Synthesis-enabled understanding of the mechanism of action of amphotericin B and the development of increased therapeutic derivatives

The polyene macrolide amphotericin B (AmB) remains a critically vital antifungal as the last line of defense against a wide range of life-threatening fungal pathogen. Despite its clinical usage for over half a century, AmB has evaded the development of clinically relevant microbial resistance. AmB has been shown to form ion channels similar to that of their protein counterparts, which has led to the proposal that AmB kills yeast cells via membrane permeabilization. The capacity for ion channel formation and cytotoxicity of AmB are thought to be dependent upon membranous sterol, but the role of sterols in this mechanism and whether membrane permeabilizaton and biological activity are even linked has remained unclear. Thus, the complete understanding of the mechanism of action of AmB would enable the development of new antifungals with an improved therapeutic index, as well as guide the pursuit of new antimicrobials that evade resistance. To elucidate the operative mechanism, we pursued a systematic functional group deletion strategy where derivatives of AmB are synthesized lacking a single protic functional group to understand its role in AmB’s activity. The C35 hydroxyl group of AmB has been proposed to be critical for ion channel formation and so we accessed the derivative lacking the C35 hydroxyl via an iterative cross-coupling (ICC) strategy. The resulting derivative maintained the capacity to bind membranous ergosterol, but could no longer cause membrane permeabilization. Despite the lack of channel activity, this derivative still demonstrated potent fungicidal activity. Deletion of the mycosamine sugar yielded a derivative that could no longer bind ergosterol and was completely inactive against yeast. Collectively, these results led us to conclude that the primary mechanism by which AmB kills yeast is the binding of the ergosterol and that channel formation is a complementary mechanism that marginally increases AmB's potency. This finding suggests that toxicity to humans is likely due to the binding of the major mammalian sterol: cholesterol. Given the importance of the mycosamine appendage on the binding of sterol, we pursued an atomistic understanding of this interaction. The axial C2' hydroxyl group of AmB has been proposed to be critical in binding both sterols. Surprisingly, derivatives lacking or epimerizing the C2' hydroxyl maintained the capacity to bind ergosterol but could no longer bind cholesterol. Consistent with sterol binding being the operative mechanism for toxicity, both derivatives exhibited potent antifungal activity but no toxicity in human cells and mice. However, synthetic access to both derivatives limited their further pursuit. We hypothesized that the sterol selectivity resulted from a ligand-selective allosteric modification and proposed that a similar effect could be achieved by derivatization of the accessible C41 carboxylate. Similar to the C2' modified analogues, the new AmB ureas also demonstrated a preferential binding for ergosterol over cholesterol. This corresponded with their potent activity against a wide range of fungal pathogens as well as their substantial decrease in toxicity to human cells and mice. Despite their decreased toxicity, the AmB ureas maintained the ability to evade resistance similar to that of the parent compound

Metastatic recurrence in a pancreatic cancer patient derived orthotopic xenograft (PDOX) nude mouse model is inhibited by neoadjuvant chemotherapy in combination with fluorescence-guided surgery with an anti-CA 19-9-conjugated fluorophore.

The aim of this study is to determine the efficacy of neoadjuvant chemotherapy (NAC) with gemcitabine (GEM) in combination with fluorescence-guided surgery (FGS) on a pancreatic cancer patient derived orthotopic xenograft (PDOX) model. A PDOX model was established from a CA19-9-positive, CEA-negative tumor from a patient who had undergone a pancreaticoduodenectomy for pancreatic adenocarcinoma. Mice were randomized to 4 groups: bright light surgery (BLS) only; BLS+NAC; FGS only; and FGS+NAC. An anti-CA19-9 or anti-CEA antibody conjugated to DyLight 650 was administered intravenously via the tail vein of mice with the pancreatic cancer PDOX 24 hours before surgery. The PDOX was brightly labeled with fluorophore-conjugated anti-CA19-9, but not with a fluorophore-conjugated anti-CEA antibody. FGS was performed using the fluorophore-conjugated anti-CA19-9 antibody. FGS had no benefit over BLS to prevent metastatic recurrence. NAC in combination with BLS did not convey an advantage over BLS to prevent metastatic recurrence. However, FGS+NAC significantly reduced the metastatic recurrence frequency to one of 8 mice, compared to FGS only after which metastasis recurred in 6 out of 8 mice, and BLS+NAC with metastatic recurrence in 7 out of 8 mice (p = 0.041). Thus NAC in combination with FGS can reduce or even eliminate metastatic recurrence of pancreatic cancer sensitive to NAC. The present study further emphasizes the power of the PDOX model which enables metastasis to occur and thereby identify the efficacy of NAC in combination with FGS on metastatic recurrence

Metastatic recurrence in a pancreatic cancer patient derived orthotopic xenograft (PDOX) nude mouse model is inhibited by neoadjuvant chemotherapy in combination with fluorescence-guided surgery with an anti-CA 19-9-conjugated fluorophore.

The aim of this study is to determine the efficacy of neoadjuvant chemotherapy (NAC) with gemcitabine (GEM) in combination with fluorescence-guided surgery (FGS) on a pancreatic cancer patient derived orthotopic xenograft (PDOX) model. A PDOX model was established from a CA19-9-positive, CEA-negative tumor from a patient who had undergone a pancreaticoduodenectomy for pancreatic adenocarcinoma. Mice were randomized to 4 groups: bright light surgery (BLS) only; BLS+NAC; FGS only; and FGS+NAC. An anti-CA19-9 or anti-CEA antibody conjugated to DyLight 650 was administered intravenously via the tail vein of mice with the pancreatic cancer PDOX 24 hours before surgery. The PDOX was brightly labeled with fluorophore-conjugated anti-CA19-9, but not with a fluorophore-conjugated anti-CEA antibody. FGS was performed using the fluorophore-conjugated anti-CA19-9 antibody. FGS had no benefit over BLS to prevent metastatic recurrence. NAC in combination with BLS did not convey an advantage over BLS to prevent metastatic recurrence. However, FGS+NAC significantly reduced the metastatic recurrence frequency to one of 8 mice, compared to FGS only after which metastasis recurred in 6 out of 8 mice, and BLS+NAC with metastatic recurrence in 7 out of 8 mice (p = 0.041). Thus NAC in combination with FGS can reduce or even eliminate metastatic recurrence of pancreatic cancer sensitive to NAC. The present study further emphasizes the power of the PDOX model which enables metastasis to occur and thereby identify the efficacy of NAC in combination with FGS on metastatic recurrence

Metastatic recurrence in a pancreatic cancer patient derived orthotopic xenograft (PDOX) nude mouse model is inhibited by neoadjuvant chemotherapy in combination with fluorescence-guided surgery with an anti-CA 19-9-conjugated fluorophore.

The aim of this study is to determine the efficacy of neoadjuvant chemotherapy (NAC) with gemcitabine (GEM) in combination with fluorescence-guided surgery (FGS) on a pancreatic cancer patient derived orthotopic xenograft (PDOX) model. A PDOX model was established from a CA19-9-positive, CEA-negative tumor from a patient who had undergone a pancreaticoduodenectomy for pancreatic adenocarcinoma. Mice were randomized to 4 groups: bright light surgery (BLS) only; BLS+NAC; FGS only; and FGS+NAC. An anti-CA19-9 or anti-CEA antibody conjugated to DyLight 650 was administered intravenously via the tail vein of mice with the pancreatic cancer PDOX 24 hours before surgery. The PDOX was brightly labeled with fluorophore-conjugated anti-CA19-9, but not with a fluorophore-conjugated anti-CEA antibody. FGS was performed using the fluorophore-conjugated anti-CA19-9 antibody. FGS had no benefit over BLS to prevent metastatic recurrence. NAC in combination with BLS did not convey an advantage over BLS to prevent metastatic recurrence. However, FGS+NAC significantly reduced the metastatic recurrence frequency to one of 8 mice, compared to FGS only after which metastasis recurred in 6 out of 8 mice, and BLS+NAC with metastatic recurrence in 7 out of 8 mice (p = 0.041). Thus NAC in combination with FGS can reduce or even eliminate metastatic recurrence of pancreatic cancer sensitive to NAC. The present study further emphasizes the power of the PDOX model which enables metastasis to occur and thereby identify the efficacy of NAC in combination with FGS on metastatic recurrence

Recurrence rate of PDOX in each treatment group.

<p>* p = 0.041, compared to FGS only.</p><p>Recurrence rate of PDOX in each treatment group.</p

Representative gross and histological images of excised tumors in each treatment group.

<p>Left panels of (A) and (B) indicate bright field (BF) images and right panels indicate fluorescence images for DyLight 650 (650). Histopathological response to GEM treatment was defined according to Evans’s grading scheme. The tumors without GEM treatment (FGS only) were comprised of viable cancer cells that formed glandular structures and judged as Grade I (C). In the tumors with GEM treatment, over 50% of cancer cells were dead and replaced by necrotic tissue or stromal cells (D). Treatment efficacy of GEM on the pancreatic cancer PDOX was judged as grade IIb - III (D). Fluorescence decreased in some areas of the tumor treated with GEM, but was sufficient for FGS (B). Scale bars: 5 mm (A and B), 250 µm (C and D).</p

Antibody labelling of the pancreatic cancer patient derived orthotopic xenograft.

<p>The patient’s pancreatic cancer was diagnosed as moderately differentiated adenocarcinoma with H&E staining (A). The tumor was strongly stained with anti-CA19-9 antibody (B), whereas the signal was very weak with anti-CEA antibody (C). Scale bars: 100 µm. (D and E) Whole body images of a subcutaneous tumor in nude mice labeled with anti-CA19-9- or anti-CEA-conjugated DyLight 650. Fifty µg anti-CA19-9 DyLight 650 or anti-CEA DyLight 650 was injected in the tail vain of the mice with subcutaneous tumors. Twenty-four hours later, whole body images were taken with the OV100 (Olympus). Yellow arrowheads indicate subcutaneous tumors. The subcutaneous tumors were brightly labeled with anti-CA19-9 DyLight 650 (D), whereas, the fluorescence signal from the tumor labeled with anti-CEA DyLight 650 was very weak (E). Scale bars: 10 mm.</p

Recurrent tumor weights for each experimental group.

<p>(A) Recurrent tumor weight in the BLS-only; BLS+NAC; FGS-only; and FGS+NAC treatment groups. The average local recurrent tumor weight for FGS-only treatment was significantly less than for BLS-only treatment (p = 0.0041). The average local recurrent tumor weight for FGS+NAC treatment was also significantly less than for BLS+NAC treatment (p = 0.008). The average metastatic recurrent tumor weight for FGS+NAC treatment was significantly less than for BLS+NAC treatment (p = 0.001). FGS+NAC treatment reduced recurrence rate significantly compared to FGS-only treatment (p = 0.041) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114310#pone-0114310-t001" target="_blank">Table 1</a>). FGS+NAC treatment tended to reduce the metastatic recurrent tumor weight compared to FGS-only treatment (p = 0.059).</p

Experimental schema and FGS imaging system.

<p>(A) Schema of the experimental design. After confirmation of tumor growth, the PDOXs were randomized to 4 groups: BLS only; BLS+NAC; FGS only; or FGS+NAC. Each treatment arm involved 8 tumor-bearing mice. The mice randomized to the NAC groups were treated with GEM (80 mg/kg) on day 8, 15 and 22. All animals underwent surgery on day 29. BLS was performed under standard bright-field using the MVX10 microscope. Fifty µg of anti-CA19-9 antibody conjugated with DyLight 650 was injected in the tail vain of mice with tumors in the FGS group 24 hours before surgery. FGS was performed using the MINI MAGLITE LED PRO flash light (MAG INSTRUMENT, Ontario, CA, USA) with excitation filter ET640/30X (Chroma Technology Corporation, Bellows Falls, VT, USA) and a Canon EOS 60D digital camera with an EF–S18–55 IS lens (Canon, Tokyo, Japan) and emission filter HQ700/75M-HCAR (Chroma Technology Corporation) under fluorescence navigation (B). Twelve weeks after surgery, animals underwent laparotomy, and the tumors were imaged, weighed and harvested for analysis. Scale bars: 2 cm (filters) and 5 cm (flash light).</p

Representative images during FGS with or without NAC.

<p>Upper panels indicate bright field (BF) images and lower panels indicate fluorescence images for DyLight 650 (650). The fluorescence in the tumors treated with GEM (B) decreased compared to untreated tumors (A), but were still clearly detected. Scale bars: 10 mm.</p