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A stable PGE1 prodrug for targeting therapy
Journal of Controlled Release, 20 (1992) 37-46
37
© 1992 ElsevierScience Publishers B.V. All rights reserved O168-3659/92t$OS.00
COREL0722
A
stable PGE1 prodrug for targeting therapy
R. Igarashi ~, Y. M i z u s h i m a ~, M. T a k e n a g a u, K, M a t s u m o t o a, Y. M o r i z a w a b a n d A. Y a s u d a b ~Division of Drug Deliver3, System. Institute of Medical Science, St. Marianna Univei~ity, 2-16-1 Sugao, Miyamae-ku. 216 KawasakL Japan; hResearch Center, Asahi Glass Co., Ltd.. 1150 llazawacho. Kanagawa.ku, 221 Yokohama, Japan
( Received 27 August 1991; accepled in revisedform 23 December 1991)
Lipo-PGE~ is a passively targetable prostaglandin E~ (PGE~) preparation, in which PGE~ is incorporated into lipid microspheres (LM); it has been widely used in Japan since 1988 for the treatment of various vascular diseases. Passive targeting results from the accumulation of LM in the vascular lesions following intravenous administration. However, this preparation has two major disadvantages. One is the chemical instability of PGE~ in the LM and the other is the rapid leakage of PGE~ from the LM in the blood, leading to a decrease in the targeting of this drug. We accordingly synthesized several PGE~ prodrugs and evaluated them to develop a better LM preparation. Among the PGE~ prodrugs tested, As-9-O-butyryl prostaglandin F~ butyl ester (AS013) was readily hydrolyzed to PGE~ in human serum. AS013 was stable as an LM preparation and 80% of this ester was recovered unchanged even after storage as LM-AS013 for 4 weeks at 40°C. In contrast, PGE~ showed a recovery & o n l y 5% from the LM under the same conditions. Moreover, AS013 was more effectively retained in the LM after incubation with human blood or serum when compared with PGE~. These results suggested that LM-AS013 would be a far superior LM preparation than lipo-PGE~ for clinical use. Key words: Drug delivery system; Lipid microsphere (LM); Stable prodrug of PGE~; LM preparation of PGEL
Introduction Prostaglandin Et (PGE~) is used for the treatment of various vascular disorders because of its antiplatelet ae.d vascdilatory action~ [ ! .2 !. We have previously developed "lipo-PGEl', a drug dellvery system ( DDS ) incorporating PGE ~ into the lipid particles of an oil-in-water emulsion. These lipid particles have a diameter of 0.2/tm, are composed of soybean oil and lecithin, and are designated as "lipid mierospheres ( L M ) " . LM Correspondence to: R. lgarashi, Ph.D., Division of Drug DeliverySystem, Institute of Medical Science, St. Marianna University,2-16-1 Sugao.Miyamae-ku,216 Kawasak~,Japan.
shows a similar tissue distribution to liposomes, and provide a stable and safe drug carrier that can be used as a DDS. Since LM largely accumulate in inflamed tissues and vascular lesions, lipo-PGE I shows a marked efficacy for treating vascular diseases such as atheroselerosis [ 3,4 ]. However, PGE~ in LM is chemically unstable and is largely converted to PGA~ after storage in brown glass ampoules at room temperature for 6 months. In addition, PGE~ is rapidly released from the LM on incubation with human blood or serum. Since the LM are used as a targeting carrier, the PGE~ has to be retained in the microspheres until after it is delivered to the target site.
3~ To develop a better LM-PGE( system that overcomes the disadvantages of lipo-PGEl, we have investigated a PGE~-prodrug which is chemically stable, shows little leakage from LM, and is readily hydrolyzed into PGE~ at the target site in the body.
OB,
compouncl
OR,
OH OH pmstaglandin E~(PGEO
Rt
R~
R~
R,
IV~aterials and Methods esterified C-l, acvlaled C-9
Materials Adenosine 5'-diphosphate (ADP) (Niko Bio Science, Tokyo, Japan), the 0.45,um membrane filter (Japan Millipore Corporation, Tokyo, Japan), bovine serum albumin (BSA) (Sigma Chemical Company, St. Louis, U.S.A. ), Sepharore 4B and Dextran T40 (MW 40,000) (Pharmacla, Uppsala, Sweden), Hionic Fluor ® scintillation cocktail (Packard Company Inc., Meriden, U.S,A.), and the other chemicals used such as acetonitrile and tetrahydrofuran (Wako Pure Chemical Industries Ltd., Osaka, Japan) were all commercially available preparations. Guinea pigs weighing 250-300 g were obtained from Japan Laboratory Animals Inc. (Tokyo, Japan). Lipo-[3H]PGE~ (167 kBq/5.0 /~g PGE~/ml), lipo-[3H]AS006 (180 kBq/6.38 #g AS006/ml), and lipo-[3H]AS013 (192 kBq/ 6.78 gg ASOI3/ml) were supplied by Green Cross Co. Ltd. (Osaka, Japan). Synthesis of PGEt derivatives (AS-9.O-acy|ated prostaglandin Ft derivatives) The chemical structures of the PGE, derivatives synthesized are shown in Fig. 1. C-l-estefifled C-9-O-aeylated compounds These were synthesized according to the method of Sih et al. [ 5 ii. To a solution of (1 E, 3S) -l-iodo- (t-butyldimethylsiloxy) -1-octene (4.99 g, 13.56 mmol) in ethyl ether ( 100 ml), tbutyl lithium (f= 1.4 hexane solution, 18.1 ml, 27.1 mmol) was added at -78°C. After 2 h, a solution of tributylphosphine-copper(I) iodide complex ( 4.63 g, 2 I. 31 mmol ) and tributylphosphine (2.92 ml, 12.I6 retool) in ethyl ether (40
AS 006
C4H~
CHACO
H
H
AS 019
C4Hg
C2H~CO
H
H
ASOla C,Hg C~H~CO esterified C-1, acylaled C-@,11
H
H
AS 002
CH3
CHACO
CHACO
H
A$012
C4H9
CHACO
CHACO
H
AS014
C.H~
C~HL~CO CHACO
H
esterified C-1, acylated C-9,15 CHACO
H
CHACO
AS O 15 C~H:~ CHACO esterified C-1, acylatecl C a, 11,15
AS 003
H
CHACO
AS 025
CFI3
CH3
CHACO
CHACO
CHACO
Fig. I. Chemicalstructuresof the PGE~derivativesusedin this study. ml) was added dropwise to the main solution and stirred for 50 min at -78°C. An ether solution ( 160 ml) of 4R-t-butyldimethylsiloxy-2- (6-earbobutoxyhexyl)-2-cyclopenten-l-one (4.75 ml, 11.3 mmol) was then added to the above solutint). The mixture was stirred at - 7 8 ° C for 20 min and warmed to - 2 3 ° C for 35 rain. Finally, 30 mmol of butyric anhydride (for AS013), acetic anhydride (for AS006), or propionic anhydride (for AS019) was added dropwise at 0°C, followed by stirring for 15 h at from 0 °C to room temperature. Saturated ammonium sulfate solution (200 ml) was added to this solution and extraction was performed with ethyl ether (100 ml). The organic layer was dried over anhydrous luagncsium sulfate and evaporated under re-. duced pressure. The residue was puriflc.I by silica gel chromatography (hexane-ethyl e.cetale=20/1 to 4/1 ) at 0°C to aflbra the protf,cted As-9-O-acylated prostaglandin F~ C-I butyl ester. The protected compound (8.25 retool) was dissolved in acelonitrile (100 ml) and 10 ml of 40% hydrofluoric acid solution was added at 0 °C.
39 After 30 min, the reaction mixture was poured into a mixture of an aqueous solution of 20% potassium carbonate (150 ml) and dichloromethane ( 150 ml). The organic layer was dried over anhydrous magnesium sulfate and the solvent was evaporated. The residue was then purified by silica gel chromatography (dichlorumethaneacetone= 2/'1 ) at 0°C to afford dB-9-O-acylatcd prostaglandin Fj butyl ester (about 80% yield). Each compound was identified by NMR spectrometry. AS013: IH-NMR (CDCI3): ff0.86 (3H, t, J=7.2 Hz), 0.93 (3H, t, J = 7 . 2 Hz), 1.2-1.9 (221-I, m), 2.28 (1H, t, J=7.7 Hz), 2.45 (IH, m), 2.9-3.1 ( IH, m), 3.2-3.3 ( 1H, m), 4.0-4.2 44I-I, m), 4.9-5.1 t i l l , m), 5.5-5.7 (2H, m). IR (neat film): 3400, 2930, 1750, 1730, 970 cm -I. [c~]:°0--61.6 ° (c=0.5, abs. ethanol) ASO06: IH-NMR (CDCla):b'0.85 (3H, t , J = 7 Hz), 0.95 (3H, t, J = 7 Hz), 1.2-2.9 (29H, m + s (b'2.15, 3H)), 3.0-3.05 (1H, m),4.1 (2H, t , J = 7 Hz), 4.9-4.2 (2H, m), 5.45 (IH, dd), 5.6 (IH, dd). ASOI9: ~H-NMR (CDCI3): t~0.88 (3H, t, J=7.2 Hz), 0.91 (31-1,t, J=7.2 Hz), 0.93 (3H, ~., J--7.2 Hz), 1.2-2.2 (23H, m), 2.28 (2H, t, J=7.2 Hz), 2.45 (2H, t, J = 7 . 2 Hz), 2.4-2.5 (IH, m), 2.86 (IH, dd, J = l S , 5.7 Hz), 3.16 (IH, D , J = 15 Hz),4.07 (2H, t , J = 6 . 6 Hz), 4.04.2 (2H, m), 5.47 (IH, dd, J=16.1, 6.6 Hz), 5.60 ( 1H, dd, J = 16.1, 9.4 Hz). C-11-O-acylated derivatives (AS002, AS012, and AS014) or C-15-O-acylated derivatives (AS003 and AS015) were transformed by acylation with acetic anhydride after the selective deprotection of hydroxyl protecting group at C11 or C-15. Diacylated compound (AS025) was synthesized by ti~ereaction of the corresponding diol with acetic anhydride. ASO02: ~H-NMR (CDCI3): ~0.88 (3H, t, J=7.2 Hz), 1.2-1.9 (19H, m), 2.05 (31-1, s), 2.15 (3H, s),2.30 (2H, t , J = 7 . 3 Hz),2.45 (IH, d, J = 16 Hz), 2.97 (IH, dd, J = 16, 6.0 Hz), 3.2¢6 (IH, d , J = 6 . 0 Hz), 3.67 (3I-I, s), 4.0-4.1 (IH, m), 4.9-5.0 ( 1H, m), 5.4-5.7 (2H, m). ASO03: ~H-NMR (CDCI3): t$0.80 (3H, t, J = 7 . 2 Hz), 1.1-1.8 (19H, m), 1.95 (3H, s),
2.08 (3H, s),2.22 (2H, t , J = 7 . 2 Hz),2.32 (IH, d, J = 1 6 Hz), 2.81 (IH, dd, J = 16, 7 Hz), 2.93.1 ( IH, m), 3.60 (3H, s), 4.0-4.1 ( IH, m), 5.15.2 (IH, m), 5.2-5.3 (2H, m). ASOI2: tH-NMR (CDCI3): t$0.86 (3H, t, J=7.2 Hz), 0.93 (3H, t, J=7.2 Hz), 1.2-1.9 (22H, m), 2.28 (Ill, t, J=7.7 Hz), 2.45 (IH, m), 2.9-3.1 ( IH, m), 3.2-3.3 ( IH, m), 4.0-4.2 (4H, m), 4.9-5.1 (IH, m), 5.5-5.7 (2H, m). ASOI4: ~H-NMR (CDCI3): J0.88 (3H, t, J = 7 . 2 Hz), 0.91 (3H, t, J=7.2 Hz), 0.93 (3H, t, J = 7 . 2 Hz), 1.2-1.9 (32H, m), 2.05 (3H, s), 2.28 (2H, t, J = 7 . 4 Hz), 2.40 (2H, t, J=7.3 Hz), 2.45 (2H, d, J = 17.0 Hz ), 2.95 ( ill, dd, J = 17.0, 7.6 Hz), 3.19 ( IH, d, J=5.5 Hz), 4.0-4.2 (4H, m), 4.95 ( 1H, m), 5.5-5.7 (2H, m). ASOIS: ~H-NMR (CDCI3): t~0.88 (3H, t, J = 7 . 2 Hz), 0.94 (3H, t, J = 7 . 2 Hz), 1.2-1.8 (23H, m), 2.04 ( 3H, s), 2.16 ( 3H, s), 2.28 (2H, t, J = 7 . 3 Hz), 2.41 ( IH, d , J = 16Hz), 2.86 ( 1H, dd, J = 1 6 , 7 . 6 H z ) , 3.06 (1H, brs),4.07 (lH, t, J = 6 . 6 Hz), 4.1-4.2 (IH, m), 5.1-5.3 (IH, m), 5.5-5.6 (2H, m). AS025: ~H-NMR (CDCI3): ~0.86 (3H, t, J = 7 . 2 Hz), 1.2-1.9 (19H, m), 2.01 (6H, s), 2.15 (3H, s), 2.30 (2H, t, J = 7 . 2 Hz), 2.41 (IH, d, J = 16 Hz), 3.00 (IH, dd, J = 16, 7.4 Hz), 3.13.2 (IH, m), 3.66 (3H, s),4.9-5,0 (IH, m), 5.25.3 (I H, m), 5.5-5.6 (2/-1, m). Hydrolysis of PGE~ derivatives to PGEa in hum~
SerUtrB
A PGE~ derivative-containing solution (2.5 gg/25 gl methanol) was evaporated to dryness under a N2 stream and the residue was incubated at 37 °C after the addition of 0.25 ml of normal human serum at a final concentration of 10 gg/ ml. Then, aeetonitfile (0.25 ml) was added to produce deproteinization, and the resulting mixtore was centrifuged for ~0 min at 3000 rpm. The sapematan~ was subjected to high-performance liquid chromatography (HPLC) after being passed through a 0.45 /.tin membrane filter. HPLC conditions were as follows: The mobile phase was a mixture of acetonitrile, tetrahydro-
40 furan, and 1% triethylamine (oH 6.4), the flow rate was 1.0 ml/min, and detection was performed at 278 nm. Assay of the inhibition of platelet aggregation The inhibitory effects of the PGE~ derivatives and their metabolites on human platelet aggregation were measured according to the method of Cardinal et al. [6]. Briefly, peripheral blood was collected using a syringe containing 10% sodium citrate, centrifuged for 10 min at 1000 rpm to obtain platelet-rieh plasma ( P R P ) , and further c,eutrifuged for 15 min at 3000 rpm to obtain plalelet-poor plasma (PPP). Solutions of the PGEt derivatives ( 10/d) were added to 215 #1 of PR??, and aggregation induced by adding 25/zl of 20 #M ADP. After 1 min, the extent of aggregation was measured using a N K K hematracer (PAC-SS, Niko Bioscience Co. Ltd., Tokyo, Japan). PPP was used as the control. The concentration of the test substance which caused 50% inhibJ tion was calculated by taking the aggregation induced by saline as 100%. Solutions of the PGE~ derivatives in ethanol (10 m g / m l ) were diluted to different concentrations with saline for use in this study. L M preparations of PGEI derivatives LM preparations of the PGE~ derivatives were made as described previously [7]. Briefly, the PGE 1 derivatives were dissolved in soybean oil and emulsified with lecithin using a French pressure homogenizer (SL/,4. Aminco, Urbana, U.S.A. ). The final concentration was equivalent to 5 pg of PGE~ per ml. Stability of P G E I derivatives in LM LM-PGE~ derivatives were stored at 40°C for 4 weeks, and the content of the derivatives was determined by H P L C before and after storage. Tetrahydrofuran (2.5 ml) was added to 0.5 ml of the LM preparation to produce decomposition of the emulsion and 10 ml of distilled water was added to allow the mixture to be adsorbed by a Sep Pak cartridge (C 18 ). After washing with
10 ml of distilled water, elution was 0erformed with 7 ml of methanol. After evaporating the methanol, the mobile phase for H P L C (a mixture of acetonitrile and 0.1% triethylamine, pH 6.3) was added, and the resultant solution was used for H P L C under the conditions mentioned above. Leakage of PGEj derivatives from L M when incubated with human serum and blood Human serum ( 900 #1 ) was added to 100 ,ul of each LM-[3H]PGE~ derivative. Sepharose 4B column chromatography (a 1 X 15 em column eluted with 1% BSA-saline, pH 7.4) was performed after incubation for 5 rain at 37°C. The amount (%) of the [3H]PGE~ derivative retained in the LM was calculated by counting the radioactivity of the eiuate of the LM fraction with the turbid void volume. The amount of [ 3H ] PGE ~ derivative initially contained in the LM preparations was defined as 100%. It was con firmed that all of the [ 3H ] PGE~ derivatives were eluted at positions different from the LM fractions. Similar experiments were also performed using rat serum. The dextran gradient method was also performed as described by Heath et al. [8]. Each LM-[3H]PGE~ derivative (100/zl) was mixed with 900 ~1 of human serum or blood in a centrifuge tube, and then 1 ml of a 30% dextran aqueous solution was added 5 rain later. The mixture was overlaid with 3.5 ml of a 10% dextran aqueous solution, the tube was topped up with distilled water, and centrifugation was performed for 20 h at 100,000 × g. Then the top LM layer was pipetted off and the radioactivity was counted after addition of a scintillation cocktail. The amount (%) of the PGE~ derivative retained in the LM was then calculated from the radioactivity. Skin irritation A modification of the passive cutaneous anaphylaxis method of Ovary et al. [9] was used. Evans blue-saline (1%) was intravenously injected into white guinea pigs weighing about 250
41 g, after the subcutaneous injection of 100 pl of each PGE~ derivative diluted to different concentrations with histamine saline (0. I #g/ml saline). Injection of the PGE~ preparations was performed at sites about 1.5 cm away from the mid line on previously depilated areas of the back. The animals were killed 30 rain later, exsanguinated, and skinned in order to measure the diameters of the pigmented spots. The degree of irritation was evaluated using the 4 categories defined in Table 5. The same evaluation was made for the LM-PGE~ derivatives.
Results Hydrolysis of PGEt derivatives to PGE~ The eompound,,~ produced from PGE~ derivatives after incubation with human serum were analyzed by HPLC. C- l-esterified C-9-O-aeylated compounds ( AS006, AS013, and AS019): AS006, AS013, and AS019 were readily hydrolyzed to yield PGE~ during incubation with human serum. The enol acetate at C-9 was initially hydrolyzed to yield the keto form and then the C-I carboxylic acid % 100 o
80
~
~ "
40
20
'
'
8'o
'
'
6'0
'
'
9b
TABLE I Hydrolysisof PGEI derivativesto PGEIin human serum Compound Incubatingtimeat 37°C 15rain 30rain 60rain 90rain 120min AS0O6 AS013 AS019 AS002 AS012 AS014 AS003 AS015 AS025
30.9 22.8 trace
65.5 50.9 22.0 -
2.0
90.5 70.9 64.4 trace trace trace 3.5 2.4
100 90.2 80.4 trace trace trace 5.6 3.6
100 95.8 trace trace trace 6,5 4.7
Mean %of 3 experiments. The amount of PGEt produced whenPGEt derivativeswere incubated with human serum was determinedby HPLC. -: not detectable. ester (an intermediate) was converted to carboxylic acid. The hydrolysis o f AS013 is shown in Fig. 2 and the total hydrolysis rate is shown in Table I. The sequence of the readiness of hydrolysis was as follows: AS006 > AS013 > AS019. C- l-esterified C-9,1 l-O-diaeylated compounds (AS002, AS012, and AS014): these compounds generated PGAt and PGB~, as well as the intermediate C-I monoester PGAt, after incubation with human serum. PGE~ was produced only in trace amounts (Table I ). C- l-esterified C-9,15-O-diacylated compounds (AS003 and AS015): the incubation of AS003 or AS015 with human serum produced PGEt at a very slow rate (Table 1). 15-OAcPGEI-Bu and 15-OAc-PGEt were their intermediates. PGAt and PGB~ are metabolites of PGE~, and PGBt is an isomer of PGAt in which a double bond shifted from between C-10 and C-l I to between C-8 and C-12 [10]. PGE~, PGA=, and PGBt are commercially available as standard substances. Inhibitory effects of PGEz derivatives on human platelet aggregation
Incubation time (rain)
Fig. 2. Hydrolysisof AS013 to PGE= in human serum (as determinedby HPLC).
As shown in Table 2, all the PGE~ derivatives inhibited human platelet aggregation much less
TABLE2 [nhibaory effect of PGEt derivativeson ADP-induced human platclctaggregationbefore and after incubation in human serum Compound
EDso (pM) n=6 No incubation
PGEj 0.057_+0.009" EsterifiedC-l, acylated C-9 AS006 1.64+ 0.79 AS013 2.00± 0.76 AS019 2.20-+0.73 EsterifiedC- 1, acylated C-9,11 AS002 > 2.5 ASO!2 >2.5 AS014 >2.5 Esterifled C-I, acylated C-9,15 AS003 > 2.5 ASOI5 >2.5 Esterifled C-1, acylated C-9~11,15 AS025 > 2.5
After 10 rain incubation at 37~C ill human serum
After 20 min incubation at 37°C in human serum 0.057-+0.012
0.30+-0,15 0.51+_0,30 0.65_+0.27
0-19± 0.0~0.22± 0.09 0.42-+0.17
> 2.5 >2.5 >2.5
> 2.g >2.5 >2.5
> 2.5 >2.5
> 2.5 >2.5
> 2.5
> 2,5
"mean ± SD. effectively than P G E I itself. The inhibitory effects of these derivatives on platelet aggregation were also evaluated after incubation with human serum. The inhibitory potency of C-I esterified C-9-O-acylated compounds (AS006, AS013, and AS019 ) increased dramatically after incubation with human serum. However, the other derivatives remained almost inactive even after incubation for 20 min. Long-term chemical stability of PGE~, AS006, and A S 0 t 3 in L M LM-AS006, and LM-A~q013 were chosen by the above studies as possible candidates for a new Iipo-PGE~ preparation, and these LM preparat[otts and LM-PGEt were stored for 4 weeks at 40°C. As shown in Table 3, the PGEt content of the LM after storage was less than 5% of that before storage. In contrast, a high percentage of the AS013 and AS006 remained in the LM (83 and 80%, respectively). PGA~ was produced when LM-PGE~ was stored, and PGA~ butyl ester and PGBI butyl ester were produced from LM-AS006 and LMAS013, respectively.
TABLE3 Chemical stabilityofPOEb AS006,and AS013when the LM preparation was stored at 40°C Compound
Amount(%) of PGEt derivative I week
2 weeks
4 weeks
Lipo-PGEL Lipo-AS006 Lipo-AS013
42.8_+0,3 g8.9_+3,3 87.8_+2.2
15,0+_2.6 87.5+2.7 80.9+_0.9
4.6+2.6 80.1+0.7 83.2_+3.7
Data are the mean results of 4 experiments (mean+ SD). The amount of the P(~Et derivatives was delermined by HPLC after storage of the LM preparations at 40~C. The amount of the PGE~ derivativesbefore storage was defined as 100%,
Leakage of PGE1 derivatives from LM in human serum or blood One volume ofLM-AS013, LM-AS006, or LMPGE I was incubated with 9 volumes of human serum or blood for 5 rain. As shown in Table 4, the amount of AS013 retained in the LM after incubation was 6.9-12.3%, which was higher than for LM-PGEL ( 0 . 5 - 2 % ) and LM-AS006 ( 1 . 8 6.9%). A large amount of the PGEt derivative
43 TABLE4 Amount of each PGEI derivativeretained in lhe LM after incubation with blood or serum Dexiran gradient method.
Lipo-PGEI Lipo-AS006 Lipo-AS013
~epharose 4B column chromatographyb
Human blood (n=6)
Human senlm (n=6)
Human serum (n=6)
Rat serum (n=3)
2.2+- I. I* 4.5 + 1.3 12.0+ 1,4
1.4+ 1.0" 6.9 + 0.4 12.3+ I.I
0.5 -+0.3" 1.8+-0.3 6.9-+0.1
trace trace trace
*% (mcan+SD). aThe dextran gradient method was used to determine the amount of each PGE~ derivativeretained in the LM after incubating the LM preparationwith human serum or blood for 5 rain at 37°C. bThe amount of each PGEI derivativeretained in the LM was determined by Sepharose 4B column chromatography afler incubating the LM preparation with human serum or rat serum for 5 min at 37°C. leaked very rapidly from the LM in tat serum. The LM content of PGE~ derivatives was taken as 100% before incubation.
Skin irritation praducedbyPGE,, AS006, and AS013 Table 5 shows the results of the guinea pig skin irritation test performed for PGE~, AS006, AS013, and their LM preparations. The diameter, area, and density of the pigmented spots produced by 1 / t g / m l PGE~, 25 p g / m l AS006, and 50 p g / ml AS013 were comparable with each TABLE5 Irritation caused by the applicationof PGEh AS006,ASOl3 and their LM preparationsto guinea pig skin Compound Concentration t pg/ml PGEI +++ AS0O6 + AS013 + Lipo-PGEt ND Lipo-AS006 ND Lipo-AS013 ND
5pg/ml
25/tg/ml
50#g/ml
>+++ ++ + >+++ ++ ++
>+++ +++ ++ ND ND ND
>+++ >+++ +++ ND ND biD
+: < 5 mm in diameter pigmented spot; + +: 5-10 mm in diameter pigmented spot; + + +: 10-15 mm in diameter pigmented spot; > + + +: > 15 mm in diameter pigmented spot; ND: not determined.
other. Moreover, the skin irritation produced by LM-AS006 and LM-AS013 was less than that due to LM-PGE1.
Discussion We developed a superior LM-PGE~ preparation which overcame two disadvantages of lipoPGEI [ 4 ] , i.e., the chemical instability of PGEI and its rapid leakage from LM in the blood. The carbonyl group of PGEt at C-9 undergoes tautomerization to its enol form under basic and even acidic conditions, which then brings about inactivation by hydrolysis to form PGA~ [ 1 0 ] . in view of the ready chemical conversion of PGE~, protection of the isomer is necessary to make it more chemically stable. This was done by acylation at C-9 and the introduction of a double bond between C-8 and C-9. In addition, it was considered that esterification at C-I a n d / or acylation at C-9, -11, and -15 would increase the solubility of PGE] derivatives in LM (which are mainly composed of soybean oil and lecithins), and result in their decreased in viva leakage from the LM preparation. In this study, we synthesized several PGE, derivatives based on these concepts. For a prodmg of PGEt to be effective for treating vascular wall lesions, it must be hydrolyzed to PGE~ in the vascular wall itself. HPLC and measurement of the inhibition o f human platelet
44 aggregation has shown that the C-9-O-acylated J8-C-I esterified compounds (AS006, AS013, and AS019) are prodrugs which are hydrolyzed to PGE~ in human serum and have almost the same potency as the parent compound. We used a very high concentration of PGE~ derivatives for accurate analysis. The hydrolysis may occur rapidly at the actual diseased site, where the drug concentration is much lower and enzymatic activity is much higher. Other PGE~ derivatives were not hydrolyzed to PGE~ in serum (or only vet3, slowly), and therefore only showed a weak activity. The ability of these derivatives to inhibit human platelet aggregation after incubation with human serum was very consistent with the amount of PGE~ produced by hydrolysis in human serum, as determined by HPLC. AS006 and AS013 are C-9-O-acylated As-C-l-estedfied prodrugs of PGE~, and were found to be much more stable in LM than the parent compound. In order to deliver PGEI to vascular lesions it is important that the drag is retained in the LM until it reaches the target site. In other words, one of the key points for increasing the therapeutic efficacy of LM preparations is to minimize the amount of the actual ingredient that leaks out during transit through the body. One method is to increase the hydrophobicity of the agent under consideration by conversion to a more hydrophobic prodrug, while another method is to improve the colloidal stability of the LM emulsion by adding cholesterol in lecithin or using a stabilizer like a polysaccharide.The latter approach will be studied in the near future, and more specific targeting using monoclonal antibody should also be studied. Since PGE, leaks from the LM due to its interaction mainly with serum albumin [ 11 ], the PGEL derivatives such as AS013 with a higher solubility in soybean oil show less leakage [ 12]. Rat serum has a stronger enzymatic activity than human serum, and the PGE~ derivatives leaked out more rapidly from the LM in rat serum (Table 4). This suggests that enzymatic hydrolysis of the ester or acyl group increases the hydrophil~city of the derivatives and accelerates their leakage from the LM.
LM preparations are ultimately diluted thousands of times after administration, with the dilution progressing gradually from the injection site. In addition, the encapsulated drugs are delivered to the target lesions rapidly. In c,,t previous study, LM were detected in "-he vascular walls by electron microscopy 5 lnin after their intravenous injection [7]. Therefore, the experiments on the leakage of the actual ingredient from the LM performed in the present study are expected to reflect the fate of LM preparations in the human body. AS006 and AS013 caused less skin irritation than PGEz, with their skin irritating potency being 1/25 and 1/50, respectively, of that of the parent compound. Skin irritation is one of the disadvantages in the clinical application ofPGE~, so AS006 and AS013 might also be useful as topieal or oral drugs.
Conclusions 38-9-O-butyryl prostaglandin F~ butyl ester (ASOI3) is a prodrug of PGE, which is readily hydrolyzed to the parent compound in human serum. It was shown to be chemically stable in lipid microspheres (LM), was efficiently retained in the LM, and caused only minimal skin irritation. An LM preparation of AS013, LMAS013, was therefore thought to be a potentially excellent LM-PGE, preparation. Since the clinical efficacy oflipo-PGE~, although the active ingredient was retained in the LM far less effectively than with LM-AS013, was more than 10 times greater than that of ordinary PGE~ [4], LM-AS013 would be expected to show a much greater efficacy in various clinical applications.
Acknowledgment The authors wish to thank Dr. S. Kawai and Dr. D. Mcquire for their helpful advice on the preparation of this manuscript.
References 1 M.F.R. Martin, Prostaglandin El in the treatmem of systemic sclerosls~ Ann. Rheum. Dis. 39 (1980) 194. 2 B.J. Pardy. M.C. Hoare, H.H.G. Eastcott, C.C. Miles, T.N. Needhman, T. Harhourne and B.W. Ellis, Prostaglandin E~ in severn Raynaud's phenomenon, S~rgery 92 ( 1932 ) 953-965. 3 Y. Mizushima, Y. Shiokawa, M. Homma, S. Kashiwazaki, Y. Ichikawa, H. Hashimoto and A. Sakuma, A multicenter double blind controlled study of lipo-PGE~, PGEt incorporated in lipid microspheres, in peripheral vascular disease secondary to connective tissue disorders, J. Rheumatol. 14 (1987) 97-101. 4 Y. Mizushima, Lipo-prostaglandin preparations ireview), Prostaglandins Leukol. Essent. Patty Acids ,12 (1991) 1-6, 5 C.J. Sih, J.B. Healhcr, R. Spud, P.P. Price, G. Peraz. zotti, L.F.H. Lee and S,S. Lee, Asymmetric total symhesis of (-)-pruslaglandin E I and (-)-prostaglandin E2, J. Am. Chem. Sue. 97 (1975) 865-874, 6 D.C. Cardinal and R.J. Flower, The electric aggregometer: a novel device for assessing platelet behavior in blood, J. Pharmcol. Melhods 3 ( 1980 ) 135-158.
7
8
9 10 Il
12
Y. Mizushima, T. Hamano and K . Yokoyama, Tissue distribution and anti-inflammatory activity of corti¢osteroids incorporated in lipid emulsion, Ann. Rheum. Dis. 41 0 9 3 2 ) 263-267. T.D. Heath, B.A. Macher and D. Papahadjopoulos, Covalent altaehment of immunoglobulins to l iposomes via glycosphingolipids, Biochim. Biophys. Acta 640 ( 1981 ) 66-gl. Z. Ovary, Immediate reactions in the skin of experimental animals provoked by antihody-amigen interaclions, ProB. Allergy 5 (1958) 459-508. B. Samue!sson, The prostaglandins, Angew. Chem. lnt. Ed. Engb4 0 9 6 5 ) 410-416. F.A. Fitzpatric, W.F. Liggett and M.A. Wynalda, Albumin-eieosanoid interactions: a model system to determine their attributes and inhibition, ]. Biol. Chem. 259 ( 1984 ) 2722-2727. M.J. Cho, Reduced hydrolytic lability of epoprostenol in the presence of cationic micelles, J. Pharm. SeL 71 (1982) 453-454.
37
© 1992 ElsevierScience Publishers B.V. All rights reserved O168-3659/92t$OS.00
COREL0722
A
stable PGE1 prodrug for targeting therapy
R. Igarashi ~, Y. M i z u s h i m a ~, M. T a k e n a g a u, K, M a t s u m o t o a, Y. M o r i z a w a b a n d A. Y a s u d a b ~Division of Drug Deliver3, System. Institute of Medical Science, St. Marianna Univei~ity, 2-16-1 Sugao, Miyamae-ku. 216 KawasakL Japan; hResearch Center, Asahi Glass Co., Ltd.. 1150 llazawacho. Kanagawa.ku, 221 Yokohama, Japan
( Received 27 August 1991; accepled in revisedform 23 December 1991)
Lipo-PGE~ is a passively targetable prostaglandin E~ (PGE~) preparation, in which PGE~ is incorporated into lipid microspheres (LM); it has been widely used in Japan since 1988 for the treatment of various vascular diseases. Passive targeting results from the accumulation of LM in the vascular lesions following intravenous administration. However, this preparation has two major disadvantages. One is the chemical instability of PGE~ in the LM and the other is the rapid leakage of PGE~ from the LM in the blood, leading to a decrease in the targeting of this drug. We accordingly synthesized several PGE~ prodrugs and evaluated them to develop a better LM preparation. Among the PGE~ prodrugs tested, As-9-O-butyryl prostaglandin F~ butyl ester (AS013) was readily hydrolyzed to PGE~ in human serum. AS013 was stable as an LM preparation and 80% of this ester was recovered unchanged even after storage as LM-AS013 for 4 weeks at 40°C. In contrast, PGE~ showed a recovery & o n l y 5% from the LM under the same conditions. Moreover, AS013 was more effectively retained in the LM after incubation with human blood or serum when compared with PGE~. These results suggested that LM-AS013 would be a far superior LM preparation than lipo-PGE~ for clinical use. Key words: Drug delivery system; Lipid microsphere (LM); Stable prodrug of PGE~; LM preparation of PGEL
Introduction Prostaglandin Et (PGE~) is used for the treatment of various vascular disorders because of its antiplatelet ae.d vascdilatory action~ [ ! .2 !. We have previously developed "lipo-PGEl', a drug dellvery system ( DDS ) incorporating PGE ~ into the lipid particles of an oil-in-water emulsion. These lipid particles have a diameter of 0.2/tm, are composed of soybean oil and lecithin, and are designated as "lipid mierospheres ( L M ) " . LM Correspondence to: R. lgarashi, Ph.D., Division of Drug DeliverySystem, Institute of Medical Science, St. Marianna University,2-16-1 Sugao.Miyamae-ku,216 Kawasak~,Japan.
shows a similar tissue distribution to liposomes, and provide a stable and safe drug carrier that can be used as a DDS. Since LM largely accumulate in inflamed tissues and vascular lesions, lipo-PGE I shows a marked efficacy for treating vascular diseases such as atheroselerosis [ 3,4 ]. However, PGE~ in LM is chemically unstable and is largely converted to PGA~ after storage in brown glass ampoules at room temperature for 6 months. In addition, PGE~ is rapidly released from the LM on incubation with human blood or serum. Since the LM are used as a targeting carrier, the PGE~ has to be retained in the microspheres until after it is delivered to the target site.
3~ To develop a better LM-PGE( system that overcomes the disadvantages of lipo-PGEl, we have investigated a PGE~-prodrug which is chemically stable, shows little leakage from LM, and is readily hydrolyzed into PGE~ at the target site in the body.
OB,
compouncl
OR,
OH OH pmstaglandin E~(PGEO
Rt
R~
R~
R,
IV~aterials and Methods esterified C-l, acvlaled C-9
Materials Adenosine 5'-diphosphate (ADP) (Niko Bio Science, Tokyo, Japan), the 0.45,um membrane filter (Japan Millipore Corporation, Tokyo, Japan), bovine serum albumin (BSA) (Sigma Chemical Company, St. Louis, U.S.A. ), Sepharore 4B and Dextran T40 (MW 40,000) (Pharmacla, Uppsala, Sweden), Hionic Fluor ® scintillation cocktail (Packard Company Inc., Meriden, U.S,A.), and the other chemicals used such as acetonitrile and tetrahydrofuran (Wako Pure Chemical Industries Ltd., Osaka, Japan) were all commercially available preparations. Guinea pigs weighing 250-300 g were obtained from Japan Laboratory Animals Inc. (Tokyo, Japan). Lipo-[3H]PGE~ (167 kBq/5.0 /~g PGE~/ml), lipo-[3H]AS006 (180 kBq/6.38 #g AS006/ml), and lipo-[3H]AS013 (192 kBq/ 6.78 gg ASOI3/ml) were supplied by Green Cross Co. Ltd. (Osaka, Japan). Synthesis of PGEt derivatives (AS-9.O-acy|ated prostaglandin Ft derivatives) The chemical structures of the PGE, derivatives synthesized are shown in Fig. 1. C-l-estefifled C-9-O-aeylated compounds These were synthesized according to the method of Sih et al. [ 5 ii. To a solution of (1 E, 3S) -l-iodo- (t-butyldimethylsiloxy) -1-octene (4.99 g, 13.56 mmol) in ethyl ether ( 100 ml), tbutyl lithium (f= 1.4 hexane solution, 18.1 ml, 27.1 mmol) was added at -78°C. After 2 h, a solution of tributylphosphine-copper(I) iodide complex ( 4.63 g, 2 I. 31 mmol ) and tributylphosphine (2.92 ml, 12.I6 retool) in ethyl ether (40
AS 006
C4H~
CHACO
H
H
AS 019
C4Hg
C2H~CO
H
H
ASOla C,Hg C~H~CO esterified C-1, acylaled C-@,11
H
H
AS 002
CH3
CHACO
CHACO
H
A$012
C4H9
CHACO
CHACO
H
AS014
C.H~
C~HL~CO CHACO
H
esterified C-1, acylated C-9,15 CHACO
H
CHACO
AS O 15 C~H:~ CHACO esterified C-1, acylatecl C a, 11,15
AS 003
H
CHACO
AS 025
CFI3
CH3
CHACO
CHACO
CHACO
Fig. I. Chemicalstructuresof the PGE~derivativesusedin this study. ml) was added dropwise to the main solution and stirred for 50 min at -78°C. An ether solution ( 160 ml) of 4R-t-butyldimethylsiloxy-2- (6-earbobutoxyhexyl)-2-cyclopenten-l-one (4.75 ml, 11.3 mmol) was then added to the above solutint). The mixture was stirred at - 7 8 ° C for 20 min and warmed to - 2 3 ° C for 35 rain. Finally, 30 mmol of butyric anhydride (for AS013), acetic anhydride (for AS006), or propionic anhydride (for AS019) was added dropwise at 0°C, followed by stirring for 15 h at from 0 °C to room temperature. Saturated ammonium sulfate solution (200 ml) was added to this solution and extraction was performed with ethyl ether (100 ml). The organic layer was dried over anhydrous luagncsium sulfate and evaporated under re-. duced pressure. The residue was puriflc.I by silica gel chromatography (hexane-ethyl e.cetale=20/1 to 4/1 ) at 0°C to aflbra the protf,cted As-9-O-acylated prostaglandin F~ C-I butyl ester. The protected compound (8.25 retool) was dissolved in acelonitrile (100 ml) and 10 ml of 40% hydrofluoric acid solution was added at 0 °C.
39 After 30 min, the reaction mixture was poured into a mixture of an aqueous solution of 20% potassium carbonate (150 ml) and dichloromethane ( 150 ml). The organic layer was dried over anhydrous magnesium sulfate and the solvent was evaporated. The residue was then purified by silica gel chromatography (dichlorumethaneacetone= 2/'1 ) at 0°C to afford dB-9-O-acylatcd prostaglandin Fj butyl ester (about 80% yield). Each compound was identified by NMR spectrometry. AS013: IH-NMR (CDCI3): ff0.86 (3H, t, J=7.2 Hz), 0.93 (3H, t, J = 7 . 2 Hz), 1.2-1.9 (221-I, m), 2.28 (1H, t, J=7.7 Hz), 2.45 (IH, m), 2.9-3.1 ( IH, m), 3.2-3.3 ( 1H, m), 4.0-4.2 44I-I, m), 4.9-5.1 t i l l , m), 5.5-5.7 (2H, m). IR (neat film): 3400, 2930, 1750, 1730, 970 cm -I. [c~]:°0--61.6 ° (c=0.5, abs. ethanol) ASO06: IH-NMR (CDCla):b'0.85 (3H, t , J = 7 Hz), 0.95 (3H, t, J = 7 Hz), 1.2-2.9 (29H, m + s (b'2.15, 3H)), 3.0-3.05 (1H, m),4.1 (2H, t , J = 7 Hz), 4.9-4.2 (2H, m), 5.45 (IH, dd), 5.6 (IH, dd). ASOI9: ~H-NMR (CDCI3): t~0.88 (3H, t, J=7.2 Hz), 0.91 (31-1,t, J=7.2 Hz), 0.93 (3H, ~., J--7.2 Hz), 1.2-2.2 (23H, m), 2.28 (2H, t, J=7.2 Hz), 2.45 (2H, t, J = 7 . 2 Hz), 2.4-2.5 (IH, m), 2.86 (IH, dd, J = l S , 5.7 Hz), 3.16 (IH, D , J = 15 Hz),4.07 (2H, t , J = 6 . 6 Hz), 4.04.2 (2H, m), 5.47 (IH, dd, J=16.1, 6.6 Hz), 5.60 ( 1H, dd, J = 16.1, 9.4 Hz). C-11-O-acylated derivatives (AS002, AS012, and AS014) or C-15-O-acylated derivatives (AS003 and AS015) were transformed by acylation with acetic anhydride after the selective deprotection of hydroxyl protecting group at C11 or C-15. Diacylated compound (AS025) was synthesized by ti~ereaction of the corresponding diol with acetic anhydride. ASO02: ~H-NMR (CDCI3): ~0.88 (3H, t, J=7.2 Hz), 1.2-1.9 (19H, m), 2.05 (31-1, s), 2.15 (3H, s),2.30 (2H, t , J = 7 . 3 Hz),2.45 (IH, d, J = 16 Hz), 2.97 (IH, dd, J = 16, 6.0 Hz), 3.2¢6 (IH, d , J = 6 . 0 Hz), 3.67 (3I-I, s), 4.0-4.1 (IH, m), 4.9-5.0 ( 1H, m), 5.4-5.7 (2H, m). ASO03: ~H-NMR (CDCI3): t$0.80 (3H, t, J = 7 . 2 Hz), 1.1-1.8 (19H, m), 1.95 (3H, s),
2.08 (3H, s),2.22 (2H, t , J = 7 . 2 Hz),2.32 (IH, d, J = 1 6 Hz), 2.81 (IH, dd, J = 16, 7 Hz), 2.93.1 ( IH, m), 3.60 (3H, s), 4.0-4.1 ( IH, m), 5.15.2 (IH, m), 5.2-5.3 (2H, m). ASOI2: tH-NMR (CDCI3): t$0.86 (3H, t, J=7.2 Hz), 0.93 (3H, t, J=7.2 Hz), 1.2-1.9 (22H, m), 2.28 (Ill, t, J=7.7 Hz), 2.45 (IH, m), 2.9-3.1 ( IH, m), 3.2-3.3 ( IH, m), 4.0-4.2 (4H, m), 4.9-5.1 (IH, m), 5.5-5.7 (2H, m). ASOI4: ~H-NMR (CDCI3): J0.88 (3H, t, J = 7 . 2 Hz), 0.91 (3H, t, J=7.2 Hz), 0.93 (3H, t, J = 7 . 2 Hz), 1.2-1.9 (32H, m), 2.05 (3H, s), 2.28 (2H, t, J = 7 . 4 Hz), 2.40 (2H, t, J=7.3 Hz), 2.45 (2H, d, J = 17.0 Hz ), 2.95 ( ill, dd, J = 17.0, 7.6 Hz), 3.19 ( IH, d, J=5.5 Hz), 4.0-4.2 (4H, m), 4.95 ( 1H, m), 5.5-5.7 (2H, m). ASOIS: ~H-NMR (CDCI3): t~0.88 (3H, t, J = 7 . 2 Hz), 0.94 (3H, t, J = 7 . 2 Hz), 1.2-1.8 (23H, m), 2.04 ( 3H, s), 2.16 ( 3H, s), 2.28 (2H, t, J = 7 . 3 Hz), 2.41 ( IH, d , J = 16Hz), 2.86 ( 1H, dd, J = 1 6 , 7 . 6 H z ) , 3.06 (1H, brs),4.07 (lH, t, J = 6 . 6 Hz), 4.1-4.2 (IH, m), 5.1-5.3 (IH, m), 5.5-5.6 (2H, m). AS025: ~H-NMR (CDCI3): ~0.86 (3H, t, J = 7 . 2 Hz), 1.2-1.9 (19H, m), 2.01 (6H, s), 2.15 (3H, s), 2.30 (2H, t, J = 7 . 2 Hz), 2.41 (IH, d, J = 16 Hz), 3.00 (IH, dd, J = 16, 7.4 Hz), 3.13.2 (IH, m), 3.66 (3H, s),4.9-5,0 (IH, m), 5.25.3 (I H, m), 5.5-5.6 (2/-1, m). Hydrolysis of PGE~ derivatives to PGEa in hum~
SerUtrB
A PGE~ derivative-containing solution (2.5 gg/25 gl methanol) was evaporated to dryness under a N2 stream and the residue was incubated at 37 °C after the addition of 0.25 ml of normal human serum at a final concentration of 10 gg/ ml. Then, aeetonitfile (0.25 ml) was added to produce deproteinization, and the resulting mixtore was centrifuged for ~0 min at 3000 rpm. The sapematan~ was subjected to high-performance liquid chromatography (HPLC) after being passed through a 0.45 /.tin membrane filter. HPLC conditions were as follows: The mobile phase was a mixture of acetonitrile, tetrahydro-
40 furan, and 1% triethylamine (oH 6.4), the flow rate was 1.0 ml/min, and detection was performed at 278 nm. Assay of the inhibition of platelet aggregation The inhibitory effects of the PGE~ derivatives and their metabolites on human platelet aggregation were measured according to the method of Cardinal et al. [6]. Briefly, peripheral blood was collected using a syringe containing 10% sodium citrate, centrifuged for 10 min at 1000 rpm to obtain platelet-rieh plasma ( P R P ) , and further c,eutrifuged for 15 min at 3000 rpm to obtain plalelet-poor plasma (PPP). Solutions of the PGEt derivatives ( 10/d) were added to 215 #1 of PR??, and aggregation induced by adding 25/zl of 20 #M ADP. After 1 min, the extent of aggregation was measured using a N K K hematracer (PAC-SS, Niko Bioscience Co. Ltd., Tokyo, Japan). PPP was used as the control. The concentration of the test substance which caused 50% inhibJ tion was calculated by taking the aggregation induced by saline as 100%. Solutions of the PGE~ derivatives in ethanol (10 m g / m l ) were diluted to different concentrations with saline for use in this study. L M preparations of PGEI derivatives LM preparations of the PGE~ derivatives were made as described previously [7]. Briefly, the PGE 1 derivatives were dissolved in soybean oil and emulsified with lecithin using a French pressure homogenizer (SL/,4. Aminco, Urbana, U.S.A. ). The final concentration was equivalent to 5 pg of PGE~ per ml. Stability of P G E I derivatives in LM LM-PGE~ derivatives were stored at 40°C for 4 weeks, and the content of the derivatives was determined by H P L C before and after storage. Tetrahydrofuran (2.5 ml) was added to 0.5 ml of the LM preparation to produce decomposition of the emulsion and 10 ml of distilled water was added to allow the mixture to be adsorbed by a Sep Pak cartridge (C 18 ). After washing with
10 ml of distilled water, elution was 0erformed with 7 ml of methanol. After evaporating the methanol, the mobile phase for H P L C (a mixture of acetonitrile and 0.1% triethylamine, pH 6.3) was added, and the resultant solution was used for H P L C under the conditions mentioned above. Leakage of PGEj derivatives from L M when incubated with human serum and blood Human serum ( 900 #1 ) was added to 100 ,ul of each LM-[3H]PGE~ derivative. Sepharose 4B column chromatography (a 1 X 15 em column eluted with 1% BSA-saline, pH 7.4) was performed after incubation for 5 rain at 37°C. The amount (%) of the [3H]PGE~ derivative retained in the LM was calculated by counting the radioactivity of the eiuate of the LM fraction with the turbid void volume. The amount of [ 3H ] PGE ~ derivative initially contained in the LM preparations was defined as 100%. It was con firmed that all of the [ 3H ] PGE~ derivatives were eluted at positions different from the LM fractions. Similar experiments were also performed using rat serum. The dextran gradient method was also performed as described by Heath et al. [8]. Each LM-[3H]PGE~ derivative (100/zl) was mixed with 900 ~1 of human serum or blood in a centrifuge tube, and then 1 ml of a 30% dextran aqueous solution was added 5 rain later. The mixture was overlaid with 3.5 ml of a 10% dextran aqueous solution, the tube was topped up with distilled water, and centrifugation was performed for 20 h at 100,000 × g. Then the top LM layer was pipetted off and the radioactivity was counted after addition of a scintillation cocktail. The amount (%) of the PGE~ derivative retained in the LM was then calculated from the radioactivity. Skin irritation A modification of the passive cutaneous anaphylaxis method of Ovary et al. [9] was used. Evans blue-saline (1%) was intravenously injected into white guinea pigs weighing about 250
41 g, after the subcutaneous injection of 100 pl of each PGE~ derivative diluted to different concentrations with histamine saline (0. I #g/ml saline). Injection of the PGE~ preparations was performed at sites about 1.5 cm away from the mid line on previously depilated areas of the back. The animals were killed 30 rain later, exsanguinated, and skinned in order to measure the diameters of the pigmented spots. The degree of irritation was evaluated using the 4 categories defined in Table 5. The same evaluation was made for the LM-PGE~ derivatives.
Results Hydrolysis of PGEt derivatives to PGE~ The eompound,,~ produced from PGE~ derivatives after incubation with human serum were analyzed by HPLC. C- l-esterified C-9-O-aeylated compounds ( AS006, AS013, and AS019): AS006, AS013, and AS019 were readily hydrolyzed to yield PGE~ during incubation with human serum. The enol acetate at C-9 was initially hydrolyzed to yield the keto form and then the C-I carboxylic acid % 100 o
80
~
~ "
40
20
'
'
8'o
'
'
6'0
'
'
9b
TABLE I Hydrolysisof PGEI derivativesto PGEIin human serum Compound Incubatingtimeat 37°C 15rain 30rain 60rain 90rain 120min AS0O6 AS013 AS019 AS002 AS012 AS014 AS003 AS015 AS025
30.9 22.8 trace
65.5 50.9 22.0 -
2.0
90.5 70.9 64.4 trace trace trace 3.5 2.4
100 90.2 80.4 trace trace trace 5.6 3.6
100 95.8 trace trace trace 6,5 4.7
Mean %of 3 experiments. The amount of PGEt produced whenPGEt derivativeswere incubated with human serum was determinedby HPLC. -: not detectable. ester (an intermediate) was converted to carboxylic acid. The hydrolysis o f AS013 is shown in Fig. 2 and the total hydrolysis rate is shown in Table I. The sequence of the readiness of hydrolysis was as follows: AS006 > AS013 > AS019. C- l-esterified C-9,1 l-O-diaeylated compounds (AS002, AS012, and AS014): these compounds generated PGAt and PGB~, as well as the intermediate C-I monoester PGAt, after incubation with human serum. PGE~ was produced only in trace amounts (Table I ). C- l-esterified C-9,15-O-diacylated compounds (AS003 and AS015): the incubation of AS003 or AS015 with human serum produced PGEt at a very slow rate (Table 1). 15-OAcPGEI-Bu and 15-OAc-PGEt were their intermediates. PGAt and PGB~ are metabolites of PGE~, and PGBt is an isomer of PGAt in which a double bond shifted from between C-10 and C-l I to between C-8 and C-12 [10]. PGE~, PGA=, and PGBt are commercially available as standard substances. Inhibitory effects of PGEz derivatives on human platelet aggregation
Incubation time (rain)
Fig. 2. Hydrolysisof AS013 to PGE= in human serum (as determinedby HPLC).
As shown in Table 2, all the PGE~ derivatives inhibited human platelet aggregation much less
TABLE2 [nhibaory effect of PGEt derivativeson ADP-induced human platclctaggregationbefore and after incubation in human serum Compound
EDso (pM) n=6 No incubation
PGEj 0.057_+0.009" EsterifiedC-l, acylated C-9 AS006 1.64+ 0.79 AS013 2.00± 0.76 AS019 2.20-+0.73 EsterifiedC- 1, acylated C-9,11 AS002 > 2.5 ASO!2 >2.5 AS014 >2.5 Esterifled C-I, acylated C-9,15 AS003 > 2.5 ASOI5 >2.5 Esterifled C-1, acylated C-9~11,15 AS025 > 2.5
After 10 rain incubation at 37~C ill human serum
After 20 min incubation at 37°C in human serum 0.057-+0.012
0.30+-0,15 0.51+_0,30 0.65_+0.27
0-19± 0.0~0.22± 0.09 0.42-+0.17
> 2.5 >2.5 >2.5
> 2.g >2.5 >2.5
> 2.5 >2.5
> 2.5 >2.5
> 2.5
> 2,5
"mean ± SD. effectively than P G E I itself. The inhibitory effects of these derivatives on platelet aggregation were also evaluated after incubation with human serum. The inhibitory potency of C-I esterified C-9-O-acylated compounds (AS006, AS013, and AS019 ) increased dramatically after incubation with human serum. However, the other derivatives remained almost inactive even after incubation for 20 min. Long-term chemical stability of PGE~, AS006, and A S 0 t 3 in L M LM-AS006, and LM-A~q013 were chosen by the above studies as possible candidates for a new Iipo-PGE~ preparation, and these LM preparat[otts and LM-PGEt were stored for 4 weeks at 40°C. As shown in Table 3, the PGEt content of the LM after storage was less than 5% of that before storage. In contrast, a high percentage of the AS013 and AS006 remained in the LM (83 and 80%, respectively). PGA~ was produced when LM-PGE~ was stored, and PGA~ butyl ester and PGBI butyl ester were produced from LM-AS006 and LMAS013, respectively.
TABLE3 Chemical stabilityofPOEb AS006,and AS013when the LM preparation was stored at 40°C Compound
Amount(%) of PGEt derivative I week
2 weeks
4 weeks
Lipo-PGEL Lipo-AS006 Lipo-AS013
42.8_+0,3 g8.9_+3,3 87.8_+2.2
15,0+_2.6 87.5+2.7 80.9+_0.9
4.6+2.6 80.1+0.7 83.2_+3.7
Data are the mean results of 4 experiments (mean+ SD). The amount of the P(~Et derivatives was delermined by HPLC after storage of the LM preparations at 40~C. The amount of the PGE~ derivativesbefore storage was defined as 100%,
Leakage of PGE1 derivatives from LM in human serum or blood One volume ofLM-AS013, LM-AS006, or LMPGE I was incubated with 9 volumes of human serum or blood for 5 rain. As shown in Table 4, the amount of AS013 retained in the LM after incubation was 6.9-12.3%, which was higher than for LM-PGEL ( 0 . 5 - 2 % ) and LM-AS006 ( 1 . 8 6.9%). A large amount of the PGEt derivative
43 TABLE4 Amount of each PGEI derivativeretained in lhe LM after incubation with blood or serum Dexiran gradient method.
Lipo-PGEI Lipo-AS006 Lipo-AS013
~epharose 4B column chromatographyb
Human blood (n=6)
Human senlm (n=6)
Human serum (n=6)
Rat serum (n=3)
2.2+- I. I* 4.5 + 1.3 12.0+ 1,4
1.4+ 1.0" 6.9 + 0.4 12.3+ I.I
0.5 -+0.3" 1.8+-0.3 6.9-+0.1
trace trace trace
*% (mcan+SD). aThe dextran gradient method was used to determine the amount of each PGE~ derivativeretained in the LM after incubating the LM preparationwith human serum or blood for 5 rain at 37°C. bThe amount of each PGEI derivativeretained in the LM was determined by Sepharose 4B column chromatography afler incubating the LM preparation with human serum or rat serum for 5 min at 37°C. leaked very rapidly from the LM in tat serum. The LM content of PGE~ derivatives was taken as 100% before incubation.
Skin irritation praducedbyPGE,, AS006, and AS013 Table 5 shows the results of the guinea pig skin irritation test performed for PGE~, AS006, AS013, and their LM preparations. The diameter, area, and density of the pigmented spots produced by 1 / t g / m l PGE~, 25 p g / m l AS006, and 50 p g / ml AS013 were comparable with each TABLE5 Irritation caused by the applicationof PGEh AS006,ASOl3 and their LM preparationsto guinea pig skin Compound Concentration t pg/ml PGEI +++ AS0O6 + AS013 + Lipo-PGEt ND Lipo-AS006 ND Lipo-AS013 ND
5pg/ml
25/tg/ml
50#g/ml
>+++ ++ + >+++ ++ ++
>+++ +++ ++ ND ND ND
>+++ >+++ +++ ND ND biD
+: < 5 mm in diameter pigmented spot; + +: 5-10 mm in diameter pigmented spot; + + +: 10-15 mm in diameter pigmented spot; > + + +: > 15 mm in diameter pigmented spot; ND: not determined.
other. Moreover, the skin irritation produced by LM-AS006 and LM-AS013 was less than that due to LM-PGE1.
Discussion We developed a superior LM-PGE~ preparation which overcame two disadvantages of lipoPGEI [ 4 ] , i.e., the chemical instability of PGEI and its rapid leakage from LM in the blood. The carbonyl group of PGEt at C-9 undergoes tautomerization to its enol form under basic and even acidic conditions, which then brings about inactivation by hydrolysis to form PGA~ [ 1 0 ] . in view of the ready chemical conversion of PGE~, protection of the isomer is necessary to make it more chemically stable. This was done by acylation at C-9 and the introduction of a double bond between C-8 and C-9. In addition, it was considered that esterification at C-I a n d / or acylation at C-9, -11, and -15 would increase the solubility of PGE] derivatives in LM (which are mainly composed of soybean oil and lecithins), and result in their decreased in viva leakage from the LM preparation. In this study, we synthesized several PGE, derivatives based on these concepts. For a prodmg of PGEt to be effective for treating vascular wall lesions, it must be hydrolyzed to PGE~ in the vascular wall itself. HPLC and measurement of the inhibition o f human platelet
44 aggregation has shown that the C-9-O-acylated J8-C-I esterified compounds (AS006, AS013, and AS019) are prodrugs which are hydrolyzed to PGE~ in human serum and have almost the same potency as the parent compound. We used a very high concentration of PGE~ derivatives for accurate analysis. The hydrolysis may occur rapidly at the actual diseased site, where the drug concentration is much lower and enzymatic activity is much higher. Other PGE~ derivatives were not hydrolyzed to PGE~ in serum (or only vet3, slowly), and therefore only showed a weak activity. The ability of these derivatives to inhibit human platelet aggregation after incubation with human serum was very consistent with the amount of PGE~ produced by hydrolysis in human serum, as determined by HPLC. AS006 and AS013 are C-9-O-acylated As-C-l-estedfied prodrugs of PGE~, and were found to be much more stable in LM than the parent compound. In order to deliver PGEI to vascular lesions it is important that the drag is retained in the LM until it reaches the target site. In other words, one of the key points for increasing the therapeutic efficacy of LM preparations is to minimize the amount of the actual ingredient that leaks out during transit through the body. One method is to increase the hydrophobicity of the agent under consideration by conversion to a more hydrophobic prodrug, while another method is to improve the colloidal stability of the LM emulsion by adding cholesterol in lecithin or using a stabilizer like a polysaccharide.The latter approach will be studied in the near future, and more specific targeting using monoclonal antibody should also be studied. Since PGE, leaks from the LM due to its interaction mainly with serum albumin [ 11 ], the PGEL derivatives such as AS013 with a higher solubility in soybean oil show less leakage [ 12]. Rat serum has a stronger enzymatic activity than human serum, and the PGE~ derivatives leaked out more rapidly from the LM in rat serum (Table 4). This suggests that enzymatic hydrolysis of the ester or acyl group increases the hydrophil~city of the derivatives and accelerates their leakage from the LM.
LM preparations are ultimately diluted thousands of times after administration, with the dilution progressing gradually from the injection site. In addition, the encapsulated drugs are delivered to the target lesions rapidly. In c,,t previous study, LM were detected in "-he vascular walls by electron microscopy 5 lnin after their intravenous injection [7]. Therefore, the experiments on the leakage of the actual ingredient from the LM performed in the present study are expected to reflect the fate of LM preparations in the human body. AS006 and AS013 caused less skin irritation than PGEz, with their skin irritating potency being 1/25 and 1/50, respectively, of that of the parent compound. Skin irritation is one of the disadvantages in the clinical application ofPGE~, so AS006 and AS013 might also be useful as topieal or oral drugs.
Conclusions 38-9-O-butyryl prostaglandin F~ butyl ester (ASOI3) is a prodrug of PGE, which is readily hydrolyzed to the parent compound in human serum. It was shown to be chemically stable in lipid microspheres (LM), was efficiently retained in the LM, and caused only minimal skin irritation. An LM preparation of AS013, LMAS013, was therefore thought to be a potentially excellent LM-PGE, preparation. Since the clinical efficacy oflipo-PGE~, although the active ingredient was retained in the LM far less effectively than with LM-AS013, was more than 10 times greater than that of ordinary PGE~ [4], LM-AS013 would be expected to show a much greater efficacy in various clinical applications.
Acknowledgment The authors wish to thank Dr. S. Kawai and Dr. D. Mcquire for their helpful advice on the preparation of this manuscript.
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