A fluorescent gemcitabine prodrug that follows a nucleoside transporter-independent internalization and bears enhanced therapeutic efficacy with respect to gemcitabine.

The multiplexity of cancer has rendered it the second leading cause of mortality worldwide and theragnostic prodrugs have gained popularity in recent years as a means of treatment. Theragnostic prodrugs enable the simultaneous diagnosis and therapy of tumors via high-precision real-time drug release monitoring. Herein, we report the development of the small theragnostic prodrug GF, based on the nucleoside anticancer agent gemcitabine and the fluorescent dye 5(6)-carboxyfluorescein. We have successfully demonstrated its efficient internalization in tumor cells, showing localization throughout both the early and late endocytic pathways. Its mechanism of cell internalization was evaluated, confirming its independence from nucleoside transporters. Its cellular localization via confocal microscopy revealed a clathrin-mediated endocytosis mechanism, distinguishing it from analogous compounds studied previously. Furthermore, GF exhibited stability across various pH values and in human blood plasma. Subsequently, its in vitro cytotoxicity was assessed in three human cancer cell lines (A549, U87 and T98). Additionally, its pharmacokinetic profile in mice was investigated and the consequent drug release was monitored. Finally, its in vivo visualization was accomplished in zebrafish xenotransplantation models and its in vivo efficacy was evaluated in A549 xenografts. The results unveiled an intriguing efficacy profile, positioning GF as a compelling candidate warranting further investigation.

Nurr1:RXRα heterodimer activation as monotherapy for Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic (DAergic) neurons in the substantia nigra and the gradual depletion of dopamine (DA). Current treatments replenish the DA deficit and improve symptoms but induce dyskinesias over time, and neuroprotective therapies are nonexistent. Here we report that Nuclear receptor-related 1 (Nurr1):Retinoid X receptor α (RXRα) activation has a double therapeutic potential for PD, offering both neuroprotective and symptomatic improvement. We designed BRF110, a unique in vivo active Nurr1:RXRα-selective lead molecule, which prevents DAergic neuron demise and striatal DAergic denervation in vivo against PD-causing toxins in a Nurr1-dependent manner. BRF110 also protects against PD-related genetic mutations in patient induced pluripotent stem cell (iPSC)-derived DAergic neurons and a genetic mouse PD model. Remarkably, besides neuroprotection, BRF110 up-regulates tyrosine hydroxylase (TH), aromatic l-amino acid decarboxylase (AADC), and GTP cyclohydrolase I (GCH1) transcription; increases striatal DA in vivo; and has symptomatic efficacy in two postneurodegeneration PD models, without inducing dyskinesias on chronic daily treatment. The combined neuroprotective and symptomatic effects of BRF110 identify Nurr1:RXRα activation as a potential monotherapeutic approach for PD.

Gemcitabine Based Peptide Conjugate with Improved Metabolic Properties and Dual Mode of Efficacy.

Gemcitabine is a clinically established anticancer agent potent in various solid tumors but limited by its rapid metabolic inactivation and off-target toxicity. We have previously generated a metabolically superior to gemcitabine molecule (GSG) by conjugating gemcitabine to a gonadotropin releasing hormone receptor (GnRH-R) ligand peptide and showed that GSG was efficacious in a castration resistant prostate cancer (CRPC) animal model. The current article provides an in-depth metabolic and mechanistic study of GSG, coupled with toxicity assays that strengthen the potential role of GSG in the clinic. LC-MS/MS based approaches were employed to delineate the metabolism of GSG, its mechanistic cellular uptake, and release of gemcitabine and to quantitate the intracellular levels of gemcitabine and its metabolites (active dFdCTP and inactive dFdU) resulting from GSG. The GnRH-R agonistic potential of GSG was investigated by quantifying the testosterone levels in animals dosed daily with GSG, while an in vitro colony forming assay together with in vivo whole blood measurements were performed to elucidate the hematotoxicity profile of GSG. Stability showed that the major metabolite of GSG is a more stable nonapeptide that could prolong gemcitabine's bioavailability. GSG acted as a prodrug and offered a metabolic advantage compared to gemcitabine by generating higher and steadier levels of dFdCTP/dFdU ratio, while intracellular release of gemcitabine from GSG in DU145 CRPC cells depended on nucleoside transporters. Daily administrations in mice showed that GSG is a potent GnRH-R agonist that can also cause testosterone ablation without any observed hematotoxicity. In summary, GSG could offer a powerful and unique pharmacological approach to prostate cancer treatment: a single nontoxic molecule that can be used to reach the tumor site selectively with superior to gemcitabine metabolism, biodistribution, and safety while also agonistically ablating testosterone levels.

GnRH-Gemcitabine conjugates for the treatment of androgen-independent prostate cancer: pharmacokinetic enhancements combined with targeted drug delivery.

Gemcitabine, a drug with established efficacy against a number of solid tumors, has therapeutic limitations due to its rapid metabolic inactivation. The aim of this study was the development of an innovative strategy to produce a metabolically stable analogue of gemcitabine that could also be selectively delivered to prostate cancer (CaP) cells based on cell surface expression of the Gonadotropin Releasing Hormone-Receptor (GnRH-R). The synthesis and evaluation of conjugated molecules, consisting of gemcitabine linked to a GnRH agonist, is presented along with results in androgen-independent prostate cancer models. NMR and ligand binding assays were employed to verify conservation of microenvironments responsible for binding of novel GnRH-gemcitabine conjugates to the GnRH-R. In vitro cytotoxicity, cellular uptake, and metabolite formation of the conjugates were examined in CaP cell lines. Selected conjugates were efficacious in the in vitro assays with one of them, namely, GSG, displaying high antiproliferative activity in CaP cell lines along with significant metabolic and pharmacokinetic advantages in comparison to gemcitabine. Finally, treatment of GnRH-R positive xenografted mice with GSG showed a significant advantage in tumor growth inhibition when compared to gemcitabine.

Combination of Doxorubicin and Antiangiogenic Agents in Drug-Eluting Beads: In Vitro Loading and Release Dynamics in View of a Novel Therapeutic Approach for Hepatocellular Carcinoma.

PURPOSE: Antiangiogenic agents have been used for many years as a first-line systemic treatment for advanced HCC. Embolization with cytostatic drugs on the other hand is the first-line treatment for intermediate HCC. The two types of drugs have not been combined for intraarterial delivery yet. The loading and release dynamics and the in vitro effect of their combination are tested in this experimental study. MATERIALS AND METHODS: Drug-eluting beads were loaded with doxorubicin, sunitinib and sunitinib analogue piperazine (SAP) alone and with their combinations. Diameter change, loading, release, and effect in cellular proliferation were assessed. RESULTS: The average microsphere diameter after loading was 473.7 µm (µm) for Doxorubicin, 388.4 µm for Sunitinib, 515.5 µm for SAP, 414.8 µm for the combination Doxorubicin/Sunitinib and 468.8 µm for the combination Doxorubicin /SAP. Drug release in 0.9% NaCl was 10% for Doxorubicin, 49% for Sunitinib, 25% for SAP, 20%/18% for the combination Doxorubicin/Sunitinib, and 18%/23% for the combination Doxorubicin/SAP whereas in human plasma it was 56%, 27%, 13%, 76%/63% and 62%/15%, respectively. The mean concentration of Doxorubicin that led to inhibition of 50% of cellular proliferation in an HCC Huh7 cell line was 163.1 nM (nM), for Sunitinib 10.3 micromolar (µΜ), for SAP 16.7 µΜ, for Doxorubicin/Sunitinib 222.4 nM and for Doxorubicin/SAP 275 nM. CONCLUSIONS: Doxorubicin may be combined with antiangiogenic drugs with satisfactory in vitro loading and release outcomes and effect on cellular lines.

Peptide and protein drugs: the study of their metabolism and catabolism by mass spectrometry.

Peptide and protein drugs have evolved in recent years into mainstream therapeutics, representing a significant portion of the pharmaceutical market. Peptides and proteins exhibit highly diverse structures, broad biological activities as hormones, neurotransmitters, structural proteins, metabolic modulators and therefore have a significant role as both therapeutics and biomarkers. Understanding the metabolism of synthetic or biotechnologically derived peptide and protein drugs is critical for pharmaceutical development as metabolism has a significant impact on drug efficacy and safety. Although the same principles of pharmacokinetics and metabolism of small molecule drugs apply to peptide and protein drugs, there are few notable differences. Moreover, the study of peptide and protein drug metabolism is a rather complicated process which requires sophisticated analytical techniques, and mass spectrometry based approaches have provided the capabilities for efficient and reliable quantification, characterization, and metabolite identification. This review article will focus on the current use of mass spectrometry for the study of the metabolism of peptide and protein drugs.

Transarterial embolization with sorafenib in animal livers: a pharmacokinetics study.

PURPOSE: To assess the safety and feasibility of the targeted delivery of the antiangiogenic drug sorafenib to the liver using transarterial chemoembolization methodology as a novel approach to hepatocellular carcinoma (HCC) therapy. MATERIALS AND METHODS: Seven healthy New Zealand white rabbits were used in the study. After placement of a catheter in the common hepatic artery, six rabbits were treated with chemoembolization of sorafenib in iodized oil (Lipiodol) (sorafenib dose 0.1 mg/kg), and one rabbit received Lipiodol only. Liquid chromatography tandem mass spectrometry was used to measure the concentration of sorafenib in the peripheral blood and liver tissue 24 hours and 72 hours after treatment. Histochemical staining of the liver sections and biochemical measurements were performed. RESULTS: The administration of sorafenib in Lipiodol emulsions by transarterial chemoembolization resulted in sorafenib concentrations of 794 ng/g ± 240 and 64 ng/g ± 15 in the liver tissue 24 hours and 72 hours after treatment. The average liver-to-serum ratios 24 hours and 72 hours after treatment were approximately 14 and 22. The histochemical staining of the liver tissue sections and aspartate aminotransferase, alanine aminotransferase, γ-glutamyltransferase and total bilirubin concentrations indicated no significant liver damage. CONCLUSIONS: Transarterial chemoembolization with sorafenib in Lipiodol is an effective methodology for the localized delivery of this drug to the liver and has possible practical implications in therapeutic interventions for the treatment of hepatocellular carcinoma.

Monitoring of Levosimendan Administration in Patients with Pulmonary Hypertension Undergoing Cardiac Surgery and Effect of Two Different Dosing Schemes on Hemodynamic and Echocardiographic Parameters.

INTRODUCTION: The perioperative management of patients with pulmonary hypertension (PH) undergoing cardiac surgery represents one of the most challenging clinical scenarios. This fact mainly depends on the relationship existing between PH and right ventricular failure (RVF). Levosimendan (LS) is an inodilator that might be an effective agent in the treatment of PH and RVF. The aim of this study was to examine the impact of the duration of cardiopulmonary bypass (CPB) on the therapeutic drug monitoring of LS and to evaluate the effect of preemptive administration of LS on perioperative hemodynamic and echocardiographic parameters in cardiac surgical patients with preexisting PH. MATERIALS AND METHODS: In this study, LS was administered in adult patients undergoing cardiac surgery before CPB in order to prevent exacerbation of preexisting PH and subsequent right ventricular dysfunction. Thirty cardiac surgical patients with preoperatively confirmed PH were randomized to receive either 6 µg/kg or 12 µg/kg of LS after the induction of anesthesia. The plasma concentration of LS was measured after CPB. In this study, a low sample volume was used combined with a simple sample preparation protocol. The plasma sample was extracted by protein precipitation and evaporated; then, the analyte was reconstituted and detected using specific and sensitive bioanalytical liquid chromatography with mass spectrometry (LC-MS/MS) methodology. The clinical, hemodynamic, and echocardiographic parameters were registered and evaluated before and after the administration of the drug. RESULTS: A fast bioanalytical LC-MS/MS methodology (a run time of 5.5 min) was developed for the simultaneous determination of LS and OR-1896, its main metabolite in human plasma. The LC-MS/MS method was linear over a range of 0.1-50 ng/mL for LS and 1-50 ng/mL for its metabolite OR-1896. Measured plasma concentrations of LS were inversely related to the duration of CPB. LS administration before CPB during cardiac surgery was effective in reducing pulmonary artery pressure and improving hemodynamic parameters after CPB, with a more pronounced and durable effect of the drug at the dose of 12 µg/kg. Additionally, administration of LS at a dose of 12 µg/kg in cardiac surgical patients with PH before CPB improved right ventricular function. CONCLUSION: LS administration decreases pulmonary artery pressure and may improve right ventricular function in patients with PH undergoing cardiac surgery.

Integrin-Mediated Targeted Cancer Therapy Using c(RGDyK)-Based Conjugates of Gemcitabine.

c(RGDyK)-based conjugates of gemcitabine (GEM) with the carbonate and carbamate linkages in the 6-OH group of GEM were synthesized for the targeted delivery of GEM to integrin αvß3, overexpressing cancer cells to increase the stability as well as the tumor delivery of GEM and minimize common side effects associated with GEM treatment. Competitive cell uptake experiments demonstrated that conjugate TC113 could be internalized by A549 cells through integrin αvß3. Among the synthesized conjugates, TC113 bearing the carbamate linker was stable in human plasma and was further assessed in an in vivo pharmacokinetic study. TC113 appeared to be relatively stable, releasing GEM slowly into blood, while it showed potent antiproliferative properties against WM266.4 and A549 cells. The encouraging data presented in this study with respect to TC113 provide a promising keystone for further investigation of this GEM conjugate with potential future clinical applications.

Laser-Induced Forward Transfer Printing on Microneedles for Transdermal Delivery of Gemcitabine.

Cancer treatment with chemotherapeutic drugs remains to be challenging to the physician due to limitations associated with lack of efficacy or high toxicities. Typically, chemotherapeutic drugs are administered intravenously, leading to high drug concentrations that drive efficacy but also lead to known side effects. Delivery of drugs through transdermal microneedles (MNs) has become an important alternative treatment approach. Such delivery options are well suited for chemotherapeutic drugs in which sustained levels would be desirable. In the context of developing a novel approach, laser-induced forward transfer (LIFT) was applied for bioprinting of gemcitabine (Gem) to coat polymethylmethacrylate MNs. Gem, a chemotherapeutic agent used to treat various types of cancer, is a good candidate for MN-assisted transdermal delivery to improve the pharmacokinetics of Gem while reducing efficiency limitations. LIFT bioprinting of Gem for coating of MNs with different drug amounts and successful transdermal delivery in mice is presented in this study. Our approach produced reproducible, accurate, and uniform coatings of the drug on MN arrays, and on in vivo transdermal application of the coated MNs in mice, dose-proportional concentrations of Gem in the plasma of mice was achieved. The developed approach may be extended to several chemotherapeutics and provide advantages for metronomic drug dosing.

Treatment and prevention of pathological mitochondrial dysfunction in retinal degeneration and in photoreceptor injury.

Pathological deterioration of mitochondrial function is increasingly linked with multiple degenerative illnesses as a mediator of a wide range of neurologic and age-related chronic diseases, including those of genetic origin. Several of these diseases are rare, typically defined in the United States as an illness affecting fewer than 200,000 people in the U.S. population, or about one in 1600 individuals. Vision impairment due to mitochondrial dysfunction in the eye is a prominent feature evident in numerous primary mitochondrial diseases and is common to the pathophysiology of many of the familiar ophthalmic disorders, including age-related macular degeneration, diabetic retinopathy, glaucoma and retinopathy of prematurity - a collection of syndromes, diseases and disorders with significant unmet medical needs. Focusing on metabolic mitochondrial pathway mechanisms, including the possible roles of cuproptosis and ferroptosis in retinal mitochondrial dysfunction, we shed light on the potential of α-lipoyl-L-carnitine in treating eye diseases. α-Lipoyl-L-carnitine is a bioavailable mitochondria-targeting lipoic acid prodrug that has shown potential in protecting against retinal degeneration and photoreceptor cell loss in ophthalmic indications.

Evaluation of a stable gonadotropin-releasing hormone analog in mice for the treatment of endocrine disorders and prostate cancer.

Gonadotropin-releasing hormone (GnRH) receptor agonists have wide clinical applications including the treatment of prostate cancer and endocrine disorders. However, such agonists are characterized by poor pharmacokinetic properties, often requiring repeated administration or special formulations. Therefore, the development of novel peptide analogs with enhanced in vivo stability could potentially provide therapeutic alternatives. The pharmacological evaluation of a bioactive peptide [Des-Gly¹°,Tyr5(OMe),D-Leu6,Aze-NHEt9]GnRH, analog 1, is presented herein and compared with leuprolide. Peptide stability was evaluated using mouse kidney membrane preparations, followed by a liquid chromatography-tandem mass spectrometry-based approach that afforded identification and quantification of its major metabolites. The analog was significantly more stable in vitro in comparison with leuprolide. In vitro and in vivo stability results correlated well, encouraging us to develop a clinically relevant pharmacokinetic mouse model, which facilitated efficacy measurements using testosterone as a biomarker. Analog 1, an agonist of the GnRH receptor with a binding affinity in the nanomolar range, caused testosterone release in mice that was acutely dose-dependent, an effect blocked by the GnRH receptor antagonist cetrorelix. Repeated dosing studies in mice demonstrated that analog 1 was well tolerated and had potency similar to that of leuprolide, based on plasma and testis testosterone reduction and histopathological findings. Analog 1 also shared with leuprolide similar significant antiproliferative activity on androgen-dependent prostate cancer (LNCaP) cells. On the basis of pharmacokinetic advantages, we expect that analog 1 or analogs based on this new design will be therapeutically advantageous for the treatment of cancer and endocrine disorders.

Discovery of a piperazine urea based compound as a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist.

We report the discovery of piperazine urea based compound 1, a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist. Compound 1 shows anti-obesity efficacy without potentiating erectile activity in the rodent models.

Oral administration of chios mastic gum or extracts in mice: quantification of triterpenic acids by liquid chromatography-tandem mass spectrometry.

Chios mastic gum, the resin obtained as an exudate from the trunk and branches of Pistacia lentiscus L var. chia, is used extensively as a constituent of herbal drugs or functional foods. The oral absorption of its major constituents still remains unclear. In the context of identifying the features of mastic gum that are responsible for either therapeutic effects or effects of nutritional value, a methodology based on high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (MS/MS) was developed and applied for the quantification of mastic gum triterpenic acids, 24Z-isomasticadienonic acid (IMNA), and 24Z-isomasticadienolic acid (IMLA) in mouse plasma. The specific compounds were selected based on their biological activity and potential against Helicobacter pylori. Concentrations were determined simultaneously in mouse plasma after oral administration of mastic gum or total mastic extract without polymer (TMEWP) in order to evaluate the role of the natural polymer, poly-ß-myrcene, in the absorption process. Following TMEWP administration in mice, circulating IMNA and IMLA plasma levels were significantly higher (approximately 10-fold) in comparison to IMNA and IMLA plasma levels following total mastic gum administration (CMG), suggesting that the polymer plays a critical role in the absorption process. More specifically following TMEWP administration, Cmax plasma values were 3300 ± 859 ng/mL for IMNA and 163 ± 58 ng/mL for IMLA. In comparison, following CMG administration, Cmax plasma values were 329 ± 57 ng/mL for IMNA and 28 ± 8 ng/mL for IMLA. The methodological approaches presented in this study, along with the findings, offer valuable information on the availability of bioactive components following ingestion of mastic and facilitate the uses of mastic either as an ingredient of functional foods or as a herbal drug.

Immunotherapy Combined with Metronomic Dosing: An Effective Approach for the Treatment of NSCLC.

Pioneering studies on tumor and immune cell interactions have highlighted immune checkpoint inhibitors (ICIs) as revolutionizing interventions for the management of NSCLC, typically combined with traditional MTD chemotherapies, which usually lead to toxicities and resistance to treatment. Alternatively, MTR chemotherapy is based on the daily low dose administration of chemotherapeutics, preventing tumor growth indirectly by targeting the tumor microenvironment. The effects of MTR administration of an oral prodrug of gemcitabine (OralGem), alone or with anti-PD1, were evaluated. Relevant in vitro and in vivo models were developed to investigate the efficacy of MTR alone or with immunotherapy and the potential toxicities associated with each dosing scheme. MTR OralGem restricted tumor angiogenesis by regulating thrombospondin-1 (TSP-1) and vascular endothelial growth factor A (VEGFA) expression. MTR OralGem enhanced antitumor immunity by increasing T effector responses and cytokine release, concomitant with dampening regulatory T cell populations. Promising pharmacokinetic properties afforded minimized blood and thymus toxicity and favorable bioavailability upon MTR administration compared to MTD. The combination of MTR OralGem with immunotherapy was shown to be highly efficacious and tolerable, illuminating it as a strong candidate therapeutic scheme for the treatment of NSCLC.

Discovery and Pharmacological Evaluation of STEAP4 as a Novel Target for HER2 Overexpressing Breast Cancer.

Breast cancer (BC) is a highly heterogeneous disease encompassing multiple subtypes with different molecular and histopathological features, disease prognosis, and therapeutic responses. Among these, the Triple Negative BC form (TNBC) is an aggressive subtype with poor prognosis and therapeutic outcome. With respect to HER2 overexpressing BC, although advanced targeted therapies have improved the survival of patients, disease relapse and metastasis remains a challenge for therapeutic efficacy. In this study the aim was to identify key membrane-associated proteins which are overexpressed in these aggressive BC subtypes and can serve as potential biomarkers or drug targets. We leveraged on the development of a membrane enrichment protocol in combination with the global profiling GeLC-MS/MS technique, and compared the proteomic profiles of a HER2 overexpressing (HCC-1954) and a TNBC (MDA-MB-231) cell line with that of a benign control breast cell line (MCF-10A). An average of 2300 proteins were identified from each cell line, of which approximately 600 were membrane-associated proteins. Our global proteomic methodology in tandem with invigoration by Western blot and Immunofluorescence analysis, readily detected several previously-established BC receptors like HER2 and EPHA2, but importantly STEAP4 and CD97 emerged as novel potential candidate markers. This is the first time that the mitochondrial iron reductase STEAP4 protein up-regulation is linked to BC (HER2+ subtype), while for CD97, its role in BC has been previously described, but never before by a global proteomic technology in TNBC. STEAP4 was selected for further detailed evaluation by the employment of Immunohistochemical analysis of BC xenografts and clinical tissue microarray studies. Results showed that STEAP4 expression was evident only in malignant breast tissues whereas all the benign breast cases had no detectable levels. A functional role of STEAP4 intervention was established in HER2 overexpressing BC by pharmacological studies, where blockage of the STEAP4 pathway with an iron chelator (Deferiprone) in combination with the HER2 inhibitor Lapatinib led to a significant reduction in cell growth in vitro. Furthermore, siRNA mediated knockdown of STEAP4 also suppressed cell proliferation and enhanced the inhibition of Lapatinib in HER2 overexpressing BC, confirming its potential oncogenic role in BC. In conclusion, STEAP4 may represent a novel BC related biomarker and a potential pharmacological target for the treatment of HER2 overexpressing BC.

Development of programmable gemcitabine-GnRH pro-drugs bearing linker controllable "click" oxime bond tethers and preclinical evaluation against prostate cancer.

Peptide-drug conjugates (PDCs) are gaining considerable attention as anti-neoplastic agents. However, their development is often laborious and time-consuming. Herein, we have developed and preclinically evaluated three PDCs with gemcitabine as the anticancer cytotoxic unit and D-Lys6-GnRH (gonadotropin-releasing hormone; GnRH) as the cancer-targeting unit. These units were tethered via acid-labile programmable linkers to guide a differential drug release rate from the PDC through a combination of ester or amide and "click" type oxime ligations. The pro-drugs were designed to enable the selective targeting of malignant tumor cells with linker guided differential drug release rates. We exploited the oxime bond responsiveness against the acidic pH of the tumor microenvironment and the GnRH endocytosis via the GnRH-R GPCR which is overexpressed on cancer cells. The challenging metabolic properties of gemcitabine were addressed during design of the PDCs. We developed a rapid (1 hour) and cost-effective "click" oxime bond ligation platform to assemble in one-pot the 3 desired PDCs that does not require purification, surpassing traditional time-ineffective and low yield methods. The internalization of the tumor-homing peptide unit in cancer cells, overexpressing the GnRH-R, was first validated through confocal laser microscopy and flow cytometry analysis. Subsequently, the three PDCs were evaluated for their in vitro antiproliferative effect in prostate cancer cells. Their stability and the release of gemcitabine over time were monitored in vitro in cell culture and in human plasma using LC-MS/MS. We then assessed the ability of the developed PDCs to internalize in prostate cancer cells and to release gemcitabine. The most potent analog, designated GOXG1, was used for pharmacokinetic studies in mice. The metabolism of GOXG1 was examined in liver microsomes, as well as in buffers mimicking the pH of intracellular organelles, resulting in the identification of two metabolites. The major metabolite at low pH emanated from the cleavage of the pH-labile oxime bond, validating our design approach. NMR spectroscopy and in vitro radioligand binding assays were exploited for GOXG1 to validate that upon conjugating the drug to the peptide, the peptide microenvironment responsible for its GnRH-R binding is not perturbed and to confirm its high binding potency to the GnRH-R. Finally, the binding of GOXG1 to the GnRH-R and the associated elicitation of testosterone release in mice were also determined. The facile platform established herein for the rapid assembly of PDCs with linker controllable characteristics from aldehyde and aminooxy units through rapid "click" oxime ligation, that does not require purification steps, could pave the way for a new generation of potent cancer therapeutics, diagnostics and theranostics.

Pathogenic mitochondrial dysfunction and metabolic abnormalities.

Herein we trace links between biochemical pathways, pathogenesis, and metabolic diseases to set the stage for new therapeutic advances. Cellular and acellular microorganisms including bacteria and viruses are primary pathogenic drivers that cause disease. Missing from this statement are subcellular compartments, importantly mitochondria, which can be pathogenic by themselves, also serving as key metabolic disease intermediaries. The breakdown of food molecules provides chemical energy to power cellular processes, with mitochondria as powerhouses and ATP as the principal energy carrying molecule. Most animal cell ATP is produced by mitochondrial synthase; its central role in metabolism has been known for >80 years. Metabolic disorders involving many organ systems are prevalent in all age groups. Progressive pathogenic mitochondrial dysfunction is a hallmark of genetic mitochondrial diseases, the most common phenotypic expression of inherited metabolic disorders. Confluent genetic, metabolic, and mitochondrial axes surface in diabetes, heart failure, neurodegenerative disease, and even in the ongoing coronavirus pandemic.

Synthesis and SAR of heterocyclic carboxylic acid isosteres based on 2-biarylethylimidazole as bombesin receptor subtype-3 (BRS-3) agonists for the treatment of obesity.

SAR around non-peptidic potent bombesin receptor subtype-3 (BRS-3) agonist lead 2 is presented. Attempts to replace the carboxylic acid with heterocyclic isosteres to improve oral bioavailability and brain penetration are described.