Formulation and Optimization of Metronidazole and Lactobacillus spp. Layered Suppositories via a Three-Variable, Five-Level Central Composite Design for the Management of Bacterial Vaginosis

Formulation and Optimization of Metronidazole and Lactobacillus Spp Layered Suppositories via a three-variable, five-level 3 central composite design for the management of bacterial vaginosis

The manuscript has been written and presented well however, the following are some points that need to be addressed by the authors before it could be considered for publication.

AUTHORS RESPONSE TO REVIEWER’S COMMENTS

Line 15

Abstract: The availability of a vaginal layered suppository with an antimicrobial agent and a probiotic has the potential to provide a synergistic effect and prevent recurrence in the management of bacterial vaginosis.

Please rephrase the title and this sentence to reflect the studies undertaken in this manuscript.

This section now reads “Bacterial vaginosis, a polymicrobial clinical syndrome characterized by a shift in healthy vaginal microbiota due to bacterial colonization is characterized by high recurrence rates after conventional treatment an antimicrobial agent.  This has necessitated the need to develop a formulation that has the potential to ensure lactobacilli viability and bacterial clearance.  

”

Line 27

This optimised formulation provides a safe, and promising alternative to conventional suppositories in the treatment and prevention of recurrence of bacterial vaginosis as it synergistically tackles the infectious microbe while restoring and maintaining the normal vaginal flora.

Please rephrase this sentence to reflect the studies undertaken in this manuscript.

Did authors prove synergistic effect?

This has been edited to read “This optimised formulation provides a safe, and promising alternative to conventional suppositories in the treatment and prevention of recurrence of bacterial vaginosis”

`Line 162

mass containing Lactobacillus sp spores was poured into the middle layer and cooled again to room temperature to produce the second layer with 150 million Lactobacillus sp

Please add details of the third layer.

This has been included

Line 176

Melting point is a measure of the time taken for the entire suppository to melt or disperse when immersed in a water bath maintained at 37 ± 1oC.

Please add relevant citation

The citation for this section has been added and the entire section re-written

Line 192

performed using the U.S.P 25 dissolution apparatus II. The paddle was rotated at 50 rpm in 900 mL of citrate buffer (pH 4.4), maintained at 37 ± 0.5°C.

Please add relevant citation.

Please describe why citrate buffer was used.

The citation for this section has been added. Citrate buffer was used to ensure a simulation with the vaginal fluid

Line 220

daily doses of 3g/kg-bwt of the formulated suppositories suppository and a marketed brand intravaginally

Please correct.

This has been corrected to read “The rats were randomized into three groups (12 test and 2 control) and were administered daily doses of 3g/kg-bwt of the formulated suppositories suppository intravaginally.”

Line 225

were humanely euthanized (via exposure to carbon dioxide gas) and vaginal tissues were excised and fixed in 10% formalin solution for histological analysis.

Please add relevant citation

This has been included

Line 232

3.1. Physicochemical Characterization of the bilayer suppositories

Its bilayer or multilayer?

This has been corrected through the entire manuscript to read layered

Line 235

The formulated suppositories met official British pharmacopoeia standard regarding weight and content uniformity.

Please add methodology details in method section with relevant citations.

This has been included in the manuscript

Line 363

stored for a period of 90 days exhibited excellent results for mechanical strength, melting point, disintegration time and drug content.

Please cite table or figures of these results.

This is reflected in the results section 3.7

“The optimized layered suppositories which were packed in suitable packaging materials and stored for a period of 90 days exhibited excellent results for mechanical strength, melting point, disintegration time and drug content. Storage did not affect any of these parameters with disintegration time of 6.05 ± 0.18 minutes; 97.77% ± 0.18% of metronidazole was optimally released within 7.5 minutes with lactobacilli viability maintained at 97.01%.”

Line 366

optimally released within 7.5 minutes with lactobacilli viability maintained at 97.01%.

Please cite table or figures of these results.

This has been included

Line 384

Physicochemical characterization of the bilayer suppositories

Bilayer or multilayer

Layered

Line 386

and opacity, odor, homogeneity, and weight variation.

Please describe how homogeneity was measured in methods.

This has been added into the manuscript

Line 390

e highest value obtained being 35 – 37.5 oC 20

Please check and correct

This has been corrected

Line 392

higher concentration of Ovucire did not show appreciably high solidification points

Please suggest why higher concentration of Ovucire did not show appreciably high solidification points

This has been included in the manuscript to read “Ovucire has an optimized concentration at which the desired solidification points is obtained increasing the concentration beyond this point created densely formed suppositories which lacked a cohesive structure when heat is applied hence the absence of an appreciably higher solidification point.”