Distribution of lipid microsphere preparations

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Distribution

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20 (1996)

of lipid microsphere

R. Igarashi”, M. Takenaga, Institute

of Medical

Science,

147-154

St. Marianna

University.

School

preparations

T. Matsuda of Medicine.

Kawasaki

216. Japan

Abstract

Lipid microspheres (LM) are a superior carrier for use as a drug delivery system (DDS) due to their high stability and safety. The distribution of LM and the incorporated drugs was investigated in vitro and in vivo. An in vitro study showed that LM were taken up by activated macrophages, neutrophils, vascular endothelial cells, tumor cells, hepatic Kupffer’s cells and splenic macrophages. LM accumulated in the vascular lesions of spontanously hypertensive rats after intravenous administration. Moreover, “‘“Tc-labeled LM accumulated in the inflammatory lesions of patients with rheumatoid arthritis or in the vascular lesions of patients with arteriosclerosis obliterans. Confocal laser imaging showed that PGE, incorporated into LM targeted the vascular lesions in a patient with Buerger’s disease after intravenous injection. Preparations in which drugs are incorporated into LM show a more potent pharmacological activity than the parent compounds because of the targeting effect of LM. Keywords: Lipid microsphere system (DDS)

(LM); Distribution;

Drug carrier: Targeting;

Oil/water

emulsion:

Drug delivery

Contents 1. Introduction.. ........................................................................................................................................................................ 2. LM and liposomes.. ............................................................................................................................................................... 3. Pharmacokinetics of LM ....................................................................................................................................................... 3.1. In vitro studies ............................................................................................................................................................... 3.2. In vivo studies ................................................................................................................................................................ 3.3. In vivo studies in humans [12]. ....................................................................................................................................... 4. Lip0 preparations .................................................................................................................................................................. 4.1. Lipo-PGE, ......................................................................................................................................... 4.2. Lipo-PGI, [17]. .............................................................................................................................................................. 4.3. Lipo steroid [20] and lipo NSAID [21] .......................................................................................................................... 4.4. Lipo TXAz receptor antagonist inhalant [22] ................................................................................................................ References .................................................................................................................................................................................

1. Introduction Lipid microspheres (LM) with a mean diameter of 0.2 pm can be produced from soybean oil and egg yolk lecithin (Fig. 1). Mizushima et al. *Corresponding

author.

0169-409X/96/$32.00 @ SSDI 0169-409X(95)00125-5

1996 Elsevier

Science

B.V. All rights

147 147 148 149 149 149 150 150 IS1 152 152 153

[l] have used LM as a carrier for drug targeting. Preparations in which a drug is incorporated into LM are known as lipo preparations. Some lipo preparations show a much more potent pharmacological activity than the parent drugs due to the targeting effect of LM. Three lipo preparations, lipo steroid (1988), lipo prostaglandin E, reserved

148

various

+o.z-0.3um

A

lipid

+

water

between

lipid microspheres

(PGE,) (1988) and lipo non-steroidal anti-inflammatory drug (1992), are now available for clinical application. Furthermore, lipo prostaglandin I, (PG12) and lipo antitumor agents are now under investigation. In this study, we compared LM with liposomes, a well-known drug carrier, and assessed the pharmacokinetics of LM as well as those of the drugs incorporated in lipo preparations.

2. LM and liposomes Liposomes have been used as a carrier for various drugs [2-41. including the use for gene Table I Comparison

of lipid microspheres

phase

(buffer,

etc.)

Li posomes

microsphere

Fig. I. Differences

lipids

and liposomes.

transfer into target cells [5,6]. Fig. 1 shows the structures of LM and liposomes, while Table 1 shows the similarities and differences between LM and liposomes. LM have a single lipid membrane surrounding soybean oil. Consequently, drugs can only be incorporated into LM if they are soluble in soybean oil or retained within the lipid membrane. Therefore. water-soluble drugs are almost impossible to incorporate into LM. which leads to limitations as a drug carrier. In contrast, liposomes have the disadvantages of instability and toxicity but can carry watersoluble drugs. LM have been administered in nutritional preparations such as Intralipos@ and most pr-oblems related to their practical applica-

and liposomes Lipid microspheres

Liposomes

Components Lipid membrane

Soybean oil, Lipid. Water Monolayer single membrane

Emulsion form Incorporahle drugs

O/W Compounds soluble in soybean oil and retainable in lipids. 200 - 300 ,,“I Safe Clinically used as mtravenous nutrition in 100 ml doses Stable for 24 months at room temperature. Lip0 preparations are stored at 4°C Suitable for mass production but a high pressure apparatus is necessary.

Lipid. Water Mono- or multi-layer double membrane WlOlW Water soluble compounds and compounds retainable in lipids. Various sizes Liposomes made of some lipids are toxic.

Particle diameter Safety in viva

Stability

Large-scale production

Rather

unstable

A special apparatus is not necessary at the research level. An apparatus for mass production has recently been developed.

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Drug Delivery Reviews 20 (1996) 147-154

149

tion for clinical use (including stability, safety, large-scale production and sterility) have already been solved. Only a very short time has elapsed between development of the concept of lipo preparation and their clinical application, mainly because LM were already proved to be a safe carrier for drug targeting.

3. Phamacokinetics

of LM

Pharmacokinetic factors have an important influence on the use of LM as a drug carrier in drug delivery systems (DDS). 3.1. In vitro studies Electron microscopy revealed that LM were taken up by endocytosis into activated inflammatory cells such as macrophages and neutrophils, vascular endothelial cells, tumor cells such as MM46 cells, Kupffer’s cells in the liver, and spleen macrophages [7,8]. Fluorescent isothiocyanate-labeled LM were shown to be taken up by vascular endothelial cells and activated T lymphocytes [9,10] using flow cytometry. 3.2. In vivo studies Five min after lipo-PGE, was injected into spontaneously hypertensive rats (SHR, 20 weeks old), the thoracic aorta was fixed and examined by electron microscopy. SHR have several vascular abnormalities, including thickening of intima and subendothelial accumulation of fibrous components. LM penetrated through endothelial cell gaps in aortic intimal lesions and many LM accumulated in the subendothelial space (Fig. 2)

Fig. 2. Electron micrograph of an aortic intimal lesion in a 20-week-old SHR at 5 min after lipo-PGE, injection. Many LM are observed in the subendothelial space. Arrows show the LM ([ll]).

In the AS0 patients, LMs were observed at sites of suspected arteriosclerosis from the ascending to descending aorta as well as the abdominal aorta and common illiac artery (Fig. 3a and 3b). LMs are an appropriate carrier for targeting drugs towards the sites mentioned above, namely inflammatory lesions, vascular lesions, tumor cells and reticuloendothelial cells. Therefore. lipo praparations of prostaglandins, anti-inflammatory agents and antitumor agents have been studied. However, when lipo preparations are administered in vivo, not all of the contents remain with the carrier LM and a moderate amount of the drug actually escapes. The rate of drug escape from the carrier LM mainly depends on its lipophilicity and the amount incorporated. Therefore, the pharmacokinetics of each ingredient incorporated in LM must be determined. The requirements for excellent lipo preparation are as follows:

[ill. 3.3. In vivo studies in humans [12] ‘““Tc-labeled LM were intravenously administered to patients with arteriosclerosis obliterans (ASO) or rheumatoid arthritis (RA). Subsequent scintigraphy showed that 99mTc-labeled LM accumulated in the reticuloendothelial system (including the liver and spleen) and in the inflammatory lesions of RA patients. The lesions with accumulation were those with pain and swelling.

Drugs incorporated into LM must have a good pharmacologic activity (if the agent is a prodrug for more stability or better lipid solubility, it must transform into an active form in the lesions). The required dose of the drug must be dissolvable in soybean oil. The amount of drug loss from the LM after administration in vivo must be small. Release of the actual ingredient from LM leads to a decrease in the targeting effect of the drug

I so

R. lgurashi

et al.

I

Advanced

Drug

Delivery

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20 (19%)

147-1.54

AS0

(K.K.

72 y.o.1

LM in patients with rheumatoid arthritis. Scintigrams were obtained 1 h after the Fig. 3. (a) Scintigraphy with “““Tc-labeled LM. Accumulation in the articular lesions is observed [12]. (b) Scintigraphy with intravenous injection of “““‘Tc-labeled “““Tc-labeled LM in a patient with arteriosclerosis oblitcrans. A general image obtained 1 h after intravenous injection 01 “‘“‘Tc-labeled LM. Accumulation was observed in the arteriosclerotic lesions [12].

carrier. so it is important drug loss.

to minimize

such

4. Lip0 preparations Fig. 4 shows the compounds that have been used as lipo preparations. The method of producing lipo preparations is outlined in Table 2. 4.1. Lipo-PGE, Prostaglandins have various physiological actions that are clinically useful. However. a short half-life means that high doses are required which leads to adverse effects and to pain at the injection site. PGE, (a vasodilator and anti-

platelet agent) was incorporated into LM so as to provide a targeting effect and inhibit the inactivation of this prostaglandin in the lungs. A study using [‘HI-labeled lipo-PGE, showed accumulation in the thoracic aorta in SHR after intravenous injection (Fig. 5) [ll]. Also, after the intravenous injection of lipo-PGE, in a 60-yearold woman with Buerger’s disease and circulatory disturbance in the legs, a vascular lesion was removed and scanned with a confocal laser imaging system after immunostaining. It was found that PGE, had targeted the vascular lesion (Fig. 6). In clinical use, a smaller dose of PGE, in lipo-PGE, had a remarkable effect [13.14] compared to the conventional peripheral circulation improving agents or PGE, cyclodextrin clathrate. Lipo-PGE, has been used for

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m

(n=4)

lipo

[‘HlPGEr

(n=3)

Fig. 5. Vascular distribution of lipo-[3H]PGE, and [3H]PGE, in 20-week-old SHR at 5 min after intravenous injection [l I]. Mean? S.E.. ** p CO.01 (vs [‘HIPGE,).

Table 2 Production

of lip0 preparations

Materials Incorporated drug Soybean oil Egg yolk lecithin Glycerine Distilled water Lip0 preparation

.r pg 100 mg 12- 18 mp 22 - 25 mg appropriate amount 1 ml

Fig. 6. A confocal imaging photograph of PGE, targeting a vascular lesion in a patient with Buerger’s disease. Ten min after lipo-PGE, was intravenously administered to a patient with Buerger’s disease and severe circulatory disturbance in the legs. a vascular lesion was extracted, immunostained and scanned with a confocal laser scanning fluorescence microscope. PGE, was found to have accumulated in the vascular lesion. The color bar indicates the intensity of PGE,. Arrow shows PGE,

Method 1 The drug is dissolved in soybean oil 2 Egg yolk lecithin is added and the solution is preliminarily emulsified at 12 000 rpm for 10 min. 3 Glycerine and distilled water are added. The solution is emulsified at 15 000 rpm for 20 min. 4 The solution is treated through French Press@ at 638 psi 5 times. Result Lipo particles

of 200 to 300 nm in diameter

are produced.

peripheral circulatory disturbances due to collagen diseases, diabetes, Raynaud’s disease or AS0 since 1988. Sixty percent of the total sales of prostaglandin preparations in Japan were accounted for by lipo-PGE, 1995. Drugs like prostaglandins which are highly lipid soluble and require small doses (at a microgram level) are appropriate for lipo preparations. However, PGE, in LM is rather unstable and much of the

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et al. I Advanced

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drug escapes from LM after lipo-PGE, administration, so a more stable and targetable new lipo-PGE, preparation is now under investigation (see [15]). The new lipo-PGE, preparation employs a chemically stable prodrug of PGE, (AS013) that is highly lipid soluble, and is expected to be better retained by LM and be more targetable. It has been already proved to be effective in preliminary clinical trials [ 161.

lesions and the spleen in rats with carrageenin foot edema. Lipo steroid was first applied clinically in 1988 for RA patients. Lipo NSAID was the first NSAID preparation available for intravenous injection. It was released in 1992 and is used postoperatively as well as to alleviate cancer pain.

4.2. Lipo-PGI,

1221

[ 171

Prostacyclin (PGI,) has a strong anti-platelet action and it is expected to have an antithrombotic effect. However, its chemical instability and adverse effects (including facial flushing, palpitations, and hypotension, have hindered clinical application [ 18,191. Accordingly the development of a stable derivative has been anticipated. Isocarbacyclin methyl ester, a chemically stable PGI, analogue, has been incorporated into LM. As the lipophilicity of isocarbacyclin is high and it is very soluble in soybean oil, an LM preparation of isocarbacyclin is stable. When lipo-isocarbacyclin is incubated with human serum, the drug is largely retained in LM, suggesting that lipo-isocarbacyclin is an excellent lipo-PGI, preparation. A pharmacological study in a hamster cheek pouch model showed that lipo preparation is 500 times more potent than free isocarbacyclin, so this agent is expected to be clinically effective at low doses. It is now under going clinical trials. 4.3. Lipo steroid [20] and lipo NSAZD [21] Lipo steroid was the first lipo preparation. Corticosteroid and non-steroidal anti-inflammatory drugs (NSAID) were investigated for use as lipo preparations. because LM are taken up by vascular endothelial cells, the spleen, inflammatory lesions, macrophages and neutrophils. Dexamethasone palmitate (corticosteroid) and flurbiprofen axetil (NSAID) were used because they were highly lipid soluble and pharmacologically active. A study using [“HI-labeled dexamethasone palmitate showed that the drug incorporated into LM was targeted to inflammatory

4.4. Lipo TXA, recqmr

untqonist

inhalant

The possibility of using a lipo-TXA, receptor antagonist as an inhalant preparation was also studied. S-1452 is a TXA, receptor antagonist [23], with a potent pharmacologic activity [24,25]. After guinea pigs inhaled lipo-[ “C]S-145-Me or [‘“CIS-1452, via a nebulizer. the distribution of radioactivity in the airway tissues was determined (Fig. 7). [‘4C]S-145-Me showed more accumulation in the lung tissues than [‘4C]S1452. suggesting that LM allowed the drug to move deeper into the airways. Moreover. the bronchodilatory effect of lipo S-145-Me was three times stronger than that of S-1452 when administered as an aerosol. Neutrophils and eosinophils are known to infiltrate into inflammatory lesions in patients with allergic diseases [26-281. Lipo S-145-Me was taken up 7-fold more by neutrophils and 3.5-fold more by eosinophils than S-1452. This remarkable uptake was suggestive of the uptake in animals administered with lipo preparations by inhalation and the significant increase of the pharmacological effect. These results suggest that a lipo-TXA, receptor antagonist inhalant may be useful for asthma. In this report. we concentrated mainly on the distribution of LM and their ingredients. Better targeting through binding antibodies to the surface of LM or changing the lipid composition and how to incorporate non-lipid-soluble drugs into soybean oil are problems still to be studied. In addition, utilization of the LM membrane as an artificial cell membrane or application of LM to deliver genes into target cells by changing the charge on the membrane surface are issues under investigation.

R. lgarashi et al. I Advanced Drug Delivery Reviews 20 (1996) 147-154

153

(dpm x 1 O- 3/tissue)

radioactivity

10

5 trachea (upper)

(upper) (lower)

trachea (lower) bronchi

0 Fig. 7. Distribution mean radioactivity

of lipo[‘“C]S-145.Me determined in three

Lip0 [ ’ 4C&l

and [‘JC]S-1452 animals is shown.

n

45Me

[‘~cIs-~ 452

in the airway tissues after administration for 30 min by aerosol. Airway tissues were cut as illustrated [22].

LM have proved to be stable and safe, and are more useful for clinical application as a DDS.

PI

Von der Leyen, HE., Gibbons, GH.. Morishita. R., Lewis, NP., Zhang, L., Nakajima, M.. Kaneda, Y.. Cooke, J.P. and Dzau, V.J. (1995) neointimal vasucular lesion:

References

The

Gene therapy in vivo transfer

thehal cell nitric oxide synthase Sci. USA 92(4), 1137-1141.

gene.

inhibiting of endo-

Proc. Nat]. Acad.

[71 Shoji, Y., Mizushima. [II Mizushima,

Y. and Igarashi, R. (1991) Lipid microspheres and lecithinized polymer and drug delivery systems. In: (Dunn and Ottenbrite Eds.) Polymeric Drugs and Drug Delivery Systems. 267-274, American Chemical Society. PI Gregoriadis, G. and Ryman, B.E. (1971) Liposomes as carriers of enzymes or drugs: a new approach to the treatment of storage disease. Proc. Biochem. Sot. 124, 58. 131 Gabizon, A.. Catane, R.. Uziely, B., Kaufman, B., Safra, T.. Cohen, R.. Martin, F., Huang, A. and Barenholz, Y. (1994) Prolonged circulation time and enhanced accumulation in malignant exudates of doxorubicin encapsulated in polyethylene glycol-coated liposomes. Cancer Res. 54(4). 987-992. L.L. and Saravolac, E.G. (1994) ]41 Wong, JP., Stadnyk, Enhanced protection against respiratory influenza A infection in mice by liposome-encapsulated antibody. Immunology 81(2). 280-284. H. and I51 Kato. K., Yoneda, Y., Okada, Y., Kiyama, Shiosaka, S. (1994) Gene transfer and the expression of a foreign gene in vivo in post-mitotic neurons of the adult rat brain using the hemagglutinating virus of the Japan-liposome method. Mol. Brain Res. 25(334). 3S9363.

Y., Yanagawa, A. and Yonaha, T. (1985) Electron microscopic studies on tissue distribution of lipid microspheres used as drug delivery carriers. Drugs Exptl. Clin. Res. 11, 601-609.

PI Shoji. Y., Mizushima.

Y. and Kametani. T. (1986) Affinity of lipid microshere for MM46 tumor cells. Jpn. J. Pharm. Sot. 106. 605-608. K., Matsuki, Y., Kawakami. M.. [91 Suzuki. K., Takahashi, Kawaguchi, Y., Hidaka, T.. Sekiyama. Y., Mizukami, Y.. Kawagoe. M. and Nakamura, H. (1992) Fluorescent probe-labeled lipid microsphere uptake by human cndothclial cells: A flow cytometric study. Jpn. J. Pharmacol. 60, 349-356.

[lOI Suzuki, K. Activated

CD4 + T cells preferentially take up lipid microspheres but resting cells do not. Clin. Exp. Immunol. in press. S.. Kiyokawa, 1111 Mizushima, Y., Hamano, T., Haramoto. A.. Yanagawa, K., Nakura. K.. Shintomc, M. and Watanabe. M. (1990) Distribution of lipid microspheres incorporating prostaglandin E, to vascular lesions. Prostagl. Leukotr. Essent. Fatty Acids 41. 2699272. S., 1121 Kiyokawa. S.. Igarashi, R., Iwayama, T., Haraxmoto, Matsuda, T., Hoshi. K. and Mizushima. Y. (1987) ““mT~labeled lipid microspheres would be useful for an imaging study of those diseases. Jpn. J. Inflamm. 7. SSl-557.

[l-i] Mizushima. Y. ( 19Yl) Lipo-proataglandin preparation. (Review,) Prostagl. Leukotr. &sent. Fatty Acids. 42. I-6. [ 141 Mizushima. Y.. Siokawa. Y.. Homma. M.. Kashiwazaki. S.. Ichikawa. Y.. Hashimoto. H. and Sakuma A. (lYX7) A multicenter double blind conti-olled study of lipo PGE,. PGE, incorporated in lipid microsphcrcs. in peripheral vascular disease secondary to connective tissue disodcrs. J. Rheumatology 14( I ). 977101. [ 151Igarashi. R.. Mizushima. Y.. Takcnaga, M.. Matsumoto. K.. Morizawa. Y. and Yasuda. A. (lYY2) A stable PGF, prodrug for targeting therapy. J. Controlled Releaqc 20. 37-46. [16] Mizushima. Y. and Hoshi. K. ( 1903)Recent advances in lipid microsphere technology for targeting prostaglandin delivery (Review). J. Drug Targeting I. 9.1~100. [ 171 Mizushima. Y.. lgarashi, R.. Hoshi. K., Sim. A. K.. Cleland. M. E.. Hayashi, H. and Goto, J. (lYX7) Marked enhancement in antithrombotic activity of isocarhacyclin following its incorporation into lipid microspheres. Proxtaglandins 33(2). IhlllhX. [IX] Grosc. R.. Greenhcrg. M.. Strain. J. and Mucllcr. H. (19X5) Intracoronar-y prostacyclin in evolving acute mvocardial infarction. Am. J. Cardiology 55, 1625-1626. [IY] Gryglcwski. R.J. (1985) Effects of prostacyclin in atherosclerotic v~escular discasc. Adv. Prostagl. ‘l’ronboxane Lrukotr. Rcs. IS. 539-542. [20] Mizushima. Y.. Hamano, 7‘. and Yokoyama. K. (10x2) Tissue distribution and anti-intlammatory activity 01 corticosteroids incorporated in lipid emulsion. Ann. Rheum. Dis. 41. 263-267. 1211 Kuriyama. K.. Hiyama. Y.. Aoyama. Y.. Ichikawa. K.. Okumura. M.. Masumoto. S.. Ito. K.. Ohtaki. Y.. Hirata. M., Hanada, S.. LJchida. T.. Fujino. Y. and Watanahc. M. (1989) Pharmacological studies of a non-steroidal anagesic and antipyretic drug of LFP83. Folia Pharmacol. Japan 9.1, 61-73.

1221 Takenaga. M.. Nakagawa. T.. Igarashi. R. and MI/ushima. Y. (199.3) Application of lipid microsphcrcs to prepare a thromhoxane A, receptor antagonist aerosol for inhalation. J. drug Targeting I. 293-301, [?.3l Nnrisada. M.. Ohtani. M.. Watanabe. F.. Ilchida. K.. Arita. H.. Doteuchi. M.. Hanasaki. K.. Kakushi, H.. Otani. K. and Ham. S. (IYXX) Synthesis and in vitro activity of various derivatives of a novel thromhoxanr reccptoiantagonist. ( i )-(5Z)-7(3-endo-phenylsullonylamino[2.1.1]bicyclohept-2-cxoyl) heptenoic acid. J. Med. (‘hem. -31. 1X47-1854. 1711 Nakajima. M. and &da. M. (1989) Actions 01 a novel thromboxanc A, receptor antagonist. S-145. on isolated monkey and cat arteries. J. C‘ardiovasc. Pharmacol. 14, .502-SOY. 1251 Hori, Y.. Hatakeyama, H., Yamada, K. and Kurosawa. A. ( 1989) Effect of a novel thromhoxanc A, receptor antagonist. S-145, on collagen-induced ECG changes and thrombocytopenia in rodents. Jpn. J. Pharmacol. 50.

I ‘JS-30. [:h] Aktnsu. I.. Fukuda. ‘1.. Majima. ( lYY2) Inhibitory cffcct of inhaled

K. and Makino, S. FK-506 on increased bronchial responsiveness and cosinophil infiltration in the airway mucosa. Jpn. J. Allergy 41. 543-546. ]?7] Az/awi. M.. Bradley. B.L.. Jeffery. P.K.. Frew. A.J., Wardlaw. A.J.. Knowles. G.. Assouf. B., Collins. J.V., Durham. SK. and Kay. A.B. (1990) Identification of activated T-lymphocytes and eosinophils in bronchial hiopsies in stable atopic asthma. Am. Rev. Respir. Dis. I-I?. 1407~-l413. 12x1 Bcntlcy. A.M.. Maestrelli. P.. Sactta. M.. Fabbri. L.M., Robinson. D.S.. Bradley, B.L.. Jcffcry. P.K., Durham. S.R. and Kay. A.B. (19Y2) Activated T-lymphocytes and cosinophils in the bronchial mucosa in iaocyanate-induccd asthma. J. Allergy Clin. Immunol. 89, 821-829. (Review concerning lipid microsphere preparations)