N-formimidoyl thienamycin (MK-787) is a new /?-lactam with potent activity against both aerobic and anaerobic gram-positive and gram-negative bacteria. Its spectrum and activity suggest it may be useful in treatment of complicated intraocular infections. Its ocular penetration was studied in New Zealand white rabbits immediately before and after the third dose of 40 mg/kg administered intravenously at q6h intervals. Plasma, aqueous humor, and vitreous humor were obtained by direct aspiration, and antibiotic levels were asayed using an agar well diffusion method. MK-787 penetrated uninflamed intraocular fluids, including vitreous humor, although vitreous concentrations achieved (0.1-0.2 Mg/ml) were significantly lower than the mean peak plasma (15 Mg/ml) and aqueous concentrations (7 Mg/ml). Nevertheless, the intraocular levels attained approached or exceeded the MIC 90   and markedly enhanced spectrum of activity against both gram-positive and gram-negative bacteria

Anthony W Chow

Catherine L Carlson

Kelvin R Finlay

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No. 8 Reports 1147
Ocular Penetration of N-Formimidoyl Thienamycin (MK-787) and
Potentiation by Dipeptidase Inhibitor (MK-791)
Kelvin R. Finlay, Catherine L. Carlson, and Anthony W. Chow
N-formimidoyl thienamycin (MK-787) is a new /?-lactam
with potent activity against both aerobic and anaerobic
gram-positive and gram-negative bacteria. Its spectrum and
activity suggest it may be useful in treatment of complicated
intraocular infections. Its ocular penetration was studied in
New Zealand white rabbits immediately before and after
the third dose of 40 mg/kg administered intravenously at
q6h intervals. Plasma, aqueous humor, and vitreous humor
were obtained by direct aspiration, and antibiotic levels were
asayed using an agar well diffusion method. MK-787 pen-
etrated uninflamed intraocular fluids, including vitreous
humor, although vitreous concentrations achieved (0.1-0.2
Mg/ml) were significantly lower than the mean peak plasma
(15 Mg/ml) and aqueous concentrations (7 Mg/ml). Nev-
ertheless, the intraocular levels attained approached or ex-
ceeded the MIC90 for most sensitive organisms including
some gram-negative bacilli important in bacterial endo-
phthalmitis. When administered in combination with the
renal enzyme inhibitor MK-791, plasma and aqueous con-
centrations of MK-787 were markedly potentiated, al-
though vitreous concentrations were minimally affected.
The potential usefulness of MK-787 in conjunction with
MK-791 in the infected eye should be examined further in
an animal model of bacterial endophthalmitis. Invest
Ophthalmol Vis Sci 24:1147-1149, 1983
N-formimidoyl thienamycin (MK-787) is a struc-
turally novel /3-lactam with extremely high potency
and markedly enhanced spectrum of activity against
both gram-positive and gram-negative bacteria.1-2 It
is a stabilized derivative of thienamycin, a naturally
occurring /3-lactam belonging to a new class of anti-
biotics known as the carbapanems. As with other
/3-lactams, its mechanism of action is through inac-
tivation of one of the transpeptidases important in
bacterial cell wall synthesis. Its broad spectrum of
activity is in part attributable to its stability against
a wide variety of bacterial /3-lactamases. Its extremely
high potency against a variety of gram-positive and
gram-negative bacteria suggests that MK-787 might
be particularly useful for the management of bacterial
endophthalmitis. Most antibiotics, with the exception
of relatively lipid-soluble agents such as chloram-
phenicol, minocycline, and doxycycline, have poor
ocular penetrations.3 Due to its potency, we reasoned
that even if the ocular penetration of this new drug
is equivalent to other /3-lactams, the antibiotic con-
centrations achieved may still be clinically effective
in the treatment of bacterial endophthalmitis. We
therefore studied its ocular penetration, especially
into vitreous humor, in a rabbit model following in-
travenous administration. Furthermore, MK-787 is
unique among /3-lactam antibiotics in that it is me-
tabolized and inactivated in the brush border of the
renal tubules by the renal dipeptidase enzyme, de-
hydropeptidase-I, which hydrolyses the j8-lactam ring
of MK-787.4 The degradation of MK-787 in vivo can
be halted, and its urinary and plasma concentrations
potentiated by the co-administration of a dipeptidase
inhibitor (MK-791, monosodium-Z-S-[6-carboxy-6-
{[(2,2-dimethyl-(S)-cyclopropyl) carbonyl]amino}5-
hexenyl]-L-cysteine) in chimpanzees and man.5 Ac-
cordingly, we also examined the effect of MK-791 on
ocular penetration of MK-787 in the rabbit.
Materials and Methods. Study animals: Groups
of New Zealand white female rabbits weighing 2-3
kg were studied. Each rabbit was adapted in individ-
ual cages for 48 hrs prior to experimentation. Anes-
thesia was attained by intramuscular injection of ke-
tamine hydrochloride (100 mg/ml) admixed with
acepromazine maleate (25 mg/ml) in a proportion of
10 ml to 1 ml, each animal requiring approximately
1 ml per injection. Animals were administered either
MK-787 alone or in combination with the enzyme
inhibitor MK-791 at 6-hr intervals by intravenous
bolus infusion through the anterior marginal ear
veins. Immediately before, and at 0.5, 1, 2, 4, and 6
h after the third dose of antibiotic, samples were ob-
tained by direct aspiration from the anterior chamber,
: vitreous cavity, and the anterior marginal ear veins.
Approximately 2.5 ml of plasma, 0.2 ml of aqueous
humor, and 0.5-0.8 ml of vitreous humor could be
obtained per animal at each sampling. Aqueous and
vitreous humor were aspirated only once per eye for
each animal. No bloody samples were accepted for
analysis. All samples were immediately stabilized in
a 50% (vol/vol) ethylene glycol: 1 M morpholino-
ethane-sulfonate buffer, pH 6.0 (MES) in equal pro-
portions (vol/vol), quick frozen in dry ice and ace-
tone, and stored at —80° C until ready for assay.
Animals were killed by intracardiac injection of so-
dium pentobarbital.
Antibiotic preparations: N-formimidoyl thiena-
mycin (MK-787) powder (Merck Sharp & Dohme
Research Laboratories, Rahway, NJ) was dissolved
in sterile normal saline and injected via an anterior
marginal ear vein at the dosage of 40 mg/kg by bolus
infusion. Antibiotic solutions containing either MK-
0146-0404/83/0800/1147/$0.95 © Association for Research in Vision and Ophthalmology
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1148 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / August 1983 Vol. 24
Table 1. Concentrations (mean ± SE, ng/m\) of N-
formimidoyl thienamycin (MK-787) after third q6h
dosage at 40 mg/kg administered intravenously
without renal enzyme inhibitor (MK-791)
Plasma
Aqueous
Vitreous
No. of
samples
5
5
5
Pre
0
0
0
0.5
15.12
± 1.31
3.10
±0.34
0.17
±0.03
Time (hrs)
1
1.98
±0.14
6.96
± 1.70
0.15
±0.06
2
0.28
±0.04
0.25
±0.01
0.06
±0.03
4
0
0
0
787 alone or combinations of MK-787 with the en-
zyme inhibitor MK-791 (Merck Sharp & Dohme
Research Laboratories, Rahway, NJ), were prepared
by admixing stock solutions in a water bath main-
tained at 80 C. The completely dissolved MK-787/
MK-791 solution was similarly injected at the final
dosage of 40 mg/kg MK-787 and 40 mg/kg MK-791.
Antibiotic assay: Concentrations of MK-787 in
plasma, aqueous humor, and vitreous humor were
assayed by a standard agar-diffusion bioassay tech-
nique using Bacillus subtilis ATCC 12432 (American
Type Culture Collection, Rockville, MD) as the test
organism.6 Preliminary studies in our laboratory in-
dicated that this bioassay technique is highly repro-
ducible with the lowest detectable concentration of
0.02 jig/ml. Furthermore, studies with standards of
ocular fluids, plasma, or MES buffer (50% ethylene
glycol: 1 M morpholino-ethane-sulfonate, pH 6.0),
spiked with known concentrations of either MK-787
alone or with MK-787/MK-791 yielded identical re-
sults, indicating that neither aqueous humor, vitreous
humor, plasma nor MK-791, interfered with the
bioassay of MK-787. Therefore, standards for both
ocular fluids and plasma were prepared in MES
buffer. Repeated measurements using spiked samples
of either aqueous humor, vitreous humor, or plasma
indicated that the standard error of this bioassay tech-
nique varied from 0 to 5%.
Pharmacokinetic determinations: The concentra-
tions of MK-787 in plasma and ocular fluids mea-
sured after the third intravenous dose were fitted to
a regression line by the method of least mean squares.
The half-life (tVi) of this agent in plasma or ocular
fluids was calculated by dividing In2 by the elimi-
nation constant k, where k = 2.3 X the slope of the
regression line. The area-under-the concentration vs
time curve (AUC) for plasma and aqueous as well as
vitreous humor were obtained by successive trape-
zoidal approximation for time = 0 to time = oo.7
Results. The intraocular and plasma concentra-
tions of MK-787 attained without and with co-ad-
ministration of the renal enzyme inhibitor MK-791
are shown in Tables 1 and Table 2, respectively. A
mean peak plasma concentration of 15.12 ii%/m\
without, and 20.4 iig/m\ with, co-administration of
MK-791 were observed at 30 min after infusion of
the third dose. Without MK-791, the plasma half-life
was 0.27 hr, and the AUC value was 9.45 ng hr/ml.
With co-administration of MK-791, the plasma half-
life was 0.45 hr, and the AUC value was 14.2 fxg hr/
ml. Mean plasma concentrations of MK-787 admin-
istered with MK-791 were significantly higher than
corresponding concentrations without MK-791 at 0.5
and 1 hr after infusion (P < 0.05, Student's t-test)
(Table 2).
The mean peak aqueous concentrations of MK-
787 in these animals were 6.96 /ag/ml without co-
administration of MK-791, and 8.66 Mg/ml with co-
administration of Mk-791, and in both instances,
mean peak aqueous concentrations were delayed un-
til 1 hr after drug administration, confirming a barrier
in intraocular penetration. Mean aqueous concentra-
tions of MK-787 co-administered with MK-791 were
significantly higher than corresponding concentra-
tions without MK-791 at 0.5 and 2 hrs after infusion
(P < 0.05) (Table 2). Based on the ratio of aqueous
AUC to serum AUC (X100), the penetration of MK-
787 into aqueous humor was estimated at 76%, both
when administered alone, and when co-administered
with MK-791.
Vitreous penetration by MK-787 in the normal
rabbit was relatively poor and was not significantly
Table 2. Concentrations (mean ± SE, /ug/ml) of N-formimidoyl thienamycin (MK-787) after third
dosage at 40 mg/kg co-administered intravenously with renal enzyme inhibitor (MK-791)
Plasma
Aqueous
Vitreous
No. of samples
5
5
5
Pre
0
0
0
0.5
20.40 ± 1.90*
4.82 ± 0.38*
0.30 ±0.13
3.66 d
8.66 :
0.13 :
Time (hrs)
1
b 0.43* 0.76
t 2.01 1.22
b 0.03 0.05
2
±0.24
± 0.43*
±0.03
4
0.06 ±
0.05 ±
0
q6h
0.05
0.03
6
0
0
0
• Significantly higher than levels achieved without renal enzyme inhibitor (P < 0.05, Student's t-test).
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No. 8 Reports 1149
affected by co-administration with MK-791 (2.9%
and 2.3%, respectively). Nevertheless, mean concen-
trations of 0.1-0.3 ng/m\ was attained. MK-787 was
cleared from all compartments at 4 hrs postinfusion
without the enzyme inhibitor and at 6 hrs postinfu-
sion when co-administered with MK-791.
Discussion. Although the intraocular penetration
of MK-787 is comparable to other 0-lactams3 and is
relatively poor in vitreous humor even after co-ad-
ministration with MK-791, the concentrations ob-
tained in the uninflamed eye, coupled with the ex-
quisite activity of this antibiotic, make its use in the
management of endophthalmitis potentially impor-
tant. In the present study, mean vitreous concentra-
tions attained (0.1-0.3 ng/m\) exceeded or ap-
proached the MIC90 (minimal antibiotic concentra-
tion inhibiting 90% of strains) for most sensitive
bacteria including Staphylococcus aureus (0.1 ng/m\),
Staphylococcus epidermidis (0.2 ixg/m\), Streptococ-
cus pyogenes (0.1 ng/m\), Streptococcus pneumoniae
(0.01 Mg/ml), Hemophilus influenzae(0. l fig/ml), and
Escherichia coli (0.1 /ig/ml).1 Other gram-negative
bacilli potentially important in bacterial endophthal-
mitis such as Pseudomonas aeruginosa (MIC90 12.5
jig/ml), Serratia marcescens (6.3 /ug/ml), and Proteus
mirabilis (1.6 ng/m\) are much less susceptible.1 It is
conceivable, however, that intraocular penetration of
MK-787 in the inflamed eye may be considerably
higher. As shown by Barza,3 inflammatory mediators
in the inflamed eye may lower the blood retinal bar-
rier and allow for greater antibiotic penetration.
Co-administration of the renal enzyme inhibitor
MK-791, known to have no antibiotic effect by itself,
resulted in higher and more prolonged plasma and
aqueous levels of MK-787. This most likely reflects
the greater systemic bioavailability, as evidenced by
the increased plasma half-life, of MK-787 due to in-
hibition of renal metabolism and inactivation of the
drug by MK-791. Our data do not suggest presence
of the inactivating dipeptidase enzyme within intra-
ocular compartments, but do suggest that the renal
metabolism of MK-787 in the rabbit may be similar
to that reported in chimpanzees and man.5
The observation that MK-787 penetrates unin-
flamed intraocular fluids at levels above the MIC90
for most sensitive bacteria, and that its intraocular
concentrations may be further augmented by the co-
administration of MK-791, strongly suggest its clin-
ical potential for the treatment of bacterial endo-
phthalmitis. Further studies of the clinical efficacy of
this new /3-lactam in experimental infective endo-
phthalmitis are clearly indicated.
Key words: endophthalmitis, ocular pharmacokinetics, N-
formimidoyl thienamycin, dipeptidase inhibitor, MK-787,
MK-791, vitreous humor, aqueous humor
Acknowledgment. The authors thank Karen H. Bartlett
for technical assistance, and Kathy Holding for preparation
of this manuscript.
From the Division of Infectious Disease, Department of Med-
icine, and the Department of Ophthalmology, University of British
Columbia and Vancouver General Hospital, Vancouver, BC, Can-
ada. Supported in part by a Medical Research Council of Canada
Development Grant (DG-189). Submitted for publication: Novem-
ber 18, 1983. Reprint requests: Anthony W. Chow, MD., Division
of Infectious Disease, GF Strong Research Laboratories, Vancou-
ver General Hospital, 700 West 10th Avenue, Vancouver, BC,
Canada V5Z 1M9.
References
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Ocular Penetration of N-Formimidoyl Thienamycin (MK-787) and Potentiation by Dipeptidase Inhibitor (MK-791)

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Ocular Penetration of N-Formimidoyl Thienamycin (MK-787) and Potentiation by Dipeptidase Inhibitor (MK-791)

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