**4. Discussion**

The rationale for the pre-clinical development of **1**, a polychlorinated pyridine cholesteryl carbonate, was based on observed antitumor activity *vs*. IC implanted human xenografts growing in mice, in comparison with standard therapy [1,2]. **1** was synthesized during an attempt to design and develop polychlorinated pyridine carbonates that could penetrate the BBB, with cytotoxic activity *vs*. intracranially growing brain tumors and without neurotoxicity [1,2].

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 advanced cancer.

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

that DM-CHOC-PEN has no effect on the function of P-gp transport.

the last 15 minutes of incubation.

256 Tumors of the Central Nervous System – Primary and Secondary

Rho during the last 15 minutes of incubation.

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

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

**4. Discussion**

neurotoxicity [1,2].

time, 60 minutes.

time – 60 minutes.

**Cell Line**

**2** – is the result from incubating cell lines with Vpml for 1 hour and then adding Rho during

**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

**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

**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

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

HeLa 19.3\*\* 15.0 15.6 18.2 16.0 17.6 A549 23.2 20.8 16.4 13.2 17.9 21.8 MCF-7 10.5 6.5 5.9 6.2 7.4 8.9

The rationale for the pre-clinical development of **1**, a polychlorinated pyridine cholesteryl carbonate, was based on observed antitumor activity *vs*. IC implanted human xenografts growing in mice, in comparison with standard therapy [1,2]. **1** was synthesized during an attempt to design and develop polychlorinated pyridine carbonates that could penetrate the BBB, with cytotoxic activity *vs*. intracranially growing brain tumors and without

**Mean Fluorescence Intensity for Total Cells (Mean) (x 103)\* Rho Vpml + Rho Vpml → DM + Rho Vpml + DM +Rho DM + Vpml + Rho DM + Rho 1 2 3 4 5 6**

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

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 were noted with the controls.

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 articlerelated effects on either coagulation or on urinalysis parameters.

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 microscopic examinations which cleared by Day-15.

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

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 triglycerides and did not demonstrate abnormal lipid profiles.

Alanine aminotransferase (ALT) in males, and γ-glutamyl transferase (GGT), and alkaline phosphatase in females were minimally to mildly elevated in the 300 mg/kg group on Day-2. All of these findings on Day-2 resolved by Day-15. Transient elevations in transaminases were considered to be 2o to hepatic clearance of the drug. Neither gross nor microscopic evidence of toxicity (other than hepatic cysts) was noted at autopsies, including CNS. Table 7 compares calculated starting therapeutic doses for humans [19].

swimming maze assesses impaired visual spatial processing, as well as memory. The obser‐ vations for 5-FU (drug control) confirmed literature reports of learning/memory impairment

Comparative Preclinical Pharmacology and Toxicology for…

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

259

The drug penetrated human glioblastoma tumor tissue growing IC in mice, with none detectable in the normal tissue. This only reinforced our interest in using the drug to treat

**1** does not have effects on the P-gp transport system. In the current study, Rho was selected as an indicator of **1**'s interaction/inhibition with the P-gp protein transport system in three (3) human cancer cell lines-A549: *lung*; MCF7: *breast*; and HeLa: *ovarian.* Verapamil (Vpml) is a known inhibitor of the P-gp pathway and was included as a positive control, providing additional support that **1** is not a substrate for the P-gp transmitter system and not rejected *via*

Both **1** and **2** bind to erythrocytes and could penetrate the blood brain barrier (BBB) and IC growing cancers attached to rbcs *via* breaks in the BBB (penetration routes of metastatic cancers) or sites of neo-angiogenic networks. However, as previously published, **2** alone is not active *vs*. IC implanted human breast and glioma tumors implanted in a mouse model; the presence of **2** in the IC implanted tumors (see Results-*Brain/tumor penetration*) must be due to

Thus, preclinical studies, conducted under GLP guidelines are presented as support for **1** to enter Phase I clinical trials as treatment for advanced cancer with CNS involvement. Table 8 reviews calculated starting doses and data satisfied the FDA's requirements for an IND (IND 68,876), which has completed a Phase I clinical trial – DTI-021 [19]. The phase I clinical trial is nearing completion with acceptable toxicities and responses noted in patients with advanced

**Drug Species Acute IV LD10 Comparable Human IV Dosage\***

(1/6th HNSTD)

DM-CHOC-PEN Mouse 136 mg/kg/d 39 mg/m2/d (10% of LD10) DM-CHOC-PEN Rat 100 mg/kg/d 60 mg/m2 (10% of LD10)

The initial level of dosing in the Phase I clinical trial has been established as 39 mg/m2 (IND 86,876) (19).

DM-CHOC-PEN Dog >30 mg/kg/d >100 mg/m2/d\*\*

\* Standard conversion \*\*Based on the highest dose – 30 mg/kg used in the dog studies

Supported by NCI/SBIR grants – 5R44CA85021 and 1R43CA132257

the high energy ATP-binding cassette (ABC) transport systems [11].

post dosing in humans [16].

patients with cancers involving the CNS.

IC steroid esterase hydrolysis of **1** [21].

cancer involving the CNS system [15].

**Table 8.** Estimated comparable human intravenous dosages

**Acknowledgements**

A single IV dose administration study was performed in adult beagle dogs employing single doses (10 – 30 mg/kg) of **1**. No treatment related fatalities occurred. Numerous clinical signs reflecting treatment-related effects were noted in both sexes of all groups. Pertinent clinical signs noted included decreased activity, autonomic hyperactivity – vomiting, decreased urination, salivation, and lacrimation. 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, and 4 in some of the animals and through the remainder of the study. Slight body weight losses noted in some animals were not dose-dependent and it could have been attributable to the clinical signs (and associated stress) caused by the vehicle. There were marked elevations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and sorbitol dehydrogenase (SDH) noted in controls and all treated groups and apparently attributable to the vehicle – a Klugel/Tween mixture that will not be used in the clinical studies. All liver functions reversed by Day-15. This acute and transient effect on liver enzymes exhibited no dose-dependent pattern and was also apparently attributable to the vehicle. The latter finding was not observed in the rat study.

No hematological deficiencies were noted in any group. Drug-related neurotoxicity was not observed. This was confirmed by second opinion (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** [19].

Based on the conditions and findings of this study, a single bolus intravenous injection of **1** to groups of beagle dogs at dose levels of 10, 20 and 30 mg/kg produced no effects that were directly related to the test article; instead they were probably attributable to the 0.3% Klucel +1.92% Tween® 80 vehicle used.

Pharmacokinetic studies were conducted in two species – rats and dogs. Parameters were obtained from Gauss Newton algorithm modeling [3]. The values are compared in Table 6. No statistical differences between male and female rats were noticed. The differences in half life can be explained in reference to administration routes – dog-IV bolus *vs*. rat – IV infusion over 3 hrs. Similarly, the clearance is higher in the bolus studies as expected with a surge of drug being filtered.

In the learning/cognitive screening study, rats treated with **3** took a longer period of time to find the pedestal *vs*. **1**, **2** and the controls. Despite normal gross appearance of the rats after 7 hr (Table 4), the **3** treated animals demonstrated impaired learning (Table 5). There were no signs of learning impairment noted in the rats treated with **1** or **2**, as was seen for **3**. **2** is a polychlorinated 4-hydroxypyridine (Fig. 1) and exists as a zwitterion that is too polar to cross the BBB. This behavior has been observed for other 4-hydroxypyridines [20]. The water swimming maze assesses impaired visual spatial processing, as well as memory. The obser‐ vations for 5-FU (drug control) confirmed literature reports of learning/memory impairment post dosing in humans [16].

The drug penetrated human glioblastoma tumor tissue growing IC in mice, with none detectable in the normal tissue. This only reinforced our interest in using the drug to treat patients with cancers involving the CNS.

**1** does not have effects on the P-gp transport system. In the current study, Rho was selected as an indicator of **1**'s interaction/inhibition with the P-gp protein transport system in three (3) human cancer cell lines-A549: *lung*; MCF7: *breast*; and HeLa: *ovarian.* Verapamil (Vpml) is a known inhibitor of the P-gp pathway and was included as a positive control, providing additional support that **1** is not a substrate for the P-gp transmitter system and not rejected *via* the high energy ATP-binding cassette (ABC) transport systems [11].

Both **1** and **2** bind to erythrocytes and could penetrate the blood brain barrier (BBB) and IC growing cancers attached to rbcs *via* breaks in the BBB (penetration routes of metastatic cancers) or sites of neo-angiogenic networks. However, as previously published, **2** alone is not active *vs*. IC implanted human breast and glioma tumors implanted in a mouse model; the presence of **2** in the IC implanted tumors (see Results-*Brain/tumor penetration*) must be due to IC steroid esterase hydrolysis of **1** [21].

Thus, preclinical studies, conducted under GLP guidelines are presented as support for **1** to enter Phase I clinical trials as treatment for advanced cancer with CNS involvement. Table 8 reviews calculated starting doses and data satisfied the FDA's requirements for an IND (IND 68,876), which has completed a Phase I clinical trial – DTI-021 [19]. The phase I clinical trial is nearing completion with acceptable toxicities and responses noted in patients with advanced cancer involving the CNS system [15].


\* Standard conversion \*\*Based on the highest dose – 30 mg/kg used in the dog studies

The initial level of dosing in the Phase I clinical trial has been established as 39 mg/m2 (IND 86,876) (19).

**Table 8.** Estimated comparable human intravenous dosages
