Discussion
With a sophisticated and comprehensive, multimodal PK-PD, safety approach we investigated a possibly minimally invasive administration method of adalimumab with a commercially available hollow microneedle. Importantly, this clinical trial shows that i.d. administration of a single dose of 40 mg adalimumab in a volume of 0.4 mL using a hollow microneedle is safe and well accepted. However, i.d. administration was associated with an increased amount of injection pain and decreased volunteer preference compared to s.c. administration. Using imaging methods, the effect of i.d. injections on the skin was thoroughly characterized. As expected, i.d. injections led to bleb formation. Notably, i.d. injection transiently increased cutaneous microcirculation as measured by LSCI. Importantly, we found that i.d. administration of adalimumab led to a higher Cmax and a higher bioavailability compared to s.c. adalimumab administration. The inhibition of ex vivo cytokine production of whole blood stimulated with LPS/Alum was similar for i.d. and s.c. adalimumab administration indicating comparable pharmacodynamic efficacy.
Protein degradation, especially aggregation, might result in increased immunogenicity of mAbs (11) and immunogenicity of mAbs is a major reason for secondary loss of response to mAbs. Therefore, we first showedin vitro that microneedle ejection of adalimumab does not substantially alters the amount of protein fragments or aggregates compared to ejection using a regular hypodermic needle.
Hollow microneedles are frequently considered a minimally invasive device to deliver parenteral drugs (4,25–28). In this study we administered a single adalimumab dose of 40 mg in 0.4 mL or 0.4 mL placebo. We systematically studied pain associated with insertion and injection in a double-blind manner. We found that insertion pain of s.c. and i.d. administration was equal. However, injection pain of i.d. administration was significantly higher than s.c. administration. The high amount of pain is in contrast with another study, which used higher volumes but detected less pain (28). Pain due to s.c. injection is generally attributed to different factors, i.e., volume of injection, site of injection, formulation, needle size, and injection depth (29).
The volume limit of s.c. injection is generally considered to be 1.5 mL (30). Several studies have found higher volumes of s.c. administration to be associated with more pain (30–32). Thus, the increased pain that was associated with i.d. administration in the clinical trial reported in this paper is likely due to the volume injected. The volume used in this trial was limited by a minimum volume which contains a regular dose of a mAb in adults. Future studies might investigate the volume-pain relationship for i.d. administration using hollow microneedles. We did not detect a significant difference in pain when comparing adalimumab with placebo after i.d. and s.c. administration, which indicates that the formulation chosen in this study did not influence pain.
Although not quantified, we observed a higher injection pressure during i.d. administration compared to s.c. administration. With OCT, we detected fluid filled cavities after i.d. injection, indicating there was no time for the compound to distribute in the skin.
We characterized the skin response to hollow microneedle administration of adalimumab using a combination of methods. The skin response following i.d. administration of adalimumab was mild and resolved within a day after injection. Using 3D photography, we showed the bleb which is typical for i.d. administration. Furthermore, using LSCI, an increase in cutaneous microcirculation after i.d. injection of adalimumab was observed. Our observations are of interest in the context of drug absorption. The increased cutaneous microcirculation might be associated with the increased adalimumab absorption following i.d. versus s.c. administration observed in our study. Yet, drugs injected s.c. may be absorbed via the lymph capillaries, or diffuse into blood capillaries, and after s.c. administration proteins with a high molecular weight, such as mAbs, are predominantly absorbed via the lymph after s.c. administration (33,34).
Various factors influence lymph flow, one being local skin temperature. During an increase in local skin temperature, both the blood flow and the lymph flow increase (35–37). We quantified local skin temperature after i.d. adalimumab administration using thermography. A limitation is that from the skin temperature measurements we cannot unequivocally conclude which type of injection (s.c. or i.d.) leads to higher skin temperature for two reasons. The temperature measurements might be confounded by difference in depth as i.d. injections are more superficial than s.c. injections. Thus, the s.c. injections might have increased the local temperature which is not apparent from our measurements.
Initial lymphatics, the part of the lymph vessels responsible for drug uptake, are located superficially, in the dermis (38). Under physiological conditions most of these lymph vessels are collapsed. Excess fluid (high hydrostatic pressure) and proteins (high local osmotic pressure) in the dermis cause high lymph flow. We used OCT to visualize epidermal penetration after i.d. injection. Qualitative analysis of OCT observations showed an increase in vessel diameter after i.d. injection compared to s.c. injection. Based on the OCT, no distinction can be made between blood and lymph vessels. Perhaps in the future a new variant of OCT, Doppler OCT (39), could be used to further characterize the physiology of mAb absorption and lymph flow.
Several studies have reported that the i.d. administration of drugs has different PK characteristics than s.c. delivery (5,7,28,40). General observations are that Tmax is decreased, Cmax is increased and that bioavailability is either equal or increased after i.d. administration compared to s.c. administration. Most studies use insulin as model drug. For i.d. injection of insulin using hollow microneedles, it has been reported that Cmax increases and Tmax decreases after i.d. administration versus s.c. administration. It has been suggested that a shift in the concentration-time profile explains why some but not all studies have reported increased bioavailability after i.d. injection (5,41). Changes in PK are generally attributed to anatomical differences in the skin: the dermis has extensive vasculature and lymphatics while the subcutis has more adipose tissue (42). When correcting for individual differences in the covariates and the titre values, this study showed a significant difference in bioavailability between s.c. and i.d. administration; i.d. administration was associated with a 29% higher bioavailability. In our study, a clear distinction in the absorption profiles over time could be observed between s.c. and i.d. administration. Adalimumab administered by microneedle injection show a short but fast absorption, whereas s.c. dosing shows a lower absorption rate. The steep drop in absorption after a microneedle injection is caused by the distribution of sampling points and an estimated mathematical time point. In reality, this transition would probably be smoother. Altogether, the PK profile of the i.d. administration of adalimumab is favourable over s.c. administration.
The immunogenicity of mAbs is a significant clinical problem hampering the treatment of autoimmune diseases with mAbs. In this study the number of healthy volunteers allows only for descriptive reporting of anti-adalimumab antibodies. The skin is a potent immune organ (42). Studies have shown an increased immunogenicity of i.d. vaccines compared to s.c. vaccines and microneedles are frequently studied as a device to deliver vaccines (43,44). On the other hand, it has been suggested that i.d. administration of mAbs might lead to less immunogenicity compared to s.c. administration due to the presence of professional antigen presenting cells in the epidermis and dermis rather than in the subcutis (33,45). Perhaps the relatively short residence time at the i.d. injection site of the (predominantly monomeric) protein might contribute to the lack of increased immunogenicity as compared to s.c. administration. It remains to be determined whether i.d. administration of biologicals alters the incidence, degree, or time of onset of anti-drug antibody formation compared to s.c. administration.
In this study the functional effect of adalimumab administration was investigated in vitro . Whole blood was stimulated with LPS/Alum and secreted cytokines were measured. We found that i.d. and s.c. adalimumab reduced ex vivo TNFα bioavailability to a similar extent.
The increased bioavailability of i.d. adalimumab in our study suggests that lower doses may be used to achieve similar concentrations and subsequent effects compared to s.c. administration. Combined with the increased elasticity of the skin of children (46) and the need for a lower (adalimumab) dose than in adults, hollow microneedles ultimately might be suitable for use in paediatric patients. However, it is of paramount importance to better understand the pain-volume relationship of i.d. injections using hollow microneedles in adults first.
In conclusion, we showed that the i.d. administration of adalimumab is feasible and leads to faster absorption and increased bioavailability compared to s.c. administration. The amount of pain reported in this study, higher for i.d. than for s.c. adalimumab administration, is likely explained by the injection volume of 0.4 mL. Understanding the relationship between pain and the administration of mAbs is essential before hollow microneedles can be investigated for use in the paediatric patient population.