Gemtuzumab Ozogamicin Treatment Results in Decreased Proliferation and Differentiation of Human Megakaryocytes but Does Not Inhibit Mature Platelet Function.

Mylotarg (Gemtuzumab ozogamicin [GO]), an antibody drug conjugate comprising a CD33-directed antibody linked to calicheamicin, is approved for use in certain acute myeloid leukemia patients. Following reports of prolonged thrombocytopenia and hemorrhagic events in a subset of patients, a detailed series of in vitro and ex vivo studies was performed at the request of regulators, both to look at the effects of GO on platelet production and to determine whether treatment with GO was likely to affect platelet aggregation under a variety of conditions. Treatment with GO resulted in cellular cytotoxicity and/or decreased differentiation during human megakaryocyte development. However, GO did not impair platelet aggregation under the experimental conditions evaluated. Ultimately, the effect of GO on megakaryocyte development observed in our studies was determined to have no impact on the risk-benefit assessment in the intended patient population, as thrombocytopenia is a known side effect of GO, and monitoring of platelet counts in patients is already strongly recommended.

To Clot or Not to Clot: Deepening Our Understanding of Alterations in the Hemostatic System.

The session on the hemostatic system focused on new developments in coagulation and platelet biology as well as how therapeutic agents may affect hemostasis. The classic cascade model of coagulation was compared with the more recent models of cell-based and vascular-based coagulation, which may provide better insight on how the coagulation cascade works in vivo. A review of platelet biology highlighted that, as platelets age, desialylated platelets form and are recognized by Ashwell-Morell receptor (AMR), leading to hepatic uptake and subsequent increase in thrombopoietin (TPO) production. Administration of therapeutics that induce thrombocytopenia was also discussed, including Mylotarg, which is an antibody-drug conjugate that was shown to decrease human megakaryocyte development but had no effect on platelet aggregation. An acetyl co-A carboxylase inhibitor was shown to cause thrombocytopenia by inhibiting de novo lipogenesis, which is critical for the formation of the megakaryocyte demarcation membrane system responsible for platelet production. It was also illustrated how preclinical translation models have been very helpful in the development of adeno-associated virus (AAV) hemophilia B gene therapy and what old and new preclinical tools we have that can predict the risk of a prothrombotic state in people.

Therapeutic Effects of FGF23 c-tail Fc in a Murine Preclinical Model of X-Linked Hypophosphatemia Via the Selective Modulation of Phosphate Reabsorption.

Fibroblast growth factor 23 (FGF23) is the causative factor of X-linked hypophosphatemia (XLH), a genetic disorder effecting 1:20,000 that is characterized by excessive phosphate excretion, elevated FGF23 levels and a rickets/osteomalacia phenotype. FGF23 inhibits phosphate reabsorption and suppresses 1α,25-dihydroxyvitamin D (1,25D) biosynthesis, analytes that differentially contribute to bone integrity and deleterious soft-tissue mineralization. As inhibition of ligand broadly modulates downstream targets, balancing efficacy and unwanted toxicity is difficult when targeting the FGF23 pathway. We demonstrate that a FGF23 c-tail-Fc fusion molecule selectively modulates the phosphate pathway in vivo by competitive antagonism of FGF23 binding to the FGFR/α klotho receptor complex. Repeated injection of FGF23 c-tail Fc in Hyp mice, a preclinical model of XLH, increases cell surface abundance of kidney NaPi transporters, normalizes phosphate excretion, and significantly improves bone architecture in the absence of soft-tissue mineralization. Repeated injection does not modulate either 1,25D or calcium in a physiologically relevant manner in either a wild-type or disease setting. These data suggest that bone integrity can be improved in models of XLH via the exclusive modulation of phosphate. We posit that the selective modulation of the phosphate pathway will increase the window between efficacy and safety risks, allowing increased efficacy to be achieved in the treatment of this chronic disease. © 2017 American Society for Bone and Mineral Research.

Inflammatory Cell Findings in the Female Rabbit Heart and Stress-associated Exacerbation with Handling and Procedures Used in Nonclinical Studies.

Despite the use of rabbits in biomedical research, including regulatory toxicology and cardiovascular studies, little data exist on heart findings in this species. This study was designed to document myocardial findings in female rabbits and the impact of study-related procedures typical for vaccine toxicology studies. One hundred and forty 6- to 8-month-old female New Zealand White rabbits were divided equally into 2 groups, high and low study procedure groups (group 1 and group 2, respectively). All animals received intramuscular (IM) injections of sterile saline every 2 weeks for 5 times and were necropsied 2 days after the final IM injection. Clinical chemistry, hematology, and urinalysis were evaluated. Blood for stress biomarkers (norepinephrine, epinephrine, cortisol, and corticosterone), C-reactive protein, cardiac troponin I, and creatine kinase were collected at time 0 (just before dose administration) and then at 4, 24, and 48 hr after dose administration in group 1 only. Hearts were assessed histologically. Focal to multifocal minimal inflammatory cell infiltrates were common (∼80%), particularly in the left ventricle and interventricular septum, and were similar to the types of infiltrates identified in other laboratory animal species. Additionally, study-related procedures elevated serum stress biomarkers and exacerbated the frequency and severity of myocardial inflammatory cell infiltrates.

Liver Microvascular Injury and Thrombocytopenia of Antibody-Calicheamicin Conjugates in Cynomolgus Monkeys-Mechanism and Monitoring.

Purpose: Adverse reactions reported in patients treated with antibody-calicheamicin conjugates such as gemtuzumab ozogamicin (Mylotarg) and inotuzumab ozogamicin include thrombocytopenia and sinusoidal obstruction syndrome (SOS). The objective of this experimental work was to investigate the mechanism for thrombocytopenia, characterize the liver injury, and identify potential safety biomarkers.Experimental Design: Cynomolgus monkeys were dosed intravenously at 6 mg/m2/dose once every 3 weeks with a nonbinding antibody-calicheamicin conjugate (PF-0259) containing the same linker-payload as gemtuzumab ozogamicin and inotuzumab ozogamicin. Monkeys were necropsied 48 hours after the first administration (day 3) or 3 weeks after the third administration (day 63).Results: PF-0259 induced acute thrombocytopenia (up to 86% platelet reduction) with nadirs on days 3 to 4. There was no indication of effects on megakaryocytes in bone marrow or activation of platelets in peripheral blood. Microscopic evaluation of liver from animals necropsied on day 3 demonstrated midzonal degeneration and loss of sinusoidal endothelial cells (SECs) associated with marked platelet accumulation in sinusoids. Liver histopathology on day 63 showed variable endothelial recovery and progression to a combination of sinusoidal capillarization and sinusoidal dilation/hepatocellular atrophy, consistent with early SOS. Among biomarkers evaluated, there were early and sustained increases in serum hyaluronic acid (HA) that correlated well with serum aspartate aminotransferase and liver microscopic changes, suggesting that HA may be a sensitive diagnostic marker of the liver microvascular injury.Conclusions: These data support the conclusion that target-independent damage to liver SECs may be responsible for acute thrombocytopenia (through platelet sequestration in liver sinusoids) and development of SOS. Clin Cancer Res; 23(7); 1760-70. ©2016 AACR.

Best practices for evaluation of bone marrow in nonclinical toxicity studies.

This manuscript is intended to provide a best practice approach to accurately and consistently assess toxicant-induced bone marrow effects of test articles. In nonclinical toxicity studies, complete blood count data in conjunction with the histological examination of the bone marrow are recommended as the foundation for assessing the effect of test articles on the hematopoietic system. This approach alone can be used successfully in many studies. However, in some situations it may be necessary to further characterize effects on the different hematopoietic lineages, either by cytological or flow cytometric evaluation of the bone marrow. Both modalities can be used successfully, and which one is selected will depend on the expertise, preference of the facility, and the nature of the change in the bone marrow. Other specialized techniques such as clonogenic assays or electron microscopy are used rarely to further characterize hematotoxicity. The indications and techniques to successfully employ histological, cytological, or flow cytometric evaluation as well as clonogenic assays and electron microscopy are reviewed.

Best practices for evaluation of bone marrow in nonclinical toxicity studies.

This manuscript is intended to provide a best practice approach to accurately and consistently assess toxicant-induced bone marrow effects of test articles. In nonclinical toxicity studies, complete blood count data in conjunction with the histological examination of the bone marrow are recommended as the foundation for assessing the effect of test articles on the hematopoietic system. This approach alone can be used successfully in many studies. However, in some situations it may be necessary to further characterize effects on the different hematopoietic lineages, either by cytological or flow cytometric evaluation of the bone marrow. Both modalities can be used successfully, and which one is selected will depend on the expertise, preference of the facility, and the nature of the change in the bone marrow. Other specialized techniques such as clonogenic assays or electron microscopy are used rarely to further characterize hematotoxicity. The indications and techniques to successfully employ histological, cytological, or flow cytometric evaluation as well as clonogenic assays and electron microscopy are reviewed.

cDNA cloning, expression, purification, distribution, and characterization of biologically active canine alanine aminotransferase-1.

Alanine aminotransferase (ALT) is a pyridoxal enzyme found mainly in the liver and kidney, but also in small amounts in the heart, muscle, fat, and brain. Serum aminotransferase activities have been used broadly as surrogate markers for tissue injury and disease in human and veterinary clinical settings and in safety assessment of chemicals and pharmaceuticals. Because of its relative abundance in liver, increased serum ALT activity is generally considered indicative of liver damage. Two ALT isoenzymes, ALT1 and ALT2, are known and have been cloned and sequenced from human, rat, and mouse. In this study, we have cloned the complementary DNA encoding the canine orthologue of ALT1 (cALT1). The complete cDNA sequence comprised 1852 bases and contained a 1485-base open reading frame, which encodes a polypeptide of 494 amino acid residues. Canine ALT1 shares 87.7, 87.2, and 87.0% amino acid identity to its human, mouse, and rat orthologues, respectively. The cDNA was expressed in Escherichia coli, with a N-terminal His (6x) tag, and the recombinant enzyme was purified using immobilized metal-affinity chromatography. The final yield of the purified recombinant cALT1 was greater than 5mg/L culture. The alanine transaminase activity of purified cALT1 was 229.81U/mg protein, which is approximately 38-fold higher than that of total soluble recombinant E. coli cell lysate, confirming that the enzyme is a functional ALT. Evaluation of various canine tissues by RT-PCR revealed that the level of ALT1 expression is in the order of: heart>liver>fat approximately brain approximately gastrocnemius>kidney. The purified cALT1 will be helpful to develop isoenzyme-specific anti-bodies, which could further improve the diagnostic resolution of current ALT assays in drug safety studies.