Impact of degradation in subcutaneous tissue and lymphatic fluid on absorption of Fc-fusion proteins following subcutaneous administration.

The Enhancement of Subcutaneous First-Pass Metabolism Causes Nonlinear Pharmacokinetics of TAK-448 after a Single Subcutaneous Administration to Rats.

Fc fusion is an effective strategy for extending the half-lives of therapeutic proteins. This study aimed to evaluate the applicability of a human pharmacokinetics prediction method for Fc-fusion proteins by extending on reported methods for monoclonal antibodies (mAbs). To predict human pharmacokinetic profiles following intravenous (IV) dosing, the pharmacokinetic data for 11 Fc-fusion proteins in monkeys were analysed by two approaches: a species-invariant time method with a range of allometric exponents in clearance (CL, 0.7-1.0) and a two-compartment model reported for mAbs. The pharmacokinetic profiles following subcutaneous (SC) dosing were predicted by simple dose normalisation from monkeys or using the geometric means of the absorption rate constant (Ka) and bioavailability (BA) for mAbs or Fc-fusion proteins in humans and compared. In the case of IV administration, the area under the curve could be predicted for more than 85% of Fc-fusion proteins within a twofold difference from the observed value using the species-invariant time method (scaling exponent for CL, 0.95). For SC dosing, incorporating the geometric means of absorption parameters for both mAbs (BA 68.2%, Ka 0.287 day-1) and Fc-fusion proteins (BA 63.0%, Ka 0.209 day-1) in humans provided better accuracy than simple normalisation from monkeys. We have successfully predicted the human pharmacokinetic profiles of Fc-fusion proteins for both IV and SC administration within twofold of the observed value from monkey pharmacokinetic data by extending on reported methods for mAbs. This method will facilitate drug discovery and development of Fc-fusion proteins.

Leukocyte recruitment and effector functions like phagocytosis and respiratory burst are key elements of immunity to infection. Pathogen survival is dependent upon the ability to overwhelm, evade or inhibit the immune system. Pathogenic group A and group B streptococci are well known to produce virulence factors that block the binding of IgA to the leukocyte IgA receptor, Fc alphaRI, thereby inhibiting IgA-mediated immunity. Recently we found Staphylococcus aureus also interferes with IgA-mediated effector functions as the putative virulence factor SSL7 also binds IgA and blocks binding to Fc alphaRI. Herein we report that SSL7 and Fc alphaRI bind many of the same key residues in the Fc region of human IgA. Residues Leu-257 and Leu-258 in domain C alpha2 and residues 440-443 PLAF in C alpha3 of IgA lie at the C alpha2/C alpha3 interface and make major contributions to the binding of both the leukocyte receptor Fc alphaRI and SSL7. It is remarkable this S. aureus IgA binding factor and unrelated factors from streptococci are functionally convergent, all targeting a number of the same residues in the IgA Fc, which comprise the binding site for the leukocyte IgA receptor, Fc alphaRI.

Antibody fragments are emerging as promising biopharmaceuticals because of their relatively small size and other unique properties. However, compared with full-size antibodies, these antibody fragments lack the ability to bind the neonatal Fc receptor (FcRn) and have reduced half-lives. Fc engineered to bind antigens but preserve interactions with FcRn and Fc fused with monomeric proteins currently are being developed as candidate therapeutics with prolonged half-lives; in these and other cases, Fc is a dimer of two CH2-CH3 chains. To further reduce the size of Fc but preserve FcRn binding, we generated three human soluble monomeric IgG1 Fcs (mFcs) by using a combination of structure-based rational protein design combined with multiple screening strategies. These mFcs were highly soluble and retained binding to human FcRn comparable with that of Fc. These results provide direct experimental evidence that efficient binding to human FcRn does not require human Fc dimerization. The newly identified mFcs are promising for the development of mFc fusion proteins and for novel types of mFc-based therapeutic antibodies of small size and long half-lives.

Fusion to an IgG Fc region is an established strategy to extend the half-life of therapeutic proteins. Most Fc fusion proteins, however, do not achieve the long half-life of IgGs. Based on findings that scFv-Fc fusion proteins exhibit a shorter half-life than the corresponding IgG molecules, we performed a comparative study of different antibody-derived Fc fusion proteins. We could confirm that fusion of single-chain Fv (scFv) and single-chain diabody (scDb) molecules to an Fc region yields in fusion proteins with substantially extended half-lives compared with the single-chain versions. However, even fusion proteins with a size similar to that of IgG, e.g., scDb-Fc, did not have a half-life as long as an IgG molecule. Binding to the neonatal Fc receptor (FcRn) under acidic and neutral conditions was similar for IgG and all Fc fusion proteins. However, we observed differences between IgG and the Fc fusion proteins for dissociation of FcRn-bound proteins induced by shifting from acidic to neutral pH, reflecting the physiological release mechanism, further supporting a contribution of the kinetics of pH-dependent release from FcRn to the pharmacokinetic properties of IgG and Fc fusion proteins.

Fc-fusion proteins are composed of Fc region of IgG antibody (Hinge-CH2-CH3) and a desired linked protein. Fc region of Fc-fusion proteins can bind to neonatal Fc receptor (FcRn) thereby rescuing it from degradation. The first therapeutic Fc-fusion protein was introduced for the treatment of AIDS. The molecular designing is the first stage in production of Fc-fusion proteins. The amino acid residues in the Fc region and linked protein are very important in the bioactivity and affinity of the fusion proteins. Although, therapeutic monoclonal antibodies are the top selling biologics but the application of therapeutic Fc-fusion proteins in clinic is in progress and among these medications Etanercept is the most effective in therapy. At present, eleven Fc-fusion proteins have been approved by FDA. There are novel Fc-fusion proteins which are in pre-clinical and clinical development. In this article, we review the molecular and biological characteristics of Fc-fusion proteins and then further discuss the features of novel therapeutic Fc-fusion proteins.

Oral bioavailability is an essential parameter in drug screening cascades and a good indicator of the capability of the delivery of a given compound to the systemic circulation by oral administration. In the present work, we report a database of oral bioavailability of 1014 molecules determined in humans. A systematic examination of the relationships between various physicochemical properties and oral bioavailability were carried out to investigate the influence of these properties on oral bioavailability. A number of property-based rules for bioavailability classification were generated and evaluated. We found that no rule was an effective predictor for oral bioavailability because these simple rules cannot characterize the influence of important metabolic processes on bioavailability. Finally, the genetic function approximation (GFA) technique was employed to construct the multiple linear regression models for oral bioavailability using structural fingerprints as the basic parameters, together with several important molecular properties. The best model is able to predict human oral bioavailability with an r of 0.79, a q of 0.72, and a RMSE (root-mean-square error) of 22.30% of the compounds from the training set. The analysis of the descriptors chosen by GFA shows that the important structural fingerprints are primarily related to important intestinal absorption and well-known metabolic processes. The predictive power of the models was further evaluated using a separate test set of 80 compounds, and the consensus model can predict the oral bioavailability with r(test) = 0.71 and RMSE = 23.55% for the tested compounds. Since the necessary molecular properties and structural fingerprints can be calculated easily and quickly, the models we proposed here may help speed up the process of finding or designing compounds with improved oral bioavailability.

Recombinant human epidermal growth factor (rhEGF), a polypeptide of 53 amino acid residues, is subject to degradation by numerous enzymes, especially proteases, when it is applied on the skin for the treatment of open wound. Amastatin, aprotinin, bestatin, EDTA, EGTA, gabexate, gentamicin, leupeptin, and TPCK were investigated for the possible protease inhibitors, which may use to protect rhEGF from degradation by the enzymes in the skin. Skin homogenates containing protease inhibitors and rhEGF were incubated at 37 degrees C for 30 minutes. After the reaction was stopped with trifluoroacetic acid, the amount of rhEGF remaining in the sample was determined with an HPLC method. The percentages of rhEGF degraded, at the skin/PBS ratio of 0.25, in the mouse, rat, and human skin homogenate were 85%, 70%, and 46%, respectively. The degree of degradation of rhEGF in the cytosolic fraction was higher than that in the membrane fraction and these enzyme reactions were completed in 30 minutes. Bestatin, EGTA, and TPCK showed significant inhibitory effects on the degradation of rhEGF in the two fractions (p<0.05), while the other protease inhibitors had no significant inhibitory effects or, even resulted in deleterious effects. Therefore, the formulation containing one or several inhibitors among these effective inhibitors would be a promising topical preparation of rhEGF for the treatment of open wound.

Dehydroepiandrosterone (DHEA) administered percutaneously by twice daily application for 7 days to the dorsal skin of the rat stimulates an increase in ventral prostate weight with approximately one third the potency of the compound given by subcutaneous injection. The doses required to achieve a 50% reversal of the inhibitory effect of orchiectomy are approximately 3 and 1 mg respectively. By the oral route, on the other hand. DHEA has only 10-15% of the activity of the compound given percutaneously. Taking the bioavailability obtained by the subcutaneous route as 100%, it is estimated that the potencies of DHEA by the percutaneous and oral routes are approximately 33 and 3% respectively. Similar ratios of activity were obtained when dorsal prostate and seminal vesicle weight were used as parameters of androgenic activity. When examined on an estrogen-sensitive parameter, namely uterine weight in ovariectomized rats, the stimulatory effect of DHEA was much less potent than its androgenic activity measured in the male animal, a 50% reversal of the inhibitory effect of ovariectomy on uterine weight being observed at the 3 and 30 mg doses of DHEA administered by the subcutaneous and percutaneous routes respectively. When measured on uterine weight, percutaneous DHEA thus shows a 10% potency compared with the subcutaneous route. The sulfate of DHEA (DHEA-S), on the other hand, was approximately 50% as potent as DHEA at increasing ventral prostate weight after subcutaneous or percutaneous administration. When the effect was measured on dorsal prostate and seminal vesicle weight, percutaneous DHEA-S had 10-25% of the activity of DHEA. DHEA decreased serum LH levels in ovariectomized animals, an effect which was completely reversed by treatment with the antiandrogen flutamide. On the other hand, flutamide had no significant effect on the increase in uterine weight caused by DHEA, thus suggesting a predominant estrogenic effect of DHEA at the level of the uterus and an estrogenic effect on the feedback control of LH secretion. The present data show a relatively high bioavailability of percutaneous DHEA as measured by its androgenic and/or estrogenic biological activity in well-characterized peripheral target intracrime tissues in the rat.

The onset, intensity and duration of therapeutic response to a compound depend on the intrinsic pharmacological activity of the drug and pharmacokinetic factors related to its absorption, distribution, metabolism and elimination that are inherent to the biological system. The process of drug transfer from the site of administration to the systemic circulation and the interspecies factors that impact this process are the scope of this review. In general, the factors that influence oral drug bioavailability via absorption and metabolism can be divided into physicochemical/biopharmaceutical and physiological factors. Physicochemical and biopharmaceutical factors that influence permeability and solubility tend to be species independent. Although there are significant differences in the anatomy and physiology of the gastrointestinal tract, these are not associated with significant differences in the rate and extent of drug absorption between rats and humans. However, species differences in drug metabolism in rats and humans did result in significant species differences in bioavailability. Overall, this review provides a better understanding of the interplay between drug physicochemical/biopharmaceutical factors and species differences/similarities in the absorption and metabolism mechanisms that affect oral bioavailability in rats and humans. This will enable a more rational approach to perform projection of oral bioavailability in human using available rat in vivo data.

The onset, intensity and duration of therapeutic response to a compound depend on the intrinsic pharmacological activity of the drug and pharmacokinetic factors related to its absorption, distribution, metabolism and elimination that are inherent to the biological system. The process of drug transfer from the site of administration to the systemic circulation and the interspecies factors that impact this process are the scope of this review. In general, the factors that influence oral drug bioavailability via absorption and metabolism can be divided into physicochemical/biopharmaceutical and physiological factors. Physicochemical and biopharmaceutical factors that influence permeability and solubility tend to be species independent. Although there are significant differences in the anatomy and physiology of the gastrointestinal tract, these are not associated with significant differences in the rate and extent of drug absorption between rats and humans. However, species differences in drug metabolism in rats and humans did result in significant species differences in bioavailability. Overall, this review provides a better understanding of the interplay between drug physicochemical/biopharmaceutical factors and species differences/similarities in the absorption and metabolism mechanisms that affect oral bioavailability in rats and humans. This will enable a more rational approach to perform projection of oral bioavailability in human using available rat in vivo data.

Highly lipophilic and poorly water soluble drug candidates are common outcomes of drug discovery programs in recent years, presenting drug development challenges through poor gastrointestinal absorption and insufficient systemic exposure after oral administration. Co-administration with lipidic excipients presents an apparent strategy to improve the oral bioavailability of these compounds by stimulating enhanced solubilisation in the gut and recruitment of intestinal lymphatic drug transport. The impact of stimulating intestinal lymphatic transport to improve oral bioavailability on systemic drug exposure, clearance and deposition has been poorly understood. The interpretation of lymphatic drug transport data is further complicated by variations in dosing excipients and dispersed states, study models, prandial (fed/fasted) states, intravenous dosing conditions and formulations used for the assessment of absolute bioavailability. The studies described in this thesis investigated various factors affecting the intestinal lymphatic transport and oral bioavailability assessment of highly lipophilic compounds using lymph-cannulated and non lymph-cannulated animals. These factors examined included subtle differences in lipophilicity and lipid solubility of chemically similar drug analogues, lipid and non-lipid based oral formulations, intravenous dosing states and dosing conditions. The impact of lymphatic drug delivery on systemic exposure, clearance and drug deposition was also examined in comparison with portal route of drug absorption in this thesis. Oral bioavailability of highly lipophilic analogues was significantly enhanced after administration in long chain lipid-based formulations via stimulation of intestinal lymphatic transport and significantly influenced by subtle differences in lipid solubility, however, not lipophilicity as indicated by log P. After delivery in lymph or the lipid-based emulsion, systemic clearance (Cl) and volume of distribution (Vd) of a highly lipophilic, lymphatically transported model drug, halofantrine (Hf) were significantly lower than when delivered in plasma or lipid-free co-solvent formulation. However, where drug and lipid entered the systemic circulation coincidentally, Cl and Vd were unaffected by the route of entry, but significantly altered by total plasma lipid levels. These findings suggest that a mismatch in plasma lipid levels after intravenous and oral administration may lead to differences in drug clearance and errors in bioavailability assessment. This thesis also investigated the influence of absorption route (lymphatics vs. blood) on drug pharmacokinetics and tissue distribution. Brain to blood ratios were found to be significantly lower after stimulation of intestinal lymphatic delivery suggesting that drug association with intestinal lymph lipoproteins might limit brain drug access. Lipophilic model compounds (DDT, Hf) and lipids were assessed following delivery to the systemic circulation in association with lymph lipoproteins or plasma, and were found to differ significantly. For DDT, Cl and Vd were higher whereas for Hf, these parameters were lower due, in particular, to differences in adipose tissue uptake and liver uptake. For compounds like DDT, changes to the route of absorption may thus directly impact on pharmacokinetics and tissue distribution, whereas for Hf, factors which influence lymphatic transport may, by altering systemic lipoprotein concentrations, indirectly impact pharmacokinetics and tissue distribution. Ultimately, careful control of dosing conditions and thus the extent of lymphatic transport may be important in assuring reproducible efficacy and toxicity for lymphatically transported drugs.

Susceptibility of cosmeceutical peptides to proteases activity: Development of dermal stability test by LC-MS/MS analysis

Therapeutic immunoglobulin G (IgG) antibodies have comparatively long half-lives because the neonatal Fc receptor (FcRn) binds to the IgG Fc at acidic pH in the endosome and protects IgG from degradation. To further prolong the half-lives, amino acid-substituted antibodies having high affinity to FcRn are being developed, and one such therapeutic antibody (ravulizumab) has been approved. In this study, we investigated the binding property to FcγR and the conformation of seven FcRn affinity-modulated adalimumab variants to clarify the impact of the amino acid substitutions on the function and conformation of IgG Fc. The amino acid substitutions in T254-P261 caused a change in deuterium uptake into some regions of Fc in HDX-MS analysis, but those at T311, M432 and N438 did not cause such a change. The conformations around F245-L255 (FLFPPKPKDTL) were particularly influenced by the amino acid substitution in M256-P261, and the conformational changes of this region were correlated with the decrease of the affinity to FcγRIIIa. Additionally, we investigated the conformational difference of Fc between a Fc fusion protein (etanercept) and a native IgG (adalimumab). Although the Fc fusion proteins were expected to have similar FcRn affinity to IgGs, the affinity of etanercept to FcRn was lower than that of adalimumab, and its half-life was shorter than those of the IgG antibodies. Differences in deuterium uptakes were observed in the two regions where they were also detected in the adalimumab variants, and the conformational differences appeared to be an important factor for the low FcRn affinity of etanercept.

To discuss the potential implications of obesity for drug administration and absorption from subcutaneous (SC) and intramuscular (IM) injection sites. The SC and IM routes are useful for the parenteral administration of medications to optimize pharmacokinetic properties such as time to onset and duration of effect, for cost considerations, or for ease of administration, such as when intravenous access is unavailable. The choice of SC or IM injection depends on the specific medication, with SC administration preferred for products such as insulin where a slower and more sustained response is desirable, while IM administration is usually preferred for products such as vaccines where more rapid absorption leads to a more rapid antibody response. Obesity has the potential to influence the rate and extent of absorption, as well as adverse effects, of medications administered by the SC or IM route through changes in SC tissue composition and depth or by inadvertent administration of IM medications into SC tissue because of improper needle length. Potential adverse effects associated with IM or SC injections in addition to pain, bruising, and hematoma formation include sciatic nerve injury, particularly with IM injection in the upper outer quadrant of the buttock; bone contusion or rarely osteonecrosis if the IM injection is excessively deep; and granulomas, fat necrosis, and calcification with SC injection. Issues related to medication absorption in obese patients are likely to become more prominent in the future with increasing approvals of a wide range of biotherapeutic agents administered by SC injection. Studies should be directed toward these and other agents to assist with dosing decisions in this challenging population.