**1. Introduction**

4-Demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN), **1**, is a polychlorinat‐ ed pyridine cholesteryl carbonate, (Fig. 1) that is a derivative of 4-demethylpenclomedine (DM-PEN, **2**) [1-4]. The latter is a non-neurotoxic metabolite of penclomedine (PEN, **3**, NSC 338720, 2-trichloromethyl-3,5-dichloro-4,6-dimethoxypyridine), that was identified during the NCI sponsored Phase I clinical trials with **3** [4-8]. **3** was found to be active *vs.* advanced cancers but possessed unacceptable neurotoxicity and discontinued from further study [5-10].

Where –**1**. DM-CHOC-PEN: R=CO2-cholesteryl, **2**. DM-PEN: R=H, **3**. PEN: R=OCH3

**Figure 1.** Penclomedine (PEN) and Analogs

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**1** was synthesized at DEKK-TEC as part of a series of polychlorinated pyridine carbonates and carbamates that are lipophilic, non-neurotoxic alkylators of human xenograft brain and breast tumors implanted intracranially (IC) in mice [1-3].

**2. Materials and methods**

used.

**2.1. Drug formulation and chemicals**

(2) 100A packing (diameter of particles=5 mµ).

Plasma and erythrocyte samples were stored at-74o

THF. All solutions were stable for at least 2 months at 5o

delivered at a flow rate of 1.0 mL/min.

100 µL of THF and analyzed as below.

**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

**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

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

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

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

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

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

and 4.63 min., respectively and for **2** and **P** were 6.6 and 18.5 min., respectively.

C until analyzed. Standard solutions were

Comparative Preclinical Pharmacology and Toxicology for…

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

241

C. Standard assays for **1** and **2** consisted

Anticancer activity for **1** has been well documented *in vivo vs.* IC implanted human xenograft glioblastoma (U-251 and D-54) and breast cancer (MX-1) mouse models at doses lower or equivalent to its LD10 [1-3]. Over 25 carbonate and carbamate analogs of **2** have been synthe‐ sized and evaluated *in vivo* and **1** was the most active of the analogs *vs.* the above IC implanted human xenograft cancer models [1-3].

X-ray crystallography studies with **1** describe a perfectly linear configuration (Fig. 2) that includes a neutral heterocyclic ring linked through a stable carbonate group to a lipophilic cholesteryl moiety [2]. These basic characteristics plus the neutralizing effects (electrophilic) of the polyhalogenated substitutions on the pyridine ring probably contribute to **1**'s ability to form micelle particles that penetrate the blood brain barrier, and accumulate in CNS tumor tissues [1, 11].

**Figure 2.** Structural characteristics of **1**

A mechanism of action has been proposed for **1** that involves cross-linking across the trichlor‐ omethyl group with tumor DNA in the major groove *via N7* -guanine cross linking in a G-X-C sequence [1]. This mechanism of action would allow **1** to be administered in combination with many of the clinically significant DNA major groove-alkylating drugs, that include methylat‐ ing agents [*e.g*. dacarbazine and temozolomide (TemodarR)] and chloroethylating agents [*e.g*. bis(chloroethyl)-nitrosourea (BCNU) and clomesone] – all of which form carbonium ionmediated adducts *via O6* -guanine in *contrast* to binding with *N7* -guanine, as does **1** [12].

The pharmacokinetics, metabolic fate and toxicology of **1** in three animal species (mice, rats and dogs) are reviewed here.

Cognitive/behavioral studies have been conducted in rats and dogs and are reviewed in depth.
