pH-sensitive Linkage Research Articles

Lysosome-oriented, dual-stage pH-responsive polymeric micelles for β-Lapachone delivery.

β-Lapachone (β-lap), a novel anticancer agent, is bioactivated by NADP(H):quinone oxidoreductase 1 (NQO1), an enzyme over-expressed in numerous tumors, including lung, pancreas, breast, and prostate cancers. Fast renal clearance and methemaglobinemia / hemolytic side-effects from the clinical formulation (β-lap-hydroxyl propyl-β-cyclodextrin complex) hindered its clinical translation. Here, we investigated a dual model pH responsive polymers for β-lap delivery. Three pH-sensitive linkages, including acylhydrazone, ketal and imine bonds for β-lap prodrug syntheses result in an aryl imine linkage the most optimal linkage. The conversion to β-lap was 2.8%, 4.5% and 100% at pH 7.4, 6.5 and 5.0 in 8 h, respectively. β-lap aryl imine prodrug conjugated ultra pH-sensitive (UPS) polymer reached high β-lap loading density (8.3%) and exhibited dual-stages responsiveness to pH variation. In pHs under pHt, at stage I, micelle immediately dissociation and subsequently entering stage II, micelles start quickly release β-lap. In vitro release study showed that the micelles constantly release β-lap (14.9 ± 0.1%) at pHs above pHt in 72 h, whereas boosted release of β-lap (79.4 ± 1.2%) at pH 5.0. Micelle intracellular distribution predominantly in the lysosome organelle guaranteed their pH responsive dissociation and subsequently β-lap controlled release. The M-P micelles retained NQO1-dependent cytotoxicity in A549 lung cancer cells, similar to free drug in both efficacy and mechanism of cell death. The lysosome-oriented dual-stage ultra pH responsive β-lap prodrug micelles potentially offer an alternative nanotherapeutic strategy for lung, as well as other NQO1+ cancer therapies.

"Stealthy" chitosan/mesoporous silica nanoparticle based complex system for tumor-triggered intracellular drug release.

Suitable protection strategies utilized in anticancer drug delivery systems enable carriers to reach their targeted positions and release drugs intracellularly more effectively. In this study, a novel "stealthy" chitosan (CHI)/mesoporous silica nanoparticle (MSN) based complex system, named DOX@MSN-SS-CHI-PEG, was developed for tumor-triggered intracellular drug release. CHI was applied to block the pores of MSNs to prevent premature drug release, whereas mPEG was grafted on the surface of the nanoparticles via a pH-sensitive benzoic imine linker to protect the carriers. As the pH of solid tumor tissues is slightly lower than that of normal tissues, mPEG could leave the nanoparticles to expose positively charged CHI at the surface, which enabled the nanoparticles to enter cancer cells more easily. The MSNs were covered by CHI via redox-sensitive disulfide bonds. As a result, the carriers could release the drug intercellularly to kill cancer cells owing to the high concentration of glutathione (GSH) in the cytosol. In vitro drug release studies at different GSH concentrations proved the redox-sensitivity of DOX@MSN-SS-CHI-PEG. mPEG leaving studies demonstrated that mPEG could leave the nanoparticles effectively at pH 6.0. The cytotoxicity and cell internalization behavior were also investigated in detail. In conclusion, the novel DOX@MSN-SS-CHI-PEG drug delivery system, which was "stealthy" in the physiological environment at pH 7.4 because of the protection of mPEG, was "activated" in weakly acidic tumor tissues to achieve tumor-triggered intracellular drug release; this system has great potential for cancer therapy.

From solution to in-cell study of the chemical reactivity of acid sensitive functional groups: a rational approach towards improved cleavable linkers for biospecific endosomal release.

pH-Sensitive linkers designed to undergo selective hydrolysis at acidic pH compared to physiological pH can be used for the selective release of therapeutics at their site of action. In this paper, the hydrolytic cleavage of a wide variety of molecular structures that have been reported for their use in pH-sensitive delivery systems was examined. A wide variety of hydrolytic stability profiles were found among the panel of tested chemical functionalities. Even within a structural family, a slight modification of the substitution pattern has an unsuspected outcome on the hydrolysis stability. This work led us to establish a first classification of these groups based on their reactivities at pH 5.5 and their relative hydrolysis at pH 5.5 vs. pH 7.4. From this classification, four representative chemical functions were selected and studied in-vitro. The results revealed that only the most reactive functions underwent significant lysosomal cleavage, according to flow cytometry measurements. These last results question the acid-based mechanism of action of known drug release systems and advocate for the importance of an in-depth structure-reactivity study, using a tailored methodology, for the rational design and development of bio-responsive linkers.

PH-Reversible Cationic RNase A Conjugates for Enhanced Cellular Delivery and Tumor Cell Killing.

Intracellularly-acting therapeutic proteins are considered promising alternatives for the treatment of various diseases. Major limitations of their application are low efficiency of intracellular delivery and possible reduction of protein activity during derivatization. Herein, we report pH-sensitive covalent modification of proteins with a histidine-rich cationic oligomer (689) for efficient intracellular transduction and traceless release of functional proteins. Enhanced Green fluorescent protein (EGFP), as model for the visualization of protein transduction, and RNase A, as therapeutic protein with antitumoral effect, were modified with the pH-sensitive bifunctional AzMMMan linker and varying amounts of cationic oligomer. The modification degree showed impact on the internalization and cellular distribution of EGFP as well as the biological effect of RNase A conjugates, which mediated considerable toxicity against cancer cells at optimal ratio. The presented conjugates demonstrate their qualification to achieve efficient intracellular delivery and controlled release without protein inactivation and potential prospective applications in protein-based therapies.

Tumor-targeted and pH-controlled delivery of doxorubicin using gold nanorods for lung cancer therapy.

BackgroundIn lung cancer, the efficacy of conventional chemotherapy is limited due to poor drug accumulation in tumors and nonspecific cytotoxicity. Resolving these issues will increase therapeutic efficacy.MethodsGNR-Dox-Tf-NPs (gold nanorod-doxorubicin-transferrin-nanoparticles) were prepared by different chemical approaches. The efficacy of these nanoparticles was carried out by cell viability in lung cancer and primary coronary artery smooth muscle cells. The receptor-mediated endocytosis studies were done with human transferrin and desferrioxamine preincubation. The GNR-Dox-Tf nanoparticles induced apoptosis, and DNA damage studies were done by Western blot, H2AX foci, and comet assay.ResultsWe developed and tested a gold nanorod-based multifunctional nanoparticle system (GNR-Dox-Tf-NP) that carries Dox conjugated to a pH-sensitive linker and is targeted to the transferrin receptor overexpressed in human lung cancer (A549, HCC827) cells. GNR-Dox-Tf-NP underwent physicochemical characterization, specificity assays, tumor uptake studies, and hyperspectral imaging. Biological studies demonstrated that transferrin receptor-mediated uptake of the GNR-Dox-Tf-NP by A549 and HCC827 cells produced increased DNA damage, apoptosis, and cell killing compared with nontargeted GNR-Dox-NP. GNR-Dox-Tf-NP-mediated cytotoxicity was greater (48% A549, 46% HCC827) than GNR-Dox-NP-mediated cytotoxicity (36% A549, 39% HCC827). Further, GNR-Dox-Tf-NP markedly reduced cytotoxicity in normal human coronary artery smooth muscle cells compared with free Dox.ConclusionThus, GNR-Dox-Tf nanoparticles can selectively target and deliver Dox to lung tumor cells and alleviate free Dox-mediated toxicity to normal cells.

Synthesis and characterisation of a pH-sensitive magnetic nanocomposite for controlled delivery of doxorubicin

We prepared a magnetic chitosan-cis-aconitic anhydride-doxorubicin nanocomposite, denoted by MCS-CAA-DOX. Chitosan (CS) was linked to magnetic nanoparticles (Fe3O4) to decrease cytotoxicity of the composite and provided a large number of reactive sites for coupling of drug molecules. DOX was attached to the magnetic chitosan (MCS) via a pH-sensitive linker, cis-aconitic anhydride, which hydrolyses in the acidic lysosomal environment to allow pH-responsive release of DOX. The prepared nanocomposites were within 15 nm and had good superparamagnetic properties. The loading rate of DOX was up to 83%. It was found that nearly 88% DOX was released within 60 h at pH 5.0, compared with only 29% at pH 7.4.

Neural Stem Cell Carriers for the Treatment of Glioblastoma Multiforme

Neural Stem Cell Carriers for the Treatment of Glioblastoma Multiforme

Spiro Diorthoester (SpiDo), a Human Plasma Stable Acid-Sensitive Cleavable Linker for Lysosomal Release

pH-sensitive linkers designed to undergo selective hydrolysis at acidic pH compared to physiological pH can be used for selective release of therapeutics selectively at targets and orthoesters have been demonstrated to be good candidates for such linkers. Following an HPLC screening, a Spiro Diorthoester (SpiDo) derivative was identified as a potent acid-labile group for the development of pH-sensitive targeted systems. After incorporation of this linker into activatable FRET-based probe and side-by-side comparison to a well-known alkylhydrazone linker, this SpiDo linker has shown a fast and pH sensitive hydrolysis for mild acidic conditions, a pH sensitive lysosomal hydrolysis, and high stability in human plasma.

Pulmonary Codelivery of Doxorubicin and siRNA by pH-Sensitive Nanoparticles for Therapy of Metastatic Lung Cancer.

A pulmonary codelivery system that can simultaneously deliver doxorubicin (DOX) and Bcl2 siRNA to the lungs provides a promising local treatment strategy for lung cancers. In this study, DOX is conjugated onto polyethylenimine (PEI) by using cis-aconitic anhydride (CA, a pH-sensitive linker) to obtain PEI-CA-DOX conjugates. The PEI-CA-DOX/siRNA complex nanoparticles are formed spontaneously via electrostatic interaction between cationic PEI-CA-DOX and anionic siRNA. The drug release experiment shows that DOX releases faster at acidic pH than at pH 7.4. Moreover, PEI-CA-DOX/Bcl2 siRNA complex nanoparticles show higher cytotoxicity and apoptosis induction in B16F10 cells than those treated with either DOX or Bcl2 siRNA alone. When the codelivery systems are directly sprayed into the lungs of B16F10 melanoma-bearing mice, the PEI-CA-DOX/Bcl2 siRNA complex nanoparticles exhibit enhanced antitumor efficacy compared with the single delivery of DOX or Bcl2 siRNA. Compared with systemic delivery, most drug and siRNA show a long-term retention in the lungs via pulmonary delivery, and a considerable number of the drug and siRNA accumulate in tumor tissues of lungs, but rarely in normal lung tissues. The PEI-CA-DOX/Bcl2 siRNA complex nanoparticles are promising for the treatment of metastatic lung cancer by pulmonary delivery with low side effects on the normal tissues.

Abstract 2904: SN-38 antibody-drug conjugates as a novel platform for solid cancer therapy: preclinical science

Abstract SN-38 is the active metabolite of the camptothecin prodrug, CPT-11 (irinotecan), which is a solid cancer therapeutic, particularly colorectal cancer (CRC). SN-38 is highly potent (low nM), but its bioavailability is hampered by a low rate of CPT-11 conversion to SN-38 (e.g., <5%), as well as its subsequent detoxification, mainly by UGT1A1 glucoronidation. In an attempt to improve SN-38's bioavailability and therapeutic index, we conjugated it to tumor-targeting humanized monoclonal antibodies (mAbs) using a novel pH-sensitive linker and a maleimide attachment to the mAb's interchain SH groups, achieving a drug-antibody ratio of 7.6. Linker coupling on the 20th position of SN-38 helps maintain SN-38 in the more active lactone form. In human serum, SN-38 is released from the linker with a half-life of ∼24 h, providing ample time for the ADC to localize in tumors. The pH-sensitive linker has the potential to release of SN-38 in the acidic tumor microenvironment by the targeted ADC in the absence of its direct internalization. Studies using two different SN-38-conjugated mAbs targeting CEACAM5 (IMMU-130) and Trop-2 (IMMU-132) show growth inhibition and regression of diverse human solid cancer xenografts in mice given repeated doses (cumulative ≤ 50 mg/kg in mice, or ∼ 4 mg/kg human equivalent dose [HED]), without defining an MTD (CPT-11 conversion is more active in mice, but mice are more tolerant to SN-38 than humans). Dosing regimens using the same total amount of the ADC given once or twice weekly for at least 2 weeks appears to be preferred over the same total amount given once every other week (2 doses). IMMU-130 is a poorly-internalizing mAb that shows good ADC activity in human CRC models expressing CEACAM5, while IMMU-132 internalizes rapidly and is active against CRC, gastric, breast, pancreatic, and prostatic cancers expressing Trop-2; both targets have antigen receptors in the 1 x 10*5 range in most cancer cell lines. Studies to assess tumor and tissue concentrations of SN-38 in xenograft models given the ADC or CPT-11 are ongoing. Toxicity studies performed in relevant animal species found the ADCs were tolerated at HED of up to 60 mg/kg, limited by neutropenia and diarrhea (also dose-limiting for CPT-11 therapy), and with no evidence of selective toxicity to antigen-expressing normal tissues. These results with an ADC having relatively modest toxicity suggest a high therapeutic index. Ongoing Phase I/II clinical trials confirm anticancer activity with a high therapeutic window (less neutropenia and GI toxicity than CPT-11) in cancers expressing the target antigens. Citation Format: David M. Goldenberg, Thomas M. Cardillo, Robert M. Sharkey, William J. McBride, Edmund A. Rossi, Chien-Hsing Chang, Serengulam V. Govindan. SN-38 antibody-drug conjugates as a novel platform for solid cancer therapy: preclinical science. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2904. doi:10.1158/1538-7445.AM2014-2904

Abstract 4481: Genetically encoded chimeric polypeptide nanoparticles for gemcitabine delivery to solid tumors

Abstract Gemcitabine (2′,2′-difluorodeoxycytidine) (dFdC) is a chemotherapeutic drug indicated for the treatment of pancreatic, bladder, NSCLC, breast and ovarian cancers. The use of gemcitabine for the treatment of other cancers, including gallbladder cancer, cervical cancer, colorectal cancer, non-Hodgkin's lymphoma, and Kaposi's sarcoma, has been clinically investigated. The cytotoxic effect of gemcitabine is due to inhibition of DNA synthesis by two mechanisms of action: (i) dFdCTP directly competes with dCTP for incorporation into DNA, inhibiting further DNA synthesis via masked chain termination; (ii) dFdCDP inhibits ribonucleotide reductase (which catalyses reactions that produce deoxynucleoside triphosphates for DNA synthesis). However, the major fraction of the administered drug undergoes rapid metabolism into dFdU by deamination, primarily in blood and then in other major organs. Therefore, the plasma half-life of gemcitabine following intravenous administration is short, ranging from 8-17 min in human plasma and 9 min in murine plasma. Like other drugs, Gemcitabine also shows a lack of selectivity towards tumor cells, sub-optimal pharmacokinetics and bio-distribution, leading to limited efficacy and significant systemic toxicity. Finally, the rapid metabolism of gemcitabine generates the need for high doses of drug, which in turn exhibits dose-limiting systemic side effects, such as hematological (myelosuppression, neutropenia, anemia, thrombocytopenia) and other toxicities (edema, cutaneous toxicity, pulmonary toxicity, dyspnea). In an effort to overcome the limited efficacy and systemic toxicity of current Gemcitabine formulations, we have designed a novel polypeptide drug delivery system where Gemcitabine is covalently attached to a genetically engineered chimeric polypeptide (CP) through a pH sensitive linker such that covalent conjugation of multiple copies of Gemcitabine at one end of the CP triggers the formation of sub 100 nm size nanoparticles that are ideal for targeting solid tumors through the enhanced permeability and retention (EPR) effect. We found that upon conjugation, CP-Gemcitabine conjugate form nanoparticles with a diameter of ∼40 nm and narrow size distribution (<10%). In a human colon cancer cell (HCT-116), the CP-Gemcitabine conjugate showed substantial cytotoxicity that was similar to free drug. These results are promising because they demonstrate the potential of rationally engineered nanoparticle drug carriers to greatly improve the efficacy of currently approved cytotoxic agents for the treatment of solid tumors. In ongoing work that will be presented at the AACR meeitng, the efficacy of CP-Gemcitabine nanoparticles in a number of tumor models in mice will be reported. Citation Format: Jayanta Bhattacharyya, Ashutosh Chilkoti. Genetically encoded chimeric polypeptide nanoparticles for gemcitabine delivery to solid tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4481. doi:10.1158/1538-7445.AM2014-4481

Abstract CT206: SN-38 antibody-drug conjugate (ADC) targeting Trop-2, IMMU-132, as a novel platform for the therapy of diverse metastatic solid cancers: Initial clinical results

Abstract IMMU-132 is an ADC of the active metabolite of CPT-11, SN-38, conjugated by a pH-sensitive linker (average drug-antibody ratio = 7.6) to the humanized anti-Trop-2 antibody, hRS7, exhibiting rapid internalization after binding to Trop-2. Trop-2 is a type I transmembrane protein expressed at high prevalence (∼1 x 10*5) and specificity by many carcinomas. We report the results of a Phase I clinical trial of 25 patients with different metastatic cancers (pancreatic, 7; triple-negative breast [TNBC], 4; colorectal [CRC], 3; gastric, 2; small-cell lung [SCLC], 2; esophageal, prostatic, ovarian, non-small-cell lung, renal, tonsillar, urinary bladder, 1 each) after failing a median of 3 prior treatments (some including topoisomerase-I and -II inhibiting drugs). IMMU-132 was administered in repeated 21-day cycles, with each treatment given on days 1 and 8. Dosing started at 8 mg/kg/dose (i.e., 16 mg/kg/cycle), and escalated to 18 mg/kg before encountering dose-limiting neutropenia, in a 3+3 trial design. Fatigue, alopecia, and occasional mild to moderate diarrhea were some of the more common non-hematological toxicities, with 2 patients also reporting a rash. Over 80% of 24 assessable patients had stable disease or tumor shrinkage (17/24 SD; 3/24 PR) among the various metastatic cancers as best response by CT. Three patients (CRC, TNBC, SCLC) have PRs by RECIST; median TTP for all patients, excluding those with pancreatic cancer, is >18 weeks. Neutropenia has been controlled by dose reduction to 8-10 mg/kg/dose (16-20 mg/kg/cycle), which is the Phase II dose. Immunohistochemistry showed strong expression of Trop-2 in most archived patient tumors, but is not detected in serum. Corresponding reductions in blood tumor marker titers (e.g., CEA, CA19-9) reflected tumor responses. No anti-antibody or anti-SN-38 antibodies have been detected despite repeated dosing. Peak and trough assessments of IMMU-132 concentrations in the serum show that the conjugate clears completely within 7 days, an expected finding based on in vitro studies showing 50% of the SN-38 is released in the serum every day. These results indicate that this novel ADC, given in doses ranging from 16-24 mg/kg per cycle, is active in diverse metastatic solid cancers. A Phase II study is ongoing to determine response rates in CRC, TNBC, and SCLC, while also evaluating its potency in other tumor types. Citation Format: Alexander N. Starodub, Allyson J. Ocean, Manish A. Shah, Linda T. Vahdat, Ellen Chuang, Michael J. Guarino, Vincent J. Picozzi, Sajeve S. Thomas, Pius P. Maliakal, Serengulam V. Govindan, William A. Wegener, Robert M. Sharkey, David M. Goldenberg. SN-38 antibody-drug conjugate (ADC) targeting Trop-2, IMMU-132, as a novel platform for the therapy of diverse metastatic solid cancers: Initial clinical results. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT206. doi:10.1158/1538-7445.AM2014-CT206

Abstract CT211: IMMU-130, an SN-38 antibody-drug conjugate (ADC) targeting CEACAM5, is therapeutically active in metastatic colorectal cancer (mCRC): Initial clinical results of two Phase I studies

Abstract IMMU-130, an ADC of the active metabolite of CPT-11, SN-38, conjugated by a pH-sensitive linker (7.6 average drug-antibody ratio) to the humanized anti-CEACAM5 antibody (labetuzumab), is completing two Phase I trials. In both, eligible patients with advanced mCRC were required to have failed/relapsed standard treatments, one being the topoisomerase-I inhibiting drug, CPT-11 (irinotecan), and an elevated plasma CEA (>5 ng/mL). IMMU-130 was administered every 14 days (EOW) at doses starting from 2.0 mg/kg in the first protocol (IMMU-130-01). Febrile neutropenia occurred in 2 of 3 patients at 24 mg/kg; otherwise at ≤16 mg/kg, neutropenia (≥ Grade 2) was observed in 7 patients, with one also experiencing thrombocytopenia. One patient [of 8 who received > 4 doses (2 cycles)] showed a 40.6% decrease in liver (starting at 7 cm) and lung target lesions (PR by RECIST) for 4.7 months, with no major toxicity, tolerating a total of 18 doses at 16 mg/kg. The study is continuing at 12 mg/kg EOW. Since SN-38 is most effective in S-phase cells, a more protracted exposure could improve efficacy. Thus, in a second Phase I trial (IMMU-130-02), dosing was intensified to twice-weekly, starting at 6 mg/kg/dose for 2 weeks (4 doses) with 1 week off, as a treatment cycle, in a 3+3 trial design. Neutropenia and manageable diarrhea were the major side effects, until dose reduction to 4.0 mg/kg twice-weekly, with early results indicating multiple cycles are well-tolerated. Currently, tumor shrinkage occurred in 3 patients, with 1 in continuing PR (-46%) by RECIST, among 6 patients who completed >4 doses (1 cycle). In both trials, CEA blood titers correlated with tumor response, and high levels did not interfere with therapy. There have been no anti-antibody or anti-SN-38 antibody reactions, based on ELISA tests. In each study, the ADC was cleared by 50% within the first 24 h, which is much longer exposure than with typical doses of the parental molecule, CPT-11. These results indicate that this novel ADC, given in different regimens averaging ∼16-24 mg/kg/cycle, shows therapeutic activity in advanced mCRC patients. Since CEACAM5 has elevated expression in breast and lung cancers, as well as other epithelial tumors, it may be a useful target in other cancers as well. A Phase II trial evaluating twice-weekly dosing and a new regimen, once-weekly x 2 every 21 days, is ongoing in mCRC. Citation Format: Neil H. Segal, Efrat Dotan, Jordan D. Berlin, Alexander N. Starodub, Michael J. Guarino, Leonard B. Saltz, Pius P. Maliakal, Serengulam V. Govindan, William A. Wegener, Robert M. Sharkey, David M. Goldenberg. IMMU-130, an SN-38 antibody-drug conjugate (ADC) targeting CEACAM5, is therapeutically active in metastatic colorectal cancer (mCRC): Initial clinical results of two Phase I studies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT211. doi:10.1158/1538-7445.AM2014-CT211

Intracellular pH-operated mechanized mesoporous silica nanoparticles as potential drug carries

A series of pH-operated mechanized mesoporous silica nanoparticles (MMSNs) were fabricated and used as drug delivery for intracellular acid-triggered release. Firstly, adamantine (AD) was anchored on the surface of mesoporous silica nanoparticles (MSNs) by a pH-sensitive intermediate linker. Then, pH-operated MMSNs were prepared by the supramolecular interaction between adamantine (AD) and β-cyclodextrin (β-CD). Doxorubicin (DOX), as a drug model, was loaded into MMSNs. The pH-dependent release behavior of loaded-DOX in vitro revealed that there was only a small amount of loaded-DOX released in PBS solution at pH 7.4, while up to about 90% of loaded-DOX could be quickly released in PBS solution at pH 5.5. Compared with pH-insensitive ones, pH-operated MMSNs displayed a faster drug release behavior and a higher cellular proliferation inhibition efficacy toward tumor cells. These features suggested that pH-operated MMSNs DOX could efficiently load and delivery DOX into tumor cells, leading to an enhanced inhibition of tumor cell proliferation. Therefore, these pH-operated MMSNs will be of advantage as promising carriers for drug acid-triggered release in cancer therapy application.

A novel triptolide-nucleolin aptamer conjugate (TMPC001) with super antitumor activity on advanced pancreatic cancer

Nucleolin aptamer was conjugated to Triptolide (TP) by a pH-sensitive linker (Vinylether bond). Then the structure was confirmed by HPLC and LC-MS. The conjugate could be stable within neutral pH environment but rupture in acidic environment (Fig 1). The conjugate could selectively identify tumor cell in vitro (Fig 2) and the anti-proliferative efficacy was superior to gemcitabine. In vivo study revealed that the conjugate could selectively target pancreatic cancer tissue (achieve antitumor activity) but reduce the distribution to normal tissues (reduce toxicity) (Fig 3).The conjugate demonstrated super antitumor and anti-angiogenesis effects (Fig 4 and 5).

IMMU-132, an SN-38 antibody-drug conjugate (ADC) targeting Τrop-2, as a novel platform for the therapy of diverse metastatic solid cancers: Clinical results.

3032 Background: IMMU-132 is an ADC of the internalizing, humanized, anti-Trop-2 antibody, hRS7, conjugated by a pH-sensitive linker, to SN-38, the active metabolite of CPT-11 (mean drug-antibody ratio = 7.6). Trop-2 is a type I transmembrane, calcium-transducing, protein expressed at high density (~1 x 10*5), frequency, and specificity by many human carcinomas, with limited normal tissue expression. Methods: We report the initial Phase I trial of 25 patients (pts) who had failed multiple prior therapies (some including topoisomerase-I/II inhibiting drugs), and the ongoing Phase II extension that focused on pts with colorectal (CRC), small-cell lung (SCLC) and triple-negative breast (TNBC) cancers. Results: Trop-2 is not detected in serum, but was strongly expressed (≥2+) in most archived tumors. In a 3+3 trial design, IMMU-132 was given on days 1 and 8 in repeated 21-day cycles, starting at 8 mg/kg/dose, then 12 and 18 mg/kg before dose-limiting neutropenia. To optimize cumulative treatment with minimal delays, phase II is focusing on 8 and 10 mg/kg (n=22 and 14, respectively). Most common non-hematological toxicities were fatigue (17 ≤G2 ,4 G3), nausea (20 ≤G2), diarrhea (12 ≤G2, 3 G3), alopecia (14), and vomiting (10 ≤G2) ; 2 pts had a rash. Homozygous UGT1A1 *28/*28 was found in 5 pts, 2 of whom had more severe hematological and GI toxicities. Over 80% of 24 assessable pts in Phase I had stable disease or tumor shrinkage (17 SD; 3 PR [CRC, TNBC, SCLC]) as best CT response; median TTP = 18 weeks for all pts, except pancreatic cancer. Of the 36 pts in the Phase II, 14 had their first response assessment by CT: 6 PD, 7 SD; 1 PR (SCLC) by RECIST1.1. Tumor markers (CEA, CA19-9) correlated with responses. No anti-hRS7 or anti-SN-38 antibodies were detected despite dosing over months. The conjugate clears within 7 days, consistent with in vivo animal studies where 50% of the SN-38 is released daily. Conclusions: These results indicate that this novel SN-38-containing ADC is active in metastatic solid cancers, with manageable diarrhea and neutropenia. The Phase II continues, while also accruing in other epithelial tumors. Clinical trial information: NCT01631552.

Self-assembled polymeric micelles based on THP and THF linkage for pH-responsive drug delivery

Developing smart nanocarriers for drug delivery system is advantageous for many kinds of successful biomedicinal therapy. In this study, we designed an amphiphilic block copolymers containing pH-responsive tetrahydropyran (THP) and tetrahydrofuran (THF) linkage. Their structures were confirmed by 1H NMR and gel permeation chromatography (GPC). The release rate of encapsulated drugs depends upon the pH value and pH sensitive linkage in the backbone of copolymers. For PLA–THP–PEG micelles the cumulative release amount of doxorubicin (DOX) was 62% at pH 5.0, which is about four times higher than that at pH 7.4. Under the same conditions the release rate for PLA–THF–PEG micelles is a little faster than that of the PLA–THP–PEG micelles. Cellular uptake study demonstrates that DOX-loaded micelles can easily enter the cells and produce the desired pharmacological action and minimizing the side effect of free DOX. These findings indicate that THP and THF linked diblock copolymer micelles is a promising candidate for drug carrier.

Comparison of pH-sensitive degradability of maleic acid amide derivatives

We synthesized five maleic acid amide derivatives (maleic, citraconic, cis-aconitic, 2-(2′-carboxyethyl) maleic, 1-methyl-2-(2′-carboxyethyl) maleic acid amide), and compared their degradability for the future development of pH-sensitive biomaterials with tailored kinetics of the release of drugs, the change of charge density, and the degradation of scaffolds. The degradation kinetics was highly dependent upon the substituents on the cis-double bond. Among the maleic acid amide derivatives, 2-(2′-carboxyethyl) maleic acid amide with one carboxyethyl and one hydrogen substituent showed appropriate degradability at weakly acidic pH, and the additional carboxyl group can be used as a pH-sensitive linker.

CD44‐Targeted Docetaxel Conjugate for Cancer Cells and Cancer Stem‐Like Cells: A Novel Hyaluronic Acid‐Based Drug Delivery System

A CD44-targeted macromolecular conjugate of docetaxel was prepared via a pH-sensitive linkage to hyaluronic acid and was characterized using NMR, gel permeation chromatography, and differential scanning calorimetry. The conjugated species were further evaluated in terms of drug release, cytotoxicity, cellular uptake, cell cycle inhibition, and subacute toxicity in mice. Cellular microscopic studies revealed that CD44-expressing cells including MCF-7 cancer stem cells and MDA-MB-231 metastatic breast cancer cells had internalized the conjugates via a selective receptor-mediated mechanism, leading to cell cycle arrest in the G2/M phase. Hyaluronic acid-docetaxel conjugates showed specific toxicity only in CD44-expressing cells in vitro, along with a decreased risk of neutropenia and dose-dependent mortality in vivo. Hyaluronic acid-drug conjugates represent a promising and efficient platform for solubilization of sparingly soluble molecules as well as active and selective targeted delivery to cancer cells and cancer stem cells.

Dendrimer-based multilayer nanocarrier for potential synergistic paclitaxel–doxorubicin combination drug delivery

A unimolecular micelle-like nanocarrier (PPDP) based on poly(amidoamine) (PAMAM) dendrimers was synthesized to develop combined drug delivery systems for intensifying the therapeutic effect on tumors. The carrier was designed as a dual drug container with oligo-poly(ε-caprolactone) (PCL) and PEGylated amidoamine dendrons as the hydrophobic and hydrophilic domains for each molecule. PPDP was characterized with a size of 10–20 nm, indicating that it mostly remained in the single molecular state rather than in aggregates. Based on PPDP, two drug delivery systems of PPDP-enc-DOX + PTX and PPDP-coj-DOX-enc-PTX were constructed. PPDP-enc-DOX + PTX was prepared by physically encapsulating doxorubicin (DOX) and paclitaxel (PTX) in the different layers of the PPDP, while PPDP-coj-DOX-enc-PTX was fabricated by conjugating DOX through the pH-sensitive linkage on the exterior and embedding PTX in the interior of the PPDP. The drug loading or conjugating and the drug release behaviors were tested. The cytotoxicity, the cell uptake and the intracellular distribution of PPDP-enc-DOX + PTX and PPDP-coj-DOX-enc-PTX were measured in vitro. For both of the systems, the release of PTX was achieved in a sustained manner during 48 h in vitro, while for the PPDP-coj-DOX-enc-PTX system, the liberation of DOX could be controlled. PPDP-enc-DOX + PTX and PPDP-coj-DOX-enc-PTX both showed the ability to simultaneously deliver two drugs into the same tumor cells and showed higher cytotoxicity toward MCF-7/ADR and MCF-7 cells than free DOX or PTX alone due to the synergistic effect of the drugs. Notably, PPDP-enc-DOX + PTX showed a better inhibition effect on MCF-7/ADR cells as it could deliver DOX into the nucleus, while PPDP-coj-DOX-enc-PTX had the stronger cytotoxicity to MCF-7 cells due to the steady sustained release manner of the two drugs.