The principle of nonspecificity and acute toxicity

T I P S - May 1984

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Vi®w oi The principle of nonspecificity and acute toxicity L. Molinengo and M. Orsetti Instinae of Pharmacology a n d Pharmacognosy. School of Pharmacy. ! !nirersitv ~[ Turin, 10125 Tormo.

Italy. it was over 80 ),ears ago that activities of general mlaesthetws ,'ere observed to be correlated with their liposoh+bili~, rather than their molecular structures. Oth~'r evidence followed #z support of this view of drug 'nottspecificio,'. Howerer. #z the last 20 years" an overwhelming amount of data has been collected, which indicates that a pharmacological effect is always the result of a specific interaction between drugs and biochemical or bir vhysical processes. How can this apparently contradictor," evidence be reconcile, ~ To what extent shouM the physical properties of drugs be disregarded in deterrnbzing their pharmacological potencies? We propose a scheme which allows specific and nonspecific drug uzteractions to coexist. It is a scheme which can account for these contradictions. It may also account for the discrepancies often found between affinity constants derived from bbzding experiments and from dose-response curves. At the turn of the century Meyer t and Overton 2 observed that the hypnotic activity of general anaesthetics was correlated with their liposolubility. This was followed by a considerable amount of experimental data in support of the concept of non-specific drug action. including data demonstrating that other physical properties of molecules are also important; these properties tend to correl:qe with liposolubility, however. Fergusson 3 offered evidence that biological activity is correlated with thermodynamic activity, which is approximately inversely proportional to the solubility in water of the drug. Mullins4 showed that the drug molal volume is of prime importance in determining drug activity, because a given effect will be obtained 'when a constant fraction of the total volume of some non-aqueous phase in the cell is occupied by the molecules'4. An increase of the molal volume is generally correlated in organic compounds with an increase of liposolubility and with a reduction of water solubility. These correlations between physical properties of the molecules and their pharmacological activity have been taken as evidence that, in certain cases, biological activity is unrelated to molecular structure. These 'nonspecific drugs' would act simply as foreign

bodies which accumulate in some vitally important part and someho,~, disorganize biological processes. The principle of 'drug nonspecificity" has been accepted for differing depressive effects, including deathS: general 5 and local anaesthesiaS'; heart muscle depression;'": spinal cord depression"; and depression of oxygen consumption"~. Nevertheless. in many cases this principle is cllearly inadequate. For example, oils and other substances with a very high oil-water partition coefficient, a low water solubility and a rather high molal volume. are not hypnotic. Indeed. the lilx~soluble camphor is a CNS stimulant. This. ma~ indicate that the principle of "nonspecificity' holds only for compouz~ds which have a molecular structure capable of eliciting the effect being studied. For example, the depression of the ..,;pinal reflexes caused by barbiturates seems to be correlated with their physical properties". However. a slight modification of their structure (methylation of lxnh nitrogen atoms), which causes practically no change in their physical properties. transforms the compounds into excitatory drugs, in fact. there is now ample evidence that the depression of the CNS caused by barbiturates is the consequence of a specific mechanism of action (a dopaminergic action at inhibitory synapses has been proposed).

Thermodynamic model To explain this apparently contradictory situation it would be re.sonable to accept that the process by which a drug reaches a standard concentration in a biologically active phase is regulated mainly by the physical properties of the drug. We may also accept that, among the several compartments reached by the drug. one may be activated by a specific drug-receptor interaction. Different drugs, according to their specific molecular structures, will activate different receptor populations, yet a common final result, sue', as death, may be observed. Depending on the experimental conditions and characteristics of the considered drugs, the principle of "non-specificity' will be valid when physical properties rather than receptor binding properties, dominate. In contrast, strict specificity will be observed when specific interaction at receptor sites prevails. We have formulated a thermodynamic model which assumes that a drug effect is the result of the co-exiqence of t~o processes, one non-specific and unrelated to molecular structure, and the other strictly specific. This model suggests that the slope of the linear correlations between activity and physical properties obtained for different effects are parallelS t: this has been verified for spinal cord depression, sartonus mu.~le contraction, local anaesthesia, cardiac inotropism and inhibition of ventricular automaticity. While this may be taken only as circumstantial evidence, the proposed ~hcme nevertheless offers an explanation which overcomes the dilficuities in accepting the principle of "non-specificity" as an alternative to a specific drug mechanism of action. .Acute toxicity Death is the common final i~int of pharmacological effects resulting from drug interactions with various biologically active sites Therefore. acute toxicity may be a good example of a pharmacological effect in which specific drug--receptor interactions and "nonspecific" drug effects may be expected. In the present discussion the method of evaluating acute toxicity of a drug based on the hyperbolic relationship between dose and time of death t2 may be of particular interest. The theoretical significance of these hyperbolae has never

© 1984. Els~'~r S,.'k-nce P u b l ~ e ~ B V . Am:a©"nhm olt~s - 0147~4,$02.00

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values are reported in Fig. 2. A good agreement between the theoretical curve, calculated from the averages of kt and k2, and the experimental points is evident. If the effect is irreversible (death for example) the second part of the curve of Fig. 2 is, of course, impossible to achieve. By omitting these 'impossible' points on the curve, we obtain a hyperbola (Fig. 3). We conclude, therefore, that the experimental hyperbola which describes' the relation between concentration and time needed to produce a selected effect is a good empirical approximation of part of the complete curve which may be derived from the equation proposed by Gehlen fol"drug distribution at recep torial sites. Considering that the physical properties of the molecules are generally determining factors in drug distribution we may infer that drug physical properties are of prime importance in determining the differences in the doses needed to kill at different times. However, this sup position cannot be used to interpret the differences among the doses which kill over a long period. After a very long time, an equilibrium condition is reached in the dynamics of drug distribution and therefore the physical characteristic of the molecules should

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Fig. !. Kinetics of ~-tubocurarme effect on frog gastrocnemius. The curre (continuous line) has been calculated according to: W = d . z fe

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been examined. We suggest that the hyperbola relati~lg dose and time needed to produce ,some selected effect is an empirical approximation to the complete curve which may be derived from the following equation proposed by Gehlen j3 for drug distribution at receptoi" sites: W = ,cI - z (e-*" - e -k;')

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in which W = effect, d = dose, t = time at which the effect (W) is obtained with the given &~.x and kt, k2 and z are constants. Wt: are interested in curves correlating do.~e with time at which a given effect is obtained. From equation (1) we obtai:r=

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in which the symbols have the same meaning as in equation 1. This equation gives t (time of onset of a constant effect W) as a function of dose (d). In order to obtain experhnental evidence to support these theoretical considerations, we measured at selected times the reduction of frog gasuocnemius con*r-actions caused by o-tubocurarine given in the frog's ventral lymphatic sac. From the experimental points, the conslants k l, k2 and z were obtained and the theoretical curve was calculated according to equation 1. In Fig. l the experimental points and the theoretical curve obtained with o - ~ (5 mg kg -! ) are r ~ . Shmlar ~u~ves have been obtained at various doses of D-tubo-

cmarine. The averages of k~ and k2 were used to calculate the curve given by equation 2. We selected as the constant effect (W) a 70% reduction of the response. The time at which the different doses caused a 70% effect were measured on the experimental tracings and these !

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The constants k l, kz and z are the means of the measures obtained from the kinetic curves of different doses of o-tubocurarine. The experimental points (0), measured on the tracings, give the time at which different doses caused a 70% effect.

TIPS- May 1984 play a less important role in determining the value of the LD.~.. in fact, we have shown =z that the LDs. values obtained from the hyperbolic relationship between dose and time of death are equivalent to the LD~0 values obtained from the linear relationship between log dose and percentage of dead animals. The LDso values obtained from the doseresponse curve may be considered, at least in first approximation, as the equilibrium constants of a specific drugreceptor reaction. Therefore, we are forced to accept that the LD~. values obtained from the time--concentration hyperbola are also determined by a specific interaction between receptor and molecules. This conclusion is also supported by inconsistent results found in correlations between activity and physical characteristics. For example, the disappearance of activity observed passing from heptanol to octanol '° or the exceptional activity of acetylcholine as an anti-autorhythmics certainly cannot be explained by taking into consideration only the physical properties of the molecules. Nevertheless, there is evidence that the physical properties of the molecules cannot be neglected when drug activity is evaluated in equilibrium conditions. In many cases a reduction of the differences between equi-active doses is observed when compounds are corrected for their physical characteristics~4. These apparent inconsistencies may be explained if we postulate that the effect is always the consequence of a highly specific interaction with a given receptor. But a given effect will only be obtained when a standard drug concentration is achieved at the receptor site.

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O-tubocu r a r i ne (rag I kg) Fig. 3. Relationship between the doses o f o-mbocurarine and the time m'eded to l,n~chwe a 70c'~ re=h,tam o f gastrocnemh~s muscle contraction. The continuotu line is redra,'n from part of the curve or Ftg. 2. Experimental points (01 correspond closely to the theoretical curre ¢continuo~L~ hnc/.

true concentration at the receptor is evaluated with the radioligand, but only the external concentration is known when the biological effect is utilized to evaluate the affinity constant of the drug-receptor reaction. In this situation the ratio between the external drug conConcluding remarks centration and the concentration at the The differences between equi-active receptor may, in certain cases, be modidoses of different compounds at equi- fied by the physical properties of the librium conditions are primarily deter- molecules. mined by differences in specific toolIn this context the unacceptable ecular structure, although in many cases 'principle of non-specificity' might the physical characteristics of the com- assume a new meaning. The considerable pounds may cause appreciable modifi- amount of evidence collected on the cations of the drug concentration neces- correlation between physical properties saw to obtain the selected effect. Such of the molecule and their biological interference of the physical properties activity cannot be' for1=',,otten and it of the molecules might be important in merits reconsideratk,n. certain cases to explain the discrepancy found between the affinity constants Reading list derived from binding experiments and ! Meyer. H. (I.~)9) Arch. E*p. Path, d. Phatvnakol, 42, l(Iq--12t~ from dose-response curves. In fact the

2 (~'cnon. E (!~11 Mmhen u/wr dw %ar/~,w. ~L Fisher. Jena 3 Fcrgu.,am. J. ( 1q51 i ,lh'cant:~me dt h,' .'~urt,,w .-'6, pp. 25-39. Colloques Intemat:~naux du CNRS. Paris 4 Mullin.~. L. ( I t ~ ) ('hem. Rev 54. 2X~LL'3 5 Fergtt~n. J. (Iq3q) Pn~'. R..~',~ tt,m/,,n~ Ser. B ;27..'~7--.1414 h Molinengo, L (It~l).4rch. Ita/..~(v. him,J,.,,,/ 25, 3118-327 7 Molincngo. L. ( I tYoS) Arch. hit i'lz,m~ha, ,, ~vn. Ther. 143. ~k-lt)7 8 Molinengo. L. (19¢~;) Fur. J, l)/t~tr,t,tcol 5. 23-28 tl Fubini. B. and Moltncngo. L I 1t1751BuU .~,,t. Ital. Biol. Spcr 51. l~14.-I~k'~ Ill Rang, H. P. (ltlO(l) Br. J Phdr,tdtol 15. 185 I I Irnbini, B. and Nlolincngo. I.. ( It~'51 B,IL 5,,t ;tul. Biol. Sper. 51. 1815 and IS22-1,~24 12 Molincngo, L. (lq,"~)]. Pharm. Plhlrmdtol 31, M3--344 13 (;chlen. W. 110331 ..trt'h Eff~ P,ah, Jl. I~harmako/. 171, 541 14 (;cro, H. (19711 in /)r/#'.~ Phar, uwoh~gy m Medicine (DiPalma, J. R.. c d ) , pp. 5 ~ =7, McGraw-Hill. New York