propylene glycol enhances anti-inflammatory effects of phenylbutazone

Gen. Pharmac. Vol. 21, No. l, pp. 131-134, 1990 0306-3623/90 $3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1990 Pergamon Press plc

PROPYLENE GLYCOL ENHANCES ANTI-INFLAMMATORY EFFECTS OF PHENYLBUTAZONE

TEREZA T. OSHIRO, CATARINA F. P. TEIXEIRA and SEmi OGA

Departamento de Farmacologia, Instituto de Ci6ncias Biom6dicas, Universidade de Silo Paulo, 05508 $3.o Paulo, SP, Brazil [Tel. (011) 210-4341]

(Received 6 June 1989)

Abstract--1. The interference of propylene glycol with anti-inflammatory effects of phenylbutazone was investigated.

2. Inhibitory effect of phenylbutazone on both carrageenin-induced edema and the cotton pellet granuloma was increased when propylene glycol was used as solvent.

3. Propylene glycol given alone inhibited carrageeninqnduced edema and pleurisy, as well as granulomatous tissue formation.

4. Some pharmacokinetic parameters of phenylbutazone were also changed by propylene glycol administered simultaneously.

5. These results suggest that propylene glycol probably increases the anti-inflammatory effect of phenylbutazone by summation and by raising the plasma half-life and the distribution volume of phenylbutazone.

INTRODUCTION

Solubilization of chemicals and drugs and preserva- tion of their therapeutical properties are a common problem in the pharmaceutical formulations, spe- cially in those of parenteral use. One of the solvents which have been largely used in drug, cosmetic and food preparat ion is the propylene glycol. For a long time it has been considered as an inactive organic solvent (Seidenfeld and Hanzlik, 1932; Braun and Cartland, 1936; Weatherby and Haag, 1938).

More recently a number of pharmacological effects of propylene glycol have been reported (Zaroslinski et al., 1971; Yasaka et al., 1979; Singh et al., 1982). These findings suggest that propylene glycol when used as a drug vehicle can determine its own pharmacological effects as well as interfering with the thera- peutic efficacy of drugs.

In the present study we investigated the possible influence of the propylene glycol on the pharmacological activities of phenylbutazone. The anti-inflammatory effects of phenylbutazone administered intraperitoneally in aqueous solutions were compared with those of phenylbutazone in propylene glycol.

MATERIAL AND METHODS

Animals

Male Wistar rats weighing 180__+10g were used. The animals were fasted overnight, with free access to tapwater before each experiment.

Carrageenin-induced paw edema

The edema was induced by injection of 0.1 ml of 1% carrageenin suspension in saline (w/v in 0.9% NaCI) into the subplantar area of the left hind paw of the unanesthetized rats. The right hind paw of these rats was injected with an equal volume of saline. Three groups of rats were estab- lished: the first one received phenylbutazone (40mg/kg) dissolved in water while the second group received phenylbutazone (40 mg/kg) dissolved in propylene glycol.

The last one was treated with propylene glycol alone (4 ml/kg). All of these treatments were done intraperitoneally 60 min before the carrageenin injection. The paw volume of the rats was determined by measuring the dis- placement of mercury in a plethysmograph connected to a Statham P23 D6 transducer and Grass Polygraph, model 79 (Van Arman et al., 1965).

Carrageenin-induced paw edema in adrenalectomized rats

The rats were bilaterally adrenalectomized or sham-operated under ether anesthesia followed by treatment after 7 days. Two groups of animals were intraperitoneally injected with 4 ml/kg of propylene glycol and 4 ml/kg of saline, 60 min before the carrageenin.

Determination o f plasma levels o f phenylbutazone

Two groups of animals were treated respectively with phenylbutazone dissolved in water and with phenylbutazone dissolved in propylene glycol. Later on, the blood samples were withdrawn by cardiac punction 3, 5, I0, 15, 30, 60, 120 and 180 min under ether anesthesia. Then, the samples were centrifuged at 2500 rpm for 8 min. The plasma concentration of phenylbutazone in the samples was determined according to slightly modified method of Burns et al. (1953). The analysis were done on a Varian Techtron u.v.-visible spectrophotometer, model 634-S, at 260 nm. Pharmacoki- netic parameters of the fl-phase were calculated as described by Gillette (1975).

Increased vascular permeability

The vascular permeability responses were studied with Evans blue dye according to the method of Wilhelm et al. (1958). An amount of 25 mg/kg of Evans blue in 2.5% aqueous solution was intravenously administered to the rats 30rain after treatment with the propylene glycol. Five minutes later, each rat received intracutaneous injection of 3 amounts (l, 5 and 10 pg) of histamine in saline solution, at a constant volume of 0.1 ml, plus 0.1 ml of saline injection. 30 rain later, the animals were killed and their skins were stripped from the subcutaneous tissue. Eventu- ally the fragments containing the Evans blue stained area were cut and immersed in formamide in order to extract the dye. The dye concentration was estimated in a Varian

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Fig. 1. Influence of propylene glycol on the anti-edema effect of phenylbutazone. 4 ml/kg of saline (O), 4 ml/kg of propylene glycol (Q), 40 mg/kg of phenylbutazone in water (I-q) and 40 mg/kg of phenylbutazone in propylene glycol ( l l ) were administered i.p. 60min prior to carrageenin injection. Each point represents the mean value and bars represents 5- SEM of 6 animals. *Significantly different

(P < 0.05) from the control.

Techtron u.v.-visible spectrophotometer, model 634-S, at 620 rim.

Carrageenin-induced pleurisy

Pleurisy was induced in rats by injecting carrageenin suspension in sterile saline into their pleural cavity. The

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Fig. 2. Effect of propylene glycol on the carrageenin-induced paw edema in adrenalectomized rats. Adrenalec- tomized animals treated with 4 ml/kg of saline (O), and with 4 mt/kg of propylene glycol (O), sham-operated animals treated with 4 ml/kg of saline (r-l) and with 4 ml/kg of propylene glycol (ll). Drugs were administered 60 rain prior to the carrageenin injection. Each point represents the mean value of 6 animals, and the bars represent+SEM.

*Significantly different (P < 0.05) from the control.

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Fig. 3. Effect of propylene glycol (PG) on the increased vascular permeability induced by histamine. 4ml/kg of saline (open bars) and 4 ml/kg of PG (hatched bars) were administered i.p. 60 min prior to i.v. injection of 2.5% Evans blue solution. Each bar represents the mean value of 10 animals. *Significantly different (P < 0.05) from the control.

intrapleural injection was given between the third and fifth ribs on the right side of the mediastinum using a procedure described by Vinegar et al. (1973). Moreover, the rats suffered 2 administrations of propylene glycol: (a) 60 min prior to the injection of 150/~g of carrageenin in order to investigate the solvent effect during the first phase of pleurisy, and (b) 4hr after the injection of 1000#g of carrageenin in order to observe the solvent effect during the second phase. The resultant total exudate volume was determined at 4th and 21st hour after carrageenin injection corresponding respectively to the first and second phases of

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Fig. 4. Effect of propylene glycol (PG) on the carrageenin- induced pleurisy. 4 ml/kg of saline (open bars) and 4 ml/kg of PG (hatched bars) were administered i.p. 60 min prior to the earragcenin injection. Pleurisy was caused by injection of earragecnin, 150 #g (lst phase) and 1000/ag (2nd phase).


Propylene glycol on inflammation

Table 1. Effect of propylene glycol on the leucocyte mobilization in the first and second phases of the carrageenin-induced pleurisy

First phase Second phase

Neutrophyl Mononuclear Neutrophyl Mononuclear Treatment ( X 10 6) ( X l0 s) ( X 106) ( x 106) Saline 69.22 + 4.51 9.58 + 1.67 133.90 + 6.31 92.05 + 8.99 Propylene glycol 57.67 + 8.86 4.23 ± 0.58* 111.71 ± 10.53 58.35 ± 7.43* Aqueous suspension of carrageenin was injected into intrapleural cavity in a constant volume of 0.25 ml.

Propylene glycol was administered in the dose of 4 ml/kg i.p. 60 rain prior to and 240 rain after carrageenin, respectively for 1st and 2nd phases of pleurisy. Each value represents the mean _+ SEM of 7 rats.


133

pleurisy. The pleural cavity of each animal was washed with 0A % EDTA solution in PBS-buffer, adjusted to pH 7.2. Exudate and washes were pooled and their volume adjusted to 5 ml with 0.1% EDTA solution in PBS-buffer. Then, total leucocyte as well as differential cell counts were done as proposed by Miale (1977).

Cotton pellet granuloma

5 mm-cut sections of dental cotton rolls (Johnson & Johnson Inc.) were prepared in lots of 4 pellets weighing 160 mg followed by sterilization and subcutaneous implantation in four symmetrical position of the rats' abdomen, under ether anesthesia. Daily, each group of animals was intraperitoneally injected with saline (4 ml/kg), propylene glycol (4 ml/kg), as well as with phenylbutazone dissolved in propylene glycol (40 mg: 4 ml/kg) respectively for 7 days. In the 8th day the animals were killed using ether. After death their granulomas were removed and dried overnight at 65°C and eventually weighted. Lastly, the weight difference between the initial pellet and the final dry one was evaluated.

Statistical assessment

Analysis of variance with 2 ways classification was used. Sequential differences among means were calculated (at a level of P < 0.05) using the Tukey's contrast analysis (Sokal and Rohlf, 1969).

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Fig. 5. Influence of propylene glycol (PG) on the inhibitory effect of phenylbutazone (FZ) on the granuloma formation. Animals were injected with 40 mg/kg of FZ dissolved in water or in PG, 0.1 mg/kg of dexamethasone (Dexa) and 4 ml/kg of PG daily for 7 days. Each bar represents the mean value + SEM of 6 animals. *Significantly different

(P < 0,05) from the control.

Drugs and solvents

Propylene glycol (Carl6 Erba), phenylbutazone calcium (Boheringer Ingelheimer), carrageenin (Sigma), histamine (Inlab), Evans Blue and formamide (Merck, Darmstadt) were used.

RESULTS AND DISCUSSION

The administration of phenylbutazone dissolved in propylene glycol caused greater inhibitory effect on the carrageenin-induced edema than the administration of the same drug in an aqueous suspension (Fig. 1). The potentiation of the phenylbutazone effect is possible, since propylene glycol itself showed significant anti-inflammatory activity. Moreover, propylene glycol in a close of 4 ml/kg i.p. inhibited significantly the carrageenin-induced edema in both adrenalectomized and sham-operated animals (Fig. 2). This finding shows that anti- inflammatory activity of propylene glycol is not mediated by glucocorti- coids secretion.

In the vascular permeability model, propylene glycol inhibited the overflowed Evans Blue dye with plasma, indicating that the solvent reduces the vascular reactivity to the histamine (Fig. 3).

In addition, in the carrageenin-induced pleurisy in rats, the propylene glycol reduced the exudate volume on the first phase while had worthless effect on the second phase, similarly to small doses of non- steroidal anti- inflammatory agents (Fig. 4). However, the propylene glycol action is different to those of anti- inflammatory drugs with regard the inhibit ion of cellular mobilization: while aspirin-like substances inhibit neutrophyl migration, propylene glycol acts especially on the monocytes migration in the both phases of pleurisy (Table 1).

As reported by Bailey et al. (1982) mononuclear cells are essential elements in the formation of granulomatous tissues. The granuloma formation, due to cotton pellet implantation, is characterized by pres- ence of the vascularized fibrous capsules which con- tain fibroblasts and infiltrant m o n o n u d e a r cells. As Fig. 5 shows propylene glycol reduces granulomatous tissue formation. This effect probably occurred by inhibiting the leucocyte migration, especially the mononuclear cells, at the inflammation site.

On the other hand, propylene glycol interferes with the pharmacokinetics parameters of phenylbutazone. Paradoxically, the phenylbutazone plasma level reached in the propylene glycol solution was lower than that one obtained in the aqueous solution. High viscosity of propylene glycol probably delays the phenylbutazone absorption. Simultaneously, the

134 TEREZA T. OSmRO et al.

Table 2. Pharmacokinetic parameters of phenylbutazone

Parameter FZ/W FZ/PG

tl/: (min) 200.7 + 8.5 253.6 _ 10.2" B 0 (#gml -I) 106.9+__4.2 77.3 +2.1" fl (min -I) (3.4__.0.1) 10 3 (2.7 +0.1) 10 -3* V d (ml kg -t ) 363.5 + 13.3 509.4 + 12.7" AUC (,u g min ml- t ) 31,945.6 + 1322.2 28,911.3 + 1369.4 C1 (mlkg -I) 1.3 _0.1 1.4-t-0.1

Animals were injected i.p. with 40 mg/kg of phenylbutazone in aqueous solution (FZ/W) and phenylbutazone in propylene glycol (FZ/PG). Each value represents the mean of 6 determinations __. SEM; tt:2, half-life of the fl-phase of the concentration of phenylbutazone in the plasma; fl, hybrid rate constant for fl-phase; V d, kinetic volume of distribution; AUC, area under the curve calculated from t = 0 to t = o~ by the trapezoidal method; CI, clearance. *Significantly different ( P < 0.05) from the control group (FZ/W).

propylene glycol delayed the e l iminat ion rate of the pheny lbu tazone and increased its p l a sma half-life (t~/2). Propylene glycol also decreased the phenylbu ta - zone e l iminat ion coefficient (fl), possibly because the propylene glycol al tered the pheny lbu tazone metabo l i sm mainly t h r ough inhibi tory effect of the solvent on the mixture- funct ion oxidase system of the liver. Such a system is responsible for b io t rans forma- t ion of mos t of l ipid-soluble compounds , as described by Dean and Stock (1974) and Sauers et al. (1980). In tu rn the area under the curve ( A U C ) of p lasma level of the pheny lbu tazone was not al tered by substi- tu t ion of water for propylene glycol, bu t the appa ren t d i s t r ibu t ion volume (Vd) was increased. A l though the mechan i sm of this Vd increasing effect is unknown , it can part ial ly explain the po ten t ia t ion of phenylbu ta - zone effects caused by the propylene g lyco l .

In conclusion, the combina t ion of pheny lbu tazone and propylene glycol leads to a po ten t i a t ion of the pheny lbu tazone ant i - inf lammatory effect. Such a po- ten t ia t ion should be related to the s u m m a t i o n of the b o t h propylene glycol and pheny lbu tazone effects, besides a l tera t ion of some pharmacokine t ic parameters of the pheny lbu tazone aroused by the solvent.

Acknowledgements--We are grateful to Mrs Inrs P. Vieira for technical assistance.

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Van Arman C. G., Begany A. J., Miller L. M. and Pless H. H. (1965) Some details of the inflammations caused by yeast and carrageenin. J. Pharmac. Exp. Ther. 150, 328-333.

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Wilhelm D. L., Mill P. J., Sparrow E. M., Mackay M. E. and Miles A. A. (1958) Enzymes-like globulins from serum reproducing the vascular phenomena of inflammation. IV Activable permeability factor and its inhibitor in the serum of the rat and the rabbit. Br. J. Exp. Path. 39, 228-250.

Yasaka W. J., Eichbaum F. W. and Oga S. (1979) Anti- arrhythmic effects of solvents: II. Effects of propylene glycol and benzyl alcohol on the effective refractory period of isolated rabbit atrio. Cardiovasc. Res. 13, 711-716.

Zaroslinski J. F., Browne R. K. and Prossley L. H. (1971) Propylene glycol as a drug solvent in pharmacologic studies. Toxic. Appl. Pharmac. 19, 573-578.

propylene glycol enhances anti-inflammatory effects of phenylbutazone

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