Considerations for the nonclinical safety evaluation of antibody drug conjugates for oncology.

Antibody drug conjugates (ADCs) include monoclonal antibodies that are linked to cytotoxic small molecules. A number of these agents are currently being developed as anti-cancer agents designed to improve the therapeutic index of the cytotoxin (i.e., cytotoxic small molecule or cytotoxic agent) by specifically delivering it to tumor cells. This paper presents primary considerations for the nonclinical safety evaluation of ADCs and includes strategies for the evaluation of the entire ADC or the various individual components (i.e., antibody, linker or the cytotoxin). Considerations are presented on how to design a nonclinical safety assessment program to identify the on- and off-target toxicities to enable first-in-human (FIH) studies. Specific discussions are also included that provide details as to the need and how to conduct the studies for evaluating ADCs in genetic toxicology, tissue cross-reactivity, safety pharmacology, carcinogenicity, developmental and reproductive toxicology, biotransformation, toxicokinetic monitoring, bioanalytical assays, immunogenicity testing, test article stability and the selection of the FIH dose. Given the complexity of these molecules and our evolving understanding of their properties, there is no single all-encompassing nonclinical strategy. Instead, each ADC should be evaluated on a case-by-case scientifically-based approach that is consistent with ICH and animal research guidelines.

Strategy for genotoxicity testing--metabolic considerations.

The report from the 2002 International Workshop on Genotoxicity Tests (IWGT) Strategy Expert Group emphasized metabolic considerations as an important area to address in developing a common strategy for genotoxicity testing. A working group convened at the 2005 4th IWGT to discuss this area further and propose practical strategy recommendations. To propose a strategy, the working group reviewed: (1) the current status and deficiencies, including examples of carcinogens "missed" in genotoxicity testing, established shortcomings of the standard in vitro induced S9 activation system and drug metabolite case examples; (2) the current status of possible remedies, including alternative S9 sources, other external metabolism systems or genetically engineered test systems; (3) any existing positions or guidance. The working group established consensus principles to guide strategy development. Thus, a human metabolite of interest should be represented in genotoxicity and carcinogenicity testing, including evaluation of alternative genotoxicity in vitro metabolic activation or test systems, and the selection of a carcinogenicity test species showing appropriate biotransformation. Appropriate action triggers need to be defined based on the extent of human exposure, considering any structural knowledge of the metabolite, and when genotoxicity is observed upon in vitro testing in the presence of metabolic activation. These triggers also need to be considered in defining the timing of human pharmaceutical ADME assessments. The working group proposed two strategies to consider; a more proactive approach, which emphasizes early metabolism predictions to drive appropriate hazard assessment; and a retroactive approach to manage safety risks of a unique or "major" metabolite once identified and quantitated from human clinical ADME studies. In both strategies, the assessment of the genotoxic potential of a metabolite could include the use of an alternative or optimized in vitro metabolic activation system, or direct testing of an isolated or synthesized metabolite. The working group also identified specific areas where more data or experiences need to be gained to reach consensus. These included defining a discrete exposure action trigger for safety assessment and when direct testing of a metabolite of interest is warranted versus the use of an alternative in vitro activation system, a universal recommendation for the timing of human ADME studies for drug candidates and the positioning of metabolite structural knowledge (through in silico systems, literature, expert analysis) in supporting metabolite safety qualification. Lastly, the working group outlined future considerations for refining the initially proposed strategies. These included the need for further evaluation of the current in vitro genotoxicity testing protocols that can potentially perturb or reduce the level of metabolic activity (potential alterations in metabolism associated with both the use of some solvents to solubilize test chemicals and testing to the guidance limit dose), and proposing broader evaluations of alternative metabolic activation sources or engineered test systems to further challenge the suitability of (or replace) the current induced liver S9 activation source.

Induction of apoptosis in estrogen receptor-negative breast cancer cells by natural and synthetic cyclopentenones: role of the IkappaB kinase/nuclear factor-kappaB pathway.

Nuclear factor-kappaB (NF-kappaB), a transcription factor with a critical role in promoting inflammation and cell survival, is constitutively activated in estrogen-receptor (ER)-negative breast cancer and is considered a potential therapeutic target for this type of neoplasia. We have previously demonstrated that cyclopentenone prostaglandins are potent inhibitors of NF-kappaB activation by inflammatory cytokines, mitogens, and viral infection, via direct binding and modification of the beta subunit of the IkappaB kinase complex (IKK). Herein, we describe the NF-kappaB-dependent anticancer activity of natural and synthetic cyclopentenone IKK inhibitors. We demonstrate that the natural cyclopentenone 15-deoxy-Delta(12,14)prostaglandin J(2) (15d-PGJ(2)) is a potent inhibitor of constitutive IkappaB-kinase and NF-kappaB activities in chemotherapy-resistant ER-negative breast cancer cells. 15d-PGJ(2)-induced inhibition of NF-kappaB function is rapidly followed by down-regulation of NF-kappaB-dependent antiapoptotic proteins cIAPs 1/2, Bcl-X(L), and cellular FLICE-inhibitory protein, leading to caspase activation and induction of apoptosis in breast cancer cells resistant to treatment with paclitaxel and doxorubicin. We then demonstrate that the cyclopentenone ring structure is responsible for these activities, and we identify a new synthetic cyclopentenone derivative, 3-tert-butyldimethylsilyloxy-5-(E)-iso-propylmethylenecyclopent-2-enone (CTC-35), as a potent NF-kappaB inhibitor with proapoptotic activity in ER-negative breast cancer cells. The results open new perspectives in the search for novel proapoptotic molecules effective in the treatment of cancers presenting aberrant NF-kappaB regulation.

A very simple, highly stereoselective and modular synthesis of ferrocene-based P-chiral phosphine ligands.

A very simple, highly stereoselective and modular synthesis of ferrocene-based P-chiral phosphine ligands has been developed. On the basis of this new methodology, several new families of ferrocene-based phosphine ligands have been prepared coupling chirality at phosphorus with other, more standard stereogenic features. The introduction of P-chirality into ferrocene-based phosphine ligands enhances the enantioselective discrimination produced by the corresponding Rh catalyst when a matching among the planar chirality, carbon chirality, and the chirality of phosphorus is achieved.

Oligopeptides as catalysts for asymmetric epoxidation.

Oligopeptides are versatile catalysts for the enantioselective epoxidation of electron deficient alkenes, (e.g. alpha,beta-unsaturated ketones) with alkaline hydrogen peroxide. This review describes optimisation of the catalyst, substrate range, synthetic applications of the products and rationalisation of the stereoselectivity by an active site model.

Mechanistic insights from reaction progress kinetic analysis of the polypeptide-catalyzed epoxidation of chalcone.

[reaction: see text] Reaction progress kinetic analysis of the poly(L)-leucine (PLL)-catalyzed epoxidation of substituted chalcones 1a-1c helps to refine an earlier mechanistic proposal by demonstrating that the reaction proceeds via reversible addition of chalcone to a PLL-bound hydroperoxide, forming a fleeting hydroperoxy enolate species. Observation of an induction period offers an alternate rationalization for effects formerly attributed to substrate inhibition. Previous clues about the origin of enantioselectivity in this system are supported by this work.

Polyamino acids as synthetic enzymes: mechanism, applications and relevance to prebiotic catalysis.

Polyamino acids, such as polyleucine, behave as synthetic enzymes in the asymmetric epoxidation of chalcone and other electron-deficient alkenes (the Julià-Colonna reaction). The influences of reaction conditions, of the molecular structure of the catalysts and of the scaling-up of the process on the enantioselectivity of the reaction have been determined. The kinetics and mechanism have been investigated using a soluble PEG-polyleucine conjugate, which behaves in a similar way to an enzyme, showing saturation kinetics for both chalcone and HOO-. Enantioselective catalysis is achieved with peptides with as few as five residues and scalemic catalysts show high chiral amplification. Here, we discuss the relevance of these-enzyme like catalysts to prebiotic processes, such as the role of small peptides in the formation of optically active cyanohydrins.

Lack of cataleptogenic potentiation with non-imidazole H3 receptor antagonists reveals potential drug-drug interactions between imidazole-based H3 receptor antagonists and antipsychotic drugs.

Since H3 receptor (H3R) antagonists/inverse agonists can improve cognitive function in animal models, they may have the potential to be used as add-on therapy in the treatment of schizophrenia, a disease with significant cognitive deficits. However, a recent study showed potentiation of haloperidol-induced catalepsy by ciproxifan, an imidazole-containing H3R antagonist/inverse agonist, suggesting there is a potential risk of exacerbating extrapyramidal symptoms (EPS) if H3R antagonists were used as adjunctive treatment [Pillot, C., Ortiz, J., Heron, A., Ridray, S., Schwartz, J.C. and Arrang, J.M., Ciproxifan, a histamine H3-receptor antagonist/inverse agonist, potentiates neurochemical and behavioral effects of haloperidol in the rat, J Neurosci, 22 (2002) 7272-80]. In order to clarify the basis of this finding, we replicated this result and extended the work with another imidazole and two non-imidazole H3R antagonists. The results indicate that ciproxifan significantly augmented the effects of haloperidol and risperidone on catalepsy. Another imidazole H3R antagonist, thioperamide, also potentiated the effect of risperidone on catalepsy. In contrast, no catalepsy-enhancing effects were observed when selective non-imidazole H3R antagonists, ABT-239 and A-431404, were coadministered with haloperidol and/or risperidone. As ciproxifan and thioperamide are inhibitors of cytochrome P450 enzymes, responsible for metabolizing risperidone and haloperidol, the possibility that the augmentation of antipsychotics by imidazoles resulted from drug-drug interactions was tested. A drug metabolism study revealed that an imidazole, but not a non-imidazole, potently inhibited the metabolism of haloperidol and risperidone. Furthermore, ketoconazole, an imidazole-based CYP 3A4 inhibitor, significantly augmented risperidone-induced catalepsy. Together, these data suggest the potentiation of antipsychotic-induced catalepsy may result from pharmacokinetic drug-drug interactions and support the potential utility of non-imidazole H3R antagonists in treatment of cognitive impairment in schizophrenia without increased risk of increased EPS in patients.

Metabolism and disposition of the HIV-1 protease inhibitor lopinavir (ABT-378) given in combination with ritonavir in rats, dogs, and humans.

PURPOSE: The objective of this study was to examine the metabolism and disposition of the HIV protease inhibitor lopinavir in humans and animal models. METHODS: The plasma protein binding of [14C]lopinavir was examined in vitro via equilibrium dialysis technique. The tissue distribution of radioactivity was examined in rats dosed with [14C]lopinavir in combination with ritonavir. The metabolism and disposition of [14C]lopinavir was examined in rats, dogs, and humans given alone (in rats only) or in combination with ritonavir. RESULTS: The plasma protein binding of lopinavir was high in all species (97.4-99.7% in human plasma), with a concentration-dependent decrease in binding. Radioactivity was extensively distributed into tissues, except brain, in rats. On oral dosing to rats, ritonavir was found to increase the exposure of lopinavir-derived radioactivity 13-fold. Radioactivity was primarily cleared via the hepato-biliary route in all species (>82% of radioactive dose excreted via fecal route), with urinary route of elimination being significant only in humans (10.4% of radioactive dose). Oxidative metabolites were the predominant components of excreted radioactivity. The predominant site of metabolism was found to be the carbon-4 of the cyclic urea moiety, with subsequent secondary metabolism occurring on the diphenyl core moiety. In all the three species examined, the primary component of plasma radioactivity was unchanged lopinavir (>88%) with small amounts of oxidative metabolites. CONCLUSIONS: Lopinavir was subject to extensive metabolism in vivo. Co-administered ritonavir markedly enhanced the pharmacokinetics of lopinavir-derived radioactivity in rats, probably due to inhibition of presystemic and systemic metabolism, leading to an increased exposure to this potent HIV protease inhibitor.

The isomerisation of (Z)-3-[2H1]-phenylprop-2-enone as a measure of the rate of hydroperoxide addition in Weitz-Scheffer and Julia-Colonna epoxidations.

(Z)-3-[2H1]-Phenylprop-2-enone is isomerised by hydroperoxide to an equimolar mixture of the (Z)- and (E)-isomers prior to epoxidation. Poly-(L)-leucine (10 mole %) accelerates the addition of hydroperoxide by an order of magnitude and sequesters hydroperoxide from THF.

The mechanism of polyleucine catalysed asymmetric epoxidation.

Catalysis in the Julia-Colonna epoxidation of alpha,beta-unsaturated ketones is due to binding of the hydroperoxide enolate intermediate by the three N-terminal amidic N-H groups of alpha-helical poly-leucine; the N-terminal pair forms an oxy-anion hole, whilst the third aids displacement of hydroxide.

Chiral amplification by polypeptides and its relevance to prebiotic catalysis.

Polyleucine prepared from scalemic Leu-NCA monomers, shows high chiral amplification in the Julia-Colonna epoxidation of chalcone.

Kinetics of chalcone oxidation by peroxide anion catalysed by poly-l-leucine.

An insight into the kinetics, mechanism and optimum reaction conditions of the Julia-Colonna epoxidation has been gained using a soluble polyleucine catalyst.

Reactions of some cyclopentenones with selected cysteine derivatives and biological activities of the product thioethers.

The conjugate addition reaction between glutathione, N-Boc-cysteine methyl ester, N-acetyl cysteine methyl ester and N-acetyl cysteine and several substituted cyclopentenones is described. The reversibility of this process was demonstrated by thio-adduct metathesis on treatment of the adduct with a different cysteinyl derivative. The levels at which these compounds inhibit the function of nuclear factor kappa B (NF-kappaB) and potentiate heat shock factor (HSF) are reported and the possible relevance of these studies concerning the antiviral and anti-inflammatory activities of the cyclopentenone prostanoids is discussed.

The conduct of in vitro and in vivo drug-drug interaction studies: a Pharmaceutical Research and Manufacturers of America (PhRMA) perspective.

Current regulatory guidances do not address specific study designs for in vitro and in vivo drug-drug interaction studies. There is a common desire by regulatory authorities and by industry sponsors to harmonize approaches, to allow for a better assessment of the significance of findings across different studies and drugs. There is also a growing consensus for the standardization of cytochrome P450 (P450) probe substrates, inhibitors and inducers and for the development of classification systems to improve the communication of risk to health care providers and to patients. While existing guidances cover mainly P450-mediated drug interactions, the importance of other mechanisms, such as transporters, has been recognized more recently, and should also be addressed. This article was prepared by the Pharmaceutical Research and Manufacturers of America (PhRMA) Drug Metabolism and Clinical Pharmacology Technical Working Groups and represents the current industry position. The intent is to define a minimal best practice for in vitro and in vivo pharmacokinetic drug-drug interaction studies targeted to development (not discovery support) and to define a data package that can be expected by regulatory agencies in compound registration dossiers.

The conduct of in vitro and in vivo drug-drug interaction studies: a PhRMA perspective.

Current regulatory guidances do not address specific study designs for in vitro and in vivo drug-drug interaction studies. There is a common desire by regulatory authorities and by industry sponsors to harmonize approaches to allow for a better assessment of the significance of findings across different studies and drugs. There is also a growing consensus for the standardization of cytochrome P450 (CYP) probe substrates, inhibitors, and inducers and for the development of classification systems to improve the communication of risk to health care providers and patients. While existing guidances cover mainly CYP-mediated drug interactions, the importance of other mechanisms, such as transporters, has been recognized more recently and should also be addressed. This paper was prepared by the Pharmaceutical Research and Manufacturers of America (PhRMA) Drug Metabolism and Clinical Pharmacology Technical Working Groups and represents the current industry position. The intent is to define a minimal best practice for in vitro and in vivo pharmacokinetic drug-drug interaction studies targeted to development (not discovery support) and to define a data package that can be expected by regulatory agencies in compound registration dossiers.