Paolo Lazzari

Development of Novel ADCs: Conjugation of Tubulysin Analogues to Trastuzumab Monitored by Dual Radiolabeling

Tubulysins are highly toxic tubulin-targeting agents with a narrow therapeutic window that are interesting for application in antibody-drug conjugates (ADC). For full control over drug-antibody ratio (DAR) and the effect thereof on pharmacokinetics and tumor targeting, a dual-labeling approach was developed, wherein the drug, tubulysin variants, and the antibody, the anti-HER2 monoclonal antibody (mAb) trastuzumab, are radiolabeled. (131)I-radioiodination of two synthetic tubulysin A analogues, the less potent TUB-OH (IC50 > 100 nmol/L) and the potent TUB-OMOM (IC50, ~1 nmol/L), and their direct covalent conjugation to (89)Zr-trastuzumab were established. Radioiodination of tubulysins was 92% to 98% efficient and conversion to N-hydroxysuccinimide (NHS) esters more than 99%; esters were isolated in an overall yield of 68% ± 5% with radiochemical purity of more than 99.5%. Conjugation of (131)I-tubulysin-NHS esters to (89)Zr-trastuzumab was 45% to 55% efficient, resulting in ADCs with 96% to 98% radiochemical purity after size-exclusion chromatography. ADCs were evaluated for their tumor-targeting potential and antitumor effects in nude mice with tumors that were sensitive or resistant to trastuzumab, using ado-trastuzumab emtansine as a reference. ADCs appeared stable in vivo. An average DAR of 2 and 4 conferred pharmacokinetics and tumor-targeting behavior similar to parental trastuzumab. Efficacy studies using single-dose TUB-OMOM-trastuzumab (DAR 4) showed dose-dependent antitumor effects, including complete tumor eradications in trastuzumab-sensitive tumors in vivo. TUB-OMOM-trastuzumab (60 mg/kg) displayed efficacy similar to ado-trastuzumab emtansine (15 mg/kg) yet more effective than trastuzumab. Our findings illustrate the potential of synthetic tubulysins in ADCs for cancer treatment.

Evaluation of selective cannabinoid CB1 and CB2 receptor agonists in a mouse model of lipopolysaccharide-induced interstitial cystitis

Interstitial cystitis is a debilitating bladder inflammation disorder. To date, the understanding of the causes of interstitial cystitis remains largely fragmentary and there is no effective treatment available. Recent experimental results have shown a functional role of the endocannabinoid system in urinary bladder. In this study, we evaluated the anti-inflammatory effect of selective cannabinoid CB1 and CB2 receptor agonists in a mouse model of interstitial cystitis. Bladder inflammation was induced in mice by lipopolysaccharide (LPS) and whole bladders were removed 24h later. LPS induced a significant increase of the contractile amplitude in spontaneous activity and a hypersensitivity to exogenous acetylcholine-induced contraction of whole-isolated bladder. Next, we evaluated the anti-inflammatory activity of cannabinoidergic compounds by pretreating mice with CB1 or CB2 selective agonist compounds, respectively ACEA and JWH015. Interestingly, JWH015, but not ACEA, antagonized LPS-induced bladder inflammation. Additionally, anti-inflammatory activity was studied by evaluation, leukocytes mucosa infiltration, myeloperoxidase activity, and mRNA expression of pro-inflammatory interleukin (IL-1α and IL-1β), tumor necrosis factor-alpha (TNF-α) and cannabinoid CB1 and CB2 receptors. JWH015 significantly decreased leukocytes infiltration in both submucosa and mucosa, as well as the myeloperoxydase activity, in LPS treated mice. JWH015 reduced mRNA expression of IL-1α, IL-1β, and TNF-α. LPS treatment increased expression of bladder CB2 but not CB1 mRNA. Taken together, these findings strongly suggest that modulation of the cannabinoid CB2 receptors might be a promising therapeutic strategy for the treatment of bladder diseases and conditions characterized by inflammation, such as interstitial cystitis.

Novel pyrazole derivatives as neutral CB1 antagonists with significant activity towards food intake

In spite of rimonabants withdrawal from the European market due to its adverse effects, interest in the development of drugs based on CB1 antagonists is revamping on the basis of the peculiar properties of this class of compounds. In particular, new strategies have been proposed for the treatment of obesity and/or related risk factors through CB1 antagonists, i.e. by the development of selectively peripherally acting agents or by the identification of neutral CB1 antagonists. New compounds based on the lead CB1 antagonist/inverse agonist rimonabant have been synthesized with focus on obtaining neutral CB1 antagonists. Amongst the new derivatives described in this paper, the mixture of the two enantiomers (±)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-(2-cyclohexyl-1-hydroxyethyl)-4-methyl-1H-pyrazole ((±)-5), and compound 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-[(Z)-2-cyclohexyl-1-fluorovinyl]-4-methyl-1H-pyrazole ((Z)-6), showed interesting pharmacological profiles. According to the preliminary pharmacological evaluation, these novel pyrazole derivatives showed in fact both neutral CB1 antagonism behaviour and significant in vivo activity towards food intake.

NESS06SM reduces body weight with an improved profile relative to SR141716A

We have recently synthesized a new series of 4,5-dihydrobenzo-oxa-cycloheptapyrazole derivatives with the aim to discover novel CB1 antagonist agents characterized by anti-obesity activity comparable to that of SR141716A but with reduced adverse effects such as anxiety and depression. Within the novel class, the CB1 antagonist 8-chloro-1-(2,4-dichlorophenyl)-N-piperidin-1-yl-4,5-dihydrobenzo-1H-6-oxa-cyclohepta(1,2-c)pyrazole-3-carboxamide (NESS06SM) has been selected as lead compound. We found that NESS06SM is a CB1 neutral antagonist, characterized by poor blood-brain barrier permeability. Moreover, NESS06SM chronic treatment determined both anti-obesity effect and cardiovascular risk factor improvement in C57BL/6N Diet Induced Obesity (DIO) mice fed with fat diet (FD mice). In fact, the mRNA gene expression in Central Nervous System (CNS) and peripheral tissues by real time PCR, showed a significant increase of orexigenic peptides and a decrease of anorexigenic peptides elicited by NESS06SM treatment, compared to control mice fed with the same diet. Moreover, in contrast to SR141716A treatment, the chronic administration of NESS06SM did not change mRNA expression of both monoaminergic transporters and neurotrophins highly related with anxiety and mood disorders. Our results suggest that NESS06SM reduces body weight and it can restore the disrupted expression profile of genes linked to the hunger-satiety circuit without altering monoaminergic transmission probably avoiding SR141716A side effects. Therefore the novel CB1 neutral antagonist could represent a useful candidate agent for the treatment of obesity and its metabolic complications.

Weight loss induced by rimonabant is associated with an altered leptin expression and hypothalamic leptin signaling in diet-induced obese mice.

This study investigates the molecular mechanisms and the center-periphery cross talk underlying the anti-obesity effect of the cannabinoid receptor 1 (CB(1)) antagonist/inverse agonist rimonabant in diet-induced obese (DIO) mice exposed to a 31 days chronic treatment with the drug. Present data showed a significant and stable weight loss both in animals treated with rimonabant 10mg/kg by oral gavage exposed to a high fat diet (SRFD) and in vehicle treated mice switched to a regular chow (VEND) with respect to vehicle fat diet fed mice (VEFD). Caloric intake was significantly lowered in SRFD and VEND during the first two and four days, respectively, then reaching the VEFD consume throughout the treatment. The drop of body weight was accompanied by leptin mRNA decrease in visceral fat tissue both in VEND and SRFD, as revealed by Real time PCR analysis. No difference in CB(1) mRNA receptor expression in hypothalamus and in visceral fat tissue among groups was observed. Leptin receptors were decreased in the hypothalamus of SRFD but not of VEND mice. Moreover, in SRFD and VEND mice the expression of orexigenic genes Neuropeptide Y and Agouti Related Protein (AGRP) was increased, while anorexigenic ones, Pro-OpioMelanoCortin (POMC) and Cocaine-and-Amphetamine-Regulated Transcript (CART) displayed no alteration in any group. This data contribute to clarify the molecular basis of the anti-obesity properties of rimonabant, underlying the role of the peripheral modulators which affect central circuits involved in the regulation of food intake and energy homeostasis.

Tricyclic pyrazoles. Part 6. Benzofuro[3,2-c]pyrazole: a versatile architecture for CB2 selective ligands.

A new series of 1H-benzofuro[3,2-c]pyrazole-3-carboxamides was synthesized. The novel compounds (15-24) were evaluated for their affinity to CB2 and CB1 cannabinoid receptors. The synthesis of the title compounds takes advantage of the acid-catalysed thermal cyclization of bicyclic hydrazone ethyl 2-(2-(2,4-dichlorophenyl)hydrazono)-2-(6-methyl-3-oxo-2,3-dihydrobenzofuran-2-yl)acetate to tricyclic ethyl 1-(2,4-dichlorophenyl)-6-methyl-1H-benzofuro[3,2-c]pyrazol-3-carboxylate. All the obtained derivatives showed high affinity to CB2 receptors. Moreover, significant selectivity for CB2 over CB1 receptors was highlighted for lead derivatives amongst the novel series. The best binding profiles were determined for homologues bearing monocyclic and bicyclic monoterpenic substituents at the carbamoyl group at 3 position of the pyrazole ring (KiCB2 < 4 nM). In particular, the isopinocampheyl-substituted derivative 22 exhibited the highest selectivity for CB2 receptors with Ki values of 3.7 and 2398 nM for CB2 and CB1 receptors, respectively. Preliminary functional assays evidenced CB2 agonism behaviour for all the assayed novel derivatives.

Efficient cell transfection with melamine-based gemini surfactants.

Gemini surfactants consisting of two melamine scaffolds connected by a n-hexyl linker and functionalized with a 1-propylammonium polar head and a lipophilic chain having variable carbon length (from C8 to C16) were synthesized. These were then used successfully for the transfection of A549, U87 MG, and Bristol 8 cell lines with maxGFP expressing plasmid. The transfection protocol was optimized appropriately (confluence, reagent/pcDNA ratio, compaction time, and transfection time) for each cell line. Under optimized conditions, the C12 and C14 melamine gemini surfactants showed little toxicity and remarkable transfection efficiency, superior to the gold-standard Lipofectamine 2000. These reagents were also able to efficiently transfect primary DRG neurons, which are notoriously difficult to transfect. The presence of serum completely inhibited the transfection capacity of these reagents. Owing to their ready availability, straightforward synthesis, high chemical stability (even in solution), ease of use (no formulation is required), improved transfection ability, and low toxicity, melamine-based gemini surfactants are very promising reagents for cellular DNA transfection.

Tricyclic Pyrazoles. Part 5. Novel 1,4-Dihydroindeno[1,2-c]pyrazole CB2 Ligands Using Molecular Hybridization Based on Scaffold Hopping.

In search of new selective CB2 ligands, the synthesis and preliminary biological evaluation of novel 1,4-dihydroindeno[1,2-c]pyrazole hybrids of the highly potent prototypicals 5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-N-fenchyl-1H-pyrazole-3-carboxamide 1 and 1-(2,4-dichlorophenyl)-6-methyl-N-(piperidin-1-yl)-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide 2 are detailed. We postulated that the introduction of those pharmacophoric elements essential for activity of 1 in the tricyclic core of 2 might provide CB2 ligands with further improved receptor selectivity and biological activity. Among the compounds, 6-chloro-7-methyl-1-(2,4-dichlorophenyl)-N-fenchyl-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide (22) exhibited low two digit nanomolar affinity for the cannabinoid CB2R and maintained a high level of CB2-selectivity.

The tubulysin analogue KEMTUB10 induces apoptosis in breast cancer cells via p53, Bim and Bcl-2

PurposeTubulysins are natural tetrapeptides that inhibit tubulin polymerisation. Tubulysins are very potent inhibitors of mammalian cancer cell growth, but restricted availability has limited their characterisation and development as anti-cancer compounds. KEMTUB10 was recently developed as a synthetic analogue of natural tubulysins.MethodsThe cell cytotoxicity of KEMTUB10 was studied in cell lines that represent the main breast cancer sub-types. The KEMTUB10 pro-apoptotic mechanism of action was studied in MCF7 and MDAMB231 cells.ResultsKEMTUB10 exerts a potent cytotoxic effect in cells representing the main breast cancer sub-types. KEMTUB10 blocks cells in the G2/M phase of the cell cycle and is a strong stimulator of apoptosis/cell death. KEMTUB10-induced apoptosis involves p53 and Bim, and to some extent Bcl-2 phosphorylation.ConclusionsKEMTUB10 is a promising new anti-mitotic that exerts a potent cytotoxic effect in breast cancer cells, blocks cells in the G2/M phase of the cell cycle and stimulates apoptosis/cell death. KEMTUB10 has a distinct mode of action to taxol, appears to be sensitive to different molecular factors in cells and is likely subject to different mechanisms of acquired resistance. KEMTUB10 has the potential to be an important addition to the anti-cancer therapeutic armoury.

Analysis of CYP2D6 Allele Frequencies and Identification of Novel SNPs and Sequence Variations in Sardinians

The CYP2D6 enzyme is involved in the metabolism of many commonly prescribed drugs. The presence of polymorphisms in the CYP2D6 gene may modulate the enzyme level and activity affecting individual responses, to pharmacological treatment in drug level, response and adverse reactions. Aims. This study aimed to analyze the determination of allele frequencies in Sardinians and the comparison to frequencies found in the Caucasian Population. Methods and Materials. We used a Long PCR strategy coupled to direct genomic DNA sequencing analysis. An amplification allele-specific was carried out to infer the correct allelic phase. The TaqMan Gene Copy Number Assay (Applied Biosystems) was used to verify the presence of gene deletions/multiplications. Results and Conclusions. Our results indicated that CYP2D6 allele frequencies in Sardinians differed from those previously detected in the Caucasian Population. Moreover, three new SNPs and four novel haplotypes were identified.