Tilorone Hydrochloride Research Articles

Method Development and Validation for Estimation of related Substances in Tilorone Dihydrochloride using RP¬-HPLC

A simple, precise and reproducible RP-HPLC method was developed for the estimation of related substances in tilorone dihydrochloride. Quantification was performed using a Zorbax SB-phenyl column (150 × 4.6mm, 5µ) with mobile phase A: 20mM potassium dihydro phosphate + 2ml of triethylamine, pH 2.30 and mobile phase B: acetonitrile, methanol and water 60: 20: 20% v/v. A gradient program was followed with a run time of 55 minutes at a flow rate of 1.0 ml/min. The column temperature was maintained at 40°C, the injection volume was 10 µl and the detection was performed at 269nm using a PDA detector. The retention time of Tilorone dihydrochloride was found to be 10.36 minutes. The proposed method has been validated according to the ICH guidelines for Linearity, Precision, Accuracy, LOD, and LOQ. The method was linear from 0.157 - 3.934μg/ml for standard, 0.153-3.820μg/ml and 0.166 - 4.140μg/ml for impurities, TLHC01 and TLHC02 respectively. The impurities TLHC01 and TLHC02 have been mapped in all stress conditions. The LOD and LOQ of TLHC01 were found at 1.757μg/ml and 5.857μg/ml and 1.919μg/ml and 6.396μg/ml respectively for TLHC02 respectively. Statistical analysis showed that the method was precision, reproducible, selective, specific and accurate for the analysis of Tilorone dihydrochloride and its impurities. The wide range of linearity, sensitivity, precision, short retention times and simple mobile phase have shown that the method is suitable for the routine quantification of mass impurities of tilorone hydrochloride and its dosage pharmaceutical forms with high precision and accuracy.

Tilorone activates NK cells and cytotoxic lymphocytes that kill HLA-negative tumor cells.

Tilorone hydrochloride, a low-molecular-weight synthetic compound, induces interferon production and has been reported to have both antiviral and antitumor activities. Here, we have demonstrated the ability of tilorone to activate NK cells and specific subpopulations of cytotoxic CD4+ and CD8+ T lymphocytes that recognize immune-evasive tumor cells and kill them via the FasL-Fas interaction. We have also performed a comparative analysis of characteristics between lymphocytes activated in the fraction of human peripheral blood mononuclear cells (PBMCs) upon treatment with different stimulants of the immune response: tilorone, innate immunity protein Tag7, and cytokine IL-2, a regulator of adaptive immunity. The results show that all the three stimulants, regardless of their nature, activate lymphocytes that are identical with respect to the spectrum of target cells, phenotype, and mechanism of cytotoxic action However, these stimulants induce different mechanisms of lymphocyte activation at early stages of the immune response. © 2018 IUBMB Life, 71(3):376-384, 2019.

Enhanced resistance to experimental systemic candidiasis in tilorone-treated mice.

Candida albicans is an increasingly important opportunistic fungal pathogen in immunocompromised patients. Natural killer (NK) cells constitute an important immune effector mechanism and are involved in the response to different pathological disorders. We wished to determine if this immune mechanism is involved in the specific response to C. albicans. Tilorone hydrochloride and related compounds have been described to display antiviral and antitumoral activity, as well as to enhance NK cell activity. In this study, we show the antimicrobial activity of different tilorone analogues and the enhanced resistance of tilorone-treated mice in experimental systemic candidiasis. We also present data suggesting that there is a correlation between NK cell activation and the resistance to experimental systemic candidiasis. Thus, it seems that the immunosurveillance of metastatic spread and the infection by C. albicans share some immune effector mechanisms, in particular activation of NK cells.

Enhanced resistance to experimental systemic candidiasis in tilorone-treated mice

Candida albicans is an increasingly important opportunistic fungal pathogen in immunocompromised patients. Natural killer (NK) cells constitute an important immune effector mechanism and are involved in the response to different pathological disorders. We wished to determine if this immune mechanism is involved in the specific response to C. albicans. Tilorone hydrochloride and related compounds have been described to display antiviral and antitumoral activity, as well as to enhance NK cell activity. In this study, we show the antimicrobial activity of different tilorone analogues and the enhanced resistance of tilorone-treated mice in experimental systemic candidiasis. We also present data suggesting that there is a correlation between NK cell activation and the resistance to experimental systemic candidiasis. Thus, it seems that the immunesurveillance of metastatic spread and the infection by C. albicans share some immune effector mechanisms, in particular activation of NK cells.

Effect of the immunomodulator tilorone on the in vivo acetylation of procainamide in the rat.

Interferon and interferon inducers have been found to inhibit cytochrome P-450-dependent metabolism in animals and man. The effect of these agents on the acetylation of drugs has not been previously reported. Since these agents stimulate the reticuloendothelial system, together with the abundance of N-acetyltransferase in the reticuloendothelial system, it was hypothesized that these immunomodulators may affect drug acetylation. To test this hypothesis, the effect of tilorone (a synthetic interferon inducer) on the in vivo acetylation of procainamide was examined in the rat. Pretreatment with tilorone hydrochloride (50 mg/kg) 48 hr prior to the administration of procainamide hydrochloride (50 mg/kg) resulted in a 32% increase in the urinary recovery of N-acetylprocainamide and a 35% increase in the metabolic clearance of procainamide to N-acetylprocainamide. These data indicate that interferon inducers increase the N-acetylation of drugs in vivo.

Effect of the Immunomodulator Tilorone on Antipyrine Disposition in the Rat

A wide variety of immunomodulators, including the synthetic interferon inducer tilorone (2, 7-bis[2-(diethylamino)ethoxy]fluoren-9-one), have been shown to inhibit cytochrome P-450-dependent drug metabolism in vitro. In an effort to determine if tilorone also inhibits drug metabolism in vivo, we examined the effect of this agent on the disposition of antipyrine in the rat. Administration of tilorone hydrochloride (50mg-kg−1.d−1) for 4 d resulted in a 42% reduction in antipyrine clearance and a 71% increase in plasma half-life. Administration of a single dose of tilorone hydrochloride (50mg/kg) 48h prior to antipyrine was found to cause a similar alteration in antipyrine clearance and half-life. Both treatment regimens were also found to produce a substantial weight loss in the pretreated animals. Preliminary studies indicated that tilorone treatment reduced food and water consumption in the rat. Therefore, the effect of a 75% decrease in food and water consumption on antipyrine disposition was also examined. This magnitude of restriction had no detectable effect on antipyrine elimination. Thus, tilorone results in a substantial reduction of the in vivo metabolic clearance of antipyrine and appears to be a useful compound for studying the dynamic interaction between the drug metabolizing and immune systems in vivo.

Tilorone hydrochloride in the treatment of T cell lymphoproliferative cutaneous disease

Tilorone hydrochloride was used to treat eleven patients with T cell cutaneous disease ranging from pre-Sézary syndrome to tumor-stage mycosis fungoides. Cutaneous histologic study, Sézary counts, delayed skin tests, patch tests, and quantitative T cell counts were monitored. The study revealed that the response to tilorone depends in part on the type and stage of disease, as well as the characteristics and responsiveness of the individual lymphocyte population. Patients with pre-Sézary syndrome are most likely to benefit from tilorone. The effect of tilorone on the T cell population is manifested by changes in responses to patch and skin tests, as well as histologic improvement. Intact immune responses and elevated levels of IgE may be important prognostic clinical parameters in these patients. Tilorone is ineffective in patients with mycosis fungoides and advanced Sézary syndrome. Keratopathy can be a limiting but reversible complication of therapy. The drug may provide an effective therapeutic step in the treatment of early T cell cutaneous disease or an adjunctive therapy to leukapheresis and chemotherapy.

Interferon induction by lymphocytic choriomeningitis viruses correlates with maximum virulence.

Lymphocytic choriomeningitis (LCM) viruses isolated from the blood of persistently infected mice, could be clearly divided into two categories by observing the disease patterns they produced after intracerebral (i.c.) injection in a number of adult inbred and outbred mice. One type (aggressive) caused the classic pattern of convulsive death in 100% of the mice 7 to 9 days after infection, while the other (docile) caused a protracted disease with deaths occurring, if at all, 2 to 4 weeks after infection. Interferon could be detected in the serum of adult mice on the 3rd and 4th day after infection with several independently cloned aggressive, but not docile, viruses. The inability of docile virus to induce interferon was not due to poor or delayed virus replication in the brain. The aggressive pattern of disease could be provoked easily in docile virus-infected mice with the interferon inducers poly(rI) . poly(rC), tilorone hydrochloride or Newcastle disease virus. The amount of interferon produced had little effect on the mean day of death. Mice that differed over 10-fold in their serum interferon levels after LCM infection, either by genetic predisposition or by stimulation with increasing amounts of poly(rI) . poly(rC), presented almost identical patterns of mortality.

An analysis of the natural killer cell defect in beige mice

Although C57BL/6 bg J/bg J mice exhibit very low or undetectable levels of endogenous natural killer cell activity, such activity can be induced by the administration of BCG or tilorone hydrochloride to these animals. This cytotoxic activity has been shown to be due to NK cells by the criteria of nylon-wool nonadherence and of effector cell sensitivity to treatment with either anti-asialo GM 1 serum or high concentrations of anti-Thy 1.2 serum, in the presence of complement. Even after the administration of inducing agents, however, beige mice continue to display significantly less NK activity than do their heterozygous littermates. In an attempt to ascertain what cell might be defective in responding to induction, we utilized an in vitro system in which the induction of NK activity by poly I:C in a nylon-wool nonadherent population is dependent upon plastic-adherent cells. We found that adherent cells from either beige or heterozygous mice were indistinguishable in their ability to restore the NK response of nylon-wool-nonadherent spleen cells stimulated with poly I:C. This was true when either beige or heterozygous mice were used as the source of responder cells. Thus, it appears that the defect in responsiveness to inducing agents may reside in the beige NK cell itself.

Antitumour effects of tilorone hydrochloride on the in vivo growth of chemically induced and spontaneously arising rat tumours.

Inhibition of subcutaneous tumour growth, following treatment with tilorone hydrochloride, was observed with two transplantable methylcholanthrene-induced rat sarcomas (Mc7, Mc4), but similar drug treatment was ineffective against two rat hepatomas induced by dimethylaminoazobenzene (D23, D30). Successful therapy of sarcoma Mc7 and Mc4 subcutaneous growth was not accompanied by the development of specific antitumour immunity, although with hepatoma D30, which grew out following tilorone hydrochloride treatment, specific immunity was demonstrated, indicating that drug treatment does not prevent the initiation of an antitumour response during progressive tumour development. Under defined experimental conditions tilorone hydrochloride also prevented pulmonary metastases from a subcutaneous tumour graft of epithelioma SP1 and mammary carcinoma SP22, as measured by an increased survival or treated rats.

Mechanism of cellular suppression induced by oral tilorone treatment of mice.

Specific-pathogen-free B6D2 F1 hybrid mice were treated orally with tilorone hydrochloride (100 mg/kg of body weight per day) and infected with sublethal doses of Listeria monocytogenes, Salmonella enteritidis, Mycobacterium bovis (BCG Pasteur), or M. tuberculosis Erdman. Daily tilorone treatment inhibited the cell-mediated response to all of the intracellular parasites, and most of the mice succumbed to the challenge. Tilorone suppressed delayed hypersensitivity responses to the microbial sensitins as well as to sheep erythrocytes. However, humoral responses (immediate hypersensitivity reactions) were stimulated. The types of growth curves obtained in the tilorone-treated mice were quite different from those observed in T-cell-depleted mice and tended to resemble those seen in sublethally irradiated (400 rads) animals. Leukocyte counts were depressed 10-fold by daily tilorone treatment. Both monocyte and granulocyte (but not large-lymphocyte) counts were depressed. There was an initial drop in small-lymphocyte counts with a later recovery phase. Tilorone treatment reduced the granulomatous response within the Salmonella-infected liver, suggesting that the drug interferes with the mobilization of the mononuclear defenses within the normal host.

Keratopathy After Oral Administration of Tilorone Hydrochloride

Two patients given tilorone HCl orally for varying periods of time had clinical and histopathologic ocular changes. Retrospective study of 14 cancer patients who were taking tilorone HCl orally revealed that three patients had similar ophthalmic findings accompanied by the appearance of blue halos around pinpoint light sources. Examination revealed a diffuse clouding of the epithelium sometimes associated with subepithelial infiltrates. Abnormalities seen histologically included cloudy swelling of the epithelium and cytoplasmic inclusions. By electron microscopy these were found to be myelinoid bodies. Gas chromatography and mass spectrophotometry showed that tilorone HCl was present in the cornea and conjuctiva. Visual acuity was not affected and these changes were slowly reversible with the cessation of therapy. Biomicroscopic and conjunctival cytologic examination may serve to indicate the drug's storage and potential damage in the body.

Therapeutic trial of tilorone in ALS

Sixteen patients with amyotrophic lateral sclerosis (ALS) participated in a double-blind, placebo-controlled study using tilorone hydrochloride, a drug active against a variety of DNA and RNA viruses in animals. On the basis of neurologic examination, pulmonary function studies, quantitative muscle examination, speech recording, and periodic filming of functional muscle strength, it was concluded that at a dose of 1 gm per week, tilorone did not alter the course of ALS.

Induction of cytokines by tilorone hydrochloride.

Tilorone hydrochloride, an interferon inducer in small laboratory animals, was demonstrated to elicit formation of macrophage migration affecting and microbial growth inhibitory cytokines after peroral drug administration to mice. Serum kinetics of the migration inhibitory cytokine resembled those of interferon, exhibiting a peak after about 24 h, whereas the bactericidal cytokine showed a steady increase up to 48 h after drug treatment. Both the factors were found to have molecular weights of 10,000--30,000 daltons as determined by Sephadex G-200 chromatography, to be stable at pH 2 and at 56 degrees C for 30 min, sensitive to chymotrypsin and resistant to RNase digestion. The migration enhancing serum activity could not finally be characterized so far. The physicochemical data are discussed in comparison to those of lymphocyte-derived cytokines. It is suggested that cytokine production may be, at least partially, responsible for the immunological effects of tilorone and possibly contribute to its antiviral action.

THE ANTIPYRETIC EFFECT OF TILORONE HYDROCHLORIDE IN THE CAT

1 The antipyretic activity of tilorone hydrochloride was studied in conscious, unrestrained cats provided with implanted jugular venous catheters, third cerebral ventricular (i.c.v.) cannulae and retroperitoneal thermocouples. 2 In afebrile animals, 10 mg/kg i.v. or 1 mg i.c.v. tilorone hydrochloride did not alter body temperature, but 20 mg/kg i.v. or 2 to 5 mg i.c.v. caused hypothermia and various behavioural responses. 3 Non-hypothermogenic doses of tilorone (i.v. or i.c.v.) antagonized hyperthermic responses to leucocytic pyrogen (i.v. or i.c.v.), bacterial pyrogen (i.c.v.) and sodium arachidonate (i.c.v.) but did not antagonize prostaglandin E1 (i.c.v.). 4 These results indicate that tilorone has an antipyretic action within the central nervous system that is distinct from its hypothermogenic action. Although there is no published evidence to indicate that tilorone can inhibit prostaglandin synthesis peripherally, its ability to reduce hyperthermic responses to arachidonate suggests that it can inhibit prostaglandin synthesis within the brain.

Further Observations on the Inhibition of Tumor Growth by Corynebacterium parvum With Cyclophosphamide. V. Comparison of the Effects of Tilorone Hydrochloride, Levamisole, Methanol-Soluble Fraction of Mycobacterium butyricum, BCG, and a Nonviable Aqueous Ether Extract of Brucella abortus Preparation in Treatment of Mice With Tumors2

Further Observations on the Inhibition of Tumor Growth by Corynebacterium parvum With Cyclophosphamide. V. Comparison of the Effects of Tilorone Hydrochloride, Levamisole, Methanol-Soluble Fraction of Mycobacterium butyricum, BCG, and a Nonviable Aqueous Ether Extract of Brucella abortus Preparation in Treatment of Mice With Tumors2

Effect of tilorone hydrochloride on hepatic disposition of sulphobromophthalein (BSP) in the rat.

1. Biliary excretion of sulphobromophthalein (BSP) was studied in male rats after oral administration of tilorone HCl, an anti-viral and anti-tumour agent. 2. The stimulation of bile flow by BSP was decreased 1 h after tilorone administration; this decrease was no longer apparent 24 h later but a fall of basal bile flow was noted at that time. No changes in bile flow were observed 7 days after this single dose. 3. Repeated dosing with tilorone produced a dose-dependent increase in hepatic concentration of unchanged and metabolized tilorone. This was associated with a dose-dependent decrease of bile formation and BSP excretion. Bile acid excretion was depressed as well. 4. BSP metabolism was not significantly affected by tilorone administration.

Accelerated mortality in young NZB/NZW mice treated with the interferon inducer tilorone hydrochloride

Tilorone, 100 mg/kg, was given once a week to two groups of NZB/NZW mice: young mice aged 3 months without overt disease, and 8-month-old mice with established autoimmune disease. Young treated animals died prematurely with severe glomerulonephritis and vasculitis, but lifespans were not shortened in old treated mice. Thymic atrophy was common in treated animals. Tilorone therapy did not affect autoantibody levels; nevertheless, terminal C 3 values were decreased significantly in young treated mice. Although interferon activity was detected in sera from young treated mice, it provided no protection from spontaneous autoimmune disease. Tilorone-induced suppression of cell-mediated immunity may have caused accelerated disease and premature death in NZB/NZW mice.

T-Lymphocyte depletion induced in rats by analogs of tilorone hydrochloride

Tilorone, a bis-basic substituted fluorene-9-one compound, and four of its analogs depleted lymphocytes in thymic-dependent areas in both the spleen and lymph nodes after both sc and oral administration to rats. Twenty-nine out of 39 analogs administered by either route depleted lymphocytes in the spleen, lymph nodes, or in both. Active analogs encompassed a wide variety of polycylic ring structures including fluorene, fluoranthene, anthraquinone, dibenzothiophene, dibenzofuran, and even a bicyclic ring (naphthyridine) and a simple pyridine ring. The two teriary amine side chains in active compounds included ether, ketone, ester, alkane, and thioether but not carboxamide, thioester, or sulfonamide linkages. The mesenteric lymph node was especially susceptible to analogs administered by the oral route. The subcutaneous route was often associated with acute necrosis of cells in the thymic cortex, probably an indirect effect of stress and adrenal stimulation, which had the consequence of blocking lymphocyte depletion in the spleen and nodes. This complicating effect of certain compounds may be partly responsible for the inability to correlate lymphocyte-depletion with antiviral activity.

Hepatic disposition of tilorone hydrochloride in the rat.

1. The hepatic disposition of tilorone HCl, an antiviral and antitumour agent, was studied in male Wistar rats after intraduodenal administration. 2. A concentrative transfer into the liver occurred, the liver/portal blood concentration ratio being 120 or higher. The relationship between dose and hepatic concentration was linear. 3. Biliary excretion of tilorone was low and dose-related; whole blood concentration was also dose-related. 4. Neither hepatic concentration nor biliary excretion exhibited saturation over the dosage studied (6--174 mg/kg). The hepatic 'depot' of tilorone lacked the characteristics of a true 'first-pass' effect.