Embed Size (px)
Citation preview
Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1395
Methods for PreclinicalEvaluation of Cytotoxic DrugsWith Special Reference to the Cyanoguanidine CHS 828
and Hollow Fiber Method
BY
SAADIA BASHIR HASSAN
ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2004
“It is only little of knowledge that is communicated to us”
To my father To Sidahmed, Alaa and MohammedTo my sisters, my brothers and to the memory of my mother.
Contents
INTRODUCTION ........................................................................................11Chemotherapy of malignant tumors .........................................................11Dosing strategy and schedule dependency ...............................................12Apoptosis and necrosis.............................................................................13Drug development process .......................................................................13
Activity profile ....................................................................................13Multicellular, three-dimensional model system...................................14Animal tumor model system................................................................14Pharmacokinetic-Pharmacodynamic modelling (PK/PD) ...................15
The transcription factor kappa B, NF-kB, and Cancer .............................16Drugs of interest in this thesis ..................................................................17
The novel cyanoguanidine CHS 828 ...................................................17Pacliex, a new paclitaxel formulation..................................................18
AIMS OF THE STUDY ...............................................................................19
MATERIAL AND METHODS....................................................................20Human Tumor Cell Lines (HTCL)...........................................................20Primary human tumor cells (PHTC) and normal lymphocytes ................20Animals ....................................................................................................20Reagents and drugs...................................................................................21Hollow fiber method (paper I, III and V).................................................21Morphology assessment and TUNEL assay (paper I) ..............................22The fluorometric microculture cytotoxicity assay procedure (FMCA) (paper II, IV and V)..................................................................................22Pharmacokinetic monitoring and chemical assay (paper III) ...................23In vivo pharmacokinetic monitoring, general and haematological toxicities (paper III)..................................................................................23The models and data analysis (paper II and III) .......................................23Effect of CHS 828 on NF-kB translocation and the ArrayScan system (paper IV) .................................................................................................24Proteasome enzyme activity (paper IV) ...................................................24
RESULTS AND DISCUSSION ...................................................................25Hollow fiber cultures................................................................................25
In vitro antitumor activity (paper I and V) ..........................................25
Morphology assessment (paper I)........................................................27In vivo antitumor activity (paper III and V) ........................................27Hollow fiber method, general discussion ............................................29
Schedule dependency ...............................................................................31In vitro modelling of CHS 828 and standard drugs effects (paper II) .31Additional studies on the in vitro time dependence of CHS 828 (Unpublished data)...............................................................................32In vivo PK modelling (paper III) .........................................................34Toxicity (paper III) ..............................................................................35In vivo PD modelling (paper III) .........................................................36General discussion on CHS 828 schedule dependence .......................36
CHS 828 and the nuclear factor kappa B, NF-kB (paper IV)...................38Pacliex activity in cell suspension cultures, FMCA (paper V) ................39
SUMMARY AND CONCLUSIONS ...........................................................41
ACKNOWLEDGEMENT ............................................................................42
REFERENCES .............................................................................................44
Abbreviations
AUC area under the concentration-time curve AUEThresh threshold cumulative effect on the cells B cell survival at infinite drug concentration C drug concentration CHS 828 N-(6-(4-chlorophenoxy)hexyl)-N´-cyano-N´´-4-
pyridylguanidine CL oral clearance CLL chronic lymphocytic leukemia DMSO dimethyl sulphoxide E cell survival in percent of control EC50, IC50 concentration giving 50 % of the maximum effect Econ control cell survival at zero drug concentration Econb cell survival at the first plateau of a double Hill curve Emax maximal effect F1 fraction absorbed on the day of administration F2 fraction absorbed on a second absorption phase observed
after a lag time FDA fluorescein diacetate FMCA fluorometric microculture cytotoxicity assay FOCE first-order conditional estimation method in NONMEM HCS high content screening HPLC high performance liquid chromatography IIV inter-individual variability IkB inhibitory kappa B IKK inhibitory kappa B kinase K exposure constantKa,1 absorpion rate constant on the day of administration Ka,2 absorpion rate constant of a second absorption phase after a
lag time Ke rate constant of decrease of EconbKF rate constant of dose-dependent decrease fraction absorbedKGrow growth rate of tumor cells MGG May-Grünwald-Giemsa MTT [3-4,5-dimethylthiaxol-2-yl]-2,5-diphenyltetrazolium bro-
mide
n concentration coefficientNCI national Cancer Institute NF-kB nuclear factor kappa B NONMEM non-linear mixed-effect model OC ovarian cancer OD optical density OFV objective function value (produced by NONMEM) OV Oasmia vehicle PBS phosphate buffered saline PD pharmacodynamic PK pharmacokinetic PVDF Polyvinylidine fluoride R1 zero-order rate input on the day of administration S slope parameter of the time-IC50 curve SCID severe combined immunodeficiency SCLC small cell lung cancer cell line SD standard deviation SEM standard error of the mean SI survival index SlopeCL coefficient of linear time-dependent decrease in clearance T exposure duration to a drug t1/2 half-lifeTNF tumor necrosis factor alpha UV ultra violet V volume of distribution XR17 Oasmia surfactant slope parameter
11
INTRODUCTION
Chemotherapy of malignant tumorsThe post World War II era witnessed the availability of chemical agents with potential value for cancer treatment. The first documented clinical use of cancer chemotherapy was in 1942 when the alkylating agent nitrogen mus-tard was used to obtain a brief clinical remission in a patient with lymphoma. Although the tumor rapidly relapsed after an initial pronounced antitumor effect [1], this experience marked the beginning of modern chemotherapy of malignant tumors. The intention of cancer treatment may be remission or rarely, cure from the disease as with certain forms of childhood leukemia that has a 90% probability of cure [2]. There are more than 40 cytotoxic drugs in clinical use for cancer treatment in Sweden. These drugs are classi-fied into main groups according to their mechanisms of action. The groups include alkylating agents such as melphalan, microtubuli active compound such as the taxane paclitaxel, antimetabolites such as methotrexate, platinum compounds such as cisplatin and topo I and topo II inhibitors such as topo-tecan and etoposide respectively [3].
The principle strategies used in the search for new compounds with anti-tumor activity falls within four general categories.
Empirical observation such as for example the observation that ni-trogen mustard was toxic to normal lymphoid tissue. Screening of various chemical compounds from chemical libraries for antitumor effect using different experimental screening systems. Analog synthesis for drugs of known activity. Rational design of new drugs interfering with a known cellular bio-chemical target.
The therapies now available for tumors often give rise to side effects so harmful that they compromise the benefits of treatment. The available anti-cancer drugs as a group, with recent few exceptions, are similar in their spec-trum of clinical activity, toxicity and mechanisms of action [4] . Accord-ingly, there is a need for new cytotoxic drugs with unique mechanisms of action and resistance patterns.
12
Dosing strategy and schedule dependency The choice of dosing strategy is an important objective when a new drug is brought into clinical development. Differences in drug efficacy and toxicity might be achieved by giving the drug in different doses and schedules. Many anticancer drugs show schedule dependence [5] where the response and/or toxicity magnitude depends on the administration rate. Several mechanisms are possibly underlying this schedule dependence including multiple mecha-nisms of drug action, time-dependent cellular repair mechanisms, cell-cycle specificity and saturable transport into or out of cells [6, 7]. For example, the antitumor activity of the topoisomerase II inhibiting drug, etoposide, is clearly schedule dependent. This is suggested to be related to the observation that the activity of topoisomerase II varies throughout the cell cycle, a cell-cycle phase specificity, and to drug interaction with DNA, preventing DNA repair [8, 9]. Apparent schedule dependence in the pharmacodynamic (PD) could be due to dose-dependencies and circadian rhythms in pharmacoki-netic (PK) behavior if the PK is not characterized.
To aid in the selection of a proper dosing schedule for clinical trials, pre-clinical studies in vitro and in vivo can be performed to compare the antitu-mor effect after different drug concentrations and drug exposure times. Until drugs with more tumor-specific effects are well established, PK/PD models are desirable for the characterization of both the antitumor effect and the toxicity, to help in decisions on the choice of doses and schedules of treat-ment of established and new chemotherapeutic drugs. Modelling of the in-fluence of exposure time and drug concentration on drug effect in vitro can be used for optimizing the administration regimens to be adopted in vivo and might be useful in providing insights on mechanisms of drug action. How-ever, the dependence of in vitro cytotoxic effect on time and concentration might be influenced by many factors including the cell system and the ex-perimental conditions used such as the artifacts from residual drug remaining for long-term incubation times after short exposure time or possible degrada-tion of drug to active or inactive metabolites.
The effect on the normal cells, the PK of the drug and regrowth of tumor cells play important roles in dose regimen selection. The in vivo search for the optimal dosing schedule might therefore be more relevant to the clinical settings than the in vitro settings. However, it is only possible to study a few schedules at a time in vivo. A more extensive search can be done in vitro. In addition, the use of computer modelling allows a more efficient detection and characterization of schedule dependence and the findings can help to guide which schedules are appropriate to investigate in vivo.
13
Apoptosis and necrosis The term necrosis, passive cell death, has been used to describe the sum of changes occurring in the cells after they have died regardless the prelethal process. Since the late 1980’s the concept of apoptosis, as a counterpoint to necrosis, has been widely accepted. Apoptosis is an active process by indi-vidual cells that lead to cell death and is characterized by cytoplasmic shrinkage and fragmentation of the nuclei [10]. A recent report shows that the cells undergo apoptosis and/or necrosis based on the consequence of events after sharing a common phase in the cell death process. [11].
Today it is widely accepted that the majority of cytotoxic compounds act primarily by inducing cancer cell death through the mechanisms of apoptosis [12]. Some cancers (primarily leukemia and lymphoma) are very susceptible to apoptosis [13] while most solid tumors are unable to activate the apoptotic machinery and may therefore be resistant to chemotherapy [14].
Drug development process Irrespective of how the antitumor activity is observed, the subsequent devel-opment relies on testing of the compound in preclinical systems. A lot of information needs to be collected before starting the clinical trials program. The preclinical program usually involves in vitro evaluation against various cell lines followed by in vivo evaluation mostly in mice bearing xenograft tumors. The toxicity evaluation is usually performed in non-tumor bearing animals before testing the new compound in man.
Activity profile Since 1959, the National Cancer Institute (NCI) has utilized various experi-mental screening models to select agents for evaluation as clinical candi-dates. During the early the 1990s, the Developmental Therapeutics Program of NCI, established a large scale anticancer drug-screening program based on drug dose-response experiments with 60 human tumor cell types grown and treated in monolayer cultures [15]. The resulting dose-response patterns are used to select potentially active compounds for further study and can also be used to predict mechanisms of drug action. Our laboratory has shown the possibility of using a panel with only 10 cell lines for preliminary classifica-tion of new compounds [16].
14
Multicellular, three-dimensional model system The conventional in vitro monolayer cultures are oversimplistic in that they do not mimic the complex and heterogeneous properties of a solid tumor. In vitro models, that take into consideration the heterogeneous nature of the tumors as they grow and the poor drug penetration in a multicellular system, may provide important information to be used in drug development.
Many three-dimensional in vitro solid tumor models have been developed and have been widely used in biomedical research to overcome the limitation of monolayer cultures such as the multicellular tumor spheroids [17, 18] and the collagen gel system [19].
In vitro hollow fiber model system Tumor cells have been shown to grow to confluence and form heterogeneous in vitro solid masses inside semi-permeable polyvinylidine fluoride (PVDF) hollow fibers. The ability of the human tumor cell lines to grow inside the hollow fibers allows drug sensitivity studies with different tumor types to be interpreted in light of differences in the microenvironment and proliferative heterogeneity [20]. The hollow fibers are easy to work with as single entities and have high molecular weight cut-off that allows the passage of nutrients, cytotoxic drugs and staining dyes. The hollow fibers are liable to standard histologic fixation and staining procedures, which makes them suitable for histological and cytological documentation.
Animal tumor model systemIn vitro experiments may give information about the nature of the drug-receptor interaction, but not on many of the other complex interrelationships that exist in vivo. Animal studies bridge much of the gap between the in vitro experiments and human investigations. The primary aims of the animal studies are to obtain further information with respect to antitumor activity, toxicity and the starting dose of the anticancer agents before commencing the first trial in man.
Development of animal models to evaluate candidate compounds has been actively pursued by the NCI since 1955 using transplanted rodent mod-els. By 1975 the Developmental Therapeutics Program at the NCI adopted the use of human tumor xenografts in immunodeficient athymic mice lack-ing T-cells and in SCID mice (severe combined immunonodeficiency) lack-ing T-and B-cells, to avoid host immune interference, with the intent of bet-ter prediction of clinical activity against solid human tumors [21]. This model is the major model system for drug development where tumor cells are grown subcutaneously and the model allows rapid quantitative assess-ment of antitumor activity relative to mouse toxicity [22]. Compounds with activity in more than 30% of NCI xenograft models showed activity in at
15
least some phase II trials [23]. However, xenograft models in immunosup-pressive mice are accompanied by limitations including high costs in labor and in time due to the special care the mice need. In addition, only one tumor type can be implanted per animal.
In vivo hollow fiber model The in vivo screening path at the NCI was modified more to include a more rapid and efficient way to select compounds with in vivo activity. Com-pounds with interesting in vitro activity are tested in the in vivo hollow fiber system in athymic mice [24]. The hollow fibers have shown to be biocom-patible with human tumor cells and suitable for supporting the growth of tumor cells within two compartments; subcutaneous and intraperitoneal sites, of the host animal. The hollow fiber technique showed the potential to be used in the field of angiogenesis in mice and this has direct implications for drug delivery and chemosensitivity [25]. Recently, the hollow fiber model was modified by our group to perform studies in immunocompetent rats [26, 27]. Using the in vivo hollow fiber model in rats, makes it possible to study the effects of anticancer drugs, in several cell lines as well as in primary human tumor cultures, the hematological toxicity and pharmacokinetics within the same animal. Non-immunocompromised animals are easy to han-dle, and the rats can host many tumor types at a time and can withstand re-peated bleedings for pharmacokinetics characterization and for simultaneous hematological toxicity evaluation.
Pharmacokinetic-Pharmacodynamic modelling (PK/PD) Pharmacokinetics and pharmacodynamics attempt to relate the interaction of a drug with a biological environment. The reasons for modelling data from in vitro, in vivo and from clinical trials are to summarize and describe the data in a simple way and to predict the expected response when a new dose is given or when a change in PK or PD characteristics occur.
Modelling of time dependency in vitro The effect of a cytotoxic drug on tumor cell growth in vitro can be described in relation to drug concentration and exposure time. For example, one can determine whether the exposure of tumor cells to high concentration of a drug for short time can produce a greater effect than a more prolonged expo-sure to lower concentrations. Different pharmacodynamic models are used to study the time-dependence of cytotoxic drug effect in vitro [28-30]. A phar-macodynamic model that relate both exposure time and drug concentration to the resulting effect was first reported by Kalns group [31]. Another mod-elling approach for quantitative assessment of the contributions of drug con-centration and exposure time on in vitro growth inhibition was introduced by Levasseur et al [32]. The approach offers the opportunity to gain information
16
that can be used to formulate hypotheses regarding the possible mechanism of action and the response heterogeneity of the tumor cells. It can also be used for optimizing the administration regimens to be adopted in vivo. Re-cently, a mathematical model has been described to predict the dependence of survival on the time course of exposure for cisplatin, taking into account the kinetics of cisplatin uptake by the cells [33] .
Modelling of time dependency in vivo (PK/PD modelling) Studying the effect of drug scheduling in vitro does not often include infor-mation about normal cell toxicity, which is an important consideration in the clinical use of cytotoxic drugs. PK/PD models that link the administration regimen and the plasma concentrations of a candidate compound to the tu-mor response have the potential to improve the preclinical development of new drugs. Optimally, a PK/PD model will allow investigation of both dose-limiting toxicity and antitumor effects. The population modelling approach involves both PK and PD modeling. The PK/PD models can provide vital aid to the drug development process by providing reliable predictions of the individualized dose exposure-effect relationship (effect refers to both effi-cacy and toxicity) which is the key to successful therapy [34].
Apparent schedule dependence in drug response could be due to dose de-pendencies of the PK of the drug, therefore characterization of the PK of a drug is important to understand the influence of different schedules on the effect of a drug. Few PK models have been established for anticancer drugs [35, 36]. Several PK/PD models that can estimate the time course of chemo-therapy-induced toxicity (myelosuppression), have been published [37, 38]; however, there is only one publication that model the time course of tumor effect in vivo [39].
The transcription factor kappa B, NF-kB, and Cancer The emerging understanding of the molecular basis for human cancer includ-ing the extensive knowledge on the regulatory events in the pathways of the cellular response may provide insight into new approaches to cancer treat-ment. Among the signal transduction pathways that have been recognized as potentially relevant targets for selective agents is the nuclear factor kappa B, NF-kB, signalling pathway [40]. The transcription factor NF-kB and the signalling pathways that are involved in its activation are important for tu-mor development. Several recent reports have shown that activation of NF-kB is part of a regulatory circle that works to prevent cell death; most likely by inducing the expression of anti-apoptotic genes [41]. However, NF-kBcan also have pro-apoptotic effects depending upon cell type and the nature of stress [42, 43]. The NF-kB subunits (p65 and p50) are kept in the cyto-plasm of the quiescent cells in a complex with an inhibitor protein known as
17
IkB , . On activation by, for example, the cytokines TNF and IL-1, dif-ferent serine kinases such as IKK complex phosphorylate IkB / , marking it for ubiqitination and degradation by proteasome. Consequently, NF-kB sub-units translocate to the nucleus and, bind to a specific sequence in the DNA, which in turn results in gene transcription. Protein degradation is an impor-tant mechanism of cellular regulation and 26S proteasomes are the proteases responsible for selective degradation of the majority of endogenous cellular proteins that are targeted by ubiquitination system [44].
Possible therapeutic targets in the signalling pathway of NF-kB could be the activity of the kinases, the cytoplasmic proteases and the binding of nu-clear NF-kB to DNA. Recently a number of drugs have been developed and proposed to interfere with specific targets in this pathway in the tumor cell. Bortezomib, PS 341, (Velcade) is a proteasome inhibitor, that has been re-cently registered for treatment of myeloma Other examples of drugs that have entered the clinical development process, are the immunormodulatory drugs and analogues such as IMiD CC-5013 (Revimid) for multiple mye-loma and metastatic myeloma, and IMiD CC-4047 (Actimid) for multiple myeloma and prostate cancer [45].
Drugs of interest in this thesis The novel cyanoguanidine CHS 828 This thesis focuses on CHS 828, N-(6-(4-chlorophenoxy) hexyl)-N´-cyano-N´´-4-pyridylguanidine. It is a novel cytotoxic agent that has shown promis-ing antitumor activity in many preclinical systems. Currently, phase II clini-cal trial in patients with chronic lymphocytic leukemia has been closed and the results are being evaluated.
In the search for new low molecular weight synthetic inhibitors of tumor cell growth, LEO Pharmaceutical Products discovered that a number of pyridyl cyanoguanidines showed antitumor activity after oral administration in a routine screening program in rats and the candidate CHS 828 was se-lected [46]. The chemical structure of CHS 828 is shown in figure 1.
NH
NN
NH
N
O
Cl
Figure 1. N-(6-(4-chlorophenoxy)hexyl)-N´-cyano-N´´-4-pyridylguanidine, CHS 828
CHS 828 was found active against many primary human tumor cells from patients with haematological and solid tumors in vitro [47], and in an in vivo
18
hollow fiber model [27]. It has an anti-proliferative effect on a variety of human cancer cell lines, both in vitro and in xenograft models in animals. CHS 828 has been found to be considerably less cytotoxic in normal fibro-blast and endothelial cells when compared to cancer cells [48]. CHS 828 has shown a pattern of antitumor activity different from clinically used antican-cer drugs, with no correlation to known mechanisms of drug resistance [48]. CHS 828 had higher antitumor activity when administered once daily for five days than giving it as a single high dose in a hollow fiber model in rats. Toxicity was found to be low irrespective of the schedule used [26]. Sched-ule dependence in toxicity was found in phase I studies since a higher total dose for a single-dose regimen was tolerated than for a five-day dosage regimen [49, 50]. Investigations made on mode of cell death induced by CHS 828 did not confirm the typical morphology of classical apoptosis al-though early apoptotic events were shown by some of the cells [51, 52]. Interference with the NF-kB pathway through down regulation of IkB kinase
was proposed as a mechanism of action of CHS 828 [53].
Pacliex, a new paclitaxel formulation The taxane, paclitaxel, is an anticancer agent that stabilizes cellular micro-tubules and is widely accepted as a component of therapy for advanced breast, lung and ovarian carcinomas. The clinically available paclitaxel for-mulation, Taxol® (Bristol-Myers Squibb Corp.), has serious limitations due to its formulation in Cremophor EL. In collaboration with Oasmia Pharma-ceutical AB we were interested in studying the preclinical cytotoxic activity of a new paclitaxel formulation. Pacliex is paclitaxel formulated in a syn-thetic derivative of retinoic acid in a form of mixed-micelles.
19
AIMS OF THE STUDY
The general aim of the doctoral project was to characterize the cytotoxic effect of the novel cytotoxic cyanoguanidine CHS 828 and to elucidate its mechanism of action. It was also meant to develop and use preclinical meth-ods to improve development of anticancer drugs.
The detailed aims were:
To develop a three-dimensional in vitro tumor model based on cul-turing tumor cells in semipermeable hollow fibers and to use this model to study the cytotoxic effect and morphological changes in-duced by CHS 828
To evaluate the in vitro time dependence of cytotoxicity of the CHS 828 and other standard cytotoxic drugs applying a modelling strat-egy.
To describe the time course and schedule dependence of the in vivo antitumor effect of CHS 828 in a hollow fiber model in immuno-competent rats.
To elucidate the role of the transcription factor kappa B, NF-kB and the proteasome in the mechanism of action of CHS 828.
To use the in vitro and in vivo hollow fiber model in immunocom-petent rats to assess tumor response to the new paclitaxel formula-tion, Pacliex.
20
MATERIAL AND METHODS
This section will describe briefly the methods used in this thesis. These methods are well established and evaluated previously. The detailed descrip-tion of individual method will be found in the relevant papers.
Human Tumor Cell Lines (HTCL) A total of fifteen human cell lines, a panel of ten cell lines [16] in addition to five cell lines representing different tumor types were used in different com-binations in the different parts of this thesis. The choice of the cell lines was based on the previous knowledge of their sensitivity to standard cytotoxic drugs and to CHS 828 as well as on their performance in the assay system used.
Primary human tumor cells (PHTC) and normal lymphocytes Tumor cell samples from six patients with chronic lymphocytic leukemia (CLL), five samples from patients with ovarian carcinoma (OC) and four normal mononuclear samples from healthy donors were used in paper I and II of this study. The patient samples were previously obtained from routine surgery, peripheral blood or lymph node and were kept frozen after cell iso-lation.
AnimalsMale Sprague Dawley rats obtained from Charles River, Uppsala, Sweden were used in paper III and V. They were acclimatized for at least one week before randomization and the start of the experiments. The rats had free ac-cess to food and water and were kept in a room lighted up for 12h from 7 a.m. to 7 p.m. throughout the study. Phenobarbital was used for euthanasia. The Animal Ethics Committee in Uppsala approved the studies.
21
Reagents and drugs CHS 828 was provided by LEO Pharmaceutical Products (Ballerup, Den-mark). Pacliex was provided by Oasmia Pharmaceutical AB (Uppsala, Swe-den). Drugs and chemicals were obtained from various commercial sources.
Hollow fiber method (paper I, III and V) The hollow fiber procedure was modified from that of Hollingshead for drug screening in vivo [24]. Polyvinylidine fluoride hollow fibers (PVDF) were filled with cell suspension. In our experimental design the inoculation densi-ties of the cells varied between in vitro and in vivo studies and between cell lines and primary human tumor cells. The fibers were then incubated at 37°C incubator in humidified atmosphere containing 95% air and 5% CO2. The incubation time ranged from 3-14 days for the in vitro and two days prior to implantation for the in vivo studies. The hollow fiber cultures were then exposed to cytotoxic drugs, either in 6-well plates for the in vitro studies, or by dosing the rats after the implantation of the fibers on the back of the ani-mals for the in vivo studies.
The living cell density was evaluated by staining with [3-4,5-dimethylthiaxol-2-yl]-2,5-diphenyltetrazolium bromide, (MTT), which is converted by metabolically active cells to insoluble blue formazan crystals. Briefly, the fibers were incubated with MTT at 37°C for 4h and washed with protamin sulphate in PBS overnight. The fibers were left to dry until the end of the experiment. The formazan was extracted with DMSO for 4h at room temperature and the absorbance was read at 570 nm in a plate reader (Dy-natec Laboratories).
The cell densities in the treated fibers from the in vitro experiments were expressed as Survival Index (SI %) defined as the absorbance of the treated fibers in percent of control fibers.
The cell densities of the retrieved fibers from the animals were expressed as net growth (%), defined as:
(absorbance on the retrieval day– mean absorbance on the implantation day) x 100 mean absorbance on the implantation day
Hence, a net growth of –100% represents total cell kill, while a value greater than 0% represents a growth of the cells in the fiber compared with implan-tation day.
22
Morphology assessment and TUNEL assay (paper I) Two different methods were used to prepare cells from the hollow fibers for morphological assessment: paraffin sectioning and cell harvesting. The cell lines were cultured inside the hollow fibers up to 4 weeks and were investi-gated at different time points. One third of the fibers were fixed in 10% buff-ered formalin; embedded in paraffin and sectioned at 5 µm intervals and the sections were then stained in hematoxylin-eosin. The second third of the fibers were used for cell harvest from inside the fiber by incubating the fi-bers with trypsin for 5 minutes and the tumor cells were extruded by flushing the fibers with trypsin followed by culture medium and cytospin slides were prepared. The third part of the fibers as well as the empty fibers remaining after the cell harvest was stained with MTT.
To assess the mode of cell death after CHS 828 exposure the cytospin slides were either May-Grünwald-Giemsa (MGG) stained or TUNEL stained. The TUNEL assay is used for detection and quantification of apop-tosis at single cell level and is based on labelling of DNA strand breaks. Cells were defined as apoptotic, TUNEL positive, if their nuclei were frag-mented with bright fluorescence. Apoptosis was judged morphologically from the MGG stained slides by the existence of intact cytoplasmic mem-brane and fragmented nuclei [54].
The fluorometric microculture cytotoxicity assay procedure (FMCA) (paper II, IV and V). The FMCA is based on measurements of fluorescence generated from hy-drolysis of FDA to fluorescein by cells with intact plasma membrane [55]. Briefly, 96-well plates were prepared by adding drug solution to each well and kept frozen at -70°C until use within 8 weeks. The cell suspension was added and the plates were incubated for 72 h. For paper II and for experi-ments for unpublished data, the plates were incubated for time ranging be-tween 2-72h, then washed 4 times with PBS, replaced by fresh medium and incubated up to 72h. At the end of the incubation time, the plates were washed with PBS and fluorescein diacetate was added for 40 minutes. The generated fluorescence from each well was measured at 538 nm in a 96-well scanning fluorometer (Fluoroscan II, Labsystems Oy, Helsinki, Finland). The fluorescence is proportional to the number of viable cells in the well. Cell survival was presented as survival index (SI), defined as the fluores-cence in experimental wells as a per cent of that in control wells, with blank values subtracted.
23
Pharmacokinetic monitoring and chemical assay (paper III) Four blood samples (250-300 l) were collected into eppendorf tubes. After 30-60 minutes at 4 C, the blood was centrifuged at 7000 rpm for 10 minutes. The serum was immediately frozen and stored at –70 C until analysis of CHS 828 concentrations.
The concentration of CHS 828 in serum was determined at Leo Pharma, by reversed-phase HPLC method with UV-detection. The rat serum sample was mixed with blank serum and an internal standard was added. Quantifica-tion was based on a CHS 828 spiked standard curve in the range of 50-20 000 ng/ml.
In vivo pharmacokinetic monitoring, general and haematological toxicities (paper III) At the start of the study, the weights of the rats were taken and they were weighed again on every subsequent day of handling. For hematological tox-icity measurements, blood was collected into EDTA-prepared Microtainer tubes on the day before surgery and followed for 4-11 days after the first dose. Leukocyte counts, platelet counts and hemoglobin values were deter-mined in a Coulter Counter (MDII series; Beckman Coulter, Luton, UK) within two hours after sampling.
The models and data analysis (paper II and III) The concentration-effect relationship of the anticancer drug studied in paper II were best described using single or double Hill models [56]. The PD mod-els for the in vitro study were selected on the basis of a graphical examina-tion of the concentration-effect curves and the pattern of change of the pa-rameters, IC50, slope and plateau, with time. Data from all experiments from the same drug and cell line were included in the analysis. For paper III, dif-ferent PD models were investigated, where all cell density profiles from all treated and controlled rats, were modelled simultaneously. Different PK compartmental models with linear and non-linear elimination were tried as well as absorption models with and without dose dependencies. Data analy-sis of data from paper II and III was performed using non-linear mixed effect models in the program NONMEM [57].
24
Effect of CHS 828 on NF-kB translocation and the ArrayScan system (paper IV) Tumor cells from different cell lines were allowed to adhere for 18-24h in standard high density black 96-well clear bottom microplates and were ex-posed to CHS 828. The NF-kB translocation was evaluated using reagents containing a primary antibody specific for NF-kB and a secondary antibody conjugated with fluorophore Alexa Fluor® 488 to identify p65 (anti-p65) and DNA-specific Hoechst 33342 to identify the nucleus.
Quantification of NF-kB activation was performed by direct measuring of the spatial translocation of NF-kB from the cell cytoplasm to the nucleus utilizing the advanced optical imaging system, ArrayScan® (HCS reader, Cellomics, Inc. USA) [58] .The procedure provides a fixed end-point assay based on visualization of protein translocation from the cytoplasm to the nucleus by immunocytolocalization. The ArrayScan® reader automatically finds, focuses, images and analyzes a predefined number of cells using suit-able filters with a 20X objectives and then translates this information to quantify the mean nucleus-cytoplasm intensity difference of the amount of p65 immunofluorescence staining.
Proteasome enzyme activity (paper IV) Tumor cells from different cell lines were exposed to CHS 828 or to the proteasome inhibitor, MG 262 in microtiter plates. The plates were washed twice with PBS before incubation with 40 µM fluorogenic proteasome pep-tide substrate (SUC-Leu-Leu-Val-Tyr-AMC). The chymotryptic peptide hydrolyzing activity of the proteasome enzyme was assayed by monitoring the hydrolysis of the fluorogenic substrate [59]. The fluorescence intensity was followed with excitation 380 nm: emission 460 nm, using a fluorescence plate reader (Fluostar Optima, BMG Labtechnologies GmbH, Germany). The enzymatic activity of the 20S proteasome in a cell-free system was measured by adding CHS 828 or a known proteasome inhibitor, lactacystin, to the pure enzyme in 96-well plate and subsequently adding SUC-Leu-Leu-Val-Tyr-AMC substrate. The assay was performed according to manufac-turer instructions. The enzymatic activity was estimated by monitoring the increase in the fluorescence generated from cleavage of the substrate over time.
25
RESULTS AND DISCUSSION
Hollow fiber culturesThe in vitro experimental designs of paper I and V are similar but, differ in the cytotoxic agent and concentrations studied. This applies to the in vivo experimental designs of paper III and V as well.
In vitro antitumor activity (paper I and V) Most human tumors have a large population of non-proliferating cells and necrosis occurs commonly in solid tumors in human as cell proliferation decreases rapidly with increasing distance from the nutrition source. Necro-sis is also observed in experimental tumors such as spheroids [60]. Casciari et al showed that an in vitro hollow fiber tumor contained a heterogeneous cell population with a necrotic core. The hollow fiber heterogeneous cultures formed were shown to be more resistant to cytotoxic drugs than monolayer cultures [20].
Paper I demonstrated that 3 day old hollow fiber cultures, high proliferat-ing cultures of CCRF-CEM and ACHN, to be more sensitive to CHS 828 than the 14 day old slow proliferating cultures (figure 2). Proliferation of the tumor cells was slowed down by day 14 in both cell lines while the tumor cells in day 3 were highly proliferating as reflected by the inset. This differ-ence in sensitivity between the 3 and 14 days cultures was observed also for the standard cytotoxic drugs studied.
CHS 828 produced similar shape of the concentration response curves with PHTC (CLL and OC) and normal lymphocytes hollow fiber cultures. The OC cells appeared to be less sensitive to CHS 828 than the CLL and normal human lymphocytes. This observation is similar to what was previ-ously reported on the in vitro activity of CHS 828 in primary cultures of human tumors in vitro using the fluorometric microculture cytotoxicity assay [47].
26
0
50
100
150
200
10 -4 10 -3 10 -2 10 -1 10 0
3 days cultures14 day cultures
A
SI%
0
50
100
150
200
10 -4 10 -3 10 -2 10 -1 10 0
B
0 3 6 9 12 15 180
1
2
day
Sign
al
0 3 6 9 12 15 180
1
2
day
Sign
al
drug concentration (µM)
Figure 2. Concentration-response curves generated after CHS 828 on 3 and 14 day old fiber cultures of CCRF-CEM (A) and ACHN (B) cells. The results are expressed as SI (cell survival in percent of control and presented as mean ± SEM of three independent experiments). Inset is the growth curve of the cell line
The observation that the 3 day old high proliferating cultures were more sensitive to cytotoxic drugs than the 14 day old, slow proliferating cultures was also confirmed by the work in paper V (figure 3) when the cytotoxic activity of three different formulations of paclitaxel was studied. The inset in figure 3 indicates the slow proliferation of 14 days cultures of RPMI 8226/S cell line compared to the 3 days cultures. The results as shown in figure 3 indicated that the new paclitaxel formulation, Pacliex, has similar in vitro cytotoxic activity as the clinically available formulation of paclitaxel, Taxol® in both culture types.
0
50
100
150
200
10 -4 10 -3 10 -2 10 -1 10 0
A
0
50
100
150
200
10-4 10 -3 10-2 10 -1 10 0
B
PacliexTaxolPaclitaxel
drug concentration (µg/ml)
SI%
0 3 6 9 12 15 180.0
0.5
1.0
1.5
day
Sign
al
Figure 3. Pacliex, Taxol and paclitaxel concentration-effect curves on 3-and 14- day in vitro hollow fiber cultures of RPMI 8226/S cell line (A and B respectively). Inset is the growth curve of the cell line (two experiments, 4 fibers mean + SEM)
27
Morphology assessment (paper I) In paper I we used two different methods to process the fibers for morpho-logical assessment. The assessment of harvested tumor cells is easy, and has advantages over formalin fixation and sectioning. The harvested tumor cells retain their original size and shape. In contrast, morphological changes were difficult to assess in the hollow fiber sections due to shrinkage of the tumor cell’s cytoplasm during the fixation step and consequently squeezed nuclei. In the present study we have adopted the harvesting procedure to character-ize the mode of cell death in CHS 828. The two staining procedures, MGG for and TUNEL, used to detect the morphological changes and apoptosis induced by CHS 828, respectively, correlate well. CHS 828 did not induce classical apoptosis in the treated tumor cells even after prolonged exposure (48h and 72h). This result is in accordance with previous studies, where it has been shown that the mode of cell death induced by CHS 828 is atypical, but share some of the features of classical apoptosis [51, 61, 62].
In vivo antitumor activity (paper III and V)
Figure 4. Net growth for different dose levels after fiber retrieval, five days after first administration (left) and net growth over time for the 375 mg/kg doses (right) for MDA-MB-231 (A, B) and CCRF-CEM (C, D). Means +/- SEM.
0 100 200 300 400 500 600-100
0
100
200
300
Single doseFive dose schedule
Control
A
0 1 2 3 4 5 6 7 8 9 10-100
0
100
200
300 B
0 100 200 300 400 500 600-100
0
100
200
300 C
0 1 2 3 4 5 6 7 8 9 10-100
0
100
200
300 D
CHS 828 total dose (mg/kg) Time (days)
Net
gro
wth
(%)
28
In paper III, the activity of CHS 828 given in two schedules, single and five-dose regimen, and at different dose levels was determined. Both dose regi-mens were effective in the leukemia cells CCRF-CEM; however, the five-dose regimen was more effective even at the lower dose levels. For the breast-cancer cells MDA-MB-231, the cell kill of the five-dose regimen increased in a dose-dependent manner whereas the single-dose regimen pro-duced effect only at the highest dose level. In the experiment in which fibers were also retrieved on days 1 and 3 after 375 mg/kg CHS 828, the single-dose regimen was not effective in the MDA-MB-231 cells while the five-dose regimen reduced the cell survival with time. In CCRF-CEM cells the two schedules showed similar cell survival (figure 4).
In paper V, the activity of Pacliex and Taxol in the in vivo hollow fiber cultures of two cell lines was determined. Pacliex and Taxol induced similar growth inhibition on CCRF-CEM and, cell killing effect on RPMI 8226/S (figure 5). The net growth in the control rats indicates a more rapid in vivo proliferation rate in CCRF-CEM leukemia cell line than in RPMI 8226/S myeloma cell line.
-50
0
50
100
150
200
Net
gro
wth
A
-50
0
50
100
150
200 B
Net
gro
wth
0 3 6 90
1
2
1
fillin
g
impl
anta
tion
retreiva
l
C
sign
al
0 3 6 90
1
2
1
fillin
g
impl
anta
tion
retreiva
lD
sign
al
PlaceboTaxolPacliex
Figure 5. Pacliex and Taxol effect on CCRF-CEM (A) and RPMI 8226/S (B) in the in vivo hollow fibers. The insets are the in vitro growth of CCRF-CEM (C) and RPMI 8226/S (D) in parallel to the in vivo study (two experiments mean + SEM.).
29
Hollow fiber method, general discussion In the present studies we confirmed the possibility of establishing slow pro-liferating hollow fiber cultures and we used these cultures for drug sensitiv-ity evaluation. We showed that slow proliferating in vitro cultures in general are more resistant to cytotoxic drugs than rapidly proliferating cultures. Such slow proliferating tumor model could be advantageous in resembling a clini-cal tumor. The in vitro hollow fiber model could be used to provide informa-tion to answer questions regarding drug penetration through tumor cell lay-ers and to understand the possible effect of tumor heterogeneity on the re-sponse of tumors to cytotoxic drugs that could not be answered from the in vitro cell line culture models. One of the problems in the clinic is that low proliferative, large tumors are difficult to treat, as cytotoxic drugs are gener-ally more effective towards highly proliferating cells. Studying the antitumor effect in hollow fiber cultures may be a way to identify new drugs and for-mulations that are effective for these tumors.
We have shown that the hollow fibers are liable to standard histologic fixation and staining procedures, thus making the hollow fiber method suit-able for histologic and cytologic documentation. The hollow fibers allow successful cell harvested, which may offer the possibility to perform investi-gations involving, for example, cell cycle analysis or apoptosis using more specific markers and new assay procedures on the hollow fiber cultured cells. The results presented in this thesis demonstrated the potential of the hollow fiber as an experimental tool to observe the characteristics of tumor cell growth inside the fibers and the morphology of the cells before and after in vitro treatment. It has previously been shown by us that the hollow fiber paraffin embedded sections offer the possibility to investigate tumor cell growth and drug effects from in vivo studies [27]. It is possible to follow the growth of the primary human tumor cells from single cells up to cell cultures forming bridges and eventually solid masses. Figure 6 illustrates the poten-tial of the hollow fibers as an experimental tool to study the morphology of the tumor cell, in vitro and in vivo. More work is needed to assess the utility of these in vitro cultures from individual patient in cytotoxicity experiments.
The hollow fibers have been shown to be suitable for in vivo drug screen-ing as they are biocompatible with human tumor cells and can support the growth of human tumor cells in mice subcutaneously and intraperitoneally [24]. The fibers attract minimal amounts of host cells which are loosely at-tached to the fiber’s exterior. They have high molecular weight cut-off (500 kDa) that allows the passage of growth factors, most proteins and drugs. The immunocompetent animals are easy to handle, cost less than immunode-ficeint ones, the rats can host many tumor types at a time and can withstand repeated bleedings for haematological toxicity and pharmacokinetics charac-terization simultaneously. This implies that the hollow fiber model is a promising tool for use in drug evaluation.
30
Figure 6. Photographs of 21 day in vitro hollow fiber cultures of ovarian carcinoma cells (A & D) and photographs of 6 day in vivo hollow fiber cultures of ovarian carcinoma cells before (B & E) and after treatment with CHS 828 (C & F). The fiber walls are visible as parallel structures with the lower magnification (upper photos) Photos from in vivo fibers were published in Cancer letters 162 (2001) 193-200.
There are some disadvantages in using the hollow fiber solid tumor model in drug screening studies: a/ the in vitro tumor growth could be limited by the geometric constraint of the fiber wall which constitutes an artificial barrier that separates the tumor cells from their surroundings. b/ Low efficiency of drug delivery to the fibers in the in vivo setting could be a limitation in using the hollow fiber model in drug screening as the short term period used for experimentation does not allow angiogenesis to occur and thus could lead to underestimation of the response [25]. It is also possible that, due to the short term period, the full drug effect of some of schedules might not have been observed. However, it was reported that tumors do not require blood vessels until they reach certain diameter which is 3 to 4 times higher than the hollow fiber diameter [63]. The limited blood supply could also be considered ad-ventitious as it might be relevant to the clinical setting where tumors have generally poor blood supply.
A B C
D E F
31
Schedule dependency In vitro modelling of CHS 828 and standard drugs effects (paper II) In this paper, we aimed at elucidating the time dependency of CHS 828 ef-fect. Data from the concentration-effect experiments of CHS 828, etoposide, topotecan and paclitaxel were modelled using Hill models. Patterns of time-dependency of IC50 and slope of concentration-effect curves were character-ized (figure 7). The drop in IC50 for CHS 828 with exposure time had a clearly sigmoid shape for all cell lines tested, as well as for CLL cells. At exposure times less than 24 h, drug potency was leveled, and plateaued at later times (48-72 h). A steep increase in drug potency occurred at interme-diate times (24-36h) where the concentration-effect curves had a shallow shape.
The shallowness of the slopes and the sharp drop in IC50 observed for CHS 828 could be interpreted as a sign of a heterogeneous cell population or multiple cellular targets with different sensitivities to the drugs [32]. A dif-ference in cell cycle phase or heterogeneous cell population response does not seem to explain the IC50 drop for CHS 828 since the same activity pat-tern was observed in primary cells from CLL and normal mononuclear cells. These cell types have been shown not to proliferate significantly under these experimental conditions, and CLL is a single cell type disease. In addition, drug remaining bound inside the cells does not seem to be the cause for the sigmoid drop in IC50 since CHS 828 showed a rapid equilibrium between the intracellular and extracellular fluid. Instead our data suggest two different mechanisms of action of CHS 828, one of lower potency independent of exposure time and one, very potent, acting only at longer exposure times.
The patterns of IC50 change for topotecan and etoposide in our study were also best described by a sigmoid relationship; however, the sigmoidicity was not very pronounced for etoposide. The log IC50 decreased linearly with the logarithm of the exposure time for paclitaxel and this might be interpreted as the cytotoxic effect of the drug is simply dependent on the concentration times time product (AUC), and is independent on the schedule used [32].
32
0.0 0.5 1.0 1.5 2.0
CHS 828U937 GTB
10
1
0.1
0.01
0.0010 25 50 75
-15
-10
-5
0
0.0 0.5 1.0 1.5 2.0
paclitaxelU937 GTB
1
0.01
0.001
10
0.1
0 25 50 75-15
-10
-5
0
0.0 0.5 1.0 1.5 2.0
etoposideU937 GTB
1
0.1
0.01
0.001
10
0 25 50 75-15
-10
-5
0
0.0 0.5 1.0 1.5 2.0
topotecanU937 GTB
10
1
0.1
0.01
0.0010 25 50 75
-15
-10
-5
0
IC50 slope
log time (h) time (h)
Figure 7. Pattern of change of IC50 (left) and slope (right) for CHS 828, paclitaxel, etoposide and topotecan on U937 GTB tumor cells.
One main mechanism of action has been proposed for each of the three stan-dard drugs, microtubule stabilization for paclitaxel, topoisomerase II inhibi-tion for etoposide and topoisomerase I inhibition for topotecan, however recently some investigators have suggested additional mechanisms of action such as phosphorylation of Bcl-2, Bcl-xL and other proteins for paclitaxel [64], DNA interaction for etoposide [9] and NF-kB activation for camptothe-cins [65]. These possible additional mechanisms of action might explain the weak sigmoidicity shown in the pattern of change of IC50 with exposure time for etoposide and topotecan. Clinical data suggest that etoposide might be more effective if given in extended schedule [8] whereas this is not dem-onstrated clearly for paclitaxel [66] or topotecan [67].
Additional studies on the in vitro time dependence of CHS 828 (Unpublished data) In an effort to explore the mechanism of action of CHS 828 we expanded the experimental work of paper II to include more cell lines. The cell lines tested
33
were Hela, PC-3 (two experiments each) and NYH SCLC (one experiment). In these studies, log IC50 estimations were performed using the built-in equation for dose-response curves with variable slope in the GraphPad Prism software. The concentration-effect curves of CHS 828 on NYH SCLC had a similar shape as for the other cell types studied in paper II and the drop in IC50 for CHS 828 with exposure time had a clearly sigmoid shape. However, for Hela and PC-3 the drop in the IC50 was clearly shallower than for the other cell types studied and the drop in IC50 with time was of lower magni-tude than for the other cell types studied (figure 8).
0.0 0.5 1.0 1.5 2.0-3.0-2.5-2.0-1.5-1.0-0.50.00.51.01.5 RPMI 8226/S
MDA 231
U937 GTB
CLLNYH SCLC
Hela
PC-3
log time
log
EC50
Figure 8. Pattern of change of IC50 for CHS 828 on all cell types studied.
The reason(s) for the less pronounced time dependency of the CHS 828 ef-fect observed in Hela and PC-3 cell lines is not known and remains to be elucidated. However, if the theory about two different mechanisms of activ-ity of CHS 828 proves to be true, it is possible that only one of the mecha-nisms is important in these less time dependent cell types. There may be a low-potency effect that is independent of the exposure time, and that is the most important for the cytotoxic action of CHS 828 in for example Hela and PC-3. However, we can not exclude an influence of the higher potency mechanism on the cytotoxicity seen.
Interestingly, the sensitivity, reflected by the IC50 at 72h exposure, of the cell lines to CHS 828 presented in the studies of in vitro time dependence of CHS 828 (paper II and unpublished data) is generally higher than in the NF-kB study (paper IV). The overall difference was around 3-5 times for the cell lines NYH SCLC, RPMI 8226/S and MDA 231 whereas the difference was
34
around 50 times for PC-3. Hela was not responding to CHS 828 in the NF-kB study (paper IV) and IC50 could not be achieved. The difference in sensi-tivity is not known, but could be attributed to experimental differences of the drug itself (different drug batches or dissolution problems) and/or biological variation in tumor response.
In vivo PK modelling (paper III) The PK model was a one-compartment linear model with the dissolution and absorption described by a zero-order rate (R1) and a consecutive first-order rate (ka,1) input. The fraction absorbed was modelled to decrease with dose, this was best described by an exponential function with the rate constant kFand fraction absorbed on the day of administration (F1) was highest for the lowest dose of 5.9 mg (F1,max). To explain the increasing serum concentra-tions on the day after administration, a fraction of the dose (F2) was mod-elled to be absorbed after a lag time and modelled to decrease exponentially with increasing dose. The total fraction absorbed (F1 + F2) for the 5.9 mg dose was set as the reference absorbed fraction, i.e. 100%. Oral clearance increased from zero time and was modelled to be linearly dependent on cu-mulative exposure, i.e. cumulative AUC (AUC0-t) (figure 9). Because of the dose-dependent fraction absorbed, the five-dose regimens produced larger AUCs than the single-dose regimens of the same total dose and for the typi-cal individual the model predicted similar AUC values for the three highest single doses.
Figure 9. Pharmacokinetic model of CHS 828 administered orally to rats.
Absorp-
Central
R1ka,
F2 = (1 - F1,max) · e – kF · (Dose –5.9)
Delayed absorption
Lag time ka,
CL(t) / V
F1 = F1,max · e – kF · (Dose –5.9)
CL(t) = CL0 · (1 + AUC0-t · SlopeCL)
35
Because of the dose-dependent fraction absorbed, the model predicts a simi-lar duration for the absorption phase (approximately 10 hours) of the three highest single dose levels. The dose-dependent duration of the input is probably because that after approximately 10 hours the drug reaches a part of the intestine where the drug is no longer dissolved and/or absorbed. Ap-proximately one third of the systemically available dose was estimated to be absorbed after a lag-time of 20 hours after administration, probably as a re-sult of coprophagy. This is a likely explanation to the second absorption phase as the gut transit time in non-fasting rats has been estimated to 21 hours [68]. Enterohepatic recycling would not show this pattern as rats lack gall bladder [69]. A large interindividual variability (IIV) in absorption was shown in our study, which also seems to be a problem in patients receiving CHS 828 capsules [49].
The phenomenon that clearance increases with exposure has not previ-ously been shown for CHS 828 and requires confirmation. An increasing clearance over time can be due to induction of metabolizing enzymes and auto-induction phenomena has been modelled for other drugs [70, 71]. CHS 828 is metabolized by CYP 3A4 in man [49]. However, more data are re-quired before a more sophisticated induction model can be applied to CHS 828.
Toxicity (paper III) Weight loss was dose- and schedule-dependent. Haematological toxicity was limited and evident only at the highest dose level of the five-dose regimen. When the dose-dependent pharmacokinetics were accounted for and the individual actual exposure (AUC) was used instead of dose, the schedule-dependent effect was not clear.
The pharmacokinetics of CHS 828 appears to be similar in rats and pa-tients, but the toxicity pattern differs. Thrombocytopenia and gastrointestinal complications were the dose-limiting toxicities of a five-dose schedule of CHS 828 in patients [49]. However, no reduction in platelet counts was seen in this study in rats. In this study, the dominating toxicity was weight loss; however, the magnitude and duration of the weight loss was only of impor-tance at the highest dose levels (500 mg/kg). More data, using higher doses if possible, would be required to establish a pharmacokinetic-pharmacodynamic model for evaluation of a “true” schedule-dependent pharmacodynamic effect of haematological toxicity.
36
In vivo PD modelling (paper III) Based on previous knowledge of the time course and the cell death kinetics of CHS 828, a threshold cumulative effect on the cells (AUEThresh) before cell kill (Kill) start was assumed in the modeling i.e. the cell viability was unaffected until AUEThresh was attained. Exponential cell growth was adopted and the differential equations of the change in OD over time were written as
dOD/dt = kGrow ·OD for AUE0-t < AUEThresh (1) dOD/dt = kGrow ·(1 - Kill) OD for AUE 0-t AUEThresh (2)
AUE0-t is the cumulative effect that CHS exerts on the cells from the time of first administration to time t
AUE0-t = E(C) dt (3)
E(C) describes the relationship between the serum concentration of CHS 828 and the effect on the cells. The final models included linear concentration – direct effect relationships, but the relationships were different for the two schedules where AUE0-t was replaced by AUC0-t and a separate AUCThresh for single and five-daily dosing was used.
The developed pharmacokinetic-pharmacodynamic model resulted in pre-dictions of the cell viability over time that are in agreement with the observa-tions. The model also clarified that CHS 828 exerts a schedule-dependent pharmacodynamic effect on the investigated tumor cell lines in vivo as de-scribed by schedule-dependent AUCThresh. The schedule-dependent antitumor effect of CHS 828 in the rat hollow fiber model is partly due to a dose-dependent fraction absorbed, likely because of low solubility in the gastroin-testinal tract, and partly due to schedule dependence in the pharmacodynam-ics of the drug. Consequently, even when formulated to avoid the dose-dependent fraction absorbed, CHS 828 will exert an antitumor effect that is dependent on the rate of administration.
General discussion on CHS 828 schedule dependence The choice of dosing strategy is an important objective when a new drug is brought into clinical development. The search for the optimal schedule for CHS 828 is part of the goal of the preclinical studies in this thesis, and is still ongoing in the clinical studies concerning this compound. The investigation of a wide time frame and a large number of drug concentrations allows a better understanding of the three-dimensional concentration-time-effect sur-face and facilitates predictions of effect at concentrations and times not stud-ied via interpolation and very cautious extrapolation. In addition it allows, through detailed examination of the shape of the concentration-effect curves
37
and the change of its parameters with time, formulation of hypotheses re-garding drug action and resistance. Using this approach, it is possible to modulate drug effect and drug resistance by altering the conditions of expo-sure to the drug. It is possible to make careful inferences from in vitro to in vivo data; however, the lack of data regarding normal cell toxicities and the drug’s PK limits its utility in suggesting doses for in vivo and clinical stud-ies.
Investigators of clinical PK/PD studies are interested in relating the PK parameters to the PD parameters of efficacy and toxicity, as the drug effect is believed to be better related to the plasma concentration than the drug dose. It has previously been shown that the hollow fiber model in immuno-competent rats has provided the utility to study cytotoxic effects, toxicity and PK in the same animal [26].
Based on the in vitro pattern of change of IC50 with time, we speculated from paper II that CHS 828 might have two mechanisms of actions and that its cytotoxicity may depend on the schedule used. The developed PK/PD model in paper III resulted in predictions of the cell viability over time that are in agreement with the observations in this study and illustrated the schedule dependence of the CHS 828 in vivo effect. The model was sug-gested to be suitable for population PKPD modelling of antitumor effects over time in vivo.
Both the in vitro and in vivo models predicted lower IC50/AUC50 after a prolonged schedule and both models predicted a sudden onset of cell-killing effect after a certain exposure. However, a similar drug model, as the one used in paper I, did not give a better fit to the in vivo data, either due to lim-ited information in the data and/or due to that the concentrations at the effect site were too low for the less potent mechanism to be significant. The in vivo PK/PD model predicted that the cell death may start at different time points for different schedules which might not seem to fit with the predictions from the in vitro model. However the in vitro model only predicts the results at the end of the observation time where all observations were after 72h. For the in vivo model, there was only limited data of the tumor effect over time which probably leads to an over-simplified model of the time-course. Knowledge of the exact cell-killing mechanisms of CHS 828 would allow for a more mechanistic PK/PD-model, where information from in vitro studies could be included. However, it could be speculated that a low-potency effect is mainly responsible for the effect of a single dose whereas only the high-potency effect of the long exposure influences the effect of a protracted dose. In contrast to the current results, a single dose/week schedule of CHS 828 was superior to a divided dose/day schedule of the same total dose in MCF-7 xenografts in nude mice, already during the first week of treatment [48]. Similar to our rat studies, it seems that a single dose schedule is less toxic than the five-dose schedule in clinical phase I trials [49, 50]. The diverging results in schedule dependence of CHS 828 certainly illustrate the need for
38
studying PK simultaneously with tumor response to enable full understand-ing of the schedule dependence phenomenon. Regimens that maximize the antitumor activity while minimizing the toxicity would be clinically pre-ferred.
CHS 828 and the nuclear factor kappa B, NF-kB (paper IV) A recent study [53] identified IKK as a target in the NF-kB pathway for CHS 828, but it was not concluded that this is the only mechanism for the anticancer effect of CHS 828. CHS 828 has been recently shown to induce a dose-dependent inhibition of the proteasome activity after 24h exposure in U-937 GTB cells [72]. Since we speculated that CHS 828 could have two mechanisms of action we wanted to confirm the inhibitory effect of CHS 828 on the NF-kB translocation and to explore the possibility that the protea-some could be targeted by CHS 828.
Our results indicated an inhibition of NF-kB activity in the tumor cell lines, PC-3, MDA 231, and NYH SCLC and a tendency to inhibit NF-kBtranslocation in RPMI 8226/S in response to a 30 hours pre-incubation pe-riod with CHS 828. The experiments proved no effect of CHS 828 on pro-teasome activity. CHS 828 was not able to inhibit NF-kB translocation in Hela, ACHN and MCF-7. CHS 828 was not able to induce cytotoxicty in these three cell lines (figure 10 presents some of these cell lines).
The current results documented that inhibition of cell growth by CHS 828 correlates with the inhibition of TNF -induced NF-kB translocation but not with proteasome activity. The results also showed no correlation between CHS 828 effect on the NF-kB and proteasome activity. The close relation between the anti-tumor effect of CHS 828 and the inhibition of NF-kB trans-location is compatible with what has been observed previously where there was a close correlation between the inhibition of NF-kB activity and the reduction of xenografts in nude mice [53].
The observed NF-kB inhibition induced by CHS 828 at 30 h but not at 24 h is in agreement with what is known about the CHS 828 dynamic process. Not until after 24h the macromolecules synthesis is affected and the viability reduced in a time dependent manner [52].
39
PC-3
10-2 10-1 100 101 1020
50
100
150NF-kB Translocation%
viable cells %Hela
10-2 10-1 100 101 1020
50
100
150
RPMI 8226/S
10-2 10-1 100 101 1020
50
100
150
CHS 828 concentration
NF-
kB tr
anslo
catio
n %
and
viab
le c
ells
%
Figure 10. Effect of CHS 828 on TNF induced NF-kB translocation and on cell viability in PC3, RPMI 828/S and Hela cell lines. The percent of NF-kB transloca-tion and of cell survival are plotted versus CHS 828 concentration (three experi-ments mean + SEM).
Our data, presented in paper IV, in a cell-free system excluded any inhibi-tory effect of CHS 828 on the proteasome activity. Recent study [72] has shown that CHS 828 has no correlation with other known proteasome inhibi-tors with respect to the activity pattern and shape of concentration-response curves in a 10 cell line panel excluding mechanistic similarity. Taken to-gether, it is unlikely that CHS 828 induces its inhibitory effect on the NF-kBactivity and on cell survival through the regulation of the proteasome activ-ity.
CHS 828 has been proposed to inhibit the respiration function of the mi-tochondria leading to an increase in glycolysis during the first hour of expo-sure [73]. At about 24 hours after start of drug exposure the ATP level was found to be reduced in cells treated with CHS 828 [74]. Given the observa-tion that the 24h exposure to CHS 828 did not induce inhibition of the NF-kB translocation while the 30h exposure did, it is likely that this transcrip-tional factor pathway represents a major site of action of CHS 828 under long-term exposure. The interference with the respiratory and glycolytic pathways of the cells could be the secondary target that trepresents by the low potency effect of CHS 828.
Pacliex activity in cell suspension cultures, FMCA (paper V) The results presented in paper V showed that Pacliex, in terms of IC50, was more effective in CCRF-CEM, RPMI 8226/S and RPMI 8226/LR5 cell lines, while the two formulations were equally active in U-937-GTB, CEM/VM-1 and H69AR cell lines. Taxol is more effective in the more resis-tant cell types, U-937-vcr, NCI-H69 and RPMI 8226/Dox40. The overall difference in the IC50s between Pacliex and Taxol was around 3 times or less in all cell lines except ACHN, and this makes both formulations simi-
40
larly active in these cell lines. XR17 was found to be non toxic in any of the cell lines tested.
Cremophor EL, the solvent for Taxol, has been shown to enhance pacli-taxel activity [75, 76] and might have its own cytotoxic effect in vitro [77]. This may explain the higher effect seen with Taxol compared to Pacliex in the resistant cell lines where higher solvent concentrations were used. It should be noted that Pacliex is somewhat more effective than Taxol® at lower Cremophor EL concentrations and even though XR17 had no cyto-toxic effect in any of the cell lines tested, a potentiating effect on paclitaxel activity cannot however, be excluded. Whether sufficiently high concentra-tions of XR17 can be reached in vivo to exert a potentiating effect is not known.
The results from the activity of three paclitaxel formulations and the sol-vent for Pacliex, XR17, on the ten cell line panel support the evidence from the in vitro and in vivo hollow fibers studies (discussed in the relevant sec-tions) that Pacliex has similar cytotoxic activity as the clinically available formulation of paclitaxel, Taxol®
In light of results from paper V it is clearly demonstrated that Pacliex; the new paclitaxel formulation developed as a mixed micelles preparation of an amphiphilic synthetic derivative of retinoic acid, is an interesting alternative paclitaxel formulation which is devoid of the Cremophor EL related toxic effects.
41
SUMMARY AND CONCLUSIONS
In this thesis, the development and application of new methods for preclini-cal evaluation of anticancer drugs highlighted the following:
The long-term in vitro hollow fiber cultures of different tumor cell lines were more resistant to CHS 828 and other cytotoxic drugs than shorter-term cultures. CHS 828 was generally more effective against the haema-tological than the solid tumors from both cell lines and patients samples. Morphological examination and characterization of the mode of cell death induced by CHS 828 did not confirm the classical features of apoptosis.
Modelling of the in vitro concentration-time effect relationships for CHS 828 suggests two different mechanisms of action of CHS 828 and that its cytotoxicity may depend on the schedule used.
The developed hollow fiber model in immunocompetent rat can be used to evaluate pharmacokinetics, tumor response and/or toxicity simultane-ously. The PKPD model developed for CHS 828 viewed the schedule de-pendency of CHS 828 is partly due to dose-dependent fraction absorbed and partly due to a schedule-dependent pharmacodynamic effect.
The NF-kB system that regulate the transcription of both pro- and anti-apoptotic genes proved to be inhibited by CHS 828 and the inhibition was correlated to the cell death induced by this cytotoxic agent. However, CHS 828 did not seem to induce the NF-kB inhibition by affecting the proteasome activity.
The in vitro and in vivo hollow fiber methods were also used success-fully to evaluate the new paclitaxel formulation, Pacliex which has shown to have a similar activity compared to that of the clinically used formulation, Taxol®.
42
ACKNOWLEDGEMENT
This work was made possible by the contribution of several people to whom I wish to express my sincere thanks and gratitude.
Elin Lindhagen, my supervisor, for your close and keen supervision; for your unlimited support, encouragement and belief in me; for continuous sharing of your deep knowledge and experience, and for your constructive criticism of my work. Without you, it would not have been easy to accom-plish this work.
Rolf Larsson, my co-supervisor, for giving me the opportunity to work in your group; for your enthusiasm and generosity. I greatly appreciate your extensive scientific knowledge, your positive attitude, care and your nice way of creating friendly atmosphere.
Mats Karlsson, my co-supervisor; I am truly grateful to you for giving me the great chance to do research in Sweden and for accepting me to join your research group. I would like to thank you for your generous financial support and for introducing me to the mystery and the fascinating field of modelling.
Thanks are extended to Marja Liisa Dahl, the head of the Clinical Phar-macology department, for providing excellent working conditions and for being kind and interested in my work.
Peter Nygren, thanks for the valuable and constructive criticism of paper I.My co-authors: Sumeer, thank you for the scientific contribution and the
valuable discussion in many subjects and not only in science, Lena Friberg, for your fruitful collaboration and discussion, and for helping me to work and handle animals and sharing the knowledge of PKPD modelling. Manuel de la Torre is gratefully acknowledged for rendering his expertise.
My colleagues in the group: some already done and having their career elsewhere, Gunnar, Petra, Sara and Peter for help and cooperation during my work. Henrik thank you for the scientific contribution in paper IV and for being ready to help with the computer matters. Joachim thanks for rendering your expertise and time for formatting this work. My current room-mates: Daniel; for the nice company in the room and for the linguistic revision of paper V, Malin and Linda for the nice company in the room and the trip to Italy, and Arzu for being friendly from the first moment of knowing one another.
The staff at the lab: Lena Lenhammar, Christina Leek, Anna Karin and Carina. Thanks for all the help and support in the lab and the discussion in the cultural matter, especially Lena and Christina.
43
I appreciate Annika Åkerström’s assistance in taking care of the administra-tive stuff and arranging all the paper work.
The Sudanese community in Uppsala thanks for making me feel home. My especial thanks go to Izzeldin and Nagat for their support since my arri-val in Sweden.My mother, sisters, brother-in-law and my big family in Sudan for their sup-port.
My father, the great man who encourages me throughout my life, my sis-ters and my brothers for their endless love and support that provides so much encouragement to me.
My very special thanks go to my sister Innam and to my brother-in-law Elsadig Kazzam for their unlimited support, encouragement, advice, ideas and time before and after coming to Sweden. Without their guidance it would not have been possible to accomplish this work and many other things during my stay in Sweden.
Alaa and Mohammed, they are the true joy of my life. Alaa, in spite of your young age you are really a man whom I can depend on, especially for being so kind and helpful to your brother Mohammed. Mohammed, the sun shine of my life; thanks for being always the glad boy and for taking the goals of my life to another new height.
Last, but not least, my husband Sidahmed, for understanding, love and endless support. Your patience and wisdom always assure me that every thing will be good and fine at the end of the road. Without your moral sup-port, which is of great value to me, it would not be easy to keep up my track and make the dream real.
44
REFERENCES
1. De Vita JV, Hellman S. and Rosenberg S. A. Priniciples in cancer management: Chemotherapy. In: CANCER -Principles & Practice of oncology 333-347, 1997.
2. Kersey JH. Fifty years of studies of the biology and therapy of childhood leu-kemia. Blood. 90 (11): 4243-51., 1997.
3. FASS. The Swedish Drug Compendium.LINFO, Läkemedelsinformation AB. Stockholm, 2004.
4. Boyd MR. The future of new drug development. In: Current therapy in oncol-ogy: Neiderhuber J (ed) 11-22, 1993.
5. Lokich J and Anderson N. Dose intensity for bolus versus infusion chemother-apy administration: review of the literature for 27 anti-neoplastic agents. Ann Oncol. 8 (1): 15-25, 1997.
6. Gurney H, Dodwell D, Thatcher N, and Tattersall MH. Escalating drug delivery in cancer chemotherapy: a review of concepts and practice--Part 1. Ann Oncol. 4 (1): 23-34, 1993.
7. Sobrero AF, Aschele C, and Bertino JR. Fluorouracil in colorectal cancer--a tale of two drugs: implications for biochemical modulation. J Clin Oncol. 15 (1): 368-81, 1997.
8. Joel SP and Slevin ML. Schedule-dependent topoisomerase II-inhibiting drugs. Cancer Chemother Pharmacol. 34 (Suppl): S84-8., 1994.
9. Gordaliza M, Castro MA, del Corral JM, and Feliciano AS. Antitumor proper-ties of podophyllotoxin and related compounds. Curr Pharm Des. 6 (18): 1811-39., 2000.
10. Majno G and Joris I. Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol. 146 (1): 3-15., 1995.
11. Zamzami N, Hirsch T, Dallaporta B, Petit PX, and Kroemer G. Mitochondrial implication in accidental and programmed cell death: apoptosis and necrosis. J Bioenerg Biomembr. 29 (2): 185-93, 1997.
12. Kaufmann SH and Earnshaw WC. Induction of apoptosis by cancer chemother-apy. Exp Cell Res. 256 (1): 42-9., 2000.
13. Schmitt CA and Lowe SW. Apoptosis and therapy. J Pathol. 187 (1): 127-37., 1999.
14. Blagosklonny MV. Cell death beyond apoptosis. Leukemia. 14 (8): 1502-8., 2000.
15. Paull KD, Hamel E. and Malspeis L. Prediction of biochemical mechanisms of action from the in vitro antitumor screen of the Vational Cancer Institute. In: Cancer Chemotherapeutic Agents. Foye WO (ed): Americal Chemical Society. Wasshington. DC 9-45, 1995.
16. Dhar S, Nygren P, Csoka K, Botling J, Nilsson K, and Larsson R. Anti-cancer drug characterisation using a human cell line panel representing defined types of drug resistance. Br J Cancer. 74 (6): 888-96., 1996.
17. Sutherland RM. Cell and environment interactions in tumor microregions: the multicell spheroid model. Science. 240 (4849): 177-84, 1988.
45
18. Frankel A, Man S, Elliott P, Adams J, and Kerbel RS. Lack of multicellular drug resistance observed in human ovarian and prostate carcinoma treated with the proteasome inhibitor PS-341. Clin Cancer Res. 6 (9): 3719-28, 2000.
19. Sourla A, Doillon C, and Koutsilieris M. Three-dimensional type I collagen gel system containing MG-63 osteoblasts-like cells as a model for studying local bone reaction caused by metastatic cancer cells. Anticancer Res. 16 (5A): 2773-80, 1996.
20. Casciari JJ, Hollingshead MG, Alley MC, Mayo JG, Malspeis L, Miyauchi S, Grever MR, and Weinstein JN. Growth and chemotherapeutic response of cells in a hollow-fiber in vitro solid tumor model. J Natl Cancer Inst. 86 (24): 1846-52., 1994.
21. Venditti JM, Wesley RA, and Plowman J. Current NCI preclinical antitumor screening in vivo: results of tumor panel screening, 1976-1982, and future direc-tions. Adv Pharmacol Chemother. 20 1-20, 1984.
22. Peterson JK and Houghton PJ. Integrating pharmacology and in vivo cancer models in preclinical and clinical drug development. Eur J Cancer. 40 (6): 837-44, 2004.
23. Johnson JI, Decker S, Zaharevitz D, Rubinstein LV, Venditti JM, Schepartz S, Kalyandrug S, Christian M, Arbuck S, Hollingshead M, and Sausville EA. Rela-tionships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials. Br J Cancer. 84 (10): 1424-31, 2001.
24. Hollingshead MG, Alley MC, Camalier RF, Abbott BJ, Mayo JG, Malspeis L, and Grever MR. In vivo cultivation of tumor cells in hollow fibers. Life Sci. 57 (2): 131-41, 1995.
25. Phillips RM, Pearce J, Loadman PM, Bibby MC, Cooper PA, Swaine DJ, and Double JA. Angiogenesis in the hollow fiber tumor model influences drug de-livery to tumor cells: implications for anticancer drug screening programs. Can-cer Res. 58 (23): 5263-6, 1998.
26. Jonsson E, Friberg LE, Karlsson MO, Hassan SB, Freijs A, Hansen K, and Lars-son R. Determination of drug effect on tumor cells, host animal toxicity and drug pharmacokinetics in a hollow-fibre model in rats. Cancer Chemother Pharmacol. 46 (6): 493-500, 2000.
27. Jonsson E, Friberg LE, Karlsson MO, Hassan SB, Nygren P, Kristensen J, Tholander B, Binderup L, and Larsson R. In vivo activity of CHS 828 on hol-low-fibre cultures of primary human tumor cells from patients. Cancer Lett. 162 (2): 193-200, 2001.
28. Knowles M and Hwang SS. The influence of the shape of the plasma drug con-centration profile upon the pharmacological effect. Pharm Res. 12 (1): 168-70, 1995.
29. Karlsson MO, Molnar V, Bergh J, Freijs A, and Larsson R. A general model for time-dissociated pharmacokinetic-pharmacodynamic relationship exemplified by paclitaxel myelosuppression. Clin Pharmacol Ther. 63 (1): 11-25, 1998.
30. Gardner SN. A mechanistic, predictive model of dose-response curves for cell cycle phase-specific and -nonspecific drugs. Cancer Res. 60 (5): 1417-25, 2000.
31. Kalns JE, Millenbaugh NJ, Wientjes MG, and Au JL. Design and analysis of in vitro antitumor pharmacodynamic studies. Cancer Res. 55 (22): 5315-22, 1995.
32. Levasseur LM, Slocum HK, Rustum YM, and Greco WR. Modeling of the time-dependency of in vitro drug cytotoxicity and resistance. Cancer Res. 58(24): 5749-61, 1998.
33. El-Kareh AW and Secomb TW. A mathematical model for cisplatin cellular pharmacodynamics. Neoplasia. 5 (2): 161-9, 2003.
46
34. Sheiner L and Wakefield J. Population modelling in drug development. Stat Methods Med Res. 8 (3): 183-93, 1999.
35. Sandstrom M, Simonsen LE, Freijs A, and Karlsson MO. The pharmacokinetics of epirubicin and docetaxel in combination in rats. Cancer Chemother Pharma-col. 44 (6): 469-74, 1999.
36. Rouits E, Guichard S, Canal P, and Chatelut E. Non-linear pharmacokinetics of irinotecan in mice. Anticancer Drugs. 13 (6): 631-5, 2002.
37. Simonsen LE, Wahlby U, Sandstrom M, Freijs A, and Karlsson MO. Haemato-logical toxicity following different dosing schedules of 5- fluorouracil and epirubicin in rats. Anticancer Res. 20 (3A): 1519-25., 2000.
38. Friberg LE and Karlsson MO. Mechanistic models for myelosuppression. Invest New Drugs. 21 (2): 183-94, 2003.
39. Simeoni M, Magni P, Cammia C, De Nicolao G, Croci V, Pesenti E, Germani M, Poggesi I, and Rocchetti M. Predictive pharmacokinetic-pharmacodynamic modeling of tumor growth kinetics in xenograft models after administration of anticancer agents. Cancer Res. 64 (3): 1094-101, 2004.
40. Gilmore TD. The Rel/NF-kappaB signal transduction pathway: introduction. Oncogene. 18 (49): 6842-4, 1999.
41. Beg AA and Baltimore D. An essential role for NF-kappaB in preventing TNF-alpha-induced cell death. Science. 274 (5288): 782-4, 1996.
42. Kaltschmidt B, Kaltschmidt C, Hofmann TG, Hehner SP, Droge W, and Schmitz ML. The pro- or anti-apoptotic function of NF-kappaB is determined by the nature of the apoptotic stimulus. Eur J Biochem. 267 (12): 3828-35, 2000.
43. Barkett M and Gilmore TD. Control of apoptosis by Rel/NF-kappaB transcrip-tion factors. Oncogene. 18 (49): 6910-24, 1999.
44. Hilt W and Wolf DH. Proteasomes: destruction as a programme. Trends Bio-chem Sci. 21 (3): 96-102, 1996.
45. Karin M, Yamamoto Y, and Wang QM. The IKK NF-kappa B system: a treas-ure trove for drug development. Nat Rev Drug Discov. 3 (1): 17-26, 2004.
46. Schou C, Ottosen, E. R., Björkling, F.,Latini, S., Hjarnaa, P. V., Bramm, E., and Binderup, L. Novel cyanoguanidine with potent oral antitumor activity. Bioorg. & Med. Chem. Lett.,. (7): 3095-3100, 1997.
47. Aleskog A, Bashir-Hassan S, Hovstadius P, Kristensen J, Hoglund M, Tholander B, Binderup L, Larsson R, and Jonsson E. Activity of CHS 828 in primary cultures of human hematological and solid tumors in vitro. Anticancer Drugs. 12 (10): 821-7, 2001.
48. Hjarnaa PJ, Jonsson E, Latini S, Dhar S, Larsson R, Bramm E, Skov T, and Binderup L. CHS 828, a novel pyridyl cyanoguanidine with potent antitumor ac-tivity in vitro and in vivo. Cancer Res. 59 (22): 5751-7, 1999.
49. Hovstadius P, Larsson R, Jonsson E, Skov T, Kissmeyer AM, Krasilnikoff K, Bergh J, Karlsson MO, Lonnebo A, and Ahlgren J. A Phase I study of CHS 828 in patients with solid tumor malignancy. Clin Cancer Res. 8 (9): 2843-50, 2002.
50. Ravaud A, Morant R, Skov T, Uiters J, Bui BN, Gerny T, Botma HJ, Wanders J, and Fumoleau P. An EORTC/ECSG Phase I Study of CHS 828, a cyanogua-nidine in Patients with Refractory Solid Tumors. Proc. Am. Assoc. Cancer Res., 2914.. 2001.
51. Martinsson P, de la Torre M, Binderup L, Nygren P, and Larsson R. Cell death with atypical features induced by the novel antitumoral drug CHS 828, in hu-man U-937 GTB cells. Eur J Pharmacol. 417 (3): 181-7., 2001.
52. Martinsson P, Liminga G, Dhar S, de la Torre M, Lukinius A, Jonsson E, Bashir Hassan S, Binderup L, Kristensen J, and Larsson R. Temporal effects of the
47
novel antitumor pyridyl cyanoguanidine (CHS 828) on human lymphoma cells. Eur J Cancer. 37 (2): 260-7., 2001.
53. Olsen LS, Hjarnaa PJ, Latini S, Holm PK, Larsson R, Bramm E, Binderup L, and Madsen MW. Anticancer agent CHS 828 suppresses nuclear factor-kappaB activity in cancer cells through downregulation of IKK activity. Int J Cancer. 111 (2): 198-205, 2004.
54. Wjllie A, Kerr J, and Currie A. Cell Death: The Significance of Apoptosis. In-ternational Review of Cytology. 68 251-306, 1980.
55. Larsson R, Kristensen J, Sandberg C, and Nygren P. Laboratory determination of chemotherapeutic drug resistance in tumor cells from patients with leukemia, using a fluorometric microculture cytotoxicity assay (FMCA). Int J Cancer. 50 (2): 177-85, 1992.
56. Hill AV. The possible effects of the aggregation of the molecules of haemoglo-bin on its dissociation curves. J. Physiol.. 40 iv-vii, 1910.
57. Beal SL and Sheiner LB. NONMEM users guide. NONMEM project group. University of California. San Francisco, 1992.
58. Ding GJ, Fischer PA, Boltz RC, Schmidt JA, Colaianne JJ, Gough A, Rubin RA, and Miller DK. Characterization and quantitation of NF-kappaB nuclear translocation induced by interleukin-1 and tumor necrosis factor-alpha. Devel-opment and use of a high capacity fluorescence cytometric system. J Biol Chem. 273 (44): 28897-905, 1998.
59. Rock KL, Gramm C, Rothstein L, Clark K, Stein R, Dick L, Hwang D, and Goldberg AL. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell. 78 (5): 761-71, 1994.
60. Tannock IF, Hill R.P. The Basic science of Oncology 134-162, 1998. 61. Hansen CM, Hansen D, Holm PK, Larsson R, and Binderup L. Cyanoguanidine
CHS 828 induces programmed cell death with apoptotic features in human breast cancer cells in vitro. Anticancer Res. 20 (6B): 4211-20., 2000.
62. Lovborg H, Nygren P, and Larsson R. Multiparametric evaluation of apoptosis: effects of standard cytotoxic agents and the cyanoguanidine CHS 828. Mol Cancer Ther. 3 (5): 521-6, 2004.
63. Hahnfeldt P, Panigrahy D, Folkman J, and Hlatky L. Tumor development under angiogenic signaling: a dynamical theory of tumor growth, treatment response, and postvascular dormancy. Cancer Res. 59 (19): 4770-5, 1999.
64. Blagosklonny MV and Fojo T. Molecular effects of paclitaxel: myths and reality (a critical review). Int J Cancer. 83 (2): 151-6., 1999.
65. Huang TT, Wuerzberger-Davis SM, Seufzer BJ, Shumway SD, Kurama T, Boothman DA, and Miyamoto S. NF-kappaB activation by camptothecin. A linkage between nuclear DNA damage and cytoplasmic signaling events. J Biol Chem. 275 (13): 9501-9., 2000.
66. Rowinsky EK. The taxanes: dosing and scheduling considerations. Oncology (Huntingt). 11 (3 Suppl 2): 7-19., 1997.
67. O'Reilly S. Topotecan: what dose, what schedule, what route? Clin Cancer Res. 5 (1): 3-5., 1999.
68. Bungay PM, Dedrick RL, and Matthews HB. Enteric transport of chlordecone (Kepone) in the rat. J Pharmacokinet Biopharm. 9 (3): 309-41, 1981.
69. Strange RC. Hepatic bile salt transport. A review of subcellular binding sites. Biochem Soc Trans. 9 (1): 170-4, 1981.
70. Boddy AV, Cole M, Pearson AD, and Idle JR. The kinetics of the auto-induction of ifosfamide metabolism during continuous infusion. Cancer Chemo-ther Pharmacol. 36 (1): 53-60, 1995.
48
71. Hassan M, Svensson US, Ljungman P, Bjorkstrand B, Olsson H, Bielenstein M, Abdel-Rehim M, Nilsson C, Johansson M, and Karlsson MO. A mechanism-based pharmacokinetic-enzyme model for cyclophosphamide autoinduction in breast cancer patients. Br J Clin Pharmacol. 48 (5): 669-77, 1999.
72. Lövborg H, Cellular Pharmacology of the Novel Antitumor Cyanoguanidine CHS 828, Faculty of Medicine, Upssala University: Uppsala, Sweden, 2004.
73. Ekelund S, Liminga G, Bjorkling F, Ottosen E, Schou C, Binderup L, and Lars-son R. Early stimulation of acidification rate by novel cytotoxic pyridyl cyano-guanidines in human tumor cells: comparison with m-iodobenzylguanidine. Biochem Pharmacol. 60 (6): 839-49, 2000.
74. Lovborg H, Martinsson P, Gullbo J, Ekelund S, Nygren P, and Larsson R. Modulation of pyridyl cyanoguanidine (CHS 828) induced cytotoxicity by 3-aminobenzamide in U-937 GTB cells. Biochem Pharmacol. 63 (8): 1491-8, 2002.
75. Csoka K, Dhar S, Fridborg H, Larsson R, and Nygren P. Differential activity of Cremophor EL and paclitaxel in patients' tumor cells and human carcinoma cell lines in vitro. Cancer. 79 (6): 1225-33, 1997.
76. Fjallskog ML, Frii L, and Bergh J. Paclitaxel-induced cytotoxicity--the effects of cremophor EL (castor oil) on two human breast cancer cell lines with ac-quired multidrug resistant phenotype and induced expression of the permeability glycoprotein. Eur J Cancer. 30A (5): 687-90, 1994.
77. Fjallskog ML, Frii L, and Bergh J. Is Cremophor EL, solvent for paclitaxel, cytotoxic? Lancet. 342 (8875): 873, 1993.
Acta Universitatis UpsaliensisComprehensive Summaries of Uppsala Dissertations
from the Faculty of MedicineEditor: The Dean of the Faculty of Medicine
Distribution:Uppsala University Library
Box 510, SE-751 20 Uppsala, Swedenwww.uu.se, [email protected]
ISSN 0282-7476ISBN 91-554-6113-1
A doctoral dissertation from the Faculty of Medicine, Uppsala University,is usually a summary of a number of papers. A few copies of the completedissertation are kept at major Swedish research libraries, while the sum-mary alone is distributed internationally through the series Comprehen-sive Summaries of Uppsala Dissertations from the Faculty of Medicine.(Prior to October, 1985, the series was published under the title “Abstracts ofUppsala Dissertations from the Faculty of Medicine”.)
