Use of sodium nucleinate and its combinations with bacterial polysaccharides for preventing the nephrotoxic action of aminoglycoside antibiotics

Possible prevention of the nephrotoxic effect of different doses of aminoglycoside antibiotics, such as monomycin, kanamycin and gentamicin was studied experimentally on chinchilla rabbits. Substances increasing the cell resistance (sodium nucleinate, prodigiosan and pyrogenal alone and sodium nucleinate combinations with the bacterial polysaccharides) were used. It was shown that sodium nucleinate prevented nephrotoxicity of the aminoglycosides in the doses 2.5 times higher than the therapeutic ones. The combined use of sodium nucleinate with pyrogenal or prodigiosan was most effective. It prevented the nephrotoxic effect of the antibiotics in the doses 5 times higher than the therapeutic ones.     

Study of the pharmacological properties and safety of a solution of levomycetin in hexamethylene tetramine for intravenous administration]

Pharmacological properties of 2 per cent levomycetin solution in 40 percent hexamethylentetramine solution, as a new pharmaceutical form of levomycetin for intravenous administration prepared at drug-stores were studied. The maximum tolerating doses of the drug for mice, rabbits, and dogs were 26-47 times higher than the therapeutic ones with respect to the content of levomycetin and hexamethylentetramine. No increase in the toxicity of levomycetin and hexamethylentetramine in the preparation was observed. The drug in the doses 16 times higher than the therapeutic ones by the content of levomycetin did not almost change the arterial pressure and the drug in the doses 3.7 times higher than the therapeutic ones did not affect the blood coagulation either in acute experiments, or on its prolong intravenous infusion. Repeated administrations of the drug to rats and rabbits for 15-18 days in doses 3.7-4.8 times higher than the therapeutic ones by the content of levomycetin were innocuous for the animals. Absorption, circulation in the blood, distribution in the tissues and excretion with the urine of levomycetin used in the above pharmaceutical form did not differ from circulation of the antibiotic on its intravenous and oral administration. The drug is recommended for use in medical practice.     

Immunogenicity of lysozyme derivatives lipid-conjugated to various degrees in mice treated with and without cyclophosphamide: dissociation of delayed-type hypersensitivity and helper function.

Lysozyme and a series of its lipid-conjugated derivatives without adjuvant were examined in mice for their abilities to induce delayed-type hypersensitivity (DTH), helper T-cell activity, and antibody formation. In addition, the effect of cyclophosphamide (CY) on the immune responses was assessed in mice immunized with these lysozyme derivatives. Precipitated lysozyme without lipid conjugation was a good inducer of both antibody and DTH responses. Lipid conjugation to lysozyme to intermediate degrees readily caused the failure only in inducing the antibody response. As lysozyme was lipid-conjugated more heavily, DTH response was also reduced and finally abolished. In contrast, the helper activity was little affected by any degree of lipid conjugation. These results indicate that the helper T-cell activity was dissociated from the both DTH response and the antibody production. CY pretreatment extensively enhanced DTH response induced by such lipid-conjugated derivatives that failed to induce antibody response. Furthermore, CY pretreatment in doses in a wide range enhanced not only DTH response but also antibody formation. It is, therefore, concluded that the enhancement of DTH response by CY does not necessarily entail suppression of antibody formation.     

Study of general toxic and organotropic properties of ampicillin combined with sulbactam]

The effect of sulacillin, a combination of sulbactam and ampicillin (1:2), on the functions of the liver and kidneys, peripheral blood count, cardiovascular and central nervous systems was studied in acute and chronic experiments on animals of various species. The allergenic and local irritating properties of the combination were also studied. It was shown that the combination was low toxic and the interaction of sulbactam and ampicillin by the lethal effect was additive. When the combination was administered intravenously to mice, its LD50 amounted to 6 g/kg. In chronic experiments on rats parenterally given the combination in doses equivalent to the therapeutic ones there were no changes in the examined systems and organs. When used in the doses exceeding the therapeutic ones, sulacillin used during long periods induced a transitory elevation of blood levels of transaminases and alkaline phosphatases, an increase in the relative weight of the liver and kidneys, elongation the typhlon and an increase in glycogen levels in the hepatocytes without morphological changes. The combination had no significant effect of sulacillin and the painful injections alleviated by local anesthesia were recorded. The allergenic properties of the combination were moderate and did not differ from those of ampicillin. The data indicate that the combined sulacillin preparation greatly resembles its foreign analogue.     

Experimental study of the organotropic properties of dactinomycin

Dactinomycin was studied pharmacologically on experimental animals. When dactinomycin was administered to the test-animals in doses close to the therapeutic ones for humans, suppression of the bone marrow blood formation was registered in spite of some increase in the number of the reticulocytes and thrombocytes in the peripheral blood and acceleration of the process of blood coagulation. In addition, the urea nitrogen blood levels increased. When the drug was administered in higher doses, suppression of the bone marrow blood formation was pronounced and the number of the leucocytes, reticulocytes and thrombocytes in the peripheral blood decreased. The rate of the blood coagulation decreased, while the biochemical values of the blood were indicative of impairement of the liver and kidney functions.     

The cyclic nucleotide content of the blood plasma in mice irradiated at different doses]

Cyclic nucleotide content of plasma was shown to increase after irradiation of mice within a wide range of doses. Compared was the dynamics of changes in the cyclic nucleotide content 24 hr following irradiation with supralethal doses.     

Targeted localized use of therapeutic antibodies: a review of non-systemic,topical and oral applications

Therapeutic antibodies provide important tools in the “medicine chest” of today’s clinician for the treatment of a range of disorders. Typically monoclonal or polyclonal antibodies are administered in large doses, either directly or indirectly into the circulation, via a systemic route which is well suited for disseminated ailments. Diseases confined within a specific localized tissue, however, may be treated more effectively and at reduced cost by a delivery system which targets directly the affected area. To explore the advantages of the local administration of antibodies, we reviewed current alternative, non-systemic delivery approaches which are in clinical use, being trialed or developed. These less conventional approaches comprise: (a) local injections, (b) topical and (c) peroral administration routes. Local delivery includes intra-ocular injections into the vitreal humor (i.e. Ranibizumab for age-related macular degeneration), subconjunctival injections (e.g. Bevacizumab for corneal neovascularization), intra-articular joint injections (i.e. anti-TNF alpha antibody for persistent inflammatory monoarthritis) and intratumoral or peritumoral injections (e.g. Ipilimumab for cancer). A range of other strategies, such as the local use of antibacterial antibodies, are also presented. Local injections of antibodies utilize doses which range from 1/10th to 1/100th of the required systemic dose therefore reducing both side-effects and treatment costs. In addition, any therapeutic antibody escaping from the local site of disease into the systemic circulation is immediately diluted within the large blood volume, further lowering the potential for unwanted effects. Needle-free topical application routes become an option when the condition is restricted locally to an external surface. The topical route may potentially be utilized in the form of eye drops for infections or corneal neovascularization or be applied to diseased skin for psoriasis, dermatitis, pyoderma gangrenosum, antibiotic resistant bacterial infections or ulcerated wounds. Diseases confined to the gastrointestinal tract can be targeted directly by applying antibody via the injection-free peroral route. The gastrointestinal tract is unusual in that its natural immuno-tolerant nature ensures the long-term safety of repeatedly ingesting heterologous antiserum or antibody materials. Without the stringent regulatory, purity and clean room requirements of manufacturing parenteral (injectable) antibodies, production costs are minimal, with the potential for more direct low-cost targeting of gastrointestinal diseases, especially with those caused by problematic antibiotic resistant or toxigenic bacteria (e.g. Clostridium difficile, Helicobacter pylori), viruses (e.g. rotavirus, norovirus) or inflammatory bowel disease (e.g. ulcerative colitis, Crohn’s disease). Use of the oral route has previously been hindered by excessive antibody digestion within the gastrointestinal tract; however, this limitation may be overcome by intelligently applying one or more strategies (i.e. decoy proteins, masking therapeutic antibody cleavage sites, pH modulation, enzyme inhibition or encapsulation). These aspects are additionally discussed in this review and novel insights also provided. With the development of new applications via local injections, topical and peroral routes, it is envisaged that an extended range of ailments will increasingly fall within the clinical scope of therapeutic antibodies further expanding this market.     

Ontogenetic development of antibody formation in response to different doses of gram-negative microorganisms in young rabbits

Rabbits at different stages of development were immunized with different doses of heatinactivated suspension ofEscherichia coli 086 andSalmonella paratyphi B. The dynamics and the site of formation of bactericidal and haemolytic antibodies during the primary reaction was investigated. An increase and an acceleration of antibody formation after increasing the dose of antigen was found in the serum and at the cellular level. The magnitude of the response and the rate of the reaction were directly proportional to the age of the experimental rabbits. The site of antibody formation depends on the character, route of administration, antigen dose and age of rabbits. After intraperitoneal and also after intravenous immunization withEscherichia coli andSalmonella paratyphi B antigens the site of antibody production in 15-day-old rabbits was the lymphatic tissue of the intestine, the appendix, and mesenteric lymph nodes. As the antigen dose was increased and the age of rabbits rose, i.e. in correlation with the increase of the antibody response, antibody formation shifted to the spleen which is the chief site of antibody production following immunization by these bacterial antigens from the first month of life of rabbits. In contrast with this type of antigen, after intraperitoneal or intravenous immunization with sheep erythrocytes of new-born or older rabbits antibody formation was concentrated in the spleen. The development of the immunological competence and the significance of intestinal lymphatic tissue as one of peripheral type is discussed.     

Induction of oocyte maturation in the white croaker Micropogonias furnieri (Pisces: Sciaenidae) by human chorionic gonadotropin.

The present work aimed to identify the best doses of human chorionic gonadotropin (hCG) needed to induce oocyte maturation of Micropogonias furnieri and to characterize ovarian dynamics during the periovulatory period. Adult M. furnieri females with fully developed ovaries were injected intraperitoneally with four different doses of hCG. The gonadotropin response was succeeded by analyzing morphologically gonadal biopsies and following the postinjection changes in follicle diameter. Oocyte maturation was induced by three doses used: 100, 300, and 500 IU of hCG kg bw-1, and was reached 48 h after treatment with 300 and 500 IU of hCG kg bw-1, and 72 h after treatment with 100 IU of hCG kg bw-1. Concerning ovarian dynamics, only 100 and 300 IU of hCG kg bw-1 mimicked the natural ones which have a synchronic group maturation. In conclusion, the dose mimicking natural ovarian dynamics and inducing oocyte maturation more quickly is 300 IU of hCG kg bw-1.     

Molecular dynamics investigation of an antibody binding site by the fluorescence-photochrome method

A combined fluorescence-photochrome approach was used for investigation of the molecular dynamics antiDNP antibody binding site and its cavity. A 4-(N-2,4-dinitrophenylamino)-4'-(N,N'-dimethylamino)stilbene (StDNP) fluorescence DNP analog was incorporated into the antibody binding site. This was followed by measurements of fluorescence and photochrome parameters such as the StDNP excitation and emission spectra, fluorescence lifetime, steady-state and time-resolved fluorescence polarization, kinetics of trans-cis and cis-trans photoisomerization, and fluorescence quenching by nitroxide radicals freely diffused in solution. In parallel, computational modeling studies on the location and dynamics of DNP/TEMPO spin-label (NslDNP) and StDNP guests within a model of the binding site were performed. When all the experimental evidence is considered (including data from the antibody X-ray study), one can conclude that wobbling of the Trp 91 L/Trp 96 H binding-site.bound-hapten moiety (StDNP), can be responsible for the label's nanosecond dynamics monitored by fluorescence polarization techniques. A similar conclusion may be reached as a result of data analysis on NslDNP mobility within the antibody binding site. The mobility of Trp 91 L and Trp 96 H moieties provides the induced fit needed for effective stacking and release of the DNP epitope. Analysis of the above-mentioned data allows one to explore the mechanism of the probe's movement within the binding site and enables one to discuss the local dynamics of the binding site region. The combined fluorescence-photochrome approach can be used for investigation of local medium molecular dynamics in the immediate vicinity of specific sites of proteins and nucleic acids, as well as for other biologically important structures and synthetic analogues.     

Unraveling what makes a monoclonal antibody difficult-to-express: From intracellular accumulation to incomplete folding and degradation via ERAD

Although most therapeutic monoclonal antibodies (mAbs) can routinely be produced in the multigram per litre range, some mAb candidates turn out to be difficult-to-express (DTE). In addition, the class of more complex biological formats is permanently increasing and mammalian expression systems like Chinese hamster ovary (CHO) cell lines can show low performance. Hence, there is an urgent need to identify any rate limiting processing step during cellular synthesis. Therefore, we assessed the intracellular location of the DTE antibody mAb2 by fluorescence and electron microscopy (EM) and revealed an accumulation of the antibody, which led to an aberrant morphology of the endoplasmic reticulum (ER). Analysis of underlying cellular mechanisms revealed that neither aggregation nor antibody assembly, but folding represented the reason for hampered secretion. We identified that the disulfide bridge formation within the antibody light chain (LC) was impaired due to less recognition by protein disulfide isomerase (PDI). As a consequence, the DTE molecule was degraded intracellularly by the ubiquitin proteasome system via ER-associated degradation (ERAD). This study revealed that with the continuous emergence of DTE therapeutic protein candidates, special attention needs to be drawn to optimization processes to ensure manufacturability.     

Toxicity of tobramycin in single and multiple administrations to laboratory animals]

The LD50 of tobramycin sulphate administered intravenously, intraperitoneally, subcutaneously and orally to albino mice was 77 (73--82), 262 (234--294), 560 (500--627) and greater than 10500 mg/kg respectively. With an increase in the rate of intravenous administration tobramycin toxicity increased. When tobramycin sulphate was administered subcutaneously daily in multiple doses equivalent to the daily therapeutic doses from humans (calculated for the body surface) and in the doses 2--3 times higher than the above therapeutic ones, the function of the kidneys, liver and the Preier's reflex did not significantly change. When the doses were 8--10 times higher than the therapeutic ones, an increase in the urea level in the blood serum, disappearance and a decrease in the Preier's reflex were observed. The impairment of the kidney function was accompanied by degenerative changes in the convoluted tubules of the kidneys, ischemia of the renal glomeruli and appearance of protein secretion in their capsule cavities. The picture of the peripheral blood did not suffer significant changes. The studies on the acute and chronic toxicity of tobramycin sulphate prepared at the Institute of New Antibiotics showed that the drug did not differ from the import tobramycin samples.     

Structural Basis for the Recognition in an Idiotype-Anti-Idiotype Antibody Complex Related to Celiac Disease

Anti-idiotype antibodies have potential therapeutic applications in many fields, including autoimmune diseases. Herein we report the isolation and characterization of AIM2, an anti-idiotype antibody elicited in a mouse model upon expression of the celiac disease-specific autoantibody MB2.8 (directed against the main disease autoantigen type 2 transglutaminase, TG2). To characterize the interaction between the two antibodies, a 3D model of the MB2.8-AIM2 complex has been obtained by molecular docking. Analysis and selection of the different obtained docking solutions was based on the conservation within them of the inter-residue contacts. The selected model is very well representative of the different solutions found and its stability is confirmed by molecular dynamics simulations. Furthermore, the binding mode it adopts is very similar to that observed in most of the experimental structures available for idiotype-anti-idiotype antibody complexes. In the obtained model, AIM2 is directed against the MB2.8 CDR region, especially on its variable light chain. This makes the concurrent formation of the MB2.8-AIM2 complex and of the MB2.8-TG2 complex incompatible, thus explaining the experimentally observed inhibitory effect on the MB2.8 binding to TG2.     

A strategy for mapping and neutralizing conformational immunogenic sites on protein therapeutics

Antibodies are highly specific recognition molecules which are increasingly being applied to target therapy in patients. One type of developmental antibody-based therapy is antibody directed enzyme prodrug therapy (ADEPT) for the treatment of cancer. In ADEPT, an antibody specific to a tumor marker protein delivers a drug-activating enzyme to the cancer. Subsequent intravenous administration of an inactive prodrug results in drug activation and cytotoxicity only within the locale of the tumor. Pilot clinical trials with chemical conjugates of the prodrug activating enzyme carboxypeptidase G2 (CPG2) chemically conjugated with an antibody to and carcinoembryonic antigen (CEA), have shown that CPG2-mediated ADEPT is effective but limited by formation of human antibodies to CPG2 (HACA). We have developed a recombinant fusion protein (termed MFE-CP) of CPG2 with an anti-CEA single chain Fv antibody fragment and we have developed methods to address the immunogenicity of this therapeutic. A HACA-reactive discontinuous epitope on MFE-CP was identified using the crystal structure of CPG2, filamentous phage technology and surface enhanced laser desorption/ionization affinity mass spectrometry. This information was used to create a functional mutant of MFE-CP with a significant reduction (range 19.2 to 62.5%, median 38.5%) in reactivity with the sera of 11 patients with post-therapy HACA. The techniques described here are valuable tools for identifying and adapting undesirable immunogenic sites on protein therapeutics.     

Effect of tetanic anatoxin and carbonic acid level in blood on biosynthesis of antibodies in rats

The paper deals with dynamics of antibody formation in rats. Tetanic anatoxin independently of doses and methods of administration manifests weak immunogenic properties. A complete Freind adjuvant stimulates considerably the biosynthesis of antitetanic antibodies in the primary and secondary immune responses. Electrophoresis of tetanic anatoxin polyacrylamide gel permits isolating two protein fractions, one of them migrates in the immunoglobulin G zone and its mass constitutes 73%, the other-more low-molecular and contains 27% of protein. Dynamics of the acid-base blood state indices and of antibody biosynthesis is studied on models of chronic compensated alkalosis and metabolic acidosis in rats. It is established that during the experiment, simultaneously with an insignificant increase in the total carbonic acid concentration blood, there occurs a 25-60% increase in the titres of antitetanic antibodies as compared to the control. In rats with acidosis there occurs a simultaneously decrease in the intensity of antibody biosynthesis as compared to the control (43-54%) and in the total CO2 content in blood.     

Detection of pneumococcal antibodies in children with acute pneumonia and pleurisy by an immunoenzyme method

The samples of sera and pleural fluid from sick children have been analyzed by means of EIA techniques. To detect the time course of antibody production, the antigenic preparations of pneumococci (monovalent capsular polysaccharides, polyvalent polysaccharide vaccine and complex pneumococcal antigen) have been used. Antibody response observed in the forms of pneumococcal infection, studied in this investigation, has proved to be highly variable. It is expedient to determine antibodies to polysaccharide antigens not earlier than on days 10-12 from the beginning of the disease. But, besides the positive dynamics of antibodies, their unchanged level is sometimes observed in the patients at the beginning of the disease. As a rule, there is a coincidence between the dynamics of antibody formation in response to polysaccharide antigens and to complex pneumococcal antigen.     

Allergenic and immunotropic properties of yeast RNA-tilorone hydrochloride molecular complex

Immunotropic properties of the interferon-inducing molecular complex (MC) yeast RNA--tilorone hydrochloride have been under study. MC was experimentally studied in vivo to establish its influence on the amount of antibody-forming cells and the level of antibody formation. The influence of MC on the oxygen-generating activity of spleen macrophages was established in the HCT test. MC in a dose of 1.25 mg/kg was shown to considerably activate immunocompetent cells, thus producing pronounced influence on humoral immunity. In addition, the study showed the dose dependence of the influence of MC on individual elements of the immune system as well as differences in the dynamics of immunomodulation caused by the use of high and low doses of MC. The data thus obtained made it possible to regard MC as a promising immunomodulator.     

Autoradiolysis of iodinated monoclonal antibody preparations

We investigated the effects of ionizing radiation on the immunointegrity of antibody fragments (Fab) because large amounts of high specific activity ¹³¹I may damage the proteins. We found that 1000 Gy of external ¹³⁷Cs γ radiation was sufficient to destroy 80–90% of the immunointegrity of the initial preparation. This effect was also produced by internally added [¹³¹I]NaI in a quantity sufficient to provide the same radiation absorbed dose. Since radioiodinated monoclonal antibodies labeled to high specific activity are being evaluated for radioimmunotherapy, the above observation is significant since high levels of internal radiation occur with therapeutic doses of ¹³¹I-labeled antibody. Human serum albumin in low concentration (2%) added to the iodinated antibody solutions was successful in preventing loss of immunoreactivity and can be used to protect and stabilize therapeutic quantities of radiolabeled monoclonal antibody preparations.     

Ontogenetic development of antibody formation in response to different doses of gram-negative microorganisms in young rabbits

Newborn and 15-day-old rabbits were immunized with different doses of heat-inactivated suspension ofEscherichia coli andSalmonella paralyphi B. The secondary immunization was performed after 4 weeks and the dynamics, magnitude and site of the secondary response studied. The magnitude of the secondary response was found to depend on the magnitude and rate of the primary response, this latter reflecting the dose used. A direct relationship was found in the range of the minimal and optimal dose: the higher the primary dose the higher the secondary response. After a rapid and pronounced primary response evoked by a high dose of 10¹⁰ microorganisms in 15-day-old rabbits, a partial inhibition of the secondary response was observed. The inhibition was pronounced primarily in the spleen; the lymphatic nodes reacted by a higher number of antibody producing cells as compared with the control nonprimed young rabbits.     

Role of natural killer cells in the modulation of primary antibody production by purine nucleosides and their analogs.

Previous results from this laboratory demonstrated that treatment of mice with the adenosine analog tubercidin (Tub) reduced natural killer (NK) cell activity while stimulating antibody production whereas the deoxyadenosine analog, 2-fluoroadenine arabinoside-5'-monophosphate (FaraAMP), produced opposite effects; i.e., it stimulated NK cell activity at doses that inhibited antibody formation (Cancer Res. 48, 4799, 1988). Since NK cells have been reported to play a suppressor role in immunoglobulin induction, it was hypothesized that the actions of Tub and FaraAMP on antibody production occurred secondary to their opposing effects on NK cells. To test this hypothesis, abilities of these nucleoside analogs to modulate primary antibody response to sheep red blood cells were evaluated in a C57BL/6 mutant mouse lacking NK cell activity (the beige mutation. C57BL/6-bg/bg). As previously found with C3H/He mice. NK cell activity was inhibited (Tub, doses 2-6 mg/kg/day for 3 days) or stimulated (FaraAMP, doses 75-250 mg/kg/day for 3 days) in heterozygous mice C57BL/6-bg/+. In support of the hypothesis, these nucleosides had no effect on primary antibody formation in the homozygous mutant mice at doses that clearly stimulated (Tub) or inhibited (FaraAMP) this immune response in heterozygous C57BL/6-bg/+ animals. This results was corroborated in C57BL/6 wild-type mice by abrogation of NK cell activity using a monoclonal antibody to the NK cell surface glycophisingolipid, ganglio-n-tetraosylceramide. We conclude that under the conditions of drug administration, modulation of primary antibody formation by Tub and FaraAMP in mice occurs indirectly via NK cells. Similar experiments using the potent ADA inhibitor, deoxycoformycin, indicated that its enhancement of primary antibody formation is independent of NK cell activity.