Development, Validation, and Application of the LC-MS/MS Method for Determination of 4-Acetamidobenzoic Acid in Pharmacokinetic Pilot Studies in Pigs.

Each drug has pharmacokinetics that must be defined for the substance to be used in humans and animals. Currently, one of the basic analytical tools for pharmacokinetics studies is high-performance liquid chromatography coupled with mass spectrometry. For this analytical method to be fully reliable, it must be properly validated. Therefore, the aims of this study were to develop and validate a novel analytical method for 4-acetamidobenzoic acid, a component of the antiviral and immunostimulatory drug Inosine Pranobex, and to apply the method in the first pharmacokinetics study of 4-acetamidobenzoic acid in pigs after oral administration. Inosine Pranobex was administered under farm conditions to pigs via drinking water 2 h after morning feeding at doses of 20, 40, and 80 mg/kg. For sample preparation, we used liquid-liquid extraction with only one step-protein precipitation with 1 mL of acetonitrile. As an internal standard, we used deuterium labeled 4-acetamidobenzoic acid. The results indicate that the described method is replicable, linear (r2 ≥ 0.99), precise (2.11% to 13.81%), accurate (89% to 98.57%), selective, and sensitive (limit of quantitation = 10 ng/mL). As sample preparation requires only one step, the method is simple, effective, cheap, and rapid. The results of the pilot pharmacokinetics study indicate that the compound is quickly eliminated (elimination half-life from 0.85 to 1.42 h) and rapidly absorbed (absorption half-life from 0.36 to 2.57 h), and that its absorption increases exponentially as the dose is increased.

The Challenging Anticoagulant Therapy in COVID19 Patient with Associated Coagulopathy.

COVID-19 became a widespread infectious disease in late 2019. Indonesia currently has the highest COVID-19 mortality rate in Asia, between 4-5 percent. Interestingly, COVID-19-associated coagulopathy characterized by an increase of several procoagulant factor levels, including fibrinogen and D-dimer, that has been associated with higher mortality and unfavorable outcomes. We report a case of a 30-year-old male admitted to the hospital with a profuse vomiting and worsening fever, cough and shortness of breath, and was diagnosed with COVID-19-associated coagulopathy. Seven days after admission, he became deteriorated with significant reduction of oxygen saturation and his coagulation parameter levels were increased with highly suspicion of pulmonary embolism. He was treated with azithromycin, isoprinosine, lopinavir, and fondaparinux with thromboprophylaxis dosage since admission. The role of increased fondaparinux dosage at the time of clinical deterioration was then followed by clinical improvement and reduced D-dimer level. Anticoagulant therapy, mainly with fondaparinux, showed a better prognosis in patients with markedly elevated D-Dimer. Fondaparinux needs to be monitored appropriately to prevent bleeding and adverse. The patient was discharged from the hospital in an improved condition and normal D-Dimer levels. There was no bleeding event nor other major side effects had been found in this case. The decision for increasing dose of anticoagulant may be determined on individual basis, considering risks, benefits, and also the most important is clinical findings.

[EFFECTIVENESS OF COMBINED TREATMENT OF HPV INFECTION].

This study evaluates the effectiveness of immunomodulating drug isoprinosine in a comprehensive treatment of genital warts in men. Most of the patients were aged 20-30 years. The combination therapy was found to have long term effectiveness. In the group of patients undergoing only destructive methods of treatment relapse after 8 month follow-up was diagnosed in 32% and in patients of the combination therapy group (destruction plus isoprinosine) - in 7% of patients. The pharmacological action of the drug (immunostimulating, antiviral) and the effectiveness of its combination with destructive therapies justify the use of inosine pranobex (isoprinosine) both in the complex therapy of genital warts and for the prevention of the disease recurrence.

[The role of human papilloma virus in development of chronic urethritis and vulvodynia in females: perspectives of immunomodulating therapy].

The article is devoted to combined affection of the lower urinary tracts and genitalia in women with human papilloma virus (HPV) infection which manifests with persistent recurrent urethritis, pelvic pain syndrome. The colposcopic and urethrocystoscopic features, disturbed microcirculation of urethral and vaginal mucosa in virus infection promoting recurrences and persistence of HPV are discussed. Immunomodulators (inosin pranobex-groprinosin) are recommended for more effective treatment.

The effect of different doses of methisoprinol on the percentage of CD4+ and CD8+ T lymphocyte subpopulation and the antibody titers in pigeons immunised against PPMV-1.

As immunosuppression in pigeons is common and results in reduced post-vaccination immunity and lower health status of the birds, studies have been taken up aimed at evaluation of the effect of three doses of methisoprinol on the percentage of CD4+ and CD8+ T lymphocyte subpopulation in peripheral blood and in the spleen and the titre of anti-NDV antibodies in the serum of pigeons in four groups (A, B, C, D), with 20 birds each. Pigeons in each group were immunised against paramyxovirosis at week 6 and 9 of life. Water for injection (group A - control) or methisoprinol at 100 mg/kg of body weight (group B), 200 mg/kg of body weight (group C) and 600 mg/kg of body weight (group D) was administered intramuscularly for 3 days before each vaccination. The immunological analyses were carried out by flow cytometry and the ELISA test. The findings indicate that methisoprinol administered intramuscularly at 100 and 200 mg/kg of body weight for 3 successive days before vaccination against paramyxovirosis mainly stimulates the mechanisms of non-specific humoral and cellular immunity, which is indicated by a higher percentage of the subpopulation of CD4+ T lymphocytes in peripheral blood and in the spleen and a higher titre of anti-NDV antibodies.

The influence of methisoprinol administered in ovo on the morphological structure of the spleen in turkeys.

This study analyzed the effect of a synthetic, low-toxic immunomodulator - methisoprinol - administered in ovo on the morphological structure of the spleen in turkeys. Experiments were conducted on three groups of 5-day-old BUT 9 turkeys (35 birds in each group) hatched from eggs which, on day 26 of incubation, had been administered methisoprinol (VetAgro, Lublin, Poland) in ovo in a dose of 5 mg (group I) or 20 mg per egg (group II). Poults hatched from eggs administered a physiological solution of NaCl in a dose of 0.1 ml per egg in ovo served as a control (group III). Samples of the spleen were collected from 5 birds selected at random from a group of decapitated 5-day-old poults and the prepared 7 fm-paraffin sections were stained with HE. A morphometric analysis of the germinal centres of the white pulp of the spleen was conducted by subjecting pictures taken with an optical microscope to a Digital Image Analysis using Axio Vision software (by Zeiss). The study demonstrated that in terms of the morphological structure, the spleen of the poults hatched from eggs administered 5 mg of methisoprinol (group I) did not differ considerably from the spleen of the control birds. In turn, spleens of the poults hatched from eggs administered 20 mg of methisoprinol per embryo were characterized by distinctively developed red pulp and within the area of the white pulp by distinct cortical section containing numerous lymphocytes. In spleens of the poults from this group, the morphometric examination also demonstrated a higher number of germinal centres of the white pulp as compared to their number in spleens of the birds from the other groups.

Selected leukocyte subpopulations in peripheral blood and uterine washings in cows before and after intrauterine administration of cefapirin and methisoprinol.

The aim of the study was to evaluate the selected lymphocyte subpopulations TCD4, TCD8, BCD21, BCD25, CD18, CD11b, and MHC II in blood and uterine flush of cows with endometritis, before and after intrauterine (i.u.) administration of cefapirin and methisoprinol. The research was carried out on 28 cows with clinical endometritis. Animals were divided into four groups, each composed of seven cows, depending on the i.u. preparation used: Group A, cefapirin; Group B, methisoprinol; Group C, cefapirin and methisoprinol simultaneously; and a control group-without medication. The study was performed using flow cytometry method. Summarizing the results of the research, i.u. infusion of cefapirin caused a weakening of the effector phase of the local uterine immune response; however, it enhanced leukocyte chemotaxis and antigen presentation. After i.u. administration of methisoprinol, the stimulation of specific uterine immunity mechanisms was mainly observed. The use of both mentioned preparations showed the strengthening of specific uterine immunological mechanisms presumably caused by methisoprinol, despite the inhibitory effect of the antibiotic. Intrauterine use of immunostimulatory substances can improve the effectiveness of the endometritis treatment in cows by improving specific local mechanisms of uterine immunity. As a consequence, it may enhance the effector function of immune competent cells and finally eliminate inflammation.

The influence of methisoprinol applied in ovo upon hatchability and health status of turkeys.

A study was undertaken to determine the effect of a synthetic immunomodulator, i.e. methisoprinol applied in ovo, upon the hatchability of turkey poults under conditions of a standard hatchery as well as on their health status evaluated based on analyses of selected biochemical indices in their blood serum. Experiments were conducted on 5 groups of BUT 9 turkeys at the age of 5 days (35 birds in each group) hatched from eggs to which methisoprinol (VetAgro, Lublin, Poland) was applied in ovo at a dose of 5 mg (group I), 10 mg (group II) or 20 mg per egg (group III) on the 26th day of incubation. Turkeys hatched from eggs to which a physiological solution of NaCl was applied on the same day at a dose of 0.1 ml per egg (group IV) as well as those hatched from eggs without in ovo injection (group V) served as controls. Five hundreds eggs were used in each group. Hatchability was evaluated based on the number of hatched poults in respect of the number of eggs with live embryos transferred from the setting compartment to the hatching compartment, that were subjected to in ovo administration of the preparations according to the experimental design. Blood serum of the 5-day-old turkey poults was analyzed for activities of AST, ALP, LDH-L, CK, lysozyme and ceruloplasmine as well as for total protein and albumin contents. Analyses were also conducted for the immune system organ index - percentage contribution of organs of the immune system (spleen, thymus and the bursa of Fabricius) in the body weight of turkeys. The study demonstrated that methisoprinol administered to turkey embryos in ovo on day 26 of incubation at doses of 5, 10 or 20 mg per embryo did not induce any disturbances in the hatching process or affect its final result. In addition, it was shown not to exert any negative effect on the health status of the reared turkey poults.

Long-term follow-up of patients with adult-onset subacute sclerosing panencephalitis.

Subacute sclerosing panencephalitis (SSPE) is a rare infectious central nervous system disease with a poor prognosis. Nineteen patients, 18 males and one female, ranging in age from 18 to 22, mean 19.6+/-1.5 years with SSPE were evaluated. We treated 9 patients with oral isoprinosine and 10 patients with alpha-interferon plus oral isoprinosine and followed up for 16 to 160 months. Of the 9 patients treated with oral isoprinosine, 7 (77.7%) died, one stabilized, and one showed progression. Seven (70%) of 10 patients treated with alpha-interferon plus oral isoprinosine died, one showed progression, and stabilization was observed in two patients. Thus, we suggest that isoprinosine alone or in combination with intraventricular interferon did not change the prognosis in long-term follow-up periods.

Phagocytic and oxidative burst activity of phagocytic cells in peripheral blood and uterine washings in cows with clinical endometritis before and after intrauterine use of cephapirin and methisoprinol.

The aim of the study was to evaluate phagocytic and killing activity of phagocytic cells in blood and uterine flush of cows with endometritis before and after intrauterine (i.u.) administration of cephapirin and methisoprinol. The research was carried out on 28 cows with clinical endometritis. Animals were divided into four groups, each composed of seven cows, depending on the i.u. treatment used: Group A-cephapirin; Group B-methisoprinol; Group C-cephapirin and methisoprinol at the same time; and a control group-without medication. Using flow cytometry technique, the phagocytic activity of granulocytes and monocytes was identified, as well as the oxidative burst activity of neutrophils in the peripheral blood and uterine washings. Summarizing the results of the research, i.u. infusion of cephapirin caused a reduction in the phagocytic and killing activity of phagocytes. The i.u. use of methisoprinol increased phagocytic and killing activity of phagocytes in the uterus. Administering both listed substances simultaneously showed a decrease in phagocytosis, presumably due to the dominating inhibitor effect of the antibiotic. However, also an increase of mean fluorescence intensity was observed, presumably caused by the methisoprinol. Intrauterine use of immunostimulatory substances, can improve the effectiveness of the treatment of endometritis in cows.

[Therapy and prognosis in subacute sclerosing panencephalitis].

Subacute sclerosing panencephalitis (SSPE) is a progressive and fatal central nervous system disorder that results from a persistent SSPE virus infection. Compound which inhibits the replication of SSPE virus might be a candidate for the specific drug for SSPE. Out of several compounds which had been tried for the treatment of SSPE, two drugs, i.e., inosiplex and interferon-a were reported to be effective. Those drugs, however, could not cure the disease. Recently, ribavirin therapy has been proposed as novel antiviral chemotherapy for SSPE. By intraventricular administration, ribavirin level in CSF reaches a concentration at which ribavirin could completely inhibit the replication of SSPE virus. Thus, intraventricular ribavirin therapy might eradicate SSPE virus from the CNS and stop the progression of SSPE syndrome. The therapeutic efficacy should be evaluated in the patients who are treated with the therapy at an early stage of SSPE.

[A case of adult onset subacute sclerosing panencephalitis; sequential clinico-radiological findings over two years].

We present a very rare case of adult onset subacute sclerosing panencephalitis (SSPE), and explain the characteristic sequential clinico-radiological findings. The patient, a twenty three-year-old man, had noticed unsteadiness in walking about two months previously. Although inosine pranobex and intrathecal interferon were administered, symptoms worsened insidiously and he became bedridden after four months. The levels of serum and CSF anti-measles IgG antibodies have not changed. Initially, supratentorial cortical atrophy was observed, especially of the left temporal lobe, but there were no other MRI signal alterations at the time. After three months, the supratentorial cortices produced low-signal intensities in T1-weighted images and in T2-weighted MRI, the cortical margin was very unclear and white matter signal intensities had become higher. Furthermore, cortices became thinner and ventricular size increased, especially for the lateral and third ventricles. SPECT examinations showed a marked reduction in cerebral blood flow and the perfusion deficits observed seemed to be closely correlated with the abnormal MRI signal patterns. Pathological examinations of biopsy samples revealed infiltration of inflammatory cells around the small vessels. As for immunohistochemical findings, CD68 positive cells were frequently observed, and this result implied the activation of microglia. Further studies are necessary to elucidate the pathogenic mechanisms of SSPE.

[Immunological and clinical study on therapeutic efficacy of inosine pranobex]. / Immunologiczne i kliniczne badania nad przydatnoscia terapeutyczna inozyny pranobeks.

Many studies in vitro and in vivo have shown immunomodulating and antiviral activities of inosine pranobex. The object of this research was to examine the potential beneficial effects of inosine pranobex (Groprinosin) on immune system in children with cellular immunodeficiency as a prophylaxis of recurrent infections, mainly of viral origin. 50 mg/kg b.w/day of inosine pranobex in divided doses was given to the group of 30 children aged 3-15 years for 10 days in 3 following months. Clinical and immunological investigations were done before and after the treatment. Statistically significant rise of CD3T lymphocytes number (p = 0.02) and in this CD4T lymphocytes number (p = 0.02) as well as statistically significant improvement of their function (p = 0.005) evaluated with blastic transformation method were found. These laboratory findings were parallel to clinical benefits. Control study was performed in the group of children completed by randomization and treated in the same way with garlic (Alliofil).

Immunomodulatory effects of inosine pranobex on cytokine production by human lymphocytes.

Inosine pranobex (inosine dimepranol acedoben, isoprinosine) (Inos) is an immunomodulatory and antiviral drug used in some viral infections, especially in patients with weakened immunity. In the present study, effects of Inos on the production of cytokines attributable to Th1 (IL-2, IFN-g, and TNF-a) or Th2 cells (IL-4, IL-5, and IL-10) were tested in human peripheral blood lymphocyte cultures stimulated with phytohemagglutinin (PHA). Inos enhanced TNF-a secretion significantly (in short-term--24-hour, and prolonged term--72-hour cultures) and IFN-g (in 72-hour cultures). Surprisingly, production of IL-10 by PHA-stimulated lymphocytes was suppressed by Inos in a dose-dependent manner in both 24-hour and 72-hour cultures. These results shed some light on immunomodulatory properties of Inos and suggest applicability of this agent in patients with a depressed function of the immune system.

Intraventricular interferon and oral inosiplex in the treatment of subacute sclerosing panencephalitis.

We treated 22 patients with subacute sclerosing panencephalitis (SSPE) with intraventricular alpha-interferon (IFN) and inosiplex PO and followed them for 2 to 54 months. Three deaths occurred. Clinical improvement, demonstrated by decreasing scores on the Neurological Disability Index, occurred in 11/22 (50%); five patients became stable, and the progression rate of the disease decreased in three. The remission rate was significantly higher than untreated controls from the same institution. Patients who had a slowly progressive disease responded best to treatment. Serious side effects were rare. We recommend intraventricular IFN, combined with oral inosiplex, in the treatment of SSPE.

Long-term follow-up of patients with subacute sclerosing panencephalitis treated with intraventricular alpha-interferon.

We treated 22 patients with subacute sclerosing panencephalitis (SSPE) with intraventricular alpha-interferon (alpha-IFN) and oral inosiplex between 1986 and 1991. The follow-up for 56 to 108 months demonstrates a higher survival rate in these patients compared with those who did not receive alpha-IFN. However, eight of 11 patients whose condition improved after alpha-IFN treatment and five of five patients whose condition stabilized after alpha-IFN experienced neurologic deterioration 6 to 90 months after treatment; three of 11 and four of five died. The use of inosiplex did not influence the prognosis. Re-administration of the same regimen was not effective in one patient. Treatment-induced remissions in SSPE can be temporary, analogous to spontaneous remissions. Longer treatment with higher doses, or combinations of drugs, may be required.

Echinococcus granulosus:isoprinosine treatment of the metacestode stage.

The efficacy of short- and long-term treatments with Isoprinosine, an immunomodulatory compound, was studied in Echinococcus granulosus cysts developed in NMRI mice intraperitoneally infected with sheep pulmonary cysts. After treatment, a reduction in the size and number of cysts with macroscopic modifications was observed. The structural alterations included damage or destruction of the protoscoleces and partial destruction of the cyst wall, which predominated at the inner germinal layer level. The efficacy of this drug was evaluated after long-term and short-term treatment. Short-term treatment with a dose of 1 g/kg/day gave better results, with a loss of infectivity of the larval tissue. The well-tolerated long-term treatment with a dose of 2 g/kg/day showed the absence of toxicity of this compound. The survival time of treated animals was greater than that of untreated controls.

Retrospective evaluation of interferon-beta treatment in subacute sclerosing panencephalitis.

BACKGROUND: Few effective treatment methods are available for subacute sclerosing panencephalitis (SSPE),an infection associated with the measles virus. Interferons have shown some benefit in previous studies and clinical practice. OBJECTIVE: The purpose of this study was to compare the efficacy of 2 different regimens of interferon-beta(IFN-beta) in the treatment of SSPE in pediatric patients. METHODS: We retrospectively compared the results obtained with 2 regimens of IFN-beta1a: 60 microg administered intramuscularly once weekly (IFN-beta 1/wk), or 22 microg administered subcutaneously 3 times per week (IFN-beta 3/wk). All patients also received oral inosiplex 50 to 100 mg/kg daily, a treatment known to have partial efficacy in SSPE. Patients who continued treatment for at least 3 months and had at least 1 year of follow-up data were evaluated. Clinical parameters included the Neurological Disability Index (NDI), a measurement of mental, motor, and sensory functions; disease stage; and mental status. Data obtained at 6 and 12 months were compared with those at the time of diagnosis, and the percent change from baseline was calculated. A satisfactory clinical response was defined as reduction or stabilization of the NDI or stage improvement at 6 or 12 months. RESULTS: Patients treated with IFN-beta 3/wk had increased survival time (P < 0.02) and higher clinical response rates compared with those treated with IFN-beta 1/wk (P < 0.05). When stage 2 and stage 3 patients were evaluated separately, survival was significantly longer (P = 0.007) and the rate of progression slower in both stage groups with IFN-beta 3/wk. CONCLUSION: The results obtained for this patient sample suggest that IFN-beta administered subcutaneously times per week combined with inosiplex may be an effective treatment option in SSPE. This treatment regimen warrants further study.

Failure of oral inosiplex treatment of recurrent herpes simplex virus infections.

A compound, a mixture of acedoben, dimepranol, and inosine (inosiplex) was used to treat recurrent local herpes simplex virus (HSV) infections in a double-blind placebo-controlled crossover study. Altogether, 58 patients with a history of frequently recurrent HSV infections were examined. Eighteen selected patients participated in the drug trial. Ten patients received both inosiplex and placebo, three received only inosiplex, and five received only the placebo. Three patients received both placebo and inosiplex twice. No substantial differences between the treatments with inosiplex or placebo could be seen in the frequency of occurrence or healing of the local lesions, nor in the results of these patients' immunologic studies. An evident placebo effect was observed, since only 15 (26%) of the 58 subjects examined continued to have an often relapsing form of the disease when followed up regularly.

Modulation of immunological reactivity in the course of allergic encephalomyelitis by isoprinosine.

It was observed that in the course of allergic encephalomyelitis (EAE) in chickens. Isoprinosine reduced the production of the specific IgA and IgM antibodies against encephalic antigen. Its influence on IgG production was slight. It was also found that isoprinosine reduced the antitrypsine activity in EAE. Its effect upon the level of antibodies and biochemical indices of inflammation was particularly pronounced on days 28-35 after immunization, i.e. at the time of the most intensive microscopic inflammatory changes in the brain. Histological analysis revealed an inhibition of plasmatic cells reproduction in the spleens of chickens receiving isoprinosine. No effect of the drug on the GvH reaction in chickens was found.