Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--University OF MICHIGAN.

Melanotan-II (MT-II) is a cyclic heptapeptide analogue of α-MSH (α-melanocyte stimulating hormone) which tans the skin rapidly and is currently being evaluated for the prevention of sunlight-induced skin cancers. In these preformulation studies, the degradation of MT-II followed apparent first-order kinetics. The degradation rate increased as the temperature or phosphate buffer concentration were increased. The pH-rate profile of MT-II degradation showed that MT-II was most stable at approximately pH 5. The degradation of MT-II was not affected by ionic strength. The pKa1 and pKa2 were estimated to be 6.53 and 11.74, respectively. The partition coefficient was studied at various pH values. The Δ log PC at pH 7.35 was 1.05 which indicated that MT-II could pass the intestinal membrane relatively easily. The preformulation data presented here can be used to help develop an appropriate dosage form for MT-II.

We now know from genomics that many disease states lead to changes in expressed proteins (adaptation/plasticity). Therefore, drug design and discovery based on normal states and single targets often is inadequate or even counter-indicated. Therefore, the "system changes" that have occurred must be considered in any treatment for the disease. Such "systems changes" are clearly evident in neuropathic pain where opioids can actually heighten pain. In these pain states there are increased levels of neurotransmitters such as cholecystokinin (CCK) in which both the peptides and their receptors are increased in pain states. To effectively treat diseases involving "systems changes" a new paradigm for the design of compounds was proposed. In this new approach single peptide or peptidomimetic molecules are designed to interact with multiple receptor targets. For the treatment of pain, a series of linear and cyclic peptides and peptidomimetics were designed based on the overlapping pharmacophores of opioid and CCK ligands. The CCK/opioid analogues were synthesized and evaluated for their biological activities. Several of the CCK/opioid analogues were found to simultaneously interact with opioid receptors as agonists and CCK receptors as antagonists. In addition, the lead compounds have been tested in several pain models and were found to be promising in the treatment of neuropathic pain. Further, the structure-activity relationships of these novel peptides have provided new insights into the requirements for binding and bioactivity at opioid and CCK receptors, as well as the overlapping pharmacophores of CCK and enkephalin.

Pentaazapentacosane pentahydrochloride (PAPC-HCl) is a synthetically produced aliphatic pentaamine that is being investigated for use as an anticancer agent. As part of this research project a rapid high-performance liquid chromatographic method for determination of the dansyl derivative of PAPC was developed. The chromatographic system uses a reverse phase C-8 column, a mobile phase of acetic acid buffer and acetonitrile and UV detection. The dansylation conditions were optimized with a pH of 11.0 and a 20 fold dansyl chloride excess. The yield of dansyl PAPC increased 10 fold as the reaction pH was changed from 9.5 to 10.5. An investigation of the products formed in the dansylation reaction revealed that, even under conditions of pH and dansyl chloride concentration most likely to produce partially dansylated products, only perdansyl PAPC is present. This unexpected finding is explained by a mechanism whereby (1) only completely unionized amine molecules will dansylate and (2) the ratio of unionized molecules to ionized molecules increases as dansylation proceeds. The proposed mechanism is verified by comparing the dansylation vs. pH profile of PAPC to that of a reference monoamine (piperidine ·HCl). After 4 hours at room temperature and pH 9.5, 100% of piperidine is dansylated while under the same conditions only 10% of PAPC is derivatized. A pH greater than 10.5 is required to completely dansylate PAPC. This difference is significantly greater than would be predicted from the pKₐ values but it is consistent with the proposed mechanism.

Carbendazim (methyl-2-benzimidazolecarbamate), a well known anti-fungal agent that may have significant anti-cancer activity, is a poorly water-soluble ampholyte. Unfortunately its projected oral dose up to hundred mg per day is far greater than its water solubility of 6 μg/ml. The overall aim of this research was to conduct preformulation studies and develop therapeutically viable oral and parenteral formulations. The solubility of carbendazim was altered by using both solute and solvent modification approaches. The solvent modification was carried out by investigating the solubilization of carbendazim by pH in combination with cosolvents, surfactants or complexants. At pH 7 the total drug solubility is 6.11 ± 0.45 μg/ml which increases by 1 to 7 fold with cosolvent, surfactant or complexant. However, at pH 2 the solubility increases by 400 times and at pH 1.3 over 3000 times. Both cosolvents and non-ionic surfactants have a negligible effect on the total drug solubility at pH 2, whereas the total drug solubility increases by combining pH 2 with anionic surfactants or complexants. The solute modification was accomplished by preparing different salts of carbendazim. In all six different salts, viz., hydrochloride dihydrate, phosphate, hemisulfate hydrate, mesylate, besylate, and tosylate were prepared. Their structures were determined using single crystal x-ray diffraction. The developability attributes (i.e. hygroscopicity, thermal behavior, aqueous solubility, solid state stability, and dissolution rate) of these compounds were evaluated. The dissolution studies showed that all the salts had better dissolution rate than the free base, the mesylate salt been the best. Based on the preformulation studies, two formulations were chosen for oral dosing in mice. The proposed oral formulation of 9.4 mg/ml carbendazim at pH 1 in phosphate buffer and 20% SBEβCD phosphate buffer, respectively, did not precipitate on in vitro static serial dilution with water and seven up. However, the formulation containing only phosphate buffer showed activity in mice by significantly reducing the tumor growth in B-16 melanoma injected mice. Also, it gave active blood levels, which were comparable to IV dosing.

A topochemical model has been derived to account for the diverse selectivity of cyclic opioid peptides related to enkephalins and for narcotic alkaloids towards the $ mu$ and $ delta$ opiate subclasses. Efforts toward demonstrating its validity have later been accomplished. The model is based on a computer molecular modeling study using the opiate pharmacophore as a minimum prerequisite to align the molecules and benefit also of informations obtained from spectroscopic study in solution concerning the macrocycles conformation. The conformational properties of N$ sp alpha$Cbz-c((D)A$ sb2$Bu-Gly-Phe-Leu) were studied by nuclear Overhauser effect which provided semi-quantitative internuclear distances. One of the important elements that the model brings out is that the eventual presence of an aromatic ring on the beta carbon of Cys of the opioid peptide HTyr-(D)Pen-Gly-Phe-Cys-NH$ sb2$ should induce an increase in selectivity toward the 6 opiate subclass. Consequently, we engaged in preparing the special amino acid, P-phenylcysteine, using a method based on the addition of mercaptan to unsaturated azlactone. The diastereoisomers obtained were separated, and their relative configurations assigned by X-ray crystallography. The pure enantiomers were obtained by resolution with the enzyme carboxypeptidase A. Several difficulties were encountered during this preparation, the main concern being the S protecting group. This protecting group should be chosen with the property that it can be quantitatively removed when desired and compatible with the conditions of peptides synthesis. Moreover, the size of the protecting group should be minimized in order to be able to realize the resolution with the enzyme. We explored, by the same token, the size of the hydrophobic pocket of the exopeptidase. Using the structural information obtained from the X-Ray diffraction study of several of the $ beta$-phenycysteine, we have determined with the help of computer molecular modeling, the

For the first time, carbon nanostructures have been designed and synthesized to form immunoconjugates with monoclonal antibodies (mAb) for use in cell-targeted cancer diagnosis and therapy. The immunoconjugates are derived from various nanoscale carbon-based building blocks, specifically fullerenes (C60 ), gadofullerenes (M C60) and ultra-short carbon nanotubes (US-tubes). The exterior of each nanostructure has been derivatized with water-solubilizing addends using Bingel-type (nucleophilic cyclopropanation) addition chemistry to facilitate biocompatibility. Initially, conjugation to the murine anti-gp240 melanoma antibody (ZME-018 mAb) was completed with two different water-soluble C60 derivatives, only one of which had the potential to covalently attach to the ZME-018 mAb. After conjugation, this covalently linked C 60-SPDP conjugate incorporated 15 C60 moieties per antibody, while retaining 80% of the antibody's target specificity. In a second experiment a non-covalently linked C60-Ser conjugate incorporated 38 fullerenes per antibody but retained only 4% of the antibody's target specificity. These findings suggest that covalent attachment of C60 derivatives to antibodies may not be essential for the development of fullerene immunotherapy (FIT), although the ratio of C60: antibody may need to be minimized so as not to inhibit antibody targeting. To study the cell internalization characteristics of the fullerene immunoconjugates, two water-soluble Gd C60 derivatives, which allowed for Gd 3+ monitoring by inductively-coupled plasma mass spectrometry (ICP-MS) at concentrations <10 ppb, have been utilized. These studies have provided evidence that the fullerene-based immunoconjugates retain the ability to effectively internalize into target cells, with approximately 20% of the available Gd 3+ internalizing into the A375m melanoma cells. These results suggest that immunoconjugates derived from C60-based chemotherapeutics may become new-targeted therapies against cancer. Of the carbon-based nanomaterials studied in this work, US-tubes are perhaps the most attractive candidates for nanomedicine platforms, due to the possibility of internally loading medically interesting materials, such as Gd3+ ions for magnetic imaging resonance (MRI) or iodine (I2) for computed tomography (CT). Toward this end, single-molecule US-tubes have been isolated by chemical reduction of the US-tubes, followed by immediate functionalization using Bingel chemistry to produce debundled and derivatized US-tube materials. Three different malonate addends have been attached to the US-tubes, including serinol, polyethylene glycol (PEG) and amide malonates. Each of the US-tube derivatives exhibited varying degrees of solubilities in water ranging from 0.25 mg/mL to 1.00 mg/mL. An n-octanol/water partition coefficient has also been determined for each derivative, with values ranging from 0.25 to 1.20, which suggest that these derivatized nanocapsules might readily internalize into cells. Loaded internally with medically-useful materials such as Gd3+ ions, I2 or radionuclides for imaging and therapeutic applications, these biocompatible carbon nanocapsules may be engineered into a universal platform for the containment and delivery of an array of medical agents in vivo.

This research presents the synthesis and spectroscopic characterization of I2-filled single-walled carbon nanotubes (I2 SWNTs) and I2-filled ultra-short carbon nanotubes (I2 US-tubes). These I2-internally-loaded SWNTs and US-tubes have applications both as a model system for the filling of SWNTs with other medically-interesting materials, such as alpha-radiotherapeutic radionuclides like At-211, and as synthons for a new class of computed-tomography (CT) X-ray contrast agents (CAs). Existing CT technology lacks the ability to diagnose critical diseases such as coronary artery vulnerable plaque, recently discovered as the cause of 70% of heart attacks, because current CAs circulate in the bloodstream rather than being targeted to specific cell-types. SWNT-based CAs offer several advantages over commercially-available CT CAs, such as the ability to sequester toxic ion and molecule imaging agents within the SWNT, to be targeted to specific cell types, and to translocate into targeted cells for intracellular molecular imaging. The synthesis of I2-SWNTs and I2-US-tubes is achieved via sublimation of molecular iodine (I2), a method proven to produce high yields for other filling materials. X-ray photoelectron spectroscopy (XPS) along with inductively-coupled plasma analysis (ICP-AE) and Raman spectroscopy have been used to define the location and quantify the amount of I2 in I2-SWNT and I2-US-tube samples. The exterior-adsorbed I2 can be removed (as I-) from I2-SWNTs by reduction with Na0/THF or by heating the I2-SWNTs to 300°C (without reduction), leaving I2 contained only within the interior of the SWNTs (I2 SWNTs). These I2 SWNTs contain ∼25 weight % of I2 and are stable without the loss of I2 even after exposure to additional reduction with Na 0/THF or upon heating to ca. 500°C. Micro CT experiments confirm that I2 SWNTs, with a radiodensity of 28,400 HU, are functional CT contrast agents. In contrast to I2-SWNTs, the internal I 2 in I2-US-tubes is unstable and is removed by either Na 0/THF reduction or temperatures of 300°C. To date, this instability has resulted in the inability to synthesize PEG-I2 US-tubes, a biocompatible water-soluble derivative of I2-US-tubes. However, initial experiments toward the synthesis of serinol-aide-I2 US-tubes have shown promising results, indicating a water-soluble derivative of I 2 US-tubes will be synthesized in the very near future. The I2 SWNTs and I2 US-tubes prepared in this research represent the first medical applications of filled SWNTs for CT imaging. I 2 SWNTs can now be used as a model system for the filling of SWNTs with other materials such as 211AtX (x = any halogen) for targeted alpha-radioimmunotherapy. They also signify the first step toward revolutionary intracellular CT imaging for the diagnosis of many diseases including coronary artery vulnerable plaque and cancer.