Preclinical safety assessment of modified gamma globin lentiviral vector-mediated autologous hematopoietic stem cell gene therapy for hemoglobinopathies.

Previously, we reported the development of a human Aγ-globin gene lentivirus (LV), GbG, which expresses high levels of HbF to correct the sickle cell anemia (SCA) phenotype in the Berkeley SCA mouse model, and then modified the γ-globin gene by substituting glycine at codon 16 with aspartic acid in the Aγ-globin gene to generate GbGM LV. In the present study, we evaluated the long-term safety of human Aγ-globin gene carrying GbGM LV in wild-type mice after primary and secondary transplants of GbGM-modified hematopoietic stem cells (HSC) over 18 months. The safety of the GbGM bone marrow transplant was assessed by monitoring the effects on body weight, hematology, histopathology, malignancy formation, and survival. Mice transplanted with Mock-transduced and spleen focus forming virus (SFFV) γ-retroviral vector (RV)-transduced HSC served as negative and positive controls, respectively. The mean donor-cell engraftment was comparable across Mock, GbGM LV, and SFFV RV groups. There were no significant differences in body weight, clinical signs, immunophenotype, or histopathology in the GbGM-treated mice compared to controls. Four SFFV RV-treated mice, but none of the GbGM-treated mice, developed donor-derived, vector-positive lymphomas as demonstrated by flow cytometry analysis and in situ hybridization. These results highlight the safety of the administration of GbGM LV-modified HSC with long-term follow-up after primary and secondary transplants in mice. This data supported the initiation of phase 1/2 first-in-human SCA clinical trial in the United States.

Pharmacokinetic/Pharmacodynamic Modeling of a Cell-Penetrating Peptide Phosphorodiamidate Morpholino Oligomer in mdx Mice.

PURPOSE: Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) have shown promise in treating Duchenne muscular dystrophy (DMD). We evaluated a semi-mechanistic pharmacokinetic (PK) and pharmacodynamic (PD) model to capture the relationship between plasma and muscle tissue exposure/response in mdx mice treated by mouse surrogate PPMO. METHODS: A single or repeated (every 4 weeks for 20 weeks) intravenous PPMO dose was administered to mdx mice (n = 6/timepoint). A PK/PD model was built to characterize data via sequential modeling. A 2-compartment model was used to describe plasma PK. A simultaneous tissue PK/PD model was subsequently developed: 2-compartment model to describe muscle PK; linked to an indirect response model describing stimulation of synthesis of skipped transcript, which was in turn linked to stimulation of synthesis of dystrophin protein expression. RESULTS: Model performance assessment via goodness-of-fit plots, visual predictive checks, and accurate parameter estimation indicated robust fits of plasma PK and muscle PK/PD data. The model estimated a PPMO tissue half-life of 5 days-a useful parameter in determining the longevity of PPMOs in tissue and their limited accumulation after multiple doses. Additionally, the model successfully described dystrophin expression after single dosing and associated protein accumulation after multiple dosing (increasing ~ twofold accumulation from the first to last dose). CONCLUSIONS: This first PK/PD model of a PPMO in a DMD disease model will help characterize and predict the time course of PK/PD biomarkers in mdx mice. Furthermore, the model framework can be used to develop clinical PK/PD models and can be extended to other exon-skipping therapies and species.

Antisense oligonucleotides: absorption, distribution, metabolism, and excretion.

INTRODUCTION: Antisense oligonucleotides (ASOs) have emerged as a promising novel drug modality that aims to address unmet medical needs. A record of six ASO drugs have been approved since 2016, and more candidates are in clinical development. ASOs are the most advanced class within the RNA-based therapeutics field. AREAS COVERED: This review highlights the two major backbones that are currently used to build the most advanced ASO platforms - the phosphorodiamidate morpholino oligomers (PMOs) and the phosphorothioates (PSs). The absorption, distribution, metabolism, and excretion (ADME) properties of the PMO and PS platforms are discussed in detail. EXPERT OPINION: Understanding the ADME properties of existing ASOs can foster further improvement of this cutting-edge therapy, thereby enabling researchers to safely develop ASO drugs and enhancing their ability to innovate. ABBREVIATIONS: 2'-MOE, 2'-O-methoxyethyl; 2'PS, 2 modified PS; ADME, absorption, distribution, metabolism, and excretion; ASO, antisense oligonucleotide; AUC, area under the curve; BNA, bridged nucleic acid; CPP, cell-penetrating peptide; CMV, cytomegalovirus; CNS, central nervous system; CYP, cytochrome P; DDI, drug-drug interaction; DMD, Duchenne muscular dystrophy; FDA, Food and Drug Administration; GalNAc3, triantennary N-acetyl galactosamine; IT, intrathecal; IV, intravenous; LNA, locked nucleic acid; mRNA, messenger RNA; NA, not applicable; PBPK, physiologically based pharmacokinetics; PD, pharmacodynamic; PK, pharmacokinetic; PMO, phosphorodiamidate morpholino oligomer; PMOplus, PMOs with positionally specific positive molecular charges; PPMO, peptide-conjugated PMO; PS, phosphorothioate; SC, subcutaneous; siRNA, small-interfering RNA; SMA, spinal muscular atrophy.

Pharmacokinetics and Catabolism of [3H]TAK-164, a Guanylyl Cyclase C Targeted Antibody-Drug Conjugate.

TAK-164 is an antibody-drug conjugate (ADC) comprising human anti-guanylyl cyclase C (GCC) monoclonal antibody conjugated to indolinobenzodiazepine DNA alkylator IGN-P1 through a cleavable alanine-alanine dipeptide linker. TAK-164 is currently being evaluated for the treatment of gastrointestinal cancers expressing GCC. The catabolism of TAK-164 was studied using 3H-labeled ADC using GCC-expressing HEK-293 (GCC-HEK-293) cells, rat tritosomes, cathepsin B, and tumor-bearing mice. Time- and target-dependent uptake of [3H]TAK-164 was observed in GCC-HEK-293 cells with approximately 12% of radioactivity associated with DNA after 24 hours of incubation. Rat liver tritosomes and cathepsin B yielded IGN-P1 aniline, sulfonated IGN-P1 (s-IGN-P1) aniline, and a lysine conjugate of IGN-P1 (IGN-P1-Lys) aniline as catabolites. In tumor-bearing mice, [3H]TAK-164 exhibited a terminal half-life of approximately 41 and 51 hours in plasma and blood, respectively, with low plasma clearance (0.75 ml/h per kilogram). The extractable radioactivity in plasma and tumor samples revealed the presence of s-IGN-P1 aniline and IGN-P1 aniline as payload-related components. The use of a radiolabeled payload in the ADC in tumor uptake investigations provided direct and quantitative evidence for tumor uptake, DNA binding, and proof of mechanism of action of the payload. SIGNIFICANCE STATEMENT: Since payload-related species are potent cytotoxins, a thorough characterization of released products of ADCs, metabolites, and their drug interaction potential is necessary prior to clinical investigations. This study characterized in vitro and in vivo DNA binding mechanisms and released products of TAK-164. The methodologies described here will be highly useful for characterization of payload-related products of ADCs in general.

Catabolism of antibody drug conjugates and characterization methods.

Antibody drug conjugates (ADCs) are large molecule therapeutics in which a cytotoxic payload is conjugated to a monoclonal antibody (mAb) via a linker. The molecules are designed to selectively bind to target-expressing cells, thus delivering therapeutic agents directly to the tumor. Chemical and enzymatic stability prior to reaching the target is an important factor for ADCs since it impacts their safety, efficacy, and pharmacokinetics (PK). One of the main reasons for off-target effects of ADCs is premature release of cytotoxic agents, either in the blood stream or at non-specific sites. Once an ADC is internalized by target-expressing cells, the cytotoxic payload and/or related catabolites are released through chemical or enzymatic cleavage within the cells. In some cases, the released payload and/or catabolites are effluxed into the systemic circulation and follow a small molecule disposition path. Since doses of ADCs are low, the concentration of cytotoxic payload and related catabolites/metabolites range from ng to µg levels in systemic circulation or tumors in clinical studies. Hence, it is challenging to identify these species without prior knowledge of the pathways of catabolism. The current review summarizes the mechanism of cleavage/catabolism of various types of linkers and available in vitro, in vivo, and bioanalytical methods for evaluation of catabolism of ADCs.

An accelerated background subtraction algorithm for processing high-resolution MS data and its application to metabolite identification.

BACKGROUND: Metabolite identification without radiolabeled compound is often challenging because of interference of matrix-related components. RESULTS: A novel and an effective background subtraction algorithm (A-BgS) has been developed to process high-resolution mass spectral data that can selectively remove matrix-related components. The use of a graphics processing unit with a multicore central processing unit enhanced processing speed several 1000-fold compared with a single central processing unit. A-BgS algorithm effectively removes background peaks from the mass spectra of biological matrices as demonstrated by the identification of metabolites of delavirdine and metoclopramide. CONCLUSION: The A-BgS algorithm is fast, user friendly and provides reliable removal of matrix-related ions from biological samples, and thus can be very helpful in detection and identification of in vivo and in vitro metabolites.

The pneumotoxin 3-methylindole is a substrate and a mechanism-based inactivator of CYP2A13, a human cytochrome P450 enzyme preferentially expressed in the respiratory tract.

3-Methylindole (3MI), a respiratory tract toxicant, can be metabolized by a number of cytochromes P450 (P450), primarily through either dehydrogenation or epoxidation of the indole. In the present study, we assessed the bioactivation of 3MI by recombinant CYP2A13, a human P450 predominantly expressed in the respiratory tract. Four metabolites were detected, and the two principal ones were identified as indole-3-carbinol (I-3-C) and 3-methyloxindole (MOI). Bioactivation of 3MI by CYP2A13 was verified by the observation of three glutathione (GSH) adducts designated as GS-A1 (glutathione adduct 1), GS-A2 (glutathione adduct 2), and GS-A3 (glutathione adduct 3) in a NADPH- and GSH-fortified reaction system. GS-A1 and GS-A2 gave the same molecular ion at m/z 437, an increase of 305 Da over 3MI. Their structures are assigned to be 3-glutathionyl-S-methylindole and 3-methyl-2-glutathionyl-S-indole, respectively, on the basis of the mass fragmentation data obtained by high-resolution mass spectrometry. Kinetic parameters were determined for the formation of I-3-C (V(max) = 1.5 nmol/min/nmol of P450; K(m) = 14 muM), MOI (V(max) = 1.9 nmol/min/nmol of P450; K(m) = 15 muM) and 3-glutathionyl-S-methylindole (V(max) = 0.7 nmol/min/nmol of P450; K(m) = 13 muM). The structure of GS-A3, a minor adduct with a protonated molecular ion at m/z 453, is proposed to be 3-glutathionyl-S-3-methyloxindole. We also discovered that 3MI is a mechanism-based inactivator of CYP2A13, given that it produced a time-, cofactor-, and 3MI concentration-dependent loss of activity toward 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, with a relatively low K(I) value of approximately 10 muM and a k(inact) of 0.046 min(-1). Thus, CYP2A13 metabolizes 3MI through multiple bioactivation pathways, and the process can lead to a suicide inactivation of CYP2A13.

Human lung epithelial cells express a functional cold-sensing TRPM8 variant.

Several transient receptor potential (TRP) ion channels sense and respond to changes in ambient temperature. Chemical agonists of TRP channels, including menthol and capsaicin, also elicit sensations of temperature change. TRPM8 is a cold- and menthol-sensing ion channel that converts thermal and chemical stimuli into neuronal signals and sensations of cooling/cold. However, the expression and function of TRPM8 receptors in non-neuronal cells and tissues is a relatively unexplored area. Results presented here document the expression and function of a truncated TRPM8 variant in human bronchial epithelial cells. Expression of the TRPM8 variant was demonstrated by RT-PCR, cloning, and immunohistology. Receptor function was characterized using the prototypical TRPM8 agonist, menthol, and exposure of cells to reduced temperature (18 degrees C). The TRPM8 variant was expressed primarily within endoplasmic reticulum membranes of lung epithelial cells and its activation was attenuated by thapsigargin, the cell-permeable TRPM8 antagonist N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide, and shRNA-induced suppression of TRPM8 expression. Activation of the TRPM8 variant in lung cells was coupled with enhanced expression of the inflammatory cytokines IL-6 and IL-8. Collectively, our results suggest that this novel TRPM8 variant receptor may function as a modulator of respiratory physiology caused by cold air, and may partially explain asthmatic respiratory hypersensitivity to cold air.

Substituents on etoposide that interact with human topoisomerase IIalpha in the binary enzyme-drug complex: contributions to etoposide binding and activity.

Etoposide is a widely prescribed anticancer agent that stabilizes topoisomerase II-mediated DNA strand breaks. The drug contains a polycyclic ring system (rings A-D), a glycosidic moiety at C4, and a pendant ring (E-ring) at C1. A recent study that focused on yeast topoisomerase II demonstrated that the H15 geminal protons of the etoposide A-ring, the H5 and H8 protons of the B-ring, and the H2', H6', 3'-methoxyl, and 5'-methoxyl protons of the E-ring contact topoisomerase II in the binary enzyme-drug complex [ Wilstermann et al. (2007) Biochemistry 46, 8217-8225 ]. No interactions with the C4 sugar were observed. The present study used DNA cleavage assays, saturation transfer difference [ (1)H] NMR spectroscopy, and enzyme-drug binding studies to further define interactions between etoposide and human topoisomerase IIalpha. Etoposide and three derivatives that lacked the C4 sugar were analyzed. Except for the sugar, 4'-demethyl epipodophyllotoxin is identical to etoposide, epipodophyllotoxin contains a 4'-methoxyl group on the E-ring, and 6,7- O, O-demethylenepipodophyllotoxin replaces the A-ring with a diol. Results suggest that etoposide-topoisomerase IIalpha binding is driven by interactions with the A- and B-rings and potentially by stacking interactions with the E-ring. We propose that the E-ring pocket on the enzyme is confined, because the addition of bulk to this ring adversely affects drug function. The A- and E-rings do not appear to contact DNA in the enzyme-drug-DNA complex. Conversely, the sugar moiety subtly alters DNA interactions. The identification of etoposide substituents that contact topoisomerase IIalpha in the binary complex has predictive value for drug behavior in the enzyme-etoposide-DNA complex.

Binding of helix-threading peptides to E. coli 16S ribosomal RNA and inhibition of the S15-16S complex.

Helix-threading peptides (HTPs) constitute a new class of small molecules that bind selectively to duplex RNA structures adjacent to helix defects and project peptide functionality into the dissimilar duplex grooves. To further explore and develop the capabilities of the HTP design for binding RNA selectively, we identified helix 22 of the prokaryotic ribosomal RNA 16S as a target. This helix is a component of the binding site for the ribosomal protein S15. In addition, the S15-16S RNA interaction is important for the ordered assembly of the bacterial ribosome. Here we present the synthesis and characterization of helix-threading peptides that bind selectively to helix 22 of E. coli 16S RNA. These compounds bind helix 22 by threading intercalation placing the N termini in the minor groove and the C termini in the major groove. Binding is dependent on the presence of a highly conserved purine-rich internal loop in the RNA, whereas removal of the loop minimally affects binding of the classical intercalators ethidium bromide and methidiumpropyl-EDTAFe (MPEFe). Moreover, binding selectivity translates into selective inhibition of formation of the S15-16S complex.