Effect of Polar Head Group Modifications on the Tumor Retention of Phospholipid Ether Analogs: Role of the Quaternary Nitrogen.

We have previously described the remarkable capacity of radioiodinated alkyl phospholipids to be sequestered and retained by a variety of tumors in vivo. We have already established the influence of certain structural parameters of iodinated alkyl phospholipids on tumor avidity, such as stereochemistry at the sn-2 carbon of alkylglycerol phosphocholines, meta-or para-position of iodine in the aromatic ring of phenylalkyl phosphocholines, and the length of the alkyl chain in alkyl phospholipids. In order to determine the additional structural requirements for tumor uptake and retention, three new radioiodinated alkylphospholipid analogs, 2-4, were synthesized as potential tumor imaging agents. Polar head groups were modified to determine structure-tumor avidity relationships. The trimethylammonio group in 1 was substituted with a hydrogen atom in 2, an ammonio group in 3 and a tertiary butyl group in 4. All analogs were separately labeled with iodine-125 or iodine-124 and administered to Walker 256 tumor-bearing rats or human PC-3 tumor-bearing SCID mice, respectively. Tumor uptake was assessed by gamma-camera scintigraphy (for [I-125]-labeled compounds) and high-resolution micro-PET scanning (for [I-124]-labeled compounds). It was found that structural modifications in the polar head group of alkyl phospholipids strongly influenced the tumor uptake and tissue distribution of these compounds in tumor-bearing animals. Phosphoethanolamine analog 3 (NM401) displayed a very slight accumulation in tumor as compared with phosphocholine analog 1 (NM346). Analogs 2 (NM400) and 4 (NM402) lacking the positively charged nitrogen atom failed to display any tumor uptake and localized primarily in the liver. This study provided important insights regarding structural requirements for tumor uptake and retention. Replacement of the quaternary nitrogen in the alkyl phospholipid head group with non-polar substituents resulted in loss of tumor avidity.

In Silico Docking of Alkylphosphocholine Analogs to Human Serum Albumin Predicts Partitioning and Pharmacokinetics.

Alkylphosphocholine (APC) analogs are a novel class of broad-spectrum tumor-targeting agents that can be used for both diagnosis and treatment of cancer. The potential for clinical translation for APC analogs will strongly depend on their pharmacokinetic (PK) profiles. The aim of this work was to understand how the chemical structures of various APC analogs impact binding and PK. To achieve this aim, we performed in silico docking analysis, in vitro and in vivo partitioning experiments, and in vivo PK studies. Our results have identified 7 potential high-affinity binding sites of these compounds on human serum albumin (HSA) and suggest that the size of the functional group directly influences the albumin binding, partitioning, and PK. Namely, the bulkier the functional groups, the weaker the agent binds to albumin, the more the agent partitions onto lipoproteins, and the less time the agent spends in circulation. The results of these experiments provide novel molecular insights into the binding, partitioning, and PK of this class of compounds and similar molecules as well as suggest pharmacological strategies to alter their PK profiles. Importantly, our methodology may provide a way to design better drugs by better characterizing the PK profile for lead compound optimization.

CLR 125 Auger Electrons for the Targeted Radiotherapy of Triple-Negative Breast Cancer.

PURPOSE: Auger electrons emitted by radioisotopes such as 125I have a high linear energy transfer and short mean-free path in tissue (<10 µm), making them suitable for treating micrometastases while sparing normal tissues. The authors developed and subsequently investigated a cancer cell-selective small molecule phospholipid ether analog to deliver 125I to triple-negative breast cancer (TNBC) cells in vivo. METHODS: A Current Good Manufacturing Practice (cGMP) method to radiolabel 125I-CLR1404 (CLR 125) with >95% radiochemical purity was established. To estimate CLR 125 in vivo dosimetry and identify dose-limiting organs, the biodistribution of the analog compound 124I-CLR1404 (CLR 124) was investigated using micro-positron emission tomography (PET)/computed tomography (CT) in conjunction with a Monte Carlo dosimetry platform to estimate CLR 125 dosimetry. In vivo antitumor efficacy was tested by injecting nude mice bearing either MDA-MB-231-luc orthotopic xenografts or lung metastases with 74 MBq (3.7 GBq/kg) of CLR 125 or an equivalent mass amount of nonradiolabeled CLR 125. Longitudinal tumor measurements using calipers and bioluminescence imaging were obtained for the xenografts and lung metastases, respectively. RESULTS: Dosimetry analysis estimated that CLR 125 would impart the largest absorbed dose to the tumor per injected activity (0.261 ± 0.023 Gy/MBq) while the bone marrow, which is generally the dose-limiting organ for CLR1404, appears to have the lowest (0.063 ± 0.005 Gy/MBq). At administered activities of up to 74 MBq (3.7 GBq/kg), mice did not experience signs of toxicity. In addition, a single dose of CLR 125 reduced the volume of orthotopic primary TNBC xenografts by ∼60% compared to control vehicle (p < 0.001) and significantly extended survival. In addition, CLR 125 was efficacious against preclinical metastatic TNBC models by inhibiting the progression of micrometastases (p < 0.01). CONCLUSIONS: Targeted radionuclide therapy with CLR 125 displayed significant antitumor efficacy in vivo, suggesting promise for treatment of TNBC micrometastases.

Alkylphosphocholine analogs for broad-spectrum cancer imaging and therapy.

Many solid tumors contain an overabundance of phospholipid ethers relative to normal cells. Capitalizing on this difference, we created cancer-targeted alkylphosphocholine (APC) analogs through structure-activity analyses. Depending on the iodine isotope used, radioiodinated APC analog CLR1404 was used as either a positron emission tomography (PET) imaging ((124)I) or molecular radiotherapeutic ((131)I) agent. CLR1404 analogs displayed prolonged tumor-selective retention in 55 in vivo rodent and human cancer and cancer stem cell models. (131)I-CLR1404 also displayed efficacy (tumor growth suppression and survival extension) in a wide range of human tumor xenograft models. Human PET/CT (computed tomography) and SPECT (single-photon emission computed tomography)/CT imaging in advanced-cancer patients with (124)I-CLR1404 or (131)I-CLR1404, respectively, demonstrated selective uptake and prolonged retention in both primary and metastatic malignant tumors. Combined application of these chemically identical APC-based radioisosteres will enable personalized dual modality cancer therapy of using molecular (124)I-CLR1404 tumor imaging for planning (131)I-CLR1404 therapy.

Quantification of CLR1401, a novel alkylphosphocholine anticancer agent, in rat plasma by hydrophilic interaction liquid chromatography-tandem mass spectrometric detection.

A rapid and specific LC-MS/MS based bioanalytical method was developed and validated for the determination of 18-(p-iodophenyl)octadecyl phosphocholine (CLR1401), a novel phosphocholine drug candidate, in rat plasma. The optimal chromatographic behavior of CLR1401 was achieved on a Kromasil silica column (50 mm x 3 mm, 5 microm) under hydrophilic interaction chromatography. The total LC analysis time per injection was 2.8 min with a flow rate of 1.5 mL/min under gradient elution. Liquid-liquid extraction in a 96-well format using ethyl acetate was developed and applied for method validation and sample analysis. The method validation was conducted over the curve range of 2.00-1000 ng/mL using 0.0500 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed < or = 5.9% relative standard deviation (RSD) and -10.8 to -1.4% relative error (RE). The method was successfully applied to determine the toxicokinetics of CLR1401 in rats from three dose groups of 0.4, 4.0, and 10.0 mg/kg/day via intravenous administration.

Synthesis and structure-activity relationship effects on the tumor avidity of radioiodinated phospholipid ether analogues.

Radioiodinated phospholipid ether analogues have shown a remarkable ability to selectively accumulate in a variety of human and animal tumors in xenograft and spontaneous tumor rodent models. It is believed that this tumor avidity arises as a consequence of metabolic differences between tumor and corresponding normal tissues. The results of this study indicate that one factor in the tumor retention of these compounds in tumors is the length of the alkyl chain that determines their hydrophobic properties. Decreasing the chain length from C12 to C7 resulted in little or no tumor accumulation and rapid clearance of the compound in tumor-bearing rats within 24 h of administration. Increasing the chain length had the opposite effect, with the C15 and C18 analogues displaying delayed plasma clearance and enhanced tumor uptake and retention in tumor-bearing rats. Tumor uptake displayed by propanediol analogues NM-412 and NM-413 was accompanied by high levels of liver and abdominal radioactivity 24 h postinjection to tumor-bearing rats. Addition of a 2-O-methyl moiety to the propanediol backbone also retarded tumor uptake significantly. A direct comparison between NM-404 and its predecessor, NM-324, in human PC-3 tumor bearing immune-compromised mice revealed a dramatic enhancement in both tumor uptake and total body elimination of NM-404 relative to NM-324. On the basis of imaging and tissue distribution studies in several rodent tumor models, the C18 analogue, NM-404, was chosen for follow-up evaluation in human lung cancer patients. Preliminary results have been extremely promising in that selective uptake and retention of the agent in tumors is accompanied by rapid clearance of background radioactivity from normal tissues, especially those in the abdomen. These results strongly suggest that extension of the human trials to include other cancers is warranted, especially when NM-404 is radiolabeled with iodine-124, a new commercially available positron-emitting isotope. The relatively long physical half-life of 4 days afforded by this isotope appears well-suited to the pharmacodynamic profile of NM-404.

Methoxypoly(ethylene glycol)-conjugated carboxypeptidase A for solid tumor targeting: part II: pharmacokinetics and biodistribution in normal and tumor-bearing rodents.

We have evaluated effects of mPEG modification on pharmacokinetic properties of carboxypeptidase A (CPA) in normal rats. Attachment of two or three mPEG chains to CPA resulted in the generation of mPEG2-CPA and mPEG3-CPA analogs with significantly enhanced plasma half-lives, especially during the distribution phase. Moreover, the assessment of real-time whole-body kinetics in CT26 tumor-bearing mice showed both mPEG2-CPA and mPEG3-CPA exhibited increased body retention at 48 h post-injection. In addition, tumor localization of mPEG3-CPA at 72 h was visualized and confirmed by fusion of the gamma-scintigraphy and microCT data sets. Results from the imaging studies support our hypothesis of a correlation between tumor uptake and enhanced circulatory half-life. Tissue distribution data indicated the combination of increased tumor extravasation and effective renal elimination observed with mPEG2-CPA at 48 h following administration led to the highest observed tumor-to-blood ratio of 4.8:1. Although the total concentration of mPEG3-CPA accumulated in tumor was higher than that of mPEG2-CPA and CPA at predetermined time intervals, a higher tumor-to-blood ratio was not obtained owing to a higher level of blood activity. Clearly, the attachment of an appropriate number of mPEG chains can facilitate tumor localization as effectively as can the use of a tumor-specific antibody.

A surface-modified chylomicron remnant-like emulsion for percutaneous computed tomography lymphography: synthesis and preliminary imaging findings.

RATIONALE AND OBJECTIVES: To assess a surface-modified emulsion as a percutaneous CT lymphographic agent in normal dogs. METHODS: An iodinated chylomicron remnant-like microemulsion was formulated with a mean particle size of 91.3 nm and an iodine concentration of 91 mg I/mL. Contrast material (2 mL) was injected into the subcutaneous tissues of the metatarsus and metacarpus of six normal dogs to enhance popliteal and cervical lymph nodes, respectively. CT images were acquired at 0, 15, 30, 45, 60, 240, 480, and 1440 minutes. RESULTS: Significant lymph node enhancement occurred in as little as 15 minutes after injection and persisted at least 8 hours. Node opacification was most pronounced at 1 to 4 hours postinjection and exceeded 200 HU in some nodes (precontrast attenuation = 45 HU). Marked enhancement of popliteal efferent lymphatics and of iliac and sacral node groups also occurred indicating distribution to second order nodes. Attenuation of enhanced nodes reverted to precontrast levels by 24 hours. CONCLUSION: The new surface-modified, chylomicron remnant-like emulsion provided marked, selective enhancement of targeted lymph nodes after subcutaneous administration. Moreover, the formulation produced significant opacification of more distant node groups from a single injection.