Manufacturing-dependent change in biological activity of the TLR4 agonist GSK1795091 and implications for lipid A analog development.

A phase I trial (NCT03447314; 204686) evaluated the safety and efficacy of GSK1795091, a Toll-like receptor 4 (TLR4) agonist, in combination with immunotherapy (GSK3174998 [anti-OX40 monoclonal antibody], GSK3359609 [anti-ICOS monoclonal antibody], or pembrolizumab) in patients with solid tumors. The primary endpoint was safety; other endpoints included efficacy, pharmacokinetics, and pharmacodynamics (PD). Manufacturing of GSK1795091 formulation was modified during the trial to streamline production and administration, resulting in reduced PD (cytokine) activity. Fifty-four patients received GSK1795091 with a combination partner; 32 received only the modified GSK1795091 formulation, 15 received only the original formulation, and seven switched mid-study from the original to the modified formulation. Despite the modified formulation demonstrating higher systemic GSK1795091 exposure compared with the original formulation, the transient, dose-dependent elevations in cytokine and chemokine concentrations were no longer observed (e.g., IP-10, IL10, IL1-RA). Most patients (51/54; 94%) experienced ≥1 treatment-emergent adverse event (TEAE) during the study. Safety profiles were similar between formulations, but a higher incidence of TEAEs associated with immune responses (chills, fatigue, pyrexia, nausea, and vomiting) were observed with the original formulation. No conclusions can be made regarding GSK1795091 anti-tumor activity due to the limited data collected. Manufacturing changes were hypothesized to have caused the change in biological activity in this study. Structural characterization revealed GSK1795091 aggregate size in the modified formulation to be twice that in the original formulation, suggesting a negative correlation between GSK1795091 aggregate size and PD activity. This may have important clinical implications for future development of structurally similar compounds.

Quantitative Systems Pharmacology Modeling of Acid Sphingomyelinase Deficiency and the Enzyme Replacement Therapy Olipudase Alfa Is an Innovative Tool for Linking Pathophysiology and Pharmacology.

Acid sphingomyelinase deficiency (ASMD) is a rare lysosomal storage disorder with heterogeneous clinical manifestations, including hepatosplenomegaly and infiltrative pulmonary disease, and is associated with significant morbidity and mortality. Olipudase alfa (recombinant human acid sphingomyelinase) is an enzyme replacement therapy under development for the non-neurological manifestations of ASMD. We present a quantitative systems pharmacology (QSP) model supporting the clinical development of olipudase alfa. The model is multiscale and mechanistic, linking the enzymatic deficiency driving the disease to molecular-level, cellular-level, and organ-level effects. Model development was informed by natural history, and preclinical and clinical studies. By considering patient-specific pharmacokinetic (PK) profiles and indicators of disease severity, the model describes pharmacodynamic (PD) and clinical end points for individual patients. The ASMD QSP model provides a platform for quantitatively assessing systemic pharmacological effects in adult and pediatric patients, and explaining variability within and across these patient populations, thereby supporting the extrapolation of treatment response from adults to pediatrics.

Practical considerations in clinical strategy to support the development of injectable drug-device combination products for biologics.

The development of an injectable drug-device combination (DDC) product for biologics is an intricate and evolving process that requires substantial investments of time and money. Consequently, the commercial dosage form(s) or presentation(s) are often not ready when pivotal trials commence, and it is common to have drug product changes (manufacturing process or presentation) during clinical development. A scientifically sound and robust bridging strategy is required in order to introduce these changes into the clinic safely. There is currently no single developmental paradigm, but a risk-based hierarchical approach has been well accepted. The rigor required of a bridging package depends on the level of risk associated with the changes. Clinical pharmacokinetic/pharmacodynamic comparability or outcome studies are only required when important changes occur at a late stage. Moreover, an injectable DDC needs to be user-centric, and usability assessment in real-world clinical settings may be required to support the approval of a DDC. In this review, we discuss the common issues during the manufacturing process and presentation development of an injectable DDC and practical considerations in establishing a clinical strategy to address these issues, including key elements of clinical studies. We also analyze the current practice in the industry and review relevant and status of regulatory guidance in the DDC field.

CSL112 enhances biomarkers of reverse cholesterol transport after single and multiple infusions in healthy subjects.

OBJECTIVE: The ability of apolipoprotein A-I (apoA-I) to transport cholesterol from atherosclerotic plaque is thought to underlie its inverse correlation with cardiovascular risk. To gauge the potential of infused apoA-I to transport cholesterol, we quantified cholesterol transport markers in human subjects infused with a novel formulation of apoA-I (CSL112). APPROACH AND RESULTS: CSL112 was infused into human subjects in single (57 subjects) and multiple (36 subjects) ascending dose trials. Pharmacokinetic and biomarker assessments were conducted before and after infusions. CSL112 caused an immediate, up to 3-fold elevation of apoA-I and subsequent movement of tissue cholesterol into plasma. Cholesterol appeared first as unesterified cholesterol in the high-density lipoprotein (HDL) fraction and was promptly esterified by lecithin cholesterol acyltransferase. HDL cholesterol increased up to 81±16.5%. Underlying this movement of cholesterol was an immediate and strong rise in the ability of plasma to promote cholesterol efflux from cells ex vivo. CSL112 had its greatest impact on the fraction of efflux mediated by ATP-binding cassette transporter A1 (ABCA1), a cholesterol transporter induced in cholesterol-loaded tissues such as plaque. ABCA1-dependent efflux capacity increased ≤630±421% and total efflux capacity by ≤192±40%. In keeping with this finding, we observed a profound rise in very small HDL, also known as preß1-HDL, the preferred substrate for ABCA1. Very small HDL increased ≤3596±941%. Elevations in apoA-I, cholesterol efflux, and very small HDL were dose-proportional over a wide range. No significant changes in atherogenic lipids were observed at any dose. CONCLUSIONS: Infusion of CSL112 elevates the ability of plasma to withdraw cholesterol from cells. Preferential elevation of ABCA1-dependent efflux may target atherosclerotic plaque for cholesterol removal, making CSL112 a promising candidate therapy for acute coronary syndrome.

A multiple ascending dose study of CSL112, an infused formulation of ApoA-I.

CSL112 is apoA-I purified from human plasma and reconstituted with phosphatidylcholine (PC) to form high-density lipoprotein (HDL)-particles suitable for infusion. CSL112 is in development for the potential treatment of acute coronary syndromes (ACS) by optimizing cholesterol efflux. This study assesses the pharmacokinetics (PK), safety and tolerability of CSL112. Repeat doses of CSL112 or placebo were administered intravenously once- (3.4 g or 6.8 g) or twice-weekly (3.4 g) to healthy subjects in a placebo-controlled, randomized (3 CSL112: 1 placebo), ascending-dose study (NCT01281774). Twenty-seven subjects received CSL112 and nine received placebo. Study endpoints included plasma apoA-I and PC concentrations and specific PK parameters. CSL112 infusions immediately produced robust increases in apoA-I concentration in a dose-proportional manner, reaching levels higher than observed with currently available or investigational HDL products. After infusion of CSL112, apoA-I levels remained above baseline for approximately 3 days. Multiple infusions of CSL112 were safe and well tolerated with no evidence of major organ toxicity or immunogenicity. CSL112 may provide a novel option to rapidly transport cholesterol from atherosclerotic plaque to the liver and reduce early recurrent events following ACS. The data presented here support continued clinical development of CSL112 in patient populations.

Loss of Akt1 in mice increases energy expenditure and protects against diet-induced obesity.

Akt is encoded by a gene family for which each isoform serves distinct but overlapping functions. Based on the phenotypes of the germ line gene disruptions, Akt1 has been associated with control of growth, whereas Akt2 has been linked to metabolic regulation. Here we show that Akt1 serves an unexpected role in the regulation of energy metabolism, as mice deficient for Akt1 exhibit protection from diet-induced obesity and its associated insulin resistance. Although skeletal muscle contributes most of the resting and exercising energy expenditure, muscle-specific deletion of Akt1 does not recapitulate the phenotype, indicating that the role of Akt1 in skeletal muscle is cell nonautonomous. These data indicate a previously unknown function of Akt1 in energy metabolism and provide a novel target for treatment of obesity.

Postprandial hepatic lipid metabolism requires signaling through Akt2 independent of the transcription factors FoxA2, FoxO1, and SREBP1c.

Under conditions of obesity and insulin resistance, the serine/threonine protein kinase Akt/PKB is required for lipid accumulation in liver. Two forkhead transcription factors, FoxA2 and FoxO1, have been suggested to function downstream of and to be negatively regulated by Akt and are proposed as key determinants of hepatic triglyceride content. In this study, we utilize genetic loss of function experiments to show that constitutive activation of neither FoxA2 nor FoxO1 can account for the protection from steatosis afforded by deletion of Akt2 in liver. Rather, another downstream target positively regulated by Akt, the mTORC1 complex, is required in vivo for de novo lipogenesis and Srebp1c expression. Nonetheless, activation of mTORC1 and SREBP1c is not sufficient to drive postprandial lipogenesis in the absence of Akt2. These data show that insulin signaling through Akt2 promotes anabolic lipid metabolism independent of Foxa2 or FoxO1 and through pathways additional to the mTORC1-dependent activation of SREBP1c.

Insulin regulates adipocyte lipolysis via an Akt-independent signaling pathway.

After a meal, insulin suppresses lipolysis through the activation of its downstream kinase, Akt, resulting in the inhibition of protein kinase A (PKA), the main positive effector of lipolysis. During insulin resistance, this process is ineffective, leading to a characteristic dyslipidemia and the worsening of impaired insulin action and obesity. Here, we describe a noncanonical Akt-independent, phosphoinositide-3 kinase (PI3K)-dependent pathway that regulates adipocyte lipolysis using restricted subcellular signaling. This pathway selectively alters the PKA phosphorylation of its major lipid droplet-associated substrate, perilipin. In contrast, the phosphorylation of another PKA substrate, hormone-sensitive lipase (HSL), remains Akt dependent. Furthermore, insulin regulates total PKA activity in an Akt-dependent manner. These findings indicate that localized changes in insulin action are responsible for the differential phosphorylation of PKA substrates. Thus, we identify a pathway by which insulin regulates lipolysis through the spatially compartmentalized modulation of PKA.

Akt2 is required for hepatic lipid accumulation in models of insulin resistance.

Insulin drives the global anabolic response to nutrient ingestion, regulating both carbohydrate and lipid metabolism. Previous studies have demonstrated that Akt2/protein kinase B is critical to insulin's control of glucose metabolism, but its role in lipid metabolism has remained controversial. Here, we show that Akt2 is required for hepatic lipid accumulation in obese, insulin-resistant states induced by either leptin deficiency or high-fat diet feeding. Lep(ob/ob) mice lacking hepatic Akt2 failed to amass triglycerides in their livers, associated with and most likely due to a decrease in lipogenic gene expression and de novo lipogenesis. However, Akt2 is also required for steatotic pathways unrelated to fatty acid synthesis, as mice fed high-fat diet had reduced liver triglycerides in the absence of hepatic Akt2 but did not exhibit changes in lipogenesis. These data demonstrate that Akt2 is a requisite component of the insulin-dependent regulation of lipid metabolism during insulin resistance.

Akt1/protein kinase Balpha is critical for ischemic and VEGF-mediated angiogenesis.

Akt, or protein kinase B, is a multifunctional serine-threonine protein kinase implicated in a diverse range of cellular functions including cell metabolism, survival, migration, and gene expression. However, the in vivo roles and effectors of individual Akt isoforms in signaling are not explicitly clear. Here we show that the genetic loss of Akt1, but not Akt2, in mice results in defective ischemia and VEGF-induced angiogenesis as well as severe peripheral vascular disease. Akt1 knockout (Akt1-/-) mice also have reduced endothelial progenitor cell (EPC) mobilization in response to ischemia, and reintroduction of WT EPCs, but not EPCs isolated from Akt1-/- mice, into WT mice improves limb blood flow after ischemia. Mechanistically, the loss of Akt1 reduces the basal phosphorylation of several Akt substrates, the migration of fibroblasts and ECs, and NO release. Reconstitution of Akt1-/- ECs with Akt1 rescues the defects in substrate phosphorylation, cell migration, and NO release. Thus, the Akt1 isoform exerts an essential role in blood flow control, cellular migration, and NO synthesis during postnatal angiogenesis.

Role for Akt3/protein kinase Bgamma in attainment of normal brain size.

Studies of Drosophila and mammals have revealed the importance of insulin signaling through phosphatidylinositol 3-kinase and the serine/threonine kinase Akt/protein kinase B for the regulation of cell, organ, and organismal growth. In mammals, three highly conserved proteins, Akt1, Akt2, and Akt3, comprise the Akt family, of which the first two are required for normal growth and metabolism, respectively. Here we address the function of Akt3. Like Akt1, Akt3 is not required for the maintenance of normal carbohydrate metabolism but is essential for the attainment of normal organ size. However, in contrast to Akt1-/- mice, which display a proportional decrease in the sizes of all organs, Akt3-/- mice present a selective 20% decrease in brain size. Moreover, although Akt1- and Akt3-deficient brains are reduced in size to approximately the same degree, the absence of Akt1 leads to a reduction in cell number, whereas the lack of Akt3 results in smaller and fewer cells. Finally, mammalian target of rapamycin signaling is attenuated in the brains of Akt3-/- but not Akt1-/- mice, suggesting that differential regulation of this pathway contributes to an isoform-specific regulation of cell growth.

Endemic babesiosis in another eastern state: New Jersey.

In the United States, most reported cases of babesiosis have been caused by Babesia microti and acquired in the northeast. Although three cases of babesiosis acquired in New Jersey were recently described by others, babesiosis has not been widely known to be endemic in New Jersey. We describe a case of babesiosis acquired in New Jersey in 1999 in an otherwise healthy 53-year-old woman who developed life-threatening disease. We also provide composite data on 40 cases of babesiosis acquired from 1993 through 2001 in New Jersey. The 40 cases include the one we describe, the three cases previously described, and 36 other cases reported to public health agencies. The 40 cases were acquired in eight (38.1%) of the 21 counties in the state. Babesiosis, a potentially serious zoonosis, is endemic in New Jersey and should be considered in the differential diagnosis of patients with fever and hemolytic anemia, particularly in the spring, summer, and early fall.

Intrinsic and extrinsic pathway signaling during neuronal apoptosis: lessons from the analysis of mutant mice.

Trophic factor deprivation (TFD)-induced apoptosis in sympathetic neurons requires macromolecular synthesis-dependent BAX translocation, cytochrome c (cyt c) release, and caspase activation. Here, we report the contributions of other intrinsic and extrinsic pathway signals to these processes. Sympathetic neurons expressed all antiapoptotic BCL-2 proteins examined, yet expressed only certain BH3-only and multidomain proapoptotic BCL-2 family members. All coexpressed proapoptotic proteins did not, however, exhibit functional redundancy or compensatory expression, at least in the Bax-/-, Bak-/-, Bim-/-, Bid-/-, and Bad-/- neurons examined. Although the subcellular distribution or posttranslational modification of certain BCL-2 proteins changed with TFD, neither transcriptional nor posttranslational mechanisms regulated the expression or subcellular localization of BID, BAD, or BAK in this paradigm. Despite modest induction of Fas and FasL expression, Fas-mediated signaling did not contribute to TFD-induced apoptosis in sympathetic neurons. Similar findings were obtained with K+ withdrawal-induced apoptosis in cerebellar granule neurons, a model for activity-dependent neuronal survival in the CNS. Thus, expression alone does not guarantee functional redundancy (or compensation) among BCL-2 family members, and, at least in some cells, extrinsic pathway signaling and certain BH3-only proteins (i.e., BID and BAD) do not contribute to BAX-dependent cyt c release or apoptosis caused by TFD.

Presenilin-1 protects against neuronal apoptosis caused by its interacting protein PAG.

Mutations in the presenilin-1 (PS-1) gene account for a significant fraction of familial Alzheimer's disease. The biological function of PS-1 is not well understood. We report here that the proliferation-associated gene (PAG) product, a protein of the thioredoxin peroxidase family, interacts with PS-1. Microinjection of a plasmid expressing PAG into superior cervical ganglion (SCG) sympathetic neurons in primary cultures led to apoptosis. Microinjection of plasmids expressing wild-type PS-1 or a PS-1 mutant with a deletion of exon 10 (PS1dE10) by themselves had no effect on the survival of primary SCG neurons. However, co-injection of wild-type PS-1 with PAG prevented neuronal death, whereas co-injection with the mutant PS-1 did not affect PAG-induced apoptosis. Furthermore, overexpression of PAG accelerated SCG neuronal death induced by nerve growth factor deprivation. This sensitizing effect was also blocked by wild-type PS-1, but not by PS1dE10. These results establish an assay for studying the function of PS-1 in primary neurons, reveal the neurotoxicity of a thioredoxin peroxidase, demonstrate a neuroprotective activity of the wild-type PS-1, and suggest possible involvement of defective neuroprotection by PS-1 mutants in neurodegeneration.