Phyto-Research

Phyto-Research

Translational Research On COVID

Covid as a pandemic has created significant morbidity and mortality asking for a solution. Atrimed Pharmaceuticals, being specialized in drug development based on plant based molecules, has a systematic approach for drug development. The steps include target identification, Lead generation, lead optimization, ligand target binding in silico studies, MD Simulation, in vitro studies, in vivo studies and human trials.

spike glycoprotein, RdRp, 3CLPro, PLPro,E-Protein and M-Protein are the drug targets which have been identified and worked upon.  Understanding of plant chemistry and computational chemistry takes significant role in lead generation. Since we have a library of plant molecules, initial screening is done by looking for simulation studies for target ligand binding. Lead optimization is carried out by making changes at chemical moieties to simplify the synthesis, reduce the size or improve the binding. Subsequent  retrospective similarity search can identify the potential plants. A poly pharmacology approach to identify a molecule for multiple targets or, multiple molecules in single plant towards same or multiple targets or multiple molecules in multiple plants for different or same targets are some of the strategies to be adopted. All these strategies need to be reconfirmed in in vitro as well as in vivo experiments before trying them in human beings.

Through good understanding of medicinal chemistry, analytical chemistry and Synthesis, team of chemists can contribute for getting physical samples for experiments.

Thus a translational work between traditional medicine practitioners, computation biologists, medicinal chemists, synthetic chemists, Microbiologists/Molecular biologists/Biotechnologists, veterinarians/Animal pharmacologists and finally clinical pharmacologists leads to drug development at Atrimed Pharmaceuticals. The drug development for Covid is currently at Phase-1 and will resume to Phase 2 & 3 soon.

Covid-19 and related elisa tests

 As COVID-19 is a new disease, new blood tests to diagnose, followup and treat paLents needed to be developed very quickly. The first tests to come up were ELISA-based tests for anLbodies against the viral surface proteins S (spike) and N (nucleocapsid). There are 2 different tests for S-protein which detect 2 different parts of the protein – S1 and RBD. As there are other coronaviruses like SARS and MERS which are very similar, it was important to make sure that these tests only detected COVID-19 specifically and not other viruses. These studies showed that the test for S1 is the best and results in least number of false posiLves. Similar studies for RBD and N- protein were also done and very specific sequences of RBD and N-protein were idenLfied to make the tests robust and dependable. Using the ELISA test for these 3 proteins, it was found that anLbody levels are higher in paLents with severe infecLon than mild infecLon and also that IgG anLbodies, an indicator that the paLent is recovering, appear in the second week ader disease onset. It is probably best to test for more than one viral protein/anLgen to avoid false-negaLve and false-posiLve results. These tests have made it possible to survey large populaLons and manage the pandemic in large countries like India. 

In silico screening of phytotherapeutics for anti psoriasis activity

Psoriasis is a noncontagious autoimmune disease. Psoriasis is an immune-mediated chronic skin disease characterized by epidermal hyperproliferation, and intraepidermal accumulation of neutrophils and dermal inflammatory cell infiltrates that are composed of dendritic cells and T cells. In psoriasis, the diseased areas are typically red, dry, itchy, and scaly. Psoriasis varies in severity from small, localized patches to complete body coverage. Injury to the skin can trigger psoriatic skin changes at that spot, which is known as the Koebner phenomenon. It has an estimated global prevalence of 2–4% [2]. Males are more likely to develop psoriasis compared to females [3]. As desquamative erythema can affect any skin site, psoriasis profoundly impairs the patients’ quality of life, treatment satisfaction and adherence, and socioeconomic stability [4].

Psoriasis is also significantly comorbid with other autoimmune diseases, such as bullous pemphigoid [5]. Psoriasis is frequently associated with cardiovascular diseases, metabolic diseases, and renal disorders [6,7,8]. Cancer risk is slightly higher in patients with psoriasis [9]. The topical application of steroids and vitamin D3 analogs inhibits psoriatic inflammation and normalizes epidermal differentiation [10]. Systemic treatments, such as methotrexate, cyclosporine, phototherapy, and the phosphodiesterase 4 inhibitor apremilast, are useful for patients with extensive lesions [11].

Proinflammatory cytokines are commonly called immunoregulatory cytokines produced predominantly by activated macrophages and are involved in the up-regulation of inflammatory reactions and hence they favor inflammation. The net effect of an inflammatory response is determined by the balance between proinflammatory and anti-inflammatory cytokines. That the tumor necrosis factor-α (TNF-α) and IL-23/IL-17A axes appear to be major drivers in the pathogenesis of psoriasis is underscored by the excellent response of
psoriasis to biologics targeting TNF-α, IL-23, and IL-17A, although a difference exists in their efficacy [11]. Anti-TNF-α/IL-23/IL-17A inhibitors successfully improve psoriatic arthritis [12]. Reductions in comorbid cardiovascular events and systemic inflammation have been reported in patients with psoriasis treated with anti-TNF/IL23/IL17 molecules.

Interleukin (IL)-1 and tumor necrosis factor (TNF) are the major proinflammatory cytokines involved, inflammation and tissue destruction, and also the inducers of endothelial adhesion molecules. Proinflammatory cytokine-mediated inflammation is a cascade of gene products usually not produced in healthy persons. IL-1, IL-6, IL-17, IL-12/23, and TNF are the major proinflammatory cytokines in psoriatic conditions, and hence, we are exclusively focused on inhibiting them using plant-derived small molecule inhibitors. The product under development for topical application is likely to contain the phyto-molecule which can significantly inhibit the pro-inflammatory cytokines IL-17, IL-12/23, and TNF and deliver the anti-psoriatic activity.

Our work on psoriasis towards attaining this polypharmacological activity, we employed computer aided drug discovery approach at Atrimed to screen the plant secondary metabolites as proinflammatory cytokine inhibitors and accelerated the classical route of drug discovery.
Drug target identification and validation Psoriasis associated protein-protein interaction (PPI) networks Target proteins of drug molecules are classified into a primary target and off-targets. The former is the desired target, whereas the latter could lead to adverse drug reactions or unexpected beneficial effects in drug repositioning. Therefore, comprehensive analysis throughout primary targets and off-targets on a genome- wide-scale is crucial in drug discovery. The in silico approach is expected to improve the research productivity in this field.
The protein-protein interactions were obtained from the STRING database and PPI networks were reconstructed around differentially expressed genes (DEGs). The visualization and topological analysis of the PPI network was performed via Cytoscape. Hub proteins were identified using the CytoHubba plugin which provides a user-friendly interface to analyze the topology of protein-protein interaction networks, such as human, yeast, fly, etc.
Apart from the identification of essential nodes, the betweenness and centrality score based subnetwork can be reconstructed utilizing CytoHubba in Cytoscape. These essential nodes in the reconstructed network may serve as candidates of drug-targets for developing novel therapy of human diseases.

Molecular Docking

As a part of the study, we have developed the plant secondary metabolites database which holds the 3D co-ordinate information of more than 4Lakh plant molecules. The the plant secondary metabolites in the database were screened against proinflammatory cytokine using molecular docking technique and the potential inhibitors are screened. The phytochemicals screened are possessing significant interaction with L-17, IL-12/23 and TNF with fairly acceptable bonded interactions. The screened molecules are isolated, purified and analysed experimentally to confirm the inhibitory potential of screened molecules.

Drug targets for topical application

References:

1.Boehncke WH, Schön MP. Psoriasis. Lancet. 2015 Sep 5; 386(9997):983-94.

2. Michalek IM, Loring B, John SM . A systematic review of worldwide epidemiology of psoriasis. J Eur Acad Dermatol Venereol. 2017 Feb; 31(2):205-212.

3. Ito T, Takahashi H, Kawada A, Iizuka H, Nakagawa H. Epidemiological survey from 2009 to 2012 of psoriatic patients in Japanese Society for Psoriasis Research. Japanese Society For Psoriasis Research. J Dermatol. 2018 Mar; 45(3):293-301.

4. Ichiyama S, Ito M, Funasaka Y, Abe M, Nishida E, Muramatsu S, Nishihara H, Kato H, Morita A, Imafuku S, Saeki H. Assessment of medication adherence and treatment satisfaction in Japanese patients with psoriasis of various severities. J Dermatol. 2018 Jun; 45(6):727-731.

5. Furue K, Ito T, Tsuji G, Kadono T, Nakahara T, Furue M. Autoimmunity and autoimmune co-morbidities in psoriasis. Immunology. 2018 May; 154(1):21-27.

6. Bayaraa B, Imafuku S. Relationship between environmental factors, age of onset and familial history in Japanese patients with psoriasis. J Dermatol. 2018 Jun; 45(6):715-718.

7. Chiu HY, Chang WL, Shiu MN, Huang WF, Tsai TF. Psoriasis is associated with a greater risk for cardiovascular procedure and surgery in patients with hypertension: A nationwide cohort study. J Dermatol. 2018 Dec; 45(12):1381-1388.

8. Furue M, Kadono T.  “Inflammatory skin march" in atopic dermatitis and psoriasis. Inflamm Res. 2017 Oct; 66(10):833-842.

9. Lee JH, Kim HJ, Han KD, Kim HN, Park YM, Lee JY, Park YG, Lee YB. Cancer risk in 892 089 patients with psoriasis in Korea: A nationwide population-based cohort study. J Dermatol. 2019 Feb; 46(2):95-102.

10. Imafuku S, Zheng M, Tada Y, Zhang X, Theng C, Thevarajah S, Zhao Y, Song HJ. Asian consensus on assessment and management of mild to moderate plaque psoriasis with topical therapy. J Dermatol. 2018 Jul; 45(7):805-811.

11. Masutaka Furue, Kazuhisa Furue, Gaku Tsuji, and Takeshi Nakahara. Interleukin-17A and Keratinocytes in Psoriasis. Int J Mol Sci. 2020 Feb; 21(4): 1275.

12. Yamamoto T, Ohtsuki M, Sano S, Morita A, Igarashi A, Okuyama R, Kawada A, working group of the epidemiological survey in the Japanese Society for Psoriasis Research. Switching biologics in the treatment of psoriatic arthritis in Japan. J Dermatol. 2019 Mar; 46(3):e113-e114.

A new approach to treatment for psoriasis

Thymoquinone (TMQ), a complex plant molecule is the main acLve ingredient of Nigella seeds. TMQ can scavenge or remove free radicals which are molecules in our body that can cause harm to us by destroying or inhibiLng the acLvity of various anL-oxidants. It has been shown by experiments in human skin cells that TMQ can help treat psoriasis. However, TMQ is easily degraded inside our body before it can act. Thus, to increase its stability inside our body so that it can act for a longer Lme, experiments were done in which they were placed inside nanoparLcle carriers. These carriers are less than 70nm in size and can enter the psoriaLc skin very efficiently and release the TMQ where they can act. Studies have been done in human skin cells as well as in mice with psoriasis, to show that this strategy works very well. Results indicate a clear reducLon in the level of nitric oxide and cytokines such as IL-2, IL-6, IL-1β, TNF-α, whereas in vivo results indicated improvement in the phenotypic, histopathological features and reduced level of IL-17 and TNF-α in psoriaLc skin. This study concludes that TMQ lipospheres could be used in the management of psoriasis.

Positive regulators of Wnt/β-catenin signaling pathway in wound healing and their applications.

The objec+ve of a recent study (Yeh et al. 2017) was to es+mate the wound healing poten+al and therapeu+c mechanism of Artocarpin (ARTO), a prenylated flavonoid purified from the plant Artocarpus communis. Immunohistochemical staining of neutrophils and macrophages and mouse cytokine array analysis demonstrated that ARTO accelerates inflammatory progression and subsequently decreases persistent inflamma+on. ARTO increases collagen produc+on and increases human fibroblast prolifera+on and migra+on by ac+va+ng the P38 and JNK pathways. Moreover, ARTO increases the prolifera+on and migra+on of human kera+nocytes through the ERK and P38 pathways and augments human endothelial cell prolifera+on and tube forma+on through the Akt and P38 pathways. The data in the study suggests that ARTO enhances skin wound healing, possibly by accelera+ng the inflammatory phase and by increasing myofibroblast differen+a+on, prolifera+on, and migra+on of fibroblasts and kera+nocytes, collagen synthesis and matura+on, re-epithelializa+on, and angiogenesis. These findings indicate that ARTO has poten+al as a therapeu+c agent for the treatment of skin wounds.

A wound healing agent from plants

 A WOUND HEALING AGENT FROM PLANTS – Healing of wounds requires high levels of molecules called prostaglandins or PGs in our body, specially one parLcular PG called PGE2. But this molecule is very quickly degraded in our body by an enzyme called 15- PGDH. So, inhibiLon of this enzyme, will lead to beSer and faster wound healing. A large study with 98 plants was conducted to look for molecules that could inhibit 15-PGDH. 5 top inhibitors were chosen from an in silico study for tesLng in human skin cells (HaCaT cells). The human skin cells were wounded by scratching them and then they were treated with the plant molecules and levels of PGE2 were checked, as well as, the Lme taken for the scratch to heal. It was found that a molecule called EEAH, from the plant Artocarpus heterophillus, the common jackfruit, has very good wound healing ability. It causes very high levels of PGE2 to be maintained both inside and outside human skin cells to heal scratches very quickly. The molecule is safe, non-toxic and can be used in a topical formulaLon. 

Inhibition of Propionibacterium acnes by Staphylococcus epidermidis in the human skin by fermentation and scope for acne probiotics

Commensal microorganisms in the skin microbiota like Staphylococcus epidermidis (S. epidermidis) are known to fight pathogens like Propionibacterium acnes (P. acnes), associated with progression of acne vulgaris. It is known that many skin microorganisms can mediate fermenta7on of glycerol, which is produced in the skin. A recent study of microbial interac7ons in skin has revealed new details on how S. epidermidis contributes to controlling the growth of P. acnes. S. epidermidis ferments glycerol to produce succinic acid, one of four short chain faOy acids produced during fermenta7on. Succinic acid can effec7vely inhibit the growth and inflamma7on caused by P. Acnes, both in vitro as well as in vivo, in mice treated either with intra-lesional injec7ons or topical applica7on over the lesions. With recent evidence of fermenta7on based control of P. acnes by S. epidermidis, the role of poten7al new probio7cs against acne and other skin diseases.

The hidden danger to implants from acne

The bacteria that cause acne live mainly on the skin, but, under the right condiLons, they can enter the skin and cause serious infecLons inside our body as well. Acne bacteria have the property to sLck to surfaces, like orthopedic implants at shoulder, knee and hip joints, cerebrovascular shunts, pacemakers and breast implants, causing serious infecLons because of their ability to form biofilms around the metal surfaces of the implants. Their increasing resistance to the anLbioLc clindamycin normally used for treatment, makes the infecLon hard to treat quickly, thus requiring surgery as well as 3-6 months of anLbioLc treatment to completely eliminate the infecLon. Recently, many studies have been done with bacterial cultures isolated from such infected implants to idenLfy the exact strain of acne bacteria and accordingly treat it with anLbioLcs like rifampicin to which it is highly suscepLble. This has decreased the treatment Lme and side-effects. However, more studies are required to prove this.

Management of burn pain naturally

 Burn pain can be severe and even cause death. The Magnolia tree, a Chinese flowering plant, produces a molecule called Honokiol, which has pain relieving properLes. To test these, mice were inflicted with burn wounds and then treated with a honokiol extract preparaLon for 3 days along with standard opiod pain medicaLon. It was found that the combinaLon of honokiol and opiod is very effecLve in reducing the pain sensaLon, heat in the wound area and oedema/swelling at the wound area. Honokiol also succeeded in reducing weight loss and spontaneous pain behaviour in the mice. It also significantly balanced blood electrolytes and general profile. Honokiol has these effects by significantly decreasing the expression of molecules called cytokines which cause inflammaLon, a process that can cause pain to persist. Honokiol treatment also maintains anL-oxidant levels in blood which aid in reducing inflammaLon and promoLng pain management. It can be concluded on the base of the results that honokiol has a significant analgesic/pain relieving acLvity through its acLon on cytokines. It also has a protecLve role against burn damage by promoLng levels of anLoxidants.