Preclinical Safety Assessment of a Highly Selective and Potent Dual Small-Molecule Inhibitor of CBP/P300 in Rats and Dogs

Abstract

Cyclic adenosine monophosphate-response element (CREB)-binding protein (CBP) and EP300E1A-binding protein (p300) are members of the bromodomain and extraterminal motif (BET) family. These highly homologous proteins have a key role in modulating transcription, including altering the status of chromatin or through interactions with or posttranslational modifications of transcription factors. As CBP and p300 have known roles for stimulating c-Myc oncogenic activity, a small-molecule inhibitor, GNE-781, was developed to selectively and potently inhibit the CBP/p300 bromodomains (BRDs). Genetic models have been challenging to develop due to embryonic lethality arising from germline homozygous mutations in either CBP or P300. Hence, the purpose of this study was to characterize the role of dual inhibition of these proteins in adult rats and dogs. Repeat dose toxicity studies were conducted, and toxicologic and pathologic end points were assessed. GNE-781 was generally tolerated; however, marked effects on thrombopoiesis occurred in both species. Evidence of inhibition of erythroid, granulocytic, and lymphoid cell differentiation was also present, as well as deleterious changes in gastrointestinal and reproductive tissues. These findings are consistent with many preclinical (and clinical) effects reported with BET inhibitors targeting BRD proteins; thus, the current study findings indicate a likely important role for CBP/p300 in stem cell differentiation.

Keywords

CREB-binding protein p300 toxicology hematopoiesis bromodomain

Introduction

Cyclic adenosine monophosphate-response element (CREB)-binding protein (CREBBP or CBP) and EP300E1A-binding protein p300 (EP300 or p300) are members of the bromodomain and extraterminal motif (BET) family. The BET proteins are involved in numerous cellular processes including cell growth, apoptosis, and immune surveillance.¹ The CBP/p300 transcriptional coactivator proteins are highly homologous proteins that can modulate transcription by altering the status of chromatin through histone acetyltransferase (HAT) activity or by serving as scaffolding for multiple transcription factors.² Similarly, CBP/p300 proteins play an important role in post-translational modification of other transcriptional regulators, illustrating why CBP/p300 proteins are often considered “master” regulators of transcription.^3,4

Several BET family members, including CBP/p300, are known to stimulate c-Myc activity.⁵ As c-Myc is a known proto-oncogene, considerable interest in downregulating c-Myc activity has arisen in the oncology field, and several BET inhibitors have entered into clinical trials.⁶ These small-molecule inhibitors have targeted the bromodomain (BRD) either through pan-inhibition of several BRD proteins or by attempting more selective inhibition of certain isoforms such as BET bromodomain-containing protein 4 (BRD4). Selective targeting of the BRD domain inhibits the protein–protein interactions between BET proteins and acetylated histones, leading to loss of “reader” function and effectively inhibiting transcription of downstream genes. GNE-781, the molecule used in the current study, is a small-molecule inhibitor of the BRD region of CBP/p300 and is also highly selective against other BET family members. However, GNE-781 retains potent c-Myc inhibitory activity.⁷

The role of CBP/p300 has been assessed in vivo through genetic deficits in either humans or rodents. Heterozygous germline mutations in either CBP or p300 have been described in humans and have been phenotypically characterized as Rubinstein-Taybi syndrome (RTS).⁸ Rubinstein-Taybi syndrome is characterized primarily by neural tube developmental defects, including mental retardation and facial deformities. Notably, these patients have significantly increased risk of brain tumors and hematologic malignancies.⁹ Defects in the cardiovascular, reproductive system, and skeletal system have also been observed.

In rodents, homozygous mutations in either CBP or p300 lead to embryonic lethality around embryonic day 11.¹⁰ Studies in heterozygous mice have generally been phenotypically similar to RTS, with notable partial embryonic or postnatal lethality. Similar to humans, effects on hematopoiesis have been described suggesting a role for CBP/p300 in early hematopoietic development.¹¹ Experiments have demonstrated that CBP^+/− mice have hypocellularity of the bone marrow reflected in decreased B- and T-cells and myeloid and erythroid subpopulations.¹² In CBP^+/− mice, altered progenitor cell development, morphology, and proliferation have been reported, including pseudo Pelger-Huët anomalies and hypersegmented granulocytes.¹³ Mouse CBP^+/− bone marrow transplantation into irradiated mice resulted in 39% of the recipients to have hematologic neoplasms between 10 and 21 months of age, whereas p300^+/− transplantation did not increase the risk, suggesting that CBP may be more critical in hematopoietic differentiation,¹² however, it should be noted that the number of animals on study was limited, and the rate of spontaneous tumor formation in the strain of mouse used was not cited. In mice with compound mutations in c-Myb and/or the KIX domain of CBP and p300, increases in platelets and decreases in B- and T-cells, and erythrocytes occurred.¹⁴ This study highlights the complexity of CBP/p300 as a scaffold for numerous transcription factors and also that inhibiting these interactions genetically or pharmaceutically can alter the hematopoietic differentiation process.

Similarly, tissue-specific conditional knockout (KO) CBP or p300 mice have been described. In general, significant compensation in the phenotype can be observed in development as long as 1 allele of CBP or p300 remains intact to preserve HAT activity.¹⁵ Genetic models have also demonstrated the critical role for CBP and p300 in organogenesis in the kidney,¹⁶ lymphocytes and T_regs,¹⁷ brain,^18,19 and likely in many other tissues. CBP and p300 are also known to have a continuing role in cognition and maintaining hematopoiesis into adulthood.^12,18

CBP and p300 are paralogous proteins that are indispensable in embryogenesis and juvenile development, however, the role of these 2 epigenetic modulators in adulthood is currently poorly understood. Due to the challenges of utilizing genetic models to ascribe function, the purpose of the current study is to offer novel insight into the function of these proteins in hematopoietic cell differentiation and tissue homeostasis in adulthood in 2 different preclinical species through utilization of a potent, selective, dual small-molecule inhibitor of the CBP/p300 BRD.

Materials and Methods

Reagents

GNE-781, a free base, has been described previously and was of the highest purity achievable for the in vivo studies (>99%).⁷

Formulation

GNE-781 was formulated for the rat and dog studies as a suspension in 0.5% (wt/vol) methylcellulose (20 to 30 cps) and 0.2% (wt/vol) polysorbate 80 (Tween 80) in reverse osmosis water. The dose volume was 5 mL/kg.

Human Ether-à-go-go-Related Gene

The in vitro effects of the compounds on the human ether-à-go-go-related gene potassium channel current (a surrogate for IKr, the rapidly activating, delayed rectifier cardiac potassium current) expressed in mammalian cells were evaluated at room temperature using the QPatch HT (Sophion Bioscience A/S, Ballerup, Denmark), an automatic parallel patch clamp system (Chantest; Cleveland, Ohio). The test articles were evaluated at 1 and 10 μmol/L; each concentration was tested in at least 2 cells (n ≥ 2). The duration of exposure to each test article concentration was approximately 3 minutes. The positive control cisapride was utilized to confirm the sensitivity of the test system.

Secondary Pharmacology

The potential for GNE-781 to elicit secondary (off-target) pharmacology was evaluated in a panel of 42 receptors, enzymes, transporters, and ion channels in vitro and reported as percentage inhibition of the binding of a radioactively labeled ligand or percentage inhibition of control enzyme activity (Eurofins Cerep; Celle-Lévescault, France) at a concentration of 10 μmol/L.

Bromodomain and Kinase Selectivity

The BRD selectivity against 32 targets (DiscoverX; Fremont, California) was determined according to previously reported methods.²⁰ Kinase selectivity against 220 targets (ThermoFisher; Waltham, Massachusetts) was also conducted as previously described.²¹

Hepatotoxicity Panel

Cytotoxicity in human primary hepatocytes (half maximal inhibitory concentration [IC50]) and inhibition of the bile salt export pump (BSEP; Solvo Biotechnologies; Budapest, Hungary) were assessed as described by the manufacturer.

Genotoxicity

Ames II assays (Bioreliance; Rockville, Maryland) and high-content (Cerep) and manual in vitro micronucleus assays including follow-up florescence in situ hybridization (FISH; Covance; Harrogate, United Kingdom) were conducted according to standard methods.

Animals

All animal experiments for this study were conducted in accordance with the Guide for the Care and Use of Laboratory Animals at an Association for Assessment and Accreditation of Laboratory Animal Care-approved facility. All procedures complied with applicable animal welfare acts and were approved by the local Institutional Animal Care and Use Committee.

The in-house non-Good Laboratory Practice (GLP) rat study was comprised of male and female Crl: CD Sprague Dawley (SD) rats (Charles River Labs; Hollister, California) to conduct the toxicokinetic (TK; n = 3/sex/TK satellite group) and toxicity (n = 6/sex/dose/group) portions of this study. The animals were acclimated for a minimum of 10 days and prior to study initiation, animals were assigned to terminal and recovery groups using a block stratified randomization procedure based on body weight. Rats were group housed in solid bottom cages with bedding, provided rodent chow (Harlan Teklad Global Diets) and water ad libitum unless otherwise specified, and were provided with environmental enrichment.

The non-GLP dog study was conducted at Covance-Madison and was comprised of male and female beagle dogs (Covance; Cumberland, Virginia). Animals were acclimated for 22 days and randomized into vehicle and treatment groups with 2/sex/group. After group assignment, the mean body weight for each group was checked using analysis of variance F probability, and dogs were housed in stainless steel cages with Tenderfoot flooring. When possible, animals were socially housed by sex, provided various cage-enrichment devices and dietary enrichment and provided the opportunity to exercise/group 3 times weekly. Animals were offered certified Canine Diet #5007 (PMI Nutrition International Certified LabDiet) and water ad libitum unless fasted for study procedures. Body weight, cageside observations, and detailed observations were conducted daily.

Study Designs

Study A: A 10-day (BID) repeat-dose oral toxicity and TK study in male and female Sprague-Dawley rats with a 7-day recovery period

Six male and 6 female rats per group were administered 0, 15, 50, or 150 mg/kg GNE-781 via oral gavage twice daily (BID) dosing daily for 10 days. At study initiation, rats were aged between 8 and 11 weeks and weighed between 244 and 261 g (males) and 235 and 265 g (females). Dose formulations were administered BID approximately 8 hours apart beginning at 7 Am and 3 Pm, respectively. On day 11, half of the rats in each dose and vehicle group were euthanized and the remaining rats in each group were followed for a 7-day drug-free recovery period and euthanized on day 18. Clinical observations were conducted BID. Body weight measurements were obtained once daily during dosing and recovery phases, and food consumption was recorded for each cage throughout the study on a 2- or 3-day cycle. Clinical and anatomic pathology and TK sample assessments were conducted as described subsequently in the corresponding sections.

Study B: A 7-day (BID) repeat-dose oral gavage tolerability and TK study in male and female dogs

Four dogs per dose level (2 per sex) were administered 0, 1.25, 3.75, or 12.5 mg/kg/d GNE-781 via oral gavage BID for 7 days. On day 8, dogs were euthanized. Dose formulations were administered BID approximately 8 hours apart beginning at 8 Am and 4 Pm, respectively. At initiation of dosing, animals were 10 to 11 months old, and their body weights ranged from 8.8 to 11.4 kg (males) and 6.8 to 8.3 kg (females). Assessment of toxicity was based on mortality, clinical observations, body weight, food consumption, ophthalmic observations, and clinical and anatomic pathology. Blood samples were collected for TK evaluations on study days 1 and 7 at predose and multiple times postfirst daily dose.

Toxicokinetic Analysis

Blood was collected into tubes containing potassium (K2) ethylenediaminetetraacetic acid (EDTA), maintained on wet ice, and centrifuged within hour of collection with plasma stored at 80°C until analysis. Plasma samples were analyzed using a liquid chromatography–mass spectrometry method. Toxicokinetic analysis included maximum observed concentration (C_max), time to maximum observed concentration, and area under the plasma concentration–time curve (AUC_0-t). Toxicokinetic parameters were determined using PhoenixWinNonlin version 6.4 or higher (Certara USA, Inc, Princeton, New Jersey). A noncompartmental approach consistent with the oral route of administration was used to calculate TK parameters.

Rat Study

Blood samples were collected via tail vein over a 24-hour period at 1, 2, 4, 8, and 24 hours postdose on study days 1 and 10.

Dog Study

Blood samples were collected via jugular vein over a 24-hour period at 0.5, 1, 4, 8, 8.5, 9, 12, and 16 hours postdose on study days 1 and 7. On study day 7, samples were collected predose, 0.5, and 1 hours after the first dose from animals euthanized at an unscheduled interval.

Clinical Pathology

Rat study

Blood samples were collected via retro-orbital sinus under isoflurane anesthesia from fasted animals for routine hematology, and clinical chemistry testing at terminal time points (day 11 or 18). Samples were collected without anticoagulant (clinical chemistry) or with EDTA anticoagulant (hematology) and tested using automated analyzers and appropriate reagents and controls according to manufacturer instructions. Whole blood samples were also collected via descending aorta at the time of scheduled necropsy for exploratory Multiplate platelet aggregometry testing on day 11 or 18.

Dog study

Blood samples for routine hematology, clinical chemistry, and coagulation testing were collected from fasted animals via jugular vein twice during the predose phase and on study day 7. In addition, hematology samples were collected on study day 3. Whole blood was collected in EDTA-containing tubes (hematology), sodium citrate (coagulation), or serum separator tubes (clinical chemistry) and analyses performed using automated analyzers and appropriate reagents and controls according to manufacturer instructions.

Exploratory multiplate platelet aggregometry testing

Blood samples were collected from female rats on days 11 and 18 in hirudin anticoagulant as per manufacturer instructions. Sample collection was limited to 3 females/group to minimize the effects of sample processing time on platelet viability and also because a sex effect on decreased platelet counts was observed in prior studies (female > male). All samples were analyzed on the Roche Multiplate Analyzer (Diapharma Group, West Chester, Ohio). Blood samples were tested in the presence of a strong (collagen) and a weak (adenosine diphosphate [ADP]) agonist using mini test-cells. All testing was performed as per manufacturer instructions including the use of appropriate experimental and quality controls. Platelet counts were measured prior to analysis and deemed acceptable for testing (>150 K platelets/μL) as per manufacturer recommendations.

Electron microscopy

Rat blood samples were collected from 3 animals per sex/group at the time of scheduled necropsy on day 11 or 18 and fixed in modified Karnovsky fixative (2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer, pH 7.2), and postfixed in 1% osmium tetroxide and stained “en block” with 0.5% (wt/vol) uranyl acetate at 4°C overnight. The samples were then dehydrated through a series of ascending ethanol concentrations, followed by propylene oxide, and embedded in Eponate 12 (Ted Pella, Redding, California). Semithin sections (300 and 1000 nm) were cut on a Leica Ultracut UCT (Leica Microsystems, Buffalo Grove, Illinois) and stained with toluidine blue for light microscopic examination. Ultrathin sections (80 µmol/L) were stained with 0.2% lead citrate and examined in a JEOL JEM-1400 transmission electron microscope at 80 kv (JEOL USA, Peabody, Massachusetts). Digital images were captured with a GATAN Ultrascan 1000 CCD camera.

Anatomic pathology

A necropsy was conducted for 3 rats per sex/group on day 11 (end of dosing phase) or 3 per sex/group on day 18 (end of recovery phase) for the rat study, or for all surviving animals on day 8, for the dog study. Macroscopic examinations were conducted for both unscheduled and scheduled euthanasias, and organ weights were recorded.

Standard tissues were collected, embedded in paraffin, sectioned, and slides were stained with hematoxylin and eosin and examined microscopically. Paired organs were weighed together. Eyes and/or testes were immersion-fixed in Davidson solution for 24 hours, then rinsed with phosphate-buffered saline or water and transferred to 10% neutral buffered formalin.

Statistical Methods

Rat study

For groups of n ≥ 3, differences in body weights, organ weights, organ weight ratios, and clinical pathology parameters were compared by one-way analysis of variance using Dunnett test as the post hoc test for any significant results (P < .05). Treated groups were compared to their corresponding controls at common time points.

Dog study

Due to the small number of animals, statistical data analyses were limited to the calculation of means and standard deviations for the following parameters: absolute body weight, body weight change, food consumption, continuous clinical pathology values, and organ weights.

Results

In Vitro Studies

GNE-781 was subjected to a multitude of in vitro screening assays to determine the selectivity and safety profile of the molecule (Figure 1). Notably, the off-target liabilities due to secondary pharmacology were minimal (Supplementary Table 1), with modest inhibition of BET proteins BRDT and WDR9 at concentrations approximately 1000-fold higher than CBP biochemical potency. No major liabilities in ion-channel inhibition were present (IC₅₀ > 10 µmol/L).

Figure 1.

In vitro safety and selectivity assays performed for GNE-781.

In regard to the hepatotoxicity panel, no potential hazards were identified in the intrinsic cytotoxicity profile in human hepatocytes (IC₅₀ > 10 µmol/L). However, a possible hazard flag in inhibiting bile acid transporters such as BSEP was identified, with an IC₅₀ < 25 µmol/L, suggesting that drug-induced liver injury with GNE-781 is a possible risk in humans that might not be easily identified in rats and dogs.²²

GNE-781 was negative in Ames assays and was not considered mutagenic, however, both the high-content and manual micronucleus assays were positive, and FISH analysis indicating that GNE-781 has clastogenic potential. However, as no definitive micronucleus testing (in vitro or in vivo) was conducted, these results should be considered only as hazard screens and should interpreted with a degree of caution. This is consistent with previous work suggesting that CBP and/or p300, due to their role in epigenetic remodeling, could be important in maintaining genomic stability.²³

Rat Studies

Toxicokinetics

The exposure in males and females was relatively similar between day 1 and day 10. Females appeared to have higher exposure than males (approximately 2- to 3-fold), and the oral exposures were less than dose proportional from 15 to 150 mg/kg BID (Figure 2).

Figure 2.

The mean toxicokinetic profile for GNE-781 in rats over a 24-hour period on day 1 and day 10 in male and female rats.

Animal fate, clinical observations, and body weight

GNE-781 administration resulted in the early euthanasia on day 9 of a single moribund female from the 150-mg/kg dose group due to body weight loss. All other rats survived until scheduled terminal (day 11) and recovery (day 18) necropsies. At 15 mg/kg/d, decreased food consumption, decreased body weight gain, and/or body weight loss were the primary clinical observations and were generally dose-dependent (Table 1).

Table 1.

Mean Body Weight and Mean Percentage Body Weight Change From Baseline in Rats Following Gdc-781 Treatment.^a

	Study Day
Treatment	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18
Males
0 mg/kg
Mean, kg	0.228	0.251	0.255	0.264	0.274	0.279	0.287	0.293	0.304	0.307	0.314	0.310	0.335	0.339	0.348	0.353	0.360
% Change	1.53	5.09	9.01	11.06	14.13	16.61	20.96	22.28	24.98	23.66	33.66	35.20	39.05	41.16	43.77	45.09	37.98
15 mg/kg
Mean, kg	0.229	0.251	0.255	0.256	0.259	0.260	0.262	0.262	0.266	0.270	0.267	0.260	0.267	0.271	0.279	0.286	0.292
% Change	1.69	1.76	3.14	3.40	4.49	4.43	5.83	7.47	6.20	3.69	6.88	8.20	11.57	14.55	16.90	19.44	14.14
50 mg/kg
Mean, kg	0.228	0.253	0.255	0.253	0.252	0.255	0.255	0.256	0.259	0.258	0.259	0.248	0.247	0.252	0.263	0.269	0.272
% Change	0.66	−0.33	−0.42	0.52	0.61	1.13	2.11	1.74	1.99	−1.96	−1.06	0.76	5.52	7.60	8.76	11.63	5.78
150 mg/kg
Mean, kg	0.227	0.252	0.256	0.254	0.252	0.254	0.256	0.256	0.255	0.254	0.251	0.244	0.246	0.248	0.262	0.270	0.275
% Change	1.66	0.99	0.04	0.94	1.86	1.58	1.31	0.75	−0.37	−3.09	−2.82	−2.20	3.25	6.58	8.39	10.26	5.34
Females
0 mg/kg
Mean, kg	0.246	0.250	0.247	0.242	0.246	0.245	0.250	0.248	0.252	0.251	0.252	0.246	0.255	0.255	0.258	0.255	0.259
% Change	−1.16	−3.16	−1.53	−1.85	−0.20	−0.98	0.88	0.18	0.73	−1.67	3.84	3.73	5.02	3.92	5.41	3.22	0.25
15 mg/kg
Mean, kg	0.245	0.250	0.247	0.244	0.243	0.243	0.244	0.245	0.244	0.243	0.240	0.234	0.244	0.245	0.252	0.254	0.256
% Change	−1.23	−2.58	−2.96	−2.61	−2.36	−1.80	−2.21	−2.65	−3.89	−6.35	−2.08	−1.47	1.19	2.06	2.84	2.26	−3.90
50 mg/kg
Mean, kg	0.246	0.249	0.249	0.243	0.242	0.242	0.243	0.241	0.240	0.238	0.237	0.231	0.243	0.244	0.251	0.249	0.252
% Change	−0.01	−2.18	−2.65	−2.83	−2.42	−2.98	−3.51	−4.42	−4.49	−7.19	−4.10	−3.73	−1.00	−1.75	−0.70	−1.41	−4.79
150 mg/kg
Mean, kg	0.247	0.251	0.247	0.238	0.237	0.237	0.235	0.232	0.228	0.226	0.233	0.228	0.232	0.237	0.242	0.245	0.249
% Change	−1.51	−4.97	−5.39	−5.60	−6.34	−7.43	−8.93	−9.72	−7.17	−8.99	−6.29	−4.15	−2.12	−0.92	1.01	0.20	−6.40

^a % Change = mean of individual percentage change from baseline body weight taken on day 1; day 11 = End of dosing period; day 18 = end of recovery period.

Clinical pathology

GNE-781-related hematology findings were present in males and females at all dosages, were generally dose-related, and included marked decreases in platelet counts which correlated with microscopic observations of decreased megakaryocytes on histologic review and mild (male) to marked (female) decreases in reticulocytes and reticulocyte percentage (Figure 3A-F). There were mild increases in absolute white blood cell (WBC) counts in females, primarily due to increased lymphocyte numbers (data not shown). All hematology findings demonstrated reversibility after a 7-day recovery period.

Figure 3.

Hematology data: Rat study. A-F, Marked decreases in platelet counts and reticulocyte-related parameters were present in males (B, D, F) and females (A, C, E) administered 15, 50 and 150-mg/kg GNE-781 at the end of the dosing phase (day 11). Values recovered after a 7-day drug-free period.

GNE-781-related clinical chemistry findings were present in rats at all dosages and included dose-dependent decreases in alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase activities and nonspecific, changes in glucose, lipid, and electrolyte parameters. All findings had evidence of reversibility at the end of the 7-day recovery period.

Multiplate platelet aggregometer testing

GNE-781-treated rats had diminished platelet aggregation responses in the presence of collagen or ADP agonists with administration of ≥15 mg/kg GNE-781 at the end of the dosing phase. Platelet aggregation effects demonstrated reversibility in all dose groups following the 7-day recovery period (Figure 4A and B).

Figure 4.

In vitro platelet aggregation study: Rat blood collected at the end of the dosing phase (day 11). Platelet aggregation was decreased at dosages of ≥15 mg/kg GNE-781 in the presence of a strong (collagen) or weak (ADP) agonist. These effects reversed following the 7-day recovery period (A and B). ADP indicates adenosine diphosphate.

Electron microscopy

Blood-derived platelets collected from control and GNE-781-treated rats demonstrated comparable ultrastructural morphology after the end of the dosing period (Figure 5A and B).

Figure 5.

Transmission electron microscope micrographs: Rat platelets. Platelets collected from the blood of GNE-781 treated (150 mg/kg) and vehicle-control animals demonstrated comparable ultrastructural morphology at the end of the dosing period (day 11). Representative images are shown, 8000× (A—control, B—GNE-781 treated).

Anatomic pathology

GNE-781 administration resulted in the early euthanasia on day 9 of a single moribund female rat from the 150-mg/kg dose group. Macroscopic findings at necropsy included red pinpoint discolorations of the stomach mucosa (no histologic correlate), a small thymus (which correlated with the associated histologic finding of decreased lymphocyte cellularity), and thin body condition (no specific histologic correlate). The histologic findings for this animal were typical of those attributed to GNE-781 in the terminal phase animals.

End of dosing phase

GNE-781-related histologic findings at the terminal time point occurred at ≥15 mg/kg and included alterations of cellularity in the bone marrow (diffuse hypercellularity and/or hypocellularity of megakaryocytes and/or hypercellularity of granulocytic series cells); decreased cellularity of lymphocytes in the spleen, thymus, and gut-associated lymphoid tissue (GALT); acinar cell degeneration in the pancreas; and degeneration of seminiferous tubules of the testes (Figure 6A-F). Additional findings likely related to GNE-781 included necrosis/inflammatory cell infiltrate in the heart, hemorrhage and/or decreased size/number of ovarian corpora lutea (CL), and decreased size/number of follicular bulbs in the skin. The histologic findings correlated with decreased spleen, testes, and thymus weights (absolute and relative) at dose levels ≥15 mg/kg. Macroscopic findings at ≥15 mg/kg correlated with the histologic findings and included small thymus, small testes, and sporadic instances of alopecia.

Figure 6.

Histology sections (A-F): Rat. A, Normocellular bone marrow with prominent large megakaryocytes; vehicle-control group rat; B, Bone marrow with depleted megakaryocytes; high dose (150 mg/kg GNE-781) group rat; C, Normocellular spleen with active lymphoid follicles; vehicle control group rat; D, Hypocellular spleen with generalized lymphoid depletion and inactive lymphoid follicles; high dose (150 mg/kg GNE-781) group rat; E, Normal testicular tubules with actively proliferating seminiferous epithelial cells; vehicle control group rat; F, Hypocellular testicular tubules with depleted seminiferous epithelial cells; mid-dose (50 mg/kg) group rat.

End of recovery phase

Most GNE-781- associated changes did not reverse or partially reversed after completion of the 7-day recovery period. Bone marrow megakaryocyte numbers were near normal, and diffuse marrow hypercellularity was still present, although limited to the 50 and 150 mg/kg groups. Hypercellularity of the granulocytic series remained frequent, although with lesser severity and with indication of partial recovery in the low-dose group (lower incidence). Decreased lymphocyte cellularity in the spleen had decreased incidence and decreased in severity, decreased lymphocyte cellularity in the thymus had nearly complete recovery, and there was no evidence of decreased cellularity of lymphocytes in GALT. Degeneration of acinar cells in the pancreas had a decrease in incidence and severity. Degeneration of seminiferous tubules of the testes was increased, being mild to marked and present in all test article-dosed males. The heart lesion was still present in dosed rats, with no change in incidence or severity, ovarian findings decreased to a single instance, and hair follicle findings had not changed. New findings at recovery included increased hematopoiesis in the spleen and extramedullary hematopoiesis in the liver.

Dog Studies

Toxicokinetics

Maximum observed concentration and AUC_0-t were greater than dose proportional from 1.25 to 12.5 mg/kg/d, and there was no difference in exposures between the males and females. Similar exposures (C_max and AUC_0-t values) were observed on study days 1 and 7 for the doses of 1.25 and 3.75 mg/kg/d while greater than 2-fold differences were observed for 12.5 mg/kg/d (Figure 7).

Figure 7.

The mean toxicokinetic profile for GNE-781 in dogs over a 24-hour period on day 1 and day 7 (no sex differences 2-fold observed, thus were sexes combined).

Animal fate, clinical observations, and body weight

GNE-781 was associated with moribundity and early euthanasia. On study day 7, 2 males administered 3.75 mg/kg/d (mid dose) and 1 male and 1 female administered 12.5 mg/kg/d (high dose) were moribund and were euthanized. The cause of moribundity was not determined.

At ≥3.75 mg/kg/d, clinical observations included lethargy, hypoactivity, thin appearance, and increased body temperature. Similarly, at ≥1.25 mg/kg/d, increased incidence of liquid and/or nonformed feces, body weight loss, and decreased food consumption were present. The severity and incidence generally increased with dose. Individual percentage body weight change data are presented in Table 2.

Table 2.

Individual Percentage Body Weight Change From Baseline in Dogs Following GNE-781 Treatment.^a

		Study Day
Treatment	Animal ID	2	3	4	5	6	7	8
Males
0 mg/kg	58	0.00	0.00	−1.14	0.00	−1.14	0.00	0.00
	59	−0.92	0.00	−0.92	−1.83	−1.83	−2.75	−2.75
0.625 mg/kg	60	0.00	−1.10	−1.10	−2.20	0.00	−2.20	−3.30
	61	0.00	−0.93	−0.93	−0.93	0.93	−0.93	−1.85
1.875 mg/kg	62	−1.00	−2.00	−4.00	−5.00	−7.00	−11.00	^b
	63	−0.88	−0.88	−1.75	−3.51	−4.39	−7.89	^b
6.25 mg/kg	64	1.02	0.00	−2.04	−3.06	−5.10	−6.12	^b
	65	0.00	−2.86	−4.76	−6.67	−8.57	−9.52	−11.43
Females
0 mg/kg	66	1.35	1.35	0.00	0.00	0.00	0.00	0.00
	67	−1.28	0.00	−5.13	−5.13	−1.28	−2.56	−2.56
0.625 mg/kg	68	0.00	−1.47	−1.47	−1.47	0.00	−2.94	−2.94
	69	0.00	−1.20	−3.61	−3.61	−3.61	−4.82	−6.02
1.875 mg/kg	70	−1.27	−3.80	−5.06	−5.06	−2.53	−5.06	−7.59
	71	0.00	−2.90	−5.80	−5.80	−5.80	−7.25	^b
6.25 mg/kg	72	−1.32	−3.95	−5.26	−5.26	−5.26	−9.21	−10.53
	73	0.00	0.00	−1.14	0.00	−1.14	0.00	0.00

^a Values are percentage change from baseline body weight taken on day 1; day 8 = end of dosing period.

^b Body weight not taken due to early euthanasia.

Clinical pathology

GNE-781-related hematology effects at scheduled necropsy were present at all dose levels. Findings included moderately decreased reticulocyte counts, reticulocyte percentage, and platelet counts (Figure 8A-F) and mildly to moderately decreased absolute lymphocyte counts at all dose levels (data not shown). Hematology findings were not present at the day 3 hematology time point. Dogs euthanized after the first dose on day 7 also had increased absolute neutrophil and monocyte counts and also increased fibrinogen concentrations (inflammatory response). There were no test article-related clinical chemistry findings observed at any dosages.

Figure 8.

Hematology data: Dog study. Platelet counts and reticulocyte-related parameters were decreased in males and females administered ≥15 mg/kg GNE-781 on day 7 (A-F).

Anatomic pathology

At the end of the dosing phase, GNE-781-related microscopic findings included decreased cellularity in sternal bone marrow in males administered ≥1.25 mg/kg/d and females administered ≥3.75 mg/kg/d, which was characterized by lower numbers of cells of myeloid and erythroid lineages.

Decreased lymphocytes were present in the gastric and rectal GALT of animals administered ≥1.25 mg/kg/d, in the GALT in the cecum and ileum of males administered ≥3.75 mg/kg/d and females administered ≥1.25 mg/kg/d, in the GALT in the jejunum of dogs administered ≥3.75 mg/kg/d, in the GALT in the colon of males administered 3.75 mg/kg/d, in the follicles of the mesenteric lymph node of males administered ≥3.75 mg/kg/d and females administered ≥1.25 mg/kg/d, and in the follicles of the mandibular lymph node and spleen of males administered ≥3.75 mg/kg/d and females administered 12.5 mg/kg/d. This was characterized by decreased numbers of lymphocytes in lymphoid follicles, decreased size or absence of germinal centers, and/or decreased numbers of lymphocytes in the follicle mantle zone. The finding of decreased lymphocytes was correlated with clinical pathology changes of decreased peripheral lymphocytes.

Ectasia of crypt/glands was present in the stomach of males administered ≥3.75 mg/kg/d, in the duodenum of males administered ≥1.25 mg/kg/d and 1 female administered 12.5 mg/kg/d, and in the rectum of 1 male administered 12.5 mg/kg/d and was characterized by focal to multifocal dilatation of glands in the mucosa, which occasionally contained necrotic cellular debris.

Decreased lymphocytes were present in the thymus of dogs administered ≥1.25 mg/kg/d which correlated with decreased thymus weight parameters in males administered ≥3.75 mg/kg/d and females administered ≥1.25 mg/kg/d. Vascular thrombi were present in villous tips of ileal mucosa in males administered ≥3.75 mg/kg/d and 1 female administered 12.5 mg/kg/d (Figure 9A-D).

Figure 9.

Histology sections (A-D): Dog. A, Normocellular bone marrow: vehicle control; B, Hypocellular bone marrow: high dose (12.5 mg/kg GNE-781); C, Normocellular mesenteric lymph node with active lymphoid follicular structures: vehicle control; D, Hypocellular mesenteric lymph node with small, inactive cortical follicular structures: high dose (12.5 mg/kg GNE-781).

Hypospermatogenesis, decreased spermatogenic cells, cell sloughing, and multinucleate cells were observed in the testicular tubular lumina in males administered GNE-781, however, the putative degenerative, compound association with these changes is equivocal due to similar changes reported in peripubertal dogs as were used in this study.²⁴

Discussion

The compound associated changes with the CBP/p300 inhibitor GNE-781 have similarities and differences from the phenotypes observed with genetic mutations of either CBP or p300. In general, GNE-781 findings were consistent with a “radiomimetic effect” in which rapidly dividing cells are the most affected, including effects on the differentiation and viability of the hematopoietic, gastrointestinal (GI), and reproductive systems. Consistent with the dosing of mature animals, the developmental effects observed with CBP-related genetic defects or gene knock-out such as neural tube defects, cardiac, or bone malformations were not present with GNE-781-mediated pharmacologic inhibition.

Consistent with murine KO phenotypes, hematopoietic effects were a predominant finding. In both dogs and rats, peripheral reticulocyte and platelet counts were moderately to markedly reduced in treated animals after treatment with GNE-781. In the rat study which incorporated a 7-day drug-free recovery period, both platelet and reticulocyte counts rebounded to normal ranges with associated microscopic evidence of increased extramedullary hematopoiesis in the spleen and liver, and bone marrow histology confirmed that megakaryocyte numbers had rebounded by the end of the recovery period. Red blood cell counts were not impacted in either species; this denotes the lack of a direct effect of GNE-781 on circulating mature erythrocytes and also the relative brevity of dosing durations. It is expected that with a longer dosing period, the circulating mature red blood cell counts would be negatively impacted by the continued lack of production of immature red blood cells.

Observations of decreased lymphocytes in various lymphoid tissues (GALT, thymus, and spleen) were also a consistent feature of GNE-781 administration in both dogs and rats. In the dog study, microscopic observations of decreased lymphoid follicles included additional findings of decreased cellularity within germinal centers and mantle zones, indicating likely B- and T-cell effects, respectively. After the 7-day recovery period in rats, decreased cellularity had partially or completely resolved in various lymphoid compartments. The myeloid phenotype was unclear due to evidence of an inflammatory process seen in both studies; inflammatory infiltrates were present in the heart in the rat study, and fibrinogen (a positive acute phase protein) was increased in dogs with elevated WBC counts. Therefore, an ability to make a conclusive attribution of myeloid findings to GNE-781 administration was not possible. Neither hypersegmented neutrophils nor pseudo Pelger-Huët anomalies, which have been observed in CBP KO mice, were identified in either species upon peripheral blood smear microscopic reviews.

Erythroid and lymphoid decreased cellularity in rats and dogs are consistent with hematopoietic findings described in CBP, p300, and/or c-Myb genetically modified mice. Decreased bone marrow cellularity and defective hemopoiesis, including decreased erythrocytes and B- and T-cells, were demonstrated in mice with monoallelic inactivation of the CBP gene compared to p300^+/− and wild type (WT) age- and genetic background-matched controls.¹² A comprehensive study utilizing mice with various compound mutations in c-Myb and/or the KIX domains of CBP and p300 led to similar erythrocyte decreases and B- and T-lymphocyte deficiencies.¹⁴ These study results firmly established the importance of the interaction between CBP/p300 KIX and c-Myb in multiple blood cell lineages and also built upon earlier c-Myb KO studies^25

–28 that had also demonstrated hematopoietic effects.

Notably decreased platelet counts were observed in both rats and dogs which demonstrated reversibility when assessed. These findings were consistent with a reversible, dose-proportional reduction in platelets reported for a 28-day preclinical study testing the small-molecule CBP/p300 inhibitor, CCS147.²⁹ Both CCS1477 and GNE-781 purportedly spare BET inhibition which has been associated with dose-limiting thrombocytopenia, neutropenia, and anemia in clinical trials.^30,31 Despite the moderate (dogs) to marked (rats) decreases in circulating platelets, there were no clinical observations of a coagulopathy observed in either our dog or rat studies. Interestingly, Morowski et al³² reduced platelet counts in mice to defined ranges between 0 and 1000 platelets/nL using platelet-depleting antibodies and demonstrated that only very low platelet counts are required to maintain hemostasis in the mouse, therefore, the situation may be similar for rats.

Platelet aggregation was also decreased in the GNE-781-treated rats in response to both a strong (collagen) and weak (ADP) agonist denoting an alteration in platelet function. These findings resolved after the 7-day recovery period. Rats typically have markedly elevated platelet counts relative to humans (eg, GNE SD rat reference range: approximately 900 to 1400 K/µL, mean approximately 1100K/µL vs human range: approximately 150 to 450 K/µL, mean approximately 300 K/µL). Therefore, despite the marked drop in platelet counts, blood from GNE-781-treated rats still met the minimum platelet count required for platelet aggregometry testing (ie, platelet counts were essentially equivalent to the “normal” range for human). It has been reported that platelets express high levels of p300 and that inhibition prevents platelet aggregate formation under shear stress, suggesting a physiological role for p300-mediated lysine acetyltransferase activity in platelet function.^33,34 The Aslan studies also support that p300 plays a role in the regulation of the platelet cytoskeleton as well as phosphatidylserine exposure to effect platelet spreading, aggregation, and thrombus formation. The current platelet aggregometry studies are consistent with this mechanistic study but do not discount the possibility of CBP-mediated contributions.

Other notable target-organ toxicities with GNE-781 include effects on the GI and reproductive systems. A weight of evidence indicated that GI effects in the dog were generally the cause of moribundity, and liquid feces was a common clinical sign throughout the study. Gastrointenstinal histologic findings were limited to gastric glandular ectasia and vascular thrombi of the ileal mucosa. In rats, there were no GI histologic findings but gross, pinpoint hemorrhagic lesions of the stomach, accompanied by dose-limiting body weight loss. Similarly, GI effects with BET inhibitors have been a commonly observed adverse clinical event and include a significant incidence of diarrhea, nausea, and vomiting; however, none of these findings were reported to be dose-limiting.³⁵ Effects on the GI system were also reported in doxycycline-inducible doxycycline-inducible transgenic BRD4 in vivo depletion/restoration studies in mice, which highlighted a loss of differentiation of secretory lineages such as Paneth and Goblet cells and depletion of the LGR5⁺ stem cell population in the intestine.³⁶ Furthermore, upon radiation challenge, these epithelia were poorly tolerant to insult compared to WT. Interestingly, cross-species studies of intestinal spheroids further clarified the discrepancies in the preclinical and clinical profiles. Notably, rats and dogs are significantly more sensitive to pharmacologic BET effects on the GI system than humans, leading to dose-limiting findings in preclinical studies such as intestinal villous atrophy, body weight loss, and inappetence.³⁷ Also, it is notable that alterations in formulation (eg, capsule, amorphous solid dispersion) were able to mitigate the moribundity, clinical signs (eg, liquid stool), and significantly increase the tolerability in dogs in a second study (Supplementary Figure 1) at exposures that would be predicted to be higher than efficacious in humans and similar to those in the initial study. Although the hematopoietic effects were similar between studies (ie, absolute platelet counts were comparably decreased (44%-62% vs 56%-65%) 7 days postdose when compared to respective individual pretest values in male dogs administered GNE-781 in a crystalline form via oral gavage in the initial study or via a capsule, amorphous solid dispersion formulation in the second study, respectively, data not shown), it is suggestive that local effects and form could also significantly influence the overall safety profile as well.

Reproductive effects in male rats included seminiferous tubular degeneration and epididymal luminal debris. In female rats, decreased size and number of CLs in the ovaries were present. This is generally consistent with literature that CBP and p300 are critical in retinoic acid receptor signaling, which is a required step for proper germ cell differentiation.³⁸ In totality, the data suggest a potential role for CBP/p300 in gametogenesis.

In summary, the current report provides novel insight into potent, pharmacologic inhibition of both CBP and p300. These pharmacologic findings are consistent with known phenotypes observed with genetic ablation of CBP and/or p300 but are devoid of the profound effects on embryogenesis and juvenile development that occur in these genetic models. Generally, the most significant effects were seen in hematopoietic, GI, and reproductive tissues; these findings are remarkably consistent with reported preclinical (and clinical) effects of BET inhibitors targeting BRD proteins. The present study findings collectively indicate a likely important role for CBP/p300 in stem cell differentiation.

Supplemental Material

Supplemental Material, CBPi_manuscript_Supplemental_Data_Table_1_(11-22-19) - Preclinical Safety Assessment of a Highly Selective and Potent Dual Small-Molecule Inhibitor of CBP/P300 in Rats and Dogs

Supplemental Material, CBPi_manuscript_Supplemental_Data_Table_1_(11-22-19) for Preclinical Safety Assessment of a Highly Selective and Potent Dual Small-Molecule Inhibitor of CBP/P300 in Rats and Dogs by Paula Katavolos, Gary Cain, Cindy Farman, F. Anthony Romero, Steven Magnuson, Justin Q. Ly, Edna F. Choo, Anand Kumar Katakam, Roxanne Andaya and Jonathan Maher in Toxicologic Pathology

Supplemental Material

Supplemental Material, maher_Supplemental_fig1 - Preclinical Safety Assessment of a Highly Selective and Potent Dual Small-Molecule Inhibitor of CBP/P300 in Rats and Dogs

Supplemental Material, maher_Supplemental_fig1 for Preclinical Safety Assessment of a Highly Selective and Potent Dual Small-Molecule Inhibitor of CBP/P300 in Rats and Dogs by Paula Katavolos, Gary Cain, Cindy Farman, F. Anthony Romero, Steven Magnuson, Justin Q. Ly, Edna F. Choo, Anand Kumar Katakam, Roxanne Andaya and Jonathan Maher in Toxicologic Pathology

Footnotes

Acknowledgments

The authors would like to acknowledge and thank Charlie Sioson (Genentech) and our colleagues at Covance-Greenfield for their diligent study oversight, and Angela Martzall (Genentech) for performing the platelet aggregation analyses. We would also like to thank Harbeen Grewel and her expert team of associates at Anshin BioSolutions Corp for their invaluable assistance in manuscript preparation. Some of the data was presented at the Applied Pharmaceutical Toxicology Meeting denoted “Dose-Limiting Thrombocytopenia in the Clinic: Strategies in the Discovery Space for Assessing Risk” on May 15, 2018.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Jonathan Maher

Supplemental Material

Supplemental material for this article is available online.

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