Central Finding of Linear Relation between ZIA and Efficacy
Chapter 5. - Central Finding of Linear Relation between ZIA and Efficacy

Chapter Executive Summary

Hydrated Zn2+ ions at pH 7.4 have been shown to have a broad range of effects in fighting common colds. In vivo, zinc gluconate lozenges with high daily ZIA values have rapidly reduced cold duration and severity; lozenges with moderate ZIA values show only late efficacy against duration; and, with a minor exception, lozenges with low, zero, and negative daily ZIA values failed in clinical studies.

Clearly, different results have occurred (as mentioned in Chapter 4), and a method to reconcile differences is needed. By organizing the data using ZIA values and changes in common cold duration, a linear relationship between ZIA and reductions in duration in colds results. A surprising increase in duration usually occurs using lozenges yielding negatively charged zinc species.

Table 7. ZIA Factors (zinc, fraction Zn2+, dissolution times, doses/day, and saliva generated)

Study Lozenge	 Amount of  Fraction  Dissolution  Doses Saliva	Lozenge 
		 zinc (mg)  Zn2+      Time (min)  /day 	(grams) (grams)
________________________________________________________________________

Eby zinc gluconate
in nonsoluble 
tablet		  23.0	    0.30	30	    9	 15.0	0.66

MRC zinc gluconate
in fructose 
tablet		  23.0	    0.30	19	    9	22.0	1.00

McNeil zinc gluconate
in mixed tablet	  11.5	    0.30	15	   10	17.5	1.56

Danish 4.5 mg zinc 
gluconate in 
maltitol lozenge   4.5	    0.30	15    	    12	17.0	3.00

Zinc orotate 
lozenge		  37.0      0.00	40	     6	18.0	3.00

Zinc aspartate 
lozenge		  24.0	    0.00	14	     9	18.0	3.00

Zinc gluconate 
and citric acid	  23.0      0.00	15	     9  35.0	4.50

Zinc acetate 
with tartaric,acid 
& sodium bicarb-
onate in mannitol 10.0	    0.00	10	     9	44.0	3.00

Zinc gluconate-
glycine	(10 mole) 23.7	    0.00	15	     9  26.3	4.50
_____________________________________________________________________

ZIA Factors

Data collected during analysis of lozenge performance and daily ZIA calculations include mg zinc, fraction of zinc as hydrated Zn2+ ions, length of time to dissolve lozenges in minutes, number of doses/day, and saliva volume produced by zinc lozenges (numerically equivalent to weight of saliva minus lozenge weight). Data are summarized in Table 7.

Data were obtained by lozenge dissolution/expectoration experimentation using lozenges based upon descriptions of the lozenges in the original clinical reports or upon lozenge formulations and data supplied by the original authors or lozenge manufacturers. Every possible effort was made to use the exact lozenges used by the trialists or replications exactly duplicating the chemical and physical properties of the lozenges.

Comparative ZIA Values

Comparison of zinc ion availability (ZIA) values for clinical studies shows close relationship to actual clinical results. The finding of reasonable linearity of those relationships represents the central finding of this report. For scientific objectivity, results from experimental zinc lozenge treatments of common colds in the various published studies must be compared in an identical manner through ZIA reduction or increase in common cold duration. ZIA is the concentration of Zn2+ ions applied to the oral mucosa over time, which equals: [0.7697 times mg zinc, times fraction as Zn2+ ion, times time to dissolve (minutes), times number of doses per day], divided by [total mg saliva minus mg lozenge weight as approximation of saliva volume in ml]. The constant 0.7697 sets the Eby and co-worker reference value of 100 for ease in comparison. (See Chapter 3, Zinc Ion Availability Values, for more information on ZIA values and their calculation.)

Table 8. Study lozenges, ZIA values, electronic charge, and reduction in duration of colds.

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Study Lozenges		ZIA values	Charges		Efficacy	  
_________________________________________________________________________

Eby (23 mg zinc) 
zinc gluconate in							
nonsoluble tablets	 100		2+,1+,0		7-day reduction in
							duration of colds
MRCC (23 mg zinc)
zinc gluconate in
fructose tablets	  44		2+,1+,0		4.8-day reduction in 							duration of colds
							

McNeil (11.5 mg zinc) 
zinc gluconate in
mixed base  		  25 		2+,1+,0		1.6-day	reduction in
							duration of colds
									
Danish 4.5 mg zinc 
gluconate in maltitol	 13.4		2+,1+,0		none
							
Zinc orotate lozenges	  0		0		none

Zinc aspartate lozenge	  0		0		none

Zinc gluconate and 
1.33 mole citric acid    -11 *		0, N-   	1-day increase in
							duration of colds
							& worsened symptoms

Zinc acetate with very 
large molar excesses	 
of tartaric acid and 					
sodium bicarbonate					 
in mannitol lozenge	-55 *		0, N-		4.4-day increase in
							duration of colds
							& worsened symptoms
Zinc gluconate-glycine 
in hard candy lozenge
(10 mole glycine 1992)	Unknown		0, N-	        1.3 day reduction in
		       (negative)			duration of colds
____________________________________________________________________________

     *  Negative ZIA values were determined by projection.

Table 8 data (excluding negative values) were analyzed to determine correlation between zinc ion availability (ZIA) and change in duration of common colds in these studies. Spearman's rank difference correlation method was used. Spearman's is most useful with samples of small size.(1) The statistic was r (rho) = 0.96. The test of the hypothesis that the population correlation is zero is significant beyond 0.02 level in a 2-tailed test. From a statistician's perspective, "one is warranted in rejecting the null hypothesis that there is no relationship between ZIA values and reduction in duration in common colds." Additional data using lozenges above ZIA value 70 would be helpful in confirming and extending these observations.

By comparing daily ZIA values and reduction in duration of common colds, a linear relationship can be established between these (Figure 19), and linearity is the central finding of this report. Reasonable linearity through the range shows that there is a good relationship between ZIA values and reduction in duration of common colds in these studies. This linearity can be used to predict the efficacy of clinically untested zinc lozenges based upon ZIA values of experimental lozenges having zinc complexed with very weak ligands and no added zinc chelators. Linearity shows that seemingly divergent results of studies by Eby, Douglas, Farr, Smith, and others are reconciled by considering zinc ionization availability (ZIA).

From the reports analyzed, ZIA and Zn2+ ion, but not zinc compounds, are correlated with the reduction in duration of common colds. Correlation is consistent with the findings of Merluzzi and co-workers for in vitro activity of zinc against rhinoviruses.(2) Reduction in duration is also correlated with Zn2+ ion-laden saliva concentration, but it is not a reliable indicator of future lozenge efficacy, as duration of oral contact by Zn2+ ions must be considered. One can also argue that efficacy is linearly correlated only with the concentration of zinc gluconate, because only zinc gluconate-derived Zn2+ ions have shown efficacy in published studies reported to date. However, unreported experimental evidence with zinc acetate lozenges in many subjects clearly demonstrates the importance of availability of Zn2+ ions in shortening colds.

Lozenges having a positive ZIA value reduce the duration of common colds in a dose-response manner.  ZIA 25 lozenges reduce duration of colds by about 1-1/2 day, while lozenges having a ZIA value of 44 reduce the duration of colds by 4.8 days, and lozenges having a ZIA value of 100 reduce the duration of colds by 7 days.  Lozenges containing tightly bound zinc complexes have a zero ZIA and do not change the duration of colds.  Lozenges releasing negatively charged zinc species at physiologic pH increase the duration of common colds in a dose-response manner.

Figure 19. Relationship of zinc ion availability (ZIA) values and reduction in duration of common colds in days (y = 0.077x - 0.16).

The highly efficacious lozenges tested by Eby and colleagues in 1984 had a ZIA value more than twice as high as the next highest lozenge (MRC), and much higher than the other lozenges. Absence of a significant effect from Zn2+ ions on the rate and amount of viruses excreted by volunteer in the study by Al-Nakib and co-workers at the Medical Research Council Common Cold Unit,(3) does not mean antirhinoviral effect is absent using lozenges with higher, more efficacious ZIA values and higher Zn2+ ion salivary concentrations. A ZIA value of 50 and a 5 mMol salivary Zn2+ ion concentration appear to be thresholds for significant early Zn2+ ion effects on the severity and duration of colds. One might expect antirhinoviral activity, interferon induction, histamine mitigation, and cell membrane stabilization and drying effects to occur at sufficiently high ZIA, perhaps at about ZIA 100 and 7.4 mMol Zn2+ ion concentration, where a much stronger clinical effect was observed.

Linearity is attributed to diffusion of hydrated Zn2+ ions across biologic membranes and through tissues according to Fick's first and second laws with electrophoretic effects. These observations are the most readily observable examples of biologically closed electric circuits, as first proposed by Nordenström.(4)

Along with the strong repelling effects of nasal mucus and cilia on foreign substances introduced to the nose, as noted by Aoki(5) and many others, the differential repels intranasally introduced Zn2+ ions from mucosal surfaces, further explaining lack of efficacy from zinc nasal sprays.

The importance of the amount of zinc available, the fraction as Zn2+ ion, lozenge dissolution times, saliva generation, and dosages per day can all be understood as equally important in this system, as no factor was emphasized over another. Within certain limits, the system demonstrates an important tool needed to design successful common cold lozenges. Dissolution times and saliva production are as important as amount of Zn2+ ions released and appear to be important areas for developmental efforts, while increasing the number of lozenges used per day is a simple method of increasing efficacy.

Even though the system shows linearity throughout the range studied, the actual curve is probably parabolic with an upward slope from negative ZIA values through about ZIA 500. At some point between about ZIA 500 and perhaps ZIA 1000, the curve may level off. At some point over 1000, the curve may develop a downward slope demonstrating mast cell degranulation and general tissue injury caused by toxic concentration of Zn2+ ions as described by in vitro and toxicity studies.

Projection of Negative ZIA Values

Negative ZIA values for 2 of the 3 zinc lozenges having excess strong zinc chelators were estimated by straight-line projection of the non-negative values using the equation found in the legend of Figure 19. The zinc gluconate-glycine data-point, actually a horizontal line at +1.27 days, would be located in the upper-left quadrant of Figure 19, but it was omitted as it would have caused confusion and it was meaningless information. No negative value was used in determining r or the curve. Lengthened colds were associated with several lozenge formulations having negative ZIA values -- that is -- lozenges releasing negatively (and neutrally) charged zinc species at pH 7.4.

One may question the validity of negative ZIA value estimates, but the fact remains that lozenges releasing negatively charged zinc species (ZnLN-) increased the duration of common colds in a dose-response manner relative to placebo.

As discussed in Chapter 2, Zn2+ ions are highly concentrated (4 to 20 mMol) in mast cell and basophil granules. Zinc2+ ions are released during degranulation of these cells during inflammation. ZnLN- released from these lozenges binds native Zn2+ ions present in oral and nasal tissues and fluids. If release of Zn2+ ions from these cells during inflammation has the function of inhibiting viral replication, stimulating T-cells, stabilizing cell membranes, regulating mast cell homeostasis, catabolizing histamine, and stimulating interferon production during common colds as they do in vitro, then one must assume that common colds would be lengthened by neutralizing native Zn2+ ions with ZnLN- to render them biologically unavailable at physiologic pH -- thus making colds worse.

Taste Scale and ZIA

Lozenge taste is a crucial criterion for commercialization of zinc lozenges. Each of the studies following Eby and co-workers used lozenges selected for pleasant tastes. Zinc gluconate lozenges known not to have a pleasant taste were not considered for testing. Even though the McNeil-sponsored lozenges had a bitter taste at the time of the study, it is safe to assume McNeil and General Nutrition representatives did not know zinc gluconate in combination with dextrose molecule would become dreadfully bitter after aging, or if they did, they had no idea how to correct the flavor-masking problem.

Had the eight studies been rated on a taste scale, relationships concerning ZIA values and taste could have been better developed. Volunteers have carefully taste-tested each of the lozenges (or nearly exact chemical copies) used in the eight clinical studies and offer the following subjective, relative ranking of zinc lozenge taste acceptability. Although taste preferences are highly subjective, differences noted were obvious. On a scale of 0 to 10 (with 0 being dreadfully bitter, 5 being acceptable but not particularly pleasant, and 10 being as pleasant as candy or no taste sensation other than astringency), the lozenges are rated in Table 9 below.

Taste acceptability is dependent upon the zinc complex, other lozenge ingredients and their reactions with zinc, but taste acceptability is not dependent upon ZIA.

The 1984 lozenges tested by Eby and co-workers had not only the taste of zinc gluconate but also the taste of dicalcium phosphate, and both active and placebo lozenges primarily tasted chalky. Lozenges used by the MRC relied upon strong, sweet Italian flavors and fructose to preserve both taste and promote efficacy through absolute compliance. Without other soluble ingredients, taste was stable and reasonably pleasant. The McNeil lozenges were so bitter from complexation of zinc gluconate with dextrose that clinical failure and lack of compliance resulted. The Australian lozenges relied upon greatly reduced dosage and chelation by tartaric acid and sodium bicarbonate to eliminate astringency, also eliminating efficacy of the modified zinc acetate lozenges. The taste associated with their lozenges was of flavored tartaric acid.

Table 9. Lozenge taste, salivary pH, near aftertaste and overnight aftertaste.

_____________________________________________________________________
 	
Study lozenges	   ZIA     Salivary  Lozenge   Near 	   Overnight 
		   values  pH	     taste     aftertaste* aftertaste  
________________________________________________________________________

Eby zinc gluconate 
(23 mg zinc)
tablets		      100     5.5	5	  6		8

MRC zinc gluconate 
(23 mg zinc)
fructose lozenge       44     5.4	9	  7		8

McNeil zinc gluconate 
(11.5 mg zinc) 3-g 
mixed sweeteners 
lozenge		       25     6.4	0	  3		5
	
Danish zinc gluconate 
(4.5 mg zinc)
maltitol lozenges     13.4     6.4	7	  8		9

Zinc orotate
(37 mg zinc) 
3.6-g lozenges         0.0      7	6	  8 	       10

Zinc aspartate 
(24 mg zinc) lozenge   0.0      7	8	 10	       10

Bristol-Myers zinc 
gluconate (23 mg zinc)
and extramolar citric 
acid hard candy	
(sucrose/corn syrup)
lozenges	       -11      4.3  	 8   	 10 	       10

Australian zinc acetate
(10 mg zinc) effervescent 
lozenges with tartaric acid,
sodium bicarbonate in 
mannitol 	       -55      4.1  	 7	  8		 9

Godfrey zinc 
gluconate-glycine    Unknown    5.0 	 9	  9		10
		    (negative)
_______________________________________________________________________

* Near aftertaste is aftertaste observed during the first 2 hours after completion of lozenge.

The Bristol-Myers lozenges relied upon chelation by citric acid and orange flavor oils to eliminate objectionable taste with a resulting loss of efficacy. Insoluble zinc orotate in lozenges was essentially tasteless but lozenges were orally abrasive. Zinc aspartate lozenges were naturally pleasant-tasting, were highly chelated, released no Zn2+ ions, and had no efficacy. Godfrey designed zinc gluconate-glycine (10 mole) lozenges that were pleasant-tasting but yielded no Zn2+ ions at physiologic pH 7.4. Although Godfrey's lozenges were pseudo-astringent, such is the result of some Zn2+ ion being available at salivary pH 5.0 but not at the higher physiologic pH capable of affecting a reduction in common cold duration. All pH tests were conducted with both Nester and Corning pH meters calibrated with fresh Ricca pH 4, 7, and 10 reference buffer solutions. Natural salivary pH varies between pH 6.2 and 7.2, with pH 6.2 to 6.5 being observed in these tests.

Concession to Taste Through Use of a Double Loading Dose

Before the Eby 1984 clinical trial, the author determined that the minimum dosage should be about 50 mg zinc from zinc gluconate every 2 hours, not the 23-mg dosage used in the trial. However, lozenge harshness suggested some patients would reject treatment, and the dosage was compromised to 23 mg doses every 2 hours. The full dosage (46 mg zinc) was retained as a loading dose because considerable anecdotal evidence was available to the author that the first dose taken was important to achieve extremely rapid treatment responses when taken sufficiently early in a cold.

Treatment responses more rapid than documented occur frequently in field use. Faster responses are possible using larger doses or more frequent dosing with zinc gluconate or zinc acetate. Extremely rapid responses occur if treatment is started upon announcement by a throat or nose tickle of an incipient cold.

No other researchers incorporated a loading dose into their protocols. The ZIA value of the loading dose described by Eby and co-workers was therefore 4.6 times higher than the ZIA value from the first lozenge used in the MRC trial, eight times the McNeil lozenge value, and so forth.

Zn2+ Ions and Zinc Compound Molar Concentration with ZIA Values

Although ZIA values are the best indicators of successful lozenges, Zn2+ ion concentrations are noteworthy as concentrations are important components of ZIA values (see Table 10). Clear distinctions can be detected in salivary Zn2+ ion concentration and zinc compound concentration. The most effective lozenges produced the highest ZIA values and salivary Zn2+ ion concentrations, while the largest concentration of zinc in saliva was found with the ineffective zinc orotate lozenges. For comparison, normal zinc serum concentration is 0.015 + 0.006 mMol; and minimum antirhinoviral- and interferon-inducing concentrations of Zn2+ ions are 0.05 mMol according to Merluzzi2 and Geist and colleagues,(6) or 0.10 mMol according to Korant and co-workers.(7) Zinc gluconate releases 30 percent of its zinc as Zn2+ ion at physiologic pH 7.4.

Table 10. Zn2+ ion and zinc compound salivary molar concentration compared with ZIA values.

___________________________________________________________________________

Study Lozenge			ZIA		Zn2+ mMol   	Zinc 
				Value		at pH 7.4	mMol	
___________________________________________________________________________ 

Eby 23 zinc gluconate in 
nonsoluble tablets		100.0		   7.4		24.7
	
MRC 23 zinc gluconate in 
fructose tablets		  44		   5.0		16.8

McNeil 11.5 mg zinc 
gluconate lozenges		  25		   3.3		11.1

Danish 4.5 mg zinc gluconate 
lozenges			  13.4		   1.5		 5.0

Zinc orotate lozenge	           0.0		   0.0		31.0

Zinc aspartate lozenge		   0.0		   0.0		20.0
	
Zinc gluconate-citrate lozenges	 -11		   0.0		10.0

Zinc acetate-tartarate
  -carbonate lozenges	   	  -55		   0.0	         3.0

Zinc gluconate-glycine lozenges  Negative	   0.0		13.9
				(unknown)
_______________________________________________________________________

In 1989, Vincent J. Merluzzi, and co-workers showed antirhinoviral effects of zinc in HeLa cells were directly related to the amount of Zn2+ ion available and unrelated to the total amount of zinc complex available.(2)

Similarly, the present author has shown the reduction in duration of common colds to depend upon ZIA, which depends upon Zn2+ ion concentration and time of contact with oral mucosal membranes, -- not the amount of zinc compound.

Salivary Zn2+ ion concentration is an oversimplified and misleading indicator of efficacy. High salivary Zn2+ ion concentrations may have a low ZIA value if lozenges dissolve too quickly. The time that Zn2+ ion is applied (as time to dissolve lozenges) and number of doses per day are important variables in determining ZIA values of experimental lozenges.

Good examples of the limited relationship in ZIA values between chemically similar 23-mg zinc as zinc acetate lozenges may be seen in Chapter 7, Table 11.

Chapter 5. References


Chapter 6. - Effects of Flavor-Masking on Efficacy