A Phase 1 Dose Escalation Study of the Pyruvate Kinase Activator Mitapivat (AG-348) in Sickle Cell Disease.

In the quantitative forensic toxicological analyses using instruments, major methods to be employed are conventional matrix-matched calibration method (MMCM). However, nowadays, the needs for using the standard addition methods (SAM) are increasing. In spite of this situation, there are no reports of the guidelines for the validations of SAM. In this review, the principle, how to perform it, advantages, disadvantages, reported application data, and the details of validation procedures for the SAM are described. Various databases such as SciFinder, Google and Google Scholar were utilized to collect relevant reports referring to the SAM. The long experiences of our research group on the SAM were also included in this review. Although the experimental procedures for the SAM are much more laborious than those of the MMCM, the SAM is essential to quantify target xenobiotic(s) in special matrices such as human solid tissues or biles, which remarkably interfere with the usual quantitative analyses. The validation methods for the SAM have been also proposed for the cases in the absence of the blank matrices. To our knowledge, this is the first presentation of detailed SAM procedure and its validation, which will facilitate the use of the SAM in forensic toxicology. Especially for its validation, new simple methods have been proposed.

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The standard addition method and its validation in forensic toxicology

Safety and efficacy of mitapivat, an oral pyruvate kinase activator, in adults with non-transfusion dependent α-thalassaemia or β-thalassaemia: an open-label, multicentre, phase 2 study

Painful vaso-occlusive crisis (VOC) remains the most common reason for presenting to the Emergency Department and hospitalization in patients with sickle cell disease (SCD). Although two new agents have been approved by the Food and Drug Administration for treating SCD, they both target to reduce the frequency of VOC. Results from studies investigating various approaches to treat and shorten VOC have so far been generally disappointing. In this paper, we will summarize the complex pathophysiology and downstream events of VOC and discuss the likely reasons for the disappointing results using monotherapy. We will put forward the rationale for exploring some of the currently available agents to either protect erythrocytes un-involved in the hemoglobin polymerization process from sickling induced by the secondary events, or a multipronged combination approach that targets the complex downstream pathways of VOC.

/pdf/vaso-occlusive-crisis-in-sickle-cell-disease-a-vicious-cycle-5aanoispdx.pdf

Vaso-occlusive crisis in sickle cell disease: a vicious cycle of secondary events.

To the Editor: Sickle cell disease (SCD) is one of the most common and debilitating inherited blood disorders affecting millions of people worldwide. SCD is characterized by a single nucleotide mutation in the β-globin gene, HBB:c.20A>T (p.Glu6Val), leading to the production of sickle hemoglobin (HbS). HbS polymerization upon deoxygenation results in poorly deformable sickled red blood cells (RBCs) with a shortened lifespan. These RBCs cause extremely painful vaso-occlusive crises (VOCs), chronic hemolytic anemia, and progressive end-organ damage. New therapeutic options have evolved very slowly despite the medical need for safe and effective therapies that both reduce VOCs and improve anemia. None of the US Food and Drug Administration (FDA) approved therapies for SCD are universally effective nor globally available, and they are limited by their drug costs and safety profiles. Mitapivat (AG-348), an investigational, first-in-class, oral, small-molecule allosteric activator of pyruvate kinase (PK), holds promise as an antisickling agent in addition to its use in PK deficiency (US FDA approved Pyrukynd USPI, 2022; NCT02476916, NCT03548220, NCT03559699, NCT03853798), and thalassemia (NCT03692052, NCT04770779, NCT04770753). PK is a key enzyme in RBC metabolism, generating adenosine triphosphate (ATP) to maintain energy homeostasis, thereby membrane integrity and deformability, and modulating 2,3-DPG levels (Figure S1). In SCD, an important metabolic feature directly associated with HbS polymerization is an increased intracellular 2,3-DPG level which promotes deoxygenation of hemoglobin (Hb) by lowering its oxygen affinity. Ex vivo treatment of RBCs of SCD patients with mitapivat restored PK activity and thermostability, increased ATP/2,3-DPG ratio, and markers of RBC sickling. Based on this and phase 1 study results (NCT04000165), we hypothesized that increased glycolytic flux through treatment with mitapivat decreases 2,3-DPG levels and increases ATP levels, thereby inhibiting HbS polymerization, RBC sickling, and ameliorating hemolytic anemia in patients with SCD. The aim of this ongoing phase 2, investigator-initiated, open-label, monocenter study (the ESTIMATE study) was to evaluate the safety and efficacy and provide proof of concept for mitapivat treatment in patients with SCD. Patients were enrolled at the Center for Benign Hematology, Thrombosis and Hemostasis Van Creveldkliniek, University Medical Center Utrecht, Utrecht, The Netherlands. Key inclusion criteria included patients ≥16 years with a diagnosis of SCD (homozygous HbS [HbSS], HbS/β or HbS/β thalassemia), prior VOCs (1–10 in the 12 months prior to study treatment) or SCD related complications (e.g., osteonecrosis, nephropathy, retinopathy, leg ulcer, acute chest syndrome, acute hemolytic crisis), and a Hb level >6.1 and ≤11.1 g/dL during screening. VOCs were defined as acute pain episodes that resulted in a healthcare provider contact and treatment with oral or parenteral narcotics, or parenteral non-steroidal anti-inflammatory drugs, acute chest syndrome, priapism, hepatic or splenic sequestration. Patients receiving hydroxyurea had to be on a stable dose for at least 3 months prior to study treatment. Major exclusion criteria were regular RBC transfusions and significant comorbidities conferring an unacceptable risk for participating. The study comprised different phases: a screening phase, an 8-week dosefinding period, a 52-week fixed-dose extension period, a 1-to-2-week taper phase to prevent hemolysis after drug interruption or withdrawal, and a safety follow-up visit 4 weeks after the last dose (Figure S2). In the here reported 8-week dose-finding period, the initial dosing of mitapivat was 20 mg twice daily. Patients received a maximum of two sequential dose escalations (i.e., from 20 to 50 mg and 100 mg twice daily) after study visit days 14 and 28 unless dosage limiting side effects or an excessive Hb response exceeding 12.9 g/dL occurred. This was based on phase 1 studies and (ongoing) phase 2–3 studies in patients with PK deficiency and thalassemia. In total nine patients were enrolled at the final analysis of the dose-finding period in December 2021 (Figure S3). Baseline characteristics were: median age of 22 years (range 16–59 years), six patients (6/9, 67%) were female and six (6/9, 67%) were on stable-dose hydroxyurea (Table S1). Seven patients (7/9, 78%) had homozygous HbSS, one patient (1/9, 11%) had HbS/β thalassemia and one patient (1/9, 11%) had HbS/β thalassemia. The median VOC episodes experienced was three (range 0–7) with a median of ten days (range 0–42 days) of hospitalization for SCD-related complications in the 24 months prior to screening. Eight patients (8/9, 89%) completed the dose-finding period, with a median compliance rate of 98% (range 94%–100%) based on diaries and pill counts. The safety analysis, evaluated by frequency and severity of adverse events according to Common Terminology Criteria for Adverse Events version 5.0 (National Cancer Institute, Bethesda, MD), included all enrolled patients. The majority of treatment-emergent adverse events (TEAEs) in the dose-finding period were grade 1 and transient (Table S2). The most common reported TEAEs included headache (n = 4, 44%), transaminase increase (alanine aminotransferase increased in n = 4 [44%]; aspartate aminotransferase increased in n = 2 [22%]) and dyspepsia (n = 2, 22%). No treatment-related TEAEs grade 3 or higher were reported. The non-treatment-related TEAE grade 4 was a serious adverse event of a urinary tract infection with Received: 10 March 2022 Revised: 25 March 2022 Accepted: 28 March 2022

Safety and efficacy of mitapivat, an oral pyruvate kinase activator, in sickle cell disease: A phase 2, open‐label study

Decreased activity and stability of pyruvate kinase in sickle cell disease: a novel target for mitapivat therapy

A double surrogate approach for the quantitation of 2-Hydroxyglutarate - An oncometabolite in human brain tumors via LC-MS/MS.

Aim: 2-Hydroxyglutarate (2-HG) is a target engagement biomarker in patients after treatment with inhibitors of mutated isocitrate dehydrogenase (mIDH). Accurate measurement of 2-HG is critical for monitoring the inhibition effectiveness of the inhibitors. Materials & methods: Human plasma samples were spiked with stable isotope labelled internal standard, processed by protein precipitation, and analyzed using LC-MS/MS. This method was validated following regulatory guidance and has been successfully applied in a clinical study for mIDH inhibition. Results: An LC-MS/MS method with a surrogate analyte approach was developed and validated to measure 2-HG in human plasma with acceptable intra- and inter-assay accuracy and precision. Conclusion: A sensitive and robust LC-MS/MS method was developed and validated for measuring 2-HG in human plasma.

Quantitation of 2-hydroxyglutarate in human plasma via LC–MS/MS using a surrogate analyte approach

Phase 1 Multiple Ascending Dose Study of Safety, Tolerability, and Pharmacokinetics/Pharmacodynamics of Mitapivat (AG-348) in Subjects with Sickle Cell Disease

Aim: Ivosidenib is a potent and selective small molecule inhibitor of mutant isocitrate dehydrogenase 1. Accurate measurement of ivosidenib is the key to ivosidenib pharmacokinetics in clinical trials. Materials & methods: Quantitation of ivosidenib was conducted by using a stable isotope labeled compound (ivosidenib-d4) as the internal standard. Results: This assay was validated and successfully applied to support multiple clinical trials. Selected clinical samples were also tested by a chiral LC-MS/MS method against four ivosidenib isomer standards to exclude the possibility of in vivo racemization of ivosidenib. Conclusion: A robust LC-MS/MS method was validated for ivosidenib in human plasma. This is the first time for ivosidenib bioanalytical method in any human matrix to be reported.

Heidi Mangus

Papers

Decreased activity and stability of pyruvate kinase in sickle cell disease: a novel target for mitapivat therapy

A double surrogate approach for the quantitation of 2-Hydroxyglutarate - An oncometabolite in human brain tumors via LC-MS/MS.

Quantitation of 2-hydroxyglutarate in human plasma via LC–MS/MS using a surrogate analyte approach

Phase 1 Multiple Ascending Dose Study of Safety, Tolerability, and Pharmacokinetics/Pharmacodynamics of Mitapivat (AG-348) in Subjects with Sickle Cell Disease

Quantitation of ivosidenib in human plasma via LC-MS/MS and its application in clinical trials.