Jerome P. Horwitz

Preclinical antitumor efficacy of analogs of XK469: sodium-(2-[4-(7-chloro-2-quinoxalinyloxy)phenoxy]propionate

A series of quinoxaline analogs of the herbicide Assure® was found to have selective cytotoxicity for solid tumors of mice in a disk-diffusion-soft-agar-colony-formation-assay compared to L1210 leukemia. Four agents without selective cytotoxicity and 14 agents with selective cytotoxicity were evaluated in vivo for activity against a solid tumor. The four agents without selective cytotoxicity in the disk-assay were inactive in vivo (T/C > 42%). Thirteen of the fourteen agents with selectivity in the disk-assay were active in vivo (T/C < 42%). Five of the agents had curative activity. These five agents had a halogen (F, Cl, Br) in the 7-position (whereas Assure® had a Cl in the 6 position). All agents with curative activity were either a carboxylic acid, or a derivative thereof, whereas Assure® is the ethyl ester of the carboxylic acid. All other structural features were identical between Assure® and the curative agents. Assure® had no selective cytotoxicity for solid tumors in the disk-assay, and was devoid of antitumor activity. The analog XK469 is in clinical development.

Preclinical efficacy of thioxanthone SR271425 against transplanted solid tumors of mouse and human origin

A highly active and broadly active thioxanthone has been identified: N-[[1-[[2-(Diethylamino)ethyl]amino]-7-methoxy-9-oxo-9H-thioxanthen-4-yl] methylformamide (SR271425, BCN326862, WIN71425). In preclinical testing against a variety of subcutaneously growing solid tumors, the following %T/C and Log10 tumor cell kill (LK) values were obtained: Panc-03 T/C = 0, 5/5 cures; Colon-38 (adv. stage) T/C = 0, 3/5 cures, 4.9 LK; Mam-16/C T/C = 0, 3.5 LK; Mam-17/0 T/C = 0, 2.8 LK; Colon-26 T/C = 0, 1/5 cures, 3.2 LK; Colon-51 T/C= 0, 2.7 LK; Panc-02 T/C = 0, 3.1 LK; B16 Melanoma T/C = 13%, 4.0 LK; Squamous Lung-LC12 (adv. stage) T/C = 14%, 4.9 LK; BG-1 human ovarian T/C = 16%, 1.3 LK; WSU-Br1 human breast T/C = 25%, 0.8 LK. The agent was modestly active against doxorubicin (Adr)-resistant solid tumors: Mam-17/Adr T/C =23%, 0.8 LK; and Mam-16/C/Adr T/C = 25%, 1.0 LK, but retained substantial activity against a taxol-resistant tumor: Mam-16/C/taxol T/C = 3%, 2.4 LK. SR271425 was highly active against IV implanted leukemias, L1210 6.3 LK and AML1498 5.3 LK. The agent was equally active both by the IV and oral routes of administration, although requiring approximately 30% higher dose by the oral route. Based on its preclinical antitumor profile, it may be appropriate to evaluate SR271425 in clinical trials.

The role of autophagy in the death of L1210 leukemia cells initiated by the new antitumor agents, XK469 and SH80

The phenoxypropionic acid derivative 2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid (XK469) and an analogue termed 2-{4-[(7-bromo-2-quinalinyl)oxy]phenoxy}propionic acid (SH80) can eradicate malignant cell types resistant to many common antitumor agents. Colony formation assays indicated that a 24 h exposure of L1210 cells to XK469 or SH80 inhibited clonogenic growth with CI90 values of 10 and 13 μmol/L, respectively. This effect was associated with G2-M arrest and the absence of any detectable markers of apoptosis (i.e., plasma membrane blebbing, procaspase 3 activation, loss of mitochondrial membrane potential, and formation of condensed chromatin). Drug-treated cells increased in size and eventually exhibited the characteristics of autophagy (i.e., appearance of autophagosomes and conversion of microtubule-associated protein light chain 3-I to 3-II). The absence of apoptosis was not related to an inhibition of the apoptotic program. Cultures treated with XK469 or SH80 readily underwent apoptosis upon exposure to the Bcl-2/Bcl-xL antagonist ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate. Continued incubation of drug-treated cells led to a reciprocal loss of large autophagic cells and the appearance of smaller cells that could not be stained with Höechst dye HO33342, had a chaotic morphology, were trypan blue–permeable, and lacked mitochondrial membrane potential. L1210 cells cotreated with the phosphatidylinositol-3-kinase inhibitor wortmannin, or having reduced Atg7 protein content, underwent G2-M arrest, but not autophagy, following XK469 treatment. Hence, the therapeutic actions of XK469/SH80 with L1210 cultures reflect both the initiation of a cell cycle arrest as well as the initiation of autophagy. [Mol Cancer Ther 2007;6(1):370–9]

Unsaturated sugars I. Decarboxylative elimination of methyl 2,3-di-O-benzyl-α-D-glucopyranosiduronic acid to methyl 2,3-di-O-benzyl-4-deoxy-β-L-threo-Pent-4-enopyranoside☆

Abstract Decarboxylative elimination of methyl 2,3-di- O -benzyl-α- D -glucopyranosiduronic acid ( 1 ) with N,N -dimethylformamide dineopentyl acetal in N,N -dimethylformamide gave methyl 2,3-di- O -benzyl-4-deoxy-β- L - threo -pent-4-enopyranoside ( 3 ). Debenzylation of 3 was effected with sodium in liquid ammonia to give methyl 4-deoxy-β- L - threo -pent-4-enopyranoside ( 4 ). Hydrogenation of 3 catalyzed by palladium-on-barium sulfate afforded methyl 2,3-di- O -benzyl-4-deoxy-β- L - threo -pentopyranoside ( 5 ), whereas hydrogenation of 3 over palladium-on-carbon gave methyl 4-deoxy-β- L - threo -pentopyranoside ( 6 ). An improved preparation of methyl 4,6- O -benzylidene-α- D -glucopyranoside is also described.

Transplantable Syngeneic Rodent Tumors: Solid Tumors in Mice

As preclinical chemotherapists, we are often asked to identify experimental tumor models that can accurately predict for the drug response characteristics of all tumors of a given cellular subtype or molecular target. Unfortunately, it is impossible to give satisfactory answers to these inquiries. Because of the unique character of each independently arising tumor (whether spontaneous or induced), it does not take very long to realize that each tumor is a unique biologic entity with its own tumor growth behavior, histological appearance, drug response and molecular expression profiles. This is true whether the tumor is an experimental animal model or one originally derived from a patient. Further, many factors can influence the tumor growth and therapy response of experimental tumor models. Still, in vivo models are needed to adequately assess pharmacodynamics, toxicity and efficacy of any potential novel therapy. Presented herein is what we hope will be useful information regarding the transplant characteristics of tumor models, with some of the “pitfalls” to look out for when using any given tumor model for chemotherapy evaluations. Although most of the examples given use syngeneic models, the methodologies for assessing the predictive worth and maintaining model usefulness can be applied to almost any given transplantable tumor system (whether syngeneic or xenograft).

Transplantable Syngeneic Rodent Tumors

For many decades, the results from transplantable tumor models have been viewed with considerable skepticism. The perception has long been that these models are excessively sensitive, and not predictive of the human disease. Although we do not intend to debate the many issues involved, it is our view that a better understanding of the transplant properties (e.g., take-rate) of the models—as well as a better understanding of the potential shortcomings in data presentation—will greatly aid the reader in the interpretation of these data. This chapter is an effort to summarize some of the basic operating characteristics of a wide range of solid-tumor models. Since this is a chemotherapy group, we can best explain some of the behavioral characteristics of these models within therapeutic experiments. Most of the data is drawn from the use of transplantable, syngeneic mouse tumors, but a few human tumors have been used for contrast.

Skeletal conformations and receptor binding of some 9,11-modified estradiols

The effect of the modification of the 9-11 positions on the skeletal conformation of estradiol (E2) has been analyzed by X-ray crystallography and MM2 molecular mechanics. The 11 beta-hydroxyl and 11-keto analogs of E2 maintained ring conformations which were similar to the natural hormone (E2). Introduction of a double bond at position 9-11 induced a flattening of the entire steroid molecule. An 11 alpha-hydroxyl group brought about significant changes in the alicyclic rings of E2. 9 beta-Estradiol and 11-keto-9 beta-estradiol formed ring conformations which were significantly bent from E2 (below the plane of the A-ring). Examination of the affinity of these C-ring analogs of E2 for the human estrogen receptor has shown extreme variations. A hydroxyl group placed either alpha or beta at the 11-position yielded ligands with vastly different and reduced affinities for the receptor. The low affinity of 11 alpha-hydroxyestradiol (1/300th of E2) may be due to the drastic structural change induced in the alicyclic portion of the molecule, as well as, to the steric or electrostatic effects of the alpha-hydroxyl group upon the receptor protein. An 11 beta-hydroxyl group diminished the receptor binding to 1/60th that of E2 without alicyclic ring distortions, whereas a 9-11 unsaturation reduced the binding to 1/5th although this steroid displayed a flattening of rings B, C, and D. The 11-keto function, which had little effect on the conformation of the estrogen nucleus, reduced the affinity of this ligand to 1/1000th that of E2. The negative bend at the C-ring of 11-keto-9 beta-estradiol and 9 beta-estradiol prevented these ligands from binding receptor. Some of the observed receptor interactions were related to structural alterations in the estrogen ring system induced by modifications on the 9-11 region.

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

XK-469 is advancing to Phase I clinicaltrials. Preclinical studies were carriedout to assist in clinical applications.Dose-schedule route testing: Singledose IV treatment with XK-469 producedlethality (LD20 to LD 100) above 142 mg/kg.Optimum treatment required total dosages of350 to 600 mg/kg. Furthermore, highindividual IV dosages (100 to 142 mg/kg)were poorly tolerated, producingsubstantial weight loss (8 to 18% of bodyweight), poor appearance, and slow recovery(8 to 12 days). A 1-hour infusion ofdosages more than 140 mg/kg, or BIDinjections 6 hrs apart, did not reducelethality. However, lower individualdosages of 40 to 50 mg/kg/injection IV werewell tolerated and could be given daily toreach an optimum total dose with minimaltoxicities. Likewise, 75 mg/kg/injection IVcould be used every other day to reachoptimal treatment. The necropsy profiles ofdeaths from toxic dosages were essentiallyidentical regardless of schedule (deaths 4to 7 days post treatment). The profileswere: paralytic ileus or gastroparesis; GIepithelial damage; and marrow toxicity.Interestingly, the key lethal events wererapidly reversible and simple to overcomewith lower dosages given daily or everyother day. Based on these results, the highdose, Q21day schedule should be avoided inclinical applications. Instead, a splitdose regimen is recommended (e.g., daily,every other day, or twice weekly). XK-469was also well tolerated by the oral route,requiring 35% higher dosages PO to reachthe same efficacy and toxicity as producedIV. Cross-resistance studies: XK-469resistance was produced by optimumtreatments of IV implanted L1210 leukemiaover seven passage generations. Thisleukemia subline (L1210/XK469) had reducedsensitivity to VP-16 (with a 4.0 log killin IV implanted L1210/XK469 compared to an8.0 log kill against IV implanted L1210/0). It also had a reduction in the sensitivityto 5-FU (with a 2.0 log kill in theimplanted L1210/XK469 compared to a 4.0 logkill against IV implanted L1210/0). Otheragents were approximately as active againstthe resistant tumor, including: Ara-C,Gemzar, Cytoxan, BCNU, DTIC, and CisDDPT. No case of collateral sensitivity wasobserved; i.e., no agent was markedly moreactive against the resistant sublineL1210/XK-469 than against the parent tumorin mice.

The conjugation of estrogen metabolites by liver slices

Abstract 1. 1. The conjugation of the metabolites of [16- 14 C]estrone in rat liver has been studied utilizing gas-liquid chromatography for the characterization of micro-quantities of estrogen metabolites. 2. 2. The sulfate (13.9%), glucosiduronate (10.3%), and phosphate (4.1%), estrogen conjugates and the unconjugated estrogen metabolites (47.4%) have been isolated from liver slices incubated under air. Incubation under an O 2 atmosphere doubles the sulfate fraction at the expense of the unconjugated estrogens. 3. 3. The phenolic estrogen metabolites in each of these fractions have been analyzed by gas-liquid chromatography. The metabolites of [16- 14 C]estrone (23.6% recovered) that have been characterized are 17β-estradiol (2.2%), estriol (0.97%), 2-methoxyestrone (6.2%), 2-methoxy-17β-estradiol (0.36%). In addition, the presence of three unidentified phenolic metabolites and an acidic component(s) has been 4. 4. Certain of the estrogen metabolites were found in only one conjugated form. For example, the sulfate fraction is the only source of 2-methoxy-17β-estradiol in addition to two unidentified phenolic estrogens. Estrone, on the other hand, is not conjugated in the form of a sulfate. Nearly the entire phosphate fraction consists of an unidentified carboxylic acid metabolite.

Estrogen-bridged purines: a new series of anti-tumor agents which alter cell membrane properties.

Abstract An estrogen-bridged adenine derivative was equitoxic to both the P388 murine leukemia and an adriamycin-resistant subline, P388 ADR . The drug rapidly altered several P388 and P388 ADR membrane properties resulting in impaired nucleoside transport and increased membrane hydrophobicity. Resistance to anthracyclines in P388 ADR is associated with an operational barrier to drug retention which was reversed by exposure to the estrogen-bridged adenine derivative. These results suggest further exploration of the estrogen-bridged purines as chemotherapeutic agents.