DRUG- CYCLODEXTRIN INCLUSION COMPOUNDS. STUDIES OF THE FORMULATION OF PHARMACEUTICAL PRODUCTS CONTAINING CYCLODEXTRINS.

Interaction of beta -cyclodextrin with ampicillin, phenobarbitone , and phenytoin was studied and complexation was observed with all three drugs . Analytical methods for the quantification of these drugs were validated. In order to obtain an inclusion compound in the solid powdered form, a variety of methods (freeze drying, spray drying, kneading, and solvent evaporation) was evaluated. Among these methods freeze dryi ng was the most feasible and reproducible method from a laboratory scale to a pilot scale and to the manufacturing level. Methods using an industrial model freeze drier were developed which resulted in an increase in yield from 53% to more than 90% . However, because of ampicillin instability in freezing conditions, it was not possible to obtain an inclusion compound in the solid powdered form . Physico-chemical properties of the inclusion complexes were evaluated by a variety of methods such as stability, solubility, X-ray diffractometry, differential scanning calorimetry, infrared and proton magnetic resonance spectroscopies, photomicrographs, etc. The stability of ampicillin in an acidic medium (pH 2.0) was considerably improved by complex formation with beta-cyclodextrin. The observed apparent pseudo-first order rate constants (hr1) of ampicillin were 1.8 x 102, 1. 2 x 10-2, and 1.1 x 10-2 for ratios of .1:0.0 and 1:1 . 0, 1:0.0 and 1:1 . 5 and 1:0.0 and 2.0 of ampicillin to beta-cyclodextrin, respectively. Furthermore, it was estimated that the complex degrades nine times slower than the drug itself, and the apparent stability constant was found to be 458.46M1. Aqueous solubility of phenobarbitone and phenytoin beta-cyclodextrin complexes were five and eleven fold larger than that of phenobarbitone and phenytoin alone, respectively. A phase solubility diagram was constructed and the apparent stability constants were calculated for phenobarbitone and phenytoin beta-cyclodextrin complexes. The calculated stability constants were 4.54 X 103M-l and 766Ml for phenobarbitone and phenytoin complexes respectively. Thus, the formation and presence of the inclusion complex were confirmed by stability and solubility analysis. Furthermore, the proton magnetic resonance spectra of phenobarbitone beta-cyclodextrin complex strongly indicated the presence of the inclusion complex in the liquid phase. The mode of interaction and elucidation of the presence of the inclusion complex in the solid phase were studied by X-ray diffraction, differential scanning calorimetry, infrared spectra photomicrographs, etc . The X-ray diffraction spectra and differential scanning calorimetry thermograms clearly suggested the presence of the inclusion complex in the freeze dried material but not in the physical mixture. The infrared spectra and photomicrographs evaluations were such that a definitive, unambiguous interpretation could not be made; however, they were useful to some extent to show the interaction with beta -cyclodextrin. A solution was prepared from the phenobarbitone beta-cyclodextrin complex. Unlike an official USP preparation, the solution prepared from the complex did not require the addition of alcohol to keep the phenobarbitone in solution. Four months' physical and one month's chemical stability data indicated a considerable potential especially for pediatric use. It was possible to prepare a pharmaceutically elegant suspension from the phenytoin beta-cyclodextrin complex. The suspensi on possessed desirable qualities of a pharmaceutically elegant suspension, such as ease of dispersion, high sedimentation volume, etc.; in addition, the ~uspension had a low viscosity. Physical stability was good even in freezing conditions and a syr ingeability study suggested a potential for intramuscular use. A flow study was designed using a recording flowmeter with small quantities of material in an attempt to predict the flow rate during large scale operations . The data indicated that the flowmeter was sensitive enough that a small quantity of material could be used to predict the flow rate, effect of formulation and processing variables on production scale quantities. The flow study, and the bulk properties of beta-cyclodextrin suggested that the material was fairly free flowing and compressible. The fre eze dried phenytoin beta-cyclodextrin complex lacked flowability and compressibility; neverthless, it was possible to compress tablets of a suitable size and weight using the wet granulation method. All the properties of tablets were measured and found to be within the USP standards. Dissolution studies in different test media strongly suggested that the dissolution rate. for phenytoin beta-cyclodextrin complex tablets was superior to all other phenytoin formulations or preparations. Furthermore, due to in situ complexation in the dissolution medium, the dissolutio n rate of the physical mixture of the freeze dried phenytoin and beta-cyclodextrin was found to be similar to the dissolution rate of the freeze dried complex.

freeze dry i ng was the most feasible and re producible method from a laboratory scale to a pilot scale and to the manufacturing level.
Methods using an ind ustrial model freeze drier were developed which resulted in an increase in yield from 53% to more than 90% . However, because of ampicillin instability in freezing conditions, it was not possible to obtain an inclusion compound in the solid powdered form .
Physico-chemical properties of the inclusion complexes were evaluated by a variety of methods such as stability, solubility, X-ray diffractometry, differential scanning calorimetry, infrared and proton magnetic resonance spectroscopies, photomicrographs, etc. The stability of ampicillin in an acidic medium (pH 2.0) was considerably improved by complex formation with beta-cyclodextrin. The observed apparent pseudo-first order rate constants (hr-1 ) of ampicillin were 1. 8 x 10-2 , 1. 2 x 10-2 , and 1.1 x 10-2 for ratios of . 1:0.0 and 1:1 . 0, 1:0.0 and 1:1 . 5 and 1:0.0 and 2.0 of ampicillin to beta-cyclodextrin, respectively. Furthermore, it was estimated that the complex degrades nine times slower than the drug itself, and the apparent stability constant was found to be 458.46M-1 . Aqueous solubility of phenobarbitone and phenytoin beta-cyclodextrin complexes were five and eleven fold larger than that of phenobarbitone and phenytoin alone, respectively. A phase solubility diagram was constructed and the apparent stability constants were calculated for phenobarbitone and phenytoin beta-cyclodextrin complexes.
The calculated stability constants were 4.54 X 10 3 M-l and 766M-l for phenobarbitone and phenytoin complexes respectively. Thus, the formation and presence of the inclusion complex were confirmed by stability and solubility analysis. Furthermore, the proton magnetic resonance spectra of phenobarbitone beta-cyclodextrin complex strongly indicated the presence of the inclusion complex in the liquid phase. The mode of interaction and elucidation of the presence of the inclusion complex in the solid phase were studied by X-ray diffraction, differential scanning calorimetry, infrared spectra photomicrographs, etc . The X-ray diffraction spectra and differential scanning calorimetry thermograms clearly suggested the presence of the inclusion complex in the freeze dried material but not in the physical mixture. The infrared spectra and photomicrographs evaluations were such that a definitive, unambiguous interpretation could not be made; however, they were useful to some extent to show the interaction with beta -cyclodextrin.
A solution was prepared from the phenobarbitone beta-cyclodextrin complex. Unlike an official USP preparation, the solution prepared from the complex did not require the addition of alcohol to keep the phenobarbitone in solution. Four months' physical and one month's chemical stability data indicated a considerable potential especially for pediatric use. It was possible to prepare a pharmaceutically elegant suspension from the phenytoin beta-cyclodextrin complex. The suspensi on possessed desirable qualities of a pharmaceutically elegant suspension, such as ease of dispersion, high sedimentation volume, etc.; in addition, the ~uspension had a low viscosity.
Physical stability was good even in freezing conditions and a syr ingeability study suggested a potential for intramuscular use.
A flow study was designed using a recording flowmeter with small quan tities of material in an attempt to predict the flow rate during large scale operations . The data indicated that the flowmeter was sensitive enough that a small quantity of material could be used to predict the flow rate, effect of form ulation and processing variables on production scale quantities. The flow study, and the bulk properties of beta-cyclodextrin suggested that the material was fairly free flowing and compressible. The fre eze dried phenytoin beta-cyclodextrin complex lacked flowability and compressibility; neverthless, it was possible to compress tablets of a suitable size and weight using the wet granulation method. All the properties of tablets were measured and found to be within the US P standards. Dissolution studies in different test media strongly suggested that the dissolution rate. for phenytoin beta-cyclodextrin complex tablets was superior to all other phenytoin formulations or preparations. Furthermore, due to in situ complexation in the dissolution medium, the dissolutio n rate of the physical mixture of the freeze dried phenytoin and beta-cyclodextrin was found to be similar to the dissolution rate of the freeze dried complex.     71 9. Effect of beta-cyclodextrin concentration on the psuedo first order rate constant for the hydrolysis of ampicillin at pH 2.0 and 37° . . . . . . . . . . 73 10. Plot of the rate data (from Fig. 9) . . . . 75 11. Solubility of phenobarbitone ad a function of betacyclodextrin in water at 25 C . . . . . 78 12. Solubility of phenytoin as a function 3f betacyclodextrin in pH 7.0 buffer at 25 C 82 13. 14.

15.
Powder X-ray diffraction patterns (Phenytoin beta-cyclodextrin systems) Powder X-ray diffraction patterns (Phenytoin beta -cyclodextrin systems) Powder X-ray diffraction patterns (P henobarbitone beta-cyclodextrin systems). 16. Powder X-ray diffraction patterns 88 89 92 (Pheno barbitone beta-cyclodextrin systems). 93 17 A--Historical background The first reference to cyclodextrin was made in a publication of "Villers in 1891 (1), who isolated a small amount of a crystalline substance from a culture of Bacillus amylobacter, grown on a medium containing starch. Villers named his crystalline product " Cellulosine " because of its similarity to cellulose. Then Schardinger (2 -4 ) observed that Villers cellulosine often formed on starch-based culture media as a product of putrefying microorganisms. Schardinger succeeded in isolating a bacillus which he named Bac illus macerans . This is th e most frequently used source of the enzyme glycosyl-transferase by which cyclodextrin is now produced.
Afte r Schardinger, it was Pringsheim (5) who played the major role in the cyclodextrin research, and he discovered the complexing power of the cyclodextrins. From the mid -thirties on, pure cyclodextrins were prepared, first by Freudenberg and his co -workers, who also elucidated the chemical structure of cyclodextrins and discovered gamma -cyclodextrin (6) . The cyclodextrins, also called cycloamyloses, Schardinger dextrins, or Cycloglycopyranoses, are cyclic oligosaccharides in which glucose units are linked by alpha 1-4 glycosidic bonds (7,8). French (7) in the mid-fifties demonstrated the existence of delta and epsilon cyclodextrins. Thoma and Stewart (9) described further homologues containing 11 and 12 glycopyranose units.

8-Properties of cyclodextrins 1. Physico-chemical properties
Since the discovery of the Schardinger dextrins, these cyclic compounds have been of special interest as they relate to starch (7) .
However, with respect to various chemical reactions, especially their biochemical behavior, the Schardinger dextrins vary from starch in important aspects (7,10). One of the rather remarkable properties of the Schardinger dextrins is their resistance to hydrolysis by the common starch -splitting enzymes; for example, it has been repeatedly reported that the Schardinger dextrins are resistant to beta amylase action.
Since the Schardinger dextrins, are cyclic, there are no reducing end groups; hence they are resistant to beta amylase attack . The Schardinger dextrins have been reported (11) to be stable to alpha type amylases.  Table I (8,12).
French and co-workers (13) found that the alpha-dextrin is essentially completely resistant to beta amylase, the beta-dextrin is attacked very slowly indeed and gamma-dextrin is attacked about l % as rapidly as is starch. So it is clear that the ring size exerts an effect; possibly the smaller rings have greater rigidity and hence can   In solution, the cyclodextrin's cavity cannot be regarded as an empty space. For example, the diameter of the cavity in alpha-cyclodextrin is 4.5A 0 and its height is 6.7A 0 and therefore the total volume is 64ml/mole. The energy required to maintain an empty space of this dimension would be approximately 40kcal/mole. This value is so high that it is hardly conceivable that the cavity could remain empty (8). The other two cyclodextrins (beta and gamma), having a larger diameter, this ' energy requirement is even higher. The viscosity of aqueous cyclodextrin solutions differs only insignificantly from that of water (14). One of the most striking properties of cyclodextrins is their ability to form inclusion complexes (8,15). This property will be dicussed in detail separately.

Cyclodextrin metabolism and toxicity
Detailed studies of metabolism and toxicity are necessary for any compound intended for use in pharmaceuticals or in food. Therefore, utilization of the cyclodextrins in the pharmaceutical or food industries depends to a large extent on knowledge of the effects they have on living organisms. In 1963 it was discovered that, in rats, 14C -labeled alpha and beta cyclodextrins are distributed into tissues and organs similarly to 14C-labeled starch (10) . It is remarkable that, on feeding 14C-starch, the 14C-level of the 14C0 2 expired reached a maximum after one hour; whereas, in the case of 14C-beta cyclodextrin, the maximum appeared after approximately nine-hours. The total quantity of expired 14C0 2 when related to the carbbohydrate consumed, was about equal in both experients. The cyclodextrins are not metabolized as rapidly as starch; this is because they are cleaved more slowly than linear dextrins (10), and are not hydrolyzed by enzymes which attack terminal groups. Szejtli et al. (16), reported a study in which they fed rats with beta-cyclodextrin and glucose, both uniformly labeled with 14C, and measured the radioactivity level of blood and exhaled air. With glucose, about 2 % of the initial radioactivity was found to be present in blood within 10-min. With labeled beta-cyclodextrin, only 0.5 % of the initial radioactivity could be recovered from blood and this value was reached only between the 4th and 10th hour following administration . The amount of exhaled radioactivity was practically identical with rats treated orally with 14C -glucose or 14C~beta cyclodextrin and followed for a 24-hr period. No significant difference was found in the tissue distribution of radioactivity (17 ) . Further studies (15,16) indicated that beta cyclodextrin is metabolized and that the intestinal flora are probably responsible for the first step in the degradation of cyclodextrin.
Attempts to detect the intact beta-cyclodextrin by high performance liquid chromatography) in blood following oral administration of 14C-labeled substance gave negative results; no significant radioactivity was found at retention times that correspond to the beta-cyclodextrin.
Chronic six months oral toxicity of the beta-cyclodextrin was studied with doses up to l.6gm./body weight kg. / day in rats, and up to 0.6gm . /body weight kg./day in dogs (18). No toxic effects were observed with respect to weight gain, food consumption, or biochemical values of clinical significance. Pathologic and histopathologic investigations following the six-month treatment did not reveal any sign of toxicity in the digestive organs, central nervous system, cardiovascular system, or in any other organ tested. In addition, the beta-cyclodextrin was also devoid of embryotoxic effect. Chromosomal tests performed in rats treated for six months did not reveal increased incidence of spontaneous abberrations or gene mutation (18). The cyclodextrins administered in high doses (above SOmg./body weight kg . ) subcutaneously, intraperitoneally, or intravenously, can induce renal damage in the rat, but at lower doses the compound can be administered without ill effects ( 20,21).
All toxicity tests have shown that the orally administered cyclodextrin is innocuous. According to the report (22) of the ·Food and Agriculture Organization (FAD), enzjmatically modified starch--this includes cyclodextrins--is also · toxicologically harmless. The FAQ report concludes that there is no need to conduct a toxicological examination in the case of enzjlllatically modified starch products.
However, considering the reduced activity of starch-degrading enzjmes towards cyclodextrins, and also their retarded resorption and slow metabolism when administered orally, it is the author's opinion that additional toxicological studies of cyclodextrins seem nevertheless to be justified, especially if the parenteral route of administration were to be used.

Cyclodextrin derivatives
Among the natural cyclodextrins, beta-cyclodextrin is widely used in many fields because of its cavity size (8 .0 A 0 }, and the ease with which it can be obtained at a relatively low cost on a large scale (8,15,23) .
The present cost of this material is about $ 500 per kilogram. Numerous derivatives of cyclodextrins have already been reported (8) . However, for biomedical applications only a small number of cyclodextrin derivatives have been tested (24).
Recently, the chemically modified cyclodextrins have received considerable attention because their physico-chemical properties are different from those of the natural cyclodextrins (25) . Among the properties that can be modified are solubility, membrane permeability, chemical reactivity, and dissociation constant (26). These properties will provide a rational basis for the design of formulations, and a means of improving the efficiency of drug activity; however, it should also be noted that modified cyclodextrins cause more hemolysis and local tissue damage than do natural cyclodextrins (27) . Hence, the practical application of modified cyclodextrins must await the results of exhaustive toxicological studies.

C-lnclusion complexes
For a half century chemists speculated that certain molecular structures might enclose other structures of suitable size and geometry.
It was not until late 1940's that this fonn of molecular architecture was actually shown to exist. Then, as so often happens, when the time is ripe, the discovery was made almost simultaneously by several research groups (15,(28)(29)(30)(31)(32)(33). The tenn " Einschlussverbindung " (i nclusion compound) was introduced by Schlenk (33) (34), depending on the architecture of the " host " structures and the shapes of the cavities they enclose (Fig. 3) . The structural arrangements that allow this snug 11 hand in glove 11 or 11 lock and key" fitting of molecular shapes differ from one family of inclusion compounds to another (34). In any event, according to John F. Brown Jr . (3 4), the discovery and detaiJed understanding of inclusion compounds have provided a powerful support for the hypothesis that such " hand in glove " or " lock and key 11

Steric requirements
The steric configurations of the molecules are such that the " host " molecule can spatially enclose the " guest " molecule, leaving unaffected the bonding systems of the components. This type of fonnation is typified by the Schardinger dextrins or cyclodextrins (35). According to Cohen and Lach (36), geometrical rather than chemical factors are decisive in detennining the kind of guest molecules which can penetrate into the cyclodextrin cavity to fonn an inclusion complex. The extent of the complex fonnation also depends on the polarity of the guest molecule.
Because of the cyclodextrins' cyclic structures and relatively large open space within each molecule ( 6.0 A 0 for alpha, 8.0 A 0 for beta, and 10.0-11 . 0 A 0 for gamma ) , they have been reported to fonn complexes with different sized molecules (36) . For example, naphthalene is too bulky for alpha-cyclodextrin, and anthracene fits only into gamma -cyclodextrin.
On the other hand, propionic acid fits well with alpha-c yclodextrin , but it is too small to fit properly in larger cavitives. Similarly the role of molecular dimensions is well demonstrated by the complex fonnation with halogenated benzenes (28) . Reaction of the cyclodextrins with halobenzenes show that 1:1 complexes may be prepared from chloro, bromo, and iodobenzenes. Chlorobenzene reacts only with alpha-cycl odextrin , brompbenzene reacts with alpha and beta-cyclodextrins, and iodobenzene reacts with beta and gamma-cyclodextrins. According to Va n Hooidonk and Breebaart-Hansen (37), the diameter of the cavity in beta-cyclodextrin is about 8.0A 0 and the size of the benzene ring is about 6.8 A 0 ; therefore a substituted benzene ring can penetrate into the ring. Certain chemical groups or substituents may promote complex fonnation or stability.
However, the stability of the complex is proportional to the hydrophobic characater of the substituents. Thus, a methyl, ethyl, or phenyl substituent will increase the complex formation or stability. A methyl group in the ortho position to a carbonyl group has a shielding effect on the hydrophilic carbonyl group, thereby increasing the hydrophobicity of the whole molecule. A similar substitu~nt in the para position has a relatively weak effect. Hydroxyl groups hinder complex formation, but their hydrophilic effects decrease in the order ortho, meta, and para (38).

Complexation by cyclodextrins
Many drugs are amenable to inclusion complex formation (36 ,39-43 ) with cyclodextrins. As shown in Fig. 1, the lining of the cyclodextrins cavity is formed by hydrogen and glucosidic oxygen atoms. Therefore, this surface is rather apolar (8). In aqueous solution the apolar cyclodextrin cavity is occupied by water molecules that are in an energetically unfavored state polar-apolar repulsion ) and are, therefore readily replaced by an appropriate • guest molecule • that is less polar than water. Schematic illustration of the complexation process is as shown in the Fig. 4   Eq. (1) Where S 0 is the equilibrium constant of the substrate S, in the absence of the ligand L, and thus S 0 is equal to the intercept of the plot in Fig. 5,A. In some cases, the phase solubility diagram shows a plateau region before the descending part of the curve (Fig.5,B), making it possible, on the basis of the length of the plateau, to estimate the stoichiometry. In such plots, the fonnation of the 1:2 com ple x is assumed and the stability constant can be calculated by using Eq. 1 and 2 according to (45,46) as follows: Eq. (2) Where Lt is the total ligand concentration and St is the equilibrium solubility of the substrate in the presence of ligand.

Diffusion
Gas diffusion provides one of the most direct demonstrations of random molecular motion. Without this phenomenon, the perfume industry would not exist, and skunks would be much less feared. The diffusion properties of cyclodextrin complexes as reported in the literature (8 ,48 ) have been used to investigate the complex fonnation by cyclodextrins.
For example, when indomethacin solution was separated by a semipenneable membrane from a buffer solution, a 1:1 equilibrium was observed (47).
Whereas, when the indomethacin solution was separated by a semipenneable membrane from a buffer solution saturated with beta-cyclodextrin a higher concentration of indomethacin was observed on the side containing cyclodextrin, because, the diffusion rate of the complex was slower due to the increase in molecular weight and extensive hydration of beta-cyclodextrin. Thus, the concentration of indomethacin was higher on the side containing cyclodextrin, indicating that the majority of the indomethacin molecules were fanned in a complex.
3. X-ray spectroscopy Takeo and Kuge (4g,5Q) published X-ray diffraction diagrams of several alpha, beta, and gamma cyclodextrins complexes. According to their studies, inclusion complexes can be detected quickly and directly by X-ray diffraction. If the diffraction pattern does not correspond to those of pure components, a true inclusion complex may exist. In the case of liquid guest molecules X-ray powder diffraction is the most useful method fo r the detection of inclusion complex fonnation . Since the liquid guest molecules produce no diffraction pattern at all, if the diffractogram differs significantly from that of uncomplexed cyclodextrin, fonnation of a crystal lattice of a new type and complex fonnation can be established. Single X-ra y structure analysis is the best method for detecting complex fonnation (15) .

Infrared (IR) spectroscopy
The complex fonnation may be proved by IR spectroscopy, the characteristic bands of cyclodextrin, which represent the overwhelming part of the complex, are influenced by complex fonnation . Bands due to the included part of the guest molecule are generally shifted or their intensities are altered (51).

Differential scanning calorimetry (DSC)
In certain cases cyclodextrins complex fonnation can be proved by DSC. Kurozumi ~ (51) have found that freeze dried mefenamic acid gives at 232°c an endothennic peak; its mechanical mixture with freeze dried beta cyclodextrin gives the same peak at the same temperature . But the beta cyclodextrin complex prepared by freeze drying or crystallization from water does not give any peak . This behavior is usually characteristic of the inclusion complexes.
6. Proton magnetic resonance (PMR ) spectroscopy Demarco and Thakkar (52) investigated cyclodextrin inclusion complex formation by PMR spectrometry. In their study, they observed that the beta-cyclodextrin spectrum was shifted upfield in the presence of guest molecules. According to Demarco and Thakkar, if the guest molecule is accommodated in the cyclodextrin cavity, then the hydrogen atoms located in the interior of the cavity (C-3-H and C-5-H) will be considerably shielded by the guest and hence the signal will be shifted upfield.
Whereas, the hydrogen atoms on the outer surface (C -2-H, C-4-H, and C-6-H) will not be affected by the guest molecule, hence the signals of those protons will remain unchanged. The cyclodextrins contain six, seven, and eight units of glucose, and due to the multiplicity of (C -3 -H and C-5 -H) protons located in the interior of the cavity, an exact evaluation was impossible.

Other methods
Inclusion of a guest molecule can also be investigated by monitoring changes in its cond uctivity {53-55), apparent changes in its basicity (56,57), changes in the optical spectra, thermodynamic parameters, etc. (8,58).
F-Present and possible future potential of cyclodextrins in pharmaceutical products.

Organoleptic properties
It is possible to improve the organoleptic properties of drug molecules by inclusion complexation with cyclodextrins (8,15,59,60). For instance, femoxetine is a selective serotonin uptake inhibitor with antidepressant properties (59}. This compound is used as a water soluble salt, but it has a very bitter taste which hinders the development of oral liquid formulations. A study was undertaken to obtain a liquid formulation of femoxetine with acceptable oraganoleptic properties through inclusion complexation with the beta -cyclodextrin (59}, and the results were favorable.

Conversion of a liquid drug to a solid form
Liquid compounds can be converted into the solid form by inclusion complexation with cyclodextrins (61). Organic nitrates have been used for a long time for the treatment of angina pectoris, and they are still widely used. For example, one of the best known drugs is nitroglycerin,which is a highly explosive liquid drug and therefore cannot be tabletted. However, by complexation with cyclodextrin, it is possible to convert the drug into solid form and tablets can be manufactured (61). In addition, complexes of unsaturated fatty acids, ascaridol, clofibrate, methyl pentynol, etc . , have been converted into a solid form by complexation with cyclodextrins (62,63). These reports suggest that the product obtained in this way can be used to man ufacture t ablets.

Physico-chemical stability
Inclusion complexation can be regarded as micro-encapsulation, because each guest molecule is surrounded by cyclodextrin molecules and is thus, from the microscopical point of view, encapsulated. This phenomenon can be exploited by industries which manufacture drugs, foodstuffs, plant protective agents, etc. The use of cyclodextrin complexes in indust r ies, particularly in the pharmaceutical field, can result in the following improvements in stability.

a. Stabilization of light or oxygen sensitive substances
The extremely labile vitamin 03 (c holecalciferol) should be mentioned here; heat, light, and oxygen all increase the degradation of vitamin 03 by oxidation, but these effects can be inhibited by the inclusion complexation (64 ,65 ). Similarly, inclusion of vitamin A in alpha-cyclodextrin increased its stability against heat (66). The sensitivity to light of clofibrate (67) and guaiazulene (68) are reduced by the inclusion complexation with the cyclodextrins.
b. Modification of the chemical activity or stability of guest molecules Lach and Chin (41) have reported that the alkaline hydrolysis of benzocaine becomes considerably slower in the presence of beta-cyclodextrin.
In 1 % beta-cyclodextrin solution the half-life of benzocaine is increased five-fold . Chin,Chung,and Lach (69) studied in detail the alkaine hydrolysis of esters of various aminobenzoic acids and acetylsalicylic acid.
It was found that the rate of hydrolysis decreased when in clusion was complete. If, however, incorporation of the guest molecule was incomplete then the hydrolysis rate increased. Since the inclusion was complete, the rate of hydrolysis of para, meta, and ortho aminobenzoates was decreased by the beta-cyclodextrin. Beta-cyclodextrin has also been shown to have a stabilizing influence on procaine, atropine, aspirin, and phenylbutazone (70) . Indomethacin is stabilized by beta-cyclodextrin but not by alpha-cyclodextrin (71) . In addition, there are many reports on the modification of the chemical activity or stability of guest molecules by inclusion complexation with the cyclodextrins (8 ,15, 72,73).  of the tablet weight is filled with the inert materials .
By the inclusion complexation technique, a drug can be dispersed at a molecular level, and usually the drug content in the inclusion complex is in the range of 15-25% (8). If the required dose is a few milligrams or less then it may not be difficult to disperse unifonnly lOOmg of the complex instead of 15 or 25mg of drug into the inert materials. By this type of molecular dispersion it is conceivable that some or part of the previously mentioned factors may be reduced in achieving a good content unifonnity of potent low dose drugs.

Dissolution
The number of papers and patents describing drug -cyclodextrin complexes, their stability, dissolution rate, bioavailability, and phannacological effects has been growing rapidly in phannaceutical research (8,15,23). Solubility changes have been observed as a function of complexatio n and substantial increases in drug stability have also been reported (78,84) . These reports als~ showed that the improved aqueous solubility by complexation resulted in an enhancement of the Interaction of digitalis glycosides with cyclodextrins has been reported (86). It has been clearly shown that the dissolution rate (in acidic medium) of the complexed fonn of digoxin is about 100-fold more than that of digoxin itself. In addition, during the dissolution experiments, the simultaneous conversions of digoxin to hydrolysis products were also follwed by HPLC. The rapid dissolving form of the complex showed the reduced decomposition compared to that of digoxin alone. The dissoltuion of menadion (vitamin k 3 ), and its beta-cyclodextrin complex in water was found to be increased by 10 to 12-fold (23). Improved dissolution characteristics of acetohexamide has been reported by complexation with cyclodextrins (87). Similarly, there are many examples which clearl y demonstrate that complexation with cyclodextrins has improved aqueous solubility (78)(79)(80)(81)(82)(83)(84) and dissolution rate of various drugs (8,71,74,88,89 Whenever a drug is administered by the oral route, there is a possibility that part of the dose may not reach the blood due to incomplete absorption (90 ,91 ). This result may arise for a variety of reasons, such as poor dissolution of the drug in the gastrointestinal fluids (92)(93)(94)(95), first-pass liver metabolism (90) , etc. In instances where bioavailability is incomplete, the ratio of oral to intravenous blood level curve areas is less than unity (96). Hence, a great deal of investigation is required to appreciate fully the role of the gastrointestinal tract in drug absorption. For example, a thorough investigation is required with respect to interactions of drug with dietary factors, physiological and physico-chemical factors (97) , etc.
In this section, enhancement of bioavailability by cyclodextrin inclusion complexation is discussed. Improving the absorption of drugs may be one of the important practical applications of cyclodextrin complexes.
Following the oral administration of the drug, practically no cyclodextrin is absorbed (23) . Cyclodextrin is onl y a carrier agent; it transports the lipophilic guest molecule through an aqueous milieu to the lipophilic membrane of cells in the gastrointestinal tract. There the guest molecule is absorbed, since the membrane has a higher affinity for a lipophilic guest molecule than the cyclodextrin itself (23). The relationship of complexation to the absorption and distribution of a drug in the body is well documented in the literature (98).
The dissociation and association reactions of cyclodextrin complexes in solution are very rapid, and the equilibrium of free and complexed drug is established instantaneously (23,99). This may perhaps be used to improve medication through the use of tablets containing both the hydrophobic drug complexed to cyclodextrin and the same drug in the free form. The former would enter the circulation very rapidly, while dissolution of the. latter would occur slowly and form a drug depot (100). Andersen,F.M.,et al. (59), reported the absorption of femoxetine (a selective serotonin uptake inhibitor with antidepressant properties ) in five human volunteers. In this study, five subjects were given six sugar-coated tablets each containing lOOmg of femoxetine salt and femoxetine beta-cyclodextrin complex formulated as a suspension. The dose of the suspension was equivalent to 600mg femoxetine salt. The study was single dose and cross -over design. Each volunteer was given the doses with an interval of at least two weeks between each administration. The blood level was determined by gas chromatography.
The area under the blood level curve (AUC) was calculated using the trapezoidal rule. The AUC for the cyclodextrin complex varied from 23 to 96 ng"h"kg"m1-1 ·mg-1 , while the corresponding values were 30 to 120 ng"h"kg"m1-1 ·mg-l for the sugar-coated tablet. There was statistically no difference between the AUCs for the two formulations. The maximum plasma concentration was reached within one to four hours. The variations were similar to those observed in AUCs . In conclusion, the bioavailability of femoxentine beta-cyclodextrin complex, formulated as a suspension, was found to be similar to that observed from a sugar -coated tablet of femoxetine salt. However, this study would have been more meaningful if the results of the above study had been compared with (a ) femoxetine tablet, preferably the uncoated tablet, or {b) with the same dosage form.
In vivo absorption studies were undertaken to find if the in vitro dissolution enhancement of digoxin from its cyclodextrin complex increases in the absorption of the drug (86). In this study, digoxin tablets containing 100/ug digoxin and its gamma -cyclodextrin complex containing 100/ug digoxin were administered orally to six dogs. The concentration of digoxin in the plasma sample was determined by enzyme immunoassay. The maximum plasma concentration of 0. 90 ~ 0. 14mg/l at 45 minutes was obtained. This concentration level was three times higher than that of digoxin alone. The area under the plasma concentrati on time curve of the complex up to 24 hours was found to be 5. 4 times as much as that of digoxin alone. In addition, the area under the curve of the complex containing 50/ug of digoxin was found to be superior to that of 100/ug digoxin alone. Thus, the improved bioavailability of digoxin by gamma-cyclodextrin complexation suggests a decrease in doses and fewer side effects in oral digitalis glycoside therapy.
Ukema, K., ~-(87), studied the hypoglycemic action of acetohexamide-beta-cyclodextrin complex with that of acetohexamide by oral administration in five male rabbits . The complex equivalent to 30mg/kg of acetohexamide was administered as a suspension in 80ml of water. In each case at least seven days were allowed between each blood glucose estimation. The reduction in the blood glucose level was obse rved in the system co ntain ing the complex. W hen results were compared using paired Student's t-test, the difference was found to be statistically significant . However, it is the opinion of the author of this thesis that, a detailed study should be made to elucidate the absorption mechanism of cyclodextrin complex . Seo,H. ,et al. (88), studied in vivo absorption of spironolactone (S P) to determine whether or not the enhanced in vitro dissolution of SP from its beta or gamma -cyclodextrin complex increased the GI absorption of the drug. SP is a steroidal aldosterone antagonist that is widely used fn the treatment of hypertension, edematous states, etc. Because of its low solubility in water, the bioavailability of the SP preparation is known to vary significantly among brands and batches (a uthors reported with proper references). In the above study, four male beagle dogs were administered orally a capsule containing (5mg/kg of body weight as SP), the drug, and its complex. The administration sequence was based on a crossover design with an interval of more than one week. The plasma concentration was determined by high pressure liquid chromatography. The standard curve range was from 20 to 200ng/ml. The beta and gamma-cyclodextrin complexes produced a maximum plasma concentration of 103_: 28"3ng/ml and 131.!_14 "7ng/ml. at 90-minutes, respectively . These concentrations were about two to three times higher than that of SP alone. Similarly, the areas under the curves of complexes up to 24 hours were found to be more than two times greater than SP alone. The authors did not report any significant difference in time to reach the maximum concentration (Tmaxl· It is the authors' opinion of the above study that the enhanced bioavailability of SP produced by beta or gamma-cyclodextrin complexation makes possible the use of a lower dose with fewer side effects in (o ral ) SP therapy .
Tsuruoka, M., et al. (9 5) , studied the absorption of freeze dried phenytoin, phenytoin and its beta -cyclodextrin complex in a group of four female beagle dogs. Dogs were given orally 300mg of phenytoin and its complex equivalent to 300mg phenytoin. The concentration of phenytoin was detennined by gas chromatography. There was a twofold increase in the area under the curve of the blood level. Further, it was reported in this study that increased bioavailability of phenytoin by means of beta-cyclodextrin complexation suggested the possibility of smaller doses and fewer side effects in phenytoin therapy. The details of the studies such as, statistical evaluations, etc., were not clearly reported.
Indomethacin and its beta -cyclodextrin complex were administered orally to rats (100). The rats were treated orally with 30mg/kg indomethacin and its complex equivalent to 30mg of indomethacin.
Indomethacin level in the blood was measured by high pressure liquid chromatography. The maximum blood level was found between 1 and 4 hours after treatment . It was approximately 25% higher in the case of the complex. The details of the experimental design, such as number of animals, statistical evaluations, sensitivity of the assay method, etc., were not reported; however, based on the reported data it appears that the beta-cyclodextrin complex may be used to increase the bioavailability of indomethacin. Tokumara,T.,et al. (101), studied the bioavailability of cinnarizine with beta-cyclodextrin in three male beagle dogs. Two tablets of cinnarizine and its complex containing 2Smg of cinnarizine in each tablet were administered orally to dogs . The concentration of the drug in the plasma was detennined by high pressure liquid chromatog raphy.
In less than an hour the complex gave the maximum plasma level of 166.9 _: 22.4ng/ml, which was 8. 6 times as high as that of cinnarizine alone.
This initial increase in drug absorption might be due (according to the authors) to the 30 times higher dissolution rate of the complex than that of intact cinnarizine alone; however, there was no significant difference between the areas under the plasma concentration-time curves (AUC). The AUC of cinnarizine and its complex up to eight hours was 267.2 .:!: 102.9 and 374.2 .:!: 97.2ng · h/ ml, respectively. This might be due (authors' opinio n) to the large stability constant of the complex, estimated to be approximately 6.2 x 10 3 M -l in water at 20°c.
Fremming and Weyermann (102), investigated the absorption of an orally administered salicylic acid and its beta -cyclodextrin complex in ten human volunteers (7 male and 3 female). The percentage of absorbed salicylic acid at a given time was reported as follows. From salicylic acid in one hour 24.8%, whereas from the complex 43.0% appeared in the blood; in two hours the values were 56 .8% and 82.5%, respectively.
Hence, a significant difference in blood level was reported .
Nambu, N. , ~· (103)  gave the highest blood level, and no double maxima phenomenon was observed. The KPF recovery in the urine was 60.5% for the freeze dried inclusion compound and 40.0% for the simply freeze-dried drug. In order to confirm the results obtained in the rabbits, a bioavailability study was carried out using beagle dogs. In the area under the curve up to four hours after administration the ratio was found to be 1.52 to 1.00 for the inclusion compound KPF against the simply freeze dried drug. There was a significant difference in the blood levels up to four hours, as determined by Student's-t-test.
Cyclodextrin complexes are not necessarily limited to oral use.
Cyclodextrin complexes in suppositories have improved dissolution and bioavailability. For instance, Iwaoku, R. ~-(10 4), studied the absorption of phenobarbital from suppositories containing beta-cyclodextrin. In this study, five male albino rabbits were used, and a test suppository containing approximatel y 50mg phenobarbital was inserted into the rectum. An interval of more than two days was all owed prior to the next experiment. Assay of phenobarbital in blood samples withdrawn from the ear vein was performed by gas chromatography; however, details of assay sensitivity, reproducibility were not reported. Blood levels of the drug containing the complex were muth higher during the initial three hour period. There was a statistically significant difference in the extent of absorption compared to the rate of absorption.
Summarizing the biological availability data, the author of this thesis feels as follows: Some of the published data demonstrate unambiguously that the cyclodextrin inclusion compounds are shown to improve bioavailability (in animals as well as in humans) of certain drugs . However, some of the reported studies are not well designed or may lack certain essential details; hence, reported results should be accepted with some reservations. However, there is a substantial number of papers that show that orally administered cyclodextrins are safe and useful in the enhancement of bioavailability. There are some cases in which it has been shown that cyclodextrins are not only useful in the enhancement of bioavailability but also in the reduction of side effects .
It has been reported that for digoxin there was a correlation between..!..!! vitro dissolution enhancement by complexation and in vivo absorption in animals.

Target organ oriented dosage forms
It is understood by the author of this thesis that certain research Because of today's myriad regulations, the NOA submission has become a compilation of information that could be compared in size to any one of the well known encyclopedias. Currently, the period f rom the time of synthesis of a compound to its release for marketing is genera l ly some ten-year or longer (122)(123)(124) . The FDA does try to minimize duplication of effort in preparing application for drugs about which some of the needed information is already available, by allowing use of that informati on with assurance that the new product will be equivalent to established marketed products. In this regard, the detailed description of the concept of an abbreviated new drug application (ANDA) was published in 1975 (125). Unlike the NOA, which requires submission of well controlled clinical studies to demonstrate effectiveness, data to show saftey, and detailed description of the manufacturing and packaging of a drug as well as stability data, the ANDA requires the following: a description of the components and composition of the dosage form to be marketed; brief statements that identify the place where the drug is to be manufactured; the name of the supplier of the active ingredients; an outline of the methods and facilities used in the manufacture and package, etc. (126).
The purpose of the ANDA proced ure is to eliminate uncessary and costly animal and human experimentaion and to make all drug substances not covered by patents readily available to the consumer in a competitve market. As discussed in the sections B and C of this introduction, detailed studies of the metabolism and toxicity have shown that the orally administered beta-cyclodextrin is innocuous. The cyclodextrins administered in high doses subcutaneously, intraperitoneally, or intravenously can induce renal damages in the rat, but at lower doses the compound can be administered without any ill effects (20,21). Discussion with people from the pharmaceutical industry suggests that several companies are working on the formulation and evaluation of pharmaceutical products containing cyclodextrins, while other companies are waiting to see a cost reduction and improved availability of cyclodextrins before proceeding with their studies . For instance, Squibb is evaluating parenteral administration of a pharmaceutical product containing cyclodextrin, while in Japan prostaglandin El is now commercially available ;n the form of its cyclodextrin complex (15,23). Applying Beer's law, the slope of the straight line obtained by the absorbance (peak height in this case) as a function of concentration is equivalent to the term ab in Eq. (4).

Technical aspects
By rearranging Eq. (4) to Eq . (5) The concentration of an unknown sample may be calculated.
A graph of log concentration of the drug as a function of time was then plotted. The hydrolytic degradation constant (kd) and half-life (tl /2) with and without beta -cyclodextrin were calculated using Eqs. (6) and (7), respectively.
Eq. (8) Where ko and kc are the pseudo -first order rate constants for the degradation of uncomplexed and complexed ampicillin, respectively. kobs is the observed pseudo-first order rate constant, and K is the apparent stability constant for the complex. A plot of ( kobs -ko ) as a function of (kobs -ko) /beta -cyclodextrin concentration was then plotted. From the slope and intercept of the plot ko, kc, and K were calculated .

b. Phenobarbitone
Interaction of beta-cyclodextrin with phenobarbitone was studied by solubility analysis. The procedure used in this study was the solubility method of Higuchi and Lach (131). v/v). A phase solubility diagram was constructed according to the method described by Higuchi and Connors (47). The stability constant was calculated from the phase solubility diagram as described in the section E, 1 (Eq. 1) of the introduction.

c. Phenytoin
Interaction of beta-cyclodextrin with phenytoin was studied by solubility analysis. The solu bility was determined in a pH 7.0 using the Therell and Stenhagen buffer system (133 ) . The method was as described in section l, b, except that the drug was assayed by ultra-violet absorption at the wavelength of 225nm. The USP (128) value for this drug is at the wavelength of 258nm in water. However, at this wavelength there was no maximum absorption observed even with the USP standard of this drug.
Hence, E 1 cm 1 % was determined as in the Isola tion and Identif icati on of Grugs edited by Clarke (13 4 were dissolved in water. It was necessary to add in some cases less than 2 % of 28 % ammonium hydroxide solution to dissolve the drug completely.
The solution was filtered through .a 100 mesh screen and then freeze dried using a laboratory model and as well as an industrial model freeze drier.
The details of the freeze drying operating conditions are given in Table   II. This freeze drying procedure was basically that described by Ku rozumi, ~{5 1 ) . d. Spray drying : As described in the freeze drying procedure, solutions were prepared and spray dried using a laboratory model spray drier. The details of the operating conditions are in Table III. 3. Evaluation of the complexes a. Stability and solubility analysis As described in the section 8, of the introduction, stability and solubility studies were carried out.  Table II -Freeze drying procedure-*1. Prefreeze the solution to be freeze dried.
2. Turn on refrigeration before turning on vacuum pump.
3. Do not proceed unless the temperature is between -40 tO -50oC or lower. ~·-Difficult to monitor the product ·temperature and chamber pressure using a laboratory freeze drier.
~·-These two parameters were monitored using an i ndustrial model freeze drier (Appendix B). e. As soon as the inlet temperature has stabilized, adjust and stabilize ~he outlet temperature.
p. Turn on compressed air pressure to the desired value.
~· When the desired values have been achieved, the unit is ready for the spraying operation.
5. Adjust the aspirator to regulate the spraying.
~-Following conditions were used for the present study.
Inlet temperature-120 to 140oC Outlet temperature -80 to 90oC Td W/V Eq. (9) and bulk density Bd = W/50 Eq. ( 10) W here Td and Bd represent the tapped and bulk densities, respective 1 y.
Compressibility was then determined as follows.
% Compressibility= ( Td -Bd )/Td X 100 Eq. ( 11) e. Moisture content The moisture content of each material was determined using an Ohaus Moisture Balance, operating at a temperature of so 0 c (4 watt light) for a period of 30-min. The percent loss was directly read from the balance.
f. Flowability studies Powder flow was determined using a rec ording flow meter (Fig . 6) .
The same procedure was used in this study. Eq. ( 12 ) Where fl is the powder flow index and r 2 is the least squares correlation coefficient .

g. Compressibility studies
Compression measurments were made using an instrumented Stokes B rotary press, located in the Department of Pharmaceutics of the University of Rhode Island. This tablet press was instrumented with four piezo-electric transducers and interfaced with an Apple Il e computer.
The software to the Apple computer (d eveloped by Mr. J. Hoblitzell) enabled the calculation of compression and ejection forces. In additi on, it also calculates mean area under the compression, ejection curves, mean area to height ratio for compression and standard deviation for all the above parameters.

Solution preparation and evaluation
The compositions of two potential f ormulations are shown in Tab les IV and V. Once the dosage form was prepared, its properties were evaluated on daily basis to weekly, and then one month. These evaluations were as follows: a. Visual examination for clarity, such as, precipitation, crystal growth, change in color, odor, etc.
b. Assay of drug content (HPLC) after samples had been stored at room temperature for one month.

Suspension preparation and evaluation
A final suspension formula is given in  ( 13) c. Measurement of redispersibility study : To standardize the evaluation of redispersibility a blender was modified ( Fig . 7), such that the cylinder would turn through 360° at a speed of 20-22 rpm. The number of revolutions necessary to restore the suspension to homogen ity was recorded. Similar methods of evaluation have been reported in the literature ( 137).

d. Photomicrographs
As described in the experimental section B, 3g .    Tablet formulation and preparation are given in Table VII . Tablets were evaluated as follows: a. Appea ranee Tablets were examined using a lOX magnifying glass for chipping cracking, picking or mottling of the surface as an in-process check.
b. Weight The weight of each individual tablet was determined after dedusting.
This procedure was repeated for twenty tablets. The data from the tablets were analyzed for sample mean and standard deviation .

c. Thickness
The thickness of ten tablets was determined by first dedusting, and then placing each of them in the jaws of a micrometer. The measurements were recorded and analyzed for mean value and standard deviation.

d. Hardness
The hardness of ten tablets was determined by placing each tablet in the hardness tester (Erweka), which recorded the breaking strength of the tablet in kilograms. This procedure was repeated and the data were analyzed for sample mean and standard deviation.

e. Friability
This test is a measure of the abrasion resistance which was determined by first weighing twenty tablets after dedusting, then placing them in a tumbling chamber for four minutes or 100 revolutions. The tablets were again dedusted and weighed after tumbling, and the percent friability was determined as follows: % Friability = ( Iw -Fw ) I Iw X 100 Eq. (14) Where Iw and Fw represent the initial and final weights, respectively. ~horoughly mixed for fifteen minutes in a Kitchen Aid Mixer, and wetted ~sing water as a gra nulating agent. The wet mass was seived (10 #) and ~ried at 40oC to approximately a moisture level of 5% as detennined by a ~oisture balance. Dried mass was reseived (20 #) to produce unifonn granules, then mixed for ten minutes with the remaining (0.75%) sodium starch glycolate and microcrystal line cellulose (app. 18%). The ~agnesium stearate was mixed in a Turbula Rapid Blender for three minute~ ~ith the tablet matrices and tablets were compressed. Punch diameter wa~ approximately 0.95cm and target weight was 312.50mg.

f. Ease of manufacturing
The noise and vibration from the tablet press were carefully monitored subjectively to identify any problems in manufacturing tablets.

g. Disintegration
Tablet disintegratio n was tested using the USP apparatus, as described in the U. S. Pharmacoepia (128). The time needed for all the palpable fragments to pass through the screen at the bottom of the cage was detected visually and was recorded. Six tablets were used in each test, and mean and standard deviations were calculated .

h. Dissolution
When a drug was to be measured for rate and extent of dissolution, a sample of the lot of drug to be used was placed in various concentrations in a spectrophotometer to measure the max for that particular drug.
Once this value was determined, the spectrophotometer was set at that wavelength, and each sample was analyzed for absorbance. The result was recorded on a Beer's plot (as described in the method section B, 1) and correlation between absorbance and concentration was recorded. The monograph, as it appeared in the USP (128) was used as a reference for determining the dissolution medium for the test . In addition, dissolution was determined in acidic (pH 2.2) and basic media (pH 7.4).
The requried acidic and basic media were prepared in accordance with the USP (128). A sample of three and six tablets wer~· used for each system.  Table {VIII) . An analysis of variance {ANOVA) was used to evaluate any statistical significance among the treatments. There was a significant difference (.£.=0. 05) between the treatments with 1:0 . 0 and 1:1.0 molar concentrations of ampicillin to beta-cyclodextrin. Furthermore, due to the single replicate nature of the data and the constant time period between observations it was deemed more appropriate to evaluate the colinearity of the degradation curves presented in Fig . 8. This analysis detected a significant difference {-<.
= 0.05) between the slopes associated with the treatments using ratios of 1:0.0 and 1:1 . 0 of ampicillin to betacyclodextrin. In order to determine the optimal ratio of beta-cyclodextrin to ampicillin, a graph was constructed by plotting the observed degradation rate constants as a function of the beta-cyclodextrin concentration. As seen in Fig. 9, the observed degradation rate constant asymptotically approaches a minimal value as the beta-cyclodextrin concentration is increased. This saturation behavior is a characteristic of reactions which proceed Where Amp., Beta-CD, and Am p. Beta-CD, represent ampicillin, beta-cyclo-dextrin and the inclusion complex of ampicillin with the beta-cyclodextrin, respectively. The pseudo-first order rate constants fa~ the deg radati on of uncomplexed and complexed ampicillin are k 0 and kc and K is the apparent stability constant for the complex. In Fig. 10 the rate constants of Fig. 9 are plotted according to Eq. (8).
For example: kobs -k 0 = -(ko bs -k 0 )/K(Beta -CD) + kc -k 0 A plot of kobs -k 0 as a function of (ko bs -k 0 )/ beta-cyclodextrin is shown in Fig. 8. From the slope and intercept of the plot k 0 , kc, and K were obtained and they are reported in Table (  The solubility method of Higuchi and Lach (131) was used to study the complex formation of phenobarbitone and phenytoin . Interactions of these two drugs were studied by solubility analysis. A phase solubility diagram of phenobarbitone was constructed according to the method described by Higuchi and Connors (47) and is depicted in Fig. 11. There was a five-fold increase in the solubility of phenobarbitone due t o complex formation. According to the authors of the phase solubility techniques, if a plot of the total molar concentration of substrate as a functio n of the total molar concentration of ligand and the complex is of a 1:1 type then a straight line with a positive slope will result. Thus, from such plots the stability constant (K) can be calculated using Eq. 1 as foll OWS: Where S 0 is the solubility of the substrate ( phenobarbitone ) in t he absence of the ligand (beta -cyclodextrin) . In other words, S 0 is the intercept as shown in Fig. 9, and using the slope from this figure, the stability constant can be obtained. However, if the slope is greater than unity, as in this case (Fig. 9), it becomes impossib l e to calculate the stability constant using the above equation. Hence, the derivation of the above equation was reexamined, and it was modified to calculate an approximate apparent stability constant as follows.
Higuchi and Connors derived the above Eq. l fr om the fol lowing equation. In order to determine the value of m, the complex was isolated (preparation is discussed in Section JIB, 2) and assayed. The results are reported in Table (X). Based on the percent drug content in the complex, the value of mis calculated as follows .
The molecular weight of phenobarbitone is 232gm/mole and that of the beta-cyclodextrin is 1135gm/mole . Hence, the number of moles in 20% of the drug is equal to 8.62 x 10 -2 and that of beta -cyclodextrin is equal  Eq. (18 ) The calculated apparent stability constant is 4.54 x 10 3 M-l by using the slope and intercept values from Fig . 9. This value is close to the va l ue (3.60 x 10 3 M-1 ) obtained by Thoma and Stewart (9). Although the stability constant value obtained by Thoma and Stewart is cl ose t o the value obtained in the present study, there is a discrepancy. This discrepancy can be explained as follows . Firstly, the reported stability constant value by Thoma and Stewart was from the interaction study carried out at 30°c, whereas, the present study was carried out at 2s 0 c.
Hence there is a temperature effect on the stability constant . Second ly , unless a solid complex is isolated and its stoichiometry analyzed, calculation of the exact stability constant may lead to an approximati on.
It is the opinion of the author of this thesis that Thoma and Stewart did not isolate the complex to detennine the stoichiometry; hence there is a discrepancy in the stability constant values. Further, Higuchi and Connors (47) also pointed out in their phase solubility techniques that the stoichiometry and the equilibrium constants may be ambiguous quantities . Hence (according to them), whenever there is an ambiguity or conflicting results, a solid complex should be isolated and its stoichiometry analyzed and compared with a graphical estimate of the stoichiometric ratio. Thus, the phenobarbitone beta-cycl odextrin comp l ex may be a molecular ratio of 1. 2:1.0, rather 1:1.
The results of the interaction of phenytoin with beta-cyclodextrin are depicted in Fig . 12. There was an el even-fold increase in the solubility of phenytoin due t o the comple x f ormati on.  Uekama (117) reported the solubilities of several drugs in water both in the presence and in the absence of beta-cyclodextrin. Among the reported drugs is phenytoin . These results are reported in Table ( A phase solubility diagram was constructed from the solubility data of phenytoin and is shown in Fig . 12. From this phase diagram, and using Eq. 1, the stability constant {766M -1 ) of the complex was obtained. As shown in Fig. 12, in this interaction study, the slope is less than unity and hence there was no need to determine a value for m (Eq . 16) to calculate the stability constant. However, a solid complex was isolated (preparation is described in Section IIB, 2) and the drug content in the complex was analyzed by a spectrophotometric method. The results of the drug content in the comple x are reported in Table XII . Based on the drug content in the complex, the ratio of the substrate concentration to the ligand concentration {ob tained as described earlier) gave a value of 1.05 . This value is an indication of the complex with a molecular ratio of 1:1.

Lach and Cohen {40) studied the interaction of nineteen compounds
with alpha and beta-cyclodextrin. The data in their study indicate that    Table   XIII, phenobarbitone shows the highest slope compared to phenytoin, since phenobarbitone is more soluble than phenytoin . However, increase in solubility of phenytoin relative to its initial solubility is eleven-fold compared to only a five -fold increase in phenobarbitone solubility. This increase in solubility, especially for phenytoin, has a considerable potential in the development of suitable dosage forms. The extent of complex formation (as evidenced by the stability constant) of phenobarbitone is greater than that of phenytoin. This stronger interaction of phenobarbitone may be due to the size of the molecule.
Since the size of the phenobarbitone molecule is smaller than the phenytoin molecule (Table XIII), smaller molecules may fit well into the beta-cyclodextrin cavity to form a more stable complex. Cohen and Lach (36) also felt that geometrical rather than chemical factors are decisive in determining the extent of complex formation.
c. X-ray diffractometry The X-ray diffractometer allows us to examine the atomic arrangement of a material, giving information on crystal geometry and structure . It has been reported (49,50) that an inclusion complex can be detected quickly and directly by the X-ray methods. As per these reported studies, a true inclusion complex may exist if the diffraction pattern does not correspond to those of pure components. Figures 13 and 14 show the X-ray diffraction patterns of phenytoin and the beta-cyclodextrin. As shown in the Fig. 13c, the diffraction Table XIII Slopes of intera ction isotherms, solu bility data an d stab ility constants .  pattern of the freeze dried material is different from that of the pure components and their physical mixture. The diffraction pattern of the physical mixture was found to be a simple superimposition of those of the compo nents. The X-ray diffraction pattern shown in the Fig. 14b, i ndicates that the beta-cyclodextrin, which was originally in a crystalline fonn (Fig . 13b), is transfonned to an amorphous state after freeze drying. Similarly, the phenytoin was originally in a crystalline fonn; which did not transfonn to an amorphous state after freeze drying of phenytoin alone (Fig. 14a); however, the complex prepared by the  Fig. 15b indicate that , like phenytoin, phenobarbitone also retains its crystalline form even after freeze drying. However, the phenobarbitone beta-cyclodextrin complex prepared by the freeze drying method is completely transformed to an amorphous state (Fig . 15c). The physical mixture pattern is a simple superimposition of the pure components (Fig . 16c) This new molecular order may be due to inclusion complexation of phenytoin with the beta-cyclodextrin. Based on the X-ra y resluts and solubility characteristics, it is possible by inclusion complexation that the axial length (A) may have changed. However, in order to reach a definitive conclusion, this question is deferred for further study.
Since, the author of this thesis could find no data reported in the literature on the measurement of the lattice paramete rs of inclusion complexes.   Cyclodextrins complex formation may be proved by DSC . Fig ure 17 shows the DSC curves of freeze dried phenytoin and the freeze dried complex. In Figs . 17 and 18 the freeze dried phenytoin and beta-cyclodextrin gave an endothermic peak aro und 29S°C {568°K) , which corresponds to the melting points of the phenytoin and beta-cyclodextrin.
The freeze dried complex and the physical mixture {Figs . 17 and 18) also gave an endothermic peak around the same temperature (Fig . 18). However, the pattern of the freeze dr ied complex thermogram is clearly different · from that of the phys i cal mixtu re. These thermograms suggest that there is some interaction in the freeze dried complex but not in the physical mixture. Because of the very narrow range of the melting points of the phenytoin and beta-cyclodextrin (Table XV) '° .....

Table XV
Melting points data on phenytoin, phenobarbitone and beta-cyclodextrin.  (ii) This phenytoin beta-cyclodextrin complex contains around 20% drug in the complex (Table XII).

Drug
For IR spectra , one or two mg of sample is used with 80 -90  to dissolve the complex, if the solvent is more polar than the included guest molecule, then a complex with the organic solvent will be formed.
In this study, since the phenytoin beta-cyclodextrin complex did not dissolve in the deuterium oxide, it was very diffcult to show the complex formation in the liquid phase . However, the phenobarbitone complex readily dissolved in the deuterium oxide, and the results are shown in Fig. 23 . The large peak at approximately 4.6ppm is common to both spectra due to small amounts of deuterium hydroxide and water as impurities. All the peaks observed in the Fig. 23a, are those of the beta-cyclodextrin . The deuterium oxide peak is fixed in both spectra; this peak was used as an internal standard for the comparison of the chemical shift observations. From the spectra of both the beta-cyclodextrin and the complex, it is clear that all of the protons of the complex located in the internal cavity of the beta-cyclodextrin are experiencing a shielding effect; hence signals are shifted upfield (Fig .   23b). These spectra strongly suggest the presence of the complex in the liquid phase. Demarco and Thakkar (52) reported ~hat the protons l ocated in the interior cavity of the beta-cyclodextrin (C -3-H, and C-5-H) will be considerably shielded by the guest molecule; hence the signals will be = ;:: shifted upfield. Whereas, the protons on the outer surface (C-2-H, C-4-H, and C-6-H) will not be affected by the guest molecule; hence the signals of those protons will remain unchanged. In addition, Frank and Cho (32) also observed similar results in the study of the complexing behavior of dinoprostone with beta-cyclodextrin in water.
h. Photomicrographs Figures 24,25 and 26 show the morphology of recrystallized beta-cyclodextrin, phenytoin, and phenobarbitone before and after freeze drying . All the photomicrographs were taken at the same magnification (125x) . As shown in the Fig. 24a, the beta-cyclodextrin is a crysatlline material, however after freeze drying it has completely transfonTied into an amorphous state. This amorphous state is also confinTied in the X-ray analysis (Fig. 14b). Whereas phenytoin and phenobarbitone ( Figs. 25b and 26b) as seen i n the X-ray analysis (Figs . 14a, and 15b) did not transfonTI into an amorphous state, the complex prepared by the freeze drying method of these two drugs as shown in Figs . 27b, and 28b, are completely transfonTied into an amorphous state. This was also previously observed in the X-ray analysis (Figs . 13c, and 15c). The complex prepared by various methods are shown in Figs. 27a, 28a, and 29. It is very diffcult to prove in an unambiguous manner the fonTiation of an inclusion complex in these photomicrographs; it can be readily shown in Fig. 29b that, some kind of interaction has taken place in the complex prepared by the solvent evaporation method. As previously discussed, a single crystal isolated from this preparat io n was taken for lattice parameters measurement and showed an increase in one of the axial length.
The results are reported in the Table (XIV) . Based on this photomicrograph, the lattice parameters data, and also from the X-ray     days to evaporate at room temperature.
(ii) . The solvent is 50% ethanol, which is expensive and without a solvent recovery system this process may not be economically feasible.
A phenytoin beta-cyclodextrin complex was prepared by this procedure with a satisfactory yield of 95%. The final end product was in a crystalline form. As discussed in Section !!IA, d., th e measurement of lattice parameters showed some interaction with the beta -cyclodextrin.
However, in order to collect a reasonable quantity of material to begin formulation work, it would take several months' time to collect a workable amount of material. Further, even in the lite rature this method of preparation of inclusion compounds has not been explored.

b. Kneading
In the preparati on of inclusion compounds the most common techinque is to stir or shake an aqueous solution of cyclodextrin with a guest molecule or its solution. In aqueous solution the apolar cyclodextrin cavity is occupied by water molecules that are in an energetically unfavorable state (polar -apolar repulsion) and are, therefore readily replaced by an appropriate guest molecule that is less polar than water (Fig. 4). In this kneading process the cyclodextrin is not dissolved, it is kneaded with a small amo un t of water to make a slurry, and then guest components are added to the slurry of cyclodextrin. Since neither the guest nor host molecules are dispersed at a molecular level, it is difficult to conceive the formation of inclusion compounds in this process. However, this method of preparation has been reported in the literature (8).
A phenytoin-beta-cyclodextrin complex was prepared by this method with a satisfactory yield of 92% . Only the X-ray-analysis (Fig. 14 ) , showed ambiguously the presence of the inclusion complex as a separate molecular species. Thus, it is interesting to see the effect of kneaded complex on formulations.
c . Spray drying Figs. 27a and 28a show the morphology of spray dried phenytoin and phenobarbitone complex with beta -cyclodextrin . It appears from the photomicrographs that the complex may be a physical mixture of the pure components (Figs. 24a, 25a and 26a). These types of results may be conceivable from the spray drying process as follows.
During the spray drying process the surface liquid is quickly evaporated and a tough shell of solids may form in its place. As drying proceeds, the liquid in the interior of the droplet must diffuse through this shell; however, the diffusion of the liquid occurs at a much slower rate than does the transfer of heat through the shell to the interior of the droplet. The resultant build-up of heat causes the liquid below the shell to evaporate at a far greater rate than it can diffuse to the surface. The internal pressure causes the droplet to swell; and if the shell is nonelastic, it ruptures, producing either fragments or ruptured hollow spheres and perhaps some intact spheres . Therefore, when the cyclodextrin solution with the drug is sprayed, if the shell is nonelastic, it may rupture producing fragments of drug and cyclodextrin or there may be fragments of both species together. Hence, if the shells of cyclodextrin solids are nonelastic it may not be possible to produce an intact cyclodextrin species containing drug molecules.
As shown in Figs. 14 and 15, only the X-ray analysis showed some kind of interaction with beta-cyclodextrin rather than a physical mixture of the pure components. The photomicrographic evaluations are highly subjective; therefore based on the X-ray analysis, it is felt that the inclusion complex exists as a separate molecular species.
However, there was only a 10% yield of the complex prepared by this method. The low yield is due to the poor design of the equipment rather than technique, because of the following reasons: (i). (ii).
The solution was sprayed as fine droplets into a moving stream of hot air, where they did not evaporate rapidly before reaching the wall of the drying chamber.
The resultant build-up of liquid droplets on the wall of the drying yield.
chamber did not dry into a fine powder to give a good (iii). The temperature of the system was fluctuating constantly during the spraying process.
(iv) The peristaltic feed pump was not functioning properly.
Therefore, because of the unsatisfactory yield,, this method was not explored further.

d. Freeze-drying
Among the various methods of preparing the inclusion complexes, this method was satisfactory at a laboratory level. However, the ampicillin inclusion compound could not be prepared by this method since it has been (114) reported that the rate of decomposition of the ampicillin increases upon freezing. Savello and Shangraw (139) showed that for a 1% sodium ampicillin solution in 5% dextrose, the percentage of degradation at four hours is approximately 14% at -20°c, compared to 10% at s 0 c. Therefore, only phenytoin and phenobarbitone beta-cyclodextrin complexes were prepared firstly by using a laboratory freeze drier.
As shown in Fig. 13c, the phenytoin beta-cyclodextrin complex prepared by the freeze drying method strongly suggested the presence of inclusion complex as a separate molecular species . In addition the OSC thermograms ( Fig. 17) also suggested that the freeze dried complex is not a physical mixture. Similarly, the DSC thermograms of the phenobarbitone complex (Figs . 19 and 20) and the NMR spectra (Fig. 23 Lach and Cohen (40) studied the interaction of nineteen compounds with cyclodextrins, but they did not report how to harvest these inclusion compounds.
In the present study, preparation of the inclusion compounds using the freeze drying method is expanded from a laboratory freeze drier to an industrial level freeze-drier. Since the laboratory freeze drier was not capable of handling more than a liter per batch, the batch size was limited to one liter. The total time required to comp lete the batch was 4-5 days. Although theoretically the yield per batch (with 2% solids) was 20 grams, the experimental yield was only 18 grams. Therefore, in order to begin formulation work by using the laboratory freeze dryer it would take months to collect a reasonable quantity of material. Hence, the success of a new drug or complex for dosage forms formulation work is dependent upon scale-up evaluation from laboratory procedure to routine production operations.
With this in mind two phenytoin beta-cyclodextrin complex batches were made using an industrial level freeze dr yer at Miles Pharamceutics in West Haven, Connecticut . The first batch yield (Ta ble XVI) was much poorer than expected (43% less). This process was carried out in an open tray freeze -drying system under a vacuum, thus, directly exposing the drying material to the applied vacuum. A large portion of the dried fluffy end product may have been inadvertently vacuumed away during the drying cycle. In order to increase the yield and decrease the percent loss, another batch was made with a minor modification to the open tray system (Fig . 30a). As shown in the Fig. 30b, a cover with an opening in the center was placed onto the open metal tray. In addition, a stainless steel screen (30 mesh) was used to cover the center opening (Fig. 30c).
This equipment resulted in a reduction in direct exposure and prevented the loss of the drying material to the applied vacuum. The yield was significantly improved, limiting the loss to 27% (Table XVI) .
The results of preliminary scale-up batches indicated that the preparation of the complex by freeze drying may be scaled-up to a Table XVI Preliminary scale-up results of the phenytion beta-cyc lode xt rin comp l ex.
Batch No .  (Table XVII) suggested that the method is also reproducible. It was felt that it may be possible to improve the yield by using finer screens to cover the center opening in Fig. 30b. A 60 mesh stainless steel screen over the center openi ng improved the yield from 73% to 90% (Table XVIII). Thus, it was clearly shown that th e preparation of the phenytoin inclusion compound can be scaled-up to manufacturing level. The total length of time required to complete the ten liters batch was little less than two days (Appendix B, 1), compared to four to five days to complete the lite r batch by using the laboratory freeze drier. In addition, in the laboratory freeze drier, it was not possible to determine the final end product temperature without breaking the vacuum system. However, as shown in Appendix B, 1, it was possible to determine quantitatively the final end product temperature.
A phenobarbitone beta-cyclodextrin complex was also prepared using the same technique to improve the yield. It was possible to get a yield of more than 90%. The total length of time required to complete the ten liter batch was less than two days (App endix B, 2).
C. Preparation and evaluation of dosag e forms

Liquid dosage forms
The compositions of two potential liquid formulations are given in Tables IV and V. These preparations were visually examined for clarity, ( precipitation, crystal growth) change in color, odor, etc. After two weeks the formulation containing 10% glycerol, 40% sorbitol, and 20% syrup (Table V) showed slight precipitation and microbial growth.
However, the formulation (Table IV) containing 40% glycerol, 20% sorbitol  and 20% syrup did not show any precipitation or visual evidence of microbial growth after samples had been stored at ambient temperature up to four months. The probable reason for an increase in stability of the formulation is the higher concentration of glycerol, since glycerol itself acts as a preservative when it is present in higher concentrations (130). This formulation was compared with an official elixir ( 128 ) with respect to physical properties as well as one month's chemical stability data. The results are in Table XIX. The official formula contains 45% glycerol, 15% syrup and 15% alcohol. The alcohol content of the official preparation is required to keep the phenobarbitone in solution; however, the alcohol content in the official elixir may not be desirable, especially for pediatric use. For the phenobarbitone beta-cyclodextrin complex the addition of alcohol was not required. It has been reported (140,141) that in the case of elixir of phenobarbitone, propylene glycol, and a combination of poly-alcohols can be used as substitutes, and formulations using these substitutes have been proposed. Peterson and Hepponen (142) , developed a formula containing 35% of propylene glycol, 20% syrup, and 0.4% of phenobarbitone with 0.1% flavoring oil . They did not report any chemical stability data, however, after three months physical stability was satisfacto ry. In or der to aid in masking the bitter taste of phenobarbitone, and since propylene glycol does not add any sweetness as does the glycerol of the official formula , they increased the syrup content from the 15% of the official formula to 20%.
However, their taste tests showed that the propylene glycol preparati ons were less acceptable than the official elixir.
The formulation (Table IV) of the present study does not contain any propylene glycol, and the concentration of the glycerol is similar to One month chemical stability of solution prepared from the phenobarbitone beta-cyclodextrin complex, stored at room temperature.
that of the official preparation. The taste was better than or equal to the official formulation. Furthermore, it is the op in ion of the author of this thesis that the complex could readily be used as a powder for reconstitution were any chemical stability problems to arise in the preparaticrn.
A phenytoin suspension was prepared (Ta ble VI) from t he complex and its physical stability up to two years did not show changes in appearance, color, odor, etc. The results of the sedimentation study {Fig. 31), indicated that the sedimentation volume was slowly decreased during the first period of the week, then decreased grad ua lly . However, even after six months, the sedimentation volume was approximately 60% of its original value. The redispersibility study showed that ten revolutions were required to restore the suspension to homogene ity. The blender rotation was 20 to 22 rpm. Hence, the suspension was easi ly redispersed within 30 seconds . In order to compare the ease of redispersability using the tech nique described in Experimental Section Ile, 2c, a light magnesium oxide suspension was introduced as a control.
The percentage of solid content was the same as in the phenytoin suspension (Table VI) in the same suspending medium. However, due to formation of a concrete cake, it was not possible to redisperse the lig ht magnesium oxide suspension, even after five minutes. In contrast, the phenytoin beta-cyclodextrin complex suspension, which contained amorphous freeze dried material, formed a flocculated suspension. A floe, or floccule, by definition (143), is a loose aggregation of individual particles that are held together by comparatively weak particle-to-particle bonding forces; the sediment is loosely packed and has a scaffold -like structure. Particles do not bond tightly to each other, and a hard, concrete cake does not form. The sediment of our suspension was easy to redisperse, so as to form the original suspension.
Generally, a typical suspension form ula contains drug, suspending agent, suspending medium, flocculating agent, electrolytes, preservatives, etc . (144 ,145 ). But the formula selected in this study (Table VI ) contains only drug or suspensoid and suspending medium. It may also be noteworthy that a 50:50 mixtu re of water and glycerol as the suspending medium was chosen because of the poor solubility of phenytoin (138 ,146) plus the low solubility of beta -cyclodextrin in the suspending medium (147). The drug complex exhibits a minimum degree of solubility and thereby it is possible to achieve a maximum chemical stability.
Hence, despite the high concentration of the solid content (10%) without any extra additives, the suspension possessed desirable qualities (144, 145 and 148) such as ease of dispersion, high sediment volume, etc.
These properties are indicative of a pharmaceutically elegant suspension.  useful for detecting changes in particle s ize and crystal form. Nash (145) also used a similar technique in the evaluation of physical stability of a suspension and also particle size measurement. However, he reported that particle size measurement using a photomicrograph gives an approximation but not an accurate value. Therefore Nash (145) suggested the use of a Coulter Counter, an electronic particle counter that measures the resistance caused by the presence of a particle in an electrolyte. Since it was not possible to use such an electronic particle counter, an estimation of the particle size was determined from  Table XX.
It is evident in Table XX that good flow rate and a high linearity index were recorded through hoppers I and II as the mass was increased up  The results are plotted in Fig. 35.   it is estimated that the total amount of the complex required would be SOOmg (e quivalent to lOOmg phenytoin). Therefore, the complex would contribute a major portion of the t otal tablet weight.
In order to enhance the flow properties especially of the freeze dried complex (poor flow, Table XX!!), different concentrations of the complex were mixed with a direct compression vehicle ( Emcompress ) and flow rates evaluated. As the concentrati on of the complex was increased ( Fig . 36), flow rate decreased drastically . An acceptable flow rate was found only when the conentration of complex was less than 5%. Thus, direct compression was not found to be feasible, since the final tablet weight would be more than a few grams. An attempt was made to prepare slugs, however, due to lack of compressibility and flowability of the complex, slugs could not be prepared. Thus wet granulation was the only method available for tabletting. Since freeze dried materials are generally highly soluble, it was thought that after addition of a granulating agent the material might agglomerate rap idly; however, no agglomeration problem was observed during the wet granulation process.
It was then possible to develop a formula (Table VII) with an acceptable weight and size. It has been reported by Szejtli,J. (8) , who is one of the authorities in the field of cyclodextrin inclusion complexes that, "if the required dose amounts to several hundred milligrams, then especially with low molecular weight drugs, it would be necessary to   disperse several grams of cyclodextrin complex tablets. Thus no practical application of the complex seems to be likely." However, the phenytoin dose is lOOmg, ( by complexation with beta-cyclodextrin) and using the wet granulation method it was possible to make a tablet of 625mg containing lOOmg phenytoin. It is in the opinion of the author of this thesis that if the drug content in the complex is less than 10%, and if the molecular weight of the drug is very low, practical application of cyclodextrin complexes for tabletting is highly unlikel y.
Table XX!!! shows the particle size distribution, bulk properties, flow characteristic and compressibility of formulation prepared from the phenytoin beta-cyclodextrin complex. The complex was prepared by the freeze drying method and then wet granulated . By wet granulati on it was possible to achieve good flow rate and compressibility. Furthermore, tablets were compressed using an instrumented tablet press and Fig. 37 depicts the results of compressibility. It is evident from Fig. 37  properties. All the examined properties meet the USP standards. Fig. 38 shows the dissolution test data performed on weighed portions of the tablets; the figure shows that at 60 minutes more than 90% of the drug had dissolved. Fig. 39 depicts the dissolution test data performed in accordance with the USP monograph dissolution medium . In this plot (Fig.   39) the dotted line represents the percent drug dissolved based on the equilibrium solubility of phenytoin (138). The dissolution results of three different products are shown in this plot. The three products are:  tablets prepared from the freeze dried beta -cyclodextrin complex; Infatabs from Parke-Davis; and phenytoin tablets prepared by Mr. S.R.
Ghanta (gra duate student). It is evident that the phenytoin beta-cyclodextrin complex showed highest percent dissolved (more than 60% at 30 minutes). Because of th~ low aqueous solubility of phenytoin (138), dissolution was conducted with half tablets. However, as shown in the same plot (Fig. 39), there was no significant improvement in the dissolution rate in water. The tablets prepared from the freeze dried complex reached the equilibrium solubility within fifteen minutes; whereas, the other two phenytoin products did not reach the equilibrium solubility even after 60 minutes. Further, it was thought that, since the Infatabs are used as chewable tablets, they may erode very slowly and for this reason showed slowest dissolution rate. However, the instructions on the Infatabs bottle indicate that the tablets can be either chewed thoroughly before being swallowed or swallowed whole. Therefore these tablets were taken for comparison . Generally, chewable tablets are hard, they erode very slowly in the mouth, so that a slow dissolution rate is expected. However, when these tablets were crushed, passed through 60 mesh sieve and filled into capsules, then dissoltuion was compared (Fig. 40) with the similar preparation of the tablets prepared from the freeze dried complex. As shown in Fig. 40, the freeze dried complex showed significant improvement in the percent drug dissolved. Figure 41 shows the dissolution of phenytoin beta-cyclodextrin complex tablets after two years' storage at ambient temperatures, compared with a sodium phenytoin capsule (Zenith Labs).
Tablets from the freeze dried complex reached the equilibrium solubility within 30 minutes. As shown in Fig. 41, the half tablets showed  * Each point is an average of three determinat i ons.
significant improvement in the dissolution rate compared to sodium phenytoin capsules.
Dissolution was also investigated in an acidic medium. The results are depicted in Fig . 42. In this plot, dissolution results of sodium phenytoin, freeze dried complex, physical mixture of phenytoin beta--cyclodextrin, and phenytoin powder alone are shown. As expected, the physical mixture and phenytoin did not improve dissolution . However, the dissolution rate of the complex and sodium phenytoin powder was by and large equal after a 30 minutes time period. Figure 43 shows the dissolution results of the freeze dried complex tablet (full and half) and sodium phenytoin capsule. As observed in Fig. 42, approximately 60% of the drug was dissolved from the complex within 60 minutes. The sodium phenytoin capsule showed a higher percent dissolution (apprx. 70%) within 60 minutes. This increase in percent dissolved may be due to the effect of formulation and processing. However, the percent drug dissolved from the half tablets was higher at the 30 minute time period than sodium phenytoin capsule. These results indicated that even in the acidic medium the dissolution of the phenytoin beta-cyclodextrin complex was better or equal to the sodium phenytoin capsules.
Since there was no significant difference between total percent drug dissolved in water and in an acidic medium, further dissolution work was carried out in water according to the USP (128) . Figure 44 shows the dissolution results of phenytoin beta-cyclodextrin complex prepared by the various methods. As indicated earlier the dotted line represents the equilibrium solubility of phenytoin in water at 37°C . In this plot the complex prepared by spray drying, kneading, etc, showed less than 40%  drug dissolved even at 60 minutes. Whereas, the complex prepared by th e freeze drying method showed more than 60% dissolv ed at 60 min utes.
This increase in percent dissolved is due to the amorphous nature of the freeze dried complex as seen earlier in X-ray analysis ( Fig. 13c) and photomicrographs ( 27b ) . However as shown in Table XXV, there was no si gnificant difference i n disintegration time of phen ytoin beta-cyclodextrin complex t ablets prepared by the va r ious methods.
Furthermore, it was thought that a decrease in partic le size of phenytoin may increase the dissolution rate similar to the freeze dried complex.
Hence, phenytoin was freeze dried alone, compressed into tablets and dissolution was carried out. The results are shown in Fig. 44. As shown in Fig. 44, the freeze dried phenytoin did not improve t he dissolution rate However, when the freeze dried phenytoi n was mixed with fre eze dried beta-cyclodextrin, the tablets prepared from th at mixture showed ( Fig. 44 ) the similar resu lts as obta ined from th e free ze dried complex.
As shown in Fig. 44, there is a slight difference at 15 minutes time period; however, after that time period, the percent drug dissolved was by and large equal to the freeze dried complex. This unique phenomenon, may be due to the in situ complex formati on in the dissolution medium.
It is the opinion of the author of this thesis that as f ar as ca n be ascertained, this unique phenomenon (i n situ complexation) is the first report of the interaction of beta-cyclodextrin wi th phenytoin. · Phenytoin is a high melting (293°C), weakly acidic (138 , 146 ) and poorl y water soluble drug (138). Because of these physicochemical properties, phen ytoiri is subject to erratic and incomp lete bioava ila bility (156 -1 59). Phen ytoin has been classified as a drug with "high risk potent ial " with respect to bioavailability problems (160 , 161). It has been reported that the rate of dissolution of phenytoin is influenced by particle size (162) , characteristics of excipients {163), manufacturing procedures and dosage form (164).
Chakrabarti ~ (162) reported that significant differences in bioavailability were observed when phenytoin crystals of different particle size were administered. In their investigation 3 dogs were administered 390 _: lOmg capsules in a cross-over design with an interval of one week between two administrations. The radioimmunoassay method was used in the analysis of drug content . The largest area under the curve (AUC) and maximum concentration (Cmaxl were observed with smallest particle size (0-32u), followed by 75 and lOOu particle size respectively . Chakrabarti ~ {162) did not report the time required to reach maximum (Tmax l plasma level. The paired t-test was used to show the significant difference between the Cmax and the AUC among the treatments. They concluded that the AUC is inve rsely related to the particle size. In addition, their results were correlated with the faster dissolution of phenytoin of smaller particle size. It is the opinion of the author of this thesis that since the phenytoin beta-cyclodextrin complex prepared by the freeze drying method produced amorphous particles (less than 25u), and therefore a smaller dose would be enough to achieve the therapeutic range of plasma level (10 -20 ug/ml c. The commercial crystals gave a very low plasma level and showed a significant difference was found at each time period. The coprecipitated powder gave 7.63-11.6 times larger AUC than the phenytoin crystals in individual volunteers and the mean was 8.97 times larger . The urinary excretion study also revealed a lower excretion rate of pheyntoin from the crystals. d. It was concluded from the above study that the phenytoin crystals (between 177-350u) gave significantly lower bioavailability than the coprecipitated powder due to poor dissolution characteristics of the fonnulation. However, the authors of this bioavailability study did not report the particle size range of the coprecipitated phenytoin powder.
They simply reported that when the same batch of phenytoin crystals was processed by solvent deposition, the particle size might have reduced in the course of the manufacture to below the critical range ( less than 50u ) of crystal size distribution. Further, they concluded that a a. At lower ratios of phenytoin to polyethylene glycol (0 .5:10, and 1:10) the bulk concentration reached a plateau of 43ug/ml (10 . 75%) in less than five minutes, while higher ratios (2:10, 3:10 and 4:10) gave rapid rises to 32ug/ml (8 . 0%) in five minutes with subsequent small increases.
b. These results suggested that a rati o of less t han l to 10 is required to disperse phenytoin completely in polyethylene glycol.
c. Further, the dissolution rate of different particle size suggested a critical particle size between 74-149u for higher dissolution rate.
d. Based on the results of the in vitro studies, a bioavailability study was conducted in five male subjects, and 300mg of ph enytoin crystals (size 44-53um), 200mg of physical mixture with polyethylene glycol, and 200mg of the solid dispersion powders were administered orally . A three-way cross -over study was used. Blood samples were taken at 2, 4, 6, 8, 12, 24, 32, 36, and 48 hour, and urine was also collected at the same i ntervals. The plasma samples were assayed for phenytoin by enzyme immunoassay and the levels of the intact phenytoin in urine were assayed by gas-liquid chromatography. The details of the assay methods are not reported. Phenytoin solid dispersion gave the highest plasma level (3.74ug/ml) in 5.6 hours, followed by the physical mixture and phenytoin alone. The physical mixture gave a plasmal level of (l.92ug/ml) in 6.8 hours, and phenytoin gave a lowest plasma level of 0.75ug/ml in 5. 7 hours.
e. The areas under the plasma level curve were: phenytoin solid dispersion 73.86h . ug/ml, phenytoin physical mixture 50.26h.ug/ml, and phenytoin crystals 18.45h . ug/ml. There was a significant difference among the treatments as shown by Student 's-t-tes t.
f . The urinary excretion rate from the solid dispersion of phenytoin in polyethylene glycol exceeded that from the physical mixture or the phenytoin crystal at any time.
g. It was concluded from the above study that the phenytoin solid dispersion showed superior dissolution and bioavailability. These characteristics of the phenytoin solid dispersion should offer the clinical advantages of quick drug release and excellent bioavailability in phenytoin therapy.
As previously mentioned in Section F, 6 (Bioavaila bility) of the Introduction to this thesis Tsurika ~ (95) studied the absorption of freeze dried phenytoin, phenytoin, and its beta-cyclodextrin complex in a group of four female beagle dogs. They reported that there was a two-fold increase in the area under the curve of the blood level. The Student's-t-test showed a significant difference in the blood level curves . Jn addition, it was reported that an increase in bioavailability of phenytoin by means of beta -cyclodextrin complexation suggested the possibility of smaller doses and fewer side effects in phenytoin th erap y.
The results of the in vitro dissolution studies of phenytoin obtained by Yakou ~ (163), Sekikawa et al (164), and Yakou et al (165) can be compared with the results obtained by the author of this thesis . These results are summarized in Anderson,~-(59), reported a preparation of an inclusion complex of femoxetine with beta-cyclodextrin by a precipitation method.
The method of preparation was time consuming and labo r ious (o ne liter  (15) 56 . 00 (15) 58.30 ( 15) batch took one week to collect the precipitate and one day to dr y in vacuum). The yield was onl y 87% . Scale-up evaluations, reproducibility, etc. were not reported. However, they had developed a suspension dosage fonn only on a small sacle (lOOml) from the complex. Physical properties of the suspension such as else of dispersion, sedimentation volume, freeze thaw study, etc., were not studied. Hence, the fonnulation feasibility and physical stability are to be investigated .
Sea, et al. (88), studied the inclusion complex fonnations of spironolactone with three cyclodextrins (alpha, beta, and gamma). They obtained the solid complex by a precipitation method, it took seven days to precipitate the complex and then 48-hr to dry the complex at 6o 0 c.
The percent yield, scale-up, dosage fonn development, etc., were not reported . It is the opinion of the author of this thesis that the method is time consuming and also that the preparation of the solid complex of aqueous labile drugs can be a problem . However, the above authors did not report any stability problems .
Iwaoka,~· (104), studied the abs orption of phenobarbitone from suppositories contaning beta-cyclodextrin . They dissolved the drug and beta-cyclodextrin in a molar ratio of 1:1 in hot water (temperature not reported), the solution was then filtered and left to crystall i ze (l ength of time not reported). In this study also details of the preparation of the complex, scale-up evaluation, etc., were not reported. However, after rectal administration of the suppository containing the beta-cyclodextrin complex to rabbits, the blood concentration of the drug was higher than that following administration of the phenobarbitone suppository. In the present thesis, the complex was prepared on a large scale; a batch of ten liters took less than two days, and the yield was more than 90%. It is the opinion of the author of this thesis that the complex obtained by the freeze drying method was amorphous; hence it may be more easily uniformly dispersed in a melted suppository base than the crystalline complex obtained by Iwaoka~- (104).
Tokumura, ~- (166), studied an inclusion complex of cinnarizine with beta-cyclodextrin in aqueous solution and in the solid state. They confirmed the inclusion formation by the solubility, powder X-ray diffractometry, differential scanning calorimetry and proton magnetic resonance spectroscopy methods. In order to prepare the complex, coprecipatation and neutralization methods were used . Scale -up evaluation, batch size, drug content in the complex, percent yield, etc., were not reported. Even though they reported that the dissolution rate of cinnarizine in the inclusion complex was 30 times larger than that of cinnarizine alone, they did not report on how one can conveniently incorporate the complex into a suitable dosage form.
The literature is replete (87, 95, 101 -103) with papers which report laboratory techniques of the preparation of inclusion complexes. It has been shown that dissolution and bioavailability of drugs improved by complexation with beta-cyclodextrin. However, how such inclusion complexes can be scaled-up from a laboratory scale to a pilot scale, formulation development, etc., have not been reported indetail.

IV Conclusions and suggestions for future work
The following are believed to be the salient conclusions of this work as reported and discussed in the previous section.
The data obtained in this thesis indicate that the beta -cyclodextrin interacts with ampicillin, phenobarbitone, and phenytoin. The mode of interaction was studied by a variety of methods, which include stability, solubility, X-ray diffractometry, differential scanning calorimetry, infrared and proton magnetic resonance spectroscopies, photomicrographs, etc. It was found that in the preparation of the complexes, the freeze drying method was the most feasible and reproducible from a laboratory scale to a pilot scale production. However, because of ampicillin insta bility under freezing conditions, it was not possible to obtain the inclusion compound in the solid powdered form. Phenobarbitone and phenytoin beta -cyclodextrin complexes were prepared using both a laboratory model freeze drier and an industrial model freeze drier.
Modifications to the techniques in the freeze drying process allowed the yield to be improved to almost 90%.
Properties of the complexes in solutions were evaluated by stability, solubility and proton magnetic resonance spectroscopy methods.
In addition, to confirm the presence of the inclusion complex in the solid phase, X-ray diffractometry and differential scanning calorimetry techniques were found to be useful. Infrared spectra and photomicrographs were difficult to interpret in an unambiguous manner.
However, these two methods are deemed to be useful to some extent; hence, they can not be ruled out in the evaluation of physico-chemical properties of the complexes. Liquid dosage forms were prepared from the phenobarbitone and phenytoin beta-cyclodextrin complexes. A solution dosage form was prepared from the phenobarbitone beta-cyclodextrin complex . Unlike an official preparati on there was no need to use ethanol to keep the phenobarbitone in solution. Hence, this dosage form may hav e a considerable potential, mainly for pediatric use. Evaluation of the physical stability of the suspension prepared from the phenytoin beta-cyclodextrin complex were indicative of a pharmaceutica lly elegant suspension . In addition, another distinct advantage is that this suspension contains freeze dried material so it is presented to the body in fine particles and therefore ready for the dissolution process immediately upon administration.
Despite the lack of flowability and compressibility of the phenytoin beta-cyclodextrin complex, it was possible to manufacture tablets of suitable size and weight. All the examined physical properties of the tablets were found to be within the USP standards. Dissolution studies of the tablets in different dissolution media indicated that the phenytoin beta-cyclodextrin had greatly improved dissolution compared to the uncomplexed drug. Due to in situ complexation of the physical mixture of the freeze dried phenytoin and beta-cyclodextrin, tablets prepared from the mixture showed a similar dissolution profile to that obtained from tablets prepared from the complex. However, a similar dissolution profile was not observed from the tablets prepared from th e nonfreeze dried phenytoin and beta-cycl odextrin. Hence, it ma y be concluded that, when the substrate disperses at a molecular level, it comes in direct contact with the molecularly dispersed ligand and in situ complexation takes place in the dissolution medium. It appears that the rate limiting step to form in situ complexation is the solubility of a substrate in the vicinity of the solution of a ligand. In order to increase the dissolution rate by in situ complexation, the following techniques may be useful: a. A physical mixture of a weakly acidic drug and cyclodextrins can be mixed with a GRAS (Generally Recognized As Safe) buffering agent to compress a tablet along with other tabletting ingredients.
b. When such tablets disintegrate in the dissolution medium, the buffering agent will cause a rise in pH in the close proximity of disintegrated tablet. When there is an increase in pH, a weakly acidic drug will dissolve an· d may fonn an in situ complex with the molecularly dispersed cyclodextrins . This technique makes it possible to increase the dissolution rate of weakly acidic drugs. However, there are some assumptions to be made to make this technique work. Firstly, weak ly acidic drugs are stable in the presence of a buffering agent in the liquid as well as solid phase . Secondly, the amount of a buffering agent incorporated is sufficient to increase the pH in the dissolution medium so as to increase the solubility of weakly acidic drugs . Furthennore, due to an increase in the pH, acidic drugs will ionize (d epending on pKa ) and the ionized species may not fonn a complex of suitable stability constant unlike unionized species . A similar technique may also be useful for basic drugs with appropriate buffering agents .
In addition, the following suggestions can be given for future work.
1. It would be interesting to investigate the interaction of an ampicillin with gamma-cyclodextrin, provided the cost is reasonable.
2. Under controlled humidity and temperature facilities, micronization or co-grinding of ampicillin with cyclodextrins might be explored to increase the dissolution rate by in situ complexation, the following techniques may be useful: a. A physical mixture of a weakly acidic drug and cyclodextrins can be mixed with a GRAS (Generally Recognized As Safe) buffering agent to compress a tablet along with other tabletting ingredients.
b. When such tablets disintegrate in the dissolution medium, the buffering agent will cause a rise in pH in the close proximity of disintegrated tablet. When there is an increase in pH, a weakly acidic drug will dissolve and may fonn an in situ complex with the molecularly dispersed cyclodextrins. This technique makes it possible to increase the dissolution rate of weakly acidic drugs. However, there are some assumptions to be made to make this technique to work. Firstly, weakly acidic drugs are stable in presence of a buffering agent in the liquid as well as solid phase. Secondly, the amount of a buffering agent incorporated is sufficient to increase the pH in the dissolution medium so as to increase the solubility of weakly acidic drugs . Furthennore, due to an increase in the pH, acidic drugs will ionize (depending on pKa ) and the ionized species may not fonn a complex of suitable stability constant unlike unionized species . A similar technique may also be useful for basic drugs with appropriate buffering agents.
In addition, following suggestions can be given for future work.
1. It would be interesting to investigate the interaction of an ampicillin with gamma -cyclodextrin, provided the cost is reasonable.
2. Under controlled humidity and temperature facilities, micronization or co-grinding of ampicillin with cyclodextrins might be expl ored to obtain the inclusion compound in solid powdered form, with subsequent evaluations of the physico-chemical properties of the complex.
3. It is planned to conduct bioavailability studies of dosage forms prepared from the phenytoin beta-cyclodextrin complex in humans. 5. Furthermore, since the aqueous solubility of alpha and gamma-cyclodextrins is higher than for the beta -cyclodextrin, the complexes prepared from them may be useful for parenteral dosage forms.
6. Although the literature reports the existence of delta, epsilon, and further homologues of cyclodex t rins containing 9-12 glycopyranose units, they are not available commercially in the pure form. However, even if l6i they become available in the future, complex formation will tend to diminish as the higher homologues of cyclodextrins increase in internal diameter. The probable reasons are as follows: The number of water molecules taken up by alpha, beta, and gamma cyclodextrins are 6, 11, and 17, respectively. As the cyclodextrins' cavity is enlarged (wit h higher homologues), the properties of the water filling it will approach those of bulk water . Hence, the lowering of the energy of the entrapped water may account for the failure of the inclusion; alternatively, the inclusion "fit" may be too "loose".   Time (Hours) . Fig . 51 Freeze drying cycle of phenobarbitone betacyclociextri n con;plex. Key: 6-Prociuct ter, 1perature, a -Heat applied, • -Condenser temperature.