Murine Toxicology and Pharmacokinetics of Lead Next Generation Galeterone Analog, VNPP433–3β

Abstract

VNPP433–3β (compound 2, (3β-(1H-imidazole-1-yl)-17-(1H-benzimidazole-1-yl)-androsta-5,16-diene), a multitarget anticancer agent has emerged as our lead next generation galeterone analogs (NGGA). Compound 2 is currently in development as potential new therapeutic for prostate and pancreatic cancers. The preliminary toxicity study reveals that the compound 2 was better tolerated by the normal male CD-1 mice than the male Nude mice. The maximum tolerated dose (MTD) in the Nude mice was estimated to be between 25 < 50 mg/kg. After oral dosing of compound 2 to male and female rats, the plasma concentration versus time curves were very consistent between animals and the AUC_last increased with dose. Many plasmas concentration versus time curves profiles were nearly flat over 24 hr., suggesting extended absorption from the GI tract. Consequently, reliable values for half-life and AUC_inf were not determined. Calculated oral bioavailability (using oral AUC_last and excluding the outlier IV animal) ranged from 32–47%. This should be considered a minimum value since the contribution to true AUC beyond 24 hr. is clearly not zero. Clearly, these toxicology and pharmacokinetics parameters pave the way for understanding the anticancer pharmacological actions and provide a meaningful basis for further preclinical development and eventual clinical development.

Graphical Abstract

1. Introduction:

New treatment modalities are urgently needed to prolong the time to development of castration-resistant prostate cancer (CRPC), attack CRPC and metastatic disease (mCRPC) and to improve the quality of life of men suffering from advanced prostate cancer (PC). The progression to CRPC, metastatic disease and resistant to therapy correlates with gain-of-function of androgen receptor (AR) [both full-length (fAR) and splice variants, especially, AR-V7 and AR^v567es] [1–8] and hyper-activation of eukaryotic translation initiation factor 4E (eIF4E), the nexus where the well-established PI3K/Akt/mTOR and RAS/MAPK oncogenic signaling pathways converge [9–13].

We recently developed clinical candidate Galeterone (Gal, 1, Fig. 1) which disrupts AR signaling via multiple mechanisms of action [reviewed in [14] ]. Gal progressed into pivotal Phase III clinical studies in patients with mCRPC [14–16]]. However, the disappointing termination of ARMOR3-SV trial and the required 2550 mg/day high therapeutic dose of Gal (because of its low oral bioavailability and short half-live) underscores the need to further systematic refinements to enable development of next generation galeterone analogs (NGGAs) with enhanced efficacies, improved pharmaceutical properties and high therapeutic indices at low dose-administration expected to result in safer, more effective PC therapy, in addition to lowering costs of drug production.

Fig. 1.

Chemical structures of Galeterone (1), VNPP433-3β (2), VNPT-155 (3) and VNPP414 (4)

During studies to develop more potent androgen receptor degraders (ARDs) to modulate AR signaling in PC models [17] , we modified structure of Gal at C3, C16 and C17 positions. Among the C3 modifications we designed the imidazole substituted compound (3β-(1H-imidazole-1-yl)-17-(1H-benzimidazole-1-yl)-androsta-5,16-diene (VNPP433–3β, 2, Fig. 1)) as an alternate moiety for the 3β-hydroxyl group of Gal to maintain hydrophilic interactions with active site of the biological targets.

We envisioned that this modification would avoid metabolic modification by steroidogenic enzyme, 3β-hydroxysteroid dehydrogenase (3βHSD). In our initial synthetic efforts to synthesize VNPP433–3β (2), we serendipitously discovered Gal 3β-imidazole carbamate (3, Fig. 1) [17]. We discovered that Gal and the novel more efficacious ARDs also effectively target oncogenic eukaryotic protein translation, via modulation of Mnk-eIF4E axis [18]. These agents also strongly suppress expression of oncogenic peIF4E via degradation of Mnk1 and 2 and as such are also referred to as Mnk1/2 degraders (MNKDs) (reviewed in [19]).

In recent studies, we successfully synthesized and evaluated VNPP433–3β and 3β-(pyridine-4-ylmethoxy)-17-(1H-benzimidazol-1-yl)androsta-5,16-diene (VNPP414, 4, Fig. 1) [20]. These two compounds exhibited improved in vitro anticancer activities over Gal [20], which led to additional studies that enabled improved procedures for their gram-scale syntheses [21]. Due to the chemical nature of the C-3 moieties (absence of Δ⁵, 3β-hydroxyl structure) of these two lead compounds (Fig. 1), we predicted that they are likely to be metabolically more stable than Gal. It is important to state here that in addition to our earlier report of the pharmacokinetics of Gal following subcutaneous (SC) administration to male SCID mice [22], there is now extensive literature on the pharmacokinetics and metabolism of Gal [23] and structurally related prostate cancer drug, abiraterone (Abi) [24, 25] in mice. These two compounds are metabolized mainly by two steroidogenic enzymes, 3βHSD and steroid-5α-reductase (SRD5A).

We have reported that compound 2 exhibited improved pharmacokinetic profile over Gal in mice and as expected, 2 (at 7.53-fold lower equimolar dose than Gal) markedly suppressed (84% vs. Gal, 47%; p < 0.01) the growth of castration-resistant PC (CRPC) CWR22Rv1 xenograft tumors, with no apparent host toxicity and was more efficacious than enzalutamide [26]. Because of the impressive and promising anti-PC activities, compound 2 was selected to receive targeted resources from National Institutes of Health’s National Cancer Institute (NCI) through a new program called ‘Stepping Stones’ to advance compound 2 towards clinical trials (https://dtp.cancer.gov/discovery_development/stepping_stones/default.htm). Recently, we reported the large-scale synthesis of Gal and VNPP433-3β (2), with support from the ‘Stepping Stones’ Program [27]. We have also reported that VNPP433-3β (2) inhibits PC stem cells by downregulating epithelial-mesenchymal transition and stem cell marked [28], and on its molecular mechanisms of action in AR-overexpressing CRPC in vitro and in vivo models [29].

In this manuscript, we report for the first time on the acute toxicity in male CD-1 normal male mice, acute and chronic toxicological profile of VNPP433-3β (2) in male Nude mice, the model used for the antitumor efficacy assessments, and the oral and intravenous pharmacokinetics and bioavailability in male and female rats.

2. Experimental section

2.1. Chemicals and reagents:

(3β-(1H-imidazole-1-yl)-17-(1H-benzimidazole-1-yl)-androsta-5,16-diene (VNPP433-3β, 2) was synthesized in our laboratory as previously reported [27, 30]. Abiraterone was purchased from Sigma-Aldrich, St. Louis, MO, USA.

2.2. Animal Studies

The studies with animals were conducted at three locations, including University of Maryland School of Medicine, Baltimore, USA, GVK Biosciences Pvt. Ltd. (now called Aragen Life Sciences Private Limited (Aragen), Hyderabad, India, and University of Pittsburgh, Pittsburgh, PA, USA

2.2.1. Animal handling at University of Maryland School of Medicine, Baltimore:

All animal studies were performed according to the guidelines approved by the Animal Care Committee of the University of Maryland, School of Medicine (UMSOM), Baltimore. Male Nude mice 5–6 weeks of age were obtained from the National Cancer Institute (Fredrick, MD) and were fed autoclaved pellets and sterile water ad libitum. Mice were allowed to acclimate to the UMSOM Animal Facility for at least 5 days before use.

2.2.2. Animal handling at GVK Bio:

The study was conducted at GVK BIO., Hyderabad, India, in accordance with Study Protocol No.: 045–16- IPH. This study was performed with approval from the Institutional Animal Ethics Committee (IAEC) in accordance with the requirement of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. The study was not performed as per Good Laboratory Practices (GLP) regulations and was not audited by Quality Assurance; however, all appropriate study documentation including study plan and raw data are maintained in the study file. Study phases, generated data and the report were verified for accuracy by the study group. Male CD-1 mice 9–10 weeks old were used for the acute toxicology dosing study. Mice were allowed to acclimate to the GVK BIO Animal Facility for at least 5 days before use.

2.2.3. Animal handling at University of Pittsburgh:

Specific pathogen-free, adult Sprague Dawley male and female rats with catheterized jugular veins were purchased from Charles River (Wilmington, MA, USA). Rats were allowed to acclimate to the UPMC Hillman Cancer Center Animal Facility for at least 5 days before being used. To minimize infection, rats were maintained in micro-isolator cages in a separate room and handled in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996) and on a protocol approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. Ventilation and airflow were set to 12 changes per hour. Room temperatures were regulated at 22 ± 1 °C, and rooms were kept on automatic 12 h light/dark cycles. Rats received Prolab ISOPRO RMH 3000 Irradiated Lab Diet (PMI Nutrition International, St. Louis, MO, USA) and water ad libitum. Rats were 8–12 weeks old at the time of dosing and were stratified into 3 groups of 3 animals each per sex. Rats received a single dose of compound 2 either 1 mg/kg IV, 10 mg/kg PO or 50 mg/kg PO (with curved blunt tip oral gavage needle) at a dosing volume of 10 ml/kg body weight. Drug was formulated as a solution in freshly prepared chremophor:ethanol:saline (1:1:8), pH 4.0 (with acetic acid). At 5, 15, 30 min, and 1, 2, 4, 6, and 24 h post-dose, EDTA blood was collected and processed to obtain plasma by centrifugation at 12,000 × g for 4 min.

2.3. Bioanalysis and Pharmacokinetics

Bioanalysis was performed with a generic assay approach as reported previously [31]. In brief, the assay utilized 50 μL of plasma and 8 potential internal standards (IS), of which abiraterone was selected for use. Preparation consisted of acetonitrile protein precipitation (150 μL) and aqueous dilution (125 μL supernatant and 500 μL water) in a 96 well-plate format. Chromatographic separation was achieved with a Kinetex C18 reverse phase (2.6 μm, 2 mm × 50 mm) column and a gradient of 0.1 % formic acid in acetonitrile and water over an 8 min run time. Mass spectrometric detection was performed on an AB SCIEX4000QTRAP with electrospray, positive-mode ionization. One microliter of supernatant was injected into the LC-MS/MS system. The approximate retention time of 2 was 2.71 min and for the internal standard (abiraterone) was 3.09 min, respectively. The MRM m/z transitions monitored were 439.2>371.2 for 2 and 350.0>156.2 for abiraterone. In both analytical runs, calibrators, and QC’s met FDA criteria. PK parameters were estimated using standard noncompartmental methods with Phoenix WinNonlin (Certara, Princeton, NJ).

3. Results and Discussions:

3.1. Acute and Chronic Toxicity Evaluation in Male Nude Mice

Prior to investigating the in vivo anti-tumor efficacy of compound 2, we evaluated the maximum tolerable doses that could be used in the study. First, we performed an acute dosing study, where male nude mice were injected intraperitoneally once with 25, 50 or 100 mg/kg body weight of 2 formulated in 40% β-cyclodextrin in saline and observed for two weeks. Considering the efficacy of 2 in vitro and the fact that it is several folds more efficacious than gal, we decided to use 100 mg/kg body weight as the highest dose. Body weights were taken twice a week and animals examined for any adverse effects (inactivity, skin changes, or bleeding). One of the three mice in the 100 mg/kg group died on the second day after injections. The rest of the animal in groups 25 and 50 mg/kg did not exhibit any signs of toxicity. From the overall average body weights and physical appearance of mice there were no apparent toxicities from the treatment (Fig. 2A).

Fig. 2:

Mean body weights of nude mice (A) following treated with one bolus of compound 2 and (B) during chronic dosing of compound 2. Note: * It should be noted that one mouse died in the 100 mg/kg group and the mean body weight in this group was of two mice. ns = not significant.

Results from the acute dosing indicated that 100 mg/kg body weight of 2 was tolerated well by the two mice in that group. Hence, we decided to use 100 mg/kg as the maximum tolerable dose (MTD) for the next phase in determining the viable dose for in vivo studies. However, two astute reviewers brough to our attention that because one mouse died in the 100 mg/kg group, our assumption of 100 mg/kg MTD was incorrect. Based on the correct definition of MTD, the MTD of compound 2 in our acute dosing study is 50 mg/kg, and we have revised Fig. 2B accordingly. Thus, we performed a chronic dosing administering 0.2, 0.5 and MTD, five days a week for two weeks. Body weights were taken as before, twice a week and any adverse conditions noted (Fig. 2B). After 14 days, experiment was terminated, and animals sacrificed. Two mice died in the 0.5 MTD group on day 7. All other groups looked healthy, including the lone mice in group MTD. Based on the acute and chronic dosing results, we estimate that the MTD for 2 in nude mice is between 25 and <50 mg/kg body weight. However, more rigorous toxicology studies are required to characterize compound 2 adverse effects.

3.2. Acute Toxicity Evaluation in Male CD-1 Mice

Because the nude mice are immunocompromised, we conducted another MTD study in normal immunocompetent male CD-1 mice. Compound 2 was formulated in 20% β-cyclodextrin in saline, resulting in milky white thick suspensions. One vehicle control group and three dose groups (3 mice per group) were in the study i.e., low (100 mg/kg), medium (300 mg/kg), and high (1000 mg/kg) dose groups via oral gavage (PO). Animals were dosed 100 mg/kg and observed for 72 hrs. Body weights were taken on day 0, 1, 2, and 3 and any adverse conditions noted (Fig. 3). This dose was well tolerated, and no mortality was observed. Mice in the medium group were dosed 300 mg/kg and observed for 72 hrs. Thereafter, the high dose was opted, and animals were dosed with 1000 mg/kg and observed till mortality. All mice in the low and medium dose groups were normal after dosing. After 72 hrs., the animals were necropsied, and no signs of toxicity were observed. This study was stopped at day 3 based on the approved rat acute toxicity study of GVK BIO, a renown Certified Research Organization (CRO).

Fig. 3:

Mean body weights of CD-1 mice on day 0, 1, 2 and 3 following treatments (oral gavage, PO) with three doses of compound 2. * Note: For the group dosed with 1000 mg.kg of compound 2, two mice died on days 2 and the third mice died on day 3.

All three animals in the 1000 mg/kg group were normal after dosing. However, after 24 hrs. animals exhibited some clinical signs like piloerection, apathy (inactive) and hypothermia. Changes in gait were observed as well. Two mice were in a moribund stage and found dead 36 hrs. post dose, the third mice died 72 hrs. post dose. Dead animals were necropsied and accumulation of compound 2 in stomach and intestine was observed. Gross necropsy of mice in all three groups postmortem revealed no evident organ toxicity, however the clinical signs at 1000 mg/kg dose seemed toxic to animals. Thus, the NOAEL (No Observed Adverse Effect Level) was greater than 300 mg/kg via oral gavage. To further study 2 organ toxicity/safety profile, repeated dose toxicity studies will be conducted in the future.

3.3. Plasma concentration pharmacokinetic parameter in male and female rats

For IV dosing, the concentration versus time profiles of VNPP433-3β in male and female rats is presented in Fig. 4 and the PK parameters are presented in Table 1. No overt toxic effects were observed during conduct of this PK study. The third male rat dosed at 1 mg/kg IV had a strikingly deviant profile relative to the other 2 rats in that group. The rise in plasma levels is slower than that observed in rats dosed PO, and therefore likely represent extravascular dosing. Summary statistics therefore excluded this rat. After IV dosing, C0 values were similar for both sexes, confirming similar initial volumes of distribution. Half-lives were in the same order of magnitude between the sexes, with large variability in the males. AUC_0-inf was moderately captured with AUC_0-last. Clearance values were generally in the 5–7 mL/min/kg range with the male clearance being higher, agreeing with the shorter half-life. Volume of distribution was ~6 L/kg, suggesting extensive tissue distribution.

Fig. 4.

Concentration vs. time profiles of compound 2 dosed intravenously to (A) female and (B) male rats at 1 mg/kg. Note: * LLOQ = lower limit of quantification.

Table 1.

PK parameters of compound 2 dosed intravenously to male and female rats at 1 mg/kg.

SEX	Dose (mg/kg)	C0 (ng/mL)	Cmax (ng/mL)	Cmax/D ng/mL / mg/kg)	Tmax (min)	T1/2 (h)	AUClast (min•μg/mL)	AUCinf (min•μg/mL)	CL (mL/min/kg)	Vz (L/kg)	Vss (L/kg)
F	1	259	296	296	15	14.1	168	248	4.04	4.93	4.96
		342	291	291	5.0	15.0	140	199	5.03	6.51	5.99
		418	350	350	5.0	13.8	126	171	5.85	6.98	6.31
GeoMean		333	311	311	7.21	14.3	144	203	4.92	6.08	5.73
GeoSD		1.27	1.11	1.11	1.89	1.0	1.16	1.20	1.20	1.20	1.13
M	1	254	237	237	5.0	2.96	46.6	64.0	15.6	4.00	4.14
		443	392	392	5.0	31.4	139	321	3.11	8.46	8.14
		29.7*	94.8*	94.8*	240*	2.32*	20.2*	30.6*	32.7*	6.55*	11.0*
GeoMean		336	305	305	5.00	9.64	80.4	143	6.97	5.82	5.81
GeoSD		1.48	1.43	1.43	1.00	5.3	2.16	3.13	3.13	1.70	1.61

Note:

^*Excluded from summary statistics. C₀: extrapolated plasma concentration at time 0; C_max: maximum observed plasma concentration; T_max: time to maximum concentration; T_1/2: elimination half-life; AUC_last: area under the concentration-time curve from dosing (time 0) to the time of the last measured concentration; AUC (0−∞): area under the concentration-time curve from time of dosing extrapolated to infinity; CL: clearance; Vz: apparent volume of distribution during the terminal phase; Vss: steady state volume of distribution

The concentration versus time profiles of 2 in male and female rats following oral dosing at 10 mg/kg or 50 mg/kg is presented in Fig. 5 and the PK parameters are presented in Table 2. After oral dosing, PK was quite consistent as suggested by low geometric SD values. All profiles suggest continued absorption, likely through the last time point of 24 h. Not enough data is available to definitively conclude it, but there may be absorption rate limited elimination. In agreement with slightly higher clearance in males, indeed, the male PO profiles peak at 6 h, while the female PO profiles peak later. Bioavailability estimates were based on PO AUC_0-last values, given that the extrapolated percentage of AUC_0-inf was so large (17–82% and beyond). Even this underestimate of oral absorption suggested robust absorption of at least 32–34% (at 10 mg/kg) to 47% (at 50 mg/kg), with exact values likely much higher. Absorption was remarkably similar between sexes.

Fig. 5.

Concentration vs. time profiles of compound 2 dosed orally to female and male rats at 10 mg/kg (A and B) or 50 mg/kg (C and D). Note: * LLOQ = lower limit of quantification.

Table 2.

PK parameters of VNPP433-3β dosed orally to male and female rats at 10 or 50 mg/kg.

SEX	DOSE_1 (mg/kg)	Cmax (ng/mL)	Cmax/D (ng/mL / mg/kg)	Tmax (min)	T1/2 (h)	AUClast (min•μg/mL)	AUCinf (min•μg/mL)	AUC% Extrap (%)	CL/F (mL/min/kg)	Vz/F (L/kg)
F	10	755	75.5	360	57	896	3870	76.9	2.58	12.7
		408	40.8	360	120	484	4230	88.6	2.37	24.1
		681	68.1	360	67	785	4090	80.8	2.45	14.3
GeoMean		594	59.4	360	76.7	698	4060	81.9	2.46	16.3
GeoSD		1.39	1.39	1.00	1.5	1.38	1.05	1.07	1.05	1.41
F	50	4820	96.4	1400	*	5560
		4320	86.4	1400	*	4300
		4380	87.6	1400	*	4610
GeoMean		4500	90.0	1440		4800
GeoSD		1.06	1.06	1.00		1.14
M	10	702	70.2	360	9.8	519	686	24.4	14.6	12.4
		460	46.0	360	6.3	329	364	9.55	27.5	15.0
		715	71.5	360	9.0	577	715	19.3	14.0	10.9
GeoMean		613	61.3	360	8.22	462	563	16.5	17.8	12.6
GeoSD		1.28	1.28	1.00	1.3	1.35	1.46	1.63	1.46	1.18
M	50	1870	37.4	1400	*	2380
		3430	68.6	360	260	4460	78400	94.3	0.638	14.4
		3160	63.2	360	37	3600	10900	66.9	4.60	14.8
GeoMean		2730	54.5	571	98.6	3370	29200	79.5	1.71	14.6
GeoSD		1.39	1.39	2.23	4.0	1.38	4.04	1.27	4.04	1.02

Note:

^*Negative half-life due to the last concentration still being higher than the preceding concentration, indicating more prolonged absorption of drug, resulting in incomplete capture of data with the sampling scheme used. C₀: extrapolated plasma concentration at time 0; C_max: maximum observed plasma concentration; T_max: time to maximum concentration; T_1/2: elimination half-life; AUC_last: area under the concentration-time curve from dosing (time 0) to the time of the last measured concentration; AUC (0−∞): area under the concentration-time curve from time of dosing extrapolated to infinity; CL: clearance; Vz: apparent volume of distribution during the terminal phase; Vss: steady state volume of distribution.

4. Conclusion

Herein, we report for the first time on the acute toxicity in male CD-1 normal male mice, acute and chronic toxicological profile of compound 2 in male Nude mice, and the pharmacokinetics in male and female rats which have human-like physiology. The PK parameters for compound 2 in rats observed in this study are like those previously reported by us in mice [26]. Given the highly potent anticancer efficacy of compound 2 [26, 28, 32], the toxicity and PK profiles reported here provides confidence that the compound can be further developed as potential new anticancer drug.

Highlights:

• A generic assay LC-MS/MS approach was utilized to detect the concentration of VNPP433-3β (2) in rat plasma.

• Pharmacokinetics of VNPP433-3β (2), a potent anti-cancer agent, in male and female rats, has been accomplished.

• The toxicity profile of VNPP433-3β (2) in CD-1 and Nude mice has been address.

• The maximum tolerated dose of VNPP433-3β (2) in Nude mice has been determined.

List of Abbreviations/Acronyms and Their Definitions

Abi

Abiraterone

AR

Androgen Receptor

ARD

Androgen Receptor Degraders

AUC

Area Under the Curve

CL

Clearance

CRPC

Castration-Resistant Prostate Cancer

EDTA

Ethylenediaminetetraacetic Acid

eIF4E

Eukaryotic Translation Initiation Factor 4E

fAR

Full-length Androgen Receptor

Gal

Galeterone

GI

Gastrointestinal

IV

Intravenous dosing

LC/MS/MS

Liquid Chromatography/Tandem-Mass Spectrometry

LLOQ

Lower Limit of Quantification

mCRPC

Metastatic Castration-Resistant Prostate Cancer

Mnk1/2

MAP Kinase-Interacting Serine/Threonine-Protein Kinase 1 and 2

MNKD

Mnk1/2 Degraders

MS

Mass Spectrometry

MTD

Maximum Tolerated Dose

NGGA

Next Generation Galeterone analogs

NOAEL

No Observed Adverse Effect Level

PC

Prostate Cancer

p-eIF4E

Phospho- Eukaryotic Translation Initiation Factor 4E

PK

Pharmacokinetics

PO

Oral Dosing

SC

Subcutaneous

SRD5A

Steroid-5α-Reductase

T_1/2

Elimination half-life

T_max

Time to Maximum Concentration

Vss

Steady state volume of distribution

Vz

Apparent volume of distribution during the terminal phase