Site-specific and hydrophilic ADCs through disulfide-bridged linker and branched PEG

Kadcyla® (T-DM1), an antibody–drug conjugates (ADCs) for HER2+ breast cancer treatment, has been approved by the Food and Drug Administration (FDA) in 2013. An ADC of random lysine conjugation, it has difficulties in DAR control and unsatisfactory PK due to uneven DAR distribution. It also gives rise to aggregation during conjugation because of the hydrophobicity nature of the cytotoxin, DM1. The linker-drug in T-DM1, SMCC-DM1 is hydrophobic and requires certain percentage of organic solvent such as DMA in the conjugation solution, limiting the manufacturing process in an organic-solvent-compatible device and adding extra costs. To address these problems, a site-specific conjugation method was developed involving full reduction of antibody and full conjugation with the bridge-like conjugator-drug, based on the work of Caddick and co-workers, to obtain a site-directed antibody-drug conjugate with DAR 4. The bridge-like conjugator was assembled with SMCC-DM1 and different lengths of hydrophilic polyethylene glycol (PEG) moiety. By applying PEG moiety in the side chain of the linker-drug, the organic solvent used in the conjugation can be reduced. When the PEG length is about 26 units, organic solvent is no longer needed in the conjugation. Reducing the amount of organic solvent in conjugation could also diminish the aggregation occurrence during the conjugation. Moreover, the conjugation configuration with the designed conjugator was also discussed in the article. The binding affinity of the resulting ADCs did not show significant decrease and the cell based assay and animal study have shown the comparable results with T-DM1.     

Improved synthesis of a protected 11-oxoestrone

An improved synthesis of 11-oxoestrone-3-acetate-17-ethyleneketal is reported. Adjustments are proposed for the oxidation of estrone by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone into 9(11)-dehydroestrone. A complete hydroboration-oxidation of the resulting ketal, by means of borane-methylsulfide complex, gives the corresponding 11-hydroxy derivative. This latter compound is then acetylated for successful oxidation with pyridinium chlorochromate on alumina. The overall yield is 30%.     

Auxin metabolism

Auxin metabolism encompasses transport, conjugation, deconjugation, conversion, and catabolism. The balance between auxin metabolism and biosynthesis determines the actual level of the hormone in a given cell and consequently plays an important role in many developmental processes from seed germination to fruit ripening. Mass spectrometry used in conjunction with stable isotope labeling studies has enabled comprehensive examination of auxin biosynthesis and turnover along with the identification of many auxin conjugate. It appears that the conjugate moiety may signal the metabolic fate (e.g. storage and eventual hydrolysis to free hormone, or catabolism). Recently identified auxin-metabolizing enzymes are encoded by gene families which vary in specificity for auxin metabolites. The expression patterns of these genes will reveal a great deal about the mechanics of auxin metabolism.     

Is the Drug-Responsive NADH Oxidase of the Cancer Cell Plasma Membrane a Molecular Target for Adriamycin?

Enhanced growth inhibition and antitumor responses to adriamycin have been observed repeatedly from several laboratories using impermeant forms of adriamycin where entry into the cell was greatly reduced or prevented. Our laboratory has described an NADH oxidase activity at the external surface of plasma membrane vesicles from tumor cells where inhibition by an antitumor sulfonylurea, N-(4-methylphenylsulfonyl)-N-(4-chlorophenyl)urea (LY181984), and by the vanilloid, capsaicin (8-methyl-N-vanillyl-6-noneamide) correlated with inhibition of growth. Here we report that the oxidation of NADH by isolated plasma membrane vesicles was inhibited, as well, by adriamycin. An external site of inhibition was indicated from studies where impermeant adriamycin conjugates were used. The EC₅₀ for inhibition of the oxidase of rat hepatoma plasma membranes by adriamycin was several orders of magnitude less than that for rat liver. Adriamycin cross-linked to diferric transferrin and other impermeant supports also was effective in inhibition of NADH oxidation by isolated plasma membrane vesicles and in inhibition of growth of cultured cells. The findings suggest the NADH oxidase of the plasma membrane as a growth-related adriamycin target at the surface of cancer cells responsive to adriamycin. Whereas DNA intercalation remains clearly one of the principal bases for the cytotoxic action of free adriamycin, this second site, possibly related to a more specific antitumor action, may be helpful in understanding the enhanced efficacy reported previously for immobilized adriamycin forms compared to free adriamycin.     

On the Synthesis of Neurotransmitter Receptor Agonists with Antagonist Potential

The synthesis of a new type of antagonist is described, capable of inactivating neuroreceptors with heretofore unattainable selectivity and permanence. These antagonists are referred to as mazek agonists (i.e. direct, inhibitory agonists) as they have the high receptor affinity and initial receptor-stimulatory effect of direct agonists and are positively coupled to effector systems. However, like direct antagonists, they have a high receptor affinity and the potential to inhibit or prevent receptor stimulation. The synthesis of the present compounds consisted of the covalent attachment of a tethered dye to three different neurotransmitter analogues, resulting in dye-neuropeptide conjugates with a high affinity for the FMRFa receptor. The dye was prepared from azure B (Az), the neurotransmitter was the neuropeptide FMRFamide (FMRFa), and the dye-neuropeptide conjugates synthesized were Az-CFMRFa; Az-CFMRF and Az-CLRFa. In this procedure, the analogues serve as carrier molecules, bound at one end to the receptor and at the other end to the dye, which is thereby brought into close contact with the receptor. The receptor can then be inactivated by singlet oxygen generated by laser irradiation of the photosensitized receptor.     

On the synthesis of cyclodextrin–peptide conjugates by the Huisgen reaction

A,D‐substituted cyclodextrin (CDX) substituted on their primary rim side are ideal scaffolds for the macromolecular assembly and formation of templated structures. Their substitution can be achieved through various reactions. However, the use of the well‐known Huisgen reaction in this context is under‐reported. We present here results of the synthesis of model conjugates formed between CDX and representative peptides by click chemistry. Notably, bis‐conjugation of peptides onto a unique scaffold promotes α‐helix formation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Gas chromatography profile of estrogens: Application to pregnancy urine

A method for the simultaneous quantitation of 7 estrogens in pregnancy urine is described. It involves enzymatic hydrolysis, extraction of free steroids, ion-exchange column chromatography and quantitation of the trimethylsilyl derivatives by gas chromatography on OV 1. Data obtained from normal and twin pregnancies and from women with anencephalic fetus or intra uterine fetal death are analysed. The sensibility of the method is about 40 μg of each estrogen by liter of urine.     

Estriol concentrations in plasma of normal, non-pregnant women.

Using a rabbit antisera directed against estriol-3-0-carboxy methyl ether complexed to BSA, an immunoassay for estriol (1) was developed. The mean plus or minus SE concentration of estriol in 18 women in days 5-7 of their cycle was 7.9 plus or minus 0.6 pg/ml which was significantly (P less than 0.01) less than the mean value of 11.1 plus or minus 0.8 pg/ml in 15 women in days 20-22 of the cycle. In 3 of 6 women in whom plasma samples were drawn frequently during their cycle, an estriol peak occurred coincident with the estradiol peak. In 3 women from whom plasma was obtained several times during the course of a day estriol levels did not appear to vary significantly. In 8 women who were on oral contraceptives the mean level of estriol was 7.6 plus or minus 1.5 pg/ml. In 8 post-menopausal women the mean level was 6.0 plus or minus 1.2 pg/ml which is significantly (P less than 0.01) less than the mean luteal phase value but not less (P greater than 0.1) than the follicular phase or oral contraceptive user values. We conclude that some of the circulating estriol is directly secreted by the ovary of normal women.     

Metabolism of [3H]gibberellin A4 in somatic suspension cultures of anise

The native gibberellin A₄ (GA₄) was fed as [1, 2-³H]GA₄ (1.3 Ci/mmol) to anise somatic cultures maintained either at a proembryo-like stage with 2,4-dichlorophenoxyacetic acid (2,4-D), or allowed to undergo embryogenic development on a - 2,4-D medium. Proembryos, although only 20% of the dry wt of embryos, absorbed 1.4-times more [³H]GA₄/g dry wt than embryos. The [³H]GA₄ was metabolized to GA₁ and GA₈, and at least six conjugates [GA₄-glucoside (GA₄-G), GA₄ glucosyl ester (GA₄-GE), GA₁-0(3)-G, GA₁-0(13)-G, GA₁-GE and a GA₈-glucosyl conjugate]. The major metabolite was GA₄-G at each of two, 204 and 348 hr harvests (56–71 %), with GA₈-G increasing from < 1 % to 13 % with harvest time. The percentage and amount of GA₄-GE was highest at 204 hr (2% and 8 %, for embryos and proembryos, respectively), dropping to < 1 % at 348 hr, thereby indicating hydrolysis (e.g. reversible conjugation). Embryos had reduced amounts and percentages of biologically active GA₄ and GA₁, and most of their conjugates, but increased amounts and percentages of GA₈ and its conjugate(s). This finding is consistent with the hypothesis (based on present and past work) that high levels of biologically active GAs, especially GA₁, inhibit somatic embryogenesis in anise and carrot. The auxin, 2,4-D, may thus derive, at least in part, its ability to maintain the proembryo-like stage by inhibiting oxidative metabolism and conjugation of biologically active GAs.     

An enzyme from lupin seeds forming alanine derivatives of cytokinins

A new enzyme, which catalyses the conversion of the cytokinin zeatin to the alanine conjugate lupinic acid, has been partly purified from developing lupin seed (Lupinus luteus). Paired-ion, reverse phase HPLC was adapted to analyse the enzyme reaction quantitatively. The enzyme used O-acetyl-l-serine as the source of the amino acid residue, and it interacted with substrates in a ping pong bi bi mechanism. A number of adenine derivatives served as substrates, but preference was shown for compounds with high cytokinin activity. The possible role of the enzyme, tentatively called β-(9-cytokinin)alanine synthase or lupinic acid synthase, in the regulation of hormone activity is discussed.