A Review on Inosine Pranobex Immunotherapy for Cervical HPV-Positive Patients

Introduction

To date, >200 human papillomavirus (HPV) types have been detected.^1–3 The most common HPV transmission route is sexual contact.² Sexually active individuals will acquire ≥ 1 anogenital HPV genotype at some point in their lives.⁴ Sensitive PCR monitoring has indicated that most HPV infections are cleared by the immune system and disappear within 1–2 y without clinical complications.^5,6 Epidemiological evidence for HPV carcinogenicity was first presented by zurHausen in 1983 and accepted in 1995 by the International Agency for Cancer Research (IARC) of the World Health Organization (WHO).⁴ HPV carcinogenicity was first established for cervical precancers and cancers. Cervical carcinogenesis is a multi-step process starting with HPV infection of the cervical epithelium. However, cancer onset occurs after persistent or recurring HPV infection.⁷ There is ample evidence to support the hypothesis that host immunological status and HPV-induced immune changes are responsible for persistent HPV infection and subsequent development of cervical neoplasia.^7–9 The immune system eliminates the virus and recognizes the tumor antigen associated with cervical carcinogenesis.^7,9 Both cellular and humoral immunity are vital for clearing cervical HPV infection.^8,9

To date, no antiviral drugs effectively kill or hinder the progress of cervical HPV.¹⁰ Hence, host immunity alone must fend off and clear genital HPV.¹¹ However, the host immune response may be inadequate and require modulation.¹² Suppressive modulations may be required when the host immune response is too strong. In contrast, immunostimulation is needed when host immunity is weak.¹²

Immunotherapy is an essential treatment modality for microbial infections, malignancies, and other forms of pathogenesis.¹³ Immunotherapeutic agents may be specific and nonspecific.¹⁴ Immunomodulators are nonspecific immunotherapeutic agents and can be immunostimulants or immunosuppressants.¹⁵ The former induce nonspecific humoral and cellular immune responses.

Inosine pranobex (IP), also known as inosine acedobendimepranol (INN) or methisoprinol, is an antiviral drug consisting of a 1:3 ratio of inosine and dimepranolacedoben. The latter is a salt of acetamidobenzoic acid and dimethylaminoisopropanol.¹⁶ Since 1971, IP has been licensed for the treatment of cell-mediated immunodeficiency associated with human papillomavirus (HPV), herpes simplex virus (HSV), varicella-zoster virus (VZV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV).¹⁶

The purpose of this review was to examine and summarize data on IP immunotherapy for cervical HPV-positive patients.

Materials and Methods

Google Scholar, PubMed/MEDLINE, Scopus, Cochrane Library, Research Gate, were searched from 1971 when IP was first licensed until 2021. The main terms used during database search were cervical human papillomavirus infection, clearance, persistence, oncogenesis, immunity, inosine pranobex, and immunomodulation.

Prospective controlled trials, observational and retrospective studies, meta-analysis, and reviews on immunotherapy against HPV cervical infection were examined. The primary outcomes were persistence and clearance of cervical HPV genotypes in women undergoing IP therapy and the onset of low- or high-grade cervical intraepithelial neoplasia (CIN) or LSIL/HSIL (low/high squamous intraepithelial lesions) in patients being monitored for a certain period of time. The overall quality of evidence for immunotherapy against cervical HPV infection was evaluated. Of 108 identified abstracts, 62 full-text articles were reviewed, and 41 studies were included in the synthesis.

Cervical HPV Infection: Self-Clearance and Persistence

Persistence of HPV infection is critical in the development of cervical precancerous lesions and cancer. The main unresolved issues regarding the natural history of HPV are the extent to which the viral infection is self-cleared and the time required for this process.¹⁷ Unlike self-clearance, persistence, or the time during which HPV infection remains detectable, is unclear.^17,18 Persistence is longer for high-risk than low-risk HPV types.^5,19,20 Only ~10% of all HPV infections persisting for several years were strongly associated with a high absolute risk of a precancer diagnosis.^21–23

Rositch et al²⁴ carried out a meta-analysis of published studies on the persistence and self-clearing of HPV infection and the risks of developing CIN and cervical cancer associated with it.²⁴ They summarized the data for 86 studies involving > 100,000 patients.²⁴ The authors concluded that ~50% of the HPV infections persisted within the last 6–12 mo.²⁴ Cervical HPV tests at 12-mo intervals can identify women at an increased risk of high-grade CIN as a result of persistent HPV infection.²⁴ The PATRICIA study comprised 4825 women with cervical HPV infections.²⁵ The aim of that trial was to determine the proportions of HPV infections that are cleared or progress to CIN.²⁵ Persistent infection with high-risk HPV genotypes more frequently leads to CIN compared to persistent infections with low-risk HPV genotypes.²⁵ High-risk HPV type infections were the least likely to be cleared.²⁵ Miranda et al²⁶ surveyed 569 women in Brazil for persistence and clearance of HPV infection over 24 mo. Eighty-nine of these patients were HPV positive.²⁶ After 24 mo, HPV persistence and clearance were observed in 59.6% and 40.4% of the women, respectively.²⁶ High-risk HPV genotypes persist over a longer period of time and play a major role in CIN and cervical cancer pathogenesis.²⁶

Cervical HPV clearance and persistence depend on host immunity.^7,27–30 Adequate immune response is vital to the self-clearance of numerous cervical HPV types.^27,28,30 Ineffective immune response is associated with insufficient activation of congenital immunity, non-inclusion of the adaptive immune response, or strong viral persistence.^7,29,31,32 Nguyen et al followed systemic and mucosal immune responses to cervical HPV in women who underwent radical hysterectomy for cervical cancer (HCC) or loop conization for cervical dysplasia.⁸ They analyzed HPV-specific antibodies through ELISA and cytokine assays performed with the Linco cytokine multiplex method using Luminex technology on vaginal washes.⁸ They found relatively lower levels of HPV16 E7-specific immunoglobulin A (IgA) in the vaginal wash of women with cervical cancer and cervical dysplasia compared with the control. Neither Th1-nor Th2-type cytokine induction was detected in the cervical cancer group.⁸ Thus, selective downregulation of local HPV-specific IgA responses occurred in the cervical cancer patients.⁸

In HPV-induced carcinogenesis, interactions between virally infected keratinocytes and the cervical local immune response can determine the course of the disease.³⁰ Elucidating these interactions could help develop innovative diagnostic immune tests to determine cervical pre-cancer progression and design novel immunotherapy approaches targeting the mechanisms and pathways involved in HPV-induced carcinogenicity.³⁰

Cervical HPV Infection and Host Immune Response

HPV is an epitheliotropic virus that exclusively attacks epithelial cells and primarily spreads and acts locally in this type of tissue. In the cervix, HPV infects the keratinocytes in the basal layer of the cervical epithelium in the transformation zone of the cervix.^27,30,33,34 Viral replications markedly increase in differentiated epithelial layers that have undergone recent desquamation.^27,35 Viral particles are assembled when differentiated keratinocytes die and the virus is released during desquamation into the ambient environment.³⁵ Downregulated protein expression in the lower epithelial layers, high non-cytolytic genomic replication, and the absence of a viremic phase enable the virus to evade immune system recognition.^33,34

Keratinocytes infected by HPV initiate an adaptive immune response³⁶ as they are part of innate immunity. Antigens may not be fully represented in keratinocytes but the cells nonetheless upregulate the cytokines TH1 and TH2 and cytotoxic responses in CD4⁺ and CD8⁺ memory T cells, respectively.^7,30,36 CD4⁺ T helper (Th1 and Th2) and CD8⁺ T cell responses were identified in patients with genital HPV-induced warts.³⁷ Comparisons of HPV6 and HPV11 antigen-specific T cell responses between venereal wart patients and healthy controls disclosed low frequencies of IFN-γ-producing CD4⁺ and CD8⁺ T cells in HPV-infected women.³⁷ The relative number of IL-4-expressing CD4⁺ T cells was significantly higher in infected women than in normal women.³⁷ The observed change from Th1 to Th2 in HPV-infected patients was a consequence of a weakened immune response caused by the HPV infection. A direct effect of depressed CD8 T cell activity is delayed or poor genital wart clearing.³⁷ In the female genital tract, keratinocytes express several Toll-like receptors (TLR) on the cell surfaces (TLR-1, TLR-2, TLR-4, TLR-5, and TLR-6) and in the endosomes (TLR-3 and TLR-9).^7,30,38 TLR are a family of immunological receptors that recognize pathogen-related molecular patterns (PAMPs) and induce congenital and adaptive immune responses.^7,28 TLRs recognize viral nucleic acids. TLR-3 recognizes one-sided-chain RNA (dsRNA) while TLR-7 and TLR-8 recognize single-chain RNA (ssRNA) and TLR-9 recognizes CGG-rich DNA.²⁸ Activated TLRs receptors induce cytokine production and strong anti-inflammatory responses.³⁹ Switching on TLR-9 in keratinocytes initiates the production of TNF-α, IL-8, CCL2, CCL20, CXCL9, and type 1 IFN.^40,41 The second level is adaptive immunity via cytotoxic T lymphocytes (CTLs) targeting HPVE2 and HPVE6 and eliminating HPV-infected cells.⁴¹ CD4⁺ and CD8⁺T lymphocytes predominate in cervical epithelium.³³ Langerhans cells are located on the surface epithelial layers.³³ When HPV attacks the epithelium, Langerhans cells switch on and activate T lymphocytes.⁴² Adequate T cell immune responses regress HPV-induced lesions and clear local HPV infection.⁴³ Inactive T cell immune responses are associated with long-term viral persistence and cervical cancer induction.³³

HPV can evade encounters with the immune system.^28,33,41 As HPV lacks a viremic phase, immune cells cannot easily enter into contact with it.^28,33 HPV attacks basal epithelial layers of the cervix as immunocompetent Langerhans cells occur only in the surface epithelial layers and avoid invoking the T cell immune response.^33,44 HPV does not cytolyse the cervical epithelium. In this way, it attenuates inflammatory and immune signal responses and can persist incognito.^41,44 HPV also avoids the immune response by downregulating its oncogenes throughout the initial life cycle and only synthesizing immunogens such as L1 and L2 capsid proteins in surface epithelial layers that undergo desquamation.^33,34,41

Evasion of a local immune response is required for the development of successful cervical HPV infection.^33,44 Therefore, the application of therapeutic agents stimulating innate immunity may effectively prevent the onset of local HPV infections.

Inosine Pranobex: Immunomodulatory Mechanism

Inosine acedobendimepranol (IAD) is a synthetic purine derivative consisting of a 3:1 ratio of p-acetamidobenzoic acid salt of N,N-dimethyl-amino-2-propanol (DiP.PAcBA) and the β polymorph of the β anomer of inosine.IAD has immunomodulatory and antiviral properties¹⁵ and is registered in numerous countries under the brand names Immunovir, Isoprinosine, Viruxan, Inosiplex, Methisoprinol, and Inosine Pranobex.¹⁵

IP was introduced in 1971 and several studies from the 1970s and 1980s showed that it did not induce serious side effects and was well tolerated by patients.^45,46 At high doses and after long-term use, the metabolites of IP may cause reversible increases in serum urinary uric acid and nausea.^45,46

Several studies have demonstrated that IP is antiviral and immunomodulatory. However, its exact mechanisms are not fully understood.⁴⁷ IP affects cellular and humoral immunity and is antiviral by immunomodulation.⁴⁷

IP stimulates a Th1 cell response characterized by proinflammatory cytokine (IL-2 and IFN-c) upregulation in mitogen- and antigen-activated cells.^48,49 Induction of IL-2 and IFN-c activates T lymphocyte maturation and differentiation and the lymphoproliferative response.^49,50 Similarly, IP modulates T lymphocyte and natural killer cell cytotoxicity and T8 suppressor and T4 helper cell function and increases the numbers of immunoglobulin G (IgG) and complement-surface markers.⁴⁷ Certain studies investigated the influences of IP on Th1- and Th2-related cytokines. When the lymphocytes were at rest, the cytokine levels remained constant.^49,51 Lymphocyte stimulation with IP upregulated IL-2, interferon c (IFN-c),and tumor necrosis factor alpha (TNF-a) and downregulated IL-4, IL-5, and IL-10.^49,51

IFN-c expression and IP block IL-10 production. Moreover, IP downregulates other anti-inflammatory cytokines and may block their effects on the immune response.⁵² IP administration increases the number and activity of natural killer cells (NK).^47,53 NK activity in eosinophils rises in response to increases in the numbers of lgG and complement-surface markers as well as macrophage, monocyte, and neutrophil chemotaxis and phagocytosis.^51,53

IP suppresses the synthesis and translational ability of lymphocyte mRNA and inhibits viral RNA synthesis by:

The humoral immune response consists of activating B lymphocyte transformation into plasma cells and initiating antibody production.⁵⁵ B lymphocyte transformation may be a response to IP action but also a consequence of the effects of IP on macrophages and T helper cells.⁴⁷ IP stimulates certain immune mediators, promotes Th1 cells, and inhibits Th2 cells.⁵⁰ Most of the 21 healthy patients undergoing daily administration of 4 g IP in a Bulgarian study presented with rapid immune responses, increases in the numbers of their lymphocytes, and circadian fluctuation of these parameters in their peripheral blood.⁵⁶ In another Bulgarian study, serum cytokine levels were measured in 10 healthy volunteers during and after IP treatment (1 g IP, 3x daily, 3 wks).⁴⁸ Increases in proinflammatory cytokine levels were observed.⁴⁸ Another trial evaluated lymphocyte subsets [CD19⁺ B cells, CD3⁺ T cells, CD4⁺ T helper cells, FoxP3hi/CD25hi/CD127lo regulatory T cells (Tregs), CD3-/CD56⁺ NK cells, and CD3⁺/CD56⁺ NKT cells], serum immunoglobulins, and IgG subclasses in ten healthy volunteers being administered 4 g IP daily for 14 d.¹⁵ NK cell counts increased over time and recurred at the end of the study. Furthermore, different high/low Treg and T helper fractions were detected during the study period.¹⁵

The effects of IP on the immune systems of patients with different diseases were also investigated. Herpes virus-infected patients had higher lymphocyte transformation rates than healthy controls.⁵⁷ In patients with autoimmune diseases and chronic active hepatitis B, IP therapy increased the numbers of active T cell rosettes and enhanced responses to mitogen activation.^58,59 High NK cell activity and elevated numbers of CD4⁺ T helper cells were observed in patients with chronic fatigue syndrome undergoing IP therapy.⁶⁰

Despite the foregoing studies and their findings, the precise immunomodulatory mechanism of IP remains to be clarified.

Cervical HPV Infection and IP Immunotherapy

The first studies on IP administration to patients with genital HPV infection were published in the 1970s and 1980s soon after the drug was launched. Most reports evaluated the clinical efficacy of IP combined with cryotherapy, surgery, or electrosurgery of genital condylomata. In most cases, clinical efficacy of the combination therapies surpassed that of IP administration alone. Sadoul and Beuret followed two groups of patients with cervical or vulvovaginal condylomata.⁶¹ They were treated either with CO₂ laser or with CO₂ laser plus IP.⁶¹ Monotherapy was less efficacious than the combination treatments.⁶¹ Combination therapy consisting of CO₂ laser plus IP significantly reduced the number of recurring condyloma acuminate.⁶¹ Mohanty and Scott compared the efficacies of IP, conventional therapy, and their combinations in 165 patients with genital warts.⁶² Whereas conventional therapy alone had 41% efficacy, the combination of IP plus conventional therapy increased efficacy to 94%.⁶² The patients undergoing IP therapy in this study presented with relatively higher numbers of B cells.⁶² In a multicenter, prospective, randomized, placebo-controlled study, Davidson-Parker et al administered 3 g IP daily for 4 wks to 55 patients with genital warts.⁶³ The IP therapy improved the relative clinical efficacy of the conventional treatment (podophyllin or trichloroacetic acid).⁶³ Nejmark et al evaluated the efficacy of combination therapy including IP for the treatment of male genital warts.⁶⁴ The combination therapy had long-term efficacy and there was only a 7% relapse rate. In contrast, destructive therapy was associated with a 32% relapse rate.⁶⁴ The authors recommended IP (isoprinosine) for both the complex therapy and prevention of the recurrence of genital warts.⁶⁴ Tay performed a randomized, placebo-controlled study on the efficacy of IP immunotherapy. They orally administered 1 g IP thrice daily for 6 wks to 55 patients with vulvar HPV infections. There were 22 patients in the IP group and 24 in the placebo group.⁶⁵ After 2 mo, IP monotherapy proved effective in 14 (63.5%) of the patients. However, only 4 patients (16.7%) in the placebo group presented with substantial improvement in vulvar epithelial morphology (P = 0.005).⁶⁵ The authors concluded that IP therapy is suitable for the treatment of vulvar HPV infections.⁶⁵

IP has been on the market since 1971.Nevertheless, our scientific literature and electronic databases searches revealed that only a few studies mainly from the last 20 y have focused on the use of IP in the treatment of patients with cervical HPV infections. It was only in 1983 that zurHausen discovered the connection between cervical HPV infection and carcinogenesis. Moreover, this finding was only accepted as irrefutable scientific fact by the global medical community in the 1990s. It has not been determined why IP immunotherapy has seldom been applied in the treatment of cervical HPV. Furthermore, some of the carcinogenetic mechanisms of HPV have been explored but there are some details that need to be completely clarified. Finally, the relationships among cervical HPV infection, host immune response, and the immune system remain to be established. For these reasons, IP immunotherapy has not been entirely adopted for the treatment of cervical HPV infection.

Most studies on IP administration for cervical HPV infection originated in Eastern Europe, especially Russia.^10,66–70 It appears that IP administration for immunotherapy against viral diseases is widespread and common in Eastern European countries. Table 1 provides a summary of the studies on the efficacy of IP in HPV genital infections.

Table 1 Summary of Studies Investigating the Efficacy of IP Therapy in HPV Genital Infections, Especially Cervical Infections

Bitsadze et al reported that combined therapy including IP eliminated HPV in 98% of all cases and lowered the relative relapse rate by threefold.⁶⁸ Rakhmatulina reviewed numerous Russian studies and concluded that IP against HPV infection had 87.5–97% clinical efficacy in combination therapy and 72.4–95% clinical efficacy in monotherapy.⁶⁶

Eliseeva et al assessed 5650 women with genital HPV infection from 33 towns in the Russian Federation before and after IP monotherapy and combined treatments.⁷¹ After 6 mo, HPV self-clearance without treatment had occurred in 22.6% of all cases.⁷¹ The rates of HPV negative cases were 35.5%,54.8%, and 84.2% after conventional, single IP, and combined IP treatments, respectively.⁷¹ Therefore, combined IP had the greatest efficacy in terms of clearing genital HPV infection and regressing its associated diseases.⁷¹ Pestrikova and Pushkar evaluated the effectiveness of IP treatment in 123 patients with cervical HPV pathology. HPV16 and HPV18 were the most frequently isolated viral types in this study.⁷² IP treatment stopped CIN1 progression in 96.23±2.62% of all patients.⁷² Cervical HPV clearance and full recovery were observed in 91.06% of all patients administered IP.⁷² Pestrikova et al used colposcopy and PAP and cervical PCR tests for HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 to assess the efficacy of IP treatment in 39 women with cervical HPV infection.⁷³ The therapeutic regimen comprised three courses of 3000 mg IP daily at 10-d intervals between courses.⁷³ At the end of the trial, viral load declined to a clinically insignificant level in 17.95% of all women and there was 74.36% cervical HPV clearance.⁷³ Over 3 y, Ordiyants et al followed 80 patients with persistent cervical HPV infection and dysplasia. They had been subjected to a combination of radio-wave conization and three courses of 1000 mg IP thrice daily for 10 d with 14 d intervals between courses.⁷⁴ At the end of the study, clearance of cervical HPV infection was confirmed for 97.5% of all patients.⁷⁴ Budanov et al assessed cervical HPV infection in 100 women aged 19–35 y.⁶⁷ Forty-four of the patients were excluded as they had mixed infections. The remaining 56 were all HPV positive and were divided into two groups.⁶⁷ The first consisted of 25 women (44.6%) who were administered 1000 mg oral IP monotherapy thrice daily for 10 d and subjected to cervical HPV PCR after 60 d.⁶⁷ The second comprised 31 women (55.4%) with confirmed CIN1treated by electro-ablation plus 1000 mg oral IP monotherapy thrice daily for 10 d. They were subjected to cervical HPV PCR, PAP tests, and colposcopy after 60 d.⁶⁷ In the first group, clearance of cervical HPV infection was established in 17 patients (68%) and persistence in eight patients (32%). The latter were administered two additional IP courses.⁶⁷ Cervical HPV PCR revealed 88% clearance of cervical HPV infection after 6 mo.⁶⁷ In the second group, the combined treatment provided 93.5% cervical HPV clearance after 6 mo.⁶⁷ Hence, both the IP monotherapy and combined therapy had strong cervical HPV clearance efficacy.⁶⁷ Kedrova et al evaluated the efficacy of combined IP treatment in women presenting with cervical HPV16 and HPV18 infection and CIN.⁷⁵ After IP treatment, HPV16 and HPV18 were cleared in 77.8% and 50% of the women, respectively.⁷⁵ Few patients required any additional IP treatment.⁷⁵ In a subsequent study, 128 patients with cervical HPV infection and LSIL were divided into three groups and followed for up for 6 mo. In the first group, 48 women were treated for 28 d with six 0.500-mg IP tablets taken at 8-h intervals. In the second group, 41 women were treated with the same IP dose over a 14-d period. In the third group, 39 women were administered no IP.⁷⁶ After 6 mo, there was no evidence of cervical HPV infection in 93.7%,78.0%, and 43.6% of the women in the first, second, and third groups, respectively.⁷⁶ Based on the foregoing results, the authors recommended IP monotherapy for rapid clearance of cervical HPV infection.⁷⁶

In our 6-y trial, 32 women with cervical HPV infection had undergone electroconization (loop electrosurgical excision) of the uterine cervix for established HSIL and were randomly divided into two groups. In the first group, ten patients (31.2%) were not administered postoperative IP immunotherapy while the 22 patients in the second group (68.8%) did receive it. All women were subjected to cervical tests after 24 mo and 48 mo to check for HPV persistence.¹¹ The patients in the second group were administered 1 g IP thrice daily at 8-h intervals for 1 mo. For the next 5 mo, the patients were administered 500 mg IP thrice daily at 8-h intervals.¹¹ At the end of the trial, five of the patients in the first group (15.6%) presented with cervical HPV infection whereas two of the patients in the second group (6.3%) had persistent HPV infection comprising a mixture of HPV16 and other high-risk genotypes.¹¹ Three patients (9.3%) from the second group had new HPV infections consisting of different genotypes.¹¹ At the end of the study period, only four patients (12.5%) in the second group had cervical HPV infections.¹⁰ Two patients (6.2%) in the second group had newly established cervical HPV infections comprising various genotypes while another two patients (6.2%) in the second group had persistent HPV infections including HPV16 and other high-risk genotypes.¹¹ By the end of the trial, there were significant differences between groups in terms of HPV incidence. Hence, long-term IP therapy reduces HSIL recurrence in HPV-positive women who have undergone surgery.¹¹ Adjuvant IP immunotherapy augmented cervical HPV infection clearance and diminished cervical dysplasia relapses in patients who were positive for cervical HPV and who underwent HSIL surgery.¹¹

Conclusion

• Genital intraepithelial neoplasia and corresponding cancers caused by HPV are associated with high morbidity and mortality rates. Thus, ongoing research on HPV infection, epidemiology, pathogenesis, treatment, and prophylaxis is required to reduce the incidence and severity of these diseases. There is a clear relationship between HPV and human immunity, but the underlying mechanisms have not been fully elucidated.
• The IP therapy has demonstrated good clinical efficacy in stimulating innate immunity, strengthening the host immune system, and promoting cervical HPV clearance.
• IP therapy shows promise at lowering the recurrence of cervical dysplasia and precancers caused by chronic cervical HPV infection.