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

Abstract

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 [ 3 H]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, [3 H]compound 1 was metabolized to 3 H-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.

Functional Loss of the Reduced Folate Carrier Enhances the Antitumor Activities of Novel Antifolates with Selective Uptake by the Proton-Coupled Folate Transporter

“…Because PCFT functions optimally at acidic pH values (Qiu et al, 2006;Umapathy et al, 2007;Zhao et al, 2008), transport of C1 and C2 by PCFT may lead to further enhancement of tumor selectivity owing to the acidic microenvironments of many solid tumors (Helmlinger et al, 1997;Gillies et al, 2002;Anderson and Thwaites, 2010;Webb et al, 2011). Our previous results established that C1 and C2 are potent inhibitors of tumor cell proliferation both in vitro and in vivo Kugel Desmoulin et al, 2011).…”

“…2A) represent a new class of antitumor agents that exhibit a lack of significant membrane transport by RFC Kugel Desmoulin et al, 2011). Cellular uptake of C1 and C2 by PCFT and FR␣ is efficient and offers a promising new strategy for solid tumor targeting (Anderson and Thwaites, 2010;Kugel Desmoulin et al, 2011).…”

“…2A) represent a new class of antitumor agents that exhibit a lack of significant membrane transport by RFC Kugel Desmoulin et al, 2011). Cellular uptake of C1 and C2 by PCFT and FR␣ is efficient and offers a promising new strategy for solid tumor targeting (Anderson and Thwaites, 2010;Kugel Desmoulin et al, 2011). Because PCFT functions optimally at acidic pH values (Qiu et al, 2006;Umapathy et al, 2007;Zhao et al, 2008), transport of C1 and C2 by PCFT may lead to further enhancement of tumor selectivity owing to the acidic microenvironments of many solid tumors (Helmlinger et al, 1997;Gillies et al, 2002;Anderson and Thwaites, 2010;Webb et al, 2011).…”

Intestinal Transport of Aminopterin Enantiomers in Dogs and Humans with Psoriasis Is Stereoselective: Evidence for a Mechanism Involving the Proton-Coupled Folate Transporter

“…Competition of [ 3 H]L-AMT (0.5 M) uptake was measured over 2 min at 37°C in R2/hPCFT4 cells at pH 5.5, 6.5, and 6.8 by unlabeled L-AMT (10 M), D-AMT (10 or 30 M), PMX (10 M; IC 50 ϭ 13.2 nM for R2/hPCFT4 cell growth), and compound 1 (10 M; IC 50 ϭ 43.4 nM for R2/hPCFT4 cell growth) Kugel Desmoulin et al, 2011). Levels of […”

Identification and Functional Impact of Homo-oligomers of the Human Proton-coupled Folate Transporter

“…In addition to proximal small intestine, PCFT is expressed in other normal tissues, such as liver and kidney, which do not experience low pH conditions (2). In terms of cancer, a prominent low pH transport route was identified in 29 of 32 human solid tumor cell lines (20), and abundant hPCFT transcripts were detected by real-time PCR in a large cohort (n ϭ 53) of human tumor sublines from an assortment of lineages (21). The interstitial pH of solid tumors is reportedly acidic (22,23), conditions under which hPCFT would provide an important route of cytotoxic antifolate uptake if expressed at sufficient levels.…”