Profiles In Monkeys Research Articles

Analysis of the pharmacokinetics and efficacy of RBD1016 – A GalNAc-siRNA targeting Hepatitis B Virus X gene using semi-mechanistic PK/PD model

Small interference RNA (siRNA) is a class of short double-stranded RNA molecules that cause mRNA degradation through an RNA interference mechanism and is a promising therapeutic modality. RBD1016 is a siRNA drug in clinical development for the treatment of chronic Hepatitis B Virus (HBV) infection, which contains a conjugated with N-acetylglucosamine moiety that can facilitate its hepatic delivery. We aimed to construct a semi-mechanistic model of RBD1016 in pre-clinical animals, to elucidate the pharmacokinetic/pharmacodynamic (PK/PD) profiles in mice and PK profiles in monkeys, which can lay the foundation for potential future translation of RBD1016 PK and PD from the pre-clinical stage to the clinic stage. The proposed semi-mechanistic PK/PD model fitted PK and PD data in HBV transgenic mice well and described plasma and liver concentrations in the monkeys well. The simulation results showed that our model has a reasonable predictive ability for Hepatitis B surface antigen (HBsAg) levels after multiple dosing in mice. Further PK and PD data for RBD1016, including clinical data, will assist in refining the model presented here. Our current effort focused on model building for RBD1016, we anticipate that the model could apply to other GalNAc-siRNA drugs.

Abstract 2362: Preclinical efficacy and safety of M9140, a novel antibody-drug conjugate (ADC) with topoisomerase 1 (TOP1) inhibitor payload targeting carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5)-expressing colorectal tumors

Abstract Background: M9140 is the first clinical-stage TOP1 inhibitor payload ADC directed against CEACAM5, which is expressed in colorectal cancer (CRC) and other cancer indications. M9140 is composed of a CEACAM5-specific antibody conjugated to eight, highly stable and hydrophilic ß-glucuronide-exatecan linker-payloads. After linker cleavage, the highly potent and membrane permeable TOP1 inhibitor exatecan is released. Methods: M9140 potency was tested in viability assays using cancer cell lines with different levels of CEACAM5 expression. Potency shift viability assays were conducted using two CRC cell lines, with zosuquidar used as multidrug resistance-1 (MDR-1) inhibitor. The M9140 bystander effect was assessed in co-culture experiments with CEACAM5-negative and -positive cancer cells. Antitumor efficacy and safety were evaluated in patient-derived xenograft (PDX) mouse models and in cynomolgus monkeys, respectively. Results: M9140 demonstrated specific binding to CEACAM5 and selective killing of target-positive cancer cells with potencies between 0.09 nM and 0.62 nM. A potent bystander effect of M9140 was demonstrated in co-culture experiments indicating the potential to treat tumors with heterogeneous target expression. Unlike other ADC payloads such as monomethyl auristatin E (MMAE) and the maytansine derivative DM4, exatecan potency was not affected by MDR-1 inhibition, indicating that this drug efflux resistance mechanism may not be relevant for exatecan. A single treatment with 10 mg/kg M9140 caused strong antitumor effects in all 14 CRC PDX models tested, with tumor stasis in 10 models and tumor regression in four models as the best response. The TOP1 inhibitor-based ADC M9140 caused strong antitumor efficacy with a tumor volume reduction of 74% and 88% as best response in two CRC PDX models where a maytansine-based CEACAM5 ADC was not effective. In cynomolgus monkeys, M9140 showed dose-dependent hematolymphoid and intestinal effects, consistent with exatecan payload, with no indication of any other toxicities such as interstitial lung disease (ILD) or ocular toxicity. Conclusions: M9140 demonstrated high potency, strong antitumor activity, and bystander effect in preclinical models. The side effect profile in monkeys was favorable and in line with the expected toxicity of exatecan. Notably, ILD and ocular toxicity, which are known adverse effects of deruxtecan and maytansine-based ADCs respectively, were absent. A first-in-human study to evaluate the safety, tolerability, pharmacokinetics, and preliminary clinical activity of M9140 in advanced solid tumors is ongoing (NCT05464030). Citation Format: Sabine Raab-Westphal, Felix Hart, Willem Sloot, Min Shan, Nicolas Rasche, Christiane Amendt, Jan Anderl. Preclinical efficacy and safety of M9140, a novel antibody-drug conjugate (ADC) with topoisomerase 1 (TOP1) inhibitor payload targeting carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5)-expressing colorectal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2362.

Raludotatug Deruxtecan, a CDH6-Targeting Antibody-Drug Conjugate with a DNA Topoisomerase I Inhibitor DXd, Is Efficacious in Human Ovarian and Kidney Cancer Models.

Cadherin-6 (CDH6) is expressed in several cancer types, but no CDH6-targeted therapy is currently clinically available. Here, we generated raludotatug deruxtecan (R-DXd; DS-6000), a novel CDH6-targeting antibody-drug conjugate with a potent DNA topoisomerase I inhibitor, and evaluated its properties, pharmacologic activities, and safety profile. In vitro pharmacologic activities and the mechanisms of action of R-DXd were assessed in serous-type ovarian cancer and renal cell carcinoma cell lines. In vivo pharmacologic activities were evaluated with several human cancer cell lines and patient-derived xenograft mouse models. The safety profile in cynomolgus monkeys was also assessed. R-DXd exhibited CDH6 expression-dependent cell growth-inhibitory activity and induced tumor regression in xenograft models. In this process, R-DXd specifically bound to CDH6, was internalized into cancer cells, and then translocated to the lysosome. The DXd released from R-DXd induced the phosphorylation of Chk1, a DNA damage marker, and cleaved caspase-3, an apoptosis marker, in cancer cells. It was also confirmed that the DXd payload had a bystander effect, passing through the cell membrane and impacting surrounding cells. The safety profile of R-DXd was favorable and the highest non-severely toxic dose was 30 mg/kg in cynomolgus monkeys. R-DXd demonstrated potent antitumor activity against CDH6-expressing tumors in mice and an acceptable safety profile in monkeys. These findings indicate the potential of R-DXd as a new treatment option for patients with CDH6-expressing serous-type ovarian cancer and renal cell carcinoma in a clinical setting.

Utilizing PK and PD Biomarkers to Guide the First-in-Human Starting Dose Selection of MTBT1466A: A Novel Humanized Monoclonal Anti-TGFβ3 Antibody for the Treatment of Fibrotic Diseases

MTBT1466A is a high-affinity TGFβ3-specific humanized IgG1 monoclonal antibody with reduced Fc effector function, currently under investigation in clinical trials as a potential anti-fibrotic therapy. Here, we characterized the pharmacokinetics (PK) and pharmacodynamics (PD) of MTBT1466A in mice and monkeys and predicted the PK/PD of MTBT1466A in humans to guide the selection of the first-in-human (FIH) starting dose. MTBT1466A demonstrated a typical IgG1-like biphasic PK profile in monkeys, and the predicted human clearance of 2.69 mL/day/kg and t1/2 of 20.4 days are consistent with those expected for a human IgG1 antibody. In a mouse model of bleomycin-induced lung fibrosis, changes in expression of TGFβ3-related genes, serpine1, fibronectin-1, and collagen 1A1 were used as PD biomarkers to determine the minimum pharmacologically active dose of 1 mg/kg. Unlike in the fibrosis mouse model, evidence of target engagement in healthy monkeys was only observed at higher doses. Using a PKPD-guided approach, the recommended FIH dose of 50 mg, IV, provided exposures that were shown to be safe and well tolerated in healthy volunteers. MTBT1466A PK in healthy volunteers was predicted reasonably well using a PK model with allometric scaling of PK parameters from monkey data. Taken together, this work provides insights into the PK/PD behavior of MTBT1466A in preclinical species, and supports the translatability of the preclinical data into the clinic.

MTX-13, a Novel PTK7-Directed Antibody-Drug Conjugate with Widened Therapeutic Index Shows Sustained Tumor Regressions for a Broader Spectrum of PTK7-Positive Tumors.

Protein tyrosine kinase 7 (PTK7) is a Wnt signaling pathway protein implicated in cancer development and metastasis. When using a potent microtubule inhibitor (Aur0101), PTK7-targeting antibody-drug conjugate (ADC), h6M24-vc0101 (PF-06647020/cofetuzumab pelidotin) is efficacious only in limited tumor types with low response rates in a phase I trial. To improve patient response and to expand responding tumor types, we designed MTX-13, a PTK7-targeting ADC consisting of a novel antibody (Ab13) conjugated to eight molecules of topoisomerase I inhibitor exatecan through T1000, a novel self-immolative moiety. MTX-13 exhibited PTK7-specific cell binding, efficient internalization, and exatecan release to cause cytotoxic activity through DNA damage and apoptosis induction, and a strong bystander killing. MTX-13 displayed potent antitumor activities on cell line-derived xenograft and patient-derived xenograft models from a wide range of solid tumors, significantly outperforming h6M24-vc0101. PTK7 was shown to be an actionable target in small cell lung cancer for which MTX-13 showed complete and durable responses. With a consistent overexpression of PTK7 in squamous cell carcinomas derived from diverse anatomic sites, strong potency of MTX-13 in this group of heterogenous tumors suggested a common treatment strategy. Finally, MTX-13 inhibited tumor growth and metastasis in an orthotopic colon cancer xenograft model. MTX-13 displayed a favorable pharmacokinetic and safety profile in monkeys with the highest non-severely toxic dose (HNSTD) of ≥30 mg/kg, significantly higher than 3-5 mg/kg of HNSTD for h6M24-vc0101. The higher therapeutic index of MTX-13 bodes well for its clinical translation with the potential to expand the responding patient population beyond that of current PTK7-targeting ADCs.

Significant Microbial Changes Are Evident in the Reproductive Tract of Pregnant Rhesus Monkeys at Mid-Gestation but Their Gut Microbiome Does Not Shift until Late Gestation.

Vaginal and rectal specimens were obtained from cycling, pregnant, and nursing rhesus monkeys to assess pregnancy-related changes in the commensal bacteria in their reproductive and intestinal tracts. Using 16S rRNA gene amplicon sequencing, significant differences were found only in the vagina at mid-gestation, not in the hindgut. To verify the apparent stability in gut bacterial composition at mid-gestation, the experiment was repeated with additional monkeys, and similar results were found with both 16S rRNA gene amplicon and metagenomic sequencing. A follow-up study investigated if bacterial changes in the hindgut might occur later in pregnancy. Gravid females were assessed closer to term and compared to nonpregnant females. By late pregnancy, significant differences in bacterial composition, including an increased abundance of 4 species of Lactobacillus and Bifidobacterium adolescentis, were detected, but without a shift in the overall community structure. Progesterone levels were assessed as a possible hormone mediator of bacterial change. The relative abundance of only some taxa (e.g., Bifidobacteriaceae) were specifically associated with progesterone. In summary, pregnancy changes the microbial profiles in monkeys, but the bacterial diversity in their lower reproductive tract is different from women, and the composition of their intestinal symbionts remains stable until late gestation when several Firmicutes become more prominent.

Abstract 2965: LBL-033, a novel bispecific antibody targeting MUC16 and CD3, for the treatment of tumors over-expressing MUC16

Abstract Background: MUC16 is a large glycoprotein overexpressed on cell membrane of solid tumors, especially in almost 80% of ovarian cancer. The extracellular part of MUC16 could be cleaved as CA125, which is well known as a diagnosis biomarker for ovarian cancer. Despite recent advances in therapies such as PARP inhibitors, advanced ovarian cancer remains a disease of high unmet need. Herein a novel bispecific antibody targeting MUC16 and CD3, LBL-033, is developed to specifically kill MUC16 positive tumor cells by engaging T cells. Methods: LBL-033 was constructed using our unique T cell engager platform, which is a 2:1 format bispecific antibody with two arms targeting MUC16 membrane-proximal domain with high affinity and one arm targeting CD3 with fine-tuned affinity. The binding affinity of LBL-033 to MUC16 and CD3 was determined with Fortebio, while the activity was measured using several cell based assays including reporter gene and TDCC assays. The anti-tumor activity of LBL-033 was investigated in C57BL/6-huCD3e mice implanted with MC38-huMUC16 cells. GLP-compliant repeat-dose toxicologic study was evaluated in cynomolgus monkeys. Results: The binding affinity of LBL-033 to MUC16 and CD3 protein was 2.67 nM and 29.6 nM, respectively. The epitope that anti-MUC16 binds was identified at the C-terminal of MUC16 extracellular domain; and the activity was not affected in the presence of soluble CA-125. In CD3 reporter gene assay, LBL-033 could activate the NFAT reporter element through MUC16 antigen-dependent CD3 cross-linking with an EC50 value of 1.068 nM. LBL-033 was shown a potent TDCC activity in OVCAR3 cells with T cell activation. In C57BL/6-huCD3e transgenic mice implanted with MC38-huMUC16 tumor cells, LBL-033 was shown a dose dependent anti-tumor activity. In addition, LBL-033 had a significant synergistic effect, when combined with anti-PD-1. LBL-033 was shown favorable pharmacokinetic properties with a good safety profile in cynomolgus monkeys. Conclusion: LBL-033, a novel bispecific antibody targeting CD3 and MUC16 with a unique epitope, which is located at the MUC16 membrane-proximal domain, with affinity differentiation between anti-MUC16 and anti-CD3. It is shown a great anti-tumor efficacy in animal models, with a good safety profile in monkeys. These data support LBL-033 as a novel therapeutic bispecific antibody for ovarian cancer and other MUC16 positive tumors. A dose escalation study in humans will begin in Q1 2023. Citation Format: Jianming Sun, Xiao Huang, Yurong Qin, XingXing Fang, Min Chen, Huan Lin, Jing Guan, Jing Huang, Tongjun Liu, Baohui Wang, Guojin Wu, Shoupeng Lai, Xiaoqiang Kang, Hong Ling. LBL-033, a novel bispecific antibody targeting MUC16 and CD3, for the treatment of tumors over-expressing MUC16 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2965.

An Engineered IFNγ-Antibody Fusion Protein with Improved Tumor-Homing Properties.

Interferon-gamma (IFNγ) is one of the central cytokines produced by the innate and adaptive immune systems. IFNγ directly favors tumor growth control by enhancing the immunogenicity of tumor cells, induces IP-10 secretion facilitating (CXCR3+) immune cell infiltration, and can prime macrophages to an M1-like phenotype inducing proinflammatory cytokine release. We had previously reported that the targeted delivery of IFNγ to neoplastic lesions may be limited by the trapping of IFNγ-based products by cognate receptors found in different organs. Here we describe a novel fusion protein consisting of the L19 antibody, specific to the alternatively spliced extra-domain B of fibronectin (EDB), fused to a variant of IFNγ with reduced affinity to its cognate receptor. The product (named L19-IFNγ KRG) selectively localized to tumors in mice, showed favorable pharmacokinetic profiles in monkeys and regained biological activity upon antigen binding. The fusion protein was investigated in two murine models of cancer, both as monotherapy and in combination with therapeutic modalities which are frequently used for cancer therapy. L19-IFNγ KRG induced tumor growth retardation and increased the intratumoral concentration of T cells and NK cells in combination with anti-PD-1.

Preclinical Pharmacokinetics and Pharmacodynamics of DISC-0998, a Humanized Anti-Hemojuvelin (HJV) Monoclonal Antibody to Suppress the Production of Hepcidin

Background Anemia of inflammation (AI) is an immune-driven disorder that displays reduced erythropoietic activity and dysregulation of iron homeostasis. It is associated with a number of underlying diseases including myelofibrosis (MF) and chronic kidney disease (CKD) and caused by inflammatory cytokines which increase hepcidin levels. Increased hepcidin leads to sequestered iron in macrophages, decreased iron absorption and decreased serum iron. Interfering with HJV function and suppressing the production of hepcidin results in increases in serum iron in healthy volunteers has been observed with another humanized monoclonal antibody (mAb, DISC-0974). PK/PD results from a Phase 1 clinical trial with DISC-0974 in healthy volunteers support monthly subcutaneous (SC) dosing. DISC-0998 is a potent and highly selective HJV mAb with an engineered Q and L mutation (QL-mutation) in the Fc region, aimed to alter binding to the FcRn receptor, resulting in an increased PK half-life. Preclinical studies have demonstrated that DISC--0998 has biological activity, low immunogenicity potential and desirable pharmacokinetic (PK) and pharmacodynamic (PD) properties. Objectives To evaluate the PK/PD relationships of DISC-0998 with hepcidin, serum iron, and TSAT in male cynomolgus monkeys to inform drug development strategy. Methods Male cynomolgus monkeys were randomized to different dose groups and received a single SC (0.1, 0.3, 1.0, and 3.0 mg/kg) or intravenous (IV, 6 mg/kg) dose of DISC-0998. Blood samples were obtained at predefined time points for PK, hepcidin-25, serum iron, and total iron binding capacity (TIBC) measurements. Concentration of serum hepcidin-25 was analyzed using a qualified LC-MS/MS method. Concentrations of serum iron and TIBC were analyzed using a HITACHI7180 Chemistry Analyzer. Serum DISC-0998 concentrations were analyzed using a qualified MSD method. For comparison purposes, the results from DISC-0974 monkey PK/PD studies following 0.3, 1, and 6 mg/kg SC were also included. WinNonlin (PhoenixTM, v8.1) was used for PK/PD calculations. Results Following a single SC or IV dose, DISC-0998 exhibited low clearance (SC CL/F 0.14 - 0.83 mL/hr/kg; IV CL 0.14 mL/hr/kg), small volume of distribution (Vz 50 -104 mL/kg), and nonlinear PK typical for a mAb. Compared to DISC-0974, DISC-0998 exhibited lower clearance (~30 -40%), higher volume of distribution (30 - 70%), and a longer half-life (~ 2 times), which translated to longer duration of downstream PD effects. Dose-dependent hepcidin-25 reduction, along with elevations in serum iron and TSAT were also demonstrated. Based on PK and serum iron response, DISC-0998 has similar or slightly better potency in monkey compared to DISC-0974 (EAUC50 of 1495 hr*ug/mL vs. 3918 hr*g/mL, respectively). Conclusion DISC-0998 demonstrated a favorable PK/PD profile in monkeys, allowing prediction of its PKPD profiles in humans. These data support further development of DISC-0998 for anemia of inflammation related disorders, with the potential of long intervals in between dosing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

DS-7300a, a DNA Topoisomerase I Inhibitor, DXd-Based Antibody-Drug Conjugate Targeting B7-H3, Exerts Potent Antitumor Activities in Preclinical Models.

B7-H3 is overexpressed in various solid tumors and has been considered as an attractive target for cancer therapy. Here, we report the development of DS-7300a, a novel B7-H3-targeting antibody-drug conjugate with a potent DNA topoisomerase I inhibitor, and its in vitro profile, pharmacokinetic profiles, safety profiles, and in vivo antitumor activities in nonclinical species. The target specificity and species cross-reactivity of DS-7300a were assessed. Its pharmacologic activities were evaluated in several human cancer cell lines in vitro and xenograft mouse models, including patient-derived xenograft (PDX) mouse models in vivo. Pharmacokinetics was investigated in cynomolgus monkeys. Safety profiles in rats and cynomolgus monkeys were also assessed. DS-7300a specifically bound to B7-H3 and inhibited the growth of B7-H3-expressing cancer cells, but not that of B7-H3-negative cancer cells, in vitro. Additionally, treatment with DS-7300a and DXd induced phosphorylated checkpoint kinase 1, a DNA damage marker, and cleaved PARP, an apoptosis marker, in cancer cells. Moreover, DS-7300a demonstrated potent in vivo antitumor activities in high-B7-H3 tumor xenograft models, including various tumor types of high-B7-H3 PDX models. Furthermore, DS-7300a was stable in circulation with acceptable pharmacokinetic profiles in monkeys, and well tolerated in rats and monkeys. DS-7300a exerted potent antitumor activities against B7-H3-expressing tumors in in vitro and in vivo models, including PDX mouse models, and showed acceptable pharmacokinetic and safety profiles in nonclinical species. Therefore, DS-7300a may be effective in treating patients with B7-H3-expressing solid tumors in a clinical setting.

Enhancing monoamine oxidase B inhibitory activity via chiral fluorination: Structure-activity relationship, biological evaluation, and molecular docking study

Parkinson's disease (PD) is a common neurodegenerative disease among the elderly. Currently, monoamine oxidase B (MAO-B) inhibitors are extensively used for PD in clinics. In this work, a series of novel chiral fluorinated pyrrolidine derivatives were designed and synthesized. In vitro biological evaluations revealed that compound D5 was the most potent, selective MAO-B inhibitor (IC50 = 0.019 μM, MAO-A/MAO-B selectivity index = 2440), which was 10-fold than that of miracle drug safinamide (IC50 = 0.163 μM, MAO-A/MAO-B selectivity index = 172). It was verified that the enhanced hydrophobic interaction of D5 improved the activity against MAO-B in molecular docking study. Besides, D5 exhibited excellent metabolic properties and pharmacokinetic profiles in monkeys and rats. Moreover, D5 displayed more efficacious than safinamide in vivo models. In the MPTP-induced PD mouse model, D5 significantly alleviated DA deficits and increased the effect of levodopa on dopamine concentration in the striatum. Meanwhile, D5 produced a prominent reduction in tremulous jaw movements induced by galantamine. Accordingly, we present D5 as a novel, highly potent, and selective MAO-B inhibitor for PD therapy.

Preclinical immunogenicity testing using anti-drug antibody analysis of GX-G3, Fc-fused recombinant human granulocyte colony-stimulating factor, in rat and monkey models

Human granulocyte colony-stimulating factor (G-CSF, this study used Fc-fused recombinant G-CSF; GX-G3) is an important glycoprotein that stimulates the proliferation of granulocytes and white blood cells. Thus, G-CSF treatment has been considered as a crucial regimen to accelerate recovery from chemotherapy-induced neutropenia in cancer patients suffering from non-myeloid malignancy or acute myeloid leukemia. Despite the therapeutic advantages of G-CSF treatment, an assessment of its immunogenicity must be performed to determine whether the production of anti-G-CSF antibodies causes immune-related disorders. We optimized and validated analytical tools by adopting validation parameters for immunogenicity assessment. Using these validated tools, we analyzed serum samples from rats and monkeys injected subcutaneously with GX-G3 (1, 3 or 10 mg/kg once a week for 4 weeks followed by a 4-week recovery period) to determine immunogenicity response and toxicokinetic parameters with serum concentration of GX-G3. Several rats and monkeys were determined to be positive for anti-GX-G3 antibodies. Moreover, the immunogenicity response of GX-G3 was lower in monkeys than in rats, which was relevant to show less inhibition of toxicokinetic profiles in monkeys, at least 1 mg/kg administrated group, compared to rats. These results suggested the establishment and validation for analyzing anti-GX-G3 antibodies and measurement of serum levels of GX-G3 and anti-GX-G3 antibodies, which was related with toxicokinetic profiles. Taken together, this study provides immunogenicity assessment which is closely implicated with toxicokinetic study of GX-G3 in 4-week repeated administrated toxicological studies.

Discovery of N-(6-(5-fluoro-2-(piperidin-1-yl)phenyl)pyridazin-3-yl)-1-(tetrahydro-2H-pyran-4-yl)methanesulfonamide as a brain-permeable and metabolically stable kynurenine monooxygenase inhibitor

Kynurenine monooxygenase (KMO) is expected to be a good drug target to treat Huntington’s disease (HD). This study presents the structure-activity relationship of pyridazine derivatives to find novel KMO inhibitors. The most promising compound 14 resolved the problematic issues of lead compound 1, i.e., metabolic instability and reactive metabolite-derived side-effects. Compound 14 exhibited high brain permeability and a long-lasting pharmacokinetics profile in monkeys, and neuroprotective kynurenic acid was increased by a single administration of 14 in R6/2 mouse brain. These results demonstrated 14 may be a potential drug candidate to treat HD.

Nonclinical Development of Next-generation Site-specific HER2-targeting Antibody-drug Conjugate (ARX788) for Breast Cancer Treatment.

Conventional antibody-drug conjugates (ADC) utilize native surface-exposed lysines or cysteines on the antibody of interest to conjugate cytotoxic payload. The nonspecific conjugation results in a mixture with variable drug-to-antibody ratios (DAR), conjugation sites, and ADCs that are often unstable in systemic circulation. ARX788 is an ADC consisting of a HER2-targeting antibody site-specifically conjugated with a potent antitubulin cytotoxic drug-linker, AS269. The site-specific conjugation is achieved by first incorporating the nonnatural amino acid, para-acetyl phenylalanine (pAF), into the antibody, followed by covalent conjugation of AS269 to the pAF to form a highly stable oxime bond resulting in a DAR 2 ADC. ARX788 exhibits significant, dose-dependent antitumor activity against HER2- expressing breast and gastric xenograft tumors. Pharmacokinetic (PK) studies in multiple species showed the highly stable nature of ARX788 with overlapping PK profiles for the intact ADC and total antibody. Metabolism studies demonstrated that pAF-AS269 was the sole major metabolite of ARX788, with no evidence for the release of free drug often observed in conventional ADCs and responsible for adverse side effects. Furthermore, ARX788 demonstrated a favorable safety profile in monkeys with a highest nonseverely toxic dose of 10 mg/kg, which was well above the efficacious dose level observed in preclinical tumor models, thus supporting clinical development of ARX788.

Species differences in the CYP3A-catalyzed metabolism of TPN729, a novel PDE5 inhibitor.

TPN729 is a novel phosphodiesterase 5 (PDE5) inhibitor used to treat erectile dysfunction in men. Our previous study shows that the plasma exposure of metabolite M3 (N-dealkylation of TPN729) in humans is much higher than that of TPN729. In this study, we compared its metabolism and pharmacokinetics in different species and explored the contribution of its main metabolite M3 to pharmacological effect. We conducted a combinatory approach of ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry-based metabolite identification, and examined pharmacokinetic profiles in monkeys, dogs, and rats following TPN729 administration. A remarkable species difference was observed in the relative abundance of major metabolite M3: i.e., the plasma exposure of M3 was 7.6-fold higher than that of TPN729 in humans, and 3.5-, 1.2-, 1.1-fold in monkeys, dogs, and rats, respectively. We incubated liver S9 and liver microsomes with TPN729 and CYP3A inhibitors, and demonstrated that CYP3A was responsible for TPN729 metabolism and M3 formation in humans. The inhibitory activity of M3 on PDE5 was 0.78-fold that of TPN729 (The IC50 values of TPN729 and M3 for PDE5A were 6.17 ± 0.48 and 7.94 ± 0.07 nM, respectively.). The plasma protein binding rates of TPN729 and M3 in humans were 92.7% and 98.7%, respectively. It was astonishing that the catalyzing capability of CYP3A4 in M3 formation exhibited seven-fold disparity between different species. M3 was an active metabolite, and its pharmacological contribution was equal to that of TPN729 in humans. These findings provide new insights into the limitation and selection of animal model for predicting the clinical pharmacokinetics of drug candidates metabolized by CYP3A4.

Absorption and Disposition of Coproporphyrin I (CPI) in Cynomolgus Monkeys and Mice: Pharmacokinetic Evidence to Support the Use of CPI to Inform the Potential for Organic Anion-Transporting Polypeptide Inhibition

Despite a recent expansion in the recognition of the potential utility of coproporphyrin (CP) as an endogenous biomarker of organic anion-transporting polypeptide (OATP) 1B activity, there have been few detailed studies of CP's pharmacokinetic behavior and an overall poor understanding of its pharmacokinetic fate from tissues and excretion. Here, we describe the pharmacokinetics of octadeuterium-labeled coproporphyrin I (CPI-d8) in cynomolgus monkeys following oral and intravenous administration. CPI-d8 has a half-life and bioavailability of 7.6 hours and 3.2%, respectively. Cynomolgus monkeys received oral cyclosporin A (CsA) at 4, 20, and 100 mg/kg which yielded maximum blood concentrations (C max) and area under the plasma concentration-time curve (AUC) values of 0.19, 2.5, and 3.8 µM, and 2.7, 10.5, and 26.6 µM·h, respectively. The apparent CsA-dose dependent increase in the AUC ratio of CPI-d8 (1.8, 6.2, and 10.5), CPI (1.1, 1.4, and 4.4), and CPIII (1.1, 1.8, and 4.6) at 4, 20, and 100 mg, respectively. In contrast, the plasma concentrations of CPI and CPIII were generally not affected by intravenous administration of the renal organic anion and cation transporter inhibitors (probenecid and pyrimethamine, respectively). In addition, tritium-labeled coproporphyrin I ([3H]CPI) showed specific and rapid distribution to the liver, intestine, and kidney after an intravenous dose in mice using quantitative whole-body autoradiography. Rifampin markedly reduced the liver and intestinal uptake of [3H]CPI while increasing the kidney uptake. Taken together, these results suggest that hepatic OATP considerably affects the disposition of CPI in animal models, indicating CPI is a sensitive and selective endogenous biomarker of OATP inhibition. SIGNIFICANCE STATEMENT: This study demonstrated that coproporphyrin I (CPI) has favorable oral absorption, distribution, and elimination profiles in monkeys and mice as an endogenous biomarker. It also demonstrated its sensitivity and selectivity as a probe of organic anion-transporting polypeptide (OATP) 1B activity. The study reports, for the first time, in vivo pharmacokinetics, tissue distribution, sensitivity, and selectivity of CPI as an OATP1B endogenous biomarker in animals. The data provide preclinical support for exploration of its utility as a sensitive and selective circulating OATP biomarker in humans.

The enhanced immunopharmacology of VIB4920, a novel Tn3 fusion protein and CD40L antagonist, and assessment of its safety profile in cynomolgus monkeys.

Inhibition of the T- and B-cell interaction through the CD40/CD40 ligand (L) axis is a favourable approach for inflammatory disease treatment. Clinical studies of anti-CD40L molecules in autoimmune diseases have met challenges because of thromboembolic events and adverse haemostasis. VIB4920 (formerly MEDI4920) is a novel CD40L antagonist and Tn3 fusion protein designed to prevent adverse haemostasis and immunopharmacology. We evaluated the pharmacokinetics, activity and toxicity of VIB4920 in monkeys. Cynomolgus monkeys received i.v. or s.c. 5-300 mg·kg-1 VIB4920 or vehicle, once weekly for 1 month (Studies 1 and 2) or 28 weeks (Study 3). VIB4920 exposure and bioavailability were determined using pharmacokinetic analyses, and immune cell population changes via flow cytometry. Pharmacological activity was evaluated by measuring the animals' capacity to elicit an immune response to keyhole limpet haemocyanin (KLH) and tetanus toxoid (TT). VIB4920 demonstrated linear pharmacokinetics at multiple doses. Lymphocyte, monocyte, cytotoxic T-cell and NK cell counts were not significantly different between treatment groups. B-cell counts reduced dose-dependently and the T-cell dependent antibody response to KLH was suppressed by VIB4920 dose-dependently. The recall response to TT was similar across treatment groups. No thromboembolic events or symptoms of immune system dysfunctionality were observed. VIB4920 demonstrated an acceptable safety profile in monkeys. VIB4920 showed favourable pharmacokinetics, dose-dependent inhibition of a neoantigen-specific immune response and no adverse effects on immune function following long-term use. Our data support the use of VIB4920 in clinical trials.

Persistent Crimean-Congo hemorrhagic fever virus infection in the testes and within granulomas of non-human primates with latent tuberculosis.

Crimean-Congo hemorrhagic fever (CCHF) is the most medically important tick-borne viral disease of humans and tuberculosis is the leading cause of death worldwide by a bacterial pathogen. These two diseases overlap geographically, however, concurrent infection of CCHF virus (CCHFV) with mycobacterial infection has not been assessed nor has the ability of virus to persist and cause long-term sequela in a primate model. In this study, we compared the disease progression of two diverse strains of CCHFV in the recently described cynomolgus macaque model. All animals demonstrated signs of clinical illness, viremia, significant changes in clinical chemistry and hematology values, and serum cytokine profiles consistent with CCHF in humans. The European and Asian CCHFV strains caused very similar disease profiles in monkeys, which demonstrates that medical countermeasures can be evaluated in this animal model against multiple CCHFV strains. We identified evidence of CCHFV persistence in the testes of three male monkeys that survived infection. Furthermore, the histopathology unexpectedly revealed that six additional animals had evidence of a latent mycobacterial infection with granulomatous lesions. Interestingly, CCHFV persisted within the granulomas of two animals. This study is the first to demonstrate the persistence of CCHFV in the testes and within the granulomas of non-human primates with concurrent latent tuberculosis. Our results have important public health implications in overlapping endemic regions for these emerging pathogens.

CB1 receptor antagonism in capuchin monkeys alters social interaction and aversive memory extinction.

The endocannabinoid system (eCS) is an important modulator of social anxiety and social reward, as well as memory functions. The present study evaluated the role of eCS in social interactions and aversive memory extinction in capuchin monkeys (Sapajus spp.) by blocking the cannabinoid type 1 receptor (CB1r). In experiment 1, spontaneous social and non-social behaviors of five capuchin males, each one living in triads with two other females, were observed after AM251 treatment (vehicle, 0.3, 1.0, and 3.0mg/kg; i.m.). In experiment 2, seven male capuchin monkeys were trained to reach for a reward inside a wooden box. After training, they were given either vehicle or a 3.0-mg/kg i.m. dose of AM251 before a single aversive encounter with a live snake in the box. The latency to return to reach the reward inside the box in subsequent trials was measured. The 3.0-mg/kg dose significantly increased the time spent performing self-directed behaviors, while decreasing that of social interactions. No changes were observed in vigilance or locomotion. AM251 increased the latency to reach the reward after the aversive encounter. Taken together, these results suggest that CB1r antagonism induces social deficits without increasing anxiety levels and impairs the extinction of aversive memories. This behavioral profile in monkeys underscores the potential involvement of eCS signaling in the deficits observed in autism spectrum disorders.

Anti-hypercholesterolemic Effects and a Good Safety Profile of SCM-198 in Animals: From ApoE Knockout Mice to Rhesus Monkeys.

Although several lipid-lowering agents have been introduced for the treatment of atherosclerosis (AS), currently marketed medications have not solved the problem completely. This study aims to investigate the effects of leonurine (SCM-198) on dyslipidemia in mammals with ApoE knockout (ApoE-/-) mice, New Zealand white rabbits and senile Rhesus monkeys fed with high fat diet were dosed daily with leonurine or atorvastatin. The serum total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), and high-density lipoprotein (HDL) were determined. Moreover, in Rhesus monkeys, bodyweight, arterial ultrasound of right common carotid artery, Apolipoprotein A1 (ApoA1) and ApoB levels, hematologic and toxicological examinations were detected. Serum TC and TG in both mice and rabbits were significantly reduced by SCM-198 and atorvastatin. In the 10 mg/kg SCM-198 group of monkeys, maximum TC reduction of 24.05% was achieved at day 150, while 13.16% LDL reduction achieved at day 60, without arterial morphologic changes or adverse events. Atorvastatin (1.2 mg/kg) showed similar effects as SCM-198 in improving lipid profiles in monkeys, yet its long-term use could induce tolerance. Furthermore, leonurine suppressed genes expression of fatty acid synthesis, such as fatty acid synthase (FASN), stearoyl-CoA desaturase (SCD-1), sterol regulatory element-binding protein (SREBF) in liver in high fat diet feeding ApoE-/- mice. SCM-198, with a reliable safety profile, is of high value in improving lipid profiles in mammals, providing an alternative to a substantial population who are statin-intolerant.