Preclinical Safety Assessment and Toxicokinetics of Apitegromab, an Antibody Targeting Proforms of Myostatin for the Treatment of Muscle-Atrophying Disease.

Myostatin is a negative regulator of skeletal muscle and has become a therapeutic target for muscle atrophying disorders. Although previous inhibitors of myostatin offered promising preclinical data, these therapies demonstrated a lack of specificity toward myostatin signaling and have shown limited success in the clinic. Apitegromab is a fully human, monoclonal antibody that binds to human promyostatin and latent myostatin with a high degree of specificity, without binding mature myostatin and other closely related growth factors. To support the clinical development of apitegromab, we present data from a comprehensive preclinical assessment of its pharmacology, pharmacokinetics, and safety across multiple species. In vitro studies confirmed the ability of apitegromab to inhibit the activation of promyostatin. Toxicology studies in monkeys for 4 weeks and in adult rats for up to 26 weeks showed that weekly intravenous administration of apitegromab achieved sustained serum exposure and target engagement and was well-tolerated, with no treatment-related adverse findings at the highest doses tested of up to 100 mg/kg and 300 mg/kg in monkeys and rats, respectively. Additionally, results from an 8-week juvenile rat study showed no adverse effects on any endpoint, including neurodevelopmental, motor, and reproductive outcomes at 300 mg/kg administered weekly IV. In summary, the nonclinical pharmacology, pharmacokinetic, and toxicology data demonstrate that apitegromab is a selective inhibitor of proforms of myostatin that does not exhibit toxicities observed with other myostatin pathway inhibitors. These data support the conduct of ongoing clinical studies of apitegromab in adult and pediatric patients with spinal muscular atrophy (SMA).

An antibody that inhibits TGF-ß1 release from latent extracellular matrix complexes attenuates the progression of renal fibrosis.

Inhibitors of the transforming growth factor-ß (TGF-ß) pathway are potentially promising antifibrotic therapies, but nonselective simultaneous inhibition of all three TGF-ß homologs has safety liabilities. TGF-ß1 is noncovalently bound to a latency-associated peptide that is, in turn, covalently bound to different presenting molecules within large latent complexes. The latent TGF-ß-binding proteins (LTBPs) present TGF-ß1 in the extracellular matrix, and TGF-ß1 is presented on immune cells by two transmembrane proteins, glycoprotein A repetitions predominant (GARP) and leucine-rich repeat protein 33 (LRRC33). Here, we describe LTBP-49247, an antibody that selectively bound to and inhibited the activation of TGF-ß1 presented by LTBPs but did not bind to TGF-ß1 presented by GARP or LRRC33. Structural studies demonstrated that LTBP-49247 recognized an epitope on LTBP-presented TGF-ß1 that is not accessible on GARP- or LRRC33-presented TGF-ß1, explaining the antibody's selectivity for LTBP-complexed TGF-ß1. In two rodent models of kidney fibrosis of different etiologies, LTBP-49247 attenuated fibrotic progression, indicating the central role of LTBP-presented TGF-ß1 in renal fibrosis. In mice, LTBP-49247 did not have the toxic effects associated with less selective TGF-ß inhibitors. These results establish the feasibility of selectively targeting LTBP-bound TGF-ß1 as an approach for treating fibrosis.

Human placenta-derived adherent cells prevent bone loss, stimulate bone formation, and suppress growth of multiple myeloma in bone.

Human placenta has emerged as a valuable source of transplantable cells of mesenchymal and hematopoietic origin for multiple cytotherapeutic purposes, including enhanced engraftment of hematopoietic stem cells, modulation of inflammation, bone repair, and cancer. Placenta-derived adherent cells (PDACs) are mesenchymal-like stem cells isolated from postpartum human placenta. Multiple myeloma is closely associated with induction of bone disease and large lytic lesions, which are often not repaired and are usually the sites of relapses. We evaluated the antimyeloma therapeutic potential, in vivo survival, and trafficking of PDACs in the severe combined immunodeficiency (SCID)-rab model of medullary myeloma-associated bone loss. Intrabone injection of PDACs into nonmyelomatous and myelomatous implanted bone in SCID-rab mice promoted bone formation by stimulating endogenous osteoblastogenesis, and most PDACs disappeared from bone within 4 weeks. PDACs inhibitory effects on myeloma bone disease and tumor growth were dose-dependent and comparable with those of fetal human mesenchymal stem cells (MSCs). Intrabone, but not subcutaneous, engraftment of PDACs inhibited bone disease and tumor growth in SCID-rab mice. Intratumor injection of PDACs had no effect on subcutaneous growth of myeloma cells. A small number of intravenously injected PDACs trafficked into myelomatous bone. Myeloma cell growth rate in vitro was lower in coculture with PDACs than with MSCs from human fetal bone or myeloma patients. PDACs also promoted apoptosis in osteoclast precursors and inhibited their differentiation. This study suggests that altering the bone marrow microenvironment with PDAC cytotherapy attenuates growth of myeloma and that PDAC cytotherapy is a promising therapeutic approach for myeloma osteolysis.

Systemic delivery of RafsiRNA using cationic cardiolipin liposomes silences Raf-1 expression and inhibits tumor growth in xenograft model of human prostate cancer.

Raf-1, a protein serine-threonine kinase, plays a critical role in mitogen-activated protein kinase kinase (MKK/MEK)- mitogen-activated protein kinase (extracellular signal-regulated kinase) (MAPK/ERK) pathways. We show here that systemically delivered novel cationic cardiolipin liposomes (NeoPhectin-AT) containing a small interfering RNA (siRNA) against Raf-1 silence the expression of Raf-1 in tumor tissues and inhibit tumor growth in xenograft model of human prostate cancer. The knockdown of Raf-1 expression by siRNA is also associated with down-regulation of cyclin D1 expression in vivo.

Preclinical safety, pharmacokinetics and antitumor efficacy profile of liposome-entrapped SN-38 formulation.

BACKGROUND: SN-38, 7-ethyl-10-hydroxycamptothecin, is a biologically active metabolite of irinotecan. Its poor solubility restricted its development as an anticancer agent. We have developed an easy-to-use liposome-entrapped SN-38 (LE-SN38) and evaluated its toxicology, pharmacokinetics and antitumor efficacy profile. MATERIALS AND METHODS: Toxicity and pharmacokinetics studies were conducted in CD2F1 mice and beagle dogs. Therapeutic efficacy studies were performed in murine leukemia (P388 and P388/ADR) and in a human pancreatic (Capan-1) tumor models. RESULTS: Multiple dose administration (i.v. x 5) of LE-SN38 indicated a maximum tolerated dose (MTD) of 5.0 and 7.5 mg/kg/day for male and female mice, respectively. The MTD of LE-SN38 in dogs was 1.2 mg/kg. The elimination half-life (t1/2) of SN-38 in mouse plasma was 6.38 h with volume of distribution (VdSS) 2.55 L/kg. In dogs, t1/2 and VdSS were 1.38-6.42 h and 1.69-5.01 L/kg; respectively. P388 tumor-bearing mice dosed with LE-SN38 at 5.5 mg/kg (i.v. x 5) showed 100% survival. LE-SN38 at 4 or 8 mg/kg (i. v. x 5) inhibited 65% and 98% tumor growth, respectively, in a human pancreatic tumor model. CONCLUSION: LE-SN38 showed a favorable pharmacokinetics profile and can be administered safely at therapeutically effective doses.

Differential expression of glutathione S-transferase isoenzymes in murine small intestine and colon.

Glutathione (GSH) S-transferase (GST) isoenzymes of the small intestine and colon of female A/J mice have been purified and characterized to determine their interrelationships with other murine GSTs. Cytosolic GST activity in the small intestine was at least due to six isoenzymes with isoelectric points (pI) of 9.5, 9.3, 9.1, 8.5, 6.2 and 5.5. Small intestine isoenzymes with pI values of 9.5, 9.3, 8.5, and 6.2 were identical to the mGSTA1-1 (Alpha class), mGSTP1-1 (Pi class), mGSTM1-1 (Mu class) and mGSTA4-4 (Alpha class), respectively, of other A/J mouse tissues on the basis of their reverse-phase HPLC elution profile, immunological cross-reactivity and/or N-terminal region amino acid sequence. Even though GST9.1 of the small intestine cross-reacted with the antibodies raised against Pi class GST, reverse-phase HPLC and N-terminal amino acid sequence analyses suggested that this isoenzyme may be structurally different from mGSTP1-1 as well as mGSTP2-2. Likewise, despite immunological similarity with the Mu class GSTs, small intestine GST5.5 appeared to be different from other Mu class murine GSTs characterized previously. Cytosolic GST activity in the colon was mainly due to four isoenzymes with pI values of 9.8, 9.4, 6.6 and 5.8. While the identity of colon GST6.6 could not be established due to its low abundance, GST9.8, GST9.4 and GST5.8 were identical to mGSTP1-1, mGSTM1-1 and mGSTA4-4, respectively, of other A/J mouse tissues including the small intestine. Isoenzymes corresponding to small intestine GST9.1 and GST5.5 could not be detected in the colon. The results of the present study indicate that the small intestine of female A/J mice is better equipped for protection against toxic effects of electrophiles than colon.

Human placenta-derived adherent cell treatment of experimental stroke promotes functional recovery after stroke in young adult and older rats.

BACKGROUND: Human Placenta-Derived Adherent Cells (PDAC®) are a novel mesenchymal-like cell population derived from normal human placental tissue. PDA-001 is a clinical formulation of PDAC® developed for intravenous administration. In this study, we investigated the efficacy of PDA-001 treatment in a rat model of transient middle cerebral artery occlusion (MCAo) in young adult (2-3 month old) and older rats (10-12 months old). METHODS: To evaluate efficacy and determine the optimal number of transplanted cells, young adult Wistar rats were subjected to MCAo and treated 1 day post MCAo with 1×10(6), 4×10(6) or 8×10(6) PDA-001 cells (i.v.), vehicle or cell control. 4×10(6) or 8×10(6) PDA-001 cells were also tested in older rats after MCAo. Treatment response was evaluated using a battery of functional outcome tests, consisting of adhesive-removal test, modified Neurological Severity Score (mNSS) and foot-fault test. Young adult rats were sacrificed 56 days after MCAo, older rats were sacrificed 29 days after MCAo, and lesion volumes were measured using H&E. Immunohistochemical stainings for bromodeoxyuridine (BrdU) and von Willebrand Factor (vWF), and synaptophysin were performed. RESULTS: In young adult rats, treatment with 4×10(6) PDA-001 cells significantly improved functional outcome after stroke (p<0.05). In older rats, significant functional improvement was observed with PDA-001 cell therapy in both of the 4×10(6) and 8×10(6) treatment groups. Functional benefits in young adult and older rats were associated with significant increases in the number of BrdU immunoreactive endothelial cells, vascular density and perimeter in the ischemic brain, as well as significantly increased synaptophysin expression in the ischemic border zone (p<0.05). CONCLUSION: PDA-001 treatment significantly improved functional outcome after stroke in both young adult and older rats. The neurorestorative effects induced by PDA-001 treatment may be related to increased vascular density and synaptic plasticity.

Neuroprotective effect of human placenta-derived cell treatment of stroke in rats.

UNLABELLED: Human placenta-derived adherent (PDA001) cells are mesenchymal-like stem cells isolated from postpartum placenta. In this study, we tested whether intravenously infused PDA001 improves neurological functional recovery after stroke in rats. In addition, potential mechanisms underlying the PDA001-induced neuroprotective effect were investigated. Young adult male rats (2­3 months) were subjected to 2 h of middle cerebral artery occlusion (MCAo) and treated with PDA001 (4x10(6)) or vehicle controls [dextran vehicle or phosphate buffer saline (PBS)] via intravenous (IV) administration initiated at 4 h after MCAo. A battery of functional tests and measurements of lesion volume and apoptotic cells were performed. Immunostaining and ELISA assays for vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) and brain derived neurotrophic factor (BDNF) were performed in the ischemic brain to test the potential mechanisms underlying the neuroprotective effects of PDA001 cell treatment of stroke. PDA001 cell treatment at 4 h post stroke significantly improved functional outcome and significantly decreased lesion volume, TUNEL, and cleaved caspase 3-positive cell number in the ischemic brain, compared to MCAo-vehicle and MCAo-PBS control. Treatment of stroke with PDA001 cells also significantly increased HGF and VEGF expression in the ischemic border zone (IBZ) compared to controls. Using ELISA assays, treatment of stroke with PDA001 cells significantly increased VEGF, HGF, and BDNF levels in the ischemic brain compared to controls. CONCLUSION: When administered intravenously at 4 h after MCAo, PDA001 cells promoted neuroprotective effects. These effects induced by PDA001 cell treatment may be related to the increase of VEGF, HGF, and BDNF expression,and a decrease of apoptosis. PDA001 cells may provide a viable cell source to treat stroke.

Structure-Based Design of Anticancer Prodrug PABA/NO.

Glutathione S-transferase (GST) is a superfamily of detoxification enzymes, represented by GSTalpha, GSTmu, GSTpi, etc. GSTalpha is the predominant isoform of GST in human liver, playing important roles for our well being. GSTpi is overexpressed in many forms of cancer, thus presenting an opportunity for selective targeting of cancer cells. Our structure-based design of prodrugs intended to release cytotoxic levels of nitric oxide in GSTpi-overexpressing cancer cells yielded PABA/NO, which exhibited anticancer activity both in vitro and in vivo with a potency similar to that of cisplatin (Findlay et al. Mol. Pharmacol. 2004, 65, 1070-1079). Here, we present the details on structural modification, molecular modeling, and enzymatic characterization for the design of PABA/NO. The design was efficient because it was on the basis of the reaction mechanism and the structures of related GST isozymes at both the ground state and the transition state. The ground-state structures outlined the shape and property of the substrate-binding site in different isozymes, and the structural information at the transition-state indicated distinct conformations of the Meisenheimer complex of prodrugs in the active site of different isozymes, providing guidance for the modifications of the molecular structure of the prodrug molecules. Two key alterations of a GSTalpha -selective compound led to the GSTpi-selective PABA/NO.