An anti-CD33 antibody-calicheamicin conjugate for treatment of acute myeloid leukemia. Choice of linker.

The anti-CD33 antibody, P67.6, has been chosen to target the potently cytotoxic calicheamicin antitumor antibiotics to acute myeloid leukemia (AML) due to the presence of CD33 on >80% of patient samples and its lack of expression outside the myeloid cell lineages, especially its lack of expression on pluripotent stem cells. Previous calicheamicin conjugates relied on the attachment of a hydrazide derivative to the oxidized carbohydrates that occur naturally on antibodies. This results in a "carbohydrate conjugate" capable of releasing active drug by hydrolysis of a hydrazone bond in the lysozomes where the pH is low. Conjugates have now been made that are formed by reacting a calicheamicin derivative containing an activated ester with the lysines of antibodies. This results in an "amide conjugate" that is stable to hydrolysis, leaving the disulfide that is present in all calicheamicin conjugates as the likely site of drug release from the conjugate. In this article, these two classes of calicheamicin-antibody conjugates are compared for potential use in AML with the anti-CD33 antibody P67.6. Conjugates of P67.6 are shown to require the site of hydrolytic release afforded by the carbohydrate conjugates in order to retain good potency and selectivity in vitro, in vivo, and ex vivo. The P67.6 carbohydrate conjugate of calicheamicin is selectively cytotoxic at <0.006 ng/mL of calicheamicin equivalents (cal equiv) toward HL-60 promyelocytic leukemia cells in tissue culture. Long-term, tumor-free survivors are seen in xenograft models when mice bearing HL-60 subcutaneous tumors are treated with the P67.6 carbohydrate conjugate at a dose of 300 microg/kg cal equiv given three times. This conjugate also selectively inhibits the formation of colonies from AML marrow samples at 2 ng/mL cal equiv. The P67.6 carbohydrate conjugate of calicheamicin therefore appears to have promise as an antibody-targeted chemotherapeutic agent for CD33-positive diseases such as AML.     

Gemtuzumab ozogamicin, a potent and selective anti-CD33 antibody-calicheamicin conjugate for treatment of acute myeloid leukemia.

CD33 is expressed by acute myeloid leukemia (AML) cells in >80% of patients but not by normal hematopoietic stem cells, suggesting that elimination of CD33(+) cells may be therapeutically beneficial. A conjugate of a calicheamicin hydrazide derivative attached via hydrazone formation to the oxidized carbohydrates of the anti-CD33 murine antibody P67.6 had been chosen for use in AML prior to humanization of this antibody. However, the CDR-grafted humanized P67.6 could not be used to make the carbohydrate conjugate because of the unexpected sensitivity of this antibody to periodate oxidation. Exploration of a series of bifunctional linkers resulted in a new class of calicheamicin conjugates, termed the hybrid conjugates, that allows for the attachment of the calicheamicin to lysines but incorporates the site of hydrolytic release, a hydrazone, previously shown to be required for activity. The optimized conjugate chosen for clinical trials, gemtuzumab ozogamicin ("gem-ozo", Mylotarg, formerly designated CMA-676), was significantly more potent and selective than the carbohydrate conjugate it replaced. It was selectively cytotoxic to HL-60 leukemia cells in tissue culture with an IC(50) in the low to sub-pg cal/mL range (cal = calicheamicin equivalents). Doses of gem-ozo as low as 50 microg cal/kg given three times to mice bearing HL-60 xenografts routinely resulted in long-term, tumor-free survivors, while a nonbinding control conjugate was relatively inactive. Gem-ozo at a concentration of 2 to 10 ng cal/mL selectively inhibited leukemia colony formation by marrow cells from a significant proportion of AML patients. Gem-ozo has also shown significant activity against AML in Phase II trials and is the first antibody-targeted chemotherapeutic agent approved by the FDA.     

A drug repurposing strategy for overcoming human multiple myeloma resistance to standard-of-care treatment

Despite several approved therapeutic modalities, multiple myeloma (MM) remains an incurable blood malignancy and only a small fraction of patients achieves prolonged disease control. The common anti-MM treatment targets proteasome with specific inhibitors (PI). The resulting interference with protein degradation is particularly toxic to MM cells as they typically accumulate large amounts of toxic proteins. However, MM cells often acquire resistance to PIs through aberrant expression or mutations of proteasome subunits such as PSMB5, resulting in disease recurrence and further treatment failure. Here we propose CuET—a proteasome-like inhibitor agent that is spontaneously formed in-vivo and in-vitro from the approved alcohol-abuse drug disulfiram (DSF), as a readily available treatment effective against diverse resistant forms of MM. We show that CuET efficiently kills also resistant MM cells adapted to proliferate under exposure to common anti-myeloma drugs such as bortezomib and carfilzomib used as the first-line therapy, as well as to other experimental drugs targeting protein degradation upstream of the proteasome. Furthermore, CuET can overcome also the adaptation mechanism based on reduced proteasome load, another clinically relevant form of treatment resistance. Data obtained from experimental treatment-resistant cellular models of human MM are further corroborated using rather unique advanced cytotoxicity experiments on myeloma and normal blood cells obtained from fresh patient biopsies including newly diagnosed as well as relapsed and treatment-resistant MM. Overall our findings suggest that disulfiram repurposing particularly if combined with copper supplementation may offer a promising and readily available treatment option for patients suffering from relapsed and/or therapy-resistant multiple myeloma.Subject terms: Myeloma, Cancer therapeutic resistance     

A paradigm for therapy-induced microenvironmental changes in solid tumors leading to drug resistance

Intrinsic alterations in the tumor microenvironment are known to contribute to various forms of drug resistance. For example, tumor hypoxia, due to abnormal or sluggish blood flow within areas of solid tumors, can result in both microenvironment-mediated radiation and chemotherapeutic drug resistance. In contrast, acquired resistance to chemotherapy is generally considered to be the result of the gradual selection of mutant subpopulations having genetic mutations and biochemical alterations responsible for the resistant phenotype. Here we present a paradigm for therapyinduced microenvironment-mediated acquired drug resistance. It is based on the results showing that tumor cells appear to be heterogeneous in their relative dependence on adjacent tumor-associated vasculature for survival. Some tumor cells are highly vessel dependent, whereas some are significantly less so, and thus can survive in more hypoxic regions of tumors, distal from such tumor vessels. Hence, it is possible that variant tumor cells that are less vessel dependent may therefore be selected for over time by successful antiangiogenic drug therapies. This results in loss of response or attenuated responses to the therapy. Preliminary evidence is summarized in support of this hypothesis, using paired human colon cancer (HCT116) cell lines that contain two copies of either the wild-type or the disrupted p53 tumor suppressor gene. The mutant cells were found to be less responsive to antiangiogenic therapy, compared to the wild-type cells, and could be progressively selected for in mixed cell populations. Because p53 inactivation can lead to resistance to hypoxia-mediated apoptosis, the results suggest that a protracted and successful antiangiogenic therapy may create more hypoxic tumor microenvironments, thereby creating the necessary conditions to accelerate the selection of mutant tumor cells that are more adept in surviving and growing in such environments. As such, consideration might be given to the combined use of bioreductive hypoxic cell cytotoxic drugs and angiogenesis inhibitors to prolong the efficacy of antiangiogenic therapeutics.     

Comparing solid tumors with cell lines: implications for identifying drug resistance genes in cancer

Gene expression arrays allow researchers to profile the differences between cell lines or tissues and they may identify genetic markers of development, organ maturation, or tumor progression. Although a primary tumor that grows in a host and a tumor-cell-line derived from that primary tumor and grown in vitro share similar gene expression profiles, there are, not unexpectedly, some important differences. In fact, Stein and colleagues have found that genes that are differentially expressed in primary tumors as compared to the specific genes expressed in their cell-line derivatives are more reliably predictive of tumor tractability. Thus, sensitivity in vitro might not reflect sensitivity in vivo. Because anti-tumor compounds are largely evaluated in cell culture assays, these compounds' therapeutic utility must be judged in light of genes described by Stein et al. that better predict tractability.     

Site-specifically traced drug release and biodistribution of a paclitaxel-antibody conjugate toward improvement of the linker structure

Tumor-directed drug delivery is a promising strategy in cancer treatment, and in this field, monoclonal antibodies constitute an important class of targeting vehicles. A critical issue in the design of targeting conjugates is the timing of the release of the cytotoxic payload, with the ideal situation being the release at the maximum tumor uptake of the targeting molecule. A site-specific radiolabeling technique was used to elucidate the biodistribution and in vivo drug release pattern of an antibody conjugate of paclitaxel (PTX, 1, Figure 1) in which the drug and the antibody moieties were connected by a succinate (SX) linker. In this new method, a metabolite of PTX, 3'-(4-hydroxyphenyl)paclitaxel (3'-OH-PTX, 2, Figure 1) was used as a tyrosine mimic for the synthesis of the drug site-labeled conjugate (DSL, [(125)I]-3'-OH-PTXSXC225). This was achieved by iodogen (125)I-labeling of 3'-OH-PTXSX and subsequent conjugation to C225. The antibody site-labeled conjugate (ASL, PTXSX-[(125)I]-C225) was prepared by direct radioiodination of PTXSXC225. Biodistribution of these compounds was studied in Balb/c nude mice bearing DU-145 human prostate carcinoma xenografts. While the 4 and 24 h tumor uptake (in percent injected dose per gram of tissue, %ID/g) for [(125)I]-3'-OH-PTXSXC225 were 3.3 +/- 1.5 and 1.7 +/- 0.6%ID/g, the PTXSX-[(125)I]-C225 showed tumor uptake values of 3.8 +/- 4.2 and 14.8 +/- 4.2%ID/g at these time points. This difference in the tumor uptake over time indicates an early cleavage of the drug with respect to the antibody tumor localization. This was further confirmed by an in vitro drug release kinetics study leading to a half-life of about 2 h for PTXSXC225 under physiological conditions. To increase the stability of the PTX-MAb bond, a new conjugate (PTXGLC225) with glutaric acid (GL) as the linker was synthesized. Under the same conditions, the PTXGLC225 showed a 16-fold increase in the half-life (t(1/2)) of the drug release. The effect of the increased t(1/2) of this compound on the antitumor activity of the conjugate was tested in a DU-145 human prostate tumor-implanted mouse model. In comparison to a previous similar experiment with PTXSXC225, better antitumor activity was observed for the PTXGLC225 conjugate as compared to controls. These results demonstrated the first time use of radioiodinated 3'-OH-PTX for in vivo tracing of a paclitaxel conjugate and application of the resulting information to the design of a therapeutically more useful PTX-MAb linker.     

Radiation induction of drug resistance in RIF-1 tumors and tumor cells

The RIF-1 tumor cell line contains a small number of cells (1-20 per 10(6) cells) that are resistant to various single antineoplastic drugs, including 5-fluorouracil (5FU), methotrexate (MTX), and adriamycin (ADR). For 5FU the frequency of drug resistance is lower for tumor-derived cells than for cells from cell culture; for MTX the reverse is true, and for ADR there is no difference. In vitro irradiation at 5 Gy significantly increased the frequency of drug-resistant cells for 5FU, MTX, and ADR. In vivo irradiation at 3 Gy significantly increased the frequency of drug-resistant cells for 5FU and MTX, but not for ADR. The absolute risk for in vitro induction of MTX, 5FU, and ADR resistance, and for in vivo induction of 5FU resistance, was 1-3 per 10(6) cells per Gy; but the absolute risk for in vivo induction of MTX resistance was 54 per 10(6) cells per Gy. The frequency of drug-resistant cells among individual untreated tumors was highly variable; among individual irradiated tumors the frequency of drug-resistant cells was significantly less variable. These studies provide supporting data for models of the development of tumor drug resistance, and imply that some of the drug resistance seen when chemotherapy follows radiotherapy may be due to radiation-induced drug resistance.     

Heterocycle amide isosteres: An approach to overcoming resistance for HIV-1 integrase strand transfer inhibitors

A series of heterocyclic pyrimidinedione-based HIV-1 integrase inhibitors was prepared and screened for activity against purified integrase enzyme and/or viruses modified with the following mutations within integrase: Q148R, Q148H/G140S and N155H. These are mutations that result in resistance to the first generation integrase inhibitors raltegravir and elvitegravir. Based on consideration of drug-target interactions, an approach was undertaken to replace the amide moiety of the first generation pyrimidinedione inhibitor with azole heterocycles that could retain potency against these key resistance mutations. An imidazole moiety was found to be the optimal amide substitute and the observed activity was rationalized with the use of calculated properties and modeling. Rat pharmacokinetic (PK) studies of the lead imidazole compounds demonstrated moderate clearance and moderate exposure.     

Euphorbia and Momordica metabolites for overcoming multidrug resistance

Multidrug resistance (MDR) is the major obstacle for cancer chemotherapy. MDR is a multifactorial phenomenon that can result from several mechanisms, including an increased drug efflux, due to overexpression of P-glycoprotein (P-gp) that transports anticancer drugs out of the cells. Thus, the role of this transporter has made it a therapeutic target and the development of P-gp modulators considered among the most realistic approaches for overcoming P-gp-mediated MDR. Many other strategies have been proposed. One of them is the identification of compounds that selectively kill multidrug resistant cells. In our search for MDR modulators from plants, the P-gp inhibition ability of a large number of compounds on resistant cancer cells was evaluated. These compounds, presented in this review, comprise mainly diterpenes, triterpenes and phenolic derivatives. The most relevant results were obtained from two sets of compounds: macrocyclic diterpenes with the jatrophane and lathyrane scaffold, and triterpenes of the cucurbitane-type isolated from Euphorbia species and Momordica balsamina L., respectively. Additionally, some of those macrocyclic diterpenes, and ent-abietane diterpenic lactones, also isolated from Euphorbia species, were found to be selectively toxic to drug-resistant phenotypes.     

Synthesis and biological evaluation of paclitaxel and vorinostat co-prodrugs for overcoming drug resistance in cancer therapy in vitro

Paclitaxel (PTX) is the first-line treatment drug for breast cancer. However, drug resistance after a course of treatment and low selectivity restricted its clinical utility sometimes. In this study, we successfully bound PTX and vorinostat (SAHA) to form co-prodrugs based on the synergistic anticancer effects. The PTX-SAHA co-prodrugs were conjugated by glycine (1a) and succinic acid (1b) respectively and the former has shown better activity in cytotoxicity, cell cycle arrest and western-blot experiments. Therefore, 1a was further prepared to nanomicelles with mPEG₂₀₀₀-PLA₁₇₅₀ as the carrier by using thin film method. PTX-SAHA co-prodrug nanomicelles were spherical with a particle size of 20–100?nm. In vitro drug release test showed 1a nanomicelles had sustained release effect, which could reduce the resistance of PTX. In vitro cytotoxicity was evaluated by SRB assay in HCT-116 cells, MCF-7 cells and drug-resistant MCF-7/ADR cells. The results showed 1a nanomicelles had comparable or even better cytotoxicity than PTX especially in the MCF-7/ADR cells. All the results suggested that PTX-SAHA co-prodrug nanomicelles were promising treatment for PTX resistance cancer.     

A calicheamicin conjugate with a fully humanized anti-MUC1 antibody shows potent antitumor effects in breast and ovarian tumor xenografts

Murine CTM01 is an internalizing murine IgG(1) monoclonal antibody that recognizes the MUC1 antigen expressed on many solid tumors of epithelial origin. Calicheamicin conjugates of this antibody have previously been shown to be potent, selective antitumor agents in preclinical models. A conjugate has now been made with a genetically engineered human version of this antibody, hCTM01. The hCTM01 is an IgG(4) isotype, has an immunoaffinity approximately 30% higher than mCTM01 by competitive RIA, and is efficiently internalized into target cells. The hCTM01-NAc-gamma calicheamicin DM amide conjugate, referred to as CMB-401, shows targeted killing of MUC1-expressing cells in vitro and produces pronounced dose-related antitumor effects over an 8-fold dose range against a MUC1-expressing, ovarian xenograft tumor, OvCar-3. The specificity of CMB-401 was confirmed by comparing its antitumor effects with those of an isotype-matched nonspecific conjugate against the MX-1 breast carcinoma. CMB-401, given either ip or iv, was highly active in these models in single and multiple dose regimens and gave complete regressions at the highest doses examined with good overall therapeutic ratios. CMB-401 also gave good antitumor effects at similar doses with a cisplatin-resistant MUC1-expressing cell line.     

Drug failure during HIV-1 treatment. New perspectives in monitoring drug resistance

Since the discovery of 3'-azido-3'deoxthymidine (zidovudine) as an effective antiretroviral agent against human immunodeficiency virus type 1 (HIV-1), drug therapy has been widely used in the treatment of AIDS. To date, new combination therapies have significantly altered the longterm prognosis for HIV-infected patients showing a reduction of plasma viral load, associated with clinical and immunological recovery. Nevertheless, in various circumstances treatment can fail for several reasons, such as patient noncompliance with the therapeutic regimen, suboptimal antiviral drug concentrations, drug pharmacokinetics, and virus resistance to one or more drugs. Virus drug resistance is the most important factor contributing to the failure of antiretroviral therapy. Since some evidence indicates that viral resistance and treatment failure are closely linked, this brief review explores the routine determination of drug resistance and its importance to shed more light on the meaning of mutations correlated to drug resistance.     

Towards treatment planning and treatment of deep-seated solid tumors by electrochemotherapy

Background  

Electrochemotherapy treats tumors by combining specific chemotherapeutic drugs with an intracellular target and electric pulses, which increases drug uptake into the tumor cells. Electrochemotherapy has been successfully used for treatment of easily accessible superficial tumor nodules. In this paper, we present the first case of deep-seated tumor electrochemotherapy based on numerical treatment planning.     

A mathematical model to study the effects of drug resistance and vasculature on the response of solid tumors to chemotherapy

A mathematical model is developed that describes the reduction in volume of a vascular tumor in response to specific chemotherapeutic administration strategies. The model consists of a system of partial differential equations governing intratumoral drug concentration and cancer cell density. In the model the tumor is treated as a continuum of two types of cells which differ in their proliferation rates and their responses to the chemotherapeutic agent. The balance between cell proliferation and death within the tumor generates a velocity field which drives expansion or regression of the spheroid. Insight into the tumor's response to therapy is gained by applying a combination of analytical and numerical techniques to the model equations.     

HIF-1α drives resistance to ferroptosis in solid tumors by promoting lactate production and activating SLC1A1

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Adjuvant treatment for complement activation increases the effectiveness of photodynamic therapy of solid tumors.

Phototoxic lesions generated in tumor tissue by photodynamic therapy (PDT) are recognized by the host as a threat to the integrity and homeostasis at the affected site. Among the canonical pathways invoked by the host for dealing with this type of challenge is the activation of the complement system, integrating proteins that serve as molecular sensors of danger signals produced by PDT and those initiating signalling cascades coupled into the network of inflammatory and immune responses. Since the activated complement system is a salient participant of the antitumor response produced by PDT, it is worth exploring whether its manipulation can be exploited for the therapeutic benefit. Using mouse tumor models, the present study examined the potential of representative complement-activating agents to act as effective adjuvants to PDT. Tumor-localized treatment with zymosan, an alternative complement pathway activator, reduced the recurrence-rate of PDT-treated tumors, markedly increasing the percentage of permanent cures. In contrast, a similar treatment with heat aggregated gamma globulin (complement activator via the classical pathway) was of no significant benefit as a PDT adjuvant. Systemic complement activation with streptokinase treatment had no detectable effect on complement deposition at the tumor site without PDT, but it augmented the extent of complement activity in PDT-treated tumors. This finding based on immunohistochemistry analysis explains the results of tumor therapy experiments, which showed that systemic treatment with streptokinase or a similar agent, urokinase, enhances the PDT-mediated tumor response. Zymosan and streptokinase administrations produced no beneficial results with PDT of tumors growing in complement-deficient mice. This study, therefore, establishes the potential of complement-activating agents to serve as effective adjuvants to PDT for cancer treatment.     

Effect of tumor shape and size on drug delivery to solid tumors

ABSTRACT: Tumor shape and size effect on drug delivery to solid tumors are studied, based on the application of the governing equations for fluid flow, i.e., the conservation laws for mass and momentum, to physiological systems containing solid tumors. The discretized form of the governing equations, with appropriate boundary conditions, is developed for predefined tumor geometries. The governing equations are solved using a numerical method, the element-based finite volume method. Interstitial fluid pressure and velocity are used to show the details of drug delivery in a solid tumor, under an assumption that drug particles flow with the interstitial fluid. Drug delivery problems have been most extensively researched in spherical tumors, which have been the simplest to examine with the analytical methods. With our numerical method, however, more complex shapes of the tumor can be studied. The numerical model of fluid flow in solid tumors previously introduced by our group is further developed to incorporate and investigate non-spherical tumors such as prolate and oblate ones. Also the effects of the surface area per unit volume of the tissue, vascular and interstitial hydraulic conductivity on drug delivery are investigated.     

Understanding drug resistance for monotherapy treatment of HIV infection

The purpose of this study was to investigate strategies in the monotherapy treatment of HIV infection in the presence of drug-resistant (mutant) strains. A mathematical system is developed to model resistance in HIV chemotherapy. It includes the key players in the immune response to HIV infection: virus and both uninfected CD4⁺ and infected CD4⁺ T-cell populations. We model the latent and progressive stages of the disease, and then introduce monotherapy treatment. The model is a system of differential equations describing the interaction of two distinct classes of HIV—drug-sensitive (wild type) and drug-resistant (mutant)—with lymphocytes in the peripheral blood. We then introduce chemotherapy effects. In the absence of treatment, the model produces the three types of qualitative clinical behavior—anuninfected steady state, andinfected steady state (latency), andprogression to AIDS. Simulation of treatment is provided for monotherapy, during theprogression to AIDS state, in the consideration of resistance effects. Treatment benefit is based on an increase or retention in CD4⁺ T-cell counts together with a low viral titer. We explore the following treatment approaches: an antiviral drug which reduces viral infectivity that is administered early—when the CD4⁺ T-cell count is ≥300/mm³, and late—when the CD4⁺ T-cell count is less than 300/mm³. We compare all results with data. When treatment is initiated during the progression to AIDS state, treatment prevents T-cell collapse, but gradually loses effectiveness due to drug resistance. We hypothesize that it is the careful balance of mutant and wild-type HIV strains which provides the greatest prolonged benefit from treatment. This is best achieved when treatment is initiated when the CD4⁺ T-cell counts are greater than 250/mm³, but less than 400/mm³ in this model (i.e. not too early, not too late). These results are supported by clinical data. The work is novel in that it is the first model to accurately simultate data before, during and after monotherapy treatment. Our model also provides insight into recent clinical results, as well as suggests plausible guidelines for clinical testing in the monotherapy of HIV infection.     

Pre-existence and emergence of drug resistance in HIV-1 infection.

Antiviral treatment of HIV-1 infection often fails because of the rapid emergence of resistant virus within weeks of the start of therapy. This raises the question of whether resistant viruses pre-exist in drug-naive patients or whether it is produced after the start of therapy. Here we compare the likelihood of pre-existence with the likelihood of production of resistant virus during therapy. We show that provided resistant virus pre-exists, then a stronger therapy may lead to a greater initial reduction of virus load, but will also cause a faster rise of resistant virus. In this case the total benefit of treatment is independent of the degree of inhibition of sensitive virus. If, on the other hand, resistant mutants do not pre-exist, then the emergence of resistance during treatment depends on the efficacy of the drug. If the drug is sufficiently potent to eradicate sensitive virus, then the probability that resistant mutants first appear during therapy is smaller than the probability that they existed before therapy. If the drug cannot eradicate the sensitive virus, then after sufficiently long time resistant mutants will appear. However, mutants that are unlikely to pre-exist may taken long time to appear.     

Synthesis of cholesterol-carborane conjugate for targeted drug delivery

The cholesterol-carborane conjugate has been designed and synthesized to selectively deliver boron to tumor cells by means of reconstituted low-density lipoprotein. The chemical stability and cytotoxicity of the new compound have been examined. Several methods have been evaluated for incorporation of the compound into LDL.