Key words 1,3-Bis[4-(2-morpholinoethoxy)phenyl]adamantane - 2,2-Bis[4-(2-morpholinoethoxy)phenyl]adamantane
- antituberculars - drug delivery
Introduction
Tuberculosis (TB) is a disease usually caused by a bacterium called Mycobacterium tuberculosis (Mtb). Once rare in developed countries, tuberculosis infections began increasing
in 1985, partly because of the emergence of HIV [1 ]. The bacteria usually attack the lungs, but Mtb bacteria can attack any part of
the body, such as the kidney, spine, and brain. If not treated properly, TB disease
can be fatal. Many strains of tuberculosis resist to the drugs mostly used to treat
the disease and as a result new drugs and regimens are urgently needed to shorten
the required duration of tuberculosis treatment [2 ]
[3 ]
[4 ]
[5 ]
[6 ]. N -Adamantan-2-yl-N' -[(E )-3,7-dimethylocta-2,6-dienyl]ethane-1,2-diamine (SQ109 , [Fig. 1 ]), is a drug candidate that is active against both drug-susceptible and drug-resistant
TB strains and affects cell wall synthesis [7 ]. It bears in its skeleton the active 1,2-ethylenediamine pharmacophore [8 ] of the most widely used antitubercular drug, ethambutol (EMB), and was found safe
and well tolerated in Phase I and early Phase II clinical trials. Based on these findings
and our previous experience, the diarylmethane moiety, which is reported in the literature
[9 ], as active against Mycobacterium tuberculosis, was introduced into the adamantane skeleton. Moreover, 2 phenolic rings, functionalised
with an aminoether side chain, were introduced onto C1, C3 (compound I ) and for comparison purposes onto the C2 position of the adamantane ring (compound
II ).
Fig. 1 Chemical structures of SQ109 and compound I : 1,3-Bis[4-(2-morpholinoethoxy)phenyl]adamantane and II : 2,2-Bis[4-(2-morpholinoethoxy)phenyl]adamantane.
Both of these compounds exhibit antitubercular activity [10 ]. Albeit the fact that the lipophilicities of compound (I ) (clogP=7.835) and (II ) (clogP=7.985) are substantially higher than that of SQ109 (clogP=6.205), they are, however, within the allowed limits for oral administration
[11 ]. Therefore, it was intriguing to probe their oral absorption profile, because this
information is of paramount importance for future in vivo studies and subsequent clinical
trials on their potential as tuberculosis treating agents. To this end, dissolution
studies, at pH values of 1.2 and 6.8, were conducted, as an initial attempt to decipher
the release characteristics of these compounds from controlled release matrix tablets.
Controlled release vs. immediate release was chosen as it is known to be more clinically
useful in treating tuberculosis suffering patients compared with immediate release
or conventional therapy [12 ].
Materials and Methods
Chemistry
The synthesis of compounds (I ) and (II ) involved the nucleophilic attack of the phenoxide, formed by adding sodium hydride
in dry DMF to the corresponding phenolic derivative, to 4-(2-chloroethyl)morpholine,
under heating [10 ].
Biological Evaluation
Bacterial strains and culture conditions
Mycobacterium tuberculosis strains H37Rv, Mmpl3 mutant and STR-starved 18b (SS18b)
were grown at 37°C with shaking in 7H9 broth (Difco) supplemented with 10% albumin-dextrose-catalase
(ADC) enrichment, 0.2% glycerol, 0.05% Tween 80. Streptomycin (STR) (50 µg/mL) was
added to SS18b culture. Replicating strain H37Rv was generated as follow. H37Rv and
MmpL3 mutant were grown to mid-logarithmic phase in medium. Non-replicating, SS18b
was generated as follows: 18b was grown to mid-logarithmic phase in STR-containing
medium and washed 3 times in phosphate-buffered saline containing 0.05% Tween 80 (PBST).
Final bacterial pellets of H37Rv, MmpL3 mutant and SS18b strains were re-suspended
in medium and frozen in 15% glycerol at -80°C in 0.5-milliliter aliquots (supplemented
with 50 µg/mL STR for SS18b). When needed, one aliquot of H37Rv, MmpL3 mutant and
SS18b was defrosted and inoculated in 7H9. SS18b culture was maintained at an optical
density of 600 nm (OD600) between 0.2 and 0.5 for 2 weeks (with the addition of fresh
medium if necessary), by which time they had stopped replicating. MmpL3 mutant was
generated by Dr. Giovanna Riccardi in the University of Pavia. This strain has a mutation
in mmpL3 genes and an amino acid change V681I. This strain is known to be resistant
to BM212 and its derivatives. (http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0056980 ).
Antimicrobials
Ethambutol (EMB) and rifampin (RIF) were purchased from Sigma-Aldrich. Adamantane
aminoethers, EMB and RIF were dissolved in dimethyl sulfoxide (DMSO).
Resazurin reduction microplate assay (REMA)
To determine the in vitro efficacy of compounds, a H37Rv culture (OD600=0.0001) and
a 2 week old SS18b culture (OD600=0.1) were used in the REMA. 2-fold serial dilutions
of each test compound were prepared in white 96-well plates containing the bacilli
in a total volume of 100 µl and then were incubated for 6 days at 37°C before the
addition of 10 µl of 0.025% resazurin. After overnight incubation, the fluorescence
of the resazurin metabolite resorufin was determined (excitation, 560 nm; emission,
590 nm; gain, 70) by using a Tecan Infinite M200 microplate reader.
Statistical analysis
Data were processed and graphs were constructed with Prism version 5.0 (GraphPad).
Materials used for the preparation of matrix tablets
Compounds I and II were provided by our colleagues in the Pharmaceutical Chemistry Division of our Department.
Low viscocity Sodium Alginate and Polyvinylpyrrolidone (PVP, M.W.: 55.000) were purchased
from SIGMA. Lactose monohydrate was purchased from Merck and Magnesium Stearate from
Riedel-De Haen.
Tablets’ preparation and dissolution experiments
The dissolution experiments involved flat tablets (10 mm diameter, 200 mg weight and
6–9 kp hardness). Each matrix tablet was comprised of compound (I ) or compound (II ) and combinations of the following excipients: low viscosity sodium alginate, lactose
monohydrate, polyvinylpyrrolidone (M.W.: 55.000) and magnesium stearate ([Table 1 ]). The tablets were stirred at 50 rpm in a USP XXIII dissolution apparatus II (Pharmatest,
Hainerp, Germany) containing for the first 3 h 500 ml of gastric (pH 1.2) and for
the next 5 h 1000 ml of intestinal fluids (pH 6.8) at 37±0.5°C. Samples (5 ml) were
withdrawn at predetermined time intervals, filtered and analyzed at λmax =223 nm for compound (I ) and λmax =245 nm for compound (II ), using a Perkin-Elmer UV spectrophotometer (Norwalk, CT). All experiments were carried
out in triplicate. The results obtained are graphically presented in [Fig. 2 ] and [Fig. 3 ].
Fig. 2 Drug release (%) vs. time of Compounds I and II in Formulation 1.
Fig. 3 Drug release (%) vs. time of Compounds I and II in Formulation 2.
Table 1 Quantitative composition of the formulants used for the preparation of compounds
(I) and (II) tablets.
Ingredients
Formulation 1 (mg)
Formulation 2 (mg)
Compound I or II
5
5
Sodium alginate
144
130
Magnesium stearate
2
2
Lactose monohydrate
49
–
PVP (M.W.: 55.000)
–
63
Total
200
200
The structures of the new compounds were sketched in Maestro 10.2 [13 ] and prepared using the LigPrep 3.4 module [14 ]. They were minimized using the OPLS3 force field. Computer program QikProp [15 ] was used to predict the physically significant descriptors of the compounds.
Comparison indices, f1 and f2 , were also used to compare the dissolution profiles of compounds I and II [16 ]
[17 ].
Results and Discussion
The 2 new adamantane isomers, 4,4'-[4,4'-[adamantane-1,3-diyl]bis(phenoxyethyl)]dimorpholine
(I ) and 4,4'-[4,4'-[adamantane-2,2-diyl]bis(phenoxyethyl)]dimorpholine (II ), have shown different tuberculocidal potencies. The analogue (I ) exhibited a significant activity in the REMA assay; the MIC value of (I ) is 7.1 µg/mL against M. tuberculosis MmpL3 mutant, which is at least 14-fold more
potent than its congener (II ). The same pattern was shown against the H37Rv strain, with derivative (I ) having a MIC value of 24.1 µg/mL, i. e., about 5-fold more active than (II ).
The solid pharmaceutical formulations include various excipients ([Table 1 ]), which both in acidic (0–3 h, pH 1.2) and in neutral pH (3–8 h, pH 6.8) environment
facilitate the controlled/extended release of these 2 new bioactive substances. It
becomes apparent from the release curves of compounds I and II , shown in [Fig. 2 ]
[3 ], that the lactose formulant present in F1 tablets leads to a faster 100% release
(180 min) than the PVP excipient in formulation F2 (240 min).
In all cases, the f1 values are higher than 15 and the f2 estimates are lower than 50%, implying a high degree of dissimilarity in the dissolution
profiles of the compounds I and II ([Table 2 ]). The difference in the overall release profiles observed between the compounds
I and II ([Fig. 2 ]
[3 ]) is possibly related to their respective degrees of lipophilicity. These differences
are also corroborated by the values of the f1 and f2 factors.
Table 2 f1 and f2 indices for Formulations 1 and 2.
Indices
Formulation 1
Formulation 2
f
1
81.49
68.42
f
2
12.40
17.32
The theoretical clogP and QPlogPo/w values suggest that compound II is more lipophilic than its isomer I (
[Table 3 ]
) . This is possibly due to the fact that the 2 phenyl rings in I are in spatial vicinity, allowing them to form a π-π stacking complex (dotted line)
([Fig. 4a ]). Conversely, the respective phenyl rings in compound II are too far away to form this type of complex ([Fig. 4b ]). As a result more water molecules are trapped in the surrounding region of I and this justifies its higher solubilisation [16 ].
Fig.4 a Compound I : 1,3-Bis[4-(2-morpholinoethoxy)phenyl]adamantane; b Compound II : 2,2-Bis[4-(2-morpholinoethoxy)phenyl]adamantane
Table 3 Calculation of significant descriptors and pharmaceutically relevant properties
for the compounds I and II.
Computed property
II
I
QPlogP0 /w1
5.382
4.773
QPlogS2
−5.271
−2.809
QPPCaco3
566.843
548.164
1 QPlogPo/w: Predicted octanol/water partition coefficient. Range or recommended values
for 95% of known drugs: −2.0 – 6.5.
2 QPlogS: Predicted aqueous solubility, log S. S in mol dm–3 is the concentration of the solute in a saturated solution that is in equilibrium
with the crystalline solid. Range: –6.5 – 0.5.
3 QPPCaco: Predicted apparent Caco-2 cell permeability in nm/s. Caco- 2 cells are a
model for the gut-blood barrier. QikProp predictions are for non-active transport.<25
Poor;>500 Great.
Conclusions
In conclusion, 2 new tuberculocidals adamantane aminoethers, although more lipophilic
than SQ109 , showed a satisfactory controlled release profile. Taking into account that the currently
used antitubercular medicines have limited efficacy against the rising threat of drug-resistant
Mtb, significant side effects, and must be given in combinations of 4–6 drugs for
at least 6 months, for drug-sensitive Mtb, and up to 24 months for drug-resistant
Mtb, new drug TB treatment with less frequent dosing and improved patient compliance,
is very important. Information about the oral absorption profile of the molecules
presented herein is very useful in future in vivo studies.
