Development and Validation of Micro-Azocasein Assay for Quantifying Bromelain.

The proteolytic activity of enzymes may be evaluated by a colorimetric method with azocasein. Hence, we developed a micro-assay to quantify bromelain using azocasein. A total of 250 µL of 1.0% azocasein in dH2O was added to 250 µL of test solution, vortexed and incubated at ambient room temperature/30 min. The reaction was terminated with 1500 µL of 5% trichloroacetic acid, vortexed and centrifuged. A total of 150 µL of 0.5M NaOH was added to 150 µL of supernatant in triplicates, and absorbance was recorded at 410 nm. The linearity of the calibration curve was tested with 200-800 µg/mL serial dilutions. The detection limit, precision, accuracy, and robustness were tested along with the substrate enzyme reaction time and solvent matrix effect. Good linearity was seen with serially diluted 200 µg/mL bromelain. The limit of quantification and limit of detection were 5.412 and 16.4 µg/mL, respectively. Intra-day and inter-day analyses showed a relative standard deviation below 2.0%. The assay was robust when tested over 400-450 nm wavelengths. The assays performed using dH2O or PBS diluents indicated a higher sensitivity in dH2O. The proteolytic activity of bromelain was enhanced with L-cysteine or N-acetylcysteine. Hence, this micro-azocasein assay is reliable for quantifying bromelain.

Intraperitoneal BromAc® Does Not Interfere with the Healing of Colon Anastomosis.

A combination of bromelain and acetylcysteine, BromAc®, is an efficient intraperitoneal mucolytic for thick mucus secreted in pseudomyxoma peritonei (PMP). Patients with PMP quite often undergo colon anastomosis. Hence, we investigated the effect of the intraperitoneal delivery of BromAc® on colon-anastomosis healing in a rat model. Sixteen Wistar rats were divided into two groups (N = 8). The controls received intraperitoneal saline after anastomosis, whilst the other group received BromAc®. They were monitored for body-weight and general health parameters. Half the rats in each group (N = 4) were culled at 4 or 13 days post-surgery for assessment. The healing process of the tissues was assessed by burst pressure and collagen density with histology to assess the integrity of the internal organs. The results indicated that there was a similar pattern of weight fluctuation during the experiment, although the rats treated with the BromAc® showed slightly greater weight loss during the first 4 days. Although the burst pressure was similar in both groups, the BromAc® group at day 13 showed a slightly higher burst pressure, which was complemented by a higher collagen density (albeit not statistically significant). The histology of the internal organs was comparable to those of the controls. This study indicates that the intraperitoneal delivery of BromAc® in a rat model does not interfere with the healing process of colonic anastomosis.

Effect of Nebulized BromAc on Rheology of Artificial Sputum: Relevance to Muco-Obstructive Respiratory Diseases.

Respiratory diseases such as cystic fibrosis, COPD, and COVID-19 are difficult to treat owing to viscous secretions in the airways that evade mucocilliary clearance. Earlier studies have shown success with BromAc as a mucolytic agent. Hence, we tested the formulation on two gelatinous airway representative sputa models, to determine whether similar efficacy exist. Sputum lodged in an endotracheal tube was treated to aerosol N-acetylcysteine, bromelain, or their combination (BromAc). After measuring the particle size of aerosolized BromAc, the apparent viscosity was measured using a capillary tube method, whilst the sputum flow was assessed using a 0.5 mL pipette. Further, the concentration of the agents in the sputa after treatment were quantified using chromogenic assays. The interaction index of the different formulations was also determined. Results indicated that the mean particle size of BromAc was suitable for aerosol delivery. Bromelain and N-acetylcysteine affected both the viscosities and pipette flow in the two sputa models. BromAc showed a greater rheological effect on both the sputa models compared to individual agents. Further, a correlation was found between the rheological effects and the concentration of agents in the sputa. The combination index using viscosity measurements showed synergy only with 250 µg/mL bromelain + 20 mg/mL NAC whilst flow speed showed synergy for both combinations of bromelain (125 and 250 µg/mL) with 20 mg/mL NAC. Hence, this study indicates that BromAc may be used as a successful mucolytic for clearing airway congestion caused by thick mucinous immobile secretions.

Physical and chemical factors affecting the loading and release of bromelain from DC beads.

Doxorubicin loaded DC beads (microspheres) has been used for treating un-resectable tumours by transarterial chemoembolization (TACE). We have shown that bromelain, an enzyme from the pineapple plant, enhances the cytotoxic effect of a number of chemotherapeutic drugs and in an earlier study we have demonstrated that it can be loaded into DC beads. Therefore, in the current study we have investigated how certain physical and chemical parameters affect its loading and release for future development of DC beads in cancer therapy. Aliquots of 40-60 µL of DC beads (100-300 µm) were treated to bromelain in distilled water and various parameters such as pH of solution, bromelain concentration, temperature, loading period, presence/absence of agitation and the cytotoxic effect of bromelain loaded beads were investigated. Further release kinetics was also studied with additional investigation of pH effect on the proteolytic activity of bromelain. Results indicate that higher loading of bromelin was achieved in the beads at lower pH, higher concentration of bromelain, with agitation, 24 hours loading and ambient room temperature. Proteolytic activity of bromelain was maximal at pH 4.5 whilst cytotoxicity was at par if not better in the bromelain loaded DC beads. Release kinetics indicated that bromelain can be delivered over several hours. Hence, we conclude that bromelain can be loaded more efficiently with manipulation of certain parameters with noticeable cytotoxicity in tumour cells.

A novel method for potentiation of chemotherapy in soft tissue sarcomas with BromAc.

Single-agent doxorubicin currently forms part of standard care for patients with sarcomas. However, efficacy is limited by the presence of dose-dependent cardiotoxicity and toxicity to renal, hepatic, and neurological systems. Therefore, there is a pressing need for novel drug regimens which can provide increased efficacy and safety. BromAc is a novel drug combination developed as a mucolytic agent which has demonstrated anticancer activity both in vitro and in vivo in several cancers. Here, we investigated the efficacy of BromAc in combination with doxorubicin for four subtypes of sarcoma. Cell proliferation, alongside western blot for a variety of cell cycle, apoptosis, and autophagy biomarkers assays was performed following treatment of cell lines in vitro at various concentrations of BromAc and doxorubicin. The impact of drug treatment on MUC1 and MUC4 levels was assessed through immune-cytological methods. Drug agent synergy was assessed through the Chou-Talalay framework. BromAc treatment in combination with doxorubicin was more efficacious than single-agent doxorubicin, with synergistic effects observed. The immuno-cytological analysis demonstrated significant mucin depletion following treatment with BromAc and doxorubicin used in combination, providing a potential mechanistic underpinning for the observed anticancer effects.

Comparison of proteolytic, cytotoxic and anticoagulant properties of chromatographically fractionated bromelain to un-fractionated bromelain.

Bromelain consisting of a number of proteolytic enzymes possess anticancer and thrombotic properties. Hence, four chromatically separated fractions were examined for their proteolytic, anticancer and antithrombotic activity. Bromelain fractions were separated using ion-exchange column chromatography. Proteolytic properties were assessed using standard azocasein assay. Anticancer properties were first assessed using four different cell lines PANC-1, HEP 2B, HEP 3G and OVCAR-3 on cells grown in 96 well plates. Subsequently, fraction 2 and fraction 3 combined with gemcitabine were tested in ASPC-1 cells. Then cytotoxicity of fraction 3 was compared to bromelain in combination with doxorubicin and N-acetylcysteine on HEP G2 and HEP 3B cells. Finally, the anticoagulation effect of fraction 3 or bromelain combined with N-acetylcysteine was evaluated using human blood. Fraction 3 showed the highest proteolytic activity (5% greater than standard bromelain) whilst others were less active. Cytotoxicity as assessed by IC50 indicated fraction 3 to be the most potent whilst the others did not follow their proteolytic potency order. OVCAR-3 was the most sensitive amongst the cell lines. Fraction 3 showed higher potency in combination with gemcitabine in ASPC-1 cells compared to fraction 2. Similarly, fraction 3 in combination with doxorubicin showed higher toxicity when compared to bromelain. Fraction 3 or bromelain only showed thrombolytic activity in combination with N-acetylcysteine. Fraction 3 may be developed for clinical use since it showed better cytotoxicity compared to bromelain.

Addition of bromelain and acetylcysteine to gemcitabine potentiates tumor inhibition in vivo in human colon cancer cell line LS174T.

The combinations of Bromelain and Acetylcysteine (BromAc®) with cytotoxics such as Gemcitabine, 5-Fluorouracil or Oxaliplatin have shown a dramatic reduction in IC50 values in a variety of cancers, including colon cancer, suggesting the possibility of effective treatment without undesired side effects. In the current study, we investigated whether a similar effect is present in vivo using the colorectal cell line LS174T. Animals after acclimatization were randomized and allocated equally in the groups for the different studies (safety, dose-escalation, and efficacy). Drugs were delivered by the intraperitoneal route and animals were monitored for wellbeing. Separately, an efficacy study was conducted with intraperitoneal drug delivery after intraperitoneal tumor induction. At the termination of the experiment, tumors and other tissues were collected for evaluation. BromAc® was safe when delivered intraperitoneally in a rat model at the concentrations used. Subsequent investigations of these adjuvants in combination with Gemcitabine, Oxaliplatin, and 5-Fluorouracil in mice were also proven to be safe. Preliminary efficacy studies with Oxaliplatin and 5-Fluorouracil on tumor growth (LS174T) were negative. Gemcitabine was assessed with BromAc® showing an almost 71% tumor inhibition compared to controls. This in vivo study indicates that Gemcitabine at 2 mg/kg in combination with BromAc® 3 mg/300 mg/Kg was effective and safe, supporting its potential for future clinical application.

The Combination of Bromelain and Acetylcysteine (BromAc) Synergistically Inactivates SARS-CoV-2.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection is the cause of a worldwide pandemic, currently with limited therapeutic options. The spike glycoprotein and envelope protein of SARS-CoV-2, containing disulfide bridges for stabilization, represent an attractive target as they are essential for binding to the ACE2 receptor in host cells present in the nasal mucosa. Bromelain and Acetylcysteine (BromAc) has synergistic action against glycoproteins by breakage of glycosidic linkages and disulfide bonds. We sought to determine the effect of BromAc on the spike and envelope proteins and its potential to reduce infectivity in host cells. Recombinant spike and envelope SARS-CoV-2 proteins were disrupted by BromAc. Spike and envelope protein disulfide bonds were reduced by Acetylcysteine. In in vitro whole virus culture of both wild-type and spike mutants, SARS-CoV-2 demonstrated a concentration-dependent inactivation from BromAc treatment but not from single agents. Clinical testing through nasal administration in patients with early SARS-CoV-2 infection is imminent.

The effect of intraperitoneal administration of BromAc on blood parameters: phase 1 study.

Intraperitoneal administration of BromAc (bromelain + acetylcysteine) is currently undergoing a phase 1 clinical trial for pseudomyxoma peritonei at our institution. This study reports on analysis of routine blood parameters before and after treatment for a series of 25 patients in this trial. Blood parameters assessed included full blood count, electrolytes, urea, and creatinine, liver function tests, coagulation studies, as well as inflammatory markers (CRP). Certain parameters such as CRP, and white cell count, were significantly elevated after treatment whilst serum albumin level was reduced indicating an inflammatory reaction. However, liver enzymes, coagulation studies, and other parameters were not affected. Therefore, there are no additional safety signals evident upon analysis of routine blood parameter testing.

Bromelain and acetylcysteine (BromAc®) alone and in combination with gemcitabine inhibit subcutaneous deposits of pancreatic cancer after intraperitoneal injection.

Gemcitabine (GEM) is commonly chosen for treating pancreatic cancer. However, its use is limited by toxicity. Earlier in vitro studies with GEM in combination with Bromelain (Brom) and Acetylcysteine (Ac) indicated a substantial reduction in IC50. In this study, immunocytochemistry and Western blot were used to explore the mechanistic effects of Brom and Ac (BromAc®) in vitro. Then, we explored the efficacy and safety of BromAc® only and with GEM in a pancreatic cancer model in vivo. Immunocytochemistry results revealed a reduction in both MUC1 and MUC4 post-treatment. There was a decrease in VEGF, MMP-9, NF-κß and cleavage of PARP. There was also a decrease in the cell cycle regulators Cyclin B and D as well as TGF-ß and the anti-apoptotic Bcl-2. In vivo, the low and high doses of BromAc® alone and with chemotherapy agents were safe. A very significant reduction in pancreatic tumour volume, weight, and ki67 were seen with BromAc® therapy and was equal to treatment with GEM alone and better than treatment with 5-FU. In addition, tumour density was significantly reduced by BromAc®. In conclusion, the anticancer effect of BromAc® is probably related to its mucin depletion activity as well as its effect on proteins involved in cell cycle arrest, apoptosis and modulation of the tumour microenvironment. The in vivo results are encouraging and are considered the first evidence of the efficacy of BromAc® in pancreatic cancer. These results also provide some mechanistic leads of BromAc®.

Enhancing the potency of chemotherapeutic agents by combination with bromelain and N-acetylcysteine - an in vitro study with pancreatic and hepatic cancer cells.

Current systemic dosages of chemotherapeutic drugs such as gemcitabine, 5-FU, cisplatin, doxorubicin are administered every 7 days over 4 cycles due to systemic toxicity. An increase in potency of the drugs will result in dosage reduction with more frequent administration and efficacy increase. Hence, we investigated how the drugs potency can be increased by combining with bromelain and N-acetylcysteine. Tumour cells (5,000/well) were seeded into a 96 well plate and treated 24 hrs later with either single agents or in combinations at various concentrations. Cell survival was assessed by the sulforhodamine B assay after 72 hours of exposure. LD 50 was determined for each treatment and the Combination Index (CI) was assessed to determine synergy using Tallarida's method. CI indicated that synergy was dependent on the concentration of the agents used and was cell line specific. For bromelain and N-acetylcysteine, certain ratio of the two agents gave very good synergy that was prevalent in almost all cell lines. Gemcitabine and 5-FU and doxorubicin reacted favourably with most concentrations of bromelain and NAC investigated. Cisplatin and oxaliplatin were not very compatible with NAC. A value of CI <0.5 indicated that the current clinical chemotherapeutic dosage can be dramatically reduced. Bromelain with NAC showed synergy in all tumour cell lines and acting synergistically with chemotherapeutic drugs. Synergistic combinations resulting in considerable dosage reduction of chemotherapeutic agents may enable more frequent treatment with higher efficacy.

Physical and chemical characteristics of mucin secreted by pseudomyxoma peritonei (PMP).

Background: Pseudomyxoma peritonei (PMP) is a rare disease with excess intraperitoneal mucin secretion. Treatment involves laparotomy, cytoreduction and chemotherapy that is very invasive with patients often acquiring numerous compromises. Hence a mucolytic comprising of bromelain and N-acetyl cystein has been developed to solubilise mucin in situ for removal by catherization. Owing to differences in mucin appearance and hardness, dissolution varies. Therefore the current study investigates the inter-mucin physical and chemical characteristics, in order to reformulate an effective mucolytic for all mucin. Method: PMP mucin, from the three categories (soft, semi hard and hard mucin) was solubilised and then various physical characteristics such as turbidity, density, kinematic viscosity were measured. The water content and the density of solid mucin were also determined. This was followed by the determination of sialic acid, glucose, lipid, Thiol (S-S and S-H) content of the samples. Lastly, the distribution of MUC2, MUC5B and MUC5AC was determined using western blot technique. Results: Both turbidity and kinematic viscosity and sialic acid content increased linearly as the hardness of mucin increased. However, density, hydration, protein, glucose, lipid and sulfhydryl and disulphide content decreased linearly as hardness of mucin increased. The distribution ratio of mucins (MUC2:MUC5B:MUC5AC) in soft mucin is 2.25:1.5:1.0, semi hard mucin is 1:1:1 and hard mucin is 3:2:1. Conclusion: The difference in texture and hardness of mucin may be due to cellular content, hydration, glucose, protein, lipids, thiol and MUC distribution. Soft mucin is solely made of glycoprotein whilst the others contained cellular materials.

Assessment of a novel mucolytic solution for dissolving mucus in pseudomyxoma peritonei: an ex vivo and in vitro study.

BACKGROUND: Pseudomyxoma peritonei (PMP) is difficult to treat. Intraperitoneal delivery of mucolytic solutions might potentially improve therapy, in addition to surgical cytoreduction and hyperthermic intraperitoneal chemotherapy. METHODS: Comparison of mucolytic effect of two formulations (control: bromelain 300 µg/mL+N-Acetylcystein 250 mM; test: bromelain 200 µg/mL+200 mM cysteamine) in vitro on a mucin producing cell lines (HT29) and ex vivo on mucus obtained from 18 PMP patients. Mucin plugs were classified according to their density into three categories: hard, semi hard and soft. Simulation of peritoneal washing ex vivo using a closed heated circulating pump. RESULTS: Solubilisation was faster with the test vs. the control formulation (90 vs. 180 min) for dissolving the soft mucin plugs (p < 0.05). The test solution was also more effective in dissolving the hard mucus plugs compared to control (82.5±2.74 % vs. 36.33±3.27 %). All mucin types disintegrated in simulated peritoneal washing. Cytotoxicity of the test solution on HT29 cell line was time-dependent. CONCLUSIONS: The test formulation is more effective and faster than the control formulation in dissolving mucus plugs of various densities. Mucus plugs were all solubilised after 40 min in simulated peritoneal washing. This novel mucolytic formulation might pave the way for an effective and less invasive therapy of PMP in the future.

Cissampelos pareira Linn. ameliorates thyroxin-induced cardiac hypertrophy in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Cissampelos pareira extract has been traditionally used in ayruveda as cardiotonic, diuretics and in heart complains but its pharmacological evaluation in thyroxin-induced cardiac hypertrophy has not yet been explored. AIM OF THE STUDY: The aim of this study was to assess the cardioprotective effect of C. pareira root extract in experimentally induced hyperthyroidism in rats. MATERIALS AND METHODS: Male Wistar rats were treated with (i) thyroxin (0.1 mg/kg/day, i.p.) for 30 days, (ii) C. pareira extract (200 mg/kg/day, p.o.) alone for 60 days, (iii) C. pareira extract (100 and 200 mg/kg/day, p.o., respectively) for 30 days then with thyroxin for another 30 days, (iv) thyroxin for 30 days then C. pareira extract (100 and 200 mg/kg/day, p.o., respectively) for another 30 days. At the end of experiment, serum calcineurin, nitric oxide, lactate dehydrogenase, and thiobarbituric acid reactive substance as well as serum and/or myocardial antioxidant enzymes activity were estimated. RESULTS: Hyperthyroid induced cardiotoxicity was characterized by a significant (P<0.001) increase in heart weight/body weight ratio, serum calcineurin, nitric oxide, lactate dehydrogenase and thiobarbituric acid reactive substance levels as well as a significant decrease in serum reduced glutathione, myocardial glutathione peroxidase, glutathione reductase and glutathione-S-transferase levels, which were significantly (P<0.05 and P<0.01) reverted by C. pareira extract treatment. Reversal of histological changes on treatment with C. pareira extract was also supported the biochemical parameters. These results were quite comparable with amlodipine, the standard drug taken in this study. CONCLUSIONS: Treatment with C. pareira extract ameliorates thyroxin-induced oxidative stress and cardiac hypertrophy, probably through amelioration of calcineurin activity and augmentation of antioxidant enzyme activities.

Inhibition of α-glucosidase by new prenylated flavonoids from euphorbia hirta L. herb.

ETHNOPHARMACOLOGICAL RELEVANCE: Euphorbia hirta L. (Euphorbiaceae) is a pantropical medicinal rhizomatous herb, traditionally used in the treatment of diabetes, respiratory and gastro-intestinal disorders. AIM OF THE STUDY: To isolate and characterize the constituents of Euphorbia hirta and evaluate their in-vitro α-glucosidase inhibitory activity. The study is also aimed at describing structural activity relationship, type of α-glucosidase inhibition and in-vivo potential to regulate post prandial hyperglycemia in Wistar rats. MATERIALS AND METHODS: Methanolic extract of whole plant was suspended in water, and sequentially fractionated with n-hexane and ethyl acetate. Further ethyl acetate fraction was subjected to medium pressure liquid chromatography (MPLC) to isolate the active molecules under the following experimental conditions, pressure (up to 5 kg/cm(2)) and flow rate (2 in./min). The structural elucidation of isolated compounds was done on the basis of detailed spectral analysis. The α-glucosidase inhibitory potential of isolated compounds was evaluated and compared with standard drug acarbose. In addition, type of inhibition was dwelled by Lineweaver-Burk plot analysis. Further, sucrose tolerance test was performed in Wistar rats pre-treated with the isolated compounds and acarbose (0.015 mM) followed by a sucrose load (2g/kg, p.o.) and blood glucose level was measured up to 120 min by the glucose oxidase method. RESULTS: The ethyl acetate fraction afforded quercetrin (1), dimethoxy quercetrin (2), along with two new prenylated flavonosides designated as hirtacoumaroflavonoside (3) and hirtaflavonoside-B (4) characterized as 7-O-(p-coumaroyl)-5,7,4'-trihydroxy-6-(3,3-dimethyl allyl)-flavonol-3-O-ß-D-glucopyranosyl-(2" → 1"')-O-α-L-rhamnopyranoside and 5, 7, 3', 4'-trihydroxy-6-(3, 3-dimethyl allyl)-8-(iso-butenyl)-flavonol-3-C-ß-d-glucopyranoside, respectively. All the isolated compounds showed dose dependent inhibition of α-glucosidase which was found to be comparable to acarbose. Maximum α-glucosidase inhibition was achieved with compound 3 (IC50 0.022 mM) followed by 4 (IC50 0.071 mM) in comparison to acarbose (IC50 0.092 mM). The results revealed that 5,7,4'- trihydroxyflavone structure is imperative for the inhibitory activity. The prenylation in the flavonoids increase the potency and p-coumaroyl substitution at C-7 further enriched the α-glucosidase inhibition. Compound 3 exhibited non-competitive inhibition while compounds 1, 2 and 4 showed mixed non-competitive inhibitory pattern. The results of sucrose tolerance test corresponded well with the in vitro studies. CONCLUSION: α-Glucosidase inhibitory activity and sucrose tolerance test demonstrated by the prenylated flavonoids present in E. hirta provide credence to the ethnomedicinal use of the plant in the management of diabetes in folk medicine.

Prognostic significance of Ki67 expression in malignant peritoneal mesothelioma.

OBJECTIVES: Although Ki67 measurement by immunohistochemistry has been widely used as a prognostic index in cancers, it has not been reported in malignant peritoneal mesothelioma (MPM). Hence, this study examines the prognostic significance of Ki67 in MPM. METHODS: Specimens from 42 MPM patients were screened for Ki67 expression using immunohistochemistry. Ki67 expression was classified into 2 groups on the basis of expression (<25%=low; ≥25%=high) using standard methods. Using Kaplan-Meier survival analysis, the significance of Ki67 was assessed in different clinicopathologic categories. RESULTS: High expression of Ki67 (≥25% by immunohistochemical evaluation) was correlated with poor survival in the overall group (P=0.001); male sex (P=0.001); female sex (P=0.001); epithelioid tumors (P=0.001): male epithelioid (P=0.001), female epithelioid (P=0.003); peritoneal cancer index (PCI): PCI<20 (P=0.001), PCI≥20 (P=0.002); and age at diagnosis (AAD): AAD<60 years (P=0.001), AAD≥60 years (P=0.004). Independent of Ki67, male sex (P=0.007), sarcomatoid histology (P=0.001), PCI≥20 (P=0.013), and AAD≥60 years (P=0.004) correlated with poor survival. Multivariate analysis showed that only AAD≥60 years (P=0.049) and high Ki67 expression for all tumors (P=0.031), male sex (P=0.038), female sex (P=0.021), epithelioid tumors (P=0.044), and AAD<60 years (P=0.029) were statistically significant. CONCLUSIONS: Ki67 expression affects prognosis in MPM patients and helps to predict survival within the various clinicopathologic categories.

A formulation for in situ lysis of mucin secreted in pseudomyxoma peritonei.

Although numerous clinical attempts have been made to disintegrate mucin secreted by pseudomyxoma peritonei (PMP), none are clinically recommended. Through examination of the pharmacologic characteristics of two novel agents, we titrated an optimized combination of bromelain and N-acetyl cysteine (NAC) that demonstrates in vitro and in vivo efficacy in the dissolution of mucinous ascites from PMP. In the in vitro experiments, 1 g of mucin was incubated in varying concentrations of bromelain (0-400 µg/ml) and NAC (0-5%) individually followed by a combination before arriving at a therapeutic combination dose of 300 µg/ml bromelain+4% NAC. This established an effective dose of bromelain 300 µg/ml+4% NAC at pH 7.0, when tested in a rat model implanted with 3 g of mucin intraperitoneally (IP). IP administration of the drug in a rat model of PMP was shown to result in mucin disintegration within 72 hr with no toxicity observed.

Tulsi oil as a potential penetration enhancer for celecoxib transdermal gel formulations.

The focus of the present study was to develop and evaluate the transdermal system of celecoxib. Transdermal gels composed of carbopol 940 in propylene glycol (PG) containing penetration enhancers. The formulations were characterized by permeation, pharmacokinetics, pharmacodynamics and histopathology. Celecoxib permeation across excised rat skins were statistically (p < 0.05) enhanced by tulsi oil compared to turpentine oil containing formulations. In comparison to orally administered formulations, the pharmacokinetic parameters of gel and control formulations were significantly higher (p < 0.05). The maximum plasma concentration (Cmax) obtained with formulations containing 4% turpentine and 6% tulsi oil was, respectively, 1.52 and 2.41 times higher than the formulations without penetration enhancer. Similarly, area under the curve (AUC) of these formulations was 1.70 and 2.40 times higher than the formulations without penetration enhancers. Anti-inflammatory studies demonstrated a statistically significant (p < 0.05) pharmacodynamics profile for the transdermal gel formulations compared to orally administered and control celecoxib formulations. Histopathological studies revealed some disruption in the epidermis without any toxic effect on the dermis layer of skin by penetration enhancers. In summary, the transdermal gel formulations of celecoxib containing penetration enhancers sustained drug level in the blood and will reduce the dose frequency as required with its conventional oral formulation.

Anticancer effect of bromelain with and without cisplatin or 5-FU on malignant peritoneal mesothelioma cells.

Malignant peritoneal mesothelioma (MPM) is a rare neoplasm of the peritoneum, causally related to asbestos exposure. Nonspecific symptoms with a late diagnosis results in poor survival (<1 year). Treatment with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy has improved survival in some patients (median 3-5 years). Hence, new therapies are urgently needed. MUC1 is a glycosylation-dependent protein that confers tumours with invasiveness, metastasis and chemoresistance. Bromelain (cysteine proteinase) hydrolyses glycosidic bonds. Therefore, we investigated the antitumour effect of bromelain on MUC1-expressing MPM cell lines. MUC1 expressions in cells were assessed using immunofluorescent probes with cells grown on cover slips and western blot analysis on cell lysates. The cell lines were treated with various concentrations of bromelain and after 4 and 72 h, their viability was assessed using standard sulforhodamine assays. The cells were also treated with combinations of bromelain and cytotoxic drugs (cisplatin or 5-FU) and their viability was assessed at 72 h. Finally, with western blotting, the effects of bromelain on cellular survival proteins were investigated. PET cells expressed more MUC1 compared with YOU cells. The cell viability of both PET and YOU cells was adversely affected by bromelain, with PET cells being slightly resistant. The addition of bromelain increased the cytotoxicity of cisplatin significantly in both cell lines. However, 5-FU with bromelain did not show any significant increase in cytotoxicity. Bromelain-induced cell death is by apoptosis and autophagy. Bromelain has the potential of being developed as a therapeutic agent in MPM.