Bo Lennernäs

Chemotherapy and Antiangiogenesis Drug-specific, Dose-related Effects

Dose-response effects of fluorouracil, paclitaxel, doxorubicin, cisplatin, methotrexate, cyclophosphamide and etoposide on VEGF 165/164 -mediated angiogenesis using the rat mesenteric-window angiogenesis assay are reported. VEGF is a pivotal pro-angiogenic factor in most tumors. Microvessel spatial extension, density, pattern formation and segment length were assessed quantitatively and objectively. A single i.v. injection of each drug was given at a low, intermediate or high dose, 7 days before sacrifice. All the drugs elicited significant responses in terms of one or more measured variables. Only paclitaxel, doxorubicin and cyclophosphamide significantly suppressed the overall angiogenic response (p h 0.0001, p h 0.0002 and p h 0.05, respectively), however. Taking toxicity into account, paclitaxel was more potent in inhibition of angiogenesis than the other agents. No clear correlation was found between drug half-life, the degree of toxic effects (in terms of body weight changes) and the antiangiogenic effect. The antiangiogenic effects were distinctly drug specific.

Sublingual administration of fentanyl to cancer patients is an effective treatment for breakthrough pain: results from a randomized phase II study

In this study we evaluated the efficacy and tolerability of sublingual fentanyl (SLF) for breakthrough pain (BTP) in adult opioid-tolerant cancer patients. Patients received one dose of placebo, SLF 100, 200 and 400 μg in random order at four pain episodes. The primary efficacy endpoint was pain intensity difference (PID) from baseline. Twenty-seven patients received study medication. Overall PID increased significantly with SLF 400 μg versus placebo (8.57 mm, p <0.0001). Improvements were statistically different from placebo at 15 min (p = 0.005). SLF 100 and 200 μg showed a numerical trend towards improved pain relief. A dose that gave a clinically important reduction in pain (PID > 20 mm) was identified by 95% of patients. Reduced use of rescue medication (p < 0.001, SLF 400 μg) and improved global assessment of treatment (p = 0.0146, SLF 400 μg) confirmed these differences as clinically important. Nausea and dizziness were the most common treatment-related adverse effects. SLF appears to be a fast, effective and well-tolerated treatment for BTP.

Chemotherapy and antiangiogenesis: drug-specific effects on microvessel sprouting.

Tumors are angiogenesis dependent. Some chemotherapeutics have been shown to be able to suppress angiogenesis and thus tumor growth in vivo at low, well‐tolerated doses. Not much is known about the angiogenesis‐modulating effects of chemotherapeutics in vivo, however. Microvessel sprouting is inherent to angiogenesis. Using the rat mesentery assay, we studied the effect of cyclophosphamide, doxorubicin and paclitaxel at a low, atoxic dose on the number of sprouts per unit tissue volume (No. SP) and their length (Le. SP) at the edge of the expanding network in VEGF165‐mediated angiogenesis. A single dose of each cytotoxic drug was administered i.v. 7 days before the animals were sacrificed. Cyclophosphamide significantly lengthened the shortest Le. SP and shortened the longest Le. SP, doxorubicin did not significantly affect Le. SP, whereas paclitaxel significantly shortened both the shortest and the longest Le. SP. No correlation was found between the present results and the distinctly drug‐specific results of microvessel segment number and length analyzed within central parts of the same expanding network (1). To our knowledge, this is the first quantitative report on the effect of chemotherapy on angiogenesis sprouting in vivo. Collectively, the data suggest that cyclophosphamide, doxorubicin and paclitaxel at a non‐toxic dose primarily target different intrinsic components of the angiogenic cascade, leading to distinctly drug‐specific effects.

FLUTAMIDE METABOLISM IN FOUR DIFFERENT SPECIES IN VITRO AND IDENTIFICATION OF FLUTAMIDE METABOLITES IN HUMAN PATIENT URINE BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY

A new metabolic scheme of flutamide is proposed in this article. Some patients treated with flutamide, a nonsteroidal antiandrogen, have developed severe hepatic dysfunction. Toxic metabolites have been proposed to be responsible for these negative effects. In this study, the qualitative aspects of the in vitro metabolism of flutamide in liver microsomes from human, dog, pig, and rat were evaluated. A direct comparison of the flutamide metabolism in liver and prostate microsomes from pig was made, and the in vivo metabolism of flutamide was investigated in urine from orally treated prostate cancer patients. Liquid chromatography/tandem mass spectrometry was used for analysis. The mass spectrometer was equipped with an electrospray interface and operated in the negative ion mode. In liver microsomes from pig, dog, and rat, extensive hydroxylation of flutamide occurred. One, two, or three hydroxy groups were attached, and isomeric forms were detected for both monohydroxylated and trihydroxylated drug. In pig liver microsomes, isomers of a third metabolite, hydroxylated 4-nitro-3-(trifluoromethyl)-aniline, were also found after incubation with either flutamide or 2-hydroxyflutamide. In human liver microsomes, the pharmacologically active 2-hydroxyflutamide was the only metabolite detected. Several phase I metabolites as well as four intact phase II metabolites could be recovered from the urine samples. For the first time in humans, glucuronic acid conjugates of hydroxylated 4-nitro-3-(trifluoromethyl)-aniline, and mono- and dihydroxylated flutamide were identified, together with hydroxylated 4-nitro-3-(trifluoromethyl)-aniline conjugated with sulfate. In addition, one mercapturic acid conjugate of hydroxylated flutamide, probably formed from flutamide via a reactive intermediate, was detected.

Antiangiogenic effect of metronomic paclitaxel treatment in prostate cancer and non‐tumor tissue in the same animals: a quantitative study

Well‐tolerated continuous or metronomic chemotherapy can exert a marked antiangiogenic and thus superior antitumor effect compared with conventional high‐dose, temporarily spaced‐out chemotherapy, as shown in preclinical studies. There is, however, no means of directly assessing the antiangiogenic effect in a tumor, a serious impediment to assessing the effects of putative antiangiogenic chemotherapeutics or treatments. In an attempt to circumvent or minimize this impediment we studied the antiangiogenic effect of well‐tolerated metronomic paclitaxel therapy in a surrogate tumor‐free tissue that allows true quantitative analysis as well as in syngeneic At‐1 prostate cancer in the same rat. This novel model allows an accurate comparison of the angiogenesis‐modulating effect of chemotherapy in the two tissues to be made. The effect of chemotherapy on VEGF‐A‐mediated angiogenesis, a characteristic of most tumors, was assessed truly quantitatively by microscopic morphometry and image analysis in the tumor‐free mesentery. The chemotherapy significantly suppressed VEGF‐A‐mediated angiogenesis in the mesentery to an extent that closely mirrored the significant increase in tumor necrosis measured morphometrically and the significant decrease in tumor growth rate. This finding opens an avenue to study quantitatively and systematically the antiangiogenic effect of chemotherapeutic modalities and treatments that approximately mirror their antiangiogenic effect in the At‐1 tumor.

External Beam Radiotherapy Of Localized Prostatic Adenocarcinoma: Evaluation Of Conformal Therapy, Field Number And Target Margins

The purpose of the present study was to identify factors of importance in the planning of external beam radiotherapy of prostatic adenocarcinoma. Seven patients with urogenital cancers were planned for external radiotherapy of the prostate. Four different techniques were used, viz. a 4-field box technique and four-, five- or six-field conformal therapy set-ups combined with three different margins (1-3 cm). The evaluations were based on the doses delivered to the rectum and the urinary bladder. A normal tissue complication probability (NTCP) was calculated for each plan using Lymans dose volume reduction method. The most important factors that resulted in a decrease of the dose delivered to the rectum and the bladder were the use of conformal therapy and smaller margins. Conformal therapy seemed more important for the dose distribution in the urinary bladder. Five- and six-field set-ups were not significantly better than those with four fields. NTCP calculations were in accordance with the evaluation of the dose volume histograms. To conclude, four-field conformal therapy utilizing reduced margins improves the dose distribution to the rectum and the urinary bladder in the radiotherapy of prostatic adenocarcinoma.

Effects of IL-2 on MMP expression in freshly isolated human NK cells and the IL-2-independent NK cell line YT.

Interleukin-2 is an important activation factor for natural killer (NK) cells but its effect on NK cell matrix metalloproteinases (MMP) production and matrix degradation is less well investigated. We have used freshly isolated human NK cells and the IL-2-independent NK cell line, YT, to investigate the effects of IL-2 stimulation on NK cell invasion of Matrigel and on MMP expression and production. In YT cells, we found opposing early and late effects of IL-2 stimulation with an early (2 h) increase in MMP-9 protein level and enhanced migration in the Matrigel invasion assay and by 30 hours a decreased mRNA expression of MMP-2, MMP-9, MMP-13, MT3-MMP, and MT6-MMP. We also found a preculture period of 48 hours with IL-2 to negatively affect YT cell migration. We furthermore found that freshly isolated human NK cells Matrigel invasion was MMP-dependent and it increased in response to IL-2. Importantly, in freshly isolated human NK cells we did not see a downregulation of MMPs after 24 hours IL-2 stimulation, but instead a significant upregulation of MT6-MMP mRNA. Because of the cellular localisation of MT6-MMP, which ensures a focalized proteolytic activity, and its high expression compared with the other MMPs in freshly isolated human NK cells makes it of interest to study further.

Human NK cell lines migrate differentially in vitro related to matrix interaction and MMP expression

Matrix metalloproteinases (MMPs) are thought to be of importance for the migratory ability of natural killer (NK) cells. Their expression and production may influence the amount of tumour‐infiltrating NK cells and thereby any therapeutic capability. In this study, we sought to investigate the importance of MMPs for human NK cells’ ability to degrade and migrate through the extracellular matrix (ECM). The two human NK cell lines, NK‐92 and YT, migratory ability, MMP expression and production as well as their morphological appearance when cultured in the ECM equivalent Matrigel were analysed and compared. The quantitatively more migratory NK‐92 cells were found to express invadopodia/podosomes at a significantly higher degree when cultured in Matrigel and gave rise to a general disintegration of the Matrigel. The NK‐92 cells had a higher mRNA expression of MMP‐2, ‐9, ‐13, MT1‐, MT3‐ and MT6‐MMP and a significantly higher production of MMP‐9 compared to YT cells. These differences could explain the substantial functional difference observed between the two cell lines with respect to migratory capacity. In addition, the number of Matrigel invading NK‐92 cells decreased significantly in the presence of the MMP inhibitor GM6001, demonstrating that MMPs have a critical function in their migration.

Pharmacokinetics of an Injectable Modified-Release 2-Hydroxyflutamide Formulation in the Human Prostate Gland Using a Semiphysiologically Based Biopharmaceutical Model

The local distribution of 2-hydroxyflutamide (2-HOF) in prostate tissue after a single intraprostatic injection of a novel parenteral modified-release (MR) formulation in patients with localized prostate cancer was estimated using a semiphysiologically based biopharmaceutical model. Plasma concentration-time profiles for 2-HOF were acquired from a phase II study in 24 patients and the dissolution of the MR formulation was investigated in vitro. Human physiological values and the specific physicochemical properties of 2-HOF were obtained from the literature or calculated via established algorithms. A compartmental modeling approach was adopted for tissue and blood in the prostate gland, where the compartments were modeled as a series of concentric spherical shells contouring the centrally positioned depot formulation. Discrete fluid connections between the blood compartments were described by the representative flow of blood, whereas the mass transport of drug from tissue to tissue and tissue to blood was described by a one-dimensional diffusion approximation. An empirical dissolution approach was adopted for the release of 2-HOF from the formulation. The model adequately described the plasma concentration-time profiles of 2-HOF. Predictive simulations indicated that the local tissue concentration of 2-HOF within a distance of 5 mm from the depot formulation was approximately 40 times higher than that of unbound 2-HOF in plasma. The simulations also indicated that spreading the formulation throughout the prostate gland would expose more of the gland and increase the overall release rate of 2-HOF from the given dose. The increased release rate would initially increase the tissue and plasma concentrations but would also reduce the terminal half-life of 2-HOF in plasma. Finally, an in vitro-in vivo correlation of the release of 2-HOF from the parenteral MR formulation was established. This study shows that intraprostatic 2-HOF concentrations are significantly higher than systemic plasma concentrations and that increased distribution of 2-HOF throughout the gland, using strategic imaging-guided administration, is possible. This novel parenteral MR formulation, thus, facilitates good pharmacological effect while minimizing the risk of side effects.

Calculated Effects of Displacement Errors in External Beam Radiotherapy of Prostatic Adenocarcinoma

In order to evaluate the impact on the biological effective dose (BED) of irradiation delivered to a tumour with large displacement errors (LDE) and to estimate the effect on local control, simulated treatment of prostatic adenocarcinoma was performed. The calculation of BED in combination with the critical-voxel model and the LQ model was used to evaluate the effect of different combinations of LDEs. The model is called the Dose Volume Inhomogeneity Corrected BED (DVIC-BED) model. The dose-response curve was assumed to follow Poisson statistics. Different combinations of radiobiological parameters were used to test the model. A simulated clinical treatment with a dose of 66-80 Gy in 2 Gy fractions was carried out to evaluate displacement errors and non-optimal dose distributions. Five random LDEs excluding 33% of the target volume corresponded to an overall dose reduction of 3-5 Gy compared with a 10 Gy reduction if 100% of the target is missed five times. A 5 Gy decrease in dose corresponds to a reduction in clinical or chemical control up to 10-25% in the interval 65-85 Gy. LDEs in different directions are less deleterious than errors occurring in the same direction. Different alpha/beta-ratios (3-15) had little effect on the DIC-BED, but the effect of different alpha values (0.05, 0.2 and 0.5) was large. However, the results depend on radiobiological parameters for prostatic adenocarcinoma, which not are well known, and further studies in the field should be encouraged.