Magnetic resonance imaging measurements of the response of murine and human tumors to the vascular-targeting agent ZD6126.

PURPOSE: ZD6126 is a novel vascular targeting agent currently undergoing clinical evaluation. It acts by destabilizing the microtubulin of fragile and proliferating neoendothelial cells in tumors. The drug leads to blood vessel congestion, the selective destruction of the vasculature, and extensive necrosis in experimental tumors. The aim of the study reported here was to assess the ability of dynamic contrast enhanced magnetic resonance imaging (MRI) to measure the antivascular effects of ZD6126 in tumors. EXPERIMENTAL DESIGN: The work was carried out in mice bearing C38 colon adenocarcinoma and in patients with advanced cancers. MRI was performed before and 6 h (human tumors) or 24 h (C38 tumors) after i.v. drug administration. Contrast agent (gadolinium diethylenetriaminepentaacetate) enhancement was characterized by the initial area under the gadolinium diethylenetriaminepentaacetate uptake versus time curve (IAUC). IAUC reflects blood flow, vascular permeability, and the fraction of interstitial space. RESULTS: The median IAUC was reduced in all C38 tumors after ZD6126 administration [by 6-48% at 50 mg/kg (n = 3)], 58-91% at 100 mg/kg (n = 4), and 11-93% at 200 mg/kg (n = 6). In contrast, the administration of vehicle only led to no consistent change in median IAUC (n = 4). The ZD6126-induced changes in median IAUC appeared to be dose dependent (P = 0.045). No ZD6126-induced changes were apparent in murine muscle. Similar effects were seen in preliminary data from human tumors (11 tumors studied, 9 patients). At doses of 80 mg/m(2) and higher, the median IAUC post-ZD6126 treatment was reduced in all of the tumors studied (8 tumors, 6 patients) to 36-72% from the baseline value. There was a significant trend of increasing reductions with increasing exposure (P < 0.01). No drug-induced changes in human muscle or spleen IAUC were apparent. The reproducibility of the median IAUC parameter was investigated in patients. In 19 human tumors (measured in 19 patients) inter- and intratumor coefficients of variation were 64 and 18%. CONCLUSIONS: The contrast enhanced-MRI measured median IAUC is a useful end point for quantifying ZD6126 antivascular effects in human tumors.

Biological evaluation of cryptophycin 52 fragment A analogues: effect of the multidrug resistance ATP binding cassette transporters on antitumor activity.

Cryptophycin 52 (LY355703) is a potent antiproliferative analogue of the marine natural product cryptophycin 1. It has been shown to have a broad range of antitumor activity against human tumor xenografts and murine tumors including tumors resistant to Taxol and Adriamycin. Its mechanism of action involves arresting cells in the G2-M phase of the cell cycle by binding to microtubules and suppressing their dynamics. This 16-membered depsipeptide can be divided into four major subunits or fragments (A-D). We reported previously on our synthetic efforts around fragment A and discovered that this region of the molecule was amenable to a structure-activity relationship study that resulted in highly active antiproliferative agents when evaluated in the CEM leukemia cell line. The synthetic analogues were designed to help improve the efficacy and aqueous solubility of the parent compound; therefore, many in this series contained ionizable functional groups such as an amino group, a hydroxy group, or a carboxylic acid. Although several of these analogues showed improvements in potency over cryptophycin 52 in drug-sensitive tumor xenograft models, many lost their activity against Adriamycin-resistant tumor lines. It was discovered on additional in vitro evaluation that these analogues became good substrates of the multidrug resistance transporter P-glycoprotein.

II. Synthesis and biological evaluation of some bioisosteres and congeners of the antitumor agent, 2-(4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy)propionic acid (XK469).

XK469 (1) is among the most highly and broadly active antitumor agents to have been evaluated in our laboratories. Subsequent developmental studies led to the entry of (R)-(+) 1 (NSC 698215) into phase 1 clinical trials (NIH UO1-CA62487). The antitumor mechanism of action of 1 remains to be elucidated, which has prompted a sustained effort to elaborate a pharmacophoric pattern of 1. The present study focused on a strategy of synthesis and biological evaluation of topologically based, bioisosteric replacements of the quinoxaline moiety in the lead compound (1) by quinazoline (4a-d), 1,2,4-benzotriazine (12a-18b), and quinoline (21a-g) ring systems. The synthetic approach to each of the bioisosteres of 1 utilized the methodology developed in previous work (see Hazeldine, S. T.; Polin, L.; Kushner, J.; Paluch, J.; White, K.; Edelstein, M.; Palomino, E.; Corbett, T. H.; Horwitz, J. P. Design, Synthesis, and Biological Evaluation of Analogues of the Antitumor Agent 2-(4-[(7-Chloro-2-quinoxalinyl)oxy]phenoxy)propionic acid (XK469). J. Med. Chem. 2001, 44, 1758-1776.), which is extended to the procurement of the benzoxazole (23a,b), benzthiazole (23c,d), pyridine (25a,b), and pyrazine (27) congeners of 1. Only quinoline analogues, bearing a 7-halo (21a,b,d,e) or a 7-methoxy substituent (21g), showed antitumor activities (Br > Cl > CH(3)O > F approximately I), at levels comparable to or greater than the range of activities manifested by 1 and corresponding analogues. At high individual dosages, the (S)-(-) enantiomers of 1 and 21b,d all produce a reversible slowing of nerve-conduction velocity in the mice, the onset of which is characterized by a distinctive dysfunction of the hind legs, causing uncoordinated movements. The condition resolves within 5-10 min. However, at higher dosages, which approach a lethal level, the behavior extended to the front legs, lasting from 20 min to 1 h. By contrast, the (R)-(+) forms of these same agents did not induce the phenomenon of slowing of nerve-conduction velocity.

A convergent approach to cryptophycin 52 analogues: synthesis and biological evaluation of a novel series of fragment a epoxides and chlorohydrins.

Cryptophycin 52 is a synthetic derivative of Cryptophycin 1, a potent antimicrotubule agent isolated from cyanobacteria. In an effort to increase the potency and water solubility of the molecule, a structure-activity relationship study (SAR) was initiated around the phenyl ring of fragment A. These Cryptophycin 52 analogues were accessed using a Wittig olefination reaction between various triphenylphosphonium salts and a key intermediate aldehyde prepared from Cryptophycin 53. Substitution on the phenyl ring of fragment A was well tolerated, and several of these analogues were equally or more potent than Cryptophycin 52 when evaluated in vitro in the CCRF-CEM leukemia cell line and in vivo against a murine pancreatic adenocarcinoma.

Pharmacokinetics and tissue distribution of cryptophycin 52 (C-52) epoxide and cryptophycin 55 (C-55) chlorohydrin in mice with subcutaneous tumors.

PURPOSE: To compare the pharmacokinetics and tissue distribution (both normal and tumor) of cryptophycin 52 (C-52) and its putative chlorohydrin prodrug cryptophycin 55 (C-55) in a murine model and to investigate a possible mechanism behind the superior activity of C-55. METHODS: Mammary adenocarcinoma 16/c tumor-bearing mice were treated with an i.v. bolus of 11 mg/kg C-52 or 38 mg/kg C-55 in Cremophor-alcohol. At predetermined time intervals, C-52 and C-55 concentrations in plasma, liver, kidney, small intestine and tumors were measured using a previously described HPLC method. Pharmacokinetic parameters were computed using noncompartmental methods. Tissue (both normal and tumor) to plasma ratios as a function of time were also calculated for comparison. RESULTS: Both C-52 and C-55 were rapidly distributed into different tissues including tumors following i.v. administration. However, the affinities of these compounds towards different tissues were different. Thus, the half-lives (minutes) of C-55 were in the decreasing order liver (725), intestine (494), tumor (206), kidney (62) and plasma (44), whereas the AUC values (microg x min/ml) were in the order tumor (9077), liver (7734), kidney (6790), plasma (2372) and intestine (2234). For C-52, the half-lives (minutes) were in the decreasing order liver (1333), kidney (718), intestine (389), tumor (181) and plasma (35), and the AUC values (microg x min/ml) were in the order kidney (1164), liver (609), intestine (487), plasma (457) and tumor (442). The relative exposures to C-52 after i.v. injection of C-55 were plasma 3.9%, tumor 80.8%, kidney 3.4%, liver 1.1% and intestine 2.8%. Although plasma exposure to C-52 following C-55 administration was relatively small, the use of C-55 to deliver C-52 increased the retention of C-52 and its AUC in tumor compared to direct injection of C-52. Simultaneously, this approach shortened C-52 retention in all normal tissues studied. CONCLUSIONS: The distribution of C-55 and its bioconversion to C-52 in different organs and tumor tissue observed in this study suggest the ability of C-55 to target tumor tissue, creating a depot of C-52 in tumor. Increased C-52 exposure of tumor, with concomitant decreased exposure of normal tissue, is a contributing factor to the superior activity of C-55 versus C-52. However, except in the case of tumor tissue in which 81% of C-55 converts to C-52, only a minor amount of C-55 may serve as a prodrug for C-52, whereas the majority is handled by the biosystem through a different route of elimination. Tissue distribution combined with rate of conversion may be an important determinant of the relative effectiveness of other epoxide-chlorohydrin pairs of cryptophycins.

Synthesis and biological evaluation of conformationally constrained analogs of the antitumor agents XK469 and SH80. Part 5.

Conformational restriction of bioactive molecules offers the possibility of generating structures of increased potency. To this end, a synthesis has been achieved of (R,S)-2-[(8-chlorobenzofurano[2,3-b]quinolinyl)oxy]propionic acid (12a), a highly rigidified, polycyclic analog of 2-[4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy]propionic acid (2a, XK469). Efforts to effect the same synthesis of the corresponding 8-bromo-derivative led to a mixture of intermediate, 8-chloro (9a), and 8-bromo-2-hydroxybenzofurano[2,3-b]quinoline (9b), generated by halogen-exchange, via an aromatic S(RN)1(A(RN)1) reaction of precursor, 8b, with pyridine hydrochloride. The presumption that conformational restriction of 1b-12a might enhance the antitumor potency of the latter has not been sustained. In fact, 12a proved to be significantly less active than 1b. However, it is apparent that virtually all of the spatial and steric properties of 12a, necessary for improved activity, including the disposition of the 2-oxypropionic acid side chain remain to be identified.

Synthetic modification of the 2-oxypropionic acid moiety in 2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid (XK469), and consequent antitumor effects. Part 4.

The criteria for the activity of 2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid (XK469) and 2-{4-[(7-bromo-2-quinolinyl)oxy]phenoxy}propionic acid (SH80) against transplanted tumors in mice established in previous studies, require a (7-halo-2-quinoxalinoxy)- or a (7-halo-2-quinolinoxyl)-residue, respectively, bridged via a 1,4-OC(6)H(4)O-linker to C(2) of propionic acid. The present work demonstrates that substitution of fluorine at the 3-position of the 1,4-OC(6)H(4)O-linker of XK469 leads to a 10-fold reduction in activity, whereas the corresponding 2-fluoro analog proved to be 100-fold less active than XK469. Moreover, the latter tolerated substitution of but a single, additional methyl group to the 2-position of the propionic acid moiety, that is, the isobutyric acid analog, without loss of significant in vivo activity. Indeed, an intact 2-oxypropionic acid moiety is a prerequisite for maximum antitumor activity of 1a.

Part 3: synthesis and biological evaluation of some analogs of the antitumor agents, 2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid, and 2-{4-[(7-bromo-2-quinolinyl)oxy]phenoxy}propionic acid.

2-{4-[(7-Chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid (X469) and 2-{4-[(7-bromo-2-quinolinyl)oxy]phenoxy}propionic Acid (SH80) are among the most highly and broadly active antitumor agents to have been developed in our laboratories. However, the mechanism(s) of action of these agents remain to be elucidated, which prompted our continued endeavor to delineate a pharmacophoric pattern, from which a putative target might be deduced. Herein, we provide additional evidence that intact quinoxaline and quinoline rings in XK469 and SH80, respectively, are fundamental to the activities of these structures against transplanted tumors in mice. The consequence of further modification of the heterocyclic ring system in XK469 and SH80, leading to [1,8]naphthyridine; pyrrolo[1,2-a]; imidazo[1,2-a]; and imidazo[1,5-a] derivatives, all deprive the parent structures of antitumor activity. Introduction of CH3, CF3, CH3O, CO2H, or C6H5 substituents at C4 of the quinoline ring of SH80 led to weakly active antitumor agents. Similarly, the phenanthridine analog of SH80 manifested only modest cytotoxicity. Lastly, XK469 and SH80 are both significantly more active than the corresponding regioisomeric structures, 2-{4-[(7-halo-4-quinolinyl)oxy]phenoxy)propionic acids.

Cryptophycins-309, 249 and other cryptophycin analogs: preclinical efficacy studies with mouse and human tumors.

Cryptophycins-1 and 52 (epoxides) were discovered to have in-vitro and in-vivo antitumor activity in the early 1990s. The chlorohydrins of these, Cryptophycins-8 and 55 (also discovered in the early 1990s) were markedly more active, but could not be formulated as stable solutions. With no method to adequately stabilize the chlorohydrins at the time, Cryptophycin-52 (LY 355073) entered clinical trials, producing only marginal antitumor activity. Since that time, glycinate esters of the hydroxyl group of the chlorohydrins have been synthesized and found to provide stability. Three of the most active were compared herein. Cryptophycin-309 (C-309) is a glycinate ester of the chlorohydrin Cryptophycin-296. The glycinate derivative provided both chemical stability and improved aqueous solubility. After the examination of 81 different Cryptophycin analogs in tumor bearing animals, C-309 has emerged as superior to all others. The following %T/C and Log Kill (LK) values were obtained from a single course of IV treatment (Q2d x 5) against early staged SC transplantable tumors of mouse and human origin: Mam 17/Adr [a pgp (+) MDR tumor]: 0%T/C, 3.2 LK; Mam 16/C/Adr [a pgp (-) MDR tumor]: 0%T/C, 3.3 LK; Mam 16/C: 0%T/C, 3.8 LK; Colon 26: 0%T/C, 2.2 LK; Colon 51: 0%T/C, 2.4 LK; Pancreatic Ductal Adenocarcinoma 02 (Panc 02): 0%T/C, 2.4 LK; Human Colon HCT15 [a pgp (+) MDR tumor]: 0%T/C, 3.3 LK; Human Colon HCT116: 0%T/C, 4.1 LK. One additional analog, Cryptophycin-249 (C-249, the glycinate of Cryptophycin-8), also emerged with efficacy rivaling or superior to C-309. However, there was sufficient material for only a single C-249 trial in which a 4.0 LK was obtained against the multidrug resistant breast adenocarcinoma Mam-16/C/Adr. C-309 and C-249 are being considered as second-generation clinical candidates.

The molecular pharmacology of symplostatin 1: a new antimitotic dolastatin 10 analog.

Symplostatin 1, an analog of dolastatin 10, was recently isolated from cyanobacteria of the genus Symploca. Symplostatin 1 is a potent inhibitor of cell proliferation with IC(50) values in the low nanomolar range and it exhibits efficacy against a variety of cancer cell types. Symplostatin 1 caused the formation of abnormal mitotic spindles and accumulation of cells in metaphase at concentrations that had only minor effects on interphase microtubules. At higher concentrations, symplostatin 1 caused the loss of interphase microtubules. Cell cycle analysis revealed that symplostatin 1 caused G(2)/M arrest, consistent with its effects on mitotic spindles. Symplostatin 1 initiated the phosphorylation of Bcl-2, formation of micronuclei and activation of caspase 3, indicating induction of apoptosis. The cellular effects of symplostatin 1 are consistent with other antimitotic tubulin-targeting drugs. Tubulin polymerization experiments indicated that symplostatin 1 potently inhibits the assembly of purified tubulin, suggesting that tubulin may be its intracellular target. Some microtubule-targeting agents are reported to have antiangiogenic activity and therefore the effects of symplostatin 1 on endothelial cell proliferation and invasion were evaluated. Symplostatin 1 was found to be a potent inhibitor of both endothelial cell proliferation and invasion. Because of its potent and broad activity in vitro, symplostatin 1 was evaluated in vivo. Symplostatin 1 was active against murine colon 38 and murine mammary 16/C; however, it was poorly tolerated and the mice were slow to recover from the toxicity. The data indicate that symplostatin 1 has a mechanism of action similar to dolastatin 10.

Discovery and preclinical antitumor efficacy evaluations of LY32262 and LY33169.

The discoveries of a new antitumor agent (LY32262) (N-[2,4-dichlorobenzoyl]phenylsulfonamide) and a close analog (LY33169) are described. For this discovery, a disk-diffusion-soft-agar-colony-formation-assay was used to screen a portion of the Eli Lilly inventory, with the evaluation of each agent against normal cells, leukemic cells and several solid tumors, including a multidrug-resistant solid tumor (with marked selective cytotoxicity for Colon-38 and Human-Colon-15/MDR compared to normal fibroblasts and L1210 leukemic cells characterizing the discovery). In mice, LY32262 and/or LY33169 had curative activity against Colon Adenocarcinoma-38, Human Colon-116, Human Prostate LNCaP, and Human Breast WSU-Br-1. In addition, many other tumors were highly sensitive: Panc-03 = 2.4 log kill (LK); Panc-02 = 2.9-4.1 LK; Squamous Lung LC-12 = 2.1 LK; Colon-26 = 2.2 LK; AML1498 = 2.7 LK; Human Sm Cell Lung DMS-273 = 6.3 LK; Human Squamous Lung 165 = 3.7 LK; Human Ovarian BG-1 = 3.7 LK; Human Colon CX-1 (H29) = 1.6 LK; Human Colon-15/MDR (a p-glycoprotein positive multidrug resistant tumor) = 2.3 LK; Human CNS-gliosarcoma-SF295 = 3.8 LK. Several tumors were only marginally responsive or totally unresponsive: Mammary Adenocarcinoma-16/C = 0.6 LK; Mammary Adenocarcinoma-17 = no kill; Colon Adenocarcinoma-11 = no kill; L1210 leukemia = 1.3 LK; Human Prostate PC-3 = 0.5 LK; Human Adenosquamous Lung H125 = no kill; and Human Breast Adenocarcinoma MX-1 = 0.9 LK. There was no absolute tissue of origin correlation with antitumor efficacy, although colon tumors were most responsive and mammary tumors least responsive. The cause of the "hit and miss" efficacy has not been determined.

Tryptophanyl phosphoramidates as prodrugs of synadenol and its E-isomer: synthesis and biological activity.

Phosphorotryptophanates 2c and 3c were synthesized and investigated as prodrugs of synadenol (2a) and its E-isomer 3a. The antiviral activity of 2c corresponds to parent analogue 2a but it is lower than that of phenylphosphoralaninate 2b. This may indicate an enzymatic cleavage of phosphorotryptophanate 2c to 2a before or after entering the host cells. The E-isomer 3c was effective only against EBV with parameters suggesting intracellular delivery of the respective phosphate. Compound 2c has a moderate but selective activity against solid tumors.

Symplostatin 3, a new dolastatin 10 analogue from the marine cyanobacterium Symploca sp. VP452.

Symplostatin 3 (1), a new analogue of dolastatin 10 (2), has been isolated from a tumor selective extract of a Hawaiian variety of the marine cyanobacterium Symploca sp. VP452. Compound 1 differs from 2 only in the C-terminal unit; the dolaphenine unit is substituted by a 3-phenyllactic acid residue. Symplostatin 3 (1) possesses IC(50) values for in vitro cytotoxicity toward human tumor cell lines ranging from 3.9 to 10.3 nM. It disrupts microtubules, but at a higher concentration than 2, correlating with the weaker in vitro cytotoxicity.

Preclinical efficacy evaluations of XK-469: dose schedule, route and cross-resistance behavior in tumor bearing mice.

XK-469 is advancing to Phase I clinical trials. Preclinical studies were carried out to assist in clinical applications. DOSE-SCHEDULE ROUTE TESTING: Single dose i.v. treatment with XK-469 produced lethality (LD20 to LD100) above 142 mg/kg. Optimum treatment required total dosages of 350 to 600 mg/kg. Furthermore, high individual i.v. dosages (100 to 142 mg/kg) were poorly tolerated, producing substantial weight loss (8 to 18% of body weight), poor appearance, and slow recovery (8 to 12 days). A 1-hour infusion of dosages more than 140 mg/kg, or BID injections 6 hrs apart, did not reduce lethality. However, lower individual dosages of 40 to 50 mg/kg/injection i.v. were well tolerated and could be given daily to reach an optimum total dose with minimal toxicities. Likewise, 75 mg/kg/injection i.v. could be used every other day to reach optimal treatment. The necropsy profiles of deaths from toxic dosages were essentially identical regardless of schedule (deaths 4 to 7 days post treatment). The profiles were: paralytic ileus or gastroparesis; GI epithelial damage; and marrow toxicity. Interestingly, the key lethal events were rapidly reversible and simple to overcome with lower dosages given daily or every other day. Based on these results, the high dose, Q21 day schedule should be avoided in clinical applications. Instead, a split dose regimen is recommended (e.g., daily, every other day, or twice weekly). XK-469 was also well tolerated by the oral route, requiring 35% higher dosages p.o. to reach the same efficacy and toxicity as produced i.v.. CROSS-RESISTANCE STUDIES: XK-469 resistance was produced by optimum treatments of i.v. implanted L1210 leukemia over seven passage generations. This leukemia subline (L1210/XK469) had reduced sensitivity to VP-16 (with a 4.0 log kill in i.v. implanted L1210/XK469 compared to an 8.0 log kill against i.v. implanted L1210/0). It also had a reduction in the sensitivity to 5-FU (with a 2.0 log kill in the implanted L1210/XK469 compared to a 4.0 log kill against i.v. implanted L1210/0). Other agents were approximately as active against the resistant tumor, including: Ara-C, Gemzar, Cytoxan, BCNU, DTIC, and CisDDPT. No case of collateral sensitivity was observed; i.e., no agent was markedly more active against the resistant subline L1210/XK-469 than against the parent tumor in mice.