Vol. 57, No. 11, November 1968
`
`1945
`
`Stilbene isothiocyanates-synthesis
`Fluorescent tagging, antibodies-tilbene
`thiocyanates
`
`iso-
`
`Keyph rases
`Quinine reference units-fluorescence
`deter -
`mination
`Fluorometry-analysis
`
`Behavior and Solubility of
`Dissolution
`Trihydrate Forms of Ampicillin
`Anhydrous and
`By JOHN W. POOLE and CHANDER KANTA BAHAL
`
`Anhydrous ampicillin and ampicillin trihydrate were compared for solubility and
`relative rates of dissolution in distilled water at temperatures ranging from 7.5 to
`50'. Differences were noted in the physical-chemical properties of these two forms
`of ampicillin. The thermodynamic properties of these compounds have been experi-
`mentally evaluated. The properties noted for the two forms of the antibiotic are
`consistent with the observed differences in the biological utilization of the two forms
`after oral administration to laboratory animals and human subjects.
`
`M capable of existing in more than one
`
`ANY ORGANIC medicinal compounds are
`
`crystalline form having different physical-chem-
`ical properties. The resulting variation in the
`thermodynamic properties associated with differ-
`ences in crystal form may be of considerable phar-
`maceutical importance as pointed out previously
`by Higuchi (1). The present report is concerned
`with studies conducted to determine the differ-
`ences in some Of the physical-chemical properties
`of two forms of ampicillin, a semisynthetic peni-
`cillin. Specifically, the solubilities and relative
`rates of dissolution in distilled water of anhydrous
`ampicillin and ampicillin trihydrate were deter-
`mined and the thermodynamic properties of these
`crystal forms were experimentally evaluated.
`Most of the past work reported on the physical-
`chemical properties of crystalline hydrates has
`been concerned with inorganic compounds. The
`studies of Taylor and Henderson (2) on the vari-
`ous hydrates of calcium nitrate and of Hill (3) on
`calcium sulfate are examples of such studies.
`More recently several investigations concerned
`with studies of organic molecules in the anhydrous
`
`Received June 26, 1968, from Pharmacy Reasearch & De-
`velopment Division, Wyeth Laboratories, Philadelphia,
`PA 19101
`Accepted for publication August 7, 1968.
`Presented to Basic Pharmaceutics Section, APAA Academy
`of Pharmaceutical Sciences, Miami Beach meeting, May
`1968.
`
`and hydrated forms have been reported. An
`anhydrous form of phenobarbital and two of its
`hydrates were examined by Eriksson (4) for ap-
`parent solubility in water as a function of time.
`The relative dissolution rates of solvated and non-
`solvated crystal forms of several types of com-
`pounds of pharmaceutical
`interest, including
`steroids and xanthines were reported by Shefter
`and Higuchi (5). These workers also determined
`the thermodynamic properties of several of these
`crystal systems.
`
`EXPERIMENTAL
`constant-temperature water bath
`Apparatus-A
`equipped with Unitherm Haake constant-tempera-
`ture circulator' and a rotating-bottle apparatus,2
`Swinney hypodermic adaptor,s Millipore filtersS
`(pore sue 0.45 p ) , amber bottles, 120 ml. with poly-
`seal caps.'
`Compounds-In
`all the experiments anhydrous
`ampicillin, (Wyeth Laboratories batch C-10575,
`m.p. 203-204O) was used. The trihydrate form of
`ampicillin was prepared from the anhydrous form
`by the method of Austin et ul. (6). IR spectra and
`differential thermal analysis curves were obtained
`for this material.
`Procedure-An excess of drug, 2 g., in the appro-
`priate form was added to 100 ml. of distilled water
`previously equilibrated to the desired temperature.
`
`1 Brinkmann Instruments, Westbury, N. Y.
`E. D. Menold Sheet Co., Lester, Pa.
`a Millipore Corp., Bedford, Mass.
`4 Erno Products, Philadelphia, Pa.
`
`Merck Exhibit 2161, Page 1
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`
`

`

`1946
`
`Joiirnal of Pharmacedical Sciences
`
`The bottles were rotated in a constant-temperature
`water bath maintained at the indicated temperature.
`Samples withdrawn at definite intervals were filtered
`through a Millipore filter and diluted immediately
`to avoid any precipitation of ampicillin in the filtered
`samples due to supersaturation. The penicillin con-
`tent was determined by means of an iodometric titra-
`tion procedure as described below. To 2.0-ml. ali-
`quots containing 1 to 3 mg. of ampicillin, 2.0 ml. of
`1 N sodium hydroxide was added and samples were
`allowed to stand at room temperature for 15 min.
`At the end of this time, 2.0 ml. of 1.2 N HCl was
`added followed by 10 ml. of 0.01 N iodine. After
`15 min., the excess of iodine was titrated using 0.01
`N sodium thiosulfate. For the blank determina-
`tions, to a 3.0-ml. sample, 10 ml. of 0.01 N iodine
`was added and titrated immediately.
`
`RESULTS AND DISCUSSION
`
`The solubility and dissolution behavior of the an-
`hydrous and trihydrate forms of ampicillin at 7.5,
`20, 30, and 40" are shown in Fig. 1. Similar data
`for the study conducted at 50" are shown in Fig. 2.
`These figures show the concentration of the anti-
`biotic attained in solution as a function of time in
`the presence of an excess of the solid phase in the
`appropriate form and under essentially constant
`agitation. One interesting feature of these studies
`is the inverse relationship noted between tempera-
`ture and solubility for the anhydrous form of the
`drug. The apparently greater dissolution rate
`observed for the anhydrous form at the lower tem-
`peratures cannot be attributed solely to the higher
`free energy content of this species since no serious
`attempt was made to maintain equal spec& surface
`areas of
`the two forms. However, microscopic
`examination of the materials employed showed the
`anhydrous and trihydrate forms to be substantially
`the same with regard to particle size and shape and
`since the anhydrous form is significantly more
`soluble than the trihydrate form, the dissolution
`rate of the two forms is in the direction that would
`be expected on solubility considerations alone.
`I n
`addition, the study a t 50°, which is above the transi-
`tion temperature of this system, shows the trihy-
`drate to have an apparently greater dissolution rate
`than the anhydrous form. This supports the con-
`tention that solubility is the dominant factor in this
`system.
`The dissolution behavior in water noted for the
`two forms of ampicillin suggest that the equilibrium
`
`4 4
`0
`
`,
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`
`
`hr
`Fig. 2-The dissolution behavior of anhydrous and tri-
`hydrate crystalline forms of ampicillin i n water at 50'.
`Key: 0, anhydrous; 0 , trihydrate.
`
`solubility observed are good approximations of the
`true solubility of these crystals. Consequently
`the measurements made at the several temperatures
`permit calculation of the thermodynamic quantities
`involved in the transition of the anhydrous form to
`the trihydrate. An extensive treatment of
`the
`thermodynamic relationship
`involving polymor-
`phism and solubility is presented in reports by
`Shefter and Higuchi (5) and Higuchi et d . (7).
`The apparent equilibrium solubilities observed
`over the temperature range 20 to 50' when plotted
`in the classical van't Hoff fashion gave a reasonably
`good linear relationship for both forms of the anti-
`biotic as shown in Fig. 3.
`The transition temperature for the trihydrate-
`anhydrous crystal system corresponds to the tem-
`perature at which the solubility of the two forms
`is equal. The transition temperature for this sys-
`tem is 42" as shown in Fig. 3. This plot also points
`up the fact noted earlier that the solubility of the
`anhydrous form decreases with an increase in tem-
`perature whereas that of the trihydrate exhibits the
`usual temperature-solubility relationship.
`The values of the heat of solution for each of the
`crystal forms was calculated from the slopes of the
`van't Hoff-type plot (Fig. 4) and were determined
`to be - 1000 and 5400 cal./mole for the anhydrous
`and trihydrate forms, respectively. The enthalpy
` the heat of solution of the ,
`of hydration ( ~ A . H )
`
`anhydrous form minus the heat of solution of the
`hydrated species, was determined to be -6400
`cal. /mole.
`At constant temperature and pressure the free
`energy difference AFT, between the anhydrous and
`
`"1
`
`TRANS TEMP
`
`.,
`
`hr
`Fig. 1-The dissolution behavior of anhydrous and tri-
`hydrate crystalline forms of amfi'cdlin in water at
`temperatures ranging from 7.5 to Poo. Key: 0, an-
`hydrous;
`trihydrate.
`
`I / T s 10'
`Fig. 3-The van't Hoff-ty#e #lot for the anhydrous and
`trihydrate forms of ampicillin in water. Key: 0,
`anhydrous; 0 , trihydrate.
`
`Merck Exhibit 2161, Page 2
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`
`

`

`I~'o1. $7, No. 1 1 , Noriemher 1968
`
`TABLE I-THERMODYNAMIC VALUES CALCULATED
`THE ANHYDROUS-TRIHYDRATE AMPICILLIN
`FOR
`SYSTEM
`
`1947
`
`cal./mol-
`Temp., Y A H ,
`A F T ,
`O C . Anhydrous Trihydrate cal./molca
`(- 1000) (5400)
`
`25
`37
`42
`
`A S T .
`e.u.O
`
`-20.0
`-20.2
`-20.3
`
`-430
`-140
`0
`
`injtuence of seeding anhydrous ampicillin
`Fig. &The
`with 1% trihydrate crystals on the dissolution behavior
`in water at 10'. Key: 0, anhydrous; 0 , trihydrate;
`A , anhydrous seeded with 1 % trihydrate.
`
`hydrated forms is determined by Eq. 1.
`C.
`(anhydrous)
`AFT = R T l n
`(Eq. 1)
`C.
`(trihydrate)
`where C. is the solubility of the form under considera-
`tion at a particular temperature T , and R is the gas
`constant. This AFT is a measure of the free energy
`the anhydrous
`change involved in conversion of
`crystal to the trihydrate crystal. The AFT at 25
`and 37O (corresponding to room and body tem-
`peratures) have been determined to be -430 and
`- 140 cal./mole, respectively.
`The entropy change, AST, for the reaction in-
`volved in hydrate formation can be calculated by
`Eq. 2.
`
`The values computed for the hydration of the an-
`hydrous to trihydrate ampicillin crystals a t 25" and
`37' were -20.0 e.u. and -20.2 e.u., respectively.
`At the transition temperature of the anhydrous-
`trihydrate crystal system AF is equal to zero and
`the entropy change can be calculated by Eq. 3.
`
`For ampicillin ASt,.,.. was determined to be -20.3
`e.u. The hydrated species in this system contains
`three molecules of water and the possible intra-
`molecular hydrogen bond formation between these
`associated water molecules may account for the
`It has been
`relatively large entropy change noted.
`suggested that a hydrated ampicillin complex of
`this type may be responsible to some extent for the
`relative stability of this compound in acidic solution
`(8).
`The thermodynamic values calculated for the
`anhydrous-trihydrate ampicillin system are sum-
`marized in Table I.
`As noted earlier the equilibrium solubilities ob-
`served in these experiments apparently correspond
`to the solubilities of the anhydrous and trihydrate
`crystalline phases for the ampicillin molecule. At
`the temperatures utilized there was no evidence of
`conversion of the more soluble anhydrous form to the
`less soluble trihydrate specks as would be expected
`from
`the
`thermodynamic considerations. This
`may be due in part to the steric factors involved in
`the association of three molecules of water in the
`crystal system.
`However, at lower temperatures (10') the seeding
`of the anhydrous form with trihydrate crystals re-
`
`Calculated for the conversion from the anhydrous to
`trihydrate form.
`
`the
`
`sulted in a relatively rapid and complete conversion
`of the anhydrous to the trihydrate form, as shown
`by the decrease in solubility. These data are shown
`in Fig. 4.
`The relative ease of conversion of anhydrous to
`trihydrate ampicillin at the lower temperatures is
`to be expected, since the rate of phase transforma-
`tion in a given system depends on the solubilities of
`the forms at that temperature, the rates of solution,
`and the diffusion rates of the molecules in solution.
`The higher the solubility and the greater the dif-
`ference in solubilities of the two phases the greater
`will be the rate of transformation. At the lower
`temperatures the anhydrous form exhibits an in-
`creased solubility due to its negative heat of solution.
`The trihydrate form shows a decrease in solubility
`at lower temperatures (positive heat of solution)
`which results in a relatively large difference in the
`solubilities for the two forms.
`Higuchi (1) pointed out that the physiological
`activity and availability of a drug is often directly
`related to its thermodynamic activity in a system
`of this type. Recently Aguiar et al. (9) reported on
`the effect of polymorphism on the absorption of
`chloramphenicol from chloramphenicol palmitate.
`From the data presented in the present investigation
`it would be reasonable to expect an enhanced
`biological utilization of
`the anhydrous form of
`ampicillin compared to the trihydrate form of this
`agent.
`In addition, since the anhydrous system is
`apparently stable to conversion at room and body
`temperature, these differences should be main-
`tained in the clinical situation. That this is the
`case was shown in a recent report by Poole ct al.
`(10). In these investigations, various pharmaceuti-
`cal formulations of the drug in each form were
`administered to laboratory animals (beagle dogs)
`and to human subjects in a series of crossover experi-
`ments. The formulations containing the anhy-
`drous form of the penicillin resulted in blood serum
`levels of
`the antibiotic which were consistently
`earlier and significantly higher than those observed
`after administration of similar formulations con-
`In every instance
`taining the hydrated material.
`the area under the serum level-time curves was
`greater for the anhydrous form of the drug than
`for the hydrated substance indicating a more ef-
`ficient biological utilization of
`this form of
`the
`medicinal agent, The results of the in vivo evalua-
`tion of the oral suspensions of the two forms of
`ampicillin are summarized in Table 11.
`
`SUMMARY
`
`The solubility and relative rates of dissolutian
`of anhydrous ampicillin and ampicillin trihydrate
`
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`

`Journal of Pharmaceutical Sciences
`REFERENCES
`
`Peak
`Serum
`Level,
`Test
`Form of
`Species mcg./ml.
`Ampicillin
`20.6
`Anhydrous' Dog
`Trihydrateb Dog
`11.0
`Anhydrousa Human
`2.2
`Trihydrateb Human
`1 . 7
`
`Area
`Under
`Curve
`(mcg./ml.
`X hr.)
`36.6
`22.8
`6.9
`5.7
`
`Peak
`Time,
`min.
`45
`90
`60
`120
`
`1948
`TABLE 11-PEAK SERUM LEVEL AND AREA UNDER
`THE BLOOD LEVEL-TIME CURVE AFTER ORAL
`ADMINISTRATION OF SUSPENSIONS OF ANHYDROUS
`(1) Hipuchi. T.. J . Am. Phnrm. Arsoc., Sci. Ed., 47,
`AMPICILLIN AND AMPICILLIN TRIHYDRATE
`RR711RRR).
`(2) Taylor. H. S., and Henderson, W. N., J. Am. Chem.
`Soc., 37, 1688(1915).
`(3) Hill, A. E., ibid., 59, 2243(1937).
`(4) Eriksson, S. O., Svensk. Farm. Tidskr., 65, 353(1961).
`( 5 ) Shefter, E., and Higuchi, T., J . Phorm. Sci., 5 2 ,
`781(1963).
`(6) Austin, K. W. B., Marshall, A. C.. and Smith, H.,
`Nafure, 208,999(1965).
`(7) Higuchi, W. I., Lau. P. K., Higuchi, T., and Shell,
`J. W., J . Phorm. Sci., 52, 150(1963).
`(8) Hou, J. P., and Poole, J. W., unpublished data.
`(9) Aguiar. A. J., Krc, J.. Kinkel, A. W., and Samyn.
`J. C., J. Pharm. Scr., 56, 847(1967).
`(10) Poole, J. W., Owen, G., Silverio, J.. Freyhof. J. N.,
`and Hosenman, S. B.. Current Therap. Res., 10,292(1968).
`
`a Administered as Omnipen for oral suspension, Wyeth
`Inc. Radnor Pa. Administered a s Poly-
`Laboratories
`cillin for or61 suspension, 'Bristol Laboratories, Syracuse,
`N. Y.
`
`in distilled water have been determined over a tem-
`perature range of 7.5 to 50". Below the transition
`temperature, 42', the anhydrous form was found to
`be significantly more water soluble than the tri-
`hydrate.
`In addition, the solubility of
`the an-
`hydrous crystal was shown to be inversely related
`to temperature.
`The thermodynamic values have been calculated
`for the anhydrous-trihydrate ampicillin system.
`The greater thermodynamic activity Qf the anhy-
`drous form correlates with the observed enhanced
`biological availability noted with this crystal form
`of the antibiotic.
`
`Keyphrases
`
`trihydrate-thermo-
`
`Ampicillin, anhydrous,
`dynamic properties
`Dissolution rate-ampicillin,
`hydrate
`Solubility-ampicillin, anhydrous, trihydrate
`Blood serum levels-ampicillin,
`anhydrous,
`trihydrate
`Iodometric titration-analysis
`
`anhydrous, tri-
`
`Potential Antitumor Agents I11
`
`Sodium Salts of a- "1-Heterocyclic Carboxaldehyde
`Thiosemicarbazones
`
`By KRISHNA C. AGRAWAL and ALAN C. SARTORELLI
`
`Sodium salts of four of the most active antineoplastic agents in a series of a-(N)-
`heterocyclic carboxaldehyde thiosemicarbazones have been prepared as a means of
`solubilizing for parenteral administration these extremely insoluble compounds.
`The sodium salt of 1-formylisoquinoline thiosemicarbazone (11) is soluble in non-
`aqueous vehicles for injection such as propylene glycol, whereas the sodium salts of
`5-hydroxy-1-formylisoquinoline thiosemicarbazone
`(HI), 3-hydroxy-~-formyl-
`pyridine thiosemicarbazone (IV), and 5-hydroxy-2-formylpyrid1ne thiosemicar-
`bazone (V) are readily soluble in water. Compounds 111 and IV, at the optimum
`effective dosage regimens, caused a greater prolongation of the survival time of mice
`bearing the L1210 lymphoma than did the parent derivatives, while I1 and V pro-
`duced antineoplastic activity a dnst sarcoma 1 8 0 and the LlZlO lymphoma, respec-
`tively, equivafent to that of the parent compounds.
`
`VARIETY OF thiosemicarbazones of a-(N)-
`
`A heterocyclic carboxaldehydes has been pre-
`
`(1-7). Several of these derivatives, especially 1-
`formylisoquinoline thiosemicarbazone (2, 3), its
`pared and tested for antineoplastic activity 5-hydroxy derivative (4), and both 3-hydroxy-2-
`formylpyridine
`thiosemicarbazone and 5-hy-
`Received June 17, 1968, from the Department of Phar-
`macology, Yale University School of Medicine, New Haven,
`droxy-2-formylpyridine thiosemicarbazone (5, 6),
`CT 06510
`Accepted for publication August 13. 1968.
`have demonstrated pronounced antineoplastic
`This investigation was supported by grant T-23 from the
`American Cancer Society and grant CA-02817 from the
`activity when tested against a relatively wide
`Institute, USPHS. The authors are
`National Cancer
`indebted to Miss Andrea F. Gorske and Mr. Karim A.
`transplanted rodent tumors. To
`spectrum of
`Alkadhi for excellent assistance.
`
`Merck Exhibit 2161, Page 4
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
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`
`