410 ml min kg(-1)) and rapidly distributed into the tissues at all doses. Tissue-to-plasma ratios were large, ranging from 8 in the liver (15 mg kg(-l)) to 600 (50 mg kg(-1)) in the spleen. Overall concentrations were ranked in the order of plasma << liver < kidney < fat < small intestine < spleen. Tumour concentrations were similar to those measured in the liver and kidney, with AUCs being 186 (MAC15A) and 94.4 microg h ml(-l)(HT-29). Plasma pharmacokinetics were linear at doses of 15-100 mg kg(-1), but disproportionate increases were seen in plasma and tissue concentrations at doses above 100 mg kg(-l). C1311 distributed unevenly in both mouse and human blood, with higher concentrations occurring in the cellular fraction than in plasma. Nucleated cells accounted for a large proportion of this localised drug. In conclusion, C1311 is quickly cleared from the plasma and rapidly distributed into the tissues, with tissue concentrations being far higher than plasma levels. The plasma pharmacokinetics are linear up to but not above doses of 100 mg kg(-1). Concentrations of C1311 are greater in the cellular fraction of the blood than in the plasma, with disproportionately high concentrations occurring in the nucleated fraction.

ThioTEPA pharmacokinetics during intravesical chemotherapy: the influence of dose and volume of instillate on systemic uptake and dose rate to the tumour.

ThioTEPA is given intravesically in a variety of schedules to treat superficial bladder cancer. In this study, the influence of the dose of ThioTEPA and the volume of instillate on the dose rate to the tumour and the systemic uptake of ThioTEPA was investigated in eight patients with pTa or pTl disease. Each patient received four courses of ThioTEPA consisting of 30 mg of drug/30 ml of distilled water, 30 mg/60 ml, 60 mg/30 ml and 60 mg/60 ml. Blood and urine samples were obtained for 8 h following instillation, and ThioTEPA and TEPA levels were measured. The AUC(infinity) values (areas under the concentration-time curve, extrapolated to infinity) in plasma were approximately 2 factors higher at the two 60-mg doses. However, the AUC value in the bladder was nearly 70% higher when 60 mg of drug was instilled in 30 ml of distilled water as compared with 60 mg in 60 ml. Thus, by decreasing the volume of instillate it is possible to increase the dose rate to the tumour without increasing the systemic toxicity.

Influence of the tissue distribution of ThioTEPA and its metabolite, TEPA, on the response of murine colon tumours.

Disposition studies in the same animals as those used for assessment of antitumor and toxic effects could increase understanding of the variation in response to cytotoxic drugs. Tissue and plasma levels of ThioTEPA and triethylenephosphoramide (TEPA) were measured to see if any correlation existed between them and the effects of the drug on a series of mouse colon tumours (MAC). The tumour panel included an ascitic form (MAC 15A), an anaplastic (MAC 13) and a well-differentiated (MAC 26) solid tumour, all grown subcutaneously. The maximum tolerated dose of ThioTEPA was 20 mg kg-1 in females bearing MAC 13 and 15 mg kg-1 in males having MAC 15A or 26. The diverse growth characteristics of the tumour cell lines necessitated the use of different methods for assessment of response. After administration of the maximum tolerated dose, the greatest response was observed in MAC 26, in which a growth delay of 15 days-twice the doubling time of the tumour volume-occurred. ThioTEPA produced 58% inhibition of MAC 13 tumour weight, but MAC 15A was unresponsive. One hour after intraperitoneal administration of Thio-TEPA (20 mg kg-1), ratios of tissue to plasma concentration were 1.13, 0.87 and 1.17 in tumours and 0.80, 0.75 and 0.73 in spleens of mice bearing MAC 13, 15A and 26 respectively. These data show greater accumulation of drug in neoplastic than in normal tissues. The pattern of distribution of the metabolite was similar, but there was a lesser degree of tissue accumulation than by the drug. Concentrations of drug and metabolite in neoplastic tissues related to their protein content were 116.0, 126.3 and 183.3 micrograms ThioTEPA/g and 57.5, 83.1 and 78.6 micrograms TEPA/g in MAC 13, 15A and 26 respectively. Combination of these chemosensitivity and pharmacokinetic data indicates that differences in response of these tumours to ThioTEPA cannot be explained by the availability of the drug and metabolite. The therapeutic effects of ThioTEPA cannot be predicted purely from a knowledge of drug and metabolite disposition.

ThioTEPA pharmacokinetics during intravesical chemotherapy and the influence of Tween 80.

A pharmacokinetic study of randomised crossover design was carried out in which eight patients with recurrent stage pTa or pT1 transitional cell carcinoma of the bladder were given thioTEPA (30 mg) in distilled water or in 10% (v/v) Tween 80 (30 ml) intravesically for 2 h, followed 3 months later by the alternative treatment. ThioTEPA and its primary metabolite, TEPA, were measured in plasma and urine using a sensitive and specific chromatographic assay. Large differences between patients were observed in the proportion of thioTEPA absorbed, ranging from 20%-78%. Peak plasma levels of thioTEPA were observed within 1 h of intravesical administration. By 2 h after administration the plasma levels of TEPA were similar to those of thioTEPA and, in contrast to those of the parent compound, remained at a similar level over the next 4 h. The rate of absorption of thioTEPA was not influenced by Tween 80, but it did cause statistically significant increases in mean peak plasma levels (from 101 to 154 ng/ml) and mean AUC values (from 0.376 to 0.496 micrograms h per ml) and a decrease in the mean half-life (from 1.83 to 1.25 h). To obtain plasma levels similar to those achieved after instillation with thioTEPA alone, the dose should be reduced with Tween 80.

Combination chemotherapy with combretastatin A-4 phosphate and 5-fluorouracil in an experimental murine colon adenocarcinoma.

The di-sodium phosphate pro-drug of combretastatin-A4(combA-4P) is undergoing Phase 1 clinical trial in the USA and UK. Its mechanism of action is thought to be related to tubulin-binding properties that result in rapid, tumour endothelial cell damage, neovascular shutdown and subsequent haemorrhagic necrosis. Drugs that work by this mechanism are unlikely to eradicate the tumour as a single agent but should potentiate standard chemotherapy. This study demonstrates that extensive necrosis occurred in a treated refractory murine colon adenocarcinoma but the damage was not accompanied by any measurable effect on tumour growth. Tumours continued to grow from the viable rim that remained. Combination chemotherapy with 5-fluorouracil (5-FU) resulted in significant (p < 0.01) anti-tumour effects. Measurement of 5-FU concentrations suggested that this was true synergism and not simply a pharmacokinetic interaction due to the vascular mechanism of combA-4P. The study suggests that if an antivascular mechanism can be demonstrated in humans, combination chemotherapy should be rapidly assessed in a clinical setting.

Mathematical and computational models of drug transport in tumours.

The ability to predict how far a drug will penetrate into the tumour microenvironment within its pharmacokinetic (PK) lifespan would provide valuable information about therapeutic response. As the PK profile is directly related to the route and schedule of drug administration, an in silico tool that can predict the drug administration schedule that results in optimal drug delivery to tumours would streamline clinical trial design. This paper investigates the application of mathematical and computational modelling techniques to help improve our understanding of the fundamental mechanisms underlying drug delivery, and compares the performance of a simple model with more complex approaches. Three models of drug transport are developed, all based on the same drug binding model and parametrized by bespoke in vitro experiments. Their predictions, compared for a 'tumour cord' geometry, are qualitatively and quantitatively similar. We assess the effect of varying the PK profile of the supplied drug, and the binding affinity of the drug to tumour cells, on the concentration of drug reaching cells and the accumulated exposure of cells to drug at arbitrary distances from a supplying blood vessel. This is a contribution towards developing a useful drug transport modelling tool for informing strategies for the treatment of tumour cells which are 'pharmacokinetically resistant' to chemotherapeutic strategies.