Copaxone (glatiramer acetate, GA), a structurally and compositionally complex polypeptide nonbiological drug, is an effective treatment for multiple sclerosis, with a well‐established favorable ...safety profile. The short antigenic polypeptide sequences comprising therapeutically active epitopes in GA cannot be deciphered with state‐of‐the‐art methods; and GA has no measurable pharmacokinetic profile and no validated pharmacodynamic markers. The study reported herein describes the use of orthogonal standard and high‐resolution physicochemical and biological tests to characterize GA and a U.S. Food and Drug Administration–approved generic version of GA, Glatopa (USA‐FoGA). While similarities were observed with low‐resolution or destructive tests, differences between GA and USA‐FoGA were measured with high‐resolution methods applied to an intact mixture, including variations in surface charge and a unique, high‐molecular‐weight, hydrophobic polypeptide population observed only in some USA‐FoGA lots. Consistent with published reports that modifications in physicochemical attributes alter immune‐related processes, genome‐wide expression profiles of ex vivo activated splenocytes from mice immunized with either GA or USA‐FoGA showed that 7–11% of modulated genes were differentially expressed and enriched for immune‐related pathways. Thus, differences between USA‐FoGA and GA may include variations in antigenic epitopes that differentially activate immune responses. We propose that the assays reported herein should be considered during the regulatory assessment process for nonbiological complex drugs such as GA.
Members of the JAK family of nonreceptor tyrosine kinases play a critical role in the growth and progression of many cancers and in inflammatory diseases. JAK2 has emerged as a leading therapeutic ...target for oncology, providing a rationale for the development of a selective JAK2 inhibitor. A program to optimize selective JAK2 inhibitors to combat cancer while reducing the risk of immune suppression associated with JAK3 inhibition was undertaken. The structure–activity relationships and biological evaluation of a novel series of compounds based on a 1,2,4-triazolo1,5-apyridine scaffold are reported. Para substitution on the aryl at the C8 position of the core was optimum for JAK2 potency (17). Substitution at the C2 nitrogen position was required for cell potency (21). Interestingly, meta substitution of C2-NH-aryl moiety provided exceptional selectivity for JAK2 over JAK3 (23). These efforts led to the discovery of CEP-33779 (29), a novel, selective, and orally bioavailable inhibitor of JAK2.
Analogues structurally related to anaplastic lymphoma kinase (ALK) inhibitor 1 were optimized for metabolic stability. The results from this endeavor not only led to improved metabolic stability, ...pharmacokinetic parameters, and in vitro activity against clinically derived resistance mutations but also led to the incorporation of activity for focal adhesion kinase (FAK). FAK activation, via amplification and/or overexpression, is characteristic of multiple invasive solid tumors and metastasis. The discovery of the clinical stage, dual FAK/ALK inhibitor 27b, including details surrounding SAR, in vitro/in vivo pharmacology, and pharmacokinetics, is reported herein.
A novel set of 2,4,8,22-tetraazatetracyclo14.3.1.13,7.19,13docosa-1(20),3(22),4,6,9(21),10,12,16,18-nonaene macrocycles were prepared as potential anaplastic lymphoma kinase (ALK) inhibitors, ...designed to rigidly lock an energy-minimized bioactive conformation of the diaminopyrimidine (DAP) scaffold, a well-documented kinase platform. From 13 analogues prepared, macrocycle 2m showed the most promising in vitro ALK enzymatic (IC50 = 0.5 nM) and cellular (IC50 = 10 nM) activities. In addition, macrocycle 2m exhibited a favorable kinase selectivity preference for inhibition of ALK relative to the highly homologous insulin receptor (IR) kinase (IR/ALK ratio of 173). The inclusive in vitro biological results for this set of macrocycles validate this scaffold as a viable kinase template and further corroborate recent DAP/ALK solid state studies indicating that the inverted “U” shaped conformation of the acyclic DAPs is a preferred bioactive conformation.
The JAK2/STAT pathway has important roles in hematopoiesis. With the discovery of the JAK2 V617F mutation and its presence in many patients with myeloproliferative neoplasms, research in the JAK2 ...inhibitor arena has dramatically increased. We report a novel series of potent JAK2 inhibitors containing a 2,7-pyrrolotriazine core. To minimize potential drug-induced toxicity, targets were analyzed for the ability to form a glutathione adduct. Glutathione adduct formation was decreased by modification of the aniline substituent at C2.
There are numerous published studies establishing a link between reactive metabolite formation and toxicity of various drugs. Although the correlation between idiosyncratic reactions and reactive ...metabolite formation is not 1:1, the association between the two is such that many pharmaceutical companies now monitor for reactive metabolites as a standard part of drug candidate testing and selection. The most common method involves in vitro human microsomal incubations in the presence of a thiol trapping agent, such as glutathione (GSH), followed by LC/MS analysis. In this study, we describe several 2,7-disubstituted-pyrrolotriazine analogues that are extremely potent reactive metabolite precursors. Utilizing a UPLC/UV/MS method, unprecedented levels of GSH adducts were measured that are 5–10 times higher than previously reported for high reactive metabolite-forming compounds such as clozapine and troglitazone.
The Maillard or browning reaction between sugar and protein contributes to the increased chemical modification and cross-linking of long-lived tissue proteins in diabetes. To evaluate the role of ...glycation and oxidation in these reactions, we have studied the effects of oxidative and antioxidative conditions and various types of inhibitors on the reaction of glucose with rat tail tendon collagen in phosphate buffer at physiological pH and temperature. The chemical modifications of collagen that were measured included fructoselysine, the glycoxidation products N epsilon-(carboxymethyl)lysine and pentosidine and fluorescence. Collagen cross-linking was evaluated by analysis of cyanogen bromide peptides using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by changes in collagen solubilization on treatment with pepsin or sodium dodecylsulfate. Although glycation was unaffected, formation of glycoxidation products and cross-linking of collagen were inhibited by antioxidative conditions. The kinetics of formation of glycoxidation products proceeded with a short lag phase and were independent of the amount of Amadori adduct on the protein, suggesting that autoxidative degradation of glucose was a major contributor to glycoxidation and cross-linking reactions. Chelators, sulfhydryl compounds, antioxidants, and aminoguanidine also inhibited formation of glycoxidation products, generation of fluorescence, and cross-linking of collagen without significant effect on the extent of glycation of the protein. We conclude that autoxidation of glucose or Amadori compounds on protein plays a major role in the formation of glycoxidation products and cross-liking of collagen by glucose in vitro and that chelators, sulfhydryl compounds, antioxidants, and aminoguanidine act as uncouplers of glycation from subsequent glycoxidation and cross-linking reactions.
Anaplastic lymphoma kinase (ALK) is a promising therapeutic target for the treatment of cancer, supported by considerable favorable preclinical and clinical activities over the past several years and ...culminating in the recent FDA approval of the ALK inhibitor crizotinib. Through a series of targeted modifications on an ALK inhibitor diaminopyrimidine scaffold, our research group has driven improvements in ALK potency, kinase selectivity, and overall pharmaceutical properties. Optimization of this scaffold has led to the identification of a potent and efficacious inhibitor of ALK, 25b. A striking feature of 25b over previously described ALK inhibitors is its >600-fold selectivity over insulin receptor (IR), a closely related kinase family member. Most importantly, 25b exhibited dose proportional escalation in rat compared to compound 3 which suffered dose limiting absorption preventing further advancement. Compound 25b exhibited significant in vivo antitumor efficacy when dosed orally in an ALK-positive ALCL tumor xenograft model in SCID mice, warranting further assessment in advanced preclinical models.
Elaboration of the SAR around a series of 2,4-diaminopyrimidines led to a number of c-Met inhibitors in which kinase selectivity was modulated by substituents appended on the C4-aminobenzamide ring ...and the nature of the C2-aminoaryl ring. Further lead optimization of the C2-aminoaryl group led to benzoxazepine analogs whose pharmaceutical properties were modulated by the nature of the substituent on the benzoxazepine nitrogen. Tumor stasis (with partial regressions) were observed when an orally bioavailable analog was evaluated in a GTL-16 tumor xenograft mouse model. Subsequent PK/PD studies suggested that a metabolite contributed to the overall in vivo response.
The Janus kinase (JAK)-signal transducer and activator of transcription pathway is an important therapeutic target because of its role in the regulation of cell growth. Aberrant, constitutive ...activation of JAK2 signaling has been implicated in myeloproliferative disorders with a single, activating somatic V617F mutation in the JH2 pseudokinase domain of JAK2 as the prevalent molecular lesion. Invitrogen has developed the CellSensor(®) cell lines interferon regulatory factor-1 (irf1)-beta-lactamase (bla) TF-1 and irf1-bla HEL for use in evaluating inhibitors of wild-type JAK2 and mutant JAK2 V617F, respectively. Both contain a bla reporter gene downstream of the irf1 response element stably integrated into either TF-1 or HEL cells. A fluorescence resonance energy transfer-based bla substrate is utilized to give a robust detection of JAK2 activity. Examination of Invitrogen's protocols for the two cell lines revealed significant differences that are not conducive to direct comparison of inhibitor activities against wild-type and mutant JAK2. Systematic changes to standardize the two assays were incorporated and evaluated for effects on assay response ratio, assay quality, and potency for a diverse series of inhibitors.