Figure 3.1: The activity of DOX niosomes against a resistant ovarian cancer cell line CHl DOX^ (B) and its susceptible parent line - CHl (A), i) Span 60, cholesterol, Solulan C24 (45: 45: 10) niosomes; ii) C16G2, cholesterol, Solulan C24 (45: 45: 10)
niosomes; iii) C16G2, cholesterol, NaDCP (47.5: 47.5: 5) niosomes; iv) DOX solution.
3.1). However DOX niosomes were generally less active than DOX solution against the parent susceptible cell line CH l. The most active formulation against the resistant cell line CH l DOX^, Span 60 niosomes was the least active formulation against the susceptible cell line| 3.3), The use of DOX niosomes against the resistant CH l DOX^ cell line resulted in a 3.5 fold increase in sensitivity with Span 60 niosomes and a 1.6 and 1.4 fold increase in sensitivity with the Ci6G2-Solulan C24 and C16G2-
NaDCP niosomes respectively. These values could have been influenced by the lipid formulation, DOX load and/or niosome size. From Table 3.3, it appears that the Span 60 niosomes, which had a higher DOX load, were also the most active against the resistant cells. Each niosome with a larger payload on association with the cells would deliver more of drug to the cell than niosomes with a smaller payload. In a similar experiment involving DOX phosphatidylcholine-cardiolipin (PC-CL) liposomes and a resistant human ovarian carcinoma SKVLB cell line DOX sensitivity was increased 9.6 fold by encapsulation in liposomes (Thierry et al., 1992). Studies with resistant Chinese hamster LZ cells and a resistant human breast cancer MCF-7/ADR cell line showed that PC-CL DOX containing liposomes increased the sensitivity of these cells to DOX by 9 and 3 fold respectively (Thierry et a/., 1993). A value of a 7 fold increase in sensitivity with the LZ cell line and DOX PC-CL liposomes was also reported (Thierry et al., 1989). The sensitization to DOX by liposomal encapsulation experienced by MCF-7/ADR cell line was comparable to that obtained in this study with Span 60 niosomes and the resistant ovarian cancer cell line CH l DOX^ (3.0 and 3.5 respectively). Sadasivan and associates (1991) have quantified the sensitization to DOX encountered on liposomal encapsulation by computing a value called the relative resistance index (RR).
RR = IC50 resistant cells/ICgo sensitive cells (3.1)
DOX liposomes prepared from phosphatidylcholine-phosphatidylserine (PC-PS) reduced this value from 80 to 32 in a resistant variant of a human preleukemic cell line HL60R (Sadasivan et a i, 1991), P-gp overexpression was confirmed in this cell line. A computation of similar RR values with the Span 60 niosomes used in this work revealed a reduction in this value from 230 to 20, clearly larger than the value obtained with DOX PC-PS liposomes. When the IC50 values of DOX PC-CL
liposomes and free DOX were compared for HL-60R cells not expressing P-gp, no sensitization to DOX was observed with the use of the encapsulated material (Thierry
et aL, 1992). On the contrary, alternative HL-60R cell lines resistant to vincristine and with attendant overexpression of P-gp showed that DOX PC-CL liposomes increased the susceptibility of these cells to DOX 5 fold. Work with a colon cancer cell line SW620/R overexpressing P-gp showed a 1.4 fold increase in sensitivity with the use of PC-CL liposomal DOX (Oudard et aL, 1991). The number of different cell lines used in these various studies make it extremely difficult for any meaningful comparisons to be made and the systematic characterisation of the exact processes that lead to multidrug resistance would help future studies in this area.
While the amplification of the multidrug resistance gene and the overexpression of P- gp were not confirmed for the cell line used in this work, it appears that in many of the earlier reports cited above, confirmation of P-gp overexpression was linked to the ability of vesicular drug delivery to modulate this resistance. In certain cases the increased susceptibility of the resistant variant was associated with an increased
accumulation of DOX within the cell (Thierry et aL, 1989; Oudard et aL, 1991; Rahman et aL, 1992). This suggests that these vesicles may interact with P-gp and in some way change its function in resistant cell lines (Thierry et aL, 1989; Oudard
et aL, 1991; Rahman et aL, 1992), preventing drug efflux. A specific binding of liposomes to P-gp has been reported (Rahman et aL, 1992; Thierry et aL, 1992). P- gp, apart from being responsible for the much vaunted energy dependant efflux mechanism (Beck, 1987), is also thought to alter membrane function by an alteration of membrane structure (Awasthi et aL, 1992). A reduced DOX accumulation in the lipid fraction of the membrane is found in some DOX resistant cells (Awasthi et aL,
1992). The interaction of surfactants with cell membranes in resistant cell lines also alters membrane fluidity and P-gp function (Woodcock et aL, 1992) while the interaction of empty PC-CL liposomes with P-gp is believed to be responsible for the increased susceptibility of these resistant cells to empty liposomes (Thierry et aL,
1992). It is possible that the surfactants present in the Span 60 niosome formulation would interact with the cell membrane increasing its retention of DOX.
In a resistant human bladder cancer cell line, resistance was associated with altered cellular distribution of the drug (Usansky et aL, 1991; Thierry et aL, 1993) and the modulation of resistance found a change in the intracellular drug distribution in favour of nuclear sites (Thierry et aL, 1993). It is not known whether the niosomes are taken up intact into the intracellular space, but the association of DOX with niosome membrane surfactants (Figure 2.6) may alter the intracellular distribution of the drug and reduce DOX resistance this way.
Largely the action of these vesicle formulations on the parent susceptible lines was unchanged by the encapsulation of DOX (Thierry et aL, 1993; Oudard et aL, 1991) and in our case, encapsulation actually decreased the sensitivity of the parent line CHl to DOX, reducing the activity on niosomal encapsulation by 3.3, 2.7 and 2.3 fold for the Span 60 niosomes, Ci6G2-Solulan C24 and Ci^Gz-NaDCP niosomes respectively.
Liposomal DOX preferentially lowers the white blood cell count of MDR-transgenic mice in vivo (Mickisch et aL, 1992). This mouse model expresses the multidrug resistance gene in the bone marrow. Measurements of white blood cell count relate directly to the level of multidrug resistant gene expression. In addition the use of the polyoxyethylene surfactant Cremophor in conjunction with DOX increased the survival time of mice bearing a resistant P388 transplantable tumour (Woodcock et aL, 1992).
These data suggest that drug delivery may have a role to play in the modulation of multidrug resistance. The encouraging results obtained with Span 60 niosomes in this report demonstrate a need for in vivo evaluation of this formulation against a multidrug resistant neoplasm. It is hoped that this may be done at some future date.