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JERP

Editor-in-ChiefProf. Ramón Cacabelos

Editor-in-ChiefEuroEspes Biomedical Research Center,

Corunna, SpainContinental University Medical School,

Huancayo, Peru

Managing EditorXiaowan DongWuhan, China

Executive EditorHua He

Houston, USA

Technical EditorHuili ZhangWuhan, China

Contact InformationEditorial Office

Executive Editor: Hua HeManaging Editor: Xiaowan Dong Telephone: +1 281-980-0553 E-mail: [email protected] Postal Address: 14090 Southwest Freeway, Suite 300, Sugar Land, Texas, 77478, USA

PublisherXia & He Publishing Inc.

Website: www.xiahepublishing.com E-mail: [email protected] Postal Address: 14090 Southwest Freeway, Suite 300, Sugar Land, Texas, 77478, USA

Aims and Scope

Journal of Exploratory Research in Pharmacology (JERP) publishes origi-nal innovative exploratory research articles, state-of-the-art reviews, editori-als, short communications that focus on novel findings and the most recent advances in basic and clinical pharmacology, covering topics from drug re-search, drug development, clinical trials and application. Topics included, but not limited to the following areas will be considered for publication in JERP: drug composition and properties; synthesis and design of drugs and potential drugs; molecular/cellular and organ/system mechanisms; signal transduction/cellular communications/interactions; toxicology; chemical biology; molecu-lar/biomarker diagnostics; therapeutics; medical applications; interventional (phases I-IV) clinical trials; observational (post-marketing) clinical studies on investigational new drugs; pharmacogenetics; pharmacoepigenetics; pharma-cokinetics; pharmacodynamics; molecular pharmacology; transgenic models.

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Journal Home: https://www.xiahepublishing.com/journal/jerp Editorial Board: https://www.xiahepublishing.com/journal/jerp/editors Archive: https://www.xiahepublishing.com/journal/jerp/archive Instructions for Authors: https://www.xiahepublishing.com/journal/jerp/instructionOnline Submission System: https://www.publinexh.com/

PUBLISHED BY XIA & HE PUBLISHING INC.eISSN: 2572-5505

Frequency: QuarterlyLaunch date: November 10, 2016 (Volume 1, Issue 1)

Current Issue: Volume 6, Issue 1Publication date: March 25, 2021

JERPBen J. GuThe Florey Institute of Neuroscience & Mental HealthParkville, Australia

Xin-Sheng GuHubei University of MedicineShiyan, China

Anastasios LymperopoulosDepartment of Pharmaceutical Sciences, College of Pharmacy, Nova Southeastern UniversityFort Lauderdale, USA

Gurpreet SinghDepartment of Pharmaceutical Sciences, Guru Nanak Dev UniversityAmritsar, India

Massimo TusconiSection of Psychiatry, Department of Medical Sci-ences and Public Health, University of CagliariCagliari, Italy

Associate Editors

Editorial Board

Editorial Board Members

Nisar AhmadSialkot, Pakistan

Idress Hamad AttitallaBayda, Libya

Zhaohui BaiShenyang, China

Tomasz BoczekStanford, USA

Sudip BanerjeeLittle Rock, India

Kishore B. ChallagundlaOmaha, USA

Malavika DeodharOrlando, USA

Yosra S. R. ElnaggarAlexandria, Egypt

Talha Bin EmranChandanaish, Bangladesh

Surampalli GurunathWarangal, India

Darlan GussoPorto Alegre, Brazil

Kadda HachemSaida, Algeria

Tahereh HosseinabadiTehran, Iran

Zhen-peng HuangXi’an, China

Tuo JiAnn Arbor, USA

Irfan Ahmad KhanAligarh, India

Weijun KongBeijing, China

Jing-Ting LiSan Diego, USA

Xiao-Hong LiMichigan, USA

Xin LiChangsha, China

Xin LiShanghai, China

Shih-Jung LiuTaiwan

Yi-Fei MiaoDuarte, USA

Saeed MohammadiTehran, Iran

Rafiullah Baig MirzaDubai, UAE

Anna Pratima NikaljeAurangabad, India

Cyprian Ogbonna OnyejiIle-Ife, Nigeria

Vaishali M. PatilGhaziabad, India

Min-Hua PengShenzhen, China

Hakim RahmouneSetif, Algeria

Md Obayed RaihanJashore, Bangladesh

Biagio RaponeBari, Italy

Celestino SarduNaples, Italy

Muhammad ShahidPeshawar, Pakistan

Laila SheriefAl-Sharkia, Egypt

Rajesh Kumar SinghJalandhar, India

Wang-Ze SongDalian, China

Bing SunWashington, D.C., USA

Hemant D. UneAurangabad, India

Srijayaprakash Babu UppadaOmaha, USA

Feyzahan UzunRize, Turkey

Sarah VascellariMonserrato, Italy

Yang WangGuangzhou, China

Karol WróblewskiRzeszów, Poland

J. Ruth Wu-WongLibertyville, USA

Wen-Rui XieGuangzhou, China

Shao-hua XieStockholm, Sweden

Yoon Yen YowSubang Jaya, Malaysia

Jianshe YangShenzhen, China

Guoxin ZhangSan Diego, USA

Lingmin ZhangGuangzhou, China

Ying-Wen ZhangWuhan, China

Wei ZhaoHong Kong, China

JOURNAL OF EXPLORATORY RESEARCH IN PHARMACOLOGY

CONTENTS 2021 6(1):1–27

Editorials

The Medicinal Importance of Annona squamosa FruitsTahereh Hosseinabadi 1

A New Vitamin D Receptor Agonist, VS-105: A Promising Path to Control of Postmenopausal Os-teoporosisDarlan Gusso 3

Original Article

Chemical Characteristics and Biological Activities of Annona squamosa Fruit Pod and Seed ExtractsJulius K Adesanwo, Akinola A Akinloye, Israel O Otemuyiwa and David A Akinpelu 5

Review Article

The Efficacy and Safety of RET-selective Inhibitors for Cancer PatientsFu-Bin Zhu, Qi-Heng Gou and Lin-Yong Zhao 16

Case Report

A Male Case of Renal AmyloidosisZiryab Imad Taha, Mohammed Elmujtba Adam Essa, Asaad Tageldein Idris Abdelhalim, Mohey Aldein Ahmed Elamin Elnour, Allaa Ahmed Osman Eltayeb, Shaza Adel Awad Mohammed Elwakeel and Abdelkareem Abdallah Ahmed 23

© 2021 The Author(s). This article has been published under the terms of Creative Commons Attribution-Noncommercial 4.0 International License (CC BY-NC 4.0), which permits noncommercial unrestricted use, distribution, and reproduction in any medium, provided that the following statement is provided. “This article has been published

in Journal of Exploratory Research in Pharmacology at https://doi.org/10.14218/JERP.2020.00039 and can also be viewed on the Journal’s website at https://www.xiahepublishing.com/journal/jerp ”.

Journal of Exploratory Research in Pharmacology 2021 vol. 6(1) | 1–2 DOI: 10.14218/JERP.2020.00039

Editorial

The Medicinal Importance of Annona squamosa Fruits

Tahereh Hosseinabadi*

Department of Pharmacognosy and Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Received: December 03, 2020 | Revised: December 27, 2020 | Accepted: January 04, 2021 | Published: January 13, 2021

Annona squamosa is a medicinal plant with edible fruits and is commonly known as the sugar apple. This plant belongs to the Annonaceae family and has been used as a traditional medicine for many years with benefits for patients with various diseases.1,2 However, there is little information regarding the medicinal basis of this plant and the action of its pods and seed oil. Recently, Ade-sanwo et al.3 investigated the chemical constituents and anti-mi-crobial activities of the fruits pods and seed oil extracts, as well as the antioxidant activity of seed oil. GC-MS analysis identified sev-eral potentially bioactive compounds, including numerous types of fatty acids and fatty acid esters. These results support previ-ous observations regarding the presence of unsaturated fatty acids and acetogenins in seed oil.4 Functional studies have also revealed that the purified fruit pod extracts and seed oil of A. squamosa ex-hibit broad-spectrum antibacterial properties. Interestingly, previ-ous antibacterial activities previously reported from A. squamosa leave extracts on some bacterial strains.5–7 In addition, the seed oil extracts of A. squamosa have been found to exhibit potent anti-oxidant activity, extending previous reports on seed,8 leave,5,9 and fruit pulp10 extracts. Such novel findings may help in understand-ing the pharmacological actions of A. squamosa and potentially open a new direction for further investigations.

Previous studies have shown that various chemical compounds, such as alkaloids, carbohydrates, tannins, phenolic compounds, isomeric hydroxyl ketones, cyclopeptides and acetogenins can be found in different parts of the A. squamosal plant.11,12 GC-MS analysis of A. squamosa fruit pod extracts has recently shown that 9,10-dehydro-isolongifolene, androsterone and spathulenol are major compounds found in the plant. These results extend previous reports as spathulenol has been reported to be present in fruit pulp extracts and essential oil.13,14 Furthermore, the determination of the chemical parameters of A. squamosa seed oil, including iodine, saponification, acid and peroxide values, as well as total phenol content is valuable for nutritional, industrial and medicinal utiliza-tion. In addition, the phenolic content of A. squamosa is associated with its anti-bacterial and antioxidant activity.5,15

It is still unclear whether the human body can digest A. squa-mosa to generate such chemical molecules in vivo and which

chemical components have beneficial biological effects in vivo. Therefore, further phytochemical evaluations of A. squamosa pods (including more polar fractions) and seed oil with different extraction and chromatography methods are necessary to identify specific compounds with potent biological activities. Given that natural products play a special role in pharmacotherapy, the find-ings from this work may help develop new medicinal therapies for clinical applications.

Acknowledgments

None.

Funding

None.

Conflict of interest

There is no conflict of interest.

References

[1] Bhattacharya A, Chakraverty R. The pharmacological properties of Annona squamosa Linn: a review. Int J Pharm Eng 2016;4(2):692–699.

[2] Ma C, Chen Y, Chen J, Li X, Chen Y. A Review on Annona squamosa L.: Phytochemicals and Biological Activities. Am J Chin Med 2017;45(5): 933–964. doi:10.1142/S0192415X17500501.

[3] Adesanwo JK, Akinloye AA, Otemuyiwa IO, Akinpelu DA. Chemical Characteristics and Biological Activities of Annona squamosa Fruit Pod and Seed Extracts. J Explor Res Pharmacol 2020. doi:10.14218/JERP.2020.00019.

[4] Chen Y, Chen Y, Shi Y, Ma C, Wang X, Li Y, et al. Antitumor activity of Annona squamosa seed oil. J Ethnopharmacol 2016;193:362–367. doi:10.1016/j.jep.2016.08.036.

[5] El-Chaghaby GA, Ahmad AF, Ramis ES. Evaluation of the antioxidant and antibacterial properties of various solvents extracts of Annona squamosa L. leaves. Arabian J Chem 2014;7(2):227–233. doi:10.1016/ j.arabjc.2011.06.019.

[6] Patel JD, Kumar V. Annona squamosa L.: Phytochemical Analysis and Antimicrobial Screening. J Pharm Res 2008;1(1):34–38.

[7] Neethu Simon K, Santhoshkumar R, Neethu SK. Phytochemical analy-sis and antimicrobial activities of Annona squamosa (L) leaf extracts.

Abbreviations: GC-MS, gas chromatography–mass spectrometry.*Correspondence to: Department of Pharmacognosy and Pharmaceutical Biotechnol-ogy, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran. ORCID: https://orcid.org/0000-0003-3127-0233. Tel: +98-21-88665694, Fax: +98-21-88665250, E-mail: [email protected]; [email protected] to cite this article: Hosseinabadi T. The Medicinal Importance of Annona squamosa Fruits. J Explor Res Pharmacol 2021;6(1):1–2. doi: 10.14218/JERP.2020. 00039.

http://creativecommons.org/licenses/by-nc/4.0/

https://doi.org/10.14218/JERP.2020.00039

https://orcid.org/0000-0003-3127-0233

https://doi.org/10.1142/S0192415X17500501



https://doi.org/10.1016/j.jep.2016.08.036

https://doi.org/10.1016/j.arabjc.2011.06.019

https://doi.org/10.1016/j.arabjc.2011.06.019

https://orcid.org/0000-0003-3127-0233

mailto:[email protected]


DOI: 10.14218/JERP.2020.00039 | Volume 6 Issue 1, March 20212

Tahereh H.: Importance of Annona squamosa fruitsJ Explor Res Pharmacol

J Pharmacogn Phytochem 2016;5(4):128–131.[8] Kothari V, Seshadri S. Antioxidant activity of seed extracts of Annona

squamosa and Carica papaya. Nutr Food Sci 2010;40(4):403–408. doi:10.1108/00346651011062050.

[9] Kalidindi N, Thimmaiah NV, Jagadeesh NV, Nandeep R, Swetha S, Ka-lidindi B. Antifungal and antioxidant activities of organic and aque-ous extracts of Annona squamosa Linn. leaves. J Food Drug Anal 2015;23(4):795–802. doi:10.1016/j.jfda.2015.04.012.

[10] Nandhakumar E, Indumathi P. In vitro antioxidant activities of methanol and aqueous extract of Annona squamosa (L.) fruit pulp. J Acupunct Meridian Stud 2013;6(3):142–148. doi:10.1016/j.jams.2012.09.002.

[11] Pandey N, Barve D. Phytochemical and pharmacological review on Annona squamosa Linn. Int J Res Pharm Biomed Sci 2011;2(4):1404–1412.

[12] Singh Y, Bhatnagar P, Thakur N. A review on insight of immense nutra-ceutical and medicinal potential of custard apple (Annona squamosa Linn.). Int J Chem Stud 2019;7(2):1237–1245.

[13] Thang TD, Dai DN, Hoi TM, Ogunwande IA. Study on the volatile oil contents of Annona glabra L., Annona squamosa L., Annona muricata L. and Annona reticulata L., from Vietnam. Nat Prod Res 2013;27(13): 1232–1236. doi:10.1080/14786419.2012.724413.

[14] Madhumitha G, Rajakumar G, Roopan SM, Rahuman AA, Priya KM, Saral AM, et al. Acaricidal, insecticidal, and larvicidal efficacy of fruit peel aqueous extract of Annona squamosa and its compounds against blood-feeding parasites. Parasitol Res 2012;111(5):2189–2199. doi:10.1007/s00436-011-2671-2.

[15] Tomar RS, Sisodia SS. Estimation of phenolic content, total flavonoids and in-vitro antioxidant activity of Annona squamosa Linn. and Bou-gainvillea glabra Choisy. J Global Pharma Technol 2013;3(5):11–14.


https://doi.org/10.1108/00346651011062050

https://doi.org/10.1016/j.jfda.2015.04.012

https://doi.org/10.1016/j.jams.2012.09.002

https://doi.org/10.1016/j.jams.2012.09.002

https://doi.org/10.1080/14786419.2012.724413

https://doi.org/10.1007/s00436-011-2671-2




Editorial

A New Vitamin D Receptor Agonist, VS-105: A Promising Path to Control of Postmenopausal Osteoporosis

Darlan Gusso*

Pontifícia Universidade Católica do Rio Grande do Sul, Escola de Ciências, Programa de Pós-Graduação em Biologia Celular e Molecular, Porto Alegre, RS, Brazil

Received: November 16, 2020 | Revised: January 27, 2021 | Accepted: January 29, 2021 | Published: February 5, 2021

Vitamin D refers to a group of fat-soluble secosteroids that are crucial for bodily functions. Increasingly evidence has shown their importance in regulating the process of cancer,1 cardiovascular diseases, and immune responses,2,3 as well as postmenopausal os-teoporosis (PMO).4 PMO is a metabolic bone disorder that is char-acterized by decreased bone mass and frequent bone fractures, and affects about 49 million women worldwide.5 Despite the variation in osteoporosis phenotypes, osteoporosis is attributed to estrogen deficiency. Clinically, osteoporosis can be diagnosed by assessing bone mineral density.6 Although there are various drugs, such as calcitriol, paricalcitol, abaloparatide and rommosozumab, avail-able for PMO treatment, the discovery of new therapeutic drugs remains an urgent need.

In recent years, a vitamin D receptor agonist, VS-105, has been developed and shows potential for the intervention of PMO.7 In fact, a recent article published in the Journal Exploratory Research in Pharmacology entitled “A Novel Vitamin D Receptor Agonist, VS-105, Improves Bone Mineral Density without Affecting Se-rum Calcium in a Postmenopausal Osteoporosis Rat Model”. The authors demonstrated that VS-105 was relatively safe and signifi-cantly improved bone mineral density, but did not change serum calcium and phosphate levels in rats. In addition, the therapeutic effects of VS-105 on bone mineral density were comparable to cal-citriol.7 These novel findings are important as a small increase in phosphate levels can be harmful.8 These findings extend previous observations in that the administration of VS-105 did not cause hypercalcemia, hyperphosphatemia, or vascular calcification in ro-dents was found to be safer than paricalcitol.9 Together, these data suggest that VS-105 may be safe and effective for the intervention of PMO, as well as chronic kidney diseases. Currently, there are ongoing clinical trials to test the safety and pharmacokinetics of VS-105 in human subjects. If successful, this drug should be rap-idly translated from the bench to the bedside for the intervention of women with PMO. Overall, VS-105 is a promising adjunctive

therapy or alternative strategy to improve bone mineral density in patients with PMO.

Acknowledgments

None.

Funding

None.


None.

References

[1] Fathi N, Ahmadian E, Shahi S, Roshangar L, Khan H, Kouhsoltani M, et al. Role of vitamin D and vitamin D receptor (VDR) in oral can-cer. Biomed Pharmacother 2019;109:391–401. doi:10.1016/j.bi-opha.2018.10.102.

[2] Manson JE, Cook NR, Lee IM, Christen W, Bassuk SS, Mora S, et al. Vitamin D Supplements and Prevention of Cancer and Cardiovas-cular Disease. N Engl J Med 2019;380(1):33–44. doi:10.1056/NEJ-Moa1809944.

[3] Baeke F, Takiishi T, Korf H, Gysemans C, Mathieu C. Vitamin D: modu-lator of the immune system. Curr Opin Pharmacol 2010;10(4):482–496. doi:10.1016/j.coph.2010.04.001.

[4] Zhang L, Yin X, Wang J, Xu D, Wang Y, Yang J, et al. Associations be-tween VDR Gene Polymorphisms and Osteoporosis Risk and Bone Mineral Density in Postmenopausal Women: A systematic review and Meta-Analysis. Sci Rep 2018;8(1):981. doi:10.1038/s41598-017-18670-7.

[5] Wade SW, Strader C, Fitzpatrick LA, Anthony MS, O’Malley CD. Es-timating prevalence of osteoporosis: examples from industrialized countries. Arch Osteoporos 2014;9:182. doi:10.1007/s11657-014-0182-3.

[6] Eastell R, O’Neill TW, Hofbauer LC, Langdahl B, Reid IR, Gold DT, et al. Postmenopausal osteoporosis. Nat Rev Dis Primers 2016;2:16069. doi:10.1038/nrdp.2016.69.

[7] Wu-Wong JR, Wessale JL, Chen YW, Chen T, Oubaidin M, Atsawasu-

Abbreviations: VS-105, vitamin D receptor agonist; PMO, postmenopausal osteopo-rosis.*Correspondence to: Darlan Gusso, Laboratório de Neuroquímica e Psicofarmaco-logia, Escola de Ciências, Pontifícia Universidade Católica do Rio Grande do Sul. Avenida Ipiranga, 6681, 90619-900, Porto Alegre, RS, Brazil. ORCID: https://orcid.org/0000-0003-0293-239X. Tel: +55-54-99958-5316, E-mail: [email protected] to cite this article: Gusso D. A New Vitamin D Receptor Agonist, VS-105: A Promising Path to Control of Postmenopausal Osteoporosis. J Explor Res Pharmacol 2021;6(1):3–4. doi: 10.14218/JERP.2020.00037.



https://orcid.org/0000-0003-0293-239X

https://doi.org/10.1016/j.biopha.2018.10.102

https://doi.org/10.1016/j.biopha.2018.10.102

https://doi.org/10.1056/NEJMoa1809944


https://doi.org/10.1016/j.coph.2010.04.001

https://doi.org/10.1038/s41598-017-18670-7

https://doi.org/10.1038/s41598-017-18670-7

https://doi.org/10.1007/s11657-014-0182-3

https://doi.org/10.1007/s11657-014-0182-3

https://doi.org/10.1038/nrdp.2016.69

https://orcid.org/0000-0003-0293-239X

https://orcid.org/0000-0003-0293-239X



Gusso D.: Vitamin D receptor agonist VS-105J Explor Res Pharmacol

wan P. A Novel Vitamin D Receptor Agonist, VS-105, Improves Bone Mineral Density without Affecting Serum Calcium in a Postmenopau-sal Osteoporosis Rat Model. J Explor Res Pharmacol 2020;5(4):73–80. doi:10.14218/JERP.2020.00020.

[8] Hong SH, Park SJ, Lee S, Kim S, Cho MH. Biological effects of inorganic phosphate: potential signal of toxicity. J Toxicol Sci 2015;40(1):55–69.

doi:10.2131/jts.40.55.[9] Fujii H, Yonekura Y, Nakai K, Kono K, Goto S, Nishi S. Comparison of

the effects of novel vitamin D receptor analog VS-105 and parical-citol on chronic kidney disease-mineral bone disorder in an experi-mental model of chronic kidney disease. J Steroid Biochem Mol Biol 2017;167:55–60. doi:10.1016/j.jsbmb.2016.11.002.



https://doi.org/10.2131/jts.40.55

https://doi.org/10.1016/j.jsbmb.2016.11.002




Original Article

Keywords: Annona squamosal; Antibacterial activity; Antioxidant activity; Octadec-9-enoic acid; Iodine value; Saponification value.Abbreviations: AV, acid value; AOAC, Association of Official Analytical Chemists’ methods; DCM, dichloromethane; DPPH, 2, 2-diphenyl-1-picryl-hydrazil; FRP, Fer-ric ion reducing power; FFA, free fatty acid; GAE, gallic acid equivalent; GC-MS, gas chromatography-mass spectroscopy; IV, iodine value; MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentrations; NIST, National Institute of Standard Technology; SV, saponification value; TP, total phenol..*Correspondence to: Julius K. Adesanwo, Department of Chemistry, Obafemi Awolowo University, Ile-Ife, Nigeria. Tel: +2348030821561, E-mail: [email protected] to cite this article: Adesanwo JK, Akinloye AA, Otemuyiwa IO, Akinpelu DA. Chemical Characteristics and Biological Activities of Annona squamosa Fruit Pod and Seed Extracts. J Explor Res Pharmacol 2021;6(1):5–15. doi: 10.14218/JERP.2020.00019.

Abstract

Background and objectives: Annona squamosa (A. squamosa) is a medicinal plant, used in ethnomedicinal treat-ment of various ailments. However, there is a dearth of information on the chemical constituents of this plant’s fruit pod and chemical parameters of the seed oil. The objectives of this study were, therefore, to determine the chemical characteristics and biological activities of extracts of the fruit pod and seed oil of A. squamosa.

Methods: Crude methanol extract of the dried and pulverized fruit pod were partitioned using n-hexane and di-chloromethane (DCM), the fractions concentrated in-vacuo to yield n-hexane and DCM fractions of the fruit pod. The n-hexane extract of the dried ground seed was concentrated in vacuo to afford the seed oil. The fractions and the seed oil were subjected to gas chromatography-mass spectroscopy (GC-MS) analysis. The seed oil was characterized for chemical properties using standard methods. The seed oil, crude methanol extract of seed pod and fractions were assayed for antibacterial properties using both Gram-positive and Gram-negative bacteria. The seed oil was also examined for antioxidant activity.

Results: The results from chemical analyses of the seed oil indicated that acid value, iodine value, saponification value and total phenol were 1.91 (as % oleic acid), 109.8 g I2/kg, 204.8 g KOH/kg and 36.2 mg gallic acid equivalent (GAE)/kg, respectively. GC-MS analysis revealed the presence of 14, 8 and 15 compounds in n-hexane and DCM fractions of the fruit pod and seed oil, respectively. Of the compounds identified, octadec-9-enoic acid, 9,10-dehy-droisolongifolene and androsterone were the most abundant. The extracts displayed broad spectrum antibacte-rial activity against the 13 bacterial strains tested, except for Bacillus polymyxa, Enterococcus faecalis and Bacillus cereus, which were resistant to the n-hexane and DCM fractions of the fruit pod.

Conclusions: The findings in this study indicated that the extracts and oil of A. squamosa contain bioactive compounds which have antibacterial and antioxidant properties, and the oil could be applied both as industrial and edible oil.

Introduction

The growing resistance of pathogenic bacterial isolates against an-tibiotics as well as resurgence of old disappeared diseases have lead researchers to focus on bioactive natural compounds that will be effective, with no side effect, in treatment of diseases.

Annona squamosa belongs to Annonaceae family, which com-prises about 135 genera and over 2,300 species.1,2 The most im-portant genera having the largest number of species are Annona, with 166 species. A. squamosa is commonly known as custard ap-ple, sweet sop and sugar apple and is cultivated in tropical areas

Chemical Characteristics and Biological Activities of Annona squamosa Fruit Pod and Seed Extracts

Julius K. Adesanwo1*, Akinola A. Akinloye1, Israel O. Otemuyiwa1 and David A. Akinpelu2

1Department of Chemistry, Obafemi Awolowo University, Ile-Ife, Nigeria; 2Department of Microbiology, Obafemi Awolowo University, Ile-Ife, Nigeria

Received: June 18, 2020 | Revised: September 9, 2020 | Accepted: September 21, 2020 | Published: October 16, 2020






Adesanwo J.K. et al: Analyses of A. squamosa fruit pod and seed J Explor Res Pharmacol

and sub-tropical regions worldwide.3 The plant is an evergreen tree which reaches 3–8 m in height. The leaves are lanceolate, 6–17 cm in length and 3–5 cm in width, while its fruits are 5–10 cm in diameter, with many round protuberances, and can be either heart-shaped, conical, ovate, or round. The seeds of the plant are 1.3–1.6 cm long; they are smooth, shiny, blackish or dark brown in color.4

The plant is traditionally used for the treatment of epilepsy, dys-entery, cardiac problem, worm infection, constipation, hemorrhage, dysentery, fever, and ulcer,5 and also reported to possess antidia-betic activity.6 Different parts of A. squamosa have been used in the treatment of various ailments and human diseases because the plant contains several bioactive compounds. The plant is said to possess biological activities, such as analgesic, anti-inflammatory, antimi-crobial, cytotoxic, antioxidant, antilipidimic, antiulcer hepatopro-tective, vasorelaxant, antitumor larvicidal insecticidal anthelmin-tic, molluscicidal properties, and genotoxic effect.7 The fruit of A. squamosa has hematinic, sedative, stimulant and expectorant prop-erties and are also useful in treating anemia and burning sensation.8 The seeds are useful in treating lice infection in the hair.9

Hopp et al.5 isolated three annonaceous acetogenins (9-hydroxy asimicinone, squamoxinone B and C) from bark of A. squamosa. In spite claims of the medicinal properties of the A. squamosa plant, there is dearth of empirical information on the chemical composi-tion and biological activities of the plant’s fruit pod and seed oil. Therefore, this study was designed to investigate the extracts of the fruit pod and seed for chemical constituents, antioxidant ac-tivity, and anti-microbial properties. The results of this study will provide empirical information that justifies the use of A. squamosa for medicinal purpose, and the possibility of harnessing its oil for nutritional and industrial purposes.

Materials and methods

Plant collection

A. squamosa fruit pod and seeds used for this study were collect-ed in Ile-Ife, southwest of Nigeria, identified at the Herbarium in the Department of Botany, Obafemi Awolowo University, Ile-Ife (voucher number: IFE-17927).

Extraction of A. squamosa seed

The seed was removed from the capsule, dried, pulverized, packed in air-tight plastic containers and kept in the freezer until use. The pulverized sample of the seed was soaked in distilled n-hexane for 72 h, after which it was filtered and concentrated using a ro-tary evaporator at 40 °C. The extract thus obtained was labeled n-hexane extract and kept in a desiccator, and subsequently used for both biological assay and gas chromatography-mass spectros-copy (GC-MS) analysis. The extraction of the seed oil for chemical analysis was carried out using soxhlet extractor and n-hexane as the extracting solvent.

Extraction and partitioning of A. squamosa fruit pod

The dried and pulverized fruit pod (42 g) was soaked in distilled methanol for 48 h, after which it was filtered. The extraction pro-cess was repeated thrice, for optimum yield. The extracts were pooled and then concentrated using a rotary evaporator at 40 °C. The crude methanol extract thus obtained was partitioned with n-

hexane and DCM to afford respective fractions, which were kept for further analysis.

GC-MS analysis of the samples

The n-hexane extract of the seed (seed oil), and n-hexane and DCM fractions of the fruit pod were taken for GC-MS analysis. The samples were analyzed using gas chromatography (19091J-413; Agilent, Santa Clara, CA, USA) coupled to a mass spectrom-eter (model 5975C) with triple-axis detector equipped with an auto injector (10 µL syringe). Helium gas was used as the carrier gas.

All chromatography was performed on a capillary column having specification length of 30 m, internal diameter of 0.2 µm, thickness of 320 µm, and treated with 5% phenyl methyl siloxane. Other GC-MS conditions were pressure of 3.2875 psi and a flow time of 1.5 mL/min. The column temperature started at 80 °C for 2 mins and increased to 280 °C at the rate of 3 °C/min for 20 mins. The total elusion time was 88.667 mins. Identification of the com-pounds was carried out by comparing the mass spectra obtained with those of the mass spectra from the National Institute of Stand-ard Technology (NIST) library (NISTII).

Determination of chemical parameters of A. squamosa seed oil

The chemical parameters were determined as reported by the Asso-ciation of Official Analytical Chemists’ methods (AOAC 920.158; AOAC 936.15; AOAC 936.16; AOAC 933.08 for iodine, saponifi-cation, acid and peroxide values, respectively).10

Biological activity

Antibacterial sensitivity testing of the extracts

The antibacterial activity of n-hexane extract of the seed, and n-hexane and DCM fractions of the fruit pod were determined using the agar-well diffusion method described by Akinpelu et al.11 The test organisms were reactivated in nutrient broth for 18 h before use. Exactly 0.1 mL of standardized test bacterial strains (106 cfu/mL of 0.5 McFarland standards) was transferred into Mueller-Hinton agar medium at 40 °C. This was thoroughly mixed together and later poured into pre-sterilized Petri dishes. The plates were allowed to set and wells were bored into the medium using a 6-mm sterile cork borer. These wells were then filled up with the prepared solutions of the extracts. Care was tak-en not to allow the solution to spill on the surface of the medium. The concentration of the extract used was 25 mg/mL, while the concentration of streptomycin used as positive control was 1 mg/mL. The plates were left on a laboratory bench for 1 h to allow proper in-flow of the solution into the medium before incubat-ing them at 37 °C for 24 h. The plates were not stock-piled, to allow even distribution of temperature around the plates in order to avoid false results. The plates were later observed for zones of inhibition, which is an indication of susceptibility of the test organisms to the extracts.

Determination of minimum inhibitory concentrations (MIC) of the extracts

The minimum inhibitory concentration (MIC) of n-hexane seed extract, and n-hexane and DCM fractions of the fruit pod were


DOI: 10.14218/JERP.2020.00019 | Volume 6 Issue 1, March 2021 7


determined according to the method described by Akinpelu and Kolawole.12 A 2 mL aliquot of different concentrations of the solution was added to 18 mL of pre-sterilized molten nutrient agar, to give final concentrations ranging between 0.39 and 12.5 mg/mL. The mixture was then poured into sterile Petri dishes and allowed to solidify. The plates were left on the laboratory bench overnight to ascertain their purity. The surfaces of the plates were allowed to dry well before striking with standard-ized inoculum of the test organisms and incubated aerobically at 37 °C for 48 h. The plates were later examined for the presence or absence of bacterial growth. The MIC was taken as the lowest concentration of the extracts that inhibited the growth of the test organisms.

Determination of minimum bactericidal concentration (MBC) of the extracts

The minimum bactericidal concentration (MBC) of the extracts were assessed by taking a sample from the streaked line of the MIC test and cultured on fresh sterile nutrient agar plates. The plates were incubated at 37 °C for 72 h. The MBC was taken as the con-centration of the extracts that did not support the bacterial growth on the medium.

Antioxidant activity assay of the seed oil

The anti-oxidant activity of the seed oil was accessed through three parameters: the total phenol, ferric ion reducing power (FRP) and 2,2-diphenyl-1-picryl-hydrazil (DPPH) assay.

Determination of total phenol

Total phenol (TP) of the seed oil was measured as previously de-scribed by Moreno et al.13 and estimated spectrophotometrically using Folin–Ciocalteu’s phenol reagent assay with gallic acid as the standard.14 The TP content was expressed as mg/kg gallic acid equivalent (GAE) and linearity range for the standard was between 0–40 mg/L GAE (R2 = 0.9928).

Measurement of free radical scavenging activity

This was determined using the DPPH reagent, according to Brand-Williams et al.15 The oil (0.5 mL) was put in screw cap test tubes, and 4 mL of methanol and 4 mL of 0.1 mmol L−1 methanol solu-tion of DPPH were added and shaken. A blank probe was obtained by mixing 4 mL of 0.1 mmol L−1 methanol solution of DPPH and 0.5 mL of deionized distilled water (ddH2O). After 30 mins of in-cubation in the dark at room temperature, the absorbance was read at 517 nm against the prepared blank. Various concentrations of standard catechin (0, 2, 4, 6, 8 and 10 mg/mL) were used to gener-ate the standard curve, and the result was extrapolated from linear curve equation (y = 0.033x, R2 = 0.995); the result was expressed as IC50 catechin equivalent.

Determination of FRP

The FRP assay was carried out according to Stratil et al.,16 with slight modifications. FRP was measured using the potassium fer-ricyanide assay. The oil (1 mL) was added to 2.5 mL phosphate

buffer (0.2M, pH 6.6) and 2.5 mL of potassium ferricyanide (1%, w/v). The mixture was incubated at 50 °C for 20 mins. After add-ing trichloroacetic acid solution (2.5 mL, 10%, w/v), the mixture was separated into aliquots of 2.5 mL and diluted with 2.5 mL of water. To each diluted aliquot, 5 mL of ferric chloride solution was added. After 30 mins, absorbance was measured at 700 nm. Ascorbic acid was used as standard and the FRP value of extracts was expressed as the ascorbic acid equivalent (mg AAE/g), and the content was calculated from a linear equation of the standard y = 5.661x and R2 = 0.988.

Statistical analysis

Results were expressed as mean and standard deviation of three determinations, and data were subjected to one-way analysis of variance to determine the levels of significant difference by per-forming a multiple comparison post-test (Tukey) and were con-sidered significant at p ≤0.05. GraphPad InStat version 3.06 for Windows 2003 was used for the analysis.

Results and discussion

GC-MS analysis: N-hexane fraction of A. squamosa fruit pod

The chromatogram of GC-MS analysis of the n-hexane fraction of the fruit pod and the chemical characteristics of compounds de-tected are presented in Figure 1 and Table 1, respectively.

This fraction contained a mixture of compounds, mainly monoterpenes, diterpenes, sesquiterpene and derivatives, fatty acids, and fatty acid esters. Fourteen compounds were identified, 9,10-dehydro-isolongifolene, a sesquiterpene is the main com-pound (20.90%) in this fraction (Table 1). Previously, 9,10-de-hydro-isolongifolene was found in the wood oil of giant sequoia (Sequoiadendron giganteum (Lindl.) Buchh) by Jerković et al.17 and reported to be one of the main constituents of the leaves essential oil of Cedrelopsis grevei which exhibited good antican-cer, anti-inflammatory, antioxidant and antimalarial activities.18

DCM fraction of A. squamosa fruit pod

The gas chromatogram and list of chemical constituent of the DCM fraction of A. squamosa fruit pod are as shown in Figure 2 and Table 2, respectively.

Eight compounds were identified in the fraction, the major ones are androsterone (7.83%) and spathulenol (6.22%). An-drosterone is a natural product which has been found in pine pollen and is well known in many animal species.19 It is an in-hibitory androstane neurosteroid,20 acting as a positive allosteric modulator of the GABAA receptor21 and exerts anticonvulsant effect.22

Spathulenol, a volatile oil, is a tricyclic sesquiterpene alcohol with basic skeleton similar to the azulenes. It occurs in waterwort distillery (Artemisia vulgaris) and tarragon (Artemisia dracun-culus), among other plants.23 It is an anesthetic and a vasodilator agent, possessing antioxidant, anti-inflammatory, antiproliferative and antimycobacterial activities.24 Selene et al.25 reported that spathulenol was identified as a major constituent in the essential oils of four Croton species, which displayed good antioxidant activity. According to them, spathulenol was active against the enzyme Leishmania infantum trypanothione reductase, showing




Fig. 1. Gas chromatogram of the n-hexane fraction of A. squamosa fruit pod.

Table 1. Chemical constituents of n-hexane fraction of A. squamosa fruit pod

S/N Compound RT in m

PA, % MF MM in

g/mol Structural formula

1 5-(propan-2-ylidene)cyclopenta-1,3-diene 7.3 1.08 C8H10 106.16

2 9,10-dehydro-isolongifolene 32.9 20.90 C15H24 204.35

3 6-((benzyloxy)methyl-2,3,4-trimethylcyclohexyl) formaldehyde

33.1 1.91 C18H26O2 274.40

4 2-methyloct-5-yn-4-yl-3-fluorobenzoate 37.6 1.11 C16H19FO2 262.14

5 Methyl palmitate 38.5 6.20 C17H34O2 270.45

6 N-hexadecenoic acid 38.7 4.68 C16H32O2 256.42

7 Trans-13-octadecenoic acid 39.8 7.28 C18H34O2 282.46

8 Octadecanoic acid 39.9 3.11 C18H36O2 284.48




excellent interaction energies, making it a promising agent for leishmaniasis control.

N-hexane extract of A. squamosa seed

The chromatogram obtained from the GC-MS analysis of the n-hexane extract of the seed of A. squamosa is shown in Figure 3. The chemical compounds identified through comparison of the mass spectra, based on ≥50% matching, with the NIST library are listed with their retention time (RT) and peak area (PA%) in Table 3.

Fifteen compounds were identified from this extract, which constitutes 86.71% of the total detected compounds in the extract, and 9-octadecenoic acid is the main compound in this extract. 2,4-decadienal and 1-dodecanol are other compounds present in appreciable proportions. 9-octadecenoic is a monounsaturated fatty acid present in human diet in the form of its triglycerides and it is said to be responsible for the hypotensive effect of olive oil.26

2,4-decadienal was implicated in the nematicidal activity exhib-ited by Ailanthus altissima methanol extract against the root knot nematode Meloidogyne javanica.27 Dodecanol or lauryl alcohol, is a fatty alcohol produced industrially from palm kernel oil or coconut oil, and it is used to make surfactants, lubricating oils, and pharmaceuticals. It is found to inhibit the activity of Candida albicans.28 Anethole, a principal component of anise oil, has been found to prolong the transient antifungal effect of dodecanol.29

Antibacterial analysis

The crude methanol extract of the fruit pod (S1) and n-hexane ex-tract of the seed inhibited the growth of all the bacterial strains tested. The other two fractions, that is n-hexane (S2) and DCM (S3) fractions of the fruit pod, inhibited 11 and 12 of the bacterial strains tested, respectively. Overall, both the extracts and fractions exhibited broad spectrum activities against the bacterial strains and compared favorably with the standard antibiotic-streptomycin


PA, % MF MM in

g/mol Structural formula

9 3-(1,1-dimethylallyl)- scopoletin 40.2 1.10 C15H16O4 260.10

10 2-[(1,2-dimethylpiperidin-3-yl)methyl]-3H-indol-3-one

40.2 2.08 C16H20N2O 256.34

11 1,3-diethyl-4-oxo-4H-benzo4,5thiazolo[3,2-a] pyrimidin-1-ium-2-olate

40.7 1.53 C14H15N2O2S+ 274.34

12 Andrographolide 40.8 2.85 C20H30O5 350.45

13 Nordextromethorphan 41.6 7.12 C17H23NO 257.37

14 (1R,4aR,4bS,7R,10aR)-methyl-1,4a,7-trimethyl-7-vinyl1,2,3,4,4a,4b,5,6,7,8,10,10a-dodecahydro phenanthrene-1-carboxylate

44.3 3.32 C21H32O2 316.48

MF, molecular formula; MM, molecular mass; PA, peak area; RT, retention time.

Table 1. Chemical constituents of n-hexane fraction of A. squamosa fruit pod - (continued)




Fig. 2. Gas chromatogram of the DCM fraction of A. squamosa fruit pod.

Table 2. Chemical constituents of DCM fraction of A. squamosa fruit pod


PA, % MF MM Structural formula

1 1,1,7-trimethyl-4-methylene decahydro-1H-cyclopropa[e]azulen-7-ol (spathulenol)

32.9 6.22 C15H24O 220.35


3 N-hexadecanoic acid 38.7 4.72 C16H32O2 256.42

4 Oleic acid 39.8 2.63 C18H34O2 282.46

5 Androsterone 41.6 7.83 C19H30O2 290.44




used as positive control (Table 4). The results obtained from the study support the usefulness of A. squamosa in folklore remedies to treat infections caused by pathogens in humans. This serves as a pointer towards the development of antimicrobial agents of natural origin for treatment of superbugs that have developed resistance against the available antibiotics.

Among the bacterial strains that were susceptible to the extracts from A. squamosa are Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus cereus and B. anthracis, which are all known to cause infections in humans.30 These pathogens are now gradually developing resistance against the available antibiot-

ics used as therapy against infections caused by these pathogens. There is an urgent need to source potent antimicrobials, especially of natural origin, to combat infections caused by these pathogens. Thus, antimicrobials produced from A. squamosa may go a long way in healthcare delivery to take care of the menace of these pathogens.

MIC and MBC exhibited by extracts against bacterial strains

The results obtained from the MIC and MBC analyses of the ex-



6 Kaur-16-ene 43.1 2.16 C20H32 272.47

7 2,3,4,6-tetramethyl-benzoic acid 44.0 3.21 C11H14O2 178.23

8 Methyl-4,11-dimethyl-8-methylenetetradecahydro-6a,9-methanocyclohepta[a]napthalene-4-carboxylate

44.4 2.48 C21H32O2 316.48


Table 2. Chemical constituents of DCM fraction of A. squamosa fruit pod - (continued)

Fig. 3. GC-MS chromatogram of n-hexane extract of A. squamosa seed.




tracts from A. squamosa against susceptible bacterial strains used for this study showed high antibacterial potency (Table 5). The lowest MIC obtained for the crude methanol extract of the fruit pod (S1) was 0.39 mg/mL, while the MBC was 1.56 mg/mL. The lowest MIC observed for the n-hexane fraction of the fruit pod was 1.56 mg/mL and the MBC was 3.13 mg/mL, while S3 and S4 showed low MIC and MBC values of 0.78 and 1.56 mg/mL, re-spectively. According to Achinto et al.,31 any plant extracts exhibit-ing low MIC and MBC against susceptible pathogens possess high antimicrobial potency. This observation in A. squamosa extracts showed this extract to exhibit high antimicrobial potency. Such a

plant can be used to produce potent antimicrobial compounds to combat the antimicrobial resistance experienced in many of these pathogenic infections.

Antioxidant activity

The TP recorded for the oil was 36.2 mg/kg (Table 6), and this value compared favorably with the 30.3 mg/kg recorded for groundnut oil32 but was higher than the 14.4 mg/kg recorded for Hibiscus rosa sinensis.33 Phenolic compounds have been associated with antioxi-

Table 3. Chemical constituents of n-hexane extract of A. squamosa seed



1 o-xylene 7.276 0.97 C9H10 106.16

2 (E)-hept-2-enal 11.1 3.77 C7H12O 112.17

3 Nonanal 17.1 1.37 C9H18O 142.24

4 9-methyl-undec-1-ene 22.4 1.09 C12H24 168.32

5 1-dodecanol 22.9 10.28 C12H25O 183.33

6 (2E,4E)-deca-2,4-dienal 24.8 17.77 C10H16O 152.23

7 (E)-oct-2-enal 26.3 3.15 C8H14O 126.20

8 8-heptadecene 35.8 1.13 C17H34 238.45


10 N-hexadecanoic acid 38.7 6.31 C16H32O2 256.42

11 2-chloroethyl linoleate 39.5 1.34 C20H35ClO2 342.94

12 (E)-methyloctadec-9-enoate

39.6 3.70 C19H36O2 296.49

13 (E)-octadec-9-enoic acid 39.8 26.37 C18H34O2 282.46

14 Octadecanoic acid 39.9 6.19 C18H36O2 248.48

15 Palmitic anhydride 40.8 1.21 C32H62O3 494.47





dant activity; this implies that the oil could be a good source of an-tioxidants, which could prevent the oil from oxidative degeneration.

The antioxidant capacity of the oil determined from DPPH radi-cal scavenging activity expressed as IC50 was 1.33; this value is higher than the 0.027 reported for rice bran34 but lower than the

5.03 recorded for Abrus precatorious seed oil.35 The IC50 value is inversely proportional to the antioxidant activity; the lower the val-ue the better the radical scavenging ability. The low DPPH (IC50) value positively correlated with high value of TP of the oil. Also, the ferric reducing power recorded for the oil was 34.8 mg AAE/g

Table 4. Sensitivity patterns of zones of inhibition exhibited by the extracts against bacterial strains

Bacterial strainsZones of inhibition in mm*

S1 (25 mg/mL) S2 (25 mg/mL) S3 (25 mg/mL) S4 (25 mg/mL) Strep (1 mg/mL)

Gram-positive

Bacillus anthracis (LIO) 15 10 12 16 20

B. cereus (NCIB 6349) 12 10 0 08 21

B. polymyxa (LIO) 10 0 08 14 18

B. stearotherphilus (NCIB 8222) 13 09 08 12 19

B. subtilis (NCIB 3610) 16 12 10 17 20

Clostridium sporogenes (NCIB 532) 14 10 09 12 15

Corynebacterium pyogenes (LIO) 12 08 10 15 18

Staphylococcus aureus (NCIB 8588) 15 12 11 10 19

Enterococcus faecalis (LIO) 10 0 08 14 16

Gram-negative

Escherichia coli (NCIB 86) 19 11 13 14 22

Klebsiella pneumoniae (NCIB 418) 13 10 07 18 16

Pseudomonas fluorescence (NCIB 3756) 16 10 12 11 17

Proteus vulgaris (NCIB 67) 20 13 10 21 22

S1, crude methanolic extract of the fruit pod; S2, n-hexane fraction of the fruit pod; S3, DCM fraction of the fruit pod; S4, n-hexane extract of the seed; 0, resistant; Strep, strep-tomycin; *, mean of three replicates.

Table 5. MIC and MBC exhibited by the extracts against susceptible bacterial strains

Bacterial strain

Extracts

S1 S2 S3 S4

MIC, mg/mL

MBC, mg/mL

MIC, mg/mL

MBC, mg/mL

MIC, mg/mL

MBC, mg/mL

MIC, mg/mL

MBC, mg/mL

Bacillus anthracis (LIO) 1.56 6.25 6.25 12.50 1.56 3.13 0.78 1.56

B. cereus (NCIB 6349) 3.13 6.25 3.13 6.25 ND ND 1.56 6.25

B. polymyxa (LIO) 3.13 6.25 ND ND 6.25 12.50 1.56 3.13

B. stearotherphilus (NCIB 8222) 3.13 6.25 6.25 12.50 6.25 12.50 3.13 6.25

B. subtilis (NCIB 3610) 1.56 3.13 3.13 6.25 3.13 6.25 1.56 3.13

Clostridium sporogenes (NCIB 532) 1.56 3.13 1.56 3.13 3.13 6.25 3.13 6.25

Corynebacterium pyogenes (LIO) 6.25 12.50 6.25 12.50 1.56 3.13 1.56 3.13

Escherichia coli (NCIB 86) 0.39 1.56 1.56 3.13 0.78 1.56 0.78 3.13

Klebsiella pneumoniae (NCIB 418) 3.13 6.25 3.13 6.25 6.25 12.50 0.78 1.56

Pseudomonas fluorescence (NCIB 3756) 0.78 1.56 3.13 6.25 1.56 3.13 3.13 6.25

Proteus vulgaris (NCIB 67) 0.78 1.56 1.56 3.13 3.13 6.25 0.78 1.56

Staphylococcus aureus (NCIB 8588) 1.56 3.13 3.13 6.25 3.13 6.25 3.13 6.25

Enterococcus faecalis (LIO) 3.13 6.25 ND ND 6.25 12.50 1.56 3.13

S1, crude methanolic extract of the fruit pod; S2, n-hexane fraction of the fruit pod; S3, DCM fraction of the fruit pod; S4, n-hexane extract of the seed; ND, not done.




(Table 6); this value falls within the range of 7.79 to 56.4 reported for fruit juices.36 The Fe(III) reduction can be used as an indicator of electron donating activity of primary antioxidants whose func-tion is to prevent oxidative damage.37 The higher the FRP value, the better the electron donating ability; therefore, from the values obtained, the oil could be said to have high antioxidant activity.

Chemical characteristics of seed oil

The results of the chemical characteristics of A. squamosa seed oil is presented in Table 7. Oil content of the seed was 19.65%; this value is lower than the 47% reported for groundnut32 but the value recorded still categorized the seed as oil seed. The iodine value (IV), which is a measure of the degree of unsaturation of vegetable oil, was observed to be 109.8 g I2/kg. This value was higher than the 91.90 g I2/kg reported for groundnut oil.32 The result shows that A. squamosa oil could be easily oxidized and may likely dry up when stored. Oil with high IV is preferred nutritionally, due to the presence of unsaturated fatty acids, but is prone to oxidative rancid-ity if not stored properly. Hence, the seed oil must be refined and protected with an antioxidant to increase storage time (shelf-life).

Saponification value (SV) provides information on the suitabil-ity or otherwise of vegetable oil for the production of soap. SV ob-served for this seed oil was 204.8 mg KOH/g, which is higher than that reported for groundnut oil (193.20 mg KOH/g).32 The high SV indicated high content of triacylglycerols, which is consistent with a high ester value (>99%); this implies that the oil could comple-ment or even substitute some conventional oils in soap making.

The acid value (AV) obtained for A. squamosa seed oil was 1.91 (as % oleic acid), which is lower than the 2.89 reported for ground-nut30 but comparable to the 1.49 reported for sunflower oil.38 The low acid value indicates that triacylglycerol had not been appreci-ably hydrolyzed, which could indicate a good stability of the oil. The percentage free fatty acid (FFA) was 3.81; this value was significant-ly higher than the 2.82% recorded for acacia seed oil.14 The high FFA value obtained in this study could be adduced to the activity of lipo-lytic enzymes during the preparation of the seed for oil extraction. The AV and FFA values provide information on the storage quality of vegetable oil. For example, FFA is more susceptible to oxidation compared to intact fatty acids. The result thus indicated that A. squa-mosa oil would have a longer shelf-life than some conventional oils, due to its high IV. However, the appropriate condition for storage should be observed. The seed oil could therefore be adjudged suit-able as food for human consumption, medicinal as well as for indus-trial purposes in view of its biological and chemical characteristics.

Conclusion

The GC-MS analysis of the extracts showed that the plant contains some bioactive compounds which can contribute towards the bio-logical activities of the plant. The extracts obtained from A. squa-

mosa exhibited appreciable antibacterial potency against the panel of bacterial strains used for this study. The extracts exhibited broad spectrum activities and thus showed a significant therapeutic action for the treatment of infections caused by pathogens. This observa-tion supported the usefulness of this plant in folklore remedies for the management of infections caused by microorganisms. The oil content of the seed (18.75%) is high enough for it to be considered as oil seed. Results from the chemical characteristics of the seed oil showed that the oil can be used both as edible and industrial oil. The seed oil also demonstrated a good antioxidant property.

Future directions

This current research is focused primarily on qualitative determi-nations on the fruit pod and seed oil of A. squamosa. Future re-search should focus on isolation of specific compounds and struc-ture elucidation. Also, other parts of the plant (leaf, stem and root back and wood) should be further examined.

Acknowledgments

None.

Data sharing statement

No additional data are available.

Funding

None.


The authors declare that there are no conflicts of interest.

Author contributions

Study design and supervisor (JKA), performance of experiments, analysis and interpretation of data (AAA, IOO, DAA), manuscript writing (AAA), critical revision (JKA).

Table 6. Antioxidant activity of A. squamosa seed oil

Parameter Value*

TP, mg GAE/kg 36.2 ± 0.3

FRP assay, mg AAE/g 34.8 ± 0.01

DPPH, IC50 1.33 ± 0.001

*mean and standard deviation of triplicate analysis.

Table 7. Chemical characteristics of A. squamosa oil

Parameters Value*

Moisture content of seed 44.3 ± 2.0

Oil content 19.6 ± 0.9

AV as % oleic acid 1.91 ± 0.02

FFA (%) 3.81 ± 0.001

IV as g I2/kg 109.8 ± 4.2

SV as mg KOH/g 204.8 ± 2.8

Ester value 203.3 ± 4.2

*mean and standard deviation of triplicate analysis.




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Review Article

Introduction

Rearrangements in the rearrangement during transfection (RET) genes that encode transmembrane receptor tyrosine kinases (RTK) are as-sociated with tumorigenesis. The RET protein forms a heterodimer complex after it is engaged by a ligand in the glial cell line-derived neurotropic factor (GDNF) family that causes autophosphorylation of the tyrosine kinase domain in the cells and activates downstream sign-aling, regulating the processes of cell differentiation, cell migration,

and proliferation.1–4 RET can regulate the development of multiorgans, cell survival, death, and migration and its mutations or gene fusion can promote spontaneous tumor proliferation, activation, and migration.

Alterations in the RET gene are associated with the pathogen-esis of many human diseases, including multiple endocrine neopla-sia type 2 (MAN-2), papillary thyroid cancer, Hirschsprung’s dis-ease, colon adenocarcinoma, invasive breast cancer, non-small cell lung cancer (NSCLC), and others.5 CDCC6-RET and KIF5B-RET, two RET fusions are common in papillary thyroid carcinoma and NSCLC, respectively. A germline mutation in the RET can cause MAN-2 syndrome.6 Germline-activated RET mutations are found in 95–98% of hereditary medullary thyroid cancer (MTC) and so-matic RET mutations are found in 25–40% of sporadic MTC. In addition, RET mutations are associated with the aggressiveness of MTC, such as distant metastasis.7 The RET fusions are detected in 1–2% of NSCLC,8 particularly for lung adenocarcinoma and RET rearrangements, are found in other histological types of NSCLC, including malignant neuroendocrine tumor and squamous cell car-cinoma.9 It was found that the RET fusion genes were detected in lung cancer, thyroid cancer, colon adenocarcinoma (CCDC6-RET) and invasive breast cancer (ERC1-RET).10

The biology and function of RET

The RET, a transforming gene, was first discovered in mouse em-

The Efficacy and Safety of RET-selective Inhibitors for Cancer Patients

Fu-Bin Zhu1#, Qi-Heng Gou1# and Lin-Yong Zhao2*

1Department of Head and Neck Oncology, Cancer Center, West China Hospital, Sichuan University, Chengdu, China; 2Department of Gastrointestinal Surgery and Laboratory of Gastric Cancer, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, China

Received: November 11, 2020 | Revised: January 13, 2021 | Accepted: February 01, 2021 | Published: February 20, 2021

Abstract

The rearrangement during transfection (RET) encodes a receptor tyrosine kinase (RTK), which is involved in the devel-opment of various tissues and cells. The rearrangements and mutations of RET contribute to the development of a variety of human malignancies. Therefore, RET alterations are novel therapeutic targets. Inhibitors for RET and other kinases have been approved for the treatment of RET-altered tumors and have demonstrated their benefits for some types of cancer patients in clinics. However, due to off-target effects, these inhibitors have some adverse effects and dose-limiting toxicity. Therefore, long-term treatment with these inhibitors has potential limitations. Novel highly selective inhibitors (pralsetinib and selpercatinib) that target the RET pathway are well tolerated and have significant and long-lasting antitumor activity. They have been accelerated for approval by the FDA. This article will focus on the role of highly selective inhibitors targeting the RET and their efficacy and safety in therapy for RET-associated cancers.

Keywords: Efficacy; Pralsetinib; Rearrangement during transfection alteration; Safe-ty; Selpercatinib; Tyrosine kinase inhibitor.Abbreviations: RET, rearrangement during transfection; MAN-2, multiple endocrine neoplasia type 2; NSCLC, non-small cell lung cancer; MTC, medullary thyroid can-cer; NIH, National Institutes of Health (USA); RTK, receptor tyrosine kinase; CNS, central nervous system; GDNF, glial cell line-derived neurotrophic factor; GFL, GDNF Family Ligands; GFRα, growth factor receptor-alfa; MKI, multi-kinase in-hibitors; PFS, progression-free survival; ORR, overall response rate; DOR, duration of response; FDA, Food and Drug Administration.*Correspondence to: Lin-Yong Zhao, Department of Gastrointestinal Surgery and Laboratory of Gastric Cancer, State Key Laboratory of Biotherapy, West China Hos-pital, Sichuan University, and Collaborative Innovation Center for Biotherapy, No. 37 Guo Xue Xiang Street, Chengdu, Sichuan Province, China. ORCID: https://orcid.org/0000-0003-0884-4657. Tel: +86-28-85422878; Fax: +86-28-85164035. E-mail: [email protected]#These authors contributed equally to this study.How to cite this article: Zhu F-B, Gou Q-H, Zhao L-Y. The Efficacy and Safety of RET-selective Inhibitors for Cancer Patients. J Explor Res Pharmacol 2021;6(1):16–22. doi: 10.14218/JERP.2020.00035.



https://orcid.org/0000-0003-0884-4657

https://orcid.org/0000-0003-0884-4657

https://orcid.org/0000-0003-0884-4657



Zhu F.B. et al: Efficacy and safety of RET-selective inhibitors J Explor Res Pharmacol

bryonic fibroblast cells that were established by the National In-stitutes of Health (NIH) transfected with DNA from human T cell lymphoma.11 The RET gene is located at 10q11.2 and contains 21 exons.12 The RET encodes an RTK, a type of transmembrane glycoprotein, which mediates signal transduction during various processes, such as cell migration, proliferation, and differentia-tion. It is required for the development and maturation of various organs and cells.13 Studies have confirmed that RET is crucial for the formation and development of the kidneys and nervous sys-tem. In addition, the RET supports the survival of hematopoietic stem cells and early spermatogenesis.14,15 Structurally, RET con-tains three domains; an extracellular domain, a transmembrane domain, and an intracellular domain that contains the tyrosine kinase domain adjacent to the transmembrane region. The large extracellular region contains a domain of four cadherin-like re-peats, a calcium-binding site, and a conserved cysteine-rich por-tion at the proximal end of the membrane.16 The C-terminal of RET has two main forms, which are formed by alternate splic-ing of G1063 residue to exon 3. There are 9 or 51 amino acids at the end of the C-terminal, respectively called RET 9 and RET 51.17 Unlike other RTKs, RET protein does not directly transmit signals after it is engaged by its ligand. The ligands of RET are members of the GDNF family, which include neuroturin, artemin, and persephin.2 These GDNF family ligands (GFLs) can bind to four types of GDNF family growth factor receptor-alfa (GFR-α) to form a coreceptor. This GFL-GFR-α binary complex can bind to the intracellular tyrosine kinase domain of RET and induce dimerization of RET.18 The formation of a homodimer between two RET will cause transphosphorylation of intracellular tyrosine residues of RET and create a docking site for the signal adapter

molecule. The phosphorylated RET will then recruit key signal adapter molecules and activate a variety of cellular signal cas-cades, including the MAPK, PI3K, JAK-STAT, PKA, and PKC pathways.3,16 (Fig. 1)

Multikinase inhibitors

Multikinase inhibitors for thyroid cancer

Multikinase inhibitors (MKIs) that target the RET pathway have been tested for their antitumor activity in patients with thyroid can-cer. Some drugs have shown clinical efficacy, such as vandetanib, cabozantinib, lenvatinib, alectinib, and sorafenib.19 Between them, cabozantinib and vandetanib have been approved for the treatment of locally advanced or metastatic MTC. Vandetanib, an oral RET kinase inhibitor, has shown therapeutic potential in a Phase III trial (ZETA, ClinicalTrials.gov number NCT00410761) in patients that have locally advanced or metastatic MTC. The results of the ZETA study indicated that treatment with vandetanib significantly prolonged the progression-free survival (PFS) in patients that had locally advanced or metastatic MTC (30.5 versus 19.3 months for patients with placebo).20 Similarly, treatment with cabozantinib significantly prolonged the PFS (11.2 versus 4.0 months) of MTC patients in a Phase III trial (EXAM, NCT00704730) with a higher objective response rate [ORR (28% versus 0%)].21 The retrospec-tive analysis of the EXAM trial in two studies revealed that treat-ment with cabozantinib for MTC patients with the RET M918T mutation achieved a better median PFS (61 versus 17 weeks).22,23

Fig. 1. The RET structure and signaling network. RET, rearrangement during transfection; GDNF, glial cell line-derived neurotrophic factor; GFRα, growth factor receptor-alfa; ART, artemin; NTN, neurturin; PSP, persephin; Ca++, calcium ion.



Zhu F.B. et al: Efficacy and safety of RET-selective inhibitorsJ Explor Res Pharmacol

MKIs for NSCLC

MKIs have made some progress in the treatment of RET-associat-ed NSCLC. A multicenter Phase II clinical trial (LURET, UMIN-CTR, UMIN000010095) revealed that treatment with vandetanib achieved an ORR of 53% [95 confidence interval (CI)% 28–77], and a median PFS of 4.7 months (95% CI 2.8–8.5) in previously treated NSCLC patients that harbored RET rearrangements. Fur-ther subgroup analysis indicated that treatment with vandetanib resulted in ORRs of 83% and 20% in patients with CCDC6-RET and KIF5B-RET fusion genes, respectively.24 Furthermore, a Phase II clinical trial (NCT01639508) reported that treatment with cabo-zantinib led to an ORR of 28% (95% CI 12–49) with a median PFS of 5.5 months (95% CI 3.8–8.4) in NSCLC patients.25 This data indicated that MKIs are effective for NSCLC patients with RET fusion, particularly for the common CCDC6-RET and KIF5B-RET fusions. However, whether the efficacy of MKIs is a result of their inhibition of these specific biomarkers needs to be further explored.

Limitations of MKIs

MKIs are usually not selective for targeting RET, and they can target other kinases, such as EGFR, VEGFR-2, KIT, and MET.26 In particular, because the domain of VEGFR-2 kinase has a high degree of homology with RET, several tyrosine kinase inhibitors that target VEGFR-2 (e.g., cabozantinib, vandetanib, and len-vatinib) have shown therapeutic potential for cancer patients with RET alterations to a certain extent.8,27–29 Due to the off-target ef-fect, the inhibitory effect of these MKIs specifically on RET might be limited. Moreover, these MKIs have drug-related toxicity, and increase the dose-reduction rates and treatment-discontinuation rates of drugs, further reducing their clinical applications.17 In addition, these MKIs have developed intrinsic resistance that has limited their clinical application in targeted therapy for RET-altered cancers. The intrinsic resistance might be caused by the fusion between the upstream partner gene KIF5B and RET.16,30 Treatment with MKIs had less efficacy in NSCLC patients that carried KIF5B-RET fusion genes than those without the KIF5B-RET fusion in the LURET study and the Phase I/Ib trail of RXDX-105.31 The acquired resistance to MKIs is probably from specific RET alterations, which result in gatekeeper mutations V804M and V804L on RET.32,33 Particularly, cabozantinib and vandetanib are not effective for NSCLC patients with V804M and V804L muta-tions.34,35

Selective RET inhibitors

Because traditional MKIs have limitations, including off-target ef-fects, treatment-related toxicity, and acquired resistance new and potent inhibitors that selectively inhibit RET have recently been developed and approved for clinical applications for some types of cancers. For example, selpercatinib (RETEVMO or LOXO-292) and pralsetinib (BLU-667) are two small molecule inhibitors with highly selective inhibition of RET and have been approved by the FDA.36,37

Selpercatinib (RETEVMO or LOXO-292)

Compared with MKIs, preclinical studies have shown that

LOXO-292 can selectively target the RET mutants, including gatekeeper resistance mutations and RET fusions compared with MKIs; LOXO-292 exhibits lower toxicity and has low activ-ity against non-RET gene alterations (i.e., VEGFR-2).37 Several clinical studies have been carried out on the treatment of cancer patients.

Selpercatinib for thyroid cancer

Selpercatinib was approved for the treatment of NSCLC and MTC patients with RET-alteration by the FDA on 8 May 2020.38 The multicohort, Phase I/II clinical trial (LIBRETTO-001, NCT03157128) reported that selpercatinib had a significant and long-lasting antitumor activity with an ORR of 69% (n = 38, 95% CI 55–81) and low-grade toxicity in advanced thyroid cancer pa-tients with RET alterations, including patients with RET-mutant MTC resistant to vandetanib or cabozantinib (Table 1). Of inter-est, some patients with RET mutations or gatekeeper resistance responded to selpercatinib although they were resistant to one or two MKIs previously. Treatment with selpercatinib for the patients with MTCs that harbored RET-alteration without previous MKI treatment achieved an ORR of 73% (n = 64, 95% CI 62–83), the median duration of response (DOR) of 22 months (95% CI Not-Estimable NE–NE) and a PFS of 23.6 months (95% CI NE–NE). Furthermore, treatment with selpercatinib for patients with thyroid cancer bearing the RET fusion observed an ORR of 79% (95% CI 54–94), median DOR of 18.4 months (95% CI 7.6–NE) and a PFS of 20.1 months (95% CI 9.4–NE). Of interest, treatment with selp-ercatinib for patients with newly diagnosed thyroid cancer without previous systemic treatment resulted in an ORR of 100% (95% CI 63–100).39,40 Selpercatinib appeared to be safe for humans and there was only grade 1 and 2 of treatment-related adverse effect in a population of 162 patients. There were few cases with a low adverse effect or discontinuer event following selpercatinib treat-ment.39 The low adverse effect of selpercatinib might be attributed to its high selectivity against the RET.

Selpercatinib for NSCLC

In the Phase I/II clinical trial, LIBRETTO-001 (NCT03157128), the therapeutic efficacy of selpercatinib in patients with NSCLC bearing advanced RET fusion was evaluated (Table 1).41 Treatment with selpercatinib 105 NSCLC patients with previous platinum-based chemotherapy obtained an ORR of 64% (n = 67, 95% CI 54–73), median DOR of 17.5 months (95% CI 12.0–NE) and a PFS of 16.5 months (95% CI 13.7–NE). In addition, treatment with selpercatinib benefited 55% of NSCLC patients who received im-munotherapy and 56% of NSCLC patients who had received ≥3 systemic therapies. Of note, 38 out of 105 patients had brain me-tastases, and 11 of them had measurable lesions. The intracranial ORR was 91% (n = 10, 95% CI 95–100), and the median central nervous system (CNS) DOR was 10.1 months (95% CI 6.7–NE). Treatment with selpercatinib obtained an ORR of 85% (n = 33, 95% CI 70–89) in 39 patients with newly diagnosed NSCLC.41 Selpercatinib has a higher therapeutic efficacy in newly diagnosed NSCLC patients than in those with NSCLC refractory common therapies.

Similar to thyroid cancer, selpercatinib treatment resulted in grade 1 and 2 drug-related adverse effects in NSCLC patients. There were a few patients that needed to reduce drug doses or treatment termination.41 Because the most common grade 3 ad-verse reactions are reversible after dose adjustment, long-term




treatment with selpercatinib is feasible. Further analysis of selp-ercatinib safety supported that selpercatinib was relatively safe in a population of 531 patients with NSCLC and thyroid cancer. The most common adverse events during selpercatinib treatment were at grade 1–2, where 30% (n = 160) of patients reduced the drug dose, and 2% (n = 12) discontinued treatment.

Selpercatinib for other cancers with RET-alteration

Preliminary studies have shown that selpercatinib benefits pedi-atric cancer patients bearing RET-alterations. A study reported that treatment with selpercatinib for 1–2 cycles achieved a par-tial response in four pediatric patients with cancers harboring RET fusions (including papillary thyroid cancer and soft-tissue sarco-mas).42 Similarly, treatment with selpercatinib resulted in a partial response in four out of five pediatric cancer patients and the re-maining one achieved stable disease. Several clinical trials are on-going, for example, LIBRETTO-431 (NCT04194944), LIBRET-TO-121 (NCT03899792), and LIBRETTO-321 (NCT04280081), and might extend selpercatinib to other types of cancers that have RET alterations (Table 1).

Pralsetinib (BLU-667)

A preclinical study has shown that BLU-667 can selectively tar-get RET with higher efficiency.36 The Phase I/II study (ARROW study) (ClinicalTrials.gov number NCT03037385) indicated that BLU-667 has better therapeutic efficacy than other MKIs in ad-vanced thyroid cancer and NSCLC.43,44 Pralsetinib was approved by the FDA for the treatment of NSCLC with RET fusion on 4 September 2020.45

Pralsetinib for thyroid cancer

According to the available data from the ARROW study, treatment with pralsetinib achieved an ORR of 65% (95% CI 53–75) in 79 MTC patients with RET mutations. Similarly, pralsetinib treatment resulted in an ORR of 60% (95% CI 46–74), 71% (95% CI 58–85) with 18 month PFS and 90% (95% CI 77–100) with DOR in 53 MTC patient’s resistant to cabozantinib, or vandetanib, or both. Furthermore, treatment with pralsetinib resulted in 74% (95% CI 49–91) of patients with ORR, 85% with 18-month PFS, and 86%

Table 1. Key Clinical trials of selpercatinib and pralsetinib

Agent Condition Phase Status Locations NCT no.

selpercatinib RET fusion-positive solid tumors, MTC, and other tumors with RET activation

II Active China NCT04280081

selpercatinib Advanced solid tumors, lymphomas, or histiocytic disorders with RET activation in pediatric patients( a pediatric MATCH treatment trial)

II Recruiting US NCT04320888

selpercatinib, cabozantinib, vandetanib

RET-mutant MTC III Recruiting Multiple countries NCT04211337

selpercatinib RET fusion-positive solid tumors, MTC, and other tumors with RET activation

I/II Recruiting Multiple countries NCT03157128

selpercatinib, carboplatin,cisplatin, pemetrexed, pembrolizumab

Advanced or metastatic RET fusion-positive NSCLC

III Recruiting Multiple countries NCT04194944

selpercatinib RET fusion-positive advanced NSCLC II Recruiting US NCT04268550

selpercatinib Solidtumors with RET activation (expanded access)

N/A Available Multiple countries NCT03906331

selpercatinib Advanced solid or primary CNS tumors in pediatric patients

I/II Recruiting US NCT03899792

selpercatinib,osimertinib,savolitinib,gefitinib,necitumumab,durvalumab,carboplatin,pemetrexed,alectinib

Advanced NSCLC II Recruiting Multiple countries NCT03944772

pralsetinib Thyroid cancer, NSCLC, and other advanced solid tumors

I/II Recruiting Multiple countries NCT03037385

pralsetinib Unresectable or metastatic MTC or NSCLC

N/A Available N/A NCT04204928

pralsetinib,carboplatin,cisplatin,pemetrexed,pembrolizumab, gemcitabin

Advanced NSCLC III Recruiting Multiple countries NCT04222972

N/A, not applicable; CNS, central nervous system; MTC, medullary thyroid carcinoma; NSCLC, non-small cell lung cancer; NCT, National Clinical Trials




with DOR in patients newly diagnosed MTC.46 Finally, 9 out of 12 patients with thyroid cancer achieved ORR and a median DOR of 14.5 months following pralsetinib treatment.47

Pralsetinib for lung cancer

In the ARROW trial, pralsetinib treatment was effective for pa-tients with NSCLC bearing RET fusion, including an ORR of 57% (95% CI 46–68) in patients with previous cisplatin chemotherapy. Furthermore, treatment with pralsetinib achieved an ORR of 59% (95% CI 42–74) in a cohort of 39 patients without anti-PD-1 or anti-PD-L1 treatment. Similarly, pralsetinib treatment resulted in an ORR of 70% (95% CI 50–86), a median DOR of 9.0 months (95% CI 6.3–NE) in the untreated cohort (n = 2 7). After 8 weeks of treatment with pralsetinib, 90% of NSCLC patients eliminated plasma ctDNA of the RET variant and 90% of them reduced plas-ma ctDNA levels by ≥50%.48

Pralsetinib for other solid tumors with RET-alteration

In addition to thyroid cancer and NSCLC, pralsetinib has been used for the treatment of other solid tumors with RET alterations. Following pralsetinib treatment, three out of five patients respond-ed, including two advanced pancreatic cancer patients with a par-tial response and a DOR of 5.5 months; two colon cancer patients with stable disease; an intrahepatic bile duct carcinoma patient with a DOR of 7.5 months.47 Several clinical trials, such as the ARROW trial and AcceleRET Lung study (NCT04222972), are ongoing to test the therapeutic efficacy and safety of pralsetinib. The analysis of 438 patients that received pralsetinib showed that the treatment-related adverse effects of pralsetinib were in grades 1–2, and only 4% of patients discontinued treatment, similar to that of selpercatinib.46

Future directions

Several problems need to be solved urgently. The emergence of po-tent and highly selective RET inhibitors has led to the development of acquired resistance. The mechanisms underlying the resistance of these inhibitors need to be explored. Precise therapy for cancers that harbor RET alterations requires accurate diagnosis. Therefore, the detection of RET alterations in tumors faster and accurately (including RET structural variants of unknown significance and uncommon RET-alteration) is a challenge for future research. In addition, the exploration of the next-generation of selective RET inhibitors and the combination of existing RET inhibitors and agents that target other pathways might provide new strategies for the clinical treatment of RET-altered tumors.

Conclusions

In the last few decades, the role of RET proto-oncogene mutations and rearrangements in the development of several malignancies have been clarified. Treatment with MKIs has achieved certain efficacy in tumor patients with RET alterations. However, due to their off-target effects, the inhibition of non-RET targets, includ-ing the VEGFR-2, leads to significant dose toxicity, limiting their long-term administration. Selpercatinib and pralsetinib are new se-lective RET inhibitors, which are well tolerated and have reproduc-

ible and significant antitumor activity. Therefore, these drugs have been approved for clinical application.

Acknowledgments

We apologize for not being able to cite all the publications related to this topic due to a space limitation.

Funding

This work was supported by the Sichuan Science and Technology Program (No.2019YJ0070; 2021YFS0111).


The authors declare no conflict of interest.


All the listed authors made substantial contributions to: conception and design, and/or acquisition of data, and/or analysis and inter-pretation of data; and all the authors gave final approval of the version to be published. Each author has participated sufficiently in the work to take public responsibility for appropriate portions of the content. The details are listed as follows: study concept and design and obtained funding (LYZ); draft manuscript and analysis and interpretation of data (FBZ, QHG).

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https://doi.org/10.1093/annonc/mdz268.093




Case Report

Introduction

Amyloidosis (AL) is a group of rare diseases and pathologically is characterized by abnormal deposition of fibril-like insolu-ble amyloid protein in body organs, causing organ damage that leads to death. There are approximately 60 heterogeneous amy-

loidogenic proteins, and 27 of these are associated with known human diseases, affecting the liver, kidney, peripheral nervous system, and heart.1 If the bone marrow is involved, the case may be linked with multiple myeloma.2 Without optimal treatment, AL has a very high death rate, of approximately 75% within a 2-year period after diagnosis.3 AL can be diagnosed pathologi-cally and classified by immunohistochemistry and mass spec-trometry.4 However, there has been no case reported from Sudan, Here, we report a male case of renal AL, possibly associated with abdominal tuberculosis (Tb). This case report should serve as an alert of clinical attention to physicians in the high-prevalence Tb regions.

Case report

A male patient, 30 years-old, was brought to Haj-Elsafi General Hospital, Khartoum, Sudan, on March 2019. He complained of systemic body swelling that had lasted for 2 months. He reported having begun to develop bilateral lower limb swelling, which was

A Male Case of Renal Amyloidosis

Ziryab Imad Taha1,2* , Mohammed Elmujtba Adam Essa2,3* , Asaad Tageldein Idris Abdelhalim4, Mohey Aldein Ahmed Elamin Elnour5, Allaa Ahmed Osman Eltayeb6, Shaza Adel Awad Mohammed Elwakeel7 and Abdelkareem Abdallah Ahmed2,8,9,10*

1Department of Internal Medicine, Faculty of Medicine, University of Bahri, Khartoum, Sudan; 2Department of Clinical Medicine, Medical and Can-cer Research Institute (MCRI), Nyala, Sudan; 3Faculty of Medicine, Alfashir University, Al Fashir, Sudan; 4Department of Clinical Immunology, Sudan Medical Specialization Counsel, Khartoum, Sudan; 5Faculty of Medicine, Omdurman Islamic University, Khartoum, Sudan; 6Faculty of Medicine, Ahfad University for Women, Khartoum, Sudan; 7Faculty of Medicine and Health Science, University of Gadarif, Gadarif, Sudan; 8Department of Physiology and Biochemistry, Faculty of Veterinary Science, University of Nyala, Nyala, Sudan; 9Biomedical Research Institute, Darfur University College, Nyala, Sudan; 10Institute of Molecular Biology, University of Nyala, Nyala, Sudan

Received: September 28, 2020 | Revised: October 29, 2020 | Accepted: November 10, 2020 | Published: November 24, 2020

Abstract

Amyloidosis is a group of rare, serious disorders caused by deposition of amyloid protein in tissues, such as the kidney, heart and brain. However, there is no case reported from Sudan. Here, we report one male case of renal amyloidosis, possibly secondary to abdominal tuberculosis (Tb). A male, 30 years of age, complained of systemic body swelling, shortness of breath, and decreased urine output with abnormal color for 2 months. He had been diagnosed with abdominal Tb 10 years prior, for which he received systemic anti-Tb treatment. Clinical examina-tion exhibited anasarca, particularly in the abdomen. Abdominal ultrasound indicated massive ascites, and echo-cardiography indicated the ejection fraction reduced to 60%. Renal biopsy revealed renal amyloidosis. The patient was treated with ceftriaxone, furosemide, prednisolone, pantoprazole, spironolactone, calcium and mycopheno-late mofetil, and his condition improved. The patient was discharged 2 weeks after treatments. Hence, this is the first case of renal amyloidosis, possibly secondary to abdominal Tb, in Sudan. This case report should serve as an alert to physicians working in high-prevalence Tb regions.

Keywords: Secondary amyloidosis; Anasarca; Renal biopsy; Tuberculosis.Abbreviations: AL, amyloidosis; Tb, tuberculosis.*Correspondence to: Ziryab Imad Taha, Department of Internal Medicine, Faculty of Medicine, University of Bahri, Khartoum, Sudan. ORCID: https://orcid.org/0000-0001-9104-1737; Mohammed Elmujtba Adam Essa, Department of Clinical Medi-cine, Medical and Cancer Research Institute, Nyala, Sudan. ORCID: https://orcid.org/0000-0002-1050-2771, Tel: 00249907009389, E-mail: [email protected]; Abdelkareem Abdallah Ahmed, Department of Physiology and Biochemistry, Fac-ulty of Veterinary Science, University of Nyala, Nyala, P.O. Box: 155 Nyala, Sudan. ORCID: https://orcid.org/0000-0003-1524-0392, E-mail: [email protected] to cite this article: Taha ZI, Adam Essa ME, Idris Abdelhalim AT, Elamin El-nour MAA, Osman Eltayeb AA, Mohammed Elwakeel SAA, Abdallah Ahmed A. A Male Case of Renal Amyloidosis. J Explor Res Pharmacol 2021;6(1):23–27. doi: 10.14218/JERP.2020.00031.



https://orcid.org/0000-0001-9104-1737

https://orcid.org/0000-0002-1050-2771

https://orcid.org/0000-0003-1524-0392

https://orcid.org/0000-0001-9104-1737

https://orcid.org/0000-0001-9104-1737

https://orcid.org/0000-0002-1050-2771

https://orcid.org/0000-0002-1050-2771


https://orcid.org/0000-0003-1524-0392



Taha Z.I. et al: A male case of renal amyloidosisJ Explor Res Pharmacol

more severe while standing and walking and which also started 2 months prior (Fig. 1). One month prior to hospital presentation, he noticed scrotal, abdominal and facial swelling (Fig. 1). He had shortness of breath with exertion and when lying down. He report-ed that his urine output was reduced and frothy in the morning, but without obvious burning or pain sensations and without symptoms related to urinary tract obstruction. He reported no fever, fatigue, no weight loss, appetite change, vomiting, abdominal pain, change in bowel habit, headache, memory functional change, nor effects of muscular movement.

The patient reported smoking tobacco moderately and drink-ing alcohol occasionally. He was allergic to penicillin. He had no diabetes, hypertension nor chronic cardiovascular disease. He had been diagnosed with abdominal Tb 10 years prior and received regular anti-Tb treatments. His family members were generally healthy, with no specific reports of illness or conditions.

Physical examination found that, in general, he was weak but not pale or jaundiced. Abdominal examination detected a distended abdomen, with full flanks, positive shifting dullness, fluid thrill and pitting edema in the lower limbs up to the knee.

Abdominal ultrasound indicated massive ascites and mild liver enlargement with low homogeneous texture. Echocardiography revealed ejection fraction of 60% and sinus tachycardia with-out abnormal valves, general work up done (Table 1), He also underwent a renal biopsy. His renal tissue sections were stained with hematoxylin-eosin, periodic acid Schiff (PAS), Mucin Stain (MS) and sliver and represented 70% renal cortex and 40% me-dulla, muscles, and adipose tissues. Pathologically, his renal tis-sues displayed a wide eosinophilic mesangial increase extended to the loops of the glomerular capillary, a hallmark of renal amy-loidosis (Fig. 2).

Given his past history of abdominal Tb, unexplained systemic body edema, particularly for massive ascites, and typical pathological characteristics of his renal tissue sections, he was diagnosed with AL, possibly secondary to previous abdominal Tb. The patient was treat-ed with 1 g ceftriaxone b.i.d for 5 days, 20 mg injectable furosemide b.i.d for 3 days, 30 mg prednisolone daily tapered by 5 mg weekly, 20 mg pantoprazole daily, 25 mg spironolactone daily, 500 mg calcium daily, and 500 mg mycophenolate mofetil b.i.d. Two weeks later, his overall condition had improved and he was discharged.

Fig. 1. Patient display of systemic edema. (a) Lower limb edema. (b) Ascites. (c) Sacral edema. (d) Facial swelling.



Taha Z.I. et al: A male case of renal amyloidosis J Explor Res Pharmacol

Fig. 2. Pathological findings of biopsied renal tissue. The section shows renal amyloidosis, wide mesangial increase by eosinophilic and noncellular material, extended to the loops of the glomerular capillary (H and E stain, original magnification ×400).

Table 1. Lab test results for the patient

Parameter Result Reference

White blood cell count 8,000 cells/mcl 4–11×109/L

Hemoglobin 13.2 g/dL 12–16g/dL

Platelet count 245 cells/mcl 150–450 cells/mcl

Erythrocyte sedimentation rate 120 mm/h normal reference up to 20 mm/h

Serum urea 24 mg/dL 5–20 mg/dL

Serum creatinine 0.8 mg/dL 0.5–1.1 mg/dL

Serum Sodium 137 mmol/L 135–145 mmol/L

Serum Potassium 3.5 mmol/L 3–3.5 mmol/L

Serum albumin 4.4 g/dL 2.4–4 g/dL

Serum globulin 2.1 g/dL 2–3.5 g/dL

Total protein 6.5 g/dL 6–8.3 g/dL

Total bilirubin 0.56 mg/dL 0.2–1.3 mg/dL

Direct bilirubin 0.23 mg/dL 0.2–0.3 mg/dL

Indirect bilirubin 0.33 mg/dL 0.2–0.3 mg/dL

Alanine aminotransferase 15 U/L 10–130 U/L

Aspartate aminotransferase 43 U/L 10–34 U/L

Alkaline phosphatase 65 U/L (24–147 UL) 24–147 UL

Urine general ++++ Protein, oval fat deposition ++

7–9 pus

Fatty cast ++

24 Urine proteins 9.990 150 mg/day



Taha Z.I. et al: A male case of renal amyloidosisJ Explor Res Pharmacol

Discussion

Renal AL is a rare disease and hard to diagnose because of its early unspecific symptoms, particularly before onset of organ failure.5 AL can display systemic or localized symptoms, such as fatigue and weight loss, which usually occur after an organ has become severely damaged. Our patient complained of symptoms similar to nephrotic syndrome lasting for 2 months. Some multiple my-eloma patients may present with similar symptoms, but for our patient this was excluded by the absent evidence of malignancy.2 Renal AL patients usually die of both renal and heart failure.6 For-tunately, our patient achieved improvement in clinical symptoms after treatments that lasted for 2 weeks. Hence, early diagnosis and treatment of AL are crucial for saving an AL patient’s life.

Secondary AL can occur during the progression of many infec-tious and chronic inflammatory diseases,7 such as familial Medi-terranean fever in Turkish people,8 juvenile idiopathic arthritis, rheumatoid arthritis, inflammatory bowel diseases and ankylosing spondylitis in western countries.9 In developing countries, Tb and other infectious diseases remain the most common predisposing factors for secondary AL, with a declining trend.10 Our patient had a history of abdominal Tb and received anti-TB therapies. When he visited our hospital, he had massive ascites accompanied by renal and liver abnormalities. Because of the lack of evidence of an abdominal solid mass, we suspected that he had a recurrent abdominal Tb, which can occur, especially in the highly endemic countries for Tb, such as Sudan.11 Unfortunately, we had no micro-biological evidence for diagnosis of abdominal Tb due to techni-cal difficulty in our hospital. It is possible that this patient may have had a renal AL secondary to abdominal Tb. Thus, physicians should pay special attention to those patients with abdominal Tb for potentially secondary AL, particularly in Tb epidemic regions.

AL is commonly diagnosed by histology and laboratory tests as well as by clinical symptoms, including evidence of apple-green birefringence in the affected tissues and findings from serum-free light chain assay.12,13 We did observe these pathological changes in renal biopsied tissues. Furthermore, our patient exhibited impaired heart function and we also detected abnormal echocardiograms.14 These findings, together with impaired renal function and systemic edema, prompted us to diagnose him with AL. Conceivably, con-sideration and performance of renal biopsy for histological exami-nation are important for accurate diagnosis of renal AL.

Although therapeutic management of renal AL has been reported for many years, there is currently no specifically effective treat-ment for AL. Suppression of inflammation is the principle strategy for treatment of AL. This will decrease early phase reactants and lead to regression or stabilization of amyloid deposition.10 In addi-tion, therapeutic treatment against interleukin-1 and tumor necrosis factor-alpha have been tried in AL patients.15,16 A more anticipated approach to treatment of AL is the targeting of amyloid deposits. Treatments of renal AL to stabilize amyloid fibrils have been devel-oped recently and have improved the prognosis for those patients.17 We treated our patient with a combination of several drugs to effec-tively ameliorate his clinical symptoms within 2 weeks. Therefore, combination of multiple arms of treatment to manage renal AL pa-tient may be valuable for improving the prognosis of AL.

Conclusion

Renal AL is a rare disease that occurs due to deposition of amyloid in tissues and organs. Its diagnosis is usually difficult, due to its unspecific symptomology. We report a case of renal AL, demon-

strated by renal tissue pathology. We found that combination of several drugs for treatment of renal AL effectively improved its clinical symptoms. Given that many secondary AL cases are ne-glected and missed for its diagnosis, this report should serve to alert clinicians to pay special attention to secondary AL while making differential diagnoses because of its potential for severe consequences without optimal treatment, particularly in high epi-demic regions of Tb, like Sudan.

Acknowledgments

The authors wish to acknowledge the support of Medical and Can-cer research Institute (MCRI).

Ethical statement

The authors have obtained the written consent from the patient to publish this case report.

Funding

This study was funded by Medical and Cancer Research Institute (MCRI), Sudan.


The authors declare no competing interests.


ZIT is the supervisor rheumatologist who diagnose and manage the patient; follow-up, data collections and manuscript writing (MEAE, ATIA, MAEE, AAOE, SAAM), AAA contributed by critical revision of the study. All authors read and approved the final manuscript.

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