Synergistic combination of metal proteinates with beta-chlorovinyl dialkyl phosphates

Metals are transported throughout mammalian tissues and across the placental barrier to the foeti by the use of a synergistic mixture comprising an effective dosage of a metal proteinate and at least one beta-chlorovinyl dialkyl phosphate.

BACKGROUND OF THE INVENTION 
This invention relates to synergistic compositions of metal proteinates 
with beta-chlorovinyl dialkyl phosphates. More particularly, this 
invention relates to the increase of metals throughout body tissues and 
increased passage across the placental barrier from the mother to the 
foeti when the mother is given a synergistic mixture comprising an 
effective dosage of a metal chelate and beta-chlorovinyl dialkyl 
phosphate, said phosphate preferably being a slow release form. 
Beta-chlorovinyl dialkyl phosphates are well known in the art, as 
cholinesterase inhibitors, and are widely used and marketed as both 
pesticides and anthelminthics. Such compounds are most effectively 
administered in the form of slow release compositions which are 
particularly disclosed and claimed in U.S. Pat. No. 3,166,472 issued Jan. 
19, 1965, U.S. Pat. No. 3,318,969 issued May 9, 1967, and U.S. Pat. No. 
3,507,956 issued Apr. 21, 1970. 
In U.S. Pat. No. 3,507,956 there is disclosed a method of increasing the 
vitality of the fetus of a mammal and a newborn animal which comprises 
administering to the pregnant female mammal an effective amount of a 
beta-chlorovinyl dialkyl phosphate. That patent purports to show an 
increase in the overall vitality of piglets that are born from sows which 
have been treated with 2,2-dichlorovinyl dimethyl phosphate (DDVP) which 
has been administered as a slow release formulation utilizing polyvinyl 
chloride as the embedding resin. The data purportedly show greater 
survivability, fewer incidents of stillborn piglets, and faster weight 
increase to market weight of piglets treated with the 2,2-dichlorovinyl 
dimethyl phosphate. Similar data is also shown for dogs. In the patent, 
the only substantial difference within the internal organs of the animal 
is that the blood glucose results are higher in animals that have been 
treated with the 2,2-dichlorovinyl dimethyl phosphate. Such is not the 
case in the present invention. 
Commercial preparations of 2,2-dichlorovinyl dimethyl phosphate commonly 
known as DDVP or dichlorvos embedded in a plastic formulation for slow 
release purposes are presently being marketed by Shell Chemical Company 
under various trade names. For example, a swine anthelminthic is marketed 
under the trade name of ATGARD..RTM. 
In any growing mammal, swine in particular, it is extremely important that 
metals, iron in particular, be readily available to the growing fetus as 
well as to the newly born piglet. 
It has been shown in copending application Ser. No. 658,243 filed Feb. 17, 
1976, which is a continuation-in-part of Ser. No. 607,370 filed Aug. 25, 
1975, now abandoned, which was in turn a continuation-in-part of Ser. No. 
420,033, now abandoned, which was a continuation-in-part of Ser. No. 
739,141 filed June 14, 1968, and now abandoned, that "metal proteinates", 
which are defined at page 89 of the Official Publication of the American 
Feed Control Officials, Inc. as "the product resulting from the chelation 
of a soluble salt with amino acids and/or partially hydrolyzed protein," 
increase the flow of metals across the placental wall as well as 
increasing the metals in tissues in mammals. The increased metal content 
is also present in the mammals milk after parturition. 
It is known that metal proteinates require at least two ligands per mole of 
bivalent metal in order to form an intact chelate which forms a 
heterocyclic ring. Such chelates carry a zero net charge and are more 
readily absorbed in the small intestine and distributed throughout the 
body than are salts of proteins or inorganic metal salts. 
OBJECTS OF THE INVENTION 
It is an object of the present invention to provide a synergistic 
composition comprising a metal proteinate and a beta-chlorovinyl dialkyl 
phosphate to increase the metal content within body tissue and also 
increase the transfer of metal across the placental barrier of a mammal. 
It is also an object of the present invention to provide novel synergistic 
composition of metal proteinates, iron proteinates in particular, with 
beta-chlorovinyl dialkyl phosphates which increase the metal in the 
tissues, increase the flow of metal across the placental barrier of the 
pregnant mammal and readily make the metal available in the milk of said 
mammal after farrowing or parturition. 
Another object of this invention is to provide a method for the increased 
uptake of metals into the feoti of a pregnant mammal by administering to 
said mammal during the final stages of pregnancy an effective dosage of a 
synergistic mixture of a metal proteinate and a beta-chlorovinyl dialkyl 
phosphate. 
A still furthr object of the present invention is to increase the uptake of 
metals into a baby mammal by administering to the mother a synergistic 
mixture of an effective dosage of a metal proteinate and a 
beta-chlorovinyl dialkyl phosphate whereby the young mammal obtains the 
synergistic mixture via the mother's milk. 
These and other objects will become apparent by the following detailed 
description of the invention. 
BRIEF SUMMARY OF THE INVENTION 
It has been unexpectedly found that when effective dosages of metal 
proteinates, such as iron proteinate, are administered to mammals and to 
pregnant mammals in particular, with an effective amount of a 
beta-chlorovinyl dialkyl phosphate, a synergistic effect is obtained in 
increasing the metal content within the tissues and in transporting said 
metal throughout the body tissues and across the placental barrier into 
the unborn fetus. 
The word synergism as applied here is a term which is applied to the usage 
of two biological preparations. The use of one, when utilized with the 
other, has a combined effect greater than the net effect of each when used 
separately. 
In U.S. Pat. No. 3,507,956 it is thought that the unexpected results 
obtained by the use of beta-chlorovinyl dialkyl phosphates is that there 
appears to be an increased concentration of glucose in the blood of the 
fetus, as compared to control animals which have not been fed the 
phosphate material. 
It is shown in Ser. No. 658,243, filed Feb. 17, 1976, and its parent 
applications, that amino acid proteinates, iron proteinate in particular, 
are superior in their ability to penetrate the placenta. This is due to 
the active transport of the intact proteinate (chelate) across the 
placental barrier. 
Apparently, the action of the beta-chlorovinyl dialkyl phosphate is to 
dilate the small blood vessels and capillaries causing a greater blood 
flow to the placenta. Therefore, it is easier to transport components in 
the blood, whether it be blood glucose or chelated proteinates, across the 
placental barrier. 
Regarding metal proteinates, it is thought that there might be a difference 
in the way different types of proteinates are metabolized. For example, a 
short-chained chelate such as a tri-glycine or a long-chained chelate such 
as tryptic digested casein would probably be transported and metabolized 
differently than a single amino acid chelate or a group of mixed amino 
acid chelates. Stated another way, the shorter the protein hydrolysate is 
the more readily it will be absorbed into the bloodstream and transported 
to the various tissues of the body. 
While it has been found that metal proteinates, and iron proteinates in 
particular, are better utilized, this application should not be construed 
as being limited to any particular metal proteinate but includes 
proteinates of all essential bivalent metals such as iron, zinc, 
magnesium, manganese, copper, calcium, and cobalt. The ligand used to make 
the proteinate may be any hydrolysate from proteins including 
polypeptides, peptides, and naturally occurring amino acids and mixtures 
thereof. As previously stated, the metal proteinate or chelate forms a 
heterocyclic ring which is a coordination complex such that there is zero 
net electrical charge on the composition. Qualitatively, such proteinates 
are somewhat soluble in acidic solutions, but are insoluble precipitates 
in basic solutions. As previously stated, it is essential that each 
proteinate or chelate contain at least two ligands. The protein used may 
be hydrolyzed under either acidic or a basic condition or may be 
enzymatically digested by known methods. The chelate is formed by adding a 
soluble metal salt to a soluble protein hydrolysate under sufficiently 
basic conditions that the protons on the protein hydrolysates are removed 
thereby leaving the electrons on the hydrolysate, which would normally be 
attached to the protons, free to form coordinate complex bonds with the 
metal ions from the metal salt. The proteinate thus formed is precipitated 
and may be washed and then fed in desired dosages. Generally speaking, the 
amount of metal proteinate fed to mammals, whether pregnant or not will 
depend upon the percentage of metal in the proteinate. This, of course, 
will depend upon the particular metal being administered and can be 
determined on an empirical basis. 
When administering an iron proteinate containing 10% by weight iron one 
will ordinarily mix from 3 to 12 pounds of iron proteinate to sufficient 
feed to make up 1 ton (2,000 pounds) of feed. Assuming the feed to be 
homogeneously mixed, each pound would therefore contain from about 0.0015 
to 0.006 pounds of iron proteinate of which 10% would be iron. The dosage 
limits may vary considerably depending upon the size, sex, whether doing 
lactation and the general condition of the mammal. 
For example, in a swine weighing 250 pounds and consuming 8 pounds of feed 
each day and using the above criteria, such swine would consume from 0.012 
to 0.048 pounds of iron proteinate a day or 0.0012 to 0.0048 pounds of 
iron each day. Translated into milligrams of iron per pound of body weight 
per day, the amount would be from about 2 to 9 mg.lb. This range need not 
be construed as limiting but would probably constitute the preferred range 
for most metals fed to most mammals. The proteinate may be administered in 
divided dosages and will preferably be mixed with the food of the mammal 
such as the 3 to 12 pounds of proteinate per ton of feed as previously 
mentioned. Other levels of metal proteinates may be used in specialized 
cases. There has been no palatability problem when utilizing metal 
proteinates in this manner. 
Numerous insecticidally active esters of phosphoric acids have been 
prepared as is illustrated by U.S. Pat. No. 2.956,073. A limited class of 
these pesticides have been shown to be useful in controlling endoparasites 
in both mammals and birds as well as being combined with resins to provide 
slow release pesticides. Such compositions are characteristically 
represented by the formula 
##STR1## 
in which R and R.sup.1 may be alkyl groups of from 1 to 4 carbon atoms and 
may be the same or different. X represents a member selected from the 
group consisting of hydrogen or chlorine. Typical of compounds of this 
class are 2-chlorovinyl phosphate; 2,2-dichlorodimethyl phosphate (DDVP); 
2-chlorovinyl phosphate; 2,2-dichlorovinyl diethyl phosphate; 
2,2-dichlorovinyl methyl ethyl phosphate; 2,2-dichlorovinyl methyl propyl 
phosphate; 2,2-dichlorovinyl methyl isopropyl phosphate; 2,2-dichlorovinyl 
methyl butyl phosphate; 2-chlorovinyl ethyl butyl phosphate. 
The compounds of this class are generally known and the methods of 
preparation are taught in U.S. Pat. Nos. 2,956,073 and 3,299,190. 
Preferably the chlorovinyl phosphates will be administered orally and may 
be conveniently mixed in the food of the animal. As with the proteinate, 
the phosphate can be administered in a single dose or in a series of doses 
over a specific period of time. Preferably, the chlorovinyl phosphate will 
be formulated in a slow release form rather than used as a neat 
composition. 
Preparations which may be used for oral use may either by liquids or solids 
such as syrups, elixers, emulsions, powders, capsules or tablets. Such 
compositions are taught in U.S. Pat. No. 3,507,956. 
As taught in the above mentioned patent, the phosphates will preferably be 
formulated in a polyvinylchloride resin for slow release. Such phosphates 
are readily soluble in such resins. The preparation of such a formulation 
may be made by warming and mixing the resin and phosphate together. 
Generally, the resin will contain from 10% to 30% by weight of the 
phosphate. 
As previously stated, the phosphate can be administered to the mammal's 
food or may be combined with a carrier which is non-reactive with the 
phosphate, such as corn cob meal, walnut shell flour, citrus meal, bone 
meal, blood meal, fish meal and the like. 
For continuous feeding for several days in the phosphate and proteinate may 
be mixed together in the mammal's feed and administered to the mammal at 
the same time. For single applications preferably the vinyl phosphte 
should be administered some 3 to 6 hours before the dose of metal 
proteinate is given. Presumably, as previously stated, the phosphate tends 
to dilate the bloof vessels. When administered to pregnant mammals, the 
vessels are diluted within the placental wall thereby enhancing the flow 
of blood across the placenta from the mother to the fetus. 
The preferred phosphate is referred to as DDVP (2,2-dichlorovinyl dimethyl 
phosphate). Both the phosphate and the proteinate may be administered at 
any time. In the case of pregnancy, the synergistic mixture may be 
administered during the entire course of pregnancy and is advantageously 
administered during the latter portions of pregnancy. The period and 
dosage will depend upon the mammal and period of gestation. Normally the 
synergistic mixture should be given during the last trimester of pregnancy 
and from at least 1 week to 1 month prior to parturition. 
While the data presented hereinafter relates primarily to laboratory 
animals, both the synergistic composition and the method of administering 
the same are applicable not only to laboratory animals such as mice, rats, 
ginuea pigs, rabbits, monkeys and the like, but also to pets such as dogs 
and cats and to fur bearing animals such as foxes, mink, and the like. The 
composition and method are also applicable to domestic animals such as 
swine, cattle, sheep, goats, horses, and the like, with emphasis being 
placed on swine. There is a particular problem with piglets in that they 
grow very rapidly and therefore require significant amounts of iron to 
avoid anemia. Oftentimes, this iron must be administered by injection to 
keep the piglet alive. It is believed that the present invention will 
solve this problem in that not only will the piglet be born with a higher 
hemoglobin content, but if the synergistic composition continues to be 
administered during lactation the young animal will continue with the 
increased uptake of metal achieved by means of the mother's milk. 
In most mammals the dosage of the beta-chlorovinyl dialkyl phosphate varies 
from about 0.5 to 5 milligrams per pound of body weight per day with 
dosages of 2 to 3.5 milligrams per pound being preferred. 
For pregnant swine the application of the synergistic mixture may begin, if 
not already in use, when the sow is placed in the farrowing house. The 
dosages previously stated may be administered. When using the phosphate in 
a slow release form it may be desirable to administer a higher dosage as 
only 20% to 50% of the phosphate may be released or made available during 
passage through the gastrointestinal tract depending upon the formulation. 
The following examples are intended to be exemplary only and are not to be 
construed as limitations to the present invention.

EXAMPLE 1 
In order to show the synergistic effect in increasing iron in tissues and 
in transporting iron across the placental wall utilizing 2,2-dichlorovinyl 
dimethyl phosphate along with the iron amino acid proteinate as a 
synergistic mixture, the following groups were set up. 
Fifteen timed pregnant Sprague Dawley White Rats were fasted overnight and 
divided randomly into five groups and given the following dosages: 
GROUP I 
There was mixed together 75 ul (microliters) of distilled H.sub.2 O and 4.4 
uc (microcuries) of Fe.sup.59 Cl.sub.3 dissolved in 10 ul of H.sub.2 O. 
GROUP II 
There was mixed together 75 ul of 2% triglycine buffered to a pH of 10 with 
a bicarbonate-carbonate buffer and 4.4 uc of Fe.sup.59 Cl.sub.3 dissolved 
in 10 ul of H.sub.2 O to form an iron triglycine chelate. 
GROUP III 
There was mixed together 71 ul of 2% tryptic peptide digest buffered to a 
pH of 10 with a bicarbonate-carbonate buffer and 4.4 uc of Fe.sup.59 
Cl.sub.3 dissolved in 10 ul of H.sub.2 O to form an iron tryptic peptide 
digest chelate. 
GROUP IV 
There was mixed together 75 ul of 2% amino acid solution (hydrolyzed high 
vegetable protein plus methionine) buffered to a pH of 10 with a 
bicarbonate-carbonate buffer and 4.4 uc of Fe.sup.59 Cl.sub.3 dissolved in 
10 ul of H.sub.2 O to form an iron amino acid chelate. 
GROUP V 
Same as GROUP IV except that 2 milligrams of 2,2-dichlorovinyl dimethyl 
phosphate in the form of a slow release polyvinyl chloride pellet (Shell 
ATGARD.RTM.) was added. 
In all occasions, except GROUP I, the buffered protein hydrolysate, whether 
it be a triglycine, tryptic peptide digest, or high vegetable 
protein-methionine mixture, immediately formed a chelate at the buffered 
basic pH. 
All animals were dosed with the above dosages on a Friday afternoon and on 
the following Monday, one day before they were to give birth, the female 
rats were asphyxiated with ether and fetus and organs of the rats were 
removed and tested for Fe.sup.59 radioactivity measured in corrected 
counts per minute (cc/min). 
The fetus, along with the cord and placenta, were counted on a 
Nuclear-Chicago 2851 Gamma Counter with a 2 inch Sodium Iodide Crystal. 
The following results were attained: 
______________________________________ 
CC/MIN .times. 100 COUNTS 
GROUP NO. OF BABIES AVERAGE FETUS 
______________________________________ 
I 34 15.50 
II 37 14.91 
III 32 19.05 
IV 32 18.07 
V 36 45.98 
______________________________________ 
The following tissues were dissected from the female rats and counted as 
above with the following cc/min .times. 100 counts: 
______________________________________ 
U- 
TER- LIV- KID- 
GROUP US ER NEY SPLEEN HEART LUNG 
______________________________________ 
I 3,333 8,167 567 134 333 1,367 
II 3,733 5,900 700 267 233 1,650 
III 2,200 5,967 467 117 67 1,150 
IV 4,925 9,675 950 325 1,425 2,925 
V 14,333 39,133 2,433 1,433 5,033 10,834 
______________________________________ 
As will be noted from the above data GROUP III which is the tryptic acid 
chelate and GROUP IV which is the amino acid chelate show about 25% better 
results in transporting iron across the placental wall than in GROUP I 
which is the control group. However, GROUP V, which is the amino acid 
chelate plus the 2,2-dichlorovinyl dimethyl phosphate synergistic mixture, 
was transported across the placental barrier about 300% better than GROUP 
I which is the control group. 
In the tissues, GROUP V which is the synergistic mixture shows an increase 
in the radioactive iron content throughout the various organs of the 
female rat which is several hundred percent better than the control GROUP 
I and proteinate GROUPS II, III and IV made from a protein hydrolysate or 
amino acid. 
In summary, the uptake of iron proteinates or chelates is markedly enhanced 
by the synergistic action of 2,2-dichlorovinyl dimethyl phosphate in the 
transport and absorption mechanisms throughout the various organs of the 
female rat and across the placenta wall into the fetus. 
EXAMPLE II 
In three separate experiments involving 36 timed pregnant 280 gram female 
rats and in excess of 340 young rats it has been demonstrated that 
mixtures of 2,2-dichlorovinyl dimethyl phosphate and iron amino acid 
chelates or proteinates are indeed synertistic. Experimental groups were 
set up in the following manner: 
GROUP VI 
Control Group 
To this group was given 4 uc of Fe.sup.59 Cl.sub.3 in 25 ul of H.sub.2 O to 
which was added 50 ul of pH 10 buffer solution (bicarbonate-carbonate 
buffer). The rats were dosed orally with a 100 ul automatic pipette after 
being partially anesthetized with ether. 
GROUP VII 
Fe.sup.59 Cl.sub.3 Plus 2,2-Dichlorovinyl Dimethyl Phosphate 
To this group was given 4 uc of Fe.sup.59 Cl.sub.3 in 25 ul of H.sub.2 O to 
which was added 50 ul of a pH 10 buffer solution as used in GROUP VI. The 
rats were dosed orally with a 100 ul automatic pipette after they had been 
partially anesthetized with ether. Following the administration of the 
radioactive ferric chloride solution the rats were force fed with 
polyvinyl chloride pellets amounting to a dosage of 2 milligrams of 
2,2-dichlorovinyl dimethyl phosphate (ATGARD.RTM.) for each rat. 
GROUP VIII 
Iron Amino Acid Chelate 
To this group was administered 4 uc of Fe.sup.59 Cl.sub.3 in 25 ul of 
H.sub.2 O which had been chelated with 50 ul of buffered amino acid 
solution. The solution added, therefore, was a buffered amino acid 
chelate. Each rat was dosed with 100 ul from an automatic pipette after 
the rats had been partially anesthetized with ether. 
GROUP IX 
Amino Acid Chelate Plus 2,2-Dichlorovinyl Dimethyl Phosphate 
This group was the same as in GROUP VIII except the dosage of the amino 
acid was followed by the force feeding of 2 milligrams of 
2,2-dichlorovinyl dimethyl phosphate in polyvinyl chloride (ATGARD.RTM.) 
to each rat. 
The dosages were given 3 days before the expected parturition date of the 
rats. Following are the results of the studies listed as an average and 
median plus or minus the standard deviation for the fetus. The whole body 
counts are given as corrected counts per minute or whole body counts 
(cc/min/rat): 
______________________________________ 
GROUP VI. Control Group (4 uc Fe.sup.59 Cl.sub.3). 
Average 18,160 .+-. 6,190 
Median 17,800 .+-. 6,200 
GROUP VII. 
Fe.sup.59 Cl.sub.3 Plus 2,2-Dichlorovinyl 
Dimethyl Phosphate. 
Average 22,340 .+-. 5,670 
Median 22,000 .+-. 5,680 
GROUP VIII. 
Iron Amino Acid Chelate. 
Average 24,070 .+-. 11,400 
Median 25,000 .+-. 11,400 
GROUP IX. Iron Amino Acid Chelate Plus 
2,2-Dichlorovinyl Dimethyl Phosphate 
Average 31,700 .+-. 16,600 
Median 30,300 .+-. 16,700 
______________________________________ 
As is obvious from the above results GROUP VII is higher up in uptake than 
is GROUP VI which is the control group. The 2,2-dichlorovinyl dimethyl 
phosphate appears to be beneficial in promoting the transport of iron 
across the placental barrier; however, the amino acid chelate in GROUP 
VIII is better than either the control GROUP VI or the 2,2-dichlorovinyl 
dimethyl phosphate GROUP VII in its ability to cross the placenta and 
enter the young rat. GROUP IX shows the synergistic action of the joint 
use of amino acid chelates and 2,2-dichlorovinyl dimethyl phosphate 
showing a 31% increase over GROUP VIII and a 42% increase over GROUP VII. 
From the above, it is imminently obvious that by combining a metal 
proteinate (chelate) with a beta-chlorovinyl dialkyl phosphate that 
synergistic results are obtained in moving essential bivalent metals into 
tissues and across the placental wall. While the above results are 
directed to the utilization of iron as the essential metal and 
2,2-dichlorovinyl dimethyl phosphate as the beta-chlorovinyl dialkyl 
phosphate they are considered to be exemplary only and the application is 
to be interpreted according to the scope of the appended claims.