5B)

5B). Open in a separate window Figure 5 (A) Percent radioactivity remaining in the cell pellets following a 1 h incubation at 37C of radioiodinated antibodies in minipig whole blood. human; (3) minipig mAb SC absorption rates are generally higher than those in human and (4) the SC bioavailability appears to correlate with systemic clearance in minipigs. Given the important role of the neonatal Fc-receptor (FcRn) in the PK of mAbs, the in vitro binding affinities of these IgGs against porcine, human and cynomolgus monkey FcRn were tested. The result showed comparable FcRn binding affinities across species. Further, mAbs with higher isoelectric point tended to have faster systemic clearance and lower SC bioavailability in both minipig and human. Taken together, these data lend increased support for the use of the minipig as an alternative predictive model for human IV and SC PK of mAbs. Key words: mAb IgG, neonatal Fc receptor (FcRn), pharmacokinetics, subcutaneous bioavailability, animal model, minipig BIBW2992 (Afatinib) Introduction The primary advantages of monoclonal antibodies (mAbs) as therapeutic molecules are their target specificity and their prolonged serum persistence. During the development of mAb therapeutics, the selection of a relevant preclinical animal model is essential for the prediction of the human pharmacokinetic (PK) profile, as well as for assessing overall safety and exposure-response relationships prior to clinical studies.1C3 Moreover, a predictive preclinical PK model is also important for understanding the impact of various molecular properties on the in vivo behavior of mAbs, thus enabling optimization of therapeutic candidates in early development. Therapeutic mAbs are typically administered via intravenous (IV) infusion or subcutaneous (SC) injection. While the PK properties of mAbs following IV administration are well predicted by studies in non-human primates,4C6 a reliable animal model to predict the PK of mAbs following SC administration has not BIBW2992 (Afatinib) been clearly established.7 Relatively little is known about the mechanism of SC absorption of mAbs in different species. The major parameters that affect this process are thought to include the role of lymph and blood capillaries in systemic absorption, cross-species differences in hypodermis morphology and physiology, drug formulation, stability of the molecule, the site of injection, the depth of injection, as well as the molecular properties of the mAbs themselves.8C11 Published data suggest that animal models are not necessarily reliable predictors of antibody SC PK in humans as there is often no apparent relationship in SC bioavailability between humans and animals.7 Indeed, for larger biotherapeutics, absolute SC bioavailability has been reported to be higher in cynomolgus monkeys than in humans, while for biotherapeutics with molecular weights <40 kDa no clear relationship was obvious between human and animal data, both in rodents and cynomolgus monkeys.7 BIBW2992 (Afatinib) Given these discrepancies, it is believed that the lack of predictability could be attributed to differences in hypodermis structure and physiology between humans and rodents or non-human primates.12 With the increasing ethical concerns regarding the use of primates in non-clinical testing, attention has been increasingly focused on the potential use of minipigs as non-rodent alternatives for pharmaceutical testing.13C17 As such, minipigs are becoming more frequently used for toxicological and PK studies of small molecules.18C20 Recently, minipigs have been used to test the immunogenicity of therapeutic proteins;21,22 however, limited data exist for the pharmacokinetics of macromolecules in minipigs.23C25 In particular, to the best of our knowledge, there Mmp23 are no studies that describe the PK of mAbs in minipigs. The combined results from previous studies have demonstrated similarities between pig/minipig and human skin and lymph architecture26, 27 that are likely key contributors to SC absorption BIBW2992 (Afatinib) and bioavailability of macromolecules. In addition, the thickness of the epidermis and the stratum corneum as well as the lipid composition of the stratum corneum is similar between human and pigs, which has led to the frequent use of the pig/minipig model for dermal administration.28 The pig also has a tight link between dermis and underlying muscle, which is similar to the human situation, and these features have been attributed to the arrangement of elastic fibers in the hypodermis/subcutis, the target.