Comparative clinical pharmacokinetics of antibody-drug conjugates in first-in-human Phase 1 studies

Although there are currently more than 30 antibody-drug conjugates (ADC) in clinical development for the treatment of blood cancers and solid tumors, comparison of their clinical pharmacokinetics (PK) is challenging because of the large number of, and differences between, the targets, ADC constructs, dosing regimens, and patient populations. In this review, we standardized the evaluation, using non-compartmental PK data reported at Cycle 1, i.e., following the first drug administration of what is usually a repeated-dose treatment, in monotherapy. We report ADC clinical PK properties, dosing regimen, determination of doses ranges and associated maximum tolerated doses. We also evaluated the effect of structural characteristics and target types (hematological vs. solid tumors) on PK. In addition, we discuss how integration of PK/pharmacodynamics approaches on top of classical dose escalation in first-in-human studies may improve dosing regimen determination for subsequent phases of clinical development.     

Prediction of non-linear pharmacokinetics in humans of an antibody-drug conjugate (ADC) when evaluation of higher doses in animals is limited by tolerability: Case study with an anti-CD33 ADC

For antibody-drug conjugates (ADCs) that carry a cytotoxic drug, doses that can be administered in preclinical studies are typically limited by tolerability, leading to a narrow dose range that can be tested. For molecules with non-linear pharmacokinetics (PK), this limited dose range may be insufficient to fully characterize the PK of the ADC and limits translation to humans. Mathematical PK models are frequently used for molecule selection during preclinical drug development and for translational predictions to guide clinical study design. Here, we present a practical approach that uses limited PK and receptor occupancy (RO) data of the corresponding unconjugated antibody to predict ADC PK when conjugation does not alter the non-specific clearance or the antibody-target interaction. We used a 2-compartment model incorporating non-specific and specific (target mediated) clearances, where the latter is a function of RO, to describe the PK of anti-CD33 ADC with dose-limiting neutropenia in cynomolgus monkeys. We tested our model by comparing PK predictions based on the unconjugated antibody to observed ADC PK data that was not utilized for model development. Prospective prediction of human PK was performed by incorporating in vitro binding affinity differences between species for varying levels of CD33 target expression. Additionally, this approach was used to predict human PK of other previously tested anti-CD33 molecules with published clinical data. The findings showed that, for a cytotoxic ADC with non-linear PK and limited preclinical PK data, incorporating RO in the PK model and using data from the corresponding unconjugated antibody at higher doses allowed the identification of parameters to characterize monkey PK and enabled human PK predictions.     

Assessing pharmacokinetic variability directly induced by drug intake behaviour through development of a feeding behaviour-pharmacokinetic model

Variability in drug intake is increasingly recognized as a major source of variability in drug response. The non-uniform access to medicated feed, influenced by swine individual feeding behaviour, is a determinant of antibiotic exposure, recalling the intrinsic similarity with human compliance to drug regimens. In this paper, we developed a feeding behaviour-pharmacokinetic (FBPK) model of in-feed chlortetracycline (CTC) and established, in a definite way, the effect of feeding behaviour and its induced pharmacokinetic (PK) variability. Based on reported animal behaviour, we mathematically formulated swine feeding behaviour by incorporating its main characteristics: intense feeding periods that repeat on a daily basis and random feeding periods of free access to feed, along with growth stage factors. This behaviour model was then integrated into a PK model of CTC. Moreover, we analysed the effect of each feeding behaviour component and assessed the corresponding PK variability. We have been able to delineate the impact of different feeding behaviour components and characterize the induced PK variability. We have compared different therapeutic assumptions to our model and shown that random features underlying the feeding behaviour have dramatic influence on the PK variability. A practical tool to adopt the dosing regimen in terms of dose and age has been proposed. The method developed here can be generalized to other therapeutic contexts and incorporated into medical practice, particularly to make long-term projections of drug-intake behaviour, to explain possible treatment failure and guide practitioners in adjusting the dosing regimen.     

Animal models in the pharmacokinetic/pharmacodynamic evaluation of antimicrobial agents

Animal infection models in the pharmacokinetic/pharmacodynamic (PK/PD) evaluation of antimicrobial therapy serve an important role in preclinical assessments of new antibiotics, dosing optimization for those that are clinically approved, and setting or confirming susceptibility breakpoints. The goal of animal model studies is to mimic the infectious diseases seen in humans to allow for robust PK/PD studies to find the optimal drug exposures that lead to therapeutic success. The PK/PD index and target drug exposures obtained in validated animal infection models are critical components in optimizing dosing regimen design in order to maximize efficacy while minimize the cost and duration of clinical trials. This review outlines the key components in animal infection models which have been used extensively in antibiotic discovery and development including PK/PD analyses.     

Combined autophagy and HDAC inhibition

We previously reported that inhibition of autophagy significantly augmented the anticancer activity of the histone deacetylase (HDAC) inhibitor vorinostat (VOR) through a cathepsin D-mediated mechanism. We thus conducted a first-in-human study to investigate the safety, preliminary efficacy, pharmacokinetics (PK), and pharmacodynamics (PD) of the combination of the autophagy inhibitor hydroxychloroquine (HCQ) and VOR in patients with advanced solid tumors. Of 27 patients treated in the study, 24 were considered fully evaluable for study assessments and toxicity. Patients were treated orally with escalating doses of HCQ daily (QD) (d 2 to 21 of a 21-d cycle) in combination with 400 mg VOR QD (d one to 21). Treatment-related adverse events (AE) included grade 1 to 2 nausea, diarrhea, fatigue, weight loss, anemia, and elevated creatinine. Grade 3 fatigue and/or myelosuppression were observed in a minority of patients. Fatigue and gastrointestinal AE were dose-limiting toxicities. Six-hundred milligrams HCQ and 400 mg VOR was established as the maximum tolerated dose and recommended phase II regimen. One patient with renal cell carcinoma had a confirmed durable partial response and 2 patients with colorectal cancer had prolonged stable disease. The addition of HCQ did not significantly impact the PK profile of VOR. Treatment-related increases in the expression of CDKN1A and CTSD were more pronounced in tumor biopsies than peripheral blood mononuclear cells. Based on the safety and preliminary efficacy of this combination, additional clinical studies are currently being planned to further investigate autophagy inhibition as a new approach to increase the efficacy of HDAC inhibitors.     

Combined autophagy and HDAC inhibition: A phase I safety,tolerability, pharmacokinetic,and pharmacodynamic analysis of hydroxychloroquine in combination with the HDAC inhibitor vorinostat in patients with advanced solid tumors

We previously reported that inhibition of autophagy significantly augmented the anticancer activity of the histone deacetylase (HDAC) inhibitor vorinostat (VOR) through a cathepsin D-mediated mechanism. We thus conducted a first-in-human study to investigate the safety, preliminary efficacy, pharmacokinetics (PK), and pharmacodynamics (PD) of the combination of the autophagy inhibitor hydroxychloroquine (HCQ) and VOR in patients with advanced solid tumors. Of 27 patients treated in the study, 24 were considered fully evaluable for study assessments and toxicity. Patients were treated orally with escalating doses of HCQ daily (QD) (d 2 to 21 of a 21-d cycle) in combination with 400 mg VOR QD (d one to 21). Treatment-related adverse events (AE) included grade 1 to 2 nausea, diarrhea, fatigue, weight loss, anemia, and elevated creatinine. Grade 3 fatigue and/or myelosuppression were observed in a minority of patients. Fatigue and gastrointestinal AE were dose-limiting toxicities. Six-hundred milligrams HCQ and 400 mg VOR was established as the maximum tolerated dose and recommended phase II regimen. One patient with renal cell carcinoma had a confirmed durable partial response and 2 patients with colorectal cancer had prolonged stable disease. The addition of HCQ did not significantly impact the PK profile of VOR. Treatment-related increases in the expression of CDKN1A and CTSD were more pronounced in tumor biopsies than peripheral blood mononuclear cells. Based on the safety and preliminary efficacy of this combination, additional clinical studies are currently being planned to further investigate autophagy inhibition as a new approach to increase the efficacy of HDAC inhibitors.     

Antimicrobial breakpoint estimation accounting for variability in pharmacokinetics

Background  

Pharmacokinetic and pharmacodynamic (PK/PD) indices are increasingly being used in the microbiological field to assess the efficacy of a dosing regimen. In contrast to methods using MIC, PK/PD-based methods reflect in vivo conditions and are more predictive of efficacy. Unfortunately, they entail the use of one PK-derived value such as AUC or Cmax and may thus lead to biased efficiency information when the variability is large. The aim of the present work was to evaluate the efficacy of a treatment by adjusting classical breakpoint estimation methods to the situation of variable PK profiles.     

抗体偶联药物的技术现状和展望

抗体偶联药物（antibody drug conjugate,ADC）通常由抗体通过链接体与毒素小分子偶联而成,同时具备抗体的高靶向性和小分子药物的高活性,使之作为一种新兴的靶向治疗手段,在肿瘤治疗领域展现出了优秀的疗效和潜力,成为药物研发领域的新热点。目前全球已有14款ADC药物获批上市,处于临床研究阶段的ADC候选药物分子超过140个。为了进一步提高ADC药物的安全性和有效性,近年来涌现出了各种新颖的技术。本文对ADC药物分子的关键元素,包括抗体、链接体、毒素小分子以及偶联技术等方面的最新研究进展进行总结,并讨论其优缺点。期望这些讨论能够帮助增加对ADC药物研究和开发更加系统的理解,为研发出更加高效和安全的ADC药物带来一些思考。     

Dose dependent pharmacokinetics,tissue distribution,and anti-tumor efficacy of a humanized monoclonal antibody against DLL4 in mice

Delta-like-4 ligand (DLL4) plays an important role in vascular development and is widely expressed on the vasculature of normal and tumor tissues. Anti-DLL4 is a humanized IgG1 monoclonal antibody against DLL4. The purpose of these studies was to characterize the pharmacokinetics (PK), tissue distribution, and anti-tumor efficacy of anti-DLL4 in mice over a range of doses. PK and tissue distribution of anti-DLL4 were determined in athymic nude mice after administration of single intravenous (IV) doses. In the tissue distribution study, radiolabeled anti-DLL4 (mixture of ¹²⁵Iodide and ¹¹¹Indium) was administered in the presence of increasing amounts of unlabeled anti-DLL4. Dose ranging anti-DLL4 anti-tumor efficacy was evaluated in athymic nude mice bearing MV522 human lung tumor xenografts. Anti-DLL4 had nonlinear PK in mice with rapid serum clearance at low doses and slower clearance at higher doses suggesting the involvement of target mediated clearance. Consistent with the PK data, anti-DLL4 was shown to specifically distribute to several normal tissues known to express DLL4 including the lung and liver. Maximal efficacy in the xenograft model was seen at doses ≥ 10 mg/kg when tissue sinks were presumably saturated, consistent with the PK and tissue distribution profiles. These findings highlight the importance of mechanistic understanding of antibody disposition to enable dosing strategies for maximizing efficacy.     

Dose dependent pharmacokinetics,tissue distribution,and anti-tumor efficacy of a humanized monoclonal antibody against DLL4 in mice

Delta-like-4 ligand (DLL4) plays an important role in vascular development and is widely expressed on the vasculature of normal and tumor tissues. Anti-DLL4 is a humanized IgG1 monoclonal antibody against DLL4. The purpose of these studies was to characterize the pharmacokinetics (PK), tissue distribution, and anti-tumor efficacy of anti-DLL4 in mice over a range of doses. PK and tissue distribution of anti-DLL4 were determined in athymic nude mice after administration of single intravenous (IV) doses. In the tissue distribution study, radiolabeled anti-DLL4 (mixture of ¹²⁵Iodide and ¹¹¹Indium) was administered in the presence of increasing amounts of unlabeled anti-DLL4. Dose ranging anti-DLL4 anti-tumor efficacy was evaluated in athymic nude mice bearing MV522 human lung tumor xenografts. Anti-DLL4 had nonlinear PK in mice with rapid serum clearance at low doses and slower clearance at higher doses suggesting the involvement of target mediated clearance. Consistent with the PK data, anti-DLL4 was shown to specifically distribute to several normal tissues known to express DLL4 including the lung and liver. Maximal efficacy in the xenograft model was seen at doses ≥ 10 mg/kg when tissue sinks were presumably saturated, consistent with the PK and tissue distribution profiles. These findings highlight the importance of mechanistic understanding of antibody disposition to enable dosing strategies for maximizing efficacy.     

Impact of drug conjugation on pharmacokinetics and tissue distribution of anti-STEAP1 antibody-drug conjugates in rats

Antibody-drug conjugates (ADCs) are designed to combine the exquisite specificity of antibodies to target tumor antigens with the cytotoxic potency of chemotherapeutic drugs. In addition to the general chemical stability of the linker, a thorough understanding of the relationship between ADC composition and biological disposition is necessary to ensure that the therapeutic window is not compromised by altered pharmacokinetics (PK), tissue distribution, and/or potential organ toxicity. The six-transmembrane epithelial antigen of prostate 1 (STEAP1) is being pursued as a tumor antigen target. To assess the role of ADC composition in PK, we evaluated plasma and tissue PK profiles in rats, following a single dose, of a humanized anti-STEAP1 IgG1 antibody, a thio-anti-STEAP1 (ThioMab) variant, and two corresponding thioether-linked monomethylauristatin E (MMAE) drug conjugates modified through interchain disulfide cysteine residues (ADC) and engineered cysteines (TDC), respectively. Plasma PK of total antibody measured by enzyme-linked immunosorbent assay (ELISA) revealed ～45% faster clearance for the ADC relative to the parent antibody, but no apparent difference in clearance between the TDC and unconjugated parent ThioMab. Total antibody clearances of the two unconjugated antibodies were similar, suggesting minimal effects on PK from cysteine mutation. An ELISA specific for MMAE-conjugated antibody indicated that the ADC cleared more rapidly than the TDC, but total antibody ELISA showed comparable clearance for the two drug conjugates. Furthermore, consistent with relative drug load, the ADC had a greater magnitude of drug deconjugation than the TDC in terms of free plasma MMAE levels. Antibody conjugation had a noticeable, albeit minor, impact on tissue distribution with a general trend toward increased hepatic uptake and reduced levels in other highly vascularized organs. Liver uptakes of ADC and TDC at 5 days postinjection were 2-fold and 1.3-fold higher, respectively, relative to the unmodified antibodies. Taken together, these results indicate that the degree of overall structural modification in anti-STEAP1-MMAE conjugates has a corresponding level of impact on both PK and tissue distribution.     

An integrated disease/pharmacokinetic/pharmacodynamic model suggests improved interleukin-21 regimens validated prospectively for mouse solid cancers

Interleukin (IL)-21 is an attractive antitumor agent with potent immunomodulatory functions. Yet thus far, the cytokine has yielded only partial responses in solid cancer patients, and conditions for beneficial IL-21 immunotherapy remain elusive. The current work aims to identify clinically-relevant IL-21 regimens with enhanced efficacy, based on mathematical modeling of long-term antitumor responses. For this purpose, pharmacokinetic (PK) and pharmacodynamic (PD) data were acquired from a preclinical study applying systemic IL-21 therapy in murine solid cancers. We developed an integrated disease/PK/PD model for the IL-21 anticancer response, and calibrated it using selected "training" data. The accuracy of the model was verified retrospectively under diverse IL-21 treatment settings, by comparing its predictions to independent "validation" data in melanoma and renal cell carcinoma-challenged mice (R(2)>0.90). Simulations of the verified model surfaced important therapeutic insights: (1) Fractionating the standard daily regimen (50 μg/dose) into a twice daily schedule (25 μg/dose) is advantageous, yielding a significantly lower tumor mass (45% decrease); (2) A low-dose (12 μg/day) regimen exerts a response similar to that obtained under the 50 μg/day treatment, suggestive of an equally efficacious dose with potentially reduced toxicity. Subsequent experiments in melanoma-bearing mice corroborated both of these predictions with high precision (R(2)>0.89), thus validating the model also prospectively in vivo. Thus, the confirmed PK/PD model rationalizes IL-21 therapy, and pinpoints improved clinically-feasible treatment schedules. Our analysis demonstrates the value of employing mathematical modeling and in silico-guided design of solid tumor immunotherapy in the clinic.     

Pharmacokinetic / pharmacodynamic relationships of liposomal amphotericin B and miltefosine in experimental visceral leishmaniasis

BackgroundThere is a continued need to develop effective and safe treatments for visceral leishmaniasis (VL). Preclinical studies on pharmacokinetics and pharmacodynamics of anti-infective agents, such as anti-bacterials and anti-fungals, have provided valuable information in the development and dosing of these agents. The aim of this study was to characterise the pharmacokinetic and pharmacodynamic properties of the anti-leishmanial drugs AmBisome and miltefosine in a preclinical disease model of VL.Methodology / Principal findingsBALB/c mice were infected with L. donovani (MHOM/ET/67/HU3) amastigotes. Groups of mice were treated with miltefosine (orally, multi-dose regimen) or AmBisome (intravenously, single dose regimen) or left untreated as control groups. At set time points groups of mice were killed and plasma, livers and spleens harvested. For pharmacodynamics the hepatic parasite burden was determined microscopically from tissue impression smears. For pharmacokinetics drug concentrations were measured in plasma and whole tissue homogenates by LC-MS. Unbound drug concentrations were determined by rapid equilibrium dialysis. Doses exerting maximum anti-leishmanial effects were 40 mg/kg for AmBisome and 150 mg/kg (cumulatively) for miltefosine. AmBisome displayed a wider therapeutic range than miltefosine. Dose fractionation at a total dose of 2.5 mg/kg pointed towards concentration-dependent anti-leishmanial activity of AmBisome, favouring the administration of large doses infrequently. Protein binding was >99% for miltefosine and amphotericin B in plasma and tissue homogenates.Conclusion / SignificanceUsing a PK/PD approach we propose optimal dosing strategies for AmBisome. Additionally, we describe pharmacokinetic and pharmacodynamic properties of miltefosine and compare our findings in a preclinical disease model to available knowledge from studies in humans. This approach also presents a strategy for improved use of animal models in the drug development process for VL.     

Ozanezumab Dose Selection for Amyotrophic Lateral Sclerosis by Pharmacokinetic-Pharmacodynamic Modelling of Immunohistochemistry Data from Patient Muscle Biopsies

Amyotrophic Lateral Sclerosis (ALS) is a rare and fatal neurodegenerative disease with a high unmet medical need. In this context, a potential therapy should be brought to patients in the most expeditious way and early exploration of pharmacology is highly beneficial. Ozanezumab, a humanised IgG monoclonal antibody against Nogo-A protein which is an inhibitor of neurite outgrowth, is currently under development for the treatment of ALS and has been recently assessed in 76 patients in a first-in-human study. Inadequate target engagement has been recognised as a major contributing reason for drug trial failures. In this work, we describe the development of a pharmacokinetic-pharmacodynamic (PKPD) model using immunohistochemistry (IHC) data of co-localization of ozanezumab with Nogo-A in skeletal muscle as a surrogate measure of target engagement. The rich plasma concentration data and the sparse IHC data after one or two intravenous doses of ozanezumab were modelled simultaneously using a non-linear mixed-effect approach. The final PKPD model was a two-compartment PK model combined with an effect compartment PD model that accounted for the delay in ozanezumab concentrations to reach the site of action which is skeletal muscle. Diagnostic plots showed a satisfactory fit of both PK and IHC data. The model was used as a simulation tool to design a dose regimen for sustained drug-target co-localization in a phase II study.     

Preclinical pharmacokinetics of MHAA4549A,a human monoclonal antibody to influenza A virus,and the prediction of its efficacious clinical dose for the treatment of patients hospitalized with influenza A

MHAA4549A is a human immunoglobulin G1 (IgG1) monoclonal antibody that binds to a highly conserved epitope on the stalk of influenza A hemagglutinin and blocks the hemagglutinin-mediated membrane fusion in the endosome, neutralizing all known human influenza A strains. Pharmacokinetics (PK) of MHAA4549A and its related antibodies were determined in DBA/2J and Balb-c mice at 5 mg/kg and in cynomolgus monkeys at 5 and 100 mg/kg as a single intravenous dose. Serum samples were analyzed for antibody concentrations using an ELISA and the PK was evaluated using WinNonlin software. Human PK profiles were projected based on the PK in monkeys using species-invariant time method. The human efficacious dose projection was based on in vivo nonclinical pharmacological active doses, exposure in mouse infection models and expected human PK. The PK profiles of MHAA4549A and its related antibody showed a linear bi-exponential disposition in mice and cynomolgus monkeys. In mice, clearance and half-life ranged from 5.77 to 9.98 mL/day/kg and 10.2 to 5.76 days, respectively. In cynomolgus monkeys, clearance and half-life ranged from 4.33 to 4.34 mL/day/kg and 11.3 to 11.9 days, respectively. The predicted clearance in humans was ～2.60 mL/day/kg. A single intravenous dose ranging from 15 to 45 mg/kg was predicted to achieve efficacious exposure in humans. In conclusion, the PK of MHAA4549A was as expected for a human IgG1 monoclonal antibody that lacks known endogenous host targets. The predicted clearance and projected efficacious doses in humans for MHAA4549A have been verified in a Phase 1 study and Phase 2a study, respectively.     

Targeting Trop-2 in cancer: Recent research progress and clinical application

The development of new antitumor drugs depends mainly upon targeting tumor cells precisely. Trophoblast surface antigen 2 (Trop-2) is a type I transmembrane glycoprotein involved in Ca²⁺ signaling in tumor cells. It is highly expressed in various tumor tissues than in normal tissues and represents a novel and promising molecular target for caner targeted therapy. Up to now, the mechanisms and functions associated with Trop-2 have been extensively studied in a variety of solid tumors. According to these findings, Trop-2 plays an important role in cell proliferation, apoptosis, cell adhesion, epithelial-mesenchymal transition, as well as tumorigenesis and tumor progression. In addition, Trop-2 related drugs are also being developed widely. There are a number of Trop-2 related ADC drugs that have demonstrated potent antitumor activity and are currently been studied, such as Sacituzumab Govitecan (SG) and Datopotamab Deruxtecan (Dato-Dxd). In this study, we reviewed the progress of Trop-2 research in solid tumors. We also sorted out the composition and rationale of Trop-2 related drugs and summarized the related clinical trials. Finally, we discussed the current status of Trop-2 research and expanded our perspectives on its future research directions. Importantly, we found that Trop-2 targeted ADCs have great potential for combination with other antitumor therapies. Trop-2 targeted ADCs can reprogramme tumor microenvironment through multiple signaling pathways, ultimately activating antitumor immunity.     

Dose-dependent functional benefit of human cardiosphere transplantation in mice with acute myocardial infarction

Despite mounting pre-clinical and clinical evidence of the beneficial effects of cell-based therapy, optimal cell dosing and delivery approaches have not been identified. Cardiospheres are self-assembling three-dimensional (3D) microtissues formed by cardiac stem cells and supporting cell types. The ability of cardiospheres to augment cardiac function has been demonstrated in animal models of ischemic cardiomyopathy. In this study, we studied the dose dependence of the benefits of human cardiospheres, delivered via intramyocardial injection, upon cardiac function and ventricular remodelling in SCID mice with acute myocardial infarction. Four doses of cardiospheres were used: 1 × 10(4), 5 × 10(4), 1 × 10(5) and 5 × 10(5) (expressed as number of plated cardiosphere-forming cells). Acute (24 hr) cell retention rates in all groups were similar. Functional assessment and quantitative heart morphometry indicated benefit from higher cell doses (≥5 × 10(4)) in terms of ejection fraction, infarct size and capillary density. Histological analysis indicated that the dose-dependent benefit was primarily because of indirect effects of transplanted cells. The results provide scalable data on cardiosphere dosing for intramyocardial injection.     

The same drug but a different mechanism of action: comparison of free doxorubicin with two different N-(2-hydroxypropyl)methacrylamide copolymer-bound doxorubicin conjugates in EL-4 cancer cell line

Doxorubicin is one of the most potent anti-tumor drugs with a broad spectrum of use. To reduce its toxic effect and improve its pharmacokinetics, we conjugated it to an HPMA copolymer carrier that enhances its passive accumulation within solid tumors via the EPR effect and decreases its cytotoxicity to normal, noncancer cells. In this study, we compared the antiproliferative, pro-survival, and death signals triggered in EL-4 cancer cells exposed to free doxorubicin and doxorubicin conjugated to a HPMA copolymer carrier via either enzymatically (PK1) or hydrolytically (HYD) degradable bonds. We have previously shown that the intracellular distribution of free doxorubicin, HYD, and PK1 is markedly different. Here, we demonstrated that these three agents greatly differ also in the antiproliferative effect and cell death signals they trigger. JNK phosphorylation sharply increased in cells treated with HYD, while treatment with free doxorubicin moderately decreased and treatment with PK1 even strongly decreased it. On the other hand, treatment with free doxorubicin greatly increased p38 phosphorylation, while PK1 and HYD increased it slightly. PK1 also significantly increased ERK phosphorylation, while both the free doxorubicin and HYD conjugate slightly decreased it. Long-term inhibition of JNK significantly increased both proliferation and viability of EL-4 cells treated with free doxorubicin, showing that the JNK signaling pathway could be critical for mediating cell death in EL-4 cells exposed to free doxorubicin. Both activation of caspase 3 and decreased binding activity of the p50 subunit of NFkappaB were observed in cells treated with free doxorubicin and HYD, while no such effects were seen in cells incubated with PK1. Analysis of the expression of genes involved in apoptosis and regulation of the cell cycle demonstrated that free doxorubicin and HYD have very similar mechanisms of action, while PK1 has very different characteristics.