Treatment of murine L1210 lymphoid leukemia and melanoma B16 with lipophilic cytosine arabinoside prodrugs incorporated into unilamellar liposomes

Abstract

Lipophilic prodrugs of I-beta-D-arabinofuranosyl cytosine (Ara-C), namely N4- and 5'-oleyl-I-beta-D-arabinofuranosyl cytosine (N4-oleyl-ara-C, 5'-oleyl-ara-C) and N4-palmitoyl-I-beta-D-arabinofuranosyl cytosine (N4-palm-ara-C) were incorporated into liposomes of various lipid compositions. The phospholipid vesicles were prepared by controlled dialysis of lipid/prodrug/detergent micelles yielding homogeneous and stable unilamellar liposomes. The liposome size ranged from 70 to 120 nm depending on the lipid composition and the amounts of prodrug incorporated. Depending on the total amount of micellized Ara-C prodrugs, a maximal incorporation rate of 250 micrograms prodrug per mg egg phosphatidylcholine was achieved. The incorporation efficiency was in the range of 85 to 97%. The in vivo antitumor activity of the liposome preparations against L1210 lymphoid leukemia was clearly superior by factors of 2-8 depending on the therapy schedule and route of drug application. The treatment of melanoma B16 with free Ara-C as well as with the prodrug-liposomes exhibit rather weak antitumor activity. Drug application by means of prodrug-liposomes yields equal or higher tumor-inhibitory effects at drug concentrations which were 2-4 times lower than those of free Ara-C. Although drug tolerance and myelosuppression studies with Swiss albino mice revealed that the prodrug-liposomes were 5-10 times more toxic than free Ara-C, a substantial improvement of efficiency could be observed. The incorporation of the ara-C prodrugs into liposomes provides protection against fast degradation and systemic elimination which might be an explanation for the improved antitumor effect of these preparations.

Low density lipoprotein and liposome mediated uptake and cytotoxic effect of N4-octadecyl-1-β-D-arabinofuranosylcytosine in Daudi lymphoma cells

“…For the cytotoxicity experiments liposomes with the same lipid composition were used but NOAC was added at 20 mM (1500 molecules NOAC per liposome). For the flow cytometry experiments liposomes with the same lipid composition, 1 mM DiI and 180 mM sodium cholate were prepared by dialysis against saline-EDTA as described by Rubas et al (1986). DiI concentration was determined with a Spectrofluorometer SFM 23 (Kontron, Zurich, Switzerland) after solubilization of an aliquot of liposomes (20 µl) with 2 ml sodium cholate (0.05%), yielding an average of 80 DiI probes per liposome.…”

Comparative pharmacokinetic and cytotoxic analysis of three different formulations of mitoxantrone in mice

“…A third technique involves the modification of hydrophilic drugs into lipophilic derivatives (prodrugs). These molecules are incorporated as lipophilic components into the liposomal membrane (Rahman et al, 1986;Rubas et al, 1986).…”

Comparative studies of the preparation of immunoliposomes with the use of two bifunctional coupling agents and investigation of in vitro immunoliposome-target cell binding by cytofluorometry and electron microscopy

“…PC, 15-3 Bq/mmol) and manganese chloride (54MnC12) were from Amersham Int, Amersham, U K N2-Palmatoyl-L-lyslne methyl ester HC1 (PL) was synthesized as described [15] The hpopinhc malelrmde derivatives N 6-(3-malelmldopropmnyl)phosphaudylethanolarmde (MP-PE) and N6-(6-malemudocaproyl) -phosphaudylethanolarmne (EMC-PE) were prepared m analogy to the pubhshed synthesis of N-(4-(pmalelrmdophenyl)butyryl)phosphatldylethanolamlne [4] N4-Oleyl-ara-C (NOAC) was prepared as described before [17] and the hpophlhc metal complexmg agent DTPA-stearate as described in Ref 19 The Inghly fluorescent hpophlhc perylene dye N, N'-bls(1-hexylheptyl)-3,4 9,10-perylenebls(dlcarboxlmlde) (BHPD) was obtained from 7-trxdecanamlne as described before [20] Before use BHPD was purified on a Sdlca-gel column with CHC13 as eluent N-Succlnlmldyl-S-acetylthloacetate (SATA) was prepared according to Ref 21 …”

“…Small undamellar llposomes were prepared by detergent dialysis as descnbed before [17] The matrix hpld composmon used throughout all hposome preparations was soy phosphatldylchohne (SPC)/cholesterol/DL-atocopherol at 1 0 2 0 01 mol parts with 20 mg SPC/ml (26 #mol/ml) lmtml hpld concentration The malemude derlvatwes MP-PL, MP-PE, EMC-PL and EMC-PE were added at 0 02 or 0 05 mol parts Nn-oleyl-ara-C (NOAC) concentraUon was 0 2 mol parts The hpophlhc fluorescent label BHPD was Incorporated at 0 006 mol parts into the hposome membranes All hplds Including the corresponding malelnude denvatwe plus NOAC and BHPD were dissolved in MeOH/CHC13 (1 1, v/v) Sodmm cholate as detergent was added at a ratio of 0 6-0 7 moles referred to the sum of the concentraUons of all membrane-fornung hplds In some preparations the llposomes were labeled by addmon of trace amounts of 3H-PC or by the mcorporaUon of 0 003 mol parts of the hpopluhc chelating agent DTPA-SA to which radioactive manganese (54Mn2+) ions were complexed after hposome formation [19] After the removal of the orgamc solvents on a rotatory evaporator (40 o C, 60 rmn) the hpld/detergent nuxtures were solub~hzed by addmon of phosphate buffer (67 mM dlsodmm hydrogen phosphate dlhydrate/67 mM potassium dlhydrogen phosphate (pH 7 4)) The resulting nucellar solutions (10-20 ml) were dialyzed against 5-10 1 of the same buffer at 40°C for the first 2 h, followed by dialysis at 25 °C for another 24-36 h The hposome preparations were filtered through 0 45 #m sterile filters (Sartonus) and stored at 4 o C Llposome s~zes and homogeneity were deternuned by laser light scattering and negattve stare freeze-fracture electron microscopy [17] The mean number of membrane-fornung molecules, wluch comprised all hpopluhc compounds constituting the membrane bdayer of one hposome and the total number of hposomes formed by the hpld concentraUon umt of 1 mg SPC/ml, were calculated from the mean vesicle diameters obtaaned from the laser hght scattenng data and from the assumptions on vesicle geometry parameters as made by Huang …”

“…The covalent couphng of Abs, Ab fragments or peptldes to hgands winch are located on the surface of hposomes can be achieved by numerous chemical methods [3][4][5][6][7][8][9][10][11][12][13][14][15][16] Bffunctmnal hgands have become useful tools for the couphng of proteins to hposomes Among a vanety of such molecules, N-succlmmldyl-3-(2-pyndyldlthlo)propinnate (SPDP) is currently one of the most used linkers, although ~t has a number of disadvantages hke its suscept~inhty to traces of reducing agents and the short half-hfe of the reactwe sulfhydryl groups [16] N-Succxnlrmdyl-S-acetyltinoacetate (SATA) is an alternatwe couphng agent [16] In tins work we present the results that were obtained by the direct comparison of Ab couphng methods using SPDP and SATA as hnkers to hposomes which contmned N4-oleylcytosme arabmoslde (NOAC), a hpopinhc denvatwe of arabmosylcytoslne, as cytostatic prodrug [17,18] In particular, the couphng of the two Abs, B8-24 3 specific for the mouse MHC class I H-2k b surface antigen on EL4 mouse T lymphoma cells and IB16-6, specific for a surface antigen on B16 mouse melanoma cells, to small umlamellar drug carrying hposomes was lnvest~gated In addmon, the new highly fluorescent hpopinhc dye N,N'-b~s(1-hexylheptyl)-3,4 9,10-perylenebls(dlcarboxlnude) (BHPD) was used as fluorescence label for the hposomes…”