Metabolism and Excretion of Therapeutic Peptides: Current Industry Practices, Perspectives, and Recommendations.

Therapeutic peptides (TPeps) have expanded from the initial endogenous peptides to complex modified peptides through medicinal chemistry efforts for almost a century. Different from small molecules and large proteins, the diverse submodalities of TPeps have distinct structures and carry different absorption, distribution, metabolism, and excretion (ADME) properties. There is no distinct regulatory guidance for the industry on conducting ADME studies (what, how, and when) for TPeps. Therefore, the Peptide ADME Working Group sponsored by the Translational and ADME Sciences Leadership Group of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) was formed with the goal to develop a white paper focusing on metabolism and excretion studies to support discovery and development of TPeps. In this paper, the key learnings from an IQ industry survey and U.S. Food and Drug Administration/European Medicines Agency submission documents of TPeps approved between 2011 and 2022 are outlined in detail. In addition, a comprehensive assessment of in vitro and in vivo metabolism and excretion studies, mitigation strategies for TPep metabolism, analytical tools to conduct studies, regulatory status, and Metabolites in Safety Testing considerations are provided. Finally, an industry recommendation on conducting metabolism and excretion studies is proposed for regulatory filing of TPeps. SIGNIFICANCE STATEMENT: This white paper presents current industry practices for metabolism and excretion studies of therapeutic peptides based on an industry survey, regulatory submission documents, and expert opinions from the participants in the Peptide Absorption, Distribution, Metabolism, and Excretion Working Group of the International Consortium for Innovation and Quality in Pharmaceutical Development. The group also provides recommendations on the Metabolites in Safety Testing considerations and metabolism and excretion studies for regulatory filing of therapeutic peptides.

Optimized two-dimensional gel electrophoresis in an alkaline pH range improves the identification of intracellular CFDA-cisplatin-protein adducts in ovarian cancer cells.

Intracellular binding of cisplatin to proteins has been associated with acquired resistance to chemotherapy. In our previous study we established an analytical method for the identification of intracellular cisplatin-binding proteins. The method used a fluorescent carboxyfluorescein-diacetate-labeled cisplatin analogue (CFDA-cisplatin), two-dimensional gel electrophoresis (2DE) and mass spectrometry, which allows detecting and identifying intracellular CFDA-cisplatin-containing protein adducts in the acidic pH range (pH 4-7). Based on this analytical method we extended the identification of intracellular cisplatin-protein adducts to the alkaline pH range (pH 6-10) giving chance to discover new important binding partners. 2DE analysis of alkaline proteins is challenging due to the difficult separation of basic proteins during the isoelectric focusing (IEF). The establishment of an optimized IEF protocol for basic proteins enabled us to identify several intracellular CFDA-cisplatin-binding proteins including enzymes of the glucose and serine metabolism like alpha enolase and D-3-phosphoglycerate 1-dehydrogenase.

A fluorescent oxaliplatin derivative for investigation of oxaliplatin resistance using imaging techniques.

Oxaliplatin is the backbone of chemotherapy for advanced colorectal cancer and undergoes clinical trials for treatment of other tumour entities. However, acquired resistance is a major hurdle. Confocal microscopy is a useful tool to get an insight into the mechanisms of resistance but it requires fluorescent compounds. This work describes the synthesis of the novel oxaliplatin derivative (CFDA-oxPt) featuring 5(6)-carboxyfluorescein diacetate and evaluation of its applicability for the investigation of oxaliplatin resistance using imaging techniques. CFDA-oxPt was somewhat less cytotoxic than oxaliplatin in sensitive colorectal cancer cells, with EC50 values of 26 and 5.8 µM, respectively. Nevertheless, the potency of the novel complex was significantly decreased to the EC50 of 711.2 µM in oxaliplatin-resistant cells, as was the case for oxaliplatin (EC50 = 81 µM). After incubation, both nuclear and cytosolic localisation was observed. Over time CFDA-oxPt concentrated near the cell membrane and in the vesicular structures, in contrast to the platinum-free label, which was rapidly excreted. These findings suggest that CFDA-oxPt can be used to study oxaliplatin resistance and open the route to new fluorophore-tethered oxaliplatin derivatives.

HIPEC ROC I: a phase I study of cisplatin administered as hyperthermic intraoperative intraperitoneal chemoperfusion followed by postoperative intravenous platinum-based chemotherapy in patients with platinum-sensitive recurrent epithelial ovarian cancer.

This phase I study tested the safety, feasibility, pharmacokinetics and pharmacodynamics of cisplatin administered as hyperthermic intraoperative intraperitoneal chemoperfusion (HIPEC) in patients with platinum-sensitive recurrent epithelial ovarian cancer (EOC) undergoing secondary cytoreductive surgery followed by postoperative platinum-based intravenous chemotherapy. Twelve patients with operable, recurrent platinum-sensitive EOC (recurrence ≥6 months after first-line therapy) were included according to the classical 3+3 dose-escalation design at three dose levels-60, 80 and 100 mg/m(2). After surgical cytoreduction, a single dose of cisplatin was administered via HIPEC for 90 min at 41-43°C. Postoperatively, all patients were treated with standard intravenous platinum-based combination chemotherapy. One of six patients experienced a dose-limiting toxicity (grade 3 renal toxicity) at a dose of 100 mg/m(2). The remaining five patients treated with 100 mg/m(2) tolerated their treatment well. The recommended phase II dose was established at 100 mg/m(2). The mean peritoneal-to-plasma AUC ratio was 19·5 at the highest dose level. Cisplatin-induced DNA adducts were confirmed in tumor samples. Common postoperative grade 1-3 toxicities included fatigue, postoperative pain, nausea, and surgical site infection. The ability to administer standard intravenous platinum-based chemotherapy after HIPEC was uncompromised. Cisplatin administered as HIPEC at a dose of 100 mg/m(2) has an acceptable safety profile in selected patients undergoing secondary cytoreductive surgery for platinum-sensitive recurrent EOC. Favorable pharmacokinetic and pharmacodynamic properties of HIPEC with cisplatin were confirmed at all dose levels, especially at 100 mg/m(2). The results are encouraging to determine the efficacy of HIPEC as a complementary treatment in patients with EOC.

Combination of two-dimensional gel electrophoresis and a fluorescent carboxyfluorescein-diacetate-labeled cisplatin analogue allows the identification of intracellular cisplatin-protein adducts.

Cisplatin is one of the most widely used anticancer agents, but a major problem for successful chemotherapy is the development of drug resistance of tumor cells against cisplatin. Resistance to cisplatin is a multifactorial problem. A method to detect and identify intracellular cisplatin-protein adducts was developed using a fluorescent carboxyfluorescein-diacetate-labeled cisplatin analogue (CFDA-cisplatin), 2DE, and ESI-MS/MS. We identified several CFDA-cisplatin-protein adducts including members of the protein disulfide isomerase family (PDI). These are the first results of the detection of intracellular CFDA-cisplatin-protein adducts, which may help to understand the resistance mechanism of cisplatin.

Optimized sample preparation strategy for the analysis of low molecular mass adducts of a fluorescent cisplatin analogue in cancer cell lines by CE-dual-LIF.

Pt-based anticancer drugs, such as cisplatin, are known to undergo several (bio-)chemical transformation steps after administration. Hydrolysis and adduct formation with small nucleophiles and larger proteins are their most relevant reactions on the way to the final reaction site (DNA), but there are still many open questions regarding the identity and pharmacological relevance of various proposed adducts and intermediates. Furthermore, the role of buffer components or additives, which are inevitably added to samples during any type of analytical measurement, has been frequently neglected in previous studies. Here, we report on adduct formation reactions of the fluorescent cisplatin analogue carboxyfluorescein diacetate platinum (CFDA-Pt) in commonly used buffers and cell culture medium. Our results indicate that chelation reactions with noninnocent buffers (e.g., Tris) and components of the cell culture/cell lysis medium must be taken into account when interpreting results. Adduct formation kinetics was followed up to 60 h at nanomolar concentrations of CFDA-Pt by using CE-LIF. CE-MS enabled the online identification of such unexpected adducts down to the nanomolar concentration range. By using an optimized sample preparation strategy, unwanted adducts can be avoided and several fluorescent adducts of CFDA-Pt are detectable in sensitive and cisplatin-resistant cancer cell lines. By processing samples rapidly after incubation, we could even identify the initial, but transient, Pt species in the cells as deacetylated CFDA-Pt with unaltered complexing environment at Pt. Overall, the proposed procedure enables a very sensitive and accurate analysis of low molecular mass Pt species in cancer cells, involving a fast CE-LIF detection within 5 min.

Preclinical efficacy and pharmacokinetics of an RNA-encoded T cell-engaging bispecific antibody targeting human claudin 6.

We present the preclinical pharmacology of BNT142, a lipid nanoparticle (LNP)-formulated RNA (RNA-LNP) encoding a T cell-engaging bispecific antibody that monovalently binds the T cell marker CD3 and bivalently binds claudin 6 (CLDN6), an oncofetal antigen that is absent from normal adult tissue but expressed on various solid tumors. Upon BNT142 RNA-LNP delivery in cell culture, mice, and cynomolgus monkeys, RNA is translated, followed by self-assembly into and secretion of the functional bispecific antibody RiboMab02.1. In vitro, RiboMab02.1 mediated CLDN6 target cell-specific activation and proliferation of T cells, and potent target cell killing. In mice and cynomolgus monkeys, intravenously administered BNT142 RNA-LNP maintained therapeutic serum concentrations of the encoded antibody. Concentrations of RNA-encoded RiboMab02.1 were maintained longer in circulation in mice than concentrations of directly injected, sequence-identical protein. Weekly injections of mice with BNT142 RNA-LNP in the 0.1- to 1-µg dose range were sufficient to eliminate CLDN6-positive subcutaneous human xenograft tumors and increase survival over controls. Tumor regression was associated with an influx of T cells and depletion of CLDN6-positive cells. BNT142 induced only transient and low cytokine production in CLDN6-positive tumor-bearing mice humanized with peripheral blood mononuclear cells (PBMCs). No signs of adverse effects from BNT142 RNA-LNP administration were observed in mice or cynomolgus monkeys. On the basis of these and other findings, a phase 1/2 first-in-human clinical trial has been initiated to assess the safety and preliminary efficacy of BNT142 RNA-LNP in patients with CLDN6-positive advanced solid tumors (NCT05262530).

Structure-Permeability Relationship of Semipeptidic Macrocycles-Understanding and Optimizing Passive Permeability and Efflux Ratio.

We herein report the first thorough analysis of the structure-permeability relationship of semipeptidic macrocycles. In total, 47 macrocycles were synthesized using a hybrid solid-phase/solution strategy, and then their passive and cellular permeability was assessed using the parallel artificial membrane permeability assay (PAMPA) and Caco-2 assay, respectively. The results indicate that semipeptidic macrocycles generally possess high passive permeability based on the PAMPA, yet their cellular permeability is governed by efflux, as reported in the Caco-2 assay. Structural variations led to tractable structure-permeability and structure-efflux relationships, wherein the linker length, stereoinversion, N-methylation, and peptoids site-specifically impact the permeability and efflux. Extensive nuclear magnetic resonance, molecular dynamics, and ensemble-based three-dimensional polar surface area (3D-PSA) studies showed that ensemble-based 3D-PSA is a good predictor of passive permeability.

Transcutaneous glomerular filtration rate measurement in a canine animal model of chronic kidney disease.

INTRODUCTION: Quantitative assessment of renal function by measurement of glomerular filtration rate (GFR) is an important part of safety and efficacy evaluation in preclinical drug development. Existing methods are often time consuming, imprecise and associated with animal burden. Here we describe the comparison between GFR determinations with sinistrin (PS-GFR) and fluorescence-labelled sinistrin-application and its transcutaneous detection (TD-GFR) in a large animal model of chronic kidney disease (CKD). METHODS: TD-GFR measurements compared to a standard method using i.v. sinistrin were performed in a canine model. Animals were treated with one-sided renal wrapping (RW) followed by renal artery occlusion (RO). Biomarker and remote hemodynamic measurements were performed. Plasma sinistrin in comparison to transcutaneous derived GFR data were determined during healthy conditions, after RW and RW+RO. RESULTS: RW alone did not led to any significant changes in renal function, neither with PS-GFR nor TD-GFR. Additional RO showed a rise in blood pressure (+68.0mmHg), plasma urea (+28.8mmol/l), creatinine (+224,4µmol/l) and symmetric dimethylarginine (SDMA™; +12.6µg/dl). Plasma sinistrin derived data confirmed the expected drop (-44.7%, p<0.0001) in GFR. The calculated transcutaneous determined Fluorescein Isothiocyanate (FITC)-sinistrin GFR showed no differences to plasma sinistrin GFR at all times. Both methods were equaly sensitive to diagnose renal dysfunction in the affected animals. DISCUSSION: Renal function assessment using TD-GFR is a valid method to improve preclinical drug discovery and development. Furthermore, TD-GFR method offers advantages in terms of reduced need for blood sampling and thus decreasing animal burden compared to standard procedures.

Assessing the contribution of the two protein disulfide isomerases PDIA1 and PDIA3 to cisplatin resistance.

Intracellular binding of cisplatin to non-DNA partners, such as proteins, has received increasing attention as an additional mode of action and as mechanism of resistance. We investigated two cisplatin-interacting isoforms of protein disulfide isomerase regarding their contribution to acquired cisplatin resistance using sensitive and resistant A2780/A2780cis ovarian cancer cells. Cisplatin cytotoxicity was assessed after knockdown of either protein disulfide isomerase family A member 1 (PDIA1) or protein disulfide isomerase family A member 3 (PDIA3). Whereas PDIA1 knockdown led to increased cytotoxicity in resistant A2780cis cells, PDIA3 knockdown showed no influence on cytotoxicity. Coincubation with propynoic acid carbamoyl methyl amide 31 (PACMA31), a PDIA1 inhibitor, resensitized A2780cis cells to cisplatin treatment. Determination of the combination index revealed that the combination of cisplatin and PACMA31 acts synergistically. Our results warrant further evaluation of PDIA1 as promising target for chemotherapy, and its inhibition by PACMA31 as a new therapeutic approach.