**3. Results**

#### **3.1. Stability studies**

Bulk drug product, **1**, as crystals, has been observed to remain stable for > four (4) years under ambient conditions [1]. The final clinical product (2 mg/mL) was stored at refrigerator temperatures (4-8o C) for 1.5 years without deterioration [15].

To further document product stability a 50:50 mixture of **1** (as the clinical product) with 10% IntralipidR (Fresenius Pharmaceuticals) was infused at room temperature over 8 h into a sterile container. Aliquots were withdrawn at 0, 0.5, 1, 2, 3, 4 h and analyzed by HPLC as described above. Decomposition of **1** during an 8-hour infusion at room temperature was 6% (mean for 4-runs). The breakdown product, **2**, could not be identified during this extended simulated infusion study [11].

#### **3.2. Toxicity**

Acute oral and IV toxicity study results for **1** in mice, rats and dogs are presented in Table 1 which presents median lethal dose values observed. The oral study with mice failed to produce toxicity at maximal administered doses of 0.8-2 g/kg. Volume restrictions prevented higher escalations. The drug (oral) was not active in the xenograft models, thus additional oral administration route studies were terminated. Two separate single IV mouse-dosing studies calculated a LD10/50 of 136/385 mg/kg (for both sexes; with 95% confidence limits) – Table 1. Specific lethal/sublethal values and details are discussed in Table 1.

Clinical signs generally reflecting the deteriorating state of both mice and rats post dosing were observed in both sexes for **1** in a dose-dependent manner and included body surface staining, decreased activity, lethargy, loss of appetite, decreased defecation, tremors, and/or wholebody edema. The lethal experiences were sedation followed by respiratory arrest. No seizures or loss of coordination was observed for the survivors.

#### **3.3. Acute single dose intravenous studies in mice**

Adult male and female mice, 10 animals per sex per dose level, were intravenously dosed with 50, 100, 200, 400 and 600 mg/kg. No animals died at 0 or 100 mg/kg, 1 animal died at both 50 and 200 mg/kg doses, 7 of 10 animals died at 400 mg/kg and 8 of 10 animals died at 600 mg/kg (Table 1). Various clinical signs reflecting treatment-related effects were noted in both sexes, oftentimes in a generally dose-dependent manner. These clinical signs included decreased activity, rapid/difficult/slow/shallow breathing, limbs splayed, tremors and skin 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 necropsy.


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

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

\*The time of the last death.

**2.7. Data analysis**

**3. Results**

**3.1. Stability studies**

temperatures (4-8o

infusion study [11].

**3.2. Toxicity**

software available from Stat soft, Tulsa, OK).

244 Tumors of the Central Nervous System – Primary and Secondary

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

Bulk drug product, **1**, as crystals, has been observed to remain stable for > four (4) years under ambient conditions [1]. The final clinical product (2 mg/mL) was stored at refrigerator

To further document product stability a 50:50 mixture of **1** (as the clinical product) with 10% IntralipidR (Fresenius Pharmaceuticals) was infused at room temperature over 8 h into a sterile container. Aliquots were withdrawn at 0, 0.5, 1, 2, 3, 4 h and analyzed by HPLC as described above. Decomposition of **1** during an 8-hour infusion at room temperature was 6% (mean for 4-runs). The breakdown product, **2**, could not be identified during this extended simulated

Acute oral and IV toxicity study results for **1** in mice, rats and dogs are presented in Table 1 which presents median lethal dose values observed. The oral study with mice failed to produce toxicity at maximal administered doses of 0.8-2 g/kg. Volume restrictions prevented higher escalations. The drug (oral) was not active in the xenograft models, thus additional oral administration route studies were terminated. Two separate single IV mouse-dosing studies calculated a LD10/50 of 136/385 mg/kg (for both sexes; with 95% confidence limits) – Table 1.

Clinical signs generally reflecting the deteriorating state of both mice and rats post dosing were observed in both sexes for **1** in a dose-dependent manner and included body surface staining, decreased activity, lethargy, loss of appetite, decreased defecation, tremors, and/or wholebody edema. The lethal experiences were sedation followed by respiratory arrest. No seizures

Adult male and female mice, 10 animals per sex per dose level, were intravenously dosed with 50, 100, 200, 400 and 600 mg/kg. No animals died at 0 or 100 mg/kg, 1 animal died at both 50 and 200 mg/kg doses, 7 of 10 animals died at 400 mg/kg and 8 of 10 animals died at 600 mg/kg (Table 1). Various clinical signs reflecting treatment-related effects were noted in both sexes, oftentimes in a generally dose-dependent manner. These clinical signs included decreased activity, rapid/difficult/slow/shallow breathing, limbs splayed, tremors and skin

C) for 1.5 years without deterioration [15].

Specific lethal/sublethal values and details are discussed in Table 1.

or loss of coordination was observed for the survivors.

**3.3. Acute single dose intravenous studies in mice**

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

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

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 the controls. No seizures were noted.

### **3.5. Acute single dose intravenous studies in rats (Table 1)**

A rat study was conducted with the objectives to evaluate and characterize acute toxicity, maximum tolerated dose (MTD), and evaluate pharmacology (including pharmacokinetic parameters) of **1** when intravenously (IV) administered over 3-hours, as an emulsion to rats. The same formulation that is being administered to patients via IV infusion in clinical trials was used for the rats (see Materials). A detailed clinical examination of each animal was performed daily and included evaluations of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, respiratory and circulatory effects, autonomic effects such as salivation, nervous system effects, including tremors, convulsions, reactivity to handling, and psychological behavior.

Initially, a dose range finding **(**DRF) study was conducted that consisted of four (4) treatment groups – each group included 3-M/3-F that were single dosed IV infusions and monitored – no deaths were observed. The IV doses of **1** administered were – 50, 100, 150 and 200 mg/kg/ dose.

**Parameter Day Vehicle 100 mg/kg 200 mg/kg 300 mg/kg (mg/dL) (M/F) (M/F) (M/F) (M/F)** Cholesterol 2 50/ 49.4 106.4a /77.8 206.4b/157.4 b 188b/165.6 b

H3CO

HO O O

HDL-variant 2 27.4/34.4 33.6/36.8 25.2/30.8 25.6/28.8

LDL-variant 2 134/5.4 66.0/31.3 210.2/142.6 183.6/156

Triglycerides 2 38.8/29 54.8/40 109.8 a/45.8 81/110.6

The triglycerides also increased in the 200 and 300 mg/kg groups on Day-2 post-dosing, which resolved by Day 15. The females demonstrated the most significant elevations in both trigly‐ cerides and LDL-cholesterol. On Day 15 the profiles for both total cholesterol and triglycerides

Alanine aminotransferase (ALT) in males, and γ-glutamyl transferase (GGT), and alkaline phosphatase in females were minimally to mildly elevated at the 300 mg/kg/dose on Day 2.

Various clinical signs reflecting treatment-related effects were noted, mostly lethargy that

Macroscopic/microscopic examinations revealed increased sizes of the livers and spleens. On Day-2, the 200 and 300 mg/kg groups possessed vacuolated macrophages in the liver (Kupffer cells) and spleen. By Day-15 the macrophages contained smaller oil aggregates and clusters

15 26.6/51.8 32.3/51.2 35.6 a /40 37.8 b/49.6

OH <sup>N</sup> Cl CCl3

Cl

+

15 6.6/4.4 7.5/5.6 7.2/4.4 6.8/4.4

15 36.6/43.8 37.3/34.4 33.8/28.4 46.2/30.8

Significantly different from control (p<0.05); bSignificantly different from control

/48.8 51.8 a/61.4

H3CO

HO

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HO OH O

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+

Cl

OH <sup>N</sup> Cl CCl3

(Total) 15 36.8/63.6 45.5/63.6 49.6a

**Table 2.** Summary of lipid profiles in rats dosed with 1 as an IV infusion (Day 1)

All of the findings noted on Day-2 had resolved by Day-15.

cleared. No behavioral alterations were noted.

had returned to WNL. Table 2 reviews cholesterol and triglyceride trends.

No of animals in each group – 5; a

O O <sup>N</sup> <sup>O</sup> Cl CCl3

**Scheme 1.** Metabolism of **1**

H3CO

Cl

(p<0.01).

The main study phase consisted of a control group (10-M/10-F) that each received the vehicle only and three groups (10-M/10-F) that each received a single IV infusion of **1** at dose levels of 100, 200 or 300 mg/kg. One male (1) rat in the 100 mg/kg group became moribund and was euthanized. No animals died in the 200 or 300 mg/kg dosed groups. No external related body effects were noted in the rats surviving the 14-day observation period. Both liver and spleens were target organs noted to be enlarged and evaluated in detail as discussed below.

The rats were divided into 2-groups that were euthanized either on Day-2 or Day-15. Complete macroscopic/microscopic examinations and complete clinical chemistry, hematology, coagu‐ lation studies and urinalysis were completed on all animals.

There were no meaningful hematological effects noted. On Day-2, erythrocytes, hemoglobin, and hematocrit tended to be higher in the 300 mg/kg/dosed group. These changes were most likely a result of fluid imbalances relative to reduced water intake. Monocytes were increased in both sexes at 200 and 300 mg/kg/dose and lymphocytes were decreased in males at 300 mg/ kg/dose. Neutrophils were elevated in all groups on Days-2 and 15 and were attributed to stress and/or route of administration. All other changes were resolved by Day-15 and all values returned to normal pre-drug limits.

There were no test article-related effects on coagulation parameters or on urine analysis values.

The most significant findings were **1**'s related effects on the clinical chemistry analytes in the lipid profile studies (Table 2). Both alterations in the cholesterol and triglyceride profiles were significantly affected post dosing with **1**. Cholesterol and **2** are formed following the metabo‐ lism of **1** by the liver and/or peripherally (Scheme 1). The total cholesterol levels significantly increased in all groups *vs*. the control vehicle group. Increased levels of LDL-cholesterol were the pre-dominant variant observed on Day-2. However, by Day-15 the total cholesterol levels had returned to within normal limits and the total cholesterol was predominantly accounted for as a HDL-variant.

Cl


**Scheme 1.** Metabolism of **1**

**3.5. Acute single dose intravenous studies in rats (Table 1)**

246 Tumors of the Central Nervous System – Primary and Secondary

handling, and psychological behavior.

returned to normal pre-drug limits.

for as a HDL-variant.

dose.

A rat study was conducted with the objectives to evaluate and characterize acute toxicity, maximum tolerated dose (MTD), and evaluate pharmacology (including pharmacokinetic parameters) of **1** when intravenously (IV) administered over 3-hours, as an emulsion to rats. The same formulation that is being administered to patients via IV infusion in clinical trials was used for the rats (see Materials). A detailed clinical examination of each animal was performed daily and included evaluations of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, respiratory and circulatory effects, autonomic effects such as salivation, nervous system effects, including tremors, convulsions, reactivity to

Initially, a dose range finding **(**DRF) study was conducted that consisted of four (4) treatment groups – each group included 3-M/3-F that were single dosed IV infusions and monitored – no deaths were observed. The IV doses of **1** administered were – 50, 100, 150 and 200 mg/kg/

The main study phase consisted of a control group (10-M/10-F) that each received the vehicle only and three groups (10-M/10-F) that each received a single IV infusion of **1** at dose levels of 100, 200 or 300 mg/kg. One male (1) rat in the 100 mg/kg group became moribund and was euthanized. No animals died in the 200 or 300 mg/kg dosed groups. No external related body effects were noted in the rats surviving the 14-day observation period. Both liver and spleens

The rats were divided into 2-groups that were euthanized either on Day-2 or Day-15. Complete macroscopic/microscopic examinations and complete clinical chemistry, hematology, coagu‐

There were no meaningful hematological effects noted. On Day-2, erythrocytes, hemoglobin, and hematocrit tended to be higher in the 300 mg/kg/dosed group. These changes were most likely a result of fluid imbalances relative to reduced water intake. Monocytes were increased in both sexes at 200 and 300 mg/kg/dose and lymphocytes were decreased in males at 300 mg/ kg/dose. Neutrophils were elevated in all groups on Days-2 and 15 and were attributed to stress and/or route of administration. All other changes were resolved by Day-15 and all values

There were no test article-related effects on coagulation parameters or on urine analysis values.

The most significant findings were **1**'s related effects on the clinical chemistry analytes in the lipid profile studies (Table 2). Both alterations in the cholesterol and triglyceride profiles were significantly affected post dosing with **1**. Cholesterol and **2** are formed following the metabo‐ lism of **1** by the liver and/or peripherally (Scheme 1). The total cholesterol levels significantly increased in all groups *vs*. the control vehicle group. Increased levels of LDL-cholesterol were the pre-dominant variant observed on Day-2. However, by Day-15 the total cholesterol levels had returned to within normal limits and the total cholesterol was predominantly accounted

were target organs noted to be enlarged and evaluated in detail as discussed below.

lation studies and urinalysis were completed on all animals.

No of animals in each group – 5; a Significantly different from control (p<0.05); bSignificantly different from control (p<0.01).

**Table 2.** Summary of lipid profiles in rats dosed with 1 as an IV infusion (Day 1)

The triglycerides also increased in the 200 and 300 mg/kg groups on Day-2 post-dosing, which resolved by Day 15. The females demonstrated the most significant elevations in both trigly‐ cerides and LDL-cholesterol. On Day 15 the profiles for both total cholesterol and triglycerides had returned to WNL. Table 2 reviews cholesterol and triglyceride trends.

Alanine aminotransferase (ALT) in males, and γ-glutamyl transferase (GGT), and alkaline phosphatase in females were minimally to mildly elevated at the 300 mg/kg/dose on Day 2. All of the findings noted on Day-2 had resolved by Day-15.

Various clinical signs reflecting treatment-related effects were noted, mostly lethargy that cleared. No behavioral alterations were noted.

Macroscopic/microscopic examinations revealed increased sizes of the livers and spleens. On Day-2, the 200 and 300 mg/kg groups possessed vacuolated macrophages in the liver (Kupffer cells) and spleen. By Day-15 the macrophages contained smaller oil aggregates and clusters within hepatic sinusoids. The findings in both the spleen and the liver showed trends toward resolution by Day-15 with both biliary hyperplasia in the liver and splenic focal necrosis resolving; vacuoles were smaller and cell cytoplasm had a more eosinophilic tint seen in both livers and spleens, albeit the vacuoles still expanded the cytoplasm of the cells. The latter changes are artifacts resulting from the extraction of drug/lipids/cholesterol from hepatic/ splenic macrophages during fixation/preparation of tissues for microscopic examination. The changes seen on Day-2 in the spleens and the livers trended towards resolution by Day-15. Controls (vehicle alone) did not demonstrate the above changes.

**Route/Schedule Dose (mg/kg) Number and Sex Observations**

There were marked elevations of alanine aminotransferase (ALT), and sorbitol dehydrogenase (SDH) in males in both controls and all treated animals on Day 2. Increased aspartate amino‐ transferase (AST) was observed in one (1) male with remarkably increased values for AST, ALT and SDH. The other male in this group exhibited only mildly increased values on Day 2 for these parameters. Similar trends were noted in females. ALT and SDH were highest on Day 2 for all dose levels, including controls. This acute and transient effect on liver enzymes exhibited no dose-dependent pattern and attributable to the vehicle. The latter finding was not observed to this extent in the rat study which used a different vehicle. The above was verified

The dog hematological studies confirmed that the 10-30 mg/kg doses do not produce myelo‐

Treatment-related neurotoxicity was not observed following the single IV bolus administra‐ tion of **1** to dogs. A second opinion review was obtained (RT), who conducted silver stains and confirmed MPI's observation that there were no microscopic pathological CNS changes

**Table 1** summarizes the toxic effects of single IV dose administrations of **1** in mice, rats and dogs. Three intravenous studies were conducted under FDA GLP guidelines. The summary of the median lethal single dose (LD50) values were calculated by combining the data from the acute single dose studies according to species and are available only for mice and dogs.

Rats in groups (5-females) received **1**, **2**, or **3** in a dose range finding (DRF) screen to identify and verify gross behavioral patterns (Table 4). Documentation of drug cognitive/learning abilities were conducted in a Morris modified water maze with adult female rats (Hsd:SD,

Impaired learning behavior has not been observed for **1** or **2** in contrast to observed data for **3** during the first 1-3 h and at 20 h periods post-dosing (Table 5). A vehicle and a 5-FU control were included for comparison. The observations noted for **3** and 5-FU support the literature reports that both drugs impair memory in patients receiving the drug [16-18]. The described

present in the brains of dogs treated with **1** (FDA IND – 68,876) [14].

175-225 g.) which were grouped 3-6 animals per drug arm (Table 5).

 2M 2 F No deaths 2 M 2 F No deaths 2 M 2 F No deaths 2 M 2 F No deaths

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IV once

**Table 3.** Acute IV toxicity in the dog

in a second group of 4M/4F.

**3.8. Acute rat behavioral studies**

suppression, thrombocytopenia or anemia.

**3.7. Summary Median Lethal Dose (Single Dose)**

Transitory changes in the hepatic profile are considered 2o to stasis of **1** in hepatic sinusoids with biliary congestion that results in cysts and shunting of blood to the spleen resulting in splenic cysts and fatty deposits.

Although the above findings resolved by Day-15, they must be considered adverse – based on the degree of elevation in triglycerides (3-fold) and LDL-cholesterol (30-fold in females) seen in some groups. The control group received the vehicle alone – soybean oil and egg yolk lecithin – both rich in triglycerides and did not demonstrate abnormal lipid profiles or the liver/spleen changes.

Based on the conditions and findings of this study, the intravenous LD10 of **1** in rats was calculated to be 100 mg/kg (95% confidence limits could not be calculated) – Table 1.

#### **3.6. Acute dog IV toxicity (Tables 1, 3)**

A single IV dose study was performed in adult Beagle dogs, which consisted of **1** administered once as an IV bolus. Sixteen (16) adult beagle dogs (8 male and 8 female) divided into three groups received a single intravenous injection of **1**. The experimental design and results are presented in Table 3.

No treatment related effects on survival, hematology, urinalysis, or macroscopic and micro‐ scopic evaluations were noted during the study. Numerous clinical signs reflecting treatmentrelated effects were noted in both sexes of all groups, including the control group, and exhibited no dose-dependent pattern, clearly suggesting that the effects were attributable to the 0.3% Klucel+1.92% Tween 80 vehicle rather than **1**. Pertinent clinical signs noted included decreased activity, emesis, impaired righting reflex, limb function impaired, breathing slow/ shallow, red skin discoloration (entire body, ears, or face), swelling (face and/or nose/muzzle), skin cold to touch, eyes swollen, slow gum capillary refill time, feces-mucoid/soft/discolored/ watery, lacrimation, salivation, sclera injected, vocalization, tremors, and urination decreased. The effects were of immediate onset (within one hour post dose), with most of the signs clearing by Day 2 of the study. However, decreased activity persisted for Days 2, 3, or 4 in some of the animals and through the remainder of the study. No clear treatment-related body weight effects were noted during the study when comparing treated groups *vs*. controls. Slight body weight losses were noted in some animals which were non-dose related. The latter observa‐ tions were in all probability attributable to the vehicle.


**Table 3.** Acute IV toxicity in the dog

within hepatic sinusoids. The findings in both the spleen and the liver showed trends toward resolution by Day-15 with both biliary hyperplasia in the liver and splenic focal necrosis resolving; vacuoles were smaller and cell cytoplasm had a more eosinophilic tint seen in both livers and spleens, albeit the vacuoles still expanded the cytoplasm of the cells. The latter changes are artifacts resulting from the extraction of drug/lipids/cholesterol from hepatic/ splenic macrophages during fixation/preparation of tissues for microscopic examination. The changes seen on Day-2 in the spleens and the livers trended towards resolution by Day-15.

with biliary congestion that results in cysts and shunting of blood to the spleen resulting in

Although the above findings resolved by Day-15, they must be considered adverse – based on the degree of elevation in triglycerides (3-fold) and LDL-cholesterol (30-fold in females) seen in some groups. The control group received the vehicle alone – soybean oil and egg yolk lecithin – both rich in triglycerides and did not demonstrate abnormal lipid profiles or the liver/spleen

Based on the conditions and findings of this study, the intravenous LD10 of **1** in rats was

A single IV dose study was performed in adult Beagle dogs, which consisted of **1** administered once as an IV bolus. Sixteen (16) adult beagle dogs (8 male and 8 female) divided into three groups received a single intravenous injection of **1**. The experimental design and results are

No treatment related effects on survival, hematology, urinalysis, or macroscopic and micro‐ scopic evaluations were noted during the study. Numerous clinical signs reflecting treatmentrelated effects were noted in both sexes of all groups, including the control group, and exhibited no dose-dependent pattern, clearly suggesting that the effects were attributable to the 0.3% Klucel+1.92% Tween 80 vehicle rather than **1**. Pertinent clinical signs noted included decreased activity, emesis, impaired righting reflex, limb function impaired, breathing slow/ shallow, red skin discoloration (entire body, ears, or face), swelling (face and/or nose/muzzle), skin cold to touch, eyes swollen, slow gum capillary refill time, feces-mucoid/soft/discolored/ watery, lacrimation, salivation, sclera injected, vocalization, tremors, and urination decreased. The effects were of immediate onset (within one hour post dose), with most of the signs clearing by Day 2 of the study. However, decreased activity persisted for Days 2, 3, or 4 in some of the animals and through the remainder of the study. No clear treatment-related body weight effects were noted during the study when comparing treated groups *vs*. controls. Slight body weight losses were noted in some animals which were non-dose related. The latter observa‐

calculated to be 100 mg/kg (95% confidence limits could not be calculated) – Table 1.

to stasis of **1** in hepatic sinusoids

Controls (vehicle alone) did not demonstrate the above changes.

Transitory changes in the hepatic profile are considered 2o

248 Tumors of the Central Nervous System – Primary and Secondary

splenic cysts and fatty deposits.

**3.6. Acute dog IV toxicity (Tables 1, 3)**

tions were in all probability attributable to the vehicle.

changes.

presented in Table 3.

There were marked elevations of alanine aminotransferase (ALT), and sorbitol dehydrogenase (SDH) in males in both controls and all treated animals on Day 2. Increased aspartate amino‐ transferase (AST) was observed in one (1) male with remarkably increased values for AST, ALT and SDH. The other male in this group exhibited only mildly increased values on Day 2 for these parameters. Similar trends were noted in females. ALT and SDH were highest on Day 2 for all dose levels, including controls. This acute and transient effect on liver enzymes exhibited no dose-dependent pattern and attributable to the vehicle. The latter finding was not observed to this extent in the rat study which used a different vehicle. The above was verified in a second group of 4M/4F.

The dog hematological studies confirmed that the 10-30 mg/kg doses do not produce myelo‐ suppression, thrombocytopenia or anemia.

Treatment-related neurotoxicity was not observed following the single IV bolus administra‐ tion of **1** to dogs. A second opinion review was obtained (RT), who conducted silver stains and confirmed MPI's observation that there were no microscopic pathological CNS changes present in the brains of dogs treated with **1** (FDA IND – 68,876) [14].

#### **3.7. Summary Median Lethal Dose (Single Dose)**

**Table 1** summarizes the toxic effects of single IV dose administrations of **1** in mice, rats and dogs. Three intravenous studies were conducted under FDA GLP guidelines. The summary of the median lethal single dose (LD50) values were calculated by combining the data from the acute single dose studies according to species and are available only for mice and dogs.

#### **3.8. Acute rat behavioral studies**

Rats in groups (5-females) received **1**, **2**, or **3** in a dose range finding (DRF) screen to identify and verify gross behavioral patterns (Table 4). Documentation of drug cognitive/learning abilities were conducted in a Morris modified water maze with adult female rats (Hsd:SD, 175-225 g.) which were grouped 3-6 animals per drug arm (Table 5).

Impaired learning behavior has not been observed for **1** or **2** in contrast to observed data for **3** during the first 1-3 h and at 20 h periods post-dosing (Table 5). A vehicle and a 5-FU control were included for comparison. The observations noted for **3** and 5-FU support the literature reports that both drugs impair memory in patients receiving the drug [16-18]. The described


**Table 4.** Rats - gross behavior patterns (5 female rats per group, 160-168 g)

assay is a simple, reproducible quantitative assessment of impaired visuospatial cerebellarlearning/memory and performance functions *via* swimming and navigating a water maze. The treated and control rats were timed to navigate to find a hidden platform – Figures 3 and 4.

**3.9. Brain/tumor penetration**

**Figure 4.** A rat swimming through the peanuts.

Adult male mice **(**athymic NCr-nu/nu – NCI-Frederick Production Area, NCI) were sedated

were divided into 5-control and 5-treated with **1**. The latter group was administered **1** (135 mg/ kg/day) IP daily for two consecutive days (q1d x 2) beginning 4-days post inoculation of cells. Four hours after the second treatment the animals were sacrificed and the brains removed

The intact frozen brains (~1.3 g) were coronal sliced into three sections in a mouse brain blocker (Kopf). The encapsulated gliomas were easily identified and separated readily from normal brain tissue with a scalpel and using microscopic 'touch finger printing' – separation verified. The tumor tissues were weighed, pooled and homogenized in 10 mL saline at 5OC. This process was repeated for the normal brain tissue. The cold homogenates were separately extracted

The residues were dissolved in dichloromethane and underwent preparative TLC on silica gel plates (Sigma-Aldrich, Milwaukee, WI) with a mobile phase – hexane/dichloromethane:10/30. The respective spots for **1** and **2** were extracted with dichloromethane, concentrated and analyzed by HPLC (for procedure, see-Methods). **1** and **2** were present in the gliomas extracts -62 and 11 ng/g, resp. (avg.) were present in the gliomas, but none in the normal brain tissue.

with 10 mL dichloromethane, the organic layer separated and evaporated to dryness.

intact (cerebellum included), flash frozen in liquid nitrogen and stored at -77 o

) from tissue culture. The mice

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C until assayed.

and intracerebrally (IC) implanted with U251 glioma cells (106

**Figure 3.** A rat on the water maze platform.

A control memory impairment agent, MK-801, is included to demonstrate complete impale‐ ment. In contrast, **1** and **2** had little or no influence on learning/memory, while **3** produced long lasting impairment.

**Figure 4.** A rat swimming through the peanuts.

#### **3.9. Brain/tumor penetration**

assay is a simple, reproducible quantitative assessment of impaired visuospatial cerebellarlearning/memory and performance functions *via* swimming and navigating a water maze. The treated and control rats were timed to navigate to find a hidden platform – Figures 3 and 4.

**Drug Dose (mg/kg) IP 1 - 4 Hours 5th Hour 7th Hour**

behavior

lethargy Less lethargy; spastic Normal behavior

Normal behavior Normal behavior

Normal behavior Normal behavior

Vehicle (Controls) 0.5 mL Alert; normal behavior Alert; normal behavior Alert; normal

Mk-801 (control) 0.05 once Lethargic Lethargic Lethargic

5-FU (Chemo Control) 78 Lethargy, eyes closed More alert Normal behavior

spasms

spasms

**<sup>3</sup>** 400-800 once Eyes closed; spastic;

250 Tumors of the Central Nervous System – Primary and Secondary

**<sup>2</sup>** 350-600 once No acute toxicity; no

**<sup>1</sup>** 100 & 300 once No acute toxicity; no

**Table 4.** Rats - gross behavior patterns (5 female rats per group, 160-168 g)

A control memory impairment agent, MK-801, is included to demonstrate complete impale‐ ment. In contrast, **1** and **2** had little or no influence on learning/memory, while **3** produced

**Figure 3.** A rat on the water maze platform.

long lasting impairment.

Adult male mice **(**athymic NCr-nu/nu – NCI-Frederick Production Area, NCI) were sedated and intracerebrally (IC) implanted with U251 glioma cells (106 ) from tissue culture. The mice were divided into 5-control and 5-treated with **1**. The latter group was administered **1** (135 mg/ kg/day) IP daily for two consecutive days (q1d x 2) beginning 4-days post inoculation of cells. Four hours after the second treatment the animals were sacrificed and the brains removed intact (cerebellum included), flash frozen in liquid nitrogen and stored at -77 o C until assayed.

The intact frozen brains (~1.3 g) were coronal sliced into three sections in a mouse brain blocker (Kopf). The encapsulated gliomas were easily identified and separated readily from normal brain tissue with a scalpel and using microscopic 'touch finger printing' – separation verified. The tumor tissues were weighed, pooled and homogenized in 10 mL saline at 5OC. This process was repeated for the normal brain tissue. The cold homogenates were separately extracted with 10 mL dichloromethane, the organic layer separated and evaporated to dryness.

The residues were dissolved in dichloromethane and underwent preparative TLC on silica gel plates (Sigma-Aldrich, Milwaukee, WI) with a mobile phase – hexane/dichloromethane:10/30. The respective spots for **1** and **2** were extracted with dichloromethane, concentrated and analyzed by HPLC (for procedure, see-Methods). **1** and **2** were present in the gliomas extracts -62 and 11 ng/g, resp. (avg.) were present in the gliomas, but none in the normal brain tissue.


and 100 µL chromatographed on silica gel plates with hexane:dichloromethane (1:1) as solvent. DM-CHOC-PEN was identified at Rf 0.74 with an additional spot – Rf 0.51. All spots were cut out, extracted with THF and analyzed by HPLC. DM-CHOC-PEN and a polar metabolite were

DM-CHOC-PEN was calculated to be present-100 ng/g (avg.) of whole brain. The more polar

fraction identified a pair of peaks at δ 5.68 & δ 5.75-consistent with loss of a methylene chlorine and binding to an NH-group, possible adduct. The material possessed a cholesteryl carbonate

Plasma concentration-time profiles for **1** in adult rats post a 3-hour single dose IV infusion of 100, 200 and 300 mg/kg are presented in Fig. 5. Mean pharmacokinetic parameters for rats summarized in Table 6, Figs. 5 and 6 were – T1/2α 15+/-7 h, T1/2β 19.1+/-1.3 h and CL 22.2+/-6.5 L/h for **1**, which could be detected 24 h post infusion. As mentioned previously and outlined in Scheme 1, **1** is metabolized to 2 and cholesterol, which are compared in Fig. 6 for the 100

H-NMR of the latter

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peak (Rf 0.51) was not DM-PEN and present at 20 ng/g of whole brain. 1

mg/kg dose. The levels of 2 and cholesterol paralleled each other, as expected.

moiety. Normal brain tissue was used as a control.

**Figure 5.** Mean plasma levels for **1** IPEOI – 4-rats per group.

**3.11. Pharmacokinetic studies in rats and dogs (Table 5)**

identified by HPLC (see below).

\*Rats were tested – six (6) separate trials – 1, 2, 3 and 20 hrs post dosing. The rats are timed to swim to the stage. The fold improvements are recorded in the table. For hours 1, 2, 3, and 20 post dosing, two values are given for each drug. The first number (X) for each hour is the difference (in seconds) between the initial and final attempts (first – sixth run). A negative number indicates complete lack of learning throughout six runs – taking a longer amount of time on the sixth try than the first. The next value (indicated with \*) is X fold performance improvement. This is calculated by initial/ final time. <1 indicates complete lack of improvement (taking longer on the last try than the first). Doses used were the therapeutic values as determined from the tumor models.

\*\*MK-801-is a NMDA (N-methyl-D-aspartate) inhibitor that produced a solemn effect that prevented the rats from swimming and learning. After 3-days the rats were equivalent *vs*. control. 5-FU has been associated with memory loss in patients treated with chemotherapy (17).

**Table 5.** Evaluation of the behavioral activity of penclomedine analogs in female rats

Five (5) control mice bearing IC implanted U251 cells (non-treated) were used as the dissection and extraction controls. No chemicals were identified in the above extraction assays or in the brains from the control tumor/normal mice.

#### **3.10. Normal brain penetration**

Adult male rats [Crl: CD1(ICR) BR] (325-350 g wt) in groups of 5 animals were dosed intra‐ peritoneally with 50 mg/kg of DM-CHOC-PEN in 0.3% Klucel/Tween80/saline daily x 2 days. On the 3nd day the rats were sacrificed and the intact brains removed (~1.9 g) and each homogenized in 10 mL saline at 5OC. To the cold homogenates, 20 mL dichloromethane was added and shaken for 30 minutes. The organic layer was removed and evaporated to dryness under vacuum at room temperature. The residues were dissolved in 1 mL of tetrahydrofuran and 100 µL chromatographed on silica gel plates with hexane:dichloromethane (1:1) as solvent. DM-CHOC-PEN was identified at Rf 0.74 with an additional spot – Rf 0.51. All spots were cut out, extracted with THF and analyzed by HPLC. DM-CHOC-PEN and a polar metabolite were identified by HPLC (see below).

DM-CHOC-PEN was calculated to be present-100 ng/g (avg.) of whole brain. The more polar peak (Rf 0.51) was not DM-PEN and present at 20 ng/g of whole brain. 1 H-NMR of the latter fraction identified a pair of peaks at δ 5.68 & δ 5.75-consistent with loss of a methylene chlorine and binding to an NH-group, possible adduct. The material possessed a cholesteryl carbonate moiety. Normal brain tissue was used as a control.

#### **3.11. Pharmacokinetic studies in rats and dogs (Table 5)**

Plasma concentration-time profiles for **1** in adult rats post a 3-hour single dose IV infusion of 100, 200 and 300 mg/kg are presented in Fig. 5. Mean pharmacokinetic parameters for rats summarized in Table 6, Figs. 5 and 6 were – T1/2α 15+/-7 h, T1/2β 19.1+/-1.3 h and CL 22.2+/-6.5 L/h for **1**, which could be detected 24 h post infusion. As mentioned previously and outlined in Scheme 1, **1** is metabolized to 2 and cholesterol, which are compared in Fig. 6 for the 100 mg/kg dose. The levels of 2 and cholesterol paralleled each other, as expected.

**Figure 5.** Mean plasma levels for **1** IPEOI – 4-rats per group.

Five (5) control mice bearing IC implanted U251 cells (non-treated) were used as the dissection and extraction controls. No chemicals were identified in the above extraction assays or in the

\*\*MK-801-is a NMDA (N-methyl-D-aspartate) inhibitor that produced a solemn effect that prevented the rats from swimming and learning. After 3-days the rats were equivalent *vs*. control. 5-FU has been associated with memory loss in

**LEARNING - IMPROVEMENT (MEAN) \*[ DIFFERENCE BETWEEN 1st AND 6th TEST – SAME TIME PERIOD]**

1 Hr 2 Hr 3 Hr 20 Hr

Control Saline 24 \_ 3.2 1.3 2.1 2.0 3.0 0.7 1.6 3.5 35.1 MK-801 0.05 3 QD X 1 0\*\* 0\*\* 0\*\* 0\*\* 0 0 0 0 0 5-FU 78 6 QD X 1 1.7 1.3 1.7 8.2 1.0 0.6 1.3 0.7 -13.5 3 400 6 QD X 1 0.8 1.3 0.6 1.2 1.4 1.7 0.6 1.0 -1.2 2 135 6 QD X 1 6.7 1.5 1.7 2.4 4.0 1.0 1.0 3.5 42.1 1 100 6 QD X 1 3.2 1.3 1.8 3.8 3.2 0.6 1.5 2.4 23.6

\*Rats were tested – six (6) separate trials – 1, 2, 3 and 20 hrs post dosing. The rats are timed to swim to the stage. The fold improvements are recorded in the table. For hours 1, 2, 3, and 20 post dosing, two values are given for each drug. The first number (X) for each hour is the difference (in seconds) between the initial and final attempts (first – sixth run). A negative number indicates complete lack of learning throughout six runs – taking a longer amount of time on the sixth try than the first. The next value (indicated with \*) is X fold performance improvement. This is calculated by initial/ final time. <1 indicates complete lack of improvement (taking longer on the last try than the first). Doses used were the

**MEMORY – IMPROVEMENT/IMPAIRMENT [Differences Between 1 – 20 hrs]**

2 Hr 3 Hr 20 Hr 1 Hr →

(Overall) Fold ↑ Time (sec)

1Hr→ 2Hr→ 3Hr→

Fold↑ Fold↑ Fold↑ Fold↑ Fold↑ Fold↑ Fold↑ 20 Hr

Adult male rats [Crl: CD1(ICR) BR] (325-350 g wt) in groups of 5 animals were dosed intra‐ peritoneally with 50 mg/kg of DM-CHOC-PEN in 0.3% Klucel/Tween80/saline daily x 2 days. On the 3nd day the rats were sacrificed and the intact brains removed (~1.9 g) and each homogenized in 10 mL saline at 5OC. To the cold homogenates, 20 mL dichloromethane was added and shaken for 30 minutes. The organic layer was removed and evaporated to dryness under vacuum at room temperature. The residues were dissolved in 1 mL of tetrahydrofuran

brains from the control tumor/normal mice.

therapeutic values as determined from the tumor models.

**Table 5.** Evaluation of the behavioral activity of penclomedine analogs in female rats

**3.10. Normal brain penetration**

patients treated with chemotherapy (17).

**TREATMENTS**

252 Tumors of the Central Nervous System – Primary and Secondary

Dose (mg/kg/ dose

#Rats Schedule

Agent

**Mean rat AUC vs Dose**

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255

**0 100 200 300 400**

**DM-CHOC-PEN (mg/kg)**

**Mean rat [C] max vs dose**

**0 100 200 300 400**

**DM-CHOC-PEN (mg/kg)**

The results of incubating Rho in the presence or absence of Vpml and/or **1** are summarized in

**0**

**0.0**

**3.12. P-glycoprotein (P-gp) transport (Table 6)**

Six reaction conditions (**1-6**) are reviewed, where:

**0.5**

**1.0**

**[C] max (mg/L)**

**1.5**

**Figure 7.** AUC *vs.* dose for **1**

**Figure 8.** [C]max*vs*. dose for **1**

Table 7.

**2**

**4**

**AUC (mg\*h/L)**

**6**

**8**

**Figure 6.** Mean plasma and rbc levels for **1, 2** and cholesterol, IPEOI for **1** after IV infusion (200 mg/kg) – 4-rats per group.

The shifts noted in the bioavailability for **1** *vs*. **2** are suggestive of an enzyme overload – a Michaelis Menten effect (Fig. 6). For rats, the AUC and Cmax values *vs*. doses of **1** administered were linear – Figs. 7 & 8**.** Dog pharmacokinetics for **1** are compared to the rat values in Table. For dogs, plasma clearance was constant between 10 and 30 mg/kg. The plasma clearance is 328.8 L/h *vs*. 346.8 L/h; demonstrating the PK linearity of **1**. Differences seen between rats and dogs are due to the fact that rats received **1** during a 3-hr IV infusion and dogs *via* an IV bolus injection – thus not comparable.


**Table 6.** Comparative PK parameters of **1** in rats and dogs

**Figure 7.** AUC *vs.* dose for **1**

**Figure 6.** Mean plasma and rbc levels for **1, 2** and cholesterol, IPEOI for **1** after IV infusion (200 mg/kg) – 4-rats per

The shifts noted in the bioavailability for **1** *vs*. **2** are suggestive of an enzyme overload – a Michaelis Menten effect (Fig. 6). For rats, the AUC and Cmax values *vs*. doses of **1** administered were linear – Figs. 7 & 8**.** Dog pharmacokinetics for **1** are compared to the rat values in Table. For dogs, plasma clearance was constant between 10 and 30 mg/kg. The plasma clearance is 328.8 L/h *vs*. 346.8 L/h; demonstrating the PK linearity of **1**. Differences seen between rats and dogs are due to the fact that rats received **1** during a 3-hr IV infusion and dogs *via* an IV bolus

**Dose Specie (N) T1/2 α (h) T1/2β (h) AUC (mg\*h/L) Cl (L/h)** 10 mg/kg Dog (4) 1.23 (Mean) 21.6 (Mean) 0.42 (Mean) 328.8 (Mean)

30 mg/kg Dog (4) 0.63 (Mean) 18.7 (Mean) 1.12 (Mean) 346,8 (Mean)

100 mg/kg Rat (5) 0.51 (Mean) 2.48 (Mean) 1.05 (Mean) 30.4 (Mean9.99

200 mg/kg Rat (5) 0.25 (Mean) 6.94 (Mean) 3.46 (Mean) 16.9 (Mean)

300 mg/kg Rat (5) 0.12 (Mean) 4.0 (Mean) 5.17 (Mean) 19.40 (Mean)

0.53 (SD) 16.00 (SD) 0.17 (SD) 221.2 (SD)

0.09 (SD) 10.7 (SD) 0.09 (SD) 54.5 (SD)

0.05 (SD) 0.8 (SD) 0.53 (SD) (SD)

0.1 (SD) 2.1 (SD) 0.46 (SD) 4.04 (SD)

0.06 (SD) 1.2 (SD) 1.74 (SD) 9.13 (SD)

group.

injection – thus not comparable.

254 Tumors of the Central Nervous System – Primary and Secondary

(Males & females combined)

**Table 6.** Comparative PK parameters of **1** in rats and dogs

**Figure 8.** [C]max*vs*. dose for **1**

#### **3.12. P-glycoprotein (P-gp) transport (Table 6)**

The results of incubating Rho in the presence or absence of Vpml and/or **1** are summarized in Table 7.

Six reaction conditions (**1-6**) are reviewed, where:

**1** – is the result from incubating cell lines with Rho for 15 minutes.

**2** – is the result from incubating cell lines with Vpml for 1 hour and then adding Rho during the last 15 minutes of incubation.

We report here the results of acute toxicity and pharmacology studies with single intravenous injections of **1** in groups of rodents and dogs. The end-point of all the studies was to identify drug toxicity and an acceptable starting dose for a Phase I clinical trial in humans with

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The IV LD10 single-dose value for mice (sexes combined) was calculated as 139 mg/m2

mouse study generally displayed a typical dose-response effect (with the exception of one death at 50 mg/kg), with **1** being slightly more toxic in males than in females at the two highest

A sub-chronic oral mouse toxicity study was conducted at MPI Research, Mattawan, MI, under GLP conditions in male/female mice. The study evaluated **1** in an emulsion administered per oral gavage 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, but no seizures. Similar body appearances

Adult rats were treated once with single IV infusions of **1** in doses of 50, 100, 150, 200, and 300 mg/kg. One (1) death occurred at the 100 mg/mL dose level. No deaths occurred in the treated vehicle group. There were no meaningful effects on hematology parameters. On Day-2 erythrocytes, hemoglobin, and hematocrit tended to be higher at the 300 mg/kg/dose. These changes were most likely a result of fluid imbalance. Monocytes were increased in both sexes at 200 and 300 mg/kg/dose and lymphocytes were slightly decreased in males at 300 mg/kg/ dose level. Neutrophils were elevated relative to expected ranges in all groups on Days-2 and 15 and were attributed to stress and/or route of administration. All other changes were resolved by Day 15 and all values returned to normal pre-drug limits. There were no test article-

The most significant abnormal findings were the statistically increased plasma values for cholesterol and triglycerides in the 200 and 300 mg/kg treated groups. LDL-cholesterol was significantly elevated in females – increased from 5.4 to 142 and 156 mg/dL for the 200 and 300 mg/kg groups, resp. This elevation is significant and considered a SLT (CTEP.v4). The triglycerides were increased by 4-fold in the 300 mg/kg group females, however, they return to normal values by Day-15. Hepatic and splenic deposits of fats were also noted on gross and

Cholesterol is released during metabolism of **1** (Table 2 & Scheme 1). The early formation of LDL-cholesterol is not a surprise since the formation of the LDL-variant is the initial natural method to 'initially encase cholesterol molecules'. The cholesterol is cleared through the liver as the HDL-variant. Triglycerides were elevated secondary to increased cholesterol and the

Although the above cholesterol and triglyceride findings resolved by Day-15, they must be considered adverse – triglycerides (3-fold) and LDL-cholesterol (30-fold in females). The control group received the vehicle alone – soybean oil and egg yolk lecithin – both rich in

related effects on either coagulation or on urinalysis parameters.

lipid character of the emulsion vehicle, which also rapidly reversed [3].

triglycerides and did not demonstrate abnormal lipid profiles.

microscopic examinations which cleared by Day-15.

. The

257

advanced cancer.

were noted with the controls.

doses.

**3** – is the result from incubating the cell lines with Vpml for 15 minutes, adding **1** after an incubation time of 30 minutes and then adding Rho during the last 15 minutes – total incubation time, 60 minutes.

**4** – is the result from treating the cell lines simultaneously with Vpml and **1** for 45 minutes and then adding Rho and continuing the incubations for an additional 15 minutes – total incubation time – 60 minutes.

**5** – is the result from incubating the cell lines with **1** for 15 minutes and then adding Vpml for additional 45 minute incubation. Rho is added during the last 15 minutes of the incubation.

Finally, **6** is the result of incubating each of the cell lines for 1 hour with **1** alone and then adding Rho during the last 15 minutes of incubation.

The results summarized in Table 7 for the 3 sensitive cell lines are coherent: The rate of incorporation of Rho is lower when cells are treated by the mixture of Vpml and DM-CHOC-PEN or Vpml alone but not when the cells are treated with DM-CHOC-PEN alone (mean fluorescence intensity of 6 roughly the same for control cells). This is interpreted as meaning that DM-CHOC-PEN has no effect on the function of P-gp transport.


\*Cell concentration per each assay. Average of triplicate assays.

**Table 7.** P-glycoprotein transport of **1**
