Efficacy and Safety of Polaprezinc-Based Therapy versus the Standard Triple Therapy for Helicobacter pylori Eradication: A Systematic Review and Meta-Analysis of Randomized Controlled Trials

Helicobacter pylori (H. pylori) is the most prevalent etiology of gastritis worldwide. H. pylori management depends mainly on antibiotics, especially the triple therapy formed of clarithromycin, amoxicillin, and proton pump inhibitors. Lately, many antibiotic-resistant strains have emerged, leading to a decrease in the eradication rates of H. pylori. Polaprezinc (PZN), a mucosal protective zinc-L-carnosine complex, may be a non-antibiotic agent to treat H. pylori without the risk of resistance. We performed a systematic review and meta-analysis to evaluate the efficacy and safety of a PZN-based regimen for the eradication of H. pylori. This study used a systematic review and meta-analysis synthesizing randomized controlled trials (RCTs) from WOS, SCOPUS, EMBASE, PubMed, and Google Scholar until 25 July 2022. We used the odds ratio (OR) for dichotomous outcomes presented with the corresponding 95% confidence interval (CI). We registered our protocol in PROSPERO with ID: CRD42022349231. We included 3 trials with a total of 396 participants who were randomized to either PZN plus triple therapy (n = 199) or triple therapy alone (control) (n = 197). Pooled OR found a statistical difference favoring the PZN arm in the intention to treat and per protocol H. pylori eradication rates (OR: 2.01 with 95% CI [1.27, 3.21], p = 0.003) and (OR: 2.65 with 95% CI [1.55, 4.54], p = 0.0004), respectively. We found no statistical difference between the two groups regarding the total adverse events (OR: 1.06 with 95% CI [0.55, 2.06], p = 0.85). PZN, when added to the triple therapy, yielded a better effect concerning the eradication rates of H. pylori with no difference in adverse event rates, and thus can be considered a valuable adjuvant for the management of H. pylori. However, the evidence is still scarce, and larger trials are needed to confirm or refute our findings.


Introduction
Helicobacter pylori (H. pylori), a virulent Gram-negative organism infecting mainly the human gastric mucosa, afflicted nearly 4.4 billion of the world's population in 2015 [1]. Chronic infection with H. pylori can lead to the emergence of some serious alimentary complications, such as chronic gastritis, irritable bowel syndrome, peptic ulcer, and gastric cancer, the third most prevalent etiology of cancer-associated mortality around the world,

Protocol Registration
Our review was prospectively registered and published in an international prospective register of health-related systematic reviews (PROSPERO) with ID: CRD42022349231.

Protocol Registration
Our review was prospectively registered and published in an international prospective register of health-related systematic reviews (PROSPERO) with ID: CRD42022349231. We performed a systematic review and meta-analysis in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [41][42][43] and the Cochrane Handbook of Systematic reviews and meta-analysis [44]. The process is documented in a PRISMA 2020 checklist (Appendix A).

Data Sources and Search Strategy
Web of Science, SCOPUS, EMBASE, PubMed (MEDLINE), Google Scholar, and Cochrane Central Register of Controlled Trials (CENTRAL) were comprehensively searched by two reviewers (A.M. and M.A.) until 25 July 2022. We used no filters. The thorough selection procedure is illustrated in (Table 1).

Eligibility Criteria
We included randomized controlled trials (RCTs) with the following PICO criteria: population (P): patients with H. pylori infection; intervention (I): PZN 150 mg plus triple therapy (amoxicillin, clarithromycin, and PPI), control (C) triple therapy only and outcome (O): the primary outcome of this study is to evaluate the eradication rate of H. pylori (patients who achieved H. pylori clearance) according to intention to treat or per protocol analysis. The secondary outcome is the safety, defined as any reported adverse events. The exclusion criteria involved animal studies, cohort, retrospective, case reports, case reports, non-randomized trials, laboratory studies, and conference abstracts.

Study Selection
After duplicates removal using the Covidence online tool [45], two investigators (A.M. and H.A.) independently checked the eligibility of titles and abstracts of the obtained records. Then, they evaluated the full texts of the relevant studies according to the previously mentioned eligibility criteria. Any discrepancies were solved via discussion to reach a consensus.

Data Extraction
Using a pilot-tested extraction form, two reviewers (A.A.S.A. and H.A.) separately extracted the following data from the included articles: study characteristics (year of publication, country, study design, total participants, used triple therapy, frequency, and dose of PZN and method by which H. pylori was diagnosed); baseline information (age, sex, number of patients in each group, and number and location of ulcers); and efficacy outcomes data (intention-to-treat H. pylori eradication rate, per-protocol H. pylori eradication rate, and adverse events including (nausea, vomiting, heartburn, diarrhea, skin rash, and total adverse events). Disagreements were resolved by another investigator (A.M.).

Risk of Bias and Quality Assessment
The Cochrane Collaboration's technique was our guide to evaluate the risk of bias in randomized trials; two reviewers (A.A.S.A. and H.A.) separately evaluated the included studies for risk of bias (ROB) [46], based on the following six items: random sequence generation (selection bias), allocation concealment (selection bias), blinding of participants and personnel (performance bias), blinding of outcome assessment (detection bias), incomplete outcome data (attrition bias), selective reporting (reporting bias), and other potential sources of bias. Disagreements were settled through discussion. Two reviewers (M.T. and B.A.) employed the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) guidelines to appraise the quality of the evidence [47][48][49]. Imprecision, indirectness, inconsistency, publication bias, and bias risk were evaluated. Our results about the quality of evidence were justified, written, and included in each outcome. Any discrepancies were handled through discussion.

Statistical Analysis
The statistical analysis was carried out with Revman software version 5.4 [50]. We used odds ratio to pool dichotomous outcomes presented with the corresponding 95% confidence interval (CI). We utilized the I-square and Chi-square tests to assess heterogeneity; while the Chi-square test tells whether there is heterogeneity, the I-square determines the depth of heterogeneity. A grand heterogeneity (for the Chi-square test) is named as an alpha level below 0.1, in accordance with the Cochrane Handbook (chapter nine) [46], while the I-square test is interpreted as: (0-40 percent: not significant; 30-60 percent: moderate heterogeneity; 50-90 percent: substantial heterogeneity). We used the fixed-effects model. We calculated the number needed to treat (NNT) via the next equation, Absolute risk reduction (ARR) = (control event rate) − (experimental event rate) and the NNT equals the inverse of the ARR.

Search Results and Study Selection
We identified 1496 records after searching the databases, then 635 duplicates were excluded. Title and abstract screening excluded 841 irrelevant records. We moved to full-text screening with 20 articles, and 17 articles were excluded. Finally, only three articles met our inclusion criteria. The PRISMA flow chart of the detailed selection process is demonstrated in (Figure 2).
We identified 1496 records after searching the databases, then 635 duplicates were excluded. Title and abstract screening excluded 841 irrelevant records. We moved to fulltext screening with 20 articles, and 17 articles were excluded. Finally, only three articles met our inclusion criteria. The PRISMA flow chart of the detailed selection process is demonstrated in (Figure 2).

Characteristics of Included Studies
We included 3 trials with a total of 396 participants who were randomized to either PZN plus triple therapy (n = 199) or triple therapy alone (control) (n = 197). Further included trials' characteristics are presented in (Table 2). PZN dose was 150 mg twice daily for seven days in two trials [26,27] and for fourteen days in one trial [28]. Male participants were a total of 122 (61.3%) in the PZN group and 124 (62.94%) in the control group. Further baseline characteristics of the participants are presented in (Table 3).

Characteristics of Included Studies
We included 3 trials with a total of 396 participants who were randomized to either PZN plus triple therapy (n = 199) or triple therapy alone (control) (n = 197). Further included trials' characteristics are presented in (Table 2). PZN dose was 150 mg twice daily for seven days in two trials [26,27] and for fourteen days in one trial [28]. Male participants were a total of 122 (61.3%) in the PZN group and 124 (62.94%) in the control group. Further baseline characteristics of the participants are presented in (Table 3).

Risk of Bias and Quality of Evidence
We appraised the quality of the included studies according to the Cochrane risk of bias tool [46], as shown in Figure 3. Regarding the selection bias, Isomoto et al. [26] had low risk in the random sequence generation and unclear risk in the allocation concealment, Kashimura et al. [27] had unclear risk in both domains, and Tan et al. [28] had low risk in both domains. Moreover, the included trials had a high risk of performance and detection biases, except Kashimura et al. [27], with a low risk of performance and detection biases. Additionally, the included trials had a low risk of attrition bias. Furthermore, all included trials had an unclear risk of reporting bias. Finally, the included trials had a low risk of other bias. Author judgments are furtherly clarified in the Appendix (Appendix B). Using the GRADE system, the included primary outcomes yielded very-low-quality evidence. Details and explanations are clarified in Table 4.

Risk of Bias and Quality of Evidence
We appraised the quality of the included studies according to the Cochrane bias tool [46], as shown in Figure 3. Regarding the selection bias, Isomoto et al. [2 low risk in the random sequence generation and unclear risk in the allocation c ment, Kashimura et al. [27] had unclear risk in both domains, and Tan et al. [28] h risk in both domains. Moreover, the included trials had a high risk of performan detection biases, except Kashimura et al. [27], with a low risk of performance and tion biases. Additionally, the included trials had a low risk of attrition bias. Furthe all included trials had an unclear risk of reporting bias. Finally, the included trial low risk of other bias. Author judgments are furtherly clarified in the appendix (Ap B). Using the GRADE system, the included primary outcomes yielded very-lowevidence. Details and explanations are clarified in Table 4.    Figure 4A, Table 4). The pooled studies were homogenous (p = 0.27, I-square = 24%). From our calculation of the NNT on average, 7.5 patients would have to receive PZN treatment (instead of control treatment) for one additional patient to have the outcome, ARR = 0.67 − 0.804 = − 0.134. NNT = 1/ARR = 1/− 0.134 = −7.5.  Figure 4A, Table 4). The pooled studies were homogenous (p = 0.27, I-square = 24%). From our calculation of the NNT on average, 7.

Specific Adverse Events
Only two trials, Isomoto et al. [26] and Kashimura et al. [27], reported specific adverse events incidence, and we found no difference between the two groups regarding the incidence of diarrhea  Figure 5B). Nutrients 2022, 14, x FOR PEER REVIEW 10 of 18 Figure 5. Forest plot of the secondary outcomes (A) total adverse events, (B) specific reported adverse events [50]. OR: odds ratio, CI: confidence interval.

Specific Adverse Events
Only two trials, Isomoto et al. [26] and Kashimura et al. [27], reported specific adverse events incidence, and we found no difference between the two groups regarding the incidence of diarrhea  Figure 5B).

Discussion
H. pylori infection and colonization of the human gastric mucosa are prevalent in over 50% of the world's population [51]. Although most cases are asymptomatic, H. pylori can lead to significant complications, including peptic ulcer disease, gastric adenocarcinoma, and mucousa-associated lymphoma [52,53] Specifically, the incidence of peptic ulcer disease is about 10 to 20% of H. pylori patients with about 1 to 3% cases complicated by gastric cancer [4]. Accordingly, the burden of H. pylori is overwhelming, and an effective H. pylori eradication strategy is required. Therefore, we evaluated the efficacy and safety of PZN

Discussion
H. pylori infection and colonization of the human gastric mucosa are prevalent in over 50% of the world's population [51]. Although most cases are asymptomatic, H. pylori can lead to significant complications, including peptic ulcer disease, gastric adenocarcinoma, and mucousa-associated lymphoma [52,53] Specifically, the incidence of peptic ulcer disease is about 10 to 20% of H. pylori patients with about 1 to 3% cases complicated by gastric cancer [4]. Accordingly, the burden of H. pylori is overwhelming, and an effective H. pylori eradication strategy is required. Therefore, we evaluated the efficacy and safety of PZN as an adjuvant muco-protective agent in adjuvant with the standard triple therapy to eradicate H. pylori.
Regarding the H. pylori eradication rate, our pooled analysis favored PZN over triple therapy alone in both ITT analysis (80.4% versus 67.01%) and per-protocol analysis (86.8% versus 70.9%), respectively. Moreover, the incidence of adverse events was similar in both groups.
The specific mechanism of the PZN role in enhancing the eradication of H. pylori is still to be investigated, with several proposed theories: first, zinc can inhibit the urease activity leading to H. pylori's growth retardation by replacing the nickel ions at the active site of urease hindering the two metal ions from the complex formation [54]. Second, zinc can decrease the expression of interleukin 1 beta (IL-1β) by the gastric mucosa, further inhibiting H. pylori growth [55]. Third, PZN has shown to scavenge the monochloramine in H. pylori-infected Mongolian gerbils [25]. Finally, zinc has been shown to form a complex with famotidine inhibiting the urease enzyme and, subsequently, H. pylori growth, which was evident in both the antibiotic-resistant and sensitive strains [56] Recently, in comparative transcriptome analysis, Fan et al. proposed multiple potential anti-H. pylori effects of zinc [57]. First, zinc can alter the composition, structure, and function of the H. pylori type IV secretion system by the downregulation of cagI gene; hence, zinc can partially block the pathogenicity of H. pylori. Second, zinc can alter the synthesis process of lipopolysaccharide (LPS), a significant virulent factor of H. pylori, by altering the biosynthesis of lipid A (a significant hydrophobic part of LPS). H. pylori's surface LPS is a significant part of its cell wall contributing to the adhesion and infection of the gastric mucosa [57,58]. Therefore, disrupting LPS synthesis can subsequently affect the infectivity and adaptability of H. pylori [57]. Third, zinc upregulated the H. pylori translation and transcription genes, subsequently leading to increased protein biosynthesis, which can be an adaptation mechanism of H. pylori; however, Fan et al. argue that the synthesis of large amounts of in vivo proteins without the help of enough chaperones can lead to accumulation of mis-and unfolded proteins, subsequently disturbing the proteostasis and hindering H. pylori growth and even cell death [57]. Finally, zinc disrupted the flagellar protein assembly, disrupting H. pylori cell motility [57].
Regarding the status of high antimicrobial agents' resistance, implementing PZN into H. pylori can be beneficial. To clarify, the H. pylori resistance to clarithromycin and metronidazole is currently reported to be ≥ 15% [18,19], leading to a significant drop in the H. pylori eradication rates of triple therapy between 50% and 70% [18,19], which is significantly lower than the recommended ITT Maastricht H. pylori eradication rate of >80% [15]. Accordingly, PZN regimen can be effectively used for H. pylori with an ITT H. pylori eradication rate of 80.4%. Moreover, in a recent RCT, PZN was adjunctly used with the bismuth quadruple therapy achieving an H. pylori eradication rate of 93.5%, which was statistically significant in comparison with the triple therapy [24].
Regarding safety, PZN was safe and well tolerable in comparison with the triple therapy. The typical PZN dose is 150 mg, containing 34 mg zinc and 116 mg L-carnosine [59]. All the included trials used the typical dose with no crucial adverse events, and the reported adverse events were minor and faded spontaneously or managed feasibly [26][27][28]. However, Tan et al. observed more adverse events associated with the high-dose PZN (300 mg); they attributed this effect to either the toxic effect of the high dosage or patients' self-hypersensitivity [28]. Accordingly, the standard dose of PZN (150 mg) can be used safely with triple therapy.

Strengths
To the best of our awareness, this is the first systematic review and meta-analysis synthesizing evidence on the efficacy and safety of PZN for H. pylori eradication; hence, this study constitutes gold standard evidence in this regard. Moreover, our review was executed and fulfilled via the guidance of the PRISMA recommendations [42,43].

Limitations
Our review has a few limitations. First, we only included three RCTs with a small sample size and limited population distribution confined to the Far East [26][27][28]. Second, the proton pump inhibitor component of the triple therapy varied across the included trials; hence, this can affect our findings. Third, multiple confounding variables can significantly affect our findings, including smoking habits, genetic predisposition of cytochrome p450 2C19, the physical status of the participants, and H. pylori strain resistance. Fourth, all the included trials had a relatively short follow-up duration ranging from one to two months only [26][27][28]. Finally, the GRADE assessment yielded very-low-quality evidence; hence, the extrapolation and the generalization of our findings is limited.

Implications for Future Research
Future trials are required to address: first, the comparative efficacy of PZN adjunctly with the bismuth quadruple therapy versus the bismuth quadruple therapy alone is still to be investigated. To clarify, bismuth quadrable therapy is currently recommended as the first-line regimen in areas with a significant prevalence of ciprofloxacin and metronidazole resistance. As such, investigating the efficacy of PZN in the settings with significant resistance is still required [60]. Second, future trials should determine the baseline clarithromycin resistance to enable health authorities to predict the H. pylori eradication rate of PZN-based regimen in areas with known rates of clarithromycin resistance using the H. pylori-nomogram [28,61]. Finally, future trials should expand the follow-up duration up to 6 or 12 months to properly investigate the improvement in the gastrointestinal symptoms [28].

Conclusions
The addition of PZN to the triple therapy yielded greater eradication rates of H. pylori with no difference in adverse event rates and thus constitutes a valuable adjuvant for the management of H. pylori. However, the evidence is still scarce, and larger trials are needed to confirm or refute our findings. As such more high-quality, multicenter randomized controlled trials are warranted to ascertain its efficacy and yield generalizable findings.

Conflicts of Interest:
The authors declare no conflict of interest.  Table 1 Selection process 8

Abbreviations
Specify the methods used to decide whether a study met the inclusion criteria of the review, including how many reviewers screened each record and each report retrieved, whether they worked independently, and if applicable, details of automation tools used in the process.

Page 3 Section 2.4
Data collection process 9 Specify the methods used to collect data from reports, including how many reviewers collected data from each report, whether they worked independently, any processes for obtaining or confirming data from study investigators, and if applicable, details of automation tools used in the process.

Results of individual studies 19
For all outcomes, present, for each study: (a) summary statistics for each group (where appropriate) and (b) an effect estimate and its precision (e.g., confidence/credible interval), ideally using structured tables or plots.

Results of syntheses 20a
For each synthesis, briefly summarise the characteristics and risk of bias among contributing studies.  Describe and explain any amendments to information provided at registration or in the protocol. Page 2 Section 2.1 Support 25 Describe sources of financial or non-financial support for the review, and the role of the funders or sponsors in the review. Page 12 Competing interests 26 Declare any competing interests of review authors. Page 12 Availability of data, code and other materials 27 Report which of the following are publicly available and where they can be found: template data collection forms; data extracted from included studies; data used for all analyses; analytic code; any other materials used in the review. Page 12