Pharmaceutical Science

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Pyracrenic acid induces oxidative stress-mediated mitochondrial dysfunction in non-small cell lung cancer (NSCLC) by modulating the PI3K/AKT/GSK3Β pathway leading to caspase-dependent and -independent cellular death
(University of Hawaii at Hilo, 2025-08) Atanu, Md Samiul Huq; Tan, Ghee T.; Pharmaceutical Sciences
Lung cancer remains one of the most prevalent and lethal malignancies worldwide, with non small cell lung cancer (NSCLC) accounting for the majority of cases. Platinum based chemotherapies—most notably cisplatin—remain central to treating advanced NSCLC; however, their usefulness is limited by off target toxicity, acquired resistance, and suboptimal efficacy. These shortcomings highlight the urgent need for more selective and less toxic therapies. Natural products have long served as a source for drug discovery, either directly as sources of pure drugs, indirectly as raw materials for semisynthetic derivatives, or as structural templates for designing lead molecules. In this context, pyracrenic acid (PA), a pentacyclic triterpenoid, was isolated from Melaleuca quinquenervia fruit by column chromatography and identified via NMR. Despite being an ester, PA showed robust stability toward cellular esterases, as verified by HPLC analysis. Functionally, PA exhibited potent cytotoxicity against the human NSCLC cell line A549 (IC₅₀ = 2.8 ± 0.02 µM; p < 0.001), outperforming its potential breakdown products, betulinic acid (IC₅₀ = 73.4 ± 1.9 µM) and caffeic acid (IC₅₀ > 100 µM). Its activity was also superior to that of cisplatin (IC₅₀ = 3.3 ± 0.03 µM; p > 0.05). Selectivity indices further indicated greater toxicity toward A549 cells than toward several non malignant cell lines, Vero E6 (SI = 2.5), NIH/3T3 (SI = 2.1), and MRC 5 (SI = 3.0).Mechanistic studies revealed that PA triggers apoptosis, evidenced by dose dependent increase in caspase 3/7 activity and enhanced RealTime Glo™ Annexin V signals, alongside elevated levels of cleaved caspase 3, caspase 9, and PARP. PA also modulated cell cycle regulators—upregulating p53 and p21 while downregulating CDK2 and cyclin D1—consistent with induction of cell cycle arrest. PA induced a dose-dependent increase in oxidative stress and mitochondrial dysfunction, as measured by the ROS-Glo™ H₂O₂ assay (Promega) and JC-1 dye, respectively. This was accompanied by upregulation of pro-apoptotic proteins Bax, Bad, and cytochrome c, along with downregulation of the anti-apoptotic protein Bcl-2. Treatment with N-acetyl-L-cysteine (NAC), an antioxidant, effectively reduced both oxidative stress and mitochondrial membrane depolarization. In contrast, cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition pore (mPTP), did not alleviate oxidative stress, suggesting that oxidative stress precedes and causes mitochondrial dysfunction, rather than resulting from it. Notably, co-treatment with either NAC or CsA significantly (p < 0.01) improved cell viability following 24 hours of exposure to PA (10 µM), compared to PA treatment alone. Although PA elevated the levels of cleaved caspase 3 and caspase 9, co treatment with the pan caspase inhibitor Z VAD FMK did not significantly (p > 0.05) restore A549 cell viability, implying that PA also induces caspase independent cytotoxicity. To explore whether autophagy was involved, we assessed autophagic markers and found that PA provoked a dose dependent increase in monodansylcadaverine (MDC) staining alongside higher LC3 II and Beclin 1 levels. Notably, blocking autophagy with 3 methyladenine (3 MA) failed to significantly (p > 0.05) reverse PA induced cytotoxicity and instead further lowered cell viability, indicating that autophagy serves a protective role rather than acting as an alternative cell death pathway. The effect of PA on the PI3K/Akt/GSK3β signaling pathway was thoroughly examined. PA was found to inhibit this pathway by promoting the dephosphorylation of PI3K and Akt, which subsequently led to the dephosphorylation and activation of GSK3β. Co-treatment with lithium chloride (LiCl), a known GSK3β inhibitor, significantly improved the viability of A549 cells exposed to PA, highlighting the critical role of GSK3β activation in PA-induced cytotoxicity. Furthermore, PA demonstrated strong anti-proliferative activity against a cisplatin-resistant A549 cell line, where it similarly inhibited the PI3K/Akt signaling pathway, underscoring its potential to overcome drug resistance in NSCLC. Collectively, these findings position PA as a promising, naturally derived lead compound for the development of more effective NSCLC therapies.
Targeted Isolation of Anti-mycobacterial Labdane Terpenes from Grindelia stricta var. platyphylla
(University of Hawaii at Hilo, 2025-08) Nealand, Sasha; Chang, Leng Chee; Pharmaceutical Sciences
This dissertation describes experimental work dealing with the targeted isolation and biological activity of grindelanes from Grindelia stricta var. platyphylla (Asteraceae), a plant variety found in areas of coastal California, USA. This species and variety has not been characterized before for its profile of labdane terpenes or for its activity against mycobacteria. In general, although Grindelia is a widely used and well-known medicinal plant genera , the more than 70 different species of Grindelia have not been much studied for the characterization of the diterpene compounds unique to each species. The last extensive study isolating diterpene compounds from a Grindelia sp. was conducted in the 1980-1990’s on Grindelia tarapacana native to Peru and Chile. The lack of studies may be due to the difficulty in separating these very similar low polarity compounds, combined with their lack of UV chromophore. Two innovations and five stated research aims from the approved research proposal defense were investigated in this dissertation work. The first stated innovation was to target and isolate diterpenes from the heretofore unstudied traditional medicinal plant variety Grindelia stricta var. platyphylla collected from the California coast. This was met by the isolation of six diterpene compounds, five known and one novel as well as a known flavonol. The second stated innovation was to the isolated grindelane compounds for antimicrobial activity not yet reported in the literature. This was met by testing for activity against mycobacteria. These innovations were met by completing the following five specific aims described and approved in the dissertation research proposal. Specific Aim 1: Identification, collection and extraction of Grindelia stricta platyphylla flower buds. Separation of mixed fractions from the ethyl acetate extract of Grindelia stricta platyphylla flower buds, by column chromatography. Specific Aim 2: Bioactivity testing of mixed fractions of Grindelia stricta platyphylla against the screening organism Mycobacterium phlei. Specific Aim 3: isolation and structural elucidation of terpenes from Grindelia stricta platyphylla. Specific Aim 4: Preliminary studies of the molecular mechanism of action by assay for anti-kinase activity of column fractions and isolated terpene compounds. Specific Aim 5: bioactivity testing of mixed fractions and single chemical terpene compounds of Grindelia stricta platyphylla against Mycobacterium tuberculosis (Mtb). In this dissertation study of Grindelia stricta var platyphylla, labdane diterpenes were targeted and seven compounds (six labdanes and one very similar-structured flavonol) were isolated by various chromatographic techniques for use in the investigation of specific biological activity (anti-mycobacterial). Of these, one is new (3) and five (1)(2)(4)(5)(6) are known labdane diterpenoids of the grindelane type. One known flavonol (7) kaempferol, was isolated and determined by x-ray crystallography. The new diterpenoid was carboxyl 3,4,4a,5,6,7,8,8a-octahydro-β-hydroxy-β,2,5,5,8a-pentamethyl-3-oxo-1-naphthalenepentanoate, (compound 3, ring opening). The chemical structure was established on the basis of spectral analysis including 1D and 2D NMR. Stereochemistry was not established however, stereochemistry of C-20 and H-5 of the structures can be predicted based on the biogenetic considerations of the grindelanes. The known grindelanes were labd-7-en-15-oic acid, 9,13-epoxy- (compound 1, grindelic acid), 2-(2',3,3,6a,7a-pentamethylspiro[1a,2,2a,4,5,6-hexahydronaphtho[2,3-b]oxirene-7,5'-oxolane]-2'-yl)acetic acid (compound 2, epoxide), spiro[furan-2(3H),1′(4′H)-naphthalene]-5-acetic acid, 4,4′a,5,5′,6′,7′,8′,8′a-octahydro-2′,5,5′,5′,8′a-pentamethyl-, ethyl ester,4′aα,8′aβ]]- (compound 4, ethyl ester), spiro[furan-2(5H),1'(4'H)-naphthalene]-5-acetic acid, 3,4,4'a,5',6',7',8',8'a-octahydro-2',5,5',5',8'a-pentamethyl- (compound 5, methyl ester), methyl 2-[4-(hydroxymethyl)-2',4,7,8a-tetramethylspiro[2,3,4a,5-tetrahydro-1H-naphthalene-8,5'-oxolane]-2'-yl]acetate (compound 6, methyl 18-hydroxy-grindelate). The known flavonol was the kaempferol, 5-Hydroxy-3,7,4'-trimethoxyflavone (compound 7, kaempferol 3,7,4'-trimethyl ether). Throughout the bioassay-guided separations, in various screening assays (disk, TLC overlay, broth) for anti-mycobacterial activity; an ethyl acetate extract of the flower resin, the majority of diterpene rich fractions and the isolated compounds showed activity. In addition, during preliminary screening, the ethyl acetate extract of the flower resin and the isolated compound grindelic acid, showed a kinase inhibitory activity and lack of cytotoxicity in a Streptomyces 85E kinase inhibition assay. This same assay simultaneously screens for possible anti-mycobacterial activity due to the eukaryotic-like kinase’s found in both Streptomyces 85E and mycobacteria. The correlation between compounds testing positive for Streptomyces 85E kinase inhibition and for anti-mycobacterial activity is described in the 2002 paper (“Identifying Protein Kinase Inhibitors Using an Assay Based on Inhibition of Aerial Hyphae Formation in Streptomyces”) by Julian Davies et al, the originator of the Streptomyces 85E kinase inhibition assay. In the final broth dilution assays, fast growing non-pathogenic models M. phlei (ATCC354) and M. smegmatis (ATCC14468) were used as screening organisms. The following MIC’s were determined for M. phlei and M. smegmatis respectively, ethyl acetate extract of the flower resin (25 and 125 µg/ml); compound 1, grindelic acid (16 and 78 µg/ml); compound 2, epoxide (63 and 250 µg/ml); compound 3 (not tested, too small amount); compound 4, ethyl ester (16 and 250 µg/ml); compound 5, methyl ester (63 and 500 µg/ml); compound 6, methyl 18-hydroxy-grindelate (250 and 125 µg/ml); and compound 7 (250 and 125 µg/ml); MIC’s of the anti-mycobacteria agent rifampin (0.16 and 1.3 µg/ml) and the antibiotic/β lactamase inhibitor combo drug, amoxicillin/clavulanate (10 and 313 µg/ml) were also determined. Of the plant compounds, grindelic acid and compound 4, the ethyl ester (MIC’s 16 µg/ml) showed the best activity against M. phlei and grindelic acid also showed favorable activity (78 µg/ml) against M. smegmatis. It was interesting to note that there was a magnitude of difference between the MIC’s determined using rifampin, against the two mycobacteria species. In a subsequent broth dilution study, the MIC of rifampin (0.13 and 1.25 µg/ml) was reduced when in combination with sub-inhibitory doses of amoxicillin/clavulanate, the ethyl acetate extract of the flower resin and some of the isolated compounds, in some cases. Results varied against the two different species of mycobacteria with no clear pattern. Grindelic acid, the most abundant isolated compound, showed the greatest effect, lowering the MIC’s of rifampin to (0.001 and 0.01 µg/ml) respectively. In an assay against M. tuberculosis (Mtb) strain: Mrb mc2 6206 (MtbAux) it was found that the crude ethyl acetate extract, the major compound (1) grindelic acid and the methyl ester compound(5) demonstrated an anti-mycobacterial MIC of 0.125mg/ml. Terpene compounds are difficult to isolate due to their low polarity, relatively low concentration in plants and the lack of a chromophore in the structure. This means that many interesting terpenes present in plants may go undetected into the waste stream when using a conventional UV detector at the end of a separating column. By taking advantage of the modern development of a universal detector, the evaporative light scattering detector (ELSD), it is now possible to more easily detect low concentration compounds that lack a UV chromophore. In the past, the ELSD was a problematic detector not widely used, as it was not robust and lacked the ability to operate at low temperature and concentrations. In this dissertation, using a modern Sedex LT-ELSD, a chromatography method was developed in which grindelanes may be detected at low concentrations and temperatures at the end of a separating column.
Calcium signaling profiles in patient-paired high-risk neuroblastoma tumors - novel mechanisms driving chemotherapy resistance and targeted therapies
(University of Hawaii at Hilo, 2025-08) Sunada, Nathan Kanoa; Jarvi, Susan; Pharmaceutical Sciences
Neuroblastoma (NB) is a malignant pediatric tumor that accounts for over 15% of all pediatric cancer-related deaths. High-risk NB tumors occur in almost 50% of all patient cases and long-term outcomes for these children remain poor, with a survival rate of only ~40%, mainly due to disease relapse, which occurs frequently. Calcium has been demonstrated to play an important role in cancer cell proliferation, cell death, and metastasis, and alterations in Ca2+ signaling have been linked to tumor progression in many cancer types. However, the relationship between altered calcium signaling and the development of chemo-resistance in NB remains unknown. It was hypothesized that the acquisition of chemo-resistance in relapse NB is accompanied by calcium signaling profile alterations and that targeting these aberrations can reduce and/or reverse chemo-resistance. In this study, we demonstrate that relapse NB cells undergo significant remodeling of calcium signaling pathways, particularly store-operated calcium entry (SOCE), contributing to therapeutic escape. Through Specific Aim 1, we profiled calcium signaling in different patient pairs of pre-chemotherapy and post-relapse NB tumors and observed elevated SOCE activity in relapse NB cells, independent of MYCN amplification. This remodeling was mediated by altered expression of STIM1/STIM2, SERCA1/SERCA3, p-IP3R1, and PMCA, alongside reduced SARAF expression, particularly in the SK-N-Be2c line. Divergent patterns in STIM and SERCA isoform expression suggest complex regulatory adaptations that contribute to chemo-resistance in relapse NB. In Specific Aim 2, we leveraged these calcium profiles to identify therapeutic vulnerabilities unique to chemo-resistant relapse NB. Pharmacological modulation of SOCE using 2-APB re-sensitized relapse NB cells to chemotherapy, validating the therapeutic potential of targeting altered calcium signaling dynamics. Furthermore, we screened and identified potent anti-NB compounds, including extracts from Juniperus oblonga, synthetic molecules (compounds 248 and 249), and traditionally-prepared Native Hawaiian plant-based therapies. Intriguingly, traditional preparations demonstrated greater efficacy than Western methods, highlighting the potential of indigenous knowledge systems in novel drug discovery. In conclusion, calcium signaling remodeling, particularly SOCE elevation, is a key adaptive mechanism in NB chemo-resistance. In addition, our results validate calcium signaling profiling as a model for studying resistance mechanisms and therapeutic development in high-risk relapse NB, warranting further exploration and compound screening efforts focused on calcium modulation.
CHEMISTRY OF SPECIALIZED METABOLITES FROM HAWAIIAN FUNGI AND THEIR ANTIBACTERIAL AND ANTIPLATELET POTENTIALS
(University of Hawaii at Hilo, 2022-05) ZAMAN, KH AHAMMAD UZ; Cao, Shugeng; Pharmaceutical Sciences
For centuries, natural products have been and still is considered as the major source of new drug molecules, as about 50% of the approved drug during the last four decades are natural products, natural product derivatives, or synthetic compounds that are inspired by natural product structures.1 Moreover, the alarming rate of microbial resistance to existing drugs necessitates the development of new antibiotics from relatively new and underexplored sources, e.g., fungi. In 2018, according to the states of the world's fungi, only 7% of the world's fungal species were characterized, which means the other 93% of the fungi kingdom remain to be discovered. Despite the mid-oceanic location of Hawaii, it provides diverse ecosystems, such as tropical rain forests, marine environments, and coastlines. However, due to the lack of research on Hawaiian fungi, especially from offshore marine species and soils of high mountains and volcanoes, there is a huge scope to explore them for their biological potential. The present study describes our continuous efforts to interrogate Hawaii-derived terrestrial and marine fungi, for their biological potentials. The first specific aim of the current project was to isolate and identify pure fungal isolates from very selective locations in the Hawaiian Islands. After detailed antibacterial and antiproliferative assays, five fungal strains from five different sources, such as Aspergillus terreus FS107, Fusarium sp. FM701, Xylaria sp. FM1005, Fusarium graminearum FM1010 and Trichoderma sp. FM652, were identified as active strains among many other isolated fungi. The second specific aim of the study was to isolate and identify compounds from different Hawaiian fungi. A significant number (34) of previously undescribed specialized metabolites such as analogs of fusidic acid, tryptoquivaline, fusaric acid, fumagillin, tyrosine, etc., were isolated from different Aspergillus, Fusarium, Xylaria, and Trichoderma species. The structures of these compounds were elucidated by spectroscopic interpretation, including HR-ESIMS and NMR, electronic circular dichroism (ECD) analysis and chemical reactions.Four previously undescribed fusidic acid analogs maunakeanolic acids A-C (1.11.2, 1.4), and 6-deacetyl-1,2-dihydrohelvolic acid (1.3), one fumagillin derivative maunakeanolin (1.8), and two tryptoquivaline analogs tryptoquivalines W-X (1.121.13) were isolated from Mauna kea soil-derived fungus Aspergillus terreus FS107. Figure A. Graphical abstract of compounds from Aspergillus terreus. From two marine Fusarium (Fusarium sp. FM701 and Fusarium graminearum FM1010) species, nine previously unknown fungal polyketides, kaneoheoic acids AI (2.12.6 and 5.35.5) and two unique fusaric acid derivatives, fusariumic acids A-B (2.102.11), two undescribed diketopiperazines, gramipiperazines A-B (5.15.2), and one new isochromanone, along with eight other related compounds were isolated. Furthermore, five new tyrosine derivatives, sinuxylamides A-E (3.13.5), one new phenylacetic acid derivative, two new quinazolinone analogues, one new naphthalenedicarboxylic acid, and one new 3,4-dihydroisocoumarin derivative, were isolated from the marine fungus Xylaria sp. FM1005, which was isolated from Sinularia densa (leather coral) collected in the offshore region of the Big Island, Hawaii. Finally, two new sorbicillinoid derivatives, 2,3-dihydro-2-hydroxy vertinolides (4.1) and (-)-trichodermatone (4.2), together with ten other related compounds were isolated from a Hawaiian marine fungal strain Trichoderma sp. FM652. Figure B. Graphical abstract of compounds from Xylaria sp. and their activity. The third specific aim of the study was to investigate the biological potential of the newly isolated compounds, especially their antibacterial activities. Fusidic acid derivatives from Aspergillus terreus exhibited promising antibacterial activities against both Gram-positive and Gram-negative bacteria. Although, polyketides from both Fusarium species did not show any activity against pathogenic bacteria, the majority of them exhibited significant antibacterial activity against Staphylococcus aureus, methicillin resistant Staphylococcus aureus and Bacillus subtilis, in the range of 10–80 μg/mL in the presence of either antibiotic chloramphenicol (half of the MIC, 1 μg/mL) or an established antibiotic adjuvant disulfiram (6 μg/mL). Moreover, these antibiotic adjuvants also increased the antibacterial activities of fusidic acid derivatives against Gram-positive bacteria by two to three folds. Moreover, this study unexpectedly discovered new tyrosine derivatives, sinuxylamides A-B, which displayed promising antiplatelet activities. The fourth specific aim of the study was to determine the underlying mechanism of the biological activities, such as antibacterial and antiplatelet activities of the newly isolated compounds. The fusidic acid analogs, maunakeanolic acids A-C (1.11.2 and 1.4), 6-deacetyl-1,2-dihydrohelvolic acid (1.3), helvolic acid (1.5), 1,2-dehydrohelvolic acid (1.6) and helvolinic acid (1.7), strongly bound to the elongation factor G (EF-G)-GDP complex of ribosome and inhibited both peptide translocation and ribosome disassembly, resulting in inhibition of protein synthesis. Although all of them bound with both S. aureus and E. coli ribosomal EF-G-GDP complex, maunakeanolic acids A-C, 6-deacetyl-1,2-dihydrohelvolic acid, and helvolinic acid showed stronger binding affinity to S. aureus EF-G-GDP, whereas helvolic acid and 1,2-dehydrohelvolic acid showed stronger affinity to E. coli EF-G-GDP. Interestingly, helvolinic acid has better effect to form complex with S. aureus EF-G-GDP than the FDA-approved drug fusidic acid (parent molecule of the newly isolated triterpenoids). Due to their structure similarity to the FDA-approved antiplatelet drug tirofiban, sinuxylamides A-E (3.13.5) were investigated to find out the mechanism for their antithrombotic activities. Like tirofiban, sinuxylamides A and B strongly inhibited the binding of fibrinogen to purified integrin IIIb/IIa in a dose-dependent manner with the IC50 values of 0.89 and 0.61 μM, respectively. Collectively, the results of this study suggest that Hawaiian fungi have great potential as new source of promising lead compounds with excellent biological activities. In addition, the mechanism of action study revealed that fusidic acid analogs and tyrosine derivatives worked by the same mechanism as the positive controls, fusidic acid and tirofiban, respectively. These findings lead to further investigations of the compounds that may lead to the new drug development.
Preclinical and clinical investigations of anthelmintics for rat lungworm disease (Neuroangiostrongyliasis)
(University of Hawaii at Hilo, 2022-05) Jacob, John; Jarvi, Susan; Pharmaceutical Sciences
Angiostrongylus cantonensis, otherwise known as rat lungworm, is an obligate, digenetic, parasitic nematode, and the causative agent for the clinical condition known as neuroangiostrongyliasis, that ultimately may result in eosinophilic meningitis. It is considered as the leading cause of eosinophilic meningitis worldwide. The treatment aspects of neuroangiostrongyliasis have historically been controversial, especially the use of anthelmintics. This is primarily due to the lack of thorough evaluation of anthelmintic efficacy against A. cantonensis. In addition, there exists a provocative theory amongst clinicians that the use of anthelmintics would kill the larvae that have migrated to the brain, and the dying larvae would induce a severe inflammatory response causing even further complications. There are few reported cases where it is thought that the use of anthelmintics has brought adverse outcomes, but in contrast, there are numerous reports describing successful treatments with anthelmintics. This ambiguity has perplexed the health care system regarding the use of anthelmintics for the management of neuroangiostrongyliasis. Although numerous researchers have investigated the specific question of using anthelmintics for the management of neuroangiostrongyliasis, their findings are scattered among multiple in vitro studies, experimental animal studies and clinical reports, across a wide variety of geographical locations, by multiple authors, and thus, a decisive conclusion has never been established. Therefore, I postulated that through a series of systematic and progressive in vitro, in vivo, and clinical investigations on anthelmintic safety, efficacy, and potency against A. cantonensis, an effective treatment algorithm could be developed for neuroangiostrongyliasis. Previous in vitro studies on anthelmintics efficacy were based on the muscular contractility patterns of adult female A. cantonensis worms using isotonic transductors. However, these results may be clinically irrelevant, since not all anthelmintics affect the motor system. Additionally, anthelmintics with vermicidal properties may not necessarily have larvicidal activity against the same species. Therefore, to determine the absolute death of the parasite after exposure to an anthelmintic, a differential staining technique using propidium iodide penetration as the indicator of death, combined with the natural autofluorescence from the collagenous cuticle of the parasite, was developed. Using this assay, the in vitro efficacy of nine clinically established anthelmintics, which included albendazole, diethylcarbamazine, levamisole, pyrantel, praziquantel, niclosamide, piperazine ivermectin and moxidectin, were evaluated. I concluded that, based on our results and the current United States FDA anthelmintic prescribing guidelines, albendazole, avermectins, and pyrantel pamoate appear to be promising candidates for the management and or prevention of neuroangiostrongyliasis. Albendazole is considered as the anthelmintic of choice for the management of rat lungworm disease (neuroangiostrongyliasis), due to its broad spectrum of nematocidal activity and its ability to cross the blood-brain barrier. While the antimitotic mechanism of albendazole has been explicated in other parasitic and non-parasitic nematodes, it has never been evaluated in A. cantonensis. Hence, I evaluated its mechanism on the microtubules of adult A. cantonensis using the tubulin polymerization assay. Statistically significant dose-dependent reduction in the band intensity of polymerized tubulins (or microtubules) was observed (P = 0.019), suggesting that albendazole imparts its antimitotic effect in a dose-dependent manner.Our in vitro studies suggested pyrantel pamoate as a potential post-exposure prophylactic for rat lungworm. Pyrantel pamoate is readily available over-the-counter in most pharmacies in the USA and possesses anthelmintic activity exclusive to the gastrointestinal tract (GIT). Since its efficacy has never been evaluated in vivo, I conducted an animal study where pharmaceutical grade pyrantel pamoate (11 mg/kg) was orally administered to experimentally infected rats at 0, 2-, 4-, 6-, or 8-hours post-infection. The rats were euthanized six weeks post-infection, and worm burden was evaluated from the heart-lung complex. This study showed that pyrantel pamoate can significantly reduce worm burden by 53-72% (P = 0.004), and I concluded that pyrantel pamoate may help reduce the severity of infection associated with worm burden. Though there are numerous animal studies and clinical reports on the efficacy and safety of benzimidazole anthelmintics, particularly albendazole for neuroangiostrongyliasis, a consensual agreement has never been established. A multilinguistic comprehensive search of databases, for clinical reports and experimental animal studies associated with neuroangiostrongyliasis and benzimidazole anthelmintic treatments, was conducted. I found and evaluated 40 experimental animals and 104 clinical studies. Among those clinical reports that described a confirmed diagnosis of neuroangiostrongyliasis in which albendazole monotherapy was used, 100% reported high efficacy (743 patients, 479 animals). In those where albendazole- corticosteroid co-therapy was used, 97.87% reported it to be effective (323 patients, 130 animals). I concluded that there is insufficient evidence-based data supporting any exacerbation of condition associated with the use of albendazole. Finally, I reviewed a cluster case of neuroangiostrongyliasis involving six individuals associated with the consumption of kava contaminated with at least one slug, presumably infected with A. cantonensis. Five out of six patients were treated with albendazole- corticosteroid co-therapy for at least two weeks, while the remaining one patient received albendazole only for two days, due to lack of insurance. Symptoms in this patient recurred on two separate occasions, requiring additional courses of corticosteroids, but eventually recovered. This suggests that albendazole-corticosteroid co-therapy is safe and beneficial in reducing the symptoms of neuroangiostrongyliasis along with shortening the duration of illness.This series of pre-clinical and clinical investigations provide evidence for the safe and effective use of albendazole for the management of neuroangiostrongyliasis. Considering its safety, early empirical treatment with albendazole-corticosteroid co-therapy based on patient history should be promoted rather than delaying the treatment by waiting for diagnostic confirmation. Our in vitro and in vivo studies show that early administration of pyrantel pamoate after a known exposure could significantly reduce the worm burden and the potentially associated severity of symptoms, giving rise to a new post-exposure prophylactic for the early management of neuroangiostrongyliasis.
THE DESIGN, SYNTHESIS, AND BIOLOGICAL EVALUATION OF CARBAZOLE DERIVATIVES AND WOLLAMIDE ANALOGS
(2017-05) Tsutsumi, Lissa Sachiko; Sun, Dianqing; Tan, Ghee T.; Pharmaceutical Sciences
Design, Development and Evaluation of Targeted Delivery System for the Treatment of Lung Cancer
(2017-02) Gandhi, Nishant; Koomoa-Lange, Dana L.; Chougule, Mahavir B.; Pharmaceutical Sciences
Lung cancer is the leading cause of cancer-related deaths in the world. Lung cancer alone causes more deaths than pancreas, colon, prostate, and breast cancer deaths combined. Only 15% of lung cancer patients survive for 5 or more years after diagnosis. Lung cancer is further divided into small cell lung cancer and non-small cell lung cancer (NSCLC). The NSCLC accounts for 85% of all lung cancer cases. The current mainstay of treatment of lung cancer is multi-drug therapy. Chemotherapy is the preferred option for the treatment of lung cancer. However, severe side effects caused by chemotherapy demands developing novel methods for the treatment. The Luteinizing hormone-releasing hormone (LHRH) is overexpressed in non-small cell lung cancer (NSCLC). This thesis is focused on investigating the ability of poly (amino ether) (PAE) polymer based formulation of small-interfering RNA (siRNA) to silence the mammalian target of Rapamycin (mTOR) in NSCLC cell lines in vitro. In the first part of this thesis, we modified and developed a bio-reducible polymer by introducing a sulfhydryl group (-SH) to PAE polymer. The modified PAE polymer (mPAE) showed decreased cytotoxicity and improved buffering capacity compared to the widely used transfection polymer poly-ethyleneimine (PEI). In the second part, cationic bio-reducible polymer modified Poly (amino-ether) was used to formulate bio-reducible nanoparticles. The mPAE was used to deliver mTOR siRNA to the non-small cell lung cancer cell lines (A549 and H460) and access their potential as a siRNA deliver carrier for lung cancer therapy. The mPAE and mTOR siRNA formed stable, bio-reducible nanoparticles (NPs) at a polymer to siRNA weight ratio of 45:1, with average diameter 114 nm and surface charge of around +27mV. The mTOR siRNA showed increase release in the presence of 10mM GSH. By optimizing the concentration of the mPAE polymer, we were able to fabricate polymeric NPs capable of efficient gene knockdown (60% and 64%) in A549 and H460 cells, respectively without significant cytotoxicity at 30µg/ml concentrations. The MS-MP-NPs showed improved cell growth inhibition (31% and 32%) in A549 and H460 cells in vitro, respectively. The MS-MP-NPs also showed time-dependent cellular uptake as determined by FACS for up to 24 hours. The results demonstrate that the mPAE polymer based NPs show strong potential for future modification with Poly (ethylene glycol) and targeting ligand to improve the gene delivery and achieve higher lung cancer growth inhibition in vitro and in vivo. In the third part, PEGylated-targeted NPs (MS-MP-PG-LR) were prepared. To attach PEG on the surface of the nanoparticles, first, the PEG was conjugated with AMAS and then allowed to attach on the MS-MP NP surface. Further, cysteine-terminated LHRH was subsequently conjugated on the surface of the PEG group through a maleimide reaction with the cysteine group. We found that PEGylation of the mPAE based nanoparticles allows increased delivery of the siRNA. 22% PEGylated prevented the cytotoxic effect of 60 µg/ml concentrations of the nanoparticle system. Western blot results confirmed the overexpression of the LHRH receptor on the non-tumor (Breast adipocytes) and the tumor cells (SKOV-3). As shown before, PEGylation also provided serum stability to the NP system. The optimal particle size of the MS-MP-PG and MS-MP-PG-LR20 nanoparticles was found to be 124 ± 5.9and 132 ± 6.8 respectively. The zeta potential of the respective nanoparticles was positive (19.5 ± 4.6 and 20.2 ± 5.4). The FACS analysis showed that the targeted MS-MP-PG-LR20 nanoparticle system selectively internalizes in the LHRH-R overexpressed A549 and H460 cells compared to the SKOV-3 cells (significantly lower LHRH-R expression). The mTOR siRNA encapsulated, targeted MS-MP-PG-LR20 NPs showed significantly increased cell growth inhibition and mTOR gene silencing compared to the SS-MP-PG-LR20 nanoparticle system. The caspase activity assay confirmed the apoptotic pathway mediated cell growth inhibition of the MS-MP-PG-LR20 nanoparticles.
Development and evaluation of polymeric hybrid a-Difluoromethylornithine (DFMO) & Etoposide loaded nanocarriers for the treatment of Neuroblastoma
(2016-08) Glasgow, Micah David Kealaka'i; Chougule, Mahavir B.; Pharmaceutical Sciences
Neuroblastoma (NB) is the most common extra-cranial solid cancer in childhood and infancy with patients having an average age of 17 months. Most are diagnosed with advanced stage NB when tumor progression is aggressive, making treatment of NB even more difficult. Up to 45% of patients are in the high-risk category with MYCN gene amplification being observed. The FDA-approved drug difluoromethylornithine (DFMO) exhibits anticancer activity against MYCN-amplified NB cells. DFMO is a suicide inhibitor of ornithine decarboxylase (ODC), a rate-limiting enzyme in the biosynthesis of polyamines. ODC gene expression is directly activated by MYCN suggesting that MYCN amplification is connected to high ODC expression. ODC expression produces high polyamine levels that contribute to the malignant phenotype and maintenance of NB tumorgenesis. This MYCN-ODC connection suggests that ODC may be a suitable new target for the treatment of NB with the administration DFMO. Etoposide, a topoisomerase inhibitor is often used in front-line therapy in the treatment of NB. The use of DFMO/Etoposide in vivo is currently limited due to the short half-lives (fast elimination/clearance) of both drugs which may explain why antitumor in vivo were not synergistic as observed in vitro. iRGD peptide-conjugated PEGylated polymeric hybrid nanocarriers loaded with synergistically acting DFMO and Etoposide drugs (iRGD-PEG-HNC-D-E) were characterized at 81ﾱ7nm in size, +12ﾱ2.5mV in zeta potential with a mean polydispersity index 0f 0.354ﾱ0.03. The developed nanocarriers had a 10 and 6-fold decrease in initial drug concentrations, DFMO and Etoposide respectively, with similar efficacy as compared to free drugs alone against various NB cell-lines over a 72h period. The current formulation shows stability suspended in phosphate buffer saline (PBS) 7.4 over 6 days. iRGD-PEG-HNC-D-E formulations can be modified (polymer: polymer ratio) to alter drug release profiles with the developed formula having a 90% release of both drugs after 72h.

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