A phase I study assessing the safety and pharmacokinetics of the thrombospondin-1-mimetic angiogenesis inhibitor ABT-510 with gemcitabine and cisplatin in patients with solid tumors.

BACKGROUND: The aim of the study was to determine the safety profile, pharmacokinetics and potential drug interactions of the angiogenesis inhibitor ABT-510 combined with gemcitabine-cisplatin chemotherapy in patients with solid tumors. PATIENTS AND METHODS: Patients with advanced solid tumors received gemcitabine 1250 mg/m2 intravenously (i.v.) on days 1 and 8 and cisplatin 80 mg/m2 on day 1 of a 3-week cycle in combination with ABT-510. ABT-510 was administered subcutaneously twice daily at doses of 50 mg or 100 mg. Plasma samples for pharmacokinetics were obtained on days 1 (gemcitabine, cisplatin as single agents), 15 (ABT-510 as single agent) and 22 (gemcitabine, cisplatin and ABT-510 as combination). RESULTS: Thirteen patients received ABT-510 as either 50 mg b.i.d. (seven patients) or 100 mg b.i.d. (six patients) in combination with gemcitabine-cisplatin. The most common reported adverse events reflected the known toxicity profile induced by gemcitabine-cisplatin without ABT-510. One episode of hemoptysis occurred in a patient with non-small-cell lung cancer (NSCLC) after 13 days of treatment. No clinically significant pharmacokinetic interactions between ABT-510, gemcitabine and platinum were observed. Three partial responses were observed in 12 evaluable patients (one head and neck cancer, one melanoma and one NSCLC). CONCLUSIONS: Combining ABT-510 at doses of 50 mg and 100 mg with gemcitabine-cisplatin is feasible. Pharmacokinetic interactions were not observed and adding ABT-510 does not appear to increase toxicity.

Phase I study of eniluracil, a dihydropyrimidine dehydrogenase inactivator, and oral 5-fluorouracil with radiation therapy in patients with recurrent or advanced head and neck cancer.

5-Fluorouracil (5-FU) is an effective enhancer of radiation therapy (RT) in head and neck cancers. Due to rapid, predominantly hepatic metabolism by dihydropyrimidine dehydrogenase (DPD) and suggested clinical benefit from prolonged drug exposure, 5-FU is commonly given by continuous infusion. Eniluracil is a novel DPD-inactivator designed to prolong the half-life of 5-FU and provide sustained plasma concentrations of 5-FU with oral dosing. We conducted a Phase I study of the safety and efficacy of eniluracil given with oral 5-FU in patients receiving concurrent RT for recurrent or advanced squamous cell carcinomas of the head and neck. Thirteen patients with recurrent, metastatic, or high-risk (defined as an expected 2-year survival rate of <10%) head and neck cancer were enrolled and treated with concomitant chemoradiotherapy on an every-other-week schedule. Eniluracil at a fixed dose [20 mg twice a day (BID)] was given for 7 consecutive days (days 1-7). 5-FU and RT were given on 5 consecutive days (days 2-6). One patient was treated with once-daily RT (2.0 Gy fractions). The remaining patients received hyperfractionated RT (1.5-Gy fractions BID). The initial dose of 5-FU was 2.5 mg/m2 given BID. Dose escalation in patient cohorts was scheduled at 2.5-mg/m2 increments, with intrapatient dose escalation permitted. Lymphocyte DPD activity and serum 5-FU and uracil concentrations were monitored during two cycles. DPD activity was completely or nearly completely inactivated in all patients. Sustained, presumed therapeutic concentrations of 5-FU were observed at a dose of 5.0 mg/m2 given BID. Cumulative dose-limiting myelosuppression (both neutropenia and thrombocytopenia) was observed during the fourth and fifth cycles following administration of 5.0 mg/m2 5-FU BID. One patient died of neutropenic sepsis during cycle 4. Other late cycle toxicities included diarrhea, fatigue, and mucositis. Grade 3 mucositis was observed in 4 patients, but no grade 4 mucositis or grade 3 or 4 dermatitis was observed. A second patient death occurred during cycle 1 of treatment. No specific cause of death was identified. The study was subsequently discontinued. Cumulative myelosupression was the significant dose-limiting toxicity of oral 5-FU given with the DPD-inactivator eniluracil on an every-other-week schedule. Clinical radiation sensitization was not observed, based on the absence of dose-limiting mucositis and dermatitis. Alternative dosing schedules need to be examined to determine the most appropriate use of eniluracil and 5-FU as radiation enhancers.

Activation and deactivation of sarcoplasmic reticulum calcium release channels: molecular dissection of mechanisms via novel semi-synthetic ryanoids.

The plant alkaloids ryanodine and dehydroryanodine are high affinity, biphasic modulators of the intracellularly located, calcium-regulated calcium release channels of a variety of cell types. To date, little is certain about the molecular basis of the interactions that prompt low concentrations of ryanodine (nanomolar to low micromolar) to activate (open) the channels and higher concentrations to deactivate (functionally close) the sarcoplasmic reticulum calcium release channel. In the present study, we approached this question using novel, semi-synthetic C10-Oeq ester derivatives of ryanodine and dehydroryanodine as molecular probes of the ryanodine binding sites on the calcium release channel. Binding affinities of these C10-Oeq ester derivatives of ryanodine and dehydroryanodine with acidic, basic and neutral side chains (Kd values > 53.9 nM, Kd values 0.3-0.7 nM and Kd values 1.3-20.4 nM, compared with 2.3 and 2.8 nM for ryanodine and dehydroryanodine, respectively) were evaluated for their ability to modulate the patency of the sarcoplasmic reticulum calcium release channel. With the exception of only two derivatives tested to date, all the semi-synthetic C10-Oeq esters selectively activate the Ca2+ release channel. That is, they produce no functional closure of the sarcoplasmic reticulum calcium release channels at the highest concentration that could be tested. Half-maximal concentrations for activation (EC50act values) ranged from 0.87-4.2 microM, compared with an EC50act of 1.3 microM for ryanodine. Using a low concentration (0.5 nM) of a high specific activity, radioiodinated derivative of ryanodine, C10-Oeq N-(4-azido-5-125iodo salicyloyl) glycyl ryanodine (1400 Ci/mmol) as the radioligand in displacement binding affinity assays, two distinct, sequential ryanodine binding isotherms were demonstrated within the normal 0-300 nM ryanodine sigmoidal displacement curve. A high affinity site had an IC50 of 0.5 nM (Kd = 0.26 +/- 0.02 nM). Above this concentration, an apparent plateau occurred between 3 and 6 nM ryanodine, and at higher concentrations a lower affinity site was revealed that demonstrated an IC50 of about 25 nM (Kd = 11.7 +/- 1.2 nM). Scatchard analysis from direct binding of C10-Oeq N-(4-azido-5-125iodo salicyloyl) glycyl ryanodine to junctional sarcoplasmic reticulum vesicles also suggests the presence of more than one class of binding sites within the nanomolar concentration range. The high affinity site demonstrated a Bmax of 3 pmol/mg protein. We were unable to saturate the lower affinity binding sites with this ligand. To evaluate the functional effects occurring among sarcoplasmic reticulum calcium release channel monomers as a consequence of ryanodine's binding, we utilized a photo-activatable derivative of ryanodine, C10-Oeq N-(4-azido salicyloyl) glycyl ryanodine that demonstrates channel modulating characteristics similar to ryanodine. Covalently labeling the sarcoplasmic reticulum calcium-release channels with this ligand, followed by measurements of rates of calcium efflux and SDS-PAGE of the labeled protein, revealed that deactivation of the sarcoplasmic reticulum calcium release channels of skeletal muscle by this ryanoid occurred at concentrations which apparently produce virtually irreversibly interactions between receptor monomers. This 'polymerization' was indicated by the progressive appearance of two higher molecular weight protein bands on SDS-PAGE, concomitant with progressive decreases in the ryanodine receptor monomer band that runs at an apparent molecular mass of 365 kDa. In summary, we have prepared and utilized novel C10-Oeq ester derivatives of ryanodine and dehydroryanodine in studies aimed at better understanding the molecular basis for the complex biphasic actions of ryanodine on the sarcoplasmic reticulum calcium release channels from rabbit skeletal muscle cells. The described studies presage correlations that may be useful in furthering our understa

High affinity C10-Oeq ester derivatives of ryanodine. Activator-selective agonists of the sarcoplasmic reticulum calcium release channel.

The plant alkaloids ryanodine and dehydroryanodine are specific and potent modulators of the sarcoplasmic reticulum calcium release channel. In the present study, acidic, basic, and neutral side chains esters of these diterpene compounds were prepared and their pharmacologic activities were assessed. Binding affinities of the novel C10-Oeq ester derivatives for the sarcoplasmic reticulum Ca2+ release channel were evaluated with sarcoplasmic reticular vesicles prepared from rabbit skeletal muscle. Kd values of the derivatives varied 500-fold, ranging from 0.5 to 244 nM. In comparison, Kd values for ryanodine and dehydroryanodine were 4.4 nM and 5.4 nM, respectively. Basic substituents at the C10-Oeq side chain terminus produced the highest affinity derivatives (Kd values from 0.5 to 1.3 nM). Neutral and/or hydrophobic side chain derivatives exhibited intermediate affinities for the high affinity ryanodine receptor site (Kd values from 2.5 to 39 nM), whereas a derivative with a terminal acidic group had the lowest affinity (Kd value > 100 nM). Certain of the higher affinity C10-Oeq derivatives were evaluated more extensively for their pharmacologic activity on the sarcoplasmic reticular Ca2+ release channel. Both channel activating (opening) and deactivating (closing) actions were assessed from the ability of the ryanoids to alter Ca2+ efflux rates from skeletal junctional sarcoplasmic reticular vesicles that had been passively loaded with Ca2+. The natural Ryania secondary metabolites ryanodine, dehydroryanodine and esters E and F, all exhibit antithetical concentration-effect curves, indicating both activator and deactivator actions. In contrast, the semi-synthetic C10-Oeq esters selectively activate the Ca2+ release channel. Half-maximal concentrations for such activation (EC50 act) ranged from 0.87 microM to 4.2 microM, compared with an EC50 act of 1.3 microM for ryanodine. These derivatives were also evaluated for their ability to augment ATP-dependent CA2+ accumulation by cardiac junctional sarcoplasmic reticular vesicles, an effect that results from deactivation of the Ca2+ release channels. None of the derivatives tested was able to significantly augment Ca2+ accumulation, further substantiating their inability to deactivate the sarcoplasmic reticular Ca2+ release channel. Additionally, these derivatives functionally antagonized the action of ryanodine to close the Ca2+ release channel. The results presented demonstrate that these C10-Oeq ester derivatives of ryanodine and dehydroryanodine bind specifically to the SR Ca2+ release channel, selectively activate the channel, and, although they fail to effect channel closure, they nevertheless functionally compete with ryanodine at its low affinity (deactivator) site(s).

Structural determinants of high-affinity binding of ryanoids to the vertebrate skeletal muscle ryanodine receptor: a comparative molecular field analysis.

Ryanodine binds to specific membrane proteins, altering the calcium permeability of intracellular membranes. In this study 19 ryanoids were isolated or synthesized and the structures correlated to the strength of binding to vertebrate skeletal muscle ryanodine receptors. Global minima were determined by employment of molecular mechanics and dynamics augmented by systematic searching of conformational space. Overall, steric and electrostatic factors contribute about equally to the differences in the experimentally determined dissociation constants. The dominant electrostatic interaction is localized to a hydroxyl group in an apolar region of the molecule. The pyrrole and isopropyl groups located together at one pole of the molecule have the greatest effect on steric interactions between ligand and receptor. We suggest ryanodine binds to the receptor with the pyrrole and isopropyl groups buried deep inside a cleft in the protein. This arrangement places special importance on the conformation of the pyrrole and isopropyl groups. In contrast, the opposite pole appears to be positioned at the entrance of the binding pocket because bulky adducts placed in the 9 position of ryanodine alter binding minimally. For example, a fluorescent ryanodine adduct was synthesized which has a dissociation constant close to that of ryanodine. Detailed examination reveals subtle interactions between ryanoid and receptor. In many cases, the major factors altering the strength of binding were found to be conformational alterations in the molecule remote from the site of covalent modification.

Differential activating and deactivating effects of natural ryanodine congeners on the calcium release channel of sarcoplasmic reticulum: evidence for separation of effects at functionally distinct sites.

Two novel natural ryanoids from extracts of the wood of Ryania speciosa Vahl were evaluated with sarcoplasmic reticulum (SR) vesicles for their binding affinities and their activating and deactivating effects on Ca2+ release channels. The new ryanoids, which are more polar than the known Ryania constituents ryanodine and didehydro-(9,21)-ryanodine, were purified using silica gel column chromatography and reverse phase high performance liquid chromatography. The new ryanoids were designated ester E and ester F, in keeping with nomenclature previously used in the literature. These compounds were identified by NMR spectroscopy and mass spectroscopy as C9ax-hydroxyryanodine and C8ax-hydroxy-C10-epi-dehydroryanodine, respectively. Binding of esters E and F to the high affinity (nanomolar Kd) site on SR Ca2+ release channels was determined from relative binding affinity assays using 6.7 nM [3H]ryanodine. Apparent Kd values of ryanodine, ester E, and ester F for binding to this domain on the skeletal muscle ryanodine receptor/SR Ca2+ release channel were 4.4 +/- 0.8, 65 +/- 10, and 257 +/- 53 nM, respectively (mean +/- standard deviation, four or more experiments). Apparent Kd values for cardiac muscle receptors were 0.51 +/- 0.01, 12 +/- 0.4, and 57 nM, respectively. As a functional indication of the effects of the ryanoids, channel-opening (activator) and channel-closing (deactivator) actions were assessed from the ability of the ryanoids to alter the rate of Ca2+ efflux from passively loaded skeletal muscle junctional sarcoplasmic reticular vesicles (JSRV). Activator actions among the ryanoids were similar, in that they exhibited apparently parallel concentration-effect curves, having a slope of 40% Ca2+ loss/decade increment in ryanoid concentration. Half-maximal values for activation (EC50 values) were 2.5, 63, and 43 microM for ryanodine, ester E, and ester F, respectively. Maximal channel opening by ester E was significantly less than that produced by the other ryanoids. The deactivator actions of the compounds on skeletal JSRV were dissimilar, in that their concentration-effect curves appeared not to be parallel. The quotient of the EC50 for deactivation and that for activation was taken as the concentration-coupling ratio (CCR). The CCR for ryanodine was 114 and that for ester F was 72, but the CCR for ester E was only 21. ATP-dependent Ca2+ accumulation by cardiac JSRV provided a second means to evaluate deactivator actions of the ryanoids. Results from cardiac JSRV assays were in general similar to those from skeletal JSRV assays.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino- and guanidinoacylryanodines: basic ryanodine esters with enhanced affinity for the sarcoplasmic reticulum Ca(2+)-release channel.

Amino- and guanidinoacyl esters of ryanodine were prepared to evaluate the effect of basicity on the binding affinity of these derivatives for the sarcoplasmic reticulum Ca(2+)-release channel (SR CRC). In the presence of DCC and DMAP Cbz-beta-alanine reacts with ryanodine in CH2Cl2 to give O10eq-Cbz-beta-alanylryanodine (3a), which on hydrogenolysis yields the beta-alanyl ester (4a). N,N'-bis-Cbz-S-methylthiourea reacts with 4a to yield beta-N,N'-bis-Cbz-guanidinopropionylryanodine (5a). O10eq-beta-guanidinopropionylryanodine (6a) is obtained on hydrogenolytic deprotection of 5a. The binding affinity of beta-alanine ester (4a) and its glycyl congener (4b) is 2-3-fold greater, and that of the beta-guanidinopropionyl ester (6a) and its acetyl congener (6b) 3-6-fold greater, than that of ryanodine. The effect of ryanodine on SR Ca2+ flux is of a biphasic nature: nanomolar levels open (activate) the channel, while micromolar levels close (deactivate) it. The base-substituted esters 4a and 6a both display a unidirectional effect: they only open the channel. An understanding of ryanodine's mode of action and the design of effective SR CRC activating and deactivating ryanoids for possible therapeutic application are major research objectives.