Research ArticleAntibiotics

Treatment of otitis media by transtympanic delivery of antibiotics

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Science Translational Medicine  14 Sep 2016:
Vol. 8, Issue 356, pp. 356ra120
DOI: 10.1126/scitranslmed.aaf4363
  • Fig. 1. Synthesis, gelation, and mechanical properties of P407-PBP.

    (A) Gelation of aqueous solutions of 18%[P407], 3CPE-18%[P407], 18%[P407-PBP], and 3CPE-18%[P407-PBP], as a function of temperature. 3CPE: 2% LIM, 1% SDS, and 0.5% BUP. Data are means ± SD (n = 4). (B) TEM images of micelle formation. Scale bars, 100 nm. (C) Synthesis steps for P407-PBP and chemical structures of the ABCBA pentablock copolymers. (D) Schematic of the gelation of P407-PBP aqueous solution, facilitated by hydrophobic interactions of PPE end groups at elevated temperature. (E) FTIR spectra of P407, P407-PBP with n-butyl groups, and P407-PBP with s-butyl groups. (F) Effect of n- or s-butyl phosphoester and DP on G′ and G″ of 18% aqueous solution of P407 derivatives, as a function of temperature. Data are means ± SD (n = 4).

  • Fig. 2. Effects of individual CPEs and polymer concentrations on formulation rheology.

    (A) Effect of individual CPEs on gelation temperature of P407-PBP. (B) Effect of P407-PBP concentration on gelation temperature. All formulations contain 3CPE. (C) Effect of P407-PBP concentration on G′ and G″ at 37°C. Data are means ± SD (n = 4).

  • Fig. 3. Cumulative in vitro release and ex vivo transfer of ciprofloxacin across the TM into a receiving chamber.

    (A) Release of ciprofloxacin under infinite sink conditions. Two milligrams of ciprofloxacin were contained in each gel and solution at time zero. (B) Permeation of ciprofloxacin across the TM. The curves for Cip-12%[P407-PBP] and Cip-18%[P407-PBP] formulations overlap almost completely. The positions of the latter are indicated by arrows. Data are means ± SD (n = 4).

  • Fig. 4. In vivo efficacy, pharmacokinetics, and impact on hearing sensitivity for Cip-3CPE-12%[P407-PBP].

    (A) Percentage of animals with OM (defined as nonzero CFU values in their middle ear fluid aspirates) before (day 0) and after receiving Cip-3CPE-12%[P407-PBP] (n = 10), Cip-3CPE-18%[P407] (n = 5), 1% ciprofloxacin ear drop (n = 8), or no treatment (n = 10). Day 0 reflects status immediately before administration of therapeutics. *P = 0.0065 by Fisher’s exact test. (B) Time course of bacterial CFU from middle ear fluid from animals with OM from NTHi treated with Cip (n = 4), Cip-3CPE-18%[P407] (n = 5), Cip-3CPE-12%[P407-PBP] (n = 10), or no treatment (n = 10). Data are means ± SD. (Log10 CFU is set to zero instead of minus infinity for the purpose of this illustration.) (C) Concentration of ciprofloxacin over time in the middle ear fluid of the same animals as in (A). The black dotted line indicates the MIC for NTHi. Inset is the magnified drug concentration range of 0 to 10 μg/ml. Data are means ± SD. (D) Shifts in ABR thresholds in response to acoustic clicks and brief (8-ms) tone bursts of various frequencies. All data here had the threshold median before the treatment subtracted from them. The red lines indicate the interquartile range of values before treatment (n = 8). Measurements after application of 200 μl of Cip-3CPE-12%[P407-PBP] formulation are in black (n = 8). Black boxes and the lines within indicate the interquartile ranges and medians, respectively. Small black squares indicate the means, and crosses indicate the range. Original data are provided in table S4.

  • Fig. 5. In vivo effect on tissue for Cip-3CPE-12%[P407-PBP].

    (A) Representative photomicrographs of hematoxylin and eosin (H&E)–stained sections of TM cross-sections in healthy TMs and TMs after 7 days of OM without or after treatment with Cip-3CPE-12%[P407-PBP]. Scale bar, 12 μm. Labeled on each panel is the TM’s average thickness ± SD (n = 4). (B) H&E-stained cross-sections of the umbo-malleus region after 7 days of OM, without or after treatment with Cip-3CPE-12%[P407-PBP]. Scale bar, 20 μm. Thickness measurements are provided in table S4.

  • Fig. 6. Cytotoxicity of 12%[P407-PBP] and Cip-3CPE-12%[P407-PBP].

    (A) Survival rates (determined by MTS assay) of hFBs, PC12 cells, and keratinocytes after incubating with 12%[P407-PBP] and Cip-3CPE-12%[P407-PBP] for 1 or 3 days. Data are means ± SD (n = 4). (B) LIVE/DEAD assay of hFB, which was done to confirm the data in (A), after incubation for 1 or 3 days with 12%[P407-PBP] or Cip-3CPE-12%[P407-PBP]. GFP (green fluorescent protein), live cells (green); TRITC (tetramethyl rhodamine isothiocyanate), dead cells (red). (C) hFB cell survival rates were obtained by counting live and dead cells in (B) and calculating % cell survival = live cells/(live cells + dead cells). Cell counting was done using ImageJ. Data are means ± SD (n = 4). For all images, paired t test was applied.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/356/356ra120/DC1

    Materials and Methods

    Fig. S1. NMR of pentablock copolymers.

    Fig. S2. Modulation of gelation of aqueous solutions of Cip-[P407-PBP].

    Fig. S3. Simulation of drug transport across human TMs.

    Table S1. Molecular weight and polydispersity indices of P407 and pentablock copolymers.

    Table S2. Systemic exposure to ciprofloxacin by Cip and Cip-3CPE-12%[P407-PBP].

    Table S3. Apparent diffusion coefficients of Cip and Cip-3CPE-18%[P407-PBP] across healthy and infected human TM.

    Table S4. Tabulated data for Figs. 4 and 5 (provided as an Excel file).

    Movie S1. Gelation of Cip-3CPE-18%[P407] and Cip-3CPE-18%[P407-PBP].

    Movie S2. Gelation of Cip-18%[P407] and Cip-18%[P407-PBP].

    Movie S3. Extrusion of Cip-3CPE-18%[P407-PBP].

    Movie S4. Extrusion of Cip-3CPE-15%[P407-PBP].

    Movie S5. Extrusion of Cip-3CPE-12%[P407-PBP].

  • Supplementary Material for:

    Treatment of otitis media by transtympanic delivery of antibiotics

    Rong Yang, Vishakha Sabharwal, Obiajulu S. Okonkwo, Nadya Shlykova, Rong Tong, Lily Yun Lin, Weiping Wang, Shutao Guo, John J. Rosowski, Stephen I. Pelton, Daniel S. Kohane*

    *Corresponding author. Email: daniel.kohane{at}childrens.harvard.edu

    Published 14 September 2016, Sci. Transl. Med. 8, 356ra120 (2016)
    DOI: 10.1126/scitranslmed.aaf4363

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. NMR of pentablock copolymers.
    • Fig. S2. Modulation of gelation of aqueous solutions of Cip-[P407-PBP].
    • Fig. S3. Simulation of drug transport across human TMs.
    • Table S1. Molecular weight and polydispersity indices of P407 and pentablock copolymers.
    • Table S2. Systemic exposure to ciprofloxacin by Cip and Cip-3CPE-12%[P407-PBP].
    • Table S3. Apparent diffusion coefficients of Cip and Cip-3CPE-18%[P407-PBP] across healthy and infected human TM.
    • Legend for table S4
    • Legends for movies S1 to S5

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S4 Tabulated data for Figs. 4 and 5 (provided as an Excel file).
    • Movie S1 (.mp4 format). Gelation of Cip-3CPE-18%[P407] and Cip-3CPE- 18%[P407-PBP].
    • Movie S2 (.mp4 format). Gelation of Cip-18%[P407] and Cip-18%[P407-PBP].
    • Movie S3 (.mp4 format). Extrusion of Cip-3CPE-18%[P407-PBP].
    • Movie S4 (.mp4 format). Extrusion of Cip-3CPE-15%[P407-PBP].
    • Movie S5 (.mp4 format). Extrusion of Cip-3CPE-12%[P407-PBP].