Lessons learned from the clinical development of oral peptides.

The oral delivery of peptides and proteins has been hampered by an array of obstacles. However, several promising novel oral delivery systems have been developed. This paper reviews the most advanced oral formulation technologies, and highlights key lessons and implications from studies undertaken to date with these oral formulations. Special interest is given to oral salmon calcitonin (CT), glucagon-like peptide-1 (GLP-1), insulin, PYY-(3-36), recombinant human parathyroid hormone (rhPTH(1-31)-NH2) and PTH(1-34), by different technologies. The issues addressed include (i) interaction with water, (ii) interaction with food, (iii) diurnal variation, (iv) inter- and intra-subject variability, (v) correlation between efficacy and exposure and (vi) key deliverables of different technologies. These key lessons may aid research in the development of other oral formulations.

Proresolving and tissue-protective actions of annexin A1-based cleavage-resistant peptides are mediated by formyl peptide receptor 2/lipoxin A4 receptor.

Endogenous mechanisms regulating the host response during inflammation resolution are critical in ensuring disposal of noxious stimuli and return to homeostasis. In this article, we engineered novel Annexin A1 (AnxA1)-based peptides, AnxA1(2-50), that displayed specific binding to the AnxA1 receptor (formyl peptide receptor 2/Lipoxin A4 receptor [FPR2/ALX]; IC50 ∼4 nM). Intravenous administration of AnxA1(2-50) markedly reduced (>60%) leukocyte adhesion to postcapillary venules in wild type and Fpr1(-/-), but not Fpr2/Alx(-/-), mice. Generation of a metabolically stable form of this peptide (CR-AnxA1(2-50)), engineered by substituting a cleavage site shared by human proteinase 3 and neutrophil elastase, yielded an agonist that was resistant to neutrophil-mediated cleavage and displayed enhanced proresolving actions: accelerated resolution of self-limited inflammation and enhanced macrophage efferocytosis after sterile injury, when compared with AnxA1(2-50). These actions were retained with human primary leukocytes where CR-AnxA1(2-50) decreased neutrophil-endothelial interactions (∼25-45%), and stimulated neutrophil apoptosis and macrophage efferocytosis (∼45%). In murine cardiac ischemia/reperfusion injury, CR-AnxA1(2-50) elicited tissue-protective actions reducing infarct size (∼60%) and incidence of 24-h death. These results identify AnxA1(2-50) and CR-AnxA1(2-50) as FPR2/ALX agonists that harness the proresolving actions of AnxA1, and thus may represent therapeutic tools for treatment of inflammatory conditions.

Pharmacokinetics of oral recombinant human parathyroid hormone [rhPTH(1-31)NH2] in postmenopausal women with osteoporosis.

BACKGROUND AND OBJECTIVES: Teriparatide [rhPTH(1-34)OH] is a subcutaneously administered bone anabolic drug that increases bone mineral density (BMD) and reduces the risk of osteoporotic fracture. Because rhPTH(1-34)OH is administered by injection, oral delivery is a desirable alternative. However, the peroral delivery of peptides is challenging due to their susceptibility to protease digestion and low permeability through the intestinal layers. The objective of this study was to assess the pharmacokinetics of a PTH analog (rhPTH(1-31)NH2) in a novel oral tablet formulation and to compare them to subcutaneously administered teriparatide in postmenopausal osteoporotic women in a phase 2 proof-of-concept clinical study. METHODS: This was a 24-week repeat-dose, randomized, double blind, parallel group phase 2 study with three once-daily treatment arms: oral rhPTH(1-31)NH2 tablets (5 mg), matching placebo tablets, and open-label teriparatide (20 µg daily subcutaneous injection). The primary endpoint of this study was to assess the change in lumbar spine BMD of orally administered rhPTH(1-31)NH2 tablets compared to baseline. This study was conducted at three sites in Denmark and at one site in Estonia. The patients included were women diagnosed with postmenopausal osteoporosis as detected by lumbar spine dual-energy X-ray absorptiometry, with exclusion of those with prior treatment with bone-active agents. The study treatment consisted of orally formulated recombinant human PTH(1-31)NH2, placebo, or subcutaneous teriparatide as an active comparator. RESULTS: The pharmacokinetic profile at first and last dose was evaluated and correlated with the primary endpoint, which was to characterize the percent change from baseline in BMD of the lumbar spine after 24 weeks of once daily treatment with rhPTH(1-31)NH2. The pharmacokinetic profile for the tablets showed a pulsatile peak with durations of at least 1 h but less than 5 h, which is consistent with the requirement for bone anabolic activity. The mean maximum (peak) plasma drug concentration (C max) values for patients receiving tablets at week 0 (n = 32) and week 24 (n = 28) were 295 and 207 pg/mL, respectively. The mean time to reach maximum (peak) plasma concentration following drug administration (t max) for both week 0 and week 24 was 3.25 h. The mean area under the concentration-time curve (AUC) for week 0 and week 24 was 178 and 141 pg·h/mL, respectively. No significant differences were observed between weeks 0 and 24 in any pharmacokinetic parameters tested, demonstrating good reproducibility, no time-dependent changes, and little or no accumulation of the study drug. The systemic exposure as measured by C max values was higher for the oral tablets formulation than for subcutaneous teriparatide. CONCLUSIONS: The observed data demonstrate that the enteric-coated tablet formulation technology was able to generate consistently robust and pulsatile levels of exposure of rhPTH(1-31)NH2. There was no apparent correlation between higher exposures and adverse events, even though the pharmacokinetic variability was somewhat higher with the tablets. These positive results recommend this orally delivered drug candidate for further clinical development.

The calcitonin and glucocorticoids combination: mechanistic insights into their class-effect synergy in experimental arthritis.

INTRODUCTION: Previous work reported the anti-arthritic synergy afforded by combining calcitonin (CT) and glucocorticoids (GC). Here we focus on the pairing of elcatonin (eCT) and dexamethasone (Dex), querying whether: i) this was a class-effect action; ii) mechanistic insights could be unveiled; iii) the synergy affected canonical GC adverse effects. METHODS: Using the rat collagen-induced arthritis model, different combinations of eCT and Dex, were administered from disease onset to peak (day 11 to 18). Macroscopic disease score was monitored throughout, with biochemical and histological analyses conducted on plasma and tissues at day 18. The effect on acute hyperglycaemia and liver enzyme message were also assessed. RESULTS: Whilst eCT alone was inactive, it synergised at 1 µg/kg with low doses of Dex (7.5 or 15 µg/kg) to yield an anti-arthritic efficacy equivalent to a 4- to 7-fold higher Dex dose. Mechanistically, the anti-arthritic synergy corresponded to a marked attenuation in RA-relevant analytes. CXCL5 expression, in both plasma and joint, was markedly inhibited by the co-therapy. Finally, co-administration of eCT did not exacerbate metrics of GC adverse effects, and rescued some of them. CONCLUSIONS: We present evidence of a class-effect action for the anti-arthritic synergy of CT/GC combination, underpinned by the powerful inhibition of joint destruction markers. Furthermore, we identify CXCL5 as a marker for the combination therapy with potential diagnostic and prognostic utility. Substantial GC dose reduction, together with the absence of exacerbated adverse effects, indicated a significant clinical potential for this co-therapy in RA and beyond.

Additive Effects of Mechanical Marrow Ablation and PTH Treatment on de Novo Bone Formation in Mature Adult Rats.

Mechanical ablation of bone marrow in young rats induces rapid but transient bone growth, which can be enhanced and maintained for three weeks by the administration of parathyroid hormone (PTH). Additionally, marrow ablation, followed by PTH treatment for three months leads to increased cortical thickness. In this study, we sought to determine whether PTH enhances bone formation after marrow ablation in aged rats. Aged rats underwent unilateral femoral marrow ablation and treatment with PTH or vehicle for four weeks. Both femurs from each rat were analyzed by X-ray and pQCT, then analyzed either by microCT, histology or biomechanical testing. Marrow ablation alone induced transient bone formation of low abundance that persisted over four weeks, while marrow ablation followed by PTH induced bone formation of high abundance that also persisted over four weeks. Our data confirms that the osteo-inducive effect of marrow ablation and the additive effect of marrow ablation, followed by PTH, occurs in aged rats. Our observations open new avenues of investigations in the field of tissue regeneration. Local marrow ablation, in conjunction with an anabolic agent, might provide a new platform for rapid site-directed bone growth in areas of high bone loss, such as in the hip and wrist, which are subject to fracture.

Dramatic increase in cortical thickness induced by femoral marrow ablation followed by a 3-month treatment with PTH in rats.

We previously reported that following mechanical ablation of the marrow from the midshaft of rat femurs, there is a rapid and abundant but transient growth of bone, and this growth is enhanced and maintained over a 3-week period by the bone anabolic hormone parathyroid hormone (PTH). Here, we asked whether further treatment with PTH or bisphosphonates can extend the half-life of the new bone formed in lieu of marrow. We subjected the left femur of rats to mechanical marrow ablation and treated the animals 5 days a week with PTH for 3 weeks (or with vehicle as a control) to replace the marrow by bone. Some rats were euthanized and used as positive controls or treated with vehicle, PTH, or the bisphosphonate alendronate for a further 9 weeks. We subjected both femurs from each rat to soft X-ray, peripheral quantitative computed tomography (pQCT), micro-computed tomography (microCT), dynamic histomorphometry analysis, and biomechanical testing. We also determined the concentrations of serum osteocalcin to confirm the efficacy of PTH. Treatment with PTH for 3 months dramatically enhanced endosteal and periosteal bone formation, leading to a 30% increase in cortical thickness. In contrast, alendronate protected the bone that had formed in the femoral marrow cavity after marrow ablation and 3 weeks of treatment with PTH but failed to promote endosteal bone growth or to improve the biomechanical properties of ablated femurs. We further asked whether calcium-phosphate cements could potentiate the formation of bone after marrow ablation. Marrow cavities from ablated femurs were filled with one of two calcium-phosphate cements, and rats were treated with PTH or PBS for 84 days. Both cements helped to protect the new bone formed after ablation. To some extent, they promoted the formation of bone after ablation, even in the absence of any anabolic hormone. Our data therefore expand the role of PTH in bone engineering and open new avenues of investigation to the field of regenerative medicine and tissue engineering. Local bone marrow aspiration in conjunction with an anabolic agent, a bisphosphonate, or a calcium-phosphate cement might provide a new platform for rapid preferential site-directed bone growth in areas of high bone loss.

Replacement of bone marrow by bone in rat femurs: the bone bioreactor.

During development and repair of bone, two distinct yet complementary mechanisms, intramembranous and endochondral, mediate new bone formation via osteoblasts. Because mechanical bone marrow ablation leads to the rapid and transient formation of new bone in the marrow cavity, we postulated that parathyroid hormone (PTH), which is a bone anabolic hormone, enhances the formation of new bone that forms after marrow ablation. We subjected the left femur of rats to mechanical marrow ablation, or sham operation, and injected the animals daily with PTH or vehicle for 1, 2, or 3 weeks in a first experiment, then with PTH, parathyroid hormone-related peptide (PTHrP), or vehicle for 3 weeks in a second experiment. We subjected both femurs from each rat to soft X-ray, peripheral quantitative computed tomography, computed tomography on a microscale, and histological analysis, and determined the concentration of serum osteocalcin. In addition, in the second experiment, we determined the serum concentration of calcium, tartrate-resistant acid phosphatase (TRAP), and receptor activator of NF-kappaB ligand (RANKL) at 3 weeks, and subjected femurs to biomechanical testing. Following treatment with PTH or PTHrP for 3 weeks, bone filled the marrow cavity of the shafts whose marrow had been ablated. PTH increased trabecular density in the right femur, but failed to induce bone formation in the medullary region of the right unoperated femoral shafts. The newly formed bone endowed left femoral shafts with improved biomechanical properties when compared to those of right femurs and left femurs from control, sham-operated, and vehicle-treated rats. PTHrP, like PTH, increased serum osteocalcin, but neither increased serum calcium, TRAP, or RANKL at 3 weeks. Our results reveal that the newly formed bone that follows marrow ablation is responsive to PTH, expand the role of PTH in bone, and might open new avenues of investigations to the field of regenerative medicine and tissue engineering. Local bone marrow removal in conjunction with pharmacologic intervention with an anabolic agent might provide a technique for rapid preferential site-directed bone growth in areas of high bone loss.

Production of salmon calcitonin by direct expression of a glycine-extended precursor in Escherichia coli.

The export of heterologous products into the conditioned medium of an Escherichia coli culture offers the advantages of a higher product yield, an increased probability of recovering an intact recombinant protein, proper folding for biological activity, and greater stability of a secreted product. In this report, we describe the development of an optimized direct expression system, designed to maximize the extracellular accumulation of recombinant glycine-extended salmon calcitonin peptide (sCTgly). We have used dual promoters, an ompA signal sequence, co-expression of homologous secretion factor genes, and multiple gene cartridges to express the sCTgly. High-density fermentation conditions have been developed that allow for the selective secretion and accumulation of the expressed sCTgly at very high levels. Purification and in vitro enzymatic conversion by peptidylglycine alpha-amidating monooxygenase yields authentic, biologically active salmon calcitonin. This recombinant production technology is applicable to a variety of amidated peptide hormones.