Free THP Research Articles

Abstract 679: TXB-001 is an upgraded conjugated anthracycline that targets tumors with reduced risk of cardiotoxicity, hand-foot syndrome and alopecia

Abstract TXB-001 is a well-optimized N-(2-hydroxypropyl)methacrylamide copolymer-based conjugated pirarubicin (THP) and a novel candidate for developing anticancer drugs. It exhibits tumor-specific accumulation due to the enhanced permeability and retention effects and prolonged circulation time compared with free THP. Its hydrazone bond is dissociated under lower pH conditions, providing selective and rapid release of THP in acidic tumor environments. Therefore, TXB-001 is expected to have high efficacy and low toxicity. Anthracycline anticancer drugs have been widely used for the treatment of several cancers, but their use is limited by several adverse effects (AEs), including cardiotoxicity, which is a life-threatening AE. Furthermore, the maximum lifetime cumulative dose is decided for each anthracycline in clinical use, which leads to discontinuation of the medication. Hand-foot syndrome (HFS) is a dose-limiting AE of DOXIL®, a liposomal formulation of doxorubicin (DOX), and although it is not a life-threatening condition, it significantly deteriorates quality of life (QOL). Alopecia is another common AE, which is relatively noninvasive but adversely affects mental health and reduces QOL. In this study, we confirmed the efficacy of TXB-001 in a mice triple-negative breast cancer (TNBC) model and evaluated the cardiotoxicity-, HFS- and alopecia-inducing effects of TXB-001 in mice and rats to compare the severity with existing anthracycline anticancer drugs, including DOXIL®. The pharmacokinetic (PK) analysis in several tissues, including plasma, tumor, heart, skin of the chest, and palmar and plantar areas, was also performed after a single intravenous administration in rodents. As a result, TXB-001 showed an equivalent or greater antitumor effects than other anthracyclines in the TNBC model, and the AEs were scarcely observed. TXB-001 did not decrease left ventricular ejection fraction, with no or little HFS-like changes compared with DOXIL®, which showed significant histopathological changes. TXB-001 also showed weaker alopecia-inducing effects than DOX, DOXIL®, and THP. In the PK analysis, TXB-001 was distributed in the tumor at a higher concentration than in other tissues, except for plasma. Hence, most of the THP, the active ingredient, was released in the tumor. The exposure of the heart in the TXB-001-treated group was one-tenth of that in the DOXIL®-treated group. Moreover, accumulation in the skin, especially in palmar and plantar areas, was observed for DOXIL®, but no accumulation in the skin was observed for TXB-001. This PK profile consistently demonstrated the antitumor activity of TXB-001, without AEs. TXB-001 reduces the chance of dose-limiting and QOL-threatening AEs, and thus, cancer patients can continue treatment, making it a more convenient anticancer drug in clinical use with a wide safety margin. Citation Format: Keiyu Oshida, Mikito Hirakata, Emi Tomikawa, Chizuka Sakai, Masashi Uchida, Rieko Shimozono, Akiko Izawa, Yoko Koga, Tsubasa Okano, Hideki Narumi, Lisa Munakata, Ryo Suzuki, Miki Nonaka, Yasuhito Uezono. TXB-001 is an upgraded conjugated anthracycline that targets tumors with reduced risk of cardiotoxicity, hand-foot syndrome and alopecia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 679.

Preparation and in Vitro Characterization of Fatty-Acid Modified Pirarubicin Nanosuspensions Stabilized by Albumin.

Pirarubicin (THP) shows more rapid intracellular uptake, more effective antitumor activity, and less cardiac toxicity, compared to doxorubicin. However, THP is distributed to both tumor and normal tissues indiscriminately. This study aimed to develop a nanosuspension to deliver THP to tumor tissues more efficiently. Fatty-acid-modified THPs (FA-THPs; octanoic acid, dodecanoic acid, palmitic acid-THPs) were synthesized to increase the hydrophobicity of THP. Nanosuspensions of these FA-THPs were then prepared using an antisolvent precipitation technique. Among the FA-THPs, the most efficiently drug-loaded nanosuspension was obtained from palmitic acid-THP (pal-THP) using an aqueous antisolvent containing bovine serum albumin as a stabilizer. The pal-THP nanoparticles in the nanosuspension were confirmed to be of optimal size (100-125 nm) for delivery to tumor tissues using dynamic light scattering and transmission electron microscopy. The pal-THP nanosuspension showed cytotoxicity in colon 26 cells. The nanosuspension was shown to disintegrate in the presence of surfactants such as lecithin, liberating pal-THP, which was converted to free THP in acidic media. It is therefore proposed that pal-THP nanoparticles that reach tumor cells after intravenous administration would exert antitumor effect by liberating pal-THP (i.e., disintegration of nanoparticles by the interaction with cell membrane), followed by the release of free THP in the acidic milieu of tumor cells. These findings indicate that FA-THP nanosuspensions, particularly pal-THP nanosuspension, hold promise as a candidate for cancer treatment. However, further in vivo studies are necessary.

Improvement of the bladder perfusion curative effect through tight junction protein degradation induced by magnetic temperature-sensitive hydrogels.

Postoperative intravesical instillation of chemotherapy is a routine procedure for non-muscular invasive bladder cancer (NMIBC). However, traditional bladder perfusion methods have insufficient exposure time, resulting in unsatisfactory therapeutic effects. In the present study, a chitosan (CS)-based in situ forming depot (ISFD) delivery system, including Fe3O4 magnetic nanoparticles (Fe3O4-MNP), CS, and β-glycerophosphate (GP) as main components, was synthesized. Pirarubicin (THP), as a chemotherapeutic drug, was loaded into the new system. Results showed that our carrier system (Fe3O4-THP-CS/GP) was converted into gel and attached to the bladder wall, possessing loose network structures with magnetic targeting and sustained release properties. Moreover, its retention time in bladder was more than 72 h accompanied by a suitable expansion rate and good degradation characteristics. The antitumor activities of Fe3O4-THP-CS/GP were more effective both in vitro and in vivo than the free THP solution. In the study of its mechanism, results showed that Fe3O4-THP-CS/GP suppressed the expression of occludin (OCLN) and affected tight junctions (TJ) between urothelial cells to promote THP absorption.

Effect of Tumor Targeted-Anthracycline Nanomedicine, HPMA Copolymer-Conjugated Pirarubicin (P-THP) against Gynecological Malignancies

Anthracyclines are important for the treatment of gynecological malignancies, but their effects are modest, and one of the major reasons is the lack of a tumor-targeting property. To overcome this drawback, a poly (hydroxypropyl meta-acrylamide) conjugated with tetrahydropyraryl doxorubicin (P-THP) has been developed, which exhibits a highly tumor-specific accumulation owing to the enhanced permeability and retention effect. The effect of P-THP has been confirmed by using various cell lines and solid tumor models, while its effect on gynecological malignancies have not been investigated. In this regard, human uterine sarcoma cell line with metastatic potential MEA-SA C9 high, epithelial ovarian cancer cell line A2780 and its cisplatin-resistant line A2780cis, and DOX-resistant line A2780ADR were used in this study, and the therapeutic effect as well as the safety profiles of P-THP were investigated compared to native THP, cisplatin, and paclitaxel, which are commonly used for gynecological malignancies, both in vitro and in vivo. Similar to native THP, a dose-dependent toxicity of P-THP was identified in all cell lines. Moreover, the IC50 values in the 3 h following P-THP were 1.5–10 times higher than those at 72 h, though the intracellular uptake of P-THP in all cells were 2–10-fold less than THP. In vivo studies using xenograft tumor models revealed that P-THP significantly suppressed the MES-SA C9 high, A2780, and A2780cis tumor growth at the dose of 15 mg/kg (THP equivalent), which is three times above the maximal tolerance dose of native THP, while no body weight loss or acute death occurred. However, in A2780ADR cells and the xenograft model, no significant difference in the therapeutic effect was observed between THP and P-THP, suggesting that P-THP exhibits its effect depending on the release of the active free THP in tumor tissues, and thus the internalization into tumor cells. These findings indicates that P-THP has the potential as a therapeutic for gynecological malignancies to improve the therapeutic outcomes and survival rates of patients, even in refractory patients.

Synthesis and In Vitro Assessment of pH-Sensitive Human Serum Albumin Conjugates of Pirarubicin

In a previous study, we reported on the development of a synthetic polymer conjugate of pirarubicin (THP) that was formed via an acid-labile hydrazone bond between the polymer and the THP. However, the synthetic polymer itself was non-biodegradable, which could lead to unexpected adverse effects. Human serum albumin (HSA), which has a high biocompatibility and good biodegradability, is also a potent carrier for delivering antitumor drugs. The objective of this study was to develop pH-sensitive HSA conjugates of THP (HSA-THP), and investigate the release of THP and the cytotoxicity under acidic conditions in vitro for further clinical development. HSA-THP was synthesized by conjugating maleimide hydrazone derivatives of THP with poly-thiolated HSA using 2-iminothiolane, via a thiol-maleimide coupling reaction. We synthesized two types of HSA-THP that contained different amounts of THP (HSA-THP2 and HSA-THP4). Free THP was released from both of the HSA conjugates more rapidly at an acidic pH, and the rates of release for HSA-THP2 and HSA-THP4 were similar. Moreover, both HSA-THPs exhibited a higher cytotoxicity at acidic pH than at neutral pH, which is consistent with the effective liberation of free THP under acidic conditions. These findings suggest that these types of HSA-THPs are promising candidates for further development.

Structure-to-Efficacy Relationship of HPMA-Based Nanomedicines: The Tumor Spheroid Penetration Study.

Nanomedicines are a novel class of therapeutics that benefit from the nano dimensions of the drug carrier. These nanosystems are highly advantageous mainly within cancer treatment due to their enhanced tumor accumulation. Monolayer tumor cells frequently used in routine preclinical assessment of nanotherapeutics do not have a spatial structural architecture that allows the investigation of the penetration of nanomedicines to predict their behavior in real tumor tissue. Therefore, tumor spheroids from colon carcinoma C26 cells and glioblastoma U87-MG cells were used as 3D in vitro models to analyze the effect of the inner structure, hydrodynamic size, dispersity, and biodegradability of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-based nanomedicines carrying anticancer drug pirarubicin (THP) on the penetration within spheroids. While almost identical penetration through spheroids of linear and star-like copolymers and also their conjugates with THP was observed, THP penetration after nanomedicines application was considerably deeper than for the free THP, thus proving the benefit of polymer carriers. The cytotoxicity of THP-polymer nanomedicines against tumor cell spheroids was almost identical as for the free THP, whereas the 2D cell cytotoxicity of these nanomedicines is usually lower. The nanomedicines thus proved the enhanced efficacy within the more realistic 3D tumor cell spheroid system.

Superior Penetration and Cytotoxicity of HPMA Copolymer Conjugates of Pirarubicin in Tumor Cell Spheroid.

N-(2-Hydroxypropyl)methacrylamide copolymer conjugates of pirarubicin (THP), P-THP, accumulates selectively in solid tumor tissue by the enhanced permeability and retention (EPR) effect. Despite of high accumulation in solid tumors, some macromolecular antitumor agents show poor therapeutic outcome because of poor tissue diffusion into the tumor as well as obstructed tumor blood flow. Here, we confirmed that cellular uptake of P-THP was 25 times less than that of free THP at 1-4 h incubation time in vitro. The passage of P-THP through the confluent tight-monolayer cells junction was 12 times higher than free THP, and P-THP penetrated deeper into the tumor cell spheroid (1.3-1.7-fold) than free THP in 4 h. In addition, P-THP showed cytotoxicity comparable to that of free THP to tumor-cells in spheroid form, despite of 7 times lower cytotoxicity of P-THP to the monolayer cells to that of free THP in vitro. These results indicate that P-THP administration can exhibit deeper diffusion into the tumor cell spheroid than free THP. As a consequence, P-THP exhibits more efficient antitumor activity than free THP in vivo, which is also supported by better pharmacokinetics and tumor accumulation of P-THP than free THP.

HPMA copolymer conjugate with pirarubicin: In vitro and ex vivo stability and drug release study

We have developed a tumor environment-responsive polymeric anticancer prodrug containing pirarubicin (THP) conjugated to N-(2-hydroxypropyl) methacrylamide copolymer (PHPMA), [P-THP], through a spacer containing pH-sensitive hydrazone bond, that showed remarkable therapeutic effect against various tumor models and in a human pilot study. Toward clinical development, here we report THP release profile from its HPMA copolymer conjugate, the conjugate stability, protein and cell-binding and solubility of P-THP. Size exclusion chromatography of P-THP (molecular weight 38 kDa) showed similar hydrodynamic volume as bovine serum albumin (BSA) in aqueous solution, with no apparent interactions with BSA, nor aggregation by itself. pH-responsive release of free THP was reconfirmed at pHs 6.5 and lower. The drug release was significantly affected by a type of used buffer. Phosphate buffer seems to facilitate faster hydrazone bond cleavage at pH 7.4 whereas higher stability was achieved in L-arginine solution which yielded only little cleavage and THP release, approx. 15% within 2 weeks at the same pH at 25 °C. Furthermore, ex vivo study using sera of different animal species showed very high stability of P-THP. Incubation with blood showed high stability of P-THP during circulation, without binding to blood cells. These findings revealed that L-arginine solution provides appropriate media for formulation of P-THP infusion solution as tumor-targeted polymeric anticancer drug based on EPR effect.

One-Step Self-Assembling Nanomicelles for Pirarubicin Delivery To Overcome Multidrug Resistance in Breast Cancer.

Tumor cells can acquire multidrug resistance (MDR) as a result of drug efflux mediated by P-glycoprotein (P-gp). Here we report a targeted delivery system to carry pirarubicin (THP) to MDR breast cancer both in vitro and in vivo. PEG-derivatized vitamin E (PAMV6) amphiphiles loaded with THP were self-assembled in a single step. The PAMV6 micelles showed unimodal size distribution and high drug loading efficiency. Cytotoxicity of PAMV6/THP was higher than that of free THP on MCF-7/ADR cells but comparable to that of THP on MCF-7 cells. PAMV6/THP was able to reverse MDR more than free THP in MCF-7/ADR cells, likely reflecting the remarkably higher intracellular THP concentration in micelle-treated cells and PAMV6-mediated inhibition of P-gp activity. PAMV6/THP micelles were internalized into MCF-7/ADR cells via macropinocytosis and caveolin-mediated endocytosis, further avoiding P-gp-mediated efflux. Mechanistic studies revealed that blank PAMV6 micelles inhibited P-gp activity but did not affect P-gp expression, by significantly reducing mitochondrial membrane potential and slightly decreasing intracellular ATP levels. In a nude mouse xenograft model, PAMV6/THP led to much greater THP accumulation in tumors and much slower tumor growth than free THP. At the same time, PAMV6/THP was associated with significantly less severe bone marrow suppression and organ toxicity than free THP. Our results indicate that this PAMV6-based micelle system holds promise for combating MDR in cancer therapy.

RETRACTED: pH-Responsive Polymer Conjugate of Pirarubicin With Styrene Maleic Acid Copolymer as a Potential Therapeutic for Ovarian Cancer

Previous studies indicated the potential of styrene maleic acid copolymer (SMA)-conjugated pirarubicin (4'-O-tetrahydropyranyldoxorubicin [THP]) for targeted anticancer therapy based on the enhanced permeability and retention effect. In this study, to achieve further improved therapeutic efficacy, a pH-responsive SMA-conjugated THP-containing hydrazone bond (SMA-hyd-THP) was synthesized and evaluated invitro and exvivo using human ovarian cancer cells and tissues. SMA-hyd-THP showed good water solubility, forming micelles with a mean particle size of 48.0 nm, which is applicable for enhanced permeability and retention-based tumor accumulation. The THP loading in this preparation was 15% (wt/wt), and release rate of free THP from SMA-hyd-THP at physiological pH (7.4) was approximately 10% in 72h. However, it increased rapidly at pH 6.5 (42%) and 5.5 (83%), which indicates that tumor environment of weak acidic condition (pH 6.5-6.9) is favorable for release of THP. This notion was partly proved by incubating SMA-hyd-THP with tumor tissues from ovarian cancer patients. In addition, release of THP was not affected by serum, suggesting that SMA-hyd-THP is relatively stable in circulation. Finally, SMA-hyd-THP showed much increased cytotoxicity against various ovarian cancer cells at acidic tumor pH (6.5). These findings may provide an option for targeted therapy against ovarian cancer.

Synthesis and therapeutic effect of styrene–maleic acid copolymer‐conjugated pirarubicin

Previously, we prepared a pirarubicin (THP)-encapsulated micellar drug using styrene–maleic acid copolymer (SMA) as the drug carrier, in which active THP was non-covalently encapsulated. We have now developed covalently conjugated SMA-THP (SMA-THP conjugate) for further investigation toward clinical development, because covalently linked polymer–drug conjugates are known to be more stable in circulation than drug-encapsulated micelles. The SMA-THP conjugate also formed micelles and showed albumin binding capacity in aqueous solution, which suggested that this conjugate behaved as a macromolecule during blood circulation. Consequently, SMA-THP conjugate showed significantly prolonged circulation time compared to free THP and high tumor-targeting efficiency by the enhanced permeability and retention (EPR) effect. As a result, remarkable antitumor effect was achieved against two types of tumors in mice without apparent adverse effects. Significantly, metastatic lung tumor also showed the EPR effect, and this conjugate reduced metastatic tumor in the lung almost completely at 30 mg/kg once i.v. (less than one-fifth of the maximum tolerable dose). Although SMA-THP conjugate per se has little cytotoxicity in vitro (1/100 of free drug THP), tumor-targeted accumulation by the EPR effect ensures sufficient drug concentrations in tumor to produce an antitumor effect, whereas toxicity to normal tissues is much less. These findings suggest the potential of SMA-THP conjugate as a highly favorable candidate for anticancer nanomedicine with good stability and tumor-targeting properties in vivo.

Comparison between linear and star-like HPMA conjugated pirarubicin (THP) in pharmacokinetics and antitumor activity in tumor bearing mice

Previously we showed that linear poly(N-(2-hydroxypropyl)methacrylamide) conjugates of pirarubicin (THP), LP-THP, with MW about 39kDa, exhibited far better tumor accumulation and therapeutic effect than that of parental free THP. To improve the pharmacokinetics of LP-THP further, high-MW conjugate of poly(amido amine) (PAMAM) dendrimer grafted with semitelechelic HPMA copolymer (PHPMA) was synthesized [star polymer (SP); 400kDa] and conjugated with THP via hydrazone bond-containing spacer (SP-THP). THP was conjugated to SP to form SP-THP via acid cleavable hydrazone bonding, which responds to acidic milieu of tumor tissue. As a consequence, it would release free THP, by active therapeutic principle. SP-THP exhibits larger hydrodynamic diameter (25.9nm) in aqueous solution than that of LP-THP (8.2nm) as observed by light scattering and size exclusion chromatography. Because of the larger size, the tumor AUC5h–72h of SP-THP was 3.3 times higher than that of LP-THP. More importantly, released free THP was retained selectively in the tumor tissue for at least up to 72h after administration of SP-THP. We found that SP-THP exhibited superior antitumor effect to LP-THP against both S-180 tumor-bearing mice in vivo, and with chemically AOM/DSS-induced colon tumor-bearing mice, most probably due to their different molecular size. In our comparison study of in vitro and in vivo behavior of SP-THP and LP-THP we concluded that SP-THP exhibited enhanced therapeutic efficacy not only in implanted tumor but also in orthotopic/spontaneous tumor despite its higher toxicity compared to LP-THP. Upon these findings further investigation using various tumors including transgenic, and metastatic tumors is going to be conducted soon.

Two step mechanisms of tumor selective delivery of N-(2-hydroxypropyl)methacrylamide copolymer conjugated with pirarubicin via an acid-cleavable linkage

N-(2-Hydroxypropyl)methacrylamide copolymer containing hydrazide groups (PHPMA) conjugated with pirarubicin (THP) via a hydrazone bond (PHPMA-hyd-THP) is a drug conjugate that releases THP in the acidic milieu of a tumor. PHPMA-hyd-THP has an apparent Mw of 40,000 and a hydrodynamic diameter of 8.2±1.7nm but no apparent plasma protein binding. PHPMA-hyd-THP possesses two mechanisms of selectivity toward solid tumors and has potent antitumor action. The first one is drug accumulation in tumors that depends on the enhanced permeability and retention (EPR) effect, which results in a 4–20 times higher concentration of drug in the tumor than in normal tissues such as the heart, lung, and intestine. This accumulation in tumor tissue is in great contrast to that of conventional low-Mw THP. The second one is pH-dependent release of drug from PHPMA-hyd-THP: this conjugate released free THP more efficiently at a lower pH, which exists in tumors, and exerts cytotoxic activity. Free THP is known for its much faster uptake into tumor cells compared with doxorubicin. Thus, in our in vitro study, PHPMA-hyd-THP showed a higher cytotoxicity at the lower pH of tumor tissue than at the neutral pH of normal tissue. Furthermore, much more THP was liberated from the conjugate in acidic tumor tissue than in normal tissue. The EPR effect-dependent accumulation of PHPMA-hyd-THP and tumor-selective THP release in the tumor tissues led to highly tumor-selective drug accumulation, which continued for more than 72h, whereas the lowest free drug concentration was detected in normal tissues at 24h and no longer at a later time. In conclusion, we determined in our study here that the acid-cleavable PHPMA-hyd-THP conjugate had an excellent antitumor effect without appreciable adverse effects.

Influence of coupling bonds on the anti-tumor activity of polymer–pirarubicin conjugates

Pirarubicin (THP) was conjugated onto the pendant carboxyl groups of poly(ethylene glycol)-block-poly(l-lactide-co-2-methyl-2-carboxyl-propylene carbonate) [PEG-b-P(LA-co-MCC)] through hydrazone, ester, and amide bonds, respectively, and the conjugates were assembled into micelles with diameters between 30 and 60nm. The in vitro THP release of the three conjugate micelles was conducted in pH 7.4 and 5.0 buffer solutions, and conjugate micelles with hydrazone linkage had the fastest THP release rate. Their in vitro cytotoxicity was tested using mouse mammary adenocarcinoma EMT6 cells and in vivo anti-tumor activity in Balb/c mice models bearing EMT6 tumors were compared with free THP and with each other. The results showed that the polymer–THP conjugates displayed higher cell-uptakes and better anti-tumor activities than free THP at 4h, and among the three micelles, those with hydrazone linkage had the highest anti-tumor activity in vivo, while those with amide linkage were the lowest.

Metabonomic study on the cumulative cardiotoxicity of a pirarubicin liposome powder

Pirarubicin (THP) is an anthracycline frequently used in the chemotherapy against acute leukemia, malignant lymphoma and several solid tumors. However, its clinical use is severely limited by the development of a progressive dose-dependent cardiomyopathy that results in irreversible congestive heart failure. To provide a strategy for constraining or minimizing the cumulative cardiotoxicity of THP, a pirarubicin liposome powder (L-THP) was appropriately prepared, and the cumulative cardiotoxicity of L-THP and free THP (F-THP) were investigated on Sprague-Dawley rats after 3 successive doses. Urinary samples for metabonomic study, serum samples for biochemical assay, and heart samples for histopathology test were collected. As a result, the metabonomics-based findings such as PLS-DA plotting showed minimal metabolic alterations in L-THP as compared to F-THP, and correlated with the changes of serum biochemical assay and cardiac histopathology as measurements of damage to heart tissue. Our results confirm that when encapsulated into liposomes, the cumulative cardiotoxicity of THP can be greatly ameliorated. Lipophilic aglycone metabolites of THP associated with redox cycling are cardiotoxic for the possibility of reactive oxygen species (ROS) formation. Also, metabonomic analysis shows that the successive doses of THP will lead to severe metabolic pathways disturbances in the cell energy production. Further, the preliminary efficacy study of L-THP on lung cancer was evaluated in the approach of in vitro cytotoxicity on A549 cells by high content screening (HCS) analysis, and L-THP was found to exhibit better therapeutic index against lung cancer than THP.

Copoly(styrene-maleic acid)−Pirarubicin Micelles: High Tumor-Targeting Efficiency with Little Toxicity1

The copolymer of styrene-maleic acid (SMA) was used to construct micelles containing pirarubicin (4'-O-tetrahydropyranyladriamycin, or THP) as a new anticancer drug formulation. The procedure for the preparation of the micelles was simple, the component consisting of only SMA and pirarubicin in a noncovalent association, possibly by hydrophobic interaction between the styrene portion of SMA and pirarubicin chromophore. This method ensures more than 80% recovery of pirarubicin by weight, and 60% of drug loading (by weight) was achieved. The micelles obtained (SMA-THP) showed high solubility in water and a constant pirarubicin release rate of about 3-4%/day in vitro. SMA-THP micelles had an average molecular size of about 34 kDa according to gel chromatography; this size is a marked increase from the 627.6 Da of free THP, which suggests the formation of a micellar structure. When albumin was added, the molecular size of the micelles increased to about 94 kDa, which indicates binding to albumin, a unique characteristic of SMA. SMA-THP micelle preparation had a cytotoxic effect (93-101%) on MCF-7 breast cancer cells and SW480 human colon cancer cells in vitro that was comparable to that of free THP. An in vivo assay of SMA-THP at doses of 20 mg/kg in ddY mice bearing S-180 tumor revealed complete tumor eradication in 100% of tested animals. Mice survived for more than 1 year after treatment with micellar drug doses as high as 100 mg/kg pirarubicin equivalent. This marked antitumor activity can be attributed to the enhanced permeability and retention (EPR) effect of macromolecular drugs seen in solid tumors, which enables selective delivery of drugs to tumor and thus much fewer side effects. Complete blood counts, liver function test, and cardiac histology showed no sign of adverse effects for intravenous doses of the micellar preparation. These data thus suggest that intravenous administration of the SMA-THP micellar formulation can enhance the therapeutic effect of pirarubicin more than 50-fold.

Preparation and pharmacokinetics of pirarubicin loaded dehydration–rehydration vesicles

Liposomes entrapped pirarubicin (THP, L-THP) were prepared by the modified dehydration–rehydration vesicle (DRV) method, and their pharmacokinetics and antitumor effects were evaluated in mice bearing M5076 liver metastasis tumor. After small unilamellar vesicles (SUVs) composed of egg lecithin, cholesterol (Ch), β-sitosterol β- d-glucoside (Sit-G) and oleic acid (OA) were freeze-dried with THP and sugars, rehydration of the lyophilized powders led to form the larger vesicles entrapping drugs, but the proper amounts of sugars and OA to lipids (sucrose/lipid=8 (w/w)) maintained the small particle size (about 340 nm) with high entrapment (80.7%) of THP. After intravenous injection of L-THP, the accumulations of THP in the liver and heart were approximately 4-fold higher and half lower, respectively, than those of free THP (F-THP). L-THP had superior antitumor effect in 10 mg/kg intravenous administration without significant body weight loss. L-THP is a potential drug dosage form of liver cancer treatment since the liposomes carry THP to the liver.