INTRODUCTION:

Oral cancer is a type of malignant neoplasia that develops on the lips or in the mouth. In most ethnic groups, men are two to three times more likely than women to get oral cancer¹. Cancers of the oral cavity and pharynx are lumped together in global reports and collectively represent the world's sixth most prevalent malignancy. Oral cancer was reported in 369,200 new cases worldwide in 2012, with two-thirds of the tumours detected in poorer nations, each year roughly 145,328 people die all over the world². In 2018, India alone had estimated 120,000 new patients diagnosed, of which about 72,000 patients died³.

In terms of anatomical distribution, the five most prevalent sites for oral cancer were the tongue (25.4%), labial/buccal mucosa (21.7%), gingiva (14.0%), palate (9.9%), and alveolar mucosa (7.9%), in that order⁴. Tobacco, betel, alcohol, a diet lacking in fresh fruits and vegetables, infectious agents (Candida, viruses), poor oral hygiene, immunological weakness, frequent oral sex, and, in the case of lip cancer, sun exposure are all etiologic factors^5,6,7. The fatality rate linked with this malignancy has historically been exceptionally high, not because it is difficult to detect or diagnose, but because it is frequently detected late in its progression.

Genistein:

Genistein is an isoflavone, isolated from brooming plant of Genistatinctoria L. of Fabaceae family. Genistein possess several biological potentials such as the anti-inflammatory, anti-angiogenesis anti-cancer, anti-bacterial, anti-metastasis, anti-viral and anti-oxidant, and pharmacological activities on diabetes, estrogen and lipid metabolism^8,9.

Fig. 1: Genistein

Mechanism of action against oral cancer:

· Genistein mainly acts by altering apoptosis, the cell cycle, and angiogenesis and inhibiting metastasis¹⁰.

· Genistein Targets caspases-3, B cell lymphoma 2 (Bcl-2)–associated X protein (Bax) and Bcl-2¹¹.

· It downregulates Kinesin-like protein 20A (KIF20A), which inhibits proliferation and metastasis¹².

· Genistein downregulates extracellular signal-regulated kinase 1/2 (ERK1/2)

· It inhibits nuclear transcription factor κB (NF-κB) by inhibiting mitogen-activated protein kinase (MAPK).

· It interrupts 1 β-catenin (Wnt/β-catenin), and phosphoinositide 3 kinase/Akt (PI3K/Akt) signalling pathways¹³.

· Genistein also decreases the levels of IL-1β, IL-6, IL-8 and TNF-α therefore reducing inflammation¹⁴.

Cinnamaldehyde:

Cinnamaldehyde is an active compound of Cinnamon which has numerous properties such as anti-inflammatory, anti-microbial, anti-fungal, anti-oxidant, anti-diabetic, anti-termites, nematicidal, mosquito larvicidal, insecticidal, anti-mycotic, and anti-cancer and also it is useful in dental problems (Halitosis, toothaches, and oral microbiota) and increases the blood circulation in the uterus^15,16.

Fig. 2: Cinnamaldehyde

Mechanism of Action against oral cancer:

· Cinnamaldehyde has the potential to stop the production of NO by inhibiting the iNOS¹⁷.

· Cinnamaldehyde inhibits activation of transcription factor nuclear factor-kappa B (NF-κB).

· It inhibits the secretion of cytokine interleukin-1β (IL-1β) which is a key mediator of the inflammatory response¹⁸.

· It also obstructs the production of tumor necrosis factor-alpha (TNF alpha)¹⁹.

· Cinnamaldehyde suppresses Interferon-gamma (IFN-γ), interleukin (IL)-6, interleukin-1alpha (IL-1α) which is produced by lipopolysaccharide (LPS) or lipoteichoic acid (LTA)²⁰.

· Cinnamaldehyde also suppresses COX-2.

· Cinnamaldehyde supresses MPAKs phosphorylation pathways in the cells which are ERK, JNK, and p38²¹.

Trans-Resveratrol:

Resveratrol is a pleiotropic phytochemical which belongs to the stilbene family. Stilbenes are the secondary metabolites which are produced by the plants in response to stressful conditions, such as fungal infection or UV radiations. It possesses anti-microbial, anti-oxidant, anti-aging, anti-inflammatory, anti-estrogenic, cardio-protective, and anti-cancer properties. Also, it has been reported that resveratrol can reverse multidrug resistance in cancer cells^22,23.

Fig. 3: Trans-Resveratrol

How Trans-Resveratrol works against cancer:

· Decreases free radical scavenging incidence.

· Resveratrol effectively hinders the development tumors by inducing apoptosis, which will be indicated by the induction of cytochrome C release, the expression of Bax, p53, and APAF-1, and the inhibition of Bcl-2²⁴.

· Decreases the expression of COX-2, and ODC (Ornithine decarboxylase)²⁵.

· It also decreases hyperplasia.

· It inhibits activation of NF-κB through inhibiting the IκB-α kinase activation, and hence down-regulating pro-proliferation genes, such as cIAP-2, survivin, cyclin D1, Bcl-xL, Bcl-2, XIAP, Bfl-1/A1, and TNF-α receptor-associated factor 2 (TRAF2)²⁶.

· It also suppresses matrix metalloproteinase (MMP)-3, MMP-9, COX-2, and vascular endothelial growth factor (VEGF).

· Resveratrol causes inhibition of signal transducers and activators of transcription 3 (STAT3)²⁷.

· It exerts an anti-oxidant effect with a reduction in H₂O₂ and lipid peroxidation in the skin.

· It also decreases the levels and expressions of hepatic TNF-α, IL-1β, and IL-6²⁸.

Formononetin:

Formononetin is an O-methylated isoflavone, it is widely present in legumes, clovers (red clovers Trifoliumpratense L.) and in Chinese herb Astragalusmembranaceus (Fisch).

It has been known to be endowed with numerous pharmacological attributes such as anticancer, anti-inflammatory, anti-oxidant, anti-allergic, anti-inflammatory, anti-proliferative, growth inhibitory, vaso-relaxant, neuroprotective, anti-apoptotic, cardio-protective, mammary gland proliferative and anti-microbial activities^29,30.

Fig. 4: Formononetin

Anti-inflammatory action:

· Formononetin inhibits the pro-inflammatory transcription factor NF-κB³¹.

· With the decrease in NF-κB (is a significant transcription factor for the induction of nitric oxide synthase) there is a decrease in the production of nitric oxide in vitro.

· Formononetin demonstrated an inhibitory effect on the MAPK signalling pathway. The MAPK pathways modulate inflammatory gene transcription through the phosphorylation of the ERK, JNK and p38 proteins which reduces inflammatory effect³².

· Formononetin reduces the NO production derived from iNOS which is induced by IL-1β³³.

· Formononetin reduces the prostaglandins by acting on COX-2.

· Formononetin also shows inhibitory effect on TNF-α and IL6 levels³⁴.

· Formononetin works as a mast cell stabilizer by inhibiting the release of histamine from the mast cells.

Preparation of API loaded in Transferosomes by Thin Film Hydration Technique/Rotary Evaporation -Sonication Method:

Fig. 5: Transferosomes Comprised of Genistein Cinnamaldehyde, Trans-Resveratrol and Formnonetin

The phosphatidylcholine component, DPPC, and the edge activator, Tween-80, are dissolved in a round-bottom flask with an appropriate (v/v) ratio of chloroform and methanol. In this stage, the lipophilic substances which are Cinnamaldehyde, Genistein and Trans-Resveratrol were introduced. A rotary vacuum evaporator is used to evaporate the organic solvent above the lipid transition temperature under reduced pressure in order to generate a thin layer. To eliminate the last residues of the solvent, keep it under vacuum. The thin film is then hydrated using a buffer solution with a pH of 7.4 and rotated for a period of time at the appropriate temperature. Formononetin, a hydrophilic drug, will be included at this step. To obtain tiny vesicles, the resultant vesicles are inflated at room temperature and sonicated in a bath or probe sonicator. Extrusion across a sandwich of 200 nm to 100 nm polycarbonate membranes homogenises the sonicated vesicles^35-38.

Material required and method of preparation^39-41

Materials:

Drug loaded in transferosomes (20mg, 4mg each API and 6mg mass of transferosomes), pullulan (2mg), sodium starch glycolate (5mg), mannitol (61mg), magnesium stearate (2mg), talc (2mg), Povidone k-30 (3mg), Croscarmellose Na (0.6mg) and mannose (2.4mg), total mass of one sample will be 100mg.

Method of preparation:

Direct compression was used to make the sublingual tablets. By passing through an 80 no screen sieve, a precise amount of the active ingredient and all additions were homogeneously combined. A twin rotating tablet compression machine with an 8 mm flat faced punch and die set was used to compress the tablets directly. All tablets had the same compression force and mass, and each tablet contained 16% (16mg) of APIs.

Evaluation of Sublingual Tablets:

Evaluation of pre-compression parameters of powder:

Powder flow and compressibility parameters were assessed prior to compression. The angle of repose method will be used to determine the flow characteristics of powder. Carr's index and Hauser ratio were used to determine the compressibility index of powder.

· Angle of repose:

Angle of repose (𝜽) will be determined using funnel method by formula:

θ = tan⁻¹h /r

Whereas, Maximum cone height (h) and radius of the heap (r).

· Bulk Density:

Bulk density is defined as the mass of powder divided by bulk volume. It is calculated according to the equation:

LBD=Weight of the powder/Volume of the packing

A 10gm sample will be poured into a 100ml graduated cylinder. The cylinder will be dropped three times from a height of one inch at 2-sec intervals onto a firm wooden platform. The bulk density will be determined once the volume will be measured.

· Tap bulk density (TBD):

At 750 taps, the TBD of the disintegrant will be evaluated using a USP tap density tester. Before and after tapping, disintegrant (10g) will be put into a calibrated measuring cylinder (100ml) and the volume change will be observed. The following equation will be used to calculate TBD:

TBD=Weight of the powder/Tapped volume of the packing

· Compressibility index:

% Compressibility that is calculated as follows:

C = (ρt − ρb) × 100

ρt

ρt - Tapped density, ρb - Untapped bulk density

· Hausner’s ratio:

Hausner’s ratio is an index of ease of powder flow; it is calculated by following formula.

H = ρt / ρb

H- Hausner’s Ratio, ρt - Tapped density, ρb -Untapped bulk density

Evaluation of post-compression parameters of tablets

· Tablet Thickness:

A calibrated dial calliper will be used to measure the dimensions of the tablets. Six tablets of each formulation were chosen at random and measured separately for thickness.

· Weight Variation Test:

Ten tablets were chosen at random from each batch and weighed on an automated scale to determine the average weight. After that, each tablet will be independently weighed, and the standard variation in weight for each batch will be calculated.

· Tablet Hardness:

Six tablets were chosen at random from each batch and tested for hardness using a Monsanto hardness tester. Each batch's mean values and standard deviation were computed.

· Tablet Friability:

Friability refers to a tablet's ability to tolerate mechanical shocks while in use. The Roche Friabilator will be used to determine the tablet's friability. Initially, ten tablets were weighed and placed in the friabilator (W_initial). The friabilator will be set to 25rpm for 4 minutes or 100 revolutions, and the tablets were weighed again (W_final). Tablet friability will be determined by the amount of weight lost owing to abrasion or fracture. The percentage friability (f) will be computed using the formula below:

f=W_initial - W_final × 100

W_initial

% Friability of less than 1 % is considered acceptable

· Wetting Time:

The timings for complete wetting of the tablets were recorded in a petridish containing 10mL water at room temperature.

· Drug Content Uniformity:

Three tablets were ground and put into a 100ml volumetric flask, weighing exactly identical to 80 mg of the original tablet. 10mL methanol will be added first, and the mixture will be agitated for 10minutes. The volume will be then increased to 100ml using methanol. The solution in the volumetric flask will be then filtered, and 1ml of the filtrate will be diluted and tested for drug concentration with a UV-spectrophotometer set at λ = 238 nm.

· Disintegration Test:

The disintegration time will be obtained using the USP tablet disintegration test device with 900mL pure water and no disc. The time it took for the tablets to completely disintegrate until there will be no mass left in the apparatus will be measured in seconds.

· In-vitro Drug Release Study:

The in-vitro drug release research of API from tablets will be performed using a USP dissolving test apparatus type II Paddle Method in 900ml of phosphate buffer pH 6.8 dissolution medium at 37±0.5°C temperature and 60 rpm rotation. At predetermined intervals, a 5mL aliquot of dissolving medium will be removed and replaced with an equivalent volume of fresh medium. Using a UV spectrophotometer set at λ=238nm, samples were diluted appropriately and % drug release will be determined. All of the tests were done three times.

· Assay Testing for Anti-inflammatory response:

In this Assay testing RAW264.7 cell line is prepared and tested for the presence of different types of LPS induced inflammatory cells. The murine macrophage RAW264.7 cell line will be maintained in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100μg/mL streptomycin and then determination of COX-2, iNOS, TNF-α, IL-1β, and IL-6 Genes by Quantitative Real-Time PCR (qRT-PCR)[42].

DISCUSSION:

A sublingual tablet formulation is prepared to treat oral cancer by mixing Genistein, Cinnamaldehyde, Trans-Resveratrol and Formononetin, in excipients like pullulan, sodium starch glycolate, mannitol, magnesium stearate, talc, Povidone k-30, Croscarmellose Na and mannose and prepared by direct compression method. Before preparation, the formulation will be tested on pre-compression parameters of powder which are angle of repose, bulk density, tap bulk density, Compressibility index and Hausner’s ratio. After preparation, the formulation will be tested on post-compression parameters which are tablet thickness, weight variation, tablet hardness, tablet friability, wetting time, drug content uniformity, disintegration test, In-vitro drug release study and assay testing on RAW264.7 cell line to check its bio-availability, stability of the formulation, content uniformity of formulation, and efficient working against cancer cells.

Several Other compounds such as EGCG, TF3, Quercetin, Curcumin, Kaempferol, Apigenin, etc can also be seen as the potential canditates for the treatment of Oral Cancer^43-49.

CONCLUSION:

A sublingual tablet formulation which comprises Genistein, Cinnamaldehyde, Trans-Resveratrol and Formononetin which possesses potent anti-inflammatory and anti-proliferative agents. Various excipients like pullulan, sodium starch glycolate, mannitol, magnesium stearate, talc, Povidone k-30, Croscarmellose Na and mannose are used to increase the efficiency, stability, and bioavailability of the formulation by several folds. The formulation can efficiently penetrate through the cell membrane because of the presence of transferosomes and reaches to the cancerous cells where it inhibits inflammation and proliferation.

Cinnamaldehyde inhibit LPS-induced IL-8, TNF-α, IL-12-induced T cell proliferation IFN-γ and COX-2. Trans-Resveratrol decreases the expression of COX-2, and ODC, inhibits activation of NF-κB, down-regulating pro-proliferation genes, such as cIAP-2, survivin, cyclin D1, Bcl-xL, Bcl-2, XIAP, Bfl-1/A1, and TNF-α receptor-associated factor 2 (TRAF2), suppresses matrix metalloproteinase (MMP)-3, MMP-9, COX-2, VEGF and STAT3. Formononetin inhibits the MAPK pathways and hence modulate the inflammatory gene transcription through the phosphorylation of ERK, JNK and p38 proteins, it reduces the NO production; also it works as a mast cell stabilizer. Genistein mainly acts by altering apoptosis, the cell cycle, and angiogenesis and inhibiting metastasis, it targets caspases-3, Bcl-2 and Bax, it downregulates KIF20A and ERK1/2, also interrupts Wnt/β-catenin and PI3K/Aktsignaling pathways. This formulation doesn’t produce any severe side effects and also it is much cheaper and effective than traditional chemotherapies. The whole treatment will be quicker than chemotherapies and also the technique is non-invasive and also lowers the chances of re-occurrence of cancer.

ACKNOWLEDGEMENT:

The authors are thankful to Department of Hospital Management and Hospice Studies, Jamia Millia Islamia for providing kind guidance and excellent opportunity as well as necessary facilities for the research.

CONFLICTS OF INTEREST:

The authors confirm that the content of the article has no conflict of interest.

DATA AVAILABILITY:

The original data that support the findings of this study are included in the article.

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Received on 11.03.2023 Modified on 13.10.2023

Research J. Pharm. and Tech 2024; 17(5):2056-2062.

DOI: 10.52711/0974-360X.2024.00326