Pharmacokinetics of imipenem after intravenous,intramuscular and subcutaneous administration to cats

Abstract

The study describes the pharmacokinetics and predicted efficacy of imipenem after intravenous (IV), intramuscular (IM) and subcutaneous (SC) administration to five adult cats at a dose of 5 mg/kg. Susceptibility to imipenem [minimum inhibitory concentration (MIC)] was determined for antimicrobial resistant Escherichia coli (n = 13) and staphylococci (n = 3) isolated from domestic cat infections (urinary system, skin and conjunctiva). Maximum plasma concentrations of imipenem were 13.45 µg/ml (IV), 6.47 µg/ml (IM) and 3.83 µg/ml (SC). Bioavailability was 93.18% (IM) and 107.90% (SC). Elimination half-lives for IV, IM and SC administration were 1.17, 1.44 and 1.55 h, respectively. All tested bacteria were susceptible to imipenem; MIC values were 0.03 µg/ml for Staphylococcus species and <0.25–0.5 µg/ml for E coli. Mean imipenem concentrations remained above a MIC of 0.5 µg/ml for approximately 4 h (IV and IM) and 9 h (SC). Imipenem would be predicted to be effective for the treatment of antimicrobial resistant bacterial infections in cats at a dosage of 5 mg/kg every 6–8 h (IV, IM), or longer for the SC route. However, clinical trials are mandatory to establish its efficacy and proper dosing.

Introduction

Imipenem (N-formimidoyl thienamycin) is a beta-lactam antibiotic of the carbapenem class with a broad spectrum of activity that includes most pathogenic bacteria. It is highly active against aerobic and anaerobic Gram-positive and Gram-negative bacteria [Staphylococcus species (with the exception of methicillin-resistant strains), Streptococcus species, Enterobacteriaceae and Pseudomonas aeruginosa] and is resistant to many beta-lactamases.¹ Susceptible bacteria from human isolates have a minimum inhibitory concentration (MIC) break-point of ≤4 µg/ml, which could be an approximate reference for veterinary pathogens.²

Beta-lactams have a time-dependent antibacterial efficacy; the cumulative percentage of the dose interval that drug concentration exceeds the MIC at steady state pharmacokinetic conditions (T>MIC) is the best predictor of efficacy. As carbapenems have greater bactericidal activity and a longer post-antibiotic effect than other beta-lactams, the T>MIC may be as low as 20–40% of the dose interval.^3,4 However, greater T>MIC is indicated to decrease the risk of resistance development.⁵

In kidney, a dihydropeptidase hydrolyses imipenem to an inactive nephrotoxic metabolite. Combination with cilastatin (1:1), a dihydropeptidase inhibitor that lacks antibacterial activity, inhibits renal metabolism. Cilastatin increases its elimination half-life and allows its renal elimination as an active drug resulting in higher urine concentrations.¹

The efficacy of imipenem for treating hospital-acquired infections in humans caused by multi-resistant Gram-negative bacteria, or mixed aerobic and anaerobic infections¹ is very high.

In veterinary medicine, imipenem is used for infections caused by bacteria resistant to other antimicrobials (especially P aeruginosa, Escherichia coli and Klebsiella pneumoniae), which are refractory to more commonly used agents.^6,7

Imipenem pharmacokinetic studies in domestic animals are scarce; they have only been described in dogs,⁸ horses⁹ and sheep.¹⁰ To our knowledge, there are not reports in cats. The objective of the present study was to describe the pharmacokinetics and to predict efficacy based upon pharmacokinetic data of imipenem after intravenous (IV), intramuscular (IM) and subcutaneous (SC) administration to cats after single doses of 5 mg/kg.

Materials and methods

Animals

Five adult mixed breed cats (one female and four males) weighing 5.54 ± 0.47 kg were assessed as healthy based on the results of physical examination, complete blood count, biochemical profile and urinalysis. Animals were housed in the University of Buenos Aires (UBA) Faculty of Veterinary Medicine facilities and allowed to acclimatize for 2 months before the experiments. Access to high quality dry food (Fit 32; Royal Canin) and water was ad libitum.

The study protocol was approved by the Institutional Animal Care and Use Committee, School of Veterinary, University of Buenos Aires, Argentina.

Dosage forms

A commercially available formulation of imipenem-cilastatin (Zienam IV Imipenem/Cilastatina Sódica; Merck Sharp & Dohme) was used. Before administration, the powder was dissolved in sterile saline solution (0.9% NaCl) to a concentration of 25 mg/ml. Imipenem was administered IV, IM and SC at a dose of 5 mg/kg.

Experimental design

A three-period, three-treatment crossover design was used, such that each of the five cats received imipenen IV, IM and SC in a randomized sequence (Microsoft Office Excel). For IV administration, the dose was given over a 2 min period via a catheter placed in the cephalic vein. For the IM route, the dose was administered in the dorsal lumbar muscles, and for SC administration the dose was injected into the loose skin over the shoulders. Two-week intervals were allowed between each period.

Sampling procedures

Blood samples (0.7 ml) were collected via an aseptically placed cephalic catheter prior to drug administration at 0.083, 0.16, 0.33, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10 and 12 h. Samples were placed immediately into tubes containing sodium heparin, mixed and placed on ice until plasma separation. Plasma was separated after centrifugation (15 min at 1500 g), stored at 4ºC and analyzed within 6 h of collection.

Plasma drug analysis

Plasma concentrations of imipenem were determined by use of microbiological assay¹¹ using Bacillus subtilis American Type Culture Collection (ATCC) 6633 as test microorganism. The lack of antimicrobial activity of cilastatin was verified by comparing the sizes of the zones of inhibition for standards concentration of imipenem with and without cilastatin. Standard curves were prepared in cat normal plasma between 0.05 and 25 µg/ml imipenem concentrations (Imipenem; Sigma-Aldrich) on the day of each study and stored at −4°C until sample analyzing (within 6 h of collection). The assay was linear from 0.05 to 25 µg/ml, with inter- and intra-assay variation of <10% within this range. The limit of detection and quantification of the method were 0.05 µg/ml and 0.10 µg/ml, respectively. The lower limit of quantification (LLOQ) was the lower limit of concentration used in the pharmacokinetics analysis.

Pharmacokinetic analysis

Pharmacokinetic analyses were performed with computer software (PCNonlin, SCI Software, v4). Initial estimates were determined using the residual method¹² and refitted by non-linear regression.

The number of exponents needed for IV, IM and SC administration data was determined by applying the Schwartz¹³ and Akaike criterions,¹⁴ and the residual distribution around the estimated concentrations.

Pharmacokinetic parameters were calculated using classic equations associated with compartmental analysis, except C_max and T_max, which were determined by visual inspection of plasma concentration time curves.¹²

Statistical analysis

All data are presented as medians and ranges. Main parameters [area under the curve_(0–∞), k, T_½, mean residence time (MRT)] were statistically compared for the three administration routes, applying a non-parametric test for repeated measures (Friedman test) and a multiple comparison post-test (Dunn’s test). Pharmacokinetic parameters related to the absorption process [C_max, T_max, k_a, T_½(a), mean absorption time (MAT) and F] were compared statistically by applying a non-parametric paired test (Wilcoxon test; GraphPad Prism, GraphPad Software). Values of P ≤0.05 were considered significant.

Susceptibility testing

The MIC of imipenem against thirteen strains of E coli (all resistant to ampicillin/sulbactam, first generation cephalosporins and trimethoprim/sulfamethoxazole; 12 resistant to gentamicin; 10 to ciprofloxacin; seven to ceftazidime; four to nitrofurantoin; and two resistant to tetracycline and chloramphenicol) and three strains of Staphylococcus species (all resistant to penicillin; two resistant to erythromycin, clindamycin and ciprofloxacin; and one resistant to tetracycline). All the strains were isolated from cat infections (urinary system, skin and conjunctiva). MICs were determined via broth macrodilution assay.² The isolates were provided by the diagnostic laboratory of the UBA Faculty of Veterinary Medicine.

Escherichia coli ATCC 25922 (imipenem MIC 0.06–0.25 µg/ml) and Staphylococcus aureus ATCC 29213 (imipenem MIC 0.015–0.06 µg/ml) were used as quality control strains.

Pharmacokinetic/pharmacodynamic integration

Time above theT>MIC for the three different administration routes was estimated by visual approximation from the plasma concentration versus time curve. The MIC value applied was the highest MIC obtained for the multi-resistant isolated microorganisms. T>MIC was expressed as percentage of the recommended interdose interval in cats (6–8 h).¹⁵

Results

No adverse effects were observed in any of the cats by physical examination. Also, there was no pain manifestation after imipenem SC administration.

Imipenem plasma concentration versus time curves after IV administration were best described by an open monocompartmental model in all the animals (Figure 1). Table 1 shows the main pharmacokinetic parameters.

Figure 1

Mean and 95% confidence interval imipenem plasma concentration–time profile after intravenous (IV) (▲), intramuscular (IM) (■) and subcutaneous (SC) (▼) administration at a dose of 5 mg/kg to cats (n = 5). MIC = minimum inhibitory concentration

Table 1

Median and range pharmacokinetic parameters of imipenem after intravenous, intramuscular and subcutaneous administration at single doses of 5 mg/kg to cats (n = 5) in a crossover design

Pharmacokinetic parameter	Intravenous administrationmedian (range)	Intramuscular administrationmedian (range)	Subcutaneous administration median (range)
AUC_(0–∞) (µg.h/ml)	23.65 (20.39–31.72)	21.33 (20.60–22.09)	22.87 (15.49–27.31)
C_p(0) (µg/ml)	13.47 (13.08–14.25)	–	–
Vss (l/kg)	0.37 (0.35–0.38)	–	–
T_max (h)	–	1.00 (0.50–2.00)	3.00 (1.50–3.00)*
C_max (µg/ml)	–	6.47 (4.77–7.47)	3.83 (3.10–4.80)*
k_a (h^-1)	–	5.23 (2.17–6.71)	0.42 (0.29–0.71)*
T_½(a) (h)	–	0.13 (0.10–0.32)	1.63 (0.98–2.42)*
MAT (h)	–	0.64 (0.49–0.90)	2.75 (2.30–3.52)*
F (%)	–	93.18 (67.25–101.00)	107.90 (70.09–115.50)
Cl_B (l/h.kg)	0.22 (0.16–0.24)	–	–
k (h^-1)	0.59 (0.43–0.66)*	0.48 (0.38–0.65)	0.45 (0.29–0.49)*
T_½ (h)	1.17 (1.06–1.62)*	1.44 (1.07–1.82)	1.55 (1.41–2.40)*
MRT (h)	1.65 (1.45–2.21)*	2.29 (2.00–2.85)	4.34 (3.75–5.73)*

Significantly different (P <0.05)

AUC _(0-∞) = area under the plasma concentration vs time curve from 0 to infinite; C_{p (0) =} plasma concentration at 0 time; Vss = volume of distribution; T_max = time of maximum concentration; C_max = maximum concentration; k_a = absorption rate constant; T_{½ (a)} = absorption half-life; MAT = mean absorption time; F = bioavailability; Cl_B = body clearance; k = elimination rate constant; T_½ = elimination half-life; MRT = mean residence time

Imipenem administered IV was eliminated rapidly with a Cl_B of 0.22 l/h/kg (range, 0.16–0.24), a T_½ of 1.17 h (range, 1.06–1.62 h) and a MRT of 1.65 h (range, 1.45–2.21 h). Imipenem plasma concentrations remained above the LLOQ for 6 h in 4/5 cats and for 8 h in the remaining cat.

Imipenem plasma disposition curves after IM and SC administration (Figure 1) were best described by a biexponential equation and explained by an open monocompartmental model with first order absorption in all the animals.

IM absorption was rapid, as reflected by the T_max [1.00 h (range, 0.50–2.00 h)], the T_½(a) [0.13 h (range, 0.10–0.30h)] and the MAT [0.64 h (range, 0.49–0.90 h)]. Bioavailability was high [93.18% (range, 67.25–101.00%)], reflecting the high extent of the process. C_max was 6.47 µg/ml (range, 4.77–7.47 µg/ml); plasma concentrations remained above the LLOQ for 8 h in three cats and for 10 h in the remaining two cats.

Imipenem absorption after SC administration was, compared with IM administration, significantly slower (P ≤ 0.05), with no statistically significant differences on bioavailability. The delayed absorption after SC administration acts as limiting factor of the elimination process (flip/flop phenomenon) leading to a longer permanence of imipenem in plasma. This finding explains the smaller k [0.45 h (range, 0.29–0.49 h)], and longer T_½ [1.55 h (range, 1.41–2.40 h] and MRT [4.34 h (range, 3.75–5.73 h)] (P ≤ 0.05) (Table 1). Imipenem plasma concentrations were above the LLOQ for 12 h in three cats and for 10 h in the other two.

All the microorganisms tested were susceptible to imipenem. MIC values were 0.03 µg/ml for all Staphylococcus species and ≤0.5 µg/ml for the E coli strains.

For susceptible microorganisms with a MIC ≤0.5 µg/ml, T>MIC was, for the IV and IM administration routes, approximately 4 h (ranging between 5 and 6 h and 4 and 6 h, respectively), representing around 50–67% of the inter-dose interval (8 h or 6 h, respectively). For the SC route, it was around 9 h (ranging between 8 and 10 h) exceeding 100% of the proposed inter-dose intervals.

Discussion

Good correlation between microbiological assay and high performance liquid chromatography has been reported for imipenem determination.^8,16 In fact, this assay has been used for measuring imipenem concentrations in plasma and other biological fluids in a number of studies in animals and humans.^{8,10,17
–20}

Imipenem plasma concentration profile and the main pharmacokinetic parameters after IV administration to cats were quite similar to, and in the range of, those described for humans and other domestic animals.^{4,8
–10,17,18,20,21} However, in dogs administered an equivalent dose, plasma concentrations declined faster (T_½ 0.80 ± 0.23 h) than in cats [T_½ 1.17 h (range, 1.06–1.62 h)].⁸

The estimated imipenem distribution value is in the order of that expected for a beta-lactam antibiotic. In fact, similar values are reported for other species.^4,8
–10 This value seems to indicate that imipenem distributes in the extracellular compartment (the site where most infections are localized).

Imipenem Cl_B in cats [0.22 l/h/kg (range, 0.16–0.24 l/h/kg)] was slightly greater than the corresponding glomerular filtration rate (0.18 l/h/kg),²² suggesting that imipenem (combined with cilastatin) is excreted mainly through renal tubular filtration with minimal involvement of other mechanisms, such as active tubular secretion. The same was proposed for humans, dogs, horses and sheep.^4,8
–10

After IM administration, imipenem absorption was faster and C_max higher than after SC administration. Moreover, after SC administration imipenem remained in the plasma longer than after IM administration. These findings could be explained through the ‘flip-flop’ phenomenon, where terminal T_½ is reflecting the k_a rather than the k. The same was observed in dogs.⁸ This pharmacokinetic phenomenon would have clinical implications as it would prolong dosage intervals.

Bioavailability of imipenem after extravascular administration in cats was high (93.18% IM and 107.90% SC) without significant differences between administration routes. The same has been reported for other species, such as ovine (65.97% IM),¹⁰ human (89% IM)²⁰ and canine (146% IM and 159% SC).⁸ The almost complete absorption of the drug and its long permanence in plasma when administered extravascularly (especially SC) yield more desirable plasma concentration profiles than after IV administration. Barker et al⁸ and Signs et al²⁰ reached similar conclusions when studying the pharmacokinetics of imipenem in dogs and humans respectively.

The isolation of bacteria resistant to many antimicrobials in patients treated previously with antibiotics is a common clinical finding.^23,24 All the bacteria strains isolated from cat infections (13 strains of E coli and three Staphylococcus species strains) tested in the present study showed resistance to commonly used antimicrobials (disk diffusion susceptibility test method²), but were highly susceptible to imipenem, with MIC values in the range of those found in other studies for the same bacterial species.²⁴

For carbapenems, a T>MIC 30–40% of the dose interval has been established as optimal for bactericidal action.^3,4 In this study, this goal is achieved for up to 4 × MIC (2 µg/ml) in 6- or 8-h dose interval regimens.

Conclusions

Although the longest permanence of imipenem plasma concentrations is achieved after SC administration, efficacy will depend on MIC for the etiological agent. The superiority of the SC route would only be valid when the MIC of the organisms to be treated is relatively low (≤0.5 µg/ml). For microorganisms with higher MIC (MIC ≤4 µg/ml²) the IV or IM routes with an interval of 6–8 h is advisable.

Footnotes

Acknowledgements

The authors are indebted to Royal Canin, Argentina for the kind provision of the animal food.

Funding

This work was supported by a Research Project UBACyT (grant number 20020100100745, 2011-2014) of Secretaría de Ciencia y Técnica, Universidad de Buenos Aires, Argentina.

Conflict of interest

The authors do not have any potential conflicts of interest to declare.

Accepted: 26 November 2012

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