Kathryn White

In Vivo Methods for Screening and Preclinical Testing

The classic question in the field of drug discovery is: Which tumor model is a satis-factory predictor for cancer in humans? The classic answer is: None of them!

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.

Treatment of human prostate tumors PC-3 and TSU-PR1 with standard and investigational agents in SCID mice

Both the PC-3 and the TSU-PR1 prostate tumor models were found to be satisfactory for chemotherapeutic investigations in ICR-SCID mice. The 30 to 60 mg fragments implanted took in all mice (as judged by 100% takes in the controls of all experiments as well as the passage mice). The tumor volume doubling time was 4.0 days for PC-3 and 2.5 days for TSU-Pr1. Nine agents were evaluated IV against early stage subcutaneous PC-3 tumors, with Nano-piposulfan being the only agent highly active (4.9 log kill). Three other agents were moderately active: Taxol (1.5 log kill), Cryptophycin-8 (1.6 log kill), Vinblastine (1.0 log kill). Five agents were inactive: VP-16, Adriamycin, CisDDPt, 5-FUra, and Cyclophosphamide. Ten agents were evaluated IV against early stage subcutaneous TSU-Pr1 tumors. Three agent were highly active, producing > 6 log kill and cures: Taxol (5/5 cures), Cryptophycin-8 (5/5 cures), Vinblastine (2/4 cures). Two other agents were moderately active: Nano-piposulfan (1.2 log kill), and Cyclophosphamide (1.1 log kill). Five agents were inactive: VP-16, Adriamycin, CisDDPt, 5-FUra, and BCNU. In part, activity was determined by the ability of the SCID mice to tolerate meaningful dosages of the agents. Agents producing granulocyte toxicity (e.g., Adriamycin) were poorly tolerated and appeared less active than expected. Vinblastine, producing little or no granulocyte toxicity was very well tolerated and appeared to be more active than expected.

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.

Discovery of Solid Tumor Active Agents Using a Soft-Agar-Colony-Formation Disk-Diffusion-Assay

The history of antitumor drug discovery has essentially been the use of two lymphocytic leukemias of mice as selection funnels through which all agents needed to pass in order to advance toward clinical development (L1210 prior to 1975 and P388 after 1975). It is thus not surprising that agents in the clinic are highly active against these tumor systems. However, none of the agents discovered by these leukemias are tumor specific (i.e., active against all tumors), and none of the agents are broadly active against solid tumors of either rodents or humans (1, 2, 3). An example contrasting the responsiveness of transplantable solid tumors of mice and the two leukemias is shown in Table-1. The lack of responsiveness of these solid tumors of mice is not unlike those seen in human lung, pancreatic, colon, and prostate tumors. The point to emphasize is that the lack of solid tumor activity of available antitumor agents is not species related. The fault does not lie with the omission of human tumors in the initial selection process, but rather with the omission of solid tumors.

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.

Tumor Models and the Discovery and Secondary Evaluation of Solid Tumor Active Agents

AbstractEach independently arising tumor is a separate and unique biologic entity with its own unique histologic appearance, biologic behavior, and drug response profile. Thus, in drug discovery, no single tumor has been a perfect predictor for any other tumor. For this reason, new agents are evaluated in a variety of tumor models which is known as breadth of activity testing. In recent years, human tumors implanted in athymic nude mice and SCID mice have also become available for breadth of activity testing. In studies carried out in these laboratories, it was found that 10 human tumors metastasized in the SCID mice, but failed to metastasize in nude mice. In addition, tumor growth and tumor takes were superior in the SCID mice. The strengths and weaknesses of xenograft model systems are discussed. For example, most human tumor xenograft models are excessively sensitive to alkylating agents as well as to a new class of DNA binders (XE840 and XP315). Using human tumor models that are the least sensitive t...