3. Description of different cytotoxicity tests

The cellular damage caused by different chemical compounds can be various and thus, the methods to measure this effect are numerous. To select the proper test, we must know: number of treated cells, number of treatments, what kind of treatment the cells got, do we need these cells later, or the chosen method can terminate them? Also, do we want to know the kinetics of the post-treatment population's changes or simply what happens with the cells after a short incubation with the selected chemical? The price, the reliability, the user-friendliness of the kit is an important question too. The mechanism of cytotoxicity can be various so a single method only gives a simple view on a chosen material. Multiple tests and methods must be used before anyone can make a solid point about the biocompatibility of a chemical compound. Also, it must be noted, that a compound can interfere with the detection mechanism of a certain assay, resulting in a false positive or negative result; thus, multiple methods are needed to avoid such cases.

MTT assay is a cheap, popular way, to measure cell death [21]. The reduction of the tetrazolium structure in the MTT dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide leads to a colored formazan product—this is the basic chemical reaction in every tetrazolium based assay. The MTT dye has a positive charge, thus it is taken up by the cells and intracellular oxidoreductases catalyze the mentioned reaction. The oxidative state of the cell and the mitochondrial respiratory chain are essential in the conversion. Basically, the concentration of NADH limits the process and any chemical compound that modifies the oxidative potential of the cell can possibly decrease or increase the signal of the assay [22]. If this effect is not linked with direct cytotoxic activity, then, it may lead to a false positive or negative result. Various reports have already indicated that certain test compounds have radically different results in tetrazolium assay, than in other methods because of the possible antioxidant capabilities [23]. The reaction starts immediately, but 1–4 h should pass before measuring the absorbance of the test system the exact time must be setup according to the parameters of the current test system. A too short amount of time might result in low signal strength; a too long may mask the difference between different treatments/concentrations. Various organic solvents might be used to dissolve final product, the insoluble, purple formazan crystals, but we suppose an isopropanol:hydrochloride acid (25:1) solution, because it is safe and cheap to use. The acidification of the system is required, to reduce the amount of the original yellow dye, thus, give us a stronger main signal. The

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XTT, MTS, WST-1, and WST-8 assays are the improved versions of the old MTT. The final product of the reaction is soluble in water/cell culture medium; yet, the solubility of the original dye is greatly reduced, they must be used at 1–2 mg/ml concentrations instead of the 0.2–0.5 mg/ml of the original MTT dye [24]. The XTT and the WST-1/8 compounds have a negative charge, so they cannot penetrate the cell membrane; their reduction takes place in the extracellular space [22]. To enhance the effectiveness of transmembrane oxidoreductases, an

absorbance must be measured at 570 nm (Figures 1 and 2).

Figure 1. MTT dye reaction, the tetrazolium ring opens as a result of the reduction.

Figure 2. Simplified mechanisms of MTT and MTS assays.

#### 3.1. Assays

Cell viability assays are usually cheap, easy-to-perform methods, where after a given incubation with the selected chemical compound, the number of the surviving cells is measured by some method. They use no antibodies or radioactive chemicals. Usually, these assays are carried out on 96- or 384-well plates, making them ideal for screening experiments. It must be noted, that in some special cases, multiple measurements can be made, but these methods are not suited for long-term, kinetical or time-dependent killing studies. We must be aware of the fact, that most methods use some kind of "signal molecule" which—in normal cases—is directly proportional with the number of cells. If there is an uncontrolled factor that increases or decreases the strength of the signal, we can get false positive of false negative results. Such factor is usually increased uptake into the cell or increased activity of the specific enzyme, responsible for the creation of the signaling molecule. Usually not a single enzyme, but multiple proteins catalyze the reaction, so the overall metabolic state of the cell must be considered. It can be said that additional filters for background measuring can greatly increase the sensitivity. Nearly all eukaryotic cells can perform these biochemical reactions, but previous research can avoid incompatibility with certain cell lines. In the following table, the most well-known cell viability assays are listed. Prices are approximating, and they mean the price of 1000 tests of the kit, according to the manufacturer (Table 2).

Table 2. Most common cytotoxicity assay methods.

MTT assay is a cheap, popular way, to measure cell death [21]. The reduction of the tetrazolium structure in the MTT dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide leads to a colored formazan product—this is the basic chemical reaction in every tetrazolium based assay. The MTT dye has a positive charge, thus it is taken up by the cells and intracellular oxidoreductases catalyze the mentioned reaction. The oxidative state of the cell and the mitochondrial respiratory chain are essential in the conversion. Basically, the concentration of NADH limits the process and any chemical compound that modifies the oxidative potential of the cell can possibly decrease or increase the signal of the assay [22]. If this effect is not linked with direct cytotoxic activity, then, it may lead to a false positive or negative result. Various reports have already indicated that certain test compounds have radically different results in tetrazolium assay, than in other methods because of the possible antioxidant capabilities [23]. The reaction starts immediately, but 1–4 h should pass before measuring the absorbance of the test system the exact time must be setup according to the parameters of the current test system. A too short amount of time might result in low signal strength; a too long may mask the difference between different treatments/concentrations. Various organic solvents might be used to dissolve final product, the insoluble, purple formazan crystals, but we suppose an isopropanol:hydrochloride acid (25:1) solution, because it is safe and cheap to use. The acidification of the system is required, to reduce the amount of the original yellow dye, thus, give us a stronger main signal. The absorbance must be measured at 570 nm (Figures 1 and 2).

XTT, MTS, WST-1, and WST-8 assays are the improved versions of the old MTT. The final product of the reaction is soluble in water/cell culture medium; yet, the solubility of the original dye is greatly reduced, they must be used at 1–2 mg/ml concentrations instead of the 0.2–0.5 mg/ml of the original MTT dye [24]. The XTT and the WST-1/8 compounds have a negative charge, so they cannot penetrate the cell membrane; their reduction takes place in the extracellular space [22]. To enhance the effectiveness of transmembrane oxidoreductases, an

Figure 1. MTT dye reaction, the tetrazolium ring opens as a result of the reduction.

Figure 2. Simplified mechanisms of MTT and MTS assays.

of treated cells, number of treatments, what kind of treatment the cells got, do we need these cells later, or the chosen method can terminate them? Also, do we want to know the kinetics of the post-treatment population's changes or simply what happens with the cells after a short incubation with the selected chemical? The price, the reliability, the user-friendliness of the kit is an important question too. The mechanism of cytotoxicity can be various so a single method only gives a simple view on a chosen material. Multiple tests and methods must be used before anyone can make a solid point about the biocompatibility of a chemical compound. Also, it must be noted, that a compound can interfere with the detection mechanism of a certain assay, resulting in a false positive or negative result; thus, multiple methods are needed to avoid such

Cell viability assays are usually cheap, easy-to-perform methods, where after a given incubation with the selected chemical compound, the number of the surviving cells is measured by some method. They use no antibodies or radioactive chemicals. Usually, these assays are carried out on 96- or 384-well plates, making them ideal for screening experiments. It must be noted, that in some special cases, multiple measurements can be made, but these methods are not suited for long-term, kinetical or time-dependent killing studies. We must be aware of the fact, that most methods use some kind of "signal molecule" which—in normal cases—is directly proportional with the number of cells. If there is an uncontrolled factor that increases or decreases the strength of the signal, we can get false positive of false negative results. Such factor is usually increased uptake into the cell or increased activity of the specific enzyme, responsible for the creation of the signaling molecule. Usually not a single enzyme, but multiple proteins catalyze the reaction, so the overall metabolic state of the cell must be considered. It can be said that additional filters for background measuring can greatly increase the sensitivity. Nearly all eukaryotic cells can perform these biochemical reactions, but previous research can avoid incompatibility with certain cell lines. In the following table, the most well-known cell viability assays are listed. Prices are approximating, and they mean the price

Name Mechanism How to detect Price (€) XTT Enzymatic activity Spectrophotometer 235 MTT Enzymatic activity Spectrophotometer 129 WST-1 Enzymatic activity Spectrophotometer 235 WST-8 Enzymatic activity Spectrophotometer 420 MTS Enzymatic activity Spectrophotometer 172 LDH Enzymatic activity Spectrophotometer 315 Resazurin Enzymatic activity Spectrofluorometer 110 Neutral Red Lysosomal uptake Spectrophotometer 339

of 1000 tests of the kit, according to the manufacturer (Table 2).

Table 2. Most common cytotoxicity assay methods.

cases.

136 Cytotoxicity

3.1. Assays

intermediate electron acceptor (IEA in figure) is required, which gets reduced by NADH and intensifies the final signal of the assay. The specific IEA (such as phenazine ethyl sulfate) is usually part of the assay kits and its concentration must be determined according to the given test system. The MTS is at least partially reduced in the intracellular compartment, thus an IEA is not essentially needed [22]. Because, the final formazan product is soluble in water, the solubilization step is unnecessary; these assays are more flexible to combine with additional methods and easier to carry out (Figures 3 and 4).

Resazurin assay (or Alamar Blue) is based on the enzymatic conversion of the cell permeable blue resazurin to the pink resorufin [25]. Both chemicals are soluble in water and the assay provides higher sensitivity than the previous methods, because the detection is based on fluorescence, not spectrophotometry. Also, it must be noted that the reagent is toxic, so the appropriate reaction time must be based on the sensitivity of the given cell line so the toxicity of the reagent can be distinguished from the tested chemical. The fluorescence should be measured with 560 nm excitation/590 nm emission filters. Also, it can be combined with other techniques such as caspase activity assay because of the different detection method and the non-interference of the respective mechanisms [26] (Figure 5).

the LDH assay or multiple wash steps are needed before applying the assay reagent on the cells. Another problem is that if the cells are dead (or growth is inhibited), but the specific cytotoxic chemical does not distort the cell membrane, then the actual number of dead cells is underestimated. A solution of it can be the usage of control compounds which have a similar

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Neutral red assay is not based on a directly enzymatic biochemical reaction, but the dye is taken up by the cells and it stains the lysosomes of the cells [29]. A weak cationic compound, the neutral red is taken up by micropinocytosis or by non-ionic diffusion and is accumulated into the lysosomes [30]. After the cytotoxic treatment with the possibly cytotoxic chemical, the cells should be washed, then the staining solution must should be added to the test system. After an appropriate time of incubation, the dye must be removed and the cells are washed again. The incubation with a solubilization solution forces the cells to excrete the neutral red dye and thus, the concentration of it can be measured at 540 nm. As damaged cells can only take up and store neutral red at a decreased rate and dead cells are not stained at all, it is a sensitive assay, but, the cells are washed, disturbed multiple times which—if not carried out smoothly enough—can decrease the cell number, destroy the monolayer or other kind of

The simple cytotoxicity assays are limited in case of kinetical or time-dependent killing experiments. Simply, multiple measurements cannot be executed, because the test system is disturbed, some xenobiotics (tetrazolium dyes, other cofactors, etc.) are added to the cells or in case of some methods, the cells are solubilized. The reagents used in the assays can be directly

way-of-killing, than the tested chemical [28] (Figure 6).

Figure 5. Mechanism of the Resazurin assay.

collateral damage can happen to the cells (Figure 7).

Figure 6. Mechanism of LDH membrane leakage assay.

3.2. RT-CES

LDH assay is based on the activity of a cytoplasmic enzyme, the lactate dehydrogenase which reduces the NAD to NADH. NADH then reduces a tetrazolium dye (or other reagent) whose concentration can be measured spectrophotometrically [27]. The assay is mainly used for the detection of membrane leakage which correlates with the cell damage. The most common reagent is the 2-p-iodophenyl-3-p-nitrophenyl tetrazolium chloride (INT) which forms a red formazan product. The LDH is quite stable for the duration of the assay in the extracellular space and its amount is dependent on the cell size and oxidative activity, but similar among the cells of the same cell line. It is excellent when compared with other tetrazolium based methods because it can measure the damaged cells as well, not just the dead ones. Also in special cases, it can be used to detect the intracellular LDH concentration. A limitation of this method is that serum has a high LDH activity on its own, so serum-supplemented mediums are not ideal for

Figure 3. Chemical formulae of the new generation tetrazolium assays from left to right: MTS, XTT, and WST-1/8.

Figure 4. Simplified mechanism of the XTT and WTS-1/8 assay.

Figure 5. Mechanism of the Resazurin assay.

intermediate electron acceptor (IEA in figure) is required, which gets reduced by NADH and intensifies the final signal of the assay. The specific IEA (such as phenazine ethyl sulfate) is usually part of the assay kits and its concentration must be determined according to the given test system. The MTS is at least partially reduced in the intracellular compartment, thus an IEA is not essentially needed [22]. Because, the final formazan product is soluble in water, the solubilization step is unnecessary; these assays are more flexible to combine with additional

Resazurin assay (or Alamar Blue) is based on the enzymatic conversion of the cell permeable blue resazurin to the pink resorufin [25]. Both chemicals are soluble in water and the assay provides higher sensitivity than the previous methods, because the detection is based on fluorescence, not spectrophotometry. Also, it must be noted that the reagent is toxic, so the appropriate reaction time must be based on the sensitivity of the given cell line so the toxicity of the reagent can be distinguished from the tested chemical. The fluorescence should be measured with 560 nm excitation/590 nm emission filters. Also, it can be combined with other techniques such as caspase activity assay because of the different detection method and the

LDH assay is based on the activity of a cytoplasmic enzyme, the lactate dehydrogenase which reduces the NAD to NADH. NADH then reduces a tetrazolium dye (or other reagent) whose concentration can be measured spectrophotometrically [27]. The assay is mainly used for the detection of membrane leakage which correlates with the cell damage. The most common reagent is the 2-p-iodophenyl-3-p-nitrophenyl tetrazolium chloride (INT) which forms a red formazan product. The LDH is quite stable for the duration of the assay in the extracellular space and its amount is dependent on the cell size and oxidative activity, but similar among the cells of the same cell line. It is excellent when compared with other tetrazolium based methods because it can measure the damaged cells as well, not just the dead ones. Also in special cases, it can be used to detect the intracellular LDH concentration. A limitation of this method is that serum has a high LDH activity on its own, so serum-supplemented mediums are not ideal for

Figure 3. Chemical formulae of the new generation tetrazolium assays from left to right: MTS, XTT, and WST-1/8.

methods and easier to carry out (Figures 3 and 4).

138 Cytotoxicity

non-interference of the respective mechanisms [26] (Figure 5).

Figure 4. Simplified mechanism of the XTT and WTS-1/8 assay.

the LDH assay or multiple wash steps are needed before applying the assay reagent on the cells. Another problem is that if the cells are dead (or growth is inhibited), but the specific cytotoxic chemical does not distort the cell membrane, then the actual number of dead cells is underestimated. A solution of it can be the usage of control compounds which have a similar way-of-killing, than the tested chemical [28] (Figure 6).

Neutral red assay is not based on a directly enzymatic biochemical reaction, but the dye is taken up by the cells and it stains the lysosomes of the cells [29]. A weak cationic compound, the neutral red is taken up by micropinocytosis or by non-ionic diffusion and is accumulated into the lysosomes [30]. After the cytotoxic treatment with the possibly cytotoxic chemical, the cells should be washed, then the staining solution must should be added to the test system. After an appropriate time of incubation, the dye must be removed and the cells are washed again. The incubation with a solubilization solution forces the cells to excrete the neutral red dye and thus, the concentration of it can be measured at 540 nm. As damaged cells can only take up and store neutral red at a decreased rate and dead cells are not stained at all, it is a sensitive assay, but, the cells are washed, disturbed multiple times which—if not carried out smoothly enough—can decrease the cell number, destroy the monolayer or other kind of collateral damage can happen to the cells (Figure 7).

#### 3.2. RT-CES

The simple cytotoxicity assays are limited in case of kinetical or time-dependent killing experiments. Simply, multiple measurements cannot be executed, because the test system is disturbed, some xenobiotics (tetrazolium dyes, other cofactors, etc.) are added to the cells or in case of some methods, the cells are solubilized. The reagents used in the assays can be directly

Figure 6. Mechanism of LDH membrane leakage assay.

cytotoxic or at least they modify the cells biochemical equilibrium and activity in a way that the results are no longer relevant for the latter in vivo experiments. This means that after a certain treatment with the probably cytotoxic compound, we can only make an end-point measurement because the test system is irreversibly changed after the addition of the given signaling molecule(s). Also, if the tested chemical can absorb light or is capable of fluorescence, it can interfere with the detection system. Because of these limitations the need for a noninvasive, yet precise method resulted in the invention of real-time cell electronic sensor.

This technique is based on the impedance changes of the cell populations [31]. The cells are seeded into special E-plates, which are available in multiple size (4-, 8-, 16-, 96-wells, etc.). These special devices have a positive and a negative electrode in every well, and a low voltage alternate current flows through the well. As the cells grow, they have a higher impedance (resistance in AC circuits); and as they die, the impedance value lowers. This effect has literally no impact on the cells, so it is a non-invasive technique. The length of the experiment is theoretically unlimited, as there is no end-point of AC current flow. For this reason, the cell growth can be measured during multiple treatments of the cells, and not just the cytotoxic or non-toxic effects, but the possible recovery of the cells can be studied as well. It is important, that first, a part of the cell medium and the solution of the screened chemical must be placed into the wells of the E-plate, thus the connected software can detect it as a background, with zero impedance, so chemicals with ionic charges do not interfere with the measured signal. The cells should be added to the wells after the background detection in a high-density suspension. Also, the whole experiment can be stopped at any point, to remove the test solution or to add a new compound to test system (Figure 8).

requires an experienced crew to execute perfectly. Also, the total protein only works, if the cells grew in the presence of the cytotoxic chemical, otherwise, the dead cells cannot be distinguished from the viable ones. However, there are several studies indicating that the sulforhodamin B results correlate well with the MTT results [34]. A slight advantage of this method is that because of the multiple wash steps, the tested chemicals can hardly interact with the dye, unlike other enzyme-based methods. The optimization with the specific cell line

Figure 8. A typical RT-CES diagram showing a time and dose dependent cytotoxicity of 2,6-dichloro-(1,4)-benzoquinone

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Calcein-AM/Hoechst 33342 and propidium iodide are dyes that stain viable and dead cells [35]. In appropriate concentration, Calcein-AM, a lipophilic derivative of calcein is capable pass through cell membranes and stains the cell, as intracellular enzymes cleave the lipophilic carbon chain from the dye [36]. Hoechst 33342 binds the A-T rich regions of the DNA. Propidium iodide stains the nucleus of the cell, but cannot penetrate the cell membrane, thus it only binds to the dead cells. As the two reagents can be detected at different wavelengths, multiple emission and excitation filters are needed. Also, every cell line has a different binding rate and the ideal concentration must be found through testing, as the cytosol of the cells can be stained by each dye and instead of spectrophotometric detection, manual counting is

is also much easier because the lack of dependence on metabolic activity (Figure 9).

needed with a microscope with the specific filters/lamps (Figure 10).

[32].

Figure 9. Sulforhodamin B.

However, this system is ideal for cell viability studies, it has some disadvantages as well. The devices and the E-plates have a high price and a limited number of slots to use. The E-plates can be used multiple times after a specific cleaning protocol, but the sensitive, microelectronic sensing arrays are easily damaged by washing and organic solvents. This means, that it is not suited for high performance screening experiments, because multiple assays can be done during the same amount of time.

#### 3.3. Other methods

Sulforhodamin B is a dye which stains the total protein amount of the cells [33]. The reagent is an aminoxanthene dye which binds stoichiometrically with the amino-acids under acidic pH. First, the cells must be fixed with trichloroacetic acid, then washed and dried and the wells respective optical density measured for background detection. The sulforhodamin B must be added after this, and it should stain the cells for 20–30 min. After a wash step, the stained cells must be solubilized and the absorbance measured at 565 nm. The protocol is quite long and

Figure 7. Neutral red dye.

Figure 8. A typical RT-CES diagram showing a time and dose dependent cytotoxicity of 2,6-dichloro-(1,4)-benzoquinone [32].

requires an experienced crew to execute perfectly. Also, the total protein only works, if the cells grew in the presence of the cytotoxic chemical, otherwise, the dead cells cannot be distinguished from the viable ones. However, there are several studies indicating that the sulforhodamin B results correlate well with the MTT results [34]. A slight advantage of this method is that because of the multiple wash steps, the tested chemicals can hardly interact with the dye, unlike other enzyme-based methods. The optimization with the specific cell line is also much easier because the lack of dependence on metabolic activity (Figure 9).

Calcein-AM/Hoechst 33342 and propidium iodide are dyes that stain viable and dead cells [35]. In appropriate concentration, Calcein-AM, a lipophilic derivative of calcein is capable pass through cell membranes and stains the cell, as intracellular enzymes cleave the lipophilic carbon chain from the dye [36]. Hoechst 33342 binds the A-T rich regions of the DNA. Propidium iodide stains the nucleus of the cell, but cannot penetrate the cell membrane, thus it only binds to the dead cells. As the two reagents can be detected at different wavelengths, multiple emission and excitation filters are needed. Also, every cell line has a different binding rate and the ideal concentration must be found through testing, as the cytosol of the cells can be stained by each dye and instead of spectrophotometric detection, manual counting is needed with a microscope with the specific filters/lamps (Figure 10).

Figure 9. Sulforhodamin B.

cytotoxic or at least they modify the cells biochemical equilibrium and activity in a way that the results are no longer relevant for the latter in vivo experiments. This means that after a certain treatment with the probably cytotoxic compound, we can only make an end-point measurement because the test system is irreversibly changed after the addition of the given signaling molecule(s). Also, if the tested chemical can absorb light or is capable of fluorescence, it can interfere with the detection system. Because of these limitations the need for a noninvasive, yet precise method resulted in the invention of real-time cell electronic sensor.

This technique is based on the impedance changes of the cell populations [31]. The cells are seeded into special E-plates, which are available in multiple size (4-, 8-, 16-, 96-wells, etc.). These special devices have a positive and a negative electrode in every well, and a low voltage alternate current flows through the well. As the cells grow, they have a higher impedance (resistance in AC circuits); and as they die, the impedance value lowers. This effect has literally no impact on the cells, so it is a non-invasive technique. The length of the experiment is theoretically unlimited, as there is no end-point of AC current flow. For this reason, the cell growth can be measured during multiple treatments of the cells, and not just the cytotoxic or non-toxic effects, but the possible recovery of the cells can be studied as well. It is important, that first, a part of the cell medium and the solution of the screened chemical must be placed into the wells of the E-plate, thus the connected software can detect it as a background, with zero impedance, so chemicals with ionic charges do not interfere with the measured signal. The cells should be added to the wells after the background detection in a high-density suspension. Also, the whole experiment can be stopped at any point, to remove the test

However, this system is ideal for cell viability studies, it has some disadvantages as well. The devices and the E-plates have a high price and a limited number of slots to use. The E-plates can be used multiple times after a specific cleaning protocol, but the sensitive, microelectronic sensing arrays are easily damaged by washing and organic solvents. This means, that it is not suited for high performance screening experiments, because multiple assays can be done

Sulforhodamin B is a dye which stains the total protein amount of the cells [33]. The reagent is an aminoxanthene dye which binds stoichiometrically with the amino-acids under acidic pH. First, the cells must be fixed with trichloroacetic acid, then washed and dried and the wells respective optical density measured for background detection. The sulforhodamin B must be added after this, and it should stain the cells for 20–30 min. After a wash step, the stained cells must be solubilized and the absorbance measured at 565 nm. The protocol is quite long and

solution or to add a new compound to test system (Figure 8).

during the same amount of time.

3.3. Other methods

140 Cytotoxicity

Figure 7. Neutral red dye.

viability or cytotoxicity assays can be performed. Cytotoxicity assays based on membrane integrity changes are positive-readout assay which are most typically indicated for shorterterm exposure models (48 h or less). These assays may not properly determine the absolute degree of early or late stage cytotoxicity since the kinetics of biomarker emergence or degradation. Viability assays measure the level of biomarker activity inversely correlated with cytotoxicity, and therefore may be used at any endpoint during a compound/cell incubation period. Each biomarker of viability and cytotoxicity has advantages and disadvantages. Moreover in early drug discovery, in vitro evaluations of new drug candidates is often met with skepticism since their reliability and in vivo correlatability. It can be concluded that however there is some validity to this argument, it is important to put in vitro toxicity data into

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Ildikó Bácskay\*, Dániel Nemes, Ferenc Fenyvesi, Judit Váradi, Gábor Vasvári, Pálma Fehér,

Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Debrecen,

[1] Horvath S. Cytotoxicity of drugs and diverse chemical agents to cell cultures. Toxicology.

[2] Diaz D, O'Brien PJ. Defining the sequence of events in cerivastatin toxicity using a high content multi-parametric cytotoxicity assay. European Pharmaceutical Review. 2006;11:38-45

[3] Bolt M, Card J, Racz W, et al. Disruption of mitochondrial function and cellular ATP levels by amiodarone and N-desethylamiodarone in initiation of amiodarone-induced pulmonary cytotoxicity. The Journal of Pharmacology and Experimental Therapeutics. 2001;298:1280-1289

[4] Buxser S, Vroegop S. Calculating the probability of detection for inhibitors in enzymatic or binding reactions in high throughput screening. Analytical Biochemistry. 2005;340:1-

[5] Ujhelyi J, Ujhelyi Z, Szalai A, Laszlo JF, Cayasso M, Vecsernyes M, Porszasz R. Analgesic and anti-inflammatory effectiveness of sitagliptin and vildagliptin in mice. Regulatory

[6] Xia M, Huang R, Witt K, et al. Compound cytotoxicity profiling using quantitative highthroughput screening. Environmental Health Perspectives. 2008;116:284-291. DOI: 10.1289/

Peptides. 2014;194-195:23-29. DOI: 10.1016/j.regpep.2014.09.006

consideration during pharmaceutical development.

\*Address all correspondence to<EMAIL_ADDRESS>
Miklós Vecsernyés and Zoltán Ujhelyi

13. DOI: 10.1016/j.ab.2005.01.034

Author details

Debrecen, Hungary

1980;16:59-66

ehp.10727

References

Figure 10. Chemical formula of the Calcein-AM (upper image), Hoechst 33342 (left image) and the propidium iodide (right image).

#### 3.4. Comparison between in vitro cytotoxicity data and in vivo data

As the whole, medical science and industry is based on the modification, repair of damaged or badly functioning cells and tissues in human or animal body, the correlation between in vitro and in vivo data is crucial. Do these artificial test systems, cell lines truly replicate how a real tissue would react to a certain treatment or compound? The answer is based on the application of multiple in vitro methods and the careful planning of the in vivo experiments. A good example of the practice is the study of Yu et al. [37]. Xantii fructus is a traditional Chinese herbal drug and clinical reports indicated its renal toxicity. The study was based on MTT and LDH assays of the main components of the herbal drug on a renal cell line, as well as acute and chronic toxicity experiments in rats. While the main component of the drug, the atractyloside potassium salt showed no cytotoxicity on the cell lines, the water extract of the fruit had an inhibitory effect in case of high concentrations on the MTT assay, but no membrane damage on the LDH assay. These results indicate that the secondary components of the water extract have cytotoxic capabilities and the exact mechanism of killing might involve the suppressed metabolic activity of the cells, but not the damage of the cell membrane. The acute in vivo toxicity showed that only high concentrations could terminate the rats and cause abnormalities in the organs and the chronic toxicity showed only minor changes in the highest concentration group. Overall, this complex study created a much more accurate, scientific point of view about the toxicity of Xanthii fructus, what chemicals are responsible for its toxicity, what are the exact dosages, and what are the side effects that are caused by the herbal drug. It could only be made by the co-application of various in vivo and in vitro methods.

#### 4. Conclusion

The importance of cytotoxicity assays in early drug development is unquestionable although it must be concluded that no assay technology for detecting cytotoxicity in vitro is perfect. Strong arguments can be made for and against using cell viability or cytotoxicity assays as a reliable model of human medication. Depending upon the objectives of the investigation, either viability or cytotoxicity assays can be performed. Cytotoxicity assays based on membrane integrity changes are positive-readout assay which are most typically indicated for shorterterm exposure models (48 h or less). These assays may not properly determine the absolute degree of early or late stage cytotoxicity since the kinetics of biomarker emergence or degradation. Viability assays measure the level of biomarker activity inversely correlated with cytotoxicity, and therefore may be used at any endpoint during a compound/cell incubation period. Each biomarker of viability and cytotoxicity has advantages and disadvantages. Moreover in early drug discovery, in vitro evaluations of new drug candidates is often met with skepticism since their reliability and in vivo correlatability. It can be concluded that however there is some validity to this argument, it is important to put in vitro toxicity data into consideration during pharmaceutical development.


**2. Clinical studies of our modified boron neutron capture therapy**

Since the 1950s, BNCT has been used to treat malignant gliomas, although the results have not been satisfactory. We modified the therapy in several ways to resolve problems previously existing, and applied this modified BNCT to malignant gliomas beginning in 2002 by using KURRI (Kawabata et al. 2003). First, we utilized an epithermal rather than a thermal beam to improve the distribution of thermal neutrons in deep sites. Second, we used both of the boron compounds that are currently available worldwide for BNCT: sodium borocaptate (BSH) and boronophenylalanine (BPA). These compounds reach different subpopulations of tumor cells and accumulate in them in a different fashion.

#### **2.1 Survival benefit from modified boron neutron capture therapy in combination with external beam fractionated X-ray treatment**

We evaluate the clinical results of a form of tumor selective particle radiation, BNCT in combination with external beam fractionated X-ray treatment (XRT) for newly-diagnosed GB patients. Between 2002 and 2006, we treated 21 patients of newly-diagnosed GB with BNCT utilizing sodium borocaptate and boronophenylalanine simultaneously. The first 10 were treated with only BNCT (protocol 1), and the last 11 were treated with BNCT followed by fractionated XRT of 20 to 30 Gy (protocol 2) to reduce the possibility of local tumor recurrence. No chemotherapy was applied until tumor progression was observed.

#### **2.1.1 Methods and the study design of our modified BNCT with XRT**

As mentioned previously, we modified the protocol of BNCT as follows: First, we started using epithermal neutrons instead of thermal neutrons to obtain good penetration for deepseated lesions. Second, we simultaneously used 2 different boron compounds (BSH and BPA) with different accumulation mechanisms to the tumor cells (Ono *et al.* 1999, Yokoyama *et al.* 2006). Third, we utilized a dose simulation work station, the Simulation Environments for Radiotherapy Applications (SERA; Idaho National Laboratory, Idaho Falls, ID) or the JAERI computational dosimetry system (JCDS). Fourth, 18F-labeled BPA positron emission tomography (18F-BPA-PET) (Fig.2) was applied for the estimation of the boron compound

been synthesized (Hiramatsu *et al.* 2011, Miyata *et al.* 2011, Wu *et al.* 2007, Yang *et al.* 2006), but only two of these currently are being used in clinically. The first, which has been used primarily in Japan, is sodium borocaptate or BSH, and the second is a dihydroxyboryl derivative of phenylalanine referred to as boronophenylalanine or BPA (Barth *et al.* 2005). The latter has been used in clinical trials in Japan, Europe and the United States, primarily for the treatment of high grade gliomas, and more recently for recurrent tumors of the head and neck region (Ariyoshi *et al.* 2007, Haginomori *et al.* 2009, Kimura *et al.* 2009). Following i.v. administration of either BPA or BSH by i.v. infusion, the tumor site is irradiated with

Recently, BNCT studies carried out by us at Osaka Medical College (OMC) and Kyoto University Research Reactor Institute (KURRI), in which BPA and BSH were administered in combination (Kawabata *et al.* 2003, Miyatake *et al.* 2005, Miyatake *et al.* 2009) for the patients with recurrent tumor after irradiation, or BNCT followed by an X-ray boost showed favorable responses in patients with newly-diagnosed glioblastoma (GB) and especially

Since the 1950s, BNCT has been used to treat malignant gliomas, although the results have not been satisfactory. We modified the therapy in several ways to resolve problems previously existing, and applied this modified BNCT to malignant gliomas beginning in 2002 by using KURRI (Kawabata et al. 2003). First, we utilized an epithermal rather than a thermal beam to improve the distribution of thermal neutrons in deep sites. Second, we used both of the boron compounds that are currently available worldwide for BNCT: sodium borocaptate (BSH) and boronophenylalanine (BPA). These compounds reach different subpopulations of tumor cells and accumulate in them in a different fashion.

**2.1 Survival benefit from modified boron neutron capture therapy in combination with**

We evaluate the clinical results of a form of tumor selective particle radiation, BNCT in combination with external beam fractionated X-ray treatment (XRT) for newly-diagnosed GB patients. Between 2002 and 2006, we treated 21 patients of newly-diagnosed GB with BNCT utilizing sodium borocaptate and boronophenylalanine simultaneously. The first 10 were treated with only BNCT (protocol 1), and the last 11 were treated with BNCT followed by fractionated XRT of 20 to 30 Gy (protocol 2) to reduce the possibility of local tumor

As mentioned previously, we modified the protocol of BNCT as follows: First, we started using epithermal neutrons instead of thermal neutrons to obtain good penetration for deepseated lesions. Second, we simultaneously used 2 different boron compounds (BSH and BPA) with different accumulation mechanisms to the tumor cells (Ono *et al.* 1999, Yokoyama *et al.* 2006). Third, we utilized a dose simulation work station, the Simulation Environments for Radiotherapy Applications (SERA; Idaho National Laboratory, Idaho Falls, ID) or the JAERI computational dosimetry system (JCDS). Fourth, 18F-labeled BPA positron emission tomography (18F-BPA-PET) (Fig.2) was applied for the estimation of the boron compound

recurrence. No chemotherapy was applied until tumor progression was observed.

**2.1.1 Methods and the study design of our modified BNCT with XRT**

**2. Clinical studies of our modified boron neutron capture therapy**

neutrons, the source of which is a nuclear reactor.

those in high risk groups (Kawabata *et al.* 2009a).

**external beam fractionated X-ray treatment**

accumulation prior to neutron irradiation (Imahori *et al.* 1998). Fifth, we filled the tumor removed cavity with air to obtain enough neutron flux, especially for the bottom of deepseated tumors (Sakurai *et al.* 2006). Sixth, we developed a central shielding method with a lithium plate at the center of the irradiation field to obtain uniform neutron distribution and increase the neutron flux relativey at the periphery in the radiation field (Ono 2006, Ono *et al.* 2000). With these modifications, even patients with deep-seated tumors can be treated by BNCT without craniotomy with a short hospital stay. In the present study, the revised protocol was used as a new protocol as follows.

Fig. 2. 18F labeled BPA positron emmision tomography (18F - BPA PET) has been applied for the estimation of the boron compound accumulation prior to BNCT. The tracer is fluororide labelled boron compound. This PET ensures the effectiveness of BNCT. 18F-BPA accumulates well and distributes precisely in the tumor lesion and the infiltrating tumor zone.

Twelve hours before the neutron irradiation, the patients were administered 100mg/kg of BSH intravenously for one hour. 700mg/kg of BPA was infused continuously to the patients for 6 hours before the irradiation, and they were positioned for neutron irradiation in the reactor (KURRI or JRR-4 (Japan Atomic Energy Agency Research Reactor 4)). Just after termination of continuous BPA infusion for 6hrs, neutrons were irradiated. We used the dose-planning workstation to calculate the radiation dose for tumors from the 18F-BPA-PET data and blood 10B concentrations obtained every 2 hours after BSH administration. We used an epithermal neutron beam. Following this, a 2 Gy daily fraction of XRT was applied, for a total of 20 to 30Gy. The total dose of XRT was decided based on the BNCT dose for the normal brain. In Protocol 1, we aimed to apply more than 30 gray-equivalent (Gy-Eq) for gross tumor volume (GTV) and less than 12 Gy-Eq for normal brain, as BNCT. In Protocol 2, we aimed to apply more than 40 Gy-Eq for GTV and less than 15 Gy-Eq for normal brain. No chemotherapy was applied for any of the patients until the tumor progression was confirmed histologically or by 18F-BPA-PET (Miyashita *et al.* 2008).

Survival time from histologically diagnosed as GB was compared with the survival time for the institutional historical controls who were treated by surgical removal followed by XRT and chemotherapy (mainly nitrosourea) from 1990 to 2006 in OMC. In this historical control

Clinical Study on Modified Boron Neutron Capture Therapy for Newly Diagnosed Glioblastoma 329

The RPA class distribution of 21 patients treated with BNCT at the initial diagnosis was as follows: Class III = 6 (29%); Class IV = 6 (29%); Class V = 8 (38%); Class VI = 1 (5%). The MSTs of the patients in classes III, IV, V, and VI were 23.5, 16.9, 13.2, and 9.8 months, respectively (Table 1). In historical control, the RPA class distribution was as follows: Class III = 3 (11%); Class IV = 14 (52%); Class V = 8 (30%); Class VI = 2 (7%). The distributions of each RPA class in BNCT group and institutional historical control group are a little bit different. We compare the survival of both groups in low risk RPA (class III and IV) and in high risk RPA (class V and IV) separately. The MST of BNCT group in low risk group was 18.5 months (n=12, 95% CI: 13.7-36.1) and that of historical control was 13.0 months (n=17, 95% CI: 8.6-18.0). There is statistical significance in log-rank test (p=0.028). The MST of BNCT group in high risk group was 12.2 months (n=9, 95% CI: 9.8-undetermined) and that of historical control was 7.4 months (n=10, 95% CI: 2.7-10.3). There is also statistical significance in log-rank test (p=0.0083). Therefore, it can be concluded that BNCT group shows the long survival in comparison with historical control not mainly by the difference of distribution of each RPA class in both groups. Our BNCT results for survival among the newly-diagnosed GB cases were favorable in comparison with those obtained from the

Fig. 4. Cumulative survival data for all newly diagnosed glioblastoma (protocol 1 and 2). External beam X-ray irradiation boost after boron neutron capture therapy (BNCT) (protocol 2, red line) was indicated for later 11 cases. This improve the median survival time as 23.5

months (vs 14.1 months for BNCT only (protocol 1, dotted red line)).

corresponding RTOG - RPA subclasses.

group, all cases were operated on to aim for the maximum tumor removal, the same as for the cases of the BNCT group. These Kaplan–Meier curves were calculated and the Log-rank test was used for statistical analysis. For the 21 patients who received BNCT, survival time was also compared with the corresponding recursive partitioning analysis (RPA) subclasses by the Radiation Therapy Oncology Group (RTOG) (Curran *et al.* 1993), as the international historical control, and European Organisation for Research and Treatment of Cancer - National Cancer Institute of Canada (EORTC-NCIC) trial (Stupp *et al.* 2009).

#### **2.1.2 Results from our past study**

Patients treated with BNCT (n=21) had a median ST of 15.6 months (95% confidence interval (CI): 12.2-23.9) after diagnosis (Fig. 3). Here the date of diagnosis is the initial debulking surgery date. This was significantly longer than the median survival time (MST) for the historical controls at our institute who were treated with surgical removal followed by XRT and chemotherapy (n=27, MST was 10.3 months (95% CI: 7.4-13.2), log-rank test p = 0.0035). The survival time from the date of diagnosis was calculated using the Kaplan-Meier method. The MST of the protocol 2 was 23.5 months (95% CI: 10.2 - undetermined) after diagnosis (n=11), and that of the protocol 1 patients (n=10) was 14.1 months (95% CI: 9.9- 18.5), although the difference was not statistically significant (Fig. 4).

Fig. 3. Cumulative survival data for all newly diagnosed glioblastoma (WHO grade 4, n=21). Blue line is our recent historical control treated by external beam X-ray irradiation. The median survival time (ST) of boron neutron capture therapy (BNCT) group (red line) is 15.6 months. There is statistical significance between both group in Log-rank test (p=0.0035).

group, all cases were operated on to aim for the maximum tumor removal, the same as for the cases of the BNCT group. These Kaplan–Meier curves were calculated and the Log-rank test was used for statistical analysis. For the 21 patients who received BNCT, survival time was also compared with the corresponding recursive partitioning analysis (RPA) subclasses by the Radiation Therapy Oncology Group (RTOG) (Curran *et al.* 1993), as the international historical control, and European Organisation for Research and Treatment of Cancer -

Patients treated with BNCT (n=21) had a median ST of 15.6 months (95% confidence interval (CI): 12.2-23.9) after diagnosis (Fig. 3). Here the date of diagnosis is the initial debulking surgery date. This was significantly longer than the median survival time (MST) for the historical controls at our institute who were treated with surgical removal followed by XRT and chemotherapy (n=27, MST was 10.3 months (95% CI: 7.4-13.2), log-rank test p = 0.0035). The survival time from the date of diagnosis was calculated using the Kaplan-Meier method. The MST of the protocol 2 was 23.5 months (95% CI: 10.2 - undetermined) after diagnosis (n=11), and that of the protocol 1 patients (n=10) was 14.1 months (95% CI: 9.9-

Fig. 3. Cumulative survival data for all newly diagnosed glioblastoma (WHO grade 4, n=21). Blue line is our recent historical control treated by external beam X-ray irradiation. The median survival time (ST) of boron neutron capture therapy (BNCT) group (red line) is 15.6 months. There is statistical significance between both group in Log-rank test

National Cancer Institute of Canada (EORTC-NCIC) trial (Stupp *et al.* 2009).

18.5), although the difference was not statistically significant (Fig. 4).

**2.1.2 Results from our past study**

(p=0.0035).

The RPA class distribution of 21 patients treated with BNCT at the initial diagnosis was as follows: Class III = 6 (29%); Class IV = 6 (29%); Class V = 8 (38%); Class VI = 1 (5%). The MSTs of the patients in classes III, IV, V, and VI were 23.5, 16.9, 13.2, and 9.8 months, respectively (Table 1). In historical control, the RPA class distribution was as follows: Class III = 3 (11%); Class IV = 14 (52%); Class V = 8 (30%); Class VI = 2 (7%). The distributions of each RPA class in BNCT group and institutional historical control group are a little bit different. We compare the survival of both groups in low risk RPA (class III and IV) and in high risk RPA (class V and IV) separately. The MST of BNCT group in low risk group was 18.5 months (n=12, 95% CI: 13.7-36.1) and that of historical control was 13.0 months (n=17, 95% CI: 8.6-18.0). There is statistical significance in log-rank test (p=0.028). The MST of BNCT group in high risk group was 12.2 months (n=9, 95% CI: 9.8-undetermined) and that of historical control was 7.4 months (n=10, 95% CI: 2.7-10.3). There is also statistical significance in log-rank test (p=0.0083). Therefore, it can be concluded that BNCT group shows the long survival in comparison with historical control not mainly by the difference of distribution of each RPA class in both groups. Our BNCT results for survival among the newly-diagnosed GB cases were favorable in comparison with those obtained from the corresponding RTOG - RPA subclasses.

Fig. 4. Cumulative survival data for all newly diagnosed glioblastoma (protocol 1 and 2). External beam X-ray irradiation boost after boron neutron capture therapy (BNCT) (protocol 2, red line) was indicated for later 11 cases. This improve the median survival time as 23.5 months (vs 14.1 months for BNCT only (protocol 1, dotted red line)).

Clinical Study on Modified Boron Neutron Capture Therapy for Newly Diagnosed Glioblastoma 331

Fig. 5. Dose planning of neutron capture therapy for the tumor (upper) and for the normal brain (lower). (Gy-Eq: gray equivalent, star: air instillation to the tumor removed cavity)

Fig. 6. Serial MRI images (T1-weighted with Gd enhancement) of the patient treated by boron neutron capture therapy combined with fractionated external beam X-ray irradiation. (A: prior to surgical removal and BNCT, B: 12 months, C: 14 months and D: 5 years after BNCT).

\*Median survival time (months). RTOG: radiation therapy oncology group, EORTC: european organisation for research and treatment of cancer, NCIC: National Cancer Institute of Canada Clinical Trials, RPA: recursive partitioning analysis, XRT: X-ray radiation therapy, TMZ: Temozolomide, BNCT: boron neutron capture therapy.

Table 1. Comparison of survival data among RPA class in the RTOG database, EORTC-NCIC (XRT/TMZ) trial, and in our cases treated with BNCT.

#### **2.2 Representative case treated by modified boron neutron capture therapy combination with fractionated X-ray irradiation**

A 63-year old female had a left parietal tumor removed partially in a hospital in November 2004. The histopathological diagnosis was GB. She was introduced to our hospital for BNCT for the remaining lesion. While she waited for the BNCT, rapid growth of the tumor caused aggravations of right hemiparesis, total aphasia and consciousness disturbance, all of which caused us to remove the tumor again in our hospital prior to BNCT. The tumor was gross totally removed, and the patient received BNCT with air instillation in tumor removed cavity, followed by XRT. At the time of BNCT, the workstation calculates two different estimated doses, one is for the tumor and the other is for the normal brain (Fig. 5).

Dose calculation shows estimated gray-equivalent (Gy-Eq) isodose lines from BNCT. This is mainly based on the decay of the irradiated neutron, boron concentration and biological effectiveness of each tissue. The deepest part of the tumor was 7.5 cm from the scalp in this case. The irradiated minimum tumor dose by BNCT was improved by the air-instilation (star in the Fig. 5) methods from 18.9 Gy-Eq (without air) to 26.9 Gy-Eq (with air). The maximum irradiated point of the normal brain was 2 cm from the scalp and was 12.7 Gy-Eq and scalp surface was 9.0 Gy-Eq. An additional 20 Gy was applied by XRT (daily fraction of 2 Gy x 10 days) aimed for the deep part of the mass but included surface of the tumor and the normal brain.

No tumor recurrence on MRI and no neurological deficit were seen for 13 months after BNCT (Fig. 6). BNCT followed by fractionated XRT could control the tumor for 12 months (Fig. 6B). However, 14 months after BNCT, the patient suffered from right hemiparesis and MRI revealed a Gd-enhanced lesion from the surface area of the tumor removed cavity which was irradiated by BNCT with enough dose and also included into the boost XRT (Fig. 6C). Radiation necrosis was suspected as the pathological condition based on L/N ratio in BPA-PET, but we could not neglect the possibility of a recurrence of the GB. We performed a re-craniotomy to remove this lesion. The pathological diagnosis was radiation necrosis; no apparent tumor cells were found by pathologists. After the surgery, the right hemiparesis was improved, while no tumor progression was observed on MRI in the follow-up for more than 5 years (Fig. 6D). The patient received best medical treatment (anti-coagulants and vitamin E) for a year to prevent a further radiation necrosis.

EORTC-NCIC

III 17.9 14.8 18.7 13.4 23.5 IV 11.1 13.3 16.3 12.8 16.9 V 8.9 9.1 10.7 7.4 13.2 VI 4.6 Not reported 3.6 9.8 \*Median survival time (months). RTOG: radiation therapy oncology group, EORTC: european organisation for research and treatment of cancer, NCIC: National Cancer Institute of Canada Clinical Trials, RPA: recursive partitioning analysis, XRT: X-ray radiation therapy, TMZ: Temozolomide, BNCT: boron neutron capture therapy. Table 1. Comparison of survival data among RPA class in the RTOG database, EORTC-

XRT XRT TMZ/XRT XRT BNCT

(XRT/TMZ) BNCT

RTOG RPA class

the normal brain.

RTOG original

NCIC (XRT/TMZ) trial, and in our cases treated with BNCT.

vitamin E) for a year to prevent a further radiation necrosis.

**combination with fractionated X-ray irradiation**

**2.2 Representative case treated by modified boron neutron capture therapy**

estimated doses, one is for the tumor and the other is for the normal brain (Fig. 5).

A 63-year old female had a left parietal tumor removed partially in a hospital in November 2004. The histopathological diagnosis was GB. She was introduced to our hospital for BNCT for the remaining lesion. While she waited for the BNCT, rapid growth of the tumor caused aggravations of right hemiparesis, total aphasia and consciousness disturbance, all of which caused us to remove the tumor again in our hospital prior to BNCT. The tumor was gross totally removed, and the patient received BNCT with air instillation in tumor removed cavity, followed by XRT. At the time of BNCT, the workstation calculates two different

Dose calculation shows estimated gray-equivalent (Gy-Eq) isodose lines from BNCT. This is mainly based on the decay of the irradiated neutron, boron concentration and biological effectiveness of each tissue. The deepest part of the tumor was 7.5 cm from the scalp in this case. The irradiated minimum tumor dose by BNCT was improved by the air-instilation (star in the Fig. 5) methods from 18.9 Gy-Eq (without air) to 26.9 Gy-Eq (with air). The maximum irradiated point of the normal brain was 2 cm from the scalp and was 12.7 Gy-Eq and scalp surface was 9.0 Gy-Eq. An additional 20 Gy was applied by XRT (daily fraction of 2 Gy x 10 days) aimed for the deep part of the mass but included surface of the tumor and

No tumor recurrence on MRI and no neurological deficit were seen for 13 months after BNCT (Fig. 6). BNCT followed by fractionated XRT could control the tumor for 12 months (Fig. 6B). However, 14 months after BNCT, the patient suffered from right hemiparesis and MRI revealed a Gd-enhanced lesion from the surface area of the tumor removed cavity which was irradiated by BNCT with enough dose and also included into the boost XRT (Fig. 6C). Radiation necrosis was suspected as the pathological condition based on L/N ratio in BPA-PET, but we could not neglect the possibility of a recurrence of the GB. We performed a re-craniotomy to remove this lesion. The pathological diagnosis was radiation necrosis; no apparent tumor cells were found by pathologists. After the surgery, the right hemiparesis was improved, while no tumor progression was observed on MRI in the follow-up for more than 5 years (Fig. 6D). The patient received best medical treatment (anti-coagulants and

Fig. 5. Dose planning of neutron capture therapy for the tumor (upper) and for the normal brain (lower). (Gy-Eq: gray equivalent, star: air instillation to the tumor removed cavity)

Fig. 6. Serial MRI images (T1-weighted with Gd enhancement) of the patient treated by boron neutron capture therapy combined with fractionated external beam X-ray irradiation. (A: prior to surgical removal and BNCT, B: 12 months, C: 14 months and D: 5 years after BNCT).

Clinical Study on Modified Boron Neutron Capture Therapy for Newly Diagnosed Glioblastoma 333

chemotherapy with TMZ. Prescription dose by BNCT is regulated as not to be more than 13Gy-Eq for normal brain. Additional XRT is given with 3 gradient such as 8, 16, 24Gy from the surface of scalp to the bottom of tumor infiltrated zone (Fig. 7). Chemotherapy with TMZ is applied concomitantly during XRT treatments and adjuvant chemotherapy with the

Fig. 7. Illustrated image of the protocol combined with boron neutron capture therapy (BNCT) and 3 gradient fractionated X-ray irradiation (XRT). Gy: gray, fr: fraction.

18F-BPA-PET study is included for the diagnosis of these pathologies.

Based on our previous clinical study, the Hazard ratio of BNCT vs. XRT was simulated as 0.4, so the total estimated number of the patients who should be included in our new study become 45 totally. Primary end point is overall survival and these patients will be followed up for 2 years after the last patient treatment. The most important point in our protocol is diagnosis and treatment of radiation effects such as swelling, radiation induced edema, transient expansion of the tumor, pseudo- progression / response, and radiation necrosis.

same agent is repeated in outpatient clinic (Fig. 8) (Stupp *et al.* 2005).

#### **2.3 Phase II clinical study of boron neutron capture therapy combined with X-ray radiotherapy / Temozolomide in patients with newly diagnosed glioblastoma multiforme**

Results of our clinical study showed the efficacy of combination therapy with external beam XRT and BNCT. For our future study, we planned the Multi-centric Phase II clinical study named "*Boron Neutron Capture Therapy, Radiation Therapy, and Temozolomide in Treating Patients with Newly Diagnosed Glioblastoma Multiforme*" in Japan (OSAKA-TRIBRAIN0902, NCT00974987). The major differences of our protocol from the other past BNCT studies were simultaneous use of both BSH and BPA, and combination with XRT.


**1. Introduction**

Salt marshes are defined by daily tidal inundation and dominated by halophytic vegetation. These ecosystems are the boundary between terrestrial and nearshore aquatic environments their unique location on the landscape and vegetation composition provides ecosystem services such as denitrification, filtration of pollutants, nursey habitat, coastal resilience, and carbon storage and sequestration [1,2]. Historically, salt marshes have displayed high rates of loss due to land reclamation and disturbances such as mosquito ditching [3,4]. Currently, salt marshes along the mid-Atlantic coastal region of the United States are at risk of loss due to sea level rise (SLR), eutrophication, nutrient enrichment, sediment availability, tidal range, and herbivory and human disturbances [5–12]. Recent studies have demonstrated regional and site-specific salt marsh changes including degradation in the Mid-Atlantic [13], proliferation of salt marsh pools in Maryland [14], loss coupled with increased Phragmites on Long Island [15], and loss driven by SLR in New England [16]. However, salt marsh change is a complex combination of persistence, migration, and loss. In the Chesapeake Bay, conversion of uplands to wetlands has mitigated past salt marsh losses [17]. Future salt marsh change is uncertain with some models predicting that salt marsh migration in response to SLR will result in increased salt marsh area [18]. Salt marsh monitoring is necessary for improved understanding of how these ecosystems are changing which in turn can inform their management.

Salt marshes are changing in a variety of ways necessitating a shared nomenclature to discuss these changes. In New England, four types of salt marsh losses have been identified channel widening, interior die-o ff, shoreline erosion, and loss in the bay head region [16]. These distinctions are dependent on the location of the change. In remote sensing and this study, two major categories were evident change along the edge and change in the interior of the salt marsh. Two types of interior salt marsh loss have been identified: sudden vegetation dieback and drowning. Sudden vegetation dieback in salt marshes is a rapid onset event that persists for a brief period (≈2 years) [19]. These die-o ffs are predominately located in the mid-marsh and have been documented across the eastern Atlantic coast [20]. In contrast, interior drowning driven by sea level rise is outside the scope of these rapid die-o ff events and represents a fundamental shift in the ecosystem [20]. Monitoring salt marsh change is further complicated by drowning appearing similar to ponding in microtidal salt marshes [21] and pools changing shape dynamically, draining, and revegetating [22]. Monitoring and di fferentiating between sudden vegetation dieback, drowning, and ponding necessitates high spatial resolution monitoring to assess expansion and recovery dynamics of interior salt marsh areas.

In this study, we focused on estuarine persistent emergent vegetation, i.e. intertidal areas with perennial salt marsh vegetation [23]. We are interested in identifying pools and pannes and how they changed. Pannes are recessed areas of the salt marsh which drain at mean lowest low water (MLLW) and can be vegetated or nonvegetated, in this study we are referring to nonvegetated pannes unless otherwise stated. In comparison pools are those areas of persistent water. Ponding, pools, and pannes are natural elements of the salt marsh landscape, however long-term and widespread loss of vegetation is not. An in situ study of Plum Island estuary, Massachusetts, found pools were in equilibrium with vegetation regrowth occurring after a few years or at most a decade [24]. However, vegetation regrowth is not guaranteed with site-specific characteristics, such as low sediment input, small tidal range, and high regional SLR, contributing to lack of vegetation growth in pannes and slow filling of pools [25]. Identifying these changes in situ is time consuming and non-tenable for large geographic areas. This study presents a method for using satellite and aerial imagery to di fferentiate between drowning and ponding by monitoring for decades, a temporal period beyond the expected recovery time.

This study is focused on Fire Island, NY, a barrier island, salt marshes on barrier islands have limited land for salt marsh migration. As a result, salt marsh persistence in place is of particular concern, and is a key component of understanding the long-term stability of barrier island systems. Storm events are critical for shaping the geomorphology of barrier islands, e.g., Hurricane Sandy caused overwash on 41 percent of Fire Island depositing an estimated 508,354 m3 of sediment [26] and breached the barrier island. Both overwash and inlet creation are essential sources of sediment accretion in bayside salt marsh environments [27,28]. Even thick overwash deposits (>50 cm) result in quick recovery of the salt marsh vegetation [29]. Mapping salt marsh change following a storm event and breach is critical for our understanding of salt marsh persistence on barrier islands. A previous breach on Fire Island was documented with radiometric dating of salt marsh cores, finding a connection between back-barrier salt marsh formation and inlet creation and the resulting processes [30]. Recent research on anthropogenic alterations of an inlet, i.e., jetty creation, demonstrated changes in local mean sea level (LMSL) and tidal range, which resulted in stabilization of salt marsh directly surrounding the inlet [31]. Similar changes to LMSL and tidal range could be a ffecting the salt marshes of Fire Island. The decision to allow the Fire Island breach to evolve naturally facilitates monitoring to understand the e ffect of this process on the surrounding salt marshes.

Remote sensing monitoring can quantify essential attributes of the salt marsh landscape. Remote sensing has been used to understand di fferences in salt marsh pond density and the total surface area between ditched and unditched marshes [32]. Imagery and Light Detection and Ranging (LiDAR) have been used to quantify pools and pannes and their landscape location [33]. Salt marsh change analysis has been used to identify complex patterns of spatial and temporal variation [14], dramatic conversions from high to low marsh [34], impacts of salt marsh restoration and Hurricane Sandy [35], and identify the e ffect of SLR on salt marsh communities [36]. The combination of very high resolution (VHR) satellite imagery and aerial imagery provided the necessary spatial and temporal resolution to understand the dynamic coastal environment.

This study mapped salt marsh habitat on Fire Island National Seashore (FIIS) utilizing object-based image analysis (OBIA) with VHR satellite imagery. Multitemporal OBIA was utilized to analyze the development and geospatial dynamics of pool and pannes with high spatial resolution aerial imagery from 1994, 2011, 2013, 2015, and 2017. Change to pannes and pools and edge erosion were analyzed to determine if they were significant components of salt marsh loss at FIIS. The use of remote sensing to determine how protected areas are changing with high accuracy is vital to better manage these areas. The objectives of this study are to (1) classify FIIS salt marsh with OBIA and VHR remote sensing imagery data; (2) determine change rates for interior salt marsh pannes and pools and edge erosion from 1994-2017; (3) determine the relationship between hydrological connectivity and panne expansion; and (4) determine if edge erosion or panne/pool expansion increased surrounding the Hurricane Sandy breach between 2011–2017.

#### **2. Materials and Methods**