COMPROBANTES DE PAGO

The ease of filtration and amount of residue recovered from drug-free dispersions converted in one stage and two stages is shovm in Table 5.1.

Table 5.1 Filtration of drug-free egg phospholipid liposome dispersions converted from pro-liposomes in one and two stages. The abbreviation IS denotes a one stage conversion and 2S denotes a two stage

conversion.

Sample Ease of filtration

Average increase in filter weight ± s.d. (mg)

Appearance o f filter under the light microscope Egg PL PRO

(IS )

II 0.0 ± 0 .0 No crystalline material. Small amount of debris Egg PL PRO

(2S)

I 0.0 ± 0.0 No crystalline material. A few long fibres and small amount of debris

The drug-free sample converted in two stages passed through the filter with somewhat more ease than the liposomes converted in one stage, perhaps this was attributable to the slightly larger average size of the liposomes converted in one stage. From previous particle size studies, discussed in section 3.5, it was evident that liposomes converted in one stage (section 3.5.2) were considerably larger than liposomes converted in two

Chapter five-Solubilisation o f cyclosporin A by liposomes

stages (section 3.5.4). The liposomes converted in two stages have diameters close to 200 nm (the same size as the pore size of the filter employed in the analytical filtration), compared to over 400 nm for the one stage converted dispersions.

Although SEM and light microscopy detected a small amount o f non-crystalline debris on the filters from both drug-free dispersions, the weight of the filters, to one d.p. of a milligram, did not increase after passage of blank liposome dispersions. This small amount of debris on the filter seemed unavoidable when non-aseptic laboratory conditions were used to manufacture and to process the pro-liposomes and the resultant liposomes. The debris mainly composed of long fibres. It was unclear whether or not this small quantity of debris was either present in starting materials and/or introduced during processing.

5.5.1.2 Analytical filtration of drug-free liposome dispersions converted in two stages The M3 Mettler balance only became available during the study investigating the factors affecting particle size and association (section 5.5.4). This particular balance enabled the filters to be accurately measured in milligrams to three d.p.

The filter weight increase in section 5.5.1.2, measured using the more accurate Mettler balance, largely mirrored the weight increase obtained in section 5.5.3, which employed the less sensitive Sartorius balance. Drug-free liposome dispersions converted in two stages yielded a weight increase of less than 0.1 mg when measured using the M3 Mettler balance. In comparison, no weight increase was detected when similar filters (Table 5.1) were measured in milligrams to only one d.p. on the Sartorius balance.

Pro-liposome weight ratio Amount of pro-liposome (g) Amount o f deionised water for hydration (g) Ease of filtration Average increase in filter weight ± s.d. (mg) Appearance of filter under LM 39.85:0.15:36:26 0.598 0.460 II 0.082 ± 0.034 Clear

5.5.1.3 Diluting cyA dissolved in ethanol and glycerol

The dilution o f cyA dissolved in ethanol and glycerol resulted in major precipitation (Table 5.3). Diluting the solvent mixes with deionised water to a cyA concentration of 2 mg/g resulted in the immediate precipitation of cyA. The diluted mixes appeared opaque and flocculates of the precipitated powder were visible to the naked eye. Over 87% or more o f the cyA was recovered from these diluted mixes. Under the light microscope this powder seemed fine and appeared as a fine dense mass. The unretained

cyA may have adhered to the surface of the filter holder during the processing of the diluted mix. Additionally, some of this cyA may have dissolved in the water used for diluting and washing the filter and some may have been in a fine precipitate capable of passing through the filter.

Ethanol: glycerol: cyA weight ratio diluted with water to give 2 mg/g cyA

Ease o f filtration

Average increase in filter weight ± s.d. (mg)

Appearance of filter under the light microscope

24:26:2 III 8.7 ± 0.4 Dense mass of powder

48:26:2 III 8.9 ± 0 .5 Dense mass o f powder

5.5.2 One stage conversion

Egg PL pro-liposomes containing cyA were converted in one stage into liposome dispersions with a lipid concentration of 60 mg/g. The amounts of the components in the individual cyA pro-liposomes are outlined in section 5.4.

5.5.2.1 Ethanol cyA pro-liposomes

One stage conversion of a cyA liposome dispersion from the pro-liposome with an egg PL: ethanol: cyA weight ratio of 60:24:2 was rapid when the pro-liposome and bulk water were handshaken vigorously. The amount of precipitation recovered from this dispersion is shown in Table 5.4.

CyA pro-liposome weight ratio

Ease of filtration

Average increase in filter weight ± s.d. (mg)

Appearance o f filter under the light microscope

60:24:36:2 (IS)

III 1.4 ± 0 .2 Vast amount o f spherical precipitation. Diameter ranged fi’om few to 50 pm. Though most were 10-25 pm in diameter

The liposome dispersions generated from this egg PL pro-liposome were difficult to filter. The large amount of force required to overcome this resistance suggested that many of the filter pores had become occluded during the filtration of the dispersions. Indeed upon visual inspection of shiny upper side o f the filter, a covering of fine white powder, which weighed approximately 1.4 mg, could be observed macroscopically by the naked eye. The precipitate on the filter was distinct from the cyA powder (Plate 5.1), but similar in shape to the cyA precipitated from an ethanol: glycerol solution (Plate 5.2). Under xlOO magnification of a light microscope, it was clear that this precipitate composed largely spherical individual particles with diameters between approximately 5 pm and 50 pm (Plate 5.3). The formation of this precipitate in this

Chapter five-Solubilisation of cyclosporin A by liposomes

dispersion demonstrated that even when there were approximately 50 molecules o f lipid to every molecule o f cyA, association o f cyA into the liposome bilayer was disappointingly poor. This contrasts with the commercially available micelle formulation (Sandimmun® IV data sheet, 1996), which fully solubilises cyA when diluted in water to a cyA concentration o f 2.5 - 0.5 mg/ml.

It is possible that the unsolubilised cyA precipitated along with phospholipid present in the dispersion. Subjective evidence for this type o f precipitation is provided by SEM, which indicates that the surface o f the spheres are “wax-like”. As a result o f the possible precipitation o f cyA with lipid, the gravimetric analysis o f the filter could not be used to accurately determine the amount o f cyA associated with the liposomes, since the lipid associated with precipitated cyA would have contributed to the increase in filter weight. Therefore, if the increase in filter weight after analytical filtration was subtracted from the total amount o f cyA in the formulation, the level o f cyA molecularly associated with the liposomes would have been underestimated. However, in this study the weights o f the solids retained by the analytical filtration can be employed to indirectly assess the minimum level o f association, if it is assumed that increase in the filter weight is entirely due to cyA. To quantitatively ascertain the degree o f unassociated cyA with a greater degree o f accuracy, it would be necessary to employ HPLC to analyse the cyA retained on the filter.

Plate 5.1 Typical SEM o f cyA powder (Bar = 2 pm), as described in section 5.5.2.1

Plate 5.2 Typical SEM o f cyA precipitated from ethanol:glyccrol (Bar = 2 pm), as described in section 5.5.2.1

mm

Plate 5.3 Typical light micrograph o f precipitate recovered from a liposome dispersion generated in one stage from an egg PL pro-liposome containing cyA (Bar = 100 pm), as

described in section 5.5.2.1

5.S.2.2 Raising the ethanol level in ethanol cyA pro-liposomes

The results for the analytical filtration o f the cyA dispersions converted in one stage from a pro-liposome with an egg PL: ethanol: cyA weight o f 60:36:2 containing a raised ethanol level are shown in Table 5.5.

Table 5.5 Association o f cyA with liposomes converted in one stage from an egg PL pro-liposome CyA pro-liposome

weight ratio

Ease o f filtration Average increase in filter weight ± s.d. (mg)

Appearance o f filter under the light microscope

60:36:2 III 2.5 ± 0 .3 Copious amount o f spherical

Chapter five-Solubilisation o f cyclosporin A by liposomes

Raising the ethanol level in glycerol-free cyA pro-liposomes generated even larger amounts of precipitate. After analytical filtration, an average weight of 2.5 mg of residue was recovered from each liposome dispersion. This indicated that a higher level of ethanol in the glycerol free cyA pro-liposomes significantly (p = < 0.05) reduced the intercalation of cyA into the liposomes, if one stage conversion was adopted. It seemed that some of the cyA dissolved in the ethanol may have precipitated out upon the addition of the cyA pro-liposome to bulk deionised water.

S.5.2.3 CyA pro-liposomes containing glycerol

The pro-liposome with an egg PL: ethanol: glycerol: cyA weight ratio of 60:24:26:2 was added to deionised water and converted into liposome dispersions by either vigorously handshaking for one minute or leaving to convert without external agitation. The amounts of residue recovered by filtration from both sets of cyA liposome dispersions are shown in Table 5.6.

CyA egg PL pro-liposome subjected to;

Ease of filtration

Average increase in filter weight ± s.d. (mg)

Appearance o f filter under the light microscope

Immediate vigorous handshaking

III 0.5 ±0.1 Moderate amount of spherical

precipitation

No agitation III 0.8 ± 0.2 High level o f spherical precipitation

Inclusion of glycerol into the cyA pro-liposome formulation had a beneficial influence on the incorporation of cyA into liposomes. As is evident from both the gravimetric analysis and inspection under the light microscope, the presence of glycerol in the cyA pro-liposome significantly (p = < 0.05) reduced the formation of the insoluble precipitate. The weight of precipitate was reduced from 1.4 mg if glycerol was omitted (Table 5.4), to about 0.5 mg if glycerol was incorporated into the cyA pro-liposome. The possible reason for this improved association is explained in section 5.5.3.8. Nevertheless, although the degree of cyA association was improved, even at this high lipid: cyA molecular ratio of 30:1, there was still a moderate amount of unsolubilised cyA in the dispersion as revealed by light microscopy (Plate 5.4). The dark circles seen in Plate 5.4 are the accumulation of fine precipitate around the unsupported areas of the filter.

It was apparent from the light microscopy results that the cyA dispersions converted in one stage without agitation had a greater amount of insoluble residue present compared

to the dispersions formed by vigorous handshaking. This indicated that the speed o f the liposome formation may have influenced the intercalation o f cyA into the bilayers o f the liposomes. Perhaps without agitation, some cyA had time to diffuse and precipitate from the cyA pro-liposome before organised bilayers could be formed. However, this difference was not significantly different when the filter weight increase o f the two sets o f dispersions was compared (p = 0.08).

Plate 5.4 Typical light micrograph of precipitate recovered from liposome dispersion generated in one stage from an egg PL pro-liposome (Bar = 100 pm), as described in

section 5.5.2.3 5.S.2.4 Short term storage of cyA dispersions

The cyA pro-liposome formulation, described in section 5.4.2.3, was converted into a liposome dispersion in one stage by vigorous handshaking and stored for 24 hours at 4°C and 20 °C. After 24 hours had elapsed, the dispersions were filtered and the filter was inspected in the usual manner. The details o f the recovered residue are shown in Table 5.7.

Table 5.7 Association o f cyA with liposomes formed in one stage from an egg PL pro-liposome after Storage conditions o f cyA

egg PL dispersion

Ease o f filtration

Average increase in filter weight ± s.d. (mg)

Appearance o f filter under the light microscope Stored for 24 hours at 4 °C III 0.6 ± 0 . 1 Moderate amount o f

precipitation Stored for 24 hours at

20 °C

III 0.5 ± 0 . 1 Moderate amount o f

precipitation

After analytical filtration, the light microscope and gravimetric examination o f the filter revealed that the precipitate weight recovered from these dispersions was not different after 24 hours o f storage at either 20 °C or 4 °C compared to those filtered immediately

Chapter five-Soluhilisation o f cyclosporin A by liposomes

after vigorous agitation (Table 5.6). This indicated that the precipitate formed during conversion did not equilibrate with the liposome dispersion within 24 hours o f their formation. Once formed, this precipitate was not solubilised by the liposome dispersions under the conditions tested.