PET Imaging of Macrophage Mannose Receptor-Expressing Macrophages in Tumor Stroma Using 18F-Radiolabeled Camelid Single-Domain Antibody Fragments.

UNLABELLED: Tumor-associated macrophages constitute a major component of the stroma of solid tumors, encompassing distinct subpopulations with different characteristics and functions. We aimed to identify M2-oriented tumor-supporting macrophages within the tumor microenvironment as indicators of cancer progression and prognosis, using PET imaging. This can be realized by designing (18)F-labeled camelid single-domain antibody fragments (sdAbs) specifically targeting the macrophage mannose receptor (MMR), which has been identified as an important biomarker on this cell population. METHODS: Cross-reactive anti-MMR sdAbs were generated after immunization of an alpaca with the extracellular domains of both human and mouse MMR. The lead binder was chosen on the basis of comparisons of binding affinity and in vivo pharmacokinetics. The PET tracer (18)F-fluorobenzoate (FB)-anti-MMR sdAb was developed using the prosthetic group N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB), and its biodistribution, tumor-targeting potential, and specificity in terms of macrophage and MMR targeting were evaluated in mouse tumor models. RESULTS: Four sdAbs were selected after affinity screening, but only 2 were found to be cross-reactive for human and mouse MMR. The lead anti-MMR 3.49 sdAb, bearing an affinity of 12 and 1.8 nM for mouse and human MMR, respectively, was chosen for its favorable in vivo biodistribution profile and tumor-targeting capacity. (18)F-FB-anti-MMR 3.49 sdAb was synthesized with a 5%-10% radiochemical yield using an automated and optimized protocol. In vivo biodistribution analyses showed fast clearance via the kidneys and retention in MMR-expressing organs and tumor. The kidney retention of the fluorinated sdAb was 20-fold lower than a (99m)Tc-labeled counterpart. Compared with MMR- and C-C chemokine receptor 2-deficient mice, significantly higher uptake was observed in tumors grown in wild-type mice, demonstrating the specificity of the (18)F tracer for MMR and macrophages, respectively. CONCLUSION: Anti-MMR 3.49 was denoted as the lead cross-reactive MMR-targeting sdAb. (18)F radiosynthesis was optimized, providing an optimal probe for PET imaging of the tumor-promoting macrophage subpopulation in the tumor stroma.

In the search for new anticancer drugs. 29. A study on the correlation of lipophilicities, ionization constants and anticancer activities of aminoxyl labeled TEPA congeners.

Ionization constants for a series of sterically hindered pyrrolidine, pyrroline and piperidine derivatives were determined by potentiometric titrations. The pKa values for the secondary amines as a group ranged from about 7.7 to 11.7, whereby the ring size had no decisive effect on the values. The corresponding hydroxylamine derivatives as a group had distinctly lower pKa values than the amine derivatives ranging between about 4.0 and 6.3. It was shown using the Henderson-Hasselbalch equation that at physiological pH, arbitrarily chosen 6, 7 and 8, the amine derivatives would exist mainly in the protonated form, whereas the hydroxylamine derivatives would be expected to be mainly in the unprotonated form. In contrast, the 4-hydroxy-2,2,6,6-tetramethylpiperdin-1-oxyl radical, under analogous conditions, was a neutral species, i.e. it could not be titrated in aqueous media. On the basis of these results, it was hypothesized that the alkylating anticancer drugs of TEPA (N,N:N'N':N",N"-tri-1,2-ethanediylphosphorictriamide) type, containing sterically hindered carrier moieties, would be expected to permeate across cell membranes, and, consequently, exhibit anticancer activities according to the following sequence: spin-labeled drugs containing no titratable components > hydroxylamine derivatives > secondary amine congeners. This assumption is confirmed by good correlations of anticancer activities of these drugs with their pKa values, and the partition coefficients. The conclusion was reiterated that, in the quest for a rational design of anticancer drugs, the aim should be to construct agents with partition coefficients approaching the logarithm of zero, either from the negative or positive side, and pKa values as low as practically possible and applicable.

In the search for new anticancer drugs. 26. A comparison of anticancer activities of several TEPA, thio-TEPA, Seleno-TEPA, and azetidine analogs, including congeners containing an aminoxyl moiety.

A series of TEPA, Thio-TEPA, Seleno-TEPA, and azetidine analogs, including congeners containing an aminoxyl moiety, were synthesized and evaluated in vivo for anticancer activity against the murine lymphocytic leukemia P388. All aziridine derivatives were found to be active with an increase in life span ranging from 42% to 272%, and all azetidine analogs were rated as inactive with one marginal exception. An attempt was made to rationalize the results on the basis of the lipophilic properties of the compounds. The most active compound (8) possessed the most balanced lipophilic properties, corresponding to a log P value near zero.

Evaluation of spin labeled TEPA and CCNU analogs against osteosarcoma and MNU-induced mammary carcinoma of the SD-rat.

The effectiveness to reduce tumor growth by 1-(2-chloroethyl)-3-(1-oxyl-2,2,6,6-tetramethylpiperidinyl)- 1-nitrosourea (SLCNU) and N,N,N,'N'- bis(1,2-ethanediyl)-N"-(1-oxyl-2,2,6,6-tetramethyl- 2-piperidinylaminocarbonyl)-phosphoric triamide (SLDU) was studied in osteosarcoma and MNU-induced mammary carcinoma in the SD-rat. Both compounds elicited neither an inhibitory effect on these tumors nor an increase in the mean/median life span as compared to the control group.

In the search for new anticancer drugs. XX: A comparison of the in vitro growth inhibition of P388 cells by TEPA and N,N;N',N'-bis (1,2-ethanediyl)-N''-(1-oxyl-2,2,6,6-tetramethyl-4- piperidinylaminocarbonyl) phosphoric triamide and congeners.

We tested the in vitro growth inhibitory activity of TEPA, and three analogs against P388 murine lymphocytic leukemia cells in culture. The analogs consist of spin-labeled TEPA and two reduced forms containing the NH and NOH groups instead of the nitroxyl function. Spin label TEPA was obtained by replacing one of the aziridine groups in TEPA with spin-labeled urea. In a concentration range of 10(-6)-10(-5) M, only the reduced analog containing the NH group was active. That is, to achieve a 50% inhibition of cell growth, a five-fold excess in concentration of this analog (IC50 = 10 X 10(-6) M) was needed as compared to the parent compound TEPA (IC50 = 2 X 10(-6) M). These results are in contrast with those obtained in vivo against the same leukemia cell line, indicating inherent discrepancies between in vivo and in vitro evaluation of antitumor agents.

In the search for new anticancer drugs. 19. A predictive design of N,N:N',N':N'',N''-tri-1,2-ethanediylphosphorothioic triamide (TEPA) analogues.

The nitroxyl-labeled analogues of N,N:N',N':N",N"-tri-1,2-ethanediylphosphoric triamide (TEPA), N,N:N',N'-bis(1,2-ethanediyl)-N"-[[(2,2,6,6-tetramethyl-1-oxypiperidi n-4- yl)amino]carbonyl]phosphoric triamide (5a) and N,N:N',N'-bis(1,2-ethanediyl)-N"-[[(2,2,5,5-tetramethyl-1-oxypyrrolid in-3- yl)amino]carbonyl]phosphoric triamide (11a), possess therapeutic indexes that are 8-12 times higher than those of thio-TEPA (1) and TEPA (2). The introduction of methyl groups into the aziridine ring, or the replacement of the nitroxyl moiety with hydroxylamine or amine derivatives, or with an adamantane moiety, results in compounds of lesser activity. An attempt is made to rationalize these results using a lipophilicity scale. A predictive design pattern is established.

In the search for new anticancer drugs. XVIII. Various mono- and bis-anthraquinone hydrazones containing the N,N;N',N'-bis(1,2-ethanediyl)phosphoric diamide moiety.

The structure-anticancer activity and the activity-lipophilicity relationship of 8 mono- and bis-anthraquinone hydrazones containing the N,N;N',N'-bis(1,2-ethanediyl) phosphoric diamide moiety were evaluated. These compounds were tested in vivo, using murine lymphocytic leukemia P388. Seven of these compounds were active and one was marginal at optimum doses. The highest activity was exhibited by the bis[N,N;N',N'-bis(1,2-ethanediyl)-[N2-(1',4',5',8'-tetrahydroxy anthracenylidene)-N1-methyl hydrazin-1-yl]]-phosphoric triamide, bis[N,N;N',N'-bis(1,2-ethanediyl)-[N2-(1',4'-dihydroxy anthracenylidene)-N1-methylhydrazin-1-yl]]phosphoric triamide and bis-[N,N;N',N'-bis(1,2-ethanediyl)-[N2-(1',4',5',8'-tetrahydroxy anthracenylidene)-N1-phenylhydrazin-1-yl]]phosphoric triamide as is evidenced by their percent T/C of 183, 175 and 172, respectively. The correlation of the anticancer activities of these compounds with their lipophilicities leads to the general hypothesis that the intercalating and alkylating capabilities of a potential drug may play only a secondary role as compared to the selective permeability of the drug through normal and cancerous membranes.

In the search for new anticancer drugs. X. N,N; N',N'-bis(1,2-ethanediyl)-N''-(1-oxyl-2,2,6,6-tetramethyl- 4-piperidinylaminocarbonyl) phosphoric triamide--a new potential anticancer drug of high activity and low toxicity.

A new nitroxyl labeled TEPA derivative 5 containing the urea bridge between the phosphorus and the nitroxyl moiety, and the congeners containing the NOH and NH groups instead of the nitroxyl function were synthesized, and tested in vivo on CD2F1 mice for anticancer activity against P388 and L1210. The nitroxyl compound is more active than the reduced forms. The nitroxyl compound 5 elicits 170% ILS at 90 mg/kg after 30 days and 439% ILSmax after 60 days against P388, and has a higher therapeutic ratio (26.4) than the clinically used Thio-TEPA (2.75). The LD50 of 5 is 270 mg/kg, while that of Thio-TEPA is 18 mg/kg. Consequently, the nitroxyl compound 5 is a promising new anticancer drug.

In the search for new anticancer drugs XI. Anticancer activity of nitroxyl labeled phosphoric N,N;N',N';N",N"-tris[1,2-ethanediyl]triamide(TEPA) and phosphorothioic N,N;N',N';N",N"-tris[1,2-ethanediyl]triamide(thio-TEPA) derivatives.

In order to further evaluate the effect of the nitroxyl moiety on the anticancer activity of nitroxyl labeled analogues of phosphoric N,N;N',N';N",N"-tris[1,2-ethanediyl]triamide (TEPA) and phosphorothioic N,N;N',N';N",N"-tris[1,2-ethanediyl]triamide (thio-TEPA), the activity of these compounds was compared in vivo, using murine lymphoid leukemia L1210, with the reduced forms of the drugs, i.e. the hydroxylamines and amine congeners. At optimum dose, all compounds were active. However, the nitroxyl containing compounds were more active than the corresponding reduced forms. An admixture of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl had no effect on the activity of thio-TEPA. Consequently, the nitroxyl moiety must be an integral part of the anticancer drug's structure in order to influence that drug's performance.

[Endolymphatic use of solutions of alkylating substances in a mixture with iodolipol in malignant neoplasms]. / Ob éndolimfaticheskom primenenii rastvorov alkiliruiushchikh veshchestv v smesi s iodolipolom pri zlokachestvennykh novoobrazovaniiakh.

The results of endolymphatic chemotherapy and lymphography in 57 cases of different malignant neoplasms in the lymphatic system are presented. Procedures for making alcohol-oil solution of benzo-TEP, ether-oil solution of tio-TEP and alcohol-oil emulsion of cyclophosphamide are discussed and the clinical evaluation of the results of their application is given. The therapeutic effect of endolymphatic infusions of the mixtures of alkylating agents and iodolipol was assessed on the basis of clinical, histological and radiological examination of malignant lesions of lymph nodes. It is shown that the efficacy of endolymphatic chemotherapy is determined by the susceptibility of neoplasms to antitumor drugs and the availability of the latter at lymph node metastases.

[Effect of diiodobenzotefa on the functionally different subpopulations of T-lymphocytes]. / Deistvie diiodbenzotéfa na funktsional'no razlichnye subpopuliatsii T-limfotsitov.

Diiodobenzo-tepa (DIB) was given orally to CBA mice in a dose of 25 mg/kg for 3 successive days. The number of nucleus-containing cells decreased 3.9 fold in the thymus and 1.4 fold in the bone marrow. In experiments on transplantation of lymphoid cells to intact or lethally irradiated (CBA X C57BL/6J)F1 mice treated with DIB this substance did not influence the helper activity of T lymphocytes but inhibited the activity or B and T lymphocytes, inducing "graft-versus-host" and T cell-suppressor functions.

[Pharmacological properties and antitumor activity of a preparation diiodo-benzo-tepa in experiments]. / Farmakologicheskie svoistva i protivoopukholevaia aktivnost' preparata diiodbenzotef v eksperimente.

Experimental data on the pharmacokinetics, toxicity, antitumor activity of the antitumor drug diiodo-benzo-TEPA are reported.

[Effect of iodobenzoteph and cytostasan on several immune responses in animals]. / Vliianie iodbenzotefa i tsitostazana na nekotorye immunnye reaktsii zhivotnykh.

Tests staged on line-bred mice immunized with sheep erythrocytes brought evidence that cystostasan and iodobenzoteph cut down the number of antibody-forming cells both with the primary and secondary immune response. The maximal effect becomes manifest following introduction of the drugs on the 1--2nd day after immunization of the animals. Iodobenzoteph brings down the number of hemopoietic stem cells colonies while cytostosan leaves them unchanged.