Over the past several years, we have evaluated various BIO 300 formulations for prophylactic dosing against H-ARS in different animal models. While parental formulations were proven safe and effective, it was essential to develop a formulation that is suitable for oral administration in an austere environment, stable at ambient temperature, and highly effective and safe upon administration. To date, the BIO 300 OP formulation appears to satisfy all these requirements. As part of the development of a medical countermeasure, it is imperative to determine the metabolic consequences of drug administration as a measure of safety and efficacy using an NHP model. Herein, we utilized UPLC QTOF-MS based metabolomics and lipidomic profiling to gain insights into metabolic consequences of BIO 300 OP treatment in NHPs. We found transient alterations in phenylalanine, tyrosine, glycerophosphocholine, and glycerophosphoserine after 4 h of drug treatment that reverted to near pre-treatment levels within 36 – 48 h after drug administration. In addition, there was an overlap in the metabolite classes including palmitamide and oleamide measured in sera from these data compared to that obtained from NHPs administered an im dose of BIO 300 IS and an equivalent dose of BIO 300 OS that we have reported previously33. This initial assessment highlighted the importance of using various bioanalytical techniques to interrogate underlying pharmacological actions of the drug and predict drug toxicity. Of note, we did not observe significant changes in the metabolic profile following a single BIO 300 im dose of 200 mg/kg. In part, this could be due to the small number of animals (2 males and 2 females) or the modulation of regulatory pathways following the higher dose of BIO 300 OP that abrogate the signals observed following the lower BIO 300 OP dose. The intended dosing regimen for BIO 300 OP is 6 days of consecutive prophylactic dosing; thus, it will be important to analyse the metabolic profile following 6 days of consecutive BIO 300 OP administration in future studies.
While PK parameters for a medical countermeasure candidate are very crucial for a better understanding of the mechanisms of absorption, distribution, and elimination of the drug, dose optimization remains a determining factor for the FDA approval process. Analysis of the PK data, obtained from measuring the serum levels of the drug throughout the study for both tested doses, indicate that there was no influence of the dose on the time to maximum plasma concentration (Tmax). Also, the time for half the maximum concentration (T1/2) remains the same. Interestingly, there is an apparent shorter Tmax in males compared to females following either of the BIO 300 OP doses. Although this observation was made in only two animals for each sex, it may be correlated with sex-specific PK parameters. The differential mean gut transit time is shorter in males compared to females which could lead to a faster absorption time in males compared to females40,41. If this is the case, the shorter Tmax in males may constitute a potential safety factor that requires further investigation. One potential implication of this could be that males may require a different dosing regimen (e.g., more frequent dosing) than females. However, one important limitation in our current study was that the PK profile was only examined in two females and two males at each of the BIO 300 OP doses. Confirmatory studies are required to fully investigate potential sex-specific differences in PK.
The average Cmax value between groups was nearly quadrupled by doubling the BIO 300 OP dose, while the AUC was only tripled by doubling the dose. Of note, this observation was driven by a single animal in the BIO 300 OP 200 mg/kg group. Overall, compared to PK parameters of BIO 300 OS in NHPs, BIO 300 OP demonstrated an enhanced bioavailability. At an equivalent dose (100 mg/kg), BIO 300 OP had a 2X higher Cmax, and 1.3X higher AUC compared to BIO 300 OS33. Interestingly, BIO 300 OP appears to be absorbed faster and eliminated more rapidly compared to BIO 300 OS, as evidenced by the 1-h shorter Tmax and the roughly 1-h shorter half-life (T1/2)33.
Both OP doses resulted in very similar metabolomic profiles compared to the pre-dose samples. However, a slight difference between the tested doses was observed in the 4 h and later time points. We found transient downregulation of several metabolites including methyl linolenate, phosphocholine, palmitamide, oleic acid, oleoyl ethylamide, and oleamide. This pattern was comparable to our previous finding in which we reported a transient perturbation around the 4 h time point in the metabolic profiles of C16 sphinganine following the administration of an equivalent BIO 300 OS dose and of glutamate, suberic acid, sphingosine-1-phosphate as well as nonandioic acid following administration of a single BIO 300 IS dose33. This could be due to a natural rebound effect caused by the drug’s elimination from the blood. This temporal effect was transient and was not correlated with the identification of any safety-related features.
A few metabolites were modestly upregulated after administration of the BIO 300 OP formulation including C18(Plasm)-20:4 PE and 16:1 (DELTA 9-CIS) PC. Meanwhile, in our earlier PK and metabolomic studies with BIO 300 when administered orally and im, we observed a slight upregulation around the same time in taurocholic acid and tauroursodeoxycholic acid which appeared after a single im dose of the drug33. This could be attributed to transient changes in lipid biosynthesis, activation, and metabolism. These findings were supported by our pathway analysis that we performed on all detected features utilizing Mummichog 2.06v software. Furthermore, statistical analyses built on the annotated metabolites indicate that there is a statistically significant difference between the doses of BIO 300 OP evaluated. Also, time seems to be crucial since most of the transient changes appear to revert to baseline by 48 h post-drug adminitration, which correlates with the clearance of the drug.
Assessment of the bioavailability of the two BIO 300 OP doses demonstrates an expected PK pattern with an expected dose dependent increase in the Cmax and AUC values at the higher dose. Longitudinal metabolomic investigations show transient metabolic changes that occur 4 – 8 h post drug administration which seem to stabilize over time (by 48 h). Moreover, no changes in the metabolome impacted safety and all noted changes were transient in nature, which reaffirms the safety profile and concurs with previously reported preclinical experimental results. BIO 300 is an ideal candidate for further validation assessments as a radiation countermeasure for the prevention of H-ARS.