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Treatment of cavitary and infiltrating pulmonary tuberculosis with and without the immunomodulator Dzherelo

S. I. Zaitzeva1, S. L. Matveeva1, T. G. Gerasimova1, Y. N. Pashkov1, D. A. Butov1, V. S. Pylypchuk2 and G. A. Kutsyna3
1) Department of Phtysiatry and Pulmonology, Kharkov National Medical University, Kharkov, 2) Ekomed LLC, Kiev and 3) Department of Xxx, Luhansk 7 State Medical University, Luhansk, Ukraine
Abstract
An open-label, 60-day trial was conducted in 75 newly diagnosed tuberculosis (TB) patients to assess the adjunctive effect of the oral immunomodulator Dzherelo with standard anti-TB chemotherapy (ATT) consisting of izoniazid, rifampicin, pyrazinamide and streptomycin (HRZS) administered as directly observed therapy. Group 1 (n = 28) with cavitary TB and group 2 (n = 17) with infiltrating pulmonary TB received 50 drops of Dzherelo twice daily in addition to HRZS. Group 3 (n = 30), which served as a control, received ATT Oonly. Liver damage indicators, bilirubin, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) decreased to normal levels in groups 1 and 2, but increased significantly in group 3. Kidney failure markers, urea and creatinine, normalized in Dzherelo recipients, but were unchanged or worsened in the ATT-only group. The changes in serum lactate dehydrogenase, catalase, malondialdehyde and diene conjugates suggested that Dzherelo downregulates TB-associated inflammation. The anti-inflammatory property of Dzherelo was further supported by a favourable haematology profile, reduced erythrocyte sedimentation rate and faster defervescence. Radiological recovery was significant in both Dzherelo groups, but not in the control group (p = 0.0085, p = 0.025 and p = 0.23, respectively). 6|These findings correlated positively with sputum smear conversion and clinical findings (r = 0.94; p < 0.05). Mycobacterial clearance at day 30 was observed in 77%, 72% and 40% of patients in groups 1, 2 and 3, respectively. After 2 months sputum conversion rates in groups 1, 2 and 3 were 93%, 89% and 70%, respectively. Sixty-day treatment outcomes in groups 1, 2 and 3 as assessed by improvement in clinical features and respiratory function attained respective p-values of 0.008, 0.25 and 0.72, and 0.013, 0.48 and 0.0015. Dzherelo is thus useful as an immunotherapeutic adjunct in the management of TB.
Keywords: Botanical, cavitary, DOT, hepatotoxicity, herbal, immunotherapy, Mycobacterium, phytoconcentrate, phytomedicine, phytotherapy, X-ray
Original Submission: 11 June 2008; Revised Submission: 30 September 2008; Accepted: 23 October 2008 Edited by M. Drancourt Clin Microbiol Infect
Introduction
Tuberculosis (TB) is a re-emerging global public health problem, the incidence of which has increased in the Ukraine, as it has in other countries [1]. The incidence of TB in the Odessa region was 178 cases per 100 000 individuals in 1962. It gradually declined to 73.0, 42.0 and 41.6 cases per 100 000 in 1972, 1982 and 1992, respectively. This trend, however, then reversed, and by 2002 the incidence and prevalence of TB had risen to 80.4 and 330 cases per 100 000, respectively. Mortality caused by TB more than doubled from 10.2 per 100 000 to 21.6 per 100 000 between 1990 and 2001 [2]. The success rates of therapy in Eastern Europe, including the Ukraine, as in Africa, are substantially below those in other regions of the world [1]. In addition, the Ukraine has worsening epidemics of drug-resistant TB, which is increasingly converging with HIV. Despite the availability of anti-TB drugs the situation is far from ideal and better therapeutic interventions are clearly needed to reverse the current trend.
The oral immunomodulator Dzherelo, which contains extracts of many plants (see below), is used in the Ukraine for the management of both TB and HIV infections, including simultaneous infections by both. Dzherelo was approved in 1997 by the Ukrainian Ministry of Health as an immunomod-ulating supplement and has been used extensively for various indications, including chronic bacterial and viral infections, autoimmune diseases and malignancy. In 1999 Dzherelo was recommended by the Ukrainian health authorities as an immune adjunct in the treatment of tuberculosis [3]. Previous clinical studies have indicated that Dzherelo can increase CD4 T-lymphocytes and decrease viral load, and that it improves the clinical response when combined with standard anti-retroviral (ART) or anti-tuberculosis therapy (ATT).
Dzherelo has been found to reduce the incidence of opportunistic infections and reverse TB-associated wasting. Dzherelo has also been found to alleviate the hepatotoxicity associated with ATT, as evidenced by improvement in liver function tests [4-10]. However, these studies have not dealt with the effect of Dzherelo on other clinical parameters associated with TB. Our study was thus aimed at defining the adjunct effect of Dzherelo on clinical and radiological symptoms as well as on selected biochemical and blood parameters among patients with cavitary and infiltrating pulmonary TB. The addition of Dzherelo to standard ATT was compared with a treatment regimen consisting of ATT alone.
Materials and Methods
Patients and ethical approval
The study involved 75 patients with newly diagnosed pulmonary TB. All patients were males aged between 19 and 68 years. All patients who presented with newly diagnosed pulmonary TB were enrolled in this study. No restrictive exclusion criteria for study enrolment were set up except that we disallowed patients who tolerated chemotherapy poorly or had resistant strains of Mycobacterium tuberculosis.
Patients were divided into three groups. Group 1 contained 28 individuals with cavitary or destructive forms of pulmonary TB; their mean ± standard deviation (SD) age was 46.5 ± 11.9 years (median 48 years). Group 2 contained 17 TB patients with the disseminated form of pulmonary TB but without cavitary lesions; their mean age was 46.4 ± 13.7 years (median 47 years). The third group, which received standard TB therapy without Dzherelo and served as a comparison population, contained 30 patients with a mean age of 42.7 ± 11.3 years (median 44 years) and included patients with the cavitary and the infiltrating forms of pulmonary TB. Patients were allocated to the Dzherelo and ATT-only groups without formal randomization. All study patients presented with the active form of pulmonary TB. The most common symptoms were prolonged heavy cough, pain in the chest, high fever, profuse night sweats, fatigue, dyspnoea, haemoptysis, and loss of weight and appetite. Active pulmonary tuberculosis was defined by a medical history and clinical findings compatible with tuberculosis, a chest X-ray showing lung involvement, and a positive sputum smear for acid-fast bacilli (AFB) and positive culture for M. tuberculosis. Participation in this study was voluntary and conduct of the trial was approved by the internal review board of the TB dispensary.
Treatment regimen
All patients received standard ATT, consisting of orally administered izoniazid (H; 300 mg), rifampicin (R; 600 mg) and pyrazinamide (Z; 2000 mg), and intramuscular injection of streptomycin (S; 1000 mg). The anti-TB drugs were procured through the Ukraine’s centralized national supply system. Patients in groups 1 and 2 also received 50 drops of Dzherelo, given in a glass of water twice daily, usually 2 h after breakfast and 30 min before supper. The treatment was administered for 60 days as directly observed therapy (DOT) to patients hospitalized in our department at Kharkov National Medical University, between March and May 2006.
The over-the-counter phytoconcentrate Dzherelo (Immunoxel) was generously supplied by Ekomed LLC (Kiev, Ukraine). It contains a concentrated aqueous alcohol extract from medicinal plants, including: aloe (Aloe arborescens); common knotgrass (Polygonum aviculare); yarrow (Achillea millefolium); centaury (Centaurium erythraea); snowball tree berries (Viburnum opulus); nettle (Urtica dioica); dandelion (Taraxacum officinale); sweet-sedge (Acorus calamus); oregano (Origanum majorana); marigold (Calendula officinalis); sea buckthorn berries (Hippophae rhamnoides); elecampane (Inula helenium); tormentil (Potentilla erecta); greater plantain (Plantago major); wormwood (Artemisia sp.); Siberian golden root (Rhodiola rosea); cudweed (Gnaphalium uliginosum); licorice (Glycyrrhiza glabra); fennel (Foeniculum vulgare); chaga (Inonotus obliquus); thyme (Thymus vulgaris); three-lobe beggarticks (Bidens tripartita); sage (Salvia officinalis); dog rose (Rosa canina), and juniper berries (Juniperus communis).Dzherelo was approved in 1997 by the Ukrainian Ministry of Health as a dietary supplement. In 2006 it received the status of a ‘functional food’, placing it in a superior category of herbal supplements which can carry medical claims substantiated by clinical evidence.
Clinical endpoints
Primary endpoints of interest in this study were the effect of prescribed therapy on clinical features and radiological and microbiological findings. The clinical manifestations of TB that were considered comprised a complex of so-called ‘intoxication’ symptoms and signs, including fever, night sweats, loss of appetite, weight loss, weakness, depression, cough, and lung crepitations, etc. [11,12]. The second aspect of clinical evaluation concerned respiratory function and covered symptoms such as dyspnoea and lung function tests, including total lung capacity (TLC), forced vital capacity (FVC), forced expiratory volume (FEV), residual volume (RV), carbon monoxide transfer factor, O2UQ and CaO2 according to the Dembo and Liberman classification [11,12]. In both cases, the features or tests were given a score ranging from 0 (severely affected) to 3 (absent or normal) and the mean of these scores was used to define the overall result as indicative of no, mild or severe abnormality. In addition, information on the potential adverse effects of Dzherelo administered in combination with chemotherapy was sought via questionnaires completed by the patients.
Laboratory evaluation
A standard microbiological examination of a sputum smear stained by the Ziehl–Neelsen method was conducted prior to study entry and at days 30 and 60 from the start of treatment. Isolates of M. tuberculosis were tested for sensitivity to first- and second-line anti-TB drugs with a commercially available kit (Tulip Diagnostics Pvt Ltd, Goa, India) [7]. These tests were supplemented by regular examination of haematological and biochemical parameters. Plasma levels of catalase, malondialdehyde (MDA), lactate dehydrogenase (LDH) and diene conjugates (DC) were measured as markers of oxidative stress, as described earlier [7].
Statistical analysis
The results were analysed with the statistical software GraphPad (GraphPad Software, Inc., La Jolla, CA, USA). Baseline quantitative values were compared with end-of-study values by paired or unpaired Student’s t-test. Non-parametric or categorical values of treatment outcomes were compared by McNemar or chisquare contingency tables. All statistical analyses were performed on an intent-to-treat basis, involving the total number of patients without subgrouping them into responders and non-responders. The resulting probability values were considered significant at p < 0.05.
Results
Lack of adverse reactions
During the entire follow-up no adverse reactions attributable to Dzherelo were identified. In particular, no dyspepsia, malaise, intolerance or allergic reactions were evident at any time (data not shown).
Effect on body temperature
The dynamics of body temperature among treated patients are shown in Fig. 1. All three groups showed a decline in mean body temperature during the investigation. Group 1, however, showed a clear tendency towards more rapid normalization: the mean axillary temperature on day 30 reached a ‘normal’ level, at 36.65 ± 1.24 C, compared with 37.17 ± 2.02 C in group 3 (p < 0.005). Group 2 had a similar rate of decline to group 1, whereas group 3 showed a slower rate of decrease.

Effect on haematological parameters
The effects of ATT and Dzherelo on blood cell counts, erythrocyte sedimentation rate (ESR) and haemoglobin are shown in Table 1. Patients in groups 1 and 2 reacted in a similar manner and displayed changes which appeared to be specific to Dzherelo intervention as opposed to the effect of ATT alone in group 3 patients. In particular, the relative content of lymphocytes almost tripled among Dzherelo-treated patients, reaching normal levels, whereas patients in the control group failed to attain normal values. Elevated counts of cells, such as neutrophils, eosinophils and total leukocytes, commonly associated with inflammation, were reduced to a greater extent in Dzherelo-treated patients, although the decrease was also statistically significant in group 3 patients. By contrast, monocyte levels were almost unchanged among patients who received Dzherelo, whereas patients in the comparison group had a greater reduction in these cells. Other cells such as erythrocytes behaved in a similar manner in all three groups. Erythrocyte sedimentation rate declined faster in the Dzherelo groups. Haemoglobin concentrations decreased at the same rate in all patients. In general, Dzherelo appeared to have greater effect in normalizing the haematology profile than ATT treatment alone.
Serum biochemistry markers of liver and kidney function improved markedly among Dzherelo recipients, whereas patients in group 3 showed a decline in liver function (Table 2). All three evaluated hepatic markers, bilirubin, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), decreased in groups 1 and 2 but increased in group 3 in a statistically significant manner. Levels of urea and creatinine normalized in Dzherelo recipients but were unchanged or worsened in ATT-alone patients.

Mobirise

Clinical validation of sublingual formulations of Immunoxel (Dzherelo) as an adjuvant immunotherapy in treatment of TB patients

The significant proportion of patients presented with newly diagnosed TB (37.7%); remaining patients had re-treated TB (RTB), treatment-failed TB, MDR-TB and extra-pulmonary TB at a 31:6:4:2 ratio. Most of the patients were treated with a standard isoniazid, rifampicin, pyrazinamide, ethambu-tol and streptomycin combination (84.9%) or were on individualized chemotherapy regimens (15.1%). The duration of chemotherapy prior to immunotherapy initiation ranged between 1 and 11 months with a mean ± standard deviation (SD) of 2.6 ± 2.4 (median: 2.0) months for the group, comprising 42% of patients who were treated for 1 month or less, and the rest were treated for a mean ± SD of 3.8 ± 2.7 (median: 2.5) months. The study group had 15 females and 54 males between the ages of 20 and 61 years with a mean ± SD age of 38.7 ± 10.5 (median: 36.0) years. The baseline bodyweight and BMI ranged between 40 and 98 kg and 15.2 and 36.0 kg/m2, respectively, with a mean ± SD of 56.9 ± 9.2 (median: 56.0) and 19.1 ± 3 (median: 18.5), respectively. At study initiation, only six out of 69 (8.7%) patients had normal 36.8°C axillary body temperature, with an overall mean ± SD of 38.0 ± 0.7 (median: 38.0)°C.
Twenty one patients in the control chemotherapy group received placebo sugar pills (Tables3 &4). The group had afemale:male ratio of 2:19 with an age range between 21 and 58 years and a mean ± SD of 36.6 ± 9.8 (median: 35.0) years. The baseline bodyweight and BMI ranged between 47 and 72 kg and 16.3 and 22.5 kg/m2 with a mean ± SD of 60.9 ± 6.7 (median: 61.0) and 19.9 ±1.6 (20.5), respectively. The baseline distribution of TB categories was according to the ratio 5:11:5:6 for newly diagnosed TB (first diagnosis), RTB, MDR-TB andTB/HIV, respectively. Most patients (57.1%) received standard first-line TB chemotherapy while the remaining patients were on an individualized TB drug regimen. At the beginning of the study, four out of 21 patients (19%) had normal body temperature with an intragroup mean ± SD of 37.7 ± 0.5 (median: 38.0)°C. Participation in this study was voluntary and patients were eligible to enroll only after signing the written consent. The conduct of the trial was approved by the internal review board of the Lisichansk TB Dispensary [101].

■ Solid formulations of Immunoxel
Four different solid formulations were designed for sublingual absorption:
■ Sugar-based dragees
■ Sugar-coated pills
■ Gelatin pastilles
■ Dried-honey lozenges

Sugar-based dragées were made by Ekomed. Sugar-coated pills were made in collaboration with Immunitor at their manufacturing plant in Chachoengsao, Thailand. The gelatin-based formulation was made by a local confectionery factory specializing in the production of gummy bear-like candies. The dried-honey lozenges were made in collaboration with Island Abbey Foods Ltd (Charlottetown, Prince Edward Island, Canada). This company has a unique proprietary technology for making dried-honey lozenges.
Each sublingual preparation contained either five or ten drops (125 or 250 pl) of Immunoxel, which was administered once per day for a month. Immunoxel, an improved and simplified version of the herbal immunomodulator Dzherelo, contains an aqueous—alcohol cold extract from medicinal plants including aloe (Aloe arborescens), licorice (Glycyrrhiza glabra), dog rose fruit (Rosa canina), oregano (Oreganum majorana), sage (Salvia officinalis), thyme (Thymus vulgaris), fennel (Foeniculum vulgare), purple coneflower (Echinacea purpurea), dandelion (Taraxacum officinale), nettle (Urtica dioica), marigold (Calendula officinalis), greater plantain (Plantago major), wormwood (Artemisia sp.), common knotgrass (Polygonum aviculare), yarrow (Achillea millefolium), cen-taury (Centaurium erythraea), elecampane (Inula helenium), tormentil (Potentilla erecta), cudweed (Gnaphalium uliginosum), three-lobe beggarticks (Bidens tripartita), Siberian golden root (Rhodiola rosea), chaga (Inonotus obliquus), snowball tree berries (Viburnum opulus), seabuckthorn berries (Hippophae rhamnoides) and juniper berries ( Juniperus communis). This multiherbal extract is manufactured by Ekomed according to international Hazard Analysis Critical Control Point (HACCP) standards and has been approved by the Ministry of Health of Ukraine as an immunomodulating herbal supplement belonging to a functional food category.

■ Laboratory evaluation
The sputum microscopy on acid-fast bacilli smears was conducted in a blinded fashion at baseline and 1 month later. TB drug resistance was determined by a commercially available kit (Tulip Diagnostics, Goa, India) in approximately a third of the patients. The failure to test every patient for drug resistance was due to a temporary lack of funds for laboratory services. MDR-TB status was assigned when resistance to both isoniazid and rifampicin, with or without resistance to other drugs, was present.
The hematology parameters such as hemoglobin content, leukocyte counts and erythrocyte sedimentation rate (ESR) were evaluated by standard routine techniques at baseline and repeated 1 month later.
■ Statistical analysis
The obtained results were analyzed with commercially available statistical software (GraphPad Software Inc., La Jolla, CA, USA). The paired Student’s t-test was used to compare before and after mean values by assuming that the distribution of the differences was in accord with the Gaussian distribution. Side-by-side comparison between two independent populations was performed by an unpaired t-test or by a Mann-Whitney U test. The Wilcoxon ranking test was used to compare paired before and after nonparametric values such as conversion rates arranged in a ‘yes’ or ‘no’ binary manner. Fisher’s exact two-tailed test was employed for analysis of data arranged in a contingency table. Comparison across multiple groups was tested either by analysis of variance or a categorical Kruskal-Wallis test. All statistical analyses were performed on an intent-to-treat basis, involving all initially enrolled patients including fatalities. The resulting probability values were considered significant at p < 0.05.

Results
■ Sugar dragées
The first trial of a solid form of Immunoxel involved small-size (~4 mm) sugar dragées to which gradually increasing doses of Immunoxel were added, so that ten of them contained a total of ten drops of Immunoxel. The test involved 21 patients who were instructed to place dragées under the tongue until fully absorbed. Half of the patients received five drops and the other half a dose of ten drops. After 1 month, every measured end point had improved significantly (p < 0.0001). These and other results described below are summarized in Tables 1 & 2 and Figure 1. The percentage of patients with negative sputum conversion was 81% — results from which the authors did not exclude the three patients who died. In two of these patients post-treatment sputum samples were not analyzed and were assumed to remain positive at the time of death. One, a female patient, was in a very unfavorable condition and died a few days after checking into the hospital. Another female patient was obese (BMI = 36 kg/m2) and had a fatal stroke before her sputum could be analyzed. The third patient, whose sputum had converted to negative, unfortunately died from an alcohol overdose after the 1-month study was concluded. If the two female patients without post-treatment sputum results are excluded, then the conversion rate in this group stands at 89.5%. No difference was seen in sputum outcome between five- and ten-drop doses.

■ Sugar-coated pills
Following the initial trial with sugar dragées, which were found to be inconvenient owing to the high moisture absorption property and cumbersome dosing procedure, conventional sugar-coated, oval-shaped pills were made, each containing the same ten-drop dose of Immunoxel and standard excipients such as lactose, talc, magnesium stéarate and menthol flavoring. Ten patients were enrolled to evaluate the effect. Of these ten patients, seven became negative after 1 month (p < 0.0001). Bodyweight and temperature, as well as BMI, demonstrated statistically significant improvements. Other end points, such as hemoglobin content, ESR and leukocyte counts, had improved as well, but statistical significance was not reached owing to sample size (Tables 1 &2 & Figure 1). This formulation had no additional active ingredients that could have biased the outcome and was essentially the same as the sugar dragées described above. If sugar-coated pill results are combined with the outcome from sugar dragées, then the overall conversion rate for sugar-based formulations is 79.8%.

■ Gelatin pastilles
The batch of Immunoxel formulated into so-called ‘gummy candies’ contained a ten-drop dose per pastille, also containing in addition sugar, glucose syrup, starch, food coloring, citric acid and porcine gelatin, according to their standard procedure. Out of 20 patients, 13 were smear-positive at baseline, of whom ten (76.9%) became negative after 1 month (p < 0.0001). All other end points except leukocyte counts had improved in a statistically significant manner (Tables 1 & 2 & Figure 1).

■ Dried-honey lozenges
This preparation was made by Island Abbey Foods Ltd. This company has a unique proprietary technology for slowly drying liquid honey into solid dried-honey lozenges. Two batches were manufactured, one with four drops and another with ten drops of Immunoxel per lozenge, which were then given to two equal groups of patients (n = 18). After 1 month, every smear-positive patient in both groups had converted. All other measured end points were also improved in a statistically significant manner (Tables 1 & 2 & Figure 1).

■ Comparison of outcomes from different Immunoxel formulations
The baseline characteristics of four groups of patients were tested by the analysis of variance or Kruskal—Wallis test, as appropriate, and were found to be matched by age (p = 0.25), BMI (p = 0.83), and hematological parameters such as hemoglobin (p = 0.36) and ESR (p = 0.12), but not by gender distribution (p = 0.03) or leukocyte counts (p = 0.03). The primary end point of the study is sputum conversion (Figures 1 & 2). When results from the four formulations were compared by the nonparametric Kruskal—Wallis test, the outcomes were not different from each other (p = 0.15), even though, at baseline, there were differences between individual groups (p = 0.02). This indicates that solid formulations have an equivalent biological activity (Tables 1 & 2 & Figure 1). The effective dose was equivalent to ten drops of liquid Immunoxel. However, it is possible that smaller doses could be equally effective, since sugar-based dragées and dried-honey lozenges tested at five- and four-drop doses produced the same favorable outcome. The stratified analysis of data comparing outcomes between newly diagnosed TB (n = 26), RTB (n = 31), TB/HIV (n = 16) and MDR-TB (n = 4) also failed to reveal any difference (p = 0.17). Similar results were obtained when outcomes of patients on standard isoniazid, rifampicin, pyrazinamide, ethambutol and streptomycin chemotherapy (n = 57) were compared with the conversion of patients receiving individualized drug regimens (n = 12). The Mann-Whitney U test shows that the two treatment regimes produced identical favorable outcomes (p =0.83). The duration of chemotherapy prior to immunotherapy also had no impact on outcome. Those who were treated for 1 month or less were converting as readily as those who were treated for longer; for example, 1 versus 3.8 months (p = 0.67). The same Mann-Whitney U test failed to reveal gender- or age-dependent differences. Males and females, as well as younger or older patients (the age threshold was equal to highest mean age + highest SD, i.e., > 48 years), converted at the same rate without any gender or age preference (p = 0.5 and p = 0.29, respectively). A baseline BMI higher or lower than the normal 18.5 kg/ m2 value had no effect on conversion rate either (p = 0.6). However, when these data were compared with outcomes from control patients, there was an obvious and highly significant difference (p < 0.0001) irrespective of formulation type, discrepancies in baseline diagnosis, gender, age, BMI or treatment regimen.

■ Treatment outcome in the placebo group
As a control, the authors used sugar-coated pills without Immunoxel, which were administered to 21 randomly chosen patients who were concurrently receiving TB drugs. Despite a sufficient sample size that was equivalent to or larger than any of the Immunoxel-treated populations, the only statistically significant results were obtained with ESR and sputum smear conversion (Tables 3 & 4). As can be seen from Figure 1, the proportion of patients responding to chemotherapy was adequate; however, the magnitude of response was smaller. For example, control group patients gained less weight than those who received various Immunoxel formulations; the mean weight and BMI accruals were 0.5 kg and 0.2 kg/m2 versus 1.9 kg and 0.6 kg/m2 in the immune intervention arm. Similar trends were observed with other end points indicating that, even though chemotherapy alone has been beneficial to TB patients, its potency was lower than that of chemotherapy with adjunct immunotherapy.

■ Discussion
This study reveals that all tested solid formulations of Immunoxel produced similar favorable outcomes. The conversion rates in patients were identical regardless of their baseline diagnosis, such as first diagnosis, RTB, MDR-TB or TB/HIV. The success of therapy was not affected by concomitant chemotherapy regimens, and both standard and individualized treatments had comparable favorable outcomes. Similarly, the duration of chemotherapy prior to immunotherapy had no impact either. Those who were treated for 1 month or less converted at the same rate as those who were receiving TB drugs for 4 months on average. Neither gender nor age was a significant factor influencing sputum conversion. Underweight patients converted at the same rate as those in the normal weight range. In addition to bacterial clearance, Immunoxel has shown clear improvement in clinical manifestations, weight gain, defervescence, reduction of inflammation such as leukocytosis and ESR, and increase in hemoglobin content. All these benefits were described in prior studies of liquid Immunoxel, except that solid formulations acted faster and much smaller and less frequent doses were required.
A bewildering range of botanical supplements, especially in traditional Chinese and Ayurvedic medicine, is used today with the intention of boosting immunity and curing various diseases including tuberculosis [7]. However, the clinical or experimental evidence is lacking in most cases [8]. To the best of the authors’ knowledge, clinical results from only three plant-derived preparations with anti-TB properties have been reported in peer-reviewed literature [9-11]. The oldest known is umck-aloabo – a traditional South African medicine from the roots of Pelargonium sidoides — used to treat TB and respiratory tract infections since the late 19th century [9]. Another report concerns inhaled tea tree oil from Australian Melaleuca alternifolia, which has produced remarkable recovery in a few cases of advanced TB [10]. Finally, a multiherbal water-infusion cocktail based on Russian folk medicine has been reported to reduce the duration of TB treatment to 6.4 months, instead of 8.6 months in controls [11]. For other phytomedicines, there is only anecdotal evidence without any systematic clinical studies [12,13]. Immunoxel stands out in this crowd since more than a dozen clinical trials involving a total of 1200 patients have been published by the authors and their colleagues [14-26]. During the past couple of years the authors have attended several conferences on TB topics and Immunoxel has become quite familiar in professional circles. While the TB research community outside of Ukraine has been slow to embrace this approach, Immunoxel clinical data have attracted genuine interest and calls have been made at various venues to test it independently [3,4].

In the course of the present study several advantages of solid Immunoxel were uncovered. First, sublingual formulations appeared to act very quickly, producing, within 1 month, the conversion rate that usually occurs after 2—4 months of dosing with liquid formulas. Second, the effective dose was equivalent to ten drops and possibly even less, given once-daily as opposed to 30—60 drops of liquid immunomodulator given twice-daily, showing at least six- to 12-fold enhancement in activity. Third, sublingual administration as opposed to liquid formulation was simple and convenient; there was no need to count drops and mix them in drinking water, which can be an issue in countries without ready access to clean water. Fourth, the elimination of an alcohol component from the preparation has improved patient compliance. Many of the authors’ patients have a tendency to abuse alcohol and were often inclined to overdose with a herbal tincture containing 50% ethanol. As a result, the monthly supply of Immunoxel was consumed at once causing treatment default. Finally, another advantage of solid formulations relates to shipping cost and logistics, especially for export purposes. These issues are now defunct owing to the elimination of bulky glass bottles and potentially flammable liquid, which complicated clearing procedures with cargo airlines and customs. In summary, the solid form of Immunoxel has major advantages over the original formulas and is more appealing as the next generation of botanical immunomodulator from Ukraine.

The evaluation of clinical results in the context of commercial feasibility of product manufacture leads us to two choices; one is sugar-based tablets and the other dried-honey lozenges. Even though Immunoxel pastilles performed no worse than other formulations they have limited appeal owing to the presence of gelatin from animal sources. Sugar-based tablets are the cheapest and simplest delivery vehicle, and in two separate tests produced reliable conversion results. However, this formulation needs to be improved, which will be achieved by adding appropriate excipients such as those found in slow-release tablets. On the other hand, dried-honey lozenges, which produced 100% conversion, may be more attractive than sugar-coated pills. We do not know whether this outcome was random or if the higher efficacy was due to synergy with honey. The antimycobacterial activity of honey has been known since ancient times and it is used as a folk medicine even today. Several in vitro studies have demonstrated the direct inhibition of tubercle bacilli growth with honey [27-30]. Although inhibitory doses of honey were rather high, there are clinical studies showing its benefit in healing tuberculous lesions and counteracting the toxicity of TB drugs [31,32]. The only drawback in this approach is the price of honey. If Immunoxellozenges can be made at a low cost, this, perhaps, will be an ideal formulation, especially considering the beneficial antimycobacterial and hepatoprotective properties of honey [27-32]. Both formulations will be pursued in future clinical studies expanding on these preliminary findings.

Executive summary
Four different versions of solid Immunoxel were tested: sugar dragées, sugar-coated pills, gelatin pastilles and dried-honey lozenges, which produced negative sputum conversion equal to 89.5% (n = 21), 70% (n = 10), 76.9% (n = 20) and 100% (n = 18) of patients, respectively. The differences in outcome were not statistically significant.
Concurrent administration of various solid forms of Immunoxel with first- or second-line TB drugs resulted in the clearance of Mycobacterium tuberculosis in sputum smears in a mean 84.1% of patients versus 19% among placebo recipients. Sputum conversion occurred very fast – only 1 month of treatment was needed.
No difference was seen when first-diagnosed TB was compared with re-treated TB, multidrug-resistant TB or TB with HIV coinfection; the proportion of converted patients and time to conversion were the same. Differences in gender, age and bodyweight had no influence on conversion rates.
A 1-month pretreatment time with chemotherapy had the same favorable outcome as a 3.8-month pretreatment time.
One sublingual dose of Immunoxel given once-daily produced the same effect as six- to 12-times higher doses of liquid formula
administered for 2-4 months.
Immunoxel reversed TB-associated wasting; the average weight gain was 1.9 kg in 89.8% of Immunoxel-treated patients versus 0.5 kg in 61.9% of placebo recipients.
Immunoxel eliminated TB-associated fever in 97.5% of patients versus 42.9% of placebo recipients.
Immunoxel demonstrated marked anti-inflammatory effects. Erythrocyte sedimentation rate and leukocyte counts reverted back to normal over 1 month. However, patients on TB drugs alone also experienced reduced inflammation.Immunoxel is affordable, easy to administer and made from renewable sources. Immunoxel is safe; it has not produced any adverse effects or caused reactivation of TB.

Conclusion
Many medicinal plants have been identified as having antimycobacterial activity, but they have to go through lengthy development processes before, and if, they are made available to patients [33]. The integration of herbal medicines into modern medical practice has to be validated through rigorous scientific and clinical research in order to be accepted by the medical establishment. Ekomed started developing herbal immune modulators in the late 1980s and today both Dzherelo and Immunoxel are commonly found in Ukrainian pharmacies. Present findings further support the value of Immunoxel and may allow us to persuade public opinion and health authorities that new sublingual formulations are worth being considered as an integral part of TB management strategy. As a result, lives of many thousands of individuals may be saved in Ukraine and elsewhere. Our dream of contributing to the reduction of the global TB burden may then materialize.

Future perspective
Despite increasing attention, the future for TB immunotherapy is uncertain. We cannot predict whether, in the next 5 years, solid forms of Immunoxel will be used widely as an adjunct to immune therapy for TB or will remain outside of mainstream TB management and control strategies, without adequate funding or interest. However, the emergence of drug-resistant TB and TB/HIV has become a great concern to TB caregivers and policymakers. If this trend continues we may see changes that will be constructive and may result in adoption of immune interventions on a larger scale than exists today.

Acknowledgements
We thank all patients who participated in this study. The assistance of Ekomed in generously providingfree supplies of Immunoxel is very much appreciated.

Financial & competing interests disclosure
This work was supported in part by the STEP Business Partnership Grant UKB1-9017-LK-09 awarded by the US Civilian Research and Development Foundation (CRDF), a nonprofit organization authorized by the US Congress and established in 1995 by the National Science Foundation. This study was conducted under the auspices of the regional health authorities of Ukraine as a part of the routine clinical care at the TB Dispensary (Lisichansk, Ukraine). IV Mospan, VS Pylypchuk, J Rowe, VJirathitikal and AS Bourinbaiar are employees of their respective companies. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research
The authors have obtained the Institutional Review Board approval of Lisichansk TB Dispensary and have followed the principles of the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved. Participation in this trial was voluntary and participants were free to withdraw from this study at any time. This study is registered with ClinicalTrials.gov, identifier NCT01061593.

References
Papers of special note have been highlighted as:
■ of interest
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■ First report in English describing the effects of herbal immunomodulator Dzherelo (Immunoxel) in TB patients.
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18 Nikolaeva LG, Maystat TV, Pylypchuk VS, Volyanskii YL, Masyuk LA, Kutsyna GA. Changes in CD4 + T-cells and HIV RNA resulting from combination of anti-TB therapy with Dzherelo in TB/HIV dually infected patients. Drug Des. Dev. Ther. 2, 87-93 (2008).
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20 Nikolaeva LG, Maystat TV, Pylypchuk VS, Volyanskii YuL, Frolov VM, Kutsyna GA. Cytokine profiles of patients with pulmonary tuberculosis resulting from adjunct immunotherapy with herbal phytoconcentrates Dzherelo and Anemin. Cytokine 44, 392-396 (2008).
21 Zaitzeva SI, Matveeva SL, Gerasimova TG et al. Efficacy and safety of phytoconcentrate Dzherelo (Immunoxel) in treatment of patients with multi-drug resistant TB (MDR-TB) in comparison to standard chemotherapy. Res. J. Med. Sei. 3, 36—41 (2009).
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32 Sharma M, Khayyam KU, Kumar V, Imam F, Pillai KK, Behera D. Influence of honey on adverse reactions due to anti-tuberculosis drugs in pulmonary tuberculosis patients. Cont. J. Pharm. Tox. Res. 2, 6-11 (2008).
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■ Website
101 Adjunct Immunotherapy With Immunoxel in
Patients With TB and TB/HIV.
http://clinicaltrials.gov/ct2/show/
NCT01061593

Mobirise

Adjuvant Immunotherapy of Extensively Drug-Resistant Tuberculosis (XDR-TB) in Ukraine

‘Nafhalia D. Prihoda, ‘Olga V. Arjanova, ‘Larisa V. Yurchenko, ‘Nina I. Sokolenko, Lyudmila A. Vihrova, Volodymyr S. Pylypchuk, Valery M. Frolov and GalynaA. Kutsyna ‘Lisichansk Regional Tuberculosis Dispensary, Lisichansk, Ukraine Lisichansk Regional Hospital, Lisichansk, Ukraine Ekomed LLC, Prospect Pravdy 80-A, Kiev, Ukraine Luhansk State Medical University, Luhansk, Ukraine

Abstract: Conventional TB chemotherapy success rates are very low in patients with Extensively Drug-Resistant Tuberculosis (XDR-TB). We treated twelve XDR-TB individuals, seven of which in addition to standard Anti-TB Therapy (ATT) received Immunoxel (Dzherelo), Svitanok and Lisorm – ove – the-counter herbal immunomodulators. All seven patients, who received adjunct immunotherapy improved clinically and radiologically and were discharged after 3.7 ± 0.8 months, with average/median time to mycobacterial clearance 28/25 days. None of five patients on TB drugs alone improved and one had died. Patients on immune intervention gained 9.6 kg (p = 0.0001) while those on ATT lost 1.4 kg. The levels of total bilirubin had decreased from 15.6 to 10.7 uM L-1, similarly the values of alanine transaminase (ALT) have declined from abnormally high 42.6 IU L”1 to normal levels 22. IU L”1 (p = 0.23). Patients on ATT had unchanged levels of bilirubin, but their ALT declined from 29.6 to 12 IU L”1 (p = 0.02). The levels of hemoglobin had risen from 104.1 to 118 g L-1 (p = 0.07), whereas, leukocyte counts descended to normal levels from 8.9 to 7.3x10s cells L-1 (p = 0.003). Conversely, inpatients on ATT leukocyte counts had risen from 8.7 to 13.8x10s cells L-1 (p = 0.21), whereas, hemoglobin declined to below normal levels from 116.4 to 96.6 g L-1 (p = 0.18). These results show that immune-modulating interventions can favorably influence the effect of TB drugs. The difference between two treatment outcomes was highly significant (Mantel Haenszel odds ratio = 11; p = 0.0009 at 95% CI). Thus, adjunct herbal immunotherapy is safe, shortens treatment duration and can overcome drug resistance even in patients with XDR-TB.

Key words: Immunomodulator, MDR-TB, XDR-TB, phytoconcentrates, Mycobacterium

INTRODUCTION
The extensively resistant TB (XDR-TB) is diagnosed whenM tuberculosis bacilli in addition to lack of sensitivity to isoniazid (H) and rifampicin (R), two most commonly-used, first-line TB drugs, are also resistant to any one of fluoroquinolones and at least one of second-line injectable drugs, e.g., capreomycin, kanamycin and amikacin (Migliori et a!., 2008). This emerging form of TB caused worldwide concern after outbreak in Kwazulu Natal province of South Africa where 52 of 53 patients with XDR tuberculosis and HIV co-infection had died within 2 weeks from the time of diagnosis (Gandhi et a!., 2006). Success rates in treating XDR-TB are significantly lower than among drugsensitive cases ranging between 29 and 67%. In additionit takes much longer (18-24 months) to achieve a cure and concerns over adverse effects of drugs became more prominent since second-line drugs are more toxic. The cost is another factor as the deployment of second-line drugs increases treatment cost by about hundred-fold. Clearly there is an urgent need to find additional therapeutic interventions that could overcome these problems.

Immunomodulators Immunoxel (Dzherelo), Svitanok and Lisorm are made from aproprietary combination of medicinal plants and are commonly used in Ukraine for the management of TB and HIV infections, including patients with dual infection (Arjanova et a!., 2009; Chkhetiany et a!., 2007; Melnik et al., 1999; Nikolaeva et al., 2008; Prihoda et al., 2007; Zaitzeva et al., 2008). They have been approved in 1997 by the Ministry of Health of Ukraine as functional supplements with therapeutic indications. Dzhereloand Svitanok were specifically recommended as immune adjuvants to the therapy of pulmonary tuberculosis (Melnik et al., 1999). So far, the phytoconcentrates we have decided to use in this study have been taken safely by several hundred thousand individuals for various indications including chronic bacterial and viral infections such as TB and HIV, autoimmune diseases and malignancy (Chkhetiany et al., 2007). In this retrospective study, conducted at Lisichansk TB Dispensary, we have compared the adjunct effect of herbal immunomodulators to outcome of treatment with conventional TB therapy.

MATERIALS AND METHODS

Patients
Lisichansk TB Dispensary is within Luhansk administrative region of the Eastern Ukraine with total population 2.5 million. Approximate population of registered TB patients in this region is 2 thousand. Lisichansk TB dispensary has turnover of about 600-800 patients per year. The dispensary has six medical doctors and approximately 15 medical nurses and lab technicians who care for hospitalized patients and perform the lab work. Twelve patients with pulmonary XDR-TB were identified retrospectively, five of which received individualized TB drugs regimen and seven who received in addition to ATT an immunomodulating phytopreparations Dzherelo, Svitanok and Lisorm. All patients were males with age range between 25 and 67 years. Five presented with first-diagnosed TB and the rest were previously treated, relapsed cases of TB. All study patients presented with acute symptoms of pulmonary TB that required hospitalization. Most common symptoms were prolonged heavy cough, pain in the chest, high fever, profuse night sweats, fatigue and loss of weight and appetite. Active pulmonary tuberculosis was certified by a medical history and clinical findings compatible with tuberculosis, a chest X-ray showing lung involvement and positive sputum smear for Acid-Fast Bacilli (AFB) and the culture of M. tuberculosis. The conduct of the study was approved by the Internal Review Board (IRB) of Lisichansk TB dispensary in accordance with the Helsinki Declaration.

Treatment Regimen
All anti-TB drugs were procured free-of-charge through centralized national supply system of Ukraine. The over-the-counter phytoconcentrates, Dzherelo, Lisorm and Svitanok were generously supplied by Ekomed LLC. Individualized, first- and second-line anti-TB drugs were administered to all hospitalized patients based on physician’s decision prior to or after results of drug susceptibility tests. In the immunotherapy group, in addition to ATT, patients received a daily dose of Dzherelo which was given as 30 drops diluted in a half-glass of water 30 min before breakfast. Some patients received Immunoxel-slightly modified form of Dzherelo. The same dose, 30 drops, of Lisorm and Svitanok were given before lunch and supper, respectively. The exact formula of phytoconcentrates has been described by Prihoda et al. (2008). Sputum smear and culture examinations for AFB were performed at monthly intervals. The decision to discharge was based on at least twice-repeated negative culture outcome and satisfactory clinical and radiological findings.

TB Drug Resistance Testing
The drug resistance to first- and second-line TB drugs was tested with commercially supplied kit (Tulip Diagnostics, Goa, India). The cultures ofM tuberculosis derived from sputum of each patient were inoculated into ready-to-use tubes containing TB drugs incorporated at manufacturer-predetermined concentrations into standard Lowenstein-Jensen agar slants. The cultures were incubated at 37°C and checked periodically until appearance of colonies in control tubes without drugs.
The calculation of the proportion of resistant bacilli was done by comparing counts on drug free and drug-containing Lowenstein-Jensen medium.

Statistical Analysis
The obtained results were analyzed with available online statistical software (GraphPad Software, Inc., La Jolla, CA). All statistical analysis were done on intent-to-treat basis, involving the total number of patients without subgrouping them into responders and non-responders. The stratification analysis of patients was conducted to reveal the difference between distinct treatment categories. Parametric baseline values relative to the end of study values were evaluated by paired or unpaired Student t-test. The categorical test was done by Mantel Haenszel’s odds ratio calculation. The probability values were considered as significant at p< 0.05 cut-off value.

RESULTS AND DISCUSSION
None of five patients on conventional TB drugs regimen had positive outcome after 9 months of treatment and one patient died after 9.5 months. The duration of treatment in immunotherapy group ranged between 10.6-20.4 weeks with average/median 15.7/16.7 weeks (Table 1). The treatment lasted until patients were discharged from the dispensary upon twice-repeated negative culture findings and clinical and radiological improvements. The time to negative culture conversion ranged between 20-37 days with mean/median 28/25 days. Mycobacterial clearance was confirmed by repeated cultures at monthly intervals.
There appears to be no difference between first-diagnosed TB cases versus chronic, previously treated TB in terms of median days to discharge, i.e., 117 vs. 105.6 or days to mycobacterial clearance, 23 vs. 30. However, sample size was too small to reveal statistically significant difference.
At the end of study every patient had gained substantial lean body mass, ranging between 6 and 13 kg. The average accrual in lean body mass was 9.6 kg, which was statistically highly significant as evidenced by a paired Student’s t-test (p = 0.0001) -an effect that was evident early as one month from initiation of the therapy. In contrast, patients on ATT had lost on average 1.4 kg (p = 0.4).
The potential hepatotoxicity of ATT combination with herbal preparations was monitored by quantitative liver function tests. Surprisingly, despite intensive chemotherapy patients have shown signs of better liver function. The level of total bilirubin had decreased from mean 15.6 to 10.7 uM L-1 -a favorable change that was not statistically significant (p= 0.16). Similarly, the values of alanine transaminase (ALT), another marker of liver damage, have declined from abnormally high (42.6 IU L-1) to normal levels (22. IU L-1) -a change that was not statistically significant (p = 0.23). Patients on ATT had same levels of bilirubin but their ALT declined from 29.6 to 12 IU L”1 (p = 0.02).Another phenomenon observed during therapy is a reversal of baseline anemic state and proinflammatory condition-symptoms very common in TB. Most patients at study entry displayed signs of anemia and had abnormally elevated leukocyte counts. At the end of treatment these parameters were improved in a statistically significant manner. The levels of hemoglobin had risen from 104.1 to 118 g L-1 (p = 0.07), whereas leukocyte counts descended to quasi-normal levels from 8.9 to 7.3x10s cells L 1 (p = 0.003). Inpatients on ATT the reverse trend was observed. Leukocyte counts had risen from 8.7 to 13.8x10s cells L-1 (p =0.21) whereas hemoglobin declined to below normal levels from 116.4 to 96.6 g L”1 (p = 0.18).
Table1: Baseline and outcome characteristics of XDR-TB patients treated with TB drugs without or in combination with Dzherelo (Immunoxel), Svitanok and Lisorm  

These results show that immune-modulating interventions can favorably influence the efficacy of TB drugs (Arjanova et al., 2009; Chkhetiany et al., 2007; Nikolaeva et ah, 2008; Prihoda et ah, 2007; Zaitzeva, 2008). All seven patients who received ATT and immunotherapy improved clinically and radiologically and were discharged after 3.7±0.8 months, with average/median time to mycobacterial clearance 28/25 days. None of five patients on TB drugs alone improved and one had died. The difference between two treatment outcomes was statistically significant (Mantel Haenszel odds ratio = 11; p = 0.0009 at 95% CI).
Present results compare favorably to XDR-TB chemotherapy outcomes reported in several recent papers. According to study by Kim et al. (2008) only 29.3% of those with XDR-TB were cured. TB therapy success rate in Russian patients with XDR-TB as reported by Keshavjee et al. (2008) was 48.3%. Earlier reported cure rates in Europe, USA, Peru and Korea were between 37.5-67% indicating that XDR-TB poses serious clinical challenge (Edward et al., 2008; Kwon et al., 2008; Migliori et al., 2008; Mitnicke/o/., 2008). In conclusion, adjunct herbal immunotherapy is safe, enhances significantly treatment outcome and can overcome drug resistance even in patients with extremely poor prognosis. Further studies are needed to confirm present findings.

ACKNOWLEDGMENTS
We thank all participants who volunteered in this study. The support of clinical staff and technicians who contributed to this study has been of tremendous help to us. We are grateful to other colleagues who shared their insight and provided helpful suggestions based on their own experience with phytoconcentrates used in present study.
Kwon, Y.S., Y.H. Kim, G.Y. Suh, M.P. Chung and H. Kim et a!., 2008. Treatment outcomes for HlV-uninfected patients with multidrug-resistant and extensively drug-resistant tuberculosis. Clin. Infect. Dis.,47: 496-502.
Melnik, V.P., O.V. Panasyuk, V.S. Pylypchuk, O.P. Moshich, N.M. Procenko and O.M. Leonenko,1999. Deployment of herbal preparations Dzherelo and Svitanok for combination therapy of pulmonary tuberculosis. Medical Institute of UkrainianAssociation of People’s Medicine. Information Bulletin of the Ministry of Health of Autonomous Republic of Crimea. UDK:616.24-002.5-085-038:615.017. 1999. Kiev, Ukraine.
Migliori, G.B., C. Lange, R. Centis, G. Sotgiu and R. Mutterlein et al., 2008. TBNET Study Group. Resistance to second-line injectables and treatment outcomes in multidrug-resistant and extensively drug-resistant tuberculosis cases. Eur. Respir. J., 31: 1155-1159.
Mitnick, CD., S.S. Shin, K.J. Seung, M.L. Rich and S.S. Atwood et al., 2008. Comprehensive treatment of extensively drug-resistant tuberculosis. New Engl. J. Med., 563: 563-574.
Nikolaeva, L.G., T.V. Maystat, V.S. Pylypchuk, Y.L. Volyanskii, L.A. Masyuk and G.A. Kutsyna, 2008. Effect of oral immunomodulator Dzherelo (Immunoxel) in TB/HIV co-infected patients receiving anti-tuberculosis therapy under DOTS. Intl. Immunopharmacol., 8: 845-851.
Prihoda, N.D., O.V. Arjanova, L.V. Yurchenko, N.I. Sokolenko, L.A. Vihrova, V.S. Pylypchuk and G.A. Kutsyna, 2007. Open label trial of adjuvant immunotherapy with Dzherelo, Svitanok and Lisorm, in MDR-TB, XDR-TB and TB/HIV co-infected patients receiving anti-tuberculosis therapy under DOT. J. Med. Plant Res., 1: 117-122. Prihoda, N.D., O.V. Arjanova, L.V. Yurchenko, N.I. Sokolenko, L.A. Vihrova, V.S. Pylypchuk and GA. Kutsyna, 2008. Adjuvant immunotherapy of tuberculosis in drug-resistant TB and TB/HIV co-infected patients. Int. J. Biomed. Pharm. Sci., 2: 59-64.
Zaitzeva, S.I., S.L. Matveeva, T.G. Gerasimova, Y.N. Pashkov, D.A. Butov, V.S. Pylypchuk V.M. Frolov and G.A. Kutsyna, 2008. Efficacy and safety of phytoconcentrate Dzherelo (Immunoxel) in treatment of patients with multi-drug resistant TB (MDR-TB) in comparison to standard chemotherapy. Res. J. Med. Sci.

Mobirise

Immune approaches in tuberculosis therapy: a brief overview

Expert Rev. Anti Infect. Ther. 10(3), 1–xxx (2012)Aldar S Bourinbaiar*1, Marina V Mezentseva 2, Dmitry A Butov 3, Peter S Nyasulu 4, Yuri V Efremenko 5, Vichai Jirathitikal 1, Vladimir V Mishchenko6 and Galyna A Kutsyna 71Immunitor Inc., Vancouver, BC, Canada, 2 Gamaleya Institute for Epidemiology & Microbiology, Moscow, Russia, 3 Department of Phtysiatry and Pulmonology, Kharkov National Medical University, Kharkov, Ukraine, 4 Faculty of Health Sciences, Witwatersrand University, Johannesburg, South Africa, 5 Lisichansk Regional Tuberculosis Dispensary, Lisichansk, Ukraine, 6 Central Institute for Tuberculosis,Moscow, Russia, 7 Luhansk State Medical University, Luhansk, Ukraine.*Author for correspondence: aldar@immunitor.com

TB is typically caused by Mycobacterium tuberculosis, a symbiotic bacterium present in one-third of the world’s population. There any many factors triggering overt clinical disease in a small proportion of humans. In our view the major role in the process is played by the host’s immune response, especially self-directed, destructive inflammation. Conventional chemotherapy produces bactericidal or bacteriostatic effects, but immunopathological changes can only be corrected by immunotherapy. Various attempts have been made to identify the optimal immune intervention. Some have shown promising effects, but many have failed. It is commonly believed that the field started in 1890: the year Robert Koch announced his tuberculin therapy. In the Pên Ts’ao Kang Mu, classical Chinese materia medica, published during Ming dynasty, Li Shi Chen (1518–1593) recommended, as a remedy for hemoptysis, to collect from the sputum “… blood lumps, roast them till they are black, and take then them as a powder”. In retrospect, this is perhaps the earliest recorded reference relating to immunotherapy of TB with heat-killed mycobacteria. Modern science is obviously geared toward more palatable approach, but without hindsight from often disdained empirical evidence no progress can be made. The clinical experience from various trial and error processes is briefly discussed in this review.

Keywords: AIDS • antibody • autoimmune • cytokine • HIV • immunomodulator • inflammation • lung •multidrug-resistant TB • mycobacterial • pulmonary • therapeutic vaccine • tubercle bacillus • tuberculin

Abundant literature exists concerning immune factors contributing to the development of TB. As Mycobacterium tuberculosis is an intracellular parasite the role of humoral, that is antibody- mediated immunity, is far less important than that of cell-mediated immunity. The survey of published studies concerning immunol- ogy of TB reveals a great deal of confusion. Contradicting opinions coexist even today and this perhaps is one of the main reasons why we still have no unanimous consensus with regard to the immunotherapy of TB. What is worse is that prevailing opinion is bent towards the incorrect concept, which postulate that TB arises from an insufficient immune response. This concept lacks logic; two main diagnos- tic tools for identifying active TB, tuberculin test and IFN-g release assay, are based on the contrary principle. Indeed, individuals with TB do mount a vigorous immune response against tubercle bacilli [1]. Progressive pulmonary TB is caused by a continuous host response to myco- bacterial products and is not due to increasing numbers of viable bacilli in a host [2]. The goal for immunotherapy is to prevent a self-damaging inflammatory response, instead of instigating more of the same. This goal is closely related to figuring out the immune response that correlates with protective immunity. Over the past 90 years, this gap in our understanding of TB immunologyhas been responsible for the failure of developing a safe and effective vaccine, since introduction of BCG in 1921. Recently, the research efforts in TB have intensified. However, it is difficult to appreciate the difference or distinguish immunopathologies that are associated with, and those that are causing TB. Without this there is an impression that we are ‘flying without a compass’ a term that Gene Shearer used in his comment with regard to the effort of developing AIDS vaccines. Conventional chemotherapy approaches are straightforward with clear goals of suppressing mycobacterial replication. Bacteriostatic or bactericidal agents seldom restore the immune status. This task cannot be achieved without proper immune intervention. The emergence of multidrug-resistant TB and TB–HIV coinfection, together with advances in immunology, has led to renewed interest of exploiting the immune response against M. tuberculosis. This review is intended to provide a short excurse into the history of TB immunotherapy and analysis of the current state-of-the-art. We hope that understanding the past and present experiences will provide valuable insight and may result in a better grasp of the problem. As Kaufmann quoted in one of his articles on immunol- ogy of TB “those who don’t remember the past are condemned to repeat it” [3].

Early days of immunotherapyRobert Koch, discoverer of M. tuberculosis, can be considered as the first modern TB immunotherapist when in 1890 he announced his tuberculin therapy. Koch’s original preparation of tuberculin was a crude extract from heat-killed cultures of M. tuberculosis. Koch’s idea was based on the assumption that the reaction of the organism to tuberculin would cause the elimination of bacilli-infected tissue. It must be said that 300 years earlier, renowned Chinese scholar, Li Shi Chen (Figure 1) in his encyclopedical treatise Pên Ts’ao Kang Mu (1595) described methods of treating TB, which were not too far from Koch’s principle. Koch’s initial results of treating skin and lymphatic forms of TB were encouraging. Unfortunately, the massive trial that followed shortly after his announcement had produced disastrous results and the hopes he raised were not fulfilled.

Figure 1. The portrait rendition of Li Shi Chen (1518-1593). Li Shi Chen is the author of most comprehensive Chinese pharmacopoeia Pên Ts’ao Kang Mu, describing the earliest recorded TB immunotherapy based on ingestion of heat- inactivated tubercle bacilli derived from sputum. Taken from [101].

The medical opinion settled firmly against immunotherapy and against the founder of modern microbiology. The implications stemming from this trial, which was clearly indicative of exaggerated and deleterious immune reaction, known today as Koch reac- tion, have been ignored. It is clear that Koch knew of this and he was calling for caution, pointing out that tuberculin should not be used in patients with virulent or acute forms of TB and he argued against high starting doses (Figure 2). Despite negative attitudes prevailing in the medical establishment, Koch’s approach has not been totally ignored. The late 19th and early 20th centuries were replete with success and failure. Several dozen tuberculin-based approaches were promoted by their proponents [4]. A wide range of ingenious modifications of tubercle bacillus were made including passage through refractory animals, attenuation and killing by various methods. Furthermore, inoculation of nonpathogenic or atypical mycobacteria came into wide use. Names of many physicians in Europe and the USA, practicing various tuberculin- derived therapies, are commonly found in the medical literature and lay media of those days. Among them were Lichtheim (1891), Hunter (1892), Klebs (1892), Trudeau (1893), Viquerat (1894), Maragliano (1895), Paquin (1895), Fisch (1897), De Scweinitz and Dorset (1897), Smith and Baldwin (1898), Murphy (1898), Cantacuzino (1901), Goetsch (1901), Armand-Delille (1902), Marmorek (1903), Wright (1903), Behring (1905), Calmette (1906), Matthew (1908), Hunter (1908), von Ruck (1908), Leber and Steinharter (1908), Vallée (1909), Webb (1911), Wolff-Eisner (1912), Sahli (1912), Friedmann (1914), Wyeth (1914), Dreyer (1919), Spahlinger (1922), Josset (1924), Dryer (1924), Reenstierna (1934) and many others. This highly active period in the history of immunotherapy had dwindled down by 1940s when first, streptomycin, and then para-aminosalicylic acid and isoniazid, were introduced as TB drugs and chemotherapy research became the next trend.

Nevertheless, Koch’s idea has greatly influenced the field. The tuberculin skin test (TST) became a critically important diagnos- tic tool in the management of TB. Von Pirquet and Schick (1907) developed a method of cutaneous scratch, Moro and Doganoff (1907) made percutaneous patch, and Calmette and Wolff-Eisner (1907) a conjunctival probe. Intracutaneous injection of tubercu- lin was refined by Mendel and Mantoux (1908) and this method became widespread because of the reproducibility of the results. In the 1930s Florence Seibert prepared purified protein derivative from ‘old tuberculin’, which now serves as a standard reference material. Jules Freund’s emulsified suspension of killed M. tuberculosis cells in mineral oil is nothing more than a super-tuberculin and most potent experimental vaccine adjuvant known, which is not used in humans owing to an extreme reactogenicity. Another outcome that remains as a tribute to Koch’s greatness is BCG vaccine, the only prophylactic TB vaccine available today. At the International Congress on TB, held in Washington, USA, in 1908, Albert Calmette stated that, “he had prepared for thera-peutic use [sic] a particularly active and relatively pure tuberculin called Tuberculin CL, which could be introduced intravenously into the body of a healthy animal…which evidently delayed the progress of the disease, and endowed the organism with resistance to the infection.” The fact that Calmette was inspired by Koch and started initially developing BCG as a therapy, which ended up as a preventive vaccine for cattle and was then applied in people is seldom known or is ignored by most TB vaccinologists.

It should be remembered that even though BCG is used widely with a relatively good safety record, it does not provoke the immune reaction that one would expect from a classical vaccine. As TB is a disease of mucosal origin, the route of administration of BCG makes a critical difference [5]. The massive transition from Calmette’s oral version to parenteral BCG may have been respon- sible for what is now blamed as a loss of vaccine’s effectiveness. It is also usually ignored that BCG is seldom administered alone as most children receive a vaccine after having been tested with the TST, which in vaccine terminology equals to priming with tuberculin. Thus, in this setting BCG is used as a booster vaccine. In neonatal direct BCG vaccination the efficacy of a vaccine is tested by TST – a situation where the prime–boost sequence is reversed. One needs to closely evaluate failed and successful BCG trials to determine whether the outcomes were influenced by these ignored but important factors. Aronson’s study in children in Indian reservations, which had shown one of the best known success rate for BCG vaccination, offers clues to that. Furthermore, depending on particular species of environmental mycobacteria, the BCG vaccination can be more efficacious or not effective at all in certain endemic areas [6,7]. The combination of BCG with tuberculin has been used with success in the treatment of TB [8]. The combination of BCG priming with heat inactivated M. vaccae therapeutic vaccine was reported to prevent TB incidence [9]. These examples imply that vaccines for therapeutic and prophylactic purposes have a common mechanism. Successes with both types of vaccines were usually associated with the lack of or lower delayed-type hypersensitivity, that is, tuberculin reactogenicity, either pre- or post-vaccination.

Tuberculin continued It is commonly held that the reputation of tuberculin therapy has been deeply affected by a large TB trial held at the Charité hos- pital in Berlin. Robert Koch’s tuberculin trial, which included 1010 patients with pulmonary TB, resulted in 1.3% cured, 36.1% improved, 4.6% dead, and the rest without apparent clinical benefit. Among 707 patients with extrapulmonary TB, including skin, bone and lymphatic forms, the outcomes were 2.1, 54.5, 1.3 and 42.1% for cured, improved, dead and without clinical benefit patients, respectively [10]. The way these finding are usually interpreted is that therapeutic vaccination against TB is ineffective and even harmful [11]. Considering that the mortality among untreated TB cases in 19th century Berlin was approximately 25%, this is clearly not an example of a failed treatment, but rather a case of misin- terpreted results, which was orchestrated by Koch’s rival, Rudolf Virchow. Today, in the 21st century, the global TB death rate has not greatly improved, even though chemotherapy is widely avail- able. Thus, even by today’s standards, if one looks at mortality as an unequivocal clinical end point, since cure and improvement end points were subject to arbitrary bias, then Koch’s trial had an outstanding outcome.

Figure 2. Robert Koch (1843–1910), discoverer of Mycobacterium tuberculosis and tuberculin immunotherapy. A portrait by unknown artist (most likely German portraitist Gustav Graef – mentor of Hedwig Freiberg – Koch’s second wife) made from the original photograph taken in 1890 [102] – the year he announced tuberculin remedy. The hastened clinical trial at Charité and misplaced interpretation of the results had tarnished his reputation. His later method of making pure tuberculin, as opposed to the original crude reactogenic tuberculin, was based on washing out the lipid fraction with ethanol and salt-precipitating proteinaceous content, which resulted in apparently safe and effective preparation. Normal healthy volunteers, including his students von Behring and Kitasato, without signs of TB, had only transient mild increase in temperature and malaise. However, one volunteer with suspected TB, had more pronounced adverse reaction and Koch had overcome this by reducing further the starting dose of tuberculin. Unfortunately, his ingenious foresight of decreasing undesirable reactogenicity by removing lipids and using small initial doses, which were gradually increased, was somehow forgotten and lost to the TB vaccinologists of today. Image courtesy of Vera Seehausen of the Institute of History of the Medicine, Centre for Human and Health Sciences, at Charité University of Medicine, Berlin, Germany. Taken from [102].

Note that Calmette’s preventive BCG trial in children resulted in an almost identical mortality rate and yet his results were hailed as a pioneering breakthrough. However, owing to Virchow’s domineering influence on medical establishment in Germany at the time, his negative opinion perpetuated and carried over to modern day. Judging from various contemporary sources, Virchow did not seem to be overly preoccupied with praising clini- cal benefits of tuberculin, in fact he did not discuss it at all – he was primarily concerned with fatal ‘injection pneumonias’ – found in approximately half of the deceased patients and attributed to Koch reaction.

Nevertheless, the renewed interest in this approach became apparent by the 1950s, perhaps due to the realization that chemo- therapy was not a panacea and doctors started seeing drug-resist- ant cases. By then most of the work had been carried out in the Soviet Union. The important clues to successfully managing tuberculin therapy outcome were revealed by several groups of Soviet phthysiatists who started using immune intervention in combination with chemotherapy. Most prominent among them was Edvard Mirzoian whose deductive work in the 1960s has laid basis to the currently used tuberculin treatment scheme common in Russia [12]. The main message resulting from their extensive work spanning over decades was simple: dilute! This concept was not entirely new, heated debates were raging in the early part of 20th century as to which dose was most optimal. Koch himself noted that smaller doses were producing tolerizing effects that could eventually heal guinea pigs and high doses were caus- ing massive, occasionally lethal, immune reactions, which have become known as the ‘Koch phenomenon’.

The Soviet school has adopted an approach consisting of a series of increasing doses of subcutaneous or intradermal tuberculin starting with initial dilutions as low as 1:10,000,000. The initial dose was one that produced minimal induration and this was incrementally scaled-up by injecting a tenfold higher dose twice every week until it reached a 1:10 dilution after a 2–3-month treatment course. The typical results were quite convincing. In a large-scale trial involving 1380 patients, sputum clearance at the end of 3 months stood at 82 versus 61.9% of those who received chemotherapy alone (Table 1). Clinical symptoms, healing of cavities were clearly better in the tuberculin group. The healing of cavities was observed in 73.7% (n = 752) as opposed to 49% (n = 422) patients in the control group. This and other trials spanning over the five decades (beginning in 1950) have been, unfortunately, totally ignored in the west.

It must be said that the use of gradually increasing doses has been known since Koch times. His contemporaries such as Ziegler, Pottenger, Petruschky, Römer, Pearson, Gilliland, Jurgens and Neumann were promoting this approach, but to no avail. It is perhaps worthwhile to reassess the value of ‘similia similibus curantur’, the term used in homeopathic practice that relies on tiny doses of antigen. It is well known that immune tolerance can be produced by two means; either by injecting very small doses or by oral administration of antigen. Interestingly, both of these approaches had, historically, higher success rates in TB immunotherapy than other modalities. Indeed, Koch’s tenth experiment, described in his seminal work Die Ätiologie der Tuberkulose (1882), refers to oral feeding of tubercular meat to rats, a proce- dure that completely protected them from subsequent challenge without provoking reactivity, but when feeding was stopped for several weeks animals became susceptible to repeated challenge. The famous tenth experiment was certainly a most curious one, as it almost defied Koch’s own Postulates, but it might have inspired another Nobelist, Charles Richet, of promoting zomotherapy and René Dubos’ eloquent example of lower TB incidence among meat-eating Masai than in vegetarian tribe Akikuyu.

Cytokines, Th1/Th2 & rational empiricism

With rapid advances in immunology resulting in the discovery of various humoral factors secreted by immune cells and domination of Th1/Th2 concept in cellular immunity, attempts have been made to apply this new knowledge to immunotherapeutic interventions. Without going into details of several clinical trials, employing the extensive range of cytokines and anticytokines, as reviewed elsewhere it can be said that the value of these immuno- modulatory molecules remained unclear and their clinical utility has been dismal [13]. Immune intervention such as inhaled IFN-g, corticosteroids, Type 1 interferons, IL-2 or anti-TNF regimens have all been quite disappointing and were largely responsible for the cautious attitude toward immunotherapy that prevails today in the field. In fact calls were made to abandon immunotherapy altogether. Thus, there is a significant gap between a priori (rational reasoning) and a posteriori (empirical evidence), a problem that has been plaguing not only the TB community but the philosophy in general, long before Immanuel Kant’s reconciliation attempt in his Critique of Pure Reason (1781). Despite the progress the correlates of protective immunity are still unknown and without it the rational science has to rely on evidence from empirical ‘poking’. Without mutual feedback and cooperation between bench versus bedside camps there will be no true progress.

Other mycobacterial vaccines

In addition to tuberculin, there are several preparations of mycobacterial origin, which have been or are still being used as adjunct therapy for TB. Among these are Mycobacterium chelonae or ‘turtle vaccine’ developed by Friedrich Friedmann, sold until recently under brand name Anningzochin (Laves-Arzeimittel Gmbh, Ronnenberg, Germany) [4]; Mycobacterium microti, which has been tested in UK and used widely in The Czech Republic and Slovakia, but mostly as a prophylactic vaccine [14]; Mycobacterium phlei from Sanum-Kehlbeck in Germany, also manufactured in China (Utilin S, Chengdu Jin Xing Sanum-Kehlbeck Medicine Co., Ltd, China) [15]; Mycobacterium w or Mycobacterium indicus originally developed as a leprosy vaccine (Immuvac, Cadila Pharmaceuticals, India) [16]; Mycobacterium bovis (standard BCG vaccine), which is also used as an adjunct immunotherapy [8,17]; and Mycobacterium vaccae, a therapeutic vaccine developed by Stanford et al., (Immodulon Therapeutics, London, UK) [18], approved in China under the Vaccae® brand name for adjuvant therapy of TB (Anhui Longcom, China) [19]. The oral tableted form of heat-killed of M. vaccae (V7) is now being tested in a Phase II trial by Immunitor Inc., (Canada), this trial is aimed to confirm beneficial clinical findings reported by Dlugovitzky et al., of their own oral formulation [20]. Preliminary results from the V7 trial are shown in Table 1. A therapeutic vaccine, RUTI®, containing delipidated fragments of M. tuberculosis, as a modern day Dreyer’s ‘defatted’ or diaplyte tuberculin [21] and (Figure 3),was developed by Archivel Farma (Spain), but no dataregarding its efficacy in humans are yet available [22].

Figure 3. Box containing ampoules with the‘defatted’ tuberculin vaccine of Georges Dreyer (1924).

Of special interest is the extremely small quantity of tubercular antigen – only 10 ng per dose. Georges Dreyer, born inShanghai from Danish parents, was,between 1907 and 1934, the first fullProfessor of Pathology at Oxford University, UK. Of note is that after Dreyer’s death his place was taken by Howard Florey – who with Chain and Heatley succeeded in producing clinicalquantities of penicillin from the sample of the original mold strain, which was actually given to Dreyer by none other than Alexander Fleming himself. The principle of action of Dreyer’s vaccine is similar to ‘delipidated’ or detoxified RUTI vaccine, currently being developed by Archivel Farma, Spain. This principle follows the proposal made by Auclair (1900) and Sternberg (1902) that adverse effects of tuberculin were due to the presence of the lipid fraction of the bacterial cell wall, which supports Koch’s description of the same phenomenon described in Über neue Tuberkulinpräparate (1896).

Image courtesy of Lorenzo Iozzi (History and Technology Department, Museum Victoria, Victoria, Australia).

Last year, Immunitor made a similar vaccine, V5; containing circulating M.tuberculosis antigens derived from heat and chemically inactivated blood of donors, which has already been through several Phase II trials [23–26]. Finally, theNational Institute for the Control of Pharmaceutical and Biological Products(Beijing, China) reported therapeutic vaccine based on acellular Mycobacteriumsmegmatis that appeared to be safe in patients with TB [27]. The efficacy of theabove vaccines is variable, ranging from unknown to excellent, but what is important is that all of them reported to be safe. Furthermore, these vaccinesappeared to be equally effective against drug-resistant TB and TB with HIV, themajor challenge for current chemotherapeutic interventions. Curiously, most ofthese vaccines, some of which have been in commercial use for decades, areknown to only small number of TB researchers. Nevertheless, they exist andresources need to be devoted to investigate them further, since ignoring ordismissing them a priori will be a disservice to patients, especially to those whohave no treatment options left.

Other immunotherapeutics This section summarizes TB trials in which variousimmuno- modulators from Russia and other former Soviet republics were used as anadjunct therapy to conventional chemotherapy. Russia has an exceptionally diverserepertoire of immunomodulating agents, with over 130 unique chemical entitiescurrently sold for a wide range of clinical indications. We have previously published areview on many of these agents and refer to it for the detailed description ofcomposition and putative mechanisms of action [28]. Most studies relating to their useagainst TB are either published in the Russian language or exist as a technicalinformation sheet or treatment recommendations from the manufacturers [29–32]. Areview article that summarizes TB trials with select immuno modulators was recentlypublished by Mezentseva et al. [33]. See also Table 1 for the outcome of some of theseimmune interventions. It can be seen that in all cases the immunomodulatorsenhance the efficacy of TB drugs and in less time than the mandatory 6 monthsrequired for chemotherapy. Admittedly, many of them are inconvenient in terms ofadministration, or have potential side effects, but one thing is clear – there is plentyof choice. This is in stark contrast to the situation in western countries, there are noimmunomodulators to choose from, since none exist.

We and our clinical collaborators in the Ukraine have worked on the immunotherapy of TB over the past 10 years resulting in nearly 20 published clinical trials involving close to 1500 indi- viduals. Initially, we have used locally-produced, herbal immu- nomodulator Dzherelo or Immunoxel [34–36]. Sputumconversion among multidrug-resistant-TB, extremely drug-resistant-TB and TB–HIVpatients after 2–4 months of treatment were approximately 85–100%, while incontrols on TB drugs alone it took 6–24 months to reach the 48–85% range.However, what was of interest to us is that inflammation and other TB symptomswere improved by Dzherelo in a manner that was strikingly similar to V5 [23–26].Similar anti-inflammatory properties were reported by our colleagues in Russia ininvestigations of their own immuno modulators [29–33]. This suggests that an effectiveimmunotherapy ought to have a beneficial effect on inflammation, which can be,for example, measured by two unsophisticated tests, that is, eryth- rocytesedimentation rate and leukocyte counts. Measuring fever reduction or bodyweightgain are even more simple tests. These simple biomarkers combined withimmunological tests evaluating cytokine profile and Th1/Th2 balance can providereliable correlates of immune protection [37]. The effective immune intervention mustpossess similar if not identical properties in terms of anti-inflam matory effects andother clinical improvements we have observed in our trials.

Conclusion The emergence of TB, especially drug-resistant TB and TB–HIV is ofgreat concern to global public health. As new prophylactic vaccines, currentlyunder investigation, are not expected before 2020, the elimination of TB must relyon existing immune inter- ventions to be used together with currently available TBdrugs. In recent times, the immunotherapy of TB has been mostly a disappointingexperience with high rates of failure, and it is not surprising that many physiciansare skeptical about this concept. We have been working with various immunetherapies during the past 10 years and can attest that this approach merits moreatten- tion than it has previously received. However, caution is required so thatprotective and not harmful traits of immunity are induced [11,38]. TB is a classicexample of chronic infectious disease char acterized by persistent inflammationwith autoimmune features. This simple concept is still struggling to gain wideracceptance [1–2,38–40]. Conventional chemotherapy cannot correct protectiveimmunity. Without adjunct immunotherapy one cannot speed up bacterialclearance and the healing process. Without immuno therapy one cannot overcomedrug resistance and HIV coinfec tion. Clinical experience with various immunomodulators has not always been encouraging, but those that have shownclinical benefit must be urgently adopted and more resources must be allocatedto understand their mechanism and introduce existing immunomodulators intowider clinical use.

Expert commentary Medical practitioners have a set of 40-year-old antibioticswhich they use according to outdated regimens and they seldom venture outsideindoctrinated confines. With expansion of drug resistant strains and HIV thetherapeutic choices are becoming even more limited. It is thus imperative thatbasic science, especially immuno logy, is brought back to iatrochemist masses.Phtysiatrists must understand that their enemy is not tubercle bacilli only, butmisdirected immune response. They often hear and read that a third of thepopulation carries M. tuberculosis, but they seldom question why 95% of them neverget sick. They know well that every 25 seconds a person dies from TB, but theynever question what the real cause of death was. Everyone has been taught in their medical school to equate mycobacterium with death. In our opinion this is wrong. The death results from self-destructive inflammatory responses triggered bymycobacterial infection. The division of TB bacilli into virulent and avirulent typesis based on in vivo assays, which is clearly dependent from the host immuneresponse, since in vitro assays based on measurement of cell death in infectedmacrophages or dendritic cells, the virulent TB strains are usually less cytotoxicthan avirulent ones [41,42]. To the best of our knowledge there is only one referencein the literature, which implies M. tuberculosis being directly cytopathic to human epithelialcells and even then this might have been an artifact due to HIV coinfection [43]. Furthermore, thesymbiotic relationship between mycobacteria and host is not necessarily harmful, onecan find in the literature examples of beneficial coexistence [44]. In order to increaseour ability to control infectious diseases and prevent epidemics, it is vital thatdoctors are properly educated. It is important to allocate more resources towardexisting immune interventions so that clinical outcomes are improved. The currentgaps in our knowledge must be identified and explored according to their relevancenot paradigmatic convenience.Five-year view Basic TB research has intensified in the last decade. The TBpandemic that started in early 1990s has shown that even the most developed countries are vulnerable. TB will continue to be a threat to the globalcommunity as it has been for mil- lennia. Every country should take measures by implementing a national preparedness and response plan, by empowering thelocal community, by training the medical personnel and out- reach health staff in the identification and management of TB, and by creating new tools fordiagnosis and treatment. The establishment of truly novel protocols for management of TB needs to be implemented. If the scarcely populated field ofimmune interventions is invigorated with renewed enthusiasm then in the next 5years we may witness the renaissance of the immune therapy that was dominating TB research prior to introduction of the chemotherapy.

Acknowledgements We would like to thank many experts whohave contributed their comments and critiques. In particular,we are indebtful to John Stanford, Graham Rook, John Grange,Ali Zumla, Gene Shearer, Feng-li Tao, Shilong Yang, Jiang Pu,Chuanyou Li, Wing-wai Yew, CC Leung, Ying Zhang, Satoshi Makino, Ray Spier, Nataliya Kozhan, Vasilyi Petrenko, ElenaRekalova, Irina Il’ inskaya, Petr Rytik, Felix Ershov, VolodymyrPylypchuk, Pere-Joan Cardona, Jim Johnston, Carl Feng,Christoph Lange, Bob Wallis, Keertan Dheda, Gavin Churchyard,Tony Hawkridge, Tom Evans, Robert Loddenkemper, StefanKaufmann and Mel Spigelman for sharing with us their views on TBissues. The authors are also grateful to patients who participated intrials of immunotherapeutics discussed in this review.

Key issues

• The immune approach in TB therapy has been promoted since 1890, starting with Robert Koch himself, but there are even earlier empirical precedents such as those found in Li Shi Chen’s Pên Ts’ao Kang Mu.

• Immunotherapy is still skeptically viewed due to the failure of variousapproaches, especially involving cytokines.

• Failures are linked to the belief that TB arises as a result of a ‘weakened’ immune response.

• We believe that TB, as a disease, occurs when the immune tolerance to mycobacterium is replaced by an exaggeratedimmune response, characterized by inflammatory reaction against own tissues harboring the tubercle bacilli.

• By consequence, TB is an autoimmune disease triggered by mycobacterial infection.

• In order to produce a favorable clinical outcome one needs to manage immunity with the aim of restoring immune tolerance.

• Immune tolerance does not mean immune suppression oranergy, it’s an active process, which is as potent as classicalimmune activation.

• If this concept is true, then immune correlates of protectiveimmunity have to be searched for in places that were overlooked bycurrent dogmas.

• There are many immunotherapeutics that have been successful and they need to be investigated so that clues emerge, which could help not only the therapy but also prophylaxis of TB.

Financial & competing interests disclosure

AS Bourinbaiar and V Jirathitikal are officers of the Immunitor company,involved in developing TB immunotherapies. Some of the trials ofimmuno- therapeutics described in this review were supported byBusiness Partnership Grant UKB1-9017-LK-09 awarded by the US Civilian Research & Development Foundation – a nonprofit organization authorizedby the US Congress and established in 1995 by the National ScienceFoundation. The authors have no other relevant affiliations or financialinvolvement with any organization or entity with a financial interest in orfinancial conflict with the subject matter or materials discussed in themanuscript apart from those disclosed. No writing assistance was utilizedin the production of this manuscript.

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Immunotherapy 1, 549–556 (2009). 36 Prihoda ND, Arjanova OV, Yurchenko LV et al. Adjuvant immunotherapyof extensively drug-resistant tuberculosis (XDR-TB) in Ukraine. Curr. Res.TB 1, 1–6 (2009). 37 Mezentseva MV, Stakhanov VA, Zakharova MV, Zotova IF, TregubovaMI, Shapoval IM. Cytokines as the markers of the infiltrative pulmonary tuberculosis progress. Infect. Immun. 1, 367–372 (2011). 38 Grange JM, Brunet LR, Rieder HL. Immune protection againsttuberculosis – when is immunotherapy preferable to vaccination?Tuberculosis 91, 179–185 (2011).39 Ellner JJ. Immunoregulation in TB; observations and implications. Clin.Transl. Sci. 3, 23–28 (2010). • Links TB with inflammatory reaction. 40 Kivity S, Agmon-Levin N, Blank M, Shoenfeld Y. Infections andautoimmunity friends or foes? Trends Immunol. 30, 409–411 (2009).41 Danelishvili L, McGarvey J, Li YJ, Bermudez LE. Mycobacteriumtuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells. Cell Microbiol. 5, 649–660 (2003).42 Ryan RC, O’Sullivan MP, Keane J. Mycobacterium tuberculosis infection induces non-apoptotic cell death of human dendritic cells. BMC Microbiol. 11, 237 (2011).43 Gadea I, Zapardiel J, Ruiz P, Gegúndez MI, Esteban J, Soriano F.Cytopathic effect mimicking virus culture due to Mycobacteriumtuberculosis. J. Clin. Microbiol. 31, 2517–2518 (1993).44 Lehmann D, Ben-Nun A. Bacterial agents protect against autoimmune disease. I. Mice pre-exposed to Bordetella pertussis or Mycobacteriumtuberculosis are highly refractory to induction of experimentalautoimmune encephalomyelitis. J. Autoimmun. 5, 675–690 (1992).• Demonstrates that tubercular infection can protect againstautoimmunity. Websites101Classics of traditional Chinese medicine. www.nlm.nih.gov/exhibition/chinesemedicine/emperors.html • Refers to Ch’en Hsueh-lou, Chung-kuo li tai ming i t’u chua: Biographiesand Portraits of Chinese Famous Doctors in Past Dynasties, Nan-ching,1987.102 The natural history of pulmonary tuberculosis, facilitator guide.www.emro.who.int/STB/media/pdf/ HARRIS+coverpage.pdf 103 Cycloferon (Polysan) St-Peterburg, Russia. www.polysan-ru.com 104 Amiksin (Pharmstandart) Tomsk, Russia. http://pharmstd.ru 105 Galavit (Medicor) Moscow, Russia. www.medicor.ru 106 Polyoxidonium (Petrovax) Moscow, Russia. www.petrovax.ru 107 Likopid (Peptec) Moscow, Russia. www.licopid.ru 108 Glutoxim (Farma VAM) St-Peterburg, Russia www.glutoxim.ru109 Neovir (Pharmsynthez) St-Peterburg, Russia. www.pharmsynthez.com110 Bestim (Verta) St-Peterburg, Russia. www:hpb-spb.com 111 Ronkoleukin (Biotech) St-Peterburg, Russia. www.biotech.spb.ru112 Dzherelo Immunoxel (Ekomed) Kiev, Ukraine. www.ekomed.com.ua113 Tuberculin (SPbNIIVS – Institute of Vaccines and Sera) St-Peterburg,Russia. www.spbniivs.ru114 V7 (Immunitor) Vancouver, Canada. www.immunitor.com

Mobirise

Effect of Immunomodulator Dzherelo on CD4 + T-Lymphocyte Counts and Viral Load in HIV Infected Patients Receiving Anti-Retroviral Therapy

Research Journal of Pharmacology 2 (1): 8-12, 2008 ISSN: 1815-9362
© Medwell Journals, 2008

1Lyudmila G. Nikolaeva, 1Tatyana V. Maystat, 2Volodymyr S. Pylypchuk, 3Yuri L. Volyanskii and 4Galyna A. Kutsyna ‘Kharkov Regional AIDS Prophylaxis and Prevention Center, Kharkov Medical Academy of Postgraduate Education, 6 Bor’by Street, Kharkov 61044, Ukraine 2Ekomed LLC., Prospect Pravdy 80-A, Kiev 04208, Ukraine 3I.I. Mechnikov Institute of Microbiology and Immunology, Kharkov 61057, Ukraine 4Luhansk Regional AIDS Center, Luhansk 91045, Ukraine.

Abstract: The phase II, randomized, clinical trial was conducted in 40 HIV infected patients to evaluate the effect of oral immunomodulator Dzherelo on immune and viral parameters. The arm A (n = 20), received standard Anti-Retroviral Therapy (ART) consisting of zidovudine, lamivudine and efavirenz (AZT/3TC/EFV) and arm B (n = 20) received 50 drops of Dzherelo twice per day in addition to ART. After 2 months the total CD3 T-lymphocytes increased in ART recipients from 664 to 819 cells |±L«1 (p = 0.06), whereas inDzherelo recipients they rose from 595 to 785 cells |±L«1 (p = 0.03). The population of CD4 T-cells expanded by 57.3% in ART (218-343; p = 0.002) and by 93.5% in Dzherelo arms (184-356; p = 0.004). The accrual in absolute and relative number of CD8+ lymphocytes in ART and Dzherelo recipients was 43.2% (2.7%) and 50.4% (-0.5%) , respectively. The CD4/CD8 ratio in Dzherelorecipients had increased from 1.495 to 1.940 (p = 0.03) but in the control group the increase was not significant, i.e., 1.418-1.613 (p = 0.14). About three-quarters (14/19) of patients on ART displayed the decrease in viral load (1718-1419 copies mL«1; p = 0.008), while 95% of patients on Dzherelo had a reduction in the number of viral copies (1793-1368 copies; p = 0.001). Dzherelo has a favorable effect on T-lymphocyte subsets and viral burden in HIV patients when given as an immunomodulating adjunct to ART.
Key words: AIDS, antiviral, ART, HAART, immunotherapy, phytotherapy.

INTRODUCTION
The antiretroviral drug resistance, drug toxicity and adherence are major concerns in clinical management of HIV infection (Pokrovskii, 2001). The combination of antiviral drugs, such as Highly Active Antiretroviral Therapy (HAART), may prevent HIV from mutating and spreading, allowing patients to rebuild their immune system to the same levels as in normal individuals (Mocroft et al., 2007). On the other hand, the immune activation caused by enhanced immune reaction to HIV, is now recognized as a cause of depletion of CD4 T-cells and resulting immunodeficiency (Appay et al., 2005). These problems are driving force for research on new therapies that can provide an answer. Most optimal therapeutic solution is an effective and safe immunotherapy that could regulate the immune response in a manner favorable to a host. There are many types of immune modulators that have been used clinically for viral infections, but for HIV the choice of immune interventions is limited (Ershov, 2003). Ukraine has the highest prevalence of HIV infection in Eastern Europe (Kelly and Amirkhanian, 2003; Vander werf et al., 2006). Oral immunomodulator Dzherelo is used in Ukraine for the management of HIV infections, including patients co-infected with TB (Chkhetiany et al., 2006, 2007; Kutsyna et al., 2003, 2005; Prihoda et al., 2006; Zaitseva, 2006). Clinical studies have indicated that Dzherelo can significantly increase CD3 and CD4 T-lymphocyte populations and helps to achieve better clinical response when combined with standard Anti-Retroviral Therapy (ART) consisting of zidovudine (AZT); lamivudine (3TC) and Efavirenz (EFV) (Chkhetiany et al., 2007, 2006; Kutsyna et al, 2003, 2005; Prihoda et al, 2006). Dzherelo has been found to decrease the incidence of opportunistic infections and reverse AIDS-associated wasting (Chkhetiany et al., 2007; Kutsyna et al., 2003,
2005).
Corresponding Author: A. Galyna Kutsyna, Luhansk Regional AIDS Center, Luhansk 91045, Ukraine
Dzherelo has also been found to decrease the hepatotoxicity associated with ART (Chkhetiany et al., 2006, 2007; Kutsyna et al, 2003, 2005; Prihoda et al,
2006; Zaitseva, 2006).

Dzherelo was approved in 1997 by the Ministry of Health of Ukraine as immunomodulatory preparation, which so far has been used by over 150,000 individuals for various indications including chronic bacterial and viral infections such as TB and HIV, autoimmune diseases and malignancy. Dzherelo contains concentrated aqueous-alcohol extract from medicinal plants such as Aloe, Common knotgrass, Yarrow, Purple coneflower, St. John’s Wort, Centaury, Snowball tree berries, Nettle, Dandelion, Sweet-sedge, Oregano, Marigold, Seabuckthorn fruit, Elecampane, Tormentil, Greater plantain, Wormwood, Siberian golden root, Cottonweed, Licorice, Fennel, Birch tree fungus, Thyme, Three-lobe Beggarticks, Sage, Dog rose fruit and Juniper fruit. Our study was aimed at evaluating the effect of Dzherelo on immune cell subsets and viral load among HIV patients treated with standard ART in comparison to a control population which received ART alone.

MATERIALS AND METHODS
Patients: The patients, aged 20-59 years, have been selected and divided into arms A and B, each consisting of 20 patients randomized by their disease progression. The average (median) age in arms A and B was 33.9 (31.5) and 32.8 (31) years. The proportion of males and females was 15/5 and 17/3 in arms A and B, respectively. Patients were in advanced clinical stage of HIV infection with average baseline CD4+ T-cell count below 200 cells/microliter. Another inclusion criterion was the lack of any form of anti-retroviral therapy prior to the trial. The diagnosis of HIV infection was established by standard ELISA test further confirmed by Western blot analysis. The participation in this trial was voluntary and patients were enrolled only after signing the written consent indicating that they were free to withdraw from the study at any time. The conduct of the trial was approved by the advisory board of the AIDS center.

Treatment regimens : None of the patients received anti-retroviral therapy prior to the trial. After initial screening, qualifying patients were randomly divided in two arms: Arm A was prescribed: zidovudine (AZT) at 300 mg doses twice-daily; lamivudine (3TC) 150 mg tablets twice-daily and Efavirenz (EFV) 600 mg dose once-daily. The arm B received, in addition to ART, twice per day dose of Dzherelo which was given as 50 drops diluted in 100 mL of water.
Immunophenotyping oflymphocyte subpopulations: The samples of peripheral blood of patients with HIV were analyzed using commercially available Clonospectr panel of monoclonal antibodies against surface antigens of lymphocytes (MedBioSpectr, Moscow, Russia). Assays were carried out at study entry and after 1 and 2 months on the therapy. The absolute and percent values of the following subpopulations were assessed in a blinded fashion by fluorescent microscopy: Total T lymphocytes (CD3+), helper T lymphocytes (CD3+CD4+) and cytotoxic T lymphocytes (CD3+CD8+). In addition the changes in the ratio between CD4 and CD8 cells were evaluated as a part of assessment of the immune status of patients. The samples of the blood from 19 healthy blood donors were analyzed as a reference for normal values.

PCR analysis: Stored frozen samples of plasma were processed in bulk by using commercially available PCR kit (AmpliSense HIV-1, Central Research Institute of Epidemiology, Moscow, Russia) designed for quantitative analysis of HIV-RNA copies. Tests were carried out at baseline and after two months of the therapy.

Statistical analysis: The obtained results were analyzed with the aid of statistical software STATMOST (Datamost, South Sandy, UT). The baseline cell numbers relative to 1st and 2nd months of follow-up were evaluated by paired Student t-test. The non-parametric values of viral load were analyzed by Wilcoxon signed-rank test. All statistical calculations were per intent-to-treat basis or the total number of available patients without subgrouping them into responders and non-responders. The resulting probability values were considered as significant at the cut-off levels of p« 0.05.
RESULTS
After one month on the therapy there was a clear distinction between recipients of ART alone and those who received ART along with the daily dose of Dzherelo . This disparity became even more evident at the end of 2nd month of therapy. The changes in viral load among HIV patients of both groups have also reached statistical significance. These findings are described in detail below.
CD3+ total T Lymphocytes: After one month on ART alone the absolute and percent (%) values of total CD3+ lymphocytes per microliter of blood have changed in a statistically discordant manner, i.e., 664 (36.5%) vs 743(38.4%) with p = 0.13 (p = 0.03), as analyzed by paired Student t-test. Similarly, at the end of the first month, in the group receiving Dzherelo there was a statistical discordance between absolute and percent CD3+ values: 595(34.2%) vs 664(40.5%); p = 0.14(p = 0.0003). After 2 months the number of total CD3+ lymphocytes increased further to 785 (43.9%) in arm B, i.e., p = 0.034 (p = 0.0002), whereas in the control it increased to 819 (39.8%) cells, with probability values p = 0.06 (p = 0.03) , respectively. The accrual in total lymphocytes from baseline to the end of follow-up, was 23.3 and 31.9% for absolute and 9 and 28.4% for relative numbers in control and Dzherelo arms, respectively (Fig. 1).
CD4+ T Lymphocytes: The trends similar to those of total CD3+ lymphocytes were observed when CD3+CD4+ lymphocyte subsets were analyzed. Significant changes were seen in ART alone arm after one month, i.e., 218(30.3%)-295(35.2%); p = 0.007 (p = 0.02). Similarly in Dzherelo arm the helper T-cell counts have risen in a significant manner from 184 (28.4%)-254 (34.8%) cells; p = 0.03 (p = 0.0002). At the end of 2nd month lymphocyte subsets have risen to 343 (35.7%) and 356 (38%) with probability values p = 0.002 (p = 0.01) and p = 0.004(p = 0.0005) for arms A and B , respectively. When study completion results of ART and Dzherelo recipients were calculated in terms of accrual in CD4+ lymphocytes relative to entry levels there was an increase of 57.3% (17.8%) and 93.5% (33.8%) in absolute and relative values.
CD8+ T Lymphocytes: The changes observed in CD3+CD8+ cytotoxic T-cell population are different from those seen with helper cells. In ART alone group absolute but not relative numbers of CD8+ cells increased in a significant manner from 155 (22.6%)-203(24%); p = 0.014 (p = 0.21), while in Dzherelo group the changes were insignificant in both categories, i.e., from 123 (19.8%)-152 (20.3%); p = 0.08 (p = 0.28). At the end of the 2nd month the CTL population in ART group was still above baseline, i.e., 222 cells (23.2%), an accrual that was statistically significant for absolute numbers p = 0.009 but not significant when relative numbers were evaluated (p = 0.39). Similarly, among Dzherelo recipients the 2nd month absolute but not relative numbers of CD8 cells have also increased in a significant manner, from baseline levels 123(19.8%)-185(19.7%), with P values being 0.013 and 0.39, respectively. When study completion results of ART and Dzherelo recipients were calculated in terms of accrual in CD8 + lymphocytes as compared to baseline levels there was an accrual corresponding to 43.2% (2.7%) and 50.4% (-0.5%) of absolute and relative values, respectively.

Fig. 1: Changes in absolute and relative numbers of T-lymphocyte subsets at 2 months post-therapy as expressed in percentage values relative to their respective baseline levels.
CD4/CD8 ratio: The differential changes in CD4 and CD8 lymphocyte numbers had affected the CD4/CD8 ratio in patients on ART alone regimen as early as one month after treatment initiation. Their ratio had increased from baseline value 1.418-1.532 but without reaching significance (p = 0.23). The CD4/CD8 ratio among Dzherelorecipients had increased from 1.495-1.671, which was also above the cut-off value (p = 0.09). The disparity between CD4 and CD8 lymphocytes had progressed further by the end of 2nd month. Among ART alone patients the ratio had increased to 1.613(p = 0.14), while in Dzherelo group the ratio had risen to 1.940 (p = 0.03). When study end results of ART and Dzherelo recipients were calculated in terms of relative accrual from baseline levels there was a gain of 13.8 and 29.3% , respectively.
Lymphocyte subsets in normal blood donors: Samples of the peripheral blood of 19 healthy individuals were analyzed to obtain the normal distribution values of peripheral blood subsets. The average number of absolute and relative (%) CD3 lymphocytes were 1,370±169 cells nL»1 (52.9±6.8). The values of CD4 and CD8 lymphocytes were 622±89 (35.9±4.3) and 349±42(19.9±2.1), respectively, with ratio being 1.76±0.19.
Viral load: The viral load, as measured by plasma RNA-PCR at baseline and at the end of 2nd month, decreased in ART group (1718-1419 copies mL*1, p = 0.008), as analyzed by Wilcoxon signed rank test. In Dzherelo arm the viral load decreased from 1793-1368 copies; p = 0.001). About three-quarters (14/19) of patients on ART alone had displayed the decrease in viral load, while the 18 out of 19 of patients on Dzherelo (95%) had a reduction in their number of viral copies (Table 1).
Table 1: Effect of 2-month ART without or with Dzherelo on HIV-RNA plasma levels

DISCUSSION
In the prior studies Dzherelo has been shown to influence positively CD3 and CD4 lymphocyte numbers (Chkhetiany et al, 2006, 2007; Kutsyna et al, 2003). Dzhereloreduced the incidence of opportunistic infections and reversed body weight loss associated with HIV (Chkhetiany et al, 2006, 2007; Kutsyna et al, 2003, 2005). It had also reduced the toxic side effects of ART, the hepatotoxicity in particular (Chkhetiany et al., 2006, 2007; Zaitseva, 2006). For example, elevated liver aminopeptidase ALT and AST levels caused by ART have been shown to return back to normal levels. However, these studies have not dealt with the effect of Dzherelo on other immune markers and viral load.
Our 2-month study conducted in prior anti-retroviral drug-naive population reveals that when Dzherelo is added to ART there are significant benefits associated with this intervention. In our hands Dzherelo appears to display the same effect as reported by independent investigators. Our results indicate that administration of Dzherelo along with ART can produce significant increase in total CD3+ lymphocytes, CD4+ helper cells, better
CD4/CD8 ratio, higher number of CD3+HLA-DR+ activated lymphocytes and NK cells. Dzherelo appears to increase absolute but not relative numbers of CD8+ T lymphocytes and reduce significantly CD20+ B lymphocyte subpopulation. Furthermore, Dzherelo appears to contribute to inhibitory effect of ART on viral replication resulting in statistically significant lower viral load in a higher proportion of patients (Table 1).
It is well established that elevated CD3 and CD4 counts and higher CD4/CD8 ratio are associated with better prognosis in patients with HIV (Bonger and Goebel, 1991). For this reason Dzherelo is likely to influence positively the outcome of treatment and disease progression in our study population. Similarly, the viral load is a predictor of HIV disease progression, its persistent elevation in HIV infected patients is indicative of poor prognosis (Arduino et al., 2001; Dybul et al., 2002). While, there were earlier indications that Dzherelo may reduce the viral burden, our study is the first to report this phenomenon in a systemic fashion. Despite the fact that the HIV RNA levels had decreased by less than a log the difference between baseline and outcome levels was highly significant (Table 1). It is likely that the observed effect on viral load is mediated by immune cells since Dzherelo does not have the direct effect on HIV replication (Chkhetiany et al., 2007).
Many studies have been conducted aimed at determining the phenotype of immune cells in HIV infection. While there is a consensus that the immune response plays a critical role in determining the clinical outcome much more has to be learned in order to have a clear picture of cellular events during the course of disease. The understanding of the immune mechanism controlling HIV may result in design of better vaccines and immunotherapies. Currently available anti-retroviral therapy is far from ideal, requiring multiple drugs to be taken in combination for the rest of life of a patient (Pokrovskii, 2001). The extended duration of therapy, coupled with the side effects, often results in poor patient adherence, treatment failure and the emergence of drug resistance with major social and economic implications. We believe that the immunotherapy is the indispensable part of therapeutic strategies against HIV. The development of novel immune-based therapies is an urgent objective for anti-HIV drug discovery. Many immune interventions are available against bacteria, protozoa, fungi and viruses (Ershov, 2003). While often effective the mechanism of many immunomodulators is poorly understood. This downside should be balanced against clinically confirmed benefits. Our study provides an early glimpse into the putative immune mechanism of Dzherelo, which has been successfully used as an immune adjunct to HIV therapy in Ukraine during last ten years (Chkhetiany et al, 2006, 2007; Kutsyna et al, 2003, 2005; Prihoda et al., 2006; Zaitseva, 2006) Additional studies need to be conducted to develop better understanding of Dzherelo properties and to enlarge the current arsenal of HIV therapies.
ACKNOWLEDGEMENT
We thank all participants who volunteered in this study. The generosity of Ekomed in supplying Dzherelo is appreciated very much. The tireless support of clinical staff and technicians who contributed to this study has been of tremendous help to bring this study to fruition. The discussion with other investigators of Dzherelo who shared their insight and provided helpful suggestions has guided our study and we are thankful to them all.
REFERENCES
Appay, V., F. Boutboul and B. Autran, 2003. The HIV infection and immune activation: To fight and burn. Curr. Infect. Dis. Rep., 7: 473-479.
Arduino, J.M., M.A. Fischl, K. Stanley, A.C. Collier and D. Spiegelman, 2001. Do HIV type 1 RNA levels provide additional prognostic value to CD4(+) T lymphocyte counts in patients with advanced HIV type 1 infection? AIDS Res. Hum. Retroviruses, 17: 1099-105.
Bogner, J.R. and F.D. Goebel, 1991. Lymphocyte subsets as surrogate markers in antiretroviral therapy. Infection, 19: 103-108.
Chehitiany, R., V. Pylipchuk, O. Argzanova, N. Prihoda, I. Vihrova, E. Zagaidanova and G. Kutsyna, 2006. Influence of an agent with immunomodulating activities, Dzherelo , on immune response and metabolic parameters in combination therapy with NRTI+NNRTI and as a monotherapy in untreated HIV-infected individuals. Probl. Ecol. Med. Gen. Clin. Immunol., 71-72: 307-318.
Chkhetiany, R., V. Pylipchuk, O. Argzanova, N. Prihoda, L. Vihrova, E. Zagaynova and G. Kutsyna, 2007. Comparative effect of an immunomodulator Immunoxel (Dzherelo)when used alone or in combination with antiretroviral therapy in drug-naive HIV infected individuals. Int. J. Biotechnol., 9: 267-276.
Dybul, M., A.S. Fauci, J.G. Bartlett, J.E. Kaplan and A.K. Pau, 2002. Panel on Clinical Practices for Treatment of HIV. Guidelines for using antiretroviral agents among HIV-infected adults and adolescents. Ann. Int. Med., 137: 381-433.
Ershov, F.I., 2003. Use of immunomodulators in viral infections. Antibiot Khimioter, 48: 27-32.
Kelly, J.A. and Y.A. Amirkhanian, 2003. The newest epidemic: A review of HIV/AIDS in Central and Eastern Europe. Int. J. STD AIDS, 14: 361-71.
Kutsyna, G., R. Chechitiany, I. Bascacov, E. Zagaydanova and I. Zaharova, 2005. Influence of a novel immunomodulator on the prevalence of new events of opportunistic infections in untreated HIV-infected individuals. 3rd European HIV Drug Resistance Workshop. Athens, Greece., 4-7. Abst 36.
Mocroft, A., A.N. Phillips, J. Gatell, B. Ledergerber, M. Fisher, N. Clumeck, M. Losso, A. Lazzarin, G. Fatkenheuer and J.D. Lundgren, 2007. EuroSIDA study group. Normalisation of CD4 counts in patients with HIV-1 infection and maximum virological suppression who are taking combination antiretroviral therapy: An observational cohort study. Lancet, 370: 407-13.
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Prihoda, N.D., O.V. Arjanova, N.I. Sokolenko and L.A. Vihrova, 2006. Clinical efficacy of phyto preparation Dzherelo in patients with co-morbid pathology: Pulmonary tuberculosis in combination with HIV infection. Probl. Ecol. Med. Gen. Clin. Immunol., 71-72: 151-161.
Van der Werf, M.J., O.B. Yegorova, N. Chentsova, Y. Chechulin, E. Hasker, V.I. Petrenko, J. Veen and L.V. Turchenko, 2006. Tuberculosis-HIV co-infection in Kiev City, Ukraine. Emerg. Infect. Dis., 12: 766-768.
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Mobirise

Synthetic and Natural Immunomodulators Acting as Interferon Inducers

Dmytro S. Silin1*, Oksana V. Lyubomska1, Feliks I Ershov2, Valeriy M. Frolov3 and Galyna A. Kutsyna3,+
Laboratory of Molecular Virology, Medical and Biology Center, School of Biomedical Sciences, Queen’s University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK; 2Gamaleya Institute of Epidemiology and Microbiology, 18 Gamaleya Str., Moscow, 123098, Russia and ^Department of Infectious Diseases and Epidemilology State Medical Univerity, 50-Years of Defence of LuhanskStr., Luhansk 91045, Ukraine
Abstract: Interferons are first immunomodulatory molecules that have been shown to display a wide range of applications due to their antiviral, antibacterial, antitumor, and inflammatory activities. Natural and recombinant interferons are among most common biologic therapeutics worldwide. Interferon inducers, however, are less known and have been mostly developed and used in former socialist countries. Despite the fact that they are virtually unknown to the Western world, they represent a substantial market share of modern pharmacopoeia in former socialist republics. This review provides a brief description of most popular interferon inducers including Amyxin, Amizon, Anandin, Arbidol, Blasten, Cy-cloferon, Galavit, Groprinosine, Hepon, Immunoxel, Dzherelo, Kagocel, Larifan, Ligfol, Likopid, Mebavin, MIGI-KLP, V-5 Immunitor, SCV-07, Milife, Neovir, Poludan, Ragocin, Ridostin, Thymogen and Savratz, some of which were in use for several decades for the same clinical indications as for interferons. The variety and choice offered by the pharmaceutical industry behind the former “iron curtain” certainly deserves the appreciation, familiarity and application prospects for medical and research investigators worldwide.

INTRODUCTION
Immune system is the main regulatory system controlling homeostasis of the body and participates virtually in all (processes) cycles of the life from birth to death. The incompetence of the immune system opens door to infectious, malignant, autoimmune, and inflammatory diseases. There are many modern interventions directed to stimulation, modulation or suppression of the immunity by various routes.
Interferons are extremely important category of protein therapeutics aiding defense against infections and malignancies carrying foreign for host genetic information. Interferons are intra- and inter-cellular signaling proteins of three classes – alpha, beta, and gamma, which differ by their activity, cell origin and cell targets. Natural and recombinant interferons are widely used in the modern therapy of acute and chronic infectious and oncological diseases and some immune disorders. Alpha interferons such as Laferon, Intron A, Welferon, Reaferon, Viferon, Viaferon, Roferon A as well as beta interferons – Betaferon, Feron, Fron, Rebif, and others represent the type I interferons which express high antiviral activity and widely applied in a complex antiviral therapy [19]. Gamma interferons such as Iimmukin, Interferonlagen, mega-D-interferon and others represent the type II interferons which increase MHC II level on antigen-presenting cells and regulates the level of inflammatory and immune responses. Gamma interferons were successfully applied for
*Address correspondence to these authors at the Laboratory of Molecular Virology, Medical and Biology Center, School of Biomedical Sciences, Queen’s University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK; E-mail: d.silin@queens-belfast.ac.uk and Department of Infectious Diseases and Epidemilology State Medical Univerity, 50-Years of Defence of Luhansk Str., Luhansk 91045, Ukraine; E-mail: kutsyna@list.ru
therapy of viral, malignant, and autoimmune diseases [10-14].
However, interferons are species-specific and for the replacement therapy species-specific proteins are necessary, which is a limiting factor for their using in the veterinary and animal experimentations. On the other hand, administration of interferons could activate negative reverse loop of regulation, inhibiting endogenous interferon production and it could be undesirable side effect, particularly in the chronic cases of diseases. To overcome both of the above restricting factors, interferonogens could be successfully applied, as they are not species-specific and stimulate endogenous production of interferon.
Generally, interferonogens have significant advantages comparing to native or recombinant interferons: single administration of interferonogens increases interferons to therapeutic level for up to several days, whilst interferons administration should be multiple and in high dosage as their semi-life is about 20-40 minutes; such high-rate administration of interferons could turn on regulatory machinery of their endogenous synthesis and severe side effects [15,16]; overdosing of interferonogens (and according side effects) is practically impossible as interferon synthesis is still controlled by organism; and, finally, interferonogens are mostly not antigenic and could be used long period repeatedly.
The first successful clinical application of experimental viral interferonogen IVS (inactivated Semliki Forest virus) in therapy of viral ocular infection was performed in the USSR by A.A.Kasparov and colleagues in 1966 [17]. Starting from that several interferonogenes were discovered and investigated, most of them by scientific groups of the former USSR [18].
Among contemporary immunomodulators with interfer-onogenic activity the physical, chemical and biological agents could be designated. Physical influence on the immune system by low-intensity laser, ultrasound, low-frequency magnetic field etc. could normalize various immunity subsystems activity, particularly phagocytosis, cellular and humoral immune response by both interferon-dependent and independent pathways [19].
As a result of many years of screening several promising interferonogenes were revealed among various kinds of natural and synthetic compounds (fluorenones, acridanones, gossypol derivatives, polynucleotides, ds-RNAs etc.) They have quite high chemotherapeutic index and could be useful for prophylaxis and treatment of viral and other diseases. By chemical synthesis and biotechnology means lowmolecular weight substances were obtained such as Neovir, Cy-cloferon, Kagocel, Amixin, as well as high molecular weight substances such as Poludan, Ridostin, Larifan, and others. Others preparations with interferonogenic properties were discovered from natural biological sources, for example, Milife – from fungi; “MIGI-KLP”- from mussel; Immunomax and Immunoxel (Dzherelo) – from medicinal plants; V5 Immunitor – from pooled blood.
In the next chapters we concentrate on some scientifically proven and industrially manufactured interferonogenes and review their properties related to clinical uses.
Hepon. Manufacturer: “Immapharma”, Russia
Hepon is synthetic immunomodulator based on tetrade-capeptide: Thr-Glu-Lys-Lys-Arg-Arg-Glu-Thr-Val-Glu-Arg-Glu-Lys-Glu, induces alpha and beta-interferons, inhibits inflammatory cytokines, stimulates humoral immunity. Experimentally demonstrated inhibition activity of Hepon on hepatitis C virus replication in human cell cultures [20], antiviral activity of Hepon was also demonstrated for rabies with dose-dependant protection of up to 40% mice [21], Herpes simplex viruses types 1 and 2 with one hundred fold reduction of viral titer in vitro and 36% protection after 10 LD50 dose challenge [22]. Hepon-treatment intensifies antibody production against HIV1-antigens [23] and increases concentration of CD4 and NK cells, functionality of neutro-phils and CD8 T-cells, and decreases virus load in the blood of HIV-infected patients [24].
Stimulation of activity of intestinal mucosal immunity was demonstrated in several clinical trials [25,26]. In the experimental and clinical studies was proved efficiency of therapy with Hepon and Immunomax (another immunocor-rector, developed by the same group) during acute purulent surgical infections [27]. There were no noted contraindications and adverse reactions associated with Hepon.
Cycloferon. Manufacturer: “Polysan”, Russia
Cycloferon is a synthetic analogue of Cytrus Grandis alkaloid, stimulates B-cells, macrophages and other cells and tissues to produce almost pure type 1 interferons. It was reported to have up to 100-fold upregulation of beta-interferon gene and 10-fold upregulation of alpha-interferon gene in the human blood samples after administration of Cycloferon without affecting essentially the activity of other genes of blood cells [28]. In the placebo controlled multicentered study on totally 16,000 children and adolescents Cycloferon demonstrated clear epidemiological benefit in the prophy-laxys of the influenza and other acute respiratory viral infections with 1.5-2.9 -fold decreased morbidity and 41-90% protection index [29]. Its efficiency was demonstrated in chronic infections of upper respiratory tract too [30]. Specific antiviral activity of Cycloferon against adenovirus type 6 in vitro [31] and herpes virus on experimental herpetic infection was demonstrated [32]. The author’s (D.S.) personal observations in the veterinary hospital have revealed antiviral efficiency of Cycloferon in cases of canine distemper and parvoviral gastroenteritis. The duration of the disease, commonly, decreases for 3-4 days when standard complex therapy was supported by Cycloferon. In the animals with normal immune status Cycloferon induced the formation of the serum interferon in high titers (up to 20,000) with the peak achieved 4-8 hours after the injection and increased survival rate in generalized herpes infection by 30-100% in comparison with the controls. Under immunosuppression caused by gamma-radiation or cyclophosphamide the titers of serum interferon were 4-8 times lower and the protective effect of this preparation was considerably milder [32]. However, in HIV-infected patients the remission period of herpes simplex virus 1 and 2 infections is prolonged after combination of antiviral treatment with Cycloferon [33]. The antibacterial activity of cycloferon was demonstrated for various pathogenic and opportunistic species [34], and correction of the immune status after anti-tumor therapy was also observed [35]. Anti-apoptotic activity of Cycloferon was seen in the hypothalamic neurosecretory centers [36].
Amyxin (Amixine). Manufacturer: “Lancepharm”, “Dalhimpharm”, “Masterlek”, Russia; and Odessa Physico-Chemical Institute, Ukraine
Amyxin (Tilorone) induces alpha, beta, and gamma interferons by intestinal epithelium, hepatocytes, and granulo-cytes.In the animal models, 4-24 hours after oral administration, maximum levels of interferon are reached in the intestine, liver and blood, resulting in efficient prevention and therapy of chronic enteritis and hepatitis [37]. Besides potent interferonogenic activity, Amyxin causes activation of NK and phagocytes in peripheral blood [38]. Interestingly, linkage of RNA-Amyxin complex to bead carriers improves in-terferonogenic properties and proves that mechanism of such activity requires the contact between the effector and the cell surface without its penetration into the cell [38]. Antiviral properties of Amyxin are well documented on a range of viruses. Thus, experimental Haemorrhagic fever studies reveals 52% protection of animals by combined Amyxin-Virosole therapy which was superior to the effect of their monotherapy [40], although some regimens of Amyxin only provided protection up to 61% with oral administration and up to 65% with subcutaneous injection [39]. Preventive Amyxin therapy in population groups with high hemorrhagic fever with renal syndrome (HFRS) risk prevents development of HFRS and acute respiratory viral infection [41]. In the same study it was shown that Amyxin in chronic viral hepatitis (CVH) improved general condition of the patients, removed jaundice of the skin and sclera, normalized activity of aminotransferases and blood bilirubin level. Virus replication was stopped in 25% cases of chronic HBV and in 1.6% cases of chronic HCV infection [39]. The 33% lethality reduction by Amyxin was demonstrated in experimental West Nile Fewer in vivo [40], while antiviral effect of RNA-Amyxin molecular complex was registered in vitro for three virus-cell systems: vesicular stomatitis virus (VSV) – murine fibroblast L929 cells, Venezuelan equine encephalitis virus (VEEV) – swine embryo kidney (SEK) cells and encephalo-myocarditis virus (EMCV) – established piglet testicular (EPT) cells [42]. The administration of Amyxin simultaneously with polyvalent vaccination of pups in context of emergency prophylaxis, seemed to reduce cases of vaccination failure, although efficiency of Amyxin in cases of developed canine distemper and parvovirosis was insignificant even at early stages of diseases. The efficacy of Amyxin for flu and acute respiratory viral infections’ prophylaxis and treatment was demonstrated in a controlled trial of the risk group of medical personnel [43]. Amyxin in combination with herpes vaccination was highly efficient (87.9-90.9%) for the treatment of herpetic keratitis and prevented the relapse of the disease [44].
Arbidol. Manufacturer: “Dalchimpharm”, “Masterlek”, Russia
Arbidol (ethyl- 6 – bromo – 4- [(dimethylamino)methyl]- 5 – hydroxy – 1 – methyl 2 – [(phenylthio)methyl] – indole – 3-carboxylate hydrochloride monohydrate) stimulates humoral and cellular immunity, posses interferonogenic and antioxi-dant activity. Arbidol was shown to have effects on nonspecific defense factors, on capacity to induce interferon and to activate phagocytes in particular. Arbidole-treated patients with lower baseline immunity showed improvement in im-munological parameters (in the counts of CD4 and CD8 lymphocytes, B lymphocytes, in the levels of serum immu-noglobulins). Arbidol produces a high preventive and therapeutical effects in influenza A and B and other acute respiratory viral infections, prevents postinfluenza complications, reduces the incidence of exacerbations of chronic diseases in postinfluenza patients [45]. In the randomized, double-blinded, placebo controlled trial was revealed that the median duration of naturally acquired influenza was 72.0 hours in Arbidol group and 96.0 hours in placebo group. The median area under the curve (AUC) of decreased total score were significantly higher in Arbidol group than in placebo group, thus Arbidol was effective and well tolerated in the treatment of early naturally acquired influenza [46]. Specifically, reproduction of human IVA antigenic strains H1N1, H2N2, H3N2, remantadin-sensitive and remantadin-resistant strains of influenza virus as well as pathogenic for humans strains of avian influenza virus H5N1 and H9N2, were inhibited in vitro by Arbidol [47]. Efficiency of arbidol against bird flu virus H5N1 isolated from wild birds and poultry in Russia was proved in vitro [48, 49], and in the treatment of humans during avian influenza outbreak [50]. The wide spectrum of antiviral activity against respiratory viruses has led to the assessment of its efficiency on hepatitis C virus in cell culture. Long-term Arabidol treatment of Huh7 cells chronically replicating a genomic length genotype 1b repli-con resulted in sustained reduction of viral RNA and protein expression, and eventually cured HCV infected cells. Besides, pre-treatment of human hepatoma Huh7.5.1 cells with 15 microM ARB for 24 to 48 hours inhibited acute infection with JFH-1 virus by up to 1000-fold [51].
Amizon. Manufacturer: “Pharmac”, Ukraine
Amizon (N-methyl-4-benzylcarbamidopyridinium jodide) – derivative of isonicotinic acid belongs to analgetic group and among anti-inflammatory, antifever and analgetic properties expresses interferonogenic activity increasing 3-4 fold endogenous interferon in plasma, enhancing humoral and cellular immunity. Antiviral and immunomodulatory activity of amizon was clinically demonstrated on patients with hepatitis B and C with renal lesions [52] and chronic toxic hepatitis [53]. Clear clinical improvement was detected in 149 patients with Mumps treated by complex therapy with amison in comparison to 177 patients obtaining the same conventional treatment without amizon [54]. Antiinflamma-tory activity of amizon enhance its positive interferonogenic influence on patients with acute infectious inflammation [55].
Neovir. Manufacturer: “Pharmavit”, Russia, “Pharm-synthez”, Russia
Neovir (for veterinary use Camedon. Manufacturer: MEDITER, Russia) – sodium 10-methylencarboxylate-9-acridone – induses high titres of endogenous interferon, particularly alpha-interferon with peak interferonogenic activity at few hours after intramuscular injection prolonging up to 16-20 hours. Antiviral activity of Neovir was demonstrated on patients with chronic viral hepatitis B and C [56], and individual therapy programs for such patients were developed [57]. Very powerful interferonogenic activity of Neovir allowed to use it successfully on the spectrum of bacterial diseases [58,59]. Some positive effect of Neovir on steroid hormones receptors in uterus cancerous tissues was shown [60]. Moreover, Neovir exerted the direct cytotoxic action on HT-29 and K-562 cells, intact and transfected with mdr1 gene. Preliminary incubation of cells with Neovir for 24 h efficiently increased the cytotoxic effect of doxorubicin and vincristine. The enhancement of toxic action of doxorubicin for HT-29 cells had, as a rule, additive character, while for HT-29 MDR1 cells the interaction was synergistic (CD(50) was decreased by 2.85- and 8.67-fold respectively). The effect of vincristine toxicity enhancement didn’t depend on mdr1 gene expression and had synergistic character. Neovir enhanced the cytotoxic effect of doxorubicin in relation to K-562 and K-562 MDR1 cells by 3.18-fold and more than by 100-fold respectively. Preincubation of HT-29 cells with Neovir has resulted in 2000-fold decrease of 5-fluorouracil
CD(50) and in 36.6-fold for HT-29 MDR1 cells. Thus, the effect of Neovir seems to have no relation to the action on the mechanisms of multiple drug resistance and may be mediated through some other pathways [61]
Kagocel. Manufacturer: “Nearmedic plus”, Russia
Kagocel – is a potent inducer of so-called “late interferons’, a mixture of alpha and beta interferons, produced by Tand B-lymphocytes, macrophages, granulocytes, fibroblasts, endothelial other cells after oral administration of one dose of Kagocel the peak titer of interferon is registered in the intestine in four hours, although peak titer in blood registered in 48 hours, and interferonogenic response lasts up to 5 days. In vitro kagocel indused production of alpha and gamma interferons and interleukin 2 by human long-term cell cultures of different origin: J-96 and J-41 (monocytic leukemia), SW-13 (adenocarcinoma), and MT-4 (T-cell leukemia) [62]. The antiviral effect of Kagocel on the reproduction of Herpes simplex virus including its mutant strains resistant to basic antiherpetic medicine Acyclovir was demonstrated. Kagocel inhibited reproduction of Herpes virus type 1 and Herpes virus type 2 in noncytotoxic concentrations. Kagocel was also demonstrated to inhibit the reproduction of Herpes virus type 1, resistant to combination of Acyclovir and phos-phonoacetic acid [63].
Poludan. Manufacturer: “Lens Pharm”, Russia
Poludan – complex of polyadenilic and polyuridilic acids in equimolar ratio – induces mostly alpha interferon and some beta and gamma interferons. Subconjunctival injection of poludan increases the level of the interferon in blood and tears more than 10-fold and 7-fold after three hours respectively. The daily injections support elevated level of interferons which, dramatically influenced on ophtalmoherpes [64]. The same group of clinical ophthalmologist developed very promising method of viral and non viral eye lesions treatment. The method of local express auto-cytokine therapy (LEACCT) consists in using an experimentally tested auto-logous complex of cytokines (alpha-, beta-, gamma-inter-ferons, interleukins 2, 8, tumor necrosis factor alpha etc.), which is produced by joining the autoblood of patients with poludan. The administration (subconjunctivally and as instillations) of the autoblood-poludan mixture was effectively used for herpes- and adenovirus keratoconjunctivitis, slow re-epithelization after laser keratectomy and in eye burns (178 patients). Apart from the external LEACCT procedures, a 1-4-time injection of the mentioned mixture into patient’s anterior chamber used in endothelial herpetic keratoirido-cyclitis, initial bullous keratopathy, severe keratoconus and in injuries of the anterior lens capsule (117 patients). The clinical-study results (main group -295 patients) show that the increased visual acuity ranging from 0.05 to 1.0 was registered in 85% of cases [65]. The epidemiological effectiveness of poludan for prevention of acute respiratory viral infections was shown on group of (101 students). The placebo group (96 students) received the distilled water. In the students receiving poludan the incidence of acute respiratory diseases was significantly lower than in the control group (p = 0.058), decreasing to two times [66]. Similar data was obtained for prophylactic activity in the cases of the poly-ethiologic group of acute respiratory viral infection during the seasonal peak of the disease, with a coefficient of efficiency of 2.1 and corresponding protection index of 52.7%. Having the same chance of getting infected, individuals protected with these drugs often have the disease in a milder or asymptomatic form [67].
Ridostin. Manufacturer: “Vecterpharm”, Russia, “Dia-pharm”, Russia
Ridostin – mixture of double – stranded and single -stranded RNA sodium solts – potently induses interferone production and stimulates phagocytosis. Intraperithoneal injection of ridostin to mice induses intensive blood accumulation of interferon with peak at 8 hours, albeit interferone level was low in the respiratory tract and brain. Contrastly, intranasal and aerogenic administration of ridostin induced interferon mainly in the upper respiratory tract and lung [68]. Intracerebral injection of ridostin induced accumulation of interferon in the brain and serum [69]. Combined treatment with killed vaccine and ridostine by the scheme of urgent prophylaxis (3 days before challenge) demonstrated 100% protection of Aujeszky’s disease infected minks, 75% protection of foot-and-moth infected pigs, and 50% protection of canine distemper infected dogs. Clinical symptoms of dogs developed canine dictemper was mild and delayed 2325 days post infection [70].
Larifan. Manufacturer: “Pharm”, Riga, Latvia
Larifan – double stranded RNA of f2-phage – potently indused interferone after systemic or local administration. Larifan demonstrated high antiviral efficacy against Omsk haemorrhagic fever virus (strain “Ondatra”) in experiments with laboratory animals. This drug prevented the death of 65% infected mice and significantly decreased infection severity in rabbits [71]. However, this virus reproduction on cell culture was suppressed mildly whilst human adenovirus serotype 2 wasn’t suppressed by larifan in vitro at all [72].
Savratz. Manufacturer: “SRIEM”, Russia
Savratz – oxybenzylamine derivative – demonstrated high interferone-inducing capacity with early and late peaks of interferone production (4-8 and 48-96 hours after administration) depending on the route of administration [73]. Savratz showed antiviral activity in vitro against hepatitis C virus on cell cultures SW-13 and MT – 4 [74].
Groprinosine. Manufacturer: “Polfa”, Poland
Groprinosine – inosine pranobex – induces interferon, stimulates macrophages activity and lymphocytes proliferation, with specific damage to viral genetic machinery. Antiviral properties of groprinosine were demonstrated in 35 patients with acute virus hepatitis of average severity, who developed, after short-term improvement of general status, a negative dynamics of clinical and laboratory indexes. The 21 patients have received traditional treatment, 14 patients additionally were prescribed groprinosine within 5-10 days. It was shown, that addition of groprinosine to combination therapy positively influenced the disease course, promoted a rapid regress of clinical symptoms, normalization of biochemical indexes of liver function and decreased duration of hospitalization [75].
Milife. Manufacturer: “Vilar”, Russia, “Dija”, Russia
Milife – biomass of Fusarium sambicium fungi strain VSB-917 – stimulate production of alpha and gamma interferons, normalize humoral, cellular immunity and cytokine homeostasis. Milife administration to mice led to rapid and significant increase in total leukocyte and lymphocyte count in peripheral blood that persisted for at least 3 weeks after a 6 days treatment. Cellularity of lymph nodes, bone marrow and thymus increased significantly at days 4 and 6 of treatment, but returned to pretreatment levels after Milife discontinuation. Though total splenocyte numbers did not change dramatically, there occurred delayed increase in CD4+ cells in the spleen 3 weeks following treatment. Preferential accumulation of CD4+ cells was also found in peripheral blood, with the peak at day 6 of treatment. As a result, CD4/CD8 ratio in blood and spleen was significantly higher in treated than in untreated mice. Splenocytes from treated mice proliferated more vigorously in response to Con A. When added in vitro, Milife also mildly co-stimulated Con A-induced proliferation of splenocytes from intact animals [76].
Mebavin. Ragosin. Manufacturer: “IBC”, Uzbekistan
Mebavine and ragosin – soluble gossypol derivatives -possess interferonogenic and inflammation-regulatory activity. Anti-inflammatory activity of mebavin was similar to prednisolone as revealed on patients with adjuvant arthritis [77], without suppression and even with stimulation of immunity [78].
Prodigiosan. Manufacturer: MBRC “Alexis”, Georgia
As a polysaccharide extracted from Serratia marcescens and other bacteria Prodigiosan activates enzymatic activity of macrophages and stimulates phagocytic processes.Like other polysaccharides compounds Prodigiosan possesses the direct antibacterial activity and increases efficiency of antibiotics in therapy of infections caused by a wide spectrum drug-resistant bacterial strains [79]. Its interferonogenic properties were demonstrated both in vivo and in vitro [80,81], and its antiviral efficiency was confirmed in complex therapy of viral respiratory diseases [82] and hepatitis B [83]. In the later research efficiency of prodigiosan combined with ibuprofen was more pronounced than monotherapy with reaferon (alpha 2-interferon) in terms of decreasing of total serum IgE levels. Interestingly, another remedy – prodigiosin isolated from the culture broth of Serratia marcescens B-1231 possessed anti-autoimmune properties by suppressing progression of autoimmune diabetes and collagen-induced arthritis [84].
Rusam. Manufacturer: “Bryntsalov A”, Russia
Extraction from thermophilic strain C of S. aureus possess antiallergic activity and stimulate cellular immunity and both type of interferon production. Clinical trials in bronchial asthma patients demonstrated high interferonogenic and anti-autoimmune activity [85].
MIGI-K. Developer: “VNIRO”, Russia
MIGI-K preparation – a result of acidic hydrolysis of mussels flesh – contains several pharmacologically active compounds: melanoidines, peptides carnosin and taurin, amino acids, polyunsaturated lipids, vitamins and minerals. MIGI-K demonstrated antitumor, immunostimulating, anti-oxidant and radioprotective properties. Preparation secured radioprotection in trials after Chernobyl accident [86] and demonstrated strong antioxidative properties on animal models significantly or completely preventing intensification of lipoperoxidation and depression of antioxidative systems (superoxide dismutase, glutathione peroxidase, nonprotein thiols, lipoantioxidants) in skin and liver of UV-irradiated rats [87]. Interferonogenic activity of MIGI-K allowed recommending it as food addidtive in viral hepatitis and respiratory infections [88].
Blasten. Manufacturer: SIC “Enzypharm”, “Enzyme”, Ukraine
Immunomodulatory preparation from cellular walls of Lactobacillus Delbrueckii demonstrated potent immunostimulation of all types of immunity with very wide therapeutic limits. Clinical trials proved efficiency of blasten in complex treatment of oncological diseases [89], respiratory and surgical infections [90]. Very low toxicity and adjuvan-ticity comparable with complete Friend’s adjuvant led to recommendation of Blasten to wide use in medical practice by health authorities of Ukraine.
Maxidin. Developer: “Niarmedic-plus”, Manufacturer: “Micro-plus”, Russia
Maxidin (germanium bis(pyridine-2,6-dicarboxylate)) potently induces interferon and normalizes immunity in secondary immunodeficient conditions. Maxidin is effectively used in immune disorders and viral diseases of animals [91].
Immunoxel (Dzherelo). Manufacturer: “Ekomed” Kiev, Ukraine
This immunomodulator contains the wide spectrum of biologically active substances derived from herbs. The preparation possesses interferonogenic and potent anti-inflammatory activities. Series of clinical trials have demonstrated that Dzherelo induces protective immune response to a broad range of bacterial and viral infections and positive immune activity in autoimmune conditions and cancer as well. Dzherelo has been recommended by the health authorities of Ukraine as an adjunct therapy for TB and seasonal flu [92]. When Dzherelo and anti-tuberculosis therapy (ATT) or antiviral therapy are combined, it improves clinical symptoms and produces higher cure rate than in patients than on chemotherapy alone. It has been shown to achieve faster and superior rate of mycobacterial clearance, reduce HIV burden, accelerate healing of pulmonary lesions, decrease inflammation markers and pro-inflammatory cytokines, liver damage, improve hematology picture, i.e., increased hemoglobin levels, CD4 counts, and enhance significantly quality of life such as weight gain, fever, respiratory function, physical fitness, well-being and better mood. Immunoxel has been shown effective even against multidrug (MDR-TB) and extensively drug-resistant TB (XDR). The details of these beneficial outcomes were published earlier [93-100].
SCV-07. Manufacturer: “Verta”, St.Petersburg, Russia. Licensee: “Sciclone”, San Mateo, USA
Scv-07 or gamma-D-glutamyl-L-tryptophan, is a synthetic dipeptide with potent immunomodulatory and antimicrobial activity. Verta and SciClone Pharmaceuticals are developing SCV-07, the lead product in a series of immnunostimulants from Verta, for the potential treatment of tuberculosis and hepatitis C virus infection. Phase II clinical trials of the compound are ongoing [101]. SCV-07 has also shown potential in treatment of herpes infection.
Immucor GA-40 and GA-47. Manufacturer: “Alexis”, Georgia
Chromatographically purified polypeptide complexes extracted from plants demonstrated antitumor and immunomodulatory activity in all arms of immunity, including stimulation of interferon production.
Likopid. Manufacturer: “Peptek”, Moscow, Russia
Likopid or N-acetyl glucosaminyl-1-4-N-acetylmuramyl-L-alanine-D-isoglutamine dipeptide, is a synthetic analogue of the fragment of cell walls of bacteria. It stimulates the functional activity of macrophages and synthesis of cytoki-nes. It is clinically used in adjuvant therapy for chronic immunodeficiency conditions, low current and recurring inflammatory infectious diseases at various sites [102]. Due to broad spectrum activity Likopid is also used for treatment of cytomegalovirus infection and pulmonary tuberculosis [103].
Galavit. Manufacturer “Medikor” Moscow, Russia
Galavit is a monosodium a-luminol or monosodium 5-amino-2-3-dihydro-1-4-phthalazine dione. Galavit inhibits production of inflammatory cytokines such as TNF-alpha, IL-1 through regulation of metabolic activity of macrophages. As such it has been found useful for various clinical indications as follows: gastrointestinal infections of various origins; viral hepatitis; herpes infections; urogenital infections, i.e., chlamidia, endometriosis and other bacterial and fungal infections [104].
V5 Immunitor. Manufacturer: “Monserum”, Mongolia
This product is made from hydrolyzed pooled blood of hepatitis B and C carriers by using unique technology. The hepatitis viruses are killed by heat- and chemical inactivation and then formulated into a tablet. The principle for production of V5 is not much different from established principles with old-fashioned killed vaccines, i.e., Hepatitis B vaccine made from pooled plasma. V-5 is available as 850 mg coated pill, ten of which are sealed in a “blister” pallet, with 30 pills per one package. The recommended dose is one-two pills per day. The preparation is stable at ambient temperature for five years. Studies in chronic hepatitis B and C patients have shown nearly 100% efficacy, without any adverse effects, and with positive outcome achievable within one month from treatment initiation [105,106].
Ligfol (Olipifat). Manufacturer: “Ligpharm”, Moscow, Russia
Ligfol is obtained as a result of hydrolysis of wood lignin that is reduced to a sterile liquid for injection and has been in veterinary use since 2000. This preparation is quite unusual, bearing in mind its origin and broadness of clinical applications. It has been found useful in the management of stress; as an antioxidant; anti-tumor agent; enhancer of healing; hepatoprotector; hematopoiesis stimulant; inducer of cellmediated immunity and interferon synthesis. These properties may appear unrelated to each other but there are published clinical studies that lend support to these claims [107109].
Anandin. Manufacturer: “Meditere”, St. Petersbourg, Russia
Anandin is an injectable and topical preparation of modified sugar, glucosamine-propyl-carbocridone, developed by Travkin and Yakovleva in 1990-1995. It has been used in Russia over the last ten years in humans but predominantly in the veterinary practice without any significant toxicity. Main indications are for acute and chronic viral and bacterial infections; inflammatory conditions; as an enhancer of healing process; and for a variety of immune disorders. In animals it is commonly prescribed for parvovirus enteric infections, pestiviruses, bovine herpes, infectious bovine rhiono-tracheitis (IBR), bovine viral diarrhea (BVD), hepatitis, and many other viral infections of unknown etiology [110,111].
Imunofan. Manufacturer: “Bionox”, Moscow, Russia
This immunomodulator consists of a short synthetic pep-tide, (Arg-a-Asp-Lys-Val-Tyr-Arg), which imitates the action of thymopoietin. It is provided as a rectal suppository, injectable solution or intranasal spray. According to Russian studies the pharmacological effect is due to three main modes of action: correction of immune response; restoration of antiodixadant/peroxidation processes; and inhibition of multidrug resistance through interaction with transmembrane pumps responsible for drug resistance. Imunofan is prescribed for a wide range of clinical conditions including as adjunct for cancer therapy; acute and chronic pyogenic infections; opportunistic infections such as Cytomegalovirus; Toxoplasma gondii; Klebsiella pneumonia; Herpes virus; Chlamydiae and Cryptococcus neoformans; HIV; acute and chronic viral hepatitis; diphtheria; as adjuvant for vaccination; and psoriasis. Although it is unlikely, Imunofan may cause inflammatory reactions in certain individuals [112114].
Thymogen. Manufacturer: “Cytomed”, Russia
This preparation is perhaps best know interferon inducing immunomodulator. It was originally discovered by Khavin-son et al., and has been sold in Russia since 1991 [115]. It is very simple dipeptide (L-Glu-L-Trp) that is orally available and has been used for innumerable clinical conditions ranging from cancer to infectious diseases and other unrelated uses especially in the neurological or neuroendocrine context. The number of references on PubMed alone is by an order of magnitude higher than for any other of above reviewed substances. Thymogen is fully synthetic but since it has been discovered by screening other preparations of thymic extract, Thymalin and Vilon, it appears to affect various immune responses by mimicking the function of the thymus.
CONCLUSIONS
There are several dozen clinically deployed immunomodulators in Russia and former Soviet block countries. Most popular ones are listed in this review. They have been used with various success rates in a large number of patients, but are practically unknown in the English-language medical literature. We hope that this review provides a glimpse into current situation and perhaps will stimulate further research in this exciting area.
Table 1. Summarized Data on Immunomodulators as Compiled from Available Literature Sources, Authors Own Clinical Observations, and Personal Communications
Category of Preparations Commercial Name Clinical Indications
Synthetic, low-molecular
Anandin

Amyxin

Arbidol

Amizon

Cycloferon

Hepon

Galavit

Groprinosine

Imunofan

Likopid

Maxidin

Neovir

Thymogen

Rabies, hepatitis A, B, C virus, TB, herpes simplex virus type 1 and 2, HIV, influenza, acute and chronic respiratory viral infections, adenovirus type 6, mumps, canine distemper, parvovirus, panleukopenia, viral hemorrhagic fever, West Nile fever, vesicular stomatitis virus, Venezuelan equine encephalitis virus, encephalo-myocarditis virus, chronic enteritis, surgical infections, keratoconjunctivitis, rhinitis, secondary immunodeficiencies, malignant diseases.
Synthetic, high-molecular Poludan Ophathalmoherpes, influenza, acute and chronic respiratory viral infections, viral hepatitis B, rabies, HIV.
Natural, low-molecular
Kagocel

Ligfol Mebavi

Ragocin

Savratz

Herpes simplex, influenza, acute and chronic respiratory viral infections, hepatitis C, rabies, enteroviruses
Natural, high-molecular
Ridostin Larifan

Prodigiosan

Influenza, acute and chronic respiratory viral infections, Aujeszky’s disease, foot-and-mouth disease, canine distemper, rabies, Omsk haemorrhagic fever, herpes
virus
Natural, complex
Blasten

Dzherelo(Immunoxel) Milife

Rusam

V5 Immunitor MIGI-K

Influenza, TB, acute and chronic respiratory viral infections, oncological and autoimmune diseases, malignancies, viral hepatitis, purulent wounds

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