1. Introduction
Nano-mupirocin is a formulation of PEGylated nano-liposomes loaded with mupirocin, an antibiotic with a unique mode of action and no cross-resistance. The specific target is the isoleucine-binding site on the bacterial isoleucyl-transfer-RNA synthetase, as demonstrated for
Staphylococcus aureus and
Escherichia coli—though the latter species is inherently resistant owing to impermeability [
1]. Mupirocin was approved by the FDA in 1997 but is limited to topical use owing to rapid systemic elimination and high protein binding [
2,
3,
4].
Computational machine-learning identified mupirocin as a highly suitable candidate for nano-liposomal delivery [
5,
6,
7]. Remote active loading protects circulating drug from metabolism and promotes accumulation at an infection site, facilitated by leaky vessels and low lymphatic clearance [
8]. Nano-mupirocin’s activity after injection has been confirmed in mouse models of necrotizing fasciitis, osteomyelitis and pneumonia, also rabbit endocarditis [
9,
10]. A mouse study showed higher plasma levels and a much longer half-life than for the free drug (4.4 h vs. 5 min), with this pharmacokinetic pattern confirmed in rabbits [
9]. In addition, we showed that mupirocin retained antibacterial activity despite being encapsulated in the intraliposomal aqueous phase. Nano-mupirocin is taken-up by macrophages, killing internalized bacteria [
10]. The antibacterial inactivity of the unloaded liposomes was previously demonstrated both in vitro and in vivo [
10].
Here, we further describe in vitro profiling of nano-mupirocin, including its effect on resistant strains and in relation to pharmacokinetic (PK) and biodistribution (BD) studies.
4. Discussion
Mupirocin has a long history of use for superficial staphylococcal skin infections and for elimination of nasal MRSA. Parenteral Nano-mupirocin, being protected from rapid metabolism, opens the novel possibility of use against deep infections and against other pathogens. Nano-mupirocin is a stable product with a loading stability of at least 2 years at 4 °C (not shown).
MICs of Nano-mupirocin were mostly 2- to 4-fold above those of the free drug. This differential is surprisingly small, given that the in vitro release of free drugs from the liposomes is slow [
17]. The explanation, based on previous results, is that the intact Nano-mupirocin interacts directly with
S. aureus and does not require drug release to achieve an antibacterial effect [
10].
Mupirocin’s unique mode of action suggests that it should retain activity against otherwise resistant strains, and this indeed was seen, with activity confirmed against MRSA resistant to vancomycin, daptomycin and linezolid, vancomycin-resistant
E.
faecium, and, previously, against
N. gonorrhoeae isolates resistant to extended-spectrum cephalosporins [
15].
The rat pharmacokinetic study performed here demonstrated a linear increase in exposure with dose, with neither accumulation nor faster clearance upon repeated administration. Exposures in this study can usefully be compared with MICs of key pathogens, as represented by the two horizontal lines in
Figure 1A,B. The 1 µg/mL line corresponds to the MIC for most Gram-positive isolates (except
E.
faecalis), and the 64 µg/mL line to the maximal MIC for MRSA with low-level mutational-type mupirocin resistance [
18]. Concentrations > 1 µg/mL were achieved for >48 h even after the lowest dose administered (10 mg/kg, equivalent to 97 mg for a 60 kg human [
19]). Mupirocin has time-dependent bactericidal activity [
16] and, in our previous in vitro study, achieved complete clearance of bacteria in three independent experiments, during 4 h of incubation in the presence of plasma [
10]. Here, 10, 30, and 100 mg/kg doses of Nano-mupirocin to male rats gave plasma drug concentrations of 49, 131, and 552 µg/mL, respectively, at 4 h post-dose, with all these levels remaining far above MBC values (
Table 4).
IM antibiotic administration is preferred for some infections, notably gonorrhea. Rat plasma concentrations after IM administration of Nano-mupirocin greatly exceeded the MIC
90 of 0.03 µg/mL for
N. gonorrhoeae [
15] at all time points tested. However, the pharmacodynamic drivers in gonorrhea are poorly defined, and plasma concentrations may not reflect concentrations at disease sites [
20]. Moreover, the female mouse model of gonococcal infection developed by Prof. Jerse’s laboratory [
21] proved to be unsuitable for testing Nano-mupirocin due to requiring pre-treatment of the animals with estrogen. This causes thickening of the vaginal mucosa by stimulating the proliferation of epithelial cells [
21,
22] which then appears to serve as a barrier for the nano-liposomes. An in-house study showed that the levels of mupirocin achieved in the vaginal secretions of estrogen-treated mice were much lower than in un-treated animals, and that the secretions were denser and thicker (not shown). Therefore, we measured drug concentrations in the vaginal secretions, showing that, over the 24 h after injection, mupirocin was present at concentrations much above the MIC
90, ranging from 11 µg/g 1 h after injection to 8 µg/g, 24 h after injection and corresponding to 267–367 times the MIC
90. Nano-mupirocin addresses many of the criteria of a Consensus Target Product Profile recently published by a gonorrhea expert group [
23] including: (i) activity against
Mycoplasma genitalium [
24]; (ii) activity against cephalosporin- and macrolide- resistant gonococci [
15]; (iii) intracellular activity [
10]; (iv) lack of cross-resistance; and (v) suitability for IM injection.
Low-level mupirocin resistance in staphylococci is well-recognized. It arises by mutation and may reflect high local usage of topical mupirocin [
18]. In the light of this concern, we undertook mutation frequency and 15-day passage studies. Very little resistance emerged, particularly for MRSA and
N. gonorrhoeae. More mutants were selected with Nano-mupirocin than with free mupirocin, which may be a result of technical differences related to drug dispersion in MHA (see
Section 3.1). High drug concentrations at infections sites should militate against selection in vivo [
9,
10], with activity predicted (above) even against
S. aureus with low-level mutational resistance. Moreover, the reported association [
18] between mupirocin use and resistance prevalence may reflect the spread of resistant strains, rather than repeated de novo selection.
Nano-mupirocin has potential for multiple indications where the causative bacteria are typically susceptible. These include gonorrhoea as well as deep infections such as pneumonia and osteomyelitis. Patients with these infections would benefit from a safe drug that is distributed at the infection site [
10]. Nano-mupirocin uses a known antibiotic and PEGylated nano-liposomes identical in lipid composition and size to those of Doxil
® [
25], which has been used in >700,000 cancer patients. The PK and toxicity of free mupirocin were evaluated after IV injection in healthy volunteers up to a dose of 252 mg/person, with good tolerability [
26]. Accordingly, Nano-mupirocin should be seen as a formulation with considerable potential and low development risks.