**2.1. Drug formulation and chemicals**

**1** and **2** were synthesized by DEKK-TEC, Inc., using GLP/GMP guidelines, as previously described [1]. **1** is very stable in the solid state under ambient temperature and was adminis‐ tered in various vehicle media for the animal studies. For the rat studies, **1** was formulated as a buffered emulsion of soybean oil (20%), egg yolk lecithin (10%), glycerin (3%), histidine (3.1%) and water emulsion (containing 2-7 mg/mL of **1**) [IND 86,876]. For the mouse and dog IV studies, **1** was formulated as a 0.3% Klucel+0.3-3.3% Tween® 80, saline suspension and for the oral mouse study an emulsion of **1** in a 8% Tween-80®/Neobee®-1053 (Squibb) solution was used.

#### **2.2. High pressure liquid chromatography (HPLC) analysis of 1 and a metabolite, 2**

HPLC analysis was performed using an Agilent Technologies (New Castle, DE) 1200 model HPLC fitted with a diode array UV detector set at a wavelength of 244 nm (λmax for **1**). A Rheodyne Model 7725 injection port (Cotati, CA, USA) with a 20µL sample loop was used to inject the samples. Chromatograms were recorded with an Agilent Technologies integrator. Samples were chromatographed using an Alltech 150 x 4.6 mµ column that contains *Luna* C8 (2) 100A packing (diameter of particles=5 mµ).

The mobile phase for **1** consisted of 80% THF: 20% water and for **2**, was 45% THF: 47% water, which was degassed, filtered through a 0.45 µm Rainin filter (Woburn, MA, USA) and delivered at a flow rate of 1.0 mL/min.

Plasma and erythrocyte samples were stored at-74o C until analyzed. Standard solutions were prepared by dissolving 6 mg of **1** or **2** in 20 mL THF. Internal standards were 20 mg of cholesteryl benzoate (**ChB**, Sigma Aldrich Co) or phenol (**P**, Sigma Aldrich Co) each in 20 mL THF. All solutions were stable for at least 2 months at 5o C. Standard assays for **1** and **2** consisted of 0.25 mL of plasma, 25 µL of **1** or **2** and internal standard (**ChB** or **P** – 2 µL) and 2 mL dichloromethane. The samples were vortexed for 10 min and frozen to separate the layers. The bottom organic layer was removed with a 25 gauge needle and glass syringe, filtered through a 0.45 mm Acrodisc syringe filter and evaporated to dryness under vacuum, reconstituted with 100 µL of THF and analyzed as below.

Control dog and rat whole blood and plasma samples were spiked with **1** in the concentration range 5-1000 ng/mL, plus 20 µL of the above **ChB**. Similar controls were prepared for **2** (ng/mL) and its int. std. – **P**. Peak-area ratios of **1**/**ChB** or (**2**/**P**) *vs*. the concentration of **1** or **2** (ng/mL) were subjected to linear regression analysis. Retention times for **1** and **ChB** were 6.41 and 4.63 min., respectively and for **2** and **P** were 6.6 and 18.5 min., respectively.

Verification of the HPLC assays included calibration curves derived from the assay of five (5) erythrocyte and eleven (11) plasma standards in duplicate prepared each with **1** and **2** (0.5 ng/ mL-600 µg/mL). Plasma and erythrocyte samples were obtained from healthy rats and dogs and spiked with **1** or **2** and their respective internal standards. Drug concentrations in all samples were calculated using the results of linear regression analysis. Reproducibility was higher than 85%. Limit of quantitation for **1** and **2** was 0.2 ng/mL.

All mice, rat and dog studies were conducted at MPI Research (Mattawan, MI) under GLP regulations as described in the Guide for the Care and Use of Laboratory Animals, Office for

Comparative Preclinical Pharmacology and Toxicology for…

http://dx.doi.org/10.5772/58353

243

For mouse toxicity studies, **1** was administered IV bolus *via* the tail vein or per oral gavage; for the dog studies, **1** was administered as an IV bolus *via* the femoral vein and for rats, administration was during a 3 h IV infusion via indwelling femoral vein catheters. The observation period for all studies was 14-days – followed with euthanasia. For the rat and dog studies, complete necropsies with complete blood pharmacokinetic parameters, hematology, coagulation profiles, clinical chemistry and urine analyses were performed. For the mouse

For the acute behavioral studies, rats received drugs and controls to identify/verify gross behavioral patterns employing a Morris modified water maze (4' x 3' x 1.5') with a single layer of white polyethylene peanuts that floated (in 6'' of water) and covered a single mounting stage [13, 14]. Adult female rats (Hsd:SD, 175-225 g.) were grouped 3-6 animals per drug arm. The test agents were dissolved in or suspended in 5% aqueous TweenR 80-hydroxypropyl cellulose in 5% saline and administered intraperitoneally (IP). Controls received the vehicle only. Swimming trials began – 1, 2, 3 and 20 hours post-dosing. For each time period postdosing, the rats were challenged on six (6)-back-to-back swim trial events to find the platform. The daily swimming times and ranges were compared to vehicle controls *vs*. chemotherapeu‐ tics alone in rats. The data (latency to find the platform) was analyzed by variance (ANOVA). Body weights and water temperature-prior to each dosing and during each FOB assessment

Groups of rats (5/sex) were administered 100, 200, or 300 mg/kg of **1** as a 3 h timed infusion and samples of blood collected (in EDTA tubes) at various time points IPEOI:-15,+10,+45 min, 1.5, 3, 6, 8, 12, 24 h and 14-days. Each animal possessed an indwelling femoral catheter for ease of the study. The catheters were flushed after each blood draw. The plasma and erythrocytes

Adult beagle dogs (8 M) were administered **1** as a slow bolus injection once through a femoral vein in doses of 10, 20, and 30 mg/kg. A 20-gauge venous catheter was inserted into a saphenous vein for bleeding and samples were withdrawn at 0, 5, 15, and 30 min. and 1, 1.5, 2, 4, 8, 12 and 24 h into EDTA containing tubes. Plasma was separated and stored at-70o C until assayed.

Model parameters were estimated using Micropharm software and nonlinear least squares regression was performed using Simplex and Gauss Newton algorithms []. An open twocompartment model provided the best fit. Clearance, volume of distribution and half-lives

C until analyzed.

Laboratory Animal Welfare, NIH, Bethesda, MD.

were monitored.

**2.5. Pharmacokinetic studies**

were separated and stored separately at -74o

Animals were anesthetized with ketamine.

**2.6. Animal pharmacokinetic data analysis**

were derived from estimates of the model parameters.

studies physical/gross necropsy examinations were conducted.

#### **2.3. P-glycoprotein (P-gp) transport studies**

**Cell lines**-three (3) human cancer cell lines-A549: *lung*; MCF7: *breast*; and HeLa: *ovarian* were obtained from ATCC, Manassas, MD, and maintained in culture in a temperature controlled (36 o C), 5% carbon dioxide, and humidity controlled incubator system. All culture transfers were in a laminar flow hood under sterile conditions. Tissue culture medium used was RPMI 1640 containing 10% FBS and penicillin/streptomycin/fungizone (1%) – all purchased from InVitrogen.

**Material preparation-**Rho was prepared in distilled water as a stock solution (1 mg/mL), stored frozen at-22o C. Rho (0.2µg/mL) was added to the culture medium in the presence or absence of Vpml and/or **1**. Vpml (2-23 µg/10 mL) was prepared in PBS solution and **1** (0.5-73 µg/10 mL) in PBS+5% DMSO.

**A typical assay**-contained: **1**-7.3 µg/mL, Vpml-2.5 µg/mL and Rho-0.2 µg/mL. Cells per test system were 0.8-1.2 x 106 /mL. Incubations occurred in the above incubator conditions for the times and schedules discussed below. Experiments were conducted in triplicate and at times as specified in the Results. Reaction incubations were 15-60 minutes in length as discussed in the Results section.

**Post incubation**-After treatment, cells were rapidly trypsinized (~2 min), washed with cold PBS and kept on ice until analyzed. Assay conducted with a Becton-Dickinson FACS-ARIA II flow cytometer with CELLQUEST software. The Rho fluorescence was measured at laser excitation 488 nm with emission at 530 nm and is expressed in arbitrary units compared to control cells (untreated). Measurements were conducted on 10,000 cells per assay.

#### **2.4. Animals**

Adult Sprague-Dawley mice [Crl: CD1(ICR) BR] (males 20-25 g and female 18-25 g) and rats [Crl: CD1(ICR) BR] (males 300-350 g and female 225-250 g) were obtained from Harlan Industries (Indianapolis, IN), housed in groups of three-five per cage in light-controlled (12 h/ day) and temperature-controlled (24o C) animal isolators with filtered vents and exhausts. They were fed a diet of Purina Laboratory Chow (Purina Feed) and received tap water *ad libitum.* The rats were fasted and 3-4 mL of blood was drawn *via* the jugular vein. The order of bleeding was alternated (one animal from each dose group, then repeating) to reduce handling and time biases.

Adult male and female beagle dogs (6.5-7.5 mo. of age, 6.5-9.49 kg) were raised and maintained at MPI Research (Mattawan, MI). They were fed a diet of Purina Dog Chow (Purina Feed), received tap water *ad libitum* and exercised per IACUC protocol. Mouse, rat and dog were euthanized with phenobarbital/ketamine anesthesia and/or carbon dioxide inhalation. The chest cavities were exposed to insure death. Institutional animal care and use committees (IACUC) reviewed and approved all the studies.

All mice, rat and dog studies were conducted at MPI Research (Mattawan, MI) under GLP regulations as described in the Guide for the Care and Use of Laboratory Animals, Office for Laboratory Animal Welfare, NIH, Bethesda, MD.

For mouse toxicity studies, **1** was administered IV bolus *via* the tail vein or per oral gavage; for the dog studies, **1** was administered as an IV bolus *via* the femoral vein and for rats, administration was during a 3 h IV infusion via indwelling femoral vein catheters. The observation period for all studies was 14-days – followed with euthanasia. For the rat and dog studies, complete necropsies with complete blood pharmacokinetic parameters, hematology, coagulation profiles, clinical chemistry and urine analyses were performed. For the mouse studies physical/gross necropsy examinations were conducted.

For the acute behavioral studies, rats received drugs and controls to identify/verify gross behavioral patterns employing a Morris modified water maze (4' x 3' x 1.5') with a single layer of white polyethylene peanuts that floated (in 6'' of water) and covered a single mounting stage [13, 14]. Adult female rats (Hsd:SD, 175-225 g.) were grouped 3-6 animals per drug arm. The test agents were dissolved in or suspended in 5% aqueous TweenR 80-hydroxypropyl cellulose in 5% saline and administered intraperitoneally (IP). Controls received the vehicle only. Swimming trials began – 1, 2, 3 and 20 hours post-dosing. For each time period postdosing, the rats were challenged on six (6)-back-to-back swim trial events to find the platform. The daily swimming times and ranges were compared to vehicle controls *vs*. chemotherapeu‐ tics alone in rats. The data (latency to find the platform) was analyzed by variance (ANOVA). Body weights and water temperature-prior to each dosing and during each FOB assessment were monitored.

#### **2.5. Pharmacokinetic studies**

samples were calculated using the results of linear regression analysis. Reproducibility was

**Cell lines**-three (3) human cancer cell lines-A549: *lung*; MCF7: *breast*; and HeLa: *ovarian* were obtained from ATCC, Manassas, MD, and maintained in culture in a temperature controlled

**Material preparation-**Rho was prepared in distilled water as a stock solution (1 mg/mL), stored frozen at-22o C. Rho (0.2µg/mL) was added to the culture medium in the presence or absence of Vpml and/or **1**. Vpml (2-23 µg/10 mL) was prepared in PBS solution and **1** (0.5-73 µg/10 mL)

**A typical assay**-contained: **1**-7.3 µg/mL, Vpml-2.5 µg/mL and Rho-0.2 µg/mL. Cells per test

times and schedules discussed below. Experiments were conducted in triplicate and at times as specified in the Results. Reaction incubations were 15-60 minutes in length as discussed in

**Post incubation**-After treatment, cells were rapidly trypsinized (~2 min), washed with cold PBS and kept on ice until analyzed. Assay conducted with a Becton-Dickinson FACS-ARIA II flow cytometer with CELLQUEST software. The Rho fluorescence was measured at laser excitation 488 nm with emission at 530 nm and is expressed in arbitrary units compared to

Adult Sprague-Dawley mice [Crl: CD1(ICR) BR] (males 20-25 g and female 18-25 g) and rats [Crl: CD1(ICR) BR] (males 300-350 g and female 225-250 g) were obtained from Harlan Industries (Indianapolis, IN), housed in groups of three-five per cage in light-controlled (12 h/

were fed a diet of Purina Laboratory Chow (Purina Feed) and received tap water *ad libitum.* The rats were fasted and 3-4 mL of blood was drawn *via* the jugular vein. The order of bleeding was alternated (one animal from each dose group, then repeating) to reduce handling and time

Adult male and female beagle dogs (6.5-7.5 mo. of age, 6.5-9.49 kg) were raised and maintained at MPI Research (Mattawan, MI). They were fed a diet of Purina Dog Chow (Purina Feed), received tap water *ad libitum* and exercised per IACUC protocol. Mouse, rat and dog were euthanized with phenobarbital/ketamine anesthesia and/or carbon dioxide inhalation. The chest cavities were exposed to insure death. Institutional animal care and use committees

control cells (untreated). Measurements were conducted on 10,000 cells per assay.

/mL. Incubations occurred in the above incubator conditions for the

C) animal isolators with filtered vents and exhausts. They

C), 5% carbon dioxide, and humidity controlled incubator system. All culture transfers were in a laminar flow hood under sterile conditions. Tissue culture medium used was RPMI 1640 containing 10% FBS and penicillin/streptomycin/fungizone (1%) – all purchased from

higher than 85%. Limit of quantitation for **1** and **2** was 0.2 ng/mL.

**2.3. P-glycoprotein (P-gp) transport studies**

242 Tumors of the Central Nervous System – Primary and Secondary

(36 o

InVitrogen.

in PBS+5% DMSO.

the Results section.

**2.4. Animals**

biases.

day) and temperature-controlled (24o

(IACUC) reviewed and approved all the studies.

system were 0.8-1.2 x 106

Groups of rats (5/sex) were administered 100, 200, or 300 mg/kg of **1** as a 3 h timed infusion and samples of blood collected (in EDTA tubes) at various time points IPEOI:-15,+10,+45 min, 1.5, 3, 6, 8, 12, 24 h and 14-days. Each animal possessed an indwelling femoral catheter for ease of the study. The catheters were flushed after each blood draw. The plasma and erythrocytes were separated and stored separately at -74o C until analyzed.

Adult beagle dogs (8 M) were administered **1** as a slow bolus injection once through a femoral vein in doses of 10, 20, and 30 mg/kg. A 20-gauge venous catheter was inserted into a saphenous vein for bleeding and samples were withdrawn at 0, 5, 15, and 30 min. and 1, 1.5, 2, 4, 8, 12 and 24 h into EDTA containing tubes. Plasma was separated and stored at-70o C until assayed. Animals were anesthetized with ketamine.

#### **2.6. Animal pharmacokinetic data analysis**

Model parameters were estimated using Micropharm software and nonlinear least squares regression was performed using Simplex and Gauss Newton algorithms []. An open twocompartment model provided the best fit. Clearance, volume of distribution and half-lives were derived from estimates of the model parameters.

#### **2.7. Data analysis**

Data analysis was performed on all plasma and *in vitro* studies and analyzed via non-linear regression using a non-weighed quasi-Newtonian/simplex fitting algorithm (Statistical software available from Stat soft, Tulsa, OK).

cold to touch. No seizures or loss of coordination was noted. The deaths at 400 and 600 were of a very immediate nature, occurring within minutes or less post-dose, with no clinical signs exhibited prior to death. While transient incidences of rapid breathing were also noted in a couple of control animals, a definitive relationship to the vehicle was unclear. No definitively clear treatment-related body effects were noted in those mice surviving the 14-day observation period when compared with controls. No macroscopic findings were noted in any animal at

**Method LD10/50 (mg/kg) Time\* (Days) Comments**

Comparative Preclinical Pharmacology and Toxicology for…

http://dx.doi.org/10.5772/58353

245

cholesterol – elevated, but

36 M 50-600 IV 132/385 2-14 days Deaths were erratic

30 F Gavage 5d – no deaths No deaths

acceptable 73 F 50-300 Infusion (LD10) 15 days

8 F 10-30 Bolus >30.0 10 days No deaths

4 F 10-30 Bolus >30.0 10 days No deaths

Based on the conditions and findings of this study, the intravenous LD10 for **1** was calculated to be 136 mg/kg (95% confidence limits could not be calculated) in mice (combined sexes),

A study was conducted in groups of 10 male/10 female mice per dose. The study evaluated **1** administered daily for five days at doses of 0, 800, 1000, 1200, 1500 and 2000 mg/kg per gavage to mice. Only one death occurred at 800 mg/kg on day 2 after dosing. All animals demonstrated some degree of lethargy and unkempt appearance. Similar body appearances were noted with

while the intravenous LD50 was calculated to be 385 mg/kg (95% confidence limits).

73 M IV 100 1-Rat died @ 100 mg/kg;

Neither aplastic bone marrow nor splenic depletion of lymphocytes was noted.

30 M 800-2000 Oral – 0.8 – 2.0 g/d x 21 days

36 F Bolus (LD10/50)

necropsy.

Mice

Mice

Rat

Dog

Dog

\*The time of the last death.

**Table 1.** Medial dose summary for **1**

**Species No/ Sex**

**Doses (mg/kg)**

8 M IV

4 M IV

**3.4. Sub-chronic oral mouse toxicity (Table 1)**

the controls. No seizures were noted.
