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Limethason as a lipid microsphere preparation: An overview
_
advanced
drug delivery reviews ELSEVIER
Advanced Drug Delivery
Limethason
Reviews
20 (1996)
195-201
as a lipid microsphere An overview K. Yokoyama”,
preparation:
M. Watanabe
The Green Cross Co., 3-3, Imabashi I-Chome,
Chuoh-Ku,
Osaka, Japan
Abstract
Limethason is an i.v. injectable lipid emulsion in which dexamethasone-21-palmitate is dissolved as an active ingredient. Limethason exhibited 2 to 5 times as potent anti-inflammatory activity as water-soluble dexamethasone phosphate on chronic inflammatory disease models. The strong anti-inflammatory activity of the drug was primarily based on a high distribution in the inflammatory lesion, a high uptake by macrophages and a suppressive effect on the macrophage function. A multicenter double-blind comparative clinical trial showed a tendency to a significantly higher rate of improvement with lower frequency of side effects in the Limethason group than the dexamethasone phosphate group. These results indicate that Limethason is more useful for rheumatoid arthritis and that the separation of the efficacy and side-effects of steroid could be clinically confirmed to some extent. Keywords:
Liposteroid;
Dexamethasone
palmitate;
Rheumatoid
arthritis; DDS; Macrophage:
Anti-inflammatory
action
Contents 1. Introduction ...................................................................................................................................................... ................... 2. Composition.. ........................................................................................................................................................................ 3. Pharmacological actions.. ...................................................................................................................................................... 3.1. Effect of Limethason on adjuvant arthritis [lo] ............................................................................................................. 3.2. Effect of Limethason on carrageenin granuloma in rat [lo] ........................................................................................... 3.3. Effect of Limethason on disc granuloma in rats [lo] ...................................................................................................... 3.4. Phagocytosis of Limethason lipid particles by macrophages [lo] ................................................................................... 3.5. Effects of Limethason on macrophages [lo] .................................................................................................................. 4. Distribution, metabolism and excretion ................................................................................................................................ 5. Clinical study ........................................................................................................................................................................ 5.1. Patients and study design.. ............................................................................................................................................. 5.2. Results.. ......................................................................................................................................................................... References .................................................................................................................................................................................
1. Introduction
One
approach
*Corresponding
to increasing
the effectiveness
Shaw and others
author.
0169-409X/96/$32.00 @ SSDl 0169-409X(95)00122-0
and to reducing systemic side effects is to the necessary amount of drugs to the disease site. Liposomes are suitable drug vehicles for such a drug delivery system
1996 Elsevier
Science
B.V. All rights
reserved
[4,5] already
reported
195 196 196 196 196 196 197 197 198 199 199 199 201
deliver target carrier
[l-3]. that
I’)6
K. Yokovama,
M. Watanabe
i
Adrunced
hydrocortisone palmitate incorporated into liposomes had a much stronger anti-inflammatory effect than free hydrocortisone given by systemic administration in animals. However, liposomes are relatively unstable and are not easily mass produced. Lipid microspheres consisting of soybean oil and lecithin are stable and are widely used in clinical medicine for parenteral nutrition. Regarding their distribution in the body, lipid microspheres. like liposomes, are easily taken up by the reticuloendothelial systems and by inflammatory cells [6-g]. On the basis of these properties of lipid microspheres. their use in a drug delivery system has been studied for dexamethasone which has excellent anti-inflammatory activities but serious side effects and dexamethasone palmitate incorporated in lipid microspheres has been developed as “Liposteroid” (LimethasonR) [9,10].
2. Composition Limethason is an i.v. injectable lipid emulsion in which dexamethasone-21-palmitate is dissolved as the active ingredient. Average particle size ranges from 0.1 to 0.3 pm in diameter and no larger particle than 1 pm is contained. An ampulla of Limethason contains the following components: 4.0 mg Dexamethasone palmitate (dexamethasone equivalent 2.5 mg): 100 mg soybean oil, purified: 12 mg Yolk lecithin. purified; 22.1 mg glycerin. concentrated: 1.0 ml distilled water for injection q.s.ad.
3. Pharmacological 3.1. Effect
actions
of Limethason
on adjuvant arthritis
[lOI An arthritis model was induced in rats by i.d. injection of Mycohacteriutn hutyricum suspended in liquid paraffin into the volar surface of the right hind paw. Drugs were given i.v. 3 times every other day from the 11th day after the adjuvant injection. The volume of the left noninjected foot was measured on days 11 and 1X. As a result, Limethason showed a significant
Drug
Delivery
Reviews
20 (1YYhj
19.5-201
Liposteroid (L) (EDso= 0.27 mg/kg)
_ 3,3 EDso of L
Dexamethasone phosphate (ED% = 0.9 mg/kg)
0-I
(D)
6
0.1
0.9
0.3
(fMk9)
Dose of steroid Fig. I. Effect
of liposteroid
on adjuvant
arthritis
in rats.
inhibition of edema at dexamethasone equivalent doses of 0.3 and 0.9 mglkg as compared with dexamethasone phosphate and the ED,, was 0.27 mg/kg for Limethason and 0.90 mg/kg for dexamethasone phosphate (Fig. 1). 3.2. Effect of Limethason on carrageenin grtrnuloma in rat [IO] Rats were injected S.C. with 2% lambda carrageenin suspended in saline into the air pouch of the back and given i.v. either Limethason or dexamethasone phosphate at 5, 6 and 7 days after injection of carrageenin. Then, 1 day after the final injection of the drug, the weight of granuloma removed was measured. The ED,,, calculated from inhibition percent to the granuloma formation was 0.072 mg/kg for Limethason and 0.38 mg/kg (Fig. 2). On the other hand, Limethason induced a dose-dependent decrease in body weight gain and in the weight of the thymus and adrenal in the model rats. although no significant difference was found as compared with those of dexamethasone phosphate at the equivalent dose level. 3.3. Effect of Limethason on disc granuloma in rats [lo] A formalin disc of filter paper was implanted S.C. into the back of rats. Drugs were injected i.v. at 5. 6 and 7 days after formalin disc implanta-
K. Yokoyama,
M. Watanabe I Advanced
Drug Delivery Reviews 20 (1996) 195-201
197
Liposteroid (L) (ED5 o- 0.072 mgkg)
W) 60 60
Liposteroid
5.3 EDw of L
Dexamethasone phosphate (EDso- 0.38 mgkg)
/
0.03
0.1
0.3
1.0
y&T-
(D)
@WW 20
Dose of steroid
Dexamethasone
Fig. 2. Effect
of liposteroid
on carrageenin
granuloma
4
IO
i----_ 0I
0
0
tion. The granuloma formed was removed and weighed at the 8th day. The ED,, calculated from the inhibition percent to granuloma formation was 0.15 mg/kg for Limethason and 0.30 mg / kg for dexamethasone phosphate. Limethason induced a dose-dependent decrease in body weight gain and in the weight of the thymus and adrenal in the model rats, although no significant difference was found as compared with those of dexamethasone phosphate at the equivalent dose level. 3.4 Phagocytosis of Limethason by macrophages [ 101
phosphate
in rats.
lipid particles
A mixture of thioglycolate-elicited macrophages with [3H]Limethason or [ 3H]dexamethasone phosphate was incubated at 37°C for 180 min. As a result, the radioactivity in the macrophages treated with [3H]Limethason rapidly increased up to 30 min of the incubation time and the steroid level reached 50.5 ng per 10’ cells, whereas that with [‘Hldexamethasone phosphate remained to be 6.5 ng per 10’ cells even at 180 min incubation (Fig. 3). The dexamethasone palmitate in Limethason entrapped by macrophages was found to be hydrolysed within the macrophages to active dexamethasone and to be released gradually into the incubation medium.
I, ,I
”
0 0 I/
I
I,
30
1.5
45
60
180 Onin)
Tlme after administration Fig. 3. Uptake
of liposteroid
3.5. Effects qf Limethason
by macrophages
on macrophages
[lo]
The effect of Limethason on the Fc receptordependent phagocytosis was studied. The macrophages prepared from rat peritoneal cavity were incubated with either Limethason or dexamethasone phosphate with sheep erythrocytes which were sensitized with rat anti-sheep erythrocyte antiserum. The uptake rate of the sensitized sheep erythrocytes by macrophages was determined microscopically on the stained section
Inhibition 0
I
Lfposterofd
50
rate (%) 100
0.03 mgimf 0.1 m@ml
Dexamethasone phosphate
0.1 mg/ml 0.3 rng/rnl
Fig. 4. Effect
of liposteroid
on phagocytosis
of macrophages.
1%
K. Yokovama.
M. Watanabe
lnhlbition
rate (%)
50
0 Liposteroid
I Advanced
100
0.03 mglml 0.1 mg/ml
Oexamethasone phosphate
0.1 mglml 0.3 mg/ml
lntralipos
13 fll/ml
Fig. 5. Effect of liposteroid by macrophages.
on superoxide
anion
production
prepared after incubation. As a result. Limethason suppressed more than 80% of the macrophage phagocytosis at the concentration of 0.03 mg/ml while dexamethasone phosphate inhibited only 30% even at the concentration 0.3 mg/ml (Fig. 4). The effect of Limethason on the superoxide anion release from macrophages was studied using opsonized zymosan as stimulant. The macrophages obtained from rat peritoneal cavity were incubated with either Limethason or dexamethasone phosphate for 60 min at 37°C. After incubation, the macrophages were treated with cytochrom C and opsonized zymozan. The superoxide anion production was determined from the rate of cytochrome c reduction by the formed OZ. As a result, Limethason strongly suppressed the superoxide anion release from the macrophages. The inhibition rate was 74.5 and 80.6% at the concentrations of 0.03 and 0.1 mg/ml. respectively. On the other hand, the suppressive effect of dexamethasone phosphate was weaker than Limethason (Fig. 5).
Drug
Delivery
Reviews
20 (1996)
1’9~5-201
circulation with the mediation of bile and was cumulatively eliminated into the urine by approximately 60% and into the faeces by approximately 40% at 48 h after injection. Principal urinary metabolites were identified to be dexamethasone, 11-keto-dexamethasone, 6-/Shydroxy-dexamethasone [11,12]. The distribution of Limethason in inflammalesions was also tory studied [lOI. [‘H]Limethason or [“Hldexamethasone phosphate prepared from [1,2.4-“Hldexamethasone was administered i.v. 0.5 mglkg as dexamethasone to the adjuvant-arthritis rat. As a result, for up to 30 min, no difference of the dexamethasone contents in the arthritis lesions of the hind paw were observed between the Limethason and dexamethasone phosphate group. In the Limethason group, however, the steroid content was maintained at the same level 2 h after injection. whereas it decreased to half in the dexamethasone phosphate group during this period. Thereafter, the steroid level decreased at a similar rate with time in both groups (Fig. 6). Limethason was administered iv. 2.5 mg per man as dexamethasone to 5 patients with rheumatoid arthritis to measure the pharmacokinetics [13]. Dexamethasone palmitate decreased rapidly after administration followed by a rather gradual decrease. Free dexamethasone produced from dexamethasone palmitate by hydrolysis reached a maximum concentration of 4.09 2 0.92 pg/dl plasma at 1.54 2 0.41 h. @g/g tissue)
3H-Liposteroid 1k-Y
/
E 4. Distribution,
metabolism
and excretion
When Limethason was injected iv. at the dose of 0.05 mg/kg in rats, it underwent hydrolysis by esterase to generate bioactive metabolite, dexamethasone which entered a comparatively slow elimination pathway with a plasma half-life of approximately 2.7 h and distributed largely in the reticuloendothelial tissues such as liver, spleen and lungs. Limethason was principally metabolized in the liver, entered the enterohepatic
s
z E
lo’3H-Dexamethasone
8
6
24
48
(h)
Time atter administration Fig. 6. Distribution of [‘Hldexamethasone phosphate rats.
‘H-labeled in adjuvant
liposteroid and arthritis lesion in
K. Yokoyama,
M. Watanabe I Advanced
0 : Dexamethasanepaimitate (D-PAL) 0 : Dexamethasone
0
2
4
Fig. 7. Changes of plasma concentration palmitate and dexamethasone following liposteroid in chronic rheumatoid arthritis
a (h)
6
of dexamethasone i.v. injection of patients.
The half-life in P-phase was 5.48 ‘_’1.77 h for free dexamethasone in contrast to 2.17 -f 0.51 h for dexamethasone palmitate (Fig. 7).
5. Clinical
study
A double-blind comparative rheumatoid arthritis was done [14].
study
in
5.1. Patients and study design
A multicenter double-blind comparative trial was performed in 138 patients with rheumatoid arthritis after biweekly intravenous or intramuscular injection of Limethason and Decadron, a dexamethasone phosphate preparation, as a reference drug. The subjects were diagnosed as classical or definite rheumatoid arthritis according to the diagnosis criteria of the American Rheumatism Association. More than 6 months after the onset of the condition, they had to fulfill at least 3 among the following 5 parameters: (1) morning stiffness persisting more than 30 min, (2) grip strength less than 139 mmHg in male and 127 mmHg in female, (3) pain in more than 6 joints, (4) swelling in more than 3 joints, (5) erythrocyte sedimentation rate (ESR) higher than 28 mm/h. The medical characteristics of subjects
Drug Delivery Reviews 20 (1996) 195-201
199
were found not to be significant by U and xz tests in groups. An i.v. dosage form containing 1 ml of Limethason in an ampul as test drug and an intramuscular dosage form containing 1 ml of physiological saline in an ampoule as placebo were prepared for the Limethason group. An intramuscular dosage form containing 1 ml of dexamethasone (4 mg of dexamethasone phosphate, equivalent to 3.3 mg dexamethasone) in an ampoule as reference drug and an intravenous dosage form containing 1 ml of 10% lipid emulsion as placebo were also prepared for the Decadron group. An intravenous Limethason real/placebo ampul paired, respectively, with an intramuscular Decadron real/placebo ampul per dose were injected by each specific route every two weeks in a total of 4 doses and the term of trial was designed to be 8 weeks. 5.2. Results Number of active joints, grip strength, duration of morning stiffness and pain score were monitored at the commencement of trial, immediately before the 2nd, 3rd and 4th dosing and at 8 weeks. Based on these monitoring results, the grade of improvement achieved was evaluated by the doctors in charge (Table 1). As compared with the Decadron group, the Limethason group produced a higher rate of ‘improved’ and ‘remarkably improved’ responses as well as a lower rate of ‘aggravated’ responses. As such, the distribution of graded final overall improvement demonstrated a trend toward a significant difference between both treatment groups, indicating the superior response rate in the Limethason group. Nevertheless, the statistical analysis of these results by means of Mann-Whitney’s U test revealed that the difference between both groups existed merely at the 10% level of significance. Further, the effectiveness evaluated by the patient’s impression with a special focus on his/ her subjective symptoms showed no statistic difference between both treatment groups. The safety was assessed based on the incidence of adverse reactions and the results of clinical laboratory tests such as general hematology,
4 3
Liposteroid Decadron
22 (36.1)h 14 (25.8)
Moderately improved
rate in double-blind
comparative
24 (69.4) 24 (62.1)
Slightly improved
in double-blind
comparative
studv
4 3
Liposteroid Decadron
26 (41.7)h 11 (21.2)
Useful
in double-blind
18 (66.7) 25 (59.1)
Slightly useful
comparative
study
IS 13
Undecided
LP < 0.05.
4 9
)
5 10
Slightly unfavorable
I 4
2 4
Unfavorable
arthritis
subjects
in rheumatoid
72 6(8.3% 3(4.2%) Incidence I 2 2 1 I 3
>iposteroid
Evaluable
arthritis
not be evaluated.
in rheumatoid
with liposteroid
with liposteroid
rate could
15 I2
arthritis
Aggravated
in rheumatoid
Slightly aggravated
with liposteroid
Unchanged
study
“ Patients dropped out so that the utility rate could not be evaluated. h Numbers in parentheses indicate accumulative percentage.
very useful
utility
Drugs
Table 3 Comprehensive
NS, not significant.
Total No. of cases No. of cases with side-effects No. of discontinued cases Clinical symptoms of side effects Stomach ache, diarrhoea. etc. Headache, anxiety, etc. Full-moon face (feeling of lacial heat. etc.) Pruritus, etc. Pyrexis Pains at the site of (intramuscular) injection
Item
Table 2 Side effects
” Patients dropped out so that the final global improvement h Numbers in parentheses indicate accumulative percentage. c PiO.10. iJ P < 0.05
Markedly improved
improvement
Drugs
Table 1 Final global
5 (7.6%) Incidence 3 5 0 2 2 3
10(15.2% )
66
Decadron
0 0
Utterly unfavorable
0 0
Markedly aggravated
2 0
Uncertain
2 0
Unknown”
72 66
Total
72 66
Total
U: L>D’ x’:L>D’
U. x’ test
NS NS NS NS NS NS
NS NS
xz test
U: L>D’ x’: L>Dd
U, x2 test
K. Yokoyama,
M. Watanabe I Advanced
serum biochemistry, endocrinology and urine analysis. The incidence of adverse reactions was 8.3% (6/72) in the Limethason group and 15.2% (10/66) in the Decadron group, although statistically not significant by the x2 test (Table 2). U-test evaluation on the safety including clinical laboratory check-ups failed to reveal a significant difference between the two groups. On the other hand, the utility of the drug treatment was comprehensively judged after the termination of the trial with reference to the precedent improvement grading, patient’s impression and safety. As shown in Table 3, the significant difference between both groups was found at a risk of 5% (U-test). In consequence, Limethason was evaluated to be clearly more useful than Dexamethasone phosphate in clinical practice.
References [l] Fendler, J.H. and Romero, A. (1977) Liposomes as drug cariers. Life Sci. 20, 1109. [2] Weinstein, J.N. (1984) Liposomes as drug carriers in cancer therapy. Cancer Treat. Rep. 68, 127. [3] Gregoriadis, G. (1984) Liposome technology. In: G. Gregoriadis and A.C. Allison (Eds), Liposomes in biological systems, Vols. I-III, Wiley, New York, NY. [4] Shaw, I.H., Knight, C.G., Thomas, D.P.P. et al. (1979) Liposome-incorporated corticosteroid. I. The interaction of liposomal cortisol palmitate with inflammatory synovial membrane. Br. J. Exp. Pathol. 60, 142.
Drug Delivery Reviews 20 (1996) [5] Phillips, N.C., Thomas, Liposome-incorporated activity 553.
in experimental
195-201
201
D.P.P., Knight, corticosteroids. arthritis.
C.G. et al. (1979) II. Therapeutic
Ann.
Rheum.
Dis. 38,
[6] Mizushima, Y., Hamano, T. and Yokoyama, K. (1982) Tissue distribution and anti-inflammatory activity of corticosteroids incorporated in lipid emulsion. Ann. Rheum. Dis. 41, 263. [7] Koga, Y., Swanson,VL. and Hayes, D.M. (1975) Hepatic ‘intravenous fat pigment’ in infants and children receiving lipid emulsion. J. Pedatr. Surg. 10. 641. [8] Hallberg, D. (1985) Elimination the bloodstream. Acta Physiol.
of exogenous lipid from Stand. 65. 254 (Suppl.).
[9] Mizushima, Y. (1985) Lipid microspheres carriers. Drugs Exp. Clin. Res. 11, 595. [lo]
[ll]
as novel drug
Yokoyama, K., Okamoto, H., Watanabe, M. et al. (1985) Development of a corticosteroid incorporated in lipid micropheres (Liposteroid). Drugs Exp. Clin. Res. 11, 611. Iwai, M., Hamano, T., Arakawa, Y. et al. (1985) Pharmacokinetics of dexamethasone palmitate, a lipophilic pro-drug. Part 1. Distribution and excretion. Kiso to Rinsho (Lab. Clin.) 19, 4085.
[12] Iwai, M., Inoue, T.. Arakawa, Y. et al. (1985) Pharmacokinetics of dexamethasone palmitate. a lipophilic prodrug. Part 2. Metabolism and identification of metabolite. Kiso to Rinsho (Lab. Clin.) 19. 4110. [13] Ii, S., Okamoto, H., Yokoyama, K. et al. (1988) Comparative pharmacokinetic study of dexamethasone disodium phosphate in chronic rheumatoid arthritis patients. Yakuri to Chiryo (Basic Pharmacol. Ther.) 16, 865. [14] Hoshi, K., Mizushima, Y., Shiokawa, Y. et al. (1985) Double-blind study with liposteroid in rheumatoid arthritis. Drugs Exp. Clin. Res. 11, 621.
advanced
drug delivery reviews ELSEVIER
Advanced Drug Delivery
Limethason
Reviews
20 (1996)
195-201
as a lipid microsphere An overview K. Yokoyama”,
preparation:
M. Watanabe
The Green Cross Co., 3-3, Imabashi I-Chome,
Chuoh-Ku,
Osaka, Japan
Abstract
Limethason is an i.v. injectable lipid emulsion in which dexamethasone-21-palmitate is dissolved as an active ingredient. Limethason exhibited 2 to 5 times as potent anti-inflammatory activity as water-soluble dexamethasone phosphate on chronic inflammatory disease models. The strong anti-inflammatory activity of the drug was primarily based on a high distribution in the inflammatory lesion, a high uptake by macrophages and a suppressive effect on the macrophage function. A multicenter double-blind comparative clinical trial showed a tendency to a significantly higher rate of improvement with lower frequency of side effects in the Limethason group than the dexamethasone phosphate group. These results indicate that Limethason is more useful for rheumatoid arthritis and that the separation of the efficacy and side-effects of steroid could be clinically confirmed to some extent. Keywords:
Liposteroid;
Dexamethasone
palmitate;
Rheumatoid
arthritis; DDS; Macrophage:
Anti-inflammatory
action
Contents 1. Introduction ...................................................................................................................................................... ................... 2. Composition.. ........................................................................................................................................................................ 3. Pharmacological actions.. ...................................................................................................................................................... 3.1. Effect of Limethason on adjuvant arthritis [lo] ............................................................................................................. 3.2. Effect of Limethason on carrageenin granuloma in rat [lo] ........................................................................................... 3.3. Effect of Limethason on disc granuloma in rats [lo] ...................................................................................................... 3.4. Phagocytosis of Limethason lipid particles by macrophages [lo] ................................................................................... 3.5. Effects of Limethason on macrophages [lo] .................................................................................................................. 4. Distribution, metabolism and excretion ................................................................................................................................ 5. Clinical study ........................................................................................................................................................................ 5.1. Patients and study design.. ............................................................................................................................................. 5.2. Results.. ......................................................................................................................................................................... References .................................................................................................................................................................................
1. Introduction
One
approach
*Corresponding
to increasing
the effectiveness
Shaw and others
author.
0169-409X/96/$32.00 @ SSDl 0169-409X(95)00122-0
and to reducing systemic side effects is to the necessary amount of drugs to the disease site. Liposomes are suitable drug vehicles for such a drug delivery system
1996 Elsevier
Science
B.V. All rights
reserved
[4,5] already
reported
195 196 196 196 196 196 197 197 198 199 199 199 201
deliver target carrier
[l-3]. that
I’)6
K. Yokovama,
M. Watanabe
i
Adrunced
hydrocortisone palmitate incorporated into liposomes had a much stronger anti-inflammatory effect than free hydrocortisone given by systemic administration in animals. However, liposomes are relatively unstable and are not easily mass produced. Lipid microspheres consisting of soybean oil and lecithin are stable and are widely used in clinical medicine for parenteral nutrition. Regarding their distribution in the body, lipid microspheres. like liposomes, are easily taken up by the reticuloendothelial systems and by inflammatory cells [6-g]. On the basis of these properties of lipid microspheres. their use in a drug delivery system has been studied for dexamethasone which has excellent anti-inflammatory activities but serious side effects and dexamethasone palmitate incorporated in lipid microspheres has been developed as “Liposteroid” (LimethasonR) [9,10].
2. Composition Limethason is an i.v. injectable lipid emulsion in which dexamethasone-21-palmitate is dissolved as the active ingredient. Average particle size ranges from 0.1 to 0.3 pm in diameter and no larger particle than 1 pm is contained. An ampulla of Limethason contains the following components: 4.0 mg Dexamethasone palmitate (dexamethasone equivalent 2.5 mg): 100 mg soybean oil, purified: 12 mg Yolk lecithin. purified; 22.1 mg glycerin. concentrated: 1.0 ml distilled water for injection q.s.ad.
3. Pharmacological 3.1. Effect
actions
of Limethason
on adjuvant arthritis
[lOI An arthritis model was induced in rats by i.d. injection of Mycohacteriutn hutyricum suspended in liquid paraffin into the volar surface of the right hind paw. Drugs were given i.v. 3 times every other day from the 11th day after the adjuvant injection. The volume of the left noninjected foot was measured on days 11 and 1X. As a result, Limethason showed a significant
Drug
Delivery
Reviews
20 (1YYhj
19.5-201
Liposteroid (L) (EDso= 0.27 mg/kg)
_ 3,3 EDso of L
Dexamethasone phosphate (ED% = 0.9 mg/kg)
0-I
(D)
6
0.1
0.9
0.3
(fMk9)
Dose of steroid Fig. I. Effect
of liposteroid
on adjuvant
arthritis
in rats.
inhibition of edema at dexamethasone equivalent doses of 0.3 and 0.9 mglkg as compared with dexamethasone phosphate and the ED,, was 0.27 mg/kg for Limethason and 0.90 mg/kg for dexamethasone phosphate (Fig. 1). 3.2. Effect of Limethason on carrageenin grtrnuloma in rat [IO] Rats were injected S.C. with 2% lambda carrageenin suspended in saline into the air pouch of the back and given i.v. either Limethason or dexamethasone phosphate at 5, 6 and 7 days after injection of carrageenin. Then, 1 day after the final injection of the drug, the weight of granuloma removed was measured. The ED,,, calculated from inhibition percent to the granuloma formation was 0.072 mg/kg for Limethason and 0.38 mg/kg (Fig. 2). On the other hand, Limethason induced a dose-dependent decrease in body weight gain and in the weight of the thymus and adrenal in the model rats. although no significant difference was found as compared with those of dexamethasone phosphate at the equivalent dose level. 3.3. Effect of Limethason on disc granuloma in rats [lo] A formalin disc of filter paper was implanted S.C. into the back of rats. Drugs were injected i.v. at 5. 6 and 7 days after formalin disc implanta-
K. Yokoyama,
M. Watanabe I Advanced
Drug Delivery Reviews 20 (1996) 195-201
197
Liposteroid (L) (ED5 o- 0.072 mgkg)
W) 60 60
Liposteroid
5.3 EDw of L
Dexamethasone phosphate (EDso- 0.38 mgkg)
/
0.03
0.1
0.3
1.0
y&T-
(D)
@WW 20
Dose of steroid
Dexamethasone
Fig. 2. Effect
of liposteroid
on carrageenin
granuloma
4
IO
i----_ 0I
0
0
tion. The granuloma formed was removed and weighed at the 8th day. The ED,, calculated from the inhibition percent to granuloma formation was 0.15 mg/kg for Limethason and 0.30 mg / kg for dexamethasone phosphate. Limethason induced a dose-dependent decrease in body weight gain and in the weight of the thymus and adrenal in the model rats, although no significant difference was found as compared with those of dexamethasone phosphate at the equivalent dose level. 3.4 Phagocytosis of Limethason by macrophages [ 101
phosphate
in rats.
lipid particles
A mixture of thioglycolate-elicited macrophages with [3H]Limethason or [ 3H]dexamethasone phosphate was incubated at 37°C for 180 min. As a result, the radioactivity in the macrophages treated with [3H]Limethason rapidly increased up to 30 min of the incubation time and the steroid level reached 50.5 ng per 10’ cells, whereas that with [‘Hldexamethasone phosphate remained to be 6.5 ng per 10’ cells even at 180 min incubation (Fig. 3). The dexamethasone palmitate in Limethason entrapped by macrophages was found to be hydrolysed within the macrophages to active dexamethasone and to be released gradually into the incubation medium.
I, ,I
”
0 0 I/
I
I,
30
1.5
45
60
180 Onin)
Tlme after administration Fig. 3. Uptake
of liposteroid
3.5. Effects qf Limethason
by macrophages
on macrophages
[lo]
The effect of Limethason on the Fc receptordependent phagocytosis was studied. The macrophages prepared from rat peritoneal cavity were incubated with either Limethason or dexamethasone phosphate with sheep erythrocytes which were sensitized with rat anti-sheep erythrocyte antiserum. The uptake rate of the sensitized sheep erythrocytes by macrophages was determined microscopically on the stained section
Inhibition 0
I
Lfposterofd
50
rate (%) 100
0.03 mgimf 0.1 m@ml
Dexamethasone phosphate
0.1 mg/ml 0.3 rng/rnl
Fig. 4. Effect
of liposteroid
on phagocytosis
of macrophages.
1%
K. Yokovama.
M. Watanabe
lnhlbition
rate (%)
50
0 Liposteroid
I Advanced
100
0.03 mglml 0.1 mg/ml
Oexamethasone phosphate
0.1 mglml 0.3 mg/ml
lntralipos
13 fll/ml
Fig. 5. Effect of liposteroid by macrophages.
on superoxide
anion
production
prepared after incubation. As a result. Limethason suppressed more than 80% of the macrophage phagocytosis at the concentration of 0.03 mg/ml while dexamethasone phosphate inhibited only 30% even at the concentration 0.3 mg/ml (Fig. 4). The effect of Limethason on the superoxide anion release from macrophages was studied using opsonized zymosan as stimulant. The macrophages obtained from rat peritoneal cavity were incubated with either Limethason or dexamethasone phosphate for 60 min at 37°C. After incubation, the macrophages were treated with cytochrom C and opsonized zymozan. The superoxide anion production was determined from the rate of cytochrome c reduction by the formed OZ. As a result, Limethason strongly suppressed the superoxide anion release from the macrophages. The inhibition rate was 74.5 and 80.6% at the concentrations of 0.03 and 0.1 mg/ml. respectively. On the other hand, the suppressive effect of dexamethasone phosphate was weaker than Limethason (Fig. 5).
Drug
Delivery
Reviews
20 (1996)
1’9~5-201
circulation with the mediation of bile and was cumulatively eliminated into the urine by approximately 60% and into the faeces by approximately 40% at 48 h after injection. Principal urinary metabolites were identified to be dexamethasone, 11-keto-dexamethasone, 6-/Shydroxy-dexamethasone [11,12]. The distribution of Limethason in inflammalesions was also tory studied [lOI. [‘H]Limethason or [“Hldexamethasone phosphate prepared from [1,2.4-“Hldexamethasone was administered i.v. 0.5 mglkg as dexamethasone to the adjuvant-arthritis rat. As a result, for up to 30 min, no difference of the dexamethasone contents in the arthritis lesions of the hind paw were observed between the Limethason and dexamethasone phosphate group. In the Limethason group, however, the steroid content was maintained at the same level 2 h after injection. whereas it decreased to half in the dexamethasone phosphate group during this period. Thereafter, the steroid level decreased at a similar rate with time in both groups (Fig. 6). Limethason was administered iv. 2.5 mg per man as dexamethasone to 5 patients with rheumatoid arthritis to measure the pharmacokinetics [13]. Dexamethasone palmitate decreased rapidly after administration followed by a rather gradual decrease. Free dexamethasone produced from dexamethasone palmitate by hydrolysis reached a maximum concentration of 4.09 2 0.92 pg/dl plasma at 1.54 2 0.41 h. @g/g tissue)
3H-Liposteroid 1k-Y
/
E 4. Distribution,
metabolism
and excretion
When Limethason was injected iv. at the dose of 0.05 mg/kg in rats, it underwent hydrolysis by esterase to generate bioactive metabolite, dexamethasone which entered a comparatively slow elimination pathway with a plasma half-life of approximately 2.7 h and distributed largely in the reticuloendothelial tissues such as liver, spleen and lungs. Limethason was principally metabolized in the liver, entered the enterohepatic
s
z E
lo’3H-Dexamethasone
8
6
24
48
(h)
Time atter administration Fig. 6. Distribution of [‘Hldexamethasone phosphate rats.
‘H-labeled in adjuvant
liposteroid and arthritis lesion in
K. Yokoyama,
M. Watanabe I Advanced
0 : Dexamethasanepaimitate (D-PAL) 0 : Dexamethasone
0
2
4
Fig. 7. Changes of plasma concentration palmitate and dexamethasone following liposteroid in chronic rheumatoid arthritis
a (h)
6
of dexamethasone i.v. injection of patients.
The half-life in P-phase was 5.48 ‘_’1.77 h for free dexamethasone in contrast to 2.17 -f 0.51 h for dexamethasone palmitate (Fig. 7).
5. Clinical
study
A double-blind comparative rheumatoid arthritis was done [14].
study
in
5.1. Patients and study design
A multicenter double-blind comparative trial was performed in 138 patients with rheumatoid arthritis after biweekly intravenous or intramuscular injection of Limethason and Decadron, a dexamethasone phosphate preparation, as a reference drug. The subjects were diagnosed as classical or definite rheumatoid arthritis according to the diagnosis criteria of the American Rheumatism Association. More than 6 months after the onset of the condition, they had to fulfill at least 3 among the following 5 parameters: (1) morning stiffness persisting more than 30 min, (2) grip strength less than 139 mmHg in male and 127 mmHg in female, (3) pain in more than 6 joints, (4) swelling in more than 3 joints, (5) erythrocyte sedimentation rate (ESR) higher than 28 mm/h. The medical characteristics of subjects
Drug Delivery Reviews 20 (1996) 195-201
199
were found not to be significant by U and xz tests in groups. An i.v. dosage form containing 1 ml of Limethason in an ampul as test drug and an intramuscular dosage form containing 1 ml of physiological saline in an ampoule as placebo were prepared for the Limethason group. An intramuscular dosage form containing 1 ml of dexamethasone (4 mg of dexamethasone phosphate, equivalent to 3.3 mg dexamethasone) in an ampoule as reference drug and an intravenous dosage form containing 1 ml of 10% lipid emulsion as placebo were also prepared for the Decadron group. An intravenous Limethason real/placebo ampul paired, respectively, with an intramuscular Decadron real/placebo ampul per dose were injected by each specific route every two weeks in a total of 4 doses and the term of trial was designed to be 8 weeks. 5.2. Results Number of active joints, grip strength, duration of morning stiffness and pain score were monitored at the commencement of trial, immediately before the 2nd, 3rd and 4th dosing and at 8 weeks. Based on these monitoring results, the grade of improvement achieved was evaluated by the doctors in charge (Table 1). As compared with the Decadron group, the Limethason group produced a higher rate of ‘improved’ and ‘remarkably improved’ responses as well as a lower rate of ‘aggravated’ responses. As such, the distribution of graded final overall improvement demonstrated a trend toward a significant difference between both treatment groups, indicating the superior response rate in the Limethason group. Nevertheless, the statistical analysis of these results by means of Mann-Whitney’s U test revealed that the difference between both groups existed merely at the 10% level of significance. Further, the effectiveness evaluated by the patient’s impression with a special focus on his/ her subjective symptoms showed no statistic difference between both treatment groups. The safety was assessed based on the incidence of adverse reactions and the results of clinical laboratory tests such as general hematology,
4 3
Liposteroid Decadron
22 (36.1)h 14 (25.8)
Moderately improved
rate in double-blind
comparative
24 (69.4) 24 (62.1)
Slightly improved
in double-blind
comparative
studv
4 3
Liposteroid Decadron
26 (41.7)h 11 (21.2)
Useful
in double-blind
18 (66.7) 25 (59.1)
Slightly useful
comparative
study
IS 13
Undecided
LP < 0.05.
4 9
)
5 10
Slightly unfavorable
I 4
2 4
Unfavorable
arthritis
subjects
in rheumatoid
72 6(8.3% 3(4.2%) Incidence I 2 2 1 I 3
>iposteroid
Evaluable
arthritis
not be evaluated.
in rheumatoid
with liposteroid
with liposteroid
rate could
15 I2
arthritis
Aggravated
in rheumatoid
Slightly aggravated
with liposteroid
Unchanged
study
“ Patients dropped out so that the utility rate could not be evaluated. h Numbers in parentheses indicate accumulative percentage.
very useful
utility
Drugs
Table 3 Comprehensive
NS, not significant.
Total No. of cases No. of cases with side-effects No. of discontinued cases Clinical symptoms of side effects Stomach ache, diarrhoea. etc. Headache, anxiety, etc. Full-moon face (feeling of lacial heat. etc.) Pruritus, etc. Pyrexis Pains at the site of (intramuscular) injection
Item
Table 2 Side effects
” Patients dropped out so that the final global improvement h Numbers in parentheses indicate accumulative percentage. c PiO.10. iJ P < 0.05
Markedly improved
improvement
Drugs
Table 1 Final global
5 (7.6%) Incidence 3 5 0 2 2 3
10(15.2% )
66
Decadron
0 0
Utterly unfavorable
0 0
Markedly aggravated
2 0
Uncertain
2 0
Unknown”
72 66
Total
72 66
Total
U: L>D’ x’:L>D’
U. x’ test
NS NS NS NS NS NS
NS NS
xz test
U: L>D’ x’: L>Dd
U, x2 test
K. Yokoyama,
M. Watanabe I Advanced
serum biochemistry, endocrinology and urine analysis. The incidence of adverse reactions was 8.3% (6/72) in the Limethason group and 15.2% (10/66) in the Decadron group, although statistically not significant by the x2 test (Table 2). U-test evaluation on the safety including clinical laboratory check-ups failed to reveal a significant difference between the two groups. On the other hand, the utility of the drug treatment was comprehensively judged after the termination of the trial with reference to the precedent improvement grading, patient’s impression and safety. As shown in Table 3, the significant difference between both groups was found at a risk of 5% (U-test). In consequence, Limethason was evaluated to be clearly more useful than Dexamethasone phosphate in clinical practice.
References [l] Fendler, J.H. and Romero, A. (1977) Liposomes as drug cariers. Life Sci. 20, 1109. [2] Weinstein, J.N. (1984) Liposomes as drug carriers in cancer therapy. Cancer Treat. Rep. 68, 127. [3] Gregoriadis, G. (1984) Liposome technology. In: G. Gregoriadis and A.C. Allison (Eds), Liposomes in biological systems, Vols. I-III, Wiley, New York, NY. [4] Shaw, I.H., Knight, C.G., Thomas, D.P.P. et al. (1979) Liposome-incorporated corticosteroid. I. The interaction of liposomal cortisol palmitate with inflammatory synovial membrane. Br. J. Exp. Pathol. 60, 142.
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in experimental
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D.P.P., Knight, corticosteroids. arthritis.
C.G. et al. (1979) II. Therapeutic
Ann.
Rheum.
Dis. 38,
[6] Mizushima, Y., Hamano, T. and Yokoyama, K. (1982) Tissue distribution and anti-inflammatory activity of corticosteroids incorporated in lipid emulsion. Ann. Rheum. Dis. 41, 263. [7] Koga, Y., Swanson,VL. and Hayes, D.M. (1975) Hepatic ‘intravenous fat pigment’ in infants and children receiving lipid emulsion. J. Pedatr. Surg. 10. 641. [8] Hallberg, D. (1985) Elimination the bloodstream. Acta Physiol.
of exogenous lipid from Stand. 65. 254 (Suppl.).
[9] Mizushima, Y. (1985) Lipid microspheres carriers. Drugs Exp. Clin. Res. 11, 595. [lo]
[ll]
as novel drug
Yokoyama, K., Okamoto, H., Watanabe, M. et al. (1985) Development of a corticosteroid incorporated in lipid micropheres (Liposteroid). Drugs Exp. Clin. Res. 11, 611. Iwai, M., Hamano, T., Arakawa, Y. et al. (1985) Pharmacokinetics of dexamethasone palmitate, a lipophilic pro-drug. Part 1. Distribution and excretion. Kiso to Rinsho (Lab. Clin.) 19, 4085.
[12] Iwai, M., Inoue, T.. Arakawa, Y. et al. (1985) Pharmacokinetics of dexamethasone palmitate. a lipophilic prodrug. Part 2. Metabolism and identification of metabolite. Kiso to Rinsho (Lab. Clin.) 19. 4110. [13] Ii, S., Okamoto, H., Yokoyama, K. et al. (1988) Comparative pharmacokinetic study of dexamethasone disodium phosphate in chronic rheumatoid arthritis patients. Yakuri to Chiryo (Basic Pharmacol. Ther.) 16, 865. [14] Hoshi, K., Mizushima, Y., Shiokawa, Y. et al. (1985) Double-blind study with liposteroid in rheumatoid arthritis. Drugs Exp. Clin. Res. 11, 621.