Pharamacokinetic Modeling for Boronophenylalanine-fructose Mediated Neutron Capture Therapy: 10B Concentration Predictions and Dosimetric Consequences

A two-compartment open model has been developed for predicting ¹⁰B concentrations in blood following intravenous infusion of the L-p-boronophenylalanine-fructose complex in humans and derived from pharmacokinetic studies of 24 patients in Phase I clinical trials of boron neutron capture therapy. The ¹⁰B concentration profile in blood exhibits a characteristic rise during the infusion to a peak of 32g/g (for infusion of 350mg/kg over 90min) followed by a biexponential disposition profile with harmonic mean half-lives of 0.32±0.08 and 8.2±2.7h, most likely due to redistribution and primarily renal elimination, respectively. The mean model rate constants k ₁₂, k ₂₁, and k ₁₀ are (mean ± SD) 0.0227±0.0064min^–1, 0.0099±0.0027min^–1, 0.0052±0.0016min^–1, respectively, and the central compartment volume of distribution V ₁ is 0.235±0.042L/kg. In anticipation of the initiation of clinical trials using an intense neutron beam with concomitantly short irradiations, the ability of this model to predict, in advance, the average blood ¹⁰B concentration during brief irradiations was simulated in a retrospective analysis of the pharmacokinetic data from these patients. The prediction error for blood boron concentration and its effect on simulated dose delivered for each irradiation field are reported for three different prediction strategies. In this simulation, error in delivered dose (or, equivalently, neutron fluence) for a given single irradiation field resulting from error in predicted blood ¹⁰B concentration was limited to less than 10%. In practice, lower dose errors can be achieved by delivering each field in two fractions (on two separate days) and by adjusting the second fraction's dose to offset error in the first.     

Lack of enhanced cytotoxicity of cultured L1210 cells using folinic acid in combination with sequential methotrexate and fluorouracil

Summary Previous studies from this laboratory have demonstrated that treatment of cultured cells with sequential methotrexate (MTX) and fluorouracil (FUra) leads to synergistic cell killing in several murine and human neoplasms in vitro. In this study leucovorin (folinic acid, LCV) was added to the MTX/FUra combination with the intention of generating elevated levels of methylenetetrahydrofolate to promote the formation of a stable fluorodeoxyuridylate-thymidylate synthetase ternary complex, thereby augmenting the cytotoxicity of the MTX-FUra sequence. The addition of 10 or 100 M LCV concurrently with or after 10 M FUra following MTX (1 M) pretreatment did not augment the inhibition of L1210 cell growth or the clonigenicity compared with MTX prior to FUra without LCV. The effects of LCV schedulling on the sequential MTX and FUra-induced inhibition of thymidylate synthesis were measured by examining the rate of [6-³H] dUrd incorporation into the acid-precipitable cell fraction and by direct quantitation of the thymidylate synthetase ternary complex. Combination of 100 M LCV with 10 M FUra after 1 M MTX resulted in significantly more ternary complex formation than did 1 M MTX before 10 M FUra alone. The inhibitory effects of FUra on thymidylate synthetase in the presence of MTX, however, could not be augmented by LCV as determined by [6-³H] incorporation into acid-precipitable material, nor did the addition of LCV result in increased cytotoxicity. Factors other than the inhibition of DNA synthesis may be critical to the cytotoxicity of sequential MTX and FUra in L1210 cells.Abbreviations MTX methotrexate - FUra 5-fluorouracil - LCV d, 1-N ⁵-formyltetrahydrofolic acid, calcium salt (folinic acid, leucovorin) - F dUMP 5-fluoro-2-deoxyuridylate - FUTP 5-fluorouidine-5-triphosphate - PRPP 5-phosphoribosyl-1-pyrophosphate - CH₂FAH₄ N ^5,10-methylenetetrahydrofolate - dUMP 2-deoxyuridylate - dTMP thymidylate - TS thymidylate synthetase - PBS phosphate-buffered saline - NaCl 8.0 g - KCl 0.2 g - Na₂HPO₄ 1.15 g - KH₂PO₄ 0.2 g in 1 1 H₂O, pH 7.4 - FdUrd 5-fluoro-2-deoxyuridine This work was supported in part by grant CH-145 from the American Cancer Society and by grants CA-27130 and CA-08341 from the National Cancer Institute. LLD was supported by Postdoctoral Training Grant S-T32-CA09085-08 from the National Institutes of Health and EC was the recipient of a Cancer Research Award from the American Cancer Society     

Histamine H1 Receptor Activation Stimulates Mitogenesis in Human Astrocytoma U373 MG Cells

In human astrocytoma U373 MG cells that express histamine H₁ receptors (180 ± 6fmol/mg protein) but not H₂ or H₃ receptors, histamine stimulated mitogenesis as assessed by [³H]-thymidine incorporation (173 ± 2% of basal; EC₅₀, 2.5 ± 0.4M). The effect of 100M histamine was fully blocked by the selective H₁ antagonist mepyramine (1M) and was markedly reduced (93 ± 4% inhibition) by the phospholipase C inhibitor U73122 (10M).The activator of protein kinase C (PKC) phorbol 12-tetradecanoyl-13-acetate (TPA, 100nM) stimulated [³H]-thymidine incorporation (270 ± 8% of basal), and this response was not additive with that to 100M histamine. The incorporation of [³H]-thymidine induced by 100M histamine was partially reduced by the PKC inhibitor Ro 31-8220 (57 ± 7% inhibition at 300nM) and by the compound PD 098,059 (30M, 62 ± 14% inhibition), an inhibitor of the mitogen-activated kinase (MAPK) kinases MEK1/MEK2.These results show that histamine H₁receptor activation stimulates the proliferation of human astrocytoma U373 MG cells. The action of histamine appears to be partially mediated by PKC stimulation and MAPK activation.     

Pharmacokinetics of methotrexate and 7-hydroxy-methotrexate in plasma and bone marrow of children receiving low-dose oral methotrexate

Summary The absorption, distribution, and elimination kinetics of low-dose p.o. methotrexate (MTX) were repeatedly studied in 19 children during maintenance treatment of childhood acute lymphoblastic leukemia. Plasma concentrations, urinary elimination, and bone marrow concentrations of MTX and 7-hydroxymethotrexate (7-OH-MTX) were monitored during 24 h following a routime p.o. dose (30 mg/m²) using high-pressure liquid chromatography. Significant interindividual variability was found in time to peak concentration (30–180 min), peak concentration (0.41–2.77 M), and to a lesser extent the half-lives (t_1/2: 32.8–86.1 min; t_1/2: 43.6–350.0 min; t_1/2 absorption: 25.2–60.3 min) and plasma area under the concentration-time curve from zero to infinity (195.6–818.5 M.min). Significant amounts of 7-OH-MTX were detected in plasma, with a mean area under the concentration-time curve from zero to infinity of 208 M.min compared with 365.6 M.min for MTX. High concentrations of 7-OH-MTX were present in bone marrow 24 h after oral MTX (15/19 patients) and were at least five fold those in plasma and three fold the concentration of MTX in bone marrow. In four patients occasionally neither MTX nor metabolite could be detected. Repeated examination of these pharmacokinetic parameters in plasma and bone marrow showed that the intraindividual variability was small.This study was supported by the Netherlands Cancer Foundation Koningin Wilhelmina Fonds     

Saturation of 2′, 2′-difluorodeoxycytidine 5′-triphosphate accumulation by mononuclear cells during a phase I trial of gemcitabine

Summary The plasma and cellular pharmacology of 2, 2-difluorodeoxycytidine (dFdC, Gemcitabine) was studied during a phase I trial. The steady-state concentration of dFdC in plasma was directly proportional to the dFdC dose, which ranged between 53 and 1,000 mg/m² per 30 min. The cellular pharmacokinetics of an active metabolite, dFdC 5-triphosphate (dFdCTP), were determined in mononuclear cells of 22 patients by anion-exchange highpressure liquid chromatography. The rate of dFdCTP accumulation and the peak cellular concentration were highest at a dose rate of 350 mg/m² per 30 min, during which steady-state dFdC levels of 15–20 M were achieved in plasma. A comparison of patients infused with 800 mg/m² over 60 min with those receiving the same dose over 30 min demonstrated that the dFdC steady-state concentrations were proportional to the dose rate, but that cellular dFdCTP accumulation rates were similar at each dose rate. At the lower dose rate, the AUC for dFdCTP accumulation was 4-fold that observed at the higher dose rate. Consistent with these observations, the accumulation of dFdCTP by mononuclear cells incubated in vitro was maximal at 10–15 M dFdC. These studies suggest that the ability of mononuclear cells to use dFdC for triphosphate formation is saturable. In the design of future protocols, a dose rate should be considered that produces maximal nucleotide analogue formation, with increased intensity being achieved by prolonging the duration of infusion.Abbreviations ara-C I--d-arabinosylcytosine - ara-C _ss steady-state concentration of ara-C - ara-CTP 5-triphosphate of ara-C - dFdC 2, 2-difluorodeoxycytidine, Gemcitabine - dFdC _ss steady-state concentration of dFdC - dFdCTP 5-triphosphate of dFdC Supported in part by grants CA28596 and CA32839 from the National Cancer Institute, Department of Health and Human Services, and by grant CH-130 from the American Cancer Society     

Interactions of vinblastine and vincristine with methotrexate transport in isolated rat hepatocytes

The accumulation of methotrexate (MTX) in the presence of vinblastine (VBL) and vincristine (VCR) was studied in isolated rat hepatocytes. In accordance with our recent study on vindesine (VDS), we found VBL and VCR to reduce net MTX accumulation significantly at 15 min after MTX addition. Drug concentrations of 100 M VBL and 500 M VCR led to 67% and 82% reductions in intracellular MTX, respectively. Since there was only a slight inhibition of MTX efflux by 100 M VBL, the accumulation data demonstrate that the major effect of VBL is on MTX influx. Dixon-plot analyses are suggestive of competitive inhibition of the MTX influx, yielding inhibition constants (K _i values) of 55 M for VBL and 110 M for VCR. Since theK _i values correspond grossly to plasma levels obtained in humans shortly after the infusion of therapeutic doses of the vinca alkaloids studied herein, the interaction with MTX uptake could serve to diminish the toxicity of MTX to nonmalignant cells.This study was supported financially by the Norwegian Cancer Society     

Lack of effect of cisplatin on i. v. L-PAM plasma pharmacokinetics in ovarian cancer patients

Summary Melphalan (L-PAM) pharmacokinetics were investigated in nine ovarian cancer patients before and after cisplatin (DDP) treatment. When L-PAM was given 24 h before DDP, the elimination half-life (t_1/2), plasma clearance (Clp), and volume of distribution (Vd) of L-PAM were, respectively: 46.4±6.7 min, 20.5±3.7 l/m², and 306.8±34.4 ml/min per square meter. When L-PAM was inoculated 24 h after DDP, t_1/2, Clp, and Vd were 47.5±6.3 min, 20.4±2.8 l/m², and 322.0±54.1 ml/min per square meter. Thus, DDP pretreatment does not significantly affect L-PAM pharmacokinetics. Regression analysis showed a significant correlation between the L-PAM elimination rate constant () and renal function assessed by creatinine clearance. One patient who received this sequence of treatment for six courses showed a threefold decrease of L-PAM clp after the last treatment. The reported high myelotoxicity of the combination of DDP and L-PAM when DDP was given 24 h before L-PAM cannot be attributed to DDP-induced changes in L-PAM kinetics but might to some extent be related to a loss of renal function consequent to many courses of treatment.This work was supported by a Grant from the Swiss League against Cancer     

Pharmacokinetics and toxicity of the antitumour agentN-[2-(dimethylamino)ethyl]acridine-4-carboxamide after i.v. administration in the mouse

Summary The pharmacokinetics, tissue distribution and toxicity of the antitumour agentN-[2-(dimethylamino)ethyl]acridine-4-carboxamide(AC) were studied after i.v. administration to mice. Over the dose range of 9–121 mol/kg (3–40 mg/kg), AC displayed linear kinetics with the following model-independent parameters: clearance (C), 21.0±1.9 l h^–1 kg^–1; steady-state volume of distribution (V_ss), 11.8±1.4 l/kg; and mean residence time (MRT), 0.56±0.02 h. The plasma concentration-time profiles for AC fitted a two-compartment model with the following parameters:C _c, 19.4±2.3 l h^–1 kg^–1; V_c, 7.08±1.06 l/kg;t _1/2 13.1±3.5 min; andt _1/2Z, 1.60±0.65 h. AC displayed moderately high binding in healthy mouse plasma, giving a free fraction of 15.9%–25.3% over the drug concentration range of 1–561 M. After the i.v. administration of 30 mol/kg [³H]-AC, high radioactivity concentrations were observed in all tissues (especially the brain and kidney), showing a hight _1/2c value (37–59 h). At 2 min (first blood collection), the AC concentration as measured by high-performance liquid chromatography (HPLC) comprised 61% of the plasma radioactivity concentration (expressed as AC equivalents/l). By 48 h, 73% of the dose had been eliminated, with 26% and 47% of the delivered drug being excreted by the urinary and faecal routes, respectively; <1% of the total dose was excreted as unchanged AC in the urine. At least five distinct radiochemical peaks were distinguishable by HPLC analysis of plasma extracts, with some similar peaks appearing in urine. The 121-mol/kg dose was well tolerated by mice, with sedation being the only obvious side effect and no significant alterations in blood biochemistry or haematological parameters being recorded. After receiving a dose of 152 mol/kg, all mice experienced clonic seizures for 2 min (with one death occuring) followed by a period of sedation that lasted for up to 2h. No leucopenia occurred, but some mild anaemia was noted. There was no significant change in blood biochemistry. A further 20% increase in the i.v. dose (to 182 mol/kg) resulted in mortality, with death occurring within 2 min of AC administration.Supported by the Auckland Medical Research Foundation and the Cancer Society of New Zealand     

Clinical pharmacokinetics of mitoxantrone after intraperitoneal administration

Summary The pharmacokinetics of intraperitoneally (i.p.) injected mitoxantrone was determined in plasma and peritoneal dialysate taken from five patients presenting with cancer confined to the peritoneal cavity over a sampling period of 1 week. The drug was given through a Tenckhoff catheter as a 15-min infusion and the peritoneal dialysate was removed after a dwell time of 4 h; the doses delivered varied between 20 and 50 mg/m². Dose-limiting local toxicity was moderate. The HPLC technique used for mitoxantrone determinations proved to be sensitive within the range of 0.3–4,000 ng/ml. Median values obtained for the pharmacokinetic parameters of mitoxantrone in peritoneal dialysate were:t _1/2 (distribution), 56.4 min (range, 16.8–235.8 min);t _1/2 (elimination), 128 h (range, 28.3–171.0 h); Vd_SS (volume of distribution at steady state), 24.8 l (range, 17.0–232.5 l); _ss (volume of distribution at steady state corrected for the body surface area in square meters), 14.4 l/m² (range, 10.6–129.2 l/m²); and clearance, 0.25 l/h (range, 0.16–0.59 l/h). For plasma the median values were:t _1/2 (absorption), 58.8 min (range, 45.6–87.0 min);t _1/2 (distribution), 2.5 h (range, 1.4–6.3 h);t _1/2 (elimination), 44.1 h (range, 9.1–91 h); Vd_SS, 2,152 l (range, 352–19,733 l); _ss, 1,345 l/m² (range, 220–11,606 l/m²); and clearance, 117 l/h (range, 51–1,609 l/h). After 168 h the median plasma concentration was 1 ng/ml. The median peak concentration in peritoneal dialysate was 490 ng/ml. Considering the moderate toxicity observed and the concentrations achieved in the peritoneal dialysate, removal of the dialysate after certain dwell times seems reasonable to be a reasonable approach for the optimization of i.p. treatment with mitoxantrone.     

Pharmacokinetic study of doxifluridine given by 5-day stepped-dose infusion

Summary Doxifluridine (5-deoxy-5-fluorouridine, 5-dFUR) metabolism has been reported to be saturable and associated with a fall in clearance of the drug as the dose is increased. The aim of the present study was to determine the disposition of 5-dFUR and 5-fluorouracil (5-FU) when 5-dFUR was given as a 5-day infusion, with the infusion rate increased stepwise every 24 h. Measurement of plasma and urinary levels of 5-dFUR and 5-FU at steadystate for each infusion rate enabled the estimation of 5-dFUR renal (Cl_R) and nonrenal (Cl_NR) clearance and 5-FU renal clearance. A total of 28 patients with histologically proven malignancy received 5-day courses of 5-dFUR ranging in dose from 3.75 to 20 g/m² per 120 h. The lowest dose given over 24 h was 0.25 g/m², and the highest was 5 g/m². Steady-state plasma levels of 5-dFUR ranged from 167 to 6.519 ng/ml. At these plasma levels there was no evidence of significant saturation of 5-dFUR metabolism; steady-state plasma levels of 5-dFUR increased approximately linearly with dose, and nonrenal clearance did not change significantly with dose. There was also no evidence of nonlinearity in 5-dFUR renal clearance. The mean (±SD) Cl_R of 5-dFUR was 108.9±53.6 ml/min per m² (range, 45.7–210 ml/min per m²), and the Cl_NR was 728±181 ml/min per m² (range, 444–1,119 ml/min per m²). Renal clearance comprised 13% of the total 5-dFUR clearance. The mean renal clearance of 5-FU was 100.8±48.6 ml/min per m² (range, 23.5–198 ml/min per m²). There was considerable interpatient variability in 5-dFUR renal and nonrenal clearance, event at the same dose level. We concluded that the administration of 5-dFUR by the infusion method described avoided the saturation of nonrenal elimination processes reported to occur with shorter infusion schedules.This study was supported by a grant from F. Hoffmann-La Roche, Basel, Switzerland     

Beta-adrenergic receptors in DMBA-induced rat mammary tumors: Correlation with progesterone receptor and tumor growth

Summary In order to gain further knowledge about the potential role of catecholamines in mammary carcinoma, we have used the potent -adrenergic antagonist cyanopindolol (CYP) as iodinated ligand to characterize -adrenergic receptors in membranes prepared from mammary tumors induced by dimethylbenz(a)anthraene (DMBA) administration in the rat. The binding of [¹²⁵I]CYP to membrane preparations of DMBA-induced rat mammary tumors is rapid at room temperature, reaching half maximal specific binding at 30 min of incubation. Scatchard analysis of the data indicates that [¹²⁵I]CYP binds to a single class of high affinity sites (114 ± 2.1 fmoles/mg protein) at an apparent K_D value of 38.0 ± 0.3 pM. The order of potency of a series of agonists to compete for [¹²⁵I]CYP binding is consistent with interaction with a ₂-subtype receptor: zinterol > (–)isoproterenol > (–)epinephrine» (–)norepinephrine. In addition, the potency of a series of specific ₁, and ₂ synthetic compounds to displace [¹²⁵I]CYP in mammary tumors is similar to their potency in typical ₂-adrenergic tissues. The binding of [¹²⁵I]CYP to DMBA-induced rat mammary tumors shows a marked stereoselectivity, the (–)isomers of isoproterenol and propranolol being 150 and 80 times more potent, respectively, than their respective enantiomers. The autoradiographic localization of [¹²⁵I]CYP performed on frozen sections revealed the presence of specific -adrenergic receptors in all the malignant cells. Spontaneous mammary tumors of aging (18–22 months) female rats have high levels of -adrenergic receptors. Castration decreased the concentration of [¹²⁵I]CYP binding sites in DMBA-induced mammary tumors. A close correlation was observed between progressing, static, and regressing tumors after ovariectomy and -adrenergic receptor concentration. The presence of -adrenergic receptors in mammary tumors as well as the modulation of their level by ovarian hormones provides a mechanism for catecholaminergic influence in mammary cancer tissue.     

Plasma kinetic study of folinic acid and 5-methyltetrahydrofolate in healthy volunteers and cancer patients by high-performance liquid chromatography

Summary A reversed-phase HPLC method is described for the simultaneous determination of folinic acid, MTX, and their plasma metabolites 5-CH₃–FH₄ and 7-OH-MTX respectively. In addition, this technique allows the separation of FA another naturally occurring folate, and of AMT, used as internal standard.Separation of these compounds was achieved on a Waters Spherical C₁₈ column at a flow rate of 0.8 ml.min^-1. Elution was carried out with 0.1 M sodium acetate buffer (pH 5.5) as solvent A and 7.5% acetonitrile 92.5% bidistilled water as solvent B. UV detection was performed at 280 nm. This method was applied in a pharmacokinetic study of folinic acid and its plasma metabolite 5-CH₃–FH₄ following two different protocols: (1) i. v. bolus injection of 50 mg calcium folinate in six healthy volunteers and (2) simultaneous i. v. bolus injections of 50 mg/m² MTX and 50 mg/m² folinic acid in four cancer patients. Mean apparent half-life values for folinic acid and its metabolite were 7.02±1.81 h and 3.90±0.86 respectively in the first protocol, 4.80±1.48 h and 4.74±1.47 h in the second protocol. MTX and 7-OH-MTX were also quantified in the second protocol and were found not to affect the pharmacokinetics of folinic acid and 5-CH₃–FH₄.Since in vitro studies on metabolism of folinic acid might be of great interest in trying to assess the mechanism of action of the folates and the potential interaction of MTX and 7-OH-MTX in this mechanism via the metabolism, the chromatographic method we describe here has been adapted for the separation of all the potential intracellular monoglutamyl metabolites of folinic acid.Abbreviations FH₂ dihydrofolate - FH₄ tetrahydrofolate - 5-CHO–FH₄ 5-formyltetrahydrofolate - 10-CHO–FH₄ 10-formyltetrahydrofolate - 5-CH₃–FH₅ 5-methyltetrahydrofolate - 5,10-CH=FH₄ 5,10-methenyltetrahydrofolate - MTX methotrexate - 7-OH-MTX 7-hydroxymethotrexate - AMT aminopterin - HPLC high-performance liquid chromatography This work was supported by the Fédérations Nationale et Départmentale des Centres de Lutte contre le Cancer, by the Ministère de la Recherche et de la Technologie and by the Association pour le Développement de la Recherche sur le Cancer     

Inhibition of 7-hydroxymethotrexate formation by amsacrine

Summary The inhibition of methotrexate (MTX) biotransformation to 7-hydroxymethotrexate (7-OH-MTX) by 4-(9-acridinylamino)-methanesulfon-m-anisidide (mAMSA) was studied in bile-drained rats in vivo and in incubates of isolated rat hepatocytes and rat-liver homogenate in vitro. In vivo, i.v. administration of 10 mg/kg mAMSA prior to [³H]-MTX infusion (50 mg/kg) led to a significant alteration in 7-OH-MTX kinetics. 7-OH-MTX peak concentrations and AUC in bile and serum were reduced by 75% and the recovery of MTX as 7-OH-MTX in bile and urine decreased by 70%, whereas MTX pharmacokinetics remained unaltered. In suspensions of isolated hepatocytes. 10 m mAMSA led to a 54% decrease in 7-OH-MTX formation. However, the hepatocellular influx and efflux of MTX was not perturbed by mAMSA. Preincubation of rat-liver homogenates with 1.25–10 m mAMSA reduced the formation of 7-OH-MTX by up to 73%. mAMSA appeared to inhibit MTX hydroxylation competitively, exhibiting aK _iof 3 m. Due to its inhibition of the MTX-oxidizing system, mAMSA may be beneficial in combination chemotherapy with MTX by reducing 7-OH-MTX-associated toxicity and, possibly, enhancing the cytotoxic effects of MTX.This study was financially supported by the Norwegian Cancer Society and the Erna and Olav Aakre Foundation for Cancer Research     

Biochemical pharmacology of 5,6-dihydro-5-azacytidine (DHAC) and DNA hypomethylation in tumor (L1210)-bearing mice

Summary Dihydro-5-azacytidine (DHAC) is a hydrolytically stable congener of 5-azacytidine, which retains antileukemic activity against experimental leukemias. The biochemical pharmacology of DHAC was studied in tumor-bearing mice in order to elucidate the mode of action of this drug. We found that after an LD₁₀ dose of DHAC, the plasma peak concentration achieved was 317 M and was eliminated biexponentially, with a t_1/2 of 1.03 h and a t_1/2 of 5 h. By 4h, an unidentified metabolite of [³H]DHAC peaked and was eliminated biexponentially, with a t_1/2 of 1.06 h and a t_1/2 of 10.6 h. [³H]DHACTP was the major anabolite in the L1210/0 cells, and was also eliminated biexponentially, with a t_1/2 of 4.3 h and a t_1/2 of 12.2 h. An unknown anabolite of [³H]DHAC that eluted 5 min after [³H]DHACTP, between UTP and ATP, peaked at 3 h and could possibly be the deoxy-derivative [³H]DHAdCTP. A tissue distribution study revealed that the liver, L1210/0, and lung accumulate the most radioactivity per gram of wet tissue. Methylation studies showed that an LD₁₀ dose of [³H]DHAC resulted in a 25.06% hypomethylation of DNA in L1210/0 cells and a 46.32% hypomethylation in a deoxycytidine kinase mutant cell line L1210/dCK(-), compared with their respective controls.Abbreviations used 5-aza-C 5-azacytidine - DHAC 5,6-dihydro-5-azacytidine - DHACTP dihydro-5-azacytidine 5-triphosphate - DHAdCTP dihydro-5-azacytidine 5-deoxytriphosphate - LD₁₀ lethal dose 10% of animals - UTP uridine 5-triphosphate - ATP adenosine 5-triphosphate - TCA trichloroacetic acid - PCA perchloric acid - RBC red blood cells - i.p. intraperitoneally - SAX strong anion exchange - SCX strong cation exchange Supported by a research grant, CA 38905, from the National Institutes of Health, NCI     

Pharmacologic studies on the dibutyl and γ-monobutyl esters of methotrexate in the rhesus monkey

Summary The pharmacokinetics and metabolism of dibutyl methotrexate (DBMTX) and -monobutyl methotrexate (-MBMTX) were studied in Rhesus monkeys. When a bolus IV dose of either [³H]DBMTX or [³H]-MBMTX was given, the principal species in serum for up to 1 h was the monoester, with MTX accounting for < 10% of the total radioactivity. Products other than -MBMTX and MTX were formed in substantial amounts with DBMTX, but not with -MBMTX. Total radioactivity recovered in the bile 5 h after [³H]DBMTX injection accounted for 32% of the administered dose, indicating high hepatic extraction for this lipophilic compound. Serum and CSF levels of unchanged -MBMTX, as well as of MTX arising via esterase cleavage, were measured by HPLC after IV infusion of -MBMTX (10 g/m²). Efflux of monoester from CSF was slower than disappearance from serum. However, -MBMTX levels in CSF were no higher than could be attained by infusing MTX itself at the same dose rate. While CSF/serum ratios were ca. 10-fold higher for -MBMTX than for MTX, this difference could be explained on the basis of the very different affinities of the two compounds for serum proteins. HPLC analysis of serum processed by methanol precipitation as opposed to ultrafiltration of the proteins showed -MBMTX to be >99% bound, whereas for MTX this value was 50% or less. When -MBMTX and MTX levels measured after ultrafiltration were corrected for this difference in serum protein binding the total amount of the two drugs in serum became almost equivalent.The abbreviations used are MTX methotrexate (4-amino-4-deoxy-N ¹⁰-methylpteroyl-l-glutamic acid) - DBMTX dibutyl methotrexate (NSC-305985) - -MBMTX -monobutyl methotrexate (NSC-305986) - DDMP 2,4-diamino-5-(3,4-dichlorophenyl)-6-methylpyrimidine (NSC-19494) - CSF cerebrospinal fluid - HPLC high-performance liquid chromatography - TLC thin-layer chromatography     

Phase II of doxorubicin/taxol in metastatic breast cancer

Purpose. To assess the response rate, survival, and toxicity of Taxol®(paclitaxel) as 1h infusion plus doxorubicin as firstline treatment for patients with metastatic breast cancer (MBC).Patients and methods. Seventysix patients with untreated MBC were recruited. All of them had measurable disease and were evaluable for toxicity. Fiftyfive percent of the patients had visceral involvement. The dose of doxorubicin was fixed at 50mg/m² as a short intravenous infusion, followed by 200mg/m² of Taxol as a 1h intravenous infusion. Doxorubicin was administered during the first seven cycles, continuing with Taxol only up to a maximum of ten cycles.Results. Neutropenia was the most important toxicity: 30% grade 3 and 18% grade 4. Only 2 patients showed a decrease in the left ventricular ejection fraction (LVEF) which caused discontinuing the treatment. No clinical congestive heart failure (CHF) was observed. Seventyfour patients were eligible for response evaluation: 10 (14%) achieved complete response (CR) and 46 (62%) achieved partial response (PR). The mean duration of response was 13.47± 1.35 months (95% confidence interval (CI): 10.82; 16.12) and the mean survival was 21.50± 1.42 months (95% CI: 18.72; 24.29).Conclusion. The overall response (OR) rate was 76%. No CHF was assessed and 2 patients stopped treatment due to LVEF decrease. Although doxorubicin 50mg/m² followed by Taxol 200mg/m² in 1h intravenous infusion presents a toxicity profile which demands a close followup, it represents a convenient outpatient schedule with similar activity rate compared to longer Taxol infusions.     

Pre-clinical and clinical study of QC12, a water-soluble, pro-drug of quercetin

Background:Quercetin is a naturally occurring flavonoid with manybiological activities including inhibition of a number of tyrosine kinases.A phase I, dose-escalation trial of quercetin defined the maximum tolerateddose (MTD) as 1700 mg/m² three weekly, but the vehicle, dimethylsulphoxide (DMSO) is unsuitable for further clinical development ofquercetin. Patients and methods:A water-soluble, pro-drug of quercetin(3(N-carboxymethyl)carbomyl-3,4,5,7-tetrahydroxyflavone), QC12has been synthesised. Six cancer patients received 400 mg of QC12 (equivalentto 298 mg of quercetin), orally on day 1 and intravenously (i.v.) in normalsaline on day 14. Results:Following oral administration of QC12 we were unable todetect QC12 or quercetin in plasma. After i.v. administration, we detectedpeak plasma concentrations of QC12 of 108.7 ± 41.67 µMolar(µM). A two-compartment model with mean t_1/2 of 0.31± 0.27 hours and mean t_1/2 of 0.86 ± 0.78 hoursbest described the concentration-time curves for QC12. The mean AUC was 44.54± 13.0 µM.hour and mean volume of distribution (Vd) of 10.0± 6.2 litres (l). Quercetin was found in all patients following i.v.infusion of QC12, with peak levels of quercetin 19.9 ± 11.8 µM.The relative bioavailability of quercetin was estimated to be20%–25% quercetin released from QC12. Conclusions:QC12 is not orally bioavailable. This water-solublepro-drug warrants further clinical investigation; starting with a formal phaseI, IV, dose-escalation study.     

Weight gain in women with breast cancer treated with adjuvant cyclophosphomide,methotrexate and 5-fluorouracil. Analysis of resting energy expenditure and body composition

Background. Weight gain is a common side effect observed in women undergoing adjuvant chemotherapy for breast cancer. Among possible causes a direct effect of chemotherapy on metabolism has been proposed. Body composition variations after adjuvant chemotherapy suggest the occurrence of sarcopenic obesity, possibly due to ovarian failure. We investigated acute and chronic effects of adjuvant chemotherapy on body weight, resting energy expenditure (REE) and plasma catecholamines in a group of menopausal women. Patients and methods. Thirty menopausal women with stage I–II breast cancer were recruited for the study. We measured REE and respiratory quotient (RQ) and body composition at the beginning and after 3 and 6 months of adjuvant cyclophosphomide, methotrexate, and 5-fluorouracil (CMF). REE, RQ, and plasma catecholamines were assessed before and after each chemotherapy session. At each session food intake was also assessed in all patients, by a food diary. Seven patients out of the group of 30 were also evaluated after a placebo infusion (saline). Results. A significant weight gain was observed in all women (70.5±3 v.s. 67.7±3kg, p<0.001), with increase in both fat-free mass (FFM) (45.2±1.5 v.s. 43.6±1.3kg, p<0.001) and fat-mass (FM) (25.3±1.7 v.s. 24.1±1.8kg, p<0.005). A decrease in REE and RQ was observed both during CMF and placebo infusion (p<0.05). During acute CMF and placebo infusion a reduction of plasma levels of noradrenaline was observed at the first and last session. REE increased progressively during the study period. Conclusions. CMF therapy apparently has no effect on REE either acutely or during a 6-month-period; the increased REE observed in the long-term is likely due to the concomitant increase in FFM. The lack of evidence of sarcopenic obesity, at variance with previous literature, is likely due to different patient selection.     

Intracavitary VEGF,bFGF, IL-8, IL-12 levels in primary and recurrent malignant glioma

Intracavitary levels of VEGF, bFGF, IL-8 and IL-12 were evaluated by ELISA in 45 patients, 7 with recurrent anaplastic astrocytoma (rAA), 12 with glioblastoma (GBM) and 26 with recurrent glioblastoma (rGBM). In 25 patients plasma levels of the molecules were also quantitated. Twenty-three healthy controls were also studied for plasma concentrations of the same molecules.Plasma levels of VEGF (mean 33.89 ± 6.71pg/ml) and bFGF (mean 11.1 ± 3.24pg/ml) were higher in patients than in controls (mean 16.78 ± 3.7pg/ml for VEGF, mean 0.21 ± 0.09pg/ml for bFGF) (p = 0.04 and p = 0.001, respectively) while plasma IL-12 levels were lower (mean 45.6 ± 1.5pg/ml in patients, mean 79.7 ± 1.3pg/ml in controls) (p = 0.009).Intracavitary VEGF levels were 5–53.307 fold higher (mean 90,900 ± 24,789pg/ml) than in the corresponding plasma. Also IL-8 concentrations were higher in intracavitary fluid (mean 6,349.76 ± 1,460.93pg/ml) than in plasma (mean 43.44 ± 24.82pg/ml). Maximum VEGF levels were found in tumor fluid of recurrent glioblastoma patients (mean 147,678 ± 39,903pg/ml), intermediate levels in glioblastoma patients (mean 20,322 ± 11,892pg/ml) and lower levels in rAA patients (mean 9,111 ± 5,789pg/ml). The data also suggest that higher intracavitary levels of VEGF and IL-8, and lower IL-12 levels, may be correlated with shorter adjunctive survival times, but more data will need to be collected to establish this correlation clearly.     

Carboplatin and etoposide pharmacokinetics in patients with testicular teratoma

Summary The pharmacokinetics of carboplatin and etoposide were studied in four testicular teratoma patients receiving four courses each of combination chemotherapy consisting of etoposide (120 mg/m² daily×3), bleomycin (30 mg weekly) and carboplatin. The carboplatin dose was calculated so as to achieve a constant area under the plasma concentration vs time curve (AUC) of 4.5 mg carboplatin/ml x min by using the formula: dose=4.5×(GFR+25), where GFR is the absolute glomerular filtration rate measured by ⁵¹Cr-EDTA clearance. Carboplatin was given on either day 1 or day 2 of each course and pharmacokinetic studies were carried out in each patient on two courses. Etoposide pharmacokinetics were also studied on two separate courses in each patient on the day on which carboplatin was given and on a day when etoposide was given alone. The pharmacokinetics of carboplatin were the same on both the first and second courses, on which studies were carried out with overall mean ± SD values (n=8) of 4.8±0.6 mg/ml x min, 94±21 min, 129±21 min, 20.1±5.41, 155±33 ml/min and 102±24 ml/min for the AUC, beta-phase half-life (t_1/2), mean residence time (MRT), volume of distribution (Vd) and total body (TCLR) and renal clearances (RCLR), respectively. The renal clearance of carboplatin was not significantly different from the GFR (132±32 ml/min). Etoposide pharmacokinetics were also the same on the two courses studied, with overall mean values ±SD (n=8) of: AUC=5.1±0.9 mg/ml x min, t_1/2=40±9 min, t_1/2=257±21 min, MRT=292±25 min, Vd=13.3±1.31, TCLR=46±9 ml/min and RCLR=17.6±6.3 ml/min when the drug was given alone and AUC=5.3±0.6 mg/ml x min, t_1/2=34±6 min, t_1/2=242±25 min, MRT=292±25 min, Vd=12.5±1.81, TCLR=43±6 ml/min and RCLR=13.4±3.5 ml/min when it was given in combination with carboplatin. Thus, the equation used to determine the carboplatin accurately predicted the AUC observed and the pharmacokinetics of etoposide were not altered by concurrent carboplatin administration. The therapeutic efficacy and toxicity of the carboplatin-etoposidebleomycin combination will be compared to those of cisplatin, etoposide and bleomycin in a randomised trial.