[New adamantane derivatives and ways of overcoming the resistance of influenza A viruses to rimantadine and amantadine].

The amino acid and peptide derivatives of 1-adamantane carboxylic acid and rimantadine (18 compounds) have been first synthesized and investigated for their activity against influenza A virus (H1N1, H1N1v). In a series of obtained adamantine derivatives, some compounds have been found to be able to inhibit rimantadine-resistant influenza A virus strains. Thus, the antiviral properties of rimantadine can be restored.

Influence of an additional 2-amino substituent of the 1-aminoethyl pharmacophore group on the potency of rimantadine against influenza virus A.

We examined whether the incorporation of a second amino group into the 1-aminoethyl pharmacophore of rimantadine 2 and into the piperidine pharmacophore of the heterocyclic rimantadine 4 was compatible with anti-influenza virus A activity. The new synthetic molecules are capable of forming two hydrogen bonds within the receptor. We identified molecules 8 and 16, bearing the adamantyl and 1,2-diaminoethyl groups, which are equipotent to rimantadine 2 bearing the adamantyl and 1-aminoethyl pharmacophore groups. Interestingly, diamino compound 16 is a 4-fold more potent inhibitor than its parent monoamino heterocyclic rimantadine 4 propably because of additional hydrogen bonding interactions with the M2 protein receptor.

Heterocyclic rimantadine analogues with antiviral activity.

2-(1-Adamantyl)-2-methyl-pyrrolidines 3 and 4, 2-(1-adamantyl)-2-methyl-azetidines 5 and 6, and 2-(1-adamantyl)-2-methyl-aziridines 7 and 8 were synthesized and tested for their antiviral activity against influenza A. Parent molecules 3, 5, and 7 contain the alpha-methyl-1-adamantan-methanamine 2 pharmacophoric moiety (rimantadine). The ring size effect on anti-influenza A activity was investigated. Pyrrolidine 3 was the most potent anti-influenza virus A compound, 9-fold more potent than rimantadine 2, 27-fold more potent than amantadine 1, and 22-fold more potent than ribavirin. Azetidines 5 and 6 were both markedly active against influenza A H2N2 virus, 10- to 20-fold more potent than amantadine. Aziridine 7 was almost devoid of any activity against H2N2 virus but exhibited borderline activity against H3N2 influenza A strain. Thus, it appears that changing the five-, to four- to a three-membered ring results in a drop of activity against influenza A virus.

Simultaneous liquid chromatographic assay of amantadine and its four related compounds in phosphate-buffered saline using 4-fluoro-7-nitro-2,1,3-benzoxadiazole as a fluorescent derivatization reagent.

Simultaneous HPLC assay of 1-adamantanamine hydrochloride (amantadine) and its four related compounds [2-adamantanamine hydrochloride (2-ADA), 1-adamantanmethylamine (ADAMA), 1-(1-adamantyl)ethylamine hydrochloride (rimantadine) and 3,5-dimethyl-1-adamantanamine hydrochloride (memantine)] in phosphate-buffered saline (pH 7.4) after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed. Phosphate-buffered saline samples were mixed with borate buffer and NBD-F solution in acetonitrile at 60 degrees C for 5 min and injected into HPLC. Five derivatives were well separated from each other. The lower limits of detection of amantadine, 2-ADA, ADAMA, rimantadine and memantine were 0.008, 0.001, 0.0008, 0.0015 and 0.01 microg/mL, respectively. The coefficients of variation for intra- and inter-day assay were less than 6.4 and 8.2%, respectively. The method presented was applied to a binding study of these compounds to human alpha(1)-acid glycoprotein. While affinity constants and capacities for ADAMA, rimantadine and memantine were calculated by means of Scatchard plots, those for the others were not determined. ADAMA, rimantadine and memantine were bound with different affinities and capacities. These results indicate that NBD-F is a good candidate as a fluorescent reagent to simultaneously determine amantadine and its four related compounds by HPLC after pre-column derivatization. Our method can be applied to binding studies for protein.

Simultaneous determination of amantadine and rimantadine by HPLC in rat plasma with pre-column derivatization and fluorescence detection for pharmacokinetic studies.

We investigated simultaneous high-performance liquid chromatographic (HPLC) determination of amantadine hydrochloride (AMA) and rimantadine hydrochloride (RIM) levels in rat plasma after fluorescent derivatization with o-phthalaldehyde and 2-mercaptoethanol. Afterwards, the method was applied to determine their pharmacokinetics. The retention times of AMA and RIM derivatives were 12.6 and 22.2 min and the lower limits of detection were 0.025 and 0.016 microg/mL, respectively. The coefficients of variation for intra- and inter-day assay of AMA and RIM were less than 5.1 and 7.6%, respectively. After i.v. administration of AMA or RIM to rats, the total body clearance and distribution volume at the steady-state of RIM were higher than those of AMA. Bioavailability of AMA and RIM was 34.9 and 37.2%, respectively. When AMA and RIM were p.o. co-administered, the area under the plasma concentration--time curve of RIM was significantly lower than that after RIM alone. On the other hand, pharmacokinetic parameters of AMA did not significantly change. These results indicate that our HPLC assay is simple, rapid, sensitive and reproducible for simultaneously determining AMA and RIM concentrations in rat plasma and is applicable to their pharmacokinetic studies. Also, co-administration of AMA and RIM may result in the lack of pharmacological effects of RIM.

Heterocyclic rimantadine analogues with antiviral activity.

2-(1-Adamantyl)pyrrolidines 6, 7, 2-(1-adamantyl)piperidines 10, 12a-c, 15a,b and 2-(1-adamantyl)hexahydroazepines 19, 21, 22 were synthesized and tested for their antiviral activity against influenza A, B viruses and the human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2). The synthetic procedure followed for the preparation of the parent piperidine 10 represents a general method for the synthesis of 2-alkyl- or cycloalkyl-substituted piperidine alkaloids. Parent aminoadamantanes 6, 10 and 19 contain the 1-aminoethyl pharmacophore group of rimantadine drug 2, extended into a saturated nitrogen heterocycle: pyrrolidine, piperidine and hexahydroazepine, respectively. The ring size effect in anti-influenza A activity was investigated. Rimantadine analogues 6 and 10 were, respectively, 6- and 4-fold more active than the drug Rimantadine 2, whereas the hexahydroazepine derivative 19 was inactive. Thus, enlargement from a 5-(pyrrolidine)- or 6-(piperidine)- to a 7-(hexahydroazepine)- membered heterocyclic ring dramatically reduced the anti-influenza virus A activity. Substitution of piperidine 10 with a dialkyaminoethyl group led to the active compounds 15a and 15b: compound 15a was active against influenza A virus whereas both 15a and 15b were active against HIV-1.

Are the 2-isomers of the drug rimantadine active anti-influenza A agents?

There is a lack of information in the medical chemistry literature concerning the anti-influenza A activity of the drug rimantadine's 2-isomer (2-rimantadine). We now present results showing that, although 2-adamantanamine (2-amantadine) 3 is only moderately active, some 2-rimantadine analogues are effective anti-influenza A virus agents in vitro. The 2-rimantadine analogues and their spirocyclobutane and spirocyclopentane congeners were synthesized through interesting routes. The 2-rimantadine analogues were 2-4 times more potent than rimantadine 2 against influenza virus A H2N2 strain; their spirocyclobutane congeners proved equally active to rimantadine 2. Two compounds exhibited a similar activity and one of the compounds was was fourfold more potent than rimantadine 2 against H3N2 strain.

Ileoanal pouch function is related to postprandial pouch tone.

BACKGROUND: Functional impairments are frequently observed in patients with an ileoanal pouch. Meal ingestion increases pouch tone and motility. Little is known, however, about the influence of meal-stimulated pouch characteristics on pouch function. The aim was to characterize basal and postprandial pouch motor and sensory characteristics in relation to clinical pouch function in patients with an ileoanal pouch. METHODS: Nineteen patients with an ileoanal pouch, without faecal incontinence but with either a high stool frequency (n = 8) or an adequate stool frequency (n = 11), underwent pressure distension of the pouch, by which pouch compliance and sensitivity characteristics were assessed using an electronic barostat. A set pressure procedure was performed to assess the influence of a meal on pouch tone and motility. RESULTS: Mean(s.d.) compliance was 10(6) and 11(4) ml/mmHg in the groups with poor and adequate pouch function respectively (P not significant). Mean(s.d.) visual analogue scale scores (0-10 cm) for urge at the highest pressure of 28 mmHg were 2.3(1.0) versus 2.3(2.4) cm respectively (P not significant); those for pain were 0.8(1.0) versus 0.5(0.7) (P not significant). Postprandially mean(s.d.) pouch volume decreased by 70(24) per cent in the group with poor pouch function and 29(25) per cent in the group with adequate pouch function (P < 0.01). The frequency and amplitude of phasic pouch contractions increased significantly postprandially, but no differences in motility characteristics were observed between the two groups. CONCLUSION: In patients with uniform pouch design and follow-up after pouch construction, pouch compliance and sensitivity were no different between patients with normal and high stool frequency; however, postprandial pouch tone was increased significantly in patients with a high stool frequency.

[The efficacy of Polirem in the prevention of influenza in servicemen]. / Effektivnost' polirema pri profilaktike grippa u voennosluzhashchikh.

The prophylactic influence of Polirem was studied during influenza A (H3N2) epidemic in cadets of military school. A considerable reduction of morbidity rate (alpha < 0.001) and the lowering of medical cases were found out in cadets who were given Polirem. There were no complications in this group of cadets. A considerable reduction of average durability of sickness was marked in this group. The authors recommend Polirem administration for coping epidemic outbreaks of influenza in military contingency.

[Comparative quantitative pharmacological-EEG analysis of the effects of psychostimulants]. / Sravnitel'nyi kolichestvennyi farmako-EEG-analiz deistviia psikhostimuliruiushchikh sredstv.

Amphetamine, caffeine, sydnocarb, meclofenoxate, adapromine, midantan, and nomifensine were studied for their effects on bioelectrical activity and Fourier EEG power spectra of the sensomotor cortex, dorsal hippocamp and lateral hypothalamus of freely behaving awake rats. The drop in the absolute power of all frequency ranges with the enhanced power of fast beta 1,2-ranges was common to the action of psychostimulants. In addition to the common properties, specific features of their action were revealed. Amphetamine, meclofenoxate, and nomifensine were found to increase the amplitude of the dominant peak in the theta-range and amphetamine shifts the frequency of the dominant peak to the region of faster ranges. The agents-induced electrophysiological changes correspond to the varying degrees of activation of the central nervous system, causing the optimization of behavioral functions, abolition of fatigue and drowsiness and enhancing physical and mental working capacity.

[The origin of resistance to chemicals of naturally occurring isolates of influenza A virus]. / Proiskhozhdenie rezistentnosti k khimiopreparatam u prirodnykh izoliatov virusa grippa A.

The mechanisms responsible for the formation of resistance of influenza A virus isolates during the natural circulation of the influenza viruses in the environment were studied. The influenza viruses H1N1 and H3N2 resistant to remantadine, adapromine, and deitiforine have been isolated in the USSR and Mongolia since 1982. The majority of natural resistant isolates appeared to be atypical both in antigenic properties and genomic structure as compared to the isolates prevalent in the common epidemic process. The nucleotide sequences of the M2 gene of some resistant strains and virus A/PR8/34 used in our country as an attenuation donor for preparation of killed recombinant vaccines. The electrophoretic mobility of genomic RNA of two resistant isolates is similar to that of the vaccine strain X-54 based on the virus A/PR/8/34. In this connection, the appearance of resistant strains in the environment may be due not only to spontaneous mutagenesis or selective drug actions, but also to the involvement into the circulation of vaccinal strains.

Functional reconstitution in lipid vesicles of influenza virus M2 protein expressed by baculovirus: evidence for proton transfer activity.

The influenza virus M2 protein was expressed from a recombinant baculovirus in Spodoptera frugiperda Sf9 cells, purified and reconstituted into artificial membrane vesicles. The specific inhibitor amantadine overcame the toxic activity of the protein and boosted the rate of M2 synthesis by a factor of 10, allowing yields of about 1 mg of purified M2 protein per g of Sf9 cells. M2 protein expressed in this system was phosphorylated and palmitoylated and displayed properties similar to the authentic virus protein. Purified wild-type M2 protein and an amantadine-resistant mutant M2 (M2 delta) with a deletion in the trans-membrane domain (amino acids 28 to 31) were incorporated into lipid vesicles, which were loaded with the fluorescent pH indicator pyranine. On imposition of an ionic gradient, M2 caused a decrease in intravesicular pH, which was susceptible to inhibition by 0.1 to 1 microM-rimantadine or N-ethyl-rimantadine. M2 delta behaved similarly but exhibited the expected drug resistance. These experiments indicate that isolated M2 functions as an ion channel and demonstrates in vitro M2-mediated proton translocation.

[Effect of various adamantane compounds on the reproduction of Sindbis virus. Isolation and properties of a resistant strain]. / Izuchenie deistviia nekotorykh soedinenii adamantana na reproduktsiiu virusa sindbis. Poluchenie i svoistva rezistnogo shtamma.

Rimantadine and its structural analogs, i. e. amide of 1-adamantane carboxylic acid (AACA) and 1-adamantane acetic acid amide, were shown to be able to inhibit reproduction of Sindbis virus in culture Vero cells. AACA had the maximum antiviral activity. Subcultures of the initial sensitive population of Sindbis virus in the presence of AACA led to formation of mutants resistant to AACA as well as to rimantadine, adamantane acetic acid amide and ammonium chloride. The Sindbis virus population was heterogenous in sensitivity to AACA, which was evident from isolation of separate clones with various levels of sensitivity to the above mentioned compounds from the population. It was found that reproduction of the AACA sensitive and resistant strains of Sindbis virus differed: the latent period of the resistant strain was 2 hours longer than that of the sensitive strain. The same effect was observed in the comparative study on synthesis of the virus-specific RNA.

[A spectral analysis of the effect of adapromine on brain bioelectrical activity]. / Spektral'nyi analiz vliianiia adapromina na bioélektricheskuiu aktivnost' mozga.

A study was made of the influence of adapromine on bioelectrical activity of the brain, sensorimotor cortex, dorsal hippocamp and lateral hypothalamus in freely moving wakeful rats. Adapromine was established to evoke a decrease of the amplitude of the dominant peak and dominant theta-activity in power spectra of the EEG in the cortex and hippocamp, with an increase of rapid wave activity in the beta 2 range in the right cortex and hippocamp. These changes attained maximum after 1 to 1.5 hour and lasted up to 4-5 hours after adapromine administration. These changes can be viewed as activation of the cortex and hippocamp, which may attest to the presence of antidepressive and psychostimulant effects in the action spectrum of adapromine. The specific influence of adapromine on catecholaminergic processes of the brain may lie at the basis of the above effects.

Synthesis and antiviral activity of metabolites of rimantadine.

The hydroxy metabolites of rimantadine (3-5) were synthesized and compared to amantadine (1) and rimantadine (2) for their ability to inhibit the replication of influenza viruses in vitro. All three metabolites were inhibitory to wild-type influenza A viruses (H3N2 and H1N1). In particular, 2-hydroxyrimantadine (3) showed similar activity to amantadine, but the 3- and 4-hydroxy metabolites (4 and 5, respectively), both of which are found in rimantadine-treated patients, showed only modest inhibitory activity. A rimantadine-resistant isolate of influenza A virus exhibited cross-resistance to amantadine and to each of the metabolites 3-5. None of the compounds were effective against influenza B virus.

[The isolation and study of the properties of current influenza A viruses (H1N1) with a natural resistance to remantadine]. / Vydelenie i izuchenie svoistv sovremennykh virusov grippa A (H1N1) s prirodnoi rezistentnost'iu k remantadinu.

Studies of influenza A viruses (H1N1) isolated in 1985-1988 revealed 4 strains with natural resistance to chemical drugs (remantadine, adapromine, deitiforin). Three of them were isolated in Mongolia (A/Mongolia/230/85, A/Mongolia/231/85, and A/Mongolia/128/86) and one in the USSR (A/Moscow/771/88). These strains differed from other isolates by the following features: (i) antigenic remoteness from reference A/Chile/1/83 and A/Taiwan/1/86; (ii) similarity in antigenic specificity with A/Khabarovsk/74/77; (iii) resistance to chemical drugs; (iv) high electrophoretic mobility of genes M and NS in PAG. The possible origin of these strains is discussed.

[Interferon and other immunological indices of influenza patients undergoing different methods of treatment]. / Interferon i drugie immunologicheskie pokazateli u bol'nykh grippom pri raznykh metodakh lecheniia.

The authors review the results of immunological examinations of patients with influenza given adapromine and virazole drugs as well as reflex therapy aimed at increasing general host resistance. Patients with influenza administered reflex therapy among other therapeutic measures developed more intensive increase of serum interferon activity in the acute period of the disease, with its more rapid decline; they had a high level of IgM and more intensive rise of specific antibody (in the HI test) as compared with the control group. In the group of patients treated with adapromine and virazole there was also a more significant increase of IgM level, early and significantly high level of IgG as compared with the patients given symptomatic drugs. No differences in the number of T-lymphocytes were found by the method of spontaneous rosette formation with sheep erythrocytes. Both in treatment with antiviral drugs and using reflex therapy, a more rapid disappearance of influenza antigen from nasal smears was observed than in patients of the control groups.