NOVEL DELIVERY APPROACHES OF CO-TRIMOXAZOLE FOR RECREATING ITS POTENTIAL USE-A REVIEW

Co-trimoxazole appropriates to category of broad-spectrum antimicrobial. They are active upon administration in vitro against an extensive collection of microorganisms. Their application in medical field has roughly spanned over decade now. There are numerous approaches that were progressed for improving their effectiveness towards their antimicrobial potency. However, routine use of this could accelerate the chance of bacterial resistance, and portrait it ineffective when required to treat infection. Consequently, newer investigations are necessary to keep the drug effective by minimise the development of resistance and maximise its safe use. Safe use is meant by safe delivery of drug in low dose, low frequency at the targeted molecule by effective ways. This can be achieved by using nanocarrier systems as they possess smart characteristics of effective drug delivery. These nanocarrier systems are including nanoparticle, liposomes, nanogels etc. Present review article deals with the historical perspectives with regards to co-trimoxazole, their mechanism of act/resistance and spectrum of activity in first section. In second portion different novel carriers, importance and application of nanogels, rational for co-trimoxazole nanogels are discussed. In conclusion, different literatures have proved the efficacy of nanogels in delivery of antimicrobial drug similar to co-trimoxazole. In the present time very less data is available for delivery of this drug with novel carriers. Therefore, this review aims to encourage researchers for creating some new findings in this perspective

Nanocarriers in Tuberculosis treatment: Challenges and delivery strategies

The World Health Organization identifies tuberculosis (TB), caused by Mycobacterium tuberculosis, as a leading infectious killer. Although conventional treatments for TB exist, they come with challenges such as a heavy pill regimen, prolonged treatment duration, and a strict schedule, leading to multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. The rise of MDR strains endangers future TB control. Despite these concerns, the hunt for an efficient treatment continues. One breakthrough has been the use of nanotechnology in medicines, presenting a novel approach for TB treatment. Nanocarriers, such as lipid nanoparticles, nanosuspensions, liposomes, and polymeric micelles, facilitate targeted delivery of anti-TB drugs. The benefits of nanocarriers include reduced drug doses, fewer side effects, improved drug solubility, better bioavailability, and improved patient compliance, speeding up recovery. Additionally, nanocarriers can be made even more targeted by linking them with ligands such as mannose or hyaluronic acid. This review explores these innovative TB treatments, including studies on nanocarriers containing anti-TB drugs and related patents.LA/P/0101/2020; LA/P/0140/2020;info:eu-repo/semantics/publishedVersio

Toxicity of pro-oxidant molecules towards macrophages at different temperature.

<p>Macrophages are either untreated or treated with Methemoglobin, β-hematin or combination of β-hemain/Methemoglobin mixture at 37°C or subpermissive temperature (10°C) for 6 hr and the macrophage survival was measured by MTT assay. The experiment is performed in triplicate and values presented were the mean ± SD of four different experiments (n = 4). The MTT absorbance (0.37±0.05) of untreated cells is considered as 100% and used to express the survival of macrophages in other conditions.</p

Phagocytic Uptake of Oxidized Heme Polymer Is Highly Cytotoxic to Macrophages

<div><p>Apoptosis in macrophages is responsible for immune-depression and pathological effects during malaria. Phagocytosis of PRBC causes induction of apoptosis in macrophages through release of cytosolic factors from infected cells. Heme polymer or β-hematin causes dose-dependent death of macrophages with LC₅₀ of 132 µg/ml and 182 µg/ml respectively. The toxicity of hemin or heme polymer was amplified several folds in the presence of non-toxic concentration of methemoglobin. β-hematin uptake in macrophage through phagocytosis is crucial for enhanced toxicological effects in the presence of methemoglobin. Higher accumulation of β-hematin is observed in macrophages treated with β-hematin along with methemoglobin. Light and scanning electron microscopic observations further confirm accumulation of β-hematin with cellular toxicity. Toxicological potentiation of pro-oxidant molecules toward macrophages depends on generation of H₂O₂ and independent to release of free iron from pro-oxidant molecules. Methemoglobin oxidizes β-hematin to form oxidized β-hematin (βH*) through single electron transfer mechanism. Pre-treatment of reaction mixture with spin-trap Phenyl-N-t-butyl-nitrone dose-dependently reverses the β-hematin toxicity, indicates crucial role of βH* generation with the toxicological potentiation. Acridine orange/ethidium bromide staining and DNA fragmentation analysis indicate that macrophage follows an oxidative stress dependent apoptotic pathway to cause death. In summary, current work highlights mutual co-operation between methemoglobin and different pro-oxidant molecules to enhance toxicity towards macrophages. Hence, methemoglobin peroxidase activity can be probed for subduing cellular toxicity of pro-oxidant molecules and it may in-turn make up for host immune response against the malaria parasite.</p></div

MetHb and β-hematin interaction generate single electron containing species (β-hematin*) to exhibit cyto-toxicity towards macrophages.

<p>(<b>A</b>) Optical spectra of β-hematin oxidation by methemoglobin. Soret spectra were recorded in 100 mM Tris-HCl buffer, pH 7.4, in a total volume of 0.8 ml. Soret spectrum (a) of MetHb (1 µM); (b) a + H₂O₂ (100 µM); (c) b + β-hematin (10 µM). Equal concentration of β-hematin (10 µM) was added in the reference cuvette to correct the absorbance in soret region. (<b>B</b>) Scavenging single electron containing species (β-hematin*) restores cellular viability of macrophages. Cells were treated with different concentration of β-hematin (0–150 µg/ml)/methemoglobin (7.75 µM) mixture in the presence of different concentration of PBN (50–300 µM) or remained untreated. Cellular viability was determined by MTT assay as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103706#s2" target="_blank">material and methods</a>”. Cells treated with incomplete media was considered as 100% viable. Data is the mean ± SD of three independent experiments (n = 3) with triplicate measurement. (<b>C</b>) Light microscopic observation of macrophages treated in (B) with 20x objective to detect cellular morphology at 0 hr and 6 hrs. (<b>D</b>) Binding of PBN to the oxidized βH. β-hematin was incubated with the different concentration of PBN (0–600 µM) in the presence of MetHb (7.75 µM), H₂O₂ and optical spectra were recorded.</p

Change in level of lipid peroxidation, protein carbonyl and GSH in J774A.1.

<p>Macrophages were treated with different agonist for 6 h and lipid peroxidation, protein carbonyl and GSH level were measured as described under “material and method section”. Untreated cells were taken as control. The values presented were the mean ± SD of three different experiments (n = 3).</p

Determination of intracellular oxidative stress indices within macrophages.

<p>(<b>A–C</b>) Level of oxidative stress indices in macrophages treated with combination of β-hematin (60 µg/ml)/methemoglobin (7.75 µM) over the course of time. Macrophages were either untreated or treated with the combination of β-hematin (60 µg/ml)/MetHb (7.75 µM) for different time points (0–6 hr) and (<b>A</b>) lipid peroxidation (<b>B</b>) protein carbonyl and (<b>C</b>) reduced glutathione is measured and expressed as mM/mg of cell lysate. Data is the mean ± SD of three independent experiments (n = 3) with triplicate measurement. Cellular viability is measured to correlate the change in oxidative stress with the viability of treated cells. The correlation factor (r²) for change in viability Vs lipid peroxidation is 0.97, change in viability Vs protein carbonyl is 0.86 and change in viability Vs GSH level is 0.97.</p

Extracellular H₂O₂ Generation is responsible for methemoglobin mediated βH toxicological potentiation towards macrophages.

<p>(<b>A</b>) Removal of extracellular H₂O₂ gives recovery from cytotoxic effects of β-hematin towards macrophages. Macrophages were pre-incubated with different amount of catalase (0–500 U) and either remains untreated or treated with combination of β-hematin (60 µg/ml)/methemoglobin (7.75 µM) for 6 hr at 37°C. Macrophage viability was determined by MTT assay as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103706#s2" target="_blank">material and methods</a>” and expressed as % viability ± SD. (<b>B</b>) Scavenging free iron has no effect on reversal of cytotoxic effects of β-hematin towards macrophages. Macrophages were pre-incubated with different amount of deferoxamine (0–500 µM) and either remains untreated or treated with combination of β-hematin (60 µg/ml)/methemoglobin (7.75 µM) for 6 hr at 37°C. Macrophage viability was determined by MTT assay as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103706#s2" target="_blank">material and methods</a>” and expressed as % viability ± SD. Data is the mean ± SD of three independent experiments (n = 3) with triplicate measurement.</p

Oxidative stress is required for interaction of different pro-oxidant molecules (β-hematin/methemoglobin) to exhibit enhanced toxicity in macrophages.

<p>(<b>A</b>) Removal of oxidative stress through anti-oxidant treatement provides recovery in macrophages from the toxicity of β-hematin/methemoglobin mixture. Macrophages were either untreated or treated with β-hematin (60 µg/ml)/methemoglobin (7.75 µM) for 6 hr at 37°C in the absence or presence of NAC (5 mM) and mannitol (5 mM) respectively. Cell viability was measured by MTT assay as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103706#s2" target="_blank">material and methods</a>”. Macrophage treated with incomplete media was considered as 100% viable. Macrophage treated with combination of β-hematin (60 µg/ml)/methemoglobin (7.75 µM) was considered as 0% recovery and the cellular viability in the presence of NAC (5 mM) or mannitol (5 mM) was calculated and expressed as % recovery ±SD. Data is the mean ± SD of three independent experiments (n = 3) with triplicate measurement. The pairwise results were analyzed with Anova & Student t-test and it was considered statistically significant with *P<0.001, #P<0.001. (<b>B</b>) Light microscopic observation of macrophages treated in (A) with 20x objective to detect cellular morphology at 0 hr and 6 hr.</p

Effect of P2 with other pro-oxidant molecules on macrophage viability and membrane integrity.

<p>Macrophage are exposed to P2 (90 µg) alone or in combination with other pro-oxidant molecules present in malaria culture supernatant for 6 hrs and viability was determined by MTT assay where as membrane integrity was measured by LDH release assay. Macrophage exposed to hemin (60 µg/ml), heme polymer (40 µg/ml) was used as control. Change in cellular viability after reconstitution was calculated considering viability of P2 exposed macrophage as 100%. NA = “Not applicable”.</p