The effect of 677C>T and 1298A>C MTHFR polymorphisms on sulfasalazine treatment outcome in rheumatoid arthritis.

Despite the availability of several new agents for the treatment of rheumatoid arthritis (RA), sulfasalazine remains the mainstay because of both cost and experience with its use. Methylenetetrahydrofolate reductase (MTHFR) is involved in folate metabolism and several polymorphisms have been described in the MTHFR gene. Of these, the 677C>T and 1298A>C polymorphisms have been associated with altered enzyme activity. To examine the association between 677C>T and 1298A>C MTHFR polymorphisms and sulfasalazine efficacy for the treatment of RA, a total of 117 RA patients treated with sulfasalazine (1 g daily; duration of treatment 17 +/- 5 months) were analyzed. The 677C>T and 1298 A>C polymorphisms were detected using a PCR-RFLP method. RA was diagnosed according to the criteria of the American College of Rheumatology (ACR). The remission of RA symptoms was evaluated according to the ACR 20% response criteria. Allele and genotype frequencies were compared by the two-sided Fisher exact test. The frequency of remission was 47.2% and 44.6% in carriers of 677T and 1298C alleles, compared to 40.7% and 42.0% in carriers of 677C and 1298A alleles, respectively. These differences were statistically non-significant. When the multivariate analysis was additionally adjusted for patients' age, gender and RA duration, the association of the MTHFR 677T allele with increased frequency of remission was statistically significant. Although RA remission rate in carriers of the MTHFR 677T and 1298C alleles was more frequently observed, it does not seem that 677C>T and 1298A>C MTHFR polymorphisms have a major influence on treatment outcome in RA patients treated with sulfasalazine.

The effect of 677C>T and 1298A>C MTHFR polymorphisms on sulfasalazine treatment outcome in rheumatoid arthritis

Despite the availability of several new agents for the treatment of rheumatoid arthritis (RA), sulfasalazine remains the mainstay because of both cost and experience with its use. Methylenetetrahydrofolate reductase (MTHFR) is involved in folate metabolism and several polymorphisms have been described in the MTHFR gene. Of these, the 677C>T and 1298A>C polymorphisms have been associated with altered enzyme activity. To examine the association between 677C>T and 1298A>C MTHFR polymorphisms and sulfasalazine efficacy for the treatment of RA, a total of 117 RA patients treated with sulfasalazine (1 g daily; duration of treatment 17 ± 5 months) were analyzed. The 677C>T and 1298 A>C polymorphisms were detected using a PCR-RFLP method. RA was diagnosed according to the criteria of the American College of Rheumatology (ACR). The remission of RA symptoms was evaluated according to the ACR 20% response criteria. Allele and genotype frequencies were compared by the two-sided Fisher exact test. The frequency of remission was 47.2% and 44.6% in carriers of 677T and 1298C alleles, compared to 40.7% and 42.0% in carriers of 677C and 1298A alleles, respectively. These differences were statistically non-significant. When the multivariate analysis was additionally adjusted for patients’ age, gender and RA duration, the association of the MTHFR 677T allele with increased frequency of remission was statistically significant. Although RA remission rate in carriers of the MTHFR 677T and 1298C alleles was more frequently observed, it does not seem that 677C>T and 1298A>C MTHFR polymorphisms have a major influence on treatment outcome in RA patients treated with sulfasalazine.

Interleukin-18 promoter polymorphism in patients with atopic asthma.

Allergic asthma is a chronic inflammatory disease in which interleukin-18 (IL-18) plays an important role. However, there are controversial reports on IL-18 promoter polymorphism as an independent marker of asthma susceptibility. The aim of the present study was to examine the IL-18 promoter polymorphism in patients with allergic asthma. Two hundred and thirty-one patients with allergic asthma from a Polish population diagnosed according to the National Heart, Lung, and Blood Institute (NHLBI)/WHO guidelines were examined. An allele-specific polymerase chain reaction was used to analyse polymorphisms at positions -137 and -607 in the promoter region of the IL-18 gene. Neither in the -607 C>A nor in the -137 G>C promoter polymorphism were there any differences observed between the total group of asthmatic patients and the controls in the frequencies of genotypes, alleles, diplotypes or haplotypes. In patients with severe asthma, the -607 CC and -137 GG genotypes were observed significantly more frequently (P = 0.03 for both), whereas in patients with mild and moderate asthma, the -137 CC genotype was more prevalent than in the former group. The strongest difference between mild to moderate and severe asthma was observed in -137 allele frequencies (P = 0.006). The results of the present study suggest that the -137 G allele and the C-G/C-G diplotype seem to be involved in the pathogenesis of the severe form of asthma.

Polymorphisms of catechol-0-methyltransferase (COMT), monoamine oxidase B (MAOB), N-acetyltransferase 2 (NAT2) and cytochrome P450 2D6 (CYP2D6) gene in patients with early onset of Parkinson's disease.

The aim of the present study was to evaluate the contribution of MAOB, COMT, NAT2 and CYP2D6 gene polymorphisms to early onset Parkinson's disease (PD). The study enrolled 134 patients with Parkinson's disease (early onset-EOPD--67 patients, and late onset--LOPD--patients), and 66 healthy individuals. Polymerane chain reaction restriction fragment length polymorphism (PCR-RFLP) methods were used for genotyping. Univariate analysis revealed a significant two-fold higher EOPD risk among carriers of MAOB allele A or AA genotype. Multivariate analysis revealed that MAOB allele A was an independent factor predisposing to EOPD. It was shown that neither NAT2, CYP2D6 nor COMT genotype was associated with PD.

Interleukin-18 promoter polymorphism in patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease in which interleukin (IL)-18 plays an important role. However, there are controversial reports on IL-18 promoter polymorphism as an independent marker of RA susceptibility. The aim of present study was to examine the IL-18 promoter polymorphism in patients with RA, and its association with disease susceptibility, activity and severity. We examined 309 patients with RA from a Polish population diagnosed according to the criteria of American College of Rheumatology. An allele-specific polymerase chain reaction was used for analysis of the polymorphisms in positions - 137 and - 607 in promoter region of IL-18 gene. A significantly decreased number of subjects with AC/AC and AG/AG diplotypes was observed among RA patients as compared with healthy controls (OR - 0.51, 95%CI 0.28-0.95, P = 0.045) and (OR - 0.12, 95% CI 0.02-0.97, P = 0.042), respectively. Nevertheless, there was no significant association with disease activity, joint erosions, extra-articular manifestations, rheumatoid factor. Above results suggest that IL-18-137 and - 607 promoter polymorphisms are not the significant factors influencing RA course and severity in a Polish population.

Association of cytokine gene polymorphisms and the release of cytokines from peripheral blood mononuclear cells treated with methotrexate and dexamethasone.

Immunosuppressive drugs are widely used in the therapy of autoimmune disorders to suppress autoreactive T cells. The immune system is regulated by the release of cytokines. Cytokine are potent immunomodulatory molecules that act as mediators of inflammation and the immune response. Primarily secreted by T cell and macrophages, they influence cellular activation, differentiation, and function. Cytokine production is under genetic control. This is evidenced by the identification of polymorphism in cytokine gene regulatory regions that correlate with intra-individual variations in actual cytokine production. The aim of the study was to examine whether the individual differences in the polymorphic cytokine genes can lead to individual variation in release of cytokines after treatment with methotrexate and glucocorticosteroids. The study was carried out on mononuclear cells isolated from peripheral blood of 72 healthy subjects. The cells were activated with PHA and incubated with increasing concentrations of methotrexate (0.1-10 microM) and dexamethasone (0.01-1 microM). Levels of IL-2, IL-4, IL-6, IL-10, and TNFalpha in the culture supernatants were quantified by flow-cytometry using Th1/Th2 kit and correlated with cytokine gene polymorphisms. The increased concentrations of DEX resulted in comparable cytokine concentrations in cultures from subjects with low and high cytokine genotypes. Despite MTX treatment, the cytokine levels were significantly increased in individuals homozygous for the high producer allele. These results suggest that the cytokine gene variants may influence the efficacy of therapy with some immunosuppressive and anti-inflammatory drugs.

Involvement of P-glycoprotein in the release of cytokines from peripheral blood mononuclear cells treated with methotrexate and dexamethasone.

P-glycoprotein (P-gp), a product of the MDR1 gene, is an important factor in the turnover of many drugs and xenobiotics. Recent reports have suggested that P-gp can also be involved in the transport of cytokines. The aim of this study was to examine the role of P-gp in cytokine release from phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (MNCs) as well as in the release of cytokines from MNCs treated with methotrexate (MTX) and dexamethasone (DEX). The study was carried out on PHA-stimulated MNC from 10 healthy subjects. Flow cytometry was applied to measure interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha levels in the culture supernatants. In the experiments verapamil (VER) and P-gp specific monoclonal antibodies (mAb) (clone 17F9) were used to inhibit P-gp function. P-gp inhibitors suppressed the release of IL-2, IL-4, IFN-gamma and TNF-alpha from PHA-stimulated MNC, whereas release of IL-6 and IL-10 remained unaffected. VER and mAb significantly decreased the release of IL-2, IL-4, TNF-alpha and INF-gamma in MNC cultures treated with MTX or DEX. The results of this study suggest that P-gp may be involved in the transmembrane transport of some cytokines. Moreover, it seems that blocking of P-gp function may influence the release of some cytokines from MNCs, displaying an additive inhibitory effect to DEX and MTX.

IL-6 promoter polymorphism in patients with rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is a chronic inflammatory disease in which cytokines play an important role. The aim of the present study was to examine the interleukin-6 (IL-6) -174 promoter polymorphism in patients with RA and its association with disease susceptibility and activity. METHODS: The study included 98 patients with RA diagnosed according to the criteria of the American College of Rheumatology. Polymerase chain reaction (PCR) amplification was used for analysis of the polymorphism at position -174 in the promoter of the IL-6 gene. RESULTS: The distribution of IL-6 genotypes in RA patients did not differ from that in control subjects. Nevertheless, in patients with a GG genotype the active form of RA was more frequently diagnosed compared with homozygous CC and GC patients. Moreover, in carriers of two G alleles the parameters of disease activity score (DAS28), erythrocyte sedimentation rate (ESR), number of swollen and tender joints] were significantly increased. CONCLUSION: We suggest that the IL-6 promoter polymorphism may be a genetic risk factor for RA activity.

IL-2 and TNF-alpha promoter polymorphisms in patients with acute kidney graft rejection.

INTRODUCTION: Proinflammatory cytokines have been implicated in the pathogenesis of acute kidney allograft rejection. The aim of the study was to examine the association between interleukin (IL)-2 -330 and tumor necrosis factor (TNF)-alpha -308 promoter polymorphisms and acute kidney allograft rejection. METHODS: The study included 72 patients with long-term stable graft function, and 57 diagnosed with acute kidney allograft rejection. RESULTS: Patients with acute kidney allograft rejection showed a prevalence of subjects with TNF-alpha T2 allele (P < .05). The risk of acute kidney allograft rejection diagnosis was 2.5-fold greater among carriers of the T2 allele than those homozygous for T1T1 (OR 2.53, 95% CI 1.19 to 5.37, P < .05) There was no statistically significant difference in the distribution of IL-2 genotypes between patients with stable graft function and acute kidney allograft rejection. CONCLUSION: The results suggest that TNF-alpha-308 promoter polymorphism is a risk factor for acute kidney allograft rejection.

IL1beta+3953 exon 5 and IL-2 -330 promoter polymorphisms in patients with rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is chronic inflammatory disease in which cytokines play an important role. The aim of present study was to evaluate the exon 5 +3953 IL-1beta and IL-2 -330 promoter polymorphisms in patients with RA in association with disease activity and severity. METHODS: In the study 93 patients with rheumatoid arthritis diagnosed according to the criteria of American College of Rheumatology were included. Polymerase chain reaction amplification was used for analysis of the polymorphisms studied. RESULTS: The distribution of IL-1beta and IL-2 genotypes in RA patients did not differ from control subjects. Nevertheless in patients with A2 allele of IL-1beta and GG genotype of IL-2, the active form of RA was more frequently diagnosed. Moreover in these patients the measurements of disease activity (DAS 28 score, ESR, number of swollen and tender joints) were significantly increased. CONCLUSION: We suggest that exon 5 +3953 IL1beta and IL-2 -330 promoter polymorphisms may be a genetic risk factor for RA severity.

The cytokine gene polymorphisms in patients with chronic kidney graft rejection.

Chronic allograft rejection remains an important cause of morbidity after kidney transplantation. The aim of the study was to examine the association between IL-2, IL-6 and TNF-alpha promoter polymorphisms and chronic kidney allograft rejection. The study included 64 patients with long-term stable graft function and 62 with chronic allograft nephropathy. Among patients with chronic allograft nephropathy a statistically significant prevalence of the IL-6 CC genotype associated with low IL-6 expression was observed (p < 0.01, OR 3.18; 95% CI 1.27-8.15). There were no statistically significant differences in distribution of IL-2 and TNF-alpha genotypes between patients with stable graft function and chronic allograft rejection. The results of present study suggest that the genetically determined low IL-6 production may be the risk factor of chronic allograft nephropathy development.

Involvement of P-gp in the process of apoptosis in peripheral blood mononuclear cells.

Multidrug resistance mediated by the drug-efflux protein P (P-gp) is one of mechanisms that cells use to escape death induced by drugs and other agents. The aim of the study was to evaluate the effect of P-gp inhibition on apoptosis of PHA-activated peripheral blood mononuclear cells (MNC) as well as apoptosis induced by methotrexate (MTX), dexamethasone (DEX), methylprednisolone (MP) and cortisone (COR). Apoptosis was quantified by flow cytometry using Annexin V/PI and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL). P-gp expression was inhibited using verapamil (VER) and P-gp specific monoclonal antibodies (mAb). VER and mAb enhanced the apoptosis of PHA-activated MNC. Moreover these agents significantly increased the apoptosis induced by MTX, DEX, MP and COR. The results of this study suggest that P-gp is involved in the process of apoptosis in peripheral blood mononuclear cells.

Lack of association between arylamine N-acetyltransferase 2 (NAT2) polymorphism and systemic sclerosis.

OBJECTIVES: It has been shown that exposure to some environmental toxins may induce scleroderma-like illness in predisposed individuals, but the etiopathogenesis of the idiopathic form of systemic sclerosis (SSc) remains obscure. The genetic background of this illness has been confirmed in multiple studies. We investigated whether patients with SSc differ from healthy subjects with regard to the enzymatic activity of polymorphic N-acetyltransferase 2 (NAT2). METHODS: The study was carried out in 39 patients with SSc; 15 fulfilled the criteria of diffuse SSc (dSSc) and 24 of limited SSc (lSSc); an ethnically matched control group consisted of 100 healthy volunteers. Acetylation phenotype was estimated using the isoniazid as a model drug. The most common mutations in the Caucasian population at positions 481T, 803G, 590A and 857A on the NAT2 gene were determined using the polymerase chain reaction-restriction fragment length polymorphism method with deoxyribonucleic acid (DNA) extracted from peripheral blood. RESULTS: In the group of patients with SSc, the frequency of fast acetylator genotypes was 38.5% (95% CI 23.4-55.4), while that for the genotypes coding slow acetylator status was 51.3% (95% CI 34.8-67.6). There was a strong correlation between NAT2 phenotype and NAT2 genotype with a concordance of 97%. We did not observe a preponderance of slow acetylators among patients with SSc and in two subsets of SSc. With the sample size analyzed in the present study, there is a 90% probability of detecting significant differences in distribution of slow, fast, and intermediate phenotypes between patients with SSc and controls, there is a difference of at least 30.3, 28.7 and 21.9% in the distribution of these phenotypes in the general population, respectively. CONCLUSION: Acetylator status does not seem to be the significant factor in the development of SSc in patients with both subsets of this autoimmune disease, but further studies are required to confirm this conclusion.

The MDR1 3435 polymorphism in patients with rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is a multifactorial disease, the pathogenesis of which involves immunological, genetic and environmental factors. P-glycoprotein (P-gp) encoded by the MDR1 gene, is an important transporter for many drugs, xenobiotics and cytokines and may be associated with many immunological processes and apoptosis. The activity of P-gp is genetically determined. Naturally occurring MDR1 polymorphisms have been described and correlated with potential clinical effects. Several mutations in the MDR1 gene have been recognized, but only some of them are associated with P-gp expression. The C3435T polymorphism was found to correlate with the activity of P-glycoprotein. The aim of the study was to evaluate the C3435T MDR1 polymorphism in patients with rheumatoid arthritis and to investigate a possible correlation with disease susceptibility, activity and severity. METHODS: The study was carried out in 92 patients with rheumatoid arthritis and 97 healthy subjects as a control group. The C3435T polymorphism was determined using the PCR-RFLP method. RESULTS: The distribution of C3435TT MDR1 genotypes in RA patients did not differ significantly from that in a control group and was as follows: 3435CC in 25 (26.9%) subjects, 3435CT in 50 (53.8%) and 3435TT in 17 (18.3%). The probability of remission of RA symptoms after therapy with methotrexate and glucocorticosteroids however, was 2.89-fold greater in patients with the 3435TT genotype compared to patients with the genotypes 3435CC and 3435CT. The risk of having an active form of rheumatoid arthritis resistant to therapy with disease-modifying antirheumatic drugs in patients with 3435CC and 3435CT genotypes was 2.89 times greater than in homozygous 3435TT subjects. CONCLUSION: We suggest that the C3435T MDR1 polymorphism is not an important genetic risk factor for RA susceptibility, but that this polymorphism may have an influence on the activity of the disease and its response to therapy with disease-modifying antirheumatic drugs.

The effect of monoamine oxidase B (MAOB) and catechol-O-methyltransferase (COMT) polymorphisms on levodopa therapy in patients with sporadic Parkinson's disease.

OBJECTIVES: The etiology of sporadic idiopathic Parkinson's disease (PD) is considered multifactorial with both genetic and environmental factors modifying the disease expression. Recent studies suggest that polymorphism in monoamine oxidase B (MAOB) and catechol-O-methyltransferase (COMT) might influence the risk and treatment of PD. The aim of the study was to evaluate the effect of MAOB and COMT genetic polymorphism on effective daily dose of levodopa applied during the first 5 years of treatment, and to find out if a relationship exists between MAOB and COMT haplotypes and motor disturbances onset in PD patients treated with levodopa preparations. MATERIALS AND METHODS: A total of 95 patients (40 females and 55 males) of Polish origin diagnosed with sporadic PD were enrolled into the study, and were divided into two groups. Group 1 - patients treated with doses of levodopa below 500 mg/day during the first 5 years of treatment. Group 2 - patients requiring levodopa doses exceeding 500 mg/24 h during the first 5 years of treatment. Low activity alleles of MAOB and COMT, i.e. MAOB allele A and COMT(L) as well as high activity ones, i.e. MAOB allele G and COMT(H), were determined using PCR-RFLP method. RESULTS: No statistically significant differences were found in MAOB and COMT allele distribution in the two groups. However, the frequency of COMT(L/L) homozygotes was higher in the group treated with low doses of levodopa when compared with the second group. MAOB and COMT AG-HH haplotype predominated in the group of females treated with high daily doses of levodopa when compared with AG-LL haplotype in the group of females treated with low daily doses of levodopa (<500 mg/24 h). CONCLUSION: The results of the study suggest that patients with COMT(L/L) genotype and possibly MAOB genotype A may benefit from more efficient and safer levodopa therapy.

The FcgammaRIIa polymorphism in patients with chronic kidney graft rejection.

The FcgammaRIIa receptors, which provide a crucial link between cellular and humoral components of the immune response, display allelic polymorphism. Individuals are homozygous for either arginine 131 (RR131) or histidine 131 (HH131) or are heterozygous for these two alleles (RH131). The HH131 genotype binds human IgG2 with high RR131 with low, and RH131 with intermediate affinity. The aim of the study was to evaluate the FcgammaRIIa polymorphism in patients with chronic kidney graft rejection. The study included 121 renal transplant recipients: 53 patients with long-term stable graft function and 68 with chronic allograft rejection. The distribution of FcgammaRIIa genotypes in patients with chronic kidney graft rejection did not differ significantly from that in patients with stable graft function. The results suggest that the FcgammaRIIa polymorphism is not an important genetic risk factor for chronic rejection of kidney allografts.

The influence of N-acetyltransferase 2 polymorphism on rheumatoid arthritis activity.

OBJECTIVE: The N-acetyltransferase polymorphism is involved in the metabolism of many xenobiotics, as well as in susceptibility to some diseases such as rheumatoid arthritis (RA). The aim of this study was to investigate the influence of NAT 2 polymorphism on disease activity in RA patients. METHODS: 70 with RA were enrolled in the study. As a measure of disease activity, the number of swollen and tender joints, the duration of morning stiffness, ESR and CRP as well as disease activity based on a global physician's assessment were evaluated. The NAT2 polymorphism was determined by a polymerase chain reaction-restriction fragment length polymorphism assay (PCR-RFLP). RESULTS: The mean number of swollen and tender joints, as well as the ESR and CRP values, did not differ significantly with the acetylation genotype. Erosive RA was diagnosed in 74.5% of the slow and 40% of the fast acetylators. The risk for the development of erosive RA was 4.39 time greater in slow acetylators than in fast acetylators. CONCLUSION: NAT2 polymorphism may be a genetic risk factor for joint destruction.

The FcgammaRIIa polymorphism in children with atopic diseases.

BACKGROUND: Fcgamma receptor II (FcgammaRIIa) is the most widely distributed of the classes of FcR and is expressed in polymorphic forms on most types of hematopoietic cells. Recent data suggest that this polymorphism may be relevant to FcgammaRIIa function. This might be linked to variability in immune response and therefore related to the pathogenesis of atopic diseases. The aim of the study was to evaluate the FcgammaRIIa polymorphism in children with atopic diseases. METHODS: In the study were included 140 atopic children, 77 with food allergy and 126 healthy subjects as the control group. The FcgammaRIIa polymorphism was determined using the polymerase chain reaction method. RESULTS: The distribution of FcgammaRIIa genotypes in atopic children did not differ from that of healthy controls. Moreover, there was no association between the FcgammaRIIa genotypes and atopic diseases. CONCLUSION: It seems that the FcgammaRIIa polymorphism does not represent an important genetic risk factor for atopic diseases susceptibility.

The expansion of CD4+CD28- T cells in patients with chronic kidney graft rejection.

CD4+CD28- T cells are oligoclonal lymphocytes rarely found in healthy subjects, but are present in high frequencies in patients with inflammatory diseases. Contrary to paradigm, they are functionally active and produce interferon gamma and cytolytic proteins, are cytotoxic in vessels and may contribute to tissue damage. The size of the peripheral blood CD4+CD28- T cell compartments was determined in 20 healthy individuals, 20 patients after renal transplantation with stable graft function, and 20 with chronic graft rejection by two-color FACS analysis. In patients with stable graft function, the median frequency of CD4+CD28- T cells was 3.1% and was significantly higher in comparison to the control group (1.4%) (P <.01). The highest subset CD4+CD28- cells was detected in patients with chronic graft rejection (10.65%). The amount of CD4+CD28- cells was significantly higher in this group in comparison to patients with stable graft function (P <.01). The evaluated number of CD4+CD28- cells in patients after renal transplantation, especially in graft recipients with chronic graft rejection, suggests a role of these cells in chronic graft destruction.

Evaluation of the efficacy of a combined formulation (Grippostad-C) in the therapy of symptoms of common cold: a randomized, double-blind, multicenter trial.

BACKGROUND: The aim of the present trial was to evaluate the efficacy of a combined product in the treatment of common cold and to examine the contribution of the separate components. In the published literature there is conflicting data on the efficacy of agents used in the treatment of common cold, especially when given in drug combinations. METHODS: A prospective, randomized, double-blind, multicenter, 4-arm, controlled trial was carried out in 1,167 patients with common cold treated with one of the following medications: Grippostad-C, a combination of acetaminophen, caffeine, chlorpheniramine and ascorbic acid (verum), ascorbic acid (control), chlorpheniramine and ascorbic acid (reference 1), as well as acetaminophen, caffeine, and ascorbic acid (reference 2). A score of common cold symptoms (headache, throat pain, extremities and joint pain, cough, blocked nose, and disturbances of sleep quality) was the primary outcome. The test drug was first compared with the control using a hierarchic test strategy, then with reference 1, followed by reference 2 with the aim of proving superiority. FINDINGS: A clinically relevant and statistically significant difference was demonstrated at each level of the hierarchy. Grippostad-C was significantly superior to all other treatment groups, the combination of acetaminophen, caffeine, and ascorbic acid was significantly superior to the control, and the combination of chlorpheniramine and ascorbic acid was not statistically different from the control. INTERPRETATION: The efficacy of Grippostad-C for the treatment of common cold was proven. The findings demonstrate that the combination is superior to each of its separate components and each of the components has its own distinctive contribution to the efficacy of the combination product.