Even apparently insignificant chemical deviations among bioequivalent generic antibiotics can lead to therapeutic nonequivalence: the case of meropenem

Abstract

Several studies with animal models have demonstrated that bioequivalence of generic products of antibiotics like vancomycin, as currently defined, do not guarantee therapeutic equivalence. However, the amounts and characteristics of impurities and degradation products in these formulations do not violate the requirements of the U.S. Pharmacopeia (USP). Here, we provide experimental data with three generic products of meropenem that help in understanding how these apparently insignificant chemical differences affect the in vivo efficacy. Meropenem generics were compared with the innovator in vitro by microbiological assay, susceptibility testing, and liquid chromatography/mass spectrometry (LC/MS) analysis and in vivo with the neutropenic guinea pig soleus infection model (Pseudomonas aeruginosa) and the neutropenic mouse thigh (P. aeruginosa), brain (P. aeruginosa), and lung (Klebisella pneumoniae) infection models, adding the dihydropeptidase I (DHP-I) inhibitor cilastatin in different proportions to the carbapenem. We found that the concentration and potency of the active pharmaceutical ingredient, in vitro susceptibility testing, and mouse pharmacokinetics were identical for all products; however, two generics differed significantly from the innovator in the guinea pig and mouse models, while the third generic was therapeutically equivalent under all conditions. Trisodium adducts in a bioequivalent generic made it more susceptible to DHP-I hydrolysis and less stable at room temperature, explaining its therapeutic nonequivalence. We conclude that the therapeutic nonequivalence of generic products of meropenem is due to greater susceptibility to DHP-I hydrolysis. These failing generics are compliant with USP requirements and would remain undetectable under current regulations.

FIG 1

Flow chart for the project design.

FIG 2

Pharmacodynamics of three generics compared with the innovator of meropenem against the WT strain P. aeruginosa GRP-0019 (MIC, 1 mg/liter) in the neutropenic guinea pig soleus infection model (panels correspond to separate experiments). The hydrolytic activity of cDHP-I against meropenem was very close to that of hDHP-I, making this species useful to model the PD in a human-like environment and without protecting the antibiotic with cilastatin. All products fit the Hill equation without faults; gMer-A and gMer-C required 91% (A) and 122% (B) greater doses to attain bacteriostasis than iMer, respectively. gMer-B, on the other hand, was indistinguishable (P = 0.88) from iMer (B).

FIG 3

Pharmacodynamics of one generic product (gMer-A) compared with the innovator of meropenem against the MDR strain P. aeruginosa GRP-0049 (MIC, 2 mg/liter) in the neutropenic mouse thigh infection model. Against this organism, a 1:1 M:C ratio was insufficient to protect meropenem from mDHP-I hydrolysis, and it was necessary to accumulate data from three identical experiments to obtain a valid nonlinear regression for gMer-A; persistent multicollinearity of both products prevented their statistical comparison, but their PD profiles looked quite different (A). With a 1:3 M:C ratio, both products fit the Hill equation with a single experiment without faults, dropping the T>MIC to 18.1% (B); increasing the M:C ratio to 1:5 made both products even more potent (T>MIC, 16.8%), suggesting additional hydrolysis of meropenem caused by this strain, probably by an enzyme other than mDHP-I but still susceptible to cilastatin inhibition (C). Increasing concentrations of cilastatin made products identical, with overlapping PD curves (compare panel A with panels B and C).

FIG 4

Pharmacodynamics of three generics compared with the innovator of meropenem against P. aeruginosa strains GRP-0019 (WT; A and B) and GRP-0049 (MDR; C and D) in the neutropenic mouse meningoencephalitis model. Without cilastatin, and using a dose-range encompassing the T>MIC from 12.4% to 100% (in serum) against GRP-0019, it was possible to obtain flawless fits for gMer-B, gMer-C, and iMer to the Hill model, demonstrating therapeutic equivalence of gMer-B and therapeutic nonequivalence of gMer-C (A). With cilastatin, only a 1:3 M:C ratio gave gMer-A a clean regression that, compared with iMer, required 47% more drug to achieve bacteriostasis (B). GRP-0049 was untreatable in this model without cilastatin (data not shown); with a 1:1 M:C ratio, gMer-A gave an accurate nonlinear regression but iMer did not (C), as seen in the thigh model with this MDR strain. A 1:3 M:C ratio gave an impeccable regression for both products, demonstrating that gMer-A required 56% more meropenem than iMer to attain bacteriostasis in vivo (D). These findings suggest that the innovator is preferentially hydrolyzed by GRP-0049, a strain that evolved in our hospital under the selective pressure of iMer (discussed in the text).

FIG 5

Pharmacodynamics of three generics compared with the innovator of meropenem against the WT strain K. pneumoniae GRP-0107 (MIC, 0.06 mg/liter) in the neutropenic mouse pneumonia model. mDHP-I is very active in this tissue, providing a good model to test meropenem under a highly hydrolytic environment. Without cilastatin, a T>MIC range from 22.8% to 100% (2.5 to 80 mg/kg/day) was not enough to reach maximal efficacy, and the data did not fit the Hill equation (A). Increasing the dose to 640 mg/kg provided much higher serum concentrations of meropenem (30- to 897-fold above the MIC) during 100% of the dosing interval, allowing gMer-B and iMer to reach their maximal effect and fit the Hill equation (P = 0.13 for comparison by CFA); gMer-C could not be included in the analysis because it did not fit the model, suggesting greater susceptibility to mDHP-I (B). When the dose range was narrowed to 5 to 160 mg/kg (%T>MIC, 42.8 to 100%), the addition of cilastatin up to a 1:3 M:C ratio did not help gMer-A or iMer (C); under a 1:5 M:C ratio, only iMer generated a flawless regression, suggesting that gMer-A is more susceptible to mDHP-I (D).

FIG 6

Meropenem hydrolysis by mDHP-I: detection by HPLC-UV (left panels) and microbiological assay (right panels) of the remaining fraction of meropenem products with time after incubation at 37°C with mDHP-I extract for 4.5 h. The control curve (dotted) corresponds to meropenem incubated without DHP-I and shows the spontaneous degradation of the carbapenem. Both products were hydrolyzed by the enzyme, but gMer-A degraded much faster than iMer at all concentrations tested.

FIG 7

LC/MS scan mode (range, m/z 100 to 1,000) of the pharmaceutical forms of one generic and the innovator of meropenem (fresh samples). The left (innovator) and right (generic) panels show above the spectrogram and, under it, the centroids graphs describing the composition masses of each peak numbered. There were no differences in the analyte signal (peak 2 in both panels), demonstrating that the active pharmaceutical ingredient (m/z 384) is present in both products at the same concentration, in compliance with current regulations. However, the generic product exhibited one additional peak, detected at 10 min (peak 3, right panel), with a main molecular mass of m/z 359 [M + 1] that was absent in the mass spectra of the innovator.

FIG 8

LC/MS scan mode (range, m/z 100 to 1,000) for samples stored for 48 h at room temperature. Spectrograms for iMer (A) and gMer-A (B) are shown, demonstrating more abundance of degradation products in the pharmaceutical form of the generic, with different mass compositions between retention times of 3 and 6 min.