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. 2026 Oct 7;11(41):62402–62411. doi: 10.1021/acsomega.5c11584

Potent Antineoplastic Activity against Breast Cancer in Vitro and in Vivo of a Chloro-Substituted Polyaromatic Platinum(II) Complex and Its Platinum(IV) Prodrug

Omar Hammoud †, Maria George Elias †,‡, Amjad Slika †, Aleen Khoury ‡, Stephanie Mehanna †, Joelle Abou Chacra †, Selim Nasser §, Janice R Aldrich-Wright ‡, Costantine F Daher †,∥, Robin I Taleb ⊥,*
PMCID: PMC13689262  PMID: 42923595

Abstract

This study investigates the chemotherapeutic potential of Pt II 5CLSS and Pt IV 5CLSS against breast cancer in vivo. Cell viability was assessed 72 h post-treatment, revealing potent cytotoxic activity with IC50 values of 0.447 μM for Pt II 5CLSS and 0.922 μM for Pt IV 5CLSS. Cellular uptake studies used ICP-MS to confirm active transport of both complexes into cells, showing significant accumulation in the nuclear/cytoskeletal fractions. A 28-day toxicity study in female Sprague-Dawley rats demonstrated that escalating doses up to 15 mg/kg (Pt II 5CLSS) and 30 mg/kg (Pt IV 5CLSS) caused no mortality nor adverse effects on liver and kidney function. In a chemically induced breast cancer rat model, both complexes significantly reduced tumor weight by 93–100% at doses of 1 and 5 mg/kg, with no observable nephrotoxicity or hepatotoxicity. Histopathological analysis confirmed the reduction or absence of tumors in the majority of the treated animals with Pt IV 5CLSS (1 mg/kg bw) being the most effective. Toxicity evaluation in sus scrofa pigs showed that Pt IV 5CLSS was well tolerated at both 1- and 2.5 mg/kg bw, with no mortality, behavioral changes, or signs of acute toxicity. Overall, both Pt II 5CLSS and Pt IV 5CLSS demonstrate strong antineoplastic activity against breast cancer with a favorable safety profile, supporting their potential as alternative platinum-based chemotherapeutic agents.

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Significance

We have synthesized and tested platinumII and a platinumIV complexes that have shown to be potent, selective, and safe chemotherapeutic agents in breast cancer treatment with no observed nephrotoxicity nor hepatotoxicity.

Introduction

Following the success of cisplatin as an anticancer agent, analogues such as carboplatin and oxaliplatin were developed to improve the efficacy and reduce toxicity. Once platinumII complexes enter the cell through passive diffusion or transport proteins, they undergo aquation, in which the chloride groups are replaced by water molecules, rendering the complex electrophilic. , This form readily binds to DNA at the N7 position of purine bases, forming intra- and interstrand cross-links, ultimately leading to cell cycle arrest and apoptosis. Cisplatin also disrupts calcium homeostasis and induces reactive oxygen species (ROS), contributing to mitochondrial damage and the release of pro-apoptotic proteins. , Other platinumII complexes may interact with DNA through intercalation or modulate cellular signaling pathways, including MAPK and PI3K, leading to further cytotoxic effects. Despite their effectiveness, platinumII drugs have significant drawbacks. Their non-selective toxicity can lead to side effects such as nephrotoxicity, ototoxicity, and neurotoxicity, with kidney damage being particularly associated with cisplatin. , Additionally, resistance to platinum-based chemotherapy is a major clinical challenge. Mechanisms of resistance include reduced drug uptake, increased efflux, and enhanced DNA repair capabilities in cancer cells.

PlatinumIV complexes differ from platinumII complexes by possessing two additional axial ligands, giving them an octahedral geometry with a general formula ML2X2R2, where R represents the axial ligands. These axial ligands contribute to the unique activity of platinumIV complexes and offer several advantages over their platinumII counterparts, including increased chemical stability, reduced reactivity with non-target molecules, and improved cellular uptake. Once inside the cell, platinumIV complexes may be reduced to platinumII, releasing their axial ligands and exerting cytotoxic effects similar to those of platinumII drugs such as DNA cross-linking, intercalation, and interference with key signaling pathways. , Notably, the released axial ligands could be tailored to induce their own biological activity. For example, mitaplatin, a platinumIV analogue of cisplatin, releases dichloroacetate (DCA) upon reduction. DCA inhibits pyruvate dehydrogenase kinase, thereby downregulating aerobic glycolysis; a pathway heavily relied upon by cancer cells, resulting in more selective apoptosis. Other platinumIV complexes have been shown to induce cell death by oxidizing guanine or generating reactive oxygen species, such as hydrogen peroxide, which can cleave double-stranded DNA.

The structural versatility of platinumIV complexes allows for fine-tuning of their pharmacokinetic and pharmacodynamic properties. Their increased stability and reduced off-target interactions can lead to fewer side effects, and some platinumIV drugs can be administered orally, improving patient convenience and lowering treatment costs. Furthermore, the tumor microenvironment characterized by lower pH, hypoxia, and elevated glutathione levels can be exploited to trigger selective drug activation in cancer cells. By designing axial ligands responsive to these conditions, platinumIV prodrugs can achieve greater tumor selectivity. Despite these advantages, several platinumIV candidates including ormaplatin, iproplatin, and satraplatin have failed to gain FDA approval due to dose-limiting toxicities, such as neutropenia, thrombocytopenia, neurotoxicity, and myelosuppression. Nevertheless, ongoing research continues to explore platinumIV complexes as promising alternatives to traditional platinum-based chemotherapeutics. Pt II 5CLSS and Pt IV 5CLSS (Figure ) were recently shown to possess potent cytotoxicity against MDA-MB-231 breast cancer cells (triple negative breast cancer-TNBC) as well as significant cancer cell selectivity when compared to rat red bone marrow mesenchymal stem cells (selectivity index: SI > 233 and SI > 97, respectively). TNBC is known to be unresponsive to advanced therapies such as targeted therapies and immunotherapies, there is hence an immediate need to explore new chemotherapy approaches that would enhance efficacy while minimizing side effects. In addition, a dose escalation study carried out on BALB/c mice treated with Pt II 5ClSS or Pt IV 5ClSS exhibited an 8- or 12.5-folds more tolerability then when treated with cisplatin. The present study investigates the chemotherapeutic potential of both complexes on DMBA-induced breast cancer in rats, as well as the toxicity of the platinumIV analogue on domestic pigs.

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Chemical structures of Pt II 5CLSS and Pt IV 5CLSS.

Methods

Platinum Complexes and Cell Lines

The platinum complexes Pt II 5CLSS and Pt IV 5CLSS were synthesized in the research laboratory of Professor Janice Aldrich-Wright (Western Sydney University) as previously described. Triple-negative breast cancer cells (MDA-MB-231, “RRID:CVCL_0062”) were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich, Missouri, USA) supplemented with 10% FBS and 1% penicillin–streptomycin, at 37 °C and 5% CO2.

Cytotoxicity Assay

MDA-MB-231 (“RRID:CVCL_0062”) cells, purchased from the American Type Culture Collection (ATCC- CRM-HTB-26; passage 21), were seeded in 96-well plates at a density of 1 × 104 cells per well. Cells were allowed to adhere overnight in a Dulbecco’s Modified Eagles Medium (DMEM) containing 10% heat-inactivated fetal bovine serum and 5% streptomycin–penicillin in humidified incubators at 37 °C and 5% CO2. Adhered cells were then treated with varying concentrations (3-fold dilution; 0.023-150 μM) of PtII5CLSS, PtIV5CLSS and cisplatin for 72 h. The cytotoxic effect was assessed at the end of the treatment period using the WST-1 cell proliferation assay Kit (Abcam; ab65473) where a 10 μL of the WST-1 reagent (4-[3-(4-iodophenyl)-2-(4-nitro-phenyl)-2H-5-tetrazolio]-1,3-benzenesulfonate) was added to each well and incubated for 4 h at 37°C. Cell viability was then assessed by measuring absorbance at 450 nm using a microplate Varioskan LUX Multimode reader. IC50 values were determined using GraphPad Prism software. Cells were plated in triplicates in three independent experiments.

Quantification of Cellular Uptake by ICP-MS

MDA-MB-231 (“RRID:CVCL_0062”) cells were seeded in 6-well plates at a final concentration of 106 cells per well. Plates were incubated at 37°C and 5% CO2 overnight for cells to adhere before treatment with either Pt II 5CLSS, Pt IV 5CLSS or cisplatin (3 μM). Procedure was carried out as per our previously published protocol. Three independent experiments were performed in triplicate, optimized following a calibration curve, and expressed as nmol/106 cells.

Cellular Fractionation

MDA-MB-231 (“RRID:CVCL_0062”) cells were cultured in 6-well plates and left to adhere overnight. Cells were then treated with either Pt II 5CLSS, Pt IV 5CLSS or cisplatin (3 μM) for 24 h, media removed and cells washed three times with cold PBS. Cell isolation and fractionation were conducted as described by the cell fractionation kit (Cell Signaling Technology, USA, #9038). The resulting solutions were filtered, and the platinum concentrations were quantified using ICP-MS with indium as internal standard (0.025 μM).

Experimental Animals

Adult Sprague-Dawley female rats (“RRID:MGI:5651135”; 120–160 g), obtained from the animal facility of the Lebanese American University were housed under optimal conditions (22 ± 2 °C, humidity 50 ± 5%, 12 h alternating light/dark cycle) and supplied with standard laboratory diet and water ad libitum. All experimental protocols were approved by the Institutional Animal Care and Use Committee (#LAU.ACUC.SAS.CD7.15/June/2022) at LAU and complied with the guide for the care and use of laboratory animals (National Research Council Committee, USA).

28-Day Toxicity Study

Weekly single IP injections (5, 15, or 30 mg/kg.b.w) of Pt II 5CLSS or Pt IV 5CLSS were administered to rats (n = 3 per group; no animal was excluded from the analysis) over a period of 28 days in order to assess toxicity. The control group (n = 3; no animal was excluded from the analysis) received weekly IP injections of saline. Physical changes, behavioral changes, and death incidences were monitored over the study period. At day 28, rats were sacrificed under CO2, blood was withdrawn from the inferior vena cava into vacuum collection tubes and sent to LAU Medical Center Saint John’s hospital (Jounieh, Lebanon) for serum biochemistry testing. Organs (brain, liver, and kidney) were washed, blot dried, weighed, and stored in 10% formalin and sent to LAU Medical Center Rizk hospital (LAUMCRH, Achrafieh, Lebanon) for histopathological analysis. The hospital analyst received blind samples that can be decoded only by the project lead. The attrition rate of the study was 100%.

Breast Cancer Induction

Using a gastric tube, a dose of 80 mg/kg bw of DMBA (7,12-Dimethylbenz[a]anthracene) dissolved in olive oil (8 mg/mL) was instilled in the stomach of 50-day old rats. , Rats were palpated to monitor tumor development, and once formed, tumor dimensions (length and width) were measured using a vernier caliper.

Breast Cancer Treatment

Once tumor diameters reached 10–13 mm, rats were randomly assigned to treatment groups (n = 4 per group; no animal was excluded from the analysis) and received weekly intraperitoneal (IP) injections of either Pt II 5CLSS or Pt IV 5CLSS at doses of 1 mg/kg (1.57 μmol/kg and 1.49 μmol/kg respectively) or 5 mg/kg (7.83 μmol/kg and 7.43 μmol/kg, respectively). A positive control group (n = 4; no animal was excluded from the analysis) was treated with cisplatin (2.5 mg/kg, 8.3 μmol/kg, IP, once weekly), while a negative control group received an equivalent volume of saline (n = 4; one animal was excluded from the analysis due to death). Tumor dimensions were measured using a vernier caliper, and body weights were recorded weekly. Tumor volume was calculated using the formula: V = (W 2 × L)/2 where V is the volume of the tumor, W its width, L its length. After 5 weeks from the initial dose (4 doses), animals were sacrificed under CO2 asphyxiation, and tumors were excised and fixed in 10% neutral-buffered formalin for histopathological evaluation at LAUMCRH. The attrition rate of the study was 95.8% (1 death in the control out of 24 animals).

Histopathology

At necropsy, organs and breast tumors were preserved in 10% neutral-buffered formalin and subsequently embedded in paraffin. Tissue sections were prepared from the paraffin blocks and stained with hematoxylin and eosin (H&E). A board-certified pathologist examined the slides using a light microscope (Zeiss, St. Louis, MO, USA). The samples were evaluated for signs of invasive carcinomas and inflammation. The hospital analyst received blind samples that could be decoded only by the project lead.

In Vivo Toxicity of PtIV5CLSS in Pigs

Twelve healthy young adult female pigs (Sus scrofa domesticus) weighing between 41 and 51 kg, were selected for the study. Animals were housed at Jamon Phoeniciano s.a.r.l. (Barka Farms, Bekaa Valley) in a controlled environment that ensured access to food, water, and clean bedding, while being fully isolated from other animals. A defined feeding program, designed in consultation with an animal production engineer, was tailored to the animals age and aimed at maintaining a steady but moderate increase in body weight. More details can be found in the supplementary section under Protocol S1. Experimental protocol was approved by the Institutional Animal Care and Use Committee (#LAU.ACUC.SON.CD2.16/January/2024) at LAU and complied with the Guide for the Care and Use of Laboratory Animals (National Research Council Committee, USA). Pigs were assigned to three groups (n = 4 per group; no animal was excluded from the analysis). The control group received a weekly dose (8 weeks) of 2 mL 5% dextrose solution via intravenous (IV) injection. The two experimental groups, were treated similarly, except they received 2 mL of Pt IV 5CLSS dissolved in 5% dextrose at doses of 1 mg/kg bw (D1) or 2.5 mg/kg bw (D2.5). The attrition rate of the study was 100%. Blood samples were collected at week 0 and 8 for biochemical and hematological analysis. Following the treatment phase, animals were monitored for an additional two months to assess any delayed adverse effect or mortality. A certified veterinarian conducted routine clinical evaluations throughout the study, including assessments of body weight and physical/behavioral changes. The veterinarian analyst (for blood samples) received blind samples that can be decoded only by the project lead.

Statistical Analysis

Values were reported as mean ± SEM. Statistical difference between groups was assessed using one-way ANOVA and was considered significant indicated by * when p-value <0.05, ** when p < 0.01, *** when p < 0.001 and **** when p < 0.0001. Data was analyzed using GraphPad prism 10 (“RRID:SCR_00278”).

Results

Cytotoxicity and Cellular Uptake

Cytotoxicity assays were performed on MDA-MB-231 cells 72 h post-treatment. The IC50 values of Pt II 5CLSS and Pt IV 5CLSS were in the submicromolar range with both complexes being more potent than cisplatin (Figure A). MDA-MB-231 cell uptake of cisplatin and Pt II 5CLSS showed a plateau at 24 h, while Pt IV 5CLSS continued to accumulate inside the cells (Figure B). Although cisplatin was initially shown to be taken up at a faster rate up until 18 h, both platinum complexes accumulated at significantly higher concentrations within the cells after 24 and 30 h.

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(A) Cytotoxic effect of Pt II 5CLSS, Pt IV 5CLSS, and cisplatin on the survival of MDA-MB-231 cells. Cells were treated with different concentrations of Pt II 5CLSS, Pt IV 5CLSS, and cisplatin over 72 h. (B) ICP-MS analysis of the uptake of Pt II 5CLSS and Pt IV 5CLSS complexes by MDA-MB-231 cells. Data points denote mean ± SEM from three independent experiments where samples were run in triplicates. The statistical difference of Pt II 5CLSS and Pt IV 5CLSS relative to cisplatin is identified by ** p < 0.01, as measured by an unpaired Student’s t-test.

Intracellular Distribution

Cellular buildup of Pt II 5CLSS, Pt IV 5CLSS, and cisplatin was explored in subcellular fractions of MDA-MB-231 cells. Both of Pt II 5CLSS and Pt IV 5CLSS accumulated mainly in the nuclear and cytoskeletal fraction (NC, 51%, 64.5%, respectively) followed by membrane/organelles fraction (MO, 30%, 22.5%, respectively) and cytoplasmic fraction (CF, 19%, 13%, respectively). Cisplatin, accumulate mainly in the membrane/organelles fraction (MO, 56%) followed by the cytoplasmic fraction (CF, 27%) and nuclear and cytoskeletal fraction (NC, 17%) (Figure ).

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Cellular accumulation Pt II 5CLSS, Pt IV 5CLSS, and cisplatin in MDA-MB-231 cells. The bar graph shows the percentage of each platinum complex in the cytoplasmic fraction (CF, blue), membrane/organelles (MO, orange), and nucleus/cytoskeletal (NC, gray). Cells were exposed to the platinum compounds for 24 h. Results are expressed as the mean ± SEM from three independent experiments where samples were run in triplicates.

28-Day Toxicity Study

After 4 weeks of treatment (a total of 4 IP injections) with 5, 15, or 30 mg/kg bw of Pt II 5CLSS, animals receiving the 5 mg and 15 mg doses exhibited a consistent increase of body weight throughout the study similar to the control group. However, the mean body weight of the animals receiving the 30 mg dose remained constant throughout the study with one death reported after the third dose. Animals treated with Pt IV 5CLSS (5, 15, or 30 mg/kg bw) showed consistent increase in body weight at all doses used with no reported deaths (Figure S2). Blood samples were collected to assess liver and kidney function, and the levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and bilirubin were shown to be in the normal range, indicating a normal liver function. Similarly, the levels of creatinine and blood urea nitrogen in all tested groups fell within the normal range, suggesting normal kidney function (Table and Figure S3).

1. Biochemistry Parameters Obtained from Blood Samples of Rats after 4 Weeks of Injections with PtII5CLSS or PtIV5CLSS.

  control PtII5CLSS 5 mg PtII5CLSS 15 mg PtII5CLSS 30 mg PtIV5CLSS 5 mg PtIV5CLSS 15 mg PtIV5CLSS 30 mg
ALT 48.3 ± 4.3 28.0 ± 0.4 36.7 ± 0.8 36.5 ± 0.7 34.7 ± 1.7 39.3 ± 2.5 35.0 ± 2.4
ALP 115 ± 16 103 ± 9 105 ± 7 91 ± 5 132 ± 10 125 ± 5 91 ± 6
AST 122 ± 6 92 ± 3 99 ± 4 100.0 ± 0.5 112 ± 2 87 ± 2 105 ± 12
total bilirubin 0.11 ± 0.01 0.10 ± 0.01 0.08 ± 0.01 0.08 ± 0.01 0.12 ± 0.01 0.09 ± 0.01 0.08 ± 0.01
creatinine 0.35 ± 0.01 0.33 ± 0.01 0.35 ± 0.01 0.37 ± 0.02 0.34 ± 0.01 0.34 ± 0.01 0.34 ± 0.01
BUN 15.6 ± 0.5 15.4 ± 0.7 19.3 ± 0.7 25.0 ± 1.7 19.2 ± 0.7 20.1 ± 0.6 16.8 ± 1.3
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a

P <0.05 with respect to the control.

Chemotherapeutic Potential of PtII5CLSS and PtIV5CLSS in a Chemically Induced Breast Cancer Model in Rats

Chemotherapeutic treatment of animals with Pt II 5CLSS (1 or 5 mg/kg bw), Pt IV 5CLSS (1 or 5 mg/kg bw), or cisplatin (2.5 mg/kg body weight) was initiated when tumor size reached 10–13 mm in diameter. Compared to control, both Pt II 5CLSS (1 and 5 mg/kg bw) and Pt IV 5CLSS (1 and 5 mg/kg bw) showed significant reduction in tumor volume after the first week of treatment until the end of the experiment (Figure A); unlike cisplatin, where no significant change in tumor volume was noted throughout the 4 weeks. After 4 weeks of treatment, 100% of animals treated with Pt II 5CLSS 1 mg/kg and cisplatin 2.5 mg/kg had tumors but with 93% and 84.5% reduction in tumor mass. Only 25% of animals (one animal) treated with Pt II 5CLSS at 5 mg/kg had tumors with a 96.5% reduction in tumor mass. On the other hand, 100% of animals treated with Pt IV 5CLSS 1 mg/kg were tumor-free. Only 25% of animals (one animal) treated with Pt IV 5CLSS at 5 mg/kg had tumors with a 93% reduction in tumor mass (Figure B). None of the control, Pt II 5CLSS, and Pt IV 5CLSS treatment groups exhibited any drops in body weights throughout the study period. However, a slight but non-significant decrease in body weight was observed with the cisplatin-treated group (Figure S4). No significant changes in organ weight (heart, lung, spleen, liver, and kidneys) were recorded in all groups (Figure S5).

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(A) Rat mammary tumor volumes (A) and weight (B) post treatment with Pt II 5CLSS, Pt IV 5CLSS, or cisplatin. Data points denote mean ± SEM, n = 4 from four rats per treatment group. **** indicates P < 0.0001 compared with control. *** indicates P < 0.001 compared with control. ** indicates P < 0.01 compared with control. * Indicates P < 0.05 compared with control.

Histopathological analyses of tumors extracted from rat mammary glands are presented in Figure and Table . The control group depicts a mammary gland with malignant cancer cells invading the dermis and subcutaneous tissue. The cisplatin-treated group shows extensive tumor formation in 50% of the animals, while the remaining animals exhibit fibro-epithelial tumors. The Pt II 5CLSS-treated groups exhibit 25% complete response (no tumor) and 75% with significant tumor persistence at the lower dose. At the higher dose, the Pt II 5CLSS-treated animals showed 50% with complete tumor inhibition with the remaining 50% of the animals exhibiting <50% persisting tumors. The Pt IV 5CLSS-treated groups exhibit almost complete response in terms of tumor inhibition at the lower dose and <10% overall tumor persistence at the higher dose.

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Histopathological analyses of DMBA-induced tumors extracted from rat mammary glands post treatment with either cisplatin, Pt II 5CLSS or Pt IV 5CLSS. Representative sections of mammary gland tissue taken at week 5 and stained with hematoxylin and eosin. All images are taken with 100× magnification.

2. Rat Mammary Tumor Response to Treatment with Either Cisplatin, PtIi5clss or PtIv5clss at the End of the Treatment Period.

  extensive tumour partial response (>50% tumour) partial response (10–50% tumour) partial response (<10% tumour) complete response (no tumour) fibroepithelial tumor
Control 100%          
Cisplatin 2.5 mg 50%         50%
Pt II 5ClSS 1 mg 25% 50%     25%  
Pt II 5ClSS 5 mg     25% 25% 50%  
Pt II 5ClSS 1 mg       25% 75%  
Pt IV 5ClSS 5 mg     25% 50% 25%  
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In Vivo Toxicity of PtIV5CLSS in Pigs

Body Weight Analysis

Pigs were treated intravenously once per week over an 8-week period with either D1 (1 mg/kg bw) or D2.5 (2.5 mg/kg.b.w) of Pt IV 5CLSS. Group D1 (Figure S7) exhibited a comparable body weight increase over the length of the injection period relative to the control group at both the end of the treatment period (week 8; p = 0.28) as well as during the wash-out period (until week 18; p = 0.89). The average body weight increase for the D1 group was 9.7 kg (21.8%) and 18 kg (40.6%) at weeks 8 and 18, respectively; this was comparable with that observed in the control group (week 8:11.6 kg, 24.7%; week 18:17.9, 38.2%). Group D2.5 showed a minor increase in body weight over the 8-week treatment period (week 8:5.1 kg, 11.3%) which was significantly less (p < 0.001) than that of the control. During the wash-out period (weeks 8–18), the D2.5 animals recovered with a higher body weight increase (14.7 kg; 34.9%) which resulted in a final body weight still significantly lower (p = 0.02) than that of the control.

Survival and Behaviour

Animals were monitored daily throughout the injection and wash-out period. None of the animals showed any behavioral or physical changes during the entire study period. In addition, a 100% survival rate was observed in both treated groups as well as in the control.

Biochemistry and Hematology

Following 8 weeks of weekly injections, a comprehensive result analysis of blood biochemistry (Table and Table S1) and hematology (Table and Table S1) was performed on the control, D1 and D2.5 groups. Albumin (25.2 ± 2.0 g/L) and Total Protein (5.1 ± 0.1 g/dL) levels were significantly reduced in the D2.5 group compared to control (32.9 ± 1.4 g/L and 5.9 ± 0.17 g/dL, respectively). Despite the statistical difference, both parameters fell within the normal range (Albumin: 22.3-45.3 g/L; Total Protein: 4.9-8.0 g/dL). Amylase activity was significantly decreased in both D1 (2253 ± 113 U/L) and D2.5 (2427 ± 78 U/L) groups relative to the control (2930 ± 82 U/L), yet both values were within the normal range (450-3594 U/L). Phosphate (phosphorus) levels increased in the D2.5 group (9.8 ± 0.5 mg/dL), yet was within the normal range (4.99–10.7 mg/dL). No significant changes were observed in liver enzymes (ALT, AST, GGT, ALP), total bilirubin, bile acids, or lipid profile (cholesterol and triglycerides), although ALT and AST were relatively higher in D2.5. No significant differences were seen in creatinine, BUN, BUN/Creatinine ratio, or uric acid (all <10 μmol/L).

3. Biochemistry Parameters Obtained from Blood Samples after 8 Weeks of Injections with PtIV5CLSS. * P < 0.05 with Respect to Control.
parameters control D1 D2.5
albumin g/L 32.9 ± 1.4 31.4 ± 1.6 25.2 ± 2.0*
total protein g/dL 5.9 ± 0.17 5.5 ± 0.1 5.1 ± 0.1*
globulin g/dL 2.6 ± 0.1 2.3 ± 0.2 2.6 ± 0.1
albumin/globulin ratio 1.3 ± 0.1 1.4 ± 0.2 1.0 ± 0.1
total bilirubin mg/dL 0.17 ± 0.03 0.18 ± 0.06 0.13 ± 0.02
gamma-glutamyl transferase U/L 29.7 ± 1.0 32.0 ± 2.6 30.8 ± 3.4
aspartate aminotransferase U/L 16.3 ± 4.1 19.8 ± 1.4 21.0 ± 2.3
alanine aminotransferase U/L 84.7 ± 5.0 77.0 ± 3.2 99.9 ± 9.3
alkaline phosphatase U/L 70.0 ± 1.4 59.3 ± 3.3 66.3 ± 5.0
total bile acids μmol/L 111.7 ± 6.8 91.9 ± 14.1 90.5 ± 12.7
amylase U/L 2930 ± 82 2253 ± 113* 2427 ± 78*
lipopolysaccharides U/L 13.0 ± 0.9 12.3 ± 0.2 12.0 ± 0.5
lactate dehydrogenase U/L 477 ± 20 440 ± 23 521 ± 6
creatine kinase U/L 449 ± 168 288 ± 51 243 ± 42
creatine mg/dL 1.56 ± 0.1 1.7 ± 0.2 1.4 ± 0.2
uric acid μmol/L <10 <10 <10
blood urea nitrogen mg/dL 15.1 ± 0.2 12.6 ± 1.1 15.8 ± 0.8
BUN/CREA 38.9 ± 3.5 32.1 ± 4.2 48.8 ± 6.2
glucose mg/dL 118 ± 3 109 ± 9 128 ± 10
total cholesterol mmol/L 3.9 ± 0.1 4.1 ± 0.1 3.5 ± 0.1
triglycerides mg/dL 50.4 ± 5.1 95 ± 14 61.6 ± 10.6
tCO2 mmol/L 20.0 ± 0.3 20.9 ± 1.3 17.5 ± 1.6
calcium mg/dL 9.1 ± 0.3 9.0 ± 0.1 9.0 ± 0.4
phosphorus mg/dL 7.3 ± 0.3 7.9 ± 0.2 9.8 ± 0.5*
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4. Hematology Parameters Obtained from Blood Samples after 8 Weeks of Injections with PtIV5CLSS. * P < 0.05 with Respect to Control.
parameters control D1 D2.5
hematocrit % 40.9 ± 2.0 37.9 ± 1.5 31.6 ± 5.0
hemoglobin g/dL 14.2 ± 0.7 13.6 ± 0.5 11.1 ± 1.8
mean corpuscular hemog. conc. g/dL 34.6 ± 0.1 35.8 ± 0.5 35.1 ± 0.2
white blood cell count 109/L 12.9 ± 0.9 16.5 ± 2.0 12.8 ± 1.5
cranulocytes, cat of WBC 109/L 8.2 ± 0.8 11.2 ± 1.4 7.3 ± 0.6
Grans percentage % 63.7 ± 2.1 67.3 ± 0.7 58.3 ± 2.4
neutrophil count 109/L 4.1 ± 0.9 5.6 ± 0.3 3.6 ± 0.1
eosinophil count 109/L 4.1 ± 0.5 2.80 ± 0.2* 4.8 ± 0.3
lymphocyte to monocyte 109/L 4.6 ± 0.2 5.4 ± 0.6 5.5 ± 1.0
L/M % 36.3 ± 2.1 32.8 ± 0.7 41.7 ± 2.4
platelet count 109/L 314 ± 36 317 ± 43 227 ± 29
retics % 1.2 ± 0.4 1.1 ± 0.1 1.1 ± 0.48
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Eosinophil count was significantly lower in D1 (2.80 ± 0.2 × 109/L) compared to the Control (4.1 ± 0.5 × 109/L) and D2.5 (4.8 ± 0.3 × 109/L). The Eosinophile count in all three groups fell higher than the kit’s expected range of (0-0.5 × 109/L). White blood cell, granulocytes, and platelet counts showed no statistically significant differences. Hematocrit and hemoglobin levels were lower in D2.5 but without statistical significance.

Discussion

Pt II 5CLSS and Pt IV 5CLSS were previously tested in our laboratory against A549 cells and rat mesenchymal cells (MCs). The promising findings (high cytotoxicity and selectivity) of these complexes against lung cancer cells led to testing Pt II 5CLSS and Pt IV 5CLSS against breast cancer both in vitro and in vivo. The complexes displayed IC50 values against MDA-MB-231 are in the sub-micromolar range for Pt II 5CLSS and Pt IV 5CLSS with a selectivity index (SI) of >232 and >100 against mesenchymal stem cells, respectively. The sub-micromolar IC50 values coupled with the very high SI values support their high antineoplastic potency against breast cancer cells.

The results of cellular uptake suggest a time-dependent mode of transport for all complexes with only Pt II 5CLSS and cisplatin showing a plateau at 24 h. Pt II 5CLSS and Pt IV 5CLSS showed a slower uptake compared to cisplatin up until 18 h. The increased rate of uptake of Pt II 5CLSS into MDA-MB-231 compared to Pt IV 5CLSS can be attributed to the structure size of the complexes with Pt IV 5CLSS boasting an additional two hydroxyl axial ligands. Compared to cisplatin, both platinum complexes exhibited higher uptake which rationalizes their significantly enhanced cytotoxicity. The observed high intracellular to extracellular concentrations of both complexes in MDA-MB-231 cells suggests that an active mode of transport is behind the intracellular accumulation of these complexes (Figure S1). The intracellular accumulation in MDA-MB-231 cells revealed that both complexes accumulated largely (51–64.5%) in the nucleus/cytoskeleton (NC) fraction unlike cisplatin which mainly accumulated in the mitochondria/organelle (MO) fraction (56%). Complexes similar to Pt II 5CLSS and Pt IV 5CLSS were previously observed to accumulate primarily in the cytoskeleton. Therefore, the present complexes may be also mainly localized in the cytoskeleton, however further studies are needed to confirm it. In the case of cisplatin, only ∼18% accumulated in the NC fraction; these results align with those reported in the literature (∼15–20%).

The toxicity study carried out on rats showed that Pt II 5CLSS and Pt IV 5CLSS had no effect on liver and kidney functions at all used doses as all the results fell within the normal range. Additionally, all experimental groups showed increased body weights throughout the study period, except for the group receiving Pt II 5CLSS at 30 mg/kg in which the mean rat body weight was constant throughout the study (with one reported death after the third dose). The rats assigned to this group were receiving singular doses (3 in total) of Pt II 5CLSS that are higher than the maximal tolerated dose (25.11 mg/kg.b.w.) extrapolated from the MTD on mice using the conversion factor published by Saadh et al. ,

Mammary tumors induced by DMBA have shown great similarity to human breast cancer. In the present study, Pt II 5CLSS and Pt IV 5CLSS exhibited potent chemotherapeutic activity, extending from significantly reducing tumor size to completely abolishing the tumors with Pt IV 5CLSS having a more pronounced antitumor activity. In our previous publication by Baz et al., we have reported the MTD (most tolerated dose) values of both Pt II 5CLSS and Pt IV 5CLSS on mice to be 50 and 75 mg/kg.bw, respectively. Using the conversion factor reported by Saadh et al. in 2020, the MTD values of both complexes in rats extrapolate to 25 and 38 mg/kg.bw. Metronomic chemotherapy employs doses that range from one-third to one-tenth of the MTD values. The dose of 5 mg/kg bw falls between the accepted range; and due to the high potency of the two complexes, we also investigated a much lower dose as our cytotoxicity results against MDA-MB-231 were sub-micromolar. Cisplatin, however, has an MTD value of approximately 6–8 mg/kg bw in rats; , and that renders the 2.5 mg/kg bw close to one-third, which is the maximum allowed value. This dose of cisplatin (administered once weekly in rats) is in line with clinically relevant human doses when scaled by body surface area. Using the standard FDA-recommended interspecies conversion factor for rats (K m = 6.2), this dose corresponds to approximately 15 mg/m 2 in humans per weekly administration. This exposure lies below the commonly used human cisplatin dose range of approximately 50-100 mg/m 2 /cycle, which is typically administered every 3-4 weeks. Therefore, the 2.5 mg/kg dose of cisplatin in rats represents a clinically reasonable dosing regimen. ,

At the 1 mg/kg bw dose of Pt IV 5CLSS, the tumor weight and volume were reduced to a minimum with 75% complete response (no tumor) and 25% partial response (<10% tumor) as per the histopathological analysis of the isolated tissue. Moreover, no drops in body weight were recorded for rats receiving each of Pt II 5CLSS and Pt IV 5CLSS throughout the treatment period. These treatment doses were significantly lower than the corresponding maximum tolerated doses of the complexes, further reinforcing the safety of each of Pt II 5CLSS and Pt IV 5CLSS. Nevertheless, the cisplatin group showed a slight nonsignificant decrease in body weight. As for the organ weights (heart, lung, spleen, liver, and kidney), no significant differences were observed in all groups. These findings additionally corroborate the selectivity of the present complexes for cancer cells.

The toxicity investigation on sus scrofa pigs was carried out using Pt IV 5CLSS since it has previously shown better tolerability by Balb/c mice as well as enhanced efficacy (at a lower dose, 1 mg/kg bw instead of 5 mg/kg bw) against chemically induced breast cancer in rats compared to Pt II 5CLSS. In the present study, two doses of 1 and 5 mg/kg.bw. were used to treat chemically induced breast cancer in rats. Using the conversion factor published by Saadh et al. (2020), the equivalent treatment doses in pigs amount to 0.18 and 0.89 mg/kg.bw. This renders the doses (1 and 2.5 mg/kg.b.w) used on pigs to be 1.12- and 2.8-folds higher than the highest dose (5 mg/g bw) used to treat breast tumour in rats. While only 4 weeks of treatment were conducted on rats, the pigs were subjected to either D1 or D2.5 over a period of 8 weeks followed by a 10-week monitoring period (wash out period). Notably, a 100% survival rate and no physical/behavioral changes were observed over the study period. The D1 group exhibited a similar body weight gain over the treatment period and the monitoring period compared to the control group. On the other hand, the D2.5 group demonstrated loss of appetite over the treatment period which resulted in only 11.3% increase in their average relative body weight compared to that of the control (24.7%). However, during the monitoring period, the D2.5 group recovered significantly and recorded a 34.9% increase in average relative body weight compared to a 38.2% increase in the control group. It is noteworthy to clarify that the animals received their treatment doses in a single IV injection at once rather than in a time release manner which is the norm in delivering chemotherapeutic agents. This may have proved challenging, especially on the D2.5 group, and could have resulted in the loss of appetite due to a high drug plasma concentration value.

The analysis of the biochemistry parameters on pigs injected with Pt IV 5CLSS suggest a dose-related physiological response with emerging signs of potential systemic toxicity at doses higher than 2.5 mg/kg bw or at prolonged treatment periods beyond 8 weeks at the D2.5 dose. The observed decrease in total protein and albumin, particularly in the D2.5 group, may point towards hepatic synthetic dysfunction or increased catabolic activity, despite the absence of significant elevation in liver enzyme markers. It is noteworthy to mention that despite the reduction in values, both D2.5 parameters for Albumin and Total protein fell within the normal range. These subtle shifts may reflect early-stage hepatic stress or altered protein turnover due to platinum exposure. The most consistent biochemical change was the significant reduction in Amylase levels in both treatment groups; notably however both Amylase values fell within the normal Amylase range (739–2882 U/L). Despite no significant differences in creatinine or urea values, a notable increase in phosphate (phosphorus) in the D2.5 group could signal subclinical renal effects or perturbed phosphate handling should higher doses be used; having said that the D2.5 phosphate parameters levels fell within the normal range. The results on hand could be considered a breakthrough since the both the D1 and even the D2.5 doses didn’t exhibit nephrotoxicity which is a common side-effect of other well know platinum agents such as cisplatin. Parameters related to red blood cells, showed no statistically significant reductions in hematocrit and hemoglobin levels in D2.5 which may hint at mild anemia or suppressed erythropoiesis with prolonged exposure or higher doses. White blood cell, platelet and reticulocytes parameters, showed no does dependent correlation with the dose of the platinum­(IV) complex as all the data fell within the normal range thus suggesting that Pt IV 5CLSS poses no risk concern to the immune system nor incurring a risk of bone marrow suppression at either dose.

In conclusion, Pt II 5CLSS and Pt IV 5CLSS were shown to exhibit potent antineoplastic activity against invasive metastatic MDA-MB-231 breast cancer cells. The cellular uptake of the two complexes was largely mediated through active transport with Pt II 5CLSS being transported at a faster rate than Pt IV 5CLSS, and both accumulated in the nuclear/cytoskeletal fraction. A toxicity study on rats revealed no signs of kidney or liver damage with doses as high as 30 mg/kg bw. Pt II 5CLSS and Pt IV 5CLSS exhibited potent chemotherapeutic activity against chemically induced breast cancer in rats, with Pt IV 5CLSS proving to be more potent and effective in the lower dose range. An in vivo toxicity investigation on Sus scrofa pigs presented Pt IV 5CLSS as a safe drug candidate with zero mortality rate and no adverse behavioral changes. Biochemistry and hematology analyses on pigs treated with Pt IV 5CLSS showed no acute toxicity at both doses. The present study, hence, provides evidence that both complexes (mainly Pt IV 5CLSS) could be considered effective and relatively safe anti-cancer adjuncts that could be used in breast cancer treatment. This is in light of the fact that TNBC is not responsive to targeted and immunotherapy treatments but instead requires chemotherapeutic approaches. Although the present study provides promising evidence regarding the anticancer activity of the newly synthesized platinum complexes, their potential anti-migratory and antimetastatic properties were not directly evaluated using functional assays. Future studies should therefore include scratch wound healing assays and migration/invasion assays to confirm whether these complexes can inhibit cancer cell migration and invasion. Such studies would provide stronger mechanistic and functional evidence supporting the possible anti-metastatic potential of these platinum complexes. Additionally, our work will also revolve around a large-scale toxicity study of Pt IV 5CLSS on GLP-certified pigs as a bridge experiment onto Phase I clinical trials.

Supplementary Material

ao5c11584_si_001.pdf (875.4KB, pdf)

Acknowledgments

We like to thank Elias Abi-Ramia and Dr. Rachel Jelwan for assisting with animal care and handling at the facilities of the Lebanese American University. We also acknowledge the financial support from the President Intramural Research Fund at the Lebanese American University (PIRF I0054).

Glossary

Abbreviations

SI

selectivity index

H

hours/hour

PtII5CLSS

[(5-chloro-1, 10-phenanthroline)­(1S,2S-diaminocyclohexane)­platinum­(II)]­dichloride

PtIV5CLSS

di-hydroxido derivative [(5-chloro-1, 10-phenanthroline)­(1S,2S-diaminocyclohexane)­(di-hydroxido)­platinum­(IV)]­dinitrate

ICPMS

inductively coupled plasma mass spectrometer

IC50

half-maximal inhibitory concentration

GLP

Good Laboratory Practice

TNBC

triple negative breast cancer

PIRF

President Intramural Research Fund

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c11584.

  • Intracellular-to-extracellular platinum concentration ratios; rat body-weight, serum biochemistry, organ-to-body-weight, and mammary tumor data; pig body-weight, hematological and biochemical data, and detailed protocol and monitoring schedule for the porcine toxicity study (PDF)

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

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Supplementary Materials

ao5c11584_si_001.pdf (875.4KB, pdf)

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