M. Perwaiz Iqbal

The effects of non-steroidal anti-inflammatory drugs on the disposition of methotrexate in patients with rheumatoid arthritis

We have studied the pharmacokinetics of methotrexate in patients with rheumatoid arthritis concurrently taking the most commonly used non‐steroidal anti‐inflammatory drugs (NSAIDs), aspirin, diclofenac, naproxen, indomethacin, and ibuprofen. The area under the curve, the total systemic clearance, the distribution volume, and the half‐life of methotrexate in patients receiving concurrent NSAID therapy did not change significantly (at p <0.05). Concurrent treatment with NSAIDs resulted in increased inter‐patient variability of methotrexate concentration, possibly as a result of biochemical interactions; however, it does not appear clinically relevant. The data suggest that the NSAIDs do not significantly affect the disposition of methotrexate, contrary to some of the earlier reports.

Interactions and associations of paraoxonase gene cluster polymorphisms with myocardial infarction in a Pakistani population.

Polymorphisms of paraoxonase gene (PON) cluster have been investigated in numerous studies for their association with myocardial infarction (MI) but the results have been conflicting. Epistasis and gene–environment interactions at this locus could possibly modulate susceptibility toward MI and account for the discrepancies. We carried out a case–control study (211 MI patients and 370 control subjects) to test association of PON cluster polymorphisms with MI, their interactions with each other and with smoking. Genotyping was performed by PCR–restriction fragment length polymorphism based assays. The Q192R, C‐108T, and A148G polymorphisms were associated with MI. Two haplotypes consisting of C‐108T, C311S, and A148G, having allele frequencies of 0.17 and 0.14 in the control population, predisposed to MI (global haplotype statistic χ2 = 34.74, df = 15, p = 0.0027). Multifactor dimensionality reduction analysis showed a significant three‐locus model (p = 0.02) involving these three polymorphisms, suggesting a potential gene–gene interaction between PON1 and PON2. These polymorphisms also interacted with smoking, in a three‐locus and a four‐locus model (p = 0.01 and p = 0.05, respectively). Additionally, the R192 allele may advance the age‐at‐onset of MI. The PON cluster appears to be a susceptibility locus for MI in Pakistani population, and the susceptibility is modulated through gene–gene and gene–environment interactions.

Urinary N-acetyl-beta-D-glucosaminidase in rheumatoid arthritis

Excretion of urinary N-acetyl β-D-glucosaminidase (NAG) and its isoenzyme patterns were studied in two groups of patients with rheumatoid arthritis (RA) and in normal control subjects. Urine samples were collected from 30 seropositive RA patients, 19 seronegative RA patients, and 15 normal healthy subjects. All the patients and normal subjects were assessed to have normal liver and kidney functions. A small portion of the urine sample was dialyzed against 0.01 M phosphate buffer, pH 7.0 and NAG activity was monitored. Mean ± SD values of urinary NAG in seropositive RA patients, in seronegative RA patients and in normal healthy subjects were found to be 4.20 ± 3.73 U/g creatinine, 2.96 ± 2.11 U/gm creatinine, and 1.71 ± 0.6 U/g creatinine, respectively. The mean urinary, NAG value in RA patients was found to be significantly higher (P < 0.05) in seropositive RA compared to the mean NAG value in normal healthy subjects and patients with seronegative RA when analyzed by one way ANOVA and Tukey-HSD test. The mean proportion of isoenzyme form B to isoenzyme form A in seropositive RA patients was also found to be significantly different (P < 0.05) from the mean proportion of these forms in normal healthy subjects and seronegative RA patients. There also appears to be a correlation between the concentration of urinary NAG and severity of the disease in seropositive RA.

Lack of association of methylenetetrahydrofolate reductase 677C>T mutation with coronary artery disease in a Pakistani population

Pakistanis belong to the South Asian population which has the highest known rate of coronary artery disease. Folic acid deficiency also appears to be highly prevalent in this population. Methylenetetrahydrofolate reductase (MTHFR) 677C>T polymorphism decreases the activity of this enzyme and can be associated with mild to moderate hyperhomocysteinemia in homozygotes, particularly when there is folic acid deficiency, as well as with coronary artery disease. To assess the value of genotyping the MTHFR 677C>T dimorphism, we carried out a case-control study of dimorphism 677C>T for putative association with myocardial infarction (MI) among Pakistani nationals. We investigated a sample population of 622 Pakistanis consisting of 225 controls and 397 patients with clinical diagnosis of acute MI (AMI). MTHFR C677T alleles were determined by assays based on polymerase chain reaction and restriction endonuclease analysis. Frequencies of C alleles were 0.87 among controls and 0.86 among AMI patients. The MTHFR 677C>T dimorphism showed no association with MI (χ2 = 0.25, 1df, P=0.62), serum levels of folate and vitamin B12 and plasma level of vitamin B6. A significant association, however, was found between homozygous 677T genotype and plasma levels of homocysteine. Multivariate analysis of the data showed that in case of log homocysteine, age and MTHFR genotypes were significantly different (P<0.001). In case of B12, smoking and age were found to be statistically significant (P<0.001), while in case of serum folate only smoking was found to be significant (P<0.001). The results indicate that MTHFR 677C>T polymorphism, though associated with homocysteine levels, confers no significant risk of coronary artery disease in the Pakistani population investigated here. We suggest that the higher incidence of AMI in South Asia occurs through mechanisms other than the MTHFR related pathways.

Association study of the angiotensin-converting enzyme (ACE) gene G2350A dimorphism with myocardial infarction

The angiotensin converting enzyme (ACE) is a strong candidate gene for myocardial infarction (MI). Insertion-deletion dimorphism in intron 16 of this gene has been inconclusively found to be associated with it. Several new polymorphisms in the ACE gene have been identified and among these, a dimorphism in exon 17, ACE G2350A, has a significant effect on plasma ACE concentrations. To assess the value of genotyping the ACE G2350A dimorphism in a genetically homogeneous population, we carried out a case-control study of dimorphism G2350A for a putative association with MI among Pakistani nationals. We investigated a sample population of 370 Pakistanis, comprising 163 controls, and 207 patients with clinical diagnosis of acute MI (AMI). ACE G2350A alleles were visualized by assays based on polymerase chain reaction and restriction endonuclease analysis. Frequencies of G alleles were 0.68 among controls and 0.72 among AMI patients. The ACE G2350A dimorphism showed no significant association with MI (χ2=0.90, 2 df, P=0.64), plasma levels of homocysteine (P=0.52) or with serum levels of folate (P=0.299). The results indicate that ACE G2350A polymorphism is not associated with risk of myocardial infarction in the Pakistani population investigated here.

N-acetyl-beta-D-glucosaminidase in acute myocardial infarction.

The objective of the study was to investigate whether the lysosomal enzyme, N-Acetyl-β-D-glucosaminidase (NAG) activity is increased in plasma of patients with acute myocardial infarction (AMI) and to determine if there is any association between plasma levels of NAG and severity of myocardial infarction (MI). NAG activity in plasma was monitored in 69 patients with AMI and 135 normal healthy subjects using a spectrofluorimetric method. A modified Aldrich ST elevation score was used to gauge the severity of MI in terms of size of the infarct. Plasma NAG levels in AMI patients and normal healthy subjects were found to be 10.92±7.5 U/l and 6.8±2.2 U/l, respectively. These two mean value when compared by Students t-test were significantly different P = 0.0001. No statistically significant differences in NAG activity were observed in patients in terms of gender, age, location of infarct, time from onset of chest pain to blood sampling in the hospital and size of the infarct.

Additive effect of indomethacin and methotrexate on suppression of growth in rats

The purpose of this study was to investigate the medium‐term effects of methotrexate (MTX) and indomethacin on the growth of young rats. Four equal groups of Sprague–Dawley male rats (four animals in each group; mean±S.D. body weight, 183±13 g, in their rapid growth phase) were subjected to the following drug treatment: one group was given MTX (0.2 mg kg−1 body weight) subcutaneously on every fourth day, another received indomethacin (2.5 mg kg−1 body weight) subcutaneously daily and the third group was given both of these drugs (MTX on every fourth day and indomethacin daily). The fourth group was injected subcutaneously with physiological saline every day to serve as a control group. Total body weight, food and water consumption by animals in each group were monitored every second day for a period of 10 weeks. After this period, liver, spleen and kidneys were excised, weighed and analysed for MTX and dihydrofolate reductase activity. Compared with the groups, which received MTX alone, indomethacin alone, or physiological saline, mean increase (17±11 g) in body weight of rats was minimal in the group receiving both MTX and indomethacin. The difference was statistically significant (p=0.001) when the values of mean increase in body weight of rats in different treatment groups after a 10‐week treatment were compared. The mean weights of liver and spleen in this group receiving both MTX and indomethacin were also found to be significantly less than the weights of these organs in the control group (p<0.01). There also appears to be a decline in food consumption in this group (p<0.05). This negative effect on growth of animals in this group appears to be not only due to decreased food consumption but also due to increased inhibition of de novo pathway of DNA synthesis. This is supported by increased accumulation of MTX and decreased dihydrofolate reductase activity in this group receiving both MTX and indomethacin, as compared with the group receiving MTX alone. The data indicate an additive effect of MTX and indomethacin on the suppression of growth in young rats, alluding to the notion that patients suffering from juvenile rheumatoid arthritis or acute lymphoblastic leukaemia receiving these two drugs concomitantly over a long period of time might be at a risk of experiencing short‐term suppression of growth. Copyright

Increased levels of multiple forms of dihydrofolate reductase in peripheral blood leucocytes of cancer patients receiving haematopoietic colony-stimulating factors: Interim analysis

The precise mechanism whereby granulocytes proliferate when haematopoietic colony stimulating factors (CSFs) are used in neutropenic cancer patients is poorly understood. The purpose of this study was to investigate whether these cytokines bring about leucocyte proliferation by increasing the levels of multiple forms of dihydrofolate reductase (DHFR). Blood samples were collected from 36 cancer patients (25 males and 11 females) with chemotherapy-induced neutropenia. One sample of blood from each patient was obtained before therapy either with CSF, such as granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) or with placebo, and another one at the time of resolution of neutropenia. Peripheral blood leucocytes in these blood samples were counted, separated and lysed. From lysates, cytoplasmic samples were prepared and analyzed for active DHFR by a methotrexate-binding assay and for total immunoreactive DHFR by an enzyme linked immunosorbent assay. The increase in total leucocyte count (TLC) was most prominent (P < 0.005) in the CSF group and less so (P < 0.05) in the placebo group. The mean ± SD concentration values of active DHFR before and after stimulation with GM-CSF found were to be 0.34 ± 0.4 ng/mg protein and 0.99 ± 0.82 ng/mg protein, respectively, and in the group treated with G-CSF, 0.24 ± 0.32 ng/mg protein and 1.18 ± 2.4 ng/mg protein, respectively. This increase in active DHFR after stimulation with CSF was statistically significant (P <0.05). Similarly, concentration values of immunoreactive but nonfunctional form of DHFR (IRE) were 110 ± 97 ng/mg protein and 605 ± 475 ng/mg protein before and after stimulation with GM-CSF, and 115 ± 165 ng/mg protein and 1,054 ± 1,095 ng/ mg protein before and after stimulation with G-CSF. This increase in concentration of IRE after stimulation with GM-CSF or G-CSF was statistically significant (P < 0.005). In the control group, there was an increase in the concentration of both active DHFR and IRE after treatment with placebo. However, this was not statistically significant. Resolution of neutropenia was quicker in the groups treated with CSF compared to the control group. Results of this study indicate that colony stimulating factors (G-CSF and GM-CSF) induce white cell proliferation by increasing the levels of multiple forms of DHFR.

N-acetyl-beta-D-glucosaminidase--a marker of reperfusion and a prognostic indicator in patients with acute myocardial infarction.

Objective — N-acetyl--D-glucosaminidase (NAG) is a lysosomal enzyme of which the activity in plasma is increased in a number of conditions including myocardial infarction. Plasma levels of cardiac proteins, such as myoglobin, troponin and creatine kinase, have been used as markers of myocardial reperfusion as well as for the prognosis of the disease.The aim of this study is to investigate whether NAG could be used as an additional biochemical marker to predict myocardial reperfusion and to find out if its release following thrombolysis has a prognostic value as well. Method — Streptokinase (SK) in a dose of 1.5 million units was administered intravenously to 75 patients with acute myocardial infarction (AMI) and the response to SK was assessed over a period of 90 minutes. Plasma NAG activity was monitored before (0 min) and 90 min after SK treatment. Results — The mean NAG activity values were found to be 8.6 ± 4.8 U/l and 10.95 ± 7.3 U/l, respectively, and when compared using paired samples t-test revealed a significant increase (p = 0.0001) following thrombolytic therapy. The increase appears perfusion related as rabbits injected with SK failed to show any increase in plasma NAG activity.There was an association between plasma NAG activity and clinical response to SK treatment (p = 0.045). A follow-up of 66 patients over a period of 18 months, revealed increased survival in AMI patients having significantly more activity/release of plasma NAG after thrombolytic treatment (p = 0.001). Conclusion — NAG appears to be another potential biochemical marker of reperfusion. Moreover, NAG release profile during thrombolysis could be of value in predicting prognosis of the disease.

Admission troponin T as a prognostic marker and it relationship to streptokinase treatment patients with acute myocardial infarction.

The relationship between the admission troponin T (TnT) level and the response to streptokinase (SK) was examined in 76 patients with acute myocardial infarction (AMI). Of 27 TnT positive patients, 10 (37%) showed a response to SK as suggested by a non-invasive criterion for reperfusion, while 24 (49%) were ‘responders’ among 49 TnT negative patients. There appeared to be a trend towards a better response to SK in the TnT negative group but the difference lacked statistical power due to the small sample size. The mean time-interval between the onset of symptoms and thrombolytic treatment among TnT positive ‘non-responders’ was significantly (P <0.005) higher than the TnT negative ‘non-responders’ (5.23 ±3.42 h versus 2.38 ± 1.37 h). An 18 month follow up on 61 patients revealed a higher mortality (33%) among TnT positive patients than TnT negative patients (10%). Mortality among TnT positive ‘non-responders’ was significantly higher (P=0.0494) than mortality among TnT-negative ‘non-responders’ (43% versus 9%), indicating that TnT positive patients, non-responsive to SK were at a greater risk of cardiac death. The data suggest that the admission TnT level can be of value in risk stratification of patients with AMI.