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Duodenal Absorption of Oxytetracycline Hydrochloride in Rabbits Following Admixture of Cholesterol Esters.

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Henderson, Ciaccio and Kincl
Arch. Pharm.
MP: uncorr. IR spectra (KBr): Unicam SP-1OOO. 'H-NMR (DMSO-D,): TMS int. stand., chemical
shifts: 6 (ppm). Analytical data: microanalytical center at Cairo University.
Reaction of cyanoacetanilide (1)with aldehydes
A mixture of 0.5g 1and 0.5g of the appropriate aldehyde was fused in the presence of 0.5g fused
sodium acetate in an oil bath for 1 hat 180"C. The reaction mixture was cooled, triturated with ethanol
and poured into cold water, the yellow solid that separated was crystallised from the proper solvent
(cf. Table 1).
Reaction of 2b with the activated methylene compounds
To a solution of 0.01 mol2b in 50 ml ethanol were added 0.01 mol of the active methylene reagent and
some drops of piperidine. The reaction mixture was heated under reflux for 5 h, then evaporated i.vac.
The remaining product was triturated with ethanol and the resulting solid product was crystallised
from the proper solvent (cf. Tablesl, 2).
Reaction of 1 with 4-benzylidene-3-methyl~oxazolin-5-one(14)
A solution of 0.01mol 1 in 50ml ethanol was treated with compound 0.01mol 14 and lml
triethylamine. The reaction mixture was heated under reflux for 8h, then evaporated i.vac. The
remaining product was acidified and the solid product that separated was crystallised from the proper
solvent (cf. Tables 1, 2).
Mohamed A. E. Khalifa and M. H. Elnagdi, Indian. J. Chem. 12,46 (1974).
M. Sammour, Egypt. J. Chem. 14,213 (1971).
J.J. Eatough, L.S. Fuller, R.H. Good and R.K. Smalley, J. Chem. SOC.C 1970, 1874.
E. Schenker and H. Herbest, Progr. Drug Res. 5, 269 (1963).
J.I.G. Cadogen, R.K. Mackie and M.J. Todd, Chem. Commun. 1966, 491.
[Ph 6581
Arch. Pharm. (Weinheim) 316, 826-831 (1983)
Duodenal Absorption of Oxytetracycline Hydrochloride in
Rabbits Following Admixture of Cholesterol Esters
The late Steven B . Henderson, Leonard A. Ciaccio and Fred A. Kincl*
Department of Biology, The College of Staten Island of the City University of New York, New
York, USA.
Eingegangen am 30. August 1982
Cholesterol acetate admixed with oxytetracycline hydrochloride in equal proportions exhibits a
maximal melting point depression. The mixture, administered orally to rabbits, provided higher and
longer lasting plasma concentrations than when the same antibiotic was given with oil as the delivery
03454233/83/101W824 $ 02.50/0
0 Verlag Chemie GmbH, Weinheim 1983
Absorption of Oxyietracycline
Duodenale Resorption von Oxytetracyclin bei Kaninchen nach Zumischung von
Cholesterolacetat bddet mit gleichen Anteilen Oxytetracyclinhydrochlond eine Mischung mit einer
maximalen Schmelzpunkterniedrigung. Bei oraler Verabreichung an Kaninchen wird eine hohere
Konzentration und Venveildauer des Antibiotikums im Plasma erzielt als bei Verabreichung mit 0 1
als Tragersubstanz.
The realization that the characteristics of intestinal absorption are a function of the type of delivery
formulation represents an important development in realm of pharmaceutical design. Consequently
there has developed a recognitionthat formulation design could be an important parameter to be used
to increase bioavailability of drugs') and this field has become one of the most important in
pharmaceutical research.
For example, steroidal lipids have been used to increase oral absorption of several drugs. The
activity of a natural hormone progesterone (which is poorly orally activewhen given in a conventional
formulation) was significantly enhanced when the hormone was deposited on lactose admixed with
cholesterol esters2) as compared to a conventional formulation. Oral activity of norethindrone
has similarly been increased'). Increased urinary excretion of the potassium salts of penicillins G and V and of erythromycin lactobionate following coating
with cholesterol or p-sitostero14)and increased urinary excretion of unchanged salicylate followed oral
administration of salicylic acid mixed with cholesteryl n-decylate') has also been reported.
The purpose of this investigation was to evaluate the influence of a cholesterol acetate
matrix on bioavailability of oxytetracycline hydrochloride. The effectiveness of the test
preparation was evaluated by monitoring plasma levels of the drug6,')
Experimental Part
Materials: The followingwere obtained from commercial sources: cholesterol (1) and oxytetracycline
hydrochloride (2); cholesterol acetate was prepared in our laboratory using routine esterification
method (3). Cholesterol acetate was used in a ratio which yielded a solution of the drug in the carrier.
The selection was made on the basis of melting point phase diagrams(4) to select the appropriate
regions of maximal melting point depression. This method does not allow one to distinguish whether
selected mixtures were true solid solutions or micellar solutions of colloidal suspensions, but did allow
a selection of mixtures where drug distribution in the carrier approached a "perfect" mixing
condition. Thus, oxytetracycline hydrochloride-cholesterol acetate were used in 1:1 w/w ratio.
Animals: Rabbits, 4-6 per group, weighing 1,2-2kg were used. The subjects were placed under
anesthesia and a cannula was placed into the duodenum and a second cannula into the cervical artery.
The outlets of both cannulas were located into a plastic chamber, with a screw cap top, cemented to
the sku1 of the animal; this permitted a direct delivery of test substancesinto the duodenum bypassing
the stomach, and a facile collection of blood without anesthesia. We have found this arrangement to
be of importance; preliminary experiments revealed that intestinal peristalsis and antibiotic
absorption was significantly decreased in animals placed under total anesthesia. The preparations
were suspended in corn oil prior to administration. All preparations were given in amounts to provide
a dose of 200mg per animal. Blood was drawn 1,2,4,8,24 and 48 h following the administration and
assayed for tetracycline, and the results were expressed as pg of oxytetracyclinelmlof plasmdg of
antibiotic given.
Henderson. Ciaccio and Kincl
Arch. Pharm.
Procedure:(5) 1ml of each plasma to be analyzed was transferred to a 15 ml centrifuge tube, 0.1 ml of
distilled water was added, followed by 0.1 ml of 1 N-HCI. The tube was capped and shaken vigorously
on a mechanical shaker (6) for 5 min. TCA reagent (7) (0.3ml) was added, the tube was capped and
shaken vigorously on a mechanical shaker for 2 min. The contents were then centrifuged at 10,OOO rpm
(8) for lOmin, the supernatant was further clarified by filtration through small plugs of glass wool
using 6" disposable glass pipets a funnels. 1 ml of filtered supernate was transferred to a clean 15 ml
centrifuge tube. 0.6ml of 1N-HCl(9) with TPA was added, the contents were mixed and the tubes
placed in a boiling water bath for 3 min. The tubes were then removed and immediately placed in an
ice bath until they reached room temp.; 0.6ml of 6 N-NaOH was then added, mixed well and
immediately fluorescence intensities at 500 nm under 390nm excitation was measured (10). Plasma
blank intensity were subtracted from each standard and sample intensity measurement and a
calibration line plotted (11).
Analysisof Data: The areasunder the curve for each rabbit in each treatment group were calculated by
the trapezoidal rule after transforming the readings to ln(x 1) in order to remove indications of
heteroscedascity (principally in the oil-based groups). Comparisons among the group areas were
performed by analysis of variance and the subsequent partitioning of the overall sums of squares into
separate comparisons along lines of interest.
Table 1: Oxytetracycline Plasma Levels in Rabbits Following IntraduodenalAdministration Using Two
Different Carriers
(Back-transformed means and 95 % confidence limits)
Time after
dose, h.
Plasma levels, pg/ml/g antibiotic
Control (oil as vehicle)
Subcutaneous injection
5.7 (2.2-13.2)
2.0 (.3-11.0)
0.6 (.2-2.3)
2.7 (.2-15.3)
0.9 (.7-9.8)
2.3 (.l-9.3)
Steroidal lipids as drug carrier
Cholesterol acetate
11.6 (1.7-57.3)
11.5 (3.9-31.0)
7.3 (1.9-22.4)
32.3 (22.8-45.5)
50.3 (37.1-68.2)
67.8 (56.2-81.4)
54.5 (42.3-70.2)
13.4 (8.4-21.1)
1.4 (.2-5.9)
6.7 (2.6-15.6)
3.8 (2.3-5.8)
4.5 (2.0-9.1)
Table 2 Calculated Areas Under the Curve Depicting Plasma Levels Following Intraduodenal
Treatment Group
Cholesterol acetate matrix
Subcutaneous injection12)
Plasma levels, h x In (pg/ml/g antibiotic f S.E.M.)
45.3 * 15.0
84.7 f 4.8
126.7 t 8.3
Absorption of Oxytetracycline
The back transformed data expressed as means and their 95 % confidence limits for each of the
intraduodenal treatment groups and the subcutaneous injection of a commercial preparation are
given in table 1. Integration of the data yielded areas under the curve for the time period studied
expressed as means and the standard errors in table 2. The large variationsin plasma levels seen in the
control group that received the antibiotic suspended in corn oil were not seen in the experimental
group, or in the animals injected subcutaneously with the drug.
Drug availability to the organism is a function of the dosage form ("drug delivery
system") and its metabolic fate ("drug removal system"). The most obvious way to obtain
a greater drug effect is to increase the dosage. However, this has the disadvantage that the
level may persist beyond the time for the next dosage and consequently leads to a drug
biuldup. Divided dosage increases drug availability and leads to improved drug efficiency.
A sustained release dosage form which represents an infinite number of daily divided doses
should theoretically prove to be the ideal drug carrier. The shorter half-life of the drug
used, the more closely will the temporal pattern of drug concentration in blood or other
body fluid follow the temporal pattern of drug administration: remove the therapeutic
system and the drug level in the body will rapidly approach zero8). Formulations which
would provide continuously steady plasma levels for a desired period of time are
theoretically possibleg,lo) albeit not as yet available.
In our previous studies'.') we have achieved increased oral bioavailability of steroidal
hormones by incorporating the drugs in a lipid matrix. The selection of the carrier is
obviously not a matter of chance, but of careful search. This was shown by a group from
England") who attempted to increase oral bioavailability of norethindrone by formulating
with various high molecular polymers (polyethyleneglycol, polyoxypropylene, and
polyoxyethylene) and acetoglyceride without achieving increases intestinal absorption.
We have elected to introduce the antibiotic directly into the duodenum since it has been
amply documented") that tetracyclines are absorbed in great part within the duodenum
and ileum. This group used both anesthetized and unanesthetized dogs but did not
comment on differences in absorption, if any. In our studies with rabbits we have seen a
significant lowering of intestinal absorption in anesthetized animals; to eliminate this
drawback all our studies were performed without the use of anesthesia.
The increase in oral absorption achieved by using steroidal-lipid matrix was significant
(table 3). The relative efficiencies, based upon the calculations of the area under the curve
was double both the cholesterol based lipid matrix which was 1.9 times more active than
oxytetraxcycline suspended in oil and given orally. The plasma levels obtained approached
plasma levelsseeninanimalsinjectedsubcutaneous1y")with an equivalent dose. Assuming
arbitrarily the efficacy of oxytetracyc1ine:cholesterol acetate in 100 than the efficacy of a
subcutaneous injection of a commercial preparation was only 1.5 times higher.
The improvement in oral bioavailability of lipid coated penicillins (4) found to be more
stable in vitro led one group to speculate that lipid coating increased the bioavailability of
penicillins by protecting the drug against the inactivation in the stomach. Our previous
Arch. Pharm.
Henderson, Ciaccio and Kind
Table 3: Relative Efficiencies of Several Oxytetracycline Preparations Tested in the Rabbit
Dosage form
Relative Potency
Control - oral
Cholesterol acetate matrix - oral
Subcutaneous injection
* Significant at p = .05
results') lead us to believe that when steroidal lipids are used in proportions to yield an
optimal drug dispersion, such drug delivery system are most likely also absorbed into the
lymphaticsystem thereby increasing the bioavailability.The present results demonstrating
increased bioavailability following introduction directly into the duodenum provide a
have reported
further support for this view. It is also of interest to note that two
decreased intestinal absorption of cortisol sulfadiazine in the presence of hypocholesterolomic acid-binding anion exchange polymers (colestipol hydrochloride).
(1) K and K Laboratories, Plainview, New York.
(2) Sigma Chemical Company, St. Louis, Missouri.
(3) Acetic anhydride in pyridine while heating.
(4) Thomas-Hoover, Arthur H. Thomas Company, Chicago, Illinois.
( 5 ) We thank Dr. J.W. Tolun, Pfizer Corporation for providing us the details of the assay.
(6) Shaker
(7) TCA Reagent (Prepare fresh immediately before use): 3.0 g reagent grade trichloroacetic acid
dissolved in about 5 ml distilledwater, then diluted to 10.0ml with distilledwater; 3.3 ml of reagent
grade hydrochloric acid was added and mixed well.
(8) IEC PR-6000
(9) 1 N-HCI with TPA: Dilute 8.3 ml of reagent grade hydrochloric acid, plus 0.08 ml reagent grade
thiopropionic acid (TPA) was diluted to 100ml with distilled water.
(10) Eppendorf 1101 M. Brickmann Corporation.
(11) Working standards contained 1, 2.5, 5 and l0ppm. The assay is sensitive to detect
0.5 ppdml.
W.H. Barr, L.M. Gerbracht, K. Letcher, M. Plaut andN. Strahl, Clin. Pharmacol. Ther. 13,97
F.A. Kincl, L.A. Ciaccio and G. Benagiano, J. Steroid Biochem. 9, 83 (1978).
J.R. Pasqualini, R. Castellet, M.C. Portois, J.L. Hill and F.A. Kincl, J. Reprod. FertiL.49, 189
S.P. Patel and C.I. Jarowski, J. Pharm. Sci. 64,869 (1975).
S.P. Patel and C.I. Jarowski, Drug Res. Commun. 2, 465 (1976).
B. Scales and D.A. Assinder, I. Pharm. Sci. 62, 913 (1973).
7 J.E. Hayes, Jr. and H.G. DuBuy, Anal. Biochem. 7, 322 (1964).
8 H.W. Rude1 and F.A. Kincl in Nobel Symposium 15,p. 39, J.Wiley&Sons, New York 1971.
9 A. Zaffaroni, Acta Endocrinol. (Copenhagen) Suppl. 185,423 (1974).
10 F.A. Kincl, Proc. Int. Congr. Pharmacol. 5 , 105 (1975).
11 K. Fotherby and R.J. Warren, Contraception 14,261 (1976).
12 M.H. Pindell, K.M. Cull, K.M. Doran and H.L. Dickison, J. Pharmacol. Exp. Ther. 125,287
13 W.A. Phillips, J.R. Schultz and W.W. Stafforo, J. Pharm., Sci. 63, 1097 (1974).
14 R.E. Kauffman and D.L. Azarnoff, Clin. Pharmacol. Ther. 14, 886 (1973).
[ Ph 6591
Arch. Pharm. (Weinheim) 316,831-834 (1983)
Dihydroisochmolinumlagerung, 34. Mitt.’)
7-All yl-6-methyl-6,7-dihydrothieno[2,3-c]pyridin
Joachim Knabe* und Joachim Lorem**
Fachrichtung Pharmazeutische Chemie der Universitat des Saarlandes, Im Stadtwald,
6600 Saarbriicken
Eingegangen am 3. September 1982
Die Titelverbindung 4 wkd, ausgehend vom 7-Chlorthienopyridh 1,uber die 7-Allylverbindung2
synthetisiert, die durch Grignard-Kupplung erhalten wird. 2 wird mit Methyhodid zum Iminiumsalz3
methyliert, das mit LiAlH4 zu 4 redudert wird. Bei Behandlung mit 0,l N-HC1 werden aus 4 die
Disproportionierungsprodukte3 und 5 gebildet. Das Hauptprodukt ist jedoch das Umlagerungsprodukt 6, das als Pseudocyanid 7 isoliert wird.
Dihydroioinoline Rearrangement, XXXIV: 7-Allyl&methyl-6,7-dU1ydmthieno[2,3-c]pyrW1e
The title compound 4 is synthesized from the 7-chlorothienopyridine 1 via the 7-ally1 compound 2
which is obtained by Grignard coupling. Compound 2 is methylated by methyl iodide to give the
iminium salt 3 which yields 4 on reduction with LiAlH4. When 4 is treated with 0,l N HCl the
disproportionation products 3 and 5 are formed. The main product, however, is the rearranged
compound 6 which is isolated as the pseudocyanide 7.
Nachdem in den vorhergehenden Mitt.’)’)die Umlagerungsfiihigkeit von 4-Allyl- und 4-Benzyl-4,S-dihydrothieno[3,2-c]pyridinen untenucht wurde, sollten nunmehr 7-Allyl- und 7-Benzyl-6,7-dihydrothieno[2,3-c]pyridinein die Untenuchungen einbezogen werden.
Das erste Thieno[2,3-c]pyridin, niimlich die 7-Methylverbindung, wurde 1951 von
Hem3)dargestellt .Die Synthese des 7-Allyl-6-methyl-6,7-dihydrothieno[2,3-c]pyridins
gelang, ausgehend von 7-Chlorthieno[2,3-c]pyridin(l), das nach Eloy und Deryckere4)’)
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0 Verlag Chemie GmbH, Weinheim 1983
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cholesterol, admixture, duodenal, rabbits, following, esters, hydrochloric, absorption, oxytetracycline
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