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Proceeding Paper

Investigation of the Effect of Selected Piperazine-2,5-Diones on Cartilage-Related Cells †

1
Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University, Ilkovicova 6, 842 15 Bratislava, Slovakia
2
Department of Pharmacology and Toxicology, Veterinary Research Institute, Hudcova 296/70, 621 00 Brno, Czech Republic
3
Department of Chemical Drugs, Faculty of Pharmacy, Masaryk University, Palackeho 1946/1, 612 00 Brno, Czech Republic
*
Author to whom correspondence should be addressed.
Presented at the 25th International Electronic Conference on Synthetic Organic Chemistry, 15–30 November 2021; Available online: https://ecsoc-25.sciforum.net/.
Chem. Proc. 2022, 8(1), 108; https://doi.org/10.3390/ecsoc-25-11650
Published: 13 November 2021

Abstract

:
Various chronic inflammatory diseases have become a problem, especially in the Western world. Whether it concerns inflammation of visceral organs, joints, bones, etc., it is always a physiological reaction of the body, which always tries to eradicate harmful substances and restore tissue homeostasis. Unfortunately, prolonged or chronic inflammation often results in damage to the affected tissues. Diseases such as osteoarthritis, rheumatoid arthritis, and arthrosis, as well as cartilage damage, are very common. In addition to suppressing inflammation in the joints and around the cartilage, it is advantageous to administer compounds that are capable of stimulating cartilage growth and regenerating damaged tissue. Variously substituted piperazine-2,5-dione derivatives were investigated as compounds with a potential effect on cartilage regeneration. A series of assays were performed to evaluate their cytotoxicity, anti-inflammatory activity, and ability to potentiate chondrocyte proliferation and suppress synovial cell growth. The compounds proved to be completely non-toxic for all used types of cells up to the concentration of 20 µM. Unfortunately, their evaluated biological activity proved to be insignificant based on the comparison with untreated cells.

1. Introduction

Degenerative diseases of the bones and joints affect millions of people. Fractures of the hands, hips, and spine caused by osteoporosis are associated with significant morbidity and mortality. Destruction and deformity of the joints and other complications caused by arthritis not only make movement difficult, but reduce the ability to perform routine activities, resulting in an overall reduced quality of life for patients, among other things [1,2,3,4,5].
Many different treatment approaches are being developed for the burning problem of increasingly common musculoskeletal degenerative diseases. Treatment options for musculoskeletal disorders are non-pharmacological, pharmacological, and surgical. These are incurable diseases for this moment, so the goal of treatment is to achieve remission or low activity of the disease. The longer the treatment takes to start, the worse the results, including irreversible damage to the joints. Non-pharmacological treatment is based on regular exercise (weight reduction and physical activity), rehabilitation, and manipulation therapy to strengthen muscles and maintain maximum mobility and joint functionality. In the advanced stages of the disease, some damaged joints can be surgically removed and replaced with artificial implants (endoprostheses of the hip, knee, shoulder, elbow, wrist, and finger joints). The surgical treatment of the patient also relieves pain in the affected joint. Pharmacological treatment includes two basic groups of drugs, which are usually combined: drugs that reduce inflammation and pain, and drugs that reduce the progression of structural damage, i.e., inhibit the destruction of articular cartilage and induce the balance of its metabolism. Non-steroidal anti-inflammatory drugs and paracetamol are used to reduce inflammation and pain. In case of acute inflammation, glucocorticoids can be given. Conventional synthetic (e.g., methotrexate), targeted synthetic JAK kinase inhibitors, or biologicals (antibodies) are used as antirheumatics. In this context, it is necessary to mention that there are also many dietary supplements on the market that are intended to prevent or alleviate diseases of the musculoskeletal system. Agents that inhibit the destruction of articular cartilage are so-called chondroprotectives. Currently recommended are glucosamine sulfate, chondroitin sulfate, hyaluronic acid, avocado-soybean unsaponifiables, diacerein, Boswellia serrata extract, curcumin, S-adenosyl methionine, methylsulfonylmethane, and rose hip. Alternatively, fish liver oil; omega-3 fatty acids; vitamins A, C, and E in combination; vitamin K; vitamin D; ginger; and collagen/gelatin are listed as beneficial dietary supplements [6,7,8,9,10,11,12,13].
The long-term administration of most of the above-mentioned drugs has negative effects on other organs; thus, in accordance with the concept of polypharmacology and multi-target drugs, efforts have been made to design agents that have the ability to regenerate both cartilage and bone while exhibiting anti-inflammatory activity. In addition, these agents must be non-toxic in order to be administered to long-term chronically ill patients [14,15,16]. Alaptide ((S)-8-methyl-6,9-diazaspiro-[4.5]decan-7,10-dione) was chosen as a model molecule, which showed high regenerative abilities on the skin and mucosa and no chronic toxicity in previous studies. In addition, this compound exhibits other remarkable biological properties. Alaptide was prepared at the Research Institute for Pharmacy and Biochemistry in Prague in the former Czechoslovakia in the 1980s [17,18,19,20,21,22,23]. A disadvantage of alaptide is its practical insolubility, so more soluble simple derivatives were designed and prepared, and all compounds were evaluated using a set of in vitro assays for the required biological activities.

2. Results and Discussion

The structures of all the investigated compounds are listed in Table 1 together with their biological activities. The preparation procedure of the compounds was described previously [19,23].
The basic safety profile of the tested compounds was evaluated based on the determined relative cell viability of different cell types related to cartilage tissue (monocytes THP-1, synovial cells SW982, and primary porcine chondrocytes). Neither alaptide (1) nor other piperazine-2,5-dione derivatives 25 significantly influenced cell viability, which was still 90–120% when cells were incubated for 72 h with the highest concentrations of the compounds (20 µM for THP-1 and 30 µM for SW982 and chondrocytes). The tested molecules did not show any cytotoxic effect or pro-proliferative action. Similarly, no toxic effect was observed for the same compounds tested up to the concentration of 50 µM on human skin fibroblast cells (BJ), a T-lymphoblastic leukemia cell line CEM, and a breast adenocarcinoma cell line MCF7 [23].
To determine the anti-inflammatory potential of the tested compounds, their effect on the lipopolysaccharide (LPS)-stimulated activation of NF-κB, one of the key pro-inflammatory transcription factors, was evaluated. In this assay, the used agents were not able to reduce the NF-κB activity in the concentration of 10 µM.
All obtained assay results showed that the tested alaptide (1) and its derivatives 25 are not able to influence the pathological features of rheumatoid arthritis. On the other hand, they have a very low cytotoxic effect on different cell types and thus are safe for further biological experiments.

3. Experimental

3.1. Synthesis

The described piperazine-2,5-diones were characterized by Pokorna et al. [23].

3.2. Cell Lines Culture

Human synovial SW982 and human monocytic leukemia THP-1 cell lines (both from ATCC, Manassas, VA, USA) were routinely cultivated in RPMI 1640 medium with glutamine supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin mixture (all from Merck, St. Louis, MO, USA). Cells were passaged twice a week, and their viability was regularly controlled by Trypan Blue staining.

3.3. Primary Porcine Chondrocytes Isolation

The cartilage tissue was obtained from porcine elbow joint from slaughtered pigs in a local slaughterhouse. Approximately 300 mg of tissue was twice washed in sterile phosphate buffered saline (PBS; Merck) and cut by a scalpel to 1 mm3 pieces approximately. Cartilage pieces were covered by the solution of 6 mg/mL of collagenase I (Merck) in DMEM/F12 medium (Biosera, Nuaille, France) and incubated at 37 °C for 2 h until all parts were completely lysed. After that, the enzyme was inactivated by adding DMEM/F12 medium containing 10% FBS and 1% penicillin/streptomycin mixture. The cell suspension was filtrated through a 70 µm nylon membrane and centrifuged at 150 g force for 5 min. Then, the supernatant was discarded, and the cells were resuspended in a fresh medium, counted using the Trypan Blue dye, and split into cultivation plates coated by collagen I (Corning; Kennebunk, ME, USA) at the density of 5 × 103/cm2. The cells were incubated at 37 °C in humidify atmosphere with 5% CO2 for 5 days. After this period, the medium was exchanged, and the cells were ready for further experiments.

3.4. Cell Viability Determination

To determine cell viability, a Cell Counting Kit 8 (CCK-8; Merck) was used according to the manufacturer’s instruction. All experiments were performed in the complete cultivation medium containing 10% FBS. SW982 cells were split into 96-well plate in the concentration of 1 × 104 cells per well and let to attach overnight. Then, the medium was exchanged. THP-1 cells were seeded in the concentration of 5 × 104 cells per well. Primary chondrocytes were used after 5-day attachment, as described above. When the cells were prepared, they were treated by the tested compounds dissolved in dimethyl sulfoxide (DMSO), and the relative cell viability (the ratio between cells treated with compounds and cells treated with DMSO only) was measured after 72 h, as we described previously [24].

3.5. NF-κB Activity Determination

The ability of the tested compounds to inhibit the transcription factor NF-κB, one of the key pro-inflammatory intracellular regulators, was evaluated on THP-1 Blue NF-κB cell line (Invivogen; San Diego, CA, USA), as we described previously [25]. NF-κB was activated by lipopolysaccharide (LPS) from E. coli 0111:B4 (Merck) and was dissolved in serum-free RPMI 1640 medium (1 g/mL) after 1 h pre-treatment with the tested compounds dissolved in DMSO in the concentration of 10 µM.

Author Contributions

Conceptualization, J.J.; methodology, J.H. and J.J.; investigation, J.H., P.B. and J.J.; writing, J.H., P.B. and J.J.; funding acquisition, J.H. and J.J. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Slovak Research and Development Agency (APVV-17-0373). The work of J.H. was supported by the Ministry of Education, Youth, and Sports of the Czech Republic under the project “FIT” CZ.02.1.01/0.0/0.0/15_003/0000495, and Czech Ministry of Agriculture grant no. RO0518.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

The authors thank Cupalova for her help with a cell viability assay and to Novobilsky for his assistance with cartilage tissue preparation.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Musculoskeletal Conditions, WHO. 2021. Available online: https://www.who.int/news-room/fact-sheets/detail/musculoskeletal-conditions (accessed on 14 September 2021).
  2. Nakamura, K.; Ogata, T. Locomotive syndrome: Definition and management. Clin. Rev. Bone Miner. Metab. 2016, 14, 56–67. [Google Scholar] [CrossRef] [PubMed]
  3. Osteoporosis, National Institutes of Health, USA. Available online: https://www.niams.nih.gov/health-topics/osteoporosis (accessed on 14 September 2021).
  4. Arthritis. Centers for Disease Control and Prevention, USA; 2021. Available online: https://www.cdc.gov/arthritis/index.htm (accessed on 14 September 2021).
  5. Osteoarthritis. Mayo Clinic, Mayo Foundation for Medical Education and Research 2021. Available online: https://www.mayoclinic.org/diseases-conditions/osteoarthritis/symptoms-causes/syc-20351925 (accessed on 14 September 2021).
  6. Abbasi, M.; Mousavi, M.J.; Jamalzehi, S.; Alimohammadi, R.; Bezvan, M.H.; Mohammadi, H.; Aslani, S. Strategies toward rheumatoid arthritis therapy; the old and the new. J. Cell. Physiol. 2019, 234, 10018–10031. [Google Scholar] [CrossRef] [PubMed]
  7. Smolen, J.S.; Landewe, R.B.M.; Bijlsma, J.W.J.; Burmester, G.R.; Dougados, M.; Kerschbaumer, A.; McInnes, I.B.; Sepriano, A.; van Vollenhoven, R.F.; de Wit, M.; et al. EULAR recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2019 update. Ann. Rheum. Dis. 2020, 79, 685–699. [Google Scholar] [CrossRef] [PubMed]
  8. Ferro, M.; Charneca, S.; Dourado, E.; Guerreiro, C.S.; Fonseca, J.E. Probiotic supplementation for rheumatoid arthritis: A promising adjuvant therapy in the gut microbiome era. Front. Pharmacol. 2021, 12, 711788. [Google Scholar] [CrossRef] [PubMed]
  9. Grassel, S.; Muschter, D. Recent advances in the treatment of osteoarthritis. F1000Research 2020, 9, 325. [Google Scholar] [CrossRef] [PubMed]
  10. Oo, W.M.; Little, C.; Duong, V.; Hunter, D.J. The development of disease-modifying therapies for osteoarthritis (DMOADs): The evidence to date. Drug Des. Devel. Ther. 2021, 15, 2921–2945. [Google Scholar] [CrossRef] [PubMed]
  11. Cao, J.H.; Feng, D.G.; Wang, Y.Z.; Zhang, H.Y.; Zhao, Y.D.; Sun, Z.H.; Feng, S.G.; Chen, Y.; Zhu, M.S. Chinese herbal medicine Du-Huo-Ji-Sheng-decoction for knee osteoarthritis: A protocol for systematic review and meta-analysis. Medicine 2021, 100, e24413. [Google Scholar] [CrossRef] [PubMed]
  12. Jampilek, J.; Kos, J.; Kralova, K. Potential of nanomaterial applications in dietary supplements and foods for special medical purposes. Nanomaterials 2019, 9, 296. [Google Scholar] [CrossRef] [PubMed]
  13. Placha, D.; Jampilek, J. Chronic inflammatory diseases, anti-inflammatory agents and their delivery nanosystems. Pharmaceutics 2021, 13, 642019. [Google Scholar] [CrossRef] [PubMed]
  14. Talevi, A. Multi-target pharmacology: Possibilities and limitations of the “skeleton key approach” from a medicinal chemist perspective. Front. Pharmacol. 2015, 6, 205. [Google Scholar] [CrossRef] [PubMed]
  15. Ramsay, R.R.; Popovic-Nikolic, M.R.; Nikolic, K.; Uliassi, E.; Bolognesi, M.L. A perspective on multi-target drug discovery and design for complex diseases. Clin. Transl. Med. 2018, 7, 3. [Google Scholar] [CrossRef] [PubMed]
  16. Micheli, L.; Bozdag, M.; Akgul, O.; Carta, F.; Guccione, C.; Bergonzi, M.C.; Bilia, A.R.; Cinci, L.; Lucarini, E.; Parisio, C.; et al. Pain relieving effect of-NSAIDs-CAIs hybrid molecules: Systemic and intra-articular treatments against rheumatoid arthritis. Int. J. Mol. Sci. 2019, 20, 1923. [Google Scholar] [CrossRef] [PubMed]
  17. Kasafirek, E.; Vanzura, J.; Krejci, I.; Krepelka, J.; Dlabac, A.; Valchar, M. 2,5-Piperazinedione Derivs. Belgian Patent 897843, 20 May 1984. Czechoslovakian Patent CS 231227, 26 January 1986. [Google Scholar]
  18. Radl, S.; Kasafirek, E.; Krejci, I. Alaptide. Drug. Future 1990, 15, 445–447. [Google Scholar] [CrossRef]
  19. Kasafirek, E.; Rybak, M.; Krejci, I.; Sturs, A.; Krepela, E.; Sedo, A. Two-step generation of spirocyclic dipeptides from linear peptide ethyl ester precursors. Life Sci. 1992, 50, 187–193. [Google Scholar] [CrossRef]
  20. Jampilek, J.; Opatrilova, R.; Coufalova, L.; Cernikova, A.; Dohnal, J. Utilization of Alaptide as Transdermal Penetration Modifier in Pharmaceutical Compositions for Human and Veterinary Applications Containing Anti-Inflammatory Drugs and/or Antimicrobial Chemotherapeutics. WO/2013/020527 A1, 14 February 2013. [Google Scholar]
  21. Jampilek, J.; Dohnal, J. Alaptide as transdermal permeation modifier. In Percutaneous Penetration Enhancers—Chemical Methods in Penetration Enhancement: Modification of the Stratum Corneum; Dragicevic-Curic, N., Maibach, H.I., Eds.; Springer: Berlin/Heidelberg, Germany, 2015; pp. 115–132. [Google Scholar]
  22. Cernikova, A.; Bobal, P.; Bobalova, J.; Dohnal, J.; Jampilek, J. Investigation of permeation of acyclovir through skin using alaptide. Acta Chromatogr. 2018, 30, 62–65. [Google Scholar] [CrossRef]
  23. Pokorna, A.; Bobal, P.; Oravec, M.; Rarova, L.; Bobalova, J.; Jampilek, J. Investigation of permeation of theophylline through skin using selected piperazine-2,5-diones. Molecules 2019, 24, 566. [Google Scholar] [CrossRef] [PubMed]
  24. Kos, J.; Kozik, V.; Pindjakova, D.; Jankech, T.; Smolinski, A.; Stepankova, S.; Hosek, J.; Oravec, M.; Jampilek, J.; Bak, A. Synthesis and hybrid SAR property modeling of novel cholinesterase inhibitors. Int. J. Mol. Sci. 2021, 22, 3444. [Google Scholar] [CrossRef] [PubMed]
  25. Hosek, J.; Kos, J.; Strharsky, T.; Cerna, L.; Starha, P.; Vanco, J.; Travnicek, Z.; Devinsky, F.; Jampilek, J. Investigation of anti-inflammatory potential of N-arylcinnamamide derivatives. Molecules 2019, 24, 4531. [Google Scholar] [CrossRef] [PubMed]
Table 1. Structure and values of viability of THP-1, SW982, and primary porcine chondrocytes (Chondr.) assays [IC50 (µM) after 72 h incubation] of investigated compounds—alaptide (1) and its derivatives 25.
Table 1. Structure and values of viability of THP-1, SW982, and primary porcine chondrocytes (Chondr.) assays [IC50 (µM) after 72 h incubation] of investigated compounds—alaptide (1) and its derivatives 25.
Chemproc 08 00108 i001
Comp.R1R2R3Tox IC50 [μM] (72 h)
THP-1SW982Chondr.
1–(CH2)4−CH3>20>30>30
2−H−H−H>20>30>30
3−H−H−CH3>20>30>30
4−CH3−H−CH3>20>30>30
5−CH3−CH3−CH3>20>30>30
THP-1 = human monocytic leukemia; SW982 = human synovial cell line; Chondr. = primary porcine chondrocytes.
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MDPI and ACS Style

Jampilek, J.; Hosek, J.; Bobal, P. Investigation of the Effect of Selected Piperazine-2,5-Diones on Cartilage-Related Cells. Chem. Proc. 2022, 8, 108. https://doi.org/10.3390/ecsoc-25-11650

AMA Style

Jampilek J, Hosek J, Bobal P. Investigation of the Effect of Selected Piperazine-2,5-Diones on Cartilage-Related Cells. Chemistry Proceedings. 2022; 8(1):108. https://doi.org/10.3390/ecsoc-25-11650

Chicago/Turabian Style

Jampilek, Josef, Jan Hosek, and Pavel Bobal. 2022. "Investigation of the Effect of Selected Piperazine-2,5-Diones on Cartilage-Related Cells" Chemistry Proceedings 8, no. 1: 108. https://doi.org/10.3390/ecsoc-25-11650

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