3.2.1. Swelling Test
A shows the swelling test profiles from CH batches. All samples underwent moderate swelling in the first 30 min of the assay, followed by an erosion process. This behaviour is due to the inability of CH to form a gel in SVF, as has been previously described in the texture analysis results. Slight differences can be seen in the swelling curves of the batches containing different amounts of CH. Batches containing a higher amount of polymer (CH2 and CH2D) take longer to erode totally (120 h) due to the difference in the tablet’s surface/polymer amount ratio (s/a). It can also be seen that, although DPV is highly hydrophobic, the presence of the drug does not modify the erosion process.
B shows the swelling curves of LB systems. The amount of polymer in each sample clearly conditions the tmax
; when the amount of polymer is increased, tmax
is also prolonged and occurs in 48 h for L1 and 96 h for L2 (Table 2
). Although the maximum swelling is similar in all cases, tablets containing a higher amount of polymer take longer to totally erode. These curves can be justified by the fact that swelling and erosion occur simultaneously, as in P- and CH-based tablets. In terms of tmax
values, erosion of a complete swelled gel occurs in L1 while a more pronounced plateau is observed in the swelling curve for L2; the erosion also takes longer to complete (312 h) and occurs concurrently with swelling. The addition of DPV to the tablets does not modify LBG swelling behaviour, suggesting that the arrangement of the polymer chains in the aqueous medium is not altered by the presence of hydrophobic particles in the drug. Finally, LB tablets disaggregate in small gel particles in the medium and gradually lose their structure.
C shows the swelling curves for P tablets. A swelling and erosion process were detected in every case. The time taken to achieve maximum swelling (tmax
) is in the same range (24–48 h), although differences can be seen in the total proportion of water imbibed and in the time required for complete erosion, quantified by the area under the curve (AUC
). The amount of P in the batches conditions the swelling behaviour, which is more pronounced the less polymer there is in the tablet (P1), although erosion is also faster. This can be attributed to the S
/a ratio, as the larger the surface of the tablet in contact with water, the more water penetration is favoured, producing a more fluid gel than for P2 and P2D. The content of DPV in P1D and P2D confers some hydrophobia on the tablets and also slightly increases the S/a ratio, reducing the surface in contact with the medium. Consequently, P1D and P2D batches imbibed a smaller amount of SVF than P1 and P2.
3.2.2. Drug Release Study
The dissolution process of pure micronized DPV was assessed in mSVF (Figure 4
A) and revealed the almost complete dissolution of the drug (93%) over a period of 4 h. DPV release profiles from polymer systems are different, and a controlled release was obtained in all cases, although the release profile clearly varies depending on the polymer used. Differences were observed in the drug release between CH batches (CH1D and CH2D) during the first six hours, but the rapid erosion of the tablets led to a quick and complete DPV release in the first 24 h for both batches (Figure 4
LBG is the polymer that most closely controls the release of DPV from the batches (L1D and LD2). In the case of batch L1D, up to 410 h are required to release the entire dose. In the case of batch L2D, the drug release extended to 792 h, or 33 days (Figure 4
Batches based on P (P1D and P2D) released the entire drug amount in 120 h in a sustained manner with overlapping profiles (Figure 4
D), indicating that P systems are sufficiently robust to release DPV in a sustained and uniform manner in the proportions evaluated in this study.
However, vaginal turnover must be taken into account. This is a physiological mechanism whose objective is to remove possible foreign elements from the environment of the vaginal epithelium (such as pathogens or any extraneous element). This represents a limitation for these systems, as this cyclical process takes place in 96 h [41
]. Vaginal leakage limits the efficiency of L tablets during such prolonged periods in in vivo studies; they would be expelled when no more than 60% of the dose had been released. In contrast, P systems release over 90% of the drug in this period, so vaginal clearance would occur after the release of almost the entire dose of the drug. It should also be noted that this is an in vitro testing of the systems, so vaginal discharge must be considered in future research stages, as this causes the loss of the drug, leading the concentration in vaginal tissue to be lower than predicted.
It should be noted that swelling results are related to the drug release profiles: the higher the AUC and tmax for a formulation, the more controlled is the release of the drug.
The results obtained when comparing the batches using an f2
statistical process (Table 3
) highlight significant differences in the release of DPV in all the systems, except between the two batches constituted by P, whose release profiles can be considered to be overlapping.
Mathematical models were used to determine the drug release mechanisms [42
]. Table 4
shows the results for these fits.
While CH batches do not fit any kinetic model, P and L batches can be adjusted to Korsmeyer‒Peppas with a good correlation coefficient. In the case of L batches, a good adjustment to this model with a nk value of between 0.45 and 0.89 implies the release of the drug through the simultaneous relaxation of the polymer chains and Fickian diffusion processes, agreeing with the data obtained from the swelling studies, as this polymer simultaneously undergoes swelling and erosion during the release process. This can be confirmed by the fact that these systems also show a good fit to Hopfenberg and Higuchi kinetics, indicating erosion and diffusion as their respective release mechanisms. P batches have higher values of nK for adjustment to the Korsmeyer‒Peppas kinetic, suggesting that the release occurs mostly through polymer chain relaxation. This is confirmed by the higher correlation coefficient observed for Hopfenberg (erosion) compared to Higuchi (diffusion). These results also agree with the swelling studies, as P erodes rapidly after reaching its maximum while the drug is still being released.
It is also worth highlighting that systems P1D and P2D release DPV in the medium at a constant rate during the first 72 h, so the dissolution of the drug from the system occurs at the same rate the drug is released. This is explained by the fact that the release occurs through an erosion process in which the pectin acts throughout this period as a purely erodible matrix (Costa & Lobo, 2001), leading to a pseudo-zero-order process where the release of the drug would lead to constant concentrations of DPV in the vagina while the formulation remains attached to the mucosa, thus avoiding toxic or ineffective concentrations.
Drug release data are related to texture behaviour: the consistency acquired by P tablets in contact with SVF is not dependent on the amount of P. The opposite occurs in the case of LBG, so an increase in the amount of polymer reduces the amount of drug released. This can be explained by the fact that gel texture analyses (Section 3.1.1
) revealed that an increase in P concentration slightly increased the consistency parameters (MPF and MDF), in contrast with LBG-based gels. The consistency of the swelled P tablets is therefore homogeneous, while in LBG-based tablets it is heterogeneous and greater at the deepest layers (Figure 5
), hence the differences between the release profiles for the L batches and the homogeneity between P batches.
Although vaginal rings that show a longer release of DPV have been reported previously [43
], the technologies and excipients required to their manufacturing make them too expensive. Taking into account that these formulations have been designed for use in developing countries, the reduction in manufacturing costs may imply greater access to pre-exposure prophylaxis therapies. According to this, studies on the effectiveness of our tablets can be justified in future research once the drug has proved capable of release from the formulation in the swollen tablets.
3.2.3. Bioadhesion Test
shows the results of the bioadhesion test. CH batches have low bioadhesion values (between 0.12 and 0.25 N), and a rise in the value of this parameter can be seen when the proportion of CH in the system is increased. It is interesting to note that the addition of the drug reduces the variability with respect to the reference tablets, as the hydrophobic drug generates more compact structures and displays more uniform erosion. In LBG systems, an increase in the amount of polymer produces a greater adhesion force in the system, and these values are greatest when the drug is incorporated. Nevertheless, both L2 batches show a wide variability between the samples due to the heterogeneity of the gel, as seen in Section 3.1.1
. Similar results for detachment force and work are obtained when analysing systems containing P, where bioadhesion forces increase as greater amounts of polymer are added to the system. The addition of DPV also enhances bioadhesion forces in all the cases. This is probably due to the S/a ratio, as an increase in the amount of polymer leads to a higher proportion per unit of area, thus impeding the penetration of water and facilitating contact between the biological surface and the polymer. Likewise, the incorporation of DPV increases the systems hydrophobicity, resulting in less hydrated gels with a greater bioadhesion capacity; the highest bioadhesion force values are obtained in the system containing DPV and a higher proportion of polymer. These results suggest that the interactions between the polymer chains and the aqueous medium after gelation compete with the interactions taking place between the bioadhesive polymer and the biological surface [44
]. The consistency of the bioadhesive bonds must also be considered, as this plays a key role. Diluted gels have a lower interaction than concentrated gels when in contact with the mucosa, as diluted gels tend to slip and become detached. The tablets containing higher amounts of polymer are therefore more bioadhesive as they generate more consistent gels. This explains why gels have poorer adhesion than solid systems.
Statistical processing indicates that there cannot be said to be any statistically significant differences between CH batches in terms of either the amount of polymer or the presence of the drug. The same is true of L batches, due to the high variability in the bioadhesion values for L or CH batches, indicating that these systems may have a somewhat erratic adhesion to the area of action. However, there are significant differences in both factors in the case of P batches, where the variability between the samples is low in all cases; this implies that the adhesion of the tablets could be highly reproducible, so P can be considered a promising candidate for the development of mucoadhesive dosage forms [45
]. No significant interaction was observed between the presence of drug and the amount of polymer in any of the batches, indicating that the drug’s contribution to the bioadhesion capacity is not conditioned by the amount of polymer.
The results of bioadhesion are a good indicator of the ability of the tablets to remain attached to the mucosa. This justifies future evaluations to check the in vivo permanence of the formulations in the vaginal environment.
The biocompatibility of the tablets was evaluated through an in vitro cell toxicity assay. All the components of the different batches were incubated with PBS 1× in humidified atmosphere at 37 °C and 5% CO2 for five days before the assay to ensure that any potential toxic component would be present in the suspension. MT-2, a lymphoblastoid cell line, and HEC-1A, a uterus-derived cell line, were seeded and treated with different dilutions of the suspensions. All the components were tested at a maximum concentration of 1000 μg/mL in base-5 serial dilutions. Experiments were performed on MT-2 cells to evaluate toxicity on the immune cells present in vaginal or uterine mucosae and on the uterine epithelial cell line (HEC-1A) to assess any potential damage to mucosae integrity. CC50 were calculated when possible.
As shown in Table 5
, no toxicity was detected at the concentrations tested for the polymers or at the higher concentration of 1000 µg/mL.
DPV showed high cytotoxicity, and significantly higher for MT-2 than for HEC-1A. However, after complete dissolution of the entire dose in the evaluation medium, and even without considering the vaginal clearage rate (as it is an in vivo parameter), it would generate a 1 mM solution, a concentration that shows good compatibility with ex vivo human cervical tissue explants, as shown in the study by Fletcher et al. [46
]. This can be attributed to the fact that vaginal tissue may protect these cells from toxic concentrations of the drug and act as a barrier to penetration. Further in vivo studies should therefore be done to determine whether these formulations present significant toxicity or not.
These results would justify the in vivo evaluation of the tablets, since the polymers showed no toxicity and the dose of dapivirine in the vaginal environment would not be able to produce tissue damage.