Journal of Vimlogical Mefhods, 19 (1988) 121-130 Elsevier

121

JVM 00683

Pharmacokinetics and chemotherapeutic efficacy of adriamycin encapsulated in immunoliposomes

against avian myeloblastosis virus infection

K.V.R. Dhananjaya1.2 and A. Antony’

‘Turnour Biology Laborato~, Fred ~~tchj~son Cancer Research Center, Sent&, W~hi~gton, U.S.A.; ~M~crob~o~ogy and Cell Siology Laboratory, lndian Institute of Science, Bangalore, India

(Accepted 19 November 1987)

Summary

Immunoliposomes were prepared using rabbit anti-AMV gp80 IgG for the tar- geted chemotherapy of avian myeloblastosis virus infection. Adriamycin was en- capsulated into immunoliposomes and used for in vivo studies. Comparative phar- macokinetics of free drug, drug encapsulated in free hposomes and of drug encapsulated in immunoliposomes in the virus-infected cells revealed that (i) the drug encapsulated in liposomes was cleared from the plasma slowly, and (ii) the drug encapsulated in immunoliposomes accumulated in the target tissue, the bone marrow, 5- and W-fold more than the drug encapsulated in free liposomes and free drug, respectively. The drug encapsulated in immunoliposomes inactivated the virus and exhibited more chemotherapeutic efficacy as compared to controls when injected up to 24 h post-infection. However, when injected 48 h post-infection the drug encapsulated in immunoliposomes did not offer any protection against the vi- rus infection. There is no detectable antibody response against immunoliposomes in the infected animals.

Immunoliposomes; Adriamycin; Pharmacokinetics; Drug targeting; Avian mye- loblastosis; Virus infection

-.._

Correspondence to: K.V.R. Dhananjaya, Tumour Biology Laboratory, Fred Hutchinson Cancer Re- search Center, 1124 Columbia Street, Seattle, WA 98104, U.S.A.

Old-0934~88/~~3.50 0 1988 Elsevier Science Publishers B.V. (B~medical Division)

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Introduction

Many anticancer agents have short biological half-lives due to rapid metabolic inactivation and excretion (Mihich and Creaven. 1982). Liposome encapsulation increases the plasma lifetime and tissue retention of several anticancer drugs (Ju- liano and Stamp, 1978) and also offers chemotherapeutic advantages (Gregoriadis and Allison, 1980). However, free liposomes are not target-specific. Antibodies raised against specific cell surface antigens serve as good ligands for the targeted delivery of drugs. Several authors have coupied the cell-specific antibodies to li- posomes and used them for in vitro targeting (Huang et al., 1983; Heath et al.. 1984; Matthay et al., 1984; Connor and Huang, 1986). It was reported that im- munoliposomes were able to bind to the target cells in vivo (Wolff and Grego- riadis, 1984).

Pharmacokinetics of daunomycin (daunorubicin) and Adriamycin (doxorubicin) in several experimental animals (Bachur et al., 1970; Yesair et al., 1972; Chan et al., 1978) and in human patients (Benjamin, 1975; Lee et al., 1980) have been de- scribed. The pharmacokinetics of these drugs were altered significantly when they were encapsulated in liposomes and used in vivo (Juliano and Stamp, 1978; Rah- man et al., 1980 and 1985).

In this paper, we report the comparative studies on the plasma clearance of free drug, drug encapsulated in free liposomes and of drug encapsulated in immuno- liposomes in chicks. The chemotherapeutic efficacy of drugs encapsulated in im- munoliposomes against avian myeloblastosis virus infection in chicks is also de- scribed.

Materials and Methods

Materials

Adenosine-5’-triphosphate, cholesterol, Coomassie brilliant blue G-250, ortho- phenylene diamine, phenol red, Steeryiamine and Triton X-100 were obtained from Sigma Chemical Co., St. Louis MO, U.S.A. Sephadex G-100 was from Pharmacia Fine Chemicals, Uppsala, Sweden. Adriamycin was a gift from Dr. Federico Ar- camone, Farmitalia Carlo Erba, Milan, Italy. [“HI Adriamycin (0.54 Ciimmol) was purchased from Bhabha Atomic Research Centre, Bombay, India. Adriamycin used in some of the experiments was purchased from Calbiochem, Los Angeles CA, U.S.A. All other chemicals used were of analytical grade.

Virus BAI-A strain of avian myeloblastosis virus (AMV), originally obtained from Dr.

J.W. Beard’s laboratory, was provided by Dr. M.R. Das, Centre for Cellular and Molecular Biology, Hyderabad, India. The virus was grown in leukosis-free white leghorn chicks (Karnataka State Poultry Farm, Bangalore, India).

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Preparation of immunoliposomes and encapsulation of drugs lmmunoliposomes were prepared using phosphotidyl choline: cholesterol and

stearylamine in a molar ratio of 51:21:15 and palmitoylated rabbit anti-AMV gp80 as described by Huang et al. (1982). Adriamycin was encapsulated into immuno- liposomes as described by Shen et al. (1982).

Pharmacokinetics of Adriamycin When [3H] Adriamycin was used, the following amounts of drugs were injected

into each chick: free Adriamycin (1.07 x 10’ cpm), Adriamycin encapsulated in free liposomes (1.05 x 10’ cpm), and Adriamycin encapsulated in immunolipo- somes (0.92 x 10’ cpm). The radioactivity in the tissue or plasma was determined using a scintillant containing Triton:Toulene: 0.1 N HCl (1:6:1) in a LKB rackbeta counter. For plasma clearance kinetics, the amount of drug present in the sample at a given time point was compared to that present in the sample isolated 2 min after injection and the percentage was calculated.

In vivo toxicity of drugs Various concentrations of drugs in 0.1 ml of PBS were injected intravenously

into chicks. In each group 6 chicks were used. The birds were observed for toxic symptoms such as: necrosis, induration and fibrosis, atrophy of the injected wings and haemorrhages in viscera for one month.

ATPase assay for virus ATPase assay was performed by the spectrophotometric method described by

Beaudreau and Becker (1958). The enzyme activity was detected by monitoring the change in absorbance at 550 nm of the substrate indicator system (60 mg di- sodium ATP, 0.05 M KCl, 0.14 mM phenol red, 0.04 M MgCl,, and 5 PM Na- HC03, pH adjusted to 7.5 with 0.2 N NaOH) after mixing with plasma samples. One unit of enzyme activity is defined as that amount which brings about change of 0.05 unit OD 550 nm. One unit of enzyme activity corresponds to 4.57 x 10’ virus particles (Beaudreau and Becker, 1958). Plasma samples from uninfected chicks were used as controls.

Analysis of anti-rabbit antibodies in chicks The y-globulins from chicken serum were prepared as described by Williams and

Chase (1967). The precipitate was dissolved in 100 ~1 of 50 mM Tris-HCl, pH 8.0 and spun through Sephadex G-100 column (1 ml) at 1000 X g for 10 min. The y- globulin sample was collected and used for sandwich ELISA. The y-globulins (20 kg/well) in 0.1 M sodium carbonate buffer, pH 9.5, were coated to ELISA plate at 37°C for 2 h. The plates were washed with 50 mM sodium phosphate buffer, pH 7.2 containing 0.8% NaCl and 0.05% Tween-20. Rabbit IgG (50 kg/well) was then added and incubated at 37°C for 3 h. The wells were washed extensively with the same buffer and incubated with goat anti-rabbit IgG-HRPO conjugate at 37°C for 3 h. The wells were washed with 50 mM sodium phosphate buffer, pH 7.2, con- taining 0.8% NaCl and a 0.05% Tween -20. The plates were developed using a 100

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Time (n)

Fig. 1. Plasma clearance kinetics of (‘HI Adriamycin A free drug n drug encapsulated in free liposomes l drug encapsulated in immunoliposomes

~1 substrate solution consisting of 0.4 mg orthophenylenediamineiml and 0.01% hydrogen peroxide in 0.1 M citrate phosphate buffer, pH 5.0. The reaction was arrested by adding equal volume of 4 N Hz Sod. The absorbance was measured in

a Biotek ELISA reader.

Effect of Adriamycin on AMV infection in chicks In order to test the effect of the drugs, two sets of experiments were carried out.

In the first set of experiments the chicken plasma containing AMV (1.2 x 10’ par-

ticles) was incubated with various preparations of drugs at 37°C for 1 h, before injecting into one-day-old chicks. In the second set, the drugs were injected intra- venously into chicks, at various time intervals after infecting with virus (1.2 x 10’ particles). The chicks were examined for one month for symptoms of AMV infec- tion (Beard, 1956). The plasma samples from surviving and moribund chicks were analysed separately for ATPase activity. The presence of anti-rabbit chicken y- globulins was analysed by sandwich ELISA as described above.

Results

In vivo toxicity of drugs The chicks were injected with Adriamycin in the range of 5 mg - 35 mgikg body

weight. The LD,, for Adriamycin was found to be 28 mg/kg body weight.

Plasma clearance kinetic of drugs Free drug had a plasma half-life of 10 min whereas the drug encapsulated in li-

posomes had a half-life of 1 h (Fig. 1). The drug encapsulated in immunolipo- somes exhibited more plasma half-life. The clearance of liposome-encapsulated drug

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TABLE 1

Inactivation of AMV by Adriamycin

Treatment

Number of birds surviving on day

18 30

Virus 016

Anti-AMV gp80 antibody (1 mg) 116

Free Adriamycin (150 kg) 216

Adriamycin encapsulated in free liposomes (150 kg) 316

Adriamycin encapsulated in immunoliposomes (100 516

CLg) Adriamycin encapsulated in immunoliposomes (150 616

I%)

016

116

l/6

116

316

416

from the plasma was biphasic. The liposome-encapsulated drugs were retained in plasma for longer periods than the free drug.

Inactivation of AMV by Adriamycin encapsulated in immunoliposomes In the case of infected chicks there was a marked increase in the number of

myeloblasts. Congestion and marked increase in the sizes of liver, spleen, kidney and bursa of Fabricius as a consequence of AMV infection were observed.

10 20 30 10 20 30

Days Days

Fig. 2. Effect of Adriamycin on AMV infection in chicks

(A) Simultaneous injection of drug (600 kg) and virus

o free drug

n drug encapsulated in free liposomes

0 drug encapsulated in immunoliposomes

(B) Controls

l chicks without virus infection

n chicks infected with virus x chicks infected with virus and empty immunoliposomes are injected

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The virus was preincubated separately with Adriamycin, Adriamycin encapsu- lated in free liposomes, and Adriamycin encapsulated in immunoliposomes and in- jected into chicks. The results of their effects on the virus multiplication in vivo are shown in Table 1. The drug encapsulated in immunoliposomes was able to in- activate the virus to a significant extent as compared to free drug. When 150 pg of Adriamycin encapsulated in immunoliposomes was used for inactivation, 416 (67%) birds survived. On the other hand, when free drug at the same concentra- tion was used, l/6 (17%) birds survived (Table 1).

100

80

20

1 II

0 I L4 I I 1 1 16 1

10 20 30 10 20 30

Days Days

Fig. 3. Effect of Adriamycin on AMV infection in chicks

Drug (600 pg) was injected at (A) 16 h (B) 24 h (C) 48 h (D) 72 h post-infection.

q free drug A drug encapsulated in free liposomes o drug encapsulated in immunoliposomes

Effect of targeted drugs on AMV infection in vivo Adriamycin encapsulated in immunoliposomes was more effective over free drug

and drug encapsulated in free liposomes. Simultaneous injection of virus and drug encapsulated in immunoliposomes into chicks has given 85% protection from AMV infection (Fig. 2). When Adriamycin encapsulated in immunoliposomes was in- jected at 16 and 24 h post-infection, 67% and 50% of the chicks survived respec- tively (Fig. 3A and B). However, when Adriamycin encapsulated in immunoli- posomes was injected at 48 h post-infection, 17% of the birds survived (Fig. 3C). At 72 h post-infection they did not offer any significant protection. However, the survival period increased by a few days (Fig. 3D). Empty immunoliposomes when injected at any of the time points after infection did not show any effect (Fig. 2B). This clearly showed the effect of drugs encapsulated in immunoliposomes on AMV infection in vivo. The results of sandwich ELISA showed no detectable antibody production against immunoliposomes in the infected chicks (data not shown).

Discussion

Liposomes are used as carriers of drugs in experimental cancer chemotherapy (Gregoriadis, 1976 and 1977). In this paper we are reporting the results of targeted chemotherapy of AMV infection in chicks. It is important to understand the site of drug delivery in targeted chemotherapy. Rahman et al. (1980, 1985) have shown that encapsulation of Adriamycin in positively charged liposomes reduced the acute cardiotoxicity due to the drug.

Positively charged liposomes remained in circulation for longer time (Juliano et al., 1978). Methotrexate encapsulated in positively charged liposomes was more effective against transplanted tumours (Kimelberg and Atchison, 1978). Vasude- vachari and Antony (1985) reported that positively charged liposomes are better carriers of antiviral agent, cupric-INH complex. The pharmacokinetics of dauno- mycin, actinomycin D, cytosine arabinoside and vinblastine (Juliano and Stamp, 1978) were altered significantly when they were encapsulated in liposomes. In the present investigation, it was shown that the free drug is cleared rapidly from plasma. These results are in agreement with the data reported by Yesair et al. (1972) and Juliano and Stamp (1978). The clearance of liposome-encapsulated drugs from the plasma was found to be slow and biphasic which correlated with the results re- ported by Juliano and Stamp (1978) and Wolff and Gregoriadis (1984). As com- pared to free drug and drug encapsulated in free liposomes, the drug encapsulated in immunoliposomes reached the target tissue, the bone marrow, in increased amount (data not shown). The accumulation of immunoliposomes in the target tis- sue has been reported (Singhal and Gupta, 1987). These results suggest the effi- cacy of liposome-targeting over other means of drug delivery.

The drug encapsulated in immunoliposomes inactivated the virus, when prein- cubated before the injection, better than the free drug and exhibited more chem- otherapeutic efficacy. Up to 24 h post-infection drug encapsulated in immunoli- posomes offered better protection than the controls. However, these

128

immunoliposomes were found to be ineffective when injected 48 h post-infection. Recently, the inhibition of transplanted bovine leukaemia tumour growth in mice by immunoliposomes carrying Adriamycin has been reported (Onuma et al., 1986). In the present studies no detectable antibody response against immunoliposomes in chicks was observed as analysed by sandwich ELISA. It was reported that re- troviral infection leads to immunosuppression in the host (Bendinelli et al., 1985). Immunosuppression was observed in patients with AIDS (Gallo et al., 1984; Mon- taigner et al., 1984). The lack of response against immunoliposomes may be due to immunosuppression by virus infection.

Recent studies have shown that pH-sensitive (Connor and Huang, 1986), heat- sensitive (Sullivan and Huang, 1986), and target-sensitive immunoliposomes (Ho et al., 1986) are better carriers of drugs. These novel liposomes may be useful in the chemotherapy of viral infection in vivo.

Acknowledgements

This work is supported by a grant from the Department of Science and Tech- nology, Government of India. KVRD is thankful to Indian Institute of Science for a fellowship.

References

Bachur, N.R., Leon Moore, A., Bernstein, J.G. and Liu A. (1970) Tissue distribution and disposition of daunomycin (NSC-82151) in mice. Fluorimetric and isotopic methods. Cancer Chemother. Rep.

Part 1, 54, 89-94.

Beard, J.W. (1956) Virus of avian myeloblastic leukosis. Poultry Sci. 35, 203-223.

Beaudreau, G.S. and Becker, C. (1958) Virus of avian myeloblastosis X. Photometric microdetermi- nation of adenosine triphosphatase activity. J. Natl. Cancer Inst. 20. 339-349.

Bendinelli, M., Matteucci, D. and Friedman, H. (1985) Retrovirus induced acquired immunodeficien- ties. Adv. Cancer Res. 45, 125-1-81. Academic Press, New York.

Benjamin, R.S. (1975) Clinical Pharmacology of Adriamycin (NSC-123127) Cancer Chemother. Rep.

Part 3, 6, 183-185.

Chan, K.K., Cohen, J.L., Gross, J.F., Himmelstein, K.J.. Bateman, J.R.. Lee. Y.T. and Marlis, A.S.

(1978) Prediction of adriamycin disposition in cancer patients using a physiologic and pharmaco- kinetic model. Cancer Treat. Rep. 62, 1161-1171.

Connor, J. and Huang, L. (1986) pH-sensitive immunoliposomes as an efficient and target specific car- rier for antitumour drugs. Cancer Res. 46, 3431-3435.

Gallo, R.C., Essex, M.E. and Gross, L. (Eds) (1984) Human T Cell LeukaemiaiLymphoma Viruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 437.

Gregoriadis, G. (1976). The carrier potential of liposomes in biology and medicine. N. Engl. J. Med.

295, 765-770. Gregoriadis, G. (1977) Targeting of drugs. Nature 265, 407-410. Gregoriadis, G. and Allison, A.C. (Eds) (1980) Liposomes in Biological Systems. John Wiley and Sons

Ltd., Chichester, U.K. Heath, T.D., Bragman, K.S., Matthay, K.K., Lopez-Straubinger, G. and Papahadjopoulos, D. (1984)

Antibody directed liposomes: the development of a cell specific cytotoxic agent. Biochem. Sot. Trans.

12. 340-342.

129

Ho, R.J.Y.. Rouse, B.T. and Huang, L. (19X6) Target sensitive immunoliposomes: preparation and

characterization. Biochemistry 25, 5500-5506.

Huang. A., Tsao. Y.S. and Kennel, S.J. (1982) Characterization of antibody covalently coupled to Ii-

posomes. Biochim. Biophys. Acta 46. 140-150.

Huang, A.. Kenel, S.J. and Huang, L. (19X3) Interactions of immunoliposomes with target cells. J.

Biol. Chem. 25, 14034-14040. Juliano. R.L. and Stamp, D. (1978) Pharmacokinetics of liposome encapsulated antitumour drugs.

Studies with vinblastine, actinomycin D, cystosine arabinoside and daunomycin. Biochem. Pharm.

27, 21-27. Juliano. R.L.. Stamp, D. and McCullough. N. (1978) Pharmacokinetics of liposome encapsulated anti-

tumour drugs and implications for therapy. Ann. N.Y. Acad. Sci. 308. 411-425.

Kimelberg, H.K. and Atchison, M.A. (1978) Effect of experiment in liposomes on disposition, de-

gradation and effectiveness of methotrexate in vivo. Ann. N.Y. Acad. Sci. 308, 395-410.

Lee. Y.N., Chan, K.K., Harris, P.A. and Cohen. J.L. (1980) Distribution of Adriamycin in cancer

patients. Tissue uptakes plasma concentration after IV and IA administration. Cancer 45, 2231-2239.

Matthay. K.K.. Heath, T.D. and Papahadjoupoulos, D. (1984) Specific enhancement of drug delivery

to AKR lymphoma by antibody targeted unilammelar vesicles. Cancer Res. 44, 18X0-1886.

Mihich, E. and Creaven, P.J. (1982) In: ‘Cancer Medicine’ (eds. Holland, J.F. and Frei. E.) p. 650. Lea and Febiger, Philadelphia.

Montaigner, L.. Gruest, J.. Chararet, S., Dauguet, S., Axler, C., Guetard, D., Nugeyre, M.T., Barre-

Sinnoussi, F.. Chermann, J.C., Burnett, J.B.. Klatzmann, D. and Gluckmann, J.C. (1984) Lymph-

adenopathy associated virus infection of a blood donor-recipient pair with Acquired Immunodefi-

ciency Syndrome. Science 225. 63-66.

Onuma, M., Odawara, T., Watarai. S., Aida. Y., Ochiai, K., Syuto. B., Matsumoto, K., Yasuda, T., Fujimoto. Y., Izawa, H. and Kawakami. Y. (19X6) Antitumour effect in liposomes conjugated with

monoclonal antibody against tumour associated antigen of bovine leukaemia cells. Jpn. J. Cancer

Res. (Gann) 77, 1161-1167.

Rahman, A., Kesseler, A., More, N., Sikic. B., Rowden. G.. Wooley, P. and Schein, P.S. (1980) Li-

posomal protection of adriamycin induced cardiotoxicity in mice. Cancer Res. 40, 1532-1537.

Rahman. A., White, G., More, N. and Schein, P.S. (19X5) Pharmacological, toxicological and thera-

peutic evaluation in mice of doxorubicin entrapped in cardiolipin liposomes. Cancer Res. 45, 796-803.

Shen, D.-F.. Huang, A. and Huang, L. (1982) An improved method for covalent attachment of an- tibody to liposomes. Biochim. Biophys. Acta 689, 31-37.

Singhal. A. and Gupta, C.M. (1986) Antibody mediated targeting of liposomes to red cells in vivo. FEBS Lett. 201, 321-326.

Sullivan, S.M. and Huang, L. (1986) Enhanced delivery to target cells by heat sensitive immunolipo-

somes. Proc. Natl. Acad. Sci. U.S.A. 83, 6117-6121.

Vasudevachari, M.B. and Antony, A. (1985) Antiviral activity of liposome encapsulated cupric com-

plex of isonicotinic acid hydrazide against AMV infection. Ind. J. Exp. Biol. 23, 393-396.

Williams, C.A. and Chase, M.W. (eds) (1967) Methods in Immunology and Immunochemistry 1, 320. Academic Press, New York.

Wolff, B. and Gregoriadis, G. (1984) Th e use of monoclonal anti-Thy,G, for the targeting of lipo- somes to AKR-A cells in vitro and in viva. Biochim. Biophys. Acta 802, 259-273.

Yesair, D.W., Schwartzbach. E., Shick, D.. Dennie, E.P. and Asbell, M.A. (1972) Comparative phar-

macokinetics of daunomycin and adriamycin in several animal species. Cancer Res. 32. 1172-l 183.