In vitro and in vivo studies on the effect of a mitochondrial fusion promoter on Leydig cell integrity and function.

Background: The interstitial testicular Leydig cells are responsible for the production of testosterone, which functionally deteriorate with normal aging. Decreased expression of mitochondrial steroidogenic interactome proteins and diminished mitochondrial function in aging Leydig cells suggest that mitochondrial dynamics play a role in maintaining adequate levels of testosterone. Optic atrophy 1 (OPA1) protein regulates mitochondrial dynamics and cristae formation in many cell types. Previous studies showed that increasing OPA1 expression in dysfunctional Leydig cells restored mitochondrial function and recovered androgen production to levels found in healthy Leydig cells. These findings suggested that mitochondrial dynamics may be a promising target to ameliorate diminished testosterone levels in aging males. Methods: We used twelve-month-old rats to explore the relationship between mitochondrial dynamics and Leydig cell function. Isolated Leydig cells from aged rats were treated ex vivo with the cell-permeable mitochondrial fusion promoter 4-Chloro-2-(1-(2-(2,4,6-trichlorophenyl)hydrazono)ethyl) phenol (mitochondrial fusion promoter M1), which enhances mitochondrial tubular network formation. In parallel, rats were treated with 2 mg/kg/day M1 for 6 weeks before Leydig cells were isolated. Results: Ex vivo M1-treated cells showed enhanced mitochondrial tubular network formation by transmission electron microscopy, enhanced Leydig cell mitochondrial integrity, improved mitochondrial function, and higher testosterone biosynthesis compared to controls. However, in vivo treatment of aged rats with M1 not only failed to re-establish testosterone levels to that of young rats, it also led to further reduction of testosterone levels and increased apoptosis, suggesting M1 toxicity in the testis. The in vivo M1 toxicity seemed to be tissue-specific, however. Conclusion: Promoting mitochondrial fusion may be one approach to enhancing cell health and wellbeing with aging, but more investigations are warranted. Our findings suggest that fusion promoters could potentially enhance the productivity of aged Leydig cells when carefully regulated.

Eyes on Lipinski's Rule of Five: A New "Rule of Thumb" for Physicochemical Design Space of Ophthalmic Drugs.

The study objective was to investigate molecular thermodynamic properties of approved ophthalmic drugs and derive a framework outlining physicochemical design space for product development. Unlike the methodology used to obtain molecular descriptors for assessment of drug-like properties by Lipinski's Rule of 5 (Ro5), this work presents a retrospective approach based on in silico analysis of molecular thermodynamic properties beyond Ro5 parameters (ie, free energy of distribution/partitioning in octanol/water, dynamic polar surface area, distribution coefficient, and solubility at physiological pH) by using 145 marketed ophthalmic drugs. The study's focus was to delineate inherent molecular parameters explicitly important for ocular permeability and absorption from topical eye drops. A comprehensive parameter distribution analysis on ophthalmic drugs' molecular properties was performed. Frequencies in distribution analyses provided groundwork for physicochemical parameter limits of molecular thermodynamic properties having impact on corneal permeability and topical ophthalmic drug delivery. These parameters included free energy of partitioning (ΔGo/w) calculated based on thermodynamic free energy equation, distribution coefficient at physiological pH (clog DpH7.4), topological polar surface area (TPSA), and aqueous solubility (Sint, SpH7.4) with boundaries of clog DpH7.4 ≤4.0, TPSA ≤250 Å2, ΔGo/w ≤20 kJ/mol (4.8 kcal/mol), and solubility (Sint and SpH7.4) ≥1 µM, respectively. The theoretical free energy of partitioning model streamlined calculation of changes in the free energy of partitioning, Δ(ΔGo/w), as a measure of incremental improvements in corneal permeability for congeneric series. The above parameter limits are proposed as "rules of thumb" for topical ophthalmic drugs to assess risks in developability.

Oral administration of VDAC1-derived small molecule peptides increases circulating testosterone levels in male rats.

Cholesterol is the precursor of all steroid hormones, and the entry of cholesterol into the mitochondria is the rate-limiting step of steroidogenesis. Voltage-dependent anion channel (VDAC1) is an outer mitochondrial protein part of a multiprotein complex that imports cholesterol. We previously reported that intratesticular administration of a 25 amino acid peptide blocking the interaction between 14-3-3ϵ with VDAC1 increased circulating levels of testosterone. This fusion peptide was composed of a HIV-1 transactivator of transcription (TAT) protein transduction domain cell-penetrating peptide, a glycine linker, and amino acids 159-172 of VDAC1 (TV159-172). Here, we describe the development of a family of small molecules that increase circulating testosterone levels after an oral administration. We first characterized an animal model where TV159-172 was delivered subcutaneously. This subcutaneous model allowed us to study the interactions between TV159-172 and the hypothalamus-pituitary-gonadal axis (HPG) and identify the biologically active core of TV159-172. The core consisted of the tetrapeptide RVTQ, which we used as a platform to design synthetic peptide derivatives that can be administered orally. We developed a second animal model to test various derivatives of RVTQ and found 11 active compounds. Dose-response experiments identified 4 synthetic peptides that robustly increased androgen levels in a specific manner. We selected RdVTQ as the leading VDAC1-core derivative and profiled the response across the lifespan of Brown-Norway rats. In summary, we present the development of a new class of therapeutics that act within the HPG axis to increase testosterone levels specifically. This new class of small molecules self-regulates, preventing abuse.

Design and Characterization of a Pyridone-Containing EZH2 Inhibitor Phosphate Prodrug.

A pyridone-derived phosphate prodrug of an enhancer of zeste homolog 2 (EZH2) inhibitor was designed and synthesized to improve the inhibitor's aqueous solubility. This prodrug (compound 5) was profiled in pharmacokinetic experiments to assess its ability to deliver the corresponding parent compound (compound 2) to animals in vivo following oral administration. Results from these studies showed that the prodrug was efficiently converted to its parent compound in vivo. In separate experiments, the prodrug demonstrated impressive in vivo tumor growth inhibition in a diffuse large B-cell lymphoma Karpas-422 cell line-derived xenograft model. The described prodrug strategy is expected to be generally applicable to poorly soluble pyridone-containing EZH2 inhibitors and provides a new option to enable such compounds to achieve sufficiently high exposures in vivo.

Structure-Based Drug Design and Synthesis of PI3Kα-Selective Inhibitor (PF-06843195).

The phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway is a frequently dysregulated pathway in human cancer, and PI3Kα is one of the most frequently mutated kinases in human cancer. A PI3Kα-selective inhibitor may provide the opportunity to spare patients the side effects associated with broader inhibition of the class I PI3K family. Here, we describe our efforts to discover a PI3Kα-selective inhibitor by applying structure-based drug design (SBDD) and computational analysis. A novel series of compounds, exemplified by 2,2-difluoroethyl (3S)-3-{[2'-amino-5-fluoro-2-(morpholin-4-yl)-4,5'-bipyrimidin-6-yl]amino}-3-(hydroxymethyl)pyrrolidine-1-carboxylate (1) (PF-06843195), with high PI3Kα potency and unique PI3K isoform and mTOR selectivity were discovered. We describe here the details of the design and synthesis program that lead to the discovery of 1.

Translational Pharmacokinetic-Pharmacodynamic Modeling for an Orally Available Novel Inhibitor of Epigenetic Regulator Enhancer of Zeste Homolog 2.

PF06821497 has been identified as an orally available small-molecule enhancer of zeste homolog 2 inhibitor. The objectives of the present study were to characterize pharmacokinetic-pharmacodynamic-disease relationships of PF06821497 in xenograft mouse models with diffuse large B-cell lymphoma (Karpas422). An indirect-response model reasonably fit dose-dependent pharmacodynamic responses [histone H3 on lysine 27 (H3K27) me3 inhibition] with an unbound EC 50 of 76 nM, whereas a signal-transduction model sufficiently fit dose-dependent disease responses (tumor growth inhibition) with an unbound tumor stasis concentration (T sc ) of 168 nM. Thus, effective concentration for 70% of maximal effect (EC70) for H3K27me3 inhibition was roughly comparable to T sc , suggesting that 70% H3K27me3 inhibition could be required for tumor stasis. Consistently, an integrated pharmacokinetic-pharmacodynamic-disease model adequately describing tumor growth inhibition also suggested that ∼70% H3K27me3 inhibition was associated with tumor stasis. Based on these results, we would propose that an EC70 estimate for H3K27me3 inhibition corresponding to tumor stasis could be considered a minimum target efficacious concentration of PF06821497 in cancer patients. SIGNIFICANCE STATEMENT: Using a mathematical modeling approach, the quantitative relationships of an orally available anticancer small-molecule enhancer of zeste homolog 2 inhibitor, PF06821497, were characterized among pharmacokinetics, pharmacodynamic biomarker inhibition, and disease responses in nonclinical xenograft models with diffuse large B-cell lymphoma. The modeling results suggest that >70% histone H3 on lysine 27 (H3K27) me3 inhibition would be required for tumor stasis (i.e., 100% tumor growth inhibition). Accordingly, we would propose that an effective concentration for 70% of maximal effect estimate for H3K27me3 inhibition could be considered a minimum target efficacious concentration of PF06821497 in cancer patients.

Ocular biopharmaceutics: impact of modeling and simulation on topical ophthalmic formulation development.

The estimation of ocular pharmacokinetics (PK) in various eye tissues is limited because of sampling challenges. Computational modeling and simulation (M&S) tools underpinning the elucidation of drug access routes and prediction of ocular exposure are essential for the mechanistic assessment of biopharmaceutics in the eye. Therefore, theoretical and experimental evaluation of ocular absorption and transit models is necessary. Biopharmaceutical parameter sensitivity analysis based on permeability and drug dose illustrates utility in ocular drug delivery assessment, which could have innovative and cost-saving impacts on ophthalmic product development and therapeutic bioequivalence (BE) evaluations.

Safety Assessment of Formulation Vehicles Following Intravitreal Administration in Rabbits.

PURPOSE: Evaluate 21 formulation vehicles administered to rabbits after intravitreal injection for tolerability and safety. METHODS: Forty-two Dutch Belted rabbits were anesthetized, and the eyes received a single intravitreal injection of the excipient formulation. Clinical signs and ocular irritation responses were recorded twice daily for 7 days and microscopic evaluation of the eyes, optic nerve, and eyelids was completed at 1-week post treatment. RESULTS: Saline (≥ 300 mOsm and ≤ 592 mOsm at pH 7.0 or 300 mOsm at pH 8.0) and 10 formulation excipients; (10% w/v PEG 3350 at pH 7.4, 1% polysorbate 21 at pH 7.4, PVA at pH 7.0, 0.2% polysorbate 80 at pH 7.2, 0.2% Pluronic F108® at pH 7.3, 2%, 100 mM sodium sulfate at pH 3.2, 2 mM sodium glycocholate at pH 7.4, and 275 mM D-mannitol pH 7.0 in sterile water, and 100 mM sodium phosphate in combination with 0.9% NaCl 300 mOsm and 0.01% or 0.05% polysorbate 80 at pH 7.4) considered as formulation vehicles for intravitreal injectables, were well-tolerated in rabbits. Clinical signs were transient and microscopic changes were not observed. CONCLUSIONS: Of the 21 formulation vehicles evaluated, 10 formulation vehicles were well-tolerated in rabbits and feasible candidates for future investigations.

Optimization of Orally Bioavailable Enhancer of Zeste Homolog 2 (EZH2) Inhibitors Using Ligand and Property-Based Design Strategies: Identification of Development Candidate (R)-5,8-Dichloro-7-(methoxy(oxetan-3-yl)methyl)-2-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3,4-dihydroisoquinolin-1(2H)-one (PF-06821497).

A new series of lactam-derived EZH2 inhibitors was designed via ligand-based and physicochemical-property-based strategies to address metabolic stability and thermodynamic solubility issues associated with previous lead compound 1. The new inhibitors incorporated an sp3 hybridized carbon atom at the 7-position of the lactam moiety present in lead compound 1 as a replacement for a dimethylisoxazole group. This transformation enabled optimization of the physicochemical properties and potency compared to compound 1. Analysis of relationships between calculated log D (clogD) values and in vitro metabolic stability and permeability parameters identified a clogD range that afforded an increased probability of achieving favorable ADME data in a single molecule. Compound 23a exhibited the best overlap of potency and pharmaceutical properties as well as robust tumor growth inhibition in vivo and was therefore advanced as a development candidate (PF-06821497). A crystal structure of 23a in complex with the three-protein PRC2 complex enabled understanding of the key structural features required for optimal binding.

Oligopeptide Transport in Rat Lung Alveolar Epithelial Cells is Mediated by Pept2.

PURPOSE: Studies were conducted in primary cultured rat alveolar epithelial cell monolayers to characterize peptide transporter expression and function. METHODS: Freshly isolated rat lung alveolar epithelial cells were purified and cultured on permeable support with and without keratinocyte growth factor (KGF). Messenger RNA and protein expression of Pept1 and Pept2 in alveolar epithelial type I- and type II-like cell monolayers (±KGF, resp.) were examined by RT-PCR and Western blotting. 3H-Glycyl-sarcosine (3H-gly-sar) transmonolayer flux and intracellular accumulation were evaluated in both cell types. RESULTS: RT-PCR showed expression of Pept2, but not Pept1, mRNA in both cell types. Western blot analysis revealed presence of Pept2 protein in type II-like cells, and less in type I-like cells. Bi-directional transmonolayer 3H-gly-sar flux lacked asymmetry in transport in both types of cells. Uptake of 3H-gly-sar from apical fluid of type II-like cells was 7-fold greater than that from basolateral fluid, while no significant differences were observed from apical vs. basolateral fluid of type I-like cells. CONCLUSIONS: This study confirms the absence of Pept1 from rat lung alveolar epithelium in vitro. Functional Pept2 expression in type II-like cell monolayers suggests its involvement in oligopeptide lung disposition, and offers rationale for therapeutic development of di/tripeptides, peptidomimetics employing pulmonary drug delivery.

Evaluation of miR-216a and miR-217 as Potential Biomarkers of Acute Exocrine Pancreatic Toxicity in Rats.

Detecting and monitoring exocrine pancreatic damage during nonclinical and clinical testing is challenging because classical biomarkers amylase and lipase have limited sensitivity and specificity. Novel biomarkers for drug-induced pancreatic injury are needed to improve safety assessment and reduce late-stage attrition rates. In a series of studies, miR-216a and miR-217 were evaluated as potential biomarkers of acute exocrine pancreatic toxicity in rats. Our results revealed that miR-216a and miR-217 were almost exclusively expressed in rat pancreas and that circulating miR-216a and miR-217 were significantly increased in rats following administration of established exocrine pancreatic toxicants caerulein (CL) and 1-cyano-2-hydroxy-3-butene (CHB) as well as in rats administered a proprietary molecule known to primarily affect the exocrine pancreas. Conversely, neither microRNA was increased in rats administered a proprietary molecule known to cause a lesion at the pancreatic endocrine-exocrine interface (EEI) or in rats administered an established renal toxicant. Compared with amylase and lipase, increases in miR-216a and miR-217 were of greater magnitude, persisted longer, and/or correlated better with microscopic findings within the exocrine pancreas. Our findings demonstrate that in rats, miR-216a and miR-217 are sensitive and specific biomarkers of acute exocrine pancreatic toxicity that may add value to the measurement of classical pancreatic biomarkers.

Design and Synthesis of Pyridone-Containing 3,4-Dihydroisoquinoline-1(2H)-ones as a Novel Class of Enhancer of Zeste Homolog 2 (EZH2) Inhibitors.

A new enhancer of zeste homolog 2 (EZH2) inhibitor series comprising a substituted phenyl ring joined to a dimethylpyridone moiety via an amide linkage has been designed. A preferential amide torsion that improved the binding properties of the compounds was identified for this series via computational analysis. Cyclization of the amide linker resulted in a six-membered lactam analogue, compound 18. This transformation significantly improved the ligand efficiency/potency of the cyclized compound relative to its acyclic analogue. Additional optimization of the lactam-containing EZH2 inhibitors focused on lipophilic efficiency (LipE) improvement, which provided compound 31. Compound 31 displayed improved LipE and on-target potency in both biochemical and cellular readouts relative to compound 18. Inhibitor 31 also displayed robust in vivo antitumor growth activity and dose-dependent de-repression of EZH2 target genes.

Axitinib inhibits retinal and choroidal neovascularization in in vitro and in vivo models.

Age-related Macular Degeneration (AMD) is the leading cause of visual impairment and blindness in the elderly in developed countries. Neovascular/exudative (wet) AMD is the aggressive form of AMD and can involve choroidal neovascularization and vascular leakage. Anti-vascular endothelial growth factor (anti-VEGF) medications have significantly improved treatment of wet-AMD. However, only approximately 40% of patients obtain full benefit from anti-VEGF therapy and the medications are given by intravitreal injection. Axitinib, a small molecule multi-receptor tyrosine kinase inhibitor used for the treatment of advanced renal cell carcinoma, is taken orally and inhibits VEGF activity by blocking VEGF receptors. Axitinib also has the advantage of blocking platelet derived growth factor (PDGF) receptors which play a role in neovascularization. Using in vitro human retinal microvascular endothelial cells (HRMVECs), human brain vascular pericytes (HBVRs), 3D co-culture vessel sprout assay, and in vivo laser induced rat choroidal neovascularization (CNV) models, the effect of axitinib on neovascularization was evaluated. Axitinib inhibited neovascularization better than anti-VEGF and/or anti-hPDGF-B mAb in the in vitro models demonstrating that combined inhibition of both VEGF and PDGF pathways may be synergistic in treating wet-AMD. Additionally, axitinib showed good efficacy at a low dose (0.875 mg/day) in laser-induced CNV model in rats. In conclusion our data shows that axitinib, an inhibitor of VEGF and PDGF-B pathways may be useful in ameliorating wet-AMD therapy.

Design, synthesis, and evaluation of NO-donor containing carbonic anhydrase inhibitors to lower intraocular pressure.

The antiglaucoma drugs dorzolamide (1) and brinzolamide (2) lower intraocular pressure (IOP) by inhibiting the carbonic anhydrase (CA) enzyme to reduce aqueous humor production. The introduction of a nitric oxide (NO) donor into the alkyl side chain of dorzolamide (1) and brinzolamide (2) has led to the discovery of NO-dorzolamide 3a and NO-brinzolamide 4a, which could lower IOP through two mechanisms: CA inhibition to decrease aqueous humor secretion (reduce inflow) and NO release to increase aqueous humor drainage (increase outflow). Compounds 3a and 4a have shown improved efficacy of lowering IOP in both rabbits and monkeys compared to brinzolamide (2).

An assessment of the ocular safety of excipient maleic acid following intravitreal injection in rabbits.

Maleic acid was formulated in 0.7% saline and injected intravitreally in rabbits in order to evaluate ocular safety and tolerability. Maleic acid was formulated within a narrow pH range (2-3), administered in a fixed volume (100 µl), and concentrations ranged from 0.00 to 2.00 mg/eye (0.00 to 12.30 mM vitreous). Ocular evaluations were conducted at 2, 4, and 8 days post injection. Ocular irritation responses were observed at doses from 0.50 mg/eye (3.07 mM vitreous) to 2.00 mg/eye (12.30 mM vitreous) and included conjunctival redness and scleral swelling. Chemosis was observed at 2.00 mg/eye (12.30 mM vitreous). Funduscopic evaluations revealed enlarged retinal blood vessels and optic disk swelling at doses ≥1.50 mg/eye (9.22 mM vitreous), retinal folds and retinal discoloration at 2.00 mg/eye (12.30 mM vitreous). Histopathologic evaluations on days 4 and 8 post injection revealed retinal degeneration at doses ≥1.0 mg/eye (6.15 mM vitreous), conjunctival inflammation at doses ≥1.5 mg/eye (9.22 mM vitreous), and retinal pigment epithelial hypertrophy, optic nerve demyelination, anterior chamber fluid, and conjunctival fibrosis at 2.00 mg/eye (12.30 mM vitreous) maleic acid. The data suggest that maleic acid formulations at ≥1.00 mg/eye (6.15 mM vitreous) were not suitable for intraocular indications.

Pharmacokinetic study of vitreous and serum concentrations of triamcinolone acetonide after posterior sub-tenon's injection.

PURPOSE: To compare a theoretical pharmacokinetic model of triamcinolone acetonide after posterior sub-Tenon's injection with experimental serum and undiluted vitreous triamcinolone acetonide concentrations obtained during pars plana vitrectomy. DESIGN: Clinical-practice, prospective, interventional case series study. METHODS: This study compared computer-modeled triamcinolone acetonide diffusion after posterior sub-Tenon's injection with triamcinolone acetonide levels in experimental undiluted vitreous and serum samples from 57 patients undergoing vitrectomy assessed via mass spectrometry and high-pressure liquid chromatography. At least 5 pairs of samples were collected at each of 7 time points (1 day, 3 days, and 1, 2, 3, 4, and 8 weeks) after triamcinolone acetonide injection, with 6 controls without injection. Cortisol levels were measured in 31 sets of samples. RESULTS: The theoretical model predicted that triamcinolone acetonide levels in systemic blood, vitreous, and choroidal extracellular matrix would plateau after 3 days at 15 ng/mL, 227 ng/mL and 2230 ng/mL, respectively. Experimental vitreous levels of triamcinolone peaked at 111 ng/mL at day 1, then reached a plateau in the range 15 to 25 ng/mL, while serum triamcinolone levels peaked at day 3 near 35 ng/mL and plateaued near 2 to 8 ng/mL. Serum triamcinolone and cortisol levels were inversely correlated (Spearman -0.42, P = .02). CONCLUSIONS: The theoretical model predicts efficient delivery of triamcinolone acetonide from the posterior sub-Tenon's space to the extracellular choroidal matrix. The experimental findings demonstrate low levels of serum triamcinolone that alter systemic cortisol levels and higher vitreous levels lasting at least 1 month. Both assessments support trans-scleral delivery of posterior sub-Tenon's triamcinolone.

Solution formulation development of a VEGF inhibitor for intravitreal injection.

PF-00337210 is a potent, selective small molecule inhibitor of VEGFRs and has been under consideration for the treatment of age-related macular degeneration. An ophthalmic solution formulation intended for intravitreal injection was developed. This formulation was designed to maximize drug properties such that the formulation would precipitate upon injection into the vitreous for sustained delivery. As a parenteral formulation with additional constraints dictated by this specialized delivery route, multiple features were balanced in order to develop a successful formulation. Some of these considerations included low dosing volumes (≤0.1 mL), a limited repertoire of safe excipients for intravitreal injection, and the unique physical chemical properties of the drug. The aqueous solubility as a function of pH was characterized, buffer stressing studies to select the minimal amount of buffer were conducted, and both chemical and physical stability studies were executed. The selected formulation consisted of an isotonic solution comprised of PF-00337210 free base in a citrate-buffered vehicle containing NaCl for tonicity. The highest strength for regulatory toxicology studies was 60 mg/mL. The selected formulation exhibited sufficient chemical stability upon storage with no precipitation, and acceptable potency and recovery through an intravitreal dosing syringe. Formulation performance was simulated by precipitation experiments using extracted vitreous humor. In simulated injection experiments, PF-00337210 solutions reproducibly precipitated upon introduction to the vitreous so that a depot was formed. To our knowledge, this is the first time that a nonpolymeric in situ-forming depot formulation has been developed for intravitreal delivery, with the active ingredient as the precipitating agent.