USOO7001892B1

(12) United States Patent (10) Patent No.: US 7,001,892 B1 Chmielewski et al. (45) Date of Patent: Feb. 21, 2006

(54) PHARMACEUTICAL MATERIALS AND Borman, Stu. Biochemical Applications of Mass METHODS FOR THEIR PREPARATION AND Spectrometry Take Flight, C&EN, Jun. 19, 1995, 23-32. USE Carpenter, J.F. and Crowe, J.H. Infrared Spectrosopic Stud ies of the Interaction of Carbohydrates with Dried Proteins, (75) Inventors: Jean A. Chmielewski, Lafayette, IN Biochemistry, 1989, pp. 3916-3922, vol. 28. (US); Bart E. Kahr, Seattle, WA (US); Carpenter, J.F, et al. Separation of Freezing- and Drying Jerry Lewis, Carmel, IN (US) induced Denaturation of Lyophilized Proteins by stress Specific Stabilization: I. Enzyme Activity and Calorimetric (73) Assignee: Purdue Research Foundation, West Studies. Arch. Biochem. BiophyS., 1993, pp. 456-464, vol. Lafayette, IN (US) 3O3. Chmielewski, Jean, et al. Single-Crystal Matrix Isolation of (*) Notice: Subject to any disclaimer, the term of this Biopolymers, J. Am. Chem. Soc., Oct. 29, 1997, pp. 10565 patent is extended or adjusted under 35 10566, vol. 119, No. 43. U.S.C. 154(b) by 0 days. Goke, R., et al. EXendin-4 is a High Potency Agonist and Truncated Exendin-(9-39)-amide an Antagonist at the (21) Appl. No.: 10/018,043 Glucagon-like Peptide 1-(7-36)-amide Receptor of Insulin (22) PCT Filed: Jun. 12, 2000 secreting Beta-cells. J.Biol. Chem., Sep. 15, 1993, pp. 19650-19655, 268 (26). (86) PCT No.: PCT/US00/16140 Hillenkamp, Franz, et al. Matrix-Assisted Laser Desorption/ lonization Mass Spectrometry of Biopolymers, Analytical S371 (c)(1), Chemistry, Dec. 15, 1991, pp. 1193A-1283A, vol. 63, No. (2), (4) Date: May 21, 2002 24. Izutsu, K., et al. The Effects of additives on the stability of (87) PCT Pub. No.: WO00/76480 freeze-dried 3-galactosidase Stored at elevated temperatures. Int. J. Pharm:, 1991, pp. 137-146, vol. 71. PCT Pub. Date: Dec. 21, 2000 IZutsu, K., et al. Decreased protein-Stabilzing effects of cryoprotectants due to crystallization. Pharm. Res., 1993, Related U.S. Application Data pp. 1232-1237, vol. 10. (60) Provisional application No. 60/138,912, filed on Jun. Kurimoto, Miki et al. Kinetic Stabilization of Biopolymers 11, 1999. in Single-Crystal Hosts: Green Fluorescent Protein in C.- Lactose Monohydrate, J. Am. Chem. Soc. 1999, vol. 121, (51) Int. Cl. pp. 6952-6953. A6 IK3I/7016 (2006.01) Malkin, A.J. et al. Mechanisms of Growth for Protein and A6 IK 47/26 (2006.01) Virus Crystals, Nature Structural Biology, vol. 2, No. 11, (52) U.S. Cl...... 514/53: 536/123.13 Nov., 1995. (58) Field of Classification Search ...... 514/53; 536/123.13 See application file for complete Search history. (Continued) Primary Examiner-Leigh C. Maier (56) References Cited (74) Attorney, Agent, or Firm-Woodard, Emhardt, U.S. PATENT DOCUMENTS Moriarty, McNett & Henry LLP 4,501,726 A 2/1985 Schroder et al. (57) ABSTRACT 4,713.249 A 12/1987 Schroder 5,015,480 A 5/1991 Childers et al. 5,075,291 A 12/1991 DuROSS Pharmaceutical compositions comprising crystals of a phar 5,506.203 A 4/1996 Backstrom et al. maceutically-acceptable crystal lattice component, and an FOREIGN PATENT DOCUMENTS active pharmaceutical ingredient different from and included EP O 052 413 * 5/1982 within the crystal lattice component in a growth-sector EP O 052 413 A2 5/1982 Specific orientation. The crystals are prepared using com EP O 119 480 A 9/1984 ponents and methods which yield crystals having Suitable EP O 314. 469 A 5/1989 purity and efficacy for use in administering the active EP O 435 450 A 7/1991 pharmaceutical ingredients to a patient. The crystals are EP O 629 393 A 12/1994 typically combined with adjuvants Such as excipients, dilu GB 2 160 100 A 12/1985 ents or carriers, and are preferably formulated into tablets, WO WO95/241.83 9/1995 capsules, Suspensions, and other conventional forms con WO WO 97/21838 A 6/1997 taining predetermined amounts of the pharmaceuticals. Also WO WO 98/42367 10/1998 provided are methods for preparing the crystals, and meth WO WOOO/076480 A3 12/2000 ods for Storing and administering the active pharmaceutical OTHER PUBLICATIONS ingredient either included within the crystals or upon recon Beavis, Ronald C. et al. Epitaxial Protein Inclusion in Stitution of the crystals to a Solution. Sinapic Acid Crystals, J. Phys. D: Appl. Phys. 26 (1993) 442-447. 8 Claims, 2 Drawing Sheets US 7,001,892 B1 Page 2

OTHER PUBLICATIONS Strupat, K. et al. 2.5-Dihydroxybenzoic Acid: A New Matrix Powers, H.E.C. Sucrose Crystals: Inclusions and Structure, For Laser Desorption-Ionization Mass Spectrometry, Sugar Technol Rev., 1 (1969/70) 85-190. International Journal of Mass Spectrometry and Ion Rasimas, J.P. et al. Measuring Self-Assembly in Solution: Processes, 111 (1991) 89-102. Incorporation and Dynamics of A“Tailor-Made Impurity” in Visser, R.A., et al. A Natural Crystal Growth Retarder in Precrystalline Glucose Aggregates, Department of Lactose. Milk Diary J., 1980, pp. 255-275, vol. 34. Chemistry and Department of Chemical Engineering, Michigan State University, East Lansing, Michigan, J. PhyS. Chem. (1996) vol. 42 pp 17034-17040. * cited by examiner U.S. Patent Feb. 21, 2006 Sheet 1 of 2 US 7,001,892 B1

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0009 0099 US 7,001,892 B1 1 2 PHARMACEUTICAL MATERIALS AND catalyzed hydrolysis at Asp (9), Asp (15) and Asp (21). HGH METHODS FOR THEIR PREPARATION AND undergoes chemical decomposition via oxidation at Met (14) USE and deamidation at Asn (149). A critical challenge of product development Science in the This Application claims the benefit of Provisional Appli pharmaceutical industry therefore has been devising formu cation No. 60/138,912 filed on Jun. 11, 1999. lations that maintain the Stability of the active pharmaceu tical ingredient over an acceptable Shelf-life. This has been BACKGROUND OF THE INVENTION especially difficult to achieve for certain API's which are unstable in Solution or with respect to many common 1. Field of the Invention formulation processes. Developing techniques for Stabiliza The present invention relates to pharmaceutical formula tion and Storage looms as a great impediment to the phar tions involving the inclusion of an active pharmaceutical maceutical industry. Formulation Scientists have conse ingredient ("API) in a pharmaceutically-acceptable single quently used a variety of techniques to enhance the Stability crystal matrix. More particularly, the crystals contain of APIs while maintaining other important product charac growth-sector Specific, oriented inclusions of active phar 15 teristics Such as biocompatibility, absorption, pharmacoki maceutical ingredients which are isolated. The active phar netics, efficacy and excretion. maceutical ingredients have higher Stability and shelf-life, One technique used in formulating biopharmaceuticals and can be delivered in conventional dosage forms. This has been lyophilization of the biopharmaceutical Solution in invention has general application to active pharmaceutical the presence of excipients, buffers and/or bulking agents. ingredients, and in one aspect has particular application to However, even lyophilized preparations must typically be biopharmaceuticals. AS used herein, the term “biopharma Stored under refrigeration, a requirement which is neither ceuticals” is used to refer to a Subset of APIs which are technically nor economically feasible in many markets and polymeric in nature, including for example, proteins, inhibits flexibility of patient use. There has therefore been a polypeptides, enzymes, immunoglobulins, polynucleic continuing demand for formulations of many biopharma acids, and plasmids. 25 ceuticals which would permit their Storage at ambient tem 2. Description of the Prior Art peratures. This would permit more rapid development of There is a continuing need for pharmaceutical composi products, increasing flexibility in Shipping, Storing and car tions which are capable of maintaining the quality and rying the drug products, and allowing introduction and use efficacy of the API during storage and delivery. The loss of of Such products in markets where refrigeration is too costly. potency of an API is a critical concern in assuring that Moreover, the increased Stabilization of biopharmaceuticals viable, effective drugs are delivered to patients. It is simi would naturally improve the general use of the biopharma larly desirable to have formulations which do not require ceuticals where shelf life is an important consideration, Special package or handling. Further, it remains a constant whether or not refrigeration or other concerns are at issue. goal to provide active pharmaceutical ingredients in a form The prior art use of excipients in the lyophilization of which facilitates their use by the consumer, Such as though 35 biopharmaceuticals has been directed away from inclusion convenient dosage forms. The present invention addresses of the biopharmaceuticals in Single crystals in the manner of these and other issues concerning pharmaceutical composi the present invention. It has been widely assumed that tions and formulations. amorphous glasses are critical in the Stabilization of biop Although not limited to biopharmaceuticals, the useful harmaceuticals by Such excipients in lyophilized form, and neSS of the present invention is well exemplified with respect 40 it has been Suggested that the drug molecules must exist in to biopharmaceuticals, many of which demonstrate the prob amorphous regions between the crystalline domains. See, lems encountered in prior-art approaches. Ensuring long e.g., M. J. Pikal, “Freeze Drying of Proteins”, to be pub term Stability and maintaining activity of biopharmaceuti lished in Peptide and Protein Delivery, 2" Ed., V. H., L. Lee, cals is a prevalent concern. The chemical complexity and Marcel Dekker, Prepint, 1995. Implicit in this reasoning is conformational fragility of protein drugs, for example, make 45 the conclusion that the biopharmaceuticals could not exist as them highly Susceptible to both physical and chemical guests within Single crystals. instabilities and threaten their emergence into the market In the process of lyophilization, typically an aqueous place. Denaturation, adsorption with container walls, aggre Solution containing a biopharmaceutical with a limited gation, and precipitation can result from non-covalent inter amount of excipient(s) is frozen and then dried under actions between a drug and its environment. Insulin, for 50 Vacuum to produce Solids of Sufficient Stability for Storage instance, has been shown to adsorb onto the Surfaces of glass and distribution. Excipients are added to prevent blow out of and plastic containers, and to have interactions at air-water the product, to provide Stability during lyophilization and/or interfaces, leading to denaturation, aggregation and precipi dissolution, and to enhance compatibility for parenteral use. tation. For example, upon demonstration human growth Various excipients used with lyophilization have included hormone (HGH) forms dimers and higher molecular weight 55 Salts, metal ions, polyalcohols, Surfactants, reducing agents, aggregates, and glucagon in Solution has been shown to chelating agents, other proteins, amino acids, fatty acids, and readily gel or aggregate when Subjected to mechanical phospholipids. The more frequently used excipient include StreSS. mannitol, alanine, glycine, Sorbitol, lactose, arginine, and AS a further example, researchers have distinguished nine maltose. The results obtained with Such excipients, however, major reaction mechanism by which proteins degrade, 60 have usually been inconsistent. Most lyophilized biophar including hydrolysis, imide formation, deamidation, isomer maceuticals are amorphous powders that have not specific ization, racemization, diketopiperazine formation, oxida Structure, and as a result, the amount and location of the tion, disulfide exchange, and photodecomposition. The rates incorporated biopharmaceutical varies widely for the prod of these deleterious processes depend in large measure on uct particles. Also, they are typically readily dissolved, the protein and its environment. The primary chemical 65 rendering them unsuitable for use as a Sustained-release degradation products of glucagon, for example, include material. Further, there is no isolation of the pharmaceutical oxidation of Met (27), deamidation of Gln (24), and acid molecules from the environment or one another, leaving US 7,001,892 B1 3 4 them Susceptible to degradation by various mechanisms. described in this article, namely phthalic acid, gentistic acid Studies have shown that lyophilization of excipients can and Sinapic acid, were not Selected or evaluated for biocom typically damage proteins rather than protect them. See, e.g., patibility, and the crystal sizes were not optimized for J. F. Carpenter, J. H. Crowe, “Infrared spectroscopic studies particular routes of administration. Therefore, the produced of the interaction of carbohydrates with dried proteins”, crystals with included biopolymers would not be suitable for Biochemistry 1989, 28, 3916-3922; J. F. Carpenter, S. administration to patients. Prestrelski, T. Arakawa, “Separation of freezing- and drying Other prior art procedures have required the use of induced denaturation of lyophilized proteins by StreSS-Spe polymers that are difficult to prepare, require harsh prepa cific Stabilization: I. Enzyme activity and calorimetric Stud ration conditions that can be harmful to the API's, and yield ies,” Arch. Biochem. Biophys. 1993, 303, 456-464. K. inconsistent results. For example, U.S. Pat. No. 5,075,291 Izutsu, S. Yoshioka, Y. Takeda, “The effects of additives on describes a proceSS for preparing a uniformly-dispersed, the Stability of freeze-dried B-galactosidase Stored at pharmaceutically-active material in a crystalline Sugar alco elevated temperatures”, Int. J. Pharm. 1991, 71, 137-146. K. hol matrix. However, this proceSS requires the addition of Izutsu, S. Yoshioka, T. Teroa, “Decreased protein-stabilizing the API into a molten sugar alcohol with considerable effects of cryoprotectants due to crystallization”, Pharm. 15 mechanical agitation. Many APIs and virtually all biophar Res. 1993, 10, 1232–1237. maceuticals would not be stable in the extreme temperature Crystallized pharmaceuticals have been used in Some of 110° C. and the physical stresses of a high-shear vortex instances, but there have been inherent limitations. Some mixer used for agitation. The present invention does not APIs, e.g. insulin, can be crystallized themselves, and are require these extremes of temperature and physical agita useful in that form for administration to patients. However, tion. Also, the process of the present invention slowly the majority of biopharmaceuticals either do not crystallize includes the API into the growing crystal lattice in Specific or the crystallization is very difficult, particularly on a growth Sectors, instead of homogeneous mixing and entrap commercial Scale. Further, crystallization procedures are ping of the active pharmaceutical ingredient in a Viscous limited to the use of pharmaceutically-acceptable ingredi melt. ents and proceSS conditions that do not adversely affect the 25 active pharmaceutical ingredient, thus further constraining SUMMARY OF THE INVENTION the ability to obtain desired microcrystalline Suspensions. The fact that macromolecules are routinely isolated in In one aspect, the present invention relates to pharmaceu Sub-millimolar concentrations in a variety of crystals is tical compositions comprising Single crystals of a pharma known. See, e.g., K. Strupat, M. Karas, F. Hillenkamp, Int. ceutically-acceptable crystal lattice component, and an J. Mass Spec. Ion Proc., 111, 89-102, 1991. Also, certain active pharmaceutical ingredient different from and included aromatic acids have been employed as hosts for biopolymer within the crystal lattice component in a growth-sector guests in crystals for use in matrix-assisted laser desorption Specific orientation. The crystals are prepared using com ionization (MALDI) mass spectrometry, but not for the ponents and methods which yield crystals having Suitable purposes of the present invention. See, Review by F. Hill 35 purity and efficacy for use in administering the API's to a enkamp, M. Karas, R. C. Beavis, B. T. Chait, Anal. Chem, patient. The crystals may be coated or combined with 63, 1193A-1203A, S. Borman, Chem. Eng. News, 23–25, adjuvants Such as excipients, diluents or carriers, and are Jun. 19, 1995. However, crystallization conditions in these preferably formulated into tablets, capsules, Suspensions, Studies were optimized for characterization of the incorpo and other conventional forms containing dosage amounts of rated biopolymers. There were no investigations into opti 40 the API's. Alternatively, the crystals are prepared as depot mizations that would be relevant to pharmaceutical prepa formulations which may be administered, as by Subcutane rations or operations Such as homogeneity of the ous injection or implantation, to provide a long-term payout concentration of the inclusions, process Scale-up, proceSS or Sustained release of the active pharmaceutical ingredient. robustness, chemical and physical Stability of the prepara The present invention further provides methods for prepar tions, Suspendability in biocompatible Solutions, preserva 45 ing the crystals and for Storing and administering the active tive requirements and compatibility, container/closure SyS pharmaceutical ingredient either in crystal form or upon tem compatibility, and pharmacokinetic profiles. reconstitution to a Solution. The difficulty in obtaining Suitable Single crystals of Some Accordingly, it is an object of the present invention to biopolymerS has encouraged Structural chemists to partially provide Single crystals which include API's in a growth orient Such molecules with electric, magnetic, or flow fields, 50 Sector Specific orientation. It is a feature of the invention that by dissolution in liquid crystals or Stretched gels, and as the API's are included at predictable, uniform concentra monolayers. In a similar effort, the isolation of biopolymers tions that permit use of the crystals in formulating dosage in a Single crystal matrix has recently been Studied in an amounts of the APIs. effort to use Such crystals for Structural analysis of the Another object of the present invention is to provide biopolymers. Such isolation technique is described in 55 compositions comprising API's included in Single crystals to “Single Crystal Matrix Isolation of Biopolymers,” J. provide improved stability and shelf-life. The active phar Chmielewski, J. J. Lewis, S. Lovell, R. Zutshi, P. Savickas, maceutical ingredients may therefore be Stored for extended C. A. Mitchell, J. A. Subramony, and B. Kahr, J. Am. Chem. periods of time prior to use either as crystals or as recon Soc. 1997, 119, 10565-10566. However, this article simply Stituted Solutions. demonstrates that certain biopolymers are oriented by the 60 It is a further object of the present invention to provide host lattice, and the article Suggests the use of Such crystals Single crystals with included API's to provide quick, for analyzing spectral anisotropies in biological molecules delayed-release or Sustained-release formulations for flex which could not otherwise be crystallized. This article does ibility in pharmacokinetic profiles in delivery of the API's to not discuss or Suggest the use of this technique for enhance patients. ment of Stability or Sustained release of pharmaceuticals, or 65 Another object of the present invention is to provide their administration to patients. Further, the proteins Studied pharmaceutical delivery units including an amount of Single were not a pharmaceutical interest, the crystal materials crystals Sufficient to provide a dosage amount of the US 7,001,892 B1 S 6 included active pharmaceutical ingredient. Alternatively, the maceutical ingredient may be reconstituted into a Solution pharmaceutical delivery units include a quantity of crystals for administration to a patient. Sufficient to provide a prolonged payout of the active phar The active pharmaceutical ingredient molecules are gen maceutical ingredient. The crystals may be coated or erally isolated from one another and are insulated from the uncoated, and may be combined with various pharmaceuti environment by the host crystal. This leads to reduce Sus cal adjuvants including excipients, diluents and carriers. ceptibility of the API to degradation, and therefore enhanced A further object of the present invention is to provide Stability and Shelf-life. Also, the use of appropriate host methods for preparing compositions comprising Single crys crystal compounds, or Selected dosage forms, permits the tals with growth-sector specific inclusions of API's. design of quick, delayed, or Sustained-release formulations It is another object of the present invention to provide for delivery of the active pharmaceutical ingredient. Sus methods for the Storage and administration of API's utilizing tained-release formulations are particularly advantageous inclusion of the API's within single crystals. for treatment of chronic conditions as they provide a con Other objects, features, and advantages of the present Sistent amount of drug delivery over a long period of time to invention will be apparent to those skilled in the art from the improve ease of use and patient compliance in administering following description and claims. 15 the API. The crystal preferentially incorporate the active pharma BRIEF DESCRIPTION OF THE DRAWINGS ceutical ingredient on certain faces, thereby providing a growth-sector Specific inclusion and orientation to the APIs. FIG. 1 is a photomicrograph illustrating fluorescence of a AS used herein, the term “growth-sector Specific inclusion Single crystal of green fluorescent protein in C-lactose and orientation,” and equivalent terminology, refers to the monohydrate (1.8 (h)x0.8 (w)x0.5 (d) mm) with an ideal fact that the API molecules are included primarily at certain ized representation of habit. The sides of the crystal in the faces of the crystal matrix. The growth-sector Specific inclu photomicrograph are bright due to internal reflection. Sion and orientation can be determined by one skilled in the FIG. 2 is a graph of the fluorescence decay of the green art, as demonstrated in the examples herein, by fluorescence fluorescent protein is 333 K. in several environments: 25 microScopy and anisotropy measurements, Single crystal mixed crystal in C-lactose monohydrate (triangle), Saturated desorption mass spectrometry, and autoradiography of "C- lactose Solution (Square), and lyophilized C.-lactose mono labeled material. In one embodiment, at least about 0.001% hydrate (diamond). (on weight/weight (w/w) basis) of the pharmaceutical is included within Specific faces of the crystal matrix, and in DESCRIPTION OF THE PREFERRED another embodiment at least about 0.1% (w/w) and up to EMBODIMENT about 10%. The crystal parameters, including the particular crystal lattice component for a given API, the concentration For the purposes of promoting an understanding of the of API, the use of crystal adjuvants, and the crystallization present invention, reference will now be made to the conditions, are Selected to achieve the growth-sector Specific embodiments described hereafter. It will nevertheless be 35 inclusion and orientation of the API within the crystals. understood that no limitation of the Scope of the invention is The method of the present invention broadly involves the thereby intended, Such modifications and applications of the including of the active pharmaceutical ingredient into the principles of the invention as described herein being con Single crystal matrix formed from a pharmaceutically-ac templated as would normally occur to one skilled in the art ceptable crystal lattice component. AS used herein, the term to which the invention relates. 40 “included in the crystals refers to the active pharmaceutical The present invention utilizes Single-crystal matrix inclu ingredient being chemically adsorbed within the crystal sion of active pharmaceutical ingredients (“API’s”) to lattice as the crystal is formed. This inclusion of the active achieve advantageous Storage and delivery of the APIs. pharmaceutical ingredient molecules is distinguished from This invention has application to a wide range of API's to crystallization of the API molecules with one another, and provide enhanced stability and/or delivery of the active 45 from simple and random entrapment of the API molecules pharmaceutical ingredients. For Some applications, Such as by the formed crystal. The crystal product of the present for many biopharmaceuticals, the invention is particularly invention is ordered, in contrast to the amorphous material advantageous in providing greater Stability over time and in produced by other approaches. The API is incorporated in providing alternative delivery and Sustained release formu the crystal in relation to its degree of affinity for the crystal lations to patients. 50 lattice molecules. The crystal lattice component is therefore The Small molecule host crystals comprise a crystal lattice Selected to be both chemically and physically compatible component which includes the API's in an oriented, growth with the API such that the API is received by the crystal Sector Specific manner. The crystals and included API's are during formation, and remains Stable and efficacious while prepared to be pharmaceutically acceptable and pure, within the crystal and upon release therefrom. thereby being useful for administration to patients to be 55 In a typical approach, the including of the active phar treated with the API's. As used herein, the term “pharma maceutical ingredient involves combining the crystal lattice ceutically-acceptable” refers to Sufficient quality to meet component, the active pharmaceutical ingredient and a phar regulatory and compendial requirements for administration maceutically-acceptable adjuvant in a liquid State. The crys to humans and/or animals. The crystals provide a regular, tal lattice component is then crystallized under pharmaceu predictable inclusion of the guest active pharmaceutical 60 tically-acceptable conditions to form the inventive crystals. ingredient, and the crystals can consequently be used for For example, one method uses Spiking of the API into a obtaining a predetermined amount of the active pharmaceu Saturated or SuperSaturated Solution of the crystal lattice tical ingredient for delivery to a patient. In one aspect, the component in a Suitable organic and/or aqueous Solvent host crystal gradually dissolves upon contact with body System. Alternatively, the Saturated or SuperSaturated Solu tissue or fluids, and is therefore useful as a System for 65 tion of the crystal lattice component may be spiked into the delivery of the active pharmaceutical ingredient into the API solution. Other components may also be added to the body. Alternatively, the crystals and included active phar Solution, including compounds which facilitate or modify US 7,001,892 B1 7 8 crystal growth or which are desired for incorporation in the The method preferably involves preparing a mixture of final formulation. The Solution may be seeded using any of crystals of Substantially uniform size. This may include a variety of conventional techniques. processing of the harvested crystals, Such as by grinding or In one approach, the Solution is allowed to evaporate milling, to reduce the crystals to a Substantially uniform size. and/or equilibrate to cooler conditions for growth of the Greater uniformity can be achieved by Sorting the processed crystals. The crystals are then grown as the Solvent is slowly crystals, Such as by Sieving. A preferred method further evaporated away and/or the Solution is cooled, with the includes obtaining crystals which have a Substantially uni evaporation and temperature gradient conditions being form concentration of pharmaceuticals, for example, about Selected dependent on Such factors as the Solvent System and the desired crystal size. The crystals containing the active 1% (w/w) of pharmaceuticals, that do not vary between pharmaceutical ingredient are harvested from the remaining crystals by more than 10 percent. Solution and are preferably washed to remove Surface con The method of the present invention may further include tamination. This procedure yields crystals which include the formulating the crystals into pharmaceutical preparations. active pharmaceutical ingredient at a predictable concentra For example, the collected crystals may optionally be coated tion and facial orientation. 15 with a Suitable composition. Coated or uncoated crystals In accordance with the present invention, crystals are may be blended with one or more pharmaceutically-accept grown under pharmaceutically-acceptable conditions. AS able adjuvants, Such as excipients, diluents, carriers or used herein, the term “pharmaceutically-acceptable condi mixtures thereof. The blended crystals and adjuvant(s) are tions” refers to the use of crystal and API compounds which then formulated into pharmaceutical delivery units. In one are pharmaceutically-pure, and for which Such pharmaceu embodiment, each unit includes a predetermined amount of tical purity is maintained in the final crystals. The crystal and the pharmaceutical. Alternatively, the crystals are combined API compounds are pharmaceutically pure, or have phar in a delivery unit intended to deliver multiple or Sustained maceutical purity, if they are of Sufficient purity to be dosing of the API over a period of time, such as by suitable for administration under applicable FDA or other Subcutaneous implantation of the delivery unit. A further administrative regulations regarding purity. The term phar 25 aspect of the method of the present invention involves maceutically-acceptable conditions further refers to the user of crystallization conditions through which the API com reconstituting the crystals to liquid form. In accordance with pounds retain pharmaceutical efficacy in the final crystals this method, the harvested crystals are dissolved in a Suitable and upon Subsequent administration to patients. diluent for the crystal lattice component. The dissolution of The present invention readily allows the inclusion of the crystals releases the API from the crystals. The resulting APIs by affinity with the small host molecules in the Solution may include other adjuvants, Such as excipients, growing crystal lattice. This overcomes many of the limi diluents or carriers, and the mixture is formulated under tations associated with approaches. The processing involved conventional procedures to desired delivery forms. In a with preparing the present crystals does not expose the API's particular aspect of the present invention, the crystals are to harsh conditions, thereby Substantially reducing or avoid 35 used to Store the pharmaceutical for a period of times, Such ing the possible degradation or disruption of the Structural as at least one month, or at least one year, and the crystals aspects of the API which could occur with prior art tech are Subsequently dissolved to use the active pharmaceutical niques. The inventive crystals have an added advantage in ingredient. that they do not interfere with normal analytical method The present invention involves the use of any of a wide ologies used for characterizing the pharmaceutical product. 40 variety of pharmaceutically-acceptable host crystal Systems The Small host molecules can be easily Separated on the that can incorporate API's in a growing crystal lattice. The basis of molecular size, which is not the case for prior art crystal lattice component is Selected to be compatible with techniques which uses polymers that interfere with analyti the guest API, and to be Suited to the use of the resulting cal methodologies. formulation for Storage and administration. Selection of the The API molecules are incorporated into the host crystals 45 crystal lattice component will involve consideration of Such typically at rates of at least about 0.001% (w/w), preferably factors as affinity for the API, crystal size distribution and at least about 0.1%, and more preferably about 1% to about morphology, and desired pharmaceutical concentration and 10% (w/w). Alternatively, the API molecules are included at delivery rate, as well as other factors well known in the art rates of at least about 0.01%, and as much as at least about of pharmaceutical delivery Systems. The crystal Systems 1% (w/w). The limited molar concentration of the active 50 must consistently incorporate the guest active pharmaceuti pharmaceutical ingredient in the host crystals means that the cal ingredient in terms of concentration and placement active pharmaceutical ingredient molecules are generally within the crystal lattice. The crystals also must grow under isolated from one another in the crystals. Isolation of the API conditions which will not degrade or otherwise adversely molecules is particularly advantageous for those molecules, effect the viability of the active pharmaceutical ingredient. Such as certain biopharmaceuticals, which could otherwise 55 Preferred host crystal materials are those that have a high react with one another (e.g., by polymerization) or the affinity for the included API. It appears that the oriented Surrounding environment. The degree of isolation can be inclusion of the API's is related to the affinity between the Verified by those skilled in the art using atomic force crystal lattice component and the API. The affinity between microScopy or reaction fluorescence energy techniques. The these materials is therefore important in obtaining the present invention has a particular application to guest-host 60 desired inclusion of the API's, and also permits control of systems in which the guest API molecules are reactive with the inclusion based upon this affinity. For example, the one another, but in which these molecules are Sufficiently concentration of the pharmaceutical in a crystal can be isolated from one another in the crystals as to Substantially controlled by Selecting the host component to have an prevent Such interaction. Consequently, the invention pro affinity for the API which yields the desired inclusion rate. vides containment of the API molecules in the Solid state 65 Also, mixtures of host materials, or of host materials and crystals and provides for the API to be comformationally other excipients, can be used to provide an affinity yielding stable. the desired inclusion level. In one aspect of the present US 7,001,892 B1 9 10 invention, the API's are incorporated at levels of at least the active pharmaceutical ingredient in the crystals, as well about 0.001% (w/w of guest:host), more preferably at least as parameters of the active pharmaceutical ingredient itself. about 0.1% (w/w). While given crystal lattice components will have associated The preferred host crystal materials will also be very inclusion and dissolution characteristics, these can be modi Stable and readily crystallizable, and will maintain their fied by including other crystal lattice components, other “order” or crystal morphology when including a guest APIs, or a variety of excipients. Thus, Single crystals having molecule, particularly large biomolecules. The use of par two different, co-crystallied lattice components will typi ticular host crystal components will also depend on Such cally be characterized by pharmacokinetic profiles different factors as how Small or large the crystals can be produced from crystals prepared with either of the crystal lattice and how readily they dissolve. For various routes of admin components alone. Similarly, including excipients or other istration, it is desirable to have very Small crystals (e.g., APIs will provide altered rates of inclusion or dissolution pulmonary), moderately sized crystals (e.g., injectable), or for the resulting crystals, providing an associated modifica very large crystals (e.g., implantation and long term payout). tion in the pharmacokinetic profile for the resulting crystals. The useful crystal sizes will therefore vary accordingly, ranging from Submicron to millimeter sizes. In one aspect of 15 In a related aspect, the present invention involves the use the present invention the preferred crystals are in the order of mixtures of crystals having different pharmacokinetics in of 5-100 microns in size. order to achieve desired payout profiles. For example, a The useful host crystal Systems are therefore diverse, and pharmaceutical product can be obtained by combining two include various Small molecule crystal Systems which meet different types of crystals, one type of crystal using a first the desired criteria. Examples of pharmaceutically-accept crystal lattice component characterized by a first pharma able crystal lattice components include Sugars, polyhydroxy cokinetic profile, and the Second type of crystal using a alcohols, Single and polyamino acids, Vitamins, Salts, metals, Second crystal lattice component characterized by a Second preservatives, aromatic compounds especially aromatic pharmacokinetic profile. The mixture of crystals will give a acids, purified natural products, and polymers. Preferred payout of API that is different from either of the individual crystal lattice components include, for example, Sucrose, 25 payouts for the two crystal types. lactose, trehalose, maltose, galactose, Sorbose, mannitol, The included API's are similarly diverse, limited simply lactitol, Sorbitol, glycine, alanine, lysine, arginine, ascorbic by the requirements of compatibility with the host crystal acid, nicotinamide, thiamine, adenine, pyridoxine hydro and the crystal growth conditions. The active pharmaceuti chloride, caffeic acid, Vanillic acid, ferulic acid, benzoate, cal ingredient cannot be unacceptably degraded or otherwise Sorbate, methyl paraben, Sodium ascorbate, Sodium Saccha adversely affected by the conditions under which the crystals rin, and potassium citrate. Also, compatible mixtures of are formed. Also, the active pharmaceutical ingredient these materials are also useful, and can be selected to obtain should remain stable for an extended period of time while the desired rate of inclusion of the pharmaceutical, or to included within the host crystal, and pharmaceutically effi achieve desired characteristics, Such as dissolution rate and cacious upon release from the crystal. pharmacokinetic profile, for the product crystals. 35 Given the foregoing criteria, examples of API's useful in The crystal lattice components are Selected to achieve the accordance with the present include: antibiotics (Such as desired pharmacokinetics for the final crystals. AS pertains dirithryomycin, loracarbef, tilmicosin, Vancomycin, tylosin, to the present invention, the term “pharmacokinetics” is monensin), fluoxetine, raloxifene, olanzapine, and nizati used to refer to the profile of the delivery of active phar dine. A more complete list of API's useful in accordance maceutical ingredient from the crystals into the circulatory 40 with the present invention would include those identified in System. This will depend primarily on the concentration of the following Table A.

TABLE A Marketed Recombinant Protein Products Tissue Plasminogen Activator, T-PA Product name: Activase (Generic name: Altepase) Produced by: Indication: Human use, Acute myocardial infarction Date of approval: November 87, Patent expires on December 2000. Formulation: Intravenous injection. Lyophilized powder which is reconstituted with sterile water (supplied) to 1 mg/mL and results in a final pH of 7.3. Can not be reconstituted with preserved water due to precipitation. The 1 mg/mL solution can be diluted 1:1 with 0.9% NaCl or D5W and help for 8 hours at room temperature. TPA is incapable with preservatives. Ingredients 100 mg vial 50 mg vial 20 mg vial TPA 100 mg 50 mg 20 mg L-Arginine 3.5 g. 1.7 g 0.7 g Phosphoric acid 1 g 0.5 g. 0.2 g Polysorbate 80 <11 mg <4 mg <1.6 mg Vacuum No Yes Yes Expression System: Mammalian cell line (Chinese Hamster Ovary cells) Refolding Conditions: Structure: Glycoprotein of 527 amino acids, sequence from human melanoma cell line, activity of 580,000 IU/mg. Additional Information: Sales > $100 million. Cost of $2,750 (100 mg). US 7,001,892 B1 11 12

TABLE A-continued Interferon Gamma-1b

Product name: Actimmune Produced by: Genentech Indication: Human use, chronic granulomatous disease Date of approval: December 1990 Formulation: Single dose solution formulation (0.5 mL), subcutaneous injection. Each 0.5 mL contains 100 tug interferon gamma-1b, 20 mg mannitol, 0.36 mg sodium succinate, 0.05 mg polysorbate-20 in sterile Wate. Expression System: E. coli Refolding Conditions: Post-Transitional Modifications: Structure: Single chain; Human sequence, 140 amino acids, 16,465 molecular weight, non-covalent dimeric form in solution, activity or 30 million units/mg. Additional Information: 14% injection site irritation vs. 2% in placebo. Cost $140 for 50 ug. Interferon alfa-n3 (natural source, not recombinant) Product name: Alferon N Produced by: Interferon Science (New Brunswick, NJ) Indication: Human use, Genital Warts Date of approval: June 90 Formulation: Preserved solution formulation (each mL contains 5 million IU of interferon alfa-n3 in phosphate buffered saline containing 3.3 mg phenol and 1 mg human albumin). Injected intralesional twice weekly for up to 8 weeks (50 uL injected into each wart, 500 uL total dose per treatment). Expression System: Natural source - human leukocytes which are exposed to an avian virus in order to produce interferon. Refolding Conditions: None Structure: Approximately 166 amino acids with a molecular weight ranging from 16 to 27 kDa, specific activity of 20,000 IU/m or greater. Additional Information: Cost $142 per mL. Beta Interferon 1 a

Product name: Avonex Produced by: (Cambridge, MA) Indication: Human use, Multiple Sclerosis Date of approval: May 95 Formulation: Lyophilized powder (stored refrigerated or at 25 C. for < 30 days) which is reconstituted with sterile water (supplied, 1.1 mL) to 30 tug/mL beta interferon 1a, 15 mg/mL human albumin, 5.8 mg/ml NaCl, 5.7 mg/ml dibasic Na phosphate, 1.2 mg/ml monobasic sodium phosphate, and has a pH of approximately 7.3 (recon solution is stable for 6 hours at refrigerated temperatures). Weekly intramuscular injection by patient or doctor (dosed for 1–2 years in clinical trials). Expression System: Mammalian cells (Chinese Hamster Ovary cells) Refolding Conditions: Structure: Glycoprotein (single N-linked complex carbohydrate), 166 amino acids with a predicted molecular weight of 22,500 daltons, human sequence, has a specific activity of 200 million units per mg protein. Additional Information: Cost $180 per vial (33 ug) Interferon beta-1b

Product name: Betaseron Produced by: Berlex Laboratories (Wayne, NJ and Chiron, Emeryville, CA) Indication: Human use, Multiple Sclerosis Date of approval: July 93. Formulation: Lyophilized product (stored refrigerated) which is reconstituted with 0.54% NaCl (supplied) to 0.25 mg/mL interferon beta-1b, 12.5 mg/mL human albumin, 12.5 mg/ml dextrose, and has a pH of approximately 7.3 (recon solution is stable for 3 hours). Injected subcutaneously every other day (chronic use). Expression System: E. coli Refolding Conditions: Structure: 165 amino acids with an approximate molecular weight of 18,500 daltons, human sequence but with a serine or cysteine substitution at residue 17. Recombinant form does not contain the carbohydrate moiety found in the natural material. Has a specific activity of 32 million units per mg protein. Additional Information: Sales > $500 million. Cost of therapy is $13,140 (based on 0.25 mg/injection, dose every other day for 1 year; equals 46 mg protein). Interferon alfa-2b

Product name: Intron A Produced by: Schering-Plough (Madison, NJ) Indication: Human use, Hairy cell leukemia, genital warts, Hepatitis, Melanoma, Kaposi's sarcoma Date of approval: June 86 Formulation: Comes in a lyophilized and a solution formulation. The lyophilized formulations when reconstituted with 0.9% benzyl alcohol (supplied) contains either 0.015, 0.025, 0.05, 0.90, or 0.125 mg/ml. Interferon alfa-2b, 20 mg/ml glycine, 2.3 mg/ml sodium phosphate dibasic, 0.55 mg/ml sodium phosphate monobasic, and 1 mg/ml human albumin. The solution formulations contain either 0.05, 0.114, or 0.125 mg/mL Interferon alfa-2b, 20 mg/ml glycine, 2.3 mg/ml sodium phosphate dibasic, 0.55 mg/ml sodium phosphate monobasic, 1 mg/ml human albumin, 1.2 mg/mL methylparaben, and 0.12 mg/ml propylparaben. These formulations be injected intramuscular, subcutaneous, or intralesional. All formulations and reconstituted products are stored at refrigerated temperatures. Expression System: E. coil US 7,001,892 B1 13 14

TABLE A-continued Refolding Conditions: Structure: Water soluble protein a molecular weight of 19,271 daltons. The Interferon alfa-2b gene is derived from human leukocytes. Additional Information: Sales > $500 Million. Cost of therapy is $16,445 (5 million units every day for 1 year, this is equal to 9 mg protein). Specific activity is 200 million units per mg protein Interferon alfa-2a

Product name: Roferon-A Produced by: Hoffmann-La Roche (Nutley, NJ) Indication: Human use, Hairy cell leukemia, Kaposi's sarcoma, myelogenous leukemia Date of approval: June 1986 Formulation: Multi-use and lyophilized formulation indented for intramuscular or subcutaneous administration. Multi-use formulation contains either 0.015, 0.045, 0.090, 0.18 mg/mL Interferon alfa-2a, 9 mg/ml NaCl, 5 mg/ml human albumin, and 3 mg/ml phenol. The lyophilized formulation reconstituted with 3 mL of supplied diluent (6 mg/ml NaCl, 3.3 mg/ml phenol) consists of 0.03 mg/ml Interferon alfa-2a, 9 mg/ml NaCl, 1.67 mg/ml human albumin, and 3.3 mg/ml phenol. Expression System: E. coli (tetracycline promoter). Refolding Conditions: Structure: Protein of 165 amino acids having a molecular weight of 19,000 daltons Additional Information: Cost of therapy is $59,200 (28 mg protein over 1 year). Specific activity is 200 million international units per mg protein. Human Growth Hormone (Somatropin) Product name: BioTropin Produced by: Bio-Technology General (Iselin, NJ) Indication: Human use, Growth Deficiency Date of approval: May 95 Formulation: Expression System: Refolding Conditions: Post-Transitional Modifications: Structure: Additional Information: Human Growth Hormone (Somatropin) Product name: Genotropin Produced by: Pharmacia and (Kalamazoo, MI) Indication: Human use, Growth Deficiency Date of approval: August 95 Formulation: Expression System: Refolding Conditions: Structure: Additional Information: Human Growth Hormone (Somatropin) Product name: Humatrope Produced by: Eli Lilly (Indianapolis, IN) Indication: Human use, Growth Deficiency Date of approval: March 87 Formulation: Lyophilized product which is reconstituted with sterile water containing 0.3% m-cresol, 1.7% glycerin (supplied) to 2 mg/mL hCGH and has a final pH of approximately 7.5, subcutaneous or intramuscular administration. Each 5 mg lyophilized vial contains 5 mg hCH, 25 mg mannitol, 1.13 mg dibasic sodium phosphate, and 5 mg glycine. Expression System: E. coli. Refolding Conditions: Structure: 191 amino acids, molecular weight of 22,125 daltons, sequence is identical to human pituitary derived material. Additional Information: Cost $210 per 5 mg hCH. Human Growth Hormone (Somatropin) Product name: Norditropin Produced by: Novo Nordisk (Princeton, NJ) Indication: Human use, Growth Deficiency Date of approval: July 91 Formulation: Expression System: Refolding Conditions: Post-Transitional M difications: Structure: Additional Information: Human Growth Hormone (Somatropin) Product name: Nutropin and Nutropin AQ Produced by: Genentech Indication: Human use, Growth Deficiency Date of approval: March 1994 Formulation: Lyophilized product which is reconstituted with bacteriostatic water (0.9% benzyl alcohol, supplied) to 5 mg/mL hCGH and has a final pH of approximately 7.4, subcutaneous or intramuscular US 7,001,892 B1 15 16

TABLE A-continued administration. Each 5 mg lyophilized vial contains 5 mg hCGH, 45 mg mannitol, 1.7 mg sodium phosphates (0.4 mg monobasic and 1.3 mg dibasic), and 1.7 mg glycine. Expression System: E. coli, expressed with a leading secretion signal precursor which directs the protein to the plasma membrane of the cell where the sequence is removed and the native protein is secreted into the periplasm so that the protein if folded appropriately as it is synthesized Refolding Conditions: None, expressed folded in E. coli. Structure: 191 amino acids, molecular weight of 22,125 daltons, sequence is identical to human pituitary derived material. Additional Information: Cost $420 per 10 mg hCH. f-Glucocerebrosidase (imiglucerase) (B-D-glucosyl-N-acylsphingosine glucohydrolase, E.C.3.2.1.45) Product name: Cerezyme Produced by: Genzyme (Cambridge, MA) Indication: Human use, Gaucher's disease Date of approval: May 94 Formulation: Lyophilized product (212 units glucocerebrosidase, 155 mg mannitol, 70 mg sodium citrate, and 0.53 mg polysorbate-80; stored refrigerated) is reconstituted with 5.1 mL of sterile water, final pH is approximately 6.1. The reconstituted material is combined with 100 to 200 mL of 0.9% NaCl and administered intravenously. Expression System: Mammalian cell culture (Chinese Hamster Ovary cells) Refolding Conditions: Structure: Monomeric glycoprotein of 497 amino acids, containing 4 N-linked glycosylation sites, molecular weight is 60,430 daltons. Recombinant protein differs from human placental glucocerebrosidase by a arginine substituted for histidine at position 495 and the glycosylation sites have been modified to terminate in mannose sugars (which are specifically recognized by endocytic carbohydrate receptors on macrophages, the cells that accumulate lipid in Gaucher disease). Additional Information: Orphan Drug, sales > $100 million, Cost of therapy is $351,130 (1 year). Hepatitis B Surface Antigen Product name: Engerix-B Produced by: SmithKline Beechman (Philadelphia, PA) Indication: Human use, Hepatitis B Date of approval: September 89 Formulation: Suspension (20 tug/mL hepatitis B surface antigen adsorbed onto 0.5 mg aluminum, 1:20,000 thimerosal, 9 mg/ml NaCl, 1.7 mg/ml sodium phosphates). Intramuscular administration. Expression System: A portion of the hepatitis B virus gene, coding for hepatitis B surface antigen, in cloned into yeast (Saccharomyces cerevisiae) Refolding Conditions: Post-Transitional Modifications: Structure: Additional Information: Formulation contains no more than 5% yeast proteins. Hepatitis B Surface Antigen Product name: Recombivax HB Produced by: Merck (Whithouse Station, NJ) Indication: Human use, Hepatitis B prevention Date of approval: July 1986 Formulation: Suspension (10 ug/ml hepatitis B surface antigen adsorbed onto 0.5 mg aluminum, 1:20,000 thimerosal). Intramuscular administration. Expression System: A portion of the hepatitis B virus gene, coding for hepatitis B surface antigen, in cloned into yeast (Sacccharomyces cerevisiae) Refolding Conditions: Structure: Additional Information: Formulation contains no more than 1% yeast proteins. Erythropoietin (rEPO) Product name: Epogen or Epoetin alfa (Also sold under the name Procrit by Ortho Biotech but manufactured by ) Produced by: Amgen (Thousand Oaks, CA) Indication: Human use, Anemia Date of approval: June 89, Patent expires in 2004 (December). Formulation: Two solution formulations, single dose and multi-dose. Single-dose is preservative free and each mL contains 2000, 3000, 4000, or 10000 units Epogen, 2.5 mg human albumin, 5.8 mg sodium citrate, 5.8 mg NaCl, and 0.06 mg citric acid in water for injection, pH 6.9 +/- 0.3. The preserved multi dose product contains 10,000 units Epogen, 2.5 mg human albumin, 1.3 mg sodium citrate, 8.2 mg sodium chloride, 0.11 mg citric acid and 1% benzyl alcohol per mL of solution, pH is 6.1 +/- 0.3. Both solutions are stored refrigerated. Expression System: Mammalian cell Refolding Conditions: Structure: Glycoprotein of 165 amino acids having a molecular weight of 30,400 daltons, sequence identical to that of the human protein. Additional Information: Sales > $500 million, Cost $120 for 10,000 units. Human Insulin

Product name: Hurnulin Produced by: Eli Lilly (Indianapolis, IN) Indication: Human use, Diabetes Date of approval: October 82 US 7,001,892 B1 17 18

TABLE A-continued Formulation: Expression System: E. Coli Refolding Conditions: Structure: Additional Information: Sales > $500 Million. Human Insulin

Product name: Novolin Produced by: Novo Nordisk (Princeton, NJ) Indication: Human use, Diabetes Date of approval: July 91 Formulation: Expression System: Refolding Conditions: Post-Transitional Modifications: Structure: Additional Information: LysPro Human Insulin Product name: Hurnulog Produced by: Eli Lilly (Indianapolis, IN) Indication: Human use, Diabetes Date of approval: June 1996 Formulation: Expression System: Refolding Conditions: Post-Transitional Modifications: Structure: Additional Information: GM-CSF (Granulocyte Macrophage-Colony Stimulating Factor) Product name: Leukine Produced by: Immunex (Seattle, WA) Indication: Human use, Bone marrow transplantation, Hodgkin's Disease, Leukemia Date of approval: March 91 Formulation: Lyophilized solution which is reconstituted with sterile water (stored at refrigerated temperatures for < 6 hours) or 0.9% benzyl alcohol (can be stored for < 20 days at refrigerated temperatures) and administered intravenous. After reconstitution, the lyophilized single use product contains either 0.25 mg/mL or 0.50 mg/mL GM-CSF, 40 mg/ml mannitol, 10 mg/ml sucrose, and 1.2 mg/ml tromethamine (final pH is 7.4 +/- 0.3). The reconstituted solution is then diluted into a 0.9% NaCl bag for IV administration (note if final GM-CSF is below 0.01 mg/mL add human albumin to 0.1% to prevent adsorption to the IV bag. Expression System: Yeast (S. Cerevisiae) Refolding Conditions: None, expressed folded. Structure: Glycoprotein of 127 amino acids characterized by 3 primary molecular species having molecular masses of 19,500, 16800, and 15500 daltons. The primary sequence differs from natural human GM-CSF by a substitution of leucine at position 23, and the carbohydrate moiety may be different from native. Additional Information: Specific activity is 5 x 10" colony forming units per mg protein. Sargramostim is the proper name for yeast-derived recombinant GM-CSF. Cost for a 0.5 mg GM-CSF vial is $178. G-CSF (Granulocyte Colony Stimulating Factor) Product name: Neupogen Produced by: Amgen (Thousand Oaks, CA) Indication: Human use, Neutropenia, bone marrow transplantation, anemia Date of approval: February 91 Formulation: Single-use solution formulation containing 0.3 mg/mL G-CSF, 10 mM sodium acetate, 5% mannitol, and 0.004% Tween-80 at a pH of 4. The product is to be stored at refrigerated temperatures and no more than 24 hours at room temperature. If required, Neupogen can be diluted with D5W (no not dilute with saline at any time; product may precipitate), at concentrations below 5 to 15 lug/mL, add human albumin to 2 mg/mL to prevent adsorption to IV bag. Expression System: E. coli. Refolding Conditions: Structure: A 175 amino acid protein with a molecular weight of 18,800 daltons. The protein has an amino acid sequence identical to the human protein except for an additional N-terminal methionine (necessary for expression in E. coli). The human protein is glycosylated but the recombinant Neupogen is not. Additional Information: Sales > $500 million. Filgrastim is the name give to recombinant methionyl human G-CSF. Cost of therapy (lung ) is $2,130 (4.2 mg protein over 14 days). Specific activity is 30 million units per mg protein. Satumomab Pendetide Product name: OncoScint CR/OV Produced by: Cytogen (Princeton, NJ) Indication: Human use, Colorectal and ovarian Date of approval: December 92 Formulation: Expression System: Refolding Conditions: Post-Transitional Modifications: US 7,001,892 B1 19 20

TABLE A-continued Structure: Additional Information: Interleukin-2 Product name: Proleukin (generic name: Aldesieukin) Produced by: Chiron (Emeryville, CA) Indication: Human use, Renal cell carcinoma Date of approval: May 1992 Formulation: Single-use lyophilized formulation which is reconstituted with 1.2 mL sterile water and administered intravenously. Each reconstituted product contains 1.1 mg/mL Proleukin, 50 mg/ml mannitol, and 0.18 mg/ml dibasic sodium phosphate (pH is 7.5 +/- 0.3). Lyophilized product is stored at refrigerated temperatures, reconstituted product is stable up to 48 hours at refrigerated to room temperatures, but should be stored in refrigerator due to lack of preservatives. Addition of preservatives results in increased aggregation, addition of human albumin alters pharmacology. Expression System: E. coli (tetracycline promoter). Refolding Conditions: Structure: Proleukin has a molecular weight of 15,300 daltons and differs from the natural human protein (is not glycosylated, the N-terminal alanine is removed, and has a serine substituted for the free cysteine at position 125) Additional Information: Specific activity is 18 million international units per 1.1 mg protein. Cost is $395 per 1.3 mg protein. Somatrem Product name: Protropin Produced by: Genentech (S. San Francisco, CA) Indication: Human use, Growth deficiency Date of approval: October 1985, patent expired on October 1992. Formulation: Lyophilized formulation which is reconstituted with 0.9% benzyl alcohol (supplied) and administered intramuscular or subcutaneous. The lyophilized vial contains 5 mg Somatirem, 40 mg mannitol and 1.7 mg sodium phosphates (0.1 mg sodium phosphate monobasic and 1.6 mg sodium phosphate dibasic) and is reconstituted with 1 to 5 mL of 0.9% benzyl alcohol. The lyophilized product is stored at refrigerated temperature, the reconstituted product is good for 14 days at refrigerated temperatures. Expression System: E. coli. Refolding Conditions: Structure: Contains 192 amino acids with molecular weight of 22,000 daltons. Identical to human sequence but contains an extra methionine at the N-terminus. Additional Information: Sales > $100 million. Cost of therapy is $13,110 (1 year, 313 mg protein) DNase (deoxyribonuclease I) Product name: Pulmozyme Produced by: Genentech (S. San Francisco, CA) Indication: Human use, Cystic fibrosis Date of approval: December 1993 Formulation: Inhalation solution (aerosol mist produced by a compressed air driven nebulizer system). Comes in a single-use 2.5 mL ampule containing 1.0 mg/mL DNase, 0.15 mg/mL calcium chloride dihydrate, and 8.77 mg/ml sodium chloride, at a pH of 6.3. The solution is stored at refrigerated temperatures and should not be exposed to light. Expression System: Mammalian cells (Chinese hamster Ovary cells) Refolding Conditions: Structure: Glycoprotein of 260 amino acids having a molecular weight of 37,000 daltons. The primary sequence is identical to that of the native human enzyme. Additional Information: Sales > $100 Million. Cost is $32 for 2.5 mg of protein (1 ampule) M-CSF (Macrophage-Colony Stimulating Factor) Product name: Leucomax (generic name: Molgramos tim) Produced by: Indication: Human use, Date of approval: FDA unapproved Formulation: Expression System: Refolding Conditions: Post-Transitional Modifications: Structure: Additional Information: Epoetin Beta (Erythropoietin) Product name: Marogen Produced by: Indication: Human use, Date of approval: Formulation: Expression System: Refolding Conditions: Post-Transitional Modifications: Structure: Additional Information: US 7,001,892 B1 21 22

TABLE A-continued Polyribonucleotide Product name: Ampligen Produced by: Indication: Human use, Date of approval: FDA Unapproved Formulation: Expression System: Refolding Conditions: Post-Transitional Modifications: Structure: Additional Information: Human Serum Albumin

Product name: Produced by: Indication: Human use, Date of approval: Formulation: Expression System: Refolding Conditions: Post-Transitional Modifications: Structure: Additional Information: Septomonab?

Product name: Gentoxin Produced by: Indication: Human use, Date of approval: Not FDA approved Formulation: Expression System: Refolding Conditions: Post-Transitional Modifications: Structure: Additional Information: Protein

Product name: Produced by: Indication: Human use, Date of approval: Formulation: Expression System: Refolding Conditions: Post-Transitional Modifications: Structure: Additional Information:

APPROVED BIOTECHNOLOGY DRUGS AND WACCINES

Product Product Name Company Category Indication Cornwax TM Merck recombinant vaccination of infants beginning at two months of age Haemophilus b Whitehouse Station, NJ. against both invasive Haemophilus influenzae type b conjugate diseases (Hib) and hepatitis B (October 1996) (meningococcal protein conjugate) and hepatitis b (recombinant) vaccine Engerix-B (R) SmithKline Beecham recombinant hepatitis B (September 1989) hepatitis B vaccine Philadelphia, PA vaccine (recombinant) EPOGEN (R) Amgen erythropoietin reatment of anemia associated with chronic renal Epoetin alfa Thousand Oaks, CA alure, including patients on dialysis and not on (rEPO) dialysis, and anemia in Retrovir (R)-treated HIV-infected patients (June 1998); treatment of anemia caused by in patients with non-myeloid malignancies (April 1993); prevention of anemia associated with surgical blood loss, autologous blood donation adjuvant (December 1996) PROCRIT (R) Ortho Biotech erythropoietin reatment of anemia associated with chronic renal Epoetin alfa Raritan, NJ ailure, including patients on dialysis and not on (rEPO) dialysis, and anemia in Retrovi (R)-treated HIV-infected patients (December 1990); treatment of anemia caused by chemotherapy in patients with non-myeloid malignancies (April 1993); prevention of anemia US 7,001,892 B1 23 24

TABLE A-continued associated with surgical blood loss, autologous blood donation adjuvant (December 1996) |PROCRIT was approved for market ing under Amgen's epoetin alfa PLA. Amgen manufactures the product for Ortho Biotech J Under an agreement bewteen the two companies, Amgen licensed to the U.S. rights to epoetin alfa for indications for human use excluding dialysis and diagnostics. Genotropin TM Pharmacia & Upjohn human short stature in children due to growth hormone somatropin Kalamazoo, MI growth deficiency (August 1995) (rDNA origin) hormone for injection Gerel (E) Serono Laboratories growth evaluation of the ability of the somatotroph of the human growth Norwell, MA factor pituitary gland to secrete growth hormone hormone (December 1990); pediatric growth hormone releasing factor deficiency (October 1997) Genal-F (R) Serono Laboratories recombinant female infertility (September 1997) recombinant human Norwell, MA fertility follicle-stimulating hormone hormone (r-FSH) Humalog TM Eli Lilly recombinant diabetes (June 1996) insulin lispro Indianapolis, IN insulin Humatrope (R) Eli Lilly human human growth hormone deficiency in children somatropin Indianapolis, IN growth (March 1987) (rDNA origin) hormone for injection Humulin (R) Eli Lilly recombinant diabetes (October 1982) human insulin Indianapolis, IN insulin (recombinant DNA origin) infergen (R) Amgen interferon treatment of chronic hepatitis C viral infection interferon alfacon-1 Thousand Oaks, CA (October 1997) Intron (R) A Schering-Plough interferon hairy cell leukemia (June 1986); genital warts interferon alfa-2b Madison, NJ (June 1988); AIDS-related Kaposi's sarcoma (recombinant) (November 1988); hepatitis C (February 1991); hepatitis B (July 1992); malignant melanoma (December 1995); follicular lymphoma in conjunction with chemotherapy (November 1997) KOGENate (R) Bayer Corporation, clotting treatment of hemophilia A (February 1993) antihemophiliac Pharmaceutical Division factor factor West Haven, CT (recombinant) Leukine TM ImmuneX colony autologous bone marrow transplantation (March 1991); Sargramostim Seattle, WA stimulating neutropenia resulting from chemotherapy in acute (GM-CSF) factor myelogenous leukemia (September 1995); allogeneic bone marrow transplantation (November 1995); peripheral blood progenitor cell mobilization and transplantation (December 1995) MyoScint (R) Centocor MAb myocardial infarction imaging agent (July 1996) imiciromab pentetate Malvern, PA Neumega (R) Genetics Institute MAb prevention of severe chemotherapy-induced oprelvekin Cambridge, MA thrombocytopenia (November 1997) NEUPOGEN (R) Amgen colony chemotherapy-induced neutropenia (February 1991); Filgrastim Thousand Oaks, CA stimulating autologeous or allogeneic bone marrow transplantation (rG-CSF) factor (June 1994); chronic severe neutropenia (December 1994); support peripheral blood progenitor cell transplantation (December 1995) Norditropin (R) Novo Nordisk human treatment of growth failure in children due to somatropin Pharmaceuticals growth inadequate of growth hormone secretion (May 1995) (rDNA origin) Princeton, NJ hormone for injection Novolin (R) 70/30 Novo Nordisk recombinant insulin-dependent diabetes mellitus (July 1991) 70% NPH human Pharmaceuticals insulin insulin isophane Princeton, NJ suspension & 30% regular, human insulin US 7,001,892 B1 25 26

TABLE A-continued injection (recombinant DNA origin) Nowolin (E) I. Novo Nordisk recombinant insulin-dependent diabetes mellitus (July 1991) Lente (E), human Pharmaceuticals insulin insulin Zinc Princeton, NJ suspension (recombinant DNA origin) Nowolin (R) N Novo Nordisk recombinant insulin-dependent diabetes mellitus (July 1991) NPH, human Pharmaceuticals insulin insulin isophane Princeton, NJ suspension (recombinant DNA origin) Nowolin (R) R Novo Nordisk recombinant insulin-dependent diabetes mellitus (July 1991) regular, human Pharmaceuticals insulin insulin injection Princeton, NJ (recombinant DNA origin) Nutropin (R) Genentech human growth failure in children due to chronic renal somatropin S. San Francisco, CA growth insufficiency, growth hormone inadequacy in children for injection hormone (March 1994); Turner's syndrome (December 1996); growth hormone inadequacy in adults (December 1997) Nutropin AQTM Genentech human growth failure in children due to chronic renal somatropin S. San Francisco, CA growth insufficiency, growth hormone inadequacy in children (liquid) hormone (December 1995); Turner's syndrome (December 1996); growth hormone inadequacy in adults (December 1997) OncoScint TM CYTOGEN MAb detection, staging and follow-up of colorectal and CR/OV Princeton, NJ ovarian cancers (December 1992) satumomab pendetide ORTHOCLONE Ortho Biotech MAb reversal of acute kidney transplant rejection OKT (R)3 Raritan, NJ (June 1986); reversal of heart and liver transplant muroimomab-CD3 rejection (June 1993) Proleukin (R) Chiron interleukin renal cell carcinoma (May 1992); metastatic melanoma aldesleukin Emeryville, CA (January 1998) (interleukin-2) ProstaScint (R) CYTOGEN MAb detection, staging and follow-up of capiromab Princeton, NJ adenocarcinoma (October 1996) beintetate Protropin (R) Genentech human human growth hormone deficiency in children SOmatrem S. San Francisco, CA growth (October 1985) or injection hormone Pulenozyme (R) Genentech recombinant cystic fibrosis (December 1993); management of domase alpha, S. San Francisco, CA DNase advanced cystic fibrosis (December 1996) recombinant Recombinate TM Baxter Healthcaref clotting hemophilia A (December 1992) antihemophilic Hyland Division factor actor recombinant Glendale, CA (IAHF) Genetics Institute Cambridge, MA RECOMBIVAX Merck recombinant hepatitis B prevention (July 1986) HB (R) hepatitis B vaccine Whitehouse Station, NJ vaccine (recombinant), MSD Refudan TM Hoechst Marion Roussel recombinant heparin-induced thrombocytopenia type II epirudin Kansas City, MO anticoagulant (March 1998) rDNA) or injection Regranex (R) Orthro-McNeil growth ower extremity disbetic neuropathic ulcers becaplemin Pharmaceuticals factor (December 1997) Raritan, NJ ReoPro (E) Centocor MAb anti-platelet prevention of blood clots in the setting of abciximab Malvern, PA high-risk percutaneous transluminal coronary angioplasty Eli Lilly (December 1994); refractory unstable angina when Indianapolis, IN percutaneous coronary unstable angina when (November 1997) Retevase TM Boehringer Mannheim tissue reatment of acute myocardial infarction (October 1996) reteplase Gaithersburg, MD plasminogen Centocor factor Malvern, PA Rituxan (R) Genentech MAb reatment of relapsed or refractory low-grade or rituximab S. San Francisco, CA ollicular CD20-positive B-cell non-Hodgkin's IDEC Pharmaceuticals ymphoma (November 1997); San Diego, CA US 7,001,892 B1 27 28

TABLE A-continued Roferon (R)-A Hoffmann-La Roche interferon hairy cell leukemia (June 1986); AIDS-related Kaposi's interferon alfa-2a, Nutley, NJ sarcoma (NPvember 1988); chronic myelogenous recombinant leukemia (November 1995); hepatitis C (November 1996) Saizen (R) Serona Laboratories human pediatric growth hormone deficiency (October 1996) somatropin Norwell, MA growth (rDNA origin) hormone for injection Serostim TM Serono Laboratories human treatment of AIDS-associated catabolism/wasting somatropin Norwell, MA growth (August 1996); pediatric HIV failure to thrive (rDNA origin) hormone (February 1998) for injection Verluma (E) Boehringer ingelheim MAb detection of small-cell lung cancer (August 1996) nofetumomab Ridgefield, CT NeoRx Seattle, WA Vistide (E) Gilead Scienced nucleotide cytomegalovirus retinitis in AIDS patients (June 1996) cidofovir injection Foster City, CA analogue Zenapax (R) Hoffman-La Roche MAb prevention of acute kidney transplant rejection daclizumab Nutley, NJ (December 1997) The content of this survey has been obtained through government and industry sources based on the latest information. The information may not be comprehensive. For more specific information about a particular product, contact the individual company directly. PhRMA internet adress: http://www.pharma.org Provided as a Public Service by PhRMA. Founded in 1958 as the Pharmaceutical Manufacturers Association. Copyright (C) 1998 by the Pharmaceutical Research and Manufacturers of America. Permission to reprint is awarded is proper credit is given.

Leadingfiewy in the arch fircuits Pharmaceutical Research and Manufacturers of America 1100 Fifteenth Street, NW Washington, D.C. 20005 http://www.phrma.org Printed on recycled paper. 4/98 Biotechnology Medicines in Development Product Product Development Name Company Category Indication Status

AIDS/HIV INFECTION AND RELATED CONDITIONS AD-439 and Biosystems MAb HIV infection, AIDS Phase II AD-519 Houston, TX combination AD-439 MAb, Tanox Biosystems MAb HIV infection, AIDS Phase II anti-HIV to V3 Houston, TX loop of gp120 protein; neutralizing AD-519 MAb, Tanox Biosystems MAb HIV infection, AIDS Phase II anti-HIV to C4 Houston, TX region of gp120 protein; neutralizing antibody Alferon LDO (R) Interferon Sciences interferon AIDS-related complex, AIDS Phase I/II interferon alfa-n3 New Brunswick, NJ Alferon N Interferon Sciences interferon HIV infection Phase III injection (R) New Brunswick, NJ (see also infectious diseases) interferon alfa-n3 co-infection (HIV/HCV) Phase II ALVAC-MN Pasteur Merieux Connaught vaccine HIV infection Phase II 12-TMG Lyons, France (vcP205) Virogenetics Albany, NY Ampligen (E) Hemispherx Biopharma interferon HIV infection Phase II New York, NY (see also cancer, infectious dis eases, other) autologous gene- SyStemix gene therapy HIV infection Phase I modified Palo Alto, CA hematopoietic stem cells gene therapy Cell Genesys gene therapy HIV infection Phase II Foster City, CA Hoechst Marion Roussel Kansas City, MO gp120 WaxGen vaccine AIDS Phase II vaccine S. San Francisco, CA US 7,001,892 B1 29 30

TABLE A-continued HIV-IT(V) Chrion Viagene gene therapy asymptomatic HIV-1 infection Phase II Retrovector TM San Diego, CA HIV-1 1118 encfrew retroviral vector HIV vaccine Chiron vaccine AIDS Phase II (gp120) Emeryville, CA interleukin-10 Schering-Plough interleukin HIV disease Phase I (IL-10) Madison, NJ (see also autoimmune, diges tive, heart, neurologic, respiratory, skin) ISIS 2922 Isis Pharmaceuticals antisense cytomegalovirus retinitis Phase III formivirsen Carlsbad, CA ISIS 13312 Isis Pharmaceuticals antisense cytomegalovirus retinitis Phase I Carlsbad, CA Leukine TM ImmuneX colony adjuvant to AIDS therapy, Phase II Sargramostim Seattle, WA stimulating HIV infection, prevention of (GM-CSF) factor infection in HIV patients (see also cancer) memantine Neurobiological AIDS dementia complex and Phase II Technologies AIDS-related neuropathic pain Richmond, CA (see also diabetes) MPL(R) Ribi ImmunoChem vaccine AIDS Phase I immunomodulator Hamilton, MT (see also infectious diseases) vaccine NEUPOGEN (R) Amgen colony treatment and prevention of application Filgrastim Thousand Oaks, CA stimulating neutropenia in HIV patients submitted (rG-CSF) factor (see also cancer, respiratory) Ovidrel (R) Ares-Serono and recombinant Kaposi's sarcoma, AIDS-re Phase I/II lated recombinant Serono Laboratories gonadotropin hypogonadism human chorionic Norwell, MA (see also infertility) gonadotropin (r-hCG) PEG interleukin-2 Chiron interleukin HIV infection in combination Phase II Emeryville, CA with Retrovir (E) PMPA nucleotide HIV infection, AIDS Phase II Foster City, CA analogue PreveOn. TM Gilead Sciences nucleotide HIV infection, AIDS Phase III adefovir dipivoxil Foster City, CA analogue PRO 367 Progenics Pharmaceuticals HIV infection Phase I Tarrytown, NY PRO 542 Progenics Pharmaceuticals HIV infection Phase I Tarrytown, NY Proleukin (R) Chiron interleukin HIV infection in combination Phase II/III aldesleukin Emeryville, CA with Retrovir (E) (interleukin-2) (see also cancer) Remune Immune Response Corp. immune HIV seropositive Phase III HIV-1 immunogen Carlsbad, CA based therapy retroviral vector Chiron gene therapy HIV infection Phase I/II with 2 ribozymes Emeryville, CA TBC-38 Therion Biologics vaccine AIDS prevention Phase I (vaccinia virus Cambridge, MA expressing HIV genes enV, gag and pal) AUTOIMMUNE DISORDERS adensoine National Cancer Institute gene therapy severe combined Phase I deaminase Bethesda, MD immunodeficiency NCITRIAL transduced autologous CD34+ PBC or umbilical cord? placental blood cells adenosine National Cancer Institute gene therapy severe combined Phase I deaminase Bethesda, MD immunodeficiency NCITRIAL transduced T cells AnergiXTM-RA Anergen functional rheumatoid arthritis Phase I Redwood City, CA antigenics immuno therapy AnervaXTM Anergen peptide rheumatoid arthritis Phase II Redwood City, CA vaccine US 7,001,892 B1 31 32

TABLE A-continued Awakine TM Centocor MAb rheumatoid arthritis hase III chimeric anti-TNF Malvern, PA (see also digestive) antibody CD40 ligand Biogen MAb lupus, immune thromobocytopenic hase II antibody Cambridge, MA purpura clenoliximab IDEC Pharmaceuticals MAb rheumatoid arthritis hase II San Diego, CA SmithKline Beecham Philadelphia, PA ConXTM Connetics recombinant scleroderma hase II relaxin Palo Alto, CA human protein Embrel ImmuneX recombinant rheumatoid arthritis hase III tumor necrosis Seattle, WA soluble factor (TNF) -Ayerst Laboratories receptor receptor Philadelphia, PA h5G1.1 MAb lupus, rheumatoid arthritis hase III New Haven, CT IDEC-131 IDEC Pharmaceuticals MAb systemic lupus erythermatosus hase I humanized MAb San Diego, CA IL-2 fusion protein Seragen fusion severe rheumatoid arthritis hase III DABss IL-2 Hopkinton, MA protein (see also cancer, skin) interleukin-10 Schering-Plough interleukin rheumatoid arthritis hase II (IL-10) Madison, NJ (see also AIDS/HIV, digestive, heart, neurologic, respiratory, skin) IRSO1 Immune Response Corp. vaccine rheumatoid arthritis hase II therapeutic Carlsbad, CA vaccine ISIS 23O2 Isis Pharmaceuticals antisense rheumatoid arthritis hase II Carlsbad, CA (see also digestive, skin, transplantation) MDX-33 Medarex MAb autoimmune diseases, idiopathic hase I Annandale, NJ thrombocytopenic purpura ORTHOCLONE Ortho Biotech MAb treatment of CD4-mediated hase II OKT4A Raritan, NJ autoimmune diseases (see also transplantation) Quadrakine Schering-Plough interleukin rheumatoid arthritis hase I interleukin-4 Madison, NJ (IL-4) SMART TM Anti-CD3 Protein Design Labs MAb autoimmune diseases hase I HM291 Mountain View, CA (see also transplantation) BLOOD DISORDERS

CPC-111 Cypros Pharmaceuticals cellular sickle cell disease hase II Carlsbad, CA therapy (see also heart) Factor VIII Transkaryotic gene therapy hemophilia A hase I Cambridge, MA GA-EPO Hoechst Marion Roussel erythropoietin anemia associated with hase II Kansas City, MO chronic renal failure Transkaryotic Therapies Cambridge, MA Kogenate-N Bayer clotting hemophilia A hase III tFVIII Berkeley, CA factor NovoSeven (E) Novo Nordisk clotting treatment of hemophilia A & B with hase III recombinant factor Pharmaceuticals factor and without against Vita Princeton, NJ factors VIII/DX Optro TM Somatogen recombinant Oxygen-carrying agent and hase II recombinant human Boudler, CO human alternative to red blood caell hemoglobin hemoglobin transfusion (rHb1.1) stimulation of red blood cell hase I formation ReFacto (R) Genetics Institute clotting hemophilia A hase III recombinant factor Cambridge, MA factor VIII YM-337 MAb Yamanouchi USA MAb platelet aggregation hase I White Plains, NY Protein Design Labs Mountain View, CA CANCER AND RELATED CONDITIONS

1311-ChTNT 1/8 Techniclone MAb malignant glioma Phase I Tustin, CA Aastrom TM Aastrom Biosciences cellular cancer immunosuppression? Phase II Cell Production Ann Arbor, MI therapy blood and System recovery for patients receiving US 7,001,892 B1 33 34

TABLE A-continued stem and progenitor ablative chemotherapy cell expansion from bone marrow and umbilical cord blood Actimmune (R) National Cancer Institute interferon colon, lung, ovarian, prostate hase II interferon Bethesda, MD cancers, melanoma NCITRIAL gamma-1b Genentech S. San Francisco, CA AFP-Scan TM Immunomedics MAb extent of disease staging of liver hase II technetium-99m Morris Plains, NJ and germ cell cancers FAb' fragment (germ cell) allogeneic SyStemix cellular advanced leukemia, lymphona, hase I hematopoietic Palo Alto, CA therapy myelodysplastic syndromes stem cell transplantation Allovectin-7 Vical gene therapy advanced metastatic melanoma, hase II DNA/lipid complex San Diego, CA non-resectable squamous cell encoding HLA-87 carcinoma of the head and neck antigen ALT Cellcor cellular metastatic renal cell carcinoma hase III (autolymphocyte Newton, MA therapy (kidney cancer) completed therapy) CYTOGEN Princeton, NJ ALVAC-87.1 National Cancer Institute gene therapy melanoma Pahse I Bethesda, MD NCITRIAL ALVAC-CEA-87.1 National Cancer Institute gene therapy advanced adenocarcinomas hase I Bethesda, MD NCITRIAL ALVAC-CEA National Cancer Institute vaccine advanced cancers hase I vaccine Bethesda, MD NCITRIAL ALVAC-IL-12 National Cancer Institute vaccine melanoma hase I vaccine Bethesda, MD NCITRIAL Pasteur Merieux Connaught Lyons, France Ampligen (R) Hemispherx Biopharma interferon renal cancer hase I/II New York, NY (see also AIDS/HIV, infectious diseases, other) anti-cancer T-cell Cell Genesys gene therapy colon cancer hase III gene therapy Foster City, CA anti-idiotype Novartis Pharmaceuticals MAb CaCe hase I monoclonal East Hanover, NJ antibody anti-Tac(Fv)-PE38 National Cancer Institute MAb + toxin leukemia, lymphoma hase I immunotoxin Bethesda, MD NCITRIAL anti-transferrin National Cancer Institute MAb advanced, refractory solid tumors hase I receptor Bethesda, MD NCITRIAL MAb anti-VEGF Genentech MAb CaCe hase I humanized MAb S. San Francisco, CA autologous SyStemix cellular hematopoietic reconstitution in hase III hematopoietic Palo Alto, CA therapy patients with multiple myleoma, stem cells for non-Hodgkin's lymphoma, autologous breast cancer hematopoietic transplantation autologous peptide National Cancer Institute cellular advanced solid tumors hase I specific activated Bethesda, MD therapy NCITRIAL lymphocytes autologous National Cancer Institute gene therapy breast cancer, myeloma hase I transduced CD34+ Bethesda, MD NCITRIAL bone marrow and peripheral blood stem cells Avicidin (R) Janssen Pharmaceutics MAb colorectal, lung, prostate cancers hase II MAb conjugate Titusville, NJ NeoRix Seattle, WA Avicine TM AVI BioPharma vaccine colorectal, pancreatic cancers hase II CTP-37 Portland, OR Avonex (R) Biogen interferon glioma hase II interferon beta-1A Cambridge, MA (see also neurologic) B7 transfected National Cancer Institute vaccine melanoma hase I allogeneic Bethesda, MD NCITRIAL melanoma cell vaccine BEC2, anti-idiotype ImClone Systems vaccine melanoma, small-cell lung cancer hase I MAb Somerville, NJ Betaseron (R) National Cancer Institute interferon non-small-cell lung cancer hase III US 7,001,892 B1 35 36

TABLE A-continued recombinant Bethesda, MD (see also neurologic) NCITRIAL beta interferon-1b Berlex Laboratories Wayne, NJ bispecific Chiron MAb CaCe hase I antibody Emeryville, CA C225, anti-EGFR ImClone Systems MAb epidermal growth factor receptor hase II chimeric MAb Somerville, NJ positive cancers Campath 1H LeukoSite Mab chronic lymphocytic leukemia in clinical Cambridge, MA trials carcinoembryonic National Cancer Institute vaccine breast, gastrointestinal tract, hase I antigen peptide-1 Bethesda, MD lung cancers NCITRIAL vaccine CEAClde TM Immunomedics MAb hase II humanized Morris Plains, NJ anti-CEA antibody (hMN14) CEA-Scan TM Immunomedics Mab extent of disease staging of breast hase II technetium-99m Morris Plains, NJ CaCe arcitumomab (breast) CEA-Scan TM Immunomedics MAb extent of disease staging of lung hase III technetium-99m Morris Plains, NJ CaCe arcitumomab (lung) CEAVac TM vaccine colorectal cancer hase II anti-idiotype S. San Francisco, CA antibody vaccine cell therapy CytoTherapeutics cellular cancer pain, untreatable?unrelieved hase II Providence, RI therapy by other forms of treatment Cereport TM recurrent malignant brain tumor hase III (RMP-7) Cambridge, MA and carboplatin chemotherapy Genetix gene therapy treatment of cancer patients hase III resistant Rye, NY requiring chemotherapy bone marrow chimeric MAb National Cancer Institute Mab melanoma, neuroblastoma hase II 14, 18 Bethesda, MD NCITRIAL CM 101 CarboMed CaCe hase III Brentwood, TN CMA-676 Wyeth-Ayerst Laboratories Mab relapsed acute myelogenous hase II/III Philadelphia, PA leukemia CMB-4O1 Wyeth-Ayerst Laboratories MAb ovarian cancer hase III Philadelphia, PA colon cancer Immune Response Corp. vaccine colon cancer hase III cell line Carlsbad, CA vaccine Sidney Kimmel Cancer Center San Diego, CA CP-358, 774 OSI Pharmaceuticals cellular CaCe hase I Uniondale, NY therapy New York, NY CT 2584 Cell Therapeutics ovarian, , hase I Seattle, WA SaCOa cytosine deaminase GenVec gene therapy colon cancer hase I gene-adenoviral Rockville, MD wector DA/Hu (gamma).4 Chiron Viagene gene therapy metastatic melanoma hase I hiFN-y(V) San Diego, CA Retrivector TM hiFN-y retroviral wector DA/Hu(gamma), 15 Chiron Viagene gene therapy stage IV hase I transduced San Diego, CA malignant melanoma autologous tumor cells and interferon gamma expressing transduced autologous tumor cells (combination therapy) DA/Hu(gamma), 15 Chiron Viagene gene therapy disseminated Phase I transduced San Diego, CA malignant melanoma autologous tumor cells; ITAT dariplestim Searle growth factor mobilization of peripheral Phase III Skokie, IL blood stem cells dendritic cell Dendreon cellular advanced prostate cancer Phase II/III therapy Mountain View, CA therapy multiple myeloma Phase I E/A lipid complex Targeted Genetics gene therapy breast, head and neck, ovarian Phase I US 7,001,892 B1 37 38

TABLE A-continued

(rgDCC-E/A) Seattle, WA CaCeS EGF fusion protein Seragen fusion protein non-small-cell lung cancer Phase I/II DABEGF Hopkinton, MA EPREX (R) National Cancer Institute erythropoietin neuroblastoma Phase II erythropoietin Bethesda, MD NCITRIAL Ortho Biotech Raritan, NJ ERB-38 National Cancer Institute fusion protein advanced stage solid tumors Phase I immunotoxin Bethesda, MD NCITRIAL fusion protein (recombinant) Ewing's sarcoma Natioanl Cancer Institute vaccine SaCOa Phase I and alveolar Bethesda, MD NCITRIALS rhabdomyosarcoma peptide vaccine FLT3 ligand National Cancer Institute growth factor melanoma, renal cell cancer Phase I Bethesda, MD NCITRIAL ImmuneX Seattle, WA G3139 antisense CaCe Phase I San Diego, CA gamma interferon Chiron gene therapy CaCe Phase I gene therapy Emeryville, CA Gastrimmune TM Aphton vaccine colorectal, pancreatic, stomach Phase I/II neutralizing G17 Woodland, CA CaCeS hormone (see also digestive) GeneVax (E) Centocor vaccine colorectal cancer Phase I gene Vaccine Malvern, PA GL-328 Genetic Therapy gene therapy glioblastoma multiforme Phase III Gaithersburg, MD GM-CSF cellular Powderject vaccine melanoma, sarcoma Phase I Madison, WI GMK Bristol-Myers Squibb vaccine prevent recurrence following surgery Phase III ganglioside Princeton, NJ to remove primary melanoma antigen Progencis Pharmaceuticals Tarrytown, NY gp100 adenovirus National Cancer Institute vaccine melanoma Phase I vaccine Bethesda, MD NCITRIAL Genzyme Molecular Oncology Cambridge, MA gp100 peptide National Cancer Institute vaccine melanoma Phase I vaccine Bethesda, MD NCITRIAL GVAXTM Cell Genesys vaccine prostate, lung cancers, melanoma Phase I/II cancer vaccine Foster City, CA HER-2/neu. National Cancer Institute vaccine breast, colorectal, ovarian, Phase I peptide vaccine Bethesda, MD prostate cancers NCITRIAL Herceptin TM Genentech MAb breast cancer Phase III trastuzumab S. San Francisco, CA completed (anti-HER-2 humanized MAb) HPV 16, E6 and E7 National Cancer Institute vaccine cervical cancer Phase I peptide vaccine Bethesda, MD NCITRIAL HPV E7 lipopeptide National Cancer Institute vaccine cervical cancer Phase I vaccine Bethesda, MD NCITRIAL Cytel San Diego, CA HPV vaccine Medimmune vaccine cervical cancer Phase I Gaithersburg, MD (see also infectious disease) SmithKline Beecham Philadelphia, PA HSPPC-96 Antigenics heat shock melanoma, pancreatic, Phase I (autologous New York, NY portein renal cell cancers tumor derived) human growth Transkaryotic Therapies gene therapy cancer cachexia (muscle wasting) Phase I hormone Cambridge, MA IDEC-88 IDEC Pharmaceuticals Mab non-Hodgkin's B-cell lymphoma Phase I/II San Diego, CA IDEC-Y288 IDEC Pharmaceuticals MAb non-Hodgkin's B-cell lymphoma Phase I/II San Diego, CA Leucotropin Cangene colony mobilization of peripheral blood Phase III GM-CSF Mississauga, Ontario stimulating stem cells in patients with factor adjuvant breast cancer Leukine TM ImmuneX colony prophylaxis and treatment of application Sargramostin Seattle, WA stimulating chemotherapy-induced neutropenia, submitted (GM-CSF) factor prophylaxis of chemotherapy-induced neutropenia in acute myelogenous leukemia (see also AIDS/HIV) US 7,001,892 B1 39 40

TABLE A-continued Lervectin Vical gene therapy prostate cancer, renal cell carcinoma, Phase I DNA/lipd complex San Diego, CA melanoma, sarcoma encoding IL-2 LP 2307 LIDAX Pharmaceuticals vaccine malignant melanoma Phase I/II La Jolla, CA Inex Pharmaceuticals antisense chronic myelogenous leukemia Phase I Hayward, CA in accelerated phase or blast crisis Techniclone MAb lymphoma Phase II/III Tustin, CA Lymphocide TM Immunomedics MAb non-Hodgkin's B-cell lymphoma Phase I/II anti-CD22 Morris Plains, NJ humanized MAb LymphoScan TM Immunomedics MAb extent of disease staging of Phase III technetium-99m Morris Plains, NJ non-Hodgkin's B-cell lymphoma, bectumomab detection of residual disease (lymphoma) following radiation/chemotherapy Mab GlaxoWellcome Mab lung, prostate cancers Phase II Rsch, Triangle Park, NC MART-1 National Cancer Institute vaccine melanoma Phase I adenvirus Bethesda, MD NCITRIAL vaccine Genzyme Molecular Oncology Cambridge, MA MART-1 National Cancer Institute vaccine metastatic melanoma Phase I melanoma Bethesda, MD NCITRAIL vaccine MDRX1 TM Titan Pharmaceuticals gene therapy enable high-dose chemotherapy Phase I S. San Francisco, CA with reduced side effects MDX-447 Medarex Mab head and neck, renal cancers Phase I/II bispecific antibody Annandale, NJ MDX-H210 Medarex MAb breast, colorectal, kidney, Ovarian, Phase I/II bispecific antibody Annandale, NJ prostate cancers Melacine (E) Ribi ImmunoChem vaccine stage IV melanoma with interferon Phase III melanoma Hamilton, MT alpha completed theraccine Ribi ImmunoChem vaccine stage II melanoma in patients with Phase III (therapeutic Hamilton, MT no evidence of disease to prevent vaccine Southwest Oncology Group recurrence following surgery to San Antonio, TX remove primary disease myeloid progenitor Human Genome Sciences interleukin chemoprotection Phase I inhibitory factor-1 Rockville, MD myeloma-derived National Cancer Institute vaccine multiple myeloma Phase I idiotypic antigen Bethesda, MD NCITRIAL vaccine NEUPOGEN (R) Amgen colony acute myelogenous leukemia application Filgrastim Thousand Oaks, CA stimulating (see also AIDS/HIV, respiratory) submitted (rG-GSF) factor Oncaspar (R) Enzon first-line treatment of acute in clinical PEG-L-asparaginase Piscataway, NJ lymphoblastic leukemia (ALL) trials Rhone-Poulenc Rorer adult ALL non-Hodgkin's lymphoma, Titusville, NJ chronic lymphocytic leukemia Oncolym (R) Techniclone MAb malignant glioma Phase I Tustin, CA Oncoltad (R) PR CYTOGEN MAb targeted radiotherapy for prostate Phase II CYT-356-Y-90 Princeton, NJ malignancies OncoScint (E) CYTOGEN MAb detection, staging and follow-up Phase II CR/OV Princeton, NJ of breast cancer satumomab pendetide ONYX-015 Onyx Pharmaceuticals oncolytic p53 deficient cancers Phase I/II Richmond, CA virus therapy p53 and RA5 National Cancer Institute vaccine solid tumors Phase I vaccine Bethesda, MD NCITRIAL p53 tumor Schering-Plough gene therapy lung cancer Phase II Suppressor gene Madison, NJ solid tumors that carry the Phase I p53 gene mutation or deletion Panorex (R) Centocor MAb adjuvant therapy for post-operative Phase III edrecolomab Malvern, PA colorectal cancer peripheral blood National Cancer Institute gene therapy ovarian cancer Phase I lymphocytes Bethesda, MD NCITRIAL transduced with a gene encoding a chimeric T-cell receptor Proleukin (R) Chiron interleukin acute myelogenous leukemia, Phase II/III aldesleukin Emeryville, CA non-Hodgkin's lymphoma (interleukin-2) (see also AIDS/HIV) promegapoletin Searle growth factor adjunctive hematopoietic therapy Phase I Skokie, IL following chemotherapy US 7,001,892 B1 41 42

TABLE A-continued Prostrac Therion Biologics vaccine prostate cancer hase III recombinant Cambridge, MA vaccinia virus RA55-17 peptide National Cancer Institute vaccine solid tumors hase I vaccine Bethesda, MD NCITRIAL rCEA Vaccine Protein Sciences vaccine breast, colon cancers hase I recombinant Meriden, CT carcinoembryonic antigen Rebi (R) Serono Laboratories interferon colorectal cancer hase III recombinant Norwell, MA (see also infectious diseases, interferon beta-1a neurologic) non-small-cell lung cancer hase III recombinant human Genetics Institute interleukin CaCe hase III interleukin-12 Cambridge, MA (see also infectious diseases) (rhIL-12) Wyeth-Ayerst Laboratories Philadelphia, PA retroviral vector Chiron gene therapy leaCa hase I with tumor necrosis Emeryville, CA factor gene rf-gp100 Therion Biologics vaccine leaCa hase I (recombinant Cambridge, MA fowlpox virus) rF-MART1 Therion Biologics vaccine leaCa hase I (recombinant Cambridge, MA fowlpox virus) RIGScan (E) CR49 Neoprobe MAb colorectal cancer application 125I-cc49 MAb Dublin, OH submitted Rituxan (R) National Cancer Institute Mab leukemia, lymphoma hase II rituximab Bethesda, MD NCITRIAL IDEC Pharmaceuticals San Diego, CA Roferon (R)-A Hoffmann-La Roche interferon malignant melanoma adjuvant hase III interferon alfa-2a, Nutley, NJ recombinant rV-gp100 Therion Biologics vaccine leaCa hase I (recombinant Cambridge, MA vaccinia virus) rV-MART1 Therion Biologics vaccine leaCa hase I (recombinant Cambridge, MA vaccinia virus) Serostim TM Serono Laboratories human cancer cachexia hase III somatropin Norwell, MA growth (see also other) (rDNA origin) hormone for injection Sigoslx (R) Ares-Serono and interleukin hematological conditions hase III recombinant Serono Laboratories (myelodysplastic syndromes, cancer) interleukin-6 Norwell, MA (r-IL-6) SMART TM M195 Protein Design Labs MAb acute myeloid leukemia hase II/III HM195 Mountain View, CA acute promyelocytic leukemia hase II advanced myeloid leukemia hase I (with Bismuth-213) stem cell factor Amgen stem cell adjunct to chemotherapy application Thousand Oaks, CA factor submitted SU101 SUGEN PDGF malignant glioma hase III Redwood City, CA receptor prostate cancer hase II tyrosine solid tumors hase III kinase inhibitor SUS416 SUGEN angiogenesis solid tumors hase I Redwood City, CA inhibitor TBC CEA Therion Biologics vaccine colorectal and lung cancers hase III (vaccinia virus Cambridge, MA expressing carcinoembryonic antigen) TCell-HDM Coulter Cellular Therapies cellular CaCe Phase I/II Boston, MA therapy Theratope (R) Biomira vaccine breast cancer Phase II synthetic Edmonton, Alberta completed carbohydrate Chiron therapeutic Emeryville, CA vaccine thrombopoietin Genentech erythropoietin thrombocytopenia related to cancer Phase II S. San Francisco, CA treatment US 7,001,892 B1 43 44

TABLE A-continued Thyrogen (R) Genzyme detection and treatment of application recombinant human Cambridge, MA thyroid cancer metastases submitted thyroid-stimulating hormone TNT Techniclone MAb non-Hodgkin's B-cell lymphoma hase II/III Tustin, CA solid tumors hase I TriABTM Titan Pharmaceuticals vaccine breast cancer hase II anti-idiotype S. San Francisco, CA antibody vaccine TriGEMTM Titan Pharmaceuticals vaccine small-cell lung cancer, melanoma hase I anti-idiotype S. San Francisco, CA antibody vaccine urate oxidase Sanoli recombinant prophylaxis for chemotherapy hase III (recombinantly New York, NY enzyme related hyperuricemia, treatment produced enzyme) of cancer-related hyperuricemia vaccine-CEA National Cancer Institute vaccine advanced colorectal cancer hase I 18OKD Bethesda, MD NCITRIAL vaccine Therion Biologics Cambridge, MA Vaxid Vical vaccine B-cell and mantle cell lymphomas hase I anti-idiotype DNA San Diego, CA vaccine Xerecept TM Neurobiological brain tumor edema hase II human Technologies corticorropin Richmond, CA releasing factor (hCRF) Zenapax (R) Hoffmann-La Roche Mab certain blood cancers Phase II daclizumab Nutley, NJ (see also eye, neurologic, skin, Protein Design Labs transplantation) Mountain View, CA DABETES AND RELATED CONDITIONS Beta Kline Genzyme Tissue Repair growth chronic diabetic foot ulcers Phase II transforming growth Cambridge, MA actor factor-beta 2 BetaRx-H VivoRX cellular insulin-dependent diabetes Phase I encapsulated Santa Monica, CA herapy human islets BetaRx-P VivoRX cellular insulin-dependent diabetes Phase I encapsulated Santa Monica, CA herapy procine islets BetaRx-Pr VivoRX cellular insulin-dependent diabetes Phase I encapsulated Santa Monica, CA herapy proliferated human islets Glucagen TM Novo Nordisk recombinant hypoglycemia Phase III recombinant human Pharmaceuticals al (see also digestive) glucagen Princeton, NJ protein (protein) glucagen Eli Lilly recombinant to treat severe hypoglycemic events application for injection Indianapolis, IN al in patients with diabetes and to aid submitted (rDNA origin) protein in gastrointestinal diagnostic procedures insulinotropin Scios type 2 diabetes hase II Mountain View, CA mermantine Neurobiological painful diabetic neuropathy hase II Technologies (see also AIDS/HIV) Richmond, CA eWe Genentech growth diabetic peripheral neuropathy hase III growth factor S. San Francisco, CA factor pirnagedine Alteon diabetic progressive kidney disease, hase III Ramsey, NJ diabetic end-stage kidney disease Genentech (see also neurologic) S. San Francisco, CA pramlintide human improved metabolic control, hase III San Diego, CA amylin which includes glucose, weight analog and lipid profiles in type 1 and insulin-using type 2 diabetes rDNA insulin Inhale Therapeutic Systems recombinant diabetes hase II Palo Alto, CA insulin Trovert TM Sensus human diabetes-related illnesses hase II Austin, TX growth (see also growth disorders) hormone DIGESTIVE DISORDERS

Awakine TM Centocor Mab Crohn's disease application chimeric anti-TNF Malvern, PA (see also autoimmune) submitted antibody

US 7,001,892 B1 47 48

TABLE A-continued for injection (see also other) Trovert TM Sensus human acromegaly Phase II Austin, TX growth (see also diabetes) hormone HEART DISEASE

AcuTect TM Diatide peptide detection of carotid thrombus Phase II Tc-99m apcitide Londonderry, NH anti-CD18 Genentech MAb acute myocardial infarction Phase II humanized MAb S. San Francisco, CA BioByPant TM CenVec gene thearpy cardiovascular disease, Phase I therapeutic Rockville, MD including cardiac artery disease angiogenesis and peripheral vascular disease, (VEGF) either as an adjunct or alternative to existing surgical approaches such as cardiac artery approaches such as cardiac artery bypass grafts and angioplasty Biostent TM NeoRx reduction of restinosis (vascular Phase I Seattle, WA remodeling) following balloon angioplasty Capiscint Centocor MAb atherosclerotic plaque imaging Phase I Malvern, PA agent Corsewin TMM Centocor MAb thrombolytic complications of Phase I 12D10-Fab Malvern, PA percutaneous transluminal Corvas coronary angioplasty, coronary San Diego, CA arterial starts, disseminates intravascular coagulation CPC-111 Cypros Pharmaceuticals cellular coronary bypass surgery Phase II Carlsbad, CA therapy (see also blood) factor VIIa Corvas deep vein thrombosis, pulmonary Phase I inhibitors San Diego, CA embolism, unstable angina, myocardial infarction FIBLAST (R) Scios growth peripheral vascular disease, Phase II trafermin Mountain View, CA factor coronary artery disease Wyeth-Ayerst Laboratories (see also neurologic) Philadelphia, PA gene therapy Collateral Therapeutics gene therapy stable exertional angina Phase I/II San Diego, CA growth factor Chiron growth coronary artery disease Phase I Emeryville, CA factor h5G1.1-SCFV Alexion Pharmaceuticals cardiopulmonary Phase II (recombinant) New Haven, CT bypass-associated inflammation Enzon using SCD (R) technology Piscataway, NJ ICOS MAb myocardial infarction Phase II MAb Bothell, WA (see also neurologic, other) integrillin TM COR Therapeutics percutaneous transluminal application eptifibatide S. San Francisco, CA coronary angioplasty, submitted (IIb/IIIa platelet Schering-Plough unstable angina aggregation Madison, NJ acute myocardial infarction Phase II inhibitor) interleukin-10 Schering-Plough interleukin ischemic reperfusion injury Phase I (IL-10) Madison, NJ (see also AIDS/HIV, autoimmune, digestive, neurologic, respiratory, skin) lanoteplase Bristol-Myers Squibb t-PA acute myocardial infarction Phase III Princeton, NJ Inex Pharmaceuticals antisense cardiovascular restinosis Phase II Vancouver, BC Schwarz, Pharma Milwaukee, WI MH1-Fab American Biogenetic MAb in vivo imaging agent for the Phase I/II imaging agent Sciences detection of cardiovascular Boston, MA thrombosis MPL(R)-C Ribi ImmunoChem prevention/amelioration of Phase II immunomodulator Hamilton, MT cardiac ischemia reperfusion injury Natrecor (R) BNP Scios acute congestive heart failure Phase III Mountain View, CA completed/ application submitted cardiovascular pulmonary surgery Phase I Novastan (R) Texas Biotechnology heparin-induced application argatroban Houston, TX thrombocytopenia submitted thrombosis syndrome ReoPro (E) Centocor MAb unstable angina Phase III abciximab Malvern, PA (see also neurologic) Eli Lilly acute myocardial infarction Phase II Indianapolis, IN rhAntithrombin III Genzyme control of blood clotting during Phase II

US 7,001,892 B1 S1 52

TABLE A-continued MAK 195F Knoll Pharmaceutical Mab sepsis Phase III Mt. Olive, NJ MED-491 Mediummune vaccine B 19 parvovirus-induced Phase I parvovirus Gaithersburg, MD miscarriage and anemia B 19 vaccine meningococcus C Chiron vaccine meningococcus C Phase II vaccine Emeryville, CA MPL(R) Ribi ImmunoChem vaccine infectious diseases in clinical immunomodulator Hamilton, MT (see also AIDS/HIV) trials (25+ antigens for adult and pediatric applications) Neuprex TM XOMA recombinant meningococcemia Phase III recombinant human Berkeley, CA human (see also genetic, other) bactericidalf protein antibiotic adjuvant in Phase II permeability intra-abdominal infections increasing protein (rBPI-21) Protovir TM Protein Design Labs MAb cytomegalovirus infections in human anti-CMV Mountain View, CA bone marrow transplant patients antibody Rebii (R) Serono Laboratories interferon viral infections Phase II/III recombinant Norwell, MA (see also cancer, neurologic) interferon beta-1a recombinant human Eli Lilly recombinant treatment of seven sepsis Phase II activated protein C Indianapolis, IN human (rhAPC) protein recombinant human Genetics Institute interleukin infectious diseases Phase I/II interleukin-12 Cambridge, MA (see also cancer) (rhIL-12) Wyeth-Ayerst Laboratories Philadelphia, PA Rotashield TM Wyeth-Lederle continuous prevention of rotaviral application rotavirus vaccine, Vaccine & Pediatrics cell line gastroenteritis in infants submitted live, oral, Philadelphia, PA vaccine tetravalent rotavirus Virus Research Institute vaccine rotavirus in infants Phase II vaccine Cambridge, MA Savvy TM Biosyn microbicide infectious disease Phase I C31G Philadelphia, PA Tenefuss (R) Hoffmann-La Roche recombinant septic shock, severe sepsis Phase III Ienercept Nutley, NJ soluble (TNF-receptor) receptor fusion protein) tifacogin Chiron issue factor sepsis Phase II Emeryville, CA pathway Searle inhibitor Skokie, IL INFERTILITY

Anfide TM Ares-Serono and OOC- female infertility Phase I gonadotropin Serono Laboratories releasing hormone-releasing Norwell, MA OOC hormone antagonist antagonist (GhRHA) Gonal-P(R) Serono Laboratories recombinant male infertility Phase III recombinant human Norwell, MA ertility ollicle-stimulating OOC OOC (r-FSH) LhaDI (E) Ares-Serono and recombinant female infertility-follicular support, Phase II/III recombinant Serono Laboratories ertility stimulation of follicular development human leutinizing Norwell, MA OOC OOle

Ovidrel (R) Ares-Serono and recombinant female infertility Phase III recombinant Serono Laboratories gonadotropin (see also AIDS/HIV) human chorionic Norwell, MA gonadotropin (r-hCG) NEUROLOGIC DISORDERS

Activase TM Genentech t-PA acute ischemic stroke within Phase III alteplase, S. San Francisco, CA 3 to 5 hours of symptom onset recombinant AnergiX (R) MS Anergen functional multiple sclerosis Phase I Redwood City, CA antigenics immuno therapy Antigen Athena Neurosciences MAb multiple sclerosis flares Phase II US 7,001,892 B1 S3 S4

TABLE A-continued natalizumab S. San Francisco, CA ATMO27 T Cell Sciences MAb multiple sclerosis hase I humanized MAb Needham, MA Avonex (R) Biogen interferon secondary, progressive hase III interferon beta-1a Cambridge, MA multiple sclerosis (see also cancer) Betaseron (R) Berlex Laboratories interferon chronic progressive multiple sclerosis hase III recombinant Wayne, NJ (see also cancer) interferon beta-1b Chiron Emeryville, CA brain-derived Amgen growth arryotrophic lateral sclerosis hase I neurotrophic factor Thousand Oaks, CA factor (BDNF) Regeneron Pharmaceuticals Tarrytown, NY CPC-211 Cypros Pharmaceuticals cellular ischermic stroke, traumatic brain hase II Carlsbad, CA therapy injury enlimomab Boehringer Ingelheim MAb Stroke hase II/III (anti-ICAM-1 MAb) Pharmaceuticals (see also other) Ridgefield, CT FIBLAST (R) Scios growth Stroke hase II/III trafermin Mountain View, CA factor (see also heart) Wyeth-Ayerst Laboratories Philadelphia, PA Hu23F2G ICOS MAb multiple sclerosis, ischemic stroke hase II MAb Bothell, WA (See also heart, other) interleukin-10 Schering-Plough interleukin multiple sclerosis hase I (IL-10) Madison, NJ (see also AIDS/HIV, autoimmune, digestive, heart, respiratory, skin) IR 208 Immune Response Corp. vaccine multiple sclerosis hase I therapeu ic vaccine Carlsbad, CA LDP-O1 LeukoSite MAb Stroke hase III Cambridge, MA (see also transplantation) MS-TCR Connetics vaccine multiple sclerosis hase III Palo Alto, CA Myotrophin (R) growth amyotrophic lateral sclerosis application rhGF-1 West Chester, PA actor submitted Chiron peripheral neuropathies hase II NeuroCelTM-FE cellular focal epilepsy hase I (cellular harlestown, MA herapy transplan ation therapy) NeuroCe iacrin cellular Huntington's disease (cellular harlestown, MA herapy transplan ation enzyme Tissue Repair therapy) ambridge, MA NeuroCe iacrin cellular Parkinson's disease (cellular harlestown, MA herapy transplan ation enzyme Tissue Repair therapy) ambridge, MA neutrophin-3 mgen growth enteric neuropathics hase III housand Oaks, CA actor egeneron Pharmaceuticals Tarrytown, NY pimagedine Alteon overt neuropathy hase III Ramsey, NJ (see also diabetes) Genentech S. San Francisco, CA prosaptide Myelos Neurosciences growth neuropathic pain and peripheral hase II TX14(A) San Diego, CA factor neuropathy Rebii (R) Serono Laboratories interferon relapsing, remitting multiple sclerosis; application recombinant Norwell, MA transitional multiple sclerosis submitted interferon beta-1a (see also cancer, infectious diseases) ReoPro (E) Centocor MAb stroke Phase II abciximab Malvern, PA (see also heart) Eli Lilly Indianapolis, IN Spheramine TM Titan Pharmaceuticals cellular Parkinson's disease Phase I S. San Francisco, CA therapy Zenapex (R) Hoffmann-La Roche MAb tropical spastic paraparesis Phase I/II daclizumab Nutley, NJ (model for multiple sclerosis) Protein Design Labs (see also cancer, eye, skin, Mountain View, CA transplantation RESPIRATORY DISEASES

AAV CFTR Targeted Genetics gene therapy sinusitis Phase I gene therapy Seattle, WA (see also genetic) acellular pertussis Chiron vaccine pediatric pertussis application vaccine Emeryville, CA (whooping cough) submitted US 7,001,892 B1 SS S6

TABLE A-continued anti-IgE Genentech MAb allergic asthma Phase III humanized MAb S. San Francisco, CA allergic rhinitis Phase II Novartis Pharmaceuticals East Hanover, NJ Tanox Biosystems Houston, TX Influenza rhAO Protein Sciences vaccine prevention of influenza Phase II Vaccine Meriden, CT influenza vaccine influenza virus Aviron vaccine prevention of influenza Phase III vaccine Mountain View, CA (live, attenuated) interleukin-4 ImmuneX recombinant asthma Phase I receptor Seattle, WA soluble receptor interleukin-10 Schering-Plough interleukin acute lung injury Phase I (IL-10) Madison, NJ (see also AIDS/HIV, autoimmune, digestive, heart, neurologic, skin) Iisolyline Cell Therapeutics acute lung injury Phase II Seattle, WA (see also other) NEUPOGEN (R) Amgen colony multilobar pneumonia, Phase III Filgrastim Thousand Oaks, CA stimulating pneumonia sepsis (rG-CSF) factor (see also AIDS/HIV, cancer) Oxsodrol (R) bio-Technology General dismutase bronchopulmonary dysplasia Phase III rhCuZr super Iselin, NJ in premature infants dismutase parainfluenza Aviron vaccine prevention of parainfluenza type-3 Phase II type-3 vaccine Mountain View, CA infection (cause of croup in infants) (live, attentuated bovine) PIV vaccine, Wyeth-Lederle continuous prevention of parainfluenza Phase I live, attentuated Vaccines & Pediatrics cell line virus-mediated lower respiratory Philadelphia, PA vaccine disease in infants Quillerimmune-F Aquila Biopharmaceuticals vaccine pneumococcal infections in Phase II Worcester, MA the elderly recombinant ICOS acute respiratory distress syndrome, Phase II platelet activating Bothell, WA asthma factor (see also digestive) acetylhydrolase (rPAF-AI-1) RSV Subunit Wyeth-Lederle continuous prevention of respiratory syncytial Phase II vaccine Vaccines & Pediatric cell line virus-mediated lower respiratory Philadelphia, PA vaccine disease in the elderly and at-risk children RSV vaccine, Wyeth-Lederle continuous prevention of respiratory syncytial Phase I live, attenuated Vaccines & Pediatrics cell line virus-mediated lower respiratory Philadelphia, PA vaccine disease in infants soluble ICAM-1 Boehringer Ingelheim recombinant prevention and/or treatment of Phase II (BIRR4) Pharmaceuticals soluble rhinovirus-induced common cold Ridgefield, CT receptor Synagis TM Mediummune MAb prevention o respiratory syncytial application MED-493 Gaithersburg, MD virus disease submitted humanized RSV MAb TP10 T Cell Sciences recombinant acute respiratory distress syndrome Phase II Needham, MA soluble (see also heart, transplantation) receptor truncated ICAM Bayer adhesion rhinovirus-associated Phase I Berkeley, CA molecule exacerbations of asthma SKINDSORDERS anti-CD11a Genentech Mab moderate to severe psoriasis Phase II humanized MAb S. San Francisco, CA (hu1124) XOMA Berkeley, CA gamma interferon Connetics interferon iceloids Phase II Palo Alto, CA ICM3 ICOS Mab psoriasis Phase I Bothell, WA IL-2 fusion protein Seargen fusion moderate to severe psoriasis Phase I/II DABIL-2 Hopkinton, MA protein (see also autoimmune, cancer) interleukin-10 Schering-Plough interleukin psoriasis Phase I (IL-10) Madison, NJ (see also AIDS/HIV, autoimmune, digestive, heart, neurologic, respiratory) IRSO2 Immune Response Corp. vaccine psoriasis Phase II therapeutic Carlsbad, CA vaccine ISIS 23O2 Isis Pharmaceuticals antisense psoriasis Phase II US 7,001,892 B1 57 58

TABLE A-continued Carlsbad, CA (see also autoimmune, digestive, transplantation) keratinocyte Human Genome Sciences growth wound healing Phase I growth factor-2 Rockville, MD factor (see also other) (KGF-2) LFA3TIP Biogen recombinant psoriasis Phase II Cambridge, MA T-cell inhibitor Regranex TM Chiron growth pressure ulcers Phase III becaplernin Emeryville, CA factor (see also other) (recombinant R. W. Johnson human Pharmaceutical platelet-derived Research Institute growth factor-88) Raritan, NJ T4N5 Liposome Applied Genetics protection against actinic keratoses Phase III Lotion Freeport, NY in patients with Xeroderma T4 endonuclease V pigmentosa encapsulated in liposomes TGF-beta3 OSI Pharmaceuticals growth impaired wound healing Phase II Uniondale, NY factor (see also other) transforming Novartis Pharmaceuticals growth wound healing Phase II growth East Hanover, NJ factor factor-beta-3 Zenapax (R) Hoffmann-La Roche MAb psoriasis Phase I/II daclizumab Nutley, NJ (see also cancer, eye, neurologic, Protein Design Labs transplantation) Mountain View, CA TRANSPLANTATION allogeneic SyStemix cellular correct genetic diseases by in utero Phase I hematopoietic Palo Alto, CA therapy transplantation of genetically stem cells unaffected cells from a sibling or parent CBL antibody Abgenix Mab graft versus host disease Phase II Foster City, CA Bristol-Myers Squibb recombinant immunosuppression PHase II Princeton, NJ soluble receptor HSD-Tk Genetic Therapy gene therapy treatment of graft versus host disease Phase I retroviral vector Gaithersburg, MD in allogeneic hematopoietic SyStemix stem cell transplantation Palo Alto, CA HSV-tk Chiron gene therapy graft versus host disease Phase I Emeryville, CA in bone marrow transplantation ISIS 23O2 Isis Pharmaceuticals antisense renal transplant rejection Phase II Carlsbad, CA (see also autoimmune, digestive, skin) LDP-O1 LeukoSite MAb kidney transplantation Phase I/II Cambridge, MA (see also neurologic) MED-507 Medimmune MAb graft versus host disease Phase II (humanized Gaithersburg, MD acute kidney transplant rejection Phase I/II MAb) BioTransplant Charlestown, MA ORTHOCLONE Ortho Biotech MAb prevention of organ transplant Phase II OKT4A Raritan, NJ rejection (see also autoimmune) Simulect Novartis Pharmaceuticals MAb transplantation application basiliximab East Hanover, NJ submitted SMART TM Anti-CD3 Protein Design Labs MAb organ transplantation Phase I HM291 Mountain View, CA (see also autoimmune) TP10 T Cell Sciences recombinant transplantation Phase I/II Needham, MA soluble (see also heart, respiratory) receptor Zenapax (R) Hoffmann-La Roche MAb liver transplantation Phase II daclizumab Nutley, NJ (see also cancer, eye, neurologic, skin) Protein Design Labs pediatric kidney transplantation Phase I/II Mountain View, CA Zenapax (R) Hoffmann-La Roche MAb kidney transplant rejection, Phase I/II daclizumab Nutley, NJ cyclosporine elimination and Protein Design Labs Cellcept (R) Mountain View, CA OTHER

Recomburmin Centeron excipient use Phase I recombinant King of Prussia, PA human albumin Regranex TM Chrion growth venous ulcers Phase III becaplermin Emeryville, CA factor (see also skin) (recombinant R. W. Johnson US 7,001,892 B1 60

TABLE A-continued human Pharmaceutical platelet-derived Research Institute growth factor-88) Raritan, NJ rhEBMP-2 Genetics Institute growth bone and cartilage repair in clinical Cambridge, MA actor trials Saizen (R) Serono Laboratories al chronic renal failure in children Phase III somatropin Norwell, MA growth (see also growth disorders) (rDNA origin) OOC post-operative recovery Phase II for injection Serostint TM Serono Laboratories al metabolic conditions Phase II somatropin Norwell, MA growth (see also cancer) (rDNA origin) OOC for injection Somatokine (R) Celtrix Pharmaceuticals growth hip fractures, severe acute burns Phase II recombinant Santa Clara, CA actor insulin-like growth factor-1/ binding protein-3 TGF-beta3 OSI Pharmaceuticals growth oral mucositis Phase II Uniondale, NY actor (see also skin) The content of this survey has been obtained through government and industry sources based on the latest information. Survey current as of Mar. 13, 1998. The information may not be comprehensive. For more specific information about a particular product, contact the individual company directly. PhRMA Internet address: http://www.phrma.org Provided as a Public Service by PhRMA. Founded in 1958 as the Pharmaceutical Manufacturers Association. Copyright (C) 1998 by the Pharmaceutical Research and Manufacturers of America. Permission to reprint is awarded if proper credit is given.

In one aspect, particular benefit is obtained with this pharmaceutical compositions, depending on the application, invention when used with biopharmaceuticals, which route of delivery, and desired pharmacological profile. Pre include, for example, any proteins, polypeptides, enzymes, ferred percentages include, for example, concentrations of immunoglobulins, polynucleic acids, and plasmids or other 0:01-1 weight percent. AS used herein, all weight percent biopolymers. Specific examples of biopharmaceuticals to be ages are given as the percent based on the weight of the included in the crystal formulations of the present invention crystal including the crystal lattice component, the active include the following: insulin, glucagon, Glucagon-Like pharmaceutical ingredient and any other components Peptide-1 (7-37)OH (GLP-1), human growth hormone, lep 35 included within the crystals, unless Stated otherwise. tin, follicle-stimulating hormone (FSH), ribozyme, and ana The crystals may be prepared at varying Size distributions, logs thereof. Similarly depending on the Subsequent formulating to be The API's useful with the present invention include those done with the crystals, or on crystal growth parameters. The which themselves may form crystalline products, as well as 40 crystals may be harvested and then Sorted directly to desired those which do not. By way of example, any proteins can be Size ranges, or may first be processed, Such as by grinding prepared as microcrystalline Suspension products, but the or milling, and then Sorted Such as by Sieving. AS will be results have frequently been unsatisfactory using existing appreciated, a desired amount of active pharmaceutical technology. However, inclusion of these biomolecules into a ingredient may be obtained simply by obtaining a deter host crystal System in accordance with the present invention 45 mined weight of crystals containing the active pharmaceu overcomes this limitation on crystallization. The invention tical ingredient at a known weight concentration. The useful further finds utility even with API's that are readily crystal Size or weight range of the crystals of the present invention lized, such as insulin. The incorporation of such API's into accordingly varies widely, depending on Such factors as the a single crystal lattice can be used to enhance Stability or inclusion level of the active pharmaceutical ingredient, the provide means of delivery that have different characteristics. 50 dosage amount for the active pharmaceutical ingredient, and Solvents for preparation of the Saturated and SuperSatu the method of delivery of the crystals. By way of example, rated crystal lattice component include, but are not limited Suitable crystals may have an average size distribution of 1 to, water alcohols (e.g., ethanol, isoproponal), other organic tim to 1 mm. Solvents, acids, bases, and buffers. The crystals of the present invention will typically be used The crystals of the present invention are prepared to have 55 in a formulation comprising a large number of crystals. It is a predetermined amount of active pharmaceutical ingredi a feature of the present invention that the active pharma ent. The desired amount of active pharmaceutical ingredient ceutical ingredient is included within the crystal lattice will depend on typical considerations, Such as the effective component in a predictable, oriented fashion. This leads to amount of API used for administering to a patient. The a uniform concentration of the active pharmaceutical ingre concentration of API in the crystal is controlled, such as by 60 dient as a molar, and therefore weight, percentage of the previously described means, to yield crystals which are crystals. In one aspect of the present invention, there is readily used in preparing pharmaceutical formulations for provided a composition of crystals having a Substantially administration. The active pharmaceutical ingredient can be uniform weight concentration of active pharmaceutical incorporated into the crystals at any of a wide variety of ingredient as between crystals. The term “Substantially uni molar or weight percentages. Preferred percentages can be 65 form weight concentration” refers to the fact that the weight easily Selected by a skilled artisan taking into account the concentration of active pharmaceutical ingredient in the usual considerations for later formulation of the desired various crystals is Sufficiently uniform that an acceptably US 7,001,892 B1 61 62 accurate weight of active pharmaceutical ingredient can be release the active pharmaceutical ingredient only or prefer obtained based on the weight of the crystals and the average ably in a particular part of the intestinal tract or other route concentration of active pharmaceutical ingredient in Such of administration, possibly over a period of time. This is crystals. In one preferred embodiment, there is provided a accomplished, in known fashion, using coatings, envelopes, composition of crystals in which the size distribution of and protective matrices made, for example, from polymeric active pharmaceutical ingredient does not vary between Substances or waxes. crystals by more than about 20 percent. However, alternate it is a feature of one aspect of the present invention that embodiments may be equally useful, including mixtures of the crystals and included API's may be packaged and different size crystals. A desired quantity of active pharma administered to patients in discrete pharmaceutical dosage ceutical ingredient is then accurately obtained by measuring forms. The crystals may be used as Such in Solid form, or a weight amount of crystals which, given the concentration may be formulated into liquid Solutions or Suspensions prior of active pharmaceutical ingredient, yields the Selected to use. The compositions may accordingly be administered weight of active pharmaceutical ingredient. by various routes, for example, by the oral, rectal, vaginal, The crystals and included API's are useful in the crystal ocular, buccal, nasal, pulmonary, iontophoretic, topical or form for both the stabilization and storage of the API and for 15 parenteral routes. Such compositions form part of the the administration of the API to a patient. As used herein, it present invention and are prepared in manners well known will be appreciated that the term patient refers to either in the pharmaceutical art. humans or non-humans, depending on the nature of the The API's of the present invention are effective over a active pharmaceutical ingredient. The crystals may be used varied dosage range. Such dosages are readily accommo as Such, and in one aspect of the present invention the dated by the present invention by permitting various sizes of crystals consist essentially of Simply the crystal lattice crystals, concentrations of API, etc. It will be understood component and the API. Alternatively, the crystals include that the amount administered will be determined in light of the crystal lattice component and the API in combination the relevant circumstances, including the condition to be with other pharmaceutically-acceptable adjuvants also con treated, the choice of API to be administered, the size of the tained within the crystals. 25 patient being treated, and the chosen route of administration. The crystals of the present invention are preferably for Therefore, Specific dosage ranges will differ accordingly, mulated as pharmaceutical materials for ultimate delivery in and are not limiting of the Scope of the invention in any way. Solid or liquid form. In Such applications, the crystals are The compositions are formulated in one embodiment as a typically formulated with common, compatible, pharmaceu unit dosage form. The term “unit dosage form” refers to tically-acceptable adjuvants, Such as excipients, diluents, physically discrete units, Such as tablets, capsules, and carriers or mixtures thereof. For purposes herein, the term Suspensions in Vials or cartridge/pen Systems Suitable as "pharmaceutically-acceptable” refers in this context to the unitary dosages, particularly as unitary daily dosages. Each excipients, diluents or carriers, as well as coatings or other discrete unit contains a predetermined quantity of active components referred to elsewhere, being compatible with pharmaceutical material calculated to produce the desired the other ingredients of the formulation and no deleterious to 35 effect, e.g., a prophylactic or therapeutic effect. The amount the recipient thereof. of active pharmaceutical ingredient contained in a given Examples of excipients, diluents, and carriers that are dosage unit can be varied depending on the manner of Suitable for Such dosage forms are well known in the art, and delivering the crystals. For example, a Single dosage unit in include the following: Suspension additives Such as tonicity tablet form may contain /4, /3, /3 or 1 times the unit dose modifiers, buffers, precipitants, and preservatives, fillers and 40 for the active pharmaceutical ingredient, according to which extenderS Such as Starch, lactose, dextrose, Sucrose, Sorbitol, 1 to 4 tablets would be administered to achieve a unit does mannitol, and Silicic derivatives, binding agents Such as of the active pharmaceutical ingredient. carboxymethyl cellulose and other cellulose derivatives, Therefore, in one aspect of the present invention, there is alginates, gelatin, and polyvinyl pyrrollidone, moisturizing provided a pharmaceutical product in dosage form compris agents Such as glycerol, disintegrating agents. Such as cal 45 ing a pharmaceutical delivery unit including a dosage cium carbonate and Sodium bicarbonate, agents for retarding amount of active pharmaceutical ingredient. The API is dissolution Such as paraffin, resorption acceleratorS Such as contained within the crystal lattice component, and a Suffi quaternary ammonium compounds, Surface active agents cient amount of crystals is included within the delivery unit Such as cetyl alcohol and glycerol monoStearate, adsorptive to constitute the dosage amount of the API. It will be carrierS Such as kaolin and bentonite; and lubricants Such as 50 appreciated that the dosage amount of pharmaceutical may talc, calcium and magnesium Stearate, and Solid polyethyl be obtained by provision of one or more crystals of the glycols. Additionally, the adjuvant may comprise crystals of present invention. One form of the product consists essen the crystal lattice component that are prepared without the tially of a dosage amount of the crystals. In an alternative included API. form, the pharmaceutical product consists of the dosage The crystals may be coated to achieve various effects. In 55 amount of the crystals. one approach, the crystals are coated with the same crystal The ultimate delivery forms may include, for example, lattice component which forms the underlying crystal, but tablets, Soft and hard gelatin capsules, pellets, granules, without the include API. This assures that the coating and the marumes, lozenges, Sachets, cachets, elixirs, Suspensions, underlying crystal have compatibility. The coating is then ointments, Suppositories, injection Solutions and Suspen applied at a thickneSS which provides the desired effect, Such 60 Sions, nonpareils, Spheres and Sterile packaged powders. The as further protection of the active pharmaceutical ingredient, crystals may be coated or uncoated, and may be combined bulking of the crystal for handling, and/or effecting a SuS with various pharmaceutical adjuvants, including excipients, tained or delayed release of the active pharmaceutical ingre diluents and carriers, as already described. One preferred dient. Alternatively, the same effects can be accomplished by form of the pharmaceutical product consists essentially of coating the crystals with other compatible coating compo 65 the crystals, and an alternate form consists of the crystals Sitions, Such as those which are well known in the pharma and the pharmaceutically-acceptable adjuvants. The delivery ceutical coating art. The crystals can also be coated So as to forms are prepared by conventional techniques Such as US 7,001,892 B1 63 64 disclosed in Remington's Pharmaceutical Sciences, 19th Enteric formulations are used to protect crystals and the Edition, Mack Publishing Company, Easton, Pa. (1995), included APIs from the strongly acidic contents of the which is incorporated herein by reference, or other treatises Stomach. Such formulations are created by coating a Solid available to the skilled artisan. dosage form with a film of a polymer which is insoluble in Compressed tablets, for example, are prepared by well acidic environments, and Soluble in basic environments. known means which are conventional in the art. The tablets Exemplary films are cellulose acetate phthalate, polyvinyl may be prepared by wet or dry granulation methods or by acetate phthalate, hydroxypropyl methylcellulose phthalate direct compression, and may be produced by any of a wide and hydroxypropyl methylcellulose acetate Succinate. variety of tabletting machines. Tablet formulations usually The crystals with included APIs may similarly be for incorporate diluents, binders, lubricating and disintegrators, mulated into capsules for administration. Such capsules are as well as the crystals with included API's. Typical diluents prepared utilizing conventional encapsulating methods. A include, for example, various types of Starch, lactose, man general method of manufacture involves preparing the crys nitol, kaolin, calcium phosphate or Sulfate, inorganic Salts tals for use in capsules, Such as by milling the crystals to a Such as Sodium chloride, and powdered Sugar. Powdered suitable size. The crystals are blended with desired excipi cellulose derivatives are also useful. Typical tablet binders 15 ents, diluents or carriers, and the resulting mixture is filled are Substances Such as Starch, gelatin, and SugarS Such as into Suitably-sized capsules, typically hard gelatin capsules, lactose, fructose, glucose and the like. Natural and Synthetic using conventional capsule-filling machines. The usual dilu gums are also convenient, including acacia, alginates, meth ents include inert powdered Substances Such as Starch of ylcellulose, polyvinylpyrrolidine and the like. Polyethylene many different kinds, powdered cellulose, especially crys glycol, ethylcellulose and waxes can also serve as binders. talline and microcrystalline cellulose, SugarS Such as fruc Certain Solid pharmaceutical dosage forms of the present tose, mannitol and Sucrose, grain flours and Similar edible invention, most notably tablets, may be coated in conven powers. tional fashion with a wide variety of materials utilizing When it is desired to administer the crystal formulations various processes. Typically, the products of the present as a Suppository, the usual bases may be used. Cocoa butter invention may be Sugar coated or film coated in accordance 25 is a traditional Suppository base, which may be modified by with well-known techniques. The coatings Serve as aesthetic addition of waxes to raise its melting point slightly. Water purpose as well as a pratical one. Coatings can mask an miscible Suppository bases comprising, particularly, poly unpleasant taste or odor, can increase ease of ingestion by ethylene glycols of various molecular weights are also in the patient, and can Serve to improve the ultimate appear wide use. ance of the dosage form. Similarly, coatings can protect the The crystals can also be similarly formulated as elixirs or product from the effects of air, moisture and light, can Suspensions for convenient oral administration or for improve product identification, and can facilitate handling in parenteral administration, for instance by intramuscular, packaging and fill lines during manufacture. Subcutaneous or intravenous routes. Various adjuvants may be included in the coating formu The inventive crystals enable the design of Sustained lations as is well known in the art. These include, for 35 release formulations based upon various factors to yield both example, permeability enhancers, plasticizers, antitacking the desired amount of active pharmaceutical ingredient and agents and the like. A discussion of coating techniques and the desired pharmacokinetic profile for delivery of the active adjuvants is presented in U.S. Pat. No. 5,015,480, issued to pharmaceutical ingredient. Selectively incorporating the Childers et al. on May 14, 1991, the pertinent portions of active pharmaceutical ingredient into the crystal lattice, e.g., 40 into a specific crystal growth Sector, modulates the release which are hereby incorporated herein by reference. Further profiles and can therefore be used to effect desired pharma information pertinent to coating processes and equipment cological properties. The choice of the crystal component may be obtained from Remington's Pharmaceutical Sci and the process used to grow the crystals of excipient host ences, Supra. and guest active pharmaceutical ingredient can be Selected Tables are often coated with Sugar as a flavorant and 45 and/or modified to adjust parameterS Such as the delivery Sealant, or with film-forming protecting agents to modify the rate of the active pharmaceutical ingredient upon use of the dissolution properties of the tablet. The compounds may also formulation. The active pharmaceutical ingredient is incor be formulated as chewable tablets by using large amounts of porated into the crystal matrix at a Selected rate, typically as pleasant-tasting Substances Such as mannitol in the formu only a Small weight percentage of the overall crystal. The lation, as is now well-established practice. Instantly dissolv 50 permits moderate and uniform rates of release. ing tablet-like formulations are also now frequently used to Various approaches may be used to accomplish a delayed assure that the Subject consumes the dosage form, and to or Sustained release of active pharmaceutical ingredient avoid the difficulty in Swallowing solid objects that bothers from the crystals. In a typical application the crystal of the Some Subjects. desired size are combined with a compatible preservative A lubricant is used in a tablet formulation to prevent the 55 and the mixture is injected Subcutaneously or Surgically tablet and punches from Sticking in the die of the tabletting implanted to provide a prolonged payout as the crystals machine. The lubricant is chosen from Such slippery Solids dissolve as a result of contact with the Surrounding body as talc, magnesium and calcium Stearate, Stearic acid and tissue and fluid. In one approach, the concentration of the hydrogenated vegetable oils. active pharmaceutical ingredient in the crystals is reduced in Tablet disintegrators are substances which Swell when 60 order to effect a Sustained release over time. Alternatively, wetted to break up the tablet and release the crystals. They larger crystals may be used to provide for more prolonged include Starches, clays, celluloses, algins and gums. More payout of the active pharmaceutical ingredient. In another particularly, corn and potato Starches, methylcellulose, agar, approach, coatings on the crystals are used to affect the rate bentonite, wood cellulose, powdered natural Sponge, cation of release of the active pharmaceutical ingredient. Such eXchange resins, alginic acid, guar gum, citrus pulp and 65 coatings may comprise the same crystal lattice component carboxymethylcellulose, for example, may be used, as well but without the included active pharmaceutical ingredient, as Sodium lauryl Sulfate. as well as other coating compositions useful for this purpose. US 7,001,892 B1 65 66 In the alternative, the crystals of the present invention can {0-11. Small (0-10) and {1-50 faces are also occasion be used to isolate and/or Store the active pharmaceutical ally present. When illuminated with a long wavelength UV ingredient for later reconstitution into Solution. The crystals lamp, the crystals exhibited a bright green fluorescence may be Stored for extended periods of time prior to recon localized within a sharply defined pyramid corresponding to stitution in view of the added stability accorded the APIs by the (010) growth sector. This indicates that GFP is selec the encompassing crystal lattice component. The crystals are tively recognized and overgrown by the (010) face in then combined with pharmaceutically-acceptable excipients, preference to the others. More importantly, it is evidence that diluents or carriers to prepare the Solutions for Subsequent the GFP is in its native conformation. The level of GFP to administration. The crystals are readily dissolved or SuS lactose is approximately 0.008% (w/w). pended in appropriate diluents, which may be Selected, for GFP fluorescence intensity was measured as a function of example, from the list previously provided with regard to time and temperature in three environments: Saturated acque diluents used to initially prepare the crystals. ous C.-lactose Solution, lyophilized C-lactose, and crystalline Such solutions of dissolved crystals provide the active C.-lactose monohydrate. As shown in FIG. 2, both the pharmaceutical ingredient free of the previously encompass Solution and lyophilized preparations lost nearly half of the ing crystal lattice component. The Solutions are useful, for 15 fluorescence intensity at 333 K. within one hour. The example, for oral administration, parenteral use, or as Sup crystal showed no change at 333 K. or even 343 K. positories. For parenteral administration, for example, the crystals may be formulated in a pharmaceutically-acceptable EXAMPLE 2 diluent such as physiological saline (0.9%), 5% dextrose, Ringer's Solution, and the like, along with other additives to To investigate the potential for incorporation of a biop reduce the Solubility of the crystals in Suspension. harmaceutical into crystals of biocompatible excipients, The resulting pharmaceutical formulations provide an Studies were conducted using rhodamine-labeled glandular active pharmaceutical ingredient which is included within glucagon and lactose. AS in the previous Studies, the the host crystal and has enhanced stability and shelf-life. The rhodamine label was used to facilitate the visualization of present invention therefore Satisfies the desire to provide 25 glucagon in the host crystals. Typical crystal growth condi certain pharmaceuticals having an acceptable, room-tem tions involved the addition of 5 volumes of a Supersaturated perature shelf-life. Depending on the circumstances, par Solution of deionized C-lactose monohydrate to 1 Volume of ticularly the API involved, the desired shelf-life can be as an approximately 1.5 mg/mL (approximately 300 to 400 little as one month, or may be at least one year, two years or almole) of rhodamine-labeled glucagon in purified water. more. The pharmaceutical molecules are generally isolated The mixed Solution was allowed to Stand at room tempera from one another and from the environment by the surround ture in a 24-well plate. Crystals were harvested between 1-3 ing crystal lattice. The containment of the API in the solid days and displayed a hatchet morphology with a broad base. crystal lattice also fixes the conformational orientation. This With the rhodamine label, glucagon inclusion was visible in eliminates most of the potential degradation mechanisms, the crystals as a well-defined pyramid corresponding to the Such as polymerization, oxidation, deamidation and pro 35 (010) growth sector. The level of inclusion was determined teolysis, that could otherwise reduce the stability of the to be approximately 0.1% (w/w). pharmaceutical. In-vitro dissolution experiments were performed on the Methods demonstrating stability include but are not lim glucagon/lactose crystals to evaluate potential for in-vivo, ited to high-performance liquid chromatography for purity Sustained-release pharmacokinetics. The release of and potency, FT-IR for Secondary Structure, in-vitro and 40 rhodamine-labeled glucagon into Solution was followed by in-vivo bioassays, and pharmacokinetic profiles. fluorescence spectroScopy. In a typical experiment, 1-2 The crystals of the present invention are readily prepared crystals were added to 100 microliters of phosphate buffered and are useful in containing the included API in an isolated, Saline Solution at room temperature and the increase in oriented position within the lattice. The utility of the present fluorescence of the Solution was monitored over time. The invention is demonstrated in the following examples, which 45 release of glucagon from the dissolving crystals was gener are illustrative in nature, and are not to be considered ally complete after 24–48 hours depending on crystal size limiting of the Scope of the present invention. and was linear until the last few hours of dissolution. Additional details are contained in the article entitled “Sta EXAMPLE 1. bilization of Proteins in Single Crystal Hosts: Green Fluo 50 rescent Protein and C.-Lactose Monohydrate,” M. Kurimoto, To demonstrate the potential kinetic Stabilization of pro P. Subramony, R. Gurney, S. Lovell, J. A. Chmielewski, B. teins, green fluorescent protein (GFP) was incorporated into Kahr, J. Am. Chem. Soc. 1999, 121, 6952-6953, which deionized C-lactose monohydrate. GFP was selected article is hereby incorporated herein by reference. because it is known to fluoresce only in its native confor mation. Upon denaturation, the interior of the B-barrel of the 55 EXAMPLE 3 molecule is exposed and the fluorescence of the p-hydroxy benzylideneimidazolinone chromophore is rapidly To demonstrate the universality of this technology for quenched. Typical crystal growth conditions involved the incorporation of a diversity of biopharmaceuticals into crys addition of 8 volumes of an approximately 1 mg/mL (ap tals of biocompatible excipients, Studies were conducted proximately 37 umole) solution of GFP in 10 mM tris-HCl, 60 using biosynthetic human insulin and insulin analogs, pH8 and 10 mM EDTA to 100 volumes of a Supersaturated V8-GLP-1 (7-37)OH, a glucagon-like insulinotropic pep aqueous Solution (approximately 1.15 M) of deionized tide-1 analog, exendin, and human growth hormone in C.-lactose monohydrate. The mixed Solution was allowed to deionized C-lactose monohydrate or phthalic acid. Informa Stand for 3–4 days at room temperature in a 24-well plate. tion regarding V8-GLP is available in U.S. Pat. No. 5,705, Crystals were harvested between 1-3 days and displayed a 65 483, issued to Galloway and Hoffman on Jan. 6, 1998, which hatchet morphology as shown in FIG. 1 with a broad base patent is hereby incorporated herein in its entirety. For (010) further bounded by 100}, {110}, {1-10}, and information regarding eXendin, See, e.g., R. Goke, H. C. US 7,001,892 B1 67 68 Fehmann, T. Linn, H. Schmidt, M. Krause, J. Eng, B. Goke, entirety: M. Windholz, (editor). Merck Index, 10" edition, p. “Exendin-4 is a High Potency Agonist and Truncated Exen 769; R. A. Visser, Neth. Milk Dairy Journal, 34, 1980, pp. din-(9-39)-amide an Antagonist at the Glucagon-like Pep 255-275; J. Chmielewski, et al., JACS, 119, 43, pp. tide 1-(7-36)-amide Receptor of Insulin-secreting Beta 105665-10566. cells,” J. Biol. Chem. 1993, Sep 15, 268(26), pp. 19650-5, What is claimed is: which reference is hereby incorporated herein in its entirety. 1. A pharmaceutical composition comprising: Typical crystal growth conditions involved the addition of a single crystal of a pharmaceutically-acceptable crystal 1 Volume of an approximately 10 mg/mL rhodamine- or lattice component; and Texas red-labeled peptide or protein in 0.1M phosphate an active pharmaceutical ingredient different from and buffered saline solution (PBS, pH7.4) to 10 volumes of a 1O included within the crystal in a growth-sector Specific SuperSaturated C-lactose Solution or phthalic acid Solution. orientation, the crystal lattice component and the active SuperSaturated Solutions of purified C-lactose were obtained pharmaceutical ingredient being pharmaceutically by adding 0.41 grams of C-lactose to 1 mL of purified water, pure, allowing to dissolve in a 50-70° C. water bath, and cooling wherein Said crystal lattice component consists essentially to room temperature. SuperSaturated Solutions of phthalic 15 acid were prepared by adding 0.05 grams of phthalic acid to of lactose. 1 mL of either 70/30 (v/v) water/acetonitrile or 90/10 2. The invention of claim 1 and further comprising a water/ethanol, allowing to dissolve in a 50-70° C. water pharmaceutically-acceptable adjuvant Selected from the bath, and cooling to room temperature. Larger Volumes of group consisting of excipients, diluents, carriers and mix SuperSaturated Solutions are obtained by using the same tures thereof. Solute-to-Solvent ratio. 3. The invention of claim 1 in which the active pharma The solutions of labeled peptide or protein with the ceutical ingredient is a biopharmaceutical. SuperSaturated C-lactose or phthalic acid were mixed by 4. A pharmaceutical material comprising: Swirling, transferred to a 24-well crystallization plate or a mixing of Single crystals, each crystal comprising a other Suitable glass or polypropylene container, and allowed 25 pharmaceutically-acceptable crystal lattice component to Stand at room temperature. Crystals were harvested in 4-5 and an active pharmaceutical ingredient different from days and rinsed with hexanes, ethanol, or methanol. All and included within the crystal in a growth-sector preparations yielded crystals with dye-labeled protein inclu Specific orientation, the crystal lattice component and Sions as determined by microscopic examination using an the active pharmaceutical ingredient being pharmaceu Olympus SZ-40 microscope with a CCD vision camera. tically pure, The shape of the crystals formed was dependent on the wherein Said crystal lattice component consists essentially solvent system used for the phthalic acid. The crystals of lactose. formed with phthalic acid in water/ethanol were long, petal 5. The pharmaceutical material of claim 4 in which the shaped clusters. The crystals formed with water/ethanol crystals comprise at least two crystal lattice components, the were smaller and rhombic. Crystals of labeled-insulin/lac 35 first crystal lattice component being characterized by first tose were dissolved in PBS and analyzed by HPLC. The pharmacokinetics and the Second crystal lattice component level of insulin inclusion was determined to be approxi being characterized by Second pharmacokinetics. mately 0.1%. This process is scalable from 100 lull to several 6. The pharmaceutical material of claim 4 in which said liters. The larger Volume crystallizations were performed mixture comprises a mixture of two different types of Said using glass beakers, or other appropriate large containers, 40 crystals, the first type of the crystal comprising a first crystal covered with watch glasses. lattice component and the Second type of the crystals com Using the same process, unlabeled insulin and eXendin prising at least one crystal lattice component different from have also been incorporated into C.-lactose monohydrate and the first crystal lattice component. phthalic acid crystals. Upon dissolution of the crystals with 7. The pharmaceutical material of claim 4 in which the 0.01N HCl, purified water and/or methanol, the level of 45 active pharmaceutical ingredient comprises discrete units peptide included in these hosts was determined by analysis and the units are included within the crystals in isolation of the sample solutions with an HPLC system in the flow from one another. injection analysis mode using a chemiluminescent nitrogen 8. The pharmaceutical material of claim 4 in which the specific detector (CLND). The level of peptide inclusions active pharmaceutical ingredient is included within the ranged from approximately 0.1% to 10% (w/w). These data 50 crystal at a concentration of about 0.001 to 1 weight percent demonstrate that the level of inclusion can be manipulated based on the weight of the crystal including the active by appropriate choice of guest and host molecules in addi pharmaceutical ingredient. tion to crystallization conditions. See also the following reference which are hereby incorporated herein in their k k k k k