Validation of a New Methodology to Create Oral Drugs beyond the Rule of 5 for Intracellular Tough Targets.

Establishing a technological platform for creating clinical compounds inhibiting intracellular protein-protein interactions (PPIs) can open the door to many valuable drugs. Although small molecules and antibodies are mainstream modalities, they are not suitable for a target protein that lacks a deep cavity for a small molecule to bind or a protein found in intracellular space out of an antibody's reach. One possible approach to access these targets is to utilize so-called middle-size cyclic peptides (defined here as those with a molecular weight of 1000-2000 g/mol). In this study, we validated a new methodology to create oral drugs beyond the rule of 5 for intracellular tough targets by elucidating structural features and physicochemical properties for drug-like cyclic peptides and developing library technologies to afford highly N-alkylated cyclic peptide hits. We discovered a KRAS inhibitory clinical compound (LUNA18) as the first example of our platform technology.

Highlights of the 13th Japan Bioanalysis Forum Symposium.

Approximately 280 people from pharmaceutical industries, contractors, academic institutions and regulatory authorities attended the 13th Japan Bioanalysis Forum Symposium. The symposium was held via web to prevent the spread of COVID-19 from the 28 February to 2 March 2022. The theme of the symposium was 'All for One Goal', and the event has provided an opportunity for open discussion among researchers with different backgrounds but who share a common goal: "to deliver more effective and safe pharmaceuticals to patients as quickly as possible". The speakers focused on hot topics in bioanalysis, including chromatography, biomarker analysis, cell and gene therapy, COVID-19 and antidrug antibody. This symposium provided a great opportunity for the participants to have meaningful discussions, even though 'on the web' was a limited space.

Highlights of the 12th Japan Bioanalysis Forum Symposium.

Approximately 300 people associated with pharmaceutical industries, contractors, academic institutions and regulatory authorities attended the 12th Japan Bioanalysis Forum Symposium. The webinar was conducted from 9 to 11 March 2021. The theme of the symposium was 'for the next generation', and the event provided 'an opportunity for young researchers in bioanalysis (including students)' and 'an opportunity to discuss new frontiers of bioanalysis'. The speakers focused on hot topics of bioanalysis, including biomarker analysis, patient centric sampling, virtual clinical trials, gene therapy, cancer genome medicine and therapeutic middle molecules. The symposium presented a platform for the discussion of the prospects and challenges facing bioanalysts working in the field of pharmacokinetics. This report presents the key issues discussed.

A Novel Total Drug Assay for Quantification of Anti-C5 Therapeutic Monoclonal Antibody in the Presence of Abundant Target.

SKY59 or RO7112689 is a humanized monoclonal antibody against complement protein C5 with pH-dependent C5-binding and neonatal Fc receptor-mediated recycling capabilities, which result in long-lasting neutralization of C5. We developed and validated a novel total drug assay for quantification of target-binding competent SKY59 in the presence of endogenous C5 in cynomolgus monkey plasma. The target-binding competent SKY59 was determined after complex formation by the addition of recombinant monkey C5 using goat anti-human IgG-heavy chain monkey-adsorbed polyclonal antibody as a capture antibody and rabbit anti-C5 monoclonal antibody (mAb) non-competing with SKY59 for detection. The total SKY59 assay was shown to be accurate and precise over the range of 0.05-3.2 µg/mL as well as be tolerant to more than 400 µg/mL of C5 (~ 3000-fold molar excess of target). We also developed and validated a total C5 assay, confirmed selectivity and parallelism, and verified the utility of recombinant monkey C5 for the total C5 assay as well as the total SKY59 assay. Furthermore, we used these validated methods to measure SKY59 and C5 concentrations in cynomolgus monkey plasma samples in a toxicology study. This total drug assay can be applied not only to other antibody therapeutics against shed/soluble targets when a non-competing reagent mAb is available but also for clinical studies when a reagent mAb specific for engineered Fc region on a therapeutic mAb is available.

Evidence of an intestinal phosphate transporter alternative to type IIb sodium-dependent phosphate transporter in rats with chronic kidney disease.

BACKGROUND: Phosphate is absorbed in the small intestine via passive flow and active transport.NaPi-IIb, a type II sodium-dependent phosphate transporter, is considered to mediate active phosphate transport in rodents. To study the regulation of intestinal phosphate transport in chronic kidney disease (CKD), we analyzed the expression levels of NaPi-IIb, pituitary-specific transcription factor 1 (PiT-1) and PiT-2 and the kinetics of intestinal phosphate transport using two CKD models. METHODS: CKD was induced in rats via adenine orThy1 antibody injection. Phosphate uptake by intestinal brush border membrane vesicles (BBMV) and the messenger RNA (mRNA) expression of NaPi-IIb, PiT-1 and PiT-2 were analyzed. The protein expression level of NaPi-IIb was measured by mass spectrometry (e.g. liquid chromatography tandem mass spectrometry). RESULTS: In normal rats, phosphate uptake into BBMV consisted of a single saturable component and its Michaelis constant (Km) was comparable to that of NaPi-IIb. The maximum velocity (Vmax) correlated with mRNA and protein levels of NaPi-IIb. In the CKD models, intestinal phosphate uptake consisted of two saturable components. The Vmax of the higher-affinity transport, which is thought to be responsible for NaPi-IIb, significantly decreased and the decrease correlated with reduced NaPi-IIb expression. The Km of the lower-affinity transport was comparable to that of PiT-1 and -2. PiT-1 mRNA expression was much higher than that of PiT-2, suggesting that PiT-1 was mostly responsible for phosphate transport. CONCLUSIONS: This study suggests that the contribution of NaPi-IIb to intestinal phosphate absorption dramatically decreases in rats with CKD and that a low-affinity alternative to NaPi-IIb, in particular PiT-1, is upregulated in a compensatory manner in CKD.

Exploitation of Elevated Extracellular ATP to Specifically Direct Antibody to Tumor Microenvironment.

The extracellular adenosine triphosphate (ATP) concentration is highly elevated in the tumor microenvironment (TME) and remains tightly regulated in normal tissues. Using phage display technology, we establish a method to identify an antibody that can bind to an antigen only in the presence of ATP. Crystallography analysis reveals that ATP bound in between the antibody-antigen interface serves as a switch for antigen binding. In a transgenic mouse model overexpressing the antigen systemically, the ATP switch antibody binds to the antigen in tumors with minimal binding in normal tissues and plasma and inhibits tumor growth. Thus, we demonstrate that elevated extracellular ATP concentration can be exploited to specifically target the TME, giving therapeutic antibodies the ability to overcome on-target off-tumor toxicity.

MHC-associated peptide proteomics enabling highly sensitive detection of immunogenic sequences for the development of therapeutic antibodies with low immunogenicity.

Immunogenicity is a key factor capable of influencing the efficacy and safety of therapeutic antibodies. A recently developed method called MHC-associated peptide proteomics (MAPPs) uses liquid chromatography/mass spectrometry to identify the peptide sequences derived from a therapeutic protein that are presented by major histocompatibility complex class II (MHC II) on antigen-presenting cells, and therefore may induce immunogenicity. In this study, we developed a MAPPs technique (called Ab-MAPPs) that has high throughput and can efficiently identify the MHC II-presented peptides derived from therapeutic antibodies using magnetic nanoparticle beads coated with a hydrophilic polymer in the immunoprecipitation process. The magnetic beads could identify more peptides and sequence regions originating from infliximab and adalimumab in a shorter measurement time than Sepharose beads, which are commonly used for MAPPs. Several sequence regions identified by Ab-MAPPs from infliximab corresponded to immunogenic sequences reported by other methods, which suggests the method's high potential for identifying significant sequences involved in immunogenicity. Furthermore, our study suggests that the Ab-MAPPs method can recognize the difference of a single amino acid residue between similar antibody sequences with different levels of T-cell proliferation activity and can identify potentially immunogenic peptides with high binding affinity to MHC II. In conclusion, Ab-MAPPs is useful for identifying the immunogenic sequences of therapeutic antibodies and will contribute to the design of therapeutic antibodies with low immunogenicity during the drug discovery stage.

Novel hyaluronic acid-methotrexate conjugate suppresses joint inflammation in the rat knee: efficacy and safety evaluation in two rat arthritis models.

BACKGROUND: Methotrexate (MTX) is one of the most widely used medications to treat rheumatoid arthritis (RA), and recent studies have also suggested the potential benefit of the drug for the treatment of osteoarthritis (OA) of the knee. MTX is commonly administered in oral formulations, but is often associated with systemic adverse reactions. In an attempt to address this issue, we have shown previously that a conjugate of hyaluronic acid (HA) and MTX exhibits potential as a drug candidate for intra-articular treatment of inflammatory arthritis. In this study, we compare the efficacy and safety of an optimized HA-MTX conjugate, DK226, with that of MTX in inflammatory arthritis rat models. METHODS: In vitro activity of DK226 was assessed in human fibroblast-like synoviocytes (HFLS) and a synovial sarcoma cell line, SW982. Release of MTX from DK226 was investigated after incubation with rabbit synovial tissue homogenate or synovial fluid. In vivo efficacy of DK226 was evaluated in antigen-induced arthritis (AIA) and collagen-induced arthritis (CIA) in the rat knee. Pharmacokinetics and hematological toxicity after treatment with oral MTX or an intra-articular injection of DK226 were compared in AIA. RESULTS: Proliferation of HFLS and SW982 cells was inhibited by DK226, and the inhibitory activity was reversed by cotreatment with excess HA or anti-CD44 antibody. MTX was released from DK226 by incubation with rabbit synovial tissue homogenate or synovial fluid at pH 4.0, but not at pH 7.4. AIA was ameliorated by intra-articular DK226, but not by HA, as potently as oral MTX. Hematological toxicity was induced by oral MTX, but not by DK226. The maximum plasma concentration of MTX after oral MTX was 40 times higher than the concentration of MTX after an intra-articular injection of DK226. Knee swelling in AIA was inhibited by intra-articular injections of DK226, but not by free MTX or a mixture of HA and MTX. In CIA, an injection of DK226 into the right knee joint significantly reduced swelling and synovial inflammation of the treated knee joint, but had no effect on the untreated contralateral knee joint. CONCLUSIONS: DK226 exerted anti-arthritic effects in two different models of arthritis. The conjugate had a wider therapeutic window than oral MTX, and could be a future drug for treatment of arthritic disorders, including inflammatory OA.

A versatile method for protein-based antigen bioanalysis in non-clinical pharmacokinetics studies of a human monoclonal antibody drug by an immunoaffinity liquid chromatography-tandem mass spectrometry.

A versatile immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to quantify the total concentration of a protein-based antigen in non-clinical pharmacokinetics (PK) studies of a human monoclonal antibody drug. The method combines using magnetic beads that have been coated with a commercial anti-human Fc region antibody to capture an immune complex of the antigen and antibody drug, with subsequent digestion and quantification of the antigen-derived tryptic peptide via LC-MS/MS. Although a typical immunoassay or an immunoaffinity LC-MS/MS assay requires an antigen-specific antibody that uses a different epitope from the antibody drug, this method requires only a commercial anti-human Fc region antibody. The method was applied to quantify total receptor activator of nuclear factor-κB ligand (RANKL) in the presence of denosumab, a humanized monoclonal antibody (mAb) specific to RANKL. The assay was validated as fit-for-purpose and found to be accurate (<115% interbatch accuracies) and precise (<15%, interbatch coefficient of variation) across a range of 3.13-200ng/mL RANKL. Commercial enzyme-linked immunosorbent assay (ELISA) kit was not able to determine the total RANKL because interference by denosumab decreased recovery. In contrast, the antibody drug had less effect on the LC-MS/MS method. The method now provides a bioanalytical platform for developing other protein-based antigen assays in the early drug stage.

Selective SGLT2 inhibition by tofogliflozin reduces renal glucose reabsorption under hyperglycemic but not under hypo- or euglycemic conditions in rats.

To understand the risk of hypoglycemia associated with urinary glucose excretion (UGE) induced by sodium-glucose cotransporter (SGLT) inhibitors, it is necessary to know the relationship between the ratio of contribution of SGLT2 vs. SGLT1 to renal glucose reabsorption (RGR) and the glycemic levels in vivo. To examine the contributions of SGLT2 and SGLT1 in normal rats, we compared the RGR inhibition by tofogliflozin, a highly specific SGLT2 inhibitor, and phlorizin, an SGLT1 and SGLT2 (SGLT1/2) inhibitor, at plasma concentrations sufficient to completely inhibit rat SGLT2 (rSGLT2) while inhibiting rSGLT1 to different degrees. Under hyperglycemic conditions by glucose titration, tofogliflozin and phlorizin achieved ≥50% inhibition of RGR. Under hypoglycemic conditions by hyperinsulinemic clamp, RGR was reduced by 20-50% with phlorizin and by 1-5% with tofogliflozin, suggesting the smaller contribution of rSGLT2 to RGR under hypoglycemic conditions than under hyperglycemic conditions. Next, to evaluate the hypoglycemic potentials of SGLT1/2 inhibition, we measured the plasma glucose (PG) and endogenous glucose production (EGP) simultaneously after UGE induction by SGLT inhibitors. Tofogliflozin (400 ng/ml) induced UGE of about 2 mg·kg⁻¹·min⁻¹ and increased EGP by 1-2 mg·kg⁻¹·min⁻¹, resulting in PG in the normal range. Phlorizin (1,333 ng/ml) induced UGE of about 6 mg·kg⁻¹·min⁻¹ and increased EGP by about 4 mg·kg⁻¹·min⁻¹; this was more than with tofogliflozin, but the minimum PG was lower. These results suggest that the contribution of SGLT1 to RGR is greater under lower glycemic conditions than under hyperglycemic conditions and that SGLT2-selective inhibitors pose a lower risk of hypoglycemia than SGLT1/2 inhibitors.

Alteration of intracellular secretory acute phase response proteins expressed in human hepatocyte induced by exposure with interleukin-6.

Interleukin-6 (IL-6) is a principal proinflammatory cytokine inducing the acute phase response in various tissues, including liver. Here, we adopt the FD-LC-MS/MS method, consisting of fluorogenic derivatization (FD), separation by liquid chromatography (LC), and identification of proteins by LC-tandem mass spectrometry (MS/MS), to reveal how exposure to IL-6 alters temporally the intracellular secretory acute phase response (sAPR) proteins expressed in human hepatocytes as compared to non-exposure. Nine altered sAPR proteins were identified in cultures in response to IL-6. Seven of them (serum amyloid A protein, haptoglobin, fibrinogen α chain, fibrinogen ß chain, fibrinogen γ chain, α(1)-acid glycoprotein and α(1)-antitrypsin) were significantly increased and two (ß(2)-glycoprotein 1 and transferrin) were significantly decreased in response to IL-6. In addition, the transmission speed of transferrin might be much faster than the other sAPR proteins. These results suggest a different molecular mechanism for protein synthesis and the secretory pathway among the sAPR proteins. In this study, we observed the simultaneously and temporally altered expression of sAPR proteins which had been induced by exposure to IL-6 in human hepatocytes, in contrast to previous reports, in all of which the proteins were tested from the time they were secreted into the medium from the cells.

Design and synthesis of novel allosteric MEK inhibitor CH4987655 as an orally available anticancer agent.

The MAP kinase pathway is one of the most important pathways involved in cell proliferation and differentiation, and its components are promising targets for antitumor drugs. Design and synthesis of a novel MEK inhibitor, based on the 3D-structural information of the target enzyme, and then multidimensional optimization including metabolic stability, physicochemical properties and safety profiles were effectively performed and led to the identification of a clinical candidate for an orally available potent MEK inhibitor, CH4987655, possessing a unique 3-oxo-oxazinane ring structure at the 5-position of the benzamide core structure. CH4987655 exhibits slow dissociation from the MEK enzyme, remarkable in vivo antitumor efficacy both in mono- and combination therapy, desirable metabolic stability, and insignificant MEK inhibition in mouse brain, implying few CNS-related side effects in human. An excellent PK profile and clear target inhibition in PBMC were demonstrated in a healthy volunteer clinical study.

Systemic administration of C-type natriuretic peptide as a novel therapeutic strategy for skeletal dysplasias.

Skeletal dysplasias are a group of genetic disorders characterized by severe impairment of bone growth. Various forms of them add to produce a significant morbidity and mortality, yet no efficient drug therapy has been developed to date. We previously demonstrated that C-type natriuretic peptide (CNP), a member of the natriuretic peptide family, is a potent stimulator of endochondral bone growth. Furthermore, we exhibited that targeted overexpression of a CNP transgene in the growth plate rescued the impaired bone growth observed in a mouse model of achondroplasia (Ach), the most frequent form of human skeletal dysplasias, leading us to propose that CNP may prove to be an effective treatment for this disorder. In the present study, to elucidate whether or not the systemic administration of CNP is a novel drug therapy for skeletal dysplasias, we have investigated the effects of plasma CNP on impaired bone growth in Ach mice that specifically overexpress CNP in the liver under the control of human serum amyloid P component promoter or in those treated with a continuous CNP infusion system. Our results demonstrated that increased plasma CNP from the liver or by iv administration of synthetic CNP-22 rescued the impaired bone growth phenotype of Ach mice without significant adverse effects. These results indicate that treatment with systemic CNP is a potential therapeutic strategy for skeletal dysplasias, including Ach, in humans.

Simple and sensitive quantification of bioactive peptides in biological matrices using liquid chromatography/selected reaction monitoring mass spectrometry coupled with trichloroacetic acid clean-up.

A simple and sensitive liquid chromatography/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method has been developed for the quantification of bioactive peptides in biological fluids. The method employs protein precipitation with 4% trichloroacetic acid (TCA) and selected reaction monitoring (SRM) using an immonium ion as the product ion. This method was applied to determine the synthetic parathyroid hormone (PTH) analog (MW 1721) in rat plasma and human hepcidin-25 (MW 2789) in human serum. TCA clean-up showed a sufficient recovery for peptides with a MW of less than 3000, and would be useful as a simple and rapid method because of direct injection of the supernatant without evaporation or dilution. In addition, TCA clean-up allowed us not only to reduce sample preparation time, but also to select an immonium ion as a product ion of SRM, which led to detection more sensitive than SRM using other types of product ions. The lower limits of quantitation (LLOQs) of the PTH analog and the human hepcidin-25 were 0.2 ng/mL and 5 ng/mL, respectively. This method was fully validated with acceptable linearity, intra- and inter-assay precisions, and accuracy. Furthermore, this simple and rapid method is applicable to pharmacokinetic studies.

Probing the unfolding and refolding processes of carbonic anhydrase 2 using electrospray ionization mass spectrometry combined with pH jump.

A novel method for proving the time course of the unfolding and refolding processes of metalloprotein bovine carbonic anhydrase 2 (CA2) is demonstrated using electrospray ionization mass spectrometry (ESI MS) combined with pH jumps between 3.6 and 4.4. The shift in mass accompanied by the release or coordination of a zinc ion and the change in the charge state distribution were measured to evaluate the folding process. The time course of the ESI mass spectra revealed the existence of four types of ions in the experimental system, i.e., lower charged apo-CA2 and holo-CA2 ions and higher charged apo-CA2 and holo-CA2 ions. The deconvolution spectrum of the ion peak ensemble for each type of ion was processed and time course plots of the relative intensities of the four ions were prepared in order to analyze the folding processes. These analyses revealed the coexistence of two folding states of the lower and higher charged apo-CA2 under the condition of pH 3.6. The lower and higher charged apoproteins spontaneously refolded to the lower charged holoprotein by a pH jump from 3.6 to 4.4 without the addition of an extra zinc ion. The higher charged holoprotein observed during both the unfolding and refolding processes was considered to be an intermediate of the change in folding. The present study indicates that ESI MS combined with pH jump would be a powerful method to probe the unfolding and refolding of proteins. This method simultaneously measures mass spectra and analyzes the folding processes as a function of time using deconvolution spectra constructed by selecting a suitable m/z range for the analysis from the peaks of charge state distributions.

Ferrocene-based Diels-Alder derivatization for the determination of 1alpha-hydroxyvitamin D3 in rat plasma by high-performance liquid chromatography-electrospray tandem mass spectrometry.

A specific and sensitive liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method has been developed for the determination of 1alpha-hydroxyvitamin D(3) (1alphaOHD(3)) in rat plasma. A new ferrocene-based Cookson-type reagent, 4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD), designed and synthesized to be highly sensitive to vitamin D analogs in ESI, considerably improved the detection limit with 250 fg (359 amol)/injection. 1alphaOHD(3) in rat plasma was extracted with acetonitrile and then purified using Oasis HLB 96-well plates. After the precolumn derivatization with FMTAD, samples were subjected to LC/ESI-MS/MS employing a column-switching system. This method achieved a lower limit of quantitation of 5 pg from 0.1-mL plasma aliquots and 200-fold sensitivity of that without derivatization. The calibration curve (0.05-15 ng/mL) exhibited acceptable linearity (r>0.9966), intraassay precision ranged from 3.8 to 9.6%, interassay precision ranged from 3.0 to 17.0%, and accuracy was within 81.4-112.0%. This FMTAD derivatization method is considered very useful for determination of vitamin D analogs in ESI and applicable for biological samples.

The determination of 2beta-(3-hydroxypropoxy)-1alpha,25-dihydroxy vitamin D3 (ED-71) in human serum by high-performance liquid chromatography-electrospray tandem mass spectrometry.

A liquid chromatography-electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method for the determination of 2beta-(3-hydroxypropoxy)-1alpha,25-dihydroxy vitamin D3 (ED-71) in human serum has been developed. ED-71 in human serum was extracted using two solid-phase extraction steps on Bond Elut C18 and NH2 cartridge. The separation of ED-71 and preED-71 isomer was attained by LC using 2 mmol/L ammonium acetate-methanol (15:85, v/v) as a mobile phase on a Symmetry C18 column (5 microm, 150 mm x 2.1mm i.d.). ESI-MS/MS analysis was operated using selected reaction monitoring (SRM) in positive ion mode. The method achieved a lower limit of quantitation of 25 pg/mL. The calibration curve (25-3200 pg/mL) gave acceptable linearity (r>0.9964). Intra-assay precision ranged from 2.3 to 9.7%. Inter-assay precision ranged from 1.0 to 3.4%. The accuracy was within 90.8-107.0%. This highly sensitive and reproducible method is able to determine only biologically active ED-71 by separating it from preED-71, which is considered to be applicable for the determination of serum samples from pharmacokinetic studies in human.