ALBERT

Description: Change in abiotic factors such as climate may lead to shifts in species’ traits across their ranges, but the impacts of biotic elements of global change on latitudinal clines in species’ traits are less well understood. We find evidence that the presence of predatory invasive fire ants alters clines in the behavior, stress responsiveness, and morphology of a native lizard within 80 years (≈40 lizard generations). These results suggest that biotic components of global change can have powerful impacts across multiple organismal systems and large geographical extents even over relatively short time periods. Abstract Understanding the processes driving formation and maintenance of latitudinal clines has become increasingly important in light of accelerating global change. Many studies have focused on the role of abiotic factors, especially temperature, in generating clines, but biotic factors, including the introduction of non‐native species, may also drive clinal variation. We assessed the impact of invasion by predatory fire ants on latitudinal clines in multiple fitness‐relevant traits—morphology, physiological stress responsiveness, and antipredator behavior—in a native fence lizard. In areas invaded by fire ants, a latitudinal cline in morphology is opposite both the cline found in museum specimens from historical populations across the species’ full latitudinal range and that found in current populations uninvaded by fire ants. Similarly, clines in stress‐relevant hormone response to a stressor and in antipredator behavior differ significantly between the portions of the fence lizard range invaded and uninvaded by fire ants. Changes in these traits within fire ant‐invaded areas are adaptive and together support increased and more effective antipredator behavior that allows escape from attacks by this invasive predator. However, these changes may mismatch lizards to the environments under which they historically evolved. This research shows that novel biotic pressures can alter latitudinal clines in multiple traits within a single species on ecological timescales. As global change intensifies, a greater understanding of novel abiotic and biotic pressures and how affected organisms adapt to them across space and time will be central to predicting and managing our changing environment.

Description: TLK199 is a novel glutathione analog that is a selective inhibitor of the enzyme glutathione S-transferase (GST) P1-1. TLK199 treatment induces hematopoietic cell proliferation and differentiation through activation of the MAP kinase signaling pathway leading to activation of JNK and ERK2. In rodent models of chemotherapy induced neutropenia, treatment with TLK199 accelerated recovery of white cell counts at rates comparable to treatment with G-CSF. We now report TLK199 treatment of myeloid progenitor cells isolated from normal human blood resulted in the increased formation of CFU-GM (46%) CFU-MK (47%) and BFU-E (142%) lineages over baseline. A corresponding increase in the percentage of cells expressing CD11b, a granulocyte and monocyte marker, was observed in the CFU-GM cells. Since TLK199 is currently being evaluated in a Phase 2a trial in patients with refractory MDS, we examined the effect of treatment on formation of BFU-E, CFU-GM and CFU-GEMM before and after TLK199 treatment at doses of 50 to 400 mg/m2. A significant increase in the number of hematopoietic progenitor cell colonies measured from patient peripheral blood and bone marrow was observed as early as Day 4 of Cycle 1 as compared to pretreatment baseline. Ten of 12 patients showed an increase in at least one colony forming type (BFU-E, CFU-GM and CFU-GEMM) and 7 of 12 had an increase in all three colony forming types following TLK199 administration. These results correlate with clinical improvement in hematological parameters in peripheral blood and bone marrow observed in MDS patients treated with TLK199. Studies are underway to define the mechanism of bone marrow and peripheral blood count recovery observed following treatment of MDS patients with TLK199 and the role of GST P1-1 as a regulatory element in myeloid proliferation and differentiation.

Description: Introduction: Glutathione S-transferase (GST) P1-1 has shown to be an important negative regulator of cellular growth and differentiation. The effect is mediated through binding to Jun kinase (JNK) which causes a decrease in kinase activity. TLK199, a novel analog of glutathione, binds selectively to GSTP1-1 resulting in its dissociation from JNK and subsequent kinase activation. Exposure of hematopoietic progenitor cells to TLK199 led to activation of JNK followed by cellular growth and maturation. TLK199 has shown significant myelostimulant activity in vitro in human bone marrow cell cultures as well as in several in vivo preclinical models of myelopoeisis. In Phase 1, TLK199 treatment resulted in hematologic improvement (HI) in MDS patients at all dose levels. Methods: The objectives of this multicenter Phase 2 study in MDS were to determine the safety (by NCI-CTC) and efficacy (by modified IWG MDS response criteria) of two dose schedules of TLK199 HCl Liposomes for Injection administered at 600 mg/m2 over 60 minutes by constant rate IV infusion daily x 3 or daily x 5 every 3 weeks. Patients (pts) were treated until lack of response or unacceptable toxicity. Results: 52 MDS pts (33 M/19 F),(29 RA, 9 RARS, 8 RAEB, 3 RAEB-t, 1 CMML, 2 UK), median age 69 years (range 22–90), received 244+ cycles (1099+ treatments), median 4 (range 1–13+). Thirty-seven pts (71%) were red cell transfusion dependent and 10 pts (19%) were platelet transfusion dependent prior to entry. Pts had failed a median of 1 prior therapy (range 0–6) including: erythropoietin (27/52%), G-CSF (9/17%), thalidomide (10/19%), azacitidine (7/14%), steroids (6/12%), hormones (2/4%), and other therapies (14/27%). Thirty-nine pts were evaluable for efficacy, 32 pts (82%) experienced HI in one or more blood cell lineages, 14 of 16 pts (88%) with trilineage dysfunction, 8 of 13 pts (62%) with bilineage dysfunction, and all 10 pts (100%) with unilineage dysfunction experienced HI. Lineage response was HI-P (14 of 22/64%), HI-N (9 of 27/33%), and HI-E (22 of 35/63%). Responses were accompanied by clinical symptom improvement, decreases in RBC and platelet transfusion requirements including transfusion independence and improvements in bone marrow maturation, differentiation, M/E ratios, and dysplastic morphology. Most common adverse events were mild to moderate acute infusion related reactions commonly seen with liposomal formulations: back pain (9/17%), nausea (8/15%), chills (8/15%), and bone pain (6/12%). Conclusions: TLK199 is well tolerated and an active agent in all FAB types of MDS. These data support the further clinical development of TLK199 in MDS as well as in other hematologic malignancies characterized by cytopenias.

Description: Introduction: Glutathione S-transferase (GST) P1-1 binds to and inhibits Jun kinase (JNK), a key regulator of cellular proliferation, differentiation and apoptosis. TLK199, a glutathione analog, binds selectively to GSTP1-1 fostering dissociation from JNK, kinase activation and the promotion of growth and maturation of hematopoietic progenitors in preclinical models, while promoting apoptosis in human leukemia cell lines. The intravenous study with liposomal TLK199 resulted in hematologic improvement (HI) in MDS patients (pts); this trial utilizes an oral formulation of TLK199 in MDS pts. Methods: The objectives of this study were to determine the safety and pharmacokinetics (PK) of TLK199 Tablets given b.i.d. at total daily doses ranging from 200 mg to 6000 mg for the first 7 days of each 3-week cycle. The design was a standard 3 by 3 (3 pts per dose level) dose escalation. Patients were treated until MDS progression or unacceptable toxicity up to a maximum of 8 cycles. Six pts underwent fed-fast PK analysis to determine the effect of food absorption. The PK was evaluated over the dose range for TLK199, and metabolites TLK236, TLK235, and TLK117. Results: 44 MDS pts (32 M/12 F), (9 RA, 11 RARS, 3 RAEB/RAEB-1, 1 RAEB-II, 7 RCMD, 2 RCMD-Rs, 2 CMML, 5 MDS-U, 4 Unknown); IPSS risk-low 14 (32%), INT-1 27 (61%), and INT-2 3 (7%); median age 72 years (range 53–84), received total 206 cycles, median 4.5 (range 1–9) cycles/pt. Ten pts (23%) completed the intended 8 cycles of therapy. Two pts had dose reductions and 4 pts had dose delays (2 due to adverse event (AE) and 2 for scheduling difficulty) at single cycle. Twenty-seven pts (61%) were red cell transfusion (tx) dependent and 5 pts (11%) were platelet tx dependent. Sixteen pts (36%) had abnormal karyotypes. Most common treatment-related AEs were non-hematologic: There were no Grade 3 or 4 toxicities; Grade 2 toxicities were diarrhea and nausea in 2 pts each (5%). Grade 1 toxicities were nausea (43%), diarrhea (25%), vomiting (18%), abdominal pain (7%) and constipation (7%). Three pts (7%) experienced pill-induced dysphagia and reflux esophagitis. Two pts (5%) had Grade 4 neutropenia and 1 pt had febrile neutropenia. There were no DLTs reported. The plasma concentration of the primary active metabolite, TLK236, increases with TLK199 Tablets dose with a mean t1/2 = 2.5 h (range 1.5–4); Cmax = 4.5 uM (range 0.4–6.3). There was no difference seen in the fed vs. fasted patients. By IWG criteria, 15 individual cell line HI responses were observed at the various dose levels of 1000–6000 mg/day with 9 HI responses at dose levels 4000–6000 mg/day. These HI responses were characterized as 1 HI-E major and 4 HI-E minor, 1 HI-N major and 2 HI - N minor, 1 HI-P major and 6 HI-P minor. One bilineage response was reported at 5000 mg/day and 2 trilineage responses at 6000 mg/day. These responses were accompanied by clinical symptoms improvement. Conclusions: TLK199 Tablets are well tolerated and HI responses in all three cell lines were observed with oral TLK199 short-course schedule. These data support the Phase 2 development of extended schedules of oral TLK199 in MDS.

Description: Phase 1 testing of ezatiostat, a glutathione S-transferase P1-1 inhibitor, for the treatment of myelodysplastic syndrome was conducted in a multidose-escalation study. Patients received 10 dose levels (200, 400, 1000, 1400, 2000, 2400, 3000, 4000, 5000, and 6000 mg) of ezatiostat tablets in divided doses on days 1 to 7 of a 21-day cycle for a maximum of 8 cycles. The safety and pharmacokinetics of ezatiostat were evaluated. Forty-five patients with low to intermediate-2 International Prognostic Scoring System risk myelodysplastic syndrome were enrolled. No dose-limiting toxicities were observed. The most common grade 1 or 2, respectively, treatment-related adverse events were nonhematologic: nausea (56%, 9%), diarrhea (36%, 7%), vomiting (24%, 7%), abdominal pain (9%, 0%), constipation (4%, 9%), anorexia (3%, 7%), and dyspepsia (3%, 7%). Concentration of the primary active metabolite, TLK236, increased proportionate to ezatiostat dosage. Seventeen hematologic improvement (HI) responses by International Working Group criteria were observed at dose levels of 200 to 6000 mg/day with 11 HI responses at doses of 4000 to 6000 mg/day. HI responses occurred in all lineages including 3 bilineage and 1 complete cytogenetic response. Decreased number of red blood cell and platelet transfusions and in some cases transfusion independence were attained. Extended dose schedules of ezatiostat tablets are under investigation. This study was registered at http://www.clinicaltrials.gov as NCT00280631.

Description: TLK199 is a novel glutathione analog inhibitor of the enzyme GST P1-1. GST P1-1 has been shown to be a negative regulator of c-Jun N-terminal kinase, which has been implicated in the control of cellular growth and differentiation. Studies in rodents have shown that TLK199 treatment increases the levels of circulating white blood cells and stimulates the production of CFU-GM in bone marrow. In a rodent model of 5-FU-induced neutropenia, treatment with TLK199 accelerated the recovery of neutrophil levels at a rate similar to that observed with G-CSF. In a recently reported Phase 2 clinical trial in patients with refractory MDS involving multilineage blood cell dysfunction, treatment with TLK199 resulted in a 65% Hematological Improvement of neutrophils, erythrocytes and/or platelets, confirming the myelorestorative effect of TLK199 in humans. The present study was designed to determine if the myelostimulative effects of TLK199 on neutrophils and their precursors are due in part to increased levels of endogenous G-CSF. TLK199 was tested in normal and neutropenic rodents for its effects on G-CSF production. A single administration of TLK199 (p.o. or i.p.) to normal mice resulted in up to 500% increase in G-CSF serum levels. In a rat model of 5-FU-induced neutropenia, TLK199 administration accelerated the recovery of circulating neutrophils, which corresponded to maximal G-CSF serum levels being observed earlier than vehicle control. The ability of TLK199 to enhance G-CSF production in primary human bone marrow stromal cells was also examined. Treatment of these cells with TLK199 potentiated IL-1b-induced production of G-CSF, GM-CSF and IL-6 from 200 to 400%. These data suggest that the enhancement of G-CSF production may contribute to the accelerated recovery of neutrophils observed with TLK199 treatment under neutropenic conditions. Together with the results from the Phase 2 clinical trial in MDS, these data support further clinical assessment of TLK199 treatment for chemotherapy-induced cytopenia.

Description: Introduction: TLK199 is a novel glutathione analog inhibitor of the enzyme GST P1-1. Exposure of cells to TLK199 results in the activation of the MAP kinase signaling pathway leading to JNK and ERK2 activation. TLK199 treatment induces hematopoietic progenitor cell growth, maturation and differentiation. TLK199 has demonstrated in vitro myelostimulant activity in human bone marrow cultures and accelerated neutrophil recovery in the chemotherapy induced neutropenia rodent model. Methods: The objectives of this ongoing multicenter study in MDS are to determine the safety, efficacy and phamacokinetics. Blood levels of TLK199 and metabolites are measured by LC-MS-MS. TLK199 is administered as an i.v. infusion over 60 minutes daily x 5 days every two weeks in the dose ranging stage, and at 600 mg/m2 x 5 days every 3 weeks in Phase 2a for up to 8 cycles or until lack of response, blast count progression, or unacceptable toxicity. Results: At interim analysis, twenty-five patients (pts) with MDS (13 RA, 1 RARS, 7 RAEB, 3 RAEB-t and 1 CMML), median age 73 years (range 22-89), received 98+ cycles (500+ treatments) with a median of 3 cycles per pt (range 1–8). Seventeen pts (70%) were transfusion-dependent for red blood cells and 8 (30%) for platelets prior to enrollment. Pts failed a median of 1 prior therapy (range 0–4) including: erythropoietin (13), G-CSF (3), thalidomide (4), IL-11 (3), azacitidine (3), and other therapies (7). Five dose levels were studied from 50–600 mg/m2. No dose-limiting toxicities were observed and 600 mg/m2 was chosen for Phase 2a based on clinical and biologic activity. Twenty pts were evaluable for efficacy, 9 (45%) experienced improvement in one or more blood cell lineages. Six pts (30%) showed Hematologic Improvement by MDS IWG response criteria. Four of 6 experienced improvement in all 3 blood cell lineages and two pts in 2 blood cell lineages. A decrease in the marrow blast count was observed in 1 pt with RAEB. The longest duration of therapy was 8 cycles. Clinical responses were accompanied by clinical symptom improvement with decrease in transfusion requirements or transfusion independence. Bone marrow examination showed improvements in maturation, differentiation, M/E ratios, and decreased dysplastic morphology. Most common adverse events were mild (grade 1 – 2): flushing (6), rigors (4), nausea (3), headache (3), vomiting (2), pain in extremity (3), back pain (2). Grade 4 back pain occurred in one pt. Conclusions: TLK199 is well tolerated. Nine pts (45%) experienced improvement in one or more blood lineages and six (30%) showed Hematologic Improvement by MDS IWG response criteria. Enrollment in Phase 2a continues. These data support further clinical development of TLK199 in myelodysplastic syndrome as well as in other hematologic disorders characterized by cytopenias.