**3. Pharmacological activity of supramolecular complexes**

The pharamacological activity of the complexes was investigated *in vivo* on females and males of outbred white mice and on Wistar rats, obtained from the SPF vivarium of the Institute of Cytology and Genetics, Novosibirsk. All animal procedures and experiments followed the 1986 Convention on Humane Care and Use of Laboratory Animals. The activity was determined using standard pharmacological tests [46]. All studied complexes, with some exceptions, are AG-Drug or GA-Drug compositions of 10:1 weight ratio, which was found out to be of greatest efficacy [22].

## **3.1. Arabinogalactan-drug complexes**

As a special study has shown, AG complexing with nonsteroidal anti-inflammatory drugs and non-narcotic analgesics of various action mechanisms can reduce the required dose for 5-100 times and, hence, avoid the side effects typical of the drugs.

For instance, complexing with indomethacin allows 10 to 20 times dose reduction relative to the standard and induces twice fewer cases of lesion to gastric mucous membrane, with the same high anti-inflammatory activity [22, 24].

Administration of phenylbutazone in a complex with AG, at ten times lower pharmacon content, stimulated analgesic activity, both in the model of chemical effect and on thermal action, which may expand its applicability scope (Table 5).


**Table 5.** Analgesic activity of AG complexes with non-narcotic analgesics

AG complexes with 10 times smaller doses of ibuprofen and metamizole sodium showed a high analgesic activity in visceral pain models (the former) and in two models of test pain.

Studies in this line were continued, with the above approach and proceeding from the obtained results, on AG complexes with drugs that involve the central nervous system. Specifically, AG complexing with diazepam allowed reducing the dose for ten times and enhanced the anxiolytic effect. AG complexes with mezapam acted as standard anxiolytics at 20 times lower doses of the phamacon. The AG-clozapine complexing provides a two-fold dose decrease at a higher sedative action.

586 The Complex World of Polysaccharides

was found out to be of greatest efficacy [22].

**3.1. Arabinogalactan-drug complexes** 

same high anti-inflammatory activity [22, 24].

Metamizole sodium:

test pain.

\*p <0.05 relative to control

**3. Pharmacological activity of supramolecular complexes** 

5-100 times and, hence, avoid the side effects typical of the drugs.

action, which may expand its applicability scope (Table 5).

The pharamacological activity of the complexes was investigated *in vivo* on females and males of outbred white mice and on Wistar rats, obtained from the SPF vivarium of the Institute of Cytology and Genetics, Novosibirsk. All animal procedures and experiments followed the 1986 Convention on Humane Care and Use of Laboratory Animals. The activity was determined using standard pharmacological tests [46]. All studied complexes, with some exceptions, are AG-Drug or GA-Drug compositions of 10:1 weight ratio, which

As a special study has shown, AG complexing with nonsteroidal anti-inflammatory drugs and non-narcotic analgesics of various action mechanisms can reduce the required dose for

For instance, complexing with indomethacin allows 10 to 20 times dose reduction relative to the standard and induces twice fewer cases of lesion to gastric mucous membrane, with the

Administration of phenylbutazone in a complex with AG, at ten times lower pharmacon content, stimulated analgesic activity, both in the model of chemical effect and on thermal

Compounds Hot plate, s Acetic acid writhing model,

Control 28.73±2.34 4.76±0.26 Phenylbutazone:AG 1:10. 120 mg/kg per os 42.20±5.20\* 2.00±0.09\* Phenylbutazone, 12 mg/kg per os 11.40±0.80 4.25±0.50 Ibuprofen:AG 1:10. 200 mg/kg per os 23.50±2.43 0.75±0.01\* Ibuprofen, 200 mg/kg per os 19.5±2.45 1.50±0.03\*

AG 1:10. 50 mg/kg per os 32.50±3.70 1.75±0.08 Metamizole sodium, 50 mg/kg per os 30.40±3.20 0.63±0.10 Metamizole sodium, 5 mg/kg per os 17.70±2.30 4.25±0.75

AG complexes with 10 times smaller doses of ibuprofen and metamizole sodium showed a high analgesic activity in visceral pain models (the former) and in two models of

Studies in this line were continued, with the above approach and proceeding from the obtained results, on AG complexes with drugs that involve the central nervous system. Specifically, AG complexing with diazepam allowed reducing the dose for ten times and enhanced the anxiolytic effect. AG complexes with mezapam acted as standard anxiolytics

**Table 5.** Analgesic activity of AG complexes with non-narcotic analgesics

amount

Another objective was to study drugs involving the blood coagulation and cardiovascular systems. The AG-warfarin (WF) complex was tested on intragastric injection in females of Wistar rats, with prothrombin time (PT, in seconds) as the principal criterion of the action. PT is the classical laboratory test of the exterior blood coagulation pathway used to evaluate the system of hemostasis in general and the efficacy of warfarin therapy in particular. The complex was administered once, in a dose of 20 mg/kg body weight, which is equivalent to 2 mg/kg warfarin. In 24 hours after the injection, PT increased considerably (to 30 s against the 11.63 s for the intact control). With free warfarin, this time was 42 s, or 28.5% longer than with the AG-warfarin complex, but it equalized (21 s) for both agents in 48 hours after a single injection (Fig. 5,6).

**Figure 5.** Prothrombin time, 24 hours after single dosing of WF:AG and WF:GA.

**Figure 6.** Prothrombin time, 48 hours after single dosing of WF:AG and WF.

The difference in pharmacokinetics between the AG:WF complex and free warfarin was further explored after a single administration of 20 and 2 mg/kg body weight, respectively. Blood was sampled in 1, 8, 10, 12, 24, 48, and 72 hours after injection on decapitation. Fig. 7 shows average plasma warfarin contents, and pharmacokinetic parameters are listed in Table 6. The concentrations of the compounds increase in a similar way but free warfarin

reaches Cmax seven hours sooner (Tmax) than in complexes with AG, and the concentrations become equal in 24 hours after single dosing. Excretion, on the contrary, is slower in pure warfarin than in the complex, which is consistent with 27 % higher clearance (CL) in the complex than in the free drug. Thus, warfarin increases more smoothly when bound with AG than in the free form and thus poses lower bleeding risks associated with its abrupt rise during dosage adjustment. Furthermore, shorter mean retention times (MRT) for the AG:WF complex may secure the following injection and accelerate warfarin excretion in the case of drug withdrawal.

Thus, complexing of warfarin with AG increases its safety and reduces unwanted bleeding risks in the case of anti-coagulant treatment.

**Figure 7.** Mean plasma concentration–time profile of WF:AG and blank WF after single oral administration at a dose of 20 mg/kg (dose of WF is equal to 2 mg/kg) and 2 mg/kg, respectively.


**Table 6.** Pharmacokinetic parameters of WF and WF:AG

Another drug in which we studied the pharmacological effect of complexing with AG was nifedipine (NF). NF is a dihydropyridine blocker of slow calcium channels that dilates coronary and peripheral vessels and reduces the oxygen demand in myocardium. Nifedipine exerts a minor negative inotropic effect and a very weak antiarrhythmic action. Intravenous injection of 3.5 mg/kg AG:NF complex (0.35 mg/kg NF) caused 26 % drop of blood pressure, measured via a carotid cannula, while 0.35 mg/kg NF can provide only a 9% decrease.

Being aware that nifedipine has a pleiotropic antiarrhythmic effect, besides the basic hypotensive action, the NF:AG complex was investigated in this respect on intravenous injection in a model of arrhythmia induced with 250 mg/kg calcium chloride. The complex administered in a dose of 0.175 mg/kg body weight (0.0175 mg/kg NF) arrested lethal heart rate disorder in 100% and 65% of cases, respectively, when applied prior to and after exposure to the arrhythmogen. Pure NF at 0.0175 mg/kg had no antiarrhythmic effect in the model of calcium chloride arrhythmia.

Thus, the NF:AG complex has demonstrated a stronger hypotensive and antiarrhythmic action than pure NF on intravenous administration, while its effective hypotensive dose is ten times smaller. Arabinogalactan itself does not induce any statistically significant decrease in blood pressure and cardiac rates. Furthermore, it is important that the new method adds another water-soluble form of NF as there is the only soluble nifedipine (adalate) available in the market.

A similar hypotensive effect was obtained with nisoldipine, another dihydropyridine.

Complexing of hypoglycemic drugs (metformin, rosiglitazone, insulin) with AG increased their solubility but allowed no dose reduction, though it improved notably the state of animals exposed to a toxic dose of alloxan, an agent simulating trial hypergclycemia.

The reported pharmacological data on the AG complexes with these pharmacons agree with the results on complexing with terpenoids (glycirrhizic acid, stevioside, and rebaudioside). In both cases, complexing increases the basic activity of drugs, allows dose reduction and forms new properties. We suggest to call this effect complexing or clathration of pharmacons with plant-derived carbohydrate-bearing metabolic agents.

#### **3.2. Glycyrrhizic acid (GA)-drug complexes**

588 The Complex World of Polysaccharides

drug withdrawal.

risks in the case of anti-coagulant treatment.

**Table 6.** Pharmacokinetic parameters of WF and WF:AG

reaches Cmax seven hours sooner (Tmax) than in complexes with AG, and the concentrations become equal in 24 hours after single dosing. Excretion, on the contrary, is slower in pure warfarin than in the complex, which is consistent with 27 % higher clearance (CL) in the complex than in the free drug. Thus, warfarin increases more smoothly when bound with AG than in the free form and thus poses lower bleeding risks associated with its abrupt rise during dosage adjustment. Furthermore, shorter mean retention times (MRT) for the AG:WF complex may secure the following injection and accelerate warfarin excretion in the case of

Thus, complexing of warfarin with AG increases its safety and reduces unwanted bleeding

**Figure 7.** Mean plasma concentration–time profile of WF:AG and blank WF after single oral administration at a dose of 20 mg/kg (dose of WF is equal to 2 mg/kg) and 2 mg/kg, respectively.

Compounds WF, 2 mg/kg WF:AG, 20 mg/kg CL, ml/h 1.52±0.03 1.93±0.18\* MRT, h 31.39±1.82 21.81±2.38\* Terminal half life, T1/2, h 5.11±0.24 6.38±2.55 Tmax, h 11.00±1.41 18.00±8.49 Cmax, μg/ml 6.47±0.91 5.64±0.19 AUC, μg h/ml 263.01±0.02 208.34±20.03\*

\*p<0.05 against WF

Another drug in which we studied the pharmacological effect of complexing with AG was nifedipine (NF). NF is a dihydropyridine blocker of slow calcium channels that dilates coronary and peripheral vessels and reduces the oxygen demand in myocardium.

#### *3.2.1. Nonsteroidal anti-inflammatory drugs (NSAID)*

GA:NSAID complexes with acetylsalicylic acid (ASA), diclofenac (OF), phenylbutazone (BD), and indomethacin (IM) were synthesized in solutions [24] and in solid state [23]. Complexing was confirmed by spectrometry. In IR spectra, the bands of hydroxyl and carbonyl groups of the glycoside were shifted to short wave numbers.

All mentioned complexes show anti-inflammatory action in smaller doses than the primary drug, and have 3-11 times larger therapeutic index (LD50 /ED50) [47,48] (Table 7).

GA complexes with aspirin and diclofenac (GA:ASA, GA:OF) exerted a prominent anti-inflammatory action in six models of acute inflammation induced by carrageenan, formalin, histamine, serotonin, Difko's agar, and trypsin, as well as in the cases of chronic inflammation (cotton and pocket granulomas) in intact and adrenalectomized animals [48].



The anti-inflammatory action of the GA:IM complex is stronger than of the free drug at equal dosing (10 mg/kg body weight). GA:NSAID complexing also potentiates other (analgesic, antipyretic) biological activities [29]. The GA:OF complex exerted a more prominent anesthetic action than diclofenac in electric and thermal stimulation (57.5±2.0 and 43.2±2.6) and exceeded the amidopyrine effect in the case of thermoalgesic stimulation (23.4±1.1 and 18.5±1.4). The anesthetic activity of the GA: metamisole sodium (GA:AN) complex is 11.4 times higher relative to metamisole sodium (AN) alone [23] while and that of the GA:ASA complex exceeds the effect of aspirin in animals exposed to thermoalgesic stimuli. The GA complexes with ASA and OF are 3 and 2.3 times more potent pain relievers than the respective NSAID in acetyl choline writhing model. The GA:ASA complex demonstrates a 4 times higher therapeutic index than aspirin in the acetic acid writhing model. The GA:ASA and GA:ОF complexes show high antipyretic activity, twice larger than in the pure pharmacons [47,48].

Thus, water-soluble GA:ASA and GA:ОF complexes evoke prominent anti-inflammatory and antipyretic effects, their spectrum of pharmacological activities and therapeutic ratio being larger than in the respective NSAID. The complexes also induce a marked membranestabilizing effect and reduce accumulation of primary and secondary products of lipid peroxidation in animals with chronic inflammation.

Complexing of glycyrrhizic acid with ibuprofen increases the analgesic action of the latter at twice lower doses.

Furthermore, GA complexes irritate less strongly the gastric mucosal membrane than their NSAID counterparts. For instance, the GA:ASA complex promotes reparation of ulcers though the ulcerogenic activity of GA:ОF is minor. Both complexes diminish Е1 and Е2 blood prostaglandins in animals with chronic inflammation. The complexes can be recommended for clinical trials as anti-inflammatory agents, including for patients that suffer from ulcer of stomach and duodenum. The acute toxic effect of GA:NSAID complexes is 2 to 14 times as low as in the respective pharmacons (Table 8) [47, 48].

GA:NSAID complexing obviously exerts a synergetic effect of higher biological activity along with lower toxicity and weaker ulcerogenic action on the gastrointestinal tract, and has a higher water solubility. It is evident on comparing the pharmacological activities of the ASA:GA and OF:GA supramolecular complexes obtained by dissolution and solid-state mehcanochemical synthesis that the latter opens a promising perspective of a technologically preferable and saving way of producing highly active NSAI drugs [23].


**Table 8.** Acute toxicity of GA:NSAID complexes in mice (per os)

The GA:Rofecoxib complex at doses 50 and 100 mg/kg body weight shows no active antiinflammatory and analgesic effects but is more highly soluble.

## *3.2.2. Prostaglandins*

590 The Complex World of Polysaccharides

animals [48].

[47,48].

[47, 48].

twice lower doses.

GA : ASA (1:1) GA: OF (1:1) GA : BD (1:1) GA : AN (1:1)

chronic inflammation (cotton and pocket granulomas) in intact and adrenalectomized

Compounds Dose range of complex\* Dose range of free NSAID

The anti-inflammatory action of the GA:IM complex is stronger than of the free drug at equal dosing (10 mg/kg body weight). GA:NSAID complexing also potentiates other (analgesic, antipyretic) biological activities [29]. The GA:OF complex exerted a more prominent anesthetic action than diclofenac in electric and thermal stimulation (57.5±2.0 and 43.2±2.6) and exceeded the amidopyrine effect in the case of thermoalgesic stimulation (23.4±1.1 and 18.5±1.4). The anesthetic activity of the GA: metamisole sodium (GA:AN) complex is 11.4 times higher relative to metamisole sodium (AN) alone [23] while and that of the GA:ASA complex exceeds the effect of aspirin in animals exposed to thermoalgesic stimuli. The GA complexes with ASA and OF are 3 and 2.3 times more potent pain relievers than the respective NSAID in acetyl choline writhing model. The GA:ASA complex demonstrates a 4 times higher therapeutic index than aspirin in the acetic acid writhing model. The GA:ASA and GA:ОF complexes show high antipyretic activity, twice larger than in the pure pharmacons

Thus, water-soluble GA:ASA and GA:ОF complexes evoke prominent anti-inflammatory and antipyretic effects, their spectrum of pharmacological activities and therapeutic ratio being larger than in the respective NSAID. The complexes also induce a marked membranestabilizing effect and reduce accumulation of primary and secondary products of lipid

Complexing of glycyrrhizic acid with ibuprofen increases the analgesic action of the latter at

Furthermore, GA complexes irritate less strongly the gastric mucosal membrane than their NSAID counterparts. For instance, the GA:ASA complex promotes reparation of ulcers though the ulcerogenic activity of GA:ОF is minor. Both complexes diminish Е1 and Е2 blood prostaglandins in animals with chronic inflammation. The complexes can be recommended for clinical trials as anti-inflammatory agents, including for patients that suffer from ulcer of stomach and duodenum. The acute toxic effect of GA:NSAID complexes is 2 to 14 times as low as in the respective pharmacons (Table 8)

GA:NSAID complexing obviously exerts a synergetic effect of higher biological activity along with lower toxicity and weaker ulcerogenic action on the gastrointestinal tract, and

1900/98=19.4 310/8=33.7 880/56=15.7 570/55=10.3

LD50 /ED50; ED50 – effective dose

4500/82=54.8 1750/12.5=140 3150/62=50.8 8000/68=117.6

**Table 7.** Anti-inflammatory effects of GA complexes and free NSAID.

peroxidation in animals with chronic inflammation.

Prostaglandins have been of broad use in human and veterinary medicine for their ability, in small doses, of stimulating womb muscles.

Veterinary uses of prostaglandins have been especially important: prostaglandin-based drugs are employed in swine breeding for farrow synchronization and are highly potent in preparing cattle and horse females for artificial insemination; these drugs allowed solving many problems with puerperal complications in cows and horses.

Kloprostenol, one main veterinary drug, is made by multistage synthesis, like other prostaglandins, which imposes its high price. It is urgent to reduce the effective dose and at the same time to increase the stability of labile prostaglandins in finished drugs. Both solutions have been found through complexing E and F prostaglandins (PGE1, PGE2, PGF2α), sulprostone (SP) and kloprostenol with GA. The complexes were synthesized and tested for uterotonic activity (Table 9).

In experiments on rats and guinea pigs, the GA:PGE1 (1:1) and GA:PGF2α (1:1) complexes changed the amplitude of uterine actions twice more strongly than the same concentration of PGE1 sodium (10-8 g/ml). SP and PGE2 as complexes with GA (1:1) induced three times greater amplitudes and increased uteric tonicity [49].

An efficient veterinary drug of klatraprostin has been developed on the basis of GA complexing with kloprostenol, a well known synthetic luteolitic prostaglandin, at doses five times as low as in the world practice [48, 49]. The drug is cheaper than its imported analogs while its action is more physiological than in the best foreign counterparts.

Klatiram, which contains an aminoacid (tyrosine) besides GA and kloprostenol [48,49], has a still greater potency. It is more effective than estrofan though bears 100 times less prostaglandin (kloprostenol).


**Table 9.** Uterotonic activity of prostaglandins and their GA complexes (1:1) in rats *ex vivo*, phosphate buffer C = 10-8 g/ml. PGE1 , PGE2, SP, PGF2α.

### *3.2.3. Cardiovascular drugs and anticoagulant warfarin*

Pharmacological activity was also investigated in GA complexes with antiarrhythmic Lappaconitine hydrobromide (LA) and antihypertensive nifedipine (NF) drugs.

Lappaconitine hydrobromide belongs to the group of clinically used antiarrhythmic drugs and is administered to patients with various rhythm disorders, especially, ventricular arrhythmia, paroxysmal ciliary arrhythmia, and monofocal atrial tachycardia, but it has a drawback of high toxicity.

In special experiments on antiarrhythmic action of LA:GA complexes, the one patented as alaglizin [50] showed the highest efficacy. Alaglizin, being ten times less toxic than LA, induced antiarrhythmic effects in models of calcium chloride and aconitine arrhythmia and had the highest antiarrhythmic therapeutic index (LD50/ED50) among all available drugs of this kind. When administered at 0.125 mg/kg and 0.250 mg/kg body weight, alaglizin causes no influence on electrocardiogram parameters, according to an extended study in models of calcium chloride and adrenal arrhythmia. Intravenous injection of 0.125 mg/kg alaglizin prior to exposure to a lethal dose of calcium chloride blocked arrhythmia in 80% of rats; 0.250 mg/kg of alaglizin applied after arrhythmogenic СаCl2 stopped the already developed arrhythmia in 50% of animals. A single injection of 0.125 mg/kg and 0.250 mg/kg alaglizin in a model of adrenal arrhythmia prevented full development of arrhythmia; 50% and 100 % of animals recovered normal ECG parameters on receiving 0.125 mg/kg and 0.250 mg/kg alaglizin, respectively. Alaglizin in the model had an ЕD50 of 0.125 mg/kg body weight against 0.290 mg/kg in LA and thus contained 14 times lower lappaconitine.

The NF:GA complex exerted hypotensive action on intravenous injection of its water solution in rats at a dose with ten times lower NF [51, 52]. The hypotensive effect of GA complexing with nisoldipine (another dihydropyridine) was similar to that of complexes with AG.

Complexing can have an important consequence of amplifying the pleiotropic effect of pharmacons (see above), such as NF. The wanted antiarrhythmic effect of NF can be only achieved with a dose that causes almost critical blood pressure drop, but the NF:GA complex induces the same effect with 29 times lower NF than the hypotensive dose.

Thus, the NF:GA complex is a promising parenteral drug with universal activity against hypertensive crises attendant with arrhythmia.

A presumable action mechanism of the NF:GA complex was studied *in vitro* on neurons isolated from peripharyngeal ganglia of *Lymnacea stagnalis* molluscs. The provoked responses are arrested completely with an NF concentration as high as 3.0 mM but with 30 times as low concentration (0.1 mM) of NF:GA. The responses to NF are blocked faster and recover sooner after neuron washing than the responses induced by the complex, which indicates stronger binding of the latter with receptors and its more prolonged action. The higher receptor affinity of the complex is corroborated by comparing the NF and NF:GA effects on calcium channels [51] (Fig. 8)

1. amplitude of calcium currents induced by Nifedipine.

592 The Complex World of Polysaccharides

Compound Change of uterine

buffer C = 10-8 g/ml. PGE1 , PGE2, SP, PGF2α.

drawback of high toxicity.

lappaconitine.

with AG.

*3.2.3. Cardiovascular drugs and anticoagulant warfarin* 

contraction amplitude, % P Change in uterus

GA: PGE1 53.4±5.0 <0.002 49.4±1.2 <0.002 PGE1 24.3±1.5 <0.05 30.7±2.2 <0.05 GA : SP 150.0±11.0 <0.001 135.0±10.0 <.001 SP 50.0±5.0 <0.001 115.0±9.5 <0.001 GA : PGE2 63.5±6.0 <0.001 40.7±4.0 <0.002 PGE2 20.0±2.8 <0.05 33.5±2.4 <0.05 GA: PGF2α 55.6±5.0 <0.001 61.0±5.6 <0.001 PGF2α 27.8±1.5 <0.05 39.4±5.3 <0.02 **Table 9.** Uterotonic activity of prostaglandins and their GA complexes (1:1) in rats *ex vivo*, phosphate

Pharmacological activity was also investigated in GA complexes with antiarrhythmic

Lappaconitine hydrobromide belongs to the group of clinically used antiarrhythmic drugs and is administered to patients with various rhythm disorders, especially, ventricular arrhythmia, paroxysmal ciliary arrhythmia, and monofocal atrial tachycardia, but it has a

In special experiments on antiarrhythmic action of LA:GA complexes, the one patented as alaglizin [50] showed the highest efficacy. Alaglizin, being ten times less toxic than LA, induced antiarrhythmic effects in models of calcium chloride and aconitine arrhythmia and had the highest antiarrhythmic therapeutic index (LD50/ED50) among all available drugs of this kind. When administered at 0.125 mg/kg and 0.250 mg/kg body weight, alaglizin causes no influence on electrocardiogram parameters, according to an extended study in models of calcium chloride and adrenal arrhythmia. Intravenous injection of 0.125 mg/kg alaglizin prior to exposure to a lethal dose of calcium chloride blocked arrhythmia in 80% of rats; 0.250 mg/kg of alaglizin applied after arrhythmogenic СаCl2 stopped the already developed arrhythmia in 50% of animals. A single injection of 0.125 mg/kg and 0.250 mg/kg alaglizin in a model of adrenal arrhythmia prevented full development of arrhythmia; 50% and 100 % of animals recovered normal ECG parameters on receiving 0.125 mg/kg and 0.250 mg/kg alaglizin, respectively. Alaglizin in the model had an ЕD50 of 0.125 mg/kg body weight against 0.290 mg/kg in LA and thus contained 14 times lower

The NF:GA complex exerted hypotensive action on intravenous injection of its water solution in rats at a dose with ten times lower NF [51, 52]. The hypotensive effect of GA complexing with nisoldipine (another dihydropyridine) was similar to that of complexes

Complexing can have an important consequence of amplifying the pleiotropic effect of pharmacons (see above), such as NF. The wanted antiarrhythmic effect of NF can be only

Lappaconitine hydrobromide (LA) and antihypertensive nifedipine (NF) drugs.

tonus, % <sup>Р</sup>

2. amplitude of calcium currents induced by Nifedipine clathrate with glycyrrhizic acid.

Upward arrows show start point of the blocker action. Downward arrows show start point of blockers washout. X axis set as time in minutes.

Y axis set as amplitude of incoming current in pA.

**Figure 8.** Averaged changes in calcium current amplitude induced by action and washout of blockers within a group of neurons (12 cells in each group).

The propranolol-GA complex studied in terms of hypotensive activity ensured 14% blood pressure drop in normotensive animals at a dose of 0.2 mg/kg body weight (minimum dose 0.0025 mg/kg gave a 11% decrease). Pure propranolol decreases blood pressure for 11% in a dose of 0.2 mg/kg; a similar pressure drop may be achieved by the minimum dose 0.0025 mg/kg, but it is statistically unconfident. Therefore, complexing enhances and stabilizes the hypotensive action of propranolol and allows 12.5-fold reduction of its dose.

Furthermore, complexing amplifies the antiarrhythmic action (pleiotropic effect) of propranolol. Namely, 100% of animals exposed to 0.3 mg/kg arrhythomogenic 0.1 % adrenalin survived on administration of 0.0025 mg/kg GA:Propranolol, while only 40 % survived among those who received 0.0002 mg/kg propranolol (this being the dose contained in the complex). Control animals responded to adrenalin by fatal disrhythmia grading into ventricular fibrillation, and 100% of them died [53].

The complex of GA with anticoagulant warfarin was investigated in a dose of 20 mg/kg body weight, which corresponds to 2 mg/kg WF. Prothrombin time was first measured six hours after a single intragastric injection. The interval was chosen proceeding from pharmacokinetics of warfarin: its plasma metabolites reach the maximum in 6-12 hours in rats [54]. In our experiments, PT has shown a confident change only in the positive control group with 2 mg/kg WF, but it is not clinically significant as it fails to ensure the required increase in coagulation time. A significant PT increase was observed on single intragastric administration of WF, while the WF:GA complex induced a smooth PT rise as late as in 30 hours (two injections) after the beginning of the experiment and the values corresponding to the positive control (WF) were reached only in 54 hours (three injections). Thus, WF:GA complexing made WF more soluble in water but slowed down its wanted anticoagulant action, possibly because GA molecules screened its active centers (Fig. 9).

**Figure 9.** Prothrombin time of WF:GA

#### *3.2.4. Psychotropic drugs*

The complexation effect was discovered in a pharmacological study of GA complexes with antidepressant fluoxetine and with anxiolytic gamma-amino-beta-phenylbutirate hydrochloride. Fluoxetine (FL), N-methyl-3-phenyl-3-[4-(trifluoromethyl)phenoxy]propan-1-amine acts as a depression antagonist by inhibiting serotonin neuronal uptake in CNS. Antidepressants are known to have many drawbacks, such as large doses, a narrow activity spectrum, high toxicity and prolonged elimination that involves liver cells and exerts deleterious effects on the renal function.

The study of fluoxetine complexes with GA (hereafter fluaglizin) aimed primarily at checking the possibility to alleviate the side effects of the pharmacon. In preliminary testing, the 1:10 FL:GA complex showed the highest activity. The complex, with 0.072 pharmacon parts in its one weight part, was patented under the name of fluaglizin (FG) [55,56]. Note that its LD50 exceeds 5000 mg/kg body weight against LD50 =248 mg/kg in fluoxetine.

Fluaglizin exerts a more prominent antidepressant effect than fluoxetine on single-dose administration in the Porsolt test and is a stronger serotonin uptake inhibitor. For instance, it suppressed the action of chloral hydrate more effectively than fluoxetine in a test with 5 hydroxytryptophan. Like fluoxetine, fluaglizin lacks anxiolytic activity.

The antidepressive effect of fluaglizin was identical to that of fluoxetine in a model of social depression in mice, namely, the animals became twice more communicative toward familiar and unfamiliar partners (had twice greater frequency and time of contacts). The dose of fluoxetine in the FL:GA complex is 1.08 mg/kg body weight against the standard 15 mg/kg. Fluaglizin, like fluoxetine, prevents blood glucose drop and attenuates peroxidation normalizing the antioxidant status of depressed individuals [55, 56].

In order to understand the mechanism of FL:GA action and compare it with fluoxetine, we studied its effect on contents of catecholamines and their precursors in different brain parts on single and therapeutic 25 mg/kg administration. The 17 times lower dose of fluoxetine in FL:GA complexes induced a weaker effect on serotonin uptake and triggered dophamine exchange in brain [57]. The nootropic activity of fluoxetine, first mentioned in [48], shows up also in the FL:GA complex.

Fluoxetine (30 μM) is known to suppress epileptiform activity which is evident in 50% lower-amplitude oscillations of electric potential (reflecting the activity of nerve cells) in response to pulse stimulation of hippocampus on the background of picrotoxin action.

Like fluoxetine, fluaglizin inhibits bikukulin-induced epileptiform activity in hippocampus sections in rats and acts as epilepsy antagonist [48].

Phenybut (FB), 4-amino-3-phenyl-butyric acid, is a nootropic and tranquilizing drug which relieves stress and anxiety and improves sleep. It is used in clinical practice against asthenia, neurotic anxiety, and sleep disorders, prior to surgery, and for preventing naupathia. It, however, has drawbacks of inducing somnolence and allergic reactions.

The FB:GA complex is twice less toxic than FB and stimulates cognitive activity in the same way as the pharmacon and GABA but, unlike the two latter, it provides a 20% increase in memorizing abilities in animals and attenuates sedative effects [48].

#### *3.2.5. Anticancer drugs*

594 The Complex World of Polysaccharides

**Figure 9.** Prothrombin time of WF:GA

deleterious effects on the renal function.

*3.2.4. Psychotropic drugs* 

6

24

30

**Time, hours**

54

survived among those who received 0.0002 mg/kg propranolol (this being the dose contained in the complex). Control animals responded to adrenalin by fatal disrhythmia

The complex of GA with anticoagulant warfarin was investigated in a dose of 20 mg/kg body weight, which corresponds to 2 mg/kg WF. Prothrombin time was first measured six hours after a single intragastric injection. The interval was chosen proceeding from pharmacokinetics of warfarin: its plasma metabolites reach the maximum in 6-12 hours in rats [54]. In our experiments, PT has shown a confident change only in the positive control group with 2 mg/kg WF, but it is not clinically significant as it fails to ensure the required increase in coagulation time. A significant PT increase was observed on single intragastric administration of WF, while the WF:GA complex induced a smooth PT rise as late as in 30 hours (two injections) after the beginning of the experiment and the values corresponding to the positive control (WF) were reached only in 54 hours (three injections). Thus, WF:GA complexing made WF more soluble in water but slowed down its wanted anticoagulant

**38,8**

**42**

**49,4**

The complexation effect was discovered in a pharmacological study of GA complexes with antidepressant fluoxetine and with anxiolytic gamma-amino-beta-phenylbutirate hydrochloride. Fluoxetine (FL), N-methyl-3-phenyl-3-[4-(trifluoromethyl)phenoxy]propan-1-amine acts as a depression antagonist by inhibiting serotonin neuronal uptake in CNS. Antidepressants are known to have many drawbacks, such as large doses, a narrow activity spectrum, high toxicity and prolonged elimination that involves liver cells and exerts

WF:GA, 20 mg/kg WF, 2 mg/kg WF Nyc, 2 mg/kg Saline

**24,88**

0 10 20 30 40 50 60

**Prothrombin time, sec**

**22,39**

grading into ventricular fibrillation, and 100% of them died [53].

action, possibly because GA molecules screened its active centers (Fig. 9).

**15**

**14,54**

**11,8 11,63**

**11,63**

**11,63**

**11,63**

GA complexes (1:1) with 5-fluorouracil, tegafur, and daunorubicin hydrochloride were synthesized in solution [58]. Complexing decreases the toxicity of the drugs and increases their solubility in water. The GA complex with fluorouracil exerts antineoplastic action with respect to Pliss lymphosarcoma, B-16 melanoma, and Geren carcinoma. The effective indices are, respectively, 3.05; 2.11, and 1.76. The tumor growth inhibition is 67.2%; 53.4 %; 87.1 %.
