Theoretical analysis of conjugate localization in two-step cancer chemotherapy

 Considerable research has been aimed at improving the efficacy of chemotherapeutic agents for cancer therapy. A promising two-step approach that is designed to minimize systemic drug toxicity while maximizing activity in tumors employs monoclonal antibody-enzyme conjugates for the activation of anti-cancer prodrugs. A mathematical model based on the biology of human 3677 melanoma xenografts in nude mice is presented, analyzed, and numerically simulated to study the biodistribution, pharmacokinetics, and intratumoral localization properties of L49-β-lactamase fusion proteins in solid tumor masses. The model predictions were compared with published experimental data and an excellent correlation was found to exist. Analytic expressions for the total concentration of conjugate in the tumor, the time at which the concentration is maximal, and the half life of conjugate in the tissue were derived. From these results, key parameters were isolated; and the effects of the tumor vasculature, binding kinetics, and administration schedule were investigated. The antibody-antigen dissociation ratio, the conjugate permeability, and the inter-capillary half distance within the tumor mass were found to strongly influence localization and retention in the tumor. The model was used to examine various dosing strategies in an attempt to determine which regimen would provide the best biodistribution results. The results of administering a uniform dose of conjugate via bolus injection, multiple injections, and continuous infusion were compared. The model predicts that when saturation of binding sites does not occur, dosing strategy has little effect on the amount of conjugate that localizes in the tumor. Received: 20 April 1998 / Revised version: 12 October 1998     

Enzyme immunoassays for ovarian steroid metabolites in the urine of Macaca fascicularis

In vivo studies using carbon 14 labeled estradiol (E₂) and progesterone (Po) were performed to characterize the time course and metabolic fate of circulating E₂ and Po. Co-chromatography of human, orangutan, and macaque luteal phase urine samples demonstrated the presence of a steroid conjugate peak in all three species that was identified as being androsterone and etiocholanolone glucuronides. An enzyme immunoassay for urinary metabolites of Po was developed subsequently for Macaca spp. using a monoclonal antibody that cross-reacted with both C-19 and C-21 metabolites.     

Computer simulation of the cyclodextrin–phenylalanine complex

The results of the molecular dynamics simulations of the complexes of α-cyclodextrin-l-phenylalanine and β-cyclodextrine- L- phenylalanine in vacuo and in aqueous solution are presented. The trajectories of the insertion angle, rotation of the aromatic ring of the phenylalanine inside the macrocycle and the dihedral angle χ² (Cα–Cβ–Cγ–C_D2) describing the relative movement of the aromatic ring with respect to the polar region give detailed information of the dynamics of the complexes. It is found that the complex with α-cyclodextrin in water is not stable, in agreement with experimental data, while in all other situations studied the complex is stable within the computational limits. Comparing the different cases and the experimental evidence it comes out that a simulation of the complexes without an explicit treatment of the solvent gives unreliable results.     

Gibberellin conjugates: an overview

This article surveys the currently isolated and identified GA conjugates, their synthesis and evaluates modern methods for analysing GA glucose conjugates. The metabolism of applied GAs in higher plant systems leading, in most cases, to GA conjugates is also considered. The enzymology of the formation and hydrolysis of GA glucose conjugates is discussed in connection with their possible physiological function.Abbreviations API = atmospheric pressure ionization - FAB = fast atom bombardment - GA-GE = gibberellin glucosyl ester - GA-O-G = gibberellin-O-glucoside - GC = gas chromatography - GC-MS = combined gas chromatography-mass spectrometry - HPLC = high performance liquid chromatography - LC-MS = combined high performance liquid chromatography-mass spectrometry - MS = mass spectrometry - NMR = nuclear magnetic resonance - PME = permethyl - SIM = selected ion monitoring - TMS = trimethylsilyl     

Synthesis and cytostatical evaluation of cytidine- and adenosine-5′-hexadecylphosphate and their phosphonate analogs

Four phospholipid conjugates containing the non-cytotoxic nucleosides cytidine and adenosine were prepared by condensation reactions, and their cytotoxic activity was tested in vitro against the human immortalized mammary epithelial cell H184 A₁N₄, the human mammary tumor cells MaTu and MCF7 and the B lymphoblast cell line Daudi. The synthesized compounds showed considerable activity towards H184 A₁N₄, MaTu and Daudi cells, but they were not effective against MCF7 cells. The phosphorus moiety—either monophosphate or monophosphonate—does not influence the effectiveness of the phospholipid derivatives in the case of the solid tumor cell lines and H184 A₁N₄. The leukemic Daudi cell line is strongly sensitive towards the different types of ester as well as to the type of the nucleoside component. Adenosine-5′-hexadecylphosphate proved to be the most potent compound among the substances prepared (IC₅₀: 9.0 μmol).     

The clinical application of 68Ga-PSMA PET/CT and regulating mechanism of PSMA expression in patients with brain metastases of lung cancer

Brain metastases (BMs) of lung cancer are common malignant intracranial tumours associated with severe neurological symptoms and an abysmal prognosis. Prostate-specific membrane antigen (PSMA) has been reported to express significantly in a variety of solid tumours. However, the clinical applications of ⁶⁸Ga-PSMA PET/CT and the mechanism of PSMA expression in patients with BMs of lung cancer have rarely been reported. Experiments with ⁶⁸Ga-PSMA PET/CT and immunohistochemical staining were conducted to evaluate the expression of PSMA from seven patients with BMs of lung cancer who accepted surgical treatment in Fudan University Shanghai Cancer Center between October 2020 and October 2021. The mechanism of PSMA expression in BMs of lung cancer was explored by using single-cell RNA sequencing. The median maximum standardized uptake value (SUVmax) in BMs was higher than that in primary lung cancer (8.6 ± 2.8 vs. 3.6 ± 1.3, P < 0.01). The mean SUVmax in BMs was 1.76-fold higher than that in the liver, which indicated the potential of PSMA radioligand therapy (PSMA-RLT) for BMs. BMs showed intense PSMA staining, while normal lung tissue had no PSMA staining and there was only faint primary lung cancer staining by immunohistochemistry (IHC). Single-cell RNA sequencing (scRNA-seq) analysis found that PSMA was mainly expressed in oligodendrocytes of BMs, whereas it was expressed at lower levels in solid cells of lung cancer. PSMA expression in oligodendrocytes might be regulated by the factors ATF3 and NR4A1, which were associated with ER stress.     

Conjugated polyamines and reproductive development: Biochemical, molecular and physiological approaches

Whole tobacco plants containing the root-inducing, left-hand transferred DNA (Ri TL-DNA) display a transformed phenotype, that includes alterations in a number of developmental processes, such as floral induction, flowering and reproduction. We show that the entire Ri TL-DNA is responsible for repression of ornithine and tyrosine decarboxylases while it exerts no effect on transferase and the methyl transferase activities. Evidence is provided that two genes from the Ri TL-DNA, rolA and rolC, alter polyamine metabolism as well as floral induction and flowering. Thus, plants transformed by the rolC gene (under the control of the 35S promoter from cauliflower mosaic virus) were male-sterile (non-viable pollen) and female fertility was reduced by approximatively 80%. A constitutive overexpression of the rolC gene may directly or indirectly cause inhibition of the accumulation of water-insoluble amine conjugates located in the anthers and all the methyl transferases, leading to increases of ornithine decarboxylase, phenylalanine ammonia lyase and putrescine caffeoyl-CoA transferase. The results suggest that male sterility is associated with catabolic processes exerted at the level of water-insoluble amine conjugates and support the view that diamine oxidase may be involved in the regulation of the amine concentration during sexual differentiation, a factor that should be considered when attempting to decipher the mechanisms of control of sexual differentiation. The rolC gene could be useful in determining the role of diamine oxidase in the physiology of flowering. These results suggest that elevated free polyamine and water-soluble polyamine levels (located in the ovaries) contribute to abnormal floral development. The transformed phenotype due to P_35S-rolA(the rolA gene fused to the 35S promoter) consisted of inhibited or delaved flowering, and altered floral morphology in the form of flower abortion. The effects of P_35S-rolA on flowering and fertility are closely correlated with limitations in the accumulation of the water-soluble and -insoluble amine conjugates and increase in accumulation of free amines, indicating that amine conjugates (via transferases) have important functions in floral induction, floral evocation and reproduction. Spermidine availability as well as tyramine availability (in conjugated forms) could be limiting factor(s) in sexual development in tobacco.     

The transport of radiolabeled indoleacetic acid and its conjugates in nodal stem segments of Phaseolus vulgaris L.

The transport of radiolabeled indoleacetic acid (IAA), and some of its conjugates, was investigated in nodal stem segments of Phaseolus vulgaris L. Donor agar blocks containing either [2-acetyl-¹⁴C]-IAA; [2-acetyl-¹⁴C]-indole-3-acetyl-L-aspartate (IAAsp); [2-acetyl-¹⁴C]-indole-3-acetyl-L-glycine (IAGly); or [2-acetyl-¹⁴C]-indole-3-acetyl-L-alanine (IAAla) were placed on either the apical or basal cut surface of stem segments each bearing an axillary bud at the midline. In some experiments, a receiver block was placed on the end opposite to the donor. After transport was terminated, the segments were divided into five equal sections plus the bud, and the radioactivity of donors, receivers and each part of the stem segment was counted.For all four substances tested, the amount of ¹⁴C transported to the axillary bud from the base was the same or greater than that from the apical end. After basipetal transport, the distribution of ¹⁴C in the segment declined sharply from apex to base. The inverse was true for acropetal transport. Transport for the three IAA conjugates did not differ substantially from each other.The IAA transport inhibitor, N-1-naphthylphthalamic acid (NPA), inhibited basipetal ¹⁴C-IAA transport to the base of the stem segment but did not alter substantially the amount of ¹⁴C-IAA recovered from the bud. Transport of ¹⁴C-IAA from the apical end to all parts of the stem segment declined when the base of the section was treated with nonradioactive IAA. Taken together with data presented in the accompanying article [Tamas et al. (1989) Plant Growth Regul 8: 165–183], these results suggest that the transport of IAA plays a role in axillary bud growth regulation, but its effect does not depend on the accumulation of IAA in the axillary bud itself.     

A specific enzyme hydrolyzing 6-O(4-O)-indole-3-ylacetyl-β-d-glucose in immature kernels of Zea mays

The purification of 6-O(4-O)-indole-3-ylacetyl-beta-D-glucose (IAGlc) hydrolase from immature kernels of maize (Zea mays) was undertaken to separate this enzyme from 1-O-IAGlc hydrolase and beta-glucosidase. Partially purified 6-O(4-O)-IAGlc hydrolase was found to be the specific enzyme catalyzing hydrolysis of stable esters of IAA and glucose. Among a range of ester conjugates tested as substrates, only 6-O(4-O)-IAA-glucose and IBA-glucose isomers were effectively hydrolyzed. No activity against p-nitrophenyl-beta-D-glucopyranoside, a synthetic substrate for beta-glucosidase, was detected in the enzyme preparation. The enzyme is probably involved in the regulation of the IAA levels by the target release of free auxin from ester-linked conjugates, its inactive storage forms.     

Seasonal variation in the regulation of testosterone levels in the eastern mud snail (Ilyanassa obsoleta)

Abstract. Some gastropods appear to utilize a strategy for regulating testosterone levels that is atypical of such processes thus far identified in other metazoans. While most animals convert testosterone largely to polar derivatives that are readily eliminated from the organism, the mud snail Ilyanassa obsoleta converts testosterone to apolar testosterone-fatty acid esters that are retained by the organism. In the present study, we tested the hypothesis that fatty acid esteri-fication serves to store testosterone and that stored testosterone-fatty acid ester pools vary through the reproductive cycle of the snail. This hypothesis was tested by 1) modulating total testosterone levels in individual snails and measuring the amount of the hormone stored as the fatty acid ester relative to the amount retained as free steroid and 2) measuring changes in esterified testosterone in a field population of snails during their reproductive cycle. Adult snails were experimentally manipulated to contain from ∼36–300% the level of total testosterone measured in unmanipulated individuals. The amount of testosterone-fatty acid ester present in these organisms increased in direct proportion to the level of total testosterone, while, free testosterone levels remained relatively constant. These observations suggest that free testosterone levels are regulated in the snail by fatty acid esterification/de-esterification processes. Among field sampled snails, testosterone existed predominantly in the free, non-esterified form at the onset and end of the egg laying period. At other times, the majority of testosterone was sequestered as fatty acid esters. This study provides compelling evidence that free testosterone levels in the mud snail are regulated through fatty acid esterification/de-esterification processes and this regulatory function contributes to seasonal fluctuations in free testosterone levels.