Juiwanna Kushner

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.

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

SummaryCryptophycins-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 × 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.

Synthesis, biological, and antitumor activity of a highly potent 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate inhibitor with proton-coupled folate transporter and folate receptor selectivity over the reduced folate carrier that inhibits β-glycinamide ribonucleotide formyltransferase

2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with a thienoyl side chain (compounds 1-3, respectively) were synthesized for comparison with compound 4, the previous lead compound of this series. Conversion of hydroxyl acetylen-thiophene carboxylic esters to thiophenyl-α-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidine compounds of type 18 and 19. Coupling with l-glutamate diethyl ester, followed by saponification, afforded 1-3. Compound 3 selectively inhibited the proliferation of cells expressing folate receptors (FRs) α or β, or the proton-coupled folate transporter (PCFT), including KB and IGROV1 human tumor cells, much more potently than 4. Compound 3 was more inhibitory than 4 toward β-glycinamide ribonucleotide formyltransferase (GARFTase). Both 3 and 4 depleted cellular ATP pools. In SCID mice with IGROV1 tumors, 3 was more efficacious than 4. Collectively, our results show potent antitumor activity for 3 in vitro and in vivo, associated with its selective membrane transport by FRs and PCFT over RFC and inhibition of GARFTase, clearly establishing the 3-atom bridge as superior to the 1-, 2-, and 4-atom bridge lengths for the activity of this series.

Synthesis and antitumor activity of a novel series of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate inhibitors of purine biosynthesis with selectivity for high affinity folate receptors and the proton-coupled folate transporter over the reduced folate carrier for cellular entry.

2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines with a thienoyl side chain and four to six carbon bridge lengths (compounds 1-3) were synthesized as substrates for folate receptors (FRs) and the proton-coupled folate transporter (PCFT). Conversion of acetylene carboxylic acids to alpha-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidines. Sonogashira coupling with (S)-2-[(5-bromo-thiophene-2-carbonyl)-amino]-pentanedioic acid diethyl ester, followed by hydrogenation and saponification, afforded 1-3. Compounds 1 and 2 potently inhibited KB and IGROV1 human tumor cells that express FR alpha, reduced folate carrier (RFC), and PCFT. The analogs were selective for FR and PCFT over RFC. Glycinamide ribonucleotide formyltransferase was the principal cellular target. In SCID mice with KB tumors, 1 was highly active against both early (3.5 log kill, 1/5 cures) and advanced (3.7 log kill, 4/5 complete remissions) stage tumors. Our results demonstrate potent in vitro and in vivo antitumor activity for 1 due to selective transport by FRs and PCFT over RFC.

Discovery of cryptophycin-1 and BCN-183577 : Examples of strategies and problems in the detection of antitumor activity in mice

Historically, many new anticancer agents were first detected in a prescreen; usually consisting of a molecular/biochemical target or a cellular cytotoxicity assay. The agent then progressed to in vivo evaluation against transplanted human or mouse tumors. If the investigator had a large drug supply and ample resources, multiple tests were possible, with variations in tumor models, tumor and drug routes, dose-decrements, dose-schedules, number of groups, etc. However, in most large programs involving several hundred in vivo tests yearly, resource limitations and drug supply limitations have usually dictated a single trial. Under such restrictive conditions, we have implemented a flexible in vivo testing protocol. With this strategy, the tumor model is dictated by in vitro cellular sensitivity; drug route by water solubility (with water soluble agents injected intravenously); dosage decrement by drug supply, dose-schedule by toxicities encountered, etc. In this flexible design, many treatment parameters can be changed during the course of treatment (e.g., dose and schedule). The discovery of two active agents are presented (Cryptophycin-1, and Thioxanthone BCN 183577). Both were discovered by the intravenous route of administration. Both would have been missed if they were tested intraperitoneally, the usual drug route used in discovery protocols. It is also likely that they would have been missed with an easy to execute fixed protocol design, even if injected IV.

Therapeutic targeting of a novel 6-substituted pyrrolo [2,3-d]pyrimidine thienoyl antifolate to human solid tumors based on selective uptake by the proton-coupled folate transporter

The proton-coupled folate transporter (PCFT) is a proton-folate symporter with an acidic pH optimum. By real-time reverse transcription-polymerase chain reaction, PCFT was expressed in the majority of 53 human tumor cell lines, with the highest levels in Caco-2 (colorectal adenocarcinoma), SKOV3 (ovarian), and HepG2 (hepatoma) cells. A novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate (compound 1) was used to establish whether PCFT can deliver cytotoxic drug under pH conditions that mimic the tumor microenvironment. Both 1 and pemetrexed (Pmx) inhibited proliferation of R1-11-PCFT4 HeLa cells engineered to express PCFT without the reduced folate carrier (RFC) and of HepG2 cells expressing both PCFT and RFC. Unlike Pmx, 1 did not inhibit proliferation of R1-11-RFC6 HeLa cells, which express RFC without PCFT. Treatment of R1-11-PCFT4 cells at pH 6.8 with 1 or Pmx inhibited colony formation with dose and time dependence. Transport of [3H]compound 1 into R1-11-PCFT4 and HepG2 cells was optimal at pH 5.5 but appreciable at pH 6.8. At pH 6.8, [3H]compound 1 was metabolized to 3H-labeled polyglutamates. Glycinamide ribonucleotide formyltransferase (GARFTase) in R1-11-PCFT4 cells was inhibited by 1 at pH 6.8, as measured by an in situ GARFTase assay, and was accompanied by substantially reduced ATP levels. Compound 1 caused S-phase accumulation and a modest level of apoptosis. An in vivo efficacy trial with severe combined immunodeficient mice implanted with subcutaneous HepG2 tumors showed that compound 1 was active. Our findings suggest exciting new therapeutic possibilities to selectively deliver novel antifolate drugs via transport by PCFT over RFC by exploiting the acidic tumor microenvironment.

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.

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).

Functional loss of the reduced folate carrier enhances the antitumor activities of novel antifolates with selective uptake by the proton-coupled folate transporter.

Uptake of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates with four or three bridge carbons [compound 1 (C1) and compound 2 (C2), respectively] into solid tumors by the proton-coupled folate transporter (PCFT) represents a novel therapeutic strategy that harnesses the acidic tumor microenvironment. Although these compounds are not substrates for the reduced folate carrier (RFC), the major facilitative folate transporter, RFC expression may alter drug efficacies by affecting cellular tetrahydrofolate (THF) cofactor pools that can compete for polyglutamylation and/or binding to intracellular enzyme targets. Human tumor cells including wild-type (WT) and R5 (RFC-null) HeLa cells express high levels of PCFT protein. C1 and C2 inhibited proliferation of R5 cells 3 to 4 times more potently than WT cells or R5 cells transfected with RFC. Transport of C1 and C2 was virtually identical between WT and R5 cells, establishing that differences in drug sensitivities between sublines were independent of PCFT transport. Steady-state intracellular [3H]THF cofactors derived from [3H]5-formyl-THF were depleted in R5 cells compared with those in WT cells, an effect exacerbated by C1 and C2. Whereas C1 and C2 polyglutamates accumulated to similar levels in WT and R5 cells, there were differences in polyglutamyl distributions in favor of the longest chain length forms. In severe combined immunodeficient mice, the antitumor efficacies of C1 and C2 were greater toward subcutaneous R5 tumors than toward WT tumors, confirming the collateral drug sensitivities observed in vitro. Thus, solid tumor-targeted antifolates with PCFT-selective cellular uptake should have enhanced activities toward tumors lacking RFC function, reflecting contraction of THF cofactor pools.

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.