Tirzepatide Immunogenicity on Pharmacokinetics, Efficacy, and Safety: Analysis of Data From Phase 3 Studies.

CONTEXT: Antidrug antibodies (ADA) can potentially affect drug pharmacokinetics, safety, and efficacy. OBJECTIVE: This work aimed to evaluate treatment-emergent (TE) ADA in tirzepatide (TZP)-treated participants across 7 phase 3 trials and their potential effect on pharmacokinetics, efficacy, and safety. METHODS: ADA were assessed at baseline and throughout the study until end point, defined as week 40 (SURPASS-1, -2, and -5) or week 52 (SURPASS-3, -4, Japan-Mono, and Japan-Combo). Samples for ADA characterization were collected at SURPASS trial sites. Participants included ADA-evaluable TZP-treated patients with type 2 diabetes (N = 5025). Interventions included TZP 5, 10, or 15 mg. ADA were detected and characterized for their ability to cross-react with native glucose-dependent insulinotropic polypeptide (nGIP) and glucagon-like peptide-1 (nGLP-1), neutralize tirzepatide activity on GIP and GLP-1 receptors, and neutralize nGIP and nGLP-1. RESULTS: TE ADA developed in 51.1% of tirzepatide-treated patients. Proportions were similar across dose groups. Maximum ADA titers ranged from 1:20 to 1: 81 920 among TE ADA+ patients. Neutralizing antibodies (NAb) against TZP activity on GIP and GLP-1 receptors were observed in 1.9% and 2.1% of patients, respectively. Less than 1.0% of patients had cross-reactive NAb against nGIP or nGLP-1. TE ADA status, ADA titer, and NAb status had no effect on the pharmacokinetics or efficacy of TZP. More TE ADA+ patients experienced hypersensitivity reactions or injection site reactions than TE ADA- patients. The majority of hypersensitivity and injection site reactions were nonserious and nonsevere, and most events occurred and/or resolved irrespective of TE ADA status or titer. CONCLUSION: Immunogenicity did not affect TZP pharmacokinetics or efficacy. The majority of hypersensitivity or injection site reactions experienced by TE ADA+ patients were mild to moderate in severity.

Donanemab Population Pharmacokinetics, Amyloid Plaque Reduction, and Safety in Participants with Alzheimer's Disease.

Donanemab is an amyloid-targeting therapy that resulted in robust amyloid plaque reduction and slowed Alzheimer's disease (AD) progression compared with placebo in the phase II TRAILBLAZER-ALZ study (NCT03367403). The objectives of the current analyses are to characterize (i) the population pharmacokinetics of donanemab, (ii) the relationship between donanemab exposure and amyloid plaque reduction (response), and (iii) the relationship between donanemab exposure and amyloid-related imaging abnormalities with edema or effusions (ARIA-E). Model development included data from participants with mild cognitive impairment or mild to moderate dementia due to AD from the phase Ib study on donanemab (NCT02624778) and participants with early symptomatic AD from the TRAILBLAZER-ALZ study. The analysis showed donanemab has a terminal elimination half-life of 11.8 days. Body weight and antidrug antibody titer impact donanemab exposure but not the pharmacodynamic response. Maintaining a donanemab serum concentration above 4.43 µg/mL (95% confidence interval: 0.956, 10.4) is associated with amyloid plaque reduction. The time to achieve amyloid plaque clearance (amyloid plaque level < 24.1 Centiloids) varied depending on the baseline amyloid level, where higher baseline levels were associated with fewer participants achieving amyloid clearance. The majority of participants achieved amyloid clearance by 52 weeks on treatment. Apolipoprotein ε4 carriers, irrespective of donanemab serum exposure, were 4 times more likely than noncarriers to have an ARIA-E event by 24 weeks.

Improved Clinical Sensitivity of a Reflexive Algorithm to Minimize False-Negative Test Results by a Urine Benzodiazepine Immunoassay Screen.

BACKGROUND: Urine drug testing is an essential component of treating patients for chronic pain and/or anxiety and is used to monitor compliance during treatment. A common algorithm is to use an immunoassay as a urine drug screen (UDS), followed by mass spectrometry to confirm all presumptive positive samples. Many UDSs, however, have significant limitations, and false-negative test results can be common due to lack of antibody specificity. METHODS: Urine samples were screened by a benzodiazepine immunoassay followed by confirmatory testing using LC-MS/MS to determine an initial false-negative test rate for the screen. Attempts to improve the false-negative test rate included hydrolysis before screening and optimization of the absorbance cutoff required for a positive result. RESULTS: Hydrolysis corrected 41% of false-negative test results in samples containing parent benzodiazepines and/or metabolites but had no effect on samples containing only clonazepam. Of the confirmed false-negative test results, 85% (17 of 20) demonstrated absorbance values between 20 and 100, with 100 being the cutoff for a positive result. Implementing an optimized absorbance cutoff of 20, rather than 100, for a reflexive confirmation testing algorithm decreases the false-negative test rate of detecting benzodiazepine from 47% to 2%. CONCLUSIONS: Hydrolyzing samples before the benzodiazepine screen provided a modest improvement in the false-negative test rate; however, the screen still missed samples containing clonazepam. Optimization of the absorbance cutoff to reflex samples to LC-MS/MS markedly improved the false-negative test rate for all benzodiazepines.

The phosphatidic acid-binding, polybasic domain is responsible for the differences in the phosphoregulation of lipins 1 and 3.

Lipins 1, 2, and 3 are Mg2+-dependent phosphatidic acid phosphatases and catalyze the penultimate step of triacylglycerol synthesis. We have previously investigated the biochemistry of lipins 1 and 2 and shown that di-anionic phosphatidic acid (PA) augments their activity and lipid binding and that lipin 1 activity is negatively regulated by phosphorylation. In the present study, we show that phosphorylation does not affect the catalytic activity of lipin 3 or its ability to associate with PA in vitro The lipin proteins each contain a conserved polybasic domain (PBD) composed of nine lysine and arginine residues located between the conserved N- and C-terminal domains. In lipin 1, the PBD is the site of PA binding and sensing of the PA electrostatic charge. The specific arrangement and number of the lysines and arginines of the PBD vary among the lipins. We show that the different PBDs of lipins 1 and 3 are responsible for the presence of phosphoregulation on the former but not the latter enzyme. To do so, we generated lipin 1 that contained the PBD of lipin 3 and vice versa. The lipin 1 enzyme with the lipin 3 PBD lost its ability to be regulated by phosphorylation but remained downstream of phosphorylation by mammalian target of rapamycin. Conversely, the presence of the lipin 1 PBD in lipin 3 subjected the enzyme to negative intramolecular control by phosphorylation. These results indicate a mechanism for the observed differences in lipin phosphoregulation in vitro.

Air bubbles and hemolysis of blood samples during transport by pneumatic tube systems.

BACKGROUND: Transport of blood samples through pneumatic tube systems (PTSs) generates air bubbles in transported blood samples and, with increasing duration of transport, the appearance of hemolysis. We investigated the role of air-bubble formation in PTS-induced hemolysis. METHODS: Air was introduced into blood samples for 0, 1, 3 or 5min to form air bubbles. Hemolysis in the blood was assessed by (H)-index, lactate dehydrogenase (LD) and potassium in plasma. In an effort to prevent PTS-induced hemolysis, blood sample tubes were completely filled, to prevent air bubble formation, and compared with partially filled samples after PTS transport. We also compared hemolysis in anticoagulated vs clotted blood subjected to PTS transport. RESULTS: As with transport through PTSs, the duration of air bubble formation in blood by a gentle stream of air predicted the extent of hemolysis as measured by H-index (p<0.01), LD (p<0.01), and potassium (p<0.02) in plasma. Removing air space in a blood sample prevented bubble formation and fully protected the blood from PTS-induced hemolysis (p<0.02 vs conventionally filled collection tube). Clotted blood developed less foaming during PTS transport and was partially protected from hemolysis vs anticoagulated blood as indicated by lower LD (p<0.03) in serum than in plasma after PTS sample transport. CONCLUSIONS: Prevention of air bubble formation in blood samples during PTS transport protects samples from hemolysis.

Smartphone monitoring of pneumatic tube system-induced sample hemolysis.

BACKGROUND: Pneumatic tube systems (PTSs) are convenient methods of patient sample transport in medical centers, but excessive acceleration force and time/distance traveled in the PTS have been correlated with increased blood-sample hemolysis. We investigated the utility of smartphones for monitoring of PTS-related variables. METHODS: Smartphones were sent through the PTS from several hospital locations. Each smartphone used 2 apps as data-loggers to record force of acceleration vs time. To relate the smartphone data to sample integrity, blood samples were collected from 5 volunteers, and hemolysis of the samples was analyzed after they were transported by hand or via 1 of 2 PTS routes. Increased sample hemolysis as measured by plasma lactate dehydrogenase (LD) was also related to the amount of transport in the PTS. RESULTS: The smartphones showed higher duration of forceful acceleration during transport through 1 of the 2 PTS routes, and the increased duration correlated with significant increases in hemolysis (H)-index and plasma LD. In addition, plasma LD showed a positive linear relationship with number of shock forces experienced during transport through the PTS. CONCLUSIONS: Smartphones can monitor PTS variables that cause sample hemolysis. This provides an accessible method for investigating specific PTS routes in medical centers.

Disabled homolog 2 controls macrophage phenotypic polarization and adipose tissue inflammation.

Acute and chronic tissue injury results in the generation of a myriad of environmental cues that macrophages respond to by changing their phenotype and function. This phenotypic regulation is critical for controlling tissue inflammation and resolution. Here, we have identified the adaptor protein disabled homolog 2 (DAB2) as a regulator of phenotypic switching in macrophages. Dab2 expression was upregulated in M2 macrophages and suppressed in M1 macrophages isolated from both mice and humans, and genetic deletion of Dab2 predisposed macrophages to adopt a proinflammatory M1 phenotype. In mice with myeloid cell-specific deletion of Dab2 (Dab2fl/fl Lysm-Cre), treatment with sublethal doses of LPS resulted in increased proinflammatory gene expression and macrophage activation. Moreover, chronic high-fat feeding exacerbated adipose tissue inflammation, M1 polarization of adipose tissue macrophages, and the development of insulin resistance in DAB2-deficient animals compared with controls. Mutational analyses revealed that DAB2 interacts with TNF receptor-associated factor 6 (TRAF6) and attenuates IκB kinase ß-dependent (IKKß-dependent) phosphorylation of Ser536 in the transactivation domain of NF-κB p65. Together, these findings reveal that DAB2 is critical for controlling inflammatory signaling during phenotypic polarization of macrophages and suggest that manipulation of DAB2 expression and function may hold therapeutic potential for the treatment of acute and chronic inflammatory disorders.

Catecholamine-induced lipolysis causes mTOR complex dissociation and inhibits glucose uptake in adipocytes.

Anabolic and catabolic signaling oppose one another in adipose tissue to maintain cellular and organismal homeostasis, but these pathways are often dysregulated in metabolic disorders. Although it has long been established that stimulation of the ß-adrenergic receptor inhibits insulin-stimulated glucose uptake in adipocytes, the mechanism has remained unclear. Here we report that ß-adrenergic-mediated inhibition of glucose uptake requires lipolysis. We also show that lipolysis suppresses glucose uptake by inhibiting the mammalian target of rapamycin (mTOR) complexes 1 and 2 through complex dissociation. In addition, we show that products of lipolysis inhibit mTOR through complex dissociation in vitro. These findings reveal a previously unrecognized intracellular signaling mechanism whereby lipolysis blocks the phosphoinositide 3-kinase-Akt-mTOR pathway, resulting in decreased glucose uptake. This previously unidentified mechanism of mTOR regulation likely contributes to the development of insulin resistance.

Lipin 2 binds phosphatidic acid by the electrostatic hydrogen bond switch mechanism independent of phosphorylation.

Lipin 2 is a phosphatidic acid phosphatase (PAP) responsible for the penultimate step of triglyceride synthesis and dephosphorylation of phosphatidic acid (PA) to generate diacylglycerol. The lipin family of PA phosphatases is composed of lipins 1-3, which are members of the conserved haloacid dehalogenase superfamily. Although genetic alteration of LPIN2 in humans is known to cause Majeed syndrome, little is known about the biochemical regulation of its PAP activity. Here, in an attempt to gain a better general understanding of the biochemical nature of lipin 2, we have performed kinetic and phosphorylation analyses. We provide evidence that lipin 2, like lipin 1, binds PA via the electrostatic hydrogen bond switch mechanism but has a lower rate of catalysis. Like lipin 1, lipin 2 is highly phosphorylated, and we identified 15 phosphosites. However, unlike lipin 1, the phosphorylation of lipin 2 is not induced by insulin signaling nor is it sensitive to inhibition of the mammalian target of rapamycin. Importantly, phosphorylation of lipin 2 does not negatively regulate either membrane binding or PAP activity. This suggests that lipin 2 functions as a constitutively active PA phosphatase in stark contrast to the high degree of phosphorylation-mediated regulation of lipin 1. This knowledge of lipin 2 regulation is important for a deeper understanding of how the lipin family functions with respect to lipid synthesis and, more generally, as an example of how the membrane environment around PA can influence its effector proteins.

Diacylglycerol kinase α is a critical signaling node and novel therapeutic target in glioblastoma and other cancers.

Although diacylglycerol kinase α (DGKα) has been linked to several signaling pathways related to cancer cell biology, it has been neglected as a target for cancer therapy. The attenuation of DGKα activity via DGKα-targeting siRNA and small-molecule inhibitors R59022 and R59949 induced caspase-mediated apoptosis in glioblastoma cells and in other cancers, but lacked toxicity in noncancerous cells. We determined that mTOR and hypoxia-inducible factor-1α (HIF-1α) are key targets of DGKα inhibition, in addition to its regulation of other oncogenes. DGKα regulates mTOR transcription via a unique pathway involving cyclic AMP. Finally, we showed the efficacy of DGKα inhibition with short hairpin RNA or a small-molecule agent in glioblastoma and melanoma xenograft treatment models, with growth delay and decreased vascularity. This study establishes DGKα as a central signaling hub and a promising therapeutic target in the treatment of cancer.

Phosphorylation of lipin 1 and charge on the phosphatidic acid head group control its phosphatidic acid phosphatase activity and membrane association.

The lipin gene family encodes a class of Mg(2+)-dependent phosphatidic acid phosphatases involved in the de novo synthesis of phospholipids and triglycerides. Unlike other enzymes in the Kennedy pathway, lipins are not integral membrane proteins, and they need to translocate from the cytosol to intracellular membranes to participate in glycerolipid synthesis. The movement of lipin 1 within the cell is closely associated with its phosphorylation status. Although cellular analyses have demonstrated that highly phosphorylated lipin 1 is enriched in the cytosol and dephosphorylated lipin 1 is found on membranes, the effects of phosphorylation on lipin 1 activity and binding to membranes has not been recapitulated in vitro. Herein we describe a new biochemical assay for lipin 1 using mixtures of phosphatidic acid (PA) and phosphatidylethanolamine that reflects its physiological activity and membrane interaction. This depends on our observation that lipin 1 binding to PA in membranes is highly responsive to the electrostatic charge of PA. The studies presented here demonstrate that phosphorylation regulates the ability of the polybasic domain of lipin 1 to recognize di-anionic PA and identify mTOR as a crucial upstream signaling component regulating lipin 1 phosphorylation. These results demonstrate how phosphorylation of lipin 1 together with pH and membrane phospholipid composition play important roles in the membrane association of lipin 1 and thus the regulation of its enzymatic activity.