Background Endothelial cells harbor specialized storage organelles, Weibel-Palade bodies (WPBs). Exocytosis of WPB content into the vascular lumen initiates primary hemostasis, mediated by von Willebrand factor (VWF), and inflammation, mediated by several proteins including P-selectin. During full fusion, secretion of this large hemostatic protein and smaller pro-inflammatory proteins are thought to be inextricably linked. Objective To determine if secretagogue-dependent differential release of WPB cargo occurs, and whether this is mediated by the formation of an actomyosin ring during exocytosis. Methods We used VWF string analysis, leukocyte rolling assays, ELISA, spinning disk confocal microscopy, high-throughput confocal microscopy and inhibitor and siRNA treatments to demonstrate the existence of cellular machinery that allows differential release of WPB cargo proteins. Results Inhibition of the actomyosin ring differentially effects two processes regulated by WPB exocytosis; it perturbs VWF string formation but has no effect on leukocyte rolling. The efficiency of ring recruitment correlates with VWF release; the ratio of release of VWF to small cargoes decreases when ring recruitment is inhibited. The recruitment of the actin ring is time dependent (fusion events occurring directly after stimulation are less likely to initiate hemostasis than later events) and is activated by protein kinase C (PKC) isoforms. Conclusions Secretagogues differentially recruit the actomyosin ring, thus demonstrating one mechanism by which the prothrombotic effect of endothelial activation can be modulated. This potentially limits thrombosis whilst permitting a normal inflammatory response. These results have implications for the assessment of WPB fusion, cargo-content release and the treatment of patients with von Willebrand disease.

Dietary supplements are used by most patients with cancer. As nutraceuticals can interact with many drugs, this study investigated the effect of herbal remedies and vitamins on the toxicity of representative cancer chemotherapeutic agents. Fisher 344 rats were fed a standard cereal-based diet or the same diet with additional vitamin E in low (50 mg/kg) or high (750 mg/kg) concentrations, or with added St. John's wort (400 mg/kg). The LD50 was determined after the administration of chemotherapy drugs. Neither low or high vitamin E supplements nor St. John's wort significantly changed the LD50 for doxorubicin, docetaxel, or cyclophosphamide. The nadir white blood cell (WBC) count was significantly higher (P = 0.004) after docetaxel in rats supplemented with low-dose vitamin E, but the drop in WBC count from initial to nadir levels (Nfall) was greater in rats fed a diet containing high vitamin E supplementation (P = 0.04). Similarly, the Nfall was greater in the standard and high vitamin E dietary groups than in the low vitamin E group after cyclophosphamide (P = 0.03). No effect of vitamin E or St. John's wort supplementation occurred on doxorubicin pharmacokinetics. Neither vitamin E nor St. John's wort had an important effect on the mitochondrial deoxyribonucleic acid (DNA) damage caused by either doxorubicin or docetaxel. These data suggest that the leucopenia caused by some chemotherapeutic agents can be modified by dietary supplementation with vitamin E, but the effect seems to be dose-dependent. St. John's wort had neither a beneficial nor a detrimental effect on chemotherapy-induced toxicity.

The effects of diet and folate status on cyclophosphamide or 5-fluorouracil toxicity were studied in Fischer 344 rats maintained on either a cereal-based diet or a purified diet (AIN-93G). The rats fed the purified diet were divided into 3 groups: folate deficient (no dietary folic acid), folate replete (2 mg folic acid/kg diet), and high folate (2 mg folic acid/kg diet plus 50 mg/kg body weight folic acid intraperitoneally daily). The LD50 for cyclophosphamide was significantly higher for the cereal diet than for the purified diets, but there was no difference among the purified diets. Deaths were predicted by dose, diet, white blood cell count, and BUN on Day 4 after treatment. In the saline-treated rats fed the purified diet, hepatic total glutathione levels increased in the following order: folate deficient < folate replete < high folate. There was no significant difference in aldehyde dehydogenase activities or of microsomal P450 levels in livers from rats on the different diets. In the rats treated with 5-fluorouracil, the high folate rats developed more severe anemia, azotemia, and leukopenia than the other groups. Weight, white blood cell count, hematocrit, and BUN were important predictors of death. The kidneys from rats fed the cereal-based diet were histologically normal, but rats ingesting the purified diet had increasing renal pathology that correlated with folate intake. These results indicate that diet has an important influence on the toxicity of cyclophosphamide and 5-fluorouracil and that folate status modulates hepatic glutathione levels, which is a major cellular defense against oxidant and alkylating agent damage.

Three carbocylic analogues of the potent cytidine deaminase inhibitor (CDA) zebularine [1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2- one-, 1a] were synthesized. The selected pseudosugar templates correspond, respectively, to the cyclopentenyl moiety of neplanocin A (compound 4), the cyclopentyl moiety of aristeromycin (compound 5), and a newly designed, rigid bicyclo[3.1,0]hexane moiety (compound 6). These three carba-nucleoside versions of zebularine were fashioned to overcome the inherent instability of the parent drug. Each target compound was approached differently using either convergent or linear approaches. The immediate precursor to the cyclopentenyl analogue 4 was obtained by a Mitsunobu coupling of pseudosugar 7 with 2-hydroxypyrimidine. The cyclopentyl analogue 5 was linearly constructed from carbocyclic amine 17, and the final target 6 was similarly constructed from the carbobicyclic amine 27. Of the three target compounds, only 5 showed a significant level of inhibition against human CDA, but it was 16 times less potent than zebularine (K-i = 38 mu M vs K-i(apparent) = 2.3 mu M). Although these carbocyclic analogues appeared to be more stable than zebularine, replacement of the electronegative CO4' oxygen for the less electronegative carbon in 4-6 presumably reduces the capacity of the pyrimidin-2(1H)-one ring to form a covalent hydrate, a step considered crucial for the compound to function as a transition-state inhibitor of the enzyme.

Select chemokine receptors act as coreceptors for HIV-1 entry into human cells and represent targets for antiviral therapy. In this report we describe a distamycin analogue, 2,2'-[4, 4'-[[aminocarbonyl]amino]bis[N,4'-di[pryrrole-2-carboxamide- 1, 1'-dimethyl]]-6,8-naphthalenedisulfonic acid]hexasodium salt (NSC 651016), that selectively inhibited chemokine binding to CCR5, CCR3, CCR1, and CXCR4, but not to CXCR2 or CCR2b, and blocked chemokine-induced calcium flux. Inhibition was not due to nonspecific charge interactions at the cell surface, but was based on a specific competition for the ligand receptor interaction sites since the inhibitory effect was specific for some but not all chemoattractant receptors. NSC 651016 inhibited in vitro replication of a wide range of HIV-1 isolates, as well as HIV-2 and SIV, and exhibited in vivo anti-HIV-1 activity in a murine model. In contrast, a distamycin analogue with similar structure and charge and the monomeric form of NSC 651016 demonstrated no inhibitory effects. These data demonstrate that molecules which interfere with HIV-1 entry into cells by targeting specific chemokine coreceptors can provide a viable approach to anti-HIV-1 therapy. NSC 651016 represents an attractive candidate for the chemotherapeutic treatment of HIV-1 infection and as a microbicide to prevent the sexual transmisssion of HIV-1. Moreover, NSC 651016 can serve as a template for medicinal chemical modifications leading to more effective antivirals.

Mice developed massive splenomegaly and polyclonal hypergammaglobulinemia within 2 days after intravenous injection of a phosphorothioate oligomer that is antisense to a portion of the rev region of the HIV-1 genome. Histologie examination of spleens from injected animals showed marked expansion of a uniform-appearing population of small lymphocytes and many mitoses. Spleen mononuclear cells (SMNCs) from injected animals showed approximately a 10-fold-increased uptake of [3H]thymidine and production of IgM and IgG. Flow cytometry analysis indicated that the responding cells were predominantly B-lymphocytes. The anti-reu oligomer also was mitogenic in vitro and stimulated immunoglobulin production by normal mouse SMNCs and human peripheral blood mononuclear cells. Similar immunologie effects were observed with an anti-rev 21-mer phosphorothioate, truncated at the 3' end, but not with a 20-mer human p53 antisense phosphorothioate or a 28-mer anti-rev phosphodiester. These observations are consistent with the possibility that DNA sequences homologous to the rev gene participate in the regulation of mammalian lymphocyte activation, proliferation and maturation.

Publisher Summary This chapter describes the biochemical pharmacology, cellular pharmacology, preclinical pharmacology, preclinical toxicology, clinical pharmacology, and clinical toxicology of antitumor bisindole alkaloids from Catharanthus . Despite the close structural resemblance of vinblastine, vincristine, and vindesine, the profiles of biological activities of the compounds are not identical. Vinblastine treatment is much less likely to result in neurotoxicity than the treatment with vincristine; on the other hand, vinblastine produces much more perturbation of bone marrow function than vinblastine. Vincristine is more effective than vinblastine in the treatment of acute leukemia in children. Vincristine and vinblastine interfere with the mitotic process by producing arrest of cell division in metaphase. These drugs interact with tubulins, dimeric cellular proteins that play several critical roles in cell structure and function, including the organization of chromosomes on a matrix (spindle), which is an essential feature of normal cell division. The toxicological profile for vindesine includes effects observed with both vinblastine and vincristine. Among the effects observed with vindesine are bone marrow depression, alopecia, and peripheral neurotoxicity.

A series of aminoethylpyrrolidine-platinum complexes were synthesized and partially characterized for chemical structure. The leaving groups in this series of complexes were varied in an attempt to identify cytotoxic, water-soluble aminoethylpyrrolidine-platinum complexes. The cytotoxic activity was tested in vitro against L1210 cells sensitive to cis-diamminedichloroplatinum(II) (L1210/0), L1210 cells resistant to cis-diamminedichloroplatinum(II) (L1210/DDP), and L1210 cells resistant to 1,2-diaminocyclohexane platinum (L1210/DACH). The complexes were also tested for in vivo antitumor activity against L1210/0 cells injected i.p. The results of these studies indicate that the aminoethylpyrrolidine-platinum complexes have moderate antitumor activity but are cross-resistant in L1210/DDP cells. The degree of antitumor activity was dependent on the characteristic leaving group of a given complex.

Chloroquinoxaline sulfonamide (CQS), a chlorinated derivative of sulfaquinoxaline (SQ), inhibited proliferation of murine B16 melanoma cells, but only when relatively high drug concentrations (1 mM) were used. The inhibition of cell growth by CQS was at least partially reversible by incubation in drug-free medium. Incubation of melanoma cells with CQS was associated with an arrest of the cell cycle in G0/G1 as measured by flow cytometry. The drug slightly decreased uptake of radiolabeled deoxyuridine and thymidine after 24- and 48-hr incubation periods but increased nucleoside incorporation at 72 hr. No evidence of intercalation with DNA was found. Because SQ previously was reported to inhibit an aspect of folate metabolism, we investigated the possibility that CQS limits tumor cell growth by altering folate homeostasis. This appears unlikely, however, in view of the following observations: (1) the cytotoxic effects of CQS could not be reversed by folinic acid; (2) deoxyuridine suppression of thymidine incorporation was not affected by CQS treatment; (3) CQS did not inhibit dihydrofolate reductase from mammalian or bacterial sources; and (4) CQS toxicity in mice was not reduced by folinic acid. Experiments performed with analogues modified in the quinoxaline and para-amino phenyl functions indicated that tumor cell inhibition did not require preservation of the conventional sulfonamide structure.

Bull.Chem.Soc.Ethiop.,2(2), 69-72 (1988).

One of the problems encountered in the use of tetrahydrouridine (THU, 2) and saturated 2-oxo-1,3-diazepine nucleosides as orally administered cytidine deaminase (CDA) inhibitors is their acid instability. Under acid conditions these compounds are rapidly converted into inactive ribopyranoside forms. A solution this problem was sought by functionalizing the acid-stable but less potent CDA inhibitor 1-beta-D-ribofuranosyl-2(1H)-pyrimidinone (1) with the hope of increasing its potency to the level achieved with THU. The selection of the hydroxymethyl substituent at C-4, which led to the synthesis of 4-(hydroxymethyl)-1-beta-D-ribofuranosyl-2(1H)-pyrimidinone (10), 3,4-dihydro-4-(hydroxymethyl)-1-beta-D-ribofuranosyl-2(1H)-pyrimidinone (7), and 3,4,5,6-tetrahydro-4-(dihydroxymethyl)-1-beta-D-ribofuranosyl-2(1H)-p yrimidinone (28) was based on the transition-state (TS) concept. The key intermediate precursor, 4-[(benzoyloxy)methyl]-1-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)-2(H) -pyrimidinone (24), was obtained via the classical Hilbert-Johnson reaction between 2-methoxy-4-[(benzoyloxy)methyl]pyrimidine (20) and 2,3,5-tri-O-benzoyl-1-D-ribofuranosyl bromide (21). Deprotection of 24 afforded compound 10, while its sodium borohydride reduction products afforded compounds 7 and 28 after removal of the blocking groups. Syntheses of 3,4-dihydro-1-beta-D-ribofuranosyl-2(1H)-pyrimidinone (9) and 3,6-dihydro-1-beta-D-ribofuranosyl-2(1H)-pyrimidinone (8), which lack the hydroxymethyl substituent, was accomplished in a similar fashion. The new compounds bearing the hydroxymethyl substituent were more acid stable than THU, and their CDA inhibitory potency, expressed in terms of Ki values, spanned from 10(-4) to 10(-7) M in a manner consistent with the TS theory. Compound 7, in particular, was superior to its parent 1 and equipotent to THU (Ki = 4 X 10(-7) M) when examined against mouse kidney CDA. The superior acid stability of this compound coupled to its potent inhibitory properties against CDA should provide a means of testing oral combinations of rapidly deaminated drugs, viz. ara-C, without the complications associated with the acid instability of THU.

Trimetrexate glucuronate (TMTX) is a methotrexate (MTX) analog that is active against transport-deficient MTX-resistant tumor cells. We performed a phase I study of TMTX administered by daily bolus for 9 consecutive days since this schedule is one of the most active in experimental murine tumor models. The drug was administered in this fashion every 4 weeks for at least two cycles. Fifteen patients with refractory metastatic cancers were studied and all had received prior chemotherapy. The dose-limiting toxicity was a rapidly reversible thrombocytopenia first seen at a daily dose of 4.0 mg/m2 which occurred 7 days after the end of TMTX administration. There was great inter-and intrapatient variability in the platelet nadirs observed in the six patients treated at 4.0 mg/m2. One patient died of massive hemoptysis during a platelet nadir at that dose level. Granulocyte counts never dropped below 1500/mm3. Only one patient had significant non-hematological toxicity: a radiation recall skin toxicity along with a self-limited maculopapular rash. One patient with melanoma and lung metastases treated at 4.0 mg/m2 had a partial response. TMTX plasma levels were measured by HPLC every 3 days prior to daily dosing in patients receiving 4 mg/m2 to determine whether drug accumulation occurred during this prolonged administration schedule. Nadir drug levels varied from less than 0.02 to 0.35 μM and did not seem to increase during the 9-day schedule in individual patients. By comparison with other phase I trials, the hematologic toxicity of TMTX seems to be schedule-dependent, with less drug being tolerated and more severe thrombocytopenia observed with more protracted treatment protocols. A firm phase II starting dose for daily bolus x 9 schedules is difficult to recommend in view of the variable toxicity observed in the patients treated at 4.0 mg/m2 daily, who, in addition, had all been extensively pretreated. A reasonable starting dose might be 3.0 mg/m2 daily with built-in dosage increases or decreases.

AbstractDie wasserlöslichen Bis‐ [monocarboxylato]‐Komplexe (III) werden ausgehend von K2PtCl4 über den Komplex (II) dargestellt.

Tetrahydrouridine (THU, 2) and other fully reduced cyclic urea ribofuranosyl nucleosides undergo a rapid, acid-catalyzed isomerization to their more stable ribopyranosyl form. This isomerization is characterized by a change in spectral properties and by a greater than 10-fold decrease in potency for those nucleosides that act as potent inhibitors of cytidine deaminase in their ribofuranose form. 1-(beta-D-Ribopyranosyl)hexahydropyrimidin-2-one (7) was synthesized and used in conjunction with its furanose isomer 6 as a model compound for more extensive 1H and 13C NMR, mass spectral, and kinetic studies of this isomerization. The 0.4 delta upfield shift and 4-Hz increase in the J1',2' coupling constant for the pyranose anomeric proton in the 1H NMR spectrum is indicative of a pyranose beta-CI conformation in which the aglycon and C-2' and C-4' hydroxyls are equatorial. The mass spectra of trimethylsilylated pyranose nucleosides also show a characteristic large shift in the m/z 204-217 abundance and the appearance of two new rearrangement ions at M-133 and M-206. For furanose 6 the rate of isomerization is pH and temperature dependent with pyranose 7 predominating by a factor of 6-9 equilibrium. At pH 1 and 37 degrees C, furanose 6 has an initial half-life of less than 12 min. Accordingly, this isomerization may explain the observed lack of enhanced ara-C levels in studies evaluating the oral administration of an ara-C and THU combination to species with an acidic stomach content.