Plasma Stability Assays Research Articles

Development of plasma stability assay for oligonucleotides

Development of plasma stability assay for oligonucleotides

4-Phenyl-thiazole-based dual inhibitors of fatty acid amide hydrolase and soluble epoxide hydrolase do not alleviate orofacial inflammatory pain in female rats

Pain arising from trigeminal systems such as headache is common, debilitating, and current treatments (e.g., sumatriptan) are limited. New treatments that target novel mechanisms of action may be required to innovate both short- and long-term pain therapy. Fatty acid amide hydrolase and soluble epoxide hydrolase are two pain-related enzymes that regulate pain and inflammation via independent pathways. We have previously demonstrated that simultaneous inhibition of these enzymes using a novel dual inhibitor alleviates acute inflammatory pain in the hindpaw and does not depress wheel running in rats. Here, we expanded on these findings and performed structure-activity relationships of our lead compound, the 4-phenyl-thiazole-based dual inhibitor SW-17, to generate 18 analogs and tested them for their inhibition at both enzymes. Conversion of the sulfonamide group to a tertiary amine led to a general decrease in the potency for the sEH enzyme, while this change was well-tolerated at the FAAH enzyme yielding several strong inhibitors. Six selected inhibitors were evaluated in mouse and rat sEH inhibition assays and results showed a species difference, i.e. 4-phenyl-thiazole-based analogs are significantly less or not active in mouse sEH compared to human and rat enzymes. The most potent inhibitor, SW-17, was evaluated in a plasma stability assay in human and rat plasma and showed moderate stability. However, SW-17 did not alleviate orofacial inflammatory pain in female rats compared to the traditional anti-migraine agent sumatriptan. Although modification of 4-phenyl-thiazole-based dual inhibitor SW-17 changes potencies at both FAAH and sEH, these approaches may not produce antinociception against trigeminal pain. Key Words: polypharmacology, formalin, inflammation, enzyme inhibition, structure-activity relationship studies

Abstract 2702: GQ1001 is a next generation HER2-targeting ADC with excellent druggability, safety and potency

Abstract Background: GQ1001 was developed to overcome the current limitation of existing HER2 ADCs by using the next generation site-specific conjugation and a stable linker technology, coined the intelligent ligase-dependent conjugation (iLDC) technology. iLDC provides a robust platform for the generation of ADC with high homogeneity and excellent stability. Our data show that GQ1001 is highly homogeneous with strong anti-tumor potency, more importantly, markedly enhanced linker stability and reduced off-target toxicity, and points to a safer HER2 ADC with a larger therapeutic window in human. Methods: In vitro and in vivo anticancer efficacy and the mechanism of action (MOA) of GQ1001 were assessed using several HER2+ cancer cell lines and animal models. Ex vivo linker stability was assessed by incubating GQ1001 with the plasma from different species. Pharmacokinetics in cynomolgus monkeys and safety profiles in rats and monkeys were evaluated. Results: GQ1001 was generated by conjugating trastuzumab to DM1 via an unique open-ring containing linker and the enzyme-based site-specific conjugation technology that significantly increases the stability of GQ1001. Optimal physiochemical property of GQ1001was demonstrated from the long-term stability study in liquid formulation. GQ1001 demonstrated similar in vitro anti-cancer activities to T-DM1 in HER2+ tumor cells. However, T-DM1 showed significant non-specific cytotoxicity in HER2- cells while GQ1001 didn’t. In animal studies, GQ1001 induced a robust dose-dependent tumor growth inhibition in multiple HER2-positive CDX and PDX models. From the combo treatment studies, GQ1001 showed synergistic anti-tumor response when combined with HER2-targeting tyrosine kinase inhibitors TKIs and chemotherapeutic agents in multiple HER2-positive models, including those resistant to anti-HER2 TKIs and/or mAbs. In ex vivo plasma stability assay, the DM1-shedding ratio of GQ1001 in human plasma was ~1/100th of that of T-DM1. This supreme linker stability was confirmed by the favorable pharmacokinetics in monkey with extremely low free DM1 exposure in circulation at a significantly lower level than T-DM1. The highest non-severely toxic dose HNSTD of GQ1001 in monkey was 45 mg/kg without any signs of peripheral neuropathy and interstitial lung disease, suggesting GQ1001 will demonstrate excellent tolerance in human. Consistent with this preclinical data, GQ1001 demonstrated superior tolerability profile in heavily pretreated HER2-positive advanced solid tumors patients in a global, open labeled multicenter phase Ia trial. Conclusions: GQ1001 is a unique HER2-targeting ADC that exhibits markedly-enhanced linker stability and safety profiles. Judging from preclinical data, GQ1001 demonstrates the potential to treat HER2-positive cancer patients, alone or in combination, who have progressed on previous HER2-targeting therapeutics with reduced toxicity. Citation Format: Lei Huang, Lili Shi, Yajun Sun, Cao Lv, Chengcheng Gong, Chong Liu, Jun Yang, Lu Jiang, Jinduo Yuan, Xuesong Li, Mingyu Hu, Xinju Gao, Yu Si, Hui Yang, Yan Shi, Bo Yang, Paul H. Song, Gang Qin, Biyun Wang. GQ1001 is a next generation HER2-targeting ADC with excellent druggability, safety and potency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2702.

Encapsulation of Temozolomide in a Calixarene Nanocapsule Improves Its Stability and Enhances Its Therapeutic Efficacy against Glioblastoma.

The alkylating agent temozolomide (TMZ) is the first-line chemotherapeutic for glioblastoma (GBM), a common and aggressive primary brain tumor in adults. However, its poor stability and unfavorable pharmacokinetic profile limit its clinical efficacy. There is an unmet need to tailor the therapeutic window of TMZ, either through complex derivatization or by utilizing pharmaceutical excipients. To enhance stability and aqueous solubility, we encapsulated TMZ in a p-sulphonatocalix[4]arene (Calix) nanocapsule and used 1H-NMR, LC-MS, and UV-Vis spectroscopy to chart the stability of this novel TMZ@Calix complex according to FDA and European Medicines Agency guidelines. LC-MS/MS plasma stability assays were conducted in mice to further explore the stability profile of TMZ@Calix in vivo The therapeutic efficacy of TMZ@Calix was compared with that of unbound TMZ in GBM cell lines and patient-derived primary cells with known O6-methylguanine-DNA methyltransferase (MGMT) expression status and in vivo in an intracranial U87 xenograft mouse model. Encapsulation significantly enhanced the stability of TMZ in all conditions tested. TMZ@Calix was more potent than native TMZ at inhibiting the growth of established GBM cell lines and patient-derived primary lines expressing MGMT and highly resistant to TMZ. In vivo, native TMZ was rapidly degraded in mouse plasma, whereas the stability of TMZ@Calix was enhanced threefold with increased therapeutic efficacy in an orthotopic model. In the absence of new effective therapies, this novel formulation is of clinical importance, serving as an inexpensive and highly efficient treatment that could be made readily available to patients with GBM and warrants further preclinical and clinical evaluation.

Tailoring acyclovir prodrugs with enhanced antiviral activity: rational design, synthesis, human plasma stability and in vitro evaluation.

Bile acid prodrugs have served as a viable strategy for refining the pharmaceutical profile of parent drugs through utilizing bile acid transporters. A series of three ester prodrugs of the antiherpetic drug acyclovir (ACV) with the bile acids cholic, chenodeoxycholic and deoxycholic were synthesized and evaluated along with valacyclovir for their in vitro antiviral activity against herpes simplex viruses type 1 and type 2 (HSV-1, HSV-2). The in vitro antiviral activity of the three bile acid prodrugs was also evaluated against Epstein-Barr virus (EBV). Plasma stability assays, utilizing ultra-high performance liquid chromatography coupled with tandem mass spectrometry, in vitro cytotoxicity and inhibitory experiments were conducted in order to establish the biological profile of ACV prodrugs. The antiviral assays demonstrated that ACV-cholate had slightly better antiviral activity than ACV against HSV-1, while it presented an eight-fold higher activity with respect to ACV against HSV-2. ACV-chenodeoxycholate presented a six-fold higher antiviral activity against HSV-2 with respect to ACV. Concerning EBV, the highest antiviral effect was demonstrated by ACV-chenodeoxycholate. Human plasma stability assays revealed that ACV-deoxycholate was more stable than the other two prodrugs. These results suggest that decorating the core structure of ACV with bile acids could deliver prodrugs with amplified antiviral activity.

High-Throughput, Multispecies, Parallelized Plasma Stability Assay for the Determination and Characterization of Antibody-Drug Conjugate Aggregation and Drug Release.

The stability of antibody–drug conjugates (ADCs) in circulation is critical for maximum efficacy and minimal toxicity. An ADC reaching the intended target intact can deliver the highest possible drug load to the tumor and reduce off-target toxicity from free drug in the blood. As such, assessment of ADC stability is a vital piece of data during development. However, traditional ADC stability assays can be manually intensive, low-throughput, and require large quantities of ADC material. Here, we introduce an automated, high-throughput plasma stability assay for screening drug release and aggregation over 144 h for up to 40 ADCs across five matrices simultaneously. The amount of ADC material during early drug development is often limited, so this assay was implemented in 384-well format to minimize material requirements to <100 μg of each ADC and 100 μL of plasma per species type. Drug release and aggregation output were modeled using nonlinear regression equations to calculate formation rates for each data type. A set of 15 ADCs with different antibodies and identical valine–citrulline–p-aminobenzylcarbamate–monomethylauristatin E linker-drug payloads was tested and formation rates were compared across ADCs and between species, revealing several noteworthy trends. In particular, a wide range in aggregation was found when altering only the antibody, suggesting a key role for plasma stability screening early in the development process to find and remove antibody candidates with the potential to create unstable ADCs. The assay presented here can be leveraged to provide stability data on new chemistry and antibody screening initiatives, select the best candidate for in vivo studies, and provide results that highlight stability issues inherent to particular ADC designs throughout all stages of ADC development.

Differential stability of therapeutic peptides with different proteolytic cleavage sites in blood, plasma and serum.

Proteolytic degradation of peptide-based drugs is often considered as major weakness limiting systemic therapeutic applications. Therefore, huge efforts are typically devoted to stabilize sequences against proteases present in serum or plasma, obtained as supernatants after complete blood coagulation or centrifugation of blood supplemented with anticoagulants, respectively. Plasma and serum are reproducibly obtained from animals and humans allowing consistent for clinical analyses and research applications. However, the spectrum of active or activated proteases appears to vary depending on the activation of proteases and cofactors during coagulation (serum) or inhibition of such enzymes by anticoagulants (plasma), such as EDTA (metallo- and Ca2+-dependent proteases) and heparin (e.g. thrombin, factor Xa). Here, we studied the presumed effects on peptide degradation by taking blood via cardiac puncture of CD-1 mice using a syringe containing a peptide solution. Due to absence of coagulation activators (e.g. glass surfaces and damaged cells), visible blood clotting was prevented allowing to study peptide degradation for one hour. The remaining peptide was quantified and the degradation products were identified using mass spectrometry. When the degradation rates (half-life times) were compared to serum derived freshly from the same animal and commercial serum and plasma samples, peptides of three different families showed indeed considerably different stabilities. Generally, peptides were faster degraded in serum than in plasma, but surprisingly all peptides were more stable in fresh blood and the order of degradation rates among the peptides varied among the six different incubation experiments. This indicates, that proteolytic degradation of peptide-based therapeutics may often be misleading stimulating efforts to stabilize peptides at degradation sites relevant only in vitro, i.e., for serum or plasma stability assays, but of lower importance in vivo.

New Oral Treatments for Asthma through Tissue-Specific Modulation of the GABAA Receptor

This study addresses the unmet need for an oral, safe, non-steroidal asthma treatment. The hypothesis is that GABAARs in airway smooth muscle (ASM) and inflammatory cells can be targeted by subtype-selective GABAAR modulators to tissue-selectively induce ASM relaxation and immunosuppression. New drug candidates were characterized by electrophysiology and microsome, S9, and blood plasma stability assays. Pharmacokinetic studies in mice are used to identify in vivo stability and distribution. Murine pharmacodynamic models are used to quantify sensorimotor effects (rotarod), disease specific airway hyperresponsiveness, airway mucus production, and airway eosinophilia. ASM muscle relaxation was tested in ex vivo lung tissue. The immune modulatory effect of subtype-selective GABAAR ligands was evaluated by flow cytometry. A α5 subtype-selective GABAAR ligand showed excellent PK when given orally. The compound did not cross the BBB, thus no sensorimotor effects were observed. The compound inhibited eosinophilia and reduced the number of inflammatory cells. Significant muscle relaxation was observed ex vivo in trachea rings and reduced airway hyperresponsiveness was observed in mice. Importantly, this is the first oral treatment for asthma specifically targeting muscle relaxation and inflammation. In addition, a novel α4 subtype-selective GABAAR ligand was identified with equally excellent stability in vitro and in vivo. Reduced airway hyperresponsiveness was observed at low concentrations of methacholine. The anti-inflammatory properties were significant but immune cell populations were regulated differently. α4 and α5-selective GABAAR modulators have a great potential as novel orally active drug candidates for asthma to alleviate symptoms of airway hyperresponsiveness mediated by ASM constriction, hypereosinophilia, and inflammation.

Design, synthesis and biological evaluation of peptide dendrimers with wound healing promoting activity

Peptide dendrimers are tree-like macromolecules composed of a peptidyl branching core and/or covalently attached surface functional units. Owing to their multimeric nature and unambiguous composition, peptide dendrimers show several significant advantages over single peptides, such as amplification of function, biocompatibility and water-solubility. Thus, dendrimer-based strategies are widely used for designing peptide agents. Using this strategy, we have designed and synthesized a series of wound healing-promoting peptide dendrimers and their efficacy was evaluated on wounded diabetic mice. The results showed that all dendrimers have wound healing promoting effects, while tetramers showed superior activity to others. Treatment with the dendrimers of AK14, AK21, and AK22 resulted in a smaller wound area percentage than any others on the 7th, 9th, and 11th days. Moreover, hemolysis assay suggested all dendrimers except AK22, AK23, and AK31 have little hematotoxicity, even at 512 μg/mL. In vitro plasma stability assay also showed an increased stability of AK21 when comparing with its parent compound. Therefore, we suggest the dendrimer-based strategy has potential for designing wound healing-promoting peptides.

Coencapsulation of alendronate and doxorubicin in pegylated liposomes: a novel formulation for chemoimmunotherapy of cancer

We developed a pegylated liposome formulation of a dissociable salt of a nitrogen-containing bisphosphonate, alendronate (Ald), coencapsulated with the anthracycline, doxorubicin (Dox), a commonly used chemotherapeutic agent. Liposome-encapsulated ammonium Ald generates a gradient driving Dox into liposomes, forming a salt that holds both drugs in the liposome water phase. The resulting formulation (PLAD) allows for a high-loading efficiency of Dox, comparable to that of clinically approved pegylated liposomal doxorubicin sulfate (PLD) and is very stable in plasma stability assays. Cytotoxicity tests indicate greater potency for PLAD compared to PLD. This appears to be related to a synergistic effect of the coencapsulated Ald and Dox. PLAD and PLD differed in in vitro monocyte-induced IL-1β release (greater for PLAD) and complement activation (greater for PLD). A molar ratio Ald/Dox of ∼1:1 seems to provide an optimal compromise between loading efficiency of Dox, circulation time and in vivo toxicity of PLAD. In mice, the circulation half-life and tumor uptake of PLAD were comparable to PLD. In the M109R and 4T1 tumor models in immunocompetent mice, PLAD was superior to PLD in the growth inhibition of subcutaneous tumor implants. This new formulation appears to be a promising tool to exploit the antitumor effects of aminobisphosphonates in synergy with chemotherapy.

Where Did the Linker-Payload Go? A Quantitative Investigation on the Destination of the Released Linker-Payload from an Antibody-Drug Conjugate with a Maleimide Linker in Plasma.

The reactive thiol of cysteine is often used for coupling maleimide-containing linker-payloads to antibodies resulting in the generation of antibody drug conjugates (ADCs). Currently, a numbers of ADCs in drug development are made by coupling a linker-payload to native or engineered cysteine residues on the antibody. An ADC conjugated via hinge-cysteines to an auristatin payload was used as a model in this study to understand the impact of the maleimide linkers on ADC stability. The payload was conjugated to trastuzumab by a protease-cleavable linker, maleimido-caproyl-valine-citruline-p-amino-benzyloxy carbonyl (mcVC-PABC). In plasma stability assays, when the ADC (Trastuzumab-mcVC-PABC-Auristatin-0101) was incubated with plasma over a 144-h time-course, a discrepancy was observed between the measured released free payload concentration and the measured loss of drug-to-antibody ratio (DAR), as measured by liquid chromatography-mass spectrometry (LC-MS). We found that an enzymatic release of payload from ADC-depleted human plasma at 144 h was able to account for almost 100% of the DAR loss. Intact protein mass analysis showed that at the 144 h time point, the mass of the major protein in ADC-depleted human plasma had an additional 1347 Da over the native albumin extracted from human plasma, exactly matching the mass of the linker-payload. In addition, protein gel electrophoresis showed that there was only one enriched protein in the 144 h ADC-depleted and antipayload immunoprecipitated plasma sample, as compared to the 0 h plasma immunoprecipitated sample, and the mass of this enriched protein was slightly heavier than the mass of serum albumin. Furthermore, the albumin adduct was also identified in 96 h and 168 h postdose in vivo cynomolgus monkey plasma. These results strongly suggest that the majority of the deconjugated mc-VC-PABC-auristatin ultimately is transferred to serum albumin, forming a long-lived albumin-linker-payload adduct. To our knowledge, this is the first report quantitatively characterizing the extent of linker-payload transfer to serum albumin and the first clear example of in vivo formation of an albumin-linker-payload adduct.

A new18F-heteroaryltrifluoroborate radio-prosthetic with greatly enhanced stability that is labelled by18F–19F-isotope exchange in good yield at high specific activity

An 18F-heteroaryltrifluoroborate was designed as a novel radioprosthetic for PET imaging. The stability of the new prosthetic was confirmed using 19F-NMR spectroscopy: the solvolytic half-life was measured to be >16 000 min. Conjugation to RGD gave a stable radiotracer precursor for kit-like labeling by 18F–19F isotope exchange in 15 min. The total radiosynthetic procedure including HPLC purification requires <45 min. A plasma stability assay showed negligible defluoridation following 150 min and stability is corroborated by in vivo imaging showing minimal bone uptake. With Curie levels of 18F–fluoride, yields of 20% and specific activities as high as 3 Ci μmol−1 (110 GBq μmol−1) are achieved (n = 3).

68Ga/DOTA- and64Cu/NOTA-Phthalocyanine Conjugates as Fluorescent/PET Bimodal Imaging Probes

In this paper, we describe the synthesis and characterization of a series of new bimodal probes combining water-soluble sulfonated zinc phthalocyanine (ZnPc) as a fluorescence imaging unit and either (68)Ga/1,4,7,10-tetraazocyclododecane-N,N'N″,N'″-tetraacetic acid (DOTA) or (64)Cu/1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) for PET imaging. The two moieties were linked through aliphatic chains of different lengths to modulate amphiphilicity. Labeling of DOTA- or NOTA-ZnPc conjugates with (68)Ga (t1/2 = 68 min) and (64)Cu (t1/2 = 12.7 h) was performed at 100 °C for 15 min with >90% efficiency for all conjugates. In vitro plasma stability assays demonstrated high stability of the (64)Cu/NOTA-ZnPc conjugate, which remained intact over a 24 h time period, and reasonably high stability of the (68)Ga/DOTA-ZnPc conjugate, which released up to 7% of free (68)Ga over a 3 h period. Based on in vitro plasma stability results, we performed biodistribution studies on two (64)Cu-labeled derivatives, which allowed us to select a single candidate for preliminary in vivo experiments. Fluorescence and PET imaging confirmed the potential of these novel conjugates to act as bimodal probes.

Hairpin ODN-based ligands as potential inhibitors of HMGB1 cytokine activity

We here report the design, synthesis, and biological activity of a kinked hairpin-loop DNA acting at low nM concentrations as a strong inhibitor of HMGB1 (High-Mobility Group Box-1), a nuclear protein with cytokine activity in a number of inflammatory diseases. Lead compound optimization has been realized by inserting different oligo-ethylene glycol spacers at the 5′-end and loop positions of the natural hairpin DNA, in order to improve its enzymatic stability and structuring capability, as well as its overall pharmacokinetic properties. Thermal stability data as well as activity assays proved that the ODN which contained two hexa-ethylene glycol spacers, one at the 5′-end and the other in the loop, was the best candidate to inhibit HMGB1. Plasma stability assays and hydrodynamic volume measurements afforded further encouraging results in view of future in vivo evaluation of the optimized ligand.

Expedient Synthesis of 1,3-Substituted Benzene Peptidomimetics

A synthetic route for replacing the central amino acid in the tripeptide Thr-Ala-Val (TAV) with a 1,3-substituted benzene ring was developed. l-Threonine was introduced into the benzene ring by a Grignard reaction with protected l-threoninal, where the nature of the side-chain protecting group was found to be of utmost importance. Subsequently, l-valine was introduced by a copper-mediated amination. Overall, the tripeptide analogue was obtained in six steps with an overall yield of 18%. An orthogonal protecting group strategy allowed attachment of the tripeptide analogue to a solid support and subsequent preparation of the corresponding pentapeptide analogue. Both compounds were tested in a plasma stability assay, showing improved stability compared to their peptide counterparts.

A Single β-Amino Acid Substitution to Angiotensin II Confers AT 2 Receptor Selectivity and Vascular Function

Novel AT(2)R ligands were designed by substituting individual β-amino acid in the sequence of the native ligand angiotensin II (Ang II). Relative ATR selectivity and functional vascular assays (in vitro AT(2)R-mediated vasorelaxation and in vivo vasodepressor action) were determined. In competition binding experiments using either AT(1)R- or AT(2)R- transfected HEK-293 cells, only β-Asp(1)-Ang II and Ang II fully displaced [(125)I]-Ang II from AT(1)R. In contrast, β-substitutions at each position of Ang II exhibited AT(2)R affinity, with β-Tyr(4)-Ang II and β-Ile(5)-Ang II exhibiting ≈ 1000-fold AT(2)R selectivity. In mouse aortic rings, β-Tyr(4)-Ang II and β-Ile(5)-Ang II evoked vasorelaxation that was sensitive to blockade by the AT(2)R antagonist PD123319 and the nitric oxide synthase inhibitor L-NAME. When tested with a low level of AT(1)R blockade, β-Ile(5)-Ang II (15 pmol/kg per minute IV for 4 hours) reduced blood pressure (BP) in conscious spontaneously hypertensive rats (β-Ile(5)-Ang II plus candesartan, -24 ± 4 mm Hg) to a greater extent than candesartan alone (-11 ± 3 mm Hg, n=7, P<0.05), an effect that was abolished by concomitant PD123319 infusion. However, in an identical experimental protocol, β-Tyr(4)-Ang II had no influence on BP (n=10), and it was less stable than β-Ile(5)-Ang II in plasma stability assays. Thus, this study demonstrated that a single β-amino acid substitution resulted in a compound that demonstrated both in vitro vasorelaxation and in vivo depressor activity via AT(2)R. This approach to the design and synthesis of novel AT(2)R-selective peptidomimetics shows great potential to provide insight into AT(2)R function.

Lipophilic derivatives of leu-enkephalinamide: In vitro permeability, stability and in vivo nasal delivery

Leu-enkephalin is an endogenous pain modulating opioid pentapeptide. Its development as a potential pharmaceutic has been hampered by poor membrane permeability and susceptibility to enzymatic degradation. The addition of an unnatural amino acid containing a lipidic side chain at the N-terminus and the modification of the C-terminus to a carboxyamide was performed to enhance the nasal delivery of the peptide. Two lipidic derivatives with varying side chain lengths (C8-Enk-NH2 (1), C12-Enk-NH2 (2)) and their acetylated analogues were successfully synthesised. Caco-2 cell monolayer permeability and Caco-2 cell homogenate stability assays were performed. C8-Enk-NH2 (1) and its acetylated analogue Ac-C8-Enk-NH2 (3) exhibited apparent permeabilities (mean±SD) of 2.51±0.75×10−6cm/s and 1.06±0.62×10−6, respectively. C12-Enk-NH2 (2) exhibited an apparent permeability of 2.43±1.26×10−6cm/s while Ac-C12-Enk-NH2 (4) was not permeable through the Caco-2 monolayers due to its poor solubility. All analogues exhibited improved Caco-2 homogenate stability compared to Leu-Enk-NH2 with t½ values of: C8-Enk-NH2 (1): 31.7min, C12-Enk-NH2 (2): 14.7min, Ac-C8-Enk-NH2 (3): 83min, Ac-C12-Enk-NH2 (4): 27min. However, plasma stability assays revealed that the diastereoisomers of C8-Enk-NH2 (1) did not degrade at the same rate, with the l isomer (t1/2=8.9min) degrading into Leu-enkephalinamide and then des-Tyr-Leu-Enk-NH2, whereas the d isomer was stable (t1/2=120min). In vivo nasal administration of C8-Enk-NH2 to male rats resulted in concentrations of 5.9±1.84×10−2μM in the olfactory bulbs, 1.35±1.01×10−2μM in the brain and 6.53±1.87×10−3μM in the blood 10min after administration.

Development and application of high throughput plasma stability assay for drug discovery

Plasma stability plays an important role in drug discovery and development. Unstable compounds tend to have rapid clearance and short half-life, resulting in poor in vivo performance. This paper examines the variables that affect the plasma stability assay results, including substrate concentration, %DMSO, plasma concentration, enzyme activity upon incubation and batch variation. The results show that plasma stability can accommodate a wide range of experimental conditions. Relatively minor differences in results are produced with major differences in conditions. Significant batch-to-batch variations were observed for rat plasma. We selected the following conditions: 1 μM substrate concentration, 2.5% DMSO, and 50% dilution of plasma in pH 7.4 buffer. Plasma stability can be used as a diagnostic assay when compounds are unexpectedly rapidly cleared, as a special assay when structural classes contain groups that may be susceptible to plasma enzyme hydrolysis, or as general screen for compounds if resources are available. Plasma stability assay has many applications in drug discovery: to alert teams to labile structural motifs, to prioritize compounds for in vivo studies and to screen prodrugs and antedrugs.

Kringle 5 peptide–albumin conjugates with anti-migratory activity

Three peptide fragments of the kringle 5 region of plasminogen and their respective N- and C-terminus maleimido derivatives conjugated to Cys34 of human serum albumin were evaluated in vitro using a human umbilical vein endothelial cell (HUVEC) migration assay and a human plasma stability assay. The N-terminus maleimido derivative of the 64 to 74 segment of kringle 5 conjugated to human serum albumin possessed remarkable anti-migratory activity.