Kathleen A. Merkler

Evaluation of Toxicity following Electrically Mediated Interleukin-12 Gene Delivery in a B16 Mouse Melanoma Model

Purpose: Interleukin-12 (IL-12) has potential as an immunotherapeutic agent for the treatment of cancer but is unfortunately associated with toxicity. Delivery of a plasmid encoding IL-12 with electroporation induces an antitumor effect in the B16 mouse melanoma model without serious side effects. To translate this observation to the clinic, an evaluation of toxicity was done in the mouse model. Experimental Design: Weight change, tumor response, blood chemistry and hematology values, and serum IL-12 levels were evaluated. Multiple tissues were analyzed histopathologically. Results: A pronounced reduction in tumor volume, including a large percentage of complete regressions, was observed after electrically mediated gene therapy. No significant increases in serum IL-12 levels were detected. Tumor-bearing mice showed an increased number of atypical hematology values when compared with normal naive controls. Statistically significant differences in chemistry and hematology values were observed sporadically in most of the standard chemistry and hematology categories in all groups. The only histopathologic abnormality specific to the animals receiving both plasmid and electroporation was inflammation associated with the kidney at the last time point. Conclusions: In general, mice that received both plasmid and electroporation showed the least abnormal histopathologic findings and were found to be in the best health, reflecting the reduced burden of disease. No significant toxic effects due to the IL-12 gene therapy were observed.

Substituted hippurates and hippurate analogs as substrates and inhibitors of peptidylglycine α-hydroxylating monooxygenase (PHM)

Peptidyl alpha-hydroxylating monooxygenase (PHM) functions in vivo towards the biosynthesis of alpha-amidated peptide hormones in mammals and insects. PHM is a potential target for the development of inhibitors as drugs for the treatment of human disease and as insecticides for the management of insect pests. We show here that relatively simple ground state analogs of the PHM substrate hippuric acid (C(6)H(5)-CO-NH-CH(2)-COOH) inhibit the enzyme with K(i) values as low as 0.5microM. Substitution of sulfur atom(s) into the hippuric acid analog increases the affinity of PHM for the inhibitor. Replacement of the acetylglycine moiety, -CO-NH-CH(2)-COOH with an S-(thioacetyl)thioglycolic acid moiety, -CS-S-CH(2)-COOH, yields compounds with the highest PHM affinity. Both S-(2-phenylthioacetyl)thioglycolate and S-(4-ethylthiobenzoyl)thioglycolic acid inhibit the proliferation of cultured human prostate cancer cells at concentrations >100-fold excess of their respective K(i) values. Comparison of K(i) values between mammalian PHM and insect PHM shows differences in potency suggesting that a PHM-based insecticide with limited human toxicity can be developed.

Glutathione, S-substituted glutathiones, and leukotriene C4 as substrates for peptidylglycine α-amidating monooxygenase

The C-terminal alpha-amide moiety of most peptide hormones arises by the posttranslational cleavage of a glycine-extended precursor in a reaction catalyzed by bifunctional peptidylglycine alpha-amidating monooxygenase (PAM). Glutathione and the S-alkylated glutathiones have a C-terminal glycine and are, thus, potential substrates for PAM. The addition of PAM to glutathione, a series of S-alkylated glutathiones, and leukotriene C(4) results in the consumption of O(2) and the production of the corresponding amidated peptide and glyoxylate. This reaction proceeds in two steps with the intermediate formation of a C-terminal alpha-hydroxyglycine-extended peptide. Amidated glutathione (gammaGlu-Cys-amide) is a relatively poor substrate for glutathione S-transferase with a V/K value that is 1.3% of that for glutathione. Peptide substrates containing a penultimate hydrophobic or sulfur-containing amino acid exhibit the highest (V/K)(app) values for PAM-catalyzed amidation. The S-alkylated glutathiones incorporate both features in the penultimate position with S-decylglutathione having the highest (V/K)(app) of the substrates described in this report.

Characterization of regulatory intronic and exonic sequences involved in alternative splicing of scavenger receptor class B gene

Scavenger receptor class B type I (SR-BI) is a major receptor of the high-density lipoprotein that mediates cholesterol efflux and reverse cholesterol transport. Alternative splicing of the scavenger receptor class B (SR-B) gene is observed and different splice forms, SR-BI and scavenger receptor class B type II (SR-BII), have been shown to function and localize in distinct ways. We have previously shown that SR-B alternative splicing regulation is associated with splicing factor ASF/SF2. In this study, using a SR-B minigene as a model, we determined the critical regulatory regions in the upstream intron, intron 11, by serial deletion and mutation analyses. We also further characterized the regulatory elements in intron 11 as well as in the skipped exon, exon 12. Moreover, we studied the interactions of these elements with the splicing factor ASF/SF2. This study provides new insights into the mechanism of SR-B splicing and it is important for further study on the mechanism of SR-B alternative splicing regulation, such as its regulation by estrogen.

A pathway for the biosynthesis of fatty acid amides.

Fatty acid primary amides are potent, bioactive molecules. Oleamide induces physiological sleep1,2, potentiates the action of 5-hydroxytryptamine on rat brain 5-HT2A and 5-HT2C receptors3, and blocks gap junction communication in glial cells4. Erucamide stimulates angiogenesis5, arachidonamide is a tight-binding inhibitor of human synovial phospholipase A2 6, and valpromide (n-propylpentanamide) is used clinically as an antiepileptic7. These studies indicate that the fatty acid primary amides are an exciting new class of mammalian, bioactive lipid. The biosynthetic pathway leading to the formation of the fatty acid primary amides is not understood and is information critical to the understanding and control of diseases related to their production.

699. Evaluation of Potential Toxicity of Electroporation Mediated Deliver of a Plasmid Encoding for IL-12 in a Mouse Melanoma Model

Previous reports from our laboratory have shown that intra-tumor injection of a plasmid encoding IL-12 (pIL-12) followed by electroporation results in an 80% cure rate of established B16.F10 tumors in mice. Twelve of twelve cured mice were resistant to subsequent challenge with B16.F10 cells. In addition, intra-muscular injection of plasmid encoding IL-12 followed by electroporation significantly reduced the formation of lung colonies following intravenous injection of B16.F10 cells. These pre-clinical studies demonstrated the therapeutic potential of this approach. To translate this to the clinic, the following study was conducted to evaluate the potential toxicity of plasmid delivery with electroporation. Mice were divided into 5 groups: intratumor injection of saline (Group 1) or injections of plasmid at 0.1 mg/ml (Groups 2, 3) or 1mg/ml (Groups 4, 5) with or without electroporation. The plasmid (50 |[mu]|l) was injected directly into the tumor and immediately followed by electroporation. Treatments were administered on days 1, 5 and 8. Each mouse was monitored for body weight and general condition on a daily basis beginning the day prior to the first treatment. On days 9, 11, 16, 23 and 30 after treatment, 10 mice from each group (5 male and 5 female) were euthanized. Blood was collected from each animal and used to perform serum chemistries and CBCs. In addition, liver, lung, lymph node, tumor, skin, heart, kidney, spleen and brain samples were collected and histologically analyzed in mice from Groups 1, 4, and 5. All mice appeared to have a generally healthy appearance. Increased weight (10-20%) was noted at the later time points. Mice in Group 5 were found to have the most pronounced reduction in tumor volume including a large percentage of complete regressions. Tumor growth was decreased in Group 3 and the most pronounced increase in tumor size was observed in Groups 1, 2 and 4. With respect to laboratory findings, Group 1 showed the maximum incidence and severity of side effects. The vast majority of ill effects were related to the growth of tumors. For example, Group 1 showed increased AST, increased BUN, decreased lymphocytes, decreased hemoglobin and increased platelets. In comparison to this group, Groups 2 and 4 had reduced magnitude of laboratory abnormalities. Group 5 showed the least laboratory abnormalities. With respect to histopathology, the untreated animals had the most profound abnormalities. Specifically, Group 1 had pronounced tumor involvement in multiple organs. In addition, Group 4 mice showed increased metastatic spread over Group 5 mice. Overall, minimal toxicity and significant tumor regression were observed when pIL-12 is delivered with electroporation. The results of this toxicity study demonstrate that it is safe to test this approach in a clinical study.

822. Efficient Delivery of Plasmid DNA to Murine Skin Using In Vivo Electroporation1

The easy accessibility of skin makes it an excellent target for gene transfer protocols. Cutaneous diseases can be treated directly. In addition, the skin is a suitable target for delivering proteins directly to the circulation for systemic therapy. To be able to fully take advantage of skin as a target for gene transfer, it is important to establish an efficient and reproducible delivery system. Electroporation as a tool for the delivery of plasmid DNA is a strong candidate to meet these delivery criteria. Previously, we demonstrated that electroporation could be used to deliver plasmid DNA to the skin and confirm that localized delivery can also result in increased serum levels of a specific protein. In addition, it was shown that several factors needed to be considered when developing an electroporation delivery protocol including electrode configuration, pulse width, field strength and DNA concentration. More recently, we have been determining optimal delivery conditions using a plasmid encoding luciferase (pLUC). Various EP parameters for delivery to murine skin were examined and included over 50 different combinations. Most of the conditions showed an increase of expression over injection alone. There were 5 combinations that showed the largest increase when compared to injection alone. With respect to plasmid DNA concentration, the highest reproducible expression was seen with 2 μg/μl. Using a plasmid encoding for green fluorescent protein, an efficiency of > 30% was obtained. The effect of multiple applications was also examined. The procedure was evaluated by using the “optimal” electroporation conditions with pLUC at either 2 or 10 days apart. Peak expression could be maintained at a later time point than with a single treatment but the overall length of expression was not extended. This protocol was also repeated using a secreted protein as a marker as well. The results presented here demonstrate that electroporation can be used to augment the efficiency of direct injection of plasmid DNA to skin.