Cyclopamine: From Cyclops Lambs to Cancer ... - ACS Publications

Apr 10, 2014 - In the late 1960s, the steroidal alkaloid cyclopamine was isolated from the plant Veratrum californicum and identified as the teratogen...
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Cyclopamine: From Cyclops Lambs to Cancer Treatment Stephen T. Lee,*,† Kevin D. Welch,† Kip E. Panter,† Dale R. Gardner,† Massoud Garrossian,‡ and Cheng-Wei Tom Chang§ †

Poisonous Plant Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 1150 East 1400 North, Logan, Utah 84341, United States ‡ Logan Natural Products, Inc., Logan, Utah 84341, United States § Department of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan, Utah 84322-0300, United States ABSTRACT: In the late 1960s, the steroidal alkaloid cyclopamine was isolated from the plant Veratrum californicum and identified as the teratogen responsible for craniofacial birth defects including cyclops in the offspring of sheep grazing on mountain ranges in the western United States. Cyclopamine was found to inhibit the hedgehog (Hh) signaling pathway, which plays a critical role in embryonic development. More recently, aberrant Hh signaling has been implicated in several types of cancer. Thus, inhibitors of the Hh signaling pathway, including cyclopamine derivatives, have been targeted as potential treatments for certain cancers and other diseases associated with the Hh signaling pathway. A brief history of cyclopamine and cyclopamine derivatives investigated for the treatment of cancer is presented. KEYWORDS: cyclopamine, Veratrum californicum, teratogen, cancer, Hh signaling pathway

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2A,B).3,4 Pregnant ewes that consumed V. californicum were also observed to have reduced birth rates due to early embryonic loss,5,7 and if the plant was consumed on days 28−31 and 31−35 of gestation, limb, foregut, and tracheal malformations, respectively, occurred.8,9 In 1968 the toxin responsible for causing the cyclopic type defects was isolated from V. californicum and named cyclopamine.10,11 The structure of cyclopamine, 1, was elucidated in 1969 and was found to be identical to 11-deoxojervine isolated from Veratrum grandiflorum four years earlier in Japan (Figure 3).11,12 Management strategies and recommendations developed by scientists at the PPRL and implemented by sheep producers, such as restricting the access of pregnant ewes to V. californicum during critical periods of gestation, have essentially eliminated the craniofacial syndrome as a poisonous plant problem in the western United States. However, sporadic cases of Veratrum-induced malformations in sheep and other species such as llamas and alpacas continue to be reported to the PPRL.

DISCOVERY OF CYCLOPAMINE: A POTENT TERATOGEN In the half century from the 1900s−1950s, ewes that grazed on the alpine ranges in southwestern and south-central Idaho during the summer gave birth to lambs with a variety of craniofacial deformities including cyclopia at an incidence of up to 25% in some individual flocks.1,2 Initially, sheep producers were reluctant to disclose the extent to which their sheep suffered from this disease, as many thought it may be genetic.1 In 1954 sheep producers in Idaho contacted scientists at the Poisonous Plant Research Laboratory in Logan, UT, USA (PPRL), and requested assistance in researching the cause of the craniofacial deformities in lambs born in central Idaho.3 Studies to determine the cause of the disease including geneticbased trials and the investigation of water, soil, and numerous plants for potentially teratogenic agents were performed without success. Although Veratrum californicum (false hellebore, skunk cabbage, corn lily) was not observed to be toxic to sheep by livestock owners and by the first scientists involved in the study of cyclopia in lambs, in the summer of 1958, a sheepherder told a summer employee hired by the PPRL that sheep become sick when they grazed V. californicum.3 This critical piece of information was shared with scientists at the PPRL, and feeding trials with V. californicum were initiated. Subsequent studies by scientists at the PPRL demonstrated that animals that consume sufficient amounts of V. californicum show clinical signs of poisoning including excessive salivation, frothing around the mouth, irregular gait, vomiting, weakness, irregular heartbeat, dyspnea, convulsions, coma, and death.4−6 In 1959 the first cyclopic lamb was born under controlled feeding conditions at the PPRL (Figure 1). In 1965 it was reported that the pregnant ewe must graze V. californicum on the 14th day of gestation to induce the cyclopean type birth defect in the lamb, often with a proboscis-like nasal structure above the single eye (Figure © 2014 American Chemical Society

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HEDGEHOG GENES: FRUIT FLIES TO HUMANS In 1995 Wieschaus and Nusslein-Volhard received the Nobel Prize in Medicine for work performed in 1978 and published in 1980. Working together, they systematically identified genes in Drosophila melanogaster (fruit flies) responsible for determining the body plan and segmentation of the fly during embryonic development.13 The inactivation of one of these genes caused the Drosophila embryo (larvae) to be covered with spiny Special Issue: Poisonous Plant Symposium, Inner Mongolia Received: Revised: Accepted: Published: 7355

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Figure 1. (A) Veratrum californicum; (B) the first cyclopic lamb born under controlled feeding conditions.

Figure 2. (A, B) Cyclopic lambs from lambs fed Veratrum californicum on day 14 of gestation. (C, D) Full brothers from a family with known mutations in the Shh gene. Images C and D reprinted with permission from ref 20. Copyright 1999 Oxford University Press.

expressed and has been the most investigated of the mammalian Hh signaling genes. In mammals, Shh is responsible for embryonic development of symmetry along the sagittal plane, the central nervous system, skeleton, limbs, eyes, teeth, intestines, lungs, skin, muscles, cartilage, and sperm. Mouse embryos that lacked a Shh gene resulted in early embryonic death, holoprosencephaly (failure of the forebrain to develop into hemispheres), cyclopia at the base of an external proboscis, limb mispatterning, and foregut abnormalities, among numer-

dendricles resembling a hedgehog, resulting in its being named the hedgehog (Hh) gene. In 1993 three mammalian Hh counterparts, Desert hedgehog (Dhh), Indian hedgehog (Ihh), and Sonic hedgehog (Shh), were identified in a mouse14 and named after two hedgehog species and the video game character, Sonic the Hedgehog. In 1995 human Ihh and Shh genes were identified and their chromosomal locations determined.15 In 1999 the human Dhh gene was identified and its location determined.16,17 Shh is the most broadly 7356

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Figure 3. Chemical structures of cyclopamine (1), 3-keto-N-(aminoethyl-aminocaproyl-dihydrocinnamoyl)cyclopamine (2) (KAAD-cyclopamine (2)), veratramine (3), N-(4-L-rhamnopyranosyl-1H-1,2,3-triazol-1-yl)-methylcyclopamine (4), Mu-SSKLQ-cyclopamine (5), IPI-926 (6), and GDC0449 (7).

ous other malformations.18,19 Loss of Shh functioning in embryonic human development has also resulted in holoprosencephaly, cyclopia, and proboscis above the eye (Figure 2C).20 In less severe human cases, flat nose absent of nasal cartilage, decreased distance between the eyes, and underdevelopment of the midface have been observed (Figure 2D).20

cyclopamine, 1, could be used to probe the Shh pathway. In 1998 Beachy and co-workers21 and Incardona et al.22 established that cyclopamine inhibits the Shh signaling pathway. A simplified characterization of the Shh signaling pathway is presented in Figure 4. In most adult cells the Shh receptor Patched interacts with Smoothened, in effect acting as a molecular brake and inhibiting Smoothened (Figure 4A). During embryonic development, when Shh is present, Shh binds to Patched and Smoothened is then released from Patched. Smoothened then initiates downstream signaling that results in the binding of Gli transcription factors to DNA and subsequent activation of gene expression (Figure 4B). Cyclop-

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CYCLOPAMINE AND THE SONIC HEDGEHOG GENE In 1996 while actively conducting research on the Shh gene pathway, Dr. Philip Beachy recalled images of the cyclopic lambs reported in Idaho in 1950−1960s and hypothesized that 7357

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Figure 4. Shh signaling pathway. (A) Patched interacts and inactivates Smoothened. (B) When Shh is present, Shh binds to Patched, Smoothened is released from Patched, and Smoothened leads to downstream signaling via binding of the Gli transcription factors to DNA and activation of gene expression. (C) Cyclopamine binds to Smoothened and inhibits the Shh pathway downstream from Patched.

xenograft models with human glioma,31 melanoma,32 colon cancer,33 pancreatic cancer,34−36 prostate cancer,37 and small cell lung cancer38 without noticeable side effects in the cyclopamine-treated adult mice.29−32,34,36 Tumor regression in human patients with basal cell carcinomas was reported after administration of a topical cream containing cyclopamine was applied directly onto the tumors, with no adverse effects to the patient, including the skin surrounding the treated tumors.39 These results highlight the potential of using cyclopamine and other inhibitors of the Shh signaling pathway as therapies for human cancers promoted by aberrant Shh signaling and argue for initiation of human clinical trials using inhibitors of the Shh signaling pathway for lethal cancers. Although cyclopamine, 1, shows promise as a human chemotherapy agent, cyclopamine per se does have some inherent limitations. Cyclopamine has low solubility in water, normal saline, and other physiological solutions. In addition, cyclopamine is not stable under acidic conditions, as would be encountered in the stomach of humans and other monogastric animals. Under acidic conditions, the ether bond in ring E is readily hydrolyzed followed by aromatization of ring D, resulting in veratramine, 3,40,41 which is inactive in the Shh signaling pathway. These characteristics limit the bioavailability and delivery options of cyclopamine. Previous success in increasing the potency of the Shh pathway inhibition with KAAD-cyclopamine, 2, suggests that other semisynthetic cyclopamine, 1, compounds could be developed, which would overcome the pharmacokinetic limitations of cyclopamine, 1, and may even have increased potency as anticancer therapies. In this regard, several different approaches have been undertaken to address the limitations of cyclopamine as a chemotherapeutic agent. One simple solution to increase the solubility of cyclopamine, 1, in physiological solutions has been achieved by the creation of a cyclopamine-tartrate salt. The solubility of cyclopamine was increased to approximately 2 mg/mL as cyclopamine-tartrate salt and up to 5 mg/mL with the addition of 1.5 mg of tartaric acid to the solution, as reported by the inventors.42 Cyclopamine-tartrate salt was used to characterize the pharmacokinetic profile of cyclopamine in sheep43 and

amine binds to Smoothened and inhibits the Shh pathway downstream from Patched and upstream from Gli (Figure 4C).23,24 Modifications of cyclopamine to improve solubility as well as other pharmacokinetic characteristics have resulted in compounds with enhanced Shh signaling pathway inhibitor activity. For example, 3-keto-N-(aminoethyl-aminocaproyldihydrocinnamoyl)cyclopamine, 2 (KAAD-cyclopamine), a semisynthetic derivative of cyclopamine, was shown to have a 10−20-fold increase in potency compared to cyclopamine.23

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CYCLOPAMINE DERIVATIVES AND CANCER TREATMENT In addition to playing a key role in embryonic development, inappropriate activation of the Shh signaling pathway in mature cells has been associated with nevoid basal cell carcinoma (Gorlins syndrome) and several cancers, such as basal cell carcinoma, medullablastoma, and rhabdomyosarcoma, that are often driven by inherited mutations in Patched and, less frequently, Smoothened.24,25 Nevoid basal cell carcinoma is an inherited condition characterized by multiple basal cell carcinomas, other malignant and benign tumors, and malformations.26 Basal cell carcinoma is the most common type of skin cancer, medulloablastoma is the most common brain tumor in children, and rhabdomyosarcoma is a relatively rare cancer that is most commonly observed in children.25,27 Other cancers such as colon, glioma, breast, esophageal, gastric, pancreatic, prostate, small cell lung, biliary tract, bladder, ovarian, liver, myeloma, leukemia, and oral cancers have been associated with autocrine- and paracrine-secreted hedgehog protein signaling.27,28 The Shh signaling pathway plays a critical role in embryonic development and appears to play an important role in a number of cancers in mouse models, because it no longer has a normal biological function in most mature cells. Consequently, inhibition of the Shh signaling pathway may be a promising cancer treatment method as it could stop tumor growth without many of the common side effects of traditional cancer therapeutics. Cyclopamine, 1, has been shown to inhibit tumor growth in a genetic mouse medulloblastoma model,29 a mouse medulloblastoma allograph model,30 and mouse 7358

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Figure 5. Agricultural, cell biology, and cancer research time line.

profile, and potency. Enlargement of ring D from a six- to a seven-member ring dramatically improved the acid stability and aqueous solubility compared to cyclopamine.47 The addition of the sulfonamide at the C-3 position further increased the metabolic stability of IPI-926 compared to cyclopamine.47 IPI926 was shown to have a longer pharmacokinetic half-life in multiple mammalian species and was more potent in a cellular Hh inhibition assay than other semisynthetic lead compounds.47 Daily oral administration of IPI-926 in a mouse medulloblastoma allograph model led to complete tumor regression during the treatment phase, and tumor regrowth was not observed 21 days post treatment.47 IPI-926 was also effective in inhibiting tumor growth in lung and pancreatic cancer xenograft models.47 A phase I clinical study of IPI-926, 6, was commenced in 2010 in adult patients with solid tumors. The objectives of this first in-human study were to determine maximum tolerated oral dose and to characterize the pharmacokinetic profile of IPI-926. In addition, an expanded portion of the study evaluated the efficacy of IPI-926 at the maximum tolerated dose in patients with advanced or metastatic basal cell carcinomas. A welltolerated dose was determined, and IPI-926 showed antitumor activity in patients with basal cell carcinomas.48 IPI-926, 6, was further evaluated in a phase 1b/2 clinical study in patients with previously untreated metastatic pancreatic cancer in 2011. In the phase 1b portion of the study, patients were orally treated with IPI-926 in combination with intravenously administered gemcitabine. This trial evaluated the safety and pharmacokinetics and determined the recommended dose of IPI-926 for phase 2 clinical trials. In this study, a partial response to the therapy was observed in 31% of the patients. The combination of IPI-926 and gemcitabine was well tolerated, and no pharmacokinetic interactions were observed between the two drugs.49 The phase 2 portion of the study compared IPI-926 in combination with gemcitabine to placebo with gemcitabine in patients with previously untreated metastatic pancreatic cancer with the

induced tumor shrinkage in two mouse basal cell carcinoma models.44 Chemists at Utah State University in collaboration with scientists at the PPRL investigated increasing the solubility of cyclopamine, 1, while retaining or improving its potency by using “click” chemistry to form a series of 11 carbohydrate− cyclopamine conjugates. Most of the conjugates were less potent than cyclopamine; however, the N-(4-L-rhamnopyranosyl-1H-1,2,3-triazol-1-yl)-methylcyclopamine, 4, conjugate showed higher potency than cyclopamine in a cytotoxicity assay with lung cancer cells.45 Although the Shh signaling pathway is inactive in most mature cells, there are select mature cells in which Shh signaling is required, such as the gonads, gastrointestinal tract, and bone marrow. To circumvent any possibility of toxicity in these cells, two cyclopamine, 1, based pro-drugs were developed to target only prostate cancer cells. High levels of enzymatically active prostate specific antigen (PSA) exist in the immediate vicinity of both normal and tumorigenic prostate cells. Enzymatically active PSAs are inactivated by serum protease inhibitors in circulation. Peptide carriers were synthesized that serve as substrates to PSA. Amino acid sequences of His-Ser-Ser-LysLeu-Gln (HSSKLQ) and Ser-Ser-Lys-Leu-Gln (SSKLQ) that could be selectively cleaved from cyclopamine in the presence of PSA were conjugated to cyclopamine. The PSA substrate pro-drugs, morpholino (Mu), Mu-HSSKLQ-cyclopamine, and Mu-SSKLQ-cyclopamine, 5, were evaluated in the presence of enzymatically active PSA, with Mu-SSKLQ-cyclopamine being more efficiently hydrolyzed. In addition, whereas the pro-drugs were minimally effective when cultured in a non-PSAexpressing prostate cancer cell line, the addition of PSA to the culture led to a dramatic 7-fold increase in the efficacy of the pro-drug.46 Several pharmaceutical companies have developed Shh signaling pathway inhibitors. For example, IPI-926, 6, is a compound with a number of synthetic modifications to cyclopamine, 1, which result in improved stability, kinetic 7359

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made by an interdisciplinary and nonlinear approach to solving important research problems.

primary end point being overall survival. However, this study was terminated after an interim analysis found that the median survival of patients on the IPI-926 plus gemcitabine arm of the study was 6 months.50 Phase 2 clinical studies were also conducted with IPI-926, 6, as a single agent in patients with myelofibrosis, an incurable cancer of the bone, and chondrosarcoma, a cancer of the cartilage, with primary end points of progression-free survival. Interim analysis of the chondrosarcoma study concluded that treatment with IPI-926 was similar to placebo and the study was terminated. At the same time, the phase 2 exploratory study with myelofibrosis was discontinued due to unsatisfactory clinical activity in initial patients.51 Other small molecules that act as hedgehog pathway inhibitors are currently being studied as anticancer therapies. However, many of these compounds are not based on the steroidal alkaloid structure of cyclopamine, 1. The most successful of these compounds to date is the synthetically derived GDC-0449, 7.52,53 GDC-0449 has completed phase 2 clinical studies in patients with basal cell carcinoma, ovarian, and colorectal cancers and received FDA approval in January 2012 for treatment of adults with metastatic and/or locally advanced basal cell carcinoma.54−56 In addition, clinical studies with GDC-0449 are active or are recruiting patients with basal cell nevus syndrome, basal cell carcinoma, myeloma, pancreatic, small cell lung, chondrosarcoma, medullablastoma, glioblastoma, prostate, glioma, leukemia, sarcoma, breast, and other solid cancers.57

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AUTHOR INFORMATION

Corresponding Author

*(S.T.L.) Phone: (435) 752-2941. Fax: (435) 797-5681. Email: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Binns, W.; James, L. F.; Shupe, J. L. Cyclopian-type malformation in lambs. Arch. Environ. Health 1962, 5, 106−108. (2) Binns, W.; James, L. F.; Shupe, J. L.; Everett, G. A congenitital− type malformation in lambs induced by maternal ingestion of a range plant Veratrum californicum. Am. J. Vet. Res. 1963, 24, 1164−1175. (3) James, L. F. Teratological research at the USDA-ARS Poisonous Plant Research Laboratory. J. Nat. Toxins 1999, 8, 63−80. (4) Binns, W.; Shupe, J. L.; Keeler, R. F.; James, L. F. Chronologic evaluation of teratogenicity in sheep fed Veratrum californicum. J. Am. Vet. Med. Assoc. 1965, 147, 839−842. (5) Keeler, R. F. Early embryonic death in lambs induced by Veratrum californicum. Cornell Vet. 1990, 80, 203−207. (6) Welch, K. D.; Lee, S. T.; Gardner, D. R.; Panter, K. E.; Stegelmeier, B. S.; Cook, D. Dose-response evaluation of Veratrum californicum in sheep. In Poisoning by Plants, Mycotoxins, and Related Toxins; Riet-Correa, F., Pfister, J. A., Schild, A. L., Wierenga, T. L., Eds.; CAB International: Wallingford, UK, 2011. (7) Van Kampen, K. R.; Binns, W.; James, L. F.; Balls, L. D. Early embryonic death in ewes given Veratrum californicum. Am. J. Vet. Res. 1969, 30, 517−519. (8) Keeler, R. F.; Stuart, L. D. The nature of congenital limb defects induced in lambs by maternal ingestion of Veratrum californicum. Clin. Toxicol. 1987, 25, 273−286. (9) Keeler, R. F.; Young, S.; Smart, R. Congenital tracheal stenosis in lambs induced by maternal ingestion of Veratrum californicum. Teratology 1985, 31, 83−88. (10) Keeler, R. F.; Binns, W. Teratogenic compounds of Veratrum californicum (Durand). V. Comparison of cyclopean effects of steroidal alkaloids from the plant and structurally related compounds from other sources. Teratology 1968, 1, 5−10. (11) Keeler, R. F. Teratogenic compounds of Veratrum californicum (Durand) − VI. The structure of cyclopamine. Phytochemistry 1969, 8, 223−225. (12) Masamune, T.; Mori, Y.; Takasugi, M.; Murai, A.; Ohuchi, S.; Sato, N.; Katsui, N. 11-Deoxojervine, anew alkaloid from Veratrum species. Bull. Chem. Soc. Jpn. 1965, 38, 1374−1378. (13) Nusslein-Volhard, C.; Wieschaus, E. Mutations affecting segment number and polarity in Drosophila. Nature 1980, 287, 795− 801. (14) Echelard, Y.; Epstein, D. J.; St-Jacques, B.; Shen, L.; Mohler, J.; McMahon, J. A.; McMahon, A. P. Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 1993, 75, 1417−1430. (15) Marigo, V.; Roberts, D. J.; Lee, S. M.; Tuskurov, O.; Levi, T.; Gastier, J. M.; Epstein, D. J.; Gilbert, D. J.; Copeland, H. G.; Seidman, C. E.; Jenkins, N. A.; Seidman, J. G.; McMahon, A. P.; Tabin, C. Cloning, expression, and chromosomal location of SHH and IHH: two human homologues of the Drosophilia segment polarity gene hedgehog. Genomics 1995, 28, 44−51. (16) Kamisago, M.; Kimura, M.; Furutani, Y.; Furutani, M.; Takao, A.; Momma, K.; Matsuoka, R. Assignment of human desert hedgehog gene (DHH) to chromosome band 12q13.1 by in situ hybridization. Cytogenet. Cell Genet. 2000, 87, 117−118. (17) Tate, G.; Satoh, H.; Endo, Y.; Mitsuya, T. Assignment of desert hedgehog (DHH) to human chromosome bands 12q12→q13.1 by in situ hybidization. Cytogenet. Cell Genet. 2000, 88, 93−94.

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PERSPECTIVE Dr. Philip Beachy’s recollection in 1996 of the research and images of the cyclopic lambs reported in the 1960s led to the correct hypothesis that cyclopamine, 1, could be used to probe the Shh pathway. This observation and hypothesis connected agricultural-based research that had taken place 30 years earlier to current cell biology research. Since this discovery, cyclopamine has served as a valuable tool in elucidating and understanding the Hh signaling pathway in embryonic development and in the investigation of the role of the Hh pathway in various cancers. In addition, cyclopamine has served as a model compound for new cancer therapy drugs. Figure 5 is a timeline that shows the relationship between agricultural research, cell biology research, and cancer research and the major advances in the research areas that relate to cyclopamine. This long timeline is reminiscent of that involved in the development of Taxol (paclitaxel), which began in 1964 with the discovery that extracts of Pacific yew (Taxus brevifolia) bark were cytotoxic.58 The Taxol time line continued with the identification of Taxol as the active principle in 1971 and the approval by the FDA of Taxol as a treatment for ovarian cancer and breast cancer, respectively, in 1992 and 1994. The use of Taxol has continued to increase and, along with its semisynthetic analogue, Taxotere, has become the best-selling anticancer drug in history.58 We anticipate that cyclopamine will continue to be an important research tool in the future and additional advances in cancer therapies and the Hh pathway will be forthcoming. In addition, other poisonous plants should be investigated as sources for anticancer agents, new drugs, and other biomedical applications.59 This review highlights the importance of agricultural/ poisonous plant research and the rapid advances that can be 7360

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