Preclinical Development of Inhalable d-Cycloserine and Ethionamide To Overcome Pharmacokinetic Interaction and Enhance Efficacy against Mycobacterium tuberculosis

We compared the pharmacokinetics and efficacy of a combination of d-cycloserine (DCS) and ethionamide (ETO) via oral and inhalation routes in mice. The plasma half-life (t_1/2) of oral ETO at a human-equivalent dose decreased from 4.63 ± 0.61 h to 1.64 ± 0.40 h when DCS was coadministered.

ABSTRACT

We compared the pharmacokinetics and efficacy of a combination of d-cycloserine (DCS) and ethionamide (ETO) via oral and inhalation routes in mice. The plasma half-life (t_1/2) of oral ETO at a human-equivalent dose decreased from 4.63 ± 0.61 h to 1.64 ± 0.40 h when DCS was coadministered. The area under the concentration-time curve from 0 h to time t (AUC_0–t) was reduced to one-third. Inhalation overcame the interaction. Inhalation, but not oral doses, reduced the lung CFU/g of Mycobacterium tuberculosis H37Rv from 6 to 3 log₁₀ in 4 weeks, indicating bactericidal activity.

TEXT

Rationale for the combination.

DCS and ETO are second-line drugs used to treat multidrug-resistant tuberculosis (MDR-TB). DCS is an analog of d-alanine and interferes with bacterial cell wall synthesis and maintenance (1). It is administered orally at 250-mg doses, two to three times daily, but is often unacceptably toxic (1). ETO is administered in three to four divided doses up to 1,000 mg/day. It forms covalent adducts with NAD in Mycobacterium tuberculosis (2). The two agents are considered bacteriostatic, and the World Health Organization does not recommend incorporating both together in a treatment regimen for MDR-TB. However, the clinical relevance of designating antimicrobial agents as bacteriostatic or bactericidal on the basis of pharmacodynamics (3) or in vitro mechanism of action (4) has been questioned in several infectious diseases (5). Both drugs are inexpensive and are preferred in resource-poor settings. Their different mechanisms of action suggest that, in combination, the two might exhibit bactericidal effects and may even give rise to pharmacokinetic synergy. Pulmonary drug delivery is often attempted to overcome poor oral bioavailability or to avoid parenteral administration. Undesirable pharmacokinetics following oral administration receives less attention but is important in the context of targeting therapeutic doses to specific anatomical sites. A further benefit of administering inhalations may lie in the fact that ETO is a prodrug that is converted into its active sulfoxide metabolites by the enzyme FMO2, available predominantly in host lung tissue. Hence, bacteria residing in the lungs would be exposed to the high concentration of the active metabolite (6). Pulmonary delivery of ETO alone has been recently reported (6). A fixed-dose inhaled combination of the two drugs might therefore be useful (7).

Potential interactions.

While considering the use of drug combinations, it is important to rule out chemical or pharmacokinetic drug-drug interactions. For example, processing isoniazid (INH) and rifampin (RIF) together led to the formation of an adduct (8). Antagonism between INH, RIF, and pyrazinamide has been observed in mice. The likely cause of the apparent antagonism is the effect of INH on the area under the concentration-time curve (AUC) and on the maximum concentration of a drug in serum (C_max) of RIF, both of which decreased in the presence of INH (9). Methods of allometric scaling permit extrapolation of results obtained with mice to human beings (10), unless species differences in absorption (11) and metabolism (12) indicate otherwise.

Scope of work.

We compared preclinical pharmacokinetics and efficacy of DCS and ETO via the oral and pulmonary routes. This otherwise routine exercise yielded unexpected results in respect of a pharmacokinetic interaction not reported earlier to our knowledge.

Inhaled dose.

Animal experiments were performed with the approval of the Institutional Animal Ethics Committee (IAEC approval number IAEC/2017/F-250). Please see the Supplemental Material for details regarding the methods. Estimation (13) of concentrations in the bronchioalveolar lavage fluid and lung homogenate obtained immediately after inhalation from three mice confirmed that 65.5 ± 10.5 μg of ETO and 33.3 ± 0.4 μg of DCS were inhaled by mice within 30 s of exposure to inhalable powder containing DCS:ETO:l-leucine (1:2:1.4) (13) administered using a hand-held inhalation apparatus (14).

Pharmacokinetics of a single oral dose in mice.

At human-equivalent doses (HED) of 2.35 mg of DCS and 4.7 mg of ETO, both drugs were rapidly absorbed and eliminated (Fig. 1, Fig. S1). The time to attain maximal concentration (T_max) was determined as 0.5 to 1 h. Although the dose of ETO was twice that of DCS, the AUC from 0 h to time t (AUC_0–t) values for both drugs were comparable (227.2 ± 43.6 μg/ml × h for ETO and 243.7 ± 18.3 μg/ml × h for DCS). A pharmacokinetic interaction between ETO and DCS was suspected, which was further investigated by administering the drugs, either alone at HED, or in combination with an excess of one of the two drugs. The concentration-time graphs of oral ETO alone or in combination with DCS highlight the differences in plasma concentrations over time as shown in Fig. 1

FIG 1.

Plasma concentration profile of ETO given alone (curve 1) or in combination with DCS (curve 2). Scatter points represent data from three individual animals at different time points. Solid lines represent a one-compartment model fitted curves to group means, and error bars show the standard deviations (SD) from fitted points (n = 3).

DCS alone versus DCS coadministered with ETO.

The pharmacokinetic profile of DCS also indicated interaction with ETO. The AUC_0–t of DCS_(alone) was 189.1 ± 35.5 μg/ml × h compared to 243.7 ± 18.3 μg/ml × h and 299.2 ± 40.8 μg/ml × h when administered at a human-equivalent dose or with excess ETO. The MRT of DCS_{(excess ETO)} increased by more than two times against DCS_(alone). As shown in Tables S1 to S5 in the supplemental material, similar trends of tissue concentrations of DCS were observed in different organs. With DCS_(alone), the AUC_{(0–t, lungs)} was 60.2 ± 8.3 μg/g × h as against 135.1 ± 5.7 μg/g × h when given as DCS_{(excess ETO)}. The AUC_{(0–t, spleen)} was 66.1 ± 6.6 μg/g × h against 193.5 ± 19.5 μg/g × h, and the AUC_{(0–t, brain)} was 31.6 ± 10.9 μg/g × h against 66.1 ± 11.6 μg/g × h when given as DCS_{(excess ETO)}.

Single inhalation.

Lung concentrations obtained after pulmonary delivery indicated that higher concentrations of drugs were achieved in the lungs compared to oral administration. The AUC_{(0–t, combi)} with respect to DCS was 111.9 ± 24.6 μg/g × h, while for ETO it was 170.3 ± 24.9 μg/g × h. It is important to reiterate that there was a large difference between the amounts (doses) of orally administered and inhaled drugs. Thus, 4.7 mg of ETO and 2.35 mg of DCS were administered orally to each mouse, while the inhaled doses were about 70 μg of ETO and 30 μg of DCS per animal.

Preclinical pharmacokinetic parameters.

Selected pharmacokinetic parameters calculated from one-compartment model fitting are shown in Table 1 . The pharmacokinetic interaction is clearly discernible. No attempts were made to establish pharmacodynamic indices.

TABLE 1.

Pharmacokinetic parameters of DCS and ETO in plasma upon oral administration of formulation variants to mice (n = 3 per time point)^a

Parameter	t1/2ka (h)	t1/2k10 (h)	Tmax (h)	Cmax (μg/ml)	AUC0–t (μg/ml × h)	AUC0–inf (μg/ml × h)	AUMC (μg/ml × h2)	MRT (h)
ETO(alone)	0.15 ± 0.17	4.63 ± 0.61	0.70 ± 0.56	86.6 ± 6.0	609.3 ± 16.9	643.2 ± 18.2	4,445.5 ± 479.3	6.91 ± 0.68
ETO(combi)	0.08 ± 0.01	1.64 ± 0.40	0.36 ± 0.02	83.3 ± 5.2	227.2 ± 43.6	229.4 ± 46.0	584.5 ± 248.8	2.48 ± 0.55
ETO(excess DCS)		2.22 ± 0.55b	0.83 ± 0.14	115.6 ± 13.2	1,075.9 ± 152.9	1,079.2 ± 152.2	6,910.5 ± 1,606.5	6.35 ± 0.63
DCS(alone)	0.09 ± 0.07	1.38 ± 0.62	0.32 ± 0.15	81.9 ± 8.8	189.0 ± 35.5	189.4 ± 35.9	420.1 ± 222.8	2.12 ± 0.79
DCS(combi)	0.05 ± 0.01	1.54 ± 0.34	0.25 ± 0.03	99.8 ± 11.5	243.7 ± 18.3	243.7 ± 18.3	562.5 ± 155.4	2.28 ± 0.47
DCS(excess ETO)	0.44 ± 0.49	3.23 ± 1.82	1.04 ± 0.53	52.2 ± 12.8	299.2 ± 40.8	304.0 ± 45.7	1,645.3 ± 821.5	5.29 ± 1.99

^aTwo-compartment analysis was carried out using PKSolver reported by Zhang et al. (17). Abbreviations: t_1/2ka, absorption half-life; t_1/2k10, elimination half-life; AUC, area under the concentration-time curve; AUMC, area under the first moment curve; MRT, mean residence time.

^bThe t_1/2 value is shown.

Findings from pharmacokinetic data.

Although no claim is made about the observed drug-DCS interfered significantly with the pharmacokinetics of ETO, but not vice versa, when both were administered orally in combination. The AUC_0–t of ETO in combination with DCS was nearly three times lower than the AUC_0–t of ETO_(alone). The effect of ETO on the AUC_0–t of DCS was not significant. Second, ETO accumulates in the liver upon oral administration (Fig. 2), escalating the associated toxicity. Upon oral administration, the liver AUC [AUC_{(0–t, liver)}] was approximately two times higher compared to the other organs (see Tables S1 to S5 in the supplemental material). When given in combination with DCS, AUC_{(0–t, liver)} of ETO_(combi) was reduced by more than 26 times. To our knowledge, there are no reports of liver enzyme induction upon a single oral dose of DCS. It is therefore intriguing to observe the apparent effect of DCS in the disposition of ETO.

FIG 2.

Comparison of liver AUC_0–t of ETO and DCS administered alone and in combination.

Tissue concentrations of ETO in the brain were depleted rapidly when DCS was coadministered orally (Fig. S2). This observation has implications, since the use of DCS has been limited due to acute neuropsychiatric toxicity, including convulsions, seizures, paralysis, and unconsciousness. The AUC_{(0–t, brain)} of DCS_{(excess ETO)} nearly doubled in comparison to DCS_(alone). Similar trends were seen in the distribution of drugs to other organs (Fig. S2, Tables S1 to S5).

Upon inhalation, as expected, high concentrations of drugs were developed and retained in the lungs (Fig. 3, Table S1). The pharmacokinetic interaction observed with orally administered drugs was not seen after pulmonary delivery in the mouse.

FIG 3.

Concentration-time profiles of DCS in blood plasma (A) and lungs (B) and of ETO in lungs (C) upon inhalation. Scatter points represent data from three individual animals at different time points. Solid lines are one-compartment model fitted curves to group means, and error bars show the SD from fitted points (n = 3).

Efficacy in H37Rv-infected mice.

Mice were infected and treated as shown in Table 2 below and as detailed in the Supplemental Material. An assay of M. tuberculosis CFU recovered from the lungs and spleens of the mice revealed that at the start of treatment the infected animals harbored 5.95 ± 0.19 and 4.38 ± 0.42 log₁₀ CFU in the lungs and spleen, respectively (Fig. 4). At the end of the treatment period, a significant (Table S6) increase in CFU in the lungs and spleen was observed in the control group receiving no drugs. Animals in the treatment groups receiving oral dose of DCS and ETO showed a reduction in the CFU by about 2 log₁₀ in the lungs but less than 1 log₁₀ in the spleen. There was significant decline in lung and spleen burden in the group receiving inhalations, with counts of 3.18 ± 0.49 and 3.41 ± 0.17 log₁₀ CFU, respectively. When inhalation-equivalent doses of DCS and ETO were administered as oral suspension, the burden in the lungs and spleen was comparable to the untreated control group, with 6.0 ± 0.4 and 4.42 ± 0.38 log₁₀ CFU/g in the lungs and spleen, respectively. Highest efficacy was obtained when mice were given HED of ETO and DCS perorally, along with 70 μg of ETO and 30 μg of DCS by inhalation. The numbers of CFU were reduced to 2.99 ± 0.06 and 2.77 ± 0.31 log₁₀ in the lungs and spleen, respectively.

TABLE 2.

Treatment regimen

Group	Intervention	Frequency (no. of daily doses)	Daily dose
A	Untreated control at the start of treatment (day 28)
B	Untreated control at the end of treatment (day 57)
C	Oral ETO and DCS at human equivalent dose for 4 weeks	20	4.7 mg ETO + 2.35 mg DCS
D	Inhaled ETO and DCS for 4 weeks	20	70 μg ETO + 35 μg DCS
E	Oral ETO and DCS at inhalation-equivalent dose for 4 weeks	20	70 μg ETO +35 μg DCS
F	Inhaled + oral to achieve human equivalent dose	20	4.63 mg ETO + 2.31 mg DCS (oral); 70 μg ETO +35 μg DCS (inhalation)

FIG 4.

CFU (means ± the SD; n = 6) in lungs (open columns) and spleen (hatched columns) of groups of mice at the start of the experiment (A); at the end of 4 weeks with no drugs administered (B); and receiving daily doses of any of the treatment variants for 4 weeks, viz., the oral HED of DCS and ETO (C); inhalations of 35 μg of DCS plus 70 μg of ETO (D); and inhalation-equivalent doses by the oral route (E) and inhalations, as well as oral doses (F). See Table S6 in the supplemental material for additional information on analysis of variance and comparison of means.

The reduction in bacterial burden in animals receiving an order of magnitude lower dose by inhalation suggests that the inhaled combination is pharmacodynamically bactericidal (>2-log kill). This inference is supported (Fig. 4) by the minor increment in efficacy (P increment to 0.02124 from 0.03058, not significant; Table S6) when oral human-equivalent doses were added to the inhaled dose.

Limitations and caveats.

The major caveat to be noted is that the interaction reported here has not been confirmed in any other species, let alone in humans. Although we have investigated dose dependence of the drug-drug interaction in a limited manner as reported here (Table 1), we do not have any information on the effect of dosing time and order of dosing on the interaction, food effects, and gender or species differences.

We can only speculate about possible mechanisms by which the observed interaction takes place and advance several hypotheses to test. First, it is likely that enzymes that metabolize ETO are rapidly induced by DCS in mice. Amino acids have not been investigated in any detail as regulators of liver enzyme systems (15). d-Serine itself is under investigation as a signaling molecule that modulates, among others, NMDA receptors (16). An investigation of mouse, rat, and human cytochromes P450 (CYPs), liver microsomal preparations, and hepatocyte cultures for the rates of metabolism of ETO in the presence and absence of DCS in vitro by liquid chromatography-tandem mass spectrometry is necessary prior to making any claim about this possibility. Ideally, the in vitro investigations should be followed up with Western blots for CYPs or other enzyme systems that may be identifiable in the in vitro studies in serial samples of lysates of liver tissue obtained from mice administered the two drugs alone or in combination.

A trivial explanation for the interaction could be that DCS interferes with the gastrointestinal absorption of ETO. However, the plasma concentration profile (Fig. 1) suggests that, if so, the interference does not affect the rate of absorption but only its extent. Unfortunately, the interanimal variation in the rate constant of oral absorption was high (0.15 ± 0.17, Table 1), and the data generated with oral administration of DCS along with ETO was not amenable to compartment analysis (Table 1). The data are of limited value if a mechanistic explanation of the interaction is to be rigorously offered.

Finally, the laboratory strain of M. tuberculosis used for the study is less sensitive to DCS and ETO in comparison to MDR strains. The efficacy study is therefore limited in its applicability to contribute to a preclinical proof of concept.

Even so, we conclude that further preclinical development of a dry powder inhalation of the combination reported here is warranted.