Harnessing ceramic hydroxyapatite as an effective polishing strategy to remove product- and process-related impurities in bispecific antibody purification.

Bispecific antibody (bsAb), a novel therapeutic modality, provides excellent treatment efficacy, yet poses numerous challenges to downstream process development, which are mainly due to the intricate diversity of bsAb structures and impurity profiles. Ceramic hydroxyapatite (CHT), a mixed-mode medium, allows proteins to interact with its calcium sites (C-sites) through metal affinity and/or its phosphate sites (P-sites) through cation exchange interactions. This dual-binding capability potentially offers unique bind and elute behaviours for different proteins of interest, resulting in optimal product purity when suitable elution conditions are employed. In this study, the effectiveness of CHT as a polishing step for bsAb purification was investigated across three model molecules and benchmarked against the traditional cation exchange chromatography (CEX). For both asymmetric and symmetric IgG-like bsAb post Protein A eluates, at least 97% product purity was achieved after CHT polishing. CHT delivered a superior aggregate clearance to CEX, resulting in low high molecular weight (HMW) impurities (0.5%) and low process-related impurities in the product pools. Moreover, CHT significantly mitigated "chromatography-induced aggregation" whereas eightfold more HMW was generated by CEX. This study illustrated the developability of CHT in effectively eliminating low molecular weight (LMW) impurities through post-load-wash (PLW) optimization, resulting in an additional reduction of up to 48% in LMW impurities. A mechanistic explanation regarding the performance of impurity removal and mitigation of the chromatography-induced aggregation by CHT was proposed, illustrating unique CHT capability is potentially driven by C-site cooperation, of which effectiveness could depend on the bsAb composition and size.

Osteoblast/osteocyte-derived interleukin-11 regulates osteogenesis and systemic adipogenesis.

Exercise results in mechanical loading of the bone and stimulates energy expenditure in the adipose tissue. It is therefore likely that the bone secretes factors to communicate with adipose tissue in response to mechanical loading. Interleukin (IL)-11 is known to be expressed in the bone, it is upregulated by mechanical loading, enhances osteogenesis and suppresses adipogenesis. Here, we show that systemic IL-11 deletion (IL-11-/-) results in reduced bone mass, suppressed bone formation response to mechanical loading, enhanced expression of Wnt inhibitors, and suppressed Wnt signaling. At the same time, the enhancement of bone resorption by mechanical unloading was unaffected. Unexpectedly, IL-11-/- mice have increased systemic adiposity and glucose intolerance. Osteoblast/osteocyte-specific IL-11 deletion in osteocalcin-Cre;IL-11fl/fl mice have reduced serum IL-11 levels, blunted bone formation under mechanical loading, and increased systemic adiposity similar to IL-11-/- mice. Adipocyte-specific IL-11 deletion in adiponectin-Cre;IL-11fl/fl did not exhibit any abnormalities. We demonstrate that osteoblast/osteocyte-derived IL-11 controls both osteogenesis and systemic adiposity in response to mechanical loading, an important insight for our understanding of osteoporosis and metabolic syndromes.

Immortalized Canine Dystrophic Myoblast Cell Lines for Development of Peptide-Conjugated Splice-Switching Oligonucleotides.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease caused by frameshift or nonsense mutations in the DMD gene, resulting in the loss of dystrophin from muscle membranes. Exon skipping using splice-switching oligonucleotides (SSOs) restores the reading frame of DMD pre-mRNA by generating internally truncated but functional dystrophin protein. To potentiate effective tissue-specific targeting by functional SSOs, it is essential to perform accelerated and reliable in vitro screening-based assessment of novel oligonucleotides and drug delivery technologies, such as cell-penetrating peptides, before their in vivo pharmacokinetic and toxicity evaluation. We have established novel canine immortalized myoblast lines by transducing murine cyclin-dependent kinase-4 and human telomerase reverse transcriptase genes into myoblasts isolated from beagle-based wild-type or canine X-linked muscular dystrophy in Japan (CXMDJ) dogs. These myoblast lines exhibited improved myogenic differentiation and increased proliferation rates compared with passage-15 primary parental myoblasts, and their potential to differentiate into myotubes was maintained in later passages. Using these dystrophin-deficient immortalized myoblast lines, we demonstrate that a novel cell-penetrating peptide (Pip8b2)-conjugated SSO markedly improved multiexon skipping activity compared with the respective naked phosphorodiamidate morpholino oligomers. In vitro screening using immortalized canine cell lines will provide a basis for further pharmacological studies on drug delivery tools.

Dystrobrevin alpha gene is a direct target of the vitamin D receptor in muscle.

The biologically active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (VD3), exerts its tissue-specific actions through binding to its intracellular vitamin D receptor (VDR) which functions as a heterodimer with retinoid X receptor (RXR) to recognize vitamin D response elements (VDRE) and activate target genes. Upregulation of VDR in murine skeletal muscle cells occurs concomitantly with transcriptional regulation of key myogenic factors upon VD3 administration, reinforcing the notion that VD3 exerts beneficial effects on muscle. Herein we elucidated the regulatory role of VD3/VDR axis on the expression of dystrobrevin alpha (DTNA), a member of dystrophin-associated protein complex (DAPC). In C2C12 cells, Dtna and VDR gene and protein expression were upregulated by 1-50 nM of VD3 during all stages of myogenic differentiation. In the dystrophic-derived H2K-mdx52 cells, upregulation of DTNA by VD3 occurred upon co-transfection of VDR and RXR expression vectors. Silencing of MyoD1, an E-box binding myogenic transcription factor, did not alter the VD3-mediated Dtna induction, but Vdr silencing abolished this effect. We also demonstrated that VD3 administration enhanced the muscle-specific Dtna promoter activity in presence of VDR/RXR only. Through site-directed mutagenesis and chromatin immunoprecipitation assays, we have validated a VDRE site in Dtna promoter in myogenic cells. We have thus proved that the positive regulation of Dtna by VD3 observed during in vitro murine myogenic differentiation is VDR mediated and specific. The current study reveals a novel mechanism of VDR-mediated regulation for Dtna, which may be positively explored in treatments aiming to stabilize the DAPC in musculoskeletal diseases.

How do we sense phosphate to regulate serum phosphate level?

Abnormal phosphate levels result in several pathological conditions such as rickets/osteomalacia and ectopic calcification indicating that there must be a system that regulates phosphate level within a narrow range. FGF23 has been shown to be an essential hormone regulating serum phosphate level. FGF23 binds to Klotho-FGF receptor complex to reduce serum phosphate level. Several reports suggested that FGF receptor is involved in the regulation of FGF23 production. It has been also shown that high extracellular phosphate can activate several intracellular signaling pathways. However, it has been unclear whether and how phosphate regulates FGF23 production in vivo. Our recent results indicate that high extracellular phosphate directly activates FGF receptor 1 and the downstream intracellular signaling enhances FGF23 production. Thus, there is a negative feedback system for the regulation of serum phosphate level involving FGF receptor and FGF23. We propose that FGF receptor works at least as one of phosphate sensors in the maintenance of serum phosphate level.

Peptide-conjugate antisense based splice-correction for Duchenne muscular dystrophy and other neuromuscular diseases.

Duchenne muscular dystrophy (DMD) is an X-linked disorder characterized by progressive muscle degeneration, caused by the absence of dystrophin. Exon skipping by antisense oligonucleotides (ASOs) has recently gained recognition as therapeutic approach in DMD. Conjugation of a peptide to the phosphorodiamidate morpholino backbone (PMO) of ASOs generated the peptide-conjugated PMOs (PPMOs) that exhibit a dramatically improved pharmacokinetic profile. When tested in animal models, PPMOs demonstrate effective exon skipping in target muscles and prolonged duration of dystrophin restoration after a treatment regime. Herein we summarize the main pathophysiological features of DMD and the emergence of PPMOs as promising exon skipping agents aiming to rescue defective gene expression in DMD and other neuromuscular diseases. The listed PPMO laboratory findings correspond to latest trends in the field and highlight the obstacles that must be overcome prior to translating the animal-based research into clinical trials tailored to the needs of patients suffering from neuromuscular diseases.

Activation of unliganded FGF receptor by extracellular phosphate potentiates proteolytic protection of FGF23 by its O-glycosylation.

Fibroblast growth factor (FGF) 23 produced by bone is a hormone that decreases serum phosphate (Pi). Reflecting its central role in Pi control, serum FGF23 is tightly regulated by serum Pi alterations. FGF23 levels are regulated by the transcriptional event and posttranslational cleavage into inactive fragments before its secretion. For the latter, O-glycosylation of FGF23 by GALNT3 gene product prevents the cleavage, leading to an increase in serum FGF23. However, the molecular basis of Pi sensing in the regulation of serum FGF23 remains elusive. In this study, we showed that high Pi diet enhanced the skeletal expression of Galnt3, but not Fgf23, with expected increases in serum FGF23 and Pi in mice. Galnt3 induction by high Pi was further observed in osteoblastic UMR 106 cells, and this was mediated by activation of the extracellular signal-regulated kinase (ERK) pathway. Through proteomic searches for the upstream sensor for high Pi, we identified one subtype of the FGF receptor (FGFR1c), which was phosphorylated by high Pi in the absence of FGFs. The mode of unliganded FGFR activation by high Pi appeared different from that of FGFR bound to a canonical FGFR ligand (FGF2) when phosphorylation of the FGFR substrate 2α and ERK was monitored. Finally, we showed that an FGFR inhibitor and conditional deletion of Fgfr1 in osteoblasts/osteocytes abrogated high Pi diet-induced increases in serum FGF23 and femoral Galnt3 expression in mice. Thus, these findings uncover an unrecognized facet of unliganded FGFR function and illustrate a Pi-sensing pathway involved in regulation of FGF23 production.

Scavenger Receptor Class A1 Mediates Uptake of Morpholino Antisense Oligonucleotide into Dystrophic Skeletal Muscle.

Exon skipping using phosphorodiamidate morpholino oligomers (PMOs) is a promising treatment strategy for Duchenne muscular dystrophy (DMD). The most significant limitation of these clinically used compounds is their lack of delivery systems that target muscles; thus, cell-penetrating peptides are being developed to enhance uptake into muscles. Recently, we reported that uptake of peptide-conjugated PMOs into myofibers was mediated by scavenger receptor class A (SR-A), which binds negatively charged ligands. However, the mechanism by which the naked PMOs are taken up into fibers is poorly understood. In this study, we found that PMO uptake and exon-skipping efficiency were promoted in dystrophin-deficient myotubes via endocytosis through a caveolin-dependent pathway. Interestingly, SR-A1 was upregulated and localized in juxtaposition with caveolin-3 in these myotubes and promoted PMO-induced exon skipping. SR-A1 was also upregulated in the skeletal muscle of mdx52 mice and mediated PMO uptake. In addition, PMOs with neutral backbones had negative zeta potentials owing to their nucleobase compositions and interacted with SR-A1. In conclusion, PMOs with negative zeta potential were taken up into dystrophin-deficient skeletal muscle by upregulated SR-A1. Therefore, the development of a drug delivery system targeting SR-A1 could lead to highly efficient exon-skipping therapies for DMD.

In Vitro Multiexon Skipping by Antisense PMOs in Dystrophic Dog and Exon 7-Deleted DMD Patient.

Antisense oligonucleotide induced exon skipping emerges as a promising therapeutic strategy for patients suffering from a devastating muscle disorder Duchenne muscular dystrophy (DMD). Systemic administration of antisense phosphorodiamidate morpholino oligomers (PMOs) targeting exons 6 and 8 in dystrophin mRNA of the canine X-linked muscular dystrophy model in Japan (CXMDJ) that lacks exon 7, restored dystrophin expression throughout skeletal muscle and ameliorated skeletal muscle pathology and function. However, the antisense PMO regime used in CXMDJ could not be considered for a direct application to DMD patients so far, because this type of mutation is quite rare. We have identified a DMD patient with an exon 7 deletion; and tried a direct translation of the antisense PMOs used in dog models to the DMD patient's cells. We converted fibroblasts obtained from CXMDJ dogs and from the DMD patient to myotubes by MyoD transduction using fluorescence-activated cell sorting (FACS). We subsequently designed antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 and administered them as a cocktail to the in vitro generated dog or human myotubes. In both cases, we observed comparable skipping efficacy of exons 6 and 8 and restoration of dystrophin protein. The accompanying skipping of exon 9, which does not alter the reading frame, varied according to the cell origin. The antisense PMOs originally administered to the CXMDJ dog model were capable of inducing multi-exon skipping of the dystrophin gene on the FACS-aided MyoD-transduced fibroblasts derived from an exon 7-deleted DMD patient. These data support the suitability of dog as a laboratory model for DMD because the similarity of dystrophin sequences allowed a successful translation of the dog's PMOs to DMD patients cells.

The Pharmacological Profile of a Novel Highly Potent Bisphosphonate, OX14 (1-Fluoro-2-(Imidazo-[1,2-α]Pyridin-3-yl)-Ethyl-Bisphosphonate).

Bisphosphonates are widely used in the treatment of clinical disorders characterized by increased bone resorption, including osteoporosis, Paget's disease, and the skeletal complications of malignancy. The antiresorptive potency of the nitrogen-containing bisphosphonates on bone in vivo is now recognized to depend upon two key properties, namely mineral binding affinity and inhibitory activity on farnesyl pyrophosphate synthase (FPPS), and these properties vary independently of each other in individual bisphosphonates. The better understanding of structure activity relationships among the bisphosphonates has enabled us to design a series of novel bisphosphonates with a range of mineral binding properties and antiresorptive potencies. Among these is a highly potent bisphosphonate, 1-fluoro-2-(imidazo-[1,2 alpha]pyridin-3-yl)-ethyl-bisphosphonate, also known as OX14, which is a strong inhibitor of FPPS, but has lower binding affinity for bone mineral than most of the commonly studied bisphosphonates. The aim of this work was to characterize OX14 pharmacologically in relation to several of the bisphosphonates currently used clinically. When OX14 was compared to zoledronate (ZOL), risedronate (RIS), and minodronate (MIN), it was as potent at inhibiting FPPS in vitro but had significantly lower binding affinity to hydroxyapatite (HAP) columns than ALN, ZOL, RIS, and MIN. When injected i.v. into growing Sprague Dawley rats, OX14 was excreted into the urine to a greater extent than the other bisphosphonates, indicating reduced short-term skeletal uptake and retention. In studies in both Sprague Dawley rats and C57BL/6J mice, OX14 inhibited bone resorption, with an antiresorptive potency equivalent to or greater than the comparator bisphosphonates. In the JJN3-NSG murine model of myeloma-induced bone disease, OX14 significantly prevented the formation of osteolytic lesions (p < 0.05). In summary, OX14 is a new, highly potent bisphosphonate with lower bone binding affinity than other clinically relevant bisphosphonates. This renders OX14 an interesting potential candidate for further development for its potential skeletal and nonskeletal benefits. © 2017 American Society for Bone and Mineral Research.

The inhibition of human farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates. Elucidating the role of active site threonine 201 and tyrosine 204 residues using enzyme mutants.

Farnesyl pyrophosphate synthase (FPPS) is the major molecular target of nitrogen-containing bisphosphonates (N-BPs), used clinically as bone resorption inhibitors. We investigated the role of threonine 201 (Thr201) and tyrosine 204 (Tyr204) residues in substrate binding, catalysis and inhibition by N-BPs, employing kinetic and crystallographic studies of mutated FPPS proteins. Mutants of Thr201 illustrated the importance of the methyl group in aiding the formation of the Isopentenyl pyrophosphate (IPP) binding site, while Tyr204 mutations revealed the unknown role of this residue in both catalysis and IPP binding. The interaction between Thr201 and the side chain nitrogen of N-BP was shown to be important for tight binding inhibition by zoledronate (ZOL) and risedronate (RIS), although RIS was also still capable of interacting with the main-chain carbonyl of Lys200. The interaction of RIS with the phenyl ring of Tyr204 proved essential for the maintenance of the isomerized enzyme-inhibitor complex. Studies with conformationally restricted analogues of RIS reaffirmed the importance of Thr201 in the formation of hydrogen bonds with N-BPs. In conclusion we have identified new features of FPPS inhibition by N-BPs and revealed unknown roles of the active site residues in catalysis and substrate binding.

The relationship between the chemistry and biological activity of the bisphosphonates.

The ability of bisphosphonates ((HO)(2)P(O)CR(1)R(2)P(O)(OH)(2)) to inhibit bone resorption has been known since the 1960s, but it is only recently that a detailed molecular understanding of the relationship between chemical structures and biological activity has begun to emerge. The early development of chemistry in this area was largely empirical and based on modifying R(2) groups in a variety of ways. Apart from the general ability of bisphosphonates to chelate Ca(2+) and thus target the calcium phosphate mineral component of bone, attempts to refine clear structure-activity relationships had led to ambiguous or seemingly contradictory results. However, there was increasing evidence for cellular effects, and eventually the earliest bisphosphonate drugs, such as clodronate (R(1)=R(2)=Cl) and etidronate (R(1)=OH, R(2)=CH(3)), were shown to exert intracellular actions via the formation in vivo of drug derivatives of ATP. The observation that pamidronate, a bisphosphonate with R(1)=OH and R(2)=CH(2)CH(2)NH(2), exhibited higher potency than previously known bisphosphonate drugs represented the first step towards the later recognition of the critical importance of having nitrogen in the R(2) side chain. The synthesis and biological evaluation of a large number of nitrogen-containing bisphosphonates took place particularly in the 1980s, but still with an incomplete understanding of their structure-activity relationships. A major advance was the discovery that the anti-resorptive effects of the nitrogen-containing bisphosphonates (including alendronate, risedronate, ibandronate, and zoledronate) on osteoclasts appear to result from their potency as inhibitors of the enzyme farnesyl pyrophosphate synthase (FPPS), a key branch-point enzyme in the mevalonate pathway. FPPS generates isoprenoid lipids utilized in sterol synthesis and for the post-translational modification of small GTP-binding proteins essential for osteoclast function. Effects on other cellular targets, such as osteocytes, may also be important. Over the years many hundreds of bisphosphonates have been synthesized and studied. Interest in expanding the structural scope of the bisphosphonate class has also motivated new approaches to the chemical synthesis of these compounds. Recent chemical innovations include the synthesis of fluorescently labeled bisphosphonates, which has enabled studies of the biodistribution of these drugs. As a class, bisphosphonates share common properties. However, as with other classes of drugs, there are chemical, biochemical, and pharmacological differences among the individual compounds. Differences in mineral binding affinities among bisphosphonates influence their differential distribution within bone, their biological potency, and their duration of action. The overall pharmacological effects of bisphosphonates on bone, therefore, appear to depend upon these two key properties of affinity for bone mineral and inhibitory effects on osteoclasts. The relative contributions of these properties differ among individual bisphosphonates and help determine their clinical behavior and effectiveness.