{"id":59162,"date":"2024-06-25T11:28:20","date_gmt":"2024-06-25T11:28:20","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=59162"},"modified":"2024-07-03T17:01:39","modified_gmt":"2024-07-03T17:01:39","slug":"improved-anti-nociceptive-anti-pyretic-and-anti-inflammatory-effects-of-orally-administered-liposome-encapsulated-piroxicam","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no2\/improved-anti-nociceptive-anti-pyretic-and-anti-inflammatory-effects-of-orally-administered-liposome-encapsulated-piroxicam\/","title":{"rendered":"Improved Anti-nociceptive, Anti-pyretic and Anti-inflammatory Effects of Orally Administered Liposome-encapsulated Piroxicam"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Piroxicam is a popular non-steroidal\nanti-inflammatory drug (NSAID) that exhibits prominent anti-nociceptive,\nanti-pyretic and anti-inflammatory activities1,2<\/sup>. This class II drug of\nBiopharmaceutical Classification System, characterized by low solubility and\nhigh permeability, is widely used for acute or long-term treatment of various\nmusculoskeletal and joint disorders3-5<\/sup>. Hence, rapid and sustainable drug\neffects to relief the signs and symptoms in these inflammatory diseases are\nhighly desirable6, 7<\/sup>. \n<\/p>\n\n\n\n

The dissolution of piroxicam in vivo<\/em>, however, is known to be an\nabsorption rate-limiting step which critically affects its therapeutic\nactivities3,8<\/sup>. Previous pharmacokinetic study has revealed that this\npoorly water-soluble drug, when orally administered, requires more than two\nhours to attain a peak plasma concentration. The slow absorption rate renders a\nlow bioavailability of drug and delayed the onset of its therapeutic effects9-11<\/sup>.\nAttempts to increase treatment efficiency by escalating the administered dose,\nhowever, increase risk of serious adverse drug reactions12<\/sup>. Due to\nthese drawbacks, it is therefore important to develop a new oral piroxicam\nformulation with enhanced onset of action and stronger therapeutic\nactivities.<\/p>\n\n\n\n

Liposomal drug delivery system\nappears to be an ideal approach to provide better therapeutic efficacy over an\nexisting drug formulation, mainly through alteration in dissolution and biodistribution of\nentrapped drug13-16<\/sup>. Liposomes-encapsulated piroxicam formulations have been previously\nreported to increase topical chronic anti-inflammatory activity in experimental\nanimal by as much as 26.3%1, 17<\/sup>. We also successfully\ndemonstrated the effectiveness of liposomal drug-encapsulation strategy to\nimprove anti-inflammatory effects of piroxicam in vitro<\/em>18<\/sup>.\nThe current study was therefore intrigued to evaluate the potential of present\nliposomal drug formulations in enhancing effectiveness of piroxicam in vivo.\nThe pharmacological properties such as anti-nociceptive, anti-pyretic and\nanti-inflammatory effects as well as the underlying mechanisms exhibited by\npiroxicam and liposome-encapsulated piroxicam administered orally were\ninvestigated using various experimental animal models.<\/p>\n\n\n\n

Materials and Methods<\/strong><\/p>\n\n\n\n

Materials<\/strong><\/p>\n\n\n\n

Pro-lipoTM<\/sup>\nDuo was from Lucas Meyer, France. Piroxicam, dimethylsulfoxide, carrageenan,\nBrewer\u2019s yeast, tribromoethanol, ter-amylalcohol, sodium chloride, acetylsalicylic acid and lipopolysaccharide from Escherichia coli<\/em> were purchased from Sigma, US.\nAcetic acid, ethanol and formalin were originated from BDH, UK. Diethyl ether\nwas obtained from R&M Marketing, UK.<\/p>\n\n\n\n

Preparation of liposomal samples<\/strong><\/p>\n\n\n\n

Liposomal\nsamples were prepared at room temperature in accordance to previously described\nprocedures19<\/sup>. Briefly, stock piroxicam solution (60 mg\/mL\ndimethylsulfoxide) was added into Pro-lipoTM Duo with moderate stirring (125\u00b125\nrpm) for 60 minutes. Concentrated piroxicam-loaded liposomal suspension was\nformed by drop-wise addition of distilled water (dH2O). This liposomal suspension\nwas continuously stirred for 10 hours before been further diluted with dH2O.\nMixture was then stirred for 30 minutes. The ratio of stock piroxicam solution:\nPro-lipoTM: dH2O (hydration): dH2O (dilution) was 1:5:9:25 w\/w\/w\/w. Blank\nliposomes were prepared according to same procedure except that\ndimethylsulfoxide was used instead of stock piroxicam solution. Final\nconcentration of dimethylsulfoxide in all prepared samples was 2.5%. The\nresulting mean liposomes diameter was around 370 nm with polydispersity index\nranging from 0.4 to 0.5.<\/p>\n\n\n\n

Experimental animals<\/strong><\/strong><\/p>\n\n\n\n

There were\n288 male Sprague Dawley rats weighing between 150 and 300 g and 48 male BALB\/c\nalbino strain mice weighing between 22 and 42 g. The mice were randomly\nassigned to normal cages with a light\/dark cycle of 12 hours, a temperature of\n25\u00b12\u00b0C, and a humidity level of 70\u201380%. Every day, the animals were given\naccess to tap water and a pelleted food. Before beginning any experimental\nmodification, animals were given a minimum of seven days to acclimate. The\nFaculty of Medicine and Health Sciences, Universiti Putra Malaysia’s Animal\nCare and Use Committee gave approval for the experiments to be conducted (Ref.\nNo. UPM\/FPSK\/PADS\/UUH\/F05).<\/p>\n\n\n\n

Anti-nociceptive\nassays<\/strong><\/p>\n\n\n\n

Acetic acid-induced abdominal writhing test<\/strong><\/p>\n\n\n\n

The test was\nconducted in accordance with Sulaiman20<\/sup> protocol description. Oral\npiroxicam or liposome-encapsulated piroxicam at 0, 0.3, 3, and 30 mg\/kg was\nadministered to mice (n = 6\/group). Mice were given a 30-minute pre-treatment\nperiod, intraperitoneal injections of 0.6% acetic acid (10 mL\/kg), and their\nenclosures made of clear Perspex. After a 5-minute delay, the presence of\ncontraction of the abdominal muscles along with elongation of the body and\nextension of the limbs (writhing effect) was cumulatively counted at 5-minute\nintervals for a duration of 30 minutes. The following ratio could be used to\nindicate the percentage of protection in terms of writhing number (W):       <\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Formalin-induced paw-licking test<\/strong><\/p>\n\n\n\n

Rats\n(n=6\/group) were given an initial 20-minute accommodation period in an\nobservation room, as per Mossadeq21<\/sup> description, before receiving\noral treatment with piroxicam or liposome-encapsulated piroxicam at 0, 0.3, 3,\nand 30 mg\/kg. Thirty minutes before the intraplantar injection of a 2.5%\nformalin solution (50 \u00b5L), all treatments were completed. The length of time\n(T) that the animal licked or bit the injected paw\u2014a sign of a pain\nreaction\u2014was noted for both the early phase (0\u20135 minutes) and the late phase\n(15\u201330 minutes). The formula used to compute the percentage inhibition of\nlicking was as follows:<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Carrageenan-induced mechanical hyperalgesia test<\/strong><\/p>\n\n\n\n

According to Fujii22<\/sup>, mechanical\nhyperalgesia in unrestrained rats was quantified as the hind limb withdrawal\nthreshold in response to a mechanical stimulus by the use of a modified\nRandall-Sellito test. A 0.1 mL intraplantar suspension of 1% carrageenan was\nused to elicit hyperalgesia in the hind paw 30 minutes after the rats (n =\n6\/group) received piroxicam or liposome-encapsulated piroxicam orally at 0,\n0.3, 3, and 30 mg\/kg. Using a dynamic plantar aesthesiometer (model 37450, Ugo\nBasile, Italy) fitted with a rounded-tip cone-shaped paw-presser that applied a\nlinearly increasing upward force (20 g\/s) to the plantar surface of the paw,\nthe nociceptive response to pressure in the injected paw was assessed. The\nforce (F) that made each animal remove its paw was the nociceptive threshold,\nmeasured in grams. Measurements were performed three times at several-second\nintervals, and mean value was taken as threshold. The reading was taken immediately after and at\n1, 2, 3, 4 and 5 hour intervals following carrageenan injection. A cut-off\nof 300 g was used to prevent mechanically induced injury. All tested rats were\nhabituated to the full procedure for three consecutive days prior to actual\nexperimentation. The percentages of inhibition of forces\nrecorded are calculated using formula stated below: <\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Carrageenan-induced thermal hyperalgesia test<\/strong><\/p>\n\n\n\n

Noxious heat stimulation of hind paw was assessed in unrestrained rats using the Hargreaves model of thermal hyperalgesia as described previously by Ortiz23<\/sup>. Rats (n=6\/group) were first treated by oral administration of piroxicam or liposome-encapsulated piroxicam at 0, 0.3, 3, 30 mg\/kg. After 30 minutes of pre-treatment, hyperalgesia was induced in the hind paw by intraplantar administration of 0.1 mL of 1% carrageenan suspension. Each animal was subjected to identical testing procedure using plantar test (model 37370, Ugo Basile, Italy). The source of the thermal nociceptive stimulus was an aluminum cylindrical vessel fitted with an infrared source. The plantar surface of the injected paw was manually targeted with a radiant infrared heat source (set to 50 i.r.) while the rat remained still. The light source automatically set off a timer, and the duration needed for a paw to exhibit an abrupt withdrawal was known as the paw withdrawal latency (L). To prevent tissue injury, a 20-second cutoff limit was placed on the stimulation duration. Paw withdrawal latencies were measured immediately following carrageenan injection, as well as at 1, 2, 3, 4, and 5 hours later. Three readings were taken at intervals of several seconds throughout each time point, and the mean value was used to determine the nociceptive threshold. Percentage of inhibition of thermal hyperalgesia was calculated using following formula:<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Anti-pyretic assay<\/strong><\/p>\n\n\n\n

Brewer\u2019s yeast-induced hyperthermia test<\/strong><\/p>\n\n\n\n

Rats were made hyperthermic via a\ntechnique outlined by Owoyele24<\/sup>. A digital thermometer with a\nlubricated probe was used to take the patient’s initial rectal temperature.\nRats were given a subcutaneous injection (10 mL\/kg) of a 20% brewer’s yeast\nslurry in the dorsum. For this investigation, only rats exhibiting a rectal\ntemperature increase of at least 0.7 \u00b0C after 18 hours were employed. Oral\npiroxicam or liposome-encapsulated piroxicam at 0, 0.3, 3, and 30 mg\/kg was\nadministered to six randomly chosen rats per group. The rectal temperature (R)\nwas taken up to five hours after the therapy was administered at one-hour\nintervals. The formula used to compute the percentage of inhibition of\nhyperthermia was as follows:<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Anti-inflammatory assays<\/strong><\/p>\n\n\n\n

Carrageenan-induced paw-edema test<\/strong><\/p>\n\n\n\n

As stated by Mossadeq21<\/sup>, this test was conducted. Oral\npiroxicam or liposome-encapsulated piroxicam at 0, 0.3, 3, and 30 mg\/kg was\nadministered to rats (n = 6\/group). Each rat’s hind paw received 0.1 mL of 1%\ncarrageenan solution intraplantarly 30 minutes after treatment, resulting in\nacute inflammation (swollen paws).  Paw\nvolume was measured using plethysmometry (model 7140 plethysmometer, Ugo\nBasile, Italy) both immediately after (V0) and at 1, 2, 3, 4, and 5 hour\nintervals (VT) after carrageenan injection. The volume displaced by the paw\nbetween final volume (VT) and initial volume (V0) was used to measure the\ndegree of inflammation. To ascertain the anti-inflammatory property, the\npercentage inhibition of edema was computed in relation to control.<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Cotton pellet-induced granuloma test<\/strong><\/p>\n\n\n\n

The test was conducted according to Panthong25<\/sup>\ninstructions. First, six rats per group (10 mL\/kg) were anesthetized with 2%\ntribromoethanol. Next, shaved dorsal portion of thoracic vertebrae region was\nsurgically implanted with 30 \u00b1 1 mg sterile cotton pellets per subcutaneous\ntissue. All rats received piroxicam or piroxicam liposome-encapsulated at 0,\n0.3, 3, and 30 mg\/kg orally throughout the seven-day study. Rats were killed on\nday eight by overdose on diethyl ether. After removing the cotton pellets,\ntheir wet weight was immediately determined. The cotton pellets were then\nweighed again after chilling and drying for an additional eighteen hours at 60\n\u00b0C. The following formulas were used to determine the test compound’s\ntransudative and granuloma weight as well as its percent transudative and\ngranuloma inhibition:<\/p>\n\n\n\n

Transudative weight (C) = Wet pellet weight \u2013 Dried pellet weight<\/p>\n\n\n\n

Granuloma weight (G) = Dried pellet weight \u2013 Initial pellet weight<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Assay of nitric oxide (NO), cytokines and cyclooxygenase (COX) activities <\/strong><\/p>\n\n\n\n

Blood\nsamples from rats in the cotton pellet-induced granuloma test model were used\nin this study. During\nthe last day of experiment (day 8), rats were anesthetized using diethyl ether\nbefore blood samples were carefully collected via cardiac puncture. All rats,\nwithout regaining consciousness, were sacrificed by overdose of diethyl ether. <\/p>\n\n\n\n

Determination of serum NO and inflammatory cytokines<\/strong><\/p>\n\n\n\n

Blood samples were allowed to coagulate for 60 minutes in sterile blank\ntubes before being centrifuged at 3000 g for 15 minutes. Subsequently, the\nsupernatant was maintained at -80 \u00b0C until commercially available\nnitrite\/nitrate colorimetric kit analysis (Roche, Germany). The concentrations\nof nitrite and nitrate were measured at 540 nm using spectrophotometry (Infinte\nM200, Tecan, Austria).Using known amounts of potassium and sodium\nnitrate, a standard curve was created. The indicator of NO level employed was\nthe sum of nitrite and nitrate. Using enzyme-linked immunosorbent assay (ELISA)\nkits, the levels of inflammatory cytokines (tumor necrosis factor (TNF)-\u03b1,\ninterleukin (IL)-1\u03b2, and IL-10) in serum samples were measured in compliance\nwith the manufacturer’s recommended methods (Thermo Scientific, US).<\/p>\n\n\n\n

Determination of blood COX-1 and COX-2 enzymes<\/strong><\/p>\n\n\n\n

A similar\nprocedure previously reported by Wallace26<\/sup> was used to measure the\nCOX-1 and COX-2 enzyme activity in blood samples.  Blood was drawn into a blank glass tube and\nincubated at 37 \u00b0C for 45 minutes in order to measure COX-1 activity. After\nthat, the samples were centrifuged for 15 minutes at 3000 g. Before being\ntested, the serum was moved to microcentrifuge tubes and kept at -80 \u00b0C. Under\nthe conditions of this experiment, thromboxane B2 generated from COX-1 in\nplatelet of blood samples was evaluated using a commercially available ELISA\nkit (Abnova, Taiwan). Each rat’s blood sample was split into two lithium\nheparin collection tubes with 10 \u00b5g\/mL of acetylsalicylic acid to measure COX-2\nactivity. Additionally, 10 \u00b5g\/mL of lipopolysaccharide from Escherichia coli, a\nbacterial endotoxin, was present in one of the tubes. Following a 24-hour\nincubation period at 37 \u00b0C in a shaking water bath, these tubes were\ncentrifuged for 15 minutes at 3000 g. Aspirating the supernatant (plasma)\nsamples into microcentrifuge tubes, they were kept at -80 \u00b0C until\nprostaglandin (PG) E2 was measured using a particular ELISA kit (Thermo\nScientific, US). The quantity of PGE2 produced by the COX-2 enzyme was equal to\nthe amount produced in the tubes containing endotoxin minus the amount in the\nother tube.<\/p>\n\n\n\n

Statistical\nanalyses<\/strong><\/p>\n\n\n\n

The analysis\nof variance was applied to the data, and then the groups were compared using\nDunnet’s multiple comparison test and the two groups were compared using\nStudent’s t-test. A significant P-value was defined as one that was less than\n0.05, or P<0.05. SPSS 16.0 was used for all statistical analyses (SPSS Inc.,\nUS).<\/p>\n\n\n\n

Results<\/strong><\/p>\n\n\n\n

Acetic acid-induced\nabdominal writhing test<\/strong><\/p>\n\n\n\n

Data obtained was stated in Table I and Figure 1. Both piroxicam and liposomes-encapsulated piroxicam exhibited dose-dependent pain inhibition effects at various experimental time points. Data showed that the highest inhibition of pain (\u226570.19%) was shown in mice treated with 30 mg\/kg liposome-encapsulated piroxicam. Lower doses of liposomes-encapsulated piroxicam (0.3 and 3 mg\/kg) were also found to be adequate in producing statistically significant anti-nociceptive activities that lasted throughout duration of this test. Contrarily, only 3 and 30 mg\/kg piroxicam produced a significant pain inhibitory activity that lasted for 10 and 30 minutes, respectively. Statistical comparisons among treatment groups with equivalent drug dosages successfully showed that liposome-encapsulated piroxicam formulations posed significantly higher antinociceptive effects than piroxicam at the dosage of 0.3, 3 and 30 mg\/kg. The total percentage inhibition was increased by 20.15%, 18.02% and 26.66% at drug dosage of 0.3, 3 and 30 mg\/kg, respectively.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: Number of abdominal writhing within 30 minutes (0-30th<\/sup> min).<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Table I: Number and percentage inhibition of abdominal writhing at different time intervals<\/strong>.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Treatment group<\/strong><\/p>\n<\/td>\n

\n

Drug dosage (mg\/kg)<\/strong><\/p>\n<\/td>\n

\n

Number of abdominal writhing [Inhibition (%)]<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

0-5th<\/sup> min<\/strong><\/p>\n<\/td>\n

\n

5-10th<\/sup> min<\/strong><\/p>\n<\/td>\n

\n

10-15th<\/sup> min<\/strong><\/p>\n<\/td>\n

\n

15-20th<\/sup> min<\/strong><\/p>\n<\/td>\n

\n

20-25th<\/sup> min<\/strong><\/p>\n<\/td>\n

\n

25-30th<\/sup> min<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Piroxicam<\/strong><\/p>\n<\/td>\n

\n

0 (Control)<\/p>\n<\/td>\n

\n

26.33 \u00b12.78<\/p>\n<\/td>\n

\n

25.67 \u00b13.46<\/p>\n<\/td>\n

\n

22.50 \u00b12.67<\/p>\n<\/td>\n

\n

22.83 \u00b13.75<\/p>\n<\/td>\n

\n

18.50 \u00b13.45<\/p>\n<\/td>\n

\n

17.33 \u00b12.23<\/p>\n<\/td>\n<\/tr>\n

\n

0.3<\/p>\n<\/td>\n

\n

17.83 \u00b11.85 [32.28]\n<\/td>\n

\n

18.83 \u00b11.91 [26.62]\n<\/td>\n

\n

20.50 \u00b11.38 [8.89]\n<\/td>\n

\n

17.67 \u00b11.74 [22.63]\n<\/td>\n

\n

14.00 \u00b12.11 [24.32]\n<\/td>\n

\n

13.83 \u00b12.04 [20.19]\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

12.83 \u00b12.69* <\/strong>[51.27]\n<\/td>\n

\n

16.00 \u00b11.29* <\/strong>[37.66]\n<\/td>\n

\n

18.17 \u00b11.40 [19.26]\n<\/td>\n

\n

17.83 \u00b13.44 [21.90]\n<\/td>\n

\n

15.50 \u00b11.52 [16.22]\n<\/td>\n

\n

11.50 \u00b11.34 [33.65]\n<\/td>\n<\/tr>\n

\n

30<\/p>\n<\/td>\n

\n

9.17 \u00b12.23* <\/strong>[65.19]\n<\/td>\n

\n

12.00 \u00b12.05* <\/strong>[53.25]\n<\/td>\n

\n

11.67 \u00b12.56* <\/strong>[48.15]\n<\/td>\n

\n

8.67 \u00b11.56* <\/strong>[62.04]\n<\/td>\n

\n

10.83 \u00b12.55 [41.44]\n<\/td>\n

\n

7.00 \u00b11.34* <\/strong>[59.62]\n<\/td>\n<\/tr>\n

\n

Liposomes-encapsulated piroxicam<\/strong><\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

20.67 \u00b12.63 [21.52]\n<\/td>\n

\n

18.00 \u00b12.07 [29.87]\n<\/td>\n

\n

20.67 \u00b10.92 [8.15]\n<\/td>\n

\n

15.83 \u00b11.35 [30.66]\n<\/td>\n

\n

15.17 \u00b11.72 [18.02]\n<\/td>\n

\n

14.33 \u00b11.87 [17.31]\n<\/td>\n<\/tr>\n

\n

0.3<\/p>\n<\/td>\n

\n

13.83 \u00b12.30* <\/strong>[47.47]\n<\/td>\n

\n

17.17 \u00b11.96* <\/strong>[33.12]\n<\/td>\n

\n

13.33 \u00b12.47*# <\/strong>[40.74]\n<\/td>\n

\n

10.83 \u00b12.27*# <\/strong>[52.55]\n<\/td>\n

\n

11.83 \u00b11.08 [36.04]\n<\/td>\n

\n

8.83 \u00b11.52* <\/strong>[49.04]\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

15.33 \u00b13.03* <\/strong>[41.77]\n<\/td>\n

\n

15.17 \u00b11.47* <\/strong>[40.91]\n<\/td>\n

\n

13.00 \u00b10.82*# <\/strong>[42.22]\n<\/td>\n

\n

9.83 \u00b11.70* <\/strong>[56.93]\n<\/td>\n

\n

9.00 \u00b12.00*# <\/strong>[51.35]\n<\/td>\n

\n

5.50 \u00b11.31*# <\/strong>[68.27]\n<\/td>\n<\/tr>\n

\n

30<\/p>\n<\/td>\n

\n

3.33 \u00b11.05*# <\/strong>[87.34]\n<\/td>\n

\n

4.00 \u00b11.44*# <\/strong>[84.42]\n<\/td>\n

\n

4.50 \u00b10.92*# <\/strong>[80.00]\n<\/td>\n

\n

2.83 \u00b11.14*# <\/strong>[87.59]\n<\/td>\n

\n

4.00 \u00b10.89*# <\/strong>[78.38]\n<\/td>\n

\n

5.17 \u00b10.95* <\/strong>[70.19]\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Values are mean \u00b1 S.E.M. (n=6\/group)<\/p>\n

*Significant difference (P<0.05) when compared to control at their respective time interval<\/p>\n

#Significant difference (P<0.05) when compared to equivalent dosage of piroxicam at their respective time interval<\/p>\n

Values in parenthesis are percentage of inhibition when compared to control at their respective time interval<\/p>\n\n\n

Formalin-induced paw-licking test<\/strong><\/p>\n\n\n\n

Result obtained was shown in Table 2. Reduction of paw-licking time, which reflected an anti-nociceptive activity, was observed in dose-dependent manner during both early and late phases. <\/s>Statistical analysis, however, showed that rats treated with 30 mg\/kg of piroxicam and 3 and 30 mg\/kg of liposome-encapsulated piroxicam only showed a significant reduction in the late phase (inflammatory phase) when compared to the control group. Their percentages of inflammatory pain inhibition were 62.88%, 47.46% and 81.36%, respectively. In addition, both 3 and 30 mg\/kg liposomes-encapsulated piroxicam also showed significant greater paw-licking time reduction when compared to their equivalent dosages of piroxicam during late phase. Percentage inhibition was increased by 25.77% and 18.48% for drug dosage of 3 and 30 mg\/kg, respectively.<\/p>\n\n\n\n

Table 2: Paw-licking time and percentage inhibition at different phases<\/strong>.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Treatment group<\/strong><\/p>\n<\/td>\n

\n

Drug dosage (mg\/kg)<\/strong><\/p>\n<\/td>\n

\n

Paw-licking time (s) [Inhibition (%)]<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Early phase<\/strong><\/p>\n<\/td>\n

\n

Late phase<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Piroxicam<\/strong><\/p>\n<\/td>\n

\n

0  (Control)<\/p>\n<\/td>\n

\n

89.83 \u00b1 8.57<\/p>\n<\/td>\n

\n

98.33 \u00b1 9.38<\/p>\n<\/td>\n<\/tr>\n

\n

0.3<\/p>\n<\/td>\n

\n

82.00 \u00b1 5.77  [8.72]\n<\/td>\n

\n

82.50 \u00b1 9.08 [16.10]\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

76.83 \u00b1 7.56 [14.47]\n<\/td>\n

\n

77.00 \u00b1 6.76 [21.69]\n<\/td>\n<\/tr>\n

\n

30<\/p>\n<\/td>\n

\n

75.33 \u00b1 5.64 [16.14]\n<\/td>\n

\n

36.50 \u00b1 5.10* [62.88]\n<\/td>\n<\/tr>\n

\n

Liposomes-encapsulated piroxicam<\/strong><\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

87.50 \u00b1 5.88  [2.60]\n<\/td>\n

\n

90.67 \u00b1 7.30  [7.80]\n<\/td>\n<\/tr>\n

\n

0.3<\/p>\n<\/td>\n

\n

71.83 \u00b1 3.97 [20.04]\n<\/td>\n

\n

73.17 \u00b1 5.26 [25.59]\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

67.00 \u00b1 6.87 [25.42]\n<\/td>\n

\n

51.67 \u00b1 4.26*# [47.46]\n<\/td>\n<\/tr>\n

\n

30<\/p>\n<\/td>\n

\n

67.67 \u00b1 6.43 [24.68]\n<\/td>\n

\n

18.33 \u00b1 6.32*# [81.36]\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Values are mean \u00b1 S.E.M. (n=6\/group)<\/p>\n

*Significant difference (P<0.05) when compared to control at their respective phase<\/p>\n

#Significant difference (P<0.05) when compared to equivalent dosage of piroxicam at their respective phase<\/p>\n

Values in parenthesis are percentage of inhibition when compared to control at their respective phase<\/p>\n\n\n

Carrageenan-induced\nmechanical hyperalgesia test<\/strong><\/p>\n\n\n\n

The acquired data was displayed in Table 3. Rats treated with 30 mg\/kg piroxicam and 3 and 30 mg\/kg liposome-encapsulated piroxicam showed a considerably higher tolerance of mechanical hyperalgesia at different experimental time points than the control group, according to statistical analyses. However, 30 mg\/kg liposome-encapsulated piroxicam was the only formulation with notable inhibitory effects that persisted throughout the entire test. Rats treated with 30 mg\/kg liposomal piroxicam sample showed the two highest percentages of inhibition (118.7% and 120.9%) in the current investigation, which were discovered two and four hours after carrageenan injection. Interestingly, at these two time points, significant differences were also found when 30 mg\/kg liposomes-encapsulated piroxicam was compared to 30 mg\/kg piroxicam. The increment in percentages of inhibition was 44.9% and 55.8%, respectively. <\/p>\n\n\n\n

Table 3: Paw pressure and percentage inhibition at different time intervals<\/strong>.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Treatment group<\/strong><\/p>\n<\/td>\n

\n

Drug dosage (mg\/kg)<\/strong><\/p>\n<\/td>\n

\n

Pressure applied on paw (g) [Inhibition (%)]<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

0h<\/strong><\/p>\n<\/td>\n

\n

1h<\/strong><\/p>\n<\/td>\n

\n

2h<\/strong><\/p>\n<\/td>\n

\n

3h<\/strong><\/p>\n<\/td>\n

\n

4h<\/strong><\/p>\n<\/td>\n

\n

5h<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Piroxicam<\/strong><\/p>\n<\/td>\n

\n

0 (Control)<\/p>\n<\/td>\n

\n

105.4 \u00b113.7<\/p>\n<\/td>\n

\n

109.2 \u00b112.4<\/p>\n<\/td>\n

\n

108.0 \u00b112.5<\/p>\n<\/td>\n

\n

106.0 \u00b114.7<\/p>\n<\/td>\n

\n

96.6 \u00b112.1<\/p>\n<\/td>\n

\n

94.9 \u00b121.7<\/p>\n<\/td>\n<\/tr>\n

\n

0.3<\/p>\n<\/td>\n

\n

135.2 \u00b114.6 [28.2]\n<\/td>\n

\n

120.4 \u00b118.2 [10.3]\n<\/td>\n

\n

123.2 \u00b115.0 [14.1]\n<\/td>\n

\n

124.8 \u00b117.4 [17.7]\n<\/td>\n

\n

110.0 \u00b118.4 [13.9]\n<\/td>\n

\n

110.2 \u00b115.5 [16.2]\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

171.3 \u00b127.0 [62.5]\n<\/td>\n

\n

153.0 \u00b119.4 [40.1]\n<\/td>\n

\n

148.8 \u00b111.1 [37.8]\n<\/td>\n

\n

151.0 \u00b119.6 [42.5]\n<\/td>\n

\n

138.9 \u00b117.1 [43.8]\n<\/td>\n

\n

127.8 \u00b114.4 [34.7]\n<\/td>\n<\/tr>\n

\n

30<\/p>\n<\/td>\n

\n

218.3 \u00b118.5* [107.1]\n<\/td>\n

\n

209.0 \u00b114.6* [91.4]\n<\/td>\n

\n

187.7 \u00b112.0* [73.8]\n<\/td>\n

\n

194.7 \u00b117.3* [83.6]\n<\/td>\n

\n

159.4 \u00b113.9 [65.1]\n<\/td>\n

\n

151.1 \u00b115.0 [59.3]\n<\/td>\n<\/tr>\n

\n

Liposomes-encapsulated piroxicam<\/strong><\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

117.7 \u00b110.9 [11.6]\n<\/td>\n

\n

117.7 \u00b122.3 [7.7]\n<\/td>\n

\n

119.6 \u00b121.0 [10.7]\n<\/td>\n

\n

121.3 \u00b121.0 [14.5]\n<\/td>\n

\n

111.2 \u00b122.3 [15.2]\n<\/td>\n

\n

110.0 \u00b115.7 [15.9]\n<\/td>\n<\/tr>\n

\n

0.3<\/p>\n<\/td>\n

\n

143.0 \u00b120.1 [35.6]\n<\/td>\n

\n

146.7 \u00b119.4 [34.3]\n<\/td>\n

\n

147.2 \u00b116.4 [36.3]\n<\/td>\n

\n

143.3 \u00b116.7 [35.2]\n<\/td>\n

\n

130.3 \u00b124.3 [35.0]\n<\/td>\n

\n

127.3 \u00b121.7 [34.2]\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

163.0 \u00b122.6 [54.6]\n<\/td>\n

\n

176.7 \u00b117.3 [61.7]\n<\/td>\n

\n

164.3 \u00b116.7 [52.2]\n<\/td>\n

\n

178.3 \u00b115.9* [68.2]\n<\/td>\n

\n

167.9 \u00b115.9* [73.9]\n<\/td>\n

\n

157.3 \u00b125.4 [65.8]\n<\/td>\n<\/tr>\n

\n

30<\/p>\n<\/td>\n

\n

213.0 \u00b111.7* [102.0]\n<\/td>\n

\n

220.8 \u00b119.1* [102.1]\n<\/td>\n

\n

236.2 \u00b117.8*# [118.7]\n<\/td>\n

\n

228.4 \u00b121.0* [115.5]\n<\/td>\n

\n

213.3 \u00b114.0*# [120.9]\n<\/td>\n

\n

199.1 \u00b119.5* [109.8]\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Values are mean \u00b1 S.E.M. (n=6\/group)<\/p>\n

*Significant difference (P<0.05) when compared to control at their respective time interval<\/p>\n

#Significant difference (P<0.05) when compared to equivalent dosage of piroxicam at their respective time interval<\/p>\n

Values in parenthesis are percentage of inhibition when compared to control at their respective time interval<\/p>\n\n\n

Carrageenan-induced thermal hyperalgesia test<\/strong><\/p>\n\n\n\n

Result was shown in Table 4. Comparing to control group, significant prolongation of paw withdrawal latencies was observed during second hour after carrageenan injection in rats treated with 30 mg\/kg piroxicam. At comparable experimental time periods, rats administered with 3 and 30 mg\/kg liposome-encapsulated piroxicam likewise showed considerable inhibition when compared to the control group, with the highest percentage inhibition being 55.71%. Regretfully, when piroxicam and liposome-encapsulated piroxicam at equal dosages were statistically compared, there was no discernible difference in the inhibitions for thermal hyperalgesia.<\/p>\n\n\n\n

Table 4: Latency time and percentage inhibition at different time intervals<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Treatment group<\/strong><\/p>\n<\/td>\n

\n

Drug dosage (mg\/kg)<\/strong><\/p>\n<\/td>\n

\n

Latency time of reaction (s) [Inhibition (%)]<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

0h<\/strong><\/p>\n<\/td>\n

\n

1h<\/strong><\/p>\n<\/td>\n

\n

2h<\/strong><\/p>\n<\/td>\n

\n

3h<\/strong><\/p>\n<\/td>\n

\n

4h<\/strong><\/p>\n<\/td>\n

\n

5h<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Piroxicam<\/strong><\/p>\n<\/td>\n

\n

0 (Control)<\/p>\n<\/td>\n

\n

7.82 \u00b10.87<\/p>\n<\/td>\n

\n

7.26 \u00b10.82<\/p>\n<\/td>\n

\n

7.10 \u00b10.57<\/p>\n<\/td>\n

\n

7.38 \u00b10.66<\/p>\n<\/td>\n

\n

7.02 \u00b10.82<\/p>\n<\/td>\n

\n

7.19 \u00b10.60<\/p>\n<\/td>\n<\/tr>\n

\n

0.3<\/p>\n<\/td>\n

\n

8.49 \u00b10.75 [8.67]\n<\/td>\n

\n

8.97 \u00b10.64 [23.66]\n<\/td>\n

\n

9.23 \u00b10.58 [30.05]\n<\/td>\n

\n

9.23 \u00b10.67 [24.98]\n<\/td>\n

\n

8.74 \u00b11.93 [24.62]\n<\/td>\n

\n

8.38 \u00b11.59 [16.53]\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

9.32 \u00b11.26 [19.19]\n<\/td>\n

\n

9.33 \u00b11.25 [28.56]\n<\/td>\n

\n

9.75 \u00b10.46 [37.32]\n<\/td>\n

\n

9.55 \u00b11.30 [29.35]\n<\/td>\n

\n

9.06 \u00b10.75 [29.14]\n<\/td>\n

\n

9.45 \u00b11.87 [31.35]\n<\/td>\n<\/tr>\n

\n

30<\/p>\n<\/td>\n

\n

10.76 \u00b11.24 [37.67]\n<\/td>\n

\n

10.42 \u00b10.93 [43.57]\n<\/td>\n

\n

10.66 \u00b10.78* [50.08]\n<\/td>\n

\n

10.46 \u00b10.60* [41.61]\n<\/td>\n

\n

10.36 \u00b10.73 [47.59]\n<\/td>\n

\n

10.82 \u00b10.83 [50.42]\n<\/td>\n<\/tr>\n

\n

Liposomes-encapsulated piroxicam<\/strong><\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

7.76 \u00b11.10 [-0.78]\n<\/td>\n

\n

8.09 \u00b10.94 [11.56]\n<\/td>\n

\n

7.97 \u00b10.57 [12.21]\n<\/td>\n

\n

8.00 \u00b10.30 [8.35]\n<\/td>\n

\n

7.79 \u00b10.56 [11.01]\n<\/td>\n

\n

7.97 \u00b10.80 [10.81]\n<\/td>\n<\/tr>\n

\n

0.3<\/p>\n<\/td>\n

\n

9.08 \u00b10.74 [16.13]\n<\/td>\n

\n

9.47 \u00b11.15 [30.47]\n<\/td>\n

\n

9.04 \u00b11.05 [27.39]\n<\/td>\n

\n

8.65 \u00b10.81 [17.16]\n<\/td>\n

\n

9.03 \u00b11.92 [28.74]\n<\/td>\n

\n

9.13 \u00b11.42 [26.87]\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

9.38 \u00b11.14 [19.97]\n<\/td>\n

\n

9.87 \u00b10.88 [35.99]\n<\/td>\n

\n

10.13 \u00b10.80* [42.64]\n<\/td>\n

\n

9.93 \u00b10.71 [34.54]\n<\/td>\n

\n

10.03 \u00b10.64 [42.91]\n<\/td>\n

\n

9.99 \u00b11.38 [38.92]\n<\/td>\n<\/tr>\n

\n

30<\/p>\n<\/td>\n

\n

10.59 \u00b11.23 [35.47]\n<\/td>\n

\n

10.53 \u00b10.85 [45.18]\n<\/td>\n

\n

11.06 \u00b10.67* [55.71]\n<\/td>\n

\n

10.60 \u00b10.76* [43.57]\n<\/td>\n

\n

10.71 \u00b10.77 [52.57]\n<\/td>\n

\n

11.01 \u00b10.51 [52.97]\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Values are mean \u00b1 S.E.M. (n=6\/group)<\/p>\n

*Significant difference (P<0.05) when compared to control at their respective time interval<\/p>\n

#Significant difference (P<0.05) when compared to equivalent dosage of piroxicam at their respective time interval<\/p>\n

Values in parenthesis are percentage of inhibition when compared to control at their respective time interval<\/p>\n\n\n

Brewer\u2019s yeast-induced hyperthermia test<\/strong><\/p>\n\n\n\n

Data from anti-pyretic assay was presented in Table 5. Significantly lowered rectal temperatures comparing to control group were observed when the hyperthermic animals were orally treated with 30 mg\/kg of piroxicam, both in non-encapsulated and liposomes-encapsulated forms. However, the former showed significant anti-hyperthermia effect at third hour following treatment whereas the latter posed its inhibitory activity after two hours. Treatment with 0.3 and 3 mg\/kg of liposomes-encapsulated piroxicam also caused significant pyretic inhibition at a few experimental time points. The percentages of inhibitions were between 1.50% and 1.99%. However, no statistically significant difference between the piroxicam and liposome-encapsulated piroxicam at equivalent dosages at all tested time points.<\/p>\n\n\n\n

Table 5: Rectal temperature and percentage inhibition at different time intervals<\/strong>.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Treatment group<\/strong><\/p>\n<\/td>\n

\n

Drug dosage (mg\/kg)<\/strong><\/p>\n<\/td>\n

\n

Rectal temperature (\u00b0C) [Inhibition (%)]<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

0h<\/strong><\/p>\n<\/td>\n

\n

1h<\/strong><\/p>\n<\/td>\n

\n

2h<\/strong><\/p>\n<\/td>\n

\n

3h<\/strong><\/p>\n<\/td>\n

\n

4h<\/strong><\/p>\n<\/td>\n

\n

5h<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Piroxicam<\/strong><\/p>\n<\/td>\n

\n

0 (Control)<\/p>\n<\/td>\n

\n

37.93 \u00b10.26<\/p>\n<\/td>\n

\n

37.98 \u00b10.06<\/p>\n<\/td>\n

\n

37.88 \u00b10.12<\/p>\n<\/td>\n

\n

37.80 \u00b10.12<\/p>\n<\/td>\n

\n

37.75 \u00b10.17<\/p>\n<\/td>\n

\n

37.57 \u00b10.17<\/p>\n<\/td>\n<\/tr>\n

\n

0.3<\/p>\n<\/td>\n

\n

37.85 \u00b10.11 [0.22]\n<\/td>\n

\n

37.73 \u00b10.10 [0.66]\n<\/td>\n

\n

37.60 \u00b10.11 [0.75]\n<\/td>\n

\n

37.53 \u00b10.13 [0.71]\n<\/td>\n

\n

37.35 \u00b10.11 [1.06]\n<\/td>\n

\n

37.27 \u00b10.16 [0.80]\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

37.97 \u00b10.18 [-0.09]\n<\/td>\n

\n

37.68 \u00b10.13 [0.79]\n<\/td>\n

\n

37.47 \u00b10.21 [1.10]\n<\/td>\n

\n

37.53 \u00b10.26 [0.71]\n<\/td>\n

\n

37.40 \u00b10.16 [0.93]\n<\/td>\n

\n

37.20 \u00b10.26 [0.98]\n<\/td>\n<\/tr>\n

\n

30<\/p>\n<\/td>\n

\n

37.98 \u00b10.26 [-0.13]\n<\/td>\n

\n

37.48 \u00b10.20 [1.32]\n<\/td>\n

\n

37.30 \u00b10.20 [1.54]\n<\/td>\n

\n

36.97 \u00b10.19* [2.20]\n<\/td>\n

\n

36.88 \u00b10.09* [2.30]\n<\/td>\n

\n

36.88 \u00b10.13* [1.82]\n<\/td>\n<\/tr>\n

\n

Liposomes-encapsulated piroxicam<\/strong><\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

37.93 \u00b10.12 [0.00]\n<\/td>\n

\n

37.83 \u00b10.21 [0.39]\n<\/td>\n

\n

37.80 \u00b10.14 [0.22]\n<\/td>\n

\n

37.78 \u00b10.10 [0.04]\n<\/td>\n

\n

37.52 \u00b10.16 [0.62]\n<\/td>\n

\n

37.47 \u00b10.09 [0.27]\n<\/td>\n<\/tr>\n

\n

0.3<\/p>\n<\/td>\n

\n

37.92 \u00b10.05 [0.04]\n<\/td>\n

\n

37.70 \u00b10.15 [0.75]\n<\/td>\n

\n

37.60 \u00b10.14 [0.75]\n<\/td>\n

\n

37.27 \u00b10.20 [1.41]\n<\/td>\n

\n

37.18 \u00b10.21* [1.50]\n<\/td>\n

\n

37.07 \u00b10.19 [1.33]\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

37.80 \u00b10.14 [0.35]\n<\/td>\n

\n

37.57 \u00b10.08 [1.10]\n<\/td>\n

\n

37.27 \u00b10.14* [1.63]\n<\/td>\n

\n

37.13 \u00b10.12* [1.76]\n<\/td>\n

\n

37.00 \u00b10.14* [1.99]\n<\/td>\n

\n

36.95 \u00b10.06* [1.64]\n<\/td>\n<\/tr>\n

\n

30<\/p>\n<\/td>\n

\n

37.90 \u00b10.12 [0.09]\n<\/td>\n

\n

37.32 \u00b10.11* [1.76]\n<\/td>\n

\n

37.23 \u00b10.16* [1.72]\n<\/td>\n

\n

36.85 \u00b10.11* [2.51]\n<\/td>\n

\n

36.75 \u00b10.08* [2.65]\n<\/td>\n

\n

36.73 \u00b10.08* [2.22]\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Values are mean \u00b1 S.E.M. (n=6\/group)<\/p>\n

*Significant difference (P<0.05) when compared to control at their respective time interval<\/p>\n

#Significant difference (P<0.05) when compared to equivalent dosage of piroxicam at their respective time interval<\/p>\n

Values in parenthesis are percentage of inhibition when compared to control at their respective time interval.<\/p>\n\n\n

Carrageenan-induced\npaw-edema test<\/strong><\/p>\n\n\n\n

Result obtained was showed in Table 6. Both piroxicam and liposomes-encapsulated piroxicam samples at 0.3, 3 and 30 mg\/kg exhibited significant anti-inflammatory effects as compared to control group at various time points following carrageenan injection. Nevertheless, in contrast to the piroxicam, 0.3 and 3 mg\/kg liposomes-encapsulated piroxicam were found to demonstrate significant paw-edema inhibitory activities that lasted until the final experimental time point (fifth hour) in present study. Further statistical analyses revealed that the 3 and 30 mg\/kg liposomes-encapsulated piroxicam successfully resulted in a significant decrement of paw-edema volumes when compared to their equivalent piroxicam dosage groups after the fifth and third hour, respectively. In term of percentages of inhibition, an increment between 23.81% and 28.57% was observed during this time frame.<\/p>\n\n\n\n

Table 6: Edema volume and percentage inhibition at different time intervals<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Treatment group<\/strong><\/p>\n<\/td>\n

\n

Drug dosage (mg\/kg)<\/strong><\/p>\n<\/td>\n

\n

Edema volume (mL) [Inhibition (%)]<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

1h<\/strong><\/p>\n<\/td>\n

\n

2h<\/strong><\/p>\n<\/td>\n

\n

3h<\/strong><\/p>\n<\/td>\n

\n

4h<\/strong><\/p>\n<\/td>\n

\n

5h<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Piroxicam<\/strong><\/p>\n<\/td>\n

\n

0 (Control)<\/p>\n<\/td>\n

\n

0.48 \u00b10.07<\/p>\n<\/td>\n

\n

0.43 \u00b10.09<\/p>\n<\/td>\n

\n

0.55 \u00b10.09<\/p>\n<\/td>\n

\n

0.50  \u00b10.11<\/p>\n<\/td>\n

\n

0.50 \u00b10.08<\/p>\n<\/td>\n<\/tr>\n

\n

0.3<\/p>\n<\/td>\n

\n

0.29 \u00b10.05* [40.00]\n<\/td>\n

\n

0.21 \u00b10.03 [50.00]\n<\/td>\n

\n

0.33 \u00b10.05 [39.13]\n<\/td>\n

\n

0.24 \u00b10.05* [52.38]\n<\/td>\n

\n

0.29 \u00b10.06 [42.86]\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

0.29 \u00b10.04* [40.00]\n<\/td>\n

\n

0.26 \u00b10.07 [38.89]\n<\/td>\n

\n

0.26 \u00b10.06* [52.17]\n<\/td>\n

\n

0.24 \u00b10.05* [52.38]\n<\/td>\n

\n

0.26 \u00b10.04 [47.62]\n<\/td>\n<\/tr>\n

\n

30<\/p>\n<\/td>\n

\n

0.29 \u00b10.05* [40.00]\n<\/td>\n

\n

0.17 \u00b10.06* [61.11]\n<\/td>\n

\n

0.21 \u00b10.03* [60.87]\n<\/td>\n

\n

0.24 \u00b10.03* [52.38]\n<\/td>\n

\n

0.17 \u00b10.04* [66.67]\n<\/td>\n<\/tr>\n

\n

Liposomes-encapsulated piroxicam<\/strong><\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0.48 \u00b10.05 [0.00]\n<\/td>\n

\n

0.36 \u00b10.08 [16.67]\n<\/td>\n

\n

0.45 \u00b10.09 [17.39]\n<\/td>\n

\n

0.38 \u00b10.05 [23.81]\n<\/td>\n

\n

0.38 \u00b10.11 [23.81]\n<\/td>\n<\/tr>\n

\n

0.3<\/p>\n<\/td>\n

\n

0.24 \u00b10.03* [50.00]\n<\/td>\n

\n

0.29 \u00b10.05 [33.33]\n<\/td>\n

\n

0.26 \u00b10.04* [52.17]\n<\/td>\n

\n

0.26 \u00b10.04* [47.62]\n<\/td>\n

\n

0.24 \u00b10.05* [52.38]\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

0.19 \u00b10.03* [60.00]\n<\/td>\n

\n

0.21 \u00b10.07 [50.00]\n<\/td>\n

\n

0.29 \u00b10.04* [47.83]\n<\/td>\n

\n

0.21 \u00b10.05* [57.14]\n<\/td>\n

\n

0.12 \u00b10.04*# [76.19]\n<\/td>\n<\/tr>\n

\n

30<\/p>\n<\/td>\n

\n

0.17 \u00b10.02* [65.00]\n<\/td>\n

\n

0.10 \u00b10.03* [77.78]\n<\/td>\n

\n

0.07 \u00b10.05*# [86.96]\n<\/td>\n

\n

0.12 \u00b10.02*# [76.19]\n<\/td>\n

\n

0.05 \u00b10.03*# [90.48]\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Values are mean \u00b1 S.E.M. (n=6\/group)<\/p>\n

*Significant difference (P<0.05) when compared to control at their respective time interval<\/p>\n

#Significant difference (P<0.05) when compared to equivalent dosage of piroxicam at their respective time interval<\/p>\n

Values in parenthesis are percentage of inhibition when compared to control at their respective time interval<\/p>\n\n\n

Cotton pellet-induced granuloma test<\/strong><\/p>\n\n\n\n

Results (Table 7) showed a dose-dependent chronic anti-inflammatory effect by piroxicam and liposomes-encapsulated piroxicam. Data obtained showed that treatment using liposomes-encapsulated piroxicam at 3 and 30 mg\/kg significantly reduced both the transudative and granuloma weights by more than 36.41%. On the contrary, only highest dose (30 mg\/kg) of non-encapsulated piroxicam resulted in statistically significant inhibitory activities as compared to control group. The percentage inhibition for transudative and granuloma weights were only 30.97% and 32.89%, respectively. Further statistical analyses proved that liposomal piroxicam samples possessed greater anti-transudative and anti-granuloma effects when compared to piroxicam of same dosage. Significantly different in transudative weight was found between piroxicam and liposomes-encapsulated piroxicam at 3 mg\/kg. The transudative weight was 18.3% higher when piroxicam was used to treat the animals in present experiment. In addition, 3 and 30 mg\/kg liposomes-encapsulated piroxicam samples respectively resulted in 33.31% and 18.90% greater inhibition of granuloma weight than the piroxicam samples of equivalent dosage.<\/p>\n\n\n\n

Table 8: Transudative weight, granuloma weights and percentage inhibition<\/strong>.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Treatment group<\/strong><\/p>\n<\/td>\n

\n

Drug dosage (mg\/kg)<\/strong><\/p>\n<\/td>\n

\n

Weight (mg) [Inhibition (%)]<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Transudative<\/strong><\/p>\n<\/td>\n

\n

Granuloma<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Piroxicam<\/strong><\/p>\n<\/td>\n

\n

0  (Control)<\/p>\n<\/td>\n

\n

429.90 \u00b1 47.52<\/p>\n<\/td>\n

\n

90.82 \u00b1 11.97<\/p>\n<\/td>\n<\/tr>\n

\n

0.3<\/p>\n<\/td>\n

\n

376.95 \u00b1 37.44 [12.32]\n<\/td>\n

\n

78.92 \u00b1 12.37 [13.10]\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

352.05 \u00b1 19.22 [18.11]\n<\/td>\n

\n

76.33 \u00b1 3.75 [15.95]\n<\/td>\n<\/tr>\n

\n

30<\/p>\n<\/td>\n

\n

296.75 \u00b1 22.48* [30.97]\n<\/td>\n

\n

60.95 \u00b1 5.59* [32.89]\n<\/td>\n<\/tr>\n

\n

Liposomes-encapsulated piroxicam<\/strong><\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

428.10 \u00b1 22.87 [0.42]\n<\/td>\n

\n

82.32 \u00b1 5.90 [9.36]\n<\/td>\n<\/tr>\n

\n

0.3<\/p>\n<\/td>\n

\n

353.03 \u00b1 19.35 [17.88]\n<\/td>\n

\n

68.98 \u00b1 3.51 [24.04]\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

273.37 \u00b1 20.95*# [36.41]\n<\/td>\n

\n

46.08 \u00b1 7.07*# [49.26]\n<\/td>\n<\/tr>\n

\n

30<\/p>\n<\/td>\n

\n

243.92 \u00b1 19.60* [43.26]\n<\/td>\n

\n

43.78 \u00b1 5.08*# [51.79]\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Values are mean \u00b1 S.E.M. (n=6\/group)<\/p>\n

*Significant difference (P<0.05) when compared to control<\/p>\n

#Significant difference (P<0.05) when compared to equivalent dosage of piroxicam<\/p>\n

Values in parenthesis are percentage of inhibition when compared to control<\/p>\n\n\n

NO activity<\/strong><\/p>\n\n\n\n

The data obtained (Table 8) demonstrated that, in comparison to the control group, repeated treatment of either piroxicam or liposome-encapsulated piroxicam at the highest dose of 30 mg\/kg resulted in a considerable reduction of NO. In comparison to the control, a lower dosage of liposome-encapsulated piroxicam (3 mg\/kg) was sufficient to cause a considerable reduction in serum NO levels. Subsequent statistical analysis showed that the piroxicam encapsulated in liposomes at a dose of 30 mg\/kg significantly inhibited NO more than the piroxicam at the same dosage. It was discovered to have an 18.72% higher percentage of inhibition than the piroxicam sample.<\/p>\n\n\n\n

Table 8: Effects of different treatment upon serum NO<\/strong>.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Treatment group<\/strong><\/p>\n<\/td>\n

\n

Drug dosage (mg\/kg)<\/strong><\/p>\n<\/td>\n

\n

Concentration (\u00b5M) [Inhibition (%)]<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Piroxicam<\/strong><\/p>\n<\/td>\n

\n

0 (Control)<\/p>\n<\/td>\n

\n

18.96 \u00b1 0.97<\/p>\n<\/td>\n<\/tr>\n

\n

0.3<\/p>\n<\/td>\n

\n

17.52 \u00b1 1.36 [7.59]\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

16.08 \u00b1 1.51 [15.17]\n<\/td>\n<\/tr>\n

\n

30<\/p>\n<\/td>\n

\n

11.58 \u00b1 1.23* [38.91]\n<\/td>\n<\/tr>\n

\n

Liposomes-encapsulated piroxicam<\/strong><\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

17.08 \u00b1 1.30 [9.90]\n<\/td>\n<\/tr>\n

\n

0.3<\/p>\n<\/td>\n

\n

14.23 \u00b1 1.65 [24.94]\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

12.14 \u00b1 1.62* [35.98]\n<\/td>\n<\/tr>\n

\n

30<\/p>\n<\/td>\n

\n

8.03 \u00b1 0.95*# [57.63]\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Values are mean \u00b1 S.E.M. (n=6\/group)<\/p>\n

*Significant difference (P<0.05) when compared to control<\/p>\n

#Significant difference (P<0.05) when compared to equivalent dosage of piroxicam<\/p>\n

Values in parenthesis are percentage of inhibition when compared to control<\/p>\n\n\n

Inflammatory cytokines activity<\/strong><\/p>\n\n\n\n

Data obtained was summarized in Table IX. Analyses showed that piroxicam only resulted in a significant change to serum TNF-\u03b1 at the highest dose (30 mg\/kg). Whereas a lower dose (0.3 or 3 mg\/kg) of liposomes-encapsulated piroxicam were adequate to cause statistically significant TNF-\u03b1 and IL-1\u03b2 inhibition. Further analyses revealed that 3 and 30 mg\/kg liposomes-encapsulated piroxicam samples possessed significantly greater inhibition of TNF-\u03b1 and IL-1\u03b2 respectively than the effects exhibited by non-encapsulated piroxicam of equivalent dosages. Contrarily, only the 30 mg\/kg liposomes-encapsulated piroxicam sample successfully resulted in significant increment of serum IL-10 when compared to control group. There was no statistical difference found between the liposome-encapsulated piroxicam and equivalent dose of piroxicam for serum IL-10.<\/p>\n\n\n\n

Table 9: Effects of different treatment upon serum TNF-\u03b1, IL-1\u03b2 and IL-10<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Treatment group<\/strong><\/p>\n<\/td>\n

\n

Drug dosage (mg\/kg)<\/strong><\/p>\n<\/td>\n

\n

Concentration (pg\/mL) [Inhibition (%)]<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

TNF-\u03b1<\/strong><\/p>\n<\/td>\n

\n

IL-1\u03b2<\/strong><\/p>\n<\/td>\n

\n

IL-10<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Piroxicam<\/strong><\/p>\n<\/td>\n

\n

0 (Control)<\/p>\n<\/td>\n

\n

55.80 \u00b1 6.38<\/p>\n<\/td>\n

\n

56.88 \u00b1 6.77<\/p>\n<\/td>\n

\n

46.18 \u00b1 3.47<\/p>\n<\/td>\n<\/tr>\n

\n

0.3<\/p>\n<\/td>\n

\n

44.27 \u00b1 4.91 [20.66]\n<\/td>\n

\n

46.58 \u00b1 6.08 [18.11]\n<\/td>\n

\n

54.76 \u00b1 5.16 [18.57]\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

45.44 \u00b1 4.04 [18.57]\n<\/td>\n

\n

39.00 \u00b1 4.03 [31.44]\n<\/td>\n

\n

50.92 \u00b1 7.15 [10.26]\n<\/td>\n<\/tr>\n

\n

30<\/p>\n<\/td>\n

\n

32.90 \u00b1 4.80* [41.03]\n<\/td>\n

\n

43.26 \u00b1 3.27 [23.95]\n<\/td>\n

\n

60.47 \u00b1 4.85 [30.93]\n<\/td>\n<\/tr>\n

\n

Liposomes-encapsulated piroxicam<\/strong><\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

53.43 \u00b1 3.89 [4.25]\n<\/td>\n

\n

53.66 \u00b1 6.62 [5.67]\n<\/td>\n

\n

50.35 \u00b1 5.02 [9.03]\n<\/td>\n<\/tr>\n

\n

0.3<\/p>\n<\/td>\n

\n

44.51\u00b1 6.93 [20.23]\n<\/td>\n

\n

34.18 \u00b1 5.59* [39.91]\n<\/td>\n

\n

52.34 \u00b1 3.20 [13.34]\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

32.55 \u00b1 3.92*# [41.66]\n<\/td>\n

\n

35.76 \u00b1 4.60* [37.14]\n<\/td>\n

\n

61.46 \u00b1 4.05 [33.09]\n<\/td>\n<\/tr>\n

\n

30<\/p>\n<\/td>\n

\n

22.85 \u00b1 4.26* [59.05]\n<\/td>\n

\n

29.63 \u00b1 4.56*# [47.92]\n<\/td>\n

\n

65.21\u00b1 4.32* [41.20]\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/strong>Values are mean \u00b1 S.E.M. (n=6\/group)<\/p>\n

*Significant difference (P<0.05) when compared to control<\/p>\n

#Significant difference (P<0.05) when compared to equivalent dosage of piroxicam<\/p>\n

Values in parenthesis are percentage of inhibition when compared to control <\/strong><\/p>\n\n\n

Blood COX-1 and COX-2 activities<\/strong><\/p>\n\n\n\n

The dose-dependent inhibition of COX enzymes by different treatment samples was summarized in Table 10. Results showed that both piroxicam and liposomes-encapsulated piroxicam significantly reduced serum concentration of COX-1 enzyme only at the highest drug dosage of 30 mg\/kg. Further analyses demonstrated no significant difference between the two treatment groups. In contrast, rats which were treated with piroxicam at 3 and 30 mg\/kg as well as liposomes-encapsulated piroxicam at 0.3, 3 and 30 mg\/kg significantly inhibited the production of COX-2 enzyme. Statistical comparisons between treatment groups with equivalent drug dosage successfully showed that liposomes-encapsulated piroxicam (3 and 30 mg\/kg) posed significantly greater COX-2 enzyme inhibition than piroxicam in non-encapsulated form. The total percentage inhibition was increased by 18.25% and 19.22% at drug dosage of 3 and 30 mg\/kg, respectively. <\/p>\n\n\n\n

Table X: Effects of different treatment upon blood COX-1 and COX-2<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Treatment group<\/strong><\/p>\n<\/td>\n

\n

Drug dosage (mg\/kg)<\/strong><\/p>\n<\/td>\n

\n

Concentration (ng\/mL) [Inhibition (%)]<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

COX-1<\/strong><\/p>\n<\/td>\n

\n

COX-2<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Piroxicam<\/p>\n<\/td>\n

\n

0 (Control)<\/p>\n<\/td>\n

\n

41.04 \u00b1 4.15<\/p>\n<\/td>\n

\n

25.40 \u00b1 2.24<\/p>\n<\/td>\n<\/tr>\n

\n

0.3<\/p>\n<\/td>\n

\n

37.33 \u00b1 5.58 [9.04]\n<\/td>\n

\n

21.83 \u00b1 1.81 [14.05]\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

26.39 \u00b1 2.97 [35.70]\n<\/td>\n

\n

16.48 \u00b1 1.43* [35.14]\n<\/td>\n<\/tr>\n

\n

30<\/p>\n<\/td>\n

\n

17.99 \u00b1 3.31* [56.16]\n<\/td>\n

\n

11.87 \u00b1 1.70* [53.29]\n<\/td>\n<\/tr>\n

\n

Liposomes-encapsulated piroxicam<\/strong><\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

40.12 \u00b1 5.65 [2.25]\n<\/td>\n

\n

24.01 \u00b1 1.70 [5.49]\n<\/td>\n<\/tr>\n

\n

0.3<\/p>\n<\/td>\n

\n

36.16 \u00b1 4.52 [11.88]\n<\/td>\n

\n

17.77 \u00b1 2.57* [30.03]\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

28.65 \u00b1 4.76 [30.18]\n<\/td>\n

\n

11.84 \u00b1 1.11*# [53.39]\n<\/td>\n<\/tr>\n

\n

30<\/p>\n<\/td>\n

\n

17.64 \u00b1 3.67* [57.02]\n<\/td>\n

\n

6.98 \u00b1 0.76*# [72.51]\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Values are mean \u00b1 S.E.M. (n=6\/group)<\/p>\n

*Significant difference (P<0.05) when compared to control<\/p>\n

#Significant difference (P<0.05) when compared to equivalent dosage of piroxicam<\/p>\n

Values in parenthesis are percentage of inhibition when compared to control<\/p>\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

Fever,\ninflammation, and pain have all been linked to the pathophysiology of a number\nof clinical disorders, including cancer, vascular illnesses, and arthritis27,\n28<\/sup>. As a result, it is thought to be crucial for the liposomal delivery\nmethod to enhance the in vivo therapeutic activities (anti-nociceptive,\nanti-pyretic, and anti-inflammatory effects) of piroxicam, especially in order\nto better manage the symptoms and indicators of these illnesses. In present study, several\nanimal models were\nemployed to evaluate the effectiveness of present liposomal formulations in\nimproving in vivo<\/em> therapeutic activities of orally\nadministered piroxicam at three different dose levels.<\/p>\n\n\n\n

It is generally known that, in animal models, the assessment of pain\nperception can be done by examining overt behavioral reactions or nociceptive\nreflexes that are directly induced by noxious stimuli (such as chemical,\nthermal, electrical, and mechanical ones).29, 30<\/sup>. An very sensitive\nassay that is frequently used to screen drugs for anti-nociceptive activity at\ndose levels that may look inert in other approaches is the acetic acid-induced\nabdominal writhing test25, 31<\/sup>. Intraperitoneal injection of acetic acid, which\nirritates serous membranes and produce peritoneal inflammation, provokes a very\nstereotyped behavior known as writhing effect32<\/sup>. Administration of\nrelatively small doses of NSAIDs or opioids is known to abolish the writhing\nresponse in a dose dependent manner33, 34<\/sup>. The present study’s data\ndemonstrated that piroxicam and piroxicam formulations encapsulated in\nliposomes both demonstrated dose-dependent pain inhibitory properties, as\nevidenced by a decrease in the number of writhing effects. The results of this\nchemonociception test also showed that, in comparison to piroxicam at equal\ndosages, piroxicam encapsulated in liposomes demonstrated a more potent and\nsustained anti-nociceptive effect. However, liposome\nencapsulation has several limitations such as poor molecular targeting,\ninstability, short circulation time in vivo and low encapsulation efficiency1,3<\/sup>.\nInterestingly, we have demonstrated the solution of these limitation of\npiroxicam encapsulation previously18,19<\/sup>.<\/p>\n\n\n\n

The acetic acid-induced abdominal\nwrithing test, however, shows poor specificity as it does not indicate whether\nan anti-nociceptive activity is central and\/or peripheral29, 35<\/sup>.\nAcetic acid itself may cause pain, while at the same time stimulates peripheral\ntissues to release endogenous substances such as serotonin, histamine, PG (e.g.\nPGE2<\/sub>, PGF2\u03b1<\/sub>), bradykinin and substance P that sensitize\npain nerve endings (nociceptors) which in turn causes pain at the location36,\n37<\/sup>. The anti-nociception activity of a compound may therefore be due to\nits action on visceral receptors that are sensitive to acetic acid, the\ninhibition of algogenic substances production or the inhibition of painful\nmessages transmission at central level31<\/sup>. Therefore, the formalin-induced paw\nlicking test was carried out to strengthen further evidences of improved\nanti-nociceptive activity that was observed in writhing test. <\/p>\n\n\n\n

A valid model of tonic pain that mimics\nclinical pain circumstances in humans is the formalin-induced paw licking test.\nThis assay has a solid track record of helping to clarify a compound’s mode of\naction24,38, 39. Formalin injected intraplantarly into the paw causes two\nseparate phases, each of which represents a different kind of pain. The early\nphase, which represents centrally mediated pain (neurogenic pain), is a direct\nresult of stimulation of nociceptors in the paw. On the other hand, the late\nphase is brought on by inflammation, which causes nociceptors to become\nsensitized and produce pain (inflammatory pain) when algogenic substances are\nreleased from damaged tissues. There is also, at least to some degree,\nsensitization of central nociceptive neurons during late phase25, 34<\/sup>.\nThus, drugs that act primarily on central nervous system (e.g. opiods) are\nknown to inhibit both phases equally, whereas peripherally acting drugs (e.g.\nNSAIDs) are more effective in inhibiting the late phase33, 35<\/sup>. Data\nin present study revealed that liposomes-encapsulated piroxicam possessed\nsignificantly greater nociception inhibition than piroxicam during the late\nphase only. This finding suggested that liposomes-encapsulated piroxicam\nresulted in a more effective suppression of inflammatory pain, hence supported\nthe fact that a stronger peripherally mediated anti-nociceptive activity could\nbe attained using the present liposomal formulations.<\/p>\n\n\n\n

One ubiquitous issue that frequently\ncauses both spontaneous pain and hyperalgesia is inflammatory pain. After\ncutaneous injury and\/or inflammation, the hyperalgesia response (heightened\nsensitivity to pain) is defined by a peripheral sensitization of nociceptors\ndue to an increase in neuronal membrane excitability to inflammatory mediators30<\/sup>.\nThe potential advantages of liposomal formulations in lowering mechanical and\nthermally induced hyperalgesia responses were assessed in the current\ninvestigation. In animal models, carrageenan, a family of linear sulphated\npolysaccharides isolated from the marine red seaweed Chondrus crispu, was\nutilized to cause transient inflammation and hyperalgesia40<\/sup>. Because\ncarrageenan-induced inflammatory models can closely mimic various real pain\nsyndromes, they have been widely used in the field of pain research41<\/sup>.\nThe results of the current hyperalgesia experiments demonstrated that piroxicam\nencapsulated in liposomes may raise animals’ nociceptive thresholds to heat and\nmechanical stimuli at both highest dosage levels, indicating that lower drug\ndosages may be necessary to achieve anti-hyperalgesia benefits. In addition,\nstatistical comparisons between treatment groups with equivalent drug dosages\nrevealed that animal\u2019s tolerance to mechanical-induced hyperalgesia response\ncould also be increased using present liposomal formulations.<\/p>\n\n\n\n

Fever is resulted during tissue\ndamage, inflammation, infection or disease states. A pathogenic fever can be\ninduced in animal model by administration of yeast. Under these conditions,\nformation of cytokines and thus the synthesis of PG are increased 32<\/sup>. NSAIDs exert\ntheir anti-pyretic effect by inhibition of PGE2<\/sub> synthesis, which is\nresponsible for triggering hypothalamus to increase body temperature42<\/sup>. Data obtained in present study\nshowed that liposomal formulation was able to result in a faster onset of\nanti-pyretic effect, even though the intensity of effect was not significantly\nincreased. Moreover, in comparison to non-encapsulated form of piroxicam, lower\ndosages of liposomes-encapsulated piroxicam were found to be sufficient in\nproducing significant anti-pyretic effects.<\/p>\n\n\n\n

The effects of the liposomal drug\nencapsulation strategy on the anti-inflammatory properties of piroxicam were\nassessed in addition to its anti-nociceptive and anti-pyretic properties. An\nindication of acute inflammatory alterations was a very reproducible edema that\nformed after an intraplantar injection of carrageenan42<\/sup>. In the\ncurrent investigation, the first- and third-hours following carrageenan\nadministration were when inflammatory edema peaked. It is widely established\nthat paw edema caused by carrageenan is a biphasic event: PG and lysosome\nenzymes are released two to three hours after carrageenan injection, while\nserotonin, histamine, and kinins are released during the initial phase (first\nhour)31<\/sup>. Data obtained in present carrageenan-induced paw edema test\nindicated that, at two highest dose levels, liposomes-encapsulated piroxicam\nexhibited stronger acute anti-inflammatory activity than piroxicam. A higher\ninhibition of paw edema during the second phase suggested that piroxicam might\nattenuate release of PG or lysosome enzymes more effectively when the drug was\nformulated using liposomes.<\/p>\n\n\n\n

On the other\nhand, chronic inflammation results from an initial response that is unable to\ncompletely remove proinflammatory chemicals, leading to neutrophil infiltration\nand exudation as well as fibroblast proliferation43<\/sup>. A common method\nfor determining how well a substance inhibits the transudative and\nproliferative aspects of chronic inflammation is the cotton pellet-induced\ngranuloma test44<\/sup>. By regulating mucopolysaccharides, decreasing\ncollagen fiber synthesis, and limiting granulocyte infiltration to foreign\nimplanted bodies, NSAIDs can reduce the growth of granuloma tissue, which is\nthe outcome of a cellular reaction. However, it is reported that the inhibitory\nactivities exerted by NSAIDs are only slight, whereas steroidal\nanti-inflammatory agents can strongly inhibit both transudative and\nproliferative phases25<\/sup>. Data from present study proved that the\npresent liposomal formulations were able to increase anti-transudative and\nanti-granuloma effects of piroxicam. <\/p>\n\n\n\n

All NSAIDs, including piroxicam, are\nknown to exhibit their therapeutic activities through inhibition of various\nmediators, which in turn result in blockage of pathways that contribute to\npathologic conditions such as pain, fever and inflammation44<\/sup>. As an\nattempt to gain further insights into the molecular basis of underlying\nmechanisms that contributed to the improved in\nvivo <\/em>therapeutic activities of piroxicam using present liposomal\nformulations, the production of different key inflammatory mediators in animal\nwas evaluated. Blood samples from rats in cotton pellet-induced granuloma test\nwere used for present evaluation since a model of chronic inflammation involved\na complex response which released numerous inflammatory mediators and resulted\nin multiple interactions39<\/sup>.<\/p>\n\n\n\n

NO is a short-lived regulatory\nmolecule which mediates diverse physiological processes such as vasodilatation\nand neurotransmission. While NO serves as a vital, multipurpose chemical\nmessenger in biological systems, inducible NOS is known to produce excessive\namounts of NO, which is linked to multiple forms of inflammation and multistage\ncarcinogenesis in inflammatory sites46<\/sup>. Considerable evidence has\ndemonstrated that NO and its synthases play pivotal roles in development and\nmaintenance of edema, pain and hyperalgesia during inflammation 2,19<\/sup>.\nBesides the NO pathway, it is also well-established that a distinct cytokines\ncascade that unfolds during inflammatory processes can contribute to the\ndevelopment of inflammatory pain and hyperalgesia. The increased production of\nTNF-\u03b1 during inflammation stimulates expression of other proinflammatory mediators\n(e.g. IL-1\u03b2, IL-6 and granulocyte-macrophage colony-stimulating factors), facilitates\ninflammatory cell infiltration and promotes induction of COX enzymes (39). Both\nTNF-\u03b1 and IL-1\u03b2 are reported to contribute to a hyperalgesia state\nthrough activation or sensitization of peripheral nociceptors, thus decreasing\nthreshold of nociceptor during inflammation47<\/sup>. However,\nanti-inflammatory cytokines (such IL-10) that are released by monocytes and\nlymphocytes can prevent the generation of proinflammatory cytokines, which in\nturn causes an anti-inflammatory effect in the test model48<\/sup>.<\/p>\n\n\n\n

Additionally, the level of PG is\nincreased during an inflammatory process. PG can intensify and extend the\nsignals generated by other molecular messengers, such as NO and proinflammatory\ncytokines, even when they do not cause inflammation on their own48<\/sup>. It\nhas been demonstrated that the inhibition of PG production by NSAIDs\neffectively reduces inflammatory symptoms such as pain and edema49<\/sup>. The\nprimary enzyme responsible for converting arachidonic acids into PG is COX. COX\ncomes in two different isoforms: COX-1 and COX-2. It is well established that\nCOX-1 supplies PG at a physiological level necessary for normal kidney,\nstomach, and platelet function; in contrast, COX-2 has been shown to be strongly\nstimulated by proinflammatory mediators, which raises PG synthesis during\ninflammation50<\/sup>. While it has been proposed that constitutively\nexpressed COX-1 contributes to inflammatory processes, it is generally\nacknowledged that reduced COX-2 synthesis or activity oversees\nanti-inflammatory effects in both localized and systemic settings37<\/sup>.\nNSAIDs inhibit both COX isoenzymes, but to different degrees7<\/sup>.<\/p>\n\n\n\n

The\ncurrent investigation found that the suppression of COX-1 in the animal model\nwas unaffected by liposomal formulation. On the other hand, at equivalent\ndosages, piroxicam encapsulated in liposomes demonstrated a higher degree of\nCOX-2 inhibition than piroxicam. Additionally, it has been shown that at lesser\ndosages, piroxicam encapsulated in liposomes effectively suppresses COX-2.\nTherefore, it can be hypothesized that current liposomal formulations enhanced\nthe piroxicam’s therapeutic effects by selectively inhibiting COX-2.<\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

In summary, present study\nsuccessfully revealed that the liposomal drug formulations improved in vivo<\/em> therapeutic activities for\npiroxicam. As compared with non-encapsulated form of piroxicam, the liposomal\npiroxicam formulations were shown to exhibit stronger and longer-lasting peripherally\nmediated anti-nociceptive effects in animal models. Lower dosages of\nliposome-encapsulated piroxicam may result in a considerable suppression of\nacetic acid-induced abdominal writhing, formalin-induced paw licking, and\ncarrageenan-induced mechanical and thermal hyperalgesia, according to results\nfrom the nociception assays. Besides enhancing latency\nthreshold for inflammatory pain, lower dosage of\nliposomes-encapsulated piroxicam was also found to be enough in resulting\nanti-pyretic effect in Brewer\u2019s yeast-induced hyperthermia test. Furthermore,\neven when lower medication dosages were employed to treat animals, the current\ninvestigation demonstrated that piroxicam encapsulated in liposomes held much\nstronger acute and chronic anti-inflammatory properties than piroxicam. The improved in vivo<\/em> therapeutic activities by present liposomal delivery system\nwere probably mediated via enhanced inhibition of proinflammatory\nmediators including NO, TNF-\u03b1\nand IL-1\u03b2. Besides, liposomal formulations were also shown to modulate the\nrelease of IL-10, an anti-inflammatory cytokine. Moreover, present liposomal\nformulation also resulted in an effective down-regulation of COX-2 but not\nCOX-1 mediated PG synthesis in animal. Thus, these may prove very useful in\nclinical settings. Regardless of these excellent results, future\nstudies are needed to fully understand the exact mechanism and extensive\ntoxicological assessment for safety.<\/p>\n\n\n\n

Acknowledgment<\/strong><\/p>\n\n\n\n

The authors thank Animal Care and Use Committee of Faculty of Medicine and Health Sciences, Universiti Putra Malaysia.<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

There is no conflict of interest<\/p>\n\n\n\n

Funding Source<\/strong><\/p>\n\n\n\n

This research was supported by\nFundamental Research Grant Scheme from the Ministry of Higher Education,\nMalaysia (04-10-07-277FR). <\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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